The Embryonic Processes And The Expression Of The RAR And Hox Gene In Rat Fetuses With Anorectal Malformations Induced By Retinoic Acid

IntroductionCongenital anorectal malformations (ARMs) are the most common alimentary deformaties and occur in 1 per 1500 to 5000 live births. Anorectal malformations represent a spectrum of anomalities from the very minor in covered anus to the more extreme cloaca and their pathologic variations are very complex. After correction of the anomalies, problems such as constipation and incontinence can prove to be continuing challenges in management. Now to the developmental processes of the normal anorectal structures and anorectal malformations , the understanding remains incomplete.The essential nutrient Vitamin A is required for several life processes, including vision, reproduction, growth, cell differentiation, immune function and embryonic development. The lack or excess of Vitamin A in diet can lead to many clinical disorders from the abnormal metabolism of Vitamin A. Vitamin A or related compounds ( retinoids) plays a crucial role in the process of normal embryonic development. Females who intaked either an excess or a deficiency of vitamin A and retinoids during pregnency may result in fetal death or congenital abnormalities in the offsprings such as anorectal malformations.ATRA is the most important functional form of retinoids. We induced anorectal malformations in rats with it and study the mechanism of ARMs. Using the animal model we can ignore the insufficiency of clinical samples and restriction of ethnics and explore the relativity of retinoids and ARMs in aspect of embryology and molecular biology.The constents of the current study are as follows: ① Animal model ofARMs: The pregnant rats were gavage - fed with over - dose ATRA on the tenth day of gestation and they would bring out offsprings with ARMs. We observed and summarize the characters of ARMs and accompanied malformations to study whether the abnormalities have the same embryonic mechanism. ? The expression of RARmRNA in rat embryos with ARMs induced by ATRA: We determine the expression pattern of RARmRNA in caudal part of the embryos with ARMs by RT - PCR and analyze the data of the control group and the ARMs group to explore the relation of the pathogenesis of ARMs and vitamin - A signaling pathway. (§)The embryonic processes and expression of Hoxd -13 gene in rat embryos with ARMs induced by ATRA: We study the development of ARMs by continuous and dynamic observation of caudal morphogenesis and detect the expression pattern of Hoxd -13 gene in rat embryos by in situ hybridization (ISH) to deeply discuss the mechanism of ARMs.Materials and MethodsMaterialsAnimals; Wistar rats were provided by Medical animal center, the second clinical college of China Medical University.Reagents: ATRA was provided by Beijing Phetx Co. , Ltd. Agar sugar was provided by Sigma Co. , Ltd. BcaBEST? RNA PCR Kit Ver. 1.1, Trizol(RNA extraction system) , RNA Marker and Bca - Optimized Taq were provided by TaKaBa Biotechnology ( Dalian) Co. , Ltd. The primers for Retinoic Acid Receptors and 3 - actin were made by Shanghai Bioasia Biotechnology Co. , Ltd. Hoxd -13 ISH Detection Kit were were provided by Boster Biological Technology (Wuhan) Co. , Ltd.Instruments: Stereomicroscope and opticmicroscope are from Olympus Co. , Ltd. The image analysis system is Universal Imaging Porporation(USA).Methods(DAnimal model of ARMs: The timed - pregnant rats were divided into two groups (the ATRA treated group and the control group) in random. The ATRA treated females were gavage - fed with ATRA ( dissolved in mineral oil) at135mg/kg b. w. on E10(day 10 of gestation). The control group received the same volume of mineral oil on the same day. Fetuses were recovered by Caesare-an section on El6, El8 and E20 from 3 ATRA treated females and 2 control females respectively. The fetuses were weighted and measured the total body length. After examining the fetuses macroscopically by stereomicroscope , the whole fetuses on E16 and the fetuses without head and limbs on El8 and E20 were fixed in 4% formaldehyda for 24 h, processed, embedded in paraflin wax and serially sliced at 6 um sagittally. The slices were stained with hemateoxylin and eosin ( HE) and were examined microscopically. All quantitative data are presented as the mean and the significance of differences assessed using by students t -test, with P <0.01 considered to indicated significance.(D The expression of RARmRNA in rat embryos with ARMs induced by ATRA: The rat embryos with ARMs were induced by ATRA as mentioned. Fetuses were recovered by Caesarean section on Ell, E12, E13, E14, E15 and El 6. We examined the fetuses of E16 macroscopically and microscopically to test the stability of the animal model. Then we determined the expression pattern of RARmRNA in caudal part of the embryos by RT - PCR. The data of the control group and the ARMs group were compared and analyzed.