Organic Osmolytes and Embryos: Substrates of the Gly and β Transport Systems Protect Mouse Zygotes against the Effects of Raised Osmolarity1

Abstract

Mouse embryo development is identically inhibited by raised osmolarity, whether produced by added NaCl or raffinose, demonstrating that high osmolarity is itself detrimental to embryos. In the face of increased osmolarity, cells in the brain and kidney, and likely many other cells, accumulate nonperturbing organic osmolytes in their cytoplasm. In the presence of any of a number of organic compounds that were proven or probable substrates of either the Gly or the beta transport systems, mouse embryo development in vitro was protected from raised osmolarity. Zygotes developed past the "2-cell block," and with most Gly or beta substrates, to the blastocyst stage. The most effective osmoprotectants were glycine, glutamine, betaine, proline, beta-alanine, and hypotaurine; several others were partially effective. A model Gly substrate, glycine, was effective at a much lower concentration (EC50 = 50 microM) than was a model beta substrate, beta-alanine (EC50 = 1.3 mM). The protective effect of these two compounds was additive, indicating a common mode of action. The various effective compounds tested do not all share metabolic pathways or other such properties in common. Thus, it is likely that cleavage-stage mouse embryos utilize them, in large part, as organic osmolytes.

Cell volume regulation in mammalian oocytes and preimplantation embryos

“…In contrast to M16, whose osmolarity was around 290 mOsM, the osmolarity of KSOM was $250 mOsM while that of CZB was $275 mOsM. Furthermore, it appeared that osmolarity alone could determine whether or not embryos arrested during culture since raising the osmolarity of KSOM by adding NaCl or the inert trisaccharide raffinose converted it into a medium in which the 2-cell block occurred again (Dawson and Baltz, 1997;Hadi et al, 2005). This increased osmolarity caused an irreversible arrest at the G2/M transition at the end of the 2-cell stage in mouse embryos (Wang et al, 2011), identical to what had been reported for the classical 2-cell block in older culture media (Goddard and Pratt, 1983).…”

“…b-alanine, however, was relatively ineffective at protecting embryos at physiological osmolarities, requiring several millimolar to have a maximal effect, while taurine, the main substrate of TAUT, was essentially ineffective (Dawson and Baltz, 1997;Hammer and Baltz, 2003). Possibly, TAUT normally mediates b-amino acid accumulation for other functions , such as maintaining a high intracellular concentration of taurine in mouse embryos (Schultz et al, 1981) to protect against oxidative stress.…”

“…Surprisingly, none of the other known organic osmolyte transporters were responsible for the uptake of the remaining compounds that protected embryos against increased osmolarity: The betaine transporter BGT1 is not expressed in preimplantation embryos (Hammer and Baltz, 2002); System A, which could transport any of these compounds, first appears in blastocysts (Jamshidi and Kaye, 1995); and SMIT transports only myo-inositol, which had no effect in embryos (Dawson and Baltz, 1997).…”

“…Glutamine, glycine, proline, betaine (N,N,Ntrimethylglycine), or b-alanine each protects mouse embryo development past the 2-cells stage at higher osmolarities (Van Winkle et al, 1990b;Lawitts and Biggers, 1992;Biggers et al, 1993;Dawson and Baltz, 1997). Such observations led Biggers and Van Winkle to independently propose that preimplantation embryos require organic osmolytes for cell volume regulation (Van Winkle et al, 1990b;Lawitts and Biggers, 1992).…”

“…One-cell mouse embryos will develop through the 2-cell block in media that are up to about 70 mOsM more hypertonic when glycine is present than when it is absent (Hadi et al, 2005). Glycine is also abundant in the embryo's environment in vivo, with 0.5-3.0 mM glycine in oviductal fluid (Guerin et al, 1995;Harris et al, 2005), far above the half-maximally effective concentration of 50 mM for protecting embryo development against increased osmolarity (Dawson and Baltz, 1997). Additionally, a single, robust glycine transport mechanism was shown to be present and active in early preimplantation embryos until compaction, and had been identified as the classical amino acid transport system designated GLY (Van Winkle et al, 1988).…”