Biomedical subjects

J C Herr

Publications and source records attributed to J C Herr.

At least 55 records · Page 3Linked to original sources

PubMed1996

Inhibition of bovine fertilization in vitro by antibodies to SP-10.

The inhibiting effect on fertilization of antibodies to the intra-acrosomal protein SP-10 was studied in a bovine in vitro fertilization model. Indirect immunofluorescence showed that antibodies to human SP-10 localized to the acrosomal region of methanol-fixed, but not live, bovine spermatozoa, confirming the intra-acrosomal localization of bovine SP-10. The presence of the SP-10 protein in bovine spermatozoa was further established by immunoblotting of extracts of bovine spermatozoa with monoclonal antibodies and polyclonal antibodies to human SP-10. The antibodies identified a pattern of immunoreactive peptides similar to human, baboon, monkey and pig SP-10. Fertilization experiments in vitro demonstrated that monoclonal antibodies, as well as polyclonal antibodies, to human SP-10 significantly reduced fertilization rates of bovine oocytes by bovine spermatozoa. SP-10 antibodies exerted their anti-fertilization effect by reducing sperm-zona secondary binding. Consistent with the accessibility of the SP-10 antigen following capacitation, SP-10 antibodies also reduced the ability of capacitated spermatozoa to complete the acrosome reaction. Furthermore, SP-10 antibodies affected the motility of capacitated spermatozoa, while not affecting the motility of noncapacitated spermatozoa.

Acrosome↗

PubMed1995

Effects of vasectomy on the epididymis.

Common principles can be discerned in the response of the epididymis to vasectomy, despite species differences. Increases in the size and number of lysosomes are the most frequent changes in the epididymal epithelium. The presence or absence of additional alterations such as changes in the height of the epithelium may be related to variations in distensibility of the vas deferens and epididymis. Direct measurements by micropuncture of epididymal and seminiferous tubule hydrostatic pressure indicate that, contrary to dogma, increased pressure in the distal epididymis after vasectomy is not generally transmitted to the seminiferous tubules. The epididymal interstitium shows microscopic changes indicative of chronic inflammation, with infiltration of macrophages, lymphocytes, and plasma cells, and rats with these lesions have higher antisperm antibody levels than animals lacking epididymal changes. Macrophages and neutrophils may enter the duct through the epididymal epithelium, at sites of rupture of the duct, and in the efferent ductules. Cyst-like spermatic granulomas occur in virtually all species where the epididymis or vas deferens ruptures with escape of spermatozoa. The sites and timing of granuloma formation may depend on the mechanical properties of the tract in different species, and they are probably important in the immune response to vasectomy. Postvasectomy sera in Lewis rats recognize a consensus repertoire of dominant autoantigens that closely resembles the antigens bound by sera from rats immunized with isologous spermatozoa. There are multiple routes for disposal of the sperm that continue to be produced after vasectomy.

Animals↗

PubMed1995

The baboon expresses the calbindin-D9k gene in intestine but not in uterus and placenta: implication for conservation of the gene in primates.

Expression of the Calbindin-D9k (CaBP-9k) gene was studied in the baboon. Northern blot analysis using a human CaBP-9k cDNA probe detected expression in duodenum but not in uterus and placenta. Reverse transcription/polymerase chain reaction (RT/PCR) confirmed this expression pattern and indicated a high degree of identity between the baboon and human CaBP-9k mRNAs. PCR was employed to amplify the intron A region of the baboon CaBP-9k gene using human-derived primers and baboon genomic DNA. The baboon intron was closely related to the human CaBP-9k intron A, including the presence a complete Alu-repetitive element. Most significantly, a 13 nucleotide long element at the 5' end of the baboon intron matched exactly the human sequence. This element represents a nonfunctional variation of an estrogen response element found at the same location in the rat CaBP-9k gene. The rat element functions as an enhancer and mediates uterine and possibly placental CaBP-9k expression in the rat and probably most other mammals. The finding of a modified ERE in baboon as in human suggests that during primate evolution the expression of this mammalian-specific gene has been eliminated in uterus and placenta. This scenario raises the question of the role of CaBP-9k in these reproductive tissues.

