FSH-stimulated Granulosa Cells Research Articles

Adverse effect of superoxide-induced mitochondrial damage in granulosa cells on follicular development in mouse ovaries

High mitochondrial oxidative phosphorylation (mt-OXPHOS) levels are required to supply the ATP necessary for follicle-stimulating hormone (FSH)-induced granulosa cell proliferation during the follicular development process. Consequently, excessive reactive oxygen species (ROS) might be generated and have an adverse effect on follicular health. This study aimed to elucidate the negative effects of ROS on mitochondrial functions in FSH-stimulated granulosa cells during the follicular development process and to investigate whether pyrroloquinoline quinone (PQQ) treatment could accelerate this process by ameliorating the adverse effects. To do this, both in vitro and in vivo experiments were performed with granulosa cells from superovulated immature (3-week-old) mice that were pretreated with or without PQQ, and a natural mating study was also performed. The ROS level in FSH-/eCG-stimulated granulosa cells was significantly increased. Moreover, high oxidative stress and mtDNA damage levels were evident in the granulosa cells. PQQ treatment not only reduced the ROS and oxidative stress levels but also ameliorated mtDNA damage, accelerated FSH-/eCG-induced ATP production, and increased the mitochondrial membrane potential and the expression levels of mitochondrial genes (Nd1, Cytb, Cox1, ATPase6) and the mt-ND1 protein. Accordingly, the proliferation and viability of granulosa cells, numbers of healthy preovulatory follicles and ovulated oocytes and serum estrogen level were significantly improved, while the apoptosis of granulosa cells was reduced. However, PQQ treatment did not change the fertility parameters in mature mice with natural cycles but did significantly increased the number of offspring born per delivery. These results revealed that ROS-associated damage in FSH-stimulated granulosa cells adversely affects their physiology and follicular health during the follicular development process. Treatment with PQQ is a beneficial tool to increase both the number of ovulated oocytes and pups per delivery.

Transcriptome profiling to identify key mediators of granulosa cell proliferation upon FSH stimulation in the goose (Anser cygnoides)

ABSTRACT1. The low reproductive performance of geese has seriously hampered the development of the industry. Reproductive performance, particularly the egg laying rate mainly depends on the development of the follicle. Previous studies have shown that follicle-stimulating hormone (FSH) plays an important role in the process of follicular development, but the exact underlying mechanism remains unclear.2. This study showed that FSH stimulated granulosa cell proliferation in a dose-dependent manner. The effect of FSH treatment on granulosa cell proliferation was greatest at a dose of 100 mIU/ml FSH for 24 h.3. Secondly, the effect of different concentrations of FSH on goose granulosa cell proliferation was investigated, and de novo transcriptome assembly and gene expression analysis performed using short-read sequencing technology (Illumina). High-throughput sequencing results yielded 62.61 M reads and 7.8 G base pairs from granulosa cells treated with 100 mIU/ml FSH. These reads were assembled into 65,757 unigenes (mean length: 705 bp) with an N50 of 903 bp. A total of 110 upregulated and 510 downregulated differentially expressed genes (DEGs) were identified by RNA-seq.4. Functional analysis by gene ontology (GO) and KEGG pathway annotation indicated that hormone biosynthesis (GO:0042446), positive regulation of hormone secretion (GO:0046887), steroid biosynthesis, oxidative phosphorylation and carbon metabolism pathways were involved in FSH-mediated proliferation of goose granulosa cells.5. After screening, a group of key responsive genes including superoxide dismutase 1, fatty acyl-CoA reductase 1, transforming growth factor-beta receptor-associated protein 1 and follistatin were tested by real-time reverse transcription PCR to confirm differential expression in granulosa cells stimulated by FSH.6. FSH-stimulated goose granulosa cells and DEG profiling data provided comprehensive gene expression information at the transcriptional level that could promote better understanding of the molecular mechanisms underlying follicle development in response to FSH stimulation.

The effect of androgens on ovarian follicle maturation: Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARγ-dependent PTEN expression.

