Assessing oestrogenic effects of brominated flame retardants hexabromocyclododecane and tetrabromobisphenol A on MCF-7 cells.

Tetrabromobisphenol A (TBBPA) is the main flame retardant used in printed circuit boards and laminates. The human population is highly exposed to TBBPA as it is used in consumer electronics as well as office and communication equipment. The main use of hexabromocyclododecane (HBCD) is in insulation foam boards, which are widely used in the construction sector. Brominated flame retardants may possess endocrine disrupting activity and thus represent a threat to the environment, including humans and their reproduction. The aim of this work was to evaluate the oestrogenic effects of TBBPA and HBCD in vitro on MCF-7 cells. We used the proliferation test (E-screen assay) in MCF-7 breast cancer cells and reverse transcription quantitative polymerase chain reaction analysis of TFF1 gene expression to analyse oestrogenicity of the studied compounds. RT-qPCR has proved to be a fast and valuable molecular technique in gene expression quantification. HBCD but not TBBPA increased cell proliferation in MCF-7 cells and up-regulated TFF1 gene expression in a concentration-dependent manner. Anti-oestrogen ICI 182,780 inhibited up-regulation of TFF1 by HBCD. We have shown that HBCD displays oestrogen- like effects on MCF-7 cells. TBBPA, on the other hand, has not shown any oestrogenic effect mediated by the oestrogen receptor α.

The influence of fluorides on mouse sperm capacitation.

Increasing infertility, due to pathological changes on sperm, has become a serious issue. Eco-toxicological effect of rising concentration of fluorides can be enhanced in the presence of aluminium ions by forming fluorometallic complexes, analogues of phosphate groups that interfere with the activity of G-proteins and P-type ATPases, which are part of several signalling pathways during sperm maturation. In order for sperm to gain fertilizing ability, they must undergo in the female reproductive tract, capacitation that includes tyrosine phosphorylation and consequent actin polymerization. The present paper reports the findings of 3-month oral toxicity in mice of fluorides at the concentrations 0, 1, 10, and 100ppm and their synergic action with aluminium at dose of 10ppm. There were no mortalities, clinical signs of discomfort or body weight loss during the experiment. The analysis revealed, for the concentrations of 10 and 100ppm, abnormalities of spermatogenesis and ability of epididymal spermatozoa to capacitate in vitro, as the result of decreased sperm head tyrosine phosphorylation and actin polymerization. The enhancing overload caused by fluorides represents a potential factor, having an impact on function of sperm, hence contributing to a growing infertility in the human population.

The optimization of the protocol for immunofluorescence on fish spermatozoa.

In comparison with mammals, the fertilization of fish occurs predominantly outside the organism in a water environment, where fish spermatozoa require specific conditions to interact with oocytes. It is evident that optimal conditions for fish and mammalian spermatozoa are quite different. This paper describes a special approach to handling fish (common carp and Siberian sturgeon) spermatozoa in comparison with the samples originating from mammals (boar). This approach concerns not only the differences in the composition of the media applied but also primarily emphasizes the concrete parts of the immunofluorescence protocol determining accurate results. Individual parts of the protocol for indirect immunofluorescence of mammalian sperm were changed step by step and modified protocols were applied to immunofluorescence experiments with carp and sturgeon spermatozoa. By evaluating the changes in the integrity of the fish sperm head and flagellum, we selected the steps and corresponding conditions that are crucial for handling the fish spermatozoa. Based on our results, it may be concluded that when working with fish spermatozoa, the cells attached to the microscopic slides must not desiccate prior to the fixation, which is a usual step when working with mammalian sperm. The second crucial step is the necessity to fix the fish spermatozoa, especially when the research is focused on the structure of the flagellum. The impact of the temperature conditions is rather low, but working at low temperatures, except for the period of incubation with antibodies, leads to a higher number of unaffected cells.

Origin, localization and binding abilities of boar DQH sperm surface protein tested by specific monoclonal antibodies.