Animals↗

PubMed1995

Characterization of alternatively spliced human SP-10 mRNAs.

Alternatively spliced mRNAs encoding the human intraacrosomal protein SP-10 were sought by the reverse transcriptase polymerase chain reaction (RTPCR). Eleven RTPCR products were identified, characterized, and found to represent authentic alternatively spliced SP-10 mRNAs. The 11 alternatively spliced SP-10 mRNAs encoded proteins ranging from 81 to 265 amino acids. The 10 smaller variants all resulted from one or two in-frame deletions in exons 2 and/or 3 of the SP-10 genomic sequence. Quantitative competitive RTPCR showed that the four largest SP-10 mRNAs represented the majority (> 99%) of the SP-10 message in testes from each of four men. The relative abundance of each of the four SP-10 mRNAs varied between individuals, but the longest SP-10 mRNA, SP10-1, which encoded a 265 amino acid protein, was consistently the most abundant, comprising 53-72% of the total SP-10 message. This was followed by the second largest SP-10 mRNA, SP10-2, which encoded a protein of 246 amino acids and comprised 15-32%. The third and fourth largest SP-10 mRNAs, SP10-3 and SP10-4, encoded proteins of 210 and 195 amino acids and accounted for 3.4-8.3% and 8.7-12.5% of the total SP-10 messages, respectively. The remaining 7 SP-10 mRNAs combined accounted for < 1% of the total SP-10 message. Within the low abundance group of mRNAs were two that deleted the entire third exon of SP-10. The present study suggests that phenomena of cryptic splicing and exon skipping occur within the SP-10 mRNA. Along with proteolysis, alternative splicing also helps to explain the heterogeneous forms of SP-10 that have been observed on Western blots of human sperm extracts.

Acrosome↗

PubMed1995

Antisperm autoantibody responses to vasectomy and vasovasostomy in Fischer and Lewis rats.

Antisperm autoantibodies were studied in Fischer and Lewis strains of rats after either vasectomy, vasectomy followed one month later by vasovasostomy, or sham operations. The time course of antibody response to sperm protein autoantigens was assayed by Western blot analysis of sera obtained at intervals up to 3 months. Rats of both strains responded to immunization with isologous spermatozoa with production of high titer hyperimmune sera. Sera from vasectomized Fischer rats showed antisperm antibodies on Western blots, but bands were stained with less intensity and frequency than for Lewis rats. In both Fischer and Lewis strains, major protein autoantigens were observed at 75-83, 68-71, 63, 57, 51, 41, and 21-23 kDa, lending support to the hypothesis that there is a set of dominant sperm autoantigens recognized by a consensus of postvasectomy rat sera. The lesser response of Fischer rats to vasectomy was not due to absence of dominant postvasectomy sperm autoantigens in Fischer sperm extracts, nor was it attributable to inability of Fischer rats to mount an immune response to these antigens, since immunization with isologous sperm was successful in raising antibodies to the dominant autoantigens. Vasovasostomy did not result in a general decrease in antisperm antibodies, and reactions to some antigens actually increased.

Animals↗

PubMed1995

Purification and characterization of a sperm antigen recognized by HSA-5 monoclonal antibody.

Among the monoclonal antibodies generated against acrosome-reacted human sperm, HSA-5 was shown to react with a sperm antigen localized predominantly to the equatorial region of the acrosome of human sperm and to the head and tail of mouse sperm. This antibody reacted with the methanol-fixed sperm, but not with fresh live sperm. When purified by immunoaffinity column, a major protein band with a molecular mass of approximately 100 kDa on SDS gel was isolated from fresh human sperm extract. The immunospecificity of isolated human sperm protein to this monoclonal antibody was verified by enzyme-linked immunosorbent assay and Western blot analysis. This antigen, designated as HSA-5, was susceptible to proteolytic degradation and revealed multiple immunoreactive bands in Western blot analyses of some preparations. Mouse sperm homogenates showed a similar polymorphic pattern to that of human samples. The tissue specificity of this antigen was examined immunohistochemically using various mouse and human tissues. HSA-5 did not cross-react with any other tissues except for sperm in adult testes and epididymis. This antibody also showed no binding activity to testicular tissue sections from mice of 13 and 21 days of age. The results of our study suggest that the sperm antigen recognized by HSA-5 monoclonal antibody is a differentiation antigen, which is expressed postmeiotically in testicular sperm but not in any somatic tissues.

