Reorganization of the mouse oocyte' cytoskeleton after cultivation under simulated weightlessness

Abstract

Female germ cells provide the structural basis for the development of a new organism, while the main molecular mechanisms of the impact of weightlessness on the cell remain unknown. The aim of this work was to determine the relative content and distribution of the main proteins of microtubules and microfilaments, to assess the relative RNA content of genes in mouse oocytes after short-term exposure to simulated microgravity, and to determine the potential for embryo development up to the 3-cell stage. Before starting the study, BALB/c mice were divided into two groups. One group received water and standard food without any modifications. Before exposure to simulated microgravity, the oocytes of these animals were randomly divided into two groups – c and µg. The second group of animals additionally received essential phospholipids containing at least 80% phosphatidylcholines, per os for 6 weeks before the start of the experiment at a dosage of 350 mg/kg of the animal's body to modify the lipid composition of the oocyte membrane. The obtained oocytes of these animals were also randomly divided into two groups – ce and µge. To determine the protein distribution and its relative content, immunofluorescence analysis was performed, and the RNA content of genes was assessed using real-time PCR with reverse transcription. After cultivation under simulated microgravity, beta-actin and acetylated alpha-tubulin are redistributed from the cortical layer to the central part of the oocyte, and the relative content of acetylated alpha-tubulin and tubulin isoforms decreases. At the same time, the mRNA content of most genes encoding cytoskeletal proteins was significantly higher in comparison with the control level. The use of essential phospholipids led to a decrease in the content of cellular cholesterol in the oocyte and leveled changes in the content and redistribution of acetylated alpha-tubulin and beta-actin after cultivation under simulated microgravity. In addition, after in vitro fertilization and further cultivation under simulated weightlessness, we observed a decrease in the number of embryos that passed the stage of the 2-cell embryo, but while taking essential phospholipids, the number of embryos that reached the 3-cell stage did not differ from the control group. The results obtained show changes in the content and redistribution of cytoskeletal proteins in the oocyte, which may be involved in the process of pronucleus migration, the formation of the fission spindle and the contractile ring under simulated weightlessness, which may be important for normal fertilization and cleavage of the future embryo.