THE ROLE OF INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR SIGNALLING IN THE MOUSE EMBRYO DURING PREIMPLANTATION DEVELOPMENT AND EARLY IMPLANTATION

Abstract

The use of assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) is increasing and today close to 4 percent of babies born in Australia, are as a result of ART. IVF procedures involve the culture of resultant embryos in medium that routinely lacks the growth factors that are present in the reproductive tract. Cultured embryos develop at a slower rate and have higher levels of developmental arrest, with fewer than 50% of embryos reaching the blastocyst stage. Furthermore, once an embryo is transferred back into the uterus, it faces the hurdle of implantation, with implantation failure being a major cause of IVF failure. This thesis examines whether the addition of growth factors to the embryo culture medium can increase blastocyst development and adhesion competency, using mouse embryos as a model system. In particular, the effect of insulin-like growth factor 1(IGF1) and insulin-like binding protein 3 (IGFBP3) on preimplantation mouse embryo development in vitro and the role of IGF1 on implantation in vitro was examined. In the present study, the culture of preimplantation embryos in the presence of a physiological concentration of IGF1 improved development from compaction onwards, resulting in improved blastocyst development. Conversely, high levels of IGF1 negatively impacted on development by decreasing hatching probably due to these high levels of IGF1 causing IGF1 receptor (IGF1R) down-regulation and apoptosis in the mouse embryo, as shown previously. Blocking the IGF1R with a neutralising antibody was shown to decrease blastocyst development, hatching and cell numbers and to increase apoptosis. Furthermore, treatment of blastocysts with IGF1 caused phosphorylation of Akt, which regulates cell survival by activating anti-apoptotic pathways. Therefore, IGF1 may act as a survival factor in the preimplantation embryo. During early implantation integrins accumulate on the surface of the blastocyst and endometrium and these interact with each other via extracellular matrix proteins such as fibronectin. These interactions are important for the attachment of blastocysts to the endometrium. In the present study treatment of blastocysts with IGF1 increased fibronectin on the surface of the blastocyst via activation of the Phosphoinositide 3 Kinase (PI3K) pathway. As a result, blastocysts had increased attachment to cultured uterine epithelial cells and increased outgrowth. In addition to IGF1, the reproductive tract produces IGFBP3, which is also thought to improve development of the embryo. However, to the best of our knowledge this is the first study to examine the effect of exogenous IGFBP3 on embryo development in vitro. IGFBP3 caused an increase rate of progression of embryos through the early stages of division (5-8cells) and activation of Akt and ribosomal protein S6 (rpS6) proteins as well as induction of calcium signalling. In the present study, it appears that IGFBP3 signalling in the embryo requires the IGF1R, as the use of an IGF1R neutralising antibody blocked IGFBP3 from enhancing early stages of division and the induction of calcium signalling. In other cell types, IGFBP3 signals through the IGF1R following a transactivation event involving the Sphingosine 1-Phosphate (S1P) pathway. The complex interactions and signalling of IGFBP3 are beginning to emerge in a number of different cell types and further investigation of IGFBP3 function is required in the embryo. As growth factors are generally absent from embryo culture media there is a potential avenue for the improvement of embryo culture and ART outcomes by addition of IGF1 and or IGFBP3 to the culture medium. The requirements of the embryo are complex and understanding the role of growth factors in embryo development is essential in order to optimise embryo culture and develop culture media for use in ART.