sperm function
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BACKGROUND: Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. METHODS: Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. RESULTS: Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. CONCLUSION: GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.
- MeSH
- akrozom metabolismus MeSH
- energetický metabolismus MeSH
- flagella metabolismus MeSH
- glyceraldehyd-3-fosfátdehydrogenasy analýza genetika fyziologie MeSH
- interakce spermie a vajíčka MeSH
- lidé MeSH
- motilita spermií MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- prasata metabolismus MeSH
- spermatogeneze MeSH
- spermie metabolismus MeSH
- zona pellucida metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The spermatozoon is the most diverse cell type known and this diversity is considered to reflect differences in sperm function. How the diversity in sperm morphology arose during speciation and what role the different specializations play in sperm function, however, remain incompletely characterized. This work reviews the hypotheses proposed to explain sperm morphological evolution, with a focus on some aspects of sperm morphometric evaluation; the ability of morphometrics to predict sperm cryoresistance and male fertility is also discussed. For this, the evaluation of patterns of change of sperm head morphometry throughout a process, instead of the study of the morphometric characteristics of the sperm head at different stages, allows a better identification of the males with different sperm cryoconservation ability. These new approaches, together with more studies employing a greater number of individuals, are needed to obtain novel results concerning the role of sperm morphometry on sperm function. Future studies should aim at understanding the causes of sperm design diversity and the mechanisms that generate them, giving increased attention to other sperm structures besides the sperm head. The implementation of scientific and technological advances could benefit the simultaneous examination of sperm phenotype and sperm function, demonstrating that sperm morphometry could be a useful tool for sperm assessment.
Cíl studie: Jednou z příčin mužské neplodnosti je snížená motilita spermií. Ukazuje se, že ve vývoji této poruchy může hrát roli snížená efektivita respirační aktivity mitochondrií. Cílem naší studie bylo komplexní stanovení respirační aktivity mitochondrií spermií s normální a sníženou pohyblivostí. Typ studie: Prospektivní studie. Název a sídlo pracoviště: Ústav histologie a embryologie, LF UK, Plzeň; Ústav fyziologie, LF UK, Plzeň; Institut reprodukční medicíny a endokrinologie, IVF Centrum Prof. Zecha, Plzeň. Metodika: Ejakuláty byly získány od 14 mužů z IVF Centra Prof. Zecha v Plzni. Podle klasifikace World Health Organization byli muži rozděleni do skupiny normozoospermatiků (n = 7) a astenozoospermatiků (n = 7). Respirační aktivitu spermií jsme měřili na dvoukanálovém oxygrafu Oroboros. Výsledky: V astenozoospermatických vzorcích byla nalezena signifikantně snížená aktivita komplexu I(p = 0,007), zvýšená respirace po aplikaci inhibitoru ATP-syntázy oligomycinu (ukazující na zvýšené rozpřažení oxidace a fosforylace; p = 0,046). Inhibice komplexu I rotenonem ukázala, že příspěvek komplexu I k celkové kapacitě oxidační fosforylace byl i u zdravých spermií relativně nižší, než je tomu typicky v somatických buňkách. Závěr: V naší studii jsme měřili respirační aktivitu mitochondrií lidských spermií permeabilizovaných digitoninem vysokoúčinnou oxygrafií, která umožňuje stanovení spotřeby kyslíku z nejmenšího možného množství zárodečných buněk. Výsledky studie potvrzují sníženou aktivitu komplexu I u astenozoospermatiků a naznačují, že na snížené pohyblivosti spermií by se mohl podílet i zvýšený únik protonů z mitochondriální matrix, který vede ke snížené efektivitě fosforylačního procesu. Lepší charakterizace mužských zárodečných buněk, ať zcela zdravých, či s postiženou motilitou, nám pomůže lépe pochopit proces fyziologického oplodnění a zároveň pomůže i ve výběru té nejvíce životaschopné spermie pro léčbu neplodnosti metodami asistované reprodukce.
