Yavuz ÖZTÜRKLER, SAVAŞ YILDIZ, ÖRSAN GÜNGÖR, ŞÜKRÜ METİN PANCARCI, Cihan KAÇAR, UMUT ÇAĞIN ARI

The effects of L-ergothioneine and L-ascorbic acid on the in vitro maturation (IVM) and embryonic development (IVC) of sheep oocytes

Bu çalışmanın amacı koyun oositlerinin in vitro olgunlaştırılması ve olgunlaşmış oositlerin embriyonal gelişimi üzerine L-Ergothiyonin (LE) ve L-askorbik asit (LAA)’in etkilerini karşılaştırmaktı. Mezbahaneden toplanan ovaryumlar % 0.9’luk ılık (30-35°C) fizyolojik tuzlu suda (1-2 saat içerisinde) labaratuvara sevkedildi. Kumulus Oosit Kompleksleri folliküllerin ensizyonu ile elde edildi. Sadece 3 katlı cumulus hücre tabakasına sahip Kumulus Oosit Kompleksleri TCM199 [%10 Fötal Buzağı Serumu (FCS) (v/v), Follikül stimüle edici hormone (FSH), Luteinleştirici Hormon (LH) ve Sodyum Piruvat içeren] içinde 24 saat süreyle in vitro maturasyona (IVM) alındı. IVM ardından, genişlemiş cumulus tabakasına sahip Kumulus Oosit Kompleksleri IVF TALP mikro-droplarına aktarıldı. Koçlardan elde edilen taze sperma Percoll-Gradientle separe edildi ve heparin (30 IU/ml) içeren Hepes TALP ile kapasite edildi. Separe ve kapasite edilen spermatozoonlar, olgunlaşmış ositleri içeren IVF TALP mikrodroplarına aktarıldı ve 20 saat süreyle inkübe edildi. Olası zigotlar TCM 199’a aktarılarak 7-8 gün in vitro kültüre edildi. Çalışmanın sonuçları, LE eklenmiş grupta, sayısal olarak daha yüksek olan yarıklanma oranı hariç (P≥0.05), 8-hücreli ve morula oranları açısından LAA ve kontrol gruplarından önemli derecede yüksekti (P

Anahtar Kelimeler:

oosit, FSH, LH, askorbik asit, kumulus ooforus, embriyonik gelişim, embriyo, koyunlar, in vitro döllenme, oosit olgunlaşması, yumurtalıklar, koyun, zigot

Koyun oositlerinin in vitro maturasyonu (IVM) ve embriyonal Gelişimi (IVC) üzerine L-Ergotiyonin ve L-askorbik asit’in etkileri

The aim of this study was to compare the effects of L-ergothioneine (LE) and L-ascorbic acid (LAA) on the IVM of immature oocytes and embryonic development of in vitro matured oocytes in sheep. The ovaries were collected from slaughterhouse, then they were transferred (within 1-2 hs) to the laboratory at warm (30-35°C) 0.9% saline solution. Cumulus Oocyte Complexes (COCs) were obtained by incision of follicles. Oocytes covered with at least three compact layers of cumulus oophorus cell were in vitro matured in TCM 199 with 10% Foetal Calf Serum (FCS) (v/v), Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Na Pyruvate with supplemented of 10 mM of LE (Group 1), 10 mM LAA (Group 2) or without any antioxidant (Group 3: Control) at 24 h. COCs with expanded cumulus layers following IVM were transferred to IVF TALP micro-drops with or without antioxidants (LE or LAA). Fresh semen from ram were separated by Percoll-Gradient and capacitated with Hepes TALP with heparine (30 IU/ml). Then, separated and capacitated spermatozoa were transferred to IVF TALP micro-drops containing matured oocytes and incubated for 20 hs. Presumptive zygotes were in vitro cultured in TCM 199 with or without antioxidants (LE or LAA) in 7-8 days. The results showed that, in LE group, the percentages of maturation, 8 -cell and morula stages were significantly higher than those of LAA and control groups (P<0.01-0.05) except for cleavage rate which had a numerically higher (P≥0.05). In conclusion, addition of L-ergothionine into IVM, IVF and IVC medium had a beneficial effect on in vitro maturation of oocytes and embryonic development, especially from cleavage to morula stages compared to LAA and Control groups.

Keywords:

oocytes, FSH, LH, ascorbic acid, cumulus oophorus, embryonic development, embryos, ewes, in vitro fertilization, oocyte maturation, ovaries, sheep, zygotes,

