Effect of Retinoic Acid on Palate Formation during Rat Embryogenesis / 대한해부학회지

In order to understand the effect of retinoic acid (RA) on the craniofacial pattern formation during embryogenesis, we injected RA intraperitoneally into the pregnant female rat on day 11 post coitum (p.c.) and then embryos of day 13 to day 17 p.c. were isolated consequently. The overall morphology and the differential gene expression patterns were analyzed by the microscopic and (DD) RT-PCR methods, respectively. For the morphological study, the retardation of craniofacial region, the shortage of crown rump length and limbs were analyzed in the RA-treated embryos. In the RA-treated embryos of day 17, it was observed that the palatogenesis was completely finished just like in the normal embryos. However, the cleft plate was observed in 36 out of 52 total samples with the distance of cleft palate being 0.80+/-0.36 mm in average. The temporal expression pattern of Hox genes through RT-PCR revealed that the expression of Hoxa7 reached its peak on day 13 then slowly declined in the normal embryos. Whereas in the RA-treated embryos, the expression peak was observed on day 15, then declined subsequently. With the Hoxc8 gene, its expression was low in all stages until the day 16 of normal embryogenesis. On the other hand, Hoxc8 gene expression was detected slightly early on day 15 in the RA-treated embryos. In the study of Bcl-2 family genes, uniformly strong expression of anti-apoptotic and pro-apoptotic genes was observed from day 13 to day 17 of normal embryos, whereas anti-apoptotic gene expressions were decreased after day 16 in the RAtreated embryos. Additionally, a dramatic decline of pro-apoptotic gene expression was observed from day 13 to day 15 of the RA-treated embryos. Therefore, we believe that RA is a potential factor that is actively involved in the cleft palate formation. Moreover, it is profoundly linked with the regulation of Hox and Bcl-2 family gene expression pattern that leads to the embryonic malformation.

Development of Model System for Radiation Induced Congenital Malformations by Whole Embryo Culture / 체질인류학회지

It is known that radiation can induce various kind of mutations, cancers and congenital malformations. Day 9 rats embryos were cultured by whole embryo culture method and irradiated 0.1, 0.5, 2 and 5 Gy at day 10 to study the effect of radiation on the development of rat embryos. Haversting after 48 hours culture, the morphological changes and apoptosis were investigated. In addition, we assessed the expression patterns of p53, WAF1, Bcl -2, Bcl -x and Bax. Compared to the control group, no remarkable morphological changes were observed in the low dosage group (0.1 and 0.5 Gy). But at high dosage group (2 and 5 Gy), growth was retarded and the heart beats were weak. The crown rump length, the number of somites, and branchial arch were decreased and the rotation of embryo and development of otic pit and lens pit, and upper limb bud was delayed significantly. Especially in the 5 Gy group the most of external morphology were difficult to discriminate. In histological observations, high dosage group showed marked increase in the number of apoptotic bodies in the optic cup, trigeminal ganglion, pharyngeal arches, heart and small intestine. In western blot analysis, Bcl -x and Bax were not expressed in all groups. Expression level of Bcl -2 was constant regardless of the amount of radiation. Expression level of p53 and WAF1 increased significantly in accordance with the increasing amount of radiation. Especially, WAF1 was expressed 7.2, 6.3, 9.9 and 11.3 folds more than the control groups when embryos were exposed to 0.1, 0.5, 2 and 5 Gy, respectively. Considering WAF1 arrests cell cycle, we concluded that cell cycle was affected most sensitively to radiation injury. From these results, radiation showed growth retardation, decrease in protein synthesis, increase in apoptosis and expression of related genetic materials. These results may be used as a standard to test the effect of drugs for reducing and protecting agents against deterious effect of radiation in developing embryo and fetus.