不育症精子乳酸脱氢酶同工酶C_4的细胞化学定位、定量研究

本文采用酶细胞化学方法,在光镜下用积分分级计数法,全自动图像分析仪检测精子ＬＤＨ－Ｃ４活性,两法测定ＬＤＨ－Ｃ４活性显著正相关（ｒ＝０．８０１和０．７４２,Ｐ＜０．０１）;ＬＤＨ－Ｃ４酶细胞化学电镜技术观察精子ＬＤＨ－Ｃ４活性,通过计数酶点可测定ＬＤＨ－Ｃ４活性。本文检查３８例不育男性和２０例已生育男性精子ＬＤＨ－Ｃ４活性,ＬＤＨ－Ｃ４主要分布于精子ＳＴＭ及胞质,其次是顶体及质膜表面。可通过胞膜外逸到精浆,ＬＤＨ－Ｃ４在各部活性比值为５∶２∶１∶１。不育组精子ＬＤＨ－Ｃ４可能通过膜破损处外漏到精浆使精子内ＬＤＨ－Ｃ４显著减少,ＬＤＨ－Ｃ４在精子顶体亦显著减少,使不育组精子ＬＤＨ－Ｃ４活性显著低于生育组精子,提示ＬＤＨ－Ｃ４活性的定位和大小直接与生育能力相关,检测ＬＤＨ－Ｃ４活性可作为检查不育症精子质量的可靠指标。ＬＤＨ－Ｃ４与精子密度显著相关（ｒ＝０．７３７和－０．４９３,Ｐ＜０．０５）;与活动性无相关性。     

男性精浆中IL-6 和sICAM-1 与免疫性不育的关系研究

目的:探究男性精浆中白细胞介素-6(IL-6)和可溶性细胞黏附分子-1(sICAM-1)与免疫性不育的关系。方法:选择2014年6月至2015年12月我院收治不育症患者189例及100例健康体检者为研究对象,根据患者精子混合抗球蛋白反应实验(MAR)结果将不育症患者分为免疫性不育组(88例)和非免疫性不育组(101例);免疫不育组患者按照精液白细胞过氧化物酶染色情况分为免疫性阳性白细胞组(WBC≥1×10~6/mL)36例和免疫性阴性白细胞阴性组(1×10~6/m L)52例,分析并比较各组间精子质量、IL-6和sICAM-1水平。结果:免疫性不育组与非免疫性不育组患者精液精子向前运动比率、存活率均低于对照组(P0.05);免疫性不育组与非免疫性不育组精液各项参数均不存在差异(P0.05);免疫性不育组患者IL-6、sICAM-1水平均高于非免疫性不育组及对照组,差异有统计学意义(P0.05);非免疫性不育组IL-6、sICAM-1水平均高于对照组,差异有统计学意义(P0.05)。相关性分析显示,精子被Ig G黏附数比例与研究对象精液IL-6、sICAM-1水平呈正相关关系(r=0.438,0.561;P0.05);免疫性阳性白细胞组患者精液IL-6与sICAM-1水平均高于免疫性阴性白细胞组(P0.05)。结论:免疫性不育症患者精子质量下降,男性精液中IL-6和sICAM-1水平表达越高,MAR阳性率越高,男性免疫性不育发生的可能性越大。     

短暂性缺血对小脑皮质影响的组织化学研究

为了探讨全脑短暂性缺血对小脑皮质蒲肯野细胞的影响 ,实验用组织化学方法对家兔全脑缺血 5分钟 (B组 )、 10分钟 (C组 )及缺血再灌 (D、 E组 )后蒲肯野细胞的酶组织化学变化进行了观察。结果显示 ,缺血 10分钟及缺血再灌后蒲肯野细胞的 SDH、Mg2 + - ATP活性及 PAS反应均降低 (P<0 .0 5 ) ,L DH增高 (P<0 .0 1)。结果提示家兔全脑缺血 10分钟和缺血再灌可损害小脑皮质蒲肯野细胞的能量代谢酶的活性     

