Developmentalgene Expression And Regulation Of Goat Preimplantation Embryos

Embryo development is one of the most important subjects in life science research. Embryogenesis represents the critical transition from the single-celled fertilized embryo to the mature and multicellular embryo by undergoing a series of controlled cell divisions and cell differentiation events. It is a process of gene expression and protein regulation with time-space alternation. Nucleus gene determined translation progress of cytoplasm protein, on the contrary gene expression during embryogenesis and development stages was controlled by protein regulation factors. The impact between nucleolus and cytoplasm was promoted the development of embryo. The oocytes, 2-cell, 4-cell, 8~16-cell, morulae and blastocyst embryos of in vitro of goat were obtained through the process of oocytes matured in vitro (IVM), fertilized in vitro (IVF) and cultured in vitro (IVC). The 2-cell, 4-cell, 8~16-cell, morulae and blastocyst embryos of in vivo in goat were obtained using the way of estrus synchronization and superovulation. Silver staining mRNA differential display and SDS-PAGE were used in this study to analysis the variety of gene expression and proteome at developmental embryo. In order to establish the pattern of developmental gene expression and to understand the molecular mechanism of developmental goat preimplantation embryos, provide the academic theory of gene expression and control mechanism about mammal. Results as follow:44 different stage specific bands in total were screened. 5, 5, 11, 8 specific bands which only displayed in in vivo 2-cell, 4-cell, 8~16-cell, morulae and blastocyst stage were screened and 4, 2, 3, 3, 1 specific bands were displayed in in vitro oocytes, 2-cell, 4-cell, 8~16-cell, morulae and blastocyst. 2 specific bands were found all stage of embryo development. The sequencing and alignment results The sequencing and alignment results suggested that 10 differential expression bands were homologous with function genes or regulatory genes which already known from the GenBank. Other 34 differential expression bands were unknown genes.The specific band S12 which was expressed in oocytes was similar to protein kinase gene (98%). Protein kinase played an important role in regulation of gene transcription and expression in the progress of oocytes mature. The specific band Y62 which was expressed in 2-cell stage, was similar to phosphatas gene (98%). Cell divisions and cell differentiation were controlled by phosphates through signal transfer. E66 was 2-cell stage specific band and it was homologous with sapiens dynein gene (95%). Dynein regulated gene transcription and expression of preimplantation stage embryo in virtue of provided energy. Y72 was 4-cell stage specific band and it was homologous with epidermal growth factor receptor gene (89%). Effect of EGF on early embryos development in goat was stimulated cell divisions and differentiation. Sg78 was 4-cell and 8~16- cell stage specific band and it was homologous with aldehyde dehydrogenase gene (97%). E8 was 8~16-cell stage specific band and it was homologous with Glycine dehydrogenase gene (97%). Glycine dehydrogenase and aldehyde dehydrogenase were essential for ATP synthesis to provide energy for cell metabolism, DNA demethylation and genomic reprogramming. S81 was 8~16- cell stage specific band and it was similar to fibronection gene (97%). Fibronection was the regulator of connection between cleavage cells, it also can promoted come into being blastula. S97 was morulae and blastocyst specific band and it was homologous with leukemia inhibitory factor gene (95%). LIF is an important cytokine in early embryo development, which regulated fetation and pregnancy. The specific band Y97 which expressed in morulae and blastocyst was homologous with growth hormone gene (91%). It was promoted embryonic cell division and differentiation by the way of influence pregnancy physiology and reproductive hormones secretion. Sg6789 band was found at all stage of embryo development and it homologous with leptin receptor-like protein (obese) gene (96%). Leptin had the effects on oocytes maturation, embryonic cell division and differentiation.The changes of embryo protein in different development stags from in vivo and in vitro were discussed in the present research. Through electrophoresis and staining about 195 protein spots were detected in the gel. 14 protein spots were detected in MII. 32 protein spots were detected in 2-cell stage, 16 in in vivo and 16 in in vitro, respectively. Match protein spots were 15, the matching rate of these two patterns reached 93.7%. 39 protein spots were detected in 4-cell stage, 21 in in vivo and 18 in in vitro, respectively. Match protein spots were 16, the matching rate of these two patterns reached 76.9%. 53 protein spots were detected in 8~16-cell stage, 29 in in vivo and 24 in in vitro, respectively. Match protein spots were 19, the matching rate of these two patterns reached 71.7%. 57 protein spots were detected in morulae and blastocyst stage, 30 in in vivo and 27 in in vitro, respectively. The matching rate of these two patterns reached 70.2%. 11 protein spots were not detected from embryo in in vitro culture, which suggested those protein spots connected with block of development. 4 protein spots were detected from 2-cell stage to blastocyst stage and 2 protein spots were detected from MII stage to blastocyst stage. No change of proteins during embryo development showed that it played an important role in cell divisions and differentiation.Through the comparative analysis of the patterns of protein and mRNA of goat embryo in different stages, the results showed as follow:The zygote genome of goat synthesized mRNA and protein at the stage when embryo was start cleavage. The gene expression and protein composing were difference in different developmental stage of goat embryo, it indicated that process of gene expression was regulation with time-space alternation. The potential ability of gene expression was determined by previous correlation gene expression. Cell divisions and differentiation was controlled by gene expression, and it also related with block of development. Research on the changes of special protein spots and mRNA appeared in each stage showed that the majority of these spots disappeared in the following contiguous stage of embryo development. The results suggested that these proteins and genes existed in the corresponding stages might be related with the development of morphological character of corresponding embryo, even with contiguous embryo. The special proteins and genes which were expressed at all stage were concerned with preimplantation embryo development. Those proteins and genes were necessary for embryo activity.This study on the molecule level of the hereditary basis provided important information for researching the mechanism of developmental embryo. However, further research on the function, structure and orientation of special expressed gene in the gel from developmental embryo is required.