miR-449b对绵羊早期胚胎发育的影响

doi:10.16431/j.cnki.1671-7236.2022.07.024

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (7): 2661-2668.doi: 10.16431/j.cnki.1671-7236.2022.07.024

miR-449b对绵羊早期胚胎发育的影响

任秀美奥^1,2, 姚旭东^1,2, 蒙亚琦^1,2, 郭延华², 唐红², 张译元², 赵兴旺³, 王立民², 周平^1,2

1. 石河子大学动物科技学院, 石河子 832000;
2. 新疆农垦科学院, 省部共建绵羊遗传改良与健康养殖国家重点实验室, 石河子 832000;
3. 石河子总场农业服务发展中心, 石河子 832000

收稿日期:2021-12-15 出版日期:2022-07-05 发布日期:2022-06-29
通讯作者: 王立民, 周平 E-mail:Wanglm1980@126.com;zhpxqf@163.com
作者简介:任秀美奥,E-mail:748946454@qq.com。
基金资助:
兵团应用基础研究计划（2015AG011）；兵团中青年科技创新人才计划（2019CB002）；转基因重大专项（2016ZX08008001）

Effects of miR-449b on Early Embryo Development in Sheep

REN Xiumeiao^1,2, YAO Xudong^1,2, MENG Yaqi^1,2, GUO Yanhua², TANG Hong², ZHANG Yiyuan², ZHAO Xingwang³, WANG Limin², ZHOU Ping^1,2

1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
2. State Key Laboratory of Sheep Genetic Improvement and Healthy Production, Xinjiang Academy of Agricultural Reclamation Sciences, Shihezi 832000, China;
3. Agricultural Service Development Center of Shihezi General Farm, Shihezi 832000, China

Received:2021-12-15 Online:2022-07-05 Published:2022-06-29

摘要/Abstract

摘要： 【目的】研究miR-449b对绵羊早期胚胎发育的影响。【方法】采用Percoll密度梯度离心法获得纯化绵羊精子，将绵羊卵丘-卵母细胞复合体（COCs）在培养箱中培养16~18 h获得成熟卵母细胞，从1月龄纯种萨福克绵羊耳组织分离成纤维细胞，通过实时荧光定量PCR比较绵羊精子、成熟卵母细胞与成纤维细胞中miR-449b的相对表达量；给成熟卵母细胞分别显微注射0.9 %生理盐水（Con组）、miR-449b模拟物对照（NC mimic）与miR-449b模拟物（miR-449b mimic），注射后进行孤雌激活，分别于激活的第2、7天统计胚胎卵裂率与囊胚率；将成熟卵母细胞进行体外受精（IVF），收集IVF、Con、NC mimic与miR-449b mimic组2-细胞胚胎，采用免疫荧光法比较组蛋白H3K9me3的变化。【结果】miR-449b在精子中的表达显著高于成熟卵母细胞和成纤维细胞（P＜0.05）；与Con组相比，miR-449b mimic和NC mimic组胚胎的卵裂率均显著降低（P＜0.05），NC mimic组胚胎卵裂率显著低于miR-449b mimic组（P＜0，05）；miR-449b mimic组胚胎囊胚率提高，但差异不显著（P＞0.05）；IVF、Con、NC mimic与miR-449b mimic组2-细胞胚胎均表达组蛋白H3K9me3，且都在细胞核表达。与Con组相比，NC mimic组2-细胞胚胎组蛋白H3K9me3的表达水平显著增加（P＜0.05），miR-449b mimic组显著降低（P＜0.05）；NC mimic组2-细胞胚胎组蛋白H3K9me3的表达水平显著高于miR-449b mimic组（P<0.05），miR-449b mimic与IVF组无显著差异（P＞0.05）。【结论】miR-449b能显著提高绵羊早期胚胎的发育率，且降低早期胚胎中H3K9me3的表达水平。

