Ovulation is the unique biological process by which a mature oocyte and surrounding cumulus cells, comprising the cumulus cell-oocyte complex (COC), are released from the surface of the ovary. This LH-induced process is similar to an inflammatory response and requires the expression of specific genes: the EGF-like factors (Areg, Ereg, Btc), matrix factors (Has2, Ptgs2, Tnfaip6, Ptx3) and cytokines (Il6). AREG potently activates ERK1/2, COC expansion, oocyte maturation and IL6 production in cultured COCs. However, the genes regulated specifically by AREG/ERK1/2 and the consequences of disrupting these signaling molecules in vivo have not been determined. Therefore, using Erk1 (Erk1-/-) null mice, Erk2fl/fl mice and mice expressing Cre driven by the aromatase (Cyp19) promoter, we have generated Erk1/2 double KO (Erk1-/-;Erk2fl/fl/;Cyp19-Cre) mice. Follicular development is normal but preovulatory follicles fail to ovulate and COC expansion is impaired in the Erk1/2 double KO mice. In addition to inflammation-related genes, LH/AREG also regulate the expression of specific genes that comprise the innate-immune cell surveillance response system, including the Toll-like receptors TLR2 and TLR4 and components of this system (C1q, Cd14, and Pdcd1) uniquely expressed in cumulus cells. TLR2 and TLR4 are functional in cumulus cells and respond to known ligands (including matrix factors) leading to the production of IL6 that can induce COC expansion in culture in the absence of AREG or PGE. Preliminary data indicate that Tlr4-/- mice are subfertile. By analyzing the signaling cascades and the expression of specific genes in Erk1/2 double KO mice and in mice null for the Areg target genes (Tlr/42, Ptx3 and Pdcd1) that are expressed in COCs, we should be able to dissect and delineate a novel genetic program that controls cumulus cell function and ovulation. Because the production of cytokines, including IL6, appears to be critical for ovulation, we propose that the ERK1/2 pathway controls an endocrine-immune-cytokine regulatory network during ovulation. Therefore we propose these specific aims:
Specific Aim I : Determine the molecular mechanisms by of ERK2 (MAPK1) and ERK1 (MAPK3) regulate granulosa cell and cumulus cell functions and ovulation in vivo. Test the hypothesis that ERK1/2 mediate specific effects of LH induced ovulation and cumulus cell function.
Specific Aim II : Determine the function of AREG (ERK1/2) target genes that are uniquely expressed at high levels in ovulated COCs and that impact ovulation. Test the hypotheses that specific matrix factors, IL6 and innate immune cell-related genes expressed in cumulus cells direct the unique fate of these cells.

Public Health Relevance

The studies being proposed in this application are designed to uncover new information about how ovarian follicles mature and ovulate in response to the gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). This information should help us understand the basis for infertility in women who have abnormal menstrual cycles, reduced ovarian function and are anovulatory.

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Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
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Yoshinaga, Koji
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Baylor College of Medicine
Anatomy/Cell Biology
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United States
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Ren, Yi A; Liu, Zhilin; Mullany, Lisa K et al. (2016) Growth Arrest Specific-1 (GAS1) Is a C/EBP Target Gene That Functions in Ovulation and Corpus Luteum Formation in Mice. Biol Reprod 94:44
Adams, Jaye; Liu, Zhilin; Ren, Yi Athena et al. (2016) Enhanced Inflammatory Transcriptome in the Granulosa Cells of Women With Polycystic Ovarian Syndrome. J Clin Endocrinol Metab 101:3459-68
Ren, Yi A; Mullany, Lisa K; Liu, Zhilin et al. (2016) Mutant p53 Promotes Epithelial Ovarian Cancer by Regulating Tumor Differentiation, Metastasis, and Responsiveness to Steroid Hormones. Cancer Res 76:2206-18
Mullany, Lisa K; Wong, Kwong-Kwok; Marciano, David C et al. (2015) Specific TP53 Mutants Overrepresented in Ovarian Cancer Impact CNV, TP53 Activity, Responses to Nutlin-3a, and Cell Survival. Neoplasia 17:789-803
Kawashima, Ikko; Umehara, Takashi; Noma, Noritaka et al. (2014) Targeted disruption of Nrg1 in granulosa cells alters the temporal progression of oocyte maturation. Mol Endocrinol 28:706-21
Zhang, Yin-Li; Xia, Yan; Yu, Chao et al. (2014) CBP-CITED4 is required for luteinizing hormone-triggered target gene expression during ovulation. Mol Hum Reprod 20:850-60
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Liu, Zhilin; Castrillon, Diego H; Zhou, Wei et al. (2013) FOXO1/3 depletion in granulosa cells alters follicle growth, death and regulation of pituitary FSH. Mol Endocrinol 27:238-52
Mullany, Lisa K; Richards, JoAnne S (2012) Minireview: animal models and mechanisms of ovarian cancer development. Endocrinology 153:1585-92
Kawashima, Ikko; Liu, Zhilin; Mullany, Lisa K et al. (2012) EGF-like factors induce expansion of the cumulus cell-oocyte complexes by activating calpain-mediated cell movement. Endocrinology 153:3949-59

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