Oocyte-derived R-spondin2 as a Follicle Stimulating Hormone

描述（由申请人提供）：卵母细胞衍生的R-spondin 2作为卵泡刺激激素摘要：无翅（Wnt）信号通路是从果蝇到哺乳动物的细胞增殖所必需的。虽然多种Wnt配体及其同源卷曲受体在卵巢体细胞中表达，但没有报道证明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-spondin 2在初级和更高级卵泡的卵母细胞中的排他性表达，但在休眠的原始卵泡的卵母细胞中不表达。在人类基因组中存在四个R-spondin旁系同源基因（R-spondin 1 -4），并且已经发现这些分泌的蛋白质通过充当细胞增殖所必需的Wnt信号传导途径的共配体而成为胃肠器官和毛囊中的成体干细胞的有效生长因子。最近的研究进一步证明了LGR 4/5/6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，在培养的卵巢体细胞中，R-spondin 2与Wnt配体协同激活Wnt信号通路的能力。R-spondin 2处理也促进培养的卵巢外植体中的卵泡发育。与FSH不同，R-spondin 2不激活cAMP-蛋白激酶A通路，并与FSH一起显示卵巢细胞增殖的附加刺激。我们进一步通过将R-spondin 1 cDNA与IgG的Fc结构域的cDNA融合来产生分泌的R-spondin激动剂R-spondin 1-Fc。我们显示了R-spondin 1-Fc在体内促进新生小鼠初级卵泡生长至次级阶段的能力。用R-spondin 1-Fc预处理导致能够对连续eCG和hCG处理作出响应的早期有腔卵泡的诱导，从而导致成熟卵母细胞的产生。这些卵母细胞可以在体外受精并发育成囊胚。虽然FSH已广泛用于治疗女性不孕症，但一个亚群的患者显示FSH反应较低，并且没有替代治疗选择。目前的R21方案将首先证明卵巢R-spondin 2配体和LGR 4/5/6受体蛋白的表达，以及卵巢LGR 4/5/6受体的介导作用。随后将产生重组R-spondin 2以刺激新生野生型小鼠的早期卵泡生长。在使用R-spondin 2促进早期卵泡发育后，将依次用eCG和hCG处理小鼠，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幼仔的衍生。然后，我们将使用低FSH反应性的啮齿动物模型（FSH受体单倍不足杂合突变体）来测试R-spondin 2促进早期卵泡发育的能力，以用于随后的促性腺激素刺激和成熟卵母细胞的产生。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，以确保本治疗方案的有效性和安全性。随后将衍生健康幼仔，作为使用R-spondin 2治疗FSH反应低的不育患者的未来临床应用的基础。

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.