Granulosa Cell Culture System Research Articles

P-678 Cabergoline has inhibitory effects on FSH-induced cell differentiation and estrogen secretion in a primary culture system of rat ovarian granulosa cells

Abstract Study question How does cabergoline have effects on cell differentiation and hormone secretion induced by FSH stimulation in ovarian granulosa cells? Summary answer Cabergoline suppresses LH receptor expression and Estradiol (E2) secretion in primary cultures of rat ovarian granulosa cells by inhibiting cAMP production induced by FSH stimulation. What is known already Cabergoline is a dopamine receptor 2 agonist that has been used to treat hyperprolactinemia and, more recently, to prevent ovarian hyperstimulation syndrome. Although it has been previously reported that cabergoline suppresses E2 and Progesterone secretion induced by FSH stimulation in a primary culture system of rat ovarian granulosa cells, but the effect on LH receptor expression has not been investigated. It has also been reported that the activation of dopamine receptor 2 suppresses VEGF secretion and reduces the incidence of early-onset OHSS by inhibiting the VEGF/VEGFR2 pathway in research using human luteinized granulosa cells. Study design, size, duration We used a primary culture system of ovarian granulosa cells obtained from immature female rats treated with diethylstilbestrol to examine the effect of cabergoline addition on the changes in ovarian granulosa cells induced by FSH stimulation. Participants/materials, setting, methods FSH and cabergoline were added alone or co-added to a primary culture system of rat ovarian granulosa cells, and the changes induced by them were measured and compared. (1) mRNA expression of Lhcgr, Cyp19a1, and Fshr by RT-qPCR (2) E2 concentration in the culture medium (3) Measurement of hCG binding on the cell surface (4) cAMP concentration Main results and the role of chance Co-addition of cabergoline suppressed each of the changes induced by FSH stimulation as follows. (1) The mRNA expression levels of Lhcgr, Cyp19a1, and Fshr decreased in a concentration-dependent manner (0.01-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, they decreased to 14%, 9%, and 49% of FSH alone, respectively. (2) E2 concentration in the culture media also decreased in a concentration-dependent manner (1-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, it decreased to 49% of FSH alone. (3) The amount of hCG binding on the cell surface also decreased in a concentration-dependent manner (0.1-10 µM) of co-added cabergoline compared to FSH alone. In the 10 µM of co-added cabergoline group, it decreased to 7% of FSH alone. (4) FSH-stimulated cAMP production decreased in a concentration-dependent manner (0.01-1µM) of co-added cabergoline compared to FSH alone. In the 1µM of co-added cabergoline group, it decreased to 50% of FSH alone. These results suggest that cabergoline suppresses LH receptor and aromatase expression and E2 production by inhibiting cAMP production induced by FSH stimulation. Limitations, reasons for caution Since this is an in vitro experiment using a rat primary culture system, the results of this research cannot be considered directly applicable to humans. Wider implications of the findings Our findings imply that cabergoline administration during the follicular phase may inhibit ovarian granulosa cell differentiation and estrogen secretion. In women taking relatively high doses of cabergoline, attention may need to be paid regarding the effects of cabergoline on follicular development and estrogen secretion. Trial registration number not applicable

Função do fator de crescimento fibroblástico 18 na regulação da cascata de eventos pré-ovulatórios em bovinos

ABSTRACT: Suboptimal LH surges can lead to delayed or absent ovulation during reproductive management programs in cattle. Several signaling pathways may enhance the LH signal, potentially including growth factors. In the present study, we determined whether Fibroblast growth factor 18 (FGF18), which mimics some actions of the LH surge on granulosa cells, may potentiate the action of a suboptimal dose of LH using an established bovine granulosa cell (GC) culture system. Addition of recombinant FGF18 increased the concentration of progesterone (P4) in the culture medium. A combination of LH and FGF18 in the culture medium increased the abundance of mRNA encoding EREG, EGR1 and EGR3, proteins that are early modulators of the LH-induced pre-ovulatory cascade. This study demonstrated that FGF18 may be a co-factor in the regulation of the pre-ovulatory cascade in bovine ovarian granulosa cells.

Ethanol potentiates follicle-stimulating hormone action in ovarian granulosa cells.

