Biomedical subjects

J C Herr

Publications and source records attributed to J C Herr.

At least 19 recordsLinked to original sources

PubMed1992

Light and electron microscopic immunolocalization of sperm proteins identified by monoclonal antibodies from the World Health Organization Task Force on Sperm Antigens.

Sperm antigens recognized by monoclonal antibodies (mAbs), S19, S69, S71, S72 and S77 submitted to the World Health Organization (WHO)-Sponsored Sperm Monoclonal Antibody Workshops were immunolocalized by light (LM) and transmission electron microscopy (TEM). S19 was surface reactive while mAbs S69, S71, S72 and S77 recognized internal antigens. Indirect immunofluorescence staining of permeabilized sperm with the mAbs revealed that S69 recognized an internal tail antigen, while S71, S72 and S77 recognized acrosomal proteins. Preservation of immunoreactivity after fixation in various combinations of glutaraldehyde, paraformaldehyde and tannic acid was evaluated for mAbs S69 to S77 using immunofluorescence microscopy. The epitopes recognized by these mAbs were adversely affected by these fixatives; therefore, pre-embedding immunogold staining was employed, prior to fixation, osmication, dehydration and embedding. Using this approach, the antigen recognized by mAb S19 was found associated with the plasmalemma of the head and tail of intact sperm. Monoclonal antibody S69 localized to the fibrous sheath. The mAbs S71, S72 and S77, which required sperm permeabilization to expose their acrosomal locus by LM, did not immunoreact with the plasmalemma at the TEM level. Ultrastructural examination of acrosome-reacted sperm revealed the localization of S71 and S77 on the inner and outer acrosomal membranes and with acrosomal matrix. The S72 antigen was associated with the inner and outer acrosomal membranes.

Antibodies, Monoclonal↗

PubMed1992

Interactions of human sperm acrosomal protein SP-10 with the acrosomal membranes.

The interaction of the human acrosomal protein SP-10 with the acrosomal membranes was analyzed by the ability of Triton X-114 (TX-114) and other agents to release SP-10 from the acrosome. Treatment of human sperm with TX-114 revealed a pool of SP-10 that was released by TX-114 and a pool of SP-10 that was TX-114-resistant. TX-114-resistant SP-10 was associated with the equatorial segment and with TX-114-resistant portions of the acrosomal matrix and the inner acrosomal membrane. Phase partitioning of TX-114-released and TX-114-resistant SP-10 pools showed that both were hydrophilic, indicating that these pools consist of proteins that are peripherally associated with, rather than integral to, the acrosomal membranes. Sequential treatments of human sperm with various agents showed that repeated washes with TX-114 or 1.5 M NaCl had little or no effect on TX-114-resistant SP-10, whereas treatment with a chaotropic salt (150 mM sodium thiocyanate) and buffers at pH extremes (pH 2.0 and 10.0) completely released this pool of SP-10 from the acrosome. Together the results suggest that SP-10 is a hydrophilic peripheral acrosomal membrane protein that may be associated with a TX-114-resistant "anchor."

Acrosome↗

PubMed1992

Purification and microsequencing of the intra-acrosomal protein SP-10. Evidence that SP-10 heterogeneity results from endoproteolytic processes.

The human sperm antigen SP-10 has been shown to be a testis-specific, intra-acrosomal protein that is associated with the membranes and matrix of the acrosomal vesicle. Sperm extracts, analyzed on Western blots with a monoclonal antibody to SP-10, have shown heterogeneity of SP-10 peptides ranging from 17.5-34 kDa. Although the entire SP-10 amino acid sequence of 265 amino acids (28.3 kDa) has been deduced from sequencing SP-10 cDNAs, the nature of multiple SP-10 peptide bands is incompletely understood. In this study, we developed a three-step purification method for SP-10 peptides using monoclonal antibody affinity chromatography, reverse-phase HPLC, and preparative gel electrophoresis. Eight SP-10 peptides separated by this protocol and sequenced using Edman degradation showed amino termini that corresponded to regions on the deduced SP-10 amino acid sequence. Peptides with progressively lower apparent mass aligned further toward the carboxy terminus. On the basis of putative cleavage sites on the SP-10 sequence, endoproteases that act at five different peptide bonds are predicted to cleave SP-10: these hydrolyze following arginine (a trypsin-like protease, possibly acrosin), and following serine, proline, glycine, and glutamic acid (previously undescribed intra-acrosomal protease specificities). The present studies 1) provide a purification method for SP-10 peptides; 2) confirm that the SP-10 cDNAs previously sequenced encode authentic SP-10; and 3) yield indirect evidence that endoproteases act to contribute to SP-10 heterogeneity.

