Rho相关激酶抑制剂Y27632对小鼠腔前卵泡体外发育的作用

张樱馨; 俞晓丽; 罗晓强; 邱意开; 裴秀英

doi:10.16098/j.issn.0529-1356.2021.06.023

PDF(15258 KB)

解剖学报 ›› 2021, Vol. 52 ›› Issue (6) : 979-985. DOI: 10.16098/j.issn.0529-1356.2021.06.023

组织学胚胎学发育生物学

Rho相关激酶抑制剂Y27632对小鼠腔前卵泡体外发育的作用

张樱馨¹ 俞晓丽¹ 罗晓强² 邱意开¹ 裴秀英^1*

作者信息 +

Effects of Rho-associated coiled-coil-forming kinase inhibitor Y27632 on mouse preantral follicles development in vitro

ZHANG Ying-xin¹ YU Xiao-li¹ LUO Xiao-qiang² QIU Yi-kai¹ PEI Xiu-ying^1*

Author information +

文章历史 +

摘要

目的探讨Rho相关激酶（ROCK）抑制剂Y27632在小鼠腔前卵泡体外发育中的作用。方法取出生12.5 d的小鼠卵巢，机械法收集单枚腔前卵泡，采用超低吸附96孔板模拟3D环境对其进行培养。设置对照组和含有Y27632的实验组，通过形态学观察、卵泡直径的变化、成熟卵泡的数量、成熟卵母细胞纺锤体的变化等来评估卵泡的整体发育情况。采用Real-time PCR技术检测颗粒细胞中卵泡刺激素受体（FSHR）、卵母细胞中骨形态发生蛋白15（BMP15）、生长分化因子9（GDF9）等以及凋亡相关基因（Bad、Bax和Caspase-3）的表达情况，同时，用细胞存活染料检测卵泡发育过程中颗粒细胞的凋亡情况。结果 ROCK抑制剂Y27632对卵泡直径增长和基本发育指标（存活数、成腔率和成熟率）无明显影响（P>0.05），但对照组卵母细胞成熟后纺锤体组装出现异常。培养8 d后，与对照组相比，实验组颗粒细胞特异性基因FSHR上调；卵母细胞特异性基因BMP15和GDF9上调，而叉头框O3（FoxO3)下调；凋亡基因Bax、Bad和Caspase3表达下调，实验组卵泡内的颗粒细胞凋亡明显少于对照组。结论小鼠卵泡体外发育过程中，ROCK抑制剂Y27632能够抑制颗粒细胞的凋亡，防止卵母细胞纺锤体组装异常，进而提高卵泡体外发育质量。

Abstract

Objective To investigate the effects of Rho-associated coiled-coil-forming kinase（ROCK）inhibitor Y27632 on the development of mouse preantral follicles in vitro. Methods The ovaries of 12.5-day-old mice were collected and single preantral follicle was isolated by mechanical method to culture Nunclon Sphera 96-well U-bottom 3D cell culture plate in vitro. Set up a control group and treated group containing Y27632, and evaluate the overall development of the follicles through follicular morphology, follicle diameter, the number of mature follicles, and changes in oocyte spindles. The expression of oocytes ［bone morphogenetic protein 15(BMP15), growth differentiation factor 9(GDF9)］, granulosa cells ［follicle stimulating hormone receptor (FSHR)］ and apoptosis related genes (Bad, Bax and Caspase-3) were also assessed by the Real-time PCR technique. Meanwhile, the follicle viability was detected by follicular activity test during follicular development. Results Rock inhibitor Y27632 had no significant effect on the growth diameter and basic development indicators (follicle survival rate, cavity formation rate and maturation rate) (P>0.05). And abnormal spindle assembly occurred during follicle development in the control group. After 8 days of culture, compared with the control group, the granulosa cell-specific gene FSHR was up-regulated in the experimental group; As well as the oocyte-specific genes BMP15 and GDF9 were up-regulated, while forkhead box O3(FoxO3) was down-regulated; The apoptosis genes Bax, Bad and Caspase-3 were also showed down-regulated. And the granulosa cell apoptosis in the experimental group was significantly less than that in the control group. Conclusion ROCK inhibitor Y27632 can inhibit the apoptosis of granulosa cells and prevents the abnormal assembly of oocyte spindles to improve the quality of follicle development in vitro.

关键词

Key words

Rho-associated coiled-coil-forming kinase / Y27632;Spindle / Follicle culture / Real-time PCR / Mouse

引用本文

EndNote

Ris (Procite)

Bibtex

导出引用

张樱馨俞晓丽罗晓强邱意开裴秀英. Rho相关激酶抑制剂Y27632对小鼠腔前卵泡体外发育的作用[J]. 解剖学报. 2021, 52(6): 979-985 https://doi.org/10.16098/j.issn.0529-1356.2021.06.023

ZHANG Ying-xin YU Xiao-li LUO Xiao-qiang QIU Yi-kai PEI Xiu-ying. Effects of Rho-associated coiled-coil-forming kinase inhibitor Y27632 on mouse preantral follicles development in vitro[J]. Acta Anatomica Sinica. 2021, 52(6): 979-985 https://doi.org/10.16098/j.issn.0529-1356.2021.06.023

中图分类号： R321.1

参考文献

［1］ Tukur HA, Aljumaah RS, Swelum AA, et al. Effects of short-term inhibition of Rho kinase on dromedary camel oocyte in vitro maturation［J］. Animals (Basel), 2020,10(5):750.

