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Aida Cancel1 *, Susan Jeffay2 , Sally Perreault2 *
Toxicology Program, UNC-Chapel Hill, NC 1
US EPA, NHEERL, RTP, NC 2
Previously we examined changes in rat spermatozoa associated with in vitro capacitation in Armstrong’s Rat Fertilization Medium (ARFM) supplemented with caffeine (CAFF, 1 mM) or heparin (HEP, 10 ug/ml). We found that the inclusion of HEP (but not CAFF) significantly enhanced fertilization of zona-free hamster eggs (both %eggs fertilized and mean number of decondensed sperm per egg). Sperm motion analysis showed increased sperm velocities with the appearance of hyperactivated motion after 4h incubation, but these changes did not, in themselves, account for the heparin-enhanced fertilization results. Therefore we hypothesized that heparin may enhance fertilization by stimulating the acrosome reaction. Because the rat sperm acrosome cannot be visualized using phase contrast microscopy, we used an FITC-peanut agglutinin (PNA) staining assay to assess rat sperm acrosome integrity. Sperm with an intact acrosome (AI) showed bright green fluorescence over the dorsal crest of the sperm head, while acrosome reacted and acrosome damaged sperm (AR) appeared dull green. Cauda epididymal sperm (0.33×105/ml) were cultured for 4h (38 C, 5% CO2) in ARFM alone, CAFF or HEP. At 0h, the average % motile and % AI were 63-70% and 70-61% respectively, suggesting that most motile sperm were AI. At 4h, the %AI spermatozoa had declined significantly in all treatments, while % motile declined <10%, suggesting that a significant proportion of sperm had undergone AR. There were no differences in %AI between HEP (24.9%), CAFF (28.1%) or ARFM alone (23.2%) at 4h. Therefore, we conclude that the heparin-enhanced fertilization seen in our previous experiments does not appear to be due to a stimulation of the acrosome reaction. Current efforts are directed at confirming these results using a dual staining method to detect AR in viable sperm. (Does not reflect EPA policy).
This abstract is being presented on Monday, August 2 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.