槲皮素在羊卵巢组织玻璃化冻存中的卵泡保护及抗氧化作用

doi:10.6040/j.issn.1671-7554.0.2020.0655

摘要/Abstract

摘要： 目的探讨在冷冻保护剂中添加槲皮素对羊卵巢组织冷冻复苏后卵泡活性的影响。方法将36只性成熟母羊的卵巢皮质组织块随机分配到新鲜对照组(CON组),玻璃化冷冻组(VIT组),添加低、中、高浓度(1、5、10 μmol/L)槲皮素的玻璃化冷冻组(VWQ1组、VWQ2组、VWQ3组),将卵巢组织玻璃化冷冻后复苏、培养,测定各组雌激素水平,分析各组的卵泡计数及形态学改变。采用免疫组织化学法进行组织增殖细胞核抗原分析,采用TUNEL法进行组织凋亡分析,采用免疫组织化学法及可见光比色法进行组织抗氧化能力检测。结果 CON组原始卵泡形态正常比例最高、凋亡细胞计数最少(84.1%,P<0.001;13.92±3.88,P<0.001);VWQ1组的原始卵泡正常形态率高于VIT组(76.5% vs 71.7%,P=0.035),凋亡细胞数少于VIT组(50.96±24.28 vs 73.44±35.47,P=0.017),锰超氧化物歧化酶(SOD-2)蛋白表达量、过氧化氢酶(CAT)含量均高于VIT组［0.54(0.53,0.55)vs 0.32(0.29,0.51),P<0.001;5.60±1.49 vs 3.48±1.21,P=0.008］;VWQ3组的原始卵泡正常形态率最低(61.2%,P<0.001),凋亡细胞计数最多(127.12±42.46,P<0.001)。结论低浓度槲皮素可以减轻卵巢组织氧化损伤进而保存卵泡活性,但过高浓度的槲皮素对卵泡活性有害。

关键词: 卵巢组织, 冷冻保护剂, 抗氧化作用, 玻璃化冷冻, 羊

Abstract: Objective To investigate the effect of quercetin in cryoprotectant on the activity of follicles after cryopreservation of ovarian tissues. Methods Cortical pieces of 36 sexually mature ewe ovaries were randomly assigned to fresh control(CON)group, vitrification(VIT)group, vitrification with low, medium and high concentration(1, 5, 10 μmol/L)of quercetin(VWQ1, VWQ2, VWQ3)groups. The cortical pieces were rewarmed and cultured after vitrification to measure the estrogen levels, count the number of follicles and observe the morphological changes. The proliferating cell nucleus antigen of ovarian tissues was analyzed with immunohistochemistry. Tissue apoptosis was detected with TUNEL, and antioxidant capacity of ovarian tissues was determined with immunohistochemistry and visible light colorimetry. Results The proportion of morphological normal follicles was the highest while the number of apoptotic cells was the lowest in CON group(84.1%, P<0.001; 13.92±3.88, P<0.001). Compared with the VIT group, the VWQ1 group had higher rate of morphological normal follicles(76.5% vs 71.7%, P=0.035), fewer number of apoptotic cells(50.96±24.28 vs 73.44±35.47, P=0.017), and higher expressions of manganese superoxide dismutase(SOD-2)and catalase(CAT)［0.54(0.53, 0.55)vs 0.32(0.29, 0.51), P<0.001; 5.60±1.49 vs 3.48±1.21, P=0.008］. The VWQ3 group had the lowest rate of morphological normal follicles(61.2%, P<0.001), but the largest number of apoptotic cells(127.12±42.46, P<0.001). Conclusion Low concentration of quercetin in cryoprotection can reduce the oxidative damage on ovarian tissues and thus preserve the follicular activity, but excessively high concentration of quercetin can be harmful.

Key words: Ovarian tissue, Cryoprotective agent, Antioxidation, Vitrification, Sheep

中图分类号:

R711.75

杨璐恺,蒋利刚,崔妍婷,刘金,韩亦龙,陈超,邓晓惠. 槲皮素在羊卵巢组织玻璃化冻存中的卵泡保护及抗氧化作用[J]. 山东大学学报 (医学版), 2020, 1(9): 1-7.

YANG Lukai, JIANG Ligang, CUI Yanting, LIU Jin, HAN Yilong, CHEN Chao, DENG Xiaohui. Follicle protection and antioxidant effect of quercetin on the cryopreservation of ewe ovarian tissues[J]. Journal of Shandong University (Health Sciences), 2020, 1(9): 1-7.

