No (40)
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Possible role of spermatogenic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) in mammalian sperm
Margaryan, Hasmik; Dorosh, Andriy; Čapková, Jana; Postlerová, Pavla; Philimonenko, Anatoly; Hozák, Pavel; Pěknicová, Jana
2015 - English
Sperm proteins are important for the structure and function of these specific, highly differentiated cells. Certain of these proteins play a role in sperm-egg recognition during primary or secondary binding at zona pellucida glycoprotein matrix. The aim of this study was to characterize the acrosomal sperm protein recognized by a monoclonal antibody (MoAb) Hs-8, prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperm, and to test the possible role of this protein in gamete interaction. MoAb Hs-8 specifically labelled a 45 kDa protein from the sperm extract in the immunoblotting test. Sequence analysis identified this Hs-8 protein as GAPDHS. In order to perform a control tests, a commercial mouse anti-GAPDHS MoAb was applied. Both antibodies showed similar staining patterns using immunofluorescence labelling, transmission electron microscopy and immunoblot analysis. Moreover, both Hs-8 and commercial anti-GAPDHS antibodies blocked the secondary sperm-zona pellucida binding. Generally, GAPDHS was considered mainly as sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility and its role in the sperm head was unknown. In this study, we confirmed the potential additional role of GAPDHS as a binding protein that is involved in the sperm-zona pellucida interaction.
Keywords:
GAPDHS; sperm glycolytic enzyme; zona pellucida glycoprotein matrix; monoclonal antibody
Available at various institutes of the ASCR
Possible role of spermatogenic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) in mammalian sperm
Sperm proteins are important for the structure and function of these specific, highly differentiated cells. Certain of these proteins play a role in sperm-egg recognition during primary or secondary ...
Book of abstract XXIst Symposium of biology and immunology of reproduction
Pěknicová, Jana; Kubátová, Alena; Elzeinová, Fatima
2015 - English
The Symposium was focused on immunology of reproduction and specific problems in reproduction (mainly in human infertility).
Keywords:
reproduction; infertility; capacitation; spermatozoa; anti-Mullerian hormone; oocytes; seminal plasma
Available at various institutes of the ASCR
Book of abstract XXIst Symposium of biology and immunology of reproduction
The Symposium was focused on immunology of reproduction and specific problems in reproduction (mainly in human infertility).
Sperm protein profiles of different mammalian species
Pohlová, Alžběta; Zigo, Michal; Jonáková, Věra; Postlerová, Pavla
2015 - English
Proteins are a substantial equipment of the spermatic cell; therefore, the characterization of sperm proteins is crucial for explanation of molecular mechanisms in the reproduction process. We isolated sperm proteins from different mammalian species - pig, bull, human, mouse, dog and cat. Extracted proteins were separated by SDS-electrophoresis and protein/glycoprotein profiles from epididymal or ejaculated sperm were compared. Additionally, we tested cross-reactivity of antibodies prepared to sperm boar proteins on spermatozoa of other mammalian species using immunofluorescent technique. Our future plan is to compare the protein profiles of sperm during their functional development (epididymal, ejaculated, capacitated) in various mammalian species and identify species-specific sperm proteins with zona pellucida binding activity.
Keywords:
SDS-electrophoresis; sperm proteins; zona pellucida binding activity; monoclonal antibody
Available at various institutes of the ASCR
Sperm protein profiles of different mammalian species
Proteins are a substantial equipment of the spermatic cell; therefore, the characterization of sperm proteins is crucial for explanation of molecular mechanisms in the reproduction process. We ...
Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla; Zigo, Michal; Pohlová, Alžběta; Jonáková, Věra
2015 - English
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various biochemical methods for a long time. We have expertise in protein extraction from spermatic cells using different approaches, and by kits for proteins from the sperm surface and distinct subcellular compartments. The proteins of reproductive organ fluids are separated by chromatographic methods, such as size exclusion chromatography, high-performance liquid chromatography with reverse phase (RP-HPLC) and affinity chromatography on matrices with various ligands. Proteins are subjected to SDS- or 2D-electrophoresis for their characterization and comparison of various extraction methods, different mammalian species, and sperm in different functional development. Electrophoretically separated proteins may be transferred onto nitrocellulose membrane (Western blot) for antibody detection or binding studies with lectin-labelled ligands (lectins, polysaccharides, zona pellucida glycoproteins). We use immunoprecipitation method with specific antibody for protein determination followed by the MALDI identification. Proteins are localized by immunofluorescent techniques on/in spermatic cells and tissue sections of reproductive organs. Isolation of proteins from reproductive tissues and fluids, and the antibody detection is crucial for the studying of reproductive protein origin.
