Studies On Associations And Mechanisms Between Azoospermia And XRCC1, XPD, TERT Genes

The World Health Organization (WHO) estimates thatapproximately 15ï¼…of couples experience some form of infertilityproblem. On a worldwide scale, this means that over 80 million peoplesuffer from infertility, male factors accounted for approximately half ofthese cases. Nearly 50ï¼…of male infertility is idiopathic azoospermia,with uncertain causes, which is generally assumed to be the result ofgenetic alterations.Spermatogenesis is a continuum of cellular differentiation in whichthree principal phases can be discerned: spermatogonia renewal andproliferation, meiosis, and spermiogenesis. Many important genes arerequired for normal spermatogenesis. The deletion or dysregulation of these critical genes will resulte in azoospermia.We estimate the association between three important genes involvedin spermatogenesis, XRCC1, XPD, TERT, and azoospermia. Furthermore,we explore the potential pathophysiologic mechanism accounting forazoospermia. The study will help us elucidate the molecular mechanisminvolving in spermatogenesis as well as raise awareness of clinicaldiagnosis and treatment of male infertility and male molecularcontraception.Part 1: A molecular epidemiological study on XRCC1 and XPD GenePolymorphisms and Susceptibility to Idiopathic AzoospermiaGenetic polymorphisms in DNA repair genes may influenceindividual variation in DNA repair capacity and further influence the riskof developing cancer. However, little information is available on thesepolymorphisms in infertile. To investigate whether polymorphisms inDNA repair gene, XRCC1 and XPD, alone or in combination, areassociated with risk of developing idiopathic azoospermia, the genotypeand allele frequencies of three observed polymorphisms (XRCC1Arg194Trp and Arg399Gln, and XPD Lys751Gln) were examined bypolymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) based on a Chinese population consisting of 171 idiopathicazoospermia patients and 247 normal-spermatogenesis fertile controls.Associations between the polymorphisms and idiopathic azoospermia riskwere estimated by logistic regression, and the Statistical Analysis Systemwas used to test the gene-gene joint effects. All observed polymorphismswere in agreement with Hardy-Weinberg equilibrium. The XPD 751 Gln allele seemed to be a risk allele for azoospermia, with its frequency11.40ï¼…in cases and 5.67ï¼…in controls (p=0.004). Compared withLys/Lys genotype, the XPD 751 Lys/Gln+Gln/Gln genotype associatedwith a moderate increased risk of azoospermia (OR=2.09, 95ï¼…CI=1.22-3.57), while the risk increased 4.100 (OR=5.100, 95ï¼…CI=1.951-13.330) or 2.064(OR=3.064, 95ï¼…CI=1.440-6.523) fold,respectively, when combined with XRCC1 194 Arg/Arg or 399 Arg/Arggenotype. In conclusion, our study provided the first evidence that XPDand XRCC1 polymorphisms contributed to the risk of developingidiopathic azoospermia in a selected Chinese population.Part 2: Study on the Role of TERT Gene in Idiopathic AzoospermiaTelomeres, composed of conserved sequences of DNA repeats, arespecialized structures at the ends of eukaryotic chromosomes. TelomericDNA is not completely synthesized by conventional DNA polymerase,but progressively shorten with each cellular division. Cellular aging ischaracterized by a decrease in telomere length, and this has beenimplicated as a mitotic clock that signals stop in cellular division whentelomeres reach a critically short length. Telomerase, a ribonucleoproteinthat synthesizes telomere repeats onto chromosome ends, is involved inmaintaining telomere length in germ line and cancer cells (＞90ï¼…). Thecatalytic subunit (TERT) appears to be the limiting component in mosttelomerase-negative cells. As TERT is expressed constitutively, the presence of telomerase activity is correlated with TERT mRNAexpression in extracts from tissue culture cells as well as normal andcancer tissues. Telomere length increases during the development of malegerm cells from spermatogonia to spermatozoa and is inversely correlatedwith the expression of telomerase activity. However, their exact functionsin male reproduction are still unclear.In the current study, telomerase repeat amplification protocol assay(TRAP), real-time quantitative PCR and in situ hybridization (ISH) wereused to detect the telomerase activity (TA) and the expression level ofTERT gene in both mouse testis and the testicular biopsies fromazoospermia patients. We further used RNA interference to knock-downthe mTERT gene in GC2-spd cell line (a cell line of mouse spermatocyte)in an attempt to explore the potential role of TERT gene in spermatocyte.Our results demonstrate that quantitative detection of hTERT mRNAexpression in testicular tissue enables a molecular-diagnosticclassification of gametogenesis. Quantitative detection of hTERT intesticular biopsies is thus well suited for supplementing thehistopathological evaluation of azoospermia.1. We found that the expression level of TERT mRNA was quite inagreement with telomerase activity (TA). We failed to detect the TA andhTERT mRNA in testicular biopsies of SCOC (Sertoli-cell-onlysyndrome) patient, while the expression of hTERT mRNA was high inOA (obstructive azoospermia) testis and low in MA (maturation arrest)testis. Thus, detection of hTERT expression in differenthistopathologically classified azoospermia enables a molecular-diagnosticconfirmation.2. Comparison of the mouse and human TERT sequences revealed 78ï¼… identity at the nucleotide level. We also found that TERT gene expressedspecificly in germ cell by ISH of mouse testis and spermatocyte cell line.3. It appeared that the knocked-down expression of mTERT in GC2-spdcell line inhibited the normal cell growth and sensitized GC2-spd cells toH₂O₂-induced DNA damage. When given the same oxidative stress, thesecells displayed more DNA damage compared with controls.