关键词: miR-449b; 早期胚胎; H3K9me3; 精子

Abstract: 【Objective】 The study was to investigate the effect of miR-449b on early embryo development in sheep.【Method】 Purified sheep sperm and mature oocytes were obtained by Percoll density gradient centrifugation and in vitro maturation of cumulus-oocyte complexes (COCs) for 16-18 h,respectively.And fibroblasts were isolated from the ear tissues of one-month-old Suffolk sheep.Firstly,the relative expression of miR-449b in sperms,mature oocytes and fibroblasts were compared by Real-time quantitative PCR.Mature oocytes were injected with 0.9% normal saline (Con group),NC mimic (NC mimic group) and miR-449b mimic (miR-449b mimic group) by microinjection,respectively.Then the embryonic cleavage rate and blastocyst rate were counted on the 2nd and 7th day after parthenogenetic activation. Mature oocytes were fertilized in vitro (IVF), 2-cell embryos in IVF, Con, NC mimic and miR-449b mimic groups were collected, and the changes of histone H3K9me3 were compared by immunofluorescence method. 【Result】 The expression of miR-449b in sperm was significantly higher than that in mature oocytes and fibroblasts (P＜0.05). Compared with Con group, the cleavage rates of embryos in miR-449b mimic and NC mimic groups were significantly decreased (P＜0.05), and the cleavage rate of embryos in NC mimic group was significantly lower than that of miR-449b mimic group (P＜0.05). The blastocyst rate of embryos in miR-449b mimic group was increased, but the difference was not significant (P＞0.05). Histone H3K9me3 was expressed in 2-cell embryos in IVF, Con, NC mimic and miR-449b mimic groups, and was expressed in the nucleus. Compared with Con group, the expression level of histone H3K9me3 in 2-cell embryos in NC mimic group was significantly increased (P＜0.05), and was significantly decreased in miR-449b mimic group (P＜0.05). The expression level of histone H3K9me3 in 2-cell embryos in NC mimic group was significantly higher than that in miR-449b mimic group (P＜0.05), but there was no significant difference between miR-449b mimic and IVF groups (P＞0.05). 【Conclusion】 miR-449b could significantly increase the development rate of early sheep embryos and reduce the expression of H3K9me3 in early embryos.

Key words: miR-449b; early embryo; H3K9me3; sperm

中图分类号:

S813.1

任秀美奥, 姚旭东, 蒙亚琦, 郭延华, 唐红, 张译元, 赵兴旺, 王立民, 周平. miR-449b对绵羊早期胚胎发育的影响[J]. 中国畜牧兽医, 2022, 49(7): 2661-2668.

REN Xiumeiao, YAO Xudong, MENG Yaqi, GUO Yanhua, TANG Hong, ZHANG Yiyuan, ZHAO Xingwang, WANG Limin, ZHOU Ping. Effects of miR-449b on Early Embryo Development in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2661-2668.

