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: 6446031
• 负责人: GABOR B HUSZAR
• 金额: $ 23.29万
• 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 2003-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6446031
• 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，都有成熟和成熟度降低的精子。 成熟人口的下降影响男性生育力和妊娠结果，可以通过复杂的客观生物化学方法来测量，目前只有少数学术中心可以使用。 本项目的目标与RFA的目标一致，是在常规精液特征之外引入生化标志物，以监测由于雄性生殖毒性导致的精子成熟度的潜在变化。通过暴露部位可用的试剂盒或保存精子以运输至中心实验室，将有助于检测的可及性。 考虑了三种范例：1）现场制备精液并将样本运送至中心实验室; 2）现场进行一些测试并将精液运送至其他测试; 3）受试者在家采集精液并运送精液。 具体目标也相应地制定。 首先，在第一年，我们将开发用于我们实验室常规检测的样本的保存和运输：精子肌酸激酶（CK）活性、CK-M亚型比率和CK-B的CK免疫染色，以突出未成熟精子，以及CK-M，以证明CK-M在成熟精子中的表达。我们还将介绍，在精子切片发送给我们的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。 第二，在进一步的测试中，需要开发便携性方面：精子与透明质酸（HA）珠的结合表明成熟，DNA切口平移和单精子DNA彗星试验，突出精子DNA片段化，这是男性不育的已知原因，并增加了流产率。 第三，针对RFA，我们正在开发新的评估方法：优化精液运输条件，研究精子活力与可见波长染色，保存在射精时的活精子的比例，CK-M抗体偶联到发色团的一步精子免疫染色，测量CK-M直接在精液中的夹心免疫测定。 由于生殖毒性可能选择性地影响精子成熟和生育能力的各种特征，我们建议用一种以上的标记物检测同一精子。 初步结果中的示例：HA珠结合和CK免疫细胞化学、HA珠结合和活力DNA切口平移和尾/头比、苯胺蓝染色质测定和CK染色。 我们将确定并提供最有效和最实用的方法，以简化对生殖毒性的监测。