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The role for insulin-like growth factor-I in preimplantation embryonic development and decidualization… Katagiri, Seiji 1996

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T H E R O L E F O R I N S U L I N - L I K E G R O W T H F A C T O R - I I N P R E I M P L A N T A T I O N E M B R Y O N I C D E V E L O P M E N T A N D D E C I D U A L I Z A T I O N F O L L O W I N G S U P E R O V U L A T I O N I N T H E R A T by S E U I K A T A G I R I D . V . M . , H o k k a i d o U n i v e r s i t y , 1987 M . S c . , H o k k a i d o U n i v e r s i t y , 1987 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R D E G R E E O F D O C T O R O F P H I L O S O P H Y i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Reproductive and Developmenta l Sciences Programme) W e accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A p r i l 1996 © Se i j i Ka tag i r i , 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) -i : 11 ABSTRACT Superovulation causes detrimental effects, including embryonic loss and implantation failure. This study determined potential roles for insulin-like growth factor (IGF-I) in uterine environment regulation and preimplantation development, in conjunction with the detrimental effects of superovulation in the rat. IGF-I may be beneficial to preimplantation embryonic development. IGF-I stimulated embryonic development and metabolism in vitro and increased the rate of implantation and fetal development when the blastocysts were transferred into a receptive uterus. However, IGF-I may be involved in embryonic loss following superovulation, by perturbing the uterine environment. Superovulation enhanced uterine IGF-I action from day 1 to 3 of pregnancy and reduced its action on days 5 and 6. Uterine luminal fluid from the uterus exposed to increased IGF-I action from day 1 to 3 was detrimental to embryonic development. This uterine luminal fluid had altered electrolyte composition that is similar to that observed following superovulation. Anti-IGF-I antibody restored superovulation-induced alterations in cations, suggesting that IGF-I may partially mediate this effect of superovulation. Superovulation-induced alterations in IGF-I action may adversely affect decidualization, a critical step in implantation. High IGF-I levels from day 1 to 3 and low levels from day 3 to 5, inhibited deciduoma formation. Alterations in IGF-I action after day 5 had no effect, suggesting a role for IGF-I in uterine sensitization. The role of IGF-I in decidualization may be complex. IGF-I cannot be substituted for growth hormone (GH) and thyroxine (T4) during decidualization, but altered deciduoma formation and alkaline phosphatase (ALP) activity in the GH and T4-dependent manner. IGF-I either stimulated or inhibited deciduoma formation and ALP activity, depending upon concentration and treatment period. IGF-I stimulated basal ALP activity Ill but inhibited prostaglandin E2-stimulated ALP activity in the endometrial stroma cells. In conclusion, IGF-I may play an important role in the maintenance of a receptive uterine environment for embryonic development and the regulation of decidualization. Embryonic loss and failure of implantation following superovulation may be partially attributed to disturbances in uterine IGF-I action as observed in this study. iv TABLE OF CONTENTS Page Abstract ii Table of Content iv List of Tables vii List of Figures viii Glossary xi Acknowledgment xiii INTRODUCTION 1 UNDERLYING HYPOTHESIS 2 OUTLINE OF THE THESIS 2 CHAPTER ONE LITERATURE REVIEW I. SUPEROVULATION 9 A. General 9 B. PMSG 11 C. Immature Rat Superovulation Model 14 D. Detrimental Effects of Superovulation 16 II. THE INSULIN-LIKE GROWTH FACTOR (IGF) SYSTEM 18 A. IGF-I 18 B. Insulin-Like Growth Factor Binding Proteins (IGFBPs) 21 C. IGF-I Receptors 23 D. The IGF System in the Uterus 25 UI. GROWTH FACTORS IN PREIMPLANTATION EMBRYONIC DEVELOPMENT 27 A. Paracrine and Autocrine Regulation 27 B. Insulin-Like Peptides in the Preimplantation Embryonic Development 29 V IV. ELECTROLYTES IN PREIMPLANTATION EMBRYONIC DEVELOPMENT 32 A. General 32 B. Electrolyte Transport in Preimplantation Embryonic Development 33 C. Preimplantation Embryonic Development In Vitro 35 D. Electrolytes in the Uterus 37 V. DECIDUALIZATION 37 A. General 37 B. Endocrine Regulation 39 C. Paracrine/Autocrine Regulation 40 D. Initiation of Decidualization 46 E. Evaluation of Decidualization 48 CHAPTER TWO THE EFFECT OF IGF-I IN THE PREIMPLANTATION RAT EMBRYONIC DEVELOPMENT I. INTRODUCTION 53 II. MATERIALS AND METHODS 54 III. RESULTS 58 IV. DISCUSSION 68 V. SUMMARY AND CONCLUSIONS 74 CHAPTER THREE THE EFFECT OF SUPEROVULATION ON THE UTERINE IGF SYSTEM I. INTRODUCTION 75 n . MATERIALS AND METHODS 76 HI. RESULTS 82 V. DISCUSSION 91 V. SUMMARY AND CONCLUSIONS 93 VI CHAPTER FOUR THE EFFECT OF IGF-I ON THE UTERINE MICROENVIRONMENT FOR PRELMPLANTATION EMBRYONIC DEVELOPMENT I. INTRODUCTION 94 H. MATERIALS AND METHODS 96 III. RESULTS 102 IV. DISCUSSION 114 V. SUMMARY AND CONCLUSIONS 121 CHAPTER FIVE THE EFFECT OF IGF-I ON DECIDUALIZATION I. INTRODUCTION 123 II. MATERIALS AND METHODS 124 III. RESULTS 129 IV. DISCUSSION 155 V. SUMMARY AND CONCLUSIONS 162 CHAPTER SIX SUMMARY AND GENERAL CONCLUSIONS I. SUMMARY 163 II. CONCLUSIONS 167 BIBLIOGRAPHY 168 V l l LIST OF TABLES Table 2-1 The developmental stages of 8-cell embryos following 36-h-culture with human recombinant (hr)-IGF-I at various concentrations Page 59 Table 2-2 The rate of implantation and development into day 18 fetuses of blastocysts obtained from cultures with human recombinant (hr)- -IGF-I at various concentrations 67 Table 4-1 Osmotic concentrations of uterine luminal flushes following the IGF-I infusion and superovulation 106 Table 4-2 The effect of uterine luminal fluids obtained from IGF-I infused or superovulated rats on 8-cell stage embryonic development 107 Table 4-3 The effect of dialysis of uterine luminal fluids obtained from IGF-I infused or superovulated rats on 8-cell stage embryo development 109 Table 4-4 Total cation content in the uterine luminal flushes 115 ( V l l l LIST OF FIGURES Page Figure 1 Outline of the thesis (Chapter two) 4 Figure 2 Outline of the thesis (Chapter three) 5 Figure 3 Outline of the thesis (Chapter four) 6 Figure 4 Outline of the thesis (Chapter five) 7 Figure 5 Outline of the thesis (Chapter five — continued) 8 Figure 2-1 The number of cells in rat blastocysts following in vitro culture with varying concentrations of IGF-I 62 Figure 2-2 The dead-cell index in the rat blastocysts following in vitro culture with varying concentrations of IGF-I 64 Figure 2-3 The levels of protein synthesis by the rat blastocysts following in vitro culture with varying concentrations of IGF-I 66 125 Figure 3-1 Competition-inhibition curves for I-IGF-I binding to the uterine membrane preparations by human recombinant (hr)-IGF-I, hr-IGF-IL and insulin 81 Figure 3-2 Profiles of the serum estradiol-17 {5 and progesterone levels of superovulated and control rats 84 Figure 3-3 Profiles of IGF-I, IGFBP and IGF-I receptor levels in the uterus of superovulated and control rats 86 Figure 3-4 Profiles of IGF-I, IGFBP and IGF-I receptor levels in the uterine endometrium of superovulated and control rats 88 IX Figure 3-5 Profiles of (A) IGF-I and (B) IGFBP levels in the serum of superovulated and control rats 90 Figure 4-1 A rat uterine IGF-I infusion model 98 Figure 4-2 The effect of IGF-I infusions (day 1-day 3) on the uterine IGF system 104 Figure 4-3 The effect of PMSG on the electrolyte composition of the uterine luminal fluids 111 Figure 4-4 The effect of IGF-I infusion on electrolyte composition of the uterine luminal fluids 113 Figure 5-1 The effect of IGF-I infusions at different time period on the uterine IGF system 132 Figure 5-2 The effect of IGF-I infusions at different time period on deciduoma formation 134 Figure 5-3 The effect of anti-IGF-I antibody infusions at different time period on deciduoma formation 136 Figure 5-4 Distribution of the uterine tissue weight of the IGF-I-infused horns 138 Figure 5-5 Distribution of the uterine horn weight of the anti-IGF-I antibody-infused horns 141 Figure 5-6 The effect of IGF-I infusions during the prestimulation period on deciduoma formation 143 Figure 5-7 The effect of IGF-I infusions during the pre- and poststimulation periods on deciduoma formation 145 Figure 5-8 The effect of IGF-I infusions during the prestimulation period on the levels of uterine alkaline phosphatase activity 147 Figure 5-9 The effect of IGF-I infusions during the pre- and poststimulation periods on the levels of uterine alkaline phosphatase activity 149 Figure 5-10 The effect of anti-IGF-I antibody on the deciduoma formation and uterine alkaline phosphatase activity 151 Figure 5-11 The effect of IGF-I on alkaline phosphatase activity in cultured uterine endometrial stroma cells 154 xi G L O S S A R Y ALP alkaline phosphatase ANOVA analysis of variance ATP adenosine triphosphate BSA bovine serum albumin BW body weight cAMP cyclic adenosine monophosphate °C degree of Celsius a curie (= 3.7 x 1 0 1 0 disintegrations per second) cm centimeter cpm radioactive counts per minute CSF colony stimulating factor DMEM:F-12 Dulbecco's modified Eagle's medium:Ham's F-12 nutrient mixture DPBS Dulbecco's phosphate buffered saline EDTA emylenediaminetetraacetic acid eFSH equine follicle stimulating hormone eLH equine luteinizing hormone EGF epidermal growth factor ET embryo transfer FCS fetal calf serum FSH follicle stimulating hormone g gram 8 gravity GH growth hormone h hour 3H tritium, a radioactive isotope of hydrogen HBSS Hank's balanced salt solution hCG human chorionic gonadotropin hMG human menopausal gonadotropin hr-IGF-I human recombinant insulin-like growth factor 125j a radio active isotope of iodine ICM inner cell mass IGF insuhn-like growth factor IGFBP insulin-like growth factor binding protein IL Interleukin X l l IU international unit rVF in vitro fertilization kDa kilodalton kb kilobase 1 liter LH luteinizing hormone LIF leukemia inhibitory factor M 16 embryo culture media M199 medium 199 mCi millicurie mg milligram mg microgram min minute ml milliliter ml microliter mm millimeter mM millimolar nM nanomolar ng nanogram PG prostaglandin pGH porcine growth hormone pH -log H + concentration in a fluid PMSG pregnant mare's serum gonadotropin PVA polyvinylalchohol RIA radioimmunoassay rpm revolutions per minute SEM standard error of means T4 thyroxine TGF transforming growth factor TSH thyroid stimulating hormone TNF tumor necrosis factor v/v volume per volume X l l l ACKNOWLEDGEMENT I would like to express my sincere appreciation: To my supervisor, Dr. Young S. Moon, and Dr. Basil Ho Yuen for providing me the opportunity to pursue this study and for their continuous support, patience, and encouragement throughout the thesis study; To members of my research supervisory committee, Dr. Rajadurai Rajamahendran, Dr. Gregory C. Y. Lee, and Dr. Josef Skala for their invaluable advice and guidance; To the staff of animal holding facility at the BC Children's Hospital Research Centre for the care of animals and technical assistance; To Mr. Murray MacKinnon for his advice in statistical analysis; To Dr. Colin MacCalman for his critical reading during the preparation of this thesis. This study was supported by the grants from the British Columbia Health Care Research Foundation. r -1 -I N T R O D U C T I O N Superovula t ion, i n association w i t h in vitro fer t i l iza t ion-embryo transfer ( I V F - E T ) , and related techniques are now c o m m o n l y used i n the treatment o f human infer t i l i ty . W i t h few exceptions, standard superovulatory protocols currently use pha rmaco log ica l dosages o f exogenous gonadotropins to obta in mu l t i p l e oocytes . F e r t i l i z e d embryos are then r e p l a c e d in to a d i s tu rbed uter ine env i ronment . M a n y studies a i m e d at i m p r o v i n g superovulatory treatments have been focused on the ovar ian funct ional aspects i n order to obtain m a x i m u m number o f fert i l izable oocytes. H e n c e , an attempt o f ovar ian s t imulat ion has typ i ca l ly been determined by the number and size o f fo l l i c l e s and the l eve l o f ovar ian steroid hormones that include estrogen and progesterone. Aspec ts o f uterine function have been largely disregarded wi th the exception o f moni tor ing o f uterine endometrial thickness. Recen t studies have c o n f i r m e d that n o r m a l l y func t ion ing e n d o m e t r i u m and a receptive uterine environment p lay a k e y role i n pre implanta t ion embryon ic development and the establishment o f successful pregnancy. T h i s i s compat ib le w i t h h igh pregnancy rates obse rved i n I V F - E T cases where the patient has not been subjected to ova r i an s t imula t ion and oocyte donat ion programs. In these cases, embryos are transferred to a recept ive uterine environment w h i c h was not been subjected to ova r i an hypers t imula t ion . T h e ab i l i ty to induce development o f mul t ip l e fo l l i c les and to manage the p h y s i o l o g i c a l status o f the uterine e n d o m e t r i u m is fundamenta l to the success o f superovula to ry treatments i n a c l i n i c a l setting. Further studies to improve superovulatory treatments wi th respect to ach iev ing n o r m a l uterine funct ion and a receptive uterine envi ronment for the preimplantation embryo is c lear ly needed. It is apparent that a large part o f ovar ian steroid hormone act ion i s mediated- by a c o m p l e x l o c a l regula tory ne twork o f g rowth factors and cy tok ines i n the uterus. T h e i n s u l i n - l i k e g rowth factor ( I G F ) system that consists o f I G F - I , I G F - I receptor, and I G F b i n d i n g proteins i s a part o f the regulatory network. Poten t ia l roles for the uterine I G F -2-sys tem i n the regula t ion o f the uterine envi ronment and p re implan ta t ion deve lopment f o l l o w i n g superovula t ion are the m a i n focus o f this study. T h e l o n g te rm goals o f this study are to determine the mechan i sms by w h i c h superovula tory treatments affect the uter ine e n v i r o n m e n t and the deve lopmen t o f the p r e imp lan t a t i on e m b r y o , and the subsequent es tabl ishment o f a successful pregnancy. T h i s s tudy was pe r fo rmed i n an attempt to detemiine changes i n the uterine I G F system f o l l o w i n g superovulat ion and their significance i n preimplantation embryonic development and implantat ion i n the rat. U N D E R L Y I N G H Y P O T H E S I S T h e u te r ine e n v i r o n m e n t fo r p r e i m p l a n t a t i o n e m b r y o n i c d e v e l o p m e n t i s p r e d o m i n a n t l y regula ted by ova r i an s teroid hormones . A d i s tu rbed o v a r i a n s teroid hormone balance, especial ly h ighly elevated estrogen levels, or estrogen/progesterone ratio, f o l l o w i n g superovulat ion results i n a var iety o f detr imental effects. S ince the uterine I G F sys tem is regula ted by ovar ian s teroid hormones , this sys tem m a y mediate detr imental effects such as ear ly e m b r y o n i c loss and fa i lure o f imp lan t a t i on by a l te r ing uterine r ecep t i v i t y . T h e d i s tu rbed I G F sys tem m a y render the uter ine m i c r o e n v i r o n m e n t detr imental to pre implanta t ion embryon ic development . T h e dis turbed I G F system may also affect uterine sensi t izat ion to a dec iduogenic s t imulus and endometr ia l s t romal c e l l decidual izat ion. T h i s i n turn results i n failure o f implantation. O U T L I N E O F T H E T H E S I S Chapter one rev iews avai lable informat ion that i s related to the topic o f this study. T h e literature r e v i e w is f o l l o w e d by chapter two that shows the benef ic ia l effect o f I G F - I o n p re implan ta t ion e m b r y o n i c deve lopment in vitro ( F i g . 1). Chapte r three examines alterations i n the uterine I G F system f o l l o w i n g superovula t ion by us ing an immature rat superovulat ion mode l (F ig . 2). Chapter four and chapter f ive determine signif icance o f the al terat ions i n the uterine I G F sys tem, caused by superovu la t ion , i n p re implan ta t ion e m b r y o n i c development and implanta t ion. T h i s is f o l l o w e d b y the study to determine the po ten t i a l m e c h a n i s m s by w h i c h the a l tered uterine I G F sys t em inc reased an ear ly embryon ic loss and impa i red blastocyst implantat ion. In particular, chapter four examines the ro le o f I G F - I i n the regula t ion o f uterine env i ronment for e m b r y o n i c development , w h i c h inc ludes the i o n i c compos i t i on o f the uterine l u m i n a l f lu ids ( F i g . 3). Chapter f ive focuses o n dec idua l iza t ion , a c ruc ia l step i n implanta t ion i n the rats. T h e role o f I G F - I i n the uterine sensitization process for the decidual reaction is determined (Figs. 4 and 5). C H A P T E R T W O Embryo culture with IGF-I I Embryo development I 1 Cell Protein Embryo count synthesis transfer ure 1 T h e effect o f I G F - I o n preimplanta t ion embryon ic development in vitro is de termined . T h e 8-ce l l stage rat embryos are cu l tu red for 36 h i n the presence o f I G F - I o f v a r y i n g concent ra t ions . T h e effect o f I G F - I on e m b r y o n i c deve lopment i s de te rmined by the deve lopmen ta l stages o f embryos , the number o f cel ls i n the resul t ing blastocysts, and the levels o f pro te in synthesis i n the blastocysts . T h e blastocysts are also transferred in to a recept ive uterus and the ab i l i ty o f the blastocysts to implan t and develop into day 18 fetuses determined. - 5 -C H A P T E R T H R E E PMSG treatments 4IU Control 40 IU Superovulation Ovarian steroid hormones estradiol-170 progesterone JL 1 IGF-I uterus, serum Total IGFBP uterus, serum IGF-I receptor uterus Figure 2 T h e effect o f superovula tory treatment o n the uter ine I G F sys tem is determined i n an immature rat superovulation mode l . Immature female rats are injected w i t h a s ingle dose o f 4 I U (control) pregnant mare's serum gonado t rop in ( P M S G ) o r 4 0 I U (superovula t ion) P M S G to ach ieve a pregnancy. L e v e l s o f I G F - I , total I G F b i n d i n g protein ( I G F B P ) , and I G F - I receptor are de termined i n the uterus and serum f r o m day 1 to day 6 o f pregnancy. The levels o f I G F - I is determined by rad io immunoassay ( R I A ) and the levels o f total I G F B P and I G F - I receptor de te rmined by l i g a n d b i n d i n g assays. T h e serum leve l s o f estradiol-17 f3 and progesterone are determined by R I A f rom the day o f the P M S G inject ion (day -2) to day 6 o f pregnancy. -6-C H A P T E R F O U R IGF-I infusion - concentration - infusion rate IGF-I infusion 1 Embryo culture Electrolyte composition with U L F of U L F Superovulation + (Anti-IGF-I antibody) Figu re 3 T h e potential mechanisms by w h i c h an altered uterine I G F system, caused by superovulat ion, increases the rate o f e m b r y o n i c loss are determined. A cond i t ion (concentration and infus ion rate) for I G F - I infus ion that achieves an increase i n uterine I G F - I ac t ion after superovu la t ion i s de termined . E i g h t - c e l l stage rat embryos are cu l tu red w i t h the uterine l u m i n a l f lu ids ( U L F ) ob ta ined f r o m the I G F - I in fused rats, superovu la ted rats, and superovula ted and a n t i - I G F - I an t ibody infused rats to de termine i f an increase i n I G F - I action i n the uterus mediates early embryon ic , loss caused by superovulation, through an alteration o f uterine environment. Elect ro lyte compos i t i on o f each U L F is determined i n an effort to ident i fy detr imental factors i n the U L F . -7-C H A P T E R F I V E Deciduoma induction IGF-I infusion Anti-IGF-I antibody infusion • J Alkaline phosphatase activity Figure 4 T h e ro le for I G F - I i n the uterine sens i t iza t ion process for the dec idua l reac t ion is de termined. F i r s t , rats are o v a r i e c t o m i z e d and treated w i t h ovar ian steroid hormones for m a x i m a l uterine sensit izat ion and are infused w i t h I G F - I or an t i - IGF- I ant ibody dur ing the different t ime periods (day 1-3, day 3-5, and day 5-9). A n ar t i f ic ia l dec iduogenic s t imulus was g i v e n o n day 5 and the degree o f dec iduoma format ion were determined o n day 9. Second ly , the potent ia l mechan isms o f I G F - I ac t ion i n the regula t ion o f uterine sensit ization i n conjunct ion w i t h g rowth hormone ( G H ) and thyro id h o r m o n e ( T 4 ) . P i t u i t a r y i n t a c t , h y p o p h y s e c t o m i z e d , a n d h y p o p h y s e c t o m i z e d - G H and T4 treated rats are ova r i ec tomized and treated w i t h ovar ian steroid hormones. Rats are infused w i t h I G F - I o r a n t i - I G F - I ant ibody du r ing the uterine sensi t izat ion pe r iod or f r o m the sensi t izat ion p e r i o d th roughout the t ime o f d e c i d u o m a f o r m a t i o n . A n a r t i f i c i a l dec iduogenic st imulus is g iven o n day 5 o f pseudopregnancy. T h e degree o f dec iduoma format ion and levels o f uterine a lka l ine phosphatase act ivi ty are determined on day 9 o f pseudopregnancy. - 8 -C H A P T E R F I V E (continued) •In vivo-IGF-I inftision/Non-infusion T Li vitro Endometrial stromal cells IGF-I PGE, Alkaline phosphatase activity Figure 5 T h e ro le for I G F - I i n the regula t ion o f a lka l ine phosphatase ac t iv i ty i s further studied i n the uterine endometr ia l s t romal ce l l s that is undergoing dec idua l iza t ion . Rats are ova r i ec tomized and treated w i t h ovar ian steroid hormones for m a x i m a l uterine sensi t izat ion. S o m e rats are infused w i t h I G F - I dur ing the uterine sensit izat ion per iod . U te r ine endometr ia l s tromal cel ls are obtained f rom the uterus after the uterine endometr ium is sensitized (day 5) and are treated w i t h v a r y i n g concentrations o f I G F - I . S o m e uterine endomet r i a l s t romal ce l l s are treated w i t h p ros tag land in E 2 (PGE2) i n addi t ion to I G F - I . A l k a l i n e phosphatase act iv i ty i n the endometr ia l s troma cells are determined after the 48 h culture per iod. - 9 -C H A P T E R O N E L I T E R A T U R E R E V I E W I. S U P E R O V U L A T I O N A . Genera l a. H i s t o r y Superovula t ion is n o w c o m m o n l y used i n human infer t i l i ty c l i n i c s , i n the l ives tock industry and i n basic reproduct ive b i o l o g y research. T h e ve ry first attempt o f ovu la t ion induct ion or superovulation was made i n the effort o f def ining the role o f the pituitary gland i n the reproductive system, us ing laboratory species. The importance o f the pituitary i n the regulat ion o f reproduct ive system became apparent dur ing the first quarter o f this century. T h i s l e d to an attempt o f us ing implants o f pi tui tary tissue to st imulate fo l l i cu l a r growth w h i c h resulted i n mul t ip l e fo l l i cu l a r growth and ovu la t ion (Engle , 1927; S m i t h and E n g l e , 1927). Implants o f p i tu i tary tissue were soon replaced b y pi tu i tary extracts or pregnant mare's serum. B i o l o g i c a l l y active elements have been separated f rom the pituitary extracts (foll icle s t imula t ing hormone: F S H and lu te in iz ing hormone: L H ) , pregnant mare's sera (pregnant mare's serum gonadotropin : P M S G ) , human placentae (human c h o r i o n i c gonadotropin: h C G ) and u r ine o f w o m e n i n the p o s t - m e n o p a u s a l p e r i o d ( h u m a n m e n o p a u s a l gonadot ropin : h M G ) . P M S G and h M G are r i c h sources o f F S H ac t iv i ty but they also conta in a considerable amount o f L H act ivi ty . In contrast, h C G o n l y contains L H act ivi ty and acts through the L H receptor. In humans and labora tory an ima l s , the standard superovulatory treatment employs gonadotropins w i t h F S H ac t iv i ty to st imulate mul t ip le f o l l i c u l a r deve lopment i n combina t ion w i t h L H ac t iv i ty to induce ovu l a t i on and oocy te maturat ion. P M S G has become one o f the most w i d e l y used sources o f F S H ac t iv i ty i n laboratory animals , w h i l e h M G is a preferred source o f F S H ac t iv i ty for c l i n i c a l use i n - 1 0 -humans. h C G is the standard cho ice as a source o f L H act iv i ty i n laboratory animals and humans. b. M e c h a n i s m T h e mechanisms used by exogenous gonadotropins to achieve mu l t i p l e fo l l i cu l a r deve lopment and ovu la t ion have been p rev ious ly descr ibed. E x o g e n o u s gonadotropins appear to recruit a non-g rowing p o o l o f sma l l fo l l ic les and transform them into a group o f ma tu r ing f o l l i c l e s (Greenwa ld , 1973). Supe rovu la t ion i s a lso b e l i e v e d to i n v o l v e the rescue o f atretic f o l l i c l e s (Peters et a l . , 1975; B y s k o v , 1978; H a y and M o o r , 1978; B y s k o v , 1979; B r a w and Tsa f r i r i , 1980; F u j i m o r i et a l . , 1987). H o w e v e r , the term o f "rescue" is ambiguous and mi s l ead ing . S o m e invest igators d o not d i s t ingu i sh between "rescue" o f fo l l i c les f rom atresia and "recruitment" o f sma l l fo l l i c l e s , as they be l ieve that most o f the s m a l l fo l l i c l e s w i l l undergo atresia unless they are s t imulated by exogenous gonado t rop ins to the o v u l a t o r y cohor t o f the present c y c l e . In contrast , severa l investigators use the term o f "rescue" based o n the evidence that a reduced ratio o f atretic fo l l i c l e s to healthy non-atretic fo l l i c les or total number o f fo l l i c l e s is observed f o l l o w i n g h o r m o n a l treatments. In this case, the te rm o f "rescue o f f o l l i c l e f r o m atresia" may be rep laced by "reversal o f atresia", w h i l e i n the former case "rescue o f f o l l i c l e f rom atresia" m a y be replaced by "prevention o f atresia" (Hirshf ie ld , 1989). Ev idence suggests that hormonal treatments c o u l d stop or reverse the atretic process (Tsafr i r i and B r a w , 1984). H o w e v e r , this i s cont rovers ia l , s ince other ev idence suggests that once a fo l l i c l e begins to degenerate in vivo, i t w i l l p robably not return to the ovulatory pa thway ( H a y et a l . , 1979; H i r s h f i e l d , 1989). A n e w in vivo l a b e l i n g technique w i t h [ 3 H ] t h y m i d i n e w h i c h a l lows us to examine the reversal o f atresia i n an i n d i v i d u a l f o l l i c l e demonstrated that f o l l i c u l a r atresia was an i r revers ib le process ( H i r s h f i e l d , 1989). A decreased rat io o f atretic fo l l i c l e s to the total number o f fo l l i c l e s f o l l o w i n g h o r m o n a l treatments m a y indicate the reversal o f atresia by the ho rmona l treatments. H o w e v e r , a - 1 1 -reduct ion i n the ratio o f atretic fo l l i c les m a y also be the result o f an increase i n the number o f n e w l y deve loped fo l l i c l e s a r i s ing f r o m a reserve o f sma l l f o l l i c l e s recru i ted b y the ho rmona l treatments. Thus , i t is unclear i f "reversal o f atresia" can be considered to be a mechanism through w h i c h exogenous gonadotropins achieve superovulat ion. B . Pregnant Mare ' s Se rum Gonado t rop in ( P M S G ) a. Gonado t rop in for Superovulat ion T h e strong F S H act ivi ty and the extraordinary l o n g half- l i fe o f P M S G a l lows us to induce fo l l i cu l a r development and superovulat ion w i t h a s ingle dose o f this hormone i n species other than the horse. T h i s is a great advantage when a large number o f animals are to be treated. Furthermore, the avai labi l i ty o f commerc ia l preparations at a l o w cost has led to a widespread use o f this gonadotropin i n basic research and i n the l ives tock industry. A l t h o u g h the ro le o f P M S G i n main ta in ing pregnancy i n the horse m a y be controvers ia l , P M S G is undoubtedly an indispensable pharmacologica l agent i n reproduct ive b i o l o g y and i n the l ives tock industry. b. O r i g i n , Secretion, and M o l e c u l a r Properties P M S G is f i rs t detected i n the se rum o f mares o n a p p r o x i m a t e l y day 4 0 o f pregnancy, peaks between days 60 and 80, and declines towards base levels o n day 130 o f p regnancy . P M S G , a l so k n o w n as equine c h o r i o n i c gonado t rop in , is p r o d u c e d by chor ion ic ce l l s i n the uterine endometr ia l cup, a pale and c i r cumsc r ibed plaques o f tissue that deve lop adjacent to the chor ion ic g i rd le o f the embryo ( C o l e and Har t , 1930; A l l e n , 1969; M o o r et a l . , 1975). E v i d e n c e suggests that synthesis and secre t ion o f P M S G appears not to be regulated by endocrine and paracrine regulatory factors (Hami l t on et a l . , 1973; Hernandez-Jauregui and G o n z a l e s - A n g u l o , 1975; Net t and Picket t , 1979; T h o m p s o n et a l . , 1982). Instead, the s ize and numbers o f the endometr ia l cups and their h i s to log ica l appearance are correlated w i t h serum P M S G levels ( M u r p h y and M a r t i n u k , 1991; Ginther , - 1 2 -1992; Squires , 1993; H o p p e n , 1994). T h e ro le for P M S G i n the horse pregnancy is not w e l l understood. H o w e v e r , this hormone is be l i eved to induce ovu la t ion and subsequent lute inizat ion o f accessory corpora lutea dur ing early pregnancy through its L H act ivi ty , and thus contributes to the maintenance o f pregnancy un t i l p lacenta l steroidogenesis reaches sufficient levels (Stewart and A l l e n , 1981). P M S G is a d i m e r i c g l y c o p r o t e i n that i s s t ruc tura l ly s i m i l a r to the p i tu i t a ry g lycopro te in hormones, w h i c h inc lude F S H , L H , and thyro id s t imula t ing hormone ( T S H ) , and composed o f an a - and P-subunit ( M u r p h y and M a r t i n u k , 1991; H o p p e n , 1994). T h e cc- and P -subuni ts o f P M S G are c o m p o s e d o f 96 and 149 a m i n o ac ids , r e spec t ive ly (Stewart and M a h e r , 1991; Sherman et a l . , 1992). T h e gene encod ing the ct-subunit o f P M S G is ident ical to that encoding the a-subunit o f a l l pi tui tary g lycoprote in hormones i n the horse (Stewart et a l . , 1987), w h i l e the P-subunit o f P M S G is encoded by the same gene that encodes equine L H ( e L H ) (Stewart and M a h e r , 1991; Sherman et a l . , 1992). S ince P M S G and e L H have ident ical a - and P-subunits, o n l y differential g lycosy la t ion appears to dis t inguish P M S G and e L H (Smi th et a l . , 1993; M a t s u i et a l . , 1994). b. D u a l F S H and L H A c t i v i t y T h e most unique feature o f P M S G may be its strong F S H act ivi ty , i n addi t ion to its L H act iv i ty , i n ndn-equid species ( M u r p h y and M a r t i n u k , 1991; H o p p e n , 1994). P M S G and e L H , as w e l l as equine F S H , b i n d to the F S H receptors i n a l l non-equid species and donkey (an e q u i d species), but not i n the horse ( L i c h t et a l . , 1979; Stewart and A l l e n , 1981; M o u d g a l and Papkoff , 1982; G u i l l o u and Combarnous , 1983; C o m b a r n o u s et a l . , 1984). S o m e poss ib le explanat ions for the unique F S H act iv i ty o f P M S G and e L H have been proposed. T h e oc-subunits o f equine gonadotropins possess a unique t ransposi t ion and non-conservat ive substitutions o f amino ac id residues, compared w i t h those o f the other species (Stewart et a l . , 1987). T h e h igh amount o f carbohydrates, especia l ly s ia l ic a c i d , has been suggested to be re spons ib le for the un ique F S H ac t i v i t y o f P M S G - 1 3 -( A g g a r w a l et a l . , 1980a,b; D a m m et a l . , 1990). T h e P-subunit o f P M S G has as h igh as 55 .3% o f carbohydrate content that is 2- to 3-fold greater than that o f F S H and L H . S i a l i c a c i d compr i se 2 1 . 