Gonadotropin Receptor Expression Research Articles

Expression of components of the insulin-like growth factor system and gonadotropin receptors in bovine cumulus–oocyte complexes during oocyte maturation

IGF system expression has been largely explored in the bovine follicular wall whereas it remains poorly studied in the COC. Using semi-quantitative RT–PCR and Western blot analysis, we have investigated spatial and temporal expression of IGF-1, IGFR-1, IGFBP-2, IGFBP-4, as well as gonadotropin receptors in bovine COC during oocyte maturation. In addition, we have compared changes in the IGF system and FSHR expression during in vitro maturation in TCM199 alone or in the presence of 10 ng/ml of EGF. The transcripts for IGFR-1 and IGFBP-2 were detected in cumulus and germinal cells whereas IGF-1, IGFBP-4 and FSHR mRNA were restricted to cumulus cells. Topography of the IGF system and gonadotropin receptor expression within COC were unaffected by the maturation step. In contrast, levels of IGFBP-2 and FSHR expression decreased ( P<0.05) in matured COC. Under defined culture conditions, IGFBP-2 and FSHR mRNA expression remained at a high level in TCM199 alone and were significantly reduced ( P<0.05) in the presence of 10 ng/ml EGF after a 24 h period of in vitro maturation. In conclusion, our results demonstrate a cell-specific pattern of IGF system member gene expression within bovine COC suggesting interaction between the somatic and germinal compartments. In addition, synchronized changes in the pattern of COC IGFBP-2 and FSHR expression during oocyte maturation suggest possible synergistic actions between IGF-1 and FSH.

Regulation of luteinizing hormone/human chorionic gonadotropin receptor expression: a perspective.

The LH/hCG receptor, a member of the G protein coupled receptor family mediates the cellular actions of LH in the ovary. A considerable amount of information regarding its structure, mechanism of activation, and regulation of expression has emerged in recent years. Here we provide a brief overview of the current information on the structural organization of the receptor and the mechanism of receptor mediated signaling as well as an in-depth discussion on recent developments pertaining to the regulation of receptor expression. Specifically, we describe studies from our laboratory showing that the posttranscriptional regulation of the receptor involves an LH/hCG receptor mRNA-binding protein. We also propose a model to explain the loss of steady-state LH/hCG receptor mRNA levels during receptor down-regulation.

Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes.

Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [(125)I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG alphabeta+); and 3) ovariectomized (OVX) hCG alphabeta+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG alphabeta+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG alphabeta+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.

The Cyclin-dependent Kinase Inhibitors p27Kip1 and p21Cip1 Cooperate to Restrict Proliferative Life Span in Differentiating Ovarian Cells

The timing of cellular exit from the cell cycle during differentiation is specific for each cell type or lineage. Granulosa cells in the ovary establish quiescence within several hours after the ovulation-inducing luteinizing hormone surge, whereas they undergo differentiation into corpora lutea. The expression of Cdk inhibitors p21(Cip1/Waf1) and p27(Kip1) is up-regulated during this process, suggesting that these cell cycle inhibitors are involved in restricting proliferative capacity of differentiating granulosa cells. Here we demonstrate that the lack of p27(Kip1) and p21(Cip1) synergistically renders granulosa cells extended an proliferative life span. Immunohistochemical analyses demonstrated that corpora lutea of p27(Kip1), p21(Cip1) double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27(Kip1) single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21(Cip1)-deficient mice ceased proliferation similarly to those in wild-type mice. Interestingly, granulosa cells isolated from p27(Kip1), p21(Cip1) double-null mice exhibited markedly prolonged proliferative life span in culture, unlike cells with other genotypes. Cultured p27(Kip1), p21(Cip1) double-null granulosa cells maintained expression of steroidogenic enzymes and gonadotropin receptors through 8-10 passages and could undergo further differentiation in responses to cAMP accumulation. Thus, the cooperation of p27(Kip1) and p21(Cip1) is critical for withdrawal of granulosa cells from the cell cycle, in concert with luteal differentiation and possibly culture-induced senescence.

Insulin-like growth factor binding protein 4 expression parallels luteinizing hormone receptor expression and follicular luteinization in the primate ovary.

