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Single cell studies of calcium as second messenger in human granulosa-lutein and embryonic kidney 293… Lee, Pearly S.N. 1999

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Single Cell Studies df Calcium as Second Messenger in Human Granulosa-Lutein and Embryonic Kidney 293 Cells Pearly S. N. Lee A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ! I in the ; j I :- : ' i : I ' | Faculty of Graduate Studies j Department of Reproductive and Developmental Sciences j We accept this thesis as conforming to the required standard University of British Columbia 1999 © Pearly S. N. Lee, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 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 Date 24 Uw DE-6 (2/88) Abstract i It is wel l established that L H action is mediated primarily by!adenylate cyc l a se / cAMP. Conversely, the role of inositol phosphate/calcium in L H signalling has only recently been investigated. We examined the effects of ! j gonadotrophins on intracellular calcium mobilisation i n HEK293 cells tran-siently transfected wi th human wi ld type or chimeric gonadotrophs receptors (n=3400). Intracellular free calcium concentration was measured using fura-2 microspectrofluorimetric techniques. H u m a n L H (2-4 pg /ml ) arid C G (lO I U / m l ) consistently evoked oscillatory calcium signals i n HEK293 cells trans-fected wi th h L H r , whereas h F S H (2-4 pg /ml) failed to elicit any calcium res-ponses. Both h L H and hFSH failed to elicit a calcium response from HEK293 cells transfected wi th hFSHr. Pre-treatment of transfected HEK293 cells with pertussis toxin (100 ng/ml) or with U-73122 (10 pM), a phospholipase C inhibi-tor, negated all gonadotrophin-evoked calcium mobilisation. Our study of chi-meric gonadotrophin receptors show that the carboxy-terminal third of the h L H r is crucial i n evoking intracellular calcium changes. Al though various subdivisions of this region is capable of stimulating calcium transients, an intact carboxy-terminal third of the receptor is required for normal and sustain-ed intracellular calcium profile. To our knowledge, this is the first demonstra-i j tion of calcium oscillations in response to the activation of the h L H receptor, and to unequivocally show that the h L H receptor is coupled to the inositol i ! phosphate/calcium signall ing pathway v ia a pertussis toxin-sensitive G protein. j j The role of extracellular A T P in the human ovary remains equivocal. We demonstrated that P2 purinoreceptor agonists evoke oscillatory intracellular calcium responses in hGLCs . The cells were responsive to A T P at concentra-tions ranging from 1-100 u M . A T P and UTP were more effective in stimulating calcium mobilisation than A D P . Neither adenosine nor A M P were capable of inducing intracellular calcium responses. The positive responses to adenosine thiotriphosphate, a non-hydrolysable A T P analogue, indicate that the calcium responses were not due to by-products from A T P hydrolysis, and that hGLCs possess P2U purinoreceptors. We have also demonstrated that these purinergic-mediated intracellular calcium responses involve both C a 2 + influx; and C a ^ + mobilisation from intracellular stores. - i i i -Table of Contents Abstract......:...; i ....1 ; i i Table of Contents... • : I i y List of Tables... '. . . . . i ] v i i " ! ' , ' '' ' • I I j I List of Figures.........: L : : . v i i i Abbreviations ... .; i '. . . . . .xi ; . '• ' I i Acknowledgements : I . . . . .xi i i Background.: 1 1! I. Gonadotrophic Hormones.: i 1 A . Luteinising Hormone/Chorionic Gonadotrophin Receptors .....3 ; 1. Desehsitisation of L H / C G Receptors 1 6 | (i) Uncoupling of L H / C G Receptors :.!..... i ....7j (ii) Down-Regulation of L H / C G Receptors ! .....8 j 2. Signal Transduction Pathways of L H / C G Receptors i ...1:9 j B. Follicle-Stimulating Hormone Receptors j ..10 1. Desensitisation of F S H Receptors . . . i i 11 2. Signal Transduction Pathways of F S H Receptors j 12 II. Intracellular Signalling in the Ovary 1 13 A . GTP-Binding Protein-Coupled Receptors I j 14 B. Adenylate Cyclase-Cyclic Adenosine Monophosphate ! Pathway i 16 C. Phospholipase C Pathway ; .18 III. Calcium and Cellular Regulation i 23 A . Modulation of Intracellular Free Calcium Concentrations..; 24 B. Calcium as Intracellular Regulator i 25 1.! Inositol 1,4,5-trisphosphate and ryanodine receptors ! .....26 C. Cytosolic Calcium Oscillations. ; i .....28 1. Characteristics of Calcium Oscillations ! 30 rV. Adenosine Triphosphate and Purinergic Agonists i ..31 Objectives: ! n.. i .33 - i v -Materials and Methods • ; i : 36 I. Reagents and Materials i 36 II. H u m a n Granulosa-Lutein Cells ! 37 III. Culture and D rug Treatments i 38 • • : : • i IV. Radioimmunoassays for Oestradiol and Progesterone J .38 A. Reagents j : i ..38 • ' ' '• • \ ! V. Microspectrofluorimetry..i j .40 •. i 'i ' , ; • • • | ]• VI. Transfection of H u m a n Embryonic K idney 293 Cells i ..41 A. Transient Transfection of H u m a n Embryonic K idney 293 '< [ Ce i l s : „ ' j. i Al B. Transfection Efficiency ....[.. ! 42 Results : 1 44 I. Gonadotrophin- Induced Ca lc ium Oscillations in HEK293 Cells ! Expressing the H u m a n Luteinising Hormone/Chor ion i c I Goriadotrophin Receptor : 44 A. Specificity of the H u m a n L H / C G Receptor : 44 B. Effect of H u m a n Chorionic Gonadotrophin on [Ca 2 + ] i ! 49 C. Ca lc ium Influx vs. Ca lc ium Mobil isation J 49 II. Ca lc ium Signalling in HEK293 Cells Transfected with the Wi ld f I Type or Chimeric H u m a n Gonadotrophin Receptors 1 54 A. Phospholipase C Involvement in Gonadotrophin- Induced \ \ Ca lc ium Responses ...i i 54 B. Effect of Gonadotrophin on Chimeric H u m a n Gonadotrophin j Receptors '. ] .58 III. P2-Purinoreceptor Agonist -Evoked Ca lc ium Oscillations in Single J H u m a n Granulosa-Lutein Cells J j i 73 A . Effects of purinergic receptor agonists on intracellular calcium j concentrations ;. .1 1 73 B. Effects of ATP7S on intracellular calcium concentrations \ 78 C. Calcium-inf lux vs. calcium-mobilisation..... '•. 1 78 D. Pertussis Toxin Pre-treatment....... 1 i 81 E. Effects of purinergic receptor agonists on steroid secretion.; 81 Discussion '• 87 I. GonadotarOphin-Induced Calcium Oscillations in HEK293 Cells Expressing the Human Luteinising Hormone/Chorionic Gonadotrophin Receptor II. Role of calcium oscillations in gonadal physiology. .87 i .93 III. Calcium Signalling in HEK293 Cells Transfected with the Wild-Type or Chimeric Human Gonadotrophin Receptors ! 96 TV. P2-Purinoreceptor Agonist-Evoked Calcium Oscillations in Single Human Granulosa-Lutein Cells : 98 V. Summary and Conclusion : i 102 VI. Future Directions. References. .103 .106 List of Tables Table 1: Control groups for transfected HEK293 cells... ; 46 Table 2A: Wild-type and chimeric human gonadotrophin receptor sche-matics and detectability of intracellular calcium mobilisa-tion L 55 Table 2B: Wild-type and chimeric human gonadotrophin receptor sche-matics and detectability of intracellular calcium mobilisa-tion "... i 56 Table 2C: Wild-type and chimeric human gonadotrophin receptor sche-matics and detectability of intracellular calcium mobilisa-tion '. ''. 57 - v i i -List of Figures Figure 1: Adenylate cyclase-cAMP pathway ; 17 j ' " • ' ' ' ' ! j Figure 2: Phospholipase C-fil and Phospholipase C-yl pathways j 19 | Figure 3: Phospholipase C-fsT pathway.. i 20 Figure 4: Effects of h C G treatment on human G L C s ; 45 i Figure 5: Effects of gonadotrophin treatment on human L H recepfors j expressed in HEK293 cells ..; j 47 j Figure 6: Human C G concentration-response relationship. L 48 Figure 7: Involvement of extracellular calcium on hCG-evoked cal-cium mobilisation . . .J • -50 Figure 8: Effects of thapsigargin pre-treatment on transfected HEK293 cells „. : ; ! 51 Figure 9: Effects of caffeine on hCG-evoked calcium signals in trans-fected H E K 293 cells 1 53 Figure 10: Effects of pFSH and D M S O on HEK293 cells transfected with ! the chimeric human gonadotrophin receptor FLR '. 59 Figure 11: Effects of forskolin treatment on HEK293 cells transfected with the wild-type human L H receptor ; 60 Figure 12: Effects of forskolin treatment on HEK293 cells transfected with the chi-meric human gonadotrophin receptor FLR . . . . j . 61 Figure 13: Effects of U-73122 treatment on HEK293 cells transfected with the Wild-type human L H receptor 62 - v i i i -Figure 14: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor LF(C)R : 63 Figure 15: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor FLR....64 Figure 16: ; Effects of gonadotropins treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor FLR....66 •V ! i ; . : I Figure 17: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor FL(C)R.. : 1 67 Figure 18: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor F(l -4)LR. '. | 68 Figure 19: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor FL(7-C)R I i 69 Figure 20: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor FL(V-i3)FR j •. : i 70 Figure 21: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor FL(V/VI)R 1 71 Figure 22: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor FL(V-VI)R 1 72 Figure 23: A T P concentration-response relationship ; 74 Figure 24: Upper panel: Efficacy profile of various pxirinergic agonists Lower panel: Comparison of the relative potencies of \ the various purinergic agonists '• 75 Figure 25: Upper panel: Effects of ATPyS, a non-hydrolysable A T P ana-logue, on hGCs; | •  L o w e r panel: Compar i s on of the relative potencies of ithe various P2U agonists.... I i i 76 Figure 26: Involvement of extracellular calcium on A T P - i n d u c e d ca l -c ium mobil isation 1 77 ,: ' '. . ' i Figure 27: Effects of verapami l on ATP-s t imu la ted ca lc ium mobi l i sa-tion..;... j 79 Figure 28: Effects of PGF2 a and A T P on intracellular calc ium mobil isa-t ion. '. ..: j 80 > . j Figure 29: Effects of purinergic agonists on basal oestradiol product ion in human G L C s i 82 Figure 30: Effects of purinergic agonists on hCG-st imulated oestradiol production in human G L C s • 83 Figure 31: Effects of purinergic agonists on basal progesterone produc-tion in human GLCs;... ''• 84 I ' . ' • !i . . ! : ; i Figure 32: Effects of purinergic agonists on h C G induced-progesterone production in human G L C s '. 85 Abbreviations adenylate cyclase : A C adenosine diphosphate • A D P j adenosine monophosphate •  A M P • adenosine 5'-o-(3-thiotriphosphate) ;. ATP7S adenosine triphosphate , A T P j 8-bromoadenosine 3':5'-cyclic monophosphate 8-Br-cAMP caffeine ; caf i carbon dioxide • C O 2 | cyclic adenosine 3'r5'-mononphosphate '• c A M P diacylglycerol : • D A G : i | dimethyl sulphoxide : J . D M S O i Dulbecco's Modified Eagle's Medium D M E M j ethylene glycol-bis(P-aminoethylether) HHN '^ '-tetraacetic acid ; E G T A foetal bovine serum, heat-inactivated FBS j follicle-stimulating hormone • F S H fura-2-acetoxymethyl ester : : fura-2-AM gonadotrophin-releasing hormone G n R H i :. ; j 1 : 1 ' i granulosa-lutein cells • G L C s | guanosine diphosphate '. • G D P - x i - ; i guano sine triphosphate i GTP human chorionic gonadotrophin hCG human granulosa-lutein cells i hGLCs inositol 1,4,5-trisphosphate : IP3 j international unit(s) IU intracellular calcium concentration [Ca 2 +]i luteinising hormone • L H oestradiol • E2 pertussis toxin • PTX phosphatidylinositol 4,5-bisphosphate PIP2 phospharidylinositol 1 PI phosphatidylinositol-4-phosphate PEP phospholipase C PLC potassium chloride KC1 progesterone • P4 prostaglandin F2a PGF201 protein kinase A PKA ; ' I protein kinase C • PKC i thapsigargin TPG uridine triphosphate UTP volume per volume (ml/100 ml) '. y / v weight per volume (gm/100 ml)... W/v - x i i -Acknowledgements I wou ld like to thank the members of my supervisory committee for their munificence, patience, and guidance in matters professional and personal. I am ever grateful to Dr. Paul E. Squires, the resident thaumaturge of calcium imaging, for his ready assistance and lambent wit. I would also like to thank my many colleagues for their ready assistance. I am eternally indebted to my kith and k in , for without their stalwart support and concern, the oft-times ' ' ' 1 ! : i fraught journey through graduate studies would have been impossible. j j i ' ' ! i • ' I i 1 ', I With deepest gratitude, PSL • ! - x i i i -Background \ \ • ': i i ' i ! Gonadotrophic Hormones : i : L H and F S H regulate gonadal function and gametogenesis, and are critical for normal sexual maturation and reproductive function; Both • • , ' • i hormones are synthesized in and secreted from pituitary gonadotrophs, under the regulation of G n R H . L H and F S H have approximate molecular weights of 28,000 and 33,000, respectively; the uncertainty i n their molecular weights result from the heterogeneity of the attached carbohydrate groups and minor differences i n amino acid composition.: These two pituitary glycoprotein hormones share chemical and structural similarities; both hormones are heterodimers composed of glycosylated subunits (a and C) tightly bound in a non-covalent association. The individual subunits appear to have no intrinsic biologic activity, and must be appropriately glycosylated and tightly associated to act as gonadotropins, j j Within a species, the a-subunits of glycoprotein hormones possess the same amino acid sequence. The oc-subunit of the L H / C G and F S H molecules, common to pituitary glycoprotein hormones, has a molecular weight of 14,000. The C-subunit of each glycoprotein hormone has a distinct amino acid sequence, and thus dictates hormone specificity [Pierce and Parsons, 1981]. The human C G 6-subunit, structurally very similar to the L H 6-subunit, shows about 80% similarity in amino acid sequence to the L H C-subunit, and confers almost identical biologic properties when associated wi th the oc-subunit. The human C G C-subunit contains an additional 32 amino acids at the carboxy-terminal, however this has no apparent role i n the biological activity or metabolism of the human C G molecule. L H and C G bind to the same receptor to initiate hormone action, but with different kinetics. j . ! The main difference in biological activity between human C G and L H is the more prolonged action of h C G in vivo, because of its slower'metabolic clearance and its somewhat higher affinity for the L H receptor sites in the testis and ovary. These features largely result from different carbohydrate compositions of the two molecules, in particular the much higher sialic acid content of h C G [Lambert, et al., 1998]. | :. i • : ! i '• The N-l inked oligosaccharides of these hormones are necessary for proper folding, assembly, secretion, metabolic clearance and biological activity. The carbohydrate content of F S H is greater than that of L H , but they share a similar structure. Specific chemical features of the L H , C G , and FSH molecules include the locations of the carbohydrate moieties: there are two oligosaccharide groups on the oc-subunit common to the glycoprotein hormones, one in the human L H fi-subunit, and two in the human FSH and C G 6-subunits. In addition to the N-linked oligosaccharides, the human C G fi-subunit also contains four O-linked oligosaccharides [Matzuk, et jal., 1990]. Deglycosylation has little effect oh hormone binding, but it does j markedly attenuate the hormones ability to activate target cells in the gonads [Sairam, 1989]. The carbohydrate moieties of the a-subunit, and not the 6-subunit, are essential for the activation of the L H receptor and its GTP-binding protein (G-protein)-coupled adenylate cyclase system [Matzuk, et al., 1989; Sairam, 1989]. The sialic acid content of the glycoprotein hormones varies from twenty residues per molecule in human C G , five residues in FSH, and only one or two in human L H . Removal of the sialic acid residues drastically shortens the - 2 -circulating half-life of the hormones, but has little effect on their ability to act ' : ' i I on their respective cellular receptor sites [Lambert, et al., 1998]. \ \ Luteinising Hormone/Chorionic Gonadotrophin Receptors \ \ L H and C G bind to, and activate, the same cell surface reporter. This receptor belongs to the large family of G-protein-coupled membrane proteins [Loosfelt, et al., 1989; McFarlandj et aL, 1989]. The L H / C G receptor is a glycoprotein consisting of a single polypeptide chain with six potential N-linked glycosylation sites [Kusuda and Dufau, 1988]. The hydrophilic amino-terminal of the receptor comprises approximately half of the total amino acids. This extracellular domain is necessary for high affinity binding to gonadotrophin. The transmembrane portion of the L H / C G receptor]has seven • i ' membrane spanning segments which form three extracellular loops; and three intracellular loops. The short intracellular carboxy-terrhinal domain contains serine, threonine, and tyrosine residues, suggesting the potential for modulation of receptor function by the 'action of serine-threonine protein kinases, and tyrosine kinases [Bousfield, et al., 1994]. j . i / , ' • • . ' . | | Structure-function relationship studies have demonstrated that the truncated extracellular amino-terminal half of the receptor is capable of high affinity ligahd binding without c A M P induction, whereas the truncated carboxy-terminal is capable of low affinity binding with c A M P induction [Ji and Ji, 1993; Segaloff, et al., 1990]. Binding of the L H ligand to its receptor results in conformational changes leading to activation of the C-terminal. Point mutation studies [Ji and Ji, 1993; Segaloff and Ascoli, 1993; Shenker, et al., - 3 -1993] have demonstrated that1 high affinity receptor binding and receptor activation with intracellular signal generation are distinct events. Receptors for L H / C G have be found on a variety of tissues in reproductive systems, including Leydig cells, granulosa cells, and luteal cells [Akamizu, et al., 1990; Ascoli and Segaloff, 1989]; they have also been detected in non-ovarian cells [Lincoln, et al., 1992]. This glycoprotein receptor consists of a single polypeptide chain, and share the same basic structure as the FSH and T S H receptors: a large amino-terminal domain, seven transmembrane spanning domains, and a short carboxy-terminal domain [Frazier, et al., 1990; Rodriguez and Segaloff, 1990; Strader, et al., 1995]. Receptors for L H / C G , FSH, and T S H belong to the large family of G-protein-coupled membrane