Mfn2减轻内质网应激抑制绵羊卵泡颗粒细胞凋亡的分子机制

doi:10.16288/j.yczz.24-247

遗传 ›› 2025, Vol. 47 ›› Issue (3): 342-350.doi: 10.16288/j.yczz.24-247

Mfn2减轻内质网应激抑制绵羊卵泡颗粒细胞凋亡的分子机制

古丽米热·阿布都热依木¹(), 陈莹¹, 唐淑红², 董红³, 汪立芹¹, 吴阳升¹, 黄俊成³(), 林嘉鹏¹()

1.农业农村部草食家畜遗传育种与繁殖重点实验室，新疆动物生物技术重点实验室，乌鲁木齐 830011
2.阿克苏地区畜牧技术推广中心，阿克苏 843000
3.新疆畜牧科学院畜牧研究所，乌鲁木齐 830011

收稿日期:2024-08-23 修回日期:2024-12-05 出版日期:2025-03-20 发布日期:2025-01-08
通讯作者: 林嘉鹏，博士，研究员，研究方向：动物遗传育种与繁殖。E-mail: linjiapeng5188@163.com;
黄俊成，博士，研究员，研究方向：家畜繁殖生物技术。E-mail: h_jc@sina.com
作者简介:古丽米热·阿布都热依木，硕士研究生，专业方向：动物遗传育种与繁殖。E-mail: gulimire127@163.com
基金资助:
新疆维吾尔自治区重大专项(2023A02011-2);新疆维吾尔自治区羊产业技术体系项目(XJARS-09-08);新疆维吾尔自治区三农骨干人才培养项目(2023SNGGNT031);“天池英才”引进计划项目资助

Molecular mechanism of Mfn2 alleviating endoplasmic reticulum stress and inhibiting apoptosis of sheep follicular granulosa cells

Gulimire·Abudureyimu ¹(), Ying Chen¹, Shuhong Tang², Hong Dong³, Liqin Wang¹, Yangsheng Wu¹, Juncheng Huang³(), Jiapeng Lin¹()

1. Key Laboratory of Genetics Breeding and Reproduction of Grass Feeding Livestock, Ministry of Agriculture and Rural Affairs, Key Laboratory of Animal Biotechnology of Xinjiang, Institute of Biotechnology, Xinjiang Academy of Animal Sciences, Urumqi 830011, China
2. Akesu Region Animal Husbandry Technology Promotion Center, Aksu 843000, China
3. Institute of Animal Sciences, Xinjiang Academy of Animal Science, Urumqi 830011, China

Received:2024-08-23 Revised:2024-12-05 Published:2025-03-20 Online:2025-01-08
Supported by:
Xinjiang Uygur Autonomous Region Major Special Project(2023A02011-2);Xinjiang Uygur Autonomous Region Sheep Agriculture Research System Project(XJARS-09-08);Xinjiang Uygur Autonomous Region Three Rural Backbone Talent Training Project(2023SNGGNT031);Tianchi Talent Introduction Plan Project

摘要/Abstract

摘要：

卵泡发育是哺乳动物生殖过程中至关重要的环节。线粒体融合蛋白2 (mitofusin 2，Mfn2)是一种参与调节线粒体融合的GTP酶，是维持线粒体网络完整性和功能的关键因素，其在卵泡发育过程中调控线粒体功能与内质网应激的具体作用尚不明确。基于此，本研究探讨了Mfn2在成年绵羊卵泡发育过程中的作用。通过收集大、中、小卵泡并分离大卵泡的颗粒细胞(granulosa cells，GCs)，利用qRT-PCR和Western blot检测Mfn2在不同卵泡中的表达水平，并通过免疫荧光技术确定Mfn2在卵泡中的定位。同时，检测了线粒体自噬相关蛋白Pink1、内质网应激蛋白(Grp78、Perk、Chop)和凋亡相关蛋白(Bcl2和BAX)的表达。进一步通过在GCs中转染siRNAs敲降Mfn2，评估细胞内Ca²⁺积累及线粒体膜电位的变化，并检测上述蛋白的表达水平。结果表明，Mfn2在大卵泡中的表达显著高于小卵泡，且主要表达于GCs。与小卵泡相比，大卵泡中Pink1、Grp78、Perk、Chop和BAX的表达显著降低，而Bcl2的表达显著增加(P<0.01)。Mfn2敲降后，细胞内Ca²⁺水平及线粒体膜电位显著降低，Pink1、Grp78、Perk、Chop和BAX的表达显著升高，而Bcl2的表达显著降低(P<0.01)。Mfn2可能通过调控线粒体功能及内质网应激，影响绵羊卵泡发育过程中的细胞凋亡。