It has been suggested that locally produced insulin-like growth factor binding protein 4 (IGFBP4) inhibits ovarian follicular growth and ovulation by interfering with IGF action. According to this hypothesis, IGFBP4-expressing follicles should demonstrate atresia, whereas healthy dominant follicles should be devoid of IGFBP4. Alternatively, according to this view, there could be constitutive expression of the inhibitory IGFBP4 but selective expression of an IGFBP4 protease in dominant follicles, allowing the follicle to mature and ovulate because of degradation of the binding protein. To examine these views concerning the role of IGFBP4 in primate follicular selection, we analyzed cellular patterns of IGFs 1 and 2, IGFBP4, and the IGFBP4 protease (pregnancy-associated plasma protein A [PAPP-A]) mRNA expression in ovaries from late follicular phase rhesus monkeys using in situ hybridization. The IGF1 mRNA was not detected, but the IGF2 mRNA was abundant in theca interna and externa of all antral follicles and was present in the granulosa of large preovulatory and ovulatory follicles. The IGFBP4 mRNA was selectively expressed by LH receptor (LHR) mRNA-positive theca interna cells of healthy antral follicles (defined by aromatase and gonadotropin receptor expression) and by LHR-expressing granulosa cells found only in large preovulatory and ovulatory follicles (defined by size and aromatase expression). The PAPP-A mRNA was abundant in granulosa cells of most follicles without obvious relation to IGFBP4 expression. Ovarian IGFBP4 mRNA levels were markedly increased after treatment with the LH analog, hCG, whereas IGF2 and PAPP-A mRNAs were not significantly altered. In summary, IGFBP4 expression appears to be associated with follicular selection, not with atresia, in the monkey ovary. The IGFBP4 is consistently expressed in healthy theca interna and in luteinized granulosa cells, likely under LH regulation. The IGFBP4 protease, PAPP-A, is widely expressed without apparent selectivity for IGFBP4-expressing follicles or for dominant follicles. These observations suggest that IGFBP4 or an IGFBP4 proteolytic product may be involved with LH-induced steroidogenesis and/or luteinization rather than with inhibition of follicular growth.

Dominant bovine ovarian follicular cysts express increased levels of messenger RNAs for luteinizing hormone receptor and 3 beta-hydroxysteroid dehydrogenase delta(4),delta(5) isomerase compared to normal dominant follicles.

The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17 alpha-hydroxylase, cytochrome P450 aromatase, 3 beta-hydroxysteroid dehydrogenase Delta(4),Delta(5) isomerase, LH, and FSH receptors and estrogen receptor-beta in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17 beta, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17 beta (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17 beta concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-beta mRNAs than other groups. In summary, increased expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17 beta concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.

Reverse transcription-polymerase chain reaction analysis of genes involved in gonadal differentiation in pigs.

In mammals, testis development is initiated in the embryo as a response to the expression of the sex-determining gene, SRY. The time course of SRY expression during gonadal differentiation in the male has been described in detail only in mice and sheep. In this study, we used reverse transcription-polymerase chain reaction analysis to define the SRY transcription profile in pig genital ridges. SRY transcripts were first detectable from 23 days postcoitum (dpc), then declined sharply after 35 dpc. None were detected at 60 dpc. In addition, we analyzed temporal expression of other genes known to be involved in mammalian sex determination: WT-1, SF-1, SOX9, and AMH. A key stage seems to be 28 dpc, in which SOX9 expression switches between the male and female, and AMH expression begins to attest to Sertoli cell differentiation and to correspond to seminiferous cord formation in the male. Expression of gonadotropin receptors and aromatase was also investigated in porcine gonads, and we showed that their transcripts were detected very early on, especially in the male: 25 dpc for the LH receptor (rLH) and aromatase, and 28 dpc for the FSH receptor (rFSH). In the female, aromatase transcripts were not detected until 70 dpc, and rFSH expression occurred later: at 45 dpc at the onset of meiosis. Moreover, no difference was observed between the sexes for the onset of rLH transcription at 25 dpc. Such a thorough study has never been performed on pigs; developmental analysis will be useful for investigating sex-reversed gonads and determining ontogeny in intersexuality, a common pathology in pigs.

