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（排卵）的释放，成熟卵子受精并经历随后的胚胎发育，这一过程尚未完全确定。系统和详细描述这些事件对于推进不孕症治疗和开发新的非激素避孕形式是必要的。在这方面，P.I.使用高通量基因组方法导致鉴定了大多数（如果不是全部的话）在排卵刺激后的排卵期间隔期间表达增加的基因。这些mRNA可能参与卵泡破裂所必需的活动，包括卵丘细胞之间富含透明质酸的细胞外基质的形成和细胞-细胞接触的丧失（卵丘-卵母细胞扩增; 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。从所得的数据库和额外的初步研究中，发现恒河猴卵泡中白血病抑制因子（LIF）mRNA和蛋白质从给予排卵刺激（人绒毛膜促性腺激素; hCG; 0 h对照）前的不可检测水平增加到排卵前（hCG后12 h）和排卵后（hCG后36 h）的峰值。此外，编码负责LIF作用的下游信号传导组分（糖蛋白130或gp 130; Janus激酶1或JAK 1;信号转导子和转录激活子3或STAT 3）的mRNA在hCG给药后12小时和hCG后36小时排卵的未破裂恒河猴卵泡中也最高。进一步将编码细胞表面LIF结合蛋白（LIF受体，或LIFR; gp 130）的mRNA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）防止排卵刺激后恒河猴卵泡破裂，而单独注射载体导致排卵。总的来说，这些发现支持了这样的假设，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必需的事件的关键调节剂（在目标1中评估）;以及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中评估）。将采用涉及卵泡内注射LIF拮抗剂（sLIFR）的新技术来评估LIF在卵泡破裂以及黄体形成（即，合成孕酮和雌二醇的能力）。从非黄素化卵泡（即，hCG前）直接暴露于LIF，以确定该细胞因子对促进C-OE、减数分裂重新开始、受精和胚胎发育的直接作用。

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.