Reproduction最新文献

Ensuring the timely transport of gametes and embryos within the oviduct is essential for the successful establishment of pregnancy. This study investigated the direct effect of estradiol-17β (E2) on bovine oviductal contractility and the differences in responsiveness to E2 during the estrous cycle. Bovine isthmic tissues from four estrous stages were analyzed using the Magnus method to assess contractile responses to E2 and related reagents. Protein expression of G-protein-coupled estrogen receptor 1 (GPER1) and components of the RhoA/Rho kinase (ROCK) signaling pathway were also evaluated. E2 and a GPER1 agonist significantly increased oviductal tonus at 1-4 days after ovulation. This effect was significantly suppressed by treatment with a GPER1 antagonist and a ROCK inhibitor. At 1-4 days after ovulation, both ROCK II expression and ROCK activity were elevated. E2 also enhanced phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin light chain (MLC), key downstream targets of ROCK. Before ovulation, when endogenous E2 levels peak, the expression of RND3-a ROCK inhibitor-was upregulated. The application of an RND inhibitor restored E2 responsiveness in oviductal tonus, ROCK activity, and the phosphorylation of MYPT1 and MLC in oviductal tissues before ovulation. These findings suggest that E2 directly increases oviductal tonus via GPER1 and ROCK/MYPT1/MLC activation at 1-4 days after ovulation. Differences in oviductal responsiveness to E2 during the estrous cycle appear to be mediated by the expression of ROCK and RND3. This mechanism can enable sperm transport within the oviduct at an appropriate time.

The pronuclei in human one-cell stage embryos (zygotes) contain several nucleoli called nucleolus precursor bodies (NPBs). Based on their number and distribution, it is possible to predict the developmental potential of the zygote. Recently, it has been demonstrated that the speed of NPBs movement in pronuclei may also indicate how the embryo will develop, as well as its chromosomal constitution (euploidy versus aneuploidy). These observations, however, do not elucidate the mechanisms behind these processes, and a deeper understanding will certainly be important for the more efficient production of healthy human embryos.

The female reproductive tract (FRT) is composed of distinct sections which all take part in the sperm selection processes that occur after copulation. Among the different sections, the uterotubal junction (UTJ) presents a significant obstacle to sperm and is a major site of sperm selection. This selection is based not only on motility and morphology, but on the presence and conformation of specific sperm surface proteins. In the mouse model, a disintegrin and metallopeptidase domain 3 and other sperm surface proteins have been identified as being critical for passage through the UTJ, however, the potential female counterparts of these proteins are still unknown. Our study takes a transcriptomics approach, through an RNAseq analysis of the FRT tissue following copulation in female mice in estrus, to uncover candidate sperm surface protein interactors on the UTJ epithelium. Our results showed 240 genes with higher expression specifically in the UTJ in response to the presence of semen. The UTJ exhibited an upregulation of extracellular matrix-related genes that was not found in the uterus or oviduct following copulation. A list of candidate genes was generated using further bioinformatics analysis, and two candidates, galectin 3 (LGALS3) and integrin alpha 8 (ITGA8), were selected for immunofluorescence and western blot analysis. Both LGALS3 and ITGA8 were identified on the surface of the UTJ epithelium, with a higher abundance of LGALS3 in the UTJ of copulated females compared to non-copulated animals. This suggests a role of these candidates in the selection processes occurring in the murine UTJ, with prospective future usage in the refinement of artificial reproduction technologies.

Trehalose is a disaccharide known for protecting cells against desiccation and freezing. However, mammalian cells lack endogenous trehalose transporters, limiting its intracellular utility. In this study, we examined whether transient expression of pvTret1, a trehalose transporter from the desiccation-tolerant midge Polypedilum vanderplanki, could enable trehalose uptake in mouse embryos without impairing development. We synthesized mRNA encoding pvTret1-eGFP and microinjected it into mouse zygotes. Fluorescence microscopy confirmed plasma membrane localization, and AlphaFold modeling indicated that eGFP fusion did not alter protein structure. Embryos expressing pvTret1 developed to the blastocyst stage and produced live offspring at rates comparable to sham-injected controls, suggesting that transient expression does not compromise -developmental potential. LC-MS analysis revealed that pvTret1-expressing embryos took up trehalose in a concentra-tion--dependent manner following 20-min exposure to trehalose-containing medium, and effectively cleared intracellular -trehalose within 20 min of removal, indicating functional, bidirectional transport. In contrast, control embryos exhibited shrinkage under hyperosmotic conditions. Embryos with intracellular trehalose also showed improved post-warming survival after vitrification in dimethyl sulfoxide-free, trehalose-based cryoprotectant solutions. Although optimization is ongoing, these findings highlight the potential utility of pvTret1-mediated trehalose uptake in developing low-toxicity embryo preservation methods. This is the first demonstration that pvTret1 can function in mammalian embryos, enabling transient intracellular trehalose accumulation without transgenesis. Future work will focus on refining trehalose loading protocols and advancing vitrification or dry-preservation strategies for reproductive and biomedical applications.

