EphB2 Expression in the development of anorectal malformations in fetal rats

Abstract

Objective To investigate EphB2 expression in anorectal development in normal ratembryos and embryos with anorectal malformations (ARMs) induced by ethylenethiourea (ETU), andto elucidate its role in the anorectal morphogenesis. Methods Twenty-four pregnant rats were admin-istrated ETU(125mg/kg) on the tenth gestational day (Gdl0) via gastric gavage. Meanwhile, another12 pregnant rats were administrated saline (125ml/kg). Cesarean section was carried out on Gd13,Gd14, Gd15, Gd16, Gd18 and Gd20, respectively. The samples were fixed, embedded and serial-sec-tioned in the sagittal and transversal planes. According to the anorectal morphology, the 309 embryoswere divided into 3 groups: ARMs Group - fetuses with ARMs born from pregnant rats that receivedETU; ETU Without ARMs Group--fetuses without ARMs born from pregnant rats that were admin-istered ETU, and Control Groupnormal fetuses from pregnant rats that were not administered ETU.The serial sections were analyzed by immunohistochemistry to quantify and localize EphB2 protein in the terminal bowl tissue. The cloaca and anorectat tissues were dissected under microscope for totalRNA extraction. Reverse transcription polymerized chain reaction techniques (RT-PCR) were carriedout to investigate EphB2 mRNA. Results EphB2 protein and mRNA were detected in all of the threegroups. On Gd13 and Gd14, EphB2 expression mostly located in the urorectal septum (URS) and clo-aeal membrane. On Gd15, increased expression was observed in the fusion tissue of URS and cloacalmembrane. On Gd16, anal membrane broke down, and EphB2 expression was confined to mucousmembrane of rectum. EphB2 expression was significantly lower in the ARMs group than that in theother two groups from Gd13 to Gd16 (P<0. 05). EphB2 mRNA increased from Ga113 to Gd16, andthen decreased gradually. EphB2 mRNA of the ARM group was lower than that in the other twogroups from Gd13 to Gd16(<P0. 05). Conclusions EphB2 may play an important role in anorectalmorphogenesis, and the decreased expression of EphB2 might be related to the development of ARMs. Key words: Receptors,EphB2; Digestive system abnormalities; Embryology; Cloaca