3 % o f the total carbohydrate weight (Papkoff , 1978). S o m e molecu la r properties o f the P-subunit o f P M S G , other than g lycosy la t ion , have also been discussed i n r e l a t ion to the dua l F S H and L H ac t iv i ty o f P M S G ( M u r p h y and M a r t i n u k , 1991) . H o w e v e r , none o f these molecu la r properties, i n c l u d i n g g lycosy la t ion , do not appear to be a s ingle factor responsible for the dua l F S H and L H ac t iv i ty o f equine gonadotropins . Thus , the mechan i sm w h i c h a l lows the dua l F S H and L H act ivi ty o f P M S G and e L H is not clear. c. L o n g H a l f - L i f e P M S G has the highest carbohydrate content a m o n g the g lycopro te in hormones . T h e heavy g lycosy la t ion o f P M S G , especia l ly by s ia l ic acids, gives an extraordinary long half- l i fe i n the c i rculat ion i n the mare and other species (Catchpole et a l . , 1935; Sasamoto et a l . , 1972; M c i n t o s h et a l . , 1975; M e n z e r and Schams , 1979; K a t a g i r i et a l . , 1991). A l t h o u g h the half- l i fe o f c i rcu la t ing F S H is general ly m u c h shorter than that o f P M S G , a v a r y i n g degree o f s i a ly l a t i on i s a lso found to be respons ib le for d is t inc t ha l f - l ives o f va r ious i so fo rms o f F S H ( C h a p p e l et a l . , 1983). T h e subuni ts o f P M S G / e L H have structural ly dis t inct A 7 - l i n k e d ol igosacchar ides and te rminal charge groups ( D a m m et a l . , 1990; S m i t h et a l . , 1993; M a t s u i et a l . , 1994). e L H has been shown to conta in mono- o r di-branched oligosaccharides terminating wi th S 0 4 - 4 - G a l N A c beta l , 4 G l c N A c that b i n d to specific receptors i n l i ve r endothel ial cel ls (Smi th et a l . , 1993). O n the other hand, P M S G P-subunits have d i - or tri-branched oligosaccharides terminating S i a alpha 2,3 or 6 G a l beta l , 4 G l c N A c that are not metabol ized by l i ve r ( D a m m et a l . , 1990; S m i t h et a l . , 1993). A s a result , P M S G remains i n the c i rcu la t ion for longer pe r iod (approximate ly 5.7-fold) than equine L H . P M S G shows remarkable heterogeneity i n the structure o f N - l i n k e d chains as w e l l as the degree o f g lycosy l a t i on ( A g g a r w a l et a l . , 1980a,b). These heterogeneity i n - 1 4 -g l y c o s y l a t i o n has been rela ted to the v a r i a b i l i t y i n b i o l o g i c a l ac t iv i ty and ha l f - l i fe o f different P M S G preparations. C . Immature Rat Superovulat ion M o d e l a. B a c k g r o u n d S m a l l labora tory an imals , w h i c h i nc lude rats, m i c e and hamsters , have been extens ive ly u t i l i z ed for basic studies i n reproduct ive b io logy . E a r l y studies used rats and m i c e i n an attempt to induce superovulat ion by the da i ly implanta t ion o f anterior pituitary t issues ( E n g l e , 1927; S m i t h and E n g l e , 1927) . P regnan t mare ' s s e r u m w a s then int roduced to induce superovulat ion. A single inject ion o f crude pregnant mare's serum or par t ia l ly pur i f i ed P M S G was capable o f i nduc ing superovulat ion and p roduc ing o f greater than n o r m a l litter sizes (Co le , 1936; C o l e , 1937; C o l e , 1940). Induct ion o f superovulat ion w i t h P M S G has become a standard cho i ce due to the s i m p l i c i t y o f the procedure and ava i l ab i l i ty o f the mater ial . Immature rats, immedia te ly p r io r to sexual maturi ty at 28-30 days o l d , were found to be more suitable than sexual ly mature rats ( C o l e , 1937; Strauss and M e y e r , 1962; M c C o r m a c k and M e y e r , 1963; Z a r r o w and Q u i n n , 1963). Immature rats and m i c e p roduced a greater number o f oocytes compared to adults ( C o l e , 1937; F o w l e r and Edwards , 1957; B igge r s et a l . , 1971). T w o poss ible explanat ions have been proposed for the better response o f immature an imals to P M S G . F i r s t , a greater number o f deve lop ing fo l l i c l e s , capable o f responding to P M S G , m a y be avai lable i n immature rats (Jones and K r o h n , 1961). Second , the t i m i n g o f P M S G inject ion i n the estrous cyc le m a y affect the results o f superovulat ion i n adults. In part icular, the t ime o f P M S G inject ion to obtain an op t ima l result m a y be ve ry l im i t ed ( M c L a r e n and M i c h i e , 1959; E d w a r d s et a l . , 1963) . b. P M S G dose T h e normal i ty o f a pregnancy induced wi th P M S G i n immature rats m a y be a matter o f concern . N u m e r o u s studies have shown that l o w doses (4-8 I U ) o f P M S G produces a - 1 5 -n o r m a l p regnancy i n immature rats. A l o w dose o f P M S G induces a s y n c h r o n i z e d ovu la t ion through e l i c i t ing an endogenous L H surge (Sorrentino et a l . , 1972; W i l s o n et a l . , 1974; K o s t y k et a l . , 1978). T h e L H surge is preceded b y an increase i n the l eve l s o f es t rad io l -170 and progesterone w h i c h are s imi l a r to those found du r ing proestrus i n the adult (Sha ikh , 1971; Bu tche r et a l . , 1974; Parker et a l . , 1976). H o r m o n a l prof i les , t i m i n g o f oocyte maturat ion, and qual i ty o f oocytes i n immature rats treated w i t h a l o w dose o f P M S G appear to be compat ib le to those observed i n the adult (Bar rac lough et a l . , 1971; L i n k i e and N i s w e n d e r , 1972; H i l l e n s j o et a l . , 1974). In addi t ion , treatment w i t h a l o w dose o f P M S G does not increase embryonic loss or fetal wastage i n immature rats (Nu t i et a l , 1975). In contrast, treatments w i t h greater doses (16-40 I U ) o f P M S G result i n reduced fer t i l i ty i n immature rats, a l though an increased number o f oocytes are ovulated. Reduced fe r t i l i za t ion rates, an increase i n ear ly embryon ic loss and fa i lure o f implan ta t ion were observed i n immature rats f o l l o w i n g a single injection w i t h a greater dose o f P M S G ( M i l l e r and A r m s t r o n g , 1981a; M i l l e r and A r m s t r o n g , 1982; W a l t o n et a l . , 1983; Y u n et a l . , 1987; Y u n et a l . , 1988; Y u n et a l . , 1989). These detr imental effects appear to become more apparent as the P M S G dose increases. Fur thermore , the detr imental effects caused by a large dose o f P M S G are also observed i n the adult (Boo th et a l . , 1975; Bet ter idge, 1977; M o o r et a l . , 1980; M o o r et a l . , 1985). c. Pract ica l Aspec t Other than the b io log ica l aspect discussed, there are some other pract ical advantages i n us ing the immature rat m o d e l . Immature rats are e c o n o m i c a l , easy to handle and are read i ly avai lable f rom a c o m m o n supplier. Immature rats are especia l ly useful i n studies associated w i t h pregnancy or pseudopregnancy. A single inject ion o f P M S G can be used to produce t imed pregnancies i n large number o f immature rats. T h i s saves labor ious and time-consuming examina t ion o f vag ina l smears required i n exper iments i n v o l v i n g adult - 1 6 -rats. Thus , immature rats appears to be a suitable m o d e l for basic studies i n reproduct ion. Treatment w i t h 4 I U P M S G c o u l d serve as a con t ro l w h i l e treatment w i t h 4 0 I U P M S G achieves superovulation and has exaggerated detrimental effects. D . Detr imenta l Effects o f Superovulation a. Genera l Remarkable progress has been made i n the management o f infert i l i ty dur ing the last decade. I V F - E T procedures have become an indispensable treatment opt ion for many types o f infer t i l i ty . S ign i f ican t and profound advances have been ach ieved i n both the c l i n i c a l and basic science studies a imed at i m p r o v i n g I V F - E T outcome. H o w e v e r , these advances have not translated into d ramat ica l ly higher pregnancy rates. A number o f investigators have attributed reduced fer t i l i ty to abnormali t ies or fa i lure i n the process o f fer t i l iza t ion, ear ly loss o r abnormal development o f preimplantat ion embryos , implanta t ion failure and h igh fetal wastage. A l t h o u g h no s ingle factor can be attributed to the detr imental effects a ssoc ia ted w i t h I V F - E T , adverse effects o f o v a r i a n h y p e r s t i m u l a t i o n caused by superovulatory treatments are undoubtedly one o f the major factors ( M o o n et a l . , 1990). T h e adverse effects o f ova r i an hypers t imula t ion , resu l t ing f r o m superovulatory treatments w i t h exogenous gonadotropins , have been r e c o g n i z e d since the ear ly use o f I V F - E T . S tandard superovula tory pro tocols current ly use p h a r m a c o l o g i c a l dosages o f exogenous gonadotropins to obtain mul t ip l e oocytes. A s a result , fe r t i l ized embryos are replaced into a disturbed uterine environment . Increased ear ly embryon ic loss and failure o f implanta t ion are c o m m o n after superovulat ion and have been l i n k e d to a non-receptive endomet r ium. A large v o l u m e o f endocr ino log ica l and h i s to log ica l studies suggests that superovula tory treatment prevents the uterine e n d o m e t r i u m f r o m b e c o m i n g recept ive through hyperestrogenemia or an increased rat io o f estrogen/progesterone. H o w e v e r , the mechanisms by w h i c h imbalance i n ovarian steroid hormones causes the detrimental effects o n p re implan ta t ion e m b r y o n i c development and the implan ta t ion process r e m a i n to be - 1 7 -elucidated. b. O v a r i a n Steroid Hormones R e d u c e d f e r t i l i t y rates after s u p e r o v u l a t i o n h a v e b e e n a t t r i b u t e d to h y p e r e s t r o g e n e m i a o r an increase i n es t rogen/proges terone ra t io i n m a n y species (Ba ranczuck and G r e e n w a l d , 1973; M o r r i s and V a n W a g e n e n , 1973; B o o t h et a l . , 1975; G r e e n w a l d , 1976; Fu j imo to and Tanaka , 1977; Saumande, 1980; M i l l e r and A r m s t r o n g , 1981a; M i l l e r and A r m s t r o n g , 1982; W a l t o n and A r m s t r o n g , 1982; W a l t o n et a l . , 1983; L a u f e r et a l . , 1986; Y u n et a l . , 1988; Safro et a l . , 1990). Es t rogen adminis te red after ovu la t ion has anti-fert i l i ty effects i n many species (Greenwald , 1961; G i d l e y - B a i r d et a l . , 1986; F o r m a n et a l . , 1988). H o w e v e r , freshly ovula ted , normal -appear ing oocytes and embryos (at least dur ing the first 36 h) after superovulation have an equal abi l i ty to ferti l ize and deve lop to the fetal stage as d o the c o n t r o l oocytes and embryos , i f they were transferred in to a n o r m a l o v i d u c t a l and uterine envi ronment , respec t ive ly ( M i l l e r and A r m s t r o n g , 1982; W a l t o n and A r m s t r o n g , 1983). Changes i n the levels o f ovar ian steroid hormones appears to render the ov iduc ta l and uterine environment detrimental to ferti l ization and embryonic development. Ov iduc ta l f lu id , obtained f rom mice that have experienced h igh estrogen levels , become detrimental to ear ly e m b r y o n i c development as determined i n cultures ( C l i n e et a l . , 1977). T h e uterine microenvironment associated wi th elevated estrogen/progesterone ratios also detrimental to the blastocyst me tabo l i sm and R N A synthesis i n m i c e (Safro et a l . , 1990). T h e rate o f fer t i l iza t ion and the ab i l i ty o f embryos to deve lop to the blastocyst stage is substantial ly impai red i n rats w h i c h have experienced h igh serum estrogen levels for extended periods o f t ime p r i o r to, and after, ovu l a t i on (Fugo and Bu tche r , 1966; B u t c h e r and Pope , 1979, M i l l e r and A r m s t r o n g , 1981a; M i l l e r and A r m s t r o n g , 1982; W a l t o n et a l . , 1983) . Superovula tory treatment, w i t h a large dose o f P M S G , increases ear ly embryon ic loss and results i n fai lure o f implanta t ion i n immature rats ( M i l l e r and A r m s t r o n g , 1981a,b). T h e d i s turbed endocr ine env i ronment asynchronizes e m b r y o n i c and uterine deve lopment - 1 8 -(Wa l ton and A r m s t r o n g , 1982). c. H i s t o l o g y A n increase i n uterine wet w e i g h t o f up to 2 - f o l d has been o b s e r v e d after superovulatory treatment i n immature rats ( M i l l e r and A r m s t r o n g , 1981a). T h i s increase is a c c o m p a n i e d b y the presence o f desquamated ce l l u l a r debr is i n the uterine f lush ings . H i s t o l o g i c a l examina t ion o f the debris suggests that i t is epi thel ia l hyperplas ia , suggesting that there is hypers t imula t ion o f the endometr ia l ep i the l ium. It has also been shown that superovulat ion m a y cause hypertrophy o f the uterine endometr ia l s t roma (Rennels , 1951; F a n g , 1988). Hyperes t rogenemia interferes w i t h the proper secretory transformation o f the endomet r ium ( M a t t e l et a l . , 1987). These structural f indings suggest that superovulatory doses o f exogenous gonadotropins m a y induce abnormal changes w h i c h results i n a non-receptive state i n the endometrium. H \ T H E I N S U L I N - L I K E G R O W T H F A C T O R ( I G F ) S Y S T E M A . I G F - I a. M o l e c u l a r Properties I G F - I is a member o f the insu l in- l ike peptide fami ly w h i c h also includes insu l in and I G F - I I . I G F - I is composed o f 70 amino acids and has structural s imi lar i t ies to p ro insu l in , I G F - I I and re l ax in (Rinderknecht and H u m b e l , 1978). Ma tu re I G F - I consists o f B (amino-terminal domain , 29 amino acids), C (12 amino acids), A (21 amino acids), and D (8 amino acids) domains . T h e B and A domains exhibi t approximate ly 4 0 % h o m o l o g y to the B and A chains o f i n s u l i n and approximate ly 6 0 % h o m o l o g y to the cor responding domains o f I G F - I I (Foy t and Rober ts , 1991). T h e amino ac id sequence o f I G F - I is h i g h l y conserved (>92%) among m a m m a l i a n species. F o r example , human, bovine , and porc ine I G F - I have ident ica l amino a c i d sequence, and difference i n three amino a c i d residues d is t inguish rat I G F - I f r o m that o f these species (Rinderknecht and H u m b e l , 1978; Shimatsu and R o t w e i n , 1987; F ranc i s , 1988; F ranc i s , 1989). A t runcated var ian t f o r m o f I G F - I l a c k i n g the N - t e r m i n a l t r ipept ide w h i c h is - 1 9 -i n v o l v e d i n interactions w i t h I G F B P s has been isola ted i n many tissues, i n c l u d i n g human brain and porcine uterus (Sara et a l . , 1986; Ogasawara et a l . , 1989). T h i s var iant is at least 5 to 10 t imes more potent i n its b i o l o g i c a l ac t iv i ty than the nat ive peptide. It has been suggested that the N - t e r m i n a l t r ipept ide i s i t s e l f b i o l o g i c a l l y ac t ive i n the b ra in and stimulates acetylcholine release (Sara et a l . , 1989). I G F - I m R N A are seen i n mul t ip le size transcripts, due to alternate sp l i c ing i n the 5'-untranslated r e g i o n and the d is t inc t l ength o f 3 ' -untranslated r e g i o n , w h i c h is i t s e l f con t ro l l ed by dif ferent ia l po lyadeny la t ion site usage ( L u n d et a l . , 1989; Rober t s et a l . , 1989; F o y t and Roberts , 1991). T h e size o f m R N A transcripts varies w i d e l y f rom 0.8 k b to 15 k b , a l though 7.0-7.5 kb appears to be the most c o m m o n size o f transcript i n many tissues. b. Endocr ine Regula t ion T h i s g r o w t h factor was i n i t i a l l y de te rmined to be a med ia to r o f the g r o w t h p romot ing actions o f g rowth hormone ( G H ) (Scholenle et a l . , 1982). A c c o r d i n g l y , I G F - I synthesis i s G H - d e p e n d e n t i n l i v e r and many other tissues and i s present i n s ignif icant quantit ies i n p lasma, due to its secretion by the l i v e r ( D ' E r c o l e et a l . , 1984; L u n d et a l . , 1986; Rober ts et a l . , 1986; M u r p h y et a l . , 1987a,b). W h i l e G H exerts a body w i d e effect i n I G F - I regula t ion, other hormones seem to exert tissue or ce l l - type specif ic effects. O f these, estrogen is the best studied and the most important regulator o f I G F - I synthesis i n the reproduct ive organs. Es t rogen stimulates I G F - I synthesis i n the uterus and granulosa ce l l s but appears to have no effect, or even inhibi t , hepatic I G F - I synthesis ( M u r p h y et a l . , 1987c; M u r p h y and Fr iesen , 1988). G H has o n l y a m i n o r effect, by compar i son , o n I G F - I synthesis i n the uterus ( M u r p h y and Fr iesen , 1988). T h e mechanisms by w h i c h G H and estrogen increase I G F - I m R N A levels appear to be different. Pro te in synthesis is required i n G H - i n d u c e d I G F - I synthesis but not i n estrogen-induced I G F - I synthesis ( M u r p h y and L u o , 1989). - 2 0 -Estrogen also increases I G F - I m R N A levels i n the bone. In this tissue, parathyroid ho rmone increases synthesis o f I G F - I at m R N A and peptide levels (Ernst et a l . , 1989; M c C a r t h y et a l . , 1989). Gonadot ropins l i k e F S H and L H stimulate gonadal I G F - I m R N A levels without an increase i n c i rcula t ing I G F - I levels (Hsu and H a m m o n d , 1987; Closset et a l . , 1989). Dexamethasone decreases I G F - I m R N A levels i n cu l tu red b ra in ce l l s and reduces the basal or G H - s t i m u l a t e d synthesis o f I G F - I i n m a n y tissues in vivo ( A d a m o et a l . , 1988; L u o and M u r p h y , 1989). T h y r o i d hormone increases hepatic and pi tui tary c e l l I G F - I m R N A levels (Fag in et a l . , 1989a; W o l f et a l . , 1989). A l t h o u g h thyro id hormone a lone d i d not affect I G F - I m R N A leve l s , pre- o r co-treatment w i t h t h y r o i d ho rmone enhances G H - i n d u c e d I G F - I synthesis i n the rat l i v e r ( W o l f et a l . , 1989) . T h y r o i d hormone act ion o n I G F - I m R N A levels i n a pituitary c e l l l ine c o u l d also be media ted by endogenous G H secretion (Fag in et a l . , 1989a). In addi t ion, i n su l in increases I G F - I m R N A levels i n cul tured hepatocytes (Johnson et a l . , 1989). M o s t studies e x a m i n i n g the effect o f i n su l i n o n the regula t ion o f I G F - I has been done w i t h respect to diabetes. In the rat diabetes m o d e l , reduced basal I G F - I levels were f o u n d i n the l i v e r and other tissues. Fur thermore , G H is no longer capable o f i n d u c i n g I G F - I synthesis ( F a g i n et a l . , 1989b) . B a s a l uterine I G F - I l eve l s are not s igni f icant ly reduced; however , estrogen-induced I G F - I synthesis i s impa i r ed i n diabetic rats ( M u r p h y , 1988). Insul in treatment i n diabetic rats restored basal I G F - I levels and G H -and estrogen-induced I G F - I synthesis ( M u r p h y , 1988; F a g i n et a l . , 1989a). T h e regulat ion o f I G F - I synthesis by I G F s is p o o r l y understood. I G F - I I secret ing tumors decreased c i r c u l a t i n g I G F - I l eve l s ; however , the same study fa i l ed to demonstrate a decrease i n hepatic I G F - I m R N A levels ( W i l s o n et a l . , 1989). In rats fed an energy-restr icted diet, I G F - I infus ion decreased hepatic I G F - I m R N A levels . Energy-res t r ic t ion o r fasting per se a lso reduces hepat ic I G F - I m R N A leve ls w h i c h is caused p r i m a r i l y b y reduced G H secret ion and hepatic resistance to G H resul t ing f r o m d o w n regula t ion i n G H receptor express ion ( E m l e r and S c h a l c h , 1987; L o w e et a l . , 1989; Straus and T a k e m o t o , 1990; - 2 1 -Th i s sen et a l . , 1990). c. B i o l o g i c a l A c t i o n A l t h o u g h I G F s and i n s u l i n share many structural and func t iona l features, these l igands have s igni f icant ly different b io log i ca l roles. Insul in is a k e y regulator o f systemic and c e l l u l a r m e t a b o l i s m w h i c h inc ludes g lucose transportat ion, and g l y c o g e n and fat b iosynthesis . In contrast, I G F - I appears to be a more potent mi togen and mediates the g rowth p romot ing actions o f G H . Fur thermore, I G F - I appears to funct ion not o n l y i n the c lass ica l endocrine manner but also i n the autocrine and/or paracrine mode. T h e abi l i ty o f I G F - I to regulate D N A synthesis, c e l l prol iferat ion and ce l lu lar metabol i sm i n an autocrine and paracrine fashion has been w e l l documented ( W i l l i a m , 1991). I G F - I has also been i m p l i c a t e d i n the con t ro l o f c e l l d i f ferent ia t ion and i n tissue repa i r and regenerat ion ( W i l l i a m , 1991). B . I n s u l i n - L i k e G r o w t h Fac tor B i n d i n g Proteins ( I G F B P s ) a. Genera l I G F B P s are a group o f structural ly homologous proteins that b i n d I G F - I and II. T h e nomencla ture o f I G F B P s is somewhat confus ing ( B a l l a r d et a l . , 1989; D r o p s and H i n t z , 1989). S o m e I G F B P s have been g iven several names accord ing to the source o f pur i f i ca t ion and the est imated mo lecu la r weight o f proteins. I G F B P s n o w g o under the nomenclature; I G F B P - x where 'x ' is the number w h i c h reflects the order i n w h i c h fu l l sequence o f c D N A was pub l i shed . Consequent ly , s ix I G F B P s ( I G F B P - 1 to I G F B P - 6 ) have been iden t i f i ed and n a m e d to date. S o m e I G F B P s have var ian ts w i t h d is t inc t molecula r weights caused by posttranslational modif icat ions that inc lude g lycosy la t ion and phosphoryla t ion (Rechler et a l . , 1989; C l e m m o n s , 1991; C l e m m o n s , 1993). I G F B P s possess c o m m o n structural features such as s m a l l prepeptides (s ignal peptides) o f s imi lar size and cysteine-rich areas i n the amino and ca rboxy l terminal regions. T h e amino a c i d sequences i n the amino and c a r b o x y l terminal cys te ine-r ich areas show a - 2 2 -h i g h degree o f h o m o l o g y across I G F B P s , whereas the centra l areas are less conserved. S i g n a l peptides and the cys te ine- r ich amino te rmina l are h i g h l y hyd rophob ic , w h i l e the r ema in ing areas are hydrophy l i c (Lee et a l . , 1988). T h e cys te ine-r ich amino and c a r b o x y l te rmina l regions appear to be responsible for l i g a n d b ind ing , al though evidence indicates that non-cysteine amino terminal residues m a y also be i n v o l v e d i n l i gand b i n d i n g (Huhtala et a l . , 1986; L e e et a l . , 1988; B r i n k m a n et a l . , 1991). E a c h b i n d i n g protein also has some un ique proper t ies . F o r e x a m p l e , I G F B P - 1 and -2 have an R G D sequence near the ca rboxy l terminal . The R G D sequence is bel ieved to mediate trie b ind ing o f matr ix proteins to structurally related receptors, k n o w n as the integrins (Rouslaht i and Pierchbaker , 1988). b. Regula t ion and Func t ion I G F B P - 1 is present at n g / m l levels i n the p lasma, and i s the o n l y I G F B P where the p lasma l e v e l varies f rom minute to minute, due to ho rmona l and metabo l ica l ly regulations (Baxte r and J . L . , 1989; H a l l et a l . , 1991; H o l l y , 1991; L e e et a l . , 1993). Insu l in , G H , I G F - I , estrogens, g lucose and g lucocor t i co ids appear to suppress I G F B P - 1 synthesis , w h i l e g lucagon, c A M P , protein kinase C , theophyl l ine , progesterone and fasting stimulate I G F B P - 1 synthesis (Baxte r and J . L . , 1989; H a l l et a l . , 1991; H o l l y , 1991; L e e et a l . , 1993). I G F B P - 3 i s the most abundant b i n d i n g prote in i n the c i r cu la t ion ex i s t ing at mg/1 levels , f o l l o w e d by I G F B P - 2 to approximately 1/10 less extent (Lee et a l . , 1993). I G F B P -3 forms a 140-150 k D a c o m p l e x w i t h an I G F - I or I G F - I I m o l e c u l e and an 88 k D a a c i d labi le subunit i n the b lood . Th i s complex is generally bel ieved to serve as a carrier and as a reservo i r for I G F s . A l t h o u g h the exact r o l e o f I G F B P s i s unclear , the t issue-specif ic h o r m o n a l r egu la t ion o f I G F B P s suggests that they func t ion i n both a paracr ine and autocrine fashion. In general , I G F B P s inh ib i t I G F - I actions, p r i m a r i l y by regula t ing the amount o f free I G F - I i n these tissues. H o w e v e r , the funct ion o f these b i n d i n g proteins can be diverse, depending upon the c e l l type and other factors. Enhancement o f I G F - I actions by I G F B P s has been reported ( E l g i n e t a l . , 1987). I G F B P - 3 appears to have a bi funct ional - 2 3 -role that c o u l d be based o n its c e l l surface association (De M e l l o w and Baxter , 1988). C e l l surface associa ted I G F B P - 3 enhanced I G F - I ac t ion o n D N A synthesis i n f ibroblas t cul tures, w h i l e free I G F B P - 3 inh ib i t ed I G F - I ac t ion i n the same sys tem. Fur thermore , I G F B P - 3 , -4 , and -5 have been s h o w n to b i n d to e x t r a c e l l u l a r m a t r i x p ro te ins . E x t r a c e l l u l a r mat r ix -assoc ia ted I G F B P - 5 enhanced c e l l g r o w t h i n response to I G F - I ( C l e m m o n s , 1993; Jones et a l . , 1993). C . I G F - I Receptors a. Genera l I G F - I b inds to I G F - I , I G F - I I , and in su l in receptors w i t h different affinit ies. L i k e their l igands, I G F - I and insu l in receptors are structurally s imi lar to each other (Bhaumick et a l . , 1981; Chernausek et a l . , 1981). In contrast, the I G F - I I receptor has a ve ry different structure w h i c h i s i den t i ca l to mannose 6-phosphate receptors ( M o r g a n et a l . , 1987; O s h i m a et a l . , 1988). I G F - I and i n su l i n receptors are tetrameric g lycopro te ins w h i c h are composed o f two ct-subunits and two p-subunits (H in t z et a l . , 1972; L e B o n et a l , 1986). T h e a-subuni t is entirely extracellular , contains a cyste ine-r ich d o m a i n and is responsible for l i gand b ind ing , w h i l e the P-subunit has a cy top lasmic tyrosine kinase d o m a i n . I G F - I and i n s u l i n receptors are members o f the subclass I I o f tyros ine k inase receptor f a m i l y (Yarden and U l l r i c h , 1988). T h e I G F - I receptor demonstrates heterogeneity through dist inct g lycosy la t ion . F o r example , a less iV-g lycosy la t ed f o r m o f the a-subuni t is found i n the bra in ( M c E l d u f f et a l . , 1988; O t a et a l . , 1988). A n o t h e r source o f heterogeneity i s fo rmat ion o f h y b r i d receptor w i t h i n su l in receptor subunits. T h e structural s imilar i t ies a l l ow insu l in and I G F - I receptors to f o r m h y b r i d receptors in vitro and in vivo (Fel tz et a l . , 1988; L a m m e r s et a l . , 1989; T r e a d w a y et a l . , 1989; W i l d e n et a l . , 1989). A m o n o c l o n a l an t ibody , w h i c h r ecogn izes the C - t e r m i n a l r e g i o n o f the i n s u l i n receptor but not the I G F - I receptor , immunoprecipi ta ted a receptor that has two phosphorylated P-subunits o f distinct molecular - 2 4 -weights (Garofa lo and R o s e n , 1989). T h e molecu la r we igh t o f each P-subunit represents that o f the phosphoryla ted P-subunits o f i n su l i n and I G F - I receptors, respect ively . Other examples o f heterogeneity o f the I G F - I receptor also can be exp la ined by in t roduc ing the h y b r i d receptor concept (Burant et a l . , 1987; I z u m i et a l . , 1987; K a d o w a k i et a l . , 1987; A l e x a n d r i d e s and S m i t h , 1989). These h y b r i d receptors have been re la ted to some pathophysio logica l condit ions such as tumor formation and diabetes. b. F u n c t i o n I G F - I receptor recognizes I G F - I I and in su l in , w h i l e I G F - I I and i n su l i n receptors i n turn recognize I G F - I . These cross-bindings are d is t inguishable by b i n d i n g assay due to dis t inct b i n d i n g affinities ( M a r s h a l l et a l . , 1974; Rech l e r et a l . , 1980; Jonas et a l . , 1982; F l i e r et a l . , 1986; L a m m e r s et a l . , 1989). A l t h o u g h the s ignif icance o f these cross-binding capabi l i t ies is not f u l l y understood, i t is suggested that they m a y be a part o f mechanisms that a l l ow I G F - I or related peptides to exert a variety o f actions i n many c e l l types. Desp i t e the structural s imi la r i t ies and l i g a n d c ross -b ind ing capabi l i t ies between in su l in and I G F - I receptors, these two receptors appear to have dist inct funct ional roles. A differential s ignal ing potential o f the cytoplasmic domain o f the P-subunit o f human insu l in and I G F - I receptors has been demonstrated i n a mouse fibroblast c e l l l ine (Lammers et a l . , 1989). B o t h in su l in and I G F - I receptor cy top lasmic domains have a compat ib le s ignal ing potent ial i n the short-term effect, such as s t imulat ion o f g lucose transportation. H o w e v e r , the cy top lasmic d o m a i n o f I G F - I receptor appears to have a greater s igna l ing potent ial i n the long- te rm effect such as mi togen ic actions (Lammers et a l . , 1989). T a k e n together, these observations demonstrate that the I G F - I receptor is more potent than insu l in receptors i n med ia t ing mi togen ic and growth p romo t ing ac t iv i ty , w h i l e the i n s u l i n receptor has a greater potent ia l i n ce l l u l a r energy m e t a b o l i s m ( H i n t z et a l . , 1972; F l i e r et a l . , 1986; L a m m e r s et a l . , 1989; W i l l i a m , 1991). - 2 5 -D . T h e I G F Sys t em i n the Uterus a. I G F - I T h e rat uterus is a major site o f I G F - I synthesis. I G F - I synthesis i n the uterus is p r i m a r i l y under estrogen regula t ion, and to a lesser extent G H regula t ion ( M u r p h y et a l . , 1987c; M u r p h y and F r i e sen , 1988; Nors ted t et a l . , 1989; C a r l s s o n and B i l l i g , 1991). Exogenous gonadotropins or estrogen injections into rats cause an increase i n the levels o f uterine I G F - I m R N A and pept ide ( M u r p h y et a l . , 1987c; M u r p h y and F r i e sen , 1988). Proges terone m a y also p l a y a ro le i n the regu la t ion o f I G F - I synthesis i n the uterus (Norstedt et a l . , 1989; C r o z e et a l . , 1990a; K a p u r et a l . , 1992). I G F - I m R N A has been l o c a l i z e d to the s troma and m y o m e t r i u m o f the rat uterus (Ghahary et a l . , 1990). I G F - I m R N A transcripts are abundant i n the ant imesometr ial s t romal tissue and has been further l o c a l i z e d to the per iepi thel ia l and per iglandular s t romal ce l l s (Croze et a l . , 1990a). I G F - I levels , both protein and m R N A , vary i n the uterus dur ing the estrous c y c l e and is m a x i m a l d u r i n g the p re imp lan ta t ion pe r iod . In contrast , there i s no change i n se rum I G F - I concentrations dur ing the same pe r iod ( M u r p h y et a l . , 1987c; K a p u r et a l . , 1992; K a t a g i r i et a l . , 1996). M u l t i p l e size transcripts o f I G F - I m R N A has been descr ibed i n the uterus. Uter ine I G F - I m R N A levels increase du r ing the pre implanta t ion p e r i o d i n rats, m i c e , p igs , sheep and c o w s (Letcher et a l . , 1989; C r o z e et a l . , 1990a; Geiser t et a l . , 1991; K o et a l . , 1991; K a p u r et a l . , 1992) . In m i c e , the 7.0 k b transcript decreases o n D a y 5 and 6, after in i t i a t ion o f implan ta t ion , w h i l e the leve ls o f the other smal le r transcripts r e m a i n h igh ( K a p u r et a l . , 1992). T h e decl ine i n the levels o f I G F - I m R N A transcripts at the t ime o f implan ta t ion has also been observed i n the rat uterus (Croze et a l . , 1990a). In p igs and sheep, the concentration o f uterine l u m i n a l I G F - I and its m R N A levels i n the uterine tissues is higher dur ing early pregnancy compared w i t h its corresponding days i n the estrous cyc le ( K o et a l . , 1991). I G F - I levels i n the uterine l u m i n a l f luids and I G F - I m R N A i n the uterus - 2 6 -increase steadily throughout the preimplantation period. T h i s is f o l l owed by a rap id decline after the ini t iat ion o f implantat ion i n the p i g (Letcher et a l . , 1989). A h igh molecula r weight variant w i t h molecula r mass o f 18 k D a , but not the 7 k D a f o r m , o f I G F - I has been detected i n the rat uterus ( M u r p h y and G h a h a r y , 1990) . A truncated I G F - I mo lecu l e has also been i so la ted f r o m the p i g uterus (Ogasawara et a l . , 1989) . T h i s variant lacks an N - t e r m i n a l tr ipeptide w h i c h is i n v o l v e d i n interactions w i t h I G F B P s , i n d i c a t i n g a greater b ioac t iv i t y . T h e s ign i f i cance o f these var iants requires clarif icat ion. b . I G F B P s I G F B P s are be l i eved to p l a y a central ro le i n the uterine I G F sys tem w h i c h is i n v o l v e d i n regula t ing uterine g rowth and funct ion. In par t icular , the ro le o f I G F B P - 1 , w h i c h has been associated w i t h implanta t ion, has d rawn m u c h attention i n the rodent and primate. H o w e v e r , the specific role o f the I G F B P s remains poo r ly characterized. I G F B P s have been detected i n the rat uterus. I G F B P - 1 has been l o c a l i z e d to the uterine epi the l ium, stroma and decidual tissue i n the rat (Croze et a l . , 1990a; M u r p h y et a l . , 1990) . I G F B P - 1 levels va ry dur ing the estrous c y c l e , w i t h m a x i m a l levels be ing observed at diestrus and nadir at proestrus (Ghahary and M u r p h y , 1989). T h i s express ion pattern is the inverse to the uterine I G F - I receptor prof i le . I G F B P - 1 m a y act as an inhib i tor and also a reservo i r for I G F - I d u r i n g diestrus. T h i s w o u l d a l l o w m a x i m a l I G F - I ac t ion d u r i n g proestrus w h e n the I G F - I receptor levels are greatest ( M u r p h y , 1991). T h i s system seems to assure a r ap id growth o f the uterine endometr ium dur ing proestrus. I G F B P - 1 m R N A is abundant i n the decidual ized uterus, indicat ing a possible role for the uterine I G F system i n decidual izat ion. The role o f the I G F system i n decidual izat ion w i l l be discussed later. I G F B P - 2 and -4 have been l o c a l i z e d to the ep i the l ium. In contrast, I G F B P - 3 has been l o c a l i z e d to the s t roma and I G F B P - 5 and -6 to the m y o m e t r i u m and the serosa ( G i r v i g i a n et a l . , 1994). T h e m R N A levels o f each I G F B P varies dur ing the estrous c y c l e - 2 7 -and early pregnancy ( G i r v i g i a n et a l . , 1994). c. I G F - I receptor I G F - I receptors have been detected i n the rat uterus (Ghahary and M u r p h y , 1989; Chandrasekhar et a l . , 1990). T h e b ind ing affinity and capaci ty o f uterine I G F - I receptors are s i m i l a r to those found i n other t issues. T h e major i ty o f I G F - I b i n d i n g bas been l o c a l i z e d to the smooth musc le layer by autoradiography (Ghahary and M u r p h y , 1989). S ince I G F - I has also been loca l i zed to the myomet r i a l and stromal layer, I G F - I m a y p lay a ro le i n m y o m e t r i a l and s t romal c e l l g rowth and funct ion i n the rat uterus (Ghahary et a l . , 1990). H o w e v e r , this does not exc lude the presence o f I G F - I receptors and their potential ro le i n other layers o f the uterus. Admin i s t r a t i on o f estrogen increases the number o f I G F - I receptors i n the uterus o f h y p o p h y s e c t o m i z e d and o v a r i e c t o m i z e d adult rats and i m m a t u r e rats (Ghaha ry and M u r p h y , 1989; Chandrasekha r et a l . , 1990). In mature c y c l i n g rats, I G F - I b i n d i n g capaci ty is greatest d u r i n g proestrus and lowes t du r ing diestrus (Ghahary and M u r p h y , 1989). A t present, it is not clear whether uterine I G F - I receptors are d i rec t ly regulated by estrogen. S ince the increase i n I G F - I m R N A levels precedes the increase i n the number o f I G F - I receptors seen i n response to estrogen adminis t ra t ion, I G F - I m a y also mediate the estrogen effect o n I G F - I receptor synthesis ( M u r p h y , 1991). HI . G R O W T H F A C T O R S I N P R E I M P L A N T A T I O N E M B R Y O N I C D E V E L O P M E N T A . Paracrine and Autocr ine Regulat ion T h e establ ishment o f a successful pregnancy requires synch ron i zed g rowth and differentiation o f the preimplantat ion embryo and the uterine endometr ium. It is apparent that ovar ian steroid hormones coordinate the synchron ized growth and differentiat ion o f embryos and the uterine endomet r ium ( F i n n and M a r t i n , 1967). These effects m a y be mediated by growth factors, der ived f rom the oviduct and uterus i n a paracrine manner. In general , p re implanta t ion embryos g r o w more s l o w l y and less successful ly in - 2 8 -vitro. Co-cu l tu re o f embryos w i t h somatic ce l l s , such as o v i d u c t a l and uterine ce l l s , o r even ce l l s not de r ived f rom the genital tract, augment embryon ic development (Bavis ter , 1988). A n adequate o v i d u c t a l and uterine envi ronment m a y o p t i m i z e p re implan ta t ion e m b r y o n i c development through mu l t i p l e mechanisms that i nc lude secret ion o f g rowth-p r o m o t i n g factors. P re implan ta t ion e m b r y o n i c deve lopment i n mos t