FSH-induced Estradiol Production Research Articles

Effects of transforming growth factor β1 on steroidogenesis of feline granulosa cells cultured

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. To determine if transforming growth factor β1 (TGFB1), activin, epidermal growth factor (EGF), follicle stimulating hormone (FSH), luteinizing hormone (LH), melatonin, and insulin-like growth factor 1 (IGF1) regulate granulosa cell steroidogenesis and proliferation in cats, three experiments were conducted in winter season. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48h after an initial 48h plating in 10% fetal calf serum. Treatment with IGF1 and FSH increased (P <0.05) estradiol production by 2.3- and 1.33-fold, respectively. In contrast, TGFB1 blocked (P <0.05) IGF1-induced estradiol production and inhibited FSH-induced estradiol production by 60%. Combined with FSH or FSH plus IGF1, TGFB1 inhibited (P <0.05) cell proliferation, whereas TGFB1 increased progesterone production by 2.8-fold in the presence of FSH plus IGF1. EGF decreased (P <0.05) FSH plus IGF1-induced estradiol production by 89% but did not affect progesterone production or cell numbers. Activin did not affect (P >0.10) cell numbers or steroidogenesis in the presence of FSH plus IGF1. Melatonin and LH decreased (P <0.05) estradiol production 53% and 59%, respectively, without affecting progesterone production or cell proliferation. The present study has identified TGFB1 as a major regulator of feline ovarian function, in addition to EGF, IGF1, melatonin, LH and FSH. These studies will provide useful information for future development of fertility control in feline species.

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.

MiRNA-143 mediates the proliferative signaling pathway of FSH and regulates estradiol production.

MicroRNAs (MiRNAs) play important regulatory roles in many cellular processes. MiR-143 is highly enriched in the mouse ovary, but its roles and underlying mechanisms are not well understood. In the current study, we show that miR-143 is located in granulosa cells of primary, secondary and antral follicles. To explore the specific functions of miR-143, we transfected miR-143 inhibitor into primary cultured granulosa cells to study the loss of function of miR-143 and the results showed that miR-143 silencing significantly increased estradiol production and steroidogenesis-related gene expression. Moreover, our in vivo and in vitro studies showed that follicular stimulating hormone (FSH) significantly decreased miR-143 expression. This function of miR-143 is accomplished by its binding to the 3'-UTR of KRAS mRNA. Furthermore, our results demonstrated that miR-143 acts as a negative regulating molecule mediating the signaling pathway of FSH and affecting estradiol production by targeting KRAS. MiR-143 also negatively acts in regulating granulosa cells proliferation and cell cycle-related genes expression. These findings indicate that miR-143 plays vital roles in FSH-induced estradiol production and granulosa cell proliferation, providing a novel mechanism that involves miRNA in regulating granulosa cell functions.

1142 Protein kinase A directly phosphorylates GSK3β, and regulates β-catenin via phosphorylation in granulosa cells

Estradiol serves an important role in female fertility and FSH drives estradiol production. Beta-catenin is a transcriptional co-factor that is required for FSH-induced estradiol production. Beta-catenin is activated via phosphorylation at Ser552 and Ser675 by protein kinase A (PKA) dependent event. Wingless-type mouse mammary tumor virus (WNT) also regulates the β-catenin pathway. Glycogen Synthase Kinase-3 β (GSK3β) is a component of the β-catenin degradation complex; canonical WNT signaling pathway phosphorylates protein kinase B (AKT) and inhibits GSK3β resulting in β-catenin accumulation in the cytoplasm. Work in our laboratory demonstrated that WNT downregulates steroidogenesis. Furthermore, AKT is required for β-catenin accumulation and FSH-induced estradiol production, suggesting convergence of the WNT and FSH pathways. We hypothesize that WNT inhibition of FSH signaling occurs through modulation of phosphorylation patterns on β-catenin. The objective of these experiments was to evaluate the phosphorylation pattern of β-catenin in response to PKA, AKT, and WNT signaling pathways. Granulosa cells (KGN cell line) were cultured and treated with vehicle control, phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY) (30 μ M; 30 min), Forskolin (FSK) (10 μ M; 1.5 h), WNT (50 ng/mL, 30 min) and the combination of these treatments. Western blot was used to detect total and phosphorylated β-catenin and phosphorylated GSK3β. Protein abundances were analyzed using densitometry software and densitometry values for treatments were analyzed using the GLM procedure of SAS. When significant model interactions were detected, means were separated using PDIFF. Treatment of FSK combined with WNT and LY enhanced GSK3β phosphorylation compared to control (P < 0.05). Similarly, FSK alone or in combination with WNT and LY enhanced (P < 0.05) phosphorylation of β-catenin at Ser675 and Ser552, but WNT did not alter β-catenin phosphorylation after FSK stimulation (P > 0.10). However, when the AKT pathway was blocked with LY, FSK, and WNT treatment increased phosphorylation of GSK3β and subsequently β-catenin at Ser552 suggesting that PKA can directly phosphorylate GSK3β. Results from these experiments demonstrate that WNT does not alter FSH-stimulated phosphorylation patterns of β-catenin indicating that WNT inhibits FSH signaling via other unknown mechanisms.

