Effect of sperm pooling with seminal plasma collected in breeding or nonbreeding season on Saanen goat sperm cryosurvival

The aim of this study was to determine the effects of both the removal of seminal plasma (SP) and the pre‐freezing addition of seminal plasma collected during the breeding or nonbreeding season on goat sperm survival after thawing. Semen samples were pooled. One aliquot of pooled semen was used as a control group. Four aliquots were then centrifuged, and the SP was removed in Group I, pipetted but not removed in Group II, removed and then pooled for animals collected in the breeding season in Group III and removed and pooled for animals collected in the nonbreeding season in Group IV. Group samples were frozen and then were assessed for rates of sperm motility, plasma membrane functional integrity hypo‐osmotic swelling test (HOST), defective acrosomes (FITC‐PSA), DNA fragmentation (TUNEL) and mitochondrial membrane damage (Rhodamine 123). The results showed that pre‐freezing addition of SP collected in breeding season maintained post‐thaw sperm characteristics at 0 hr better than SP removal group, but removing seminal plasma showed positive effects on spermatozoa, as incubation time increased to 5 hr. In conclusion, the pre‐freezing addition of seminal plasma did not maintain post‐thaw goat sperm characteristics as successfully as in the groups with seminal plasma removed after an incubation period.     

Evaluation of deoxyribonuclease activity in seminal plasma of ejaculated chicken semen

Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents. Methods: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40℃for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light.Results: The DNA substrate was completely diminished after incubation with seminal plasma. However, the sub-strate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L-5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents. Conclusion: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex. ( Asian JAndro12003 Sep; 5: 213-216)     

精索静脉曲张不育患者的精液常规、精子动态参数和精浆生化指标分析

目的观察精索静脉曲张（VC）伴不育患者的精液质量和精浆生化水平的变化。方法利用计算机辅助精液分析系统（CASA）及酶免法分别检测52例VC不育患者、85例非精索静脉曲张不育患者和21例正常生育男性的精液常规、精子动态参数以及精浆酸性磷酸酶、精浆锌（Zn）、果糖、α-糖苷酶的含量。结果与对照组相比,VC组患者A级精子率、（A＋B）级精子率、精子直线速度（VSL）、直线性（LIN）、侧摆幅度（ALH）、精浆锌含量、α-糖苷酶的含量与对照组相比显著下降（P〈0.05）。与非VC不育组相比,VC不育组（A＋B）级精子率、精子直线速度（VSL）、侧摆幅度（ALH）、精浆锌含量、α-糖苷酶的含量也明显降低（P〈0.05）。并且随着精索静脉曲张程度的增加,VC患者A级精子率、（A＋B）级精子率、精子曲线速度VCL、直线速度VSL、直线性LIN、平均路径速度VAP、前向性STR、侧摆幅度ALH、精浆中锌含量、α-糖苷酶的含量有下降趋势。结论 VC能够降低精子活动率、精子动态活动能力以及改变了精浆微环境,从而导致男性生育力下降。     

Influence of seminal plasma on cryopreservation of human spermatozoa in a biological material-free medium: study of normal and low-quality semen

The objective was to evaluate the efficiency of a biological material-free medium and the role of seminal plasma (SP) in the cryopreservation of human spermatozoa. Normal semen samples and low-quality semen samples were used for this study. After centrifugation of 300 microL fractions of whole semen, pellets were resuspended either in autologous SP or in a chemically defined medium (BM) supplemented or not with 3% bovine serum albumin (BSA); after 15 min at 37 degrees C, the samples were diluted (V/V) with cryoprotective medium (30 mM NaCl; 22 mM sodium citrate, 19.4 mM fructose; 80 mM glutamine; 14%, V/V, glycerol) and maintained for 15 min at room temperature before freezing. Assessment of viability and motility was performed using fresh semen (T0), after centrifugation and resuspension prior to adding the cryoprotectant (T15), after adding the cryoprotectant (T30) and after freezing and thawing (Tpost). In all three resuspending media used, sperm viability and motility (forward and total) decreased (p < 0.05) during both the equilibration period especially before addition of the cryoprotective medium (between T0 and T15) and during the freeze-thaw process comparison between T30 and Tpost. The recovery of viable and motile spermatozoa (post-thaw values/values of fresh samples) was higher (p < 0.05) in normal semen than in low-quality semen. In both groups, the recovery was slightly, but significantly, higher with SP than with BM and the presence of BSA has no beneficial effect. To conclude, these data suggest that SP may reduce the deleterious effects of cryopreservation. Nevertheless cryopreservation of spermatozoa in a medium containing neither SP nor biological substances could offer an acceptable cryoprotection of spermatozoa to be used in assisted fertilization procedures, especially for intracytoplasmic sperm injection.     

