Calcium-dependent actions of gonadotropin-releasing hormone agonist and luteinizing hormone upon cyclic AMP and progesterone production in rat ovarian granulosa cells

The Ca2+ dependency of the direct stimulatory effect of the gonadotropin-releasing hormone (GnRH) agonist analog [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide (GnRHa) on progesterone production was investigated and compared to that of luteinizing hormone (LH) in rat granulosa cells from preovulatory follicles. Removal of extracellular Ca2+ by EGTA, or the use of the Ca2+ channel blockers verapamil and La3+, resulted in complete inhibition of GnRHa-induced progesterone production and a partial inhibition of LH-stimulated progesterone production (80, 80 and 50% inhibition respectively for EGTA, verapamil and La3+). Removal of extracellular Ca2+ increased the ED50 for LH-induced cAMP production by four-fold (from 80 to 330 ng/ml) and decreased maximal nucleotide formation by 44%. LH-induced cAMP production was also inhibited partially by verapamil (35%) at 10(-4) M drug concentration. GnRHa had no effect on cAMP production in the presence or absence of Ca2+. GnRHa and LH were found to have maximal effects on progesterone production at about 0.5 mM of Ca2+ in the incubation medium. On the other hand the stimulatory effect of dibutyryl cAMP [Bu)2cAMP) on progesterone production showed little dependency on extracellular Ca2+. The calmodulin antagonist trifluoperazine (TFP) caused concentration-dependent inhibition of the stimulatory action of GnRHa and LH on progesterone production with IC50 values of 3 and 8 microM, respectively. The stimulatory effect of (Bu)2cAMP on progesterone synthesis was attenuated by verapamil and TFP. These results indicate that the direct stimulatory effect of GnRH on ovarian progesterone production is absolutely dependent on Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of gonadotropin-releasing hormone antagonists on steroidogenesis in human granulosa lutein cells

OBJECTIVE: GnRH antagonists have recently been introduced for the prevention of premature LH surges during controlled ovarian hyperstimulation (COH). We have here investigated whether the GnRH antagonists cetrorelix and ganirelix exert effects on ovarian steroidogenesis. Since there is some controversy about the action of GnRH agonists in the human ovary we also tested the effect of triptorelin on steroid production in cultured human granulosa lutein cells. METHODS: Cells were obtained from patients treated with different protocols of COH. In addition to gonadotropins they received triptorelin, cetrorelix, ganirelix or no GnRH analogue. RESULTS: Such in vivo treatment did not result in significant effects of triptorelin or the two GnRH antagonists on spontaneous or human chorionic gonadotropin (hCG)-stimulated steroidogenesis. To exclude the possibility that the in vivo treatment might not affect in vitro steroid production because of low or absent peptide activity, we performed in vitro treatments with triptorelin, cetrorelix and ganirelix for up to 96 h. However, these treatment paradigms did not influence basal or hCG-stimulated steroid production. CONCLUSIONS: We conclude that GnRH antagonists do not exert any significant effects on ovarian steroidogenesis in vitro and therefore their introduction into protocols of COH is unlikely to impair ovarian function.     

Luteinizing hormone induces progesterone receptor gene expression in cultured porcine granulosa cells.

We have examined the effect of LH on the regulation of the progesterone receptor (PR) in cultured porcine granulosa cells. In this study we used the RNase protection assay to evaluate the PR mRNA levels with a porcine cDNA clone isolated by the polymerase chain reaction (PCR) method. This clone was regarded as part of the porcine PR cDNA because of its 98.3% and 95.7% homology to the hormone-binding domain of human PR cDNA in amino acid and nucleotide sequences, respectively. Treatment with LH (500 ng/ml) increased porcine PR mRNA to a maximum level of 8.6 +/- 1.1-fold (mean +/- SE) after 3-h exposure. This induction was mimicked by (Bu)2cAMP as well as by FSH and hCG, and the increased PR caused by LH and (Bu)2cAMP occurred in a dose-dependent manner. Basal and LH-induced PR mRNA levels were not affected by progesterone (100 ng/ml), estrogen (100 ng/ml), and RU 486 (10 ng/ml) at 3 h. The mechanism of the increased PR mRNA levels was studied in the presence of actinomycin-D and cycloheximide. While inhibition of RNA synthesis with actinomycin-D blocked LH-induced PR mRNA expression, inhibition of protein synthesis with cycloheximide increased basal and LH-induced PR mRNA levels. These results indicate that the expression of PR mRNA is positively regulated by LH, and this induction does not require ongoing protein synthesis. There may be a cycloheximide-sensitive mechanism that modulates PR mRNA stability. From our results we suspect that progesterone modulates ovarian function through LH-induced PR in granulosa cells.     

