Genes Associated With Ovarian Development and Premature Ovarian Failure - test

与卵巢发育和卵巢早衰相关的基因 - 测试

• 批准号: 9341858
• 负责人: David Schlessinger
• 金额: $ 66.62万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9341858
• 关键词: Ablation; Activins; Adult; Affect; Age; Age-Months; Aging; Aging-Related Process; Animals; Apoptosis; Attention; Attenuated; Autophagocytosis; Biological Assay; Cell Culture Techniques; Cell Cycle Regulation; Cell Lineage; Cell Survival; Cells; Cessation of life; Complex; Cornea; Data; Development; Developmental Delay Disorders; Diagnosis; Embryonic Development; Endocrine; Ensure; Environmental Risk Factor; Epithelial Cells; Eye; Eyelid structure; FOXL2 gene; Female; Fertility; Fertility Study; Formulation; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Germ Cells; Goals; Gonadal structure; Gonadotropins; Growth; Human; Impairment; In Vitro; Inherited; Intervention; Joints; Knockout Mice; Left; Life; Longevity; Maintenance; Measures; Menopause; Mesenchymal; Methods; Modeling; Mus; Mutant Strains Mice; Mutate; Nutrient; Oocytes; Organ; Ovarian; Ovarian Follicle; Ovarian Tissue; Ovary; Oxidative Stress; Pathway interactions; Perimenopause; Pharmacologic Substance; Phosphorylation; Pituitary Gland; Population; Premature Ovarian Failure; Premenopause; Primordial Follicle; Process; Production; Ptosis; Puberty; Regulation; Reproductive system; Research; Role; SF1; Signal Transduction; Smooth Muscle; Spermatogenesis; Starvation; Sterility; Surface; Syndrome; System; Testing; Testis; Transgenes; Transgenic Mice; Transgenic Organisms; Untranslated RNA; Up-Regulation; Vascularization; Woman; Work; base; cell type; cohort; comparative; fertility improvement; fetal; folliculogenesis; forkhead protein; gain of function; leydig interstitial cell; male; mouse model; mutant; notch protein; overexpression; prevent; promoter; reproductive; research study; response; sertoli cell; sex; sex determination; sry Genes; transcription factor

Our work began with our findings that the forkhead tanscription factor 2 (Foxl2) is important in preventing POF; most strikingly, we showed that Foxl2 hereditary deficiency provokes POF in women with the Blephariphomisis-Ptosis-Epicanthus Inversus Syndrome (BPES). We have then shown over the last 6 years that Foxl2 is critical throughout female reproductive life, vitally involved in the ovary in somatic sex determination and maintenance as well as in the development and stabilization of ovarian follicles. The studies have led to a significant change in the paradigm for somatic sex determination and maintenance, from a formulation in which the ovary was a default pathway when the male-determining gene Sry was absent or inactive to a model in which Sry remains male-determinative but several other genes, and especially Foxl2, are actively required to determine ovary formation and stability, continually repressing the male pathway. We have continued to analyze the action of Foxl2 by generating mouse models in which Foxl2 is ablated or overexpressed, either alone or in combination with other important ovarian factors. We have now progressed further in the elucidation of the role of Foxl2 in the formation and maintenance of follicles and in germ cell survival, and are extending the studies to the comparative action of other master genes affecting follicle dynamics and the results of joint ablation of several of them. In extensions of studies, we demonstrated that Cyp26b1, as well as transcription factor genes steroidogenic factor-1 (Sf1) and Sox9 are co-expressed in Sertoli cells in mouse fetal testes (and Cyp26b1 and Sf1 are co-expressed in Leydig cells). Furthermore, qRT-PCR showed that Cyp26b1 up-regulation by Sox9/Sf1 was attenuated by Foxl2, whereas in Foxl2-null mice, Cyp26b1 expression in XX gonads was increased 20-fold relative to WT controls. These data support the