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Pasqualino Loi, Josef Fulka Jr, Thomas Hildebrand, and Grazyna Ptak

Reproductive technologies have been often used as a tool in research not strictly connected with developmental biology. In this study, we retrace the experimental routes that have led to the adoption of two reproductive technologies, ICSI and somatic cell nuclear transfer (SCNT), as biological assays to probe the ‘functionality’ of the genome from dead cells. The structural peculiarities of the spermatozoa nucleus, namely its lower water content and its compact chromatin structure, have made it the preferred cell for these experiments. The studies, primarily focused on mice, have demonstrated an unexpected stability of the spermatozoa nuclei, which retained the capacity to form pronuclei once injected into the oocytes even after severe denaturing agents like acid treatment and high-temperature exposure. These findings inspired further research culminating in the production of mice after ICSI of lyophilized spermatozoa. The demonstrated non-equivalence between cell vitality and nuclear vitality in spermatozoa prompted analogous studies on somatic cells. Somatic cells were treated with the same physical stress applied to spermatozoa and were injected into enucleated sheep oocytes. Despite the presumptive fragile nuclear structure, nuclei from non-viable cells (heat treated) directed early and post-implantation embryonic development on nuclear transfer, resulting in normal offspring. Recently, lyophilized somatic cells used for nuclear transfer have developed into normal embryos. In summary, ICSI and SCNT have been useful tools to prove that alternative strategies for storing banks of non-viable cells are realistic. Finally, the potential application of freeze-dried spermatozoa and cells is also discussed.