Production of mice through intracytoplasmic injection of sperm or spermatogenic cells

Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.

Freeze-dried mouse spermatozoa are all motionless and dead in the conventional sense. When injected into oocytes, however, their nuclei can support normal embryonic development even after three month preservation in a dried state. Although the freeze-drying protocol reported here will need further improvement, the results suggest that it may be possible to store the male genomes at room temperature.

Intracytoplasmic sperm injection (ICSI) was successful in the mouse when a piezo-driven micropipette was used instead of a mechanically driven conventional pipette. Eighty percent of sperm-injected oocytes survived, and approximately 70% of them developed into blastocysts in vitro. When 106 embryos at the 2- to 4-cell stage were transferred to eight naturally mated foster mothers, 30% of the embryos (25-43%, depending on the host) reached the full term. Except for two that were cannibalized soon after birth, all of the young (30 pups) grew into normal adults. Studies of this type on the mouse may increase understanding of the fertilization process and of how ICSI works.