Effect of the knockout of mitofusins on mouse oocyte: implications to fertility and mitochondrial inheritance

Abstract

Mitochondria play a fundamental role during oocyte growth. As a result, the existence of dysfunctional mitochondria or mutant mitochondrial DNA (mtDNA) in oocytes may lead to infertility syndromes and transmission of mitochondrial diseases. Compared to other cell types, oocyte mitochondria distinguish by their increased number, smaller size and rounded shape. These characteristics are dependent on mitochondrial fusion and fission, which is determinant to mitochondrial function and inheritance. Mitochondrial fusion is coordinated by mitofusins (Mfn1 and Mfn2). These proteins are critical to regulation of mitochondrial and endoplasmic reticulum (RE) function, as Mfn2 is also present on the RE membrane. Recently, we found that Mfn1 is essential to oocyte development and fertility. However, Mfn2 deficiency in Mfn1-null oocytes partially reversed these effects in double-mutant (DM) oocytes. On the other hand, Mfn2 deficiency interfered with elimination of NZB/BINJ (NZB) mutant mtDNA, resulting in metabolic abnormalities in the progeny. Hence, we propose to investigate these findings aiming to explain i) the molecular mechanism connecting Mfn1 deficiency in oocytes to folliculogenesis arrest; ii) the effect of Mfn2 deficiency on energetic metabolism in oocytes, cumulus cells (CC) and progeny; iii) the reason why double knockout of Mfn1 + Mfn2 (DM) has a milder effect on oocyte development and folliculogenesis than the single knockout of Mfn1; and, iv) the role of Mfn2 on elimination of NZB mtDNA in the oocyte. In this regard, Mfn1 and/or Mfn2 will be conditionally knocked out in oocytes using heteroplasmic mice (containing NZB and C57BL/6 mtDNA). Oocytes that are wild-type (WT) and homozygous knockout for Mfn1, Mfn2 and Mfn1 + Mfn2 will be compared by RNA sequencing (RNAseq) considering the abundance of gene transcripts. This experiment is expected to give important evidences about the effects of the knockouts on oocytes. Nonetheless, considering that some hypotheses were raised based on the above findings, these will be addressed in parallel by using alternative approaches (metabolic analyses, biochemical evaluations, fluorescency microscopy, transmission electron microscopy, real-time PCR, western blot, etc). Since these analyses should consider the use of oocytes, CC and progeny, they are expected to complement the RNAseq data as well as speed up publication of our findings. We expect the results from this project will enable a better understanding of the role of mitochondria, RE and mitofusins on oocyte, fertility and mitochondrial inheritance. (AU)