Steroidogenesis In Human Granulosa Cells Research Articles

DEHP Decreases Steroidogenesis through the cAMP and ERK1/2 Signaling Pathways in FSH-Stimulated Human Granulosa Cells.

DEHP is an endocrine disruptor that interferes with the function of the female reproductive system. Several studies suggested that DEHP affects steroidogenesis in human and rodent granulosa cells (GC). Some studies have shown that DEHP can also affect the FSH-stimulated steroidogenesis in GC; however, the mechanism by which DEHP affects hormone-challenged steroidogenesis in human GC is not understood. Here, we analyzed the mechanism by which DEHP affects steroidogenesis in the primary culture of human cumulus granulosa cells (hCGC) stimulated with FSH. Cells were exposed to DEHP and FSH for 48 h, and steroidogenesis and the activation of cAMP and ERK1/2 were analyzed. The results show that DEHP decreases FSH-stimulated STAR and CYP19A1 expression, which is accompanied by a decrease in progesterone and estradiol production. DEHP lowers cAMP production and CREB phosphorylation in FSH but not cholera toxin- and forskolin-challenged hCGC. DEHP was not able to decrease steroidogenesis in cholera toxin- and forskolin-stimulated hCGC. Furthermore, DEHP decreases FSH-induced ERK1/2 phosphorylation. The addition of EGF rescued ERK1/2 phosphorylation in FSH- and DEHP-treated hCGC and prevented a decrease in steroidogenesis in the FSH- and DEHP-treated hCGC. These results suggest that DEHP inhibits the cAMP and ERK1/2 signaling pathways, leading to the inhibition of steroidogenesis in the FSH-stimulated hCGC.

Epigallocatechin-3-gallate stimulates StAR expression and progesterone production in human granulosa cells through the 67-kDa laminin receptor-mediated CREB signaling pathway.

Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechins extracted from green tea. The health benefits of EGCG have been extendedly studied. Ovarian steroidogenesis plays a pivotal role in maintaining normal reproductive function. Granulosa cells in the ovary are essential for steroid hormone production. To date, the effect of EGCG on steroidogenesis in human granulosa cells remains unclear. In the present study, we examine the physiological concentrations of EGCG on steroidogenesis in a steroidogenic human granulosa-like tumor cell line, KGN. Our results demonstrate that treatment with EGCG upregulates steroidogenic acute regulatory protein (StAR) expression and increases progesterone (P4) production. EGCG does not affect the expression levels of other steroidogenesis-related enzymes, such as P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. In addition, we identify the expression of 67-kDa laminin receptor (67LR) in KGN cells. Moreover, EGCG-induced StAR expression and P4 production require the 67LR-mediated activation of the PKA-CREB signaling pathway. These results provide a better understanding of the function of EGCG on ovarian steroidogenesis, which may lead to the development of alternative therapeutic approaches for reproductive disorders.

Hypo-glycosylated hFSH drives ovarian follicular development more efficiently than fully-glycosylated hFSH: enhanced transcription and PI3K and MAPK signaling.

