Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Demonstration of GnRH-R in bovine uterus and oviducts and GnRH agonist (buserelin) induced in vitro regulation… Singh, Ravinder 2009

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2010_spring_singh_ravinder.PDF [ 3.03MB ]
Metadata
JSON: 24-1.0072233.json
JSON-LD: 24-1.0072233-ld.json
RDF/XML (Pretty): 24-1.0072233-rdf.xml
RDF/JSON: 24-1.0072233-rdf.json
Turtle: 24-1.0072233-turtle.txt
N-Triples: 24-1.0072233-rdf-ntriples.txt
Original Record: 24-1.0072233-source.json
Full Text
24-1.0072233-fulltext.txt
Citation
24-1.0072233.ris

Full Text

DEMONSTRATION OF GnRH-R IN BOVINE UTERUS AND OVIDUCTS AND GnRN AGONIST (BUSERELIN) INDUCED IN VITRO REGULATION OF STEROID HORMONE RECEPTORS AND APOPTOSIS IN BOVINE ENDOMETRIUM  by  RAVINDER SINGH B.V.Sc. & AH, H.P. Agriculture University Palampur, India, 1990 M.V.Sc. (Hons.) H.P. Agriculture University, Palampur, India, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Animal Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2009 © Ravinder Singh, 2009  ABSTRACT The presence of GnRH-R in bovine uterus and oviducts and the local modulatory role of the GnRH, GnRH-R system in the uterine physiology is not known. This dissertation reports studies designed to determine the presence of GnRH-R in the bovine uterus and oviducts and further studied GnRH agonist, buserelin induced steroid hormone receptor regulation (ERct, ERf3 and PR) and apoptosis in the follicular and luteal phase bovine endometrium. RT-PCR and immunoblotting revealed GnRH-R mRNA (920 bp) and protein (60 kD), respectively in both the follicular and luteal phase endometrium and oviducts. Immunohistochemistry further localized GnRH-R to the endometrial and oviductal epithelial cells. In the second experiment (Chapter 3) RT-PCR studies on in vitro endometrial explant cultures showed that the GnRH, agonist, buserelin (200 ng/mL) had a stimulatory response (P  0.05) on ERct mRNA in the luteal phase endometrium after 6 h of treatment. The PR  mRNA levels were not changed in these experiments. The last experiment (chapter 4) investigated the effect of buserelin on endometrial apoptosis at the molecular (Bax, Bcl-2, caspase3, Fas mRNA) and cellular level. This part used endometrial explant culture, RT PCR, endometrial epithelial cell culture, immunofluoroscence and apoptotic assay. Buserelin induced an apoptotic response (P  0.05) in the follicular phase endometrium by stimulating  Bax (200 ng/mL) and caspase3 mRNA (200, 500 ng/mL) after 6 h of treatment. The Bcl-2 and Fas mRNA expressions remained unchanged. The buserelin  (200 ng/mL) induced  epithelial cell apoptosis in the follicular phase endometrium at the cellular level, when the cells were treated for 6 h and further allowed to grow for 24 h. The GnRH antagonist-antide did not induce similar effects on ERa mRNA and apoptosis in these experiments. In conclusion, this study a) demonstrated GnRH-R in the follicular and luteal phase bovine  II  endometrium and oviducts, b) elucidated that GnRH agonist, buserelin up-regulates ERcL mRNA in the luteal phase endometrium and, c) induced apoptosis in the follicular phase endometrium. These findings indicate that GnRH supports reproductive process at the endometrial level. These are new findings in the field of reproductive biology. The local modulatory role of GnRH, GnRH-R system in oviducts needs further investigation.  111  TABLE OF CONTENTS  ABSTRACT  .  TABLE OF CONTENTS  ii iv  LIST OF TABLES  viii  LIST OF FIGURES  ix  LIST OF PLATES ABBREVIATIONS  xii  ACKNOWLEDGEMENTS  xiv  CO-AUTHORSHIP STATEMENT  xv  CHAPTER 1- INTRODUCTION AND BACKGROUND INFORMATION 1.1 INTRODUCTION 1.2 GnRH, GnRH-R SYSTEM 1.2.1 GnRH 1.2.2 GnRH structure 1.2.3 GnRH agonists and antagonists 1.2.4 GnRH-R 1.2.4.1 GnRH-R structure and function 1.2.4.2 GnRH agonist and antagonist binding to GnRH-R 1.2.4.3 G-protein coupling 1.2.4.4 GnRH-R internalization 1.2.5 The GnRH and GnRH-R interactions in pituitary 1.2.6 The GnRH, GnRH-R mediated cell signaling 1.2.7 Transcriptional regulation of GnRH-R 1.2.8 The GnRH and GnRH-R system in extra-pituitary reproductive tissues 1.2.8.1 GnRH, GnRH-R system at the gonadal level 1.2.8.2 Oocytes, sperm and embryos 1.2.8.3 Uterus and oviducts 1.2.8.4 Placenta 1.2.8.5 Mammary gland 1.2.8.6 GnRH and GnRH-R in ovarian and endometrial carcinomas 1.2.8.7 Prostate and prostate cancer 1.2.9 Extrapituitary GnRH and GnRH-R interactions and biological actions 1.2.9.1 Gonadal steroidogenesis  1 1 4 4 5 7 8 10 13 15 16 17 18 19 22 22 25 26 27 27 28 29 29 31 iv  1.2.9.2 Steroid hormone receptor regulation 1.2.9.3 Apoptosis 1.2.9.4 Cell proliferation 1.2.9.5 Fertilization and embryo implantation 1.3 GnRH IN BOVINE REPRODUCTIVE MANAGEMENT 1.3.1 Early embryonic sustainability and pregnancy outcome 1.3.2 Estrous and ovulation synchronization 1.3.3 Postpartum cyclicity 1.3.4 Cystic ovarian condition 1.4 DISSERTATION RATIONALE 1.5 OBJECTIVES 1.6 BIBLIOGRAPHY  33 34 37 37 39 40 41 42 43 44 45 46  CHAPTER 2- GONADOTROPIN RELEASING HORMONE RECEPTOR GENE AND PROTEIN EXPRESSION AND IMMUNOHISTOCHEMICAL LOCALIZATION IN BOVINE UTERUS AND OVIDUCTS 2.1 INTRODUCTION 2.2 MATERIALS AND METHOD 2.2.1 Tissue collection and processing 2.2.2 RNA extraction 2.2.3 RT-PCR 2.2.4 Protein extraction and quantification 2.2.5 Immunoblotting 2.2.6 Immunohistochemistry 2.3 RESULTS 2.3.1 GnRH-R mRNA expression 2.3.2 GnRH-R protein expression 2.3.3 Immunolocalization of GnRH receptors 2.4 DISCUSSION 2.5 CONCLUSIONS 2.6 BIBLIOGRAPHY  67 67 69 69 69 70 72 72 73 74 74 74 75 75 79 83  CHAPTER 3- GnRN AGONIST (BUSERELIN) INDUCED STEROID HORMONE RECEPTOR mRNA REGULATION IN BOVINE ENDOMETRIUM, IN VITRO 3.1 INTRODUCTION 3.2 MATERIALS AND METHODS 3.2.1 Collection and processing of tissues 3.2.2 Endometrial explant culture and treatment 3.2.3 RNA extraction  86 86 88 88 88 89 v  3.2.4 Semiquantative RT-PCR 3.2.5 Statistical analyses 3.3 RESULTS 3.3.1 RT-PCR 3.3.2 GnRH agonist induced regulation of ERa mRNA 3.3.3 GnRH agonist effect on PR mRNA 3.3.4 Follicular vs. luteal phase mRNA levels of ERcL, ERj3 and PR 3.4 DISCUSSION 3.5 CONCLUSIONS 3.6 BIBLIOGRAPHY  .90 92 92 92 92 93 93 93 96 102  CHAPTER 4- GnRH AGONIST (BUSERELIN) INDUCED APOPTOSIS IN BOVINE ENDOMETRIUM, IN VITRO 106 4.1 INTRODUCTION 106 4.2 MATERIALS AND METHODS 108 4.2.1 Collection and processing of tissues 108 4.2.2 Endometrial explants culture and treatment 108 4.2.3 RNA extraction 109 4.2.4 Semiquantative RT-PCR 110 4.2.5 Endometrial epithelial cell isolation, culture and treatment 111 4.2.6 Immunofluroscence 112 4.2.7 Apoptotic assay 113 4.2.8 Statistical analyses 114 4.3 RESULTS 114 4.3.1RT-PCR 114 4.3.2 GnRH agonist induced Baz, Bcl-2 and caspase-3 mRNA regulation 114 4.3.3 Fas mRNA regulation in follicular phase endometrium 115 4.3.4 Immunofluroscence 115 4.3.5 Apoptotic assay 115 4.4 DISCUSSION 115 4.5 CONCLUSION 120 4.6 BIBLIOGRAPHY 128  CHAPTER 5- GENERAL DISCUSSION AND CONCLUSIONS 5.1 GnRH-R IN ENDOMETRIUM AND OVIDUCTS 5.2 GnRH INDUCED ERa REGULATION 5.3 GnRH AND APOPTOSIS 5.4 DISSERTATION STRENGTHS 5.5 DISSERTATION LIMITATIONS  132 133 134 136 138 139 vi  5.6 FUTURE RESEARCH DIRECTIONS 5.7 CONCLUSIONS 5.8 BIBLIOGRAPHY  .139 140 142  APPENDIX I BUSERELIN INDUCED ERcz REGULATION IN LUTEAL PHASE ENDOMETRIUM -  146  vii  LIST OF TABLES  Table 3.1 Primer sequences and RT-PCR conditions for amplifications of ERa, ERf3 and PR nRNA Table 4.1 Primer sequences and RT-PCR conditions for amplifications of Bax, Bcl-2, Caspase-3 and Fas mRNA  97  121  viii  LIST OF FIGURES  Figure 1.1 Schematic representation of molecular structure of mammalian GnRI-I and precursor protein encoded by the GnRH gene  6  Figure 1.2 Schematic representation showing GnRH agonists and antagonists in clinical use and relative substitution of amino acids in the native GnRH sequence in these agonists and antagonist  9  Figure 1.3 Diagrammatic representation of GnRH-R gene and the corresponding regions of the GnRH-R encoded by different exons  12  Figure 1.4 Pictorial representation of two dimensional structure of human GnRH-R  14  Figure 1.5 Schematic representation of cell signalling pathways induced by the GnRH, GnRH-R interaction and biological effects thereof  20  Figure 1.6 Schematic representation showing presence of GnRH-R in various extra-pituitary reproductive tissues  24  Figure 2.1 GnRH-R mRNA expressions in bovine endometrium and oviducts  81  Figure 2.2 GnRH-R protein expressions in bovine endometrium and oviducts  82  Figure 3.1 Representative autoradiograms showing intensities of bands obtained after agrose gel electrophoresis for ERci, ERj3 and PR. G3PDH was used as a housekeeping gene in the experiments 98 Figure 3.2 GnRH agonists, buserelin induced ERcL mRNA expression in luteal phase bovine endometrium  99  Figure 3.3 GnRH agonist, buserelin effect on PR mRNA expression in follicular and luteal phase bovine endometrium  100  Figure 3.4 Relative ERcL, ERI3 and PR mRNA expression in follicular and luteal phase bovine endometrium during in vitro explants culture conditions employed in experiments ..1 01 . .  Figure 4.1 GnRT-1 agonist, buserelin induced regulation of Bax mRNA in follicular and luteal phase endometrium 123 Figure 4.2 GnRI-1 agonist, buserelin effect on Bcl-2 mRNA regulation in follicular and luteal phase endometrium 124  ix  Figure 4.3 GnRH agonist, buserelin induced regulation of caspase-3 mRNA in follicular and 125 luteal phase endometrium Figure 4.4 GnRH agonist, buserelin induced regulation of Fas mRNA in the follicular phase 126 endometrium Figure 4.5 GnRH agonist, buserelin induced apoptosis in endometrial epithelial cells  127  x  LIST OF PLATES  Plate 2.1 Immunohistochemical staining showing localization of GnRH-R in bovine endometrial and oviductal epithelial cells  80  Plate 4.1 Characterization of primary cell monolayers as endometrial epithelial cells  122  Plate 4.2 Acridine-orange-ethidium-bromide stained endometrial epithelial cells to study GnRH agonist, buserelin induced apopotosis at cellular level in bovine endometrium 122  xi  ABBREVIATIONS Al BSA cAMP cDNA CL DAG DEPC DMEM  Artificial insemination Bovine serum albumin Cyclic adenosine monophosphate Complimentary DNA Corpus luteum 1, 2-diacylglycerol Diethylpyrocarbonate Dulbecco’s modified Eagle’s medium  DMSO DNA 2 E EC1,2,3 EGF ER ERK 1/2 FSH FSHI3 GAP G3PDH GPCR GnRH GnRH- 1, 11, 111 GnRH-R GSE  Dimethyl sulfoxide Deoxyribonucleic acid Estradiol-17f3 Extracellular loopl,2,3 Epidermal growth factor Estrogen receptor Extracellular-signal-regulated kinase 1/2 Follicle stimulating hormone Follicle stimulating hormone beta Gonadotropin releasing hormone- associated proteins Glyceraldehydes 3- phosphate-dehydrogenase G protein-coupled receptor Gonadotropin releasing hormone Gonadotropin releasing hormone type 1, 11, 111 Gonadotropin releasing hormone receptor Gonadotrope specific elements  HBBS hCG IC 1,2,3 IM 1P3 JNK LH LHI3 LHRH MAPK MEK mRNA ng  Hank’s balanced salt solution Human chorionic gonadotropin Intracellular loopi,2,3 Intramuscular Inositol 1 ,4,5-trisphosphate c-Jun amino-terminal kinase Luteinizing hormone Luteinizing hormone beta Luteinizing hormone releasing hormone Mitogen-activated protein kinase Mitogen-activated protein kinase kinase Messenger ribonucleic acid Nanogram xii  P 4 P45Oscc PBS 2 PIP PKA PKC PR RAF1 RGS RT-PCR TM 1, 2,3,4,5,6,7  Progesterone Cytochrome P450 side chain cleavage enzyme Phosphate buffered saline Phosphatidylinositol 4, 5-biphosphate Protein kinase A Protein kinase C Progesterone receptor v-raf-1 murine leukemia viral oncogene homolog 1 Regulators of G-protein signalling Reverse transcription polymerase chain reaction Transmembrane domain 1,2,3,4,5,6,7  xlii  ACKNOWLEDGEMENTS I extend my deepest gratitude to Dr. R. Rajamahendran for his mentorship, supervision, encouragement, constructive criticism, and support during this search project. I would especially thank my committee members, Dr. Kim Cheng and Dr. Calvin Roskelley for their valuable inputs and guidance during the course of this thesis. I would always remember Dr. Cheng’s open door policy and valuable advice from time to time. I appreciate Dr. Calvin Roskelley for his benevolence for letting me come to his lab and learn western blotting and immunohistochemistry. I am thankful to Dr. Keith Choi for his support during the most part of this research project. I greatly appreciate the support received from Dr. Mahesh Upadhyaya, Associate Dean, Faculty of Land and Food Systems, and staff graduate program office. I would always be indebted to Dr. Jim Thompson, Associate Dean, Faculty of Graduate Studies for helping me with editing of this manuscript and bringing it to the current shape. I greatly appreciate the financial assistance received from the Elizabeth R. Howland Fellowship, Jim Shelford memorial scholarship and Dr. R. Rajamahendran’s NSERC grants during the course of this project. I am thankful for the assistance rendered by my past and current colleagues in the lab including- Dr. G. Giritharan, Anusha Balendran, Pretheeban, Ruwanie and Miriam. Also the contributions of Manoja in managing RNA extraction work are deeply appreciated. Support and assistance received from Sylvia Leung in procuring research material is highly appreciated. Gill Gaizi’s help in laboratory equipment maintenance is greatly acknowledged. I also acknowledge my friend Mrigank Sharma for keeping me a good company in the later parts of my stay at UBC. My heart is full of gratitude to Graduate Student Society, which was other place of involvement during most part of my stay at UBC. To conclude, I appreciate and thank my family and relatives, who helped and encouraged me to achieve academic excellence. My special thanks to Rajesh Sood and family for giving me shelter and good company in the beginning of this program. My heart is full of gratitude to my mother, who didn’t see me for more than five years, when I was engaged in this research project in the farthermost corner of the world. I have tried to fulfill the dreams that inspired me to come across seven seas. “Needless to mention, all errors and omissions are mine.”  xiv  CO-AUTHORSHIP STATEMENT  R. Singh identified the research problem, designed experiments, conducted research, compiled and analyzed results, prepared and revised manuscripts and wrote this thesis. M.L. Graves assisted in experimental design (Chapter 2). C.D. Roskelley assisted in experimental designs and manuscript review (Chapter 2). G. Giritharan assisted in manuscript preparation (Chapter 2). T. Pretheeban assisted in manuscript preparation (Chapter 3). R. Rajamahendran provided funds for research, assisted in experimental design and manuscript review.  xv  CHAPTER 1- INTRODUCTION AND BACKGROUND INFORMATION 1.1 INTRODUCTION Gonadotropin-releasing hormone (GnRH) or mammalian GnRH-l is a neuronal secreted decapeptide, produced primarily in the hypothalamus and plays a central role in mammalian reproduction (Fink, 1988). This hormone is synthesized by about 1000 hypothalamic neurones, carried by the axonal processes to the level of the median eminence in the brain, and released in a pulsatile manner into the hypothalamo-hypophyseal portal circulation. GnRH pulses vary in amplitude and frequency and the hormone is released every 20-120 mm to stimulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary. LH and FSH enter into the systemic circulation and regulate the synthesis and release of estrogen and progesterone (P ). GnRH 4 pulses of higher amplitude and over a shorter period of time result in synthesis and release of LH, whereas lower amplitude and prolonged pulses are responsible for biosynthesis and release of FSH. GnRH synthesis in the hypothalamus is under the control of catecholaminergic neurons such as noradrenaline or dopamine and its activity is modulated partly by auto-stimulation of its own receptor and partly by short feedback loops from the pituitary by the retrograde blood flow, but mainly by the ovarian steroids progesterone (P ) and estrogen (E 4 ) through the long 2 loop feedback mechanism. The biological half-life of GnRH is only a few minutes (4-5 mm) and during this time it must travel to the pituitary via the hypothalamo-pituitary portal vein, bind to its receptors on the surface of gonadotrophs and exert its action (Millar et al., 2004 ;Cheng and Leung, 2005; Ramakrishnappa et al., 2005) During the last decade, several researchers reported the presence of a GnRH and GnRH-R system in extrapituitary reproductive tissues. It has been shown to be present in the ovaries, endometrium, placenta, testes, prostate, preimplantation embryos, oocytes and sperm  across species ranging from rodents to humans (Janssens et al., 2000, Cheng and Leung, 2005, Ramakrishnappa et aL, 2005). Additionally, GnRH and its receptor system has been found in ovarian and endometrial carcinomas, uterine fibroids, prostate cancer, pituitary adenomas, mononuclear blood cells, T lymphocytes and certain cancer cell lines in humans. From both in vivo as well as in vitro model studies in rodents, primates or in humans, it is increasingly becoming evident that GnRH or its synthetic analogues could exert direct effect(s) through an autocrine and or paracrine manner, eliciting a variety of responses depending on the type of target tissue and the physiological conditions (Janssens et al., 2000; Leung et a!., 2003). Thus, in recent years this extra-pituitary role of GnRH has generated interest in the field of reproductive biology and physiology. It is well established that the hypothalamus and pituitary are the principal source and target site of GnRH. GnRH plays a central role in mammalian reproduction by acting through the hypothalamo-pituitary-gonadal axis. But the extra-hypothalamic origin of GnRH and the extra-pituitary presence of GnRH-R suggest that GnRH may act as a local modulator of different biological and physiological processes. Ramakrishnappa et al. (2003) demonstrated GnRH and GnRH-R mRNA in bovine ovaries but, the presence of GnRH-R in the bovine tubular reproductive tract including the uterus and oviducts was still to be tested. The local modulatory role of the GnRH, GnRH-R system is comparatively well elucidated in normal and neoplastic ovaries where it acts as a negative autocrine/paracrine growth factor modulating cellular proliferation and apoptosis. There are relatively few reports showing that this hormone induced similar effects in endometriosis and uterine carcinomas. The local modulatory role of GnRH in normal uterine physiology is not understood. GnRH is extensively used in bovine reproductive management for estrous and ovulation synchronization, induction of postpartum ovarian cyclicity, treatment of cystic ovarian disease 2  and to increase pregnancy rates. Increase in pregnancy rates was observed with the administration of GnRH or its analogues at the time of Al, 2-3 days after Al or in mid luteal phase post Al in cattle (Peters, 2005). It is believed that GnRH produces these effects by acting through the hypothalamo-pituitary-gonadal axis. It can be postulated that this hormone might act as a local modulator of steroid hormone receptor regulation and apoptosis at the level of uterus. There are a few reports showing that GnRH and steroid hormone receptor systems can interact with each other at the pituitary and ovarian levels. The uterus is a dynamic structure and is under the influence of the ovarian steroids, estrogen and P 4 which regulate uterine cyclicity by acting through its own nuclear receptors. GnRH regulates apoptosis and acts as a local modulator of ovarian physiology in rats and humans. This hormone also induces pro apoptotic effects in endometriosis and, uterine and ovarian cancers. Apoptosis plays an important role in maintaining tissue homeostasis and acquires more significance in renewable structures such as ovaries and uterus, which undergo changes in cellular turnover every estrous/ menstrual cycle (Harda et al., 2004). In light of growing evidence about the presence of the GnRH, GnRH-R system in extrapituitary reproductive tissues in mammals, there was a dearth of information on the presence of GnRH-R and the local modulatory role of the GnRH, GnRH-R system in bovine extrapituitary reproductive tissues. Therefore, it was imperative to investigate the presence of the GnRH-R in the bovine uterus and oviducts, and to further study the GnRH agonist induced steroid hormone receptor regulation and apoptosis in the uterus.  3  1.2 GnRH, GnRH-R SYSTEM 1.2.1 GnRII The hypothalamic GnRH or GnRH-I, was first isolated and sequenced by Dr A. Schally, Dr R. Guillemin, Dr R. Yalow and co-workers in the early seventies. These researchers were awarded a Nobel prize for this discovery in 1977. Mammalian GnRH was considered to be a single and unique structure with a primary role in regulating synthesis and release of LH and FSH (Fink, 1988). Now it is known that diverse forms of this hormone exist and so far 23 structural variants of GnRH have been identified. These are distributed in a wide range of tissues in protochordates and vertebrates and perform diverse functions (Millar et al., 2004). Generally, most vertebrate species have at least two and usually three forms of GnRH, which differ in their amino acid sequences, localization and embryonic origins. All these forms are of classical 10 amino acid peptides, (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Ser-Pro-Gly-NH2) with a pyro-glutamyl modified amino-terminus, an amidated carboxy-terminus, and conserved amino acids in positions 1, 2, 4, 9, and 10 (Powell et a!., 1994). The most widely recognized and common structural variation among the different forms of GnRH resides in amino acids between positions 5 and 8 in the sequence. The second type, a midbrain GnRH or GnRH-II was first identified in the chicken brain and is referred to as chicken GnRH-II or cGnRH-II (Millar et al., 2001). It is structurally conserved in species ranging from teleost fish to humans. The GnRH-II differs from GnRH-1 by three amino acid residues at positions 5, 7, and 8 8 5 (His T 7 ) rp yr . GnRH-II specifically serves as a potent inhibitor of K channels in the amphibian sympathetic ganglion. Inhibition of these ion channels facilitates rapid excitatory transmission of conventional neurotransmitters and may provide a general neuromodulatory mechanism for GnRH-II in the nervous system (Millar et al., 2004; Ramakrishnappa et al., 2005). The third type, a telencephalic GnRH, also known as type III GnRJ-I or GnRI-I-III, 4  preferentially localizes in the terminal part of the olfactory neuronal cell in the brain (Millar et al., 2001) 1.2.2 GnRII structure The coding region of hypothalamic GnRH cDNA consists of an open reading frame of 276 bp followed by a 162 bp 3’ UTR. The reading frame encodes for a GnRH precursor protein of 92 amino acids and the first 23 amino acids of this precursor protein form the signal sequence. Two serine residues separate the decapeptide GnRH sequence from the signal sequence. The GnRH sequence is further followed by a 3 amino acid signal processing site (GKR) and a 56 amino acid-GnRH-associated peptide (GAP). The GKR sequence is responsible for signal amidation of the carboxy terminal and cleavage of GnRH peptide from the precursor protein. The GnRH gene is 5 kb and consists of four exons and three introns. The first exon is untranslated and the second exon encodes the signal sequence, the GnRH decapeptide, the GKR processing signal and first 11 residues of GAP. The third exon codes for the next 32 GAP residues. The fourth exon encodes for the remaining 13 GAP residues, has a translational terminus codon and an entire 3’ UTR (Cheng and Leung, 2005). GAP is secreted along with GnRH and is perceived to play a role in processing and packaging of the decapeptide into the GnRH neurons. Figure 1.1 shows schematic representation of GnRH gene. The conservation of the GnRH decapeptide, NH 2 (pGlu-His-Trp-Ser) and COOH (Pro ) termini suggests that these sequences are important for receptor binding and 2 Gly.NH activation. The variation in position 8 is generally tolerated by the decapeptide, but mammalian GnRH-R requires Arg in position 8 for high-affinity binding (Seaflon et al., 1997). It seems that the residue in position 8 plays an important role in ligand-selectivity of GnRH-R (Guarnieri and Weinstein, 1996).  5  5 Uiitrans1ated  Signal sequence niLJT  32 A.P  13 AP 3’—Untranslatcd  11 AP  Figure 1.1 Schematic representation of the molecular structure of mammalian GnRH. The  GnRH gene is comprised of four exons (rectangles) and three introns (lines joining the rectangles). Downward arrows show the components of precursor peptide encoded by different exons.  6  The initial studies on GnRH structure by conformational energy analysis of the NH 2 terminal 1—6 and carboxyl-terminal 6—10 amino acids identified a low energy CC conformer that featured a B-IT’ type turn. The B-TI’ turn involves 8 6 Ty?-G1y 7 Leu in such a way that Arg the NH 2 and COOH termini are closely apposed. The 13-TI’ type turn conformation of GnRH appears to be induced in part by the interaction of Arg 8 with an acidic residue (Asp ) in 302 extracellular loop three (EC 3) of the mammalian GnRH-R. Replacement of Gly 6 in the GnRH decapeptide, with a D-amino acid appears to enhance the B-IT’ type turn conformation and results in an increased activity of Arg 8 at mammalian receptors. The D-amino acid substitution overcomes the deleterious effects of Arg 8 substitution such that binding affinity for the mammalian receptor is increased almost 1000-fold (Fromme et al., 2001; Petry et al., 2002) 1.2.3 GnRH agonists and antagonists  Knowledge on peptide sequence and awareness on its shorter half life in the general circulation led to the developments of synthetic GnRH analogues with greater stability against enzymatic degradation, increased receptor affinity and biological potency. The original concept was to use the potent GnRH agonist (GnRH-a), for the treatment of hypogonadism due to insufficient endogenous secretion of GnRH. Later on the multidimensional properties of GnRH analogues and their potential application in the reproductive physiology and medicine were quickly realized. Structural differences in GnRH agonist and the natural sequence of GnRH are substitution of glycine with a D-amino acid (e.g. D-tryptophan) at position 6 and removal of glycine from the amino terminus. Substitution with a D-amino acid at position 6 increased the half-life of agonists in circulation, and removal of the amino terminal glycine increases affinity for the GnRH-R (Karten and River, 1986). The GnRH agonists, currently in use forclinical or experimental purposes in human and bovine medicine are: gonadorelin (native-like GnRH;  7  gonadorelin diacetate tetrahydrate or gonadorelin hydrochloride), buserelin, deslorelin, lupron, zoladex Supprelin, Synarel and triptorelin ( Millar et al., 2004). ,  GnRH antagonists have substitutions at position 1 to 3 and position 6 and 10 as compared to GnRJ-1 sequence. GnRH antagonists commonly in use for experimental and clinical purposes are- antide, abarelix, cetrorelix and ganirelix. The development of nonpeptide GnRH antagonists has received considerable attention from the pharmaceutical industry. The first described nonpeptide GnR}1 antagonist was a fused tetracyclic benzodiazepine that blocks ovulation in rats when given at a dose of 0.5 mg/kg. The antifungal drug ketoconazole (Nizoral, Janssen Pharmaceutica, Beerse, Belgium) binds and inhibits the rat pituitary GnRH-R (Millar et al., 2004). Figure 1.2 shows schematic representation of GnRH, GnRH agonist and antagonist sequences. 1.2.4 GnRH-R GnRH-R has been cloned from rats (Eidne et al., 1992), cows (Kakar et al., 1993), humans (Kakar et al., 1992; Chi et al., 1993), and sheep (Brooks et al., 1993; hung et al., 1993). These findings revealed that the GnRH-R is a member of large superfamily of G-protein coupled receptors (GPCR) having seven transmembrane helical domains. Since two other forms of GnRH, i.e. GnRH-II and GnRH-III do exist, it suggests that separate and cognate receptor types may be present in vertebrates (Troskie et al., 1998). The Type II GnRH-R from the marmoset and human (Millar et al., 2001; Neill et al., 2001) is highly selective for GnRH-hI and is widely expressed in reproductive tissues and in the central nervous system. In addition, expression of GnRH-II receptor in the majority of gonadotrophs is indicative of its role in the regulation of gonadotropin secretion. The Type II receptor is rapidly internalized in contrast to the Type I receptor and has distinctly different signaling pathways and preferentially stimulates  8  [Rporbirig  GrRH  pG  2  3  4  His  1  S  5  5  7  T  Gty  Lsu  Ar r  P  L  Ms Ms Ass Mg Ass Ass  Pro Pro Pro Pro Pro Pro  ty  NH:  Agouists pGh pGk pGh pGh pGhs Gb  His His His His His His  DNsI DNa1 DNaI DNa1  DCp DCpa DCpa DCp  ZDbdex Sepprsii  Tq Tp Tq Tp Tp Tp  S S S S S  T tr E i I’r Tw  I I I I  L Ls’. Ls  1  (My G1 GI Gt  NET NET NH. NH NH NH  Aa-& ts AbeJix Gtix Cete [  4  OPal OPal OPal OPal  1  1)-amino acid subtibitiG±i in anLaon  Lys NM: 1jT  DQt DAs  Lsu L Ls  I  Ar  Pro Pro Pro Pro  I I  I I  NH NH.. NH: NH:  13-amino acid ubtibiion in aoni.sts  Figure 1.2 Amino acids sequence of GnRH, GnRH agonists and antagonists with receptor  binding and activation regions. GnRH sequence is depicted in light shade showing the relative position of amino acids in the decapeptide. Substitution at positions 6 and 10 results in production of agonists. Dark shaded areas show the substitutions for different agonists and antagonists relative to the GnRH sequence.  9  FSH secretion. This has led to the suggested hypothesis that GnRH-II and the Type II receptor have a specific role in the regulation of the gonadotrope function (Millar, 2002). The localization of the Type II receptor to the brain region affects sexual arousal and suggests a role in reproductive behaviors. Also, the presence of the Type II receptor in reproductive tissues (gonads, prostate, and mammary glands) is an indication of the diverse functions this molecule could have (Cheng and Leung, 2005). 1.2.4.1 GnRH-R structure and function Mammalian GnRH-R shares more than 80% amino acid homology amongst different species. The GnRH-R was first sequenced from mouse pituitary oT3 gonadotrope cell lines. The mouse GnRH-R sequence contains 327 amino acids as compared to 328 in human, sheep and bovines. The GnRH-R gene spans more than 15 kb and is composed of three exons separated by two introns (Figure 1.3). Exon 1 contains 5’-UTR and the first 522 nucleotides open reading frame. This sequence encodes for first three transmembrane domains and a part of fourth transmembrane domain of the receptor. Exon 2 contains 220 nucleotides of reading frame and encodes for the rest of the fourth transmembrane domain, the fifth transmembrane domain and a part of third extracellular loop. Exon 3 encodes for the rest of the receptor  th, 5 (  th, t7 6 h transmembrane domains, extra and intracellular loops) and contains 3’-UTR (Millar et al., 2004, Cheng and Leung 2005). The molecular weight of GnRH-R protein is 37, 680. Three consensus sequences for N linked glyocosylation are present in the extracellular domain as well as sites for phosphorylation by cAMP-dependent protein kinase. PKC sites have been shown to be present in the first intracellular loop, and an additional site in the third extracellular loop has also been suggested. Most of the GPCRs contain a signature sequence at the beginning of the second intracellular loop and the third transmembrane domain. It is a conserved sequence containing 10  the Asp-Arg-Tyr triplet which is implicated in G-protein coupling. But, this sequence of GnRJ-1-R varies from other GPCRs signature sequences by the replacement of two residues conserved at position 140 (serine for tyrosine). The GnRH-R differs from other GPCR as an aspartate residue (position 87) in the second transmembrane domain is replaced with asperagine and a conserved asparagine residue at position 318 in the seventh transmembrane domain is exchanged by aspartic acid. In addition, GnRH-R has relatively longer first intracellular ioop with high contents of basic amino acids and phenyl-alanine rich seventh transmembrane domain. Ala ’ in the third intracellular loop is important for G-protein coupling and receptor 26 internalization, Asp , Trp’°’, Asn’° 98 , and Asp 212 , Lys’ 2 , Asn 21 302 are crucial for ligand binding and, Iys’ ’ is associated with expression and internalization of human GnRH-R (Cheng and 9 Leung 2005). The GnRH-R structure is stabilized by a disulfide bond formed by conserved cysteines in the first and second extracellular loops. The disulfide bridges between C114 (numbering relative to N-terminal amino acid) and C196 of the mouse receptor and Cl4 and C200 of the human receptor are important for ligand and receptor binding. The bovine, ovine and human GnRH-R have two potential sites for N-linked glyocosylation at Asn’ 8 and Asn’° . The rodent 2 GnRH-R contains the same potential glycosylation sites with an additional site in the amino terminus, Asn . These glyocosylation sites of the GnRH-R appear to provide receptor stability 4 rather than receptor transport to the cell surface (Rispoli and Nett, 2005). Figure 1.4 shows schematic representation of mammalian GnRH-R.  11  .Exon 1  Exon2  Exon 3  2 NH  IC 2 328 ammo acids  Figure 1.3 Diagrammatic representations of the GnRH-R gene and parts of the receptor  encoded by three exons. The mammalian GnRH-R has seven transmembrane domains (TM 17), three intracellular loops (IC 1-3) and three extracellular loops (EC 1-3) with an extracellular amidated and intracellular carboxy terminal. Downward arrow show parts of GnRH-R encoded by respective exons. Exon 3 encodes for EC 1, EC 2, EC 3 and IC 1, IC 2, IC 3 of receptor are not depicted by arrows in the picture.  12  1.2.4.2 GnRH agonist and antagonist binding to GnRH-R The two common modifications in synthetic GnR}I agonists are substitution of G1y 6 with a D-amino acid and Gly’°-NH 2 with ethylamide. GnRH agonists with D-amino acid substitutions for G1y 6 and without substitution of Gly’°.NH 2 are dependent on Asp , Asn’° 98 , 2 ’ for receptor binding and activation (Flanagan et a!., 2000; Hoffman et a!., 2000). 2 and Lys’ Since, Asp 302 interacts with Arg 8 for configuring the native ligand at the GnRH-R, it is no longer required in agonists with the D-amino acid substitution of G1y . The pituitary 6 membranes pre-treated with proteolytic enzymes reduced binding of antagonist as compared to agonist, suggesting that the agonist binding site is less accessible and more buried within the GnRH-R molecule than the antagonist binding site (Hazum E 1981; Wormald et a!., 1985). The tryptic digestion of photoaffinity labelled GnRH-R with agonist or antagonist attached to D-Lys in position 6 of the ligands yielded different size fragments. It suggested distinct binding sites for agonist and antagonist ligands or distinct configurations of the agonist and antagonist bound GnRH-R. On the contrary, when identical receptor fragments were labelled with both a photoactive agonist (Davidson et a!., 1997) and a photoactive antagonist (Assefa et al., 1999), the mutation in Lys’ ’ to Gin showed differential effects on agonist and antagonist interactions. 