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ObjectiveTo construct a single-cell transcriptomic map of testicular tissue under hypobaric hypoxia exposure and perform diversity analysis of supportive cells, aiming to provide new insights into the mechanisms of reproductive toxicity for future research.MethodsTwenty healthy male mice were randomly divided into a control group (n=10) and a hypobaric hypoxia group (n=10). The control group was raised under normal conditions, while the hypobaric hypoxia group was exposed to a low-pressure hypoxic environment(pressure=14 kPa, oxygen content=14.5%). After 6 weeks, testicular tissue from both groups of mice was collected, and the transcriptomic map was obtained using the Singleron MatrixTM single-cell platform and Illumina NovaSeq second-generation sequencing technology. Clustering, pseudo-temporal trajectory analysis, functional analysis, transcription factor and cellular communication research were conducted.ResultsA single-cell transcriptomic map of testicular tissue was successfully constructed, including 6 samples with a total of approximately 49 027 cells covering 11 cell types. Supportive cells were clustered into 4 subgroups using non-negative matrix factorization algorithm. Subgroup 3 may be more sensitive to hypoxic stimulation and could reduce reproductive cell function damage caused by hypobaric hypoxia by affecting PTN expression levels and regulating the cell cycle of spermatogonia. The PTN-PTPR pathway may be an important regulatory node for supportive cells to regulate spermatogonia. Subgroup 4 could regulate the cell cycle of spermatogonia after exposure to hypobaric hypoxia through the KITLG-KIT signaling pathway, thus affecting sperm development.ConclusionsBased on single-cell sequencing technology, the molecular basis and regulatory signals of supportive cells under hypobaric hypoxia exposure is revealed for the first time. It provides an in-depth exploration of the mechanisms of reproductive toxicity induced by hypobaric hypoxia at the single-cell level, offering a new perspective for future clinical research in this field.

Objective The process of spermatogenesis is complex involving multiple regulatory mechanisms and requiring complex interactions between germ cells and somatic cells. The mechanisms of Sertoli cells involving in spermatogenesis remain to be explored. Methods We collected single-cell RNA sequencing data from a high-throughput gene expression database (GEO: GSE113293) from wild-type C57BIL6J male mice with normal spermatogenesis and four different mutant mouse strains (knockout Mlh3, Hormadl, and Cul4a; Cnp transgene). Then bioinformatics analyses such as gene function enrichment analysis (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched pathway analysis, protein- protein interaction (PPI) network analysis, transcription factor regulation analysis (SCENIC), and gene set variation analysis (GSVA) on the Sertoli cells were further indentified. Results We identified nine subtypes of spermatogenic cell clusters (undiferentiated spermatogonia, diferentiating sperrmatogonia, leptotene/zygotene, pachytene, diplotene, round sperrmatids, elongating spermatids, and spermatozoa), and two types of somatic cell clusters (Leydig cells, and Sertoli cells), and one unknown type of cell cluster. Compared to the normal spermatogenesis model, 276 genes were upregulated and 234 genes were downregulated in the Sertoli cells of the mouse spermatogenesis defect model. GO analysis showed that differential genes in Sertoli cells were significantly enriched in spermatogenesis and energy metabolism. KEGG enrichment analyses showed that the homogeneous ribosomal pathway, glycolytic pathway, and oxidative phosphorylation pathway were significantly enriched in Sertoli cells. Protein-protein interaction (PPI) network analysis of diferentially expressed genes identified 20 hub genes, all of which belonged to ribosomal genes, and GSVA analysis revealed that the upregulated genes in Sertoli cells were closely related to the Notch signaling pathway. Conclusion The hub genes and the Notch signaling pathway in Sertoli cells are involved in the process of spermatogenesis. This study provides new perspectives on infertility caused by spermatogenesis dis orders. [ABSTRACT FROM AUTHOR]

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