Intraovarian hyperandrogenism is one of the determining factors of follicular arrest in women with polycystic ovary syndrome (PCOS). Using androgenized rat models, we investigated the effects of androgens on metabolism, as well as on factors involved in follicular arrest and the reduced number of estrus cycles. The dihydrotestosterone (DHT)-treated rats had fewer estrus cycles, higher numbers of large arrested follicles and an increased in body weight gain compared with the dehydroepiandrostenedione (DHEA)- and placebo-treated rats. In cultured rat granulosa cells, DHT suppressed follicle stimulating hormone (FSH)-induced granulosa cell proliferation and increased the accumulation of cells in the G2/M phase. DHT decreased phosphorylated Akt (p-Akt) and cyclin D1 levels through increasing PTEN. DHT-promoted PTEN expression was regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in granulosa cells. Meanwhile, in the large follicles of the DHT-treated rats, the expressions of PPARγ and PTEN were higher, but the expression of p-Akt and proliferating cell nuclear antigen (PCNA) were lower. Conclusively, DHT and DHEA produced differential effects on metabolism in prepubertal female rats like clinical manifestations of women with PCOS. DHT treatment may affect ovarian follicular maturation by altering granulosa cell proliferation through the regulation of enhancing PPARγ dependent PTEN/p-Akt expression in the granulosa cells.

Defective CFTR-regulated granulosa cell proliferation in polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) is one of the most frequent causes of female infertility, featured by abnormal hormone profile, chronic oligo/anovulation, and presence of multiple cystic follicles in the ovary. However, the mechanism underlying the abnormal folliculogenesis remains obscure. We have previously demonstrated that CFTR, a cAMP-dependent Cl(-) and HCO3 (-) conducting anion channel, is expressed in the granulosa cells and its expression is downregulated in PCOS rat models and human patients. In this study, we aimed to investigate the possible involvement of downregulation of CFTR in the impaired follicle development in PCOS using two rat PCOS models and primary culture of granulosa cells. Our results indicated that the downregulation of CFTR in the cystic follicles was accompanied by reduced expression of proliferating cell nuclear antigen (PCNA), in rat PCOS models. In addition, knockdown or inhibition of CFTR in granulosa cell culture resulted in reduced cell viability and downregulation of PCNA. We further demonstrated that CFTR regulated both basal and FSH-stimulated granulosa cell proliferation through the HCO3 (-)/sAC/PKA pathway leading to ERK phosphorylation and its downstream target cyclin D2 (Ccnd2) upregulation. Reduced ERK phosphorylation and CCND2 were found in ovaries of rat PCOS model compared with the control. This study suggests that CFTR is required for normal follicle development and that its downregulation in PCOS may inhibit granulosa cell proliferation, resulting in abnormal follicle development in PCOS.

Non-canonical WNT5A is a potential regulator of granulosa cell function in cattle

The WNT family has been implicated in follicular development in rodents, however, the role of WNTs in the follicle of monovulatory species is poorly understood. The objective of this study was to determine the potential roles of WNTs in bovine granulosa cell function. Cells cultured in serum-free medium expressed mRNA encoding WNT2B, WNT5B and WNT5A. Levels of WNT5A, but not of WNT2B or WNT5B mRNA, were down-regulated by FSH. Addition of WNT5A to cultured cells suppressed FSH-stimulated estradiol and progesterone secretion, and levels of mRNA encoding the steroidogenic enzymes CYP19A1, CYP11A1 and the FSH receptor, but had no effect on cell proliferation or apoptosis. Immunoblot experiments showed that WNT5A reduced activation of CTNNB1 and stimulated phosphorylation of MAPK8 and JUN proteins. We conclude that WNT5A is a negative regulator of FSH-stimulated granulosa cell steroidogenesis, and that it acts by suppressing canonical WNT signaling activity and inducing the non-canonical MAPK8/JUN pathway.

Methoxychlor and its metabolite HPTE inhibit cAMP production and expression of estrogen receptors α and β in the rat granulosa cell in vitro.

