60KD的精子膜抗原对抗精子抗体阳性精子顶体酶活性的影响

目的观察60KD精子膜抗原对抗体阳性精子顶体酶活性的影响。方法用全胶电洗脱结合连续电洗脱的方法提取60KD精子膜抗原,使用60KD精子膜抗原体外处理抗精子抗体阳性精子。精子顶体酶活性的检测采用固定明胶底物薄膜法。结果抗精子抗体阳性精子经60KD抗原处理后,精子的凝集率由处理前的(35.115.1)%下降到了(13.03.3)%(P<0.001)。精子顶体酶阳性反应率由处理前的(30.37.3)%升高到(56.49.1)%(P<0.001)。结论60KD精子膜抗原可以有效去除精子结合的抗体,并提高精子的顶体酶活性。     

Analysis of sperm membrane antigens relevant to antisperm antibody using Western blot

To identify the sperm membrane antigens associated with antisperm antibody. Methods: The antisperm antibody in serum was tested by ELISA. Antisperm antibody positive sera from 18 infertile men and 15 infertile women were used. The molecular weight (MW) of sperm membrane antigens associated with the antisperm antibody was analyzed with antisperm antibody positive serum using Western blot. Results: Eight kinds of MW of sperm membrane antigens were identified. The ratio of identification on the 78 KD(60.7 %), 60KD (71.4 %), 51 KD (14.9 %) and 23 KD (14.29 %) sperm antigen was higher than others. Conclusion: Sperm membrane antigens with MW of 78 KD, 60 KD, 51 KD and 23 KD were associated with antisperm antibody and immunological infertility. (Chin J Andro12002; 16: 345)     

抗精子抗体相关精子膜抗原的免疫印迹分析

目的 筛选抗精子抗体相关的精子膜抗原。方法 使用ELISA试验检测不育病人血清的抗精子抗体,选取抗精子抗体阳性血清,通过免疫印迹的方法来筛选抗精子抗体相关精子膜抗原。结果 通过ELISA试验共收集到抗精子抗体阳性男子血清标本18份、女子血清标本15份。有8种分子量的精子膜抗原被抗精子抗体阳性血清所识别,其中78KD、60KD、51KD、23KD的精子膜抗原的识别率较高,分别为71.43％、60.71％、14.29％和14.29％。结论 78KD、60KD、51KD、23KD的精子膜抗原与抗精子抗体的生成和免疫不育密切相关。     

An acrosomal antigen of human spermatozoa and spermatogenic cells characterized with a monoclonal antibody

Monoclonal antibodies were raised against acrosomal antigens of human sperm by immunizing BALB/CA mice with purified ejaculated human spermatozoa. An ELISA-assay, employing glutaraldehyde-fixed spermatozoa as antigen, was used to screen the hybridomas producing anti-human sperm antibodies. Two hybridoma cell-lines produced antibodies which bound to the acrosomal region of spermatozoa. Both gave identical results in preliminary tests and therefore only one was chosen for further experiments. This antibody stained the acrosomal region of fixed but not living spermatozoa by indirect immunofluorescence, indicating an intra-acrosomal localization of the antigen. In acetone-fixed frozen sections of human testis this antigen was expressed only in germ cells in the adluminal compartment of seminiferous tubules. The antigen was clearly visible in round spermatids from the beginning of the cap phase of acrosome development and was also present in premature germ cells which were present in ejaculates and which were in the early stages of acrosome development. By immunochemical analysis this antibody recognized a molecule of 50 K MW as well as other components of 24 to 34 K. The pattern of staining for the antigen was similar in the presence or absence of beta-mercaptoethanol in the sample buffer. The species specificity of the antigen was studied by indirect immunofluorescence using acetone-fixed spermatozoa and the antigen was found to be present in mouse, bovine, ram and boar spermatozoa. This antibody may be useful as an acrosomal marker.     

