高温应激下湖羊卵巢颗粒细胞m6A甲基化修饰差异研究

doi:10.11843/j.issn.0366-6964.2025.04.020

摘要/Abstract

摘要：

旨在研究高温应激对湖羊卵巢颗粒细胞中m⁶A甲基化修饰的影响，为揭示高温应激下m⁶A甲基化修饰对湖羊卵巢颗粒细胞发育和功能的调控机制奠定基础。本研究采集2岁龄左右湖羊母羊新鲜卵巢，收集卵巢表面3~5 mm卵泡中的颗粒细胞，随机分为2组，对照组(37 ℃)和高温应激组(42 ℃，每天2 h，连续3 d)。通过甲基化RNA免疫共沉淀测序(MeRIP-seq)鉴定m⁶A峰，获得基因表达数据并进行分析。MeRIP-seq在对照组和高温应激组共有135个具有显著差异的m⁶A峰，映射到130个差异甲基化基因，主要富集到PI3K-Akt信号通路、谷胱甘肽代谢和Wnt信号通路等。RNA-seq鉴定出359个显著差异表达基因，主要富集在类固醇合成、胆固醇生物合成等信号通路。MeRIP-seq和RNA-seq数据联合分析结果显示，DTX3L、MAGEF1、MAN1C1、CCDC77、RAD51共计5个基因m⁶A修饰和基因表达水平同时存在显著差异，主要富集在Notch信号通路等。本研究结果表明，高温应激会改变湖羊卵巢颗粒细胞mRNA中m⁶A修饰水平，最终可能影响细胞增殖、凋亡、雌激素合成等相关通路，为进一步探究m⁶A修饰在高温应激下的具体作用机制提供了理论基础。

关键词: 高温应激, 卵巢颗粒细胞, 湖羊, m⁶A甲基化修饰, MeRIP-seq

Abstract:

The aim of this study was to investigate the effect of heat stress on m⁶A methylation modification in Hu sheep ovarian granulosa cells, so as to lay the foundation for revealing the regulatory mechanism of m⁶A methylation modification on the development and function of Hu sheep ovarian granulosa cells under heat stress. In this study, fresh ovaries of 2-year-old Hu ewes were collected, and granulosa cells in 3-5 mm follicles on the surface of ovaries were collected. They were randomly divided into two groups: control group (37 ℃) and heat stress group (42 ℃, 2 h per day for 3 consecutive days). The m⁶A peak was identified by methylated RNA immunoprecipitation sequencing (MeRIP-seq), and the gene expression data were obtained and analyzed. There were 135 significantly different m⁶A peaks in MeRIP-seq between the control group and the heat stress group, which were mapped to 130 differentially methylated genes, mainly enriched in the PI3K-Akt signaling pathway, glutathione metabolism and Wnt signaling pathway. RNA-seq identified 359 significantly differentially expressed genes, which were mainly enriched in steroid synthesis, cholesterol biosynthesis and other signaling pathways. The combined analysis of MeRIP-seq and RNA-seq data showed that there were significant differences in m⁶A modification and gene expression levels of five genes, DTX3L, MAGEF1, MAN1C1, CCDC77 and RAD51, which were mainly enriched in the Notch signaling pathway. This study results shows that heat stress can change the mRNA m⁶A modification level of Hu sheep ovarian granulosa cells, which may eventually affect cell proliferation, apoptosis, estrogen synthesis and other related pathways, providing a theoretical basis for further exploring the specific mechanism of m⁶A modification under heat stress.

Key words: heat stress, ovarian granulosa cells, Hu sheep, m⁶A methylation modification, MeRIP-seq

中图分类号:

S826.3

李笑微, 田微, 刘媛, 李惠侠. 高温应激下湖羊卵巢颗粒细胞m⁶A甲基化修饰差异研究[J]. 畜牧兽医学报, 2025, 56(4): 1712-1721.

LI Xiaowei, TIAN Wei, LIU Yuan, LI Huixia. Study on the Difference of m⁶A Methylation Modification in Ovarian Granulosa Cells of Hu Sheep under Heat Stress[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1712-1721.

