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用牛血清白蛋白或胎牛血清培养的牛卵丘细胞中DNA修复机制相关关键基因的表达谱

Expression profile of key genes involved in DNA repair mechanisms in bovine cumulus cells cultured with bovine serum albumin or fetal calf serum.

作者信息

Goetten André Lucio Fontana, Koch Júlia, Rocha Cecília Constantino, Mezzalira Alceu, Price Christopher Allan, Portela Valério Marques, Barreta Marcos Henrique

机构信息

Centro de Ciências Rurais, Universidade Federal de Santa Catarina (UFSC), Curitibanos, SC, Brazil.

Department of Animal Sciences, University of Florida, Gainesville, FL, USA.

出版信息

Reprod Biol. 2023 Mar;23(1):100709. doi: 10.1016/j.repbio.2022.100709. Epub 2022 Nov 17.

Abstract

Cumulus cells from cumulus-oocyte complexes (COC) matured in vitro in serum-free medium show high incidence of apoptosis and DNA double-strand breaks (DSB). This study aimed to characterize the transcript expression profile of selected genes involved in DNA repair mechanisms in bovine cumulus cells cultured with bovine serum albumin (BSA) or fetal calf serum (FCS). Briefly, bovine cumulus-oocyte complexes were in vitro matured with either, 0.4% BSA or 10% FCS for 3, 6, 12 or 24 h. The total RNA of cumulus cells was used for real-time PCR analysis. Transcript abundance of XRCC6, XRCC5, DNAPK, GAAD45B, TP53BP1, RAD50, RAD52, ATM and BRCA2 target genes changed as the IVM proceeded (P < 0.05). However, an interaction between protein source (FCS or BSA) and time was not detected (P ≥ 0.05). Cumulus cells from COCs matured with BSA presented higher mRNA expression of two genes compared to FCS group: TP53BP1 at 6 h and BRCA1 at 3, 6, 12 and 24 h (P < 0.05). In summary, our results showed for the first time the expression profile of the key genes involved in DSB repair mechanisms in cumulus cells obtained from bovine COCs matured with FCS or BSA. The higher mRNA expression of BRCA1 and TP53BP1 and lower mRNA expression of TNFAIP6 suggests an increase in apoptosis rate and DNA damage in cumulus cells cultured in BSA-supplemented medium and may explain, at least to some extent, the reduced developmental potential of bovine oocytes matured in serum-free medium.

摘要

在无血清培养基中体外成熟的卵丘-卵母细胞复合体(COC)中的卵丘细胞显示出高凋亡率和DNA双链断裂(DSB)。本研究旨在表征在用牛血清白蛋白(BSA)或胎牛血清(FCS)培养的牛卵丘细胞中,参与DNA修复机制的选定基因的转录表达谱。简要地说,牛卵丘-卵母细胞复合体在含有0.4% BSA或10% FCS的培养基中体外成熟3、6、12或24小时。卵丘细胞的总RNA用于实时PCR分析。随着体外成熟过程的进行,XRCC6、XRCC5、DNA-PK、GAAD45B、TP53BP1、RAD50、RAD52、ATM和BRCA2靶基因的转录丰度发生变化(P < 0.05)。然而,未检测到蛋白质来源(FCS或BSA)与时间之间的相互作用(P≥0.05)。与FCS组相比,用BSA成熟的COC中的卵丘细胞在两个基因上呈现出更高的mRNA表达:6小时时的TP53BP1以及3、6、12和24小时时的BRCA1(P < 0.05)。总之,我们的结果首次显示了从用FCS或BSA成熟的牛COC中获得的卵丘细胞中,参与DSB修复机制的关键基因的表达谱。BRCA1和TP53BP1的较高mRNA表达以及TNFAIP6的较低mRNA表达表明,在添加BSA的培养基中培养的卵丘细胞的凋亡率和DNA损伤增加,这可能至少在一定程度上解释了在无血清培养基中成熟的牛卵母细胞发育潜力降低的原因。

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