There are many previous reports on the effects of ethanol on physiological function, including reports of elevated blood estrogen levels in women who drank alcohol. However, the mechanism of ethanol's effects on ovarian functions, such as follicle development and hormone secretion, has not been fully clarified. Therefore, in this study, we investigated the impacts of ethanol on these phenomena and their mechanisms using a primary culture system of rat ovarian granulosa cells (GCs). In the present experiment, groups were created in which follicle-stimulating hormone (FSH) or ethanol was added alone or FSH and ethanol were co-added, and mRNA and protein expression in each group was measured for luteinizing hormone receptor (LHR) and sex steroid hormone synthase, as well as for estradiol (E2) production, cAMP production, and FSH receptor (FSHR) internalization rate. The addition of FSH induced mRNA expression of LHR and aromatase, which led to membrane LHR expression and E2 production. The coexistence of ethanol enhanced all these responses. The action of FSH is exerted via cAMP, and the co-addition of ethanol enhanced this cAMP production. Ethanol alone did not induce cAMP production. The enhancing effect of ethanol was also observed for cAMP induced by cholera toxin. Ethanol had no significant effect on the internalization rate of FSHR. In conclusion, ethanol increased FSH-stimulated cAMP production by increasing the activity of adenylyl cyclase, which enhanced FSH actions in rat GCs. Alcohol is an exacerbating factor in several female hormone-related diseases, and the mechanism of ethanol-induced increase in estrogen secretion revealed in this study may be involved in the pathogenesis of these diseases.

Synergistic Regulatory Effect of Inhibin and Anti-Müllerian Hormone on Fertility of Mice.

Inhibin (INH) and anti-Müllerian hormone (AMH) are essential in ovarian folliculogenesis and play an inhibitory role in mammalian fertility. However, the interactive effect of INH and AMH on the animal reproduction remains unknown. This study aimed to determine the possible interaction and synergy between INH and AMH in steroidogenesis by primary granulosa cells, and investigate their synergistic effect on fertility in mice. In in vitro granulosa cell culture system, we found that the treatment of either INHA or AMH had no significant effect on basal estradiol and progesterone production, whereas both significantly attenuated FSH-induced steroid hormone secretion. Importantly, combined treatment with INHA and AMH showed additive inhibitory effect on FSH-induced estradiol and progesterone production, accompanying a significant downregulation in the expression of FSH-stimulated CYP19A1, HSD3B, CYP11A1, StAR transcripts. The interrelationship of INH and AMH combinations was further investigated through active immune neutralization strategy. Female mice were immunized against INH and AMH eukaryotic expression plasmids, and the litter size was recorded after successfully mating. We observed that both INH and AMH plasmids were able to induce either anti-AMH or anti-INH antibodies in the immunized mice. In comparison with the control group, co-immunization with INH and AMH plasmids induced higher levels of estradiol, resulting in more litter size. Moreover, there was no significant difference on the offspring's weight between each group. Collectively, the results of the present study suggest that INH and AMH have synergistic effect in regulating steroidogenesis and the litter size in mice.

Effects of selected hormones and their combination on progesterone and estradiol production and proliferation of feline granulosa cells cultured in vitro

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. The present study aimed to develop a hormone responsive granulosa cell culture system to measure steroidogenic and cell proliferation responses to help identify factors that might regulate ovarian function in queens. Five experiments were conducted each with 75 or more ovaries, three in spring and two in fall seasons. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48 h after an initial 48 h plating in 10% fetal calf serum. In granulosa cells isolated from spring and fall collected feline ovaries, IGF1 alone and combined with FSH stimulated (P < 0.05) cell proliferation, whereas FSH alone had no effect (P > 0.10) on cell proliferation. Also, in granulosa cells collected in spring and fall, IGF1 alone and FSH alone increased (P < 0.05) estradiol production by severalfold, and a combination of FSH and IGF1 increased (P < 0.05) estradiol production above either FSH or IGF1 treatment alone. The FSH plus IGF1 treatment increased (P < 0.05) CYP19A1 mRNA abundance by 27-fold. In contrast, EGF decreased (P < 0.05) FSH plus IGF1-induced estradiol production by over 80% in granulosa cells of both spring and fall collected ovaries. In granulosa cells isolated from spring and fall collected ovaries, IGF1 plus FSH inhibited (P < 0.05) progesterone production. Melatonin increased (P < 0.05) FSH plus IGF1-induced cell proliferation and amplified (P < 0.05) the FSH plus IGF1-induced inhibition of progesterone production. However, melatonin and GH had no effect (P > 0.10) on estradiol production either alone or in combination with FSH plus IGF1 in both spring and fall. Prolactin, FGF9 and activin had no effect (P > 0.10) on cell proliferation or steroidogenesis. FGF2 decreased (P < 0.05) estradiol production without affecting progesterone production or cell numbers. Growth differentiation factor 9 (GDF9) increased (P < 0.05) progesterone production but had no effect (P > 0.10) on granulosa cell proliferation or estradiol production. In conclusion, the in vitro system described herewithin may be useful to assess and evaluate ovarian function in feline species and has identified EGF, FSH and IGF1 as major regulators of feline ovarian follicular function.