Acrosome↗

PubMed1991

Localization of post-vasectomy sperm autoantigens in the Lewis rat.

Major rat sperm autoantigens of 86, 63, 43, 28 and 20 kDa are recognized by post-vasectomy and hyperimmunization antisera from the Lewis rat (Handley et al., Biol. Reprod. 39 (1988) 1239-1250). In the present study, affinity purified monospecific isoantibodies to each autoantigen were produced by elution from antigens which had been separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis confirmed a singular specificity for the 63, 28 and 20 kDa antisera and demonstrated some cross reactivity between the 86 kDa and the 43 kDa antisera. The polyclonal antiserum from which the monospecific antisera were produced stained the entire spermatozoon, while monospecific antibodies bound only to the sperm tail, staining the proximal portion (43 and 28 kDa), a distal domain (63 kDa), or the entire tail (86 kDa). Immunohistochemically stained sections of normal rat testes revealed that the 63, 43 and 28 kDa autoantigens were synchronously expressed in the cytoplasm of spermatids in the apical portions of seminiferous tubules during stages II-VIII in the cycle of the seminiferous epithelium. The 86 kDa autoantigen showed little or no staining in testis sections, implying that this autoantigen appeared on mature sperm following spermiation. These and other data suggest that a highly polymeric structure, possibly within the outer dense fibers of the tail, is a dominant sperm autoimmunogen following vasectomy of the Lewis rat.

Animals↗

PubMed1991

Localization of sperm antigen SP-10 during the six stages of the cycle of the seminiferous epithelium in man.

The distribution of the sperm protein SP-10 was investigated in plastic-embedded samples of human testes by light and electron microscopy. An immunogold and silver enhancement technique, in conjunction with a monoclonal antibody (MHS-10) raised against SP-10, was used to localize the protein. SP-10 was detected in spermatids at each of the six stages of the cycle of the seminiferous epithelium. Light microscopy showed immunoreactive material at the circumference of developing acrosomes in the early steps of spermiogenesis. As differentiation proceeded and cell shape changed from round to elongated, immunoreactive material appeared in an arc, which gradually became a V shape bordering the spermatid nucleus. The area of the immunoreactive material and its shape corresponded to that of the developing acrosome. At the electron microscopic level, gold particles indicative of the presence of SP-10 were detected on electron-dense material found within the developing acrosomal vesicle in early steps of spermiogenesis. As the electron density of the acrosome increased, a high concentration of gold particles was seen in the vesicle matrix. The gold particles gradually became associated with the inner and outer acrosomal membranes of the most mature spermatids.

Acrosome↗

PubMed1991

Factors that influence fertility after vasovasostomy in rats.

OBJECTIVE: To determine if fertility after vasovasostomy of immunologically responsive Lewis rats differs from that of the less responsive Sprague-Dawley strain and to relate fertility to antisperm antibodies, fluid flow in the vas deferens, and testicular structure. DESIGN: Male rats received: (1) bilateral vasectomies; (2) vasectomies followed 3 months later by vasovasostomy; or (3) sham operations. SETTING: Research laboratory. MAIN OUTCOME MEASURES: Fertility was assessed by caging males with three females for 2 weeks and subsequently counting implantation sites. Antisperm antibodies were measured with an enzyme-linked immunosorbent assay, fluid flow through vas deferens segments was tested in vitro, and testicular structure was studied microscopically. RESULTS: Nearly all vasovasostomized Lewis rats were infertile (33 of 34), whereas 62% (18 of 29) Sprague-Dawley rats were fertile after vasovasostomy (P less than 0.001). In fertile Sprague-Dawley males, significant correlations existed between: (1) implantation sites or females impregnated; and (2) antisperm antibodies early after vasectomy, vas flow, and testicular morphology. CONCLUSIONS: Genetic differences affect fertility after vasovasostomy. Fertility after vasovasostomy is also influenced in a multifactorial manner by the immune response, mechanical elements, and structural changes in the reproductive tract.

Animals↗

PubMed1990

Early testicular changes after vasectomy and vasovasostomy in Lewis rats.