镉、铅对蟾蜍精巢毒作用的酶学研究

为研究镉、铅对精巢的生化毒作用机理 ,应用氯化镉、硝酸铅溶液对成年雄性蟾蜍进行腹腔染毒 (按镉计 0 .1、0 .2、0 .4、0 .8mg/ kg体重 ;按铅计 1、2、4、8mg/ kg体重 ) ,连续染毒 7d后活体解剖 ,测定分析精巢中各种酶的活性。结果显示 ,在镉染毒下 ,精巢乳酸脱氢酶 (L DH)和酸性磷酸酶 (ACP)的活性随镉染毒剂量的增加而降低 ,而碱性磷酸酶 (AL P)未发现明显变化 ;在铅染毒下 ,乳酸脱氢酶 (L DH)和碱性磷酸酶 (AL P)的活性随铅染毒剂量的增加而降低 ,而酸性磷酸酶 (ACP)未发现明显变化 ;乳酸脱氢酶 (L DH)同工酶在镉、铅染毒下则主要表现为酶带 L DH1 、L DH2 的抑制或缺失和 L DH5活性的增强。因此 ,镉、铅对蟾蜍的雄性生殖毒性机理可能与酶的活性存在着一定的关系 ,L DH可以考虑作为反映镉、铅中毒对精巢功能影响程度的一种有价值的生化指标     

Y染色体CDY1基因CDY1b复制的缺失对男性不育的影响

本研究的目的是研究中国男性中染色质蛋白的不同类型、Y染色体(CDY1)基因缺失及其对男性不育和精子形成的影响。病例对照研究241例不同的精原性损伤的不育男性和115例有生育能力的男性。利用PCR-RFLP技术采用Pvu II作为限制性内切酶,确定了CDY1a和CDY1b复制缺失的流行情况。在356个研究案例中,有93.25%存在2种CDY复制基因(CDY1a和CDY1b),其中91.2%为不育患者,97.3%为正常生育男性。研究还发现,不育患者(少精症/生精正常)中CDY1b的缺失率明显高于生育男性,分别为7%和1.7%(p=0.02)。然而,CDY1a复制的缺失是非常罕见的,并仅在1个生育男性和4个不育患者中检测到。CDY1b复制基因的缺失是独立于精子浓度影响男性不育的因素,而CDY1a基因的缺失似乎对中国人的生育能力没有影响。     

度他雄胺对大鼠附睾精子成熟和生育的影响

目的通过度他雄胺对大鼠附睾精子和生育的影响,探索调节雄性生育的睾丸后作用靶点。方法使用度他雄胺20和40 mg/(kg.d)大鼠灌胃给药,连续2周。给药结束后雄雌鼠按1∶2合笼,计算生殖指数;采用计算机辅助精子分析系统分析精子活力和形态;采用SYBR-14和PI双重荧光染色计算精子存活率;采用Elisa法测定大鼠睾酮(T)和双氢睾酮(DHT)血清浓度;采用HE染色法对各组睾丸、附睾进行组织学分析。结果度他雄胺低、高剂量组双氢睾酮浓度均显著下降,分别为0.54和0.28 nmol/L(P<0.01),精子活力明显降低,分别为39.0%和28.7%(P<0.01),畸形率分别增加为10.3%和15.6%(P<0.05),最后受孕率分别降为62.5%和38.4%。而睾酮水平和交配指数均无明显变化(P>0.05),睾丸和附睾亦无明显病理学改变。结论度他雄胺通过抑制DHT生成,影响附睾精子成熟而导致大鼠不育,为今后男性避孕和不育药物研发提供了新思路。     

游离及固定化果糖基转移酶部分酶学性质的比较研究

 从诱变、筛选的米曲霉GX0 0 10菌株所产生的果糖基转移酶 ,经过纯化和固定化操作分别制备游离酶和固定化酶 ,对两者的酶学性质进行了比较研究 .结果表明 ,两者在蔗糖转化为蔗果低聚糖的酶促反应中 ,最适pH为 5 5,在pH5 0～ 7 5之间酶活性相对稳定 .游离酶和固定化酶的适宜温度范围分别是 4 5～ 52℃和 4 0～ 55℃ .在 55℃保温 60min ,酶活性保存率分别是 61 6%和 87 5% .固定化酶的热稳定性提高 .0 1mmol LHg2 +和 1mmol LAg+能完全抑制游离酶的活性 ,但只能部分抑制固定化酶的活性 ,1mmol L的Ti2 +能完全抑制两者的活性 .以蔗糖为底物时 ,游离酶的米氏常数Km=2 15mmol L ,而固定化酶Km =386mmol L .游离酶只能使用一次 ,固定化酶反复使用 54次后 ,剩余活力为 55 2 % .用 55% (W V)蔗糖溶液与固定化酶在pH5 0 ,4 6℃下作用 12h ,可获得61 5% (总低聚糖 总糖 )产物 ,其中蔗果五糖含量达到 7 2 % .     

