Involvement Of Homeobox Genes In The Effects Of Vitamin A Deficiency On Rat Embryonic Congenital Malformation

Objectives:Through the establishment of different levels of vitamin A deficiency animalmodel, we observed the the teratogenicity of complete vitamin A deficiency andmarginal vitamin A deficiency and the preventive effects of vitamin A supplement inearly pregnancy on embryonoic growth and developmet. We detected the effects ofdifferent levels of vitamin A deficiency and supplement on the expreesion ofhomeobox genes (Hox genes) at mRNA levels; we also detected the expression ofretinoic acid receptors (RARs) at mRNA and protein levels; meanwhile, we examinedthe DNA methylation pattern in the regulatory region of Hoxa5 gene. This studyaimed to elucidate the the molecular mechanism of the mediation of Hox genes in theembryonic teratogenicity of vitamin A deficiency and offered theoretical foundationof the prevention of congential malformation induced by vitamin A deficiency.Materials and Methods:1. We developed vitamin A deficient and marginal vitamin A deficient female ratmodel and experiment assignment. Weanling female SD rats were assigned into 3groups and given experiment diet containing different doses of vitamin A. Thevitamin A concentrations were 0 IU/g diet, 0.4 IU/g diet and 4 IU/g diet, respectively.10 weeks later, serum retniol concentration of female rat was examined. Then the rat were mated (female: male=2: 1) and the morning of vaginal plug positive was takenas embryonic day 0.5 (E0.5). The pregnant rats were then subdivided into 5 groups:vitamin A normal group (AN), completely vitamin A-deficient group (AD), vitamin Adeficient-supplement at E0.5d group (DS), marginally vitamin A-deficient group(AM), and marginally vitamin A deficient-supplement at E0.5d group (MS). Pregnantrats in group DS and group MS were fed diet containing vitamin A 10 IU/g dietstarted from E0.5 day.2. We observed the growth and development of both female rats and embryos ofE12.5d and E19.5d, including the conception rate, abortion rate and embryonicdevelopment. At E19.5d, gross weight of placenta, average placenta weight, the bodyweight, body length and tail length of the fetuses were measured; the malformation ofexternal appearance, internal organs and skeleton of the fetuses were examined.Meanwhile, the survival and morphogenesis of E12.5d and E19.5d embryos wererecorded.3. In order to shed the light on the important mediation of Hox genes in theeffects of vitamin A deficiency on embryonic development, E12.5 embryos were usedfor the analysis of Hox genes mRNA expression by real time RT-PCR.4. We use the real time RT-PCR and western blot to detect the mRNA andprotein expression of retinoic acid receptors in order to elucidate the possibility thatvitamin A deficency might disturb the regularly expression of RARs and thusdown-regulated the important target genes, Hox genes' expression.5. To seek the association between the changes of DNA methylation pattern ofHox genes and the teratogenicity of vitamin A deficiency, we use the bisulfitesequence PCR to detect the DNA methylation of regulatory region of Hoxa5.Results:1. After 10 weeks, female rats of CVAD group exhibited an array of vitamin Adeficient symptoms, as were appetite loss, weight loss, loss of whiskers, patches offur, ocular porphyrin deposits and depression in response to external stimuli, whilerats of normal group and group MVAD exhibited normal appearance. Serum retinol concentration of pregnant rats of group CVAD and MVAD group was less than 0.7μmol/, indicating that both vitamin A-deficient and marginal vitamin A-deficient ratmodels were successfully developed.2. At 10 weeks, an obvious reduce of gross weight of placenta, average placentaweight, body weight, body length gain, food utilization rate and conception rate wasobserved in CVAD female rat (P＜0.01, P＜0.05). There was no significantdifference in other growth and development index. No significant difference wasobserved in MVAD group compared to normal group. Compared to normal group,abortion rate of pregnant rat, incidence rate of dead or absorption of E12.5d andE19.5d embryos in CVAD group was significantly higher; while survival rate wassignificantly lower (P＜0.01). There was no difference in supplement groupcompared to normal group. At E12.5d, the embryos in VAD group weremaldeveloped than normal group. At E19.5d, the embryos in CVAD group were allabsorbed. There was significant difference in the developmental index including bodyweight, body length and tail length, incidence rate of dead embryos and abnormalembryos in MVAD group compared to normal group and supplement group (P＜0.01, P＜0.05), and obvious malformation of external appearance, internal organsand skeleton of the fetuses were observed, such as encephaledema, exophthalmos,microphthalmos, subcutaneous hemorrhage, internal organs hemorrhage, bonedeformity (dysostosis, short rib, et al), scoliolosis. There was no significant differencein the abnormity rate of MVAD group compared to normal group. The embryos innormal group and supplement group appeared normal.3. The results of real tiome RT-PCR demonstrated that Hox genes near 3' endmRNA expression level of E12.5 embryos was down-regulated by complete vitaminA deficiency and marginal vitamin A deficiency (P＜0.01, P＜0.05), while the Hoxgenes near 5' end exhibited no response to vitamin A deficiency.4. The results of real tiome RT-PCR and western blot demonstrated thatcomplete vitamin A deficiency could suppressed the mRNA and protein expression ofRARs (P＜0.01, P＜0.05) while the expression of RARβand RARγwere down-regulated by marginal vitamin A deficiency (P＜0.01, P＜0.05) while RARα'sexpression had no difference relative to vitamin A normal and supplement groups.5. The distrubition and level of DNA methylation in near 500bp nucleotide basesin regulatory region of Hoxa5 of complete vitamin A deficiency and marginal vitaminA deficiency were significantly different from the vitamin A normal and supplementgroups. The DNA methylation site in 34 methylated sites of vitamin A deficnecygroups were focused on 9^th-10^th,12^th-16^th,20^th-21^st regions.ConclusionsComplete vitamin A deficiency induces growth retardation, lower reproductivecapacity and teratogenicity of embryonic development. Marginal vitamin Adeficiency also induces embryonic congential malformation. Supplement withvitamin A at early stage of pregnancy could reverse these changes. Thedown-regulation of embryonic Hox genea near 3' end mRNA expression is animportant molecular mechanism of abnormal embryonic development by vitamin Adeficiency. The interruption of mRNA and protein expreesion of RARs and DNAmethylation pattern of Hox genes at regulatory region might be further explanation ofthe down-regulation of Hox genes.