Localization of binding sites of naturally occurring antisperm antibodies on human spermatozoa by immunofluorescence

Antisperm antibodies (ASA) may affect sperm motility, acrosome reaction, sperm penetration of cervical mucus, binding to the zona pellucida, and sperm-egg fusion. We investigated the localization of ASA of infertile men or men after vasectomy bound on the sperm surface using an immunofluorescence method. Binding occurred in the acrosomal region, midpiece, and tail. Most of the ASA in both groups of patients bound to the midpiece alone or in combination with other regions of spermatozoa. Only few ASA samples showed binding to all the three sperm regions. A combination of binding to the acrosomal region and to the midpiece was never observed. In infertile patients with ASA, the binding site was compared with sperm parameters. ASA binding to the sperm head influenced the acrosome reaction. Binding of ASA on tail and/or midpiece was not associated with a significant alteration of viability and motility. Immunofluorescence appears to be a valuable tool in the diagnosis of immune infertility, in particular when impairment of the acrosome activity is suggested.     

The mouse sperm glycine receptor/chloride channel: cellular localization and involvement in the acrosome reaction initiated by glycine

Previously, we reported that glycine initiates the in vitro acrosome reaction (AR) of porcine and human sperm by a mechanism that includes the glycine receptor/Cl- channel (GlyR) and that this receptor/channel is required for the zona-pellucida-initiated AR. Because mouse sperm are important tools in the study of fertilization, we investigated whether glycine initiated the mouse sperm AR and whether the sperm GlyR was involved in that initiation. Glycine (250 microM to 1 mM) initiated the AR of capacitated but not noncapacitated mouse sperm. The glycine-initiated AR was significantly inhibited by 50 nM strychnine, a neuronal GlyR antagonist. The neuronal GlyR agonists taurine and beta-alanine did not initiate the AR at 1 mM or 5 mM. A monoclonal antibody against the rat spinal cord GlyR significantly inhibited the glycine-initiated AR but not the spontaneous AR. Indirect immunofluorescence localization studies with that monoclonal antibody and postfixed live sperm detected 3 patterns of immunoreactivity involving 2 sites in the periacrosomal plasma membrane. These patterns were as follows: type A localization on the plasma membrane overlying the tip of the anterior acrosomal region; type B localization on the plasma membrane overlying the posterior part of the acrosomal equatorial segment and/or, in acrosome-reacted sperm, the posterior part of the modified equatorial segment; and type C localization that included both type A and type B. Type A and type C localization were only observed on the acrosome-intact sperm. During capacitation, the number of the sperm showing type A localization increased. Our results demonstrate that mouse sperm provide an excellent model for studying the role of the GlyR in the acrosome reaction.     

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.     

ConA-binding proteins of the sperm surface are conserved through evolution and in sperm maturation

Summary.  Lectin-binding glycoconjugates present on the surface of spermatozoa are believed to play a crucial role in sperm maturation, capacitation, acrosome reaction, or sperm-egg interaction. We have studied ConA-binding surface proteins on spermatozoa from different mammalian species. First, ConA-binding proteins were isolated from boar spermatozoa by affinity chromatography. ConA-binding ability was confirmed by Enzyme-linked Lectin assay (ELLA). Monoclonal (MAb436/10) and polyclonal antibodies were raised against chromatography fractions containing purified ConA-binding proteins of boar spermatozoa. MAb436/10 (IgG_2a) recognizes a 40 kD ConA-binding antigen. Indirect immunofluorescence on fixed and unfixed boar spermatozoa with MAb436/10 indicated a plasma membrane localization of antigen 436/10 in the acrosomal macrodomain. Interspecies cross-reactivity with MAb436/10 was found by whole cell ELISA and immunocytochemistry. MAb436/10 cross-reacted with human, horse, guinea-pig, bull, and ram spermatozoa in both assays. Expression of ConA-binding antigen 436/10 on guinea pig sperm surface was detectable during spermiogenesis and in early stages of sperm maturation. Change of regionalization of the antigen did not occur during the epididymal passage. ConA-binding antigen 436/10 was also detectable in testis and caudal segments of the epididymis. These findings suggest that ConA-binding proteins located in the acrosomal region are highly conserved through evolution as well as in sperm maturation indicating an important role for the physiology of spermatozoa.     

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.     