衣原体感染不孕不育患者血清中自身抗体的检测

目的:探讨衣原体感染与血清中抗精子抗体(AsAb)、抗心磷脂抗体(ACA)、抗子宫内膜抗体(EMAb)之间的关系.方法:用ELISA法检测120例衣原体感染不育男性(男性衣原体感染组)和55例正常生育男性(男性正常时照组)的血清中AsAb、ACA;检测142例衣原体感染不孕女性(女性衣原体感染组)和60例正常生育女性(女性正常对照组)的血清中AsAb、ACA、EMAb.结果:男、女衣原体感染组AsAb、ACA检出率高于相应对照组,具有显著性差异(P值均＜0.05);女性衣原体感染组EMAb检出率高于女性对照组,具有显著性差异(P＜0.05).结论:生殖道衣原体感染和与不孕不育有着密切的相关性,且AsAb、ACA、EmAb的产生与衣原体感染有关.     

邻苯二甲酸二丁酯(DBP)胁迫对三疣梭子蟹亚急性毒性

研究不同浓度邻苯二甲酸二丁酯(DBP)对三疣梭子蟹相关酶活的影响及组织损伤。设置了三个处理浓度(0.03 mg/L、0.2 mg/L和0.4 mg/L)分别在第0、5、10、15、20 d进行取样。结果表明,随着DBP浓度的增大,三疣梭子蟹死亡率升高。在0.03 mg/L浓度组胁迫下,血清超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和碱性磷酸酶(AKP)活性呈现升高-下降交替波动,过氧化物酶(POD)活性在第10 d低于对照组(P<0.05),其余时间与对照组持平,酸性磷酸酶(ACP)活性在第5 d较对照组显著升高(P<0.05),此后一直保持对照组水平;在0.2 mg/L浓度组胁迫下,血清SOD活性呈现先升高后下降的趋势,POD活性变化不明显,CAT活性先下降后升高,此后恢复到对照组水平,ACP活性在第15 d出现最高值,其余时间与对照组持平,AKP活性呈现先升高后恢复的波动趋势;0.4 mg/L浓度组SOD、CAT和AKP活性呈现先升高后下降的趋势,POD活性和ACP活性在第20 d较对照组低(P<0.05),其余时间与对照组持平。组织切片观察可见,DBP胁迫损伤了三疣梭子蟹肝胰腺和鳃组织,DBP浓度越大,胁迫时间越长,损伤越明显。     

萌发绿豆种子中的一种大豆胰蛋白酶抑制剂钝化酶

通过 30 %～ 6 0 % (NH4 ) 2 SO4 分级沉淀、DEAE_SepharoseCL_6B离子交换层析、SephacrylS_2 0 0凝胶过滤层析和WatersAP_1离子交换层析 ,从萌发的绿豆 (Vignarabiata (L .)Wilczek)种子中分离纯化出一种可降解大豆胰蛋白酶抑制剂 (STI)的蛋白酶。SDS_PAGE测定该酶的分子量为 2 9.8kD。该酶催化降解STI的Km 值为 76 9.2BAEE/mL ,Vmax为 115 .3BAEE·mL-1·min-1。该酶在 5 0℃、pH 8.0、相对酶活力 5 0 0 0BAEE/mL和 4h的反应时间时可将脱脂大豆粉中的STI活性钝化 90 .91%。该酶在温度低于 5 0℃及pH 6 .5～ 8.5时能保持其活性。     

