Published papers

An endogenous retrovirus-derived membrane protein, syncytin (SYN), contributes to placental function via trophoblast fusion. Multinuclear trophoblasts (syncytiotrophoblasts) physically and functionally mediate the interaction between fetal and maternal vessels in various ways. Suncus murinus (suncus) is a small mammalian species with a pregnancy duration of approximately 30 days, 1.5 times longer than mice. However, the molecular basis for the longer pregnancy duration is unknown. In this study, we first isolated two genes that encoded putative SYN proteins expressed in the suncus placenta, which were named syncytin-1-like proteins 1 and 2 (SYN1L1 and SYN1L2). When their expression vectors were introduced into cultured cells, suncus SYN1L2 was found to be active in cell fusion. Moreover, the SYN1L2 protein was homologous to a SYN1-like protein identified in greater mouse-eared bats (bat SYN1L) and was structurally compared with bat SYN1L and other SYN proteins, implying the presence of structural features of the SYN1L2 protein.

In both men and women, pathogenic bacteria enter the reproductive tract and cause harmful symptoms. Intrauterine and oviductal inflammation after copulation may have severe effects, such as infertility, implantation failure, oviduct obstruction, and robust life-threatening bacterial infection. Human seminal plasma is considered to be protective against bacterial infection. Among its components, Semenogelin-I/-II proteins are digested to function as bactericidal factors; however, their sequences are not conserved in mammals. Therefore, alternative antibacterial (bactericidal and/or bacteriostatic) systems may exist across mammals. In this study, we examined the antibacterial activity in the seminal plasma of mice lacking a gene cluster encoding Semenogelin-I/-II counterparts. Even in the absence of the majority of seminal proteins, antibacterial activity remained in the seminal plasma. Moreover, a combination of gel chromatography and liquid chromatography coupled with tandem mass spectrometry revealed that the prostate and testis expressed 4 protein as a novel antibacterial (specifically, bacteriostatic) protein, the sequence of which is broadly conserved across mammals. Our results provide the first evidence of a bacteriostatic protein that is widely present in the mammalian seminal plasma.

Background: Pathogenic mitochondrial (mt)DNA mutations, which often cause life-threatening disorders, are maternally inherited via the cytoplasm of oocytes. Mitochondrial replacement therapy (MRT) is expected to prevent second-generation transmission of mtDNA mutations. However, MRT may affect the function of respiratory chain complexes comprised of both nuclear and mitochondrial proteins. Methods: Based on the literature and current regulatory guidelines (especially in Japan), we analyzed and reviewed the recent developments in human models of MRT. Main findings: MRT does not compromise pre-implantation development or stem cell isolation. Mitochondrial function in stem cells after MRT is also normal. Although mtDNA carryover is usually less than 0.5%, even low levels of heteroplasmy can affect the stability of the mtDNA genotype, and directional or stochastic mtDNA drift occurs in a subset of stem cell lines (mtDNA genetic drift). MRT could prevent serious genetic disorders from being passed on to the offspring. However, it should be noted that this technique currently poses significant risks for use in embryos designed for implantation. Conclusion: The maternal genome is fundamentally compatible with different mitochondrial genotypes, and vertical inheritance is not required for normal mitochondrial function. Unresolved questions regarding mtDNA genetic drift can be addressed by basic research using MRT.

