Biomedical subjects

J C Herr

Publications and source records attributed to J C Herr.

At least 37 records · Page 2Linked to original sources

PubMed1999

Post-obstruction rat sperm autoantigens identified by two-dimensional gel electrophoresis and western blotting.

Although antisperm autoantibody responses to obstruction of the male reproductive system have been documented, information on the nature of the cognate sperm autoantigens has been limited. In the present study, the patterns of sperm autoantigens recognized by sera from rats after obstruction of the vas deferens or epididymis were studied by high resolution two-dimensional (2-D) gel electrophoresis and western blotting. Comparisons of patterns of autoantigens stained on 2-D western blots of sera from prepubertal vasectomy, prepubertal epididymal ligation and adult vasectomy groups revealed both similarities and differences. Sera from sham-operated animals showed no detectable reaction or much lighter staining of a small number of spots. Visualization of sperm autoantigens on 2-D western blots supported the hypothesis that there is a relatively small set of sperm proteins that can be regarded as dominant post-obstruction sperm autoantigens because they are recognized by multiple post-obstruction sera. The 2-D analysis revealed previously undetected distinctions in the autoantigens recognized after adult and prepubertal vasectomy, as well as variations with the site of obstruction. These differences in the response may be due in part to changes in antigens of spermatozoa in different parts of the tract and at different ages, as well as variations in exposure of sperm cell proteins to the immune system resulting from the sites of spermatic granulomas. Preparative 2-D gels and western blotting with post-obstruction sera are now being used to identify specific sperm autoantigens by microsequencing of selected proteins.

Animals↗

PubMed1999

Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.

The objective of this study was to identify those immunodominant sperm antigens recognized by antisperm antibodies (ASA) in the serum samples of infertile men and women. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing or nonequilibrium pH gradient electrophoresis, followed by PAGE. Serum samples from 15 infertile male subjects and 6 infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT) were analyzed by Western blotting followed by enhanced chemiluminescence (ECL). Serum samples from 10 fertile subjects (5 males and 5 females) that were ASA negative by IBT were used as controls. The ECL blots were analyzed by computer scanning to compare the immunoreactivity between serum samples from fertile and infertile subjects and to identify the antigens unique to the sera of the infertile subjects; 98 sperm auto- and iso-antigenic protein spots were recognized by sera from infertile males and females but not from fertile subjects. Based on vectorial labeling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility.

Antigens, Surface↗

PubMed1999

Sperm mitochondria-associated cysteine-rich protein (SMCP) is an autoantigen in Lewis rats.

A common repertoire of rat sperm antigens have previously been identified by Western blotting of sperm proteins with sera obtained after vasectomy or isoimmunization with sperm. Aside from a determination of their apparent masses, however, the biochemical characteristics of these antigens have remained unknown. In this study, a rat testis cDNA expression library was screened with polyclonal antibodies obtained from rats immunized with isologous spermatozoa to identify and sequence a full-length clone encoding rat sperm mitochondria-associated cysteine-rich protein (SMCP). The open reading frame of SMCP was expressed in the pET22b vector, and recombinant SMCP (rec-SMCP) was purified. Sera from rats that had been vasectomized or hyperimmunized with isologous sperm specifically recognized rec-SMCP whereas preimmune sera from these experimental groups did not react. Rabbit antiserum produced to rec-SMCP recognized rec-SMCP on Western blots and precisely immunolocalized SMCP to the mid-piece of rat sperm. On Western blots against sperm extracts, the rabbit antibody recognized a major protein band of approximately 22-25 kDa that co-migrated with bands of identical mass that were recognized by sera from hyperimmune or vasectomized rats. These findings demonstrate that SMCP is a sperm autoantigen, recognized following vasectomy, and an isoantigen, recognized by antibodies generated through isologous immunization with sperm.

Amino Acid Sequence↗

PubMed1999

FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa.

Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.

Amino Acid Sequence↗

PubMed1999

Round spermatid-specific transcription of the mouse SP-10 gene is mediated by a 294-base pair proximal promoter.

