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Y.Y. Yuan1 , L.Z. Mao1 , Q.X. Shi1 *, E.R.S. Roldan2 *, S.Q. Yu1
Zhejiang Academy of Medical Sciences, 60 Tian Mu Shan Rd, Hangzhou 310013, P.R. of China. 1
Instituto de Bioquimica (CSIC), Univ. Complutense 28040-Madrid, Spain. 2
This study was designed to investigate whether GABA stimulates PPI breakdown and its role during the AR. Spermatozoa were precapacitated in MCM-Ca2+-deficient medium with 3.5 mg BSA/ml for 4.5 h at 38.5° under 5% CO2 in air, and then labeled with 500 Ci [32P]Pi/ml for 1 h. Spermatozoa were then washed through a 30-50-75% Percoll gradient and exposed to 2 mM Ca2+ and 5 M GABA, 5 M progesterone, or 20 M A23187. Lipids were separated by t.l.c., and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The main results were as follows: (a) When spermatozoa were treated with GABA, 32P label diminished rapidly in phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). Loss of label from PPIs was almost complete by 5 min. The time-course of the AR was much slower: at 5, 10 and 15 min, about 20%, 25% and 45% of sperm have undergone the AR. (b) The pattern of PPI hydrolysis and the stimulation of AR was similar for the three agonists tested; their potency followed the order A23187 > progesterone GABA. (c) GABA-induced PIP2 hydrolysis and rise in PA was prevented by inclusion of 10 mM neomycin. In addition, neomycin blocked the AR in response to GABA, without affecting cell viability (control: 6.0±2.8% AR, GABA: 46.5±19.1% AR, GABA+neomycin: 5.0±2.4% AR). In conclusion, PPI breakdown is an essential event in the series of changes leading to membrane fusion during the AR of guinea-pig spermatozoa (Supported by “9.5” PDPC and NSFC No. 39870364).
This abstract is being presented on Monday, August 2 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.