Microinsemination with first-wave round spermatids from immature male mice

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.