Proteomics of Mouse Testicular Organoids on Day 6
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Xiaoyang Zhao
PROVIDER: PXD075301 | iProX | Thu Mar 05 00:00:00 GMT 2026
REPOSITORIES: iProX
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Project description:Testicular organoids that support spermatogenesis and generate functional haploid germ cells are still lacking. Here, we developed a formation-differentiation culture approach to generate optimized testicular organoids (O-Torgs) derived from neonatal mouse primary testicular cells. These O-Torgs could essentially recapitulate the seminiferous tubule morphogenesis, Sertoli cell maturation and testosterone secretion. Importantly, O-Torgs not only support spermatogenesis but also sustain proliferative undifferentiated spermatogonia and continuous generation of functional haploid cells for up to three months, with offspring derived from these haploid cells displaying normal growth and reproductive capability to F2 generation. Mechanistically, we found that the formation stage enhanced the reconstruction of tubule-like structures and subsequent spermatogenesis by providing a more conducive extracellular matrix niche. Finally, O-Torgs were proved to be effective in modeling male infertility and drug screening, with BTT-3033 acting as a potential drug in protecting busulfan-mediated germ cell loss. Overall, our work establishes a strategy to obtain functional testicular organoids, offering promising avenues for male infertility modeling and drug discovery.
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Project description:Testicular organoids that support spermatogenesis and generate functional haploid germ cells are still lacking. Here, we developed a formation-differentiation culture approach to generate optimized testicular organoids (O-Torgs) derived from neonatal mouse primary testicular cells. These O-Torgs could essentially recapitulate the seminiferous tubule morphogenesis, Sertoli cell maturation and testosterone secretion. Importantly, O-Torgs not only support spermatogenesis but also sustain proliferative undifferentiated spermatogonia and continuous generation of functional haploid cells for up to three months, with offspring derived from these haploid cells displaying normal growth and reproductive capability to F2 generation. Mechanistically, we found that the formation stage enhanced the reconstruction of tubule-like structures and subsequent spermatogenesis by providing a more conducive extracellular matrix niche. Finally, O-Torgs were proved to be effective in modeling male infertility and drug screening, with BTT-3033 acting as a potential drug in protecting busulfan-mediated germ cell loss. Overall, our work establishes a strategy to obtain functional testicular organoids, offering promising avenues for male infertility modeling and drug discovery.
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Project description:This study explores the role of porcine spermatogonia germ cells and testicular fibroblast in testis development and spermatogenesis through xenotransplantation. Previous research demonstrated the importance of Sertoli cells in maintaining testicular structure and function during testis development. However, the contribution of spermatogonia to these processes remains unclear. H&E staining revealed that FO tissues, no testicular structures were formed; instead, heterogeneous tissues with simple tubular formations were observed. Conversely, in GF tissues, structures resembling testicular tissue developed, displaying various stages of testis development including elongated spermatid. Immunofluorescence staining showed seminiferous tubules expressing PGP 9.5, VASA, and ACR2, confirming the presence of spermatogenesis. Interestingly, FO tissues were dominantly expressed ESR1, indicating bipotent gonadal property. RNA-seq analysis further revealed that GF tissue showed spermatogenesis-linked GOBPs with gene expression of mature Sertoli cells, including NR5A1, AR, and DMRT1. However, FO tissues displayed early tissue formation processes with expression of BMP4, WNT5A, ESR1, TGFBR3 and ACVR, generally inhibited in normal testis. These findings highlight the critical role of germ cells in formation of testicular structures through xenotransplantation and suggest the potential for spermatogenesis within these structures. However, further research is needed to verify the integrity and fertilizing capacity of the sperm, as these aspects have not been confirmed in this study.
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Project description:ITo derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine, we developed a method to produce a cytocompatible decellularized testicular matrix (DTM) while maintaining the native tissue-specific characteristics and components. The potential benefits of tissue-specific scaffolds consisting of naturally-derived extracellular matrix (ECM) have been demonstrated using a wide variety of animal and human tissue sources. However, so far, testis scaffolds have never been considered for constructive remodelling purposes. We have therefore developed a protocol for the preparation of cell-free extracellular matrix and characterized the material extensively using a combination of proteomics, immunohistochemsitry, and cell population assays. To prepare cell-free matrix, human cadaveric testicular tissue was exposed for 24 h or 48 h to 1% Triton X-100 and/or 1% sodium dodecyl sulfate (SDS). The extent of decellularization was evaluated by histology. Confirmation of cell removal in DTM was done by a DNA quantification technique. The protein composition was analysed by LC-MS/MS. The retention of testicular tissue-specific characteristics was evaluated by immunohistochemistry, Alcian blue staining and scanning electron microscopy. Soluble toxicity and testicular cell attachment was assessed to check the cytocompatibility of DTM scaffolds. Histological analysis showed that DTM could be obtained by mechanical agitation in 1% SDS for 24 h. The resulting DTM was found to be clear of cells while retaining the typical three-dimensional structure. Proteomics analysis revelaed the presence of the major components of the native tissue scaffold, including collagen type I and IV, fibronectin, laminin and glycosaminoglycans. In addition, numerous additional ECM proteins in DTM were detected, indicating its complex nature. Importantly, we demonstrated that DTM scaffolds are not cytotoxic, as evidenced by MTT assay showing a normal fibroblast proliferation activity after indirect exposure, and support testicular cell attachment and infiltration.
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