Fertilization and Early Embryogenesis
(W348) LEUKEMIA INHIBITORY FACTOR (LIF) AND ITS RECEPTOR ARE EXPRESSED AND FUNCTIONAL DURING GV TO MII IN VITRO TRANSITION OF OVINE OOCYTES.
Brevini, Tiziana A1, Matsukawa, Kazutsugu2, Lopes, Federica2, Cillo, Fabiana1, Antonini, Stefania1, Gandolfi, Fulvio 1, Loi, Pasqualino2, Ptak, Grazyna2, 1 University of Milan, Milan, Italy2 University of Teramo, Teramo, Italy
ABSTRACT- LIF is transiently expressed in the mouse uterus, first at the time of ovulation and again just prior to implantation. Its production is under the control of ovarian hormones. The presence of LIF and its receptors in the ovary has been reported at both mRNA and protein level in human, mouse and rat at different stages of follicular development. Studies have demonstrated a beneficial influence of this cytokine on pre-implantation embryo development in several species, however the effect of LIF on the oocyte has not been studied yet. The aim of this research was to localize the expression of this cytokine specifically in GV stage sheep oocytes, its surrounding cumulus cells and metaphase II stage oocytes following maturation in vitro. Further, we studied the effect of LIF on oocyte development in vitro, mimicking its physiological expression prior to ovulation. For this purpose, RNA was extracted from cumulus cells, GV and MII denuded oocytes and subsequently reverse transcribed. PCR was carried out with primers that were designed based on bovine and human deposited sequences. GV stage oocytes were matured in different conditions: a) in the presence of LIF, b) LIF together with anti-LIF antibody c) control. Following maturation, part of the oocytes was assessed for LIF receptor immunolocalization, while the remaining eggs were activated and cultured to the blastocyst stage in standard media. The proportion of developed embryos was evaluated for each group. PCR studies demonstrated the presence of the LIF/LIF receptor system in sheep oocytes as well as in cumulus cells, both at GV stage and at the end of maturation. The system appears to be biologically active and responsive since LIF putative action on cumulus oocyte complexes was inhibited by the antibody against this cytokine. This inhibition seems to involve a reduction of LIF receptor density on the oocyte membrane, as evidenced by immunostaining. As a consequence, very low oocyte development was observed (7% vs. 29% of blastocysts). Conversely, the enrichment of medium with recombinant human LIF significantly enhanced embryo development rate (39% vs. 29%). We report for the first time the presence of the LIF/LIF receptor system in ovine cumulus oocyte complexes and its beneficial influence on the oocyte, suggesting a role for LIF in the determination of oocyte competence.
KEY WORDS: LIF/LIF receptor, oocyte, sheep, developmental competence