Study On The Relationship Between Bcl-2 Variants And Dyszoospermia And Their Related Mechanisms

The World Health Organization (WHO) estimates that approximately 8-15% of couples were attacked by some forms of infertility problems. On a worldwide scale, male factors attributed to approximately half of sterility problems. Among many causes which lead to male infertility, previous reports from clinicals confirmed that dyszoospermia was a significant cause, which is responsible for about 10% of the cases, typified by non-obstructive azoospermia or oligozoospermia. Gamete maturation is the first step for men who are of fertility. Spermatogenesis is indispensable and complex, and multiple genes are implicated in the process of spermatogenesis. In about 30% of male infertility subjects could represent dyszoospermia which caused by genetic defection including single gene mutation and chromosomal aberration. Polymorphisms especially single nucleotide polymorphisms (SNPs) are the most common genetic variants which can change the expression protein of the gene. Recently, several studies have focused on the association of SNPs with spermatogenic failure. In mammals, germ cell death is conspicuous during spermatogenesis and occurs spontaneously at various phases of germ cell development. It is well known that mature sperm are the production of a precisely regulated network in which mammal germ cell proliferation, differentiation, and apoptosis are carefully controlled. During normal spermatogenesis, more than half of the germ cells undergo apoptosis, and 25-75% of the expected sperm yield is thought to be lost indeed. Many regulation factors can affect the germ cell apoptosis, of which, the Bcl-2 and its protein owing to its death inhibition function in cancer developments, may be most wildly studied and thought to be the key regulator. Considering its pivotal role in modification of apoptosis, the functional alteration of Bcl-2 could certainly damage the normal spermatogenesis, resulting in oligozoospermia and azoospermia. Previous reports had revealed that SNPs in the coding regions of Bcl-2 gene conferring an negative effect on anti-apoptotic viability associated some diseases, i. e. cancers and autoimmune diseases. However, little information is obtained on Bcl-2 polymorphisms in male spermatogenesis impairment. In current study, we carried out a case-control study between infertility male and normal controls to scrutinize the polymorphisms in coding regions of Bcl-2 gene, and analyzed their potential association with the risk of idiopathic azoospermia or severe oligozoospermia in a Han-Chinese population. We further evaluated its potential molecular mechanism via an in vitro functional assay. The study will help to elucidate the molecular mechanism involving in germ cell apoptotic regulation during spermatogenesis as well as raise awareness of clinical diagnosis and treatment of male infertility .Part 1: A Molecular Epidemiological Study on Bcl-2 Gene Polymorphisms and Susceptibility to Idiopathic DyszoospermiaMethodsA hospital based case-control study was conducted in the affiliated hospitals of Nanjing Medical University between April 2004 and July 2007, We used a short questionnaire to obtain demographic and risk factor information and frequency-matched the controls to the cases by age (±5 years) . Over 85% of eligible cases and controls agreed to participate in the study and also donated 5 ml of blood to be used for genomic DNA extraction. Before recruitment, a signed informed consent form was obtained from each of the eligible subjects. All patients underwent at least twice semen analysis according to the WHO guidelines, those with a history of orchitis, obstruction of vas deferens, chromosomal abnormalities or micro-deletions of AZF region on Y chromosome were excluded. Subjects who had special occupational exposure which was suspected to be associated with semen quality (such as pesticides or other agents) were precluded. The total 274 idiopathic infertility patients with abnormal sperm count were divided into two groups according to WHO semen parameters: 198 with non-obstructive azoospermia (no sperm in semen even after centrifugation) and 76 with severe oligozoospermia (sperm count<10×106/ml). The controls were frequency-matched to the cases on age (±5 years old), including 183 fertile men who had fathered at least one child without assisted reproductive measures and had normal spermatogenesis (sperm count >20×106/ml) with average count of 53.6×106/ml.We amplified the whole DNA sequences of CDS in Bcl-2 gene, and genotyped the polymorphisms of Bcl-2 using polymerase chain reaction based-sequencing analysis in all the recruited subjects. Multi-norminal and multi-variant logistic regression ananlysis were employed to investigate the risk of idiopathic azoospermia and severe oligozoospermia simultaneity for controlling multiple comparison and the