Sialoglycoproteins of ejaculated human spermatozoa and seminal plasma

Ejaculated human sperm were found to possess three major sialoglycoproteins with molecular weights of 30 000, 14 000 and 12 000 and one minor species of 18 000. Liquefied seminal plasma from normal donors contain two major sialoglycoproteins with molecular weights of 17 000 and 15 000 and two minor species of 70 000 and 54 000. In contrast, the liquefied sperm-free semen of vasectomized men had two major species of sialoglycoproteins of 20 000 and 18 000 daltons plus two minor species of 54 000 and 40 000.     

The presence of seminal plasma,especially derived from stallion semen,helps preserve chilled Asian elephant (Elephas maximus) sperm motility

The effects of seminal plasma (SP), derived from autologous, homologous and heterologous species (stallion, boar and dog) on chilled Asian elephant sperm quality, were determined. Semen was collected from eight males and samples with ≥30% motile spermatozoa were used in the study. Semen was diluted with Tris–glucose–egg yolk extender, supplemented with different SP types and preserved at 4°C for 48 hr. Experiment 1 (n = 31), showed that the presence of SP (autologous) helped to preserve sperm quality in terms of sperm motility and acrosome integrity (p < .05). Homologous SP did not result in better sperm quality than autologous SP. Heterologous SP from stallion provided higher sperm motility and velocities compared to autologous SP (p < .05). Experiment 2 (n = 14) determined the effect of different SP from four stallions. All stallion SP gave higher (p < .05) results for motile spermatozoa and sperm velocities than autologous SP. In conclusion, the presence of SP helps preserve Asian elephant sperm quality and stallion SP supports the motility of Asian elephant spermatozoa during cold storage.     

Glycerophosphocholine in seminal plasma of fertile and infertile men

Glycerophosphocholine (GPC) was measured in seminal plasma from 65 fertile men, 276 infertile men and 10 men before and after vasectomy, using a new enzymatic method. Extra-epididymal excretion of GPC accounted for 30% of the total seminal levels of GPC. From a diagnostic point of view, GPC determination did not appear to be a specific tool which could discriminate between secretory and excretory azoospermia. Although the seminal content of GPC was related positively to the total sperm count in both fertile and infertile men, there was an inverse relationship between the level of GPC and sperm motility when considering classes displaying the same total sperm count. This was observed in all classes from infertile men as well as in fertile men with a total sperm count lower than 200 x 10(6) sperm/ejaculate. These results suggest a possible role of GPC in the regulation of human sperm motility, which warrants further investigation.     

精浆弹性蛋白酶水平与精液主要参数和精子功能的相关性分析

目的:探讨精浆弹性蛋白酶与精液主要参数和指标的关系。方法:用酶联免疫吸附法(ELISA)检测精浆中的弹性蛋白酶,按照WHO人类精液实验室手册要求进行精液常规分析、精子形态分析,检测精子顶体酶活性、精浆抗体(AsAb)、解脲支原体等,分析弹性蛋白酶与男性不育相关因素的关系。结果:209例男性不育患者中,43例患者精浆弹性蛋白酶≥290ng/ml,设为炎症组;166例患者精浆弹性蛋白酶<290ng/ml,设为非炎症组。炎症组的精子密度、精子活动率、a+b级活力精子率、精子顶体酶阳性率均低于非炎症组(P<0.05);而精子畸形率、精浆抗体(AsAb)、解脲支原体阳性率均高于非炎症组(P<0.05)。两组的精液量、PH值和液化时间差异无统计学意义。结论:精浆弹性蛋白酶水平与精液质量有密切的关系,生殖道感染是导致男性不育的重要原因。     

