Monoclonal antibody from vasectomized mouse identifies a conserved testis-specific antigen TSA70

Vasectomy results in the occlusion of testicular outflow, leading to autoimmunity characterized by the production of antisperm antibodies (ASA). Reports on the rise in ASA following vasectomy in several species are available; however, not much is known about the specific sperm autoantigens to which postvasectomy antibodies are directed. In the present study, monoclonal antibodies were generated using a vasectomized mouse. One of the monoclonal antibodies, D5E5, identified an approximately 70-kd antigen localized on the principal piece of the tail and also on the tip of the acrosome of mouse sperm. The cognate antigen was expressed postmeiotically in a stage-specific manner during spermiogenesis, starting from step 8 of elongating spermatids during spermiogenesis up to mature spermatozoa. The protein was conserved across the species, as observed by its presence in rat, bull, marmoset, and human sperm. Following capacitation, the antigen on the head was seen to shift to the acrosomal region and was lost after the acrosome reaction. However, the localization on tip of the acrosome still persisted, which indicates that the antigen may play a role post-acrosome reaction in sperm egg interaction. Resistance to Triton X-100 solubilization indicates that TSA70 could be an acrosomal matrix protein. In addition, we observed a significant reduction in forward progressive motility of mouse sperm treated in vitro with D5E5. In view of its testis specificity, acrosome and tail localization, and conserved nature, TSA70 is likely to play an important role in sperm function.     

SPERM ACROSOME STATUS AND SPERM ANTIBODIES IN INFERTILITY

Purpose

We studied whether the spermatozoa from sperm autoimmune infertile men undergo premature acrosomal loss and whether this relates to the presence of sperm antibodies in wives.

Materials and Methods

We evaluated acrosome status of live washed native and overnight capacitated spermatozoa from 17 sperm nonautoimmune fertile and 23 sperm autoimmune infertile men using an immunofluorescent peanut lectin binding assay. We used cytotoxic and immunobead binding assays to prescreen the serum and seminal plasma of these men, and serum and cervical mucus of the wives for immunological infertility. We performed immunofluorescent sperm antibody assays on all study samples to ascertain sperm antibody isotype levels in each sample. Levels of acrosomal loss in husband native and capacitated spermatozoa were correlated with levels of IgG, IgA and IgM sperm antibodies in the study samples.

Results

Sperm autoimmune infertile men had a significantly larger percentage of sperm (p <0.0001) that had lost the acrosome and a lower percentage of sperm with intact acrosome (p <0.0001) in native and capacitated preparations in contrast to those of fertile controls. Levels of cytotoxic and IgA antibodies, especially in seminal plasma and cervical mucus, correlated significantly with percentages of sperm with a total loss of acrosome in native and capacitated sperm preparations (p [less than =] 0.01).

Conclusion

Infertile men with sperm antibodies in serum and seminal plasma undergo premature acrosome loss. This loss may expose the reproductive tract immune system, especially that involving IgA, in autoimmune infertile men and the wives to high immunogenic levels of sperm acrosome membrane antigens, thereby rendering them immunologically infertile.     

Role of C-type natriuretic peptide in the function of normal human sperm

C-type natriuretic peptide (CNP) is a newly discovered type of local regulatory factor that mediates its biological effects through the specific, membrane-bound natriuretic peptide receptor-B (NPR-B). Recent studies have established that CNP is closely related to male reproductive function. The aims of this study were to determine the distribution of CNP/NPR-B in human ejaculated spermatozoa through different methods (such as immunolocalization, real time polymerase chain reaction and Western Blot), and then to evaluate the influence of CNP on sperm function in vitro, such as motility and acrosome reaction. Human semen samples were collected from consenting donors who met the criteria of the World Health Organization for normozoospermia. Our results show that the specific receptor NPR-B of CNP is localized in the acrosomal region of the head and the membrane of the front-end tail of the sperm, and there is no signal of CNP in human sperm. Compared with the control, CNP can induce a significant dose-dependent increase in spermatozoa motility and acrosome reaction. In summary, CNP/NPR-B can affect sperm motility and acrosome reaction, thus regulating the reproductive function of males. CNP may be a new key factor in regulating sperm function.     