Does hypo-glycosylated human recombinant FSH (hFSH18/21) have greater in vivo bioactivity that drives follicle development in vivo compared to fully-glycosylated human recombinant FSH (hFSH24)? Compared with fully-glycosylated hFSH, hypo-glycosylated hFSH has greater bioactivity, enabling greater follicular health and growth in vivo, with enhanced transcriptional activity, greater activation of receptor tyrosine kinases (RTKs) and elevated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling. Glycosylation of FSH is necessary for FSH to effectively activate the FSH receptor (FSHR) and promote preantral follicular growth and formation of antral follicles. In vitro studies demonstrate that compared to fully-glycosylated recombinant human FSH, hypo-glycosylated FSH has greater activity in receptor binding studies, and more effectively stimulates the PKA pathway and steroidogenesis in human granulosa cells. This is a cross-sectional study evaluating the actions of purified recombinant human FSH glycoforms on parameters of follicular development, gene expression and cell signaling in immature postnatal day (PND) 17 female CD-1 mice. To stimulate follicle development in vivo, PND 17 female CD-1 mice (n = 8-10/group) were treated with PBS (150 µl), hFSH18/21 (1 µg/150 µl PBS) or hFSH24 (1 µg/150 µl PBS) by intraperitoneal injection (i.p.) twice daily (8:00 a.m. and 6:00 p.m.) for 2 days. Follicle numbers, serum anti-Müllerian hormone (AMH) and estradiol levels, and follicle health were quantified. PND 17 female CD-1 mice were also treated acutely (2 h) in vivo with PBS, hFSH18/21 (1 µg) or hFSH24 (1 µg) (n = 3-4/group). One ovary from each mouse was processed for RNA sequencing analysis and the other ovary processed for signal transduction analysis. An in vitro ovary culture system was used to confirm the relative signaling pathways. The purity of different recombinant hFSH glycoforms was analyzed using an automated western blot system. Follicle numbers were determined by counting serial sections of the mouse ovary. Real-time quantitative RT-PCR, western blot and immunofluorescence staining were used to determine growth and apoptosis markers related with follicle health. RNA sequencing and bioinformatics were used to identify pathways and processes associated with gene expression profiles induced by acute FSH glycoform treatment. Analysis of RTKs was used to determine potential FSH downstream signaling pathways in vivo. Western blot and in vitro ovarian culture system were used to validate the relative signaling pathways. Our present study shows that both hypo- and fully-glycosylated recombinant human FSH can drive follicular growth in vivo. However, hFSH18/21 promoted development of significantly more large antral follicles compared to hFSH24 (P < 0.01). In addition, compared with hFSH24, hFSH18/21 also promoted greater indices of follicular health, as defined by lower BAX/BCL2 ratios and reduced cleaved Caspase 3. Following acute in vivo treatment with FSH glycoforms RNA-sequencing data revealed that both FSH glycoforms rapidly induced ovarian transcription in vivo, but hypo-glycosylated FSH more robustly stimulated Gαs and cAMP-mediated signaling and members of the AP-1 transcription factor complex. Moreover, hFSH18/21 treatment induced significantly greater activation of RTKs, PI3K/AKT and MAPK/ERK signaling compared to hFSH24. FSH-induced indices of follicle growth in vitro were blocked by inhibition of PI3K and MAPK. RNA sequencing of mouse ovaries. Data will be shared upon reasonable request to the corresponding author. The observations that hFSH glycoforms have different bioactivities in the present study employing a mouse model of follicle development should be verified in nonhuman primates. The gene expression studies reflect transcriptomes of whole ovaries. Commercially prepared recombinant human FSH used for ovarian stimulation in human ART is fully-glycosylated FSH. Our findings that hypo-glycosylated hFSH has greater bioactivity enabling greater follicular health and growth without exaggerated estradiol production in vivo, demonstrate the potential for its development for application in human ART. This work was supported by NIH 1P01 AG029531, NIH 1R01 HD 092263, VA I01 BX004272, and the Olson Center for Women's Health. JSD is the recipient of a VA Senior Research Career Scientist Award (1IK6 BX005797). This work was also partially supported by National Natural Science Foundation of China (No. 31872352). The authors declared there are no conflicts of interest.

Toll-Like Receptor 4 Inhibits Estradiol Secretion via NF-κB Signaling in Human Granulosa Cells.

Toll-like receptor 4 (TLR4) may play a critical role in regulating follicular development. Data are scarce on the role of TLR4 in the follicle. This study investigated the effects of TLR4 on steroidogenesis in human granulosa cells. Immunohistochemical analysis revealed stage-specific expression of TLR4 in the mouse ovarian cycle, and immunofluorescence showed TLR4 expression in the human granulosa-like tumor cell line (KGN). TLR4 agonist lipopolysaccharides (LPS) significantly inhibited follicular development and synthesis of estradiol (E2) in mice. In KGN cells, TLR4 activation significantly inhibited CYP19A1, FSHR and StAR, and TLR4 inhibition reversed these effects. TLR4 activation also inhibited forskolin-induced secretion of E2 by inhibiting CYP19A1, with no effect on progesterone. Further studies showed activation of p38, JNK and NF-κB signaling after TLR4 activation. Subsequent analyses showed that an NF-κB antagonist reversed the inhibitory effects on CYP19A1 expression and E2 secretion. Together, our results suggest that TLR4 activation may suppress CYP19A1 expression and E2 secretion via NF-κB signaling in human granulosa cells, with important implications for the regulation of ovarian pathophysiology.

