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.
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