Purification and characterization of sperm-coating antigen, identified by a monoclonal antibody

Summary. A procedure was designed for purification of a human seminal plasma-specific antigen (HSP-antigen) identified by a monoclonal antibody produced in this laboratory (mAb 4E6). Pooled human seminal plasma was fractionated by consecutively applied methods: affinity chroma-tography on Lentil lectin sepharose, gel chromatography on Ultrogel AcA 34 and immunoaffinity on mAb 4E6 coupled CNBr-Spharose 4B. The antigen-containing fraction obtained after this procedure was proved to be homogeneous when analysed by electrophoresis in polyacrylamide gel. After the consecutive purification procedures the degree of purification was 128 times as compared to the starting material. Electrophoretic analysis of the purified 4E6 antigen under reducing conditions showed that it consisted of 3 polypeptide subunits with molecular weight 70 kDa, 64 kDa and 60 kDa respectively. On the basis of the data obtained from competitive elisa testing of sera from infertile patients it has been suggested that the identified antigen may be involved in pathogenesis of immunologic infertility.     

抗人精子膜抗原的单克隆抗体

用与人类不育有关的人精子膜关键抗原免疫小鼠制得抗人精子单克隆抗体。这些单克隆抗体为IgG1亚类,可以在免疫印迹中与16KD分子量的精子膜蛋白反应。免疫过氧化物酶组织化学研究证明这些抗体的组织特异性较好,其中2A9C1与包括肝、肺、肾、脾、胰、胃、肠、心肌和骨骼肌在内的人体重要组织无交叉反应。这些抗体在免疫荧光染色中特异地结合列入射出精子核后帽靠近颈部的月牙形区域,这是一个在受精过程中有重要意义的部位。     

Anti-tumor effects and lack of side effects in mice of an immunotoxin directed against human and mouse prostate-specific membrane antigen

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a transmembrane protein that is largely restricted to prostatic epithelial cells in humans and is strongly upregulated on prostatic carcinoma cells. It is also expressed on the endothelium of tumor vasculature in humans, but not on the vasculature of normal tissues. Expression of low levels of PSMA has also been found on non-vascular cells in several normal tissues, most prominently on the brain and kidney in humans. PSMA is an excellent candidate for targeting prostate cancer or targeting tumor vasculature of various solid tumors. The high potential clinical benefit of these agents has prompted the search for an animal model in which to assess the efficacy and safety of anti-PSMA monoclonal antibody (mAb)-based therapies. METHODS: A rat monoclonal antibody, E6 that recognizes both mouse and human PSMA was generated using conventional hybridoma techniques. The antibody was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry. An immunotoxin composed of E6, antibody and deglycosylated ricin A-chain (dgA) was prepared chemically. The anti-tumor effects of the immunotoxin were determined in vitro and in mice bearing subcutaneous LnCaP human prostate tumors, which express PSMA on the tumor cell surface. RESULTS: E6 recognizes the extracellular domain of both human and mouse PSMA in ELISA, immunoblot and by immunohistochemistry. E6 strongly stained the vascular endothelium of tumors from humans but not from mice. E6 stained proximal tubules in mouse and human kidneys, and neurons in the mouse and human hippocampus but, unlike the human, did not detectably stain epithelial cells in mouse prostate or small intestine. An E6-dgA immunoconjugate strongly inhibited the growth of LnCaP tumor xenografts without causing apparent toxicity to the mice. Histological observation indicated that the anti-tumor effects were mediated through direct cytotoxic effects on the tumor cells. CONCLUSIONS: We have generated and characterized a rat mAb (E6) that reacts specifically with both human and mouse PSMA and have demonstrated that an immunotoxin constructed from E6 is safe and effective against human prostatic carcinoma cells growing subcutaneously in nude mice.     

Identification of sperm antigens that regulate fertility

Polyclonal antisera were generated in rabbits against human sperm extracts. Out of many such antisera, one was selected (antiserum I) because it recognized relatively few antigens on Western blots. Antiserum I identified immunostainable material on the acrosome of human, monkey, rabbit, hamster, rat and mouse sperm. A detailed histochemical study using rat testes showed that the antigen was detectable on early spermatids and sperm, but not on less mature germ cells. Immunohistochemistry at the electron microscope level localized the antigen on the plasma membrane of rat sperm. In Western blots using human sperm extracts, the antibody recognized a major band with an apparent molecular weight (MW) of 40,000 and minor bands at 43,000 and 72,000. With extracts of monkey sperm and rat testicular cytosol, antiserum I recognized antigens of 69,000 and 23-24,000, respectively. Functionally, antiserum I produced strong agglutination of human sperm. It also prevented attachment of mouse sperm to mouse oocytes in vitro and reduced fertility when administered to female mice. These results suggest that antiserum I can be used as a possible reagent for selecting sperm antigens as components of a contraceptive vaccine.     