Sleep deprivation enhances peripheral serotonin secretion to regulate the large follicle steroidogenesis of rats

ObjectiveSleep deprivation (SD) leads to the disturbance of the estrous cycle. Serotonin, the levels of which increase with SD, has been shown to inhibit luteinizing hormone production and the receptor has been found in the follicles. In this study, the serotonin effect on preovulatory follicular steroidogenesis is investigated and the underlying mechanisms are elucidated. Materials and methodsFemale rats were subjected to SD for a time span of 1–4 days using the dish-over-water-method with a Rechtschaffen apparatus. Serum estradiol and serotonin concentrations were assessed; thereafter, they were evaluated with the effect of serotonin on the estradiol production and steroidogenic acute regulatory (StAR) protein expression in a serum-free culture system. Preovulatory follicles were dissected mechanically from the ovaries of 21-day-old rats, which induced follicle growth, and cultured for 24 hours with or without recombinant human follicle-stimulating hormone (FSH) in the presence or absence of serotonin. ResultsSD, led to a significant decrease in serum estradiol concentrations, while serotonin concentrations were significantly elevated (all p < 0.05). Follicles were cultured with a constant dose of FSH (50 mIU/mL) and increasing doses of serotonin, estradiol production was reduced by 20%. The inhibitory effect of serotonin was concentration dependent. The addition of serotonin (0.1 μg/mL) decreased FSH-induced estradiol production and attenuated FSH-stimulated follicular StAR protein expression. The inhibitory effects of serotonin could be reduced by the serotonin receptor antagonist ketanserin. ConclusionThese findings suggest that decreased serum estradiol concentrations in SD rats may be the result of serotonin-related inhibition of estradiol production and decreased large follicle expression of StAR protein.

Effect of Anti-Müllerian hormone (AMH) and bone morphogenetic protein 15 (BMP-15) on steroidogenesis in primary-cultured human luteinizing granulosa cells through Smad5 signalling.

To determine if there is any effect of AMH and BMP-15 on estradiol and progesterone production from primary-cultured human luteinizing granulosa cells, to delineate what is the effect of FSH on their actions and which are the possible mechanisms involved. Luteinizing granulosa cells (GCs), obtained from follicular fluid of 30 women undergoing in vitro fertilization, were cultured, after a short 24-h preincubation period, in serum-free medium for 24 or/and 48 h in the presence/absence of various concentrations of AMH, BMP-15 and FSH alone or in combinations. Estradiol and progesterone production, SMAD5 phosphorylation and StAR expression were studied in parallel. Steroids were measured in culture-supernatant using enzyme-immunoassays, while Smad5-signaling pathway activation and StAR protein expression were assessed immunocytochemically. We found that the treatment of AMH in GCs for 24/48 h attenuated FSH-induced estradiol production (p < 0.001), had no effect on basal estradiol levels, decreased basal progesterone production (p < 0.001) and FSH-induced StAR expression (p < 0.001). On the other hand, BMP-15 decreased basal estradiol levels (p < 0.001) and attenuated FSH-induced estradiol production (p < 0.001). Furthermore, BMP-15 reduced progesterone basal secretion (p < 0.001), an effect that was partially reversed by FSH (p < 0.01), probably via increasing StAR expression (p < 0.001). FSH-induced StAR expression was also attenuated by BMP-15 (p < 0.001). FSH, AMH and BMP-15 activated Smad-signaling pathway, as confirmed by the increase of phospo-Smad5 protein levels (p < 0.001 compared to control). AMH and BMP-15 by interacting with FSH affect the production of estradiol and progesterone from cultured luteinizing-granulosa cells possibly via Smad5-protein phosphorylation.

