Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm-egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.
During natural fertilization, mammalian spermatozoa must pass through the zona pellucida before reaching the plasma membrane of the oocyte. It is assumed that this step involves partial lysis of the zona by sperm acrosomal enzymes, but there has been no unequivocal evidence to support this view. Here we present evidence that acrosin, an acrosomal serine protease, plays an essential role in sperm penetration of the zona. We generated acrosin-knockout (KO) hamsters, using an in vivo transfection CRISPR/Cas9 system. Homozygous mutant males were completely sterile. Acrosin-KO spermatozoa ascended the female genital tract and reached ovulated oocytes in the oviduct ampulla, but never fertilized them. In vitro fertilization (IVF) experiments revealed that mutant spermatozoa attached to the zona, but failed to penetrate it. When the zona pellucida was removed before IVF, all oocytes were fertilized. This indicates that in hamsters, acrosin plays an indispensable role in allowing fertilizing spermatozoa to penetrate the zona. This study also suggests that the KO hamster system would be a useful model for identifying new gene functions or analyzing human and animal disorders because of its technical facility and reproducibility.
- MeSH
- akrosin genetika metabolismus MeSH
- akrozom metabolismus MeSH
- fertilizace in vitro MeSH
- genový knockout MeSH
- interakce spermie a vajíčka * MeSH
- křečci praví genetika metabolismus MeSH
- spermie enzymologie fyziologie MeSH
- zona pellucida metabolismus MeSH
- zvířata MeSH
- Check Tag
- křečci praví genetika metabolismus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
High levels of reactive oxygen species (ROS), which may be associated with reduced sperm quality, can be detected during cryopreservation of sperm of some species. Our objective was to investigate whether the addition of antioxidants to cryopreservation extenders influenced post-thaw sperm characteristics and fertility in Acipenser dabryanus, Acipenser sinensis and Acipenser baerii. Prior to freezing, sperm samples were diluted with a base extender as control or in extender supplemented with catalase (CAT), glutathione (GSH), cysteine (NAC), ascorbic acid (VC) or their paired combinations. Protective concentrations of CAT, GSH and VC in the three species were 25 U/ml, 0.25-0.5 mg/ml and 0.5 mg/ml, respectively. Cysteine showed no protective effect against ROS. The addition of CAT, GSH and VC positively affected either acrosome or membrane integrity of post-thawed sperm in the three species, as well as spermatozoan motility in A. sinensis. The combination of antioxidants did not show a positive synergistic effect. This study suggested that the use of antioxidants in the cryopreservation of sturgeon sperm has potential to decrease intracellular ROS, and consequently preserve acrosome and membrane integrity, as well as spermatozoan motility.
- MeSH
- akrozom účinky léků MeSH
- antioxidancia farmakologie MeSH
- buněčná membrána účinky léků MeSH
- cystein farmakologie MeSH
- glutathion farmakologie MeSH
- katalasa farmakologie MeSH
- kryoprezervace metody veterinární MeSH
- kryoprotektivní látky farmakologie MeSH
- kyselina askorbová farmakologie MeSH
- motilita spermií účinky léků MeSH
- reaktivní formy kyslíku analýza MeSH
- ryby * MeSH
- spermie účinky léků MeSH
- uchování spermatu metody veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Nitric oxide (NO) is a major gasotransmitter involved in several physiological processes of male reproduction. There is, nevertheless, little information concerning the role of NO during semen storage. The aim of this study was to evaluate the effect of NO on boar semen stored at 17oC for 72 h. For this purporse, sperm samples were treated with 0.625, 1.25, 2.5, 5, and 10 mM aminoguanidine (AG) or Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a selective and non-selective NO synthase (NOS) inhibitor, respectively. Moreover, sodium nitroprusside (SNP), a NO donor, was used at the dose of 18.75, 37.5, 75, and 150 μM. Sperm motility, membrane integrity, and acrosomal status were evaluated at 0, 4, 24, 48, and 72 h of semen storage. A significant increase of the amplitude of lateral sperm head displacement (ALH), and both curvilinear and straight-line velocity (VCL and VSL, respectively) was observed at 72 h of semen storage in samples treated with 0.625 mM AG, probably because of the antioxidant properties of this NOS inhibitor. Contrarily, 0.625 mM L-NAME showed no effect on boar sperm parameters during the entire period of semen storage. Moreover, AG and L-NAME at 10 mM negatively affected sperm kinetics and acrosome integrity, which may provide further support to the notion that low NO levels are necessary for a normal sperm function. The concentrations of SNP used in this study had mostly no or negative effects on boar sperm parameters during semen storage. In conclusion, the results from this study increase the understanding of the role of NO on boar sperm physiology.
