PIWIs maintain testis apoptosis to remove abnormal germ cells in Eriocheir sinensis

Abstract

PIWI proteins play important roles in germline development in the mammals. However, the functions of PIWIs in crustaceans remain unknown. In the present study, we identified three Piwis from the testis of Eriocheir sinensis (E. sinensis). Three Piwi genes encoded proteins with typical features of PIWI subfamilies and were highly expressed in the testis. Three PIWIs could be detected in the cytoplasm of spermatocytes and spermatids, while in spermatozoa, we could only detect PIWI1 and PIWI3 in the nucleus. The knockdown of PIWIs by dsRNA significantly affected the formation of the nuclei in spermatozoa, which resulted in deviant and irregular nuclei. PIWI defects significantly inhibited the apoptosis of abnormal germ cells through the caspase-dependent apoptosis pathway and p53 pathway. Knockdown of PIWIs inhibited the expression of caspase3, 7, 8, and p53 without affecting Bcl2 (B-cell lymphoma gene 2), Bax (B-cell lymphoma-2-associated X) and BaxI (B-cell lymphoma-2-associated X inhibitor), which further significantly increased abnormal spermatozoa in the knockdown-group crabs. These results show a new role of PIWI proteins in crustaceans that is different from that in mammals. In summary, PIWIs play roles in the formation of the germline nucleus and can maintain apoptosis in abnormal germ cells to remove abnormal germ cells in E. sinensis.

Transcriptome analysis of gonads and brain of giant freshwater prawn (Macrobrachium rosenbergii): screening and validation of genes related to germ cell development

“…Therefore, screening and identifying key genes that play a role in germ cell development, then clarifying the developmental molecular mechanisms of germ cells is an important prerequisite for the future development of sterility induction methods in M. rosenbergii. Recently, related research has made certain progress on other crustacean species: Wei et al (2021) found that PIWI plays a role in the formation of germ cell nucleus of E. sinensis, and can maintain the apoptosis of abnormal germ cells to remove abnormal germ cells, which is different from its function in mammals; In Penaeus vannamei, a newly discovered maternal gene MnTdrd has been proved to function in the formation and differentiation of reproductive cells in P. vannamei, and can be used as a new germ cell marker to track the origin and migration of germ cells (Dong et al, 2021); Meanwhile, KIFC1 was found to be possibly involved in mitosis of spermatogonia, meiosis of spermatocytes, and acrosomal formation during spermatogenesis in Penaeus japonicus, and its reduced expression may induce abnormal assembly of microtubules and lead to apoptosis of spermatogonia and spermatocytes (Hao and Yang, 2019). Additionally, the research of foxl2 (Wan et al, 2022), IAG2 (Liu et al, 2021) and DDX52 genes in the germ cells of crustaceans has also made considerable achievements.…”