Functional Studiy Of Gs? In Reproductive System Via Conditional Gene Knockout

G protein also known as guanosine nucleotide-binding proteins,which is a G protein family.G protein which had been found were more than 20 species.All types of G protein are composed of three different subunits,namely the differences ?,?,?subunits.Different G protein structure and function were mainly decided by the a subunit.The G protein with the function of activating the effector(such as phospholipase C,adenylate cyclase)is called stimulate G protein,and its a subunit is called Gsa.Gsa mainly via regulate adenylate cyclase activity of target cells to regulate the level of the second messenger's cAMP,thereby affecting the downstream signal pathway events.Therefore Gsa plays an important role in the development of mouse.It has been known that Gs activity in the oocyte is required to maintain meiotic arrest within the ovarian follicle and the follicle may keep thecell cycle arrested by activating Gs.Now,we are not quite understand the Gs?'s function in mouse spermatogenesis and oogenesis(such as recruitment,activation and development of primordial follicles)and in embryonic development after fertilization.Since the complete knockout mice stop development at E10.5 and prenatal death,which is not conducive to further research,we decided to use the conditional knockout technology to build germ cells specially knockout Gsa model to study its function in the reproductive system.We mating Gs?flox/flox mice with Zp3-Cre and Ddx4-Cre mice respectively to get conditional knockout mouse in the reproductive system.Zp3-Cre recombinase is specific expression in the oocyte,Ddx4-Cre recombinase is specific expression in ovarian and testicular germ cells,we validated the expression sites of these two recombinase in Cre transgenic mice and then using the Cre-LoxP system to knockout the Gsa gene specificity,analytical the specific knockout phenotype to study the role of Gsa in the reproductive system.Method:To test their fertility,eight-week-old female mice were housed with wild-type males for a long time or check vaginal plugs every morning.In addition,paraffin sections,HE staining,isolation and culture of oocytes,collection of embryos,BrUTP incorporation assays et al were used to research the function of Gsa in germ cell via these two animal models.Result:(1)The Gs?-/-(Zp3-Cre)females produced no litters,but the ovaries had a normal complement of primordial follicles and all stage of follicular development were represented.Corpora lutea were also present.There were no significant difference in the rate of GVBD and extrusion of PB1,indicated the normal follicular development and oocyte maturation.Sexually mature homozygous females had regular 4?6 day oestrus cycles.However,the embryos from Gsa-null females remained at the two-cell stage or had begun to degenerate.We evaluated transcription in homozygous and control embryos using BrUTP incorporation.De novo RNA transcription was decreased in the two-cell embryos lacking Gsa.Thus,the loss of Gsa in oocytes led to a suppressed ZGA in two-cell embroys and ultimately resulted in the females' sterility.(2)When mating the Gs?-/-(Ddx4-Cre)males with wildtype females,vaginal plugs were never checked,but it's unclear whether their mating behaviors abnormal or can't form vaginal plugs.Testis histology of homozygous male indicate that the seminiferous tubules had all stage of spermatogenic cell.(3)Gs?-/-(Ddx4-Cre)females could form vaginal plugs early after Sexually mature but no litter.Four months after birth,there were no vaginal plugs and maintain dioestrum all the time.Ovarian histology of homozygous females indicated that follicular development is not affected by deletion of Gsa in ovary.Conclusion and Prospects:Based on the experimental results we can get a preliminary report:In Gs?-/-(Zp3-Cre)mice's oocyte lack of Gsa results in the cAMP and PKA contents decreased,which affects zygotic gene activation(ZGA)occurs appropriately,then leads to 2-cell embryos can't continue cleavage and Gsa?-/-(Zp3-Cre)mice infertility.In the follow-up study,we plan to do some supplements and complements to the existing research results.Paraffin section of Gs?-/-(Ddx4-Cre)mice ovaries and testes shows that eggs and sperm were normal.We plan to detect the reproductive ability of mice,and explore the effects of Gsa in the mice's reproductive system based on the experiment results.