参考文献

[1] RANDO O J.Daddy issues:Paternal effects on phenotype[J].Cell,2012,151:702-708.
[2] 贾丹.牛精子RNA对早期胚胎发育影响的初步研究[D].杨凌:西北农林科技大学,2016. JIA D.Effect of sperm-borne RNAs on the early development of bovine embryos[D].Yangling:Northwest A & F University,2016.(in Chinese)
[3] LEWIS B P,BURGE C B,BARTEL D P.Conserved seed pairing,often flanked by adenosines,indicates that thousands of human genes are microRNA targets[J].Cell,2005,120(1):15-20.
[4] CARRELL D T.Contributions of spermatozoa to embryogenesis:Assays to evaluate their genetic and epigenetic fitness[J]. Reproductive Biomedicine Online,2008,16(4):474-484.
[5] WANG M,GAO Y,QU P,et al.Sperm-borne miR-449b influences cleavage,epigenetic reprogramming and apoptosis of SCNT embryos in bovine[J].Scientific Reports,2017,7(1):13403.
[6] BAO J,LI D,WANG L,et al.microRNA-449 and microRNA-34b/c function redundantly in murine testes by targeting E2F transcription factor-retinoblastoma protein (E2F-pRb) pathway[J].Journal of Biological Chemistry,2012,287(26):21686-21698.
[7] WU J,ZHU H,SONG W,et al.Identification of conservative microRNAs in Saanen dairy goat testis through deep sequencing[J].Reproduction in Domestic Animals,2014,49:32-40.
[8] WANG C,LIU X,GAO Y,et al.Reprogramming of H3K9me3-dependent heterochromatin during mammalian embryo development[J]. Nature Cell Biology,2018,20(5):12.
[9] OKAMOTO I,OTTE A P,ALLIS C D,et al.Epigenetic dynamics of imprinted X inactivation during early mouse development[J].Science,2004,303:644-649.
[10] FELDMAN N,GERSON A,FANG J,et al.G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis[J].Nature Cell Biology,2006,8(2):188-194.
[11] FUKUDA A,TOMIKAWA J,MIURA T,et al.The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice[J].Nature Communications,2014,5(1):5564.
[12] TACHIBANA M.G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis[J].Genes & Development,2002,16(14):1779-1791.
[13] MESSERSCHMIDT D M,SOLTER D,KNOWLES B B,et al.Trim28 is required for epigenetic stability during mouse oocyte to embryo transition[J].Science,2012,335:1499-1502.
[14] YUAN S,SCHUSTER A,TANG C,et al.Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development[J].Development,2016,143(4):635-647.
[15] CONINE C,SUN F,SONG L,et al.Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice[J].Developmental Cell,2018,46(4):470-480.
[16] GRIVNA S T,PYHTILA B,LIN H.MIWI associates with translational machinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(36):13415-13420.
[17] KRAWETZ S A.Paternal contribution:New insights and future challenges[J].Nature Reviews Genetics,2005,6(8):633-642.
[18] LIN,S,GREGORY R I.microRNA biogenesis pathways in cancer[J].Nature Reviews Cancer,2015,15(6):321-333.
[19] CHENG C J,BAHAL R,BABAR I A,et al.microRNA silencing for cancer therapy targeted to the tumour microenvironment[J].Nature,2015,518(7537):107-110.
[20] 潘虹,陈蓓丽,李腾龑,等.miR-449b多态性位点rs10061133差异调控LHCGR表达[J].生殖医学杂志,2019,28(12):5. PAN H,CHEN B L,LI T Y,et al.miR-449b polymorphism rs10061133 differentially regulates LHCGR expression[J].Journal of Reproductive Medicine,2019,28(12):5.(in Chinese)
[21] LIU W M,PANG R T,CHIU P C,et al.Sperm-borne microRNA-34c is required for the first cleavage division in mouse[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(2):490-494.
[22] SINHA P B,TESFAVE D,RINGS F,et al.microRNA-130b is involved in bovine granulosa and cumulus cells function,oocyte maturation and blastocyst formation[J].Journal of Ovarian Research,2017,10(1):37.
[23] WANG M,DU Y,GAO S,et al.Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling[J].Development,2021,148(5):1-13.
[24] WANG L,ZHANG J,DUAN J,et al.Programming and inheritance of parental DNA methylomes in mammals[J].Cell,2014,157(4):979-991.
[25] YAMANAKA K I,SUGIMURA S,WAKAI T,et al.Acetylation level of histone H3 in early embryonic stages affects subsequent development of miniature pig somatic cell nuclear transfer embryos[J].Journal of Reproduction and Development,2009,55(6):638-644.
[26] ZHOU L Q,DEAN J.Reprogramming the genome to totipotency in mouse embryos[J].Trends in Cell Biology,2015,25(2):82-91.
[27] YONG J C,LIN C P,RISSO D,et al.Deficiency of microRNAmiR-34a expands cell fate potential in pluripotent stem cells[J].Science,2017,355(6325):581-582.
[28] MACFARLAN T S,GIFFORD W D,DRISCOLL S,et al.Embryonic stem cell potency fluctuates with endogenous retrovirus activity[J].Nature,2012,487(7405):57-63.

miR-449b对绵羊早期胚胎发育的影响

Effects of miR-449b on Early Embryo Development in Sheep

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