Derivation of Mature Oocytes from Human Primordial Follicles

从人类原始卵泡中衍生出成熟卵母细胞

• 批准号: 7964577
• 负责人: AARON JW HSUEH
• 金额: $ 23.77万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7964577
• 关键词: Adult; Animal Model; Animal Testing; Antral; Cancer Patient; Clinical; DNA; DNA Microarray Chip; Data; Derivation procedure; Development; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Evaluation; Exclusion; Exploratory/Developmental Grant for Diagnostic Cancer Imaging; Female; Fertility; Fertilization; Freezing; Future; Gene Expression Profile; Generations; Genes; Genetic; Germ Cells; Gonadotropins; Growth; Growth Factor; Hormonal; Human; Immune; In Vitro; Incubated; Individual; Infertility; Kidney; Malignant Neoplasms; Malignant neoplasm of ovary; Menopause; Mus; Mutant Strains Mice; Neonatal; Nuclear; Nude Mice; Oocytes; Ovarian; Ovarian Follicle; Ovarian Tissue; Ovary; Patients; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Population Heterogeneity; Pregnancy; Premature Ovarian Failure; Primordial Follicle; Procedures; Proteins; Protocols documentation; Public Health; RNA; Radiation therapy; Recruitment Activity; Reporting; Research Project Grants; Residual state; Retrieval; Rodent; SCID Mice; Safety; Secondary to; Signal Pathway; Staging; System; Testing; Tissues; Transplantation; Tumor Suppressor Proteins; Vesicle; Woman; Xeno; base; blastocyst; capsule; comparative genomic hybridization; genome-wide; in vivo; inhibitor/antagonist; innovation; intraovarian; mature animal; neonatal exposure; paracrine; premature; programs; public health relevance; response; success; tensin

DESCRIPTION (provided by applicant): Derivation of mature oocytes from human primordial follicles The majority of ovarian follicles stay dormant at the primordial stage for decades in women and the depletion of this dormant pool leads to menopause. Due to genetic and environmental influences, some women suffer from premature ovarian failure/insufficiency associated with a diminishing follicle reserve. Under physiological conditions, uncharacterized intraovarian mechanisms activate ~1,000 dormant primordial follicles to initiate growth per month, whereas the remaining follicles stay quiescent for years or decades. Once recruited into the growing pool, primordial follicles continue growth to the early antral stage with minimal loss. At the early antral stage, select follicles respond to gonadotropins and one preovulatory follicle release the mature oocyte for fertilization. Studies using mutant mice indicated that oocyte-specific deletion of the PTEN (Tumor-suppressor phosphatase with TENsin homology) gene or the downstream transcriptional factor Foxo3 promoted the growth of all primordial follicles. The PTEN gene encodes a phosphatase enzyme that negatively regulates the phosphoinositol 3-kinase (PI3K) and Akt signaling pathway. Deletion of PTEN in the oocyte increases the phosphorylation of Akt and the nuclear exclusion of the downstream Foxo3 proteins, leading to the activation of all dormant primordial follicles. Taking advantage of the availability of PTEN inhibitors, we activated primordial follicles in mice. Neonatal mouse ovaries exposed transiently to PTEN inhibitors in vitro showed marked increases in follicle growth after transplantation into the kidney capsule of FSH-treated adult recipients. Mature oocytes could be retrieved from these ovaries for IVF and blastocyst formation. Our preliminary data also demonstrated the efficacy of the PTEN inhibitor in activating dormant HUMAN primordial follicles in cortical strips donated by a cancer patient to yield mature oocytes. The Fertility and Cancer program at Stanford have stored frozen human ovarian cortical strips containing mainly primordial follicles from cancer patients. Here, we propose to incubate human follicles in vitro with PTEN inhibitors and paracrine factors, followed by transplantation into immune-deficient mice to optimize the follicle activation approach for the derivation of mature human oocytes. Chromosomal integrity will be investigated for individual mature oocytes generated after transplantation to determine the safety of the present procedure. In addition, we will use a two-step in vitro culture approach to treat mouse neonatal ovaries and human cortical strips with PTEN inhibitor and ovarian paracrine growth factors for the activation of dormant follicles, followed by the in vitro promotion of their growth to secondary and preantral follicles using stage-specific paracrine factors. During the second step, we will isolate preantral follicles and culture them with diverse ovarian paracrine factors to promote their development into antral follicles containing mature oocytes. We will further investigate the chromosomal integrity of individual mature oocytes derived from in vitro cultures and further amplify RNA from individual mature oocytes for DNA microarray analyses to reveal transcriptome changes. Although fertility is compromised in patients with premature ovarian failure and peri-menopausal women, their ovaries still contain many primordial follicles. The present in vitro follicle activation approach, followed by auto- transplantation, could allow retrieval of functional mature oocytes for infertile women with diminishing ovarian reserve and for cancer patients. Future development of a two-step culture approach to allow the development of primordial follicles to the antral stage, followed by the generation of mature oocytes, could have important clinical implications for the treatment of ovarian infertility in diverse population of women. PUBLIC HEALTH RELEVANCE: Patients with premature ovarian failure or female cancer patients following chemo- or radiation therapy suffer from the loss of ovarian germ cells and infertility. The present application deals with the use of enzyme inhibitors and key ovarian growth factors to initiate the growth of dormant human ovarian follicles for subsequent derivation of mature oocytes. Once optimized, the present approach could allow the activation of residual primordial follicles from patients with premature ovarian failure or peri-menopausal women to generate mature oocytes for infertility treatment. Frozen ovaries from cancer patients before chemo- or radiation therapy could also be used to generate mature oocytes for infertility treatment.

