: 31 3 2004 Kor. J. Fertil. Steril., Vol. 31, No. 3, 2004, 9 Melatonin Effects of Melatonin on the Meiotic Maturation of Mouse Oocytes in vitro Hee-Jin Ahn, In-Ha Bae Department of Biology, Graduated School, Sungshin Women's University Objective: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. Material and Method: Immature mouse oocytes were obtained from the ovarian follicles of 3~4 weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at 37, 5% CO 2 and 21% O 2 (95% air) or 5% CO 2, 5% O 2 and 90% N 2. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of 0.0001 µm, 0.01 µm or 1.0 µm. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcamp) and/or hypoxanthine were included in the medium. Results: Under 21% oxygen condition, oocytes cultured in the presence of 0.01 µm melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with 0.0001 µm or 1.0 µm melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of 0.01 µm melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcamp alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcamp alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcamp. :, ) 136-742 3249-1, Tel: (02) 920-7171, Fax: (02) 927-5565, e-mail: ihbae@sungshin.ac.kr * 2001. - 155 -
Conclusion: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcamp or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals. Key Words: Mouse oocyte, Melatonin, dbcamp, Hypoxanthine, Oxygen (pineal gland) melatonin (N-acetyl-5-methoxytryptamine) / seasonal rhythm (ovarian function) (reproduction). melatonin (vaginal), (estrus cycle). 1 melatonin (offspring), (cattle), 14 (bovine), 15, 16 17, (age). 2 Melatonin (atresia) antral follicle. 3 melatonon, luteinizing hormone (LH). melatonin (serum) 3melatonin LH, 21 melatonin. 4 Brzezinski 5 melatonin progesterone melatonin. melatoninantigonadotropin gonadotropin-releasing hormone (GnRH). 6, Romero 7 system H 2 O 2 OH membrane-bound calmodulin melatoninbinding site melatonincalmodulinantagonist. Melatonin (melanophores) seasonal breeding mela- melatonin cyclic AMP (camp) Ca 2+. 8., 5% 21% 2-. 9, 10, 11,12 (sheep) 13 18,19. H 2 O 2. 20 super oxide dismutase (SOD),, hydroxyl radical, H 2 O 2. 10 (follicular fluid).,. 22 melatonin glutathione mannitol in vivo. 25 circadian rhythm tonin. 5% 21% melatonin - 156 -
. 1. 14, 10, (ICR strain) 3~4. 2. 5 I.U. (international unit)pregnant mare's serum gonadotropin (Sigma), 44. 0.2 mmdibutyryl cyclic AMP (dbcamp) M2 (M5A Wild, Swiss)26G. mouth-controlled micro- pipette (germinal vesicle; GV). 3. microdroplet.. 4.15 mm NaHCO 3, BSA (0.4%), (60 15 mm, Falcon) melatonin (N-acetyl-5-methoxytryptamine) 40 µlnew modified hank's balanced salt Solution (New MHBS) equilibrated mineral oil (light oil) 37, 5% CO 2 95% 100%, 37, 5% CO 2, 5% O 2 90% N 2 100% solution (-20), (Forma Scientific, Model 3130) 2, 20~25. 160 121, 15 Lb/inch 2 15. 4. Sigma (St Louis, MO). MHBS 280 mosm New MHBS. 98.70 mm NaCl, 5.37 mm KCl, 0.81 mm MgSO 4, 0.34 mm Na 2 HPO 4, 0.15 mm KH 2 PO 4, 1.71 mm CaCl 2, 5.56 mm glucose, 2.50 mm Na-Lactate, 0.30 mm Napyruvate, 10 mg/l phenol red, 100 units/ml penicillin-g, 52 mg/l streptomycin, 25.0 mm NaHCO 3, 0.4% bovine serum albumin (BSA) 3 1. 23 M2. 20.85 mm N-2-(hydroxyethyl) piperazine- N'-2-ethanesulfonic acid (HEPES) ph, 94.66 mm NaCl, 4.78 mm KCl, 1.19 mm MgSO 4, 1.19 mm KH 2 PO 4, 5.56 mm glucose, 100 units/ ml penicillin-g, 52 mg/l streptomycin 3 10 stock solution, 1.711 mm CaCl 2, 23.28 mm Na-Lactate, 0.33 mm Napyruvate100 stock solution stock solution 1. ph7.3~7.4 280~290 mosm. 24 dbcampdulbecco's phosphate-buffered salined (PBS) 20stock. Melatonin (0.0001 µm, 0.01 µm 1 µm). hypoxan- 90 thine10 stock solution - 157 -
