Oocyte-derived R-spondin2 as a Follicle Stimulating Hormone

卵母细胞来源的 R-spondin2 作为卵泡刺激激素

• 批准号: 8368062
• 负责人: AARON JW HSUEH
• 金额: $ 23.84万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-10 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8368062
• 关键词: Adult; Adverse effects; Agonist; Animal Model; Animals; Antral; Bioinformatics; Biological Assay; Cell Line; Cell Proliferation; Cell membrane; Clinical; Clinical Research; Complementary DNA; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Sequence; Data; Derivation procedure; Development; Embryonic Development; Epigenetic Process; Fc domain; Female infertility; Fertility; Fertilization; Follicle Stimulating Hormone; Follicle Stimulating Hormone Receptor; Future; GTP-Binding Proteins; Generations; Genes; Gonadotropins; Growth; Growth Factor; Hair follicle structure; Human Chorionic Gonadotropin; Human Genome; Immunoglobulin G; In Situ Hybridization; In Vitro; Infertility; Injection of therapeutic agent; Ligands; Mammals; Mediating; Mediation; Mitochondria; Mitochondrial DNA; Mus; Mutant Strains Mice; Neonatal; Oocytes; Oogonia; Organ; Ovarian; Ovarian Follicle; Ovarian Granulosa Cell; Ovarian Stimulations; Ovary; Ovulation; Pathway interactions; Patients; Phase; Phenotype; Physiology; Pituitary Gonadotropins; Pregnancy; Premature Ovarian Failure; Primordial Follicle; Principal Investigator; Production; Proteins; Recombinants; Reporter; Reporting; Reverse Transcription; Rodent Model; Role; Safety; Secondary to; Signal Pathway; Signal Transduction; Somatic Cell; Staging; Subgroup; T cell activating factor; TCF Transcription Factor; Testing; Therapeutic; Therapeutic Agents; Transcriptional Activation; Treatment Protocols; Wild Type Mouse; Wnt proteins; abstracting; adult stem cell; base; beta catenin; blastocyst; clinical application; fly; gastrointestinal; granulosa cell; high reward; high risk; hybrid protein; in vivo; mutant; novel; novel strategies; novel therapeutics; paracrine; paralogous gene; patient population; programs; promoter; pup; receptor; response

DESCRIPTION (provided by applicant): Oocyte-derived R-spondin2 as a Follicle Stimulating Hormone Abstract: The Wingless (Wnt) signaling pathway is essential for cell proliferation from flies to mammals. Although multiple Wnt ligands and their cognate Frizzled receptors are expressed in ovarian somatic cells, there is no report demonstrating the ability of Wnt ligands in the promotion of ovarian follicle growth. Based on bioinformatic and in situ hybridization analyses, we demonstrated the exclusive expression of R-spondin2 in oocytes of primary and more advanced follicles but not in oocytes of dormant primordial follicles. There are four R-spondin paralogous genes (R-spondin1-4) in the human genome and these secreted proteins have been found to be potent growth factors for adult stem cells in gastrointestinal organs and hair follicles by serving as co-ligands for the Wnt signaling pathway essential for cell proliferation. Recent studies further demonstrated the role of LGR4/5/6 proteins as the cognate receptors for R-spondin ligands. Our preliminary data demonstrated the ability of R-spondin2 to synergize with Wnt ligands in the activation of the Wnt signaling pathway in cultured ovarian somatic cells. R-spondin2 treatment also promoted ovarian follicle development in cultured ovarian explants. Unlike FSH, R-spondin2 did not activate the cAMP-protein kinase A pathway and showed additive stimulation of ovarian cell proliferation together with FSH. We further generated a secreted R- spondin agonist, R-spondin1-Fc, by fusing R-spondin1 cDNA with that for the Fc domain of IgG. We showed the ability of R-spondin1-Fc to promote the growth of primary follicles to the secondary stage in neonatal mice in vivo. Pre-treatment with R-spondin1-Fc led to the induction of early antral follicles capable of responding to sequential eCG and hCG treatment, leading to the generation of mature oocytes. These oocytes could be fertilized in vitro and developed into blastocysts. Although FSH has been used extensively for the treatment of female infertility, a sub-population of patients showed low FSH responses and has no alternative therapeutic options. The present R21 proposal will first demonstrate the ovarian expression of R-spondin2 ligand and LGR4/5/6 receptor proteins, together with the mediatory role of ovarian LGR4/5/6 receptors. This will be followed by the generation of recombinant R-spondin2 to stimulate early follicle growth in neonatal wild type mice. Following the promotion of early ovarian follicle development using R-spondin2, mice will be treated sequentially with eCG and hCG to stimulate the final phase of follicle development and to derive mature oocytes for fertilization and the derivation of pups. We will then use a rodent model of low FSH responsiveness (FSH receptor haploinsufficient heterozygous mutants) to test the ability of R-spondin2 in the promotion of early follicle development for subsequent gonadotropin stimulation and the generation of mature oocytes. We will evaluate epigenetic changes, mitochondrial integrity and early embryonic development of mature oocytes to insure the efficacy and safety of the present treatment regimen. This will be followed by the derivation of healthy pups as the basis for future clinical application using R-spondin2 to treat infertile patients with low FSH responses. PUBLIC HEALTH RELEVANCE: The present application represents a new approach for female infertility treatment by demonstrating the ability of oocyte-derived R-sponind2 to promote early ovarian follicle development using wild type and FSH receptor haploinsufficient mice. The generation and use of recombinant R-spondin2 molecules could provide a new therapeutic agent for the treatment of infertile patients with low FSH responses.

