Presence of PSA auto-antibodies in men with prostate abnormalities (prostate cancer/benign prostatic hyperplasia/prostatitis).

Prostate-specific antigen (PSA), produced by the prostate, liquefies post-ejaculate semen. PSA is detected in semen and blood. Increased circulating PSA levels indicate prostate abnormality [prostate cancer (PC), benign prostatic hyperplasia (BPH), prostatitis (PTIS)], with variance among individuals. As the prostate has been proposed as an immune organ, we hypothesise that variation in PSA levels among men may be due to presence of auto-antibodies against PSA. Sera from healthy men (n = 28) and men having prostatitis (n = 25), BPH (n = 30) or PC (n = 29) were tested for PSA antibody presence using enzyme-linked immunosorbent assay (ELISA) values converted to standard deviation (SD) units, and Western blotting. Taking ≥2 SD units as cut-off for positive immunoreactivity, 0% of normal men, 0% with prostatitis, 33% with BPH and 3.45% with PC demonstrated PSA antibodies. One-way analysis of variance (anova) performed on the mean absorbance values and SD units of each group showed BPH as significantly different (P < 0.01) compared with PC and prostatitis. All others were nonsignificant (P < 0.05). Men (33%) with BPH had PSA antibodies by ELISA and Western blot. These discoveries may find clinical application in differential diagnosis among prostate abnormalities, especially differentiating BPH from prostate cancer and prostatitis.

Acute fasting-induced repression of the hypothalamic-pituitary-gonadal axis is reversed by RF-9 administration in the adult male macaque.

Recently, hypothalamic RFRP-3 (a mammalian ortholog of avian GnIH) signaling has been proposed as an important negative modulator of the reproductive axis. The current study examined whether repression of reproductive hormonal expression during short-term fasting conditions in higher-order primate is influenced by altered RFRP-3 signaling. Eight intact postpubertal male macaques (Macaca mulatta) were administered a single intravenous bolus of RF-9 (n = 4), a potent and putative RFRP-3 receptor antagonist, or vehicle (n = 4) following a 48-h fasting condition. Intermittent blood samples were collected every 30 min during the 4-h post-bolus period, and blood glucose, plasma cortisol, and testosterone concentrations were measured. Relative to fed conditions, fasting reduced glucose and testosterone levels (p < 0.005) and increased cortisol levels (p < 0.05). Relative to baseline, mean testosterone levels were elevated 150 min after RF-9 (p < 0.05) but not vehicle administration. In addition, elevated mean plasma testosterone levels following RF-9 administration were equivalent to levels observed in normal fed monkeys. These results suggest an important role for RFRP-3 signaling in conveying metabolic state information to the reproductive axis in higher primates.

Do men with prostate abnormalities (prostatitis/benign prostatic hyperplasia/prostate cancer) develop immunity to spermatozoa or seminal plasma?

Prostate is an immunocompetent and not an immunoprivileged organ. It has an active immunologic armamentarium. There are three major prostate abnormalities namely, prostatitis, benign prostatic hyperplasia (BPH) and prostate cancer. In all these abnormalities, infection/inflammation has been implicated. As infection/inflammation of the male genital tract can also be involved in induction of antisperm antibodies (ASA), this study was conducted to examine if these prostate abnormalities lead to the formation of ASA. Sera were obtained from normal healthy men (n = 20), men with chronic prostatitis (n = 20), men with BPH (n = 25), men with prostate cancer (n = 25) and immunoinfertile men (n = 10). The presence of antisperm antibodies against lithium diiodosalicylate (LIS)-solubilized human sperm extract (HSE), seminal plasma and synthetic peptides based upon sperm-specific antigens namely fertilization antigen (FA-1) and YLP(12), were analysed using the sperm immobilization technique (SIT), tray agglutination technique (TAT), enzyme-linked immunosorbent assay (ELISA) and indirect immunobead binding technique (IBT). All the sera from normal men and men with prostate abnormalities (chronic prostatitis/BPH/prostate cancer) were found to be negative in SIT and TAT. In ELISA, a few sera from men having prostate abnormalities (4-24%) showed a weak positive immunoreactivity (2-3 SD units) with some of the spermatozoa/seminal plasma antigens. Majority of the samples did not show any immunoreactivity (<2 SD units) in ELISA. Even the samples that showed a weak positive immunoreactivity in ELISA did not bind to live human sperm in IBT, indicating lack of sperm binding antibodies in these sera. In all these assays, the sera from immunoinfertile men were positive. Our findings indicate that chronic prostatitis, BPH and prostate cancer do not induce antibodies to spermatozoa, sperm-specific antigens and seminal plasma components. Although prostate is an immunologically competent organ, and its abnormalities cause a rise in circulating prostate-specific antigen (PSA), it appears that there is no concomitant induction of immunity to spermatozoa/seminal components including sperm-specific fertility-related antigens, thus not causing ASA-induced immunoinfertlity. This is the first study to our knowledge reporting the absence of ASA in men with BPH and prostate cancer.