A stomach oncofetal antigen recognized by monoclonal antibody GC302

A monoclonal antibody, GC302, was established by fusing murine myeloma NS/1 cells with the splenocytes of a BALB/c mouse immunized with a human gastric cancer cell line, NU-GC-3. The serological specificity of GC302 was analyzed by an anti-mouse Ig mixed-hemadsorption (MHA) test on a panel of human cell lines, and an immunoperoxidase method using the frozen sections of tumors and normal tissues of adult and fetus. GC302 reacted with cancers of the stomach and colorectum but did not react with hepatocellular carcinomas, melanomas, or astrocytomas in the MHA tests. By the immunoperoxidase method, GC302 was found not to react with normal adult gastric mucosa, but to react with the mucosa in the fetal stomach, intestinal metaplasia, and almost all of the cancer of the stomach. GC302 also reacted with the normal mucosa of the intestine, colon, and rectum as well as with cancers of these origins. In normal liver sections, the antibody reacted with the bile ducts, but not with the hepatic cells. These results indicate that the antigen detected by GC302 is characterized as an oncofetal antigen in the stomach, and also as a differentiation antigen whose localization discriminates between the gastrointestinal tracts of the forgut origin and those of the midgut and hindgut origin. The molecular weight of the GC302 antigen was estimated to beca. 40,000 by the Western blot analysis. Periodic acid treatment on the antigen suggested that the antigenic determinant is a carbohydrate.     

Wheat germ agglutinin receptors on human sperm membranes and sperm morphology

Summary. Sperm membrane components play an important role in the determination of sperm fertilizing ability. During spermatogenesis, epididymal transit, and capacitation, the sperm membrane undergoes various subtle changes which are important maturational events for the production of viable spermatozoa and hence partly determine fertility. Localization and intensity of wheat germ agglutinin (WGA) receptors are reported to be directly associated with male fertility. We examined the correlations and differences between the distribution patterns and intensities of WGA receptors of 75 semen samples, classified according to the strict criteria described for sperm morphology, namely P-pattern (0–4% normal forms, n = 19), G-pattern (5–14% normal forms, n = 41) and normal samples (>15% normal forms, n = 15). Results indicate distinct differences in WGA receptor intensity in all of the three groups as well as significantly ( P < 0.05) lower per-cent staining of the equatorial segment amongst the P-pattern group when compared to the other two groups. It would appear that there is a close relationship between WGA receptors on human sperm membranes and sperm morphology.     

Influence of enterococci on human sperm membrane in vitro

Aim： To study the influence of enterococci on human sperm membrane in vitro. Methods： Ejaculated human sperm were artificially infected with β-hemolytic or non-β-hemolytic enterococci at the bacteria： sperm ratio of 50：1 at 37℃. Sperm membrane integrity was examined after incubation for 1, 3 and 5 h by hypoosmotic swelling （HOS） test and electron microscopy. Results： Sperm infected with β-hemolytic enterococci had lower HOS scores compared with non-β-hemolytic strains or uninfected control （P 〈 0.01）. The HOS test scores of sperm infected with β-hemolytic enterococci increased in the presence of phosphatidylcholine, an inhibitor of hemolysin. Non-β-hemolytic strains showed no significant difference in swelling rate, compared with the control group （P 〉 0.05）. It was shown by electron microscopy that β-hemolytic enterococci caused significant rupture of human sperm membrane. Conclusion： β-hemolytic enterococci caused human sperm membrane injury, and might be mediated by the hemolysin of enterococci.     

Purification of an acrosomal antigen recognized by a monoclonal antibody and antifertility effects of isoimmune serum

A highly conserved acrosomal antigen reactive to a monoclonal antibody (HS-63), generated against human sperm, was purified to homogeneity with a combination of conventional procedures and immunoaffinity chromatography using a soluble extract of mouse and rabbit testes. The molecular weight of the purified antigen was 42-50 kD when analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis. The high specificity of the purified antigen to monoclonal antibody HS-63 was shown by indirect immunofluorescent inhibition assay, enzyme-linked immunosorbent assay, Western blot analysis and radioimmunosorbent assay. The purified antigen was used for isoimmunization of mice and rabbits. Following successive immunizations, antisera of high titres were raised and reacted specifically with antigen on the sperm acrosome and in testes of several mammalian species, but not with somatic tissues. These isoimmune sera exhibited strong inhibition on mouse in-vitro fertilization and human sperm penetration of zona-free hamster eggs. The results of this study suggest that the sperm-specific acrosomal antigen reacting with HS-63 could be a good candidate for the development of immunocontraceptive vaccines in humans and in other animals.     