1 mm. 5. 4 (germinal vesicle breakdown; GVBD) 17 1 (first polar body; PB) (inverted phase contrast microscope, Labovert, Leitz, Germany). (germinal vesicle; GV), (GVBD) Figure 1. Effects of melatonin on the meiotic maturation of mouse oocytes under 21% O 2 in vitro. The above results were obtained by pooling of ten replicates. *p<0.05, **p<0.005 Figure 2. Effects of melatonin on the meiotic maturation of mouse oocytes under 5% O 2 in vitro. The above results were obtained by pooling of seven replicates. - 158 -
, 1 (PB) (perivitelline space). 6. Stu- dent's t-test. 1. melatonin 21%5% Figure 3. Effects of melatonin on the polar body formation of mouse oocytes cultured for 17 hours under 21% O 2 or 5% O 2 in vitro. **p<0.005 Figure 4. Effects of melatonin on the meiotic maturation of mouse oocytes under 21% O 2 or 5% O 2 in vitro. **p<0.005-159 -
(0.0001 µm, 0.01 µm 1 µm)melatonin 4 (germinal vesicle breakdown; GVBD) 17 1 (first polar body; PB). 21% melatonin, 0.01 µm 1 (Figure 1). 5% (Figure 2, 3). Figure 5. Effects of melatonin on the germinal vesicle breakdown of mouse oocytes in the presence of dbcamp cultured for 4 hours under 21% O 2. The above results were obtained by pooling of nine replicates. *p<0.05, **p<0.005 Figure 6. Effects of melatonin on the polar body formation of mouse oocytes in the presence of dbcamp cultured for 17 hours under 21% O 2 in vitro. The above results were obtained by pooling of nine replicates. *p<0.05, **p<0.005-160 -
2. melatonin 21% Figure 1 0.01 µm melatonin, 5% melatonin., 21% melatonin 0.01 µm 1 5% melatonin Figure 7. Effects of melatonin on the germinal vesicle breakdown of mouse oocytes in the presence of dbcamp cultured for 4 hours under 5% O 2 in vitro. The above results were obtained by pooling of seven replicates. Figure 8. Effects of melatonin on the polar body formation of mouse oocytes in the presence of dbcamp cultured for 17 hours under 5% O 2 in vitro. The above results were obtained by pooling of seven replicates. *p<0.05, **p<0.005-161 -
1 (p<0.005), (Figure 4). 3. dbcamp melatonin dibutyryl cyclic AMP (dbcamp) melatonin. 21% 0.1 mm dbcamp melatonin Figure 9. Effects of melatonin on the polar body formation of mouse oocytes in the presence of dbcamp cultured for 17 hours under 21% O 2 or 5% in vitro. *p<0.05, **p<0.005 Figure 10. Effects of melatonin on the germinal vesicle breakdown of mouse oocytes in the presence of hypoxanthine cultured for 4 hours under 21% O 2 in vitro. The above results were obtained by pooling of eight replicates. - 162 -
melatonin dbcamp (Figure 5, 6)., 5% 21% (Figure 7, 8, 9). 4. Hypoxanthine mela- tonin hypoxanthine Figure 11. Effects of melatonin on the polar body formation of mouse oocytes in the presence of hypoxanthine cultured for 17 hours under 21% O 2 in vitro. The above results were obtained by pooling of eight replicates. *p<0.05 Figure 12. Effects of melatonin on the germinal vesicle breakdown of mouse oocytes in the presence of hypoxanthine cultured for 4 hours under 5% O 2 in vitro. The above results were obtained by pooling of seven replicates. - 163 -
melatonin. 21%, 10.0001 µm0.01 µmmelatonin melato- ninhypoxanthine GV arrest (Figure 10, 11). hypoxanthinegybd 5% O 2 (Figure 12). Figure 13. Effects of melatonin on the polar body formation of mouse oocytes in the presence of hypoxanthine cultured for 17 hours under 5% O 2 in vitro. The above results were obtained by pooling of seven replicates. *p<0.05, **p<0.005 Figure 14. Effects of melatonin on the polar body formation of mouse oocytes in the presence of hypoxanthine cultured for 17 hours under 21% O 2 or 5% O 2 in vitro. *p<0.05, **p<0.005-164 -
Hypoxanthine 4 (17 ) (Figure 13) 21% (Figure 14). (free radical oxygen, OH) (toxity)dna (death). mannitol, glutathione melatonin. 6 melatonin melatonin, 26 melatonin. 27 21% 5% melatonin., 21% 0.01 µmmelatonin (GVBD)1 (PB) (Figure 1). 5% melatonin. 1 21% 0.01 µmmelatonin (Figure 2, 3)., melatonin21%, mrna oxygenstress 5% 21% - 165 - melatonin 0.01 µmmelatonin, 5% melatonin. 21% 0.01 µm melatonin 5% melatonin 1 (Figure 4). melatoninoxygenstress 5% 21% 0.01 µm oxygen stress. (21%). Dumoulin 12. melatonin rat lipid peroxiation anti- oxidant status, 28 β-cells OH alloxan. 29,30 OH stress melatonin oxidative DNA damage, 32 brain cortexantioxidant enzyme DNA. 33,34, (in vitro fertilization)melatonin '2-cell block'. 31
