In situ polyethylene sebacate particulate carriers as an alternative to Freund's adjuvant for delivery of a contraceptive peptide vaccine--A feasibility study.

The present study evaluates the feasibility of particulate carriers of a biodegradable polymer polyethylene sebacate (PES) as an alternative to Freund's adjuvant in the design of a peptide vaccine formulation. The vaccine formulation comprised of PES and the antigen KLH conjugated 80kDa HSA peptide-1 dissolved in N-methyl-2-pyrrolidone (NMP)/NMP-water as solvent. The antigen revealed good stability and the formulations were readily syringeable. Intradermal injection of the formulations resulted in the formation of PES particulates in situ at the site of injection. The NMP formulations revealed larger particulates which elicited no immunogenic response when injected in rabbits. On the other hand the NMP-water formulation revealed formation of microparticles which were significantly smaller in size, in combination with a small fraction of nanoparticles. It elicited an antibody titer up to 1:3200 in rabbits following intradermal injection. Western blot confirmed generation of antibodies specific to the peptide. Contraceptive efficacy was confirmed by loss of sperm motility and head-to-head agglutination of sperms in the treatment group. Unlike the severe reactions observed with administration of Freund's adjuvant, only mild hypersensitivity reaction was observed with the PES formulations. The mild reaction coupled with the contraceptive efficacy observed suggested PES particulates as a viable alternative to Freund's adjuvant.

Development of antifertility vaccine using sperm specific proteins.

Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4), protein hyaluronidase (PH-20), and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80 kDa HSA) is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80 kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80 kDa HSA (Peptide NT) and its peptides (Peptides 1, 2, 3 and 4) obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH) conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80 kDA HAS suggest that the synthetic Peptide-1 of 80 kDa HSA is the promising candidate for development of male contraceptive vaccine.

Differential pathogenicity of SHIV infection in pig-tailed and rhesus macaques.

BACKGROUND: Differential pathogenicity has been observed in cynomolgus and rhesus macaques following primate lentivirus infection. However, little is known about the comparative susceptibility of pig-tailed macaques to lentivirus infection and diseases. METHODS: We compared the in vivo infectivity and pathogenicity of a CCR5-tropic SHIV(SF162 P4) after intravenous, intravaginal or intrarectal inoculation in rhesus and pig-tailed macaques. Plasma viral load, peripheral blood CD4(+) T cell counts and clinical signs were monitored. RESULTS: Both rhesus and pig-tailed macaques are similarly susceptible to SHIV(SF162 P4) infection by intravenous and mucosal routes. However, infection was significantly more robust in pig-tailed macaques than in rhesus, resulting in persistent viremia in 9/21 pig-tails vs. 2/24 rhesus (P < 0.013) and severe CD4(+) T-cell depletion in 2/21 pig-tails (vs. none in rhesus). CONCLUSIONS: Together with earlier observations, our findings underscore the importance of considering host genetic and immunological factors when comparing vaccine efficacy in different macaque species.

HOXBES2: a novel epididymal HOXB2 homeoprotein and its domain-specific association with spermatozoa.

The sperm from the testis acquires complete fertilizing ability and forward progressive motility following its transit through the epididymis. Acquisition of these characteristics results from the modification of the sperm proteome following interactions with epididymal secretions. In our attempts to identify epididymis-specific sperm plasma membrane proteins, a partial 2.83-kb clone was identified by immunoscreening a monkey epididymal cDNA library with an agglutinating monoclonal antibody raised against washed human spermatozoa. The sequence of the 2.83-kb clone exhibited homology to the region between 1 and 1097 bp of the homeobox gene, Hoxb2. This sequence was found to be species conserved, as revealed by RT-PCR analysis. To obtain a full-length clone of the sequence, 5' RACE-PCR (rapid amplification of cDNA ends PCR) was carried out using rat epididymal RNA as the template. It resulted in a full-length 1.657-kb cDNA encoding a 32.9-kDa putative protein. The protein designated HOXBES2 exhibited homology to the conserved 61-amino acid homeodomain region of the HOXB2 homeoprotein. However, characteristic differences were noted in its amino and carboxyl termini compared with HOXB2. A putative 30-kDa protein was detected in the tissue extracts from adult rat epididymis and caudal spermatozoa, and a 37-kDa protein was detected in the rat embryo when probed with a polyclonal antibody against HOXB2 protein. Multiple tissue Western blot and immunohistochemical analysis further indicated its expression in the cytoplasm of the principal and basal epithelial cells, with maximal expression in the distal epididymal segments. Northern blot analysis detected a single approximately 2.5-kb transcript from the adult epididymis. Indirect immunofluorescence localized the protein to the acrosome, midpiece, and equatorial segments of rat caudal and ejaculated human and monkey spermatozoa, respectively. In conclusion, we have identified and characterized a novel epididymal homeoprotein different from HOXB2 protein and hereafter referred to as HOXBES2, (HOXB2 homeodomain containing epididymis-specific sperm protein) with a probable role in fertilization.

