雄性民猪寒冷压力性状跨代遗传机制研究

Cold tolerance traits are important characters of Min pig. According to the study of epigenetics, ancestral environmental exposures can be inherited by germline(environmental epigenetic transgenerational inheritance). This project proposes a scientific hypothesis. Namely, male Min pig’s cold tolerance related microRNAs can be transferred to sperms from the epididymosomes, then to embryos, influencing the DNA methylation of embryos; these kinds of DNA methylation modifications escaped the two waves of reprogramming during the development of embryos, causing the cold tolerance related epigenetic transgenerational inheritance through male germline. To verify the hypothesis, the sperm produced in the cold environment was sequenced by Small RNA-seq, and the microRNAs related to cold tolerance was identifed. Through the experiment of the fusion between the epididymosomes and the sperms, the path of microRNAs from somatic cell to sperm was verified. We injected cold tolerance related multi- microRNAs into zygotes which have not experienced cold environment ,with the non-homologous oligonucleotides injected embryos as negative control, then transplanted these embryos. We analyse the difference of DNA methylation in ICM of blastocysts, PGCs in gastrulation and PGCs in gonads prior to sex determination in order to elucidate the effect of microRNAs on epigenetic in offspring. The cold tolerance related indicators were detected to confirm the cold tolerance traits transmission in individual offspring. This project can reveal the mechanism of cold tolerance related epigenetic transgenerational inheritance. Furthermore, it provides a theoretical basis for the protection and utilization of excellent characters of local livestock and poultry.

抗寒性状是民猪重要性状，表观遗传研究表明，父代环境压力表型可通过雄性跨代遗传。本项目提出科学假设，即“雄性民猪寒冷压力性状相关的microRNAs经由附睾小体传递至精子，进而传递至胚胎，并影响胚胎DNA甲基化修饰，该种修饰逃逸发育过程中的两次重编程，使寒冷压力表观遗传信息经由雄性生殖系跨代传递”。为验证该假设，本项目对寒冷环境下产生的精子进行Small RNA测序，挖掘出寒冷压力相关的microRNAs；通过附睾小体与精子融合实验，验证microRNAs从体细胞传递至精子的路径；通过构建寒冷压力相关microRNAs显微注射胚胎并进行胚胎移植，与对照组比较,分析重编程过程几个关键时间点及后代精子的甲基化差异，解析microRNAs对后代甲基化的影响；检测后代个体寒冷压力相关指标，确定抗寒性状传递。本项目可揭示抗寒性状跨代遗传的机制，进而为促进地方畜禽品种优异性状的保护与利用提供理论依据。

成年雄性民猪寒冷压力性状通过何种渠道将表观遗传信息传递给精子以及这种表观遗传信息在早期胚胎内发挥作用的机制仍不清楚。因此，在本项目中，为了明确从个体环境压力表型至精子的 miRNAs 表观遗传信息流传递路径，我们探讨了不同环境温度下ST细胞中外泌体中的miRNA差异，且阐明了，ST细胞中特定miRNA的表达量高低可直接影响外泌体中miRNA的表达量。同时，通过PKH67染色实验，进一步证实了精子可吸收ST细胞释放的外泌体，最终明确了，ST细胞外泌体作为ST细胞和精子间的通讯工具，可将环境温度变化导致的mir-148a传递给精子，进而导致精子中相应mir-148a的表达量改变。继而，将mir-148a的模拟物注射入早期胚胎，我们发现精子来源的mir-148a可促进早期胚胎发育。对4-细胞期胚胎进行RNA-seq，并对差异表达基因进行分析，我们发现，mir-148a注射入早期胚胎后，参与DNA甲基化调控的酶表达没有发生显著变化，同时，注射mir-148a也未影响早期胚胎的基因组DNA甲基化水平；对差异表达的基因进一步分析显示，差异表达的基因显著富集在核糖体生物合成通路上。我们认为，注射mir-148a通过抑制rRNA的生物合成，改变核仁的组成及结构，进而释放先锋转录因子，并促进了早期胚胎发育。继而，我们使用CX-5461瞬时干扰了rRNA的生物合成及核仁的液-液相分离，进而破坏了核仁的完整结构，随后发现，干扰核仁的组装也可促进早期胚胎发育，并促进了ZGA标志性基因的表达。总之，本研究发现，ST细胞释放的外泌体作为睾丸支持细胞和精子间的通讯工具，可将环境温度变化导致的mir-148a传递给精子，进而导致精子中 mir-148a 的表达量改变；且mir-148a可通过抑制早期胚胎内核糖体的生物合成来促进早期胚胎发育；本研究为促进民猪优异性状的保护与利用提供基础数据和理论依据。

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