TEKT4蛋白在特发性弱精子症患者精子中的表达

目的:探讨TEKT4蛋白在特发性弱精子症发生机制中的作用。方法:采用非连续密度梯度离心分离和纯化特发性弱精子症患者和正常男性精子,采用RT-PCR和Western印迹方法,从mRNA和蛋白水平检测TEKT4的表达及其差异。结果:RT-PCR结果表明,TEKT4 mRNA在特发性弱精子症患者精子中的表达水平与正常男性相比显著降低(0.59±0.13 vs 0.75±0.15,t=4.325,P<0.05);Western印迹结果与RT-PCR结果一致,TEKT4蛋白在特发性弱精子症患者精子中的表达水平与正常男性相比亦显著降低(0.48±0.14 vs 0.69±0.13,t=5.939,P<0.05)。结论:特发性弱精子症患者精子中TEKT4表达水平显著降低,可能是导致弱精子症发生的重要环节之一。     

SEPT4蛋白在特发性弱精子症患者精子中的表达

目的:探讨SEPT4蛋白在特发性弱精子症发生机制中的作用。方法:采用非连续密度梯度离心分离和纯化精子,免疫细胞化学技术检测SEPT4在特发性弱精子症患者和正常男性精子中的定位及表达变化,同时用RT-PCR和W estern印迹技术,从基因和蛋白水平检测SEPT4的表达及差异。结果:免疫细胞化学表明,SEPT4定位于精子环并且在特发性弱精子症患者精子中的表达显著低于正常男性(94.75±19.95vs111.51±17.57,t=3.452,P<0.01);RT-PCR显示,特发性弱精子症患者精子中SEPT4 mRNA的表达水平与正常男性相比显著降低(0.56±0.15vs0.71±0.16,t=3.521,P<0.05);W estern印迹结果与RT-PCR结果一致,SEPT4蛋白在特发性弱精子症患者精子中的表达亦显著低于正常男性(0.48±0.20vs0.77±0.18,t=5.872,P<0.05)。结论:SEPT4在特发性弱精子症患者精子中的表达水平显著降低,可能是导致弱精子症发生的原因之一。     

碳酸酐酶Ⅱ在人睾丸、精子中的表达及其临床意义

目的:观察碳酸酐酶Ⅱ(CA2)在人睾丸、精子中的表达定位及比较CA2在生育男性和弱精子症患者精子中的表达差异。方法:通过免疫组化观察CA2在人睾丸中的定位;通过免疫荧光观察CA2在人精子中的定位;收集健康生育男性和弱精子症患者的精液标本各16份,60%Percoll分离精液标本,排除生精细胞和白细胞,采用Western印迹方法,从蛋白水平检测CA2的表达。结果:免疫组化结果显示CA2在人睾丸中特异性定位于长形精子的尾部;免疫荧光结果显示CA2在人精子的尾部有较强表达。Western印迹结果显示弱精子症患者精子中CA2的表达显著高于生育男性(1.84±0.32vs1.41±0.26,P<0.05)。结论:CA2在睾丸生精周期的长形精子阶段才表达,定位于精子的尾部。CA2在弱精子症患者精子中表达显著增高,提示其可能与精子活动力下降有关。     

