229 EFFECT OF CULTURE CONDITIONS ON AQUAPORIN mRNA ABUNDANCE IN MOUSE BLASTOCYSTS

Abstract

It is known that culture conditions can alter gene expression of the pre-implantation embryo. We have previously shown that aquaporins (AQPs) are expressed in the mouse embryo and that they are involved in the passage of water across the trophoblast cells during blastocyst formation. This study was conducted to investigate whether AQP mRNA abundance is altered by culturing embryos in vitro compared to in vivo developed embryos. Furthermore we wanted to investigate if AQP mRNA abundance was influenced by the osmolality of the media. It is possible to compare the effect of hyperosmolality that the embryo may be able to compensate for by adding glycerol which can cross some AQPs, compared to the addition of sucrose which can not cross the membranes. Mouse embryos were obtained by superovulating B6D2F1 mice followed by culture of the flushed presumptive zygotes in KSOM to the blastocyst stage (in vitro) or by flushing blastocysts from the uterus (in vivo). For the study of the influence of osmolality on AQP mRNA abundance, zygotes were flushed and cultured to the compacted 8-cell stage and then placed in media of increasing osmolality, using either glycerol or sucrose. The osmolalities of the media were 243 (control), 300, 350, and 400 mOsm. Embryos were cultured to the blastocyst stage and frozen in liquid nitrogen. Embryonic RNA was extracted using a Dynabeads mRNA Capture kit (Dynal, Oslo, Norway). Real time PCR was performed on embryonic cDNA on a Lightcycler (Roche Diagnostics, 2650 Hvidovre, Denmark) using aquaporin-specific primers and primers for β-actin and GAPDH. The results of the quantitative RT-PCR analysis showed that in vitro-cultured embryos had a lower mRNA abundance for AQP 8, 9, and 11 compared to the in vivo-developed embryos but that the AQP 3 mRNA abundance was unaltered. Analysis of the housekeeping genes showed that GAPDH mRNA levels were unchanged in vitro, whereas β-actin was up-regulated in vitro. The osmotically challenged embryos showed the following blastocyst rates compared to the controls: glycerol 300: 100%; glycerol 350: 100%; glycerol 400: 100%; sucrose 300: 100%; sucrose 350: 78%; and sucrose 400: 0%. Thus, glycerol up to 400 mOsm had no effect on blastocyst rates, whereas addition of sucrose reduced blastocyst formation, with a total inhibition at 400 mOsm. Analysis of the mRNA abundance showed a reduction of AQP 8 in the glycerol solutions. The level was reduced to 30% of the control group at 300 mOsm, to 27% at 350 mOsm and to 8% at 400 mOsm. There was no corresponding reduction of AQP 8 mRNA abundance in sucrose solutions. Further, AQP 3, 7, 9, and 11 mRNA levels as well as β-actin and GAPDH mRNA levels were unaltered in the osmotically challenged embryos. In conclusion, this study shows that embryonic culture affects the abundance of several AQPs and that compensation of a glycerol-induced osmotical challenge induces down-regulation of AQP 8 expression. Embryos tolerate high glycerol concentrations better than high sucrose concentrations but the possible role of AQP 8 in this process is unclear at present.