(blastocyst). 22 dbcamp melatonin, melatonindbcampgv arrest (Figure 5, 6) 21% 5% H 2 O 2 (Figure 7, 8, 9). dbcamp. 37~40 camp adenylate cyclase dbcamp dbcamp campmimic camp. melatonin (melanophores) camp. 8 dbcamp melatonin. melatonindbcamp camp 5% oxygen stress melato- nin (synergic effect). hypoxanthine dbcamp. 1 mmhypoxnathine melatonin 0.0001 µm0.01 µmmelatonin 1 (Figure 10, 11, p<0.05). (5%) (21%) - 166 - hypoxanthine melatonin (syner- gic effect) (Figure 12, 13, 14). Hypoxanthine xanthine oxi- dase uric acid, uric acid (blastomere). 35 Hypoxanthine, 36 dbcamp phosphodiesteraseactivity camp melatoninhypoxanthine camp H 2 O 2., melatonin oxygen stress dbcamp hypoxanthine. Melatonin. 1. Villanua MA, Agrasal C, Esquifino AI. Meonatal melatonin administration advances rat vaginal open- ing and disrupts estrous cyclicity and estrogendependent regulatory mechanisms of luteinzing hormone and prolactin. J Pineal Res 1989; 7: 165-74. 2. Fernandez B, Diaz E, Colmenero MD, Diaz B. Maternal poneal gland participates in prepubertal rat's ovarian oocyte development. Anat Rec 1995; 243: 461-5. 3. Spanel-Borowski K, Richardson BA, King TS, Pet- terborg LJ, Reiter RJ. Follicular growth and intrao- varian and extraovarian oocyte release after daily injections of melatonin and 6-chloro-melatonin in the Syrian hamster. Am J Anat 1983; 167: 371-80. 4. Ying SY, Greep RO. Inhibition of ovulation by me-
latonin in the cyclic rat. Endocrinology 1973; 92: 333-5. 5. Brzezinski A, Fibich T, Cohen M, Schenker JG, Laufer N. Effects of melatonin on progesterone production by human granulosa lutein cells in culture. Fertil Steril 1992; 58: 526-9. 6. Hadley M. Endocrinology (fourth edition). Department of anatomy, university of arizona. 1996; 458-76. 7. Romero MP, Garcia-Perganeda A, Guerrero JM, Osuna C. Membrane-bound calmodulin in Xenopus laevis oocytes as a novel binding site for melatonin. FASEB J 1998; 12: 1401-8. 8. Martensson LG, Andersson RG. Is Ca 2+ the second messenger in the response to melatonin in cuckoo wrasse melanophores? Life Sci 2000; 66: 1003-10. 9. Auerbach S, Brinster RL. Effect of oxygen concentration on the development of two-cell mouse embryos. Nature 1968; 3; 217: 465-6. 10. Noda Y, Matsumoto H, Umaoka Y, Tatsumi K, Kishi J, Mori T. Involvement of superoxide radicals in the mouse two-cell block. Mol Reprod Dev 1991; 28: 356-60. 11. Dumoulin JC, Vanvuchelen RC, Land JA, Pieters MH, Geraedts JP, Evers JL. Effect of oxygen concentration on in vitro fertilization and embryo culture in the human and the mouse. Fertil Steril 1995; 63: 115-9. 12. Dumoulin JC, Meijers CJ, Bras M, Coonen E, Geraedts JP, Evers JL. Effect of oxygen concentration on human in-vitro fertilization and embryo culture. Hum Reprod. 1999; 14: 465-9. 13. Thompson JG, Simpson AC, Pugh PA, Donnelly PE, Tervit HR. Effect of oxygen concentration on invitro development of preimplantation sheep and cattle embryos. J Reprod Fertil 1990; 89: 573-8. 14. Watson AJ, Watson PH, Warnes D, Walker SK, Armstrong DT, Seamark RF. Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere. Biol Reprod 1994; 50: 715-24. 15. Liu Z, Foote RH. Development of bovine embryos in KSOM with added superoxide dismutase and taurine and with five and twenty percent O 2. Biol Reprod 1995; 53: 786-90. 16. Li J, Foote RH. Culture of rabbit zygotes into blastocysts in protein-free medium with one to twenty per cent oxygen. J Reprod Fertil 1993; 98: 163-7. 17. Johnston LA, Donoghue AM, O'Brien SJ, Wildt DE. Influence of temperature and gas atmosphere on in-vitro fertilization and embryo development in domestic cats. J Reprod Fertil 1991; 92: 377-82. 