potential confouding factors. Hardy–Weinberg Equilibrium was tested by a goodness-of-fit chi-square test. The PHASE 2.0 software was used to reconstruct haplotypes based on the detected Bcl-2 genotypes. All statistical analyses were conducted with Statistical Analysis System software. Statistical significance was assumed to p≤0.05.ResultsAccording to Bcl-2 expression in vivo, we designed two primer sets to amplify the corresponding CDS in Exon 2 and Exon 3. The PCR products using the two primer sets were 639bp and 185bp length, respectively, and confirmed by DNA sequencing. The results revealed that the sequences were consistent with the wild-type Bcl-2 (RefSeq: AY220759). We further Blast the NCBI database with the sequencing results to find the DNA variants.Three possible SNPs were detected in this assay, which are A21G (rs1801018, Thr7Thr), G127A (rs1800477, Ala43Thr), and C300T (rs61733416, Ala100Ala). All three SNPs are located in Exon 2, including two synonymous mutations (i.e. Thr7Thr: A>G and Ala100Ala: C>T) and one missense mutation (Ala43Thr: G>A). In normal controls, the frequencies of variants were 14.7% for A21G, 15.3% for G127A, and 7.7% for C300T, among which, the C300T was first reported in Han-Chinese population.Genotype frequencies of the detected polymorphisms in patients and controls and their association with risk of azoospermia and severely oligozoospermia were analyzed. The results showed that the variant frequencies in severe oligozoospermia were 14.5% for A21G, 14.5% for G127A, and 6.6% for C300T, which were very similar to the controls, while in the azoospermia the frequencies of variants were relative low, there were 8.6%, 7.1% and 3.0% for three SNPs, respectively. The significance was found between the azoospermia and the controls (P=0.010) for the GA+AA genotype, P=0.034 for the GA genotype. By logistic regression analysis, individuals carrying heterozygote GA and GA+AA genotypes of Ala43Thr (G>A) was significantly different between azoospermia and controls (adjusted OR = 0.403, 95℅CI = 0.189-0.857 and adjusted OR = 0.448, 95℅CI = 0.226-0.889, respectively).We further combined these three SNPs based on the number of variants, and found that individuals with 1-2 variants had a significant lower risk of azoospermia (adjust OR=0.37, 95%CI=0.23-0.60) than those with no variant when the combined genotypes were dichotomized into two groups. We performed haplotype inference on these three polymorphisms, and revealed that, the AAC (21A-127A-300C), GGC (21G-127G-300C) and AGT (21A-127G-300T) haplotypes showed significantly decreased risk of idiopathic azoospermia compared with AGC (21A-127G-300C) (adjusted OR=0.43, 95%CI=0.24-0.80, OR=0.48, 95%CI=0.26-0.89 and OR=0.35, 95%CI=0.13-0.92, respectively). ConclusionsThere are three SNPs screened in our Han-Chinese population, which are A21G (rs1801018, Thr7Thr), G127A (rs1800477, Ala43Thr), and C300T (rs61733416, Ala100Ala). It was novelly detected the C300T polymorphism in Han-Chinese population.The frequencies of three variants in Bcl-2 gene were similar between oligozoospermia and controls, while they were relative high compared to azoospermia. The frequency of G127A variant was significant lower than that in controls, as well as number of variants and haplotypes reconstruction. Subjects carrying the G127A genotype had a low risk of azoospermia. Keywords: idiopathic azoospermia, idiopathic oligozoospermia, spermatogenic failure, single nucleotide polymorphisms, Bcl-2 genePart 2 Functional Study of Ala43Thr Bcl-2 VariantTo date, researches on Bcl-2 gene polymorphisms were focus on autoimmune diseases and cancers, however, the results still controversial. Our epidemiologic study had revealed that there was a potential functional SNP in Bcl-2 gene, which was Ala43Thr (G127), and it is a minsense mutation(Ala→Thr). Owing to its significant different frequencies distribution between controls and azoospermia, we further explored the underlying functional alteration of Ala43Thr variant of Bcl-2 protein, and the rationale of the variant affecting the normal spermatogenesis, resulting in dyszoospermia, especially azoospermia. MethodsBoth wild-type and Ala43Thr variant Bcl-2 cDNA were cloned into pEGFP-C1 expression vectors, and confirmed by directed sequencing. Different vectors (pEGFP-C1 vector, wild-type, and Ala43Thr Bcl-2 variant) were transfected into HeLa cells for 24h, then the transfectional efficiency were measured both by fluorescence microscope and Bcl-2 proteins expression. To evaluate the effect of different genotypes of Bcl-2 proteins on the Paclitaxel-induced cytotoxicity in HeLa cells, cells which were transfected with corresponding vectors for 24h, were incubated with 0.0, 0.05, 0.1, 0.5, 1.0, and 2.0μM of Paclitaxel for another 24h, and the cell viability were measured by MTT assay.To examine the different anti-apoptotic effects of wild-type and Ala43Thr variant Bcl-2 