【作者】 马骏；

【导师】 王守林； 李忠； 刘起展；

【作者基本信息】 南京医科大学 ， 劳动卫生与环境卫生学， 2009， 硕士

【摘要】 世界卫生组织(WHO)的统计数据表明,全世界约有8-15%的育龄夫妇被不孕不育所困扰。在所有的不孕不育中,由男方因素造成的约占50%,其中一个重要的原因是精子生成障碍,约占男性不育的10%,主要表现为无精子症或少精子症。产生成熟的精子是男性具有生育能力的首要条件,许多与生精相关的基因参与了精子发生这一复杂过程。近年来的研究发现,如果精子生成过程中的调控基因出现缺失、突变或表达异常都有可能引起精子生成障碍,形成少精子症或无精子症,导致男性不育症。由遗传缺陷包括单基因突变和染色体异常所引起的精子发生障碍估计占男性不育的30%。其中基因多态性尤其是单核苷酸多态性(Single Nucleotide Polymorphisms, SNPs)是人类最常见的遗传变异,它可以引起基因产物或功能的改变。越来越多的研究表明,精子生成障碍与某些特定基因的多态性存在相关性。在哺乳动物的正常精子生成过程中,生精细胞的退化死亡是一个重要的环节。据估计,约占潜在成熟精子数量25-75%的生殖细胞将会在精子生成过程中退化,而现在普遍认为这些生殖细胞的自发性死亡主要是通过凋亡实现的,因此生殖细胞凋亡是精子发生和成熟过程中关键的环节。体内许多调节凋亡的因素参与了精子细胞的凋亡过程,其中Bcl-2家族成员发挥了重要的调节功能,而Bcl-2基因是最早发现具有抗凋亡活性的调节因子。因此,其活性改变很大程度上会影响精子发生,导致少精子症和无精子症的发生。已有的研究显示,Bcl-2编码区基因多态性可通过影响其抗凋亡活性而与某些疾病的发生(如肿瘤和自身免疫性疾病)相关,但目前未见Bcl-2多态性与男性生精障碍关系的报道。本研究从分子流行病学和分子生物学角度,通过人群研究和体外功能研究,初步探讨了Bcl-2编码区基因多态性与精子生成障碍(无精子症和严重少精子症)的相关性及其可能的分子机制。研究结果将为深入阐明男性精子发生的凋亡调控机制并进一步为临床男性不育症的个体化诊疗提供一定的理论和实践依据。第一部分Bcl-2基因多态性与原发性生精障碍关系的分子流行病学研究方法1、开展基于医院的病例-对照研究,根据流行学调查要求将已确诊的不孕不育门诊病人和已有一个孩子出生的正常男性作为研究对象,完成统一印制的调查表,采集血液等生物样本,并适当保存备用。2、不孕不育男性病人要求留置精液,按照WHO的要求用CASA仪进行精液质量分析。3、从研究对象的血液白细胞中用酚-氯仿抽提法提取基因组DNA,保存备用。4、按WHO精液中精子数量标准在收集的病例中挑选精液离心后检测没有精子(无精子症)和精子密度少于10×106/mL的(严重少精子症)个体作为病例。共有198例原发性非梗阻性无精子症病人和76例原发性严重少精子症病人以及183例生育正常的男性对照组成为本课题的研究对象。5、针对Bcl-2基因的结构,设计了两对用于扩增Bcl-2基因全长编码区的引物,进行PCR扩增后产物送公司进行序列测定。6、测序结果与野生型Bcl-2基因序列进行同源性比对,判别存在于Bcl-2基因编码区内的变异,查找可能的单核苷酸多态性位点(SNPs)。7、运用统计学方法分析Bcl-2不同基因型在病例和对照中的分布差异,探讨Bcl-2编码区基因多态性与无精子症和严重少精子症的相关性。结果1、根据Bcl-2蛋白表达的特性,设计了针对扩增Exon 2和Exon 3全长序列的引物,通过PCR和凝胶电泳,分别得到了639bp和185bp的扩增产物。以此产物经测序后,通过网上序列比对,获得编码区基因变异位点,确认Bcl-2基因变异类型。