图/表 10

表 1

表 2

图 1

图 2

图 3

图 4

图 5

图 6

图 7

图 8

参考文献 33

1	ZHOU R , LIU D . The function of exosomes in ovarian granulosa cells[J]. Cell Tissue Res, 2023, 394 (2): 257- 267. doi: 10.1007/s00441-023-03820-3
2	RUSSELL D L , ROBKER R L . Molecular mechanisms of ovulation: co-ordination through the cumulus complex[J]. Hum Reprod Update, 2007, 13 (3): 289- 312. doi: 10.1093/humupd/dml062
3	HSUEH A J , KAWAMURA K , CHENG Y , et al. Intraovarian control of early folliculogenesis[J]. Endocr Rev, 2015, 36 (1): 1- 24. doi: 10.1210/er.2014-1020
4	ZHANG C H , LIU X Y , WANG J . Essential role of granulosa cell glucose and lipid metabolism on oocytes and the potential metabolic imbalance in polycystic ovary syndrome[J]. Int J Mol Sci, 2023, 24 (22): 16247. doi: 10.3390/ijms242216247
5	WANG S J , LIU W J , WU C J , et al. Melatonin suppresses apoptosis and stimulates progesterone production by bovine granulosa cells via its receptors (MT1 and MT2)[J]. Theriogenology, 2012, 78 (7): 1517- 1526. doi: 10.1016/j.theriogenology.2012.06.019
6	CARABATSOS M J , SELLITTO C , GOODENOUGH D A , et al. Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence[J]. Dev Biol, 2000, 226 (2): 167- 179. doi: 10.1006/dbio.2000.9863
7	VAN WETTERE W H E J , CULLEY S , SWINBOURNE A M F , et al. Heat stress from current and predicted increases in temperature impairs lambing rates and birth weights in the Australian sheep flock[J]. Nat Food, 2024, 5 (3): 206- 210. doi: 10.1038/s43016-024-00935-w
8	VAN WETTERE W H E J , KIND K L , GATFORD K L , et al. Review of the impact of heat stress on reproductive performance of sheep[J]. J Anim Sci Biotechnol, 2021, 12 (1): 26. doi: 10.1186/s40104-020-00537-z
9	MANABE N , GOTO Y , MATSUDA-MINEHATA F , et al. Regulation mechanism of selective atresia in porcine follicles: regulation of granulosa cell apoptosis during atresia[J]. J Reprod Dev, 2004, 50 (5): 493- 514. doi: 10.1262/jrd.50.493
10	MU H , CAI S , WANG X , et al. RNA binding protein IGF2BP1 meditates oxidative stress-induced granulosa cell dysfunction by regulating MDM2 mRNA stability in an m6A-dependent manner[J]. Redox Biol, 2022, 57, 102492. doi: 10.1016/j.redox.2022.102492
11	ZHU L , ZHANG H , ZHANG X , et al. RNA m6A methylation regulators in sepsis[J]. Mol Cell Biochem, 2023, 479 (9): 2165- 2180.
12	SAMMAD A , LUO H , HU L , et al. Transcriptome reveals granulosa cells coping through redox, inflammatory and metabolic mechanisms under acute heat stress[J]. Cells, 2022, 11 (9): 1443. doi: 10.3390/cells11091443
13	LU Z , LIU J , YUAN C , et al. m(6)A mRNA methylation analysis provides novel insights into heat stress responses in the liver tissue of sheep[J]. Genomics, 2021, 113 (1 Pt 2): 484- 492.
14	LU Z , MA Y , LI Q , et al. The role of N(6)-methyladenosine RNA methylation in the heat stress response of sheep (Ovis aries)[J]. Cell Stress Chaperones, 2019, 24 (2): 333- 342. doi: 10.1007/s12192-018-00965-x
15	CHEN B , YUAN C , GUO T , et al. Molecular mechanism of m6A methylation modification genes METTL3 and FTO in regulating heat stress in sheep[J]. Int J Mol Sci, 2023, 24 (15): 11926. doi: 10.3390/ijms241511926
16	ZHANG Y , YAN C , XIE Q , et al. Exposure to bisphenol A affects transcriptome-wide N6-methyladenine methylation in ovarian granulosa cells[J]. Ecotoxicol Environ Saf, 2024, 272, 116071. doi: 10.1016/j.ecoenv.2024.116071
