Investigation of possible cholinergic mechanisms in fertilization ofXenopuseggs

Abstract

The hypothesis that cholinergic mechanisms play a role in fertilization was tested in Xenopus laevis. Acetylcholine (ACh) analyses were done with gas chromatography -- mass spectrometry. Eggs and testis-derived sperm contained material identified as acetylcholine (ACh, 0.19 pmol per egg and 0.6 pmol per 10$^{6}$ sperm). ACh release from oocytes was studied after immersion in hypotonic solutions, simulating events during natural oviposition. With or without sperm present, eggs released 30-40% of their ACh content in the first 2-3 min after hypotonic immersion, after which release decreased to low levels. Cholinesterase inhibition had no effect on gamete ACh content or release. The release of radioactivity from eggs preloaded with [$^{3}$H]ACh was not accelerated by immersion of eggs in hypotonic solution. Low levels of choline acetyltransferase were found in eggs and sperm. Cholinesterase activity was similar in intact and disintegrated sperm, but intact eggs showed negligible hydrolysis of external ACh compared to homogenized eggs. Cholinesterase activity of gametes was inhibited by neostigmine, diisopropylfluorophosphate (DFP) and diethyldimethyl-pyrophosphonate (DEPP). Nicotinic or muscarinic receptor-blocking agents, cholinesterase inhibitors or added electric eel acetylcholinesterase had no substantial effect on the percentage of eggs fertilized. Exposure of sperm to blockers of ACh receptors or to ACh in the highest concentrations likely to be encountered during fertilization had no effect on motility. We conclude that it is unlikely that cholinergic mechanisms are involved in sperm--egg interactions during fertilization of Xenopus, but the transient release of ACh on immersion of eggs into hypotonic solution may have biological significance.