［2］ An L, Liu J, Du Y, et al. Synergistic effect of cysteamine, leukemia inhibitory factor, and Y27632 on goat oocyte maturation and embryo development in vitro［J］. Theriogenology, 2018,108:56-62.

［3］ Lian R,Chen X,Zhang YJ,et al.Effects of homocysteine on histone modification during oocyte development in mice［J］. Acta Anatomica Sinica, 2017,48(5):585-589.(in Chinese)

梁蓉, 陈曦, 张育军, 等. 同型半胱氨酸对小鼠卵母细胞成熟过程中组蛋白表观遗传修饰的影响［J］. 解剖学报, 2017,48(5):585-589.

［4］ Baerwald AR, Adams GP, Pierson RA. Ovarian antral folliculogenesis during the human menstrual cycle: a review［J］. Hum Reprod Update, 2012,18(1):73-91.

［5］ Palma GA, Arganaraz ME, Barrera AD, et al. Biology and biotechnology of follicle development［J］. ScientificWorldJournal, 2012,2012:938138.

［6］ Gutierrez CG, Campbell BK, Webb R. Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle-stimulating hormone, and morphological characteristics［J］. Biol Reprod, 1997,56(3):608-616.

［7］ Xiang L, Yin Y, Zheng Y, et al. A developmental landscape of 3D-cultured human pre-gastrulation embryos［J］. Nature, 2020,577(7791):537-542.

［8］ Duan X, Liu J, Dai XX, et al. Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis［J］. Biol Reprod, 2014,90(2):37.

［9］ Zhang Y, Duan X, Xiong B, et al. ROCK inhibitor Y-27632 prevents porcine oocyte maturation［J］. Theriogenology, 2014,82(1):49-56.

［10］ Arayatham S, Tiptanavattana N, Tharasanit T. Effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 inhibitor on the meiotic and developmental competence of feline oocytes［J］. J Reprod Dev, 2017,63(5):511-517.

［11］ Pan MH, Wang F, Lu Y, et al. FHOD1 regulates cytoplasmic actin-based spindle migration for mouse oocyte asymmetric cell division［J］. J Cell Physiol, 2018,233(3):2270-2278.

［12］ Hwang IS, Hara H, Chung HJ, et al. Rescue of vitrified-warmed bovine oocytes with rho-associated coiled-coil kinase inhibitor［J］. Biol Reprod, 2013,89(2):26.

［13］ Artini PG, Tatone C, Sperduti S, et al. Cumulus cells surrounding oocytes with high developmental competence exhibit down-regulation of phosphoinositol 1,3 kinase/protein kinase B (PI3K/AKT) signalling genes involved in proliferation and survival［J］. Hum Reprod, 2017,32(12):2474-2484.

［14］ Li L, Ji SY, Yang JL, 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.

［15］ Mikaeili S, Rashidi BH, Safa M, et al. Altered FoxO3 expression and apoptosis in granulosa cells of women with polycystic ovary syndrome［J］. Arch Gynecol Obstet, 2016,294(1):185-192.

［16］ Matsuda F, Inoue N, Maeda A, et al. Expression and function of apoptosis initiator FOXO3 in granulosa cells during follicular atresia in pig ovaries［J］. J Reprod Dev, 2011,57(1):151-158.

［17］ Cui C, Han S, Yin H, et al. FOXO3 is expressed in ovarian tissues and acts as an apoptosis initiator in granulosa cells of chickens［J］. Biomed Res Int, 2019,2019:6902906.

［18］ Wu Y, Shu J, He C, et al. ROCK inhibitor Y27632 promotes proliferation and diminishes apoptosis of marmoset induced pluripotent stem cells by suppressing expression and activity of caspase 3［J］. Theriogenology, 2016,85(2):302-314.

［19］ Shoorei H, Banimohammad M, Kebria MM, et al. Hesperidin improves the follicular development in 3D culture of isolated preantral ovarian follicles of mice［J］. Exp Biol Med (Maywood), 2019,244(5):352-361.

［20］ Shoorei H, Khaki A, Ainehchi N, et al. Effects of matricaria chamomilla extract on growth and maturation of isolated mouse ovarian follicles in a three-dimensional culture system［J］. Chin Med J (Engl), 2018,131(2):218-225.

［21］ Liu L, Rajareddy S, Reddy P, et al. Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Fox03a［J］. Development, 2007,134(1):199-209.