receptors, but are unusual in that they have large extracellular domains (300-400 amino acids) arid bind large ligands (23-38 kDa) [McFarland, et al., 1989]. Other members of the receptor family have small amino-terminal extracellular domains (30-50 amino acids) and bind small ligands (200-300 Da) [Dohlman, et al., 1991; Jackson, 1991; Savarese and Fraser, 1992]. It is the large extracellular domains of the L H / C G receptors which are responsible for the recognition and high affinity binding of the respective glycoproteins [Braun, et al., 1991; Xie, et al., 1990]. Despite the i . | wide range of ligands that activate these receptors, the receptors themselves share a surprising amount of structural homology. j I The L H / C G receptor is highly conserved, with the highest• degree of conservation in the transmembrane domains and connecting loops| followed by the extracellular amino-terminal domains. The lowest degree of conservation occurs in the he intracellular carboxy-terminal cytoplasmic tails [Segaloff and Ascoli, 1993]. The human receptor is 85% identical to the rat L H / C G receptor and 87% identical to the porcine L H / C G receptor [Minegishi, - 4 -et al., 1990]. Despite the high homology between human, rat, and porcine, trie human L H / C G receptor has a high degree of species specificity; it does not bind equine L H and C G , rat L H or ovine L H [Jia, et al., 1991]. The large extracellular hydrophil ic domain of the L H / C G ; receptors comprises about half the total number of amino acids, and contains 6 potential sites for N - l i n k e d glycosylation [Loosfelt, et al., 1989; McFarland, et al., 1989; Minegishi , et al., 1989]. This extracellular domain contains 14 copies of an imperfectly repeated sequence of about 25 amino acids, similar to a repeated motif called "leucine rich repeat" [Leong, et al., 1992]. These repeats allow for the formation of amphipathetic helices or C-sheets, which can interact with both hydrophilic and hydrophobic surfaces [Krantz, et al., 1991], thus providing,a basis for the possible interaction of the hydrophilic extracellular domain with the hydrophobic transmembrane domain of the L H / C G , and also the FSH, receptors [Segaloff and Ascol i , 1993]. Leucine-rich repeats 1-6 have also been shown to be involved in hormone binding [Thomas, et al., 1996]. Involvement of the carbohydrate moieties of L H / C G receptors, in the recognition and high ! i affinity binding, remains equivocal. While some have indicated that at least • : ! •' I one of the carbohydrate chains is required for ligand binding [Minegishi, et al., 1989; Zhang, et al., 1995; Zhang, et al., 1991], others have reported that deglycosylation of the L H / C G receptor does not compromise its binding ability [Davis, et al., 1997; Ji, et al., 1990; Petaja-Repo, et al., 1991]. The seven transmembrane spanning domains of the L H / C G receptor are highly homologous wi th other receptors belonging to the family of G-proteih-coupled membrane receptors [Baldwin, 1994]. The seven hydrophobic transmembrane spanning domains are connected by hydrophilic extracellular and intracellular loops. The transmembrane domain of the L H / C G receptor has also been implicated in hormone binding. Several studies have shown that this region contains a low affinity hormone binding site [Ji and Ji, 1991a; Roche, et al., 1992], and that it may be important in the activation of the adenylate cyclase [Abell and Segaloff, 1997; Ji and Ji, 1991a; Ji and Ji, 1991b] The short carboxy-terminal cytoplasmic tail, along with the cytoplasmic loops connect ing the t ransmembrane domains , conta in 'potent ia l phosphorylation sites, and thus may be a further site for the regulation of • i i • •  ! : hormone-receptor function. Two potential kinase C phosphorylation: sites have been identified, along with a third domain [Loosfelt, et al., 1989]. Mutations of the carboxy-terminal cytoplasmic tail resulted i n the non-expression of rat L H / C G receptors on the plasma membrane, suggesting that the carboxy-terminal cytoplasmic tail is important for the trafficking of receptors to the plasma membrane [Rodriguez, et al., 1992; Sanchez-Yague, et al., 1992], and for receptor desensitisation [Sanchez-Yague, et al., 1992]. ; Desensitisation of LH/CG Receptors Ligand binding to the L H / C G receptor results in uncoupling and down-regulation. Uncoupl ing is defined as the agonist-induced change in the functional properties of the receptor without a change in the number of receptors. This relatively fast phenomenon occurs wi th in minutes of the administration of the agonist, is thought to be due to phosphorylation of intracellular amino acid residues, thereby attenuating its ability to activate the effector system(s) (i.e. adenylate cyclase, phospholipase C) [Segaloff and Ascoli , 1993]. Down regulation is defined as the actual reduction in the density of the receptors at the plasma membrane. This slower phenomenon occurs wi th in minutes to hours of addition of the agonist, and can be caused by a decrease in the synthesis of the receptors, an increase in the degradation of recep'tors, or by a combination of both [Segaloff and Ascoli , 1993]. : ' • ' • I : ; ' .' ! I j ! . : : ! i Uncoupling of L H / C G Receptors ; \ j Uncoupl ing of the L H / C G receptor leads to a reduction in!hormonal responsiveness without a concomitant reduction in the number of L H / C G receptors [Rebois and Fishman, 1986], and without changes to the functional • ' : • • i : properties of G s or the catalytic subunit of adenylate cyclase [Rebois arid Fishman, 1986; Sanchez-Yague, et al., 1993]. Uncoupling of the {^-adrenergic receptor involves phosphorylation of different regions of the receptor catalysed by the cAMP-dependent and 62-adrenergic receptor kinases [Dohlman, et al., 1991; Lefkowitz, et al., 1990]. As both the {^-adrenergic and L H / C G receptors belong to the same family of G-protein-coupled membrane receptors, they w i l l undoubtedly possess similarities; however, the cAMP-dependent protein kinase is unl ikely to be involved in the phosphorylation and /o r uncoupling of L H / C G receptors because: (1) increases in c A M P levels elicited by agents other than L H / C G do not uncouple the L H / C G receptor [Rebois and Fishman, 1986]; and (2) there are only weak consensus sites for the cAMP-dependent protein kinase-catalysed phosphorylation in the intracellular regions of the rat, : ! • 1 ! porcine, mouse, or human L H / C G receptor [Kennelly and Krebs, 1991]. M a x i m a l uncoupl ing of the L H / C G receptor also requires guanosihe triphosphate (GTP) [Ekstrom and Hunzicker -Dunn, 1989a; Ekstrom and Hunzicker-Dunn, 1989b; Ezra and Salomon, 1980]. i - 7 -Studies have demonstrated that the carboxy-terminal cytoplasmic tail is involved in the uncoupling of the L H / C G receptor [Sanchez-Yague, et al., 1992; Wang, et al., 1996]. They have shown that truncation of the carboxy-terminal i ; cytoplasmic tail results in a higher maximal c A M P response than that observed w i t h wi ld- type receptors. S imi lar ly , the magnitude of hCG- induced uncoupl ing : is more pronounced i n cells expressing wi ld- type L H / C G ! ' i ! receptors, than those expressing receptors wi th truncated cytoplasmic; tails. The fact that L H / C G receptors, wi th truncated cytoplasmic tails, still lose hormonal responsiveness upon prolonged exposure to its l igand, suggests that uncoupling is not the only mechanism involved in desensitisation. Truncation studies [Rodriguez, et al., 1992; Sanchez-Yague, et al., 1992] have shown that receptor uncoupling and receptor internalisation are separate phenomena, with different determinants. • Down-Regulation of L H / C G Receptors ; j i i ' i ; H u m a n CG-induced reduction i n the density of L H / C G receptors is elicited by both an increase in receptors internalisation, and by decreased transcription of the receptor gene. Exposure of L H / C G receptors to their ligands results i n a time-dependent decrease i n the number of membrane receptors, without changes in receptor affinity [Freeman and Ascol i , 1982; Rebois and Fishman, 1984]. There is an actual decrease i n the number of receptors, and not a mere redistribution of receptors from the cell surface to an intracellular compartment [Ascoli, 1985]. Studies have shown that the entire ligand-receptor complex is internalised into endocytic vesicles and transferred into lysosomes without ligand dissociation [Ascoli, 1982; Ascol i , 1984; Freeman i i : and Ascol i , 1982]. Although only about 50% of internalised receptors follow this route, the accumulation of these internalised receptors in lysosomes : ; i • • ! I prevent receptor recycling, promotes receptor degradation and is ultimately responsible for receptor down-regulation [Ascoli, 1982; Ascoli, 1984; Freeman and Ascoli, 1982; Segaloff and Ascoli, 1993]. j ! Wang er al. [Wang, et al., 1991] demonstrated that ligand-induced down-regulation of L H / C G receptors, in MA-10 cells, consists of 2 distinct phases: (1) the first phase, lasting 3-4 hrs following ligand exposure, is characterised by an 80% reduction in the levels on L H / C G receptors with little or no changes in the level of L H / C G mRNA. Quantitatively the most important phase, it involves an increased rate of receptor degradation. This in turn seems to be due to internalisation and lysosomal accumulation of the receptor that occurs during receptor-mediated endocytosis of L H / C G . (2) a further reduction of L H / C G receptor levels that is accompanied by a 40-60% reduction in L H / C G receptor mRNA levels. ! ; ! ! . : ] ' : 1 Thus, the process of LH/CG-induced down-regulation of the L H / C G receptor involves an increase in receptor degradation and a decrease in receptor synthesis, that is secondary to a decrease in mRNA [Segaloff and Ascoli, 1993]. It is unknown whether the decrease in receptor synthesis during the first phase, is die to LH/CG-induced changes in the rate of translation of trie L H / C G ' • • ' i receptor mRNA. ; j ' ! ' 1 : ' ' , : i Signal Transduction Pathways of LH/CG Receptors 1 i ! • i • • i i It is well-established that the L H / C G receptor is coupled to the adenylate cyclase/cAMP pathway [Dufau and Catt, 1978; Hunzicker-Dunn and Bimbaumer, 1985; Leung and Steele, 1992]. Alternatively, it has been reported that the murine and rat L H receptors are coupled to the phospholipase C/ inos i to l 1,4,5-trisphosphate (IP3) pathway [Davis, 1994; Gudermann, et al., 1992a; Herrlich, et al., 1996; Hipkh% et al., 1993]. The ability of L H to stimulate phospholipase C activity is not associated wi th the accumulation' of c A M P , indicating that the activation of phospholipase C is not secondary to the activation of adenylate cyclase, jit has been reported that L H increases IP3 arid [ C a 2 + ] i in isolated bovine luteal cells [Davis, et al., 1987]. Likewise, inositol ; i ; '• ' ' i phosphates accumulation are increased in porcine granulosa cells following L H treatment [Dimino, et al., 1987]. In porcine granulosa cells isolated from 5.0 nm and 1.0 mm diameter ovarian follicles, L H induces a rapid and transient [Ca2+]i increment, which is similar to that induced by endothelin-1 [Flores, et al., 1992b] These data lend support to the notion of a novel signalling pathway in L H action, involving adenylate cyclase and phospholipase C. \ Follicle-Stimulating Hormone Receptors : \ F S H is necessary for gonadal development and maturation at puberty [Chappel arid Howies , 1991]. \ F S H acts by binding to specific 'receptors, localised exclusively i n the gOnads. The F S H receptor is synthesized in granulosa [Hsueh, et al., 1984] and Sertoli cells [Reichert and Dattatreyamurty, • • ' j 1 1989], and transported to the membrane surface. ; j '•; ' • I ' ! Like the L H / C G receptor, the F S H receptor belongs to the large family of G protein-coupled membrane proteins [Abou-Issa and Reichert, 1976]. Unl ike the L H / C G and T S H receptors, the F S H receptor has;not been comprehensively investigated. Like the L H / C G receptor, the human F S H receptor is a single polypeptide chain [Sprengel, et al., 1990] wi th four potential 1 I TV-linked glycosylation sites [Minegishi, et al., 1991]. Although deglycosylatidn of the F S H receptor does not seem to affect l igand binding, glycosylation is necessary for the proper folding of the glycoprotein hormone, and for its expression on the plasma membrane [Davis, et al., 1995; Rozzell , et al., 1995]. Mutations that prevent receptor folding and/or transportation result in tne retention of the receptor protein in the cell. : i i : As aforementioned, the F S H receptor also comprises a large amino-terminal domain, seven transmembrane spanning domains, and a short carboxy-terminal domain. The extracellular amino-terminal domain contains 14 leucine-rich repeats, s imilar to those described for the L H receptor [Bousfield, et al., 1994]. Ligand specificity is conferred by the extracellular domain, and not by the transmembrane domain [Braun, et al., 1991]. The structure of the seven transmembrane spanning domains is typical of members belonging to the superfamily of G-protein-coupled membrane; receptors [Baldwin, 1994]. ^ ! f ; .'. • : ' ' i I , . • ' ; ! • 1 Desensitisation of FSH Receptors ' As wi th the L H receptors, desensitisation of the F S H receptors can be distinguished into two phases: uncoupling and down-regulation. Uncoupling of the F S H receptor from the G-protein occurs shortly after ligand-receptpr bind.[Grasso and Reichert, 1989]. This process occurs v ia enzymatic ; , | i phosphorylation of the carboxy-terminal, intracellular domain of the G -protein-coupled receptors and may be due to receptor-specific kinases or to effector kinases typical of the receptor system (i.e. protein kinase A or protein kinase C) [Simoni, et al., 1998]. The down-regulation of receptors involves;a decrease i n receptor number through internalisation and sequestration of hormone receptor complexes in lysosomes or reduced receptor protein synthesis as a result of both decreased transcription and/or reduced m R N A half-life. Themmen et al. [Themmen, et al., 1991] have shown that the FSH-induced decrease in F S H receptor m R N A is due! to a cAMP-dependent, post-transcriptional mechanism. Signal Transduction Pathways of FSH Receptors Unlike the L H receptor, in which dual signalling pathways have been demonstrated [Davis, 1994; Gudermann, et al., 1992a; Herrl ich, et al., 1996; Hipk in , et al:, 1993], the F S H receptor seems to be almost exclusively mediated by the adenylate cyclase-cAMP pathway [Flores, et al., 1992a; Gorczynska, et al., 1994]. Sertoli cells possess the protein kinase C pathway, and exposure of trie cells to stimulators of the protein kinase C pathway inhibits FSH-dependent c A M P production [Monaco, et al., 1988; Monaco and Conti , 1987]. F S H neither activates [Quirk and Reichert, 1988] nor inhibits [Monaco, et al., 11988] the phosphatidyl , inositol pathway, receptors in HEK293 cells indicate Studies wi th chimeric human; L H / F S H that inositol production upon activation of F S H receptors is weak [Hirsch, et al., 1996]. j i F S H increases intracellular calcium concentrations i n Sertoli cells i i , ; j [Gorczynska and Handelsman, 1991] and granulosa cells [Flores, et al., 1990]. The possibility that F S H receptors might act as ligand-gated calcium channels was deemed unlikely by Shibata et al. [Shibata, et al., 1992]; however] F S H may increase intracellular calcium by stimulating other calcium channels pre-existing on granulosa and Sertoli cells [Grasso,. et al., 1991]. FSH-induced elevations in intracellular calcium concentrations are independent of protein kinase C [Flores, et al., 1992a]. j ! | j • ' . . ; . ' ! : • ' \ ! ' • ! ' Intracellular Signalling in the Ovary i N o r m a l ovarian function is dependent on diverse hormones acting through endocrine, paracrine, autocrine, and intracrine processes. Hormonal • 1 • ' • ! .• • • ' I signals are often translated into cellular activities via signal transduction ; i pathways. Ovar ian hormones exert their effects through complex signal transduction mechanisms, and some may even stimulate mult iple secorid messenger pathways. | - i ! Many signalling pathways comprise a series of proteins, including: specific receptors, GTP-b ind ing proteins, second messenger-generating . . . , i enzymes, protein kinases, target functional proteins, and regulatory proteins. Molecular cloning analysis has revealed that almost al l of these signalling proteins show extensive heterogeneity and differential tissue expression wi th specific intracellular localisation. However, the biological significance of this heterogeneity has not always been clear. There are diverse interactions between s ignal l ing systems. These interactions include potentiation, cooperation, synergism, antagonism, and co-transmission. The regulation of cellular functions by hormones and growth factors are dependent upon the ability of the target cells to differentially recognise and respond to the i i I i individual: effector molecules. Such responses can be rapid (e.g. contraction, transmission, secretion, etc.) or long-term (e.g. differentiation, proliferation, death, etc.).. j j • GTP'-Binding Protein- Coupled Receptors \ 1 , ' i : 1 1 G-protein-coupled receptors comprise the largest known family of cell ' surface receptors, and are defined by their similarities in structure and function. These surface receptors mediate cellular responses to a diverse array of s ignal l ing molecules, including: peptide [Flanagan, et al., 1997] and glycopeptide [Davis, et al., 1987] hormones , neurotransmitters [Jose, et al., 1990], phospholipids [Onorato, et al., 1995], odorants [Firestein and Shepherd, 1992], and photons [LeVine, et al., 1990]. Despite the myriad ligands wi th which they interact, G-protein-coupled receptors share a surprising amount of primary and tertiary structural homology [Strader, et al., 1994]. G-proteiri-coupled receptors may be further classified into three subfamilies: rhodopsin/C-adrenergic, secretin/vasointestinal, and metabotrophic receptors [Strader, et al., 1995]. ; glutamate G-protein-coupled receptor signalling comprises three components: the surface membrane receptor w h i c h binds the extracellular l igand, the heterotrimeric G-protein, and the effector system. Surface membrane receptors known to function v ia G-protein mediation are characterised by seven : ; 1 . ' I i I transmembrane spanning domains joined by extracellular and intracellular • •• I loops [Dohlman, et al., 1991]. Through their intracellular domains, these I ! receptors interact wi th heterotrimeric G-proteins, which in turn modulate the activity of various effector systems. These effectors generate the intracellular second messengers which ultimately evoke cellular responses to event of receptor activation by the ligand. the init ial ! 