EFFECTS OF AMINO ACIDS AND THEIR METABOLITES ON THE DEVELOPMENT OF ORGANOTYPICAL TISSUE CULTURE OF TESTES IN RATS

THE AIM of the investigation was to study effects of extra low doses (10⁻¹⁰ – 10⁻¹⁴ mol/l) of 20 coded amino acids and their metabolites on the development of organotypical culture of rat testes. MATERIAL AND METHODS. The testes of 3 months old Wistar rats were divided into explants (1 mm³ fragments). The explants were placed in a Petri dish onto collagen and cultured in a CO₂-incubator in nutrient medium. Three days later the growth zone consisting of Leydig cells, Sertoli cells and spermatozoa was formed. For a quantitative assessment the area index was determined as the ratio of the total explant area to the central zone area. RESULTS. Reliable effects on the area index more than in one concentration was noted in 6 out of 20 coded amino acids: isoleucine (31±6%), lysine (25±4%), arginine (26±5%). None of the structural analogues of isoleucine (leucine, norleucine, valine) proved to be similarly effective. From all metabolites of L-arginine the testes growth is stimulated by L-ornitine (26±4%), urea (31±4%), creatine (20±3%); and L-citrulline (22±3%); and inhibited by nitric oxide (nitroglycerine – 100%). Glutatione eliminates antiproliferative effects of nitric oxide (25±7% vs 13±3% in concentration 10⁻¹³ mol/l) and thus increases effectiveness of L-arginine. In forming the mixture of the most active amino acids the arginine concentration must be 10 times more than the concentration of L-ornitine and creatine, but 10 times less that the concentration of L-lysine, L-isoleucine and L-citrulline. CONCLUSION. L-isoleucine, L- arginine and L-lysine are the most perspective amino acids for treatment of male infertility in clinical practice. It was first proved that L-isoleucine is maximally active for growth of testes. Nitric oxide is the only L-arginine metabolite with antiproliferative effect. A combination of L-arginine with glutathione eliminates the toxic effect of nitric oxide. A share correlation of amino acids necessary for maximal stimulation of the testes is established.

1. Западнюк ВИ, Купраж ЛП, Заика МУ, Безверхая ИС. Аминокислоты в медицине. «Здоров’я», Киев, 1982: 12-14

2. Боровец СЮ, Закуцкий АН, Субботина ТФ. Биохимические и клинические аспекты влияния L-аргинина на функциональную активность мужской половой системы. Учёные записки 2005; XII (3): 12-17

3. Roulet V, Denis H, Staub C. Human testis in organotypic culture: application for basic or clinical research. Hum Reprod 2006; 21 (6): 1564-1571

4. Lambrota R, Liveraa G, Coffignya H. A new method for toxicity assays on human and mouse fetal testis. Biochimie 2006; 88 (11): 1831-1839

5. Чалисова НИ, Князькин ИВ, Кветной ИМ. Нейроэндокринные механизмы действия пептидов и аминокислот в тканевых культурах. ДЕАН, СПб., 2005: 26-30

6. Чалисова НИ, Пеннияйнен ВА, Харитонова НВ, Ноздрачев АД. Динамика стимулирующих и ингибирующих эффектов в органотипической культуре нервной и лимфоидной ткани. Доклады Академии наук 2001; 380 (3): 418-421

7. Шевченко ИН. Международный симпозиум «Механизмы действия сверхмалых доз». Радиационная биология. Радиоэкология 2003; 43 (3): 261-384

8. Belougne-Malfatti E, Aguejouf O, Doutremepuich F et al. Combination of two doses of acetyl salicylic acid: experimental study of arterial thrombosis. Thromb Res 1998; 90 (5): 215-221

9. Lucas KA, Pitari GM, Kazerounian S et al. Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev 2000; 52 (3): 375-414

10. Gaston BM, Carver J, Doctor A, Palmer LA. S-Nitrosylation signaling in cell biology. Mol Intervent 2003; (3): 253-263

11. Salonia A, Rigatti P, Montorsi F. Sildenafil in erectile dysfunction: a critical review.Curr Med Res Opin 2003; 19 (3): 241-262

12. Bustamante JC, Setchell BP. The uptake of amino acids, in particular leucine, by isolated perfused testes of rats. J Androl 2000; 21 (3): 452-463

13. Сastillo L, Beaumier L, Ajami AM, Young VR. Whole body nitric oxide synthesis in healthy men determined from [15N]arginine-to-[15N]citrulline labeling. Proc Natl Acad Sci 1996; (93): 11460-11465

14. Белик ЯВ, Гриненко АГ, Смерчинская ЛС. Определение протеолитической активности тканей с использованием протамина в качестве субстрата. Укр Биохим Журнал 1968; 40 (4): 532-536

15. Solomonson LP, Flam BR Pendleton LC et al. The caveolar nitric oxide synthase/arginine regeneration system for NO production in endothelial cells. J Exp Biol2003; (206): 2083-2087

16. Брюне Б, Сандау К, фонКнетен А. Апоптическая гибель клеток и оксид азота: механизмы активации и антагонистические пути. Биохимия 1998; (63): 966-975