Paraben concentrations found in human body fluids do not exert steroidogenic effects in human granulosa primary cell cultures

In cosmetics and food products, parabens are widely used as antimicrobial agents. Reports have suggested that parabens may be linked to infertility, owing to their effects on basal steroidogenesis properties or their capacity to inflict mitochondrial damage. Despite growing concerns about parabens as endocrine disruptors, it is unclear whether they affect any of these actions in humans, particularly at environmentally relevant concentrations. In this work, an in vitro primary culture of human granulosa cells was used to evaluate steroidogenesis, based on the assessment of progesterone production and regulation of critical steroidogenic genes: CYP11A1, HSD3B1, CYP19A1, and HSD17B1. The effects of two commercially relevant parabens, methylparaben (MPB) and butylparaben (BPB), were screened. Cells were exposed to multiple concentrations ranging from relatively low (typical environmental exposure) to relatively high. The effect was assessed by the parabens’ ability to modify steroidogenic genes, progesterone or estradiol production, and on mitochondrial health, by evaluating mitochondrial activity as well as mtDNA content. Neither MPB nor BPB showed any effect over progesterone production or the expression of genes controlling steroid production. Only BPB affected the mitochondria, decreasing mtDNA content at supraphysiological concentrations (1000 nM). Prolonged exposure to these compounds produced no effects in neither of these parameters. In conclusion, neither MPB nor BPB significantly affected basal steroidogenesis in granulosa cells. Although evidence supporting paraben toxicity is prevalent, here we put forth evidence that suggests that parabens do not affect basal steroidogenesis in human granulosa cells.

Bisphenol S Impaired Human Granulosa Cell Steroidogenesis in Vitro.

Bisphenol S (BPS) is a structural analog of the endocrine disruptor bisphenol A (BPA); it is the main BPA replacement in the plastics industry. Previous studies have shown that BPA and BPS exhibit similar effects on reproduction in fish and rodent species. BPS reportedly alters steroidogenesis in bovine granulosa cells. Luteinised granulosa cells collected from 59 women who were undergoing an in vitro fertilization procedure were cultured for 48 h in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). BPS exposure was investigated by assessing follicular fluids from these 59 women for their BPS content. Culture medium, cells, total messenger RNA (mRNA) and total protein extracted from the luteinised granulosa cells were examined for oestradiol and progesterone secretion, cellular proliferation, viability, gene expression, steroidogenic enzyme expression and cell signaling. BPS was measured in follicular fluids using mass spectrometry. Exposure of granulosa cells to 10 or 50 µM BPS for 48 h induced a 16% (p = 0.0059) and 64% (p < 0.0001) decrease, respectively, in progesterone secretion; 50 µM BPS decreased oestradiol secretion by 46% (p < 0.0001). Ten µM BPS also tended to reduce CYP11A1 protein expression by 37% (p = 0.0947) without affecting HSD3B1 and CYP19A1 expression. Fifty µM BPS increased ERRγ expression. Environmental levels of BPS (nanomolar range) did not induce changes in steroidogenesis in human granulosa cells. The effects of BPS were observed after only 48 h of BPS exposure. These acute effects might be similar to chronic effects of physiological BPS levels.

Exposure to Mono-n-Butyl Phthalate in Women with Endometriosis and Its Association with the Biological Effects on Human Granulosa Cells.

