The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

DNA methylation could take part in the gene expression and acts an important role in muscle development. In this study, DNA methylation and expression in adipose and muscle tissues were examined at the same time to evaluate the extent of epigenetic modifications and gene expression on the differentially methylated region (DMR) in SERPINA3. Chain reaction of bisulfite sequencing polymerase (BSP) was used to compared difference among DNA methylation patterns. The result of quantitative real-time PCR (qPCR) analysis showed that there was an extensive expression of SERPINA3 gene in tissue and there was a significant difference existing in muscle and adipose between Jiaxian cattle and individual of other breeds with increasing hybridization (p < 0.05). The statistic analyses indicated that DNA methylation patterns had a significant influence to the level of mRNA in tissue of fat and muscle. This study may be an important reference for investigating development of muscle tissue in cattle, and may promote the process of cattle molecular breeding.

Numerous dynamic and complicated processes characterize development from the oocyte to the embryo. However, given the importance of functional transcriptome profiles, long non-coding RNAs, single-nucleotide polymorphisms, and alternative splicing during embryonic development, the effect that these features have on the blastomeres of 2-, 4-, 8-, 16-cell, and morula stages of development have not been studied. Here, we conducted a scRNA-seq survey of cells from sheep from the oocyte to the blastocyst developmental stages. We then carried out experiments to identify and functionally analyze the transcriptome profiles, long non-coding RNAs, single-nucleotide polymorphisms (SNPs), and alternative splicing (AS). We founded that between the oocyte and zygote groups significantly down-regulated genes and the second-largest change in gene expression occurred between the 8- and 16-cell stages. We used various methods to construct a profile to characterize cellular and molecular features and systematically analyze the related GO and KEGG profile of cells of all stages from the oocyte to the blastocyst. This large-scale, single-cell atlas provides key cellular information and will likely assist clinical studies in improving preimplantation genetic diagnosis.

Objective The objective of this study is to explore the protective mechanisms of proanthocyanidin B2（PB2）when incorporated into the maturation medium of bovine oocytes under heat stress conditions.This investigation aims to provide a scientific foundation for enhancing the quality of bovine oocytes exposed to high temperatures and improving their capacity for embryonic development in vitro. Method A total of 103 pairs of ovaries were collected from 6-8 years old Holstein cattle.The cumulus oocyte complex（COCs）in ovarian follicles were aspirated in the laboratory and randomly divided into 3 groups for in vitro maturation：control group（cultured at 38.5 ℃ for 24 h）；heat stress group（cultured at 41 ℃ for 8h and transferred to 38.5 ℃ incubator for 16 h）；proanthocyanidin group（25 µmol/L PB2，cultured at 41 ℃ for 8h and transferred to 38.5 ℃ incubator for 16 h）.After the oocytes were treated by hyaluronidase，the oocyte maturation rate of each group was measured under stereomicroscope.After parthenogenetic activation of oocytes by chemical agent，the cleavage rate and blastocyst rate were recorded at 48 h and 168 h，respectively.The ROS and GSH of oocytes and the total number of blastocysts were measured by fluorescence staining.The expression levels of antioxidant genes（SOD1，SOD2，CAT）and maturation genes（GSS，GPX1，CAS6）were detected by quantitative real-time PCR（qPCR）. Result （1）COCs matured in vitro for 24 h.Compared with the heat stress group，the proanthocyanidin group increased the oocyte maturation rate，but there was no significant difference between the two groups（P>0.05）；（2）After parthenogenesis activation，compared with heat stress group，the cleavage rate of proanthocyanidin group was significantly increased（P<0.05），the blastocyst rate was not significantly different（P>0.05），but the total number of blastocyst cells was significantly increased（P<0.05）；（3）After 24h maturation in vitro，compared with heat stress group，ROS content in proanthocyanidins group was significantly decreased（P<0.01），GSH content was significantly increased（P<0.05），the expression levels of maturation genes and antioxidant genes were significantly increased（P<0.05）. Conclusion By adding proanthocyanidin B2 to in vitro maturation medium，oxidative stress of bovine oocytes induced by heat stress can be effectively alleviated，thus improving the maturation process of oocytes in vitro and enhancing the development potential of embryos as well as embryo quality.