Heterogeneity in the presence of CD4-like molecules on human spermatozoa

Summary. The object of the present study was to study if there are differences in the presence of CD4-like molecules on human ejaculated spermatozoa in fertile donors and infertile patients (with globozoospermia). Indirect and absorption enzyme-linked immunosorbent assay and indirect immunofluorescence were applied. The enzyme-linked immunosorbent assay data showed that monoclonal anti-human CD4-antibody recognizes an epitope common to the human spermatozoa with normal morphology and round-headed spermatozoa. Localization of the antigenic determinants, identified by anti-human CD4-monoclonal antibody, in the acrosomal region, including equatorial segment, postnuclear cap and tail was determined in normozoospermic samples. A positive reaction was found on the sperm head both in the acrosomal and postacrosomal region of some round-headed spermatozoa in the samples with globozoospermia. The tails of the normozoospermic spermatozoa and of some round-headed spermatozoa were weakly immunopositive. The results of the experiments carried out are evidence of heterogeneity in the presence of CD4-like antigen determinants on human spermatozoa. These data increase the information about the CD4-antigen characteristic of the spermatozoa from fertile donors and infertile patients.     

Identification of swine and primate cellular adhesion molecules (CAM) using mouse anti-human monoclonal antibodies

Abstract: A series of adhesion molecules of swine and Cynomolgus monkeys were identified by screening for cross-reactivity with a panel of monoclonal murine anti-human adhesion molecule antibodies obtained from the 5th International Workshop and Conference on Human Leukocyte Differentiation Antigens (Boston, MA, USA, 1993). Of 162 antibodies tested, 25 were found that cross-react significantly with swine cells, while 67 were found to react strongly with cells of Cynomolgus monkeys. Cross reactivities to swine were found with antibodies to CD 18 (β2 integrin), CD29 (β1 integrin), β7 integrin, CD49d (α4 integrin) CD49b(α2 integrin), and to a lesser extent to CD62p(P-selectin), CD62L(L-selectin), CD102(ICAM-2), CD11b, and CD49c (α3 integrin). Cross-reactivities to primate cells also included CD18, CD29, CD49d, and CD49b. In addition, reactivities to Cynomolgus monkey cells were detected with antibodies to CD11 (a, b, and c), CD31, CD44, CD49e, CD49f, CD50, CD54, CD56, CD62p, CD 102 and CD56. The tissue distribution and molecular weight of the swine antigens are similar to their human counterparts. These findings provide a spectrum of monoclonal antibodies that react with shared epitopes on homologous adhesion molecules of human, swine, and monkey cells, thus facilitating study of the role of these molecules in the immunobiology of monkeys and swine. These reagents may be useful to dissect the role of adhesion molecules in both alio- and xenoreactivity.     

Actin and actin-binding proteins in bovine spermatozoa: potential role in membrane remodeling and intracellular signaling during epididymal maturation and the acrosome reaction

The actin cytoskeleton influences a wide range of functions in nonmuscle somatic cells, including shape, movement, and interactions with extracellular matrices. The role of actin in mammalian male germ cells, however, particularly during post-testicular development, is not well understood. In this paper, we examine 1) the distribution of 3 actin-regulatory proteins (thymosin beta10, destrin, and a testis-specific actin capping protein) involved in controlling the balance between actin monomers (G-actin) and actin filaments (F-actin), and 2) the distribution and polymerization status of actin in bull spermatozoa during epididymal maturation and following acrosomal exocytosis. Results show that in fixed, permeabilized testicular spermatozoa all 3 regulatory proteins (as determined by binding of specific antibodies) are localized primarily to the acrosomal domain but during epididymal maturation they become confined to the equatorial segment. Following ejaculation, however, they extend back into the acrosomal region. In spermatozoa induced to undergo an acrosome reaction with the calcium ionophore, A23187, further rearrangement occurs with destrin, thymosin beta10, and TS-ACP appearing in the postacrosomal domain. Actin is also found over the acrosome of testicular spermatozoa with both G- and F-actin present, although the 2 forms show slightly different patterns of distribution. Subsequently, actin in the sperm head is largely confined to the equatorial segment until F-actin appears in the postacrosomal domain of acrosome-reacted spermatozoa. This redistribution of actin and actin-regulatory proteins, coupled with changing levels of actin polymerization, suggest a continuing role for actin in both post-testicular sperm maturation and acrosomal exocytosis.     