proteins, cells transfections were done as aforementioned, following treatments with 0.01μM of Paclitaxel for another 24h, and the apoptotic cells was measured by Hoechst 33258 staining assay and flow cytometry. At the same time, the expression of Bcl-2 and its phosphorylational levels, the Bcl-2 related proteins (Bax and P-53), as well as Cleaved-PARP were measured via Weastern Blotting assay. Data obtained from in vitro assays were presented as the means±SD, Two-way analysis of variance procedures were used to assess significant difference among different groups. P<0.05 was used as statistical significance.To further predict the potential difference of structure between wild-type and Ala43Thr Bcl-2 variant proteins, we reconstructed the comformations of the two proteins, and analyzed the binding alteration to pro-apoptotic proteins, i. e. Bax. ResultsThe expression vectors were confirmed to contain the wild-type and Ala43Thr variant Bcl-2 cDNA. After transfection with wild-type, Ala43Thr variant Bcl-2, as well as vector control for 24h, the exogenous Bcl-2 expression were both high, and there was no difference between wild-type and Ala43Thr variant, while the expression of Bcl-2 were very slight in vector controls cells.The identified tranfectional cells were pre-treated with different concentration of Paclitaxel, i.e. 0.0 (control), 0.01, 0.05 0.1, 0.5, 1.0, and 2.0μM, for another 24h, and cell viability was found significantly decreased in cells with different plasmids incubated at 0.5μM or higher concentration of Paclitaxel at 24h. However, there was no difference on cell viability at 0.1μM or lower concentration of Paclitaxel treatment among three groups of cells. The results showed that Paclitaxel induced a significant inhibition of HeLa cell toxicity in a concentration-dependent manner, while cells with wild-type Bcl-2 represented a striking difference in cell viability from Ala43Thr Bcl-2 or controls at >0.5μM concentration, i. e. the relative viability of cells were 80% for wild-type and 73% for Ala43Thr at 0.5μM by MTT assay.To further evaluate the potential effects of different genotypes of Bcl-2 on cell anti-apoptosis, we generated the cell models with previous same transfection and 0.1μM Paclitaxel-induced cell apoptosis for 24h, the Hoechst staining assay results showed that the relative apoptosis were 44.4% for wild-type Bcl-2, 53.5% for Ala43Thr Bcl-2, and 57.7% for controls. These were consistent with the results examined by Flow cytometry assay, namely, the relative apoptosis were 28.6% for wild-type Bcl-2, 36.2% for Ala43Thr Bcl-2, and 42.1% for controls., cells bearing wild-type Bcl-2 were more resistant to Paclitaxel-induced apoptosis compared to Ala43Thr Bcl-2.To explore the potential molecular mechanisms involving in the functional changes of Bcl-2 Ala43Thr polymorphism, we detected the expression levels of Bcl-2 related proteins such as Bax and P-53. The results found the levels of Bax and P-53, which can interact with Bcl-2 to regulate the apoptotic balance, were not altered. However, the p-Bcl-2 were different between wild-type and Ala43Thr Bcl-2, in which Ala43Thr Bcl-2 were more phosphorylated than wild-type Bcl-2. Consistent with previous observations, the Cleaved-PARP were much lower for wild-type than Ala43Thr.Bio-informatic prediction analysis showed that the Ala43Thr which was located in flexible loop domains (FLD) of Bcl-2, could change the conformation of Bcl-2, and alter the interaction with Bax. This findings may provide some clues for the reduced anti-apoptotic viability of Ala43Thr Bcl-2. ConclusionsAla43Thr variant Bcl-2 represented a significant decreased anti-apoptotic potential, and cells bearing variant Bcl-2 were more sensitive to Paclitaxel-induced apoptosis. The expression of Bax and P-53 were not changed, while the phosphorylation alteration were observed in variant Bcl-2 proteins, which may be associated with the potential phosphorylate residue Ala43Thr, thus affected the anti-apoptotic function of Bcl-2.Another underlying mechanism for the functional difference of Ala4Thr variant Bcl-2 may involve in the protein conformational changes especially in the FLD region of Ala43Thr Bcl-2, which may affect the interaction between Bcl-2 and the pro-apoptotic members, i.e. Bax.In conclusion, we found that Ala43Thr Bcl-2 polymorphism significantly decreased the risk of idiopathetic azoospermia rather than severe oligozoospermia, and its potential molecular mechanism was in the azoospermia, the relative low frequency of Ala43Thr variants caused more resistace to apoptosis of germ cells, resulting in abnormal accumulation of spermatogonia was more apparent during first wave of spermatogenesis. This accumulation of germ cells in turn led to vacuolization of seminiferous tubules, and azoospermia, as a result. However, certain ratio of Ala43Thr Bcl-2 can confer the balance of apoptosis during spermatogenesis.