Non-obstructive azoospermia (NOA) is a major cause of male infertility, and its pathogenic mechanisms remain incompletely elucidated. This study aims to systematically analyze the expression profiles of SOX family genes in normal human testes and in different types of NOA testes, and to explore the role and mechanism of SOX30 in NOA.

The single-cell RNA sequencing (scRNA-seq) datasets from the GEO database (GSE120508) were used to analyze the transcriptional profiles of SOX family genes in different cell types of the human testis (n=3). The microarray dataset GSE45885, comprising a normal spermatogenesis group (n=4), a post-meiotic arrest NOA group (n=11), a meiotic arrest NOA group (n=7), a pre-meiotic arrest NOA group (n=2), and a Sertoli cell-only syndrome group (n=7), was used to analyze the expression of SOX family genes in clinical testicular biopsy samples to screen for the potential key regulatory factors of NOA in the SOX family. Sox30 knockout mice were generated, and histopathological morphology of the testis and epididymis was observed by HE staining in wild-type and knockout mice. Transcriptome sequencing analysis was performed on pooled samples from 3 mice per group. By integrating NOA-related genes from public datasets, testicular transcriptome data from Sox30 knockout mice and potential SOX30 target genes identified by ChIP-seq results from public datasets, target molecules through which SOX30 participates in NOA were screened, and the expression of these target genes was validated in the testicular tissues of Sox30 knockout mice.

Single-cell data showed high expression of SOX4-6, SOX17-18 and SOX30 in normal human testicular germ cells. Microarray data analysis indicated that SOX30 expression was significantly downregulated in all 4 different types of NOA (P<0.01). In the animal model, Sox30 knockout led to impaired spermatogenesis in mouse testis and no sperm in the epididymis. A total of 256 overlapping genes were identified between NOA differential genes and testicular differential genes of Sox30 knockout mice, and these genes were mainly enriched in spermatogenesis pathways. Combined with target genes identified from ChIP-seq data, the results suggested that SOX30 may play a role in NOA by transcriptionally regulating the expression of key genes such as Odf1 involved in sperm development. RT-qPCR confirmed that the expression of Odf1 and other related genes was significantly downregulated in the testicular tissues of Sox30 knockout mice (P<0.005).

SOX30 contributes to the pathogenesis of NOA by transcriptionally regulating key spermatogenesis-related genes, such as Odf1.

Polystyrene microplastics (PS-MPs), as an emerging environmental pollutant, have attracted considerable attention regarding their toxic effects on the male reproductive system and underlying mechanisms. This study aims to investigate whether PS-MPs promote the expression and translocation of thioredoxin interacting protein (Txnip) by inducing oxidative stress, and thereby activate the apoptosis signal-regulating kinase 1 (ASK1)-cysteinyl aspartate specific proteinase9/3 (Caspase9/3) signaling pathway, ultimately mediating the apoptosis of mouse spermatocytes.

Mouse testicular spermatocyte line GC-2 was used as a model and treated with 5 μm PS-MPs (0, 10, 20, 40 μg/mL). Cell morphology and viability were analyzed after 48 and 72 h of exposure, and cell morphology was observed by optical microscopy, and cell viability was detected by CCK-8 assay (n=3). The remaining mechanistic studies were based on PS-MPs exposure for 48 h. Flow cytometry was performed to determine the levels of reactive oxygen species (ROS) (n=7, 9) and apoptosis rates (n=11, 13). Transcriptome sequencing and GO function enrichment analysis were conducted to screen differentially expressed genes (DEGs) and signaling pathways. RT-qPCR and Western blotting were utilized to detect Txnip expression at mRNA and protein levels (n=3), and immunofluorescence assay was adopted to observe Txnip subcellular translocation. The protein levels of downstream apoptosis-related molecules, such as ASK1, p-ASK1, pro-cysteinyl aspartate specific proteinase 3 (pro-Caspase3), cleaved-cysteinyl aspartate specific proteinase 3 (Cleaved-Caspase3) and Caspase9 were detected by Western blotting. Small interfering RNA was used to knock down Txnip expression; RT-qPCR and Western blotting were employed to verify the knockdown efficiency, and flow cytometry and Western blotting were utilized to detect apoptosis rates (n=12, 13) and the expression of downstream apoptosis-related molecules. CCK-8 assay was conducted to screen the appropriate intervention concentration of N-Acetylcysteine (NAC), and the expression of Txnip was detected by RT-qPCR and Western blotting (n=3). The apoptosis rate (n=7, 8), Txnip translocation and expression of downstream apoptosis-related molecules were detected by flow cytometry and Western blotting.

After 48 h of PS-MPs exposure, the number of cells was decreased and altered morphology was observed in all exposure groups. Compared with 0 μg/mL group, Except for the 10 μg/mL group, with no significant difference in cell viability, Compared with 0 μg/mL group, all other exposure groups demonstrated significantly decreased viability (P<0.0001). After 72 h of PS-MPs exposure, all exposure groups showed reduced cell numbers, altered morphology, and significantly decreased cell viability (P<0.001). Compared with 0 μg/mL group, exposure to PS-MPs for 48 h significantly increased ROS levels (P<0.001) and promoted cell apoptosis (P<0.01). Transcriptome analysis showed that DEGs were significantly enriched in oxidative stress and apoptosis regulatory pathways, with Txnip showing the most significant expression. After 48 h of PS-MPs exposure, Compared with 0 μg/mL group, the mRNA and protein levels of Txnip were mildly increased in the 10 μg/mL group, while the levels in all other exposure groups were increased significantly (P<0.05). Furthermore, knockdown of Txnip significantly inhibited cell apoptosis (P<0.0001). PS-MPs exposure induced the translocation of Txnip from the nucleus to mitochondria and accumulate in the cytoplasm, thereby promoting ASK1 phosphorylation, Caspase9 activation, and Caspase3 cleavage. Knockdown of Txnip significantly inhibited the activation of the ASK1-Caspase9/3 pathway. Co-exposure of PS-MPs with 1 mmol/L NAC for 48 h also inhibited Txnip expression (P<0.01) and translocation, thereby blocking downstream pathway activation and ultimately reducing apoptosis (P<0.0001).

PS-MPs can upregulate Txnip expression and promote its translocation through oxidative stress, thereby activating the ASK1-Caspase9/3 signaling pathway and finally inducing apoptosis in mouse GC-2 cells.