To study the association between urinary phthalate metabolite levels, endometriosis, and their effects on human granulosa cells, we recruited patients who underwent laparoscopy to confirm endometriosis (n = 123) and control patients (n = 78). Liquid chromatography–tandem mass spectrometry was used to measure the following five urinary phthalate metabolites: mono-n-butyl phthalate (MnBP), mono(2-ethylhexyl) phthalate, monobenzyl phthalate, mono(2-ethyl-5-oxo-hexyl) phthalate, and mono(2-ethyl-5-hydroxyhexyl) phthalate. Urinary MnBP levels were higher in patients with endometriosis than in controls after multivariable logistic regression including the number of deliveries, body mass index, and use of medicine as covariables. MnBP correlates with other phthalate metabolites. Previous studies found that endometriosis was a detrimental condition for granulosa cells. In our study, we observed whether MnBP affected granulosa cells. MnBP treatment altered the gene expression of BIRC5, BUB1B, CDC20, cyclin B1, IL-1β, TNF-α, inhibin-B, StAR, and P450ssc and attenuated the ratio of the mitochondrial membrane potential in human granulosa cells. Moreover, MnBP decreased the expression of the anti-Mullerian hormone. These findings suggest that MnBP concentration is associated with endometriosis and may affect the health and steroidogenesis of human granulosa cells.

Vitamin D attenuates the effect of advanced glycation end products on anti-Mullerian hormone signaling

Vitamin D3 (1,25-dihydroxyvitamin D3, VD3) in vitro attenuates the effect of the pro-inflammatory advanced glycation end products (AGEs) on steroidogenesis in human granulosa cells (GCs) by downregulating the receptor for AGEs (RAGE). It has been shown that VD3 alone downregulates anti-Mullerian hormone (AMH) type 2 receptor (AMHR-2) gene expression and suppresses AMH-induced SMAD 1/5/8 phosphorylation in granulosa cells. However, the effect of AGEs, in the absence or presence of VD3, on AMH action in GCs has not been studied. Using human GCs, this study showed that human glycated albumin (HGA), an in vitro representative for AGEs, upregulated AMHR-2 mRNA but did not alter AMH mRNA expression levels. VD3 inhibited the HGA-induced increase in AMHR-2 mRNA expression levels. In KGN granulosa cell line, recombinant AMH induced SMAD 1/5/8 phosphorylation. HGA augmented the recombinant AMH-induced SMAD 1/5/8 phosphorylation while the addition of VD3 to HGA attenuated the recombinant AMH-induced SMAD 1/5/8 phosphorylation. Thus, AGEs could potentially affect folliculogenesis as reflected by changes in AMH signaling. These findings have significant implications for women with polycystic ovary syndrome who have significantly elevated serum and ovarian AGEs.

Activin A, B and AB decrease progesterone production by down-regulating StAR in human granulosa cells

Activins are homo- or heterodimers of inhibin β subunits that play important roles in the reproductive system. Our previous work has shown that activins A (βAβA), B (βBβB) and AB (βAβB) induce aromatase/estradiol, but suppress StAR/progesterone production in human granulosa-lutein cells. However, the underlying molecular determinants of these effects have not been examined. In this continuing study, we used immortalized human granulosa cells (SVOG) to investigate the effects of activins in regulating StAR/progesterone and the potential mechanisms of action. In SVOG cells, activins A, B and AB produced comparable down-regulation of StAR expression and progesterone production. In addition, all three activin isoforms induced equivalent phosphorylation of both SMAD2 and SMAD3. Importantly, the activin-induced down-regulation of StAR, increase in SMAD2/3 phosphorylation, and decrease in progesterone were abolished by the TGF-β type I receptor inhibitor SB431542. Interestingly, the small interfering RNA-mediated knockdown of ALK4 but not ALK5 reversed the activin-induced suppression of StAR. Furthermore, the knockdown of SMAD4 or SMAD2 but not SMAD3 abolished the inhibitory effects of all three activin isoforms on StAR expression. These results provide evidence that activins A, B and AB down-regulate StAR expression and decrease progesterone production in human granulosa cells, likely via an ALK4-mediated SMAD2/SMAD4-dependent pathway. Our findings provide important insights into the molecular mechanisms underlying the regulatory effects of activins on human granulosa cell steroidogenesis.

Effects of recombinant activins on steroidogenesis in human granulosa-lutein cells.