正常人及不育者精子核DNA的定量研究

用流式细胞术,Feulgen显微分光光度法,荧光显微分光光度法测定正常人精子核DNA相对含量:结果表明,上述方法所测得的精子核DNA相对含量稳定,变化范围小。同时用Feulgen显微分光光度法测定不育者精子核DNA相对含量。显示不育者精子核DNA相对含量高于正常人,提示精子核DNA核蛋白复合物异常可能是某些男性不育症的原因。此项研究为诊断男性不育症提供了新方法,在男性学精子核分子研究方面提供了依据。     

DAZL 260A > G and MTHFR 677C > T variants in sperm DNA of infertile Indian men

DAZL (deleted in azoospermia-like) 260A > G and MTHFR (methylene tetrahydrofolate reductase) 677C > T are two important autosomal variants associated with impaired spermatogenesis. In this study, we investigated DAZL 260A > G and MTHFR 677C > T variants in sperm DNA and their frequency in oligozoospermic infertile men of Indian origin. The study on sperm DNA was performed, since it is more prone to oxidative stress-induced damage and mutation. One hundred oligozoopsermic infertile men having normal chromosomal complement with intact Y chromosome and 100 age- and ethnically-matched fertile controls were investigated for these variants in their sperm genome. Spermatozoa were separated by gradient centrifugation and DNA was isolated and analyzed for the single nucleotide polymorphisms (SNPs) by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed no significant differences in the frequency of DAZL AG (P = 0.58) and MTHFR CT (P = 0.44) between oligozoospermic infertile men and controls. However, 8% (8/100) oligozoospermic infertile men harbored both the variants and showed significantly (P < 0.0001) lower sperm count (3.28 +/- 1.1 vs 12.50 +/- 4.09) compared to infertile men with either of the single variant. None of the fertile controls showed the presence of the both variants. In conclusion, the combined effect of both DAZL 260A > G and MTHFR 677C > T variants may have role in compromised sperm count. However, further studies are required to find the pathological role of these combined variants in male infertility.     

精子端粒长度与特发性男性不育相关

端粒是真核生物染色体末端的多功能特异性DNA-蛋白结构,覆盖在染色体末端,保护基因组的稳定性。端粒在减数分裂过程中起到了十分重要的作用,协助染色体配对、联会、同源重组和分离。精子中的端粒可能在精子的受精能力和胚胎发育中起到重要作用。近年来,端粒与生殖的相关性研究成为一个新的热点,但精子端粒与男性不育间的相关性并不明确。本文采用实时荧光定量PCR方法检测中国特发性男性不育人群(126例)和正常可育男性人群(138例)的精子相对端粒长度,结果发现,特发性男性不育病例的精子平均相对端粒长度(2.894±0.115)低于正常对照组(4.016±0.603),差异具有统计学意义(P=5.097×10^-5);并且精子相对端粒长度与精子密度、精子总数和精子活力都有显著的相关性：精子数量较多和/或精子活力较高,精子相对端粒长度较长。研究结果提示,在中国人群中,精子端粒长度与特发性男性不育具有相关性,精子的端粒长度可能影响精子发生和精子的功能,精子端粒的缩短导致精子数目及活力的降低从而导致男性不育。     

Study of the effects of oral zinc supplementation on peroxynitrite levels, arginase activity and NO synthase activity in seminal plasma of Iraqi asthenospermic patients

Background

Low concentrations of nitric oxide (NO) are necessary for the biology and physiology of spermatozoa, but high levels of NO are toxic and have negative effects on sperm functions. Although several studies have considered the relationship between infertility and semen NO concentrations, no study on the effects of asthenospermia treatments such as oral zinc supplementation on concentrations of NO, which are important in fertility, has been reported. Studies have shown that oral zinc supplementation develops sperm count, motility and the physical characteristics of sperm in animals and in some groups of infertile men. The present study was conducted to study the effect of zinc supplementation on the quantitative and qualitative characteristics of semen, along with enzymes of the NO pathway in the seminal plasma of asthenospermic patients.

Methods

Semen samples were obtained from 60 fertile and 60 asthenozoospermic infertile men of matched age. The subfertile group was treated with zinc sulfate; each participant took two capsules (220 mg per capsule) per day for 3 months. Semen samples were obtained (before and after zinc sulfate supplementation). After liquefaction of the seminal fluid at room temperature, routine semen analyses were performed. The stable metabolites of NO (nitrite) in seminal plasma were measured by nitrophenol assay. Arginase activity and NO synthase activity were measured spectrophotometrically.