1 Heterotrimeric G-proteins belong to the superfamily of GTP-bindirig proteins that includes ras and ras-like proteins, as wel l , as elongation arid -14-initiation factors of ribosomal protein synthesis . This trimeric unit consists df: an oc-subunit which contains a guanine nucleotide binding site and intrinsic GTPase activity, and a fiy-subunit complex [Neer, et al., 1990]. The family of G -proteins comprises over 20 isofoirms, wi th four classes of cc-subunits, five of the C-subunits, and at least six of the y-subunit [Coleman and Sprang, 1996]. G-protein-mediated signal transduction begins with the activation of an ligand-specific surface membrane receptor. Ligand binding to the receptor which results in a conformational change that exposes a high-affinity binding site for the G-prote in , i n its guanosine diphosphate (GDP)-bound heterotrimeric form, the receptor [Rens-Domiano and Hamm, 1995]. ! Multi-site interactions between the ligand-receptor complex and G-protein leads to the exchange of the oc-subunit-bound G D P for guanosine triphosphate (GTP) [Dratz, et al., 1993; Hamm, 1991]. Once GTP-bound, the oc-subunit of the j G-protein dissociates from the ligand-receptor-fiy complex, and regulates the appropriate effector system. The system is inactivated when the intrinsic GTPase]activity of the oc-subunit hydrolyses G T P back to G D P ; the oc-subunit reverts to its prior conformation and regains high affinity for the fiy-complex, and the system returns to its resting state. Formation of the heterotrimer is required for high affinity coupling of G-protein to receptor [Cerione, 1991; Fung, 1983].1 G-protein oc-subunits interact wi th a diverse array of second messenger enzymes and ionic channels, including: adenylate cyclase, phosphodiesterase, phospholipase C, and potassium and calcium channels [DeVivo ancl Iyengar, 1994]. It was once thought that only the oc-subunit regulates second messenger effector systems, but studies have demonstrated that the Cy-complex is also important in the regulation of many second messenger systems, solely, or in conjunction wi th the oc-subunit [Clapham and Neer, 1993; Spiegel, et al., 1992; -15 Tang and Gilman, 1991]. The fiy-complex regulates the yeast mating response; both the a-subunit and the fiy-complex act independently on muscarine-gated potassium channels, phospholipase C - a isoforms, type T adenylate cyclase, ras-mediated extracellular signal-regulated kinases activation, and PI-3-kinase in platelet cytosol; and synergistically in activating adenylate cyclase types II and IV [Clapham and Neer, 1993; Crespb, et al., 1994; Thomason, et al., 1994]. ! • 1 ' I !' Adenylate Cyclase-Cyclic Adenosine Monophosphate Pathway J | Intracellular signalling via the adenylate cyclase-cAMP pathway (Figure 1) is ubiquitous in eukaryotic cells regulating myriad vital functions: energy metabolism, gene transcription, proliferation, differentiation, reproductive functions, secretion, neuronal activity, memory, contractility, and motility. The adenylate cyclase-cAMP signal transduction pathway comprises a cascade of ; i . i ' i regulatory proteins, and many of them have the potential to modulate the magnitude and/ or the duration of signalling events. ' ' •• ! ' • ! j Activation of the agonist-specific plasma membrane receptor, which coupled to a heterotrimeric G-protein, elicits a conformational change in the receptor. Two classes of G-protein may be associated with plasma membrane receptors: G s , a stimulatory G-protein responsible for the activation of adenylate cyclase; Gi, an inhibitory G-protein responsible for the inhibition of the enzyme.; Agonist-induced conformational changes to the receptor catalyses the exchange of bound guanosine diphosphate (GDP) for guanosine trisphos-phate (GTP). Once GTP-bound, the a-subunit of the G-protein dissociates from the fiy-complex, and activates the catalytic unit of the adenylate cyclase. The j - : , -16- : ! Figure 1: Adenylate Cyclase - c A M P Pathway I I - 1 7 -enzyme hydrolyses A T P to c A M P , which then either activates the c A M P -! I dependent protein kinase A , or is degraded to 5 ' A M P by phosphodiesterases. The activated protein kinase A can then phosphorylate Other proteins [Hanley and Steiner, 1989]. I j ' ; i i Phospholipase C Pathway i I The association of a calcium-mobil is ing agonist w i t h its receptor activates a phosphodiesterase, phospholipase C . This enzyme preferentially hydrolyses inositol-containing phospholipids. Phosphoinositides present in : i membranes include phosphatidylinositol and its phosphorylated derivatives, , | j polyphosphoinositides such as phosphatidylinositol-4-phosphate (PIP) and phosphat idyl inosi tol 4,5-bisphosphate (PIP2)- The polyphosphoinositides : 1 : ! I • • 1 I result from the phosphorylat ion of phosphatidylinosi tol by A T P i n the presence of specific kinases at the plasma membrane to form PIP and subsequently, PIP2. These reactions are reversible through the hydrolytic activities of specific phosphatases. These polyphosphoinositides are the ; ; • i preferred substrates of the phospholipase C enzyme. Phosphoinositol is also ' • ' 1 ' j I hydrolysed by phospholipase A2 to form phosphatidic acid and free fatty acid, usually arachidonic acid. Arachidonic acid is the precursor for the biosynthesis of various eicosanoids. j ' ' ' i ! The two isoforms of phospholipase C trigger different pathways (Figure 2). Whi le phospholipase C - f i l hydrolyses membrane-bound PIP2 to generate IP3 and diacylglycerol, phospholipase C - y l appears to act exclusively on phos-phatidylcholine, the most abundant phospholipid in mammalian membrane, to produce diacylglycerol and phosphocholine [Berridge, 1993]. i J - 1 8 - ! i Figure 2: Phospholipase C - f i l and Phospholipase C - y l Pathways tyrosine kinase-linked receptor G protei n-linked receptor : cellular activities - 1 9 -When a ligand binds to a receptor, the resulting ligand-receptbr complex activates the receptor-coupled G-protein (Figure 3). Once the G-protein is activated, the a-subunit-bound G D P is released, allowing G T P to bind in its place and the a-subunit dissociates from the fiy-complex. The GTPrbound a-subunit in turn activates phospholipase C- f i l . The a-subunit exhibits intrinsic GTPase activity capable of hydrolysing G T P to G D P . Once inactivated (i.e. GDP-bound), the a-subunit re-associates wi th the Sy-complex [Berridge, 1993; Hanley and Steiner, 1989]. Phospholipase C - f i l hydrolysis membrane-bound PIP2 to produce IP3 and D A G which act as second messengers for the mobilisation of calcium and activation of protein kinase C, respectively. IP3 is released into the cytoplasm where it binds IP3 receptors and mobilises internal calcium stores. D A G remains membrane-bound and activates protein kinase C. D A G can also be hydrolysed to arachidonic acid. Protein kinase C and the increased intracellular calcium levels then promote cellular activities [Berridge, 1993; Hanley and Steiner, 1989]. : ! j The tyrosine kinase-linked receptor directly activates phospholipase C-y l (Figure 2). The tyrosine kinase-linked receptor consists of a single transmembrane protein containing a cytoplasmic tyrosine kinase.; When a ligand binds to the receptor, if induces receptor dimerisation, allowing two kinase domains to phosphorylate each other at specific tyrosine residues; this action provides a docking site for the SH2 domain of phospholipase G-yl . Once phospholipase C - y l is phosphorylated, it can then hydrolyse PIP2 to yield IP3 and D A G . : '.' \ A t least nine distinct protein kinase C isoenzymes have been identified, and differ in their tissue expression as wel l as in their mode of activation and their substrate specificities. The individual enzymes w i l l probably prove to have distinct functions in signal transduction and in the control of metabolism, secretion, differentiation, and proliferation [Hug and Sarre, 1993].! The nine isoenzymes can be subdivided into the conventional calcium-dependent isoforms (a, 61, 611, and y) and the calcium-independent isoforms (5, e, rj , 9, arid Q. The former are single polypeptide chains wi th catalytic domains containing the A T P and substrate binding sites located in the carboxy-terminal half of the molecule, and regulatory domains containing the calcium, phospholipid, and D A G / phorbol ester binding sites in the amino-terminal half. The regulatory domains are similar among the calcium-dependent a , 6, and 7 enzymes, but the calcium-independent 8-£ enzymes lack the calcium binding domain, and £ is not activated by D A G or phorbol ester [Nishizuka, 1988]. j I The major l i p i d activator of protein kinase C is D A G , acting in conjunction wi th PS as a cofactor. Fol lowing ligand-activation, the calcium released from InsP3-sensitive stores binds to the conventional protein kinase C isoenzymes and promotes their translocation to the plasma membrane, where they are activated by the PS present in the l ip id bilayer and the D A G produced from phosphoinositide hydrolysis. Phorbol esters act by mimicking the action of D A G , and lowering the calcium requirement for enzyme activation. In the case of calcium-independent protein kinase Cs, phosphoserine and D A G or other l ip id derivatives are required for activation. Several of the protein kinase C isoenzymes are activated by other phospholipid metabolites including cis-unsaturated fatty acids, arachidonic acid and its derivatives, and PIP2. Differential act ivat ion can also result from D A G produced dur ing phosphat idylchol ine breakdown st imulated by certain hormones and cytokines, and from PIP3 formed during activation of growth factor receptors. I In this manner, the several protein kinase C isoenzymes could be differentially activated by specific s t imul i to phosphorylate their substrates at defined cellular locations. - I Calcium and Cellular Regulation \ Calcium is the fifth most abundant element in the human body and the most common of the mineral ions [Lehninger, 1982]. It is also the most important structural element, occurring not only in combinat ion w i t h phosphate in bone and teeth, but also wi th phospholipids and proteins in cell membranes where it plays a vi ta l role i n the maintenance of membrane integrity and i n controlling the permeability of the membrane to many ions including calcium itself. It is involved i n a myr iad of physiological and biochemical processes [Lehninger, 19821: blood coagulation, coupl ing of muscle excitation and contraction [Ebashi, et al., 1978], regulation of nerve excitability [Katz, 1966], sperm moti l i ty , fertilisation [Epel, 1982], cell reproduction [Hepler, 1994; Mor r i l l and Kostellow, 1986], control of enzymatic reactions, and as the second! messenger i n the many hormone-induced pathways [Berridge, 1993; Rasmussen, 1989]. Because of its importance, many mechanisms have evolved to preserve body stores of the ion and to ensure:a • • .: : i : : I j sufficient supply to the organism so that it can maintain relatively constant concentrations of both intra- and extracellular calcium. It is so vi tal to the ' • • i : body's normal functioning that if the plasma levels of ionised calcium falls below 0.6-0.7 m m o l / L (normal range being 1.10 - 1.30 m m o l / L ) then trie neuromuscular system ceases to function normally and bone fails to mineralise properly. O n the other hand, abnormally high levels of ionised calcium (> 1.6 m m o l / L ) are toxic to many enzyme systems so that the level must also be kept : • ( i ' • ' ' • i ! ! - 2 3 - • i • ! below this critical upper l imit to ensure the continuance of normal cellular function. Thus a finely tuned mechanism for calcium homeostasis has evolved i to maintain a constant extracellular f luid (ECF) concentration of the cation ' ' ! I [Lehninger, 1982]. 1 i • 1 1 i • : ' ' 1 i Extracellular fluid calcium homeostasis is achieved by the steady-state 1 ; ' i ' ^ ' control of calcium fluxes into arid out of the E C F by a number of hormones, < ' i ! namely parathyroid hormone, calcitonin, and the active metabolites of vitamin D. These act on the main target organs for calcium, namely kidney, intestine and bone, of which the kidney is by far the most important regulatory organ for calcium homeostasis. Deviations from the normal E C F level of calcium I : : ' I occur in certain disease states, particularly those involving alterations in the circulating concentrations of the aforementioned hormones. : ; i In order to fulfill its various functions, calcium must often be transferred from one body compartment to another or from one cellular compartment to another. The cells involved in the translocation of calcium must be able to protect themselves against a surfeit of the cation, which, although necessary for some intracellular activities is toxic to many others. To achieve both objectives, highly specific transport and buffering mechanisms for calcium have had to be developed within these cells. I j Modulation of Intracellular Free Calcium Concentrations '•, I Being a cri t ical mediator in a myr iad of cellular responses, the concentration of free, ionised calcium in the cytosol is carefully! regulated • ; ; i [Berridge, 1993]. Basal intracellular calcium levels are approximately 0.1 u.M and can rise over 100-fold in response to influx of extracellular calciuiri (~1 mM) or mobilisation of intracellular calcium stores in the endoplasmic reticulum. The elevation of cytosolic free calcium concentrations in a hormone-responsive tissue can be due to several mechanisms [Meldolesi and Pozzan, 1987]: (i) influx • • • : i . I of extracellular calcium by the activation and opening of second messenger-activated channels, receptor-operated calcium channels and /o r voltage-dependent calcium channels;' (ii) release of calc ium from intracellular membrane-bound stores; (iii) inhibition of calcium extrusion systems such as the calcium pump and the N a + / C a 2 + antiport; and (iv) release of calcium from intracellular binding proteins. These mechanisms may also work in synergy, as i n the process of calcium-induced calcium response. Each of these mechanisms has been implicated in the different tissues in response ;to various calcium-mobilising agents. The return of calcium concentrations to resting levels after stimulation is brought about in essence by the reversal of these events; i.e. by the release of the hormone from its receptor, the destruction of intracellular second messengers, the active extrusion of calcium from the cell and the sequestration of calcium by intracellular organelles and binding prote ins . M u c h of the calcium that enters the cytoplasm dur ing agonist • • • ,; ' stimulation is rapidly re-sequestrated into the endoplasmic reticulum via C a 2 t -ATPase pumps. In addition, agonist-induced elevations in cytosolic calcium often activate the Ca 2 + -ca lmodul in-sens i t ive enzyme, C a 2 + - M g 2 " t - A T P a s e , which extrudes calcium from the cell [Berridge, 1992], • •. . • r :'i '. . ' ; ..• I I Calcium as Intracellular Regulator The importance of calcium as an intracellular messenger has long been recognised, but only in the last decade has the complexity of this signalling system been fully appreciated. Most of the signalling actions of calcium are dependent upon its interaction wi th binding proteins such as calmodulin and regulatory enzymes such as protein kinase C. The calcium-calmodulin complex regulates the activities of numerous enzyme systems, including adenylate arid guanylate cyclase, cyclic nucleotide phosphodiesterase, C a 2 + - M g 2 + - A T P a s e , and calcineurinj By influencing the cytoplasmic levels of cyclic nucleotides and calcium, calmodul in links the intracellular messenger systems as we l l as controlling enzymes involved in signalling, secretion, arid contractility. I ; | j '• i 1 i ' i • • • ' • i Calcium also binds directly to several calcium-dependent enzymes, the most important of which is protein kinase C, a calcium- and phospholipid-dependent phosphokinase. The phospholipase C pathway is the predominant mechanism of calcium-mobilising receptors (Figure 3). Both the G-protein-linked receptor and the tyrosine kinase-linked receptor stimulate release of IP3 , . • • i - the G-protein-linked receptor v ia phospholipase C-61, while the tyrosine kinase-linked receptor works through phospholipase C - y l [Jayaraman, et al., 1996]. Once PIP2 is converted to IP3 and D A G , the latter acts by activating protein kinase C, while IP3 diffuses into the cytosol to release calcium from intracellular reservoirs. IP3 acts as the intracellular second messenger by binding to the specialised tetraineric IP3 receptor that spans the endoplasmic • . . , j i reticular membrane and triggers the release of calcium from the ER; [Li, et al., 1995]. . ; \ ' • 1 ! . , '. 