Protein Kaynaklarının İn Vitro Olgunlaştırılmış Sığır Oositlerinin Glutatyon Peroksidaz Enzim Aktivitesi Üzerine Etkisi

Year 2024, , 575 - 579, 15.05.2024

Nisa Nur Yılmaz

https://doi.org/10.34248/bsengineering.1439699

Abstract

Bu çalışma, sığır oositlerinin in vitro olgunlaştırma (IVO)’sında kültür medyumuna ilave edilen protein kaynaklarının glutatyon peroksidaz (GPx) enzim aktivitesi üzerine etkisinin belirlenmesi amacıyla yapılmıştır. Sığır ovaryumlarından elde edilen oositler %10 fötal buzağı serum (FBS), 4 mg/mL sığır serum albümin (SSA), 1 mg/mL polivinil alkol (PVA) içeren ve protein kaynağı içermeyen bikarbonat tamponlu doku kültür medyumlarında (TCM–199) 38,5 °C sıcaklıkta ve %5 CO2 içeren nemlendirilmiş ortamda 22 saat IVO’ya alınmıştır. Olgunlaşma süresi sonunda elde edilen kumulus-oosit kompleksleri (KOK'ler), kumulus hücrelerinden ayırmak için işleme tabi tutulmuştur. Kumulus hücrelerinden ayrılan oositler, polivinil alkol içeren ve Ca2+ ve Mg2+ içermeyen fosfat tamponlu tuz çözeltisi (PBS) ile yıkanmıştır. Daha sonra, oositler belirlenmiş örnek boyutlarına bölünerek mikrotüplerde saklanmıştır. IVO kültürü sonrasında oositlerdeki glutatyon peroksidaz (GPx) enzim aktivitesi için ticari kit kullanarak 340 nm dalga boyunda spektrofotometrik olarak belirlenmiştir. Çalışmada, her bir deneme grubundaki oositlerden izole edilen hücre ekstraktlarındaki GPx enzim aktivitesinin seviyelerinin zamanla değişmediği, kültür medyumuna PVA eklenmiş oositlerinde GPx enzim aktivitesinin diğer deneme gruplarındaki oositlerden daha düşük olduğu tespit edilmiştir (P>0,05). Bu çalışma sonucunda farklı protein kaynaklarının antioksidan aktivitenin göstergelerinden biri olan GPx enzim seviyelerinin sığır oositlerinde üzerine etkileri belirlenmiş ve FBS ve BSA’nın protein kaynağı olarak sığır oosit gelişimi üzerine daha etkili olabileceği tespit edilmiştir.

Keywords

Oosit, Sığır, Antioksidan aktivitesi, Protein, Olgunlaşma

Effect of Protein Sources on Glutathione Peroxidase Enzyme Activity of In Vitro Maturated Bovine Oocytes

Abstract

This study was conducted to determine the effect of protein sources added to the culture media during in vitro maturation (IVM) on the glutathione peroxidase (GPx) enzyme activity of bovine oocytes. Oocytes obtained from bovine ovaries were IVM cultured in bicarbonate buffered tissue culture media (TCM-199) containing 10% fetal calf serum (FBS), 4 mg/mL bovine serum albumin (SSA), 1 mg/mL polyvinyl alcohol (PVA) and without protein source for 22 hours in a humidified environment containing 38.5 °C and 5% CO2. Following the maturation period, cumulus-oocyte complexes (COCs) obtained were subjected to a procedure to separate the cumulus cells from the oocytes. The oocytes separated from the cumulus cells were washed with phosphate-buffered saline (PBS) containing polyvinyl alcohol and devoid of Ca2+ and Mg2+. Subsequently, the oocytes were divided into predetermined sample sizes and stored in microtubes for further processing. After IVM culture, using a commercial kit, glutathione peroxidase (GPx) enzyme activity in oocytes was determined spectrophotometrically at a wavelength of 340 nm. In the study, the levels of GPx enzyme activity in the cell extracts isolated from the oocytes in each experimental group did not change over time, and the GPx enzyme activity in the oocytes with PVA added to the culture medium was lower than the oocytes in the other experimental groups (P>0.05). As a result of this study, the effects of different protein sources on GPx enzyme levels, one of the indicators of antioxidant activity in bovine oocytes, were determined, and FBS and BSA, as protein sources, could be more effective on bovine oocyte development.

Keywords

Oocyte, Bovine, Antioxidant activity, Protein, Maturation

References

• Ali A, Sirard MA. 2002. Effect of the absence or presence of various protein supplements on further development of bovine oocytes during ın vitro maturation. Biol Reprod, 66(4): 901-905.
• Brackett B, Zuelke K. 1993. Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology, 39(1): 43-64.
• Cetica PD, Pintos LN, Dalvit GC, Beconi MT. 2001. Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation. IUBMB Life, 51(1): 57-64.
• Cevik M, Kocyigit A, Sen U, Kuran M. 2014. Can commercial human embryo culture media be used in bovine embryo culture? J Fac Vet Med, Univ Kafkas, 20(1): 149–153.
• Cevik M, Sen U, Kocyigit A, Soydan E, Kuran M. 2011. Effects of serum, gonadotropins, epidermal growth factor and estradiol 17-Beta on cumulus expansion and nuclear maturation of bovine oocytes. J Fac Vet Med, Univ Kafkas, 17(6): 1009–1014.
• Flood L, Shirley B. 1991. Reduction of embryotoxicity by protein in embryo culture media. Molec Reprod Develop, 30: 226-231.
• Genç S, Soysal Mİ. 2018. Parametric and nonparametric post hoc tests. BSJ Eng Sci, 1(1): 18-27.
• Gordon I. 1994. Laboratory produstion of cattle embryo. Wallingford: CAB International, Newyork, USA, pp: 30-142.
• Halli̇wel B, Gutteri̇dge J. 1990. Role of free radicals and catalytic metal ions in human disease. Anoverview Methods Enzymol, 186: 1-8.
• Halliwell B. 1996. Antioxidants in human health and disease. Annu Rev Nutr, 16: 33-50.
• Kocyigit A, Cevi̇k M, Sen U, Kuran M. 2015. The effect of macromolecule and growth factors combinations on in vitro development of bovine embryos. Turkish J Vet Anim Sci, 39: 308-313.
• Sen U, Kuran M. 2018. Low incubation temperature successfully supports the in vitro bovine oocyte maturation and subsequent development of embryos. Asian-Australas J Anim Sci, 31(6): 827-834.
• Sen U, Şirin E, Onder H, Özyürek S, Kolenda M, Sitkowska B. 2022. Macromolecules Influence Cellular Competence and Expression Level of IGFs Genes in Bovine Oocytes In Vitro. Animals, 12(19), 2604.
• Sen U. 2015. Effects of sperm from different bulls on developmental competence, blastosist quality and cell number of bovine embryos in vitro. J Fac Vet Med, Univ Kafkas, 21(3): 339–344.
• Sen U. 2021. Maturation of bovine oocytes under low culture temperature decreased glutathione peroxidase activity of both oocytes and blastocysts. Polish J Vet Sci, 24: 93-99.
• Sen U. 2022. In vitro maturation culture temperature alters the enzymatic antioxidant activity of bovine oocytes and embryos. 37th International Conference on “Chemical, Agriculture, Biological & Environmental Sciences”, December 12-14, Lisboni, Portugal, pp: 1-4.
• Sturmey RG, Hawkhead JA, Barker EA, Leese HJ. 2009. DNA damage and metabolic activity in the preimplantation embryo. Hum Reprod, 24: 81-91.