2 There was a marked effect on agonist potency without affecting antagonist affinity in these experiments. It suggested that Lys’ 21 may be important for agonist binding but not for antagonist binding (Zhou et a!., 1995). Mutations in Asp Glu, Trp 98 Ala, Asn 101 Ala, 212 2‘ Asn Gln, Tyr 2 Ala, and Asp 290 Asn also showed differential effects on agonist and antagonist 302 interactions (Hoffman et a!., 2000; Fromme et al., 2001).  The Asp Glu mutation disrupts 98  interaction with His 2 of GnRH, a residue that is substituted with D-amino acids in antagonists (Flanagan et al., 2000) and it seems that Asp 98 side chain may not be interacting with  13  ExtraceHular  Figure 1.4 Pictorial representation of the human GnRH-R showing TM domains (boxed), extracellular loops (ECs) and intracellular loops (ICs). Ligand binding sites (red) and residues considered to be important structure or binding pocket formation for GnRH-R are shown in green letters, including disulfide bond formation and glycosylation sites. Residues shown in blue are involved in receptor activation and residues in squares are highly conserved throughout the rhodopsin family of GPCRs. Residues in orange are involved in G protein coupling and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites are indicated at their corresponding locations (Millar et al., 2005, Endo. Rev.).  14  antagonists. The Trp’°’Ala mutation decreased agonist potency and also decreased antagonist binding affinity 23-fold. There is possibility that this response on antagonist binding might be a 212 to Gln mutation result of disruption of a direct interaction of Trp’°’ with the antagonist. Asn had minimal effects on antagonist interactions, but the Ala mutation decreased antagonist affinity 86-fold (Hoffhiaim et al., 2000; Hovelmann et al., 2002). Froamme et a!. (2001) suggested that this mutation at the Asn 212 stabilized the antagonist binding site configuration. 1.2.4.3 G-protein coupling Gqiii is the predominant G-protein coupled to the GnRH-R in most of the cellular departments (Hsieh and Martin, 1992). But GnRH-R coupling to 0 and G proteins can also mediate GnRH-R actions in ovarian carcinomas  (Grundker et al.,  2001),  uterine  leiomyosarcomas (Imai et al., 1996), uterine endometrial carcinomas (Imai et al., 1997), and human prostate cancer cells (Limonta et al., 1999). The intracellular loops (ICs) and carboxyl terminal tail have been implicated in coupling of GPCRs to 0-proteins. Conservation of the carboxyl-terminal sequence of 1C3 in vertebrate GnRH receptors indicates that this region might be crucial for coupling to the primary mediator, Gquii and Ala ’ is 26  important for  coupling. In Id there is a G recognition motif (BBxxB, where B is a basic amino acid) and mutation in these residues leads to uncoupling of cAMP production but not of inositol phosphate production (Arora et al., 1998). On the basis of available literature it can be summarized that GnRH-R is able to couple to several 0-proteins and activate a number of effectors via different elements of the three ICs. It appears that coupling to  q/11 0  occurs through  1C2 and 1C3, and to G through IC1. Coupling to G is less understood, but may be related to the cell-type, stage of the cell cycle, or availability of the 0 protein. Recently, it has been suggested that G activation could be the reason for antiproliferative effects of GnRH in many cancers (Grundker et al., 2000; Everest et al., 2001). Inactive 0-proteins are heterotrimers and 15  contain the  -,  B-, and 7-subunits. GTP displaces GDP from binding to the Ge-subunit and is  subsequently hydrolyzed to GDP by intrinsic GTPase activity. This promotes re-association of Gc-subunit and B-dimers, resulting in the inactive form of heterotrimers (Castro-Fernandez and Conn, 2002). 1.2.4.4 GnRII-R internalization The classical GPCR internalization pathway involves GPCR kinases, 13-arrestin, clathrin-coated pits, and the GTPase dynamin (Pitcher et a!., 1998). Receptor activation targets G-protein coupled receptor kinases to the receptor. This leads to separation of B7-subunits from the receptor. Activated GPCR kinases phosphorylate the receptor at specific serine and threonine residues. This phosphorylation enhances the binding of B-arrestin to the receptor. The B-arrestin binding prohibits further G-protein coupling to GPCR and subject the receptor to clathrin-coated pits for internalization. Dynamin is considered important for the detachment of clathrin-coated vesicles from the plasma membrane. The subcellular localization of certain GPCRs to smooth non-coated membrane structures and vesicles suggest that GPCRs use internalization pathways different from clathrin-coated vesicles. (Raposo et al., 1989). The internalization pathways for GnRH-R appear to be cell-type and receptor subtype dependent. Most of available information on internalization process of mammalian GnRH-R comes from the studies conducted in rats. The rat GnRH-R internalizes in a B-arrestin-independent manner, but probably uses dynamin-dependent clathrin-coated vesicle mechanism. The rat GnRH-R can colocalize with transferrin receptors. The transferring receptors are known to internalize in a clathrin-coated manner (Vrecl et al., 1998). The lack of a carboxyl-terminal in the mammalian GnRH-R appears to account for their B-arrestin independency for internalization. It seems that GnRI-I-R utilizes clathrin-mediated internalization process.  16  1.2.5 GnRH and GnRH-R interactions in the pituitary Once, GnRH binds to its receptors on pituitary gonadotrophs, ligand receptor binding produces conformational changes in the receptor resulting in activation of the Gq/G1 1 subfamily of G-proteins. This in turn causes an increase in phospholipase C (PLC) activity that results in breakdown of phosphoinositide into inositol 1 ,4,5-trisphosphate (1P3) and diacyiglycerol (DAG). 1P3 mobilizes intracellular Ca 2 and DAG activates protein kinase C (PKC). These events lead to synthesis and release of gonadotrophins, LH and FSH (Stojilkovic and Catt 1995). The varying pulse frequencies and amplitude of GnRH release from hypothalamic neuronal cells is a critical and rate-limiting step for the control and maintenance of gonadotrophins secretion from pituitary gonadotrophs. The GnRH pulse and amplitude changes in turn depend on feedback exerted by sex steroids and gonadal peptides produced throughout the reproductive cycle. Both GnRH-R and LH synthesis is favored at high GnRH pulse frequencies of one pulse every 20-30 mm, whereas FSH synthesis is favored at low GnRH pulse frequencies of one pulse every 120 mm (Shupnik, 1996; Kaiser et al., 1997; Millar et al., 2004). Kaiser et al. (1995) postulated that modulation of the GnRH pulse frequency could regulate multiple physiological effects resulting in the differential activation of signal transduction pathways and initiating different cellular processes. These workers demonstrated that the GnRH stimulated LH promoter activity was at the optimal point of stimulation coincident with that of relatively high GnRH-R numbers, while FSH promoter activity was optimally stimulated at relatively low GnRH-R numbers. Haisenleder et al. (1997) further suggested that in addition to GnRH-R numbers, the modulation of the frequency of intracellular Ca pulses may play a role in the differential regulation of LH and FSH mRNA synthesis.  17  1.2.6 GnRH, GnRH-R mediated cell signalling Though Gq/1 1 is the major G-protein coupled to GnRH-R, but the GnRH-R could also couple to a 41 kDa Gi protein in human ovarian cancer cells to activate the MAPK cascade (Kimura et al., 1999; Luttrell 2002). The GnRH, GnRH-R interaction activates MAPK cascades, including ERK1/2, c-Jun amino-terminal kinase (INK), p38 MAPK, and big MAPK (BMK1/ERK5) via Ca and PKC mediated cell signalling pathways (Johnson & Lapadat, 2002). ERK1/2 are key proteins regulating cellular proliferation and involve two different pathways that converge at RAF1. GnRH-R induced mechanisms leading to MAPK cascade activation and the regulation of intracellular loops in coupling to MAPK cascades are still not understood.  INK has been shown to play an important part in cancer cell survival,  proliferation, apoptosis, and invasion. INK activation is highly dependent on cytosolic Ca and is regulated through serial stimulation of PKC, c-Src, CDC42/RAC1, and MAPK kinase (MEK). Another enzyme, p38 MAPK is involved in the PKC-dependent cascade and selectively induces apoptosis with a pro-apoptotic role in some cells but anti-apoptotic in others. This protein (p38 MAPK) induces similar effects in cell cycle regulation (Mulvaney & Roberson 2000; Bradham & McClay 2006; Weston & Davis 2007). There are variations in GnRH mediated signalling in extrapituitary tissues and pituitary gonadotrophs in spite of the fact that GnRH-R are similar in extrapituitary tissues and pituitary gonadotrophs. The MAPK cascade activation by GnRH-R in extrapituitary tissues/cells is attributed to intracellular mechanisms that seem to lead to transactivation of the epidermal growth factor (EGF) receptor. GnRH analogues have been shown to reduce growth factor receptor expression and growth factor-induced tyrosine kinase activity. GnRH analogues are considered to neutralize growth factor mediated tyrosine phosphorylation through the activation of the phosphotyrosine phosphatase (PTP) and it is speculated that PTP is coupled to the GnRH-R through a Gaj 18  protein in human reproductive tissue cancers (Imai  Ct  a!. 1996; Grundker et al. 2002; Shah et al.  2003). It is evident that GnRH, GnRH-R induced cell signalling pathways are different in pituitary and extra pituitary tissues. It could be postulated that the varied GnRH signalling between pituitary gonadotrophs and extra-pituitary tissues/cells is due to coupling of GnRH-R to different G-proteins. In human endometrial cancer cells, GnRH agonist activated activator protein-i (AP- 1) through the pertussis toxin-sensitive Gai-protein, but could also activate AP- 1 by simulating INK. Also, agonist suppressed growth factor-induced MAPK activity without activating PLC and PKC. It seems that GnRH agonist might not involve PKC and MAPK to activate the JNKJAP-1 pathway. It needs to be further established that GnRH agonist induced AP- i activation and the subsequent antiproliferative effect is mediated through its ability to control the cell cycle (Grundker et al. 2002). Figure 1.5 summarizes the GnRH induced signalling pathways. 1.2.7 Transcriptional regulation of GnRH-R  GnRH itself, gonadal steroids, activins and inhibins are the main regulators of GnRH-R in pituitary gonadotrophs. GnRH has a known ability to regulate its own receptors and its ability is substantiated by the findings that the continuous administration of GnRH in rat pituitary cell cultures does not result in a detectable increase in GnRH-R mRNA, but when administered in a pulsatile manner it causes several fold increase in its receptor mRNA (Fauser, 1999). It has also been shown that lower concentrations of GnRH upregulate GnRH-R expression whereas higher concentrations downregulate these receptors and a maximal stimulatory response is achieved at a pulse frequency of every 30 mm in rat pituitary cell cultures. GnRH induces a biphasic effect on  its receptor expression in human granulosa luteal cells, ovarian surface  epithelial cells, ovarian cancers and peripheral blood mononuclear cells. In these tissues/cells,  19  PLC  Ir /\  P38  2 breakdown 1P  3 1P  \9  C-j ‘in Jun D  DAG  411t  JNK  1. 1  MAP.K  ERK1JZ  ii  4.  PKC  Synthesis an  rele:seofFSH  Apoptosis  Figure 1.5 Schematic representation of cell signalling pathways induced by GnRH, GnRH-R interactions in pituitary and extra pituitary reproductive tissues. GnRI-I, GnRH-R interaction can result in activation of receptor through G proteins and involve differential cell signalling pathways in pituitary and extrapituitary tissues.  20  lower GnRH doses would  up-regulate whereas, higher doses down-regulate GnRH-R  expression levels. Contrarily, chronic GnRH administration results in increased GnRH-R expression in extravillous trophoblast cells. There seems to be a consensus that gonadal steroids estrogen and P 4 have an inhibitory effect on pituitary GnRH receptor expression. To study the transcriptional regulation of GnRH-R, aT3-1 cells have emerged as a novel model. Isolation of the human GnRH-R, 5’-flanking sequence intensified research on transcriptional regulation of GnRH-R gene and a proximal 173-bp flanking region has attained significance for its role in directing GnRH-R gene expression in gonadotrope. This 173-bp regulatory region consists of two putative gonadotrope specific elements (GSE) and the core sequence 5’ TGA/TCC-3’ at 143/ (at  —  -  —  135 and -131-5. Site directed mutagenesis studies further clarified that the upstream GSE  143/ 135) is required for gonadotrope-specific transcription of GnRH-R gene as mutation -  of this element selectively inhibited the promoter function of ctT3-l cells. The steroidogenic factor-i (SF-i) binds specifically to upstream GSE and its second, fifth, sixth, and the ninth nucleotides are important for the interaction and the functionality of the SF-i gene in regulating human receptor gene transcription in gonadotrophs. These observations are supported by the findings that sense and anti sense SF-i mRNA overexpression can stimulate and repress the native promoter. Since SF-i is also expressed in extrapituitary tissues including gonads, it is further postulated that GSE might not be the only mediator in gonadotropic-specific GnRH-R gene expression.  Studies from the mouse model have revealed the importance of tripartite  enhancer element in targeting gonadotrope-specific GnRH-R expression (Cheng and Leung, 2005).  21  1.2.8 The GnRH and GnRH-R system in extra-pituitary reproductive tissues The major function of GnRH is regulation of mammalian reproduction through the hypothalamo-hypophyseal portal circulation and through the controlled synthesis and release of LH and FSH. This action is produced by binding of the hormone to its receptors in pituitary gonadotrophs, but in last decade or so, there has been an explosion of information on the extrapituitary presence of GnRH, GnRH-R system across different tissues and in different species of animals including humans. This extrapituitary extrahypothalamic GnRH, GnRH-R system played a local modulatory role by regulating various biological processes. Figure 1.6 shows presence of GnRH-R in extrapituitary tissues. 1.2.8.1 The GnRH, GnRH-R system at the gonadal level The initial evidence for the presence of ovarian GnRH-R in rodent species dates back to the late seventies. Through radioligand binding assays, ligand specific binding sites were demonstrated on rat granulosa and luteal cells (Clayton et al., 1979; Harwood et a!., 1980; Reeves et al., 1980; Jones et al., 1980; Pieper et al., 198 1).These findings were further verified by several other researchers (Latouche et al., 1989; Whitelaw et a!., 1995). Expression of GnRH mRNA was demonstrated in rat gonads (Oikawa et al., 1990; Bahk et al., 1995). In-situ hybridization studies showed that GnRH mRNA is localized in granulosa cells of primary, secondary, and tertiary follicles in the ovary (Clayton et al., 1992; Whitelaw et al., 1995). Then GnRH-R mRNA was demonstrated in human granulosa luteal cells (hGLC) by using reverse transcription polymerase chain reaction (RT-PCR) techniques (Minaretzis et al., 1995; Olofsson et a!., 1995; Kang et al., 2000). Ramakrishnappa et al. (2003) demonstrated the GnRH-R mRNA transcripts in bovine follicles and bovine corpus luteum (CL) by using RT PCR technique. The earliest reports about the presence of the GnRH, GnRH-R system in the rat testicular tissues date back to early eighties. Through radioligand binding assays, ligand 22  specific binding sites were demonstrated in interstitial testicular tissue (Boume et al., 1980 and 1982) and leydig cells (Lefebyre et al., 1980; Sharpe and Fraser, 1983). Presence of GnRH mRNA in the testicular tissue, in both fetal (Botte et al., 1998) and mature rats as well as in adult human seminiferous tubular cells was observed, Also the reports on the presence of GnRH-R in these tissues date back to early eighties. Through radioligand binding assays, ligand specific binding mRNA has been observed in interstitial cells, including leydig cells (Bahk et al., 1995; Clayton et al., 1980). Nucleotide sequence analysis verified that both rat and human ovarian and testicular GnRH-R have sequence identical to that found in the pituitary (Kakar et al., 1992; Peng et al., 1994; Moumni et al., 1994; Olofsson et al., 1995; Ramakrsihnappa et. al., 2001). On the other hand, the knowledge of hypothalamic GnRH structure and its physiological concentrations (Nett et al., 1974) or its shorter half life in the general circulation (Eskay et al., 1977; Hsueh and Jones, 1982) resulted in exploring the endogenous presence of GnRH or GnRH like molecules in gonads. Several researchers successfully demonstrated the existence of such molecules in sertoli cells by competitive binding studies and immuno-chemistry (Sharpe and Fraser 1983; Paull et al., 1981). GnRH or GnRH-like molecules were detected in human and bovine follicular fluid (Ying et al., 1981; Aten et al., 1987a; Ireland et al., 1988), human ovaries (Aten et al., 1987b), in human seminal plasma (Izumi et al., 1985), in testicular interstitial fluid of hCG-treated rats (Sharpe and Fraser, 1980), and in rat germ cells (Paull et al., 1981). It was suggested that these molecules are probably being synthesized in the gonads (Oikawa et al., 1990; Sharpe and Cooper, 1982a) and in the prostate (Azad et al., 1993).  23  Uterus Uteilne fibroids Uterine carcinoma Endometriosis  Liver  Kidney Heart  Figure 1.6 Schematic representation showing the presence of GnRH-R in extra-pituitary  reproductive tissues.  24  1.2.8.2 Oocytes, sperms and embryos Through radioligand binding assays, Dekel et al. (1988) showed ligand specific binding sites for GnRH in rodent oocytes. In bovine species Funston and Seidel (1995) demonstrated the presence of GnRH-R mRNA by RT-PCR in matured cumulus-oocyte complexes and verified these findings through Southern hybridization with cDNA for the ovine GnRH receptor. Lee et al. (2000) showed the presence of GnRH-R in human sperm by using immunohistochemical staining and inferred that GnRH-R was localised in the acrosomal region. By using Northern hybridization procedures, three distinctive, different sized GnRH-R mRNA transcripts were demonstrated in rat and mouse testicular germ cells (Bull et al., 2000). In addition, type II GnRH-R exon 1-containing transcripts were detected by in situ hybridization in human mature sperm and in postmeiotic germ cells, and were considered to be closely related with spermatogenesis, sperm maturation, and fertilization (van Biljon et al., 2002). Seshagiri and Wahlstron (1994) showed GnRH release in preimplantation rhesus monkey embryos. GnRH is expressed in the developing mouse embryos from the morula to the hatching blastocyct stages at the mRNA and protein levels. Preimplantation embryonic development is significantly enhanced with GnRH agonist treatment (Raga et. al., 1998). Furthermore, the presence of GnRH mRNA and protein was confirmed in human embryos (Casan et al., 1999). The GnRH and GnRH-R system is believed to play a role in early embryonic development, and in endometrial preparation and implantation. After GnRH agonist treatment, an in vitro increase in the proportion of mouse embryos reaching the hatching blastocyst stage was observed. GnRH antagonist had inhibited preimplantation embryonic development, which was totally reversed with agonist treatment, indicating a specific action on 25  embryonic development rather than toxic or non-specific effects (Raga et al., 1998). Recently, Nam et al. (2005) demonstrated the presence of GnRH and GnRH-R mRNA in porcine preimplantation embryos and further showed that, in vitro supplementation of embryo culture media with GnRH agonist increased blastocyst production rates. 1.2.8.3 Uterus and oviducts Li et al. (1993), for the first time, demonstrated the presence of an immunoreactive GnRH in porcine endometrial tissue. Subsequently Ikeda et al. (1996) reported expression of GnRH mRNA in rat endometrial stromal cells. Then, Imai et al. (1994) through RT-PCR and binding assays demonstrated the presence of GnRH-R mRNA in both normal human endometrium and endometrial carcinomas. In humans, dynamic expression patterns for GnRH mRNA were observed in both the endometrium and isolated endometrial cells, and these levels were significantly increased in the secretory phase of the menstrual cycle (Dong et al., 1998; Raga et al., 1998). In addition, GnRH-I immunoreactivity was noticed in all endometrial cell types and it has been observed that staining patterns were more intense during the luteal phase as compared to follicular phase of the menstrual cycle (Raga et al., 1998). The spatiotemporal expression of GnRH-II was investigated in the human endometrium and it was shown that throughout the entire reproductive cycle, two splice variants of GnRH mRNA are expressed with the shorter transcript carrying a 21 bp deletion, which reduces the length of the gonadotropin-associated peptide (GAP) from 77 to 70 amino acids. Like GnRH-I, GnRH-II immunoreactivity was dynamically expressed in stromal and epithelial cells such that stronger signals were detected in the early and mid secretory phases than in the proliferative and late secretory phases (Cheon et al., 2001). In human fallopian tubes, Casan et al. (2000) showed GnRH mRNA and protein expression by using RT-PCR and immunohistochemical  26  techniques in the luteal phase of the menstrual cycle and inferred that GnRH immunostaining was localized in the tubal epithelium. 1.2.8.4 Placenta Human placenta synthesises GnRH- 1 in vitro, which was immunologically identical to its pituitary counterpart. GnRH-II is also released in a pulsatile fashion from human placenta and is more resistant to enzymatic degradation as compared to GnRH-I. Both GnRH-I and GnRH-II mRNA are expressed in first trimester human placenta, whereas GnRH-I could be expressed by full term human placenta. Immunohistochemistry showed that both forms localize in the mononucleate villus and in distinct subpopulations of the extravillous cytotrophoblast. GnRH-I localizes in the outer multinucleated syncytiotrophoblast layer and allowed differentiation and fusion in vitro, in cytotrophoblast cultures (Cheng and Leung, 2005). 1.2.8.5 Mammary gland Specific ligand binding sites have been shown to be present in both breast carcinomas and MCF-7 mammary cancer cell lines. MCF-7 cell lines express two different binding sites, one specific for GnRH-I having high affinity and the other one for GnRH antagonists (Eidne et al., 1985; Segal-Abramosan et al., 1992). Other researchers have shown the presence of GnRJJ R immunoreactivity and mRNA having a sequence identical to pituitary receptor mRNA in both normal and carcinogenic breast tissues (Kakkar et al., 1994; Kottler et al., 1997., Moriya et a!., 2001). However, unlike its ligand, expression of GnRH-R is not up-regulated in the breast cancer cells. Mixed opinion exists regarding the presence of the GnRH, GnRH-R system and its autocrine or paracrine role in the mammary system. GnRH mRNA was detected in the mammary gland of pregnant and lactating rats, but not in that of virgin rats (Palmon et al., 1994; Ikeda et al., 1995). Since it is present during the period of pregnancy or lactation, it was hypothesized that GnRH expression might be regulated by prolactin (Palmon et al., 1994). The 27  presence of biologically active GnRH peptides in milk suggests that the possible target of mammary GnRH may be the offspring (Gore, 2002). 1.2.8.6 GnRH and GnRH-R in ovarian and endometrial carcinomas GnRH is believed to play an autocrine or paracrine regulatory role in the growth of ovarian and endometrial carcinomas. Recent findings demonstrated that GnRH is more abundant in ovarian and endometrial carcinomas as compared to normal ovarian and endometrial tissues (Furui et al., 2002). In general, GnRH peptide expression was observed in certain reproductive tissue tumors including ovarian (Arencibia and Schally, 2000; Kang et al., 2000; Irmer et al., 1995; Ohno et a!., 1993), endometrial (Irmer et al., 1994), prostate (Limonta et a!., 1993; Bahk et al., 1998; Lau et al., 2001) and breast cancers (Harris et al., 1991; Kottler et al., 1997). GnRH and GnRH-R expression has been observed in both endometrial and ovarian cancers; therefore, an autocrine growth-regulatory system resulting in cancer inhibition has been suggested through both in vivo and in vitro model studies. In these studies it was shown that GnRH has direct effects on the ovary and endometrium in these cancer types (Imai et al., 2000; Arencibia and Schally, 2000; Emons and Schulz, 2000; Grundker et al., 2002b). Grundker et al. (2002b) demonstrated that a second GnRH system exists in primates. It was observed that endometrial cancer cell lines (HEC-1A and Ishikawa) and the ovarian cancer cell lines (EFO-2 1, and NIH:OVCAR-3) are positive for GnRH-II receptor mRNA expression. The anti-proliferative effect of GnRH-II or GnRH-I agonist on the cell cycle and apoptosis were studied in endometrial (HEC- 1 A and Ishikawa) and the ovarian cancer cell lines (EFO-2 1 and NIH: OVCAR-3). The proliferation of GnRH-II receptor positive cell lines was observed to be dose- and time-dependently reduced by authentic GnRH-II and the antiproliferative effect with GnRH-II was significantly higher than with equimolar doses of GnRH-I agonist (triptorelin). The available literature indicates that the antiproliferative effect of GnRH 28  analogues could prove to be the basis of novel therapeutic remedies (Grundker et al., 2002a; Volker et al., 2002). 1.2.8.7 Prostate and prostate cancer Through RT-PCR studies, both nonnal and neoplastic prostatic tissues have shown the presence of GnRH-R mRNA (Kakkar et a!., 1994; Bahk et a!., 1998; Limonta et al 1999; Halmos et al., 2000; Tieva et aL, 2001), whereas immunoreactivity against GnRH-R could only be detected in tumorous prostate tissue and intraprostatic lymphocytes (Tieva et aL, 2001 ).The presence of the specific binding sites for GnRH-I were demonstrated in human prostate cancer and certain prostatic cancer lines (Feket et al., 1989;Limonta et a!., 1992; Ravenna et al., 2000) with a lower affinity than that of the pituitary receptors (Limonta et al., 1992; Ravenna et a!., 2000). Expression of the GnRH-R in the prostate was verified through western blotting and by detecting a 64 kDa band, which corresponds to the molecular weight of the pituitary GnRH-R (Limonta et al., 1999). 1.2.9 Extrapituitary GnRB and GnRH-R interactions and biological actions  In summary, there are at least two distinctive types of GnRH that have been fully characterized in mammals and seem to have been structurally conserved for over 500 million years. The wide tissue distribution, particularly of GnRH-II, suggests that it may have a variety of reproductive and non-reproductive functions that are yet to be identified (Millar, 2002; Leung et al., 2003).  The existence of distinctive GnRH forms suggests the presence of  distinctive cognate receptor types in vertebrates and is potential area of research in coming years. Like both GnRH-I and GnRH-II, even GnRH-III may also play a role in the management of autocrine and paracrine effects within reproductive tissues (Grundker et al., 2002a). The existence of additional GnRH systems in brain, pituitary and reproductive tissues, as well as different mechanisms of action in these tissues, may contribute to the development of a new 29  generation of GnRH analogues (agonists and antagonists) with highly selective and controlled actions on these different types of receptors. Nonetheless, it is much easier to delineate different mechanisms of action in these tissues; therefore, target-specific GnRI-I analogues could be developed in the near future that allow a better control over manipulation of various reproductive processes. For example, target- specific GnRH analogues that only act either at the pituitary, the gonadal or uterine level could be developed. Although much remains to be elucidated with regard to the significance of different GnRH, GnRH-R systems in reproductive tissues, it is certain that the future looks promising for research and development of innovative and novel therapeutic measures utilizing the GnRH system. In addition, it is possible that additional roles for GnRH governing one or more physiological events in the body could be unveiled in the future (Ramakrishnappa et a!., 2005). In addition to its pivotal role in stimulating gonadotrophin synthesis and secretion from pituitary gonadotrophs, GnRH-I functions as an autocrine and/or paracrine factor in a number of extrapituitary compartments. Other than gonadal steroidogenesis this hormone may regulate the processes of spermatogenesis, fertilization, apoptosis, cell proliferation, and embryo implantation. In tumours derived from various reproductive tissues, there is evidence showing that GnRH-R couples to pertussis toxin-sensitive Ga protein (most probably Gal) and mediates its biological effects via pathways that are distinct from the classical cascade operated in gonadotrophs. Extra pituitary actions elicited by GnRH-II have also been demonstrated in certain mammalian peripheral tissues (Millar et al., 2004; Ramakrishnappa et al., 2005; Cheng and Leung, 2005).  30  1.2.9.1 Gonadal steroidogenesis  In the ovary, GnRH induces inhibitory and stimulatory responses affecting ovarian function (Sharpe, 1982; Janssens et al., 2000). GnRH is believed to exert its direct effects on gonadal steroidogenesis either on its own or in conjunction with other factors such as PGF2a, angiotensin II or luteinizing hormone. Stimulation of one or more signalling pathways such as phospholipase C (PLC), phospholipase A2 (PLA2) and phospholipase D (PLD), activate protein kinase C (PKC)- causing either inhibitory or stimulatory effects on gonadal cellular steroid output. These dual effects were demonstrated in in vivo experiments in adult males or female hypophysectomized rats where exogenous GnRH agonist administration either stimulated or inhibited gonadal functions in terms of steroidogenesis (Hsueh and Jones, 1982). In adult male rats, GnRH agonist administered at lower dose of for a short-term stimulated testosterone secretion (Sharpe et al., 1982), whereas the effect was the opposite when the agonist was administered at a higher dose or for long-term durations (Arimura et a!., 1979, Hsueh and Erickson, 1979). Similarly, other reports demonstrated that GnRH modulates both basal and gonadotrophin-stimulated steroidogenesis (Olofsson et al., 1995) in the ovary. The inhibitory action of GnRH or its agonists on gonadal steroidogenesis involved down regulation of gonadotrophin receptors or intermediary enzymes involved in steroidogenic pathway. Reports suggesting GnRH agonist induced down regulation of FSH and LH receptors (Tilly et al., 1992; Piquette et al., 1991; Guerrero et a!., 1993), reduced gonadotrophin-induced cAMP levels (Richards, 1994; Knecht et a!., 1985) or steroidogenic enzyme activity such as peripheral-type benzodiazepine, steroidogenic acute regulatory protein, P45Oscc enzyme, and 3I3HSD (Sridaran et a!., 1999a; Sridaran et al., 1999b) or no effect (Casper et al., 1984) of GnRH on P 4 production in human granulosa-lutein cells (hGLCs).  31  In bovine species, GnRH agonist, buserelin inhibited P 4 secretion from in vitro cultured luteal cells (Milvae et al., 1984). In vivo studies by D’Occhio et al. (2000) suggested that the suppressed ovarian function in heifers treated for long-term with GnRH agonist may have been due, in part, to a direct action of deslorelin (GnRH implant) on the ovaries. On the other hand, there are several reports, both from in vitro and in vivo studies, in rodents, primates and from in vitro human granulosa cell culture models providing sufficient evidence to contradict the above mentioned inhibitory effects of GnRH agonist at the gonadal level. Liu et al. (1991) reported dose-related stimulatory effects of GnRH agonist on aromatase activity and P 4 production in monkey granulosa cell cultures. They also demonstrated GnRH antagonist suppresses the stimulatory effect of GnRH agonist on granulosa cell steroidogenesis in culture. Similar findings demonstrating GnRI-1 agonist induced steroidogenesis in cultured human granulosa cells were reported (Ranta et al., 1982; Parinaud et al., 1992; Bussenot et al., 1993). Parinaud et al. (1998) suggested that GnRH agonist could modulate steroidogenesis by a direct ovarian action. The agonist, buserelin, increased basal and decreased LH-induced P 4 secretion in vitro. Guerrero et al. (1993) found an increase in P 4 production and decrease in E 2 production and that could be related to a decrease in LH receptor numbers and aromatase activity in GnRH agonisttreated cells. In our lab, GnRH agonist, buserelin treatment resulted in a dose dependent stimulatory effect on steroid hormone output from both granulosa cells and luteal tissues in vitro. From further investigations it was concluded that there is an increase in the mRNA expression for both steroid acute regulatory protein and cytochrome P450 side chain cleavage (P450) enzymes (Ramakrishnappa, 2004).  In male gonads GnRH induced a direct stimulatory effect on basal steroidogenesis and an inhibitory effect on gonadotrophin-stimulated androgen biosynthesis in rats (Hsueh and Jones, 1982). The short-term in vitro treatment of adult rat leydig cells with GnRH resulted in 32  increased testosterone production (Sharpe and Copper, 1 982b; Moicho et al., 1984), while longterm incubation decreased their response to hCG (Browning et al., 1983). GnRH agonist also stimulated testosterone production and sprematogonial multiplication in frogs (Minucci et al., 1986; Zerani et al., 1991). In contrast, a high dose of long acting GnRH agonist had a direct inhibitory effect on testosterone secretion in hypophysectomized rats. In immature and hypophysectomized rats, in vivo administration of GnRH and GnRH agonist had inhibitory effects on reproductive function (Bambino et al., 1980; Kerr and Sharp, 1986), and GnRH agonist inhibited the basal and LH-stimulated steroidogenesis in rat fetus and cultured testicular cells (Dufau and Knox, 1985; Habert, 1992). The above studies suggest that GnRH agonist has a direct effect on testis without the action of gonadotrophin (Arimura et al., 1979). Therefore, an understanding of the physiological functions induced by GnRH or its agonist may be derived by studying the direct effects of GnRH and/or indirect effects of GnRH via monitoring the variations of gonadotrophin and testosterone levels (Botte et al., 1999). 