OBJECTIVE ANALYSIS OF SPERM MOTION AND MEMBRANE FUNCTION

MULTIPARAMETER ANALYSIS OF SPERMATOZOAL FUNCTION IS NECESSARY FOR ACCURATE ASSESSMENT OF VIABILITY. BY ADAPTINGAN OBJECTIVE, AUTOMATED SPERM MOTION ANALYZER (HTM-2000) TO MEASURE FLUORESCENCE INTENSITY OF VARIOUS SUPRAVITAL STAINS AND MEMBRANE PROBES, AN ASSAY WILL BE DEVELOPED WHICH SIMULTANEOUSLY EVALUATES MOTILITY PARAMETERS (MOTILE FRACTION, VELOCITY, LINEARITY, ETC.) AND MEMBRANE INTEGRITY ON A LARGE NUMBER OF INDIVIDUAL SPERMATOZOA. SPECIFIC AIMS FOR THIS PHASE I PROPOSAL ARE: 1. DEVELOP SUITABLE FLUOROCHROMES FOR PROBING SPERM MEMBRANES USING THE EQUINE AS A PRIMARY MODEL. 2. USE SELECTED FLUOROCHROMES TO DEVELOP SYSTEM FOR SIMULTANEOUSLY MEASURING MOTILITY PARAMETERS AND FLUORESCENTINTENSITY OF SPERMATOZOA. 3. CONDUCT EXPERIMENTS TO DETERMINE CORRELATION BETWEEN VARIOUS KNOWN TYPES OF CELL DAMAGE WITH FLUORESCENT INTENSITY AND SPERM MOTILITY PATTERNS. EFFICIENCY OF FLUOROCHROME LABELLING AND DETECTION WILL BE EVALUATED BY INTENTIONALLY DISRUPTING SPERM MEMBRANES WITH SNAP FREEZING, DIGITONIN OR TRITON X 100 TREATMENT. MIXTURES OF INTACT AND DAMAGED SPERM WILL BE EXAMINED AND MOTILITY AND FLUORESCENT INTENSITY WILL BE CORRELATED WITH MEMBRANE INTEGRITY. THE TECHNOLOGY DEVELOPED WOULD HAVE MANY RESEARCH AND COMMERCIAL APPLICATIONS, INCLUDING: 1) POTENTIAL FERTILITY EVALUATION, AND INFERTILITY DIAGNOSIS, AND 2) METHOD FOR EVALUATION OF VARIOUS SEMINAL TREATMENTS, SUCH AS IN VITRO CAPACITATION AND ACROSOME REACTION MEDIA, CRYOPRESERVATION TECHNIQUES, AND RESPONSE TO POSSIBLE TOXINS.