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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.

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.

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.

The biological activity of methanolic and aqueous extracts from dehydrated hypocotyls of Lepidium meyenii (Brassicaceae, vernacular name "maca"), was studied on rat hepatocytes and human breast cancer MCF-7 cells. The extracts did not exhibit cytotoxicity in hepatocyte primary cultures up to 10 mg/ml as measured by the MTT viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 h, extracts inhibited LDH and AST leakage from the hepatocytes. When hepatocytes were intoxicated by t-butyl hydroperoxide, neither extract prevented oxidative damage. Both extracts showed weak antioxidant activity in the DPPH radical scavenging test with IC(50) values of 3.46 +/- 0.16 and 0.71 +/- 0.10 mg/ml, for aqueous and methanolic extracts, respectively. Thus, the observed effect on spontaneous enzyme leakage is probably mediated through mechanisms other than antioxidant activity. Both methanolic and aqueous extracts have shown estrogenic activity comparable with that of silymarin in MCF-7 cell line. Maca estrogenicity was exhibited in the range from 100 to 200 mug of extract per ml. The findings in the present study show that maca does not display in vitro hepatotoxicity. In contrast, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. Maca extracts exhibited estrogenic activity comparably to the effect of silymarin in MCF-7 cells.

Spermadhesins, proteins secreted by the boar sexual accessory glands, are believed to play an important role in sperm capacitation and primary contact of sperm and egg. We have previously found human seminal plasma proteins immunobiochemically related to boar AQN and AWN spermadhesins. In this study, we characterized further the AQN spermadhesin-related proteins, here designated as hSA (human spermadhesin-like) proteins. On Western blot, we immunodetected 14, 16 and 18 kDa forms of hSA proteins (hSA-14, hSA-16 and hSA-18, respectively) cross-reacting with rabbit antibody against AQN spermadhesins. Each relative molecular-mass form of hSA comprised three isoelectric isoforms (6.0, 6.8 and 8.4) as shown by 2D-PAGE. Glycoprotein analysis revealed that all hSA-16 and hSA-18 isoforms were N-glycosylated, and those of hSA-14 were non-glycosylated. Two isoforms of hSA-14 (pI 6.0 and 8.4) had affinity to heparin. Size-exclusion chromatography of human seminal plasma indicated that hSA proteins formed high molecular-mass complexes either with other hSA proteins or with seminal plasma lactoferrin and/or its fragments. Similarity of biochemical properties (relative molecular masses, isoelectric points and existence of non- and N-glycosylated forms) of hSA proteins and those of boar AQN spermadhesins, together with a previously described N-terminal amino acid sequence of one hSA protein identical to AQN spermadhesins, imply that hSA proteins are structurally related to boar AQN spermadhesins. However, localization of hSA proteins on the sperm tail and neck suggests that their biological role differs from that of boar AQN spermadhesins located on the sperm head.

Objective: Comparison of the positive intra-acrosomal proteins and spermagglutinating antibodies in human semen samples from various groups of patients., Design: Prospective study., Setting: Department of Gynecology and Obstetrics and Faculty Hospital, Charles University, Pilsen, Institute of Molecular Genetics, Czech Academy of Science, Prague., Methods: Monoclonal antibodies Hs-8 and Hs-14 (prepared in the Institute of Molecular Genetics, Prague) were used for detection of intra-acrosomal sperm proteins. Microscopic immunofluorescent methods detected the incidence, the character and the percentage of the spermatozoa specified by above-mentioned monoclonal antibodies. Direct mixed anti-immunoglobulin reactions test (MAR-test) for IgG, IgM, IgA, IgE was used for detection of spermagglutinating antibody. We examined 315 infertile patients from Special Consultation for Immunology of Reproduction and from the IVF programme, and sperm healthy donors (January 2002-March 2003)., Results: Native donor's sperm cells had excellent positive intra-acrosomal proteins stained with monoclonal antibodies Hs8 and Hs14 and after thawing as well as. No spermagglutinating antibodies were found. In the group with normal sperm count and light microscopic morphology we found the presence of seminal spermagglutinating antibodies in 11% (IgG), in 14.5% (IgA), in 3.6% (IgM), in 5.2% (IgE). Significant positivity of intra-acrosomal protein stained with Hs8 monoclonal antibody was reached in 68.4%, and with Hs14 monoclonal antibody in even 81.3% of men. On the other hand, in oligoasthenospermatic patients we found significant increasing of spermagglutinating antibodies (for IgG 40.5%, for IgA 28.6%, for IgM 9.5%, for IgE 11.9%). Dominant good staining of intra-acrosomal proteins were seen only in 15.5% of men (for Hs8) and in 20.2% (for Hs14)., Conclusion: The quantitative detection of intra-acrosomal sperm proteins and spermagglutinating antibodies are used as important properties of human semen and serve for evaluation of acrosomal state, and male fertility together.

