141 EXPRESSION STATUS OF AQUAPORINS 3, 7, AND 9 IN BOVINE PRE-IMPLANTATION EMBRYOS

Abstract

The aquaporins (AQP) are a family of small integral membrane proteins that work as channels for rapid water transport. In mouse embryos, AQP3, AQP7, and AQP9 were found to play important roles in the pre-implantation development or adaptive cellular response to osmotic stress. In addition, because AQP3, AQP7, and AQP9 permeate not only water but also cryoprotectant, such as glycerol, these AQP thought to be involved in the process of cryopreservation. However, little information is available for AQP in bovine embryos. The understanding of the mechanisms mediated by AQP that embryos utilise to survive during culture and the process of cryopreservation will contribute to development of in vitro culture systems and cryopreservation procedures for bovine embryos. The aims of present study were to clarify the expression status of AQP3, AQP7, and AQP9 in bovine pre-implantation embryos, and to evaluate the expression level of AQP3 in in vivo-derived (IVD) and IVF bovine embryos. For production of IVF embryos, cumulus-oocyte complexes (COC) were aspirated from ovaries collected at a local slaughterhouse. The COC were in vitro matured, fertilized, and then cultured for 7 days. The IVD embryos at early blastocyst (EB) and blastocyst (BC) stage were obtained from donor cows treated with superovulation and AI. In experiment 1, in order to clarify the expression of AQP3, AQP7, and AQP9 mRNA in bovine pre-implantation embryos, total RNA was extracted from pools of 30 IVM oocytes, pools of 15 IVF embryos at 2- to 4-cell, 8- to 16-cell, compaction morula (CM), EB, BC, and expanded blastocyst (ExBC) stage, and RT-PCR was performed followed by agarose gel electrophoresis. In experiment 2, in order to clarify the expression status of AQP3, AQP7, and AQP9 during bovine pre-implantation development after zygotic gene activation, total RNA was extracted from pools of 15 IVF embryos at 8- to 16-cell, CM, EB, BC and ExBC stage (n = 5), and relative quantifications of AQP mRNA were performed using real-time RT-PCR. Data were analysed by Scheffé’s method. In experiment 3, AQP3 transcript levels in single IVD and IVF embryos at EB and BC stages (n = 10) was evaluated by real-time RT-PCR. Data were analysed by Mann-Whitney’s U test. In experiment 1, AQP3 and AQP7 transcripts were detected in IVM oocytes and all stages of embryos. AQP9 mRNA was detected in IVM oocytes and in 2- to 4-cell, 8- to 16 cell, CM, and EB stage embryos, but was not detected in BC and ExBC stage embryos. In experiment 2, AQP3 and AQP7 transcript levels were significantly increased from 8- to 16-cell to CM and EB stage, and significantly decreased from EB to BC and ExBC stages (P < 0.05). AQP9 transcript level was significantly decreased from 8- to 16-cell to CM and EB stage (P < 0.05). In experiment 3, AQP3 transcript level in IVF embryos was significantly lower than that in in vivo embryos at the BC stage (P < 0.05). Our results indicate that AQP3 and AQP7 may have specific roles at morula and EB stage in bovine embryos. In addition, AQP3 expression is influenced by developmental condition of bovine embryos.