Animals↗

PubMed1995

Epitope analysis of a sperm acrosomal antigen defined by HSA-5 monoclonal antibody.

Among the monoclonal antibodies recommended by the WHO Sperm Antigen Workshop for immunocontraceptive vaccine development, HSA-5 showed a high degree of sperm specificity and significantly inhibited in vitro fertilization in both humans and mice. Using a Western blot assay, HSA-5 was found to recognize a sperm antigen designated as HSAg-5 (human) or MSAg-5 (mouse) which ranged in molecular weight from 18 to 100 kDa. This monoclonal antibody was used as the probe for the immunoscreening of mouse testis cDNA libraries constructed in the lambda gt-11 expression vector. One of the positive cDNA clones was shown to have a cDNA insert of approximately 1 kb and to encode a recombinant fusion protein containing 77 amino acid residues in the C-terminal region of MSAg-5. This 1 kb cDNA insert was engineered in a pGEX vector to express a recombinant glutathione S-transferase fusion protein (GST-5). Using an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, both anti-GST-5 sera and the monoclonal antibody were shown to react with GST-5. The Northern blot of a mouse testis RNA preparation revealed that the isolated cDNA probe hybridized with a 4.0 kb mRNA. Several oligopeptides were synthesized based on the predicted C-terminal hydrophilic regions of the recombinant fusion protein. Using ELISA and a dot blot assay, peptide regions containing the immunogenic epitopes recognized by HSA-5 monoclonal antibody were identified.

Amino Acid Sequence↗

PubMed1995

Oral immunization with attenuated Salmonella expressing human sperm antigen induces antibodies in serum and the reproductive tract.

Induction of immune responses in the reproductive tract will be crucial for a functional gamete antigen-based antifertility vaccine. Here we describe the construction and development of an avirulent Salmonella as an oral vaccine delivery vector to elicit sperm-specific immune responses in reproductive tract secretions. A cDNA sequence encoding the human sperm antigen SP10 was cloned on an asd+vector and expressed to a high level in an avirulent delta cya, delta crp, and delta asd vaccine strain of Salmonella typhimurium. Oral immunization of female BALB/c mice with this recombinant Salmonella elicited high-titer anti-SP10 IgG antibodies in serum and IgA antibodies in vaginal secretions. Anti-SP10 antibody titers could be increased by secondary and tertiary oral administrations of the recombinant Salmonella. Induction of sperm-specific antibodies in the reproductive tract following oral administration of a recombinant Salmonella could lead to the development of a simple, safe, efficient, and easy-to-use antifertility vaccine.

Acrosome↗

PubMed1995

Complementary deoxyribonucleic acid cloning and characterization of mSP-10: the mouse homologue of human acrosomal protein SP-10.

Complementary DNA encoding the putative mouse homologue for human acrosomal protein SP-10, a candidate contraceptive vaccinogen, was cloned and sequenced. The entire open reading frame (amino acids 18 to 261) of the mouse SP-10 (mSP-10), with the exception of the signal peptide (amino acids 1 to 17), was placed under the influence of inducible T7 RNA polymerase/promoter system to overproduce recombinant protein (re-mSP-10) in Escherichia coli. A six-histidine tag, which was coexpressed at the carboxyl terminus of re-mSP-10, provided the means for purification of re-mSP-10 by immobilized metal chelation affinity chromatography technique. The level of purity of re-mSP-10 thus obtained was determined by 2-dimensional gel electrophoresis to be 98%. Immunoblotting with monoclonal and polyclonal antibodies previously generated against human or baboon SP-10 showed that mSP-10 shared significant antigenic similarity with its primate counterparts. The position of mSP-10 in the mouse genome was next mapped through segregation analysis of an interspecific backcross panel of 96 animals. Acrv1 (assigned gene symbol for mSP-10) was localized in the proximal portion of mouse chromosome 9 in a region that exhibits synteny with human 11q23, the region to which ACRV1 (gene symbol for human SP-10) was previously mapped. These characterizations by combined immunological and gene mapping techniques established the cloned mSP-10 to be the mouse homologue of SP-10.