Identification and characterization of oviductal glycoprotein-binding protein partner on gametes: epitopic similarity to non-muscle myosin IIA, MYH 9.

The mammalian estrogen induced oviductal glycoprotein (OGP) has been known to associate with capacitated sperm, oocytes and developing embryos. This study aimed to identify the putative binding partner of OGP on gametes using N-terminal peptide of bonnet monkey (Macaca radiata) OGP, Nmon, as bait. A protein(s) of molecular size approximately 54 kDa was detected by far-western blot analysis of detergent solubilized human sperm proteins. MALDI-TOF mass spectra analysis of approximately 54 kDa tryptic peptides gave a significant hit to non-muscle myosin heavy chain. Biochemical characterization of approximately 54 kDa was done with antibodies specific to non-muscle myosin IIA, MYH9. The approximately 54 kDa protein, possible breakdown product of MYH9, immunoreacted with MYH9 antibody in western blot analysis. OGP binding to approximately 54 kDa could also be demonstrated in far-western blot analysis of detergent solubilized human sperm proteins and nuclear matrix intermediate filament (NM-IF) preparations from human sperm and mouse oocytes. Far-western blot analysis of MYH9 enriched by immunoprecipitation identified the native approximately 220 kDa protein as OGP-binding partner. The identical and characteristic immunogold localization pattern of Nmon and MYH9 on sperm NM-IF preparation substantiated these findings. The results suggest that OGP binds to both gametes through its interaction with MYH9 through the non-glycosylated N-terminal conserved region of OGP, spanning the residues 11-137.

Effect of Lomodex-MgSO(4) in the prevention of reperfusion injury following unilateral testicular torsion: an experimental study in rats.

Fertility in patients treated for unilateral testicular torsion has been shown to be significantly reduced in all the reported series to date, implying that the present-day treatment requires further refinement in the form of adjunct pharmacotherapeutic intervention (Lomodex and MgSO(4)) in addition to scrotal exploration. Prepubertal Holtzman strain rats (35 days old) were used for our study. Two sets were formed with six groups of rats in each set. Rats were treated as follows: group 1, sham-operated group; group 2, torsion (4 h); group 3, torsion + detorsion (1 h); group 4, torsion + ATP-MgCl(2) + detorsion; group 5, torsion + Lomodex-MgSO(4) + detorsion; group 6, torsion + normal saline + detorsion. Whereas the first set of animals was sacrificed immediately at the end of experiment, animals in set 2 were sacrificed 8 weeks after the end of the experiment to look for the development of antisperm antibodies. Parameters studied were thiobarbituric acid reductase (TBAR) assay, histology of testicular tissue, and sperm agglutination test. Student's t-test was used for significance. With detorsion (149.95+/-30.68) there was a significant rise in the TBAR values (P<0.05) compared with torsion (57.39+/-14.47). Treatment with both Lomodex-MgSO(4) (40.74+/-6.39) and ATP-MgCl(2) (48.30+/-18.35) yielded TBAR levels comparable to those in the sham group (31.35+/-11.96). Similar injury was also seen on the contralateral testis, with detorsion (114.28+/-10.68) much more detrimental than torsion (40.59+/-15.02) and rescue seen following treatment with Lomodex-MgSO(4) (27.55+/-8.64) as well as ATP-MgCl(2) (38.61+/-12.23). Regarding th histology, with detorsion there was evidence of severe distortion of tubules, with almost all the tubules showing maturation arrest and a few tubules completely devoid of any germinal cells. Treatment with Lomodex-MgSO(4) as well as ATP-MgCl(2) showed preservation of tubular morphology. Our study failed to document the presence of agglutinating antibodies (antisperm antibodies) in any of the groups. Unilateral testicular torsion has bilateral effects and is a form of ischemia-reperfusion injury. Treatment of torsion by detorsion alone does not prevent testicular damage. The results of the present study show that administration of Lomodex + MgSO(4) prior to detorsion results in prolonged testicular salvage with a potential of subsequent improvement in semen quality and fertility and reduction in long-term morbidity. The presence of agglutinating antibodies could not be detected in the present study.