关键词: 绵羊卵泡, 颗粒细胞, Mfn2, 内质网应激, 细胞凋亡

Abstract:

Follicle development is a crucial step in mammalian reproductive processes, the specific role of Mfn2 in regulating mitochondrial function and endoplasmic reticulum stress in this process is still unclear, this study aimed to investigate the role of Mfn2 in the follicular development of adult sheep. Large, medium, and small follicles were collected, and granulosa cells (GCs) were isolated from large follicles. The expression levels of Mfn2 in different follicles were detected using qRT-PCR and Western blot, and the localization of Mfn2 in follicles was determined through immunofluorescence. Additionally, the expression levels of the mitochondrial autophagy-related protein Pink1, endoplasmic reticulum stress proteins (Grp78, Perk, Chop), and apoptosis-related proteins (Bcl2 and BAX) were detected. Furthermore, siRNAs were transfected into GCs to knock down Mfn2 expression, and changes in intracellular Ca²⁺ accumulation and mitochondrial membrane potential were evaluated, along with the expression levels of the aforementioned proteins. The results showed that Mfn2 expression was significantly higher in large follicles compared to small follicles and was primarily localized in GCs. Compared to small follicles, the expression levels of Pink1, Grp78, Perk, Chop, and BAX were significantly lower in large follicles, while Bcl2 expression was significantly increased (P<0.01). After Mfn2 knockdown, intracellular Ca²⁺ levels and mitochondrial membrane potential were significantly reduced, while the expression levels of Pink1, Grp78, Perk, Chop, and BAX were significantly increased, and Bcl2 expression was significantly decreased (P<0.01). Mfn2 may influence cell apoptosis during sheep follicular development by regulating mitochondrial function and endoplasmic reticulum stress.

Key words: sheep follicles, granulosa cells, Mfn2, endoplasmic reticulum stress, cell apoptosis

古丽米热·阿布都热依木, 陈莹, 唐淑红, 董红, 汪立芹, 吴阳升, 黄俊成, 林嘉鹏. Mfn2减轻内质网应激抑制绵羊卵泡颗粒细胞凋亡的分子机制[J]. 遗传, 2025, 47(3): 342-350.

Gulimire·Abudureyimu , Ying Chen, Shuhong Tang, Hong Dong, Liqin Wang, Yangsheng Wu, Juncheng Huang, Jiapeng Lin. Molecular mechanism of Mfn2 alleviating endoplasmic reticulum stress and inhibiting apoptosis of sheep follicular granulosa cells[J]. Hereditas(Beijing), 2025, 47(3): 342-350.

