Characterization of boar sperm cytoskeletal cylicin II as an actin-binding protein.

The presence of actin-binding proteins in the perinuclear theca of boar spermatozoa has been investigated, using stepwise extractions of proteins from sperm heads. Proteins extracted with the alkaline buffer 1M Na(2)CO(3), pH 11, were found to contain a 66kDa protein that binds F-actin in actin pelleting assays. Sequence studies and immunological characterization with antibodies specific for human cylicin II identified the 66kDa protein as the homologue of bovine and human cylicin II.

Actin-binding properties and colocalization with actin during spermiogenesis of mammalian sperm calicin.

The nucleus of mammalian spermatozoa is surrounded by a rigid layer, the perinuclear theca, which is divided into a subacrosomal layer and a postacrosomal calyx. Among the proteins characterized in the perinuclear theca, calicin is one of the main components of the calyx. Its sequence contains three kelch repeats and a BTB/POZ domain.

Y-autosome translocation and infertility: usefulness of molecular, cytogenetic and meiotic studies.

An apparently balanced reciprocal translocation 46,X,t(Y;6) (q11.23 approximately q12;p11.1) was observed in an infertile man with severe oligozooteratozoospermia.

Distribution of gelsolin in human testis.

Gelsolin, an actin-binding and severing protein present in many mammalian cells, was characterized in human testis. Although abundant in testicular extracts, gelsolin was not detected in purified spermatogenic cells by immunoblot analysis. Immunofluorescence studies of testis sections showed that gelsolin has two main localizations: peritubular cells and the seminiferous epithelium.

Molecular mapping of a Yq deletion in a patient with normal stature.

The proximal long arm of the Y chromosome probably contains a gene (GCY) involved in stature determination. Recent reports have proposed the critical region extends from interval 4B to interval 5G (or 5E). In the present study, the deletion breakpoint in a male adult patient of normal height with a 46,X,del(Yq) karyotype was defined by the use of sequence-tagged site markers.

Molecular localization of free thiols in human sperm chromatin.

The presence in human sperm nuclear proteins of a limited amount of unoxidized thiol groups, stabilized by reversible binding to zinc ions, has been presumed to play a role in the decondensation of sperm within the oocyte. In the present study, the number and molecular localization of free sulfhydryls in the major proteins of human sperm chromatin, protamines P1 and P2, were determined by alkylation of reactive thiols with 14C-iodoacetamide, isolation of protamines, and peptide mapping. Less than 1.

Interaction of human P1 and P2 protamines with DNA.

The interaction of two classes of human sperm protamines, P1 (HP1) and P2 (HP2, HP3, HP4), with DNA was investigated. Gel mobility shift assays with a range of DNA fragments of defined sizes show that, whatever its length, all the DNA is complexed with protamines at arginine to phosphate ratio of 0.1.

Interaction of mammalian sperm nuclear protamines and peptides derived thereof with immobilized zinc.

The interaction of mammalian and human protamines with zinc was studied by immobilized metal ion affinity chromatography (IMAC). The affinity of protamines containing blocked cysteine residues was found to correlate in part with the presence and number of histidine residues in the protamine structure: absence or low affinity of P1 protamines containing 0 or 1 histidine residue; high affinity of human P2 protamine containing 9 histidines. Nevertheless a fraction strongly retained on an IDA-Zn(II) column was observed for P1 protamines with one histidine in the N-terminal sequence (ram and boar protamines).

Abnormal expression of protein 4.1 in spermatozoa of infertile men with teratospermia.

Molecular defects responsible for morphologically abnormal spermatozoa (teratospermia) associated with male sterility are largely unknown. We report defective expression of protein 4.1, a cytoskeletal protein initially recognised in red cells.

Auto-antibodies to human sperm basic nuclear proteins in infertile and vasectomized men: characterization of antigens and epitopes recognized by antibodies.

The sera of vasectomized men and of patients with immune infertility were used to study the antigens and epitopes of sperm nuclear proteins that bind antibodies in these sera. No reaction with sperm histones was observed except for one serum. P1, P2 protamines and pro-P2 protamines were recognized by auto-antibodies.

P2 protamines from human sperm are zinc -finger proteins with one CYS2/HIS2 motif.

P1 (HP1) and P2 (HP2, HP3, HP4) protamines were isolated from human sperm nuclei in the reduced form and their interaction with zinc and cobalt was studied. One zinc atom per molecule of P2 protamines but not of P1 protamine was found. Absorption spectra of P2 protamines with cobalt were characteristic of a tetrahedral complex involving two histidine and two cysteine residues and with one cobalt per molecule.

The immune response to synthetic peptides of human protamines HP1 and HP2.

Peptides representing the amino-terminal sequence of protamines HP1 (sequence 1-12) and HP2 (sequence 1-11), the two major nuclear proteins of human sperm, have been synthesized. Rabbits were immunized either with peptide conjugated with a carrier or with free peptide. The resulting antisera were examined for their capacities to bind the homologous peptide, other peptides from protamines HP1, HP2, from ram protamine, a protein resembling HP1, and finally with the whole protamine.

Fractionation of basic nuclear proteins of human sperm by zinc chelate affinity chromatography.

