Mammalian fertilization remains a poorly understood event with the vast majority of studies done in the mouse model. The purpose of this review is to revise the current knowledge about semen deposition, sperm transport, sperm capacitation, gamete interactions and early embryonic development with a focus on the porcine model as a relevant, alternative model organism to humans. The review provides a thorough overview of post-ejaculation events inside the sow's reproductive tract including comparisons with humans and implications for human fertilization and assisted reproductive therapy (ART). Porcine methodology for sperm handling, preservation, in vitro capacitation, oocyte in vitro maturation, in vitro fertilization and intra-cytoplasmic sperm injection that are routinely used in pig research laboratories can be successfully translated into ART to treat human infertility. Last, but not least, new knowledge about mitochondrial inheritance in the pig can provide an insight into human mitochondrial diseases and new knowledge on polyspermy defense mechanisms could contribute to the development of new male contraceptives.
- MeSH
- fertilita fyziologie MeSH
- fertilizace fyziologie MeSH
- kapacitace spermií fyziologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Estrogens are steroid hormones that affect a wide range of physiological functions. The effect of estrogens on male reproductive tissues and sperm cells through specific receptors is essential for sperm development, maturation, and function. Although estrogen receptors (ERs) have been studied in several mammalian species, including humans, they have not yet been described in bull spermatozoa and reproductive tissues. In this study, we analyzed the presence of all types of ERs (ESR1, ESR2, and GPER1) in bull testicular and epididymal tissues and epididymal and ejaculated spermatozoa, and we characterize them here for the first time. We observed different localizations of each type of ER in the sperm head by immunofluorescent microscopy. Additionally, using a selected polyclonal antibody, we found that each type of ER in bull sperm extracts had two isoforms with different molecular masses. The detailed detection of ERs is a prerequisite not only for understanding the effect of estrogen on all reproductive events but also for further studying the negative effect of environmental estrogens (endocrine disruptors) on processes that lead to fertilization.
- MeSH
- epididymis metabolismus MeSH
- receptory pro estrogeny metabolismus MeSH
- receptory spřažené s G-proteiny metabolismus MeSH
- rozmnožování * MeSH
- skot metabolismus MeSH
- spermie metabolismus MeSH
- testis metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot metabolismus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We studied the participation of the ubiquitin-proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8-16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P < 0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P > 0.99) but showed the accumulation of DQH (P = 0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. The activity of sperm UPS modulates de-aggregation of boar spermadhesins and DQH sperm surface protein/binder of sperm1 (BSP1) during the sperm capacitation.
- MeSH
- kapacitace spermií fyziologie MeSH
- membránové glykoproteiny metabolismus MeSH
- prasata MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteiny semenné plazmy metabolismus MeSH
- ubikvitin metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tetraspanins are multifunctional molecules located in specific microdomains on the plasma membrane. Thanks to their ability to form networks with other proteins they can participate in many cellular functions. Tetraspanins are part of the interactive network in gametes; however, their precise role in fertilization is not yet clear. The aim of this study was to compare the localization of CD9 and CD81 tetraspanins during oocyte maturation and early development of the embryos in bovine and porcine model. CD9 was detected on the oocyte plasma membrane and vesicles in the perivitelline space of bovine oocytes and embryos. We suggest that CD9 could be a component involved in transzonal projections. Based on the results of in vitro fertilization assay, CD9 and CD81 seem to be part of a more complex fusion network on the plasma membrane of bovine oocytes. On the other hand, both tetraspanins showed a clustered expression pattern on the plasma membrane and inner margin of zona pellucida (ZP) in porcine oocytes and embryos. We found a new species-specific pattern of CD9 and CD81 distribution in ZP which could reflect their specialized role in processes associated with cell adhesion and intercellular communication upon fertilization.
