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Publications

Journal Article Epublish

PubMed: 40787197 DOI: 10.3389/fbioe.2025.1631542

Engineering a 3D platform for testis bioengineering: generation and proteomic profiling of decellularized fish testicular scaffolds.

Front Bioeng Biotechnol 13:1631542

Authors: Ivana Felipe Rosa, Beatriz Marques Souza, Lucas Benites Doretto, Maira da Silva Rodrigues, Caroline Nascimento Barquilha, Matheus Naia Fioretto, Luiz Marcos Frediani Portela, José Carlos Souza Vieira, Luis Antonio Justulin, Pedro de Magalhães Padilha, Changwei Shao, Rafael Henrique Nóbrega

Affiliations

  • Reproductive and Molecular Biology Group, Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, Brazil.
  • State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong, China.
  • Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao, Shandong, China.
  • State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong, China. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao, Shandong, China.
  • Laboratory of Extracellular Matrix Biology, Department of Structural and Functional Biology, Institute of Biosciences of Botucatu, Sao Paulo State University (UNESP), Botucatu, Brazil.
  • Bioanalytics and Metalloproteomics Laboratory, Department of Chemistry, Institute of Biosciences of Botucatu, Sao Paulo State University (UNESP), Botucatu, Brazil.

Abstract

Decellularization represents a robust strategy for generating biologically derived scaffolds that retain the native architecture and biochemical complexity of the extracellular matrix (ECM), thereby providing a conducive microenvironment for germ cell adhesion, proliferation, and differentiation-processes fundamental to the reconstitution of testicular function. While decellularized ECM (dECM) scaffolds have been extensively utilized in mammalian organoid systems for in vitro spermatogenesis and fertility-related research, the development of standardized protocols tailored to teleost models remains largely unexplored. In the present study, we established an efficient decellularization protocol for testicular tissue derived from Astyanax lacustris, employing 0.1% sodium dodecyl sulfate (SDS) in conjunction with physical agitation. The efficacy of cellular removal was confirmed by DNA quantification, histological evaluation and DAPI staining, whereas the preservation of ECM integrity was validated through immunofluorescence, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and proteomic profiling. SDS treatment effectively eliminated cellular components while preserving key ECM proteins, including Collagen I, Fibronectin, and Laminin α1. Notably, critical ultrastructural features-such as the basal lamina, seminiferous tubules, and the D-periodic banding pattern of collagen fibrils-were retained post-decellularization. Proteomic analyses revealed enrichment of proteins associated with ECM organization, cell adhesion, and collagen biosynthesis, while proteins involved in glycolysis and metabolic pathways were downregulated. Moreover, the decellularized matrix retained a comprehensive repertoire of matrisome components, including multiple collagen subtypes (Col1, Col2, Col4, Col5, Col6, and Col7), glycoproteins (Fibronectin, Laminin), proteoglycans (Heparan sulfate), ECM-affiliated proteins (Integrins), secreted factors (Collagen- and calcium-binding EGF), and ECM regulators (Glycosaminoglycans). Collectively, these findings demonstrate that our protocol effectively preserves the structural and functional hallmarks of the testicular ECM, underscoring its potential as a biologically relevant scaffold for future applications in fish reproductive biology. Further investigations are warranted to optimize hydrogel formulations and assess their capacity to support the in vitro proliferation and differentiation of spermatogonial stem cells (SSCs).

MeSH

Cells, Tissue Adhesions, Bioengineering, Microscopy, Electron, Scanning Transmission, Collagen, Microscopy, Electron, Microscopy, Cell Adhesion, Testis