Spermiogenesis is the terminal phase of male germ cell differentiation during which haploid spermatids engage in coordinate expression of a number of testis-specific genes, including those specifying acrosomal proteins. To begin to understand the transcriptional regulation during acrosomal biogenesis, we initiated promoter analysis of the gene encoding the acrosomal protein SP-10. SP-10 was previously shown to be transcribed within Golgi-phase round spermatids in the human. The present study characterizes SP-10 gene expression during spermiogenesis in the mouse and identifies regions of the mouse SP-10 (mSP-10) promoter that are capable of driving round spermatid-specific transcription in vivo. Expression of mSP-10 mRNA was initiated in early round spermatids coincident with acrosomal biogenesis and was terminated prior to nuclear elongation. The core promoter of mSP-10 lacked a TATA box but contained a canonical initiator (Inr) element surrounding the transcription start site. Using transgenic mice, we showed that the -408 to +28-base pair (bp) or the -266 to +28-bp mSP-10 5' flanking region is sufficient to direct round spermatid-specific expression of a green fluorescent protein reporter gene. On the other hand, the -91 to +28-bp mSP-10 gene fragment lacked promoter activity in vivo. This is the first functional characterization of a testis-specific gene promoter active in early round spermatids.

Acrosome↗

PubMed1999

N-linked glycan of a sperm CD52 glycoform associated with human infertility.

In a benchmark study, Isojima and colleagues established H6-3C4, the first successful heterohybridoma immortalized from the peripheral blood lymphocytes of an infertile woman who exhibited high sperm-immobilizing antibody titers. The present report demonstrates the identity between the glycoprotein antigens recognized by the human H6-3C4 monoclonal antibody (mAb) and the murine S19 mAb, generated in our laboratory to sperm agglutination antigen-1 (SAGA-1). Both mAb's recognize N-linked carbohydrate epitopes on the 15-25 kDa, polymorphic SAGA-1 glycoprotein that is localized to all domains of the human sperm surface. Treatment with phosphatidylinositol-specific phospholipase C demonstrated that SAGA-1 is anchored in the sperm plasmalemma via a GPI-lipid linkage. Immunoaffinity purification and microsequencing indicated that the core peptide of the SAGA-1 glycoprotein is identical to the sequence of CD52, a GPI-anchored lymphocyte differentiation marker implicated in signal transduction. Comparison of anti-SAGA-1 and anti-CD52 immunoreactivities revealed that the sperm form of CD52 exhibits N-linked glycan epitopes, including the epitope recognized by the infertility-associated H6-3C4 mAb, which are not detected on lymphocyte CD52. Thus, the two populations of the CD52 glycoprotein on lymphocytes and spermatozoa represent glycoforms, glycoprotein isoforms with the same core amino acid sequence but different carbohydrate structures. Furthermore, mAb's to the unique carbohydrate epitopes on sperm CD52 have multiple inhibitory effects on sperm function, including a cytotoxic effect on spermatozoa in the presence of complement. These results are the first to implicate unique carbohydrate moieties of a sperm CD52 glycoform as target epitopes in the anti-sperm immune response of an infertile woman. Furthermore, localization of CD52 on all domains of the sperm surface coupled with the multiple sperm-inhibitory effects of antibodies to its unique carbohydrate moieties suggest opportunities for immunocontraceptive development.

Antibodies, Monoclonal↗

PubMed1999

Evidence for a unique N-linked glycan associated with human infertility on sperm CD52: a candidate contraceptive vaccinogen.

A major objective in developing a sperm antigen-based contraceptive vaccine for humans is the discovery of sperm surface immunogens that are functionally relevant and sperm specific. The latter criterion is deemed essential to avoid the possibility of inducing autoimmune disease upon vaccination. This review presents evidence that a unique carbohydrate epitope is synthesized in the human epididymis, is attached to the core peptide of CD52, a lymphocyte differentiation marker, and is subsequently inserted into the sperm membrane via a glycosylphosphatidylinositol anchor. This unique CD52 glycoform is localized to the entire sperm surface, functions as a potent target for agglutinating and cytotoxic antibodies, and is one of the few well-defined sperm surface glycoproteins indicated in human antibody-mediated infertility.

Antibodies, Monoclonal↗

PubMed1997

Immunological response in the primate oviduct to a defined recombinant sperm immunogen.