species requires supplementat ion o f g rowth-p romot ing factors such as serum to the culture m e d i u m . It appears that g rowth factors and cy tok ines i n se rum and f r o m co-cu l tu red ce l l s m a y be l a rge ly responsible fo r the g rowth-promot ing effect on e m b r y o n i c development . M a n y g r o w t h factors and c y t o k i n e s that are f o u n d i n the o v i d u c t and uterus have been demonstrated to promote embryon ic development , but some o f them have been shown to inhib i t or to have no effect on embryonic development (Br igs tock et a l . , 1989; S i m m e n and S i m m e n , 1991; A d a m s o n , 1993). T h e presence o f funct ional receptors for g rowth factors i n preimplantat ion embryos is compatible to these observations (Rappolee et a l . , 1991). In contrast, preimplantat ion mouse embryos grow to the blastocyst stage i n a s imple de f ined cu l ture m e d i a , suggest ing that the deve lopmen ta l process o f p re implan ta t ion embryos m a y be autonomous. Deve lopmen t o f 2 -ce l l mouse embryos and the number o f ce l l s per blastocyst are superior i n cultures w i t h mul t ip l e embryos to i n cultures w i t h a single embryo (Par ia and D e y , 1990). T h i s suggests that embryon ic autocrine factors may be important i n embryon ic development, and that concentrations o f these factors i n culture media m a y not reach effective concentrations i f cultured i n relat ively large culture m e d i u m droplet. T h e presence o f l i gand and receptor for many growth factors supports this concept. E x p r e s s i o n o f m R N A s for a var ie ty o f g rowth factors and their receptors have been detected i n the pre implanta t ion e m b r y o (Rappolee et a l . , 1988; Rappo lee et a l . , 1991). E v i d e n c e suggests that these m R N A s are translated in to funct ional proteins (Rappolee et a l . , 1991; A d a m s o n , 1993; S c h u l t z and H e y n e r , 1993). T a k e n together, these f indings suggest that these g rowth factors regulate pre implanta t ion development i n an autocrine - 2 9 -manner. Immediate ly after fer t i l izat ion, embryonic development appears to be cont ro l led by the express ion o f m R N A transcripts o f maternal o r i g i n . T h e transi t ion f rom maternal to embryon ic cont ro l i n ear ly development occurs at a certain stage between the 2 -ce l l stage and the 16-ce l l stage, depending upon the species (Te l fo rd et a l . , 1990). T h e transi t ion occurs at the 2-ce l l stage i n m ice (Schultz, 1986), between the 4-ce l l and 8-cel l stages i n p i g and humans (Tesar ik et a l . , 1986; Tesar ik et a l . , 1987a; Braude et a l . , 1988), between the 8-ce l l and 16-ce l l stages i n cows and sheep (Camous et a l . , 1986; C r o s b y et a l . , 1988; F r e i et a l . , 1989), and at the 16-cel l stage or later i n rabbits (Manes , 1973; M a n e s , 1977). In m i c e , ref lect ing the transition o f m R N A expression f rom maternal to embryonic o r i g i n , abundance o f m R N A transcripts s h o w four d i s t inc t t e m p o r a l patterns: (1) undetectable unt i l the blastocyst stage; (2) present as maternal transcripts w h i c h decrease i n l eve l s due to the des t ruc t ion o f materna l t ranscripts and reexpressed as e m b r y o n i c transcripts; (3) t ranscribed o n l y as embryon ic transcripts; and (4) detectable throughout preimplantat ion development (Rappolee et a l . , 1991). The temporal expression patterns o f growth factors and their receptors i n preimplantation embryos may give insight into the role o f these growth factors i n the paracrine and autocrine regulation o f their development. B . Insu l in -L ike Peptides i n the Preimplantation E m b r y o n i c Development a. G e n e Expre s s ion Ini t ia l observations suggested that I G F - I m R N A transcripts were not present i n the preimplantat ion mouse embryos p r io r to the blastocyst stage (Werb , 1990; Rappolee et a l . , 1992). H o w e v e r , I G F - I m R N A transcripts have recently been detected i n mouse embryos (Doher ty et a l . , 1994). The m R N A transcript levels decrease f r o m oocytes to 8-cel l stage e m b r y o s and then increase f r o m the 8 -ce l l to the b las tocys t stages. I G F - I m R N A transcripts are a lso detectable at a l l stages o f p re implan ta t ion deve lopmen t i n bov ine embryos (Schul tz et a l . , 1992; W a t s o n et a l . , 1992). H o w e v e r , I G F - I m R N A transcripts -30-have not been detected at all stages of preimplantation development in the rat (Zhang et al., 1994). The pattern of IGF-I expression in embryonic development appears to be different across the species. In contrast, information about IGF-II and insulin in preimplantation embryos is consistent across all species. The presence of IGF-II mRNA and the absence of insulin mRNA have been reported in mouse, rat and bovine embryos (Rappolee et al., 1992; Watson et al., 1992; Zhang et al., 1994). Expression of IGF-I and insulin receptors has been detected from oocytes to blastocysts throughout preimplantation development (Zhang et al., 1994). The levels of mRNA transcripts for both receptors; however, show a temporal decline at the 4-cell and 8 cell stages (Zhang et al., 1994). Expression of IGF-I receptor mRNA becomes detectable at the 8-cell stage in the mouse (Werb, 1990; Rappolee et al., 1992; Schultz et al., 1992). Insulin receptor mRNA, which also mediates IGF-I actions, is detected in the mouse embryo at the 8-cell stage, the time when embryos enter the uterus (Werb, 1990; Rappolee etal., 1992; Schultz et al., 1992). IGF-II receptor mRNA is detectable at the 2-cell stage and at later stages in the mouse and rat (Werb, 1990; Rappolee et al., 1992; Schultz et al., 1992; Zhang et al., 1994). The stage-specific expression of these receptors has been evidenced by detection of these receptor proteins using an antibody against the receptors and gold- or radioisotope-labeled ligands (Mattson et al., 1988; Heyner et al., 1989; Harvey and Kaye, 1991a,b; Smith et al., 1993). In the cow, IGF-I, IGF-II, and insulin receptor mRNA transcripts have been detected throughout preimplantation development (Schultz et al., 1992; Watson et al., 1992). The preimplantation embryos appear to translate these mRNAs into functional receptors which allows the embryos to respond to exogenous IGF-I, IGF-II, and insulin (Harvey and Kaye, 1988; Harvey and Kaye, 1990; Zhang and Armstrong, 1990; Harvey and Kaye, 1991c; Harvey and Kaye, 1992a,b; Kaye et al., 1992; Rappolee et al, 1992; Smith et al., 1993). Expression of IGFBPs have been detected in preimplantation mouse embryos (Hahnel and Schultz, 1994). The mRNA for IGFBP-2, -3, and -4 are detectable -31-throughout preimplantation development, and IGFBP-6 mPvNA at the blastocyst stage. In contrast, IGFBP-5 mRNA is not detectable at any preimplantation stage. IGFBP-1 has not been examined in this study. Information for IGFBPs in preimplantation rat embryos is not available at this time. b. Growth Promoting Effects The effect of insulin-like peptides on the preimplantation embryonic development has been demonstrated in the mouse. IGF-I and insulin have been shown to be internalized through receptor-mediated endocytosis in the mouse embryo (Heyner et al., 1989; Smith et al., 1993). Insulin and IGF-I enhance the development of embryos, increase the number of cells in the embryo and stimulate protein synthesis in vitro (Harvey and Kaye, 1988; Harvey and Kaye, 1990; Harvey and Kaye, 1992a; Schultz et al., 1992). Although expression of embryonic IGF-II receptor mRNA is detectable as early as the 2-cell stage, there is no evidence that IGF-II plays a critical role in embryonic development prior to the compaction stage when the expression of insulin and IGF-I receptors becomes detectable (Rappolee et al., 1992). IGF-II accelerates blastocyst formation, increases the number of cells in the embryo and stimulated protein synthesis (Rappolee etal., 1992). Insulin, in the presence of amino acids, stimulates 8-cell stage embryos to develop to the blastocyst stage in the rat (Zhang and Armstrong, 1990). Embryos cultured to the blastocyst stage in the presence of insulin are capable of developing to day 18 fetuses at a greater rate than blastocysts cultured in the absence of insulin, when transferred to the receptive uterus (Zhang and Armstrong, 1990). The presence of mRNA transcripts for IGF-I receptor, but not for IGF-I, has been detected in the preimplantation rat embryos (Zhang et al., 1994). Together, along with information regarding the IGF system in the uterus, it appears that the uterine IGF system plays a role in preimplantation embryonic development in a paracrine manner in the rat. -32-IV. ELECTROLYTES IN PREIMPLANTATION EMBRYONIC DEVELOPMENT A. General The difference in electrolyte composition between cells and their microenvironment is a universal source of energy for cell growth and function. In particular, the sodium gradient across the plasma membrane has great importance. Sodium gradients across the plasma membrane are maintained by Na+/K+-ATPase which transports Na + and K + across the plasma membrane against their electrochemical gradient, using energy obtained from the hydrolysis of ATP. The sodium gradient, in turn, provides energy to transport protons, chloride, phosphate, glucose, amino acids and other substances by co- and countertransport mechanisms across the plasma membrane (Lechene, 1988; Cohen and Lechene, 1989; Biggers et al., 1991). These co- and countertransports are required to maintain cellular homeostasis and regulate cellular metabolisms. Furthermore, the proper intracellular pH and other electrolyte composition are necessary for optimal enzyme function and actions of growth factors that play a central role in preimplantation embryonic development (Pouyssegur et al., 1985; Somero, 1985; Moolenaar et al., 1988). The intracellular concentrations of Na +, K + , Cl", and H + have been determined in preimplantation mouse embryos (Powers and Tupper, 1977; Lee, 1987). These values are conflicting between the studies. For example, the intracellular concentration of Na + of the 2-cell stage embryos is reported to be 143-151 mM and 25 mM (Lee, 1987). The extremely high Na + concentration of the former study (Powers and Tupper, 1977) suggests that the 2-cell stage embryos of the study may be damaged to some extent. However, the same study has demonstrated that the plasma membrane permeabilites to Na + and K + of the 2-cell stage mouse embryo are compatible to those of other cell types (Powers and Tupper, 1977; Jain and Wagner, 1980). Furthermore, the Na+/K+-ATPase activity in the 2-cell stage mouse embryo is intact, suggesting that the embryos are normal (Powers and Tupper, 1977). The intracellular concentrations for electrolytes including these four ions need to be determined. -33-B. Electrolyte Transport in Preimplantation Embryonic Development a. Regulation of Intracellular pH The 2-cell stage mouse embryos appear to highly permeable to H + , being several orders of magnitude larger than that of other cell types (Baltz et al., 1990). The extremely high H + permeability in the 2-cell stage mouse embryo is of particular interest with respect to the intracellular pH regulation. Evidence suggests that the 2-cell stage mouse embryo lacks Na+-dependent intracellular pH regulatory systems, the Na + /H + antiporter and Na+-dependent HCOy-Cl" exchanger, that are the most common systems in other cell types (Baltz et al., 1990; Baltz et al., 1991a). The 2-cell stage mouse embryo appears to have no specific mechanism for alleviating intracellular acid loads, but possess a Na+-independent HCOy/Cl- exchange mechanism to recover from alkaline loads (Baltz et al., 1991a,b). The oviductal fluid has a high pH and K + concentration, both of which make the cells of the embryo alkaline (Leese, 1988; Biggers et al., 1991). Therefore, the specific mechanism for acid load relief may not be necessary in this particular stage of development. The extremely high H + permeability was diminished in the trophectoderm, but still present in the inner cell mass (ICM) (Baltz et al., 1993). Taken together, the pH and concentrations of K + , H C O 3 - , and Cl - in the oviductal and uterine environment may be critical for the intracellular pH regulation of preimplantation embryos; and therefore be critical to embryonic development. b. Compaction Loosely associated blastomeres in the embryos of the early cleavage stages become flattened against one another during the compaction process. The morphological change during the compaction process in the mouse embryo is believed to be caused by an extensive increase in Ca+-dependent cell-cell adhesion that is mediated by the surface glycoprotein, uvomorulin (Hyafil etal., 1980; Hyafil et al., 1981; Peyrieras et al., 1983; Johnson et al., 1986). Calcium appears to regulate proteolytic cleavage of uvomorulin that -34-is identical to E-cadherin (Yoshida and Takeichi, 1982; Shirayoshi et al., 1983; Yoshida-Noro et al., 1984), and homologous with cell adhesion molecule 120/80 in the human (Damsky et al., 1983). One of the essential features of compaction is the polarization of blastomeres, so that the blastomeres show distinct apical and basolateral membrane. Changes in the distribution pattern of uvomorulin is one of many examples of cell polarization. The protein is evenly distributed over the entire surface of the blastomere prior to compaction, while it becomes no longer detectable on apical cell surface of outer cells of compacted morulae, but remains entirely distributed on the inner cell surface (Vestweber et al., 1987). The distribution patterns of Na + /K +-ATPase in both uncompacted and compacted morulae are similar to those of uvomorulin (Vestweber et al., 1987; Watson and Kidder, 1988). The polarized distribution of Na +/K +-ATPase is particularly important with respect to electrolyte and fluid transport. Together with the formation of tight junctions between the cells of the outer surface of the embryos, the preimplantation embryos establishes the polarity of a plasma membrane and the transport mechanisms that are essential to subsequent blastocyst formation (Wiley, 1987; Biggers et al., 1991). Although the prevention of compaction and Ca+-dependent cell-cell adhesion do not prevent blastomeres from developing cell surface polarity (Ziomek et al., 1982), evidence suggests that uvomorulin plays a central role in the formation of tight junction and the polarization of Na +/K+-ATPase distribution (Johnson et al., 1986; Watson et al., 1990; Larue et al., 1994). These events were inhibited by an anti-uvomorulin (or E-cadherin) antibody in the 8-cell mouse embryos. c. Formation of Blastocoelic Cavity The polarized distribution of Na +/K +-ATPase to the basolateral plasma membrane is a commonly used marker of the basolateral domain of epithelial cells (Cereijido et al., 1989; Rodriguez-Boulan and Nelson, 1989). Expression of the protein is restricted to the -35-basolateral plasma membrane of the trophectoderm cells but remains evenly distributed on the surface of the ICM cells (Watson and Kidder, 1988). Indeed, the trophectoderm is believed to be the first epithelium developed during embryonic development (Wiley et al., 1990). Evidence suggests that Na+/K+-ATPase plays a central role in formation of the blastocoele; i.e. formation of blastocysts (Wiley, 1987). Blastocoele formation is inhibited or delayed by ouabain, a inhibitor of Na+/K+-ATPase, in the rabbit and mouse (DiZio and Tasca, 1977; Biggers et al., 1978; Wiley, 1984; Manejwala et al., 1989). The rate of blastocoele formation is inversely related to the concentration of extracellular K + (Wiley, 1984; Wiley, 1987). Evidence suggests that the effect of varying concentrations of K + is exerted through its ability to modulate Na+/K+-ATPase activity (Wiley, 1987; Dumoulin et al., 1993). Extracellular K + levels affect Na+/K+-ATPase activity by changing plasma membrane potential and passive ion fluxes that are caused by the current of Na + (Cohen et al., 1976; Wiley, 1984). Furthermore, substitution of either Na + or Cl - , but not K+, in embryo culture media reduced the rate of blastocoele expansion in the mouse, suggesting the role of Na+/K+-ATPase in blastocoele formation (Manejwala et al., 1989). The levels of Na+/K+-ATPase mRNA increase after compaction in the mouse and rabbit preimplantation embryos (Gardiner et al., 1990a,b). Interestingly, IGF-I has been shown to stimulate Na+/K+-ATPase synthesis (Madsen and Bern, 1993; Matsuda et al., 1993). The temporal expression patterns of mRNAs for the IGF-I and insulin receptors in the mouse and rat embryos appear to coincide with that of Na+/K+-ATPase (Rappolee et al., 1991; Zhang et al., 1994). These observations suggest that IGF-I may stimulate the formation of the blastocoele in the preimplantation embryo. C. Preimplantation Embryonic Development In Vitro The electrolyte composition of the embryo culture media has been well defined for laboratory animals; mostly in the mouse and hamster (Whitten, 1971; Whittingham, 1971; Bavister et al., 1983). The composition of the most commonly used culture media for -36-preimplantation embryos are based on Krebs-Ringer solution. All of these media are very simple and have similar electrolyte compositions. Conventional complex cell culture media, such as Ham's F-10 and tissue culture medium 199, have been chosen for embryo culture in most other species to satisfy the potentially complex but largely unknown nutritional requirements of the embryo. Classical culture media for the preimplantation mouse embryos allow 2-cell stage mouse embryos to develop into blastocysts which are capable of implantation and subsequent fetal development (Biggers, 1987). However, with the exception of some inbred and F l strains, the culture of 1-cell stage embryos has met with limited success due to a phenomenon termed the '2-cell block'. Modification of the culture media in compositions of energy substrates and ions, and supplementation of EDTA largely improved development of 1-cell embryos to blastocysts (Cross and Brinster, 1973; Abramczuk et al., 1977; Chatot et al., 1989; Gardner and Leese, 1990; Lawitts and Biggers, 1991). Some ions and energy substrates, which are commonly found in the defined culture media for preimplantation development, have been shown to be detrimental to preimplantation embryonic development. High NaCI and K+concentration in culture media is detrimental on mouse embryonic development, causing cell block and inhibition of blastocyst formation (Wiley, 1984; Lawitts and Biggers, 1991; Dumoulin et al., 1993). Conversely, low levels of NaCI allow glutamine to impair embryonic development, although glutamine appears to be a preferred energy substrate during early development in the mouse (Chatot et al., 1989; Lawitts and Biggers, 1992). In contrast, glucose is detrimental to embryonic development at early stages especially prior to the morula stage (Seshagiri and Bavister, 1989a; Lawitts and Biggers, 1991). Phosphate plays a role in the toxic effects of glucose on early preimplantation embryonic development (Schini and Bavister, 1988; Seshagiri and Bavister, 1989b; Seshagiri and Bavister, 1991). Thus, the electrolyte environment affects preimplantation embryonic development. -37 -Although our knowledge is still limited, the electrolyte environment appears to play a central role in preimplantation embryonic development. D. Electrolytes in the Uterus Changes in electrolyte composition of uterine luminal fluid has been examined during delayed implantation in the rat and mouse, in which diapause of embryos at the blastocyst stage occurs as a normal part of reproduction. A decrease in N a + levels has been found in the uterine luminal fluids of the rat and mouse (Setty et al., 1973; Van Winkel, 1977; Van Winkle et al., 1983). This has been interpreted as being part of the mechanism that regulates embryo metabolism, since embryos become metabolically less active during the delayed implantation period (McLaren, 1973; Weitlauf et al., 1979; Weitlauf and Kiessling, 1980). Low levels of N a + have been shown to suppress embryo metabolism (Van Winkel, 1977; Van Winkel, 1981). However, low levels of N a + in culture media may impair viability of embryos in vitro. During the delayed implantation period, the detrimental effect of the low levels of N a + appears to be compensated against by some unknown mechanism. As progesterone is the dominant steroid hormone in the uterus at this time, progesterone may be involved in this regulatory mechanism. V. DECIDUALIZATION A. General Decidualization is a conspicuous and critical part of implantation in species that have an true placenta, such as Carnivora, Rodenta and Primates. Since Loeb's original studies in 1908 (Loeb, 1908a,b), the process of decidualization has been studied in many species. Decidualization involves many cytological events and has been likened to the inflammatory reaction. Localized changes associated with decidualization are characterized by cell proliferation and differentiation, reorganization of the extracellular matrix, and infiltration of macrophages from the circulation (Aplin, 1991; Abrahamsohn and Zorn, 1993). -38-Decidual transformation of the stromal cells in the endometrium results in apparent increases in the size and weight of the uterus. The embryo becomes embedded in the enlarging mass of decidual tissue. a. Deciduoma induction Model These histological changes are triggered by implanting embryos in naturally occurring decidualization. However, formation of the decidua can also be obtained in response to many different types of artificial stimuli (Loeb, 1907; Loeb, 1908a; De Feo, 1963a). This artificially induced decidua is usually referred to as deciduoma, to distinguish it from the naturally occurring decidua. Formation of the deciduoma and naturally occurring decidua are compatible, except for subtle differences in the timing of development and morphology (Deanesly, 1971; Lundkvist and Nilsson, 1982; Welsh and Enders, 1985). Induction of deciduoma has been used as a model to study the physiology of decidualization. Decidualization can take place when a deciduogenic stimulus is applied during a very limited time, although this period varies with the nature of stimuli. The grossly traumatic stimuli such as cutting or crushing the uterus, can induce decidualization during day 3 to day 4 of pseudopregnancy. Less traumatic or non-traumatic stimuli such as intrauterine instillation of different chemical substances and intraperitoneal injection of pyrathiazin is only effective for a few hours during early pregnancy (De Feo, 1963a,b; Finn and Hinchliffe, 1965; Hetherington, 1968; Finn and Martin, 1972). This very limited period of sensitivity to non-traumatic stimuli seems to correspond to the period of uterine receptivity for implanting embryos (McLaren and Michie, 1956; Dickmann and Noyes, 1960; Noyes and Dickmann, 1960). -39-b. Function The function of the decidua is not fully understood. The decidual tissue may play a role in controlling trophoblast invasion and serve as a source of nourishment for the embryo (Shelesnyak, 1962; Kirby, 1965). The decidua may also isolate each embryo and thus ensure the development of individual embryonic vascular systems, protecting embryos from the deleterious effects of adjacent implantation sites (De Feo, 1967). Furthermore, decidual tissue may provide a cleavage zone for placental separation at the time of delivery (Kirby, 1965). Prostaglandins (PGs) and oxytocin which appears to be important regulators in parturition is synthesized in the placenta of some species that include the human. The decidua plays a central role in steroidogenesis, as a part of the fetoplacental unit during pregnancy. Decidual tissue has been shown to produce many cytokines and growth factors in response to hormonal stimulation (Abrahamsohn and Zorn, 1993; Clark, 1993). Therefore, it is likely that decidual tissue plays a role in the endocrine and paracrine regulation of the implantation, maintenance of pregnancy and parturition. Rodents, especially the rat, are the most commonly used animal model for studying decidualization. Therefore, the following literature review is based largely on the information obtained using this model. However, it is noteworthy that the degree of decidualization, as well as many details of this process, differ among species (De Feo, 1967). B. Endocrine Regulation a. Ovarian Steroid Hormones The requirements of ovarian steroid hormones for decidualization have been well established using deciduoma induction in ovariectomized-steroid hormone primed animals (De Feo, 1967; Finn and Martin, 1972). It appears that the uterus needs to be exposed to progesterone for a minimum of 48 h, followed by a single, small dose of estrogen. The uterus becomes responsive to a deciduogenic stimulus some 24 h after exposure to estrogen -40-(Psychoyos, 1976). Maximal decidualization is achieved with three to four days of daily treatments of progesterone followed by a small dose of estrogen. Excessive or low doses of estrogen diminished the decidual reaction in response to deciduogenic stimuli. The process in which the uterus acquires the capability of responding to a deciduogenic stimulus is called uterine sensitization. b. Pituitary Hormones Evidence suggests that that the pituitary gland is also involved in the regulation of decidualization (Kennedy and Doktorcik, 1988). A poor decidual response has been described in hypophysectomized rats. Treatment with GH and thyroid hormone, a substitute for TSH, restored the decidual response in ovariectomized-hypophysectomized rats (Kennedy and Doktorcik, 1988). Interestingly, the treatment with GH and thyroid hormone was able to restore the decidual response in hypophysectomized rats during the predecidualization period. Continuous treatment from the prestimulation (predecidualization) period throughout the poststimulation (decidual tissue formation) period had no further effect on decidual tissue formation. This may indicate that these hormones play a role in the uterine sensitization process rather than decidual tissue formation itself. However, the mechanisms through which pituitary hormones act in the uterus have not been defined. Pituitary hormones may restore the poor decidual reaction following hypophysectomy by improving impaired general metabolism caused by hypophysectomy, or may directly regulate uterine function in a specific manner. C. Paracrine/Autocrine Regulation There is a growing body of evidence to suggest that a variety of local regulators, such as cytokines and growth factors, regulate uterine growth and function (Tabibzadeh, 1991; Clark, 1993; Murphy and Ballejo, 1994; Weitlauf, 1994). It is believed that these local regulators form a complex regulatory network that contains intrinsic redundancy, -41-thereby assuring each step of the implantation process. These local regulators not only interact with each other but also interact with endocrine regulators such as ovarian steroid hormones. The spatio-temporal expression of these local regulators in the uterus appears to be tightly regulated mainly by ovarian steroid hormones (Murphy and Ballejo, 1994; Weitlauf, 1994). In contrast, litUe is known about their roles, in the implantation or decidualization. However, recent evidence suggests these local factors which include such as epidermal growth factor (EGF), leukemia inhibitory factor (LIF), colony stimulating factor-1 (CSF-1) and interleukin-ip (IL-1P) may play a critical role in the process of implantation (Pollard et al., 1991; Stewart etal., 1992; Johnson and Chatterjee, 1993a,b; Simon et al., 1994a,b; Stewart, 1994a,b). This section reviews available information regarding these four cytokines and the IGF system in the uterus during the periimplantation period. a. Epidermal growth factor (EGF)/Transforming Growth Factor (TGF)-a EGF and its receptor have been detected in the uterus of mouse, rat and human. EGF has been further localized to the luminal and glandular epithelium (Gonzalez et al., 1984; Hoffmann et al., 1984; Mukku and Stancel, 1985a,b; Sheets et al., 1985; Chakraborty et al., 1988; DiAugustine et al., 1988; Lingham et al., 1988; Huet-Hudson et al., 1990). Estrogen appears to stimulate both ligand and receptor synthesis in the uterus of the mouse and rat (Gonzalez et al., 1984; Mukku and Stancel, 1985a,b; Lingham et al., 1988; Gargener et al., 1989), although there is little evidence to suggest that estrogen regulates the levels of the EGF receptor in the human uterus (Chengini et al., 1986; Berchuck et al., 1989). A localized increase in EGF binding at implantation sites has been shown in the mouse uterus, even before the blastocyst attachment (Brown et al., 1989). Furthermore, it has been demonstrated that EGF is capable of inducing implantation in the absence of nidatory estrogen during the experimentally-induced delayed implantation period in rats (Johnson and Chatterjee, 1993a,b). Expression of TGF-cc mRNA, a cytokine that -42-binds to EGF receptor, increases with the progression of decidualization in the rat (Bonvissuto et al., 1992). EGF and TGF-a have also been demonstrated to mediate or even replace estrogen actions in the female genital tract in the mouse (Nelson et al., 1991; Nelson et al., 1992). EGF stimulates PG synthesis in the mouse and rat uterus which has been sensitized to decidual reaction (Paria et al., 1991; Bany and Kennedy, 1995). The EGF-mediated implantation can be reversed by a large dose of indomethacin, an inhibitor of PG synthase, and phospholipase A 2 (Johnson and Chatterjee, 1995). Together, along with the established roles of PGs in decidualization (Hoffman et al., 1977; Kennedy and Lukash, 1982; Kennedy, 1986a,b; Yee and Kennedy, 1988; Hamilton and Kennedy, 1994), EGF appears to initiate the decidualization and subsequent implantation through PG synthesis and/or activation of the arachidonic acid cascade in the uterine endometrial stroma. b. Leukemia Inhibitory Factor (LIF) The levels of LIF mRNA transcripts in the uterus increase, coinciding with the onset of blastocyst implantation, in the mouse, rabbit, and human (Bhatt et al., 1991; Charnock-Jones et al., 1994; Kojima et al., 1994; Yang et al., 1994). LIF expression is greater at implantation sites than other part of of endometrium. The time-specific expression of LIF appears to be under maternal control and precedes blastocyst implantation. Further studies using LIF-knockout mice revealed that LIF is essential for blastocyst implantation (Stewart et al., 1992). The LIF-deficient female mice are unable to become pregnant due to the failure of the blastocyst implantation. Infusion of recombinant LIF reversed the defect of implantation in LIF-deficient mice. LIF does not affect embryonic development up to blastocyst hatching and LIF-deficient embryos are capable of implanting in a normal recipient following embryo transfer. Thus, LIF undoubtedly plays an essential role in the implantation process, although the mechanism through which LIF regulates implantation has not been determined. -43-c. Colony Stimulating Factor (CSF)-l CSF-1 mRNA transcripts have been localized to the uterine epithelium as early as day 3 of pregnancy in the mouse (Arceci et al., 1989). The levels of CSF-1 mRNA and protein dramatically increase during pregnancy in response to ovarian steroid hormones in the mouse and human (Muller et al., 1983; Muller et al., 1983; Arceci et al., 1989; Regenstreif and Rossant, 1989; Kauma et al., 1991; Pampfer et al., 1992). The spatio-temporal expression of this cytokine and its receptor has led to the hypothesis that this cytokine may play an important role in placentation and placental function (Pollard et al., 1987; Pollard, 1990). A role for CSF-1 in the regulation of implantation has been demonstrated in osteopetrotic (op/op) mice that lack CSF-1 synthesis due to homologous recessive mutation in the 5'-region of the CSF-1 gene (Pollard et al., 1991). These mice have severe osteopetrosis, various skeletal defects, a markedly diminished number of macrophage, are toothless and infertile. It has also been demonstrated that the injection of a small amount of CSF-1 during the preimplantation period results in a complete inhibition of implantation from certain genetic crosses (Tartakovsky et al., 1991). Thus, CSF-1 appears to be an essential local regulator of implantation. It is still not clear whether this cytokine plays a specific role in decidualization. d. Interleukin (LL)-lp IL-ip is found in human decidual tissue and has been localized to macrophages and trophoblast cells. In the mouse, IL-ip has been detected in the macrophage-like cells of the subepithelial stromal layer and placenta (Takacs et al., 1988; Kauma et al., 1990; De et al., 1992a,b; De et al., 1993; Simon et al., 1994a). Evidence suggests that ovarian steroid hormones may regulate this cytokine in the uterus (Polan et al., 1988; Kauma et al., 1990; De et al., 1992b; Simon et al., 1993). In the mouse, LL-la and LL-lp mRNA levels and IL-1 activity increase from day 3 of pregnancy, peak between day 4 and day 5 when blastocyst implantation initiates, and then decrease on day 7 and day 8 (De et al., 1993). - 4 4 -T h e type I receptor is found predominant ly i n the uterine l u m i n a l ep i the l ium i n the human and mouse, especial ly at the site o f implantat ion ( S i m o n et a l . , 1994a,b). Blas tocyst implantat ion or, more specif ical ly, the attachment o f the blastocyst to the endometr ium, can be b l o c k e d by I L - 1 receptor antagonists i n the mouse, i nd ica t ing a ro le for EL-1 i n mouse implanta t ion ( S i m o n et a l . , 1994a). I L - 1 (3 and - a have been shown to stimulate L I F and C S F - 1 synthesis (Lubbert et a l . , 1991; Ha r ty and K a u m a , 1992). I L - l a also stimulates the synthesis o f P G s , I L - 6 , and class II major h is tocompat ib i l i ty complexes (Tab ibzadeh et a l . , 1989; T a b i b z a d e h et a l . , 1990a,b; Jacobs et a l . , 1992; B a n y and K e n n e d y , 1995). T h e abi l i ty o f I L - 1 to stimulate P G synthesis m a y suggest a ro le for this c y t o k i n e i n d e c i d u a l i z a t i o n . H o w e v e r , i t i s not k n o w n i f E L - 1 a lone can induce implan ta t ion o r dec idua l i za t ion . Fur thermore , I L - 1 has been l i n k e d to apoptosis ( V a n D a m m e et a l . , 1989). T h i s m a y be an interest ing func t ion o f this c y t o k i n e , s ince the implan ta t ion process contains apoptosis o f the uterine endometr ia l ep i the l ium (Parr et a l . , 1987) . e. T h e I G F system Ev idence suggest a potential role for the uterine I G F system i n the regulat ion o f the decidual iza t ion process. B o t h estrogen and G H , w h