The testes of Lewis rats were studied at intervals from 2 weeks to 3 months after bilateral vasectomy, vasectomy followed 1 month later by vasovasostomy, or sham operations. Aims were to determine the nature of early alterations after vasectomy, and to determine whether vasovasostomy after 1 month would result in reversal of vasectomy-induced changes. Approximately one-fourth of the testes in the vasectomy and vasovasostomy groups displayed histological changes, which consisted mainly of depletion of germ cells. The extent of the depletion varied greatly in different seminiferous tubules. In testes altered in this way, no abnormal infiltrations of lymphocytes, macrophages, or other cells were observed in the seminiferous epithelium or in the interstitium. The rete testis and straight tubules were normal in testes with altered seminiferous epithelium. A few testes in the vasectomy and vasovasostomy groups had necrotic centers. The results suggest that depletion of germ cells occurred as a result of shedding from the seminiferous epithelium into the lumen of the tubules. A cellular immune response, such as occurs in experimental allergic orchitis in other species, did not appear to be responsible for the observed loss of germ cells. This suggests a possible role for humoral antibody in this model, since there is an association between testicular changes and serum antisperm antibodies at longer intervals after vasectomy. Testicular alterations were not reversed by performance of a vasovasostomy 1 month after vasectomy.

Animals↗

PubMed1990

Biphasic production of antisperm autoantibodies follow vasectomy of the Lewis rat.

Temporal changes in the specificity of post-vasectomy autoantibodies to SDS-PAGE separated sperm antigens were investigated in Lewis rats. Sera were obtained from nine vasectomized animals prior to vasectomy, every two weeks for 14 weeks, and less frequently thereafter, up to 41 weeks. Changes in antisperm autoantibodies over time were assessed by ELISA and western blot assay and compared to antisperm isoantiserum and normal Lewis rat serum. A "biphasic" pattern of autoantibody production over time was observed in a majority of individuals. This pattern was characterized by early phase autoantibodies, produced between 0 and 6 weeks after vasectomy, which bound antigens at the stacking, separating and ionic fronts and by late phase autoantibodies, produced after 4 weeks following vasectomy which bound antigens at 86, 63, 52, 43, 31 and 26 kDa. Previous work suggested that some high molecular weight autoantigens were disulfide-bonded polymers of the polypeptides at 86, 63, and 43 kba (Handley, et al., 1988). Indirect immunofluorescence with monospecific isoantisera to the 86 kDa autoantigen suggested that its corresponding high molecular weight polymer was located in the tail of cauda epididymal spermatozoa. This polymer possessed several characteristics of T cell independent autoantigens. These data show a change in the specificity of autoantibodies produced over time after vasectomy which may reflect a shift from T cell independent to T cell dependent autoantibody production by the Lewis rat.

Animals↗

PubMed1990

Differential diagnosis of immature germ cells in semen utilizing monoclonal antibody MHS-10 to the intra-acrosomal antigen SP-10.

The monoclonal antibody (mAb) MHS-10 (IgG1) is a mouse antihuman sperm antibody which recognizes a polymorphic sperm protein, (SP-10), which has previously been localized within the acrosomal matrix and the acrosomal membranes. The SP-10 antigen has been shown to be sperm-specific and is not found in somatic tissues. It is stage specific, having been immunohistologically localized to Golgi phase spermatids and all subsequent phases of spermiogenesis. In the present study, acetone-dried smears from washed human semen containing significant numbers of round cells were probed with mAb MHS-10. Monoclonal antibody-labeled cells were visualized by a standard streptavidin-biotin immunoperoxidase method using a light microscope. The MHS-10 mAb immunoreacted with mature sperm and with a subset of round cells diagnosed as developing spermatids, which had been sloughed off from the testis at varying stages of acrosome formation. To rule out possible cross-reactivity of the mAb with leukocytes in semen, a leukocyte surface marker (anti-HLe-1) was used in conjunction with MHS-10. Round cell populations staining with MHS-10 did not stain with anti-HLe-1. The mAb MHS-10 is thus a promising probe for the identification and quantitation of immature germ cells in human semen.

Antibodies, Monoclonal↗

PubMed1990

Biochemical and morphological characterization of the intra-acrosomal antigen SP-10 from human sperm.

The human sperm protein SP-10 was previously defined as a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. By one- and two-dimensional immunoblots, we show that SP-10, extracted from ejaculated human sperm, demonstrated a polymorphism of immunogenic peptides from 18 to 34 kDa, a pattern that was conserved from individual to individual and was not altered by reducing agents. The majority of the antigenic peptides possessed isoelectric points of approximately 4.9. Immunocytochemistry on testis sections indicated that SP-10 was localized to round spermatids and spermatozoa within the adluminal compartment of the seminiferous epithelium. Immunofluorescence showed that SP-10 was not associated with the surface of acrosome-intact, ejaculated sperm. Light and electron microscopic immunocytochemistry localized SP-10 throughout the acrosome, and electron microscopic evidence demonstrated a bilaminar array in association with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After induction of the acrosome reaction with the ionophore A23187, SP-10 remained displayed on the sperm head in association with the inner acrosomal membrane and equatorial segment. The results indicate that the MHS-10 monoclonal antibody may be used as a marker of acrosome development in the human and as a probe to evaluate acrosome status. The results also support the hypothesis that inhibition of sperm-egg interaction by anti-SP-10 monoclonal antibody may occur as a result of antigen exposure following the acrosome reaction.

