β-谷甾醇对猪卵母细胞体外成熟和胚胎发育的影响

doi:10.11843/j.issn.0366-6964.2024.04.025

摘要/Abstract

摘要： 旨在探索一种自然植物提取的抗氧化分子——β-谷甾醇(β-sitosterol，SITO)对猪卵母细胞体外成熟效率和早期胚胎体外发育质量的影响。本研究以在猪卵母细胞体外成熟培养基中添加不同浓度SITO为试验组，不添加SITO(含0.16% DMSO)的培养基为对照组，对屠宰场所得猪卵巢中收集到的卵母细胞进行体外培养，试验组浓度分别为10、20、40 μmol·L^-1，不同浓度各重复3次，每组培养卵母细胞150~200枚。培养46 h后，对卵母细胞成熟率进行统计，并收集成熟卵母细胞进行活性氧、细胞凋亡、线粒体膜电位染色以及相关基因的实时荧光定量PCR；对成熟卵母细胞进行孤雌激活，2 d后统计卵裂率，7 d后统计囊胚率，并统计囊胚细胞总数。结果发现，20 μmol·L^-1 SITO处理显著提高了猪卵母细胞的体外成熟率，且促进了孤雌激活后囊胚的形成并增加了囊胚细胞数，降低了卵母细胞中ROS含量，提高了抗氧化应激基因SOD和CAT的表达；同时，SITO降低卵母细胞的凋亡水平，增加抗凋亡基因BCL-2的表达，降低促凋亡基因Caspase 3和BAX的表达。此外，SITO可增强线粒体膜电位(ΔΨ m)，增加TFAM基因的表达。综上所述，本研究结果表明，在猪卵母细胞体外成熟培养基中添加20 μmol·L^-1SITO能够显著促进猪卵母细胞的体外成熟率、提高胚胎的体外发育能力和胚胎质量。

关键词: β-谷甾醇, 猪卵母细胞, 胚胎发育, ROS, 凋亡

Abstract: The aim of this study was to investigate the effects of β-sitosterol (SITO), a natural plant-derived antioxidant molecule, on the in vitro maturation efficiency of porcine oocytes and the quality of early embryo development in vitro. In this study, SITO with different concentrations were added into porcine oocytes in vitro maturation medium, which was the experimental group, and the medium without SITO (containing 0.16% DMSO) was the control group, the concentrations in experimental groups were 10, 20 and 40 μmol·L^-1 respectively, and they were used to culture the oocytes in vitro collected from porcine ovaries in slaughterhouse. Different concentrations were repeated 3 times, and 150-200 oocytes were cultured in each group. After 46 h of culture, measured the maturation rate of oocytes, and mature oocytes were collected for reactive oxygen species, apoptosis, mitochondrial membrane potential staining and qRT-PCR of related genes; the adult oocytes were activated by parthenogenesis, the cleavage rate was calculated after 2 days, the blastocyst rate was calculated after 7 days, and the total number of blastocysts was calculated. The results showed that 20 μmol·L^-1 SITO treatment significantly improved the maturation rate of porcine oocytes in vitro, promoted the formation of blastocysts after parthenogenetic activation, decreased the ROS in oocytes, and increased the expression of anti-oxidative stress genes SOD and CAT. At the same time, SITO decreased the apoptosis level of oocytes, increased the expression of anti-apoptotic gene BCL-2, and decreased the expression of pro-apoptotic genes Caspase 3 and BAX. In addition, SITO could enhance mitochondrial membrane potential (ΔΨ m) and increase the expression of TFAM gene. In summary, the results of this study showed that adding 20 μmol·L^-1 SITO to porcine oocyte in vitro maturation medium can significantly promote the rate of porcine oocytes, and improve the in vitro development ability and embryo quality.

Key words: β-sitosterol, porcine oocyte, embryonic development, ROS, apoptosis

中图分类号:

S828.3

蓝昕蕊, 赵宝宝, 张碧菡, 林晓语, 马会明, 王勇胜. β-谷甾醇对猪卵母细胞体外成熟和胚胎发育的影响[J]. 畜牧兽医学报, 2024, 55(4): 1629-1637.

LAN Xinrui, ZHAO Baobao, ZHANG Bihan, LIN Xiaoyu, MA Huiming, WANG Yongsheng. Effects of β-sitosterol on Porcine Oocyte Maturation and Embryonic Development in Vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1629-1637.