Humans and rhesus macaques are known to express two molecular forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II), which appear to differentially contribute to the regulation of the menstrual cycle. Specifically, there is evidence to suggest that GnRH-I is the primary mediator of negative estrogen feedback to the hypothalamus and pituitary gland, while GnRH-II is the primary mediator of the positive feedback that stimulates the preovulatory surge of luteinizing hormone. Therefore, it is plausible that selective silencing of GnRH-II would block ovulation and lay the platform for development of a novel contraceptive. To test this possibility female rhesus macaques were actively immunized against GnRH-II (and/or GnRH-I), and serum estradiol and progesterone concentrations were monitored for an additional ∼2.5 years. Despite multiple booster immunizations every ∼6 weeks, and elevated GnRH antibody titers, none of the animals ceased ovulating (i.e., revealed by monthly peaks of serum progesterone concentrations followed by menstruation). Taken together, these findings question the efficacy of GnRH vaccines as a stratagem for selectively blocking ovulation in humans. However, they do not negate the potential value of pharmacological interventions aimed at selectively silencing GnRH-II function and its involvement in stimulating the preovulatory luteinizing hormone surge.

Diisononyl phthalate (DiNP), a plasticizer increasingly replacing di(2-ethylhexyl) phthalate, is an endocrine-disrupting chemical linked to female reproductive harm. Ingestion is the most common route of DiNP exposure, making the gastrointestinal tract and gut microbiome a direct target for endocrine-disrupting chemical exposure. This study examined the effects of acute DiNP exposure either in the absence or presence of a gut microbiome on uterine development. Female C57Bl/6 germ-free (-microbiome) 40-day-old mice were orally dosed, over 3 days, with either sterile phosphate-buffered (n = 8) to remain germ-free (GF, -microbiome) or with colon contents (n = 10) to develop a gut-microbiome (+microbiome). This was followed by a 10-day period where half of the -microbiome and +microbiome mice were orally dosed with corn oil while half were orally dosed with 200 μg/kg/day DiNP. The control group were specific pathogen-free conventionally housed mice born with a microbiome. Mice were euthanized in diestrus at the end of the 10 days. Uteri were collected for histological analyses. Uterine development was significantly delayed in GF mice, regardless of later microbiome reintroduction or DiNP exposure. Key findings included reduced uterine diameter, stroma area, and gland number, and thinner myometrial layers. Endometrial stromal cell proliferation was also lower in GF mice. DiNP exposure alone showed no significant effects. Estradiol levels and ovarian follicle counts were similar across groups, but GF mice had fewer, smaller litters in fertility tests. The study highlights that the gut microbiome critically influences postnatal uterine development, with its absence leading to persistent structural deficits. DiNP, at the tested dose, did not exacerbate these effects.

Glucose transport across the placenta is essential for fetal growth and development. Glucose transporter 1, encoded by the SLC2A1 gene, plays a central role in mediating maternal-fetal glucose exchange. Dysregulation of placental glucose transport is implicated in pregnancy-related complications, such as preeclampsia and fetal growth restriction; however, the mechanistic role of SLC2A1 in trophoblast function remains poorly defined. To functionally validate the role of SLC2A1 in human trophoblasts, we used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated knockout of the SLC2A1 gene, enabling complete and permanent loss of SLC2A1 expression. In the resulting SLC2A1 knockout human trophoblast HTR8/SVneo cells, SLC2A1 depletion induced a metabolic shift from glycolysis to oxidative phosphorylation, leading to increased mitochondrial respiration, ATP production, mitochondrial calcium overload, and elevated mitochondrial reactive oxygen species generation. These changes were accompanied by enhanced endoplasmic reticulum stress, as shown by the upregulation of p-PERK, IRE1α, and GRP78, as well as increased autophagic activity indicated by LC3B-II and p62 accumulation. Notably, mTOR signaling was also upregulated, suggesting a feedback loop that regulates autophagy. The loss of SLC2A1 impaired the PI3K/AKT pathway, reduced trophoblast migration and 3D spheroid formation, and disrupted epithelial-mesenchymal transition-like properties. These findings demonstrate that SLC2A1 is essential for maintaining trophoblast energy homeostasis, redox balance, and invasive capacity; its deficiency triggers mitochondrial and endoplasmic reticulum stress responses that may contribute to placental dysfunction during early pregnancy.