Exerting a broad range of biological effects in various tissues, activins are homo- or heterodimers of activin/inhibin β-subunits (βA, βB, βC, and βE in humans). Although activins A (βAβA), B (βBβB), AB (βAβB), and AC (βAβC) have been demonstrated in the female reproductive system, little is known about their individual functions in the ovary. To investigate the biological roles and activities of activins in regulating steroidogenesis in human granulosa cells. Human granulosa-lutein cells obtained from 32 patients undergoing in vitro fertilization were used to investigate the effects of activin A, B, AB, and AC on the expression of steroidogenic enzymes and steroid production. An academic research center. mRNA and protein levels were examined by reverse transcription quantitative real-time PCR and Western blot analysis, respectively. The production of estradiol and progesterone was measured by enzyme immunoassay. P450 aromatase, FSH receptor, and estradiol levels were increased, whereas steroidogenic acute regulatory protein (StAR), LH receptor, and progesterone levels were decreased after treatment with activin A, B, and AB, but not activin AC. FSH or LH induced the production of aromatase/estradiol and StAR/progesterone; however, pretreatment with activin A, B, or AB enhanced the effects of gonadotropins on aromatase/estradiol, but suppressed their effects on StAR/progesterone. Treatment with activin A, B, or AB induced the phosphorylation of SMA- and MAD-related proteins (SMAD2 and 3), whereas activin AC had no such effects. Furthermore, co-culture of activin AC (1-100 ng/mL) with activin A (25 ng/mL) did not alter the effects of activin A on P450 aromatase or StAR mRNA levels. Activin A, B, and AB have similar effects on steroidogenesis in human granulosa cells. In contrast, activin AC is not biologically active and does not act as a competitive antagonist.

Resistin decreases insulin-like growth factor I–induced steroid production and insulin-like growth factor I receptor signaling in human granulosa cells

To identify resistin in human ovarian follicles and investigate the effect and the molecular mechanisms associated with resistin on steroidogenesis in human granulosa cells (GCs). The effects of recombinant human resistin on the secretion of progesterone (P) and estradiol (E2) by cultured human GCs were investigated. Academic institutions. Twenty infertile and healthy women undergoing IVF. Primary human GC cultures stimulated with recombinant human resistin (10 ng/mL). Determination of messenger RNA (mRNA) and protein expression of resistin in fresh human GCs by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblot and immunohistochemistry, respectively; measurement of P and E2 levels in the conditioned media by radioimmunoassay; determination of cell proliferation by tritiated thymidine incorporation; and analysis of signaling pathways activation by immunoblot analysis. Human GCs and theca cells express resistin. In primary human GCs, resistin decreases P and E2 secretion in response to insulin-like growth factor I (IGF-I). This was associated with a reduction in the P450 aromatase and P450scc (cholesterol side-chain cleavage cytochromes P450) (P450scc) protein levels but not those of 3β-hydroxysteroid dehydrogenase (3β-HSD) or steroidogenic acute regulatory protein (StAR) and with a decrease in IGF-I-induced IGF-I receptor and mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Resistin treatment does not affect IGF-I-induced cell proliferation and basal steroidogenesis (there is no IGF-I or follicle-stimulating hormone stimulation). In the basal state, resistin rapidly stimulates Akt and MAPK ERK1/2 and p38 phosphorylation in primary human GCs. Resistin is present in human GCs and theca cells. It decreases P and E2 secretion, P450scc and P450 aromatase protein levels, and IGF-IR signaling in response to IGF-I in primary human GCs.

Thyroglobulin gene is associated with premature ovarian failure

Variants of the thyroglobulin gene were significantly associated with premature ovarian failure in a Korean population. Three single nucleotide polymorphisms and one haplotype were found to be associated with a significant increase in the risk for premature ovarian failure.

Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP) signaling to inhibit steroidogenesis in human granulosa cells