Immobilized metal affinity chromatography was investigated for the fractionation of basic nuclear proteins of human sperm. Human sperm nuclei essentially contain two classes of protamines: a protamine of type P1 (HPl), rich in cysteine but with only one histidine, and three protamines of type P2 (HP2, HP3, HP4), rich in cysteine and histidine (nine in protamine HP2), potential ligands for transition metal ions. The critical conditions for metal affinity chromatography were defined: choice of metal, protein material and buffer, type of elution and sample loading.

Antibodies to sperm basic nuclear proteins detected in infertile patients by dot-immunobinding assay and by enzyme-linked immunosorbent assay.

The auto-antibody response in infertile men was investigated by means of immunoenzymatic methods, dot-immunobinding assay (DIBA), and ELISA, using, as antigens, human sperm basic nuclear proteins. Comparison was made, for the same patients, with antibody response to membrane antigens, detected by tray agglutination test (TAT), spermotoxic test (STT), and immunobead binding test (IBT). A very good agreement was observed between the two kinds of antibody responses.

Isolation and characterization of a VH domain fragment from monoclonal rat IgG of IgG1 and IgG2a subclasses.

Digestion with trypsin of monoclonal rat IgG of IgG1 and IgG2a subclasses produced two fragments, isolated only in dissociating media. The larger fragment (mol. wt.

Nuclear protein transitions in cuttle-fish spermiogenesis: immunocytochemical localization of a protein specific for the spermatid stage.

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis.

Studies of the IgE binding sites to rat mast cell receptor with proteolytic fragments and with a monoclonal antibody directed against epsilon heavy chain: evidence that the combining sites are located in the C epsilon 3 domain.

The binding sites of rat IgE to mast cell receptor were investigated by the use of proteolytic fragments and a monoclonal antibody to epsilon chain (MARE-1). Three main fragments were characterized by short-time papain digestion of IgE: F(ab')2-E, a fragment related to the C, 4 domain, and an asymmetric fragment corresponding probably to an IgE molecule with one proteolyzed C, 3 domain. Neither F(ab')2-E nor C, 4 could interfere with the binding of IgE to rat mast cells.

Differential reduction of the inter-chain disulfide bonds of rat immunoglobulin E: relation to biological activity.

Monoclonal rat IgE was reduced over a range of dithiothreitol (DTT) concns. The number of disulfide bonds reduced and their location in the IgE molecule were studied. One millimolar DTT was found to split the two inter-heavy-chain disulfide bonds of the C epsilon 2 domain while increasing DTT concn to 10 mM split the two inter-heavy-light-chain disulfide bridges.

Influence of vitamin A on the immune response of Schistosoma mansoni-infected rats.

Nutritional (vitamin A levels, weights), parasitological (adult worm burden, count of eggs in liver, stool examination) and immunological (IgE serum levels, anti-Schistosoma mansoni antibodies, lymphocyte stimulation by concanavalin A and S. mansoni antigenic extract) parameters were studied in three groups of rats, a non-infected and normally fed control group, a S. mansoni-infected but normally fed group, and a S.

Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses.

The optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal rat IgG of different subclasses are described. Digestion of IgG for 2-4 h at 37 degrees C with 1% (w/w) papaïn at pH 7.0 in the presence of 0.

Proteolysis of rat IgG subclasses by Staphylococcus aureus V8 proteinase.

Monoclonal IgG belonging to the four rat IgG subclasses (IgG1, IgG2a, IgG2b, IgG2c) and some IgG subclasses from normal rat serum were subjected to enzymatic degradation with Staphylococcus aureus V8 proteinase. The results show that only one subclass, IgG2b, is significantly cleaved by the enzyme, with the release of two main products identified as F(ab)2 and Fc-like fragments. This unique susceptibility of the IgG2b subclass represents therefore an easy means of identification and also offers a simple procedure for a preparation of F(ab)2 fragments from monoclonal IgG2b antibodies.

Subclass restriction of the antiidiotypic antibody response in the rat.

Antiidiotypic antibodies were induced in LOU/M rats by immunization with two myeloma proteins of LOU origin: IR-162 (IgE) and IR-418 (IgG2a). Antibodies to IR-162 were easily obtained after a limited number of immunizations with protein in soluble form; polymerization with glutaraldehyde did not enhance immunogenicity. Antibodies to IR-418 appeared only after a large number of immunizations with protein in polymerized form or with protein copolymerized with rabbit IgG.

Formation of biologically inactive polymers is responsible for the thermal inactivation of rat IgE.

The structural changes induced by heating rat IgE at 56 degrees C and relationship with loss of cytotropic activity were examinated in the present study. Circular dichroism spectrum of IgE heated at 56 degrees C showed irreversible changes in the peptide bond spectral regions: increase in beta-sheet structure, but no significant modifications in the aromatic side chain region. Thus, circular dichroism studies did not suggest important perturbations of the tertiary structure of the IgE molecule.

Conformational studies of rat monoclonal immunoglobulin E.

Two monoclonal IgEs (IR 2 and IR 162) were studied in terms of their conformational features by circular dichroism and differential u.v. absorption.