Control of antral follicle development and selection in sheep and cattle

The development of antral follicles in sheep and cattle is dependent on FSH, but large antral follicles can shift their gonadotrophic dependence from FSH to LH. The mechanisms that result in the selection of at least one ovulatory follicle from identical follicular cohorts, exposed to the same endocrine environment, still remain to be elucidated. The aim of this research was to extend in vitro results from the rodent to sheep and cattle and, using both in vivo and in vitro models, to identify factors that can enhance or attenuate the action of gonadotrophins in stimulating follicle development. Using sheep with ovarian autotransplants, we have obtained evidence to show that a number of factors inhibit ovarian function in vivo, whereas only insulin-like growth factor I (IGF-I) has a stimulatory effect. Further study of the mechanism of action of these factors at a cellular level has been made possible by the development of a serum-free granulosa cell culture system for both sheep and cattle that allows induction and maintenance of oestradiol production. Using this model system, we have confirmed many of the results from out studies in vivo and have shown that IGF-I and insulin interact at physiological concentrations to influence both cellular proliferation and oestradiol production. Overall, these data support the hypothesis that the physiological basis of follicle selection is the differential expression of factors that modulate the action of gonadotrophins on follicular cells at key points during the process of follicle development.

Comparison of Medium Supplementation on Proliferation and Hormone Production of Bovine Granulosa Cells in a Defined Culture System

Aims: The objective of this study was to develop a serum -free bovine granulosa cell (GC) culture system in which estradiol (E2) production could be maintained in a defined media, with polyvinyl alcohol (PVA), insulin and insulin

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

BackgroundSince noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance.MethodsPrimary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs.ResultsGCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose–response study. The highest tested concentration of NE (10 (−7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (−8) M), a non-selective beta-adrenergic antagonist.ConclusionsThe present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

Angiotensin II Signaling Promotes Follicle Growth and Dominance in Cattle

It is generally understood that angiotensin II (AngII) promotes follicle atresia in rats, although recent data suggested that this may not be true in cattle. In this study, we aimed to determine in vivo whether AngII alters follicle development in cattle, using intrafollicular injection of AngII or antagonist into the growing dominant follicle or the second largest subordinate follicle. Injection of saralasin, an AngII antagonist, into the growing dominant follicle inhibited follicular growth, and this inhibitory effect was overcome by systemic FSH supplementation. Injection of AngII into the dominant follicle did not affect follicular growth, whereas injection of AngII into the second largest follicle prevented the expected atresia of this subordinate follicle, and the treated follicle grew at the same rate as the dominant follicle for the next 24 h. Inhibition of AngII action in the dominant follicle decreased estradiol concentrations in follicular fluid and the abundance of mRNA encoding aromatase, 3β-hydroxysteroid dehydrogenase, LH receptor, and cyclinD2 in granulosa cells, with minimal effects on theca cells. The effect of AngII on aromatase mRNA levels was confirmed using an in vitro granulosa cell culture system. In conclusion, these data suggest that AngII signaling promotes follicle growth in cattle and does so by regulating genes involved in estradiol secretion and granulosa cell proliferation and differentiation.

Regulation of Inducible Nitric Oxide Synthase Expression in Bovine Granulosa Cells and Its Involvement in Follicular Dominance.

Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Nitric oxide (NO), a free-radical gas, has been implicated in follicular growth in rodents, but the cow is an intriguing enigma: NO is produced by bovine granulosa cells and is regulated by FSH, but the enzymes responsible are reported to be absent. Three genes encode NO synthase proteins, one of which - iNOS - is widely considered to be regulated by extracellular ligands. We hypothesize that granulosa cells express the gene encoding iNOS, and that the expression of this gene is under hormonal regulation. The objectives of the present study were to determine the abundance of iNOS mRNA in growing and non-growing follicles in cattle, to determine the regulation of iNOS expression by FSH and growth factors, and to determine the action of NOS in bovine granulosa cells. The two largest follicles on days 1 to 5 of the first follicular wave of the cycle were collected from each pair of ovaries from six cows. Messenger RNA encoding iNOS was detected in granulosa and theca cells of bovine follicles by real-time PCR. The granulosa cells of dominant follicles contained higher levels of mRNA for iNOS compared with subordinate follicles (P<0.01). The regulation of iNOS expression was evaluated in a serum-free granulosa cell culture system. FSH (P<0.05) and IGF1 (P<0.01) stimulated E2 secretion iNOS mRNA abundance in a dose-dependent manner. Moreover, both FGF2 and EGF (P<0.01) decreased iNOS expression and E2 secretion in FSH and in IGF1-treated cells. Owing to the parallel changes in estradiol secretion and iNOS mRNA, we assessed the role of estradiol in the regulation of iNOS mRNA; replacement of estrogen precursor with a non-aromatizable androgen (DHT) or the addition of an estrogen receptor antagonist inhibited FSH-stimulated iNOS mRNA abundance, and addition of E2 alone stimulated NO production in vitro. To assess the role of endogenous iNOS, cells were treated with an iNOS-selective inhibitor, which resulted in increased FasL mRNA levels (P<0.01), induction of caspase-3 activation (P<0.01) and an increase in the incidence of cell death (P<0.05). In conclusion, bovine graulosa cells express iNOS, and increased iNOS mRNA levels are associated with growth of the dominant follicle. Gonadotropic factors stimulate iNOS mRNA levels through increased E2 secretion, and physiological levels of NOS activity may contribute to growth and survival of granulosa cells. Supported by NSERC Canada and CNPq Brazil. (poster)

Expression of Fibroblast Growth Factors 17 and 18 (Fgf-17 And Fgf-18) in Bovine Antral Follicles.

Fibroblast growth factors (FGF) have been recognised as important local regulators of follicle development. FGF-17 and FGF-18 belong to the FGF-8 subfamily and exhibit similar amino acid sequences and receptor affinity. They induce cell proliferation and differentiation through activation of FGFR-3c and FGFR-4, recently detected (mRNA) in granulosa (GC) and theca cells (TC) of bovine antral follicles. The first aim of this study was to determine the spacial and temporal patterns of FGF-17 and FGF-18 expression in bovine antral follicles. As FGF-17 mRNA was found to be altered by follicle health status, a granulosa cell culture system was used to test the effect of FSH on GC FGF-17 expression. Antral follicles greater than 5mm in diameter were dissected from abattoir ovaries, GC and TC separated and total RNA extracted. Estradiol (E) and progesterone (P) concentrations of the follicular fluid were measured by RIA and follicles were grouped according to E:P ratios of <0.01 (highly atretic), 0.01-1 (transitional) and >1 (healthy). Cumulus oocyte complexes were aspirated from antral follicles, cumulus cells were removed and total RNA extracted from pools of 50 oocytes. To investigate regulation of FGF-17 mRNA by FSH, GC from small follicles (2-5 mm) were placed into serum-free medium supplemented with insulin and bovine FSH (0, 0.1, 1, 10 or 100 ng/ml). Expression of FGF-17 and FGF-18 mRNA was investigated by real time RT-PCR using bovine-specific primers (TaqMan Assay by Design, ABI) and GAPDH as the endogenous control. Relative expression was determined by the Pfaffl equation. FGF-17 mRNA was detected in pools of oocytes, and in TC and GC, where expression was higher in highly atretic follicles compared with transitional and healthy follicles. FGF-17 protein was localized to oocytes, GC and TC by immunohistochemistry. FSH inhibited FGF-17 mRNA expression in cultured GC in a dose-dependent manner. FGF-18 mRNA was detected in GC and TC, where it did not change with follicle status, but was absent in the oocyte. In conclusion, the present data suggest a role for FGF-17 in the control of folliculogenesis. FGF-17 expression is low in growing follicles and is inhibited by FSH, which indicates a role in the control of follicle atresia. Supported by FAPESP.

