Expression of androgen receptors and steroidogenic enzymes in relation to follicular growth and atresia following ovulation in pigs

Abstract

Among the new antral follicles that develop after ovulation in pigs, the incidence of atresia, based on granulosa cell apoptosis, increases between Days 5 and 7 of the estrous cycle. The purpose of this study was to determine how follicular growth and atresia affected the expression of some key enzymes regulating follicular steroidogenesis and androgen receptor on Days 3, 5, and 7 after the onset of estrus. Ovaries were frozen in liquid propane for subsequent sectioning and immunohistochemical analysis. Ninety-six follicles were classified according to size as small (< 3 mm), medium (3-5 mm), or large (> 5 mm). Follicles in the active stages of the cell cycle were identified by the presence of the cell proliferation-associated nuclear antigen Ki-67 in granulosa cells. Follicles with apoptotic cells were identified by in situ 3'-end labeling of DNA. Staining in tensity of antigens on sections was assigned a numeric value (0- 3). Follicles assigned a value > 1 for 3'-end labeling in their granulosa cells were classified atretic. The percentage of atretic follicles increased (p ≤ 0.05) from 5% on Days 3 and 5 to 41% on Day 7. Expression of Ki-67 in granulosa cells was more strongly (p ≤ 0.05) associated with nonatretic follicles (98% expressing) than with atretic follicles (41% expressing). Aromatase cytochrome P450 (P450(arom)) was localized predominantly in granulosa cells of nonatretic follicles and was undetectable in atretic follicles. Androgen receptor in granulosa cells and expression of P450 17α-hydroxylase/C17-20 lyase (P450(c17)) in theca interna were lower (p ≤ 0.001) in atretic follicles than in non atretic follicles. The expression of 3β-hydroxysteroid dehydrogenase (3βHSD) was localized to the theca interna and was un affected by follicle atresia. In nonatretic small follicles, the expression of P450(arom) and P450(c17) decreased (p < 0.01) between Days 3 and 7 while expression of Ki- 67 was unchanged. In non atretic follicles, increased follicle size was associated with a de crease (p < 0.01) in androgen receptor expression and increases (p < 0.01) in P450(arom), P450(c17), and 3βHSD expression. In conclusion, increased expression of steroidogenic enzymes was associated with follicular growth. Loss of P450(arom) expression in vivo is an early event in atresia and is followed by decreased cell proliferation, and decreased expression of androgen receptor and P450(c17).