Tissue inhibitors of metalloproteinases 1 and 2 in human seminal plasma and their association with spermatozoa

A 24-kDa heparin binding protein recently identified as tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) in bovine seminal fluid was suggested to play an important role in bull fertility. As no data are present for men, the concentrations of tissue inhibitors of metalloproteinases 1 (TIMP-1) and TIMP-2 were quantified in human seminal plasma of normozoospermic and azoospermic men using enzyme-linked immunosorbent assay methods. TIMP-1 and 2 were not significantly different in both groups and there were no relationships between the concentrations of both TIMPs and other sperm characteristics. It is assumed that TIMPs are released from accessory sex glands.     

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.     

Leptin and leptin receptor in human seminal plasma and in human spermatozoa

Leptin, a 167 amino acid peptide, is known to influence the gonads via direct and indirect effects. Recent studies provide contradictory proposition about the peripheral impact of leptin in the male gonads. Thus, we examined leptin and its receptors in human seminal plasma and in human ejaculated spermatozoa by Western blot technique and fluorescence microscopy. In seminal plasma we found a free leptin band (16 kDa) by an anti-leptin polyclonal antibody. Incubation of seminal plasma with recombinant leptin caused a statistically significant increase in the amount of free leptin (p < 0.01) and supports this finding. Furthermore, a soluble leptin receptor (145 kDa) was found in human seminal plasma in the same specimen. We also detected a 145-kDa leptin receptor isoform in ejaculated spermatozoa as a possible target of leptin action in the male genital tract, which was localized at the tail of spermatozoa by immunofluorescence microscopy only. This receptor was significantly associated with the intactness of sperm plasma membranes. Spermatozoa with deteriorated membranes contained 49.2 +/- 6.9% leptin receptor signal intensity compared with spermatozoa having intact membranes (p < 0.01). This finding is difficult to interpret and may be caused by a leakage of OB-R due to loss of membrane integrity. In conclusion, these data provide further hints for a peripheral role of leptin in the male genital tract, possibly, by an interaction between leptin and spermatozoa via sperm leptin receptors.     

Lack of HLA-molecules on human spermatozoa and in seminal plasma

Summary. The expression of human leukocyte antigens (HLA) on ejaculated spermatozoa and on lymphocytes was compared by flow cytometry using monoclonal antibodies towards HLA class I (pan-HLA-A, -B, -C) and class II (DR) antigens. Soluble antigens of HLA class I (s HLA-A, -B, -C) in seminal plasma and in blood plasma were monitored with an elisa technique. Lymphocytes showed specific fluorescence after incubation with the antibodies against HLA class I and class II (DR), whereas, on spermatozoa no positive immunofluorescence could be detected. No antibodies were bound to any significant extent either after modifications of sperm preparation (density gradient centrifugation, swim up-technique, addition of azide, foetal calf serum or benzamidine chloride) or after treatment of spermatozoa with detergens. Furthermore, different concentrations of soluble HLA-A, -B, -C in seminal plasma and in blood plasma were detected. The latter one showed soluble HLA about four-fold more concentrated than the seminal plasma (x ± SD: 262.5 ± 144.4 nmol 1-¹vs. 62.5 ± 27.1 nmol 1-¹). These results suggest, that the HLA-expression differs between human spermatozoa and somatic cells.     

Immunolocalisation of ghrelin and obestatin in human testis,seminal vesicles,prostate and spermatozoa

The role of ghrelin and obestatin in male reproduction has not completely been clarified. We explored ghrelin and obestatin localisation in the male reproductive system. Polyclonal antibodies anti‐ghrelin and anti‐obestatin were used to detect the expression of these hormones in human testis, prostate and seminal vesicles by immunocytochemistry, while in ejaculated and swim up selected spermatozoa by immunofluorescence. Sertoli cells were positive for both peptides and Leydig cells for ghrelin; germ cells were negative for both hormones. Mild signals for ghrelin and obestatin were observed in rete testis; efferent ductules were the most immune reactive region for both peptides. Epididymis was moderately positive for ghrelin; vas deferens and seminal vesicles showed intense obestatin and moderate ghrelin labelling; prostate tissue expressed obestatin alone. Ejaculated and selected spermatozoa were positive for both peptides in different head and tail regions. This study confirms ghrelin localisation in Leydig and Sertoli cells; the finding that ghrelin is expressed in rete testis, epididymis, vas deferens and seminal vesicles is novel, as well as the localisation of obestatin in almost all tracts of the male reproductive system. This research could offer insights for stimulating other studies, particularly on the role of obestatin in sperm physiology, which is still obscure.     