Partial characterization of an intra-acrosomal protein, human acrin1 (MN7)

Acrin1 (MN7), a 90-kd glycoprotein, is localized in the sperm acrosome of several rodents. The purpose of this study was to determine the molecular size and subcellular localization of acrin1 (MN7) in human sperm, and its organization in spermatids during spermiogenesis. Immunocytochemical and biochemical analyses revealed that acrin1 (MN7) is localized in the anterior acrosome and its molecular size is 90 kd, the same as in rodents. Based on molecular size, acrin1 (MN7) is likely to be a novel human acrosomal protein. During spermiogenesis, acrin1 (MN7) is initially localized in the head-cap portion of spermatids from the cap phase to the end of the acrosomal phase, and then relocates from the posterior to the anterior region of the acrosome in the maturation phase in spermatids. Such a morphological organization mechanism is also basically the same as that in rodents. Thus, acrin1 (MN7) is a common acrosomal protein of 90 kd in rodents and humans.     

Stage-specific expression of a protein of the acrosome of baboon sperm during spermiogenesis detected with a monoclonal antibody

A single monoclonal antibody, BSA4, raised against baboon epididymal sperm was used to study the ontogeny of the baboon sperm acrosome region during testicular spermiogenesis. This antibody is not species-specific but is restricted to the acrosome region in all other sperm examined (human, rat, and mouse). In the baboon, treatment of epididymal sperm with 0.05% Triton-X results in complete loss of anterior acrosome staining. Such treated sperm display a distinct equatorial staining. Antibody BSA4 reacts with a determinant (molecular weight, 43,000 d) that first appears in postmeiotic round spermatids during spermiogenesis. When tested for an effect on the fertilization process in vitro, the antibody BSA4 displayed significant inhibition, indicating a possible functional role for the determinant on mouse sperm. Using the avidin-biotin immunoperoxidase method, several stages of acrosome development were recognized: ie, cap, acrosome, and maturation stages of spermiogenesis. The antibody staining was restricted to the developing acrosome at all stages, indicating that the equatorial region is part of the acrosome and is expressed with temporal specificity during spermatogenesis in the baboon.     

The fate of acrosomal staining during the acrosome reaction of human spermatozoa as revealed by a monoclonal antibody and PNA-lectin

A monoclonal anti-human sperm antibody, raised against an acrosomal antigen and indicated to recognize in boar sperm the serine protease, acrosin, stained in human spermatozoa a 50 Kd antigen and several others in the region 24-34 Kd by immunoblotting. The 50 Kd band and the region of 30-34 Kd showed proteolytic activity by zymographic enzyme detection. The fate of the antigen was studied in the acrosome reaction induced by the calcium ionophore A23187. In control incubations 69.5 +/- 14.2% (mean +/- SD) of the spermatozoa had intact acrosomal staining according to indirect immunofluorescence using this antibody whereas in acrosome-reacted samples only 21.0 +/- 2.0% of the sperm were stained. Another marker for the acrosome, peanut agglutinin-lectin (PNA), was used to detect the acrosome with similar results. Acrosome reactions were verified by electron microscopy. The present results indicate that the corresponding antigen, evidently acrosin, and PNA-positive material are liberated during the acrosome reaction which suggests that they are not bound to the inner acrosomal membrane but are components of the acrosomal matrix.     

The Sperm Acrosome: Immunological Analysis using Specific Polyclonal and Monoclonal Antibodies Directed Against the Outer Acrosomal Membrane of Boar Spermatozoa

An antiserum to the purified porcine outer acrosomal membrane (OAM) was raised in female Balb/c mice and was characterized by means of an indirect ELISA. The hyperimmune serum reacted selectively with the acrosomal cap of the sperm head and showed an extremely good cross reactivity with bull and human spermatozoa when assayed by indirect immunofluorescence. Immunoelectron microscopy using the protein A-gold method further confirmed the specificity of the anti-OAM-antiserum for the OAM. In an effort to identify the OAM antigens recognized by the hyperimmune serum and to analyse the extent of cross reactivity on a molecular level, the SDS-extractable proteins were separated by SDS-PAGE, transblotted and immunoprinted using an 125J-conjugated anti-mouse-antibody. To facilitate functional and structural analysis of distinct OAM-proteins monoclonal antibodies were generated by hybridization of mouse myeloma cells with the splenocytes of female Balb/c mice immunized with the purified OAM. One fusion resulted in about 100 anti-OAM-antibodies secreting hybridoma cultures, of which about 30% showed cross reaction with human and bull spermatozoa. Four stable cell lines were selected for this study secreting antibodies directed against the outer acrosomal membrane of boar spermatozoa. Whereas the polyclonal immune mouse serum stained the entire acrosomal cap, the four hybridoma antibodies generated a patch-work-like immunofluorescence pattern over the acrosome. HPLC-ELISA of the solubilized OAM revealed first information on the nature of the corresponding membrane antigen.     

