Regulation of acrosome reaction by Liprin α3, LAR and its ligands in mouse spermatozoa.

Zona pellucida-based induction of acrosome reaction (AR) is a popular and well-accepted hypothesis. However, this hypothesis is being challenged in recent years and it has been proposed that the cumulus cells might be the site of AR. In our previous study, we reported the presence of a synaptic protein Liprin α3 on sperm acrosome, and proposed its role in AR. This study was designed to understand the role of Liprin α3 and its interacting proteins in regulation of AR. It is observed that the presence of anti-Liprin α3 antibody inhibits the process of AR. Colocalization experiments demonstrate the coexistence of leucocyte antigen related (LAR) protein, Rab-interacting molecule (RIM) and Liprin α3 on sperm acrosome thereby completing the identification of all the members of RIM/MUNC/Rab3A/liprinα complex required for membrane fusion. This study demonstrates the effect of LAR ligands such as Syndecans, Nidogens and LAR wedge domain peptide on AR. We could see an increase in AR in presence of these ligands. On the basis of these data, we speculate that in presence of ligands or wedge peptide, LAR undergoes dimerization leading to inhibition of phosphatase activity and increase in AR. The presence of one of the ligands Syndecan-1 on cumulus cells led us to hypothesize that it is Syndecan which induces AR in vivo and thus another site of AR could lie in cumulus.

Identification and validation of novel serum markers for early diagnosis of endometriosis.

BACKGROUND: Non-invasive diagnosis of endometriosis is urgently required to prevent the long delay between the onset of symptoms and diagnosis. A biomarker that possesses both high sensitivity and specificity is greatly required. Here, we describe the use of a proteomic approach to identify potential novel endometrial antigens using sera from endometriosis patients and healthy controls, with evaluation of biomarkers for non-invasive diagnosis of endometriosis. METHODS: A cross-sectional study was conducted to identify specific endometrial antigens using 1D and 2D western blots in women with early endometriosis (n = 17), advanced endometriosis (n = 23) and without endometriosis (n = 30). Five immunoreactive spots were analyzed using matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry with MASCOT analysis. ELISAs were established for specific epitopes and autoantibody titres were estimated in an independent cohort comprising women with early endometriosis (n = 18), advanced endometriosis (n = 32) and without endometriosis (n = 27) for validation. RESULTS: The 2D western blot analysis resulted in the identification of three endometrial antigens, tropomyosin 3 (TPM3), stomatin-like protein 2 (SLP2) and tropomodulin 3 (TMOD3). Serum levels of antibodies against the epitopes from the immunodominant region of proteins TPM3, SLP2 and TMOD3 were significantly elevated in endometriosis patients when compared with controls. Sensitivity and specificity of serum anti-TPM3a-autoAb (61%, 93%), anti-TPM3c-autoAb (44%, 93%), anti-TPM3d-autoAb (78%, 89%), anti-SLP2a-autoAb (50%, 96%), anti-SLP2c-autoAb (61%, 93%), anti-TMOD3b-autoAb (61%, 96%), serum anti-TMOD3c-autoAb (78%, 93%) and anti-TMOD3d-autoAb (78%, 96%) were better than those of serum CA125 levels (21%, 89%) in the detection of early stages of endometriosis. CONCLUSIONS: Serum anti-TPM3a-autoAb, anti-TPM3c-autoAb, anti-TPM3d-autoAb, anti-SLP2a-autoAb, anti-SLP2c-autoAb, anti-TMOD3b-autoAb, anti-TMOD3c-autoAb and anti-TMOD3d-autoAb could be new markers for the early diagnosis of endometriosis.

Multiple endometrial antigens are targeted in autoimmune endometriosis.

Endometriosis is defined as the growth of endometrial glands and stroma in ectopic locations. Its aetiology is multifactorial, but autoimmunity has been shown to play a role in its onset and development. The present study aimed to investigate the presence of both IgG and IgM anti-endometrial antibodies in sera of endometriosis patients in comparison with age-matched controls, and to also investigate the cognate endometrial proteins involved. Sera from these groups were screened by western blot and immunohistochemistry. Thirteen out of the 40 sera tested were positive for IgG isotype, and 10/27 IgG negative patients were positive for IgM isotype. These findings indicate that endometrial antibodies of IgG and IgM classes could be detected in almost 60% of endometriosis patients. Of the various identified endometrial antigens, 30 and 45 kDa antigens were immunodominant in both IgG and IgM positive endometriosis patients. With immunohistochemistry, positive sera showed reactivity in luminal epithelium, glandular epithelium and stroma. These anti-endometrial antibodies might be partially responsible for failure of implantation leading to infertility. Identification of specific targets would be a help in understanding the pathophysiology of endometriosis, and would also help in setting up a non-invasive test for the diagnosis of endometriosis.