17	LIU K , ZHOU X , LI C , et al. YTHDF2 as a Mediator in BDNF-Induced Proliferation of Porcine Follicular Granulosa Cells[J]. Int J Mol Sci, 2024, 25 (4): 2343. doi: 10.3390/ijms25042343
18	CAO M , CHEN X , WANG Y , et al. The reduction of the m(6)A methyltransferase METTL3 in granulosa cells is related to the follicular cysts in pigs[J]. J Cell Physiol, 2024, 239 (6): e31289. doi: 10.1002/jcp.31289
19	CAO M , YUAN C , CHEN X , et al. METTL3 deficiency leads to ovarian insufficiency due to IL-1beta overexpression in theca cells[J]. Free Radic Biol Med, 2024, 222, 72- 84. doi: 10.1016/j.freeradbiomed.2024.05.048
20	LIU Z , ZHOU L , LI D , et al. N6-methyladenosine methyltransferase METTL3 modulates the cell cycle of granulosa cells via CCND1 and AURKB in Haimen goats[J]. FASEB J, 2023, 37 (11): e31289.
21	DING H , LI Z , LI X , et al. FTO Alleviates CdCl(2)-induced apoptosis and oxidative stress via the AKT/Nrf2 pathway in bovine granulosa cells[J]. Int J Mol Sci, 2022, 23 (9): 4948. doi: 10.3390/ijms23094948
22	LU J , ZHAO P , DING X , et al. N-acetylcysteine stimulates the proliferation and differentiation in heat-stressed skeletal muscle cells[J]. J Therm Biol, 2024, 124, 103958. doi: 10.1016/j.jtherbio.2024.103958
23	GAO M , SHEN H , LI Q , et al. Perfluorooctane sulfonate (PFOS) induces apoptosis and autophagy by inhibition of PI3K/AKT/mTOR pathway in human granulosa cell line KGN[J]. Environ Pollut, 2024, 344, 123333. doi: 10.1016/j.envpol.2024.123333
24	DENG X , NING Z , LI L , et al. High expression of miR-22-3p in chicken hierarchical follicles promotes granulosa cell proliferation, steroidogenesis, and lipid metabolism via PTEN/PI3K/Akt/mTOR signaling pathway[J]. Int J Biol Macromol, 2023, 253 (Pt 7): 127415.
25	ZHANG T Y , SUN X F , LI L , et al. Ochratoxin A Exposure Impairs Porcine Granulosa Cell Growth via the PI3K/AKT Signaling Pathway[J]. J Agric Food Chem, 2019, 67 (9): 2679- 2690. doi: 10.1021/acs.jafc.8b06361
26	FRANCO R , CIDLOWSKI J A . Apoptosis and glutathione: beyond an antioxidant[J]. Cell Death Differ, 2009, 16 (10): 1303- 1314. doi: 10.1038/cdd.2009.107
27	WANG H X , LI T Y , KIDDER G M . WNT2 regulates DNA synthesis in mouse granulosa cells through beta-catenin[J]. Biol Reprod, 2010, 82 (5): 865- 875. doi: 10.1095/biolreprod.109.080903
28	HABARA O , LOGAN C Y , KANAI-AZUMA M , et al. Self-activation of Wnt signaling in pre-granulosa cells is required for ovarian folliculogenesis[J]. Development, 2020, 148 (9): dev198846.
29	LI L , JI S Y , YANG J L , et al. Wnt/beta-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components[J]. Mol Cell Endocrinol, 2014, 382 (2): 915- 925. doi: 10.1016/j.mce.2013.11.007
30	WANG L J , XUE Y , HUO R , et al. N6-methyladenosine methyltransferase METTL3 affects the phenotype of cerebral arteriovenous malformation via modulating Notch signaling pathway[J]. J Biomed Sci, 2020, 27 (1): 62. doi: 10.1186/s12929-020-00655-w
31	JING J , JIANG X , CHEN J , et al. Notch signaling pathway promotes the development of ovine ovarian follicular granulosa cells[J]. Anim Reprod Sci, 2017, 181, 69- 78. doi: 10.1016/j.anireprosci.2017.03.017
32	FLORKE GEE R R , CHEN H , LEE A K , et al. Emerging roles of the MAGE protein family in stress response pathways[J]. J Biol Chem, 2020, 295 (47): 16121- 16155. doi: 10.1074/jbc.REV120.008029
33	LIU X L , WU R Y , SUN X F , et al. Mycotoxin zearalenone exposure impairs genomic stability of swine follicular granulosa cells in vitro[J]. Int J Biol Sci, 2018, 14 (3): 294- 305. doi: 10.7150/ijbs.23898

