The Expression And Function Of EGF, TGFα And EGFR In Ovine Preimplantation Embryos Development

There is increasing evidence that development of preimplantation embryo is regulated by maternally and embryonically derived growth factors. Studies have shown a number of growth factors and their receptors are expressed in embryos and reproductive tracts. Furthermore, the fate and metabolism function of embryo cells can be improved by growth factors supplemented in culture medium. Growth factors act on embryo by autocrine, paracrine and intracrine to regulate the cell function. This study aimed to assess location and relative amounts of transforming growth factor alpha(TGFα), epidermal growth factor(EGF) and their receptor (EGFR) in ovine oocytes, preimplantation embryos and female reproductive tracts by using immunohistochemical and RT-PCR technique. Maturation and development medium supplemented with different concentration of TGFαand EGF were used to evaluate the effects on oocyte maturation and embryo development. The function of TGFαand EGFR on preimplantation embryo development was further discussed by RNA interference(RNAi).1. Expression of EGF, TGFαand EGFR mRNA in oocyte, preimplantation embryo and female reproductive tract by RT-PCR1.1 Expression of EGF, TGFαand EGFR mRNA in oocyte, preimplantation embryoEGF mRNA was not detected in oocytes and preimplantation embryos. There were expression of TGFαand EGFR mRNA in unmatured oocyte, matured oocyte, embryo and the expression of TGFαand EGFR in matured oocytes were comparatively more abundant than that in unmatured oocytes. Maternal TGFαmRNA was gradually reduced from oocyte to 8-cell stage and maintaining lower TGFαmRNA level was to morula stage, assumably TGFαmRNA transcription was begun from 8-cell stage with the zygote genome activation. The EGFR mRNA was gradually reduced from oocyte to morula stage and increased in blastocyst.1.2 Expression of EGF, TGFαand EGFR mRNA in female reproductive tractEGF mRNA was not detected in ovary, oviduct and uterus epithelium, maternal placental cotyledon. Transcript level of EGFR mRNA was higher in uterus epithelium after ovulation 7-8 day and cotyledon than that in ovary cells and oviduct epithelium. TGFαmRNA was more abundant in oviduct than that in uterus epithelium and cotyledon. It was presumed that the higher level of TGFαin oviduct could be beneficial to preimplantation embryo development, and EGFR mRNA in uterus and cotyledon could further promote the proliferation and differentication of epithelium, which provide the suitable micro-environment for embryo implantation and gestation.2. Expression of TGFαand EGFR protein in oocytes, preimplantation embryos and reproductive tracts by immunohistochemy2.1 Expression of TGFαand EGFR protein in oocytes and preimplantation embryosThe results showed that expression of EGFR and TGFαwere in immatured and matured oocytes, cumulus cells, fertilized oocytes and different stage of preimplantation embryos. However, the staining for TGFαslightly increased in the fertilized oocytes as compared to immature and mature oocytes. The relative amounts of EGFR in inner cell mass(ICM) was less than that in trophectoderm(TE) cells at the blastocyts stage. TGFαwas mainly detected in the cytoplasm close to the membrane in all cells of embryo at different stage and abundant in both ICM and TE cells at blastocyst stage. The patterns of expression of EGFR and TGFαin embryos in vivo were similar to the embryos in vitro produced.2.2 Conformations of EGFR in oocytes and embryosTwo conformations of EGFR(wild 170KD and variation 145KD) were detected in oocytes and preimplantation embryos by western-blotting. Most of EGFR in unmatured and matured oocytes was in variation, and there was obviously more expression of variation in matured oocytes than that in unmatured oocytes. Furthermore, EGFR expression levels of two conformations were increased from oocytes to embryos of day 4.2.3 Expression of TGFαand EGFR protein in reproductive tractsThe distinct staining of TGFa was observed in the granulose cells of growing follicles and oviduct luminal epithelium on day 0, 2-3, 4-5 and 6-7 after ovulation. Strong staning was observed in the luminal and glandular epithelium of uteri on day 6-7. However, no staining was detected in degraded follicles. The stainig of EGFR was detected in the granulose cells of primary follicles and luminal epithelium of uteri on day 6-7, but no distinct staining can be observed in the oviduct luminal epithelium on day 0, 2-3, 4-5 and 6-7 after ovulation.3. Effects of EGF and TGFαon oocyte maturationOvine oocytes cultured in TCM199+serum, TCM199+BSA+EGF, TCM199+BSA+TGFαand TCM199+BSA, respectively were investigated by immunofluorescence and orcein staining. The results showed that the rates of mature oocyte were 75.2％, 73.1％, 69.8％, 63.5％and oocyte at telophase of MI were 16.3％, 15.9％, 16.9％, 27.0％, respectively. The rates of maturation of oocytes cultured in TCM199 with serum, EGF and TGFαwere significantly higher than that of with BSA team (P＜0.05) and the number of oocyte stayed at telophase of MI in TCM199 with serum, EGF