参考文献 26

[1]	Donnez J, Dolmans MM. Fertility preservation in women [J]. N Engl J Med, 2018, 378(4): 400-401.
[2]	Affdal AO, Grynberg M, Hessissen L, et al. Impact of legislation and public funding on oncofertility: a survey of Canadian, French and Moroccan pediatric hematologists/ oncologists [J]. BMC Med Ethics, 2020, 21(1): 25. doi: 10.1186/s12910-020-00466-6.
[3]	Wallace WH, Kelsey TW, Anderson RA. Fertility preservation in pre-pubertal girls with cancer: the role of ovarian tissue cryopreservation [J]. Fertil Steril, 2016, 105(1): 6-12.
[4]	Donnez J, Dolmans MM. Fertility preservation in women [J]. N Engl J Med, 2017, 377(17): 1657-1665.
[5]	Shahsavari MH, Alves KA, Alves BG, et al. Impacts of different synthetic polymers on vitrification of ovarian tissue [J]. Cryobiology, 2020, S0011-2240(20)30078-X. doi: 10.1016/j.cryobiol.2020.04.007.
[6]	Shi X, Hu H, Ji G, et al. Protective effect of sucrose and antioxidants on cryopreservation of sperm motility and DNA integrity in C57BL/6 mice [J]. Biopreserv Biobank, 2018,16(6): 444-450.
[7]	Kasai N, Mera H, Wakitani S, et al. Effect of epigallocatechin-3-o-gallate and quercetin on the cryopreservation of cartilage tissue [J]. Biosci Biotechnol Biochem, 2017, 81(1): 197-199.
[8]	Xu D, Hu MJ, Wang YQ, et al. Antioxidant activities of quercetin and its complexes for medicinal application [J]. Molecules, 2019, 24(6):1123. doi: 10.3390/molesules24061123.
[9]	Nikiforov D, Russo V, Nardinocchi D, et al. Innovative multi-protectoral approach increases survival rate after vitrification of ovarian tissue and isolated follicles with improved results in comparison with conventional method [J]. J Ovarian Res, 2018, 11(1): 65.
[10]	Lundy T, Smith P, Oconnell A, et al. Populations of granulosa cells in small follicles of the sheep ovary [J]. Journal of reproduction and fertility, 1999, 115(2): 251-262.
[11]	Gandolfi F, Paffoni A, Papasso Brambilla E, et al. Efficiency of equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue: comparative analysis between human and animal models [J]. Fertil Steril, 2006, 85(Suppl 1): 1150-1156. doi: 10.1016/j.fertnstert.2005.08.062.
[12]	Lee J, Kong HS, Kim EJ, et al. Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury [J]. Hum Reprod, 2016, 31(8): 1827-1837.
[13]	Marques LS, Fossati AAN, Rodrigues RB, et al. Slow freezing versus vitrification for the cryopreservation of zebrafish(Danio rerio)ovarian tissue [J]. Sci Rep, 2019, 9(1): 15353. doi: 10.1038/s41598-019-51696-7.
[14]	Massignam ET, Ferreira M, Sanguinet E, et al. Antioxidant defense capacity of ovarian tissue after vitrification in a metal closed system [J]. JBRA Assist Reprod, 2018, 22(3): 199-204.
[15]	Silva LM, Mbemya GT, Guerreiro DD, et al. Effect of catalase or alpha lipoic acid supplementation in the vitrification solution of ovine ovarian tissue [J]. Biopreserv Biobank, 2018, 16(4): 258-269.
[16]	Dos Santos Morais MLG, De Brito DCC, Pinto Y, et al. Natural antioxidants in the vitrification solution improve the ovine ovarian tissue preservation [J]. Reprod Biol, 2019, 19(3): 270-278.
[17]	Melo MA, Oskam IC, Celestino JJ, et al. Adding ascorbic acid to vitrification and IVC medium influences preantral follicle morphology, but not viability [J]. Reprod Domest Anim, 2011, 46(4): 742-745.
[18]	Hemadi M, Abolhassani F, Akbari M, et al. Melatonin promotes the cumulus-oocyte complexes quality of vitrified-thawed murine ovaries; with increased mean number of follicles survival and ovary size following heterotopic transplantation [J]. Eur J Pharmacol, 2009, 618(1-3): 84-90.
[19]	Song X, Wang Y, Gao L. Mechanism of antioxidant properties of quercetin and quercetin-DNA complex [J]. J Mol Model, 2020, 26(6): 133. doi: 10.1007/s00894-020-04356-x.
[20]	Güller P, Karaman M, Güller U, et al. A study on the effects of inhibition mechanism of curcumin, quercetin, and resveratrol on human glutathione reductase through in vitro and in silico approaches [J]. J Biomol Struct Dyn, 2020, 1-10. doi: 10.1080/07391102.2020.1738962.
[21]	Adedara IA, Ego VC, Subair TI, et al. Quercetin improves neurobehavioral performance through restoration of brain antioxidant status and acetylcholinesterase activity in manganese-treated rats [J]. Neurochem Res, 2017, 42(4): 1219-1229.
[22]	Chen BH, Park JH, Ahn JH, et al. Pretreated quercetin protects gerbil hippocampal CA1 pyramidal neurons from transient cerebral ischemic injury by increasing the expression of antioxidant enzymes [J]. Neural regeneration research, 2017, 12(2): 220-227.
[23]	Akkoyun DC, Akyuz A, Dogan M, et al. Quercetin inhibits heart injury in lipopolysaccharide-induced endotoxemia model by suppressing the effects of reactive oxygen species [J]. Anal Quant Cytopathol Histopathol, 2016, 38(3): 183-188.
[24]	Succu S, Pasciu V, Manca ME, et al. Dose-dependent effect of melatonin on post warming development of vitrified ovine embryos [J]. Theriogenology, 2014, 81(8): 1058-1066.
[25]	Hashemzaei M, Delarami Far A, Yari A, et al. Anticancer and apoptosis inducing effects of quercetin in vitro and in vivo [J]. Oncol Rep, 2017, 38(2): 819-828.
[26]	Jafarabadi M, Abdollahi M, Salehnia M. Assessment of vitrification outcome by xenotransplantation of ovarian cortex pieces in γ-irradiated mice: morphological and molecular analyses of apoptosis [J]. Journal of Assisted Reproduction and Genetics, 2014, 32(2): 195-205.