1 i Inositol 1,4,5-trisphosphate and ryanodine receptors ! IP3 receptors are located on the nuclear membrane and on certain parts • • • ; 1 i of the ER. IP3 appear to release only a portion (usually 30-50%) of the calcium from the non-mitochondrial stores [Berridge and Irvine, 1984]. Calcium that is -26-j " • ' I i-' • • 1 '• I I sequestered in IP3-insensitive stores may not necessarily be inert, but may be released by the processes of calcium-induced calcium release [Endo, et al., 1970]. ! ' I • '• i 1 IP3 and ryanodine receptors are the two principal intracellular calcium channels involved in mobilisation of stored calcium [Coronado, et al., 1994; L i , et al., 1995]. Both receptors are tetramers composed of large subunits (300 and 550 kDa, respectively), and share considerable structural and functional similarities [Tsien and Tsien, 1990]. A significant degree of homology exists in the domain located toward the carboxy-terminal, that spans the membrane and participates in the assembly of the calcium channel. The remainder of the molecule, where no homology is evident, protrudes into the cytosol [Pozzan, et al., 1994]. • : ! ' IP3 receptor channel activity is influenced by a number of cellular factors: c A M P and guanosine 3',5'-cyclic monophosphate (cGMP) protein kinases [Danoff, et al., 1991; Komalavilas and Lincoln, 1994], protein kinase C [Ferris, et al., 1991], calcium/calmodulin-dependent protein kinase II [Hanson, et al., 1994], A T P [Bezprozvanny and Ehrlich, 1993], p H , etc. Upon binding to the ligand, the IP3 receptor undergoes a conformational change that is thought to be related to the coupling process leading to channel opening. Gating of the IP3 receptor channel by IP3 and intracellular calcium concentrations are key 1 i factors in calcium signalling. The probability of channel opening increases wi th the concentration of IP3, and saturates at very high levels of IP3. ! ; . ' ' ' ! Enhancement of H V i n d u c e d channel opening is associated wi th higher oscil lat ion frequency i n cell types exhibi t ing agonist-induced calcium oscillations [Berridge, 1990]. ] ; i i : I i i . • 1 ' i ' ; . - 2 7 - I : ' , • • • ! i i 1 r i • • ; : ' j I ' Ligands known to open the ryanodine receptor channel and stimulate ! I calcium release include: micromolar calcium concentrations, mill imolar A T P , and caffeine [Berridge, 1993; Coronado, et al., 1994]. These receptors contribute to calcium signalling in many different cell types: skeletal muscle, cardiac muscle, neurons, chromaffin cells, smooth muscle, pituitary cells, and sea urchin eggs [Berridge, 1993]. ; ; | '. I I I ; 1 | Calcium induced calcium release is one of the most interesting aspect of the ryanodine receptor. This positive feedback process allows calcium to trigger its own release. A small influx of calcium through voltage-operated calcium channels can trigger an larger release of stored intracellular calcium. | : This process allows for the amplification of the calcium signal, and possibility results in the generation of repetitive calcium spikes [Berridge, 1993]. This calcium-induced calcium release property of ryanodine receptors is also exhibited by IP3 receptors. i Apart from the calcium-sensitive regenerative ability of IP3 ' receptors, the other intriguing aspect of IP3-induced calcium mobilisation is; its all-or-none effect. This property is manifested as a sudden or near-maximal release of calcium if the level of IP3 is gradually increased. L o w concentrations IP3 w i l l elicit small intermittent bursts of calcium release; these calcium bursts continue ' I unti l a threshold concentration of IP3 is attained, after which an; explosive release of stored calcium occurs [Berridge, 1993]. ! , • ; i i i ; ! Cytosolic Calcium Oscillations | ' i !' i ' I I Given the multiplicity of receptors which stimulate InsP3 turnover, it j remains unclear how specific signal information is transmitted to different : 1 i i • ! . | • cells, or how single cells distinguish between different receptor inputs. ' • ' i : 1 ! ' i Cytosol ic calcium oscillations are widespread, occurring in both undifferentiated (e.g. mouse oocytes and hamster eggs) and specialised cells (e.g. gonadotrophs and GLCs) . The oscillations are based upon fluctuations in cytosolic free calcium, and are classified by their source of calciurn influx: (i) membrane oscillators originate from the influx of extracellular calcium, and (ii) cytosolic oscillators arise from the mobilisation of intracellular calcium stores [Berridge and Galione, 1988]. Membrane oscillators depend upon trie opening and closing of voltage-dependent calcium channels in the plasma membrane. Examples of such oscillators are sinoatrial node cells and various pacemaker neurones in the brain where oscillations are set. Cytosolic oscillators depend upon the periodic release of calcium from intracellular reservoirs. Such cytosolic calcium oscillators are frequently associated wi th st imuli that act through the phosphoinositide signalling pathway, and they probably reflect the complex feedback interactions responsible for regulating intracellular calcium. Although considerable progress has been made in understanding the mechanism of membrane oscillators, less is known about the cellular basis of the cytosolic oscillators. j Intracellular calcium oscillations can be triggered by a variety !of stimuli. ' : : ; : • • • ; j j Of the natural stimuli (neurotransmitters, hormones, and growth factors), many are calcium-mobilising agents that hydrolyse phosphoinositides to generate both diacylglycerol and IP3 [Berridge, 1987]. The significance of receptor activation is supported by observations that GTP7S can trigger oscillatory activity when injected into hamster eggs or H e L A cells [Berridge and Galionje, 1988]. In both cases, the GTPYS-induced oscillations were different from those -29-• ' • • ' : ! ! : ! ' ! : i | produced by the natural s t imul i of fertil isation or histamine. These experiments, nevertheless, indicate that the activation of a G-protein can initiate oscillatory activity, most l ikely by stimulating the hydrolysis of phosphoinositides. ; ' ! : , : ; , : : i ; ' : : I i Characteristics of Calcium Oscillations I ' '• i i ' i | • I • i Calcium oscillations appear in various forms. Al though they may be specific for any given cell type, they can vary depending upon the agonist. The two major oscillation patterns are: transient and sinusoidal oscillations. Transient calcium oscillations are characterised by a series of discrete spikes separated by quiescent phases when the level of calcium remains close to basal concentrations. Sinusoidal oscillations are calcium fluctuations whereby the : ; : i I oscillatory cycles are continuous with each other and are usually found riding • - • j on the elevated plateau level of calcium. Sinusoidal oscillations also display; a high frequency, that is independent of agonist concentration [Berridge, 1992]. ! Calcium transient profiles remain relatively constant, in spite of agonist-induced changes in frequency. Most calcium profiles may be divided into three ' • • ' ' '• i separate phases: the initial slow pacemaker rise, which then leads into the rapid upstroke of the spike, followed by the recovery phase. Although the1 pattern of the calcium spikes in response to different agents may vary considerably, the calcium transient profile should remain constant for any given cell [Berridge, 1992]. ' j j ! • ; I ' The rapid upstroke of calcium spikes suggests that there is a mechanism for synchronising the indiv idual calcium stores distributed throughout the cytosol. Calcium imaging studies have revealed that each calcium spike has;a • . : . '< • • i i - 3 0 - \ precise spatial organisation. A calcium response is often initiated at one point and then spreads throughout the cell in the form of a wave or tide ; [Berridge, 1990; M i y a z a k i , et al., 1986]J Cur ious ly , there appears to be a loss of synchronisation shortly after the initial response. This is manifested by the rapid dampening of calcium spikes, accompanied by a broadening of the spikes. j Adenosine Triphosphate and Purinergic Agonists ! Adenosine triphosphate is a ubiquitous nucleotide and serves as the principal immediate donor of free energy in biological systems. Intracellular A T P is present in mill imolar concentrations, while the micromolar-nanomolar concentrations of extracellular A T P are maintained by ectonucleotidases and ectophosphafases [Dubyak, 1991). The source of extracellular A T P is thought to be mainly neuronal in origin; either from purinergic terminals or co-released wi th traditional neurotransmitters such as acetylcholine and noradrenaline [Gordon, 1986; More l and Meunier, 1981; Morley, et al., 1994]. Extracellular A T P and its metabolites have been implicated i n a myr iad of biological systems: cardiovascular function [Olsson and Pearson, 1990], neurotransmission • 1 • • i I' [Edwards] et al., 1992], muscle contraction [Satchell, 1990], and insulin secretion ' '• . i . : ' | i [Squires, et al., 1994]. 1 I It has long been established that the ovaries are wel l innervated. The nerves of the ovaries are derivatives of the ovarian plexus and uterine nerves. A l l vessels and nerves enter the ovary through the hilum. Most of the nerves are non-myelinated and sympathetic and supply the muscular coats of arterioles. Some non-myelinated fibres form plexuses around multilaminar follicles. Whether nerves are associated also wi th generalised smooth muscles cells i n the ovary is unknown. A few sensory nerve endings have been described i i i the ovarian stroma. I The purinergic receptors' can be divided into two main categories: F/i purinoreceptors (adenosine receptors), and P2 purinoreceptors (ATP receptors) [Burnstock, 1978]. P i purinoreceptors are more responsive to adenosine arid A M P than to A D P and A T P . P2 purinoreceptors, conversely, are more responsive to A T P and A D P than to A M P and adenosine [Burnstock, 1978; Burnstock and Buckley, 1985; Dalziel and Westfall, 1994]. P2 purinoreceptors are heterogeneous [Burnstock, 1978; Dalziel and Westfall, 1994; Kennedy arid Burnstock, 1985; Kennedy, et al., 1985; White, et al., 1985] subtypes of P2 purinoreceptors characterised thus far include: P2T, P2U/ P2X/ P2Y/ and P2Z [Dalziel and Westfall, 1994]. The P2T/ ^ 2U> and P2Y purinoreceptors are coupled to G-proteiris [Dalziel and Westfall, 1994; Lustig, et al., 1993; Webb, et al., 1993]. : j i The P2X purinoreceptor is an intrinsic ion channel [Bean, 1992]; while the P2Z • . ; • • ' purinoreceptor remains to be fully elucidated [Cockcroft and Gomperts, 1979; Cockcroft and Gomperts, 1980; Dalziel and Westfall, 1994]. i ! ; j i Stimulation of the G protein-coupled P2 purinoreceptor; activates phosphol ipase C and phospha t idy l inos i t ide hydro lys i s , generatirig diacylglycerols and IP3, which activate protein kinase C and mobilisation of intracellular calcium [Berridge, 1984]. Stimulation of the cation channel-coupled P2 purinoreceptors also activate calcium mobilisation. The role of A T P in the human ovary remains equivocal. -32- 1 Objectives The primary objective of this thesis was to examine the role of calcium as messenger in human ovarian cells. Over the last two decades, it has become evident that the concentration of intracellular calcium is critical to the regula-tion of normal cellular activities. Calcium plays a pivotal role in mediating the contraction of muscles, the secretion of exocrine, endocrine, and neurocrine products/ the metabolic processes of glycogenolysis and gluconeogenesis, the transport and secretion of fluids and electrolytes, and the growth of cells [Rasmussen, 1986]. ! ! I Various events occurring over the course of the menstrual cycle are me-diated by the two female sex hormones. The L H / C G and F S H receptors belong to the large gene family k n o w n as the seven transmembrane-guanine nucleotide regulatory (G) protein-coupled receptors [Berridge and Galione, 1988; Loosfelt, et al., 1989; McFarland, et al., 1989; Minegishi , et al., 1993; Minegishi, et al., 1990; Segaloff, et al., 1990; Tsai-Morris, et al., 1990]. It has been established that both the L H / C G and F S H receptors are coupled to the adenylate cyc lase /cAMP pathway [Dufau and Catt, 1978; Hunzicker-Dunn arid Bimbaumer, 1985; Leung and Steele, 1992]. That the hormones mediate various events, suggests that they may also act via other signal transduction pathways; i • i It has long been established that the ovaries are we l l innervated. i Adenosine triphosphate is a ubiquitous nucleotide and serves as the principal immediate donor of free energy in biological systems. Intracellular A T P is present in mi l l imola r concentrations, whi le the micromolar-nanomolar concentrations of extracellular A T P are maintained by ectonucleotidases and i j 1 •; -33-: : i ectophosphatases [Dubyak, 1991]. The source of extracellular A T P is thought to be mainly neuronal in origin; either from purinergic terminals or cb-released wi th traditional neurotransmitters such as acetylcholine and noradrenaline i i [Gordon, 1986; More l and Meunier, 1981; Morley, et al., 1994]. Extracellular • . j ! A T P and its metabolites have been implicated in a myr iad of jbiological ' ^ ! systems: cardiovascular function [Olsson and Pearson, 1990], neurotransmission [Edwards, et al., 1992], muscle contraction [Satchell, 1990], and insulin secretion [Squires, et al., 1994]. The role of A T P in the human ovary remains equivocal. The role of calcium was investigated i n human granulosa-lutein cells (GLCs) acquired from the University of British Columbia In Vitro Fertilisation ' • • ' i Programme. The cells were obtained from women wi th fertility, [ including endocrine, problems, and who recently have received sufficient amounts of h C G to simulate the natural L H surge. As some of the studies involved the monitoring of intracellular calcium concentrations in response to the: activation of gonadotrophic receptors, human embryonic kidney 293 (HEK293) cells transfected wi th wild-type and chimeric gonadotrophic receptors were used in lieu of the human GLCs . The following were studies were conducted: 1. To investigate the possibility that the phospholipase pathway is also coupl-ed to the human L H / C G receptor in human granulosa-lutein cells (GLCs) and in HEK293 cells expressing the human L H / C G receptor. , ; • • : ; I ; . ; . •. I I 2. To investigate the possibility that the phospholipase C pathway is also coupled to the human F S H receptor, in HEK293 cells expressing the human F S H receptor. j -34 -' 3. To investigate the segments of the h L H receptor involved in signal trans-duction, in HEK293 cells expressing the wild-type and chimeric human gonadotrophin receptor. \ 1 ! i 4. To investigate the segments of the hFSH receptor involved in signal trans-1 . 1 • i i duction, in HEK293 cells expressing the wild-type and chimeric human : r gonadotrophin receptor. 5. To investigate the effects of A T P and other purinergic agonists oh intracel-lular calcium signalling in single human G L C s . I ' i 6. To investigate the effects of A T P and other purinergic agonists on steroid i , • ' ; j production in single human GLCs . Materials and Methods I. Reagents and Materials • : 1 : : • ! . i : i ! ! Adenosine diphosphate (ADP) , adenosine monophosphate ( A M P ) , adenosine 5'-o-(3-thiotriphosphate) (ATP7S), adenosine triphosphate (ATP), 4-androstene-3,17-dione, 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-c A M P ) , caffeine, dantrolene, ethylene glycol-bis(fi-aminoethylether) N , N , N ' , N ' -tetraacetic acid (EGTA), human chorionic gonadotrophin (hCG), N,N-bis(2-hydroxyethyl)-2-aminoethanesulphonic acid (BES), N-2-hydroxyethylpipera-' ' : ' • ! i I zine-N-2-ethanesulphonic acid (HEPES), nifidepine, 176-oestradiol, Percoll, ! ' : | ! potassium chloride (KC1), progesterone, prostaglandin F2« (PGF2a)> thapsi-gargin (TPG), verapamil (VP) were obtained from Sigma (St. Louis, M O , U.S.A.). • ! ! 5-bromo-4-chloro-3-indolyl-C-D-galactoside (X-gal), Dulbecco's j Modi f i ed Eagle's M e d i u m ( D M E M ) , Hanks ' balanced salt solution C a 2 + - , M g 2 + - f r e e (HBSS), penicil l in-streptomycin, t rypsin were obtained from Gibco-BRL (Burlington, j O N , Canada). Tritiated oestradiol-17C and progesterone were obtained from Amersham (Oakville, O N , Canada). Heat-inactivated foetal bovine serum (FBS) was obtained from Professional Diagonistics (xxx, xxx, U.S.A.). Fura-2-AM was obtained from Molecular Probes (Eugene, OR, U.S.Al). i i Scintran Cocktai l EX was obtained from Fisher Scientific (Vancouver, B C , : • • ' ' 1 I i Canada). i Rabbit anti-oestradiol-17p and anti-progesterone antisera were obtained ' ' i from Dr. D. T. Armstrong. Human luteinising hormone (hLH) and follicle-stimulating hormone (hFSH) were obtained from N I H (Maryland, U.S.A.). Falcon culture plates (48-wells), 25 mm circular coverglasses, and 12 x 75 ' ' '• I j mm borosilicate glass tubes were obtained from Fisher Scientific (Edmonton, . , : i i A B , Canada). Simport Plastics polyethylene scintallation vials wi th snap-on caps from VWR-Canlab (Edmonton, A B , Canada) II. Human Granulosa-Lutein Cells! • • : j :• The use of human G L C s was approved by the U B C Clinical:Screening Committee for Research and Other Studies Involving Human Subjects. ' ; • I Fol l icular development was stimulated by us ing one of several protocols. One of the more commonly used protocol involved administering! a G n R H analogue to down-regulate pituitary function. Once pituitary down-regulation is achieved, human menopausal gonadotrophin was administered to stimulate follicular growth. Serum oestradiol levels and ultrasound measure-ments of follicular size and number were used as indicators of oocyte maturity. Once at least three follicles exceed 17mm in diameter, 10,000 IU of h C G was administered and oocyte retrieval was performed 34 to 36 hours later. H u m a n G L C s were harvested from the follicular aspirate collected during oocyte retrieval. Harvested human G L C s were centrifuged (1000 g;|5 min) and re-suspended in D M E M containing 2% penicillin-streptomycin (v/y) . The cell suspension was then layered onto a PercolkHBSS (40:60, v / v ) column, and centrifuged (1000 g; 5 min). After centrifugation, cells on the surface of the Percoll:HBSS column were collected and suspended in D M E M . This suspension ; . j ' was centrifuged (1000 g; 5 min) and re-suspended in D M E M containing 5% FBS and 2% penicillin-streptomycin ( D M E M / F B S ) . Cel l viability was determined to be ~ 95% by trypan blue exclusion. ' III. Culture and Drug Treatments ! j < . • : i • ' i 1 : • i i i H u m a n G L C s were seeded onto 48-well plates at a density of 50,000 cel ls /well , and cultured in D M E M / F B S at 37°C in humidified air with 5% C 0 2 . Medium was replaced after the initial 24 hrs, and then every 48 hrs thereafter. The cells were incubated wi th serum-free D M E M at least 6 hrs prior to drug treatments. Cells were cultured for 7 days prior to drug treatment. Treatment periods ranged from 22-26 hrs. Treatments were made up in serum-free D M E M containing androstehedione (0.5 pM). 1 , • .' ' 1 . i ! IV. Radioimmunoassays for Oestradiol and Progesterone Oestradiol content was determined using a classical competitive binding radioimmunoassay. The rabbit anti-oestradiol antisera was raised against 1,3,5 (10)-estratiene-3,17fi-diol-6-one-6-carboxy-methyl-oxime:BSA conjugate (Stera-• • • j ' loids, Wilton, N H ) . This antisera was used at a final dilution of 1:200,000 (v/v) , with approximately 60% binding of label. j ' ' ! • ' •. ; : ; : •! ' : i Progesterone content was determined using a classical competitive ' ' ' ' binding radioimmunoassay. The rabbit anti-progesterone antisera were raised 4-pregnen-6C-ol-3,20-dione hemisuccinate:bovine serum albumin. This antisera was used at a final dilution of 1:10,000 (v/v) , wi th approximately 50% binding of label. ' ! | • • . j ' A. Reagents \ ; • • : \ ' • • • i The assay buffer used was a 0.1 M phosphate buffered saline (PBS; 4.3 m M N a H 2 P 0 4 H 2 0 , 1 1 . 