Association between total globozoospermia and sperm chromatin defects

Globozoospermia is a severe sperm morphological anomaly leading to primary infertility and low fertilisation following intracytoplasmic sperm injection (ICSI). This phenotype is observed in less than 0.1% of infertile men and is determined by small, round‐headed spermatozoa with absence of an acrosomal cap, acrosome protease and also cytoskeletal proteins. Failure of oocyte activation is considered as the main cause of fertilisation failure in these individuals post‐ICSI. Therefore, artificial oocyte activation (AOA) along with ICSI is commonly implemented. However, based on previous report, fertilisation rate remains low despite implementation of ICSI‐AOA. Therefore, other mechanisms like sperm chromatin packaging and DNA fragmentation may account for low fertilisation and development post‐ICSI‐AOA. Therefore, this study aims to assess and compare the degree of sperm protamine deficiency and DNA fragmentation in large population of infertile men with total globozoospermia (30 globozoospermic men presenting with 100% round‐headed spermatozoa) with 22 fertile individuals using chromomycin A3 and TUNEL assay respectively. Results clearly show that mean of sperm concentration and percentage of sperm motility were significantly lower, while percentage of sperm abnormal morphology, protamine‐deficient and DNA‐fragmented spermatozoa were significantly higher in infertile men with globozoospermia compared to fertile men. Therefore, increased sperm DNA damage in globozoospermia is likely related to defective DNA compaction and antioxidant therapy before ICSI‐AOA could be recommended as an appropriate option before ICSI‐AOA.     

Sperm—oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa

Summary. Successful sperm-oocyte interaction depends, among other things, on sperm capacitation, which is defined by acrosomal and motility alterations. In the study described here the authors evaluated different aspects of this gamete interaction in humans. Specifically, the authors studied (1) the relationship between the number of spermatozoa bound to the zona pellucida and sperm concentration and incubation period, (2) the capacitation status and kinetics of acrosome reaction among the zona-bound spermatozoa, and (3) the effect of human follicular fluid on the zona-binding potential and acrosome status of spermatozoa from different men. The results indicated a concentration of 10⁷ cells ml⁻¹ after 15 min of coincubation to be the optimum for zona binding. The number of sperm bound after 0, 3 and 5 h of incubation was the same. In addition, spermatozoa incubated for 3 or 5 h underwent the acrosome reaction (range 9–43%) on the zona surface within 15 min of binding. The maximum percentage of acrosome-reacted spermatozoa was reached after 60 min of binding. Follicular fluid affected the sperm populations selectively, since it did not influence zona binding capacity in all cases. The data enhances the authors' understanding of critical events occurring before fertilization.     

Hemizona assay (HZA) demonstrates effects of characterized mouse antihuman sperm antibodies on sperm zona binding.

Characterized antihuman sperm monoclonal antibodies from mice were evaluated using the hemizona assay (HZA) to determine whether sperm:zona binding was effected. The seven monoclonal antibodies were characterized using human sperm in agglutination, immobilization, and penetration assays. Semen was provided by four fertile men and used in the HZA to determine if the presence of a monoclonal antibody would affect tight binding of the sperm to the zona pellucida. Pre-incubation of MA-14 for 1 h with the sperm induced a 33-54% reduction of the number of tightly bound sperm. This antibody reacts to an antigen located on the acrosome and midpiece. Experiments in which there was no pre-incubation of the antibody with sperm, resulted in no significant reduction in the number of sperm bound in the HZA. These findings suggest that an anti-human sperm antibody produced in mice can modulate sperm:zona binding. Reduction in zona binding could indicate a cause of immune-related infertility and this test may be useful in selecting an antigen for contraceptive vaccine development.     