18. Pabon JE Jr, Findley WE, Gibbons WE. The toxic effect of short exposures to the atmospheric oxygen concentration on early mouse embryonic development. Fertil Steril 1989; 51: 896-900. 19. Nonogaki T, Noda Y, Narimoto K, Umaoka Y, Mori T. Effects of superoxide dismutase on mouse in vitro fertilization and embryo culture system. J Assist Reprod Genet 1992; 9: 274-80. 20. Nasr-Esfahani MH, Aitken JR, Johnson MH. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development 1990; 109: 501-7. 21. Yie SM, Brown GM, Liu GY, Collins JA, Daya S, Hughes EG, Foster WG, Younglai EV. Melatonin and steroids in human pre-ovulatory follicular fluid: seasonal variations and granulosa cell steroid production. Hum Reprod 1995; 10: 50-5. 22. Ishizuka B, Kuribayashi Y, Murai K, Amemiya A, Itoh MT. The effect of melatonin on in vitro fertilization and embryo development in mice. J Pineal Res 2000; 28: 48-51. 23. Bae IH, Channing CP. Effect of cakium in the maturation of cumulus enclosed pig follicular oocytes isolated from medium-sized graafian follicles. Biol Reprod 1985; 33: 79-87. 24. Bae IH, Foote RH. Maturation of rabbit follicular oocytes in a defined medium of varied osmolality. J Reprod Fert 1980; 59: 11-3. - 167 -
25. Reiter RJ, Tan DX, Poeggeler B, Menendez-Pelaez A, Chen LD, Saarela S. Melatonin as a free radical scavenger: implications for aging and age-related diseases. Ann N Y Acad Sci 1994; 719: 1-12. Review. 26. Acuna-Castroviejo D, Martin M, Macias M, Escames G, Leon J, Khaldy H, Reiter RJ. Melatonin, mitochondria, and cellular bioenergetics. J Pineal Res 2001; 30: 65-74. Review 27. Wakatsuki A, Okatani Y, Shinohara K, Ikenoue N, Kaneda C, Fukaya T. Melatonin protects fetal rat brain against oxidative mitochondrial damage. J Pineal Res 2001; 30: 22-8. 28. Vural H, Sabuncu T, Arslan SO, Aksoy N. Melatonin inhibits lipid peroxidation and stimulates the antioxidant status of diabetic rats. J Pineal Res. 2001; 31: 193-8. 29. Bromme HJ, Morke W, Peschke D, Ebelt H, Peschke D. Scavenging effect of melatonin on hydroxyl radicals generated by alloxan. J Pineal Res 2000; 29:201. 30. Ebelt H, Peschke D, Bromme HJ, Morke W, Blume R, Peschke E. Influence of melatonin on free radical-induced changes in rat pancreatic beta-cells in vitro. J Pineal Res 2000; 28: 65-72-8. 31. Bandyopadhyay D, Biswas K, Bandyopadhyay U, Reiter RJ, Banerjee RK. Melatonin protects against stress-induced gastric lesions by scavenging the hydroxyl radical. J Pineal Res 2000; 29: 143-51. 32. Morioka N, Okatani Y, Wakatsuki A. Melatonin protects against age-related DNA damage in the brains of female senescence-accelerated mice. J Pineal Res 1999; 27: 202-9. 33. Kotler M, Rodriguez C, Sainz RM, Antolin I, Menendez-Pelaez A. Melatonin increases gene expression for antioxidant enzymes in rat brain cortex. J Pineal Res 1998; 24: 83-9. 34. Qi W, Reiter RJ, Tan DX, Manchester LC, Siu AW, Garcia JJ. Increased levels of oxidatively damaged DNA induced by chromium (III) and H 2 O 2 : protection by melatonin and related molecules. J Pineal Res 2000; 29: 54-61. 35. Loutradis D, John D, Kiessling AA. Hypoxanthine causes a 2-cell block in random-bred mouse embryos. Biol Reprod 1987; 37: 311-6. 36. Nasr-Esfahani MM, Johnson MH. The origin of reactive oxygen species in mouse embryos cultured in vitro. Development 1991; 113: 551-60. 37. Downs SM, Coleman DL, Ward-Bailey PF, Eppig JJ. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid. Proc Natl Acad Sci U S A 1985; 82: 454-8. 38. Warikoo PK, Bavister BD. Hypoxanthine and cyclic adenosine 5'-monophosphate maintain meiotic arrest of rhesus monkey oocytes in vitro. Fertil Steril 1989; 51: 886-9. 39. Tornell J, Brannstrom M, Magnusson C, Billig H. Effects of follicle stimulating hormone and purines on rat oocyte maturation. Mol Reprod Dev 1990; 27: 254-60. 40. Downs SM. Purine control of mouse oocyte maturation: evidence that nonmetabolized hypoxanthine maintains meiotic arrest. Mol Reprod Dev 1993; 35: 82-94. - 168 -