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Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation related genes that impact oocyte maturation in vivo and in vitro

I Kawashima, Hiroshima University, Higashi-Hiroshima, Japan
T Okazaki, Hiroshima University, Higashi-Hiroshima, Japan
N Noma, Hiroshima University, Higashi-Hiroshima, Japan
M Nishibori, Hiroshima University, Higashi-Hiroshima, Japan
Y Yamashita, Hiroshima University, Higashi-Hiroshima, Japan
M Shimada, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, 739-8528, Japan

Correspondence: Masayuki Shimada, Email: mashimad{at}hiroshima-u.ac.jp

Abstract

In this study, we collected follicular fluid, granulosa cells and cumulus cells from antral follicles at specific time intervals following eCG and hCG treatment of gilts. The treatment with eCG increased production of estrogen coordinately with up-regulated proliferation of granulosa cells and cumulus cells. eCG also induced the expression of Lhcgr and Pgr in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and PGR were critical for FSH induced Lhcgr expression in cumulus cells in culture. The expression of Lhcgr mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of EGF like factors and ADAMTS-1 expression, promoting COCs expansion and oocyte maturation. Based on the unique expression and regulation of Pgr and Lhcgr in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 hr at which time progesterone was added for another 10 hr. After 20 hr, COCs were moved to fresh medium containing LH, EGF and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.