- MeSH
- antigeny CD81 metabolismus MeSH
- antigeny CD9 metabolismus MeSH
- buněčné linie MeSH
- embryo savčí cytologie metabolismus MeSH
- fertilizace in vitro účinky léků MeSH
- metafáze účinky léků MeSH
- myši inbrední BALB C MeSH
- oocyty cytologie metabolismus MeSH
- partenogeneze účinky léků MeSH
- prasata MeSH
- protilátky farmakologie MeSH
- skot MeSH
- stadium rýhování vajíčka cytologie účinky léků MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.
- MeSH
- buněčná membrána metabolismus MeSH
- kapacitace spermií * MeSH
- prasata MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteomika MeSH
- spermie cytologie fyziologie 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
Integrins are transmembrane cell receptors involved in two crucial mechanisms for successful fertilization, namely, mammalian intracellular signaling and cell adhesion. Integrins α6β4, α3β1 and α6β1 are three major laminin receptors expressed on the surface of mammalian cells including gametes, and the presence of individual integrin subunits α3, α6, β1 and β4 has been previously detected in mammalian sperm. However, to date, proof of the existence of individual heterodimer pairs in sperm and their detailed localization is missing. The major conclusion of this study is evidence that the β4 integrin subunit is expressed in mouse sperm and that it pairs with subunit α6; additionally, there is a detailed identification of integrin heterodimer pairs across individual membranes in an intact mouse sperm head. We also demonstrate the existence of β4 integrin mRNAs in round spermatids and spermatogonia by q-RT-PCR, which was further supported by sequencing the PCR products. Using super-resolution microscopy accompanied by colocalization analysis, we located integrin subunits as follows: α6/β4-inner apical acrosomal membrane and equatorial segment; α3, α6/β1, β4-plasma membrane overlaying the apical acrosome; and α3/β1-outer acrosomal membrane. The existence of α6β4, α3β1 and α6β1 heterodimers was further confirmed by proximity ligation assay (PLA). In conclusion, we delivered detailed characterization of α3, α6, β1 and β4 integrin subunits, showing their presence in distinct compartments of the intact mouse sperm head. Moreover, we identified sperm-specific localization for heterodimers α6β4, α3β1 and α6β1, and their membrane compartmentalization and the presented data show a complexity of membranes overlaying specialized microdomain structures in the sperm head. Their different protein compositions of these individual membrane rafts may play a specialized role, based on their involvement in sperm-epithelium and sperm-egg interaction.
- MeSH
- biologické modely MeSH
- integriny chemie metabolismus MeSH
- kompartmentace buňky * MeSH
- multimerizace proteinu * MeSH
- myši inbrední C57BL MeSH
- podjednotky proteinů metabolismus MeSH
- proteinové domény MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Phosphorylation, or dephosphorylation, is one of the most frequent post-translational modifications regulating protein-protein activity in eukaryotic cells. Whereas mature spermatozoa (as specialized cells) are transcriptionally inactive and do not synthesize new proteins, phosphorylation of sperm proteins is very important for the regulation of the sperm function. Although the post-testicular maturation of spermatozoa is a process common to all mammals, comparative studies showed significant differences in sperm surface proteins and the mechanisms of protein modification during the epididymal maturation. In our study, the evaluation of tyrosine phosphorylation, represented by the fluorescent patterns of used anti-phosphotyrosine antibodies (P-Tyr-01 and 4G10), in spermatozoa isolated from different regions of the epididymis - caput, corpus and cauda - was performed. Although in general both antibodies detected almost the same reaction patterns, we observed some dissimilarity associated with the binding specificity of the antibodies and also the segment-dependent manner of phosphorylated protein localization. These data were filled up by immunohistochemical analysis of testes and epididymides cryosections. Additionally, our phosphoproteomic study focused on evaluation of the changes in the pattern of tyrosine-phosphorylated proteins during the post-testicular maturation of bull spermatozoa (PY20 antibody). To summarize the results, an increasing trend of tyrosine phosphorylation of proteins during the maturation of bull sperm in the epididymis was consistently observed in all the methods/experiments.