Acrosome↗

PubMed1990

Identification of human acrosomal antigen SP-10 in primates and pigs.

The intra-acrosomal human sperm protein SP-10 was previously designated a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. In the present study, a monoclonal antibody to SP-10 (MHS-10) was employed on Western blots to identify immunoreactive SP-10 in sperm extracts from baboon (Papio cyanocephalus anubis) and two macaques (Macaca mulatta and Macaca fascicularis). In each of these primates, the MHS-10 monoclonal antibody recognized a polymorphic pattern of immunoreactive peptides similar to that in humans. Immunoreactive SP-10 was also demonstrated in pig sperm. Using purified preparations of the previously described intra-acrosomal molecules acrosin and sperminogen in the pig, we observed that the MHS-10 monoclonal antibody did not react with these proteins, indicating SP-10 is distinct from these known acrosomal components. Sperm from several common species including the rabbit, bull, rat, guinea pig and cat did not immunoreact with the MHS-10 monoclonal antibody. By use of a radioactive probe spanning 628 nucleotides of the open reading frame for SP-10 on Northern blots of poly A + RNA obtained from testes of Macaca fascicularis, Papio papio, and Papio cyanocephalus anubis, a 1.35-kb mRNA of identical size to the mRNA from human testes was identified. These results indicate that baboons, macaques, and pigs may be appropriate models for testing an SP-10-based contraceptive vaccine.

Acrosome↗

PubMed1990

Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10.

cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.

Acrosome↗

PubMed1990

Inflammatory changes in the epididymis after vasectomy in the Lewis rat.

The epididymides of Lewis rats were studied at intervals up to 7 months after vasectomy, vasectomy followed 3 months later by vasovasostomy, or sham operations. Epididymal histology was related to testicular alterations and to serum antisperm antibodies as determined with an enzyme-linked immunosorbent assay. In vasectomy and vasovasostomy groups. 13 of 33 rats had testicular alterations, which consisted mainly of pronounced depletion of germ cells. Over half of the rats with testicular alterations also had severe epididymal lesions that included interstitial changes characteristic of an inflammatory response. These consisted of aggregates of mononuclear cells, including lymphocytes, plasma cells, and macrophages. The lumina of epididymides with interstitial changes contained polymorphonuclear leukocytes and/or macrophages. All animals with altered testes had greatly decreased numbers of epididymal sperm. In many instances, the lumen of the proximal cauda epididymidis was collapsed, and columnar cells of the epididymal epithelium contained many very large lysosomes. The distal cauda epididymidis was distended with sperm and debris. None of the rats that lacked testicular alterations showed epididymal changes. Mean serum antisperm antibody levels were significantly higher for rats with epididymal interstitial changes than for animals without such epididymal alterations. Infiltrations of inflammatory cells into the epididymal interstitium and lumen are part of the constellation of changes that occurs after immunization with testicular homogenates to produce experimental allergic orchitis. The observations reported here support the hypothesis that reproductive tract alterations after vasectomy in this model have an immune basis.

Animals↗

PubMed1990

Dynamics of a human seminal vesicle specific protein.

The present paper is concerned with the temporal alterations and tissue localization of a seminal antigen secreted by the human seminal vesicle. This antigen is recognized by antibody MHS-5, which is one of a set produced in mice by immunization with human sperm. The respective clone produced an antibody of the IgG1 subtype, which reacted with seminal fluid from over 400 normal donors and 21 semen samples from vasectomized men. Incubation of seminal vesicle secretion with either prostatic fluid or prostate specific antigen (PSA) resulted in degradation on the antigen. The experiments showed that MHS-5 antigen is a substrate for the serine protease PSA: Immunohistochemical studies suggested that MHS-5 is a "sperm-coating" antigen and is exclusively synthesized and secreted by the seminal vesicle.

Antigens↗

PubMed1990

Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T-6 antibody.

