Cell volume regulation is initiated in oocytes at ovulation

It was previously shown that the early preimplantation mouse embryo regulates cell volume by a novel mechanism involving the accumulation of glycine as an organic osmolyte via the glycine transporter GLYT1. GLYT1-mediated cell volume control is present in fertilized eggs and persists only until the 4- to 8-cell stage of embryo development. However, it was unknown when during development this mechanism first becomes active. Therefore, I have investigated the mechanisms of cell volume regulation in oocytes prior to fertilization.;GLYT1 was quiescent in GV oocytes freshly removed from follicles. However, transport developed within several hours of ovulatory stimulation. Endogenous free cytosolic glycine, the fraction that contributes intracellular osmotic support, was measured at high levels only after ovulation, in ovulated MII oocytes and 1- and 2-cell embryos, where GLYT1 was active. Once active, GLYT1 activity was necessary for protecting against volume decreases in oocytes. Thus, GLYT1-mediated cell volume-regulatory glycine transport was initiated in oocytes at ovulation. This activation was independent of meiotic maturation and did not involve de novo synthesis. The inhibitory action of unidentified follicular factor(s) over a constitutively active GLYT1 is most likely responsible for the quiescence of GLYT1 before ovulation.;Prior to GLYT1 activation, the oocyte was unable to independently control its volume. Instead, its size was determined by a firm adherence between the oocyte's membrane and its extracellular matrix, the zona pellucida (ZP). Electron microscopy studies indicated that oocyte-ZP adherence occurs at the tip of the oocyte microvilli. When freed from the follicle or following ovulation, the oocyte released this adhesion, decreased in volume, and a perivitelline space appeared. This release from adherence and change in preferred volume coincided with GLYT1 activation.;Thus, the onset of cell volume regulation occurs at ovulation when the mammalian oocyte switches from a fixed cell volume determined by the ZP to active GLYT1 and glycine-dependent volume control.;To determine when glycine transport is initiated in oocytes and the mechanisms controlling its activation, I used the mouse as a model and various techniques including embryo and oocyte culture, 3H-glycine transport measurements, amino acid microanalysis, RT-PCR, Western blot and immunocytochemistry.