Conditional inactivation of Miwi2 reveals that MIWI2 is only essential for prospermatogonial development in mice

Abstract

The PIWI-piRNA pathway serves as a critical defense mechanism through which the genome of the male germline is protected from invasion by transposable elements (TEs). MIWI2/PIWIL4, a member of the murine PIWI subclade of the Argonaute family, has been shown to be expressed in primordial germ cells (PGCs) and prospermatogonia in fetal and prepubertal testes. Global inactivation of Miwi2 leads to male sterility due to an early meiotic arrest, which correlates with retrotransposon desuppression. However, it remains unclear whether MIWI2 functions beyond the PGC stage and whether MIWI2 has a role beyond TE suppression during male germ line development. Through conditional inactivation of Miwi2, we demonstrate herein that MIWI2 function is restricted to a narrow time window during male PGC reprograming and that Miwi2 is dispensable for postnatal male germline development and testicular function in mice. Moreover, persistent activation of LINE1 and IAP retrotransposons caused by Miwi2 inactivation is compatible with mitotic cell cycle progression of spermatogonia during the first wave of spermatogenesis, but can cause zygotene to pachytene arrest in early meiosis due to multiple defects including enhanced DNA double-strand breaks, aberrant histone modifications and altered mRNA transcriptome. Our data not only validate those from global Miwi2 KO studies, but also suggest that MIWI2 and MIWI2-associated piRNAs have functions beyond TE suppression. Cell Death and Differentiation (2014) 21, 783-796; doi:10.1038/cdd.2014; published online 24 January 2014The Argonaute family of proteins contains two subclasses, argonaute (AGO) and p-element induced wimpy testis (PIWI), both of which are highly conserved from plants to animals. 1The AGO subfamily members (for example, AGO1-4) are expressed ubiquitously in a wide range of tissues/cell types and function through associations with miRNAs and siRNAs.

A Novel Class of Somatic Small RNAs Similar to Germ Cell Pachytene PIWI-interacting Small RNAs

“…In comparison with the somatic tissues, testis samples had the largest decrease in expression between KOs and controls. This is possibly due to the decrease in germ cell content in KO testis, a phenomenon observed previously in MIWI2 KO lines (19). Despite the fact that there was some differential pilRNA expression between control and KO lines in all somatic tissues, pilRNAs were still detected at variable levels, suggesting that their biogenesis in somatic cells does not rely on the known piRNA biogenetic pathways.…”

“…Mov10l1 universal knock-out mice were purchased from the Jackson Laboratory. Miwi2 knock-out mice were generated as described (19).…”

Exploiting RNA-sequencing data from the porcine testes to identify the key genes involved in spermatogenesis in Large White pigs

“…PIWIL4 belongs to the Argonaute family of proteins, which function in development and maintenance of germline stem cells (Aravin et al, 2008). Conditional inactivation of PIWIL4 (MIWI2) reveals that PIWIL4 (MIWI2) is only essential for prospermatogonial development in mice (Bao et al, 2014). Mouse Piwi-like proteins associate with piRNAs of characteristic size-26 nt for MILI, 28 nt for PIWIL4 (MIWI2), and 30 nt for MIWI.…”

UPF2, a nonsense-mediated mRNA decay factor, is required for prepubertal Sertoli cell development and male fertility by ensuring fidelity of the transcriptome

“…The next day, testicular samples were washed in 10% sucrose solution three times for 1 h each and then embedded in OCT (Sakura Finetek) medium (50% OCT plus 10% sucrose). Cryosections (8 µm) were subjected to indirect immunofluorescence staining as described (Bao et al, 2014). Antibodies used were: WT1 (rabbit, 1:50; Santa Cruz, sc-192), purified anti-VASA/DDX4 (human, 1:500; BD, 560189), GCNA (rat, 1:10; a kind gift from Dr George Enders, University of Kansas Medical Center, Kansas City, KS, USA) (Enders and May, 1994), SOHLH1 (rabbit, 1:200; a kind gift from Dr Aleksandar Rajkovic, Magee-Womens Research Institute, Pittsburgh, PA, USA) (Suzuki et al, 2012) and CYP17 (rabbit, 1:100; a kind gift from Dr A. J. Conley, University of California, Davis, CA, USA) (Peterson et al, 2001).…”

“…Paraffin sections (5 µm) were subjected to a standard Hematoxylin and Eosin staining protocol using an automated histology station (Leica Autostainer XL Staining System ST5010) as described (Bao et al, 2014).…”