Phosphodiesterase regulation is critical for the differentiation and pattern of gene expression in granulosa cells of the ovarian follicle

Feedback regulations are integral components of the cAMP signaling required for most cellular processes, including gene expression and cell differentiation. Here, we provide evidence that one of these feedback regulations involving the cyclic nucleotide phosphodiesterase PDE4D plays a critical role in cAMP signaling during the differentiation of granulosa cells of the ovarian follicle. Gonadotropins induce PDE4D mRNA and increase the cAMP hydrolyzing activity in granulosa cells, demonstrating that a feedback regulation of cAMP is operating in granulosa cells in vivo. Inactivation of the PDE4D by homologous recombination is associated with an altered pattern of cAMP accumulation induced by the gonadotropin LH/human chorionic gonadotropin (hCG), impaired female fertility, and a markedly decreased ovulation rate. In spite of a disruption of the cAMP response, LH/hCG induced P450 side chain cleavage expression and steroidogenesis in a manner similar to wild-type controls. Morphological examination of the ovary of PDE4D-/- mice indicated luteinization of antral follicles with entrapped oocytes. Consistent with the morphological finding of unruptured follicles, LH/hCG induction of genes involved in ovulation, including cyclooxygenase-2, progesterone receptor, and the downstream genes, is markedly decreased in the PDE4D-/- ovaries. These data demonstrate that PDE4D regulation plays a critical role in gonadotropin mechanism of action and suggest that the intensity and duration of the cAMP signal defines the pattern of gene expression during the differentiation of granulosa cells.     

Genetic dissection of epidermal growth factor receptor signaling during luteinizing hormone-induced oocyte maturation

Recent evidence that luteinizing hormone (LH) stimulation of ovulatory follicles causes transactivation of the epidermal growth factor receptor (EGFR) has provided insights into the mechanisms of ovulation. However, the complete array of signals that promote oocyte reentry into the meiotic cell cycle in the follicle are still incompletely understood. To elucidate the signaling downstream of EGFR involved in oocyte maturation, we have investigated the LH responses in granulosa cells with targeted ablation of EGFR. Oocyte maturation and ovulation is disrupted when EGFR expression is progressively reduced. In granulosa cells from mice with either global or granulosa cell-specific disruption of EGFR signaling, LH-induced phosphorylation of MAPK3/1, p38MAPK, and connexin-43 is impaired. Although the LH-induced decrease in cGMP is EGFR-dependent in wild type follicles, LH still induces a decrease in cGMP in Egfr(delta/f) Cyp19-Cre follicles. Thus compensatory mechanisms appear activated in the mutant. Spatial propagation of the LH signal in the follicle also is dependent on the EGF network, and likely is important for the control of signaling to the oocyte. Thus, multiple signals and redundant pathways contribute to regulating oocyte reentry into the cell cycle.     

Role of the epidermal growth factor network in ovarian follicles

The LH surge causes major remodeling of the ovarian follicle in preparation for the ovulatory process. These changes include reprogramming of granulosa cells to differentiate into luteal cells, changes in cumulus cell secretory properties, and oocyte maturation. This review summarizes published data in support of the concept that LH stimulation of ovarian follicles involves activation of a local epidermal growth factor (EGF) network. A model describing this property of LH signaling and its branching to other signaling modules is discussed. According to this model, LH activation of mural granulosa cells stimulates cAMP signaling, which, in turn, induces the expression of the EGF-like growth factors epiregulin, amphiregulin, and betacellulin. These growth factors function by activating EGF receptors in either an autocrine/juxtacrine fashion within the mural layer, or they diffuse to act on cumulus cells. Activation of EGF receptor signaling in cumulus cells, together with cAMP priming, triggers oocyte nuclear maturation and acquisition of developmental competence as well as cumulus expansion. This model has important implications for ovarian physiology and for the development of new strategies for the pharmacological control of ovulation and for gamete maturation in vitro.     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.     

