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

在美国，8%-15%的夫妇会受到不孕症的影响。男性因素可能导致高达50%的不育症 一对情侣。男性因素不育症的病因很复杂，对许多男性(高达45%)来说，没有病因是 已确认身份。这项研究旨在重新探讨精子染色质在生育中的作用，并评估其作为 特发性男性不育的生物标志物。具体地说，在这项提案中，我们的目标是评估精子特异性 精蛋白及其在生育力正常和异常情况下的修饰。鱼精蛋白很小， 高度碱性的蛋白质，对父亲DNA的适当紧凑是必不可少的。在人类中，鱼精蛋白有两种形式 正确包装DNA和正常生育所必需的：鱼精蛋白1(P1)和鱼精蛋白2(P2)。之前 利用凝胶电泳法进行的研究表明，维持P1：P2的比例对正常是至关重要的 然而，以前使用的方法无法解决不同的生育和精子发生 鱼精蛋白亚型。此外，在2014年，老鼠的精蛋白被发现带有一些翻译后的 修改(PTM)。然而，这些新发现的PTM的功能尚不清楚，但初步数据 这表明它们对小鼠的正常生育能力很重要。在人体内，鱼精蛋白的全谱 PTMS尚不清楚。在这项提案中，我们旨在进一步探讨鱼精蛋白的存在和意义 正常生育和异常生育环境中的异构体和PTMS。我们假设人类鱼精蛋白 将带有一些翻译后修饰，鱼精蛋白异构体和PTM将是相似的 在精子正常的生育男性中。我们进一步假设鱼精蛋白亚型和PTM的一个子集 会在不育和精子发生异常的男性身上发生改变。为了检验这些假设，我们将利用 高精度定量纳液相色谱质谱分析鱼精蛋白和鱼精蛋白 异构体和修饰。为了开始了解新发现的鱼精蛋白PTM的收购，我们将 也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不同阶段。这些试点实验将提供一个重要的第一个 理解鱼精蛋白异构体和PTMS在生育中的作用的步骤，并将为未来的研究奠定基础 评估功能意义和大规模临床调查。

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.