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

Öz

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

Anahtar Kelimeler

• Mitochondrion
• Mitochondrial Fission
• Mitochondrial Fusion
• Mitophagy
• Ovarian Vitrification

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

Öz

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

Kaynakça

1. 1.Donnez, J, Introduction: fertility preservation, from cancer to benign disease to social reasons: the challenge of the present decade, Fertility and Sterility, 2013, 99, 1467–1468.
2. 2.Navarro-Costa, P, Correia, SC, Gouveia-Oliveira, A, Negreiro, F, Jorge, S et al, Effects of mouse ovarian tissue cryopreservation on granulosa cell-oocyte interaction, Human Reproduction, 2005, 20, 1607–1614.
3. 3.Edgar, DH, Gook DA, A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos, Human Reproduction Update, 2012, 18, 536–54.
4. 4.Wang, Y, Xiao, Z, Li, L, Fan, W, Li, SW, Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation,Human Reproduction, 2008, 23, 2256–2265.
5. 5.Demant, M, Trapphoff, T, Fröhlich, T, Arnold, GJ, EichenlaubRitter, U, Vitrification at the pre-antral stage transiently alters inner mitochondrial membrane potential but proteome of in vitro grown and matured mouse oocytes appears unaffected, Human Reproduction, 2012, 27 (4), 1096-1111.
6. 6.El Shourbagy, SH, Spikings, EC, Freitas, M, St John, JC,Mitochondria directly influence fertilisation outcome in the pig,Reproduction, 2006, 131 (2), 233-245.
7. 7.Santos, TA, El Shourbagy, SH, John, JCS, Mitochondrial content reflects oocyte variability and fertilization outcome, Fertility and Sterility, 2006, 85 (3), 584-591.
8. 8.Van Blerkom, J, Davis, P, Alexander, S, Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence, Human Reproduction, 2000, 15 (12), 2621–2633.

