Atsuo Ogura

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.

In vitro fertilization (IVF) and artificial insemination (AI) using frozen–thawed rat sperm have been established to enable the production of viable offspring. However, the current standard cryopreservation solution, which contains chicken egg yolk and Orvus ES Paste (OEP; sodium lauryl sulfate and other undefined components), poses the risk of microbial contamination and limits the experimental reproducibility of IVF and AI. In this study, we developed a chemically defined cryopreservation solution that does not contain chicken egg yolk or OEP and evaluated its efficacy in IVF. After examining the effects of various cryoprotective agents on sperm motility, we found that OptiPrep (a chemically defined reagent) and ethylene glycol in a lactose solution best preserved sperm motility after freezing and thawing. In addition, sericin, a cell membrane protective agent, effectively reduced damage to the plasma and acrosomal membranes of the sperm head. Although the fertilization rate of the sperm cryopreserved in a chemically defined solution (71.4%) was significantly lower than that of fresh sperm (90.5%), fertilized oocytes were successfully obtained. Approximately 60% of the fertilized oocytes developed to the blastocyst stage in vitro. Furthermore, when two-cell embryos were transferred to the oviducts of pseudopregnant females, 36% developed into offspring. Taken together, our results suggest a new strategy for the cryopreservation of rat strains using a chemically defined sperm-freezing solution, enabling the safe and reproducible production of offspring.

Cellular interactions between germ cells and gonadal somatic cells are essential for the progression of gametogenesis. Here, we report a culture method for generating fetal testicular somatic cell–like cells (fTeSLCs) from embryonic stem cells. These fTeSLCs exhibit a transcriptomic profile closely resembling that of their in vivo counterparts, including distinct cell populations corresponding to Sertoli cells and interstitial cells. For functional assessment, interstitial cell–like cells (ICLCs) and Sertoli-like cells (SerLCs) were isolated from fTeSLCs. ICLCs differentiated into Leydig cells when cocultured with testes lacking endogenous Leydig cells, thereby restoring androgenic support. SerLCs reconstituted the seminiferous epithelium following selective ablation of endogenous Sertoli cells. Both cell types supported spermatogenesis and generated spermatids reaching the elongating stage. Notably, round spermatids derived from these reconstructed systems produced viable offspring by round spermatid injection. These findings demonstrate that fTeSLCs can generate functional testicular somatic cells, providing a valuable platform for studying testis development and spermatogenesis.

In all vertebrates studied to date, a rise(s) in intracellular calcium is indispensable for successful fertilization and further embryonic development. Recent studies demonstrated that zinc is ejected to the extracellular milieu, the ‘zinc spark’, and follows the first few calcium rises of fertilization. However, the role of the zinc sparks in fertilization and development, as well as the supporting influx mechanism(s), remains unknown. In this study, we focused on zinc transporters Slc39a10/Zip10 which were expressed in mouse oocytes through follicular development and investigated the oocyte-specific deficient mice for Slc39a10/Zip10 ( Slc39a10 cKO: Slc39a10 ^flox/flox Gdf9 ^Cre/+ ). Slc39a10 mRNA or SLC39A10/ZIP10 protein was expressed throughout folliculogenesis in the oocyte or plasma membrane, respectively. The number of ovulated oocytes was examined in Slc39a10 cKO mice, and no change from the number of oocytes was observed. Slc39a10 cKO oocytes decreased zinc level in the oocytes but did not affect maturation and metaphase II spindle formation. Fertilization-induced calcium oscillations were present in Slc39a10 cKO oocytes, but zinc sparks were not observed. Despite other events of egg activation proceeding normally in Slc39a10 cKO oocytes, embryo development into 4 cells and beyond was compromised. We show here for the first time that the zinc transporter ZIP10 contributes to zinc homeostasis in oocytes and embryos, highlighting the role of labile zinc ions in early development.