- MeSH
- akrozom účinky léků MeSH
- buněčná membrána účinky léků MeSH
- časové faktory MeSH
- guanidiny farmakologie MeSH
- motilita spermií účinky léků MeSH
- nitroprusid farmakologie MeSH
- oxid dusnatý aplikace a dávkování farmakologie MeSH
- prasata * MeSH
- spermie účinky léků MeSH
- synthasa oxidu dusnatého, typ II antagonisté a inhibitory metabolismus MeSH
- uchování spermatu veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This study was conducted to investigate Brachymystax lenok tsinlingensis spermatozoa cell morphology and ultrastructure through scanning and transmission electron microscopy. Findings revealed that the spermatozoa can be differentiated into three major parts: a spherical head without an acrosome, a short mid-piece, and a long, cylindrical flagellum. The mean length of the spermatozoa was 36.11±2.84μm, with a spherical head length of 2.78±0.31μm. The mean anterior and posterior head widths were 2.20±0.42μm and 2.55±0.53μm, respectively. The nuclear fossa was positioned at the base of the nucleus that contained the anterior portion of flagellum and a centriolar complex (proximal and distal centrioles). The short mid-piece was located laterally to the nucleus and possessed just one spherical mitochondrion with a mean diameter of 0.65±0.14μm. The spermatozoa flagellum was long and cylindrical, and could be separated into two parts: a long main-piece and a short end-piece. The main piece of the flagellum had short irregular side-fins. The axoneme composed the typical '9+2' microtubular doublet structure and was enclosed by the cell membran e. This study confirmed that B. lenok tsinlingensis spermatozoa can be categorized as teleostean "Type I" spermatozoa; 'primitive' or 'ect-aquasperm type' spermatozoa. To the best of the authers knowledge, this was the first study conducted on the morphology and ultrastructure of B. lenok tsinlingensis spermatozoa.
- MeSH
- akrozom ultrastruktura MeSH
- axonema ultrastruktura MeSH
- buněčné jádro ultrastruktura MeSH
- centrioly ultrastruktura MeSH
- flagella ultrastruktura MeSH
- mikroskopie elektronová rastrovací MeSH
- mitochondrie ultrastruktura MeSH
- Salmonidae anatomie a histologie růst a vývoj MeSH
- spermie ultrastruktura MeSH
- transmisní elektronová mikroskopie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Anti-Müllerian hormone (AMH) is a factor most associated with female fertility and especially with the ovarian reserve. AMH is also used as a parameter of fertility in men as it arises from the epithelium of the seminiferous tubules that contain Sertoli cells which produce the AMH. To investigate the relationship between AMH production and sperm related parameters we compared the AMH levels in serum and seminal plasma between a group of healthy males (n=65) and male patients (n=68) of infertile couples with semen pathology. We assessed the following fertility parameters: sperm count (SC), presence of intra-acrosomal enzymes (IAE), and antispermatozoal antibodies (ASA). Infertile men were divided into four subgroups according to: SC less than 15 million, SC less than 15 million and lack of IAE, SC less than 15 million and presence of ASA, presence of all three pathological parameters. The mean AMH serum level in the healthy group was 6.95 ng/ml and no significant difference was observed in serum AMH levels. The mean AMH seminal plasma level in the healthy group was 14.21 ng/ml. We observed a statistically significant decrease in the group with a SC with less than 15 million (3.29 ng/ml, p=0.0001) sperm, in the group with SC less than 15 million sperm and lack of IAE (3.95 ng/ml, p=0.0046), and in the group with all three pathological parameters (2.65 ng/ml, p=<0.0001). No significant difference was observed in the group with SC less than 15 million sperm and ASA positivity (11.41 ng/ml, p=0.3171). In conclusion AMH serum levels do not correlate with any of the observed parameters. AMH levels in seminal plasma positively correlate with the pathological SC and with SC pathology and IAE together.