A testis specific auto-antigen TSA70 belongs to Odf2/Cenexin family.

The occlusion of testicular outflow following vasectomy leads to autoimmunity which is characterised by the production of antisperm antibodies. Although post-vasectomy autoimmune response has been reported in several species, very little is known about the sperm auto-antigens that are targeted. Using a vasectomised mouse, a number of monoclonal antibodies were generated with the aim of characterising the targeted sperm specific antigens. All the monoclonal antibodies were found to react with testicular proteins. One of the antibodies, D5E5 was then used for immunochemical characterisation of its cognate antigen that was found to be a testis specific autoantigen of - 70 kDa, termed TSA70. TSA70 is expressed postmeiotically in a stage specific pattern during spermiogenesis. TSA70 was observed to be conserved across the species as seen by its presence on rat, bull, marmoset and human spermatozoa. On mouse spermatozoa it was localised at the tip of acrosome and sperm tail as seen by indirect immunofluorescence. Following capacitation it was seen to spread all over the acrosome. However, the localisation on the acrosomal tip persisted even after acrosome reaction, which suggests that the antigen is likely to play a physiological role post acrosome reaction. Solubilisation with Triton X100, revealed the acrosomal component of TSA70 to be a matrix protein, whereas its counterpart on the tail had both a soluble as well as a particulate form. In vitro studies showed that the monoclonal antibody significantly reduced progressive motility of mouse spermatozoa. Preliminary sequence analysis showed that TSA70 belongs to the Odf2/ Cenexin family. These characterisation studies suggest that TSA70 is a conserved, testis specific sperm autoantigen with a definitive physiological role in reproduction.

Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization.

A rat epididymal protein of 27 kDa was identified using neonatal tolerization. This study reports the production and characterization of a polyclonal antiserum to this protein. ELISA was used to demonstrate that this antiserum reacts strongly with epididymal sperm proteins, but has little or no reactivity with testicular proteins. Western blot analysis revealed that this polyclonal antiserum recognized a 27 kDa protein extracted from the corpus epididymidis as well as from spermatozoa from the corpus and cauda epididymides, and immunostaining revealed the presence of the protein in the corpus to cauda epididymides. Stronger reactivity was observed in the supranuclear region and stereocilla of principal cells of the corpus epididymidis and in the luminal content of the corpus and cauda epididymides. The testicular section showed no reactivity. Treatment with the antiserum resulted in time- and dose-dependent agglutination of rat spermatozoa. By indirect immunofluorescence, the antiserum localized proteins in the mid-piece region of rat spermatozoa. Studies were carried out to determine the age at which the protein first became apparent during postnatal development. The protein was expressed from day 40 onwards, as demonstrated by western blot analysis. The androgen regulation of this protein was ascertained by castration and supplementation studies. Expression of this protein showed a decline starting at day 14 after castration and by day 21 the protein was absent; however, androgen replacement resulted in the reappearance of the protein. The results of these studies indicate that the protein identified is specific to the epididymis, and is regulated by development and androgens. The importance of epididymis-specific proteins that are regulated by androgens in sperm maturation is discussed, and the need to ascertain the sequence of the protein and clone the cognate gene is indicated.

Immunochemical and functional characterization of a polyclonal antibody to human sperm antigen.

A polyclonal antibody was raised against a 16 kDa human sperm protein identified by a monoclonal antibody to human sperm. The antibody showed significant reactivity with mouse spermatozoa as seen by ELISA. Immunohistochemical analysis showed that the antibody reacted with antigens from mouse testis, prostate as well as seminal vesicle. In both mouse and human testis the antibody localized antigens in round as well as elongated spermatids and mature spermatozoa. By SDS-PAGE and Western blot analysis the antibody reacted with a 16 kDa protein in the testis and seminal vesicle, whereas in the prostate it identified two proteins, one at 20 kDa and another at 25 kDa. Immunofluorescent localization by the antibody showed reactivity with acrosomal and/equatorial and midpiece region of human spermatozoa. The antibody showed extensive agglutination both in mouse and human spermatozoa. The results indicate that the antigen may be a conserved antigen. Cross reactivity of the antibody with mouse spermatozoa enabled us to carry out antifertility trials. Passive immunization of female mice with this antibody caused 67% reduction in fertility. It is likely that the antifertility effect could be partly due to agglutinating nature of the antibody which may have caused inhibition of all processes that depend on forward motility such as cervical mucus penetration and possibly preventing sperm egg interaction. Such well characterized and functionally relevant antibodies will enable to identify sperm antigens relevant for fertility. Identification of such antigens may also help in diagnosis of immuno infertility.

