Analysis of the pathways of androgen metabolism in the porcine ovary: An examination of the role of androgens during follicular development and maturation

Abstract

The follicle is the compartment of the ovary responsible for estrogen production. In the pig follicular estrogen production is accomplished by a concerted cooperation between the two cell types of the follicle, the granulosa cells, and the thecal cells, under the control of the pituitary gonadotropins Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH). Under the influence of LH stimulation, the thecal cells increase their production of the androgen, androstenedione. FSH stimulation of the enzyme aromatase, increases granulosa cell estradiol production from androstenedione provided by the thecal cells. Granulosa cells from large preovulatory follicles luteinize in vitro when placed in culture. This process is characterized by a decline in estrogen production and by increased progesterone production. To better define the steroid metabolic activity of granulosa cells during this period of in vitro luteinization, studies were performed to assess the metabolism of (A-dione) by granulosa cells in culture. The results of these studies demonstrated that aromatase activity of the granulosa cells declined substantially between 12 and 48 hours of culture resulting in the loss of estrogen production. In addition to this finding, an alternative pathway of A-dione metabolism was discovered. This pathway involves the production of the novel acidic steroid 19-oic-androstenedione (19-oic-A), and two additional novel metabolites, the C{dollar}\sb{lcub}18{rcub}{dollar} neutral steroids 5(10)estrene-3{dollar}\beta,\ 17\beta{dollar}-diol (estrenediol) and 19-nor-testosterone (19-nor-T). Evaluation of the time course of A-dione metabolism suggested that aromatase was responsible for the production of 19-oicA from A-dione. The data also suggested that changes in metabolism associated with luteinization of the granulosa cells resulted in the metabolism of 19-oic-A to estrenediol and 19-nor-T. Further investigation confirmed that aromatase was the enzyme responsible for the production of 19-oic-A from A-dione. The metabolism of 19-oic-A by granulosa cells was assessed, and the results demonstrated that this steroid was indeed the metabolic precursor of the two C{dollar}\sb{lcub}18{rcub}{dollar} neutral steroids, estrenediol and 19-nor-T. Isolation of 19-oic-A, estrenediol, and 19-nor-T in pig ovarian follicular fluid provided evidence that the metabolic pathways observed in culture were reflective of in vivo follicular steroidogenesis. The effects of metabolites of A-dione were tested for their ability to modulate parameters associated with follicular development and maturation. Granulosa cells isolated from prepubertal pigs were cultured with various combinations of FSH and steroids to assess the effects of these hormone combinations on the induction of LH receptors and aromatase activity. 19-nor-T significantly augmented the ability of FSH to induce LH receptors on granulosa cells, while estradiol and estrenediol were without effect. Likewise, the androgens A-dione, testosterone(T), 19-nor-T and all significantly enhanced the FSH stimulation of granulosa cell aromatase activity, while estradiol was without effect.;To determine if the regulation of LH receptor and aromatase induction by androgens, but not estrogens could be explained by the expression of steroid hormone receptors, immunohistochemical studies were performed to localize androgen and estrogen receptors in the pig ovary. Granulosa cells from small preantral and antral follicles expressed androgen receptors, but were devoid of estrogen receptors. As follicles increased in size as a result of follicular development and maturation, the expression of androgen receptors decreased. Weak expression of estrogen receptors was observed only in mature follicles and the developing corpus luteum. Collectively, these results were interpreted to indicate that androgens are potentially important modulators of FSH action during follicular development and maturation.