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.

GLYT1, a glycine transporter belonging to the neurotransmitter transporter family, has recently been identified as a novel cell volume-regulatory mechanism in the earliest stages of the mouse preimplantation embryo. It apparently acts by regulating the steady-state intracellular concentration of glycine, which functions as an organic osmolyte in embryos, to balance external osmolarity and thus maintain cell volume. GLYT1 in embryos was the first mammalian organic osmolyte transporter identified that appears to function in cell volume control under conditions of normal osmolarity, rather than being a response to the stress of chronic hypertonicity. Its maximal rate of transport was shown to be regulated by osmolarity. However, it was not known whether this osmotic regulation of the rate of glycine transport is sufficient to account for the observed control of steady-state intracellular glycine levels as a function of osmolarity in embryos. Here, we show that the intracellular accumulation of glycine in embryos is a direct function of the rate of glycine uptake via GLYT1. In addition, we have shown that the rate of efflux, likely via the volume-regulated anion and organic osmolyte channel in embryos, is also under osmotic regulation and contributes substantially to the control of steady-state glycine concentrations. Together, control of both the rate of uptake and rate of efflux of glycine underlies the mechanism of osmotic regulation of the steady-state concentration of glycine and hence cell volume in early embryos.

Regulation of intracellular pH (pH(i)) by two-cell-stage embryos derived from female mice of three different strains (CF-1, Balb/c, and BDF) was investigated. Embryos recovered at a slow rate from intracellular acidosis produced by a pulse of NH(4)Cl; the rate did not differ significantly among strains. Recovery was reversibly inhibited by amiloride or the absence of Na(+), implicating Na(+)/H(+) antiporter activity. The threshold pH(i) (setpoint) below which Na(+)/H(+) antiporter activity was elicited was approximately 7.15 for each strain. No recovery from induced acidosis occurred in the absence of external Na(+) in any strain, and thus embryos could be maintained in acidosis for an extended period. Upon reintroduction of Na(+), embryos derived from either CF-1 or BDF females recovered at a slow rate comparable to that measured in embryos not maintained for a period in Na(+)-free medium, but embryos derived from Balb/c females consistently recovered at a highly accelerated rate. This accelerated recovery appeared to be due, in part, to an activation of the Na(+)/H(+) antiporter in Balb/c-derived embryos, which did not occur in CF-1- or BDF-derived embryos. Thus, embryos derived from different strains of female mice differ in their control of mechanisms for pH(i) regulation.