Results

Peroxynitrite levels, arginase activity, NO synthase activity and various sperm parameters were compared among fertile controls and infertile patients (before and after treatment with zinc sulfate). Peroxynitrite levels and NO synthase activity were significantly higher in the infertile patients compared to the fertile group. Conversely, arginase activity was significantly higher in the fertile group than the infertile patients. Peroxynitrite levels, arginase activity and NO synthase activity of the infertile patient were restored to normal values after treatment with zinc sulfate. Volume of semen, progressive sperm motility percentage and total normal sperm count were increased after zinc supplementation.

Conclusions

Treatment of asthenospermic patients with zinc supplementation leads to restored peroxynitrite levels, arginase activity and NO synthase activity to normal values and gives a statistically significant improvement of semen parameters compared with controls.

Trial registration

ClinicalTrials.gov identifier: NCT01684059     

Comparison of sperm binding potential of uninseminated, inseminated-unfertilized, and fertilized-noncleaved human oocytes under hemizona assay conditions.

In this study, human oocytes obtained after ovarian hyperstimulation for in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) were utilized to evaluate sperm/zona pellucida binding potential. Three groups of oocytes were evaluated: 1) uninseminated; 2) inseminated-unfertilized; and 3) fertilized-uncleaved. All oocytes had undergone germinal vesicle breakdown at the time of retrieval and were salt-stored (pH 7.2) for not more than 30 days. Sperm binding was recorded under hemizona assay (HZA) conditions using spermatozoa from eight fertile men (HZA control) and from 1) four teratozoospermic (HZA test) and 2) four normozoospermic (HZA test) infertile men. First, the mean numbers (+/- SD) of sperm tightly bound for fertile controls and teratospermic men to hemizonae from uninseminated oocytes were 69.7 +/- 16 and 14.5 +/- 7, respectively (P = 0.02). Likewise, hemizonae from uninseminated oocytes bound 102.0 +/- 19 and 114.0 +/- 28, respectively, for fertile controls and normospermic men (P = 0.5). Second, hemizonae obtained from inseminated-unfertilized IVF oocytes bound 44.2 +/- 12 and 19.7 +/- 6 for fertile controls and teratospermic men, respectively (P = 0.02). This category of oocytes bound 100.5 +/- 7 and 108.5 +/- 11 sperm, respectively, for fertile controls and normospermic semen (P = 0.3). Third, HZA results of fertilized but uncleaved oocytes showed a mean number of tightly bound sperm of 6.0 +/- 4 compared with 65.0 +/- 1 in control, uninseminated oocytes using fertile sperm. These results demonstrate that uninseminated and inseminated-unfertilized human oocytes, salt-stored under controlled pH conditions, give reliable information regarding sperm binding potential under HZA conditions.     

Comparative proteomic network signatures in seminal plasma of infertile men as a function of reactive oxygen species

Background

Reactive oxygen species (ROS) plays a major role in the pathology of male infertility. It is an independent biomarker of sperm function. Seminal plasma is a natural reservoir of antioxidants responsible for the nourishment, protection, capacitation, and motility of sperm within the female reproductive tract resulting in successful fertilization and implantation of the embryo. A comparative proteomic analysis of seminal plasma proteins from fertile men and infertile men with varying levels of ROS was carried out to identify signature proteins involved in ROS-mediated reproductive dysfunction.

Methods

A total of 42 infertile men presenting with infertility and 17 proven fertile donors were enrolled in the study. ROS levels were measured in the seminal ejaculates by chemiluminescence assay. Infertile men were subdivided into Low ROS (0–<93 RLU/s/10⁶ sperm; n = 11), Medium ROS (>93–500 RLU/s/10⁶ sperm; n = 17) and High ROS (>500 RLU/s/10⁶ sperm; n = 14) groups and compared with fertile men (4–50 RLU/s/10⁶ sperm). 4 subjects from fertile group and 4 each from the Low, Medium and High ROS were pooled. 1D gel electrophoresis followed by in-gel digestion and LC/MS–MS in a LTQ-Orbitrap Elite hybrid mass spectrometer system was used for proteome analysis. Identification of differentially expressed proteins (DEPs), their cellular localization and involvement in different pathways were examined utilizing bioinformatics tools.

