The primary spermatocytes used were male germ cells at prophase I. The present study was undertaken to see whether bivalent chromosomes of mouse primary spermatocytes can undergo meiotic divisions within maturing oocytes and participate in subsequent embryonic development. Primary spermatocytes (pachytene to diplotene) freshly collected from the testes of mature males were electrofused with immature oocytes shortly before or after germinal vesicle breakdown. After culture in MEM-α medium for 15 h, most (> 90%) of the oocytes containing spermatocyte chromosomes underwent maturation and arrested at metaphase II (Mil). Among 23 Mil oocytes examined, 17 (74%) had one group of chromosomes and one polar body, indicating that male chromosomes had intermingled with those of the females and completed the first meiotic division. Chromosome analyses of these Mil oocytes demonstrated their diploidy. The metaphase chromosomes were transferred to enucleated Mil oocytes freshly recovered from superovulated mice. After artificial activation, the reconstructed Mil oocytes resumed meiosis and developed to the morula/blastocyst stage. However, no pups were born following embryo transfer into recipient females. These findings indicate that the chromosomes of primary spermatocytes undergo meiotic divisions in maturing oocytes and participate in the formation of diploid embryos.

There are many reports about the in vitro culture of spermatogenic cells, but no-one has succeeded in inducing the differentiation from spermatogonia to intact sperm. Also the study of in vitro testicular tissue culture has hardly advanced. We studied the culture of mouse immature testicular tissue derived from 5-day-old mice. We aimed to achieve the differentiation of spermatogenic cells in order to observe spermatogenesis in testicular tissue in vitro. We also froze mature testicular tissue and immature testicular tissue cultured for 2 weeks. Furthermore, spermatogenic cells differentiated by culturing were injected into metaphase II oocytes to determine whether these differentiated cells and frozen-thawed testicular tissue have fertilising and developmental ability. Under the culture conditions employed, secondary spermatocytes and a few round spermatids differentiated from spermatogonia were observed in the immature testicular tissue cultured for 2 weeks. When spermatogenic cells derived from cultured immature testicular tissue, cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were injected into ooplasm, the oocytes were fertilised and fertilised oocytes developed to the 8-cell stage. We suggest that spermatogenic cells derived from cultured immature testicular tissue have fertilising and developmental abilities equivalent to that of sperm. Also these abilities of spermatogenic cells obtained from cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were better than those of the same cells before freezing.

Cytoplasts from single spermatocytes of NZB/BinJ mice were separated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 × DNBA/2J) hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%) survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnant recipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA (mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished from the host embryo following PCR amplification. We were unable to detect the transferred mtDNA in blastocysts on day 4–5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, nor in tissues from mice born to host mothers following the transfer of blastocysts that developed from injected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cell stage in mice and is apparently dependent on mutual recognition between the mitochondria and the nuclear genome. We conclude that this mechanism has probably already developed by the time the germ cells have become committed to meiosis.