The synergic impact of decellularized testis scaffold and extracellular vesicles derived from human semen on spermatogonial stem cell survival and differentiation

Abstract

Introduction: Decellularized scaffolds create a biomimetic niche to support spermatogonial stem cell (SSC) function and engraftment. Semen-derived extracellular vesicles (SEVs), containing proteins, lipids, and microRNAs with various functions, facilitate intercellular communication, enhance sperm maturation, and regulate the testicular microenvironment. This study explored the combined effects of rat decellularized testicular scaffolds and human SEVs on SSC survival and differentiation.

Materials and methods: The experimental approach involved decellularizing rat testis using detergents, followed by histological, immunohistochemical, DNA quantification, and scanning electron microscopy analyses to confirm extracellular matrix (ECM) preservation and cellular removal. SEVs were isolated from human seminal plasma via ultracentrifugation and characterized for size, morphology, and uptake by testicular cells. Whole testicular cells, including Dolichos Biflorus Agglutinin (DBA)-positive SSCs, were seeded onto scaffolds with or without SEVs, and the gene expression and cell viability were evaluated.

Results: DNA quantification and histochemical examinations revealed that the cell debris was removed, while the ECM constitution retained properly. Flow cytometery revealed 20% of the isolated cells from testis was SSCs. In vitro results demonstrated that SEV-enriched scaffolds significantly enhanced cell viability and upregulated DAZL and PIWI expression, indicating improved SSC survival and functionality, though meiosis (SCP1 expression) was not achieved.

Conclusions: The findings underscore the potential of integrating SEV-laden decellularized scaffolds to partially promote SSC differentiation for fertility restoration in spermatogenic failure.

Keywords: Decellularized scaffold; Extracellular vesicles; Semen; Spermatogonial stem cells; Testis.

Fig. 1

Characterization of decellularized scaffolds. Macroscopic image of the fresh (A) and lyophilized (B) decellularized scaffold. DNA quantification (C) showing a significant reduction in DNA content in decellularized scaffolds compared to intact tissue (*P < 0.001). Hematoxylin and eosin staining of sections from the decellularized scaffold (D) and the intact testis (E) confirmed the effective removal of cellular components. In addition, comparison of Hoechst-stained sections from the decellularized (F) and intact (G) testis verified the absence of nuclei in the decellularized scaffold, indicating successful decellularization

Fig. 2

Histological and immunohistochemical analysis of decellularized scaffolds. The left series shows the decellularized scaffolds, while the right series depicts the intact testis, both stained using corresponding methods. Alcian Blue staining (A) demonstrates partial preservation of acidic glycosaminoglycans. Aldehyde Fuchsin staining (B) highlights the retention of elastic fibers. PAS staining (C) indicates the preservation of glycogen and neutral mucopolysaccharides. Immunostaining for Collagen I (D) confirms the presence of collagen fibers within the scaffold structure

Fig. 3

Characterization of the semen-derived extracellular vesicles (SEVs). Transmission electron microscopy image shows the spherical morphology of SEVs. Internalization test demonstrating SEV uptake by the cells. Size distribution histogram indicates the diameter range of SEVs. Zeta potential distribution, showing the surface charge of SEVs is -2.92 mV

Fig. 4

Cell morphology (left) and flow cytometric analysis (right) of cultured spermatogonial stem cells (SSCs). Flow cytometry data shows 20.5% was DBA-positive

Fig. 5

Results of the MTT assay indicated that both scaffold types, with and without SEVs, exhibited no cytotoxic effects and actively supported cell proliferation. Across all assessed timepoints, cell viability was consistently and significantly higher in the 3D culture groups compared to the standard 2D monolayer cultures, demonstrating the superior biocompatibility and proliferative support provided by the 3D scaffold environment

Fig. 6

Scanning electron microscopy (SEM): The decellularized scaffold, which maintained the native extracellular matrix (ECM) architecture, was successfully preserved after the removal of cellular components. The SEV-loaded scaffold exhibited uniform incorporation of semen-derived extracellular vesicles within the ECM network, indicating effective loading. In addition, spherical cells situated within the seminiferous tubules were visible in the cell-loaded scaffold, suggesting that the scaffold supports cellular organization similar to native testicular tissue

Fig. 7

Expression level of the DAZL (a) and PIWI (b) was significantly increased in the cells that cultured in SEV-loaded decellularized scaffold; however, they could not initiate meiosis as they could not express SCP1 (c). A representative section of the cell-loaded scaffold (d). D, decellularized scaffold; D + SEV, decellularized scaffold + seminal extracellular vesicles; D + C, decellularized scaffold + cells; D + SEV + C, decellularized scaffolds + Seminal extracellular vesicles + cells