Testosterone Induces Porcine Primordial Follicle Development In Vitro.

Abstract

Mammalian ovaries contain a large stock of oocytes enclosed in primordial follicles. A limited number of primordial follicles start to develop, while the remaining large numbers of them are quiescent. The mechanisms controlling the initiation of oocyte growth and primordial follicle development remain to be elucidated. Recent studies have shown that testosterone regulates early stages of folliculogenesis and follicle recruitment in vivo and in vitro. The objective of this study was to examine the effect of testosterone on initiation of primordial follicle development in cultured porcine ovarian tissues and subsequent xenotransplantation. Small pieces (1 mm × 1 mm × 1 mm) of ovarian cortex containing primordial follicles were dissected from ovaries of prepubertal pigs, and cultured in α-minimum essential medium supplemented with bovine plasma, insulin-transferrin-selenium, 2-mercaptoethanol, sodium pyruvate and different concentrations of testosterone (0, 10-7, 10-6, 10-5 and 10-4 M) or estradiol-17β (0, 10-7, 10-6 and 10-5 M) for 7 days. They were then fixed to examine the development of primordial follicles histologically. Ovarian tissues before culture contained primordial follicles (85 ± 6%) and small proportion of degenerated follicles (15 ± 6%). After 7 days of culture without hormone (control), no primordial follicle developed to the intermediate (early primary) or primary stage, and 56 ± 2% follicles were degenerated. On the other hand, in the tissues cultured with 10-6 M testosterone, higher percentages of intermediate follicles and small proportion of primary follicles were observed (17 ± 4% and 1 ± 1%, respectively). Percentages of degenerated follicles were significantly lower in 10-6 M (24 ± 1%) and 10-5 M (34 ± 1%) testosterone groups compared to the control. Estradiol-17β at any concentrations did not affect follicle development, although 10-6 M estradiol-17β diminished the percentage of degenerated follicles (40 ± 4%) compared to the control (61 ± 6%). Addition of an androgen receptor antagonist, cyproterone acetate (10-7 and 10-6 M) inhibited the stimulatory effect of testosterone (10-6 M) on primordial follicle development, suggesting the receptor-mediated action of testosterone. We next examined the presence of the androgen receptor in primordial follicles. Immunohistological examination of ovarian cortical sections revealed that androgen receptor signals were localized in oocytes in primordial follicles. In addition, in the Western blot analysis, anti-androgen receptor antibody recognized a band around 100 kDa in isolated oocytes from primordial follicles. Finally, the effect of testosterone on primordial follicle development was confirmed by xenotransplantation. Ovarian cortical pieces cultured for 7 days with and without 10-6 M testosterone were transplanted under the kidney capsule of female severe combined immune deficient mice for two months. Histological analysis of testosterone-treated xenografts showed that higher percentages of follicles developed to the primary, secondary and antral follicles compared to the control grafts in which no follicular development was observed. In conclusion, testosterone induces porcine primordial follicle development in vitro. (poster)