DNA Methylation Analysis in Immature Testicular Sperm Cells at Different Developmental Stages

Abstract

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.

Reproductive medicine and inheritance of infertility by offspring: the role of fetal programming

“…This is consistent with those studies that found that methylation patterns in relation to imprinted alleles are already defined at the spermatogonia stage (79,80).…”

Evaluation of DNA methylation at imprinted DMRs in the spermatozoa of oligozoospermic men in association withMTHFRC677T genotype

“…Spermatozoa isolated from micromanipulation were fewer and thus subjected to alkaline lysis (Manning et al, 2001). Bisulfite conversion was conducted with the EZ DNA Methylation-Gold Kit (Zymo Research, Orange County, CA, USA) according to the manufacturer's protocols.…”

Methylation status of the SNRPN and HUMARA genes in testicular biopsy samples

“…This fact may explain the deviation between the results of HUMARA and SNRPN analysis (Table 2) and the very low percentage of positive results observed for HUMARA locus methylation in nonobstructive men with spermatozoa. The SNRPN analysis was informative about the stages of spermatogenesis before and after the pachytene spermatocytes evolved from the diploid cells, whereas HUMARA analysis was informative only for the pachytene spermatocytes when X-inactivation occurred (4,10). Therefore, SNRPN methylation analysis seems to confer different information than does HUMARA in terms of spermatogenesis and concordance with microscopy.…”

“…This outcome is probably due to the presence of the unmethylated paternal allele in almost all spermatogenic stages, including the round-spermatid stage (10). Germ cells of all preceding stages have not yet established the paternal imprint.…”