精浆胸苷激酶1与精液参数关系的初步分析

目的 探讨精浆胸苷激酶1(Thymidine kinase 1,TK1)的浓度与精子参数的关系.方法 利用增强化学发光法检测无精子症和少弱精子症及已生育健康人精浆TK1的浓度.同时利用计算机辅助精液分析系统分析各实验组精子密度及活力.各实验组精浆酸性磷酸酶、α-葡糖苷酶、精浆果糖和锌的浓度也一并检测.分析精浆TK1浓度变化和精子参数、精浆相关参数的相关性.结果 弱精于组(2.41±0.21)及无精子组(2.72±1.16)患者精浆TK1的浓度显著高于正常对照组(1.53±0.22)(P<0.01),少精子组(1.60±0.13)和少弱精子组(1.68±0.24)患者精浆TK1的浓度与对照组相比差异没有统计学意义(P>0.05).精浆TKl与精浆生化指标即精浆酸性磷酸酶、α-葡糖苷酶、果糖和锌的浓度都没有显著的相关性(P>0.05).结论 TKl可能与精子活动质量有一定相关性,高浓度的TKl是引起弱精子症的原因之一,与精子数量的变化关系不密切.TKI在无精子症患者中浓度偏高的原因有待进一步研究.     

Zinc uptake in human seminal spermatozoa: characterization and effects on cell membranes

Human ejaculated spermatozoa take up zinc in a time- and concentration-dependent manner. The kinetics of 65Zn2+ uptake is suggestive of an at least partly carrier-mediated transport. The lack of effect of the respiratory chain inhibitor antimycin A could mean that mitochondrial ATP is not required as an energy source for the uptake. The failure of nonpermeant SH reagent mersalyl to modify zinc uptake indicates that functional membrane sulfhydryl groups are not involved in the process. A dose-dependent inhibition of 65Zn2+ uptake was induced by the "anticalmodulin" drug trifluoperazine, suggesting that the calcium-binding protein calmodulin could have a role in zinc transport. In in vitro experiments this cation brought about a powerful effect in protecting the spermatozoa from being damaged by hypo-osmosis.     

Clusterin在人精子膜表面的表达及其意义

目的 探讨clusterin在人类精子中的表达及其意义.方法 从精子表面用1%TritonX-100提取及氯仿/甲醇分离疏水性的膜表面蛋白,用免疫印迹法检测精子表面是否存在大然clusterin蛋白.用免疫荧光方法确定clusterin在人精子膜表面的分布特点.结果 人精子膜表面及精浆中含有大量的clusterin,且clusterin仅分布于精子头部及顶体区域,而阴性对照组没有发现天然clusterin的存在.结论 人精子头部及顶体表面分布大量的clusterin,可能与精子成熟及精卵结合有关.     

Detection of DNA fragmentation in human spermatozoa: correlation with semen parameters

To determine the prevalence of high levels of sperm DNA damage among infertile men with normal and abnormal semen parameters, 90 patients were subdivided into the following three groups. Group A ( n  = 30): men with normal semen parameters who acted as the controls. Group B ( n  = 30): asthenozoospermic men and group C ( n  = 30): teratozoospermic men, suffering from male infertility. DNA damage was evaluated by the rate of DNA fragmentation index (DFI) as assessed by the terminal desoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. It was found that the difference was not significant between the percentage of DFI in patients with asthenozoospermia and the normospermic men (9.46% ± 8.68 and 8.19 ± 6.84 respectively, P- value not significant). The patients with teratozoospermia showed a significantly higher percentage of DNA fragmentation compared with the controls (respectively 21.37 ± 17.26% and 8.19 ± 6.84%, P  < 0.001). There was a positive correlation between abnormal sperm morphology and the DFI ( r  = 0.44, P  < 0.01) in group C. It is concluded that the impairments of sperm parameters were associated with an increase of DNA fragmentation; this association was strictly related to atypical forms.     

Seminal plasma transferrin concentration: relationship with seminal parameters and plasma hormone levels.