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     

Quantitative ultramorphological evaluation of swim-up spermatozoa used in human in vitro fertilization and transcervical intrauterine insemination

Ultramorphological changes that occur on the sperm head during in vitro incubation of human spermatozoa was investigated using transmission electron microscopy (TEM). Motile spermatozoa that swim-up were processed for TEM. Washed but unincubated sperm heads had all of the fine structural characteristics of normal spermatozoa: intact plasma membrane, acrosome, equatorial segment, postacrosomal sheath, subacrosomal space filled with fine granular material, dense nucleus containing conspicuous nuclear vacuoles of differing sizes and numbers, and faintly discernible nuclear membrane. The procedure of washing and centrifugation did not alter the structural integrity of spermatozoa. Any evidence of ultramorphological changes in the incubated spermatozoa appeared to be confined to the surface of the anterior two-thirds of the sperm head. These changes were characterized by detachment of plasma membrane, fusion of plasma and acrosomal membranes, vesiculation of membranes, and exposure of acrosomal contents. There was a significant (p less than 0.001) time-dependent increase in the proportion of spermatozoa with such changes. The anterior border of these denuded sperm heads were bound only by the acrosome that appeared as electron-dense granular material on the outer margin and attached on its inner border to the inner acrosomal membrane. Furthermore, in vitro incubation of washed spermatozoa did not lead to any time-dependent degenerative changes.     

Lichtmikroskopischer Nachweis von Lektin-Rezeptoren an der Oberfläche humaner Spermatozoen/Demonstration of Lectin-receptors on the Surface of Human Spermatozoa by Light-microscopy

The topography of lectin binding sites on human spermatozoa has been examined by using particular series of peroxydase labeled lectin-konjugates (GS-1, GS-2, WGA, PNA, SBA, DBA, BPA, MPA, UEA-1, LPA, Con-A) which demonstrated typical patterns of lectin-receptor distributions concerning acrosomal and equatorial as well as postacrosomal areas.     

Characterizing mouse male germ cell-specific actin capping protein α3 (CPα3): dynamic patterns of expression in testicular and epididymal sperm

Aim: To characterize mouse capping protein α3 (CPα3) during spermatogenesis and sperm maturation. Methods: We produced rat anti-CPα3 antiserum and examined the expression of CPα3 in various mouse tissues using Western blot analysis and the localization of CPα3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPα3 and β-actin (ACTB) in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPα3 antiserum and anti-actin antibody. Results: Western blot analysis using specific antiserum revealed that CPα3 was expressed specifically in testes. Interestingly, the molecular weight of CPα3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPα3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPα3 and ACTB localization was virtually identical to the localization before the acrosomal reaction. Conclusion: CPα3 might play an important role in sperm morphogenesis and/or sperm function.     

Immunolocalization and possible functional role of PSP-I/PSP-II heterodimer in highly extended boar spermatozoa

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma which is able to preserve, in vitro, the viability, motility, and mitochondrial activity of highly extended boar spermatozoa for at least 5 hours. However, little is known about the binding pattern of the heterodimer to the sperm plasma membrane and its eventual relation with the maintenance of the sperm functionality. The present study investigated the effect of exposing highly extended boar spermatozoa (1 million/mL) to 1.5 mg/mL of PSP-I/PSP-II for 0.5, 5, and 10 hours at 38 degrees C on sperm characteristics and the changes in PSP-I/PSP-II localization as a result of both the addition of PSP-I/PSP-II to the extender and the incubation time. Exposure of the spermatozoa to PSP-I/PSP-II preserved sperm viability, motility, and mitochondrial activity when compared to nonexposed spermatozoa. This protective effect lasted for 10 hours (P < .05). After immunolabeling of highly extended semen with rabbit monospecific polyclonal antibody against PSP-I/PSP-II, the percentage of immunopositive spermatozoa declines over time from 71% (0.5 hours) to 49% (10 hours). However, more than 80% of spermatozoa remained labeled during the 10-hour incubation period if PSP-I/PSP-II was added. Scanning electron microscopy revealed 4 different binding patterns. The heterodimer was mainly localized to the acrosomal area, being redistributed to the postacrosomal area or lost during in vitro incubation. In conclusion, the protective effect of the heterodimer appears to be related to its adhesion to the acrosomal area, and the loss of this protective effect coincides with a stepwise redistribution of PSP-I/PSP-II during incubation.     

