Monday, August 2, 2004
10:30 AM–12:30 PM
(226) THE FIRST EVIDENCE FOR NITRIC OXIDE AS A SPERM CHEMOATTRACTANT IN THE FATHEAD MINNOW.
McAuliffe, Maureen1, Wilson, Desiree1, Scott, Condra1, Ren, Qun1, Lyon, Beth1, Atherton, Robert, 1 University of Wyoming, Laramie, WY
ABSTRACT- Our previous data demonstrated that fathead minnow oocytes stain positively for NADPH-d (nicotinamide-adenine dinucleotide phosphate diaphorase), confirmation for the existence of nitric oxide synthase (NOS) and that nitric oxide (NO) stimulates teleost sperm motility. The aims of the current studies were to determine the specific isoform of NOS, to quantify NO production by each gamete, and to examine the evidence for NO as a sperm chemoattractant. RT-PCR demonstrated that eggs contain the message for iNOS and nNOS, while sperm contain the message for iNOS and eNOS. An SEM analysis revealed the oocyte micropyle to contain eight ridges and grooves that direct sperm to the egg surface. NOS was then localized to the micropyle by immunocytochemistry. Using quantitative chemiluminescent spectroscopy, eggs on a per cell basis, produced significantly more NO than sperm and produced a gradient of NO during the time period in which fertilization usually occurs. The gradient was abolished with pre-incubation using a NOS inhibitor, aminoguanidine hemisulfate (AGHS). Finally, chemotaxis studies demonstrated that sperm numbers are significantly higher (P< 0.05) near capillary tubes containing egg water or an NO donor when compared with the control buffers. Capillary tips containing the supernatant from eggs incubated with AGHS did not have sperm concentration significantly different from controls. These observations establish the NOS isoform in both teleost gametes and expand the family of chemoattractant molecules to include the diatomic gas, NO.
KEY WORDS: nitric oxide, fish, fertilization, gametes