Seminal plasma transferrin concentrations were determined in 155 infertile male patients and in 15 pregnancy-proven fertile males (control group); then the relationship between these concentrations and seminal parameters and plasma hormone levels was investigated. The concentrations of seminal plasma transferrin in patients with a sperm concentration below 20 x 10(6)/ml were significantly lower than those in the control group (p < 0.01). There was no significant difference in seminal plasma transferrin concentration between patients with a sperm concentration of 40 x 10(6)/ml or more and the control patients. A positive correlation was observed between sperm concentration and seminal transferrin content (r = 0.56; p < 0.05). However, correlations between seminal transferrin concentration and sperm motility and between seminal plasma transferrin content and sperm morphology did not show any significance, nor did the seminal transferrin content correlate with plasma LH, FSH, prolactin or testosterone levels. It, therefore, seems that while transferrin is indicative of certain physiopathological conditions in the germ cells, this protein is not a distinctive marker of the fertility potential of an individual.     

A human seminal plasma protein blocks the motility of human spermatozoa

An inhibitor of the motility of demembranated spermatozoa has been shown to be present in human seminal plasma. This seminal plasma motility inhibitor (SPMI) was purified to apparent homogeneity and tested on intact human spermatozoa. Motility parameters of spermatozoa incubated with the sperm motility inhibitor were evaluated with the video automated Cell Soft system. SPMI decreased the percentage of motile spermatozoa in a dose-dependent manner and motility was completely blocked in the presence of 1600 units/ml. Sperm velocity and beat/cross frequency showed a similar progressive decrease as the inhibitor was augmented. However, linearity was essentially not affected. The effects of SPMI on the percentage of motile spermatozoa increased with the time of contact between the inhibitor and spermatozoa. After 120 min., the IC50 was 35% lower than that observed at five min. The presence of seminal plasma did not prevent the inhibitory effects of the seminal plasma factor on sperm motility parameters. On the contrary, a potentiating effects was observed. The data suggest that the SPMI could play a significant role in cases of infertility caused by asthenospermia.     

精浆弹性硬蛋白酶与精液主要参数及精子功能相关性分析

目的 探讨精浆弹性硬蛋白酶与精液主要参数及精子功能指标的关系.方法 按照WHO<人类精液及精子-宫颈粘液相互作用实验室检验手册>要求,对170例不孕夫妇中的男性患者进行精液常规检测及精子功能分析,根据精浆弹性硬蛋白酶的检测结果将所有患者分为A、B、C3组.A组为精浆弹性硬蛋白酶含量＞1 000ng/ml;B组为精浆弹性硬蛋白酶含量290～1 000 ng/ml;C组为精浆弹性硬蛋白酶含量＜290ng/ml.通过比较三组的精液主要参数及精子功能指标来分析精浆弹性硬蛋白酶与男性不育各因素的相关性.结果 170例患者中A组56例(32.9%)、B组38例(22.4%)、C组76例(44.7%).与C组相比A组在精子活率、活力、头部畸形、顶体酶活性指标方面均显著低下(P＜0.01),而B组与C组相比上述指标差异无统计学意义(P＞0.05),各组间精子密度及DNA完整性的差异无统计学意义(P＞0.05).结论 精浆弹性硬蛋白酶与精液质量密切相关,精浆弹性硬蛋白酶作为男性生殖道感染的指标,在评估男性生育力、男性不育症的诊断和治疗,以及精子功能等方面有着重要的临床意义.     

Localization of Hsp60 and Grp78 in the human testis, epididymis and mature spermatozoa

Molecular chaperones of the heat shock proteins (HSP) family are important in numerous cellular processes. In this study, the expression of Hsp60 and Grp78 proteins was investigated in the male reproductive tract. The cellular distribution of Hsp60 and Grp78 proteins was analysed in the human testis and epididymis by immunohistochemical approaches. DNA microarray technology was used to analyse HSP60 and GRP78 gene expression along human epididymis. The cellular localization of these chaperone proteins in ejaculated spermatozoa was investigated by indirect immunofluorescence and by Western blot following sperm sub-cellular fractionation. In the human testis, Hsp60 was detected in spermatogonia, whereas a strong Grp78 staining was observed in spermatocytes and round spermatids. Grp78 protein was also observed in the epididymal epithelium, whereas no Hsp60 staining was observed in this organ by immunohistochemistry. The presence of both Hsp60 and Grp78 RNA in human epididymis was confirmed by microarrays. In ejaculated spermatozoa, Hsp60 was localized in the mid-piece, whereas Grp78 was detected in the neck region. These results indicate that in addition to being expressed in human testis spermatogenic cells, both Hsp60 and Grp78 proteins persist in ejaculated spermatozoa. These findings are in agreement with the involvement of Hsp60 and Grp78 during spermatogenesis and in sperm functions such as fertilization.     

