Sexing mammalian spermatozoa and embryos--state of the art

Methods for sexing preimplantation embryos range from karyotyping to recording speed of development in vitro. The only method used routinely on a commercial scale is to biopsy embryos and amplify Y-chromosome-specific DNA using the polymerase chain reaction. This method is effective for more than 90% of embryos and is > 95% accurate. Within males, spermatozoa are essentially identical phenotypically due to: (1) connection of spermatogenic cells by intercellular bridges, (2) transcriptional inactivation of sex chromosomes during meiosis and spermiogenesis, (3) severe limitation of all gene expression during the later stages of spermiogenesis, and (4) coating all spermatozoa with common macromolecules during and after spermiogenesis. One consequence is that no convincing phenotypic difference has been detected between X- and Y-chromosome-bearing spermatozoa. The only consistently successful, nondestructive approach to sexing spermatozoa is to quantify DNA in spermatozoa using a fluorescing DNA-binding dye followed by flow cytometry and cell sorting. X-chromosome-bearing ruminant spermatozoa have about 4% more DNA compared with Y-chromosome-bearing spermatozoa; accuracy of sorting can exceed 90% routinely, and sorting rates currently exceed 10(3) live spermatozoa of each sex chromosome composition s-1. Hundreds of apparently normal offspring from a number of species have been produced from sexed semen, some via intrauterine artificial insemination.