Keywords:
HPLC; lectins; zona pellucida glycoproteins; monoclonal antibody
Available at various institutes of the ASCR
Biochemical methods as tool for study of reproductive proteins
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various ...
Diabetes mellitus negatively affects male reproductive parameters in vivo
Valášková, Eliška; Žatecká, Eva; Pavlínková, Gabriela; Bohuslavová, Romana; Dorosh, Andriy; Elzeinová, Fatima; Kubátová, Alena; Margaryan, Hasmik; Pěknicová, Jana
2015 - English
According to the World Health Organization (WHO), 15% of couples in reproductive age suffer from infertility problems, and up to 60% of cases are caused by male factor. This could be caused by genetic background, environmental factors and various diseases, including diabetes mellitus (DM). However, the impact of DM on male fertility is not fully understood. . The aim of this study was to investigate the effects of DM on reproductive parameters and sperm quality, using mouse model. DM (type 1) was induced by Streptozotocin in FVB inbred mouse strain. Mice with blood sugar levels higher than 13.9 mmol/L were considered diabetic. After 4 weeks of diabetes exposure, diabetic males were bred with wild type females and transgenerational effect of DM was assessed. Selected morphological, cellular, and molecular parameters of diabetic males and their male offspring were compared to appropriate controls. There was an increased in sperm fragmentation and abnormalities of sperm morphology in diabetic mice in both generations. An increased staining with apoptotic marker annexin V was also detected in the diabetic groups. Furthermore, a presence of protamines as major sperm nuclear proteins was analysed. Protamine 1 to protamine 2 ratio (P1/P2), a marker of male fertility, was altered in sperms of experimental diabetic animals in both generations. Our findings indicate that DM type 1 negatively affects sperm quality and P1/P2 ratio and this negative effect is transmitted to the progeny
Keywords:
diabetes melitus; Sterptozotocin; apoptosis; protamination
Available at various institutes of the ASCR
Diabetes mellitus negatively affects male reproductive parameters in vivo
According to the World Health Organization (WHO), 15% of couples in reproductive age suffer from infertility problems, and up to 60% of cases are caused by male factor. This could be caused by genetic ...
Fluorescent analysis of the differential protein expression in normozoospermic and asthenozoospermic sperm samples
Děd, Lukáš; Čapková, Jana; Kubátová, Alena; Teplá, O.; Pěknicová, Jana
2015 - English
Asthenozoospermia is one of the main seminal pathologies underlying male infertility. Previous proteomic studies have demonstrated the significant differences in the protein profiles between normozoospermic and asthenozoospermic sperm samples. Since these studies were primarily focused on the identification of differentially expressed proteins by mass spectrometry, we aimed to evaluate the ability of our diagnostic antibodies to detect the differential expression of selected protein markers by fluorescent microscopy and flow cytometry techniques. Therefore, we analyzed sperm samples from 30 men with normal and 30 men with astheno spermiograms, average by the panel of our diagnostic anti-human sperm (Hs) antibodies. Fluorescent microscopy and flow cytometry analysis revealed quantitative differences in the protein abundances between normo and astheno sperm samples, namely, in GAPDHs, evaluated with Hs-8 MoAb, VCP, evaluated with Hs-14 MoAb, and ATP synthase, evaluated with MoAb Hs-36. From the methodological point of view, we observed very high correlation between the data obtained by fluorescent microscopy and flow cytometry techniques and therefore both methods are useful for evaluation of protein differences associated with asthenozoospermia. From the clinical point of view, we observed the strong association of the low sperm motility in the sample with the expression of proteins, playing an important role in sperm energy metabolism (expected), but also with the expression of all tested intra-acrosomal proteins. These findings further demonstrate asthenozoospermia as a complex semen disorder frequently associated with other semen pathologies, which are not diagnosed by basic semen analysis, and the possibility to use monoclonal antibodies as a tool for diagnosis of protein associated sperm pathologies in the semen with the low sperm motility.