弱精子症患者精子外周致密纤维2基因及蛋白的表达

目的:探讨弱精子症患者精子中外周致密纤维2(ODF2)mRNA及蛋白的表达水平,比较ODF2在健康男性与弱精子症患者中的表达差异。方法:根据WHO第5版,收集身体健康无疾病正常男性精液(n=15)和弱精子症患者精液(n=45)共60份,利用计算机辅助精液分析系统(CASA)进行精子统计,将弱精子症分为轻、中、重度3组,采用实时定量PCR和Western印迹对4组标本进行ODF2基因的表达量研究。结果:与正常组相比,轻度弱精子症组精子中ODF2基因表达无明显统计学差异(1.1120±0.5255 vs 0.6880±0.3720,P0.05);中度弱精子症组ODF2基因表达降低,有显著的统计学差异(1.1120±0.5255 vs 0.4833±0.1863,P0.05);重度弱精子症组ODF2基因表达明显降低,有极显著的统计学差异(1.1120±0.5255 vs 0.448 3±0.3408,P0.01)。Western印迹中ODF2的OD值(ODF2/β-actin)在正常组和轻、中、重度弱精子症组分别为:0.4587±0.0521、0.3261±0.0714、0.1454±0.0536和0.1227±0.0457,与正常组相比,轻度弱精子症组无明显统计学差异(P0.05),中度弱精子症组有显著的统计学差异(P0.05),重度弱精子症组有极显著的统计学差异(P0.01)。结论:ODF2基因及蛋白在弱精子症患者精子中的表达下调与精子活动力下降有密切联系,呈正相关性,提示ODF2基因可能参与精子活力的调控。     

Ropporin在正常男性和弱精子症患者精液精子中的表达差异

目的 比较Ropporin在健康男性和弱精子症患者精液精子中的表达差异.方法 收集健康男性和弱精子症患者的精液精子,为了排除生精细胞和白细胞的污染,精液标本经非连续性Percoll梯度离心和分离纯化.采用荧光定量RT-PCR和免疫细胞化学方法,从基因和蛋白水平检测精子Ropporin的表达.结果 荧光定量RT-PCR显示,Ropporin mRNA在正常男性和弱精子症患者精液精子中均有表达.与正常男性相比,Ropporin在弱精子症患者精子中的表达显著降低.免疫细胞化学结果显示,Ropporin蛋白在正常男性精子的表达水平明显高于弱精子症患者,该结果与荧光定量RT-PCR结果相一致.结论 Ropporin在弱精子症患者精子中的表达显著降低,可能是导致精子运动能力低下的原因之一.     

弱精子症患者精子中CRISP2基因及蛋白的初步研究

目的:探讨弱精子症患者精子中富含半胱氨酸的分泌蛋白2(CRISP2)mRNA及蛋白的表达水平,探讨其与精子活力的关系及相关的分子机制。方法:收集成年男性弱精子症患者精液78例,活力正常精液70例作为对照组,50% Percoll离心分离纯化后,提取精子总RNA及总蛋白,采用RT-PCR,SYBR Green实时定量PCR和Western印迹技术检测CRISP2 mRNA及蛋白相对表达量。结果:CRISP2基因在弱精子症患者精子中的表达量明显低于其在正常对照组精子中的表达量,下降达4.3倍;Western印迹发现CRISP2蛋白在弱精子症组较正常对照组下降达1.71倍,两组之间的表达量有明显的统计学差异(P<0.05)。结论:CRISP2基因及蛋白在弱精子症患者精子中的表达下调可能与精子活力下降有密切联系,提示CRISP2基因及蛋白可能成为弱精子症发病机制研究的一个分子靶标。     

溶质载体家族22成员14和精子相关抗原6在特发性弱精子症患者精子中的表达

目的:探讨溶质载体家族22成员14(SLC22A14)和精子相关抗原6(SPAG6),在特发性弱精子症患者精子中的表达情况。方法:收集精子库合格捐精者(正常对照组)和特发性弱精子症患者(弱精子症组)各50例精子样本,采用非连续密度梯度离心纯化精子,分别用RT-PCR和Western印迹检测SLC22A14、SPAG6 mRNA及蛋白的表达。结果:RT-PCR检测结果显示,弱精子症组的SLC22A14、SPAG6 mRNA表达均显著低于正常对照组(SLC22A14:0.53±0.10 vs 0.77±0.08,t=12.834,P0.01;SPAG6:0.52±0.10 vs 0.77±0.06,t=13.755,P0.01),Western印迹检测结果显示,弱精子症组的SLC22A14和SPAG6蛋白表达量同样均显著低于正常对照组,分别为(SLC22A14:0.55±0.10 vs 0.80±0.09,t=12.884,P0.01;SPAG6:0.56±0.09 vs 0.78±0.09,t=12.257,P0.01)。结论:在特发性弱精子症患者中SLC22A14和SPAG6表达降低可能是导致弱精子症的重要原因之一。     