参考文献

[1] JAROUDI K A, HOLLANDERS J M G, ELNOUR A M, et al. Embryo development and pregnancies from in-vitro matured and fertilized human oocytes[J]. Hum Reprod, 1999, 14(7):1749-1751.
[2] TELFER E E, ANDERSEN C Y. In vitro growth and maturation of primordial follicles and immature oocytes[J]. Fertil Steril, 2021, 115(5):1116-1125.
[3] ITO K, TAKAE S, NAKAMURA K, et al. The study of the efficiency of in vitro maturation of ovarian tissue oocytes in pediatric patients[J]. J Assist Reprod Genet, 2023, 40(12):2787-2797.
[4] WANG L, TANG J H, WANG L, et al. Oxidative stress in oocyte aging and female reproduction[J]. J Cell Physiol, 2021, 236(12):7966-7983.
[5] LI Y, ZHANG Z Z, HE C J, et al. Melatonin protects porcine oocyte in vitro maturation from heat stress[J]. J Pineal Res, 2015, 59(3):365-375.
[6] ZHANG M Q, MIAO Y L, CHEN Q, et al. BaP exposure causes oocyte meiotic arrest and fertilization failure to weaken female fertility[J]. FASEB J, 2018, 32(1):342-352.
[7] XU P X, CHEN H, WANG H Z, et al. Research progress and detection methods of reactive oxygen species in embryo and germ cells[J]. Chemistry of Life, 2023, 43(10):1599-1608. (in Chinese)
徐沛欣, 陈红, 王惠增, 等. 活性氧在胚胎和生殖细胞中的研究进展及其检测方法[J]. 生命的化学, 2023, 43(10):1599-1608.
[8] IWATA H. Resveratrol enhanced mitochondrial recovery from cryopreservation-induced damages in oocytes and embryos[J]. Reprod Med Biol, 2021, 20(4):419-426.
[9] GHORBANMEHR N, SALEHNIA M, AMOOSHAHI M. The effects of sodium selenite on mitochondrial dna copy number and reactive oxygen species levels of in vitro matured mouse oocytes[J]. Cell J, 2018, 20(3):396-402.
[10] HUANG Z T, YUAN Y G, ZHU J Q. Effect of cisplatin nanoparticles on in vitro maturation of oocytes in mice[J]. Progress in Veterinary Medicine, 2023, 44(11):47-52. (in Chinese)
黄竹涛, 袁玉国, 朱家桥. 纳米顺铂对小鼠卵母细胞体外成熟的影响[J]. 动物医学进展, 2023, 44(11):47-52.
[11] MENDES A F, PUELKER R Z, DE SOUZA L F A, et al. In vitro maturation in synthetic oviductal fluid increases gene expression associated with quality and lipid metabolism in bovine oocytes[J]. Zygote, 2023, 31(6):582-587.
[12] LI Y, MA S H, PENG H, et al. Effects of exogenous antioxidants on in vitro maturation of oocytes[J]. The Chinese Livestock and Poultry Breeding, 2023, 19(7):114-120. (in Chinese)
李一, 马述海, 彭华, 等. 外源性抗氧化剂对卵母细胞体外成熟的影响[J]. 中国畜禽种业, 2023, 19(7):114-120.
[13] IMAI S, MURATA T, FUJIOKA S, et al. Isolation of β-sitosterol-β-D-glucoside from the leaves of Lycium chinense mill[J]. Yakugaku Zasshi, 1963, 83(11):1092.
[14] ZHOU X, XU G, WANG Q. Chemical constituents in the roots of Lycium chinense Mill[J]. Zhongguo Zhong Yao Za Zhi, 1996, 21(11):675-676.
[15] KHAN Z, NATH N, RAUF A, et al. Multifunctional roles and pharmacological potential of β-sitosterol:emerging evidence toward clinical applications[J]. Chem Biol Interact, 2022, 365:110117.
[16] HAH Y S, LEE W K, LEE S, et al. β-sitosterol attenuates dexamethasone-induced muscle atrophy via regulating FoxO1-dependent signaling in C2C12 cell and mice model[J]. Nutrients, 2022, 14(14):2894.
[17] FAN Y T, SHEN J L, LIU X L, et al. β-sitosterol suppresses lipopolysaccharide-induced inflammation and lipogenesis disorder in bovine mammary epithelial cells[J]. Int J Mol Sci, 2023, 24(19):14644.
[18] GUMEDE N M, LEMBEDE B W, BROOKSBANK R L, et al. β-sitosterol shows potential to protect against the development of high-fructose diet-induced metabolic dysfunction in female rats[J]. J Med Food, 2020, 23(4):367-374.
[19] CHEN C, SHEN J L, LIANG C S, et al. First discovery of beta-sitosterol as a novel antiviral agent against white spot syndrome virus[J]. Int J Mol Sci, 2022, 23(18):10448.
[20] WANG H Y, WANG Z, ZHANG Z H, et al. β-sitosterol as a promising anticancer agent for chemoprevention and chemotherapy:mechanisms of action and future prospects[J]. Adv Nutr, 2023, 14(5):1085-1110.
[21] VIVANCOS M, MORENO J J. β-Sitosterol modulates antioxidant enzyme response in RAW 264.7 macrophages[J]. Free Radic Biol Med, 2005, 39(1):91-97.
