Effect of cytokine supplementation on the development and quality of in vitro cultured sheep oocytes and embryos

Abstract

The optimization of pre-implantation embryo culture conditions is essential for newlineimproving the use of embryo technologies for breeding and production in livestock. newlineDespite the advances in in vitro embryo production in many species, use of this newlinetechnology is limited due to the lower developmental competence of in vitro derived newlineembryos as compared to the in vivo derived counterparts. However, optimization of newlineindividual culture systems for oocytes and embryos is an important goal, an area of newlineintensive current research in both animal and human. The current study aimed to assess newlinethe effect of exogenous cytokines on the maturation and development of ovine oocytes newlineand embryos, to develop a specific culture system for in vitro production of quality sheep newlineembryos. newlineInterleukin-7 (IL-7) is an important cytokine that is essential for cell survival and newlineproliferation of lymphocytes. Insulin like growth factor-1 (IGF-1) is a well-recognized newlinegrowth factor and it is also considered as a cytokine. The IL-7 and IGF-1 were newlinesupplemented to the in vitro maturation (IVM) medium to assess their effect on the newlinematuration of ovine cumulus-oocyte complexes (COC). It was evident that the newlinesupplementation of 1ng/ml of IL-7 to IVM medium significantly improved the newlinematuration rate of ovine COC, but the higher doses (2, 5 and 10ng/ml) of IL-7 were newlinefound detrimental. Further, the IL-7 (1ng/ml) mediated improvement in the oocyte newlinecompetence resulted better in vitro embryo development (cleavage and formation of 4-8 newlinecell embryos, morula and blastocysts) following in vitro fertilization (IVF) and culture newline(IVC). It was also demonstrated in the current study that the beneficial and detrimental newlineeffects of IL-7 were mediated through its influence on the production of intracellular newlinereactive oxygen species (ROS). The supplementation of IGF-1 (100ng/ml) to IVM newlinemedium also significantly improved the maturation rate of COC and in vitro embryo newlinedevelopment up to the stage of morula following IVF and IVC in ovine. newlineInterleukin-6 (IL-6) is a multifunctional cytokine that regulates various aspects of newlinethe immune response, acute phase reaction and haematopoiesis and has some functional newlineredundancy with IL-11 and LIF. It is a glycoprotein, produced by multiple types of newlinelymphoblasts and non-lymphocytic cells that participate in a variety of biological newline newlinefunctions. In the current study, the effect of exogenous IL-6 on the in vitro development newlineof ovine embryos was assessed. It was evident that the supplementation of IL-6 (10ng/ml) newlineto IVC medium during the 72 to 192h of culture significantly improved the blastocyst newlineformation, but the higher dose of IL-6 (25ng/ml) was found detrimental for embryo newlinedevelopment. Further, the supplementation of IL-6 (10ng/ml) to IVC medium during the newline0 to 192h of culture was also found detrimental for the in vitro development of ovine newlineembryos. The results suggest that 10ng/ml of IL-6 is beneficial when supplemented at the newlinelater stages of embryo development. newline newlineThe assessment of gene expressions in ovine COC matured with 1 and 5ng/ml IL- newline7 supplementation revealed that 1ng/ml IL-7 mediated increase in the maturation rate was newline newlinedue to the favourable level of intracellular ROS and antioxidant defence mechanisms. In newline newlinecontrast, the detrimental effects of 5ng/ml IL-7 were arbitrated through the ROS- newlinemediated oxidative stress, compromised antioxidant mechanism and increased apoptotic newline newlinesignalling. Gene expression analysis in the matured oocytes and cumulus cells treated newlinewith 1ng/ml of IL-7 indicated that the increase in oocyte competence by IL-7 was newlinemediated through the stimulated TGFand#946; and PI3K/AKT/MTOR signalling and the newlinemaintenance of appropriate levels of antioxidant mechanisms in ovine oocytes and newlinecumulus cells. newlineThe assessment of gene expressions in ovine oocytes matured with 100ng/ml of newlineIGF-1 treatment revealed that IGF-1 improved the development ability of ovine oocytes newlineby stimulating the PI3K-AKT signalling pathway that in turn enhanced oocyte quality newlinethrough the BCL2 mediated protection from apoptosis. newlineThe assessment of gene expressions in the ovine 8-16 cell embryos and compact newlinemorula revealed that the favourable level (10ng/ml) and time (72 to 192h of IVC) of IL-6 newlinesupplementation enhanced the JAK/STAT signalling pathway in the 8-16 cell embryos newlineimmediately after supplementation. Further, at the compact morula stage, IL-6 stimulated newlinethe expression of pluripotency related genes and reduced the expression of JAK-STAT newlinesignalling pathway related genes. The results indicated that the IL-6 mediated increase in newlinethe development of ovine embryos was mediated through the expression of JAK/STAT newlinesignalling and pluripotency related genes. newline newlineFrom the findings of the dissertation it is concluded that the supplementation of newlineIL-7, IGF-1 and IL-6 to the maturation and culture media at suitable dose and time point newlinecan significantly enhance the developmental competence of ovine COC and embryos. newlineThe supplementation of 1ng/ml IL-7 and 100ng/ml IGF-1 to IVM medium improves the newlinedevelopmental ability of ovine COC and can be used for in vitro maturation of ovine newlineCOC for better outcome. The supplementation of 10ng/ml IL-6 to IVC medium during newlinethe 72 to 192h of in vitro culturing improves the yield of ovine blastocysts, and therefore newlinecan be used for the in vitro production of ovine embryos with better efficiency. newline