Spermatogenesis starts with the onset of puberty within the seminiferous epithelium of the testes. It is a complex process under intricate control of the endocrine system. Physiological regulations by steroid hormones in general and by estrogens in particular are due to their chemical nature prone to be disrupted by exogenous factors acting as endocrine disruptors (EDs). 17α-Ethynylestradiol (EE2) is an environmental pollutant with a confirmed ED activity and a well-known effect on spermatogenesis and chromatin remodeling in haploid germ cells. The aim of our study was to assess possible effects of two doses (2.5ng/ml; 2.5 μg/ml) of EE2 on both histone-to-protamine exchange and epigenetic profiles during spermatogenesis performing a multi/transgenerational study in mice. Our results demonstrated an impaired histone-to-protamine exchange with a significantly higher histone retention in sperm nuclei of exposed animals, when this process was accompanied by the changes of histone post-translational modifications (PTMs) abundancies with a prominent effect on H3K9Ac and partial changes in protamine 1 promoter methylation status. Furthermore, individual changes in molecular phenotypes were partially transmitted to subsequent generations, when no direct trans-generational effect was observed. Finally, the uncovered specific localization of the histone retention in sperm nuclei and their specific PTMs profile after EE2 exposure may indicate an estrogenic effect on sperm motility and early embryonic development via epigenetic mechanisms.

• Klíčová slova
• 17α-Ethynylestradiol, DNA methylation, EE2, Endocrine disruptors, Histone-to-protamine exchange, Post-translational modifications, Sperm, Testis, Transgenerational study,
• MeSH
• endokrinní disruptory farmakologie   toxicita   MeSH
• epigeneze genetická * účinky léků   MeSH
• ethinylestradiol * farmakologie   MeSH
• histony * metabolismus   MeSH
• myši MeSH
• posttranslační úpravy proteinů účinky léků   MeSH
• protaminy * metabolismus   genetika   MeSH
• spermatogeneze * účinky léků   genetika   MeSH
• spermie účinky léků   metabolismus   MeSH
• testis * účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endokrinní disruptory MeSH
• ethinylestradiol * MeSH
• histony * MeSH
• protaminy * 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.

• Klíčová slova
• bovine, epididymis, plasma membrane, reproduction, steroid hormones, testes,
• 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
• Názvy látek
• receptory pro estrogeny MeSH
• receptory spřažené s G-proteiny MeSH

BACKGROUND: Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry. RESULTS: All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions). CONCLUSIONS: Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.

• Klíčová slova
• Acrosin staining, Acrosome reaction, Chlortetracycline assay, Flow cytometry, Fluorescent microscopy, Phalloidin staining, Tyrosine phosphorylation,
• MeSH
• akrozomální reakce fyziologie   MeSH
• faloidin MeSH
• fluorescein-5-isothiokyanát MeSH
• fluorescenční barviva * MeSH
• fluorescenční mikroskopie * metody   MeSH
• fluorescenční protilátková technika MeSH
• kapacitace spermií fyziologie   MeSH
• průtoková cytometrie * metody   MeSH
• spermie fyziologie   MeSH
• Sus scrofa fyziologie   MeSH
• vápník analýza   MeSH
• zona pellucida fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faloidin MeSH
• fluorescein-5-isothiokyanát MeSH
• fluorescenční barviva * MeSH
• vápník 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
• Názvy látek
• ATP dependent 26S protease MeSH Prohlížeč
• proteasomový endopeptidasový komplex MeSH

17β-estradiol (estradiol) is a natural estrogen regulating reproduction including sperm and egg development, sperm maturation-called capacitation-and sperm⁻egg communication. High doses can increase germ cell apoptosis and decrease sperm count. Our aim was to answer the biological relevance of estradiol in sperm capacitation and its effect on motility and acrosome reaction to quantify its interaction with estrogen receptors and propose a model of estradiol action during capacitation using kinetic analysis. Estradiol increased protein tyrosine phosphorylation, elevated rate of spontaneous acrosome reaction, and altered motility parameters measured Hamilton-Thorne Computer Assisted Semen Analyzer (CASA) in capacitating sperm. To monitor time and concentration dependent binding dynamics of extracellular estradiol, high-performance liquid chromatography with tandem mass spectrometry was used to measure sperm response and data was subjected to kinetic analysis. The kinetic model of estradiol action during sperm maturation shows that estradiol adsorption onto a plasma membrane surface is controlled by Langmuir isotherm. After, when estradiol passes into the cytoplasm, it forms an unstable adduct with cytoplasmic receptors, which display a signalling autocatalytic pattern. This autocatalytic reaction suggests crosstalk between receptor and non-receptor pathways utilized by sperm prior to fertilization.

• Klíčová slova
• 17β-estradiol, CASA, HPLC MS/MS, acrosome reaction, autocatalysis, capacitation, kinetics, sperm,
• MeSH
• akrozomální reakce účinky léků   MeSH
• estradiol metabolismus   farmakologie   MeSH
• kapacitace spermií účinky léků   fyziologie   MeSH
• kinetika MeSH
• motilita spermií účinky léků   MeSH
• myši inbrední C57BL MeSH
• progesteron farmakologie   MeSH
• signální transdukce * MeSH
• sperma účinky léků   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• estradiol MeSH
• progesteron MeSH

The crucial role that oestrogens play in male reproduction has been generally accepted; however, the exact mechanism of their action is not entirely clear and there is still much more to be clarified. The oestrogen response is mediated through oestrogen receptors, as well as classical oestrogen receptors' variants, and their specific co-expression plays a critical role. The importance of oestrogen signalling in male fertility is indicated by the adverse effects of selected oestrogen-like compounds, and their interaction with oestrogen receptors was proven to cause pathologies. The aims of this review are to summarise the current knowledge on oestrogen signalling during spermatogenesis and sperm maturation and discuss the available information on oestrogen receptors and their splice variants. An overview is given of species-specific differences including in humans, along with a detailed summary of the methodology outcome, including all the genetically manipulated models available to date. This review provides coherent information on the recently discovered mechanisms of oestrogens' and oestrogen receptors' effects and action in both testicular somatic and germ cells, as well as in mature sperm, available for mammals, including humans.

• Klíčová slova
• humans, mice, oestrogen receptors, oestrogen-like compounds, oestrogens, pigs, rats, signalling, sperm, testes,
• MeSH
• aromatasa nedostatek   genetika   MeSH
• estrogeny farmakologie   MeSH
• lidé MeSH
• receptory pro estrogeny metabolismus   MeSH
• signální transdukce MeSH
• spermatogeneze účinky léků   MeSH
• testis účinky léků   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• aromatasa MeSH
• estrogeny MeSH
• receptory pro estrogeny 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
• Názvy látek
• monoklonální protilátky MeSH
• proteiny MeSH