Regulatory role of BMP-9 in steroidogenesis by rat ovarian granulosa cells

BMPs expressed in the ovary differentially regulate steroidogenesis by granulosa cells. BMP-9, a circulating BMP, is associated with cell proliferation, apoptosis and differentiation in various tissues. However, the effects of BMP-9 on ovarian function have yet to be elucidated. Here we investigated BMP-9 actions on steroidogenesis using rat primary granulosa cells. BMP-9 potently suppressed FSH-induced progesterone production, whereas it did not affect FSH-induced estradiol production by granulosa cells. The effects of BMP-9 on FSH-induced steroidogenesis were not influenced by the presence of oocytes. FSH-induced cAMP synthesis and FSH-induced mRNA expression of steroidogenic factors, including StAR, P450scc, 3βHSD2 and FSHR, were suppressed by treatment with BMP-9. BMP-9 mRNA expression was detected in granulosa cells but not in oocytes. BMP-9 readily activated Smad1/5/8 phosphorylation and Id-1 transcription in granulosa cells. Analysis using ALK inhibitors indicated that BMP-9 actions were mediated via type-I receptors other than ALK-2, -3 and -6. Furthermore, experiments using extracellular domains (ECDs) for BMP type-I and -II receptor constructs revealed that the effects of BMP-9 were reversed by ECDs for ALK-1 and BMPRII. Thus, the functional receptors for BMP-9 in granulosa cells were most likely to be the complex of ALK-1 and BMPRII. Collectively, the results of the present study showed that BMP-9 can affect luteinization and that there are two possible sources of BMP-9, serum and granulosa cells in the ovary.

Involvement of bone morphogenetic protein activity in somatostatin actions on ovarian steroidogenesis

Somatostatin is expressed in the hypothalamus, pancreas and gastrointestinal tracts and it inhibits the secretion of various hormones in vivo. In the rodent ovary, somatostatin receptor (SSTR) subtypes 2 and 5 are expressed in granulosa cells and oocytes. Somatostatin analogs have been clinically used for treatment of endocrine tumors. For this purpose, relatively high-dose or long-term treatments of somatostatin analogs are necessary; however, the direct and continuous impact of somatostatin analogs on gonadal functions has yet to be elucidated. In the present study, we investigated the effects of somatostatin analogs (octreotide and pasireotide) on ovarian steroidogenesis by rat primary granulosa cell culture. The expression levels of SSTR2 and SSTR5 in granulosa cells were upregulated by FSH treatment. Treatment with somatostatin analogs decreased FSH-induced estradiol production with reduction in aromatase mRNA expression, while the treatment also suppressed FSH-induced progesterone production with reduction of mRNAs levels of StAR, P450scc and 3βHSD2 in granulosa cells. This trend was also observed in a granulosa/oocyte co-culture condition. The effect of pasireotide was more potent than that of octreotide. FSH-induced synthesis of steroids and cAMP was also suppressed by somatostatin analog treatment. Notably, pretreatment with a BMP-binding protein, noggin reversed the suppressive effects of somatostatin analogs on progesterone and cAMP production, suggesting that the endogenous BMP system is functionally involved in the SSTR effects in granulosa cells. Treatment with BMP-2, -4, -6 and -7 decreased the mRNA expression of inhibitory Smads6 and 7, leading to enhancement of BMP actions detected by Id-1 transcription in granulosa cells. Collectively, the results revealed that SSTR activation modulates ovarian steroidogenesis by upregulating endogenous BMP activity in growing follicles.

