SCOS is one of the most serious causes of male infertility[14]. Its pathological mechanisms and treatment are difficult study points in the clinic[15–18]. Apart from some known factors, such as Klinefelter syndrome, genomic AZF deletions, cryptorchidism, and mumps orchitis, which can lead to SCOS, there are still many unknown causes [11]. The human testis transcriptome has been studied extensively using microarray assays in recent years, but no systematic study has reported mRNA profiling in human testicular tissue via high-throughput sequencing technology[19–21].
This study explored the gene expression profiles of human testicular tissues from OA and SCOS patients using RNA-seq. We explored DEGs between these testicular tissues, aiming to discover key SCOS genes that underlie the molecular mechanisms of male infertility. In total, 6865 downregulated and 2541 upregulated genes were identified in the first round of analysis. In previous studies, FANCM, TEX14, NR5A1, and WT1 were implicated as monogenic causes of SCOS, and NANOS2, PLK4, WNK3, FANCA were reported in a single study, which requires additional cases to establish a monogenic causative link to SCOS[15]. Furthremore, our results also showed significant differences in the expression of the above genes between OA and SCOS patients (Table 2).
Table 2
Expression of previously identified SCOS-related genes in our study.
Symbol | log2(FC) | P-value | FDR | Significance |
FANCM | -1.94022 | 0.000628 | 0.004049 | down |
TEX14 | -8.18966 | 3.22E-49 | 1.15E-46 | down |
NR5A1 | 2.146846 | 0.014571 | 0.052201 | up |
WT1 | 1.227602 | 0.032359 | 0.095337 | up |
NANOS2 | -5.52878 | 7.48E-05 | 0.000612 | down |
PLK4 | -4.22917 | 1.53E-18 | 6.02E-17 | down |
FANCA | -3.92755 | 1.18E-15 | 3.54E-14 | down |
Considering the large number of DEGs screened in the first analysis, we conducted a second core DEG analysis based on the interaction network. Our study identified 21 downregulated and 3 upregulated key genes. RNA-seq data accuracy is the basis of all downstream pathway analyses[22–24]. Q-PCR analysis of all key genes showed consistent patterns with the RNA-seq data in this study.
Then, GO enrichment analysis showed that key genes were mainly involved in the cell cycle, macromolecule metabolic process, cellular nitrogen compound metabolic process, and organelle organization for BP. Key genes primarily clustered in the nuclear part, intracellular organelle part, intracellular membrane-bounded organelle, and intracellular for CC. For MF, enzyme binding and nucleic acid binding were most enriched. Similarly, analysis of associated KEGG pathways showed that downregulated core genes were mainly involved in the cell cycle, progesterone-mediated oocyte maturation, and oocyte meiosis. Upregulated core genes were mainly related to cell pyroptosis and inflammation. There were 11 downregulated key genes in cell cycle passways and 9 downregulation key genes in progesterone-mediated oocyte maturation and oocyte meiosis passways. Two more downregulated core genes were not concentrated in the same signal pathway and were mainly involved in the activation of biological enzymes (Table 3).
Table 3
Functional analysis results of down-regulated core differential genes
Function | Key genes |
Cell cycle | CDC6, CDC20, TUBA4A, TUBG1, CCNA1, CCNB1, CCNA2, CDT1, CDK1, CCNE1,MCM4 |
Progesterone-mediated oocyte maturation and oocyte meiosis passway | BRCA1, E2F1, CHEK1, TOPBP1,MAD2L1, HIST3H3, BLM, UBE2C,MCM4 |
Biological enzyme activation | BIRC5, RAD51 |
Downregulated genes were recently investigated, but no further research has been performed. It was generally believed that downregulated genes were likely caused by the loss of spermatogenic cells in SCOS. However, some scholars believed that downregulated genes could induce spermatogenesis dysfunction. This causal relationship was difficult to prove under experimental conditions. Therefore, this study mainly focused on upregulated key genes for further analysis.