Interactive effects of hydrogen sulphide and silicon enhance drought and heat tolerance by modulating hormones, antioxidant defence enzymes and redox status in barley (Hordeum vulgare L.).

Recent changes in climate have reduced crop productivity throughout much of the world. Drought and heat stress, particularly in arid and semi-arid regions, have seriously affected barley production. This study explored the separate and interactive effects of silicon (Si) and hydrogen sulphide (H2 S) on plant growth and mitigation of the adverse effects of heat stress (DS) and drought stress (HS) in a barley pot experiment. The impacts of simultaneous DS + HS were more severe than individual stresses due to increased ROS production, malondialdehyde (MDA) content and higher electrolyte leakage (EL), thereby leading to reduced water, protein and photosynthetic pigment content. Exogenously applied Si and H2 S alleviated the DS-, HS- and DS + HS-induced effects on barley by reducing ROS production, MDA and EL. A single application of H2 S or Si + H2 S increased plant biomass under all stress conditions, which can be ascribed to higher Si accumulation in barley shoots. A single application of Si or H2 S significantly increased plant biomass. However, Si + H2 S was the most effective treatment for metabolite accumulation and elevating activity of antioxidant enzymes to prevent toxicity from oxidative stress. This treatment also modulated osmolyte content, enhanced antioxidant activity and regulated the stress signalling-related endogenous hormones, abscisic acid (ABA) and indole acetic acid (IAA). Exogenous treatments regulated endogenous H2 S and Si and resulted in higher tolerance to individual and combined drought and heat stress in barley.

Assessment of phytoremediation potential of native plant species naturally growing in a heavy metal-polluted industrial soils.

The present study was carried out in Hayat Abad Industrial Estate located in Peshawar to assess the levels of cadmium (Cd) that were present in the soil as well as the plant parts (Roots and shoots). To evaluate the phytoremediation potential of the plants different factors i.e. Bioconcentration Factor (BCF), Translocation Factor (TF), and Bioaccumulation Coefficient were determined. These plants were grown in their native habitats (BAC). We have analysed, cadmium concentration from soil which are collected from 50 different locations ranged from 11.54 mg/Kg (the lowest) to 89.80 mg/Kg (highest). The maximum concentration (89.80 mg/Kg) of cadmium was found in HIE-ST-16L Marble City and HIE-ST-7 Bryon Pharma (88.51 mg/Kg) while its minimum concentration (12.47 mg/Kg) were detected in the soil of Site (HIE-ST-14L Royal PVC Pipe) and (11.54 mg/Kg) at the site (HIE-ST-11 Aries Pharma). Most plant species showed huge potential for plant based approaches like phyto-extraction and phytoremediation. They also showed the potential for phyto-stabilization as well. Based on the concentration of cadmium the most efficient plants for phytoextraction were Cnicus benedictus, Parthenium hysterophorus, Verbesina encelioides, Conyza canadensis, Xanthium strumarium, Chenopodium album, Amaranthus viridis, Chenopodiastrum murale, Prosopis juliflora, Convolvulus arvensis, Stellaria media, Arenaria serpyllifolia, Cerastium dichotomum, Chrozophora tinctoria, Mirabilis jalapa, Medicago polymorpha, Lathyrus aphaca, Dalbergia sissoo, Melilotus indicus and Anagallis arvensis. The cadmium heavy metals in the examined soil were effectively removed by these plant species. Cerastium dichotomum, and Chenopodium murale were reported to be effective in phyto-stabilizing Cd based on concentrations of selected metals in roots and BCFs, TFs, and BACs values.