Leptin exists in tubuli seminiferi and in seminal plasma

Leptin is a 167-amino acid protein that stimulates gonadotrophin-releasing hormone secretion and exerts indirect effects on the gonads via neuropeptide Y, NPY. Recent research has suggested that leptin may also have an effect on testosterone secretion. To investigate the role of leptin in reproduction, leptin in testicular tissue and seminal plasma was examined in relation to leptin in serum, semen sample qualities and vasectomy. Seminal plasma and serum of 64 infertility patients, and 15 individuals after vasectomy, were assayed for leptin using a competitive 'in house' radioimmunoassay. The concentration of leptin in seminal plasma was significantly lower in the 'normal' semen sample group than in the 'pathological' group (Mean +/- SEM; 1.45 +/- 0.18 vs. 3.19 +/- 0.57 ng ml-1; P < 0.05), and showed a significantly negative correlation with percentage of motile spermatozoa (r = -0.46; P = 0.0005) and with the velocity straight line, VSL, (r = -0.30; P = 0.029). In contrast, leptin concentration in serum did not show any relationship with the spermiogram parameters. In testicular tissue, leptin was preferentially found within the tubuli seminiferi using anti-leptin polyclonal antibody, Ob A-20 Sc 842. The amount of leptin per ejaculate did not significantly change after vasectomy, and was not correlated to fructose, zinc or neutral alpha glucosidase in seminal plasma (P > 0.05). These results suggest that the amount of leptin in the genital tract, including the tubuli seminiferi, may influence the mechanisms involved in the motility development of spermatozoa.     

精浆转铁蛋白含量与不育的关系

为探讨精浆转铁蛋白 (Tf)含量与男性生育力的关系 ,采用速率散射比浊法 ,对 2 0例正常生育男性和 96例不育男性精浆Tf含量进行了测定 ,同时做精子密度、精子活动率及精子顶体完整率 (PIA)分析。结果表明少精子组 (精子密度 <2 0× 10 6/ml)精浆Tf含量较生育组与正常密度组 (精子密度≥ 2 0× 10 6/ml)显著低 (P <0 .0 1) ;精浆Tf含量与精子活动率无关 ;正常精子密度的不育患者精子PIA <80 %组精浆Tf的含量显著低于PIA≥ 80 %组(P <0 .0 1)。提示精浆Tf含量的下降与男性生育力有关     

Incubation of frozen-thawed llama sperm with seminal plasma

Seminal plasma is intimately connected to sperm physiology and particularly in South American Camelids, has demonstrated to be involved in multiple physiological reproductive events. Different percentages of seminal plasma (0%, 10% and 50%) were added to thawed llama semen samples with the objective of evaluating the interaction with cryopreserved sperm over time (0, 1.5 and 3 hr at 37°C). A total of 20 ejaculates from five adult llama males (n = 5; r = 4) were evaluated. A significant decrease in sperm motility, membrane function and live sperm was observed in all thawed samples (0%, 10% and 50%) at 0 hr when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > .05), but a significant increase in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples versus raw semen. When evaluating thawed samples over time, a significant decrease of motility and membrane function was observed, while the percentages of total live sperm were preserved over the 3 hr of incubation in all final concentrations evaluated. To conclude, the addition of 10% or 50% of seminal plasma was incapable of preserving motility or membrane function of frozen-thawed llama sperm during 3 hr of incubation.     

Proteomic analysis of seminal plasma from asthenozoospermia patients reveals proteins that affect oxidative stress responses and semen quality

Asthenozoospermia （AS） is a common cause of human male infertility. In one study, more than 80% of the samples from infertile men had reduced sperm motility. Seminal plasma is a mixture of secretions from the testis, epididymis and several male accessory glands, including the prostate, seminal vesicles and Cowper＇s gland. Studies have shown that seminal plasma contains proteins that are important for sperm motility. To further explore the pathophysiological character of AS, we separated the seminal plasma proteins from AS patients and healthy donors using sodium dodecyl sulfate polyacrylamide gel electrophoresis and in-gel digestion, and then subjected the proteins to liquid chromatography-mass spectrometry （LC-MS/MS） analysis. A total of 741 proteins were identified in the seminal plasma, with a false discovery rate of 3.3%. Using spectral counting, we found that 45 proteins were threefold upregulated and 56 proteins were threefold downregulated in the AS group when compared with the control. Most of these proteins originated from the epididymis and prostate. This study identified a rich source of biomarker candidates for male infertility and indicates that functional abnormalities of the epididymis and prostate can contribute to AS. We identified D J-1--a protein that has been shown elsewhere to be involved in the control of oxidative stress （OS）-as a downregulated protein in AS seminal plasma. The levels of D J-1 in AS seminal plasma were about half of those in the control samples. In addition, the levels of reactive oxygen species were 3.3-fold higher in the AS samples than in the controls. Taken together, these data suggest that downregulation of DJ-1 is involved in OS in semen, and therefore affects the quality of the semen.     