BackgroundObesity has been linked to an increased risk of female infertility. Leptin, an adipocytokine which is elevated during obesity, may influence gonadal function through modulating steroidogenesis in granulosa cells.MethodsThe effect of leptin on progesterone production in simian virus 40 immortalized granulosa (SVOG) cells was examined by Enzyme linked immunosorbent assay (ELISA). The effect of leptin on the expression of the steroidogenic enzymes (StAR, P450scc, 3betaHSD) in SVOG cells was examined by real-time PCR and Western blotting. The mRNA expression of leptin receptor isoforms in SVOG cells were examined by using PCR. SVOG cells were co-treated with leptin and specific pharmacological inhibitors to identify the signaling pathways involved in leptin-reduced progesterone production. Silencing RNA against leptin receptor was used to determine that the inhibition of leptin on cAMP-induced steroidogenesis acts in a leptin receptor-dependent manner.Results and ConclusionIn the present study, we investigated the cellular mechanisms underlying leptin-regulated steroidogenesis in human granulosa cells. We show that leptin inhibits 8-bromo cAMP-stimulated progesterone production in a concentration-dependent manner. Furthermore, we show that leptin inhibits expression of the cAMP-stimulated steroidogenic acute regulatory (StAR) protein, the rate limiting de novo protein in progesterone synthesis. Leptin induces the activation of ERK1/2, p38 and JNK but only the ERK1/2 (PD98059) and p38 (SB203580) inhibitors attenuate the leptin-induced inhibition of cAMP-stimulated StAR protein expression and progesterone production. These data suggest that the leptin-induced MAPK signal transduction pathway interferes with cAMP/PKA-stimulated steroidogenesis in human granulosa cells. Moreover, siRNA mediated knock-down of the endogenous leptin receptor attenuates the effect of leptin on cAMP-induced StAR protein expression and progesterone production, suggesting that the effect of leptin on steroidogenesis in granulosa cells is receptor dependent. In summary, leptin acts through the MAPK pathway to downregulate cAMP-induced StAR protein expression and progesterone production in immortalized human granulosa cells. These results suggest a possible mechanism by which gonadal steroidogenesis could be suppressed in obese women.

Regulation of Proliferation and Steroidogenesis in Human Granulosa Cells

Background and Aims : Human follicles selected for further development are thought to receive precise signals from gonadotropins and locally produced growth factors for survival. In this experiment, the mechanisms of the regulation of proliferation and steroidogenesis by GDF-9 and Br-cAMP in human ovarian cells were investigated. Methods : SVOG cells were cultured and treated with GDF-9 and Br-cAMP. The expression of HOXA7 and cofactors were examined by Western blot analysis and/or RT-PCR. The cell proliferation was measured by thymidine incorporation assay. Results : Treatment with GDF-9 resulted in a significant increase of cell proliferation. Although the treatment with Br-cAMP increased the expression of StAR protein, this treatment resulted in a significant decrease of cell proliferation compared with the control and no change in HOXA7 or its cofactors. Conclusion : The present study demonstrated that GDF-9 had a stimulatory effect and cAMP had an inhibitory effect on the proliferation of SVOG cells. These results may reflect the characteristics of this human cell line as luteinized granulosa cell. Therefore, this culture system will be useful for studying the effects of local factors and gonadotropin on the regulation of human luteinized granulosa cells in the future.

Direct effect of gonadotropin-releasing hormone agonist and antagonist on the growth, apoptosis and steroidogenesis in human granulosa cells from women undergoing in vitro fertilization

To evaluate the direct effect of a GnRH-a (leuprolide acetate, LA) and a GnRH-ant (antide, ANT) on the steroidogenesis, growth and programmed cell death (apoptosis) of human granulosa-luteal cells (GLC) obtained from patients undergoing ART.

Embryo implantation and GnRH antagonists: GnRH antagonists do not activate the GnRH receptor.

Recent suggestions that gonadotrophin-releasing hormone (GnRH) antagonists activate the GnRH receptor are discussed. Most of the studies cited in support of this suggestion are in-vitro studies, testing supra-pharmacological doses of GnRH analogues in cancer cell lines, whereas GnRH antagonists, e.g. ganirelix or cetrorelix, do not affect the steroidogenesis of human granulosa cells in vitro. In patients treated with GnRH antagonists prior to IVF or intracytoplasmic sperm injection (ICSI), oocyte maturity and fertilization rates are equal to those achieved following a long protocol of GnRH agonists. Although there is a tendency towards a lower pregnancy rate (not statistically significant) in the initial trials using GnRH antagonist with either recombinant FSH or human menopausal gonadotrophin (HMG) for ovarian stimulation, this new treatment option of GnRH antagonists facilitates short and simple treatment and improves the convenience and safety for the patient. As with GnRH agonists in the past, the clinical outcome of GnRH antagonist treatment will improve with time as more clinical experience is gained (learning curve) and the treatment protocol is optimized. Moreover, a GnRH agonist instead of human chorionic gonadotrophin (HCG) may be used for triggering ovulation and will decrease the cancellation rate and minimize the risk for developing ovarian hyperstimulation syndrome (OHSS).

