Theriogenology最新文献

HOXA10 is a crucial marker gene for endometrial receptivity in mammals, with significantly increased expression in endometrial epithelial cells during the implantation window. However, the molecular mechanisms by which HOXA10 regulates bovine endometrial epithelial cells remain unclear. In this study, we constructed RNA interference fragments targeting the bovine HOXA10 gene, transfected them into bovine endometrial epithelial cells, and performed RNA-seq analysis. We conducted EdU staining, flow cytometry, RT-qPCR, and Western blot to assess apoptosis and the FOXO1 signaling pathway. Additionally, RT-qPCR, Western blot, and immunofluorescence staining were used to analyze the expression of genes associated with receptivity (ITGB3, ITGB6, ITGAV, and MUC1). The results showed that HOXA10 interference induced apoptotic changes in bovine endometrial epithelial cells. Metabolic pathways differentially affected included the cell cycle, cellular senescence, the FOX signaling pathway, and DNA replication. The expression levels of FOXO1, CDKN1A, CCND1, CDK4, and BAX were significantly upregulated, while CCNE1, CCNB1, CDK2, and BCL-2 were significantly downregulated. Furthermore, the mRNA expression of ITGB3, ITGB6, and ITGAV was significantly reduced, while MUC1 mRNA expression was significantly increased. Therefore, these findings indicate that the HOXA10 interference activates the FOXO1 pathway, leading to apoptosis in bovine endometrial epithelial cells.

Current porcine embryo culture relies on Porcine Zygote Medium 3 (PZM3) supplemented with fetal bovine serum (FBS), which causes batch-to-batch variability due to undefined components. Here, we evaluated N2B27-a chemically defined serum-free medium-as an alternative and compared the developmental outcomes of culture in PZM3 (PZM), PZM3 with 10% (v/v) FBS (PZM + FBS), and N2B27 in parthenogenetic embryos. N2B27 increased the total cell number, blastocyst and hatched blastocyst formation rates, and epiblast, primitive endoderm, and trophectoderm cell counts compared to PZM and PZM + FBS. Epiblast allocation increased relative to PZM but decreased relative to PZM + FBS, whereas primitive endoderm allocation increased relative to PZM and did not differ from PZM + FBS. Ungulates undergo rapid expansion of extraembryonic lineages during pre-implantation; thus we examined the effects of fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4) on extraembryonic lineage specification. FGF2 increased primitive endoderm cells and decreased epiblast cells, and their allocation. BMP4 increased the number of primitive endodermal cells and their allocation. FGF2 and BMP4 demonstrated combined effects, reducing the epiblast and increasing the primitive endoderm in both cell number and allocation. These results suggest that N2B27 can serve as a serum-free alternative for porcine parthenogenetic in vitro culture and that FGF2 and BMP4 can influence lineage specification toward the primitive endoderm.

The aberrant modification level of Histone H3 lysine 9 trimethylation (H3K9me3) is a major barrier for somatic cell nuclear transfer (SCNT) embryo development. The removal of H3K9me3 from the embryonic genome via microinjection of KDM4 mRNA can improve the developmental efficiency. However, this strategy is limiting because of its invasive and time-consuming. In the present study, through comparative transcriptomic analysis with in vivo embryos, we found that porcine SCNT embryos exhibit insufficient zygotic genome activation (ZGA) transcription, along with aberrantly elevated expression of the SUV39H2 and SUV39H1. Then we investigated an alternative approach by inhibiting SUV39H2 and SUV39H1 via their specific inhibitor OTS186935 (targeting SUV39H2) and F5446 (targeting SUV39H1). Individual treatment with either inhibitor significantly reduced H3K9me3 levels and improved blastocyst formation rates. Notably, combined treatment with OTS186935 and F5446 synergistically further enhanced developmental outcomes. This combination treatment potently decreased H3K9me3 levels, rescued transcriptional activity during ZGA, and improved gene expression profiles, making them more closely resemble those of in vivo embryos. Consequently, compared to the control group, the combined treatment significantly increased the blastocyst rate (Treatment: 25.49 ± 4.47% vs. Control: 15.15 ± 2.69%, P < 0.001), blastocyst diameter (Treatment: 116.9 ± 4.37 μm vs. Control: 75.4 ± 2.87 μm, P < 0.001), and total cell number (Treatment: 33.00 ± 3.74 vs. Control: 28.75 ± 6.55, P < 0.05). Our findings demonstrate that dual inhibition of SUV39H1 and SUV39H2 effectively corrects aberrant H3K9me3 modification, ameliorates ZGA defects, and provides a simplified and efficient strategy to enhance the developmental competence of porcine SCNT embryos.