Objective: One of causes of male infertility is reduced sperm motility. It turns out that the reduced efficiency of the mitochondrial respiratory activity may play a role in the development of this disorder. The aim of our study was to comprehensively determine mitochondrial respiratory activity of sperm with normal and reduced motility. Design: Prospective study. Setting: Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague; Department of Physiology, Faculty of Medicine in Pilsen, Charles University in Prague; Institute of Reproductive Medicine and Endocrinology, IVF Centers Prof. Zech, Plzeň. Methods: Ejaculates of 14 men were obtained from IVF Center Prof. Zech, Pilsen. According to the World Health Organization classification, samples were divided into normozoospermatic (n = 7) and asthenozoospermatic(n = 7) groups. Respiratory activity of sperm was measured on two-chamber oxygraph Oroboros. Results: In asthenozoospermatic samples, significantly reduced activity of complex I (p = 0.007) and increased respiration after application of ATP-synthase inhibitor oligomycin (showing increased uncoupled oxidation and phosphorylation, p = 0.046) were found. Inhibition of complex I by rotenone showed that complex I contribution to the total capacity of oxidative phosphorylation of healthy sperm was relatively lower than it is typical for somatic cells. Conclusion: In our study, we measured mitochondrial respiratory activity of human sperm, permeabilized by digitonin, by high-resolution oxygraphy, which allows the determination of oxygen consumption from the smallest possible number of germ cells. The study results confirm reduced activity of complex I in asthenozoospermatics and suggest that increased leakage of protons from the mitochondrial matrix, which leads to reduced efficiency of phosphorylating process, could participate in the reduced sperm motility. Better characterization of male germ cells, either completely healthy or with affected motility, will help us to understand better the physiological process of fertilization and also to choose the most viable sperm for infertility treatment by methods of assisted reproduction.
- MeSH
- astenozoospermie metabolismus MeSH
- klinické laboratorní techniky MeSH
- lidé MeSH
- mitochondriální proteiny * fyziologie metabolismus MeSH
- motilita spermií fyziologie MeSH
- mužská infertilita MeSH
- permeabilita buněčné membrány MeSH
- postup MeSH
- reaktivní formy kyslíku * metabolismus MeSH
- sperma * metabolismus MeSH
- spermie * fyziologie patologie MeSH
- spotřeba kyslíku MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
Reaktivní sloučeniny kyslíku jsou ve vhodné koncentraci důležité pro fyziologickou funkci spermií. Pokud je ale jejich množství v buňce vysoké nebo hladina antioxidantů nedostatečná, může dojít k oxidativnímu poškození buněk. Volné radikály napadají především polynenasycené mastné kyseliny ve fosfolipidové membráně spermií. Důsledkem peroxidace lipidů může být zhoršená kvalita spermií, tedy problémy s plodností. Přehledový článek popisuje složení membrány spermatických buněk, význam mastných kyselin a možnosti prevence oxidativního poškození buněk.
One of causes of male infertility is reduced sperm motility. Reactive oxygen species (ROS) play significant role for physiological sperm function. Oxidative stress occurs when the production of potentially destructive ROS exceeds the natural antioxidant defences, resulting in cell damage. Sperm phospholipid polyunsaturated fatty acids are particularly susceptible to peroxidative damage by free radicals. Detrimental effects of lipid peroxidation should decrease sperm quality and be responsible of fertility problems. The review deals with sperm membrane composition, importance of fatty acids and prevention possibilities of oxidative cell damage.