Studies with Synthetic Peptides of 80 kDa Human Sperm Antigen (80 kDa HSA).

PROBLEM: The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1-4) and endoproteinase Glu-C (peptides 5-6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies. METHOD OF STUDY: Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro. RESULTS: Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract. CONCLUSION: Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives.

Spermicidal activity of seed oil of Pongamia glabra.

The medicinal properties of seed oil of Pongamia glabra are well known in traditional Indian medicine. It has antimicrobial activity against several organisms. It is used in the treatment of herpes and scabies and, systemically, it is also used in the treatment of dyspepsia with sluggish liver. The present study demonstrates that in vitro, Pongamia oil has strong spermicidal activity.

Partial amino acid sequencing of 80-kDa human sperm antigen (80-kDa HSA).

An 80-kDa human sperm antigen (80-kDa HSA) has been identified as a sperm protein responsible for inducing immunoinfertility. Immunization with the purified protein induced infertility in male and female rats. Immunohistochemical and immunofluorescent studies have demonstrated that the antigen is specific to spermatozoa. The present study describes the partial amino acid sequencing of 80-kDa HSA. The homogeneous protein was electrophoretically transferred onto a PVDF membrane and the excised band of 80-kDa HSA was used to determine the partial N-terminal amino acid sequence. The protein was then subjected to enzymatic digestion with endoproteinase Lys-C and endoproteinase Glu-C. The partial amino acid sequence of the major peptides thus obtained was determined. The digestion with endoproteinase Lys-C generated 4 major peptides, two of which showed partial sequence homology with lactoferrin. Endoproteinase Glu-C digestion produced 3 major peptides. The sequences of the 2 peptides were determined for which no matches were found in the databank. These results confirmed earlier observations that 80-kDa HSA is a sperm-specific protein that is chemically distinct from any other protein involved in normal physiological process. Earlier studies have demonstrated that it is antigenic, efficacious, conserved, and could be a promising candidate for the development of an antifertility vaccine.

Characterization of 80 kDa human sperm antigen responsible for immunoinfertility.

PROBLEM: An 80 kDa human sperm antigen (80 kDa HSA) has been identified by western blot technique using serum of an immunoinfertile woman as a probe. The 80 kDa HSA has been subsequently purified from sperm extract and investigated for antifertility effects. METHOD OF STUDY: The purified 80 kDa HSA was used to immunize adult male and female rats. Rabbit anti 80 kDa HSA antibodies were used for immunofluorescent and immunohistochemical staining to demonstrate the presence of 80 kDa HSA on the sperm and to investigate its tissue distribution. The N-terminal sequence of native 80 kDa HSA and the peptides obtained by its endoproteinase Lys-C was determined. RESULTS: Active immunization of male and female rats with 80 kDa HSA caused infertility in all the immunized animals. Immunofluorescent staining showed its localization on the head region of the human and rat spermatozoa. While immunohistochemical studies showed its localization in the testes and epididymis but not in other somatic tissues. Partial amino-acid sequence analysis showed no sequence homology with any of the known protein in the database. CONCLUSIONS: 80 kDa HSA is a promising candidate antigen for immunocontraception as it is characterized and found to cause infertility upon active immunization. specific to spermatozoa and is conserved.

The antigonadotropic action of testosterone but not 7alpha-methyl-19-nortestosterone is attenuated through the 5alpha-reductase pathway in the castrated male rat pituitary gland.

