Functions and signaling mechanisms of the membrane androgen receptor ZIP9 in Atlantic croaker (Micropogonias undulatus) and zebrafish (Danio rerio) ovaries




Converse, Aubrey Koch

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The overall goal of this research was to further characterize and verify the role of ZIP9 in mediating androgen-induced apoptosis of teleost granulosa/theca (G/T) cell co-culture. In addition, a global ZIP9 knockout zebrafish model was used to infer other roles of the receptor in the ovary, in vivo. While ZIP9 was first identified and found to mediate apoptosis of croaker G/T cells, little was known on the signaling mechanism mediated by the receptor. Therefore, stimulatory G protein alpha subunit-mediated signaling, MAP kinase (MAPK) activity, and the role of extracellular zinc were investigated in ZIP9-mediated apoptosis of croaker G/T cells. Additionally, the role of ZIP9 in mediation of testosterone-induced anti-apoptotic response observed in croaker G/T cells from fish outside of the reproductive peak was examined. To determine if ZIP9 mediates androgen-induced apoptosis in G/T cells of other teleosts, a zebrafish primary G/T culture system was used to examine androgen-mediated events. Finally, a global ZIP9 knockout zebrafish strain was developed to examine the effects of global ZIP9 knockout on female reproduction. The ZIP9-mediated apoptotic pathway in croaker G/T cells was found to require stimulatory G protein signaling and MAPK activity. Additionally, extracellular zinc is essential for the induction of ZIP9-mediated apoptosis in this model, and downstream responses to these signaling cascades include increased mRNA expression of pro-apoptotic protein members and caspase 3 activity. Pro- and anti-apoptotic effects of testosterone on croaker G/T cells was confirmed, and both responses were found to be mediated by ZIP9 in an ovarian follicle stage-dependent manner. ZIP9 activation of stimulatory and inhibitory G proteins elicits the pro- and anti-apoptotic responses, respectively. In pooled zebrafish G/T cells, testosterone treatment results in an increased incidence of apoptosis, caspase 3 activity, upregulation of pro-apoptotic protein member mRNA, and a rapid rise in intracellular free zinc. This testosterone-mediated response was attributed to ZIP9 activity, thus, ZIP9 mediates apoptosis through a similar mechanism in zebrafish G/T cells as observed in croaker. Finally, examination of ZIP9 global knockout zebrafish demonstrated that ZIP9 mutation results in reduced fecundity and abnormal egg phenotypes, even though no abnormalities in ovarian morphology were apparent through histological examination.



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