Regulation of prostaglandin E, progesterone, and cyclic adenosine monophosphate production in ovarian granulosa cells by luteinizing hormone and gonadotropin-releasing hormone agonist: comparative studies

The early direct effects of GnRH on the ovary were investigated using cultured granulosa cells from preovulatory rat follicles, and compared to the known stimulatory effects of LH. Stimulation of ovarian functions by a GnRH agonist include a rapid receptor-mediated phosphatidylinositol turnover (approximately 5 min). On the other hand, LH action on granulosa cells is initiated by increased cAMP production (approximately 10-15 min), consisting of an indomethacin-resistant and indomethacin-sensitive pools (40% and 60%, respectively). The GnRH agonist [D-Ala6] des-Gly10 N-ethylamide (GnRHa) at concentrations of 10(-12)-10(-8) M had no effect on basal or LH-stimulated cAMP production during a 4-h incubation test. Both LH and GnRHa increase progesterone formation (30 and 120 min, respectively) with ED50 values of 2.5 ng/ml and 10(-9) M, respectively and the stimulatory effect is not blocked by indomethacin. LH and GnRHa increase also prostaglandin E (PGE) formation (180 and 120 min, respectively) and the ED50 values were 0.1 microgram/ml and 10(-9) M, respectively. No inhibitory effect of GnRHa on LH actions was observed during 4 h of incubation. It is concluded that: 1) GnRH mimicks LH stimulation of ovarian PGE and progesterone production; 2) cAMP does not play a role in mediating the direct stimulatory effects of GnRH agonists on ovarian PGE and progesterone production; 3) PGE is not involved in mediating GnRH and LH stimulation of progesterone formation. 4) LH-induced cAMP production consists of indomethacin-sensitive and indomethacin-resistant pools.     

The source of cholesterol for progesterone synthesis in cultured preovulatory human granulosa cells

There are three possible sources of cholesterol for immediate use in progesterone production by preovulatory human granulosa cells: follicular fluid high-density lipoprotein, de novo synthesis of cholesterol, and performed intracellular cholesteryl ester stores. In the present study these three alternatives were investigated. First, an in vitro model was established that mimics the preovulatory environment, including short-term cultures and use of autologous follicular fluid in the culture medium, instead of serum. Using this model it was found that the presence of high-density lipoprotein from follicular fluid in the culture medium did not affect the synthesis of progesterone by the granulosa cells. Next, addition of inhibitors of de novo sterol synthesis, like low-density lipoprotein, 25-OH cholesterol and compactin to the culture medium, did not reduce [14C]acetate incorporation into sterols and steroids by the cells. The sterol synthesis was accordingly interpreted to be at a low and therefore uninhibitable level. Finally, the content of free and esterified cholesterol in freshly isolated granulosa cells was found to be 50 +/- 7 and 52 +/- 13 pmol/mg cell protein, respectively. We suggest that neither follicular high-density lipoprotein nor endogenous synthesis is the immediate cholesterol source for the progesterone production in preovulatory human granulosa cells. However, granulosa cells have a large store of cholesteryl esters that may provide free cholesterol for the preovulatory progesterone production.     

Luteinizing hormone synthesis in cultured fetal human pituitary cells exposed to gonadotropin-releasing hormone.