hypothesis that Sox9 and Sf1 ensure the male fate of germ cells in part by up-regulating Cyp26b1, and that Foxl2 antagonizes Cyp26b1 expression (and Sox9) in ovaries. We have now examined the effect of over-expression of Foxl2. We created transgenic mice over-expressing Foxl2 under the control of an Amhr2 promoter, active starting early in embryonic development. The transgene up-regulates genes downstream of Foxl2 in the ovary and down-regulates male-determining genes in the testis. A larger follicle pool was also seen in transgenic ovaries. By contrast, the maturation of follicles was normal, with the decrease of follicle numbers during aging proceeding at the same rate as in the wild-type. The over-expression of Foxl2 thus seems to have a primary effect on the initial formation of follicles. This finding is especially relevant to the role of Foxl2 in determining the ovarian reserve and thus, the age of menopause. In the reproductive system, Foxl2 is the only gene expressed uniquely in the ovary. However, Foxl2 is also expressed in the pituitary and in the eye of both sexes, and we have investigated its action in these extra-ovarian tissues as well. In the pituitary, we found that Foxl2 is not required for the specification of gonadotropes, which secrete LH and FSH, the endocrine signals that regulate folliculogenesis in the ovary and spermatogenesis in the testis. By contrast, in Foxl2-ablated mice FSH secretion is dramatically reduced and activin is unable to activate Fshb expression. However, a small number of gonadotropes in the ventromedial pituitary of Foxl2 mutant mice still maintain some FSH expression, suggesting that an auxiliary mechanism independent of Foxl2 and activin can drive some Fshb transcription. Nevertheless, Foxl2 function aeems required in the pituitary for normal expression of FSH.In the eye, we investigated the role of Notch signaling in the formation of corneal and eyelid stroma in mice. We found that over-expression of Notch1 intracellular domain (N1-ICD) impaired eyelid levator smooth muscle formation by down-regulating Foxl2. This is similar to the effect of haploinsufficiency of FOXL2 in the human eye. These data strongly indicate that low levels of Notch1 are crucial for proper Foxl2 expression in periocular mesenchymal cells, which are in turn essential for normal eyelid development. In an independent line of research, we have investigated the role of another forkhead transcription factor, Foxo3, which is not involved in the formation of ovarian follicles but acts to maintain them. Foxo3 is known from work in other systems to act in several biologic processes including cell cycle control, maturation, survival and apoptosis. In the ovary Foxo3 has a specific role in controlling the activation of primordial follicles, governing the size of the ovarian reserve. In its absence, others have shown that the entire cohort of primordial follicles activates and grows uncontrollably, leaving the ovary empty and female mice completely sterile by 15 weeks of age. We have now found that in young females the expression of a mutated form of Foxo3 that cannot be inactivated by phosphorylation causes a delay of follicular growth, with longer survival of primordial follicles, a lower expression of factors involved in follicle maturation, and increased fertility. We also studied adult, premenopausal, and perimenopausal mice, and showed that when active Foxo3 is present, aging processes are slowed and the ovary retains a larger pool of primordial follicles. Furthermore, gene expression levels of markers for further development and aging are consistent with either a developmental delay or slower aging in transgenic ovaries. In addition, we measured the production of gonadotropins, whose levels increase as menopause approaches (an assay method used clinically in humans to diagnose the onset of