Paracrine regulation of insulin-like growth factor I (IGF-I) an IGF-II on prostaglandins F2alpha and E2 synthesis by human corpus luteum in vitro: a possible balance of luteotropic and luteolytic effects.

The existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins has been demonstrated as well as the ability of IGF-I to positively affect steroidogenesis in human granulosa cells. Furthermore, we recently showed that IGF-I and IGF-II stimulate progesterone secretion by human luteal cells. As the PGs, PGE2 and PGF2alpha, are classically known to have luteotropic and luteolytic effects, we wanted to determine whether the IGFs could affect the human luteal phase by influencing the PG system. For this reason, human luteal cells were cultured for different times (12, 24, and 48 h) with IGF-I, IGF-II (10-100 ng/mL), and GH (100 ng/mL), and both PGs were assayed in the medium culture. We found that both IGF-I and IGF-II were able to stimulate PGE2 synthesis in a time- and dose-dependent way, whereas they both inhibited PGF2alpha production. GH, too, significantly reduced PGF2alpha synthesis; this effect was IGF-I mediated because it was reverted by increasing dilutions of an anti-IGF-I antibody. On the contrary, no GH effect was observed on PGE2 production. In conclusion, based on these data and on our previous results, we speculate that IGFs could influence luteal steroidogenesis through PG system.

Effect of leptin on basal and follicle-stimulating hormone stimulated steroidogenesis in human granulosa cells

Effect of leptin on basal and follicle-stimulating hormone stimulated steroidogenesis in human granulosa cells

Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines.

We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 microM of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMP-induced progesterone synthesis only moderately. Estradiol, insulin-like growth factor I, and insulin showed no significant effect on cAMP-induced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK- and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK- or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells.

Effects of GnRH on protein kinase C activity, Ca2+ mobilization and steroidogenesis of human granulosa cells.

Gonadotropin-releasing hormone (GnRH) and its agonists have been known to directly affect steroid hormone production in human granulosa cells. In this study, we examined effects of GnRH on Ca2+ mobilization, protein kinase C activity and steroidogenesis of human granulosa cells. Human granulosa cells were harvested by aspiration of follicles during oocyte retrieval for IVF. Test substances, that is, GnRH agonist ([D-Ser(TBu)6]-LHRH-EA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), A23187, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and nordihydroguaiaretic acid (NDGA) were added to HTF medium with human granulosa cells. At the end of a 5-h incubation period, progesterone and estradiol in the medium were extracted and measured. GnRH agonist (10(-9)M and 10(-8)M) significantly stimulated (P < 0.05) progesterone and estradiol accumulation in the medium. TPA (10(-7)M, 10(-8)M and 10(-9)M) also significantly stimulated (P < 0.05) progesterone accumulation. A23187 (10(-6)M) significantly stimulated progesterone accumulation (P < 0.05) and significantly enhanced (P < 0.05) the stimulatory effect of TPA on progesterone production. H-7 (10(-3)M, 10(-4)M and 10(-5)M) and NDGA (10(-3)M, 10(-4)M and 10(-5)M) significantly inhibited (P < 0.05) the effect of GnRH agonist on progesterone and estradiol production by the granulosa cells. The granulosa cells were loaded with 4 microM Fura 2-AM, and the ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 and 380 nm was calculated at 100-ms intervals. GnRH agonist (10(-7)M and 10(-6)M) increased (P < 0.05) [Ca2+]i significantly as compared with the control. These results suggested that the effects of GnRH on progesterone and estradiol production in human granulosa cells were mediated partially by calcium mobilization and partially by C-kinase activity, and protein kinase C, [Ca2+]i and lipoxygenase were suspected of being important intracellular messages for GnRH in human granulosa cells.