Calpain and calpastatin are located between the plasma membrane and outer acrosomal membrane of cynomolgus macaque spermatozoa

Mammalian sperm must undergo an acrosome reaction prior to penetration of the zona pellucida and subsequent fusion with an oocyte. Sperm gain the capability to acrosome react after a period of capacitation, which primarily involves biochemical changes in the sperm membranes. The morphological events of the acrosome reaction have been well-documented, but the underlying cellular mechanisms that regulate capacitation and the acrosome reaction remain unclear. Antibodies to the 2 ubiquitous calpains, mu and m, as well as the small subunit, which associates with both calpains, were localized at the ultrastructural level to the region between the plasma membrane and the outer acrosomal membrane of cynomolgus macaque sperm. After the acrosome reaction, all of the anti-calpain antibodies labeled the acrosomal shroud, suggesting that calpains are located throughout the cytoplasmic area between the 2 outer sperm membranes. Calpastatin is an endogenous modulator of calpain activity and is also localized within the same cytoplasmic region as calpains. The antibodies used for ultrastructural localization were also used to probe Western blots of sperm extracts. Antibodies to either the mu- or m-calpain recognized an 80-kd protein, which is similar to the molecular weights of other ubiquitous calpains described. The small subunit (30 kd) was also recognized with a specific monoclonal antibody. An antibody to calpastatin recognized a major band at 78 kd and a lighter band at 45 kd, while the antibody to the testis-specific isoform of calpastatin (TCAST) recognized a 110-kd protein. We hypothesize that this cysteine protease system may be functional in cynomolgus macaque sperm during capacitation, the acrosome reaction, or both.     

Preparation and characterization of monoclonal antibodies against sperm-specific lactate dehydrogenase C4

LDH-C4, the testis-specific isozymes of lactate dehydrogenase (LDH), is the predominant LDH isozyme in mammalian spermatozoa. Nine monoclonal antibodies against mouse LDH-C4 have been developed. These antibodies were tested for cross reactivity with LDH-C4 from human testis and with LDH-1-5 from mouse and human testes by immunoelectrophoresis, bio-dot, and western blot assays. The results showed that all monoclonal antibodies were specific to LDH-C4 only: they did not react with LDH-1-5 from mice, nor from humans. The immunologic localization of the monoclonal antibodies on capacitated sperm was observed by indirect immunofluorescent assay. On mouse sperm the antibodies were bound to the tail only, but on human sperm eight antibodies were bound to the postacrosome, some to the neck and to the mid-piece. Most of the antibodies belonged to the Ig G class.     

Migration and ultrastructural localization of the c-kit receptor protein in spermatogenic cells and spermatozoa of the mouse

PURPOSE: The c-kit receptor is a proto-oncogene important in germ cell migration and maturation and has also been demonstrated on the acrosomal region of mature sperm. The purpose of the present study was to examine the ultrastructural location of the c-kit receptor in mouse testis and sperm. MATERIALS AND METHODS: Testis and sperm from mature male mice were examined for the c-kit receptor utilizing electron microscopy and Western blot analysis techniques. Thin sections of mouse testis and sperm were stained with immunogold-labeled anti-c-kit antibodies. The protein from these testes and sperm was also utilized for Western blot analysis. RESULTS: The c-kit protein was localized within the mouse testes to the type A spermatogonia, the round spermatids, and the mature testicular spermatozoa. The c-kit receptor was noted to migrate from the lumen of the acrosomal vesicles in the early spermatids to the plasma membrane of the late spermatids. It was also noted in the acrosomal region of the testicular spermatozoa, as well as the sperm from the epididymis. Sperm undergoing the acrosome reaction demonstrated association of the c-kit receptor with the plasma membrane of the acrosome, but not on the acrosomal membrane itself. Western blot analysis demonstrated protein bands of 150 kDa in testis and intact sperm. CONCLUSIONS: The present study confirms the presence of the c-kit receptor in mouse testis and sperm. It also demonstrates that this receptor is localized to the region of the developing acrosome.     

