Epigenetic regulation of histone eviction in spermatogenesis

摘要 在美国，超过15%的夫妇患有不孕不育。这些病例中有一半是由于男性 诸如精子数低、精子形态和活力异常或原因不明的不良等因素 胚胎发生。哺乳动物精子表现出高度致密的核和染色质结构，仅有5- 正常细胞组蛋白补体的15%。这种转变发生在与核小体相关的组蛋白 在减数分裂后的雄性生殖细胞中，组蛋白乙酰转移酶将其乙酰化，以打开结构， 反过来，允许随后的驱逐和替换为鱼精蛋白-导致在 成熟的精子。组蛋白在精子中的异常滞留可能导致不育和胚胎发育不良，因此 这意味着雄配子的表观遗传调控是生殖的关键因素。我的初步数据 描述组蛋白翻译后修饰的动态变化，如乙酰化 精子发生。我的发现进一步证明，减数分裂前组蛋白的有条件删除 乙酰转移酶Gcn5导致精子异常，组蛋白滞留增加。建议的目标是 本研究旨在探讨组蛋白乙酰化在核小体清除过程中的功能和机制。 减数分裂雄性生殖细胞，并确定超额保留的父系组蛋白是否影响 胚胎转录、发育编程和胚胎发生。为了进行这些实验，我将 利用条件性小鼠模型通过去除组蛋白对组蛋白乙酰化进行功能性干扰 减数分裂前雄性生殖细胞中的乙酰转移酶Gcn5(Gcn5cKO)。在特定的目标1中，我将使用Gcn5cKO小鼠 确定组蛋白乙酰化缺失导致染色质重塑改变的机制 和组蛋白在成熟精子中的过量保留，以及组蛋白乙酰化减少的后果 在精子发生过程中影响基因转录。在具体目标2中，我将检查和早期的生育 Gcn5cKO雄性小鼠胚胎发育和转录的影响 父性组蛋白过多和相关的父性染色质可及性改变。据我们所知，这将是 是第一个有条件地去除减数分裂前生殖细胞中组蛋白乙酰转移酶的研究 测试并调查功能后果。作为这些实验的结果，我们希望进一步发展我们的 了解组蛋白残基的乙酰化和组蛋白驱逐之间的联系，并确定 父亲过多的组蛋白的传递会影响后代的发育。此外，作为老鼠和人类， 精子发生和精子功能是高度保守的，这些实验的结果将是 为了解、诊断和治疗男性不育提供了宝贵的信息来源。

ABSTRACT Over 15% of couples in the United States suffer from infertility. Half of these cases are due to male factors such as low sperm count, abnormal sperm morphology and motility, or unexplained poor embryogenesis. Mammalian sperm exhibit a highly compacted nuclear and chromatin structure with only 5- 15% of the normal cellular histone complement. This transition occurs when nucleosome-associated histones within post-meiotic male germ cells are acetylated by histone acetyltransferases to open the structure to, in turn, allow subsequent eviction and replacement with protamines - leading to the profound compaction in mature sperm. Abnormal histone retention in sperm can lead to infertility and poor embryogenesis, thus implicating epigenetic regulation of the male gamete as a critical factor in reproduction. My preliminary data depict dynamic changes in histone post-translational modifications, such as acetylation, during spermatogenesis. My findings further demonstrate that pre-meiotic conditional deletion of the histone acetyltransferase Gcn5 results in abnormal sperm with increased histone retention. The goal of the proposed study is to investigate the functional and mechanistic role of histone acetylation in nucleosome eviction in post- meiotic male germ cells, and to determine whether contribution of excess retained paternal histones affects embryonic transcription, developmental programming and embryogenesis. To conduct these experiments I will use the conditional mouse model to functionally perturb histone acetylation through ablation of the histone acetyltransferase Gcn5 in pre-meiotic male germ cells (Gcn5cKO). In specific aim 1, I will use the Gcn5cKO mice to determine the mechanisms through which loss of histone acetylation leads to altered chromatin remodeling and excess histone retention in mature sperm, and how the consequences of reduced histone acetylation affects gene transcription during spermatogenesis. In specific aim 2, I will examine fertility of, and early embryonic development and transcription, resulting from the Gcn5cKO male mice to investigate the effect of excess paternal histones and associated altered paternal chromatin accessibility. To our knowledge, this will be the first investigation to conditionally ablate a histone acetyltransferase in pre-meiotic germ cells in the testes and investigate the functional consequences. As a result of these experiments we expect to further our understanding of the link between acetylation of histone residues and histone eviction and determine if transmission of excess paternal histones affects offspring development. Furthermore, as mouse and human spermatogenesis and sperm function are highly conserved, the findings of these experiments will be an invaluable source of information for understanding, diagnosing, and treating male infertility.