Regulation of protamine incorporation and nuclear reorganization during Drosophila spermatogenesis

果蝇精子发生过程中鱼精蛋白掺入和核重组的调节

• 批准号: 10594419
• 负责人: Jun Il Park
• 金额: $ 3.44万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10594419
• 关键词: Alleles; Architecture; Attention; Biological Models; Biological Process; Cell Nucleolus; Cell Nucleus; Cell Proliferation; Cells; Chromatin; Cytology; DNA; DNA-Directed RNA Polymerase; Data; Defect; Development; Differentiated Gene; Dominant-Negative Mutation; Drosophila genus; Drosophila melanogaster; Failure; Fertility; Fertilization; Gene Expression; Genes; Genetic Transcription; Genome; Growth; Histones; Homologous Gene; Human; Literature; Male Infertility; Mediating; Molecular Biology; Morphogenesis; Nuclear; Ontology; Phenotype; Process; Protamines; Protein Isoforms; Protein Sequence Analysis; Protein Splicing; Proteins; RNA; RNA Splicing; Regulation; Role; Shapes; Somatic Cell; Sperm Maturation; Spermatids; Spermatocytes; Spermatogenesis; Spermiogenesis; Sterility; Testing; Testis; Y Chromosome; chromatin remodeling; clinically relevant; genome integrity; insight; interest; mutant; novel; nucleolin; offspring; protein expression; protein protein interaction; sperm cell; transcription factor; transcriptome sequencing; transmission process

ABSTRACT In many species, sperm maturation requires a process of nuclear compaction, which occurs via dramatic chromatin reorganization from histone to protamine-based architecture. Nuclear compaction is essential for sperm fertility, functioning to enhance hydrodynamics and genomic integrity. While several genes have been identified to be involved in this process, it is unknown how these genes are regulated. Here, we have utilized the Drosophila testis model system to identify Modulo, the Drosophila homolog of Nucleolin, as a key player in sperm nuclear reorganization. In somatic cells, modulo has been shown to be involved in chromatin remodeling, cell proliferation, and morphogenesis. Viable modulo mutant allele that specifically influences testis-specific isoform has been known to be sterile, but the reason underlying this sterility remained unknown. We find that modulo mutants display nuclear compaction defects during late spermatogenesis alongside decreased protamine protein incorporation. Importantly, modulo’s unique phenotype cannot be explained simply by lack of protamine incorporation: compound mutant that lacks protamines Prot A/B and Mst77F causes only mild nuclear deformation rather than complete nuclear decompaction as observed in modulo mutant. Indeed, in addition to decreased incorporation of main protamines in mutant, we have detected increased expression of Mst77Y, a lesser-studied protamine gene with numerous duplications on the Y-chromosome. Thus, we hypothesize that modulo mediates sperm nuclei compaction by coordinating expression of multiple genes that are specifically involved in nuclear compaction during spermiogenesis. To test this hypothesis, we will conclusively establish the cytological defects of modulo mutant by inducing expression of mst77Y in wild-type nuclei and assessing protamine protein expression in whole testis vs. in spermatid nuclei. Additionally, we will investigate transcription and/or splicing as modulo’s mechanism for regulating its target genes. Previous literature has identified modulo to interact with testis-specific transcription factors while protein sequence analysis and gene ontology have predicted modulo as being involved in splicing. Therefore, we further hypothesize that modulo regulates its target genes at the RNA level either by functioning during transcription or splicing. If our hypothesis is true, it would result in the identification of a previously uncharacterized regulatory network governing nuclear reorganization in late stage spermatogenesis and have implications for male infertility.

摘要 在许多物种中，精子成熟需要一个核致密化的过程，这是通过以下方式发生的： 从组蛋白到基于鱼精蛋白的结构的显着染色质重组。核 压实对于精子生育力是必不可少的，其功能是增强流体动力学和基因组学。 完整虽然已经确定了几个基因参与这一过程，但尚不清楚 这些基因是如何被调控的在这里，我们利用果蝇睾丸模型系统， 确定Modulo，果蝇核仁素的同源物，作为精子核的关键参与者 重组。在体细胞中，modulo已被证明参与染色质重塑， 细胞增殖和形态发生。有活力的模突变等位基因， 已知睾丸特异性同种型是不育的，但这种不育的原因是 仍然未知。我们发现，模突变体显示核压缩缺陷，在后期 精子发生以及鱼精蛋白掺入减少。重要的是，模 独特的表型不能简单地用缺乏鱼精蛋白掺入来解释：化合物 缺乏鱼精蛋白Prot A/B和Mst 77 F的突变体仅引起轻微的核变形， 而不是在模突变体中观察到的完全核解压缩。事实上，除了 在突变体中主要鱼精蛋白的掺入减少，我们检测到表达增加 Mst77Y是一种研究较少的鱼精蛋白基因，在Y染色体上有许多重复。 因此，我们假设modulo通过协调表达介导精子核致密化 在精子发生过程中，多个基因特异性地参与了核致密化。到 为了验证这一假设，我们将最终确定模突变体的细胞学缺陷， 在野生型细胞核中诱导mst77Y的表达，并在 整个睾丸与精子细胞核。此外，我们将研究转录和/或剪接 作为modulo调节其靶基因的机制。以前的文献已经确定模 与睾丸特异性转录因子相互作用，而蛋白质序列分析和基因 本体论已经预测模是参与拼接的。因此，我们进一步假设 它在RNA水平上调节其靶基因， 或拼接。如果我们的假设是正确的，它将导致识别以前的 核重组后期监管网络不完善 精子发生和对男性不育的影响。