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     

Monoclonal antibody labelling of human spermatozoa: an electron microscope study

The antigenic determinants recognized by six anti-human sperm monoclonal antibodies were localized at the subcellular level using an indirect peroxidase immunoelectron microscopic method. Labelling was performed using fresh spermatozoa, and after cell permeabilization (by osmotic shock or freeze-thawing) or detergent demembranation. Two antibodies bound to distinct regions of the plasma membrane, one over the acrosome and the other on the tail, but both also bound to intracellular sites on damaged cells. The internal organelles labelled by the other four antibodies were identified as the acrosomal membrane of the equatorial segment, structures in the connective piece, mitochondrial membranes and axonemal microtubules, respectively. These results are compared with those of a previous immunofluorescence study (Villarroya & Scholler, 1986) and the advantages of joint light and electron microscopy for sperm immunocytochemistry are discussed.     

Electron microscopic immunolocalization of a conserved sperm acrosomal antigen recognized by HS-63 monoclonal antibody

Summary. HS-63 monoclonal antibody was shown to react with a sperm-specific acrosomal antigen from spermatozoa of a variety of mammalian species. Indirect immunofluorescence showed that HS-63 was associated with acrosome-intact sperm after sperm had been fixed with methanol. Electron microscopy (EM) was employed to determine the ultrastructural localization of this sperm antigen. When the immunogold labelled goat anti-mouse IgG was used as a probe, we demonstrated that HS-63 monoclonal antibody did not bind to the freshly prepared human spermatozoa. However, gold particles were observed in the intra-acrosomal region, when the spermatozoa had been pre-treated with 0.5% Triton X-100 prior to incubation with HS-63. We further observed that the immunogold did not stain the inner acrosome membrane when the spermatozoa became acrosome-reacted. A good correlation was obtained between the percentage of spermatozoa which did not react with HS-63 as determined by indirect immunofluorescence assay and that of acrosome-reacted spermatozoa as quantitated by electron microscopy. The results of this EM study were consistent with those obtained by indirect immunofluorescence assay and both indicated that HS-63 reacts only with the capacitated and acrosome-intact spermatozoa. Therefore, HS-63 monoclonal antibody is a useful probe for rapid evaluation of acrosomal status in human spermatozoa.     

Detection of the mouse acrosome reaction by acid phosphatase. Comparison with chlortetracycline and electron microscopy

The sperm acrosome is a uniquely regulated secretory vesicle containing several hydrolase enzymes, including acid phosphatase (AP). The exocytotic event that releases these enzymes, the acrosome reaction, is required for fertilization in mammals. Different methods have been described in the scientific literature for detection of the acrosome reaction: double and triple stains, fluorescent-lectin stains, monoclonal antibodies against acrosomal antigens (immunodetection techniques), Coomassie blue, differential interference contrast or phase contrast, flow cytometry, and chlortetracycline (CTC). In contrast, only 1 method to detect AP released by live and reacted sperm has been described in the literature thus far. In this work we compare 2 classical methods, CTC and transmission electron microscopy (TEM), with the assay of AP released from the acrosome. AP released during the acrosome reaction was measured in the culture medium. Enzyme remaining in nonreacted sperm cells was released by Triton X-100 treatment. This enzyme-based methodology shows an increase of AP in the culture media after the acrosome reaction and a corresponding decrease in the detergent-releasable enzyme. The AP assay thus permits the detection of the mouse acrosome reaction and compares well with the CTC and TEM methods. This method is performed on the whole sperm population and so avoids the observer error that is inherent in light microscopic methods.     