Comparison of two cooling protocols for llama semen: with and without collagenase and seminal plasma in the medium

Seminal plasma (SP) of South American Camelids could interfere with the interaction of spermatozoa with the extenders; therefore it becomes necessary to improve semen management using enzymatic treatment. Our objective was to compare two cooling protocols for llama semen. Twelve ejaculates were incubated in 0.1% collagenase and then were divided into two aliquots. One was extended in lactose and egg yolk (LEY) (Protocol A: collagenase and SP present). The other aliquot was centrifuged, and the pellet was resuspended in LEY (Protocol B: collagenase and SP absent). Both samples were maintained at 5°C during 24 hr. Routine and DNA evaluations were carried out in raw and cooled semen. Both cooling protocols maintained sperm viability, membrane function and DNA fragmentation, with Protocol A showing a significantly lowered total and progressive motility (p < .05) and Protocol B showing a significant increase in chromatin decondensation (p < .05). Protocol A avoids centrifugation, reducing processing times and making application in the field simpler. However, as neither protocol showed a significant superiority over the other, studies should be carried out in vivo to evaluate the effect on pregnancy rates of the presence of collagenase and SP in semen samples prior to either cooling or freeze‐thawing.     

Effects of seminal plasma from normal and asthenozoospermic men on the progressive motility of washed human sperm

Human seminal plasma stimulated the progressive motility of human sperm in a dose- and time-related manner. Serum exhibited a similar capacity for stimulation. Seminal plasma from different normozoospermic men showed marked variation in its capacity to stimulate sperm motility. The stimulatory effect was maintained after heat-treatment, indicating the action of a low molecular weight substance or metal ion. No differences could be observed in the capacity for stimulation between seminal plasma from normozoospermic (n = 23) and asthenozoospermic (n = 22) men, when tested at the same dilution and under identical conditions. It is concluded that in general, differences in seminal plasma composition cannot account for the reduced sperm motility in asthenozoospermic men. Furthermore, the stimulatory effects of seminal plasma may be a property shared by other biological fluids.     

Altered malondialdehyde, protein carbonyl and sialic acid levels in seminal plasma of microscopically abnormal semen

Microscopically abnormal (n = 26) semen showed significantly higher levels of malondialdehyde (MDA), protein carbonyl (PC) and protein-bound sialic acid (SA) in seminal plasma as compared with normal semen (n = 24). The percentage of nonmotile spermatozoa showed significant (P < 0.01) positive correlation with MDA (r = 0.5) and PC (r = 0.49). Sperm counts showed a significant negative correlation with MDA (r = -0.63, P < 0.001) level of seminal plasma. SA correlated (r = 0.56, P < 0.01) with MDA. The receiver operating characteristic (ROC) curve of MDA and SA showed that MDA of 3.15 micromol l(-1) and SA of 3.85 micromol l(-1) were optimum cut-off limits to discriminate abnormal semen from normal. In conclusion, high SA might be a protective response against prevailed oxidative stress in abnormal semen. Seminal plasma MDA and SA may act as potential markers of abnormal semen.     