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.

Problem: To determine the influence of an endocrine disruptor [bisphenol-A (BPA)] on the integrated reproductive process as well as on individual reproductive organs and gametes in order to select suitable markers for testing sperm damage., Method of Study: The effect of BPA on fertility in vivo in multigenerational studies in an outbred stock of mice was studied. Damage of reproductive organs was assessed by histochemical methods and damage of spermatozoa by means of a panel of monoclonal antibodies (MoAbs) against intra-acrosomal sperm proteins., Results: BPA had a negative influence on offspring born of mice, on reproductive organs, and on acrosome integrity of mice spermatozoa. Selected MoAbs against intra-acrosomal mammalian sperm proteins, cross-reacted with mouse spermatozoa, were used for determination of the acrosome integrity. BPA had no effect on body weight and testicle weight of males., Conclusions: The present results demonstrate that BPA has a negative effect on in vivo fertility of mice, with impact on spermatogenesis and sperm quality. Monoclonal antibodies against intra-acrosomal sperm proteins can be used for detecting sperm damage.

In previous studies a series of Mabs against boar capacitated sperm have been produced. One of these Mabs--4B12--was found to recognize a surface membrane-associated protein located in the acrosome portion of the spermatozoa that became accessible to antibody after capacitation. In biological experiments it was shown that Mab 4B12 significantly inhibited boar sperm-porcine ZP binding. In attempts to investigate the mechanisms by which Mab 4B12 affected sperm-ZP binding, the role of the cognate protein on some functional parameters such as sperm motility and ability of the capacitated spermatozoa to undergo AR was studied. Experimental models of premature AR and AR physiologically induced with ZP were applied to study the effect of Mab 4B12 on boar sperm AR using PSA staining to estimate the acrosome-reacted state of spermatozoa. Sperm motility characteristics were determined by the time-exposure photokinesigraphic method. The results obtained in the present study, together with previously established inhibition of sperm-ZP binding by Mab 4B12, documented the participation of the 4B12 protein in primary sperm-ZP binding. The protein is not connected with sperm motility and secondary sperm-ZP binding.

In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.

Certain morphological changes such as rearrangement of cytoskeletal proteins of the mammalian spermatozoa are detectable during the AR. The type of changes differs according to the studied sperm species and follows the course of AR. Relocation of cytoskeletal structures was previously observed especially in the case of actin-, alpha-, gamma-tubulin- and spectrin-containing structures. To prove these findings we used specific inhibitors of cytoskeletal proteins (eg. colcemide, cytochalasine B, nocodazole and vinblastine). It has been shown that the AR is influenced by cytoskeletal inhibitors, but the obtained results also document that cytoskeletal proteins actin, tubulin and spectrin play a significant role in the course of AR in vitro. Our results of confocal and electron microscopy also demonstrate visible changes of actin-, tubulin- and spectrin-containing structures after the AR. Our data indicate that specific cytoskeletal inhibitors influence the AR and they prove the role of cytoskeletal proteins in this process.

A new monoclonal antibody designated Hs-14 was generated after immunization of BALB/c mice with the acid extract of human sperm. In indirect immunofluorescence Hs-14 mAb binds to the acrosome of permeabilized sperm cells and consequently recognizes some intra-acrosomal protein. Western blotting analysis revealed that under non-reducing conditions the Hs-14 mAb detects a protein with a molecular mass of 220 kDa. Under reducing conditions the Hs-14 recognizes several peptide bands within the range from 55 kDa to 110 kDa. Beside human sperm the antibody positively reacts also with sperm of some other mammalian species. Using Hs-14 mAb it is possible to evaluate the acrosomal integrity of spermatozoa and to reveal sperm pathology.

