Satomi Kuramochi-Miyagawa

Germ cells can give rise to teratomas and express pluripotency-associated genes, yet they remain intrinsically resistant to reprogramming by Yamanaka factors. The mechanisms governing this resistance are poorly understood. Here we identify PIWIL2 as a key suppressor of pluripotency in the male germline. While spermatogonial stem cells (SSCs) normally lack SOX2 expression, Piwil2 - but not Piwil4 -deficient SSCs aberrantly express SOX2 and readily acquire pluripotency upon concurrent loss of Dmrt1 and Trp53 . piRNAs associated with the Sox2 locus are detected in embryonic gonocytes but not in SSCs. Piwil2 deficiency reduces DNA methylation at the Sox2 promoter and increases DMRT1 and the RNA-binding protein DAZL, which jointly restrict Sox2 expression. These findings reveal a previously unrecognized role of PIWIL2 in repressing germline pluripotency and underscore the importance of coordinated transcriptional and translational regulation in maintaining germ cell identity.

Background: Adenosine deaminase domain containing 2 (ADAD2) is a testis‐specific protein composed of a double‐stranded RNA binding domain and a non‐catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive.Objectives: This study aimed to investigate the cause of male sterility inAdad2mutant mice and to understand the molecular functions of ADAD2.Materials and methods: Adad2homozygous mutant mouse lines,Adad2^–/–andAdad2^Δ/Δ, were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co‐immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2‐interacting proteins in mouse testes. RNA‐sequencing analyses were carried out to analyze the transcriptome and PIWI‐interacting RNA (piRNA) populations in wildtype andAdad2mutant testes.Results: Adad2^–/–andAdad2^Δ/Δmice exhibit male‐specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA‐binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co‐localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster‐derived pachytene piRNAs, and increases expression of ping‐pong‐derived piRNAs.Discussion and conclusion: In collaboration with RNF17 and other RNA‐binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

PIWI-interacting RNAs (piRNAs), which are germ cell-specific small RNAs, are essential for spermatogenesis. In fetal mouse germ cells, piRNAs are synthesized from sense and antisense RNAs of transposable element sequences for retrotransposon silencing. In a previous study, we reported that transgenic mice expressing antisense-Dnmt3L under the control of the Miwi2 promoter (Tg-Miwi2P-asDnmt3L) exhibited piRNA-mediated DNMT3L down-regulation. In this study, two transgene integration loci (B3 and E1) were identified on chromosome 18 of the Tg-Miwi2P-asDnmt3L mice; these loci were weak piRNA clusters. Crossbreeding was performed to obtain mice with the transgene cassette inserted into a single locus. DNMT3L was silenced and spermatogenesis was severely impaired in mice with the transgene cassette inserted at the B3 locus (Tg-B mice). In contrast, spermatogenesis in mice bearing the transgene at the E1 locus (Tg-E mice) was normal. The number of piRNAs for Dnmt3L in Tg-B mice was eightfold higher than that in Tg-E mice. Therefore, both gene silencing and impaired spermatogenesis depended on the transgene copy number rather than on the insertion loci. Additionally, the endogenous Dnmt3L promoter was not methylated in Tg mice, suggesting that Dnmt3L silencing was caused by post-transcriptional gene silencing. Based on these data, we discuss a piRNA-dependent gene silencing mechanism against novel gene insertions.

PIWI-interacting RNAs (piRNAs), a subclass of germ cell-specific noncoding small RNAs, are essential for de novo DNA methylation of retrotransposon genes in embryonic testes. PIWIL2/MILI, one of three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably via piRNA, and normal spermatogenesis. In vitro analysis using germline stem cells (GS cells) revealed that glycerol-3-phosphate acyltransferase 2 (GPAT2), which is a mitochondrial outer membrane protein involved in generation of lysophosphatidic acid (LPA) and highly expressed in testes, plays important roles in spermatogenesis. Namely, GPAT2 binds to PIWIL2 and is closely involved in the biogenesis of piRNAs; this process is independent of its enzymatic activity on LPA. However, GS cells recapitulate only a limited phase of spermatogenesis and the biological functions of GPAT2 remain largely unknown. In this study, we generated GPAT2-deficient mice and conducted comprehensive analyses. The deficient mice showed defective piRNA production and subsequent de-silencing of IAP and Line-1 retrotransposons in fetal testes. In addition, apoptosis of pachytene spermatocytes was observed. These abnormalities were all common to the phenotype of PIWIL2-deficient mice, in which piRNA production was impaired. GPAT2-deficient mice exhibited apoptosis in spermatogonia at the neonatal stage, which was not observed in PIWIL2-deficient mice. These data show that GPAT2 plays a critical role in preventing apoptosis in spermatogonia.

PIWI‐interacting RNAs (piRNAs) are germ cell‐specific small RNAs essential for retrotransposon gene silencing and male germ cell development. In piRNA biogenesis, the endonuclease MitoPLD/Zucchini cleaves long, single‐stranded RNAs to generate 5′ termini of precursor piRNAs (pre‐piRNAs) that are consecutively loaded into PIWI‐family proteins. Subsequently, these pre‐piRNAs are trimmed at their 3′‐end by an exonuclease called Trimmer. Recently, poly(A)‐specific ribonuclease‐like domain‐containing 1 (PNLDC1) was identified as the pre‐piRNA Trimmer in silkworms. However, the function of PNLDC1 in other species remains unknown. Here, we generate Pnldc1 mutant mice and analyze small RNAs in their testes. Our results demonstrate that mouse PNLDC1 functions in the trimming of both embryonic and post‐natal pre‐piRNAs. In addition, piRNA trimming defects in embryonic and post‐natal testes cause impaired DNA methylation and reduced MIWI expression, respectively. Phenotypically, both meiotic and post‐meiotic arrests are evident in the same individual Pnldc1 mutant mouse. The former and latter phenotypes are similar to those of MILI and MIWI mutant mice, respectively. Thus, PNLDC1‐mediated piRNA trimming is indispensable for the function of piRNAs throughout mouse spermatogenesis.

The piRNA pathway is a piRNA‐guided retrotransposon silencing system which includes processing of retrotransposon transcripts by PIWI‐piRNAs in secondary piRNA biogenesis. Although several proteins participate in the piRNA pathway, the ones crucial for the cleavage of target RNAs by PIWI‐piRNAs have not been identified. Here, we show that GTSF1, an essential factor for retrotransposon silencing in male germ cells in mice, associates with both MILI and MIWI2, mouse PIWI proteins that function in prospermatogonia. GTSF1 deficiency leads to a severe defect in the production of secondary piRNAs, which are generated from target RNAs of PIWI‐piRNAs. Furthermore, in Gtsf1 mutants, a known target RNA of PIWI‐piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective. Our findings indicate that GTSF1 is a crucial factor for the slicing of target RNAs by PIWI‐piRNAs and thus affects secondary piRNA biogenesis in prospermatogonia. Synopsis Mouse GTSF1 is essential for MILI‐directed secondary piRNA biogenesis in prospermatogonia and associates with the MIWI2 complex probably to transcriptionally silence its targets. Mouse GTSF1 associates with MILI, MIWI, and MIWI2 complexes. Mouse GTSF1 deficiency leads to loss of MIWI2‐bound piRNAs and to a severe defect in secondary piRNA biogenesis, whereas it has no impact on primary piRNA biogenesis. In Gtsf1 mutants, a known target RNA of PIWI‐piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective.

PiRNA (PIWI-interacting RNA) is a germ cell–specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.

VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.