The cellular and molecular processes occurring within the primate follicle resulting in the release of a mature ovum (ovulation) that fertilizes and undergoes subsequent embryonic development are incompletely defined. Systematic and detailed characterizations of such events are necessary for advancing infertility treatments and developing novel, non-hormonal forms of contraception. In this regard, studies conducted by the P.I. using a high-throughput genomic approach led to the identification of most, if not all, genes whose expression increases through the periovulatory interval following an ovulatory stimulus. Such mRNAs are likely involved in activities necessary for follicle rupture, which include the formation of a hyaluronan-rich extracellular matrix between cumulus cells and the loss of their cell-cell contacts (cumulus-oocyte expansion;C-OE), as well as the cytoplasmic and nuclear maturation of the oocyte required for subsequent fertilization and embryonic development. From the resultant database and additional preliminary studies, it was discovered that leukemia inhibitory factor (LIF) mRNA and protein increased in the rhesus macaque follicle from undetectable levels before administration of an ovulatory stimulus (human chorionic gonadotropin;hCG;0 h controls) to peak values prior to (12 h post-hCG) and following ovulation (36 h post-hCG). Furthermore, mRNAs encoding downstream signaling components responsible for LIF action (glycoprotein 130, or gp130;janus kinase 1, or JAK1;signal transducer and activator of transcription 3, or STAT3) were also highest in unruptured rhesus macaque follicles 12 h after hCG administration and those that had ovulated 36 h post-hCG. The mRNAs encoding both cell surface LIF binding proteins (LIF receptor, or LIFR;gp130) were further localized to isolated oocytes and granulosa cells. Lastly, in pilot studies, intrafollicula injection of a LIF antagonist (the extracellular LIF binding portion of the LIFR;referred to as soluble LIFR or sLIFR) prevents rupture of the rhesus macaque follicle following an ovulatory stimulus, whereas injection of vehicle alone results in ovulation. Collectively, these findings support the hypothesis that LIF synthesis and signaling in the primate ovary is a critical regulator of events necessary for ovulation and formation of the corpus luteum (assessed in Aim 1);as well as for C-OE, reinitiation of oocyte meiosis, fertilization, and early embryonic development (assessed in Aim 2). Novel techniques involving intrafollicular injection of a LIF antagonist (sLIFR) will be employed to assess the role LIF plays in follicle rupture as well the formation of the corpus luteum (i.e., the ability to synthesize progesterone and estradiol). Isolated rhesus macaque cumulus-oocyte complexes (COCs) from non-luteinized follicles (i.e., pre-hCG) will be directly exposed to LIF to determine a direct effect of this cytokine on promoting C-OE, reinitiation of meiosis, fertilization, and embryonic development.
The experiments outlined in this proposal will provide valuable information regarding the role leukemia inhibitory factor plays in the release of a mature oocyte capable of undergoing fertilization and proper embryonic development, as well as in the formation of the corpus luteum. The knowledge gained from this project should lead to improved infertility treatment protocols through the identification of events occurring in the primate ovarian follicle required for the ovulation of high-quality oocytes that yield embryos with maximal developmental potential.