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Expression of estrogen receptor beta (ERβ) In murine male reproductive tract and sperm
Dostálová, Pavla; Děd, Lukáš; Dorosh, Andriy; Elzeinová, Fatima; Pěknicová, Jana
2014 - English
Estrogens are steroid hormones that play an important role in reproduction of both sexes. In male, the main source of estrogens are testes where both somatic and germ cells are responsible for testosteron conversion to estrogens. Estrogens are involved in control of spermatogenesis, fluid reabsorption in rete testis and epididymis, and in later maturation steps that sperm undergo in female genital tract (capacitation, acrosome reaction). Generally, estrogen action is mediated through binding to estrogen receptors (ERs) which than lead to classical genomic or rapid non-genomic signaling. Nowadays, two classical estrogen receptors are known – ERα and ERβ. ERβ is a predominant variant in testes, while ERα is more abundant in rete testis and initial segment of epididymis. In addition to classical ERs, several splice variants that can differ in their ligand- or DNA-binding properties were detected in different tissues and cell lines. ERs mostly work as a dimer (homo- and hetero-) and splice variants often „only“ modulate function of classical full-length ERs. Therefore, estrogen action seems to be a very complex. To contribute to understanding of estrogen action in male, we detected ERβ and its potential splice variants in mice testis, epididymis and sperm. According to our results, two variants are present in all analysed tissues and cells. These variants differ in one exon in ligand binding domain which leads to different affinity for estrogens. To analyse these variants also at a protein level, we prepared specific monoclonal antibodies recognizing particular variant of ERβ. Both atibodies detected band(s) in protein extracts from testes or epididymis. Taking together, there are at least two variants of ERβ in mice testes, epididymis and sperm and it seems that both variants are similar in abundance within the same organ or sperm.
Keywords:
estrogene receptor beta; spermatozoa; splice variant
Available at various institutes of the ASCR
Expression of estrogen receptor beta (ERβ) In murine male reproductive tract and sperm
Estrogens are steroid hormones that play an important role in reproduction of both sexes. In male, the main source of estrogens are testes where both somatic and germ cells are responsible for ...
Panel of monoclonal antibodies – alternative tool For monitoring of sperm–zona pellucida receptors localization and identification
Zigo, Michal; Dorosh, Andriy; Pohlová, Alžběta; Jonáková, Věra; Šulc, Miroslav; Maňásková-Postlerová, Pavla
2014 - English
Primary binding of the sperm to the zona pellucida (ZP) is one of the many steps necessary for successful fertilization in all sexually reproducing species. Sperm bind ZP by means of membrane receptors which recognize carbohydrate moieties on ZP glycoproteins according to a well-precised sequential process. Primary-binding receptors are localized throughout the acrosomal region of the sperm surface of which many have been disclosed in various mammals. For the monitoring sperm-zona pellucida receptors in terms of localization and characterization - panel of monoclonal antibodies against proteins from the sperm surface was prepared. Antibodies were screened by immunofluorescence and Western blotting for protein localizations and competence of antibodies, respectively. Antibodies recognizing proteins localized on the sperm head and simultaneously detected by Western blot were further studied by means of immunolocalization in reproductive tissues and fluids, binding to ZP, immunoprecipitation and protein identification using MS analysis. Out of 17 prepared antibodies, 8 antibodies were simultaneously recognizing proteins localized on the sperm head and detecting proteins of interest by Western blotting. Further only 3 antibodies recognized proteins which also coincided in binding to ZP. These 3 antibodies were used for immunoprecipitation, and further protein identification of immunoprecipitates revealed that the antibodies distinguish acrosin precursor, RAB2A protein, and lactadherin P47. Acrosin and lactadherin P47 have been already detected on the sperm surface and their physiological functions in reproduction have been proposed. To our knowledge, this is the first time RAB2A has been found on the surface of sperm and its physiological function in the process of fertilization remains undisclosed.
Keywords:
monoclonal antibody; zona pellucida; acrosin precursor; RAB2A; lactahedrin P47
Available at various institutes of the ASCR
Panel of monoclonal antibodies – alternative tool For monitoring of sperm–zona pellucida receptors localization and identification
Primary binding of the sperm to the zona pellucida (ZP) is one of the many steps necessary for successful fertilization in all sexually reproducing species. Sperm bind ZP by means of membrane ...
