286
FULL-LENGTH DAZ MRNA EXTRACTED FROM MATURE EJACULATED SPERMATOZOA.
Sprague, David1,2, Conley, Patrick1, Brown, Monica2, Ficht, Allison1, Kuehl, Thomas1,2, 1 2
ABSTRACT- The deleted in azoospermia (DAZ) gene has been implicated as important in sperm development. Reduced fertility in human males has been associated with spontaneous deletions of the portions of the Y-chromosome that contain the DAZ gene and/or other genes of the AZFa, AZFb, and AZFc (azoospermia factor) regions. Most current molecular diagnostics focus on detecting microdeletions in the genomic complement of DNA. We speculate that point mutations, like microdeletions, can frequently occur spontaneously in the DAZ gene located on the human Y-chromosome and contribute to reduced spermatogenesis. Alternatively, transcripts of the chromosome 3 DAZ gene may salvage spermatogenesis in the absence of non-expressed DAZ transcripts from the Y locus. We extracted RNA from mature ejaculated spermatozoa using a method adapted from Chirgwin using guanidinium HCl and multiple precipitation steps in one single container. Because the extraction of RNA without DNA contamination is very difficult, we designed primers for our PCR reaction that would not amplify genomic DNA. RT-PCR was performed using MMLV reverse transcriptase coupled with specific PCR primers to produce DAZ-specific transcripts. The resulting cDNA was sized and sequenced. Ejaculated spermatozoa were found to contain two DAZ transcripts. One was 1062bp and 1200bp and in agreement with the putative Y-chromosome DAZ products while the other was 750bp and displayed sequence homology with the putative chromosome 3 product. Methods were replicated in 2 samples from 2 different male subjects without a detectable microdeletion in the coding sequence of Y-chromosome DAZ locus. These results confirm that messages from both sources of DAZ transcripts can be found in ejaculated sperm cells. Further analysis will be performed to identify types of mutations that are compatible with normal, and reduced levels of spermatogenesis as reflected by characteristics of semen analyses.
KEY WORDS: daz, rt-pcr, mature spermatozoa, spermatogenesis