精子功能检测的研究——Ⅰ.底物膜技术对精子顶体酶的测定

本研究应用健康人和不育症患者的精液各30人份,用底物膜技术检测精子顶体酶,同时进行精子形态学和多次曝光摄片分析。结果表明,在精子头部周围,顶体蛋白酶和透明质酸酶活性区域呈现亮区或晕。不育症患者顶体蛋白酶反应率和反应区的平均直径显著降低(P<0.01),但是透明质酸酶无明显变化(P>0.05).不育症患者精子畸形率高于健康人.不育症患者和健康人精子运动平均速度相似,但直线运动方式的精子在前者明显减少.本文对精子的顶体酶、形态异常和运动等相互关系作了讨论.     

Paramagnetic beads coated with Pisum sativum agglutinin bind to human spermatozoa undergoing the acrosome reaction

Summary. For the evaluation of sperm functions it is important to assess the acrosome reaction after induction with various stimuli. Acrosome reaction tests normally include the capacitation of spermatozoa, treatment with an inducer, and detection of acrosomal loss by dyes, lectins or antibodies. Since most of these methods are time-consuming or require expensive equipment, paramagnetic beads coated with Pisum sativum agglutinin (PSA) were investigated for their usefulness in facilitating the detection of human sperm acrosome reaction.
Binding of PSA beads to the acrosomal region increased significantly after incubation of capacitated spermatozoa with 10 μM A23187 (20.3±6.7% [mean±SD, absolute binding], n = 21), 1 mM dibutyryladenosine cyclic monophosphate (17.1±8.5%, n = 25) and 10 mM phorbol myristate acetate (21.1±12.5%, n = 10). Bead binding was significantly reduced by preincubation with a protein kinase inhibitor. Beads bound to Concanavalin A (ConA) were also attached to the acrosomal region after induction of the acrosome reaction by A23187 or dbcAMP, but a lower number of spermatozoa were bound to ConA-beads than to PSA beads. Pre-treatment of spermatozoa with α-methyl-D-mannoside before addition of the PSA beads markedly decreased bead binding, which indicates its mannose-specificity. Electron microscopic examinations demonstrated that PSA beads mainly bound to membrane structures of spermatozoa that were undergoing, but had not completed the acrosome reaction.     

Morphological sperm defects analyzed by light microscopy and transmission electron microscopy and their correlation with sperm motility

Objectives: To compare sperm defects as assessed by light microscopy (LM) and transmission electron microscopy (TEM), and to correlate them with sperm motility. Methods: A cohort of 40 male partners of infertile couples was selected. Group 1 (n = 31) included subjects with motility >5 and <50%, group 2 (n = 9) included those with motility <5% and the control group consisted of 10 normospermic subjects. Semen analysis of morphological parameters was carried out by LM and TEM. Results: A linear correlation between LM and TEM regarding head defects and excess residual cytoplasm (r = 0.87 and 0.90) was found, whereas there was a poor correlation between tail and midpiece anomalies (r = 0.46 and 0.21). No significant variations were detected by LM and TEM regarding sperm head defects and excess residual cytoplasm, whereas TEM showed a significantly greater percentage of tail and midpiece alterations compared with LM in groups 1 and 2, as well as controls (P < 0.05). The microtubular pattern ‘<9 + 2’ represented the most frequent axonemal morphological alteration. Conclusions: TEM might represent an additional diagnostic tool in the presence of severe sperm hypomotility or absence of motility.     

人精子质膜孕激素受体研究

目的 :观察人精子表面孕激素受体 (PR)的定位及阳性表达率。　方法 :精子体外获能后 ,应用异硫氰酸荧光素标记的牛血清白蛋白 孕酮复合物 (P BSA FITC)染色 ,荧光显微镜观察及流式细胞术 (FCM )定量分析法 ,分别观察与孕酮 (P)结合精子形态及标记精子所占比例。　结果 :P BSA FITC染色精子的形态主要表现为 2种类型 :整个顶体区域或仅赤道区呈绿色荧光 ,顶体后区及尾部不着色。与P结合精子的百分率为 (30 2± 2 4 ) %。　结论 :人精子顶体表面有PR表达 ,且这种表达呈选择性     

