Researchers at Osaka University and Yokohama City University have achieved a groundbreaking milestone in reproductive biology by creating the world's first mouse testicular organoids from pluripotent stem cells that support the production of viable sperm precursors. Published in Science on February 26, 2026, this study demonstrates the full reconstitution of the testicular niche, including sex determination processes, seminiferous tubule formation, and germ cell differentiation up to spermatogonial stem cells capable of generating functional spermatozoa.

Led by Professor Katsuhiko Hayashi and Associate Professor Takashi Yoshino from Osaka University, in collaboration with Professor Takehiko Ogawa from Yokohama City University, the team used mouse embryonic stem (ES) cells to generate testicular somatic-like cells (TesLCs) in a Y chromosome-dependent manner. These cells, when combined with primordial germ cell-like cells (PGCLCs), self-assembled into organoids mimicking the natural testicular structure and function.

Understanding Testicular Organoids: Miniature Testes in a Dish 🧬

Testicular organoids are three-dimensional (3D) structures derived from stem cells that replicate the architecture and cellular interactions of a developing testis. The testis, or testicle, is the male gonad responsible for spermatogenesis—the process of sperm production—and androgen hormone synthesis, such as testosterone, essential for male reproductive development and secondary sexual characteristics.

Prior to this breakthrough, organoid technology had successfully modeled ovarian development and oogenesis in mice, but reconstituting the testicular niche proved more challenging due to complex cell-cell interactions and signaling pathways required for sex determination. Sex determination in mammals begins with the bipotential gonad differentiating into testis or ovary based on genetic cues, primarily the Sry gene on the Y chromosome activating Sox9 expression in Sertoli cells, which orchestrate tubule formation and germ cell support.

This Japanese innovation builds on Hayashi's lab's prior success in generating oocytes from iPS cells, shifting focus to male gametes and addressing a critical gap in in vitro gametogenesis (IVG)—the production of gametes from stem cells outside the body.

Step-by-Step Methods: Engineering the Testicular Niche

The researchers employed a multi-step protocol starting with mouse ES cells harboring sex-specific fluorescent reporters: Sox9-GFP for testicular fate and Foxl2-tdTomato for ovarian. Here's how they achieved it:

• Induction of Bipotential Gonadal Progenitors: ES cells differentiated into a bipotential state mimicking embryonic day 11.5 (E11.5) gonads via modulation of BMP and WNT signaling pathways.
• Sex-Specific Differentiation: In XY cells, activation of testicular pathways yielded TesLCs expressing Sertoli (SOX9), Leydig (NR5A1), and peritubular myoid markers, forming tubule-like structures. XX cells defaulted to ovarian fates.
• Organoid Assembly: TesLCs co-cultured with PGCLCs in hanging drop followed by rotation culture, resulting in organoids with interstitial tissues and seminiferous tubules after 40-50 days.
• Germ Cell Progression: PGCLCs differentiated sequentially into prospermatogonia, spermatogonia, and pre-meiotic spermatocytes, confirmed by single-cell RNA sequencing matching in vivo trajectories.

Single-cell transcriptomics validated the organoids' fidelity to natural testis development, revealing Y chromosome dependency for SOX9+ Sertoli cells.

Key Results: From Stem Cells to Functional Germline Stem Cells

The organoids supported advanced germ cell differentiation, producing germline stem cell-like cells (GSCLCs) that could be expanded indefinitely in vitro. Critically, these GSCLCs, when transplanted into the testes of infertile busulfan-treated recipient mice, colonized seminiferous tubules, underwent full spermatogenesis, and generated mature spermatozoa.

The proof of functionality came via intracytoplasmic sperm injection (ICSI)-like fertilization: offspring were born healthy, fertile, and produced a second generation, confirming no genetic or epigenetic abnormalities.

This marks the first time pluripotent stem cell-derived testicular organoids have enabled the generation of a self-renewing germline capable of producing viable sperm precursors.

Confirming Viability: Transplantation and Offspring Production

While full spermatogenesis to sperm occurred post-transplantation, the organoids themselves drove progression to meiotic spermatocytes—a major advance over prior cultures limited to pre-meiotic stages. Transplanted GSCLCs restored fertility in sterile mice, yielding pups at rates comparable to controls.

• Pups from organoid-derived sperm: Healthy, normal litter sizes.
• Second-generation fertility: Fully validated.

This validates the organoids as a functional testicular niche, bridging IVG gaps.

Addressing Male Infertility: A Game-Changer for Japan and Beyond

Male infertility affects approximately 50% of infertile couples globally, with spermatogenic dysfunction accounting for 82.4% of cases in Japan per Japanese Urological Association guidelines. Japan's total fertility rate hit a record low of 1.20 in 2023, exacerbating demographic pressures.

This breakthrough offers hope for non-obstructive azoospermia (NOA) patients lacking sperm, potentially via iPS-derived organoids for autologous gamete production. However, human translation faces hurdles: ethical regulations, complete IVG, and safety validation.

Ogawa's lab at Yokohama has pioneered spermatogonial transplantation, complementing Hayashi's stem cell expertise at Osaka.

Challenges in Organoid Maturation and Human Application

Current organoids halt at pre-leptotene spermatocytes; full meiosis requires transplantation. Scalability, vascularization, and long-term stability remain challenges. For humans, ethical concerns around embryo creation from IVG gametes loom large in Japan, where iPS pioneer Yamanaka's legacy emphasizes safety.

• Risks: Epigenetic errors, mosaicism.
• Solutions: Refined signaling, bioengineering scaffolds.

Prospects: 10+ years to human sperm production, per researchers.

Broader Implications: Conservation, Toxicology, and Drug Screening

Beyond infertility, organoids enable:

• Modeling endocrine disruptors' effects on spermatogenesis.
• Endangered species preservation via IVG.
• High-throughput toxicity testing for male reproductive health.

Japan's investment in regenerative medicine, via AMED funding, positions Osaka and Yokohama as leaders.

Japan's Leadership in Stem Cell Reproductive Research

Osaka University's Institute for Frontier Life and Medical Sciences and Yokohama City University's Urology Department exemplify Japan's prowess. Hayashi's team previously generated mice from iPS oocytes (2021), advancing IVG globally.

This aligns with national goals to combat declining fertility amid aging demographics.

Expert Reactions and Global Impact

Reproductive biologists hail it as a "major leap" toward IVG. Potential to revolutionize treatments for genetic infertilities like Klinefelter syndrome.

Global collaborations could accelerate human trials.

Photo by Steve Johnson on Unsplash

Future Outlook: Toward Human Clinical Applications

Next steps: Human iPS-derived organoids, complete in vitro meiosis, clinical safety trials. Ethical frameworks must evolve.

For aspiring researchers, this underscores opportunities in regenerative medicine. Check Rate My Professor for insights on faculty like Hayashi.

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