The major metabolite of the estrogenic pesticide methoxychlor (MXC) HPTE is a stronger ESR1 agonist than MXC and acts also as an ESR2 antagonist. In granulosa cells (GCs), FSH stimulates estradiol via the second messenger cAMP. HPTE inhibits estradiol biosynthesis, and this effect is greater in FSH-treated GCs than in cAMP-treated GCs. Therefore; we examined the effect of MXC/HPTE on FSH-stimulated cAMP production in cultured GCs. To test involvement of ESR-signaling, we used the ESR1 and ESR2 antagonist ICI 182,780, ESR2 selective antagonist PHTPP, and ESR2 selective agonist DPN. ESR1 and ESR2 mRNA and protein levels were quantified. Both HPTE and MXC inhibited the FSH-induced cAMP production. ICI 182,780 and PHTPP mimicked the inhibitory action of HPTE. MXC/HPTE reduced FSH-stimulated Esr2 mRNA and protein to basal levels. MXC/HPTE also inhibited FSH-stimulated Esr1. The greater inhibition on FSH-stimulated GCs is likely due to reduced cAMP level that involves ESR-signaling, through ESR2.

Atrazine enhances progesterone production through activation of multiple signaling pathways in FSH-stimulated rat granulosa cells: evidence for premature luteinization.

Premature luteinization is a possible cause of infertility in women. It is currently unknown whether environmental chemicals can induce changes associated with premature luteinization. Using rat granulosa cells (GC) in vitro, we demonstrated that exposure to atrazine (ATR), a widely used herbicide, causes GC phenotype that resembles that of human premature luteinization. At the end of the 48-h stimulation with FSH, ATR-exposed GC showed (1) higher levels of progesterone, (2) overexpression of luteal markers (Star and Cyp11a1), and (3) an increase in progesterone:estradiol ratio above 1. Mechanistic experiments were conducted to understand the signaling events engaged by ATR that lead to this phenotype. Western blot analysis revealed prolonged phosphorylation of protein kinase B (AKT) and cAMP response element-binding protein (CREB) in ATR- and FSH-exposed GC. An increased level of ERK1/2-dependent transcriptional factor CCATT/enhancer-binding protein beta (CEBPB) was observed after 4 h of ATR exposure. Inhibitors of PI3K (wortmannin) and MEK (U0126) prevented ATR-induced rise in progesterone level and expression of luteal markers in FSH-stimulated GC. Atrazine intensified AKT and CEBPB signaling and caused Star overexpression in forskolin-stimulated GC but not in epidermal growth factor (EGF)-stimulated GC. In the presence of rolipram, a specific inhibitor of phosphodiesterase 4 (PDE4), ATR was not able to further elevate AKT phosphorylation, CEBPB protein level, and Star mRNA in FSH-stimulated GC, suggesting that ATR inhibits PDE4. Overall, this study showed that ATR acts as a FSH sensitizer leading to enhanced cAMP, AKT, and CEBPB signaling and progesterone biosynthesis, which promotes premature luteinization phenotype in GC.

Involvement of ERK1/2 signaling pathway in atrazine action on FSH-stimulated LHR and CYP19A1 expression in rat granulosa cells

Worldwide used herbicide atrazine is linked to reproductive dysfunction in females. In this study, we investigated the effects and the mechanism of atrazine action in the ovary using a primary culture of immature granulosa cells. In granulosa cells, follicle-stimulating hormone (FSH) activates both cyclic adenosine monophosphate (cAMP) and extracellular-regulated kinase 1/2 (ERK1/2) cascades, with cAMP pathway being more important for luteinizing hormone receptor (LHR) and aromatase (CYP19A1) mRNA expression. We report that 48h after atrazine exposure the FSH-stimulated LHR and CYP19A1 mRNA expression and estradiol synthesis were decreased, with LHR mRNA being more sensitive to atrazine than CYP19A1 mRNA. Inadequate acquisition of LHR in the FSH-stimulated and atrazine-exposed granulosa cells renders human chorionic gonadotropin (hCG) ineffective to stimulate amphiregulin (Areg), epiregulin (Ereg), and progesterone receptor (Pgr) mRNA expression, suggesting anti-ovulatory effect of atrazine. To dissect the signaling cascade involved in atrazine action in granulosa cells, we used U0126, a pharmacological inhibitor of ERK1/2. U0126 prevents atrazine-induced decrease in LHR and CYP19A1 mRNA levels and estradiol production in the FSH-stimulated granulosa cells. ERK1/2 inactivation restores the ability of hCG to induce expression of the ovulatory genes in atrazine-exposed granulosa cells. Cell-based ELISA assay revealed that atrazine does not change the FSH-stimulated ERK1/2 phosphorylation in granulosa cells. The results from this study reveal that atrazine does not affect but requires ERK1/2 phosphorylation to cause decrease in the FSH-induced LHR and CYP19A1 mRNA levels and estradiol production in immature granulosa cells, thus compromising ovulation and female fertility.