Regulatory role of kit ligand–c-kit interaction and oocyte factors in steroidogenesis by rat granulosa cells

Although kit ligand (KL)–c-kit interaction is known to be critical for oogenesis and folliculogenesis, its role in ovarian steroidogenesis has yet to be elucidated. We studied the impact of KL–c-kit interaction in regulation of steroidogenesis using rat oocyte/granulosa cell co-culture. In the presence of oocytes, soluble KL suppressed FSH-induced estradiol production and aromatase mRNA expression without affecting FSH-induced progesterone production. The KL effect on steroidogenesis was interrupted by an anti-c-kit neutralizing antibody, suggesting that KL–c-kit interaction is involved in suppression of estrogen by granulosa cells through oocyte c-kit action. The cAMP-PKA pathway activity was not directly involved in the estrogen regulation by KL–c-kit action. It was of note that KL treatment increased the expression levels of oocyte-derived FGF-8, GDF-9 and BMP-6, while it reduced the expression levels of oocyte-derived BMP-15 in the oocyte-granulosa cell co-culture. Given the findings that FGF-8, but not GDF-9, BMP-6 or -15, suppressed FSH-induced estrogen production by granulosa cells, oocyte-derived FGF-8 is linked to suppression of FSH-induced estrogen production through the KL–c-kit interaction. Furthermore, the suppression of FSH-induced estrogen production by KL in the co-culture was reversed by a FGF receptor kinase inhibitor and the effect of the inhibitor was enhanced in combination with extracellular-domain protein of BMPRII, which interferes with BMP-15 and GDF-9 activities. Thus, the actions of endogenous oocyte factors including FGF-8 and BMP-15/GDF-9 were involved in the KL activity that inhibited FSH-induced estradiol production. Collectively, the results indicate that KL–c-kit interaction plays a role in estrogenic regulation through oocyte-granulosa cell communication.

Mutual Regulation of Growth Hormone and Bone Morphogenetic Protein System in Steroidogenesis by Rat Granulosa Cells

GH induces preantral follicle growth and differentiation with oocyte maturation. However, the effects of GH on ovarian steroidogenesis and the mechanisms underlying its effects have yet to be elucidated. In this study, we investigated the actions of GH on steroidogenesis by rat granulosa cells isolated from early antral follicles by focusing on the ovarian bone morphogenetic protein (BMP) system. We found that GH suppressed FSH-induced estradiol production with reduction in aromatase expression and, in contrast, GH increased FSH-induced progesterone level with induction of steroidogenic acute regulatory protein, side chain cleavage cytochrome P450, and 3β-hydroxysteroid dehydrogenase. The effects of GH on steroidogenesis by granulosa cells were enhanced in the presence of the BMP antagonist noggin. Coculture of GH with oocytes did not alter GH regulation of steroidogenesis. Steroid production induced by cAMP donors was not affected by GH treatment and the GH effects on FSH-induced steroid production were not accompanied by changes in cAMP synthesis, suggesting that GH actions were not directly mediated by the cAMP-protein kinase A pathway. GH exerted synergistic effects on MAPK activation elicited by FSH, which regulated FSH-induced steroidogenesis. In addition, GH-induced signal transducer and activator of transcription phosphorylation was involved in the induction of IGF-I expression. GH increased IGF-I, IGF-I receptor, and FSH receptor expression in granulosa cells, and inhibition of IGF-I signaling restored GH stimulation of FSH-induced progesterone production, suggesting that endogenous IGF-I is functionally involved in GH effects on progesterone induction. BMP inhibited IGF-I effects that increased FSH-induced estradiol production with suppression of expression of the GH/IGF-I system, whereas GH/IGF-I actions impaired BMP-Sma and Mad related protein 1/5/8 signaling through down-regulation of the expression of BMP receptors. Thus, GH acts to modulate estrogen and progesterone production differentially through endogenous IGF-I activity in granulosa cells, in which GH-IGF-I interaction leads to antagonization of BMP actions including suppression of FSH-induced progesterone production. Mutual balance between GH/IGF-I and BMP signal intensities may be a key for regulating gonadotropin-induced steroidogenesis in growing follicles.

Anti-Müllerian Hormone Inhibits FSH-Induced Adenylyl Cyclase Activation, Aromatase Expression, and Estradiol Production in Human Granulosa-Lutein Cells.

Anti-Müllerian Hormone Inhibits FSH-Induced Adenylyl Cyclase Activation, Aromatase Expression, and Estradiol Production in Human Granulosa-Lutein Cells.

