Genes and Biomolecular Pathways/Mechanisms and Factors Involved in Regulating Male Fertility of Mice that Underwent Juvenile Chemotherapy

Introduction:
Busulfan (BU) is an often used systemic cytotoxic drug. It damages proliferating cells, often causing sub-fertility or even sterility.

Aims:
To investigate the mechanisms underlying BU-induced sterility following injection at juvenile age.

Methodology:
Immature mice (IM) were BU injected (45 mg/kg). Testicular tissue was analyzed 6 weeks post-treatment with RNAseq, single cell RNA-seq (scRNAseq), histological evaluation of seminiferous tubules (STs) and cauda sperms counting. A mating test with normal females was also conducted.

Results:
Injection of BU to IM led to sterility at maturity according to all examined parameters. Testicular RNA-seq showed a significant change in gene expression (heatmap) in mature BU-Sterile mice compared to fertile (DMSO). Enrichment analysis suggests several BU effected pathways. A heatmap of differentially expressed genes (DEGs) in 12 identified clusters of testicular cells was analyzed. scRNAseq revealed two subpopulations of Sertoli cells and of spermatocytes that showed different gene expression in the same group. BU leads to differentially expressed genes in some cell types (e.g. Somatic cells) but not in others (e.g. Spermatogonia) Furthermore, injection of IM treated with BU left 20-30% fertile. RNA-seq identified significant gene expression differences between sterile and fertile mice in the busulfan-treated group, enriched in specific pathways.

Conclusion:
Our study identified for the first-time specific genes and pathways as candidates involved in male infertility following chemotherapy treatment in the prepubertal age. Furthermore, we were able to show that some of the busulfan-treated animals were infertile, similar to human-treated with aggressive chemotherapy.