The enzyme 5alpha-reductase plays a significant role in the prostate to amplify the action of testosterone (T) by converting it to a more potent androgen, dihydrotestosterone (DHT). The role of 5alpha-reductase in the testosterone feedback inhibition of gonadotropin secretion from the pituitary has not been elucidated. Therefore, we investigated the role of 5alpha-reductase on T action in in vitro and in vivo models. Castration has been reported to increase the 5alpha-reductase activity in pituitary glands. Hence, the effect of castration duration on the conversion of T to DHT by pituitary homogenates and the responsiveness of pituitary monolayer cell cultures to gonadotropin-releasing hormone (GnRH) challenge exposure were investigated. Incubation of [3H]-T with pituitary homogenates showed that the conversion of T to 5alpha-reduced metabolites was two- to threefold greater in pituitaries from rats who had been castrated for 14 days compared with those castrated for 1 day. In addition, the GnRH-stimulated release of LH from monolayer cell cultures of pituitaries from rats castrated for 1 day was twofold greater, whereas that from rats castrated for 2 weeks was six- to sevenfold greater compared with basal luteinizing hormone (LH) release. Hence we used rats castrated for 2 weeks to elucidate the role of 5alpha-reductase in T feedback inhibition. The inhibitory effects of the androgens T, 19-nortestosterone (19-NT), and 7alpha-methyl-19-nortestosterone (MENT) at 3 different concentrations (10(-9), 10(-7), and 10(-5) mol/L) on GnRH-stimulated LH release from monolayer cell cultures of pituitaries from rats castrated for 2 weeks were examined. All 3 androgens showed dose-dependent inhibition of LH release. MENT showed the greatest inhibition, followed by 19-NT and T. In the presence of finasteride (a 5alpha-reductase inhibitor), the inhibition of LH released by T and 19-NT were significantly greater. The inhibitory effect of MENT, which does not undergo 5alpha-reduction, was not altered by finasteride. In an in vivo study, rats castrated for 2 weeks received T with or without finasteride. There was a significantly greater suppression of serum LH in rats receiving T plus finasteride compared with those receiving T alone. These results suggested that 5alpha-reductase in the pituitary is not obligatory for the inhibitory action of T on gonadotropin secretion in the castrated rat. The action of MENT, a nonreducible androgen, on the pituitary is not affected by 5alpha-reductase.

Antifertility effects in rats actively immunized with 80 kDa human semen glycoprotein.

A 80 kDa human sperm antigen has been identified using the serum of an infertile woman having circulating antisperm antibodies. The antigen was then purified to homogeneity by gel permeation chromatography using HPLC (protein PAK-125 column) system and on FPLC (superose-12 column) system. The antigen was found to be a glycoprotein. The antigen was mainly localized in the postacrosomal region of the human sperm, while it was localized in the head region of the rat sperm as demonstrated by immunofluorescent staining. The presence of this antigen was also demonstrated in the human prostate and endometrium and in the rat testis; epididymis and the prostate by immunocytochemical staining. The purified protein upon active immunization in female rats caused infertility in 100 percent animals. While in male rats it caused infertility in 90 percent animals. On morphometric analysis of testicular tissue it was observed that there was no significant change in spermatogonia and spermatocytes, but significant decrease in spermatids and sperm number as well as daily sperm production in the immunized male rats. The epididymal spermatozoa were markedly reduced in number and were largely found to be agglutinated. The results suggest that 80 kDa human sperm antigen appears to be a suitable candidate for immunocontraception both in male and female.

Antifertility effects of human sperm antigen in female rats.

The sperm antigens responsible for inducing infertility were identified by Western blot technique using sera from an infertile woman with circulating antisperm antibodies. The 80 Kda was prepared from human sperm by extraction with 0.05% sodium deoxycholate in 0.01 M Tris-HCl buffer, pH 8.4 and fractionation with ammonium sulphate. The supernatant after 40% saturation ammonium sulphate extraction was separated by gelpermeation chromatography, using HPLC (Protein PAK 125 column) and FPLC (superose 12 column) systems. The homogeneity of the protein was established by SDS-PAGE and its molecular size was estimated to be 80 Kda and its isoelectric point was 4.5. The purified protein upon active immunization in female rats caused infertility in 100 percent animals. The data suggest that 80 Kda human sperm antigen has the potential for use as a contraceptive vaccine.

Modulation of lipid peroxidation in human spermatozoa and human prostate by prostatic inhibin.