1.2.9.2 Steroid hormone receptor regulation  The mammalian endometrium is a dynamic structure and is one of the primary target sites for the action of the ovarian steroids; estrogen and P . These steroids exert their actions 4 through corresponding receptors: estrogen (ER) and progesterone receptors (PR), respectively. So far two isoforms of ER namely ERa and ERI3 have been identified and it was shown that ERa is the predominant estrogen receptor in the mammalian uterus (Moutsatsou and Sekeris, 1997; DeMayo et al., 2002), whereas ERf3 is the major receptor in the ovaries. The presence of ER and PR was elucidated in ovarian and uterine compartments across different species ranging from mice to humans (Couse et al., 2006). Their presence was demonstrated in bovine ovaries and endometrium (Walther et al., 1999; Meikie et al., 2001). Estrogen and P 4 maintain normal uterine physiology by regulating endometrial proliferation, gamete transport, uterine 33  receptivity, embryonic development, implantation, decidualization and maintenance of pregnancy during the course of normal reproduction (Graham and Clark, 1997; Ulbrich et al., 2003; Vasudeven and Plaff, 2007). There is limited information available regarding the interaction of GnRH, GnRH-R and the steroid hormone receptor system in reproductive tissues. There are a few reports studying GnRH agonist induced regulation of steroid hormone receptors in pituitary and ovarian tissues. GnRH up-regulated ERcL and PR in pituitary cells and further suggesting that possible cell signalling pathway involved in producing these differential effects is PKC (Demay et al., 2001; Tasende et al., 2002). On the contrary, Chiang et al. (2000) revealed that GnRH down-regulate ERCL and ERI3 in human granulosa luteal cells, in vitro. 1.2.9.3 Apoptosis Apoptosis is a vital biological process of programmed cell death. This noninflammatory phenomenon is characterized by cellular blebbing, DNA fragmentation, formation of apoptotic bodies, and the elimination of unwanted cells (Elmore, 2007). Two major pathways named the mitochondrial pathway (intrinsic) and the death receptor pathway (extrinsic) govern apoptosis at molecular and cellular levels. The mitochondrial pathway is regulated by Bc1 2 group of genes with pro- and anti- apoptotic molecules (Bax and Bcl-2) as its important members. Fas, which belongs to the family of tumor necrosis factor (TNF) receptors is another important mediator inducing apoptosis through extrinsic pathway (death receptor pathway). Both intrinsic and extrinsic pathways converge to activate caspases to execute the process of programmed cell death (Riedi and Shi, 2004). Apoptosis in ovarian follicles and CL is considered a physiological process that selectively eliminates unwanted cells. In all the species studied so far, the initiation of apoptosis in granulosa cells is one of the earliest signs of follicular demise (Tilly and Hsueh, 1993; Yuan 34  and Giudice, 1997). The occurrence of apoptosis in granulosa cells of atretic follicles was documented based on morphological (Palumbo and Yeh, 1994; Yang and Rajamahendran, 2000a; Saito et al., 2000) and biochemical criteria (Hughes and Gorospe, 1991; Nahum et al., 1996; Manikkam and Rajamahendran, 1997; Yang and Rajamahendran, 2000a and b). Evidence from previous studies suggests a role for GnRH in inducing follicular atresia (Hsueh et al., 1984; Piquette, et a!., 1991; Billig et al., 1994). Atretic follicles in the rat during the follicular phase showed higher levels of GnRH-R mRNA expression (Whitelaw et al., 1995), whereas, during in vitro culture, GnRH inhibited DNA synthesis (Saragueta et al., 1997) or induced apoptosis in rat granulosa cells (Billig et al., 1994). During early pregnancy in rats, GnRH agonist reduced serum P 4 levels, which was associated with an increased degree of DNA fragmentation in the CL (Sridaran et a!., 1998). Zhao et al. (2000) showed in human granulosa cells (obtained during oocyte retrieval for in vitro fertilization) that GnR}I agonist treatment resulted in increased numbers of apoptotic bodies in these cells. GnRH induced a dosedependent stimulation of Fas ligand expression in reproductive tissue cancers. Fas, expression in GnRH-R positive tumors indicates that GnR}1 could be an autocrine pro-apoptotic factor in Fas-positive cancers (Imai et a!., 1998). The GnRH analogues induced anti-apoptotic and antiproliferative effects through different signalling mechanisms activated by the same Gct protein in ovarian cancer cells (Cheung et a!., 2006). In a recent study, Clementi et al. (2009) showed that the GnRH agonist, leuprolide and antagonist, cetrorelix induced expression of Bax and Bcl-2 and the activation of caspase-9 (intrinsic pathway), caspase-8 (extrinsic pathway), and caspase-3 in prostate cancer cells. These researchers further studied the mRNA level, protein expression and phosphorylation of p53 in response to the same agonist and antagonist. In these experiments leuprolide and cetrorelix treatments resulted in activation of caspase-8 and -3, but not -9. Bax protein levels increased after cetrorelix treatment. Both leuprolide and cetrorelix 35  increased p53 expressions and phosphorylation in prostate cancer cells cultures. These findings indicate that GnRH analogs could induce apoptosis in prostate cancer through the extrinsic pathway involving p53 phosphorylation. The mammalian uterus is a dynamic structure and undergoes series of cellular proliferation and demise every estrous or menstrual cycle. Apoptosis acquires more significance in rapidly renewable structures like the endometrium where cellular turnover has to be tightly controlled (Harda et al., 2004). There are a few reports from the rats and humans, investigating the role of GnRH in uterine apoptosis. Murdoch et al. (1995) suggested that in the rat endometrium, apoptosis is mediated by GnRH-R. In humans, GnRH analogues in vivo or in vitro suppressed myometrial cell growth by activating apoptosis (Wang et al., 2002). A contradictory report showed that GnRH analogues can suppress the expression of some pro apoptotic factors while up-regulating the anti-apoptotic factor, Bcl-2 protein, in vivo (Huang et a!. 2002). It was demonstrated that GnRH augmented apoptosis in the epithelial cells obtained from endometriosis and the proliferative phase human endometrium (Meresman et al., 2003; Bilotas et al., 2007). Kwon et al. (2005) showed that GnRH promoted cellular arrest in uterine leomyomas by stimulating apoptosis through the death receptor pathway. It appears that there is no consensus on GnRH agonist induced apoptotic mechanisms in mammalian reproductive tissues. Morgan et al. (2008) observed that the reasons for these differences could be in the levels of GnRH-R expression in different cell types. This variable response could be due to cell passages in vitro, culture conditions and tissue type (normal or carcinogenic).  36  1.2.9.4 Cell proliferation The role of GnRH-I as a negative autocrine growth factor has been well reported in cell lines derived from human malignant tumors including those of the ovary, endometrium, breast, prostate gland and melanoma cells (Emons et al., 1998; Millar et al., 1985; Grundker et al., 2002 a; Kang et al., 2003; Moretti et al., 2003). This antiproliferative action is mediated via high affinity GnRH- 1 binding sites and supported by the notion that the nucleotide sequence of the GnRH-R is identical in tumor and pituitary cells (Kakkar et al., 1994; Grundekar et al., 2001). Sometimes, high doses of GnRH analogoues (1-10 1 iM) are needed to demonstrate a significant growth inhibitory response in cancer cell lines (Kimura et al., 1999; Grundker et al., 2002b; Wells et al 2002; Moretti et al., 2003). Several reports show that GnRH analogoues can also inhibit cell proliferation in human uterine leiomyoma and endometriosis, where GnRH induced leiomyoma regression appears to occur predominantly through the inhibition of gonadotrophins and gonadal steroids and  the suppression may involve alteration in the  expression of growth factor (Di Lieto et al., 2002), cytokines (Senturk et al., 2001), cell cycle regulator (Kobayashi et al., 1997) and steroid hormone receptors (Vu et al., 1998). 1.2.9.5 Fertilization and embryo implantation Further, it was suggested that GnRH and its analogues may modulate spermatozoa-zona pellucid binding in humans. Short time exposure (5 mm) of spermatozoa to a 20 nM GnRH agonist, buserelin caused a significant increase in the number of zona bound sperm (by 300 to 350 fold). This effect was completely preventable by prior exposure of spermatozoa to GnRH antagonist (Morales and Llanos, 1996). These findings suggest that spermatozoa may interact with GnRI-1, or GnR}1-like molecules that they may come in contact with during their journey through the male and female reproductive tracts (Morales, 1998; Bull et al., 2000). The interaction may occur: (a) during spermatogenesis by local, intra-testicular production (Hsueh 37  and Schaeffer, 1985; Verhoeven and Cailleau, 1985); (b) during sperm maturation in the epididymis; and (c) during ejaculation, upon mixing with seminal plasma, during transport to the site of fertilization in the oviduct. In the oviduct, the spermatozoa may interact with GnRH secreted locally or transported by the products of ovulation (follicular fluid, granulosa cells) from the ovary (Ying et al., 1981; Aten et a!., 1 987a and b; Ireland et al., 1988; Oikawa et a!., 1990). In addition, it was suggested that GnRH is involved in the process of fertilization. GnRH agonist increased the cleavage rate of bovine oocytes in vitro (Funston and Seidal, 1995). GnRH agonist in supraphysiological and long-acting doses appears to exert an inhibitory effect on each step of spermatogenesis, which is thought mediated by suppressing FSH, LH and intratesticular testosterone levels. Interestingly, GnRH agonist or GnRI-I antagonist treatment enhanced the regeneration of spermatogenesis from damaged testes in the irradiated rat (Meistrich and Kangasniemi, 1997; Shuttlesworth et al., 2000), cryptorchid rat (Koichi et al., 2002), cytotoxic therapy rat (Meistrich, et a!., 1999), and juvenile spermatogonial depletion (jsd) mutant mouse (Matsumiya et a!., 1999). It is believed that GnRH agonist treatment would stimulate spermatogonial proliferation, and then regeneration of spermatogenesis occurred. The exact mechanism for this is still uncertain, however, reduced intratesticular testosterone levels by GnRH agonist may play a role as intratesticular testosterone level was elevated after irradiation or chemical insult (Meistrich and Kangasniemi, 1997). Testosterone is necessary for spermatogenesis but suppressive to spermatogonial proliferation (Koichi et al., 2002). Previous studies indicated that in early pregnancy both GnRH-1 and GnRH-II stimulated the mRNA and protein levels of urokinase-type plasminogen activator (uPA) in human extravillous, cytotrophoblasts and decidual stromal cells, in vitro. These findings suggest a regulatory role for hormone in proteolytic degradation of the extracellular matrix of the endometrial stroma which is prerequisite for the decidualization and trophoblast invasion 38  (Tabibzadeh and Babakania, 1995; Paria et al., 2002). Also, both GnRH-I and GnRH-II suppressed the trophoblastic expression of plasminogen activator inhibitor (PAT-I) in a dose and time dependent manner (Cheng and Leung, 2005). 1.3 GnRH IN BOVINE REPRODUVTIVE MANAGEMENT The availability of more potent, long acting synthetic GnRI-I analogues and enhanced knowledge about the multifunctional roles of GnRH has broadened the scope for applications of GnRH in the field of reproductive biology and medicine. Now it is a common belief that there is not a system in clinical medicine that would not utilize GnRH or its analogues in one or other way.  In the veterinary practice, the versatile functional properties of GnRH were  exploited to manage a variety of reproductive disorders with varying degrees of success (Thatcher et al., 1993; D’Occhio and Aspden, 1999; Rajamahendran et al., 2001). The fact that GnRH causes endogenous release of LH and FSH is an important factor being employed in manoeuvring ovarian activity. GnRH induced LH release is associated with numerous physiological effects such as an altered CL function, ovulation or luteinization of ovarian follicles, and induction and development of new follicles. Further, GnRH or its analogues are routinely employed in various treatment regimens including prevention of early embryonic mortality, synchronization of estrous and suppression of ovarian activity for extended periods (D’Occhio and Aspden, 1999; Peters, 2005). The later treatment has become much easier since the availability of slow releasing subcutaneous implants of GnRH analogues. Furthermore, feature appears to be a promising tool for development of safer methods of contraception through temporary suppression of ovarian function. The following sections provide the overview on the roles for GnRH or its analogues in the field of bovine reproductive management.  39  1.3.1 Early embryonic sustainability and pregnancy outcome  Peters (2005) observed that approximately 25% of bovine embryos were lost during the first 3 weeks of life and untimely luteolysis is a major reason for this as maintenance of P 4 levels by a viable CL is very important for the establishment of early pregnancy. Use of a single injection of GnRH on the day of insemination improves overall pregnancy rates and particularly in repeat breeders. The reason for it could be that GnRH induced ovulation at an appropriate time relative to insemination and stimulated luteinization, increasing the chance for successful fertilization and embryo survival. Heifers treated with GnRK agonist from day 4 to day 6 of the estrous cycle can ovulate and develop an accessory CL which may be a contributory factor for increased P 4 levels in plasma (Rajamahendran and Siangama, 1992; Schmitt et al., 1996; Rajamahendran et al., 1998). Post insemination administration of GnRH agonist during the early estrous cycle seems to enhance the likelihood of conception, pregnancy recognition, and embryo survival, by increasing plasma P 4 concentration (Ullah et al., 1996). A number of studies have been conducted to determine if GnRH administration at the time of insemination can improve pregnancy rates in cattle. In general, the GnRH doses used were 100 ig for native GnRH and 8-10 ig for its synthetic agonist, buserelin. There were studies in which these treatments were carried out during the luteal phase of the estrous cycle with the main intent to enhance luteal function for sustaining the embryos. The majority of these studies showed increased P 4 production after GnRH administration, whereas some studies have demonstrated increased pregnancy rates. Mee et al. (1990) and Stevenson et al. (1990) observed an overall 6-7% improvement in pregnancy rate in first service, in about 14,000 cows studied with GnRH administration at estrus. From the available literature it can be concluded that administration of GnRH and its agonistic analogues at the time of artificial insemination or during the luteal phase increases the pregnancy rates but the physiological and environmental 40  variables affecting fertility are still to be assessed properly. Peters et a!. (2000) carried out a meta analysis on published data on the use of GnRH and its analogues administered between day 11 and day 14 after first insemination and concluded that the response to GnRH treatment in terms of pregnancy outcome varies with cow type (beef or dairy), parity (cow or heifer), use of estrus synchronisation (synchronised or natural) and, pregnancy diagnosis (method and timing). Therefore, it is imperative to study the effect of GnRH agonist on fertility under laboratory conditions where the experimental conditions can be controlled and kept uniform, providing a much better chance to determine whether these treatments can really support reproductive process at both the molecular and cellular levels. 1.3.2 Estrus and ovulation synchronization  Since the emergence of the concept of estrus synchronization in farm animals, researchers have been working out an efficient hormonal treatment regimen for induction of synchronized follicular development, ovulation, and an effective formation of CL in cattle. Recent reports have shown the direction for effective manipulation of synchronized ovulation through usage of GnRH or its synthetic analogues. “Ovsynch” (Pursley et al., 1995) is one of such recently developed and promising method of estrus synchronization involving GnRH treatment on Day 0, PGF a on Day 5-7 and a second GnRH treatment on Days 7-9 followed by 2 timed insemination. This method is widely practiced in bovine reproductive medicine but, due to limited knowledge of the multifunctional roles of GnRI-1 or its analogues in the reproductive tissues, there is still no appropriate treatment regimen for GnRH or GnRH agonist in both the human and domestic animal reproductive medicine. GnRH administration during the luteal phase resulted in LH release and caused either ovulation or atrasia of the dominant follicle (Webb et al., 1992; Peters et al., 1999) depending upon the follicular status at the time of administration. Administration of PGF2c thereafter resulted in emergence of a new follicular 41  wave in a synchronized manner. Studies showed that low GnRH pulse frequency supported synthesis and release of FSH without increasing LH levels. High GnRH pulse frequency inhibited FSH synthesis and release (Vizcarra et a!., 1999). The size of the largest ovarian follicle was greater in heifers treated with GnRH agonist than cows (Pursley et al., 1997; Taponen et al., 1999), and it was associated with increased E 2 plasma concentrations (Bergfeld et al., 1996; Dufour et al., 1999). A second dose of GnRH agonist administered 24 h after the 2 secretion in the preovulatory mature PGF2a injection, appeared to cause LH surge, stopping E or immature follicle (Taponen et al., 1999, 2002, 2003). Peters et al. (1999) have shown that the second injection of GnRH subsequent to PGF a in the “Ovysynch” protocol significantly 2 advanced the timing of ovulation and increased plasma P 4 concentrations. 1.3.3 Post partum ovarian cyclicity  Resumption of post partum ovarian activity is important for the re-establishment of pregnancy in dairy cows. It is desirable for the dairy industry that ovarian cycles begin as soon as possible after parturition. During the last three decades researchers have used large doses of native GnRH (100-500 tg) or its analogues e.g. buserelin (10-20 fig) to stimulate ovarian activity. It was more successful in dairy cows than in beef cows. It resulted in a small, but significant reduction in days open (2.75 days) and in the number of services per conception with the use of a single large dose of GnRH in post partum dairy cows (Bracket and Lean, 1997; Peters, 2005). The preovulatory gonadotropin surge induced within 30 mm of injection of large doses ( 100 jig) of GnRH may be considered to be unphysiological. Some researchers used lower doses of GnRH to stimulate the more physiological episodic secretion of endogenous GnRH and gonadotrophins to influence follicular development and maturation. But smaller doses (1-5 jig) were not very successful in larger groups of animals (Peters, 2005). The advent of real time 42  ultrasonography for on farm diagnosis of ovarian follicular status in the post partum period provided opportunities for specifically targeting fertility treatments. A few studies were conducted thereafter with inconclusive findings about induction of post partum ovulation. There has been more focus on nutritional management and purpureal infections which may be a part of the possible etiology of post partum anovulation (Yavas and Walton, 2000). 1.3.4 Cystic ovarian condition GnRH use in treatment of cystic ovarian disease dates back to late 1 970s. The definition of a cystic follicle is persistence of a follicle- like structure greater than 25 mm in diameter, in the absence of a CL for at least 7-10 days, whereas luteal cysts have thicker walls and have undergone some luteinization. Real time ultrasonographic procedures are useful techniques for the diagnosis of ovarian cysts at the fann level (Peters, 2005). It is assumed that there is an interaction between genetics and environment in the development of cysts and cows are susceptible to the formation of cysts as a result of an environmental stressors’ effect on the neuroendocrine system. This inhibits the GnRH and/or gonadotrophin secretions in such a way that ovulation is inhibited (Cole et al., 1986) and it makes GnRH use logical in the treatment of cysts. To summarise, GnRH treatments in cystic ovarian disease appears effective in a way that it stimulated a new ovulation or luteinization similar to non-cystic cows (Kesler et al., 1981; Nanda et al., 1989; Hooijer et al., 2001). On the other hand, a group of researchers observed that cysts may luteinize, but not regress with GnRH treatment (Kesler et al., 1981; Jeffcoate and Ayliffe, 1995). Cairioli et al. (2002) suggested a better response to GnRH treatment than the other conventional treatments.  43  1.4 DISSERTATION RATIONALE The extra-pituitary presence of GnRH-R is matter of ongoing interest in mammalian reproductive biology. GnRH-R are present in normal and carcinogenic human ovarian, uterine, testicular and prostatic tissues. The GnRH, GnRH-R system has a growth inhibitory effect in carcinogenic extrapituitary reproductive tissue by acting in a pro-apoptotic manner. Recently, the local modulatory role of the GnRH, GnRH-R system in human and rat ovarian physiology has been elucidated and GnRH regulates apoptosis in ovarian tissues/cells. There are a few reports showing the presence of GnRH-R mRNA in human uterine carcinomas, leomyomas and endometriosis. In bovines, GnRH and GnRH-R mRNA has been demonstrated in ovarian structures (follicle and CL). The presence GnRH-R in bovine uterus and oviducts is still to be determined. The local modulatory role of the GnRH, GnRI-I-R system in uterine/oviductal physiology remains to be elucidated. Ovarian steroids (estrogen and P ) and apoptosis regulate 4 the reproductive cyclicity and cellular turnover, respectively. Estrogen and P 4 induce their actions through their corresponding estrogen receptors (ERcL, ERf3) and progesterone receptors (PR). There are few contradictory reports from pituitary and ovarian cells showing that GnRH either stimulates or inhibits estrogen receptors in these tissues. The possibility of cross talk between GnR}I and steroid hormone receptor systems in the bovine uterus cannot be ruled out. Apoptosis acquires greater significance in rapidly renewable tissues like the endometrium, which undergoes changes in terms of cellular turnover every estrous cycle. The role of GnRH as a regulator of apoptosis in the bovine uterus is still not investigated. Therefore, this research investigated the presence of GnRH-R in the bovine endometrium and oviducts. The current research was also designed to study the local modulatory role of GnRH, GnRH-R system in steroid hormone receptor regulation and in the induction of endometrial apoptosis.  44  These studies will strengthen our knowledge about the presence of extra-pituitary GnRH-R in bovines. This research will further elucidate the local modulatory role of GnRH, GnRH-R system in the bovine uterine physiology. These findings will be an addition of knowledge to the field of mammalian reproductive biology/physiology and could form the basis for the rationalization of the usage of GnRH, or its potent synthetic analogues, in cattle. The current research could further broaden the basis for additional studies with respect to proand anti-fertility effects, as well as therapeutic role(s), for GnRH or its analogues in the field of reproductive medicine. 1.8 OBJECTIVES The objectives of this study were: 1. To determine if GnRH-R are present in the follicular and luteal phase bovine endometrium and oviducts (Chapter 2); 2. To find out if GnRH agonist, buserelin induces regulation of estrogen receptors (ERa and ERf3) and progesterone receptor (PR) mRNA in the follicular and luteal phase bovine endometrium (Chapter 3); and 3. To investigate buserelin induced regulation of apoptotic genes (Bax, Bcl-2, caspase-3 and Fas) mRNA and epithelial cell apoptosis in the bovine endometrium (Chapter 4).  45  1.9 BIBLIOGRAPHY Arencibia J, Schally A. 2000. Luteinizing hormone-releasing hormone as an autocrine growth factor in ES-2 ovarian cancer cell line. mt. J. Oncol. 16: 1009-1013. Arimura A, Serafini P, Talbot S, Schally AV. 1979. Reduction of testicular luteinizing hormone/human chorionic gonadotropin receptors by [D-Trp6]-luteinizing hormone releasing hormone in hypophysectomized rats. Biochem. Biophys. Res. Commun. 90: 687-693. Arora KK, Krsmanovic LZ, Mores N, O’Farrell H, Catt KJ. 1998. Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor. J. Biol. Chem. 273: 2558 1—25586. Assefa D, Pawson AJ, McArdle CA, Millar RP, Flanagan CA, Roeske R, Davidson JS. 1999. A new photoreactive antagonist cross-links to the N-terminal domain of the gonadotropin-releasing hormone receptor. Mol. Cell. Endocrinol. 156: 179—188. Aten FR, Ireland JJ, Weems CW, Behrman HR. 1987a. Presence of gonadotropin releasing hormone-like proteins in bovine and ovine ovaries. Endocrinology. 120: 1727-1733 Aten RF, Polan ML, Bayless R, Barman HR. 1987b. A gonadotropin-releasing hormone (GnRH)-like protein in human ovaries: similarity to the GnRH-like ovarian protein of the rat. J. Clin. Endocr. Metab. 64: 1288-1293. Azad N, Uddin S, La Paglia N, Kirsteins L, Emanuele NV, Lawrence AM, Kelley M R. 1993. Luteinizing hormone-releasing hormone (LHRH) in rat prostate: characterization of LHRH peptide, messenger ribonucleic acid expression, and molecular processing of LHRH in intact and castrated male rats. Endocrinology. 133: 1252-1257. Backett SD, Lean U. 1997. Gonadotropin releasing hormone in postpartum dairy cattle: a metaanalysis of effects on reproductive efficiency. Anim. Reprod. Sci. 48: 93-1 12. Bahk J, Hyun JS, Lee H, Kim M, Cho G, Lee B, Choi W. 1998. Expression of gonadotropin-releasing hormone (GnRH) and GnRJ-I receptor mRNA in prostate cancer cells and effect of GnRH on the proliferation of prostate cancer cells. Urol. Res. 26: 259-264. Bahk JY, Hyun JS, Chung SH, Lee H, Kim MO, Lee BH, and Choi WS.1995. Stage specific identification of the expression of GnRH mRNA and localization of the GnRH receptor in mature rat and adult human testis. J. Urol. 154: 1958-1961. Bambino TH, Schreiber JR, Hsueh A.JW. 1980. Gonadotropin-releasing hormone and its agonist inhibit testicular luteinizing hormone receptor and steroidogenesis in immature and adult hypophysectomized rats. Endocrinology. 107: 908-917. Bergfeld EG, D’Occhio MJ, Kinder JE. 1996. Pituitary function, ovarian follicular growth, and plasma concentrations of 17 B-estradiol and P4 in prepubertal heifers during and after treatment with the luteinizing hormone-releasing hormone agonist deslorelin. Biol. Reprod. 54: 776-782. 46  Billig H, Furuta I, Hsueh AJ. 1994. Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: biochemical in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinology. 134: 245-252. Bilotas, M., Baraflo, R.I., Buquet, R., Sueldo, C., Tesone, M. And Meresmas, G. 2007. Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls. Hum. Reprod. 22: 644-653. Botte MC, Chamagne AM, Carre MC, Counis R, Kottler ML. 1998. Fetal expression of GnRH and GnRH receptor genes in rat testis and ovary. J. Endocr. 159: 179-189. Botte MC, Lerrant Y, Lozach A, Berault A, Counis R, Kottler ML. 1999. LH downregulates gonadotropin-releasing hormone (GnRH) receptor, but not GnRH, mRNA levels in the rat testis. 3. Endocr. 162: 409-4 15. Bourne GA, Dockrill MR, Regiani S, Marshall JC, Payne AH. 1982. Induction of testicular gonadotropin-releasing hormone (GnRH) receptors by GnRH: effects of pituitary hormones and relationship to inhibition of testosterone production. Endocrinology. 110: 727-33. Bourne GA, Regiani S, Payne AH, Marshall JC. 1980. Testicular gonadotropin-releasing hormone receptor-characterization and localization on interstitial tissue. 3. Clin. Endocr. Metab. 51: 407-409. Bradham C and McClay DR. 2006. p 38 MAPK in development and cancer. Cell Cycle. 5: 824—828. Brooks J, Taylor PL, Saunders PT, Eidne KA, Struthers WJ, McNeilly AS. 1993. Cloning and sequencing of the sheep pituitary gonadotropin-releasing hormone receptor and changes in expression of its mRNA during the estrous cycle. Mol. Cell. Endocr. 94: 23-27. Browning JY, D’Agat, R, Steinberger A, Grotjan Jr. HE, Steinberger E. 1983. Biphasic effect of gonadotropin-releasing hormone and its agonist analog (H0E766) on in vitro testosterone production by purified rat Leydig cells. Endocrinology. 113: 985-991. Bull P, Morales P, Huyser C, Socias T, Castellon EA. 2000. Expression of GnRH receptor in mouse and rat testicular germ cells. Mo!. Hum. Reprod. 6: 5 82-586. Bussenot I, Azoulay-Barjonet C, Parinaud J. 1993. Modulation of the steroidogenesis of cultured human granulosa—lutein cells by gonadotropin-releasing hormone analogues. J. Clin. Endocr. Metab. 76: 1376—1379. Catt KJ. 1999. Gonadotropin releasing hormone and the GnRH receptor. In: Molecular biology in reproductive medicine. The Parthenon Publishing Group: 131-146. Cairioli F, Vigo D, Battochio M, Faustini M, Veronesi MC, Maffeo G. 2002. 17 beta estradiol, P4 and testosterone concentrations in cystic fluids and response to GnRH treatment after emptying of ovarian cyst in cattle. Reprod. Domest. Anim. 37: 294-298. 47  Casan EM, Rag, F, Bonilia-musoles F, Polan ML. 2000. Human oviductal gonadotropin releasing hormone: Possible implications in fertilization, early embryonic development, and implantation. J. Clin. Endocr. Metab. 85: 1377-1381. Casan EM, Raga F, Polan ML. 1999. GnRH mRNA and protein expression in human preimplantation embryos. Mol. Hum.Reprod. 5: 234-239. Casper RF, Erickson GF, Yen SS. 1984. Studies of the effect of GnRH and its agonist on human luteal steroidogenesis in vitro. Fertil. Steril. 42: 39-43. Castro-Fernandez C, Conn PM. 2002. Regulation of the gonadotropin-releasing hormone receptor (GnRHR) by RGS proteins: role of the GnRHR carboxyl-terminus. Mol. Cell. Endocrinol. 191: 149—156. Cheng CK, Leung PCK. 2005. Molecular Biology of Gonadotropin- Releasing Hormone (GnRH)-1 and GnRH-11 and Their Receptors in Humans. Endocr. Rev. 25: 1-108. Cheon KW, Lee HS, Parhar IS, Kang IS. 2001. Expression of the second isoform of gonadotropin-releasing hormone (GnRI-1-H) in human endometrium throughout the menstrual cycle. Mol. Hum. Reprod. 7: 447-452. Chi L, Zhou W, Prikhozhan A, Flanagan C, Davidson JS, Golembo M, hung N, Millar RP, Sealfon SC. 1993. Cloning and characterization of the human GnRH receptor. Mol. Cell. Endocr. 91: 1-6. Cheung LW, Leung PC, Wong AS. 2006. Gonadotropin-releasing hormone promotes ovarian cancer cell invasiveness through c-Jun NH2-terminal kinase-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9. Cancer Res. 66: 10902—10910. Chiang CH, Cheng KWA, Igarashi S, Nathwani PS, Leung PCK. 2000. Hormonal regulation of estrogen receptor c and 13 Gene Expression in human granulosa- luteal cells in vitro. J. Clin. Endocr. Metab. 85: 3828-3839. Clayton RN, Eccieston L, Gossard F, Morel G. 1992. Rat granulosa cells express the gonadotropin-releasing hormone gene: evidence from in situ hybridization. J. Mol. Endocr. 9: 189-195. Clayton RN, Katikieneni M, Chan V, Dufau ML, Catt, CJ. 1980. Direct inhibition of testicular function by by gonadotropin-releasing hormone: mediation by specific gonadotropin releasing hormone receptors in interstitial cell. Proc. Natl. Acad. Sci. U.S.A. 77: 4459-4463. Clayton RN, Shakespear RA, Duncan JA, Marshall JC, Munson PJ, Rodbard D. 1979. Radioiodinated nondegradable gonadotropin-releasing hormone analogues: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors. Endocrinology. 105: 1369-76. Clementi M, Sanchez C, Benitez DA, Contreras HR, Huidobro C, Cabezas J, Acevedo C, Castellón EA. 2009. Gonadotropin releasing hormone analogs induce apoptosis by extrinsic pathway involving p53 phosphorylation in primary cell culture of human prostatic adenocarcinomas. Prostate. 69: 1025-1033. 48  Cole WJ, Bierschwal CJ, Youngquist RS, Braun WF. 1986. Cystic ovarian disease in a herd of Holstein cows: a hereditary correlation. Theriogenology. 25: 813-820. Couse JF, Hewitt SC, Korach KS. 2006. Steroid receptors in the ovary and uterus. Pages 593677, In: Jimmy D Neill edited knobil and Neill’s Physiology of Reproduction Volume 1 (Third Edition), Elsievier Academic Press. D’Occhio M J, Aspden, W J. 1999. Endocrine and reproductive responses of male and female cattle to agonists of gonadotropin-releasing hormone. J. Reprod. Fert. 54: 101-114. D’Occhio MJ, Fordyce G, Whyte TR, Aspden WJ, Trigg TE. 2000. Reproductive responses of cattle to GnRH agonists. Anim. Reprod. Sci. 60-6 1: 433—442. Davidson JS, Assefa D, Pawson A, Davies P, Hapgood J, Becker I, Flanagan C, Roeske R, Millar R. 1997. Irreversible activation of the gonadotropin-releasing hormone receptor by photoaffinity cross-linking: localization of attachment site to Cys residue in the N-terminal segment. Biochemistry 36: 12881—12889. Dekel N, LewysohnO, Ayalon D’ Hazum E. 1988. Receptors for gonadotropin releasing hormone are present in rat oocytes. Endocrinology. 123: 1205-7. Demay F, De Monto M, Tiffoche C, Vaillant C, Thieulant M. 2001. Steroid- independent activation of ER by GnRH in gonadotrope pituitary cells. Endocrinology. 142: 3340-3347. DeMayo FJ, Zhao B, Takamoto N, Tsai SY. 2002. Mechanisms of action of estrogen and progesterone. Ann. N.Y. Acad. Sci. 955: 48-59. Di Lieto A, De Rosa G, De Falco M, lannotti F, Staibano S, Polilo F, Scaramellino M, Salavatore C. 2002. Relationship between platelet-derived growth factor expression in leiomyomas and uterine volume changes after gonadotropin-releasing hormone agonist treatment. Hum. Pathol. 33: 220-224. Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, Roberts JL. 1998. Expression of gonadotropin releasing hormone (GnRH) gene in human uterine endometrial tissue. Mo!. Hum. Reprod. 4: 893-898. —  Dufau ML, Knox GF, 1985. Fetal Leydig cell culture-an in vitro system for the study of trophic hormone and GnRH receptors and actions. J. Steroid. Biochem. 