(§)The embryonic processes and expression of Hoxd -13 gene in rat embryos with ARMs induced by ATRA: The rat embryos with ARMs were induced by ATRA as mentioned. The fetuses of both groups ( ARMs group and control group) were recovered by Caesarean section on E12, E13, E14, E16, E18 and E20. All samples were fixed in 4% formaldehyda ( \%o DEPC) for 2 ~4 hours, processed, embedded in paraflin wax and serially sliced at 6 um sagittally. Some slices were stained with HE and were examined microscopically. We selected 2 ~ 3 slices to determine the expression of Hoxd -13 by ISH according to the results of HE stained slices. The images were collected with Universal Imaging Porporation and analysed with Meta Morph software. Average gray value ( G -value) represents the level of gene expression and the data were analysed by ANOVA with SPSS11.5 software, with P <0.05 considered to indicated significance.Results(DAnimal model of ARMs and accompanied malformations:96 fetuses were obtained from the ATRA treated rats. Among the 96 fetuses, 10 were dead and 86 were lively. 90.7% (78/86) had anorectal malformation , 52. 3% (45/86) had spinal bifida, 79.1% (68/86)had no tail and 11.6(10/86) had curly tail or shorter tail. In the control group 65 fetuses were obtained and they were normal in appearance. The total body lengthes(TBL) and body weights (BW) on E16 ,E18 and E20 of the treated fetuses were compared with the data of the control group. There were significant difference between the treated group and the control group ( P < 0. 01) and the treated group were poorer than the control group. On serial histological slices of the control group on E16, the well developed sacral vertebrae and coccygeal vertebra can be seen, spinal cord was continuous and tail was normal. Hie rectum and anus had come into being in front of the spine, and there was urinary bladder or uterus in front of the rectum. On the slices of the ATRA treated group on El6, there were severe malformations of the sacral and Coccygeal vertebra such as spinal bifida and manifesta myelocele. In front of the spine the rectum and bladder or uterus were less mature than the control group. The slices showed that the fetuses with an imperforate anus had associated recto - urethral fistula. We only find more mature caudal structure on trie slices on El 8 and E20 than on E16 ignoring they were normal or abnormal.(2)The expression of RARmRNA in rat embryos with ARMs induced by ATRA : In the ATRA treated group 80% embryos have anorectal malformations and trie control group embryos were normal. The expression level of RARot mRNA in trie ATRA treated group was lower than the control group on Ell, E12, E13 and E15. The top of RARa mRNA expression curve of the ATRA treated group came latter than the control group. The expression level of RARfJ mRNA in the ATRA treated group was lower than the control group on E12 and E14, while between the expression levels of RAR7 mRNA of the control group and of the ATRA treated group there was no statistical significance.?The embryonic processes and expression of Hoxd -13 gene in rat embryos with ARMs induced by ATRA: Histological observation of control embryos re-presented as follows: On E12 the anterior folds and posterior folds formed and there was a" Y" shaped cavity in the middle part of the embryos. On E13 the caudal structures such as sacrum, coccyx and tail had started to develop and cloaca and cloacal membrane appeared. On E14 the caudal structures developed well. The tails and spinal centra were normal and the line - liked notochord lied in the lumbosacral centra. The distance between urorectal septum and cloacal membrane was very short and anal membrane was not ruptured. On E16 the caudal structures include spine, tail, rectum, anus, bladder and urethra had well shaped. Histological observation of ATRA treated embryos represented as follows.- On El2 the posterior folds had not formed. On El3 the caudal ventral folds was clear while the tail was blunt. The cloaca and cloacal membrane appeared. On E14 the caudal part of the embryo represented a mass made of turbulent tissure. The posterior neural pore was not closed. The cloaca took on a " U" shaped narrow cavity. The distance between urorectal septum and cloacal membrane was more than in the control embryo. On E16 the ARMs, spinal bifi-da and tail deformaties came into being.As to the regions of the Hoxd - 13 gene expression, the difference is the regions in the control embryos included the endothelium of cloaca and hindgut before the anorectal and urogenital system formed while in the ATRA treated embryos did not include them. As to the level of the Hoxd - 13 gene expression, the Average gray values (G - value) analysed by AN OVA showed F value was 23. 165. It represented P < 0.05 and these means were not equal. Compared these means of G - values each other showed that in the control embryos the G -value of E12 more than other time groups and they decreased with the pregnant days increased while in the ATRA treated embryos the G - values were not so regular as in the controls. The results indicated that the expression trend was ascendant with the pregnant days.Conclusions(pATRA is an excellent teratogen used to induce anorectal malformations in rats. ATRA can disturb the differentiation of the tail bud in embryonic development and lead to malformations of its derived tissures. As the results the tail de-...