Testis cell pyroptosis mediated by CASP1 and CASP4: possible sertoli cell-only syndrome pathogenesis

Abstract

Background: Sertoli cell-only syndrome (SCOS) is the most serious pathological type of non-obstructive azoospermia. Recently, several genes related to SCOS have been identified, including FANCM, TEX14, NR5A1, NANOS2, PLK4, WNK3, and FANCA, but they cannot fully explain the pathogenesis of SCOS. This study attempted to explain spermatogenesis dysfunction in SCOS through testicular tissue RNA sequencing and to provide new targets for SCOS diagnosis and therapy.

Methods: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of nine patients with SCOS and three patients with obstructive azoospermia and normal spermatogenesis. We further explored the identified genes using ELISA and immunohistochemistry.

Results: In total, 9406 DEGs were expressed (Log2|FC|≥ 1; adjusted P value < 0.05) in SCOS samples, and 21 hub genes were identified. Three upregulated core genes were found, including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. ELISA verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic, Sertoli, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli and interstitial cells because of the loss of spermatogonia and spermatocytes. CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenisis. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in control patients. ELISA also showed that inflammatory factors (IL-1 β, IL-18, LDH, and ROS) were significantly increased in the SCOS group.

Conclusions: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS. We also observed many inflammatory and oxidative stress reactions in SCOS. Thus, we propose that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development.

Keywords: CASP1; CASP4; Pyroptosis; Sertoli cell-only syndrome (SCOS).

Fig. 1

OA vs. SCOS. Volcano plot of differentially expressed genes. OA: obstructive azoospermia with normal spermatogenic function; SCOS: Sertoli cell-only syndrome

Fig. 2

A OA vs. SCOS key genes (UP). OA: obstructive azoospermia with normal spermatogenic function; SCOS: Sertoli cell-only syndrome. B OA vs. SCOS key genes (DOWN) OA: obstructive azoospermia with normal spermatogenic function; SCOS: Sertoli cell-only syndrome

Fig. 3

A Q-PCR verification of downregulated core differential genes (P < 0.05). B Q-PCR verification of upregulated core differential genes (P < 0.05)

Fig. 4

A Gene ontology (GO) enrichment of the differential expressed genes. GO analysis covered three types: biological process (BP), cellular component (CC), and molecular function (MF); red indicates upregulated genes; blue indicates downregulated genes. B Kyoto Encyclopedia of Genes and Genomes pathway enrichment in the differentially expressed genes. Dots represent term enrichment with color coding: red indicates high enrichment; blue indicates low enrichment. Dot sizes represent the percentage of each row

Fig. 5

GSDMD and GSDME pyroptosis results (P < 0.05)

Fig. 6

A Differences in CASP1 and CASP4 activity in the testicular tissues of patients with normal spermatogenesis and SCOS. OA: Normal spermatogenesis group; SCOS: Sertoli cell-only syndrome group; Asterisks (**) indicate significant differences at P < 0.01 between the treated and control groups. B CASP1 expression in the testes of patients with normal spermatogenesis (up) and Sertoli cell-only syndrome (down). (Left: 10 × ; Right: 20 ×). C CASP4 expression in the testes of patients with normal spermatogenesis (up) and Sertoli cell-only syndrome (down). (Left: 10 × ; Right: 20 ×)

Fig. 7

Differential analysis of protein expression of key genes in cell pyroptosis. OA: Normal spermatogenesis group; SCOS: Sertoli cell-only syndrome group; Asterisks (*) and (**) indicate significant differences at P < 0.05 and P < 0.01 between the MCeLR-treated and control groups, respectively (gels and blots in the original image made tailoring necessary; the relevant original documents can be seen in the Supplementary Materials)

Fig. 8

A, B Differential analysis of cell membrane-specific markers and inflammatory factors in cell pyroptosis. OA: Normal spermatogenesis group; SCOS: Sertoli cell-only syndrome group; Asterisks (**) indicate significant differences at P < 0.01 between the treated and control groups