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
- MeSH
- akrosin antagonisté a inhibitory terapeutické užití MeSH
- apolipoproteiny B izolace a purifikace krev normy MeSH
- deriváty kyseliny fibrové terapeutické užití MeSH
- diabetes mellitus 1. typu * diagnóza etiologie terapie MeSH
- diabetes mellitus 2. typu * diagnóza etiologie terapie MeSH
- dieta s omezením tuků metody využití MeSH
- dyslipidemie * diagnóza etiologie terapie MeSH
- ezetimib terapeutické užití MeSH
- farmakoterapie metody využití MeSH
- klinické laboratorní techniky metody normy využití MeSH
- kyseliny mastné omega-3 terapeutické užití MeSH
- LDL-cholesterol izolace a purifikace krev normy MeSH
- lidé MeSH
- poruchy metabolismu lipidů diagnóza etiologie terapie MeSH
- směrnice pro lékařskou praxi jako téma normy MeSH
- společnosti lékařské normy MeSH
- statiny terapeutické užití MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Epididymal sperm maturation represents a key step in the reproduction process. Spermatozoa are exposed to epididymal fluid components representing the natural environment essential for their post-testicular maturation. Changes in sperm membrane proteins are influenced by proteolytic, glycosylation and deglycosylation enzymes present in the epididymal fluid. Accordingly, the occurrence of inhibitors of these enzymes in the epididymis is very important for the regulation of sperm membrane protein processing. In the present study, we monitored acrosin inhibitor distribution in boar epididymal fluid and in spermatozoa from different segments of the organ. Using specific polyclonal antibody we registered increasing signal of the acrosin inhibitor (AI) from caput to cauda epididymis. Mass spectroscopy examination of the immunoprecipitated acrosin inhibitor (12 kDa) unequivocally identified sperm-associated acrosin inhibitor (SAAI) in the epididymal tissue. Lectin staining showed N-glycosylation in AI from boar epididymis. Protein detection of AI was supported by the results of semi-quantitative RT-PCR showing the presence of mRNA specifically coding for SAAI and similarly increasing throughout the epididymal duct, from its proximal to distal part. Additionally, the immunofluorescence technique showed the AI localization in the secretory tissue of caput, corpus and cauda epididymis, and in the acrosome region and midpiece of the sperm.
- MeSH
- akrosin antagonisté a inhibitory MeSH
- epididymis metabolismus MeSH
- exprese genu MeSH
- glykosylace MeSH
- hmotnostní spektrometrie MeSH
- inhibitory enzymů krev chemie metabolismus MeSH
- molekulární sekvence - údaje MeSH
- prasata MeSH
- proteolýza MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- spermie metabolismus MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví 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
BACKGROUND: High-throughput studies provide a wide spectrum of genes for use as predictive markers during testicular sperm extraction (TESE) in combination with ICSI. In this work, we used the specimens from testicular biopsies of men with non-obstructive azoospermia who underwent TESE to investigate the expression of spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR. METHODS: Testicular biopsy specimens were subdivided into three groups: hypospermatogenesis (HS); maturation arrest (MA); and Sertoli cell-only syndrome (SCO). The levels of expression of the spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR in the testes were compared among these three groups using the reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: Analysis of the expression of spermatogenic genes in human testes with abnormal spermatogenesis showed different expression patterns in patients from different groups. Fertilization rate for studied set of patients was 66% and pregnancy rate 29%. For HS group fertilization rate was 72% and pregnancy rate 32%, while for MA group fertilization and pregnancy rates were 54% and 26%, respectively. Fertilization rates in relation to the studied genes were uniformly around 70%, pregnancy rates for ACR and GAPDHS genes were surprisingly low at 6% and 8% correspondingly. CONCLUSIONS: Analysis of the expression of genes involved in spermatogenesis can be a fast additional test for the level of spermatogenesis in testicular samples.
- MeSH
- akrosin genetika MeSH
- azoospermie genetika patologie MeSH
- biopsie MeSH
- dospělí MeSH
- fertilizace MeSH
- glyceraldehyd-3-fosfátdehydrogenasy genetika MeSH
- intracytoplazmatické injekce spermie MeSH
- lidé středního věku MeSH
- lidé MeSH
- nemoci varlat genetika patologie MeSH
- odběr spermií MeSH
- oligospermie genetika patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteiny buněčného cyklu genetika MeSH
- Sertoli cell only syndrom genetika patologie MeSH
- spermatogeneze genetika MeSH
- stanovení celkové genové exprese MeSH
- těhotenství MeSH
- testis metabolismus patologie MeSH
- úhrn těhotenství na počet žen v reprodukčním věku MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- akrosin analýza fyziologie MeSH
- cytoskeletální proteiny analýza fyziologie MeSH
- exocytóza fyziologie MeSH
- imunoanalýza MeSH
- lidé MeSH
- spermie fyziologie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
