剪接因子hnRNPF对蒙古马精子生成的影响

doi:10.11843/j.issn.0366-6964.2024.02.017

摘要/Abstract

摘要： 旨在探究可变剪接因子核不均一核糖核蛋白F(heterogeneous nuclear ribonucleoprotein F,hnRNPF)对蒙古马精子生成的影响。本研究提取并培养3岁性成熟蒙古马睾丸支持细胞;构建hnRNPF过表达慢病毒载体,将载体转染至蒙古马睾丸支持细胞中;每组3个重复,分别在转染0、24、48、72 h时利用CCK-8检测转染后细胞增殖情况并确定最佳转染时间;提取转染后与未转染的蒙古马睾丸支持细胞RNA,设计已知与精子生成相关基因的引物进行qRT-PCR试验,检测精子生成相关基因在两种细胞中的表达情况。琼脂糖凝胶电泳结果显示,hnRNPF慢病毒过表达载体构建成功;CCK-8检测发现,细胞经过表达慢病毒载体转染72 h后活性最高,并且转染后细胞活性要高于未转染细胞活性,说明hnRNPF过表达会促进蒙古马睾丸支持细胞的增殖;qRT-PCR结果显示,精子生成相关基因(PKMYT1、CDC25C、YWHAZ、BUB1、BTRC、CCNE1、CALM1、PLK1、REC8、MAPK3、ADCY7)在两种细胞中的表达量差异显著,均在转染hnRNPF过表达载体细胞中含量较高。本试验结果表明,剪接因子hnRNPF可能对于蒙古马精子生成具有直接或间接的促进作用,为提高蒙古马精子数量和精液品质提供了新的思路。

关键词: 蒙古马, hnRNPF, 精子生成, 可变剪接

Abstract: The aim of this study was to investigate the effect of alternative splicing factor heterogeneous nuclear ribonucleoprotein F (hnRNPF) on spermatogenesis in Mongolian horses. Testicular sertoli cells were isolated and cultured from 3-year-old Mongolian horse. The hnRNPF overexpression lentiviral vector was constructed and transfected into the sertoli cells of Mongolian horse testis. CCK-8 was used to detect the proliferation of cells at 0 h, 24 h, 48 h and 72 h after transfection, and the optimal infection time was determined with 3 duplication per group. The RNA of transfected and untransfected Mongolian horse sertoli cells was extracted, and the primers of genes known to be related to spermatogenesis were designed for qRT-PCR to detect the expression of spermatogenesis-related genes in the two types of cells. The results of agarose gel electrophoresis showed that the hnRNPF lentivirus overexpression vector was successfully constructed. CCK-8 assay showed that the cells transfected with the expression lentiviral vector had the highest activity at 72 hours, and the activity of transfected cells was higher than that of untransfected cells, indicating that the overexpression of hnRNPF could promote the proliferation of Mongolian horse sertoli cells.qRT-PCR results showed that the expression levels of spermatogenesis-related genes (PKMYT1, CDC25C, YWHAZ, BUB1, BTRC, CCNE1, CALM1, PLK1, REC8, MAPK3, and ADCY7) were significantly different between the two types of cells. All were highly expressed in cells transfected with hnRNPF overexpression vector. The results suggest that hnRNPF may directly or indirectly promote spermatogenesis in Mongolian horses, which provides a new idea for improving the sperm count and semen quality of Mongolian horses.

Key words: Mongolian horse, hnRNPF, spermatogenesis, alternative splicing

中图分类号:

S821.3

翁雅娟, 李蓓, 芒来, 薛嘉宁, 特日格乐, 宋代玲, 王国庆, 蔺雅楠. 剪接因子hnRNPF对蒙古马精子生成的影响[J]. 畜牧兽医学报, 2024, 55(2): 598-606.

WENG Yajuan, LI Bei, DUGARJAVIIN Manglai, XUE Jianing, Terigele, SONG Dailing, WANG Guoqing, LIN Ya'nan. The Effect of Splicing Factor hnRNPF on Spermatogenesis in Mongolian Horse[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 598-606.

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