Kriyoprezervasyon Uygulanmış Balık Sperm Hücrelerinde DNA Hasarının Tespitinde Farklı Comet Analizi (Tek Hücre Jel Elektroforezi) Metotlarının Uygulanmaları

Year 2016, Volume: 1 Issue: 1, 6 - 13, 11.12.2016

Burak Evren İnanan , Nazlı Yıldırım Ekin Demirkaya

Abstract

Kriyoprezervasyon,
hem insan hem de hayvan gametlerinin, hatta embriyolarının, başarılı bir
şekilde uzun süreli saklanması için yaygın olarak kullanılan yardımcı üreme
tekniklerindendir. Ayrıca, sperm kriyoprezervasyonun birçok avantajı vardır.
Örneğin, balıklarda suni döllemenin geliştirilmesinde, genetik
manipülasyonların kolaylaştırılmasında, erkek anaç stokunun azaltılmasında
kullanılır. Ancak kriyoprezervasyon sonrası, sperm hücrelerinde sonrasında
dölleme ve yumurtadan çıkış oranlarının azalmasına sebep olacak motilite kaybı
ve DNA hasarı gibi problemler ile oluşabilir. Comet analizi, DNA sarmalındaki
kırıkları tek bir sperm hücresi seviyesinde tespit edebilen basit ve hassas bir
araçtır. 

Bu çalışmada, farklı kimyasal maddeler ve uygulama
zamanlarına sahip üç farklı Comet analiz metodu balık sperm hücrelerine
uygulanmıştır. Olgun sazan balığı (Cyprinus carpio)  bireylerinden alınan sperm örnekleri bu çalışmada
kullanılmıştır. Öncelikle, taze sperm örnekleri kullanılarak, her metot için
liziz solüsyonuna 12, 25 ve 50 µM H₂O₂ eklenerek DNA
hasarı sınıflandırılmıştır. Böylece, DNA hasarı, her hücre DNA’sının
oluşturduğu kuyruk uzunluğuna göre floresan mikroskop altında incelenerek
görsel olarak, 1. 2. ve 3. dereceden hasarlı ve H₂O₂
eklenmemiş örnekler 0 (hasarsız) olarak ölçeklendirilebilir. Sonrasında,  kriyoprezervasyon uygulanmış sperm örnekleri
çözündürülerek, üç farklı Comet analiz metodu uygulanmıştır. Üç metoda ait
yüzde hasarsız DNA sonuçları (%67,7±2,0; % 69,3±1,8 ve %68,2±2,8)  arasında ve toplam hasarlı DNA sonuçları
(%32,3±2,0; % 30,7±1,8 ve %31,8±2,8) arasında istatistiki olarak önemli derece
fark saptanamamıştır. Buna karşın, DNA hasar sınıflandırmaları arasında bazı
istatistiksel farklılıklar bulunmaktadır. Sonuç olarak, her üç metot hasarsız
ve toplam hasarlı DNA tespitinde benzer başarıyı göstermekte iken DNA hasar
sınıflarının tanımlanmasında farklılıklar olabilmektedir.

Keywords

Kriyoprezervasyon, COmet Analizi, DNA Hasarı, Balık Spermi, Tek Hücre Jel Elektroforezi

Applications of Different Comet Assay (the Single Cell Gel Eelectrophoresis) Methods for Detecting DNA Damage in Cryopreserved Fish Sperm

Abstract

Cryopreservation
as an assisted reproduction method is commonly used for successful long-term
storage of not only human but also animal gametes, even their embryos.
Moreover, cryopreservation of sperm offers many advantages. In fish, for
instance, it is useful for improving artificial reproduction, facilitating
genetic manipulation, and reducing the amount of male breeders stocks. However,
after cryopreservation, sperm cell could have some problems such as loss in
motility and DNA damage, leading to lower fertilization and hatching rates. The
comet assay is a simple, but sensitive tool for detecting strand breaks in DNA
in single sperm cells. In this study, three comet assay methods which contain
different chemical agents and have different application times were carried out
on fish sperm. The mixed sperm samples obtained from mature carp (Cyprinus carpio) males were used in the
experiments. Firstly, using fresh sperm samples, DNA damage is classified by
means of lysis solutions of each used method without H₂O₂
and with 12, 25 and 50 µM H₂O₂. Thus, DNA damage is
visually scored by measuring the DNA tail of each sperm cell in fluorescent microscope
images as positive control (undamaged DNA), 1, 2, and 3 classes of damage,
referring from the less to more damaged sperm cells. Secondly, after thawing,
cryopreserved sperm samples were applied by the comet assay methods. The
results of three methods have shown no significant differences among the
percentages of undamaged DNA (67.7±2.0 %, 69. 3±1.8 % and 68.2±2.8 %) and total
damaged DNA (32.3±2.0 %, 30.7±1.8 % and 31.8±2.8 %) of sperm cells as. On the
contrary, there were some statistical differences among the classes of damage.
Consequently, all methods have been found successful in detection of undamaged
and total damaged DNA but not in the recognition of the damage classes.