We found that CASP4, CASP1, and PLA2G4A were upregulated in SCOS based on interaction networks. We compared upregulated core genes and found that both CASP1 and CASP4 are part of the caspase family, which participates in cell pyroptosis. Thus, we showed for the first time that cell pyroptosis might be involved in the SCOS pathological process.
In addition, immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic cells, Sertoli cells, and interstitial cells. In the SCOS group, CASP1 and CASP4 were mainly expressed in the nuclei of Sertoli cells and interstitial cells due to the loss of spermatogonia and spermatocytes in SCOS. At the same time, CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. We hypothesize that testis cell pyroptosis mediated by CASP1 and CASP4 could play an important role in modulating Sertoli cell functions and spermatogenesis.
Cell death occurs at any time in the process of individual growth and development. It is an ordinary life phenomenon in biology that plays an essential role in multicellular organism growth, development, and dynamic balance. There are two types of cell death: cell necrosis and programmed cell death, the latter including programmed cell necrosis and apoptosis. In addition, researchers have discovered new programmed cell death types in recent years, including cell swelling, autophagy, and pyroptosis. Unlike apoptosis and necrosis, pyroptosis depends on inflammatory caspases (CASP1 and CASP4/5/11), accompanied by an inflammatory response[35]. Therefore, pyroptosis plays an essential role in infectious, cardiovascular, and central nervous system diseases, as well as tumors[36–38]. Pyroptosis lies between apoptosis and necrosis. Pore formation in the cell membrane leads to cell integrity loss, cell content release, increase in permeability, and inflammatory responses[39].
On one hand, moderate pyroptosis contributes to cell homeostasis and may effectively prevent excessive cell proliferation, which may protect the host. During pyroptosis, IL-1β and IL-18 secretion promotes leukocyte infiltration and activation. Cell lysis releases inflammatory mediators into the extracellular space, including ATP, IL-1β, and other heat-shock proteins that stimulate pro-inflammatory cytokine production by activating pattern-recognition receptors. This contributes to the control and ultimate resolution of microbial infection and allows tissues to return to their homeostatic state. On the other hand, high pyroptosis levels may induce inflammation, which is unfavorable to homeostasis maintenance in vivo. Excessive CASP1 and CASP4 activation can result in pyroptosis. Cells with excessive pyroptosis increase the levels of inflammatory mediators IL-1β and IL-18, which can be detrimental to the host and may lead to disease if not controlled. Pyroptosis has been widely reported in various cell types, including macrophages, neutrophils, dendritic cells, endothelial cells, and cardiomyocytes. Mechanistically, pyroptosis is well preserved across cell types, involving the activation of canonical CASP1 and non-canonical CASP4/5/11 (human CASP4/5 and murine CASP11).
It is worth noting that canonical pyroptosis mediated by CASP1 and non-canonical pyroptosis mediated by CASP4 share the same downstream pathway regulated by GSDMD and GSDME. In our study, differential analysis of sequencing data also demonstrated high GSDMD and GSDME expression in SCOS. Furthermore, quantitative PCR results of testicular tissues also confirmed the increased expression of GSDMD and GSDME genes in SCOS patients. To further explain the involvement of cell pyroptosis in SCOS occurrence and development, we also analyzed the protein expression level of pyroptosis-related genes. We found that the protein expression levels of CASP1, CASP4, GSDMD, and GSDME were significantly upregulated in the testicular tissues of patients with SCOS. Furtremore, CASP1 and CASP4 activity was significantly higher than that in patients with normal spermatogenesis. We also found that LDH, ROS, IL-1β, and IL-18 levels in the testicular tissue of SCOS patients in our study were significantly increased. GSDMD and GSDME can induce inflammatory factors IL-1β and IL-18, cause cell membrane damage in testis cells due to pyroptosis. LDH and ROS can be rapidly released out of the cell when the membrane is damaged. Therefore, GSDMD and GSDME may be activated in patients with SCOS through CASP1 and CASP4 overexpression to promote testis cell pyroptosis.