To investigate whether GnRH regulates LH synthesis during human development, pituitary cells from second trimester fetuses were incubated with [35S]methionine ([35S]met) and [3H]glucosamine ([3H]gln) for 48 h with 0, 10(-9), and 10(-7) mol/L GnRH. Immunoassayable (i) LH was measured in media and cellular lysates, and dual label scintillation analysis was used to quantitate incorporation of radiolabeled precursors into cells, trichloroacetic acid-precipitable proteins, and immunoprecipitated LH subjected to electrophoresis. Exposure of cells to GnRH did not affect cellular uptake or incorporation of precursors into proteins, but specifically increased total (secreted plus cellular) LH synthesis. Both GnRH concentrations significantly increased iLH release and enhanced secreted and cellular [3H]gln-LH. The secretion of [35S] met-LH was stimulated only by 10(-7) mol/L GnRH. The proportion of newly synthesized LH that was secreted and the 3H/35S ratio of secreted and cellular LH were uninfluenced by GnRH. Although basal LH synthesis was not sex dependent, total iLH content and GnRH-stimulated LH translation were greater in cells from females than in those from males. Therefore, GnRH regulates LH synthesis by second trimester fetal human gonadotrophs without influencing the proportion of total radiolabeled LH that is secreted. The existence of a sex difference in total iLH content and GnRH-stimulated LH translation is consistent with the sexual dimorphism in pituitary LH content occurring during human development.     

Down-regulation of proliferation and up-regulation of apoptosis by gonadotropin-releasing hormone agonist in cultured uterine leiomyoma cells

OBJECTIVE: To elucidate the direct effects of gonadotropin-releasing hormone agonist (GnRHa) on the growth of human uterine leiomyoma cells, cell proliferation and apoptosis in cultured leiomyoma cells treated with GnRHa were investigated. METHODS: Isolated leiomyoma cells were subcultured in DMEM supplemented with 10% FBS for 5 days and stepped down to serum-free conditions for an additional 6 days in the presence or absence of graded concentrations of GnRHa (10(-9) mol/l to 10(-12) mol/l). The effects of GnRHa on the number of viable cells, expression of proliferating cell nuclear antigen (PCNA), Fas and Fas ligand, and apoptosis in cultured leiomyoma cells were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) assay, immunocytochemical analysis, Western blot analysis and TUNEL assay respectively. RT-PCR was performed to detect the expression of GnRH receptor mRNA in cultured leiomyoma cells. RESULTS: Treatment with GnRHa resulted in a decrease in the number of cultured viable leiomyoma cells assessed by MTT assay in a dose-dependent manner compared with that in control cultures (P<0.01). The growth inhibition of cultured leiomyoma cells treated with GnRHa in concentrations higher than 10(-10) mol/l was associated with the suppression of the proliferative potential characterized by a decrease in PCNA-positive rate of the cultured cells (P<0.01) and an increase in the apoptosis-positive rate assessed by TUNEL assay (P<0.05 and P<0.01). GnRHa markedly increased the expression of Fas and induced the expression of Fas ligand in the cultured leiomyoma cells on the basis of Western blot analysis. These direct effects of GnRHa on the number of viable cultured leiomyoma cells, PCNA-positive rate, apoptosis-positive rate and Fas/Fas ligand expression in the cultured leiomyoma cells were only attained after the 4-day treatment. RT-PCR analysis revealed that GnRH receptor mRNA was expressed in cultured leiomyoma cells. CONCLUSIONS: The present results demonstrate that GnRHa directly inhibits the growth of human uterine leiomyoma cells by suppressing cell proliferation and inducing apoptosis, which might be associated with the increase in Fas expression and the induction of Fas ligand expression in the cells.     