图/表 7

表1

图1

图2

图3

图4

图5

图6

参考文献 20

[1]	Telfer EE, Grosbois J, Odey YL, Rosario R, Anderson RA. Making a good egg: human oocyte health, aging, and in vitro development. Physiol Rev, 2023, 103(4): 2623-2677.
[2]	Chen XH, He HZ, Long BC, Wei BL, Yang P, Huang XY, Wang Q, Lin J, Tang HL. Acupuncture regulates the apoptosis of ovarian granulosa cells in polycystic ovarian syndrome-related abnormal follicular development through LncMEG3-mediated inhibition of miR-21-3p. Biol Res, 2023, 56(1): 31. doi: 10.1186/s40659-023-00441-6 pmid: 37303036
[3]	Arslan NP, Taskin M, Keles ON. Nicotinamide mononucleotide and nicotinamide riboside reverse ovarian aging in rats via rebalancing mitochondrial fission and fusion mechanisms. Pharm Res, 2024, 41(6): 921-935.
[4]	Wang ZH, Wang ZJ, Liu HC, Wang CY, Wang YQ, Yue Y, Zhao C, Wang GY, Wan JP. Targeting mitochondria for ovarian aging: new insights into mechanisms and therapeutic potential. Front Endocrinol (Lausanne), 2024, 15: 1417007.
[5]	Liao XH, Zhu SQ, Qiu SM, Cao H, Jiang WW, Xu HL, Sun Y, Zheng BH. Mfn2 regulates mitochondria-associated ER membranes to affect PCOS oocyte development. Endocr Connect, 2024, 13(1): e230343.
[6]	Basha EH, Eltokhy AKB, Eltantawy AF, Heabah NAE, Elshwaikh SL, El-Harty YM. Linking mitochondrial dynamics and fertility: promoting fertility by phoenixin through modulation of ovarian expression of GnRH receptor and mitochondrial dynamics proteins DRP-1 and Mfn-2. Pflugers Arch, 2022, 474(10): 1107-1119.
[7]	Liu WJ, Li LS, Lan MF, Shang JZ, Zhang JX, Xiong WJ, Lai XL, Duan X. Zinc deficiency deteriorates ovarian follicle development and function by inhibiting mitochondrial function. J Ovarian Res, 2024, 17(1): 115.
[8]	Zhu MK, Yan M, Chen JF, Li HY, Zhang YS. MicroRNA- 129-1-3p attenuates autophagy-dependent cell death by targeting MCU in granulosa cells of laying hens under H₂O₂-induced oxidative stress. Poult Sci, 2023, 102(10): 103006.
[9]	Esencan E, Beroukhim G, Seifer DB. Age-related changes in folliculogenesis and potential modifiers to improve fertility outcomes—a narrative review. Reprod Biol Endocrinol, 2022, 20(1): 156.
[10]	Wang TR, Xiao Y, Hu Z, Gu JK, Hua RW, Hai Z, Chen XL, Zhang JV, Yu ZY, Wu T, Yeung WSB, Liu K, Guo CX. MFN2 deficiency impairs mitochondrial functions and PPAR pathway during spermatogenesis and meiosis in mice. Front Cell Dev Biol, 2022, 10: 862506.
[11]	Xie CC, Lu H, Zhang XJ, An Z, Chen T, Yu WB, Wang SS, Shang DD, Wang XY. Mitochondrial abnormality in ovarian granulosa cells of patients with polycystic ovary syndrome. Mol Med Rep, 2024, 29(2): 27.
[12]	Paula VG, Sinzato YK, Gallego FQ, Cruz LL, de Aquino AM, Scarano WR, Corrente JE, Volpato GT, Damasceno DC. Intergenerational hyperglycemia impairs mitochondrial function and follicular development and causes oxidative stress in rat ovaries independent of the consumption of a high-fat diet. Nutrients, 2023, 15(20): 4407.
[13]	Yang C, Luo P, Yang YT, Fu XL, Li BX, Shen X, Xu DN, Huang YM, Tian YB, Liu WJ. Drp1 regulated PINK1- dependent mitophagy protected duck follicular granulosa cells from acute heat stress injury. Poult Sci, 2024, 103(1): 103247.
[14]	Martinez A, Lamaizon CM, Valls C, Llambi F, Leal N, Fitzgerald P, Guy C, Kamiński MM, Inestrosa NC, van Zundert B, Cancino GI, Dulcey AE, Zanlungo S, Marugan JJ, Hetz C, Green DR, Alvarez AR. c-Abl phosphorylates MFN2 to regulate mitochondrial morphology in cells under endoplasmic reticulum and oxidative stress, impacting cell survival and neurodegeneration. Antioxidants (Basel), 2023, 12(11): 2007.
[15]	Olaniyi KS, Areloegbe SE. Acetate ameliorates ovarian mitochondrial dysfunction in letrozole-induced polycystic ovarian syndrome rat model by improving mitofusin-2. J Physiol Sci, 2024, 74(1): 22. doi: 10.1186/s12576-024-00908-5 pmid: 38561673
[16]	Wang S, Sang XH, Li SH, Yang WJ, Wang SH, Chen HX, Lu C. Increased Ca²⁺ transport across the mitochondria- associated membranes by Mfn2 inhibiting endoplasmic reticulum stress in ischemia/reperfusion kidney injury. Sci Rep, 2023, 13(1): 17257.
[17]	Chen XY, Shi CR, He MH, Xiong SQ, Xia XB. Endoplasmic reticulum stress: molecular mechanism and therapeutic targets. Signal Transduct Target Ther, 2023, 8(1): 352.
[18]	Lounas A, Breton Y, Lebrun A, Laflamme I, Vernoux N, Savage J, Tremblay MÈ, Pelletier M, Germain M, Richard FJ. The follicle-stimulating hormone triggers rapid changes in mitochondrial structure and function in porcine cumulus cells. Sci Rep, 2024, 14(1): 436.
[19]	Ju WH, Zhao YW, Yu Y, Zhao S, Xiang S, Lian F. Mechanisms of mitochondrial dysfunction in ovarian aging and potential interventions. Front Endocrinol (Lausanne), 2024, 15: 1361289.
[20]	Zhang M, Bener MB, Jiang ZL, Wang TR, Esencan E, Scott R, Horvath T, Seli E. Mitofusin 2 plays a role in oocyte and follicle development, and is required to maintain ovarian follicular reserve during reproductive aging. Aging (Albany NY), 2019, 11(12): 3919-3938.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

