Modulation by hypoxia of membrane steroid receptor expression and functions in ovaries of Atlantic croaker (Micropogonias undulatus)
Hypoxia is an endocrine disruptor, altering estrogen, testosterone, and progestin hormone levels and stunting gonadal growth in Atlantic croaker. Steroids act through specific hormone receptors to alter reproductive functions, and the hormonal response is dependent on the concentrations of these receptors. However, information is currently lacking on the effects of hypoxia on expression and functions of membrane receptors mediating rapid, non-genomic steroid actions such as final oocyte maturation and apoptosis. Atlantic croakers were exposed to normoxia (7.0 mg DO/L) or hypoxia (1.7 mg DO/L) for 6 weeks during their period of gonadal recrudescence (October-December). Relative gene expression was quantified using quantitative real-time PCR (qRT-PCR). mRNA expression of the membrane androgen receptor, ZIP9, was increased in hypoxia-exposed fish compared to normoxia-exposed controls, whereas mRNA expression of the membrane estrogen receptor, GPER, and membrane progestin receptor, mPRα, was decreased in hypoxia-exposed fish compared to controls. mRNA expression of pro-apoptotic factors Bax and p53 was also measured and expression of both genes was increased in hypoxia-exposed fish compared to controls. Relative protein expression of these receptors was quantified using Western blotting and the results were consistent with the qRT-PCR findings. Oocytes from both hypoxia-exposed and control fish were tested in an in vitro final oocyte maturation (FOM) assay to examine possible alterations in receptor functions. When oocyte maturation was stimulated with progestin, which acts through mPRα, significantly fewer oocytes of hypoxia-exposed fish underwent FOM compared to oocytes of normoxia-exposed controls. These results are consistent with the decrease in mPRα expression following hypoxia exposure. Ovaries were sectioned and stained with hematoxylin and eosin, and the proportions of perinucleolar stage, tertiary yolk stage, and atretic oocytes were determined. Ovaries from fish exposed to hypoxia showed an increase in the proportion of perinucleolar stage and atretic oocytes and a decrease in the proportion of tertiary yolk stage oocytes compared to controls. Finally, apoptotic cells in ovarian tissue sections were labeled using in situ TUNEL staining. Ovaries from fish exposed to hypoxia showed an increased proportion of TUNEL-positive ovarian follicle cells compared to controls. Collectively, these results show the concurrence of increased ZIP9 expression and apoptotic follicle cells in ovaries of Atlantic croaker exposed to hypoxia in vivo.