- MeSH
- epididymis cytologie MeSH
- fluorescence MeSH
- fosforylace MeSH
- fosfotyrosin metabolismus MeSH
- proteiny metabolismus MeSH
- skot MeSH
- spermie cytologie metabolismus MeSH
- testis cytologie MeSH
- zrání spermie * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The G protein-coupled estrogen receptor (GPER) is a transmembrane receptor considered as a mediator of rapid non-genomic responses. GPER has been found in the male reproductive tract of many mammalian species. However, in adult boars, GPER has been reported only in ejaculated spermatozoa. Therefore, we focused on GPER detection in testicular and epididymal tissues and sperm cells in adult boars. We found GPER in Leydig cells and seminiferous tubules of boar testes and in the secretory epithelium of epididymis. A weaker signal was visible in smooth muscle cells and spermatozoa in the epididymal tubule. In spermatozoa isolated from epididymal parts, GPER was found to localize mainly in the sperm acrosome and flagellum. We immunodetected several protein bands in the extracts of the tissues and epididymal spermatozoa. A significantly higher amount of GPER mRNA was detected in the spermatozoa from caput epididymis, whereas the mRNA expression was lower in tissues of testes and caput epididymal. Our results showed the first evidence of GPER in boar epididymal spermatozoa. Moreover, the GPER localization in adult boar testes, epididymis, and mature spermatozoa suggests the involvement of estrogens via transmembrane receptor and rapid non-genomic signaling in both the sperm development and post-testicular maturation.
- MeSH
- epididymis metabolismus MeSH
- messenger RNA metabolismus MeSH
- prasata MeSH
- receptory spřažené s G-proteiny metabolismus MeSH
- spermie metabolismus MeSH
- testis metabolismus MeSH
- zrání spermie fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Proteins CD9 and CD81 are members of the tetraspanin superfamily and were detected in mammalian sperm, where they are suspected to form an active tetraspanin web and to participate in sperm⁻egg membrane fusion. The importance of these two proteins during the early stages of fertilization is supported by the complete sterility of CD9/CD81 double null female mice. In this study, the putative mechanism of CD9/CD81 involvement in tetraspanin web formation in sperm and its activity prior to fertilization was addressed. Confocal microscopy and colocalization assay was used to determine a mutual CD9/CD81 localization visualised in detail by super-resolution microscopy, and their interaction was address by co-immunoprecipitation. The species-specific traits in CD9 and CD81 distribution during sperm maturation were compared between mice and humans. A mutual position of CD9/CD81 is shown in human spermatozoa in the acrosomal cap, however in mice, CD9 and CD81 occupy a distinct area. During the acrosome reaction in human sperm, only CD9 is relocated, compared to the relocation of both proteins in mice. The structural modelling of CD9 and CD81 homologous and possibly heterologous network formation was used to propose their lateral Cis as well as Trans interactions within the sperm membrane and during sperm⁻egg membrane fusion.
- MeSH
- akrozomální reakce * MeSH
- antigeny CD81 analýza metabolismus MeSH
- antigeny CD9 analýza metabolismus MeSH
- fertilizace MeSH
- fúze membrán MeSH
- kapacitace spermií * MeSH
- lidé MeSH
- mapy interakcí proteinů MeSH
- molekulární modely MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- spermie cytologie metabolismus ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-l-fucosidase, α-d-galactosidase, β-d-galactosidase, β-D-N-acetylhexosaminidase, and α-d-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-d-mannosidase, which was increased (P < 0.001), and β-d-galactosidase, which was decreased (P < 0.001) at neutral pH, and of α-d-galactosidase and β-N-acetylhexosaminidase, which were decreased (P < 0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both β-d-galactosidase and to a lesser degree α-d-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.
- MeSH
- biotinylace MeSH
- čipová analýza proteinů MeSH
- folikulární tekutina chemie MeSH
- glykosidhydrolasy krev chemie metabolismus MeSH
- oocyty MeSH
- prasata * MeSH
- proteiny semenné plazmy chemie metabolismus MeSH
- zona pellucida chemie fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
