Repeated implantation failure is a major cause of infertility among healthy women. Uterine β-catenin (CTNNB1) plays a critical role in implantation. However, the role of embryonic CTNNB1 during implantation remains unclear. We addressed this topic by analyzing mice carrying Ctnnb1-deficient (Ctnnb1Δ/Δ) embryos. Ctnnb1Δ/Δ embryos were produced by intercrossing mice bearing Ctnnb1-deficient eggs and sperms. We found that Ctnnb1Δ/Δ embryos developed to the blastocyst stage; thereafter, they were resorbed, leaving empty decidual capsules. Moreover, leukemia inhibitory factor, a uterine factor essential for implantation, was undetectable in Ctnnb1Δ/Δ blastocysts. Furthermore, CDX2, a transcription factor that determines the fate of trophectoderm cells, was not observed in Ctnnb1Δ/Δ blastocysts. Intrauterine injection with uterine fluids (from control mice) and recombinant mouse leukemia inhibitory factor proteins rescued the uterine response to Ctnnb1Δ/Δ blastocysts. These results suggest that embryonic CTNNB1 is required for the secretion of blastocyst-derived factor(s) that open the implantation window, indicating that the uterine response to implantation can be induced using supplemental materials. Therefore, our results may contribute to the discovery of a similar mechanism in humans, leading to a better understanding of the pathogenesis of repeated implantation failure.

To generate an effective embryo prediction model and identify a non-invasive evaluation method by analyzing microRNAs (miRNAs) in embryo culture medium. Analysis of microRNA profiles from spent culture medium of blastocysts with good morphology that did or did not result in pregnancy. Clinical and experimental research. Sixty patients who underwent thawed embryo transfer of blastocysts after intracytoplasmic sperm injection. None. The association of miRNA abundance levels secreted by blastocysts in culture medium and implantation success. Our RNA sequencing analysis found a total of 53 differentially expressed miRNAs in the culture media of pregnancy and non-pregnancy groups. Twenty-one miRNAs were analyzed for their potential to predict implantation success. Eight miRNAs (hsa-miR-191-5p, hsa-miR-320a, hsa-miR-92a-3p, hsa-miR-509-3p, hsa-miR-378a-3p, hsa-miR-28-3p, hsa-miR-512-5p, and hsa-miR-181a-5p) were further extracted from the results of a logistic regression analysis of qPCR Ct values. A prediction model for high-quality blastocysts was generated using the eight miRNAs, with an average accuracy of 0.82 by 5-fold cross validation. We isolated blastocyst miRNAs that may predict implantation success and created a model to predict viable embryos. Increasing the number of investigated cases and further studying the effect of each miRNA on embryonic development is needed to refine the miRNA-based predictive model.

Reproductive capacity in women starts to decline beyond their mid-30s and pregnancies in older women result in higher rates of miscarriage with aneuploidy. Age-related decline in fertility is strongly attributed to ovarian aging, diminished ovarian reserves, and decreased developmental competence of oocytes. In this review, we discuss the underlying mechanisms of age-related decline in oocyte quality, focusing on oxidative stress (OS) in oocytes. The primary cause is the accumulation of spontaneous damage to the mitochondria arising from increased reactive oxygen species (ROS) in oocytes, generated by the mitochondria themselves during daily biological metabolism. Mitochondrial dysfunction reduces ATP synthesis and influences the meiotic spindle assembly responsible for chromosomal segregation. Moreover, reproductively aged oocytes produce a decline in the fidelity of the protective mechanisms against ROS, namely the ROS-scavenging metabolism, repair of ROS-damaged DNA, and the proteasome and autophagy system for ROS-damaged proteins. Accordingly, increased ROS and increased vulnerability of oocytes to ROS lead to spindle instability, chromosomal abnormalities, telomere shortening, and reduced developmental competence of aged oocytes.

Mitochondria are the powerhouses of eukaryotic cells and their positioning contributes to fertilization and early developmental processes. We report that sperm fusion triggers Ca2+ oscillations and mitochondrial movement toward fused sperm (mitochondrial chemotaxis) in mouse eggs. Mitochondria functioned in Ca2+ storage and were colocalized with endoplasmic reticulum (ER) during Ca2+ oscillations. Mitochondria then moved toward the fused sperm. Sperm extracts lacking nuclei induced Ca2+ oscillations, but did not promote mitochondrial chemotaxis. Our results suggest that sperm fusion motivates Ca2+ 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.

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.

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.

Autophagic recycling of cell parts is generally termed as the opposite of cell death. Here, we explored the relation between cell death and autophagy by examining granulosa cell layers that control oocyte quality, which is important for the success of fertilization. Granulosa cell layers were collected from infertile women and morphologically divided into four types, viz., mature (MCCs), immature (ICCs), and dysmature cumulus cells (DCCs), and mural granulosa cells (MGCs). Microtubule-associated protein light chain 3 (LC3), which is involved in autophagosome formation, was expressed excessively in DCCs and MGCs, and their chromosomal DNA was highly fragmented. However, autophagy initiation was limited to MGCs, as indicated by the expression of membrane-bound LC3-II and autophagy-related protein 7 (ATG7), an enzyme that converts LC3-I to LC3-II. Although pro-LC3 was accumulated, autophagy was disabled in DCCs, resulting in cell death. Our results suggest the possibility that autophagy-independent accumulation of pro-LC3 proteins leads to the death of human granulosa cells surrounding the oocytes and presumably reduces oocyte quality and female fertility.

The ubiquitin-proteasome system, which is initiated by a single ubiquitin-activating enzyme E1 (UBE1), is involved in male reproduction via spermatogenesis and function in mammals. Here we explored the influence of UBE1-specific inhibitor, 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (pyrazone-41 or PYR-41) in female reproduction. UBE-1 was detected by immunoblotting and immunocytochemistry in mouse eggs and was localized mainly under the egg plasma membrane. PYR-41 pretreatment suppresses the development of eggs into two-cell embryos. Specifically, pretreatment retarded sperm enlargement and meiotic chromosomal division after sperm-egg fusion. PYR-41 pretreatment disturbed β-catenin, a well-known target protein for ubiquitination, localization under the egg plasma membrane and on spindle microtubules in wild-type eggs. Otherwise, PYR-41 treatment had no effect on the two-cell development of eggs lacking β-catenin. Our results raise the possibility that inhibition of the ubiquitin-proteasome system suppresses sperm enlargement through impaired β-catenin-mediated mechanism.