Development of Normal Mice from Oocytes Injected with Secondary Spermatocyte Nuclei1

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Abstract
This study shows that the nucleus of the secondary spermatocyte can participate in syngamy and normal embryonic development. Spermatogenic cells were released from the seminiferous tubules of adult mice, and the secondary spermatocytes were selected according to the size of the whole cell and nucleus. The accuracy of this selection, evaluated by chromosome analysis, was 86%. Nuclei of presumptive secondary spermatocytes were freed from the cytoplasm and then injected individually into mature oocytes. This process itself did not activate the oocytes. The oocytes were electroactivated about 2 h after injection, at which time prematurely condensed chromosomes of the spermatocyte had become associated with the microtubules of a spindle. Following activation, the chromosomes of both the oocyte and spermatocyte completed their second meiotic division, culminating in the extrusion of two separate polar bodies and the formation of one male and one female pronucleus in about 75% of the oocytes into which spermatocyte nuclei had been injected. Two- or four-cell embryos arising from such oocytes were randomly selected and transplanted to foster mothers. Twenty-four percent developed into normal fertile offspring. The young born later to these offspring were all normal. The results of this study indicate that gametic imprinting of mouse spermatogenic cells is completed either in the testis before the second meiotic division or within the cytoplasm of a mature oocyte after artificial nuclear transfer.

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