Köpeklerde nano-ozon solüsyonunun in vitro nükleer oosit maturasyonu üzerine etkisi
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Date
2023-12-01
Authors
Bari, Özge
Journal Title
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Publisher
Bursa Uludağ Üniversitesi
Abstract
Köpek oositlerinin in vitro maturasyon (IVM) oranları diğer türlere göre oldukça düşüktür. IVM’deki önemli problem, reaktif oksijen türlerinin (ROS) oluşumu olup, bunun önlenmesinde en basit yaklaşım, medyumların antioksidan madde ile hazırlanmasıdır. Bu çalışmada, hem oksidan hem de antioksidan özelliğe sahip nano-ozon solüsyonu kullanıldı. Nano-ozon solüsyonunun köpek oositlerinde nükleer morfolojiye etkisi, kültür medyumunun oksidan/antioksidan durumu ve maturasyon ilişkili genlerin (Siklin bağımlı kinaz 1 (CDK1) ve Siklin B1)ekspresyon seviyeleri değerlendirildi. Ek olarak, antioksidan kapasitenin değerlendirilmesi için demir iyonu indirgeyici antioksidan güç (FRAP) testi kullanıldı. Kumulus-oosit kompleksi (COCs) 130 köpekten toplandı. COCs farklı konsantrasyonlarda nano-ozon solüsyonu eklenen maturasyon medyumunda inkübe edildi (0, 0,5, 1, 2, ve 5 µg/ml). Kültür medyumları toplanarak Malondialdehit (MDA), Glutatyon (GSH), Süperoksit-dismutaz (SOD) ve FRAP ölçümü için saklandı. Parteno genetik aktivite sonrasında, nükleer morfolojiler mikroskop altında germinal vezikül (GV), germinal vezikül parçalanması (GVBD), metafaz I (MI), metafaz II (MII), ve dejenere (DEG) oosit olarak değerlendirildi. Aktivasyon sonrası toplanan oositlerde, CDK1 ve Siklin B1 gen ekspresyonları Real-Time PCR ile değerlendirildi. Nükleer morfoloji oranları gruplar arasında farklı bulundu (p<0,05). Mayotik süreci tamamlayan oosit oranı (MI+MII) en yüksek Grup B ve C’de bulunurken, MII aşamasına ulaşan en yüksek oosit sayısı Grup C’de gözlendi. MDA ve SOD konsantrasyonları arasında fark bulunamadı (p>0,05) ancak GSH ve FRAP düzeyleri istatistiksel olarak farklı bulundu (p<0,05). Bunun ötesinde CDK1 ve Siklin B1 gen ekspresyonları gruplar arasında farklı bulundu (p<0,05). En yüksek oran Grup B ve C’de belirlendi. Sonuçlarımız nano-ozon’un IVM’de oksidatif stres parametrelerini hafifleterek MII aşamasına gelişimi indüklemiş ve maturasyon-ilişkili genekspresyonunu artırmıştır. En uygun doz olarak 0,5 µg/ml, 1 µg/ml nano-ozon katkısının IVM’de kullanılması önerilebilir.
In vitro maturation rates of canine oocytes are rather lower than other species. Long incubations and insufficient conditions of maturation culture affect negatively the success of in vitro maturation (IVM). The most important problem during the maturation process is the production of high amount of reactive oxygen species (ROS) that is the end-point of mitochondrial lipid peroxidation. The basic approach to prevent ROS production is to prepare an in vitro culture medium with antioxidant substances. In this study, a nano-ozone solution, which has both oxidant and antioxidant properties, was used. The effect of nano-ozone on the nuclear morphology of canine oocytes, oxidant and antioxidant status of maturation culture medium, and maturation-related (Cyclin Dependent Kinase 1 (CDK1) and Cyclin B1) gene expression levels were investigated. Additionally, the Ferric ion-reducing antioxidant power (FRAP) method was used to determine antioxidant capacity. The cumulus-oocytes complex (COCs) was collected from 130 dogs after the ovariohysterectomy operation. The COCs were incubated in in vitro maturation culture media, supplemented with nano-ozone solution at different concentrations (0, 0.5, 1, 2, and 5 µg/ml). After maturation of 72 hours, the culture medium was stored in Eppendorf tubes in order to determine Malondialdehyde (MDA), Glutathione (GSH), Superoxide Dismutase (SOD), and FRAP concentrations. Oocytes were parthenogenetically activated and nuclear morphology was classified as Germinal Vesicle intact (GV), Germinal Vesicle Break Down (GVBD), Metaphase I (MI), Metaphase II (MII), and degenerated (DEG) oocytes under light microscope. Additionally, cultured oocytes were collected after activation. The oocytes were used for mRNA isolation and Real-Time PCR for expression of CDK1 and Cyclin B1 genes. The rate of oocyte nuclear morphology (GVBD, MI, MII, and DEG) was statistically different between groups (p<0.05). The rate of oocytes that completed the meiotic process (MI+MII) was higher in Group B and Group C, whereas the rate of oocytes reaching the MII stage was highest in Group C. No difference was observed between the groups in terms of MDA and SOD (p>0.05). GSH and FRAP levels were statistically different between groups (p<0.05). Compared to the control group, GSH and FRAP levels were higher in nano-ozone-supplemented groups. Furthermore, CDK1 and Cyclin B1 gene expressions were also statistically different between groups (p<0.05). Expression levels of both genes were high in Group B and Group C. Our results suggested that nano-ozone solution could alleviate oxidative stress parameters during IVM, induce development to MII stage, and increase the expression levels of maturation-related genes. Low doses of nano-ozone supplementation during canine IVM studies could be advised and the most appropriate and effective doses are 0.5 µg/ml and 1 µg/ml could be suggested.
