Effects Of Different Hormones On Superovulation And Embryo Implantation In Mice

Superovulation is an important animal reproduction technology and an important part of animal reproduction regulation and assisted reproductive technology.In order to further study the role of exogenous gonadotropins in animal reproduction regulation,this study compared the application of human chorionic gonadotropin(hCG)and gonadotropin-releasing hormone agonist(GnRH-a)on superovulation and their effect on embryo implantation in mice.The control group was spontaneous ovulation,and the treatment groups 1,2,and 3(called Trial 1,2,3)were injected with equine chorionic gonadotropin(eCG)10 IU and then injected with GnRH-a 0.25 μg,0.50 μg,and 1.00 μg at an interval of 48 hours,respectively.The treatment group 4(called Trial 4)was injected with eCG 10 IU and then injected with hCG 10 IU at an interval of 48 hours.The embryo recovery of each group was compared on 3.5 d,and the pregnancy rate and embryo implantation site of the control group,Trial 2 and Trial 4 were compared on 7.5 d.In this experiment,a mouse model of superovulation transplantation was established.One fallopian tube of eCG 10 IU+hCG 10 IU superovulation mice was ligated on 0.5 d,and normal blastocysts were transplanted to the ligated side with operation on 2.5 d,and the pregnancy rate of two sides were compared on 7.5 d.In order to analyze the effect of eCG+hCG on embryo implantation in mice,this study compared the gene expression of the ovaries and uteri in superovulation and normal pseudopregnancy mice on 3.5 d,4.5 d,and 5.5 d.The sequencing results were verified by qRT-PCR.Result:(1)Compared with the control group,the total number of embryos and the number of available embryos in all treatment groups were significantly increased,of which Trial 2 was the highest,with 33.6 and 29.2 respectively.The blastocyst rate in all treatment groups were decreased,and there was no significantly difference in the blastocyst rate among the treatment groups.Compared with Trial 1 and 3,Trial 2 had better superovulation effect,and there was no significant difference compared with Trial 4.(2)Compared with the control group,the pregnancy rate of Trial 4 was significantly reduced,which was 44.83%.The mean of implantation sites of Trial 4 was significantly increased,which was 18.4.The pregnancy rate of Trial 2 and the control group both were 100%.There was no significant difference in the mean of implantation sites between the two groups.(3)In the superovulation transplanted mice,when the transferred side was pregnant,the control side also was pregnant.When the control side was not pregnant,the transferred side was not pregnant,too.There was no significant difference in the uterine horns on both sides.(4)The number of differentially expressed genes(DEGs)were 221,121 and 94 in ovaries of normal and superovulated pseudopregnant mice at different periods,respectively,which are mainly enriched in biological processes such as cell adhesion,cell cycle regulation and oxidative phosphorylation.The DEGs were mainly involved in signal pathways related to cell senescence,proteasome and steroid biosynthesis.There were no same geen in the top 10 up and down regulation DEGs in the ovarian tissues of normal and superovulated pseudopregnant mice at different periods.In the same period,the DEGs of ovaries such as Bmp15,Agtr2,Adcyap1,Timp1,Fos,Egr1 were affected by superovulation.(5)The number of DEGs were 1097,640 and 407 in uteri of normal and superovulated pseudopregnant mice at different periods,respectively,which are mainly enriched in biological processes such as cell migration,immune system,steroid metabolism.They were mainly involved in FoxO,cell cycle and cell adhesion molecules and other related signaling pathways.Klk1 was included in the top 10 up and down regulation DEGs in the uterine tissues of normal and superovulation pseudopregnant mice at different periods.In the same period,the DEGs of uteri such as Lif,Spp1,Prss28,Prss29 were affected by superovulation.(6)The qRT-PCR results showed that the relative expression differences of the target gene in each ovaries and uteri were consistent with the sequencing results.In summary,GnRH-a can be used to replace hCG in superovulation in mice and achieve a good superovulation result.The use of eCG+hCG to superovulate mice reduces the pregnancy rate and caused pregnancy failure by affectting the genes expression in ovaries and uteri in peri-implantation period.