Seminal plasma proteins bind the sperm surface at ejaculation and may modulate several aspects of sperm activity during reproduction. DQH sperm surface protein, present in boar seminal plasma, shows affinity to phoshorylcholine, acidic polysaccharides, oviductal epithelium and zona pellucida glycoproteins. Monoclonal antibodies (MAbs) against DQH protein were prepared and used for determination of the DQH protein origin in boar reproductive organs, its localization on boar spermatozoa, and for investigation of its binding abilities in the porcine oviduct and to the zona pellucida of the oocyte. The mRNA transcript of DQH protein was found in seminal vesicles and not in the testis, epididymis and prostate. Its translated products were immunodetected by MAbs in seminal vesicle extract and fluid, in seminal vesicle tissue sections and on the membrane-associated acrosomal part of ejaculated spermatozoa. These results confirm the ability of DQH protein to bind the sperm surface at ejaculation and to participate in formation of the sperm reservoir in the porcine oviduct. Moreover, monoclonal antibodies reduced binding of sperm to oocytes and proved the role of DQH protein in the sperm-zona pellucida primary binding.

Ultrastructure and morphology of spermatozoa in Chinese sturgeon (Acipenser sinensis Gray 1835) using scanning and transmission electron microscopy.

The Chinese sturgeon (Acipenser sinensis Gray 1835) is an endangered anadromous sturgeon inhabiting the Yangtze River in China. In this study, the ultrastructure and morphology of spermatozoa was studied using transmission and scanning electron microscopy with a cryo-holder. The spermatozoon consisted of an elongated head with a distinct acrosome and nucleus region, a midpiece and a flagellum. The mean length of the head and midpiece, the flagellum and total length of spermatozoon were 4.48, 33.3 and 37.8 microm, respectively. The nucleus was an elongated trapezoid shape with anterior (acrosome) end narrower than the posterior. Granular material and an actin filament were observed within the anterior acrosome. Three to five endonuclear canals were present. The midpiece was eudipleural along its longitudinal axis. Compared to other sturgeon species, the data from the present study suggest a more recent evolutionary linkage between Chinese sturgeon and white sturgeon (Acipenser transmontanus Richardson 1836).

[Inhibin B and intraacrosomal proteins in men from the couples with fertility disorders]. / Inhibin B a intraakrozomální proteiny u muzu z páru s poruchou fertility.

THE AIM: To monitor the basic andrologic and immunologic sperm factors and the levels of inhibin B in serum and in seminal plasma in men from the couples with infertility disorders. SETTING: Department of Gynecology and Obstetrics, Medical School, Charles University and University Hospital, Plzen, Institute of Molecular Genetics, AV CR, Prague, Institute of Clinical Immunology and Allergology, LF UK a FN, Plzen. METHODS: We used conventional methods for estimation of sperm quality according to WHO and we detected the intra-acrosomal proteins by monoclonal antibodies (Hs8 and Hs14, immunofluorescent method), spermantibodies by direct mixed antiimunoglobulin reaction (MAR) test, and we examined inhibin B in serum (< or =400 pg/ml= A) and in seminal plasma (< or = 600 pg/ml= N) by ELISA in 355 men aged 21-52 years (ø 34 years) with normal levels of FSH, LH and testosterone. The control group was created by 56 health sperm donors. RESULTS: We found 65% normospermatics in the group of 355 patients, 34.9% men with various kind of pathologies. Predominance of spermagglutinating antibodies was found in 15.77% in IgG, in 19.44% in IgA, in 8.44% in IgA and IgG together. Normal intraacrosomal proteins were reached in 74.65% for Hs8, in 20.85% pathologic, in 86.2% normal findings for Hs14, in 4.23% pathologic. The immunological results in control group were completaly negative. Pathological levels of inhibin B in seminal plasma was found in 37.2% (152 men), in 25% in serum, and in 5.6% in serum and in seminal plasma together. In 54.7% of patients we found physiological levels of inhibin B in both biological fluids. We also compared physiological 109/152 (71.71%), and pathological spermiogrammes 43/152 (28.29%) with abnormal levels of inhibin B in seminal plasma, with intraacrosomal proteins to levels of inhibin B in serum. Our detailed study shows high interidividual results, which must be studied in complex with diagnosis of decreased fertility in man. CONCLUSION: Andrologic and immunologic analysis in the group of 355 men showed normal parameters of spermiogrammes in 231 patients (65%), in the rest of men the immunologic profil was in various parts pathologic. Only 105 men have got excellent spermiogrammes. Inhibin B as hormon regulates in back the secretion of FSH, and serves as good indicator in male reproductive failures.