影响人类精子耐冻性的蛋白质分析

目的:利用比较蛋白质组学技术研究影响人类精子耐冻性的蛋白质。方法:将10例供精志愿者的31份精液标本依据冷冻复苏率的不同分为高冷冻复苏率组和低冷冻复苏率组,应用二维电泳技术和质谱分析鉴定技术寻找两组标本的精子蛋白和精浆蛋白在表达量上的差异。结果:在精子蛋白和精浆蛋白中共发现了22个差异表达蛋白。其中精浆蛋白12个,分别为细胞角蛋白1、神经元导向因子3、Rho2样蛋白、Krueppel样因子10、FERM域蛋白8、GMP合成酶、鸟嘌呤结合蛋白G、溶质载体家族12、ATP结合盒亚家族D1、6-磷酸果糖-2-激酶、肌钙蛋白C、肌球蛋白轻链;精子蛋白9个,分别为卷曲螺旋结构域蛋白113、RAF原癌基因丝氨酸/苏氨酸蛋白激酶、Ras相关蛋白Rab-31、α微管蛋白3C/D、锌指蛋白879、胆固醇26-羟化酶、DNA修复和重组蛋白、细胞角蛋白73、钙磷蛋白;精浆和精子共有蛋白1个,为细胞周期依赖性蛋白激酶1。文献调研发现这些差异蛋白主要与精子的能量代谢、成熟、运动、DNA修复等有关。结论:鉴定了多种可能影响人类精子耐冻性的蛋白质,为以后从分子水平上揭示精子冷冻复苏原理提供了依据。     

精索静脉曲张不育患者的精浆生化分析

目的 探讨精索静脉曲张不育患者精浆中酸性磷酸酶、果糖、锌和α-糖苷酶水平的变化.方法 分别检测120例精索静脉曲张不育患者、180例非精索静脉曲张不育患者和36例正常男性的精浆中酸性磷酸酶、果糖、锌和α-糖苷酶含量.结果 精索静脉曲张不育组和非精索静脉曲张不育组精浆中酸性磷酸酶含量均显著低于正常对照组（P＜0.01）,但精索静脉曲张不育组和非精索静脉曲张不育组之间的差异无显著性意义（P＞0.05）;各组精浆果糖活性无显著性差异（P＞0.05）;精浆中锌和α-糖苷酶含量随精索静脉曲张程度的增加而降低,且明显低于正常对照组（P＜0.05）,但与非精索静脉曲张不育组之间的差异无显著性意义（P＞0.05）.结论 精索静脉曲张可通过某些因素引起精浆中酸性磷酸酶、锌和α-糖苷酶含量降低,从而造成男性不育.     

Effect of transient scrotal hyperthermia on sperm parameters,seminal plasma biochemical markers,and oxidative stress in men

In this experimental prospective study, we aimed to analyze the effect of transient scrotal hyperthermia on the male reproductive organs, from the perspective of sperm parameters, semen plasma biochemical markers, and oxidative stress, to evaluate whether different frequencies of heat exposure cause different degrees of damage to spermatogenesis. Two groups of volunteers (10 per group) received testicular warming in a 43°C water bath 10 times, for 30 min each time: group 1: 10 consecutive days; group 2: once every 3 days. Sperm parameters, epididymis and accessory sex gland function, semen plasma oxidative stress and serum sex hormones were tested before treatment and in the 16-week recovery period after treatment. At last, we found an obvious reversible decrease in sperm concentration (P = 0.005 for Group 1 and P= 0.008 for Group 2 when the minimums were compared with baseline levels, the same below), motility (P = 0.009 and 0.021, respectively), the hypoosmotic swelling test score (P = 0.007 and 0.008, respectively), total acrosin activity (P = 0.018 and 0.009, respectively), and an increase in the seminal plasma malondialdehyde concentration (P = 0.005 and 0.017, respectively). The decrease of sperm concentration was greater for Group 2 than for Group 1 (P = 0.031). We concluded that transient scrotal hyperthermia seriously, but reversibly, negatively affected the spermatogenesis, oxidative stress may be involved in this process. In addition, intermittent heat exposure more seriously suppresses the spermatogenesis compared to consecutive heat exposure. This may be indicative for clinical infertility etiology analysis and the design of contraceptive methods based on heat stress.     

Detection of immunosuppressive activity in human seminal plasma by an immunobioassay

Recent evidence has shown that under in-vitro conditions human seminal plasma can interfere directly or indirectly with the function of cells of the immune system. It is however, questionable whether the results generated in vitro can be related directly to in-vivo activity. We have therefore standardized an in-vivo immunobioassay to detect the immunosuppressive property of human seminal plasma using adoptive transfer of contact sensitivity to a specific antigen such as dinitrofluorobenzene. Our results indicate that when sensitized lymphoid cells were incubated in vitro with human seminal plasma, their ability to transfer the delayed hypersensitivity in non-sensitized mice was suppressed or inhibited in comparison with the controls. The percentage suppression varied with different samples but the results indicate clearly that the immunosuppressive properties of human seminal plasma can be demonstrated using an in-vivo immunoassay.