Effect of danazol on gonadotropin and cAMP stimulated progesterone synthesis in cultured human granulosa cells

The effect of danazol on progesterone formation was studied in cultured human granulosa cells obtained from 20 follicles in the mid- to late follicular phase of normally menstruating women. The cells were cultured for 2 to 6 days in the presence of danazol (0.01 to 5 mg/l) alone and in combination with one of several progesterone stimulatory agents. The medium was changed every other day and analysed for progesterone with radioimmunoassay. In all cases progesterone synthesis was markedly stimulated by human FSH (10 micrograms/l), human LH (10-100 micrograms/l), hCG (1000 IU/l), forskolin (1 mumol/l) or 8-Bromo-cAMP (1 mmol/l). In the presence of danazol (1 mg/l) the FSH-, LH- and hCG-stimulated progesterone synthesis was partially inhibited by 55 to 60%. The response to forskolin or 8-Bromo-cAMP was also inhibited by danazol although to a somewhat lower extent (35 to 50%). Basal progesterone synthesis was inconsistently inhibited in some cases. It was concluded that not only gonadotropin-stimulated steroid synthesis, as previously demonstrated for hCG, but also forskolin and 8-Bromo-cAMP-stimulated steroidogenesis is sensitive to danazol inhibition. This suggests that the effect of danazol in human granulosa cells is due, at least partly, to interference with steps distal to cAMP generation.     

Mechanisms of gonadotropin-releasing hormone agonist action in the human male

Agonist analogs of GnRH are being increasingly utilized to induce medical castration for treatment of a variety of hormonally responsive clinical disorders. However, the mechanism/s of their paradoxical antigonadal action in the human male remain poorly understood. Basal and integrated concentrations of immunoreactive LH after intermediate term (4-16 weeks) GnRH agonist treatment are only modestly decreased and cannot fully account for the far greater decline in serum testosterone (T) concentrations. Bioassayable LH concentrations, however, decrease markedly and parallel the fall in serum T suggesting secretion of qualitatively different LH species with diminished biological activity while the circulating concentrations of beta-subunit parallel the measured bioassayable LH concentrations, free alpha-subunit secretion remains persistently and disproportionately elevated during chronic GnRH agonist treatment. Cross-reactivity of free alpha-subunits in the human LH RIA contributes to this disparity between the LH immuno and bioactivity. Chromatography of serum LH during GnRH agonist treatment suggests secretion of a qualitatively different LH species. Unlike the rat, in which the antifertility effects of the agonist are mediated predominantly by direct inhibition of testicular steroidogenesis, significant direct gonadal effects have not been demonstrated in man. Thus the bulk of evidence points to a predominant pituitary site of action in the human male. The molecular basis of the heterogeneity of LH during GnRH agonist, however, remains to be elucidated. The hypothesis that co- or posttranslational modification by the agonist may attenuate biologic activity of LH has not yet been directly tested.     

Androgen inhibition of follicle-stimulating hormone-stimulated luteinizing hormone receptor formation in cultured rat granulosa cells

Since LH receptors are decreased in atretic follicles known to contain high androgen levels, we have studied the androgen modulation of LH receptor formation in vitro. Granulosa cells from hypophysectomized, diethylstilbestrol-treated rats were cultured for 3 days with FSH in the presence or absence of nonaromatizable androgens, dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol, or a synthetic androgen, R1881 (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one). FSH increased LH receptor content in granulosa cells, while concomitant androgen treatment decreased LH receptor content in a dose- and time-dependent manner, without changing the equilibrium dissociation constant (Kd) for human CG. R1881 (10(-7) M), dihydrotestosterone (10(-6) M), and 5 alpha-androstane-3 alpha, 17 beta-diol (10(-6) M) inhibited LH receptor content by 68%, 65%, and 65%, respectively. Similar to earlier findings, these androgens enhanced FSH-stimulated progesterone biosynthesis and aromatase activity in the same cells. To study their LH responsiveness, androgen-treated cells were washed and reincubated for 2 more days with or without LH. Although basal progesterone production was elevated by R1881 pretreatment, the androgen-pretreated cells were less responsive to LH. Treatment with cyanoketone, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, did not alter the inhibitory effects of R1881 on LH receptors, indicating that the androgen action is not mediated by endogenous progestins. Furthermore, R1881 inhibited the stimulation of LH receptor formation by forskolin, cholera toxin, and 8-bromo-cAMP, suggesting that androgens may inhibit LH receptor induction by affecting post-cAMP events. Estrogen treatment enhanced the FSH induction of LH receptor content, while concomitant addition of R1881 also suppressed the estrogen action. Thus, androgens inhibit FSH-induced functional LH receptors in cultured rat granulosa cells. The androgen effect is exerted, at least partially, at post-cAMP sites and is independent of changes in progestin biosynthesis.     

