Journal
BIOLOGY OF REPRODUCTION
Volume 89, Issue 1, Pages -Publisher
SOC STUDY REPRODUCTION
DOI: 10.1095/biolreprod.113.108613
Keywords
meiosis; reconstruction; seminiferous tubules; spermatogenesis; spermatogonial stem cells; testis
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Funding
- AKUA (Asahi Kasei Pharma Urological Academy, Asahi Kasei Pharma Co.)
- Yokohama City University
- Research Center for Clinical Proteomics of Post-Translational Modifications
- [20116005]
- [24390371]
- Grants-in-Aid for Scientific Research [24390371, 24770216] Funding Source: KAKEN
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Cells of testicular tissues during fetal or neonatal periods have the ability to reconstruct the testicular architecture even after dissociation into single cells. This ability, however, has not been demonstrated effectively in vitro. In the present study, we reconstructed seminiferous tubules in vitro that supported spermatogenesis to the meiotic phase. First, testicular cells of neonatal mice were dissociated enzymatically into single cells. Then, the cells formed aggregates in suspension culture and were transferred to the surface of agarose gel to continue the culture with a gas-liquid interphase method, and a tubular architecture gradually developed over the following 2 wk. Immunohistological examination confirmed Sertoli cells forming tubules and germ cells inside. With testicular tissues of Acr-GFP transgenic mice, the germ cells of which express GFP during meiosis, cell aggregates formed a tubular structure and showed GFP expression in their reconstructed tissues. Meiotic figures were also confirmed by regular histology and immunohistochemistry. In addition, we mixed cell lines of spermatogonial stem cells (GS cells) into the testicular cell suspension and found the incorporation of GS cells in the tubules of reconstructed tissues. When GS cells derived from Acr-GFP transgenic mice were used, GFP expression was observed, indicating that the spermatogenesis of GS cells was proceeding up to the meiotic phase. This in vitro reconstruction technique will be a useful method for the study of testicular organogenesis and spermatogenesis.
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