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Estrogen receptor beta (ERβ) in testicular cells and sperm
Dostálová, Pavla ; Žatecká, Eva ; Děd, Lukáš ; Dorosh, Andriy ; Postlerová, Pavla ; Jonáková, Věra ; Dvořáková-Hortová, Kateřina ; Pěknicová, Jana
Estrogen is a steroid hormone that plays an important role during sperm development in the male and female reproductive tract. Estrogen signalling is a complex process that depends on cell milieu and presence of receptors. Thanks to the steroid nature of estrogens, they can pass through the plasmatic membrane and bind to the intracellular estrogen receptors (ERs). Within the cell, there are several pools of ERs. One of them is localized to the cell nucleus and their activation leads to direct or indirect binding to DNA and ultimately to alternation in gene expression (genomic pathway). Other pools of ERs are associated with plasma membrane or are located in cytosol. Activation of membrane associated ERs leads to rapid non-genomic responses. Nowadays, two classical estrogen receptors are known – ERα and ERβ. Since ERβ is a predominant variant in testes, we focused our study on expression of ERβ variants in murine testes and sperm. We detected two variants of ERβ at mRNA level in both, testes and sperm. These variants differ in 54 nucleotids within the ligand binding domain and this variability results in different affinity to estrogen. We analyzed individual testicular cell types (spermatogonia, spermatocytes, spermatids, Sertoli cells) by RT-qPCR. Our results suggest that both ERβ variants are coexpressed in the same cell type and may therefore interact together. This may have consequences in mediating of estrogen signalling. Moreover, ERβ is expressed more in the later stages of spermatogenesis suggesting the role of ERβ in these stages or alternatively in spermatozoa alone. At the protein level, we detected ERβ in nuclear, membrane and cytosolic fraction prepared from testicular tissue suggesting the involvement of both, genomic and non-genomic, pathways of estrogen signaling in testes. In sperm, anti-ERβ antibodies localized ERβ in acrosome region and tail which is in accordance with the known role of estrogen on capacitation, acrosome reaction and motility.
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Dynamics of mouse sperm capacitation and acrosome reaction
Dvořáková-Hortová, Kateřina ; Frolíková, Michaela ; Děd, Lukáš ; Šebková, Nataša
Capacitation followed by the acrosome reaction (AR), is a very complex event of molecular changes, including acrosome matrix rearrangement and actin polymerization, which mammalian sperm must undergo in the female reproductive tract in order to obtain the ability to penetrate and fertilize the egg. CD46 and β1-integrin belong to specific proteins, which are predicted to interact during molecular reorganization of capacitating sperm. The IZUMO1 as the primary fusion protein of the mammalian sperm is also involved in this dynamic network. We investigated the relationship between the Izumo, CD46 and β1 integrin relocation in the sperm head during the capacitation and AR in vitro. We have already successfully monitored by immunofluorescent labelling the dynamics of proteins CD46 and β1-integrin. The changes in the localization of these proteins associated with the AR and their mutual co-localization was observed. The original β1-integrin location in the freshly released epididymal sperm is in the acrosome and it relocates during the AR further through the sperm head compartments into the equatorial segment and over the whole sperm head. Its density over the equatorial segment is decreasing with the extended time of the AR. Also its presence in the perforatorium of the mouse sperm head is very prominent. The pattern for protein CD46 is extremely similar if not identical in both aspects such as compartment localization and time progress during capacitation and AR in vitro. The molecular interaction of CD46 and β1-integrin was investigated using the Proximity Ligation Assay and Super resolution microscopy STED. The data were statistically analysed. The newly obtained results from CD46 and β1-integrin relocation are in correlation with IZUMO1 dynamics and giving a substantial knowledge on the studied protein network rearrangement during capacitation and AR in mouse spermatozoa.
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Fluorescent analysis of the differential protein expression in normozoospermic and asthenozoospermic sperm samples
Děd, Lukáš ; Čapková, Jana ; Kubátová, Alena ; Teplá, O. ; Pěknicová, Jana
Asthenozoospermia is one of the main seminal pathologies underlying male infertility. Previous proteomic studies have demonstrated the significant differences in the protein profiles between normozoospermic and asthenozoospermic sperm samples. Since these studies were primarily focused on the identification of differentially expressed proteins by mass spectrometry, we aimed to evaluate the ability of our diagnostic antibodies to detect the differential expression of selected protein markers by fluorescent microscopy and flow cytometry techniques. Therefore, we analyzed sperm samples from 30 men with normal and 30 men with astheno spermiograms, average by the panel of our diagnostic anti-human sperm (Hs) antibodies. Fluorescent microscopy and flow cytometry analysis revealed quantitative differences in the protein abundances between normo and astheno sperm samples, namely, in GAPDHs, evaluated with Hs-8 MoAb, VCP, evaluated with Hs-14 MoAb, and ATP synthase, evaluated with MoAb Hs-36. From the methodological point of view, we observed very high correlation between the data obtained by fluorescent microscopy and flow cytometry techniques and therefore both methods are useful for evaluation of protein differences associated with asthenozoospermia. From the clinical point of view, we observed the strong association of the low sperm motility in the sample with the expression of proteins, playing an important role in sperm energy metabolism (expected), but also with the expression of all tested intra-acrosomal proteins. These findings further demonstrate asthenozoospermia as a complex semen disorder frequently associated with other semen pathologies, which are not diagnosed by basic semen analysis, and the possibility to use monoclonal antibodies as a tool for diagnosis of protein associated sperm pathologies in the semen with the low sperm motility.
