The zinc transporter ZIP9 (Slc39a9) regulates zinc dynamics essential to egg activation in zebrafish

The zinc transporter ZIP9 (SLC39A9) was recently characterized as a membrane androgen receptor in various teleost and mammalian cell models. ZIP9 shows the highest expression in ovaries of teleosts, a tissue in which both androgen signaling and zinc dynamics have significant roles. To examine the role of ZIP9 in ovarian physiology, we generated a ZIP9-mutant zebrafish strain using a CRISPR/Cas9 system. zip9-/- females showed significant reductions in fecundity, embryo viability, and growth of their offspring compared to wildtype (WT) fish. Furthermore, a high proportion of zip9-/- eggs failed to undergo normal chorion elevation during activation. In WT eggs, zinc was detected in cortically-localized vesicles which underwent exocytosis upon activation. zip9-/- eggs showed abnormal cortical vesicle development and had a significantly depressed activation-induced zinc release compared to WT eggs. Moreover, pharmacologically sustained elevation of zinc in WT eggs prior to activation resulted in abnormal chorion elevation similar to that observed in zip9-/- eggs. These results indicate that ZIP9 is essential for proper zinc modulation during zebrafish egg activation and presents the first evidence of zinc modulation during egg activation in a non-mammalian species.

Implication of Membrane Androgen Receptor (ZIP9) in Cell Senescence in Regressed Testes of the Bank Vole

Here, we studied the impact of exposure to short daylight conditions on the expression of senescence marker (p16), membrane androgen receptor (ZIP9) and extracellular signal-regulated kinase (ERK 1/2), as well as cyclic AMP (cAMP) and testosterone levels in the testes of mature bank voles. Animals were assigned to groups based on an analysis of testis diameter, weight, seminiferous tubule diameter and the interstitial tissue area: group 1, not fully regressed (the highest parameters); group 2 (medium parameters); or group 3, regressed (the lowest parameters). Cells positive for p16 were observed only in the seminiferous tubule epithelium. However, in groups 1 and 2, these were mostly cells sloughed into the tubule lumen. In group 3, senescent cells resided in between cells of the seminiferous epithelium. Staining for ZIP9 was found in Sertoli cells. Western blot analysis showed a trend towards a decreased expression of p16 and ZIP9 in the testes of the voles in groups 2 and 3, compared to group 1. In addition, a trend towards an increased expression of ERK, as well as an increase of cAMP and testosterone levels, was revealed in group 2. In the regressed testes, a functional link exists between senescence and androgen levels with implication of ZIP9 and cAMP/ERK signaling pathways.

SUN-736 Knockout of Membrane Androgen Receptor ZIP9 Results in Reduced Female Fecundity and Abnormal Egg Activation in Zebrafish

Recently, our research group cloned and characterized a putative membrane androgen receptor from teleost ovarian tissue that was found to be homologous with the zinc transporter protein ZIP9 (Slc39a9). To date, ZIP9 is the only zinc transporter that is known to be ligand activated or possess steroid receptor activity. Since the discovery of its androgen receptor activity, ZIP9 has been found to mediate androgen actions in a variety of tissues including teleost ovarian follicle cells, human cancer cell lines, and murine Sertoli cells. However, ZIP9 has not been examined in an in vivo model so the precise physiological functions of this receptor remain unclear. A ZIP9-mutant strain of zebrafish was developed using a CRISPR-Cas9 system in order to examine the role of the protein in teleost reproduction. While ZIP9-mutant males had similar breeding occurrence and fertilization rates to wild-type fish, mutant females exhibited severe reductions in fecundity compared to wild-type fish. ZIP9-mutant females spawn significantly fewer eggs of which a high proportion failed to undergo chorion elevation, a characteristic of normal egg activation. Eggs that showed this failed chorion elevation phenotype had significantly lower fertilization rates and produced larvae that exhibit a high incidence of pericardial/yolk sac edema and reduced growth compared to larvae hatched from wild-type eggs. However, no differences were observed in the proportions of oocytes at later stages of development between ZIP9-mutant and wild-type fish, suggesting the observed phenotypes are not related to abnormal oogenesis. We observed that mature wild-type eggs have numerous cortically located vesicles that are autofluorescent under ultraviolet light and decrease in number when the eggs undergo activation, suggesting they undergo exocytosis during the cortical reaction. While zinc is known to be stored in vesicles that undergo exocytosis in mammalian eggs, the role of zinc in teleost egg activation is currently unknown. In eggs from wild-type fish, we observed an increase in extracellular zinc levels upon egg activation and treatment with a zinc ionophore (zinc pyrithione) significantly reduced the number of eggs that undergo normal chorion elevation when activated. This suggests a role for zinc in zebrafish egg activation similar to that observed in mammals. Of interest, ZIP9-mutant eggs that did not undergo chorion elevation had significantly smaller vesicles than those found in wild-type fish eggs. This abnormal vesicle morphology and failure to undergo chorion elevation suggest a role of ZIP9 in egg activation. Additional insight into the role of zinc in zebrafish egg activation and the mechanism by which ZIP9 disruption leads to abnormal cortical vesicles and egg activation will help determine if ZIP9 plays a role in zinc transport and flux in zebrafish eggs during activation.