Targeted Disruption of Nrg1 in Granulosa Cells Alters the Temporal Progression of Oocyte Maturation

Abstract

Neuregulin 1 (NRG1) is induced in granulosa cells by LH and acts on granulosa and cumulus cells during ovulation. In this study, we sought to determine the role of NRG1 in oocyte maturation by generating a granulosa cell-specific Nrg1 knockout mouse (Nrg1 flox/flox ;Cyp19a1Cre mice [gcNrg1KO]). In the gcNrg1KO mice, meiosis was induced 2 hours earlier than in control mice. More than 60% of the oocytes in the mutant mice spontaneously re-resumed meiosis beyond the MII stage. The percentage of successful fertilization was comparable in oocytes of both genotypes collected at 14 or 16 hours after human chorionic gonadotropin injection but was significantly lower in oocytes of the gcNrg1KO mice at 18 or 20 hours. The number of pups per litter was significantly decreased in gcNrg1KO mice. To determine the molecular events associated with the abnormal progression of meiosis in the gcNrg1KO mouse oocytes, the defects of cumulus/granulosa cell functions were analyzed. The expression of genes involved in luteinization and cumulus expansion was significantly higher at 2 hours after human chorionic gonadotropin injection in the gcNrg1KO mice; this was related to abnormal activation of protein kinase C (PKC) and phosphorylation of connexin-43 in cumulus cells. Changes in connexin-43 by PKC might lead to early meiotic resumption of oocytes in gcNrg1KO mice. We conclude that NRG1 is induced by LH in mural granulosa cells and exerts an important regulatory role in oocyte meiotic maturation and competence by reducing PKC activation in cumulus cells and preventing premature progression to the MII stage that leads to abnormal fertilization and fertility. (Molecular Endocrinology 28: 706 -721, 2014) T he surge of LH stimulates ovulation, a process that permits the release of the cumulus cell-oocyte complex (COC) from the ovarian surface. The LH surge also leads to the reinitiation of meiosis and the development of oocytes competent for fertilization. Recent studies have revealed that the effects of LH on ovulation and oocyte maturation are mediated, in part, by the induced expression of the epidermal growth factor (EGF)-like factors, amphiregulin (AREG), epiregulin (EREG), and -cellulin (BTC) in mural granulosa cells (1). These ligands then bind ERBB1, the EGF receptor (EGFR) present in granulosa and cumulus cells, leading to activation of a RAS-MEK1-ERK1/2 pathway (2-5). In our previous study (4), we showed that targeted disruption of ERK1/2 in granulosa cells and cumulus cells did not affect follicular development to the preovulatory stage but did suppress events initiated by the LH surge: granulosa cell luteinization, cumulus cell differentiation, and oocyte meiotic maturation. These results indicated that activation of ERK1/2 is required for the induction of the ovulation process. Tar-