Improving the process of spermatogenesis in azoospermic mice using spermatogonial stem cells co-cultured with epididymosomes in three-dimensional culture system

Aims: This study aimed to explore the effect of epididymosomes on the proliferative efficiency of spermatogonial stem cells (SSCs) in vitro and the resumption of spermatogenesis in the azoospermic mice. Main methods: The epididymosomes were extracted from the epididymis and characterized. SSCs were cultured in 2D (two-dimensional) and hydrogel-based 3D culture in the presence of 20 mu g/mL epididymosome or 10 ng/mL GDNF. After two weeks of culture, the proliferation and purity of the separated SSCs were evaluated using the MTT test and flow cytometry, respectively. qRT-PCR was used to analyze PLZF, caspase-3, TGF-beta, miR-10b, and miR-21 expression levels. Then, SSCs grown in the 3D culture system were labeled by DiI and transplanted into azoospermic mice via the efferent duct. After 2 weeks, tracing of DiI and cell homing were evaluated. Subse-quently, histomorphometric studies and immunohistochemistry analysis were performed in testes after eight weeks of transplantation. Key findings: The expression of PLZF, TGF-beta, miR-10b, and miR-21 increased significantly (*p < 0.05) in the 3D + GDNF and 3D + epididymosomes groups than in the 2D group. Transplanted SSCs migrated into the seminiferous tubules of recipient mice and the number of spermatogenic cells and protein expression of PLZF, SCP3 and ACRBP in the 3D + GDNF and 3D + epididymosomes groups were considerably higher (* * * p < 0.001) compared to the azoospermic group. Significance: This finding indicates that culturing SSCs on decellularized testicular matrix (DTM) hydrogel with 10 ng/mL GDNF or 20 mu g/mL epididymosomes could lead to an increase in SSCs proliferation which provides a sufficient number of SSCs for successful transplantation in azoospermic mice.