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

描述(由申请人提供)：灵长类卵泡内发生的细胞和分子过程导致成熟卵子的释放(排卵)，然后受精并经历随后的胚胎发育，这一定义还不完全。系统和详细地描述这些事件对于推进不孕症治疗和开发新的非激素形式的避孕措施是必要的。在这一点上，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)基因的识别，这些基因的表达在排卵刺激后的排卵间隔期间增加。这种mRNAs可能参与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，失去细胞与细胞的接触(卵丘-卵母细胞扩张；C-OE)，以及随后受精和胚胎发育所需的卵母细胞的细胞质和核成熟。从结果数据库和其他初步研究中发现，恒河猴卵泡中白血病抑制因子(LIF)的mRNA和蛋白水平从给予排卵刺激(人绒毛膜促性腺激素；hCG；0h对照)之前的检测不到的水平增加，达到高峰之前(hCG后12h)和排卵后(hCG后36h)。此外，编码负责LIF作用的下游信号成分(糖蛋白130或gp130；Janus kinase1或JAK1；信号转导和转录激活子3或STAT3)的mRNAs也在hCG注射后12h未破裂的猕猴卵泡和hCG后36h排卵的猕猴卵泡中最高。编码细胞表面LIF结合蛋白(LIF受体，或LIFR；gp130)的mRNAs进一步定位于分离的卵母细胞和颗粒细胞。最后，在试验性研究中，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)可防止猕猴排卵刺激后卵泡破裂，而单独注射赋形剂可导致排卵。总之，这些发现支持这样的假设，即灵长类卵巢中的LIF合成和信号是排卵和黄体形成所必需的事件的关键调节因子(在目标1中进行了评估)，以及对C-OE、卵母细胞减数分裂的重新启动、受精和早期胚胎发育(在目标2中进行了评估)。新的技术包括卵泡内注射LIF拮抗剂(SLIFR)，以评估LIF在卵泡破裂和黄体形成(即合成孕酮和雌二醇的能力)中所起的作用。从非黄体化卵泡中分离的猕猴卵丘-卵母细胞复合体(COCs)将直接暴露于LIF，以确定该细胞因子在促进C-OE、重新启动减数分裂、受精和胚胎发育方面的直接作用。

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.