描述(申请人提供)：从人类原始卵泡衍生成熟卵母细胞在女性中，大多数卵泡在原始阶段处于休眠状态长达数十年，这种休眠池的耗尽会导致更年期。由于遗传和环境的影响，一些妇女患有卵巢早衰/功能不全与卵泡储备减少有关。在生理条件下，未知的卵巢内机制每月激活约1000个休眠的原始卵泡开始生长，而其余的卵泡则保持几年或几十年的静止。一旦被招募到生长池中，原始卵泡就会继续生长到胃窦早期，损失最小。在胃窦早期，选定的卵泡对促性腺激素有反应，一个排卵前卵泡释放成熟的卵母细胞进行受精。使用突变小鼠的研究表明，卵母细胞特异性的PTEN(肿瘤抑制同源磷酸酶)基因或下游转录因子Foxo3的缺失促进了所有原始卵泡的生长。PTEN基因编码一种磷酸酶，该酶负向调节PI3K和Akt信号通路。在卵母细胞中PTEN的缺失增加了Akt的磷酸化和下游Foxo3蛋白的核排斥，导致所有休眠的原始卵泡的激活。利用PTEN抑制剂的可用性，我们激活了小鼠的原始卵泡。在体外短暂暴露于PTEN抑制剂的新生小鼠卵巢，在移植到FSH治疗的成年受者的肾被膜中后，卵泡生长显著增加。成熟的卵母细胞可以从这些卵巢中取出，用于体外受精和囊胚形成。我们的初步数据也证明了PTEN抑制剂在激活癌症患者捐赠的皮质条中的休眠人类原始卵泡以产生成熟卵母细胞的效果。斯坦福大学的生育和癌症项目储存了冷冻的人类卵巢皮质条，其中主要包含癌症患者的原始卵泡。在这里，我们建议在体外用PTEN抑制剂和旁分泌因子孵育人卵泡，然后移植到免疫缺陷小鼠体内，以优化卵泡激活方法来获得成熟的人卵母细胞。将对移植后产生的单个成熟卵母细胞进行染色体完整性调查，以确定本程序的安全性。此外，我们将使用两步体外培养方法处理小鼠新生卵巢和人皮质条，使用PTEN抑制剂和卵巢旁分泌生长因子激活休眠卵泡，然后使用阶段特定的旁分泌因子促进其体外生长到次级和腔前卵泡。在第二步中，我们将分离腔前卵泡，并将其与多种卵巢旁分泌因子一起培养，以促进其发育为含有成熟卵母细胞的腔前卵泡。我们将进一步研究来自体外培养的个体成熟卵母细胞的染色体完整性，并进一步从个体成熟卵母细胞中扩增RNA，用于DNA微阵列分析，以揭示转录组的变化。尽管卵巢早衰患者和围绝经期妇女的生育力受到影响，但她们的卵巢中仍含有许多原始卵泡。目前的体外卵泡激活方法，然后进行自体移植，可以为卵巢储备减少的不孕不育妇女和癌症患者取回功能成熟的卵母细胞。未来两步培养方法的发展，使原始卵泡发育到胃窦阶段，随后产生成熟的卵母细胞，可能对不同人群中卵巢不孕症的治疗具有重要的临床意义。 公共卫生相关性：卵巢早衰患者或接受化疗或放射治疗的女性癌症患者遭受卵巢生殖细胞丧失和不孕症的困扰。本申请涉及使用酶抑制剂和关键的卵巢生长因子来启动休眠的人卵巢卵泡的生长，以便随后获得成熟的卵母细胞。一旦优化，本方法可以激活来自卵巢早衰患者或围绝经期妇女的剩余原始卵泡，以产生用于不孕症治疗的成熟卵母细胞。癌症患者化疗或放射治疗前的冷冻卵巢也可以用来产生用于不孕不育治疗的成熟卵母细胞。