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Mouse Round Spermatids Developed In Vitro from Preexisting Spermatocytes Can Produce Normal Offspring by Nuclear Injection into In Vivo-Developed Mature Oocytes¹

The Institute for Biogenesis Research,² Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822 Department of Cell Biology and Anatomical Sciences,³ The Sophie Davis School of Biomedical Education/The City University of New York Medical School, New York, New York 10031

It has been shown that mature oocytes injected with nuclei from round spermatids collected from mouse testis can generate normal offspring and that round spermatids can develop in vitro. An undetermined issue is whether spermatids developed in vitro are capable of generating fertile offspring by nuclear injection into oocytes. Herein, we report the production of normal and fertile offspring by nuclear injection using haploid spermatid donors derived from mouse primary spermatocyte precursors cocultured with Sertoli cells. Cocultured spermatogonia and spermatocytes were characterized by their nuclear immunoreactive patterns determined by an antibody to phosphorylated histone H2AX (-H2AX), a marker for DNA double-strand breaks. Cocultured round spermatid progenies display more than one motile flagellum, whose axonemes were recognized by antitubulin immunostaining. Flagellar wavelike movement and flagellar-driven propulsion of round spermatids developed in vitro were documented by videomicroscopy (http://www.sci.ccny.cuny.edu/kier). We also show that breeding of male and female mouse offspring generated by spermatid nuclear injection produced fertile offspring. In addition to their capacity to produce fertile offspring, cocultured, flagellated round spermatids can facilitate the analysis of the mechanisms of centriolar polarity, duplication, assembly, and flagellar growth, including the intraflagellar transport of cargo proteins.

¹ This work was supported by grants from the National Institutes of Health HD36477 (L.L.T.) and HD37282 (A.L.K.) and from the Harold Castle and Kosasa Family Foundation (R.Y.).

⁴ Correspondence: Abraham L. Kierszenbaum, Department of Cell Biology and Anatomical Sciences, CUNY Medical School, 138th St. and Convent Ave., Harris Hall, Suite 306, New York, NY 10031. FAX: 212 650 6812; kier{at}med.cuny.edu