Vitamin E was discovered in 1922 as a lipid soluble factor necessary for reproduction and named ?-tocopherol from the Greek words ????? for """"""""birth"""""""" and ?????? for """"""""to carry"""""""". To date, ?- tocopherol's role in reproduction has been impossible to approach experimentally because ?- tocopherol-deficient (E-) mothers do not produce embryonic tissues due to failed implantation. To circumvent this barrier, we have chosen to use the premier vertebrate model for studying development, the zebrafish, because they lay eggs;thus, implantation is not necessary. The zebrafish model is ideal for our studies because it, like humans, has a preference for ?- tocopherol, expresses the ttp gene and requires vitamin C, an important determinant in ?- tocopherol antioxidant function in humans. An additional strength of this model over traditional rodent transgenic models is that any gene can be knocked down during embryogenesis, a feature we plan to exploit. We have succeeded in developing the first defined zebrafish diet. Using our E- defined diet, we are able to produce E- eggs. By 48 hours post-fertilization (hpf), many E- embryos exhibit severe developmental malformations, which establishes for the first time that ?-tocopherol is required for fetal neurological and cardiovascular development, not just implantation. We also found that the ?-tocopherol transfer protein (?-TTP) is abundantly expressed by the 48 hpf zebrafish embryo and its expression increases with oxidative stress. Severe vitamin E deficiency with progressive neurologic degeneration occurs in humans with ttp gene defects, emphasizing the critical importance of this protein. ?-TTP expression in the zebrafish embryo suggests that during development ?-TTP directs ?-tocopherol from the yolk sac to specific sites in the developing embryo where it is especially required. We propose that ?-tocopherol is required to protect key oxidized lipid mediators from further oxidative degradation during specific developmental steps, especially those in the nitive functions. To obviate these problems, we propose to use vitamin E-depleted zebrafish (Danio rerio) as a model system. Zebrafish are vertebrate animals with genes generally homologous to those of humans, large numbers of animals can be raised, diets can easily be manipulated, targeted genes can be readily modified, and embryonic stages can be studied over the course of development. Critically, the ?-tocopherol transfer protein (?-TTP) is expressed in human yolk sac [2], is abundantly expressed by the 48 hour postfertilization (hpf) zebrafish embryo and its expression increaso. We hypothesize that ?-tocopherol provides antioxidant protection for specific, key lipid mediators necessary for embryonic development, including cell loss via programmed cell death. To explain the key developmental effects of ?-tocopherol deficiency, we propose the following Aim 1. Define the roles of oxidant and antioxidant apoptosis regulators during embryonic development;
Aim 2. Define the role of ?-TTP during embryonic development. Completion of these specific aims will allow us to determine specific ?-tocopherol molecular functions and key antioxidant/oxidant signaling mechanisms. Defining the molecular targets altered by ?- tocopherol-deficiency in the zebrafish will provide a solid basis for understanding why humans require ?-tocopherol for reproduction as well as to maintain a healthy nervous system

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Vitamin E (?-tocopherol) was discovered in 1922;nearly 100 years later, we still do not know why it is required. Our goal is to define molecular targets altered by ?-tocopherol- deficiency using zebrafish embryos, which provide unique experimental advantages to finally answer why vitamin E is essential for reproduction and for maintaining a healthy nervous system.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Integrative Nutrition and Metabolic Processes Study Section (INMP)
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Henken, Deborah B
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Oregon State University
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McDougall, Melissa Q; Choi, Jaewoo; Stevens, Jan F et al. (2016) Lipidomics and H2(18)O labeling techniques reveal increased remodeling of DHA-containing membrane phospholipids associated with abnormal locomotor responses in ?-tocopherol deficient zebrafish (danio rerio) embryos. Redox Biol 8:165-74
Choi, Jaewoo; Leonard, Scott W; Kasper, Katherine et al. (2015) Novel function of vitamin E in regulation of zebrafish (Danio rerio) brain lysophospholipids discovered using lipidomics. J Lipid Res 56:1182-90
Miller, Galen W; Truong, Lisa; Barton, Carrie L et al. (2014) The influences of parental diet and vitamin E intake on the embryonic zebrafish transcriptome. Comp Biochem Physiol Part D Genomics Proteomics 10:22-9
Lebold, Katie M; Traber, Maret G (2014) Interactions between ?-tocopherol, polyunsaturated fatty acids, and lipoxygenases during embryogenesis. Free Radic Biol Med 66:13-9
Motorykin, Ievgen; Traber, Maret G; Tanguay, Robert L et al. (2014) Proteome-driven elucidation of adaptive responses to combined vitamin E and C deficiency in zebrafish. J Proteome Res 13:1647-56
Lebold, Katie M; Lohr, Christiane V; Barton, Carrie L et al. (2013) Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior. Comp Biochem Physiol C Toxicol Pharmacol 157:382-9
Lebold, Katie M; Kirkwood, Jay S; Taylor, Alan W et al. (2013) Novel liquid chromatography-mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos. Redox Biol 2:105-13
Miller, Galen W; Labut, Edwin M; Lebold, Katie M et al. (2012) Zebrafish (Danio rerio) fed vitamin E-deficient diets produce embryos with increased morphologic abnormalities and mortality. J Nutr Biochem 23:478-86
Kirkwood, Jay S; Lebold, Katie M; Miranda, Cristobal L et al. (2012) Vitamin C deficiency activates the purine nucleotide cycle in zebrafish. J Biol Chem 287:3833-41
Miller, Galen W; Ulatowski, Lynn; Labut, Edwin M et al. (2012) The ?-tocopherol transfer protein is essential for vertebrate embryogenesis. PLoS One 7:e47402

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