and TGFαteam were significantly less than that with BSA team(P＜0.05); Significant higher rates (66.6％, 66.6％, 73.6％) of normalα-tubulin distribution in oocytes cultured in TCM199+ serum, EGF and TGFαwere compared to that of TCM199+BSA(43.3％) (P＜0.05). The rates of oocyte with cortical granules in cortex were 58.8％, 33.9％, 54.7％and 47.9％respectively, there was significant difference between oocytes cultured with serum, EGF and BSA(P＜0.05). In conclusion, TGFαand EGF can promote the oocyte nuclear transition from telophaseⅠto metaphase of meiosisⅡ, and improved the expression and distribution ofα-tubulin during the ovine oocytes maturation; EGF and serum could promote oocyte cytoplasm maturation. The results suggested that EGF and TGFαmight substitute some substance in serum to improve the quality of oocyte nucleus maturation in vitro, but EGF might be more functional than TGFαto promote the maturation of ovine oocyte ooplasm.4. Effects of EGF and TGFαon preimplantation development4.1 Effects on development of preimplantation embryo cultured with EGF and TGFαEmbryos were cultured in Chemically-defined culture medium supplemented with different concentration of TGFαand EGF. The results showed that the rates of 8-cell stage embryos and blastocysts cultured with 5ng/ml TGFα, 25ng/ml EGF, 5ng/ml TGFα+25ng/ml EGF and control were 88.0％, 75.8％, 76.8％, 77.8％and 26.7％, 19.2％, 23.5％, 18.6％, respectively. The developmental rates of 8-cell stage embryos cultured with 5ng/ml TGFa was increased as compared to other treatments (P＜0.05) and there was no significant difference between the rates of blastocyst of respective teams. In addition, the average cell numbers in blastocysts were 73, 74, 70 and 74 respectively. There was no significant difference in the number of cells in blastocyst stage as compared with different treatments (P＞0.05). The rates of dead cell in blastocyst were 4.3％, 18.5％, 10.4％and 23％respectively, and TGFa alone enhanced cell survival rates(P＜0.01). From above all, it was concluded that TGFa could reduce dead cells of blastocyst in vitro produced and improve in vitro development of ovine preimplantation embryos.4.2 Effects of dsRNA injection on gene expression of preimplantation embryosEGFR dsRNA, TGFαdsRNA and water were injected into oocyte respectively. Control team was no injection. The results indicated the amounts of EGFR mRNA and TGFαmRNA in matured oocyte were obviously decreased compared to injection with water and control team. However, EGFR mRNA expression level was significantly decreased in oocytes injected with TGFαdsRNA. The EGFR protein in matured oocytes injected with TGFαdsRNA was higher than that of water injection team by wester-bloting. The results of immunohistochemistry shown that the expression levels of EGFR and TGFαin embryos after dsRNA injection were decreased compared to that of control and water injection. The mRNA expression levels ofβ-actin, connexcin-43 and 45 were not influenced by dsRNA and water injection.4.3 Effects of TGFαdsRNA injection on preimplantation embryo developmentOocytes were treated with: injected with TGFαdsRNA, injected with water, no injection(control), and then were cultured for maturation, fertilization and development in vitro, the oocytes injected with TGFαdsRNA were cultured in development medium without and with 10ng/ml TGFαrespectively. The rates of development to 2-cell and blastocyst in the all treatments were 48.1％, 56.3％, 56.4％, 73.8％and 12.9％, 18.9％, 16.1％, 32.2％respectively. There was significantly difference between the no injection and injection treatments(P＜0.05) and no difference between injection teams.Cells of the blastocyst in control and water injection treatments were nearly no apoptosis in TE and frequence of apoptosis in ICM was comparatively low. Frequence of apoptosis in TE and ICM cells was obviously ascendant in the blastocyst derived from the treatment of TGFαdsRNA injection. However the apoptosis was reduced in TE when TGFαwas supplied in culture medium, but no influence on the ICM. These results shown that there was no influence on the rates of embryo development but increased the number of apoptosis cell when TGFαreduced. Supplement with TGFαrescued the cell apoptosis in TE and improved the embryo development.4.4 Effects of EGFR dsRNA injection on embryo developmentThe developmental rates of fertilized oocytes, which were treated with: injected with EGFR dsRNA, injected with water, and no injection(control), to 2-cell stage, blastocyst and hatched blastocyst were 55.8％, 69.7％, 70.0％; 18.1％, 22.7％, 30.7％and 18.2％, 33.7％, 34.1％respectively. There was significant difference between EGFR dsRNA injection treatment and the other two treatments on the rates of cleavage, blastocyst and hatched blastocyst(P＜0.05) and no differenc between water injection and control group(P＞0.05). The average of cell numbers in blastocyst were 80, 90, 94 and 22, 24, 23 in ICM and 58, 66, 71 in TE respectively, there was no difference among them(p＞0.05). These results indicated that embryo development to 2-cell stage, blastocyst and hatched blastocyst could be improved by the EGFR signal pathway.