Single-Cell RNA Sequencing Analysis of Steroidogenesis and Spermatogenesis Impairment in the Testis of db/db Mice

Abstract

Objective: The mechanism of steroidogenesis and spermatogenesis impairment in men with type 2 diabetes remains unclear. We aimed to explore the local changes of steroidogenesis and spermatogenesis in the testis of db/db mice. Research Design and Methods. We performed single-cell RNA sequencing analysis in the testis of db/db and C57BL/6J mice. The differentially expressed genes were then confirmed by real-time PCR. The histopathological characteristics of testis in db/db mice and C57BL/6J control were also performed.

Results: The 20-week-old db/db mice had significantly higher blood glucose and body weight (both p < 0.001). The serum testosterone levels (4.4 ± 0.8 vs. 9.8 ± 0.7 ng/ml, p=0.001) and weight of the testis (0.16 ± 0.01 vs. 0.24 ± 0.01 g, p < 0.001) were significantly lower in db/db mice than that in C57BL/6J controls. db/db mice had a lower cross-sectional area of seminiferous tubules and thickness of the cell layer (both p < 0.05). The numbers of Sertoli cells and Leydig cells decreased in db/db mice (both p < 0.01). Single-cell RNA sequencing analysis showed that compared with the control group, the percentage of spermatogonia was significantly higher in the db/db mouse (p < 0.001), while the proportions of spermatocytes, round and elongating spermatids, and sperms were all lower in the db/db mouse (p all < 0.001). The most differentially expressed genes were found in round spermatids (n = 86), which were not found in spermatogonia, spermatocyte, and sperm. Igfbp5 was the most significantly decreased gene in Leydig cells of the db/db mouse, while the expression of Cd74, H2-Aa, and H2-Eb1 was elevated. Ccl7 and Ptgds were the most significantly increased and decreased genes in Sertoli cells of the db/db mouse.

Conclusions: The present study indicates spermiogenesis and steroidogenesis defects in db/db mice. The mechanism of steroidogenesis impairment in the testis of db/db mice deserves further investigation.

Figure 1

The histopathological characteristics of testis in db/db mice. (a) Testis tissues from four C57BL/6J mice (CON) and four db/db mice (db/db) were stained with hematoxylin and eosin. The red arrow indicates a Leydig cell, and the yellow arrow indicates a Sertoli cell. The green arrow shows a spermatid, which together with spermatogonia and spermatocyte are used to count the total number of cells in the tubule. The wall thickness of the tubule was measured at 3 locations in each picture (blue lines). (b) Cross-sectional area (CSA) of seminiferous tubules, the thickness of the cell layer of seminiferous tubules, and total cell number, Sertoli cells, and Leydig cells number per square millimeter in CON and db/db groups.

Figure 2

Cell clusters in db/db and C57BL/6J mice. Cell type analysis visualized with UMAP. Each dot represents a single cell. (a) Cells from db/db (blue) and C57BL/6J testis (orange). (b) Somatic cells (purple) and germ cells (grey). (c) Cell clusters were distinguished through marker genes and shown in different colors. (d) The proportion of each cell in db/db testis (blue) and C57BL/6J testis (orange), respectively. ^∗p < 0.05.

Figure 3

Differentially expressed genes in germ cells between db/db and C57BL/6J mice. (a) The numbers of differentially expressed genes (DEGs) between db/db and C57BL/6J mice in each cell cluster. Red represents upregulated DEGs, and blue represents downregulated DEGs in db/db testis compared with controls. (b–e) KEGG, GO molecular function, biological process, and cellular components enrichment analysis on the DEGs in spermatids.

Figure 4

Differentially expressed genes between db/db and C57BL/6J mice. (a) Hbb-bs, Jun, Akr1b7, Igfbp5, Ctla2a, Ccl7, and Ptgds mRNA expression in the testicular tissue from db/db (n = 3) and C57BL/6J (n = 3) mice was tested using reverse transcription PCR; ^∗∗p < 0.01. (b) Ligand-receptor interactions of Sertoli cells and other cells. The color (log 2 mean) of the circle represents the log of average expression level, and the size of the circle represents the p value; a larger point is more reliable. (c) Ligand-receptor interactions of Leydig cells and other cells. (d) The differentially expressed genes in Macrophages and T cells. The size of the circle represents the p value.