PORTABILITY OF BIOCHEMICAL MARKERS OF SPERM MATURITY

精子成熟度生化标记物的可移植性

• 批准号: 6344562
• 负责人: GABOR B HUSZAR
• 金额: $ 24.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2002-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344562
• 关键词: antibody; biomarker; cell age; clinical research; creatine kinase; fertility; gene expression; human subject; immunocytochemistry; in situ hybridization; male; male reproductive system; method development; protein binding; protein isoforms; sperm; sperm analysis; spermatogenesis

In each ejaculate there are populations of mature and diminished maturity sperm. The decline of the mature population, which affects male fertility and pregnancy outcome, may be measured by sophisticated objective biochemical methods which, at the present time, are available only in a few academic centers. The goal of the present project, in line with the goals of the RFA, is the introduction of biochemical markers in addition to the conventional semen profile, in the monitoring of potential changes in sperm maturity, due to male reproductive toxicity. The accessibility of assays will be facilitated by kits usable at the exposure sites, or by conservation of sperm for transport to a central laboratory. Three paradigms are considered: 1) Semen preparation on site and shipping the samples to a central laboratory; 2) Performance of some tests on-site and shipping semen for others 3) Home collection by subjects and shipping of semen. The specific aims are structured accordingly. First, in the first year we will develop the preservation and shipping of samples for tests routinely used in our laboratory: sperm creatine kinase (CK)-activity, CK-M isoform ratio, and CK- immunostaining for CK-B in order to highlight immature sperm, and for CK-M in order to demonstrate CK-M expression in mature sperm. We will also introduce, on sperm slides sent to us the morphometric probes of sperm maturity, including sperm tail length, tail/head ratio, and midpiece area and shape. Second, in further tests, aspects of the portability need development: sperm binding to hyaluronic acid(HA)-beads indicating maturity, DNA nick translation and the single sperm DNA comet assay, which highlight sperm DNA fragmentation, a known cause of male infertility and increased rates of pregnancy loss. Third, in response to this RFA, we are developing new assessment approaches: optimization of semen shipping in conditions, study of sperm viability with visible wavelength stains, preservation of the proportion of viable sperm at time of ejaculation, CK-M antibody coupled to chromophores for one-step sperm immunostaining, measurement of CK-M directly in semen by a sandwich immuno-assay. Because reproductive toxicity may selectively affect various features of sperm maturation and fertility, we propose to test the same sperm with more than one marker. Examples in preliminary results: HAbead-binding and CK- immunocytochemistry, HAbead-binding and viability DNA nick translation and tail/head ratio, aniline blue chromatin assay and CK-staining. We will identify and make accessible the most effective and practical methods, in order to simplify the monitoring of reproductive toxicity.

在每一次射精中都有成熟度和成熟度降低的精子。影响男性生育力和妊娠结局的成熟人口的下降，可以通过复杂的客观生化方法来衡量，目前只有少数几个学术中心有这种方法。根据RFA的目标，本项目的目标是在常规精液图谱的基础上引入生化标记物，以监测由于男性生殖毒性导致的精子成熟度的潜在变化。通过在暴露地点使用试剂盒或保存精子以便将精子运送到中心实验室，将促进分析的可及性。考虑了三种模式：1)现场精液准备，并将样本运送到中心实验室；2)现场进行一些测试，并为其他测试运送精液；3)受试者在家收集精液，并运送精液。具体的目标是相应地构建的。首先，在第一年，我们将发展样品的保存和运输，用于我们实验室常规的测试：精子肌酸激酶(CK)活性、CK-M亚型比率，以及CK-B的CK-B免疫染色以突出未成熟精子，以及CK-M的CK-M以显示成熟精子中CK-M的表达。我们还将在寄给我们的精子载玻片上介绍精子成熟度的形态测量探针，包括精子尾长、尾头比、中段面积和形状。其次，在进一步的测试中，可携带性的一些方面需要开发：精子与透明质酸(HA)珠子的结合，DNA缺口翻译和单精子DNA彗星试验，这些试验突出了精子DNA碎片，这是男性不育和妊娠失败率增加的已知原因。第三，针对这种RFA，我们正在开发新的评估方法：优化精液运输条件，用可见波长染色研究精子存活率，保存射精时精子存活的比例，将CK-M抗体与生色团偶联一步法精子免疫染色，用夹心免疫分析法直接测定精液中的CK-M。由于生殖毒性可能选择性地影响精子成熟和生育的各种特征，我们建议用多个标记检测同一精子。初步结果中的例子：HAbead结合和CK免疫细胞化学，HAbead结合和活性DNA缺口平移和尾头比，苯胺蓝染色质分析和CK染色。我们将确定并提供最有效和实用的方法，以简化对生殖毒性的监测。