7 m M N a 2 H P 0 4 - 7 H 2 0 , 1 3 m M NaCl , 0.01% (w/v) thime-rasol), supplemented wi th 0.1% gelatin (PBS-G; p H 6.9). ! The tritiated oestradiol (1 | i l) , wi th an initial activity of 1 | i C i / j l L was dissolved in 1 m l of pure ethyl alcohol. The ethanol was evaporated, and the label was then reconstituted in 15 m l PBS-G, yielding -17,000 cpm/100 ul. The tritiated progesterone (1 uT), with an initial activity of 1 [ iCi/nt , was1 dissolved in 1 m l of pure ethyl alcohol. The ethanol was evaporated, and the label was then reconstituted in 15 ml PBS-G, yielding -17,000 cpm/100 \ • • • i The steroid standards were serially diluted with PBS from an initial 0.32 m M stock solution which was reconstituted i n distilled absolute ethanol. A standard curve was set up with 8 reference concentrations ranging from 1 to 128 • : ! ' i n g / m l . | i ' > i 1 ; I The separation reagent comprised charcoal (0.25%, w / v ) and dextran (0.025%, w / v ) in PBS-G. This reagent is prepared 24 hrs prior to the assay, arid was continuously stirred at 4°C. ; ; ' ! i Scintran Cocktail EX was the scintillation cocktail used. ! • : ' . j . I B. Protocol: . \ Standards were assayed i n triplicate, whi le the samples were in duplicate. A l l assays were performed in: 12 x 75 mm borosilicate glass tubes. ' i ! The assays were counted in polyethylene scintillation vials wi th snapron caps.: • . • i • 1. PBS-G was added to all tubes: 300 (il buffer into each of the total counts (TC) and non-specific binding (NSB) tubes; 200 (il into each maximum binding (Bmax) tubes; and 100 Lll into each of the sample and remaining reference tubes. j 2. Diluted antibody solution (100 pi) was added to all tubes except the TC arid NSb* tubes. > . : • ' . ! 3. Tritiated oestradiol (100 |il) was added to every tube in the assay. | 4. A l l tubes were vortex gently, and incubate at 4°C for 16-24 hrs. I j • i ! 5. Oestradiol assay: following the overnight incubation at 4°C, 1 m l charcoal-dextran separating reagent was added to all but the T C tubes. The tubes were gently vortexed and incubated at 4°C for 15 min. Progesterone assay: fol lowing the overnight incubation at 4°C, 0.5 m l charcoal-dextran separating reagent was added to all but the TC tubes. The ; , i : ( tubes were gently vortexed and incubated at 4°C for 15 min. ! ; 6. A l l tubes, except the TC, were centrifuged at 16,000 g for 15 min, at 4°C. A l l tubes were decanted into scintillation vials immediately after centrifuga-tion. I • i 7. Scintillation cocktail (3 ml) was added to all tubes, mixed, and then allowed to equilibrate in the counter (LKB Wallace) for 1 hr prior to counting. j V . Microspectrofluorimetry j Cells were seeded onto 25 mm circular coverglasses and incubated in D M E M / F B S at 37°C in humidified air wi th 5% C 0 2 before microfluorimetric measurements. | | ' . \ • ' ' ' | • j 1 * ! i . . . , , , -40- j j Intracellular calcium concentrations were measured using established fluorimetric techniques [Buchan and Meloche, 1994]. A l l fura-2 ratio measure-ments were performed using the Attofluor™ Digital Fluorescence Microscopy System (Atto Instruments, Rockville, M D , U.S.A.) . The temperature-controlled perifusion chamber was connected to a six channel perifusion system with a flow rate of 1-2 m l / m i n . A l l experiments were completed using the Zeiss 40x i Fluar™ oil immersion objective lens. The cells were il luminated alternately i ! with light at 340 and 380 nm. Measurements of intracellular free calcium levels were collected at 1-2 sec intervals. A l l data presented have been corrected for background fluorescence, as determined from cell-free regions of the cover-glass. Changes in the fluorescence ratio recorded at 340 and 380 nm correspond to changes in cytosolic free calcium. i ; The cells were incubated wi th fura-2-AM loading buffer (5 pM) for 15 min at 37°C in humidified air wi th 5% CO2. The coverglass was mounted onto the temperature-controlled perifusion chamber and equilibrated for 10 min prior to the start of the experiment. Fura-2-loaded cells were perifused wi th a balanced salt solution (BSS; 137 m M N a C l , 5.36 m M KC1, 1.26 m M C a C l 2 , 0.81 m M M g S 0 4 - 7 H 2 0 , 0.34 m M N a 2 H P 0 4 - 7 H 2 0 , 0.44 m M K H 2 P 0 4 ; 4.17 m M N a H C 0 3 , 10 m M HEPES, 2.02 m M glucose; p H 7.4). The treatment intervals ranged from 2-10 min, whereas the wash intervals varied from 2-15 min, depending upon the magnitude of the preceding calcium response. I '.' '•  i ' ; • < ! i V I . Transfection of Human Embryonic Kidney 293 Cells ! • • I • i ' ! • ' ' i I A. Transient Transfection of Human Embryonic Kidney 293 Cells j Human gonadotrophin receptor c D N A was subcloned into the pcDNA3, 1 j ' • • ' • ' i ! ' • ; -41- i vector [Hirsch, et al., 1996; Kudo, et al., 1996] and transiently transfected into 293 cells derived from human embryonic kidney fibroblasts (HEK293) by the calcium phosphate method [Raymond, et al., 1996]. The HEK293 cells were cul-tured until 80% confluency, then trypsinised (0.0625% in calcium- and magne-sium-free HBSS) and re-seeded at a density of 1 x 10 6 cells per 100 mm culture dish. The HEK293 cells were incubated wi th D M E M / F B S at 37°C in luimidifidd air wi th 5% CO2 for 24 hr prior to transfection. Thirty minutes prior to tranis-fection, the HEK293 cells were incubated at 37°C i n humidified air wi th 3% CO2. Ten to twenty micro-grams of c D N A per 100 mm culture dish were used. ... ; ^ '.. i : • '• , I j The 10-20 |xg of c D N A was precipitated with 3 M sodium acetate (1% v / v ) and 100% ethanol (1 ml). The c D N A solution was centrifuged at 4°C at 14,000 rpm for 15 min. The supernatant was discarded and the cells were washed with 1 ml of 100% ethanol. The c D N A was re-suspended in O.lx TE solution (450 and 2.5 m M C a C l 2 (50 uj) and 2X BES (500 [il). Following a 20 min incubation at room temperature, the c D N A solution was introduced into the HEK293 cell culture. ! i I 1 j i Following a 14 hr incubation at 37°C in humidified air wi th 3% CO2, the HEK293 cells were washed twice wi th D M E M and then trypsinised (0.0625% i • ' '• • i 1 trypsin), as aforementioned. The cells were centrifuged, re-suspended in D M E M / F B S , and seeded onto 25 mm circular coverglasses. The transiently transfected cells were assayed 45-80 hr post-transfection. ; ! B. Transfection Efficiency ! | To monitor transfection efficiency, the RSV-C-gal plasmid was routinely , [ j 1 included in the transfection mixture, and C-galactosidase activity was deter-mined by X-gal staining. Transfected HEK293 cells were washed wi th phos-- 4 2 -phate buffer solution (PBS), incubated at room temperature wi th fixative for 15 min, and washed again (see Appendix B for formulation for PBS and fixative). The fixed cells were then incubated at 37°C wi th the X-gal stain for approxi-mately 12 hrs. ; I Results Figure 4 shows that h C G does evoke calcium oscillations in human G L C s cells. As aforementioned, human G L C s were obtained from the U B C In Vitro Fertilisation Programme. The cells were obtained from women wi th fertility, including endocrine, problems, and who have recently received pharmacologi-cal doses of h C G to simulate the natural L H surge. To facilitate the study of LH-induced intracellular calcium mobilisation, human wild-type ancl chimeric receptors where transfected into HEK293 cells. I I. Gonadotrophin-Induced Calcium Oscillations in HEK293 Cells Express-irig the Human Luteinising Hormone/Chorionic Gonadotrophin Receptor ; • • , . i A. Specificity of the Human LH/CG Receptor ; • ' i i . "; i ! We have examined the effects of gonadotrophins in transfected HEK293 cells using single-cell dual-excitation microfluorimetry. The control groups were untransfected HEK293 cells, and HEK293 cells transfected wi th lac-Z c D N A and/or p c D N A 3 plasmid (Table 1). Gonadotrophin treatment failed to elicit cal-cium signals in all four control groups. Figure 5 shows the specificity of the human L H / C G receptor. Both human F S H and L H were administered at a dose of 4 H g / m l for a duration of 180 sec. Human F S H failed to elicit ia calcium response from the transfected cells (n=42, #=2). Under j the same conditions, human L H consistently evoked oscillatory calcium signals (n=42, • . i j #=2). The on-set of the [Ca 2 + ] i oscillations was rapid, wel l within 15 sec of the L H treatment. ! ! - 4 4 -1.0n E c o 00 CO O 0 . 5 CO (0 DC O.O-I 180 sec hCG(10 IU) Figure 4: Effects of h C G treatment on human GLCs . Single-cell microfluorime-tric studies demonstrated that h C G successfully evoked mobilisation of intracellular calcium in human G L C s . The cells were loaded wi th Fura-2-AM, and perifused wi th a balanced salt solution. A l l micro-fluorimetric studies were conducted i n a temperature-controlled (37°C) chamber. The agonist was administered at a concentration of 10 I U / m l , for a duration of 180 sec. 1 •45-control groups cells imaged (n) I number of transfectibns (#) ' HEK293 : i • 57 '' 2 1 j HEK293/fi-gal 1 45 2 HEK293 /pcDNA3 ; 72 2 | ' | HEK293/ f i -ga l /pcDNA3 | 42 1 ! ' 1 ! i Table 1: Mobil isat ion of intracellular calcium i n response to gonadotrophin treatment was investigated in HEK293 cells transfected wi th gonado-trophic receptors. Several control groups were established to demon-strate that the intracellular calcium response was due to activation of the transfected gonadotrophic receptors. The control groups were all treated wi th human F S H and L H (2-4 | l g / ml) for a duration of 180 sec. Figure 5: Effects of gonadotrophin treatment on human L H receptors'expressed in HEK293 cells. Only h L H was capable of eliciting an intracellular calcium response. The cells were loaded wi th Fura-2-AM; and peri-fused wi th a balanced salt solution. A l l microfluorimetric studies were conducted in a temperature-controlled (37°C) chamber. Trans-fected cells were treated wi th both human F S H (4 (ig/ml) and L H (4 j i g / m l ) for a duration of 180 sec. ! 0.0 J Figure 6: Human C G concentration-response relationship. Human C G was administered at the various concentrations for a duration of 180 sec. The calcium oscillations lasted throughout the entire treatment period, arid persisted for at least 25 min after the cessation of L H treatment. B. Effect ofHuman Chorionic Gonadotrophin on [Ca2+]i Figure 6 shows the concentration-response relationship between h C G and [Ca 2 + ] j . Human C G was administered at 1, 5 and 10 I U / m l , for a duration of 180 sec. A t 1 and 5 I U / m l (n=54 and 10, respectively; #-2 and 1, respectively) h C G elicited baseline calcium oscillations which were; sustained even after treatment withdrawal . A t 10 I U / m l , h C G evoked a rise i n [ C a 2 + ] i , w i th oscillations superimposed on the quasi-sustained plateau phase (n=81, #=3). The cessation of the oscillations is l ikely due to the depletion of internal calcium stores. ! • ! ! C. Calcium Influx vs. Calcium Mobilisation \ j To determine the relative contribution of calcium influx vs. calcium mobilisation of cytosolic stores i n the init iation and maintenance of the gonadotrophic response, h C G was administered in the absence of extracellular calcium. Under calcium-containing conditions, h C G (1 I U / m l ) reproducibly evoked calcium oscillations, sustained even after treatment withdrawal (Figure 6). Under calcium-free conditions, in the presence of 1 m M E G T A , h C G still evoked calcium oscillations, but the response now was transient (n=64, #=2; Figure 7). The second calcium 1 elevation in Figure 7 is due to the influx of extracellular calcium into the cell following a return to calcium-containing conditions. CO 0.5J hCG (1 lU/ml) Figure 7: The invo lvement of extracellular ca lc ium on h C G - e v o k e d calc ium : . • | i mobilisation. In the absence of extracellular calcium, the hCG- induc -ed calcium response was not sustained beyond the treatment period. The calcium-free buffer contained 1 m M E G T A . The cells were loaded wi th Fura -2 -AM, and perifused wi th a balanced salt solution. A l l microfluorimetric studies were conducted in a temperature-control-led (37°C) chamber. ! j ! - 5 0 -3.0-2.0-1 o 00 CO • • o CO o ~ 1.0> CO CC o.oJ 180 sec TPG (1 nM) h C G ( 1 , u / m | ) Figure 8: Effect of thapsigargin (TPG) pre-treatment 1 on transfected HEK293 cells. Human C G fails to elicit intracellular calcium mobilisation when cells are depleted of their endoplasmic reticular calcium stores. The cells were loaded wi th Fura-2-AM, and perifused wi th a balanced salt solution. A l l microfluorimetric studies were conducted inja temperature-controlled (37°C) chamber. 51 -To identify the internal calcium stores mobil ized in the LH-evoked calcium response, transfected HEK293 cells were pre-treated wi th thapsigargin (n=83, #=2). Thapsigargin is a plant-derived lactone, whose mode of action appears to result from the emptying of intracellular calcium jstores by inhibi t ing • sequestration pathways [Thastrup, et al., 1989]. Thapsigargin specifically inhibits al l members of the endoplasmic and sarcoplasmic reticulum calcium pump family [Lytton, et al., 1991]. Following thapsigargin pre-treatment (1 ±iM), h C G failed to elicit a calcium response (Figure 8). The cells in this, and all experiments, were co-transfected wi th fi-gal c D N A ; ergo, the presence of the human L H / C G receptor was indirectly determined by X-gal staining. .; \ : To determine the involvement of intracellular IP3-sensitive calcium stores in the hCG-evoked calcium signals, caffeine was used. H i g h conceh-; • • ' • i trations of caffeine have been shown to inhibit the mobilisation of noh-' ; • • ! I. mitochondrial, IP3-sensitive calcium stores [Toescu, et al., 1992]. In Figure 9, h C G treatment produces the usual oscillatory calcium signals; the introductibn of 20 m M caffeine eradicates the calcium oscillations to almost baseline levels. The wi thdrawal of caffeine resulted i n an elevation in [ C a 2 + ] i , but the oscillations are not restored (n=33, #=2). | To determine whether the human L H / C G receptor is coupled to calcium signalling through the Gj-protein, transfected HEK293 cells were pre-treated wi th pertussis toxin (PTX). Fol lowing a 16 hr pre-treatment with! PTX (100 ng/ml) , h C G failed to elicit a calcium response (n=163, #=4). Again, the cells were co-transfected wi th fi-gal c D N A ; ergo, the presence of the human L H / C G receptor was indirectly determined by X-gal staining. j ! 1 ' '• ' i ' I ; : ' : - 52 - I ' i 270 sec caffeine (20 mM) 0.0J Figure 9: Effect of caffeine on hCG-evoked calcium signals in transfected HEJC 293 cells. H i g h concentrations of caffeine (20 mM) inhibits; the mobi-lisation of non-mitochondrial, IP3-sensitive calcium stores.] The cells were loaded wi th Fura-2-AM, and perifused wi th a balanced salt solu-• j I tion. A l l microfluorimetric studies were conducted in a temperature-controlled (37°C) chamber. ] | | -53-II. Calcium Signalling in HEK293 Cells Transfected with the Wild-Type or i Chimeric Human Gonadotrophin Receptors j The twelve chimeric and the two wild-type gonadotropin* receptors were individual ly transfected into HEK293 cells. Gonadotrophin j treatment failed to elicit calcium mobilisation in five of the fourteen receptor types (Table 2): FFR (n=212, #=5); LFR (n=189, #=4); LF(5-C)R (n=177/#=3); FL(1-4)FR (n=202; ' ' I #=4); FL(i3-yi)FR (n=143, #=4).; Various agents used in the e x p e r i m e n t s were dissolved i n ' D M S O (20%, v / v ) . Figure 10 shows that the vehicle, D M S O , did not elicit a calcium response in the transfected HEK293 cells (Figure 10; n=87, #=2). . , , ! ; . ; • . ; I A. Phospholipase C Involvement in Gonadofrophin-Induced Calcium Responses j ' • • ! ! I To determine whether adenylate cyclase plays a role in gonadotrophin-stimulated intracellular calcium mobilisation, HEK293 cells transfected wi th 1 i i' either the wild-type human L H receptor or the chimeric human gonadotrophin • , ' ! ' ; - . ] j 1 receptor FLR were treated with 50 |nM forskoHn, an adenylate cyclase stimula-tor. Figures 11 and 12 show that forskolin failed to elicit intracellular calcium signals in HEK293 cells transfected with either the wild-type human ; L H recep-tor (Figure 11; n=98, #=2) or the chimeric human gonadotrophin receptor FLR (Figure 12; n=93) #=3). Conversely, U-73122 (10 uM), a phospholipase C activa-tor, was clearly shown to degrade hCG-induced intracellular calcium mobilisa-tion (Figure 13; n=107, #=3). ' j j Table 2A: Wi ld - type and chimeric human gonadotrophin receptor schematics and detectability of intracellular calcium mobilisation. Ca lc ium re-sponse results are from the experiments documented in this section. FSH receptor region LH receptor region Receptor Schematic Calc ium Response F F R /•> V S / ~ \ w w v > ^--^ no F(1-4)LR yes FL(1-4)FR no FL(7-C)R ^ r\ r\ r\ yes - 5 5 -Table 2B: Wi ld - type and chimeric human gonadotrophin receptor schematics and detectability of intracellular calc ium mobil isation. Ca l c ium re-sponse results are from the experiments documented in this section. FSH receptor region LH receptor region Receptor Schematic Calcium Response FL(C)R yes FL(i3-VI)FR no FL(V-i3)FR yes FL(V-VI)R yes FL (V/V I )R i l l yes - 5 6 -Table 2C: Wi ld - type and chimeric human gonadotrophin receptor schematics and detectability of intracellular calc ium mobilisation. Ca lc ium re-sponse results are from the experiments documented in this section. FSH receptor region LH receptor region Receptor Schematic Calcium Response F L R yes LF(5-C)R no LF(C)R yes LFR r\ /*\ 1 1 1 : 1 1 1 1 1 I W M •!: i Ii; W 1 0; m :M no L L R yes - 5 7 -B. Effect of Gonadotrophin on Chimeric Hu?nan Gonadotrophin Receptors ; j LF(C)R (Figure 14; n=131, #=3): the intracellular carboxy-terminal of the human L H receptor has been replaced wi th that of the human F S H receptor. • ' : : ' I ' ' ' ' ' • i I This alterations results in an altered hCG-induced calcium profile. The calcium oscillations are lost and the signal is only sustained for the duration of the gonadotrophin treatment. Porcine F S H (40 p.g/ml) fails to elicit la calcium response from this chimeric receptor. FLR (Figure 15 and 16; n=292, #=5): the transmembrane and i n t r a c e l l u l a r portions of the human F S H receptor has been replaced wi th that of the human L H receptor. The FSH-induced calcium profile is less consistent! than that observed i n the wild-type receptor. There is a marked hysteresis in the various calcium profiles of these altered human F S H receptors. Human C G (10 IU/ml ) failed to elicit a calcium response. j I • ; . . !