Expression and regulation of SNAP-25 and synaptotagmin VII in developing mouse ovarian follicles via the FSH receptor

Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.

Effect of follicle stimulating hormone on the secretion of anti-mullerian hormone in the granulosa cells in patients with polycystic ovarian syndrome

To study the effect of follicle stimulating hormone (FSH) on the secretion of anti-mullerian hormone (AMH) in granulosa cells in patients with polycystic ovarian syndrome (PCOS). From Aug. 2008 to Dec. 2009, 33 patients with PCOS in Reproductive Medicine Research Center, Sixth Affiliated Hospital of Sun Yat-sen University were collected from 8-10 mm antral follicles and classified into the following three groups: FSH-unstimulated granulosa cells (unstimulated group, n = 12), exogenous FSH-stimulated granulosa cells (exo-stimulated group, n = 12) and internal FSH-stimulated granulosa cells (int-stimulated group, n = 21). ELISA and real-time PCR were relatively used to measure protein and mRNA level of AMH. Luciferase reporting vector was constructed to detect the promoter activity of AMH. The levels of AMH secretion in PCOS granulosa cells were (11.4 ± 4.0) µg/L in unstimulated group, (7.9 ± 1.1) µg/L in exo-stimulated group and (5.6 ± 1.7) µg/L in int-stimulated group. Both the external and internal stimulation of FSH may suppress AMH secretion significantly (P < 0.05). The mRNA level of AMH in PCOS granulosa cells were 2.5 ± 1.2 in unstimulated group, which were higher than 1.5 ± 0.5 in exo-stimulated group and 1.1 ± 0.7 in int-stimulated group significantly (P < 0.05). The luciferase activity of AMH in PCOS granulosa cells were 11.5 ± 2.3 in unstimulated group, 8.7 ± 2.4 in exo-stimulated group and 6.8 ± 2.4 in int-stimulated group. The luciferase activity of AMH in unstimulated group was significantly higher than those in exo-stimulated and int-stimulated groups (P < 0.05). FSH may inhibit the excessive secretion of AMH and stimulate follicle growth in PCOS granulosa cells by suppressing activity and expression of promoter.

Granulosa Cell-Specific Inactivation of β-Catenin Alters Aromatase Production and Normal Folliculogenesis

