Biology of Reproduction最新文献

For a successful pregnancy, decidualization is a crucial process involving the significant transformation of endometrial stromal cells surrounding the implanting blastocysts. Disruption of this process can lead to the breakdown of the fetomaternal interface and result in early pregnancy loss. However, the precise mechanisms governing this process remain incompletely understood. This study aimed to elucidate the impact of citrullination, a post-translational modification, on decidualization in mice and humans. Immunohistochemical analysis and immunoprecipitation followed by immunoblotting were performed on mouse and human uterine tissues. In this study, we showed that ezrin/radixin/moesin (ERM) proteins are expressed in decidual and endothelial cells within the decidua during pregnancy, with their expression patterns significantly influenced by two factors: citrullination mediated by peptidylarginine deiminase enzymes and sphingosine kinase activity. Notably, ERM proteins were found to undergo citrullination exclusively in the decidua during pregnancy, but not in interimplantation tissues during pregnancy or in nonpregnant uteri in mice. Similar findings were observed in human decidual tissues from cases of spontaneous abortion and elective termination. In conclusion, this study demonstrates that ERM proteins undergo citrullination in the decidua during early pregnancy in both mice and humans. Our findings provide important insights into the molecular changes involved in pregnancy, particularly decidualization.

Concentrations of interferon-tau (IFNT) are likely the best proxy for early pregnancy diagnosis in cattle. Objectives were to compare the concentration of IFNT at the vaginal fornix and cervical ostium 18 days after artificial insemination (AI) and evaluate the use of IFNT for early identification of non-pregnant lactating dairy cows. Holstein cows (n = 251) from two dairies were enrolled. Expression of interferon-stimulated gene 15 (ISG15), myxovirus 2 (MX2), and viperin (RSAD2) were assessed in peripheral blood mononuclear cells (PBMC) by RT-qPCR. Expression of interferon-stimulated genes (ISG) and IFNT concentration in swab samples collected at the cervical ostium and vaginal fornix were measured by RT-qPCR and sandwich ELISA validated for bovine IFNT, respectively. Cervical IFNT concentration was 7.5-fold greater than the vaginal fornix on day 18 after AI in cows diagnosed as pregnant by ultrasonography on day 35 after AI. Concentrations of IFNT in the cervical ostium and vaginal fornix were positively correlated with ISG expression in cervical cytology of pregnant cows, however, ISG expression in PBMC was not correlated with cervical IFNT concentration. Assessment of cervical IFNT concentrations for identification of non-pregnant cows on day 18 after AI yielded reasonable specificity (0.84; 95% CI = 0.78-0.90) and negative predictive value (0.87; 95% CI = 0.81-0.93) based on ultrasound examination on day 35 after AI. These results support the feasibility of using cervical IFNT measurement for identification of non-pregnant cows, providing the basis for early resynchronization and increased insemination rates in dairy farms.

Unexplained recurrent pregnancy loss (URPL) is complex and has unknown etiologies, simultaneously endangering the patient's physical and mental health. Despite significant advancements in shifting from conventional treatments to cell therapy, clinical research and application in cell therapy have been constrained by its immunogenic properties and biosafety. As a new cell-cell communication pathway, extracellular vesicles (EVs) can transfer their bioactive cargos to other cells by endocytosis, ligand-receptor engagement, or direct fusion, thereby executing diverse biological functions both in proximity and across great distances. It is noteworthy that the role of EVs as a cell-free therapy in the form of a drug transporter has garnered increased attention, and some studies have demonstrated EVs involvement in the key pathology of URPL. This review methodically summarizes the functions of EVs from various sources in URPL, including mesenchymal stem cells, placenta, and other sources of EVs. Furthermore, we discuss the significance of EVs as possible diagnostic biomarkers and therapeutic strategies for URPL and the key questions that need to be addressed in future research.