• i j : i-FL(C)R (Figure 17; n=156, #=3): the intracellular carboxy-terminal of the human F S H receptor has been replaced wi th that of the human L H receptor. Porcine F S H (40 ug/ml) elicits a single calcium spike. The calcium oscillations and sustained calcium mobilisation is not evident in the FSH-induced calcium responses for this receptor. ] F(1-4)LR (Figure 18; n=143, #=3): the latter segment (from part of extracellular loop two to the end of the carboxy-terminal) of the human F S H receptor has been replaced wi th that of the human L H receptor. Porcine F S H (40 | i g /ml ) elicits a 'similar calcium profile to that normally observed i n the human L H / C G activation of the wild-type human L H receptor. 58-I 3.<H c2.<H o 00 CO o CO, DC o.o-« 180 sec pFSH (40 ng/ml) DMSO pFSH (20% v/v) (40 ng/ml) Figure 10: Effects of pFSH and D M S O on HEK293 cells transfected wi th the chimeric human gonadotrophin receptor FLR. Porcine F S H (40 pg/ml) and D M S O (20%, v / v ) were both administered for a du-ration of 180 sec. The cells were loaded wi th Fura-2-AM, and i perifused wi th a balanced salt solution. A l l microfluorimetric studies were conducted i n a temperature-controlled (37°C) chamber. - 5 9 -2.0n E c o 00 CO o 1.0-1 CO to cc 0.0-1 180 sec forskolin ( 5 0 L I M ) hCG (10 IU/ml) A T P (10ixM) Figure 11: Effecta of forskolin treatment on HEK293 cells transfected wi th the wild-type human L H receptor. Forskolin, an adeny-late cyclase stimulator, was administered at a concentration : of 50 | i M for a duration 360 sec, while h C G was administered at 10 I U / m l for a duration of 180 sec. The cells were loaded with Fura-2-AM, and perifused wi th a balanced salt solution. • A l l micro-fluorimetric studies were conducted in a teinpera-: ture-controlled (37°C) chamber. -60-3.GS 12.0-1 o 00 CO o CO o (0 GC 0.0J 180 sec forskolin (50 \i.M) pFSH (40 fxgVml) A T P : (10 M-M) Figure 12: Effects of forskolin treatment on HEK293 cells transfected • , i : i i with the chimeric human gonadotrophin receptor FLR. Fors-kolin, an adenylate cyclase stimulator, was administered at a concentration of 50 p:M for a duration 360 sec, while h C G was administered at 10 I U / m l for a duration of 180 sec. The cells ; were loaded wi th Fura-2-AM, and perifused wi th a balanced salt solution. A l l microfluorimerric studies were conducted in a temperature-controlled (37°C) chamber. j -61-i 0 0-1 hCG U73122 ATP j | (10 IU/ml) (10|iM) (10jiM) | | : I i • : •• • I Figure 13: Effects of U-73122 treatment on HEK293 cells transfected wi th ; the wild-type human L H receptor. U-73122, a P L C stimula- j tor, was administered at a concentration of 10 ( iM for a dura- ; tion 360 sec, while h C G was administered at 10 I U / m l for a duration of 180 sec. The cells were loaded wi th Fura-2-AM, : ; . . . i 1 and perifused wi th a balanced salt solution. A l l microfluori-metric studies were conducted in a temperature-controlled (37°C) chamber. ! ; • i - 6 2 -3.0 I2.0H o 00 CO o CO o.o-J 180 sec pFSH (40 jig/ml) hCG (10IU/ml) A T P (10ixM) Figure 14: Effects of gonadotrophin treatment on HEK293 cells trans-fected wi th the chimeric human gonadotrophin receptor LF(C)R. Porcine F S H (40 | ig /ml ) and h C G (10 I U / m l ) were both administered for a duration of 180 sec. The cells were loaded wi th Fura-2-AM, and perifused wi th a balanced salt solution. A l l microfluorimetric studies were conducted in a i " temperature-controlled (37°C) chamber. j ; -63-I2.0H o CO CO © CO 0.0J hFSH (0.2 Lig/ml) ATP (10 nM) 180 sec hFSH (0.2 ng/ml) ATP (10 nM) Figure 15: Effects of gonadotrophin treatment on HEK293 cells; trans-fected wi th the chimeric human gonadotrophin receptor FLR. Human F S H was administered at a concentration of 0.2 (ng/ ml for a duration of 180 sec. A l l microfluorimetric studies were conducted in a temperature-controlled (37°C) chamber. - 6 4 -FL(7-C)R (Figure 19; n=97, #=2): the third extracellular loop, the seventh transmembrane segment, and the carboxy-terminal of the human F S H receptor have been replaced with those of the human L H receptor. F S H is still capable of elicit ing a calcium response, but the sustained oscillatory pattern i n essentially lost, although a few oscillations may occasionally be observed. There is also a marked hysteresis in the calcium responses in these receptors, j FL(V-i3)FR (Figure 20; n=174, #=3): part of the second extracellular loop, the fifth transmembrane segment, and the third intracellular loop of the human F S H receptor have been replaced with those of the human L H receptor. F S H is still able to elicit calcium transients in HEK293 cells transfected ; w i th this chimeric receptor; however, the calcium transients are severely attenuated, and no oscillations were observed. ! [ \ F L ( V / V I ) R (Figure 21, n=173, #=3): the fifth and sixth transmembrane .; • i . ' | ' i segments of the human F S H receptor have been replaced by those of the human L H receptor. F S H is again able to elicit calcium transients i n HEK293 cells transfected wi th this chimeric receptor; however, the calcium response is attenuated. i j • ' I ! FL(V-VI)R (Figure 22; n=167, #=3): the fifth and sixth transmembrane • : I |. segments and the third intracellular loop of the human F S H receptor lhave been replaced by those of the human L H receptor. The ligand-induced calcium mobilisation profile is similar that normally expected for hCG-induced calcium mobilisation; however, the calcium oscillations have 1 j 2.0 E c o 00 CO o 1.0-1 CO CO rr 0.0 J 180 sec ^ ^ ^ ^ hCG (10 IU/ml) pFSH (40 ng/ml) A T P (10 uM) | Figure 16: Effects of gonadotrophin treatment on HEK293 cells trans-'• fected with the chimeric human gonadotrophin receptor FLR. Porcine FSH (40 jig/ml) and hCG (10 IU/ml) were adminis-tered for a duration of 180 sec. The cells were loaded with • • . i • Fura-2-AM, and perifused with a balanced salt solution. A l l microfluorimetric studies were conducted in a temperature-controlled (37°C) chamber. ! | i -66-2.0-1 180 s e c o CO CO oi.oH CO CO 0 . 0 J pFSH (40 \ig/m\) pFSH (40 (ig/ml) A T P ! (10M.M) Figure 17: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor FL(C)R. Porcine FSH was administered at a concentration of 40 jLLg/ml for a duration of 180 sec. The cells were loaded with Fura-2-AM, and perifused with a balanced salt solution. A l l microfluorimetric studies were conducted in a tempera-ture-controlled (37°C) chamber. j - 6 7 -2.0-, 180 sec E c o 00 CO O1.0 CO CO DC o.o-i pFSH (40 fig/ml) pFSH (40 jig/ml) ATP I (10 nM) Figure 18: Effects of gonadotrophin treatment on HEK293 cells trans-fected with the chimeric human gonadotrophin receptor F(l -4)LR. Porcine FSH was administered at a concentration of 40 |Lig/ml for a duration of 180 sec. The cells were loaded with Fura-2-AM, and perifused with a balanced salt solution. A l l microfluorimetric studies were conducted in a temperature controlled (37°C) chamber. j - 6 8 -| 3.0-, c2.0H o CO CO o 0.0 J 180 sec pFSH (40 \ig/m\) pFSH (40 |ig/ml) ATP (10 uM) Figure 19: Effects of gonadotrophin treatment on HEK293 cells trans-fected wi th the chimeric human gonadotrophin receptor FL(7-C)R. Porcine F S H (40 pg/ml) and h C G (10 IU/ml ) were administered for a duration of 180 sec. The cells were loaded wi th Fura-2-AM, and perifused wi th a balanced salt solution. A l l microfluorimetric studies were conducted i n a tempera-ture-controlled (37°C) chamber. i j -69 -4.0-, c o 00 CO 2 2 - 0 CO (0 o.o-J 180 sec pFSH (40 M.g/ml) pFSH (40 Lig/ml) A T P (10 \iM) Figure 20: Effects of gonadotrophin treatment on HEK293 cells trans-fected wi th the chimeric. human gonadotrophin receptor FL(V-i3)FR. Porcine F S H was administered at a concentra-tion of 40 |LXg/ml for a duration of 180 sec. The cells were loaded wi th Fura-2-AM, and perifused wi th a balanced salt solution. A l l microfluorimetric studies were conducted in a temperature-controlled (37°C) chamber. ] i i 2.0-, E c o CO CO o1.CN CO DC 0.0J 180 sec pFSH (40 (ig/ml) pFSH (40 |xg/ml) A T P (10 uM) Figure 21: Effects of gonadotrophin treatment on HEK293 cells transfected wi th the chimeric human gonadotrophin receptor F L ( V / V I ) R . Porcine F S H was administered at a concentration of 40 p g / m l for a duration of 180 sec. The cells were loaded wi th Fura-2-A M , and perifused with a balanced salt solution. A l l microfluo-rimetric studies were conducted in a temperature-controlled (37°C) chamber. 1 j - 7 1 -2.0n E c o CO CO 6 1.0 CO (0 CC 0.0 180 sec pFSH (40 jig/ml) pFSH (40 pig/ml) A T P (10|iMJ Figure 22: Effects of gonadotrophin treatment on HEK293 cells transfected : wi th the chimeric human gonadotrophin receptor FL(V-VI)R. Porcine F S H was administered at a concentration of 40 j i g / m l for a duration of 180 sec. The cells were loaded wi th Fura-2-A M , and perifused with a balanced salt solution. A l l microfluo-rimetric studies were conducted i n a temperature-controlled (37°C) chamber. ; ! III. P2-Purinoreceptor Agonist-Evoked Calcium Oscillations in Single Human Granulosa-Lutein Cells We have examined the effects of purinergic receptor agonists A T P , A D P , A M P , adenosine, U T P , and the non-hydrolysable analogue ATPyS on intracellular calcium concentration over a range of concentrations (1-100 p,M) in isolated human G L C s , using the techniques of single-cell dual-excitation microfluorometry. The data presented are representative of the changes in intracellular calcium, and are reported as the total number of cells imaged (TC) and number of patients (n) for each protocol. j •. ' ! ; ! • ' ! I i ! : '" i • . ! ' . ' • ! ' . ! i ' . ' ••• j ' ' I j A. Effects of purinergic receptor agonists on intracellular calcium concentrations ' J A T P consistently evoked a marked increase in cytosolic calcium (TC=75P, n=l l ) . A s shown in Figure 23, above micromolar levels, the response to A T P was concentration dependent. No change in intracellular calcium concentration was observed at submicromolar concentrations, whils t the plateau phase produced by 100 u M A T P exhibited partial run-down; a phenomenon consistent wi th desensitisation at the level of the receptor. The desensitisation jeffect was independent of the order of administration. The patterns of intracellular calcium rises were generally characterised as either non-oscillatory (25%; TC=187, n = l l ) , or oscillatory calcium transients (75%; TC=5j63, n = l l ) originating from a plateau of elevated intracellular calcium (Figures 23 and 24). In a efficacy profile experiment of UTP, A T P , A D P , A M P , and adenosine (Figure i | 24), cells were exposed to 10 ( iM concentrations (TC=75, n=4). The data in Fig- j Figure 23: : A T P concentration-response relationship. Submicromolar concen-trations of A T P were incapable of calcium mobilisation.! Note the oscillatory pattern at 10 | i M A T P . The cells were loaded with Fura-2 - A M , and perifused wi th a balanced salt solution. A l l microfluori-metric studies were conducted in a temperature-controlled (37°G) ! ! i chamber. 2.0-O C <D ; o c3 1.0-M 0 . 5 ' o.o. a •X-Adenosine AMP ADP A T P U T P Figure 24: Upper panel: Efficacy profile of various purinergic agonists. A l l agonists were.used at a concentration of 10 | i M . j Lower panel: Comparison of the relative potencies; of the various P 2 u agonists, (a^b^c, p < 0.05). •75-0.4 ed •' <u ; O G' CD O t« 0). o 0.2 u • a-u 0.0 U T P A T P A T P y S Figure 25: Upper panel: Effects of ATPyS, a non-hydrolysable A T P ana-logue, on hGLCs. Agonists were used at a concentration of 10 • M M - I I Lower panel: Comparison of the relative potencies; of the various purinergic agonists. (a*b, p < 0.05). 76 Ca 2 + (1 .3mM) | C a 2 + -free . Ca 2 + (1.3mM) ATP ATP ATP Ca2+-free Ca2+(i.3mM) Ca2+-fiee Figure 26: Upper panel: The involvement of extracellular calcium on ATP: ", .] -induced calciunY mobilisation. A T P was used at a concentra-; tion of 10 p M . The calcium-free buffer contained 1 m M E G T A . | : : i l i Lower panel: The involvement of extracellular calcium on! ATP-induced calcium mobilisation. A T P was used at a concen-: tration of 10 p;M. The calcium-free buffer contained 1 mM; EGTA. -77-ure 24 shows that while 10 [iM A T P evokes a substantial signal, there is no effect of either A M P or adenosine. Under the same experimental conditions, A D P (10 [iM) consistently evoked smaller changes i n intracellular calcium concentration than A T P (TC=50, n=4), whilst UTP was equipotent (TC=75, n=4):. ; ' ' • . j B. Effects of ATPyS on intracellular calcium concentrations \ Figure 25 is a representative profile of the, effects of ] the noh-hydrolysable analogue ATP7S on intracellular calcium levels in human G L C s (TC=75, n=4). The response to 10 |LlM ATP7S mirrors that evoked by; A T P both in the time course of the onset of the response and the oscillatory nature of the sustained plateau phase. The amplitude of the change in calcium is comparable for both A T P and ATPyS. Figure 25 Lower panel shows that the effects of ' ' • I i ATP7S are greater than those of A T P and UTP. i i . • . . • i ' . • ' ! : I ; ; • : \ • \ • j C. Calcium-influx vs. calcium-mobilisation \ i In order to determine the relative contribution of calcium-influx vs. calcium-mobilisation from cytosolic stores in the initiation and maintenance of the purinergic response, A T P was added in either the presence or absence of extracellular calcium. Under calcium-containing conditions, A T P (10 [iM) reproducibly evokes a sharp rise in cytosolic calcium, which is maintained as either an oscillatory (Figure 26 Upper panel) or smooth (Figure :26 Lower panel) plateau in the continued presence of the agonist. In calcium-free experiments, i n the presence of the selective calcium chelator E G T A (1 mM), A T P evokes an initial rise i n intracellular calcium levels, but the response is now transient, returning to basal levels in the continued presence of A T P . 1.5 -, 0.0-1 2.0 n 120 sec Figure 27: Upper, Middle , and Lower panels: Effects of verapamil (VP) oh ATP-st imulated calcium mobilisation. Reagents were used at a concentration of 10 p M . j -79-' I i 5 - i 1.0 A o 0 0 r o o c-> 0.5 cd 120 sec o.o-i 2 .0- , 1.5 A o 0 0 m o 1.0-1 m 0.0 J 120 sec PGF 2a ATP PGF 2a ATP Figure 28: Upper panel: Effects of P G F 2 a and A T P on intracellular cal-cium mobilisation. Reagents were used at a concentration of 10 p M . | Lower panel: Effects of PGF2 a and A T P on intracellular cai- j cium mobilisation in human G L C s pretreated wi th PTX (100 j ng /ml) for 18 h prior to experiment. P G F 2 a and A T P were j used at a concentration of 10 p,M. ' . i I 8 0 However, note how the response still exhibits oscillations (TC=60, n=3; Figure 26 Upper panel). | • To determine whether V D C C were involved in the influx component of the A T P evoked change, we used the broad acting blocker, verapamil (10 (iM), in an attempt to inhibit the plateau phase of the response. As shown in Figure 27 Upper panel (TC=18, n=4) and 27 Middle panel (TC=36, n=4), verapamil was : ' ; j able to block the maintained phase of the response in 33% of the cells. In addition, verapamil d id not prevent an ATP-evoked rise in cytosolic calcium when added prior to the P2-purinergic receptor agonist (TC=75 and 75, n=4 arid 3; Figure 27 Lower panel). j j • : ' "' ! ' j, ' ' i t I ! \ D. Pertussis Toxin Pre-treatment To determine whether the A T P evoked calcium response was coupled to a PTX sensitive G protein, we pretreated the cells wi th PTX (100 ng/ml) for 18 ' ' • • • \ • \ h. PTX failed to alter the profile of the A T P evoked calcium response (TC=75, n=3; Figure 28 Lower panel). PGF201 was used as the control in determining the i ' effectiveness of PTX. , j ! ; j 1 ! i 1 ' '. ' ' ' ' '• j E. Effects of purinergic receptor agonists on steroid secretion \ : •- • i j Human G L C s were treated after a 7 day incubation. The data presented are representative of the steroidal responses elicited by the various reagents; the data are presented in this manner because the basal steroidal concentrations varied, at time considerably, amongst the patients. ] I Figure 29: Effects of purinergic agonists on basal oestradiol production in human G L C s . The cells were incubated wi th serum-free D M E M at least 6 hrs prior to drug treatments. Cells were cultured for 7 days prior to drug treatment. Treatment periods ranged from 22-26 hrs. Treatments were made up in : serum-free D M E M containing androstenedione. (ayb, p > ' o.o5) ; 82-o o o c CD < o o Q_ Q. < + + o o Figure 30: Effects of purinergic agonists on hCG-stimulated oestradiol production in human G L C s . The cells were incubated wi th serum-free D M E M at least 6 hrs prior to drug treatments. Cells were cultured for 7 days prior to drug treatment. Treatment periods ranged from 22-26 hrs. Treatments were made up in serum-free D M E M containing androstenedione. (a*b, p < 0.05) j -83 -400 m o300 c g200 o o c O i -0 (/> Q) O) O 100J Figure 31 Effects of purinergic agonists on basal progesterone produc-tion in human G L C s . The cells were incubated wi th serum-free D M E M at least 6 hrs prior to drug treatments.' Cells were cultured for 7 days prior to drug treatment. Treatment periods ranged from 22-26 hrs. Treatments were madje up in serum-free D M E M containing androstenedione. (afb, p > 0.05) j I -84-Figure 32: Effects of purinergic agonists on hCG-induced progesterone production in human G L C s . The cells were incubated wi th serum-free D M E M at least 6 hrs prior to drug treatments. Cells were cultured for 7 days prior to drug treatment. Treatment periods ranged from 22-26 hrs. Treatments were made up in serum-free D M E M containing androstenedione. : (a*b, p < 0.05) I -85 -1 I . : • • . ' ! • j : [ i ; To determine whether purinergic agonists have any effect on basal steroid secretion, human G L C cultures were treated wi th 1 [ iM concentrations • • • I j of adenosine, A M P , A D P , A T P , and UTP. The cells were incubated wi th serum-; • ' • i ! free D M E M at least 6 hrs prior to drug treatments. Cells were cultured for!7 days prior: to drug treatment. Treatment periods ranged from 