We recently reported that FSH signaling to induce Cyp19a1 (aromatase), the rate-limiting enzyme in estrogen biosynthesis, requires the co-activator β-catenin (Ctnnb1) in a primary rat granulosa cell model. The purpose of the following investigation was to ascertain the physiological significance of β-catenin to granulosa cell maturation and folliculogenesis in an in vivo mouse model. To achieve this purpose, Ctnnb1flox/flox mice were intercrossed with mice that express Cre recombinase under the control of Amhr2 (Ctnnb1flox/flox;Amhr2Cre/+). Amhr2 is expressed along the Mûllerian ducts and in developing ovaries at embryonic day 13.5. Postnatally granulosa cells express Amhr2 specifically in developing follicles. Conditional deletion of the β-catenin gene in 8 week-old females resulted in lower uterine weights when compared to their wild-type counterparts, consistent with the recognized requirement for β-catenin in reproductive tract development. However, conditional deletion of the β-catenin gene did not affect the time to puberty (d 33). No obvious ovarian phenotype was detected between conditional knockouts and wild-type mice at 8 weeks of age. Follicles appeared to undergo maturation to the preovulatory phenotype and to form normal numbers of corpora lutea compared to wild-type mice. Ctnnb1flox/flox;Amhr2Cre/+ mice were found to be infertile after they failed to produce a litter within six months when paired with proven male studs, a result most likely attributable to aberrant reproductive tract development. Unexpectedly, pituitary Fshb mRNA but not Lhb mRNA levels were greater in conditional knockouts compared to wild-type mice. Similarly, the FSH-regulated ovarian genes Inhα and Lhr were also up-regulated in conditional knockout mice compared to wild-type mice. Serum estradiol concentrations and Cyp19a1 mRNA expression also tended to be elevated in knockout compared to wildtype mice. However, total ovarian β-catenin mRNA levels were not reduced in conditional knockout mice. To focus on the effect of β-catenin in granulosa cells we therefore used an in vitro knockout model for β-catenin in primary cultures of mouse granulosa cells. Granulosa cells from Ctnnb1flox/flox were isolated and exposed to a Cre recombinase adenovirus, resulting in a marked reduction of β-catenin mRNA and protein. Deletion of β-catenin in granulosa cells resulted in loss of the ability of FSH to induce Cyp19a. Consequently, estradiol concentrations in cell culture media were higher in FSH-stimulated control granulosa cells compared to FSH-stimulated granulosa cells in which β-catenin was deleted (15.4 ± 3.2 pg/ml and 7.6 ± 2.3 pg/ml, respectively). Collectively, the data herein indicate that β-catenin plays a role in folliculogenesis in the developing ovary, specifically enabling granulosa cells to respond to FSH by expressing Cyp19a1. In addition, our results indicate that β-catenin, normally viewed as an essential component of the canonical WNT signaling pathway, is also required for mediating hormonal signaling through G-protein coupled receptors that regulate the production of estrogen. Supported by a minority fellowship to JAHG (P01 HD21921 to MHD).

C.05.02 Molecular mechanisms of the mammalian circadian clock: identifying the clock genes

Okadaic acid is a potent inhibitor of type-2A (PP2A) and type-1 (PP1) protein phosphatases and has been proved to be a valuable tool for studies on the protein phosphlrylation. We have investigated the effects of okadaic acid on rat granulosa cells in order to determine whether the regulation of ganglioside synthesis involves protein phosphorylation via inhibition of PP2A and PP1. Granulosa cells expressed luteinizing hormone (LH) receptors, measured as the binding of 125I-deglycosylated human chorionic gonadotropin (hCG) to intact cells, and synthesized the gangliosides NeuAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer (GM3) and Galβ1 → 3GalNAcβ1 → 4[NeuAcα2 → 3]Galβ1 → 4Glcβ1 → 1Cer(GM1), demonstrated by metabolic labeling of glycosphingolipids with [3H]galactose, in response to follicle-stimulating hormone (FSH). When FSH-stimulated granulosa cells were treated with 10 nM okadaic acid for 15 h, down-regulation of LH receptors, dissociation of LH receptor-effector coupling and significant decreases of intracellular and extracellular 3′,5′-cyclic adenosine monophosphate (cAMP) levels were observed. The okadaic acid-induced desensitization to gonadotropin in granulosa cells was accompanied by increased ganglioside synthesis. The amount of 3H-labeled ganglioside GM3, the major ganglioside (about 95% of the total) synthesized by mature granulosa cells, was enhanced in okadaic acid-desensitized cells (to 215% of the control value) and in those desensitized by hCG (by 354%), forskolin (by 190%) and 12-O-tetradecanoylphorbol 13-acetate (by 143%). The results of this study suggest that an increase in the phosphorylation state of cells is accompanied by enhancement of ganglioside synthesis.

Effects of galectin-1 on regulation of progesterone production in granulosa cells from pig ovaries in vitro.