Spermatid-specific expression of the novel X-linked gene product SPAN-X localized to the nucleus of human spermatozoa.

Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%-37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88-6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27. 1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15-20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

Identification of epididymis-specific antigens using neonatal tolerization.

PROBLEM: Conventional immunization using whole sperm containing multiple antigens as the immunogen followed by hybridoma technology usually gives antibodies to antigens invariably of testicular origin, probably because of the strong immunogenic nature of these antigens. Therefore, an alternate approach of neonatal tolerization or subtractive immunization has been utilized to raise antibodies specific to epididymis by suppressing immune response to testicular antigens. METHOD OF STUDY: Neonatal mice were tolerized with testicular sperm proteins on days 0 and 5. These animals were then immunized with epididymal sperm proteins on day 21, followed by two boosters at biweekly intervals. Sera from these mice were used to localize epididymis-specific antigens. RESULTS: Sera from mice that were tolerized to testicular sperm proteins and later immunized with epididymal sperm proteins reacted only with epididymal proteins. CONCLUSION: The results of this study demonstrate that neonatal tolerization with testicular sperm proteins, followed by immunization with epididymal sperm proteins, enhances the production of antibodies to proteins exclusively of epididymal origin.

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.

Modification of radiosensitivity of chlorpromazine with biological metal ions in Thiobacillus ferrooxidans.

Effect of chlorpromazine with biological metal ions, viz. calcium, magnesium, zink and copper was studied on T. ferrooxidans cell system. Chlorpromazine, calcium and magnesium alone could produce radioprotection. Maximum radioprotection was exhibited by chlorpromazine at lower concentration while copper and zink offered radiosensitization. However, combination of chlorpromazine with all biological metal ions exhibited radiosensitization. Dose modifying factor by chlorpromazine at lower concentration (0.025 mM) was 0.754 while in combination with Ca2+, Mg2+, Cu2+ and Zn2+ was 1.08, 1.25, 1.37 and 1.389 respectively. The possible interaction between chlorpromazine and biological metal ions is discussed at cellular membrane level.

Antigenic stimulation and blocking of pregnancy in mice by an anti-idiotypic antibody to progesterone.

PROBLEM: The present study was carried out to see if the anti-idiotypic (anti-Id) antibody (Ab2) to progesterone could mimic the immunogenicity of the steroid hormone by giving rise to antiprogesterone antibodies (Ab3) and whether these antiprogesterone antibodies (Ab3) were biologically active. METHOD: Twenty virgin female Balb/C mice were actively immunized with the anti-Id antibody (Ab2). The antiprogesterone antibody (Ab3) titres in the serum were determined and the animals were used for fertility studies. In the passive immunization studies Balb/C female mice were injected i.p. with 100 micrograms of anti-Id antibody to see its effect on pregnancy. RESULTS: The actively immunized animals when mated showed 80% reduction in their fertility rate. The duration of infertility (20-121 days) in these animals could be directly correlated with the concentration of antiprogesterone antibody (Ab3). The anti-Id antibody (Ab2) blocked pregnancy in 80% of the passively immunized mice. CONCLUSION: The studies show that anti-Id antibody to progesterone could mimic the immunogenicity of progesterone and give rise to antiprogesterone antibodies (Ab3). The anti-Id antibody successfully blocked pregnancy in mice both after active and passive immunization.

Toxicities, LD50 prediction and in vivo neutralisation of some elapid and viperid venoms.