i c h regulate the uterine I G F system are i n v o l v e d i n the regu la t ion o f dec idua l i za t i on i n rats ( P s y c h o y o s , 1976; K e n n e d y and D o k t o r c i k , 1988). T h e express ion o f I G F - I , I G F B P s , and I G F - I receptors co inc ides w i t h the t ime o f dec idual iza t ion i n the mouse and rat (Chandrasekhar et a l . , 1990; C r o z e et a l , 1990a; K a p u r et a l . , 1992; Sadek et a l . , 1994). T e m p o r a l changes i n I G F - I and I G F B P - 1 m R N A express ion are o f par t icu la r interest. E x p r e s s i o n o f I G F - I m R N A is predominant i n s t romal ce l l s o n days 3 and 4, p r io r to decidual iza t ion, and i n decidual ce l ls . The levels o f I G F - I m R N A i n dec idua l cel ls dec l ine o n days 7 o r 8 o f pregnancy ( K a p u r et a l . , 1992). I G F - I m R N A transcripts are p a r t i c u l a r l y abundant i n the an t imesomet r i a l s t romal t issue and i s l o c a l i z e d to the - 4 5 -i per iep i the l ia l and per ig landular s t romal ce l l s (Croze et a l . , 1990a). I G F - I leve ls increase dur ing the preimplantat ion per iod (Katagi r i et a l . , 1996). T h e levels o f I G F B P - 1 and I G F B P - 1 m R N A transcripts are first detected o n day 5 o f pregnancy. M a x i m a l levels are observed o n day 6 o f pregnancy i n the rat uterus (Croze et a l . , 1990a; Sadek et a l . , 1994). T h e majori ty o f I G F B P - 1 and its m R N A transcripts are l o c a l i z e d to the l u m i n a l and glandular ep i the l ium i n the ant imesometr ia l r eg ion and i n the uterine l u m i n a l cavi ty . Interestingly, I G F B P - 1 is not detected i n any type o f dec idua l ce l l s i n the rat (Sadek et a l . , 1994). T h i s i s i n d i rec t contrast to the express ion pattern o f I G F B P - 1 i n the h u m a n uterus where the major i ty o f I G F B P - 1 m R N A express ion is l oca l i zed to the dec idual cel ls and pre-decidual ized stromal cel ls o f the late secretory phase (Seppala et a l . , 1994). In addi t ion, I G F - I receptor levels increase du r ing the sensit izat ion pe r iod and at the t ime o f dec idual iza t ion i n the rat (Chandrasekhar et a l . , 1990; K a t a g i r i et a l . , 1996). T h e spat io- temporal express ion pattern o f I G F B P - 1 , i n con junc t ion w i t h I G F - I m R N A express ion i n the adjacent pe r i ep i the l i a l and pe r ig landu la r s t romal ce l l s that sur round the r e g i o n o f I G F B P - 1 express ion ( C r o z e et a l . , 1990a), suggests a ro le for I G F B P - 1 i n the pe r i - implan ta t ion process . F i r s t , I G F B P - 1 m a y i n h i b i t the mi togen ic ac t ion o f I G F - I and I G F - I I i n the endomet r ium w h i c h a l l ows s t romal ce l l s to differentiate in to dec idua l ce l l s (Croze et a l . , 1990a; Sadek et a l . , 1994). Second , i t m a y be poss ib le that I G F B P - 1 acts as a carrier system w h i c h transports I G F - I f r o m the adjacent s t roma to the t rophoblast p r i o r to, o r du r ing , the inters t i t ia l i nvas ion o f the mesomet r i a l dec idua (Sadek et a l . , 1994). T h e express ion o f m R N A for I G F - I receptor, but not I G F - I , has been demonstrated i n preimplantat ion rat embryos (Zhang et a l . , 1994). A paracrine role o f uterine I G F - I i n p re implan ta t ion e m b r y o n i c deve lopment and imp lan t a t i on has been proposed i n the mouse ( K a p u r et a l . , 1992). Thus , the uterine I G F system appears to p lay a ro le i n the decidual izat ion process i n the rat. T h e role for the uterine I G F system i n the regulat ion o f dec idua l iza t ion remains to -46-be determined and will be examined in the following chapters. D. Initiation of Decidualization a. General The uterine sensitization for a deciduogenic stimulus appears to be regulated by maternal endocrine and paracrine regulation as discussed above. However, the exact mechanisms that initiate the decidual reaction are not fully understood. Interestingly, once the uterine endometrium has been sensitized for the decidual response, some changes occur before the trophoblast invades the endometrium. The most obvious of these are the increases in alkaline phosphatase activity levels and vascular permeability in the stroma, immediately adjacent to embryos (Finn and Hinchliffe, 1964; Kennedy, 1979; Milligan and Mirembe, 1984). However, the formation of decidual tissue and the major changes in the uterine vascular system do not occur in the absence of implanting embryos in a naturally occurring pregnancy, or unless a deciduogenic stimulus is applied in pseudopregnancy. This suggests that an embryonic factor or the implanting embryo itself may trigger decidualization. Many factors, as well as physical stimuli by the implanting embryos, have been proposed to be the embryonic signal required for implantation. These substances appear to vary between species (Weitlauf, 1994). It seems probable that in many cases there is more than one embryonic signal. Furthermore, even if the same signal was present, the mode of action may be quite different between species (Weitlauf, 1994). Carbon dioxide, steroids, PGs, histamine, and some proteins of embryonic origin have been proposed to be the signal secreted by the embryo. Although none of these factors have been shown to play a critical role, one or more of these factors may act as the trigger which initiates decidualization in the sensitized endometrium. Many artificial deciduogenic stimuli may also trigger the same mechanism in the sensitized uterine endometrium during deciduoma formation. - 4 7 -b. Prostaglandins ( P G s ) P G s are b e l i e v e d to p l ay an impor tant ro le i n d e c i d u a l i z a t i o n ( K e n n e d y and A r m s t r o n g , 1981; K e n n e d y , 1983a; K e n n e d y , 1986b). U te r ine P G levels increase dur ing dec idua l i za t ion (Kennedy , 1977; K e n n e d y , 1979; R a n k i n et a l . , 1979; K e n n e d y , 1980a; H o f f m a n n et a l . , 1984; M a l a t h y et a l . , 1986). Indomethacine inh ib i t s and/or delays an increase i n vascu la r permeabi l i ty , dec idua l tissue f o r m a t i o n and blastocyst implanta t ion ( L a u et a l . , 1973; K e n n e d y , 1977; E v a n s and K e n n e d y , 1978; H o f f m a n , 1978; K e n n e d y , 1979; M i l l e r and O ' M o r c h o e , 1982). P G s , w h i c h inc lude P G E 2 and P G F 2 c t , ove r come the indomethac ine i n h i b i t i o n ( K e n n e d y , 1979; K e n n e d y and L u k a s h , 1982; M i l l e r and O ' M o r c h o e , 1982; K e n n e d y , 1986a,b). P G s also increase the a lka l ine phosphatase ( A L P ) act ivi ty w h i c h is associated wi th decidual izat ion i n the rat uterus ( Y e e and Kennedy , 1988; Y e e and K e n n e d y , 1991). E v i d e n c e suggests that the act ion o f P G s is probably mediated by P G E 2 receptors i n the uterine endometr ium. P G E 2 receptors, but not P G F 2 r x receptors, have been detected i n the rat uterus (Kennedy et a l . , 1983a,b; M a r t e l et a l . , 1985). A single dose o f P G F 2 c t is not effective i n increas ing vascular permeabi l i ty . H o w e v e r , a constant infus ion o f P G F 2 c c appears to be as effective as P G E 2 (Kennedy , 1979; K e n n e d y and L u k a s h , 1982). T a k e n together, these observations suggest that P G F 2 c c m a y be converted to P G E 2 or that P G F 2 o c m a y interact w i th P G E 2 receptors to exert its effect i n the rat uterus. Furthermore, evidence has l i n k e d some post receptor s igna l ing mechanisms associated w i t h P G E 2 receptors to dec idual iza t ion i n the rat, mouse and hamster. Uter ine c A M P levels dramat ica l ly increase f o l l o w i n g the appl icat ion o f a deciduogenic s t imulus and treatment w i t h P G s (Le roy et a l . , 1974; R a n k i n et a l . , 1977; R a n k i n et a l . , 1979; K e n n e d y , 1983b; Johnston and K e n n e d y , 1984; Y e e and K e n n e d y , 1991). T h e ins t i l la t ion o f cholera t ox in resulted i n an increase i n vascu la r permeabi l i ty and dec idua l tissue fo rmat ion ( R a n k i n et a l . , 1977; A l l e u a et a l . , 1983; Johnston and K e n n e d y , 1984). - 4 8 -c. Ffistamine Hi s t amine may p lay a central role i n the mechanisms that initiate dec idual iza t ion i n response to the " t r igger ing" factor w h i c h i s p resumably o f e m b r y o n i c o r i g i n ( D e F e o , 1967; Wei t l au f , 1994). F i r s t , his tamine m a y be released by the mast ce l l s i n the uterus i n response to the n ida tory estrogen surge (Spaz ian i and Szego , 1958; She lesnyak , 1959; Spaz i an i and Szego , 1959). A n in t ra lumina l his tamine inject ion and systemic inject ion o f h i s t a m i n e re leaser i n d u c e d e c i d u o m a f o r m a t i o n ( S h e l e s n y a k , 1952 ; K r a i c e r and Shelesnyak, 1958). H i s t a m i n e antagonists prevent the format ion o f dec idua l tissue and reduce the number o f implan ta t ion sites (Shelesnyak, 1952; B r a n d o n and W a l l i s , 1977; D e y et a l . , 1978). S o m e objections have been raised based on evidence that his tamine and h i s t amine antagonists have f a i l ed to s t imulate and inh ib i t , r e spec t ive ly , the dec idua l response i n some studies ( F i n n and K e e n , 1962a,b; B a n i k and K e t c h e l , 1964; Harper , 1965; D e F e o , 1967; H u m p h r e y and M a r t i n , 1968). H o w e v e r , evidence suggests a c r i t i ca l ro le for uterine h is tamine release i n response to the n idatory estrogen surge d u r i n g the ini t ia t ion o f the decidual process. E . Eva lua t ion o f Decidual iza t ion At tempts have been made to quantitate the dec idua l response us ing many markers associated w i t h dec idua l iza t ion . M a r k e r s inc lude m o r p h o l o g i c a l and funct ional changes and var ious l o c a l products whose levels increase i n the uterine endomet r ium w h i c h has been sensi t ized to the dec idual response. H o w e v e r , some o f these changes are noted even i n the absence o f implant ing embryos or before any artificial st imulation to the endometr ium w h i c h is sensi t ized to the dec idua l react ion. S o m e o f these l o c a l products are unique to dec idua l ce l l s , others are not un ique but are present i n dec idua l ce l l s , w h i l e others are l o c a l i z e d o n l y to non-dec idua l ce l l s . Interestingly, the pattern o f synthesis o f these l o c a l products a m o n g c e l l types var ies among species. Fur thermore , the ro le o f these l o c a l products i n d e c i d u a l i z a t i o n are l a rge ly u n k n o w n . Never the less , the spa t io - tempora l - 4 9 -expression pattern o f these factors i n the uterus m a y provide a good basis for them to serve as markers for uterine sensi t izat ion and subsequent dec idua l i za t ion . In add i t ion , i f the uterine horn is treated l o c a l l y rather than sys temica l ly , m o r p h o l o g i c a l and func t iona l changes and increases i n the levels o f l o c a l products occur on ly i n the treated uterine horn, pa r t i cu la r ly i n species that have the dup lex uterus l i k e most o f rodents. T h i s a l l o w s investigators to use the other uterine horn as a control . A s d iscussed above, P G s , h is tamine, and other l o c a l factors p lay a c r i t i c a l ro le i n the in i t ia t ion o f dec idual iza t ion . Determina t ion o f increases i n uterine P G s and histamine levels m a y serve as a marker for early decidual iza t ion . S o m e other factors serve as good markers for dec idua l i za t ion due to their appearance or an increase i n the l eve l s i n the dec idual iz ing uterus and avai labi l i ty o f s imple, yet sensitive, detection methods. a. D e c i d u a l Tissue M a s s S i n c e dec idua l tissue o n l y forms i n the presence o f i m p l a n t i n g blastocysts o r an a r t i f i c i a l s t imulus , dec idua l tissue mass m a y be the most straight f o r w a r d marker for dec idua l iza t ion . T h i s marker can be used to determine the effect o f var ious treatments on uterine sensi t izat ion, and the effects o f different s t imul i on the dec idua l response. M o s t studies have used who le uterine tissue rather than separated dec idua l tissue to estimate the size o f dec idua l tissue mass. Such attempts consist o f subjective gradings based o n v i sua l inspect ion ( A s t w o o d , 1939; Shelesnyak, 1952), measurement o f the diameter o f the uterine horn (Ro thch i ld et a l . , 1940; H i s a w and V e l a r d o , 1951), est imation o f the length o f uterine h o m part icipat ing i n the reaction (Shelesnyak and Kra ice r , 1961), and determination o f the weight o f the uterus (De F e o and R o t h c h i l d , 1953; V e l a r d o et a l . , 1953; Shelesnyak, 1957; D e F e o , 1963a,b). De te rmina t ion o f the s ingle uterine horn weight has become a standard method. A l t h o u g h some studies examined the separated decidual tissue weight (Barka i et a l . , 1992), the majori ty have determined the weight o f the who le uterine horn. A l t h o u g h w e i g h i n g the -50-separated decidual tissue mass has some advantages, decidual tissue separation from the rest of the uterus may increase variability of the tissue weight. This artifact appears to be critical if decidual tissue mass is relatively small. b. Vascular Permeability and Extracellular Fluid Volume An increase in vascular permeability is one of the earliest signs of the decidual reaction (Finn and McLaren, 1967; Brandon, 1980; Kennedy, 1980b). The first detection of an increase in vascular permeability may vary, largely due to the nature of stimuli and the detection methods employed (Psychoyos, 1961; Lundkvist et al., 1977; Kennedy, 1979; Lundkvist and Nilsson, 1982). However, the increase in vascular permeability in the appropriately sensitized uterus becomes detectable as early as 15 min after an artificial stimulation (Milligan and Mirembe, 1984). Vascular permeability peaks 9 h after the stimulation, followed by a sharp decline at 12 h (Milligan and Mirembe, 1984). Changes in uterine vascular permeability associated with the decidual response are determined by measuring the degree of leakage of molecules from the circulation that have been injected systemically. Earlier studies injected dyes such as Evans Blue and Pontamine Sky Blue (Psychoyos, 1973). Since dyes injected in the circulation are readily visible when vascular permeability increases, they have been used to identify the site of decidualization and implantation (Psychoyos, 1961; Psychoyos, 1973; Lundkvist and Nilsson, 1982). However, injection of dye may not be suitable for quantitation of changes in vascular permeability. Instead, the injection of radioisotope^ labeled large molecules, in particular 125I-dbumin, may serve as a better marker for changes in vascular permeability at the time of the decidual reaction. Changes in the uterine vascular system associated with the decidual response can also be determined by an increase in the tissue blood space and a decrease in the extracellular space (Milligan and Edwards, 1990). The uterus has a large extracellular tissue compartment, the volume of which changes dramatically in response to the hormonal - 5 1 -s t imula t ion (Spaz ian i , 1975). H i s t o l o g i c a l changes associated w i t h the dec idua l react ion are i n c lose cor re la t ion w i t h an increase i n b l o o d tissue space and a decrease i n the extracel lular space ( F i n n , 1977). H o w e v e r , since changes i n the vascu la r permeabi l i ty is more obvious and precedes these changes, the significance o f these changes as a marker for the decidual reaction may be marginal . c. A l k a l i n e Phosphatase ( A L P ) A c t i v i t y T h e A L P ac t iv i ty is a w i d e l y used marker for the dec idua l react ion. T h e levels o f A L P a c t i v i t y i n the uter ine e n d o m e t r i u m , e s p e c i a l l y i n the s t roma, increase w i t h dec idua l i z a t i on i n m a n y species ( F i n n and H i n c h l i f f e , 1964; C h r i s t i e , 1966; F i n n and M c L a r e n , 1967; H a f e z and W h i t e , 1967; M u r d o c h , 1970; Y e e and K e n n e d y , 1988). T h e endomet r i a l s t romal ce l l s sensi t ized to the d e c i d u a l response appear to spontaneously dec idual ize in vitro (Sananes et a l . , 1978). T h i s provides a convenient system to study the cel lu lar phys io logy o f decidual izat ion such as intracellular s ignal transduction pathways for factors that m a y be i n v o l v e d i n the regulat ion o f the dec idua l react ion ( Y e e and K e n n e d y , 1991) . P G s stimulate uterine A L P act iv i ty as w e l l as dec iduoma tissue mass and vascular permeabi l i ty (Kennedy and A r m s t r o n g , 1981; Kennedy , 1983a; K e n n e d y , 1986b; Y e e and K e n n e d y , 1988). Therefore, the determination Of PG-s t imu la t ed A L P ac t iv i ty , i n addi t ion to basal A L P act ivi ty , m a y a l l o w us to examine the uterine response to P G s after different treatments for uterine sensit ization. T h i s m a y be o f part icular importance, when the study is performed i n cultured endometrial stromal cel ls , since the other two markers; the decidual tissue mass and vascular permeabil i ty, are not available i n the in vitro system. M o s t reports have de te rmined uterine A L P ac t iv i ty by the m e t h o d o f L o w r y ( L o w r y , 1957). T h i s c l a s s i ca l assay is s imple and sensi t ive enough to determine A L P activity i n the rat uterine tissue. - 5 2 -d . Other Marke r s S o m e o f other factors have been found to be potential markers for dec idua l iza t ion . H o w e v e r , the l im i t ed avai labi l i ty and complex i ty o f the assay system or detection methods compared to the aforementioned markers m a y prevent many o f these factors f r o m be ing used as markers for the decidual reaction. I G F B P - 1 , also c a l l e d placenta l prote in 12, has d r awn a lo t o f attention due to the h i g h leve ls found i n the dec idua l tissue o f humans (Seppala et a l . , 1994). A s discussed above, I G F B P - 1 has also been suggested to p lay a ro le i n the dec idual iza t ion process i n the rat (Croze et a l . , 1990a; Sadek et a l . , 1994). Interestingly, I G F B P - 1 i s l o c a l i z e d to non-dec idua l ce l l s i n the antimesometrial region i n the rat, w h i l e most I G F B P - 1 expression has been l o c a l i z e d to dec idua l ce l l s i n the human ( C r o z e et a l . , 1990a; Sadek et a l . , 1994; Seppala et a l . , 1994). Other potential markers for decidual izat ion i n the rat are pro lac t in- l ike proteins and c t 2 - m a c r o g l o b u l i n ( G i b o r i et a l . , 1974; B e l l , 1979; Jaya t i l ak et a l . , 1989; T h o m a s and Schreiber , 1989; C r o z e et a l . , 1990b; G u et a l . , 1992). Synthesis o f p ro lac t in - l ike proteins has been l o c a l i z e d to the an t imesomet r ia l r eg ion o f the uterus d u r i n g dec idua l i za t i on (Jayatilak et a l . , 1989; C r o z e et a l . , 1990b). P ro lac t in - l ike proteins appear to stimulate a 2 -m a c r o g l o b u l i n synthesis i n the mesomet r i a l r eg ion where the t rophoblast invades the endomet r ium ( G u et a l . , 1992; G u and G i b o r i , 1995). S ince c ^ - m a c r o g l o b u l i n i s a potent protease inhibi tor , this system may play a role i n the regulat ion o f trophoblast invas ion ( G u et a l . , 1992). T h e l o c a l i z e d express ion o f p ro lac t in - l ike proteins and a 2 - m a c r o g l o b u l i n suggests that the detect ion o f m R N A s for these factors m a y serve as markers for the antimesometrial and mesometr ial cel ls , respectively ( G u et a l . , 1992). - 5 3 -O I A P T E R T W O T H E E F F E C T O F I G F - I I N T H E P R E I M P L A N T A T I O N R A T E M B R Y O N I C D E V E L O P M E N T I. I N T R O D U C T I O N I G F - I a n d o ther r e l a t ed pep t ides such as i n s u l i n and I G F - I I s t imu la t e pre implanta t ion embryon ic development i n the mouse (Harvey and K a y e , 1988; H a r v e y and K a y e , 1990; H a r v e y and K a y e , 1992a; S c h u l t z et a l . , 1993; S c h u l t z and H e y n e r , 1993). Insul in , i n the presence o f amino acids, stimulates m or pho log i ca l development o f the 8-ce l l stage rat embryo to the blastocyst stage and later, i f the resul t ing blastocysts are rep laced i n a recept ive uterus (Zhang and A r m s t r o n g , 1990). E m b r y o n i c development is great ly i m p a i r e d i n d iabet ic rats and m i c e as de te rmined by decreased c e l l number , espec ia l ly i n the I C M , and increased c e l l death i n morulae and blastocysts , suggesting a ro le for maternal i n su l i n and I G F - I du r ing pre implanta t ion development (Pampfer et a l . , 1990; Beebe and K a y e , 1991). These observations suggest that I G F - I possesses a growth p r o m o t i n g ac t ion i n the p re implan ta t ion embryos . H o w e v e r , the a b i l i t y o f I G F - I to st imulate embryon ic development m a y d i m i n i s h after the blastocyst format ion (Paria and D e y , 1990; H a r v e y and K a y e , 1992a). I G F - I has been detected i n the ov iduc ta l and uterine l u m e n (Letcher et a l . , 1989; Geiser t et a l . , 1991; K o et a l . , 1991; W i s e m a n et a l . , 1992; S m i t h et a l . , 1993). T h e levels o f I G F - I i n the uterus and uterine l u m i n a l f lu ids vary du r ing the estrous c y c l e and ear ly pregnancy pe r iod (Letcher et a l . , 1989; M u r p h y and Ghaha ry , 1990; Geiser t et a l . , 1991; K o et a l . , 1991; M u r p h y , 1991; K a p u r et a l . , 1992). T h e levels o f uterine I G F - I increase d u r i n g the p re implan ta t ion p e r i o d and reaches m a x i m a l l eve l s p r i o r to imp lan t a t i on . Transcr ip t s for the I G F - I receptor have been detected i n oocytes and embryos o f a l l -54-preimplantation stages in the rat (Zhang et al., 1994). However, mRNA transcripts for IGF-I have not been detected in the rat embryo throughout preimplantation development (Zhang et al., 1994). Taken together, an emerging concept is that IGF-I, derived from the uterus, regulates preimplantation embryonic development in a paracrine manner in the rat. This chapter examines the effect of IGF-I on preimplantation embryonic development in the rat. Eight-cell stage rat embryos are cultured in the presence of IGF-I at various concentrations to determine if IGF-I stimulates embryonic development. Blastocysts are also transferred to a receptive uterus and their development examined to determine if the presence of IGF-I, during the preimplantation development period, improves the viability of blastocysts during implantation and subsequent fetal development. H. MATERIALS AND METHODS Preparation of IGF-I Free-Fetal Calf Serum Fetal calf serum (FCS, GIBCO, Burlington, ON) was treated with anion exchange resin and charcoal, as previously described (Smith et al., 1988). Briefly, one liter of FCS was mixed with 56 g of AG 1-X8 (CL-) resin (Bio-Rad, Mississauga, ON) and gently shaken for 16 h. The mixture was centrifuged at 10,000 x g for 15 min and adsorbed FCS was collected. The procedure was repeated with fresh AG 1-X8 (CL") resin. The FCS was mixed with 100 g of acid-washed charcoal and gently shaken for a further 16 h. The mixture was centrifuged at 10,000 x g for 15 min and adsorbed FCS was collected. The FCS was then passed through an AE/1 glass fiber filter (Gelman Science, Montreal, PQ), followed by two 0.2 Ltm filters (Gelman Science). The FCS which was considered to be IGF-I free-FCS was aliquoted and stored at -70°C. -55-Animal Treatments All animals were obtained from the Animal Care Centre (the University of British Columbia, Vancouver, BC) and maintained at the animal care facility of British Columbia Children's Hospital Research Centre (Vancouver, BC). Experiments were performed according to the guidelines of the Animal Care Committee of the University of British Columbia. Animals were kept under conditions of controlled lighting (14 h light: 10 h dark, light on at 6:00 h) and temperature (23 ± 2°C) with free access to food and water. Immature female Sprague-Dawley rats were injected intraperitoneally (ip) with a single dose of 20 IU PMSG (Equinex, Ayerst, Montreal, PQ) prepared in 0.2 ml of 0.9% NaCI at 10:00 h at 26 to 29 days of age. Immature rats were housed overnight with a fertile male (1:2, male:female) 60 h after the PMSG injection, and examined for the presence of a vaginal plug at 8:00 h in the following morning (day 1 of pregnancy). Adult female rats (180-200 g body weight, BW) were mated with a fertile male (1:2, male:female) during the night of the proestrus or estrus stage, as determined by daily vaginal smear examinations, and examined for the presence of a vaginal plug in the following morning. Embryo Collection Embryos were collected at the 8-cell stage from superovulated immature rats at 2:00-3:00 h on day 4 of pregnancy by flushing the oviduct and uterine lumen with Dulbecco's phosphate buffered saline (DPBS, GIBCO), pH 7.4, containing 1 mg/ml polyvinylalchohol (PVA, Sigma, St. Louis, USA). Eight cell embryos were pooled from at least four rats in each experiment. Blastocysts were collected from adult rats, ongoing normal pregnancy, at 11:00-12:00 h on day 5 (in v/vo-blastocysts). Embryo Culture Eight cell embryos were washed twice in medium 199 (GIBCO) supplemented with 5% IGF-I free-FCS. This was used as the basic culture medium (M199). Fifteen to - 5 6 -twenty embryos were cul tured i n a 2 0 u l droplet o f M 1 9 9 w i t h 0 (control cul ture) , 0.02, 0 .2, o r 2 .0 n M h u m a n r e c o m b i n a n t - I G F - I ( h r - I G F - I , S i g m a ) c o v e r e d w i t h m i n e r a l o i l (F i sher Sc i en t i f i c , F a i r L a w n , U S A ) for 36 h at 3 7 ° C w i t h 5% C 0 2 i n h u m i d i f i e d air. E a c h culture condi t ion was repeated at least seven times. Developmental Stages of Embryos T h e deve lopmenta l stages o f cu l tured embryos were scored at the end o f 36-h-culture under a phase-contrast mic roscope (200 x magni f ica t ion) . A l l cu l tured embryos were c l a s s i f i ed in to four categories: blastocysts , moru lae , uncompac ted e m b r y o s and degenerated embryos . T h e blastocyst stage was def ined by the presence o f a def in i t ive b las tocoele c a v i t y and an I C M . T h e m o r u l a stage was def ined by the c o m p a c t i o n o f blastomeres wi thout the blastocoele. B o t h o f these stages were cons idered as g r o w i n g embryos . E m b r y o s at the morulae stage, wi thout compac t ion , were cons idered to be non-g r o w i n g embryos , but were scored as uncompac ted embryos , a category d is t inc t f rom degenerated embryos . E m b r y o degeneration was j udged by the presence o f mul t ip l e c e l l fragments w i t h marked var ia t ion i n s ize and shape, and a hazy blastomere out l ine ( M i l l e r and A r m s t r o n g , 1981a). Cell Count T h e number o f ce l l s i n the I C M and trophectoderm o f twenty blastocysts f rom 36-h-culture and in v/vo-blastocysts were determined by the differential c e l l count ing method (Handys ide and Hunte r , 1984). F o r d i f ferent ia l c e l l s ta in ing, the z o n a p e l l u c i d a was r e m o v e d by an approx ima te ly 10 m i n incuba t ion i n 0 .5% pronase (S igma) i n D P B S . E m b r y o s wi thout zona were incubated for 30 m i n i n 10% heat-inactivated rabbit anti-rat l ymphocy te ant iserum (Sigma) i n D P B S . E m b r y o s were then incubated for 30 m i n i n 5% guinea p i g se rum i n D P B S con ta in ing 20 p g / m l b i s b e n z i m i d e (S igma) and 10 u .g /ml p r o p i d i u m iodide (Sigma). E m b r y o s were examined under fluorescent microscope to count -57-the number of nuclei in the ICM cells that stained blue, and those in trophectoderm cells which stained red. The number of cells in the ICM and trophectoderm were scored as the number of nuclei in each moiety of blastocysts. The number of cells in twelve in vivo-blastocysts were counted by the air-drying method (Tarkowsky, 1966). Cell death was judged by the presence of scattered nuclear fragments of various sizes. The dead-cell index was defined as a percentage of the number of dead cells in the total number of cells in whole blastocysts, or in the ICM and trophectoderm (Pampfer et al., 1990). Protein Synthesis by Blastocysts Blastocysts cultured for 36 h and in v/vo-blastocysts were washed in serum free M199 medium and incubated for 3 h in the same medium. A group of ten embryos were incubated for 2 h in a 20 pi droplet containing 5 u,M [4,5-3H] leucine (1 Ci/1, Amersham Canada, Oakville, ON), amino acids premixture (GIBCO) and 1 mg/ml PVA. Blastocysts were washed three times with ice-cold DPBS without radioactive leucine. Acid-insoluble material from blastocysts was subjected to scintillation counting. Protein synthesis was determined four times in each blastocyst group. Implantation Rate and In Vivo Development Adult female rats were mated with a vasectomized male during the night of the proestrus or estrus stage, as determined by the daily vaginal smear examination, and examined for the presence of a vaginal plug in the following morning (day 1 of pseudopregnancy). These rats were used as recipients for cultured embryos. Twelve blastocysts cultured for 36 h (six/uterine horn) were transferred to the recipient on day 5 of pseudopregnancy. Five rats/treatment group were used in these studies. Under anesthesia, the ovary and tubal end of the uterine horn was exposed through an incision (approximately 1 cm) on the back of the rat. Six blastocysts were washed twice in DPBS containing 5% IGF-I free FCS, loaded into a transfer pipette (150-180 |im - 5 8 -internal diameter) and transferred w i t h a m i n i m u m amount o f m e d i u m into the uterine h o m through a puncture made by a 25 G needle near the utero-tubal j unc t i on o f the uterus. A further s ix blastocysts were transferred to the other uterine h o m us ing the same p ro toco l . A l l recipients were sacr if iced on day 18, and the number o f deve lop ing fetuses, resorption sites, and the weight o f each fetus and placenta determined. T h e number o f implanta t ion sites was estimated as a total o f the number o f deve lop ing fetuses and resorption sites. Statistical Analysis T h e number o f cel ls , [4 ,5 - 3 H]leuc ine incorporat ion, and the weight o f the fetus and placenta were compared by A N O V A , f o l l o w e d by the Tukey ' s test. T h e number o f ce l l s present i n in vivo-blastocysts , determined by different coun t ing methods, were compared by the two- ta i led unpaired Student's r-test. T h e dead-ce l l indexes were compared by the M a n n - W h i t n e y Test . A l l other results, expressed as proport ions , were compared by C h i -square test. Di f fe rence o f the means was def ined by a P - v a l u e o f 0 .01 . A n a l y s i s was conducted us ing the computer software ' S Y S T A T ' ( S Y S T A T , Inc., Evans ton , U S A ) . III. R E S U L T S Developmental Stages of Embryos T h e rate o f embryon ic development to the blastocyst stage increased i n embryos treated w i t h h r - I G F - I (Table 2-1). In contrast, the rate o f uncompacted embryos decreased i n embryos cul tured i n the presence o f h r - I G F - I (Table 2-1). h r - I G F - I had no effect o n the rate o f embryo degeneration. Cell Count T h e total c e l l number o f in vt'vo-blastocysts counted by the differential c e l l s taining method was equivalent to the convent ional air d ry ing method (33.1 ± 1 . 0 v s . 33.4 ± 1.2, -59-* oo a GO c a f > "8 «-» «J o c 1) •s 4-* e d oo O TO a o o O 1 C T3 4-* O o a 3 2 CCJ 1 a s -s c o •a s c o o c o o O N 00 «n V O cn r i H m •>—w — ^ / OO T t < N V O T—i O N O N oo od 1-H od w w w W O N r- >n <n r~ ts T-H .—I 0 ^ 0 0 ^ od « - H od <n <s m >n vo *• ' N ^ S s N. " oo vO O «n r*> r~ O vo O vo O vo O N >-H (O (S N N —' 1—^ —^  w oo oo vo vo cn cn cn t~ cn T-H r> O ^ cn vo f N | r - H T—I T-H s c o o c 0 c 1 CS O CN O © O O CN -60-mean ± SEM). The number of cells in the ICM and whole blastocysts of the 0.2 and 2.0 nM hr-IGF-I groups increased, compared to those of the control group, and was comparable to that of in vj'vo-blastocysts (Fig. 2-1). The presence of hr-IGF-I in the culture medium had no effect on the number of cells in the trophectoderm, at any concentration of hr-IGF-I. The dead cell index of all the culture groups was greater than that of in v/vo-blastocysts, in both the ICM and trophectoderm (Fig. 2-2). The dead-cell index of the ICM in blastocysts of the 0.2 and 2.0 nM hr-IGF-I groups was less than the other two culture groups; however, the dead-cell index of the trophectoderm of all culture groups was equal. Protein Synthesis Protein synthesis by blastocysts of the 0.2 and 2.0 nM hr-IGF-I groups was the same as that of in v/voblastocysts (Fig. 2-3). However, the levels of protein synthesis by blastocysts of the 0.2 nM hr-IGF-I group were not significandy different from those of the control and 0.02 hr-hr-IGF-I groups whose protein synthesis was significantly lower than that observed in the in v/w-blastocysts (Fig. 2-3). Implantation Rate and In Vivo Development All the transfer results were pooled by culture group. All recipients, but one in the control culture group, became pregnant. The rate of the implantation and live fetuses in the 0.2 and 2.0 nM hr-IGF-I groups was greater than those of the other two culture groups (Table 2-2). Although the implantation rate was greater in the 0.02 nM hr-IGF-I group, the rate of live fetuses was the same as the control group. There was no difference in fetal or placental weight across all the groups (Table 2-2). - 6 1 -F igu re 2-1 T h e number o f ce l l s i n the rat blastocysts ob ta ined f r o m cul tures w i t h v a r y i n g concentrations o f human recombinant (h r ) - IGF-I . E i g h t - c e l l stage rat embryos were cul tured for 36 h i n the presence o f 0 (control) , 0.02, 0.2, or 2.0 n M h r - I G F - I . Blas tocys ts at the equivalent stage that had g r o w n in vivo were obta ined f lesh ly . T h e number o f ce l l s i n the inner c e l l mass ( I C M ) and t rophec tode rm o f b las tocys ts i n each cu l tu re g roup and blastocysts grew in vivo were determined by the different ial c e l l count ing method. V a l u e s represent the means and S E M for twenty blastocysts i n each group. Letters o n the top o f bars indicate statistical differences o f the means (a>b, P<0.01) across the different groups. 