Induction of capacitation and the acrosome reaction of boar spermatozoa by L-arginine and nitric oxide synthesis associated with the anion transport system.
The aim of this study was to determine the effect of L-arginine on nitric oxide (NO) synthesis, capacitation and acrosome reaction of boar spermatozoa. Ejaculated boar spermatozoa were washed and then cultured in a bicarbonate:CO(2)-buffered medium, modified NCSU-37, for 2 h. At the end of the culture, the status of spermatozoa was determined. The presence of (0.1-2.0 mmol l(-1)) L-arginine in the culture medium induced an acrosome reaction as determined by fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and increased intracellular NO content, as quantified by a fluorescent indicator, diaminofluorescein-2 diacetate (DAF-2 DA). This stimulatory effect of L-arginine was neutralized by supplementation with an NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (1 mmol l(-1)). However, the inactive enantiomorph, N(omega)-nitro-D-arginine methyl ester, did not affect the stimulatory effect of L-arginine. These results indicate that L-arginine induces an acrosome reaction through the NO signal pathway in boar spermatozoa. Furthermore, the stimulatory effect of L-arginine was inhibited in the presence of an anion transport inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS; 0.1 mmol l(-1)), whereas any responses of spermatozoa to caffeine were not inhibited by SITS. A stimulatory effect of L-arginine on capacitation and acrosome reaction of spermatozoa was also observed in modified NCSU37 medium by using a chlortetracycline fluorescence assay, but not in supplemented bicarbonate-free Tris-buffered medium. These results indicate that the presence of L-arginine induces nitric oxide synthesis and stimulates capacitation and acrosome reaction of boar spermatozoa only when active sperm anion transport is present as a result of bicarbonate supplementation.
Funahashi
H
H
Department of Animal Science, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan. hirofun@cc.okayama-u.ac.jp
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D020101:Acrosome Reaction; D000818:Animals; D000838:Anions; D017920:Antiporters / Q000037:antagonists & inhibitors; D001120:Arginine / Q000494:pharmacology*; D001639:Bicarbonates; D002478:Cells, Cultured; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000502:physiology*; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D013268:Stimulation, Chemical; D034421:Sus scrofa
D000838:Anions; D017920:Antiporters; D001639:Bicarbonates; D003470:Culture Media; D004791:Enzyme Inhibitors; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D019331:NG-Nitroarginine Methyl Ester