Effect of diabetes mellitus on reproductive parameters in mice
Margaryan, Hasmik; Elzeinová, Fatima; Kubátová, Alena; Strolená, Eva; Pěknicová, Jana
2014 - English
The decrease in population fertility has become a major concern in many developed countries. Recent studies show that infertility is affecting an estimated 15% of all couples (World Health Organization, WHO, 2010). Male infertility is the primary or contributing cause in 60% of cases. Male infertility is caused by a number of factors, such as genetic background, various environmental factors and disease. Diabetes mellitus (DM), a serious health problem on its own, is also suspected to be a contributing factor to male infertility. The aim of this project was to analyze the cellular, molecular and genetic effects of diabetic environment on spermatogenesis and sperm quality and to determine the impact of DM on the in vivo reproduction, using the mouse model (Mus musculus) inbred FVB. Diabetes was induced using streptozotocin. We used our knowledge and tools (unique monoclonal antibodies developed by our group) to determine the status of reproductive organs, anogenital distance, and the quality of sperms. Genetic analysis was performed by a quantitative Reverse Transcription Polymerase Chain Reaction (qPCR). We tested selected genes which are expressed in testicular tissue and thus can influence process of spermatogenesis and consequently the sperm quality. Our preliminary data strongly suggest that DM impairs male fertility. We have found significant changes in the body and reproductive organ weight of mice with DM. We have identified qualitative and quantitative changes in the expression of proteins in epididymal fluid and sperms. We have also detected an increased number of apoptotic cells in sperm of diabetic mice compared to the control group. To our knowledge, there is no study assessing the correlation between DM and “unexplained infertility”. In view of this, it is essential to analyze the effects of DM on male fertility, sperm quality, and reproduction parameters.
Keywords:
diabetes mellitus; monoclonal antibody; reproduction
Available at various institutes of the ASCR
Effect of diabetes mellitus on reproductive parameters in mice
The decrease in population fertility has become a major concern in many developed countries. Recent studies show that infertility is affecting an estimated 15% of all couples (World Health ...
Changes in the expression of selected testicular genes in mice
Valášková, Eliška; Margaryan, Hasmik; Žatecká, Eva; Pěknicová, Jana
2014 - English
The decrease in population fertility has become a major concern in many developed countries. Recent studies show that infertility is affecting an estimated 15% of all couples (World Health Organization, WHO, 2010). Male infertility is the primary or contributing cause in 60% of cases. Male infertility is caused by a number of factors, such as genetic background, various environmental factors and disease. Diabetes mellitus (DM), a serious health problem on its own, is also suspected to be a contributing factor to male infertility. The aim of this project was to analyze the cellular, molecular and genetic effects of diabetic environment on spermatogenesis and sperm quality and to determine the impact of DM on the in vivo reproduction, using the mouse model (Mus musculus) inbred FVB. Diabetes was induced using streptozotocin. We used our knowledge and tools (unique monoclonal antibodies developed by our group) to determine the status of reproductive organs, anogenital distance, and the quality of sperms. Genetic analysis was performed by a quantitative Reverse Transcription Polymerase Chain Reaction (qPCR). We tested selected genes which are expressed in testicular tissue and thus can influence process of spermatogenesis and consequently the sperm quality. Our preliminary data strongly suggest that DM impairs male fertility. We have found significant changes in the body and reproductive organ weight of mice with DM. We have identified qualitative and quantitative changes in the expression of proteins in epididymal fluid and sperms. We have also detected an increased number of apoptotic cells in sperm of diabetic mice compared to the control group. To our knowledge, there is no study assessing the correlation between DM and “unexplained infertility”. In view of this, it is essential to analyze the effects of DM on male fertility, sperm quality, and reproduction parameters.
Keywords:
diabetes mellitus; gene expression; reproduction
Available at various institutes of the ASCR
Changes in the expression of selected testicular genes in mice
The decrease in population fertility has become a major concern in many developed countries. Recent studies show that infertility is affecting an estimated 15% of all couples (World Health ...