The role of ezrin-associated protein network in human sperm capacitation

Membrane modifications in sperm cells represent a key step in sperm capacitation; however, the molecular basis of these modifications is not fully understood. Ezrin is the best-studied member of the ezrin/radixin/merlin family. As a cross-linker between the cortical cytoskeleton and plasma membrane proteins, ezrin contributes to remodeling of the membrane surface structure. Furthermore, activated ezrin and the Rho dissociation inhibitor, RhoGDI, promote the formation of cortical cytoskeleton-polymerized actin through Rho activation. Thus, ezrin, actin, RhoGDI, Rho and plasma membrane proteins form a complicated network in vivo, which contributes to the assembly of the structure of the membrane surface. Previously, we showed that ezrin and RhoGDI1 are expressed in human testes. Thus, we sought to determine whether the ezrin-RhoGDIl-actin-membrane protein network has a role in human sperm capacitation. Our results by Western blot indicate that ezrin is activated by phosphorylation of the threonine567 residue during capacitation. Co-immunoprecipitation studies revealed that, during sperm capacitation, the interaction between ezrin and RhoGDI1 increases, and phosphostaining of two dimensional electrophoresis gels showed that RhoGDI 1 is phosphorylated, suggesting that RhoGDI 1 dissociates from RhoA and leads to actin polymerization on the sperm head. We speculate that activated ezrin interacts with polymerized actin and the glycosylated membrane protein cd44 after capacitation. Blocking sperm capacitation using ezrin- or actin-specific monoclonal antibodies decreases their acrosome reaction （AR） rate, but has no effect on the AR alone. Taken together, our results show that a network consisting of ezrin, RhoGDI1, RhoA, F-actin and membrane proteins functions to influence the modifications that occur on the membrane of the sperm head during human sperm capacitation.     

Characterization of human sperm N-acetylglucosaminidase

N-acetylglucosaminidase (NAG) is particularly active in mammalian spermatozoa and appears to be involved in fertilization. Although it is assumed that this enzyme is acrosomal, previous results from our laboratory suggest the presence of NAG at the sperm plasma membrane level. The present study attempted to analyse the subcellular distribution of this enzyme in human spermatozoa. Sperm were incubated under different conditions and NAG activity measured in the soluble extracts and cell pellets using a specific fluorometric substrate. A significant proportion of NAG activity was released when sperm were incubated in culture medium, suggesting a weak association with the plasma membrane. This location was confirmed by western blot analysis of plasma membrane fractions and immunofluorescence on non-permeabilized sperm, which showed a positive signal mainly on the acrosomal domain. The distribution of NAG activity between plasma membrane and acrosome was analysed after cell disruption by freezing and thawing. Triton X-100 stimulated sperm and epididymal NAG activity but not the enzyme obtained from other sources. In addition, biotinylated human recombinant NAG was able to bind to human sperm. Finally, after sperm incubation under capacitating conditions, NAG total activity increased and the sperm enzyme lost its ability to be stimulated by Triton X-100. The possible connection of these results with sperm maturation, capacitation and NAG participation in primary binding to the zona pellucida, was discussed.     

Monoclonal antibodies against a soluble cytoplasmic antigen in human prostatic epithelial cells

Antibodies against a soluble cytoplasmic protein contained in prostate epithelial cells were developed using the hybridoma technique. This was done to provide markers for these cells that can be used to identify acinar cells in culture and to follow their development and/or differentiation over long periods of time. The antibodies do not recognize prostatic acid phosphatase nor the prostate antigen. They do recognize prostate acinar cells in formalin fixed tissue sections. The basal cells underlying the prostate epithelial acinar cells do not appear to contain the antigen. The antigen detected is believed to be a product of differentiated prostatic acinar cells.     

