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Reconstitution of spermatogenesis and continuous generation of functional haploid germ cells in mouse testicular organoids

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 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE283341 | GEO | 2026/03/08

REPOSITORIES: GEO

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Project description:Sry is sufficient to induce testis formation and subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth due to germ cell autonomous defects. Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here we demonstrated that the testicular somatic environment of XX/Sry males is defective in the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, entering meiosis and differentiating into the round spermatid stage. XY donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, thereby preventing further progress beyond the elongated spermatid stage. In contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed missing expression of several Y-linked genes and alterations in the expression profile of genes associated with spermatogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, supporting our hypothesis that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice. Experiment Overall Design: Whole testes and seminiferous tubules of XX/Sry and W/Wv males were used for microarray expression analysis using the Affymetrix GeneChip system (Affymetrix, CA). In order to isolate the seminiferous tubules, the tunica was carefully removed from the testes which were then incubated in the medium with 5 mg/ml collagnease at 37oC for 40 min. The remaining seminiferous tubules were washed several times with PBS using a 70-Âºm cell strainer to remove interstitial cells. After total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD), double-stranded cDNA and biotin-labeled cRNA were synthesized using One-Cycle cDNA Synthesis and IVT Labeling kits (Affymetrix, CA), respectively. Twenty micrograms of fragmented biotin-labeled cRNA was hybridized to the Affymetrix Mouse Expression Array MOE 430A for 16 hr at 45oC. The chips were washed, stained, and then scanned with the GeneArray Scanner (Hewlett Packard, CA) in accordance with the manufacturer's standard protocols. Finally, the microarray data were analyzed using Microarray Suite ver. 5.0 (Affymetrix). Differential expression was defined as a difference of 2-fold or more in both whole testis and seminiferous tubule samples between two recipient males. Mouse 430A Affymetrix Genome Array IDs were used to query the NetAffx data mining tool for gene annotations.

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Reconstitution of spermatogenesis and continuous generation of functional haploid germ cells in mouse testicular organoids

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