Investigation Of The Cryopreservation For Dormant Mouse Embryo

In order to investigate the potential endurance of delayed mouse embryos to resist the freezing and thawing treatment, the delayed and normal hatched murine blastocysts were frozen by cryopreservation and rapid thawing process. In addition, the special cellular structure and disposition of those dormant embryos, were also revealed as well by transmission electron microscope observation.There are total three experiments in it. Experiment I: In order to improve the collecting efficiency of mouse dormant embryos, the normal superovulation combined with anti-PMSG serum (A-PMSG) was applied to it. According to the different doses of A-PMSG injected into the mouse after injection of PMSG, the disposal animals were divided into five groups: 1) injecting A-PMSG before mating, 2) injecting A-PMSG after finding a vaginal plug, 3) injecting A-PMSG after ovariectomy in the morning of day 4 (day 1= vaginalplug), 4) superovulating without A-PMSG, and 5) the control one. It was shown that the average recovery number of delayed embryos in the group injected with the A-PMSG after finding a vaginal plug, is significantly higher than the others (P<0.01).Experiment II: To test the developmental potentiality of mouse dormant embryos after freezing-thawing treatment, the delayed embryos, blastocysts and normal hatched blastocysts, were frozen and thawed separately, then, they were cultured in vitro and transferred into suitable recipients after thawing. Meanwhile, the cell number of inner cell mass (ICM) and trophectoderm of both dormant and activated embryos, were counted by double label fluorescent staining. Then the specific cellular structure and disposition of cell organelle of those delayed embryos was revealed by transmission electron microscope. It was shown that the freeze-thawing recovery rate of hatched blastocyst (80.8%) was significantly higher than the others'(p<0.01). The freeze-thawing recovery rate of dormant embryos(72.1%)was extremely significantly higher than normal blastocysts(50.2%). There was no significantly difference on developmental rate between dormant ones(94.2%)and normal blastocysts (90.9%), but they were all extremely significantly higher than that of the hatched blastocyst group (42.1%). The pregnancy rate in group of blastocysts (49.7%), was significantly higher than that in the dormant one (40.8%). The average cell number of ICM from the delayed embryos was 27.83, which was significantly higher than that in hatched ones (19.53). The cell number of trophectcderm kept stablely among all the groups. After thawing and in vitro culture, the cell number of ICM for delayed embryos was 25.18, which was significantly higher than that for hatched ones (14.68). Their average cell number of trophectcderm (114.9) was also significantly higher than that of hatched ones (73.88) The ultrastructure of embryos in different groups, was also observed through transmission electron microscope respectively. The result implied that there were some significant differences of the shape of nucleus, number of lipid droplet and the morphogenesis of endoplasmic reticulum , especially of the significant change of cell-cell connection and cell organelle compared with the pre-freezing one. It indicated that the delayed embryos might restore their developmental potentiality during in vitro culture.Experiment III: To investigate the potential transcriptional level of cx31 and cx43 between the dormant embryos and mormal hatched blastocyst, the RT- PCR method was applied to evaluate the semi-quantity of those two genes. The results showed that there is no significant difference of mRNA level of cx43 and cx31 between the dormant embryos and the hatched blastocysts.In general, it can be concluded as following: 1) The collecting efficiency of the dormant mouse embryos can be improved by superovulation with anti-PMSG serum. 2) The different morphological change and the disposition of cell organelle in delayed mouse embryos from that in normal ones, might sustain the dormant embryos to defeat the freezing shock . 3) . The dormant mouse embryos might initiate another way to play a better role on anti-freezing shock than those hatched blastocysts , but not depending on the signal of cx43 and cx31.