A Novel Antagonistic Effect of the Bone Morphogenetic Protein System on Prolactin Actions in Regulating Steroidogenesis by Granulosa Cells

To investigate the mechanism by which prolactin (PRL) regulates follicular steroidogenesis in the ovary, we examined the functional roles of PRL in steroidogenesis using rat oocyte/granulosa cell coculture and focusing on the bone morphogenetic protein (BMP) system. The expression of long and short forms of PRL receptor (PRLR) were detected in both oocytes and granulosa cells, and PRL effectively up-regulated PRLR expression in granulosa cells in the presence of FSH. PRL suppressed FSH-induced estradiol production and increased FSH-induced progesterone production in granulosa cells. The PRL effects on FSH-induced progesterone were blocked by coculture with oocytes, implying roles of oocyte-derived factors in suppression of progesterone production in PRL-exposed granulosa cells. In accordance with the data for steroids, FSH-induced aromatase expression was suppressed by PRL, whereas FSH-induced steroidogenic acute regulatory protein, P450scc (P450 side-chain cleavage enzyme), and 3β-hydroxysteroid dehydrogenase type 2 levels were amplified by PRL. However, forskolin- and N(6),O(2)-dibutyryl cAMP-induced steroid levels and FSH- and forskolin-induced cAMP were not affected by PRL, suggesting that PRL action on FSH-induced steroidogenesis was not due to cAMP-protein kinase A regulation. Treatment with a BMP-binding protein, noggin, facilitated PRL-induced estradiol reduction, and noggin increased PRL-induced progesterone production in FSH-treated granulosa cells cocultured with oocytes, suggesting that endogenous BMPs reduce progesterone but increase estradiol when exposed to high concentrations of PRL. PRL increased the expression of BMP ligands in oocyte/granulosa cell coculture and augmented BMP-induced phosphorylated mothers against decapentaplegic 1/5/8 signaling by reducing inhibitory phosphorylated mothers against decapentaplegic 6 expression through the Janus kinase/signal transducer and activator of transcription (STAT) pathway. In addition to STAT activation, PRL enhanced FSH-induced MAPK phosphorylation in granulosa cells, in which ERK activation was preferentially involved in suppression of FSH-induced estradiol. Furthermore, noggin treatment enhanced PRLR signaling including MAPK and STAT. Considering that BMPs suppressed PRLR in granulosa cells, it is likely that the BMP system in growing follicles plays a key role in antagonizing PRLR signaling actions in the ovary exposed to high concentrations of PRL.

Functional relationship between fibroblast growth factor-8 and bone morphogenetic proteins in regulating steroidogenesis by rat granulosa cells

Bone morphogenetic proteins (BMPs) have been recognized as crucial molecules in regulating ovarian physiology, with different BMPs having differential actions in FSH-induced estradiol production. To identify the roles of oocyte factors that modulate steroidogenesis controlled by BMPs, we here investigated the effects of FGF-8 in rat granulosa/oocyte co-cultures. FGF-8 potently suppressed FSH-induced estradiol production, but did not affect cAMP-induced estradiol produced by rat granulosa cells. FGF-8 had no effects on progesterone and cAMP production induced by FSH and forskolin. The inhibitory effects of FGF-8 on FSH-induced estradiol production were not altered by BMP-2, -4, -6 or -7. In the presence of FGF-8, BMPs suppressed FSH-induced progesterone by reducing cAMP, suggesting that FGF-8 and BMP independently regulate FSH receptor signaling. Notably, FGF-8-induced ERK and SAPK/JNK phosphorylation in granulosa cells, in which ERK activation was further enhanced by FSH and oocytes. Inhibition of ERK and SAPK/JNK reduced FSH-induced progesterone and cAMP levels, suggesting that the activation of these pathways enhances FSH-induced cAMP signaling. In addition, ERK inhibition upregulated FSH-induced estradiol synthesis, indicating that ERK pathway is also involved in suppressing aromatase activity in granulosa cells. Interestingly, FGF-8 enhanced BMP-induced Smad1/5/8 and Id-1-promoter activities with decreased expression of Smad6/7. Since the SAPK/JNK inhibitor inhibited FGF-8 effects in upregulating Id-1 transcription, SAPK/JNK appears to be involved in the mechanism by which FGF-8 enhances BMP–Smad signaling. Furthermore, in the presence of oocytes, the inhibition of endogenous FGF receptor signaling suppressed FSH- and forskolin-induced progesterone and cAMP, showing that endogenous FGF system is involved in activation of FSH-induced cAMP–PKA signaling via ERK and SAPK/JNK. Thus, the oocyte factor, FGF-8, not only suppresses FSH-induced estradiol production by activating ERK, but also enhances BMP–Smad signaling in granulosa cells. This interaction between FGF-8 and BMPs may play a key role in regulating steroidogenesis through oocyte-granulosa cell communication.

