211 TRANSCRIPTS EXPRESSION RELATED TO MAPK/SMAD2 PATHWAY IN OVIDUCT CELL, CUMULUS CELL, AND OOCYTE DERIVED FROM DIESTRUS BITCHES

Abstract

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are known to play an essential role in regulating ovarian follicular development and ovulation rate via SMAD pathway in several species and have different functions depending on the species. However, their expression and function have not yet been reported in dogs. Therefore, the aim of present study is to analyse expressions of transcripts related to the MAPK/SMAD2 pathway in oviduct cell, cumulus cell, and oocyte derived from diestrus bitches. Ovariohysterectomy was performed in 5 large, mixed breed dogs 3 days after serum progesterone reached 5–10 ng mL–1. Oviduct cells were obtained by scraping the superficial oviduct tissue and oocytes were obtained by mincing the ovary. Both cumulus cells derived from non-ovulated oocytes (IM-cumulus) and ovulated oocytes (M-cumulus) were obtained. Oviduct, IM-cumulus, and M-cumulus cells were cultured using RCMEP media and cryopreserved using FBS containing 10% DMSO. Total RNA was extracted from Oviduct, IM-cumulus, and M-cumulus cells and oocytes. After cDNA synthesis, transcript expression of β-actin, GDF9, MAPK1, BMP6, and SMAD2 genes were analysed. The data was analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). The threshold for statistical significance was set at P < 0.05. In results, mRNA expression of MAPK1 was not significantly different among the cells. However, expressions of BMP6, GDF9, and SMAD2 were significantly higher in oocytes compared to oviduct, IM-cumulus, and M-cumulus cells. Although oviduct cells showed increased GDF9 and SMAD2 transcripts similar to that of oocytes, there was no significant difference between them. In conclusion, MAPK/SMAD2 pathway may have important roles in growth of immature oocytes in dogs and oviduct cells may help the maturation of ovulated immature oocytes. Further studies will be conducted to analyse MAPK/SMAD2 pathway in anestrus, proestrus, and oestrus periods.This research was supported by RDA (#PJ008975022014), IPET (#311062-04-3SB010), Research Institute for Veterinary Science, the BK21 plus program, and global PhD Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014H1A2A1021187).