Effect of gonadotrophins on progesterone secretion by cultured granulosa cells obtained from human preovulatory follicles

Granulosa cells were obtained from human preovulatory follicles in 31 women undergoing in vitro fertilization and embryo transfer due to tubal infertility. Follicular maturation was stimulated and synchronized by treatment with Clomiphene or human menopausal gonadotrophin (hMG), or both, plus human chorionic gonadotrophin (hCG). Follicles were aspirated by ultrasound guided puncture approximately 34-36 h after the hCG injection. The granulosa cells were washed and suspended in modified medium 199 containing 10% foetal bovine serum and cultured as monolayers for 6-8 days in the absence and presence of hormones and reactants. Progesterone formation was analyzed by RIA. In general, the cells underwent morphological luteinization and secreted high amount of progesterone. Under basal conditions the secretion of progesterone was highest during the first 2 days in culture and then gradually declined. Progesterone secretion was stimulated by human LH, hCG and the adenylate cyclase stimulator forskolin, with a maximal effect between days 2-6. The beta-adrenergic agonist isoproterenol in preliminary experiments potentiated the stimulatory effect of hCG but had no own stimulatory effect. No clear differences in progesterone secretion or responsiveness to in vitro stimulation relating to the various in vivo stimulation protocols were found.     

Increase in luteinizing hormone content occurs in cultured human fetal pituitary cells exposed to gonadotropin-releasing hormone

To investigate the mechanisms by which GnRH regulates LH production during intrauterine life, dispersed pituitary cells from second trimester human fetuses were cultured on extracellular matrix-coated plates for 48 h. Exposure of cells to 3 x 10(-10) mol/L GnRH for 1-48 h significantly increased cumulative LH secretion compared to that in respective controls (P less than 0.01). The rate of GnRH-stimulated LH release was accelerated during the first 6 h, after which it declined to a level similar to that of basal release. This phenomenon was associated with a decrease in the GnRH concentration of the medium. Exposure of cells to GnRH (3 x 10(-10) to 10(-6) mol/L) for 48 h induced a dose-dependent elevation of total LH which correlated with an increase in releasable, but not cellular, LH. Desensitization to GnRH (10(-7) mol/L) occurred when cells were cultured with pharmacological amounts of GnRH for 48 h. These results indicate that GnRH induces the increase in total and releasable LH in human fetal pituitary cells. These cells also appear to inactivate GnRH. Thus, GnRH may increase LH production in the human fetal pituitary and the pituitary receptor mechanism may be involved in GnRH action on LH release during intrauterine life.     

Inhibin and activin modulate the release of gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells.

Although it is clear that human chorionic gonadotropin (hCG) and progesterone play fundamental roles in pregnancy, the regulation of placental production of these hormones remains to be defined. Recent evidence suggests that the human placenta expresses proteins related to inhibin (alpha beta subunits) or activin (beta beta subunits). Inhibin and activin (follicle-stimulating hormone-releasing protein) possess opposing activities in several biological systems including pituitary follicle-stimulating hormone (follitropin) secretion, erythroid differentiation, and gonadal sex-steroid production. The actions of purified inhibin and activin on hormonogenesis by primary cultures of human placental cells were studied. The addition of activin increased gonadotropin-releasing hormone (GnRH) and progesterone production and potentiated the GnRH-induced release of hCG. Inhibin by itself did not modify placental immunoreactive GnRH, hCG, and progesterone secretion but reversed the activin-induced changes. Neither inhibin nor activin influenced the release of human placental lactogen. Furthermore, transforming growth factor beta, structurally related to inhibin/activin, did not significantly influence hormone release from cultured placental cells. These results support the hypothesis that inhibin and activin may play a role in regulating the release of GnRH, hCG, and progesterone from placenta and implicate inhibin-related proteins in the endocrine physiology of human pregnancy.     