Relationship between molecular conversions of acrosin and the progression of exocytosis in the calcium ionophore-induced acrosome reaction.

In this study we used a previously characterized monoclonal antibody to analyze the molecular conversions of acrosin during the acrosomal exocytosis induced by ionophore A23187. Before sperm exposure to the ionophore, most of the sperm acrosin was in the form of proacrosin (55-kDa and 53-kDa forms). Upon exposure to the ionophore, the concentration of proacrosin in sperm samples decreased rapidly and was negatively correlated with the progression of exocytosis. After 1 h of ionophore treatment, proacrosin was quantitatively converted into the two active acrosin forms, alpha-acrosin (49 kDa) and beta-acrosin (36 kDa). However, products of further acrosin conversions were not found after this treatment. As compared with the speed of acrosin activation during sperm contact with the ionophore, the ionophore-induced release of acrosin from the sperm cells into the soluble fraction was apparently delayed, and only the active acrosin forms (49 kDa and 36 kDa) were found in sperm incubation media. External Ca2+ influenced the speed of proacrosin conversion in a concentration-dependent manner. The ionophore-induced activation of proacrosin and acrosome reaction were partially inhibited by trypsin inhibitors. The results suggest that proacrosin activation is an essential step in the mechanism of the acrosomal exocytosis.

Mechanism of maturation and nature of carbohydrate chains of boar sperm acrosin.

The acrosin zymogen proacrosin exists in two molecular forms which are believed to be single-chain polypeptides. During autoactivation in a cell-free system, the 55 and 53 kDa zymogens are sequentially converted into the 49, 36, 31 and 25 kDa forms. A similar mechanism of maturation was revealed, when the calcium ionophore A23187 was added to suspensions of boar spermatozoa. The 49 kDa form has been identified as the first active acrosin form in the maturation cascade. However, this form is indistinguishable from the 53 kDa zymogen in SDS-PAGE at nonreducing conditions. Two carbohydrate chains were evidenced on the acrosin molecule. The chain attached to the Asn3 of the acrosin light chain was enzymatically cleaved without loss of acrosin activity. By contrast, the carbohydrate chain linked to the acrosin heavy chain could be cleaved only after acrosin denaturation. Based on the susceptibility of acrosin to endoglycosidases F and H, a biantennary structure of both carbohydrate chains is proposed.

Identification of 17-18 kDa zona pellucida binding proteins from boar spermatozoa.

Zona pellucida (ZP) binding proteins from boar spermatozoa were compared with antigens recognized by ACR.2 and ACR.3 monoclonal antibodies. The ZP binding proteins of 55, 53, 45 and 38 kDa are identical with various forms of boar acrosin immunologically detected by ACR.2 antibody. The ZP binding proteins of 17 and 18 kDa are recognized by ACR.3 antibody. The N-terminal amino acid sequence of the 17 kDa protein revealed that it is not derived from an acrosin molecule.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Identification and isolation of boar sperm specific antigens with potential role in sperm-egg interaction.

Investigations on specific and functionally active sperm antigens would bring about the elucidation of the mechanisms of gamete interaction and help the search to new approaches for prognosis and regulation of fertility. Previously, we have produced a polyclonal rabbit anti-boar spermatozoa antibody (RABSA) that might affect the fertilizing capacity of boar spermatozoa. The sperm specificity of RABSA was demonstrated by double immunodiffusion, immunoelectrophoresis and ELISA against boar spermatozoa, as well as against saline extracts of boar reproductive and somatic organs. Using indirect immunofluorescence (IIF) test, here we provide evidence that RABSA stained the acrosomes of ejaculated and capacitated boar and human spermatozoa, the fluorescence being intensified on the equatorial region after the acrosome reaction. The RABSA cognate antigen/s is a subject of interest because of their specific localization in sperm structures, which is shown to be a binding and/or fusion competence region. Using ion-exchange (Heparin-Sepharose) chromatography, we eluted an antigen with molecular mass 60 kDa (Ag60) in SDS-PAGE from NP40 extracts of capacitated boar spermatozoa. In Western blot, RABSA recognized specifically this antigen. The Ag60 did not affect the sperm-ligand activity of zona pellucida in a porcine sperm-zona binding assay. IIF experiments showed that zona-free porcine oocytes preincubated with Ag60 and RABSA presented fluorescent labeling over the entire egg surface. The biological and IIF experiments provide evidence supporting the involvement of Ag60 in functional steps required for sperm-egg binding and/or fusion, but not sperm-zona pellucida binding.

