Sexual dimorphism (SDM) is regulated by sex chromosomes, yet the specific contribution of individual genes remains unclear. To address this, we conducted a comprehensive phenotyping analysis to investigate the roles of Y chromosome-linked genes in SDM formation in mice. Using C57BL/6J mice, we identified 49 SDM traits across 14 biological systems. To assess gene-specific effects, we generated knockout (KO) mice for 10 unique Y-linked genes using CRISPR/Cas9. As expected, Sry KO resulted in feminization of most SDM traits, including gonadal sex. However, certain body size-related traits remained male-like, suggesting the involvement of additional Y-linked genes. Consistently, KOs of Uty and Usp9y significantly altered traits related to body and organ size. We further applied a dimensionality reduction approach to quantitatively capture SDM variation at the individual level, enabling visualization of phenotypic shifts in each KO mouse. Our findings demonstrate that non-Sry Y-linked genes contribute to SDM and introduce a generalizable framework for quantifying sex differences across individuals and species.

Somatic cell nuclear transfer (SCNT) in non-human primates is highly inefficient, as many reconstructed embryos arrest during preimplantation development. Here, we provide a protocol for performing SCNT in the common marmoset. We describe steps for oocyte collection, in vitro maturation, enucleation, and nuclear transfer using fibroblast or cumulus donors. We then detail procedures for oocyte activation, triple epigenetic enhancement, and embryo culture up to the blastocyst stage.For complete details on the use and execution of this protocol, please refer to Matoba et al.1

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.

Organ culture systems enabling in vitro spermatogenesis from neonatal mouse testes exist, but differentiation from fetal testes shortly after sex determination remains unsuccessful. Here, we report the in vitro generation of fertile haploid cells from E12.5 fetal testes. While optimizing in vitro spermatogenesis protocols for neonatal testes, we find that supplementing the culture medium with reverse transcriptase inhibitors (RTIs) significantly improves the efficiency of spermatogenesis, by suppressing retrotransposon activity and protecting genomic integrity. Applying this approach, we successfully induce spermatogenesis through to the elongating spermatid by culturing E12.5 fetal testes under hypoxic conditions in RTI-supplemented medium. Notably, microinsemination using in vitro-derived spermatids produces healthy and fertile offspring, confirming their functional competence. These findings demonstrate the faithful in vitro recapitulation of testicular development and complete spermatogenesis from an early fetal stage, providing a valuable platform for investigating early germ cell development and reproductive biology.

Male infertility often results from impaired interactions between germ cells and Sertoli cells. While in vitro fertilization and intracytoplasmic sperm injection are widely used, their success depends on the presence of haploid germ cells. Gene therapy remains challenging due to concerns about germline transmission. The mRNA offers a safer option, as its short-life reduces this risk. Here, we show that mRNA delivery into mouse testes restores fertility in a genetic model of infertility. Injected mRNA was specifically expressed in Sertoli cells; although it triggered an innate immune response, spermatogenesis resumed without major side effects. Delivery of naked Cldn11 mRNA into Cldn11-deficient mice, which have meiotic defects due to defective blood-testis barrier, allowed progression from spermatocytes to spermatids. Fertile offspring with normal imprinting were produced via microinsemination. These findings demonstrate the potential of mRNA-based therapy for treating male infertility by targeting testicular somatic cells, without introducing genetic material into the germline.