The human gonadotropin-inhibitory hormone ortholog RFamide-related peptide-3 suppresses gonadotropin-induced progesterone production in human granulosa cells

RFamide-related peptide-3 (RFRP-3), a mammalian ortholog of avian gonadotropin-inhibitory hormone, has pronounced inhibitory effects on reproduction in a number of species. RFRP-3 suppresses gonadotropin release at the hypothalamic and/or pituitary levels; however, increasing evidence also suggests putative functions within the ovary. We have now demonstrated the expression of both RFRP and its receptor (GPR147) in primary cultures of human granulosa-lutein cells. Immunohistochemical analysis of normal human ovaries from premenopausal women showed that RFRPs and GPR147 were primarily localized in the granulosa cell layer of large preovulatory follicles as well as in the corpus luteum. Treatment of human granulosa-lutein cells with RFRP-3 reduced FSH-, LH- and forskolin-stimulated progesterone production and steroidogenic acute regulatory protein expression but did not affect basal or 8-bromoadenosine 3'5'-cyclic monophosphate stimulated levels. In addition, RFRP-3 inhibited gonadotropin- and forskolin-induced intracellular cAMP accumulation, and these effects were abolished by pretreatment with an inhibitor of inhibitory G(i/o) proteins (pertussis toxin). Importantly, the effects of RFRP-3 on FSH-, LH-, and forskolin-induced cAMP and progesterone accumulation were completely eliminated by cotreatment with the bifunctional GPR147/GPR74 antagonist RF9 or by pretreatment with GPR147 small interfering RNA. These results suggest that RFRP-3 is expressed in human granulosa cells in which it acts via its receptor, GPR147, to inhibit gonadotropin signaling at the level of adenylyl cyclase via activation of a pertussis toxin-sensitive Gα(i/o) protein. This leads to reduced gonadotropin-stimulated cAMP accumulation and progesterone synthesis, likely via reduced steroidogenic acute regulatory protein expression. Thus, ovarian RFRP-3/GPR147 signaling could contribute to normal ovarian function.     

Effects of RU486 on progesterone secretion by human preovulatory granulosa cells in culture

The effects of RU486 on progesterone synthesis were studied in human preovulatory granulosa cells in culture. No effect was observed at 1 and 10 micrograms/mL, but at 100 micrograms/mL, RU486 inhibited the simulation of progesterone secretion induced by LH and cAMP. It is suggested that the main target of RU486 is the cytochrome P450scc function [catalyzing the formation of pregnenolone (D5P) from cholesterol], since no accumulation of D5P or hydroxy derivatives of progesterone was observed. As RU486 is an antiglucocorticosteroid and antiprogesterone agent, the effects of dexamethasone and progesterone were also investigated. Dexamethasone did not modify progesterone secretion, but progesterone inhibited its own synthesis in both the presence and absence of LH. Thus, under these experimental conditions RU486 displayed a progesterone-like effect. However, since the effect of RU486 was observed only at a concentration around 10(-4) M, the mechanism of action may not involve a receptor pathway and may not apply to most clinical circumstances.     

Cytodifferentiation of granulosa cells induced by gonadotropin-releasing hormone promotes fibronectin secretion