Control of FSH receptor mRNA expression in rat granulosa cells by 3′,5′-cyclic adenosine monophosphate, activin, and follistatin

FSH is required to maintain FSH and LH/hCG receptors at elevated steady-state levels after receptor induction. Although this function of FSH is mediated by cAMP, how cAMP level is related to the maintenance of gonadotropin receptors is unknown. To investigate cAMP’s effect on changes in the levels of FSH receptor mRNAs in rat granulosa cells, total RNA from cells was prepared and analyzed by Northern blots. Incubation with 8-Br-cAMP for 24 h produced a dose-related increase in FSH receptor mRNA in granulosa cells of DES-primed immature rats. On the other hand, 8-Br-cAMP, washed at 24 h, exerted inverse dose-related effects on FSH receptor mRNA levels at 96 h. The addition of 1 mM cAMP resulted in higher levels of FSH receptor mRNA than that induced by 0.2 mM cAMP at 24 h, while 0.2 mM cAMP is as effective as 1–2 mM cAMP for the induction of FSH receptor mRNA at 96 h. To further analyze cAMP’s role in the production of activin in granulosa cells, we measured activin levels in the culture medium after the addition of 8-Br-cAMP. The levels of activin A were suppressed by the addition of 8-Br-cAMP in a dose–dependent manner. In addition, the procedure by which 8-Br-cAMP was removed after 24 h incubation showed that the level of activin in the medium increased after medium change. With regard to the actions of activin A on gonadotropin receptors, our laboratory has demonstrated that activin A increases the levels of FSH receptor mRNAs. Therefore, cAMP has a negative effect on FSH receptor expression by suppressing the activin level. Since follistatin production is up-regulated by cAMP in this system, we examined the effect of follistatin on FSH receptor mRNA level, which is induced by activin and FSH. Cotreatment with follistatin (0–100 ng/ml) and activin (50 ng/ml) in the presence of FSH (30 ng/ml) caused a significant reduction in FSH receptor mRNA levels induced by activin. Based on these observations, it is possible that cAMP has both stimulatory and inhibitory effects on the expression of gonadotropin receptors, and the overall influence of cAMP on their expression might be determined by the integration of such opposing effects.

Regulation of luteinizing hormone-receptor and follicle-stimulating hormone-receptor messenger ribonucleic acid levels during development in the neonatal mouse ovary.

Normal gonadal development is dependent upon stimulation by the gonadotropic hormones, and it is likely that control of ovarian development is regulated by expression of gonadotropin receptors. In this study, quantitative changes in LH- and FSH-receptor mRNA levels were measured in the ovary during development in normal mice and in hypogonadal (hpg/hpg) mice, which lack circulating gonadotropins. The relative abundance of alternate transcripts encoding LH and FSH receptors was also determined. Results show that shortened transcripts of the receptors were abundant at all ages. Full-length transcripts of the LH receptor were not detectable until postnatal Day 5 although shortened transcripts encoding the extracellular domain of the receptor were present from birth. Between Days 5 and 7, LH-receptor transcript levels showed a marked increase in normal animals but no change in hpg/hpg animals. FSH-receptor transcripts encoding all domains of the receptor were detectable at low levels at birth, increased in concentration between Days 3 and 5, and peaked at Day 10. In hpg/hpg animals, FSH-receptor mRNA levels were normal up to Day 7 but failed to increase thereafter. These results show that early development of both LH- and FSH-receptor mRNA levels in the ovary is gonadotropin-independent. This coincides with early folliculogenesis to the primary stage. Further development of LH-receptor mRNA levels is gonadotropin-dependent although FSH-receptor mRNA levels continue to increase independently until early secondary follicles are present.

Messenger ribonucleic acid for insulin-like growth factors-I and -II, insulin-like growth factor-binding protein-2, gonadotropin receptors, and steroidogenic enzymes in porcine follicles.

