热应激对牛卵子及其胚胎表观遗传修饰与发育能力的影响

doi:10.11843/j.issn.0366-6964.2024.06.018

摘要/Abstract

摘要：

旨在探究热应激对牛卵子及其胚胎表观遗传修饰与发育能力的影响。本研究将卵子置于体外热应激条件下培养(41℃ 12 h+38.5℃ 12 h)，进行体外成熟(in vitro maturtion，IVM)和体外受精(in vitro fertilization，IVF)，检测对照组(38.5℃ 24 h)和热应激组牛卵子和胚胎的发育能力及表观遗传修饰组蛋白H1、组蛋白H2、组蛋白H4和DNA甲基化的修饰水平。本研究还检测了卵子活性氧(reactive oxygen species，ROS)水平、线粒体膜电位(mitochondrial membrane potential，ΔΨm)水平及与表观遗传修饰和发育能力相关基因的表达水平。结果表明，热应激处理组卵子成熟率((59.21±4.29)%)、卵裂率((57.78±4.58)%)和囊胚率((22.31±1.67)%)均显著低于对照组((85.10±6.75)%、(78.64±2.46)%、(42.64±1.38)%，P < 0.05)；热应激组牛卵子及各阶段胚胎表观遗传修饰组蛋白H1、组蛋白H2、组蛋白H4、DNA甲基化水平显著低于对照组(P < 0.05)；热应激组牛卵子ΔΨm水平显著低于对照组(P < 0.05)；热应激组牛卵子ROS水平显著高于对照组(P < 0.05)；热应激组牛卵子表观遗传修饰相关基因DNMT1、DNMT3A、DNMT3B、Histone H2A、SMYD3、IGF-2R的mRNA表达水平显著低于对照组(P < 0.05)；热应激组牛卵子发育能力相关基因C-MOS、GDF-9和POU5F1的mRNA表达水平显著低于对照组(P < 0.05)。本研究表明，热应激显著降低了牛卵子和胚胎表观遗传修饰组蛋白H1、组蛋白H2、组蛋白H4、DNA甲基化水平，显著降低了牛卵子ΔΨm水平及与表观遗传修饰和发育能力相关基因的mRNA表达水平，显著提高了ROS水平，降低了卵子质量。

关键词: 热应激, 牛, 卵子, 表观遗传修饰, 发育能力

Abstract:

The aim of this study was to investigate the effects of heat stress on epigenetic modifications and developmental capacity of bovine oocytes and their embryos. In this study, oocytes were cultured under in vitro heat stress conditions (41 ℃ for 12 h+38.5 ℃ for 12 h) for in vitro maturation (IVM) and in vitro fertilization (IVF). The developmental ability of oocytes and embryos and the modification levels of epigenetic modifications histone H1, histone H2, histone H4, and DNA methylation were examined in control (38.5 ℃ for 24 h) and heat-stressed cattle. In this study, we also examined oocyte reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm) levels, and expression levels of genes associated with epigenetic modifications and developmental competence. The results showed that the maturation rate ((59.21±4.29)%), oocyte cleavage rate ((57.78±4.58)%) and blastocyst rate ((22.31±1.67)%) of the heat stress treatment group were significantly lower than those of the control group ((85.10±6.75)%, (78.64±2.46)%, and (42.64±1.38)%, P < 0.05); The levels of epigenetic modifications histone H1, histone H2, histone H4, and DNA methylation of bovine oocytes and embryos at all stages in the heat stress group were significantly lower than those of the control group (P < 0.05); the level of ΔΨm of bovine oocytes in the heat stress group was significantly lower than that of the control group (P < 0.05); the level of ROS in bovine oocytes of heat stress group was significantly higher than that of the control group (P < 0.05); the mRNA expression levels of genes related to epigenetic modification of bovine oocytes DNMT1, DNMT3A, DNMT3B, Histone H2A, SMYD3, and IGF-2R were significantly lower in the heat stress group than in the control group (P < 0.05); the mRNA expression levels of the genes C-MOS, GDF-9 and POU5F1 related to oocyte developmental competence were significantly lower in the heat stress group than in the control group (P < 0.05). The results showed that heat stress significantly reduced the levels of epigenetic modifications histone H1, histone H2, histone H4, and DNA methylation, significantly decreased the levels of ΔΨm and mRNA expression of genes related to epigenetic modifications and developmental competence, and significantly increased the levels of ROS and reduced the quality of bovine oocytes and embryos.

Key words: heat stress, bovine, oocyte, epigenetic modifications, developmental competence

中图分类号:

S823.3

冯肖艺, 张培培, 张航, 郝海生, 杜卫华, 朱化彬, 崔凯, 赵学明. 热应激对牛卵子及其胚胎表观遗传修饰与发育能力的影响[J]. 畜牧兽医学报, 2024, 55(6): 2460-2473.

Xiaoyi FENG, Peipei ZHANG, Hang ZHANG, Haisheng HAO, Weihua DU, Huabin ZHU, Kai CUI, Xueming ZHAO. Effects of Heat Stress on Epigenetic Modifications and Developmental Competence of Bovine Oocytes and Their Embryos[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2460-2473.

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基因 Gene	引物序列(5′→3′) Primers sequence	产物长度/bp Length	登录号 GenBank accession No.
GAPDH	F：GGGTCATCATCTCTGCACC	177	NM_001034034
	R：GGTCATAAGTCCCTCCACG
DNMT1	F: AGTGGGGGACTGTGTTTCTG	218	XM_015471992.2
	R: TGCTGTGGATGTACGAGAGC
DNMT3A	F: AGCACAACGGAGAAGCCTAA	245	NM_001206502.2
	R: CAGCAGATGGTGCAGTAGGA
DNMT3B	F: GAGAATAAGACGCGGAGACG	146	NM_181813.2
	R: ACATCCGAAGCCATTTGTTC
Histone H2A	F: GCGGTCTTGGAGTACCTGAC	204	BF076713.1
	R: AGTCTTCTTCGGGAGCAACA
IGF-2R	F: GTCGTGCAGATCAGTCCTCA	152	NM_174352.2
	R: TCGTTCTGGAGCTGAAAGGT
SMYD3	F: GACCATGGAGCCTTACAGGA	135	NM_001076406.2
	R: TCAAAAGCCAGCCTCAGATT
C-MOS	F：TCTACTCCTTCGCCATCACC	260	XM_002692654.4
	R：GAGTCCTCCCAGCTCCTCTT
GDF-9	F：CTTTGCCTGGCTCTGTTTTC	116	NM_174681.2
	R：GAGTCCTCCCAGCTCCTCTT
POU5F1	F：GTTTTGAGGCTTTGCAGCTC	182	NM_174580.3
	R：CTCCAGGTTGCCTCTCACTC

组别 Group	卵子数/个 No. of oocytes	成熟率/% Maturity rate	卵裂率/% Cleavage rate	囊胚率/% Blastocyst rate
对照组 Control group	396	85.10±6.75(337/396)^a	78.64±2.46(265/337)^a	42.64±1.38(113/265)^a
热应激组 Heat stress group	760	59.21±4.29(450/760)^b	57.78±4.58(260/450)^b	22.31±1.67(58/260)^b

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