The detection of galectin-1 (gal-1) in pig granulosa cell lysates by immunoblotting and its cytosolic as well as membrane-associated localization prompted us to study its effects on cell proliferation and regulation of progesterone synthesis. The lectin stimulated the proliferation of granulosa cells from pig ovaries cultured in serum-free medium. Gal-1 inhibited the FSH-stimulated progesterone synthesis of granulosa cells. This inhibitory effect was strongly reduced by the disaccharidic competitor lactose at 30 mM. The absence of inhibitory effects on dibutyryl-cAMP (db-cAMP), forskolin, and pregnenolone-enhanced cellular progesterone synthesis suggests that gal-1interferes with the receptor-dependent mechanism of FSH-stimulated progesterone production. In FSH-stimulated granulosa cells, western blot analysis revealed the gal-1-mediated suppression of the cytochrome P450-dependent cholesterol side chain cleavage enzyme (P450(SCC)) that catalyzes the conversion of cholesterol to pregnenolone. In the presence of 30 mM lactose, the gal-1-reduced P450(SCC) expression was prevented. Strongly reduced mRNA levels were recorded for P450(SCC) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) when FSH-stimulated granulosa cells were cultured in the presence of gal-1. We conclude that gal-1 exerts its inhibitory effect on steroidogenic activity of granulosa cells by interfering the hormone-receptor interaction resulting in decreased responses to FSH stimulation.

Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation.

FSH-stimulated granulosa cell differentiation is associated with the induction of the LH receptor (LHr) as well as induction of the estrogen and progesterone biosynthetic pathways. Although activation of the cAMP-protein kinase A pathway is sufficient to stimulate progesterone production, additional pathways are required for the induction of the LHr and p450 aromatase. The orphan nuclear receptor, liver receptor homolog-1 (LRH-1), is expressed in granulosa cells and has been shown to synergize with the cAMP signaling system to regulate the gonadal type II aromatase promoter in transient transfection assays. To determine whether LRH-1 can interact with the cAMP pathway in the induction of aromatase and the LHr, we examined the effects of an adenoviral vector that directs the expression of human LRH-1 (Ad-LRH-1) on FSH-stimulated granulosa cell differentiation. Infection of undifferentiated granulosa cells with LRH-1 alone had no effect on estrogen production, progesterone production, or the expression of the LHr. However, combination of FSH stimulation and Ad-LRH-1 infection led to significantly greater progesterone production and increases in mRNA for p450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase than granulosa cells stimulated by FSH alone. However, infection with Ad-LRH-1 did not stimulate estradiol production or increases in mRNA for p450 aromatase or the LHr above that seen with FSH treatment alone. Moreover, infection with Ad-LRH-1 was able to overcome H-89 inhibition of FSH-stimulated progesterone but not estrogen production. Collectively, these observations support a direct role for LRH-1 in the induction of the progesterone but not the estrogen biosynthetic pathway during granulosa cell differentiation.

Synergistic effects between FSH and 1,1-dichloro-2,2-bis(P-chlorophenyl)ethylene (P,P'-DDE) on human granulosa cell aromatase activity.

1,1-Dichloro-2,2-bis(P-chlorophenyl)ethylene (P,P'-DDE, DDE), a metabolite of DDT, is a persistent hormonally active environmental toxicant which has been found in human serum and follicular fluid. The objective of this study was to investigate the interaction between FSH and ppDDE on aromatase activity in primary cultures of human granulosa cells. Granulosa cells were obtained at the time of oocyte retrieval for IVF procedures and cultured in defined medium containing FSH and environmentally relevant concentrations of DDE. Aromatase activity was measured by incubating the cells with 1beta-[(3)H]androstenedione and measuring the release of (3)H(2)O. The granulosa cell response to FSH was highly dependent on the basal level of aromatase activity (r = -0.703, P = 0.001, n = 17) with the highest activity occurring at low basal levels of aromatase activity. Enzyme activity was significantly stimulated at 100 ng DDE/ml. A synergistic effect on aromatizing activity was observed when cells were co-cultured with DDE and FSH. Concentrations of DDE similar to those present in human follicular fluid enhance basal and FSH-stimulated granulosa cell aromatizing enzyme activity.

