The Expression And Function Of IGF-Ⅰ/Ⅱ And IGF-IR/IIR In Development Of Bovine Preimplantation Embryos

Insulin-like growth factors and its relatives constitute a family of growth factors that might be implicated and important in growth regulation of early embryos.This study aimed to assess expression and location of insulin-like growth factorⅠ(IGF-Ⅰ), insulin-like growth factorⅡ(IGF-Ⅱ) and their receptors insulin-like growth factor receptor typeⅠ(IGF-ⅠR),insulin-like growth factor receptor typeⅡ(IGF-ⅡR) in bovine oocytes and preimplantation embryos by using RT-PCR and immunofluorescence label technique.IGF-Ⅰ/Ⅱsupplemented in maturation and development medium with different concentration were used to evaluate their effects on oocyte maturation and embryo development.The function of IGF-ⅠR on preimplantation embryo development was further discussed by RNA interference (RNAi). 1.Expression of IGF-Ⅰ/Ⅱand IGF-ⅠR/ⅡR mRNA in bovine oocyte and preimplantation embryoIGF-Ⅰ/Ⅱand IGF-ⅠR/ⅡR mRNA expression in oocytes and preimplantation embryos was detected by using RT-PCR.The results indicated that there was expression of IGF-Ⅱand IGF-ⅠR/ⅡR mRNA in immatured oocytes,matured oocytes and embryos.The IGF-ⅠmRNA was not detected in any stage of embryos and oocytes.2.Expressions of IGF-Ⅱand IGF-ⅠR/ⅡR protein in bovine oocytes and preimplantation embryosThe protein's localization of IGF-Ⅱ,IGF-ⅠR and IGF-ⅡR in bovine preimplantation embryo were first observed in this research.The results showed that expression of IGF-Ⅱand IGF-ⅠR/ⅡR was detected in immatured and matured oocytes,cumulus cells,fertilized oocytes and different stage preimplantation embryos.The expression patterns of IGF-Ⅱand IGF-ⅠR/ⅡR in embryos were similar. They were near the membrane in oocytes and 2-cell stage embryos,and in the whole cell in COC's cumulus,8-cell stage embryos and morula,and they can be observed in trophectoderm(TE) cells but not in inner cell mass(ICM) at the blastocyts stage.3.Effects of IGF-Ⅱon bovine oocyte maturationThe effect of IGF-Ⅱsupplemented in maturation medium on embryo development was observed first in this research.COCs were cultured in M199+ 0.6%BSA supplemented with 0,10,20,50 and 100ng/ml IGF-Ⅱrespectively for maturation.After in vitro fertilization and culture,the rates of embryo development and the dead cells in blastocysts were counted.The results indicated that there was no significant difference in cleavage rates between treated groups and control. There was a significant difference in 8-cell stage embryo development rates between 20ng/ml treatment(58.2%) and control(44.5%,P＜0.05).And the best blastocyst development rate was obtained in 20ng/ml treatment(37.0%) compared with others(25.0%,33.3%,24.8%,21.2;P＜0.05).Cell number of blastocysts in 20 ng/ml treatment(126) was significant higher than that in control(103,P＜0.05). There was no significant difference on the rates of dead cells in blastocysts in different treatments.4.Effects of IGF-Ⅰand IGF-Ⅱon bovine preimplantation development4.1 Effects of IGF-Ⅰon bovine preimplantation embryo developmentEmbryos derived from IVF were cultured in SOF+PVA added with 0,1,5,10 ng/ml IGF-Ⅰrespectively.The results showed that the rates of 8-cell stage embryos and blastocyst were 55.1%,55.1%,62.9%,60.9%and 18.9%,19.6%,24.2%,22.7% respectively.There was no siginificant difference between different treatments.The best development rate was observed in 5ng/ml treatment.4.2 Effects of IGF-Ⅱon bovine preimplantation embryo developmentThe effect of IGF-Ⅱsupplemented in developmental culture medium on embryo development was observed first in this research.Embryos derived from IVF were cultured in SOF+PVA added with 0,50,100,150ng/ml IGF-Ⅱrespectively.The rates of 8-cell stage embryo and blastocyst were 63.6%,68.9%,61.3%,63.9%and 19.2%, 21.7%,30%,25.9%respectively.There was no significant difference on rates of 8-cell stage embryo between different treatments.The rate of blastocysts in 100 ng/ml treatment was higher than that in control(P＜0.05).5.Effects of IGF-ⅠR siRNA injection on bovine preimplantation embryo development5.1 IGF-ⅠR siRNA design and effect detectionAs the first time,siRNA was used on bovine embryo development in this research.IGF-ⅠR siRNA designed by software was used to detect the interference effect.Immatured oocytes were injected with IGF-ⅠR siRNA or control siRNA,and then cultured in M199 added with maturation inhibitor.After 6 and 24hours,the IGF-ⅠR mRNA was detected by real-time PCR.The result showed that the IGF-ⅠR mRNA reduced to 72%and 54%.The mRNA and protein reduced both in cleavage embryos injected with IGF-ⅠR siRNA 24hours later.4.2Effects of IGF-ⅠR siRNA injection on preimplantation embryo developmentThe fertilized oocytes with no injection(control) or injected with control siRNA and IGF-ⅠR siRNA were cultured in culture medium respectively.The rates of 8-cell stage embryo and blastocyst in the all treatments were 48.8%,46.7%, 38.9%and 35.4%,33.0%,23.3%respectively;and the cell number of blastocysts were 116,81,64 respectively.The results showed that there was no significant difference on 8-cell satge embryo rates among them.Blastocyst rates of IGF-ⅠR siRNA injected treatment was siginificant lower than that of other two treatments (P＜0.05).Cell number of blastocysts in no injected treatment was significant higher than that of injected treatment(P＜0.05),and it was significant higher in injected IGF-ⅠsiRNA treatment than that of injected control siRNA treatment.The results in this research suggested that the expression of IGF-Ⅱand IGFIR/ⅡR mRNA and protein were detected in bovine oocytes and preimplantation embryos.But IGF-ⅠmRNA could not be detected.Added IGF-Ⅱin maturation and culture medium could improve the rates of embryo development.The cell number of blastocyst was higher when IGF-Ⅱwas added in maturation medium,but there was no difference on the rate of dead cell in blastocyst.Embryo development rates and cell number in blastocysts were decreased by IGF-ⅠR siRNA injection.