The Expression And Function Of Interleukin-6and Its Recptor In Development Of In Ovine Preimplantation Embryos

In recent years, the transcription and expression patterns of cytokines and their receptors in mammalian embryos and the dams during embryonic development, have been extensively studied. Significant progress has then also been made toward understanding the functions and regulation during embryogenesis. Interleukin-6(IL-6) is a multifunctional cytokine, being involved in the regulation of ovulation, implantation, embryonic development, and other reproductive processes under normal physiological conditions. In this study, the expression of IL-6and IL-6R was evaluated during early ovine embryonic development, using immunohistochemistry, real-time quantitative PCR and protein blotting. The impacts of IL-6on embryonic development were evaluated via the supplementation with IL-6, using different concentrations in the embryo culture medium. The roles of IL-6and IL-6R in early ovine embryonic development were further investigated through RNAi-mediated inhibition. The present study could thus provide an experimental basis for the study of the mechanisms underlying early ovine embryonic development.1. The expression patterns of IL-6and IL-6R during the development of the ovine oocytes and IVF embryosThe real-time quantitative PCR results illustrate the expression patterns of the IL-6and IL-6R mRNAs at different developmental stages of the ovine oocytes and IVF embryos. IL-6and IL-6R were expressed significantly at higher levels in the maturation around4h, and then decreased dramatically. However the level slightly elevated at16h-20h. IL-6R was expressed at a high level in the early maturation oocytes from Oh to8h and significantly higher levels at4h oocytes, and then showed lower expression levels. IL-6was expressed at a low level in the2-cell embryos-which level was set at1, and reached its peak expression at the morula and blastocyst stages. IL-6R showed a similar expression pattern to IL-6, with a lower expression level at the2-cell stage and significantly higher levels at the morula and blastocyst stages.The immunohistochemistry results demonstrate the expression of IL-6and IL-6R at different developmental stages of the ovine oocytes and IVF embryos. The cellular distribution of IL-6and IL-6R expression was similar in the ovine oocytes at each maturation stages. Their expression was mainly localized on the cell membrane, with little expression in the cytoplasm and none in the nucleus. However in the IVF-derived ovine embryos, the expression of IL-6was mainly localized on the cytoplasm, IL-6R was concentrated much more on the cell membrane. Protein blotting showed that IL-6and IL-6R were expressed in the IVF-derived ovine embryos at each developmental stage. The expression patterns were consistent with the levels of transcription. IL-6protein showed higher expression levels at4h-8h and then decreased at12-16h. However the level slightly elevated at20h-24h of the immature stage. The level of IL-6R protein is significantly higher at4h, and then showed lower expression. Both proteins showed lower expression levels at the early embryonic stages, and significantly higher levels at the morula and blastocyst stages. The levels of IL-6and IL-6R were higher at the blastocyst than at the morula stage. In conclusion, IL-6may influence the initiation of oocytes meiotic and the subsequent meiotic process, thereby act on the ovine oocytes maturation.2. Effects of IL-6on embryonic developmentIL-6was added to the oocyte maturation medium at a concentration of10,50and100ng/mL respectively, the samples without exogenous IL-6were used as a control, and in another control10%ovine estrus serum (OES) was instead of BSA The results showed that the maturation rates significantly increased in the10ng/mL IL-6group compared with the BSA control group (P<0.05), and were similar to the OES control group, but did not affect the rates of development of the subsequence IVF ovine embryos. No significant difference was observed in the50ng/mL IL-6groups. While the maturation rates, cleavage rates, blastocyst rates, and hatching blastocyst rates significantly decreased in the100ng/mL IL-6group.Recombinant IL-6was added to the development culture medium at a concentration of10,50, or100ng/mL, while samples without exogenous IL-6served as the control. The cleavage rate significantly increased in the10ng/mL IL-6group (p<0.05), while the cleavage rate, blastocyst rate, and hatching blastocyst rate significantly decreased in the100ng/mL IL-6group (p<0.05). This finding suggests that low concentrations of IL-6may promote embryonic development._However, indicating the toxicity of high concentrations bf IL-6on embryonic development. After the micro-injection of IL-6dsRNA, no significant differences in the cleavage rate or blastocyst rate were recorded between the siRNA-injected group, the Non-Targeting siRNA-injected group, and the uninjected group.3. Effects of supplementation IL-6to the development culture medium on the relative expression levels of IL-6and IL-6R.IL-6was added to the development culture medium at a concentration of10,50, or100ng/mL. The IL-6mRNA level was significantly higher in the embryos of the IL-6groups, compared to the control group (p<0.05). The IL-6R mRNA level did not increase significantly, until the morula and blastocyst stages (p<0.05) were reached. Protein blotting experiments showed that compared to the control group, IL-6and IL-6R protein levels were increased in the embryos of the IL-6groups, with the most significant increase being in the50ng/mL IL-6group.4. Effects of micro-injected IL-6siRNA on the relative expression levels of IL-6and IL-6RThe IVF ovine oocytes were micro-injected with IL-6siRNA or Non-Targeting siRNA and cultured along with the uninjected control oocytes. Real-time PCR showed that compared to the control group, the IL-6mRNA level decreased by69.6%in the IL-6siRNA-injected morula embryos. The levels of IL-6and IL-6R mRNA decreased by87.5%and71.8%in the IL-6siRNA-injected blastocyst embryos, respectively. The IL-6and IL-6R mRNA levels of Non-Targeting siRNA-injected group were similar to those of the control (uninjected) group. Meanwhile, the mRNA levels in the β-actin internal reference gene were similar between groups. These results suggest that the in vitro-synthesized siRNA can effectively and specifically decrease the expression of the target gene, without affecting the mRNA expression of other reference genes.Protein blotting results illustrate that the micro-injection of siRNA effect was especially evident in the8-cell and blastocyst stages. IL-6R protein levels decreased as the IL-6level decreased. Therefore, the injection of IL-6siRNA into IVF ovine oocytes can decrease the mRNA and protein levels of IL-6and IL-6R, until the blastocyst stage.Therefore, the mRNA and protein expression of IL-6and IL-6R was effectively decreased.In summary, IL-6and IL-6R were present at all the developmental stages of IVF-derived ovine embryos and exhibited distinct expression patterns. The addition of IL-6in the oocyte culture medium at10ng/mL promoted oocytes nuclear maturation, but did not affect the subsequence development of the IVF ovine embryos. The supplementation of the embryonic culture medium with10ng/mL IL-6promoted the cleavage of early stage ovine embryos. The micro-injection of IL-6siRNA also effectively reduced the embryonic mRNA and protein expression of IL-6and IL-6R, without affecting the development rate of the IVF-derived ovine embryos. IL-6as such likely plays an important role in the ovine oocytes maturation and early embryos development..