Results

The results indicate that proteins involved in biomolecule metabolism, protein folding and protein degradation are differentially modulated in all three infertile patient groups in comparison to fertile controls. Membrane metallo-endopeptidase (MME) was uniformly overexpressed (>2 fold) in all infertile groups. Pathway involving 35 focus proteins in post-translational modification of proteins, protein folding (heat shock proteins, molecular chaperones) and developmental disorder was overexpressed in the High ROS group compared with fertile control group. MME was one of the key proteins in the pathway. FAM3D was uniquely expressed in fertile group.

Conclusion

We have for the first time demonstrated the presence of 35 DEPs of a single pathway that may lead to impairment of sperm function in men with Low, Medium or High ROS levels by altering protein turn over. MME and FAM3D along with ROS levels in the seminal plasma may serve as good markers for diagnosis of male infertility.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9094-5) contains supplementary material, which is available to authorized users.     

Human male infertility may be due to a decrease of the protamine P2 content in sperm chromatin

Basic chromosomal proteins were extracted from the sperm of fertile and infertile human males. The relative proportions of protamine 1, 2, and 3 were determined by scanning microdensitometry following electrophoresis of total protamine in polyacrilamide gels. The findings were as follows: (1) The proportion of protamine P(2 + 3) in sperm obtained from infertile males was lower than that in fertile males. (2) Protamine P(2 + 3) in infertile human males showed reduced affinity to DNA. The possibility that some cases of human male infertility may be due to mutation within the protamine P2 gene is discussed. © 1993 Wiley-Liss, Inc.     

Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen

Cryopreservation of human semen can cause DNA damages, which compromise the fertilization and normal embryo development. The present study showed that the antioxidant resveratrol prevents these damages both in fertile and infertile men. The addition of ascorbic acid before cryopreservation can reduce DNA damages only in infertile men. Although further studies are needed, the present work showed that resveratrol could be considered in human cryopreservation procedures to avoid/minimize DNA damages and preserve sperm integrity.     

Is there a relationship between sperm chromosome abnormalities and sperm morphology?

This review explores the relationship between sperm chromosomal constitution and morphology. With the advent of techniques for obtaining information on the chromosome complements of spermatozoa, this relationship has been studied in fertile men and in men with a high frequency of chromosomal abnormalities. Using human sperm karyotype analysis, no relationship between sperm chromosome abnormalities and morphology was found in fertile men, translocation carriers or post-radiotherapy cancer patients. Fluorescence in situ hybridization (FISH) analysis has not generally revealed a specific association between morphologically abnormal sperm and sperm chromosome abnormalities, but has indicated that teratozoospermia, like other forms of abnormal semen profiles (aesthenozoospermia, oligozoospermia) is associated with a modest increase in the frequency of sperm chromosome abnormalities. However, FISH studies on some infertile men and mouse strains have suggested that certain types of morphologically abnormal spermatozoa, such as macrocephalic multitailed spermatozoa, are associated with a very significantly increased frequency of aneuploidy. Thus, there may be an association between sperm morphology and aneuploidy in infertile men with specific abnormalities.     

Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.

The objective of this study was to identify those immunodominant sperm antigens recognized by antisperm antibodies (ASA) in the serum samples of infertile men and women. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing or nonequilibrium pH gradient electrophoresis, followed by PAGE. Serum samples from 15 infertile male subjects and 6 infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT) were analyzed by Western blotting followed by enhanced chemiluminescence (ECL). Serum samples from 10 fertile subjects (5 males and 5 females) that were ASA negative by IBT were used as controls. The ECL blots were analyzed by computer scanning to compare the immunoreactivity between serum samples from fertile and infertile subjects and to identify the antigens unique to the sera of the infertile subjects; 98 sperm auto- and iso-antigenic protein spots were recognized by sera from infertile males and females but not from fertile subjects. Based on vectorial labeling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility.