Mustafa ÖZTATLICI, Kemal ÖZBİLGİN, Sevinç İNAN, Merve TEMEL, H. Seda VATANSEVER

Over Vitrifikasyonunun Mitokondriyal Füzyon (MFN-1, MFN-2 ve OPA-1), Fisyon (DNM-1), Mitofaji (PARKIN, PINK-1) ve Transport (MIRO-1, MILTON) Proteinleri Üzerindeki Etkileri

Giriş ve Amaç: Ovaryan kriyoprezervasyonu, yardımcı üreme teknolojilerinde doğurganlığın korunması için yararlı bir alternatiftir. Vitrifikasyon prosedüründeki birçok ilerlemelere rağmen bu teknik hala deneysel olarak kabul edilmektedir. Bu nedenle bu çalışmada, vitrifikasyon sonrası over dokularında mitokondriyal füzyon (MFN1, MFN2 ve OPA1), fisyon (DRP1), mitofaji (PARKIN, PINK1) ve transport (MIRO-1, MILTON) proteinlerinin ifadelerini qPCR tekniği ile araştırmayı amaçladık. Gereç ve Yöntemler: Vitrifikasyon sonrası mitokondriyal dinamikleri araştırmak için, overler 6-8 haftalık sağlıklı dişi farelerden (No: 12) alındı ve vitrifikasyon ve kontrol gruplarına ayrıldı. Vitrifikasyon, etilen glikol, dimetilsülfoksit ve sukroz kullanılarak gerçekleştirildi. Kontrol ve vitrifikasyon gruplarındaki overlerden total RNA izolasyonu yapıldıktan sonra hedef genlerin ifade oranlarını belirlemek için qPCR tekniği kullanıldı. Hedef genlerin relatif gen ifadeleri 2 −∆∆Ct yöntemine göre değerlendirildi. Bulgular: Histolojik değerlendirme kontrol grubundaki overlerin normal morfoloji gösterdiği, vitrifikasyon grubundaki overlerin ise doku bütünlüğünün bozulduğunu; bazı foliküllerin dejenere olduğunu ve granüloza hücrelerinin antruma döküldüğünü ortaya koydu. qPCR sonuçlarımıza göre vitrifikasyon grubunda kontrol grubuna kıyasla dış membran füzyon proteini MFN1 gen ifadesinin 1,12 kat azaldığı ve iç membran proteini olan OPA-1’in ifadesinin 1,36 kat arttığı saptandı. Mitokondriyal fisyon proteini DRP-1 gen ifadesinin vitrifikasyon grubunda 1,20 kat arttığı bulundu. Mitofaji proteinleri olan PINK-1 ve PARKIN gen ifadelerinin vitrifikasyon grubunda sırasıyla 1,34 ve 3,75 kat azaldığı tespit edildi. Kontrol grubuna göre karşılaştırıldığında transport proteinlerinin; MIRO-1 gen ifadesinin 1,16 kat azaldığı ancak MILTON (TRAK-1) gen ifadesinin 2,28 kat arttığı saptandı. Sonuç: Mitokondriyal dinamikler ile ilişkili gen ifadelerindeki değişimler, ovaryan vitrifikasyonu sırasında mitokondriyal fonksiyonda bir azalmaya yol açabilir ve oosit maturasyonu ve embriyo gelişimi potansiyelini azaltabilir

Anahtar Kelimeler:

Mitokondri, Mitokondriyal Fisyon, Mitokondriyal Füzyon, Mitofaji, Ovaryan Vitrifikasyon

The Effects of Ovarian Vitrification on Mitochondrial Fusion (MFN-1, MFN2 and OPA-1), Fission (DNM-1), Mitophagy (PARKIN, PINK-1) and Transport (MIRO-1, MILTON) Proteins

Objective: Ovarian cryopreservation is a useful alternative for fertility preservation in assisted reproductive technologies. In spite of many advances in the vitrification procedure, this technique is still considered experimental. Therefore in this study, we aimed to investigate the expressions of mitochondrial fusion (MFN1, MFN2 and OPA1), fission (DRP1), mitophagy (PARKIN, PINK1) and transport (MIRO-1, MILTON) proteins in ovarian tissues by qPCR technique after vitrification. Materials and Methods: To investigate the mitochondrial dynamics after vitrification, the ovaries were recovered from 6-8 week old healthy female mice (No: 12) and were divided into vitrification and control groups. Vitrification carried out using ethylene glycol, dimethylsulfoxide and sucrose. After total RNA isolation from ovaries in control and vitrification groups, qPCR technique was performed to determine the expression rate of target genes. The relative gene expressions of the target genes were evaluated according to 2−∆∆Ct method. Results: Histological evaluation revealed that ovaries in the control group were shown normal morphology while the tissue integrity of the ovaries in the vitrification group is disrupted, some follicles are degenerated and granulosa cells were shed into antrum. According to our qPCR results, outer membran fusion proteins MFN1 gene expression decreased 1,12 fold and inner membran protein OPA-1 increased 1,36 fold in the vitrification group compared the control group. The mitochondrial fission protein DRP-1 gene expression increased 1,20 fold in the vitrification group. The mitophagy proteins PINK-1 and PARKIN genes expressions decreased 1,34 and 3,75 fold respectively in the vitrification group. The transport proteins; MIRO-1 gene expression decreased 1,16 fold but MILTON (TRAK-1) gene expression sharply increased 2,28 fold compared the control group. Conclusion: The alternation of the mitochondrial dynamics related gene expressions may lead a decrease in the mitochondrial function during the ovarian vitrification and may reduce the potential of oocyte maturation and embryo development

Keywords:

Mitochondrion, Mitochondrial Fission, Mitochondrial Fusion, Mitophagy, Ovarian Vitrification,

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