Gas6 is a reciprocal regulator of mitophagy during mammalian oocyte maturation

Kyeoung-Hwa Kim,Jung-Jae Ko,Kyung-Ah Lee,Eun-Young Kim

doi:10.1038/s41598-019-46459-3

Abstract

Previously, we found that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation through mitochondrial overactivation with concurrent failure of pronuclear formation after fertilization. In this study, we report that Gas6 regulates mitophagy and safeguards mitochondrial activity by regulating mitophagy-related genes essential to the complete competency of oocytes. Based on RNA-Seq and RT-PCR analysis, in Gas6-silenced MII oocytes, expressions of mitophagy-related genes were decreased in Gas6-silenced MII oocytes, while mitochondrial proteins and Ptpn11, the downstream target of Gas6, was increased. Interestingly, GAS6 depletion induced remarkable MTOR activation. Gas6-depleted MII oocytes exhibited mitochondrial accumulation and aggregation caused by mitophagy inhibition. Gas6-depleted MII oocytes had a markedly lower mtDNA copy number. Rapamycin treatment rescued mitophagy, blocked the increase in MTOR and phosphorylated-MTOR, and increased the mitophagy-related gene expression in Gas6-depleted MII oocytes. After treatment with Mdivi-1, a mitochondrial division/mitophagy inhibitor, all oocytes matured and these MII oocytes showed mitochondrial accumulation but reduced Gas6 expression and failure of fertilization, showing phenomena very similar to the direct targeting of Gas6 by RNAi. Taken together, we conclude that the Gas6 signaling plays a crucial role in control of oocytes cytoplasmic maturation by modulating the dynamics and activity of oocyte mitochondria.

Highlights

Growth arrest-specific gene 6 (GAS6) is a ligand of receptor tyrosine kinases of the TAM (Tyro[3], Axl, and Mertk) receptors, and its role as a potential therapeutic target in human cancer has been recently emphasized[1]
We found that Gas[6] depletion led to a greater suppression of mitophagy through MTOR signaling activation and a reduction in the mtDNA copy number and levels of mitochondria-encoded mRNA in oocytes, supporting a novel role of Gas[6] in the maintenance of mitochondrial contents and activity during oocyte cytoplasmic maturation
We asked whether cytoplasmic maturity in oocytes is reflected at the level of transcription, and, if so, what patterns of gene expression are characteristic of GFP-dsRNA treated or Gas6-silenced metaphase II (MII) oocytes

Summary

Introduction

Growth arrest-specific gene 6 (GAS6) is a ligand of receptor tyrosine kinases of the TAM (Tyro[3], Axl, and Mertk) receptors, and its role as a potential therapeutic target in human cancer has been recently emphasized[1]. Depletion of Gas[6] in oocytes results in failure of sperm chromatin remodeling and pronuclear formation through insufficient cytoplasmic maturation[7]. Researchers have reported that, in mice, mitochondrial activity is important for oocyte maturation and subsequent embryo development but that an altered mtDNA copy number does not affect nuclear maturation[9,10]. Fertilization failure observed in oocytes with a low mtDNA number and mitochondrial dysfunction was resulted from defective oocyte cytoplasmic maturation and decreased oocyte quality. It has been reported that an increase in the number of healthy mitochondria and/or improvement in mitochondrial function www.nature.com/scientificreports/. ATP production and mtDNA copy number decrease and mitochondrial mutations increase with aging[18]. The clearance of dysfunctional mitochondria via mitophagy screens the damaged mtDNA and prevents its transmission to the generation[21]

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Results

Conclusion