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Embryonic exposure to ethanol reduces cholesterol levels in a chick embryo model of Fetal Alcohol Syndrome
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Abstracts / Reproductive Toxicology 64 (2016) 29–49
P-39
P-40
Investigating the effects of early embryonic ethanol exposure on primary cilium morphology
Embryonic exposure to ethanol reduces cholesterol levels in a chick embryo model of Fetal Alcohol Syndrome
Abdubadie Kutubi ∗ , Seamus Giles, Deirdre Brennan
Deirdre Clissman 1,∗ , Seamus Giles 2 , Deirdre Brennan 2
School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland
1 Centre for Anatomy and Human Identification, University of Dundee, Dundee, Scotland, UK 2 School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland
Introduction: Primary cilia are unique sensory organelles which project from the cell surface. The primary cilium acts as a major signaling hub for the cell, regulating key signaling networks involved in embryogenesis including the sonic hedgehog (Shh) pathway. Consequently, any defect or dysfunction of the primary cilium can lead to Shh perturbation and interruption to normal developmental processes. Fetal Alcohol Syndrome (FAS) is a condition which directly results from maternal ethanol exposure during pregnancy. Using a chick embryo model of FAS, our previous studies have shown the effect of ethanol on early development involves a significant decrease in Shh protein expression [1,2]. The present study therefore aims to investigate whether this ethanol-induced Shh disruption may be mediated through interference with primary cilia morphology. Methods: Chick embryos (Gallus gallus domesticus) were pre-incubated in a forced air incubator (Shell Lab 1555) at 38 ◦ C with 65–75% relative humidity until they had reached Hamburger–Hamilton stage 7–8 of development. For ethanol treatment, embryos were injected in ovo with 125 L of a 20% ethanol solution directly into the yolk-sac. Controls received identical treatment via injection of 125 l of chick saline (0.73% NaCl). Embryos were dissected from their vitelline membranes 24 h post-treatment and processed for wax histology. Immunohistochemistry was carried out on 7 m sections through the optic vesicle region using a monoclonal anti-acetylated tubulin antibody (T6793 Sigma). Results: Analysis of the ciliary marker acetylated alpha tubulin revealed that cilia were localised to the luminal surface of the neuroepithelial cells of the optic vesicle and telencephalon in both control and ethanol treatment groups (n = 6 per treatment group). However, in ethanol-exposed specimens a more abundant and intense staining for the ciliary marker was noted in comparison to control counterparts. At higher magnifications, a denser distribution of the cilia within the neuroepithelial lumen was observed, with an associated elongation of cilia apparent in the ethanol group by comparison to controls. Conclusions: This study suggests that embryonic exposure to ethanol is inducing some structural alterations to the overall morphology of the primary cilium. Due to the localisation of the Shh pathway to the primary cilium, this finding of an ethanol-induced ciliary disturbance may help to explain why Shh disruption was found in our previous molecular studies. Examining the effects of ethanol on the primary cilium could have wider implications in understanding the aetiology of fetal ethanol-related defects ciliaryrelated diseases [3].
[1] D. Brennan, S. Giles, Sonic hedgehog expression is disrupted following in ovo ethanol exposure during early chick eye development, Reprod. Toxicol. 41 (November) (2013) 49–56. [2] D. Brennan, S. Giles, Ocular involvement in fetal alcohol spectrum disorder: a review, Curr. Pharm. Des. 20 (34) (2014) 5377–5387. [3] K. Kennelly, D. Brennan, K. Chummun, S. Giles, Histological characterization of the ethanol-induced microphthalmia phenotype in a chick embryo model system, Reprod. Toxicol. 32 (September (2)) (2011) 227–234.
