The aim of this study was to investigate whether the
androgens testosterone and
dihydrotestosterone (DHT) and the antiandrogenic fungicide
vinclozolin (Vnz) exert proapoptotic effects on porcine granulosa cells (GCs), and to examine the roles of these compounds in follicular atresia. Granulosa cells isolated from pig follicles were cultured for 24 hours, and then exposed to 0.1 μM
testosterone, 0.1 μM DHT, 14 μM Vnz, or the equivalent concentrations of
testosterone and Vnz or DHT and Vnz for a further 24 hours. Apoptosis and
necrosis of the GCs were determined via Hoechst staining and flow cytometry analyses of
annexin V-stained cells. Whole porcine follicles were also exposed to the same compounds and combinations of compounds for 24 hours. The sections were stained with
hematoxylin and
eosin for morphologic assessments, and a
Terminal deoxynucleotidyl Transferase Biotyn-dUTP Nick-End Labeling (TUNEL) assay was performed to determine the number of apoptotic cells. The
progesterone and
estradiol concentrations secreted into the
culture media by isolated GCs and follicles were also measured. Exposure to the
androgens resulted in an increased number of apoptotic GCs both in vitro and in the organotypic model.
Vinclozolin exposure increased and decreased the number of necrotic and apoptotic GCs, respectively. Furthermore, compared with control follicles, those exposed to
testosterone, DHT, or Vnz displayed enhanced atresia, and coadministration of Vnz attenuated the promotive effect of these
androgens on atresia.
Estradiol secretion was stimulated by the combination of
testosterone and Vnz, whereas exposure to Vnz alone reduced it.
Progesterone production declined after the combined addition of
androgens and the
antiandrogen. In summary, Vnz caused massive
necrosis of GCs in vitro and induced apoptosis of GCs in whole follicles. The
androgens testosterone and DHT enhanced these effects. The results presented here suggest that selective destruction of porcine follicles is a serious consequence of exposure to Vnz, and may lead to
premature ovarian failure in affected animals.