The membrane androgen receptor ZIP9 (SCL39A9) maintains ovarian homeostasis by mediating post-ovulatory follicle breakdown in zebrafish

Abstract

ZIP9 was recently characterized as a membrane androgen receptor in Atlantic croaker granulosa/theca (G/T) cells where it mediates androgen-induced apoptosis in vitro, but the physiological significance of this action has remained unclear. In the current study, we utilized ZIP9 knockout (zip9-/-) zebrafish to investigate the role of ZIP9-mediated androgen-induced G/T cell apoptosis in vivo. We first confirmed ZIP9 mediates apoptosis of zebrafish G/T cells in vitro. Testosterone increased apoptosis, intracellular free zinc, and expression of pro-apoptotic members bax and p53 in wildtype and zip9+/+ zebrafish G/T cells, but not in ZIP9 knockout and knockdown cell models. We hypothesized ZIP9-mediated G/T cell apoptosis may be involved in post-ovulatory follicle (POF) breakdown in vivo. Post ovulation, zip9, bax, and p53 were upregulated in zip9+/+ but not in zip9-/- ovaries. Immunoreactivity of cleaved caspase 3 was also higher in POFs from zip9+/+ ovaries compared to zip9-/-, and POF breakdown was significantly delayed in zip9-/- fish compared to zip9+/+ counterparts. To determine the detrimental consequences of delayed POF breakdown in the zip9-/- model, fish were challenged with repeated ovulation induction. After the challenge, zip9-/- fish exhibited abnormal ovarian lesions that contained debris consistent with atretic or necrotic cellular material. However, no abnormalities were observed in zip9+/+ fish ovaries, indicating that the abnormal phenotype is due to the loss of ZIP9. This study demonstrates an important role for ZIP9 in mediating POF breakdown and maintaining tissue remodeling and homeostasis in the teleost ovary and indicates a role for the ZIP9-mediated androgen-induced apoptotic response in vivo.