- MeSH
- akrozom enzymologie MeSH
- analýza spermatu MeSH
- antimülleriánský hormon krev MeSH
- autoprotilátky imunologie MeSH
- dospělí MeSH
- fertilita * MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužská infertilita krev imunologie MeSH
- počet spermií MeSH
- sperma metabolismus MeSH
- spermie imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Ultrastructure of spermatozoa of redclaw Cherax quadricarinatus and yabby Cherax destructor were described and compared. The acrosome complex and nucleus are located at the anterior and posterior region of the spermatozoon, respectively. The acrosome is a complex vesicle divided into two parts: the main body of the acrosome appears as a dense cup-shaped structure in longitudinal sagittal view, with the subacrosome zone occupying the central area of the vesicle. The acrosome is larger in C. quadricarinatus (width 2.37±0.27μm, length 1.31±0.23μm) than in C. destructor (width 1.80±0.27μm, length 1.01±0.15μm). There was no significant difference in L:W ratios of the studied species. The subacrosome zone in both species consists of two areas of different electron density. The nucleus is substantially decondensed and irregular in shape, with elaborate extended processes. The examined species exhibited a well-conserved structure of crayfish spermatozoon, similar to those of Cherax cainii and Cherax albidus. Small acrosome size, the absence of radial arms, and an extracellular capsule seem to be the morphological features that mostly distinguish Cherax from the Astacidae and Cambaridae.
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
In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.
- MeSH
- akrosin metabolismus MeSH
- akrozom enzymologie MeSH
- analýza rozptylu MeSH
- histologické techniky veterinární MeSH
- imunoelektronová mikroskopie veterinární MeSH
- inhibitory proteas farmakologie MeSH
- motilita spermií účinky léků fyziologie MeSH
- neparametrická statistika MeSH
- proteasy farmakologie MeSH
- rosanilinová barviva MeSH
- ryby fyziologie MeSH
- sperma enzymologie MeSH
- spermie účinky léků enzymologie fyziologie MeSH
- tosylfenylalanylchlormethylketon farmakologie MeSH
- tosyllysinchlormethylketon farmakologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The ultrastructure and morphology of beluga spermatozoa (Huso huso) (Acipenseridae, Chondrostei) were studied by scanning and transmission electron microscopy. Spermatozoa of 51.27 ± 4.71 μm total length (mean ± SD) were typical of sturgeon and paddlefish spermatozoa. Each consisted of an elongated nucleus (length: 5.84 ± 0.46 μm) with distinct acrosome, a cylindrical midpiece (length: 2.10 ± 0.42 μm), and a single flagellum (length: 42.21 ± 3.82 μm). The acrosome was umbrella shaped (length: 1.12 ± 0.14 μm, width: 0.87 ± 0.10 μm) with seven to nine posterolateral projections (length: 0.49 ± 0.09 μm). Three endonuclear canals, spirally arranged, traversed the nucleus length from the acrosome to the implantation fossa. The flagellum comprised an axoneme with the typical 9+2 organization of microtubules. Two flat fins were present along the flagellum parallel to the plane of the central doublet of microtubules. These fins arose at different positions, 0.57 ± 0.30 and 4.06 ± 1.32 μm from the midpiece and terminated 4.18 ± 1.09 and 4.92 ± 1.34 μm from the distal end of the flagellum. Principal component analysis revealed spermatozoan ultrastructure and morphology were similar among sturgeon species, and, although assigned to genus Huso, beluga may be closely related to the genus Acipenser.
- MeSH
- akrozom ultrastruktura MeSH
- analýza hlavních komponent MeSH
- flagella ultrastruktura MeSH
- mikroskopie elektronová rastrovací veterinární MeSH
- ryby anatomie a histologie MeSH
- spermie ultrastruktura MeSH
- transmisní elektronová mikroskopie veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