The International Mouse Phenotyping Consortium (IMPC) has established a large-scale functional genomics resource by systematically generating and phenotyping knockout mouse lines, linking gene function to mammalian phenotypes. However, interpreting disease-associated non-coding variants remains particularly challenging due to their abundance, context-dependent activity, and the complexity of gene regulation. Genome-wide association studies (GWAS) have shown that many disease-associated loci map to non-coding regions. In addition, recent large-scale consortia have catalogued millions of candidate cis-regulatory elements (CREs) across mammalian genomes; however, predicting which elements contribute to disease-relevant gene regulation and organismal phenotypes remains difficult. Together, these observations highlight disease-relevant CREs as an important but still underexplored component of human disease mechanisms. In this white paper, we outline strategies to address this challenge: (i) prioritization of disease-relevant candidate CREs, (ii) genome editing in mice to functionally evaluate CREs, and (iii) the establishment of interdisciplinary working groups. By extending its activities beyond protein-coding sequences, the IMPC has a unique opportunity to define the functional and phenotypic impact of disease-relevant CREs in vivo at scale, thereby improving our understanding of how non-coding regulatory elements contribute to mammalian phenotypes and human disease.

Retroelement silencing factor 1 (Resf1) is involved in retroelement silencing in cooperation with H3K9 methyltransferase SETDB1 by regulating H3K9 methylation in mouse embryonic stem cells (mESCs). However, it remains unknown whether Resf1 functions in retroelement silencing in vivo and has a role in development. Here, we established Resf1-deficient mice, which exhibit developmental delay, partial embryonic lethality, and placental defects. Notably, retroelements are also upregulated in the Resf1-deficient placenta, correlating with increased expression of nearby genes. To further assess whether Resf1 functions within the trophoblast lineage, we generate Resf1-deficient trophoblast stem cell (TSC) lines. Both undifferentiated TSCs and differentiated TSCs (D-TSCs) display increased retroelement expression along with elevated levels of genes associated with placental development. Moreover, Resf1-deficient TSCs exhibit compromised maintenance of H3K9me3 domains in a manner independent of SETDB1. Collectively, our findings reveal that Resf1 plays multifaceted roles beyond retroelement silencing, underscoring its importance in development and its critical function in trophoblast lineage regulation.

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.

Intracytoplasmic sperm injection (ICSI) is widely used to treat human infertility, yet its effects on offspring fertility remain unclear. As infertile gametes are used in human ICSI procedures, it has remained unclear whether the procedure per se influences fertility of the resulting offspring. Here, we report on increased abortion rates and the induction of immunotolerance breakdown in mice following ICSI. Although sperm concentration was normal, sperm from ICSI-derived males showed higher curvilinear velocity and greater amplitude of lateral head displacement, suggesting a potentially increased fertilization capacity. However, when ICSI-produced males were naturally mated with wild-type females, 27.1% of the fetuses were aborted, compared to a loss of 7.5% of fetuses in control pregnancies, despite identical H2 haplotypes. Analysis of the placenta revealed infiltrating macrophages and granulocytes, as well as increased levels of 8-oxoguanine. This was accompanied by activation of innate immunity and significant downregulation of microRNAs located in the Dlk1-Dio3 microRNA cluster. Several organs of the grand-offspring exhibited inflammation. Therefore, ICSI procedure using wild-type gametes impairs the fertility of ICSI-derived male offspring by activating innate immunity during pregnancy.

Mouse embryonic stem cell (ESC) cloning via nuclear transfer is a potential platform for direct generation of genetically modified mice. However, the low G1 cell population in ESCs hampers their broader use in nuclear transfer, as S-phase donors induce 1-cell arrest in reconstructed oocytes. This study aimed to enrich the G1-phase population of ESCs to improve ESC-based cloning efficiency. We demonstrated that EB3 ESCs derived from the 129/Ola strain and C57BL/6×129/Sv hybrid ESCs exhibited superior traits for nuclear transfer in terms of cleavage rate and blastocyst formation but showed reduced efficiency in development to term. Transcriptomic analyses revealed that this failure was likely due to aberrant imprinted gene expression in critical clusters, such as H19-Igf2 or Dlk1-Dio3. In contrast, although cloned offspring were successfully generated at a promising rate (approximately 6%) using EGR-R01 (BDF1×129/Sv) ESCs, early cleavage to the 2-cell embryo was inefficient. To address this, we induced cell cycle synchronization by culturing ESCs to overconfluency, which markedly increased the G1 proportion, as confirmed via FACS analysis. This alteration led to a significant improvement in first cleavage efficiency without compromising the developmental potential of ESC-derived cloned embryos to live birth. Overall, our study highlights the importance of both cell cycle synchronization and imprinting maintenance in improving ESC cloning efficiency to facilitate broad applications, including the direct generation of genetically modified mice.

