Micro-proteomics reveals distinct protein profiles and SPARC/FGF2/CDH1 regulation of human Sertoli cells between Sertoli cell-only syndrome and normal men

Abstract

Sertoli cell-only syndrome (SCOS) is one of the most severe non-obstructive azoospermia (NOA) types, since only Sertoli cells with not any male germ cells exist with the seminiferous tubules. As such, it is of particular significance to elucidate molecular mechanisms underlying SCOS for improving the diagnosis and treatment strategies for this disease. Due to the difficulties in obtaining sufficient human testicular tissues and the limited availability of human cells, the traditional proteomics is inadequate for comparing the differences in large scale of protein expression patterns of human Sertoli cells between SCOS and normal men. To solve this issue on the requirement of large amount of cell numbers, we employed micro-proteomics to reveal distinct global protein expression profiles of human Sertoli cells between SCOS and obstructive azoospermia (OA) with normal spermatogenesis utilizing single human Sertoli cells. We found a significant downregulation of proteins involved in cell adhesion pathways in SCOS Sertoli cells, whereas proteins related to apoptosis were markedly upregulated. Interestingly, we identified the lower expression of SPARC (secreted protein acidic and rich in cysteine) and the higher expression of FGF2 (fibroblast growth factor 2) in human Sertoli cells of the SCOS compared to normal men. SPARC silencing led to upregulation of FGF2 in human Sertoli cells, and SPARC may be associated with the occurrence of SCOS and serves as a reliable marker for the diagnosis of this disease. This study thus comprehensively offers the proteomic landscape of human Sertoli cells in the testes of SCOS patients and it sheds a novel insight into the pathogenesis of SCOS.

Keywords: CDH1; FGF2; Human Sertoli cells; Micro-proteomics; SPARC; Sertoli cell-only syndrome.

Fig. 1

Isolation and identification of human Sertoli cells. A, Overview of the method used for differential protein analysis of human Sertoli cells in SCOS and OA patients using micro-proteomics.B, H&Estaining images of cross-sections of testicular tissues from SCOS patients and OA.C, Immunocytochemical staining of the isolated cells expressing Sertoli cell markers SOX9, BMP4, and GATA4.Scale bars in (B)= 20 μm; scale bars in (C) = 50 μm.

Fig. 2

Single-cell proteomics of global protein expression profiles in human Sertoli cells between SCOS patients and OA. A, PCA of proteins expressed in human Sertoli cells from SCOS and OA patients. B, Volcano plot of differentially expressed proteins in human Sertoli cells between SCOS and OA patients. C, Heatmap of the differentially expressed proteins in human Sertoli cells between SCOS and OA patients. D, GO analysis of the upregulated expression proteins in human Sertoli cells between SCOS and OA patients. E, GO analysis of the downregulated expression proteins in human Sertoli cells between SCOS and OA patients. F, PPI analysis of the top five downregulated GO pathways in human Sertoli cells between SCOS and OA patients

Fig. 3

Figure 3.Downregulation of SPARC protein in SCOS Sertoli cells. A, Relative levels of proteins intensity of SPARC, FGF2, and CDH1 in human Sertoli cells in SCOS and OA patients by micro-proteomics. B, The mRNA expression of SPARC in the OA and SCOS Sertoli cells. C-D, Western blots revealed the SPARC protein expression in the OA and SCOS Sertoli cells.E, Immunohistochemistry showed the SPARC protein expression in the seminiferous tubules of OA and SCOS patients. MAGEA4 was used for the expression of human spermatogonia. F, Real‐time PCR showed SPARCmRNA levels in human Sertoli cells with the treatment of control siRNA and SPARCsiRNA1-3. G-I, Real‐time PCR and Western blots displayed the mRNA (G) and protein (H-I) expression of FGF2, GDNF, SOX9, and SPARC in human Sertoli cells after SPARC knockdown. J, Immunocytochemistry showed co-localization of SPARC with FGF2 in human Sertoli cells of OA patients. Scale bars in (D) = 50 μm, Scale bars in (H) = 25 μm. *p< 0.05.

Fig. 4

Downregulation of SPARC in human Sertoli cells affects the biological functions of human Sertoli cells. A-B, Changes in cell apoptosis detected by flow cytometry in human Sertoli cellsafter SPARC knockdown. C, Relative changes in mRNA levels of cell adhesion pathway genes in human Sertoli cellsafter SPARC knockdown. D-F, Immunocytochemistry displayed the expression of GJA1 (D), CDH1 (E), and ZO-1 (F) proteins in human Sertoli cells with control siRNA andSPARCsiRNAs. Scale bars = 25 μm.G-H, Changes in the number of cell adhesionin human Sertoli cells bySPARC silencing. *p< 0.05.

Fig. 5

Alterations in marker proteins in human Sertoli cells of SCOS and OA. A-C, Immunocytochemistry displayed the expression of FGF2 (A), GDNF (B), and WT1 (C) proteins in SCOS and OA Sertoli cells. Scale bars = 50 μm

Fig. 6

Expression changes of marker genes and proteins in human Sertoli cells from SCOS and OA patients and FGF2 affects CDH1 expression in human Sertoli cells. A, Relative expression of marker genes in human Sertoli cells from SCOS and OA patients. B-C, Expression of marker proteins in human Sertoli cells from SCOS and OA patients; D, Transcripts of cell adhesion-related genes in human Sertoli cells by overexpression and FGF2 knockdown. E-F, Overexpression of FGF2 affects CDH1 and Occludin protein expression in human Sertoli cells. G-H, Western blot showed the changes in CDH1 and Occludin protein levels in human Sertoli cells treated without or with FGF2 knockdown. I, Immunofluorescence showed changes in CDH1 protein expression in human Sertoli cells with FGF2 overexpression and knockdown. J-K, Changes in cell adhesion in human Sertoli cells by FGF2 overexpression and knockdown. *P < 0.05

Fig. 7

Apoptosis was increased in SCOS Sertoli cells. A-B, Detection of human Sertoli cell apoptosis by flow cytometry. C-D, Detection of human Sertoli cell apoptosis by TUNEL assay. E, Ultra-structure of human Sertoli cells’ apoptosis under transmission electron microscopy (TEM). F, The JC-1 staining assay showed the mitochondrial membrane potential (MMP) levels in human Sertoli cells from OA and SCOS patients. G, GSEA analysis of apoptosis-related protein expression in human Sertoli cells of OA and SCOS. H, The qPCR detection of the expression of apoptosis-related genes in human Sertoli cells of OA and SCOS patients. * p< 0.05