Assessment of immune responses in the oviduct is of importance in understanding reproductive tract responses to infections, vaccination against reproductive tract pathogens, or contraceptive immunogens. This review discusses a technique that permits repeated sampling of oviductal fluid from the same monkey at intervals spanning up to several years, and the analysis of antigen-specific immunoglobulins in the fluid. This technique is important to immunocontraceptive development because previous studies in primates have lacked information on oviductal immune responses and contraceptive efficacy may not correlate well with serum antibody titers. Thus, a reliable method of sampling oviductal fluid before and after immunization with a defined antigen is required to determine the quantity and type of local immune responses necessary to achieve contraceptive effects. Implantation of access ports proved useful for repeatedly aspirating oviductal fluid in vivo from cynomolgus monkeys that was free from artifactual contaminants and with no observable changes in the behavior or health of the animals. Subsequent assays of relative and absolute concentrations of antibodies in oviductal fluid and serum demonstrated the presence of IgA and IgG specific for the recombinant sperm immunogen SP-10 in fluid collected from the periovulatory oviduct of primates after intramuscular inoculations. The antibodies evoked by the recombinant sperm vaccinogen recognized the endogenous antigen target on both human and macaque sperm, lending support for the possibility of developing a contraceptive immunogen that prevents fertilization.

Acrosome↗

PubMed1997

Distribution of leukocytes in the epithelium and interstitium of four regions of the Lewis rat epididymis.

BACKGROUND: Leukocytes expressing different surface markers were studied in four regions of the epididymis of Lewis rats. Cells resembling lymphocytes or monocytes had been described in the epididymis, but previous studies differed as to their nature and immunologic significance. METHODS: Frozen sections were immunocytochemically stained with monoclonal antibodies W3/25, OX-8, OX-42, and RLN-9D3, which are directed toward markers on CD4+ cells, CD8+ cells, macrophages, and B lymphocytes, respectively. The concentration of stained cells in the epithelium and interstitial tissue of the initial segment, caput, proximal cauda, and distal cauda regions was determined by a procedure based on the optical disector method. RESULTS: CD4+ leukocytes were present in greater concentration than CD8+ cells or macrophages in both the epithelium and interstitial tissue of all four regions. In the epithelium, the concentration of CD8+ leukocytes was greater than that of macrophages in the initial segment, caput, and distal cauda. In the interstitium, however, the concentration of macrophages exceeded that of CD8+ cells in both parts of the cauda. Macrophages and T lymphocytes were generally present in greater concentrations in the interstitium than in the epithelium, especially in the more proximal parts of the epididymis. In contrast to T cells, B lymphocytes were not detected in the interstitium or epithelium of any of part of the epididymis, despite prominent staining of B cells in other locations. CONCLUSIONS: The epididymal epithelium of the Lewis rat contains many T lymphocytes, which may correspond to 'halo' cells. CD4+ leukocytes predominate in all regions of the epididymis. The interstitium may function as a reservoir of leukocytes for the epithelial compartment. The epididymis is not normally a site for local immunoglobulin synthesis.

Animals↗

PubMed1997

Human fertilin beta: identification, characterization, and chromosomal mapping of an ADAM gene family member.

Fertilin alpha/beta (PH30 alpha/beta) is a heterodimeric sperm surface protein containing binding and fusion domains with potential for interaction with integrin receptors on the oocyte. We report the cDNA cloning, deduced amino acid sequence, tissue specificity, and chromosomal mapping of human fertilin beta. Encoded by a 2205 nucleotide open reading frame, the deduced amino acid sequence of human fertilin beta contains pro-, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like (EGF) repeat, transmembrane, and cytoplasmic domains. Due to this domain organization, human fertilin beta has been identified as a member of the ADAM family, which is composed of membrane-anchored proteins having A Disintegrin And Metalloprotease domain. The amino acid sequence of human fertilin beta shares 90%, 56%, and 55% identity, respectively, to monkey, guinea pig, and mouse fertilin beta homologs. A phenylalanine-glutamate-glutamate (FEE) binding tripeptide within the disintegrin-like domain of human fertilin beta, homologous to other fertilin beta RGD-like (arginine-glycine-aspartic acid) tripeptides, could compete for recognition by integrins and other receptors. Northern analysis from 16 human tissues revealed human fertilin beta's 2.9 kb message only in testis, which raises interest in possible clinical applications of this molecule as a contraceptive vaccinogen. Human fertilin beta maps to chromosome 8, band p11.2, by fluorescence in situ hybridization and mouse/human somatic cell hybrid Southern hybridization.

