PARENT SESSION
Preimplantation Embryo/Fetal Development

OSMOSENSITIVE GLYCINE ACCUMULATION IN MOUSE ZYGOTES.

Steeves, Candace^1,2,4, Hammer, Mary-Anne¹, Baltz, Jay^1,2,3,4, ^{1 2 4 3}

ABSTRACT- Cells preferentially accumulate organic compounds as major intracellular osmolytes. Glycine, a substrate of the highly concentrative, Na⁺- and Cl^--dependent transporter GLY in pre-compaction embryos, has previously been shown to be effective as an osmoprotectant, and thus the GLY transporter is postulated to be an organic osmolyte transporter in embryos. The objective of this study is to determine the mechanisms which regulate the osmosensitive accumulation of glycine in mouse zygotes. We have found that as external osmolarity was increased (range: 250-360mOsm/kg) there was a proportional increase in intracellular ³H-glycine in embryos when measured at 24h. Only a small fraction of this glycine had been incorporated into protein at 24h, as shown by TCA precipitation, indicating that almost all accumulated glycine was likely to be osmotically active. The timecourse of glycine accumulation during 24h starting at the zygote stage was determined by measuring total intracellular ³H-glycine accumulated at external osmolarities of 250, 310, and 350mOsm/kg as a function of time, measured at 2h intervals. Glycine accumulation reached a plateau between 4-6h, 8-10h, and 10-12h at 250, 310, and 350mOsm/kg, respectively, and then remained essentially constant up to at least 24h. The initial rate of glycine accumulation was similar for each osmolarity, suggesting that the greater final amount of glycine accumulated by embryos at higher osmolarities is due mainly to the longer period during which net uptake occurs. To begin to determine how osmosensitive accumulation of glycine in embryos is regulated, we are measuring the rates of uptake and efflux separately. At 4h after the glycine accumulation was initiated, net glycine influx was greater than efflux, and efflux was very low at 350 mOsm/kg but significant at 310 and 250mOsm/kg, indicating that glycine accumulation by zygotes may be regulated at least in part by control over the efflux rate. Thus, glycine functions as an organic osmolyte in early preimplantation embryos, and data indicate that its intracellular concentration is regulated in response to changes in osmolarity. (Supported by Canadian Institutes of Health Research grant MT12040; CLS holds a CIHR studentship).

KEY WORDS: pre-implantation embryo, glycine, osmolarity