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

白血病抑制因子作为灵长类动物排卵的调节剂

• 批准号: 8443168
• 负责人: Jon D Hennebold
• 金额: $ 26.25万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-28 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8443168
• 关键词: Animals; Area; Assisted Reproductive Technology; Binding; Binding Proteins; Bolus Infusion; Cell physiology; Cell surface; Cells; Complex; Contraceptive methods; Data; Databases; Development; Embryo; Embryonic Development; Estradiol; Evaluation; Event; Extracellular Matrix; Family member; Fertilization; Fertilization in Vitro; Follicle Stimulating Hormone; Follicular Fluid; Gene Expression; Genes; Genomics; Glycoproteins; Gonadotropins; Guidelines; Histologic; Human; Human Chorionic Gonadotropin; Hyaluronan; In Vitro; Individual; Infertility; Injection of therapeutic agent; JAK1 gene; Janus kinase; Janus kinase 1; Knowledge; LIFR gene; Lead; Length; Luteal Phase; Luteinizing Hormone; Macaca; Macaca mulatta; Measures; Mediator of activation protein; Meiosis; Menstruation; Messenger RNA; Modeling; Molecular; Monitor; Multiple Birth Offspring; Mus; Nuclear; Oocytes; Ovarian Follicle; Ovarian Stimulations; Ovary; Ovulation; Ovulation Induction; Ovum; PTGS2 gene; Pathway interactions; Phosphorylation; Pilot Projects; Pituitary Gland; Play; Primates; Process; Progesterone; Protein Tyrosine Kinase; Proteins; Protocols documentation; Publishing; Recombinants; Risk; Role; Rupture; STAT3 gene; Signal Transduction; Stat3 protein; Stigmata; Stimulus; TYK2; Techniques; Testing; Time; Treatment Protocols; Tumor Necrosis Factor-alpha; corpus luteum; cyclooxygenase 2; cytokine; extracellular; granulosa cell; implantation; improved; in vivo; leukemia inhibitory factor; leukemia inhibitory factor receptor; novel; oocyte maturation; paracrine; prevent; receptor; reproductive success; research study; social stigma; transcription factor

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. PUBLIC HEALTH RELEVANCE: 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.

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。 公共卫生关系：本提案中概述的实验将提供有关白血病抑制因子在成熟卵母细胞释放中的作用的有价值的信息，成熟卵母细胞能够进行受精和适当的胚胎发育，以及在黄体的形成。从该项目中获得的知识应通过识别灵长类动物卵泡中发生的事件来改善不孕症治疗方案，这些事件是产生具有以下特征的胚胎的高质量卵母细胞排卵所需的。 最大的发展潜力