Cell volume regulation is initiated in mouse oocytes after ovulation

Abstract

Fertilized mouse eggs regulate their size principally by accumulating glycine as an intracellular osmolyte using the GLYT1 (SLC6A9) transporter, a mechanism of cell volume homeostasis apparently unique to early embryos before the morula stage. However, nothing was known of cell volume regulation in oocytes before fertilization. We show here that GLYT1 is quiescent in mouse germinal-vesicle-stage oocytes but becomes fully activated within hours after ovulation is triggered. This initiates accumulation of substantial amounts of intracellular glycine in oocytes during meiotic progression, reaching a maximal level in mature eggs. Measurements of endogenous free glycine showed that there were nearly undetectable levels in ovarian germinal-vesicle-stage oocytes, but high levels were present in mature ovulated eggs and in preimplantation embryos through the two-cell stage, but not in morulae. Furthermore, intracellular glycine was regulated in response to changes in external tonicity in eggs and embryos through the two-cell stage, but not in oocytes or embryos after the two-cell stage. Before activation of GLYT1, oocytes were unable to independently regulate their volume. As GLYT1 became active, however, oocyte volume decreased substantially and oocytes gained the ability to regulate their size, which required GLYT1 activity. Before ovulation, oocyte size was instead determined by a strong adhesion to the rigid extracellular matrix of the oocyte, the zona pellucida, which was released coincident with GLYT1 activation. The ability to acutely regulate cell size is thus acquired by the oocyte only after ovulation, when it first develops glycinedependent cell volume regulation.

Cell volume regulation in mammalian oocytes and preimplantation embryos

“…The overwhelming majority of the accumulated glycine is freely soluble (Tartia et al, 2009), as expected if it provides osmotic support. These amounts of soluble, intracellular glycine correspond to very high intracellular concentrations through the 2-cell stage, calculated to be in the range of 20-30 mM and therefore contributing $20-30 mOsM to the total osmotic pressure, consistent with glycine being accumulated as an organic osmolyte in vivo (Tartia et al, 2009). …”

“…Baltz, unpublished). A highly selective GLYT1 inhibitor, ORG23798, blocked virtually all glycine transport by cleavage stage embryos (Steeves et al, 2003;Tartia et al, 2009), confirming that glycine transport is overwhelmingly via GLYT1 in preimplantation mouse embryos prior to compaction. Glycine transport via GLYT1 is also present in human embryos (Hammer et al, 2000), implying a similar role there, although this remains to be confirmed.…”

“…Glycine is very high in eggs and early preimplantation embryos, with $4-10 pmoles of glycine in eggs and 2-cell embryos, decreasing to 0.1-0.3 pmoles in morulae and blastocysts (Schultz et al, 1981;Van Winkle and Dickinson, 1995;Tartia et al, 2009). The overwhelming majority of the accumulated glycine is freely soluble (Tartia et al, 2009), as expected if it provides osmotic support.…”

“…Glycine is very high in eggs and early preimplantation embryos, with $4-10 pmoles of glycine in eggs and 2-cell embryos, decreasing to 0.1-0.3 pmoles in morulae and blastocysts (Schultz et al, 1981;Van Winkle and Dickinson, 1995;Tartia et al, 2009). The overwhelming majority of the accumulated glycine is freely soluble (Tartia et al, 2009), as expected if it provides osmotic support. These amounts of soluble, intracellular glycine correspond to very high intracellular concentrations through the 2-cell stage, calculated to be in the range of 20-30 mM and therefore contributing $20-30 mOsM to the total osmotic pressure, consistent with glycine being accumulated as an organic osmolyte in vivo (Tartia et al, 2009).…”

“…Furthermore, glycine transport via GLYT1 is required for eggs and embryos to maintain their normal size, directly demonstrating a role for GLYT1 in cell volume homeostasis (Steeves et al, 2003;Tartia et al, 2009). Cell volume regulation by GLYT1 is apparently unique to early embryos since GLYT1-mediated uptake of glycine as an organic osmolyte has not been found in mammalian somatic cells, which instead use one of the four identified organic osmolyte transporters discussed above.…”