Spermatogenesis is a complex process of cellular division and differentiation that begins with spermatogonia stem cells and leads to functional spermatozoa production. However, many of the molecular mechanisms underlying this process remain unclear. Single-cell RNA sequencing (scRNA-seq) is used to sequence the entire transcriptome at the single-cell level to assess cell-to-cell variability. In this study, more than 33,000 testicular cells from different scRNA-seq datasets with normal spermatogenesis were integrated to identify single-cell heterogeneity on a more comprehensive scale. Clustering, cell type assignments, differential expressed genes and pseudotime analysis characterized 5 spermatogonia, 4 spermatocyte, and 4 spermatid cell types during the spermatogenesis process. The UTF1 and ID4 genes were introduced as the most specific markers that can differentiate two undifferentiated spermatogonia stem cell sub-cellules. The C7orf61 and TNP can differentiate two round spermatid sub-cellules. The topological analysis of the weighted gene co-expression network along with the integrated scRNA-seq data revealed some bridge genes between spermatogenesis’s main stages such as DNAJC5B, C1orf194, HSP90AB1, BST2, EEF1A1, CRISP2, PTMS, NFKBIA, CDKN3, and HLA-DRA. The importance of these key genes is confirmed by their role in male infertility in previous studies. It can be stated that, this integrated scRNA-seq of spermatogenic cells offers novel insights into cell-to-cell heterogeneity and suggests a list of key players with a pivotal role in male infertility from the fertile spermatogenesis datasets. These key functional genes can be introduced as candidates for filtering and prioritizing genotype-to-phenotype association in male infertility.

精子发生是一个复杂的细胞分裂和分化过程，从精原干细胞开始并导致功能性精子的产生。然而，这一过程背后的许多分子机制仍不清楚。单细胞 RNA 测序 (scRNA-seq) 用于在单细胞水平对整个转录组进行测序，以评估细胞间的变异性。在这项研究中，整合了来自不同 scRNA-seq 数据集且精子发生正常的 33,000 多个睾丸细胞，以更全面地识别单细胞异质性。聚类、细胞类型分配、差异表达基因和假时间分析在精子发生过程中表征了 5 种精原细胞、4 种精母细胞和 4 种精子细胞类型。UTF1 和 ID4 基因被引入作为可以区分两个未分化的精原细胞干细胞亚细胞的最具特异性的标记。C7orf61和TNP可以区分两个圆形的精子细胞亚细胞。加权基因共表达网络的拓扑分析以及整合的 scRNA-seq 数据揭示了精子发生主要阶段之间的一些桥接基因，例如 DNAJC5B、C1orf194、HSP90AB1、BST2、EEF1A1、CRISP2、PTMS、NFKBIA、CDKN3 和 HLA- DRA。这些关键基因的重要性在之前的研究中通过它们在男性不育症中的作用得到了证实。可以说，这种整合的生精细胞 scRNA-seq 提供了对细胞间异质性的新见解，并从可育精子发生数据集中提出了在男性不育症中具有关键作用的关键参与者列表。