menopause). We found a higher level of gonadotropins in wild-type compared to age-matched transgenic mice, supporting the interpretation of slower aging in the transgenics. Finally, we studied the fertility of aging mice up to 12 months of age. With aging and approach of menopause, transgenic females showed a greater fertility (31-49%) compared with wild-type animals. This is the first gain-of-function change in a gene that gives greater fertility throughout the reproductive lifespan, and suggests a possible point for pharmaceutical intervention. We also compared the effects of loss of another transcription factor, Lhx8, highly expressed in oocytes. Lhx8 loss causes germ cell loss that is complete by puberty (21 days of age), but the mechanism of death of oocytes and follicles was poorly understood. We found that in Lhx8 mutant ovaries apoptosis does not seem to be involved in the death of oocytes. Germ cells die by activation of autophagy, involving impairment of vascularization and the formation of a nutrient starvation/oxidative stress microenvironment. These results have all been referenced in two extensive review articles on the ovarian reserve and genetic and environmental factors that affect it. Finally, we are now conducting in vitro experiments on primary isolated ovarian cell cultures to identify their contribution to the ovarian function. So far, we have assessed the gene expression profile and uniquely expressed genes in endothelial, myoid, and surface epithelial cell types, with special attention to specific noncoding RNAs.

我们的工作始于我们的发现，即叉头转录因子 2 (Foxl2) 对于预防 POF 很重要；最引人注目的是，我们发现 Foxl2 遗传性缺陷会导致患有眼睑下垂-内眦赘皮综合症 (BPES) 的女性出现 POF。我们在过去的六年中证明，Foxl2 在整个女性生殖生命中至关重要，在卵巢的体细胞性别决定和维持以及卵泡的发育和稳定中发挥着至关重要的作用。这些研究使体细胞性别决定和维持的范式发生了重大变化，从当雄性决定基因Sry缺失或失活时卵巢是默认途径的配方，到Sry仍然是雄性决定性的模型，但其他几个基因，特别是Foxl2，是决定卵巢形成和稳定性的积极需要，持续抑制雄性途径。 我们通过生成小鼠模型来继续分析 Foxl2 的作用，其中 Foxl2 单独或与其他重要卵巢因子组合被消除或过度表达。我们现在在阐明 Foxl2 在卵泡形成和维持以及生殖细胞存活中的作用方面取得了进一步进展，并将研究扩展到影响卵泡动力学的其他主基因的比较作用以及其中几个基因联合消融的结果。 在扩展研究中，我们证明了 Cyp26b1 以及转录因子基因类固醇生成因子 1 (Sf1) 和 Sox9 在小鼠胎儿睾丸的支持细胞中共表达（并且 Cyp26b1 和 Sf1 在 Leydig 细胞中共表达）。此外，qRT-PCR显示，Foxl2减弱了Sox9/Sf1对Cyp26b1的上调，而在Foxl2缺失小鼠中，XX性腺中的Cyp26b1表达相对于WT对照增加了20倍。这些数据支持这样的假设：Sox9和Sf1部分通过上调Cyp26b1来确保生殖细胞的雄性命运，并且Foxl2拮抗卵巢中Cyp26b1（和Sox9）的表达。 我们现在已经检查了 Foxl2 过度表达的影响。我们创建了在 Amhr2 启动子控制下过表达 Foxl2 的转基因小鼠，该小鼠在胚胎发育早期就开始活跃。该转基因上调卵巢中 Foxl2 下游的基因，并下调睾丸中的雄性决定基因。在转基因卵巢中还发现了更大的卵泡池。相比之下，卵泡的成熟是正常的，衰老过程中卵泡数量的减少速度与野生型相同。因此，Foxl2 的过度表达似乎对卵泡的初始形成具有主要影响。这一发现与 Foxl2 在确定卵巢储备进而确定绝经年龄中的作用尤其相关。 在生殖系统中，Foxl2是唯一在卵巢中独特表达的基因。然而，Foxl2 也在两性的垂体和眼睛中表达，我们也研究了它在这些卵巢外组织中的作用。 在垂体中，我们发现 Foxl2 不是促性腺激素规范所必需的，促性腺激素分泌 LH 和 FSH，这两种内分泌信号调节卵巢中的卵泡发生和睾丸中的精子发生。相比之下，在Foxl2消除的小鼠中，FSH分泌显着减少，并且激活素无法激活Fshb表达。然而，Foxl2突变小鼠腹内侧垂体中的少数促性腺激素仍保持部分FSH表达，这表明独立于Foxl2和激活素的辅助机制可以驱动部分Fshb转录。然而，Foxl2 功能是垂体中 FSH 正常表达所必需的。在眼睛中，我们研究了 Notch 信号在小鼠角膜和眼睑基质形成中的作用。我们发现Notch1胞内结构域（N1-ICD）的过度表达通过下调Foxl2来损害眼睑提肌平滑肌的形成。这类似于人眼中 FOXL2 单倍体不足的影响。这些数据强烈表明，低水平的Notch1对于眼周间充质细胞中Foxl2的正确表达至关重要，而Foxl2又对于正常眼睑发育至关重要。 在一项独立的研究中，我们研究了另一种叉头转录因子 Foxo3 的作用，它不参与卵泡的形成，但起着维持卵泡的作用。 Foxo3 在其他系统中的研究表明其在多种生物过程中发挥作用，包括细胞周期控制、成熟、存活和凋亡。在卵巢中，Foxo3 在控制原始卵泡的激活、控制卵巢储备的大小方面具有特定作用。其他研究表明，在它缺失的情况下，整个原始卵泡群会不受控制地激活和生长，导致卵巢空虚，雌性小鼠在 15 周龄时完全不育。我们现在发现，在年轻女性中，不能被磷酸化灭活的 Foxo3 突变形式的表达会导致卵泡生长延迟，原始卵泡存活时间更长，卵泡成熟相关因子的表达降低，并提高生育能力。我们还研究了成年、绝经前和围绝经期小鼠，结果表明，当存在活性 Foxo3 时，衰老过程会减慢，卵巢会保留更多的原始卵泡。此外，进一步发育和衰老标志物的基因表达水平与转基因卵巢中的发育延迟或衰老较慢一致。此外，我们还测量了促性腺激素的产生，其水平随着更年期的临近而增加（一种临床上用于人类诊断更年期开始的测定方法）。我们发现与年龄匹配的转基因小鼠相比，野生型小鼠的促性腺激素水平更高，这支持了转基因小鼠衰老较慢的解释。最后，我们研究了 12 个月大的衰老小鼠的生育能力。随着衰老和更年期的临近，转基因雌性与野生型动物相比表现出更高的生育能力（31-49%）。这是基因中的第一个功能获得性变化，可在整个生殖寿命中提供更高的生育能力，并表明药物干预的可能点。 我们还比较了另一种在卵母细胞中高表达的转录因子 Lhx8 丢失的影响。 Lhx8 缺失会导致生殖细胞丧失，并在青春期（21 天龄）时完全丧失，但卵母细胞和卵泡死亡的机制尚不清楚。我们发现，在 Lhx8 突变体卵巢中，细胞凋亡似乎并不参与卵母细胞的死亡。 生殖细胞通过自噬激活而死亡，涉及血管化受损和营养饥饿/氧化应激微环境的形成。 这些结果均在两篇关于卵巢储备以及影响卵巢储备的遗传和环境因素的广泛评论文章中引用。 最后，我们现在正在对原代分离的卵巢细胞培养物进行体外实验，以确定它们对卵巢功能的贡献。到目前为止，我们已经评估了内皮细胞、肌样细胞和表面上皮细胞类型的基因表达谱和独特表达的基因，特别关注特定的非编码RNA。