Loss of sperm motility as a result of the production of hydrogen peroxide by lipid peroxidation is regulated by as yet unidentified prostatic factor(s). Inhibinlike peptide of prostatic origin isolated from human seminal plasma, with a molecular size of about 10,400 daltons, was studied for its effect on ascorbate-induced lipid peroxidation in human spermatozoa. Dose-related suppression of lipid peroxidation was observed at dose levels of 0.25, 0.5, and 1.0 micrograms. The data suggest that inhibinlike peptide could be one of the factors involved in the regulation of lipid peroxidation and thereby of sperm motility. Inhibinlike peptide also exhibited local action in both normal and benign hyperplastic human prostate tissue by enhancing the rate of lipid peroxidation. These findings have implications in the pathophysiology of the prostate.

Hormonal modulation of invitro biosynthesis of inhibin like peptide by human prostate.

The hormonal regulation of inhibin biosynthesis by human benign hyperplastic prostate tissue was studied by determining the incorporation of 3H-leucine. 3H-inhibin, synthesized de novo, was immunoprecipitated from the culture media using specific antibodies to human prostatic inhibin. Testosterone and estradiol had no effect on inhibin biosynthesis whereas hFSH and PMSG had stimulatory effect. A decrease in inhibin biosynthesis was noticed on the addition of LHRH, TRH, hCG, hLH and human prolactin.

Prostatic inhibin-like peptide quantified in urine of prostatic cancer patients by enzyme-linked immunosorbent assay.

This is a highly specific enzyme-linked immunosorbent assay (ELISA) for measuring prostatic inhibin-like peptide (PIP) in urine, in which we use penicillinase (EC 3.5.2.6) conjugated with PIP and, as solid phase, a polystyrene microtiter plate. We used this ELISA to measure PIP in 24-h urine specimens from men with prostatic cancer (PCa) and from age-matched controls. For prostatic cancer patients the mean +/- SEM urinary PIP of 36.1 +/- 5 micrograms/24 h was significantly (P less than 0.001) lower than the mean of 127.1 +/- 9 micrograms/24 h for the age-matched controls. PIP values for 30 samples measured by both ELISA and RIA correlated well (r = 0.985). We could detect as little as 1.56 ng of PIP in a sample. Analytical recovery of added PIP ranged from 91% to 104%. Mean CVs were 8.9% within-assay and 12.7% between-assay. We believe that this ELISA will be useful in assessing the status of PIP in men with normal and diseased prostates and in examining the function of the hypothalamus-pituitary-prostate axis.

Effects of prostatic inhibin and thyroid releasing hormone on lipid peroxidation in rat prostate.

Effects of prostatic inhibin and thyroid releasing hormone (TRH) on lipid peroxidation in rat prostate was studied in an in vitro system. It was found that both inhibited the lipid peroxidase activity thus having a protective role in the prostate.

Flow cytometric analysis of human spermatozoa treated with antiserum to human seminal inhibin.

Inhibin from human seminal plasma is structurally identical to sperm coating antigen. Using the flow cytometric technique it has been demonstrated that there is a positive correlation between initial motility of sperm and the amount of inhibin coated on the spermatozoal surface.

Modulation of prolactin release by a 25 kD peptide of human seminal plasma.

Prolactin-regulating factor (PRF) of molecular size of 25 kD has been isolated from human seminal plasma. This 25 kD factor inhibits circulating PRL levels in intact adult male rats to the extent of about 48% at dose level of 10 micrograms. Furthermore, in vitro incubation of pituitary demonstrated that PRF inhibits the release of PRL in the medium. PRF also interferes with the binding of I125 PRL to its receptors in liver, prostate, and spermatozoa. However, I125 PRF itself does not bind to these receptors. PRF seems to modulate PRL release as well as its binding to receptors. A sensitive, specific RIA was developed for PRF. Using the RIA, levels of PRF in seminal plasma were measured. PRF levels were low in vasectomized subjects as compared with controls. A negative correlation was noted with seminal plasma PRF levels and sperm count.

Suppression of lipid peroxidation in human spermatozoa by prostatic inhibin.

Loss of sperm motility owing to the production of hydrogen peroxide by lipid peroxidation is regulated by yet unidentified prostatic factor(s). Inhibinlike peptide (HSPI) of prostatic origin isolated from human seminal plasma and having a molecular weight of about 10,400 daltons was studied for its effect on ascorbate-induced lipid peroxidation in human spermatozoa. Dose-related suppression of lipid peroxidation occurred at a dose level of 0.25, 0.5, and 1.0 micrograms. HSPI may be one of the factors involved in the regulation of lipid peroxidation and therefore sperm motility.