The influence of insulin -like growth factor-I on maturation and development in equine oocytes.

摘要

In vitro production of embryos in the horse has been of limited success. One of the problems is due at least in part to inadequate IVM procedures. Gonadotropins are the main regulators of nuclear maturation of oocytes; however, recent observations suggest that gonadotropins are only part of a complex system of autocrine and paracrine factors that may influence maturation. Growth factors have a regulatory role in ovarian function. Among them, insulin-like growth factor-I (IGF-I) has been implicated in the regulation of follicular somatic cell functions stimulating oocyte maturation in several species. The first objective was to determine the relevance of IGF-I during equine follicular development by evaluating the presence of IGF-I, insulin-like growth factor binding proteins (IGFBP), IGF-I receptors (IGFR) and IGF-I mRNA levels. Follicular growth was associated with a decrease in IGFBP-3 concentration. The IGFR was localized by immunocytochemical studies in the oocyte, cumulus and granulosa cells. Finally, the levels of IGF-I mRNA in the large follicle group demonstrated a significant increase compared with the others (p < 0.05). The second objective was to test the role of IGF-I and its interaction with gonadotropins, estradiol (E2) and fetal calf serum (FCS) on IVM and in equine oocytes using parthenogenic cleavage as an indirect measure of cytoplasmic maturation. We concluded that IGF-I increases nuclear maturation rate of IVM equine oocytes. Moreover, the addition of IGF-I to a culture medium containing hormones and FCS increased the cytoplasmic maturation rate measured by parthenogenic cleavage. The final objectives of this research were to describe and quantify the cortical granule (CG) distribution of equine oocytes during in vitro meiotic maturation and after activation with ionomycin by laser confocal microscopy using this distribution as a measurement of cytoplasmic maturation. The results of this study indicated that CG are distributed throughout the cytoplasm of oocytes at the germinal vesicle stage. As maturation proceeds, a progressive centripetal migration of CG to the oocyte cortex occurs starting by the end of the 30-hour incubation period and increasing significantly after 36 hours. After activation, significant reduction in the number of CG was observed (p < 0.001). Taken together, these results suggest that the IGF system plays an important role in vivo and in vitro maturation of equine oocytes. It also demonstrated an equine IVM system supported with nuclear and cytoplasmic maturation. In conclusion, this dissertation research has provided new information on in vivo and in vitro maturation of equine oocytes, revealing a new potential IVM system in this specie. Moreover, it represents a foundation to improve results in future attempts for assisted reproductive techniques in the horse.