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。 女性生殖老化的特征是由于生殖功能的显著下降， 构成有限卵巢储备的卵泡数量和质量的连续下降。的 卵巢网（RO）是一种保守的上皮结构，分为3个区域，卵巢内网（IOR），位于 在卵巢内，卵巢外网（EOR），位于卵巢周围组织，和连接网（CR）， 连接EOR和IOR。RO已经在解剖学背景下描述了几十年，并且是一种可能的 然而，它的功能从未被彻底研究过。生殖 女性的寿命取决于三个主要因素：1）原始卵泡池的大小，2） 排卵率和闭锁在受精窗口，和3）扰动，如代谢的变化， 状态或激素水平，影响卵巢功能和卵泡生长。本申请的目的是 以阐明RO对这些决定因素中的每一个的贡献。为了实现可视化和 为了对卵巢进行原位功能研究，我开发了组织清除和3D成像方法， 网在卵巢细胞组装成卵巢索和随后的特化中的作用 髓质和皮质区域。我发现RO始终与卵巢密切相关 成年期，和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的连接。我们的RO的转录组数据显示， 和代谢途径与注入EOR的右旋糖酐被转运到 卵巢这些数据导致了中心假设，即RO在建立中起着关键作用 卵巢储备和调节卵泡生长的反应生理线索。 这一假设将通过追求三个具体目标来检验：（1）（K99）描述 （2）（K99/R00）确定卵巢储备功能的指标， EOR和IOR在成人稳态中的功能差异和（3）（R00）研究EOR和IOR在成人稳态中的变化。 RO响应于生理线索。发现卵巢网在卵巢发育中的作用 卵巢储备和在稳态或扰动条件下成年卵泡生长的调节 在寻找女性生育力和生殖能力的决定因素方面， 中心blog此外，我开发的方法将在未来的相互作用研究中发挥作用 在卵巢和其他外部组织之间。追求这些目标需要单细胞RNA的训练 测序、小鼠手术和用于操纵基因表达的病毒介导的方法。我有 我组建了一个由卡佩尔博士担任主席的咨询委员会，我们设计了一个培训计划， 拥有运行一个高效、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.