Several aspects of growth, development, and steroidogenesis in ovarian follicles are controlled by the interaction of insulin-like growth factors (IGF-I and IGF-II) and gonadotropins (LH and FSH). The objective of this study was to determine the developmental stage and location at which mRNA for IGF, IGF-binding protein (IGFBP)-2, gonadotropin receptors, and steroidogenic enzyme genes are expressed within porcine follicles. Ovaries from pigs on Days 10 (n = 3; midluteal phase) and 19 (n = 3; preovulatory phase) of the estrous cycle were collected, frozen, and sectioned. The mRNA for IGF-I, IGF-II, IGFBP-2, LH receptor, FSH receptor, cytochrome P450 side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase (P45017 alpha), and aromatase (P450arom) were detected by in situ hybridization, and relative amounts were determined by image analysis. Expression of mRNA for IGF-I and IGF-II was cell specific (granulosa and theca interna cells, respectively) and increased with follicular size. The IGFBP-2 mRNA was expressed in greater amounts for theca interna cells than for granulosa cells. The pattern of expression for LH and FSH receptors diverged as follicles grew. For LH receptor, mRNA was detected in the theca interna cells of small antral follicles and in both the theca interna and granulosa cells of large antral follicles. Amount of LH receptor mRNA increased with follicular diameter. For FSH receptor, mRNA increased as follicles grew to a small antral size. However, FSH receptor mRNA disappeared in large antral follicles on Day 19 of the estrous cycle. Steroidogenic enzyme mRNA for 3 beta-HSD (theca interna cells), P450scc (theca interna cells), P45017 alpha (theca interna cells), and P450arom (granulosa cells) increased with follicular diameter. In summary, coordinated expression of IGF, IGFBP-2, gonadotropin receptors, and steroidogenic enzyme mRNA throughout different stages of follicular development was observed. Changes in mRNA suggested increased IGF-I and -II action and a switch from FSH to LH dependence in porcine preovulatory follicles.

Trigone of human urinary bladder is a novel site of luteinizing hormone / chorionic gonadotropin receptor expression.

Trigone of human urinary bladder is a novel site of luteinizing hormone / chorionic gonadotropin receptor expression.

3' untranslated region-mediated regulation of luteinizing hormone/human chorionic gonadotropin receptor expression.

Multiple luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor mRNAs (6.7, 4.4, 2.6, and 1.8 kb) have been identified in the rat ovary. Our laboratory has previously cloned a 3.5 kb cDNA that corresponds to the 3' untranslated region (3' UTR) of the 6.7 kb transcript, the major LH/hCG receptor mRNA in rat ovary. In order to determine the effects of the 3' UTR on receptor expression, we have constructed cDNAs corresponding to the open reading frame of LH/hCG receptor or luciferase, plus these constructs with the addition of the 3' UTRs associated with the short (4.4 kb) and long (6.7 kb) LH/hCG receptor transcripts, and measured receptor or luciferase expression in 293 cells transformed with large T antigen (293T). Ligand binding analysis with 125I-hCG revealed that the 3' UTR inhibited receptor expression, which occurs through posttranscriptional events. First, the 3' UTRs reduced receptor mRNA half-life in actinomycin D-arrested cells, as compared to the open reading frame alone. Second, LH/hCG receptor mRNAs with the long 3' UTR associated with significantly fewer ribosomes. The effect of the LH/hCG receptor 3' UTRs on luciferase expression was also determined. The short 3' UTR increased luciferase activity, whereas the long 3' UTR decreased luciferase expression. Thus, the short 3' UTR exerts opposite effects on receptor and luciferase expression. However, sequences in the long 3' UTR are sufficient to inhibit both receptor and luciferase expression in 293T cells.

Secretion of steroids, growth factors, and cytokines by immortalized mouse granulosa cell lines.