样品 Sample	原始序列/条 Raw reads	原始测序量/G Raw bases	有效序列/条 Valid reads	有效测序量/G Valid bases	有效碱基比例/% Valid base proportion	Q20/%	Q30/%	GC%
CON_IP	47 365 524	7.10	41 455 642	6.21	87.52	98.29	94.97	52.09
CON_input	54 530 712	8.18	49 710 036	7.46	91.16	98.54	95.56	53.01
HS_IP	48 938 176	7.34	42 650 390	6.40	87.15	98.31	95.02	51.66
HS_input	51 379 578	7.71	46 873 704	7.03	91.23	98.61	95.73	52.66

样品 Sample	有效序列/条 Valid reads	比对序列/条 Mapped reads	唯一比对序列/条 Unique mapped reads	多比对序列/条 Multi mapped reads	PE比对序列/条 PE mapped reads
CON_IP	41 455 642	37 148 104 (89.61%)	34 601 443 (83.47%)	2 546 661 (6.14%)	35 690 352 (86.09%)
CON_input	49 710 036	44 628 418 (89.78%)	41 778 005 (84.04%)	2 850 413 (5.73%)	43 358 814 (87.22%)
HS_IP	42 650 390	38 034 618 (89.18%)	35 149 335 (82.41%)	2 885 283 (6.76%)	36 482 296 (85.54%)
HS_input	46 873 704	41 651 352 (88.86%)	38 810 303 (82.80%)	2 841 049 (6.06%)	40 355 184 (86.09%)

样品 Sample	序列比对到正义链/条 Reads map to sense strand	序列比对到负义链/条 Reads map to antisense strand	非拼接序列/条 Non-splice reads	拼接序列/条 Splice reads
CON_IP	17 289 541 (41.71%)	17 311 902 (41.76%)	18 036 150 (43.51%)	16 565 293 (39.96%)
CON_input	20 876 469 (42.00%)	20 901 536 (42.05%)	20 787 178 (41.82%)	20 990 827 (42.23%)
HS_IP	17 561 443 (41.18%)	17 587 892 (41.24%)	18 303 008 (42.91%)	16 846 327 (39.50%)
HS_input	19 373 947 (41.33%)	19 436 356 (41.47%)	19 136 673 (40.83%)	19 673 630 (41.97%)