腺苷酸激酶家族在弱精子症患者精子中的表达特征

目的:探讨腺苷酸激酶(AK)家族在弱精子症患者精子中的表达特征。方法:收集弱精子症患者和正常男性(对照组)精子样本各30例,采用RT-PCR和Western印迹技术检测AK mRNA及蛋白相对表达量。免疫荧光、免疫组化检测目标AK组织定位,并检测其与精子运动能力的关系。结果:RT-PCR显示,AK家族中AK1-AK9在精子中均有表达,且AK1、AK6和AK7的表达在特发性弱精子症患者精子中显著低于对照组。Western印迹结果表明,弱精子症患者精子中AK1、AK6和AK7蛋白与对照组相比也显著降低。在睾丸中AK1和AK7广泛表达于精原细胞、精母细胞和圆形精子细胞的细胞质中,AK6表达于精母细胞和圆形精子的细胞核中。在精子中,AK1和AK7蛋白定位于精子鞭毛部,AK6定位于精子头部。抗体阻断实验显示,AK1、AK6或AK7抗体单独与精子共孵育对精子活动率和前向运动无显著影响,3种抗体一起与精子共孵育后与对照组相比可以显著降低精子活动率[(80.5±2.4)%vs(87.6±3.3)%,P0.05]和前向运动精子百分率[(60.6±3.6)%vs(70.2±2.3)%,P0.05]。结论:AK1、AK6和AK7 mRNA及蛋白在弱精子症患者精子中的表达下调可能与精子活力下降有密切联系。     

弱精子症患者精子中电压依赖性钙通道基因表达的变化

目的:探讨弱精子症患者精子中电压依赖性钙通道基因的功能性亚单位α1mRNA表达与弱精子症的关系。方法:首先根据WHO标准用计算机辅助精液分析(CASA)方法对供精者提供的精液进行筛选,然后用Per-coll非连续梯度离心法对精子进行优化,最后应用半定量反转录酶-聚合酶链式反应(RT-PCR)技术分别检测50例正常组成熟运动精子和50例弱精子症患者精子中各类型电压依赖性钙通道的α1mRNA相对表达量。结果:与正常成熟运动精子相比,弱精子症患者精子中电压依赖性钙通道的α1B(39.40±9.47)、α1C(48.30±11.60)、α1E(3.20±0.78)、α1G(8.40±2.03)、α1H(5.70±1.47)mRNA表达的差异具有极显著意义(P<0.01)。结论:L型和/或非L型电压依赖性钙通道的基因表达异常可能是导致人类精子运动能力下降的原因之一,为人类弱精子症病因提供了新的研究方向。     

CATSPER1蛋白与特发性弱精子症关系的研究

目的:探讨CATSPER1蛋白在特发性弱精子症发病中的作用。方法:采用非连续密度梯度离心分离精子,免疫细胞化学技术检测CATSPER1蛋白在特发性弱精子症患者精子中的定位及表达变化;同时用蛋白印迹技术检测CATSPER1蛋白在正常对照组及轻度弱精子症组、中度弱精子症组和重度弱精子症组中表达的差异,进行统计学分析。结果:CATSPER1蛋白定位于精子鞭毛主段;与正常对照组相比,CATSPER1在弱精子症患者中的表达下降,差异具有显著性(t=2.188,P=0.042);CATSPER1蛋白在正常对照组、轻度弱精子症组、中度弱精子症组和重度弱精子症组中的相对含量分别为(0.806±0.266)、(0.669±0.207)、(0.505±0.214)、(0.295±0.162),与对照组相比,CATSPER1蛋白相对表达量在各弱精子症组均存在不同程度的下降,差异存在显著性(P<0.05)。前向运动精子(a+b级)百分率与CATSPER1蛋白的含量成正相关(r=0.633,P=0.000)。结论:CATSPER1蛋白表达下降或异常可能是导致特发性弱精子症发生的一个环节。     

