Study On Expression Of IGFs And Their Receptors In Ovine Preimplantation Embryos And Reproductive System

Insulin-like growth factors (IGFs) family is one kind of multi-purpose cell proliferation regulatory factor, which mainly include IGF-â… , IGF-â…¡and their receptors IGF-IR, IGF-â…¡R. These factors act as a key role in cell differentiation, reduplication, embryonic growth and development, individual growth and development. The aim of this study was to illuminate expression patterns and differences of IGFs in ovine IVF embryos and in vivo embryos. First of all, purified RT-PCR product from IGFs genes was cloned into pGEM-T easy Vector. After PCR and restriction endonuclease digestion analysis of recombinant plasmid, the cDNAs was sequenced and the fragment was identified as IGFs cDNAs. Then, the expression of IGF-â… , IGF-IR, IGF-â…¡ and IGF-â…¡R in ovine oocytes, in vitro and in vivo fertilized preimplantation embryos in different developmental stages were detected in mRNA and protein levels with indirect immunofluorescence staining, hybridization in situ, laser scanning confocol microscopy and RT-PCR. At last, the expression of those four factors in sheep reproductive system was tested using immunostaining. All the results were listed below:1. Results of hybridization in situ indicated:Expression of IGF-â…  mRNA in immature oocytes, in vitro matured oocytes, IVF embryos in 2-cell, 4-cell, 8-cell stage and blastocysts were not detected, as same as the results detected in in vivo matured oocytes and embryos.The detected expression of IGF-â…¡ mRNA in matured oocytes and embryoscultured in vitro were as similar as that in vivo. IGF-II mRNA located in cell plasma in immatured and matured oocytes, and largely located in membrance of apical surface of embryos from 2-cell to 8-cell stage in a disproportional way, and also expressed in trophectoderm and inner cell mass of blastocyst.IGF-IR and IGF-IIR mRNA were detected in oocytes and in vitro and in vivo embryos. Immunostaining largely located around germinal vesicle of oocytes, and in membrance of apical surface of embryos from 2-cell to 8-cell stage. The expression was detected only in trophectoderm but not in inner cell mass at blastocyst stage.2. Results of RT-PCR indicated that 262bp band of IGF-IR, 304bp band of IGF-II, 415bp band of IGF-IIR were amplified respectivly with the IGFs specific primer in oocytes and in vitro embryos. The band corresponded to IGF-I was not detected. The results further confirmed that it was IGF-IR, IGF-II, IGF-IIR but IGF-I that expressed in ovine oocytes and in vitro embryos.3. Immunofluorescence staining of results:Proteins of IGF-I were located in the regions under oocytes membrance but not expressed in IVF embryos of 2-cell, 4-cell, 8-cell stage and blastocysts. The staining results showed that IGF-I were expressed in in vivo matured oocytes and embryos, but the model of expression had no difference between in vivo and vitro matured oocytes. The expression of IGF-I was observed only in part of cytoplast close to membrane and on apical domain of blastomere at 4 and 8-cell stage embryos, furthermore both in ICM and TE at blastocyst stage.The expression of IGF-II protein was observed in similar situation from oocyte to different stage of in vivo and in vitro preimplantation embryo. In unmatured oocytes the distribution of IGF-II was in membrane and cytoplast closed to membrane. Expression of IGF-II was detected clearly under the membrane near thenucleus in matured oocytes, and the fluorescence staining was much slighter on the contrary side. The fluorescence staining of IGF-II was on the apical domain of blastomere in 2-8cell stage embryos and could be observed both in ICM and TE at blastocyst stage.IGF-IR and IGF-IIR were detected in oocytes, in vitro and in vivo embryos. The staining of IGF-IR and IGF-IIR were observed in oocyte and every stage of preimplantation embryos. The location and density of staining were no difference between in vitro and in vivo embryos. Their expression mostly distributed on the whole membrane in oocytes, and on the apical domain/under membrane in 2-8cell stage embryos. At blastocyst stage, the staining could be observed in TE but in ICM.4. The expression of IGFs was detected in ovary/uterus/oviduct during estrus cycle in same model. The proteins of IGFs mostly distributed in granular cell of follicle and the staining was weak in interstitial cells in ovary. The staining was positive in oviductal epithelia cells. The staining density in epithelia and glandular cells was stronger than that in lamina propria of uterus. Additionally, The mRNAs of IGFs were detected by RT-PCR in ovary, uterus and oviduct.