Specific Protein Kinase C Isoforms α And βI Are Involved in Follicle-Stimulating Hormone-Induced Mouse Follicle-Enclosed Oocytes Meiotic Resumption

Jianwei Wang,Yingying Han,Jing Wen,Fenghua Bian,Ge Li,Xinyi Mu,Guoliang Xia,Meijia Zhang,Jinlian Zhou,Qian Chen,Hongmei Wang

doi:10.1371/journal.pone.0045043

Abstract

Protein kinase C (PKC) is involved in gonadotrophin-induced oocyte maturation. In the present study, we investigated the role of specific PKC isoforms in the process of follicle-stimulating hormone (FSH)-induced oocyte meiotic resumption. Small antral follicles (200–300 µm in diameter) were isolated from immature mice and cultured in vitro. FSH significantly induced follicle-enclosed oocytes (FEOs) meiotic resumption after 8 hr culture. However, the induced effect of FSH was dose-dependently inhibited by the specific PKC α and βI inhibitor Gö6976, and 100 nM Gö6976 completely blocked FSH function in oocyte meiotic resumption. Furthermore, FSH dramatically induced the expression of transcripts encoding epidermal growth factor (EGF)-like growth factors Areg, Btc, and Ereg mRNA levels, and up-regulated tyrosine phosphorylation level of EGF receptor (EGFR) in granulosa cells. Blocking the function of EGFR by AG1478 eliminated the effect of FSH-induced FEOs meiotic resumption, suggesting that FSH induced oocyte maturation through the activation of EGFR. FSH-induced phosphorylation of EGFR could also be inhibited by Gö6976. Next, we examined the effect of FSH on the expression and phosphorylation PKC α and βI. FSH induced the expression of PKC α at mRNA and protein level, and also up-regulated its phosphorylation level in granulosa cells after 8 hr culture. However, FSH had no effect on the expression of PKC βI but down-regulated its phosphorylation level. In conclusion, FSH-induced activation of PKC α alone, or together with the inactivation of PKC βI in granulosa cells, participates in mouse oocyte meiotic resumption, possibly by the activation of EGFR signaling pathway.

Highlights

Fully-grown mammalian oocytes are maintained in meiotic prophase arrest until the preovulatory surge of luteinizing hormone (LH) triggers the resumption of meiosis and ovulation
During the follicle-enclosed oocytes (FEOs) culture, the expression of transcripts encoding epidermal growth factor (EGF)-like growth factors Areg, Btc, and Ereg mRNA levels was increased in granulosa cells, but dramatically increased after folliclestimulating hormone (FSH) treatment (Figure 2A)
In order to examine whether these EGF-like growth factors were involved in FSH-induced FEOs meiotic resumption, the special inhibitor of EGF receptor (EGFR) AG1478 was added to the medium

Summary

Introduction

Fully-grown mammalian oocytes are maintained in meiotic prophase arrest until the preovulatory surge of luteinizing hormone (LH) triggers the resumption of meiosis and ovulation. The preovulatory gonadotropin surge induces oocyte meiotic resumption by overcoming the environmental maintenance of meiotic arrest [1], via increasing epidermal growth factor (EGF)-like growth factors [4], and the activation of mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) [5,6]. Additional studies suggested that the LHR and FSHR could provoke twice increase in ERK1/2 phosphorylation at high densities and the delayed activation of ERK1/2 require PKC activation and inhibits aromatase [16].More recently, gonadotrophins (FSH + LH) could up-regulate tumor necrosis factor-aconverting enzyme (TACE) protein and protease activity in porcine CEOs [17], and PKC participates in gonadotrophinsinduced oocyte maturation possibly by shed EGF and/or EGF-like factors [6]. The classical PKC (cPKC) isoforms are activated by calcium and diacylglycerol in a phospholipid-dependent manner, while novel

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Conclusion