Objective: To explore a morphometric grading system for blastocysts that is associated with ongoing pregnancy. Design: Cross-sectional study. Setting: None. Patients(s): All consecutive vitrified blastocysts at our center from July 2018 to November 2021 that were transferred in single blastocyst transfer cycles until January 2022. Intervention(s): None. Main Outcome Measure(s): The ongoing pregnancy rate after a single vitrified-warmed blastocyst transfer. Interobserver agreement on morphometric values among embryologists. Result(s): Three morphometric variables (blastocyst diameter, area of inner cell mass [ICM], and the estimated trophectoderm cell count) were used to evaluate the expansion, ICM, and trophectoderm morphology. During the study period, 585 blastocysts were involved in this study. Of the 3 morphometric variables, ICM area (per 500 μm2, adjusted odds ratio, 1.19; 95% confidence interval, 1.09−1.30) and estimated trophectoderm cell count (per 10 cells, adjusted odds ratio, 1.25; 95% confidence interval, 1.12−1.39) were significantly associated with the ongoing pregnancy rate after adjustment for confounding factors. The ongoing pregnancy rate was 2.0% (1/49) with an ICM area of <2,500 μm2 and the estimated trophectoderm cell count <70. The ongoing pregnancy rate reached 47.8% (22/46) when the ICM area and the estimated trophectoderm cell count were >3,500 μm2 and >110, respectively. Interobserver agreement on the blastocyst diameter, ICM area, and the estimated trophectoderm cell count was excellent-to-good among 5 embryologists (intraclass correlation coefficients: 0.99, 0.87, and 0.91, respectively). Conclusion(s): Morphometric values of ICM and trophectoderm are promising predictors of pregnancy success. The high reproducibility suggests that the morphometric variables will contribute to identifying blastocysts with the highest developmental potential as well as those that will not result in a successful pregnancy.

In some non-mammalian eggs, the fusion of one egg and multiple sperm (polyspermy) induces a robust rise in intracellular calcium ion (Ca2+) concentration due to a shortage of inducers carried by a single sperm. Instead, one of the sperm nuclei is selected inside the egg for normal embryogenesis. Polyspermy also occurs during the in vitro fertilization of human eggs; however, the fate of such eggs is still under debate. Hence, the relationship between polyspermy and repetitive Ca2+ increases (Ca2+ oscillation) in mammals remains unknown. To address this issue, we used mouse sperm lacking extramitochondrial citrate synthase (eCS), which functions as a Ca2+ oscillation inducer; its lack causes retarded Ca2+ oscillation initiation (eCs-KO sperm). Elevated sperm concentrations normalize Ca2+ oscillation initiation. As expected, eCS deficiency enhanced polyspermy in both zona pellucida (ZP)-free and ZP-intact eggs despite producing the next generation of eCs-KO males. In conclusion, similarly to non-mammalian eggs, mouse eggs may develop normally under polyspermy conditions caused by problematic Ca2+ oscillation.

In bacteria, polymers of inorganic phosphates, particularly linear polyphosphate, are used as alternative phosphate donors for adenosine triphosphate production. A six-chain form of sodium metaphosphate, sodium hexametaphosphate (SHMP), is believed to have no physiological functions in mammalian cells. In this study, we explored the possible effects of SHMP on mammalian cells, using mouse oocytes, which are useful for observing various spatiotemporal intracellular changes. Fertilization-competent oocytes were isolated from the oviducts of superovulated mice and cultured in an SHMP-containing medium. In the absence of co-incubation with sperm, SHMP-treated oocytes frequently formed pronuclei and developed into two-cell embryos owing to the increase in calcium concentration in the cytoplasm. We discovered an intriguing role for SHMP as an initiator of calcium rise in mouse oocytes, presumably in a wide variety of mammalian cells.

Purpose: In 2017, the first guidelines for fertility preservation in cancer patients were published in Japan. However, the impact of the guidelines remains unknown. Therefore, the authors conducted a nationwide survey on cryopreservation procedures in the period from shortly before to after publication of the guidelines (2016–2019) and compared the results with our previous survey (2011–2015). The authors also surveyed reproductive specialists’ awareness of the guidelines and implementation problems. Methods: The authors sent a questionnaire to 618 assisted reproductive technology facilities certified by the Japanese Society of Obstetrics and Gynecology. Results: The authors received responses from 395 institutions (63.8%). Among them, 144 institutions conducted cryopreservation for cancer patients (vs. 126 in 2011–2015) and performed 2537 embryo or oocyte and 178 ovarian tissue cryopreservation procedures (vs. 1085 and 122, respectively). Compared with the previous period, indications were more varied and protocols for controlled ovarian stimulation were more standardized. Reproductive specialists’ interest in oncofertility was high, but many reported three main difficulties: selecting a treatment method, storing samples in the long term, and securing the necessary human resources. Conclusions: The practice of fertility preservation in cancer patients in Japan has been considerably affected by the first Japanese guidelines.

