Dysregulation of Sphingolipid Metabolism in Non-Obstructive Azoospermia: Insights from Bioinformatics and a Busulfan-Induced Mouse Model.

Abstract

Sphingolipid metabolism plays an important role in regulating essential cellular processes, including apoptosis, proliferation, differentiation, and cellular signaling, which are critical for normal spermatogenesis. However, the contribution of sphingolipid metabolic alterations to male infertility, particularly azoospermia, remains insufficiently understood. This study aimed to investigate the involvement of sphingolipid metabolism-related genes and metabolites in spermatogenic failure and their potential association with azoospermia. A publicly available human microarray dataset (GSE9210), consisting of testicular tissue samples from patients with obstructive azoospermia (OA; n = 11) and non-obstructive azoospermia (NOA; n = 47), was analyzed to identify differentially expressed sphingolipid metabolism-related genes. Candidate genes identified through bioinformatic analysis were further evaluated in a busulfan-induced mouse model of azoospermia (control group, n = 8; busulfan-treated group, n = 8) using quantitative real-time PCR, immunohistochemical analysis, and high-performance liquid chromatography (HPLC). Analysis of the GSE9210 dataset identified 23 differentially expressed sphingolipid metabolism-related genes between OA and NOA samples. Validation in busulfan-treated testes indicated significant dysregulation of key sphingolipid-related genes, including decreased expression of Asah1, Asah2, Acer1, Acer2, Cerk, Elovl1, Sgms1, and Galc, and increased expression of Smpd 1,2 & 3 (p < 0.05). HPLC analysis revealed significant reductions in ceramide species (Cer14, Cer16, Cer18, and Cer20), sphingosine, and sphingosine-1-phosphate levels in busulfan-treated testes (p < 0.05). Furthermore, immunohistochemical analysis showed increased expression of the inflammatory markers MMP-2 and MMP-9, accompanied by elevated levels of cleaved Caspase-3 and reduced expression of the anti-apoptotic protein Bcl-2 in the busulfan-treated group (p < 0.05). Overall, these findings suggest that disruption of sphingolipid metabolism may contribute to impaired spermatogenesis and azoospermia. Altered sphingolipid-related gene expression and metabolite profiles provide new insights into the molecular mechanisms underlying male infertility and may represent potential targets for future therapeutic investigation.