Differential Regulation of Oocyte Maturation and Cumulus Expansion in the Mouse Oocyte– Cumulus Cell Complex by Site-Selective Analogs of Cyclic Adenosine Monophosphate

In the present study, we have examined how differential distribution of cyclic adenosine 5′-monophosphate (cAMP)-dependent protein kinase isozymes within the mouse oocyte–cumulus cell complex might influence the physiological response of the complex to cAMP, by determining the actions of site-selective cAMP analogs on oocyte maturation and cumulus expansion. Five different analogs of cAMP were utilized: 8-thiomethyl-cAMP and 8-bromo-cAMP, which bind to site 1 on the type II regulatory subunit (RII) of cAMP-dependent protein kinase A (PKA); 8-aminohexylamino-cAMP, which binds to site 1 on the type I regulatory subunit (RI) of PKA; N6-monobutyryl cAMP, which binds to site 2 on either RI or RII; and 8-piperidino-cAMP, which binds to either site 1 on RII or site 2 on RI. These analogs were tested alone or in paired combinations that synergistically activate either the type I or type II PKA isozyme. When tested alone, analogs that can bind to, and presumably activate, type I PKA were the most potent inhibitors of germinal vesicle breakdown (GVB) in both cumulus cell-enclosed and denuded oocytes. Consistent with this result was the finding that paired combinations of analogs that selectively activate type I PKA were also most effective in preventing GVB. On the other hand, pulsing meiotically arrested cumulus cell-enclosed oocytes with high concentrations of analogs that bind to PKA II, or with paired combinations of analogs that selectively activate type II PKA, led to induction of GVB; stimulation with analogs or combinations thereof that presumably stimulate type I PKA was less effective. Cumulus expansion in response to PKA stimulation showed similar selectivity in that type II PKA-stimulating treatments were considerably more effective in provoking expansion than type I PKA-stimulating treatments. 8-N3-[32P]cAMP photoaffinity labeling of PKA regulatory subunits revealed that only RI was present in oocyte extracts, while extracts from oocyte–cumulus cell complexes contained both RI and RII. These results support the hypothesis that type II PKA mediates cAMP-stimulated cumulus expansion and resumption of meiotic maturation, while direct elevation of type I PKA within the oocyte is instrumental in maintaining meiotic arrest

FSH signaling pathways in immature granulosa cells that regulate target gene expression: Branching out from protein kinase A

Follicle-stimulating hormone (FSH) is necessary and sufficient to induce maturation of ovarian follicles to a mature, preovulatory phenotype in the intact animal, resulting in the generation of mature eggs and production of estrogen. FSH accomplishes these actions by inducing a complex pattern of gene expression in target granulosa cells that is regulated by input from many different signaling cascades, including those for the extracellular regulated kinases (ERKs), p38 mitogen-activated protein kinases (MAPKs), and phosphatidylinositol-3 kinase (PI3K). The upstream kinase that appears to be responsible for initiating all of the signaling that regulates gene expression in these epithelial cells is protein kinase A (PKA). PKA not only signals to directly phosphorylate transcription factors like cAMP response element binding protein and to promote chromatin remodeling by phosphorylating histone H3, this versatile kinase also enhances the activity of the p38 MAPK, ERK, and PI3K pathways. Additionally, accumulating evidence suggests that activation of a single signaling cascade downstream of PKA is not sufficient to activate target gene expression. Rather, cross-talk between and among signaling cascades is required. We will review the signaling cascades activated by FSH in granulosa cells and how these cascades contribute to the regulation of select target gene expression

Membrane organization of luteinizing hormone receptors differs between actively signaling and desensitized receptors

Signaling by the luteinizing hormone/choriogonadotropin receptor (LHR) is of considerable interest because of its requirement for successful reproduction. Time-resolved phosphorescence anisotropy and fluorescence resonance energy transfer were used to investigate the organization of endogenous LHRs in porcine follicular membranes in two distinct signaling states, active and desensitized. Desensitized LHRs exhibited approximately 3-fold slower rotational correlation times compared with active LHRs (59 +/- 4 and 21 +/- 9 mus, respectively), suggesting that with agonist-dependent desensitization the receptors are organized into larger protein complexes. Incubation of membranes with inhibitors of LHR desensitization, such as neutralizing anti-arrestin antibodies, a synthetic peptide corresponding to the third intracellular loop of the LHR but not the corresponding scrambled peptide, or catalytically inactive ARNO, resulted in faster rotational diffusion times equivalent to those of actively signaling LHRs. Furthermore, desensitized LHRs exhibited a 2.4-fold increase in fluorescence resonance energy transfer between LHRs suggesting that the larger protein aggregates formed during desensitization contain more self-associated LHRs. These results indicate that agonist-dependent LHR desensitization precedes organization of LHRs at the cells surface into larger protein aggregates

Conditional deletion of beta-catenin mediated by Amhr2cre in mice causes female infertility

