2-氨基乙基联苯基硼酸酯对玻璃化冷冻牛成熟卵母细胞发育能力的影响

doi:10.16431/j.cnki.1671-7236.2022.07.025

摘要/Abstract

摘要： 【目的】研究内质网IP3受体（IP3R）抑制剂2-氨基乙基联苯基硼酸酯（2-APB）对玻璃化冷冻-解冻牛MⅡ期卵母细胞发育能力的影响。【方法】将体外成熟24 h的卵丘-卵母细胞复合体（COCs）用0.1%透明质酸酶消化去除卵丘颗粒细胞，获得的卵母细胞分为对照组和2-APB组，采用OPS法进行玻璃化冷冻，对照组直接进行玻璃化冷冻，2-APB组在玻璃化冷冻前先用10 μmol/L 2-APB预处理10 min。2 d后解冻卵母细胞，统计解冻后（0 h）及恢复培养2 h时卵母细胞的存活率，用FITC-PNA荧光探针检测恢复培养2 h时卵母细胞皮质颗粒（CG）的分布，用2'，7'-DCHFDA和Cell Tracker Blue CMF2HC分别检测胞内活性氧（ROS）和谷胱甘肽（GSH）含量。将成熟培养24 h未冷冻卵母细胞（Fresh组），与解冻后恢复2 h的对照组和2-APB组卵母细胞同时进行孤雌激活，于培养的第2、7天分别统计孤雌胚胎的卵裂率和囊胚率。【结果】与对照组相比，2-APB组冷冻-解冻卵母细胞0和2 h的存活率均显著增加（P＜0.05）；2-APB组皮质颗粒皮质区分布比例显著提高（P＜0.05），损伤型分布比例显著降低（P＜0.05）；2-APB组卵母细胞内GSH含量显著增加（P＜0.05），ROS含量显著降低（P＜0.05）；2-APB组卵母细胞孤雌激活胚胎的卵裂率、囊胚率均显著增加（P＜0.05），且与Fresh组无显著差异（P＞0.05）。【结论】2-APB预处理可通过增加卵母细胞内GSH含量，降低卵母细胞皮质颗粒损伤型分布比例和胞内ROS含量，提高玻璃化冷冻保存牛MⅡ期卵母细胞质量及早期胚胎发育能力。

关键词: 玻璃化冷冻; 2-氨基乙基联苯基硼酸酯; 卵母细胞; 发育能力; 牛

Abstract: 【Objective】 The study was aimed to investigate the effects of endoplasmic reticulum IP3 receptor (IP3R) inhibitor 2-aminoethyl diphenylborinate (2-APB) on the developmental capacity of frozen-thawed bovine MⅡoocytes.【Method】 Oocytes matured in vitro for 24 h were digested with 0.1% hyaluronidase to remove cumulus cells,and were randomly divided into control group and 2-APB group,the open pulled straw (OPS) method was used for vitrification.The control group was vitrified directly,2-APB group was pretreated with 10 μmol/L 2-APB for 10 min before vitrification.Oocytes were thawed after 2 d,the survival rate of oocytes after thawed (0 h) and warmed for 2 h were counted.After recovering for 2 h, cortical granules (CG)were labeled with FITC-PNA fluorescent probe to detect distribution,intracellular reactive oxygen species (ROS) and glutathione (GSH) contents were deteced by 2',7'- DCHFDA and Cell Tracker Blue CMF2HC respectively.The non-vitrified oocytes (Fresh group)matured in vitro for 24 h were parthenogenetic activated as well as those of the control and 2-APB groups,the cleavage and blastocyst rates were counted at 2 and 7 d,respectively.【Result】 Compared with control group,the survival rate of frozen-thawed oocytes at 0 and 2 h in 2-APB group was significantly increased (P＜0.05),the proportion of CG distributed in cortical area in 2-APB group was significantly increased (P＜0.05),the proportion of CG injury type was significantly decreased (P＜0.05),the content of GSH was significantly increased (P＜0.05),and the content of ROS was significantly decreased (P＜0.05),the cleavage and blastocyst rates of parthenogenetic activated embryos in 2-APB group were significantly increased (P＜0.05),and there was no significant difference with the Fresh group (P＞0.05).【Conclusion】 Pretreatment of bovine MⅡ oocytes with 2-APB before vitrification could increase GSH content,decrease the proportion of CG injury type and ROS content,improve the frozen-thawed oocyte quality and early embryo developmental capacity.

Key words: vitrification; 2-aminoethyl diphenylborinate; oocyte; development capacity; bovine

中图分类号:

S823

莫显红, 岳凯平, 孙丽瑶, 李冰, 赵冰, 郭成, 徐振军. 2-氨基乙基联苯基硼酸酯对玻璃化冷冻牛成熟卵母细胞发育能力的影响[J]. 中国畜牧兽医, 2022, 49(7): 2669-2676.

