ICSI and Nuclear Transfer Using Male Germ Cells.

Abstract

In vitro fertilization (IVF) in mammals was achieved in the middle of the last century by the pioneers of fertilization studies, Drs. Austin, Chan, and Yanagimachi (Yana). Soon after the success of IVF, a sperm injection technique (later called ICSI, or microinsemination in a broad sense) was developed to simplify the fertilization process and determine what happens within the oocyte. In its first decade, ICSI provided invaluable information on the mechanisms of mammalian fertilization including sperm nucleus stability. Then, around 1990, normal ICSI offspring were reported in rabbits, cattle, and humans. The first reliable application of mouse ICSI was achieved by Yana's group in 1995. Since then, the mouse model has held a leading role in the field of microinsemination. We have also employed the technique in mouse genetic studies and found that elongated and round spermatids, and even primary spermatocytes, could be used to fertilize oocytes to produce normal pups. The consistent fecundity enables us to apply the mouse microinsemination technique to transgenesis, gene therapy, germline stem cell, and gene-targeting studies. In contrast, in other mammals, immaturity of male germ cells significantly decreases the efficiency of microinsemination. No, or very few, offspring have been born following ICSI using spermatids in these species. This is not due to immaturity of their genome, but to their physically unstable chromatin structure and low oocyte-activating capacity. Our recent ICSI study was also undertaken to simplify the cryopreservation technique for mouse male germ cells. Whole mouse testes and even whole bodies can be frozen and then used as a source of sperm. Spermatozoa retrieved from male bodies frozen for 15 years at −20°C support full-term development following ICSI, indicating unexpectedly high stability of sperm nucleus integrity. Younger male germ cells include spermatogonia and primordial germ cells (PGCs). Their nuclei are diploid and therefore cannot be used for microinsemination. Construction of diploid embryos with these early male germ cells requires a cloning technique using nuclear transfer (NT). Cloning spermatogonia or PGCs has two scientific significances: analysis of genomic imprinting status and genomic reprogrammability (not reprogramming ability). The former is based on the fact that the imprinting memory of the donor cell genome is maintained after transfer into MII oocytes under well-controlled experimental conditions. By analyzing the fetuses thus produced, we clearly demonstrated that the major erasing process for the imprinting memory occurred around day 11.5 in mouse PGCs and that the PGC genome before imprinting erasure could support full-term development after NT. As to genome reprogrammability, we found that the some proportion of male PGCs gradually acquired the ability to be reprogrammed normally following nuclear transfer, as revealed by the gene expression patterns of the reconstructed embryos. We hope that this experimental model may allow identification of some keys or epigenetic markers that permit differentiation between somatic cell and germ cell genomes. Thus, ICSI and nuclear transfer using male germ cells have great potential as tools for a broad range of genetic and epigenetic research in mammals.