The molecular bridges that link the LH surge with functional changes in cumulus cells that possess few LH receptors are being unraveled. Herein we document that epidermal growth factor (EGF)-like factors amphiregulin (Areg), epiregulin (Ereg), and betacellulin (Btc) are induced in cumulus oocyte complexes (COCs) by autocrine and paracrine mechanisms that involve the actions of prostaglandins (PGs) and progesterone receptor (PGR). Areg and Ereg mRNA and protein levels were reduced significantly in COCs and ovaries collected from prostaglandin synthase 2 (Ptgs2) null mice and Pgr null (PRKO) mice at 4 h and 8 h after human chorionic gonadotropin, respectively. In cultured COCs, FSH/forskolin induced Areg mRNA within 0.5 h that peaked at 4 h, a process blocked by inhibitors of p38MAPK (SB203580), MAPK kinase (MEK) 1 (PD98059), and PTGS2 (NS398) but not protein kinase A (PKA) (KT5720). Conversely, AREG but not FSH induced Ptsg2 mRNA at 0.5 h with peak expression of Ptgs2 and Areg mRNAs at 4 h, processes blocked by the EGF receptor tyrosine kinase inhibitor AG1478 (AG), PD98059, and NS398. PGE2 reversed the inhibitory effects of AG on AREG-induced expression of Areg but not Ptgs2, placing Ptgs2 downstream of EGF-R signaling. Phorbol 12-myristate 13-acetate (PMA) and adenovirally expressed PGRA synergistically induced Areg mRNA in granulosa cells. In COCs, AREG not only induced genes that impact matrix formation but also genes involved in steroidogenesis (StAR, Cyp11a1) and immune cell-like functions (Pdcd1, Runx1, Cd52). Collectively, FSH-mediated induction of Areg mRNA via p38MAPK precedes AREG induction of Ptgs2 mRNA via ERK1/2. PGs acting via PTGER2 in cumulus cells provide a secondary, autocrine pathway to regulate expression of Areg in COCs showing critical functional links between G protein-coupled receptor and growth factor receptor pathways in ovulating follicles.

Ovulation is a complex process initiated by the preovulatory LH surge, characterized by cumulus oocyte complex (COC) expansion and completed by the release of a mature oocyte. Although many ovarian genes that impact ovulation have been identified, we hypothesized that genes selectively expressed in COCs would be overlooked by approaches using whole ovary or granulosa cell samples. RNA isolated from COCs collected from preovulatory follicles of equine chorionic gonadotropin (CG) primed mice and at selected times after human CG treatment was subjected to microarray analyses and results confirmed by RT-PCR analyses, Western blotting, and immunofluorescent studies. A remarkable number of genes were up-regulated in COCs including Areg, Ereg, and Btc. Several genes selectively expressed in cumulus cells compared with granulosa cells were related to neuronal (Mbp, Tnc, Nts) or immune (Alcam, Pdcd1, Cd34, Cd52, and Cxcr4) cell function. In addition to Sfrp2, other members of the Wnt/Fzd family (Sfrp4, Fdz1 and Fdz2) were expressed in COCs. Thus, there is a cumulus cell-specific, terminal differentiation process. Furthermore, immunofluorescent analyses documented that cumulus cells are highly mitotic for 4-8 h after human CG and then cease dividing in association with reduced levels of Ccnd2 mRNA. Other down-regulated genes included: Cyp19a1, Fshr, Inhb, and the oocyte factors Zp1-3 and Gja4. In summary, the vast number of matrix, neuronal, and especially immune cell-related genes identified by the gene- profiling data of COCs constitutes strong and novel evidence that cumulus cells possess a repertoire of immune functions that could be far greater than simply mediating an inflammatory-like response.

FSH stimulates in ovarian granulosa cells diverse, differentiation-dependent responses that implicate activation of specific cellular signaling cascades. In these studies three kinases were investigated to determine their relationship to FSH, cAMP, and A kinase signaling: protein kinase B (PKB/Akt), serum and glucocorticoid-induced kinase (Sgk), and p38 mitogen-activated protein kinase (p38MAPK). The phosphorylation (activation) of these kinases was analyzed by using selective agonists/inhibitors: forskolin/H89 for cAMP-dependent protein kinase (A kinase), insulin-like growth factor I (IGF-I)/LY294002 and wortmannin for phosphatidylinositol-dependent kinase (PI3-K), and phorbol myristate (PMA)/GF109203X for diacylglycerol and Ca++-dependent kinases (C kinases). An inhibitor (PD98059) of MEK1, which regulates extracellular regulated kinases (ERKs), and SB203580, which inhibits p38MAPK, were also used. In addition, we analyzed the expression of the recently described, cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFI and GEFII) that impact Ras-related GTPases and Raf kinases, known regulators of various protein kinase cascades. We provide evidence that FSH, forskolin, and 8-bromo-cAMP stimulate phosphorylation of PKB by mechanisms involving PI3-K (LY294002/wortmannin sensitive) not A kinase (H89 insensitive), a pattern of response mimicking that of IGF-I. In contrast, FSH induction and phosphorylation of Sgk protein requires A kinase (H89 sensitive) but also involves PI3-K (LY294002 sensitive) as well as p38MAPK (SB203580 sensitive) pathways. PMA (C kinase) abolished FSH-mediated (but not IGF-I-mediated) phosphorylation of PKB at a step(s) upstream of PI3-K and independent of A kinase. Lastly, FSH-mediated phosphorylation of p38MAPK is negatively affected by A kinase and PI3-K, suggesting that it may be downstream of specific members of the cAMP-GEF/Rap/Raf pathway. We propose that cAMP activation of A kinase is obligatory for transcription of Sgk in granulosa cells whereas cAMP (IGF-I-like)-mediated phosphorylation (activation) of PKB and Sgk (via PI3-K), as well as p38MAPK, involves other cellular events. These results provide new and exciting evidence that cAMP acts in granulosa cells by A kinase-dependent and -independent mechanisms, each of which controls specific kinase cascades.

The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has two serine residues and one threonine residue mutated to alanines rendering this protein constitutively active and nuclear and FOXOA3-mutant DNA-binding domain (mDBD) in which the DBD is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis, and maintained FOXO1 protein at levels of the endogenous protein observed in cells before exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24 h. Affymetrix microarray and database analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol, and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1, and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2), were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3-mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (equine choriogonadotropin) and LH (human choriogonadotropin), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development.