Functions of the rete ovarii in ovary development, adult homeostasis, and female reproductive longevity

摘要 迫切需要了解决定女性生殖寿命的因素。 女性生殖衰老的特征是生殖功能显著下降，原因是 构成有限的卵巢储备的卵泡的数量和质量依次减少。这个 卵巢网状结构(RO)是一种保守的上皮性结构，分为3个区域，即卵巢内网状结构(IOR 在卵巢内，位于卵巢膜组织中的卵巢外网(EOR)和连接网(CR)， 它将EOR和IOR联系在一起。在解剖学的背景下，RO已经被描述了几十年，并且是一种可能的 是妇科疾病的始作俑者，但其功能从未被彻底研究过。生殖方面的 雌性的寿命主要由三个因素决定：1)原始卵泡池的大小；2)原始卵泡池的大小 在生育窗口期的排卵率和闭锁率，以及3)代谢变化等干扰 影响卵巢功能和卵泡生长的状态或激素水平。此应用程序的目标是 阐明RO对这些决定因素的每一个的贡献。以允许可视化和 对卵巢的原位功能研究，我开发了组织清除和3D成像方法，提示 网在卵巢细胞组装成卵母细胞索中的作用和随后的规范 髓质区和皮质区。我发现RO自始至终都与卵巢密切相关 成年期，初步的卵巢移植实验表明，宿主EOR/CR重建了一个 与移植卵巢的IOR相连。我们的RO转录数据显示分泌丰富 代谢途径与发现注入EOR中的葡聚糖被输送到 卵巢。这些数据导致了一个中心假设，即RO在建立过程中扮演着关键角色 在卵巢储备和调节卵巢卵泡生长对生理信号的反应中。 这一假设将通过追求三个具体目标来检验：(1)(K99)表征 Ro对卵巢发育和卵巢储备的建立；(2)(K99/R00)定义 成人动态平衡状态下EOR和IOR的功能差异及(3)(R00)研究 RO对生理信号的反应。发现卵巢网在建立卵巢的过程中的作用 卵巢储备在动态平衡或扰动条件下对成年卵泡生长的调节作用 在寻找女性生育和生殖的决定因素方面，将把RO定位为新的候选对象 长寿。此外，我开发的方法在未来的相互作用研究中将是有用的 在卵巢和其他外在组织之间。追求这些目标需要训练单细胞RNA 测序、小鼠手术和病毒介导的基因表达操纵方法。我有过 组建了一个由卡佩尔博士担任主席的咨询委员会，我们设计了一项培训计划，将为我提供 具备运营一个多产、独立的发育和生殖生物学实验室所需的技能。

Abstract There is an urgent need to understand the factors determining female reproductive longevity. Female reproductive aging is characterized by a significant decline in reproductive function due to the sequential decrease in the number and quality of ovarian follicles that constitute the finite ovarian reserve. The rete ovarii (RO) is a conserved epithelial structure divided into 3 regions, the intraovarian rete (IOR), located within the ovary, the extraovarian rete (EOR), located in the periovarian tissue, and the connecting rete (CR), which links the EOR and IOR. The RO has been depicted for decades in anatomical context, and is a possible contributor to gynecological disease, yet its function has never been thoroughly investigated. The reproductive longevity of females is determined by three main factors: 1) the size of the initial primordial follicle pool, 2) the rate of ovulation and atresia during the fertile window, and 3) perturbations, such as changes in metabolic status or hormone levels, which affect ovarian function and follicle growth. The objective of this application is to elucidate the contribution of the RO to each of these determining factors. To allow visualization and functional investigation of the ovary in situ, I developed tissue clearing and 3D imaging methods that suggest a role for the rete in the assembly of ovarian cells into ovigorous cords and subsequent specification of medullary and cortical domains. I found that the RO remains closely associated with the ovary throughout adulthood, and preliminary ovary transplantation experiments suggest that the host EOR/CR reestablishes a connection to the IOR of the grafted ovary. Our transcriptomic data of the RO shows enrichment of secretory and metabolic pathways consistent with the finding that Dextran injected into the EOR is transported to the ovary. These data have led to the central hypothesis that the RO plays critical roles in the establishment of the ovarian reserve and in the regulation of ovarian follicle growth in response to physiological cues. This hypothesis will be tested by pursuing three specific aims: (1) (K99) Characterize the contribution of the RO to the development of the ovary and the establishment of the ovarian reserve; (2) (K99/R00) Define the functional differences between EOR and IOR in adult homeostasis and (3) (R00) Investigate the changes of the RO in response to physiological cues. Discovering a role for the rete ovarii in the establishment of the ovarian reserve and in the regulation of adult follicle growth under homeostatic or perturbed conditions will situate the RO as a new candidate in the search for determinants of female fertility and reproductive longevity. In addition, the approaches I develop will be useful in future investigations of interactions between the ovary and other extrinsic tissues. Pursuit of these aims requires training in single-cell RNA sequencing, mouse surgery and virally-mediated approaches for manipulating gene expression. I have assembled an advisory committee chaired by Dr. Capel, and we designed a training plan that will provide me with the skills required to run a productive, independent developmental and reproductive biology laboratory.