Supporting cells of oocytes, i.e., cumulus cells, control oocyte quality, which determines fertilization success. Therefore, the transformation of mature and immature cumulus cells (MCCs and ICCs, respectively) into dysmature cumulus cells (DCCs) with dead characteristics deteriorates oocyte quality. However, the molecular basis for this transformation remains unclear. Here, we explored the link between autophagic decline and cumulus transformation using cumulus cells from patients with infertility, female mice, and human granulosa cell-derived KGN cell lines. When human cumulus cells were labeled with LysoTracker probes, fluorescence corresponding to lysosomes was enhanced in DCCs compared to that in MCCs and ICCs. Similarly, treatment with the autophagy inhibitor chloroquine elevated LysoTracker fluorescence in both mouse cumulus cells and KGN cells, subsequently suppressing ovulation in female mice. Electron microscopy analysis revealed the proliferation of abnormal lysosomes in chloroquine-treated KGN cells. Conversely, the addition of an autophagy inducer, trehalose, suppressed chloroquine-driven problematic lysosomal anomalies and ameliorated ovulation problems. Our results suggest that autophagy maintains the healthy state of the supporting cells of human oocytes by suppressing the formation of lysosomes. Thus, our results provide insights into the therapeutic effects of trehalose on female fertility.

We recently demonstrated that the zygotic gene Zscan5b is involved in maintaining the genomic integrity of pluripotent stem cells (PSCs) during mitosis and that Zscan5b gene expression can prevent chromosomal abnormalities. Here, we provide a detailed protocol for the transfection of mouse embryonic stem cells (mESCs) and mouse-induced PSCs (miPSCs) with a Zscan5b constitutive expression plasmid, using the piggyBac transposon gene expression vector system. For complete details on the use and execution of this protocol, please refer to Ogawa et al. (2019).

© 2019 The Authors Zygotic genome activation (ZGA) begins after fertilization and is essential for establishing pluripotency and genome stability. However, it is unclear how ZGA genes prevent mitotic errors. Here we show that knockout of the ZGA gene Zscan5b, which encodes a SCAN domain with C2H2 zinc fingers, causes a high incidence of chromosomal abnormalities in embryonic stem cells (ESCs), and leads to the development of early-stage cancers. After irradiation, Zscan5b-deficient ESCs displayed significantly increased levels of γ-H2AX despite increased expression of the DNA repair genes Rad51l3 and Bard. Re-expression of Zscan5b reduced γ-H2AX content, implying a role for Zscan5b in DNA damage repair processes. A co-immunoprecipitation analysis showed that Zscan5b bound to the linker histone H1, suggesting that Zscan5b may protect chromosomal architecture. Our report demonstrates that the ZGA gene Zscan5b is involved in genomic integrity and acts to promote DNA damage repair and regulate chromatin dynamics during mitosis. In this article, Yamada and colleagues show that Zscan5b deficiency increases DNA stress, compromises chromosomal structure during mitosis, and leads to the development of early-stage cancers. Zscan5b deficiency may offer a murine model of human chromosomal breakage syndromes.

© 2018 The Authors Mitochondria are the powerhouses of eukaryotic cells and their positioning contributes to fertilization and early developmental processes. We report that sperm fusion triggers Ca 2+ oscillations and mitochondrial movement toward fused sperm (mitochondrial chemotaxis) in mouse eggs. Mitochondria functioned in Ca 2+ storage and were colocalized with endoplasmic reticulum (ER) during Ca 2+ oscillations. Mitochondria then moved toward the fused sperm. Sperm extracts lacking nuclei induced Ca 2+ oscillations, but did not promote mitochondrial chemotaxis. Our results suggest that sperm fusion motivates Ca 2+ oscillation-independent mitochondrial chemotaxis. This phenomenon indicates that egg mitochondria interact with sperm materials, presumably nuclear substances, and their network tethers egg and sperm nuclei at the early stage of zygote formation.

© 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a “molecular fossil” due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

© 2018, United States & Canadian Academy of Pathology. Tetraspanin CD9 is essential for sperm–egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.