329 EXPRESSION AND REGULATION OF GENOMIC AND NON-GENOMIC PROGESTERONE RECEPTORS IN BOVINE CUMULUS–OOCYTE COMPLEXES DURING MATURATION IN VITRO

Abstract

The steroid hormone, progesterone (P4) plays an essential role in the regulation of female reproductive events. It acts by binding to specific receptors classified as genomic (nPR-A, nPR-B) and non-genomic (mPRα, mPRβ, PGRMC1, PGRMC2) progesterone receptors (PRs). The aim of the present work was to identify and characterize the PRs that are expressed in bovine COCs during IVM. Immature COCs collected from the ovaries of slaughtered animals and either allocated to analysis at the immature, germinal vesicle stage (GV) or processed for IVM and collected at the metaphase II stage (MII) of meiosis. Groups of up to 50 COCs were placed in 500 mL of maturation medium (TCM-199) supplemented with 10% (v/v) FCS and 10 ng mL^-1 epidermal growth factor without or with FSH (100 ng mL^-1), LH (100 ng mL^-1), or P4 (10, 100, 500 ng m^-1), in 4-well dishes and cultured at 39°C for 24 h in a humidified atmosphere containing 5% CO₂. GV and MII COCs were stripped of their cumulus cells and 100 oocytes and 10 μg of protein from cumulus cells were resolved by SDS-PAGE using specific antibodies against the different PRs. In addition, the pattern of PR expression was characterized in wholemount preparations of GV and MII COCs by immunocytochemistry using the same antibodies. nPR-A, mPRα, and mPRβ were expressed in both bovine oocytes and their surrounding cumulus cells, while expression of nPR-B, PGRMC1, and PGRMC2 was detected solely in the cumulus cells. Cumulus cell expression of nPR-A and nPR-B, mPRα, and mPRβ increased following IVM, while PGRMC1 and PGRMC2 expression decreased. In contrast to the expression profile of the cumulus cells, nPR-A, mPRα, and mPRβ expression was downregulated following IVM in the corresponding oocytes. Supplementation of the IVM media with FSH or LH resulted in an increase in the expression of nPR-A and nPR-B in cumulus cells and a decrease of nPR-A in oocytes. In contrast, P4 supplementation (500 ng mL^-1) during IVM resulted in increased cumulus cell nPR-B expression and decreased nPR-A, mPRα, and mPRβ expression in oocytes. Oocyte mPRβ expression was also downregulated by LH and FSH. PGRMC1 and PGRMC2 were upregulated by 500 and 10 ng mL^-1 of P4 respectively. In conclusion, the dynamic changes observed in the protein expression of genomic and non-genomic P4 receptors in bovine COCs after IVM and after treatment with LH, FSH, and P4 suggest that some or all of these receptors may be involved in oocyte maturation in cattle.

Supported by Science Foundation Ireland (07/SRC/B1156). IMAparicio was supported by a postdoctoral contract (POS07011) from Consejeria de Economia, Comercio e Innovacion, Junta de Extremadura, Spain.