ASZ1 Directs the Precise Localization of piRNA Biogenesis Factors in Mouse Spermatogenesis

Chao Wei¹, Xiaoyuan Yan¹, Chen Chen^{1, 2, 3}

¹ Department of Animal Science, Michigan State University, East Lansing, USA.

² Reproductive and Developmental Sciences Program, Michigan State University, East Lansing, USA.

³ Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids, USA.

Abstract Text: The intermitochondrial cement (IMC) and the chromatoid body (CB) are well characterized germ granules that serve as critical sites for the biogenesis and function of PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs essential for silencing transposable elements and protecting germline genome integrity. During mouse spermatogenesis, a precisely controlled sequence of events ensures that piRNA biogenesis factors are correctly localized and assembled. However, the molecular mechanism underlying the recruitment of piRNA factors to the IMC and the subsequent translocation to the CB precursors have remained largely obscure. Here we identify the mitochondria-anchored protein ASZ1 as a critical mediator of this process. ASZ1 functions as a molecular scaffold that specifically recruits key piRNA biogenesis factors MILI and MOV10L1 to the IMC in vitro. Conditional deletion of Asz1 in postnatal germ cells in mice leads to a failure to recruit MILI and MOV10L1 to the IMC in pachytene spermatocytes, which in turn results in a near-complete loss of pachytene piRNAs. This dramatic reduction in piRNA production triggers spermatogenetic arrest and male infertility. Unexpectedly, ASZ1 deficiency does not lead to obvious upregulation of transposable element LINE1, indicating that vast majority of pachytene piRNAs are dispensable for transposon silencing. Additionally, ASZ1-mediated piRNA factor recruitment is essential not only for initiating the production of piRNAs but also for facilitating the translocation of piRNA biogenesis factors to the CB precursors. piRNA biogenesis factors such as MIWI, TDRD1, and TDRD5 are aberrantly retained in the IMC in the absence of ASZ1 and fail to translocate to the CB precursors during the late pachytene stage. This mislocalization disrupts the normal progression of piRNA biogenesis, emphasizing the tightly coordinated dynamics of germ granule formation and piRNA factor translocation. Collectively, these findings underscore the complex interplay between membrane and non-membrane molecular factors that govern the piRNA pathway and provide critical insights into the developmental regulation of piRNA biogenesis that is critical for male fertility.