Leukemia Inhibitory Factor As a Mediator of Primate Ovulation & Oocyte Maturation

描述（由申请人提供）：灵长类动物卵泡内发生的细胞和分子过程导致成熟卵子（排卵）的释放，受精并经历随后的胚胎发育尚未完全定义。这些事件的系统和详细的特征是必要的，以推进不孕症治疗和发展新的，非激素形式的避孕。在这方面，P.I.使用高通量基因组方法进行的研究导致了大多数（如果不是全部）基因的鉴定，这些基因的表达在排卵刺激后的排卵期增加。这些mrna可能参与了卵泡破裂所必需的活动，包括在卵丘细胞之间形成富含透明质酸的细胞外基质和失去细胞-细胞接触（卵丘-卵母细胞扩张；C-OE），以及卵母细胞的细胞质和核成熟，这是随后受精和胚胎发育所必需的。从由此产生的数据库和额外的初步研究中，我们发现恒河猴卵泡中的白血病抑制因子（LIF） mRNA和蛋白从给予排卵刺激（人绒毛膜促性腺激素；hCG； 0小时对照）之前的不可检测水平增加到排卵前（hCG后12小时）和排卵后（hCG后36小时）的峰值。此外，编码LIF作用的下游信号成分（糖蛋白130，或gp130； janus激酶1，或JAK1；信号传感器和转录激活因子3，或STAT3）的mrna在给药后12小时和促性腺促性腺激素后36小时的未破裂猕猴卵泡中也最高。编码两种细胞表面LIF结合蛋白（LIF受体，或LIFR； gp130）的mrna进一步定位于分离的卵母细胞和颗粒细胞。最后，在初步研究中，卵泡内注射LIF拮抗剂（LIFR的细胞外LIF结合部分，称为可溶性LIFR或sLIFR）可防止猕猴卵泡在排卵刺激后破裂，而单独注射载体可导致排卵。总的来说，这些发现支持这样的假设，即灵长类动物卵巢中的LIF合成和信号传导是排卵和黄体形成所需事件的关键调节器（在Aim 1中评估）；以及C-OE，卵母细胞减数分裂，受精和早期胚胎发育的重新启动（在Aim 2中评估）。包括卵泡内注射LIF拮抗剂（sLIFR）在内的新技术将被用于评估LIF在卵泡破裂和黄体形成（即合成黄体酮和雌二醇的能力）中的作用。将从非黄体化卵泡（即hcg前期）中分离的恒河猴卵母细胞复合物（COCs）直接暴露于LIF中，以确定该细胞因子对促进C-OE、减数分裂重新启动、受精和胚胎发育的直接影响。

DESCRIPTION (provided by applicant): 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.