ADAM Proteins↗

PubMed1997

A novel negative imaging technique for accurate localization of stainable proteins on complex two-dimensional autoradiograms.

This paper describes a fast, simple, and accurate method for localization of protein antigens in complex two-dimensional (2-D) autoradiograms where the precise position and identity of the protein is required. The method involves the creation of negative images on an autoradiogram through arrangement of image-intensifying screens. By placing a chromogenically stained 2-D gel or blot between the intensifying screen and the film, photons emitted from the intensifying screen are obstructed by the stained spots, thus creating a negative image on the film. The technique can be used in autoradiograms of proteins labeled with either 32P or (125)I radioisotopes. The technique permits analysis of radiolabeled, gold-stained and immunoreacted proteins on a single film and offers versatility by combining analysis of total protein patterns with specific identification of radiolabeled and/or immunoreacted protein spots. The technique is especially useful when selecting a subset of specifically radiolabeled proteins from the total protein pattern in 2-D gels or membranes for microsequencing.

Animals↗

PubMed1997

Appearance of 'natural' antisperm autoantibodies after sexual maturation of normal Lewis rats.

Serum antisperm antibodies were assessed quantitatively with an ELISA in normal male Lewis rats at intervals between ages 10 and 128 days, spanning the onset of puberty. Antisperm antibodies rose between 56 and 91 days, and were significantly higher in 91- and 128-day old rats than at earlier intervals. The animals underwent normal pubertal development as indicated by increases in weights of the seminal vesicles and ventral prostate. The rise in antisperm antibodies correlated temporally with events in the postnatal development of the male reproductive system, with the increase in antisperm antibodies most closely following the time when spermatozoa reach the epididymis and proximal vas deferens at approximately 56 days. The observation that serum antisperm antibodies increased only after sexual maturation suggests that some differentiation antigens of sperm are processed and presented to the immune system under normal circumstances in this strain. Western blot analysis showed that the sera from normal postpubertal Lewis rats bound several proteins, including bands of > 100, 82-75, 78, 68, 65, 63, 54-55, 42, 37, 35, 26, and 20-22 kDa. The majority of these autoantibodies were sperm-specific as shown by the absence of comigrating bands in western blots of somatic tissue extracts, although antibodies in postpubertal sera recognized certain other proteins in somatic tissues. Several protein autoantigens, defined by sera from postpubertal animals, matched dominant autoantigens recognized by antibodies produced in response to vasectomy, prepubertal vas obstruction, or immunization with spermatozoa. This finding indicates that the antisperm antibody responses following sperm immunization, vasectomy or prepubertal vasal obstruction represent accentuation of an autoantibody response to sperm that develops normally following puberty.

Age Factors↗

PubMed1997

Two-dimensional gel electrophoretic analysis of vectorially labeled surface proteins of human spermatozoa.

The objective of this study was to identify the repertoire of proteins exposed on the surface of ejaculated human spermatozoa. High-resolution two-dimensional gel systems for separation of human sperm and seminal plasma proteins were developed using both isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) followed by polyacrylamide gel electrophoresis (IEF/PAGE, NEPHGE/PAGE). Proteins were visualized by silver staining of gels and by electroblotting followed by gold staining. The protein patterns were analyzed by computer after laser or camera scanning. One thousand three hundred ninety-seven sperm proteins with a molecular mass between 5 and 160 kDa and isoelectric points (pI) from 4 to 11 were catalogued from silver-stained gels loaded with approximately 0.25 mg of NP-40/urea extracts of sperm harvested by Percoll density gradient centrifugation, and 1191 proteins were resolved following extraction with SDS/3-[3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate/urea. Analysis of seminal plasma proteins obtained from vasectomized patients revealed over 300 silver-stained proteins, which aided the identification of sperm-coating proteins acquired from secretions of the accessory sex organs. Sperm surface proteins accessible to vectorial labeling with 125I or N-hydroxysuccinimide biotin were identified; 181 protein spots were radiolabeled with 125I, while 228 protein spots were biotinylated, including several groups of protein isoforms. Cytoskeletal and intra-acrosomal control proteins were not iodinated or biotinylated, thus verifying the surface specificity of both labeling methods. Ninety-eight sperm surface proteins were labeled by both iodine and biotin, and 22 sperm surface proteins, representing five groups of protein isoforms, were shown to contain phosphotyrosine. A composite computer image showing the position of the dually vectorially labeled sperm surface proteins was constructed, together with a table of the proteins' molecular weight, pI, and relative concentration. In addition, novel isoforms of actin, beta-tubulin, PH-20, and several phosphotyrosine-containing proteins were identified in human sperm.