MO Xianhong, YUE Kaiping, SUN Liyao, LI Bing, ZHAO Bing, GUO Cheng, XU Zhenjun. Effect of 2-APB on Development Capacity of Vitrified Bovine Mature Oocytes[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2669-2676.

参考文献

[1] SOMFAI T,NGUYEN H T,NGUYEN M T,et al.Vitrification of porcine cumulus-oocyte complexes at the germinal vesicle stage does not trigger apoptosis in oocytes and early embryos,but activates anti-apoptotic Bcl-XL gene expression beyond the 4-cell stage[J]. Journal of Reproduction and Development,2020,66(2):115-123.
[2] ZHANG C H,QIAN W P,QI S T,et al.Maternal diabetes causes abnormal dynamic changes of endoplasmic reticulum during mouse oocyte maturation and early embryo development[J]. Reproductive Biology and Endocrinology,2013,11:31.
[3] MARTÍN-ROMERO F J,ORTÍZ-DE-GALISTEO J R,LARA-LARANJEIRA J,et al.Store-operated calcium entry in human oocytes and sensitivity to oxidative stress[J].Biology of Reproduction,2008,78(2):307-315.
[4] TIAN S J,YAN C L,YANG H X,et al.Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration[J].Animal Reproduction Science,2007,101(3-4):365-371.
[5] BOGLIOLO L,LEDDA S,INNOCENZI P,et al.Raman microspectroscopy as a non-invasive tool to assess the vitrification-induced changes of ovine oocyte zona pellucida[J].Cryobiology,2012,64(3):267-272.
[6] LARMAN M G,KATZ-JAFFE M G,SHEEHAN C B,et al.1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology[J].Human Reproduction,2007,22(1):250-259.
[7] MICARONI M.The role of calcium in intracellular trafficking[J].Current Molecular Medicine,2010,10(8):763-773.
[8] ZHANG A,CHENG T P,ALTURA B M.Ethanol decreases cytosolic-free calcium ions in vascular smooth muscle cells as assessed by digital image analysis[J].Alcoholism Clinical and Experimental Research,1992,16(1):55-57.
[9] LARMAN M G,SHEEHAN C B,GARDNER D K.Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes[J].Reproduction,2006,131(1):53-61.
[10] SUCCU S,BERLINGUER F,LEONI G G,et al.Calcium concentration in vitrification medium affects thedevelopmental competence of in vitro matured ovine oocytes[J].Theriogenology,2011,75(4):715-721.
[11] WANG N,HAO H S,LI C Y,et al.Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes[J].Scientific Reports,2017,7(1):10652.
[12] SANAEI B,MOVAGHAR B,VALOJERDI M R,et al.An improved method for vitrification of in vitro matured ovine oocytes;Beneficial effects of ethylene glycol tetraacetic acid,an intracellular calcium chelator[J].Cryobiology, 2018,84:82-90.
[13] CARRASCO-POZO C,PASTENE E,VERGARA C,et al.Stimulation of cytosolic and mitochondrial calcium mobilization by indomethacin in Caco-2 cells:Modulation by the polyphenols quercetin,resveratrol and rutin[J].Biochimica et Biophysica Acta-Biomembranes,2012,1820(12):2052-2061.
[14] 李松岩,于洋,刘师兵,等.2-APB对顺铂诱导人卵巢癌SKOV3细胞内质网应激途径凋亡的影响[J].毒理学杂志,2018,32(2):224-228. LI S Y,YU Y,LIU S B,et al.Effects of 2-APB on apoptosis of endoplasmic reticulum stress in cisplatin-induced human ovarian cancer SKOV3 cells[J].Journal of Toxicology,2018,32(2):224-228.(in Chinese)
[15] 薛玉环.钙离子介导的卵母细胞冻融性损伤的初步研究[D].南宁:广西大学,2018. XUE Y H.Preliminary study on the influence of freezing-thawing damage on oocytes mediated by calcium ions[D].Nanning:Guangxi University,2018.(in Chinese)
[16] 莫显红,郭成,李冰,等.2-氨基乙基联苯基硼酸酯对绵羊卵母细胞体外成熟及早期胚胎发育的影响[J].中国畜牧杂志,2022,58(3):115-118. MO X H,GUO C,LI B,et al.2-APB enhances ovine oocytematuration and early embryo development[J].Chinese Journal of Animal Science,2022,58(3):115-118.(in Chinese)
[17] ORRENIUS S,ZHIVOTOVSKY B,NICOTERA P.Regulation of cell death:The calcium-apoptosis link[J]. Nature Reviews of Molecular and Cellular Biology,2003,4(7):552-565.
[18] TAKAHASHI T,IGARASHI H,DOSHIDA M,et al.Lowering intracellular and extracellular calcium contents prevents cytotoxic effects of ethylene glycol based vitrification solution in unfertilized mouse oocytes[J].Molecular Reproduction and Development,2004,68(2):250-258.