6. W/A - 6 2 -f3 In vivo • Control S 0.02 nM M 0.2 nM 0 2.0 nM ICM Trophec toderm T O T A L - 6 3 -F igu re 2-2 T h e dead-ce l l i ndex i n the rat b las tocysts ob ta ined f r o m cul tures w i t h v a r y i n g concentrations o f human recombinant (h r ) - IGF-I . E i g h t - c e l l stage rat embryos were cul tured for 36 h i n the presence o f 0 (control) , 0.02, 0.2, o r 2.0 n M h r - I G F - I . Blas tocys ts at the equivalent stage that had g r o w n in vivo were obtained f leshly . C e l l death was j u d g e d by the presence o f the scattered nuc lea r fragments o f va r ious s izes . T h e dead-ce l l i ndex was def ined as a percentage o f the number o f dead ce l l s i n the total number o f ce l l s i n w h o l e blastocysts ( T O T A L ) , or i n the inner c e l l mass ( I C M ) and trophectoderm. Va lues represent the means and S E M for twenty blastocysts i n each group. Letters o n the top o f bars indicate statistical differences o f the means (a<b<c, P<0.01) across the different groups. -64-^ . „ . N o - o f fragmented nuclei Dead cell-index = — . x j QQ No. of total nuclei • In vivo Control 0.02 nM 0.2 nM 2.0 nM ICM Trophec toderm - 6 5 -F igu re 2-3 The levels o f protein synthesis by the rat blastocysts obtained f rom cultures w i t h v a r y i n g concentrations o f human recombinant (h r ) - IGF-I . T h e 8-ce l l stage rat embryos were cu l tured for 36 h i n the presence o f 0 (control) , 0.02, 0.2, or 2.0 n M h r - I G F - I . Blas tocys ts at the equivalent stage that had g r o w n in vivo were obta ined f l esh ly . A group o f ten blas tocysts were incubated for 2 h i n the presence o f 5 m M [ 4 , 5 - 3 H ] l e u c i n e (1 Ci/1) and ac id - inso lub le mate r ia l f r o m blastocysts were subjected to sc in t i l l a t ion count ing . V a l u e s represent the means and S E M for four exper iments i n each group. Letters o n the top o f bars indicate statistical differences o f the means (a>b, P<0.01) across the different groups. -66-15 i 10 A 5H 0 In vivo • Control Q 0.02 nM H 0.2 nM 0 2.0 nM -67-o CS* ! xi - r-H r-H oo r-H ! cn © © © o 41 (68. 48 (80. 1.07 ± 0.63 ± CS o c § 1 o § I—I VO cn X I u o r-H cn cn © © cn od +1 -H ^—' r- r-H vo 5 T t O r—1 VO d Ov r-H 03 x ^ o CS cn cn d d cn od -H +1 cn T t CS © Ov Ov CS CS d d 03 03 —N / V VO r4 cs cn * * CA <u co 3 a s 13 I o fc # e o oo o\ O - H d d -H +1 cn m VO ON P ^ r-H T-H r-H © v29 rC d fc # *-» •a •53 c Q 00 +1 S •4—» s CA a CA <u > * © OH, 2 X3 U o V x> V - 6 8 -rv. D I S C U S S I O N T h e presence o f h r - I G F - I i n the cu l tu re m e d i u m s t imu la t ed m o r p h o l o g i c a l deve lopment o f the rat embryo in vitro. I G F - I appears to st imulate blastocyst format ion and c o m p a c t i o n o f moru lae . h r - I G F - I at a l l e x a m i n e d concentra t ions (0 .02-2.0 n M ) increased the rate o f e m b r y o n i c g rowth in to the blastocyst stage, and decreased that o f u n c o m p a c t e d e m b r y o s (Table 2-1) . T h e present f i nd ings are supported by p rev ious studies. Insul in , a c lose ly related peptide to I G F - I , enhances the g rowth p romot ing act ion o f a m i n o acids o n pre implanta t ion rat e m b r y o n i c deve lopment (Zhang and A r m s t r o n g , 1990). I G F - I s t imulates p re implan ta t ion deve lopment , by inc reas ing c o m p a c t i o n and b las tocys t fo rma t ion i n the mouse e m b r y o ( H a r v e y and K a y e , 1992a). I n su l i n also st imulates blastocyst fo rmat ion but has no effect o n the c o m p a c t i o n o f mouse embryos ( H a r v e y and K a y e , 1990; G a r d n e r and K a y e , 1991) . F u r t h e r m o r e , re tardat ion i n preimplantat ion embryon ic development has been found associated w i t h diabetes i n the rat and mouse , suggesting a ro le for i n su l i n i n pre implanta t ion development (Pampfer et a l . , 1990; Beebe and K a y e , 1991). I G F - I m a y modula te blastocyst fo rmat ion through s t imula t ing N a + / K + - A T P a s e synthesis. I G F - I has been shown to stimulate N a + / K + - A T P a s e synthesis i n many species ( M a d s e n and B e r n , 1993; M a t s u d a et a l . , 1993). A s p r e v i o u s l y d i scussed , ev idence suggests that N a + / K + - A T P a s e p lays a central ro le i n format ion o f the blastocoele i n the rabbit and mouse ( D i Z i o and Tasca , 1977; B igger s et a l . , 1978; W i l e y , 1984; Ves tweber et ( a l . , 1987; M a n e j w a l a et a l . , 1989). T h e levels o f m R N A transcript and protein express ion o f N a + / K + - A T P a s e subunits increase f rom about the t ime o f compact ion unt i l the expanded blastocyst stage (Gardiner et a l . , 1990a,b). T h e increase i n the levels o f N a + / K + - A T P a s e subunit co inc ides w i t h the increase i n leve ls o f m R N A encod ing I G F - I receptor i n the preimplantat ion embryo (Rappolee et a l . , 1991). It has been suggested that m o r p h o l o g i c a l l y def ined blastocysts (embryos w i t h a - 6 9 -b las tocoe le ) m a y no t a l w a y s be capab le o f i m p l a n t a t i o n a n d p o s t - i m p l a n t a t i o n a l deve lopment . T h i s argument i s based o n at least two l ines o f observat ions . F i r s t , the fo rma t ion o f a b las tocoe le c a v i t y m a y s i m p l y ref lect a d i f ference i n the a b i l i t y o f blastomeres to transport f l u id ( W i l e y , 1984). The development o f mechanisms i n v o l v e d i n f l u i d transport is related to the number o f c e l l cyc les , or t ime after fer t i l iza t ion, rather than the ac tual number o f ce l l s i n embryos o r the d i f ferent ia t ion o f the I C M ( S m i t h and M c L a r e n , 1977; Pratt et a l . , 1981; C h i s h o l m et a l . , 1985; W i n s t o n et a l . , 1991). It has been demonstrated that a substantial proport ion o f blastocysts w i t h a fewer number o f cel ls m a y also have a numer ica l ly deficient I C M (Pratt et a l . , 1981; H a r d y et a l . , 1989). These blastocysts m a y be less capable or incapable o f subsequent development. Second ly , ev idence suggests that m o r p h o l o g i c a l l y n o r m a l blastocysts , e spec ia l ly those cul tured in vitro, often contain mult inucleated cel ls (Tesarik et a l . , 1987b; W i n s t o n et a l . , 1991). T h e most c o m m o n method to determine the number o f ce l l s i s to count the number o f n u c l e i rather than the true c e l l number ( T a r k o w s k y , 1966; H a n d y side and Hunte r , 1984). A n increase i n the number o f nuc l e i does not reflect an increase i n c e l l n u m b e r i n the presence o f m u l t i n u c l e a t e d c e l l s . A l t h o u g h f o r m a t i o n o f b i - o r mul t inuc lea ted ce l l s i n the trophoblast appears to be a part o f the n o r m a l deve lopmenta l process ( B a r l o w and Sherman, 1972; L o n g and W i l l i a m s , 1982), failure o f cytokines is and kar iokines is m a y result i n the format ion o f mult inucleated ce l l s dur ing the earlier stages o f e m b r y o n i c development (Tesar ik et a l . , 1987b; W i n s t o n et a l . , 1991). T h e format ion o f mul t inucleated cel ls , especial ly i n the I C M , m a y be detrimental to embryon ic development ( W i n s t o n et a l . , 1991). Therefore, incorporat ion o f other examinat ions , i n addi t ion to the sco r ing o f deve lopmenta l stages and coun t ing the number o f ce l l s (nucle i ) , are h i g h l y desirable to determine v iab i l i ty o f embryos f o l l o w i n g in vitro treatments. T h e present study examined the v i a b i l i t y o f blastocysts f r o m an in vitro cul ture us ing mul t ip l e cr i ter ia . h r - I G F - I , especia l ly at concentrations o f 0.2 and 2.0 n M , appears to be benef ic ia l to the quali ty o f blastocysts. h r - I G F - I had a mi togenic effect on the cel ls i n - 7 0 -the I C M ( F i g . 2-1) and decreased the dead-ce l l index i n the I C M o f the same h r - I G F - I concent ra t ion groups ( F i g . 2-2) . T h u s , h r - I G F - I appears to increase i n the number o f l i v i n g ce l l s i n the I C M w h i c h develop into the fetus. T h i s m a y contribute to the observed increase i n the v i ab i l i t y o f blastocysts w h i c h , i n turn, increases the rate o f implanta t ion and subsequent fetal development (Table 2-2). Furthermore, blastocysts o f the 2.0 n M h r - I G F -I group was metabol ica l ly more active than blastocysts o f the control and 0.02 n M h r - I G F - I groups ( F i g . 2-3). Thus , the improvement i n the deve lopmenta l stage o f embryos by 0.2 and 2 .0 n M h r - I G F - I appears to be a c c o m p a n i e d by an i m p r o v e m e n t i n v i a b i l i t y o f embryos . In contrast, h r - I G F - I at 0.02 n M i m p r o v e d developmenta l stage o f embryos and increased the implanta t ion rate to a lesser extent (Tables 2-1 and 2). Other var iables , such as the number o f ce l l s , dead-ce l l index , pro te in synthesis, and the rate o f d e v e l o p i n g fetuses, remained constant (Figs . 2 - 1 , 2 and 3, and Tab le 2-2). These results suggest that a large por t ion o f blastocysts o f the 0.02 n M group m a y be less capable or incapable o f implantat ion or postimplantation development. T h e mi togen ic action o f h r - I G F - I and i m p r o v e d dead-cel l index by h r - I G F - I were observed i n the I C M but not i n the trophectoderm (Figs. 2-1 and 2). The selective effect o f I G F - I and i n s u l i n o n the ce l l s o f the I C M has been shown to occu r i n the mouse embryo ( H a r v e y and K a y e , 1990; H a r v e y and K a y e , 1992a; S m i t h et a l . , 1993), w i t h a s ingle except ion i n w h i c h i n s u l i n increased the number o f ce l l s i n the I C M and t rophectoderm ( S m i t h et a l . , 1993). T h e effects o f I G F - I and i n s u l i n o n the i so la ted I C M f r o m the trophectoderm by immunosurgery technique are compat ible to those observed i n the who le blastocyst (Harvey and K a y e , 1990; H a r v e y and K a y e , 1992a). T h i s suggests that I G F - I and insu l in act direct ly o n the cel ls o f the I C M rather than exerting their effects through the act ivat ion o f paracrine o r paracel lular functions o f the t rophectoderm ce l l s . H o w e v e r , the trophectoderm appears to p lay a ro le i n transferring these peptides f rom their surroundings to the I C M . T h e ce l l s o f the t rophectoderm o f mouse blastocysts in ternal ize I G F - I and i n s u l i n v i a receptor-media ted endocytos i s , and then transfer the pept ides to the I C M -71 -(Heyner et al., 1989; Smith et al., 1993). hr-IGF-I had no effect on fetal and placental weights on day 18 of pregnancy (Table 2-2). Treatment of the preimplantation rat embryo with insulin in the presence of amino acids has increased the rate of implantation and the number of live fetuses. However, insulin had no effect on fetal and placental weights determined on day 18 of pregnancy under these culture conditions (Zhang and Armstrong, 1990). It is apparent that exceptionally large or small fetuses are not beneficial for reproduction. Accordingly, evidence suggests that fetal growth is strictly regulated by unknown mechanisms that optimize the size of fetuses in a given species. The 'giant' or 'miniature' blastocyst produced by aggregation of two or multiple embryos and by bisection of embryos, respectively, result in the fetus having similar weight by the middle or end of gestational period. The present study indicates that the effect of IGF-I on the rat embryo is mediated by the IGF-I receptor. It is believed that IGF-I can exert its biological effect through IGF-I, IGF-II, or insulin receptors. Expression of IGF-I receptor mRNA has been detected in oocytes and embryos throughout preimplantation development stages in rats (Zhang et al., 1994). Transcripts of the IGF-I receptor are readily detectable in oocytes, 1-cell and 2-cell stage embryos and in blastocysts. The transcripts are also detectable in 4-cell and 8-cell stage embryos but at highly reduced levels (Zhang et al., 1994). In contrast, levels of IGF-II receptor mRNA has been detected at consistent levels in rat oocytes and embryos of all stages. In addition, insulin receptor mRNA has been detected in a similar pattern to that of the IGF-I receptor mRNA expression in the rat (Zhang et al., 1994). The temporal decline in the levels of IGF-I and insulin receptor mRNA expression may reflect the transition of the regulation of gene expression from maternal to embryonic transcripts (Telford et al., 1990; Rappolee et al., 1991). It is not known if the levels of functional receptors for IGF-I and insulin fluctuate in a similar pattern to the levels of mRNA transcripts. IGF-I appears to exert its effect through the IGF-I and insulin receptors in the - 7 2 -pre implanta t ion mouse embryo (Harvey and K a y e , 1991c; H a r v e y and K a y e , 1992b). B -10 F a b fragments o f I g G f r o m an t i - in su l in receptor autoant iserum, a spec i f ic i n s u l i n receptor antagonist, have comple t e ly b l o c k e d the effect o f I G F - I and i n s u l i n o n prote in synthesis i n the mouse embryo (Harvey and K a y e , 1991c; H a r v e y and K a y e , 1992b). B -10 Fab fragments have also inhibi ted the growth promot ing action o f insu l in , w h i c h include mitogenic action on the I C M and the st imulation o f morpholog ica l development, but had no effect o n the g rowth p r o m o t i n g act ions o f I G F - I ( H a r v e y and K a y e , 1992b) . These observations suggest that the growth promot ing action o f I G F - I i n the mouse embryo m a y be media ted by the I G F - I and/or I G F - I I receptors. Fur thermore , a l though the I G F - I I receptor is expressed i n the preimplantat ion mouse embryo as ear ly as the 2 -ce l l stage, the I G F - I I receptor does not appear to mediate the g rowth p r o m o t i n g ac t ions o f I G F - I I (Rappolee et a l . , 1992). Instead, the growth p romot ing actions o f I G F - I I m a y be mediated b y the I G F - I and i n s u l i n receptors (Rappo lee et a l . , 1992) . There fore , the g rowth p romot ing act ion o f I G F - I i n the mouse embryo may be mediated by the I G F - I receptor. T a k e n together, the presence o f the I G F - I receptor i n the rat embryo , the growth promot ing action o f h r - I G F - I i n the present study appears to be mediated by the I G F - I receptor. In contrast, the act ion o f I G F - I on metabol ic s t imulat ion l i ke protein synthesis may be mediated b y the in su l in receptor. A s discussed above, evidence suggests that the effect o f I G F - I on protein synthesis i n the preimplantat ion mouse embryo appears to be mediated by the insu l in receptor (Harvey and K a y e , 1991c; H a r v e y and K a y e , 1992b). T h e levels o f protein synthesis o f the 0.2 n M h r - I G F - I group was not statistically different f rom those o f the cont ro l group and the 2.0 n M h r - I G F - I group, where protein synthesis was greater than that o f the con t ro l group, and were at intermediate leve ls o f the t w o groups ( F i g . 2-3). H o w e v e r , most o f the actions o f h r - I G F - I were apparent at the 0.2 n M h r - I G F - I group. T h e h igh levels o f h r - I G F - I w h i c h were required to stimulate protein synthesis suggest that this act ion is be ing mediated by receptors other than the I G F - I receptor, such as the insu l in and I G F - I I receptors. T h e I G F - I I receptor does not appear to mediate the act ion o f I G F - I I - 7 3 -o n prote in synthesis i n the mouse (Rappolee et a l . , 1992). T h e i n s u l i n receptor m a y be responsible for the observed act ion o f h r - I G F - I on protein synthesis i n the preimplantat ion rat embryo observed i n the present study. T h e concept that the g rowth p romot ing act ion and metabol ic s t imula t ion o f I G F - I are media ted through the I G F - I and i n s u l i n receptors, respec t ive ly , is consis tent w i t h previous observations. A differential s ignal ing potential o f the cy top lasmic d o m a i n o f the P-subunit o f human insu l in and I G F - I receptors has been demonstrated us ing transfection techniques i n the mouse N E H - 3 T 3 fibroblast c e l l (Lammers et a l . , 1989). T h e cy toplasmic d o m a i n o f the I G F - I receptor appears to have a greater s igna l ing potential i n the long- term effect, such as mi togen ic actions ( L a m m e r s et a l . , 1989). T h e I G F - I receptor is more potent than the i n s u l i n receptor i n media t ing mi togen ic and g r o w t h p r o m o t i n g ac t iv i ty , w h i l e the insu l in receptor has a greater potential i n ce l lu lar energy metabol i sm i n many c e l l types (H in t z et a l . , 1972; L a m m e r s et a l . , 1989; W i l l i a m , 1991). F i n a l l y , the rat uterus is one o f the major sites o f I G F - I synthesis ( M u r p h y et a l . , 1987a,c; Norstedt et a l . , 1989). It has been shown that the uterine l u m i n a l f lu ids contain I G F - I i n var ious species (Letcher et a l . , 1989; Geiser t et a l . , 1991; K o et a l . , 1991; S m i t h et a l . , 1993). Internalization o f maternally der ived I G F - I v i a receptor-mediated endocytosis by pre implan ta t ion embryos has been demonstrated i n the mouse ( S m i t h et a l . , 1993). E x p r e s s i o n o f I G F - I , I G F - I I , and i n s u l i n receptors , but not that o f I G F - I , have been detected i n the pre implan ta t ion rat embryo (Zhang et a l . , 1994). These , together w i t h observed benef ic ia l effects o f h r - I G F - I o n preimplantat ion development i n the rat, suggest that I G F - I m a y be i n v o l v e d i n the maternal-to-fetal s igna l ing mechan i sms that mediate synchronized development between the uterus and preimplantation embryos. -74 -V . S U M M A R Y A N D C O N C L U S I O N S T h e present study demonstrated a potential role for I G F - I i n the development o f the pre implanta t ion rat embryo . I G F - I appears to promote m o r p h o l o g i c a l development o f rat embryos to the blastocyst stage. T h e improvement i n the developmenta l stage o f embryos b y I G F - I appears to be a c c o m p a n i e d b y an i m p r o v e m e n t i n v i a b i l i t y o f embryos , as de termined by an increase i n the number o f l i ve ce l l s i n the I C M , an increase i n prote in synthesis, and a greater rate o f implantat ion and fetal development. T h e effects o f h r - I G F - I were observed at the concentrat ions at w h i c h I G F - I has been shown to effect other c e l l types through the I G F - I receptor. These experimantal observations support the hypothesis mat I G F - I m a y be i nvo lved i n the maternal-to-fetal s igna l ing mechanisms . T h i s i n turn m a y mediate synchron ized deve lopmen t be tween the uterus and p re implan ta t ion e m b r y o s . A l t e r n a t e l y , a w e l l -regulated uterine I G F system may be required for synchron ized development between the uterus and embryos . Therefore, factors that disturb the regula t ion o f uterine I G F system m a y be d e t r i m e n t a l to p r e i m p l a n t a t i o n e m b r y o n i c d e v e l o p m e n t and subsequent posurnplantational development i n the rat. - 7 5 -C H A P T E R T H R E E T H E E F F E C T O F S U P E R O V U L A T I O N O N T H E U T E R I N E I G F S Y S T E M I. I N T R O D U C T I O N T h e I G F system is one o f a number o f regulatory systems w h i c h mediate steroid hormone actions i n the uterus i n an autocrine/paracrine manner. I G F - I i s a k e y member o f the I G F system. Other components o f this system are I G F B P s and I G F - I receptors. I G F -I synthesis i n the uterus i s regula ted p r i m a r i l y by estradiol-17(3 rather than G H , the p r inc ipa l regulator o f I G F - I synthesis i n most other tissues ( M u r p h y et a l . , 1987c; M u r p h y and Fr iesen , 1988; Norstedt et a l . , 1989). Other components o f the I G F system are also under ova r i an steroid ho rmone regula t ion i n the uterus (Ghaha ry and M u r p h y , 1989; G i r v i g i a n et a l . , 1994). Therefore, i t is l i k e l y that changes i n the levels o f ovar ian steroid hormones f o l l o w i n g superovula t ion w i l l disturb the regula t ion o f the I G F system i n the uterus. S u c h disturbances i n the uterine I G F system may , i n turn, be responsible for the detrimental effects seen w i t h superovulation, such as early embryonic loss. T h e object ive o f this chapter i s to determine whether the uterine I G F system is changed by superovulation. The study is performed using an immature rat mode l , i n w h i c h the reproduct ive p h y s i o l o g y and the detr imental effects o f superovula t ion have been w e l l characterized. T h i s chapter examines the modificat ions i n the uterine I G F system fo l l owing superovulat ion. In the immature superovulat ion m o d e l , 40 I U P M S G has been used as a superovulatory dose, w h i l e 4 I U P M S G used as a cont ro l ( M i l l e r and A r m s t r o n g , 1981a; Y u n et a l . , 1987; Y u n et a l . , 1989). -76-II. MATERIALS AND METHODS Animal Treatments All animals were obtained from the Animal Care Centre of the University of British Columbia. Experiments were performed according to the guidelines of the Animal Care Committee of the University of British Columbia. Animals were kept under conditions of controlled lighting (14 h light: 10 h dark, light on at 6:00 h) and temperature (23 + 2°C) with free access to food and water. At 28 days of age, immature female Sprague-Dawley rats were injected (ip) with either 4 or 40 IU PMSG prepared in 0.2 ml of 0.9% NaCI at 9:00-10:00 h. Females were mated at 30 days of age. Day 1 of pregnancy was determined by the presence of a vaginal plug. Six females from each group were sacrificed daily at 9:00-10:00 h, from day 1 to day 6, for IGF-I and IGFBP assays. Another six rats from each group were sacrificed for IGF-I receptor assays at every time point. After decapitation, trunk blood was collected and the uterus dissected. The oviductal and uterine lumen were flushed with DPBS containing 3 mg/ml polyvinylpyrrolidone (Sigma) for embryo collection. In separate experiments, uterine endometrial IGF-I, IGFBP, and IGF-I receptor levels were determined in rats given 4 or 40 IU PMSG as above. Rats were sacrificed on days 1, 3, 5, or 6 and the uterine horns dissected. The uterine horns were opened longitudinally and the endometrial tissue separated from the myometrium by mechanical scraping. The endometrial tissue from 4 to 6 rats were pooled and used for the IGF-I and IGFBP assays. The endometrium from another 4 to 6 rats were used for the IGF-I receptor assay. Experiments were repeated three times. Four rats from each PMSG treatment group were sacrificed at 9:00-10:00 h daily from the day of PMSG treatments (day -2) to the day of mating (day 0) to collect the blood samples for estradiol-170 and progesterone assays. Serum ovarian steroid levels from day 1 to day 6 of pregnancy were determined by using blood samples from 4 out of 6 rats used -77-for the IGF-I and IGFBP assays. RIA for Ovarian Steroid Hormones Estradiol-17P and progesterone serum levels were determined by RIA (Yun et al., 1987). The cross-reactivity of the estrogen antiserum was: estrone, 2.9%; estriol, 0.5%; other major ovarian steroids, <0.2%. The cross-reactivity of the progesterone antiserum was: 5P-pregnane-3,20-dione, 35.5%; 5ct-pregnane-3,20-dione, 15.7%; 3oc-hydroxy-5P-pregnan-20-one, 2.0%; 20P-hydroxy-4-pregnen-3-one, 1.3%; 17-hydroxyprogesterone, 1.2%; other major ovarian steroids, <0.2%. The intra-assay coefficients of variation for both assays were <10% throughout the effective range of the standard curves. The inter-assay coefficients of variation for both assays were <15% throughout the effective range of standard curves. Sample Preparation for IGF-I and IGFBP Assays The uterus was weighed, minced and homogenized in 1 M acetic acid (1 g/5 ml) and centrifuged at 3,000 x g for 30 min at 4°C. The supernatants were saved and the pellets re-extracted. Supernatants from both centrifugation steps were combined, concentrated, and applied to a Sephadex G-75 (Sigma) column. The Sephadex G-75 column was previously calibrated with molecular weight markers (Sigma); aprotinin (6.5k), cytochrome c (12.4k), carbonic anhydrase (29k), and bovine albumin (66k). The void volume was determined by blue dextran. Fractions containing a molecular weight range between 20k and 60k were considered to contain IGFBP. Fractions with a molecular weight range below 10k were considered to contain IGF-I. In a preliminary study, 91 to 96% of IGF-I and 89 to 93% of IGFBP were collected in these fractions, respectively. An aliquot of each fraction was subjected to protein assay (Lowry et al., 1951). Fractions were lyophilized and stored at -20°G until assayed. The serum was mixed with the same volume of 1 M acetic acid and the mixture applied to a Sephadex G-75 column to separate -78-IGF-I from IGFBP. IGF-I and IGFBP fractions were collected, lyophilized and stored as above. RIA for IGF-I The concentration of IGF-I in the IGF-I fractions of the serum and uterine protein extract was determined by RIA using a IGF-I 125I-RIA kit (INCSTAR, Stillwater, USA). The minimum detectable concentration of the IGF-I RIA kit is <2.0 nM (15.4 ng/ml). Cross-reactivity to peptides; IGF-II, human GH, fibroblast growth factors, TGF, and platelet derived growth factor was determined to be < 1%. The intra-assay coefficients of variation were 8.4%, 10.1%, and 9.1% at the low, medium and high ends of standard curve, respectively. The inter-assay coefficients of variation were 12.5%, 10.3%, and 15.2% at the low, medium and high ends of standard curves. The lyophilized uterine protein extracts were reconstituted in 200 u.1 of RIA buffer. Ligand Binding Assay for Total IGFBP IGFBP levels in the serum and uterine protein extracts were determined by a ligand binding assay using the IGFBP fractions from Sephadex G-75 columns. For ligand binding assays (IGFBP and IGF-I receptor), 1 (ig of hr-IGF-I was iodinated with 1 mCi Na125I (Amersham Canada) and 2.5 mg Iodogen (Pierce, Rockford, USA). The 1 2 5I-IGF-I, with a specific activity of 220-280 (iCi/mg, was purified on a Sephadex G-50 column and aliquots stored at -20°C until used. The lyophilized IGFBP fractions were reconstituted at a protein concentration of 1 mg/ml in 50 mM tris-HCl buffer, pH 7.2, containing 500 mM NaCI. One hundred ul of the reconstituted IGFBP fraction was blotted on to a nitrocellulose membrane and incubated with 2% BSA (Sigma) in the same buffer at room temperature for 3 h. The membranes were then incubated with 0.5 nM ^ 1-IGF-I in 100 ul of the tris-HCl buffer containing 0.5% BSA at 4°C for 12 h The nitrocellulose filters were washed twice with 0.05% Tween-20 (Bio-Rad) in tris-HCl buffer and the -79-radioactivity levels determined using a y-counter. The non-specific binding, determined by using 100 pg BSA, was always <3%. The specific binding was calculated by subtracting the non-specific binding from the total binding for each sample. Membrane Preparation for IGF-I Receptor Assay Uteri were dissected, minced and homogenized in 50 mM tris-HCl buffer (1 g/5 ml), pH 7.4, containing 250 mM sucrose and 1 mM CaCl2. Homogenates were centrifuged at 700 x g for 10 min. Pellets were re-homogenized in fresh buffer and centrifuged. Supernatants from both centrifugations were combined and centrifuged at 40,000 x g for another 40 min. The pellets were then washed twice and resuspended in 50 mM tris-HCl, pH 7.8, containing 0.5% BSA at 2 ml/g starting tissue weight. All steps were carried out at 4°C. An aliquot of the membrane preparation was subjected to protein assay (Lowry et al., 1951). Ligand Binding Assay for IGF-I Receptor Each binding assay contained 0.1 nM 125I-IGF-I and 50 |ig membrane protein in 50 mM tris-HCl buffer, pH 7.8, containing 0.1% BSA in a volume of 100 |il. Assays were incubated for 20 h at 4°C after which 300 ul of 25% polyethylene glycol, previously chilled on ice was added to the reaction mixture. Assays were centrifuged at 3,000 x g for 15 min at 4°C and the pellets washed with 100 ul of 12.5% polyethylene glycol. The levels of radioactivity in the pellets were determined using a y-counter. Non-specific binding, determined by adding 100-fold excess of unlabeled IGF-I, was <2%. The specific binding was calculated by subtracting the non-specific binding determined in each measurement from the total binding for each sample. Fifty percent displacement of 125I-IGF-I binding by unlabeled IGF-I, human recombinant IGF-II (Sigma), and insulin (Sigma) was observed at peptide concentrations of approximately 2, 8, and 850 nM, respectively (Fig. 3-1). -80-Figure3-1 Competition-inhibition curves for 1Z3I-IGF-I binding to the uterine membrane preparations by human recombinant (hr)-IGF-I, hr-IGF-II, and 125 insuhn. Each competition-inhibition assay contained 0.1 nM I-IGF-I and 50 u.g membrane protein in a volume of 100 pi with varying concentrations of each peptides. The competition-inhibition curves were determined in three separate membrane preparations for each peptides. The competition-inhibition curves in all membrane preparations were similar to each other for 125 each peptides. The means of fifty percent displacement of I-IGF-I binding by hr-IGF-I, hr-IGF-II, and insulin was observed at peptide concentrations of 2, 8, and 850 nM, respectively. -81-0 J — • . -11 -10 -9 -8 -7 -6 -5 L o g [Peptide] M - 8 2 -Statistical Analysis D a t a are expressed as the mean ± S E M . T h e means o f I G F - I , I G F B P and I G F - I receptor levels between treatment groups were compared us ing the Student's t-test. M e a n s o f I G F - I , I G F B P , and I G F - I receptor l eve ls between different days were c o m p a r e d by A N O V A , f o l l o w e d by T u k e y ' s test. Di f fe rences were cons ide red to be s ta t i s t ica l ly s igni f icant at the 9 5 % conf idence l e v e l (P<0.05). A n a l y s i s was conduc t ed u s ing the computer software ' S Y S T A T ' . III. R E S U L T S Serum Steroid Levels T h e l eve l s o f e s t r ad io l -170 and proges terone were greater th roughout the exper imenta l pe r iod i n an imals undergoing superovula t ion c o m p a r e d w i t h those o f the con t ro l group ( F i g . 3-2). T h e increase i n the levels o f progesterone i n the superovulated rats become more apparent dur ing the postovulatory period. A secondary peak o f estradiol-170 was observed o n day 2 o f pregnancy i n the superovulated rats, i n addi t ion to the first peak o n the day o f ovula t ion ( F i g . 3-2). Uterine and Serum IGF System Tota l I G F B P and I G F - I receptor levels are presented as a percentage o f the levels o f the cont ro l group o n day 1 (F igs . 3-3, 4, and 5). T h e I G F B P levels (100%) o f the w h o l e uterus, separated endomet r ium and serum were 61 ± 2.0 , 83 ± 11, and 140 ± 10 p m o l I G F - I / m g pro te in , respec t ive ly ( F i g . 3-3, 4, and 5). T h e I G F - I receptor l eve l s o f the w h o l e uterus and separated endomet r ium were 58 ± 2.9 and 86 ± 12 f m o l I G F - I / m g membrane protein (Figs . 3-3 and 4). I G F - I l eve l s i n the w h o l e uterine tissue homogenates ob ta ined f r o m the con t ro l group increased throughout the preimplantation per iod. Peak levels were observed o n day -83-Figure 3-2 Profiles of the serum levels of estradiol-17(3 (A) and progesterone (B) in superovulated and control rats. Immature rats were injected with a single dose of 40 IU PMSG (superovulation) or 4 IU PMSG (control). Rats were mated 60 h after the PMSG injection and sacrificed on the indicated days (day 1: day of vaginal plug). The serum levels of estradiol-17(3 and progesterone were determined by RIA. Values represent the means and SEM for four animals in each point. Day of pregnancy - 8 5 -F i g u r e 3-3 P ro f i l e s o f I G F - I ( A ) , I G F B P ( B ) , and I G F - I receptor ( C ) leve ls i n the uterus o f superovulated and control rats. Immature rats were injected wi th a s ingle dose o f 4 0 I U P M S G (superovulat ion) o r 4 I U P M S G (control ) . Ra ts were mated 60 h after the P M S G in jec t ion and sac r i f i ced o n the ind ica ted days (day 1: day o f v a g i n a l p lug ) . T h e leve ls o f I G F - I i n the uterine extracts were determined by R I A and the levels o f I G F B P and I G F - I receptor determined by l i g a n d b i n d i n g assay us ing i 2 5 I - I G F - I . T h e levels o f I G F B P and I G F - I receptor are presented as a percentage o f the levels o f the con t ro l group observed o n day 1 o f pregnancy. V a l u e s represent the means and S E M for s ix animals i n each point . As te r i sks indicate statistical difference o f the means between two groups (P<0.05). -86-oT 3 1000-1 (0 CO 800-o> 600-IGF-I 400-120-i Co" 100-Q. m u. 80-g 60-180-160-0s- 140-o 120-0) o a> cc 100-80-60-' Day of pregnancy -87-r Figu re 3-4 P ro f i l e s o f I G F - I ( A ) , I G F B P ( B ) , and I G F - I receptor ( C ) l eve l s i n the uterine endometr ium o f superovulated and control rats. Immature rats were injected wi th a single dose o f 4 0 I U P M S G (superovulation) or 4 I U P M S G (control). Rats were mated 60 h after the P M S G inject ion and sacrif iced on the i nd i ca t ed days (day 1: day o f v a g i n a l p l u g ) . E n d o m e t r i a l t issue, separated f r o m the rest o f the uterus, o f four to s ix rats were poo led . T h e levels o f I G F - I i n the uterine extracts were determined by R I A and the levels o f I G F B P and I G F - I receptor de termined b y l i g a n d b i n d i n g assay us ing 1 2 5 I - I G F - I . T h e levels o f I G F B P and I G F - I receptor are presented as a percentage o f the l eve l s o f the c o n t r o l g roup o b s e r v e d o n day 1 o f pregnancy. V a l u e s represent the means and S E M for three pools o f the endometrial tissues i n each point. As ter i sks indicate statistical difference o f the means between two groups (P<0.05). -88-Day of pregnancy -89-Figu re 3-5 Prof i les o f I G F - I ( A ) and I G F B P (B) levels i n the serum o f superovulated and con t ro l rats. Immature rats were injected w i t h a s ingle dose o f 40 I U P M S G (superovulat ion) o r 4 I U P M S G (control) . Rats were mated 6 0 h after the P M S G injection and sacrificed on the indicated days (day 1: vagina l p lug ) . T h e leve ls o f I G F - I were de te rmined by R I A and the leve ls o f I G F B P determined by l i gand b ind ing assay us ing 1 2 5 I - I G F - I . T h e levels o f I G F B P are presented as a percentage o f the l eve l s o f the con t ro l group observed o n day 1 o f pregnancy. V a l u e s represent the means and S E M for s ix animals i n each point. -90-Day of pregnancy - 9 1 -5, the day o n w h i c h blastocyst implantat ion begins ( F i g . 