Sperm-egg interactions observed by scanning electron microscopy

Successful in vitro fertilization requires mature oocytes in which the first polar body has been extruded and capacitated sperm capable of penetrating the zona pellucida. In this study we made a time sequential observations on human sperm-egg interactions by SEM in two experimental systems. Human sperm-human zona pellucida interaction: Cytoplasmic processes of corona cell extend around sperm head. Spermatozoa took different angles in attaching or penetrating to the zona pellucida. The head of some spermatozoa bound to the zona were vesiculated, suggesting the progression of the acrosomal reaction. Initially, the anterior part of the sperm head penetrates from the pore of the zona pellucida. Human sperm-zona-free hamster egg interaction: Most spermatozoa lie flat on the vitellus surface covered with numerous microvilli, but a few are oriented perpendicular to the vitellus surface. Most bound sperm had lost their acrosomal caps, because a ridge exists at the leading edge of the equatorial segment. Initially most microvilli appeared to grasp and immobilize the anterior tip of the sperm head. But as gamete interaction proceeded, microvilli were overlying the postacrosomal region and were observed adjacent to the plasma membrane of the postacrosomal region. The postacrosomal region is first incorporated into the ooplasma, the anterior tip of sperm head being the last portion to be incorporated. The microvilli of the oolemmal surface where sperm penetrated did not show major changes in size or in appearance, and the so-called incorporation cone was not observed.     

The binding patterns of antisera to sex steroids and human gonadotropins on human and rhesus monkey spermatozoa

The presence of different hormones on the surface of ejaculated spermatozoa was determined by immunofluorescence studies of the binding patterns of specific antisera to these hormones. There were striking similarities in the binding pattern of antisera to steroid hormones found on human and monkey spermatozoa. Assuming the intensity of fluorescence is proportional to the concentration of the hormone, concentrations of testosterone on the acrosomal and the postacrosomal regions were higher than levels of progesterone and estrogens. Spermatozoa with a "tapering head" had more hCG bound on the acrosomal and postacrosomal regions than spermatozoa with "normal head" (oval shaped). Correlating these findings to the functions of spermatozoa will require further studies.     

Extraction of human and rabbit acrosomes: a comparison of sequential and sonication methods

Two methods for the extraction of acrosomal membranes and enzymes from both human and rabbit spermatozoa were compared. Treatment of spermatozoa with hypotonic MgCl2 (0.05 M) solution causes removal of the plasma membrane, vesiculation, disruption and removal of the outer acrosomal membrane posterior to the equatorial segment with accompanying loss of soluble acrosomal material. Subsequent exposure to Hyamine 2389 and Triton X-100 removes acrosomal material bound to the inner acrosomal membrane with concomitant solubilization of this membrane. The MgCl2 extract from rabbit spermatozoa contained a higher yield of hyaluronidase, acrosin, and total proteinase activities, whereas the subsequent detergent extracts contained higher yields of both arylsulfatase A and B activities. By comparison, after 4 minutes of sonication to separate heads and tails, both rabbit and human spermatozoa when viewed by transmission electron microscopy showed alterations of plasma and outer acrosomal membranes with considerable loss of the acrosomal contents. Analysis of acrosomal enzymes indicates the greatest percentage of all the enzymes assayed was located in the extract obtained by sonication in contrast to either the separated head or tail fractions used for further subcellular extraction. Subsequent treatment with Hyamine and Triton yields only minimal amounts of enzyme activity.     

