Molecular Regulation of the Perinatal Male Germ Cell Niche

围产期男性生殖细胞生态位的分子调控

基本信息

批准号：
9477061
负责人：
Marie-Claude Catherine Hofmann
金额：
$ 33.2万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2021-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9477061
关键词：
Address Affect Binding Birth CYP26B1 gene Carcinoma in Situ Cell Count Cell Differentiation process Cell Maintenance Cell Maturation Cell Proliferation Cell Transplants Cell physiology Cells Clinical Research Coculture Techniques Country Development Discipline of Nursing Down-Regulation Environment Enzymes Event Failure Feedback Fetus Foundations Germ Cells Growth Factor Homeostasis Human Hyperplasia Incidence Knock-out Lead Ligands Link Maintenance Male Contraceptive Agents Male Sterility Malignant Neoplasms Malignant neoplasm of testis Meiosis Modeling Molecular Mus NOTCH1 gene Notch Signaling Pathway Pathology Pathway interactions Perinatal Phenotype Play Predisposition Regulation Reporting Research Role Seminiferous tubule structure Sertoli cell only syndrome Signal Pathway Signal Transduction Spermatogenesis Stem cells Sterility Suggestion Testing Testis Time Transcription Repressor/Corepressor Transgenic Mice Tumor Suppressor Proteins Undifferentiated United States National Institutes of Health Wild Type Mouse base carcinogenesis daughter cell experimental study fetal gain of function glial cell-line derived neurotrophic factor in vitro testing in vivo infertility treatment loss of function male mouse model novel overexpression premature promoter public health relevance self-renewal sertoli cell transcription factor tumor

项目摘要

DESCRIPTION (provided by applicant): Gonocytes (or prospermatogonia) are the precursors to spermatogonial stem cells (SSCs), which provide the foundation for spermatogenesis through their ability to both self-renew and generate daughter cells. Despite their relative importance, th regulatory mechanisms that govern gonocyte maintenance in the fetus and transition to SSCs after birth are poorly understood. Using transgenic mice, we established that constitutive activation of NOTCH1 signaling in Sertoli cells causes gonocyte loss-the first suggestion of the potential role of this signaling pathway in the testis. We then inhibited NOTCH activation in mouse Sertoli cells and observed an increase in germ cell numbers and testicular size. Therefore dysregulation of NOTCH signaling induces either sterility (NOTCH overactivation) or hyperplasia that could enhance predisposition to testicular cancer (NOTCH downregulation). This proposal will test the hypotheses that NOTCH activity, through its target effectors HEY1 and HEYL, downregulates two crucial molecules that maintain the undifferentiated states of germ cells: GDNF and CYP26B1. We will use NOTCH overactivation, NOTCH lack of function and wild type mouse models to test whether the transcriptional repressors HEY1 and/or HEYL directly influence the expression of GDNF and CYP26B1 when NOTCH is activated. In Aim1, we will investigate the temporal expression of Hey1 and HeyL transcription factors by qPCR, and use ChIP-PCR to demonstrate direct binding of these repressors to the Gdnf promoter. Further, we will test whether failure of maintaining gonocyte quiescence in our NOTCH lack-of-function model leads to a carcinoma-in-situ-like (CIS-like) phenotype. In Aim 2, we will investigate the role of NOTCH signaling on the expression of CYP26B1, an enzyme that blocks germ cell differentiation. Using ChIP-PCR analysis, we will demonstrate that HEY1/HEYL transcription factors directly bind to the Cyp26b1 promoter to downregulate its expression. Finally, we will test whether overexpression of NOTCH signaling truly leads to a Sertoli cell-only syndrome through inhibition of CYP26B1. In Aim 3, using germ cell-Sertoli cells co-cultures, we will test the hypothesis that germ cells regulate NOTCH activity in Sertoli cells and therefore can regulate their own numbers. Altogether, this proposal will demonstrate for the first time that NOTCH signaling modulates the expression of two molecules essential for germ cell proliferation and maintenance of the undifferentiated state, and is a component of normal germ cell homeostasis. Dysregulation of this pathway will induce sterility or germ cell hyperplasia.

描述（由申请人提供）：糖细胞（或Prospermatogonia）是精子干细胞（SSC）的前体，它们通过其自我更新和产生子细胞的能力为精子发生提供了基础。尽管具有相对重要性，但对胎儿中的受伤细胞维持并在出生后过渡到SSC的调节机制知之甚少。使用转基因小鼠，我们确定了Sertoli细胞中Notch1信号传导的本构激活导致性腺损失 - 首先建议该信号传导途径在睾丸中的潜在作用。然后，我们抑制小鼠Sertoli细胞中的Notch激活，并观察到生殖细胞数量和睾丸大小的增加。因此，Notch信号传导失调会诱导不育（Notch过度激活）或增生，从而增强对睾丸癌的易感性（Notch下调）。该提案将测试Notch活性通过其目标效应子Hey1和Heyl下调的假设下调了两个至关重要的分子，这些分子维持了未分化的生殖细胞状态：GDNF和CYP26B1。我们将使用Notch过度活化，缺乏功能和野生型小鼠模型来测试转录阻遏物HEY1和/或HEYL是否直接影响Notch激活Notch时GDNF和CYP26B1的表达。在AIM1中，我们将通过qPCR研究HEY1和HEYL转录因子的时间表达，并使用CHIP-PCR来证明这些阻遏物与GDNF启动子的直接结合。此外，我们将测试在缺乏功能模型中维持GONOCYTE静止的失败是否导致类似于位于原位的（顺式）表型的表型。在AIM 2中，我们将研究Notch信号传导对CYP26B1表达的作用，CYP26B1是阻断生殖细胞分化的酶。使用CHIP-PCR分析，我们将证明HEY1/HEYL转录因子直接与CYP26B1启动子结合以下调其表达。最后，我们将通过抑制CYP26B1来测试Notch信号的过表达是否真正导致仅Sertoli细胞综合征。在AIM 3中，使用生殖细胞辛硫代酚细胞共培养，我们将测试生殖细胞调节Sertoli细胞的凹口活性的假设，因此可以规范他们自己的数字。总而言之，该提案将首次证明Notch信号传导调节对未分化状态的生殖细胞增殖和维持必不可少的两个分子的表达，并且是正常生殖细胞稳态的组成部分。该途径的失调将诱导不育或生殖细胞增生。