DNA methylation analysis in immature testicular sperm cells at different developmental stages

Testicular sperm extraction and intracytoplasmic sperm injection (TESE-ICSI) is a frequently used therapeutic option in azoospermic males. Genetic imprinting is a mechanism of gene regulation - mediated by the methylation of deoxyribonucleic acids (DNA) - by which only one of the parental copies of a gene is expressed. Whether the establishment of the genetic paternal imprint in this immature haploid sperm cell is complete and stable at this stage of maturation has never been analysed. In the present study, a highly sensitive heminested methylation-specific polymerase chain reaction for the target region 15q11-13 was developed for imprinting analysis at the single-cell level. Imprinting analysis was then carried out on DNA extracted from single diploid leucocytes (n = 25), ejaculated spermatozoa (n = 88), elongated testicular spermatids (n = 30), and round spermatids (n = 25). Amplification was obtained in 57% of the ejaculated spermatozoa, in all elongated spermatids and in 20% of the round spermatids. In the amplified samples, only the paternal imprint was detected. The maternal imprint was not found at all in any of the sperm cells; in 56% of the diploid leucocytes, the maternal and paternal imprints were detected at the same time (complete failure in the other 44%). The data reveal the completed establishment of the correct paternal imprint in ejaculated spermatozoa, elongated spermatids and amplified round spermatids. However, the high rate of amplification failure in round spermatids remains a factor of uncertainty.