Lipid nanoparticles (LNPs) have emerged as a nonviral mRNA delivery vehicle for both basic and clinical applications. In the present study, we showed that the LNPs passed through the zona pellucida, the extracellular matrix surrounding the egg, and efficiently transduced preimplantation embryos. We first tested different types of surface polarity in LNPs carrying Enhanced Green Fluorescent Protein (EGFP) (LNP-EGFP) by changing cholesterol and found that the LNPs composed with neutral or anionic cholesterol are less toxic and more suitable for mRNA delivery into zygotes. Next, we transferred Cre mRNA using neutral LNP (LNP-Cre) and saw the floxed allele recombination in reporter mTmG transgenic embryos. After treatment with LNP-Cre at a 10 ng/μL concentration for 20 h at the zygote stage, about 76% mTmG embryos expressed the reporter EGFP at the blastocyst stage, and about 82% of embryos expressed EGFP in both the placenta and fetus at E12.5. Finally, we treated mTmG blastocyst stage embryos with LNP-Cre at a 50 ng/μL concentration for 20 h and found that about 86% of them expressed the reporter EGFP. Intriguingly, the EGFP fluorescence was only observed in trophectoderm cells, but not in the inner cell mass. Subsequently, we observed placenta-specific reporter EGFP expression in about 65% of the later-stage embryos (E12.5). Collectively, our study shows that LNPs provide a novel mRNA delivery system into preimplantation embryos that can be used for studying gene functions in pre-, peri-, and post-implantation development.

Background: Metabolic remodeling is crucial for successful decidualization, but the mechanisms coordinating the tricarboxylic acid (TCA) cycle, fatty acids, and oxylipins with energy demands and pro-inflammatory responses during peri-implantation are unclear.

Methods: This study analyzed temporal metabolic changes and pro-inflammatory factors at the implantation site (IS) and inter-implantation site (IIS) from days 5 to 7 post-implantation in mice using GC-MS, UPLC-MS/MS, immunofluorescence, quantitative real-time PCR and ELISA, ANOVA, Tukey HSD, mixed-effects models, and Pearson correlation were used for statistical analysis.

Results: Our findings reveal dynamic metabolic changes in the IS and IIS from days 5 to 7 of pregnancy. During transition to the secondary decidual zone (SDZ), the IS exhibited metabolic adaptation, with higher TCA cycle activity compared to the IIS. On day 7, the IS also showed significantly elevated activity in the citric acid cycle, glycolysis, and pyruvate metabolism compared to day 5. Additionally, during the SDZ phase, n-3 and n-6 fatty acids and their oxylipins, particularly prostanoids like PGE2 from arachidonic acid, were upregulated in the IS, exhibiting a similar expression pattern of pro-inflammatory factors such as IL-6 and IL-8. The mRNA expression of COX-2 and mPGES-1, key enzymes related to PGE2 biosynthesis, showed a sustained increase from day 5 to day 7 of pregnancy. This suggests that PGE2 plays a key role in maintaining the pro-inflammatory environment necessary for decidualization.

Conclusion: This study highlights the importance of metabolic adaptations and fatty acid-derived oxylipins in ensuring energy supply and sustaining the pro-inflammatory environment during successful decidualization.

In 2021, Ken McNatty, Danielle Monniaux, and I published a review essay illustrating how ovarian folliculogenesis can sometimes be bizarre, amazing, or even almost incomprehensible. Examples included the mechanisms underlying intra- and inter-species differences in ovulation rates; the possibility of awakening human primordial follicles in vitro, maturing and fertilizing them to produce viable offspring; and a model in which inactivation of a single oocyte gene results in sterile mice with follicular growth blocked at the primary stage but normal steroid cyclicity maintained. The aim of this second essay is to present further examples of the extraordinary diversity of ovarian function across animal species and, where possible, to propose hypotheses that may explain them. These concern: 1) the presence of oogonial stem cells in the ovaries of invertebrates and non-mammalian vertebrates, and their very probable absence in mammals; 2) the many and varied strategies of ovarian development and oogenesis in teleosts; 3)the metabolic dialog between cumulus cells and oocytes across mammalian species; 4) the presence of numerous germline genes, specifically or even exclusively expressed in the mammalian oocyte, whose invalidation has no phenotypic consequence on fertility in the mouse; 5) the unique features of ovarian function in the dog, particularly the frequent presence of polyovular follicles and the distinctive mode of post-ovulatory oocyte maturation; and 6) the absence of an intra-ovarian dominance factor in mono-ovulating species, disproving an old hypothesis: the selection of a single follicle is instead due to a succession of negative and then positive feedback between follicles and the hypothalamic-pituitary axis. This essay is also a final tribute to Ken McNatty, who liked to say that the ovary could be crazy.

