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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
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
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Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla ; Zigo, Michal ; Pohlová, Alžběta ; Jonáková, Věra
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.
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Sperm protein profiles of different mammalian species
Pohlová, Alžběta ; Zigo, Michal ; Jonáková, Věra ; Postlerová, Pavla
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.
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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
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.
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CD46 and β1integrin interaction in mouse sperm head
Šebková, Nataša ; Frolíková, Michaela ; Dvořáková-Hortová, Kateřina
CD46 protein plays an important role during fertilization and its role is associated with acrosome stability. CD46 is probably involved in signalling pathways triggering the acrosome reaction. Integrins interact with many cytoskeletal proteins such as actin, therefore changes in the actin cytoskeleton before and after AR may lead to changes in the association and localization of CD46 and β1integrin. Our aim was to monitor mutual CD46 and β1integrin interaction detected by the proximity ligation assay. It generates a localized signal in a form of spots revealing the exact position of the recognition event. Proteins interaction was study in freshly released sperm and sperm during the acrosome reaction, during which there is a gradual relocation of these proteins towards the equatorial segment and the whole sperm head. Proteins α and β tubulin were used as a positive control, α tubulin and β1 integrin as a negative control. In situ PLA showed a distinct spotted signal indicating the mutual interaction of CD46 and β1integrin. A positive response was demonstrated not only in freshly released sperm but also in sperm during the acrosome reaction. Freshly released sperm were distinctively labelled in the acrosome region and the neck, similarly to the positive control. Sperm during the acrosome reaction showed the signal across the whole sperm head region. No signal or sporadic nonspecific staining was detected in the case of the negative control. In summary, our results deliver new information that proteins CD46 and β1 integrin interact with each other. These results suppose the theory that β1 integrin can mediate a connection between CD46 and sperm cytoskeleton thereby molecules of signalling pathways leading to activation of the acrosome reaction.
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Dynamics of mouse sperm capacitation and acrosome reaction
Dvořáková-Hortová, Kateřina ; Frolíková, Michaela ; Děd, Lukáš ; Šebková, Nataša
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.
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Fluorescent analysis of the differential protein expression in normozoospermic and asthenozoospermic sperm samples
Děd, Lukáš ; Čapková, Jana ; Kubátová, Alena ; Teplá, O. ; Pěknicová, Jana
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.
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