2、通过测序,共发现3种Bcl-2基因变异类型,都发生在Exon2,分别为A21G (rs1801018)、G127A(rs1800477),和C300T (rs61733416),可见有2种同义突变(Thr7Thr和Ala100Ala)和1种错义突变(Ala43Thr)。A21G、G127A和C300T这3种基因型在正常生育男性人群中的比例分别为14.7%(杂合型AG为13.6%,纯合型GG为1.1%)、15.3%(杂合型GA为13.7%,纯合型AA为1.6%)和7.7%(全部为杂合型CT)。其中C300T(rs61733416)在中国汉族人群中系首次报道。3.分析了上述3种基因型在病例和对照人群中的分布情况,结果显示3种基因型在严重少精症中的比例分别为14.5%、14.5%和6.6%,与对照接近,两组无显著性差异;无精症组中3中基因型的比例分别为8.6%、7.1%和3.0%,都低于对照组和严重少精症组,且G127A与对照组相比有明显差异(P=0.010),主要体现于杂合型(P=0.034)。携带GA基因型的个体罹患原发性无精子症的风险减少(调整后OR=0.403,95%CI=0.189-0.857),基因型GA和AA合并后结果改变不大(调整后的OR=0.448,95% CI=0.226-0.889)。4、从Bcl-2编码区的变异数量来分析,结果发现在对照和无精子组中变异个数是有差异的,携带1-2个变异的个体罹患无精子症的风险明显降低(调整后的OR=0.37,95%CI=0.23-0.60)。三个多态性位点的单倍型和危险位点联合分析发现与野生型相比,21G-127A-300C、21G-127G-300C和21A-127G-300T显著降低了原发性无精子症患病的风险性,OR分别为0.43(95%CI=0.24-0.80)、0.48(95%CI=0.26-0.89)和0.35(95%CI=0.13-0.92)。结论1、在中国汉族男性中共发现了3种Bcl-2基因变异类型,分别为A21G、G127A和C300T,其中C300T为汉族人群中首次报道。2、各基因型在严重少精子症患者和对照人群中的分布基本接近,但都高于无精子症人群,且G127A在无精子症中的比例显著低于对照组,可显著降低罹患原发性无精子症的风险。此外,无论是变异数量还是单倍型分析,这种趋势都依然存在。第二部分Bcl-2 Ala43Thr变异体的功能研究关于Bcl-2基因多态性的研究主要集中于自身免疫疾病和肿瘤,但是报道的结果不一致。本研究选择第一部分有显著意义且属错义突变的Bcl-2变异体G127A(Ala43Thr)为研究对象,通过体外功能学研究,探讨该位点变异对Bcl-2蛋白活性的影响因而改变精子发生特别是无精子症发生的分子机制。方法1、构建含Bcl-2野生型和Ala43Thr变异体的表达载体:pEGFP-C1-Bcl-2(Wild-type)和pEGFP-C1-Bcl-2(Ala43Thr),PCR、酶切及测序鉴定含有不同基因型的Bcl-2 cDNA序列。2、将不同基因型Bcl-2重组质粒转染到HeLa细胞中,在培养液中孵育24h后提取细胞总蛋白用Western Blot检测Bcl-2转染效率,确认获得Bcl-2基因高表达的细胞模型。3、用0.0、0.05、0.1、0.5、1.0、2.0μM Paclitaxel(紫杉醇)分布处理转染细胞,用MTT检测细胞的活性,观察不同基因型Bcl-2蛋白对Paclitaxel所致细胞毒性的影响。4、以0.1μM Paclitaxel处理转染细胞24h,用Hoechst 33258和流式细胞仪分别检测细胞的凋亡情况。5、以0.1μM Paclitaxel处理转染细胞24h,用Western blot分析细胞中Bcl-2表达情况和磷酸化水平,以及与Bcl-2相关的Bax和P53蛋白的表达情况。同时分析Cleaved-PARP的情况。