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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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Fish sperm motility parameters and total proteins profiles in seminal plasma during in vivo and in vitro storage
KOLEŠOVÁ, Anna
The effect of gamete storage on sperm quality has received considerable attention in recent years. Previous studies have shown that spermatozoa stored in vivo or in vitro for a long time can lost their motility and fertilization capacity. Moreover, it have been concluded that male fertilization potential is highly dependent, not only on spermatozoa motility parameters, but also on organic components including protein composition of seminal plasma. On the other hand, spermatozoa motility and protein profiles of seminal plasma are highly dependent on conditions of storage. Therefore, additional data about effects of in vivo and in vitro storage on quality sperm parameters and protein composition of seminal plasma are essential for development of fish artificial reproduction methods. In the current work the influence of in vitro and in vivo storage on parameters of sperm motility, DNA integrity, antioxidant defensive system and seminal plasma protein composition were studied. As a conclusion, the results of this study provide new data on sperm quality and quantity parameters of chondrostean and teleost fish species with respect to in vivo and in vitro storage capacities, which should be beneficial for the development of aquaculture of these species. The data confirmed that protein patterns in seminal plasma varied during in vivo storage, depending on time of sperm collection. Furthermore, the altered proteins are probably involved in enzymatic pathways that regulate spermatozoa movement. In practice, the results presented in this thesis should help to improve management and optimize the development of protocols for artificial reproduction.
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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
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.
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The effect of low dose of vinclozolin on reproductive tract development in CD1 outbred mice
Pěknicová, Jana ; Elzeinová, Fatima ; Nováková, Vendula ; Buckiová, Daniela ; Kubátová, Alena
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.
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Role of sperm surface proteins in reproduction
Jonáková, Věra ; Postlerová, Pavla ; Tichá, M. ; Pěknicová, Jana
Seminal plasma proteins bind to the sperm surface at ejaculation and may modulate sperm properties during reproduction. DQH sperm surface protein shows affinity to oviductal epithelium and zona pellucida (ZP) glycoproteins. The mRNA transcript of DQH protein was found in seminal vesicles (SV). DQH was immunodetected by monoclonal antibodies (MAbs) in SV extract and fluid, on SV tissue sections and on the membrane-associated acrosome part of ejaculated spermatozoa. These results confirmed the ability of DQH protein to bind to the sperm surface at ejaculation and to participate in the formation of the sperm reservoir in the porcine oviduct. Moreover, monoclonal antibodies reduced binding of sperm to oocytes and proved the role of DQH protein in the sperm-ZP primary binding.
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