Abstract
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 the 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 proven 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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Data availability
Data reported in this paper have been deposited and made publicly available in the Gene Expression Omnibus (GEO) at NCBI under accession number GSE323391. All raw sequence data reported in this paper have been deposited in the Genome Sequence Archive of the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences. The accession numbers are listed as below: the scRNA-seq raw data are under CRA019639, and the processed data are under OMIX007881 in OMIX. Bulk-seq and bisulfite sequencing raw data are under CRA034967. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE61 partner repository with the dataset identifier PXD075407. ScRNA-seq datasets of previous publications are publicly available: Brian P. Hermann et al. (GSE109033)62, Green et al. (GSE112393)63, Christina Ernst et al., (E-MTAB-6946)64, Chen Yao et al. (GSE107644)30. All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files. Source data are provided with this paper.
Code availability
The codes utilized in this manuscript rely on public library packages, which are detailed in the Methods section.
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Acknowledgements
This work was supported by the National Key R&D Program of China (2022YFA0806303 and 2022YFC2702603 to Xiao-Yang Zhao, 2022YFA0806301 to Fang Luo, 2024YFA1802400 to Gang Chang, 2025YFA0922000 to Cong Wan, 2022YFA1106200 to Jiexiang Zhao); The National Natural Science Foundation of China (U22A20278 and 82530051 to Xiao-Yang Zhao, 32370911, 32571017 and 32170869 to Gang Chang, 32200670 to Cong Wan, 32200689 to Jiexiang Zhao, 82271631 to Zhaoting Liu, 32570969 to Fang Luo); Guangzhou Key Research and Development Program (2024B03J0991 to Xiao-Yang Zhao); Shenzhen Medical Research Fund (B2402013 to Gang Chang); Guangdong-Hong Kong Joint Laboratory for Psychiatric Disorders (2023B1212120004 to Xiao-Yang Zhao); Guangdong Major Program of Basic and Applied Basic Research (2025B0303000014 to Xiao-Yang Zhao); The Postdoctoral Fellowship Program of CPSF (GZB20240180 to Zhaokai Yao); The Guangdong Basic and Applied Basic Research Foundation (2025B0303000014 to Xiao-Yang Zhao, 2025A1515012332 to Fang Luo, 2023A1515030255 to Zhaoting Liu); The Natural Science Foundation of Shenzhen (JCYJ20250604182428038 and JCYJ20230808105421043 to Gang Chang); The Open Project of National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences (NSCRC-25-98 to Fang Luo). We thank Beckman for supporting the FACS.
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X.Z., F.L., and K.M. conceived and supervised the project. C.W., Z.Yao, Z.Ye, J.Z., S.R., X.X., Q.R., Y.W., H.X., W.W., C.Z., X.L., M.P., M.C., L.M., D.C., C.L., Z.Z., Y.Z., Z.L., performed the experiments. Q.L., K.T., and X.Y. performed bioinformatics analysis. X.Z., F.L., K.M., G.C., C.W., Q.L., Z.Yao, and J.Z. wrote the manuscript with the help of all the authors.
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X.Z., C.W., Z.Yao, F.L., Q.R., S.R., C.L., and G.C. have filed a patent related to this work. The remaining authors declare no competing interests.
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Wan, C., Li, Q., Yao, Z. et al. Reconstitution of spermatogenesis and continuous generation of functional haploid germ cells in mouse testicular organoids. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71254-w
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DOI: https://doi.org/10.1038/s41467-026-71254-w