Monoclonal antibody to a human germ cell membrane glycoprotein that inhibits fertilization.

A monoclonal antibody to an antigen in the human germ cell membrane did not agglutinate or immobilize sperm but inhibited binding and penetration of zona-free hamster ova by human sperm and blocked murine fertilization in vitro. The antibody, of the 2a subclass of immunoglobulin G, was germ cell-specific but not species-specific. It recognized a single antigen of 23 kilodaltons that has been isolated from human germ cells. This fertilization antigen, located on the postacrosome , midpiece, and tail of human sperm, is a glycoprotein of testicular origin associated with some types of human involuntary immunoinfertility .

Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development.

BACKGROUND: Contraceptive vaccines can provide valuable alternatives to current methods of contraception. We describe here the development of sperm-reactive human single chain variable fragment (scFv) antibodies of defined sperm specificity for immunocontraception. METHODS: Peripheral blood leukocytes (PBL) from antisperm antibody-positive immunoinfertile and vasectomized men were activated with human sperm antigens in vitro, and the complementary DNA prepared and PCR-amplified using primers based on all the variable regions of heavy and light chains of immunoglobulins. The scFv repertoire was cloned into pCANTAB5E vector to create a human scFv antibody library. RESULTS: Panning of the library against specific sperm antigens yielded several clones, and the four strongest reactive were selected for further analysis. These clones had novel sequences with unique complementarity-determining regions. ScFv antibodies were expressed, purified and analyzed for human sperm reactivity and effect on human sperm function. AFA-1 and FAB-7 scFv antibodies both reacted with fertilization antigen-1 antigen, but against different epitopes. YLP20 antibody reacted with the expected human sperm protein of 48 +/- 5 kDa. The fourth antibody, AS16, reacted with an 18 kDa sperm protein and seems to be a human homologue of the mouse monoclonal recombinant antisperm antibody that causes sperm agglutination. All these antibodies inhibited human sperm function. CONCLUSIONS: This is the first study to report the use of phage display technology to obtain antisperm scFv antibodies of defined antigen specificity. These antibodies will find clinical applications in the development of novel immunocontraceptives, and specific diagnostics for immunoinfertility.

Involvement of fertilization antigen (FA-1) in involuntary immunoinfertility in humans.

Sera from immunoinfertile patients (n = 32) and fertile controls (n = 20) were analyzed for cross-reaction with a purified and characterized sperm-specific glycoprotein, the fertilization antigen (FA-1), employing an enzyme-linked immunosorbent assay. The immunoinfertile sera demonstrated a strong reaction with FA-1 when compared with fertile control sera. There was no correlation between the reaction of sera with FA-1 and the titers obtained through the sperm agglutination technique and the sperm immobilization technique. Immunoinfertile sera showed binding with the protein bands in the regions corresponding to FA-1 on Western blots involving sodium deoxycholate-solubilized human sperm. Antigens isolated with immunoaffinity chromatography involving immunoinfertile sera also demonstrated antigen bands corresponding to FA-1 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the seven immunoinfertile couples, three that had antibodies to FA-1 in the male as well as female partners demonstrated a block of fertilization (IVF) due to antibodies bound on the sperm surface. The anti-FA-1 antibody activity was detected in serum as well as in follicular fluid and seminal plasma. Immunoinfertile sera that showed an inhibition of human sperm penetration of zona-free hamster ova showed a significant (P less than 0.001) increase in penetration rates after absorption with FA-1. These results indicate that sera from immunoinfertile patients had antibodies reacting with FA-1, and these antibodies are involved in the fertilization process.

Antiidiotypic antibodies to sperm in sera of fertile women that neutralize antisperm antibodies.

