Intracellular Betaine Regulation in Developing Mouse Oocytes and Preimplantation Embryos

Abstract

The organic osmolyte betaine (N, N, N-trimethylglycine) has two key roles in mouse oocytes and preimplantation embryos, as a cell volume regulator and a major methyl donor. However, several aspects of the mechanisms of intracellular betaine regulation have not been fully elucidated. The origin of endogenous betaine in immature oocytes and the amount accumulated over oogenesis is unknown. Betaine levels are maintained at nearly constant levels through preimplantation embryo development but decrease at the blastocyst stage. The maintenance of betaine levels is hypothesized to be regulated by limiting its efflux through the swelling-activated, volume-sensitive organic osmolyte and anion channels (VSOACs). Which isoforms of VSOAC that are expressed in preimplantation embryos has not been determined, and it is unknown if VSOAC induces betaine efflux. In the inner cell mass of the blastocyst, betaine supplies the methyl pool by serving as the substrate for the enzyme betaine homocysteine methyltransferase (BHMT). There may be a link between metabolism by BHMT and the decrease in betaine by the blastocyst stage. Here, I investigate the mechanisms of betaine accumulation, retention, and loss from oogenesis to the blastocyst stage. Low levels of endogenous betaine were present in small growing oocytes from postnatal day 5 (P5) ovaries, and the amount remained nearly constant through the rest of oogenesis. Growing follicles contained a large reservoir of betaine and choline by the time they were fully grown. Saturable betaine transporter activity was observed in growing follicles (from P17 ovaries) and fully-grown antral follicles (P21). However, denuded oocytes did not exhibit any saturable betaine transport on any postnatal day. In preimplantation embryos, endogenous betaine levels were conserved from the two-cell stage to the morula stage both in vivo and in vitro. However, there was a swelling-induced loss of betaine when one-cell embryos were transferred to hypotonic media, which indicated the presence of VSOACs. We therefore attempted to elucidate the subunits of the channel that were expressed. We determined by conventional PCR that transcripts for four of the five Lrrc8 isoforms (Lrrc8a, b, d, and e) were variously expressed during oocyte and preimplantation embryo development. We confirmed by quantitative RT-qPCR that Lrrc8a mRNA is present at the highest levels from the GV oocyte to the two-cell embryo stage and then decreases. Since suitable primers for the other isoforms for RT-qPCR could not be found, we used an available RNAseq data set to determine the expression patterns for all five Lrrc8 isoform transcripts. Finally, it was anticipated that Bhmt⁻ᐟ⁻ blastocysts would have endogenous betaine levels comparable to morulae instead of exhibiting the normal decrease, because betaine would not be metabolized for its methyl group. However, blastocysts isolated from both Bhmt⁻ᐟ⁻ and Bhmt⁺ᐟ⁺ mice had nearly the same levels of endogenous betaine, indicating that the decrease in betaine must be through another mechanism. To conclude, I have built on previous work to provide further insight into the mechanisms by which betaine is accumulated and stored in the mouse oocyte and preimplantation embryo.