A monoclonal antibody, T-6, useful for detecting acrosome-reacted sperm based on an immunofluorescent assay, was employed to evaluate acrosomal status of human sperm that were tightly bound to hemisected human zonae pellucidae (hemizona assay). Over 90% of the bound sperm evaluated exhibited immunofluorescent patterns indicative of acrosome reaction. This staining method for evaluating the acrosomal status of sperm bound to the zona pellucida may enable definition of a group of male infertility patients heretofore not recognized.

Acrosome↗

PubMed1989

Purification of low molecular weight forms of seminal vesicle specific antigen by immunoaffinity chromatography on bound monoclonal antibody MHS-5.

A method has been developed for purification of the low molecular weight forms of seminal vesicle specific antigen (SVSA). Pooled, liquified seminal fluid was fractionated by CM cellulose chromatography followed by two cycles of monoclonal antibody affinity chromatography. Analysis of the final product shows microheterogeneity of the purified immunoreactive peptides in the range of 9-12 kDa. In one run, from 1138 mg starting material, 2.78 mg of SVSA protein was obtained, a recovery of 0.24% of the total protein in the starting material. The purified material as assessed by scanning densitometry of Coomassie stained gels is 99% pure. These findings indicate that the three-step chromatographic method is useful for purifying the low molecular weight forms of SVSA.

Antibodies, Monoclonal↗

PubMed1989

Electron microscopic immunolocalization of seminal vesicle-specific antigen in human seminal vesicle.

Seminal vesicle-specific antigen (SVSA) has been shown to be a polymorphic antigen represented by multiple immunoreactive peptides when fresh human semen is probed with monoclonal antibody (MHS-5) on Western blots. Semen samples collected directly into sodium dodecyl sulfate (SDS) demonstrate major immunoreactive peptide bands at 69-71 kDa and 58 kDa as well as a series of peptides of lower molecular mass. As semen liquefies, the higher molecular mass forms of SVSA are transformed into lower molecular mass bands, with 10-13 kDa immunoreactive peptides predominating after 8 h of liquefaction (McGee and Herr, Biol. Reprod. 37:431-439, 1987). In the present study, the 10-13 kDa form of SVSA was purified by preparative electrophoresis from SDS gels and a polyclonal antibody was generated in guinea pigs. Human seminal vesicle was fixed by immersion in combinations of glutaraldehyde and paraformaldehyde and embedded in Araldite or LR Gold. Both the guinea pig polyclonal antibody and the murine monoclonal antibody MHS-5 were employed to localize SVSA in human seminal vesicle by immunoelectron microscopy using Protein-A gold complexes. Gold particles were quantified in various subcellular compartments by a Videoplan computer. With either antibody probe, SVSA was found predominantly in the central electron-dense cores of secretory granules, with no staining evident over the electron lucent halo surrounding the granule core. With preimmune serum, the mean number of gold particles overlying secretory granules was 3/microns2; with polyclonal anti-SVSA, the mean number of particles observed over secretory granules was 182/microns2. This study represents, to our knowledge, the first fine-structural localization of a specific secretory protein to the electron-dense cores of secretory granules in principal cells of the human seminal vesicle.

Aged↗

PubMed1989

The influence of vasovasostomy on antisperm antibodies in rats.

Serum antisperm antibodies were studied in Sprague-Dawley rats after vasectomy and vasovasostomy. Animals received a bilateral vasectomy, a vasectomy followed 3 mo later by vasovasostomy, or sham operations. Blood samples were obtained at 1, 3, 4, and 7 mo, and antisperm antibodies were assayed by an enzyme-linked immunosorbent assay. After vasectomy reversal was performed at 3 mo, antisperm antibodies were significantly higher in rats in the vasovasostomy group at 4 mo than in animals that had a persisting vasectomy or sham operations. At 7 mo, the antisperm antibody level for the vasovasostomy group was approximately double that for the vasectomized rats. Spermatic granulomas occurred in 76% of rats after vasovasostomy. Antisperm antibody levels were higher in vasovasostomized animals with granulomas than in those lacking granulomas. The results suggest that vasovasostomy may stimulate an antibody response to sperm rather than lead to a reduced response, as was anticipated upon removal of the obstruction. Spermatic granulomas may serve as sires for continued antigenic challenge. The observed increase in antisperm antibodies after vasovasostomy in Sprague-Dawley rats may be related to their relatively low immunologic responsiveness to vasectomy, with vasovasostomy serving as a second major immunologic challenge, aided by the formation of an additional granuloma. In the more responsive Lewis strain, we previously observed a rise in antisperm antibodies after the initial vasectomy, with no further increase after vasovasostomy.

Animals↗