Capacitated and acrosome reacted spermatozoa of goat (Capra indicus): a fluorescence and electron microscopic study

Summary. Plasma membrane alterations accompanying in vitro capacitation and acrosome reaction of goat spermatozoa were studied using lectin labelling, scanning electron microscopy, and freeze-fracture methods. Fluorescein isothi-ocyanate linked lectins namely; Canavalia ensiformis (ConA), Maclura pomifera (MPA), Arachis hypogaea (PNA), Glycine max (SBA) and Triticum vulgaris (WGA) agglutinin were used to examine the distribution of surface carbohydrates during these two events. The head and the sperm tail reveal altered lectin labelling features after capacitation and acrosome reaction. After capacitation the surface coat components for MPA, SBA, and WGA are shed from the spermatozoon head. ConA receptors on the head are retained after capacitation but are partially shed in the acrosome reacted spermatozoa. SBA receptor sites appear on the sperm tail of the capacitated spermatozoa. Unusual morphological changes attending capacitation involve the sperm tail-end which develops a novel entity, which we have termed 'spatula'. The 'spatula' shows strong binding with ConA and WGA only. In the acrosome reacted spermatozoa the spatulated tail-end unwinds with a concomitant loss of lectin labelling. Highly ordered membrane particles, 'ladders' of the middle piece of the epididymal sperm tail, disappear and IMP clearings appear on the middle piece and in the spatulated ends of the capacitated spermatozoa. But in the acrosome reacted sperm IMPs reappear and are randomly disposed on the middle-piece and are clustered in small patches on the principal-piece. IMP free areas appear on the plasma membrane covering the acrosome and the outer acrosomal membrane (OAM) of the capacitated spermatozoa. The plasma membrane and OAM fuse at multiple foci and appear as acrosomal 'ghosts' which remain associated with the sperm head even after acrosome reaction.     

Cellular localization of GABA and GABAB receptor subunit proteins during spermiogenesis in rat testis

The GABAergic system, a major inhibitory regulator in the central nervous system, may also play important roles in peripheral nonneuronal tissues and cells. Recent studies showed that GABAB receptor is expressed in testis and sperm. To understand the role of the GABAergic system in spermiogenesis, we examined cellular localization of GABA and GABAB receptor subunits in rat spermatids by immunocytochemistry. Immunoreactivity for GABA was detected around acrosomal granules of spermatids during the Golgi and cap phases. GABAB1 immunoreactivity was observed in the acrosomal vesicle of spermatids in Golgi phase, and during cap phase, this reactivity expanded to the entire region of the acrosome covering the nuclear membrane. The level of reactivity decreased gradually with maturation of spermatids. In contrast, GABAB2 immunoreactivity was not observed in spermatids during Golgi phase but was detected in the equatorial region during cap phase. Both GABA immunoreactivity and GABAB2 immunoreactivity were transferred to the residual cytoplasm during the release of spermatozoa. Electron microscopic immunocytochemistry revealed that, during cap phase, GABA and GABAB1 were distributed within the whole acrosomal vesicle but not in the acrosomal granule. GABAB2 immunoreactivity was observed in the narrow space between the inner acrosomal and nuclear membrane and was limited to the equatorial region of the spermatid head. These results indicate that the GABAergic system might be involved in regulation of spermiogenesis.     

Characterization of human sperm antigens reacting with anti-sperm antibodies from an infertile female patient's serum

Identification of sperm antigens that elicit immunoglobulin (Ig) production and knowledge of their roles in sperm transport and fertilization may enhance diagnosis and treatment of immunologic infertility. Sperm antigens recognized by a female patient's serum anti-sperm antibodies were characterized using an indirect immunobead-binding test, immunoblot analysis, and immunochemical labeling. The anti-sperm antibodies' effect on sperm function was evaluated by acrosome induction by calcium ionophore. Immunobeads specific for IgG were bound to the head of 79% of motile donor sperm. Immunochemical labeling of antibody-binding sites was restricted to the plasma membrane over the acrosomal crescent. No labeling was observed on the inner acrosomal membrane of acrosome-reacted sperm. The antibodies reacted with 35-, 40-, 47-, and 65-kd proteins extracted from acrosome-intact donor sperm. Sperm incubated in 1:4, 1:8, 1:16, and 1:32 dilutions of anti-sperm antibody-positive serum had similar rates of spontaneous acrosome reaction and significantly lower rates of ionophore-induced acrosome reaction compared with sperm incubated in control serum. These results suggest that sperm antigens recognized by the patient's serum anti-sperm antibodies are restricted to the acrosomal region of the plasma membrane. The antibodies may impair fertility by compromising the sperm's ability to undergo capacitation and/or acrosome reaction.