Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion-related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo-/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.

Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mice lacking Ppp3cc or Ppp3r2 genes (knockout mice) are infertile, with reduced sperm motility owing to an inflexible midpiece. Treatment of mice with cyclosporine A or FK506 creates phenocopies of the sperm motility and morphological defects. These defects appear within 4 to 5 days of treatment, which indicates that sperm-specific calcineurin confers midpiece flexibility during epididymal transit. Male mouse fertility recovered a week after we discontinued treatment. Because human spermatozoa contain PPP3CC and PPP3R2 as a form of calcineurin, inhibition of this sperm-specific calcineurin may lead to the development of a reversible male contraceptive that would target spermatozoa in the epididymis.

Mammalian male preimplantation embryos develop more quickly than females . Using enhanced green fluorescent protein (EGFP)-tagged X chromosomes to identify the sex of the embryos, we compared gene expression patterns between male and female mouse blastocysts by DNA microarray. We detected nearly 600 genes with statistically significant sex-linked expression; most differed by 2-fold or less. Of 11 genes showing greater than 2.5-fold differences, four were expressed exclusively or nearly exclusively sex dependently. Two genes (Dby and Eif2s3y) were mapped to the Y chromosome and were expressed in male blastocysts. The remaining two (Rhox5/Pem and Xist) were mapped to the X chromosome and were predominantly expressed in female blastocysts. Moreover, Rhox5/Pem was expressed predominantly from the paternally inherited X chromosome, indicating sex differences in early epigenetic gene regulation.