Identification of a piwi1 homologue gene in the orange-spotted grouper (Epinephelus coioides) and its expression pattern in the germ cell lineage.

The protogynous orange-spotted grouper (Epinephelus coioides), a sequentially hermaphroditic teleost, relies on dynamic regulation of germ cell development and sex reversal mechanisms to achieve reproductive plasticity. The piwi gene family, pivotal for germ cell development and transposon silencing across metazoans, remains poorly characterized in hermaphroditic species. Here, we investigate Ecpiwi1, a piwi1 homologue in the orange-spotted grouper (E. coioides), to elucidate its role in germ cell fate determination and sex reversal. We cloned a 3084-bp Ecpiwi1 cDNA encoding an 855-amino-acid protein containing conserved PAZ and PIWI domains, phylogenetically clustering with vertebrate Piwi proteins. Real-time PCR showed that the expression level of Ecpiwi1 mRNA increases during sex reversal, transitioning from ovary to testis. Spatial expression analysis by in situ hybridization and immunohistochemistry demonstrated Ecpiwi1 localization to oogonia, previtellogenic oocytes, and spermatogonia-spermatocyte transition zones. During natural and 17α-methyltestosterone (MT)-induced sex reversal, Ecpiwi1 mRNA exhibited progressive upregulation during sex reversal. Notably, MT-treated gonads showed identical Ecpiwi1 expression dynamics and subcellular localization patterns compared to natural sex change, validating the MT induction model. These findings establish Ecpiwi1 as a germline-specific regulator coordinating oogenesis, spermatogenesis, and sex reversal in protogynous hermaphrodites. The conserved expression patterns under exogenous androgen induction position Ecpiwi1 as a robust molecular marker for teleost germline stem cell studies and advance our understanding of reproductive plasticity in hermaphroditic teleosts.