RNA ISOLATION AND DETECTION OF CELLULAR RNA QUANTITY OF SPERMATOZOA AND EMBRYOS PRIOR TO GENE EXPRESSION ANALYSES

Öz

DOI: 10.26650/IUITFD.416666

Objective: Biological samples that are analyzed in reproductive biology are generally rare, difficult to obtain, and a nonreplicable group of cells. Furthermore, investigating low numbers of cells requires modifications to the routine methods used in genetic analyses. The aim of our study was to improve RNA isolation methods for obtaining a sufficient amount of total RNA from spermatozoa and embryo samples for downstream gene expression analyses.

Materials and Methods: Excess spermatozoa samples (that had been prepared in the scope of assisted reproduction treatment) were purified of any contaminant cells and then frozen. Preimplantation stage embryos that had not been selected for embryo transfer were frozen in a phosphate-buffered saline–polyvinyl alcohol (PBS–PVA) solution. Modifications were done to obtain the optimal amount of total RNA from the spermatozoa and embryos, and subsequently both the quality and the quantity of the total RNA samples were assessed. Because of the selective degradation of 28S RNA samples in the spermatozoa, the total RNA quality was evaluated by gene amplification using quantitative real-time polymerase chain reaction (qRT-PCR) in addition to Bioanalyzer analysis.

Results: Following the modifications to the isolation technique, a sufficient quality and quantity of total RNA were obtained from the spermatozoa and embryo samples, which could be used in downstream gene expression analyses. Furthermore, the amount of cellular total RNAs was consistent to that reported in previous studies.

Conclusion: Results obtained from these experiments - conducted as a preliminary work for further transcript profiling studies - indicate that the modifications used in the RNA isolation techniques were effective.

Anahtar Kelimeler

• Spermatozoon,preimplantation stage embryo

SPERMATOZOA VE EMBRİYOLARIN GEN EKSPRESYON ANALİZİ ÖNCESİNDE RNA İZOLASYONU VE HÜCRESEL RNA MİKTARLARININ BELİRLENMESİ

Öz

DOI: 10.26650/IUITFD.416666

Amaç: Üreme biyolojisi araştırmalarında kullanılan örnekler genellikle, zor ulaşılabilir, nadir ve tekrarı olmayan hücre gruplarıdır. Diğer yandan, az sayıda hücre ile çalışıldığından dolayı, genetik analizlerde standart olarak kullanılan yöntemlerde modifikasyonlara ihtiyaç duyulmaktadır. Bu çalışmada amacımız, gen ekspresyonu araştırmalarında kullanılabilecek optimal düzeydeki total RNA’nın elde edilebilmesi amacı ile spermatozoa ve embriyo örneklerinden RNA izolasyon yönteminin geliştirilmesidir.

Gereç ve Yöntem: Yardımla üreme tedavisi kapsamında kullanılmak amacı ile hazırlanan ve fertilizasyon işlemi gerçekleştirildikten sonra geriye kalan spermatozoa örneği diğer kontaminant hücrelerden saflaştırılarak dondurulmuştur. Diğer yandan, embriyo transferinden sonra geriye kalan preimplantasyon dönem embriyolar fosfat tamponlu tuz – polivinil alkol (PBS-PVA) karışımı içerisinde dondurularak saklanmıştır. Spermatozoa ve embriyolardan optimal düzeyde total RNA elde edilmesi amacı ile çeşitli optimizasyonlar yapılmıştır. Elde edilen total RNA’lar kaliteleri ve miktarları açısından değerlendirilmiştir. Spermatozoonda 28S ribozomal RNA (rRNA)’nın seçimli olarak degrade olmasından dolayı total RNA kalitesi, Bioanalyzer cihazı ile gerçekleştirilen analize ek olarak gerçek zamanlı kantitatif PCR ile gen amplifikasyonu yapılarak da kontrol edilmiştir.

Bulgular: İzolasyonda yapılan uyarlamalardan sonra, takip eden gen ekspresyonu çalışmalarında kullanılabilecek kalitede ve miktarda total RNA elde edilmiştir. Ek olarak, örneklerden elde edilen hücresel total RNA miktarlarının önceki çalışmalar ile uyumlu olduğu belirlenmiştir.

Sonuç: Daha sonra yapılması planlanan transkript profilleme araştırmaları için ön bir çalışma olarak gerçekleştirilen bu deneylerin sonucunda elde edilen bulgular, RNA izolasyon protokollerinde yapılan uyarlamaların etkin olduğunu göstermektedir. 

Anahtar Kelimeler

• spermatozoon,preimplantasyon dönem embriyo

Kaynakça

1. 1. Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, Sullivan E, Vanderpoel S. International Committee for Monitoring Assisted Reproductive Technology; World Health Organization. The International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) Revised Glossary on ART Terminology. Human Reproduction 2009;24:2683–2687.
2. 2. Ebner T., Moser M., Sommergruber M., Tews G. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review. Hum Reprod Update. 2003;9:251–262.
3. 3. Siristatidis C, Komitopoulou MA, Makris A, Sialakouma A, Botzaki M, Mastorakos G, Salamalekis G, Bettocchi S, Palmer GA. Morphokinetic parameters of early embryo development via time lapse monitoring and their effect on embryo selection and ICSI outcomes: a prospective cohort study.J Assist Reprod Genet. 2015 Apr;32(4):563-70.
4. 4. Pribenszky C, Nilselid AM, Montag M. Time-lapse culture with morphokinetic embryo selection improves pregnancy and live birth chances and reduces early pregnancy loss: a meta-analysis. Reprod Biomed Online. 2017 Nov;35(5):511-520.
5. 5. Bromer JG., Seli E. Assesment of embryo viability in assisted reproductive technologies: shortcomings of current approaches and the emerging role of metabolomics. Curr Opin Obstet Gynecol 2008;20:234-241.
6. 6. Evsikov AV, de Evsikova CM. Gene expression during the oocyte-to-embryo transition in mammals. Mol Reprod Dev. 2009 September ; 76(9): 805–818.
7. 7. Katz-Jaffe MG., Schoolcraft WB., Gardner DK. Analysis of protein expression (secretome) by human and mouse preimplantation embryos. Fertil Steril 2006;86:678–685.
8. 8. Seli E., Robert C., Sirard M. OMICS in assisted reproduction: possibilities and pitfalls. Mol Hum Reprod 2010;16:(8), 513-30.