Golden hamsters (Mesocricetus auratus) have been extensively used in biomedical research. With the advent of genome-editing technology, it is now possible to generate gene-knockout hamsters, providing unique research models that cannot be achieved with mice or rats. Therefore, the development of cryopreservation techniques for hamster embryos is in high demand. In this study, we present a simplified vitrification protocol for hamster embryo preservation. In vivo-derived 8-cell or morula embryos (Day 3) were vitrified using Cryotop in modified HECM-3 medium containing ethylene glycol, DMSO, and sucrose. After warming, the embryos were transferred into the uteri of Day 3-pregnant females with a different coat color. The results showed that 21-26% of the transferred embryos developed to the term. The experiments were conducted in a conventional laboratory setting, avoiding direct light exposure. Given the reproducibility of our vitrification protocol, it has broad applicability in laboratories that use hamsters.

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. Abstract: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.

A limited number of female germ cells support reproduction in many mammals. The follicle, composed of oocytes and supporting granulosa cells, forms the basis of oogenesis. Crosstalk between oocytes and granulosa cells is essential for the formation, dormancy, re-awakening, and maturation of oocytes. The oocyte expresses c-KIT and growth differentiation factor-9 (GDF-9), which are major factors in this crosstalk. The downstream signalling pathways of c-KIT and GDF-9 have been well-documented; however, their intra-oocyte trafficking pathway remains unclear. Our study reveals that the exocyst complex, a heterotetrameric protein complex important for tethering in vesicular transport, is important for proper intra-oocyte trafficking of c-KIT and GDF9 in mice. We found that depletion of oocyte-specific EXOC1, a component of the exocyst complex, impaired oocyte re-awakening and cyst breakdown, and inhibited granulosa cell proliferation during follicle growth. The c-KIT receptor is localised on the oocyte plasma membrane. The oocyte-specific Kit conditional knockout mice were reported to exhibit impaired oocyte re-awakening and reduced oocyte cyst breakdown. GDF9 is a protein secreted extracellularly in the oocyte. Previous studies have shown that Gdf9 knockout mice impaired proliferation and granulosa cell multilayering in growing follicles. We found that both c-KIT and GDF9 abnormally stuck in the EXOC1-depleted oocyte cytoplasm. These abnormal phenotypes were also observed in oocytes depleted of exocyst complex members EXOC3 and EXOC7. These results clearly show that the exocyst complex is essential for proper intra-oocyte trafficking of c-KIT and GDF9. Inhibition of this complex causes complete loss of female fertility in mice. Our findings build a platform for research related to trafficking mechanisms of vital crosstalk factors for oogenesis.

A definitive understanding of intrinsic functions of endogenous melatonin may require genetic manipulation or modification of its synthesizing enzymes. Here, we established Syrian hamsters, a seasonal mammal, carrying loss of function of aralkylamine N-acetyltransferase (AANAT), a rate-limiting enzyme in melatonin biosynthesis. Mutants showed a normal circadian period but an accelerated entrainment to rescheduled light-dark cycles. We next focused on the role of melatonin in autumn/winter anticipation, given the strict increase in levels during short days. On exposure to cold after habituation to short days, all controls maintained normal core body temperature (T b), whereas many mutants showed a decrease in T b. Food shortage after this cold exposure induced hibernation in all controls and mutants; however, all mutants failed to continue a normal hibernation cycle, probably due to impaired T b elevation during arousal from deep hibernation. These failures were accompanied by a decreased volume of lipid droplets in the interscapular brown adipose tissue (iBAT). Histological analyses of the pars tuberalis suggested a defect in photoperiodic responsiveness in mutants. Taken together, these findings demonstrate that defective photoperiodic responsiveness caused delayed remodeling of the iBAT in mutants and that sudden exposure to autumn/winter conditions caused severe defects in T b homeostasis and interbout arousal, in both of which iBAT-mediated nonshivering thermogenesis plays a major role. AANAT-mediated melatonin biosynthesis appears to be indispensable to the survival of wild seasonal mammals in natural settings.