Premature follicle luteinization as a paracrine regulator of androstenedione (A4) in prenatally androgenized (PA) female rhesus monkeys receiving follicle-stimulating hormone (FSH) therapy for in vitro fertilization (IVF)

Objective: Early prenatal androgenization in female rhesus monkeys receiving FSH therapy for IVF induces premature follicle luteinization, reduces intrafollicular A4 and estradiol (E2) levels, and impairs blastocyst development (Dumesic et al. JCEM 2002;87:1111). Such premature follicle luteinization may reduce follicle fluid (FF) A4 levels by impairing paracrine regulation of theca A4 synthesis by granulosa cells, since granulosa cell-derived factors in rats enhance luteinizing hormone (LH)-responsive theca androgen production (Smyth et al. Endocrinology 1993;133:1532). The present study examines whether FSH-stimulated granulosa cells induce paracrine signalling of theca androgen production in female rhesus monkeys undergoing FSH therapy for IVF and, if so, whether such FSH-stimulated signalling is impaired by prenatal androgenization. Design: Prospective study. Materials/Methods: Five early-treated (E) and 4 late-treated (L) PA female (e.g., 15–35 days of testosterone (T) propionate exposure starting on gestational days 40–44 or 100–115, respectively) and 5 control (C) monkeys were studied. All females received recombinant (r) human (h) FSH injections (30 IU, IM twice daily) for 7–9 days (without human chorionic gonadotropin); oocyte retrieval was performed 1 day later. Serum FSH, LH, E2, progesterone (P4), A4, T and dihydrotestosterone (DHT) were measured basally, during rhFSH therapy and at retrieval. Sex steroids in FF also were analyzed. Results: Age, weight and serum FSH were similar in all female groups; serum LH bioactivity was unchanged during rhFSH therapy. Serum E2 were unaffected by female type and rose significantly during rhFSH therapy to reach maximal levels at retrieval. Serum P4 levels also were similar in all females and decreased on day 2 of rhFSH therapy before returning to basal values during rhFSH therapy and at retrieval. Serum A4, T and DHT levels were unaffected by female type. Serum A4 levels remained at basal values through day 4 of rhFSH therapy, and rose on day 6 of rhFSH therapy (P ≤0.025 vs day 4), reaching maximal levels at retrieval (P ≤0.01 vs day 6). Serum T and DHT also rose above basal values by day 6 of rhFSH therapy (P ≤0.005) and increased further at retrieval (P ≤0.01). The FF/serum P4 ratio was significantly higher in EPA females than in LPA or C females (P ≤0.025). Conversely, the FF/serum A4 ratio was significantly lower in EPA females than in LPA females or C females (P ≤0.01). FF/serum E2 and T ratios were similar in all female groups; FF/serum DHT levels were elevated in all PA females (P ≤0.025). While the number of oocytes retrieved and incidences of oocyte nuclear maturation and fertilization were similar among female types, 15.3% of zygotes developed to blastocysts in C females. No zygote developed to blastocyst in any PA female. Conclusions: Paracrine signalling of theca A4 production by granulosa cells occurs in female rhesus monkeys receiving rhFSH for IVF. Early prenatal androgenization impairs this phenomenon through premature follicle luteinization and reduces the amounts of aromatizable androgen and E2 in the follicle during oocyte developmental competence. Supported by: NIH grants RR14093/RR13635.

Interaction of interleukin-6 on human granulosa cell steroid secretion.

