The glycine neurotransmitter transporter GLYT1 is an organic osmolyte transporter regulating cell volume in cleavage-stage embryos

Abstract

Cells subjected to sustained high osmolarity almost universally respond by accumulating compatible organic osmolytes that, in contrast to inorganic ions, are not deleterious even at high intracellular concentrations. Their accumulation from the external environment by known organic osmolyte transporters, such as the four identified in mammals, occurs only slowly in response to sustained high osmolarity, by synthesis of new transporter proteins. Most cells, however, are not subject to high or varying osmolarity, and it is not clear whether organic osmolytes are generally required at normal osmolarities or how they are regulated. The fertilized egg of the mouse is protected in the oviduct from perturbations in osmolarity. However, deleterious effects of osmotic stress were evident in vitro even at normal oviductal osmolarity. Glycine was found to protect development, indicating that early mouse embryos may use glycine as an organic osmolyte at physiological osmolarity. We have now found that GLYT1, a glycine transporter of the neurotransmitter transporter gene family, functions as the organic osmolyte transporter that mediates the osmotically regulated accumulation of glycine and regulates cell volume in early embryos. Furthermore, osmotic stimulation of GLYT1 transport was immediate, without a requirement for protein synthesis, implying regulation different from known organic osmolyte transporters. Thus, GLYT1 appears to have a previously unidentified role as an organic osmolyte transporter that functions in acute organic osmolyte and volume homeostasis near normal osmolarity.

Cell volume regulation in mammalian oocytes and preimplantation embryos

“…This was demonstrated directly using 3 H-glycine, showing that glycine was indeed accumulated by 1-and 2-cell mouse embryos and that the steady-state level of intracellular glycine varied proportionally with the osmolarity of the external medium (Dawson et al, 1998;Steeves et al, 2003). Such linear dependence of intracellular glycine content on external osmolarity was precisely the relationship expected if the accumulated intracellular glycine is balancing external osmolarity.…”

“…Baltz, unpublished).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.…”

“…Inhibition of GLYT1 completely reversed the ability of glycine to support development of mouse embryos from the 1-cell stage at increased osmolarity and also completely blocked osmosensitive glycine accumulation (Steeves et al, 2003), establishing that GLYT1 is the main mechanism for osmoprotection by glycine. 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).…”

“…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.…”

“…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. Furthermore, the mechanism is different, since organic osmolyte accumulation in somatic cells requires de novo transcription and translation of transporter proteins whereas glycine accumulation in embryos begins immediately and does not require protein synthesis (Steeves et al, 2003).…”