Keywords

Cryopreservation, Comet Assay, DNA damage, Fish sperm, Single Cell Gel Eelectrophoresis

References

• Barbas JP, Mascarenhas RD ( 2009): Cryopreservation of domestic animal sperm cells. Cell Tissue Bank 10(1): 49-62.
• Baumber JB, Barry A, Jennifer J, Stuart A (2003): Reactive Oxygen Species and Cryopreservation Promote DNA Fragmentation in Equine Spermatozoa. J Androl 24(4): 621-28.
• Cabrita E, Alvarez R, Anel L, Rana KJ, Herráez MP (1998): Sublethal damage during cryopreservation of rainbow trout sperm, Cryobiology 37: 245-53.
• Cabrita E, Robles V, Rebordinos L, Sarasquete C, Herráez MP (2005): Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm. Cryobiology 50: 144-53.
• Chao N, Liao IC (2001): Cryopreservation of finfish and shellfish gametes and embryos. Aquaculture 197: 161-189.
• Chao NH, Chao WC, Liu KC, Liao IC (1987): The properties of tilapiasperm and its cryopreservation, J Fish Biol 30: 107-18.
• Diwan AD, Ayyappan S, Lal KK, Lakra WS (2010): Cryopreservation of fish gametes and embryos. Indian Journal of Animal Sciences 80(4): 109-24.
• Fraser L, Strzeżek J (2005): Effects of Freezing–Thawing on DNA Integrity of Boar Spermatozoa Assessed by the Neutral Comet Assay. Reprod Domest Anim 40: 530-36.
• Hughes CM, Lewis SEM, McKelvey-Martin VJ, Thompson W (1997): Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay. Mutat Res 374: 261-68.
• Kopeika J, Kopeika E, Zhang T, Rawson DM, Holt WV (2003): Detrimental effects of cryopreservation of loach (Misgurnus fosilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine. Cryobiology 46: 43-52.
• Magyary I, Urbanyi B, Horvath L (1996): Cryopreservation of commoncarp (Cyprinus carpio L.) sperm II. Optimal conditions for fertilization. J Appl Ichthyol 12: 117-19.
• Nossoni F (2008): Single-Cell Gel Electrophoresis (Comet Assay): Methodology, Potential Applications, and Limitations in Cancer Research. MMG 445 Basic Biotechnology eJournal 4: 30-5.
• Öğretmen F, İnanan BE (2014): Effect of butylated hydroxytoluene (BHT) on thecryopreservation of common carp (Cyprinus carpio) spermatozoa. Anim Reprod Sci 151: 269-74.
• Ostling O, Johanson KJ (1984): Microelectrophoretic study on radiation-induced DNA migration from individual cells. Biochem Biophys Res Commun 123: 291-98.
• Peris SI, Morrier A, Dufour M, Janice L (2004): Bailey Cryopreservation of Ram Semen Facilitates Sperm DNA Damage: Relationship Between Sperm Andrological Parameters and the Sperm Chromatin Structure Assay. J Androl 25(2): 224-33.
• Shen HM, Ong C ( 2000): Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med 15: 529-36.
• Singh NP, McCoy MT, Tice RR, Schneider EL (1988): A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1): 184-91.
• Suquet M, Dreanno C, Petton B, Normant Y, Omnes MH, Billard R. (1998): Long term effects of the cryopreservation of turbot (Psetta maxima) spermatozoa. Aquat Living Resour 11: 45-8.
• Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2008). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35: 206-21.
• Zilli L, Schiavone R, Zonno V, Storelli C, Vilella S (2003): Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation. Cryobiology 47: 227-35.