P-960: The role of human chorionic gonadotropin in the stimulation of cumulus granulosa cell production of inhibin A, progesterone and estradiol in culture: A predictor of functional viability

Hormone manipulation in cell culture may improve cell secretion in vitro. In this study the culture system of human cumulus granulosa cells (CGC) was established to express functional viability of secretion of dimeric inhibins, progesterone, and estradiol. The CGC were treated with sustained high dosage of Human Chorionic Gonadotropin (HCG) in a time dependent manner.

ラット卵巣か粒膜細胞におけるプロテオグライカンの生合成に与えるｐ‐ｎｉｔｒｏｐｈｃｎｙｌ‐ｘｙｌｏｓｉｄｅの影響―Ｇｏｌｇｉ装置の糖合成機能に関する解析―

Biosynthesis of proteoglycans and glycosaminoglycans in the presence of p-nitrophenyl-xyloside was studied using a primary rat ovarian granulosa cell culture system. Addition of p-nitrophenyl-xyloside into cell culture medium caused about a 700% increase of [³⁵S]sulfate incorporation (ED50 at 0.03 mM) into macromolecules, which included free chondroitin sulfate chains initiated on xyloside and native proteoglycans. Free chondroitin sulfate chains initiated on xyloside were almost exclusively secreted into the medium. The molecular size of chondroitin sulfate chains decreased from 40,000 to 21,000 as the total [³⁵S]sulfate incorporation was enhanced, suggesting that enhanced synthesis of chondroitin sulfate perturbed the normal mechanism of glycosaminoglycan chain termination. Biosynthesis of heparan sulfate proteoglycans was reduced by approximately 50%, likely due to competition at the level of UDP-sugar precursors. [³⁵S]Sulfate incorporation was shut down by the addition of cycloheximide with an initial half time of approximately 2 hr in the presence of xyloside, while that in the absence of xyloside was about 20 min. The difference likely reflects the turnover rate of glycosaminoglycan synthesizing capacity as a whole. The turnover rate of glycosaminoglycan synthesizing capacity observed in ovarian granulosa cells was much shorter than that observed in chondrocytes, reflecting the relative dominance of proteoglycan biosynthetic activity in the total metabolic activity of the cells.

Metformin alters insulin signaling and viability of human granulosa cells

To study whether insulin signaling pathways in the ovary are altered by metformin. In vitro human granulosa cell culture system. Academic research environment. Infertility patients undergoing oocyte retrieval for IVF/ICSI. Cell viability and phosphorylated protein kinase B (PKB/AKT) and p44/42 mitogen-activated protein kinase (MAPK) expression of human primary and HGL5 granulosa cells. Basal cell viability of primary granulosa cells was significantly increased relative to control by metformin preincubation, without an additional stimulatory effect of insulin or IGF. Phosphorylated AKT expression in lysates of the human granulosa cell line HGL5 was significantly increased in contrast to decreased phosphorylated MAPK expression by metformin preincubation. Besides systemic effects, the ovulation inducing action of metformin may at least partially be due to direct effects on insulin signaling intermediates and follicular growth patterns in the ovary.

Activin A and follicle-stimulating hormone control tight junctions in avian granulosa cells by regulating occludin expression.