In vitro maturation rates of canine oocytes are rather lower than other species. Long incubations and insufficient conditions of maturation culture affect negatively the success of in vitro maturation (IVM). The most important problem during the maturation process is the production of high amount of reactive oxygen species (ROS) that is the end-point of mitochondrial lipid peroxidation. The basic approach to prevent ROS production is to prepare an in vitro culture medium with antioxidant substances. In this study, a nano-ozone solution, which has both oxidant and antioxidant properties, was used. The effect of nano-ozone on the nuclear morphology of canine oocytes, oxidant and antioxidant status of maturation culture medium, and maturation-related (Cyclin Dependent Kinase 1 (CDK1) and Cyclin B1) gene expression levels were investigated. Additionally, the Ferric ion-reducing antioxidant power (FRAP) method was used to determine antioxidant capacity. The cumulus-oocytes complex (COCs) was collected from 130 dogs after the ovariohysterectomy operation. The COCs were incubated in in vitro maturation culture media, supplemented with nano-ozone solution at different concentrations (0, 0.5, 1, 2, and 5 µg/ml). After maturation of 72 hours, the culture medium was stored in Eppendorf tubes in order to determine Malondialdehyde (MDA), Glutathione (GSH), Superoxide Dismutase (SOD), and FRAP concentrations. Oocytes were parthenogenetically activated and nuclear morphology was classified as Germinal Vesicle intact (GV), Germinal Vesicle Break Down (GVBD), Metaphase I (MI), Metaphase II (MII), and degenerated (DEG) oocytes under light microscope. Additionally, cultured oocytes were collected after activation. The oocytes were used for mRNA isolation and Real-Time PCR for expression of CDK1 and Cyclin B1 genes. The rate of oocyte nuclear morphology (GVBD, MI, MII, and DEG) was statistically different between groups (p<0.05). The rate of oocytes that completed the meiotic process (MI+MII) was higher in Group B and Group C, whereas the rate of oocytes reaching the MII stage was highest in Group C. No difference was observed between the groups in terms of MDA and SOD (p>0.05). GSH and FRAP levels were statistically different between groups (p<0.05). Compared to the control group, GSH and FRAP levels were higher in nano-ozone-supplemented groups. Furthermore, CDK1 and Cyclin B1 gene expressions were also statistically different between groups (p<0.05). Expression levels of both genes were high in Group B and Group C. Our results suggested that nano-ozone solution could alleviate oxidative stress parameters during IVM, induce development to MII stage, and increase the expression levels of maturation-related genes. Low doses of nano-ozone supplementation during canine IVM studies could be advised and the most appropriate and effective doses are 0.5 µg/ml and 1 µg/ml could be suggested.
Description
Keywords
Antioksidan, CDK1/Siklin B1 gen ekspresyonu, İn vitro maturasyon, Köpek, Nano-ozon, Antioxidant, CDK1/Cyclin B1 gene expression, In vitro maturation, Canine, Nano-ozone
Citation
Bari, Ö. (2023). Köpeklerde nano-ozon solüsyonunun in vitro nükleer oosit maturasyonu üzerine etkisi. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.