P38-Mitogen-Activated Protein Kinase Stimulated Steroidogenesis in Granulosa Cell-Oocyte Cocultures: Role of Bone Morphogenetic Proteins 2 and 4

Roles of the p38-MAPK pathway in steroidogenesis were investigated using coculture of rat granulosa cells with oocytes. Activin and FSH readily phosphorylated p38 in granulosa cells. Activin effect on p38 phosphorylation was abolished by a selective activin receptor-like kinase-4, -5, and -7 inhibitor, SB431542. SB431542 decreased FSH-induced estradiol but had no effect on progesterone production with a marginal cAMP reduction, suggesting that endogenous activin is primarily involved in estradiol synthesis. FSH-induced p38 activation was not affected either by SB431542 or follistatin, suggesting that FSH activates p38 not through the endogenous activin. Bone morphogenetic protein (BMP)-2 and BMP-4 also enhanced FSH-induced p38 phosphorylation, which was augmented by oocyte action. A specific p38 inhibitor, SB203580, decreased FSH-induced estradiol production. However, FSH-induced cAMP accumulation was not changed by SB203580, suggesting that p38 activation is linked to estradiol synthesis independently of cAMP. BMP-2 and BMP-4 inhibited FSH- and forskolin (FSK)-induced progesterone and cAMP synthesis regardless of oocyte action. BMP-2, BMP-4, and activin increased FSH-induced estradiol production, which was enhanced in the presence of oocytes. In contrast to activin that enhanced FSK-induced estradiol, BMP-2 and BMP-4 had no effects on FSK-induced estradiol production, suggesting that BMP-2 and BMP-4 directly activate FSH-receptor signaling. Given that activin increased, but BMP-2 and BMP-4 decreased, FSH-induced cAMP, the effects of BMP-2 and BMP-4 on estradiol enhancement appeared to be diverged from the cAMP-protein kinase A pathway. Thus, BMP-2 and BMP-4 differentially regulate steroidogenesis by stimulating FSH-induced p38 and suppressing cAMP. The former is involved in estradiol production and enhanced by oocyte action, whereas the latter leads to reduction of progesterone synthesis.

Cocaine- and Amphetamine-Regulated Transcript Accelerates Termination of Follicle-Stimulating Hormone-Induced Extracellularly Regulated Kinase 1/2 and Akt Activation by Regulating the Expression and Degradation of Specific Mitogen-Activated Protein Kinase Phosphatases in Bovine Granulosa Cells

Pleiotropic actions of cocaine- and amphetamine-regulated transcript (CART) are well described in the central nervous system and periphery, but the intracellular mechanisms mediating biological actions of CART are poorly understood. Although CART is not expressed in mouse ovaries, we have previously established CART as a novel intracellular regulator of estradiol production in bovine granulosa cells. We demonstrated that inhibitory actions of CART on estradiol production are mediated through inhibition of FSH-induced cAMP accumulation, Ca(2+) influx, and aromatase mRNA expression via a G(o/i)-dependent pathway. We also reported that FSH-induced estradiol production is dependent on Erk1/2 and Akt signaling, and CART may regulate other signaling proteins downstream of cAMP essential for estradiol production. Here, we demonstrate that CART is a potent inhibitor of FSH-stimulated Erk1/2 and Akt signaling and the mechanisms involved. Transient CART stimulation of bovine granulosa cells shortens the duration of FSH-induced Erk1/2 and Akt signaling whereas a prolonged (24 h) CART treatment blocks Erk1/2 and Akt activation in response to FSH. This CART-induced accelerated termination of Erk1/2 and Akt signaling is mediated both by induced expression and impaired ubiquitin-mediated proteasome degradation of dual specific phosphatase 5 (DUSP5) and protein phosphatase 2A. Results also support existence of a negative feedback loop in which CART via a G(o/i)-MAPK kinase dependent pathway activates Erk1/2, and the latter induces DUSP5 expression. Moreover, small interfering RNA mediated ablation of DUSP5 and/or protein phosphatase 2A prevents the CART-induced early termination of Erk1/2 and Akt signaling. Results provide novel insight into the intracellular mechanism of action of CART in regulation of FSH-induced MAPK signaling.