Acrosome↗

PubMed1995

Testicular development and the formation of spermatic granulomas of the epididymis after obstruction of the vas deferens in immature rats.

PURPOSE: Aims were to determine the effects of obstruction of the vas deferens prior to sexual maturation on testicular growth and the formation of spermatic granulomas. MATERIALS AND METHODS: The vas deferens was ligated and divided bilaterally in 10-day-old Lewis rats. RESULTS: Testis weight and volume did not differ significantly between obstructed rats and sham controls at the majority of the intervals studied, including the end of the experiment at 128 days, although some temporary changes were observed. Testes of obstructed animals showed normal histological differentiation. Nearly all obstructed animals developed spermatic granulomas of the epididymis once they matured. CONCLUSIONS: When obstructed prior to sexual maturation, the testes are affected only transiently, but significant epididymal alterations occur. Spermatic granulomas are located predominantly in the epididymis rather than the vas deferens.

Animals↗

PubMed1994

Production in Escherichia coli, purification and immunogenicity of acrosomal protein SP-10, a candidate contraceptive vaccine.

The testis-specific human sperm antigen, SP-10, has been designated a 'primary vaccine candidate' by the World Health Organization Taskforce on Contraceptive Vaccines. Molecular cloning and sequencing of the cDNAs coding for human (h) and baboon (b) SP-10 have been reported. To produce large amounts of pure antigen for ongoing studies of the immunogenicity and anti-fertility effects of SP-10, we used an efficient Escherichia coli expression system. The full-length open reading frames for hSP-10 and bSP-10 were placed under the inducible T7 bacteriophage RNA polymerase/promoter system. An in-frame fusion was made such that a His6 stretch was produced at the C terminus of SP-10. Upon induction of gene expression, large amounts of hSP-10 or bSP-10 were synthesized and the recombinant (re-) protein segregated into an insoluble fraction. The protein was then solubilized in 6 M guanidine.HCl and purified by immobilized metal affinity chromatography (IMAC). The yield of purified bSP-10 preparation was approx. 20 micrograms/ml of culture. Immunoreactivity of the purified re-SP-10 with MHS-10, a monoclonal antibody specific to SP-10, and rabbit polyclonal sera raised against SP-10, indicated that the synthesized antigen was suitable for immunization studies. Four female baboons were then immunized with the re-bSP-10 antigen. Immunoblots using pre-immune and immune sera from these animals indicated that all four baboons produced antibodies that reacted with native SP-10 extracted from human sperm in a manner identical to that of MHS-10, the positive control. Immune sera also stained the acrosome region of human and baboon sperm heads by immunofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗

PubMed1994

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction.

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatozoa by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization.

Acrosome↗

PubMed1994

Temporal recognition of sperm autoantigens by IgM and IgG autoantibodies after vasectomy and vasovasostomy.