Toxicity levels of elapid (Naja naja and Naja oxiana) viperid (Vipera lebetina and Vipera russelli) venoms for mice and rat for intraperitoneal intravenous and intramuscular routes have been determined. The data have been analysed using a mathematical expression to calculate lethal venom concentrations in human snake bite cases. Further, in vivo neutralisation of snake venom potency (after experimental injection) using high voltage-low current electric shock treatment has been attempted. This treatment postponed the death further by 60-90 min in mice in case of elapid envenomation. In case of viperid envenomation such a postponement of death time was not noticed. The death postponement induced by the shock treatment probably refers to structural impairments that occur at molecular level in venom components and their consequent altered interactions with the target tissue or system.

Monoclonal anti-idiotypic antibody to progesterone with internal image properties.

An auto-anti-idiotypic approach was used to generate mouse monoclonal anti-idiotypic antibody E9F11F6 (Ab2) to progesterone. A conjugate of 4-pregnane 3,20 dione and bovine serum albumin was used as the immunogen. E9F11F6 bound to a rabbit anti-progesterone antibody in a linear concentration-dependent manner. It inhibited the binding of [3H]progesterone to a monoclonal anti-progesterone antibody, E9D5 (Ab1); this inhibition was concentration dependent. Results from chase experiments showed that this inhibitory activity of Ab2 was not due to steric hindrance but was a result of direct binding to the ligand combining site. Indirect immunofluorescence studies also showed that Ab2 and progesterone bound to similar binding sites on Ab1. Overall, the results suggest that E9F11F6 contains an internal image of at least part of the progesterone molecule, and that it can be classified as an Ab2 beta antibody. This anti-idiotypic antibody may be of value in further studies of endocrine and reproductive physiology.

Immunochemical localization and characterization of proteins from mouse cauda epididymis using human sperm specific monoclonal antibody.

Monoclonal antibodies (mAbs) to sperm specific antigens were generated by fusion of mouse myeloma cells (SP2/0) with splenocytes of female BALB/c mice hyperimmunized with washed human spermatozoa. The resultant hybridomas producing antisperm antibodies were first screened by ELISA. Out of these, one of the mAbs (D2G4) which recognized target antigens restricted to the acrosomal cap was chosen for these studies. The mAb D2G4 was found to be an agglutinating antibody and was also found to cross-react with mouse epididymal spermatozoa in ELISA and indirect immunofluorescence. The origin of antigens reacting with monoclonal antibody D2G4 was investigated. When frozen sections of murine testis and various regions of epididymis were reacted with mAb D2G4, only the cauda epididymal region was stained. Western blot of proteins from the epididymal spermatozoa and fluid indicated the presence of two bands of mol. wt 45 and 26 kd. These bands were identical under reducing and non-reducing conditions. These observations suggest that the two proteins are structurally similar or at least have a common epitope. These data indicate that the proteins recognized by D2G4 are acquired by spermatozoa during their passage and storage in the cauda epididymis.

Production and characterisation of a monoclonal antibody to progesterone and its effect on fertility.

A monoclonal antibody reacting with progesterone has been raised by fusion of mouse myeloma cells (SP20) and splenocytes of BALB/c mice hyperimmunized with 4-pregnane 3,20 dione conjugated to bovine serum albumin. Association constant of this antibody for binding with progesterone was 0.22 x 10(9) l/mole. The antibody was highly specific for progesterone. A single ip injection of this antibody brought about an antifertility effect which is influenced by genotype. Antibody treatment brought about a significant decrease in the fetal weight and a slight decrease in the plasma progesterone levels. The antifertility effect could be reversed only up to day 3 by exogenous administration of progesterone.

Interaction of central Asian cobra (Naja naja oxiana Eichwald) venom cytotoxins with phospholipase D.

Effect of cytotoxins from the venom of Naja naja oxiana Eichwald on the hydrolytic function of phospholipase D has been further analysed. Cytotoxins in the absence of Ca2+ activated the enzyme, whereas in its presence they inhibited it. Inhibition is shown to be related to the interaction of cytotoxins with the enzyme which blocks the absorption of the enzyme at the surface of the substrate phase. Synergism in the action of cytotoxin and phospholipase D was not noticed.

Effect of defoliant (butiphose) on morpho-physiological properties and enzyme systems of natural membranes.

Butiphose (Tributyltritiophosphate, (C4H9S)3PO) a commonly used defoliant in cotton growing regions of USSR, caused extensive alterations in morphological features of erythrocyte and nuclear membranes and affected the permeability properties of rat liver mitochondrial membrane. It disrupted Ca2+ transport system and other energy dependent processes in mitochondria. A reduction in the activity of cytochrome-c-oxidase and NAD.H-oxidase was also observed.