Numerous studies have shown that a variety of cytokines are involved in the regulation of ovarian function. Interleukin-6 (IL-6), has been found in follicular fluid. In this report we show by immunocytochemical methods the localization of the extracellular domain of the IL-6-receptor and its associated signal transducer glycoprotein gp 130 on the surface of granulosa cells (GCs). The possibility that IL-6 may also influence the basal and FSH-stimulated production of estradiol (E2) and progesterone (Prog) by GCs in vitro was also investigated. GCs were obtained from infertile patients undergoing in vitro fertilization and embryo transfer treatment and cultured for 72 h or were given increasing concentrations of human recombinant IL-6 (8--128 pg/ml) in the absence or presence of FSH (96 U/ml). For the time-course studies, FSH-stimulated GCs were treated in the absence or presence of IL-6 (128 pg/ml) and supernatants were assayed at 24 h intervals (24-96 h) for E2 and Prog productions. The results show that increasing amounts of IL-6 significantly inhibit E2 production in the absence or presence of FSH vs untreated controls (P=0.025 at IL-6=128 pg/ml and P=0.016 at IL-6=16 pg/ml respectively). IL-6 also inhibited FSH-stimulated but not unstimulated Prog release (P=0.038 at IL-6=8 pg/ml). These findings suggest that IL-6 may be one of the factors that plays a local regulatory role in the course of FSH-stimulated E2 and Prog release. The time-course studies of the effect of the absence or presence of IL-6 demonstrated a significant inhibitory effect on both E2 and Prog secretion (P<0.001) by FSH-stimulated GCs. As infections of the female reproductive tract are often accompanied by elevated local IL-6 levels, this factor may be one of the links leading to endocrine reproductive dysfunction during genital infections.

Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries.

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.

Differential responses of granulosa cells from small and large follicles to follicle stimulating hormone (FSH) during the menstrual cycle and acyclicity: effects of tumour necrosis factor-alpha.

This study determined effects of follicle stimulating hormone (FSH) alone and in combination with tumour necrosis factor (TNF), on granulosa cells from small (5-10 mm diameter) and large (>10-25 mm) follicles during follicular and luteal phases of the cycle and during periods of acyclicity. Granulosa cells were collected from ovaries of premenopausal women undergoing oophorectomy. The cells were cultured with human FSH (2 ng/ml) and testosterone (1 microM) in the presence or absence of human TNF-alpha (20 ng/ml). Media were removed at 48 and 96 h after culture and progesterone, oestradiol and cAMP in media were measured by radioimmunoassays. FSH stimulated the accumulation of oestradiol from granulosa cells of small follicles during the follicular and luteal phases but not during acyclicity; and TNF reduced oestradiol accumulation in the presence of FSH. Interestingly, in granulosa cells from small follicles, progesterone and cAMP secretion increased in response to FSH and neither was affected by TNF. Thus, TNF specifically inhibited the conversion of testosterone to oestradiol in granulosa cells from small follicles. FSH stimulated oestradiol production by granulosa cells of large follicles obtained only during the follicular phase of the cycle and TNF inhibited the FSH-induced oestradiol secretion. Granulosa cells obtained from large follicles during the luteal phase and during acyclicity did not accumulate oestradiol in response to FSH. However, FSH increased progesterone and cAMP secretion by granulosa cells obtained from large follicles during the follicular and luteal phases. During the luteal phase alone, TNF in combination with FSH increased progesterone accumulation above that of FSH alone. FSH did not increase progesterone, oestradiol or cAMP secretion by granulosa cells obtained from large follicles during acyclicity. Thus, FSH increases progesterone, oestradiol and cAMP secretion by granulosa cells of small follicles during the follicular and luteal phases and TNF appears to inhibit FSH-induced oestradiol secretion specifically in those cells. In large follicles, FSH-stimulated granulosa cell secretion of oestradiol is limited to the follicular phase and this effect can be inhibited by TNF. In addition, when granulosa cells of large follicles do not increase oestradiol secretion in response to FSH, TNF stimulates progesterone secretion.

Effect of the protein phosphatase inhibitor okadaic acid on FSH-induced granulosa cell steroidogenesis.

To address a possible role of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0.01-1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimulated steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa cells were stimulated with the cAMP-inducing agents cholera toxin (1 microgram/ml), forskolin (15 mumol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time course experiments also revealed that a minimal time period of 12 h was necessary for OA (0.1 and 1 nmol/l) to significantly enhance FSH-induced progesterone and 3 beta-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.