[19] CAJAS Y N,CANÓN-BELTRÁN K,LADRÓN DE GUEVARA M,et al.Antioxidant nobiletin enhances oocyte maturation and subsequent embryo development and quality[J].International Journal of Molecular Sciences,2020,21(15):5340.
[20] BARBERET J,BARRY F,CHOUX C,et al.What impact does oocyte vitrification have on epigenetics and gene expression?[J].Clinical Epigenetics,2020,12(1):121.
[21] MIAO Y,ZHOU C,CUI Z,et al.Dynein promotes porcine oocyte meiotic progression by maintaining cytoskeletal structures and cortical granule arrangement[J].Cell Cycle,2017,16(21):2139-2145.
[22] KULUS M,KRANC W,JESETA M,et al.Cortical granule distribution and expression pattern of genes regulating cellular component size,morphogenesis,and potential to differentiation are related to oocyte developmental competence and maturational capacity in vivo and in vitro[J].Genes (Basel),2020,11(7):815.
[23] 王寒阳,李捷鑫,李文瑞,等.不同直径的绵羊卵泡中卵母细胞核相及皮质颗粒分布特征分析[J].中国畜牧兽医,2016,43(5):1255-1262. WANG H Y,LI J X,LI W R,et al.The nuclear morphology and cortical granule distribution of oocytes from different diameter follicles of sheep[J].China Animal Husbandry & Veterinary Medicine,2016,43(5):1255-1262.(in Chinese)
[24] DE PAOLA M,GARRIDO F,ZANETTI M N,et al.VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes[J].Experimental Cell Research,2021,405(1):112629.
[25] MARANGOS P,CARROLL J.Fertilization and InsP3-induced Ca²⁺ release stimulate a resistant increase in the rate of degradation of cyclin B1 specifically in mature mouse oocytes[J].Developmental Biology,2004,272(1):26-38.
[26] MARCO-JIMÉNEZ F,CASARES-CRESPO L,VICENTE J S.Effect of cytochalasin B pre-treatment of in vitro matured porcine oocytes before vitrification[J]. Cryoletters,2012,33(1):24-30.
[27] MARQUES C C,SANTOS-SILVA C,RODRIGUES C,et al.Bovine oocyte membrane permeability and cryosurvival:Effects of different cryoprotectants and calcium in the vitrification media[J].Cryobiology,2018,81:4-11.
[28] SAUNDERS C M,LARMAN M G,PARRINGTON J,et al.PLCz:A sperm-specific trigger of Ca²⁺ oscillationsin eggs and embryo development[J].Development,2002,129(15):3533-3544.
[29] GAMBARDELLA J,MORELLI M B,WANG X,et al.The discovery and development of IP3 receptor modulators:An update[J].Expert Opinion on Drug Discovery,2021,16(6):709-718.
[30] KHARKOVSKAYA E Е,OSIPOV G V,MUKHINA I V.Ventricular fibrillation induced by 2-aminoethoxy diphenylborate under conditions of hypoxia/reoxygenation[J]. Minerva Cardioangiologica,2020,68(6):619-628.
[31] RAJANI S,CHATTOPADHYAY R,GOSWAMI S K,et al.Assessment of oocyte quality in polycystic ovariansyndrome and endometriosis by spindle imaging and reactive oxygen species levels in follicular fluidand its relationship with IVF-ET outcome[J].Journal of Human Reproductive Sciences,2012,5(2):187-193.
[32] ZHAO Z,YANG L,ZHANG D,et al.Elevation of MPF and MAPK gene expression,GSH content and mitochondrialdistribution quality induced by melatonin promotes porcine oocyte maturation and development in vitro[J].PeerJ,2020,8:e9913.
[33] HOOL L C,CORRY B.Redox control of calcium channels:From mechanisms to therapeutic opportunities[J]. Antioxidants & Redox Signaling,2007,9(4):409-435.
[34] MARTIN-ROMERO F J,ORTIZ-DE-GALISTEO J R,LARA-LARANJEIRA J,et al.Store-operated calcium entry inhuman oocytes and sensitivity to oxidative stress[J].Biology of Reproduction,2008,78(2):307-315.
[35] MATEO-OTERO Y,YESTE M,DAMATO A,et al.Cryopreservation and oxidative stress in porcine oocytes[J].Research in Veterinary Science,2021,135:20-26.
[36] APPELTANT R,SOMFAI T,SANTOS E C S,et al.Effects of vitrification of cumulus-enclosed porcine oocytes at the germinal vesicle stage on cumulus expansion,nuclear progression and cytoplasmicmaturation[J].Reproduction,Fertility,and Development,2017,29(12):2419-2429.