Temporal patterns of IgM and IgG autoantibodies to sperm proteins were studied by western blot analysis at intervals after bilateral vasectomy, vasectomy followed one month later by vasovasostomy, or sham operations. Responses were detected to eight major autoantigens at 21-23, 36, 41, 51, 57, 63, 68-71 and 75-83 kDa, by study of staining patterns of sequential serum samples from individual animals and by analysis of the incidence of reaction to each protein. The four lower molecular weight antigens (21-23, 36, 41 and 51 kDa) provoked mainly IgG responses. The strongly stained set of higher molecular weight antigens (57, 63, 68-71 and 75-83 kDa) tended to show more clearly defined temporal patterns of IgM followed by IgG response, including a high incidence of IgM antibody at the 2-week interval. Three of the larger peptides (57, 63 and 68-71 kDa) appeared highly immunogenic, since some reactions were detected even in sham-operated rats. The classical patterns of IgM and IgG antibody responses to the majority of the dominant sperm autoantigens are in accord with the hypothesis that vasectomy mimics immunization with spermatozoa. The high incidence of IgM antibodies in the earliest sample, taken 2 weeks after vasectomy, suggests that the initial immunizing event takes place within about a week after the operation. Vasovasostomy did not bring about a decrease in antisperm antibodies. Instead, some animals demonstrated an increased reaction to certain antigens after reversal of vasectomy, even though the vasovasostomies were anatomically successful.

Animals↗

PubMed1994

Early antibody response following vasectomy is related to fertility after vasovasostomy in glucocorticoid-treated and untreated Lewis rats.

The influence of treatment with a glucocorticoid on antisperm antibodies and fertility after vasectomy and vasovasostomy was studied in Lewis rats. Animals received a bilateral vasectomy followed 4 weeks later by bilateral vasovasostomy. Treatment with methylprednisolone for two months beginning at the time of the vasovasostomy resulted in a decrease in antisperm antibodies compared with nontreated vasovasostomized animals, but there was no difference in fertility between treated and nontreated vasovasostomized groups. However, when fertile vasovasostomized animals from treated and nontreated groups were compared with infertile vasovasostomized animals, antisperm antibodies were found to be significantly lower in fertile rats 2, 4 and 8 weeks after vasectomy, while antibodies did not differ between fertile and infertile animals at the end of the study (12 weeks). The observation that differences in antisperm antibodies appeared shortly after vasectomy, preceding either vasovasostomy or treatment, suggests that changes occurring very early after vasectomy have far-reaching effects and are among the factors that influence future fertility after vasovasostomy.

Analysis of Variance↗

PubMed1994

Tissue specificity of the acrosomal protein SP-10: a contraceptive vaccine candidate molecule.

The tissue specificity of the intra-acrosomal protein SP-10 was examined by Northern blot and polymerase chain reaction (PCR) analysis. Messenger RNA from 36 tissues in the female baboon (Papio papio) was isolated, separated on agarose gels, transferred to nylon, and probed with either SP-10, beta-actin, or cyclophilin cDNA. Northern blots, which were processed at both low and high stringency, showed SP-10 to be expressed exclusively in the testis. The mRNA from each tissue was also reverse transcribed, and both SP-10 and beta-actin were amplified by PCR from the resulting cDNA. Ethidium bromide-stained agarose gels of the SP-10 PCR products showed three clear bands from the testis but no co-migrating bands from the other tissues. Southern blots of the PCR products showed that only the three bands in the testis were related to SP-10. The data demonstrate that the SP-10 gene products are testis specific, a characteristic essential for a contraceptive vaccine candidate molecule.

Acrosome↗

PubMed1994

Human SP-10: acrosomal distribution, processing, and fate after the acrosome reaction.