23: 743-755. Dufour JJ, Mermillod P, Mariana JC, Romain RF. 1999. The effect of a GnRH agonist on follicular dynamics and response to FSH stimulation in prepubertal calves. Reprod. Nutr. Dev. 39: 133-44. Eidne KA, Flangan CA, Millar RP. 1985. Gonadotropin-releasing hormone binding sites in human breast carcinoma. Science. 229: 989-991.  49  Eidne KA, Sellar RE, Couper G, Anderson L, Taylor PL. 1992. Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. Mol. Cell. Endocr. 90: 5-9. Elmore S. 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35: 495-5 16. Emons G, Muller V, Ortmann 0, Schultz KD. 1998. Effects of LHRH-analogues on mitogenic signal transduction in cancer cells. J Steriod Biochem. Mol. Biol. 65: 199-206. Emons G, Schulz K. 2000. Primary salvage therapy with LHRH analogues in ovarian cancer. Recent Results Cancer Res. 153: 83-94 Eskay RL, Mical RS, Porter JC. 1977. Relationship between luteinizing hormone releasing hormone concentration in hypophysial portal blood and luteinizing hormone release in intact, castrated, and electrochemically-stimulated rats. Endocrinology. 100: 263-270. Everest HM, Hislop JN, Harding T, Uney JB, Flynn A, Millar RP, McArdle CA. 2000. Signalling and anti-proliferative effects mediated by GnRH receptors after expression in breast cancer cells using recombinant adenovirus. Endocrinology 142: 4663—4672. Fekete A, Redding TW, Comaru-Schaily AM, Pontse JE, Connelly RW, Srakalovic G, Schally AV. 1989. Receptors for lutenizing hormone-releasing hormone, somatostatin, prolactin, and epidermal growth factor in rat and human prostrate cancers and in benign prostrate hyperplasia. Prostate. 14: 19 1-208. Fink, G. 1988.Gonadotropin secretion and its control. In: Knobil E, Neill J.D. eds. The physiology of reproduction. New York: Raven Press: 1349-1377. Flanagan CA, Rodic V, Konvicka K, Yuen T, Chi L, Rivier JE, Miflar RP, Weinstein H, Sealfon SC. 2000. Multiple interactions of the Asp(2.61(98)) side chain of the gonadotropin releasing hormone receptor contribute differentially to ligand interaction. Biochemistry 39: 8133—8141. Fromme BJ, Katz AA, Roeske RW, Millar RP, Flanagan CA. 2001. Role of aspartate 7.32 (302) of the human gonadotropin-releasing hormone receptor in stabilizing a high-affinity ligand conformation. Mol. Pharmacol. 60: 1280—1287. Funston R N, Seidel G E. Jr. 1995. Gonadotropin-releasing hormone increases cleavage rates of bovine oocytes fertilized in vitro. Biol. Reprod. 53: 541-545. Furui T, Imai A, Tamaya T. 2002. Intratumoral level of gonadotropin-releasing hormone in ovarian and endometrial cancers. Oncol. Rep. 9: 349-352. Gore AC. 2002. GnRH cells out side the nervous system. In: Gore, A.C. (Eds.), GnRH: the master molecule of reproduction. Kluwer Academic Publishers, Boston. Goto T, Endo T, Henmi H, Kitajima Y, Kiaya T, Nishikawa A, Manase K, Sato H, Kudo R. 1999. Gonadotropin-releasing hormone agonist has the ability to induce increase matrix 50  metalloproteinase (MMP)-2 membrane type 1 -MMP expression in corpora lutea, structural luteolysis in rats. J. Endocr. 161: 393-402. Graham, JD, Clark CL. 1997. Physiological action of P4 in target tissues. Endo. Rev. 18 502-517. Grundker C, Grunthert AR, Westphalen S, Emons G. 2002a. Biology of the gonadotropin releasing hormone (GnRH) system in gynecological cancers. Eur. J. Endocr. 146: 1-14. Grundker C, Gunthert AR, Millar RP, Emons G. 2002b. Expression of gonadotrotropin releasing hormone II (GnRH-II) receptor in human endometrial ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. J. Clin. Endocr. Metab. 87: 1427-1430. Grundker C, Schulz K, Gunthert AR, Emons G. 2000. Luteinizing hormone-releasing hormone induces nuclear factor RB-activation and inhibits apoptosis in ovarian cancer cells. J. Clin. Endocrinol. Metab. 85: 3815-3820. Grundker C, Volker P, Emons G. 2001. Antiproliferative signalling of luteinizing hormonereleasing hormone in human endometrial and ovarian cancer cells through G protein (I) mediated activation of phosphotyrosine phosphatase. Endocrinology 142: 2369-2380. Guarnieri F, Weinstein H. 1996. Conformational memories and the exploration of biologically relevant peptide conformations: an illustration for the gonadotropin-releasing hormone. J. Am. Chem. Soc. 118: 5580-5589. Guerrero HE, Stein P, Asch RH, de Fried EP, Tesone M. 1993. Effect of a gonadotropin releasing hormone agonist on luteinizing hormone receptors and steroidogenesis in ovarian cells. Fertil. Steril. 59: 803-808. Habert R, 1992. Effect of decapitation and chronic in-vivo treatment with a gonadotrophin releasing hormone agonist on testicular steroidogenesis in the rat fetus. J. Endocr. 133: 245251. Haisenleder, DJ, Yasin M, Marshall JC. 1997. Gonadotropin subunit and gonadotropin releasing hormone receptor gene expression are regulated by alterations in the frequency of calcium pulsatile signals. Endocrinology. 138: 5227-5230. Halmos G, Arencibia JM, Schally AV, Davis R, Bostwick DG. 2000. High incidence of receptor for leutinizing hormone-releasing hormone (LHRH) and LHRH receptor gene expression in human prostate cancers. J. Uro. 163: 623-629. Harda T, Kaponis A, Iwabe T, Taniguchi F, Makrydimas G, Sofikitis N, Paschopoulos M, Parskevaidis E, Terakawa N. 2004. Apoptosis in human endometrium and endometriosis. Hum. Reprod. Update. 10: 29-38. Harris NC, Daltow C, Eiden KA, Dong KW, Millar RP. 1991. GnRH gene expression in MDA-MB-231 ZR-75-l breast carcinoma cell lines. Cancer Research. 51: 2577-258 1. 51  Harwood JP, Clayton RN, Catt KJ. 1980. Ovarian gonadotropin-releasing hormone receptors. I. Properties and inhibition of luteal cell function. Endocrinology. 107: 407-413. Hazum E. 1981. Some characteristics of GnRH receptors in rat-pituitary membranes: differences between an agonist and an antagonist. Mol. Cell. Endocrinol. 23: 275-281. Hislop JN, Everest HM, Flynn A, Harding T, Uney JB, Troskie BE, Millar RP, McArdle CA. 2001. Differential internalization of mammalian and non-mammalian gonadotropin releasing hormone receptors: uncoupling of dynamin-dependent internalization from mitogen activated protein kinase signalling. J. Biol. Chem. 276: 39685-39694. Hoffmann SH, ter Laak TT, Kuhne R, Reilander H, Beckers T. 2000. Residues within transmembrane helices 2 and 5 of the human gonadotropin-releasing hormone receptor contribute to agonist and antagonist binding. Mol. Endocrinol. 14: 1099-1115. Hooijer GA, Oijen MAAJ, van Franken K, Vaiks MMII. 2001. Fertility parameters of dairy cows with cystic ovarian disease after treatment with gonadotropin-releasing hormone. Vet. Rec. 149: 383-386. Hovelmann S, Hoffmann SH, Kuhne R, ter Laak T, Reilander H, Beckers T. 2002. Impact of aromatic residues within transmembrane helix 6 of the human gonadotropin-releasing hormone receptor upon agonist and antagonist binding. Biochemistry 41: 1129-1136. Hsieh KP, Martin TF. 1992. Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and Gil. Mol. Endocrinol. 10: 1673-1681. Hsieh KP, Martin TF. 1992. Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and Gil. Mol. Endocrinol. 10: 1673-1681. Hsueh AJ, Jones PB. 1982. Regulation of ovarian granulosa and luteal cell functions by gonadotropin releasing hormone and its antagonist. Adv. Exp. Med. Biol. 147: 223-62. Hsueh AJ, Adashi EY, Jones PB, Welsh TH Jr. 1984. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endo. Rev. 1: 76-127. Hsueh AJ, Erickson GF. 1979. Extra-pituitary inhibition of testicular function by luteinising hormone releasing hormone. Nature. 281: 66-67. Hsueh AJ, Schaeffer JM. 1985. Gonadotropin-releasing hormone as a paracrine hormone and neurotransmitter in extra-pituitary sites. J. Steroid Biochem. 23: 757-764. Hsueh AJW, Schreiber JR, Erickson GF. 1981. Inhibitory effect of gonadotropin-releasing hormone upon cultured testicular cells. Mol. Cell. Endocr. 21: 43-49.  52  Huang SC, Tang MJ, Hsu KF, Cheng YM, chou cv. 2002. Fas and its ligand, caspases, and bcl-2 expression in gonadotropin-releasing hormone agonist-treated uterine leiomyoma. J. Clin. Endocr. Metab. 87: 4580-4586. Hughes FM Jr., Gorospe wc 1991. Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology. 129: 2415-22 Ikeda M, Taga M, Sakakibara H, Minaguchi H, Vonderhaar BK. 1995. Detection of messenger RNA for gonadotropin-releasing hormone (GnRH) but not for GnRFT receptors in mouse mammary glands. Biochem. Biophys. Res. Commun. 207: 800-806. hung N, Jacobs GF, Becker II, Flanaga, CA, Davidson JS, Eales A, Zhou W, Sealfon SC. Millar RP. 1993. Comparative sequence analysis and functional characterization of the cloned sheep gonadotropin-releasing hormone receptor reveal differences in primary structure and ligand specificity among mammalian receptors. Biochem. Biophys. Res. Commun. 196: 745751. Imai A, Horibe S, Takagi A, Tamaya T. 1997. Gi protein activation of gonadotropin releasing hormone-mediated protein dephosphorylation in human endometrial carcinoma. Am. J. Obstet. Gynecol. 176: 371-376. Iinai A, Takagi A, Horibe 5, Takagi H, Tamaya T. 1998. Evidence for tight coupling of gonadotropin-releasing hormone receptor to stimulated Fas ligand expression in reproductive tract tumors: possible mechanism for hormonal control of apoptotic cell death. J. Clin. Endocr. d Metab. 83: 427-43 1. Imai A, Takagi A, Tamaya T. 2000. GnRH analog repairs reduced endometrial cell apoptosis in endometriosis in vitro. Am. J. Obstet. Gynecol. 182: 1142-1 146. Imal A, Takagi H, Horibe S, Fuseya T, Tamaya T. 1996. Coupling of gonadotropin releasing hormone receptor to Gi protein in human reproductive tract tumors. J. Clin. Endocr. Metab. 81: 3249-3253. Ireland, JJ, Aten, FRO, Barman HR. 1988. GnRH-like proteins in cows: concentrations during corpora lutea development and selective localization in granulosal cells. Biol. Reprod. 38: 544-550. Irmer G, Burger C, Muller R, Ortman 0, Peter U, Emons G. 1995. Expression of the mRNAs for luteinizing hormone-releasing hormone and its receptor in human ovarian epithelial carcinoma. Cancer Research. 55: 8 17-822. Irmer G, Burger C, Ortmann 0, Schulz K, Emons G. 1994. Expression of luteinizing hormone releasing hormone and its mRNA in human endometrial cancer cell lines. J. Clin. Endocr. Metab. 79: 9 16-919.  53  Islami D, Chardonnens D, Campana A, Bischof P. 2001. Comparison of the effects of GnRH-I and GnRH-II on HCG synthesis and secretion by first trimester trophoblast. Mo!. Hum. Reprod. 7: 3-9. Izumi S, Makino T, lizuka R. 1985. Immunoreactive luteinizing hormone-releasing hormone in the seminal plasma and human semen parameters. Fertil. Steril. 43: 6 17-620. Janovick JA, Haviv F, Fitzpatrick TD, Conn PM. 1993. Differential orientation of a GnRH agonist and antagonist in pituitary GnRH receptor. Endocrinology. 133: 942-945. Janssens RMJ, Brus L, Cahill DJ, Huirne JA, Schoemaker J, Lambalk CB. 2000. Direct ovarian effects and safety aspects of GnRH agonists and antagonists. Hum. Reprod. Update. 6: 505-518. Jeffcoate IA, Ayliffe TR. 1995. An ultrasonographic study of bovine cystic ovarian disease and its treatment. Vet. Rec. 136: 406-4 10. Johnson GL and Lapadat R. 2002. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 298: 1911-1912. Jones PB, Conn PM, Marian J, Hsueh AJ. 1980. Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells. Life Sci. 27: 2 125-32 Kaiser UB, Conn PM, Chin WW, 1997. Studies of gonadotropin-releasing hormone action using gonadotropin-releasing hormone receptor expressing pituitary cell lines. Endocr. Rev. 18: 46-70. Kaiser UB, Sabbagh E, Katzenellenbogen RA, Conn PM, Chin WW. 1995. A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin releasing hormone. Proc. Natl. Acad. Sci. USA 92: 12280-12284. Kakar SS, Grizzle WE, Neill JD. 1994. The nucleotide sequence of human GnRH receptors in breast and ovarian tumors are identical with that found in pituitary. Mo!. Cell. Endocr. 106: 145-149. Kakar SS, Musgrove LC, Devor DC Sellers JC, Neill JD. 1992. Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor. Biochem. Biophys. Res. Commun. 189: 289-295. Kakar SS, Rahe CH, Neill JD. 1993. Molecular cloning, sequencing, and characterizing the bovine receptor for gonadotropin releasing hormone (GnRH). Domest. Anim. Endocr. 10: 335342. Kang SK, Cheng KW, Ngan ES, Chow BK, Choi KC, Leung PC. 2000. Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells. Mo!. Cell. Endocr. 162: 157-166.  54  Kang SK, Choi KC, Yang HS, Leung Pc. 2003. Potential role of gonadotropin-releasing hormone (GnRH-I) and GnRH-II in the ovary and ovarian cancer. Endocr. Related Cancer. 10: 169-177. Karten MJ, River JE. 1986. Gonadotropin-releasing hormone analog design. Structure function studies towards the development of agonists and antagonists: rationale and perspective. Endocr. Rev. 7: 44-66.  —  Kerr JB, Sharpe RW. 1986. Effects and interactions of LH and LHRH agonists on testicular morphology and function in hypophysectomized rats. J. Reprod. Fertil. 76: 175-192. Keslar DJ, Elmore RG, Brown EM, Garverick HA. 1981. Gonadotropin releasing hormone treatment of dairy cows with ovarian cysts. I. Gross ovarian morphology and endocrinology. Theriogenology. 16: 359-361. Kimura A, Ohmichi M, Kurachi H, Ikegami H, Hayakawa J, Tasaka K, Kanda Y, Nishio Y, Jikihara H, Mastsuura N, Murata Y. 1999. Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone induced growth inhibition of a human ovarian cancer cell line. Cancer Res. 59: 5133-5142.  —  Kimura A, Ohmichi M, Kurachi H, Ikegami H, Hayakawa J, Tasaka K,Kanda Y, Nishio Y, Jikihara H, Matsuura N. 1999. Role of mitogenactivatedprotein kinase/extracellular signal-regulated kinase cascade ingonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line. Canc.Res. 59: 5133—5 142. Knecht M, Ranta I, Feng P, Shonohara 0, catt KJ. 1985. Gonadotropin-releasing hormone as a modulator of ovarian function. J. Steroid. Biochem. 23: 771-778. Kobayashi Y, Zhai YL, linuma M, Hoeriuchi A, Nikaido T, Fujii S. 1997. Effects of GnRH analogue on human smooth muscle cells cultured from normal myometrial and from uterine leiomyomal tissues. Mol. Hum. Reprod. 3: 91-99. Koichi U, Mitsumasa T, Masahiko H, Yoshinobu K, Takehiko 0. 2002. Recovery of spermatogenesis by high dose gonadotropin-releasing hormone analogue treatment in rat cryptorchid testis after orchiopexy. J. Urol. 168: 1279-1283. Kottler M, Starzec A, Carre M, Lagarde J, Martin A, Counis R. 1997. The genes for gonadotropin-releasing hormone and its receptor are expressed in human breast with fibrocystic disease and cancer. mt. J. Cancer. 71: 595-599. Kwon, JY, Park, 1(11, Park NY, and Cho NH. 2005. Effect of cetrorelix acetate on apoptosis and apoptosis regulatory factors in cultured uterine leiomyoma cells. Fertil. Sterli. 84: 15261528. Latouche J, crumeyrolle-Arias M, Jordan D, Kopp N, Augendre-Ferrante B, Cedard L, Haour F. 1989. GnRH receptors in human granulosa cells: anatomical localization and characterization by auto radiographic study. Endocrinology. 125: 1739—1741. 55  Lau HL, Zhu XM, Leung PC, Chan LW, Chen GF, Chan PS, Yu KL, Chan FL. 2001. Detection of mRNA expression of gonadotropin-releasing hormone and its receptor in normal neoplastic rat prostates. Tnt .J. Oncol. 19: 1193-201. Lee CY, Ho J, Chow SN, Yasojima K, Schwab C, McGeer PL. 2000. Immunoidentification of gonadotropin-releasing hormone receptor in human sperm, pituitary and cancer cells. AM. J. Reprod. Immunol. 44: 170-177. Lefebvre FA, Reeves JJ, Seguin C, Massicotte J, Labrie F. 1980. Specific binding of a potent LHRH agonist in rat testis. Mol. Cell. Endocr. 20: 127-134. Leung PC, Cheng CK, Zhu XM. 2003. Multi-factorial role of GnRH-I and GnRH-IT in the human ovary. Mol. Cell. Endocr. 202: 145-153. Li W, Jiao S, Chi PP. 1993. Immunoreactive gonadotropin-releasing hormone in porcine reproductive tissues. Peptide. 14: 543-549. Limonta P, Dondi D, Moretti R, Fermo B, Garattini E, Motta M. 1993. Expression of luteinizing hormone-releasing hormone mRNA in the human prostatic cancer cell line LNCaP. J. Clin. Endocr. Metab. 76: 797-800. Limonta P, Dondi D, Moretti RM, Maggi R, Motta M. 1992. Antiproliferative effects of leutinizing hormone releasing hormone agonists on the human prostatic cancer cell line LNCaP. J. Clin. Endocr. Metab. 75: 207-2 12. —  Limonta P, Moretti RM, Marelli MM, Dondi D, Parenti M, Motta M. 1999. The luteinizing hormone-releasing hormone receptor in human prostate cancer cells: messenger ribonucleic acid expression, molecular size, and signal transduction pathway. Endocrinology 140: 5250— 5256. Limonta P, Moretti RM, Marelli MM, Dondi B,, Parenti M, Motta M. 1999. The leutinizing hormone —releasing hormone receptor in human prostate cancer cells: messenger ribonucleic acid expression, molecular size, and signal transduction pathway. Endocrinology. 140: 5250-256. Liu YX, Hu ZY, Feng Q, Zou RJ. 1991. Paradoxical effect of a GnRH agonist on steroidogenesis in cultured monkey granulosa cells. Sci. China. 34: 1452-60. Luttrell LM. 2002. Activation and targeting of mitogen-activated protein kinases by G protein-coupled receptors. Can. J. Physiol. Pharmacol. 80: 375-382. Macmillan KL, Taufa VK, Day AM. 1986. Effeccts of an agonist of GnRH (buserelin) in cattle.ITI. Pregnancy rates after a post-insemination injection during metoestrous or dioestrous. Anim. Reprod. Sci. 11: 1-10. Manikkam M, Rajamahendran R. 1997. P4-induced atresia of the proestrous dominant follicle in the bovine ovary: changes in diameter, insulin-like growth factor system, aromatase activity, steroid hormones, and apoptotic index. Biol. Reprod. 57: 580-587. 56  Matsumiya K, Meistrich ML, Shetty G, Dohmae K, Tohda A, Okuyama A, Nishimune Y. 1999. Stimulation of spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mutant mice by gonadotropin-releasing hormone antagonist treatment. Endocrinology. 140: 4912-4915. Mee MO, Stevenson JS, Scoby RK, Folman Y. 1990. Influence of gonadotropin-releasing hormone and timing of insemination relative to estrus on pregnancy rates of dairy cattle at first insemination. J. Dairy Sci. 73: 1500-1507. Meikie A, Sahlin L, Ferraris A, Masironi B, Blanc JE, Rodriguez-Irazoqui M, Rodriguez Pinon, Kindahl H, Forsberg M. 2001. Endometrial mRNA expression of oestrogen receptor cr, P4 receptor and insulin-like growth factor-I (IGF-I) throughout the bovine oestrous cycle. Anim. Reprod. Sci. 68: 45-56. Meistrich ML, Kangasniemi M. 1997. Hormone treatment after irradiation stimulates recovery of rat spermatogenesis from surviving spermatogonia. J. Androl. 18: 80-87. Meistrich ML, Wilson G, Huhtaniemi I. 1999. Hormonal treatment after cytotoxic therapy stimulates recovery of spermatogenesis. Cancer Res. 59: 3557-3560. Meresman, CF, Bilotas MA, Lombardi E, Tesone CS, Rosa lB. 2003. Effect of GnRH analogues on apoptosis and release of interleukin- 113 and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum. Reprod. 18: 1767-1771. Millar R, Lowe S, Conklin B, Pawson A, Maudsley 5, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A. 2001. A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc. Natl. Acad. Sci. USA. 98: 9636-9641. Millar RP, Lu ZL, Pawson AJ, Adam J, Flanagan CA, Morgan K, Maudsley SR. 2004. Gonadotropin- releasing hormone receptors. Endocr. Rev. 25 : 23 5-275. Millar RP. 2002. GnRH II and type II GnRH receptors. Trends Endocr. Metab. 14: 35-43. Millar, R. P., Lu, Z. L., Pawson, A.J., Adam, J., Flanagan, C. A., Morgan, K. and Miller WR, Scott WN, Morris R, Fraser HM, Sharp RM. 1985. Growth of human breast cancer cells inhibited by a leutinizing hormone- releasing hormone agonist. Nature. 313: 23 1-233. Milvae RA, Murphy BD, Hansel W. 1984. Prolongation of the bovine estrous cycle with a gonadotropin-releasing hormone analogue. Biol. Reprod. 31: 664-670. Minaretzis D, Jakubowski M, Mortola JF, Pavlou SN. 1995. Gonadotropin-releasing hormone receptor gene expression in human ovary and granulosa-lutein cells. J. Clin. Endocr. Metab. 80: 430-434.  57  Minucci S, Di Matteo L, Pierantoni R, Varriale B, Rastogi RK, Chieffi G. 1986. In vivo and in vitro stimulatory effect of GnRH analog (HOE 766) on spermatogonial multiplication in the frog, Rana esculenta. Endocrinology. 119: 731-736. Moicho J, Zakut H, Naor Z. 1984. Stimulation of prostaglandin E and testosterone production in rat interstitial cells by a gonadotropin-releasing hormone agonist. Endocrinology. 114: 23 822387. Morales M, Lianos M. 1996. Interaction of human spermatozoa with the zona pellucida of oocyte: Development of the acrosome reaction. Front. Bio. 1: 146-160. Morales P. 1998. Gonadotropin-releasing hormone increases ability of the spermatozoa to bind to the human zona pellucida. Biol. Reprod. 59: 426-430. Moretti R1VI, Marelli MM, Van Groeninghen JC, Motta M, Limonta P. 2003. Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression. Endocr. Realt. Cancer. 10: 161-167. Morgan K, Stewart AJ, Miller N, Mullen P, Muir M, Dodds M, Medda F, Harrison D, Langdon S, Millar RP. 2008. Gonadotropin-releasing hormone receptor levels and cell context affect tumor cell responses to agonist in vitro and in vivo. Cancer Res. 68: 6331—6340. Moriya T, Suzuki T, Pilichowaska M, Ariga N, Kimura N, Ouchi N, Nagura H, Susano H. 2001. Immunohistochemical expression of gonadotropin-releasing hormone receptor in human breast carcinoma. Pathol. mt. 51: 333-337. Moumni M, Kottler ML, Counis R. 1994. Nucleotide sequence analysis of mRNA predicts that rat pituitary and gonadal gonadotropin-releasing hormone receptor proteins have identical primary structure. Biochem. Biophys. Res. Commun. 200: 1359-1366. Moutsaatsou, P and Sekeris E. 1997. Estrogen and P4 Receptors in the Endometrium. Annals of New York Academy of Science. 816: 99-115. Mulvaney JM and Roberson MS. 2000. Divergent signaling pathways requiringdiscrete calcium signals mediate concurrent activation of two mitogenactivatedprotein kinases by gonadotropin-releasing hormone. J. Biol. Chem. 275: 14182-14189. Murdoch WJ. 1995. Immunolocalization of a gonadotropin-releasing hormone receptor site in murine endometrium that mediates apoptosis. Cell. Tissue. Res. 282: 527-529. Nahum R, Beyth Y, Chun SY, Hsueh AJ, Tsafriri A. 1996. Early onset of deoxyribonucleic acid fragmentation during atresia of preovulatory ovarian follicles in rats. Biol. Reprod. 55: 1075-1080. Nam DH, Lee SH, Kim HS, Lee GS, Jeon YW, Kim 5, Kim JH, Kang SK, Lee BC, Hwang WS. 2005. The role of gonadotropin-releasing hormone(GnRH) and its receptor in development of porcine preimplantation embryos derived from in vitro fertilization. 2005. Theriogenology. 63: 190-201. 58  Nanda AS, Ward WR, Williams PCW, Dobson H. 1989. Retrospective analysis of the efficacy of different hormone treatments of cystic ovarian disease in cattle. Vet. Rec. 122: 155158. Neill JD, Duck LW, Sellers JC, Musgrove LC. 2001. A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem. Biophys. Res. Commun. 282: 1012-1018. Nett TM, Akbar AM, Niswender GD. 1974. Serum levels of luteinizing hormone and gonadotropin-releasing hormone in cycling, castrated and anestrous ewes. Endocrinology. 94: 713-718. Ohno T, Imai A, Furni T, Takahash K, Tamaya T. 1993. Presence of gonadotropin releasing hormone its messenger ribonucleic acid in human ovarian epithelial carcinoma. Am. J. Obstet. Gynecol. 169: 605-610. Oikawa M, Dargan C, Ny T, Hsueh AJ, 1990. Expression of gonadotropin-releasing hormone and prothymosin-alpha messenger ribonucleic acid in the ovary. Endocrinology. 127: 2350-2356. Olofsson JI, Conti CC, Leung PCK. 1995. Homologous and heterologous regulation of gonadotropin-releasing hormone receptor gene expression in preovulatory rat granulosa cells. Endocrinology. 136: 974-980. Palmon A, Ben Aroya N, Tel-Or 5, Burstein Y, Fridkin M, Koch V. 1994. The gene for the neuropeptide gonadotropin-releasing hormone is expressed in the mammary gland of lactating rats. Proc. Natl. Acad. Sci. USA. 91: 4994-4996. Palumbo A, Yeh J. 1994. In situ localization of apoptosis in the rat ovary during follicular atresia. Biol. Reprod. 51: 888-95. Paria BC, Reese J, Das 5K, Dey SK. 2002. Decipphering the cross talk of implantation: advances and challenges. Science. 296: 2185-2188. Parinaud J, Oustry P, Bussenot I, Tourre A, Perineau M, Monrozies X, Reme JM, Pontonnier G. 1992. Paradoxical ovarian stimulation in course of treatment by LH-RH analogues. Eur. J. Obstet. Gynecol. Reprod. Biol. 46: 117-122. Parinaud J, Vieitez G, Beaur A, Pontonnier G, Boureau E. 1998. Effect of luteinizing hormone-releasing hormone agonist (buserelin) on steroidogenesis of cultured human prevoulatory granlosa cells. Fertil. Steril. 50: 597-602. Paull WK, Turkelson CM, Thomas CR, Arimura A. 1981. Immunohistochemical demonstration of a testicular substance related to luteinizing hormone-releasing hormone. Science. 213: 1263-1264.  59  Peng C, Fan NC, Ligier M, Vaananen J. 1994. Expression and regulation of GnRH and GnRH receptor mRNA in Human Granulosa Luteal Cells. Endocrinology. 135: 1740-1746. Peters AR, Martinez TA, Cook, AJC. 2000. A metaanalysis of studies of the effect of GnRH 11-14 days after insemination on pregnancy rates in cattle. Theriogenology. 54: 1317-1326. Peters AR, Mawhinney I, Drew SB, Ward SJ, Warren MJ, Gordon PJ. 1999. Development of a gonadotropin-releasing hormone and the prostaglandin regimen for the planned breeding of dairy cows. Vet. Rec. 145: 516-521. Peters AR. 2005. Veterinary clinical application of GnRH-questions of efficacy. Anim. Reprod. Sci. 88: 155-167. Petry R, Craik D, Haaima G, Fromme B, Kiump H, Kiefer W, Palm D, Millar R. 2002. Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor. J. Med. Chem. 45: 1026-1034. Pieper BR, Richards JS. Marshall JC. 1981. Ovarian gonadotropin-releasing hormone (GnRH) receptors: characterization, distribution, and induction by GnRI-1. Endocrinology. 108: 1148-1155. Piquette GN, LaPolt PS, Oikawa M, Hsueh AJW. 1991. Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, gonadotropin releasing hormone in cultured rat granulosa cells. Endocrinology. 128: 2449-2456. Pitcher JA, Freedman NJ, Lefkowitz RJ 1998 G protein-coupled receptor kinases. Annu. Rev. Biochem. 67: 653-692. Powell JF, Zohar Y, Elizur A, Park M, Fischer WH, Craig AG, Rivier JE, Lovejoy DA, Sherwood NM. 1994. Three forms of gonadotropin-releasing hormone characterized from brains of one species. Proc. Natl. Acad. Sci. USA. 91: 12081-12085. Pursley JR, Kosorok MR, Wiltbank MC. 1997. Reproductive management of lactating dairy cows using synchronization of ovulation. J. Dairy Sci. 80: 301-6. Pursley JR, Mee MO, Wiltbank MC. 1995. Synchronization of ovulation in dairy cows using PGF2 alpha and GnRH. Theriogenology. 44: 1317-1326. Raga F, Casan EM, Wen Y, Huang H, Nezhat C, Polan ML 1998. Quantitative gonadotropin- releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol. Reprod. 59: 661-669. Rajamahendran R, Ambrose DJ, Small JA, finn N. 2001. Synchronization of estrus and ovulation in cattle. Arch. Animal. Breed. 44: 58-67. Rajamahendran R, Ambrose JD, Schmitt EJ, Thatcher MJ. Thatcher WW. 1998. Effects of buserelin injection and deslorelin (GnRI-1-agonist) implants on plasma P4, LH, accessory CL 60  formation, follicle and corpus luteum dynamics in Holstein cows. Theriogenology. 50: 11411155. Rajamahendran R, Sianangama Pc. 1992. Effect of human chorionic gonadotrophin on dominant follicles in cows: formation of accessory corpora lutea, P4 production and pregnancy rates. J. Reprod. Fertil. 95: 577-84. Ramakrishnappa N, Merwe GKVD, Rajamahendran R. 2001. Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in bovine ovary. Biol. Repro. 64 (Suppi): 229.Abstr. Ramakrishnappa N, Rajamahendran R, Em YM, Leung PCK. 2005. GnRH in nonhypothalamic reproductive tissues. Anim. Reprod. Sci. 88: 95-113. Ramakrishnappa, 2004. A study on GnRH-R and GnRH mRNA expression, direct effects of GnRH-a in bovine ovary and influence of post breeding GnRH administration on corpus luteum function and pregnancy in dairy cattle. Thesis submitted to University of British Columbia, Canada. Ramakrishnappa, N, Giritharan G, Aal, M, Madan P, Rajamahendran R. 2003. GnRH receptor messenger ribonucleic acid expression in bovine ovary. Can. J. Anim. Sci. 83: 823826. Ranta T, Knecht M, Kody M, call KJ. 1982. GnRH receptors in cultured rat granulosa cells: mediation of the inhibitory and stimulatory actions of GnRH. Mol. Cell. Endocr. 27: 233-40. Raposo G, Dunia I, Delavier-Klutchko c, Kaveri S, Strosberg AD, Benedetti EL. 1989. Internalization of B-adrenergic receptor in A43 1 cells involves non-coated vesicles. Eur. J. Cell. Biol. 50: 340-352. Ravenna L, Salvatory L, Morrone S, Lubrano c, Cardillo MR, Sciarra F, Frati L, DI Silverio F, Petrangeli E. 2000. Effects of triptorelin, a gonadotropin-releasing hormone agonist, on the human prostatic cell lines PC3 and LNCaP. J. Androl. 21: 549-557. Reeves JJ, Seguin C, Lefebvre FA, Kelly PA, Labrie F. 1980. Similar luteinizing hormonereleasing hormone binding sites in rat anterior pituitary and ovary. Proc. Natl. Acad. Sci. USA. 77: 5567-5571. Richards JS. 1994. Hormonal control of gene expression in the ovary. Endocr. Rev. 15: 725751. Rispoli L.A, Nett TM 2005. Pituitary gonadotropin-releasing hormone (GnRH) receptor: Structure, distribution and regulation of expression. Ani. Reprod. Sc. 88: 57-74. Saito H, Wang X, Saito T, Kaneko T, Hiroi M. 2000. Effects of gonadotropin-releasing hormone agonist on the incidence of apoptosis in porcine and human granulosa cells. Gynecol. Obstet. Invest. 49: 52-56. 61  Saragueta PE, Lanuza GM, and Raranao JL. 1997. Inhibitory effect of gonadotrophin releasing hormone (GnRH) on rat granulosa cells deoxyribonucleic acid synthesis. Mo!. Reprod. Dev. 47: 170-174. Schmitt EJ, Barros CM, Fields PA, Fields MJ, Diaz T, Kiuge JM, Thatcher WW. 1996. A cellular and endocrine characterization of the original and induced corpus luteum after administration of a gonadotropin-releasing hormone agonist or hun-ian chorionic gonadotropin on day five of the estrous cycle. J. Anim. Sci. 74: 19 15-1929. Sealfon SC, Weinstein H, Millar RP. 1997. Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr. Rev. 18: 180—205. Segal-Abramosan T, Kitroser H, Levy J, Schally AV, Sharoni Y. 1992. Direct effects of leutinizing hormone-releasing hormone agonists and antagonists on MCF-7 mammary cancer cells. Proc. Nat!. Acad. Sci. USA. 89: 2336-2339. Senturk LM, Sozen I, Arici A.2001. Interleukin 8 production and inetrleukin 8b receptor expression in human myometrium and !eiomyoma. Am. J. Obstet. Gynecol. 184: 559-566. Seshagiri PB, Wahistron T. 1994. Identification of luteinizing hormone- releasing-hormone by pen-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotropin. Hum. Reprod. 9: 1300-1307. Shah BH, Soh JW, Catt KJ. 2003. Dependence of gonadotropin-releasinghormone-induced neuronal MAPK signaling on epidermal growth factorreceptor transactivation. J. Biol. Chem. 278: 2866-2875. Sharpe 1982. Cellular aspects of the inhibitory actions of LH-RH on the ovary and testis. J. Reprod. Fertil. 64: 517-527. Sharpe RM, Cooper I, 1982a. Stimulatory effect of LHRH and its agonists on leydig cell steroidogenesis in.vitro. Mo!. Cell. Endocr. 26: 141-150. Sharpe RM, Cooper I. 1982b. Variation in the steroidogenic responsiveness of isolated rat Leydig cells. J. Reprod. Fertil. 65: 475-48 1. Sharpe RM, Doogan DG, Cooper I, 1982. Stimulation of leydig cell testosterone secretion in vitro and in vivo in hypophysectomized rats by an agonist of luteinizing hormone releasing hormone. Biochem. Biophys. Res. Commun. 106: 1210-1217. Sharpe R1’1, Fraser HM. 1980. HCG stimulation of testicular LHRH-like activity. Nature. 287: 642-643. Sharpe R1’1, Fraser HM. 1983. The role of LH in regulation of Leydig cell responsiveness to an LHRH agonist. Mo!. Cell. Endocr. 33: 13 1-146.  62  Shupnik MA, 1996. Gonadotropin gene modulation by steroids and gonadotropin-releasing hormone. Biol. Reprod. 54: 279-286. Shuttlesworth GA, de Rooij DG, Huhtaniemi I, Reissmann T, Russell LD, Shetty G, Wilson G, Meistrich ML. 2000. Enhancement of A spermatogonial proliferation and differentiation in irradiated rats by gonadotropin-releasing hormone antagonist administration. Endocrinology. 141: 3 7-49. Sridaran R, Hisheh S, Dharmarajan AM. 1998. Induction of apoptosis by a gonadotropin releasing hormone agonist during early pregnancy in the rat. Apoptosis. 3: 5 1-57. Sridaran R, Lee MA, Haynes L, Srivastava RK, Ghose M, Sridaran G, Smith CJ, 1999a. GnRH action on luteal steroidogenesis during pregnancy. Steroids. 64: 6 18-623. Sridaran R, Philip GH, Li H, Culty M, Liu Z, Stocco DM, Papadopoulos V. 1999b. GnRH agonist treatment decreases P4 synthesis, luteal peripheral benzediazepine receptor mRNA, ligand binding, steroidogenic acute regulatory protein expression during pregnancy. J. Mol. Endocr. 22: 45-54. Stevenson JS, Call EP, Scoby RK, Pathak AP. 1990. Double insemination and gonadotropin releasing hormone treatment in repeat-breeding dairy cattle. J. Dairy Sci. 73: 1766-1772. Stojilkovic SS, Catt KJ. 1995. Expression and signal transduction pathways of gonadotropin releasing hormone receptors. Rec. Prog. Horm. Res. 50: 16 1-205. Tabibzadeh S, Babakania A. 1995. The signal and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Hum. Reprod. 10: 1579-1602. Taponen J, Hjerppe P, Kopra E, Rodriguez-Martinez H, Katila T, Kindahl H. 2003. Premature prostaglandin F2alpha secretion causes luteal regression in GnRH-induced short estrous cycles in cyclic dairy heifers. Theriogenology. 60: 379-93. Taponen J, Katila T, Rodriguez-Martinez H. 1999. Induction of ovulation with gonadotropin-releasing hormone during proestrus in cattle: influence on subsequent follicular growth and luteal function. Anim. Reprod. Sci. 55: 91-105. Taponen J, Kulcsar M, Katila T, Katai L, Huszenicza G, Rodriguez-Martinez H. 2002. Short estrous cycles and estrous signs after premature ovulations induced with cloprostenol and gonadotropin-releasing hormone in cyclic dairy cows. Theriogenology. 58: 1291—302. Taponen J, Rodriguez-Martinez H, Katila T. 2000. Administration of gonadotropin releasing hormone during metoestrus in cattle: influence on luteal function and cycle length. Anim. Reprod. Sci. 64: 161-9. Tasende C, Meikie A, Rodrguez- Pinon M, Forsberg M. and Garofalo G. 2002. Estrogen and P4 receptor content in the pituitary gland and uterus of P4-primed and gonadotropin releasing hormone-treated anestrous ewes. Theriogenology. 57: 1719-1731. 63  Thatcher WW, Drost M, Savio JD, Macmillan KL, Entwistie KW, Schmitt RL, de La Sota RL, Morris GR. 1993. New clinical uses of GnRH and its analogues in cattle. Anim. Reprod. Sci. 33: 27-49. Tieva A, Stattin P, Wilkstrom P, Bergh A, Damber JE. 2001. Gonadotropin-releasing hormone receptor expression in the human prostate. Prostate. 47: 276-284. Tilly JL, Hsueh AJ. 1993. Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J. Cell. Physiol. 154: 519-526. Tilly JL, Lapoit PS, Hsueh AJW. 1992. Hormonal regulation of follicle-stimulating hormone receptor messenger ribonucleic acid levels in cultured rat granulosa cells. Endocrinology. 