Low expression of glycoprotein subunit 130 in ejaculated spermatozoa from asthenozoospermic men

Previous studies showed that interleukin-6 (IL-6) was expressed in human Leydig and Sertoli cells and that it inhibited sperm motility. The aim of this study was to compare the expression of IL-6, IL-6R, and GP130 in ejaculated spermatozoa between normozoospermic and asthenozoospermic men. Human spermatozoa in the semen were purified by Percoll gradient technique to separate the seminal plasma and other round cells. RT-PCR, immunocytochemistry, and Western blot were used to detect the expression of IL-6, IL-6R, and GP130 in spermatozoa. With RT-PCR, only GP130 mRNA but not IL-6 and IL-6R mRNA was expressed in human ejaculated spermatozoa. The expression of GP130 mRNA was significantly lower in asthenozoospermic men than in normozoospermic men. The protein expression of GP130 was further confirmed by both immunocytochemistry and Western blot. Again, GP130 protein levels were significantly lower in asthenozoospermic men than in normozoospermic men. The results suggested that the decreased expression of GP130 in ejaculated spermatozoa could be associated with low sperm motility in asthenozoospermic men.     

The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa

目的:研究精子特异性钙离子通道(CATSPER1)蛋白和 mRNA 在人类睾丸和射出精子中的表达,检测抗人 CATSPER1 抗体体外对人类精子前向运动的影响以初步探讨人 CATSPER1的功能及其作为免疫避孕靶点的可能性。方法:用逆转录聚合酶链反应(RT-PCR)和间接荧光免疫组化法分别检测液氮冻存人睾丸组织和12例正常精液标本中 CATSPER mRNA 和蛋白的表达。12例正常精液标本经 Percoll 不连续密度梯度离心法上游,上游后精子分别与终浓度为20 mg/mL、4 mg/mL、0.8 mg/mL 的抗人 CATSPER1抗体孵育,以抗体的储存液(0.01mol/L PBS,pH7.4)为对照,1 h、2 h、6 h 后用计算机辅助精液分析仪(CASA)分析前向运动精子百分率(WHO 际准,a 级 b 级)和快速前向运动精子百分率(WHO 标准,a 级)。结果:CATSPER1的 mRNA 存在于人类睾丸组织和射出精子中。CATSPER1蛋在人类睾丸主要表达于精子细胞,并定位于射出成熟精子尾部的主段。实验中用所有浓度的抗人 CATSPER1机体体外在1 h、2 h、6 h 时段均使前向运动精子百分率和快速前向运动精子百分率下降,都与对照组差异显著。结论:CATSPER1在人类睾九中呈减数分裂及减数分裂后式表达,存在于射出精子中的 mRNA 将是比睾丸穿刺方便、更易接受的用于研究 CATSPER1和不育检查的靶点。这些结果也提示人 CATSPER1是免疫避孕的有效靶点。     

Pentoxifylline stimulates various sperm motion parameters and cervical mucus penetrability in patients with asthenozoospermia