9. 9.Ni, HM, Williams, JA, Ding, WX, Mitochondrial dynamics and mitochondrial quality control, Redox Biology, 2015, 4, 6-13.
10. 10.Taguchi, N, Ishihara, N, Jofuku, A, Oka, T, Mihara, K, Mitotic phosphorylation of dynamin-related GTPase Drp1 participates in mitochondrial fission, Journal of Biological Chemistry, 2007, 282 (15), 11521-11529.
11. 11.Kitada, T, Asakawa, S, Hattori, N, Matsumine, H, Yamamura, Y et al, Mutations in the Parkin gene cause autosomal recessive juvenile Parkinsonism, Nature, 1998, 392 (6676), 605–608.
12. 12.Gegg, ME, Mitofusin1 and mitofusin2 are ubiquitinated in a PINK1/ parkin-dependent manner upon induction of mitophagy, Human Molecular Genetics, 2010, 19 (24), 4861–4870.
13. 13.Konc, J, Kanyó, K, Kriston, R, Somoskői, B, Cseh, S, Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction, Biomed Research International, 2014, Article ID: 307268- 9 pages.
14. 14.Chang, HJ, Moon, JH, Lee, JR, Jee, BC, Suh, CS et al, Optimal condition of vitrification method for cryopreservation of human ovarian cortical tissues, Journal of Obstetrics Gynaecology Research, 2011, 37 (8), 1092-1101.
15. 15.Valojerdi, MR, Salehnia, M, Developmental potential and ultrastructural injuries of metaphase II (MII) mouse oocytes after slow freezing or vitrification, Journal of Assisted Reproduction and Genetics, 2005, 22 (3), 119-127.
16. 16.Salehnia, M, Moghadam, EA, Velojerdi, MR, Ultrastructure of follicles after vitrification of mouse ovarian tissue, Fertility and Sterility, 2002, 78 (3), 644-645.
17. 17.Zhao, XM, Fu, XW, Hou, YP, Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN) embryos, Molecular Reproduction and Development, 2009, 76 (11), 1056–1063.
18. 18.Gualtieri, R, Iaccarino, M, Mollo, V, Prisco, M, Iaccarino, S, Talevi,R, Slow cooling of human oocytes: ultrastructural injuries and apoptotic status, Fertility and Sterility, 2009, 91 (4), 1023-1034.
19. 19.Nagai, S, Mabuchi, T, Hirata, S, Shoda, T, Kasai, T et al, Correlation of abnormal mitochondrial distribution in mouse oocytes with reduced developmental competence, Tohoku Journal of Experimental Medicine, 2006, 210 (2), 137-144.
20. 20.Chen, H, Chomyn, A, Chan, DC, Disruption of fusion results in mitochondrial heterogeneity and dysfunction, Journal of Biological Chemistry, 2005, 280 (28), 26185-92. 21.Wakai, T, Harada, Y, Miyado, K, Kono, T, Mitochondrial dynamics controlled by mitofusins define organelle positioning and movement during mouse oocyte maturation, Molecular Human Reproduction, 2014, 20 (11), 1090-100.
21. 22.Chen, H, Chan, DC, Mitochondrial dynamics in mammals, Current Topics in Developmental Biology, 2004, 59, 119-44.
22. 23.Dai, J, Wu, C, Muneri, CW, Niu, Y, Zhang, S, Rui, R et al, Changes in mitochondrial function in porcine vitrified MII-stage oocytes and their impacts on apoptosis and developmental ability, Cryobiology, 2015, 71 (2), 291-8.
23. 24.Chen, H, Detmer, SA, Ewald, AJ, Griffin, EE, Fraser, SE et al,Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development, Journal of Cell Biology, 2003, 160 (2), 189-200.
24. 25.Olichon, A, Baricault, L, Gas, N, Guillou, A, Belenguer, P et al, Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis, Journal of Biological Chemistry, 2003, 278 (10), 7743-7746.
25. 26.Boucret, L, Chao De La Barca, JM, Moriniere, C, Desquiret, V, Ferre-L'Hotellier, V et al, Relationship between diminished ovarian reserve and mitochondrial biogenesis in cumulus cells, Human Reproduction, 2015, 30 (7), 1653-1664.
26. 27.Conca Dioguardi, C, Uslu, B, Haynes, M, Kurus, M, Gul, M et al, Granulosa cell and oocyte mitochondrial abnormalities in a mouse model of fragile X primary ovarian insufficiency, Molecular Human Reproduction, 2016, 22 (6), 384-396.
27. 28.Corti, O, Brice, A, Mitochondrial quality control turns out to be the principal suspect in parkin and PINK1-related autosomal recessive Parkinson's disease, Current Opinion in Neurobiology, 2013, 23 (1), 100-108.
28. 29.Correia-Melo, C, Ichim, G, Tait, SW, Passos, JF, Depletion of mitochondria in mammalian cells through enforced tophagy, Nature Protocols, 2017, 12 (1), 183.
29. 30.Heeman, B, Van den Haute, C, Aelvoet, SA, Valsecchi, F, Rodenburg, RJ et al, Depletion of PINK1 affects mitochondrial metabolism, calcium homeostasis and energy maintenance, Journal of Cell Science, 2011, 124, 1115-1125.
30. 31.Gupta, A, Anjomani-Virmouni, S, Koundouros, N, Dimitriadi, M,Choo-Wing, R et al, PARK2 depletion connects energy and oxidative stress to PI3K/Akt activation via PTEN S-nitrosylation, Molecular Cell, 2017, 65 (6), 999-1013.
31. 32.Lee, CA, Chin, LS, Li, L, Hypertonia-linked protein Trak1 functions with mitofusins to promote mitochondrial tethering and fusion, Protein Cell, 2018, 9 (8), 693-716.
32. 33.Brough, D, Schell, MJ, Irvine, RF, Agonist-induced regulation of mitochondrial and endoplasmic reticulum motility, Biochemical Journnal, 2005, 392, 291–297.
33. 34.Magrane´, J, Cortez, C, Gan, WB, Manfredi, G, Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models, Human Molecular Genetics, 2013, 23 (6), 1413–1424.
34. 35.Wang, AW, Zhang, H, Ikemoto, I, Anderson, DJ, Loughlin, KR, Reactive oxygen species generation by seminal cells during cryopreservation, Urology, 1997, 49, 921-925.
35. 36.Cao, X, Li, J, Xue, H, Wang, S, Zhao, W et al, Effect of vitrification on meiotic maturation, mitochondrial distribution and glutathione synthesis in immature silver fox cumulus oocyte complexes, Theriogenology, 2017, 91, 104-111.