Placental enlargement in somatic cell nuclear transfer–derived mice is attributed to biallelic expression of noncanonical (H3K27me3-dependent) imprinted genes owing to loss of imprinting (LOI). Here, we investigated whether a similar mechanism underlies placental enlargement in intersubspecific hybrids between BDF1 (Mus musculus domesticus) and HMI (M. m. castaneus) mice. Quantitative and allelic expression analyses revealed gene-specific LOI in (BDF1 × HMI)F1 placentas: Jade1 (Phf17) and Slc38a4 showed LOI in all placentas regardless of expression levels, whereas Gab1 and Sfmbt2 exhibited LOI only when expression levels were elevated. Notably, Jade1 and Slc38a4 also showed biallelic expression at lower levels in normal-sized (BDF1 × JF1 [M. m. molossinus])F1 placentas. Maternal knockout of Jade1, Slc38a4, Sfmbt2, or the Sfmbt2 miRNA cluster restored monoallelic expression and significantly reduced the weight of (BDF1 × HMI)F1 placentas, indicating that these genes were collectively responsible for placental enlargement in intersubspecific hybrid placentas. Transcriptomic analysis revealed that LOI of noncanonical imprinted genes occurred after implantation. These findings suggest that placental enlargement in (BDF1 × HMI)F1 hybrids is driven by overexpression of multiple noncanonical imprinted genes, resulting from LOI after implantation and additional hybrid-specific, yet unidentified, upregulation mechanisms.

In all vertebrates studied to date, a rise(s) in intracellular calcium is indispensable for successful fertilization and further embryonic development. Recent studies demonstrated that zinc is ejected to the extracellular milieu, the ’zinc spark’, and follows the first few calcium rises of fertilization. However, the role of the zinc sparks in fertilization and development, and the supporting influx mechanism(s) are unknown. In this study, we focused on zinc transporters Zip10/Slc39a10 which was expressed in mouse oocytes through follicular development, and investigated the oocyte-specific deficient mice for Zip10 (Zip10d/d: Zip10flox/flox Gdf9Cre/+). Zip10 mRNA or ZIP10 protein was expressed throughout folliculogenesis in the oocyte or plasma membrane, respectively. The number of ovulated oocytes was examined in Zip10d/d mice, and no change from the number of oocytes was observed. Zip10d/d oocytes decreased zinc level in the oocytes, but did not affect maturation and metaphase II spindles formation. Fertilization-induced calcium oscillations were present in Zip10d/d oocytes, but zinc sparks were not observed. Despite other events of egg activation proceeding normally in Zip10d/doocytes, embryo development into 4-cells and beyond was compromised. We show here for the first time that the zinc transporter ZIP10 contributes to zinc homeostasis in oocytes and embryos, highlighting the role of labile zinc ions in early development. Submission information The zinc transporter, Slc39a10/Zip10, is required for the zinc sparks of fertilization in mice.