6、生物信息学软件分析预测Wild-type和Ala43Thr Bcl-2蛋白结构的差异以及与Bax结合能力的改变。结果1、pEGFP-C1-Bcl-2(Wild-type)和pEGFP-C1-Bcl-2(Ala43Thr)质粒经双酶切、PCR以及测序鉴定:野生型Bcl-2与GenBank中AY220759序列一致,而Ala43Thr则含有一个碱基突变,即127位碱基G→A(Ala43Thr)。2、在细胞转染质粒24h时用WesternBlot检测Bcl-2蛋白表达情况,结果此时在HeLa细胞内Bcl-2的表达量相比空载细胞提高了约20倍,并且Wild-type和Ala43Thr的表达量接近,说明在细胞内的Bcl-2转染效率相近,两者具有可比性。3、转染不同载体HeLa细胞活性在0.1和0.5μM Paclitaxel处理24h时已有显著性差异(P<0.05),在0.5、1.0和2.0μM处理后转染Wild-type Bcl-2的HeLa细胞活性均显著高于转染Ala43Thr Bcl-2或Vector Control细胞(P<0.05)。4、不同基因型Bcl-2对细胞凋亡的影响。Hoechst33258染色观察细胞凋亡的形态学改变,凋亡的细胞核呈致密浓染、碎片状,未凋亡的细胞核呈圆形或椭圆形。0.1μM Paclitaxel诱导细胞凋亡24h后,Wild-type Bcl-2的HeLa细胞凋亡率是44.4%,而Ala43Thr Bcl-2细胞的凋亡率则为53.3%,与空白细胞接近,差异显著(p<0.05);利用流式细胞仪测定相同条件处理的细胞凋亡情况结果也同样证实:Wild-type Bcl-2的HeLa细胞凋亡率是28.6%,而Ala43Thr Bcl-2细胞的凋亡率则为36.2%,差异显著(p<0.05)。5、从Bcl-2总表达量分析,Wild-type和Ala43Thr Bcl-2是表达量之比为1.1:1.2,但是Bcl-2的磷酸化水平Ala43Thr Bcl-2高于Wild-type(1.53:1.00);与凋亡的效应指标一致,Cleaved-PARP的量在Wild-type细胞内显著低于Ala43Thr和VectorControl细胞(P<0.05)。但是与Bcl-2结合有关的蛋白比如Bax和P-53等的表达却未受Bcl-2不同基因型高表达的影响。6、生物信息学软件预测Wild-type和Ala43Thr Bcl-2蛋白结构,结果显示,Ala43Thr位于Bcl-2柔性结构区域(FLD),此多态性位点软件预测可以改变Bcl-2蛋白FLD的空间构像,一定程度上改变了与Bax的结合能力,可能与凋亡调节功能有关。结论1、Ala43Thr Bcl-2可显著降低其抗凋亡能力,增加细胞对紫杉醇的敏感性;2、虽然Ala43Thr Bcl-2未影响Bax和P53的表达,但其磷酸化水平发生改变,可能是其活性改变的潜在机制;3、Ala43Thr改变了Bcl-2蛋白的FLD区域的空间构象,可能影响Bcl-2与Bax等结合能力进而影响了其抗凋亡的活性。总之,综合人群和体外功能研究结果,我们初步得出结论:Bcl-2基因Ala43Thr多态性可显著降低原发性无精子症的发生风险,可能的分子机制是:正常人群中存在一定比例的Bcl-2 Ala43Thr变异,可以诱导正常的生精细胞凋亡,促进精子的正常发生;而在无精子症中,低发生率的Ala43Thr变异相对增强了Bcl-2蛋白抗凋亡的活性,破坏了机体维持正常生殖细胞凋亡的平衡状态,结果导致大量堆积的生精细胞发生退化,并进而在生精小管中发生空泡化,最终导致无精子症的发生。更多还原

【Abstract】 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.更多还原