- MeSH
- buněčná membrána * fyziologie chemie MeSH
- kyseliny dokosahexaenové MeSH
- lidé MeSH
- mastné kyseliny * MeSH
- membránové lipidy * fyziologie MeSH
- metabolismus lipidů fyziologie MeSH
- mužská infertilita MeSH
- oxidační stres MeSH
- plasmalogeny MeSH
- spermie * fyziologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
Primary binding of the sperm to the zona pellucida (ZP) is one of the many steps necessary for successful fertilization. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-defined sequential process. Primary binding receptors, many of which have been disclosed in various mammals, are localized throughout the acrosomal region of the sperm surface. A panel of monoclonal antibodies against proteins from the sperm surface was prepared. Antibodies were screened by immunofluorescence for protein localization and Western blotting. Proteins localized on the sperm head and simultaneously detected by Western blotting were further studied in terms of immunolocalization in reproductive tissues and fluids, binding to ZP, immunoprecipitation and sequencing. Of 17 prepared antibodies, 8 recognized proteins localized on the sperm head and also detected proteins of interest by Western blotting. Only three other antibodies recognized proteins that also coincided in binding to ZP. These three antibodies were used for immunoprecipitation, and further protein sequencing of immunoprecipitates revealed that these antibodies distinguished acrosin precursor, RAB-2A protein, and lactadherin P47. This is not the first time we have detected acrosin on the surface of ejaculated and capacitated sperm. However, to our knowledge, this is the first time RAB-2A has been detected on the sperm surface. Lactadherin P47 has already been characterized and its physiological function in reproduction has been proposed.
- MeSH
- fluorescenční protilátková technika MeSH
- interakce spermie a vajíčka * MeSH
- molekulová hmotnost MeSH
- monoklonální protilátky metabolismus MeSH
- prasata MeSH
- receptory buněčného povrchu metabolismus MeSH
- spermie metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.
- MeSH
- akrozom imunologie fyziologie MeSH
- akrozomální reakce MeSH
- benzoáty farmakologie MeSH
- exocytóza MeSH
- fertilizace * účinky léků MeSH
- fosfotyrosin imunologie MeSH
- furany farmakologie MeSH
- glykoproteiny analýza imunologie MeSH
- interakce spermie a vajíčka * MeSH
- kapacitace spermií * MeSH
- prasata metabolismus fyziologie MeSH
- proteiny semenné plazmy analýza imunologie MeSH
- protilátky imunologie MeSH
- pyrazoly farmakologie MeSH
- spermatocyty metabolismus MeSH
- spermatogonie metabolismus MeSH
- spermie metabolismus MeSH
- ubikvitin aktivující enzymy metabolismus MeSH
- ubikvitin imunologie MeSH
- ubikvitinace MeSH
- zona pellucida metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Fish sperm cryopreservation is a well-established technique allowing for artificial insemination on a commercial scale. The extent of proteome alterations in seminal plasma and sperm due to cryopreservation, however, is not known. This study was conducted to evaluate the effect of cryopreservation on motility variables of sterlet Acipenser ruthenus sperm and to detect the differences in protein profiles of fresh and cryopreserved sterlet sperm and seminal plasma. Fresh sperm had 89 ± 3% motility and 160 ± 14 μm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ± 5%) was less at 15 s post-activation. No difference (ANOVA; P > 0.05) in mean curvilinear velocity of fresh and cryopreserved sperm was detected. The protein profiles of seminal plasma and sperm were characterized using comparative proteomics to determine the influence of cryopreservation. Six altered protein spots in seminal plasma and thirteen altered spots in sperm were detected in fresh and thawed sperm. Subsequent protein characterization suggested that the proteins identified were involved in sperm metabolism, cytoskeleton, and stress response. The results broaden the understanding of the effects of cryopreservation and identify the proteins associated with cryo-injury. These data may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation.
- MeSH
- kryoprezervace veterinární MeSH
- motilita spermií * MeSH
- proteom * MeSH
- ryby fyziologie MeSH
- spermie fyziologie MeSH
- transkriptom MeSH
- uchování spermatu veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In the current study, the sperm of Russian sturgeon (Acipenser gueldenstaedtii) was used to evaluate the roles of Verapamil (VRP), a calcium channel blocker, against cadmium (Cd)-induced stress. Sturgeon sperm were exposed for 2h at 50μg/L VRP, 5.0μg/L Cd, the mixture of 50μg/L VRP+5.0μg/L Cd, 50μg/L Cd and the mixture of 50μg/L VRP+50μg/L Cd. After exposure, the sperm motility parameters (motility and velocity), oxidative stress levels (lipid peroxidation [LPO] and carbonyl protein [CP]) and antioxidant enzyme activities (superoxide dismutase [SOD], glutathione reductase [GR], glutathione peroxidase [GPx]) were measured in sturgeon sperm. Compared to the control, Cd-induced stress was apparent as reflected by depressed motility parameters, induced oxidative stress and inhibited antioxidant enzyme activities at both Cd concentrations. In the presence of VRP, Cd-induced stress was reduced in sturgeon sperm, especially all the measured parameters in the sperm exposed at 5.0μg/L Cd returned to control levels, expect for the sperm motility. The present results indicate that VRP can reduce the Cd-induced stress in sturgeon sperm and suggest that using of sperm in vitro assays may provide a novel and efficient means for evaluating the effects of residual metals in the aquatic environment of sturgeon.
