Steroidogenic enzyme expression during preovulatory follicle maturation in pigs

Abstract

During preovulatory maturation, follicles destined to ovulate exhibit a marked increase in steroidogenesis, while nonovulatory, steroidogenically inactive follicles are lost by atresia. The purpose of this study was to assess the expression of the steroidogenic enzymes cytochrome P450 aromatase (P450(arom)), cytochrome P450 17α-hydroxylase/C17-20 lyase (P450(c17)), and 3β hydroxysteroid dehydrogenase (3βHSD) and that of the cell proliferation-associated nuclear antigen Ki-67 by immunohistochemistry in both nonatretic and atretic follicles developed during altrenogest- synchronized preovulatory maturation. Fifteen cyclic gilts were slaughtered on Days 1, 3, 5 (n = 3 per day), and 7 (n = 6) following the withdrawal of altrenogest. Staining intensity was assigned a numeric value and was related to follicle size and day following altrenogest withdrawal. Follicle atresia was assessed by the extent of in situ 3' end-labeling of DNA in apoptotic cells. The overall incidence of atresia was 41%, 17%, and 7% in small (< 3 mm), medium (3-5 mm), and large (> 5 mm) follicles, respectively. Expression of granulosa cell P450(arom), Ki-67 antigen, and thecal P450(c)17 was significantly less (p < 0.001) in atretic compared to nonatretic follicles. Thecal 3βHSD was expressed predominantly in medium and large nonatretic follicles; expression was nondetectable in 88% of small follicles irrespective of follicle health. Ki-67 expression in granulosa cells was greater in small and medium nonatretic than in large nonatretic follicles (p < 0.005). In large presumptive ovulatory follicles, P450(arom) was maximally expressed on Days 3 and 5 and decreased on Day 7 (p < 0.005), while P450(c)17 was unchanged between Days 3 and 7. In contrast, 3βHSD increased linearly between Days 3 and 7 (p < 0.005). Staining intensities of P450(arom), P450(c)17, and 3βHSD were correlated with each other and with follicular fluid concentrations of the steroids estradiol, androstenedione, and progesterone, whose production they catalyzed. These data show that selection of ovulatory follicles is associated with a low incidence of apoptosis, a reduction in cell proliferation, maintenance of high levels of P450(arom) and increased expression of 3βHSD to provide substrate for androgen and estrogen production. During the period of development investigated in this study, changes in follicular steroid production in vivo are explained in large measure by changes in steroidogenic enzyme expression.