Androgens are one of the most important agents influencing folliculogenesis. They can modulate follicular function by interactions with various factors and promote granulosa cells differentiation. On the other hand, androgens can antagonize follicular development by inducing apoptosis in granulosa cells. Therefore, androgens can promote follicular atresia. The purpose of the present study was to test the hypothesis that androgen receptor antagonists (2-hydroxyflutamide (2-Hf) or vinclozolin (Vnz)) in the presence of T inhibit granulosa cell apoptosis. Granulosa cells, isolated from mature pig follicles (68 mm in diameter), were cultured for 24 h in McCoys 5A medium supplemented with 10% fetal bovine serum to cause attachment cells to the plastic. After that, media were changed and T, 2-Hf, Vnz, or both T+2-Hf and T+Vnz were added to the culture media. Finally, 24 h later cells were fixed for morphological assessment of apoptotic cells utilizing a Hoechst staining technique and measurement of caspase-3 activity in cultured granulosa cells were performed.
It was shown that the addition of 2-Hf or Vnz to the culture media caused an increase in the incidence of apoptotic bodies and caspase-3 activity. Furthermore, testosterone treatment also enhanced apoptosis in granulosa cells. On the other hand apoptotic bodies were very sparse among granulosa cells cultured with T+2-Hf and T+Vnz. Moreover, caspase-3 activity was suppressed in cultures treated with combination of androgen receptor agonist and antagonists.
In conclusion, our results indicate that both 2-hydroxyflutamide and vinclozolin appear to be atretogenic, but when tested with high level of testosterone they trigger anti-apoptotic effects. The nature of this protective mechanism as yet is unknown and requires further research.
Supported by grant N N303 538838.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details unavailable.
05 - 09 May 2012
European Society of Endocrinology