Genomic stability of mouse spermatogonial stem cells in vitro.

Germline mutations underlie genetic diversity and species evolution. Previous studies have assessed the theoretical mutation rates and spectra in germ cells mostly by analyzing genetic markers and reporter genes in populations and pedigrees. This study reported the direct measurement of germline mutations by whole-genome sequencing of cultured spermatogonial stem cells in mice, namely germline stem (GS) cells, together with multipotent GS (mGS) cells that spontaneously dedifferentiated from GS cells. GS cells produce functional sperm that can generate offspring by transplantation into seminiferous tubules, whereas mGS cells contribute to germline chimeras by microinjection into blastocysts in a manner similar to embryonic stem cells. The estimated mutation rate of GS and mGS cells was approximately 0.22 × 10-9 and 1.0 × 10-9 per base per cell population doubling, respectively, indicating that GS cells have a lower mutation rate compared to mGS cells. GS and mGS cells also showed distinct mutation patterns, with C-to-T transition as the most frequent in GS cells and C-to-A transversion as the most predominant in mGS cells. By karyotype analysis, GS cells showed recurrent trisomy of chromosomes 15 and 16, whereas mGS cells frequently exhibited chromosomes 1, 6, 8, and 11 amplifications, suggesting that distinct chromosomal abnormalities confer a selective growth advantage for each cell type in vitro. These data provide the basis for studying germline mutations and a foundation for the future utilization of GS cells for reproductive technology and clinical applications.

Mouse GTSF1 is an essential factor for secondary piRNA biogenesis.

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.

PARI Regulates Stalled Replication Fork Processing To Maintain Genome Stability upon Replication Stress in Mice.

DNA replication is frequently perturbed by intrinsic, as well as extrinsic, genotoxic stress. At damaged forks, DNA replication and repair activities require proper coordination to maintain genome integrity. We show here that PARI antirecombinase plays an essential role in modulating the initial response to replication stress in mice. PARI is functionally dormant at replisomes during normal replication, but upon replication stress, it enhances nascent-strand shortening that is regulated by RAD51 and MRE11. PARI then promotes double-strand break induction, followed by new origin firing instead of replication restart. Such PARI function is apparently obstructive to replication but is nonetheless physiologically required for chromosome stability in vivo and ex vivo Of note, Pari-deficient embryonic stem cells exhibit spontaneous chromosome instability, which is attenuated by differentiation induction, suggesting that pluripotent stem cells have a preferential requirement for PARI that acts against endogenous replication stress. PARI is a latent modulator of stalled fork processing, which is required for stable genome inheritance under both endogenous and exogenous replication stress in mice.

Roles of MIWI, MILI and PLD6 in small RNA regulation in mouse growing oocytes.

The mouse PIWI-interacting RNA (piRNA) pathway produces a class of 26-30-nucleotide (nt) small RNAs and is essential for spermatogenesis and retrotransposon repression. In oocytes, however, its regulation and function are poorly understood. In the present study, we investigated the consequences of loss of piRNA-pathway components in growing oocytes. When MILI (or PIWIL2), a PIWI family member, was depleted by gene knockout, almost all piRNAs disappeared. This severe loss of piRNA was accompanied by an increase in transcripts derived from specific retrotransposons, especially IAPs. MIWI (or PIWIL1) depletion had a smaller effect. In oocytes lacking PLD6 (or ZUCCHINI or MITOPLD), a mitochondrial nuclease/phospholipase involved in piRNA biogenesis in male germ cells, the piRNA level was decreased to 50% compared to wild-type, a phenotype much milder than that in males. Since PLD6 is essential for the creation of the 5΄ ends of primary piRNAs in males, the presence of mature piRNA in PLD6-depleted oocytes suggests the presence of compensating enzymes. Furthermore, we identified novel 21-23-nt small RNAs, termed spiRNAs, possessing a 10-nt complementarity with piRNAs, which were produced dependent on MILI and independent of DICER. Our study revealed the differences in the biogenesis and function of the piRNA pathway between sexes.

GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

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.