COMPARISON OF THE SEMEN SWIM-UP AND SOMATIC CELL LYSIS PROCEDURES FOR RAM SPERM RNA EXTRACTION
Keywords:
ram semen, swim-up, somatic cell lysis buffer, total RNA extractionAbstract
Male infertility is an important aspect of animal reproduction, which has a high economic impact on the livestock industry. From rapidly increasing list of different sperm fertility biomarkers, the sperm RNA could serve as a promising diagnostic tool to assess male fertility or could have prognostic value for fertilization and embryo development. The aim of this preliminary study was to compare swim-up and somatic cell lysis buffer (SCLB) procedures for extraction of high-quality ram sperm RNA suitable for downstream molecular biology applications.
A modified TRI REAGENT RT procedure with glycogen and lysis step at 65 °C was carried out in order to extract total RNA. Spectrophotometric measurement of quality and quantity of extracted RNA showed A260/280 ratio 1.8 – 1.9, indicating the absence of contaminants and the amount of RNA 24 ± 3.9 µg (unpurified sperm), 0.9 ± 0.11 µg (swim-up) and 1.5 ± 0.2 µg (SCLB). Sperm RNA quality was further validated by RT-qPCR using primers for WBP2NL and MKRN1 genes. The CD18 and CDH1 markers for leucocytes and endothelial cells, respectively, have been used to check a successfull removal of somatic cells from ram sperm by both procedures. Unlike comparable relative amount of WBP2NL and MKRN1 transcripts among unpurified and swim-up or SCLB purified sperm RNA samples, relative amounts of CD18 and CDH1 transcripts were significantly lower in purified sperm RNA samples (p < 0.001), confirming an effective removal of leucocytes and endothelial cells from sperm by both purification techniques. Further investigations could reveal a potential of sperm RNA as a novel biomarker and promising diagnostic tool to assess ram fertility.
References
<br>Bianchi, E., Stermer, A., Boekelheide, K., Sigman, M., Hall, S. J., Reyes, G., Dere, E. & Hwang, K. (2018). High-quality human and rat spermatozoal RNA isolation for functional genomic studies. Andrology, 6, 374−383.
<br>Boerke, A., Dieleman, S. J. & Gadella, B. M. (2007). A possible role for sperm RNA in early embryo development. Theriogenology, 68, Suppl 1, S147−S155.
<br>Chen, X., Yue, Y., He, Y., Zhu, H., Hao, H., Zhao, X., Quin, T. & Wang, D. (2014). Identification and characterization of genes differentially expressed in X and Y sperm using suppression subtractive hybridization and cDNA microarray. Molecular Reproduction and Development, 81(10), 908−917.
<br>Geisinger, A., Wettstein, R. & Benavente, R. (1996). Stage-specific gene expression during rat spermatogenesis: application of the mRNA differential display method. The International Journal of Developmental Biology, 40(1), 385−388.
<br>Goodrich, R. J., Anton, E. & Krawetz, S. A. (2013). Isolating mRNA and small noncoding RNAs from human sperm. Methods in Molecular Biology, 927, 385−396.
<br>Hwang, K., Walters, R. C. & Lipshultz, L. I. (2011). Contemporary concepts in the evaluation and management of male infertility. Nature Reviews Urology, 8 (2), 86−94.
<br>Jodar, M., Selvaraju, S., Sendler, E., Diamond, M. P. & Krawetz, S. A. (2013). The presence, role and clinical use of spermatozoal RNAs. Human Reproduction Update, 19(6), 604−624.
<br>Kawano, M., Kawaji, H., Grandjean, V., Kiani, J. & Rassoulzadegan, M. (2012). Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos. PLoS One, 7, e44542. DOI: 10.1371/journal.pone.0044542.
<br>Kennedy, Ch. E., Krieger, K. B., Sutovsky, M., Xu, W., Vargovič, P., Didion, B. A., Ellersieck, M. R., Hennessy, M. E., Verstegen, J., Oko, R. & Sutovsky, P. (2014). Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination. Molecular Reproduction and Development, 81(5), 436−449.