Introduction: Fetal Alcohol Syndrome (FAS), is a condition which occurs in the offspring of alcohol abusing mothers. It is characterised by pre- and postnatal growth retardation, neurodevelopmental deficits and distinct midline craniofacial abnormalities. Many of the phenotypic FAS-related abnormalities of the brain and midline facial structures also commonly occur in the genetic condition Smith–Lemli–Opitz syndrome (SLOS). The SLOS disorder is directly caused by mutations in the enzymes responsible for the final steps of cholesterol biosynthesis and ultimately leads to an overall deficiency of cholesterol within the body. Therefore, due to the overlapping syndromic presentation of both FAS and SLOS, the aim of the present study was to further investigate whether the detrimental effects of embryonic ethanol exposure may be associated with changes in cholesterol homeostasis. Methods: Fertile chicken eggs (Gallus gallus domesticus) were pre-incubated at 37 ◦ C until embryos had reached Hamburger–Hamilton stage 7–8 of development. Subsequently, chick embryos were explanted onto a filter paper carrier using a modification of the early chick EC method and were treated with a range of ethanol doses (0%, 1%, 5%, 10%, 20%) for 2 min. Treated embryos were placed on an agar-albumen substrate within a humidity chamber and returned to the incubator to continue development for a further 24 h post-treatment. Embryos were dissected from their filter paper carriers and were homogenised in assay buffer (T25 basic homogeniser, Ika Labortechnik). To determine the cholesterol content of individual weight-matched embryos (n = 4 per treatment group), an assay kit was used according to manufacturer’s instructions to give a quantitative measure of the total concentration of free cholesterol in each group (ECCH-100 EnzyChrom cholesterol assay kit, Universal Biologicals Cambridge Ltd.). The assay was read at 340 nm using the SpectraMax Plus384 microplate reader and SoftMax Pro5.2 software (Molecular Devices). Results: Analysis of the total free cholesterol content of embryos treated with 1–20% ethanol was found to be significantly reduced by comparison to control counterparts (*p < 0.05, n = 4 per group). An average value of 0.466 g/dL of cholesterol was obtained in the control group in comparison to 0.214 g/dL at 1% ethanol, 0.267 g/dL at 5% ethanol, 0.271 g/dL at 10% ethanol and 0.207 g/dL at 20% ethanol. Conclusions: These findings confirm that one of the potential mechanisms underlying the teratogenicity of ethanol involves disruption to cholesterol homeostasis within the developing embryo. A reduction in total free cholesterol post-ethanol exposure may explain why FAS has many similar characteristics to the cholesterol-linked genetic disorder SLOS. As cholesterol plays a crucial role in activating many of the key signalling pathways which govern normal embryonic development, the relationship between ethanol and such cholesterol-dependent molecular pathways would be an interesting area of further study.
http://dx.doi.org/10.1016/j.reprotox.2016.06.100
http://dx.doi.org/10.1016/j.reprotox.2016.06.101
References
Abstracts / Reproductive Toxicology 64 (2016) 29–49
P-39
P-40
Investigating the effects of early embryonic ethanol exposure on primary cilium morphology
Embryonic exposure to ethanol reduces cholesterol levels in a chick embryo model of Fetal Alcohol Syndrome
Abdubadie Kutubi ∗ , Seamus Giles, Deirdre Brennan
Deirdre Clissman 1,∗ , Seamus Giles 2 , Deirdre Brennan 2
School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland
1 Centre for Anatomy and Human Identification, University of Dundee, Dundee, Scotland, UK 2 School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland
Introduction: Primary cilia are unique sensory organelles which project from the cell surface. The primary cilium acts as a major signaling hub for the cell, regulating key signaling networks involved in embryogenesis including the sonic hedgehog (Shh) pathway. Consequently, any defect or dysfunction of the primary cilium can lead to Shh perturbation and interruption to normal developmental processes. Fetal Alcohol Syndrome (FAS) is a condition which directly results from maternal ethanol exposure during pregnancy. Using a chick embryo model of FAS, our previous studies have shown the effect of ethanol on early development involves a significant decrease in Shh protein expression [1,2]. The present study therefore aims to investigate whether this ethanol-induced Shh disruption may be mediated through interference with primary cilia morphology. Methods: Chick embryos (Gallus gallus domesticus) were pre-incubated in a forced air incubator (Shell Lab 1555) at 38 ◦ C with 65–75% relative humidity until they had reached Hamburger–Hamilton stage 7–8 of development. For ethanol treatment, embryos were injected in ovo with 125 L of a 20% ethanol solution directly into the yolk-sac. Controls received identical treatment via injection of 125 l of chick saline (0.73% NaCl). Embryos were dissected from their vitelline membranes 24 h post-treatment and processed for wax histology. Immunohistochemistry was carried out on 7 m sections through the optic vesicle region using a monoclonal anti-acetylated tubulin antibody (T6793 Sigma). Results: Analysis of the ciliary marker acetylated alpha tubulin revealed that cilia were localised to the luminal surface of the neuroepithelial cells of the optic vesicle and telencephalon in both control and ethanol treatment groups (n = 6 per treatment group). However, in ethanol-exposed specimens a more abundant and intense staining for the ciliary marker was noted in comparison to control counterparts. At higher magnifications, a denser distribution of the cilia within the neuroepithelial lumen was observed, with an associated elongation of cilia apparent in the ethanol group by comparison to controls. Conclusions: This study suggests that embryonic exposure to ethanol is inducing some structural alterations to the overall morphology of the primary cilium. Due to the localisation of the Shh pathway to the primary cilium, this finding of an ethanol-induced ciliary disturbance may help to explain why Shh disruption was found in our previous molecular studies. Examining the effects of ethanol on the primary cilium could have wider implications in understanding the aetiology of fetal ethanol-related defects ciliaryrelated diseases [3].