Keywords:
monoclonal antibody; asthenozoospermia; flow cytometry
Available at various institutes of the ASCR
Fluorescent analysis of the differential protein expression in normozoospermic and asthenozoospermic sperm samples
Asthenozoospermia is one of the main seminal pathologies underlying male infertility. Previous proteomic studies have demonstrated the significant differences in the protein profiles between ...
Dynamics of mouse sperm capacitation and acrosome reaction
Dvořáková-Hortová, Kateřina; Frolíková, Michaela; Děd, Lukáš; Šebková, Nataša
2015 - English
Capacitation followed by the acrosome reaction (AR), is a very complex event of molecular changes, including acrosome matrix rearrangement and actin polymerization, which mammalian sperm must undergo in the female reproductive tract in order to obtain the ability to penetrate and fertilize the egg. CD46 and β1-integrin belong to specific proteins, which are predicted to interact during molecular reorganization of capacitating sperm. The IZUMO1 as the primary fusion protein of the mammalian sperm is also involved in this dynamic network. We investigated the relationship between the Izumo, CD46 and β1 integrin relocation in the sperm head during the capacitation and AR in vitro. We have already successfully monitored by immunofluorescent labelling the dynamics of proteins CD46 and β1-integrin. The changes in the localization of these proteins associated with the AR and their mutual co-localization was observed. The original β1-integrin location in the freshly released epididymal sperm is in the acrosome and it relocates during the AR further through the sperm head compartments into the equatorial segment and over the whole sperm head. Its density over the equatorial segment is decreasing with the extended time of the AR. Also its presence in the perforatorium of the mouse sperm head is very prominent. The pattern for protein CD46 is extremely similar if not identical in both aspects such as compartment localization and time progress during capacitation and AR in vitro. The molecular interaction of CD46 and β1-integrin was investigated using the Proximity Ligation Assay and Super resolution microscopy STED. The data were statistically analysed. The newly obtained results from CD46 and β1-integrin relocation are in correlation with IZUMO1 dynamics and giving a substantial knowledge on the studied protein network rearrangement during capacitation and AR in mouse spermatozoa.
Keywords:
CD46 monoclonal antibody; acrosome reaction; sperm capacitation; IZUMO 1; Proximity ligation assay
Available at various institutes of the ASCR
Dynamics of mouse sperm capacitation and acrosome reaction
Capacitation followed by the acrosome reaction (AR), is a very complex event of molecular changes, including acrosome matrix rearrangement and actin polymerization, which mammalian sperm must undergo ...
One more drop for decreasing reproduction
Dvořáková-Hortová, K.; Šídlová, A.; Děd, Lukáš; Hladovcová, D.; Vieweg, M.; Weidner, W.; Steger, K.; Stopka, P.; Paradowska-Dogan, A.
2014 - English
Toxoplasma gondii is a common protozoan parasite that infects warm-blooded animals throughout the world, including mice and humans. During infection, both, the parasite and the host, utilize various mechanisms to maximize their own reproductive success. Mice and humans are both the intermediate hosts for Toxoplasma gondii, which forms specialized vacuoles containing reproductive cysts in the formers’ tissue. As half of the human population is infected, developing a disease called toxoplasmosis, along with an ever-growing number of couples suffering with idiopathic infertility, it is therefore surprising that there is a lack of research on how T.gondii can alter reproductive parameters. In this study, a detailed histometric screening of the testicular function along with the levels of the pituitary luteinizing hormone (LH) were analysed in infected mice. Data on relative testis and epididymis weight, and sperm count were also collected. Based on the results obtained, the level of LH in the urine of Toxoplasma gondii infected mice was lower compared to the control. In direct correlation with the hormone level, testicular function and sperm production was also significantly lower in T. gondii positive group using sperm count and histometric analysis as a marker. Not only were the number of leptotene primary spermatocytes and spermatids lowered, but the number of Sertoli cells and the tubule diameter were elevated. In parallel, a pilot epigenetic study on global testicular methylation, and specific methylation of Crem, Creb1 and Hspa1genes essential for successfully ongoing spermatogenesis was performed. Global methylation was elevated in Toxoplasma infected mice, and differences in the DNA methylation of selected genes were detected between the Toxoplasma positive and control group. These findings demonstrate a direct relation between T. gondii infection and the decrease of male reproductive fitness in mice, which may contribute to an increase of infertility in humans.