<br>Kim, J. H., Park, S. M., Kang, M. R., Oh, S. Y., Lee, T. H., Muller, M. T. & Chung, I. K. (2005). Ubiquitin ligase MKRN1 modulates telomere length homeostasis through a proteolysis of hTERT. Genes & Development, 19, 776−781.
<br>Krawetz, S. A. (2005). Paternal contribution: new insights and future challenges. Nature Reviews Genetics, 6(8), 633−642.
<br>Kulikova, B., Kovac, M., Bauer, M., Tomkova, M., Olexikova, L., Vasicek, J., Balazi, A., Makarevich, A. V. & Chrenek, P. (2019). Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification. Acta Histochemica, 121, 491−499.
<br>Lalancette, C., Platts, A. E., Johnson, G. D., Emery, B. R., Carrell, D. T. & Krawetz, S. A. (2009). Identification of human sperm transcripts as candidate markers of male fertility. Journal of Molecular Medicine, 87(7), 735−748.
<br>Lambard, S., Galeraud-Denis, I., Martin, G., Levy, R., Chocat, A. & Carreau, S. (2004). Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors: relationship to sperm motility and capacitation. Molecular Human Reproduction, 10(7), 535−541.
<br>Liu, T., Cheng, W., Gao, Y., Wang, H. & Liu, Z. (2012). Microarray analysis of microRNA expression patterns in the semen of infertile men with semen abnormalities. Molecular Medicine Reports, 6, 535−542.
<br>Miller, D. (2000). Analysis and significance of messenger RNA in human ejaculated spermatozoa. Molecular Reproduction and Development, 56 (2 Suppl), 259−264.
<br>Miller, D., Briggs, D., Snowden, H., Hamlington, J., Rollinson, S., Lilford, R. & Krawetz, S. A. (1999). A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis. Gene, 237(2), 385−392.
<br>Ostermeier, G. C., Miller, D., Huntriss, J. D., Diamond, M. P. & Krawetz, S. A. (2004). Reproductive biology: Delivering spermatozoan RNA to the oocyte. Nature, 429(6988), 154.
<br>Ostermeier, G. C., Goodrich, R. J., Diamond, M. P., Dix, D. J. & Krawetz, S. A. (2005). Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility. Fertility and Sterility, 83(6), 1687−1694.
<br>Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29(9), 2002−2007.
<br>Platts, A. E., Dix, D. J., Chemes, H. E., Thompson, K. E., Goodrich, R., Rockett, J. C., Rawe, V. Y., Quintana, S., Diamond, M. P., Strader, L. F. & Krawetz, S. A. (2007). Success and failure in human spermatogenesis as revealed by teratozoospermic RNAs. Human Molecular Genetics, 16(7), 763−773.
<br>Qian, X., Wang, L., Zheng, B., Shi, Z. M., Ge, X., Jiang, C. F., Qian, Y. C., Li, D. M., Li, W., Liu, X., Yin, Y., Zheng, J. T., Shen, H., Wang, M., Guo, X. J., He, J., Lin, M., Liu, L. Z., Sha, J. H. & Jiang, B. H. (2016). Deficiency of Mkrn2 causes abnormalspermiogenesis and spermiation, and impairs male fertility. Scientific Reports, 6, 39318. https://doi.org/10.1038/.
<br>Schuster, A., Tang, C., Xie, Y., Ortogero, N., Yuan, S. & Yan, W. (2016). Spermbase: a database for sperm-borne RNA contents. Biology of Reproduction, 95(5), 99, 1−12.
<br>Sieme, H. & Oldenhof, H. (2015). Sperm cleanup and centrifugation processing for cryopreservation. Methods in Molecular Biology, 1257, 343−352.
<br>Sutovsky, P., Aarabi, M., Miranda Vizuete, A. & Oko, R. (2015). Negative biomarker based male fertility evaluation:sperm phenotypes associated with molecular – level anomalies. Asian Journal of Andrology, 17, 554−560.
<br>Vašíček, J. Tvarožková, K., Uhrinčať, M., Mačuhová, L., Hleba, L. & Tančin, V. (2019). Distribution of leucocytes and epithelial cells in sheep milk in relation to somatic cell count and bacterial occurence. A preliminary study. Slovak Journal of Animal Science, 52(4), 160−165.</div>
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