蛋白激酶C对精子活力和顶体反应的影响

蛋白激酶C(PKC)位于精子头部赤道段和尾部主段。外源性PKC激动剂可增强精子鞭毛的活力 ,而PKC抑制剂如癌基因抑活药 (staurosporine)则能抑制这种作用。精子中PKC的含量与精子活力呈显著正相关 (r =0 .9,P <0 .0 0 1)。精子与透明带 (ZP)结合刺激顶体反应的发生 ,导致顶体释放多种水解酶以及精子暴露出新的膜区域。ZP与精子质膜上的受体结合后 ,可以调节腺苷酸环化酶 (AC)的活性 ,使cAMP浓度升高并激活蛋白激酶A(PKA)。PKA能激活位于顶体外膜的电压依赖性钙通道 ,后者使顶体内部的Ca2 + 释放至胞质中 ,磷脂酶C(PLC)因Ca2 + 浓度升高而激活并水解磷脂酰肌醇二磷酸 ,其水解产物激活PKC ,后者使精子质膜上电压依赖性钙通道 (L型 )开放 ,胞质中第二次较大幅度Ca2 + 浓度的上升导致质膜溶解及顶体反应发生。推测PKC参与调节精子的活力和顶体反应     

流式细胞计检测人精子顶体及其与精液其他参数的关系

运用流式细胞计及人精子顶体特异性标记物结合异硫氰酸荧光素的花生凝集素检测了３４例精液精子顶体结构的完整性。其中正常生育精液１２例，弱精症精液１２例，畸形弱精症精液１０例。并且进一步检测了２０例精液在体外诱导下精子发生顶体反应的能力，其中正常生育精液１０例，不育精液１０例。结果表明人精子顶体结构的完整性与精于正常形态百分率，存活率，前向运动百分率显著正相关（Ｐ＜０．００１）。不育组精子在体外诱导下发生顶体反应的能力明显低于生育组（Ｐ＜０．０１），说明流式细胞计能用于预测生育力。     

Fine structure of spermatozoa retrieved from retrograde ejaculates

The proportion of viable, motile spermatozoa retrieved from retrograde ejaculates has been consistently low. Electron microscopic studies of spermatozoa of retrograde ejaculates collected in Baker's buffer and washed in Ham's F10 medium supplemented with 2% human serum albumin revealed considerable ultrastructural lesions to the sperm head and the midpiece. Swelling and/or loss of plasma membrane was observed in most of the sperm. In others, the membrane continuity was disrupted, and tattered remnants of it remained attached to a grossly abnormal acrosome. The acrosomal cap in the majority of the spermatozoa was swollen and irregular in shape. Some of the spermatozoa lacked the acrosome, and the sperm heads of these were lined by the inner acrosomal membrane. Subcellular derangement in the midpiece of retrograde spermatozoa was characterized by mitochondrial swelling and lysis, indicating definite cytotoxic injury to the spermatozoa. These EM observations are consistent with the findings of high sperm wastage in semen from men with retrograde ejaculation.     

Studies on human spermatozoal acrosome using spermac stain in fertile and infertile men including two cases of round-headed spermatozoa

Using a new acrosome stain named "Spermac stain", the relationship between the staining rate of sperm acrosome and conventional semen parameters, i.e., sp. density and sp. motility efficiency index (SMEI) was studied. The mean positive staining rate of sp. acrosome in ten fertile men was 81.5%. In 72 infertile men, a significantly higher (p less than 0.001) correlation was noted between the positive staining rate and sp. density and SMEI. 5 out of 8 male partners of unexplained infertility couples showed low staining rates (40-60%), in spite of normal sp. density and SMEI. Ejaculates from two infertile men with "round-headed" spermatozoa were examined by Spermac stain and electron microscopy. The staining rates of sp. acrosome in these two cases were 4.5% and 16.5%. The fine structures of the sperm and the spermatid were presented. Because Spermac stain is quick, reliable and easy to use, this staining method is recommended for evaluating sp. acrosomal morphology in practical use.     