Cocaine- and Amphetamine-Regulated Transcript Regulation of Follicle-Stimulating Hormone Signal Transduction in Bovine Granulosa Cells

Regulation of estradiol production, central to ovarian follicular development and reproductive function, is mediated by a complex interaction of pituitary gonadotropins such as FSH with locally produced regulatory molecules. We previously demonstrated a negative association of expression of cocaine-and amphetamine-regulated transcript (CART) with follicle health status and a novel local negative role for CART in regulation of basal estradiol production by bovine granulosa cells. However, effects of CART on FSH-induced estradiol production and the underlying mechanism(s) mediating the physiological actions of CART on granulosa cells are not known. Objectives of the present study were to determine effects of CART on basal and FSH-induced intracellular cAMP levels, aromatase mRNA, estradiol accumulation, calcium signaling, and the intracellular signaling pathways involved using primary cultures of bovine granulosa cells. CART treatment potently inhibits the FSH-induced rise in granulosa cell cAMP levels, estradiol accumulation, and aromatase mRNA. Furthermore, results show that calcium is essential for FSH-induced cAMP and estradiol accumulation, and CART significantly inhibits FSH-induced calcium influx. Select G protein and protein kinase inhibitors were used to elucidate pathways involved in CART actions. The inhibitory actions of CART on FSH signaling and estradiol production are mediated via a G(o/i)-dependent pathway, whereas none of the other signaling inhibitors had any effect on CART actions. Results demonstrate novel potent inhibitory effects of CART on multiple components of the FSH signaling pathway linked to estradiol production and follicular development and shed new insight into the mechanism of action of CART potentially pertinent within and beyond the reproductive system.

Differential Regulation of Steroidogenesis by Bone Morphogenetic Proteins in Granulosa Cells: Involvement of Extracellularly Regulated Kinase Signaling and Oocyte Actions in Follicle-Stimulating Hormone-Induced Estrogen Production

In the present study, we investigated the cellular mechanism by which oocytes and bone morphogenetic proteins (BMPs) govern FSH-induced steroidogenesis using rat primary granulosa cells. BMP-6 and BMP-7 both inhibited FSH- and forskolin (FSK)-induced progesterone synthesis and reduced cAMP synthesis independent of the presence or absence of oocytes. BMP-7 also increased FSH-induced estradiol production, and the response was further augmented in the presence of oocytes. In contrast, BMP-6 had no impact on estradiol synthesis regardless of the presence of oocytes. Because BMP-7 changed neither FSK- nor cAMP-induced estradiol production, the BMP-7 action was mediated through a FSH receptor signaling mechanism that was independent of cAMP-protein kinase A pathway. Treatment with FSH but not cAMP activated ERK1/2 phosphorylation in granulosa cells, which was further accelerated by oocytes. A specific ERK inhibitor, U0126, increased estradiol production and decreased FSH- and FSK-induced progesterone production and cAMP synthesis. This suggests that ERK activation is directly linked to inhibition of estradiol synthesis and amplification of cAMP. Moreover, FSH-induced ERK1/2 phosphorylation was inhibited by BMP-7 but not influenced by BMP-6. In contrast, BMP signaling including Smad1/5/8 phosphorylation and Id-1 transcription was up-regulated by FSH and oocytes in granulosa cells through inhibition of Smad6/7 expression. Collectively, oocytes enhance FSH-induced MAPK activation and BMP signaling in granulosa cells, which leads to differential regulation of steroidogenesis elicited by BMPs in the presence of FSH in developing follicles.