130: 1296-1302. Troskie B, Tiling N, Rumbak E, Sun YM, Hapgood J, Sealfon 5, Conklin D, Millar R. 1998. Identification of three putative GnRH receptor subtypes in vertebrates. Gen. Comp. Endocr. 112: 296-302. Ulbrich SE, Kettler A, Einspanier R. 2003. Expression and localization of estrogen receptorc, estrogen receptor f3 and P4 receptor in the bovine oviduct in vivo and in vitro. J. Steroid Biochem. Mol. Biol. 85: 279-289. Ullah G, Fuquay JW, Keawkhong T, Clark BL, Pogue DE, Murphey EJ. 1996. Effect of gonadotropin-releasing hormone at estrus on subsequent luteal function and fertility in lactating Hoisteins during heat stress. J. Dairy Sci. 79: 1950-1953. Van Biijon W, Wykes S, Scherer S, Krawetz SA, Hapgood J, 2002. Type II gonadotropin releasing hormone receptor transcripts in human sperm. Biol. Reprod. 67: 1741-1749. Vasudeven N, Piaff DW. 2007. Membrane-initiated actions neuroendocrinology: emerging principles. Endocr. Rev. 28: 1-19.  of  estrogens  in  Verhoeven G, Caiiieau J. 1985. A factor in spent media from Sertoli-cell-enriched cultures that stimulates steroidogenesis in Leydig cells. Mol. Cell. Endocr. 40: 57-68. Vizcarra JA, Wettemann RP, Morgan GL. 1999. Influence of dose, frequency, and duration of infused gonadotropin-releasing hormone on secretion of luteinizing hormone and folliclestimulating hormone in nutritionally anestrous beef cows. Dom. Anim. Endocr. 16: 171-81. Volker P, Grundker C, Schmidt 0, Schulz KD, Emons G. 2002. Expression of receptors for luteinizing hormone-releasing hormone in human ovarian endometrial cancers: frequency, auto regulation, correlation with direct antiproliferative activity of luteinizing hormone releasing hormone analogues. Am. J. Obstet. Gynecol. 186: 171-179. Vrecl M, Anderson L, Hanyaloglu A, McGregor AM, Groarke AD, Milligan G, Taylor PL, Eidne KA. 1998. Agonist-induced endocytosis and recycling of the gonadotropin releasing hormone receptor: effect of 13-arrestin on internalization kinetics. Mol. Endocrinol. 12: 1818-1829. 64  Vu K, Greenspan DL, Wu TC, Zacur HA, Kurman RJ. 1998. Cellular proliferation, estrogen receptor, P4 receptor, and bcl-2 expression in GnRH agonist-treated uterine leiomyomas. Hum. Pathol. 29: 359-363. Waither N, Lioutas C, Tiliman G, Ivell R. 1999. Cloning of bovine estrogen receptor beta (ER f3): expression of novel deleted isoform in reproductive tissues. Mol. Cell. Endocr. 152: 3745. Wang Y, Matsuo H, Kurachi 0 & Maruo T 2002. Down-regulation of proliferation and upregulation of apoptosis by gonadotropin-releasing hormone agonist in cultured uterine leiomyoma cells. Europ.J. Endo. 146: 447-456. Webb R, Gong JG, Law AS, Rusbridge SM. 1992. Control of ovarian function in cattle. J. Reprod. Fertil. 45: 141-156. Wells A, Souto JC, Solava J, Kassis J, Baily KJ, Turner T. 2002. Leutinizing hormonereleasing hormone agonist limits DU-145 prostate cancer growth by attenuating epidermal growth factor signaling. Clin. Cancer Res. 8: 125 1-1257. Weston CR and Davis RJ. 2007. The INK signal transduction pathway. Current Opinion in Cell Biol. 19: 142-149. Whitelaw PF, Eidne KA, Sellar R, Smyth CD, Hillier SG. 1995. Gonadotropin-releasing hormone receptor ribonucleic acid expression in rat ovary. Endocrinology. 136: 172-179. Wormald PJ, Eidne KA, Millar RP. 1985. Gonadotropin-releasing hormone receptors in human pituitary: ligand structural requirements, molecular size, and cationic effects. J. Clin. Endocrinol. Metab. 61: 1190-1194. Yang MY, Rajamahendran R. 2000a. Involvement of apoptosis in the atresia of nonovulatory dominant follicle during the bovine estrous cycle. Biol. Reprod. 63: 1313-21. Yang MY, Rajamahendran R. 2000b. Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells. Biol. Reprod. 62: 1209-1217. Yavas Y, Walton JS. 2000. Induction of ovulation in post partum suckeled beef cows: a review. Theriogenology. 54: 1-23. Ying SY, Ling N, Bohien P, Guillemin R. 1981. Gonadocrinins: peptides in ovarian follicular fluid stimulating the secretion of pituitary gonadotropins. Endocrinology. 108: 12061215. Yuan W, Giudice LC. 1997. Programmed cell death in human ovary is a function of follicle corpus luteum status. J. Clin. Endocr. Metab. 82: 3 148-3155.  65  Zerani M, Gobetti A, Mosconi G, Novara C, Botte V. 1991. Mammalian GnRH and possible paracrine regulation of the gonad in the green frog, Rana esculenta. Boll. Zool. 58: 77-79. Zhao S, Saito H, Wang X, Saito T, KanekoT, Hiroi M. 2000. Effects of gonadotropin releasing hormone agonist on the incidence of apoptosis in porcine human granulosa cells. Gynecol. Obstet. Invest. 49: 52-56. Zhou W, Rodic V, Kitanovic 5, Flanagan CA, Chi L, Weinstein H, Maayani S, Millar RP, Sealfon SC. 1995. A locus of the gonadotropin-releasing hormone receptor which differentiates agonist and antagonist binding sites. J. Biol. Chem. 270: 18853-18857.  66  CHAPTER 2- GONADOTROPIN RELEASING HORMONE RECEPTOR GENE AND PROTEIN EXPRESSION AND IMMUNOHISTOCHEMICAL LOCALIZATION IN BOVINE UTERUS AND OVIDUCTS 1  2.1 INTRODUCTION Gonadotropin-releasing hormone (GnRI-I) or LHRH or mammalian GnRH- 1 is a neuronal secreted decapeptide, produced primarily in the hypothalamus and plays a central role in mammalian reproduction by acting through the hypothalamo-pituitary-gonadal axis (Fink, 1988). This hormone is synthesised and secreted in a pulsatile manner by about 1000 hypothalamic neurones with pulse frequencies varying from 20-120 mm, taken up by the hypothalamo-hypophyseal portal circulation and carried to pituitary gonadotrophs resulting in biosynthesis and secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH). LH and FSH enter into systemic circulation from the anterior pituitary and regulate gonadal steroidogenesis (estrogens, progesterone and testosterone) and gametogenesis (Millar et al., 2004; Cheng and Leung, 2005). GnRH acts by binding to its receptors on pituitary gonadotrophs. GnRH-R belong to the superfamily  of G-protein coupled  receptors  (GPCR).  These receptors have  seven  transmembrane domains and extra and intracellular loops. It is well recognized that GnRH acts through the hypothalamo-pituitary-gonadal axis. Recent reports have confirmed the extrapituitary extra-hypothalamic presence of GnRH and GnRH-R systems in reproductive tissues in many mammalian species (Ramakrishnappa et al., 2005). It has been shown to be present in normal and neoplastic reproductive tissues including the ovary, oviduct, endometrium,  A version of this chapter has been published. Singh R., Graves M., Roskelley C., Giritharan G. and Rajainahendran, R. (2008) Gonadotropin releasing hormone receptor gene and protein expression and immunohistochemical localization in bovine uterus and oviducts. Domes. Ani. Endo. 34: 3 19-326.  67  placenta, testes, prostate, preimplantation embryos, oocytes and spermatozoa across species ranging from rodents to humans. GnRH is considered to be a local modulator of different biological processes such as gonadal steroidogenesis, apoptosis, and cellular proliferation; acting in an autocrine or paracrine manner (Janssens et al., 2000; Lee et al., 2000, van Bilijon et al., 2002; Cheng and Leung, 2005; Nam et a!., 2005; Ramkrishnappa et a!., 2005). The presence of GnRH has also been elucidated in porcine, rat and human endometrium (Li et al., 1993; Ikeda et al., 1996; Dong et al., 1998; Raga et al., 1998) and oviducts (Casan et al., 2000), whereas its receptor mRNA has been shown in the normal and carcinogenic human endometrium (Imai et al., 1994; Borroni et al., 2000). This extra-pituitary extra-hypothalamic presence of the GnRH, GnRH-R system is an indication of its direct modulatory role in peripheral reproductive tissues (Harrison et a!., 2004). Studies from our laboratory have demonstrated the presence of GnRH-R mRNA (Ramakrishnappa et al., 2003) in different stages of bovine ovarian follicles and corpus luteum, while, there is no evidence for the presence of the GnRH-R in the bovine uterus and oviducts. bovine  reproductive  management  including  estrus  and  GnRH is extensively used in ovulation  synchronization  (Rajamahendran et a!., 2001, Ambrose et a!., 2005), induction of post partum ovulation (Lewis et al., 2001) and increasing pregnancy rates post AT (Peters, 2005). GnRH is believed to act through the hypothalamo-pituitary-gonadal axis in inducing these effects. The possible extra pituitary target sites for this hormone could be ovaries, uterus and oviducts, where it might act in an autocrine or paracrine manner and affect reproductive processes. Therefore, we investigated the presence and localization of GnRH-R in the bovine uterus and oviducts at both mRNA and protein levels during the follicular and luteal phases of the estrous cycle.  68  2.2 MATERIALS AND METHODS 2.2.1 Tissue collection and processing Bovine reproductive tracts were collected from culled Holstein Friesian dairy cows from a local abattoir. The reproductive tracts were collected within 20 mm of exsanguination of the animals and were classified as belonging to either the follicular or luteal phases of the estrous cycle based on ovarian morphology (Yang and Rajamahendran, 2000; Bogacki et a!., 2002). Briefly, the ovulatory follicle and the 11-16 Day fully developed corpus luteum (CL) were considered as characteristic features of the follicular and luteal phases of the estrous cycle, respectively. Uterine horns ipsilateral to CL or follicle were longitudinally incised; endometrial slices were cut with a scalpel blade, washed in ice cold phosphate buffer saline (PBS), and immediately snap frozen in liquid nitrogen. Similarly, oviducts in both the follicular and luteal phases of the estrous cycle were excised from the extraneous and ovarian attachments, cut in to smaller pieces, washed with PBS, and snap frozen. Anterior pituitaries were collected and cut in two equal halves. One part of it was snap frozen for immunoblotting and the other was preserved in 10% formal saline for immunohistochemistry serving as positive control. Endometrial and oviductal tissues were similarly collected for immunohistochemistry and preserved in 10% formal saline until further use. Five reproductive tracts in the follicular phase and five in the luteal phase were used for RT-PCR study. Three reproductive tracts in the follicular and three in the lutea! phase of the estrous cycle were utilized for immunoblotting and immunohistochemistry experiments. 2.2.2 RNA extraction Total RNA was extracted using a single step RNA extraction method (Chomczynski and Sacchi, 1987). Tissues were briefly thawed at room temperature and 100 mg of each tissue sample was homogenized under liquid nitrogen by using a mortar and pestel. One mL of Tn 69  Reagent solution (Sigma Aldrich, Oakville, ON, Canada) was added to homogenates in a 2.0 mL RNAse and DNAse free microfuge tube, vortexed, and allowed to stand for 10 mm at room temperature. Then 200 iL of chloroform was added to each tube. The samples were agitated vigorously for 30 s, allowed to stand at room temperature for 15 mm, and then centrifuged at 12,000 rpm for 15 mm at 4°C. The top transparent layer of the solution containing the total RNA was transferred into new set of sterile tubes. Then 0.75 mL of isoproparanol was added to each tube containing total RNA. The tube contents were gently mixed and the tubes were allowed to stand for 30 mm at room temperature before centrifugation at 12,000 rpm for 10 mm at 4°C. The resultant RNA pellet was washed twice in ice cold 75% ethanol and centrifuged at 12,000 rpm for 5 mm, air dried and dissolved in 100 pi of sterile DEPC treated water. The quality and quantity of RNA was assessed spectrophotometericaly and ethidium bromide stained agrose gel (1%) electrophoresis. The gel electrophoresis revealed clear bands of 28S, and 1 8S, ribosomal RJNJA species when visualized under UV light. Total RNA was either used immediately or stored at —80°C for cDNA synthesis. 2.2.3 RT- PCR Reverse transcription-polymerase chain reaction (RT-PCR) was performed by using commercially available first strand cDNA synthesis kits (Cells-to-cDNA II kit, Ambion Inc. The RNA Company, Austin, Texas, USA) and following the manufacturer’s protocol. Briefly, 2ig of the total RNA was reverse transcribed by using kit supplied random decamer primers in 20 jiL of total reaction volume. The reverse transcription reaction comprised of 2 1 iL of i OX RT buffer (pH 7.4), 1 iL dNTP containing 0.5 m moles of each nucleotide, 1 iL of M-MLV reverse transcriptase (10 U), 1 iL RNAase inhibitor, 5  t  moles of random decamers, 2 ig  RNA and nuclease free water to make the final volume to 20 iL. The reaction was performed by incubating the contents in a thermal cycler at 42°C for 60 mm  followed by 10 mm 70  incubation at 95°C to inactivate the reverse transcriptase enzyme. cDNA produced was stored at -20°C for future PCR amplification. JumpStart RED Taq Ready Mix PCR reaction mix (Jumpstart; Sigma-Aldrich, Oakville, ON, Canada) and gene specific primers for GnRH-R and G3PDH (Ramakrishnappa, 2003) were used for PCR. Initially PCR conditions were standardized by exponential amplifications of the genes of interest to achieve optimal amplification. The negative controls without gene specific primers were employed to check for non specific binding. To eliminate the possibility of non genomic amplifications, negative controls without template cDNA were used during the experiments. To further test the PCR conditions G3PDH was used as a housekeeping gene and internal control in the experiments. The primer sequence for GnRH-R was- sense (5’- GAG TGA CAG TTA CTT TCT TCC -3’)- antisense (5’- AGG AAG AAG CGT AAC ATT ACC -3’) and for G3PDH wassense (5’- TGT TCC AGT ATA GAT TCC ACC -3’) and antisense (5’- AGG AGG CAT TGC TGA CAA TC -3’). Briefly, gene specific primers, nuclease free water, and 2 1 iL of cDNA template solution were added to 12.5 iL of Jumpstart reaction mixture to get 25 iL of final reaction mixture. The reaction contained 10 m moles Tris-HC1, 50 mM KCL, 2.5 m moles , 0.0001% gelatin, 0.2 m moles of each dNTP (dATP, dCTP, dGTP, dTTP), inert dye 2 MgC1 stabilizers, 0.03 U/pL taq DNA polymerase, Jumpstart Taq antibody, 200 n moles of each gene specific primer, cDNA template and nuclease free water. The PCR steps followed for GnRH-R and G3PDH were initial denaturation at 95°C for 2 mm, denaturation at 94°C for 1 mm, annealing at 60°C for 1 mm, extension at 72°C for 1 mm and final extension at 72°C for 10 mm. PCR amplification was carried out for 33 cycles. PCR products were run on ethidium bromide stained agrose gels (2%) and visualized under ultraviolet illumination to observe expected size products (approximately 920 bp for GnRH-R and 320 bp for G3PDH). 71  2.2.4 Protein extraction and quantification Endometrial, oviductal, and pituitary tissues stored in liquid nitrogen were taken out from the liquid nitrogen container and placed on a sterile bench top. The tissues were then thawed briefly at room temperature, homogenized (100 mg of each tissue under liquid nitrogen in a pestel and mortar) and transferred to sterilized microfuge tubes kept on ice, gently mixed (2-3 times) with 300 pL of RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaC1, 1% Np-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10 iL of stock aprotinin (Sigma Cat# A6279), 1 jiL of 10 mg/mL leupeptin (Sigma Cat# L-9783), 10 iL of 10 mM PMSF (Sigma Cat# P-7626), 10 tL of 1 mg/mL PepA (Sigma Cat# P53 18) and 5 jiL of 500 mM EDTA and kept on ice for 20 mm, centrifuged at 12,000 rpm at 4°C for 15 mm  and supernatant containing  proteins was transferred to new sets of tubes. Total proteins were measured using NanoDrop spectrophotometer (NanoDrop Technologies, Inc. Wilmington, USA) and samples were stored at —80°C for immunoblotting. 2.2.5 Immunoblotting After thawing the samples on ice, 40 ig of total protein from each tissue was mixed with sample buffer, boiled at 100°C for 10 mm, cooled on ice, centrifuged and subjected to 10% SDS  -  PAGE at 70V for 2-3 h along with a protein molecular weight marker (SeaBlue  Plus2 Pre-Stained Standard, Invitrogen Life Technologies, Carlsbad, CA, USA). Gels were rinsed with tap water and proteins transferred to methanol soaked PVDF membranes overnight at 30 V and 4°C. The membranes were then washed with TBS (3 X 5 mm each), blocked with 5% skimmed milk in 0.2% TBS-T for 5 h at 4°C, washed in 0.2% TBS-T, 3 X 5 mm each and incubated with GnRH-R mouse monoclonal antibody (1.5 ig/mL, Lab Vision Corporation, Fremont, CA, USA) in 1% skimmed milk and 0.2% TBS-T at 4°C overnight and washed in 0.2% TBS-T. Goat antimouse HRP (1:5000, Chemicon Upstate, Temecula, CA, USA) in 1% -  72  milk and 0.2% TBS-T was used as secondary antibody with 2 h incubation at room temperature. The membrane was again washed with TBS-T (3 X 10 mi, TBS (3 X 5mm) followed by a final washing step with TBS (3 X 10 mm) and the antibody binding was determined using ECL detection system (Amersham Biosciences, Little Chalfont, UK). The non-specific binding to GnRH-R antibody was determined using concentration-matched IgG controls. 2.2.6 Immunohistochemistry Immunohistochemistry was performed with slight modifications to the protocol described by Casan et a!. (2000). After overnight fixation in 10% formalin buffered saline at 4°C the tissues were transferred to 70% ethanol, paraffin embedded and cut into 5 im sections, that were mounted on clean glass slides and dried and dehydrated overnight at 37°C on a hot plate. Tissue sections were deparaffinized by passing through xylene (3 changes X 5 mm each), quickly rehydrated by passing through graded alcohol (95%, 80% and 70% ethanol) and finally rinsed in a large volume (appx 200 mL) of double distilled water. Antigen retrieval was done by placing the tissue sections in lOX citrate buffer (preheated to 90-98°C, pH 6.0) at 98°C for 30 mm, cooled to room temperature for 20 mm and rinsed with PBS (pH 7.2-7.4) three times at 5 mm  each and immunostained by using EnVision  +  Dual Link System-HRP kit (Dako  Cytomation, Carpinteria, CA, USA). Briefly, endogenous peroxidase activity was quenched by incubating the sections with 3% H 0 for 10 mm, and then sections were washed with PBS 2 three times at 5 mm each, blocked with Dako protein block (Dako Cytomation) and incubated with 1:100 primary antibody (GnRH-R mouse monoclonal antibody, Lab Vision Corporation, Fremont, CA) in PBS overnight at 4°C, washed in PBS for three times at 5 mm  each and  applied labelled polymer-HRP in sealed immunochamber for 30 mm, rinsed with PBS for three times at 5 mm, developed using Nova Red (Vector Lab, Burlingame, CA), rinsed twice with 73  double distilled water for 5 mm each, counterstained with hematoxylin, washed in running tap water, dehydrated by passing quickly through graded alcohol (70%, 80% and 95%), cleared in xylene (three times at 5 mm  each), mounted in a resinous mounting medium, covered with  coverslip and examined under microscope. Tissue sections treated with PBS in place of primary antibody were used as negative controls and sections of bovine anterior pituitaries were used as positive controls. The IgG controls were used to check non-specific binding in the beginning of experiments. 2.3 RESULTS 2.3.1 GnRH-R mRNA expression RT-PCR studies revealed expression of GnRH-R mRNA in the bovine uterus and oviducts at an expected molecular weight of 920 bp. To validate the PCR conditions G3PDH was used as housekeeping gene and was expressed at 320 bp by all the samples. Negative controls without the template cDNA were also co-amplified and did not yield any product after agrose gel electrophoresis, ruling out genomic DNA contamination of the samples used in the study. Bovine uterus and oviducts in both the follicular and luteal phases of the estrous cycle showed expression for the GnRH-R mRNA (Figure 2.1). 2.3.2 GnRH-R protein expression  Immunoblotting with mouse GnRH-R monoclonal antibody resulted in detection of GnRH-R protein at a molecular weight of 60 kD in endometrial and oviductal tissues in follicular as well as luteal phases of the estrous cycle. In this experiment proteins extracted from bovine pituitaries were used as positive controls and expressed GnRH-R protein at same molecular weight. Molecular weights of these products were validated against the molecular weight marker used in the experiment (Figure 2.2).  74  2.3.3 Immunolocalization of GnRH receptors Immunohistochemistry was used to localize the GnRH receptors at the cellular level in both the follicular and luteal phases of the estrous cycle. Microscopic examination revealed specific staining for GnRH receptors in both luminal and glandular epithelium of the endometrium and in the luminal epithelium of the oviducts in both the phases of the estrous cycle (Plate 2.1). Negative controls showed no reactivity to the antibody used and about 3040% of the cells in the anterior pituitary had positive staining. 2.4 DISCUSSION This research demonstrated the presence of GnRH-R in the bovine uterus and oviducts at both the mRNA and protein levels. In this study RT-PCR was employed for GnRH-R mRNA expression (Figure 2.1). The G3PDH was used as an internal control and negative controls without template cDNA were used to rule out genomic DNA contamination. To further test the expression of these receptors at protein level, we used immunoblotting (Figure 2.2) and immunohistochemistry (Plate 2.1) techniques. Bovine anterior pituitaries were used as positive controls in these experiments, as this tissue is well known to express the GnRH-R and 30- 40% of the pituitary cells showed positive staining to the monoclonal antibody used in the experiment. IgG controls were used at the start of the experiment to check the non-specific binding. This study shows that endometrial and oviductal epithelial cells express GnR}I receptors in both the follicular and luteal phases of the bovine estrous cycle. The presence of GnRH and GnRH-R system has been documented in ovaries (Ramakrishnappa et a!., 2003; Harrison et al; 2004), breast (Kottler et al., 1997), testes (Botte et al., 1998), prostate (Bono et al., 2002) and placenta (Cheng et a!., 2000). GnR}I treatment in these reproductive tissues has been shown to regulate vital reproductive processes including gonadal steroidogenesis, apoptosis, and cellular proliferation. In bovines, Ramakrishnappa et 75  a!. (2003) elucidated the presence of GnRH-R mRNA in different stages of ovarian follicles and corpus luteum by using RT-PCR and commented that demonstration of a functional form of GnRH-R protein would be an imperative step to study the direct effects of GnRH or its agonists. In the human endometrium, Imai et al. (1994) detected GnRH-R mRNA by using RT PCR and GnRH binding assays. Raga et a!. (1998) recorded that GnRH-R mRNA is present in the follicular and luteal phases of the menstrual cycles in the human endometrium and further observed that the GnRH-R is expressed at low levels and is present throughout the menstrual cycle. Furthermore, Borroni et al. (2000) observed that the GnRH-R gene is also expressed in human ovarian endometriomas. These were the only recorded evidences showing the presence of the GnRH-R at mRNA levels and this study demonstrated that these receptors are present at both the mRNA and protein levels in bovine endometrium. So far there has been no recorded evidence of the presence of GnRH-R in bovine oviductal tissue and this study for the first time demonstrated their presence at both mRNA and protein levels in the oviducts. Through immunohistochemistry we further localized these receptors to the luminal and glandular endometrial epithelium and oviductal epithelial cells. In addition to the demonstration of extrapituitary GnRH-R mRNA, Li et al. (1993) demonstrated the presence of an immunoreactive GnRH in porcine endometrial tissue and there after expression of GnRH mRNA has also been observed in rat endometrial stromal cells (Ikeda et al., 1996). In humans a dynamic expression pattern for GnRH mRNA has been observed in the endometrium and in isolated endometrial cells, and these levels were significantly increased in the secretory phase of the menstrual cycle (Dong et al., 1998 and Raga et al., 1998). Further, GnRH immunoreactivity was noticed in all endometrial cell types in humans and it was observed that staining patterns are more intense during the luteal phase as compared to the follicular phase of the menstrual cycle (Raga et a!., 1998). Casan et al. (2000) also showed the 76  presence of GnRH mRNA and protein in human fallopian tubes during the luteal phase of the menstrual cycle by using RT-PCR and immunohistochemical techniques, and inferred that GnRH immunostaining was localized in the tubal epithelium. The finding that GnRH and its analogues may modulate sperm-zona pellucida binding in humans (Morales and Llanos, 1996) indicates that the spermatozoa may interact with GnRH, during their journey through the male or female reproductive tracts (Morales, 1998; Bull et al., 2000). Furthermore, it was suggested that GnRH is involved in the process of fertilization as GnRH agonist increased the cleavage rate of bovine oocytes in vitro (Funston and Siedal, 1995). Further, in vitro studies showed that in early pregnancy GnRH stimulated urokinase type plasminogen activator (uPA) mRNA and protein levels in human placenta. These findings are suggestive of GnRH’ s regulatory role in proteolytic degradation of the extracellular matrix of the endometrial stroma which is prerequisite for the decidualization and trophoblast invasion (Tabibzadeh and Babakania, 1995; Paria et al., 2002). GnRH has been shown to suppress the trophoblastic expression of plasminogen activator inhibitor (PAl-I) in a dose and time dependent manner (Cheng and Leung, 2005) and to rapidly induce hCG secretion in human placental explants and cytotrophoblasts (Islami et al., 2001; Siler-Khodr and Garyson 2001). The presence of GnRH and GnRH-R has been recorded in carcinogenic ovaries, breasts and prostates (Ramakrishnappa et al., 2005). The anticarcinogenic role of this hormone is assuming greater significance in the treatment of ovarian and prostrate cancers (Cheng and Leung, 2005). GnRH-R mRNA expression has been elucidated in the endometrial carcinoma and endometriosis (Imai et al., 1994; Borroni et al., 2000) and GnRH had antiproliferative effect in these tissues. Therefore, the available literature provides evidence that GnR}I and GnRH-R system seems to have acquired different roles in different tissue/cell types acting in an autocrine and or paracrine manner (Cheng and Leung 2005; Ramakrishnappa et al., 2005). 77  Though there are some studies showing antiproliferative activity of this hormone in endometrial carcinomas, there is no information on its local physiological role in the endometrium or oviducts. Therefore, the presence of this system in normal uterus and oviducts is intriguing. It may be an indication that a functional GnRH, GnRH-R system exists at this level and that the local regulatory role of this hormone in relation to bovine reproduction needs to be investigated. Since GnRH might have additional roles in the reproductive axis (Pask et al., 2005) and GnRH-R were expressed in both the phases of the bovine estrous cycle, further studies are required to determine the direct roles of this system in gamete transport in the female reproductive tract, fertilization, and early embryonic development. The uterine functions are regulated by the steroid hormones acting through their own receptors. The co existence of GnRH, GnRH-R and the steroid hormone receptor systems in the endometrium is interesting and it would be exciting to study the interactions in these two systems at this level. It would be imperative to further research the direct effects of this hormone in regulation of genes associated with vital reproductive processes including apoptosis, endometrial remodelling, uterine receptivity, luteolytic mechanism and implantation in the bovine species. Furthermore, after fertilization at the ampullary isthmus junction, bovine embryos spend considerable time in the oviducts before migrating into the uterus for attachment and further development. It would be interesting to examine whether this system contributes to early embryonic development by regulating expression of growth factors in the oviductal environment. Also, it would be exciting to know whether the GnRH is produced locally in the bovine uterus and oviducts or if it comes from some other source/sources to act on these tissues.  78  2.5 CONCLUSIONS In conclusion, this study has demonstrated the presence of GnRH-R in the follicular and luteal phases of the bovine uterus and oviducts and localized these receptors to endometrial and oviductal epithelial cells. Investigations into the local modulatory and regulatory role of this hormone in these tissues in terms of its interaction with steroid hormone receptors, endometrial apoptosis, fertilization, embryonic development, implantation and maintenance of pregnancy, would further add to the existing knowledge in the field of reproductive biology and may result in the development of improved methods to increase female fertility.  79  Al  .  B  1I  •.  ••.  —  •  •.  -. -  ‘  —  .•  C  —  1.••.  -  •.  --—----• •  —‘...-.-  S  L  I  h.  bIi..  •.b..4P1t -  •  E  —  .?  F  1  L.  •  •  ‘I,  4;  J  -:  .  -  —  rq  -  -  ,_1,..  -  f  -  •..  __,w ...-.‘  ,.-  •‘  ..;  m$  ‘. •.•-•--•:••  G  •)  :-,.  .5  Plate 2.1 Immunohistochemical staining showing localization of GnRH-R in bovine  endometrial and oviductal epithelial cells. Bovine pituitary was used as positive control and 3040% of pituitary cells showed immunoreactivity (indicated by arrows) to the monoclonal antibody used in the experiment (A), negative controls without antibody for endometriurn (B) and oviduct (C) did not show any staining, endornetrial epithelial cells in follicular (D) and luteal phase (E) and oviductal epithelial cells in follicular (F) and luteal phase (G) of estrous cycle showing positive staining. Pituitary, endometrium and oviducts are shown at 400, 250  and 100 times magnification, respectively. The arrows indicate positive cells in pituitary (A) and glandular and luminal epithelium in endometrium (E) and luminal epithelium in oviduct (G) 80  A GnRH-R 920 bp  900bp  EF  EL  OF  OL  NEG  MW  B  G3PDH 320 bp  300bp  EF  EL  OF  OL  NEG  MW  Figure 2.1 GnRH-R mRNA expressions in bovine endometrium and oviducts (Panel A). PCR products, when run on 2% ethidiurn bromide stained agrose gel and visualised under UV illumination resulted in GnRH-R mRNA expression at 920 bp in follicular phase endometrium (EF), luteal phase endometrium (EL), follicular phase oviduct (OF), luteal phase oviduct (OL). G3PDH (Panel B) was co amplified as housekeeping gene and expressed at 318 bp. Negative controls without cDNA template did not yield PCR product. MW stands for molecular weight marker.  81  60kB  GnRH-R  EF  EL  OF  OL  PIT  Figure 2.2 GnRH-R protein expressions in bovine endometrium and oviduct. The GnRH-R protein was expressed at 60 kD in follicular phase endometrium (EF), luteal phase endometrium (EL), follicular phase oviduct (OF), luteal phase oviduct (OL) and bovine pituitary (PIT), used as positive control in the experiment.  82  2.7 BIBLIOGRAPHY Ambrose DJ, Kastelic JP, Rajamahendran R, Aali M, finn N. 2005. Progesterone (CIDR) based timed Al protocols using GnRH, porcine LH or estradiol cypionate for dairy heifers: ovarian and endocrine responses and pregnancy rates. Theriogenology. 64: 1457-1474 Bogacki M, Silvia J, Rekawiecki R, Kotwica, J. 2002. Direct inhibitory effect of progesterone on oxytocin-induced secretion of prostaglandin F2c from bovine endometrial tissue. Biol. Reprod. 67: 184-188. Bono AV, Salvadore M, Celato N. 2002. Gonadotropin-releasing hormone receptors in prostrate tissue. Analytical and Quantitative Cytology and Histology. 24: 22 1-227. Borroni R, Blasio AMP, Gaffari B, Santorsola R, Busacca M, Vigano P, Viganali M. 2000. Expression of GnRH receptor gene in human ectopic endometrial cells and inhibition of their proliferation by leuprolide acetate. Mol. Cell. Endocr. 159: 37-43. Botte MC, Chamagne AM, Carre MC, Counis R, Kottler ML. 1998. Fetal expression of GnRH and GnRH receptor genes in rat testis and ovary. J. Endocr. 159: 179-189. Bull P, Morales P, Huyser C, Socias T, Castellon EA. 2000. Expression of GnRH receptor in mouse and rat testicular germ cells. Mo!. Hum. Reprod. 6: 582-586. Casan EM, Raga F, Bonilia-musoles F, Polan ML. 2000. Human oviductal gonadotropin releasing hormone: Possible implications in fertilization, early embryonic development, and implantation. J. Clin. Endocr. Metab. 85: 1377-138 1. Cheng CK, Leung PCK. 2005. Molecular Biology of Gonadotropin- Releasing Hormone (GnRH)-l and GnRH-11 and Their Receptors in Humans. Endocr. Rev. 25: 1-108. Cheng KY, Nathwani PS, Leung PCK. 2000. Regulation of Human Gonadotropin-Releasing Hormone Receptor Gene Expression in Placental Cells. Endocrinology. 141: 2340-2349. Chomczynski P, Sacchi N. 1987. Single step method of RNA isolation by acid guanidinium thoicynate-phenol-chloroform extraction. Anal Biochem. 162: 156-159. —  Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, Roberts JL. 1998. Expression of gonadotropin releasing hormone (GnRH) gene in human uterine endometrial tissue. Mo!. Hum. Reprod. 4: 893-898. —  Fink G.1988. Gonadotropin secretion and its control. In: Knobil E, Neill J.D. eds. The physiology of reproduction. New York: Raven Press. 1349-1377. Funston RN, Seidel GE Jr. 1995. Gonadotropin-releasing hormone increases cleavage rates of bovine oocytes fertilized in vitro. Biol. Reprod. 53: 541-545. Harrison GS, Wierman ME, Nett TM, Glode LM. 2004. Gonadotropin— releasing hormone and its receptor in normal and malignant cells. Endocr. Relat. Cancer. 11: 725-748. 83  Ikeda M, Taga M, Sakakibara H, Minaguchi H, Ginsburg E, Vonderhaar BK. 1996. Gene expression of gonadotropin-releasing hormone in early pregnant rat and steroid hormone exposed mouse uteri. J. Endocr. Invest. 19 :708-713. Imai A, Ohno T, lida K, Fuseya T, Furui T, Tamaya T. 1994. Presence of gonadotropin releasing hormone receptor and its messanger ribonucleic acid in endometrial carcinoma and endometrium. Gynecol. Oncol. 55: 144-148. Janssens RMJ, Brus L, Cahill DJ, Huirne JA, Schoemaker J, Lambalk CB. 2000. Direct ovarian effects and safety aspects of GnRH agonists and antagonists. Hum. Reprod. Update. 6: 505-518. Kottler M, Starzec A, Carre M, Lagarde J, Martin A, Counis R. 1997. The genes for gonadotropin-releasing hormone and its receptor are expressed in human breast with fibrocystic disease and cancer. Int. J. Cancer. 71: 595-599. Lee CY, Ho J, Chow SN, Yasojima K, Schwab C, McGeer PL. 2000. Immunoidentification of gonadotropin-releasing hormone receptor in human sperm, pituitary and cancer cells. Am. J. Reprod. Immunol. 