Evolution and Development of the Outer Acrosomal Membrane (OAM) and Evidence that Acrosin-lnhibitors are Proteins of the OAM

An antiserum to the purified porcine outer acrosomal membrane (OAM) was raised in rabbits and the IgG fraction isolated by ammonium sulphate precipitation and ion exchange chromatography. The antibodies reacted exclusively with the acrosomal cap of the sperm head as revealed by indirect immunofluorescence. In addition they cross-reacted not only with the acrosomal part of the spermatozoa of all mammalian species tested (bull, horse, rabbit, rat, mouse, hamster, mole, antelope, monkey, man) but also with the spermatozoa of the cock (Class: birds) and the rainbow trout (Class: fish). All the species exhibited similar development of the acrosomal cap during spermatogenesis, with the appearance of the immunofluorescent stain in early round spermatids. In the mole the localization of the acrosome in elongated testicular spermatids differed from that in all other species: Instead of prominent fluorescence over the apical part of the sperm an equatorial belt was formed. The cross-reactivity of the anti-boar OAM antibody with the acrosomes of different vertebrate species at the morphological level was supported by the results of Western blotting experiments with purified boar OAM proteins and the SDS-extractable proteins of bull and human spermatozoa, respectively. Using anti-OAM antibodies and antibodies against the acrosin inhibitors I and II described recently by Tschesche et al. (1982), in absorption and Western blotting experiments, it was demonstrated that the acrosin inhibitor proteins are integrated in the outer acrosomal membrane.     

Functional Down-Regulation of β1 and β2 Integrins on Lamina Propria Lymphocytes (LPL) and Tumor-Infiltrating Lymphocytes (TIL) in Colorectal Cancer Patients

Integrins play an important role in various lymphocyte functions. In this study, we isolated lamina propria lymphocytes (LPL) and tumor-infiltrating lymphocytes (TIL) from normal and malignant tissues in patients with colorectal cancer, and examined the expression of 1 and 2 integrins on these lymphocytes quantitatively with two-color flow cytometry. Both LPL and TIL expressed a lower level of common 1 chain (CD29) in CD4 and CD8 subpopulations than did peripheral blood lymphocytes (PBL). Among the associated chains, the expression levels of 1 (CD49a) and 2 (CD49b) were slightly higher, whereas those of 4 (CD49d) and 6 (CD49f) were markedly reduced in LPL and TIL. No significant differences were observed in expressions of any 1 integrin chains between these two lymphocytes populations. Similarly, both L (CD11a) and 2 (CD18) were down-regulated in TIL and LPL with CD8⁺ cytotoxic phenotype, but not in those with CD4⁺ phenotype. CD8⁺ TIL expressed a slightly but significantly higher level of L2 than did CD8⁺ LPL. CD8⁺ LPL and CD8⁺ TIL consistently showed significantly decreased binding to purified ICAM-1, VCAM-1 and HT29 colon cancer cells as compared with CD8⁺ PBL. Although CD8⁺ TIL showed a slightly higher level of adhesion to these substrates than did CD8⁺ LPL, the level was much lower than that in PBL. The expression pattern and functional down-regulation of these integrins may be one of the reasons why TIL cannot eradicate the cancer cells in colorectal cancer.     

人精子tPA和PAI-1的测定及精子上tPA的定位研究

目的 :人精子上组织型纤溶酶原原激活因子 (tPA)的分布及精子中tPA、纤溶酶激活物抑制因子 (PAI 1)活性研究。　方法 :应用底物显色法测定 6 1例人精子中tPA及其PAI 1的活性 ,其中正常对照组 2 0例 (已婚生育男子 ,经 2次精液分析正常者 ) ,少弱畸精症组 41例 ;用免疫组化法进行精子表面膜上tPA定位分析。　结果 :正常对照组精子中tPA和PAI 1的活性分别为 (0 2 4± 0 0 4)IU/ 10 6精子和 (0 2 3± 0 0 3)AU/ 10 6精子 ,少弱畸精症组的tPA和PAI 1活性分别为 (0 12± 0 0 3)IU/ 10 6精子和 (0 35± 0 0 9)AU/ 10 6精子 ,经分析二者之间有极显著差异 (P <0 0 1) ;tPA主要分布在精子的顶体膜、赤道板、颈部和尾部。　结论 :正常健康人精子中的tPA和PAI 1活性有别于弱精症者 ,提示精子上的tPA和PAI 1活性可能与精子活力、精子形态以及精子数有关 ;精子的顶体膜、赤道板、颈部及尾部存在着tPA ,推测其与精子运行、获能等有关。     

Sperm surface fibronectin. Expression following capacitation.

