121 EXPRESSION OF STEROID RECEPTORS IN THE CUMULUS-OOCYTE COMPLEX AROUND OVULATION IN THE BITCH

Abstract

In the bitch, oocytes are ovulated at an immature stage (prophase I) and resume meiosis in the oviduct, 3 to 4 days after ovulation while they are still surrounded by 2 to 3 layers of cumulus cells. Canine cumulus-oocyte complexes (COC) are exposed to high and changing plasma concentrations of 17β-oestradiol (E2) and progesterone (P4) during the periovulatory period. In order to explore whether oocyte maturation may be regulated by steroids in this species, the expression of E2 (ERα, ERβ) and P4 (nuclear: PR; membrane: PGRMC1, PGRMC2, mPRα, mPRβ, mPRγ) receptors was studied in COC at precise times around ovulation. Ovaries were collected from Beagle bitches during anestrus (n = 4), after the beginning of proestrus, and before the LH peak (Pre-LH, n = 7), after the LH peak and before ovulation (Pre-ov, n = 8), and at Day 1 (n = 11) and Day 4 (n = 8) post-ovulation. Anoestrus COC were recovered from follicles smaller than 1 mm in diameter. The COC at the Pre-LH and Pre-ov stages were aspirated from preovulatory follicles (4.5–6 mm in diameter). Such follicular COC were partially denuded to leave the 2 to 3 innermost cumulus layers firmly attached to the zona pellucida. Post-ovulatory COC, naturally surrounded by 2 to 3 cumulus layers, were recovered by oviductal flushing. Total RNA was extracted from 3 batches of 10 COC per stage, then reverse transcribed. The expression of steroid receptors was assessed in duplicate by qPCR (LightCycler® 480, Roche Diagnostics) using the relative standard curve method and normalized by the geometric mean value of the two most stable reference genes (BGLR and RPS5; NormFinder software) chosen among four genes previously tested. Relative amounts of mRNA levels were compared between stages by ANOVA followed, when necessary, by a Tukey test. The ERα and ERβ expression did not vary significantly with the stage. In contrast, a significant variation between stages in nuclear and 4 membrane P4 receptor expression was observed (P < 0.0001 for PR; P < 0.001 for PGRMC1 and mPRβ; P < 0.05 for PGRMC2 and mPRγ). The PR mRNA levels were significantly higher at Pre-ov than at any other stage. PGRMC1 expression was significantly higher at Pre-ov and Day 4 compared with anestrus and Pre-LH, and was at an intermediate level at Day 1. The expression of PGRMC2, mPRβ, and mPRγ remained low from anestrus to Day 1 and increased significantly at Day 4. Lastly, mRNA levels of mPRα were below the detection limit at all stages. This is the first report of steroid receptor expression in canine COC at precise times around ovulation. The stage-specific variation in expression of nuclear and of several membrane P4 receptors around ovulation suggests a role for P4 in canine oocyte maturation. The exact localisation of these receptors in cumulus cells, oocytes, or both remains to be determined.