Pentoxifylline (PTX) was incubated in vitro with human spermatozoa to examine its effects on sperm motility characteristics and bovine cervical mucus penetrability (BCMP). Sperm motion parameters were assessed by computer-assisted motion analysis (CASA) using HTM-IVOS and BCMP was evaluated using the Penetrak kit. In vitro incubation with PTX (1  mg  ml⁻¹; 3.6  mm, 30  min) did not significantly change percentage motility, average path velocity (VAP), straight-line velocity (VSL) or beat cross frequency (BCF) of spermatozoa from normozoospermic or asthenozoospermic samples. However, it significantly increased curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and hyperactivated motility (HA), and significantly decreased linearity (LIN) of spermatozoa from both samples. Pentoxifylline was found to increase BCMP scores for spermatozoa from asthenozoospermic samples, but did not affect scores for spermatozoa from normozoospermic samples. Bovine cervical mucus penetrability (BCMP) was found to be positively and significantly correlated with the percentage motility of both non-PTX-treated and PTX-treated spermatozoa for asthenozoospermic samples. These results demonstrated that PTX enhanced several motion sperm parameters as well as BCMP in asthenozoospermic samples and suggest a potential use of the methylxanthine in infertile patients with motility defects undergoing artificial insemination.     

Capacitation-associated changes in membrane fluidity in asthenozoospermic human spermatozoa

The fertilizing potential of human spermatozoa relies on their ability to capacitate as they travel through the female reproductive tract. During this process, cholesterol is released from the plasma membrane, altering its architecture and dynamics. Using ISolate^® gradients, we obtained high (L90)- and low (L45)-quality spermatozoa from asthenozoospermic human semen samples. We tested the hypothesis that the lower fertilizing ability of asthenozoospermic L90 cells could be related to a lower ability to increase their membrane fluidity during capacitation. We assessed two sets of fluorescent probes: (i) DPH, TMA-DPH and PA-DPH which senses the hydrophobic core, cytosolic and exofacial leaflets of the bilayer, respectively and (ii) Laurdan, sensitive to the amount of water molecules intercalated between lipid moieties of the membrane (membrane hydration). Before capacitation, membrane fluidity of asthenozoospermic sperm populations was similar to the corresponding fractions of normozoospermic cells when evaluated with DPH, TMA-DPH or PA-DPH. Asthenozoospermic whole samples displayed lower plasma membrane hydration than normozoospermic cells as evidenced with Laurdan. After capacitation, asthenozoospermic L45 and L90 cells failed to increase their membrane fluidity in opposition to normozoospermic cells. Interestingly, membrane hydration significantly correlated with the main sperm motion parameters analysed, being a low membrane hydration associated with poor sperm movement. These results show that low-motility spermatozoa are unable to respond to capacitation with the necessary changes in membrane fluidity. This defect in sperm plasma membrane rheology may be responsible for their poor functional quality and low fertilizing ability.     

ICSI中不同来源精子对临床结局的影响

目的:分析不同来源精子在卵细胞胞质内单精子注射(ICSI)后的临床结局。方法:将682个ICSI治疗周期分为:射出精子组(598例)、经皮附睾精子抽吸术(PESA)得到精子组(58例)、睾丸精子获取术(TSE)得到精子组(26例),比较三组的受精率、临床妊娠率、种植率、流产率、异位妊娠率以及分娩率之间的差别。结果:TSE组受精率明显低于射出精子组和PESA组(81.06%vs87.95%,87.82%,P<0.05);射出精子组、PESA组和TSE组的妊娠率(39.46%,48.28%,34.62%)、种植率(19.80%,23.80%,18.34%)、流产率(13.13%,17.86%,11.11%)、异位妊娠率(5.51%,7.14%,11.11%)、分娩率(32.11%,36.21%,26.92%),均没有显著差异(P>0.05)。结论:虽然TSE来源精子对ICSI受精率有所影响,但射出精子和手术(TSE和PESA)来源精子对妊娠结局没有影响。     

Decreased polyunsaturated and increased saturated fatty acid concentration in spermatozoa from asthenozoospermic males as compared with normozoospermic males