We found that testicular cell pyroptosis might be a pathogenic basis of SCOS, but the specifics of the process remain unclear. It is well known that cell pyroptosis is closely related to inflammation and oxidative stress. Inflammation and oxidative stress are the most common activating factors and direct manifestations of cell pyroptosis. We speculated that local inflammation and oxidative stress responses could promote testicular cell pyroptosis.
In addition, we identified a lot of inflammatory factors and ROS in the testicular tissues of SCOS patients in this study. The inflammasome is upstream of the cell pyroptosis signaling pathway, which mainly contains receptor protein, adaptor ASC, and downstream CASP1\4\5\11. Activated CASP1\4\5\11 can promote the maturation and secretion of IL-1β and IL-18, leading to pyroptosis. Testicular inflammation can secrete various inflammatory factors, resulting in changes in spermatogenesis, sperm transport, and sperm function. IL-1β can activate intracellular inflammatory responses and further hinder the division and differentiation of spermatogonial stem cells. Previous studies have shown that ROS can promote pyroptosis-related genes and initiate cell pyroptosis. At the same time, oxidative stress produces ROS-activated inflammatory bodies, the most common upstream inflammatory response mechanism. Oxidative stress is an essential pathological factor in male infertility. The dynamic balance between ROS and antioxidants is related to male fertility. Thus, inflammation and oxidative stress could promote cell pyroptosis in SCOS.
In the case of external stimulation, moderate testis cell pyroptosis can clear the stimulus source. In contrast, excessive cell pyroptosis can lead to testis cell death and release inflammatory factors to increase the inflammatory response. Oxidative stress and inflammation in the testes of patients with SCOS could upregulate CASP1/CASP4 expression and promote pyroptosis. Overexpressed CASP1 could synthesize inflammatory mediators, such as IL-1β and IL-18, which could produce a testicular inflammatory response and disturb the differentiation and maturation of spermatogenic cells.
In this study, we ruled out some known factors, including chromosome disorders, Y chromosome microdeletions, cryptorchidism, radiation, cytotoxic drug intake, and mumps orchitis. Furthermore, we explored the pathogenic genes and pathogenesis of SCOS caused by other idiopathic factors based on RNA-seq. We did not exclude varicocele, testicular hydrocele, testicular microlithiasis, and other unknown factors when screening testicular tissues. Abnormal spermatogenesis in cryptorchidism is mainly caused by temperature-induced apoptosis and a large number of inflammatory factors. In mumps orchitis, local inflammation of the testes destroys spermatogenic cell structure and thus significantly reduces spermatogenic function. Varicocele mainly affects the secretory function of Sertoli cells through ROS and other injuries, which can change the spermatogenic microenvironment and induce spermatogenic cell loss. Testicular hydrocele and testicular microlithiasis mainly produce local inflammatory reactions and induce spermatogenesis dysfunction. The testis cell pyroptosis mediated by CASP1 and CASP4 found in this study was limited to the testicular tissue of SCOS patients, possibly caused by known factors, such as varicocele, testicular microlithiasis, and testicular hydrocele or other unknown factors. Accumulated inflammatory factors, oxidative stress, and possible testis cell pyroptosis were also observed in the testicular tissue of SCOS patients, caused by known factors, such as cryptorchidism and mumps orchitis.
In conclusion, for the first time, we found that the activities and expressions of CASP1 and CASP4, their key downstream proteins GSDMD and GSDME, and the inflammatory factors IL-1β, IL-18, LDH, and ROS were significantly increased in the testes of patients with SCOS. In turn, this could promote testis cell pyroptosis and further reduce spermatogonia, spermatocytes, and even spermatogonial stem cells during spermatogenesis in SCOS. Hence, we speculated that testis cell pyroptosis mediated by CASP1 and CASP4 might be a cause of SCOS pathogenesis.