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

不孕症影响美国8-15%的夫妇。男性因素可能导致高达50%的不孕症 情侣。男性因素不育的病因是复杂的，对于许多男性（高达45%），没有原因是 鉴定本研究旨在重新审视精子染色质在生育中的作用，并评估其作为一种潜在的 特发性男性不育症的生物标志物。具体来说，在这项提案中，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的生育能力中的修饰。鱼精蛋白很小， 高碱性蛋白质，对父亲DNA的正确压缩至关重要。在人类中，有两种形式的鱼精蛋白， 正确包装DNA和正常生育力所必需的：鱼精蛋白1（P1）和鱼精蛋白2（P2）。之前 利用凝胶电泳的研究表明，维持P1：P2比例对于正常的 然而，以前使用的方法无法解决不同的生育力和精子发生， 鱼精蛋白同种型。此外，在2014年，发现小鼠鱼精蛋白携带许多翻译后修饰。 修改（PTM）。这些新发现的PTM的功能尚不清楚，然而，初步数据显示， 表明它们对小鼠的正常生育能力很重要。在人体内， PTM未知。在本提案中，我们旨在进一步探索鱼精蛋白的存在和意义 在正常和异常生育力的情况下，我们假设人类鱼精蛋白 将带有许多翻译后修饰，并且鱼精蛋白同种型和PTM将是相似的 精子正常的有生育能力的男性我们进一步假设鱼精蛋白亚型和一个PTM亚群 在不育和精子发生异常的男性中会发生改变。为了验证这些假设，我们将使用 高精度和定量的纳米液相色谱质谱法来评估鱼精蛋白 同种型和修饰。为了开始了解新发现的鱼精蛋白PTM的获取，我们将 还产生修饰特异性抗体，并在人睾丸样品中测定其存在， 生精小管上皮细胞周期的各个阶段。这些试点实验将提供重要的第一次 进一步了解鱼精蛋白亚型和PTM在生育中的作用，并将允许未来的研究 评估功能意义和大规模临床研究。

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.