3 - 3 A ) . T o t a l I G F B P levels i n the con t ro l group r ema ined constant f r o m day 1 to day 3, decreased o n days 4 and 5, then increased o n day 6 ( F i g . 3 - 3 B ) . T h e patterns o f I G F - I and I G F B P prof i les f o l l o w i n g superovula t ion were the inverse o f the con t ro l group ( F i g . 3 - 3 A , B ) . I G F - I l eve ls were e levated d u r i n g the first three days o f pregnancy and decreased o n days 5 and 6. In contrast, superovula t ion suppressed I G F B P levels by 20 -40% d u r i n g the first three days and then increased. M a x i m u m levels i n animals f o l l o w i n g superovulat ion were observed o n day 5. T h e levels o f I G F - I receptor i n the uterus were s imi la r i n both groups unt i l day 5 ( F i g . 3 - 3 C ) . O n day 6, I G F - I receptor l eve l s i n the c o n t r o l g roup inc reased by approx imate ly 6 0 % f r o m those o f day 5, w h i l e the receptor levels o f the superovula t ion group d i d not increased dur ing the same per iod ( F i g . 3 -3C) . T h e patterns o f I G F - I , I G F B P , and I G F - I receptor prof i les i n homogenates o f the separated uterine endomet r ium were s imi l a r to those observed i n the w h o l e uterine tissue homogenates ( F i g . 3-4). A superovulatory dose o f P M S G had no effect on serum I G F - I and I G F B P levels throughout durat ion o f this experiment (F ig . 3-5). I V . D I S C U S S I O N T h e levels o f ovar ian steroid hormones observed i n this study are comparab le to those found i n previous reports. Hyperes t rogenemia dur ing the preovula tory pe r iod and a postovulatory es t radiol-170 peak f o l l o w i n g superovulatory treatment has been p rev ious ly described ( M i l l e r and Arms t rong , 1981a; G i d l e y - B a i r d et a l . , 1986). A decl ine i n the levels o f es t rad io l -170 between day 2 and day 3 preceded changes i n the uterine I G F system, w h i c h were not obse rved u n t i l day 4. These observa t ions are consis tent w i t h the hypothesis that superovulatory treatment induces hyperestrogenemia w h i c h i n turn alter the uterine I G F system i n the rat uterus. Changes i n the uterine I G F system f o l l o w i n g superovulat ion appear to be biphasic . -92-Changes in the first three days of pregnancy were characterized by elevated IGF-I levels and suppressed IGFBP levels, while suppressed IGF-I and enhanced IGFBP levels were found during the second half of preimplantation period (Fig. 3-3). Since IGFBPs, in general, suppress IGF-I action, these changes may indicate enhanced IGF-I action during the first half of preimplantation period and suppressed IGF-I action during the second half of preimplantation period, and at the time of implantation. Although the daily examination of the IGF system used whole uterine tissue homogenates due to the small amount of tissue, these changes appear to be consistent with observations made using homogenates prepared from the endometrium (Fig. 3-4). Changes in the IGF system in the endometrium may have a greater impact on the environment for embryonic development. Embryos arrive in the uterus late on day 3 or early on day 4 of pregnancy. Enhanced IGF-I actions from day 1 to day 3 may render the uterine environment detrimental to preimplantation embryonic development by the time that the embryos enter the uterus. This will be examined in the following chapter. Superovulatory treatments may also affect blastocyst implantation by interfering with uterine function. This may be caused, at least in part, by changes in the uterine IGF system following superovulation. The endocrine requirements for implantation, more particularly for decidualization, have been well characterized in the rat (Psychoyos, 1976). After the uterus has been primed with progesterone for a minimum of 48 h, a single injection with a small amount of estradiol-17 (3 sensitizes the uterus for a deciduogenic stimulus 24 h later. Thus, the sensitization process for a deciduogenic stimulus appears to initiate as early as day 2 of pregnancy when a secondary estradiol-17(3 peak was observed in superovulated rats. The uterine IGF system was perturbed throughout the uterine sensitization period (Figs. 3-3 and 4). Superovulatory treatments also changed the uterine IGF system between day 5 and day 6, i.e. around the time of implantation. Changes in the uterine IGF system following superovulation were characterized by a failure to increase receptor levels (approximately -93-60%) and IGFBP levels (approximately 25%) that were observed in the control group. It has been suggested that the uterine IGF system is possibly involved in the regulation of the decidual reaction (Chandrasekhar et al., 1990; Croze et al., 1990a; Yallampalli et al., 1992). Hence, disturbances in the uterine IGF system may perturb uterine preparation for implantation. This will be examined in further detail later in this thesis. V. SUMMARY AND CONCLUSIONS In summary, treatment with a pharmacological dose (40 IU) of PMSG creates superphysiological levels of estradiol-170 during the periovulatory period and a secondary estradiol-170 peak during the postovulatory period. Changes in the levels of estradiol-170 result in an alteration in the uterine IGF system which can be divided into two distinct phases. The first phase is observed in the first three days of pregnancy which is characterized by enhanced IGF-I action. This may be the result of both increased IGF-I levels and decreased IGFBP levels. The second phase is observed at the periimplantation period or at the time of implantation and is characterized by suppressed IGF-I action. This may be caused by a reduction in the levels of IGF-I and/or an increase in IGFBP levels. These changes may affect preimplantation embryonic development and subsequent implantation. Chapter four will examine the effect of enhanced IGF-I action on preimplantation embryonic development. Chapter five will address the significance of these changes in the uterine IGF system in the decidualization process. -94-CHAPTERFOUR THE EFFECT OF IGF-I ON THE UTERINE MICROENVIRONMENT FOR PREIMPLANTATION EMBRYONIC DEVELOPMENT I. INTRODUCTION Increased early embryonic loss and failure of implantation after superovulation have been associated with a hostile maternal endocrine environment (Moon et al., 1990). Ovarian hyperstimulation generates elevated serum levels of estradiol-17(3 that may jeopardize the establishment of a successful pregnancy (Miller and Armstrong, 1981a; Gidley-Baird et al., 1986). Estrogens administered after ovulation have caused anti-fertility effects; hyperestrogenemia probably interferes with the normal secretory transformation of the endometrium which, in turn, causes a failure in implantation (Morris and Van Wagenen, 1973; Martel et al., 1987). However, the precise mechanisms through which postovulatory estrogen causes detrimental effects on preimplantation embryonic development and implantation have not been defined. A better understanding of such mechanisms is needed if we are to improve the outcome of IVF-ET in human medicine and ET in the livestock industry. The IGF system is one of a number of regulatory systems mediating steroid hormone actions through autocrine/paracrine mechanisms in the uterus. IGF-I is a key member of the uterine IGF system. Other components of this system are IGFBPs and IGF-I receptors. Synthesis of IGF-I is primarily regulated by estrogen, rather than GH, the principal regulator of IGF-I synthesis in most other tissues (Murphy et al., 1987c; Murphy and Friesen, 1988; Norstedt et al., 1989). Other components of the IGF system are also under ovarian steroid hormone regulation in the uterus (Ghahary and Murphy, 1989; Girvigian et al., 1994). Therefore, it is likely that changes in the levels of ovarian -95-steroid hormones following superovulation could disturb the regulation of the IGF system in the uterus. Disturbances in the uterine IGF system may be responsible for the detrimental effects of superovulation, which include early embryonic loss. Electrolytes in the uterine microenvironment appear to have a great impact on preimplantation embryonic development (Biggers et al., 1991). The sodium gradient across the cellular plasma membrane provides the energy to maintain cellular homeostasis and regulate cellular function and metabolism (Lechene, 1988; Cohen and Lechene, 1989). A normal intracellular pH and electrolyte composition is necessary for optimal enzyme function and actions of growth factors that play a central role in preimplantation embryonic development (Pouyssegur et al., 1985; Somero, 1985; Moolenaar et al., 1988). Electrolyte composition in the uterus and uterine luminal fluids may vary under the regulation by ovarian steroid hormones (Kao, 1967; Setty et al., 1973; Van Winkle et al., 1983). For example, in rats and mice, sodium ion content in the uterine luminal fluids decreases during delayed implantation which is experimentally induced by controlling the levels of ovarian steroid hormones (Setty et al., 1973; Van Winkle et al., 1983). Furthermore, the decreased levels of sodium ion in the uterine luminal fluids has been related to decreased levels of metabolism of the mouse embryos (Van Winkel, 1977; Van Winkel, 1981). The previous chapter demonstrated changes in the uterine IGF system following superovulation. This chapter contains two studies. The objective of study one is to determine whether changes in the uterine IGF system, following superovulation, are responsible for an increase in early embryonic loss. It has been hypothesized that increased IGF-I action during the first three days of pregnancy may render the uterine environment hostile to preimplantation embryonic development. Secondly, it has been hypothesized that an alteration in the electrolyte composition of the uterine luminal fluid may be one of mechanisms through which the IGF system mediates embryonic loss associated with superovulation. Thus, study two examines the effect of varying doses of PMSG, between the control and superovulatory doses, on -96-electrolyte composition of the uterine luminal fluids. These studies led to the determination of the effect of IGF-I on the electrolyte composition of the uterine luminal fluids. II. MATERIALS AND METHODS Study One IGF-I Infusion Model Adult female Sprague-Dawley rats (340-360 g BW) were mated overnight with males at estrus. Females were implanted with an Alzet osmotic pump (Model 1003D or 1007D, Alza Co., Palo Alto, CA) at 10:00-11:00 h on the day of vaginal plug (day 1 of pregnancy). Rats were anesthetized and a 4 to 5 cm medial incision was made in the xiphoid process and the pubic tubercle. Rats were unilaterally ovariectomized on the right and the distal end of the delivery tubule of the osmotic pump was introduced into the right ovarian artery, proximal to the uterine artery branch. The left ovary and artery were maintained intact as the control (Fig. 4-1). The incision in the abdominal wall and the skin was sutured leaving the osmotic pump in the abdominal cavity. The osmotic pump was filled with hr-IGF-I in Ringer's solution containing 0.1% of gelatin and 20 IU heparin, prior to implantation. Ten nM hr-IGF-I was infused at 0.5 and 1.0 ul/h using the osmotic pump models 1007D and 1003D, respectively. Twenty five and 50 nM hr-IGF-I, as well as the vehicle alone, were infused at 1.0 jxl/h using the 1003D model. Four rats were used in each group including the control (non-infusion) group, in which animals were unilaterally ovariectomized but not infused. Rats were sacrificed at the end of the 48 h infusion period (day 3 of pregnancy). The uterus and blood were collected for IGF-I, IGFBP and IGF-I receptor assays. The levels of IGF-I, IGFBP and IGF-I receptor in the left and right uterine horns were determined separately. -97-Figure 4-1 A rat uterine IGF-I infusion model. Adult rats (340-360 g BW) were mated at estrus and implanted with an alzet osmotic pump. Rats were anesthetized and a 4 to 5 cm incision was made on the median line in the middle of the xiphoid process and the pubic tubercle. Rats were unilaterally ovariectomized on the right and the distal end of the delivery tubule of the osmotic pump was introduced into the right ovarian artery proximal to the uterine artery branch. The left ovary and artery were maintained intact as control. The incision in the abdominal wall and the skin was sutured leaving the osmotic pump in the abdominal cavity. -98-Osmotic pump Uterine artery Ovary Uterine horns Aorta Ovarian artery Ovary -99-Anti-IGF-I Antibody Preparation Rabbit anti-IGF-I antiserum (Amersham Canada) was diluted with 100 mM phosphate buffer, pH 7.0, (1:1 v/v) and applied to a protein A column (Bio-Rad). Antibody was eluted with 1 M acetic acid and the eluate from the protein A affinity column was then applied to a Sephadex G-25 column. Antibody solution eluted in 100 mM phosphate buffer, pH 7.4, was concentrated by using a microcentrifuge filter, aliquoted and stored at -30°C. An aliquot was subjected to protein assay, in which a standard curve was prepared using rabbit IgG (Sigma). Uterine Luminal Fluid Collection Eight adult female rats were unilaterally ovariectomized on the right hand side, and implanted with an osmotic pump (Model 1003D) at 10:00-11:00 h on day 1 of pregnancy as described above. Four rats were infused with hr-IGF-I at a concentration of 10 nM (at an infusion rate of 1 u.l/h for 48 h) from day 1 to day 3 (IGF-I group). The other four rats were infused with vehicle alone (vehicle group). As a control, four adult rats were unilaterally ovariectomized on day 1 but not infused (non-infusion group). Four immature rats were induced to superovulate by injecting with 40 IU PMSG (superovulation group). Four immature rats were induced to superovulate with 40 IU PMSG and were implanted with an osmotic pump (Model 1003D), subcutaneously on the back, at 10:00-11:00 h on day 1. Immature rats were infused with 1 mg/ml anti-IGF-I antibody for 48 h from day 1 to day 3 at an infusion rate of 1 pil/h (superovulation + IGF-I antibody group). All uterine luminal fluids were collected at 10:00-11:00 h on day 3 of pregnancy. The uterus was dissected to separate the uterine horns. Uterine luminal fluids were collected separately from the left and right uterine horns by flushing the uterine lumen with 0.25 ml of M199 (Catalogue Number 11150, GIBCO) within 5 min of being sacrificed. In the superovulation and superovulation + IGF-I antibody groups, the uterine luminal fluid from the left and right uterine horns of the same animal were combined. Collected media were centrifuged at 3,000 x g for 10 min to remove cell debris. One hundred p.1 aliquots of -100-each supernatant was mixed with M199 (1:1 v/v) containing 10% fetal bovine serum (GIBCO) and equilibrated with 5% CO2 in humidified air. The osmotic concentration of the collected media was determined using an osmometer. Two 20 (il-droplets were prepared from each sample. An aliquot of the uterine luminal fluids collected from the superovulation group and those from the right uterine horns of the non-infusion and IGF-I groups were dialyzed (molecular weight cutoff < 1,000) against fresh M199 for 6 h at 4°C and used for embryo culture. Superovulation and Embryo Collection Immature rats were injected with a single dose of 20 IU PMSG at 10:00 h at 26 to 29 days of age and mated with fertile males, 60 h after the PMSG injection. Rats were mated overnight and the presence of vaginal plugs was examined at 8:00 h on the following day (day 1 of pregnancy). Embryos at the 8-cell stage were collected at 2:00-3:00 h on day 4 of pregnancy by flushing the oviduct and uterine lumen with Dulbecco's PBS containing 1 mg/ml PVA. Eight-cell stage embryos were pooled from at least four rats in each experiment and randomly assighed to droplets of culture media that contained the uterine luminal fluids. Embryo Culture Embryos were washed three times in M199 containing 5% FCS and transferred to 20 ul droplets that contained the uterine luminal fluids. Ten embryos were cultured in each droplet (a total of eighty embryos for each uterine luminal fluid group) for 48 h at 37°C in humidified air containing 5% CO2. The developmental stages of embryos were scored and the number of cells in each blastocyst was counted by the air-drying method (Tarkowsky, 1966). -101 -Study Two Animal Treatments and Uterine Luminal Fluid Collection Immature rats were injected with 4 (control), 10, 20, or 40 (superovulation) IU PMSG at 9:00-10:00 h on the day of 28-day-old and mated 60 h after the PMSG injection. Some immature rats that had been injected with 40 IU PMSG were infused with anti-IGF-I antibody from day 1 (day of vaginal plug) to day 3 of pregnancy as described above. Adult rats were treated for IGF-I infusion, superovulation, and superovulation + IGF-I antibody groups as described in study one. Control rats were unilaterally ovariectomized on day 1 but not infused (non-infusion group). Five immature or adult rats were used in each treatment group. All the rats were sacrificed at 11:00 h on day 3 and the uterine horns removed. Uterine luminal fluids were collected by flushing the uterine lumen with degassed 0.3 M mannitol solution (0.25 ml/uterine horn) and the uterine flushes were kept away from air contact to avoid changes in free C O 2 levels. The uterine flushes centrifuged at 3,000 x g for 10 min. The uterine flushes from the left and right uterine horns in the same animals, except for the infusion groups, were combined and the electrolyte composition determined. For cation assays, an aliquot of each supernatant was saved and frozen at -70°C until assayed. Determination of Electrolyte Composition The levels of all electrolytes were determined by at Biochemical Laboratory of Vancouver General Hospital (Vancouver, BC). Frozen supernatants were thawed, and 15 mM LaCl 3 , 4 mM CsCl, and 100 mM HC1 were added. The levels of Na+' K+ C a 2 + , and M g 2 + were determined by atomic absorption photospectroscopy (Sanui, 1971; Sanui and Rubin, 1982). The levels of Cl" were determined by the coulometric-amperometric method (Dietz and Bond, 1982). The levels of H P O 4 2 - and H C O 3 - were determined, as total inorganic phosphorus and total C O 2 , respectively, by using Kodak Ektachem Clinical Chemistry Slides (PHOS and E C 0 2 , Kodak, Eastman Kodak, Rochester, NY). -102 -Statistical Analysis Data are expressed as the mean ± SEM, except for Figure 4-3 where 95% confidence limit was used for error bar. The mean levels of the uterine IGF-I and IGFBP in the right and left horns of the same animals were compared by the paired Student's t-test. The mean levels of IGF-I, IGFBP, and IGF-I receptor in the same side of uterine horns across different infusion groups were compared by ANOVA, followed by Tukey's test. The developmental rates of the embryos were compared by Chi-square analysis. The mean number of cells in embryos cultured with uterine luminal fluids from the left and right uterine horns within animals were compared by the paired Student's t-test, while those obtained from the different groups were compared by ANOVA, followed by the Tukey's test. Total cation contents of uterine luminal flushes were compared by ANOVA followed by the Tukey's test. Statistical analysis was performed using the computer software 'SYSTAT'. III. RESULTS Study One The IGF-I Infusion Model Uterine IGFBP and IGF-I receptor levels are presented as a percentage of the levels in the left uterine horn of the non-infusion group. Serum IGFBP levels are presented as a percentage of the levels in the non-infusion group (Fig. 4-2). The control values for the levels of IGFBP in the uterus and serum were 79 ± 4.7 and 131 ± 2 6 pmol IGF-I/(ig protein, respectively, while the levels of the IGF-I receptor in the uterus were 64 + 3.8 fmol IGF-I /(ig membrane protein. Vehicle alone had no effect on the levels of IGF-I as compared to the non-infusion control group (Fig. 4-2A). IGF-I infusions increased the levels of IGF-I in the infused uterine horn but had no effect on IGF-I levels in the control horn of the same animal. -103-Figure 4-2 The effect of IGF-I infusions on the uterine IGF system. IGF-I was infused at the indicated conditions from day 1 to day 3 of pregnancy by an osmotic pump that was implanted in the abdominal cavity (Fig. 4-1). The levels of uterine and serum IGF-I (A) was determined by RIA and the levels of IGFBP (B) and IGF-I receptor (C) determined by ligand binding assays using 125I-IGF-I. The levels of IGF-I, IGFBP and IGF-I receptor in the left (control) uterine horn and the right (infused) uterine horn were determined separately. The levels of IGFBP and IGF-I receptor are presented as percentage of the levels of the control group that was not received an infusion. Values represent the means and SEM for four animals in each group. Letters on the top of bars indicate statistical differences of the means (a<b<c) across the treatment groups. Asterisks indicate statistical differences (*P<0.05, **P<0.01) of the means between the control and infused uterine horns within the animals. -104-(ng/g tissue) «§> <sr <s Uterus Control horn Infused horn * (A) IGF-I Non-infusion Vehicle-1.0 ul/h 10nM-0.5 (.il/h 10 nM-1 ul/h 25 nM-1 ul/h 50 nM-1 ul/h © <v ^ co ^ ^ H Serum r H H H (%)^ ^ <§> <§> (B) IGFBP o <§> # £ <%> Non-infusion Vehicle-1.0 ul/h 10nM-0.5 ul/h 10 nM-1 ul/h 25 nM-1 ul/h 50 nM-1 ul/h H H H H 1 (%)^ #<§><§> £ (C) IGF-I receptor Non-infusion Vehicle-1.0 ul/h 10 nM-0.5 ul/h 10 nM-1 ul/h 25 nM-1 ul/h 50 nM-1 ul/h -105 -When IGF-I was infused at 1 ul/h at 10 nM or at greater concentrations, IGF-I levels in the infused horns were similar to those observed in the uterus on days 2 and 3 of pregnancy, following superovulation (Fig. 3-3A and Fig. 4-2A). The levels of IGF-I in the control horn were, however, not affected by IGF-I infusions and were not significantly different from the levels observed in the non-infusion or vehicle group (Fig. 4-2). IGF-I infusions at 10 nM at 0.5 pl/h did not alter uterine IGFBP levels in either uterine horn (Fig. 4-2B). However, IGF-I infusions at 10 and 25 nM at 1 (il/h suppressed uterine IGFBP levels by 30% (P<0.001) in the infused uterine horn. The suppressed IGFBP levels were equivalent to approximately 60% on the scale of Fig. 3-2B and were similar to the levels of IGFBP observed on day 3 of the superovulation group (Fig. 3-2B and 4-2B). IGFBP levels in the control horn of the same animal were not significantly different form the levels observed in the non-infusion group. Interestingly, IGF-I infusion at 50 nM at 1 u.l/h increased the levels of IGFBP by approximately 18% (P<0.01) in the infused horn compared with the vehicle and non-infusion controls (Fig. 4-2B). The levels of IGFBP remained at base level in the control horn. IGF-I infusions had no effect on the serum levels of IGF-I and IGFBP or uterine IGF-I receptor levels (Fig. 4-2). Embryo Culture with Uterine Luminal Fluids The osmotic concentration of the uterine flushes containing uterine luminal fluid was between 275-295 mOsm/kg H20. The mean concentrations of flushes between groups were not significantly different from each other (Table 4-1). Uterine luminal fluids from the non-infusion group did not affect embryonic development and the number of cells in blastocysts as compared to those of embryos that were cultured with media alone (Table 4-2). In contrast, uterine luminal fluids of the superovulation group inhibited embryonic development. The rate of blastocyst formation (P<0.001) and the number of cells in blastocysts decreased (P<0.01). In addition, the rate of embryo degeneration during the 48-hour-culture increased in the superovulation group (P<0.001), compared to those of the -106 -Table 4-1. Osmotic concentrations of uterine luminal flushes following the IGF-I infusion and superovulation Uterine flushes Uterine hom Osmotic concentration (mOsm/kgH20)a Flushing medium - 285b Non-infusion Left 288 ± 2.9 Right 287 ±4.1 Vehicle Left 286 ± 4.9 Right 287 ± 4.4 IGF-I Left 286 ± 5.0 Right 285 ± 5.5 Superovulation Bothc 284 ± 3.5 Superovulation + IGF-I antibody Bothc 288 ± 1.7 aValues represent the mean and SEM for four repeated experiments. bThe osmotic concentration of flushing medium (Medium 199, Cat. No. 11150, M199, Gibco) was adjusted 285 ± 3 mOsm/kg H2O. cUterine luminal flushings of both left and right horns were combined prior to determination of the osmotic concentration. -107 -w 00 +1 a a, 3 fc i o 00 Tt 1 •a CA % S> a ii a o 5 * a ~-, G S S3 -a CA 3 ^ O to o  1 2 ii G & -8 CA > , o O CA I CA •a 3 «G <n <N CN VO Tt Tt 00 <—I T-H (-» m CO Tt r - l CS vo cn T-H +1 CS »n Tt r- cn CS T-H' +1 +1 CS Tt vd cn Tt Tt oo r-^  T-H T-H +1 +1 Tt VO T-H cn Tt Tt m Tt 1-J cs +1 +1 T-j Tt Tt cn +1 CS Tt cn vq T-H +1 cn 5' cn cn in ,—s oo in o "5k Tt © cs cs vo cs — ' vd CS cs cn cn cs Ov >n cs' >n © ' cn *—' 00 T-H T-H cs T-H cs cs cs cs cs cs 00 Tt T3 CS Tt Tt CS >n cs >n F~ cn O oo •a 00 >n cs' w od >n T-H vo cs' w od Tt •>—' vd >n cn cs (28. (52. o >n Os Tt cs Tt Os cn >n Tt Os T-H cn CS cs Tt p p >n © «n cn cn 00 «n m" T-H >n T-H cs T-H © ' cs i> T-H vd T-H vd T-H 00 T-H T-H cs CS © VO Tt cn cn >n Tt <*H "W) * H *S) <4H *S) 3 2 3 2 3 2 1 cd s C 0 c 1 G O fc 1 PH o 'a •3 I 3 oo •o PH P. G I e §• CO I 1 £ 1 - i CA 8 3 U 8 a CA I *s I I bX) 3 •fl .a o E o g e o CJ 1 f o o 3 > CH CH o o e •2 I CA CO 3 oo § c 0 1 X5 2 2 o 8 8 53 u 3 13 > 00 T H <U o S .a a 53 O {in O J4H 53 p £ 13 00 ii 3 8 8 CA U 3 13 > o ? 0H 13 > © © V OH I 3 sr 4 i oo oo 13 ^ 2 3 CA ST "g 'A 'A '§ -S 1 a o 0 % CA 1 T C I 3 • f l O £> M 0 oo 1 13 J 3 - 1 0 8 -non- infus ion group. T h e an t i - IGF- I ant ibody infus ion, f o l l o w i n g superovulat ion, restored the rate o f blastocyst fo rmat ion and the number o f ce l l s i n blastocysts to n o r m a l levels (Table 4-2) . V e h i c l e infusions d i d not alter e m b r y o n i c development and the number o f cel ls i n blastocysts. I G F - I infusions selectively rendered uterine lumina l f luids detrimental to embryonic development . E m b r y o s cul tured w i t h the uterine l u m i n a l f lu ids obtained f r o m the infused uter ine ho rn o f the I G F - I i n fus ion group decreased the rate o f b las tocys t fo rma t ion (P<0.001), the number o f ce l l s i n the blastocysts (P<0.01), and an increased the rate o f embryo degeneration (P<0.001). T h e developmental rate o f embryos and the mean number o f ce l l s i n blastocysts cul tured w i t h the uterine l u m i n a l f lu ids f r o m the cont ro l horn o f the I G F - I in fus ion group were compa t ib le to those o f the non- in fus ion and v e h i c l e groups (Table 4-2). D i a l y s i s o f the uterine l u m i n a l f luids o f the superovulat ion and I G F - I infusion groups i m p r o v e d embryon ic development and the number o f ce l l s i n blastocysts (Table 4-3). T h e percentage o f embryos degenerating decreased (P<0.001), whereas the rate o f blastocyst formation (P<0.001) and the mean number o f cel ls i n blastocysts increased. Study T w o T h e leve ls o f each electrolyte is presented as a percentage o f the total amount o f cat ions o r anions (F igs . 4-3 and 4) . P M S G , at the superovulatory dose (40 I U ) , altered both ca t ion and anion compos i t ions o f the uterine l u m i n a l flushes o n day 3 o f pregnancy. In the cations, the percentage o f N a + i n the superovula t ion group decreased by 2 6 % and that o f K + increased 2 7 % , as compared to those o f the cont ro l (4 I U P M S G ) group (F ig . 4-3). In anions, the percentage o f C l " decreased by approximate ly 15% and that o f H C O 3 " increased to a s imi la r extent. A n t i - I G F - I antibody infusion restored the alterations o f cation c o m p o s i t i o n , f o l l o w i n g superovula t ion , to the con t ro l l eve ls ( F i g . 4-3) . In contrast, the an t i - IGF-I antibody infusion had no effect o n the altered anion composi t ion . Compos i t ions o f cations and anions i n the a l l P M S G groups, except for the superovulat ion group, were -109-w +i .5 oo s? 8 S "8 J * i 00 I* OO 0 1 cd C 1 1 & -8 O o OO CS ! • i - H 3 r- r- cn »—1 00 3, i — i CN cn ~ — ' COO oa*> cn CO »-H CN i - H +1 +1 +1 +1 +1 +1 00 m CN Cn J—l d Ov cn cn cn cn o as cn in vd in Ov CN CN CN CN in cn m cn »-H o 00 m CN vo CN CN CN CN -H- -H-CN 00 00 in r—1 od cn cn od v© CN in CN Ov cn cn oo f-H CN cn in m CO p oo o CN vo VO >n od d T—1 »—i l - H CN o cn CN in VO + c o • r H § fc PL, o c 0 •s 1 co I XI 3 •I * I I o * 8 ! s a *s <D «H S2 . oo o V CM m o sa ? ."§> 3 S § W fc U j . - 1 1 0 -F i g u r e 4-3 T h e effect o f P M S G o n electrolyte compos i t ion o f the uterine l u m i n a l f lu id . Immature rats were injected w i t h P M S G at the indicated doses. A group o f rats that had been in jected w i t h 4 0 I U P M S G was in fused a n t i - I G F - I ant ibody f r o m day 1 to day 3. U te r ine l u m i n a l f lu ids were co l l ec t ed by f lu sh ing the uterine l umen w i t h degassed 0.3 M mann i to l so lu t ion (0.25 ml/uter ine horn) o n day 3 o f pregnancy. T h e levels o f Na + > K + , C a 2 + , and M g 2 + were determined by atomic absorption photospectroscopy. T h e levels o f C l " were de termined b y the cou lome t r i c - amperomet r i c method . T h e l e v e l s o f H P O 4 2 " and H C O 3 " w e r e d e t e r m i n e d , as to ta l i n o r g a n i c phosphorus and total CO2 , respectively, by us ing K o d a k E k t a c h e m C l i n i c a l Chemis t ry Sl ides . Elect ro lyte composi t ions are presented as a percentage o f each component to the total anion or cation. Va lues represent the means and S E M for f ive rats i n each group. - Ill -- 1 1 2 -F igu re 4-4 T h e effect o f I G F - I in fus ion o n e lec t ro ly te c o m p o s i t i o n o f the uterine l u m i n a l f l u id . A d u l t rats were mated at estrus and infused w i t h 10 n M I G F -I at 1 m l / h f rom day 1 to day 3 by an osmotic pump . Uter ine l u m i n a l f luids were col lec ted by f lushing the uterine lumen w i t h degassed 0.3 M manni to l solut ion (0.25 ml/uterine horn) o n day 3 o f pregnancy. T h e levels o f Na + > K + , C a 2 + , a n d M g 2 + w e r e d e t e r m i n e d b y a t o m i c a b s o r p t i o n photospectroscopy. T h e levels o f C l " were determined by the coulomet r ic -amperometric method. T h e levels o f H P O 4 2 " and H C O 3 " were determined, as total inorganic phosphorus and total C O 2 , respect ively , by us ing K o d a k E k t a c h e m C l i n i c a l Chemis t ry Sl ides . E lec t ro ly te compos i t i on is presented as the mean percentage o f each component for f ive rats to the total anion or cat ion. The data for the superovulated rats were taken f r o m F i g . 4-3. -113-100 80 H 60 40 20 0-L mm, • Mg++ M Ca++ m K+ • Na+ 100 \ 80 60 40 20 o-^  Wm mm mm mmm. mm El HP04-H HC03-• Cl-- 1 1 4 -s i m i l a r to that o f the n o n - i n f u s i o n g roup ( F i g . 4 -3 ) . T o t a l c a t i o n content i n the superovula t ion group increased b y 2 .1 - fo ld compared to that o f the non- infus ion cont ro l (P<0.01, Tab le 4-4) . T o t a l ca t ion content i n the other P M S G (4, 10, and 20 I U ) groups were not statistically different f rom that o f the non-infusion group. U n i l a t e r a l o v a r i e c t o m y o r v e h i c l e i n f u s i o n had n o effect o n the e lec t ro ly te compos i t ions and total ca t ion content i n the infused and con t ro l horns . I G F - I in fus ion altered the electrolyte compos i t i on o f the uterine l u m i n a l f l u i d i n the infused uterine horns but d i d not change the electrolyte compos i t ion o f the cont ro l horns compared to that o f the non- infus ion group ( F i g . 4-4). T h e alterations i n electrolyte compos i t ions o f both cations and anions were compa t ib l e to those observed i n superovulated immature rats. C a 2 + , M g 2 + , and FIPO4 2 " levels were constant throughout these experiments . I G F - I infus ion or an t i - IGF- I infus ion f o l l o w i n g superovulat ion had no effect o n total ca t ion contents i n the uterine l u m i n a l flushes (Table 4-4). I V . D I S C U S S I O N T h i s study demonstrated that enhanced I G F - I actions i n the uterus, resul t ing f rom superovu la to ry treatments, causes an increase i n the rate o f ea r ly e m b r y o n i c loss . Superovula to ry treatments elevated serum ovar ian steroid leve ls and the pos tovula tory estradiol-17(3 peak ( F i g . 3 - l ) ( M i l l e r and A r m s t r o n g , 1981a; G i d l e y - B a i r d et a l . , 1986). Changes i n the I G F system i n the uterus dur ing the first three days o f pregnancy f o l l o w i n g superovulation were characterized by increased I G F - I actions i n the uterus (Chapter Three). T h e I G F - I in fus ion m o d e l ach ieved these changes i n the uterine I G F sys tem f o l l o w i n g superovulat ion, l oca l l y i n the infused uterine horn, without changing the I G F system i n the cont ro l uterine horn and i n the c i rcu la t ion at cont ro l levels ( F i g . 