Effect Of Shugoshin1on Bovine Oocyte Maturation、Early Embryo Development And Somatic Cell Mitosis

The chromosomal separation in both mitosis and meiosis is time-dependent and space-dependent; ensuring the faithful delivery of genetic materials.Sister chromatid cohesion plays an important role in chromosomal separation, which is mediated by cohesin, a circular multi-subunit protein complex.It is reported that Shugoshin(Sgo/SGO) involves in chromosomal separation in fruit fly,maize,rice,yeast,xenopus,mouse and Hela cells.The major role of such protein family is to protect cohesin at centromere and maintain the cohesion between sister chromatids,especially in meiosis I.Besides,Sgo/SGO functions in several other process and is essential for cell division and chromosomal separation.It is hard to see the relevant researches about mammalian Sgo/SGO in normal somatic cells and oocytes, if any, just in mouse.In this research, the amount of SGO1/Sgo1mRNA in different tissues of bovine, rat and mouse was detected firstly. The roles of SGO1was proved in bovine somatic cells subsequently.We also confirmed the distribution of SGO1during oocytes meiotic maturation.The roles of SGO1during bovine oocyte meiosis and early embryo development were further discussed via its over expression and low expression. The results as follows provide an important reference for researches in vertebrate animals, especially for mammals.1. The expression levels of SGO1/Sgol in bovine, rat and mouse tissuesThe mRNA levels of SGO1/Sgol in three species were detected by real time RT PCR. The expression of SGO1/Sgol was different among species, tissues and genders, but regularities were drawed The expression levels of SGO1/Sgol were lower in pancreas, brain, kidney, heart, liver, muscle, medulla oblongata and lung of rat and mouse, but higher in spleen of mouse, male rat and female bovine, as well as the most detection in small intestine of female rat and testis of male bovine.2. Roles of SGO1in bovine embryonic fibroblastssiRNA was transfected to the synchronized cells to clarify the function of SGO1in mitosis of mammalian cells.Depletion of SGO1promoted the premature loss of sister chromatid cohesion along chromosome arms and at centromeres and caused mitotic arrestment.The chromosome aligned in pair although with no cohesion.Then they hypercondensed and turned to short and thick rods dispersed disorderly without being pulled to the opposite spindle poles. Such polyploid cells failed to enter anaphase increased over time.3. Localization of SGO1during bovine oocyte meiotic maturationThe localization of SGO1on meiotic chromosomes was analysed by injection of marked SGO1mRNA to the cytoplasm of GV oocytes. SGO1anchored on chromosomes from prometaphase I to metaphase II, however, not lapped entirely, with several enrichment spots. SGO1signals were detected on every chromosome in meiosis Ⅰ, but appeared only at nucleus and presented some strong staining spots when entry to meiosis Ⅱ, not detected in polar body.4. Effect of SGO1on bovine oocyte meiosis and early embryo developmentSGO specific siRNA or mRNA was microinjected to oocyte cytoplasm in order to explore the influence of over expression and depletion of SGO1on oocyte meiotic division and early embryo development. SGOl was involved in chromosomal separation. The homologous chromosomes could separate apart from each other after exogenous overexpression of SGO1but not be pulled to the opposite poles of the spindle, resulting a metaphase-like appearance. Depletion of SGO1at GV stage caused oocyte division arrestment, lower blastocyst rate and blastocyst with fewer cells.