22-26 hrs. i • i i Treatments were made up in serum-free D M E M containing androstenedione. The purinergic; agonists d id not appear to have any effects on basal steroid production, nor: on hCG-stimulated steroid production (Figures 29-32). \ i j -86-l Discussion I. Gonadotrophin-Induced Calcium Oscillations in HEK293 Cells Express-ing the Human Luteinising Hormone/Chorionic Gonadotrophin Receptor • To bur knowledge, this is the; first report of sustained calcium ' • ' . ' • • ' • i. oscillations in response to the activation of human L H / C G receptors. Previous studies have reported L H / C G - i n d u c e d calcium elevations in Xenopus oocytes [Gudermann, et al., 1992b] and in HEK293 cells transfected with the rat L H / C G • • • . | .. | receptor [Lakkakorpi and Rajaniemi, 1994]. The type of calcium! response elicited by the activation of the rat L H receptor was dependent upon hormone concentration and the presence of extracellular calcium. Lakkakorpi et al. [Lakkakorpi and Rajaniemi, 1994] observed calcium oscillations in 72% of the cells in the presence of extracellular calcium, and only 33% of cells in the absence of extracellular calcium. We observed human CG-induced calcium oscillations in all HEK293 cells expressing the human L H receptor, the pattern ' . ' ! i and frequency of oscillation altered wi th increasing concentrations of the hormone. ! In the absence of' extracellular calcium oscillations jwere stil l • ' ' •'• • • i • i observed although the frequency was reduced. Thus is appears that the human L H receptor affects intracellular calcium levels in a manner significantly : I 1 1 different to the rodent receptor. Hirsch et al. [Hirsch, et al., 1996] have ' i l l previously reported that HEK293 cells transiently transfected wi th the humaln : ; . • • . • • I i L H / C G receptor do exhibit human LH-induced elevations in intracellular IP3 andcAMP. j j • •1 j j The induction of intracellular oscillations by transfection of the human L H receptors into HEK293 cells was similar to the effect of human C G on human granulosa cells maintained in short term culture. These results indicate that the coupling of the human L H receptor to both adenylate cyclase- and IPs-mediated oscillations is a response of the normal target cells as we l l as transfected cell lines. Thyrotrophin, structurally related to L H . FSH; and h C G , appears to share the ability to activate multiple signaling pathways wi th L H / h C G . Treatment of human thyroid cells wi th thryotrOphin results i n the generation of calcium oscillations [D'Arcangelo, et al., 1995]. The thyrotrophin receptor is wel l known to couple to the adenylate cyc lase /cAMP cascade inja manner similar to L H and FSH, however, thyrotrophin has been reported to stimulate the inositol phosphate/calcium signaling pathway i n primary cultures of thyroid cells, thyroidal cell lines, and transfected cell lines [Corda and Kohn, 1986; D'Arcangelo, et al., 1995; Hidaka, et al., 1994]. D'Arcangelo et al. [D'Arcangelo; et al., 1995] These results suggest that the inositol phosphate/ •; . • ' . • •: ' i • • . !' intracellular calcium cascade plays an integral role i n the complex signal transduction pathways in both gonadal and thyroidal cells. I I ; ; • .: • I i Intracellular calcium oscillations were first described by Prince arid Berridge [Prince and Berridge, 1972], but not appreciated unti l Woods et al. [Woods, et al., 1986] described the linkage between surface membrane receptors • : ' - : : i j and cellular function. Calcium oscillations are involved in the potentiation of j a l igand response [Alkon and Rasmussen, 1988; Berridge and Galione, 1988]. Calc ium oscillations have been reported following the activation |Of several receptors in the reproductive system, notably P2-purinoreceptor in human ovarian cells [Lee, et al., 1996; Squires, et al., 1997] and G n R H receptor in gonadotrophs [Stojilkovic and Catt, 1995]. Gonadotrophin releasing hormone and endothelin-1 induce biphasic intracel lular ca lc ium transients i n gonadotroph cell suspensions (measured using the cuvette technique), but -88-oscillatory intracellular calcium responses in single gonadotrophs [Stojilkovic and Cart, 1995]. The pattern of oscillations reported in the latter study resemble : " . . • : ! I the LH- induced intracellular calcium oscillations observed in the present studies. A l t h o u g h much remains to be explained w i t h regard: to their significance, i it has been suggested that the calcium oscillations may code for multiple signals in their amplitudes and frequencies. It is believed that the various calcium profiles differentially activate signaling pathways such as gene transcription by activating the calmodulin pathway and intracellular protein 1 \ phosphorylation by activating the calcium sensitive protein kinase C: isozymes. The data presented in this thesis clearly indicates that the binding of I • i L H / C G to the human L H / C G receptor activates the phospholipase C pathway. In order to determine the source of the calcium dr iv ing the oscillatory behaviour a number of pharmacological manipulations of the transfected cells were undertaken. H i g h concentrations of caffeine have been shown to inhibit the mobilisation of non-mitochondrial, IP3-sensitive calcium stores [Toescu, et al., 1992]. The data obtained using both the primary granulosa cells and the transfected H E K cells show that the calcium oscillations ini t iated by stimulation of the L H receptor were inhibited by caffeine. These results would be consistent wi th activation of the phospholipase C pathway and generation of IP3 and diacylglycerol. \ j. i I Inositol 1,4,5-trisphosphate stimulates the release of calcium from intracellular stores by binding' to and opening the IP3 receptor, ;a calcium release channel i n the endoplasmic reticulum. Inositol trisphosphate receptor activity is not only sensitive to intracellular IP3 concentrations, but also to i intracellular calcium concentrations [lino, 1987]. The IP3-induced rate of calcium release depends on a feed forward mechanism whereby the initial calcium released through the channel stimulates opening of additional IP3 • i ! • receptors and a further increase in calcium levels [lino, 1990; lino and Endo, j j 1992]. Studies have shown that IP3 is only a partial agonist of the IP3 receptor; for the full activation of the receptor, both calcium and IP3 are required [Bezprozvanny, et al., 1991; Finch, et al., 1991; lino, 1990; lino and Ehdo, 1992]. The L H / C G - i n d u c e d calcium oscillations observed were most] probably generated by the process of calcium-induced calcium release. The IP3 receptor : • • • i ' • functions as a calcium-induced calcium release channel i n the continued presence of IP3 [lino, 1999]. j i : i j ' 1 Addi t ional confirmation of the stimulation of phospholipase C by the L H receptor was obtained using thapsigargin to deplete ER calcium stores. After thapsigargin pre-treatment neither L H nor human C G were capable of initiating calcium oscillations. In addition if thapsigargin was given after stimulation of the L H receptors additional calcium release was] observed ! i indicating that human C G d id not completely empty the IP3-sensit ive endoplasmic reticular calcium stores. When thapsigargin was applied first, twin peaks of calcium release were observed suggesting that more than one isoform of the IP3 receptor is expressed on the endoplasmic reticulum. The expression of mult iple IP3 is not unusual and many cells types have been '• 1 i !' . j :' reported to contain at least two of the three known IP3 receptors [Cardy, et al., 1997; Fissore, et al., 1999]. The data obtained from the H E K cells]would be consistent wi th the initial activation by thapsigargin of the high affinity IP3 receptor (most probably IP3 type II) . The presence of thapsigargin in the perfusion medium prevents the re-sequestration of released calcium into the -90-endoplasmic reticulum, and i n the continued presence of IP3 in the cytosol would result in activation of the second lower affinity IP3 receptor, j j Multiple subtypes of IP3 receptors (types I, II, and 111) are expressed in 1 a tissue- and development-specific manner [Rosemblit, et al., 1999]! Calcium 1 ' ' i ! signalling patterns are dependent on the IP3 receptor subtypes, which differ 1 • • : ! I significantly in their responses to agonists (i.e. IP3, calcium, and ATP) . Type! I IP3 receptors are highly sensitive to A T P , and mediate irregular calcium oscillations [Miyakawa, et al., 1999]. The type II IP3 receptors form channels wi th permeation properties similar to type I. However, IP3 and calcium are : ! 1 ' i more effective at activating type II IP3 receptors, therefore, these channels mobilise substantially more calcium than type I channels [Ramos-Franco, et al., 1998]. Type IIIP3 receptor are the most sensitive to IP3 and are required for the •  ! • : • . l i t long-lasting; regular calcium Oscillations that occur upon surface receptor activation [Miyakawa, et al., 1999]. H i g h cytoplasmic calcium concentrations inactivate type I, but not type II, IP3 receptors, indicating that calcium is not inherently self-limiting thus calcium passing through an active t y p e l l channel cannot feed back and turn the channel off [Ramos-Franco, et al., 1998]. Type III i • i j IP3 receptors are the least sensitive to IP3 and calcium, and tend to generate monophasic. calcium transients [Miyakawa, et al., 1999]. It forms calcium '• i ' channels similar to those of type I receptors; however, the open probability increases monotonically wi th increased intracellular calcium concentration, whereas the type I isoform has a bell-shaped dependence on cytoplasmic calcium. Type III IP3 receptors provide positive feedback as calcium is released; the lack of negative feedback al lows complete ca lc ium release from intracellular stores [Hagar, et al., 1998] Differential expression of IP3 receptor subtypes helps to encode IP3-mediated calcium signalling; thus the complement -91 -of IP3 receptors in the cell defines the spatial and temporal nature of calcium signalling in response to stimulation of phospholipase C. The significance of intracellular calcium oscillations remains to be elucidated. The unravel l ing of the mechanisms that give rise of these oscillations w i l l provide insights into the mechanisms that regulate and set cytosolic calc ium levels w i t h i n physiological l imits . Baseline calcium • I I i oscillations are defined as rapidly rising transient increases in intracellular concentrations • from close to baseline levels. 1 These oscillations are characterised by increased oscil lation frequencies, wi thout concomitant increases in spike amplitude, in response to increasing agonist concentrations. Sinusoidal oscillations are intracellular calcium oscillations superimposed on, a sustained plateau of intracellular calcium. Increased agonist concentrations increases the overall amplitude of the sinusoidal oscillations, but not the frequency of the oscillations. Baseline oscillations may continue throughout prolonged periods of stimulation, whi le s inusoidal oscillations tend to diminish wi th time, generally lasting for only a few minutes [Putney, 1992]. Clearly the response to stimulation of the L H receptor generates baseline rather than sinusoidal oscillatory responses in the cells examined in the present studies. ; j j Several investigators have suggested that calcium oscillations encode hormone signals. Because oscillation frequencies can vary w i t h agonist concentrations, calcium transients might be part of a frequency-encoded I i signall ing system [Berridge and Galione, 1988; Putney, 1992]. I Ca lc ium oscillations might encode information to be detected over a broader range than wi th just sustained, tonic increases. This may be of especial importance for hormones of very low concentrations. Prolonged exposure to extremely low concentrations of some phospholipase C- l inked hormones, can induce biological responses such as changes i n gene expression [Stachowiak, et al., 1990; Suh, et al., 1992] L o w agonist concentrations could evoke agoniist : ' > • ' i concentration-sensitive calcium oscillations; these low frequency oscillations 1 • ' ' ' I could, over a prolonged interval, could be integrated into a biological response. Meyer et al. [Meyer, et al., 1992] suggested that the kinetic behaviour of the calmodulin/calcium-calmodulin-dependent protein kinase interaction can detect and respond to calcium oscillations. If calcium oscillations are capable of activating calmodul in/calc ium-calmodul in-dependent protein kinase, then a sustained series of oscillations wou ld be sufficient to alter the expression of proteins encoded by genes regulated by C A M activity, j ; II. Role of calcium oscillations in gonadal physiology It has long been established that L H action is mediated primarily via the adenylate cyclase signalling pathway [Dufau and Catt, 1978; Hunzicker-Durin and Bimbaumer, 1985; Leung and Steele, 1992]. However, recent reports have raised the possibility that the phospholipase C signal transduction pathway is ; I | also involved in L H action [Davis, 1994; Gudermann, et al., 1992a; Herrlich, et : ! i al., 1996; H i p k i n , et al., 1993]. Al though the mechanism underlying the i i bifurcating signal transduction remains unknown/ i t has been shown that in bovine corpora lutea and L cells stably expressing the murine L H receptor, L H stimulation can couple to both G s and Gj, and the Cy-subunits released from • : • . . . . ; i | either G protein contribute to the stimulation of phospholipase CC isofornis [Herrlich, et al., 1996] H i p k i n et al. [Hipkin, et al., 1993] have shown that trie L H / C G receptor phosphorylation is induced by a phorbol ester, but not with a calcium ionophore] Al though the phorbol ester-induced phosphorylation of the L H / G G receptor can be correlated wi th uncoupling, other experiments indicate that human CG-induced uncoupling of the L H / C G receptor can occur under conditions where the cAMP-media ted receptor phosphorylation is greatly reduced or abolished [Hipkin, et al., 1993]. Gudermann et al. have shown that L cells stably expressing murine L H receptors respond!to human C G wi th an increase in their rate of phosphoinositide hydrolysis, and an increase in intracellular calcium concentrations [Gudermann, et al., 1992a]. j It has been suggested that a single L H / C G receptor can couple to both adenylate cyclase and phospholipase C, and the ability of L H / C G to activate phospholipase C is independent of c A M P accumulation [Davis, 1994]. The concentrations of L H and human C G used in the present study were in the same range as those employed to induce IP3 accumulation associated wi th the human L H receptor [Hirsch, et al., 1996]. Interestingly, it has been reported that higher concentrations of L H are required to activate the phospholipase G pathway than that which is required for the adenylate cyclase pathway, at least i n rodents. In light of the data from Zhu et al. [Zhu, et al., 1994], the activation of the phospholipase C pathway appears to be associated wi th events surrounding ovulation and pregnancy, when circulating levels of L H and human C G are high. This notion is corroborated by the increase i n the number of L H receptors dur ing fol l icular maturation [Kammerman and Ross, 1975]. Moreover, recent studies have shown that the ability of L H to induce ovulation is impaired by protein kinase C inhibitors [Shimamoto, 1993 #70; i Kaufman, 1992 #311], further supporting a role for the phospholipase C pathway in L H action during the periovulatory period. i j Calc ium signals generated by the LH-act ivated phospholipase t pathway during ovulatory process may also act via protein kinase C i Cutler et al. [Cutler, et al., 1993] reported that a calcium-independent protein kinase C was involved i n ovulation in the rat; however, this does not negate. the possibility that a calcium-dependent protein kinase C is involved i n the ovulatory process in other species. Rodents do not produce a dominant follicle during the follicular phase and give birth to between 12 - 15 pups] thus, the endocrinological dynamics regulating ovulation w i l l differ significantly from animals producing only a single offspring by ovulating one follicle (such as humans). I I : j i " - . i i Our study shows that the human L H / C G receptor is specific for L H and human C G . While purified human F S H failed to elicit Cjalcium signals, human L H and C G consistently evoked calcium oscillations that were sustained even after treatment wi thdrawal . The ini t ia l phase of the human CG-evoked increase in intracellular calcium concentrations results from the mobilisation of cytosolic calcium stores, and is then sustained by an influx of extracellular calcium. The intracellular calcium stores in question were the intracellular IPs-sensitive calcium stores in the endoplasmic reticulum. The human L H / C G receptor also appears to be coupled to calcium signalling through the G i -protein, as is the case in the murine L H receptor [Herrlich, et al., 1996] Taken together w i t h recent report of increased IP3 accumulation fol lowing the . ! I ! i ! i 1 i' • activation of the human L H / C G receptor [Hirsch, et al., 1996], the present : : j _ j results support the concept that in addition to adenylate cyclase, activation of phospholipase C is a parallel signalling pathway coupled to the human L H / C G receptor. I j III. Calcium Signalling in HEK293 Cells Transfected with the Wild-Type or Chimeric Human Gonadotrophin Receptors : : ' ' ' i ! • i j_ Follicle stimulating hormone, L H / C G , and T S H receptors belong to the large gene family known as the seven transmembrane spanning, G-proteih-coupled receptors [Lefkowitz and Caron, 1988; McFar land, et al., 1989]-Molecular cloning of c D N A s and genes for proteins of this family has revealed that they share a common structure, consisting of seven a-helical hydrophobic putative transmembrane regions, joined by three extra- and intracellular loops that display significant homology wi th in the family. The glycoprotein hormone receptors for L H , F S H , and T S H represent a small subclass of this superfamily that wi th a large extracellular amino-terminal region responsible for high affinity binding of their large (28-38 kDa) ligands [Segaloff and Ascoli , . : \ ' ; i : i 1993; Segaloff, et al., 1990; Xie, et al., 1990]. The extracellular region of these ' ' j • I I receptors is encoded by multiple exons and comprises approximately one-half of the full-length protein. The transmembrane carboxy-terminal half of these receptors is encoded by a single exon and represents the signal-transducing component of the glycoprotein [Segaloff and Ascoli , 1993]]. > . j To investigate the segments of the human L H receptor involved in signal transduction, we studied the response of intracellular calcium concentrations to gonadotrophin treatment, in HEK293 cells expressing the wildftype arid chimeric human gonadotrophin receptor. The chimeric receptor approach was originally used to investigate the functional domains of adrenergic receptors [Kobilka, et al., 1988]. The advantage of studying gonadotrophic receptors is that binding and i activation ! I • I • • !