The Role of KDM5D in Azoospermia

KDM5D 在无精症中的作用

• 批准号: 10611951
• 负责人: Eden Ann Dulka
• 金额: $ 7.18万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10611951
• 关键词: Brain; Cells; Chromatin; Chromosome Deletion; Chromosome Mapping; Chromosome Pairing; Chromosomes, Human, Y; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; DNA Repair Gene; Data; Diagnostic tests; Excision; Exhibits; Failure; Fellowship; Future; Gene Targeting; Genes; Genetic; Genetic Recombination; Germ Cells; Goals; Histones; Homologous Gene; Human; In Vitro; Individual; Infertility; Knockout Mice; Link; Lysine; Male Infertility; Meiosis; Mentors; Methylation; Missense Mutation; Modeling; Modification; Molecular; Mus; Mutant Strains Mice; Mutation; Phenotype; Point Mutation; Positioning Attribute; Postdoctoral Fellow; Process; Prophase; Proteins; Reproductive Physiology; Reproductive Sciences; Research Personnel; Role; Site; Sorting; Specific qualifier value; Spermatogenesis; Stains; Techniques; Testing; X Inactivation; career; cell type; chromatin modification; factor A; histone demethylase; human male; insight; lens; loss of function; male; male fertility; men; molecular marker; mouse model; mutant mouse model; nuclease; repaired; reproductive; sperm cell

Non-obstructive azoospermia (NOA) is a common cause of male infertility characterized by the absence of sperm due to spermatogenic failure. NOA is frequently associated with deletions in Azoospermia factor (AZF) regions a, b and c of the human Y chromosome. Although AZF deletions are the most common genetic cause of male infertility, the individual AZF gene(s) responsible for NOA remain unknown. Kdm5d is a single copy gene located in the AZFb deleted region and encodes a histone demethylase expressed in pre-meiotic/early meiotic testicular germ cells. My preliminary data in mice lacking Kdm5d demonstrates that these mice have azoospermia and meiotic arrest of testicular germ cells, mirroring observations in men carrying AZFb deletions. The goal of my studies is to uncover the molecular basis of Kdm5d-dependent NOA in Kdm5d null mice and determine if disruption of KDM5D is responsible for NOA in humans. I will achieve this goal through the completion of three independent aims. In Aim 1, I will assess the necessity of Kdm5d for male fertility, spermatogenesis and in specific meiotic processes by characterizing reproductive phenotypes and meiotic progression in mice lacking Kdm5d. In Aim 2, I will test if Kdm5d is necessary for removal of chromatin methylation modifications at programmed meiotic double strand break sites (DSB). I will utilize cleavage under targets and release using nuclease (CUT & RUN) sequencing combined with FACS sorting of distinct staged meiotic cells to determine whether a chromatin modification that specifies meiotic DSBs remains present in the absence of Kdm5d. Finally, in Aim 3 I will determine if loss of KDM5D underlies NOA in men carrying potential loss of function point mutations in KDM5D. I will generate mouse models of Kdm5d missense mutations found in men with NOA using a conditional gene targeting by inversion (“COIN”) approach to determine if these mutant mouse lines recapitulate phenotypes found in Kdm5d null male mice. Overall my studies will determine the role of Kdm5d in NOA and will provide insight into the elusive genetic underpinnings of AZFb deletions in infertile men. These studies also have potential to inform our understanding of the propensity for single copy Y- linked genes located within AZF regions to contribute to male infertility and may thus provide a new gene for diagnostic testing of men with NOA.

非梗阻性无精子症（NOA）是男性不育的常见原因，其特征是由于生精障碍而导致精子缺乏。NOA通常与人类Y染色体的无精子因子（AZF）区域a、B和c的缺失相关。虽然AZF缺失是男性不育最常见的遗传原因，但负责NOA的单个AZF基因仍然未知。Kdm 5d是位于AZFb缺失区的单拷贝基因，编码在减数分裂前/减数分裂早期睾丸生殖细胞中表达的组蛋白去甲基化酶。我在缺乏Kdm 5d的小鼠中的初步数据表明，这些小鼠具有无精子症和睾丸生殖细胞减数分裂停滞，反映了携带AZFb缺失的男性的观察结果。本研究的目的是揭示Kdm 5d基因敲除小鼠中Kdm 5d依赖性NOA的分子基础，并确定KDM 5D的破坏是否是人类NOA的原因。我将通过完成三个独立的目标来实现这一目标。在目标1中，我将评估的必要性Kdm 5d的男性生育力，精子发生和特定的减数分裂过程中的特征生殖表型和减数分裂进程中缺乏Kdm 5d的小鼠。在目标2中，我将测试Kdm 5d是否是去除程序性减数分裂双链断裂位点（DSB）处染色质甲基化修饰所必需的。我将利用靶下的切割和使用核酸酶（CUT & RUN）测序结合不同阶段减数分裂细胞的FACS分选的释放来确定指定减数分裂DSB的染色质修饰是否在Kdm 5d不存在的情况下仍然存在。最后，在目标3中，我将确定KDM 5D缺失是否是携带KDM 5D功能点突变潜在缺失的男性NOA的基础。我将使用条件基因反向靶向（“COIN”）方法生成在NOA男性中发现的Kdm 5d错义突变的小鼠模型，以确定这些突变小鼠系是否重现在Kdm 5d缺失雄性小鼠中发现的表型。总的来说，我的研究将确定Kdm 5d在NOA中的作用，并将提供对不育男性AZFb缺失的难以捉摸的遗传基础的深入了解。这些研究也有可能为我们了解位于AZF区域内的单拷贝Y连锁基因导致男性不育的倾向提供信息，因此可能为NOA男性的诊断测试提供新的基因。