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1. Guerin P, El Mouatassim S, Menezo Y: Oxidative stress and protection against reactive oxygen species in the pre- implantation embryo and its surroundings. Hum Reprod Update, 7, 175-189, 2001.
2. Iwata H, Akamatsu S, Minami N, Yamada M: Effects of antioxidants on the development of bovine IVM/IVF embryos in various concentrations of glucose. Theriogenology, 50, 365-375, 1998.
3. Rieger D: Relationships between energy metabolism and development of early mammalian embryos. Theriogenology, 3, 75-93, 1992.
4. Matos De DG, Gasparrini B, Pasqualini SR, Thompson JG: Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content. Theriogenology, 57, 14431451, 2002.
5. Birler S, Pabuçcuoğlu S, Alkan S, Özdaş ÖB,Atalla H, İleri İK: Development of in vitro derived sheep embryos to the blastocyst stage. Turk J Vet Anim Sci, 26, 891-894, 2002.
6. Olson SE, Seidel GE JR: Culture of in vitro -produced bovine embryos with vitamin E improves development in vitro and after transfer to recipients. Biol Reprod, 62, 248252, 2000.
7. Tarin JJ, Perez-Albala S, Garcia-Perez MA, Cano A: Effect of dietary supplementation with a mixture of Vitamins C and E on fertilization of tertiary butyl hydroperoxide-treated oocytes and parthenogenetic activation in the mouse. Theriogenology, 57, 869-881, 2002.
8. Cetica PD, Pintos LN, Dalvit GC, Beconi MT: Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation. IUBMB Life, 51, 57-64, 2001.
9. Urdaneta A, Jimenez-Macedo AR, Izquierdo D, Paramio MT: Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test. Zygote, 11, 347-354, 2003.
10. Rodriguez-Gonzalez E, Lopez-Bejar M, Izquierdo D, Paramio MT: Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation. Reprod Nutr, 43, 179-187, 2003.
11. Matos De DG, Furnus CC: The importance of having high glutathione level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine. Theriogenology, 53, 761-771, 2000.
12. Tatemoto H, Ootaki K, Shigeta K, Muto N: Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic asit 2-O-alphaglucoside during in vitro maturation. Biol Reprod, 65, 18001806, 2001.
13. Mayor P, Lopez-Bejar M, Rodriguez-Gonzalez E, Paramio M: Effects of the addition of glutathione during maturation on in vitro fertilisation of prepubertal goat oocytes. Zygote, 9 323-330, 2001.
14. Feugang JM, de Roover R, Moens A, Leonard S, Dessy F, Donnay I: Addition of beta-mercaptoethanol or trolox at the morula/blastocyst stage improves the quality of bovine blastocysts and prevents induction of apoptosis and degeneration by prooxidant agents. Theriogenology, 161, 171-190, 2004.
15. Akanmu D, Cecchini R, Aruoma OI, Halli B: The antioxidant action of ergothionine. Arch Biochem Biophys, 288, 10-16, 1991.
16. Aruoma OI, Spencer JP, Mahmood N: Protection against oxidative damage and cell death by the natural antioxidant ergothionine. Food Chem Toxicol, 37, 1043-1053, 1999.
17. Mitsuyama H, May JM: Uptake and antioxidant effects of ergothionine in human erythrocytes. Clin Sci, 97, 407-411, 1999.
18. Hartman PE: L-ergothionine as antioxidant. Meth Enzymol, 186, 310-318, 1990.
19. Aruoma OI, Whiteman M, England TG, Hallikuyucuklu B: Antioxidant action of ergothionine assessment of its ability to scavenge peroxynitrite. BBRC, 231, 389-391, 1997.
20. Franzoni RC, Galeta F, Laurenza I, Barsotti M, Di Stefano R, Bocchetti R, Regoli F, Carpi A, Balbarini A, Migliore L, Santoro G: An in vitro study on the free radical scavenging capacity of ergothionine: Comparison with reduced glutathione, uric acid and trolox. Biomed Pharmacother, 60, 453-457, 2006.
21. Mann T, Leone E: Studies on the metabolism of semen, Lergothionine as a normal constituent of boar seminal plasma. Biochem J, 53, 140-148, 1953.
22. Haag FM, Mc Leod J: Relationship between nonprotein sulfhydryl concentration of seminal fluid and motility of spermatozoa in man. J Appl Physiol, 14, 27-30, 1959.
23. Yıldız S, Daskın A: Koç spermasının farklı antioksidan içeren sulandırıcılarla kısa süreli saklanması. Kafkas Univ Vet Fak Derg, 10, 155-159, 2004.
24. Metcalf ES, Dideon BA Blehr R , Schlimgen T, Bertrand W, Varner DD, Sheila Teague S, Hausman MS: The effect of DMSO and L-ergothionine, on postthaw semen parameters in stallions: Preliminary results. Anim Reprod Sci, 107, 332-333, 2008.
25. Guler A, Poulin N, Mermillod P, Terqui M, Cognie Y: Effect of growth factors, EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes. Theriogenology, 54, 209218, 2000.
26. Birler S, Pabuççuoğlu, Atalla H, Alkan S, Özdaş ÖB, Bacınoğlu S, Cirit Ü, Zazar İ: Transfer of in vitro produced sheep embryos. Turk J Vet Anim Sci, 26, 1421-1426, 2002.