The expression and function of gonadotropin receptors, and the secretion of steroids, transferrin, and cytokines were investigated in three immortalized (single transfection with v-myc) mouse granulosa cell lines (GRM01, GRM01L, and GRM02). A dose-dependent increase in progesterone production was obtained in GRM01 and GRM02 cells after addition of LH, FSH, modulators of the adenylate cyclase enzyme system, and cAMP analogues. The LH-induced release of progesterone was already detectable in GRM02 cells after 8 h and was related to incubation time and cell number. Both epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) induced the secretion of progesterone in GRM02 cells, while no effect was obtained with TGF beta. LH receptor concentration was highest in the GRM02 cell line. FSH receptor mRNA was visualized in GRM01 and GRM02 cells. Aromatase activity in GRM02 cells was induced by androgens and inhibited by aromatase inhibitors. Whereas all cell lines were able to secrete transferrin, only in GRM01 cells was transferrin secretion increased significantly by LH. FSH did not affect transferrin secretion in the three cell lines, in contrast to forskolin or 8-bromo-cAMP. The immortalized mouse granulosa cell lines were able to express and release several growth factors. The expression and secretion of activin, inhibin, TGF beta, EGF, TGF alpha, insulin-like growth factor II, fibroblast growth factor (acidic and basic), platelet-derived growth factor, and interleukin-6 suggest an autocrine or paracrine role for these factors in follicular differentiation and function. In conclusion, these cells, derived from mural granulosa cells and immortalized in a preovulatory state, can be used to study granulosa cell physiology or to study the role of granulosa cells and their derivatives in the process of follicular maturation, fertilization, and early embryonic development.

Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone-dependent steroidogenesis.

We have recently succeeded in immortalizing rat granulosa cells by co-transfection with SV-40 DNA and the Ha-ras oncogene. These cells lost their response to gonadotropins, but expressed the cytochrome P450scc mitochondrial system enzymes and produced progesterone and 20 alpha-hydroxy-4-pregnan-3-one (20 alpha-OH-P) upon cAMP stimulation (Suh, B. S., and A. Amsterdam. 1990. Endocrinology. 127:2489-2500; Hanukoglu, I., B. S. Suh, S. Himmelhoch, and A. Amsterdam. 1990. J. Cell Biol. 111:1973-1981). In an attempt to restore the steroidogenic response to gonadotropins in immortalized cells, lutropin/choriogonadotropin (LH/CG-R) receptor expression plasmid was prepared by introducing the complete coding region of LH receptor cDNA (McFarland, K. C., R. Sprengel, H. S. Phillips, M. Köhler, N. Rosemblit, K. Nikolics, D. L. Segaloff, and P. H. Seeburg. 1989. Science (Wash. DC). 245:494-499) into a SV-40 early promoter based eucaryotic expression vector. Granulosa cells from preovulatory follicles were transfected with this LH receptor expression plasmid, together with SV-40 DNA and the Ha-ras oncogene. Cell lines obtained after this triple transfection accumulated cAMP in a dose-dependent manner in response to hCG. Moreover, they produced progesterone and 20 alpha-OH-P upon hCG stimulation with an ED50 of 125 pM and 75 pM, respectively, which is within the physiological range. Concomitantly with hCG induced differentiation, inhibition of cell proliferation was evident following stimulation with hormone concentrations as low as 40 pM. The number of hCG receptor sites per cell after numerous passages and several freezing and thawing cycles was 1.9 x 10(4), they showed a Kd of 180 pM. Stimulation with hCG induced pronounced morphological and biochemical changes in these cells including formation of mitochondrial located adrenodoxin, a marker enzyme for enhanced steroidogenesis. These findings make possible the expression in immortalized granulosa cells, of selectively mutated receptor molecules which preserve their steroidogenic potential, thereby opening the way to analysis of structure-function relationships of the receptor molecule.

Isolation and characterization of native activin B.

To examine whether activin binds to follistatin, an activin-binding protein, to form a complex in vivo, we attempted to purify activin-follistatin complex from porcine follicular fluid. Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B are present as a complex with follistatin in the follicular fluid. Reverse-phase high performance liquid chromatography of the purified complex yielded follistatin and activins A, AB, and B. The activity of the purified activin B was found to be significantly lower than those of other activins in various assay systems such as stimulation of follicle-stimulating hormone secretion, induction of erythrodifferentiation, and potentiation of expression of gonadotropin receptors on ovarian cells. Moreover, binding of 125I-activin A to erythroleukemic cells which are activin-responsive was competed by activin B with approximately 10-fold lower potency compared with other activins. In contrast to these results, activin B was proved to have a potent Xenopus mesoderm-inducing activity, comparable with that of other activins. This indicates that, unlike activins A and AB, activin B can only elicit mesoderm-inducing activity and cannot function in other biological systems, suggesting a specific role of activin B in early development and unknown biological functions.

Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin.

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.

Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.