Placental lactogen (PL) and pregnancy-associated glycoproteins (PAGs), secreted by trophoblast binucleate cells, are central to fetal development in ruminants. While nutritional effects on PAGs are documented, impacts on maternal PL and its relationship with PAGs remain unclear. This study examined the effects of maternal feed restriction on ovine placental lactogen (oPL)concentrations and their correlation with PAGs in pregnant ewes. Fifty-nine ewes (singletons or multiples) were pair-matched to a control group (100 % energy requirements) or a feed-restricted group (50 % requirements) from day 24-100 of gestation. Blood was collected on Days 24 (T1), 50 (T2), 80 (T3), 110 (T4), and 140 (T5) to measure oPL, PAGs, and metabolic indicators (NEFA, glucose, total protein). Feed restriction did not significantly affect oPL (p > 0.1). oPL varied with time and litter size (p < 0.001), showing a nadir at T3 and a peak at T5; ewes with multiples had consistently higher levels. In singleton pregnancies, oPL at T5 positively predicted lamb birth weight (p = 0.05). Although oPL and PAGs displayed similar biphasic patterns (decline at T3, peak at T5), no significant correlation was found between them. oPL showed a weak, negative correlation with NEFA at T1 and moderate, negative correlation with total protein at T5; no association was observed with glucose. In conclusion, moderate maternal undernutrition during early gestation does not compromise oPL secretion, highlighting its resilience and role in supporting fetal growth. The similar temporal pattern but absence of correlation between oPL and PAGs suggest distinct regulatory mechanisms, with both hormones acting protectively to ensure fetal well-being.

Efficient in vitro maturation (IVM) of mammalian oocytes is essential for breeding and genetic improvement of elite lines. However, porcine embryos generally show lower developmental potential than those of other species. Oocyte maturation and early embryonic development occur within a dynamically changing follicular microenvironment, in which hypoxia critically shapes developmental competence. Redox homeostasis in follicles is indispensable for oocyte development, yet how exosome-derived non-coding RNAs modulate oxidative-stress balance in porcine oocytes remains unclear. Here, high-throughput sequencing and bioinformatic analysis identified the long non-coding RNA (lncRNA) ENSSSCG00000049656 (lnc49656). We then characterized its function using dual-luciferase reporter assays, RNA immunoprecipitation (RIP), quantitative real-time PCR (qRT-PCR), and Western blotting (WB). Overexpressing lnc49656 significantly enhanced cumulus expansion, cleavage, and blastocyst formation (p < 0.05). Comparative cultures under different oxygen tensions showed that, under normoxia, lnc49656 overexpression significantly reduced expression of downstream HIF-1 target genes-Vascular Endothelial Growth Factor (VEGF), Lactate Dehydrogenase A (LDHA), and Glucose Transporter 1 (GLUT1) (p < 0.05); inhibiting lnc49656 or overexpressing ssc-miR-500-3p (miR-500) reversed these effects. Mechanistically, lnc49656 sequestered miR-500, thereby relieving miR-500-mediated repression of Egl nine homolog 2 (EGLN2). As a core component of the oxygen-sensing system, EGLN2 hydroxylates hypoxia-inducible factor-1α (HIF-1α) to promote its degradation, thereby affecting redox homeostasis in the follicular microenvironment. Collectively, these findings indicate that the lnc49656/miR-500/EGLN2 axis is a key regulator of hypoxic responses and oxidative-stress balance during porcine oocyte maturation and early embryonic development, offering a new perspective for dissecting the molecular mechanisms of the follicular microenvironment.