PIWIs Regulate Spermatogonia Self-Renewal and Differentiation by Wnt/β-Catenin Signaling Pathway in Eriocheir sinensis

The roles of PIWI in mammalian spermatogenesis have been well-studied but are largely unknown in invertebrates such as the Chinese mitten crab (<i>Eriocheir sinensis</i>), which produces non-flagellar sperm. Here, we demonstrate that knockdown of PIWIs significantly promotes the proliferation of spermatogonia and the transformation into spermatocytes. Expression of PIWIs in HEK 293T significantly inhibits cell proliferation through the Wnt-signaling pathway. PIWIs suppress transcriptional activity of the Wnt pathway to down-regulate Cyclin D and Cyclin E by inhibiting β-catenin and the phosphorylation of β-catenin at Ser552. The intracellular structure of the adherens junction is destroyed by PIWIs due to downregulated α-catenin, β-catenin, and ZO1. Overall, our results suggest that PIWIs regulate spermatogonia self-renewal and differentiation through inhibiting the Wnt-signaling pathway and stabilize the structure of the adherens junction by regulating the expression and location of α-catenin, β-catenin, and ZO1 in <i>E. sinensis</i>, which are different from the functions in mammals. Our findings revealed novel functions and molecular mechanisms of PIWIs in regulating spermatogonia self-renewal and differentiation during the Crustacea spermatogenesis.