4-2) . In this m o d e l , on ly the uterine l u m i n a l f luids f rom the infused uterine horn, i n w h i c h the I G F system f o l l o w i n g superovulation had been m i m i c k e d , became detrimental to embryonic development (Table -115-Table 4-4. Total cation content in the uterine luminal flushes Treatments Uterine horn Total cation concentration (nM)a PMSG (IU) 4 10 20 40 (Superovulation) Superovulation + IGF-I antibodyd Infusionse Non-infusion Vehicle IGF-I Bothb 1.33 ±0.30 Both 1.28 ±0.22 Both 1.34 ±0.26 Both 2.52±0.29c Both 2.58 ± 0.32c Left 1.21 ±0.21 Right 1.22 ±0.18 Left 1.19 ±0.21 Right 1.23 ±0.31 Left 1.23 ±0.33 Right 1.31 ±0.26 aValues represent the means and SD for five rats. bUterine luminal fluid from the left and right horn in the same rat were combined. cTotal cation concentration is greater than that of the other groups (P<0.01). dAnti-IGF-I antibody was systemically infused from day 1 to day 3. eInfusions were performed on the right uterine horn. -116 -4-2). Furthermore, the uterine luminal fluids of the superovulation group, which was detrimental to embryonic development, could be reversed by anti-IGF-I antibody (Table 4-2). It appears that locally enhanced IGF-I actions in the uterus, caused by superovulatory treatments, may inhibit embryonic development and increase early embryonic loss through alterations in the uterine environment. Dialysis of the uterine luminal fluids obtained from the superovulation group and the uterine luminal fluids of the IGF infused horns restored embryonic development to normal levels (Table 4-3). One possible explanation for this result is that the osmotic concentration of these uterine luminal flushes may be unsuitable for embryonic development. However, this explanation can be excluded, since osmotic concentration of all uterine luminal flushes are within an acceptable range for embryonic development and the mean concentration of the uterine flushes of each group were not statistically different (Table 4-1). An alternative explanation is that some molecules in the uterine luminal fluids are responsible for the detrimental effect on embryonic development. These molecules are not likely to be proteins, since these molecules must be small enough (molecular weight < 1,000) to be removed by dialysis. The detrimental nature of the uterine luminal fluids in the uterus that have been exposed high levels of IGF-I may be attributed, at least in part, to an alteration in electrolyte composition in the uterine luminal fluids observed in sudy two. Alterations in electrolyte composition following IGF-I infusion were observed only in the infused horns and is compatible to that observed in superovulated immature rats (Fig. 4-4). Alterations in cation composition caused by superovulation were restored by anti-IGF-I antibody infusion to control levels (Fig. 4-3). These observations suggest that exposure to high levels of IGF-I causes changes in cation composition. In contrast, the role of IGF-I in the alteration in anion composition is less clear. Although IGF-I infusions mimicked the alterations in anion composition observed following superovulatory treatments, anti-IGF-I antibody failed to restore these alterations (Figs. 4-3 and 4). However, this does not rule out a role - 117-for IGF-I in regulating levels of anions in uterine luminal fluids. Alterations in anion composition of the uterine luminal fluids may be regulated by multiple factors which include IGF-I. Therefore, inhibition of IGF-I action alone may not be able to restore the altered anion composition, caused by the superovulatory treatment. This is in direct contrast to the regulation of cation composition, where IGF-I appears to play a major regulatory role. It is unclear how superovulation or IGF-I causes alterations in the electrolyte composition of uterine luminal fluid. The uterine flushings from superovulated rats often contains desquamated cellular debris (Miller and Armstrong, 1981a). In the present study, a visible amount of cellular debris was observed in some of the uterine luminal flushes from the superovulation and the IGF-I infusion groups; the amount of cellular debris in the IGF-I group was considerably less than that observed in the superovulation group. Intracellular fluid has a greater K+/Na+ ratio than extracellular fluid. Together, cell disruption of the endometrial epithelial cells may be, at least in part, responsible for the increase in the K+/Na+ ratio caused by superovulation. IGF-I may mediate this detrimental effect of superovulation. However, the levels of HPO42", determined as total inorganic phosphate, were constant throughout these experiments (Figs. 4-3 and 4). Since intracellular fluid contains high levels of inorganic phosphate, this may suggest that disruption of the uterine luminal epithelial cells may not contribute significantly to the increase in the K+/Na+ ratio observed in the present study. Superovulation may increase uterine luminal fluid volume. Generally, the sum of the four cations that were examined in this study can represent total cation levels in body fluid, and total anion levels are equivalent to those of cations. Thus, changes in the total cation contents of uterine luminal flushes, determined in the present study, may reflect changes in the volume of uterine luminal fluid. Total cation levels of the superovulation group increased by approximately 2-fold compared to those of the non-infusion or 4 IU PMSG groups (Table 4-4). This may indicate a comparable increase in the volume of - 1 1 8 -uterine l u m i n a l fluid. T h e increase i n the uterine l u m i n a l fluid v o l u m e is consistent w i th an increase i n uterine wet we igh t o f up to 2 - f o l d that i s obse rved after superovula tory treatment i n immature rats ( M i l l e r and A r m s t r o n g , 1981a). Superovula t ion m a y increase f l u id accumulat ion i n the uterus and the uterine lumen . S u c h an increase i n uterine l u m i n a l fluid vo lume may adversely affect embryonic development. In contrast, total cat ion content i n the uterine l u m i n a l f lu id o f the I G F - I infusion and superovulat ion + an t i - IGF- I ant ibody groups were indis t inguishable f r o m that o f the non-infus ion group. T h i s m a y suggest that the increase i n the v o l u m e o f uterine l u m i n a l fluid caused by superovulatory treatment i s not mediated by I G F - I . L o c a l regulators, other than I G F - I , m a y mediate this change f o l l o w i n g superovulation. T h e ra t io o f N a + to K + i n the non- in fus ion c o n t r o l group o f the present study (5.2:1) is different f r o m that observed i n uterine flushes obtained on day 5 o f pregnancy (1.4:1, Setty et a l . , 1973). T h i s m a y be caused by differences i n the exper imenta l designs o f the t w o studies. In the present study, rats were unilateral ly ovar iec tomized o n day 1 and uterine l u m i n a l f l u id col lected o n day 3 o f pregnancy. Differences i n the method o f uterine l u m i n a l f l u i d co l l e c t i on m a y also be responsible for differences i n the rat io o f the two cations. T h e previous experiment (Setty et a l . , 1973) used de ion ized water (0.5 ml /horn) , w h i l e the present study used an i so ton ic so lu t ion (0.25 ml /ho rn ) . L a r g e v o l u m e s o f hypo ton ic so lu t ion m a y increase K + content by caus ing c e l l d i s rup t ion d u r i n g uterine lumina l fluid col lect ion. E l e c t r o l y t e env i ronment appears to be c r i t i c a l for p re implan ta t ion e m b r y o n i c development as prev ious ly discussed i n chapter one. Superovulatory treatments and I G F - I infusions decreased the percentage o f N a + and C l " , and increased those o f K + and H C O 3 " (F igs . 4-3 and 4). These changes have been shown to be detr imental to pre implanta t ion embryon ic development . H i g h levels o f K + reduce the rate o f blastocyst format ion i n the mouse ( W i l e y , 1984; W i l e y , 1987). Substitution o f either N a + o r C l " i n the embryo culture m e d i a reduced the rate o f blastocoele expans ion i n the mouse ( M a n e j w a l a et a l . , 1989). -119-Low levels of NaCI in the culture medium allow glutamine, a preferred energy substrate for early preimplantation embryonic development, to impair embryonic development (Chatot et al., 1989; Lawitts and Biggers, 1992). Evidence suggests that the inhibitory effects of K +, Na+, and Cl- on the formation of blastocoele are exerted through their ability to modulate Na+/K+-ATPase activity (Cohen et al., 1976; Wiley, 1984; Wiley, 1987; Dumoulin et al., 1993). Na+/K+-ATPase plays a central role in formation of the blastocoele that morphologically defines the blastocyst stage (Wiley, 1987). This is consistent with the observation that the uterine luminal fluids obtained from superovulated rats decreased the rate of embryonic development to the blastocyst stage (Table 4-2). Thus, the observed alterations in electrolyte composition following superovulation appear to be critical for preimplantation embryonic development. Locally enhanced actions of IGF-I, caused by superovulatory treatments, may affect implantation and the post-implantational development of embryos. Uterine luminal fluids from the IGF-I infused uterine horns and uterine luminal fluids of the superovulation group not only reduced the developmental rate of embryos to the blastocyst stage but also reduced the number of cells in the blastocysts (Table 4-2). As discussed in chapter two, blastocoele cavity formation appears to reflect the development of fluid transport systems, which is related to time after fertilization (Smith and McLaren, 1977; Winston et al., 1991). Therefore, embryos with blastocoelic cavity that have fewer cells are not necessary capable of implanting. Furthermore, a substantial proportion of blastocysts which contain a fewer number of cells may have a numerically deficient or absent ICM and are not capable of post-implantation development (Hardy et al., 1989). Data from IGF-I infusion experiments performed in study one of this chapter led to some interesting observations. IGF-I, infused at 1 |il/h at all concentrations examined, elevated uterine IGF-I levels comparable to those found after superovulation (Fig. 4-2A). The total amount of IGF-I infused during a 48-hour period at 10 to 50 nM hr-IGF-I, at 1 u,l/h, is approximately 3.7 to 18.5 ng, respectively. These amounts are not enough to - 1 2 0 -achieve increases i n uterine I G F - I levels by s imple accumulat ion o f infused h r - I G F - I . T h i s m a y indicate that I G F - I up-regulates i t o w n synthesis i n the uterus. Synthesis o f I G F - I i n the uterus is p r i m a r i l y under estrogen con t ro l ( M u r p h y et a l . , 1987c) . G H , a p r i n c i p a l regulator o f I G F - I synthesis i n most o f organs, has been reported to stimulate, inhibi t , or to have no effect o n uterine I G F - I synthesis under var ious condi t ions ( M u r p h y et a l . , 1987b; M u r p h y and Fr iesen , 1988; Norstedt et a l . , 1989). Fur ther evidence indicates that I G F - I m a y self-regulate its o w n synthesis. I G F - I infusions in to rats decreased hepatic I G F - I m R N A leve l s w h e n rats were main ta ined o n an energy-rest r ic ted diet (Scha l ch et a l . , 1989). Insu l in , a c lose ly related peptide w h i c h shares its receptors w i t h I G F - I , enhanced I G F - I synthesis i n the l i v e r and other tissues (Fag in et a l . , 1989b; Johnson et a l . , 1989). C lea r ly , further investigations are needed to determine whether I G F - I self-regulates its o w n synthesis i n the uterus. E x a m i n i n g the levels o f I G F - I m R N A i n the I G F - I infus ion mode l m a y g i v e us ins igh t in to the mechan i sms regu la t ing I G F - I express ion i n the uterus. A n o t h e r explanat ion for the increase i n I G F - I levels after I G F - I infusions i s that the I G F - I infus ions m a y increase b l o o d f l o w by some m o d i f i c a t i o n i n the l o c a l vascu la r system. S ince serum I G F - I l eve ls are cons iderab ly greater than extractable-uterine tissue I G F - I leve ls (F igs . 3-3 and 4) , an increase i n b l o o d f l o w may result i n a s ignif icant increase i n I G F - I tissue levels . T h e effect o f I G F - I on b lood f l o w regulat ion has not been determined. A n o t h e r interest ing f ind ing i n this study is that I G F - I seems to d i rec t ly regulate I G F B P levels i n the rat uterus. S o m e hormones , such as ovar ian steroid hormones , that stimulate the secretion o f specific I G F B P subtypes can inhibi t the secretion o f other I G F B P subtypes ( G i r v i g i a n et a l . , 1994). A l t h o u g h the effect o f I G F - I and its related peptides on I G F B P secretion also differs between tissues and c e l l types, I G F - I and i n s u l i n have been reported to inh ib i t I G F B P - 1 secretion i n human dec idua l ce l l s ( T h r a i l k i l l et a l . , 1990). In the present study, the abi l i ty o f I G F - I to regulate total I G F B P levels differed depending on the concentrat ion o f h r - I G F - I ( F i g . 4-2) . W h e n h r - I G F - I was infused at 1 u l / h , I G F - I at l o w e r concentrat ions (10 and 25 n M ) suppressed I G F B P leve l s , w h i l e I G F - I at h igher - 121 -concentration (50 nM) increased IGFBP levels (Fig. 4-2B). These results are interesting since uterine IGF-I levels were at the same level in these three infusion groups at the end of the 48-h infusion period. There are at least two possible explanations for this apparent discrepancy. First, there may be differences in the time course of IGF-I regulating its expression in the different IGF-I infusion groups. For example, uterine IGF-I levels in the 10 nM-1 u,l/h group might achieve the elevated IGF-I levels at the end of the 48 h infusion period, while uterine IGF-I levels of the 50 nM-1 ui/h group might reach the same levels earlier and maintain the elevated levels for an extended period. Elevated IGF-I levels over an extended period of the time may be responsible for switching the IGF-I regulation on IGFBP levels from a inhibitory or stimulatory mode. Second, IGFBP levels were determined as a total amount of binding proteins, rather than the amount of specific IGFBPs. As discussed above, the levels of IGFBP subtypes appear to vary differendy to changes in ovarian steroid hormone levels (Girvigian et al., 1994). Subtypes of IGFBP would respond differently to IGF-I. Such differential regulation may also be responsible for the biphasic response in the total uterine IGFBP levels observed in the different IGF-I infusion groups. Examination of the effects of IGF-I on the levels of the specific IGFBP subtypes at different time points would provide insight into the role of IGF-I in the regulation of IGFBPs in the uterus. V. SUMMARY AND CONCLUSIONS This chapter demonstrates a role for the uterine IGF system in increasing early embryonic loss following superovulatory treatments. It is likely that pharmacological dosages of exogenous gonadotropins create superphysiological levels of estradiol-170 in the circulation. Superphysiological levels of estradiol-170 enhance IGF-I actions in the uterus by increasing IGF-I levels and by decreasing IGFBP levels. Enhanced IGF-I actions render a uterine environment hostile to preimplantation embryonic development. - 122-The alterations in electrolyte composition of the uterine luminal fluids may reflect, at least in part, changes in uterine microenvironment for preimplantation embryonic development following superovulation. These findings also suggest that the uterine IGF system is an important mediator of estrogen action in the regulation of uterine function. Superovulatory treatment may change a volume of uterine luminal fluid. This may also adversely affect embryonic development. This detrimental effect of superovulation appears to not be mediated by IGF-I. Apparently, IGF-I is one of a number of autocrine and paracrine factors that regulate uterine function. Studies involving other growth factors and cytokines may provide further understanding on the mechanisms through which superovulatory treatment causes the changes in uterine environment observed in this study. - 1 2 3 -C H A P T E R F I V E T H E E F F E C T O F I G F - I O N D E C I D U A L I Z A T I O N I. I N T R O D U C T I O N D e c i d u a l i z a t i o n , l i k e other uterine funct ions, i s p r i m a r i l y regula ted b y ova r i an s te ro id hormones . E n d o c r i n e requirements that achieve uterine sens i t i za t ion for the dec idua l response have been w e l l characterized i n ovar iec tomized rats (De F e o , 1967; F i n n and M a r t i n , 1972; P s y c h o y o s , 1976). It i s no tewor thy that o n l y a s m a l l amount o f estrogen, after the uterus has been exposed to progesterone for at least 2 days, is required for uterine sens i t iza t ion (Psychoyos , 1976). T h i s s m a l l amount o f estrogen is c a l l e d "nidatory estrogen" i n natural ly occur r ing pregnancy. A role for the pi tui tary hormones i n dec idua l i z a t i on has also been demonstrated i n h y p o p h y s e c t o m i z e d rats ( K e n n e d y and D o k t o r c i k , 1988). H y p o p h y s e c t o m y impa i r ed the dec idua l response i n ova r i ec tomized-steroid hormone treated rats. Treatment w i t h G H or thyroxine (T4, a substitute o f T S H ) res tored the p o o r dec idua l response after h y p o p h y s e c t o m y ( K e n n e d y and D o k t o r c i k , 1988). Thus , both estrogen and G H , w h i c h regulate the uterine I G F system, are required to achieve a m a x i m a l decidual response. I G F - I has been shown to mediate the act ion o f G H . T h i s has l e d to the hypothesis that I G F - I mediates the s t imulatory ac t ion o f G H and T4 o n dec idua l t issue format ion ( K e n n e d y and D o k t o r c i k , 1988). H o w e v e r , ev idence suggests that express ion o f I G F - I m R N A dur ing dec idua l iza t ion is not dependent o n pi tui tary hormone act ion (Croze et a l . , 1990a). It has been also suggested that I G F - I m a y inh ib i t the different iat ion o f s t romal ce l l s , s ince dec idua l tissue contains l o w levels o f I G F - I m R N A . Fur thermore , h igh levels o f I G F B P - 1 , w h i c h inhibi ts I G F - I act ion, is found i n the dec idua l i zed uterus. In contrast, G H and T4 appear to st imulate uterine I G F - I express ion du r ing the sensi t izat ion p e r i o d (Croze et a l . , 1990a). Treatments w i t h G H and T4 dur ing the sensitization per iod stimulate - 124-decidual tissue formation (Kennedy and Doktorcik, 1988). No further effect was observed by continuous treatment with GH and T4 during the formation of decidual tissue. In addition, IGF-I receptor levels increase during the sensitization period and of the time of decidual tissue formation (Chandrasekhar et al., 1990; Katagiri et al., 1996). These observations suggest that IGF-I may mediate the action of GH and T4 in the uterine sensitization process and at the onset of the decidual cell reaction. Chapter three demonstrated that there are changes in the uterine IGF system following superovulation. This has led to the hypothesis that changes in the uterine IGF system, caused by superovulation, may perturb the process of uterine sensitization for a deciduogenic stimulus. This may partially explain the failure of implantation associated with superovulation. This chapter first examines the effect of enhanced and suppressed IGF-I action on decidualization. Then, the effect of IGF-I on deciduoma formation and alkaline phosphatase (ALP) activity, a well used marker for decidualization, in relation to the GH and T4 actions in hypophysectomized-ovariectomized rats will be examined. Since PGs have been shown to stimulate the ALP activity in the rat uterus (Daniel and Kennedy, 1987; Yee and Kennedy, 1988), the effect of IGF-I on the PG-stimulated ALP activity was determined in cultured endometrial stroma cells. II. MATERIALS AND METHODS Study One Animal Preparation Female Sprague-Dawley rats (180-210 g BW) were purchased from the Animal Care Centre of The University of British Columbia. All rats were bilaterally ovariectomized and kept for 5 to 7 days prior to steroid hormone treatment. Rats were primed with three daily injections of 300 ng estradiol-17(3 (Sigma), followed by no steroid treatment on the following day (day 1 of pseudopregnancy). The rats were then given five -125-daily injections of 6 mg progesterone (Sigma). On the third day of progesterone treatments, females were given a single dose of 100 ng estradiol-17(3 in addition to progesterone at 13:00 h. Al l steroid injections were prepared in 0.1 ml sesame oil and given subcutaneously at 9:00 h unless otherwise stated. IGF-I and Anti-IGF-I Antibody Infusions Infusions were performed with an Alzet osmotic pUmp model 1003D or model 2001. For IGF-I infusions, reservoirs were filled with 1 and 10 nM hr-IGF-I in 100 mM phosphate buffer, pH 7.4, containing 0.1% gelatin and 20 IU heparin. The vehicle alone was infused in the control group for IGF-I infusions. Anti-IGF-I antibody was prepared as described in chapter four and infused at a protein concentration of 100 ug/ml in 100 mM phosphate buffer, pH 7.4 containing 20 IU heparin. As a control, anti-IGF-I antibody was replaced by 100 u,g/ml of rabbit IgG in the control group for the anti-IGF-I antibody infusion. Seven rats were used in each infusion group. The osmotic pump was implanted as described in chapter four (Fig. 4-1). Infusions were from 9:00 h on day 1 to 9:00 h on day 3 and from 9:00 h on day 3 to 11:00 h on day 5 (the time that the deciduogenic stimulus was applied) with a model 1003D pump and from 11:00 h on day 5 to 11:00 h on day 9 (the time that the degree of the decidual response was determined) with a model 2001 pump. The osmotic pump and delivery tubule were removed at the end of each infusion period. The ability of the IGF-I infusion to increase IGF-I action in the uterus was demonstrated for the infusion period from day 1 to day 3 in chapter four (Fig. 4-2). The ability of the IGF-I infusion to increase uterine IGF-I action during the other infusion periods was also examined. Groups of three rats were infused with 10 nM hr-IGF-I from day 3 to day 5 and from day 5 to day 9. Rats were sacrificed at the end of each infusion period. Uterine and serum levels of IGF-I, total IGFBP and IGF-I receptor were determined as described in chapter four. The levels of IGF-I and total IGFBPs in the -126-serum of rats which had not received any treatment were used as base Une values. Deciduoma Induction The formation of deciduomal tissue was induced by a bilateral intrauterine injection of 100 u.1 mineral oil at 11:00 h on day 5 of pseudopregnancy. Five rats were ovariectomized, treated with ovarian steroid hormones, and deciduoma formation induced without receiving an infusion (non-infusion group). Another group of five rats were treated with steroid hormones but were not given an infusion or a deciduogenic stimulus (non-stimulated group). All the rats were sacrificed at 11:00 h on day 9 to examine the extent of deciduoma formation. Each uterine horn was removed, cleaned and weighed. The degree of decidual tissue formation was determined by the weight of each uterine horn. Uterine horns were longitudinally opened for visual observation of uterine endometrium to confirm decidual tissue formation. Study Two Animal Preparation Hypophysectomized female Sprague-Dawley rats (180-220 g BW) were purchased from Charles River Canada Inc. (St-Constant, PQ) and pituitary intact Sprague-Dawley rats (180-210 g BW) purchased from the Animal Care Centre of The University of British Columbia. Hypophysectomized rats were given 2% glucose water throughout the experimental period. The pituitary intact and hypophysectomized rats were bilaterally ovariectomized and primed with steroid hormones for maximal uterine sensitization for a deciduogenic stimulus as described above (intact and HYPOX groups, respectively). Some hypophysectomized rats were subcutaneously injected with 200 ug porcine growth hormone (pGH, Sigma) twice daily at 9:00 h and 21:00 h and with 1 p,g thyroxine (T4, Sigma) daily at 9:00 h from day 1 to day 5 of pseudopregnancy (HYPOX-GH, T 4 group). - 127-IGF-I and Anti-IGF-I Antibody Infusions hr-IGF-I was prepared at concentrations of 0.1, 1, and 10 nM in 100 mM phosphate buffer, pH 7.4, containing 0.1% gelatin and 20 IU heparin. Fourteen rats of each treatment group; intact, HYPOX, and HYPOX-GH, T 4 groups, were infused with hr-IGF-I at an infusion rate of 1 u.l/h at each hr-IGF-I concentration from 9:00 h on day 3 to 11:00 h on day 5 (prestimulation period) with an osmotic pump model 1003D. Seven rats from each treatment group were infused with hr-IGF-I at an infusion rate of 1 |il/h from 9:00 h on day 3 till rats were sacrificed at 11:00 h on day 9 (pre- and poststimulation period) with an osmotic pump, model 2001. The vehicle alone was infused in five rats of each treatment group as controls. Anti-IGF-I antibody was infused in five HYPOX-GH, T 4 rats from 9:00 h on day 3 to 11:00 h on day 5 with a pump model 1003D. Anti-IGF-I antibody was replaced by 100 (ig/ml of rabbit IgG in the control group. Deciduoma Induction and ALP Activity Assay Deciduoma formation was induced by a bilateral intrauterine injection of 100 pi mineral oil at 11:00 h on day 5 of pseudopregnancy. Seven out of the fourteen rats that received hr-IGF-I infusions during the prestimulation period alone, were sacrificed 14:00 h on day 5. The other seven rats and seven rats given hr-IGF-I infusions throughout the pre-and poststimulation period were sacrificed at 11:00 h on day 9. All rats given anti-IGF-I antibody and IgG infusions were induced to undergo deciduoma formation as above, and sacrificed 11:00 h on day 9 of pseudopregnancy. The uterine horns were weighed and the endometrial tissue removed from the rest of the uterus by mechanical scraping. Separated endometrial tissue was homogenized in 0.25 M sucrose solution at 4°C and stored at -20°C until being used for ALP activity assay. ALP activity was determined by the method of Lowry (Lowry, 1957). The ALP activity is presented as unit activity which is defined by an amount (nmole) of substrate, /?-nitrophenol phosphate (Sigma), hydrolyzed/hour/|j.g protein in each sample. -128 -Study Three Uterine Endometrial Stromal Cell Culture All media and regents for cell culture were purchased from GIBCO except for indomethacin and prostaglandin (PGE2) which were purchased from Sigma. Pituitary intact rats were ovariectomized and received a series of ovarian steroid hormone treatment for uterine sensitization as above. hr-IGF-I (1 nM) was infused into the right uterine horn through the ovarian and uterine artery from day 3 to day 5 of pseudopregnancy. Rats were sacrificed on day 5, at the end of the infusion, and the uterus removed. As IGF-I infusion showed no systemic effect in vivo (Figs. 4-2 and 5-1), cells were prepared from the right uterine horns. Uterine horns were opened longitudinally and washed in three changes of Hanks' balanced salt solution supplemented with 20 mM HEPES, 100 units/ml penicillin, 100 [ig/ml streptomycin and 1.25 [ig/ml fungizone (HBSS). Uterine horns of the same group (usually from 4 to 6 rats) were pooled and treated with 0.5% trypsin and 0.25% pancreatin in HBSS for 2 h at 4°C, followed by 30 min digestion at room temperature. The endometrial tissue was mechanically removed with forceps and incubated in HBSS containing 0.05% trypsin and 0.02% EDTA for 5 min at 37°C. Tissue digestion was terminated by adding an excess amount of Dulbecco's modified Eagle's medium:Ham's F-12 nutrient mixture supplemented with 20% IGF-I free FCS, 100 units/ml penicillin, 100 (ig/ml streptomycin and 1.25 p.g/ml fungizone (DMEM:F-12). Endometrial cells were dispersed by pipetting and centrifuged at 800 x g for 5 min. All pellets were resuspended in DMEM:F-12 at a cell density of 5 x 105 cells/ml and seeded in 24-well cell culture plate containing 1 ml of culture medium. Cells were cultured at 37°C with 5% CO2 in humidified air. The culture medium was changed with fresh DMEM:F-12 2 h after seeding to remove most of the epithelial cells from the cell culture. Cell Treatments and Harvest Uterine endometrial stroma cells were cultured for 12 h after which they were -129-treated with 0.01, 0.1, 0.5, 1.0 or 10 nM hr-IGF-I in DMEM:F-12 for 48 h. The medium was changed after the first 24 h of the culture period to minimize the degradation of hr-IGF-I. Some cells were treated with 10 U.M indomethacin for the first 12 h of the treatment period, followed by treatment with 1 uM PGE2 plus hr-IGF-I for 48 h in the presence of 10 U.M indomethacin. At the end of the treatment period, cells were washed with HBSS and lysed with deoxycholate, pH 9.5, and stored at -20°C before being used for the ALP activity assay. At least four independent cell preparations were used in each treatment groups. To examine the effect of IGF-I on cell proliferation, the DNA content of 3-5 wells in each treatment group were determined by the method of LaBarca and Paigen (LaBarca and Paigen, 1980). Statistical analysis The uterine hom weight and ALP activity, both in vivo and in vitro, were logarithmically transformed prior to statistical analysis. Data are presented as the mean ± SEM for each group. For the ALP activity assay, decidual tissues from two animals were combined but considered as one sample in some treatment groups due to a limited tissue volume. The means among different treatment groups were compared by ANOVA, followed by Tukey's test. The means between the control and infused horns within animals were compared by paired Student's t-test. Difference of means was defined by a P-value of 0.05 unless otherwise stated. Analysis was conducted using the computer software 'SYSTAT'. III. RESULTS Study One Infusion of 10 nM hr-IGF-I increased uterine IGF-I levels in the infused hom by approximately 40% and 35% from day 3 to day 5 and from day 5 to day 9 of pregnancy -130-period, respectively (Fig. 5-1 A). Uterine levels of total IGFBP were suppressed in the infused horn by approximately 20% in both infusion periods (Fig. 5-IB). The hr-IGF-I infusion did not affect the levels of IGF-I and total IGFBP in the control horns or in the serum of the IGF-I infused rats. IGF-I receptor levels in both the control and infused horns were not affected by IGF-I infusions (Fig. 5-1C). Infusion with hr-IGF-I from day 1 to day 3 inhibited deciduoma formation in the infused horns, compared with that of the control hom in the same animal (P<0.001, Fig. 5-2). The mean weight of the infused horns was less than that of the non-infusion group but greater than that of the non-stimulated group (P<0.001). The same IGF-I infusion had no effect on deciduoma formation in the control horns of the infusion group or in the control . horns of all the groups (Fig. 5-2). The hr-IGF-I infusion had no effect on deciduoma formation in the other two infusion periods. Vehicle infusions had no effect on the deciduoma formation in any infusion period (Fig. 5-2). When anti-IGF-I antibody was infused during the prestimulation period (between day 1 and day 3, and between day 3 and day 5), the antibody infusions suppressed decidual tissue formation in the infused hom, but had no effect on the control horn in the same animal (P<0.001, Fig. 5-3). The weight of the infused hom of both infusion groups was less than that of the non-infusion group but greater than that of the non-stimulated group (P<0.001). Anti-IGF-I antibody infusion during the poststimulation period had no effect on decidual tissue formation. Infusions with IgG had no effect on deciduoma formation at any infusion period (Fig. 5-3). The degree of inhibition of the decidual response by the hr-IGF-I infusion during day 1 to day 3 greatly varied from almost no effect to complete inhibition (Fig. 5-4). Visible decidual tissue formation was not observed in the infused horn of three rats. The tissue weight of the infused horns, in these three rats, were 94, 115 and 120 mg, respectively (Figs. 5-4). These weights were not significantly different from those observed in the non-stimulated group (Figs. 5-2 and 4). The degree of inhibition of -131 -Figure 5-1 The effect of IGF-I infusions at different time period on the uterine IGF system. IGF-I (10 nM, 1 jil/h) was infused into the right uterine horn during the indicated time period by an osmotic pump that was implanted in the abdominal cavity (Fig. 4-1). The levels of uterine and serum IGF-I (A) were determined by RIA and the levels of IGFBP (B) and IGF-I receptor (C) determined by ligand binding assays using 125I-IGF-I. The levels of IGF-I, IGFBP, and IGF-I receptor in the control uterine horn and the infused uterine hom were determined separately. The levels of IGFBP and IGF-I receptor are presented as percentages of the levels of the control horn. The levels of IGF-I and IGFBP in the serum of rats which had not received any treatment were used as the control. Values represent the means and SEM for three animals in each group. Asterisks indicate statistical differences (P<0.05) of the means between the control and infused uterine horns within the animals. -132-Uterus Control horn Infused horn I I Non-infusion Infusion Serum (ng/g feu.) < # # # # # (^A) IGF-I ^ j ^ Q WMI) day 3-day 5 day 5-day 9 (B) IGFBP Ci $ $ $ (%) (C) IGF-I receptor 1 day 3-day 5 day 5-day 9 -133-Figure 5-2 The effect of IGF-I infusions at different time period on deciduoma formation. IGF-I (10 nM, 1 (il/h) or vehicle alone was infused into the right uterine horn in the ovariectomized-steroid hormone treated rats during the indicated time period. A bilateral deciduogenic stimulus was applied on day 5 of pseudopregnancy. The degree of deciduoma formation was determined separately on the infused and control horns by weighing each uterine hom on day 9. A group of five rats were treated as above and deciduoma formation induced without receiving an infusion (non-infusion). Another five rats were treated as above but were not given an infusion or a deciduogenic stimulus (non-stimulation). Values represent the means and SEM of the uterine hom weight in each group. Letters on the top of bars indicate statistical differences across the different infusion periods within the same uterine hom group (a>b, P<0.01). -134 -• Non-infusion • Non-stimulation H Vehicle-control horns M Vehicle-infused horns • IGF-I-control hons • IGF-I-infused horns a x T V: day 1-day 3 day 3-day 5 day 5-day Infusion periods -135-Figure 5-3 The effect of anti-IGF-I antibody infusions at different time period on deciduoma formation. Anti-IGF-I antibody (100 ug/ml, 1 p,l/h) or rabbit IgG (100 u.g/ml, 1 ul/h) was infused into the right uterine horn in the ovariectomized-steroid hormone treated rats during the indicated time period. A bilateral deciduogenic stimulus was applied on day 5 of pseudopregnancy. The degree of deciduoma formation was determined separately on the infused and control horns by weighing each uterine hom on day 9. A group of five rats were treated as above and deciduoma formation induced without receiving an infusion (non-infusion). Another five rats were treated as above but were not given an infusion or a deciduogenic stimulus (non-stimulation). Values represent the means and SEM of the uterine horn weight for seven rats in each group. Letters on the top of bars indicate statistical differences across the different infusion periods within the same uterine horn group (a>b, P<0.01). -136-• Non-infusion • Non-stimulation S IgG-control horns day 1-day 3 day 3-day 5 day 5-day Infusion periods -137-Figure 5-4 Distribution of the uterine tissue weight of the IGF-I-infused horns on day 9 of pseudopregnancy. IGF-I (10 nM, 1 pl/h) or vehicle alone was infused into the right uterine horn in the ovariectomized-steroid hormone treated rats during the indicated time period. A bilateral deciduogenic stimulus was applied on day 5 of pseudopregnancy. The weight of the infused uterine hom was determined on day 9 and shown on the logarithmic scale. - 1 3 8 -E "S c u o JS « "E o o 3l t i l l ! V ^ ^ ^ ^ ft* V V v v ^ ^ ^ ^ IGF-I Vehic le - 1 3 9 -dec idua l response by the an t i - IGF- I antibody infus ion was re la t ive ly consistent i n the two in fus ion per iods ( F i g . 5-5). A v i s i b l e dec idua l tissue format ion was observed i n a l l rats. T h e smal les t uterine weigh t o f the infused horn was 267 and 335 m g for the in fus ion between day 1 and day 3, and between day 3 and day 5, respect ively ( F i g . 