Effect Of Retinoic Acid On Mouse Blastocysts Development

Objective: Recent years, With increasing of the rate of abortion, more and more stress was laid on developmental toxicology in peri-implantation and implantation embryos. Some research demonstrated pre- -implantation embryos exposed to some chemicals can show embryo abnormality or death, which called the traditional theory that preimplantation period was not susceptible period in question. This study was designed to examine apoptosis in ICM and TE of mouse blastocyst induced by all-trans retinoic acid (ATRA) and subsequent change of total cell number and ICM and TE number assessment, and to detected expression of TGF-β1 protein and Fas protein in mouse blastocysts in vitro and its possible roles on preimplantation mouse embryo development. Thus ,To investigate the developmental toxicology of ATRA on peri-implantation embryo which can provide experimental foundation for teratologenic mechanism.Methods: By superovulated, mouse 3.5 d blastocysts were obtained ,Expanded blastocysts from different females were pooled and randomly selected for one control and two experiments,which exposed to doses of 0μmol/l,0.1μmol/l,lμmol/l and 10μmol/l all-trans retinoic acid for 24h Blastocysts were stained by fluorochrome propidium iodide(PI) and bibenzimide(Hoechst 33258)to count number of inner cell mass(ICM)and trophectoderm(TE) cells Differentially. Fixed blastocysts observed for their apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)and detected apoptosis and quantitated by laser scanning confocal microscopy. Expression of TGF-β1 and Fas proteins were detected by immunohistochemical S—P staining. The average optical density and the rate of positive area of expression of TGF-pi and Fas proteins were analyzed using image analysis Image HPIAS-2000 Analysis System.Results: The intense pink colour in all photomicrographs represents the chromatin in nuclei of TE cells that are stained both red (PI) and blue (Hoechst 33258),ICM nuclei remain blue only. Compared with the findings for the control and experiment 1 blastocysts ,experiment 2 resulted in a significant reduction in the average number of total cell in blastocysts and the trophectoderm/inner cell mass lineage (p<0.01). No significant difference was found between the control and experiment blastocysts (P>0.05). The fluorochrome quantity in experiment 2 was significantly higher than that in the control and experiment 1 blastocysts.But there were no significant difference between the control and experiment 1 blastocysts (P>0.05). The expression of TGF-pl in experiment 2 was significantly higher than that in the control and experiment 1 blastocysts (p<0.01) . No significant difference was found between the expression of TGF-β1 in the control and experiment 1 blastocysts (P>0.05). The expression of Fas in experiment 2 was significantly higher than that in the control and experiment 1 blastocysts (p<0.01) . No significant difference in the expression of Fas was found between the control and experiment 1 blastocysts (P>0.05).Conclusion: ATRA can induce apoptosis and inhibits cells proliferation in TE and ICM cells of mouse blastocysts . This study examined the cytotoxic effect of ATRA on mouse embryos. TGF-pl and Fas may play criticaHl regulating roles in mouse embryo development.