Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/1997
Publication Date: N/A
Citation: Interpretive Summary: Reproductive efficiency in the pig is critically dependent upon the number and fertilizability of follicular oocytes. The number of follicles formed during preovulatory follicle maturation determine the number of oocytes that ovulate. Steroid hormones are produced by the action of specific enzymes in the ovarian follicle, and these hormones set the timing of ovulation and prepare the reproductive tract for subsequent pregnancy. In this experiment, we measured the amounts of three steroidogenic enzyme proteins and a marker of cell proliferation in growing and dying follicles by immunohistochemistry using frozen tissue sections. In addition, we used a new histochemical procedure for identifying atretic (dying) follicles by measuring the extent of apoptosis (programmed cell death). The three enzymes, aromatase 17alpha -hydroxylase, and 3beta hydroxysteroid dehydrogenase, and the cell proliferation marker, Ki-67, were less in dying gthan growing follicles. In fact, aromatase was completely absent in dying follicles showing that these follicles could not produce estradiol. Furthermore, we showed that the expression of 3beta hydroxysteroid dehydrogenase controlled the overall rate of follicular steroidogenesis, therefore indirectly controlling the timing of ovulation. The results of this basic research will be used by scientists as a basis for studies on the molecular regulation of follicular growth and atresia in swine. These studies will provide knowledge required to better regulate ovulation rate.
Technical Abstract: The purpose of this study was to assess the expression of the steroidogenic enzymes, aromatase (P450arom), 17alpha hydroxylase C17-20 lyase (P450c17), and 3beta hydroxysteroid dehydrogenase (3betaHSD), and the cell proliferation antigen, Ki-67 by immunohistochemistry in follicles developed during altrenogest synchronized preovulatory maturation. Staining intensity ywas assigned a numeric value, and related to follicle size and day following altrenogest withdrawal. Follicle atresia was assessed by the extent of in situ 3' end labeling in apoptotic cells. Granulosa cell P450arom and Ki-67 antigen and thecal P450c17 and 3betaHSD were significantly less (p<0.001) in atretic compared to nonatretic follicles. Ki-67 expression in granulosa cells was greater in small and medium nonatretic follicles compared to large nonatretic follicles (p<0.005 ). In large follicles, P450arom was maximally expressed on Days 3 and 5 and decreased on Day 7 (p<0.005), while P450c17 was unchanged between Days 3 and 7. In contrast, 3betaHSD increased linearly between Days 3 and 7 (p<0.005). Staining intensities of P450arom, P450c17, and 3betaHSD 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 P450arom, and increased expression of 3betaHSD to provide substrate for androgen and estrogen production.