5-5). Study T w o A l l infusions o f h r - I G F - I and an t i - IGF- I ant ibody, as w e l l as the cont ro l infusions, had no effect o n d e c i d u o m a fo rmat ion and uterine A L P ac t i v i t y i n the c o n t r o l horn . Therefore , the remainder o f this section w i l l o n l y describe the effect o f infus ions i n the infused horn, unless otherwise stated. H y p o p h y s e c t o m y suppressed d e c i d u o m a format ion and A L P act iv i ty i n the endometr ia l tissue (P<0.01, F i g s 5 - 6 , 7 , 8, and 9). Treatment w i t h p G H or T 4 i n hypophysec tomized rats restored the format ion o f dec idua l tissue and A L P ac t iv i ty to the same levels o f pi tui tary intact rats (P<0.01). H o w e v e r , h r - I G F - I infusions were not capable o f restoring the reduced dec iduoma format ion and A L P ac t iv i ty , caused by hypophysec tomy , at any concentrat ion (F igs 5-6, 7, 8, and 9). A n t i - I G F - I ant ibody infus ions had no effect o n the ab i l i t y o f p G H or T 4 to restore the r educed d e c i d u o m a format ion and A L P activity i n hypophysectomized rats ( F i g . 5-10). h r - I G F - I infusion o f 0.1 n M dur ing the prest imulat ion per iod st imulated dec iduoma format ion i n the intact and H Y P O X - G H , T 4 groups (P<0.01, F i g . 5-6). H o w e v e r , when the in fus ion was pe r fo rmed throughout the pre- and pos t s t imula t ion per iods , h r - I G F - I infusion at the same concentration suppressed dec iduoma format ion i n the t w o groups (F ig . 5-7). T h e h r - I G F - I in fus ion at greater concentrat ions (1 and 10 n M ) had no effect o n d e c i d u o m a fo rma t ion . Interest ingly, h r - I G F - I in fus ions had no effect o n d e c i d u o m a format ion i n the H Y P O X group (Figs . 5-6 and 7). T h e effect o f h r - I G F - I infusions dur ing the pres t imula t ion p e r i o d o n uterine A L P activi ty was dependent o n the concentration o f infused h r - I G F - I and the day o f examinat ion (Figs . 5-8). T h e h r - I G F - I infusions at 1 and 10 n M increased A L P act ivi ty i n intact and -140-Figure 5-5 Distribution of the uterine horn weight of the anti-IGF-I antibody-infused horns on day 9 of pseudopregnancy. Anti-IGF-I antibody (100 [ig/ml, 1 ul/h) or rabbit IgG (100 u.g/ml, 1 |il/h) was infused into the right uterine horn in the ovariectomized-steroid hormone treated rats during the indicated time period. A bilateral deciduogenic stimulus was applied on day 5 of pseudopregnancy. The weight of the infused uterine horn was determined on day 9 and shown on the logarithmic scale. -141 -A n t i - I G F - I ant ibody I g G -142 -Figure 5-6 The effect of IGF-I infusions during the prestimulation period on deciduoma formation. Seven rats in the pituitary intact (intact), hypophysectomized (HYPOX), and hypophysectomized-porcine GH and T4 treated (HYPOX-GH, T4) groups were infused with 0 (vehicle), 0.1, 1, or 10 nM IGF-I into the right uterine hom from day 3 to day 5, till a bilateral deciduogenic stimulus was applied. Rats were sacrificed on day 9 of pseudopregnancy. Since IGF-I infusions had no effect on deciduoma formation on the control hom in all infusion groups, only the weights (mean ± SEM, n=7) of the infused hom are presented. Letters on the top of bars indicated statistical difference across the experimental groups (a>b, P<0.01). -143-IGF-I (nM) - 1 4 4 -F i g u r e 5-7 T h e effect o f I G F - I infusions dur ing the pre- and postst imulat ion periods on d e c i d u o m a f o r m a t i o n . S e v e n rats i n the p i t u i t a r y in tac t ( in tac t ) , hypophysec tomized ( H Y P O X ) , and hypophysec tomized-porc ine G H and T4 treated ( H Y P O X - G H , T4) groups were infused w i t h 0 (vehicle) , 0.1, 1, or 10 n M I G F - I throughout the pre- and pos ts t imula t ion periods in to the r ight uterine horn. A bilateral deciduogenic s t imulus was appl ied on day 5 and sacrif iced on day 9 o f pseudopregnancy. S ince I G F - I infusions had no effect o n dec iduoma format ion o n the con t ro l h o m i n a l l in fus ion groups, o n l y the weights (mean ± S E M , n=7) o f the infused h o m are presented. 1 Le t t e r s o n the top o f bars i n d i c a t e d s ta t i s t ica l d i f fe rence across the exper imental groups (a>b, P<0.01). -145 -| Intact Eg HYPOX H HYPOX-GH, T4 Vehic le 0.1 1 10 I G F - I ( n M ) -146 -Figure 5-8 The effect of IGF-I infusions during the prestimulation period on the levels of uterine alkaline phosphatase (ALP) activity. Fourteen rats in the pituitary intact (intact), hypophysectomized (HYPOX), and hypophysectomized-porcine GH and T4 treated (HYPOX-GH, T4) groups were infused with 0 (vehicle), 0.1, 1, or 10 nM IGF-I into the right uterine horn from day 3 to day 5 of pseudopregnancy. Seven rats in each group were sacrificed on day 5 (A), and the other seven rats were given a bilateral deciduogenic stimulus on day 5 and sacrificed on day 9 (B). Since IGF-I infusions had no effect on ALP activity on the control uterine horn, only the levels of ALP activity (mean ± SEM, n=7) on the infused horn are presented. Letters on the top of bars indicated statistical difference across the experimental groups (a>b>c, P<0.01). - 147-2.5 2.0 1.5 1.0 0.5+-J (A) Intact HYPOX HYPOX-GH, T4 Vehic le 0.1 1 IGF-I (nM) -148 -Figure 5-9 The effect of IGF-I infusions during the pre- and poststimulation periods on the levels of uterine alkaline phosphatase (ALP) activity. Seven rats in the pituitary intact (intact), hypophysectomized (HYPOX), and hypophysectomized-porcine GH and T 4 treated (HYPOX-GH, T4) groups were infused with 0 (vehicle), 0.1, 1, or 10 nM IGF-I into the right uterine horn from day 3 to day 9. Rats were given a bilateral deciduogenic stimulus on day 5 and sacrificed on day 9 of pseudopregnancy. Since IGF-I infusions had no effect on ALP activity on the control uterine horn, only the levels of ALP activity (mean + SEM, n=7) on the infused horn are presented. Letters on the top of bars indicated statistical difference across the experimental groups (a>b>c>d, P<0.01). - 1 4 9 -| Intact g HYPOX ^ HYPOX-GH, T4 I G F - I ( n M ) -150 -Figure 5-10 The effect of anti-IGF-I antibody on the deciduoma formation (A) and uterine alkaline phosphatase (ALP) activity (B). Anti-IGF-I antibody was infused in five hypophysectomized-porcine GH and T4 treated rats from day 3 to day 5. Rats were given a bilateral deciduogenic stimulus on day 5 pseudopregnancy. Anti-IGF-I antibody was replaced by rabbit IgG in the control group. The degree of deciduoma formation (the uterine horn weight) and ALP activity were determined on day 9. The uterine horn weights and ALP activity of the non-infusion/stimulation and non-infusion groups from study one (Figs. 5-2 and 3) were adapted for comparison. Values represent the means and SEM for five rats in each group. -151 --152-HYPOX-GH, T 4 groups on day 6 (P<0.01, Fig. 5-8 A). ALP activity in the 1 nM hr-IGF-I infusion group remained high until day 9. However, ALP activity levels in the 10 nM hr-IGF-I infusion group decreased below base levels by day 9 (P<0.01, Fig. 5-8B). Continuous infusions with hr-IGF-I (0.1 and 1 nM) throughout the pre- and poststimulation period increased ALP activity, but decreased ALP activity at 10 nM, compared to that of the vehicle infusion group in intact and HYPOX-GH, T 4 groups (P<0.01, Fig. 5-9). In intact and HYPOX-GH, T 4 groups, the continuous infusion of hr-IGF-I (0.1 and 1 nM) achieved greater levels of ALP activity than those of the corresponding infusion groups of the prestimulation period (P<0.01, Figs. 5-8 and 9). The infusion of hr-IGF-I had no effect on ALP activity in the HYPOX group. Study Three All of the hr-IGF-I treatments had no effect on total DNA and protein content in cultured endometrial stromal cells. Treatments of endometrial stromal cells with hr-IGF-I stimulated basal ALP activity at 0.1 and 0.5 nM (P<0.01). In contrast, higher levels of IGF-I had no effect on ALP activity in endometrial cells in vitro (Fig. 5-11). The uterine hr-IGF-I infusion during the sensitization period, prior to the cell culture preparations, enhanced the effect of IGF-I on basal ALP activity in the cultured endometrial stromal cells (P<0.01). PGE2 increased the unit ALP activity in cultured endometrial stromal cells (P<0.01, Fig. 5-11). Treatments of endometrial stromal cells with hr-IGF-I suppressed PGE2-stimulated ALP activity at 0.5, 1, and 10 nM (P<0.01, Fig. 5-4). hr-IGF-I infusion during the sensitization period, prior to the cell culture preparations, did not alter the effects of the IGF-I treatment on PGE2-stimulated ALP activity in endometrial stromal cells. -153-Figure5-ll The effect of IGF-I on alkaline phosphatase (ALP) activity in cultured uterine endometrial stroma cells. All the rats were ovariectomized and treated with estradiol-170 and progesterone to sensitize the uterus to a deciduogenic stimulus. Some rats were given additional IGF-I infusion from day 3 to day 5 of pseudopregnancy (IGF-I infused). Endometrial stroma cells were obtained from the uterus on day 5 of pseudopregnancy. The cells were treated with IGF-I at the indicated concentrations in the absence (Basal) and presence of indomethacin and prostaglandin E2 (PGE2-stimulated). Values represent the means and SEM of ALP activity for at least four preparations of cells in each point. -154-IGF-I (nM) -155-rv. DISCUSSION Changes in the uterine IGF system following superovulation appear to be detrimental to decidualization in rats. Changes in the uterine IGF system following superovulation are characterized by an enhanced uterine IGF-I action between day 1 and day 3 of pregnancy and by a suppressed IGF-I action thereafter (Fig. 3-3). Deciduoma formation was suppressed in uterine horns which had been exposed to high levels of IGF-I from day 1 to day 3 and reduced IGF-I action from day 1 to day 3 and from day 3 to day 5 of pseudopregnancy (Figs. 5-2 and 3). Comparisons of the time and degree of inhibition of deciduoma formation by hr-IGF-I and anti-IGF-I antibody infusions give rise to some interesting explanations. First, the increased and reduced IGF-I actions may disturb the sensitization process which in turn inhibits decidual tissue formation. Rats were given a series of ovarian steroid hormone treatment to sensitize the uterus for a maximal decidual response in the present study. The hr-IGF-I and anti-IGF-I antibody infusions during the prestimulation period inhibited deciduoma formation, while both infusions during the poststimulation period had no effect on deciduoma formation (Fig. 5-2 and 3). The exact timing that the antibody infusion becomes ineffective to inhibit the decidual response was not examined in the present study. However, it appears that anti-IGF-I infusions become ineffective when the uterine sensitization for the decidual response has been achieved. IGF-I action may no longer be necessary or become less critical for decidual tissue formation, once the uterine endometrium has been sensitized. This hypothesis is comparable to the previous observations. Expression of IGF-I mRNA and extractable IGF-I levels in the uterus increases towards the time of decidualization, followed by a decline during the decidualization period (Fig. 3-3A) (Croze et al., 1990a; Kapur et al., 1992). Secondly, the mode of inhibition by the enhanced and suppressed IGF-I action may -156-be different. The enhanced IGF-I action may be capable of inhibiting decidual tissue formation completely. However, suppressed IGF-I action may only partially inhibit the decidual response (Figs. 5-4 and 5). The visual inspection of the uterine luminal surface found no deciduoma formation in the infused hom in three out of seven rats following hr-IGF-I infusion from day 1 to day 3. In contrast, all rats given the antibody infusion during the prestimulation period developed a visible decidual tissue mass in the infused horn. The uterine IGF system is an integral part of the growth factor/cytokine network which regulates uterine growth and differentiation. Other members of the regulatory network may respond differently to increased or reduced IGF-I action. The distinct responses of the other growth factors and cytokines may cause inhibition of the decidual reaction at a different step of the decidualization process and to a various extent. Decidualized endometrial tissue has low levels of IGF-I mRNA transcripts but has high levels of IGFBP-1 mRNA (Croze et al., 1990a). IGFBP-1 has been localized to the luminal and glandular epithelium in the antimesometrial region, but not the decidual cells (Croze et al., 1990a; Sadek et al., 1994). Since IGFBPs, in general, inhibit IGF-I actions, these observations led to the hypothesis that IGF-I may even inhibit decidual tissue formation (Croze et al., 1990a; Sadek et al., 1994). The ability of IGF-I to stimulate cell multiplication may prevent uterine endometrial stroma cells from differentiating into decidual cells. Therefore, suppressed IGF-I actions, as the result of increased IGFBP-1 levels and reduced IGF-I expression in the endometrial tissue may enhance differentiation of the uterine endometrial stromal cells into decidual cells (Croze et al., 1990a). During the poststimulation period, the inhibition of decidual tissue formation appears to occur at relatively low levels of IGF-I. The continuous infusion of 0.1 nM hr-IGF-I throughout the pre- and poststimulation period suppressed deciduoma formation (Fig. 5-7). It is noteworthy that the levels of IGF-I receptor increase during the sensitization period and at the time of decidualization (Chandrasekhar et al., 1990). In the present study, uterine IGF-I receptor levels increased by 60% between day 5 and day 6 in the control ras (Fig. 3-3C). -157 -Together with the reduction in IGF-I mRNA and the high levels of IGFBP-1 mRNA transcripts, a small increase in IGF-I levels, accompanied by a decline in IGFBP-1 levels, may serve as an effective inhibitory mechanism. The effect of GH and T4 on decidual tissue formation in hypophysectomized rats has been previously demonstrated (Kennedy and Doktorcik, 1988). The present study confirmed the effect of GH and T 4 on decidual tissue formation. In addition, the present study showed that GH and T4 may also be required for maximal stimulation of ALP activity, associated with decidualization. Treatment with pGH and T4 may increase total uterine A L P activity due to an increase in decidual tissue mass. However, more importantly, the treatment appears to increase ALP activity/total protein content of the decidual tissue (Figs. 5-8 and 9). This leads to the hypothesis that the effect of GH may be exerted through the GH-IGF-I axis, since IGF-I mediates GH action in many tissues. One of objectives of the present study was to determine whether IGF-I mediates the effect of GH and T4 on decidual tissue formation. However, IGF-I appears not to mediate the action of GH and T4 as it does not restore decidual tissue formation and ALP activity in hypophysectomized rats. Infusions with hr-IGF-I failed to restore decidual tissue formation and ALP activity in hypophysectomized rats (Figs. 5-6, 7, 8, and 9). Furthermore, anti-IGF-I infusions in hypophysectomized-pGH and T 4 treated rats did not suppress decidual tissue formation (Fig. 5-10). Although IGF-I may not mediate action of GH and T4 in deciduoma formation in hypophysectomized rats, IGF-I appears to modulate GH and T4 action on decidual tissue formation and ALP activity levels. IGF-I may also require the presence of GH and T4 to exert its effect on decidualization. The hr-IGF-I infusions modulated formation of decidual tissue and ALP activity in the presence of GH and T4 (intact and HYPOX-GH, T 4 groups) but had no effect in the absence of GH and T 4 (HYPOX group, Figs. 5-6, 7, 8, and 9). In addition, hr-IGF-I increased ALP activity levels in cultured endometrial stroma cells obtained from the sensitized uterus of pituitary intact rats in the absence of GH and T 4 (Fig. -158-5-11). Together, GH and T4 may render the uterus capable of responding to IGF-I during the prestimulation period (i.e. the sensitization period). The exact period of time when the uterus requires the actions of GH and T4 for maximal decidual response remains to be determined. The mechanisms through which GH and T4 enable IGF-I to regulate the process of decidualization has not been determined. One possible mechanism may be the regulation of IGF-I receptor levels in the endometrial cells by GH and T4. The effects of GH and T4 on IGF-I receptor levels in the uterus have been studied (Yallampalli et al., 1992). Hypophysectomy increased uterine IGF-I receptor levels and treatment with GH, but not T4, reversed the increase in receptor levels. This study, however, utilized hypophysectomized-ovariectomized rats without ovarian steroid hormones. Ovarian steroid hormones are known regulators of IGF-I receptors in the uterus (Ghahary and Murphy, 1989) and as a primary regulator of uterine sensitization for decidualization. The uterine IGF-I receptor response to GH regulation may be different, if rats were treated with ovarian steroid hormones for maximal uterine sensitization, as in the present study. Nevertheless, this finding may suggest that GH plays a role in maintaining an optimal IGF-I receptor level in the uterus during the sensitization period, preceding the deciduogenic stimulus. Such regulation of IGF-I receptor levels by GH may be involved in the process of uterine preparation that enables IGF-I to modulate the decidualization process. IGF-I appears to stimulate ALP activity throughout the pre- and poststimulation period. Continuous infusion of hr-IGF-I throughout the pre- and poststimulation period increased the unit ALP activity to a greater extent than the infusion of hr-IGF-I during the prestimulation period in both intact and HYPOX-GH, T4 groups (Figs. 5-8 and 9). A differential effect of IGF-I on ALP activity was observed in cultured endometrial cells. Endometrial stroma cells isolated from uteri previously exposed to hr-IGF-I during the sensitization period showed a greater response in basal ALP activity than their non-infusion counterparts following IGF-I treatment in vitro (Fig. 5-11). It is likely that the hr-IGF-I -159 -infusion during the sensitization period enhances the subsequent response of endometrial stromal cells to IGF-I. Total DNA and protein content/well were not significandy different in any of the groups, regardless of in vitro treatment conditions. The prolonged effect of the IGF-I infusion during the prestimulation period may be specific to the regulation of ALP activity rather than the result of an altered cell metabolism. IGF-I has been reported to either stimulate, inhibit, or have no effect on basal ALP activity in bone cells in vitro and in vivo (Ohlsson et al., 1992; Fournier et al., 1993; Pirskanen et al., 1993; Tanaka et al., 1994). IGF-I also suppressed tri-iodothyronine-stimulated and carcitriol-stimulated ALP activity in cultured rat epiphyseal chondrocytes and human osteosarcoma cells, respectively (Ohlsson et al., 1992; Pirskanen et al., 1993). In contrast, IGF-I had no effect on the levels of basal ALP activity in cultured rat epiphyseal chondrocytes, but decreased basal ALP activity in human osteosarcoma cells. Thus, the effect of IGF-I on ALP activity appears to be diverse and under the influence of other factors. The mechanisms by which IGF-I exerts these effects on ALP activity remain to be elucidated. In the present study, the effect of IGF-I on uterine PGE2-stimulated ALP activity in vivo was unable to be determined due to a technical difficulty. The decidual response varied to a great extent in the vehicle group during the preliminary study. This was probably due to a high degree of intervention associated with dual pump implantation for simultaneous infusions with IGF-I and PGE2. However, IGF-I appears to inhibit PGE2-stimulated ALP activity in cultured endometrial stroma cells (Fig. 5-11). The exact mechanism of this IGF-I action is not known. IGF-I may down-regulate receptors for PGE2 or interfere with an intracellular signaling mechanisms, such as the cAMP pathway, which may mediate the effect of PGE2 on ALP activity in the rat uterus (Yee and Kennedy, 1988; Yee and Kennedy, 1991). Further studies are needed to define the regulatory mechanism that allows IGF-I to stimulate basal ALP activity and simultaneously suppress PGF^ -stimulated ALP activity in the rat uterus. -160 -PGs have drawn a large amount of attention in the regulation of ALP activity and other processes in decidualization. PGs increase ALP activity, vascular permeability, and extracellular fluid volume in the uterus and stimulate subsequent decidual tissue formation (Hoffman et al., 1977; Kennedy and Lukash, 1982; Yee and Kennedy, 1988; Hamilton and Kennedy, 1994). Modulation of PG synthesis by local uterine products has also been described. IL-lct, EGF, and platelet activating factor stimulate PG synthesis in uterine endometrial stromal cells (Smith and Kelly, 1988; Paria et al., 1991; Bany and Kennedy, 1995). These local factors may play a role in the regulation of ALP activity through PG synthesis. Leukotrienes may also have a potential regulatory role on the uterine ALP activity associated with decidualization in rats. The levels of leukotrienes in the uterus increase prior to implantation in a similar production pattern observed with PGs in response to a nidatory estrogen surge (Malathy et al., 1986; Tawfik et al., 1987). Leukotrienes inhibit ALP activity in cultured uterine endometrial stromal cells (Cejic and Kennedy, 1991a). The inhibition of ALP activity by leukotrienes may be due to a mechanism independent of PG production (Cejic and Kennedy, 1991b). The present study demonstrated an inhibitory effect of IGF-I on PGE2-stimulated ALP activity (Fig. 5-11). This may indicate that IGF-I plays a role in the regulation of ALP activity by counteracting the stimulatory action of other regulatory factors that act through PG synthesis. However, interactions between these factors and IGF-I in the regulation of ALP activity remains to be determined. The present study highlights the complexity of the regulatory mechanisms involved in the decidualization process. The effect of IGF-I infusions on deciduoma formation and ALP activity often did not correlate in the present study. For example, in intact and HYPOX-GH, T4 rats, the continuous infusion of 0.1 nM hr-IGF-I throughout the pre- and poststimulation periods inhibited deciduoma formation and stimulated ALP activity, which is often used for a marker of decidualization (Figs. 5-7 and 9). These observations, however, do not necessarily conflict with each other. An increase in ALP activity was -161 -observed before the penetration of the blastocyst of the endometrial epithelium and occurs without application of an artificial deciduogenic stimulus in the pseudopregnant rat uterus, sensitized for the decidual reaction (Fig. 5-10). In contrast, decidual tissue forms in response to either naturally occurring, or artificial deciduogenic stimuli. Thus, these two events are regulated differently. The infusion of 10 nM hr-IGF-I during the prestimulation period increased the levels of uterine ALP activity on day 6 in intact and HYPOX-GH, T4 treated rats (Fig. 5-8). However, when examined on day 9, ALP activity of the same infusion group as well as ALP activity following the infusion of 10 nM hr-IGF-I throughout pre- and poststimulation periods were lower than that of the vehicle group (Figs. 5-8 and 9). These observations suggest that excessive IGF-I action first increases and then decreases ALP activity in the uterus. However, deciduoma formation in these infusion groups remained at control levels (Figs. 5-6 and 7). This is another example of disagreement in the IGF-I regulation of the two markers associated with decidualization. Examination of the decidual tissue mass may be the simplest, single criterion available to evaluate the degree of uterine sensitization for the decidual reaction. However, the observations made in the present study strongly suggest that the use of multiple makers may be necessary to evaluate the effect of the treatments on decidual tissue mass in a practical setting. Together with the aforementioned concentration- and time-dependent effects of IGF-I on the decidual tissue formation, and the differential effect of IGF-I on basal and PG-stimulated ALP activity, the potential role for IGF-I in the regulation of the decidualization process appears to be complex. Clearly, further studies are needed to define the role for the IGF system in this complex regulatory mechanism. -162-V. SUMMARY AND CONCLUSIONS Studies in this chapter demonstrated that IGF-I may regulate decidualization at different levels such as decidual tissue formation and A L P activity. In particular, the uterine IGF system appears to play an important role in the uterine sensitization process required for the decidual response. Enhanced and suppressed IGF-I actions during the sensitization period may be detrimental to subsequent decidual tissue formation. Therefore, changes in the uterine IGF system during the preimplantation period following superovulation appears to have a significant effect on the decidual response. This may, at least in part, be responsible for the failure of implantation following superovulation in the rat. The present study also demonstrated that GH and T4 are involved in the regulation of uterine ALP activity during the uterine sensitization period, in addition to the decidual tissue formation that has been demonstrated previously. However, IGF-I was shown not to mediate the actions of GH and T4 on the decidual tissue formation and ALP activity. Instead, IGF-I appears to regulate the decidualization process in the GH and T4-dependent manner. The mechanisms by which GH and T4 enable IGF-I to regulate the decidualization process remains to be determined. -163 -CHAPTER SIX SUMMARY AND GENERAL CONCLUSIONS I. SUMMARY The present study demonstrated that IGF-I plays a central role in the establishment of a successful pregnancy. IGF-I appears to be beneficial to preimplantation embryonic development in the rat. IGF-I stimulated embryonic development to the blastocyst stage in vitro and improved the viability of blastocysts (chapter two). This is compatible with the hypothesis that maternally derived IGF-I plays a role as a signaling factor. In this role, IGF-I regulates the coordinated growth observed between the uterine endometrium and the preimplantation embryos. The role of IGF-I in the uterine function was determined in conjunction with the adverse effects of superovulation. Superovulatory treatments altered the uterine IGF system in the immature rat superovulation model (chapter three). Alterations of the uterine IGF system following superovulation are characterized by: (1) enhanced IGF-I actions by increased IGF-I levels and decreased total IGFBP levels during the first three days of pregnancy, and (2) reduced IGF-I actions by decreased IGF-I levels and increased total IGFBP levels during the second half of the preimplantation period. Alterations in the levels of IGF-I action appears to mediate the detrimental effects of superovulation on the establishment of pregnancy through several distinct mechanisms. First, an increase in IGF-I action in the uterus during the first three days of the preimplantation period render the uterine luminal fluids detrimental to embryonic development in vitro (chapter four). This may result in an increase in early embryonic loss which is associated with superovulation. The detrimental effect of the uterine luminal fluid -164-on embryonic development may be attributed, at least in part, to a distortion of electrolyte balance of the uterine luminal fluids, obtained from the uterus that has been exposed to high levels of IGF-I (chapter four). Secondly, enhanced IGF-I action is also likely to have detrimental effects on uterine sensitization to the deciduogenic stimulus. Decreased IGF-I action during the second half of the preimplantation period (the uterine sensitization period), may adversely affect decidualization. IGF-I promoted the uterine sensitization process and inhibition of IGF-I action at this time, by an anti-IGF-I antibody, resulted in a poor decidual response (chapter five). Blastocysts obtained from the culture with the uterine luminal fluids, obtained form the IGF-I infused horn and superovulated rats, contained a fewer number of cells, suggesting that these blastocysts may have reduced capability of implanting and developing into fetuses (chapter four). These detrimental effects on decidualization and viability of embryos, caused by alterations in the uterine IGF system, may partially explain the failure of implantation associated with superovulation. Furthermore, superovulatory treatment may affect the levels of IGF-I and IGFBPs in the uterine luminal fluids in the same manner that is found in the uterine endometrium. If this happens, the levels of IGF-I action on preimplantation embryonic development is reduced during the blastocyst formation period. Since IGF-I appears to stimulate blastocyst formation and improve viability of the blastocysts, a decrease in the levels of IGF-I action may reduce the rate of blastocyst formation and their viability. This will result in a pregnancy with a smaller liter size. Superovulation may also increase uterine luminal fluid volume (chapter four). Although this effect may not be mediated by IGF-I, increases in the volume of uterine luminal fluid may be detrimental on embryonic development. It is not known how superovulatory treatment causes this change in the rat uterus. Many cytokines and growth factors, including IGF-I, are believed to interact with each other, form a complex regulatory network in the uterine endometrium, and provide -165-redundant systems to increase the chances for a successful pregnancy. Expression of these cytokines and growth factors in the uterus is regulated primarily by ovarian steroid hormones, such as estrogen and progesterone. Therefore, hyperestrogenemia, caused by superovulatory treatment, affects the entire local regulatory system for the regulation of uterine function. The present study focused on the role for IGF-I, a member of the regulatory network, in mediating detrimental effects of hyperestrogenemia caused by superovulatory treatment. IGF-I appears to mediate, at least in part, the detrimental effects of hyperestrogenemia, which include an increase in early embryonic loss and a failure of implantation. It is apparent that IGF-I exerts these effects by interacting with other local regulators and modulating actions of hormonal regulators. For example, electrolyte composition of the uterine luminal fluids, especially that of anions, appears to be regulated by multiple factors that include IGF-I (chapter four). IGF-I modulated the actions of GH, T4, and PG in the uterine sensitization process for the decidual reaction (chapter five). However, mechanisms of these interactions remain to be defined. Evidence suggests that the role for IGF-I in the implantation process is not as critical as those for some growth factors and cytokines, such as IL-1 (3, EGF, LIF, and CSF-1 (Pampfer et al., 1991; Pollard et al., 1991; Arceci et al., 1992; Johnson and Chatterjee, 1993b; Cross et al., 1994; Simon et al., 1994a). These cytokines have been shown to be regulated by ovarian steroid hormones in the uterus and play a critical role in the process of blastocyst implantation. However, the present study demonstrated that alterations in the uterine IGF system have a great impact on blastocyst implantation, by regulating the viability of implanting blastocysts and the uterine sensitization process for deciduogenic stimuli. This suggests that many local regulators other than these four cytokines can also affect implantation. The present study also demonstrated the complexity in the regulatory mechanisms of the uterine IGF system in the uterus. For example, IGF-I infusions (10-50 nM, 1 ul/h) -166 -up-regulated the levels of IGF-I in the uterus. In contrast, these IGF-I infusions decreased or increased the levels of total IGFBPs, depending upon the concentration of IGF-I (chapter four). As discussed previously, each subtype of IGFBPs may play a different role in the uterine IGF system, and therefore respond differendy to IGF-I. IGF-I at varying concentrations may regulate expression of IGFBPs in a inhibitory or stimulatory mode A condition of IGF-I infusion that up-regulates some subtypes of IGFBPs may down-regulate other subtypes. Levels of total IGFBPs are determined as the sum of these changes in all IGFBP subtypes. These differential regulations of IGF-I on expression of IGFBPs may partially explain the biphasic response of IGFBP levels to IGF-I infusions observed in the present study. Examination of the effects of IGF-I on the levels of the specific IGFBP subtypes would provide insight into the role of IGF-I in the regulation of IGFBPs in the uterus. The present study showed that superovulatory treatment has a great impact on the functional aspect of uterine endometrium. Implantation is one of critical steps for the establishment of successful pregnancy. The adverse effects of ovarian hyperstimulation, resulting from conventional superovulatory treatments with exogenous gonadotropins, have been recognized since the early use of IVF-ET. Many studies aimed at improving superovulatory treatments have been focused on the ovarian functional aspects, in order to obtain maximum number of fertilizable oocytes. Aspects of uterine function have been largely disregarded. The present study examined the role for IGF-I in mediating the detrimental effects of superovulation with special reference to uterine endometrial function. The results from this study are consistent with the hypothesis that alterations in uterine function following superovulation are mediated by growth factors and cytokines in the uterus. This emphasizes that examination of function of the uterine endometrium is essential for improving superovulatory treatment. -167-n. CONCLUSIONS The uterine IGF system appears to play a central role in the regulation of uterine functions which include maintenance of a receptive uterine environment for preimplantation embryonic development and uterine sensitization for the decidual reaction. In addition, IGF-I derived from the uterus appears to promote embryonic development during the preimplantation period in the rat. Therefore, a normally functioning uterine IGF system is essential for successful preimplantation embryonic development, blastocyst implantation, and subsequent fetal development. 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