Molecular Reproduction and Development最新文献

Cumulus-oocyte complexes (COCs) used for in vitro production (IVP) of bovine embryos originate from antral follicles of different sizes, leading to variations in developmental competence. To address this, pre-in vitro maturation (pre-IVM) allows oocytes with additional time to acquire developmental competence. Given the role of follicular fluid-derived extracellular vesicles (EVs) in ovarian follicle communication, which has been shown to vary in content and function across folliculogenesis, we investigated whether EVs from early versus late antral follicles influence COCs during pre-IVM. EV supplementation significantly altered gene expression in cumulus cells and oocytes. In cumulus cells, affected pathways included MAPK signaling, Gap junctions, Cytokine-cytokine receptor interaction, Axon guidance, cAMP, and Cushing syndrome. In oocytes, fewer genes were altered, with effects on Inositol phosphate metabolism, p53 signaling and Cholesterol metabolism. Despite these changes, no significant effects of the EV treatment were noted on oocyte chromatin configuration and developmental competence, except for a significant increase of mitochondrial membrane potential (Δψm) in blastocysts. In conclusion, EV supplementation during pre-IVM significantly altered the transcriptional profile of COCs, with EVs from early follicles modulating the expression of genes regulating cumulus cell proliferation and gap junctions, while EVs from late follicles impacted pathways associated with meiotic resumption, cumulus cell expansion, and apoptosis. Along with improved Δψm in blastocysts, these results support a positive effect of EVs on bovine COCs, but further research is needed to better characterize the functional consequences, mainly in terms of the effects of early versus late follicle-derived EVs on oocyte developmental potential.

Embryo genome activation (EGA) is a crucial event implicated in proper embryonic development. Similarly, the DNA Damage Response (DDR) is essential for correcting or preventing occasional errors during embryonic cell division that could result in embryo development arrest or the propagation of genetic abnormalities. Although both EGA and DDR are regulated by epigenetic mechanisms, the role of microRNAs (miRNAs) in these processes has not been explored in pig embryos. Herein, we assessed the abundance of miRNAs linked to embryo development and DDR across four developmental stages: Day 2 (D2), Day 3 (D3), and Day 4 (D4), and at the blastocyst stage, as well as after DNA damage induction. mRNA levels of EGA-related genes confirmed our timepoints as representing EGA timeframe, while immunofluorescence for γH2AX validated DNA damage induction. Significant decrease in blastocyst rate and total cell number per blastocyst was detected in UV-exposed embryos. Analysis of miRNA abundance revealed increased levels of miR-200a-5p on D3, which were partially maintained on D4. Both miR-15a and miR-24-3p increased on D4, but their levels were downregulated in UV-exposed embryos at the same stage of development. In blastocysts, UV exposure upregulated miR-29a-3p and miR-344b-3p. Together, these findings provide the first characterization of miRNAs expression in porcine embryos during EGA and following DNA damage induction.

To identify the optimal in vitro maturation (IVM) duration for bovine oocytes with different nuclear maturation speeds (NMS), this study assessed how varying IVM durations (24, 28, and 32 h) affect developmental competence and embryo quality in oocytes with fast- or slow-predicted NMS classified via machine learning. Developmental competence was evaluated through cleavage rates, first cleavage timing and patterns, and blastocyst formation under individual culture. Embryo quality was assessed via differential staining of inner cell mass and trophectoderm and expression analysis of quality-related genes in formed blastocysts. For oocytes with slow-predicted NMS, extending IVM to 28 h increased cleavage rates and accelerated first cleavage timing (p < 0.01). The lower blastocyst formation rates of oocytes with slow-predicted NMS matured for 24 h improved when IVM reached 28 h, becoming comparable to fast-predicted NMS oocytes. However, extended IVM decreased expression of pluripotency-related genes (e.g., NANOG and OCT4; p < 0.01) regardless of predicted NMS. In conclusion, extending IVM duration to 28 h improved developmental competence of slow-predicted NMS oocytes, highlighting the importance of fertilization timing relative to nuclear maturation completion, though it reduced expression of key pluripotency genes. Individualized IVM protocols based on predicted NMS can enhance bovine embryo production efficiency.

Dairy cows often experience a period of negative energy balance (NEB) during the post-calving period, which can significantly impact economic outcomes due to extended calving-to-conception intervals and overall reduced fertility. This reduction is due, in part, to the impact on uterine biology by high nonesterified fatty acids (NEFA) and beta-hydroxybutyrate concentration. The uterine fluid (UF) contains small extracellular vesicles (UF-EVs) that, through their cargo, including microRNAs (miRNAs), respond to metabolic stress, affecting the uterine environment. This study aimed to assess the long-term impact of NEB intensity on the uterine environment of dairy cows. Post-partum dairy cows were classified based on NEFA concentrations in their blood during the 3 weeks post-calving as having either Low or High NEB. At 30 and 60 DPC, the synchronization protocol was started, and UF samples were collected (corresponding to ~15 days after initiation of the synchronization protocol) to isolate UF-EVs and uterine epithelial cells for miRNA and transcriptome profiling. We also investigated whether UF-EVs could modulate epithelial uterine naïve cells. Our results indicate that the uterine environment of dairy cows experiencing a High NEB post-calving is unfavorable for embryo development at 60-day post-calving. Importantly, we show that UF-EVs can reproduce this phenotype in epithelial uterine naïve cells, suggesting that UF-EVs may act as modulators of the uterine response to metabolic challenges.

Small extracellular vesicles (sEVs) function as critical regulators of ovarian follicular development. Although several pathways, including one involving neutral sphingomyelinase (nSMase), contribute to sEV production, the specific pathway active in ovarian follicles has not been clearly identified. In this study, we investigated GW4869, a specific inhibitor of nSMase activity, to determine its impact on sEV production by mouse mural granulosa cells (MGCs), the primary source of follicular sEVs. We also examined how nSMase inhibition affects the in vitro growth of oocyte‒granulosa cell complexes (OGCs) derived from secondary follicles. Transcripts encoding nSMases (Smpd2 and Smpd4) were detected in MGCs, and GW4869 treatment significantly reduced sEV production in MGC monolayer cultures. Control OGCs developed into antral follicle-like structures, with the antrum-like structure separating granulosa cells into cumulus-like and MGC-like cells. However, GW4869 treatment impaired OGC development. MGC-like cells from GW4869-treated OGCs exhibited significantly lower Cyp19a1 levels, whereas adding MGC-derived sEVs promoted Cyp19a1 expression. These results suggest that nSMase activity, likely involving Smpd2 and Smpd4, is required for sEV production by MGCs and that follicular sEVs may regulate Cyp19a1 expression in MGCs.