Assessment of the acrosomal status of ram spermatozoa by RCA lectin-binding and partition in an aqueous two-phase system

The acrosome reaction is an important marker for sperm function. Because different laboratory techniques may be used to detect this exocytotic process, the objective of this study was to investigate the use of fluoresceinated lectins to assess the acrosomal status of nonpermeabilized ram spermatozoa. In addition, we used centrifugal countercurrent distribution (CCCD) in an aqueous 2-phase system to assess the sperm surface modifications associated with the acrosome reaction by observing changes in their partition behavior. We analyzed the binding of 5-fluorescein isothiocyanate (FITC)-conjugated lectins to ram sperm to select a lectin that bound preferentially to the acrosomal region, which would allow differentiation of acrosome-intact from acrosome-damaged ram spermatozoa. Ricinus communis agglutinin (RCA) bound intensely to the anterior and weakly to the equatorial acrosomal regions. Acrosomal labeling changed when spermatozoa were induced to acrosome-react with calcium ionophore A23187. RCA acrosomal labeling significantly increased (P < .0001) after incubation (84% versus 28% in control samples). To determine if RCA lectin labeling could be used to assess the acrosomal status of fresh ram spermatozoa in suspension, we compared the percentage of acrosome-reacted sperm detected by the carboxyfluorescein diacetate/propidium iodide (CFDA/PI) double-fluorescent staining with the percentage detected by FITC-RCA labeling. The incidence of acrosome-reacted spermatozoa detected by CFDA/PI was not significantly different (P = .704; 13 comparisons in 6 different experiments) from the incidence of spermatozoa detected by FITC-RCA staining. The evaluation of the spontaneous acrosome reaction by RCA labeling (5.83%) was not significantly different (P = .644) from that assessed by CFDA/PI (6.88%). The percentage of induced acrosome reactions detected by CFDA/PI staining (56%) significantly correlated (P < .0001; r = 0.876) with that detected by RCA labeling (56.67%). We simultaneously carried out a comparative CCCD in an aqueous 2-phase system to analyze sperm surface changes associated with the acrosome reaction. Results revealed that sperm surface hydrophobicity decreased in samples that had been incubated with ionophore compared with the untreated-control samples. Likewise, RCA binding after CCCD showed that all acrosome-reacted cells were stained, whereas only 42% of cells were lectin-labeled in the untreated semen sample. This change in lectin reactivity of acrosome-reacted spermatozoa signals the presence of some deep membrane or intracellular residues that would affect partitioning. Therefore, the FITC-RCA-labeling procedure can be used to accurately assess the acrosomal status of ram spermatozoa in suspension.     

伊红Y水试验法评价精子膜未损率及其与精液参数关系

本文建立并应用伊红Ｙ水试验法，对６１例生育男子和７２例不育男子进行精子膜完整性检测。结果表明，精子膜未损率生育组明显高于不育组。生育组和不育组的用子膜未报率均与精子尾部肿胀率、活精子百分率及精子活动率呈高度正相关，与精子正常形态百分率呈低度正相关。本法较为准确、全面、可靠，并具有简便、快速的特点，可作为精液分析的一项常规检测方法。     

Effect of pH on the development of acrosomal responsiveness of human sperm

Human sperm incubated in vitro gradually become responsive to inducers of the acrosome reaction. The roles of constituents of the incubation medium are not well understood. These experiments tested the effect of the extracellular pH on sperm acrosomal responsiveness. Sperm were incubated 24 h in media with pH varying between 6.7 and 7.6 and then exposed to progesterone to determine the number of sperm that had become acrosomally responsive. The number of responsive sperm was very low following incubation at pH 6.7-7.0, and increased with the pH over the range 7.0-7.6. Sperm incubated at low pH were not damaged as assessed by motility or viability, and if they were transferred to medium of high pH for 8 h, the inhibition of acrosomal responsiveness was reversed. Inhibition of acrosomal responsiveness at low pH was not due to impaired loss of sperm cholesterol, but was correlated with a reduced intracellular pH. The inhibition of acrosomal responsiveness by media of low pH may result from preventing the normal capacitation-related rise in intracellular pH.     