Within the avian ovarian follicle, the oocyte is surrounded by a monolayer of granulosa cells, which exhibit pronounced epithelial properties. Here we demonstrate the presence of the major tight junction protein occludin in granulosa cells. As shown by immunohistochemistry, occludin localizes to the oocyte-facing granulosa cell surface. Occludin and thus tight junctions are dynamically regulated in a developmental stage-specific manner. Small white follicles, which have not yet started yellow yolk incorporation, show pronounced occludin expression in vitro and in vivo. By contrast, yellow yolk-incorporating small yellow follicles exhibit much lower levels of occludin, and hierarchical, preovulatory follicles are virtually devoid of this essential tight junction component. Using a primary granulosa cell culture system, we demonstrate that concerted action of two well-established ovarian growth regulators, follicle-stimulating hormone and activin A, leads to strong induction of occludin expression in vitro. We suggest that the stage-dependent decrease in the granulosa cell growth factor responsiveness triggers the disruption of tight junctions, enabling rapid and high capacity transport of macromolecules into the oocyte through a paracellular pathway. Such a high-capacity transport for yolk components may represent a crucial prerequisite for rapid oocyte growth once follicles have entered the follicular hierarchy.

Synergistic effects of activin and FSH on hyperphosphorylation of Rb and G1/S transition in rat primary granulosa cells

Activin is produced in mammalian ovarian follicles and is known to function as a paracrine as well as autocrine factor for folliculogenesis and oogenesis. We investigated the functional mechanism of activin using a hormone-supplemented serum-free culture system of granulosa cells isolated from diethylstilbestrol (DES)-primed 21-day-old rats. Recombinant human-activin A appeared to induce CycD2 and to act synergistically with FSH to promote G1/S transition and cell proliferation starting from 12 h after stimulation, accompanied by an increase of the hyperphosphorylated retinoblastoma protein (ppRb). Cells from unprimed rats gave similar results. FSH, in contrast, showed no CycD2-inducing activity, but turned out to modulate CycD2/cdk4 complex formation and enhance ppRb formation in conjunction with activin. These findings showed that the induction of CycD2 by activin and the synergistic effect of activin with FSH on ppRb formation play important roles in promoting G1/S transition in rat primary granulosa cells.

Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 receptor pathway.

Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the transforming growth factor beta superfamily, progesterone receptor, cyclooxygenase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E(2) (PGE(2)) receptor (EP2; Ptgerep2) are required for fertility in female but not male mice. To define the interrelationship of these factors, we used a preovulatory granulosa cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimulated Cox2 mRNA within 2 h, and PGE(2) within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of GDF-9 but that progesterone synthesis required an intermediate. To determine whether prostaglandin synthesis was required for progesterone production, we analyzed the effects of PGE(2) and cyclooxygenase inhibitors on this process. PGE(2) can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fold increase over 24 h, respectively). Furthermore, indomethacin or NS-398, inhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE(2) to cultures containing both GDF-9 and NS-398 overrides the NS-398 block in progesterone synthesis. To further define the PGE(2)-dependent pathway, we show that butaprost, a specific EP2 agonist, stimulates progesterone synthesis and overrides the NS-398 block. In addition, GDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone-independent pathway. Thus, GDF-9 induces an EP2 signal transduction pathway which appears to be required for progesterone synthesis in cumulus granulosa cells. These studies further demonstrate the importance of oocyte-somatic cell interactions in female reproduction.

Mechanisms underlying the steroidogenic synergy of insulin and luteinizing hormone in porcine granulosa cells: joint amplification of pivotal sterol-regulatory genes encoding the low-density lipoprotein (LDL) receptor, steroidogenic acute regulatory (StAR) protein and cytochrome P450 side-chain cleavage (P450scc) enzyme

