Identification of DEGs in the transcriptome dataset
7844 DEGs were obtained in the GSE103905 dataset, containing 4606 down-regulated genes and 3238 up-regulated genes (Fig.1A), while 4824 DEGs were obtained in the GSE103613 dataset, containing 2966 down-regulated genes and 1858 up-regulated genes (Fig.1B). The Venn diagram showed 700 DEGs overlapping between the two datasets(Fig.1C).
GO and KEGG enrichment analysis
DAVID(https://david.ncifcrf.gov/)online analysis of GO functional enrichment and KEGG signalling pathway analysis of DEGs. In the BP category, significant enrichment was found in stabilization of membrane potential, memory and methylation (S.1A). In the MF category, significant enrichment was found in potassium ion leak channel activity, ATPase activity (S.1B). In the CC category, the mitochondrion, extracellular matrix and mitochondrial intermembrane space were significantly enriched (S.1C). In the KEGG enrichment analysis, it was found that the signalling pathways that may affect spermatogenesis are: metabolic pathways, huntington's disease and nicotinamide metabolism (S.1D).
Construction of PPI networks and identification of Hub genes
The testicular tissue-specific PPI networks of DEGs were analysed through the NetworkAnalyst website, and then, these networks were imported into Cytoscape to form a PPI of 254 nodes and 340 edges (Fig.2A). We used the Degree, DMNC, MCC and MNC algorithms in the cytoHubba plugin to obtain the Top 10 key genes for each algorithm (Fig.2 B-E). Finally, 4 genes (KIF2C,MRPS2,RPS15,TSFM) were repeated in more than 3 algorithms and they were considered as hub genes (Fig.2 F).
Expression of Hub genes in sperm cells
To further investigate the expression of the four Hub genes in sperm cells, we integrated scRNA-seq data from testicular tissues of normal human and KS patients and constructed a single-cell atlas formed by clustering 20 cells (Fig. 3 A). A total of 12 sperm cells were identified by similarity heatmap of cell clustering and marker genes (Fig. 3 B-C). They were: Leydig cell (LC), Myoid cell (MC), Macrophage, Sertoli cell (SC), Endothelial cell (EC) Spermatogonial stem cell (SSC), Early primary spermatocyte (EPS), Differentiating spermatogonia (DS), Late primary spermatocyte (LPS), Round spermatid(RS), Elongated spermatid(ES) and sperm (Fig. 3 D). Only KIF2C and RPS15 were expressed in the cell clusters, with RPS15 being expressed in all cells, whereas KIF2C gene expression was cell-specific and only expressed in the DS, ES, RS, EPS and sperm (Fig. 3 E-F).
Differential expression of Hub genes in KS and normal groups
In the single-cell dataset, where sperm cells were rare in KS patients, testicular somatic cells (SC,MC,EC,LC) and immune cells were predominant, whereas normal human testicular cells contained 12 species of testicular cells covering the whole process of spermatogenesis (S.2 A,B). The analysis showed that the RPS15 gene in KS was expressed at higher levels in all testicular cells than in normal subjects, whereas the expression level of the KIF2C gene was only expressed in the normal group and almost absent in the KS group (S.2 C,D).
Pseudotime analysis of KIF2C and RPS15 in spermatocytes
We verified that KIF2C and RPS15 were differentially expressed in sperm cells from the KS and the normal group, and it was necessary to accurately capture the developmental trajectories of these two genes in sperm cells. The developmental trajectory of sperm cells was constructed using monocle 3 software, and the results showed that a total of 11 branches were present in the sperm cells, and the branches were mainly distributed throughout the development from ES to sperm and in SC cells (Fig.4 A). During spermatogenesis, we successfully identified the initiating cell as SSC and the terminal cell as sperm (Fig. 4 B). We constructed trajectories of KIF2C and RPS15 in sperm development, and the results showed that RPS15 showed a downward trend from SSC to sperm formation. The expression of KIF2C showed an upward trend from SSC to sperm formation, indicating that KIF2C plays a crucial role in sperm development (Fig.4 C,D).