- MeSH
- blokátory kalciových kanálů farmakologie MeSH
- chemické látky znečišťující vodu toxicita MeSH
- glutathionperoxidasa metabolismus MeSH
- glutathionreduktasa metabolismus MeSH
- kadmium toxicita MeSH
- oxidační stres MeSH
- peroxidace lipidů účinky léků MeSH
- ryby metabolismus fyziologie MeSH
- spermie účinky léků metabolismus MeSH
- superoxiddismutasa metabolismus MeSH
- verapamil farmakologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Motility is a characteristic function of the male gamete, which allows spermatozoa to actively reach and penetrate the female gamete in organisms with internal and external fertilization. Sperm motility is acquired under the control of many extrinsic and intrinsic factors and is based on a specialized structure of the sperm flagellum called "axoneme". An overview of how the sperm flagellum is organized, and it operates to support cell motility is presented, with special focus on the molecular mechanisms and factors involved in the development, maintenance and control of motility. Data obtained in aquatic organisms with external fertilization, such as sea urchins, ascidians or fishes are critically analyzed because they constitute model species on which most of the present day understanding of sperm motility function is based. In most animal species, sperm motility is dependent on a long appendage called flagellum. Flagella are essential organelles found in most eukaryotic cells; their basic structure is the axoneme, which consists of a scaffold of microtubules and is responsible for movement in an autonomous manner if ATP-energy is present. Flagellar beat propels the cell through the medium which surrounds sperm cells and is responsible of the translational drive of spermatozoa. The present paper includes: (1) an introduction to typical sperm morphology and ultrastructure in most aquatic species, (2) the motility apparatus or axoneme of the spermatozoa: the axoneme, (3) the structural and biochemical composition of the axoneme, (4) the axonemal motor or dynein, and its operation, (5) the regulation of motility at axoneme and cell membrane levels, including several effectors such as Ca2+ ions, (6) biophysical features of the wave propagation mechanism in motile spermatozoa, (7) the energy production and consumption, and (8) the building of a flagellum. Flagellar beating in aquatic animals is illustrated using several examples in figures and video-clips. These types of data are also used for computer simulation of various aspects of the modulation of sperm motility of marine animals.
Propolis is a natural product that honeybees collect from various plants. It is known for its beneficial pharmacological effects. The aim of our study was to evaluate the impact of propolis on human sperm motility, mitochondrial respiratory activity, and membrane potential. Semen samples from 10 normozoospermic donors were processed according to the World Health Organization criteria. Propolis effects on the sperm motility and mitochondrial activity parameters were tested in the fresh ejaculate and purified spermatozoa. Propolis preserved progressive motility of spermatozoa in the native semen samples. Oxygen consumption determined in purified permeabilized spermatozoa by high-resolution respirometry in the presence of adenosine diphosphate and substrates of complex I and complex II (state OXPHOSI+II) was significantly increased in the propolis-treated samples. Propolis also increased uncoupled respiration in the presence of rotenone (state ETSII) and complex IV activity, but it did not influence state LEAK induced by oligomycin. Mitochondrial membrane potential was not affected by propolis. This study demonstrates that propolis maintains sperm motility in the native ejaculates and increases activities of mitochondrial respiratory complexes II and IV without affecting mitochondrial membrane potential. The data suggest that propolis improves the total mitochondrial respiratory efficiency in the human spermatozoa in vitro thereby having potential to improve sperm motility.
- Publikační typ
- časopisecké články MeSH