The lipid composition of the sperm membrane has been shown to exert a significant effect upon the functional quality of spermatozoa. We have studied fatty acid composition of the phospholipids in spermatozoa in asthenozoospermic and normozoospermic men and determined the ratio of polyunsaturated fatty acids (PUFAs) to saturated fatty acids of spermatozoa of these two groups. Fatty acid concentration of spermatozoa was determined in 15 asthenozoospermic and eight normozoospermic semen samples by thin layer chromatography and gas chromatography. The most abundant polyunsaturated and saturated fatty acids in normozoospermic samples were docosahexaenoic acid (DHA 22 : 6 omega3, 98.5 +/- 4.5 nmol per 10(8) spermatozoa, mean +/- SE) and palmitic acid (103 +/- 17 nmol per 10(8) spermatozoa) respectively. The mean +/- SE values of DHA and palmitic acid in asthenozoospermic samples were 53.9 +/- 11.6 and 145 +/- 14.7 nmol per 10(8) spermatozoa respectively. Compared with normozoospermic samples, asthenozoospermic samples showed lower levels of PUFA and higher amount of saturated fatty acids. The mean +/- SE ratios of sperm PUFA/saturated fatty acids in asthenozoospermic and normozoospermic samples were 0.66 +/- 0.06 and 1.45 +/- 0.16 (P < 0.001) respectively. This study demonstrates that spermatozoa of asthenozoospermic men have lower levels of PUFA compared with saturated fatty acids. This may be contributory to the poor motility noted in samples from these men.     

Comparison of intracytoplasmic sperm injection outcomes between spermatozoa retrieved from testicular biopsy and from ejaculation in cryptozoospermic men

The infrequent presence of spermatozoa in cryptozoospermic men ejaculate is a limiting factor in the treatment of them. Sometimes, this consideration impels us to apply meticulous microscopic search in ejaculate or testicular sperm extraction (TESE) method. The aim of this study was to assess putative effectiveness of sperm origin, ejaculated or testicular, in cryptozoospermia treatment. In this context, were evaluated intracytoplasmic sperm injection (ICSI) outcomes in two parameters including fertilisation rate (2PN) and embryo quality, independently. We compared the outcome in two groups: patients who underwent ejaculate/ICSI and ones who underwent TESE/ICSI process. Nineteen ICSI cycles performed with testicular spermatozoa and the rest of cycles (n = 208) carried out with ejaculated spermatozoa. Result analysis showed similar fertilisation rate between testicular and ejaculated spermatozoa (respectively, 60% versus 68%, P ≥ 0.05). Also, on the other hand, embryo quality did not show significant differences between two groups, except grade A with low significance. With regard to almost equal performance of both methods in results and being invasive of TESE as surgical sperm retrieval method, the use of ejaculated sperm more than testicular sperm should be recommended in patients with cryptozoospermia whenever possible.     

Testicular versus ejaculated spermatozoa in ICSI cycles of normozoospermic men with high sperm DNA fragmentation and previous ART failures

As a part of male assessment, conventional sperm parameters including morphologic features have been dedicated as major factors influencing fertilisation and pregnancy rates in assisted reproductive technology (ART). Genomic integrity of spermatozoa has also been found to influence fertility prognosis, and hence, sperm DNA fragmentation index (DFI) has been adopted by many centres to document this entity. Despite several suggested approaches, there is lack of universal consensus on optimising fertility outcomes in males with high sperm DFI. In this context, the results from cycles using testicular spermatozoa (TESA) obtained by aspiration were compared with those of ejaculated spermatozoa (EJ) in normozoospermic subjects with high sperm DFI and previous ART failures. Clinical (41.9% versus 20%) and ongoing pregnancy rates (38.7% versus 15%) were significantly better and miscarriages were lower in TESA group when compared to EJ group. Sperm DFI should be a part of male partner's evaluation following unsuccessful ART attempts. When high DFI is detected (>30%), ICSI using testicular spermatozoa obtained by TESA seems an effective option particularly for those with repeated ART failures in terms of clinical, ongoing pregnancies and miscarriages even though conventional sperm parameters are within normal range.