Uterine epithelial estrogen receptor α (ERα) deficiency in epiERα-/- (Esr1f/-Wnt7aCre/+) mice leads to dysregulated environment of uterine lumen, which is lined by uterine luminal epithelium (LE). We hypothesized that transcriptomes in the LE layer held molecular keys to understanding ERα in regulating preimplantation uterine environment. Day 0.5 post-coitum (D0.5) and D3.5 LE layer (isolated via enzymatic digestion), digested uterus (DU), and uterus (U) from Esr1f/- (control) and epiERα-/- mice were processed for mRNA-seq. There were minimal differentially expressed genes (DEGs: transcripts per million > 1, fold-change > 2, false discovery rate < 0.05) between DU and U. Between D0.5 and D3.5 Esr1f/- LE layer, the top upregulated and downregulated gene ontology biological process (GOBP) pathways were on cellular processes and innate immune responses, respectively. Compared to Esr1f/- LE layer, the most upregulated and downregulated pathways in D0.5 epiERα-/- LE layer were on innate immune responses and on biosynthesis and metabolism, respectively; while those in D3.5 epiERα-/- LE layer were on cell division and on signaling and metabolic processes, respectively. Na+ transmembrane transport was among shared upregulated pathways in D0.5 and D3.5 epiERα-/- LE layer. Between Esr1f/- and epiERα-/-, most DEGs in U were also DEGs in the LE layer, and limited DEGs in U only with higher expression than in the LE layer were related to immune responses, implying potential paracrine effects of uterine epithelial ERα. Selected DEGs were verified by realtime PCR and immunohistochemistry. This mRNA-seq dataset provides molecular keys to understanding temporal (e.g., innate immunity) and constituent (e.g., uterine fluid movement) functions, and potential paracrine effects of uterine epithelial ERα in regulating the preimplantation uterine environment.

Chromosomal abnormalities are the most common cause of developmental arrest in mammalian embryos. They can be present consistently in all cells of the embryo or occur as admixtures of karyotypically distinct lineages (mosaics). The estimated incidence of mosaicism ranges from 14% to 82% in human embryo biopsies at the blastocyst stage. In cattle, mosaicism is not well described at a whole-genome level, with findings limited to sex chromosomes. Here, we conducted a retrospective analysis of published data spanning three studies from our laboratory to establish the incidence and nature of mosaicism in 2,045 bovine blastocysts genotyped using single nucleotide polymorphism-based approaches. We classified mosaic embryos as those where the inner cell mass and trophectoderm differed in ploidy and/or where embryos had a percentage of cells with aneuploidy ranging from 20% to 80%. We report an aneuploidy incidence of 15.2% (n = 311/2,045), with 25.6% of the aneuploid embryos (80/311) being mosaic. Mosaicism was particularly common (87.5%, n  = 7/8) in embryos affected by multiple types of chromosomal errors and in embryos affected only by segmental aneuploidies (50.0%, n = 9/18). The chromosomal abnormalities with the highest incidence of mosaicism were segmental aneuploidies (48.1%, n = 13/27). Most errors leading to mosaicism had a paternal origin (44.9%, n = 22/49), followed by post-zygotic errors (37.3%, n = 19/51). Our results reveal an incidence of mosaicism in bovine embryos similar to that of human embryos. Additionally, we demonstrate that ploidy and mosaicism screening can be performed in embryos using the same single nucleotide polymorphism genotyping data obtained to calculate genomic estimated breeding values.

Accurate testicular tissue characterization is critical for diagnosing reproductive disorders and malignancies. We present a rapid, label-free 3D imaging technique that leverages endogenous autofluorescence and a fast, non-toxic optical clearing protocol to reveal the full 3D architecture of thick mouse testis samples, without sectioning. This approach clearly distinguishes structures like epithelial tubules, interstitial tissue, and key cell types (germ, Sertoli, Leydig) with high precision. Autofluorescence serves as a powerful structural counterstain, enhancing multiplexed analysis alongside immunofluorescent or transgenic markers. Compatible with high-resolution techniques like image-scanning confocal microscopy (AiryScan), our approach captures fine intracellular details while avoiding the artefacts of traditional histology. Accessible, affordable, and equally usable on standard confocal microscopes, this method democratizes advanced testis imaging and accelerates progress in reproductive research.