An analysis of the hormonal regulation of fibronectin production by granulosa cells showed that GnRH stimulated fibronectin secretion. GnRH increased fibronectin production 2.5- to 5-fold over that of control untreated cultures and greater than 20-fold over that of FSH-treated cultures. The GnRH concentration required for a minimal response was 10(-10) M, and that required for a maximal response was 10(-8) M. In contrast to the effects of GnRH, FSH suppressed fibronectin production to low or undetectable levels. In addition, FSH abolished the actions of GnRH on fibronectin production. Treatment of cultured granulosa cells with either (Bu)2cAMP or methylisobutylxanthine to influence cellular cAMP levels mimicked the actions of FSH on fibronectin secretion. Similar to FSH, both (Bu)2cAMP and methylisobutylxanthine abolished the actions of GnRH on fibronectin production. These results indicated that an increase in cAMP levels resulted in suppression of fibronectin secretion by granulosa cells and inhibition of the actions of GnRH. Insulin treatment of granulosa cells also suppressed fibronectin secretion, but to a lesser extent than FSH. In addition, insulin inhibited the actions of GnRH on fibronectin production by approximately 40-50% of stimulated levels. GnRH promoted a state of cytodifferentiation of the granulosa cell which had a high level of fibronectin production and, as shown previously, a low level of steroidogenesis. In contrast, FSH promoted a state of cytodifferentiation which had a low level of fibronectin production and a high level of steroidogenesis Both fibronectin and steroidogenic enzymes (e.g. aromatase) provide useful markers for an analysis of the cytodifferentiation of granulosa cells between these two distinct differentiated states. Results are discussed in relation to the possible mechanisms controlling granulosa cell cytodifferentiation and the possible functions of fibronectin in the ovary.     

Gonadotropin-releasing hormone stimulates phospholipid labeling in cultured granulosa cells

Cultured ovarian granulosa cells from preantral and preovulatory follicles were incubated with [32P]Pi to label endogenous phospholipids. Labeled cells were then incubated with FSH, GnRH, or a GnRH agonist analog [D-Ala6]GnRH (GnRHa), cellular phospholipids were separated by two-dimensional thin layer chromatography, and the radioactivity was determined. Phosphatidylcholine was the major labeled phospholipid accounting for 64% of the total radioactivity. The remaining labeling was distributed among choline plasmalogen (8.4%), phosphatidylinositol (6.3%), lyso phosphatidylcholine (3.7%), phosphatidylethanolamine (3.4%), phosphatidic acid (1.75%), phosphatidylserine (1.65%), and cardiolipin (1.3%). GnRH and its agonist analog GnRHa, but not FSH, increased 32P incorporation into phospholipids by 2-fold. Analysis of the several phospholipids revealed that GnRHa (10(-7) M) increased 32P labeling of phosphatidylcholine and lyso phosphatidylcholine by 1.5- and 2.5-fold respectively, and that of phosphatidic acid and phosphatidylinositol by 5- and 7-fold, respectively, during 60 min of incubation. The natural decapeptide GnRH was 30 times less potent than its agonist analog. Labeling of other phospholipids was not affected by GnRHa treatment, and FSH had no effect on 32P incorporation under similar conditions. The stimulatory effect of GnRHa was blocked by the potent GnRH antagonist [D-pGlu1,pClPhe2, D-Trp3,6]GnRH. The minimal stimulating dose of GnRHa was 10(-12) M, and increased phospholipid labeling could be detected after 10 min of incubation with the analog. These results indicate that phospholipids, in particular phosphatidylinositol and phosphatidic acid, might be involved in the mechanism by which GnRH exerts its gonadal effects.     

Desensitization of luteinizing hormone release in cultured pituitary cells by gonadotropin-releasing hormone

Preincubation of cultured pituitary cells with GnRH caused a marked decrease in subsequent LH release. The rate of desensitization increased when the preincubating concentration of GnRH and the preincubation time were increased. Pituitary cells obtained from male rats were not as sensitive to GnRH as cells obtained from female rats and the extent of desensitization was also smaller in cells from male rats. Densensitization was found to be a long-lasting effects, without any change in the viability of the cells. A superactive analogue of GnRH (D-Phe6-GnRH) caused almost complete desensitization of LH secretion, while a competitive inhibitory analogue of GnRH caused a much smaller decrease in LH response which could be overcome by increasing the concentration of GnRH used for reincubation. These data suggest that the desensitization is closely related to the biological activity of GnRH and does not correlate with receptor binding. High concentrations of potassium also induced desensitization, although to a lower extent than GnRH. Since K+ induces LH release by a different mechanism than GnRH, our data suggest that the desensitization phenomenon cannot be explained only at the receptor level. The time curve of desensitization supports the idea that GnRH action has two-phases: an acute effect which cannot be desensitized, and a secondary phase which can be densensitized.