The presence of antiidiotypic antibodies (ab-2) to sperm was investigated in the sera of fertile, infertile, and virgin women using sperm-specific anti-FA-1 monoclonal antibody Fab'.ab-2 were detected in 71% (17/24) of sera from fertile women and in none (0/12) of the sera from virgin females by the enzyme-linked immunosorbent assay, Western blot procedure, and immunoprecipitation procedure. Sera from infertile women that had antisperm antibodies showed a minimal presence of ab-2, with only three sera (13%, 3/23) demonstrating the presence of low levels of ab-2. The ab-2 present in fertile women were capable of neutralizing the fertilization-inhibitory activity of anti-FA-1 antibody in a concentration-dependent manner in a human sperm penetration assay (SPA) of zona-free hamster oocytes. ab-2 were also capable of inhibiting the binding of antisperm antibodies to the sperm surface as determined by the immunobead binding technique. This is the first report demonstrating the presence of ab-2 in the sera of fertile women that are capable of neutralizing antisperm antibodies present in sera of infertile women. These findings suggest that the inability to detect antisperm antibody activity in the sera of fertile women may be due to higher levels of ab-2 present in these sera than levels found in sera of infertile women, although both groups may be producing antisperm antibody response after sexual exposure to sperm.

Human cleavage signal-1 protein; cDNA cloning, transcription and immunological analysis.

The cleavage signal-1 protein (CS-1), a doublet antigen comprised of approx. 14-kDa and 18-kDa proteins has been shown to be present on the surface of sperm of various mammalian species including humans. Polyclonal antibodies to CS-1 inhibit the early cleavage of fertilized eggs without apparently affecting sperm penetration and pronuclear formation. We report here the cloning of the human CS-1 cDNA and its expression in vitro to obtain the recombinant protein (reCS-1) molecule. The CS-1 cDNA clone was isolated by immunological screening of a human testis lambda gt11 cDNA library with mono-specific polyclonal antibody against CS-1. The cDNA is 1828 bp long; the start codon assigned to the first ATG (bp 98-100) encodes a protein with 249 amino acid residues terminating at TAA (bp 845-847). The cDNA isolated has a 97-bp 5' and a 984-bp 3' untranslated region. The potential polyadenylation signal (5'-AATAAA) is at bp 1803-1808. An extensive computer search of the GenBank database did not indicate any extensive homology with any known sequence, indicating that CS-1 is a unique protein. The CS-1 cDNA was cloned in the transcription vector, pGEM-11Zf, to obtain high-level in vitro transcription by SP6 and T7 RNA polymerase. The transcribed CS-1 RNA was translated in a rabbit reticulocyte in vitro translation system and produced a 33-kDa reCS-1 protein, as assessed by migration in a SDS-polyacrylamide gel.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of protein tyrosine phosphorylation of human sperm in capacitation/acrosome reaction and zona pellucida binding.

The aim of this article is to review the surface molecules that are involved in capacitation/acrosomal exocytosis and zona pellucida (ZP) binding in context of tyrosine phosphorylation leading to signal transduction in human sperm. During capacitation, at least 7 proteins (200, 112, 104, 48, 42, 31 and 25 kD) are phosphorylated as studied by the 32P metabolic labeling assay, and 14 proteins (122, 105, 95, 89, 73, 62, 48, 46, 40, 33, 30, 28, 25 and 22 kD) are autophosphorylated as demonstrated in the in vitro kinase assay. Of the 7-14 proteins, two proteins of 95 and 51 kD molecular identities were phosphorylated at tyrosine residues. Treatment with Talpha1 enhanced and anti-FA-1 monoclonal antibody completely blocked phosphorylation of all the relevant proteins. Sperm proteins belonging to four molecular regions, namely 95 kD (double band), 63 kD (one band), 51 kD (one band) and 14-18 kD (three bands) were involved in ZP binding. Three of these, namely 95 kD, 51 kD and 14-18 kD proteins demonstrated the presence of tyrosine phosphorylation, and the 51 kD protein (that is FA-1 antigen) also showed autophosphorylating activity. These findings, along with the other available data, indicate a vital role of protein tyrosine phosphorylation in sperm capacitation, acrosomal exocytosis and zona pellucida binding in humans. Since tyrosine phosphorylation is a primary/even exclusive indication of signal transduction, it appears that a signal transduction pathway is involved in fertilizability of human sperm.

Application of sperm antigens in immunocontraception.