27. Ord T, Patrizio P, Marello E, Balmaceda JP, Asch RH: Mini-Percoll: A new method of semen preparation for IVF in severe male factor infertility. Hum Reprod, 58, 987-989, 1990.
28. Parrish JJ, Susho-Parrish JL, Leibfried- Rutledge ML, Critser ES, Eyestone WH, First NL: Bovine in vitro fertilization with frozen-thawed semen.Theriogenology, 25, 591-600, 1986.
29. Rho GJ, Hahnel AC, Betteridge KJ: Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro. Theriogenology, 56, 503-516, 2001.
30. Clark SG, Haubert K, Bebe DJ, Ferguson CE, Wheeler MB: Reduction of polyspermic penetration using biomimetic microfluidic technology during in vitro fertilization. Lab Chi, 5, 1229-1232, 2005.
31. SAS: SAS/STAT User’s Guide, Version 6, Fourth ed., 2, SAS Institute Inc, Cary, NC, USA, 1990.
32. Dong KK, Damaghi N, Kibitel J, Canning MT, Smiles KA, Yarosh DB: A comparison of the relative antioxidant potency of L-ergothionine and idebenone. Cosmet Dermatol, 6, 183188, 2007.
33. Guijarro MV, Indart A, Aruoma OI, Viana M, Bonet B: Effects of ergothionine on diabetic embriyopathy in pregnant rats. Food Chem Toxicol, 40, 1751-1755, 2002.
34. Obayashi K, Kurihara K, Okano Y, Masaki H, Yarosh DB: L-ergothionine scavenges superoxide and singlet oxygen and suppresses TNF-alpha and MMP-1 expression in UV-irradiated human dermal fibroblasts. J Cosmet Sci, 56, 17-27, 2000.
35. Kitagawaa Y, Suzukib K, Yoneda A, Watanabe T: Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology, 62, 1186-1197, 2004.
36. Széll AZ: The effect of glutamine on the development of sheep embryos in vitro. Theriogeneology, 44, 674-680, 1995.
37. Moor RM: Ram specific effects on in vitro fertilization and cleavage of sheep oocytes matured in vitro. J Reprod Fertil, 82, 337-340, 1988.
38. Fukui Y, Glew AM, Gandolfi F, Moor RM: Ram-specific effects on in vitro fertilization and cleavage of sheep oocytes matured in vitro. J Reprod Fertil, 82, 337-340, 1988.
39. Madan ML, Naqvi SMK, Chauhan MS, Singla SK, Manik RS: In vitro production of ovine preimplentation embryos from in vitro matured oocytes using epididymal and frozen thawed spermatozoa. Proceedings of the 12th International conference on Animal Reproduction, 3, 1318-1320, 1992.
40. Morton KM, Rowe AM, Maxwell WMC, Evans G: In vitro and in vitro survival of bissected sheep embryos derived from frozen-thawed unsorted, and frozen-thawed sex-shorted and refrozen-thawed ram spermatozoa. Theriogenology, 65, 1333-1345, 2006.
41. Li F, Pi WH, Zhu HZ, Zhang SS, Liu SR, Xue JL: The effect of oestrous ewe serum and heparin on in vitro fertilization and subsequent embryonic development in sheep. Small Rumin Res, 63, 226-232, 2006.
42. O’Meara CM, Hanrahan JP, Donovan A, Fair S, Rizos D, Wade M, Boland MP, Evans ACO, Lonergan P: Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen- thawed ram semen. Theriogenology, 64, 1797-1808, 2005.
43. Farin CE, Hasler JF, Martus NS, Stokes JE: A comparison of Menezo’s B2 and tissue culture medium-199 (TCM199) for in vitro production of bovine blastocysts. Theriogenology, 48, 699-709, 1997.
44. Abdulhadi AA, Ahmed RA: In vitro maturation IVM and fertilization IVF of sheep ova cultured in two different media. Agric Sci, 1, 15-24, 2004.
45. Rao BS, Naidu KS, Amarnath D, Vagdevi R, Rao A S, Brahmaiah KV, Rao VH: In vitro maturation of sheep oocytes in different media during breeding and non-breeding seasons. Small Rumin Res, 43, 31-36, 2002.
46. Ozawa M, Nagai T, Fahrudin M, Kurniani Karja W, Kaneko H, Noguchi J, Ohnuma K, Kikuchi K: Addition of glutathione or thioredoxin to culture medium reduces intracellular redox status of porcine IVM/IVF embryos, resulting in improved development to the blastocyst stage. Reprod Biol Endocrinol, 4, 10-11, 2006.
47. Bucak MN, Satılmış M, Kızıl SH, Karaşahin T, Akyol N: Sığır embriyolarının in vitro gelişiminde kültür medyumlarına katılan antioksidanların etkisi. Kafkas Univ Vet Fak Derg, 16, 69-74, 2010.
48. Dahl TA, Midden WR, Hartman PE: Some prevalent biomolecules as defenses against singlet oxygen damage. Photeochem Photobiol, 47, 357-362, 1988.
49. Han JS: Effects of various chemical compounds on spontaneous and hydrogen peroxideinduced reversion in strain TA 104 of Salmonella typhimurium. Mutation Res, 266, 77-84, 1992.
50. Hartman PE, Hartman, Z, Auctk, T: Scavenging of singlet molecular oxygen by imidazole compounds: High and sustained activities of carboxy terminal histidine dipeptides and exceptional activity of imidazole-4-acetic acid. Photochem Photobiol, 51, 59-66, 1990.