44: 170-177. Lewis GS, Caidwell DW, Rexroad CE, Dowlen HH, Owen JR. 2001. Effects of gonadotropin releasing hormone and human chorionic gonadotropin on pregnancy rate in dairy cattle. J. Dairy Sci. 73: 66-72. Li W, Jiao 5, Chi PP. 1993. Immunoreactive gonadotropin-releasing hormone in porcine reproductive tissues. Peptide. 14: 543-549. Millar RP, Lu ZL, Pawson AJ, Adam J, Flanagan CA, Morgan K, Maudsley SR. 2004. Gonadotropin Releasing Hormone receptors. Endocr. Rev. 25: 235-275. -  Morales P. 1998. Gonadotropin-releasing hormone increases ability of the spermatozoa to bind to the human zona pellucida. Biol. Reprod. 59: 426-430. Morales M, Llanos M. 1996. Interaction of human spermatozoa with the zona pellucida of oocyte: Development of the acrosome reaction. Front. Biol. 1: 146-160. Nam DH, Lee SH, Kim HS, Lee GS, Jeon YW, Kim S, Kim JH, Kang SK, Lee BC, Hwang WS. 2005. The role of gonadotropin-releasing hormone (GnRH) and its receptor in development of porcine preimplantation embryos derived from in vitro fertilization. Theriogenology. 63: 190-201. Paria BC, Reese J, Das SK, Dey SK. 2002. Deciphering the cross talk of implantation: advances and challenges. Science. 296: 2185-2188. Pask JA, Kanasaki H, Kaiser UB, Conn PM, Janovick JA, Stockton DW, Hess DL, Justice MJ, Behringer RR. 2005. A novel mouse model of hypogonadotrophic hypogonadism: N 84  Ethyl-N Nitrosourea-induced gonadotropin-releasing hormone receptor gene mutation. Mol. Endocr. 19: 972-981. Peters AR.2005. Veterinary clinical application of GnRH-questions of efficacy. Anim. Reprod. Sci. 88: 155-167. Raga F, Casan EM, Wen Y, Huang H, Nezhat C, Polan ML. 1998. Quantitative gonadotropin- releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol. Reprod. 59: 66 1-669. Rajamahendran R, Ambrose DJ, Small JA, Dinn N. 2001. Synchronization of estrus and ovulation in cattle. Arch. Animal Breeding 44: 58-67. Ramakrishnappa N, Giritharan G, Aali M, Madan P, Rajamahendran R. 2003. GnRH receptor messenger ribonucleic acid expression in bovine ovary. Can. J. Ani. Sci. 83: 823-826. Ramakrishnappa N, Rajamahendran R, Lin YM, Leung PCK. 2005. GnRH in nonhypothalamic reproductive tissues. Anim .Reprod. Sci. 88: 95-113. Siler-Khodr TM, Grayson M. 2001. Action of chicken II GnRH on the human placenta and deciduas. J. Clin. Endocr. Metab. 89: 1459-1466. Tabibzadeh S, Babakania A. 1995. The signal and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Hum. Reprod. 10: 1579-1602. van Biljon W, Wykes 5, Scherer 5, Krawetz SA, Hapgood J. 2002. Type II gonadotropin releasing hormone receptor transcripts in human sperm. Biol. Reprod. 67: 1741-1749. Yang MY, Rajamahendran R. 2000. Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells. Biol. Reprod. 62: 1209-12 17.  85  CHAPTER 3- GnRI-I AGONIST (BUSERELIN) INDUCED STEROID HORMONE RECEPTOR mRNA REGULATION IN BOVINE ENDOMETRIUM, IN VITRO 2  3.1 INTRODUCTION GnRH is extensively used in bovine reproductive management (Rajamahendran et al., 2001, Lewis et al., 2001; Ambrose et al., 2005; Peters, 2005) and is believed to exert its actions through the hypothalamo-pituitary-gonadal axis. The extrapituitary presence of GnRH-R is a matter of ongoing interest in the field of reproductive biology. The local modulatory role of the GnRH, GnRH-R system in extrapituitary reproductive tissue has not been clearly defined. It is well established that GnRH is a central regulator of mammalian reproduction and that it controls synthesis and release of FSH and LH, which further regulate gonadal steroidogenesis and gametogenesis. Extrapituitary GnRT-I-R have been identified in rat, human and bovine ovaries (Ramakrishnappa et al., 2003) and testes, prostate, uterus and placenta in several mammalian species (Cheng and Leung, 2005). Recent research in mammalian reproductive biology has demonstrated that GnRH modulates gonadal steroidogenesis, cellular proliferation, apoptosis and implantation (Ramakrishnappa et al., 2005). Recently, GnRH-R were also demonstrated in both the follicular and luteal phase bovine endometrium and oviducts (Singh et al., 2008). The endometrium is a dynamic structure and is always under the influence of ovarian steroids, estrogen and progesterone (Moutsatsou and Sekeris, 1997). Estrogen and progesterone mediate their actions through estrogen (ERcL and ERI3) and progesterone receptors (PR), respectively.  ERci is the predominant estrogen receptor in the mammalian uterus  (Rosenfeld et al., 1999; DeMayo et al., 2002; Walker and Korach, 2004), whereas ERI3  2 version of this chapter has been published. Singh R., Pretheeban T. and Rajamahendran, R. (2009) GnRH agonist A (buserelin) up regulates Estrogen Receptor c in Luteal Phase, but not Estrogen Receptor f3 and Progesterone Receptor mRNA in Bovine Endometrium, in vitro. Can. J. Ani. Sci. 89: 467-473.  86  expression is higher in the ovaries (Koehier et al., 2005). The ER and PR have been identified in ovarian follicles, CL, uterine stromal and epithelial compartments in species ranging from mice to humans (Couse et al., 2006). In bovine species, these receptors were found in the endometrium (Waither et al., 1999; Meikle et al., 2001), oviducts (Ulbrich et a!., 2003) and placentomes (Schular et a!., 2002). Estrogen and P , maintain uterine functions by regulating 4 endometrial proliferation, gamete transport, uterine receptivity, embryonic development, implantation, decidualization and pregnancy during the course of mammalian reproduction (Graham and Clark, 1997; Ulbrich et al., 2003; Vasudeven and Plaff, 2007). Classically, GnRH binds to 0-protein coupled receptors (GPCR) on pituitary gonadotrophs and activates Ca and protein kinase C (PKC) mediated cell signalling pathways. GnRH down-regulates ERCL and ERf3 in human granulosa luteal cells in vitro (Chiang et al., 2000) by activating PKC/MAPK cell signalling pathways. In contrast, this hormone up- regulates ERCL and PR in pituitary gonadotrophs (Demay et al., 2001; Tasende et al., 2002) acting through PKA pathways. The findings that GnRH mRNA is expressed in rat, human and bovine ovaries (Ramakrishnappa et al., 2003) and in mouse, human and porcine preimplantation embryos (Casan et al., 1999; Raga et al., 1999; Nam et al., 2005), indicate that the possible targets for GnRH could be extra-pituitary reproductive tissues including the uterus. Therefore, we studied the GnRH agonist (buserelin) induced regulation of steroid hormone receptors (ERa, ERf3 and PR) mRNA in both the follicular and the luteal phase endometrium.  87  3.2 MATERIALS AND METHODS 3.2.1 Collection and processing of tissues Bovine reproductive tracts were collected from Holstein Friesian culled dairy cows from a local abattoir. The reproductive tracts were collected within 20 mm of exsanguination of the animals and were classified as belonging to either the follicular or luteal phases of the estrous cycle based on ovarian morphology (Ireland et al., 1980; Yang and Rajamahendran, 2000; Ramakrishnappa et al., 2003). The tracts were transported on ice to the laboratory within 1 h of collection. Briefly, large follicles in the absence of CL or protruding fully developed CL were considered characteristic features of the follicular and the luteal phases of the estrous cycle, respectively. The reproductive tracts were obtained from different cows. Five reproductive tracts in the follicular and five tracts in the luteal phase were used in this study. 3.2.2 Endometrial explant culture and treatment Endometrial explants were cultured with slight modifications to previously described methods (Bogacki et al., 2002; Catalano et al., 2003). Briefly, uterine horns ipsilateral to follicles or CL were excised from the base and uterotubal junction, cut open along the longitudinal axis, washed three times with normal saline containing penicillin (100 IU mU’, Sigma #204-038-0) and streptomycin (100 jtg mU , Sigma # S-9137) and placed on a clean 1 bench top. Endometrial explants were then cut into small pieces (1-2 mm ) with a scalpel, 2 washed again with sterile saline, placed in 5 mL of Ca and Mg free Hank’s balanced salt solution (HBSS; Sigma Aldrich, Oakville, Canada; Cat # H9394) containing penicillin (100 IU mU’) and streptomycin (100 ig mU’). One hundred mg of explant tissues were weighed and incubated at 37°C in 5% CO 2 in a 12 well tissue culture plate (Becton Dickinson, Mississauga, Canada) in 500 iL of Dulbecco’s modified eagle’s medium (DMEM; Sigma Cat # D6046) containing 0.1% BSA (Sigma Cat # A3311), penicillin (100 IU mU ) and streptomycin(100 hg 1 88  mLj. After 20 h, the media were replaced and the explant tissues treated with the GnRH agonist, buserelin at concentrations of 0, 200, 500, and 1000 ng mL’ GnRH antagonist- antide ,  (500 ng mL’) and antide  +  buserelin (500  +  200 ng mL ) for 6 h under similar incubation 1  conditions. The antagonist treatments (500 ng mL’) were applied for 30 mm, media changed and then tissues were treated with the agonist (200 ng mL’) in fresh media. Endometrial explants from each treatment group were then stored at —80°C until RNA extraction. In the beginning of the experiments, buserelin dose response was studied by employing doses from 50-200 ng mL . A dose of 200 ng mL’ was noticed to induce an observable effect. 1 Higher doses were applied in the experiments to further study the dose response. The GnRH antagonist was used at doses of 500 ng mL’ (Ramakrishnappa, 2004) to block the GnRH-R. The explant cultures were treated for 6 h, with antagonist, antide (500 ng mL’) agonist or combination of both, to assess the transcriptional regulation within the biological half life of the GnRH agonist used in the experiment. GnRH agonist and antagonists have a half life of 4-8 h. 3.2.3 RNA extraction Total RNA was extracted using a single step RNA extraction method (Chomczynski and Sacchi, 1987; Ramakrishnappa et al. 2003). Tissues were briefly thawed at room temperature and each tissue sample was homogenized under liquid nitrogen by using pestel and mortar. One mL of Tn Reagent solution (Sigma Aldrich) was added to homogenates in a 2.0 mL RNAse and DNAse free microfuge tube, vortexed and allowed to stand for 10 mm at room temperature. Then 200 iL of chloroform was added to each tube, samples were agitated vigorously for 30 s, allowed to stand at room temperature for 15 mm and then centrifuged at 12,000 rpm for 15 mm  at 4°C. The top transparent layer containing the total RNA was  transferred into a new set of sterile tubes, 0.75 mL of isoproparanol was added to these tubes, gently mixed by inverting three times. The tubes were allowed to stand for 30 mm  at room 89  temperature before centrifugation at 12,000 rpm for 10 mm at 4°C. The resultant RNA pellet was washed twice in ice cold 75% ethanol and centrifuged at 12000 rpm for 5 mm. The RNA pellet was air dried and dissolved in 100 tL of sterile DEPC treated water. The quality and quantity of RNA was assessed spectrophotometericaly. The quality of RNA was also checked by ethidium bromide stained agrose gel (1%) electrophoresis and visualizing clear bands of 28S and 1 8S ribosomal RNA species under UV light. Total RNA was either used immediately or stored at —80°C for cDNA synthesis. 3.2.4 Semiquantative RT- PCR Reverse transcription-polymerase chain reaction (RT-PCR) was performed using commercially available first strand eDNA synthesis kits (Cells-to-cDNA II kit, Ambion Inc. The RNA Company, Austin, Texas, USA) and following the manufacturer’s protocol. Briefly, 2 jig of the total RNA was reverse transcribed by using a kit supplied random decamer primers in 20 jiL of total reaction volume. The reverse transcription reaction mixture was comprised of 2 jiL of lox RT buffer (pH 7.4), 1 jiL dNTP containing 0.5 m moles of each nucleotide, 1 jiL of M-MLV reverse transcriptase (lOU), 1 jiL RNAase inhibitor, 5  ji  moles of random  decamers, 2 jig RNA and nuclease free water to make the final volume to 20 jiL. The reaction was performed by incubating the contents in a thermal cycler at 42°C for 60 mm followed by 10 mm  incubation at 95°C to inactivate the reverse transcriptase enzyme and the cDNA  produced was stored at -20°C for future PCR amplification. JumpStart RED Taq Ready Mix PCR reaction mix (Jumpstart; Sigma-Aldrich, Oakville, Canada) and gene specific primer sequences for ERa (Horn et al., 1998), ERf3 (Ulbrich et al., 2003) and PR (Horn et al., 1998) were used in the PCR. G3PDH (Giritharan et al., 2007) was used as a housekeeping gene and internal control in the experiments. Initially, the PCR conditions were optimized by exponential amplification of PCR products with primers for 90  individual genes of interest. The genomic DNA contamination and non-specific amplifications were checked using negative controls without template cDNA and excluding the gene specific primers from the reaction mixture. Briefly, gene specific primers, nuclease free water, and 2 iL of cDNA template solution were added to 12.5 iL of Jumpstart content to obtain 25 iL of final reaction mixture. The reaction mixture contained 10 m moles Tris-HC1, 50 m moles KC1, 2.5 m moles MgC1 , 2 0.0001% gelatin, 0.2 m moles of each dNTP (dATP, dCTP, dGTP, dTTP), inert dye stabilizers, 0.03 U/jiL taq DNA polymerase, Jumpstart Taq antibody, 200 nano mole of each gene specific primer, cDNA, and nuclease free water. PCR steps followed for G3PDH amplification were an initial denaturation at 95°C for 2 mm, extension of 72°C for 45 mm with a final extension step of 72°C for 5 mm. A touch down PCR with annealing temperatures ranging from 58°C to 45°C, with a drop of 0.5°C every PCR cycle spanning over 40 cycles was performed for ERa. PCR conditions with an initial denaturation temperature of 95°C for 2 mm for ERa, ERI3 and PR, extension of 70°C for 45 s for ERa and 40 s for ERI3 and PR, and final extension step of 70°C for 10 mm  were used in the experiment. ERf3 and PR were amplified for 40 cycles each,  whereas G3PDH was amplified for 33 cycles. Denaturation (Td), annealing (Ta) temperatures, and product size for individual genes are shown in Table 3.1. Final PCR products were run on ethidium bromide stained agrose gel (2%) and visualized under ultraviolet illumination to observe expected size of the PCR products. The optical density of individual bands (inverse image) was analyzed by using computerized densitometry software, Scion Image Beta 4.02 (Scion Corporation, Fredrick, Maryland, USA). The optical density of individual treatment groups and genes was normalized to the corresponding level of the house keeping gene and expressed as relative mRNA levels.  91  3.2.5 Statistical analyses Statistical analyses of the relative ERct and PR mRNA expressions from each treatment group were performed on the mean ± SEM. The relative mRNA expressions in these tissues were compared to the control by one- way ANOVA and using Student “t” test at 95% confidence interval. Differential expression of ERct, ER3 and PR mRNA during present in vitro culture conditions was studied by comparing the relative mRNA levels of ERcL, ERI3 and PR from the follicular and luteal phase controls used during the experiment. The statistical analysis on ERf3 mRNA expressions with different treatments used in the experiments was not performed due to variability observed in the experiments. 3.3 RESULTS 3.3.1 RT-PCR RT-PCR amplification of cDNA obtained from endometrial explants treated with GnRH agonist (buserelin), GnRH antagonist (antide) and a combination of agonist and antagonist resulted in desired molecular weight transcripts for ERa, ER3 and PR mRNA (Table 3.1; Figure 3.1). G3PDH was used as a housekeeping gene and internal control in the experiments. The negative controls without template cDNA and without gene specific primers did not yield any PCR product ruling out the possibilities of genomic DNA contamination and non-specific amplification. 3.3.2 GnRH agonist induced regulation of ERL mRNA The GnRH agonist (buserelin) treatment at a dose of 200 ng mL 1 resulted in stimulatory response on ERa in the luteal phase endometrium (P0.047; Figure 3.2) as compared to vehicle treated control. Appendix 1 shows the gel images for the luteal phase ERa and G3PDH expressions in different cows. The stimulatory response with the same dose was less appreciable in the follicular phase endometrium. 92  3.3.3 GnRH agonist effect on PR mRNA  The GnRH agonist treatment of the explant cultures for 6 h did not affect PR expression in both the follicular (P=0.833) and luteal phase (P=0.975) endometrium during the course of the experiments (Figure 3.3) when compared to the control. 3.3.4 Follicular vs. luteal phase mRNA levels of ERz, ER and PR after in vitro culture  The relative mRNA expressions for ER c, ER f3 and PR mRNA were compared between follicular and luteal phase endometrium and from the vehicle treated controls used during the experiments. This study revealed that the relative ERa expression levels under the current in vitro culture conditions were higher in the follicular phase endometrium (P= 0.002) as compared to luteal phase endometrium. Whereas, ERI3 (P=0.254) and PR (PO.895) expression levels did not differ between the follicular and luteal phase endometrium during the experimental period (Figure 3.4). 3.4 DISCUSSION  This in vitro study explored the GnRH agonist (buserelin) induced regulation of steroid hormone receptor mRNA expression at the uterine level in the bovines. This research revealed that GnRH agonist, buserelin (200 ng mL’) induced a stimulatory response on the ERa mRNA in the luteal phase bovine endometrium. The GnRH antagonist (antide) and a combination of antide and GnRH agonist did not alter ERa mRNA expression, when compared to the control, suggesting it to be a receptor mediated response. Recently, the presence of GnRH-R in both the follicular and the luteal phase bovine endometrium was demonstrated (Singh et al., 2008). The endometrium is one of the primary target sites for the action of the ovarian steroids, estrogen and P . These hormones act through their corresponding estrogen (ERa, ERI3) and progesterone 4 receptor (PR) to maintain uterine cyclicity during the course of mammalian reproduction. The  93  role of GnRH in activation of steroid hormone receptors in the bovine endometrium was not investigated. It was elucidated previously that GnRH treatment in vitro for 8 h activated ERcL in pituitary gonadotrophs (Demay et al., 1996; 2001) but down-regulated ERa and ERI3 genes in human granulosa luteal cells during 24 h in vitro treatment (Chiang et al., 2000). In ewes, long term in vivo treatment with the GnRH agonist up-regulated ER and PR in the pituitary gland (Tasende et al., 2002). In the current study we treated endometrial explant cultures, under in vitro conditions for 6 h since the biological half life of GnRH agonists is between 4-8 h (Tarlatzis and Kolibianakis, 2007). Our findings support previous evidence that short term GnRH  exposure  has  a  stimulatory response  in the  normal  reproductive  process  (Ramakrishnappa et al., 2005), but are in contradiction with Chiang at al. (2000) demonstrating that GnRH agonist down- regulated ER in human granulosa luteal cells over 24-48 h treatment periods. In the same study these researchers inferred that similar treatments could not affect ER mRNA levels before 24 h. The possible reason for this could be that the GnRH exposure over a prolonged period of time results in inhibitory effects. These dual effects have clearly been explained in the in vivo experiments conducted by Hsueh and Jones (1981, 1982) showing that exogenous GnRH or GnRI-I agonist treatment in adult male and female hypophysectomized rats either stimulate or inhibit gonadal functions. Estrogen mediates its actions through its receptors and ERa is the predominant ER isoform in the mammalian uterus. It is evident that ERa knockout mice are infertile with hypoplastic uteri and implantation failures whereas, ERf3 knockout mice have reduced fertility and ovulatory defects (Cooke et al., 1998; Walker and Korach, 2004). It is known that the GnRH agonist administration increases pregnancy rates in the bovine (Peters, 2005) and that there is ERa up-regulation prior to implantation which makes the endometrium more 94  responsive to estrogenic actions (Paria et al., 2002). Human, murine, porcine preimplantation embryos (Casan et al., 1999; Raga et al., 1999; Nam et al., 2005) and human placenta (Wolfahrt et al., 1998) have been shown to express GnRH either at mRNA or protein levels indicating that the potential target for this GnRH could be the endometrium. The interactions between GnRH, GnRH-R and ER might play a role in uterine receptivity, implantation and maintenance of pregnancy. These findings did not reveal significant changes in these receptors in the tissues treated with GnRH antagonist (antide) or a combination of antide and GnRH agonist and suggest that this ERcL stimulatory response is a GnRH-R mediated phenomenon. The additional evidence gained from this study that GnRH agonist could not alter the mRNA levels of PR tends to suggest that GnRH agonist actions in the bovine endometrium could be ERcL mediated and support the observations that GnRH could differentially regulate molecular mechanisms in reproductive tissues (Cheng and Leung, 2005). This study provides evidence that ERL mRNA expressions are higher in follicular as compared to luteal phase bovine endometrium and support similar findings from the bovine oviducts (Ulbrich et al. 2003). The findings from the current research are based on the mRNA levels of the steroid hormone receptors. It would be pertinent to further confirm this response at the protein level. Also, it is still to be conclusively established that these ERcL mRNA changes are mediated through GnRH-R. The variations observed during the current experiments could be attributed to the stage of the endometrium at the time of tissue collection. Animal factors might also contribute to these transcript variations. The tissues were collected during the follicular and luteal phases with the follicles and the mid luteal phase CL as characteristic features, respectively. The follicular phase lasts for about 3 d (day 18-21 of the estrous cycle). The mid luteal phase CL exists from day 11-16 in the bovine luteal phase. There could be a lack of precision regarding the exact day of the estrous cycle and corresponding physiological stage in 95  this highly renewable tissue. Therefore, these findings would need further substantiation using estrous synchronized animals at the same stage of lactation/age to avoid these variations due to the current experimental approaches. 3.5 CONCLUSIONS This study demonstrates that in vitro GnRH agonist (buserelin) treatment (200 ng mL’) results in a stimulatory response on ERcL mRNA in the luteal phase endometrium. The GnRH agonist did not alter PR mRNA expression in either the follicular or luteal phase endometrium. Furthermore, this study showed that ERcL mRNA expressions are higher in the follicular phase than in the luteal phase endometrium. It is believed that the estrogenic effects in the uterus are ERct mediated and this differential regulation of ERcL in terms of regulation of normal uterine functions including endometrial apoptosis, proliferation, uterine receptivity, embryonic implantation and establishment of pregnancy needs to be further examined.  96  Table 3.1 Primer sequences, denaturation temperature (Td), annealing temperature (Ta) and the product size obtained by RT-PCR amplifications during the experiments.  Gene ERa  Primer sequence F 5’- TTG ACC CTC CAT OAT CAG GT-3 R 5’-CAG GTG GAT CAA AGT GTC TG-3  ER  PR  G3PDH  *  Td  Ta  Product size (bp)  94°C  58-45°C  360  *(45)  (45s)  F 5’-GCT TCG TGG AGC TCA GCC TG-3’  94°C  58°C  R 5’-AGG ATC ATG 0CC TTG ACA CAGA-3’  (40s)  (40s)  F 5’-TTC AAT AAA OTT AGA OTT AT-3’  94°C  58°C  R 5’- TCC GAA AAC CTG GCA GTG AT-3’  (40s)  (40s)  F5’-TGTTCCAGTATAGATTCCACC-3’  94°C  58°C  R 5’- AGG AGG CAT TGC TGA CAA TC -3’  (40s)  (3 Os)  262  227  318  Figures in parenthesis show the time in seconds used in Td and Ta steps during the PCR amplifications.  97  _____  B200 B500 B1000 A  BO ERL  r  A+B  BO  $Z2_Y==r  B200 B500 B1000 A  A+B  $Zi  ER  G3PDH  —  Follicular  Luteal  Figure 3.1 Representative autoradiograms showing PCR products for ERcL, ERf3 and PR mRNA transcripts. Follicular and luteal phase endometrial explants were treated  in vitro  with  vehicle-control (BO), GnRH agonist (buserelin)- 200, 500 and 1000 ng mL’ (B200, B 500 and B 1000 respectively), GnRH antagonist (antide)- 500 ng mL’ (A) and a combination of antide 500 ng mL’  +  buserelin- 200 ng mL’ (A  +  B). The PCR products obtained from each  treatment were electrophoresed on 2% ethidium bromide stained agrose gel and visualized under ultraviolet illumination. G3PDH was used as the housekeeping gene and the internal control in the experiments.  98  2  *  1. 0.5 —  0 BO  B200  B500  B 1000  A500  A500±B200  Trei tnient  Figure 3.2 GnRH agonist, buserelin induced ERcL mRNA expression in luteal phase bovine  endometrium during in vitro culture (n5). Endometrial explants were treated in vitro with vehicle-control (BO), GnR}1 agonist (buserelin)- 200, 500 and 1000 ng mL 1 (B200, B500 and B 1000, respectively), GnRH antagonist (antide)- 500 ng mL’ (A) and combination of antide 500 ng mL 1  +  buserelin-200 ng mL’ (A + B). The mRNA levels from untreated controls were  normalized to corresponding levels of the housekeeping gene G3PDH and statistically analyzed. The value with an asterisk (*) shows significantly higher abundance (P  0.01) of  ERa mRNA in the luteal phase endometrium. Error bars in the histogram are standard error on mean (SEM)  99  7  •  • Follicular o Luteal  is.  1•  S 05  BO  B200  B500  B1000  A500  A500+B200  Treatment  Figure 3.3 GnRH agonist, buserelin effect on PR mRNA expression in follicular and luteal  phase bovine endometrium during in vitro culture. Follicular and luteal phase endometrial explants were treated in vitro with vehicle-control (BO), GnRH agonist (buserelin)- 200, 500 and 1000 ng mL’ (B200, B500 and B 1000 respectively), GnRH antagonist (antide)- 500 ng mL’ (A) and combination of antide- 500 ng mL’  +  buserelin -200 ng mL 1 (A  +  B). The  mRNA levels from untreated controls were normalized to corresponding levels of the housekeeping gene G3PDH and statistically analyzed. The experiments was repeated five times (n=5) on tissues obtained from different animals. Error bars in the histogram are standard error on mean (SEM)  100  2  • Follicular  2  flLitea1  ‘I)  a)  1  x a)  **  z  E a) >  0.5 H  j 0  ERi  ER  PR  Figure 3.4 Relative ERcL, ERf3 and PR mRNA expression in the untreated (control) follicular  and luteal phase bovine endometrium during in vitro culture (n5). The mRNA levels from untreated controls were normalized to corresponding levels of the housekeeping gene G3PDH and statistically analyzed. The value with asterisk (**) shows higher abundance (P  0.01) of  ERci mRNA in follicular endometrium as compared to luteal endometrium. The levels of ERj3 and PR were not different between the follicular and luteal phase endometrium. The experiment was repeated five times (n=5) on tissues obtained from different animals. Error bars in the histogram are standard error on mean (SEM)  101  3.6 BIBLIOGRAPHY Ambrose DJ, Kastelic JP, Rajamahendran R, Aali M, Dinn N. 2005. Progesterone (CIDR) based timed Al protocols using GnRH, porcine LH or estradiol cypionate for dairy heifers: ovarian and endocrine responses and pregnancy rates. Theriogenology. 64: 1457-1474.  -  Bogacki M, Silvia J, Rekawiecki R, Kotwica J. 2002. Direct inhibitory effect of progesterone on oxytocin-induced secretion of prostaglandin F2a from bovine endometrial tissue. Biol. Reprod. 67: 184-188. Casan EM, Raga F, Polan ML. 1999. GnRH mRNA and protein expression in human preimplantation embryos. Mol. Hum. Reprod. 5: 234-239. Catalano RD, Yanaihara A, Evans AL, Rocha D, Prentice A, Print CG, Charnock-Jones DS, Sharkey AM, Smith SK. 2003. The effect of RU 486 on the gene expression profile in an endometrial expaint model. Mol. Hum. Reprod. 9: 465-473. Cheng CK, Leung PCK. 2005. Molecular Biology of Gonadotropin- Releasing Hormone (GnRH)-1 and GnRH-1 1 and Their Receptors in Humans. Endocr. Rev. 26: 283-306. Chiang CH, Cheng KWA, Igarashi 5, Nathwani PS, Leung PCK. 2000. Hormonal Regulation of Estrogen Receptor n and f3 Gene Expression in Human Granulosa- Luteal Cells in Vitro. J. Clin. Endocr. Metab. 85: 3828-3839. Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. Cooke PS, Buchanan DL, Lubahan DB, Cunah GR. 1998. Mechanism of estrogen Action: Lessons from Estrogen Receptor- a Knockout Mouse. Biol. Reprod. 59: 470-475. Couse JF, Hewitt SC, Korach KS. 2006. Steroid Receptors in the Ovary and Uterus. Pages 593-677 in Jimmy D Neill edited knobil and Neill’s Physiology of Reproduction Volume 1 (Third Edition) Elsievier Academic Press. Demay F, De Monto M, Tiffoche C, Vaillant C, Thieulant M. 2001. Steroid- Independent activation of ER by GnRH in gonadotrope pituitary cells. Endocrinology. 142: 3340-3347. Demay F, Tiffoche C, Thieulant ML. 1996. Effect of Gonadotropin-Releasing Hormone on Estrogen Receptor Messanger Ribonucleic Acid Level in Perifused Pituitary Cells. Cell. Mol. Neurobiol. 16: 397-402. DeMayo FJ, Zhao B, Takamoto N, Tsai SY. 2002. Mechanisms of Action of Estrogen and Progesterone. Ann. N. Y. Acad. Sci. 955: 48-59. Giritharan C, Ramakrishnappa N, Aali M, Madan P, Balendran A, Singh R, Rajamahendran R. 2007. Paternal influence on apoptosis, and expression of Bc12, Bax, p53, heat shock protein 70 and interferon tau genes in bovine perimplantation embryos Can. J. Anim. Sci. 87: 157-165. -  102  Graham JD, Clark CL. 1997. Physiological action of progesterone in target tissues. Endocr. Rev. 18: 502-517. Horn S, Bathgate R, Lioutas C, Bracken K, Ivell R. 1998. Bovine endometrial epithelial cells as a model system to study oxytocin receptor regulation. Hum. Reprod. Update. 4: 605614. Hsueh AJ, Jones PB. 1981. Extra pituitary actions of gonadotropin-releasing hormone. Endocr. Rev. 2: 437-461. Hsueh AJ, Jones PB. 1982. Regulation of ovarian granulosa and luteal cell functions by gonadotropin releasing hormone and its antagonist. Adv. Exp. Med. Biol. 147: 223-62. Ireland JJ, Murphee RL, Colulson PB. 1980. Accuracy or predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. J. Dairy Sci. 63: 155-160. Koehier KF, Helguero LA, Haldosen LA, Warner M, Gustafsson JA. 2005. Reflections on the discovery and significance of estrogen receptor f3. Endocr. Rev. 26: 465-478. Lewis GS, Caidwell DW, Rexroad CE, Dowlen HH, Owen JR. 2001. Effects of gonadotropin releasing hormone and human chorionic gonadotropin on pregnancy rate in dairy cattle. J. Dairy Sci. 73: 66-72. Meikie A, Sahlin L, Ferraris A, Masironi B, Blanc JE, Rodriguez-Irazoqui M, Rodriguez Pinon Kindahi H, Forsberg M. 2001. Endometrial mRNA expression of oestrogen receptor n’, progesterone receptor and insulin-like growth factor-I (IGF-I) throughout the bovine oestrous cycle. Anim. Reprod. Sci. 68: 45-56. Moutsaatsou P, Sekeris E. 1997. Estrogen and Progesterone Receptors in the Endometrium. Ami. Ny. Acad. Sci. 816: 99-115. Nam PH, Lee SH, Kim HS, Lee GS, Jeon YW, Kim S, Kim JH, Kang SK, Lee BC, Hwang WS. 2005. The role of gonadotropin-releasing hormone (GnRH) and its receptor in development of porcine preimplantation embryos derived from in vitro fertilization. Theriogenology. 63: 190-201. Paria BC, Reese J, Das SK, Dey SK. 2002. Deciphering the cross talk of implantation: advances and challenges. Science. 296: 2185-2188. Peters AR. 2005. Veterinary clinical application of GnRH-questions of efficacy. Anim. Reprod. Sci. 88: 155-167. Raga F, Casafl EM, Kruessel J, Wen Y, Bonilla-Musoles F, Polan ML. 1999. The role of gonadotropin-releasing hormone in murine preimplantation embryonic development. Endocrinology. 140: 3705-12. Rajamahendran R, Ambrose DJ, Small JA, Dinn N. 2001. Synchronization of estrus and ovulation in cattle. Arch. Anim. Breed. 44: 58-67. 103  Ramakrishnappa, 2004. A study on GnRH-R and GnRH mRNA expression, direct effects of GnRH-a in bovine ovary and influence of post breeding GnRI-I administration on corpus luteum function and pregnancy in dairy cattle. Thesis submitted to University of British Columbia, Canada. Ramakrishnappa N, Rajamahendran R, Lin YM, Leung PCK. 2005. GnRH in nonhypothalamic reproductive tissues. Anim. Reprod. Sci. 88: 95-113. Ramakrishnappa N, Giritharan G, Aali M, Madan P, Rajamahendran R. 2003. GnRH receptor messenger ribonucleic acid expression in bovine ovary. Can. J. Anim. Sci. 83: 823826. Rosenfeld PS, Yuan X, Manikkam M, Calder MD, Garverick HA, Lubahn DB. 1999. Cloning, sequencing and localization of bovine estrogen receptor 13 within the ovarian follicle. Biol. Reprod. 60: 691- 697. —  Schular G, With C, Teichman, U, Failing K, Leiser R, Thole H, Hoffman B. 2002. Occurrence of Estrogen Receptor c In Bovine Plancentomes Throughout Mid and Late Gestation and at Parturition. Biol. Reprod. 66: 972-982. Singh R, Graves ML, Roskelley CD, Giritharan G, Rajamahendran R. 2008. Gonadotropin releasing hormone receptor gene and protein expression and immunohistochemical localization in bovine uterus and oviducts. Domest. Anim .Endocrin. 34: 319-326. Tarlatzis BC, Kolibianakis EM. 2007. GnRH agonists vs antagonists. Best. Pract. Res. Cl. Ob. 21: 57-65. Tasende C, Meikie A, Rodrguez- Pinon M., Forsberg M, Garofalo G. 2002. Estrogen and progesterone receptor content in the pituitary gland and uterus of progesterone-primed and gonadotropin releasing hormone-treated anestrous ewes. Theriogenology. 57: 1719-1731. Ulbrich SE, Kettler A, Einspanier R. 2003. Expression and localization of estrogen receptor, estrogen receptor f3 and progesterone receptor in the bovine oviduct in vivo and in vitro. J. Steroid Biochem. 85: 279-289.  Vasudeven N, Plaff DW. 2007. Membrane- Initiated Actions Neuroendocrinology: Emerging Principles. Endocr. Rev. 28: 1-19.  of Estrogens  in  Walker VR, korach KS. 2004. Estrogen Receptor Knockout Mice as a Model for Endocrine Research. Inst. Lab. Anim. Res. J. 45: 455-461. Walther N, Lioutas C, Tiliman G, Iveli R. 1999. Cloning of bovine estrogen receptor beta (ER f3): expression of novel deleted isoform in reproductive tissues. Mol. Cell. Endocrin. 152: 37-45. Wolfahrt S, Kleine B, Rossmanith WG. 1998. Detection of gonadotrophin releasing hormone and its receptor mRNA in human placental trophoblasts using in-situ reverse transcription polymerase chain reaction. Mol. Hum. Reprod. 4: 999-1006.  104  Yang MY, Rajamahendran R. 2000. Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells. Biol. Reprod. 62: 1209-1217.  