Development of a vaccine(s) based on sperm antigens represents a promising approach for contraception. The utility of an antigen in immunocontraception is contingent upon its tissue specificity, involvement in human fertility, and immunogenicity. A number of antigens have been characterized from the sperm surface. Notable among these are LDH-C4, RSA antigens, PH-20, SP 1U, HSA-63, FA-1, FA-2 and CS-1. These antigens have been proposed as potential candidates for the development of contraceptive vaccine(s). Their current status, application, relative merits, and immunogenicity in immunocontraception are discussed in this review.

Prostate-specific genes: present status and future direction.

Prostate cancer (prostatic adenocarcinoma) is the second highest cause of cancer mortality in men and the benign prostatic hyperplasia (BPH) affects 80% of men by age 80. The current diagnosis of prostate cancer relies on the serum levels of the well-known molecule designated as prostate-specific antigen (PSA). PSA, however, has limited sensitivity and specificity in appropriately detecting the earlier stages of abnormal prostate growth. Additional molecules need to be identified that are prostate-specific and have better sensitivity and specificity that can detect prostate cancer and BPH at an earlier stage for clinical management. Presently, several laboratories are actively engaged in searching for such molecules. The aim of this article is to review the current status of various prostate genes reported in the literature that have been claimed to be prostate-specific with a function in normal and abnormal prostate growth and development. The long-term objective is to define the lacunae that exist in the literature in our search for an ideal antigen.

Comparison of ZP3 protein sequences among vertebrate species: to obtain a consensus sequence for immunocontraception.

The deduced ZP3 amino acid (aa) sequences of 13 vertebrate species namely mouse, hamster, rabbit, pig, porcine, cow, dog, cat, human, bonnet, marmoset, carp, and frog were compared using the PILEUP and PRETTY alignment programs (GCG, Wisconsin, USA). The published aa sequences obtained from 13 vertebrate species indicated the overall evolutionarily conservation in the N-terminus, central region, and C-terminus of the ZP3 polypeptide. More variations of ZP3 polypeptide sequences were seen in the alignments of carp and frog from the 11 mammalian species making the leader sequence more prominent. The canonical furin proteolytic processing signal at the C-terminus was found in all the ZP3 polypeptide sequences except of carp and frog. In the central region, the ZP3 deduced aa sequences of all the 13 vertebrate species aligned well, and six relatively conserved sequences were found. There are 11 conserved cysteine residues in the central region across all species including carp and frog, indicating that these residues have longer evolutionary history. The ZP3 aa sequence similarities were examined using the GAP program (GCG). The highest aa similarities are observed between the members of the same order within the class mammalia, and also (95.4%) between pig (ungulata) and rabbit (lagomorpha). The deduced ZP3 aa sequences per se may not be enough to build a phylogenetic tree.

Testis-specific proteins and their role in contraceptive vaccine development.

Development of a vaccine(s) based on sperm antigens represents a promising approach for contraception. The utility of an antigen in immunocontraception is contingent upon its testis/sperm specificity and involvement in spermatogenesis and/or fertilization. The aim of the present article is to review the information regarding the proteins that have been reported to be testis/sperm-specific and may have an important function in spermatogenesis and/or fertilization. The potential role of these proteins in the development an antisperm contraceptive vaccine(s) is discussed.

The role of carbohydrates in mammalian sperm-egg interactions: how important are carbohydrate epitopes?

Evidence implicating the involvement of carbohydrates in fertilization has been reported for decades in species which span the phylogenetic scale. The exact nature and role of these ligands in fertilization, however, has eluded investigators. Here, such investigations are reviewed as they relate to mammalian fertilization, with the principle focus on reviewing the role of carbohydrates involved in the primary binding event between sperm cell and egg.

Effects of sperm-reactive antibodies present in human infertile sera on fertility of female rabbits.

Sera (n = 19) from immunoinfertile patients were analyzed for cross-reaction with lithium diiodosalicylate (LIS)-solubilized human sperm extract (HSE), protamine and fertilization antigen (FA-1) using an enzyme-linked immunosorbent assay (ELISA). Among the sera tested, 63% reacted with HSE, 58% with protamine and 63% with FA-1. None of the sera from male or female infertile patients was found to contain immune complexes, indicating the antibodies were present in free form. The seven sera that reacted strongest with HSE inhibited human sperm function in sperm penetration of zona-free hamster ova and were associated with fertilization failure in human in vitro fertilization (IVF) technique. The six of these sera that showed binding to rabbit sperm, especially in the head region, also inhibited fertility in female rabbits. Antibodies reactive with FA-1 and not those reactive with protamine reduced fertility in female rabbits. These results indicate that mammalian sperm have several fertilization-related antigens that are evolutionarily conserved. These data also indicate that the rabbit can provide an animal model for studying antibody-mediated human infertility.