基因	引物序列(5′→3′)	产物大小(bp)
Mfn2	F: TGGGAGGCTAGGAAGGTGAA	125
Mfn2	R: TCATGAACATGGGCTGGGTC	125
Grp78	F: TCACCTCCAATCCGGAGAAC	78
Grp78	R: CTTTGTTTGCCCACCTCCAA	78
Pink1	F: TTTAGCGCAATGAGCCAGGA	131
Pink1	R: ACCCGGATGATGTTGGGATG	131
Perk	F: GCAGTGGCAATGAGAAGTGG	200
Perk	R: CCAATCTGCAACGGAGACCT	200
Chop	F: GCAGCGACAGAGCCAAAATC	98
Chop	R: TCAGGCTCTGCTTTCAGGTG	98
β-actin	F: ATCGGCAATGAGCGGTTCC	108
β-actin	R: CGTGTTGGCGTAGAGGTCTT	108

Mfn2减轻内质网应激抑制绵羊卵泡颗粒细胞凋亡的分子机制

Molecular mechanism of Mfn2 alleviating endoplasmic reticulum stress and inhibiting apoptosis of sheep follicular granulosa cells

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 20

相关文章 15

编辑推荐

Metrics

[1]	杨剑, 石国娟, 彭昂惠, 徐清波, 王睿琪, 薛雷, 喻昕阳, 孙艺昊. Tip60-FOXO调节果蝇JNK信号通路介导的细胞凋亡【已撤稿】[J]. 遗传, 2024, 46(6): 490-501.
[2]	王翠玲, 刘信燚, 王亚会, 张争, 王治东, 周钢桥. MCM2通过抑制p53信号通路促进胆管癌细胞的增殖、迁移和侵袭[J]. 遗传, 2022, 44(3): 230-244.
[3]	秦中勇, 石晓, 曹平平, 褚鹰, 管蔚, 杨楠, 程禾, 孙玉洁. 细胞凋亡反应中NOXA基因启动子发挥增强子功能调节BCL2基因表达[J]. 遗传, 2020, 42(11): 1110-1121.
[4]	余同露,蔡栋梁,朱根凤,叶晓娟,闵太善,陈红岩,卢大儒,陈浩明. CSN4基因干扰对乳腺癌MDA-MB-231细胞增殖和凋亡的影响[J]. 遗传, 2019, 41(4): 318-326.
[5]	杨鑫宇,贾振伟. 颗粒细胞EGF类因子信号通路在调控卵母细胞成熟和发育中的作用[J]. 遗传, 2019, 41(2): 137-145.
[6]	赵建元,丁寄葳,米泽云,周金明,魏涛,岑山. HIV-1初始传播病毒Vpr基因遗传变异对诱导G₂期阻滞及细胞凋亡的影响[J]. 遗传, 2015, 37(5): 480-486.
[7]	毕丹,徐扬,逄越,李庆伟. 质膜组分磷脂酰丝氨酸外翻的分子调控机制[J]. 遗传, 2015, 37(2): 140-147.
[8]	张蕾, 隋御, 王婷, 李利坚, 李元杰, 金彩霞, 徐方. hMMS2基因对结肠癌细胞耐药逆转的影响[J]. 遗传, 2014, 36(4): 346-353.
[9]	周菁华，韩晓凤，刘艳娜，张鹏，郭风劲. BiP调控IRE1a启动子转录活性及蛋白表达的效应[J]. 遗传, 2013, 35(3): 343-351.
[10]	王师尧，金巍娜，吴丹. 青少年型神经元蜡样脂褐质沉积病(JNCL)的发病机制[J]. 遗传, 2009, 31(8): 779-784.
[11]	马向东，马兴，吴小明，陈必良，王德堂 . 胚胎神经管缺陷大鼠模型差异基因的表达[J]. 遗传, 2009, 31(3): 280-284.
[12]	郭芬，刘兆宇，李月琴，李弘剑，周天鸿. Prosaposin对细胞增殖和凋亡的调控及其分子机制[J]. 遗传, 2009, 31(12): 1226-1232.
[13]	付浩，赵丹懿，孙秀菊，滑君，阎杨，邱广蓉，尚超，富伟能，孙开来. B-RAF基因特异的siRNA干扰对胃癌BGC823细胞的影响[J]. 遗传, 2007, 29(5): 537-537―540.
[14]	王琳，梁旭方，廖婉琴，周天鸿. 斑马鱼胚胎发育中细胞凋亡的研究进展[J]. 遗传, 2006, 28(8): 1009-1014.
[15]	王振兴，王璞，姚玲，曾智东，徐汉福，段军，王根洪，夏庆友. 家蚕细胞凋亡相关基因BmICAD的克隆、序列和功能分析及其在精巢中特异表达的初步研究[J]. 遗传, 2006, 28(7): 838-844.