[1] D. Brennan, S. Giles, Sonic hedgehog expression is disrupted following in ovo ethanol exposure during early chick eye development, Reprod. Toxicol. 41 (November) (2013) 49–56. [2] D. Brennan, S. Giles, Ocular involvement in fetal alcohol spectrum disorder: a review, Curr. Pharm. Des. 20 (34) (2014) 5377–5387. [3] K. Kennelly, D. Brennan, K. Chummun, S. Giles, Histological characterization of the ethanol-induced microphthalmia phenotype in a chick embryo model system, Reprod. Toxicol. 32 (September (2)) (2011) 227–234.
Introduction: Fetal Alcohol Syndrome (FAS), is a condition which occurs in the offspring of alcohol abusing mothers. It is characterised by pre- and postnatal growth retardation, neurodevelopmental deficits and distinct midline craniofacial abnormalities. Many of the phenotypic FAS-related abnormalities of the brain and midline facial structures also commonly occur in the genetic condition Smith–Lemli–Opitz syndrome (SLOS). The SLOS disorder is directly caused by mutations in the enzymes responsible for the final steps of cholesterol biosynthesis and ultimately leads to an overall deficiency of cholesterol within the body. Therefore, due to the overlapping syndromic presentation of both FAS and SLOS, the aim of the present study was to further investigate whether the detrimental effects of embryonic ethanol exposure may be associated with changes in cholesterol homeostasis. Methods: Fertile chicken eggs (Gallus gallus domesticus) were pre-incubated at 37 ◦ C until embryos had reached Hamburger–Hamilton stage 7–8 of development. Subsequently, chick embryos were explanted onto a filter paper carrier using a modification of the early chick EC method and were treated with a range of ethanol doses (0%, 1%, 5%, 10%, 20%) for 2 min. Treated embryos were placed on an agar-albumen substrate within a humidity chamber and returned to the incubator to continue development for a further 24 h post-treatment. Embryos were dissected from their filter paper carriers and were homogenised in assay buffer (T25 basic homogeniser, Ika Labortechnik). To determine the cholesterol content of individual weight-matched embryos (n = 4 per treatment group), an assay kit was used according to manufacturer’s instructions to give a quantitative measure of the total concentration of free cholesterol in each group (ECCH-100 EnzyChrom cholesterol assay kit, Universal Biologicals Cambridge Ltd.). The assay was read at 340 nm using the SpectraMax Plus384 microplate reader and SoftMax Pro5.2 software (Molecular Devices). Results: Analysis of the total free cholesterol content of embryos treated with 1–20% ethanol was found to be significantly reduced by comparison to control counterparts (*p < 0.05, n = 4 per group). An average value of 0.466 g/dL of cholesterol was obtained in the control group in comparison to 0.214 g/dL at 1% ethanol, 0.267 g/dL at 5% ethanol, 0.271 g/dL at 10% ethanol and 0.207 g/dL at 20% ethanol. Conclusions: These findings confirm that one of the potential mechanisms underlying the teratogenicity of ethanol involves disruption to cholesterol homeostasis within the developing embryo. A reduction in total free cholesterol post-ethanol exposure may explain why FAS has many similar characteristics to the cholesterol-linked genetic disorder SLOS. As cholesterol plays a crucial role in activating many of the key signalling pathways which govern normal embryonic development, the relationship between ethanol and such cholesterol-dependent molecular pathways would be an interesting area of further study.
http://dx.doi.org/10.1016/j.reprotox.2016.06.100
http://dx.doi.org/10.1016/j.reprotox.2016.06.101
References