Subcellular immunochemical localization of acrosin in human spermatozoa during the acrosome reaction and zona pellucida penetration.

The changes in acrosin immunoreactivity in human spermatozoa undergoing spontaneous or chemically induced acrosome reactions were studied by electron microscopic immunocytochemistry with an acrosin-specific monoclonal antibody. Migration of limited amounts of acrosin to the sperm surface was the earliest event characterizing the beginning of the acrosome reaction. The acrosome of such spermatozoa remained morphologically intact, swelled, or showed intraacrosomal vesiculation without any disruption of the plasma and acrosomal membrane integrity. Massive release of acrosin coincided with the fusion of the plasma and outer acrosomal membranes. However, even fully acrosome-reacted spermatozoa always retained some acrosin on the exposed inner acrosomal membrane and in the equatorial segment of the acrosome. This residual acrosin was also detected on spermatozoa within the zona pellucida of human oocytes inseminated in vitro, while the previously released bulk of acrosin remained attached to the surface of the zona pellucida at the site of sperm entry. These findings are compatible with multiple functions of acrosin in human sperm-egg interaction, including sperm-zona pellucida binding, dispersal of acrosomal contents, and facilitation of zona pellucida penetration.

Reverse effect of indomethacin on the immunosuppressive activity of boar seminal immunosuppressive fraction.

The inhibitory activity of seminal immunosuppressive fraction (ISF) on mitogen-stimulated lymphocyte proliferation and on production of antibody to a soluble antigen was modified by indomethacin or monoclonal antibody to ISF. The ability of indomethacin or monoclonal antibody to ISF to reverse the ISF-induced inhibition of mitogen-stimulated lymphocyte proliferation was estimated by measuring bromodeoxyuridine incorporation into replicated DNA. Splenocytes from mice treated with indomethacin or monoclonal antibody to ISF prior to the application of ISF were tested. The ability of indomethacin or monoclonal antibody to ISF to reverse ISF-induced suppression of antibody production was estimated by measuring antibody titers by ELISA in the blood sera from mice immunized with keyhole limpet hemocyanin (KLH). These animals were treated with indomethacin or monoclonal antibody to ISF prior to the application of ISF. The results showed that both indomethacin and monoclonal antibody to ISF reversed the inhibitory effect of ISF on mitogen-stimulated lymphocyte proliferation as well as on antibody production.Recently, we have identified ISF as a complex of the major seminal glycoproteins PSP I and PSP II. PSP II is the part that is responsible for immunosuppressive properties of the complex. To learn whether the ISF immunosuppressive effect is associated with its protein or saccharide part, we examined the deglycosylated PSP II for its antiproliferative effect on mitogen-stimulated mouse lymphocytes. The results suggest that deglycosylation of PSP II did not affect its antiproliferative activity. This suggest that PSP II immunosuppressive properties are associated with the protein and not the saccharide part of the molecule.

Antibody against 28-kDa intra-acrosomal sperm protein as a tool for evaluation of acrosomal integrity in bull spermatozoa.

Monoclonal antibody ACR.4 recognizing specifically the 28-kDa intra-acrosomal protein was prepared by immunization of mice with acetic acid extract of boar spermatozoa, but cross-reacted also with bull intra-acrosomal protein. This monoclonal antibody was used for immunostaining analysis of bull spermatozoa before and during capacitation and ionophore-induced acrosome reaction. Immunostaining analysis showed changes of 28-kDa protein in the acrosome during capacitation and loss of this protein after induced acrosome reaction by ionophore A23187. Therefore, this monoclonal antibody can be used in the bull spermatozoa as an immunological test for detection of the acrosome state after manipulation with spermatozoa or after freezing/thawing. This test could be useful (apart from morphology and motility) for the selection of suitable spermatozoa for insemination or in vitro fertilization.

Two-step freezing of cells used in hybridoma technology.