105  CHAPTER 4- GnRH AGONIST (BUSERELIN) INDUCED APOPTOSIS IN BOVINE ENDOMETRIUM, IN VITRO 3 4.1 INTRODUCTION Gonadotropin releasing hormone (GnRH) plays a central role in mammalian reproduction. Once released from the hypothalamus, GnRH binds to its receptors on pituitary gonadotrophs and regulates synthesis and release of the gonadotrophins; follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH enter into the systemic circulation and control synthesis and release of estrogen and progesterone and regulate gametogenesis. During the last decade, the presence of the GnRH, GnRH-R system has been elucidated in the extrapituitary reproductive tissues including the ovaries, uterus, mammary glands, testes, and prostate (Ramakrishnappa et al., 2005). The local modulatory role of the extrapituitary GnRH, GnRH-R system in these tissues is an active area of research in mammalian reproductive biology. GnRH could differentially modulate diverse biological processes including gonadal steroidogenesis, cellular proliferation, and apoptosis in extra-pituitary reproductive tissues (Cheng and Leung, 2005). Apoptosis is a highly regulated biological process of programmed cell death. It does not invoke an immune response and is characterized by cellular blebbing, DNA condensation and fragmentation, formation of apoptotic bodies, and the elimination of unwanted cells (Elmore, 2007). The mitochondrial pathway (intrinsic) and the death receptor pathway (extrinsic) are two major pathways regulating apoptosis at the molecular and cellular level. The mitochondrial pathway is regulated by the Bcl 2 group of genes with pro- and anti- apoptotic molecules (Bax and Bcl-2, respectively) as its important members. The Bax/Bcl-2 ratio (Bax: Bcl-2) is an important checkpoint for cell fate decisions and an increase in this ratio results in cell death  A version of this chapter will be submitted for publication. Singh R. and Rajamahendran, R. GnRH agonist, Buserelin induced Apoptosis in Bovine Endometrium, in vitro.  106  signals from mitochondria to cytoplasm. Fas is another important mediator of apoptosis and belongs to the family of tumour necrosis factor (TNF) receptors and is involved in the extrinsic pathway (death receptor pathway). Both intrinsic and extrinsic pathways converge to activate caspases to execute the process of programmed cell death (Riedl and Shi, 2004). GnRH and GnRH-R have been found in both normal and carcinogenic human uterine tissues (Irmer et al., 1994; Imai et al., 1994; Dong et a!., 1998; Borroni et al., 2000; Cheon et al., 2001 and Grunduker et al., 2002). GnRH is an important regulator of apoptosis in the rat and human ovaries (Billing et al., 1994; Parborell et al., 2008; Hong et al., 2008), ovarian carcinomas (Tang et al., 2002), testes (Andreu cancer, uterine leiomyoma  —  Vieyra et al., 2005), prostate and prostate  ( Kwon et a!., 2005), and endometriosis (Meresman et al., 2003;  Bilotas et al., 2007). Recently Singh et al. (2008) demonstrated GnRH-R in the follicular and the luteal phase bovine endometrium at both the mRNA and protein levels, and further localized these receptors to endometrial epithelial cells. The endometrium is a dynamic structure undergoing changes in every estrous/menstrual cycle (Couse et al., 2006). Apoptosis maintains tissue homeostasis by tightly regulating cellular turnover and seems to acquire more significance in rapidly renewable tissues like the endometrium (Harda et al., 2004). The local modulatory role of the GnRH, GnRH-R system in regulation of endometrial apoptosis is still not well explored. Therefore, this study investigated i) the GnRH agonist (buserelin) induced regulation of apoptotic genes (Bax, Bcl-2, Caspase-3 mRNA), ii) buserelin induced endometrial epithelial cell apoptosis in the follicular and luteal phase bovine endometrium and, iii) agonist induced regulation of Fas mRNA in the follicular phase endometrium.  107  4.2 MATERIALS AND METHODS 4.2.1 Collection and processing of tissues Bovine reproductive tracts were collected from culled Holstein Friesian dairy cows, from a local abattoir. The reproductive tracts were collected within 20 mm of exsanguination of the animals. The tracts were classified as belonging to either the follicular or the luteal phases of the estrous cycle based on ovarian morphology (Ireland et al., 1980; Yang and Rajamahendran, 2000; Ramakrishnappa et al., 2003). Briefly, the ovulatory follicles and fully developed mid luteal phase CL were considered characteristic features of the follicular and luteal phases of the estrous cycle, respectively. The uteri were transported on ice to the laboratory within 1 h of collection. Six reproductive tracts in the follicular phase and six tracts in the luteal phase, obtained from different animals were used in these experiments. 4.2.2 Endometrial explant culture and treatment Endometrial slices were cultured as per previously described (Bogacki et al., 2002; Catalano et al., 2003). In the laboratory, uterine horns ipsilateral to the follicle or CL were excised from the base and uterotubal junction, cut open, washed three times with nonnal saline (200 mL) containing penicillin 0 (100 IU/mL; Sigma # 204-038-0  )  and streptomycin (100  jig/mL; Sigma # S 9137), and placed on a clean bench top. Endometrial explants were cut into -  smaller pieces (1-2 mm ) with a scalpel and again washed with sterile saline and placed in a 2 petri dish containing 5 mL Ca and Mg free HBSS (Sigma-Aldrich, Oakville, Canada; Cat # H 9394) supplemented with penicillin and streptomycin. A hundred milligram of tissues were weighed and incubated in 12 well tissue culture plate (Becton Dickinson, Mississauga, Canada) containing Dulbecco modified eagle’s medium (DMEM; Sigma # D 6046) supplemented with 0.1% BSA (Sigma # A 8806), 100 IU/mL penicillin G and 100 j.tg/mL streptomycin in an incubator at 37°C in air with 5% CO 2 in 500 iL DMEM. After 20 h incubation, the media was 108  replaced with fresh media and tissues were treated with GnRI-1 agonist, buserelin (0, 200, 500, 1000 ng/mL, respectively), GnRH antagonist-antide (500 ng/mL) and antide (500 ng/mL)  +  buserelin (200 ng/mL) for 6 h and stored at -80°C until RNA extraction. In the antide  +  buserelin treatment group, tissues were first incubated with antide (500 ng/mL) for 30 mm, and then the tissues were incubated with buserelin (200 ng/mL) in fresh culture media. Vehicle only treated tissues were included as controls. 4.2.3 RNA extraction Total RNA was extracted using a single step RNA extraction method (Chomczynski and Sacchi, 1987; Ramakrishnappa et al. 2003). Briefly, tissues were thawed, then homogenized under liquid nitrogen using a pestel and mortar, and then transferred to 2 mL RNAse and DNAse free microfuge tubes. Thereafter, lmL of Tn Reagent solution (SigmaAldrich) was added to each tube, which were vortexed, and allowed to stand for 10 mm at room temperature. After that, 200 tL of chloroform was added to each tube, agitated vigorously for 30 s, allowed to further stand at room temperature for 15 mill and centrifuged at 12,000 rpm for 15 mm at 4°C. The top clear transparent layer containing the total RNA was transferred into a new set of sterile tubes, 0.75 mL of isoproparanol was added, the tube contents were gently mixed inverting three times and again allowed to stand for 30 mm at room temperature and centrifuged at 12,000 rpm for 10 mm at 4°C. The resultant RNA pellet at the bottom of tube was washed two times in ice cold 75% ethanol by centrifugation at 5,000 rpm at 4°C for 5 mm and air dried. The pellet was then dissolved in 100 iL of sterile DEPC treated water. The quality and quantity of RNA was assessed spectrophotometericaly. The quality of RNA was also checked by ethidium bromide stained agrose gel (1%) electrophoresis and visualizing clear bands of 28S and 1 8S ribosomal RNA species under ultra violet illumination. Total RNA was either used immediately or stored at —80°C for cDNA synthesis. 109  4.2.4 Semiquantative RT- PCR Reverse transcription-polymerase chain reaction (RT-PCR) was performed using commercially available first strand cDNA synthesis kits (Cells-to-cDNA II kit, Ambion Inc. The RNA Company, Austin, Texas, USA) following the manufacturer’s protocol. Briefly, 2 tg of total RNA was reverse transcribed using a kit supplied random decamer primers in 20 tL reaction volume. The reverse transcription reaction comprised of 2 1 iL of i OX RT buffer (pH 7.4), 1 jiL dNTP containing 0.5 m moles of each nucleotide, 1 iL of M-MLV reverse transcriptase (10 IU), 1 iL RNAase inhibitor, 5  t  moles of random decamers, 2 tg RNA and  nuclease free water to make the final volume to 20 iL. The reaction was performed by incubating the contents in a thermal cycler at 42°C for 60 mm followed by 10 mm incubation at 95°C to inactivate the reverse transcriptase enzyme. The cDNA produced was stored at -20°C for future PCR amplification. JumpStart RED Taq Ready Mix PCR reaction mix (Jumpstart; Sigma-Aldrich, Oakville, Canada) and gene specific primer sequences for Bax, Bcl-2 (Giritharan et al., 2007), Caspase-3 and Fas (Okuda et a!., 2004) were used in the PCR amplifications. G3PDH (Giritharan et al., 2007) was used as a housekeeping gene and as an internal control in the experiments. The PCR conditions were standardized in the beginning of the experiments by testing different concentrations of primers. The PCR products were exponentially amplified to achieve optimal amplification. Non-specific amplification was checked by eliminating gene specific primers from the reaction mixture and genomic DNA contamination was checked during the experiments by including negative controls without template cDNA. Gene specific primers, nuclease free water, and 2 tL cDNA solution were added to 12.5 iL of Jumpstart contents to obtain a 25 jiL of final reaction mixture. The reaction contained 10 1 m moles Tris-HC1, 50 m moles KC1, 2.5 m moles MgC1 , 0.0001% gelatin, 0.2 m moles of each 2 110  dNTP (dATP, dCTP, dGTP, dTTP), inert dye, stabilizers, 0.03 U/j.il taq DNA polymerase, Jumpstart Taq antibody, 200 nano mole of each gene specific primer, eDNA template and nuclease free water. RT-PCR steps involved an initial denaturation at 94°C for 2 mm, 94°C for 30 s, extension of 72°C for 45 s with a final extension step of 72°C for 5 mm. Primer sequences, PCR cycles and product size for the individual genes of interest are shown in Table 4.1. Final PCR products were electrophoresed on ethidium bromide stained agrose gel (2%) and visualized under ultraviolet illumination to observe expected size products. The optical density of individual bands (inverse image) was analyzed by using computerized software, Scion Image Beta 4.02 (Scion Corporation, Fredrick, Maryland, USA) and normalized to the corresponding levels of the housekeeping gene G3PDH for each treatment group. 4.2.5 Endometrial epithelial cell isolation, culture and treatment Endometrial epithelial cells were isolated and cultured as previously described (Bogacki et al., 2002). Briefly, the follicular and luteal phase bovine uteri were collected from a local abattoir. The uteri were transported on ice to the laboratory within 1 h of collection. The uterine horns ipsilateral to ovulatory follicles or CL were then excised from the cervical and ovarian attachments. The uterine lumen was then washed three times with 30-50 mL of Ca and Mg free HB SS containing 0.1% B SA, penicillin (100 IU/mL) and streptomycin (100 ig/mL). After the initial washing steps, the uterine horns were filled with HBSS supplemented with 0.05% collagenase (Sigma # C 0130), 0.005% deoxyrinonuclease I (Sigma # D 5025) and 0.1% BSA. The uterine ends were then sealed on both sides with haemostats and the horns were incubated in a shaking water bath at 37°C for 45 mm. The enzymatic mixture containing free cells was collected and the uterine lumen again filled with 30-50 mL fresh mixture and incubated for additional 30 mm. The enzyme mixture was collected again and pooled with the previous one and filtered through a sieve (100 im pore size) to separate cells from dissociated tissue 111  fragments. The cells collected were washed three times with DMEM containing antibiotics (penicillin and streptomycin) and BSA (0.1%). The cell viability was assessed by the trypan blue (0.2%) exclusion method (Ramakrishnappa, 2004). Isolated cells were then cultured in 500 jiL of 1:1 DMEM/Ham F- 12 medium (Sigma # D 8437) supplemented with 10% heat inactivated calf serum and 20 mg/mL gentamycin. The cells were then seeded at a density of 100,000 cells/chamber coated with 10 Ig/cm 2 rat tail collagen (Type 1, Sigma # C 3867), in eight chamber glass slides (Lab -Tek II, VWR, Mississauga, ON, Canada). The chamber slides containing cells were then incubated at 37°C, 5% CO 2 in a humidified atmosphere. After 48 h incubation media was discarded and cultures were replaced with fresh media containing 1% calf serum and gentamycin. The cells were then treated with GnRH agonist, buserelin (200 ng/mL), antagonist- antide (500 ng/mL), and a combination of antide  +  buserelin (500  +  200 ng/mL) for 6 h. The cells were allowed to grow  for an additional 24 h after each treatment. 4.2.6 Immunofluorescence  In the present culture system, cells were characterized as epithelial cells (Meresman et al., 2003) using a primary antibody reacting against cytokeratin 4, 5, 6, 8, 10, 13 and 18. Briefly, isolated cells were seeded at a density of 100,000 cells/chamber in a collagen coated eight chamber cell culture slide. The cells were then incubated for 48 h at 37°C and 5% CO . 2 Then the culture media was removed and cells were fixed in ice cold methanol for 20 mm at  -  20°C. The primary monolayers were then permeabilized with Triton X (0.02%) for 5 mm, rinsed with PBS (100 iL) and, incubated with 2% goat serum in PBS for 1 h at room temperature. After this, the cells were washed with 100 jiL PBS and incubated overnight with mouse monoclonal anti-cytokeratin antibody (abcam 49779; 1:50 in PBS) at 4°C, again washed and then incubated with FITC labelled goat polyclonal to mouse IgG as secondary antibody 112  (abeam 6785; 1:500 in PBS) at room temperature for 1 h. The cells were examined with a fluorescent microscope using an excitation filter of 450-490 nm and a barrier filter of 520 nm wavelength. Cells incubated with PBS instead of primary antibody were included as negative controls in the experiments. 4.2.7 Apoptotic assay Endometrial epithelial cells were seeded at a density of 100,000 cells/chamber and treated with GnRH agonist, buserelin (200 ng/mL), antide (500 ng/mL) and antide (500  +  +  buserelin  200 ng/mL) for 6 h. Apoptosis was assessed by acridine orange-ethidium-bromide  staining techniques (Bilotas et al., 2007). Acridine-orange is a specific dye that is excluded from viable cells and it specifically labels apoptotic cell nuclei, rather than cells undergoing a necrotic form of death. Ethidium-bromide labels live cell nuclei. After 6 h of agonist or antagonist treatment, culture media were replaced and cells were further allowed to grow for additional 24 h and then stained with acridine-orange-ethidium-bromide mix (2 ig/mL each) for 10 mm at room temperature. The cells were then visualized with a fluorescent microscope under 400 X magnification using an excitation filter of 450-490 nm and a barrier filter of 520 nm wavelength. Apoptotic cell nuclei stained orange; whereas, live cell nuclei stain green, when examined under fluorescent light. The total number of cells and the number of apoptotic cells under three randomly selected fields/chamber were counted under the microscope. The percent apoptotic cells were calculated for control and treatment groups. 4.2.8 Statistical analyses The statistical analyses were performed at 95% confidence interval (CI) using ANOVA and Student’t’ test for Bax, Bcl-2 and Fas mRNAs and apoptotic assays. Statistical significance for caspase-3 mRNA expression was achieved by using ANOVA and Dunett test at the 95% CI. Data were tested to fit into a normal distribution prior to employing test of significance. 113  4.3 RESULTS 4.3.1 RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) amplification resulted in the mRNA transcripts of expected molecular weight for Bax, Bcl-2, caspase-3 and Fas mRNA. G3PDH was used as a housekeeping gene in the experiment and yielded a product of 320 bp in the experiments. The optical density of individual genes for each treatment was normalized to the corresponding level of housekeeping gene. The negative controls without cDNA and primers did not yield any PCR products during the experiments. 4.3.2 GnRH agonist induced Bax, Bcl-2 and caspase-3 mRNA regulation  The follicular and luteal phase endometrial tissues were treated in vitro with 0 (control), 200, 500, and 1000 ng/mL of GnRH agonist (buserelin), GnRH antagonist- antide (500 ng/mL) and a combination of antide ng/ mL) up-regulated (P  +  buserelin (500+200 ng/mL). The GnRH agonist, buserelin (200  0.05) Bax expression in the follicular phase endometrium (Figure 4.  1A). The luteal phase mRNA levels were not altered (P=0.617; Figure 4.1B) with similar treatments during the experiment. Bcl-2 expression remained unchanged during the study in both the follicular (P=0.8434) and luteal phase (P=0.661) endometrium with similar treatments as used to study Bax expressions (Figure 4.2A and B). GnRH agonist, buserelin treatments (200, 500 ng/mL) up-regulated (P  0.05) caspase3  mRNA levels in the follicular phase endometrium (Figure 4.3A). In contrast, there was no effect on caspase3 mRNA levels in the luteal phase endometrium with similar doses of agonist or antagonist or a combination of both (Figure 4.3B).  114  4.3.3 Fas mRNA regulation in follicular phase endometrium The Fas gene is involved in the apoptotic process acting through the death receptor pathway and mediates cell death by binding to its ligand. GnRH agonist treatment did not affect (P0.0162) Fas expression in follicular endometrium (Figure 4.4). 4.3.4 Immunofluorescence Endometrial epithelial cells were characterized by immunofluorescence using anticyokeratin antibody. Cytokeratin is a marker of the epithelial cell cytoskeleton. Cells grown in vitro showed clear green cytoplasmic labelling against cytokeratin in the present culture  system. This confirmed that the cells isolated and grown in vitro during the experiments were endometrial epithelial cells (Plate 4.1). 4.3.5 Apoptotic assay Apoptotic cell nuclei stained orange and live cell nuclei stained green with the acridine orange-ethidium-bromide stain. The GnRI-I agonist, buserelin (200 ng/mL) induced (P  0.05)  apoptosis in the follicular phase endometrial epithelial cell. Similar agonist or antagonist treatments did not result in a similar (P=0.270) apoptotic response in the luteal phase endometrial epithelial cells during the experimental periods (Plate 4.2; Figure 4.5). 4.4 DISCUSSION Apoptosis is a highly regulated process of selective cellular deletion and plays an important role in rapidly renewable tissue homeostasis such as endometrium (Tabibzadeh, 1995). This study employed an endometrial explant culture model to study the effect of buserelin on mRNA regulation of genes associated with endometrial apoptosis (Bax, Bcl-2, caspase-3 and Fas). This research confirmed the pro-apoptotic effect observed at mRNA level, by using the acridine-orange-ethidium-bromide staining technique on isolated endometrial epithelial cell monolayers from the follicular and the luteal phase endometrium. Isolated cells 115  were characterized in the experiments using immunofluoroscence and anticyokeratin primary antibody (Plate 4.1). The findings show that buserelin (200 ng/mL) up-regulated Bax, and that buserelin (200 and 500 ng/mL) up-regulated caspase-3 mRNA in the follicular phase endometrium. Similarly, GnR}I agonist induced apoptosis in epithelial cells obtained from the follicular phase endometrium. The antagonist (antide) and a combination of antide and buserelin could not produce an apoptotic effect indicating it to be a receptor mediated response. In the luteal phase endometrium similar treatments did not change mRNA levels of these genes. At the cellular level, buserelin (200 ng/mL) increased the percent apoptotic cells in the follicular phase endometrial epithelial cells. 2 family of genes includes pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) The BC! molecules and induce cell death through the mitochondrial pathway. The main function of the 2 family appears to be regulating the release of pro-apoptotic factors, particularly BC1 cytochrome C, from the mitochondria into the cytosol and to send apoptotic signals (Antonsson aid Martinou, 2000). The BC1 2 family members are located on the outer mitochondrial membrane, and can bind to each other in different pair-wise conditions. In most cases, the Bax: Bcl-2 ratio within a cell determines cell fate decisions (Antonsson et al., 1997). Fas is an important member of TNF-receptors, expressed on the cell surface, binds to Fas-L and a balance of expression between Fas and Fas-L predisposes the cells to undergo apoptosis through the death receptor pathway (Bilotas et al., 2007). Caspases are cytosolic proteases, and are synthesized as inactive precursors that are cleaved by other caspases, or autocatalytically cleave to become activated. An important function of caspases is to activate caspase-activated DNase (CAD), the endonuclease responsible for inter-nucleosomal DNA fragmentation; one of the most frequently used hallmarks of apoptosis. CAD and its inhibitory subunit, inhibitor of caspase-activated DNase (ICAD), are constantly expressed in the cells. Caspase-mediated 116  cleavage of the inhibitory subunit results in release and activation of the endonuclease (Yuang, 1997; Nagata, 1997). Both intrinsic and extrinsic cell death pathways converge to activate caspases to execute the process of programmed cell death (Riedi and Shi, 2004). GnRH induced apoptosis and its role in rat and human ovarian physiology is comparatively well elucidated. In vitro or in vivo GnRH treatment of rats promotes apoptosis in granulosa cells obtained from preovulatory follicles by inducing DNA fragmentation (Billing et al., 1994; Yano et al., 1997). Similarly in vitro buserelin treatment induces apoptosisin human granulosa luteal cells (Zhao et a!., 2000). Furthermore, GnRH induces.apoptosis in rat CL by up- regulating Bax gene expression with the possibility of involvement of the mitochondrial pathway (Papadopoulos et al., 1999).  In contrast, in the goldfish testes GnRH controls  spermatogenesis and induced apoptosis by increasing levels of Fas and Fas-L proteins (Andreu —  Vieyra et al., 2005). Most recently, it has been demonstrated in rat and human ovaries that  GnRH agonist treatment in vitro and in vivo stimulated ovarian apoptosis by activation of caspase-3. Caspase-3 activation was blocked by treatment with GnRH antagonist- antide or a combination of antagonist and agonist (Parborell et al., 2008; Hong et at., 2008), suggesting that this effect is mediated through GnRH-R. There are few studies designed to investigate the role of GnRH in uterine apoptosis. Recently, Bilotas et al. (2007) studied GnRH agonist and antagonist induced apoptosis in endometrial epithelial cells obtained from endometriosis and proliferative phase human endometrium. These researchers demonstrated that GnRH agonist and antagonist induced apoptosis in endometrial epithelial cells from endometriosis and proliferative phase human endometrium by up- regulating Bax and down-regulating Bcl-2 protein expression. They further substantiated these findings by studying apoptosis at cellular levels using the acridine-orange-ethidium-bromide staining technique. These researchers did not notice any increase in Fas and Fas-L protein levels in response to GnRH agonist or 117  antagonist treatment in the same experiments and suggested the involvement of mitochondrial pathway in GnRH induced endometrial apoptosis without ruling out the possibility of involvement of the death receptor pathway. In another study it was shown that GnRH agonist induced apoptosis in endometrial epithelial cells obtained from endometriotic objects and from the proliferative phase human endometrium (Meresman et a!., 2003) and concluded that it was a GnRH receptor mediated response, as GnRH antagonist blocked the apoptotic effect produced by agonist. In contrast, Imai et al. (1998) demonstrated that buserelin induced expression of Fas-L mRNA and Fas-L protein in ovarian and endometrial cancer cells and cell lines. Also, GnRH agonists can induce Fas expression in cells from uterine leomyomas (Wang et al., 2002). Interestingly the GnRH antagonist, cetrorelix, up-regulated Fas and Fas-L mRNA and protein expression in uterine leiomyoma and myometrial cells (Kwon et al., 2005). It is clear that there is no consensus whether GnRH agonists induce apoptosis through GnRH-R and there are also conflicting claims concerning the involvement of the mitochondrial (intrinsic) versus the death receptor pathways (extrinsic) in agonist induced cell death. The current study shows that the GnRH agonist, buserelin, induced endometrial apoptosis by up-regulating Bax and caspases-3 mRNA expression. It was also observed that Bcl-2 expression was not affected during the experiments, and that Fas mRNA did not change in the follicular phase endometrium. This suggests that the pathway involved in GnRH agonist induced endometrial apoptosis could be the mitochondrial pathway (Papadopoulos et al., 1999; Bilotas et a!. 2007). In the current experiments, the antagonist-antide did not induce apoptotic response which supports recent findings from endometrial and ovarian cells (Meresman et al., 2003; Parborell et al., 2008; Hong et al., 2008), demonstrating that GnRH induced apoptosis is a receptor mediated response. This study did not reveal similar apoptotic effects in the luteal phase endometrium as observed in the follicular phase. At this point there is no research 118  elucidating GnRH agonist induced apoptosis in the follicular and/or in the luteal phase endometrium separately. The reasons for this differential effect are not clear, but Okuda et al. (2004) have shown that progesterone is a suppressor of apoptosis in bovine luteal cells. Exposure of luteal phase endometrial tissue or cells to ovarian progesterone cannot be ruled out. It is possible that progesterone induced transcriptional and translational activity might have been initiated at the time of tissue collection. This could have countered the pro-apoptotic effect produced in the follicular endometrium at the molecular and cellular level. In this study the transcriptional up-regulation of Bax and caspase-3 mRNA in the in vitro endometrial explant cultures was observed after 6 h treatment. This could be considered a physiological response since the biological half life of the GnRH agonists and antagonists is between 4-8 h (Tarlatzis and Kolibianakis, 2007). When studying GnRH agonist induced apoptosis at the cellular level, treatments were withdrawn after 6 h and the cells were further grown for additional 24 h to allow translational changes in monolayers. Although there is no documented evidence as yet showing the presence of GnRH in the bovine uterus, this hormone is locally expressed in porcine, rat and human endometrial tissues (Li et a!., 1993; Ikeda et al., 1996; Dong et al., 1998; Raga et al., 1998) and in human, rat and bovine ovaries (Ramakrishnappa et al., 2003). The possibility that ovarian or uterine GnRH could act in an autocrine or paracrine manners in vivo cannot be ruled out and, needs further investigation. There is no consensus on the GnRH induced cell death pathways and the current study tends to support earlier evidence indicating the involvement of the mitochondrial pathway in GnRH agonist induced endometrial apoptosis (Bilotas et al., 2007). The results from this study do not eliminate the possibility of the involvement of the death receptor pathway. These are new findings in the field of reproductive biology and physiology elucidating a differential effect of GnRH agonist on endometrial apoptosis. These results indicate that GnRH treatment may 119  support the reproductive process at both the cellular and molecular levels in the bovine endometrium, which corroborates the previous reports that post AT GnRH agonist administration results in improved fertility in bovines (Rajamahendran et al., 2001, Peters, 2005). 4.5 CONCLUSIONS This study concludes that GnRH agonist, buserelin, differentially regulates apoptosis in the bovine endometrium with a pro-apoptotic effect in the follicular phase endometrium. GnRH agonist, buserelin, treatment up-regulated Bax (200 ng/ mL) and caspase-3 mRNA (200 and 500 ng/mL) without affecting Bcl-2 and Fas mRNA in the follicular phase endometrium. GnRH agonist treatment resulted in similar effects at the cellular level and induced endometrial epithelial cell apoptosis. Furthermore, this study indicates that GnRH induces apoptosis in bovine endometrium through the mitochondrial cell death pathway.  120  Table 4.! Primer sequence, PCR cycles used and the product size obtained for the genes of interest during the RT-PCR amplifications in the experiments. Gene  Primer sequence  PCR Cycles  Product size (bp)  34  223  37  156  34  278  34  206  33  318  .  Bax  F 5’- TGC TTC AGG GTT TCA TCC AG-3’ R 5’- AAC ATT TCA GCC GCC ACT C-3’  Bcl-2  F 5’- TTC GCC GAG ATG TCC AGT CAG C-3’ R5’- GTT GAC GCT CTC CAC ACA CA-3’  caspase-3  F 5’- AGC AAA CCT CAG GGAAAC CT-Y R5’- GGC AGG CCT GAA TAA TGA AA-Y  Fas  F 5’- ATG GGC TAG AAG TGG AAC AAA AC-Y R5’- CAG GAG GGC CCA TAA ACT GTT TGC-3’  G3PDH  F 5’- TGT TCC AGT ATA GAT TCC ACC -3’ R 5’- AGG AGG CAT TGC TGA CAA TC -3’  121  Plate 4.1 A representative microphotograph showing characterization of primary monolayers  as endometrial epithelial cells. The cells were isolated from follicular or luteal phase endometrium and stained and using immunofluoroscence. Cells showing clear labelling against cytokeratin when immunostained using anticyokeratin primary and FITC labelled secondary antibody and were viewed under 400 X magnification using an excitation filter of 450-490 nm and a barrier filter of 520 nm wavelength.  Plate 4.2 Endometrial epithelial cells subjected to the acridine-orange-ethidium-brornide  staining procedure and viewed under 400 X magnification with a fluorescent microscope using an excitation filter of 450-490 nm and a barrier filter of 520 nm wavelength. Apoptotic cell nuclei stained orange and live cell nuclei stained green. 122  G3PDH Bax 1  S A  —  —  0.5 .—  —i  Ip  B200  B  B500  B1000  A500  A500+B200  A500  A500+B200  Treatment  G3PDH Bax I  —  —  .  BO  B200  B500  B1000  Trea fluent Figure 4.1 GnRH agonist, buserelin (0, 200, 500, and 1000 ng/mL respectively), antagonist, antide- 500 ng/mL(A500) and a combination of antide  +  buserelin- 500+200 ng/mL  (A500+B200) induced regulation of Bax mRNA in the follicular phase endometrium (A) and the luteal phase endometrium (B). The asterisk (*) shows statistically significant values (P 0.05) when compared to control (BO). Error mars in the histograms are standard error on mean (SEM).  123  A 1  2  —  0.5  0 BO  B200  B500  Bi000  MOO  A500+B200  Tri trnent  B 1  •  0.5  —  0 B1)  B200  B500  I  MOO  B1000  A500+B200  Treitment Figure 4.2 GnRH agonist, buserelin (BO-control, 200, 500, and 1000 ng/mL, respectively), antagonist antide- 500 ng/mL (A500) and a combination of antide  +  buserelin- 5 00+200 ng/mL  (A500+B200) induced regulation of Bcl-2 mRNA in the follicular phase endometrium (A) and in the luteal phase endometrium (B). GnRH agonist treatment did not alter Bcl-2 mRNA expressions either in follicular or luteal phase endometrium. Error bars in the histograms are standard error on mean (SEM).  124  zzr  A 1  h  0,5  — —  =  0  I  B200  B500  B1000  A500A500+B200  Treatment  B 1  0.5  zc  0 BO  B200  I  B500  B1000  A500  A500+B200  Treatnient  Figure 4.3 GnRH agonist, buserelin (BO-control, 200, 500, and 1000 ng/mL, respectively),  antagonist- antide (A-500 ng/mL) and a combination of antide  +  buserelin- 500+200 ng/mL  (A+B) induced regulation of caspase-3 mRNA in the follicular phase endometrium (A) and in the luteal phase endometrium (B). The asterisk (*) shows statistically significant values  (P  0.05) when compared to control (BO). Error bars in the histogram are standard error on mean (SEM)  125  1-  -I -  1 —  BO  B200  B500  B1000  A500  A500+B200  Treatment  Figure 4.4 GnRH agonist (buserelin), induced regulation of Fas mRNA in follicular phase endometrium buserelin when treated with control (BO), and 200, 500 and 1000 ng/mL of buserelin (B200, B500, and B1000, respectively), antagonist antide- 500 ng/mL (A500) and a combination of antide + buserelin- 5 00+200 ng/mL (A500+B200). Error bars in the histogram are standard error on mean (SEM).  126  60 *  T  50  • Follicular Luteil  I  40.  c-)  Ti  30  I  0  10  0 BO  B200  iii  .4500+B200  Treatment Figure 4.5 GnRH agonist, buserelin-200 ng/mL (B200) induced apoptosis in the follicular phase endometrial epithelial cells. GnRH antagonist, antide- 500 ng/ mL (A500) and a combination of antide-500 ng/mL  +  buserelin- 200 ng/mL (A500+B200) did not induce  apoptosis when compared to vehicle treated control (BO). The value with asterisk (*) shows statistically significant value (P  0.05) when compared to control (BO). Error bars in the  histogram are standard error on mean (SEM).  127  4.6 BIBLIOGRAPHY Andreu-Vieyra CV, Buret AG, Habibi HR. 2005. Gonadotropin-releasing hormone induction of apoptosis in the Testes of Goldfish ( Carassius auratus). Endocrinology. 146: 1588-1596. Antonsson B, Conti F, Ciavatta AM, Montessuit S, Lewis S, Martinou L, Bernasconi L, Bernard A, Mermod JJ, Mazzei G, Kinsey Maundrell K, Gambale F, Sadoul R, Martinou JC. 1997. Inhibition of Bax channel-forming activity by Bcl-2. Science. 277: 370372. Antonsson B, Martinou JC. 2000. The Bcl-2 protein family. Exp. Cell Res. 256: 50-57. Billing H, Furuta I, Hsueh AJW. 1994. Gonadotropin-Releasing Hormone Directly Induces Cell Death in the Rat Ovary: Biochemicl and in situ Detection of Deoxyribonucleic Acid Fragmentation in Granulosa Cells. Endocrinology. 134: 245-252. Bilotas M, Baraflo RI, Buquet R, Sueldo C, Tesone M, Meresmas G. 2007. Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls. Hum. Reprod. 22: 644653. Bogacki M, Silvia J, Rekawiecki R, Kotwica J. 2002. Direct inhibitory effect of progesterone on oxytocin-induced secretion of prostaglandin F2a from Bovine Endometrial Tissue. Biol. Reprod. 67: 184-188. Borroni R, Blasio AMD, Gaffuri B, Santorsola R, Busaca M, Vigano P, Vignali M. 2000. Expression of GnRI-I receptor gene in human ectopic endometrial cells and inhibition of their proliferation by leuprolide acetate. Molecular and Cellular Endocrinology. 159: 37-43. Catalano RD, Yanaihara A, Evans AL, Rocha D, Prentice A, Print CG, Charnock-Jones DS, Sharkey AM, Smith SK. 2003.The effect of RU 486 on the gene expression profile in an endometrial expalnt model. Mol. Hum. Reprod. 