Adenomyosis is a common gynecological disorder characterized by the presence of endometrial tissue in the myometrium, causing chronic pelvic pain and abnormal bleeding. Although dysregulated microRNAs (miRNAs) in stromal cells of adenomyosis patients have been implicated in disease-associated extracellular matrix (ECM) remodeling, their role in regulating endometrial epithelial cell (EEC) behavior remains poorly understood. This study investigated the effect of selected adenomyosis- and endometriosis-associated miRNAs on EEC migration and matrix metalloproteinase (MMP) expression. Ishikawa cells, a human endometrial epithelium-like cell line, were transfected with mimics and inhibitors of Let-7b, miR-451a, miR-125b, miR-7 and miR-150. Cell migration was assessed using wound healing assays. MMP-2 and MMP-9 mRNA expression were quantified by quantitative real-time polymerase chain reaction, while protein production and secretion were evaluated by enzyme-linked immunosorbent assay. Transfection with Let-7b-5p inhibitor and miR-451a, miR-125b-1 and miR-150 mimics significantly enhanced cell migration and led to increased MMP-2 and MMP-9 mRNA expression, intracellular protein levels and secretion. These findings suggest that these miRNAs promote EEC migration and ECM remodeling through MMP upregulation, pointing to a potential mechanism for lesion formation in adenomyosis. Future research should seek to validate these findings in primary EECs and explore the therapeutic potential of targeting miRNA-MMP pathways for adenomyosis treatment.

During epididymal transit, spermatozoa undergo crucial morphological, biochemical, and molecular modifications that enable the acquisition of motility, acrosomal remodeling, membrane reorganization, and chromatin stabilization, culminating in full maturation. This study investigated sperm motility, sperm head and acrosome remodeling, chromatin compaction (protamination), DNA integrity, and nuclear morphology in sperm collected from different epididymal regions (caput, corpus, and cauda) and the vas deferens in three closely related mouse species exhibiting varying levels of sperm competition. Using microscopy, fluorescent staining (Hoechst 33342, chromomycin A3), sperm chromatin structure assay (SCSA), and nuclear morphology software, we characterized these parameters. Results demonstrated a gradual acquisition of total and progressive motility from the caput onwards and a shift toward a more compacted acrosome, especially between corpus and cauda. Minor species-specific variations in nuclear shape and dimensions were observed. Chromatin compaction and DNA integrity significantly increased, evidenced by decreased chromomycin A3 positive cells and reduced DNA fragmentation indices (tDFI and HDS). A correlation between protamine status and DNA fragmentation appeared when data from all species were pooled. Mus musculus showed lower overall motility and a faster decline in loose acrosomes. M. spicilegus exhibited the most rapid sperm head compaction, whereas M. spretus had the highest sperm nuclear width and the lowest tDFI values. These findings reveal distinct changes in sperm maturation markers along the epididymis and suggest associations between these changes and sperm competition levels of these species. This enhances our understanding of sperm maturation mechanisms and may inform advances in reproductive technologies.

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.

Transgenic animals are invaluable tools in genetic studies, disease modeling, drug discovery, and biotechnology. However, the low efficiency of transgenic animal generation can be an obstacle to their application. Here, we report the generation of transgenic mice using PBatase, the piggyBat transposase from the little brown bat (Myotis lucifugus). PBatase exhibits detectable transposition activity in fertilized mouse eggs within a limited concentration range, although the overall activity was lower than that of PBase, the piggyBac transposase from the cabbage looper moth (Trichoplusia ni). Transgenic animals carrying low transgene copy numbers were successfully generated with high efficiency using PBatase, and the transgene was subsequently transmitted to the next generation. This technique will be useful for the generation of transgenic animals carrying single copies of a transgene.