Varicocele and fertility: relationship between testicular volume and seminal parameters before and after treatment

Varicocele is a condition of varicosity and tortuosity of the pampiniform plexus that is often associated with a reduction in the volume of the affected testicle. Today there is much debate about how much the varicocele actually damages the reproductive system and the mechanism through which this occurs. Furthermore, it has not yet clearly been established if treatment is truly useful to restore testicular function. The goal of this study was to evaluate changes in the volume of the affected testis after treatment and to examine any correlations between volume and seminal parameters. We evaluated 43 patients with left idiopathic varicocele with ultrasound scan of the testis before and after surgery; testicular volume was obtained using the ellipsoid formula. We also examined semen parameters before and at an average time of 1 year after the procedure, using the WHO indications. We performed 2 statistical analyses, comparing changes in testicular volume before and after surgery, and volume with seminal parameters. Statistical analysis shows a significant increase of testicular volume after varicocele treatment (P < .05). Furthermore, the total number of spermatozoa and fast progressive spermatozoa rates significantly increased after surgery (respectively P < .05 and P < .01) (Figure 1). The Spearman correlation coefficient shows a good relationship between testicular volume and total number of spermatozoa (r = .445; P = .01). Our data point to the possibility that the affected testicle could benefit in terms of trophism and function after varicocele treatment. Ultrasound scan at follow-up permits assessment of not only the presence of recurrence, but it is also useful for evaluating trophism.     

Localization of adhesion molecules on human spermatozoa by fluorescence microscopy

Summary.  The expression of adhesion molecules on human spermatozoa of healthy probands was analysed. The localization patterns of adhesion molecules (AM) on the spermatozoal surface were documented by fluorescence microscopy. Spermatozoa were incubated with antibodies against α1 (CD49a), α2 (CD49b), α3 (CD49c), α4 (CD49d), α5 (CD49e), α6 (CD49f) chains of β1 integrins, β1 (CD29), β2 (CD18), αV (CD51), β3 (CD61) and β4 integrin chains, the LFA-3 (Lymphocyte function antigen, CD58) from the immunoglobulin superfamily and the extracellular matrix proteins laminin, fibronectin and collagen IV. For collagen IV, α1 and α2 chains no expression could be noticed. Laminin was detected at the acrosomal membrane, fibronectin and β4 chain mainly at the equatorial membrane. The fibronectin receptors α3, α4 and α5 chains of the β1 integrins were mainly located on acrosomal and equatorial membrane areas. Laminin receptor α6 chain was located postacrosomal and less frequently acrosomal. β2 chain and vitronectin receptors αV and β3 chains had a mainly postacrosomal localization pattern. LFA-3 was found constantly on postacrosomal membrane areas. Double staining technique was used to prove the simultaneous occurrence of fibronectin and its integrin receptors α3, α4 and α5 chains and of αV and β2 chains on spermatozoa. The localization patterns of integrins on double stained spermatozoa were similar to the patterns described for single stained spermatozoa. The localization of fibronectin appeared to be influenced by the presence of integrins: the typical equatorial fibronectin band disappeared in case of an equatorial localization of integrins.     

Distribution of alpha-, gamma (+beta)- and delta-tocopherol in the seminal plasma, spermatozoa and seminal vesicles of rabbit

Dietary vitamin E supplementation plays a key role in animal reproduction by protecting germ cells from oxidative damage. Recently, alpha-tocopherol homologues (namely, beta-, gamma- and delta-tocopherol) have been the object of increasing research because of their peculiar nonantioxidant properties. We found that these tocol-derived compounds are not homogeneously distributed among semen components. Alpha-T was the major vitamin E homologue found in all semen fractions. Half of the total gamma (+beta)-T was found in germ cells, while more than 50% of total delta-T was preferentially accumulated in seminal plasma. The concentration of various tocol-derived compounds depended on their relative amounts in diet and the competition for saturable enzymes implicated in their metabolism. A higher concentration of delta-T in seminal plasma may be related to its more polar nature. However, the biological function of this compound in semen remains to be cleared. To our knowledge, this is the first study aimed at identifying alpha-tocopherol homologues in rabbit semen fractions.