9. 9. Uyar A, Seli E. Metabolomic assessment of embryo viability. Semin Reprod Med. 2014;32(2):141-52.
10. 10. Ventura-Juncá P, Irarrázaval I, Rolle AJ, Gutiérrez JI, Moreno RD, Santos MJ. In vitro fertilization (IVF) in mammals: epigenetic and developmental alterations. Scientific and bioethical implications for IVF in humans. Biol Res. 2015;48:68.
11. 11. Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T. Transcriptomics technologies. PLoS Comput Biol. 2017;13(5):e1005457.
12. 12. Moreno JM, Núñez MJ, Quiñonero A, Martínez S, de la Orden M, Simón C, Pellicer A, Díaz-García C, Domínguez F. Follicular fluid and mural granulosa cells microRNA profiles vary in in vitro fertilization patients depending on their age and oocyte maturation stage. Fertil Steril. 2015 Oct;104(4):1037-1046.e1
13. 13. Burnik Papler T, Vrtacnik Bokal E, Maver A, Kopitar AN, Lovrečić L. Transcriptomic Analysis and Meta-Analysis of Human Granulosa and Cumulus Cells. PLoS One. 2015;10(8):e0136473.
14. 14. Montjean D, De La Grange P, Gentien D, Rapinat A, Belloc S, Cohen-Bacrie P, Menezo Y, Benkhalifa M. Sperm transcriptome profiling in oligozoospermia. J Assist Reprod Genet. 2012;29(1):3-10.
15. 15. Krawetz SA. Paternal contribution: new insights and future challenges. Nat Rev Genet. 2005;6(8):633-42.
16. 16. Miller D, Ostermeier GC, Krawetz SA. The controversy, potential and roles of spermatozoal RNA. Trends Mol Med. 2005;11(4):156-63.
17. 17. Cappallo-Obermann H, Schulze W, Jastrow H, Baukloh V, Spiess AN. Highly purified spermatozoal RNA obtained by a novel method indicates an unusual 28S/18S rRNA ratio and suggests impaired ribosome assembly. Mol Hum Reprod 2011 Nov;17(11):669-78.
18. 18. WHO laboratory manual for the examination and processing of human semen. 5th Ed. WHO, 2010
19. 19. Ostermeier GC, Dix DJ, Miller D, Khatri P, Krawetz SA. Spermatozoal RNA profiles of normal fertile men. Lancet 2002; 360: 772–77.
20. 20. Goodrich RJ, Anton E, Krawetz SA. Isolating mRNA and small noncoding RNAs from human sperm. Methods Mol Biol. 2013;927:385-96.
21. 21. Mao S, Sendler E, Goodrich RJ, Hauser R, Krawetz SA. A comparison of sperm RNA-seq methods. Syst Biol Reprod Med. 2014;60(5):308-15.
22. 22. Wells D, Bermúdez MG, Steuerwald N, Malter HE, Thornhill AR, Cohen J. Association of abnormal morphology and altered gene expression in human preimplantation embryos. Fertil Steril. 2005;84(2):343-55.
23. 23. Kleijkers SH, Eijssen LM, Coonen E, Derhaag JG, Mantikou E, Jonker MJ, Mastenbroek S, Repping S, Evers JL, Dumoulin JC, van Montfoort AP. Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media. Hum Reprod. 2015;30(10):2303-11.
24. 24. Mantikou E, Jonker MJ, Wong KM, van Montfoort AP, de Jong M, Breit TM, Repping S, Mastenbroek S. Factors affecting the gene expression of in vitro cultured human preimplantation embryos. Hum Reprod. 2016;31(2):298-311.
25. 25. Dobson AT, Raja R, Abeyta MJ, Taylor T, Shen S, Haqq C, Pera RA. The unique transcriptome through day 3 of human preimplantation development. Hum Mol Genet 2004;13(14):1461-70.
26. 26. Zhang SD, Gant TW. Efect of pooling samples on the efficiency of comparative studies using microarrays. Bioinformatics 2005;21(24):4378-83.
27. 27. Peng X, Wod CL, Blalock EM, Chen KC, Landfield PW, Stromberg AJ. Statistical implications of pooling RNA samples for microaaray experiments. BMC Bioinformatics 2003;4:26.
28. 28. Zhang P, Zucchelli M, Bruce S, Hambiliki F, Stavreus-Evers A, Levkov L, Skottman H, Kerkela E, Kere J, Hovatta O. Transcriptome Profiling of Human Pre-implantation Development. Plos One 2009;4:11-e7844.
29. 29. Zeng F. and Schultz R.M. Gene expression in mouse oocytes and preimplantation embryos: use of suppression subtractive hybridization to identify oocyte- and embryo-specific genes. Biol. Reprod 2003;68:31–9.
30. 30. Carter MG, Hamatani T, Sharov AA, Carmack CE, Qian Y, Aiba K, Ko NT, Dudekula DB, Brzoska PM, Hwang SS, Ko MSH. In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling. Genome Res 2003;13:1011–21.