Identification of calcitonin expression in the chicken ovary: influence of follicular maturation and ovarian steroids

Calcitonin (CALCA), a hormone primarily known for its role in calcium homeostasis, has recently been linked to reproduction, specifically as a marker for embryo implantation in the uterus. Although CALCA expression has been documented in several tissues, there has been no report of production of CALCA in the ovary of any vertebrate species. We hypothesized that the Calca gene is expressed in the chicken ovary, and its expression will be altered by follicular maturation or gonadal steroid administration. Using RT-PCR, we detected Calca mRNA and the calcitonin receptor (Calcr) mRNA in the granulosa and theca layers of preovulatory and prehierarchial follicles. Both CALCA and Calca mRNA were localized in granulosa and thecal cells by confocal microscopy. Using quantitative PCR analysis, F1 follicle granulosa layer was found to contain significantly greater Calca mRNA and Calcr mRNA levels compared with those of any other preovulatory or prehierarchial follicle. The granulosa layer contained relatively greater Calca and Calcr mRNA levels compared with the thecal layer in both prehierarchial and preovulatory follicles. Progesterone (P(4)) treatment of sexually immature chickens resulted in a significantly greater abundance of ovarian Calca mRNA, whereas estradiol (E(2)) or P(4) + E(2) treatment significantly reduced ovarian Calca mRNA quantity. Treatment of prehierarchial follicular granulosa cells in vitro with CALCA significantly decreased FSH-stimulated cellular viability. Collectively, our results indicate that follicular maturation and gonadal steroids influence Calca and Calcr gene expression in the chicken ovary. We conclude that ovarian CALCA is possibly involved in regulating follicular maturation in the chicken ovary.     

Actions of epidermal growth factor receptor/mitogen-activated protein kinase and protein kinase C signaling in granulosa cells from Gallus gallus are dependent upon stage of differentiation

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.     

Connective tissue growth factor is required for normal follicle development and ovulation

Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-β superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-β family intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-β and progesterone signaling cascades and is necessary for normal follicle development and ovulation.     

Stage-specific expression of Smad2 and Smad3 during folliculogenesis

Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor beta (TGFbeta) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFbeta signaling molecules known as Smads have not been evaluated during follicle development. We have demonstrated that two Smad family members that function as mediators for both activin and TGFbeta are expressed in granulosa cells of preantral follicles but not in large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in response to treatment with either TGFbeta or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFbeta stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFbeta family ligands using the same signaling pathways over the course of follicular development.     

The Expression Pattern of microRNAs in Granulosa Cells of Subordinate and Dominant Follicles during the Early Luteal Phase of the Bovine Estrous Cycle

This study aimed to investigate the miRNA expression patterns in granulosa cells of subordinate (SF) and dominant follicle (DF) during the early luteal phase of the bovine estrous cycle. For this, miRNA enriched total RNA isolated from granulosa cells of SF and DF obtained from heifers slaughtered at day 3 and day 7 of the estrous cycle was used for miRNAs deep sequencing. The results revealed that including 17 candidate novel miRNAs, several known miRNAs (n = 291–318) were detected in SF and DF at days 3 and 7 of the estrous cycle of which 244 miRNAs were common to all follicle groups. The let-7 families, bta-miR-10b, bta-miR-26a, bta-miR-99b and bta-miR-27b were among abundantly expressed miRNAs in both SF and DF at both days of the estrous cycle. Further analysis revealed that the expression patterns of 16 miRNAs including bta-miR-449a, bta-miR-449c and bta-miR-222 were differentially expressed between the granulosa cells of SF and DF at day 3 of the estrous cycle. However, at day 7 of the estrous cycle, 108 miRNAs including bta-miR-409a, bta-miR-383 and bta-miR-184 were differentially expressed between the two groups of granulosa cell revealing the presence of distinct miRNA expression profile changes between the two follicular stages at day 7 than day 3 of the estrous cycle. In addition, unlike the SF, marked temporal miRNA expression dynamics was observed in DF groups between day 3 and 7 of the estrous cycle. Target gene prediction and pathway analysis revealed that major signaling associated with follicular development including Wnt signaling, TGF-beta signaling, oocyte meiosis and GnRH signaling were affected by differentially expressed miRNAs. Thus, this study highlights the miRNA expression patterns of granulosa cells in subordinate and dominant follicles that could be associated with follicular recruitment, selection and dominance during the early luteal phase of the bovine estrous cycle.     

