Chromatin remodeling, including histone post-translational modifications, during spermatogenesis can affect sperm quality and fertility, and epigenetic marks may therefore be useful for clinical evaluations of sperm. Together with histone hyperacetylation, the dimethylation of histone H3 on lysine K4 (H3K4me2) is also required during protamination. Accordingly, we evaluated the utilization of this epigenetic mark for the identification of sperm with decrease quality and immature chromatin. In this study, 99 semen samples, including 22 normozoospermic (N), 63 asthenozoospermic (A), and 14 oligoasthenozoospermic (OA) samples, were comprehensively analyzed with respect to H3K4me2 levels, DNA damage (DNA fragmentation index, DFI), and sperm immaturity (high DNA stainability, %HDS), as determined by a sperm chromatin structure assay using flow cytometry. We detected a significant relationship between H3K4me2 and %HDS (r = 0.47; p < 0.001). Furthermore, we observed negative correlations between H3K4me2 and sperm concentration, motility, and mitochondrial activity (p < 0.05). The increase in immaturity as semen quality decreased (N > A > OA) indicates the importance of chromatin immaturity and histone code deviations in sperm evaluations. Using various approaches, our study elucidated H3K4me2 as a molecular marker of sperm quality with potential use in reproductive medicine.Abbreviations: A: asthenozoospermic; AO: acridine orange; ART: assisted reproductive therapy; BWW: Biggers-Whitten Whittingham; DAPI: 4',6' -diamidino-2-phenylindole; DFI: DNA fragmentation index; H3K4me2: dimethylation of lysine K4 on histones H3; HDS: high DNA stainability; HRP: horseradish peroxidase; MACS: magnetic-activated cell sorting; N: normospermic; NGS: normal goat serum; OA: oligoasthenozoospermic; PTM: post-translational modification; SCSA: sperm chromatin structure assay; SUTI: sperm ubiquitin tag assay; TBS-T: TBS with 0.5% Tween-20.
- MeSH
- analýza spermatu MeSH
- astenozoospermie metabolismus MeSH
- biologické markery metabolismus MeSH
- dospělí MeSH
- histonový kód * MeSH
- histony metabolismus MeSH
- lidé MeSH
- metylace MeSH
- oligospermie metabolismus MeSH
- restrukturace chromatinu * MeSH
- spermie metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry (FCM) and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies (MoAbs), which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase (cAMP-dependent protein kinase II, PRKAR2A), evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.
- MeSH
- astenozoospermie imunologie metabolismus MeSH
- fertilizace in vitro MeSH
- intracytoplazmatické injekce spermie MeSH
- lidé MeSH
- monoklonální protilátky imunologie MeSH
- proteiny metabolismus MeSH
- průtoková cytometrie MeSH
- spermie imunologie metabolismus MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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