The Arg-Gly-Asp (RGD) amino acid sequence plays a role in many cell-to-cell and cell-to-matrix adhesion systems, as a recognition sequence for cell membrane receptors termed integrins. Receptors of the VLA subfamily of integrins recognize fibronectin, laminin, and collagen. Given the authors' findings that fibronectin-derived, RDG-containing peptides competitively inhibit sperm-oolemmal adhesion and penetration in both heterologous (human-hamster) and homologous (hamster-hamster) gamete interactions, the expression of fibronectin on the surface of fresh, capacitated, and acrosome-reacted human spermatozoa was studied. The majority of fresh spermatozoa did not display fibronectin on their plasma membrane (0 to 16% positive), as demonstrated by the lack of binding of both monoclonal and polyclonal anti-fibronectin antibodies. In contrast, a significantly greater proportion of spermatozoa (varying between 18% to 100% for different donors) incubated overnight under capacitating conditions reacted with anti-fibronectin antibodies. The induction of an acrosome reaction with progesterone did not alter the proportion of sperm displaying fibronectin or its distribution on the sperm surface. A physiologic role of fibronectin in sperm-oolemmal interaction was suggested by the effects of anti-fibronectin antibodies on sperm oolemmal adhesion and penetration of hamster eggs by human spermatozoa, which were both significantly reduced (P less than 0.001).     

Immunocytochemical localization of a cell adhesion molecule, integrin α5β1, in nerve growth cones

It is well-known that nerve growth cones, the growing tips of nerves, play a central role in determining the direction taken by regenerating peripheral nerves though neurotropism and contact guidance. In order to identify the molecules expressed on growth cones that are responsible for contact guidance, we investigated the possible involvement of integrins as sensors for the extracellular matrix. In cultured rat PC-12i cells and chick dorsal root ganglion cells, we found that integrin α5β1 was concentrated in the filopodia and central regions of the growth cones. These integrin α5β1-rich regions corresponded well with the sites where the growth cones came into contact with and adhered to the extracellular matrix. Integrin α5β1 has been reported to bind with fibronectin in the extracellular matrix. When examined by triple staining and confocal laser scanning microscopy, we found that the distribution of integrin α5β1 in the growth cones was very similar to that of actin filaments. Integrin α5β1 was also expressed by Schwann cells. On immuno-electron microscopy, integrin α5β1 was also identified in regenerating axons in vivo. These results suggest that integrin α5β1 expressed on growth cones may function as a sensor for the extracellular matrix and Schwann cells, and thus mediate functionally important interactions in the development and regeneration of peripheral nerves. Received for publication on Oct. 30, 1997; accepted on April 5, 1999     

人精子膜甘露糖结合蛋白的研究--蛋白的定位

作者运用生物素标记的山羊抗人巨噬细胞甘露糖受体（ＧｏａｔＡｎｔｉ－ｈｕｍａｎＭａｃｒｏｐｈａｇｅＭａｎｎｏｓｅＲｅｃｅｐｔｏｒ,ＡＭＭＲ）ＩｇＧ对人获得能精子膜上的甘露糖结合蛋白进行定位,经激光共聚焦显微镜分析结果显示：ＡＭＭＲＩｇＧ组阳性率显著高于运用正常山羊（ＮｏｒｍａｌＧｏａｔ,ＮＧ）ＩｇＧ的对照组：ＡＭＭＲＩｇＧ组的阳性率大约在７０％（６９．４１％）左右,而ＮＧ ＩｇＧ对照组中除少量精子显