Problem: The role of Ala-Trp-Asn (AWN) and Ala-Gln-Asn (AQN) families of spermadhesive sperm proteins in fertilization., Method of Study: The preparation and characterization of polyclonal antibodies against AWN and AQN spermadhesins and one monoclonal antibody (MAb), designated Bo.5, against AWN spermadhesin. The use of biochemical and immunocytochemical methods for characterization of spermadhesins on the sperm membrane of boar spermatozoa and in the cryostat sections of boar reproductive organs., Results: Polyclonal anti-AWN and anti-AQN antibodies specifically reacted with AWN and AQN proteins, respectively. MAb Bo.5 detected the 17-, 16-, and 14-kDa protein members of AWN subfamily. The monoclonal, as well as the polyclonal, AWN antibodies remarkably decreased the sperm binding to the egg surface in an in vitro sperm zona pellucida binding assay., Conclusions: Presented results demonstrate that polyclonal antibodies and MAb Bo.5 against spermadhesins specifically recognize the membrane-associated antigens and inhibit the binding of sperm to oocytes. Reduced binding of sperm to oocytes, due to the antibodies, indicates the role of these spermadhesins in sperm-egg primary binding.

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.

Changes of protein tyrosine phosphorylation in ejaculated boar sperm incubated in vitro were examined with the use of antiphosphotyrosine antibodies and immunoblotting. The intracellular levels of cAMP were modulated by treatment with various combinations of caffeine, 3-isobutyl-1-methylxanthine (IBMX), and dibutyryl cyclic AMP (dbcAMP), and acrosome reactions (ARs) were induced via treatment with divalent cation ionophore A23187. Proteins of Mr 34, 38, 40, and 44 (p34 ... p44) were strongly phosphorylated on tyrosine residues in freshly prepared sperm samples and at the same level during all subsequent treatments. Incubation of sperm in vitro for various periods of time induced an increase of tyrosine phosphorylation of p20, p93, and p175. The tyrosine phosphorylation of p93, p175, and several other sperm proteins was up-regulated in a concentration-dependent manner following treatment of the sperm with dbcAMP, caffeine, or IBMX alone, or with combinations of caffeine and IBMX, respectively, with dbcAMP; the tyrosine phosphorylation of p20 was not correlated with treatment of sperm with cAMP-elevating reagents. The percentage of sperm cells undergoing spontaneous ARs was not affected by the manipulation of cAMP levels and was not correlated with protein tyrosine phosphorylation. In contrast, the addition of calcium to the incubation media decreased protein tyrosine phosphorylation and elevated percentage of spontaneous ARs. The induction of ARs with A23187 caused a significant decrease of tyrosine phosphorylation of p93, p175, and p220/230, indicating that dephosphorylation on protein tyrosine residues might be associated with calcium influx during physiological ARs as well. Proteins p93 and p175 were effectively solubilized in greater than 9M urea/1% triton and in SDS sample buffer, but to only a small extent in triton, while p20 was virtually completely extractable with triton. In conjunction with the previously reported isolation of active tyrosine kinase sp42 from triton extracts of noncapacitated boar sperm cells (Berruti and Porzio, 1992: Biochim Biophys Acta 1118: 149-154), our results suggest that a cAMP-dependent event is required for tyrosine phosphorylation of triton-insoluble proteins such as p93 and p175. On the other hand, the tyrosine phosphorylation of p20 (and potentially other triton-soluble substrates) might not strictly require such cAMP up-regulation. We discuss the differences in the regulation of cAMP-dependent tyrosine phosphorylation in mouse, human, and boar sperm, and suggest that sensitivity to calcium and distinct basal levels of cyclic nucleotide PDE might correspond to species-specific reproduction strategies in mammals.

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.

Monoclonal antibodies specific for acrosomal proteins were prepared and characterized. Two of these monoclonal antibodies, ACR.3 and ACR.11, reacted with the same molecular specificity of acrosomal antigens (18-20 kDa), antibody ACR.4 reacted with the 25-27 kDa antigens, and antibody ACR.2 reacted with several forms of boar acrosin (55, 53, 45, and 38 kDa). All monoclonal antibodies labelled the acrosomes of undamaged spermatozoa in immunofluorescence test. This test should be convenient as an immunological test of the sperm quality.