人精子顶体反应的检测及其与穿卵试验的相关性

选择正常生育男子（生育组）、输精管结扎再吻合（吻合组）、不育症患者（不育组）各２５例的精液进行人精子顶体蛋白酶及酸性磷酸酶测定,顶体组织化学三色染色和人精子穿透去透明带金黄仓鼠卵试验,比较不同生育条件下精子功能和生育能力的相关性。结果显示,精子获能后顶体反应率明显高于获能前（Ｐ＜０．０１）。生育组顶体反应率和穿透试验高于吻合组和不育组（Ｐ＜０．０１）。结果表明,精子穿卵试验结合精子顶体反应测定可作为客观评价人类精子受精能力的依据     

Zona pellucida mediated acrosome reaction and sperm morphology

Summary. Sperm samples from 29 men randomly selected from the andrology laboratory, were used to evaluate acrosome reaction response to solubilized human zona pellucida. Capacitated sperm samples were exposed to a solution containing 2 zona pellucidae (ZP) per μl for 60 min, after which acrosomal status were recorded using a PSA-FITC technique. Controls included samples supplied by fertile sperm donors. After completion of acrosome reaction studies, patient samples were divided according to the percentage of morphologically normal spermatozoa. Three basic groups were identified, namely, fertile donors, teratozoo-spermic (normal sperm morphology 5–14%; n = 25) and severely teratozoospermic (normal sperm morphology <4%; n = 4) groups. The mean percent normal sperm were 15.8 ± 0.9, 10.4 ± 0.7 and 2.7 ± 0.7, respectively, for normozoospermic donors, teratozoospermic and severely teratozoospermic men. The mean percentage (± SE) ZP mediated acrosome reacted sperm among teratozoospermic and severely teratozoospermic cases was 25.8% ± 0.9 and 19.0% ± 0.9 (P = 0.001), compared to 36.8% ± 0.9 for the donor controls. Results were analysed and expressed as correlations between sperm morphology and acrosomal response to human solubilized zona pellucida, spontaneous and calcium ionophore induced acrosome reaction. Predictive values for acrosome responsiveness were depicted with ROC curve analyses. Sperm morphology evaluated by strict criteria correlated positively and highly significantly with the responsiveness of the acrosome reaction (r = 0.91, P = 0.0001). At a morphology cut-off value of 4%, the ROC curve analysis showed sperm morphology to be highly predictive of zona pellucida induced acrosome responsiveness with a sensitivity of 100% and negative predictive value of 100%. Spontaneous and calcium ionophore induced acrosome reactions revealed no correlation with sperm morphology. It was concluded that (i) morphological features of human spermatozoa are indicative of specific functional characteristics; (ii) zona pellucida induction of the acrosome reaction is superior, as a predictor of sperm morphology, compared to calcium ionophore induced and spontaneous acrosome reactions.     

Morphology of spermatozoa in infertile men with and without varicocele

This study was carried out to evaluate the morphology of spermatozoa in infertile men with and without varicocele. A series of 285 ejaculates were fully evaluated for seminal volume, sperm count, motility, and morphology, and classified into fertile (165 subjects), infertile without varicocele (93 subjects) and infertile with varicocele (27 subjects). Sperm morphology was classified by multiple entry criteria and recorded as normal, abnormal with head, midpiece, or tail single anomaly or abnormal with simultaneous multiple abnormalities. Semen volume was almost identical in the three groups. Among the infertile men, sperm count was lower in those having a varicocele, but conversely those with varicocele had a higher percentage of motile spermatozoa, higher percentage of spermatozoa with forward movement and higher sperm velocity. There were higher proportions of spermatozoa with abnormal morphology, total number of anomalies, and multiple anomalies in infertile men, both with and without varicocele, than in fertile men. The percentage of abnormal spermatozoa was higher in infertile men with varicocele than in those without varicocele. The pattern of sperm morphology differed between the infertile and the fertile group, and with the presence or absence of varicocele. In the presence of varicocele, only the incidence of elongated (tapered) forms was significantly increased.