9: 465-473. Cheng CK, Leung PCK. 2005. Molecular biology of gonadotropin-releasing hormone (GnRH)-l and GnRH-l 1 and their receptors in humans. Endocr. Rev. 26: 283 306. -  Cheon KW, Lee HS, Parhar IS, Kang IS. 2001. Expression of the second isoform of gonadotropin- releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol. Hum. Reprod. 7: 447-452. Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem 162: 156-159. Couse JF, Hewitt SC, Korach KS. 2006. Steroid receptors in the ovary and uterus. In: Jimmy D Neil! edited knobi! and Neill’s physiology of reproduction volume 1 (Third Edition) Elsievier Academic Press 1: 5 93-677. 128  Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, Roberts JL. 1998. Expression of gonadotropin-releasing hormone (GnRH) gene in human endometrial tissue. Mo!. Hum.Reprod. 4: 893-898. Elmore S. 2007. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 35: 495-5 16. Giritharan G, Ramakrishnappa N, AaIi M, Madan I?, Balendran A, Singh R, and Rajamahendran R. 2007. Paternal influence on apoptosis, and expression of Bc12, Bax, p53, heat shock protein 70 and interferon tau genes in bovine perimplantation embryos Can. J. Ani. Sci. 87: 157-165. -  Grundker G, Gunhert AR, Milla, RP, Emons G. 2002. Expression of gonadotropin-releasing hormone II (GnRJ-1-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. J. Clin. Endocr. Metab. 87: 1427-1430. Harda T, Kaponis A, Iwabe T, Taniguchi F, Makrydimas G, Sofikitis N, Paschopoulos M, Parskevaidis E, Terakawa N. 2004. Apoptosis in human endometrium and endometriosis. Hum. Reprod. Update. 10: 29-38. Hong IS, Chung AP, Leung PCK. 2008. Gonadotropin-releasing hormone I and II induce apoptosis in human granulosa cells. J. Clin. Endocr. Metab. 93: 3 179-3185. Ikeda M, Taga M, Sakakibara H, Minaguchi H, Ginsburg E, Vonderhaar BK. 1996. Gene expression of gonadotropin-releasing hormone in early pregnant rat and steroid hormone exposed mouse uteri. J. Endocr. Invest. 19: 708-713. Imai A, Ohno T, lida K, Fuseya T, Furui T, Tamaya, T. 1994. Presence of gonadotropin releasing hormone receptor and its messenger ribonucleic acid in endometrial carcinoma and endometrium. Gynecol. Oncol. 55: 144-148. Imai A, Takagi A, Horibe S, Takagi H, Tamaya T. 1998. Evidence for tight coupling of gaonadotropin-releasing hormone receptor to stimulated Fas ligand expression in reproductive tract tumors: possible mechanisms for hormonal control of apoptotic cell death. J. Clin. Endocr. Metab. 83: 427-43 1. Irmer G, Burger C, Ortman 0, Schulz KD, Emons G. 1994. Expression of leutinizing hormone releasing hormone and its mRNA in human endometrial cancer lines. J. Clin. Endocr. Metab. 79: 9 16-919. Ireland JJ, Murphee RL, Colulson PB. 1980. Accuracy or predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. J. Dairy Sci. 63:155-160. Kwon, JY, Park, KH, Park NY, Cho NH. 2005. Effect of cetrorelix acetate on apoptosis and apoptosis regulatory factors in cultured uterine leiomyoma cells. Fertil. Sterli. 84: 1526-1528. Li W, Jiao S, Chi PP. 1993. Immunoreactive gonadotropin-releasing hormone in porcine reproductive tissues. Peptide. 14: 543-549. 129  Meresman CF, Bilotas MA, Lombardi E, Tesone CS, Rosa lB. 2003. Effect of GnRH analogues on apoptosis and release of interleukin- 1 f3 and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum. Reprod. 18: 1767-1771. Nagata S. 1997. Apoptosis by death factor. Cell. 88: 355-365. Okuda K, Korzekwa A, Shibaya M, Murakami S, Nishimura R, Tsubouchi M, Woclawek Potocka I, Skarzynski DJ. 2004. Progesterone is a supressor of apoptosis in bovine luteal cells. Biol. Reprod. 71: 2065-2071. Papadopoulos V, Dharmrajam AM, Li H, Culty M, Lemay M, Sridaran R. 1999. Mitochondrial peripheral-type benzodiazepine recepror expression. Correlation with gonadotropin-releasing hormone (GnRH) agonist-induced apoptosis in the corpus luteum. Biochem. Pharmacol. 58: 1389-1393. Parborell F, Irusta G, Celin AR, Tesone M. 2008. Regulation of ovarian angiogenesis and apoptosis by GnRI-I-1 analogues. Mol. Reprod. Dev.75: 623-63 1. Peters AR. 2005. Veterinary clinical application of GnRH-questions of efficacy. Anim. Reprod. Sci. 88: 155-167. Raga F, Casan EM, Wen Y, Huang H, Nezhat C, Polan ML. 1998. Quantitative gonadotropin- releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol. Reprod. 59: 661-669. Rajamahendran R, Ambrose DJ, Small JA, Dinn N. 2001. Synchronization of estrus and ovulation in cattle. Arch. Animal. Breed. 44 (special issue): 58-67. Ramakrishnappa, 2004. A study on GnRH-R and GnRH mRNA expression, direct effects of GnRH-a in bovine ovary and influence of post breeding GnRH administration on corpus luteum function and pregnancy in dairy cattle. Thesis submitted to University of British Columbia, Canada. Ramakrishnappa N, Rajamahendran R, Lin YM, Leung PCK. 2005. GnR}I in nonhypothalamic reproductive tissues. Anim. Reprod. Sci. 88: 95-1 13. Ramakrishnappa N, Giritharan G, Aali M, Madan P, Rajamahendran R. 2003. GnRH receptor messenger ribonucleic acid expression in bovine ovary. Can J Ani Sci 83: 823-826. Riedi SJ, Shi V. 2004. Molecular mechanism of caspase regulation during apoptosis. Mol. Cell. Biol. (Nature Reviews). 5: 897-907. Singh R, Graves ML, Roskelley CD, Giritharan G, Rajamahendran R. 2008. Gonadotropin releasing hormone receptor gene and protein expression and immunohistochemical localization in bovine uterus and oviducts. Domes. Anim. Endocrinol. 34: 319-326. Tang X, Yano T,Osuga Y, Matsumi H, Yano N, Xu J, Wada 0, Koga K, Kugu K, Tsutsumi 0, Schally AV, Taketani V. 2002. Cellular mechanisms of growth inhibition of 130  human epithelial ovarian cancer cell line by LH-releasing hormone antagonist cetrorelix. Clin. Endocr.Metb. 87: 3721-3727. Tabibzadeh S. 1995. Signals and molecular pathways involved in apoptosis, with special emphasis on human endometrium. Hum.Reprod. Update. 1: 303-323. Tarlatzis BC, Kolibianakis E.M. 2007. GnRH agonists vs antagonists. Best pract. Clin. Obstet. Gynaecol. 21: 57-65. Wang Y, Matsuo H, Kurachi 0, Maruo T. 2002. Down-regulation of proliferation and upregulation of apoptosis by gonadotropin-releasing hormone agonist in cultured uterine leiomyoma cells. Eur. J. Endocr. 146: 447-456. Yang MY, Rajamahendran R. 2000. Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells. Biol. Reprod. 62: 1209-1217. Yano T, Yano N, Matsumi H, Morita Y, Tsutsumi 0, schally AV, Taketani Y. 1997. Effect of letinizing hormone releasing hormone analogues on the rat ovarian follicle development. Horm. Res. 48: 35-41. -  Yuang J. 1997. Transducing signals of life and death. Current Opinions in Cell Biology. 9: 247-251. Zhao 5, Saito H, Wang X, Saito T, Kaneko T, Hiroi M. 2000. Effects of gonadotropin easing hormone agonist on the incidence of apoptosis in porcine and human granulose cells. Gynecol. Obstet. Invest. 49: 52-56.  131  CHAPTER 5- GENERAL DISCUSSION AND CONCLUSIONS It is well established that gonadotropin-releasing hormone (GnRH) acts through the hypothalamo-pituitary- gonadal axis and plays a central role in mammalian reproduction (Fink, 1988). This hormone binds to GnRH-R on pituitary gonadotrophs and controls synthesis and release of LH and FSH. Detection of the GnRH, GnRH-R system in extrapituitary reproductive tissues is a new development in mammalian reproductive biology (Ramakrishnappa et a!., 2005). Extrapituitary GnRH-R are present in normal and neoplastic reproductive tissues, including ovary, testes, endometrium, placenta, prostate, preimplantation embryo, oocytes and sperm, across mammalian species ranging from rodents to humans. In these tissues, GnRH acts as a local modulator of different biological processes including gonadal steroidogenesis, cellular proliferation, apoptosis, fertilization, early embryonic development and implantation (Janssens et al., 2000; Lee et al., 2000, van Bilijon et al., 2002; Cheng and Leung, 2005; Nam et al., 2005). Immunoreactive GnRH and GnRH mRNA has been shown to be present in porcine, rat and human endometrium (Li et al., 1993; Ikeda et al., 1996; Dong et al., 1998; Raga et al., 1998) and oviducts (Casan et al., 2000). On the other hand, GnRH-R mRNA has been detected in the normal and the carcinogenic human endometrium (Imai et al., 1994; Borroni et al., 2000). The extrapituitary presence of the GnRH, GnRH-R system is an indication that it may play a local modulatory role in peripheral reproductive tissues (Harrison et a!., 2004). In bovines, Ramakrishnappa et al. (2003) detected GnRH-R mRNA in different stages of ovarian follicles and CL. GnRH is extensively used in bovine reproductive management (Rajamahendran et a!., 2001; Lewis et al., 2001; Ambrose et a!., 2005; Peters, 2005) and is believed to act through the hypothalamo-pituitary-gonadal axis. The extrapituitary reproductive tissues including the uterus and oviducts could also be the possible targets for the exogenous, ovarian, uterine, embryonic and/or placental GnRH. But, the presence of GnRET-R 132  and the local modulatory role of the GnRI-1, GnRH-R system in the bovine uterus and oviducts was still to be established. 5.1 GnRH-R IN ENDOMETRIUM AND OVIDUCTS The current study demonstrated the presence of GnRH-R in both the follicular and luteal phase bovine endometrium and oviducts. The receptors were further localized to endometrial and oviductal epithelial cells by immunohistochemistry.  In the human  endometrium, GnRH-R transcripts were previously demonstrated using RT-PCR and southern hybridization (Imai et al., 1994; Borroni et al., 2000). These findings at the transcriptional levels are still to be substantiated at the protein level. The non-availability of GnRH-R specific antibodies 10 years ago or lack of appropriate experimental techniques could be cited as possible reasons for these researchers not being able to detect GnRI-1-R at the protein level. Immediately after the current research reported GnRH-R in bovine uterus and oviducts, Sengupta and Sridaran (2008) used real time PCR and western blotting, respectively and detected GnRH-R in rat oviducts at both the mRNA and protein levels. This group further observed intense staining in epithelial cells which inferred that GnRH-R are confined to oviductal epithelial cells. Interestingly, Sengupta and Sridaran (2008) used similar experimental approaches and the same monoclonal primary antibody targeting GnRH-R, as used in the current study. The presence of GnRH-R in normal endometrium and oviducts is intriguing and indicates that a functional GnRH, GnRH-R system might exist in these tissues. The endometrium is a dynamic structure and one of the maj or target sites for the action of ovarian steroids, estrogen and P 4 to regulate uterine cyclicity (Couse et al., 2006). The oviduct is another important reproductive structure and responsible for ovum transport, fertilization and early embryonic development (Casan et al., 200). Considering that the endometrium is involved with a variety of reproductive processes, further studies were carried out to investigate the local 133  modulatory role of the GnRH, GnRH-R system in the uterus by studying the GnRH agonist, buserelin induced regulation of steroid hormone receptors and apoptosis in both the follicular and luteal phase bovine endometrium. 5.2 GnRH INDUCED ERa REGULATION The ovarian steroids estrogen and P , mediate their actions through estrogen receptors 4 (ERc, ERI3) and progesterone receptor (PR), respectively. The co-existence of GnRH-R and steroid hormone receptor systems at the endometrial level was interesting. It was decided to investigate GnRH agonist (buserelin) induced regulation of steroid hormone receptors (ERct, ERI3 and PR) at the mRNA level in both the follicular and luteal phase bovine endometrium. The results showed that GnRH agonist, buserelin could up-regulate ERcL mRNA expression in the luteal phase endometrium. ERci is the predominant estrogen receptor in the mammalian uterus (Moutsatsou and Sekeris, 1997). The presence of ER was elucidated earlier in the uterine compartments across various species, ranging from mice to humans (Couse et al., 2006). Their presence was also demonstrated in bovine ovaries, endometrium and oviducts (Walther et al. 1999; Meikle et al. 2001; Ulbrich et al. 2003). Estrogen and P 4 maintain uterine physiology by regulating endometrial proliferation, gamete transport, uterine receptivity, embryonic development, implantation, decidualization, and maintenance of pregnancy (Graham and Clark, 1997; Ulbrich et al., 2003; Vasudeven and Plaff, 2007). Classically, after binding to G-protein coupled receptors (GPCR) on pituitary gonadotrophs, GnRH induces the Ca and protein kinase C (PKC) mediated cell signalling pathways. However, after binding to its receptors this hormone could also activate alternative cell signalling pathways, including protein kinase A (PKA) and cyclic adenosinemonophosphate (cAMP) in extrapituitary reproductive tissues (Chiang et al., 2000; Cheng and Leung, 2005). GnRH can down regulate ERci and ERf3 in human granulosa luteal cells in vitro (Chiang et al., 2000) through the PKC cell signalling 134  pathway, but up- regulate ERcL and PR in pituitary cells by activating the PKC and PKA cell signalling pathways (Demay et al., 2001; Tasende et al., 2002). The ERUL is up-regulated prior to implantation, making the endometrium more responsive to estrogen actions. There are implantation failures in ERci knockout mice (Paria et a!., 2002; Walker and Korach, 2004). Thus, ERa up-regulation seems to corroborate evidence that the mid luteal phase GnRH agonist, administration results in increased pregnancy rates in bovines (Peters, 2005). There are a few conflicting reports showing the effect of GnRH agonists on steroid hormone receptor regulation in the pituitary and ovaries. In pituitary gonadotrophs, GnRH treatment in vitro for 8 h activated ERa (Demay et at., 1996; 2001). In contrast, GnRH downregulated ERa and ERI3 genes in human granulosa luteal cells during 24 h in vitro treatment (Chiang et al., 2000). In ewes, long term in vivo treatment with GnRI-1 agonist up-regulated ERa and PR in the pituitary gland (Tasende et al., 2002) and in the same experiments receptor levels remained unchanged in the uterus. Most recently, Chen et a!. (2009) demonstrated that GnRH treatment of pituitary cells resulted in GnRH-R mediated phosphorylation of ERa during 6-12 h of the treatment period. In the same study GnRH antagonist-antide did not result in the similar activation, and ERa phosphorylation was blocked by treating pituitary cells with SiRNA targeted against GnRH-R. These results clearly showed that GnRH agonist could activate ERa and mediate this action through GnRH-R. The current study also appears to support observations by Chen et al. (2009) with pituitary cells showing that buserelin up regulated ERa mRNA and further indicated it to be a receptor mediated response. The findings from this research are in agreement with both the in vivo and in vitro reports from pituitary gonadotrophs, but do not support the evidence from human granulosa luteal cells that GnRH down-regulates ERa and ERI3 (Chiang et al., 2000) and the possible explanation for it could be the prolonged exposure of granulosa luteal cells to GnRH. The molecular transcriptional 135  mechanisms and cell signalling pathways resulting in this up regulation of ERa in bovine endometrium are yet to be defined and would need future research attention. There are limited reports studying GnRH-R and steroid hormone receptor interactions and on the basis of the available evidence, it would be too early to draw conclusions on GnR}I induced regulation of steroid hormone receptors (ER and PR) in extrapituitary reproductive tissues. 5.3 GnRH AND APOPTOSIS Apoptosis is a vital biological process maintaining tissue homeostasis and is of greater significance in rapidly renewable tissues like the endometrium (Harda et al., 2004). Therefore, additional studies reported in this dissertation were focused on the role of GnRH agonist, buserelin, in modulating endornetrial apoptosis (chapter 4). This study employed an in vitro endometrial explant culture model to elucidate buserelin induced apoptosis at the mRNA level. The findings at the mRNA level were further substantiated at the cellular level by using endometrial epithelial cell monolayer cultures. The acridine-orange-ethidium-bromide staining technique was used to identify cells undergoing an apoptotic form of death. In this study buserelin differentially regulated apoptosis at both the mRNA and cellular levels with a pro apoptotic role in follicular phase endometrium. An up-regulation of Bax mRNA was observed when endometrial explants were incubated with 200 ng/mL buserelin and the up-regulation of caspase-3 mRNA was observed with 200 and 500 ng/mL of buserelin in the follicular phase endometrium. The Bax and caspase3 mRNA levels were not changed with similar treatments in the luteal phase endometrium. The Bcl-2 mRNA expression was not affected in either the follicular or the luteal phase endometrium. The response observed at the mRNA level was further confirmed at the cellular level in which buserelin induced apoptosis in the follicular phase endometrial epithelial cells. The cells isolated from the follicular and the luteal phase endometrium were characterized as epithelial cells after 48 h in vitro culture by using 136  anticyokeratin monoclonal antibody and immunofluoroscence. GnRH antagonist, antide, or a combination of antagonist and agonist did not result in an apoptotic response, indicating it to be a GnRH-R mediated phenomenon. Furthermore, it was observed that Fas mRNA levels were not changed in the same in vitro treatment model in the follicular phase endometrium, suggesting the involvement of the mitochondrial cell death pathway (intrinsic), which is governed by Bax and Bcl-2 genes. The current findings are in agreement with previous reports showing that GnRH up-regulates apoptosis in rat and human ovaries (Billing et al., 1994; Parborell et al., 2008; Hong et al., 2008) through the up-regulation of Bax and caspase-3 genes. In the uterus, GnRH agonist induces apoptosis in the proliferative phase endometrium and in endometriotic epithelial cells through the up-regulation of Bax protein (Meresman et al., 2003; Bilotas et al., 2007). These researchers further suggested the involvement of the mitochondrial cell death pathway in GnRH agonist induced apoptosis in endometriosis. In the current study, buserelin induced up-regulation of Bax and caspase-3 mRNA and supported the previous evidence gained from ovarian and endometrial cells at the cellular level (Meresman et al., 2003; Bilotas et al., 2007, Paraborell et al., 2008, Hong et al., 2008). In contrast, Imai et al. (1998) demonstrated that buserelin induced expression of Fas-L mRNA and Fas-L protein in ovarian and endometrial cancer cells and cell lines. Also, GnRH agonists can induce Fas expression in cells from uterine leomyomas (Wang et al., 2002). The GnRH antagonist, cetrorelix up-. regulated Fas and Fas-L mRNA and protein expression in uterine leiomyoma and myometrial cells (Kwon et al., 2005), ovarian carcinomas (Tang et al., 2002), testes (Andreu-Vieyra et al., 2005), prostate and prostate cancer (Kwon et a!. 2005). Majority of the studies conducted with GnRH agonists and antagonists in reproductive tissue cancers and testes suggest that GnRH agonists/antagonists induced up-regulation of Fas and Fas ligand (Tang et a!., 2002; Kwon et a!., 2005; Andreu  —  Vieyra et al., 2005). On the basis of the present study, the possibility of the 137  involvement of death receptor pathway governed by Fas and Fas-L genes in bovine endometrial epithelial apoptosis cannot be eliminated. The available evidence is not clear on the involvement of GnR}1 agonist or antagonists in the apoptotic response observed in the uterus. The role of GnRH-R in mediating apoptosis in the human endometrium is also not conclusive. There is also no consensus with regard to the involvement of a specific cell death pathway in GnRH induced apoptosis in extrapituitary reproductive tissues including ovaries, testes, prostate and uterus. It appears that GnRH induced apoptosis in reproductive tissue carcinomas through up-regulation of Fas and Fas-L genes, but involves Bax, Bcl-2 in the ovarian and uterine tissue. It is also possible that GnRH antagonists induce apoptosis in uterine leomyomas through different binding sites on GnRH-R. 5.4 DISSERTATION STRENGTHS This dissertation advances the current knowledge in mammalian reproductive biology by demonstrating GnRH-R in the follicular and the luteal phase bovine endometrium and oviducts at both the mRNA and protein levels. The current research provides initial evidence for the presence of GnRH-R at the protein level in the uterus and oviducts, and localizes these receptors to the endometrial and oviductal epithelial cells. The evidence provided in the dissertation that GnRH up-regulates ERcL mRNA in the luteal phase bovine endometrium provides new insights that GnRH and steroid hormone receptor systems could interact locally at the uterine level. Also the pro-apoptotic role of GnRH, GnRH-R system in the follicular phase endometrium indicates that this system might have an important role in maintaining the endometrial cyclicity. Overall, this dissertation for the first time demonstrated the local modulatory role of the GnRH, GnRH-R system in the bovine uterine physiology. In the current research, GnRI-1 induced ERc up-regulation in the luteal phase endometrium and a pro apoptotic effect in the follicular phase bovine endometrium. These observations are indicative 138  of GnRH’s role in supporting reproductive process by preparing the endometrium for estrogenic actions in the luteal phase and eliminating unwanted cells in the follicular phase endometrium. The absence of a pro-apoptotic response in the mid luteal phase endometrium is suggestive that GnRH might have a role in the establishment of pregnancy in bovines since the luteal phase is characterized by early embryonic development, uterine receptively and implantation. 5.5 DISSERTATION LIMITATIONS  Though this study demonstrated GnRH-R in the follicular and the luteal phase bovine endometrium but could not quantify the receptors in these tissues. This research does not prove conclusively that buserelin induced responses (ERcL regulation and apoptosis) were GnRH-R mediated. The transcriptional regulation or ERo and apoptotic genes (Bax, Caspase) mRNA could not be verified at the protein level. Also it would have been more appropriate to measure the caspase-3 concentrations in response to GnRH agonist or antagonist treatments. There were variations in data on account of age, stage of estrous cycle, experimental and culture conditions employed during these experiments. 5.6 FUTURE DIRECTIONS  This study conclusively demonstrates the presence of GnRH-R in bovine endometrium and oviducts. It is one of the initial studies undertaken to investigate the local modulatory role of the GnRH, GnRH-R system in uterine physiology. In future, it would be of considerable interest to conduct in vivo experiments using the estrous synchronized cows to confirm the current findings. The role of the GnRH, GnRH-R system in oviducts needs to be further investigated, particularly since bovine embryos spend considerable time in the oviducts before their migration into the uterus. Future studies could be planned to study the local modulatory role of this hormonal system in sperm capacitation, gamete transport, fertilization and early 139  embryonic development. The presence of the GnRH, GnRH-R system in endometrium, early embryonic developmental stages and placenta is inspiring enough to specifically study the local modulatory role of GnRH, GnRH-R system in the uterine receptivity and implantation. It would also be interesting to use GnRH administration in the window of uterine receptivity and implantation to find out if this ERa up-regulation could increase pregnancy rates in dairy cows. The local presence of GnRH in bovine uterus, oviducts, embryos and placenta is still to be tested. To conclusively demonstrate that GnRH induced biological actions are mediated through its receptor, the experimental approaches using SiRNA and targeting GnRH-R need to be performed. This would lead to understanding the role and usage of GnRH agonists and antagonists in bovine reproductive management. 5.7 CONCLUSIONS This study demonstrates GnRH-R at both the mRNA and protein levels in the follicular and luteal phase bovine endometrium and oviducts. These receptors are localized in the endometrial and oviductal epithelial cells. This study further showed that GnRI-I agonist, buserelin (200 ng/mL) up-regulates ERa in the luteal phase endometrium. GnRH agonist does not alter ERj3 or PR expression in either the follicular or the luteal phase endometrium. The research reported in this dissertation also confirms that ERa expressions are higher in the follicular phase endometrium as compared to luteal phase endometrium as reported in other species. Furthermore this study provides evidence that GnRH agonist, buserelin differentially regulates endometrial apoptosis in bovines with a pro-apoptotic effect in the follicular phase endometrium. Buserelin up-regulates Bax and caspase-3 mRNA in the follicular endometrium as compared to the luteal endometrium, with no changes in Bcl-2 mRNA expressions. Buserelin induces apoptosis at the cellular level in the follicular endometrial epithelial cells. GnRH agonist treatment in vitro did not alter Fas mRNA expression in the follicular 140  endometrium indicating that GnRJ-1 agonist, buserelin, induces apoptosis in bovine endometrium through the mitochondrial (intrinsic) cell death pathway. These findings from this dissertation are new contributions to the field of mammalian reproductive biology and have elucidated the local modulatory role of GnRH, GnRH-R system in uterine physiology.  141  5.7 BIBLIOGRAPHY Andreu-Vieyra CV, Buret AG, Habibi HR. 2005. Gonadotropin-Releasing Hormone Induction of Apoptosis in the Testes of Goldfish (Carassius auratus). Endocrinology. 146: 1588-1596. Ambrose DJ, Kastelic JP, Rajamahendran R, Aali M, Dinn N. 2005. Progesterone (CIDR) based timed AT protocols using GnRH, porcine LH or estradiol cypionate for dairy heifers: ovarian and endocrine responses and pregnancy rates. Theriogenology. 64: 1457-1474 Billing H, Furuta I, Hsueh AJW. 1994. Gonadotropin-Releasing Hormone Directly Induces Cell Death in the Rat Ovary: Biochemicl and in situ Detection of Deoxyribonucleic Acid Fragmentation in Granulosa Cells. Endocrinology. 134: 245-252. Bilotas M, Baraflo RI, Buquet R, Sueldo C, Tesone M, Meresmas G. 2007. Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls. Hum. Reprod. 22: 644653. Borroni R, Blasio AMD, Gaffari B, Santorsola R, Busacca M, Vigano P, Viganali M. 2000. Expression of GnRH receptor gene in human ectopic endometrial cells and inhibition of their proliferation by leuprolide acetate. Mol. Cell. Endocr. 159: 37-43. Casan EM, Raga F, Bonilia-musoles F, Polan ML. 2000. Human oviductal gonadotropin releasing hormone: Possible implications in fertilization, early embryonic development, and implantation. J. Clin. Endocr. Metab. 85: 1377-1381. Chen J, An BS, Cheng L, Hammond GL, Leung PC. 2009. Gonadotropin releasing hormone-mediated phosphorylation of estrogen receptor contributes to fosB expression in mouse gonadotrophs. Endocrinology. In Press. Cheng KY, Nathwani PS, Leung PCK. 2000. Regulation of human gonadotropin-releasing hormone receptor gene expression in placental cells. Endocrinology. 141: 2340-2349. Cheng CK, Leung PCK. 2005. Molecular biology of gonadotropin-releasing hormone (GnRH)-1 and GnRH-1 1 and their receptors in humans. Endocr. Rev. 26: 283-306. Chiang CH, Cheng KWA, Igarashi S, Nathwani PS, Leung PCK. 2000. Hormonal regulation of estrogen receptor UL and 13 gene expression in human granulosa-luteal cells in Vitro. J. Clin. Endocr. Metab. 85: 3828-3839. Couse JF, Hewitt SC, Korach KS. 2006. Steroid Receptors in the Ovary and Uterus. In Jimmy D Neil! edited knobil and Neill’s physiology of reproduction volume 1 (Third Edition) Elsievier Academic Press. 593-677. Demay F, Be Monto M, Tiffoche C, Vaillant C, Thieulant M. 2001. Steroid- independent activation of ER by GnRH in gonadotrope pituitary cells. Endocrinology. 142: 3340-3347. 142  Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, Roberts JL. 1998. Expression of gonadotropin- releasing hormone (GnRH) gene in human uterine endometrial tissue. Mol. Hum. Reprod. 4: 893-898. Fink G. 1988. Gonadotropin secretion and its control. In: Knobil E, Neill J.D. eds. The physiology of reproduction. New York: Raven Press. 1349-1377. Harda T, Kaponis A, Iwabe T, Taniguchi F, Makrydimas G, Sofikitis N, Paschopoulos M, Parskevaidis E, Terakawa N. 2004. Apoptosis in human endometrium and endometriosis. Hum. Reprod. Update. 10: 29-38. Harrison GS, Wierman ME, Nett TM, Glode LM. 2004. Gonadotropin-releasing hormone and its receptor in normal and malignant cells. Endocrine Related Cancer .11: 725-748. Hong IS, Chung AP, Leung PCK. 2008. Gonadotropin-releasing hormone I and II induce apoptosis in human granulosa cells. 2008. J. Clin. Endocr. Metab. 93: 3179-3185. Ikeda M, Taga M, Sakakibara H, Minaguchi H, Ginsburg E, Vonderhaar BK. 1996. Gene expression of gonadotropin-releasing hormone in early pregnant rat and steroid hormone exposed mouse uteri. J. Endocr. Invest. 19: 708-7 13. Imai A, Ohno T, lida K, Fuseya T, Furui T, Tamaya T. 1994. Presence of gonadotropin releasing hormone receptor and its messanger ribonucleic acid in endometrial carcinoma and endometrium. Gynecol. Oncol. 55: 144-148.  —  Janssens RMJ, Brus L, Cahill DJ, Huirne JA, Schoemaker J, Lambalk CB. 2000. Direct ovarian effects and safety aspects of GnRH agonists and antagonists. Hum. Reprod. Update. 6: 505-518. Kwon JY, Park KH, Park NY, Cho NH. 2005. Effect of cetrorelix acetate on apoptosis and apoptosis regulatory factors in cultured uterine leiomyoma cells. Fertil. Sterli. 84: 1526-1528. Lee CY, Ho J, Chow SN, Yasojima K, Schwab C, McGeer PL. 2000. Immunoidentification of gonadotropin-releasing hormone receptor in human sperm, pituitary and cancer cells. Am. J .Reprod. Immunol. 44: 170-177. Lewis GS, Caidwell DW, Rexroad CE, Dowlen HH, Owen JR. 2001. Effects of gonadotropin releasing hormone and human chorionic gonadotropin on pregnancy rate in dairy cattle. J. Dairy Sci. 73: 66-72. Li W, Jiao S, Chi PP. 1993. Immunoreactive gonadotropin-releasing hormone in porcine reproductive tissues. Peptide. 14: 543-549. Meikle A, Sahlin L, Ferraris A, Masironi B, Blanc JE, Rodriguez-Irazoqui M, Rodriguez Pinon, Kindahl H, Forsberg M. 2001. Endometrial mRNA expression of oestrogen receptor c, progesterone receptor and insulin-like growth factor-I (IGF-I) throughout the bovine oestrous cycle. Anim. Reprod. Sci. 68: 45-56. 143  Meresman CF, Bilotas MA, Lombardi E, Tesone CS, Rosa lB. 2003. Effect of GnRH analogues on apoptosis and release of interleukin- 113 and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum. Reprod. 18: 1767-177 1. Moutsaatsou P, Sekeris E. 1997. Estrogen and progesterone receptors in the endometrium. Ann. N.Y. Acad. Sci. 816: 99-115. Nam DH, Lee SH, Kim HS, Lee GS, Jeon YW, Kim 5, Kim JH, Kang SK, Lee BC, Hwang WS. 2005. The role of gonadotropin-releasing hormone (GnRH) and its receptor in development of porcine preimplantation embryos derived from in vitro fertilization. Theriogenology. 63: 190-201. Parborell F, Irusta G, Celin AR, Tesone M. 2008. Regulation of ovarian angiogenesis and apoptosis by GnRH-l analogues. Mol. Reprod. Dev. 75: 623-631. Paria BC, Reese J, Das SK, Dey SK. 2002. Deciphering the cross talk of implantation: advances and challenges. Science. 296: 2185-2188. Peters AR. 2005. Veterinary clinical application of GnRH-questions of efficacy. Anim. Reprod. Sci. 88: 155-167. Raga F, Casan EM, Wen Y, Huang H, Nezhat C, Polan ML. 1998. Quantitative gonadotropin- releasing hormone gene expression and immunohistochemical localization in human endometriurn throughout the menstrual cycle. Biol. Reprod. 59: 661-669. Rajamahendran R, Ambrose DJ, Small JA, Dinn N. 2001. Synchronization of estrus and ovulation in cattle. Arch. Anim. Breed. 44 (special issue): 58-67. Ramakrishnappa N, Giritharan G, Aali M, Madan P, Rajamahendran R. 2003. GnRH receptor messenger ribonucleic acid expression in bovine ovary. Can. J. Anim. Sci. 83: 823826. Ramakrishnappa N, Rajamahendran R, Em YM, Leung PCK. 2005. GnRH in nonhypothalamic reproductive tissues. Anim. Reprod. Sci. 88: 95-113. Sengupta A, and Sridaran R. 2008. Expression and localization of gonadotropin-releasing hormone receptor in the rat oviduct during Pregnancy. J. Histochem. Cytochem. 56: 25-31. Tang X, Yano T, Osuga Y, Matsumi H, Yano N, Xu J, Wada 0, Koga K, Kugu K, Tsutsumi 0, Schafly AV, Taketani Y. 2002. Cellular mechanisms of growth inhibition of human epithelial ovarian cancer cell line by LH-releasing hormone antagonist cetrorelix. Clin. Endocr. Metab. 87: 3721-3727. Tasende, C, Meikie A, Rodrguez- Pinon M, Forsberg M, Garofalo G. 2002. Estrogen and progesterone receptor content in the pituitary gland and uterus of progesterone-primed and gonadotropin releasing hormone-treated anestrous ewes. Theriogenology. 57: 1719-1731.  144  Ulbrich SE, Kettler A, Einspanier R. 2003. Expression and localization of estrogen receptor c, estrogen receptor f3 and progesterone receptor in the bovine oviduct in vivo and in vitro. J. Steroid Biochem. Mol. Biol. 85: 279-289. van Biljon W, Wykes S, Scherer S, Krawetz SA, Hapgood J. 2002.Type II gonadotropin releasing hormone receptor transcripts in human sperm. Biol. Reprod. 67: 174 1-1749. Vu K, Greenspan DL, Wu TC, Zacur HA, Kurman RJ. 1998. Cellular proliferation, estrogen receptor, P4 receptor, and bcl-2 expression in GnR}I agonist-treated uterine leiomyomas. Hum. Pathol. 29: 359-363. Vasudeven N, Plaff, DW. 2007. Membrane-initiated actions neuroendocrinology: emerging principles. Endocr. Rev. 28: 1-19.  of  estrogens  in  Walker VR, korach KS. 2004. Estrogen receptor knockout mice as a model for endocrine research. Inst. Lab. Anim.Res. J. 45: 455-461. Walther N, Lioutas C, Tiliman G, Ivell R. 1999. Cloning of bovine estrogen receptor beta (ER 13): expression of novel deleted isoform in reproductive tissues. Mol.Cell. Endocr. 152: 3745.  145  Appendix 1: Buserelin induced regulation of ERa in the luteal phase endometrium. G3PDH was used as an internal control and housekeeping gene in the experiments  ERc (luteal)  G3PDH (luteal)  Cowl  Cow2  Cow3  Cow4  Cow5  MW 1  2  3  4  5  6  NEG  MW  1  2  3  4  5  6  NEG  MW; molecular weight marker 1; untreated control. 2; buserelin 200 ng/mL 3; buserelin 500 ng/mL 4; buserelin 1000 ng/mL 5; antide 500 ng/mL 6; antide (500 ng/mL) + buserelin (200 ng/mL) NEG; negative control without cDNA  146  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0072233/manifest

Comment

Related Items