Luteinizing Hormone Activates Chloride Currents in Hen Ovarian Granulosa Cells

Luteinizing hormone (LH) induces progesterone production in hen ovarian granulosa cells, and this induction is inhibited when chloride ions are removed from the culture medium. This suggests that chloride channels may be involved in the signal transduction pathway responsible for the LH-induced progesterone production. In this report, we examined effects of LH on plasma membrane ion currents in single granulosa cells isolated from the largest preovulatory follicle (F₁) of the hen (Gallus domesticus). Using the perforated patch whole cell voltage clamp technique, we found that addition of LH rapidly activated a chloride current in these cells. This chloride current was present at all voltages tested (−90 to +50 mV), showed outward rectification and showed no obvious time or voltage dependence. Its magnitude was 3.5-fold that of the total resting membrane current measured before LH treatment. LH is known to elevate cyclic AMP in these cells. We found that addition of the cAMP analog Sp-cAMPS mimicked LH in inducing chloride currents in these cells. We conclude that LH can activate a chloride conductance in granulosa cells, and that this action may be mediated by cAMP.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the zebrafish ovary: evidence for potentially dual roles of PACAP in controlling final oocyte maturation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.     

Induction and inhibition of in vitro oocyte maturation and production of steroids in fish follicles by forskolin

The effects of forskolin (FK) on in vitro oocyte maturation and production of steroids were examined in Oryzias latipes. When oocytes within preovulatory follicles were preincubated in the presence of FK for 2-10 hr, they matured normally after additional incubation for 10-20 hr in plain culture medium. Naked (follicle cell-free) oocytes did not mature under these conditions. FK stimulated dose-dependent production of steroids (estradiol-17 beta, E2, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta-diOHprog) and cAMP in follicle (granulosa) cells. On the other hand, exposure to FK within 2 hr after 17 alpha,20 beta-diOH prog stimulation caused reversible inhibition of gonadotropin (PMS)- or 17 alpha,20 beta-diOH prog-induced maturation of the intrafollicular oocytes in vitro. FK also significantly inhibited the 17 alpha,20 beta-diOHprog-induced maturation of naked oocytes, suggesting the existence of adenylate cyclase in fish oocytes. These data indicate that in Oryzias latipes, FK induces oocyte maturation by stimulating follicular production of maturation-inducing steroid (MIS), probably 17 alpha,20 beta-diOH prog, via an increase in cAMP, and that it may inhibit oocyte maturation by increasing ooplasmic cAMP and some inhibitory interaction between the granulosa cells and the oocyte through intercellular communication.     

Identification and expression analyses of BAMBI mediated by FSH in swine luteinizing granulosa cells

Transforming growth factor-β and related growth factors are essential regulators for the development of follicles. Bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI) was reported as a key factor participating in the transforming growth factor-β signal pathway. To investigate the role of BAMBI in porcine granulosa cells, the full length of the BAMBI was cloned from porcine ovarian cDNA. The results of bioinformatics analyses showed that the signaling peptide was located in between positions 20 and 21. The results of online prediction on phosphorylation sites indicate that the sites of Ser, Thr, and Tyr are 9, 1, and 1, respectively. In addition, BAMBI was highly homologous in rodent and livestock. Real-time quantitative polymerase chain reaction (qPCR) indicated that BAMBI was widely expressed in porcine tissues. Immunofluorescence showed that BAMBI was located in both nucleus and cytoplasm. Stimulating the granulosa cells with FSH in vitro could alter BAMBI expression level in a time-dependent manner. Moreover, the expression level declined after treatment with FSH. These results indicated that BAMBI is an FSH-repressed gene in porcine luteinizing granulosa cells and it may be involved in the regulation of ovarian follicle development and oocyte maturation.     

HDAC6 regulates primordial follicle activation through mTOR signaling pathway

Primordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-β, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up-regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction.Subject terms: TOR signalling, Cell proliferation, Endocrine reproductive disorders