[1]	王莹, 张姣姣, 王鲜忠, 权富生. 卵巢颗粒细胞自噬研究进展[J]. 畜牧兽医学报, 2025, 56(4): 1508-1517.
[2]	何雨, 王翔宇, 狄冉, 储明星, 梁琛. BMP4/SMAD4通过下调GJA1基因表达影响绵羊卵巢颗粒间隙连接活性[J]. 畜牧兽医学报, 2025, 56(2): 679-688.
[3]	李玮, 吴禧龙, 赵兴瑞, 许兰娇, 杨小斌, 宋小珍. 中药健脾四胃方剂对断奶湖羊生长性能、瘤胃发酵及菌群组成的影响[J]. 畜牧兽医学报, 2025, 56(1): 466-478.
[4]	何明亮, 吕晓阳, 蒋永清, 宋正海, 王叶青, 杨会国, 王善禾, 孙伟. 基于转录组测序分析SOX18在湖羊毛囊毛乳头细胞中的功能[J]. 畜牧兽医学报, 2024, 55(6): 2409-2420.
[5]	刘阳光, 章会斌, 文浩宇, 谢帆, 赵世明, 丁月云, 郑先瑞, 殷宗俊, 张晓东. 猪卵泡液外泌体处理卵巢颗粒细胞的SNP/Indel筛选分析[J]. 畜牧兽医学报, 2024, 55(2): 576-586.
[6]	张丽娃, 岳耀敬, 安雪姣, 李建烨, 杨博辉, 徐振飞, 张金霞, 耿智广, 郭艳丽, 张瑞. 湖羊及其不同杂交组合肌肉氨基酸、脂肪酸和挥发性风味物质比较分析[J]. 畜牧兽医学报, 2024, 55(10): 4428-4442.
[7]	段香茹, 康佳, 杨若晨, 单新雨, 李太春, 赵雯, 张英杰, 刘月琴. L-半胱氨酸对绵羊卵巢颗粒细胞增殖、凋亡和类固醇激素分泌的影响[J]. 畜牧兽医学报, 2024, 55(1): 179-191.
[8]	刘林丽, 彭雪兰, 李博, 程乐凡, 次仁拉姆, 张恩平. 过表达UCP3基因对萨湖羊前体脂肪细胞分化的影响[J]. 畜牧兽医学报, 2023, 54(8): 3275-3285.
[9]	熊程坤, 张道亮, 杨悦, 丁红研, 赵杰, 李玉, 王希春, 冯士彬, 赵畅, 汤继顺, 吴金节. 芦丁对围产期湖羊瘤胃发酵、瘤胃菌群结构及抗氧化性能的影响[J]. 畜牧兽医学报, 2023, 54(7): 2898-2909.
[10]	胡亚美, 宋湘容, 黄亮, 张璐通, 高磊, 庞卫军, 杨公社, 褚瑰燕. FGF21增强线粒体功能抑制猪卵巢颗粒细胞凋亡[J]. 畜牧兽医学报, 2023, 54(3): 1034-1045.
[11]	叶倩文, 陈卓, 李鑫, 孙亚伟, 金肖叶, 李紫仟, 吾买尔江·牙合甫, 钟旗, 马雪连, 姚刚. 一月龄吮乳羔羊肠道菌群组成及其预测物质代谢功能的动态变化研究[J]. 畜牧兽医学报, 2023, 54(3): 1095-1108.
[12]	孙美杰, 曹力文, 郑文金, 申军士, 朱伟云. 基于转录组学研究日粮添加尿素对育肥湖羊肝组织氨代谢的影响[J]. 畜牧兽医学报, 2023, 54(3): 1148-1159.
[13]	王雅涵, 郑宇婧, 钟沛, 李晓丹, 王锋. YAP1在发情期不同繁殖力湖羊子宫内膜中的表达模式及功能分析[J]. 畜牧兽医学报, 2023, 54(1): 189-200.
[14]	韦肖, 张建童, 龙唐晖, 李开嵘, 李艳娇, 欧阳克蕙, 邱清华. 日粮能量水平对湖羊瘤胃发酵特性和微生物组成的影响[J]. 畜牧兽医学报, 2022, 53(9): 3042-3051.
[15]	李宇, 段春辉, 宋志攀, 岳思聪, 王媛, 张英杰, 刘月琴. 黄体期注射PGF2α对母羊血清生殖激素及相关细胞因子的影响[J]. 畜牧兽医学报, 2022, 53(6): 1807-1818.

高温应激下湖羊卵巢颗粒细胞m⁶A甲基化修饰差异研究

Study on the Difference of m⁶A Methylation Modification in Ovarian Granulosa Cells of Hu Sheep under Heat Stress

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 33

相关文章 15

编辑推荐

Metrics

本文评价