The use of tandem repeat markers to detect Y chromosome mutation in infertile human spermatozoa

Abstract

OBJECTIVE: Oxidative stress is a cause of male infertility, and contributes to the high rate of DNA fragmentation. Effects of oxidative damage are mediated by the highly active reactive oxygen species (ROS). ROS is generated by endogenous metabolism, inflammatory and by environmental agents. The Y chromosome is susceptible to such DNA fragmentation due to presence of repetitive elements. The instability of Y chromosome is a precursor of mutagenic changes in the germ line, leading to infertility and cancer. We hypothesize that repetitive DNA sequences in the Y chromosome in infertile patients are highly susceptible to increased mutations. DESIGN: We tested repetitive DNA sequences in the Y chromosome from infertile spermatozoa and testicular tissue to identify the loci most sensitive to oxidative stress induced mutations. MATERIALS AND METHODS: Sperm DNA from both fertile (control n=5) and infertile (n=25) were analyzed for the presence of mutations by using small-pool PCR. DNA purified from semen or testicular samples from both groups were used to detect the mutations. Moreover, DNA from matching somatic cell (blood) from these samples were analyzed to determine the size of constitutive alleles for each locus and to determine background levels of the mutations. A microsatellite marker panel was used including 12 tri- tetra- and pentanucleotide repeats on the Y chromosome(Promega). Strand breaks in spermatozoa from both groups were measured by using the sperm Chromatin Structure Assay (SCSA). RESULTS: The SP-PCR data on the infertile germ line showed a significant increased mutation frequency (P<0.05) compared to controls. No mutation was seen in matched infertile blood samples from these individuals. Likewise, none of the fertile germ line and soma samples tested displayed mutation. Our results also showed that the pentanucleotide locus on the Y chromosome is amongst the most sensitive to mutation (>30%). The most sensitive repeat motifs for germ line specific mutation are tetranucleotide repeats on the Y-chromosome which exhibited higher mutation frequency (18% to 32%). Moreover, mononucleotide repeats exhibit a wider range of sensitivity to mutation (15% to 47%). The SCSA indicate a significant increase in DNA strand breaks in the infertile group compared to the controls (P<0.05). CONCLUSIONS: Our data demonstrate the sensitivity of tandem repeat markers in the Y chromosome for detection of mutations in infertile spermatozoa. These markers may also be useful for monitoring mutagenic changes in the germ line.