The fertilization antigen (FA-1) causes a reduction of fertility in actively immunized female rabbits.

Female rabbits were actively immunized against the fertilization antigen (FA-1) isolated from lithium diiodosalicylate (LIS)-solubilized murine testis. Three trials were performed in order to check the effect of immunization on fertility. In all of these trials, there was a significant (P less than 0.001) reduction of fertility as determined by the percentage of 9-day implants/corpora lutea ratio (FA-1, 0-26.3%; adjuvant control, 79.4-100%). A complete block was observed in animals which received intravenous booster immunization with the antigen. Antisera collected from FA-1-immunized rabbits were negative in the agglutination and the immobilization techniques, and demonstrated modal titers of greater than or equal to 1:2560 in the enzyme linked immunosorbent assay (ELISA) using FA-1. Antisera were tissue-specific and showed binding to the specific protein bands of 47,000 and 23,000 Mr, dimeric and monomeric forms of FA-1, respectively, in the Western blot procedure. Ova collected from rabbits inseminated with sperm which had been treated with antiserum from immunized rabbits showed reduced fertilization rates (anti-FA-1, 3.9-27.7%; control rabbit serum, 87.8%). There was again a reduction in percentage of the 9-day implants/corpora lutea ratio in the rabbits inseminated with treated sperm (anti-FA-1, 10.7%; control rabbit serum, 72.7%). It is concluded that active immunization with FA-1 resulted in a tissue-specific immune response which caused a reduction of fertility in rabbits, by a mechanism(s) involving an inhibition of the fertilization process.

Antibodies to sperm-specific human FA-1 inhibit in vitro fertilization in rhesus monkeys: development of a simian model for testing of anti-FA-1 contraceptive vaccine.

The feasibility of using the rhesus monkey as a non-human primate model for testing the efficacy of a contraceptive vaccine based on FA-1 antigen was evaluated. Affinity-purified anti-FA-1 polyclonal antibodies (Fab' fragments) and anti-FA-1 monoclonal antibody were used as probes in these studies. Anti-FA-1 antibodies (polyclonal Fab' as well as monoclonal IgG) predominantly reacted with the postacrosomal, mid-piece and tail regions of rhesus monkey sperm, as with human sperm, by an indirect immunofluorescence technique (IFT). These antibodies also specifically recognized a single protein band of 51 +/- 2 kDa, corresponding to the dimeric form of FA-1 antigen, on a Western blot of lithium diiodosalicylate (LIS)-solubilized monkey sperm. Anti-FA-1 antibodies, when present in the insemination mixture, inhibited the in vitro fertilization (IVF) of monkey oocytes. These results indicate that FA-1 antigen in rhesus monkey sperm is similar in subcellular localization, molecular identity and function to that in human sperm, and that the rhesus monkey represents a permissible non-human primate model in which the efficacy of a contraceptive vaccine based on FA-1 antigen can be tested.

Presence of expression products of c-erbB-1 and c-erbB-2/HER2 genes on mammalian sperm cell, and effects of their regulation on fertilization.

The present study was conducted to investigate the presence of expression products of c-erbB-1 and c-erbB-2/HER2 genes on mammalian sperm cell, and study the effects of their antibodies on fertilization. The mature sperm cells from various mammalian species (human, mouse, rabbit and rat) were found to have EGF-receptors but not the p185HER2 molecules by indirect immunofluorescence technique (IFT) and Western blot procedure. Though the EGF-receptors present on sperm cells were functionally active and responded to ligand binding, their activation by EGF or blocking by antibodies did not affect the sperm cells in acquiring their fertilization potential. These results indicate that the products of c-erbB-1 and c-erbB-2/HER2 genes, though they have been shown to have tyrosine kinase enzyme activity, do not seem to play a major role in the development of the fertilizing capacity of sperm cells.