The role of the IGF system in somatic cell and oocyte development during early bovine follicular development

The insulin-like growth factor (IGF) family has been identified as having a key role in regulating follicular growth in mammals. IGFs have been shown to act as survival factors that stimulate somatic cell proliferation and differentiation, as well as being involved in the final stages of oocyte maturation. Insulin-like growth factor binding proteins (IGFBPs) regulate the availability of IGFs as they can sequester extracellular IGFs and hence reduce their bioavailability. The aims of this thesis were to use a serum-free culture system to investigate the role of the IGF system during early bovine follicular development. The study focused on investigating the actions and regulation of IGF in three key stages of follicle growth. These key stages were: primordial follicle initiation, the transition from preantral to early antral stages and early antral follicle growth. To begin with, this study looked at the effect of IGF-I on primordial initiation and preantral follicle growth and oocyte health, in the presence or absence of androgen. Androgens can have a direct action via androgen receptors (AR), but it has also been suggested that androgens may have an indirect action by influencing the actions of IGF-I through its IGF receptors. No effects of IGF-I on primordial follicle initiation or activated follicle growth were evident, but IGF-I that was not regulated by IGFBPs was found to have a negative effect on the oocyte health of follicles after 6 days of culture. Hence, during the early stages of follicle development regulation of the bioavailability of IGF is crucial to maintain the health of the oocyte. AR immunoreactivity was found in the oocyte and granulosa cells of bovine preantral and antral follicles. Supplementation of culture medium with androstenedione, either on its own or in combination with IGF-I, also failed to affect follicle activation and growth. So the detrimental effect of IGF-I on oocyte health was not altered by the presence of androstenedione. Previous in vitro preantral follicle culture studies have demonstrated a positive effect of IGF-I on follicle growth but a negative effect on oocyte size and granulosa differentiation. In the present study a 6 day culture of bovine early antral follicles in the presence of IGF-I was found to stimulate both follicle proliferation in the early stages of development and differentiation at all developmental stages, as exhibited by oestradiol production. Furthermore, these effects were found to occur in a dose and stage dependent manner. Additionally, oocyte health was improved by the addition of recombinant IGF-I in the more mature antral follicles. These results highlight the importance of follicle developmental stage when deciding the best in vitro culture conditions. It is important to investigate the mechanisms involved in regulating the expression of IGFBPs at different stages of follicle growth, as changes in their expression will affect the bioavailability of IGF to its receptors. IGFBP-2 mRNA and protein were shown to be expressed in the oocyte and granulosa cells, but not theca cells, of early antral follicles. In addition, IGF-I was found to influence the expression of IGFBP-2 in granulosa cells and oocytes of the largest size range of early antral follicles. A decrease in IGFBP-2 immunoreactivity was observed when follicles were cultured in the absence or in a low dose of IGF-I compared to follicles cultured in a high dose of IGF-I. These results suggest that IGF-I may regulate the expression of IGFBP-2 through a negative feedback mechanism, which again is stage dependent. The biological actions of IGF depend in part on the ability of specific proteases to break down the IGF/IGFBP complex. The secretion of proteases capable of degrading IGFBP-2 by different bovine follicular compartments was identified, and the effect of IGF-I or follicle stimulating hormone (FSH) on modulating IGFBP-2 proteolytic degradation was also investigated. Proteolysis of IGFBP-2 caused by the incubation of IGFBP-2 with oocytes was not detected. However, incubation of IGFBP-2 with granulosa cells did cause a small level of IGFBP-2 degradation. IGF-I and FSH were not found to enhance or inhibit the degradation of IGFBP-2 after incubation with bovine oocytes or granulosa cells. The use of a serum-free culture system in this study has improved our understanding of how the complex IGF system is regulated throughout bovine follicular development. It has highlighted that the control of IGF availability to a growing follicle is governed by different levels of regulation, such as the level of expression and degree of proteolysis of IGFBPs, which are follicle stage dependent. A fuller understanding of the mechanisms that control the access and regulation of local growth factors will allow us to move closer to developing an in vitro culture system capable of producing large numbers of viable oocytes for use in reproductive technologies.