The effect of tetrabromobisphenol A on protamine content and DNA integrity in mouse spermatozoa.

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant of increasing concern to human health because of its action as an endocrine disruptor. We have previously demonstrated that TBBPA is able to increase apoptosis of testicular cells and other changes in the first and second generations of mice exposed to TBBPA. However, the potential effects of TBBPA on mouse epididymal spermatozoa have not yet been investigated. Therefore, we initiated this study to determine whether TBBPA exposure could also result in increased DNA fragmentation in epididymal spermatozoa and whether it had an effect on the protamines as the major nuclear proteins. C57Bl/6J mouse pups (n = 10) were exposed to TBBPA (experimental group) during the gestation, lactation, pre-pubertal and pubertal periods up to the age of 70 days as previously described and compared to control mouse pups (n = 10) that were not exposed. The results demonstrate that TBBPA treatment results in a significantly decreased protamine 1/protamine 2 ratio (0.362 vs. 0.494; p < 0.001), increased total protamine/DNA ratio (0.517 vs. 0.324; p < 0.001) and increased number of terminal deoxynucleotidyl transferase dUTP nick end labelling positive spermatozoa (39.5% vs. 21.2%; p < 0.05) observed between TBBPA and control mice respectively. These findings indicate that TBBPA exposure, in addition to the resulting increased sperm DNA damage, also has the potential to alter the epigenetic marking of sperm chromatin through generation of an anomalous content and distribution of protamines. The possibility is now open to study whether the detected altered protamine content and DNA integrity are related to the previously observed second-generation effects upon TBBPA exposure.

Effect of zearalenone on reproductive parameters and expression of selected testicular genes in mice.

We tested the effect of two different concentrations (150µg/l and 0.15µg/l) of mycotoxin zearalenone (ZEA) on the reproductive parameters and expression of testicular genes in male mice. In adult males, no reduction of body or reproductive organ weight was observed, and the seminiferous tubules were morphologically normal with ongoing spermatogenesis. However, we found decreased sperm concentration, increase of morphologically abnormal spermatozoa and increased binding of apoptotic marker annexin V. This study was also focused on the evaluation of gene expression profiles of 28 genes playing important roles during the processes occurring in the testicular tissue. We detected changes in the expression of genes important for proper spermatogenesis. Surprisingly, we observed a stronger effect after exposure to the lower dose of ZEA.

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.

Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L.

Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.

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.

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.

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).

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.

Monoclonal antibody FsC-47 against carp sperm creatine kinase.

The enzyme creatine kinase (CK) plays a key role in energy homeostasis of cells with high and fluctuating energy requirements. As for spermatozoa, the activity of phosphocreatine shuttle, which directs energy from the mitochondria to sites of ATP consumption, is dependent on individual species. High activities of CK are observed in spermatozoa of nonvertebrate, fish, and birds, contrary to the low-level CK activity in mammalian spermatozoa. A new monoclonal antibody (MAb) to carp sperm creatine kinase was prepared. This antibody is applicable to large-scale immunochemical techniques. In this study, it was applied to the study of carp sperm motility, and the evaluation of the influence of CK on the quality and fertilization ability of carp spermatozoa.

[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.

The in vitro biological activity of Lepidium meyenii extracts.

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.

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.

[Significance of determination of intra-acrosomal proteins and sperm antibodies in human reproduction]. / Význam urcování intra-akrosomálních proteinu a protilátek proti spermiím v lidské reprodukci.

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.

[Successful treatment of azoospermia diagnosed as "Sertoli cells only syndrome with maturation arrest"--developing a quality embryo]. / Uspesné resení azoospermie s diagnózou "Sertolli cells only syndrome" (SCO sy.) a Maturation Arrest (MA)--získání kvalitního embrya.

OBJECTIVE: The transfer of good quality embryo in the program of assisted reproduction in the case of azoospermia, dg. Sertolli cells only syndrome (SCO sy) + maturation arrest (MA). Testes were assessed and found to have a high occurrence of Sertolli cells and very low occurrence of germinal cells, which were arrested at the round spermatid level. The histological evaluation was hypospermatogenesis gr. 3 (minimum 1 spermatid/sample). DESIGN: Case report. SETTING: Laboratory IVF, Iscare, a. s., Department of Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Czech Academy of Sciences, Prague. SUBJECT AND METHOD: The successful integration of three methods provides a solution for this case of azoospermia. Immunology and histology can more exactly diagnose the degree of azoospermia. Detection and visualisation of spermatids using monoclonal antibodies against sperm proteins predicts the eventual occurrence of spermatogenesis, and histological evaluation confirms these immunological findings. Using the information of both methods it is possible to use special in vitro cultivation of testicular cells and so obtain injectable spermatozoa, or precursors of sperm, for the ICSI method. CONCLUSION: The probability of acquisition of good-quality embryo in the program of assisted reproduction is higher when these three methods are applied in combination.

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.

Role of a capacitation-related protein on some sperm functional parameters.

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.

Differential subcellular distribution of tubulin epitopes in boar spermatozoa: recognition of class III beta-tubulin epitope in sperm tail.

The exposure of tubulin epitopes was studied in ejaculated boar spermatozoa using a panel of four monoclonal antibodies specific to the N-terminal or C-terminal structural domains of tubulin and three monoclonal antibodies against class III beta-tubulin. The specificity of the antibodies was confirmed by immunoblotting. Immunocytochemical staining showed that antibodies discriminated between various parts of a spermatozoon, and that epitopes of class III beta-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of beta-tubulin was detected in the triangular segment of the postacrosomal part of the sperm head. Its distribution changed after an A23187 ionophore-induced acrosome reaction, indicating that tubulin participates in the early stages of fertilization. Three monoclonal antibodies, TU-20, SDL.3D10, and TUJ1 directed against epitopes on the C-terminal end of neuron-specific class III beta-tubulin that is widely used as a neuronal marker, stained the flagella. The reactivity of TU-20 was further confirmed by absorbing the antibody with the immunizing peptide and by immunoelectron microscopy. Immunoblotting after two-dimensional electrophoresis revealed that the corresponding epitope was not present on all beta-tubulin isoforms. These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.

Monoclonal antibodies to intra-acrosomal proteins inhibit gamete binding in vitro.

The sperm-zona pellucida-binding assay in vitro was used as a functional test for zona pellucida-binding ability of boar spermatozoa after co-incubation with monoclonal antibodies against intra-acrosomal proteins. The effect of monoclonal antibodies ACR.2 against boar acrosin (55, 53, 45 and 38 kDa), and Hs-8 against boar intra-acrosomal protein (230, 110, 88, 60, 48 kDa) on boar spermatozoa-porcine oocyte binding was examined. The sperm-zona pellucida-binding was reduced when medium was supplemented with monoclonal antibodies during sperm-oocyte co-incubation, but not when capacitated spermatozoa were pretreated with monoclonal antibodies before incubation with oocytes. Our results show that the monoclonal antibodies (ACR.2, Hs-8) against intra-acrosomal proteins reduce the secondary sperm-zona pellucida-binding with statistically significant difference. This suggests the role of these proteins in the early phases of fertilization.

The effect of various capacitation active compounds and capacitation time on the in vitro fertility and protein tyrosine phosphorylation profiles of bovine sperm.

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.