Keywords:
Toxoplasma gondii; luteinizing hormone; spermatocytes; methylation
Available at various institutes of the ASCR
One more drop for decreasing reproduction
Toxoplasma gondii is a common protozoan parasite that infects warm-blooded animals throughout the world, including mice and humans. During infection, both, the parasite and the host, utilize various ...
Enzymatic and inhibiting activity in boar epididymal fluid
Davidová, Nina; Ren, Š.; Liberda, J.; Jonáková, Věra; Maňásková-Postlerová, Pavla
2014 - English
Sperm maturation, represents a key step in the reproduction process. Spermatozoa, particularly the plasma membrane, are exposed to epididymal fluid (EF) components representing the natural environment essential for their post-testicular maturation. Changes in the sperm membrane proteins are influenced by proteolytic and glycosidic enzymes present in the EF. Accordingly, the occurrence of inhibitors in this reproductive organ is very important for the regulation of sperm membrane protein processing. In present study, we monitored protease and glycosidase activities, and inhibitors of metallo- and serine proteinases in boar EF. Additionally, we studied acrosin inhibitor in fluid, spermatozoa and tissue along the epididymis. We chromatographically separated boar EF into several fractions. These fractions were subjected to SDS-electrophoresis and the separated proteins were either studied by zymographic methods or transferred to nitrocellulose membranes for detection of metallo- and serine proteinases and their inhibitors, and acrosin inhibitor by specific antibody, respectively. Acrosin inhibitor was monitored also in the sperm and tissue of the boar epididymis. In boar epididymal fluid, several metallo- and serine proteinases with different molecular masses, and inhibitors of metalloproteinase MMP-9 and acrosin were found. We measured strong activity of mannosidase in this fluid. Using specific antibody, we registered the increasing signal of acrosin inhibitor from caput to cauda epididymis in the spermatozoa, fluid and also tissue. Proteinases and their inhibitors in reproductive fluids may play a significant role in reproduction processes. Especially, acrosin inhibitor in the reproductive tract inactivates prematurely released sperm acrosin and protects spermatozoa and reproductive epithelium against proteolytic degradation. High mannosidase activity in boar EF suggests evident role of mannose structures in the sperm interaction during reproductive events.
Keywords:
enzymatic activity; inhibiting activity; epididymal fluid; proteinase
Available at various institutes of the ASCR
Enzymatic and inhibiting activity in boar epididymal fluid
Sperm maturation, represents a key step in the reproduction process. Spermatozoa, particularly the plasma membrane, are exposed to epididymal fluid (EF) components representing the natural environment ...
Flow cytometry (FCM) sperm assessment In normozoospermic and asthenozoospermic men using monoclonal antibodies against sperm proteins
Čapková, Jana; Kubátová, Alena; Děd, Lukáš; Teplá, O.; Pěknicová, Jana
2014 - English
Recent studies have shown that infertility affects an estimated 15% of all couples. Male infertility is the primary or contributing cause in 60% of these cases. Consequently, application of methods of assisted reproduction is increasing. These methods would benefit from extended evaluation of the sperm quality. For this purpose, we analyzed sperm proteins in men with normal spermiograms and with asthenozoospermia. Ejaculates of both groups were tested with a set of well-characterized monoclonal antibodies (MoAbs) to human sperm. No statistically significant differences were found between normospermics and asthenospermics in the expression of sperm surface proteins clusterin, evaluated by Hs-3 MoAb, and semenogelin, evaluated by Hs-9 MoAb. On the other hand, flow cytometry revealed quantitative differences between normozoospermic and asthenozoospermic men in GAPDHS (glyceraldehyde phosphate dehydrogenase human sperm-specific glycolytic enzyme), evaluated by Hs-8 MoAb, VCP (valosin-containing protein), detected with Hs-14 MoAb, and PRKAR2A (cAMP-dependent protein kinase type II – alpha regulatory subunit) detected by MoAb Hs-36. Asthenozoospermic men displayed significantly reduced expression of intra-acrosomal proteins with a likely decrease in sperm quality, and thus a negative impact on successful reproduction.
Keywords:
monoclonal antibody; flow cytometry; spermatozoa; asthenozoospermia
Available at various institutes of the ASCR
Flow cytometry (FCM) sperm assessment In normozoospermic and asthenozoospermic men using monoclonal antibodies against sperm proteins
Recent studies have shown that infertility affects an estimated 15% of all couples. Male infertility is the primary or contributing cause in 60% of these cases. Consequently, application of methods of ...
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