REGULATION OF ANGIOTENSIN TYPE 2 RECEPTOR IN BOVINE GRANULOSA CELLS IN VITRO

Abstract

Angiotensin II (AngII) is best known for its role in blood pressure regulation, but it also has some documented actions in the reproductive system. There are two AngII receptors, AT1 and AT2. The AT1 receptor mediates a number of well-known AngII effects on smooth muscle contraction, aldosterone secretion and blood pressure regulation, while the AT2 receptor has been shown to mediate reproductive functions including steroidogenesis, oocyte maturation and ovulation in some species. In cattle, expression of AT2 mRNA in granulosa cells is correlated with follicle health and estradiol content. The objective of the present study was to determine if granulosa cell AT1 and AT2 receptor expression is regulated by gonadotropins and growth factors. First, we confirmed that AT2 protein is present in bovine follicles in vivo by immunohistochemistry; the AT2 receptor was localized in granulosa (and theca) cells from follicles of different sizes. Second, bovine ovaries were collected at a local abattoir and small follicles (2–5mm) were isolated for harvesting granulosa cells. The cells were cultured for 6 days in free serum medium in non-luteinizing conditions without FSH (control group) or with graded doses of FSH (0.01, 0.05, 0.1, 0.5 or 1 ng/ml) or insulin-like growth factor 1 (IGF1). In other cultures, cells were cultured with or without IGF1 (15ng/ml) and bone morphogenetic protein-7 (BMP-7; 50 ng/ml), epidermal growth factor (EGF; 10ng/ml) and fibroblast growth factor-2 (FGF-2; 10ng/ml). Abundance of AT1 and AT2 mRNA was measured by semiquantitative RT-PCR. Treatment with FSH, IGF1 and BMP-7 increased (P<0.05) estradiol secretion and AT2 mRNA expression relative to control cultures. In contrast, none of these treatments affected AT1 receptor expression. Addition of EGF and FGF2 significantly decreased estradiol secretion but did not affect AT1 or AT2 expression. Third, cells were cultured with FSH plus graded doses of FGF-7 or FGF-10, and the effects of these factors on AT2 protein levels were measured by Western blot. AT2 protein levels decreased in the groups treated with FGF-7 (10 and 100ng/ml) and FGF-10 (all concentrations; P<0.05), and estradiol secretion was significantly inhibited by the highest dose of each FGF (P<0.05). In conclusion, AT2 receptor is present in granulosa bovine cells, and mRNA and protein are regulated by FSH, IGF-1, BMP-7, FGF-7 and FGF-10 in bovine granulosa cells in vitro. Supported by NSERC Canada and CNPq, Brazil. (poster)