The ubiquitin–proteasome system is involved in the regulation of activity of spermadhesin aqn1 and acrosin inhibitor, the two sperm surface proteins, during porcine fertilization
Jonáková, Věra; Yi, Y.J.; Postlerová, Pavla; Pěknicová, Jana
2014 - English
The spermadhesin AQN1and acrosin inhibitor (AI/SPINK2) proteins bind to the sperm plasma membrane at ejaculation. The AQN1 has been implicated in sperm binding to zona pellucida (ZP) of the oocyte as well as in sperm interactions with the epithelium of the oviductal sperm reservoir. The SPINK2 protects spermatozoa from proteolytic degradation during their trip up the female genital tract toward the oocyte. This study examined the role of two components of the 19S proteasome regulatory complex, the ubiquitin C-terminal hydrolase UCHL3 and PSMD8 in the AQN1-mediated boar sperm binding to zona pellucida. Interaction of PSMD4 subunit with the acrosomal surface-associated acrosin inhibitor AI/SPINK2 provided another line of evidence for the presence of 26S proteasomes on the sperm surface. Detection of the ubiquitinated forms of SPINK2 supports the hypothesis that SPINK2 activity is controlled by ubiquitin-proteasome system (UPS). The activity of the porcine AQN1, and thus the efficiency of sperm-oocyte recognition/binding, may be controlled by elements of the sperm surface-bound UPS, in particular by UCHL3, and by proteasomal regulatory complex subunit PSMD8. Ubiquitinated isoforms of AQN1 were also detected in boar sperm extracts. The UCHL inhibitor ubiquitin aldehyde and the antibodies against UCHL3 or PSMD8 increased the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). In contrast, the addition of recombinant UCHL3 to fertilization medium significantly reduced polyspermy rates, while maintaining satisfactory rate of monospermic fertilization (~50%). These results are significant for production agriculture. The high level of polyspermy that hinders porcine IVF for commercial embryo transfer could be mitigated by the modulation of the UCHL3 and/or PSMD8 activity.
Keywords:
ubiquitin-proteasome system; acrosin inhibitor; spermadhesin AQN1
Available at various institutes of the ASCR
The ubiquitin–proteasome system is involved in the regulation of activity of spermadhesin aqn1 and acrosin inhibitor, the two sperm surface proteins, during porcine fertilization
The spermadhesin AQN1and acrosin inhibitor (AI/SPINK2) proteins bind to the sperm plasma membrane at ejaculation. The AQN1 has been implicated in sperm binding to zona pellucida (ZP) of the oocyte as ...
Endocrine disruptors induce transgenerational alterations of the male reproductive parameters and mirna expression profiles in mouse primordial germ cells
Děd, Lukáš; Brieno-Enríquez, M.A.; García-López, J.; Cárdenas, D.B.; Guibert, S.; Hourcade, J.de D.; Pěknicová, Jana; Weber, M.; del Mazo, J.
2014 - English
Primordial germ cells (PGCs) are the embryonic precursors of the germ cell linage, which are restricted to form only sperm and oocytes following their specification from pluripotent cells. PGC precursors are specified in the epiblast around 6.25 days post coitum (dpc), and around 7.25 dpc become identifiable in a 40 cell-cluster. In the present study, we used a mouse model to evaluate the trans-generational (F1-F3) effects of vinclozolin (VCZ) administrated in two doses on male reproductive parameters. We observed decreased fertility rate, higher apoptotic rate and histopathologic alterations in adult testis, PGC number reduction, increments of PGCs apoptosis and changes in PGCs gene expression among all three generations. In the attempt to clarify the trans-generational transmition of the altered phenotypes, we performed the microRNA expression and DNA methylation analysis. We observed the significant alteration in the expression of multiple microRNA and microRNA-regulated genes which are important for PGCs specification, including LIN28, let-7 and BLIMP1. Trans-generational deregulation in the expression of factors involved in the Lin28-let-7-Blimp1 pathway can lead to specific VCZ-induced phenotype observed in our study.
Keywords:
endocrine disruptor; miRNA expression; primordial germ cells; vinclozolin; DNA methylation
Available at various institutes of the ASCR
Endocrine disruptors induce transgenerational alterations of the male reproductive parameters and mirna expression profiles in mouse primordial germ cells
Primordial germ cells (PGCs) are the embryonic precursors of the germ cell linage, which are restricted to form only sperm and oocytes following their specification from pluripotent cells. PGC ...
The effect of low dose of vinclozolin on reproductive tract development in CD1 outbred mice
Elzeinová, Fatima; Pěknicová, Jana; Nováková, Vendula; Buckiová, Daniela; Kubátová, Alena
2008 - English
The effect of a low dose of vinclozolin within the development of the reproductive tract during gestation (VIN-GD 15-22) and puberty (VIN-PND 23-44) in CD1 mice was tested. We found a decrease in the anogenital distance, prostate weight and pathology of testes in both experimental groups. Sperm counts decreased to 46% (VIN-GD) and to 81% (VIN-PND), and also the acrosomal state (evaluated by antiacrosomal antibody) decreased in both groups to 89% in comparison to the control group (100%). Sperm head abnormalities increased by approximately 18% and 13%, respectively. In this connection, the expression of some genes was changed (arosome-related gene (Acr), apoptosis related genes (p53, p21)). In conclusion, a low dose of vinclozolin affected the reproductive tract, sperm parameters and expression of selected genes in both experimental groups.
