亚硒酸钠对猪卵母细胞体外成熟及其胚胎发育潜能的影响

doi:10.3864/j.issn.0578-1752.2024.17.013

摘要/Abstract

摘要：

【目的】探究亚硒酸钠（sodium selenite，SS）对猪卵母细胞体外成熟及其胚胎发育潜能的影响并对其机制进行初步分析，为猪卵母细胞体外成熟体系的完善提供理论参考。【方法】将猪卵丘卵母细胞复合物（cumulus oocyte complexes，COCs）随机放在不同浓度亚硒酸钠（0、20、40、60、80 nmol·L^-1）的成熟培养液中成熟培养44 h后，分别观察卵丘细胞扩展及卵母细胞第一极体（first polar body, PB1）排出情况，收集卵丘细胞并提取其RNA，同时对卵母细胞进行孤雌激活（parthenogenetic activation，PA）处理并进一步对孤雌胚胎进行体外培养，分别于48、168 h统计卵裂率及囊胚率。利用实时荧光定量PCR及观察法对卵丘细胞扩展指数（CEI）、卵丘扩展相关基因（Has2、Ptgs2）表达、卵母细胞第一极体（PB1）排出率和孤雌胚胎的卵裂率及囊胚率进行检测，以探究不同浓度亚硒酸钠对卵母细胞成熟、早期胚胎发育及卵丘细胞扩展的影响，并据此确定亚硒酸钠的最适添加浓度；通过光吸收法及RT-qPCR检测COCs的总抗氧化能力、卵母细胞中谷胱甘肽（GSH）含量和丙二醛（MDA）水平及抗氧化基因（CAT、PRDX2）表达，探讨体外成熟液中添加亚硒酸钠对卵母细胞抗氧化能力的影响；结合免疫荧光技术对囊胚内细胞总数和囊胚多能性基因（Nanog，Sox2）表达进行检测，探究亚硒酸钠对孤雌胚胎发育潜能的影响。【结果】QRT-PCR结果显示不同浓度亚硒酸钠添加均可显著促进PTGS2基因的表达（P<0.05），且40 nmol·L^-1浓度下HAS2基因的表达、CEI、卵母细胞PB1、早期胚胎的囊胚率都得到显著促进（P<0.05）；40、60、80、nmol·L^-1浓度下亚硒酸钠添加都可促进卵母细胞的卵裂，但40 nmol·L^-1 浓度下卵裂促进效果显著（P<0.05），据此确定猪卵母细胞体外成熟液中亚硒酸钠添加浓度为40 nmol·L^-1；由抗氧化能力检测结果显示亚硒酸钠可显著提高COCs的总抗氧化能力、卵母细胞的GSH水平、抗氧化基因CAT、PRDX2的表达、显著降低MDA含量（P<0.05）；免疫荧光及PCR结果显示，亚硒酸钠组中囊胚的内细胞团数量升高、Nanog表达升高（P<0.05）。【结论】猪卵母细胞体外成熟液中添加适宜浓度的亚硒酸钠可促进卵丘细胞扩展，提高卵母细胞的成熟率，改善卵母细胞成熟后的发育潜能。亚硒酸钠对卵母细胞体外成熟的这一有利影响可能与其抗氧化作用有关。

关键词: 亚硒酸钠（SS）, 猪卵母细胞, 体外成熟（IVM）, 氧化应激, 孤雌激活

Abstract:

