A quantitative assessment of sperm protamine isoforms and post-translational modifications in the setting of normal and abnormal fertility

正常和异常生育情况下精子鱼精蛋白亚型和翻译后修饰的定量评估

• 批准号: 10118508
• 负责人: Samantha Beth Schon
• 金额: $ 7.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10118508
• 关键词: Acetylation; Affect; Anatomy; Antibodies; Biochemical; Biological Markers; Catalogs; Cell Nucleus; Chromatin; Cohort Studies; Complex; Counseling; Couples; DNA; DNA Packaging; Data; Defect; Development; Disease; Embryo; Endocrine; Epithelial; Epithelium; Etiology; Fertility; Fertilization; Fertilization in Vitro; Foundations; Future; Genetic; Genetic Transcription; Genome; Grant; Histones; Human; Hypogonadism; Iatrogenesis; Individual; Infertility; Instruction; Investigation; Maintenance; Male Infertility; Mammals; Mass Spectrum Analysis; Methodology; Methylation; Modification; Mus; Oocytes; Outcome; Patients; Phosphorylation; Play; Population; Post-Translational Protein Processing; Process; Protamines; Protein Isoforms; Proteins; Reproduction; Resolution; Role; Sampling; Seminal fluid; Seminiferous tubule structure; Specificity; Sperm Head; Spermatogenesis; Testing; Testis; Transgenic Mice; Translational Research; United States; Ursidae Family; Variant; Varicocele; Vasectomy; Western Blotting; base; clinical investigation; clinical practice; experimental study; father role; gel electrophoresis; idiopathic infertility; inter-individual variation; liquid chromatography mass spectrometry; male; men; mouse model; nano-liquid chromatography; novel diagnostics; novel marker; protamine 1; sperm cell; sperm function; tandem mass spectrometry; tool; transmission process

Infertility affects 8-15% of couples in the United States. A male factor may contribute to infertility in up to 50% of couples. The etiology of male factor infertility is complex and for many men (up to 45%), no cause is identified. This study aims to revisit the role of sperm chromatin in fertility and assess its potential as a biomarker of idiopathic male infertility. Specifically, in this proposal we aim to evaluate sperm-specific protamines and their modifications in the setting of both normal and altered fertility. Protamines are small, highly-basic proteins essential for proper compaction of paternal DNA. In humans, two forms of protamine are necessary for correct packaging of DNA and normal fertility: protamine 1 (P1) and protamine 2 (P2). Prior studies utilizing gel electrophoresis have suggested that maintenance of the P1:P2 ratio is critical for normal fertility and spermatogenesis, however, previously used methodologies are unable to resolve different protamine isoforms. Furthermore, in 2014 mouse protamines were found to bear a number of post-translational modifications (PTMs). The function of these newly identified PTMs is unknown, however, preliminary data suggests that they are important for normal fertility in the mouse. In the human, the full spectrum of protamine PTMs is not known. In this proposal, we aim to further explore the presence and significance of protamine isoforms and PTMs in the setting of both normal and abnormal fertility. We hypothesize that human protamines will bear a number of post-translational modifications and that protamine isoforms and PTMs will be similar among fertile men with normal sperm. We further hypothesize that protamine isoforms and a subset of PTMs will be altered in men with infertility and abnormal spermatogenesis. To test these hypotheses, we will utilize highly accurate and quantitative nano-liquid chromatography mass spectrometry to assess both protamine isoforms and modifications. To begin to understand the acquisition of newly identified protamine PTMs we will also generate modification-specific antibodies and determine their presence in human testicular samples at various stages of the seminiferous tubule epithelial cycle. These pilot experiments will provide an important first step in understanding the role of protamine isoforms and PTMs in fertility and will allow for future studies assessing functional significance and large-scale clinical investigation.

在美国，8-15% 的夫妇患有不孕不育症。男性因素可能导致高达 50% 的不孕不育 的情侣。男性因素不育的病因很复杂，对于许多男性（高达 45%）来说，没有任何原因可以解释。 确定。本研究旨在重新审视精子染色质在生育能力中的作用，并评估其作为精子染色质的潜力。 特发性男性不育症的生物标志物。具体来说，在本提案中，我们的目标是评估精子特异性 鱼精蛋白及其在正常和改变生育能力的情况下的修饰。鱼精蛋白很小， 高碱性蛋白质对于正确压缩父本 DNA 至关重要。在人类中，鱼精蛋白有两种形式： DNA 正确包装和正常生育能力所必需的：鱼精蛋白 1 (P1) 和鱼精蛋白 2 (P2)。事先的 利用凝胶电泳的研究表明，维持 P1:P2 比例对于正常情况至关重要 然而，以前使用的方法无法解决生育和精子发生的不同问题 鱼精蛋白亚型。此外，2014 年，小鼠鱼精蛋白被发现带有许多翻译后蛋白。 修饰（PTM）。这些新发现的 PTM 的功能尚不清楚，但初步数据显示 表明它们对于小鼠的正常生育能力很重要。在人类中，全谱鱼精蛋白 PTM 未知。在本提案中，我们旨在进一步探讨鱼精蛋白的存在和意义 正常和异常生育情况下的亚型和 PTM。我们假设人类鱼精蛋白 将进行许多翻译后修饰，并且鱼精蛋白亚型和 PTM 将相似 在具有正常精子的生育能力男性中。我们进一步假设鱼精蛋白亚型和 PTM 的子集 患有不育症和精子发生异常的男性会发生改变。为了检验这些假设，我们将利用 高精度和定量的纳米液相色谱质谱法来评估鱼精蛋白 同工型和修饰。为了开始了解新鉴定的鱼精蛋白 PTM 的获取，我们将 还产生修饰特异性抗体并确定它们在人类睾丸样本中的存在 生精小管上皮周期的各个阶段。这些试点实验将提供重要的第一 进一步了解鱼精蛋白亚型和 PTM 在生育力中的作用，并将为未来的研究做好准备 评估功能意义和大规模临床研究。