Growth of ovarian Graafian follicles and cytodifferentiation of granulosa and theca cells are regulated by gonadotropins, sex steroids and peptidyl growth factors. For example insulin and intraovarian insulin-like growth factor type I (IGF-I) may amplify the actions of both follicle stimulating hormone (FSH) and luteinizing hormone (LH) in promoting biochemical luteinization and enhancing steroidogenesis. To explore further the notion of interactions between insulinomimetic peptides and LH and to examine the associated mechanisms, we have established porcine granulosa cells in monolayer culture for 48 h in 3% serum with insulin (1 μg/ml), estradiol (0.5 μg/ml), and follicle stimulating hormone (FSH, 5 ng/ml) to allow cell anchorage, facilitate in vitro cytodifferentiation and confer LH responsiveness. To limit any carry-over effects of serum, granulosa cells were stabilized overnight in serum-free medium. Studies were then initiated to assess the impact of insulin on the dose-responsive actions of LH. A maximally effective concentration of insulin (1 μg/ml) synergistically augmented LH’s dose-dependent ampilification of progesterone and cAMP accumulation; viz. by ≈twofold (progesterone) and ≈2.5-fold (cAMP) above that observed in maximally LH-stimulated cultures ( P<0.001). Mechanistically, insulin significantly enhanced the sensitivity of granulosa cells to LH’s drive of cAMP accumulation [ED 50 for LH 61±14 ng/ml (control) vs. 10±1.0 ng/ml (insulin) ( P<0.01)]. Insulin also augmented the maximal stimulatory effect of LH; i.e. LH efficacy rose from 6.5±0.4 to 17±1.4 (pmole cAMP/μg DNA/48 h; P<0.001). Insulin dose-response analysis showed that insulin alone minimally elevated basal, but significantly heightened LH's stimulation of progesterone and cAMP accumulation at (insulin) concentrations as low as 3–10 ng/ml. The molecular mechanisms underlying insulin and LH’s synergy were assessed by RNase protection assays with (porcine) cRNA probes encoding the low density lipoprotein receptor (LDL-R), Steroidogenic Acute Regulatory Protein (StAR), P450 cholesterol sidechain cleavage enzyme (P450scc) and (as a possible negative control) Sterol Carrier Protein 2 (SCP-2) [data normalized to constitutive 18S rRNA]. Non linear least-squares analysis was applied to confirm or refute an hypothesis of interactive synergy between LH and insulin on gene expression. LH and insulin alone exerted no effect on StAR message accumulation, and LH alone minimally stimulated P450scc and LDL-R mRNA’s accumulation at 48 h. In contrast, insulin in combination with LH augmented StAR mRNA concentrations by ≈5–10-fold and stimulated LDL-R message levels by threefold above the respective maximally LH-driven values ( P<0.01). Maximal P450scc mRNA expression was enhanced twofold by cotreatment with LH and insulin compared with maximal LH-treated cultures. In contrast SCP-2 mRNA accumulation remained unaffected by any treatment. In summary, we have used a serum-free, in vitro differentiated porcine granulosa cell culture system to assess regulatory interactions between the disparate first messengers, LH and insulin. We observe marked LH-insulin steroidogenic synergy after 48 h of joint hormonal stimulation, and further clarify that the mechanism(s) of synergy include augmentation of cAMP production and increased steady-state concentrations of transcripts of key sterol-regulatory genes; namely, LDL-R, StAR, and P450scc, but not SCP-2. Since the encoded products of these genes variously control sterol substrate uptake, delivery to and utilization in mitochondrial steroidogenesis, we speculate that the concerted actions of insulin-like peptides and LH may contribute to steroidogenic differentiation during the later stages of follicular maturation and the granulosa-luteal cell transition.

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.

Expression and effect of adrenomedullin on rat granulosa cell.

Adrenomedullin (AM) is a potent hypotensive peptide recently discovered in extracts of human pheochromocytoma. To elucidate the regulation of AM production in the ovary, the effect of gonadotropin on the production of AM was studied in the cultured rat granulosa cells. A Northern blot analysis of the LH receptor and adrenomedullin yielded a major hybridizing band at 5.4 kb and 1.6 kb, respectively. In our culture system of rat granulosa cells, without any stimulus, the LH receptor mRNA was undetectable and the AM mRNA level was stably expressed for 6 days. FSH significantly induced LH receptor mRNA and suppressed AM mRNA for 4 days of culture and with the addition of hCG after 2 days of pretreatment with FSH, AM mRNA levels were markedly suppressed. FSH and 8-Br-cAMP significantly increase LH receptor mRNA and suppress AM mRNA in a dose-dependent manner. These data indicated that the differentiation of granulosa cells mediated by gonadotropins were associated by suppression in AM expression through a cAMP-dependent mechanism. On the other hand, AM stimulated a rapid rise in intracellular cAMP levels, which peaked within 15 min of addition, in a dose-dependent manner with a maximal response seen at 100 nM. Additionally, AM enhanced the effects of FSH, acting additionally to produce cAMP in the cells. AM may play a role in the process of granulosa cell differentiation as a local regulator through an autocrine/paracrine mechanism.