Keywords:
vinclozolin; CD1 mouse; spermatozoa
Available at various institutes of the ASCR
The effect of low dose of vinclozolin on reproductive tract development in CD1 outbred mice
The effect of a low dose of vinclozolin within the development of the reproductive tract during gestation (VIN-GD 15-22) and puberty (VIN-PND 23-44) in CD1 mice was tested. We found a decrease in the ...
Differences in the strategies of mammalian and fish fertilization and the optimization of the protocol for immunofluorescence on fish spermatozoa
Koubek, Pavel; Králová, Alena; Pšenička, M.; Pěknicová, Jana
2008 - English
This lecture describes a special approach to handling fish spermatozoa in comparison with the symplex originating from mammals. This approach primarily emphasizes the concrete parts of immunofluorescence protocol determining accurate results. Vzhledem ke struktuře rybích spermií, která je jednoduchá a snadno poškoditelná, bylo třeba vypracovat protokol pro imunofluorescenční značení, který by minimalizoval jejich poškození a vznik nespecifických artefaktů. Cílem práce tak bylo především zamezit mylné interpretaci výsledků imunofluorescenčních experimentů na rybích spermiích a zvýšit vzájemnou porovnatelnost těchto experimentů.
Keywords:
fish spermatozoa; mammalian spermatozoa; immunofluorescence
Available at various institutes of the ASCR
Differences in the strategies of mammalian and fish fertilization and the optimization of the protocol for immunofluorescence on fish spermatozoa
This lecture describes a special approach to handling fish spermatozoa in comparison with the symplex originating from mammals. This approach primarily emphasizes the concrete parts of ...
Analysis of male sterility caused by T(16,17)43H chromosomal translocation
Čapková, Jana; Homolka, David; Ivánek, Robert; Jansa, Petr; Forejt, Jiří
2008 - English
Heterozygosity for certain mouse and human chromosomal rearrangements is characterized by male-limited sterility. Our model of a male-sterile chromosomal rearrangement represents mouse T(16;17)43H (T43) chromosomal translocation. The effect of the T43H translocation on spermatogenesis was studied. We analyzed meiosis in B10.T43/+ heterozygous male mice and we have obtained direct evidence on the transcriptional silencing of unsynapsed autosomal chromatin of the translocation quadrivalent and the subsequent failure of the X chromosome transcriptional inactivation at the mid-late pachytene of spermatogenesis. Po některých chromosomálních přestavbách vzniká heterozygosita, která vede ke sterilitě samců. Náš model chromosomální přestavby vedoucí samčí sterilitě představuje myší T(16,17)43H chromosomální translokace. Na tomto modelu jsme studovali účinek translokace na spermatogenezu. Analyzovali jsme meiosu u heterozygotních T43/+ samců a prokázali jsme transkripčním umlčení nesynaptického autosomálního chromatinu v translokačním kvadrivalentu a následnou neschopnost transkripční inaktivace X chromosomu v pachytenním stadiu spermatogeneze.
Keywords:
male sterility; chromosomal translocation; spermatogenesis
Available at various institutes of the ASCR
Analysis of male sterility caused by T(16,17)43H chromosomal translocation
Heterozygosity for certain mouse and human chromosomal rearrangements is characterized by male-limited sterility. Our model of a male-sterile chromosomal rearrangement represents mouse T(16;17)43H ...
Swim-up in assisted reproduction
Teplá, O.; Pěknicová, Jana; Mrázek, M.; Margaryan, Hasmik; Cibulková, E.; Králová, Alena; Strnadová, J.
2008 - English
To compare the effect on density-gradient centrifugation and swim-up on sperm preparation in assisted reproduction. Quality of sperm was evaluated by monoclonal antibodies.
Keywords:
swim-up; IVF; human spermatozoa
Available at various institutes of the ASCR
Swim-up in assisted reproduction
To compare the effect on density-gradient centrifugation and swim-up on sperm preparation in assisted reproduction. Quality of sperm was evaluated by monoclonal antibodies.
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