Biotin↗

PubMed1997

Biochemical characterization of sperm agglutination antigen-1, a human sperm surface antigen implicated in gamete interactions.

The anti-sperm monoclonal antibody (mAb) S19 was previously demonstrated to agglutinate human spermatozoa, inhibit sperm penetration of cervical mucus, and inhibit sperm-zona pellucida binding. These results implicated the cognate S19 antigen, designated sperm agglutination antigen-1 (SAGA-1), in gamete interactions and identified SAGA-1 as an attractive candidate for immunocontraceptive development. In the present study, evaluation of sperm agglutination with video microscopy showed that the S19 mAb rapidly and completely agglutinated human spermatozoa in a "tangled" pattern of agglutination. One- and two-dimensional immunoblot analyses identified SAGA-1 as a highly acidic, polymorphic sperm protein with an apparent molecular mass of 15-25 kDa and an isoelectric point of 2.5-3.0. Periodate treatment abolished this immunoreactivity, demonstrating that the S19 mAb reacted with a carbohydrate epitope and indicating that SAGA-1 is a glycoprotein. Absence of S19 immunoreactivity in postvasectomy seminal fluid implicated the testis, epididymis, and/or proximal vas deferens in the expression of SAGA-1. In solubility and phase partitioning assays, SAGA-1 was extracted from spermatozoa in Triton X-114 and exhibited the hydrophobic characteristics of integral and glycosylphosphatidyl inositol-anchored membrane proteins. These results identify SAGA-1 as a hydrophobic, highly acidic sperm glycoprotein that is localized on the entire sperm surface and has potential significance as a target for antibodies that inhibit sperm function and gamete interactions.

Antibodies, Monoclonal↗

PubMed1997

Oviductal antibody response to a defined recombinant sperm antigen in macques.

Macaque oviductal fluids were assayed for specific antibodies to the intra-acrosomal sperm protein SP-10 after immunizations with recombinant macaque SP-10 (re-mqSP-10), a candidate contraceptive vaccinogen. Access ports, consisting of a subcutaneous collecting reservoir and a catheter to cannulate the oviduct, were implanted into monkeys for repeated aspiration of oviductal fluid. Monkeys were inoculated i.m. once a month with an emulsion consisting of 2 mg re-mqSP-10 in a vehicle of squalene and mannin monooleate. Oviductal fluids and serum were collected during the periovulatory period for six menstrual cycles, and IgG and IgA antigen-specific antibodies in preimmune and immune fluids were compared by ELISA. Both relative and absolute concentrations of SP-10-specific immunoglobulins (Ig) were determined. Oviductal fluids from immunized animals showed significant increases in anti-SP-10 IgG at cycle 2 and at all subsequent intervals. Anti-SP-10 IgA significantly increased in oviductal fluid at cycles 4, 5, and 6. Serum anti-SP-10 IgG increased at cycle 2 and remained significantly elevated through cycle 6, while serum anti-SP-10 IgA was higher than in preimmune samples at cycle 4. Serum antibodies generated to the recombinant SP-10 recognized SP-10 extracted from macaque sperm on Western blots. Immunocytochemical staining of macaque and human sperm showed acrosomal immunofluorescence with both immune oviductal fluids and serum using both anti-IgG and anti-IgA secondary antibodies. This study demonstrates for the first time 1) IgG and IgA antibodies to a defined recombinant sperm-specific antigen in primate oviductal fluids after systemic immunization and 2) the recognition by primate oviductal fluid IgG and IgA of the endogenous contraceptive target on both human and macaque sperm.

Acrosome↗

PubMed1997

Sperm antigens and their use in the development of an immunocontraceptive.

