Rescue of Postcompaction-Stage Mouse Embryo Development from Hypertonicity by Amino Acid Transporter Substrates That May Function as Organic Osmolytes1

Abstract

Early preimplantation embryos are sensitive to external osmolarity and use novel mechanisms to accumulate organic osmolytes and thus control their cell volumes and maintain viability. However, these mechanisms are restricted to the cleavage stages of development, and it was unknown whether postcompaction embryos use organic osmolytes. Mouse embryos developing from the 8-cell stage formed blastocoel cavities in vitro at osmolarities up to 360 mOsM. Above this range, several putative organic osmolytes (alanine, glutamine, glycine, and beta-alanine) rescued blastocyst development, but several effective osmoprotectants in cleavage-stage embryos (such as betaine and proline) did not. At physiological osmolarities, each of these compounds resulted in significantly larger blastocysts. This was not due to increased cell numbers, which were unaffected in blastocysts by osmolarity in the range where blastocyst size was rescued by potential organic osmolytes, although cell number was decreased at higher osmolarities and was rescued by each osmolyte. The effective osmolytes were accumulated intracellularly by embryos developing in vitro from the 8-cell stage to blastocysts. However, unlike conventional organic osmolytes in somatic cells or those in cleavage-stage embryos, their intracellular concentrations were not increased with increasing external osmolarity. With the exception of beta-alanine, which is taken up via the beta-amino acid transport system, the effective osmolytes were transported by the B(0,+) system, which becomes highly active in blastocysts. The intracellular accumulation of these osmolytes in postcompaction embryos thus appears to support optimal development and blastocyst expansion at physiological osmolarities and may contribute to the embryo's ability to withstand stress.

Dipeptide forms of glycine support mouse preimplantation embryo development in vitro and provide protection against high media osmolality

“…For example, a prior study demonstrating that glycyl-glutamine was superior to alanyl-glutamine in supporting mouse embryo development [25] could simply be due to the fact that glycine is a more beneficial amino acid than alanine. Indeed, glycine has been shown to be superior in hamster embryos and is a superior osmolyte compared to alanine [4,31]. In our study, alanylglycine supported slightly higher embryo development in high osmolality media compared to glycyl-glycine.…”

“…This osmo-regulatory role is dependent upon glycine transport into the cleavage stage embryos by the GLTY 1 transporter system [9,11,12,14,[32][33][34]. In post-compaction mouse embryos, glycine transport can utilize both the GLY1 and B + ,0 transporter [31,35]. This is relevant because we have now demonstrated that dipeptide forms of glycine (alanyl-and glycyl-glycine) can also function as osmolytes in preimplantation mouse embryos.…”

“…b Further analysis of type of blastocyst formation at 96 h was performed. Different superscripts within a time point/represents a statistically significant different, p<0.05 glycyl-glutamine, can support embryo development in normal culture media [16,25,26,28], no studies exist examining efficacy of dipeptides in supporting specific physiologic roles of amino acids in embryos, such as acting as osmolytes [5,9,10,[29][30][31]. We have reported here for the first time that the dipeptides alanyl-glycine and glycylglycine can function as organic osmolytes in developing embryos; however, use of individual amino acids counterparts function more efficiently.…”

Cell volume regulation in mammalian oocytes and preimplantation embryos

“…Endogenous glycine levels decrease substantially by the morula stage, reaching intracellular concentrations resembling those of somatic cells (Tartia et al, 2009). Yet, it appears that post-compaction embryos may be able to utilize organic osmolytes, since alanine, glutamine, glycine, and b-alanine each rescued development of blastocysts from 8-cell embryos and supported greater cell numbers at increased osmolarities, while betaine and proline were not effective (Richards et al, 2010). Except for b-alanine, which is carried by TAUT as discussed above, the remaining of these putative organic osmolytes that can be used by post-compaction embryos are carried by the amino acid transport system designated B 0,þ (Richards et al, 2010), which is related to GLYT1 (Sloan and Mager, 1999) and becomes active in preimplantation embryos after compaction (Van Winkle et al, 1990a).…”

“…Yet, it appears that post-compaction embryos may be able to utilize organic osmolytes, since alanine, glutamine, glycine, and b-alanine each rescued development of blastocysts from 8-cell embryos and supported greater cell numbers at increased osmolarities, while betaine and proline were not effective (Richards et al, 2010). Except for b-alanine, which is carried by TAUT as discussed above, the remaining of these putative organic osmolytes that can be used by post-compaction embryos are carried by the amino acid transport system designated B 0,þ (Richards et al, 2010), which is related to GLYT1 (Sloan and Mager, 1999) and becomes active in preimplantation embryos after compaction (Van Winkle et al, 1990a). Acute cell volume homeostasis by coupled Na þ /H þ and HCO 3 À /Cl À exchangers is likely present at least through the morula stage (Zhou and Baltz, 2012), and thus acute protection against cell volume regulation is functional through preimplantation embryo development.…”