Follicle-stimulating hormone (FSH) regulation of aromatase gene expression in vitro requires the transcriptional coactivator beta-catenin. To ascertain the physiological significance of beta-catenin in granulosa cells during folliculogenesis, mice homozygous for floxed alleles of beta-catenin were intercrossed with Amhr2cre mice. Conditional deletion of beta-catenin in 8-wk-old females occurred in derivatives of the Müllerian duct, granulosa cells and, surprisingly, in brain, pituitary, heart, liver, and tail. Female mice deficient for beta-catenin were infertile, despite reaching puberty and ovulating at the expected age, indications of apparently normal ovarian function. In contrast, their oviducts were grossly distended, with fewer but healthy oocytes. In addition, their uteri lacked implantation sites. Together, these two phenotypes could explain the complete loss of fertility. Nevertheless, although the ovary appeared normal, with serum estradiol concentrations in the normal range, there was marked animal-to-animal variation of mRNAs encoding beta-catenin and aromatase. Similarly, inhibin-alpha and luteinizing hormone receptor mRNAs varied considerably in whole ovaries, whereas pituitary Fshb mRNA was significantly reduced. Collectively, these features suggested cyclization recombination (CRE)-mediated recombination of beta-catenin may be unstable in proliferating granulosa cells, and therefore may mask the suspected steroidogenic requirement for beta-catenin. We tested this possibility by transducing primary cultures of granulosa cells from mice homozygous for floxed alleles of beta-catenin with a CRE-expressing adenovirus. Reduction of beta-catenin significantly compromised FSH stimulation of aromatase mRNA and subsequent production of estradiol. Collectively, these data suggest that FSH regulation of steroidogenesis requires beta-catenin, a role that remains hidden when tested through Amhr2cre-mediated recombination in vivo

Lhcgr Expression in Granulosa Cells: Roles for PKA-Phosphorylated β-Catenin, TCF3, and FOXO1

Ovarian follicles lacking FSH or FSH receptors fail to progress to a preovulatory stage, resulting in infertility. One hallmark of the preovulatory follicle is the presence of luteinizing hormone/choriogonadotropin receptors (LHCGR) on granulosa cells (GCs). However, the mechanisms by which FSH induces Lhcgr gene expression are poorly understood. Our results show that protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/AKT pathways are required for FSH to activate both the murine Lhcgr-luciferase reporter and expression of Lhcgr mRNA in rat GCs. Based on results showing that an adenovirus (Ad) expressing a steroidogenic factor 1 (SF1) mutant that cannot bind β-catenin abolished FSH-induced Lhcgr mRNA, we evaluated the role of β-catenin in the regulation of Lhcgr gene expression. FSH promoted the PKA-dependent, PI3K-independent phosphorylation of β-catenin on Ser552 and Ser665. FSH activated the β-catenin/T-cell factor (TCF) artificial promoter-reporter TOPFlash via a PKA-dependent, PI3K-independent pathway, and dominant-negative (DN) TCF abolished FSH-activated Lhcgr-luciferase reporter and induction of Lhcgr mRNA. Microarray analysis of GCs treated with Ad-DN-TCF and FSH identified the Lhcgr as the most down-regulated gene. Chromatin immunoprecipitation results placed β-catenin phosphorylated on Ser552 and Ser675 and SF1 on the Lhcgr promoter in FSH-treated GCs; TCF3 was constitutively associated with the Lhcgr promoter. Transduction with an Ad-phospho-β-catenin mutant (Ser552/665/Asp) enhanced Lhcgr mRNA expression in FSH-treated cells greater than 3-fold. Finally, we identified a recognized PI3K/AKT target, forkhead box O1, as a negative regulator of Lhcgr mRNA expression. These results provide new understanding of the complex regulation of Lhcgr gene expression in GCs

A direct role for arrestins in desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes

The luteinizing hormone/choriogonadotropin (LH/CG) receptor (R) is a heptahelical R that, upon agonist binding, activates the stimulatory guanine nucleotide-binding protein (G s ) and the downstream effector adenylyl cyclase (AC). Like other G protein-coupled Rs, the LH/CG R subsequently exhibits reduced agonist-dependent effector activity, or desensitization, in response to saturating agonist. Unlike desensitization of many other G protein-coupled Rs, the in vivo desensitization response of LH/CG R-stimulated AC activity of ovarian follicles to the preovulatory surge of LH can be mimicked under cell-free conditions. Based on evidence that porcine ovarian follicular membranes unexpectedly contained β-arrestin-1, the role of arrestins in desensitization of the LH/CG R was investigated. Results showed that neutralizing arrestin antibodies blocked the development of desensitization and that desensitization was rescued with a synthetic peptide corresponding to the antibody-binding epitope on β-arrestin-1. These results suggest that endogenous β-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R. Addition of recombinant purified β-arrestin-1 mimicked human chorionic gonadotrophin to promote desensitization of human chorionic gonadotrophin-stimulated AC activity, in the presence of the ATP phosphorylation antagonist adenylyl-imidodiphosphate, with an ED 50 of ≈0.1 nM. Increased levels of an 87-kDa protein reactive with glycoprotein hormone R-reactive antibody, consistent with the LH/CG R, coimmunoprecipitated with follicular membrane β-arrestin-1 in response to LH/CG R activation compared with unactivated R. Taken together, these results show that ovarian follicles contain membrane-associated β-arrestin-1, that β-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R, and that the trigger for β-arrestin-1 binding to the LH/CG R appears to be R activation