【Objective】The aim of this study was to investigate the effects of sodium selenite (SS) on the in vitro maturation of porcine oocytes and their embryonic developmental potential, and to conduct a preliminary analysis of its mechanism, so as to provide the theoretical references for the improvement of the in vitro maturation system of porcine oocytes.【Method】After culturing porcine cumulus-oocyte complexes (COCs) in maturation medium containing different concentrations of SS (0, 20, 40, 60, and 80 nmol·L^-1) for 44 hours, observations were made, and cumulus cell expansion and oocyte maturation status were collected. Cumulus cell RNA was extracted, and oocytes were subjected to parthenogenetic activation (PA) treatment. The cleavage rate and blastocyst rate of parthenogenetic embryos were statistically analyzed at 48 and 168 hours of in vitro culture. Real-time fluorescence quantitative PCR and observation were used to detect cumulus cell expansion index (CEI), expression of cumulus expansion-related genes (Has2, Ptgs2), first polar body (PB1) extrusion rate, and cleavage rate and blastocyst rate of parthenogenetic embryos, to investigate the effects of different concentrations of SS on oocyte maturation, early embryo development, and cumulus cell expansion, and to determine the optimal concentration of SS. Total antioxidant capacity of COCs, glutathione (GSH) content, malondialdehyde (MDA) levels, and expression of antioxidant-related genes (CAT, PRDX2) in oocytes were detected by spectrophotometry and RT-qPCR to explore the effect of adding SS to the maturation medium on oocyte antioxidant capacity. Immunofluorescence technology was combined to detect the total number of cells in blastocysts and the expression of pluripotency genes (Nanog, Sox2) in blastocysts, to investigate the effect of SS on the developmental potential of parthenogenetic embryos.【Result】The RT-qPCR results showed that the addition of different concentrations of SS significantly promoted the expression of the PTGS2 gene (P<0.05), and at a concentration of 40 nmol·L^-1, the expression of the HAS2 gene, CEI, PB1 extrusion rate of oocytes, and blastocyst rate of early embryos were all significantly promoted (P<0.05). SS at concentrations of 40, 60, and 80 nmol·L^-1 promoted oocyte cleavage, with significant promotion observed at a concentration of 40 nmol·L^-1 (P<0.05). Based on those results, the optimal concentration of SS adding to the in vitro maturation medium for porcine oocytes was determined to be 40 nmol·L^-1. The results of antioxidant capacity testing showed that SS significantly increased the total antioxidant capacity of COCs, the level of GSH in oocytes, and the expression of antioxidant genes CAT and PRDX2, while significantly reducing MDA content (P<0.05). Immunofluorescence and PCR results showed that the number of inner cell mass in blastocysts and the expression of Nanog were increased in the SS group (P<0.05).【Conclusion】The addition of SS to the in vitro maturation medium of oocytes promoted cumulus cell expansion, enhanced oocyte maturation rates, and improved the developmental potential of matured oocytes. The beneficial effects of SS on the in vitro maturation of oocytes might be associated with its antioxidant properties.

Key words: sodium selenite (SS), porcine oocytes, in vitro maturation (IVM), oxidative stress, parthenogenetic activation

张冰, 杨燕燕, 冯前会, 施雯, 方肄臻, 黄佳宝, 石德顺. 亚硒酸钠对猪卵母细胞体外成熟及其胚胎发育潜能的影响[J]. 中国农业科学, 2024, 57(17): 3482-3493.

ZHANG Bing, YANG YanYan, FENG QianHui, SHI Wen, FANG YiZhen, HUANG JiaBao, SHI DeShun. Effects of Sodium Selenite on the in vitro Maturation of Porcine Oocytes and Their Embryonic Development Potentials[J]. Scientia Agricultura Sinica, 2024, 57(17): 3482-3493.

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https://www.chinaagrisci.com/CN/Y2024/V57/I17/3482

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基因 Gene	引物序列 Primer sequence	长度 Length (bp)	GenBank登录号 Accession No.
GAPDH	F: TATGATTCCACCCACGGCAAG R: CCACAACATACGTAGCACCAG	145	NM_001206359.1
PTGS2	F: CAGGCTGATACTGATAGGAGAAA R: CTGGTTGAAAAGCAGCTCTGG	112	NM_214321.1
HAS2	F: ATGACAGGCATCTAACGAAC R: TTGTACAGCCACTCTCGGAA	157	NM_214053.1
CAT	F: GCTGAGTCCGAAGTCGTCTA R: GTCAGGATATCAGGTTTCTGCG	173	NM_214301.2
PRDX2	F: TTCGGGCCGAGCATAAAAGG R: AGGCCATGACTGAAAGCTGC	119	NM_001244474.1
NANOG	F: TCCACAAGCCCCAGAGTAAA R: GCCTCTGAAATCTGTCGTTG	145	NM_001129971.2
SOX2	F: AAGAACAGCCCAGACCGAGT R: CCGTCTCCGACAAAAGTTTCCAC	154	NM_001123197.1

浓度 Concentration (nmol·L^-1)	卵母细胞数 No. of oocytes	卵裂数 No. of cleavage	卵裂率 Rate of cleavage	囊胚数 No. of blastocyst	囊胚率 Rate of cleavage
0	369	295	79.47±1.44c	73	24.74±1.88b
20	362	307	82.03±1.94bc	86	28.22±1.34ab
40	406	365	87.7±1.59a	114	31.49±1.19a
60	372	320	84.49±1.00b	89	28.23±0.59ab
80	376	323	84.41±1.59b	82	26.27±2.81b