不同获能时相对人精子膜完整性的影响

本研究用人精子低渗肿胀试验（ＨＯＳ－ｔｅｓｔ）分析了１２例正常育龄男子的精液标本。精子分别用常规洗涤法、上游法和加入钙离子载体（Ａ２３１８７）诱导获能等三种不同方法处理后，分别在液化后、上游１ｈ后、获能３ｈ、６ｈ、２４ｈ后测定精子的低渗肿胀率，结果显示经不同方法处理、在不同获能时相之后的精子，在２４ｈ之内其肿胀率保持在８７．８±１４．６％～７３．３±１１．９％之间，统计学分析表明各标本组肿胀率之间无明显差异。本研究提示在２４ｈ内精子膜的完整性不受获能时相及不同处理方法的影响。     

A germ cell-specific nuclear antigen recognized by a monoclonal antibody raised against mouse testicular germ cells

A monoclonal antibody (mAb TRA104) raised against mouse testicular germ cells was able to recognize the nuclei of testicular germ cells at all the stages of differentiation from embryonic gonocytes to spermatids and did not react with any somatic cells. The antigen recognized by mAb TRA 104 was exclusively present in testicular extracts. The molecular weights and isoelectric point (pI) of the antigens determined by Western blotting analysis were 60–110 kDa and 7.2, respectively. This antigen(s) is referred to as a germ cell-specific nuclear antigen(s) (GENA) since GENA was first detected specifically in the genital ridge at around 12 days of gestation by Western blotting analysis. In the testis, the expression increased gradually until adulthood whereas it was lost in the ovary by postpartum day 5. Thus, GENA is a molecule(s) exclusively present in the nuclei of germ cells and may be a useful marker with which to study the mechanism of germ cell development and differentiation at the molecular level.     

The partial characterization and clinical evaluation of pancreas cancer-associated antigen from the ascites fluid of a patient with pancreatic cancer

Monoclonal antibodies against pancreas cancer-associated antigen (PCAAp) were produced by established hybridoma cells. Two monoclonal antibodies, 3F1 and 3B6, were selected and these two monoclonal antibodies were found immunohistologically to react strongly with cancer cells and intraductal mucin-like products in well-differentiated pancreatic cancer tissues, but weakly, if at all, with gastric, colorectal and other cancers and at all not with normal adult or fetal pancreatic tissue. PCAAp is usually expressed in normal colonic mucosa (PCAAc), but the two monoclonal antibodies scarcely reacted with normal colonic mucosa. A sandwich enzyme immunoassay was developed to measure circulating PCAAp. Thirty-two normal subjects and 271 patients comprised of 210 with malignant disease and 61 with benign disease were surveyed. The cut-off value of PCAAp levels in the 32 normal subjects was 2.06 μg/ml, being the mean+2SD, while PCAAp levels of more than this were observed in 72 per cent of the patients with pancreatic cancers, 65 per cent of those with bile duct cancers, 60 per cent of those with hepatomas, 0–30 per cent of those with other malignant diseases, and 10 per cent of those with benign hepatobiliary diseases. The susceptibility of isolated PCAAp and PCAAc to various enzymes and chemical reagents were also studied. The results of this study suggest that the assay for PCAAp might be useful in the diagnosis of pancreatic cancer.     

Monoclonal antibody that defines the prostate specific antigen

A monoclonal antibody to a protein with a single band in the 30-kD region was obtained from fusion of Balb/C mouse spleen cells immunized with epithelial fractions of BPH, with the myeloma cell line P3 × 63 Ag8 6.5.3 by standard procedures. This antigen was characterized in immunobinding studies with various cellular and target antigens and in immunoperoxidase staining.     

不同获能时间对人类精子膜完整性的影响

本研究用人精子低渗肿胀试验（ＨＯＳ－ｔｅｓｔ）分析了１２例正常育龄男子的精液标本。精子分别用常规洗涤法、上游法和加入钙离子载体（Ａ２３１８７）诱导获能等３种不同方法处理后，分别在液化后、上游１小时后、获能３小时、６小时和２４小时后测定了精子的低渗肿胀率。结果显示经不同方法处理、在不同获能时间之后的精子，在２４小时之内其肿胀率保持在８７．８±１４．６％～７３．３±１１．９％之间。统计学分析表明各实验组肿胀率之间无明显差异。本研究提示在２４小时之内精子膜的完整性不受获能时间及不同处理方法的影响。