PROBLEM: Overpopulation is a global problem of significant magnitude, with grave implications for the future. World population is predicted to reach 10 billion people by the year 2050, an increase of more than 75% over the current population. Development of new contraceptives is necessary, since current forms of birth control are unavailable to many individuals due to sociological, financial, or educational limitations. METHODS: In an effort to identify new contraceptive alternatives, sperm-specific antigens are under the investigation as the basis for immunological regulation of fertility through contraceptive immunization. Anti-sperm antibodies (ASA) inhibit sperm function in vitro, induce infertility in experimental models, and have been implicated in some cases of clinical infertility. Furthermore, the development of ASA in vasectomized men does not lead to physiological complications despite the persistence of these antibodies for years; thus, ASA induced by immunization of men and women may be similarly harmless. Immunization has several conceptual advantages as a method of fertility regulation. Employment of non-pharmacologically active agents, convenience of administration, low cost, and the potential for relatively long-lasting yet reversible effects. RESULTS: Various approaches have been taken to identify candidate sperm antigens for immunocontraceptive development. Studies that utilized monoclonal antibodies and polyclonal antisera generated against sperm preparations have identified such promising candidates as PH-20, fertilin, SP-17, and SP-10. In animal model, 100% contraceptive effects were induced following PH-20 immunization and the effects were reversible. More recently, ASA from the sera of infertile patients were employed to identify several candidates as expressed by testis cDNA libraries. Perhaps the most extensively characterized and effective immunogen candidate identified thus far is lactate dehydrogenase-C4 (LDH-C4). Active immunization with LDH-C4 suppressed fertility in a variety of mammalian species, including primates, and the reversibility of these effects was demonstrated. CONCLUSION: The successful results obtained thus far support the feasibility of an effective immunocontraceptive and indicate the importance for continued investigation of additional sperm antigens as contraceptive immunogen candidates.

Contraception, Immunologic↗

PubMed1996

Refinement of the differentiated phenotype of the spermatogenic cell line GC-2spd(ts)

A transformed spermatogenic cell line GC-2spd(ts), recently reported to express a protein marker of spermiogenesis, was tested for the presence of several mRNAs encoded by genes transcribed specifically in the testis and at precise stages of spermatogenesis. Northern blotting and reverse-transcriptase polymerase chain reaction techniques showed that mRNAs for the stage-specific marker proteins LDH-C4 (preleptotene), acrosin (premeiotic), protamine-2 (postmeiotic), and SP-10 (postmeiotic round spermatid stage) were not detected in GC-2spd(ts) cells. Flow cytometric analysis of GC-2spd(ts) failed to detect a peak indicative of the presence of haploid chromosomes. Furthermore, the HS-63 monoclonal antibody, employed in an earlier report to demonstrate putative proacrosomal granules, failed to recognize the SP-10 protein in extracts of human or mouse sperm or in GC-2spd(ts) cells and instead recognized proteins of different masses. In view of interest in this line as a model for analyzing molecular events of spermatogenesis, this refinement of the GC-2spd(ts) phenotype may aid others considering these cells for studies of terminal stages of sperm differentiation.

Acrosin↗

PubMed1996

Update on the Center for Recombinant Gamete Contraceptive Vaccinogens.

The overall goal of the Center for Recombinant Gamete Contraceptive Vaccinogens is to develop a contraceptive vaccine which will induce antibodies in the female reproductive tract at sufficient levels to block fertilization. It is envisioned that antibodies developed by this vaccine will act to agglutinate, immobilize or coat either the egg or the sperm in the oviduct, uterus, cervix or vagina. Thus, the vaccine will exert its effects before the fertilization event, as a "prefertilization contraceptive." The Center has focused its activities over the past 4 years on four areas: 1) sperm antigens: the identification and characterization of novel sperm surface molecules which are attractive candidates for inclusion in a vaccine; 2) zona antigens: the identification of synthetic peptides derived from the zona pellucida which possess contraceptive potential without adverse pathology; 3) oral vaccine delivery: the development of live Salmonella vectors which deliver vaccine antigens through the oral route and which will stimulate secretory immunity in the female reproductive tract; and 4) animal testing: the testing of vaccine formulations in in vitro fertilization models, in small animals, in monkeys and in baboons. In addition, preparations for human testing are underway through preparation of an Investigational New Drug Application to begin Phase I human trials of a LDH-C4 peptide vaccine.

Animals↗