[22] SHARMA M, PUNETHA M, SAINI S, et al. Mito-Q supplementation of in vitro maturation or in vitro culture medium improves maturation of buffalo oocytes and developmental competence of cloned embryos by reducing ROS production[J]. Anim Reprod Sci, 2024, 260:107382.
[23] TAYLOR C T. Antioxidants and reactive oxygen species in human fertility[J]. Environ Toxicol Pharmacol, 2001, 10(4):189-198.
[24] PANG Y W, ZHAO S J, SUN Y Q, et al. Protective effects of melatonin on the in vitro developmental competence of bovine oocytes[J]. Anim Sci J, 2018, 89(4):648-660.
[25] GRZEGORZEWSKA A K, WOLAK D, HRABIA A. Effect of tamoxifen treatment on catalase (CAT) and superoxide dismutase (SOD) expression and localization in the hen oviduct[J]. Theriogenology, 2024, 214:73-80.
[26] XING X P, ZHANG J J, WU T, et al. SIRT1 reduces epigenetic and non-epigenetic changes to maintain the quality of postovulatory aged oocytes in mice[J]. Exp Cell Res, 2021, 399(2):112421.
[27] SHARMILA R, SINDHU G. Evaluate the antigenotoxicity and anticancer role of β-sitosterol by determining oxidative DNA damage and the expression of phosphorylated mitogen-activated protein kinases', C-fos, C-jun, and endothelial growth factor receptor[J]. Pharmacogn Mag, 2017, 13(49):95-101.
[28] LIN W S, CHEN J Y, WANG J C, et al. The anti-aging effects of Ludwigia octovalvis on Drosophila melanogaster and SAMP8 mice[J]. Age (Dordr), 2014, 36(2):689-703.
[29] WANG L Y, WANG D H, ZOU X Y, et al. Mitochondrial functions on oocytes and preimplantation embryos[J]. J Zhejiang Univ Sci B, 2009, 10(7):483-492.
[30] HUANG X R, JIANG Z Y, CHEN Y L, et al. Effects of different doses of ethanol administration on cardiac function and Bcl-2/Bax in rats[J]. Journal of Guangxi Medical University, 2023, 40(11):1802-1806. (in Chinese)
黄循铷, 蒋赵艳, 陈亚乐, 等. 不同剂量乙醇灌胃对大鼠心功能和Bcl-2/Bax表达的影响[J]. 广西医科大学学报, 2023, 40(11):1802-1806.
[31] ELMORE S. Apoptosis:a review of programmed cell death[J]. Toxicol Pathol, 2007, 35(4):495-516.
[32] MOON D O, LEE K J, CHOI Y H, et al. β-Sitosterol-induced-apoptosis is mediated by the activation of ERK and the downregulation of Akt in MCA-102 murine fibrosarcoma cells[J]. Int Immunopharmacol, 2007, 7(8):1044-1053.
[33] LI Y J, HE H H, YANG L X, et al. Advances in regulation of mammalian embryonic development by mitochondrial autophagy[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3):905-912. (in Chinese)
李钰浚, 何翃闳, 杨丽雪, 等. 线粒体自噬调控哺乳动物胚胎发育的研究进展[J]. 畜牧兽医学报, 2024, 55(3):905-912.
[34] JIN J X, QI X Y, LIU Z Y, et al. H₂O₂-induced mitochondrial damage rescued by Mito-Tempo in porcine oocytes[J]. Journal of Northeast Agricultural University, 2023, 54(6):28-34. (in Chinese)
金君学, 綦新月, 刘子瑜, 等. Mito-Tempo修复过氧化氢诱导的猪卵母细胞线粒体损伤机制研究[J]. 东北农业大学学报, 2023, 54(6):28-34.
[35] MAY-PANLOUP P, BOUCRET L, CHAO DE LA BARCA J M, et al. Ovarian ageing:the role of mitochondria in oocytes and follicles[J]. Hum Reprod Update, 2016, 22(6):725-743.
[36] SUN L C, XU X L, BAI J H, et al. Progress in research on mitochondrial regulation mechanism of mammalian in vitro maturation oocyte[J]. Animal Husbandry & Veterinary Medicine, 2023, 55(10):116-120. (in Chinese)
孙立晨, 许晓玲, 白佳桦, 等. 哺乳动物体外成熟卵母细胞的线粒体调控机制研究进展[J]. 畜牧与兽医, 2023, 55(10):116-120.
[37] ANGELOVA P R, ABRAMOV A Y. Role of mitochondrial ROS in the brain:from physiology to neurodegeneration[J]. FEBS Lett, 2018, 592(5):692-702.
[38] ROTH Z. Symposium review:reduction in oocyte developmental competence by stress is associated with alterations in mitochondrial function[J]. J Dairy Sci, 2018, 101(4):3642-3654.
[39] ZHAO M, WANG Y Z, LI L, et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance[J]. Theranostics, 2021, 11(4):1845-1863.
[40] ROSS P J, GOISSIS M D, MARTINS J P N, et al. Blastocyst cell number and allocation affect the developmental potential and transcriptome of bovine somatic cell nuclear transfer embryos[J]. Stem Cells Dev, 2023, 32(17-18):515-523.