SP-10 is a testis-specific acrosomal protein that has been detected in several species including humans. Extracts from whole human testis and epididymal, ejaculated, and capacitated sperm were analyzed by Western blot for SP-10 polypeptides. The testis extracts contained a full-length SP-10 protein at approximately 45 kDa as well as other immunoreactive SP-10 peptides at 32, 30, 28, and 26 kDa. Extracts from epididymal, ejaculated, and capacitated sperm contained several immunoreactive SP-10 peptides that co-migrated with the 32-26-kDa SP-10 peptides in the testis extracts. Epididymal, ejaculated, and capacitated sperm extracts did not contain the 45-kDa SP-10 peptide observed in testis extracts, but did contain immunoreactive SP-10 peptides from 25 to 18 kDa that were not detected in testis extracts. These results indicate that a full-length 45-kDa SP-10 precursor protein is present in the testis and that SP-10 peptides of 32, 30, 28, and 26 kDa result from proteolytic processing of the SP-10 precursor protein in the testis and/or alternative splicing. In addition, SP-10 peptides of 25-18 kDa were first detected in extracts of caput epididymal sperm and probably resulted from the proteolytic processing of the 45- and 32-26-kDa SP-10 peptides in the initial segment or caput epididymidis. Also, no additional SP-10 bands were detected in extracts of cauda epididymal, ejaculated, or capacitated sperm, suggesting that no further processing of the 32-18-kDa SP-10 peptides occurred during epididymal transit, ejaculation, and capacitation. Electron microscopic immunocytochemical observations of epididymal, ejaculated, and capacitated sperm revealed that colloidal gold labeling of SP-10 was most abundant within the principal segment and posterior bulb of the equatorial segment of the acrosome, while the colloidal gold labeling of SP-10 was sparse in the anterior equatorial segment of the acrosome. After a follicular fluid-induced acrosome reaction, SP-10 was detected on the inner acrosomal membrane in the equatorial segment and was associated with hybrid vesicles. This localization after the acrosome reaction is consistent with the hypothesis that SP-10 may be involved in sperm-zona binding or penetration.

Acrosome↗

PubMed1993

Ultrastructure of epididymal interstitial reactions following vasectomy and vasovasostomy.

The response of the male reproductive tract to vasectomy includes inflammation of the interstitial tissue of the epididymis. The pathogenesis of epididymal interstitial reactions and characteristics of the responding cells were studied by electron microscopy in Lewis rats at intervals following bilateral vasectomy, vasectomy followed 1 month later by vasovasostomy, or sham operations. In areas of interstitial reaction, numerous macrophages, monocytes, lymphocytes, neutrophils, and plasma cells occupied the connective tissue. Macrophages, containing many lysosomes and vesicles, aggregated and assumed the appearance of epithelioid cells. Processes of adjacent macrophages interdigitated with one another and closely approached the surfaces of lymphocytes. Many plasma cells with distended rough endoplasmic reticulum appeared in the interstitium. The majority of animals in the vasectomy and vasovasostomy groups exhibited epididymal interstitial changes by 2-3 months; the cauda epididymidis was the region most often affected. The ultrastructural features were indicative of chronic granulomatous inflammation and were consistent with an immune response that includes antigen presentation by macrophages to lymphocytes, lymphocyte differentiation, and local antibody production by plasma cells. The nearly complete absence of sperm or recognizable parts thereof in the interstitial tissue in the areas of the reactions suggests that these lesions formed in response to soluble antigens leaking from the duct. Vasovasostomy was not effective in reversing or retarding epididymal inflammation at the intervals studied.

Animals↗

PubMed1993

Stage-specific detection of mRNA for the sperm antigen SP-10 in human testes.

SP-10 is a sperm-specific, intra-acrosomal protein that is considered to be a vaccine candidate for immunocontraception. In the present study, in situ hybridization with biotin and 35S labeled riboprobes was used to determine the pattern of SP-10 mRNA expression in human testes. Both methods demonstrated SP-10 mRNA primarily in round spermatids found in stages I, II, and III of the seminiferous cycle. Morphometric analysis of silver grains with the 35S-labeled probe showed less SP-10 mRNA in spermatids at stages IV, V, and VI than in previous stages, and rarely was label found in spermatogonia or spermatocytes. The expression of SP-10 mRNA first appeared at stage I coincident with the appearance of the protein, which was shown previously to persist in the acrosomal matrix throughout spermiogenesis. The decrease in SP-10 mRNA occurred when spermatids underwent polarization, nuclear condensation, and elongation. The appearance of SP-10 mRNA in round spermatids suggests that increases in SP-10 transcription or SP-10 mRNA stability or both occur as spermatids develop from the Golgi phase to the cap phase. The subsequent decline of SP-10 mRNA, despite the persistence of the SP-10 protein in all spermatids, suggests that a decrease in SP-10 transcription or an increase in mRNA degradation occurs when spermatids elongate.

Acrosome↗