STUDY QUESTION: At which arrest stage can spermatocytes be rescued by injection into meiotic oocytes? SUMMARY ANSWER: In mice, spermatocytes arrested at the diplotene stage, but not at the pachytene stage, can resume meiosis within immature oocytes and support full-term embryonic development. WHAT IS KNOWN ALREADY: In mice, at least some of the spermatocyte arrest mutations can be overcome by injecting spermatocytes into immature oocytes. STUDY DESIGN, SIZE, DURATION: The study was carried out from October 2019 to April 2025. Adult azoospermic mice (at 4–26 weeks of age) from nine strains carrying spermatocyte arrest mutations were used as spermatocyte donors. Adult B6D2F1 females at 9–12 weeks of age were used as oocyte donors for spermatocyte injection. Adult ICR strain pseudopregnant females at 9–12 weeks of age were used as recipients for embryo transfer experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: The most advanced stage of spermatocytes from each mutant strain was assessed by chromosome spread analysis. These most advanced spermatocytes of each strain were injected into metaphase I (MI) oocytes. About half a volume of the ooplasm had been removed from the recipient oocytes to ensure more stable chromosome behaviours during meiosis. The spermatocyte-injected oocytes were allowed to mature in vitro to the metaphase II (MII) stage, and their ooplasm was refreshed with the ooplasm from intact MII oocytes. After activation with SrCl2, the reconstructed oocytes that reached the 2-cell stage were transferred into the oviducts of pseudopregnant females. On day 19.5, recipient females were euthanized and their uteri were examined for live fetuses. MAIN RESULTS AND THE ROLE OF CHANCE: Based on spermatocyte spread analysis, sperm mutants were categorized into three classes: Class 1, arrest at mid-diplotene or later stage; Class 2, arrest at early diplotene stage; and Class 3, arrest at pachytene stage. All four Class 1 mutants could resume normal meiosis following injection into MI oocytes, as evidenced by births of normal offspring. Similarly, one of two Class 2 mutants could be rescued, but the other could not. By contrast, three Class 3 mutants did not support embryo development to term because of complete implantation failure, indicating that reconstructed embryos carried severe chromosomal aberrations. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The number of mutant strains examined was limited. Nevertheless, the findings were consistent: the more advanced the arrest stage of spermatocytes, the higher the likelihood of a successful rescue. WIDER IMPLICATIONS OF THE FINDINGS: In humans, a considerable proportion of spermatogenic arrest occurs at the primary spermatocyte stage. Spermatocyte injection might be an option to treat human male-factor infertility due to azoospermia in the future. However, numerous ethical and technical challenges remain to be addressed, and the reproductive physiological differences between mice and humans must be carefully taken into account. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science to A.O. (grant number: JP19H05758), K. I. (grant number: 23H04956), M.I. (grant number: JP23K20043) and N.O. (grant number: 25H01372), and 2023 and 2025 grants of the University of Castilla-La Mancha for stays in foreign universities and research centres to E.C.-E. The authors declare that they have no conflicts of interest.

In brief: The non-genomic factors responsible for developmental arrest in SCNT embryos remain poorly understood. Using live-cell fluorescence imaging, we revealed that autophagic activity is impaired in preimplantation SCNT embryos, possibly due to ectopic activation of the mTORC1 signaling pathway, providing new insights into cytoplasmic barriers to cloning efficiency. Activation of autophagy after fertilization is essential for mammalian embryonic development, as it supplies embryos with nutrients and energy. Somatic cell nuclear transfer (SCNT) embryos frequently exhibit developmental arrest, largely because of incomplete genomic reprogramming; however, the role of non-genomic factors remains unclear. Here, we investigated autophagy dynamics in mouse SCNT embryos using immunostaining and live-cell fluorescence imaging. In fertilized embryos, autophagy increased markedly from the late 2-cell stage and peaked at the morula stage. SCNT embryos followed a similar timeline but consistently showed reduced autophagic activity. Notably, the autophagic activity levels varied among SCNT embryos and positively correlated with their developmental potential. Attempts to enhance genomic reprogramming, including the removal of somatic histone methylation, did not restore autophagy. Instead, transcriptome analysis revealed ectopic activation of mTORC1 signaling as a likely cause of impaired autophagy. Consistently, treatment with an mTORC1 inhibitor successfully rescued autophagic activity in SCNT embryos. These findings identify a persistent autophagy defect during preimplantation development in SCNT embryos and suggest that modulation of non-genomic pathways, such as mTORC1 signaling, could improve SCNT efficiency.

The structure-specific endonuclease, XPF–ERCC1, plays a central role in DNA damage repair. This nuclease is known to be important for nucleotide excision repair, interstrand crosslink repair, and DNA double-strand repair. We found that the arginine methyltransferase, CARM1/PRMT4, is essential for XPF stabilization and maintenance of intracellular protein levels. Loss of CARM1 results in a decrease in XPF protein levels and a concomitant decrease in ERCC1 protein. A similar destabilization of XPF protein was observed in cells expressing a mutant in which XPF arginine 568 was replaced by lysine. Loss of CARM1 impaired XPF–ERCC1 accumulation at the site of damage and delayed removal of cyclobutane pyrimidine dimers by UV. As a result, CARM1-deficient cells showed increased UV sensitivity. Our results provide insight into the importance of CARM1 not only in the mechanism of XPF–ERCC1 complex stabilization but also in the maintenance of genome stability.