相关文章 15

[1]	王潇,孔文茹,崔伟亮,王姝麒. 羊角棉总生物碱增强氟尿嘧啶对结直肠癌化疗敏感性[J]. 山东大学学报 (医学版), 2024, 62(6): 30-37.
[2]	于婷,李媛,吴梅. 超声诊断羊膜带综合征并胎头离断1例[J]. 山东大学学报 (医学版), 2023, 61(8): 122-124.
[3]	阮祥燕,程姣姣,杜娟,谷牧青. 卵巢组织冷冻保存[J]. 山东大学学报 (医学版), 2022, 60(9): 24-30.
[4]	石玉华,潘烨,谢燕秋. 胚胎冷冻保存技术及进展[J]. 山东大学学报 (医学版), 2022, 60(9): 12-18.
[5]	林芸,谢燕秋. 乳腺癌患者生育力保护及保存[J]. 山东大学学报 (医学版), 2022, 60(9): 42-46.
[6]	朱序理,周亮,王跃,孙庆云,曹明雅,杜元杰,曹金凤,赵志明,郝桂敏. 不同精子来源质量冷冻方式与妊娠结局的关联性分析[J]. 山东大学学报 (医学版), 2021, 59(6): 86-93.
[7]	王冉冉,朱天瑞,张凤,王敏,闵傲雪,李恒,李晓红. 长期淫羊藿苷治疗对APP/PS1小鼠神经炎症的影响[J]. 山东大学学报 (医学版), 2020, 58(4): 71-77.
[8]	阮祥燕,程姣姣,杜娟,李扬璐,金凤羽,Alfred O. Mueck. 生育力保护方法研究进展[J]. 山东大学学报 (医学版), 2019, 57(2): 29-34.
[9]	戴建国, 王中立, 陈琳, 黄玉芳, 赵玉男. 六种助阳类药物抗抑郁作用动物实验研究[J]. 山东大学学报（医学版）, 2014, 52(S2): 3-4.
[10]	文燕1，赵淑芹2，沈彦军1，晁岚1，宋长征3，邓晓惠1. 抗冷冻蛋白III对小鼠胚胎冷冻保护的作用[J]. 山东大学学报（医学版）, 2014, 52(5): 35-38.
[11]	慈倩倩，李梅，吴克良，马水英，王苗苗，刘辉，高芹，石玉华，陈子江. 玻璃化冷冻对人体外成熟卵母细胞微丝分布及发育潜能的影响[J]. 山东大学学报（医学版）, 2014, 52(4): 26-29.
[12]	王红燕, 王燕蓉, 徐仙, 赵君利, 哈灵侠, 贾韶彤, 刘春莲. 改良的玻璃化液冻存乳鼠卵巢异体原位移植后生殖功能的恢复[J]. 山东大学学报（医学版）, 2014, 52(11): 27-32.
[13]	曹立军1，王进2，孙春丽1，陈玉国1. 乌司他丁对脑缺血再灌注后大鼠海马区神经功能保护作用的机制研究[J]. 山东大学学报(医学版), 2012, 50(2): 6-.
[14]	李志会，李鹏，宋楠楠，岳盈盈，纪璇，赵元昊，孟红. 致敏绵羊红细胞在EV71细胞嗜性研究中的应用[J]. 山东大学学报(医学版), 2010, 48(11): 33-.
[15]	高髻云,张伟 . 羊膜匀浆液对兔小梁切除术后滤过泡及TGFβ1、CTGF表达的影响[J]. 山东大学学报(医学版), 2008, 46(2): 163-166.

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed