The Cytoophidium

   The cytoophidium is a micron-scale intracellular filament that can be found in a wide range of species, from prokaryotes to eukaryotes, and even in archaea. The composition of the cytoophidium is largely elusive and likely varies on a case-by-case basis. The major components of the cytoophidium are fibrillar polymers of specific proteins, metabolic enzymes in particular. To date, dozens of cytoophidium-forming proteins have been identified, yet their functions and physiological importance remain largely unclear. Unlike some irreversible protein aggregates, such as aggresomes and β-amyloid, the cytoophidium is a dynamic and reversible structure that may be present only under specific conditions.

  • Cytoophidium

   The polymerization of cytoophidium-forming enzymes is often regulated by the binding of their ligands and can modulate their catalytic activities. For some cytoophidium-forming enzymes, such as P5CS and PRPS, catalytic reactions cannot be achieved without polymerization. In other cases, such as CTPS and IMPDH, polymerization is not required for catalysis but can modulate sensitivity to their inhibitors. Apart from the regulation of enzyme activity, the formation of the cytoophidium may also influence other protein properties by controlling their intracellular distribution, stability, or accessibility. Therefore, polymerization and assembly of cytoophidia may provide an additional layer of regulation for metabolism and cellular functions under various physiological conditions.

   Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo GTP biosynthesis. In vitro, polymerization of IMPDH can enhance its catalytic activity under conditions in which GTP acts as an inhibitory regulator. In mammalian cells, the assembly of IMPDH cytoophidia is highly correlated with cell proliferation and the upregulation of nucleotide synthesis, suggesting a role in boosting nucleotide production to support active cellular metabolism. Several studies have demonstrated the presence of IMPDH cytoophidia in various normal tissues, including the retina, thymus, spleen, ovarian follicles, and pancreatic islets, as well as in many cancers, implying important physiological roles in vivo.

   Recently, we developed a genome-editing strategy to effectively impair IMPDH polymerization in cultured cells and discovered that IMPDH cytoophidia play important roles not only in the regulation of nucleotide synthesis but also in modulating glycolysis and the pentose phosphate pathway (PPP) in mouse embryonic cells and human cancers. These findings suggest that IMPDH cytoophidia contribute to a more complex network of metabolic regulation. We are currently establishing cellular and animal models to further investigate the tissue-specific functions of IMPDH cytoophidia and their roles in distinct physiological contexts.

Spermatogonial Stem Cell (SSC)

   Pigs are regarded as ideal donor species for xenotransplantation due to their anatomical and physiological similarities to humans. Recent advances in genome editing have enabled the production of genetically modified pigs with reduced immunogenicity and improved compatibility for human organ transplantation. However, current methods for transgenic pig production, such as pronuclear microinjection and somatic cell nuclear transfer (SCNT), remain technically demanding, low in efficiency, and difficult to scale. As xenotransplantation progresses toward clinical application, the need for more sophisticated, precise, and heritable genome modifications will increase substantially. Developing a renewable platform for genetic engineering is therefore essential for advancing both xenotransplantation and large-animal biotechnology.

  • Spermatogonial Stem Cell (SSC)

   Spermatogonial stem cells (SSCs) offer a particularly promising foundation for such a platform. SSCs are the origin of spermatogenesis and are responsible for the lifelong production of sperm through a balance between self-renewal and differentiation. Located on the basement membrane of seminiferous tubules, SSCs constitute a small subset of undifferentiated spermatogonia capable of maintaining the stem cell pool while generating differentiating progeny that ultimately give rise to mature spermatozoa. This dual capacity for self-renewal and genetic transmission makes SSCs a unique target for germline modification.

   In rodents, SSC research has a well-established history beginning with the landmark transplantation experiments by Brinster and colleagues in the mid-1990s, which first demonstrated that SSCs could reinitiate spermatogenesis and transmit genetic information to offspring following transplantation into infertile testes. Subsequent advances enabled the in vitro propagation of mouse SSCs, with long-term cultures capable of continuous self-renewal for years while maintaining full germline competency. Moreover, SSCs have been successfully genetically modified in vitro, and germline transmission of these modifications has been confirmed through the generation of transgenic offspring. Together, these studies establish SSCs as a powerful experimental system for investigating stem cell self-renewal, spermatogenesis, and heritable genome engineering.

   Extending SSC culture and manipulation to large animals such as pigs holds great promise for advancing both reproductive biotechnology and biomedical research. We have previously established a protocol for isolating and purifying porcine SSCs from neonatal piglets. Currently, we are working to develop a comprehensive porcine SSC manipulation platform, encompassing in vitro culture and maintenance, genome editing, and functional transplantation leading to sperm production, with the goal of enabling efficient and heritable genetic modification in pigs.