• -96- | ; are inter-related but separate phenomena [Fernandez and Puett, 1996; Ji and Ji, 1991b]. Ryu et al. [Ryu, et al., 1996] have reported that human C G binding at the . • . ! • i h igh affinity site i n the amino-terminal half of the receptor induces conformational adjustments. This leads to low affinity secondary contacts of the complex of the human CG-amino terminal end of the receptor wi th trie carboxy-terminal end of the receptor. This low affinity secondary | contact is responsible for activating the receptor. This property allows the generation of chimeric receptors wi th alterations in the signal-transducing transmembrane domains without perturbation of ligand binding. j j It has been confirmed that al l the chimeric human gonaldotrophic -., \ ; . . ; j :. • ' '•. • •  . i ! receptors are efficiently synthesized, recognise the appropriate ligands, arid •' i : • • | ! respond to ligand activation with increases in c A M P production. There were no major differences in cell surface expression and kd values of various| wild-type I i and chimeric receptors, ruling out promiscuous coupling due to changes in receptor number [Hirsch, et al., 1996]. j I i Our findings support the work of Kudo , et al. [Kudo, et jal., 1996]. Replacing the extracellular domain of the human L H receptor wi th that of the human F S H receptor did not alter receptor activation. Apart from the delay in • j 1 i ' calcium response onset, the intracellular calcium profile elicited by the binding ; j " | of F S H to the human chimeric gonadotrophin FLR receptor is very! similar to that evoked by L H stimulation of the human L H receptor. This would suggest that the extracellular domain of the receptor is important for l igand binding, • : i j but is not involved in the activation of the intracellular signalling pathways. It has prev ious ly been reported that receptor act ivat ion results i n a • •• ' I | ' . j conformational change to the L H receptor that promotes the binding of the . • i I cytoplasmic tail to the main body of the receptor. The data obtained using tfie ' ' •' ; • : ! i--97- : • • | j ' ' ' ! ! ! FLR chimeric receptors indicate that switching the external binding site to that of the F S H receptor resulted i n F S H binding initiating the conformational change required for C-terminal attachment. Alterations in the transmembrane regions of the human L H receptor ' . . . . : ' i ; results in perturbations of the agonist-induced intracellular calcium profile. In all cases this resulted i n the ablation of the basal calcium oscillations. Our findings suggest that the transmembrane regions V through VII are crucial in retaining a normal intracellular calcium profile for gonadotrophih-induced ' ' ' •' ' • ! I activation. A n y alteration to these regions resulted in a significantly:perturbed : • • : • • ! ! calcium profile. These data wou ld be consistent wi th the conclusion that the • ' • ! carboxy-terminal third of the human L H receptor mediates the activation of phospholipase C. Although the chimeric receptors containing transmembrane regions V and V I were capable of initiating a transient increase in intracellular calcium levels these were not equivalent to the sustained basal oscillations produced by stimulation of the native receptor. These results indicate that the intact carboxy-terminal third of the receptor is required to achieve the normal intracellular calcium oscillation profile. . j IV. P2-Purinoreceptor Agonist-Evoked Calc ium Oscillations i n Single H u m a n Granulosa-Lutein Cells Adenosine trisphosphate and other agonists of purinergic receptors are known to be potent stimulators of hormone secretion. Several reports in the literature have demonstrated that A T P w i l l cause a marked increase in cytosolic ; ' 1 i ! I ; calcium concentration from endocrine tissues [Bertrand, et al., 1990; Fil ippini , et al., 1994; Kamada, et al., 1994; Squires, et al., 1994]. It is known that A T P is co-! 1 ' ' ' ' I = . • i ! - 98 - ! ! localised wi th neurotransmitters at concentrations in excess of 100 m M and is co-released wi th both adrenaline and acetylcholine [Dubyak and el-Moatassim, ' i i 1993; el-Moatassim, et al., 1992; Gordon, 1986]. The sympathetic innervation of the ovaries is extensive [Dissen, et al., 1993] which would provide the source of extracellular A T P within the follicles. :" • • : : I i i 1 The work presented in this thesis clearly demonstrates that human G L C s i i possess functional purinoreceptors. Oscillatory changes in intracellular calcium •; • . ' ; :. j I ' j concentrations can be triggered equipotently by either A T P or U T P and toja lesser extent by A D P . The Pi-purinergic receptor agonists, adenosine and A M P , failed to alter basal levels of intracellular calcium, whilst the non-hydrolysable analogue ATPyS evoked a rise in calcium with a similar potency to A T P . These data suggest the existence of the P 2 U class of receptor where : • ! i A T P = A T P y S = U t P > A D P » A M P = a d e n o s i n e . Al though the existence of P 2 U receptors has been previously reported in single chicken granulosa cells [Morley, et al., 1994] and in dissociated human granulosa cells using the cuvette ' • I i > measuring technique [Kamada, et al., 1994], this is the first study to address the activity of P2-purinergic receptor agonists in single isolated human GLCs . In chicken granulosa cells, A T P has been shown to evoke calcium oscillations, which return to basal values between successive spikes] whilst in the one study in human cells to; date, the use of populations of cells! precludes the identification of oscillatory behaviour. Single human G L C s respond to A T P '. : i 1 i ! I and other P2U-purinoreceptor agonists with calcium oscillations and; that these transients differ from those previously reported in the chicken. In human, the calcium transients originate from an elevated plateau of intracellular calcium. Moreover, it appears that the mechanism dr iv ing these transients differs amongst species. Unlike the changes observed in chicken where only an initial 99 spike of reduced amplitude was observed under calcium-free conditions, the calcium oscillations seen in the present study still occurred in the absence of extracellular calcium. However, there is a gradual decline in the Oscillatory pattern as the cytosolic calcium stores are depleted. It therefore appears that the calcium transients in human G L C s originate from the release of intracellular stores of calcium arid are maintained by the influx of calcium from the extracellular media. However, in the chicken, oscillations are only seen • ! . • • ': ; i when extracellular calcium is present, although the initial response involves mobilisation of intracellular stores. The effect of verapamil on the sustained phase of the calcium response was variable with a small proportion of the cells showing a decrease which returned to plateau levels after removal of the drug. In the majority of the cells, verapamil had no effect on the alteration in intracellular calcium levels suggesting that voltage-dependent calcium channels are not involved. I i I :• <:- ! : : i i I ! The P2U purinoreceptors may be coupled to pertussis toxiri-sensitive .- ' • I ' • • • !' and/or -insensitive G proteins [Dubyak and el-Moatassim, 1993; Rhee, et al., 1989; Sternweis and Smrcka, 1992]; both pertussis toxin-sensitive arid -insensitive pathways are capable of activating P L C . The failure of pertussis toxin to alter the A T P evoked calcium changes in human G L C s would suggejst that these cells possess P2U purinoreceptors which are coupled to pertussis toxin-insensitive G proteins. This observation differs from that of the P21J purinoreceptor of rat Sertoli cells which is pertussis toxin-sensitive as reported by Fi l ippini et al. [Filippini, et al., 1994]. The P2-evoked oscillatory changes in intracellular calcium are distintt from the calcium transients previously reported for PGF2a in these cells - 1 0 0 - I • i [Currie, et al., 1992]. The effect of P G F 2 c t on human and rat ovarian cells also involved inositol phosphate metabolism, but was pertussis toxin sensitive [Davis, et al., 1989; Leung, et al., 1986; Rodway, et al., 1991]. Recent cloning and : i • • • '• '< i expressions studies of the P G F 2 a receptor confirmed that, like A T P , it acts via the phospholipase C-mediated phosphoinositide hydrolysis/calcium; signalling pathway [Abramovitz, et al., 1994; Kitanaka, et al., 1994; Lake, et al., 1994] Prostaglandin F2 a -stimulated changes in intracellular calcium are' transient, returning to baseline levels despite the continued presence of the agonist. In ! • ' : • i • |: addit ion/the concentration related effects of A T P differ from the all-or-norie i !' effects P G F 2 a on human G L C s [Currie, et al., 1992]. j j The pattern of change in intracellular calcium levels and the ability of • ' i human G L C s to instigate and maintain sophisticated calcium oscillations '• • | . i' clearly have important implications to ovarian physiology; however,; the The present study has the existence of P2U-precise role of these changes have yet to be elucidated, confirmed and extended a previous report suggesting: purinoreceptors on human G L C s . Intracellular calcium signalling was achieved via both influx and mobilisation and, for the first time, the cytosolic release of calcium has been identified as the source of calcium oscillations in single human GLCs . , Based on the cell culture experiments, P2 agonists are unlikely to be directly involved i n steroidogenesis (Figure 29-32). However, they may be i ' ' involved, in the mobil isat ion of steroidogenic precursors, e.g. v ia the ' I steroidogenic acute regulatory (StAR) protein. The StAR protein is deemed essential for the transfer of: cholesterol from the outer to the inner mitochondrial membrane, where the cytochrome P 4 5 0 cholesterol side chain ! i : ' I I cleavage enzyme is located [Ferguson, 1963; Garren, et al., 1965]. Clark et al. 1 0 1 have established a temporal relationship between levels of StAR expression and steroidogenesis [Clark, et al., 1995b] and have shown that agonists which increase intracellular calcium also increase the level of the StAR protein [Clark, et al., 1995a]. j ' • > j j V. Summary and Conclusion Activation of the human L H receptor by L H or human C G results i n the ; i • i j I stimulation of at least 2 signal transduction pathways. The data \presented concerning intracellular calcium dynamics in stimulated cells were consistent wi th the stimulation of the phospholipase C pathway i n addit ion to tne established linkage wi th adenylate cyclase. The human receptors were more responsive to low levels of agonist than was previously reported in rat models suggesting that low L H levels early i n the follicular period may play an important role in the function of granulosa cells. ! The response of both human granulosa and transfected H E K cells to low agonist levels was characterised by the presence of long lasting trains of basal calcium oscillations. This pattern of calcium mobilization could be l inked to modulation of gene transcription in both cell types i • j The studies wi th the chimeric receptors showed that the sequence of the long extracellular portion of the receptor was not critical for stimulation of phospholipase C activity but maintained the specificity of agonist binding. The C-terminal sequence of the receptor is clearly important for the generation of the basal oscillations but the precise extent of the critical sequence has yet to be identified. Stimulation of both the purinergic and L H receptors in human granulosa ' • • • I i' cells resulted in calcium oscillations, although, wi th clearly different dynamics. These results strongly suggest that the precise spatial and temporal regulation of intracellular calcium in these cells w i l l be important in the regulation of ; i cellular function. Clearly further studies into the physiological significance of these oscillations w i l l be required. , j V I . Future Directions . , 1 : ' i i i ' '• 1 • . ' I : - ! • . Women differ from the majority of experimental animal species in that late i n the follicular phase a single follicle becomes dominant and the j !• remaining follicles undergo atrophy. The precise factors regulating dominance have yet to be identified, however, there is strong support for the follicle that develops L H receptors first becoming the Graffian follicle. If this is true then the ability of human Granulosa cells to respond to low L H levels by initiating calcium transients could play a critical role in the development of the dominant i : follicle. : . .;' j ! • ; ' i I The dominant follicle is characterised by an increased output of oestradiol resulting from the ability to produce the steroid de-novo without the requirement of androstenedione secreted from the adjoining Thee a \ cells. The ability to synthesize oestradiol is dependent on the Granulosa cells expressing significant levels of P450 side chain cleavage and StAR. Clearly an important extension of the present studies w i l l be to determine the effect of L H with and without concomitant A T P on expression of the two proteins in Granulosa cells'. 103 From the two-cell theory of ovarian follicular steroidogenesis] we know that: F S H receptors are present on granulosa cells, and increased levels are induced by F S H itself; L H / C G receptors are present on theca cells and initially absent on granulosa cells; as the follicle matures, F S H induces the expression of . : . • i i L H / C G receptors on the granulosa cells; and F S H induces aromatase! activity in granulosa cells. \ j : . . : i ' i : : ' • ' i Theca cells are characterised by LH- induced androgen production. During the middle of the follicular phase prior to selection of the | dominant follicle while L H concentrations are low, it is possible that a sufficient number of L H / C G receptors are activated to evoke intracellular calcium transients. The •' • i • ' calcium spikes would form part of a frequency-encoded signalling system, arid . • ! '• ' ! ! over a period of several days, these signals may integrate into a biological ' | I response such as the expression of the StAR protein. Activation of the calcium-1 ' . ' j ' i sensitive StAR gene in the theca cell results in the transfer of cholesterol frofn the outer to the inner mitochondrial membrane, where cytochrome P450 side chain cleavage enzyme is located. \ j The cytochrome P450 side chain cleavage enzyme converts cholesterol to ! ! • ! pregnenolone, and is the key steroidogenic intermediate common to; all classes , j j of steroid hormones. Both L H and c A M P regulate transcription of the cytochrome P450 gene but c A M P is the more potent of the two. Interestingly, F S H receptors activate adenylate cyclase resulting i n an increase! i n c A M P ' , ! • j levels, however, in the early follicular stage in the absence of L H j receptors, i j F S H is incapable of stimulating the expression of P450 side chain cleavage. This suggests that a transcriptional repressor rhust be present in the granulosa cells and that the actions of this repressor protein are reversed once the cells express L H receptors. 104-There is at present no evidence that A T P has a direct j effect on steroidogenesis in human G L C s ; but it may be another candidate for regulation of the StAR protein. The possible effect of P 2 agonists in this regard warrants further investigation. ; j ! F inal ly , the phospholipase C pathway, and protein kinase C in particular, has been implicated i n the ovulatory processes in several animals, therefore, protein kinase C could play a role in ovulation i n women. Protein kinase Cs are subdivided into calcium-dependent and calcium independent isoforms. There is no information available concerning which of the] 12 known isoforms of protein kinase C are: expressed in human G L C s . If the cells possess a calcium-dependent protein kinase C, LH-induced calcium transients w o u l d function as a co-activator of this enzyme along w i t h the diacylglycerol generated by phospholipid hydrolysis. The involvement of protein kinase C in the ovulatory process suggests that the enzyme may be involved i n the increased expression and secretion of proteolytic enzymes required for rupture of the follicle. In order to test the hypothesis that the LH-induced oscillations activate a calcium-sensitive protein kinase C , w h i c h i n turn j increases • • • • ' ' 1 ' 1 ! ' I proteolytic enzyme production, the effect of L H on tissue type plasminogen activator activity must be investigated in the presence and absence of inhibitors protein kinase C. I ' ]•• • • ; ; '•  ; ' I • ^ A n extension of these studies wou ld be to identify the subtypes of protein kinase Cs present in human G L C s ; and if a calcium-dependent isoform is present, to inhibit it and to examine the effects of L H stimulation. I -105-i References i . i i-[1] Abel l A N and Segaloff D L (1997) Evidence for the direct involvement of transmembrane region 6 of the lutropin/choriogonadotropin recep-J tor in activating Gs. Journal of Biological Chemistry 272(23):14586-91 j [2] Abou-Issa H and Reichert L , Jr. (1976) Properties of follitropin-receptor interaction. Characterization of the interaction of follitropin: wi th re-| ceptors in purified membranes isolated from mature rat testes tubules.' Journal of Biological Chemistry 25l(ll):3326-37 • ! I < . i ! • ' i I [3] Abramovitz M , Boie Y , Nguyen T, Rushmore T H , Bayne M A ; Metters K M , Slipetz D M and Grygorczyk R (1994) Cloning and expression of a c D N A for the human prostanoid FP receptor. 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