Alterations in steroid hormone production by porcine ovarian granulosa cells caused by bisphenol A and bisphenol A dimethacrylate

We have investigated the effects of bisphenol A (BPA) and BPA-dimethacrylate (BPA-DMA), endocrine disruptors used as plasticizers, on steroid hormone production by porcine ovarian granulosa cells after 72 h incubation. BPA at 10 −8 M to 10 −5 M increased basal progesterone levels, while the same concentration range of BPA-DMA did not cause any changes. After FSH-stimulation of the cells, BPA-DMA showed a tendency to inhibit progesterone production. BPA, however, at 10 −7 M and 10 −6 M concentrations was even able to amplify FSH-stimulated progesterone synthesis. BPA as well as BPA-DMA inhibited FSH-induced estradiol production in the whole concentration range. LH-stimulated progesterone production was not altered by BPA in 10 −8 M to 10 −5 M, while BPA-DMA decreased progesterone levels in the cultured media. Significant inhibitory effect of both tested agents at 10 −4 M concentrations was observed specifically on progesterone production, basal as well as gonadotropin-stimulated. The results indicate that ovarian steroidogenesis might be one of the possible sites afflicted by the endocrine disrupting action of BPA and BPA-DMA.

Activation of the p38 MAPK pathway by follicle-stimulating hormone regulates steroidogenesis in granulosa cells differentially

In the present study, we started out to test whether the follicle-stimulating hormone (FSH)-activated p38 MAPK signaling cascade was involved in the regulation of steroidogenesis in granulosa cells (GCs). GCs were prepared from the ovaries of DES-treated immature rats and cultured in serum-free medium. Treatment of GCs with FSH (50 ng/ml) induced the phosphorylation of p38 MAPK rapidly with the phosphorylation being observed within 5 min and reaching the highest level at 30 min. Such activation was protein kinase A-dependent as indicated by the results using specific inhibitors. FSH stimulated the production of progesterone and estradiol as well as the expression of the steroidogenic acute regulatory protein (StAR) in a time-dependent manner, with a maximum level being observed in the production of progesterone and StAR at 48 h. Moreover, the potent p38 MAPK inhibitor SB203580 (20 microM) augmented FSH-induced progesterone and StAR production, while reduced FSH-induced estradiol production at the same time (P<0.01). RT-PCR data showed that inclusion of SB203580 in the media enhanced FSH-stimulated StAR mRNA production, while decreased the FSH-stimulated P450arom mRNA expression (P<0.05). Immunocytochemical studies showed that FSH treatment together with the inhibition of p38 MAPK activity resulted in a higher expression of StAR in mitochondria than FSH treatment alone. FSH also significantly up-regulated the protein level of LRH-1, a member of the orphan receptor family that activates the expression of P450arom in ovaries and testes. p38 MAPK inactivation down-regulated the basal and FSH-induced LRH-1 expression significantly. The intra-cellular level of DAX-1, another orphan receptor that inhibits StAR expression, also decreased upon p38 MAPK being inactivated. For the first time, the present study suggests that FSH-activated p38 MAPK signal pathway regulates progesterone and estrogen production in GCs differentially, and that the transcription factors LRH-1 and DAX-1 might play important roles in the process.

Role of pituitary adenylate cyclase-activating peptide (PACAP) in the cyclic recruitment of immature follicles in the rat ovary

Following the midcyclic gonadotropin surge, PACAP is transiently expressed for approximately 12 h in the cyclic adult rat ovary. PACAP is observed in granulosa/lutein cells of the large mature follicles destined to ovulate and is believed to be a regulator of acute progesterone production and luteinization in these follicles. PACAP is also observed in solitary theca cells of immature follicles and in interstitial glandular cells intimately surrounding immature follicles. To examine if PACAP could be involved in the process of cyclic recruitment of such immature follicles, we primed immature granulosa cells from prepubertal ovaries with PACAP (1 nM and 100 nM) for 12 h. The treatment significantly stimulated the subsequent 24 h FSH-induced estradiol production (2.2 and 2.4 fold, respectively). The response seemed to be caused by a stimulation of aromatase activity. Estradiol production induced by testosterone was increased 2.4 and 2.6 fold, respectively, whereas functional FSH-receptors (cAMP production following FSH stimulation) or spontaneous apoptosis (immunohistochemical detection of DNA fragments) was unaffected. We conclude that PACAP priming of immature rat granulosa cells for 12 h increases subsequent FSH induced estradiol production and that PACAP could be involved in the cyclic recruitment of immature follicles in the adult rat ovary.