基于单细胞转录组测序的公牛精子发生过程中动态转录图谱及新标记基因分析

doi:10.1016/j.jia.2023.04.036

摘要/Abstract

摘要： 睾丸是精子发生的重要器官，正常的睾丸发育是种公牛优良繁殖性能的基础，此过程涉及到生殖细胞和体细胞之间复杂的相互作用。然而，睾丸细胞的异质性阻碍了牛睾丸发育机制的研究。本研究以安格斯公牛为研究对象，利用 scRNA-seq技术对性成熟前后安格斯牛睾丸细胞进行测序分析，探讨公牛睾丸发育的转录机制。本研究利用Seurat包对数据进行过滤质控、标准化、降维、聚类，对青春期前后的睾丸细胞进行差异基因分析, 利用KOBAS进行富集分析, 利用Monocle包对生精细胞进行拟时分析, 最后利用FISH对标记基因进行验证。睾丸样本经过质控筛选后得到青春期前及青春期牛睾丸细胞共11083个；经过降维、聚类等分析过程后，共得到12个睾丸细胞亚群，其中包括9个体细胞亚群（未成熟支持细胞、成熟支持细胞、间质祖细胞、间质细胞、管周肌细胞、T细胞、巨噬细胞、平滑肌细胞、内皮细胞）和3个生精细胞亚群（精原细胞、精母细胞、精子细胞），不同样本之间具有明显的细胞异质性；进一步将睾丸生精细胞进行聚类分析，获得13个亚群（精原干细胞1期、精原干细胞2期、正在分化的精原细胞、已分化精原细胞、细线期、偶线期、粗线期、双线期、次级精母细胞期、圆型精子、延长型精子1期、延长型精子2期、未成熟精子）；通过Monocle算法，成功构建了生精细胞系的拟时分化轨迹；并筛选出牛睾丸不同类群细胞的特异标记基因，例如：精原干细胞标记基因GRAF2和MORC1，分化的精原细胞标记基因HJURP和TCF19，代表未成熟支持细胞标记基因ARSE，成熟支持细胞标记基因CLEC12B，间质细胞标记基因LOC112441470等。综上所述，此研究揭示了牛性成熟过程中生殖细胞的动态变化，并提出牛睾丸细胞独特的标记基因，为解释牛睾丸发育和精子生成的机制提供了理论依据，为牛青春期的生殖细胞及体细胞的发育提供了新的见解，同时也为后期研究提供了宝贵的数据资源。

Abstract:

Testicular development is an important biological process in male and requires interaction between the male germ cells and somatic cells. However, the mechanisms of testicular development in livestock, particularly in cattle, are poorly understood. Furthermore, cellular heterogeneity hinders the profiling of different cell types at different developmental stages. In this study, we first performed a single-cell transcriptomic study of the bovine testis development during puberty by using 10^× genomics single-cell RNA sequencing (scRNA-seq). By collecting the scRNA-seq data from 11,083 cells from prepubertal and pubertal bovine testes, a high-resolution scRNA-seq atlas was described, identifying 9 somatic and 13 spermatogenic clusters. We also distinguished several stage-specific marker genes for bovine germ cells and somatic cells, such as GRAF2 and MORC1 for SSC (spermatogonial stem cells), HJURP and TCF19 for differentiating spermatogonia, ARSE for immature Sertoli, CLEC12B for mature Sertoli, LOC112441470 for Leydig. In conclusion, we have examined the transcription levels and constructed the single-cell developmental maps of germ cells and somatic cells during testicular development in Angus cattle. The datasets provided new insights into spermatogenesis and testicular somatic cell development in cattle.

Key words: scRNA-seq , cattle , spermatogenesis , testis development

Yuan Gao, Fuxia Bai, Qi Zhang, Xiaoya An, Zhaofei Wang, Chuzhao Lei, Ruihua Dang. 基于单细胞转录组测序的公牛精子发生过程中动态转录图谱及新标记基因分析[J]. Journal of Integrative Agriculture, 2024, 23(7): 2362-2378.

Yuan Gao, Fuxia Bai, Qi Zhang, Xiaoya An, Zhaofei Wang, Chuzhao Lei, Ruihua Dang. Dynamic transcriptome profiles and novel markers in bovine spermatogenesis revealed by single-cell sequencing[J]. Journal of Integrative Agriculture, 2024, 23(7): 2362-2378.

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