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: Slc39a10flox/flox Gdf9Cre/+). 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.

Spermatogonial stem cells (SSCs) colonize nonablated recipient testes. Autotransplantation of SSCs is expected to restore fertility in boys who become infertile due to cancer therapy. However, since the number of SSCs recovered from testis biopsies is small, increasing SSC number is a prerequisite. Additionally, residual spermatogenesis in the recipient testes may hinder colonization. Here, we evaluated the effects of SSC culture and endogenous spermatogenesis in syngeneic and allogeneic recipient mouse testes. While cultured SSCs colonized chemically-castrated mature testes, they showed limited colonization in nonablated testes. However, successful colonization occurred in immature nonablated testes. Although cultured SSCs from DBA/2 mice failed to colonize mature C57BL/6 testes, they colonized immature C57BL/6 testes without immunoconditioning. Offspring were produced via microinsemination using DBA/2 sperm or elongated spermatids generated in C57BL/6 testis. Therefore, the recipient's age is crucial for SSC autotransplantation, and our results also underscore dramatic changes in the immunological environment during testis maturation.

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 foetuses. 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 INTERESTS: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.

Central functions of histone modifications in germ cell and embryonic development have been documented. Accumulating evidence suggests that oocytes possess unique profiles of histone modifications, among which histone H3 lysine 4 trimethylation (H3K4me3) is broadly spread on the mouse oocyte chromosomes at the metaphase II (MII) stage, unlike later embryonic stages. However, the characteristics and developmental roles of H3K4me3 on MII chromosomes are unclear. Here, we discovered that H3K4me3 was abundantly localized on some of the MII oocyte chromosomes facing the cortical side. Using multicolor FISH and CRISPR-Sirius-based labeling of chromosomes, we revealed that the X chromosome tended to be localized at the cortical side with strong H3K4me3 signals. Anchoring oocyte chromosomes to the cortex may play a role in the asymmetric H3K4me3 distribution. Furthermore, we found that the forced removal of H3K4me3 through the overexpression of a specific lysine demethylase in MII oocytes resulted in abnormal chromosome-spindle structure and impaired preimplantation development after in vitro fertilization. These findings highlight the developmental function of H3K4me3 in transcriptionally silent MII oocytes.

The common marmoset (Callithrix jacchus) is a genetically modifiable non-human primate increasingly used in biomedical research. Here, we established a method for deriving embryonic stem cells (ESCs) from blastocysts generated by somatic cell nuclear transfer (SCNT) in the marmoset. Injection of histone demethylase Kdm4d mRNA enabled efficient reprogramming of somatic nuclei, allowing blastocyst formation in 14.5% from fibroblasts. Combining this method with a G9a/EHMT2 histone methyltransferase inhibitor improved blastocyst quality and allowed derivation of nuclear transfer ESCs (ntESCs), including wild-type and GFP-transgenic lines. These ntESCs exhibited normal karyotypes and pluripotency. Nuclear and mitochondrial DNA analyses confirmed their nuclear donor origin and cytoplasmic inheritance from recipient oocytes. Transcriptome analysis identified abnormally expressed genes in ntESCs present in a line-dependent and independent manner, suggesting partial reprogramming resistance. Our study establishes a marmoset SCNT method enabling derivation of ntESCs and provides a new platform for preserving and engineering marmoset genetic resources.