PARENT SESSION
GAMETE BIOLOGY AND GAMETOGENESIS - C

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(722) THE EXPRESSIONS OF 14-REDUCTASE AND 7-REDUCTASE IN CUMULUS CELLS DURING MEIOTIC RESUMPTION OF PORCINE OOCYTES.

Yamashita, Yasuhisa¹, Nishibori, Masahide ¹, Terada, Takato¹, Shimada, Masayuki¹, ¹ Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan

ABSTRACT- We show that progesterone is produced from de novo-synthesized cholesterol in cumulus cells during meiotic resumption of porcine oocytes. Although the up-regulation of HMG-CoA reductase or 14 -dimethylase expression has been reported in FSH-stimulated granulosa cells, the gene expressions of 14-reductase (14RD) and 7-reductase (7RD) which catalyze the conversion form FF-MAS to cholesterol remain unclear. In the present study, we investigated the time-dependent changes of 14RD mRNA level and 7RD mRNA level in cumulus cells, and the role of cAMP-PKA pathway in the expression of both genes. Cumulus oocyte complexes (COCs) were isolated from porcine follicles and the expressions of specific genes were analyzed by RT-PCR. The lower levels of 14RD mRNA and 7RD mRNA were detected in cumulus cells of COCs prior to and after culture in defined medium up to 20 h. However, the expressions of them were increased by 16-h cultivation with FSH, and the levels were reached maximum at 20 h. The additional LH to FSH-containing medium increased the level of cAMP as well as message for 14RD in cumulus cells at 12 h, whereas the up-regulation of LH in the gene expression was not observed at 20 hr. When COCs were cultured with FSH, LH, and PKA inhibitor (H89), the mRNA levels were significantly lower than those in cumulus cells cultured without the drug. The cultivation of COCs with forskolin stimulated both 14RD and 7RD gene expressions in cumulus cells. PKC inhibitor, Calphostin C did not affect the gene expressions. Thus, the expressions of both 14RD and 7RD are increased in cumulus cells stimulated by FSH and/or LH via a cAMP-PKA dependent pathway. The suppression of the reductase activity decreased progesterone production in cumulus cells and meiotic resumption of oocytes. The addition of progesterone overcame the inhibitory effect on meiotic resumption of oocytes. These results suggest that FSH and/or LH stimulate the expression of enzymes which are involved in conversion to cholesterol via cAMP-PKA dependent pathway, and may help for a high level of progesterone production in cumulus cells during meiotic resumption of oocytes.

KEY WORDS: cumulus cells, gene expression, oocyte maturation, progesterone