Maternal control of early mammalian embryogenesis

Abstract

This thesis explores and discusses biochemical changes during mammalian oogenesis with the aim of providing novel information, at the transcriptomic and proteomic levels, about events and processes that help to support, direct and protect the genomic integrity of the early mammalian embryo. PIWIs are crucial guardians of genome integrity, particularly in germ cells. While mammalian PIWIs have been primarily studied in mouse and rat, a homologue for the human PIWIL3 gene is absent in the Muridae family, and hence the unique function of PIWIL3 in germ cells cannot be effectively modeled by mouse knockouts. Herein, in chapter 2, we investigated the expression, distribution and interaction of PIWIL3 in bovine oocytes. We localized PIWIL3 to mitochondria, and demonstrated that PIWIL3 expression is stringently controlled both spatially and temporally before and after fertilization. Moreover, we identified PIWIL3 in a mitochondrial-recruited three-membered complex with TDRKH and PNLDC1, and demonstrated by mutagenesis that PIWIL3 N-terminal arginine modifications are required for complex assembly. Finally, we sequenced the piRNAs bound to PIWIL3-TDRKH-PNLDC1 and report here that about 50% of these piRNAs map to transposable elements, recapitulating the important role of PIWIL3 in maintaining genome integrity in mammalian oocytes. In chapter 2, microinjection was used for the investigation of PIWIL3 localization and function. While siRNA introduction by microinjection can be useful to study the function of maternally expressed genes in oocytes, injection is a rather invasive procedure in that it damages the plasma membrane and disturbs the cell organelle distribution. Therefore, in chapter 3 we examined the effect of introducing non-specific small RNAs into oocytes by microinjection, at the single cell transcriptome level. Injection of non-specific siRNA resulted in differential expression of 119 transcripts, of which 76 were down-regulated. Gene ontology analysis revealed that the differentially regulated genes were enriched in the biological processes of ATP synthesis, molecular transport and regulation of protein polyubiquitination. This study establishes a background effect of the microinjection procedure that should be borne in mind by those using microinjection to manipulate gene expression in oocytes. Based on the results of chapter 3, a group of mRNAs with functions related to plasma membrane interaction are significantly altered by microinjection, which raises the question of the importance of plasma membrane proteins during oocyte maturation and fertilization. In chapter 4, we used an optimized plasma membrane protein purification method followed by phospho enrichment and mass spectrometry to investigate the oolemma phospho(proteome) in cattle GV and MII stage oocytes and zygotes. We identified a number of protein complexes that connect with the plasma membrane, including the Arp2/3, ERM and SCMC complexes. Moreover, we detected multiple new phospho-sites that were not previously reported. In addition, we found that PALM3, PB41L2 and TACC3 increased significantly at both the protein and phospho-protein levels during oocyte maturation. Our research validated an effective method for plasma membrane protein identification from samples of limited protein content, and also provided general knowledge on plasma membrane proteins activated during oocyte maturation and fertilization.