Hormonal and basement membrane markers for immunoidentification of cultured human trophoblast cells

Improvement of human chorionic villi cells in vitro culture has been obtained by supplementing the medium with 30% human fetal cord serum, instead of fetal calf serum. Expression of human chorionic gonadotropin, estrogen and progesterone receptors, laminin, laminin receptors, and type IV collagen has been studied on first subculture passages (4–7 weeks) by immunoperoxidase method. A minority of the cultured cells were positive for estrogen receptors, the majority were positive for progesterone receptors, while all cells were negative for human chorionic gonadotropin. Cultured cells showed variable positive immunostaining for basement membrane markers like laminin, and type IV collagen, and for laminin receptors. Detection of both progesterone receptors and laminin, or type IV collagen, excluded fibroblast contamination and could then be useful for quick identification of cultured trophoblast cells.

Inhibitory actions of adenosine on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells.

To determine the effects of adenosine on follicle-stimulating hormone (FSH)-induced differentiation, granulosa cells isolated from the ovaries of diethylstilbestrol-treated immature rats were cultured with increasing concentrations of the nucleoside and modulators of adenosine action. Although adenosine had no effect on basal granulosa cell function during 48 h of culture, concentrations of the nucleoside from 10 microM to 1 mM progressively inhibited FSH-induced responses, including progesterone production and expression of FSH and luteinizing hormone (LH) receptors. Adenosine had biphasic effects on FSH-stimulated cAMP accumulation, causing inhibition of cAMP production at 10 to 100 microM and stimulation at higher concentrations. The enhancement of cAMP production by 1 mM adenosine occurred during the first 24 h of culture, while both 100 microM and 1 mM adenosine reduced FSH-stimulated cAMP production from 24 to 48 h. The inhibitory effects of adenosine were prevented by adenosine deaminase and dipyridamole, an inhibitor of adenosine transport, and were antagonized by 1-methyl-3-isobutylxanthine. The inhibition of cAMP and progesterone production by adenosine was partially overcome when cells were washed and reincubated with forskolin, but not with FSH. Adenine, guanosine, and inosine at concentrations of 100 microM did not modify FSH-induced cAMP formation or LH receptor induction. These results indicate that adenosine exerts predominantly inhibitory actions on hormone-induced granulosa cell differentiation, as manifested by prominent reductions in steroidogenesis and gonadotropin receptor expression.

GnRH agonist-induced inhibitory and stimulatory effects during ovarian follicular maturation

The in vivo regulation of ovarian gonadotropin and prolactin receptors and adenylate cyclase activity by FSH, and the potent GnRH agonist [D-Ala 6]des-Gly 10-GnRH N-ethylamide (GnRHa), was studied in immature hypophysectomized diethylstilbestrol-implanted rats. During FSH treatment over a 48 h period, FSH receptors increased 2-fold with the maximum response during the first 12 h, whereas LH and prolactin receptors increased by 10-fold and 6-fold with the maximum response from 12 to 48 h. Administration of GnRHa at any time during the 48 h period of FSH treatment inhibited the subsequent development of gonadotropin and PRL receptors. In contrast, administration of a single dose of 10 μg GnRHa after 48 h of FSH treatment stimulated follicular luteinization and caused increases in basal adenylate cyclase activity, ovarian weight and PRL receptor content, and concomitant decreases in gonadotropin receptors and adenylate cyclase responses. In the immature follicles of animals not primed with FSH, GnRHa caused progressive inhibition of FSH-sensitive adenylate cyclase activity, with a decrease in FSH receptors, but increased both basal and GMP-P(NH)P-stimulated adenylate cyclase activities. These results demonstrate that GnRHa causes marked inhibition of gonadotropin receptor expression in the basal and FSH-stimulated ovary. This decrease in gonadotropin receptors is an important component of the mechanism by which GnRH agonists inhibit ovarian gonadotropin-sensitive adenylate cyclase activity. In addition, these peptides exert stimulatory effects upon ovarian weight and basal adenylate cyclase activity, and cause an increase in PRL receptors and luteinization of mature ovarian follicles. The stimulatory actions of GnRH agonists upon ovarian function are more prominent with increasing maturation of the granulosa cell.