描述（由申请人提供）：卵母细胞来源的 R-spondin2 作为卵泡刺激激素 摘要：Wingless (Wnt) 信号通路对于从果蝇到哺乳动物的细胞增殖至关重要。尽管多种Wnt配体及其同源Frizzled受体在卵巢体细胞中表达，但尚无报道证明Wnt配体促进卵泡生长的能力。基于生物信息学和原位杂交分析，我们证明了 R-spondin2 在初级和更高级卵泡的卵母细胞中唯一表达，但在休眠原始卵泡的卵母细胞中不表达。人类基因组中有四个 R-spondin 旁系同源基因 (R-spondin1-4)，这些分泌蛋白已被发现是胃肠道器官和毛囊中成体干细胞的有效生长因子，可作为细胞增殖所必需的 Wnt 信号通路的辅助配体。最近的研究进一步证明了 LGR4/5/6 蛋白作为 R-spondin 配体的同源受体的作用。我们的初步数据表明，R-spondin2 能够与 Wnt 配体协同激活培养的卵巢体细胞中的 Wnt 信号通路。 R-spondin2 治疗还促进培养的卵巢外植体中的卵泡发育。与 FSH 不同，R-spondin2 不会激活 cAMP-蛋白激酶 A 通路，并且与 FSH 一起显示出对卵巢细胞增殖的附加刺激。我们通过将 R-spondin1 cDNA 与 IgG Fc 结构域的 cDNA 融合，进一步生成了分泌型 R-spondin 激动剂 R-spondin1-Fc。我们在体内展示了 R-spondin1-Fc 促进新生小鼠初级卵泡生长至次级阶段的能力。 R-spondin1-Fc 预处理可诱导早期窦卵泡能够对连续的 eCG 和 hCG 治疗做出反应，从而产生成熟卵母细胞。这些卵母细胞可以在体外受精并发育成囊胚。尽管 FSH 已广泛用于治疗女性不孕症，但仍有一部分患者的 FSH 反应较低，并且没有其他治疗选择。目前的R21提案将首先证明R-spondin2配体和LGR4/5/6受体蛋白的卵巢表达，以及卵巢LGR4/5/6受体的介导作用。随后将产生重组 R-spondin2，以刺激新生野生型小鼠的早期卵泡生长。使用 R-spondin2 促进早期卵泡发育后，将依次用 eCG 和 hCG 治疗小鼠，以刺激卵泡发育的最后阶段并获得成熟的卵母细胞用于受精和衍生幼仔。然后，我们将使用低 FSH 反应性的啮齿动物模型（FSH 受体单倍体不足杂合突变体）来测试 R-spondin2 促进早期卵泡发育、随后促性腺激素刺激和成熟卵母细胞生成的能力。我们将评估成熟卵母细胞的表观遗传变化、线粒体完整性和早期胚胎发育，以确保当前治疗方案的有效性和安全性。随后将衍生出健康幼犬，作为未来使用 R-spondin2 治疗 FSH 反应低的不孕患者的临床应用的基础。 公共健康相关性：本申请通过使用野生型和FSH受体单倍体不足的小鼠证明卵母细胞来源的R-sponind2促进早期卵泡发育的能力，代表了一种治疗女性不孕症的新方法。重组R-spondin2分子的产生和使用可以为治疗FSH反应低的不孕患者提供新的治疗剂。