Hybridoma technology requires freezing of parental myeloma cells, continuous freezing and thawing of clones and stable hybridoma cells. A modification of the method of two-step freezing is described. The cryoprotective agent (5% dimethyl sulphoxide) is added to the cells at room temperature for 10 min. Cells are then transferred directly to the -25 degrees C bath, held at this temperature for 10 min, and stored directly in liquid nitrogen. Thawing is rapid in a water bath warmed to 60-80 degrees C. Hybridoma cells retain high viability and the production of specific monoclonal antibody after thawing.

Two-step freezing of hybridoma cells in 96-well microculture plates.

Stabile hybridoma cells, colonies of hybridoma cells 14 days after fusion of immune spleen and myeloma cells, myeloma cells and fibroblasts cultured in 96-well microculture plates were frozen by the method of two-step freezing. The culture medium was aspirated, and 50 microliter of the medium containing a cryoprotectant (5% dimethyl sulphoxide) was added for 10 min at room temperature. The plates were put into microtene bags, placed at -25 degrees C in a freezer for 30 min and then stored at -100 degrees C in liquid nitrogen vapour. Plates with cells were thawed rapidly in a 50 degree C water bath. After thawing the hybrid cells were viable and continued to produce the specific antibody.

Capacity of contact cytotoxic reaction to distinguish targets presented by F1 and somatic hybrid cells of the same H-2 parentage.

51Cr-release from prelabelled target cells caused by contact with effector lymphocytes was greater when the H-2d/H-2k cells used for sensitization (i.e. production of effector cells) were also phenotypically similar to the target cells. The contact cytotoxic reaction thus turned out to be sensitive enough to distinguish a putative phenotypic difference between F1 and somatic hybrid cells of the same H-2 parentage. A possible involvement of non-H-2 differences was controlled and ruled out.

On the mechanism of intercellular adhesion.

Using a 2 X 2 design, the collection rates of cells from radioactively labelled single-cell suspension on cell-coated collecting surfaces were tested for a possible H-2 effect on intercellular adhesiveness. In five donor-host combinations differing in the H-2 complex, no straight advantage of syngeneic over allogeneic relationship could unequivocally be proved. Trypsin was deliberately omitted in preparing the single-cell suspension; it was shown that the cell suspension processed in this way affected the mechanism of intercellular adhesiveness to a degree at least comparable to the role played by the properties of the collecting layer cells.

Changes in immunochemical localization of cytoskeletal proteins in human and boar spermatozoa before and after acrosome reaction.

Several cytoskeletal proteins (alpha-tubulin, beta-tubulin, actin, spectrin, tropomyosin, vimentin and cytokeratin) were studied in human and boar spermatozoa. Their localization was observed by means of specific antibodies using indirect immunofluorescence technique. Immunocytochemical results were confirmed by the Western blot technique. Cytoskeletal proteins were examined in ejaculated spermatozoa before and after acrosome reaction induced by the ionophore A23187. The immunofluorescence assay revealed that localization of the studied cytoskeletal proteins in human and boar spermatozoa differ remarkably. In human spermatozoa, the localization of actin and spectrin changed after acrosome reaction; on the other hand, in boar spermatozoa, the changes of localization concerned alpha-tubulin, beta-tubulin, actin and spectrin. The type of changes differs in the studied species and follows probably the course of acrosome reaction. The results suggest that cytoskeleton participates in the process of acrosome reaction of mammalian spermatozoa.

The effects of glycosaminoglycans on surface-mediated interactions of lymphoid cells.

The effect of chondroitin sulphate (CS) was studied in two cell surface-mediated interactions, the complement-independent contact reaction and antibody-induced capping of H-2 antigens. CS inhibited contact cytotoxicity when present in the reaction medium, but preincubation in CS of either killer cells or target cells had no effect. The capacity of CS-pretreated target cells to absorb H-2 antibodies was not reduced. However, a similar pretreatment resulted in an increased readiness to capping as induced by H-2 antibody followed by an anti-mouse immunoglobulin; in control cells time taken for the same percentage of caps to be formed was considerably longer than in CS-pretreated cells. A possible mechanism of this CS effect and the general biological role of glycosaminoglycans (particularly in immune interactions) is discussed.