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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.
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.
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One more drop for decreasing reproduction
Dvořáková-Hortová, K. ; Šídlová, A. ; Děd, Lukáš ; Hladovcová, D. ; Vieweg, M. ; Weidner, W. ; Steger, K. ; Stopka, P. ; Paradowska-Dogan, A.
Toxoplasma gondii is a common protozoan parasite that infects warm-blooded animals throughout the world, including mice and humans. During infection, both, the parasite and the host, utilize various mechanisms to maximize their own reproductive success. Mice and humans are both the intermediate hosts for Toxoplasma gondii, which forms specialized vacuoles containing reproductive cysts in the formers’ tissue. As half of the human population is infected, developing a disease called toxoplasmosis, along with an ever-growing number of couples suffering with idiopathic infertility, it is therefore surprising that there is a lack of research on how T.gondii can alter reproductive parameters. In this study, a detailed histometric screening of the testicular function along with the levels of the pituitary luteinizing hormone (LH) were analysed in infected mice. Data on relative testis and epididymis weight, and sperm count were also collected. Based on the results obtained, the level of LH in the urine of Toxoplasma gondii infected mice was lower compared to the control. In direct correlation with the hormone level, testicular function and sperm production was also significantly lower in T. gondii positive group using sperm count and histometric analysis as a marker. Not only were the number of leptotene primary spermatocytes and spermatids lowered, but the number of Sertoli cells and the tubule diameter were elevated. In parallel, a pilot epigenetic study on global testicular methylation, and specific methylation of Crem, Creb1 and Hspa1genes essential for successfully ongoing spermatogenesis was performed. Global methylation was elevated in Toxoplasma infected mice, and differences in the DNA methylation of selected genes were detected between the Toxoplasma positive and control group. These findings demonstrate a direct relation between T. gondii infection and the decrease of male reproductive fitness in mice, which may contribute to an increase of infertility in humans.
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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
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.
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The effect of tetrabromobisphenol a on protamination and DNA quality of mouse sperm
Žatecká, Eva ; Castillo, J. ; Elzeinová, Fatima ; Kubátová, Alena ; Děd, Lukáš ; Pěknicová, Jana ; Oliva, R.
Tetrabromobisphenol (TBBPA) is a widely used brominated flame retardant, currently its consumption is 210,000 tons / year and is still growing. In our previous multigenerational in vivo study we have demonstrated that TBBPA is able to induce apoptosis of testicular cells and changes in the expression of genes important for proper spermatogenesis. However the potential effect of TBBPA on epidydimal spermatozoa had not yet been investigated. Therefore, we performed further study to evaluate the effect of on sperm DNA integrity and on the protamines as the major nuclear proteins. C57Bl/6J mice pups (n=10) were exposed to TBBPA (experimental group) during the gestation, lactation, pre-pubertal and pubertal periods up to the age of 70 days and compared control mice pups (n= 10) which were not exposed. Our results demonstrate that TBBPA treatment results in a significantly decreased P1/P2 ratio, increased total protamine/DNA ratio and increased DNA fragmentation observed between TBBPA and control mice, respectively. Protamines have recently been connected to the epigenetic marking of sperm chromatin in human and mouse spermatozoa. Thus, our findings suggest that TBBPA exposure, in addition to result in increased sperm DNA damage, may also alter the epigenetic marking of sperm chromatin.
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Flow cytometry (FCM) sperm assessment In normozoospermic and asthenozoospermic men using monoclonal antibodies against sperm proteins
Čapková, Jana ; Kubátová, Alena ; Děd, Lukáš ; Teplá, O. ; Pěknicová, Jana
Recent studies have shown that infertility affects an estimated 15% of all couples. Male infertility is the primary or contributing cause in 60% of these cases. Consequently, application of methods of assisted reproduction is increasing. These methods would benefit from extended evaluation of the sperm quality. For this purpose, we analyzed sperm proteins in men with normal spermiograms and with asthenozoospermia. Ejaculates of both groups were tested with a set of well-characterized monoclonal antibodies (MoAbs) to human sperm. No statistically significant differences were found between normospermics and asthenospermics in the expression of sperm surface proteins clusterin, evaluated by Hs-3 MoAb, and semenogelin, evaluated by Hs-9 MoAb. On the other hand, flow cytometry revealed quantitative differences between normozoospermic and asthenozoospermic men in GAPDHS (glyceraldehyde phosphate dehydrogenase human sperm-specific glycolytic enzyme), evaluated by Hs-8 MoAb, VCP (valosin-containing protein), detected with Hs-14 MoAb, and PRKAR2A (cAMP-dependent protein kinase type II – alpha regulatory subunit) detected by MoAb Hs-36. Asthenozoospermic men displayed significantly reduced expression of intra-acrosomal proteins with a likely decrease in sperm quality, and thus a negative impact on successful reproduction.
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