Shogo Matoba

Sex in mammals is unidirectionally determined by the configuration of sex chromosomes. In contrast, several species, including fishes, can switch sexes in response to environmental cues, illustrating the reproductive advantages of sexual plasticity. Thus, technologies that can bypass the unidirectional nature of mammalian sex determination could open new avenues in reproductive biology. Here, by establishing a method that enables robust Y-chromosome elimination in embryos, we provide a strategy to regulate sex in mice. We show that this approach efficiently induces male-to-female sex reversal by releasing the Y chromosome as micronuclei during early development. When integrated with optimized somatic cell nuclear transfer, it enabled the parallel production of both male and sex-reversed female mice from a single male genome source. Offspring were successfully obtained from crosses between these male and sex-reversed female clones, demonstrating that this “dual-sex cloning” strategy can initiate sexual reproduction solely from a male somatic genome. These findings highlight dual-sex cloning as a valuable platform for securing mammalian reproduction and biodiversity from limited genetic resources.

Resf1 (Retroelement silencing factor 1) 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.

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.

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.

Resf1 (Retroelement silencing factor 1) is involved in retroelement silencing cooperated 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 exhibited developmental delay, partial embryonic lethality, and placental defects. Notably, retroelements were 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 generated 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.