Maternal Proteins That Are Phosphoregulated upon Egg Activation Include Crucial Factors for Oogenesis, Egg Activation and Embryogenesis inDrosophila melanogaster

Abstract

Egg activation is essential for the successful transition from a mature oocyte to a developmentally competent egg. It consists of a series of events including the resumption and completion of meiosis, initiation of translation of some maternal mRNAs and destruction of others, and changes to the vitelline envelope. This major change of cell state is accompanied by large scale alteration in the oocyte’s phosphoproteome. We hypothesize that the cohort of proteins that are subject to phosphoregulation during egg activation are functionally important for processes before, during, or soon after this transition, potentially uniquely or as proteins carrying out essential cellular functions like those they do in other (somatic) cells. In this study, we used germline-specific RNAi to examine the function of 189 maternal proteins that are phosphoregulated during egg activation in Drosophila melanogaster. We identified 53 genes whose knockdown reduced or abolished egg production and caused a range of defects in ovarian morphology, as well as 51 genes whose knockdown led to significant impairment or abolishment of the egg hatchability. We observed different stages of developmental arrest in the embryos and various defects in spindle morphology and aberrant centrosome activities in the early arrested embryos. Our results, validated by the detection of multiple genes with previously-documented maternal effect phenotypes among the proteins we tested, revealed 15 genes with newly discovered roles in egg activation and early embryogenesis in Drosophila. Given that protein phosphoregulation is a conserved characteristic of this developmental transition, we suggest that the phosphoregulated proteins may provide a rich pool of candidates for the identification of important players in the egg-to-embryo transition.

Identification of New Regulators of the Oocyte-to-Embryo Transition inDrosophila

“…Overall, our study complements recent studies aimed at identifying regulators of the oocyte-to-embryo transition in Drosophila and other organisms (Guo et al, 2015;Zhang et al, 2018). All of these studies have not only led to the discovery of new regulators, but have served to highlight important cellular and molecular processes during this transition.…”

“…Ovary depletion of mod(mdg4) and mei-P26 resulted in characteristic early embryonic arrest (Büchner et al 2000) and developmental arrest in early oogenesis (Page et al 2000;Neumüller et al 2008), respectively. mod(mdg4) was also found through a recent genetic screen for factors involved in oogenesis, egg activation and embryogenesis (Zhang et al 2018). Reduction of the activity of carbonic anhydrase 2 (cah2) led to embryos exhibiting multiple arrested nuclei, though we decided to not pursue this gene because of the multiple ways that perturbing metabolism by affecting the function of this gene could indirectly impact mitosis.…”

“…Thus, key regulators must be controlled by posttranslational modifications or control of protein levels. Although recent studies have addressed posttranslational modifications to understand the regulation of known players and to screen for potential new regulators of the oocyte-to-embryo transition (Krauchunas et al 2013;Guo et al 2015;Zhang et al 2018), we instead focused on changes in protein levels across the transition. We reasoned that changes in protein levels could be diagnostic of proteins with pivotal roles during the events of oocyte maturation and egg activation in Drosophila.…”

Calcineurin-dependent Protein Phosphorylation Changes During Egg Activation in Drosophila melanogaster

“…In Drosophila, more than 300 proteins are subject to phosphoregulation during egg activation (20), and orthologs of more than 55% of these proteins are also phosphoregulated during sea urchin egg activation (18). These deeply conserved phosphoproteomic changes suggest their functional importance during this transition, and our genetic data show that these phosphoregulated proteins include numerous factors required for oogenesis and embryogenesis (22). These results support the idea that phosphoregulation is an important mechanism that remodels the oocyte proteome to prepare the cell to undertake development.…”

“…RT-PCR-Ovaries were dissected from virgin females that had been aged for 3-5 days. RNA extraction and RT-PCR were performed as described in Zhang et al (22). The CanB2 cDNA was amplified using the primers CanB2_F: 5Ј-AGCGCGTGATCGACATTT and CanB2_R: 5Ј-CTTGCCATCCTCATCCTTATCC.…”