Oocyte-derived R-spondin2 as a Follicle Stimulating Hormone

摘要：无翅（Wnt）信号通路在果蝇向哺乳动物细胞增殖过程中起着至关重要的作用。尽管多种Wnt配体及其同源的frizzed受体在卵巢体细胞中表达，但尚未有报道表明Wnt配体促进卵巢卵泡生长的能力。基于生物信息学和原位杂交分析，我们证实了R-spondin2在初级和晚期卵泡的卵母细胞中只表达，而在休眠的原始卵泡的卵母细胞中不表达。人类基因组中有4个R-spondin1-4同源基因（R-spondin1-4），这些分泌的蛋白被发现是胃肠道器官和毛囊成体干细胞的有效生长因子，作为细胞增殖必需的Wnt信号通路的共配体。最近的研究进一步证实了LGR4/5/6蛋白作为R-spondin配体同源受体的作用。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处理也促进了体外培养卵巢外植体的卵泡发育。与FSH不同，R-spondin2不激活cAMP-protein kinase A通路，并与FSH一起表现出对卵巢细胞增殖的累加性刺激。我们进一步将R-spondin1 cDNA与IgG Fc结构域的cDNA融合，生成了一个分泌的R-spondin1-Fc激动剂。我们在体内证明了R-spondin1-Fc能够促进新生小鼠初级卵泡向次级卵泡的生长。用R-spondin1-Fc预处理可诱导能够对序贯eCG和hCG治疗有反应的早期窦腔卵泡，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。尽管卵泡刺激素已被广泛用于治疗女性不孕症，但有一小部分患者表现出较低的卵泡刺激素反应，没有其他治疗选择。本R21提案将首先证明卵巢R-spondin2配体和LGR4/5/6受体蛋白的表达，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。在使用R-spondin2促进早期卵泡发育之后，小鼠将依次接受eCG和hCG治疗，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崽的衍生。然后，我们将使用低FSH反应性（FSH受体单倍体不足杂合突变）的啮齿动物模型来测试R-spondin2促进早期卵泡发育的能力，从而促进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，以确保目前治疗方案的有效性和安全性。随后将衍生健康的幼崽，作为未来临床应用R-spondin2治疗低FSH反应的不育患者的基础。

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. 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.