Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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PPIA, HPRT1, and YWHAZ are suitable reference genes for quantitative polymerase chain reaction assay of the hypothalamic-pituitary-gonadal axis in sows

  • Kim, Hwan-Deuk (College of Veterinary Medicine, Kyungpook National University) ;
  • Jo, Chan-Hee (College of Veterinary Medicine, Gyeongsang National University) ;
  • Choe, Yong-Ho (College of Veterinary Medicine, Gyeongsang National University) ;
  • Lee, Hyeon-Jeong (College of Veterinary Medicine, Gyeongsang National University) ;
  • Jang, Min (College of Veterinary Medicine, Kyungpook National University) ;
  • Bae, Seul-Gi (College of Veterinary Medicine, Kyungpook National University) ;
  • Yun, Sung-Ho (College of Veterinary Medicine, Kyungpook National University) ;
  • Lee, Sung-Lim (College of Veterinary Medicine, Gyeongsang National University) ;
  • Rho, Gyu-Jin (College of Veterinary Medicine, Gyeongsang National University) ;
  • Kim, Seung-Joon (College of Veterinary Medicine, Kyungpook National University) ;
  • Lee, Won-Jae (College of Veterinary Medicine, Kyungpook National University)
  • Received : 2022.03.03
  • Accepted : 2022.04.25
  • Published : 2022.12.01
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Abstract

Objective: The quantitative reverse transcription polymerase chain reaction (qPCR) is the most accurate and reliable technique for analysis of gene expression. Endogenous reference genes (RGs) have been used to normalize qPCR data, although their expression may vary in different tissues and experimental conditions. Verification of the stability of RGs in selected samples is a prerequisite for reliable results. Therefore, we attempted to identify the most stable RGs in the hypothalamic-pituitary-gonadal (HPG) axis in sows. Methods: The cycle threshold values of nine commonly used RGs (18S, HPRT1, GAPDH, RPL4, PPIA, B2M, YWHAZ, ACTB, and SDHA) from HPG axis-related tissues in the domestic sows in the different stages of estrus cycle were analyzed using two RG-finding programs, geNorm and Normfinder, to rank the stability of the pool of RGs. In addition, the effect of the most and least stable RGs was examined by normalization of the target gene, gonadotropin-releasing hormone (GnRH), in the hypothalamus. Results: PPIA, HPRT1, and YWHAZ were the most stable RGs in the HPG axis-related tissues in sows regardless of the stages of estrus cycle. In contrast, traditional RGs, including 18S and ACTB, were found to be the least stable under these experimental conditions. In particular, in the normalization of GnRH expression in the hypothalamus against several stable RGs, PPIA, HPRT1, and YWHAZ, could generate significant (p<0.05) elevation of GnRH in the preovulatory phase compared to the luteal phase, but the traditional RGs with the least stability (18S and ACTB) did not show a significant difference between groups. Conclusion: These results indicate the importance of verifying RG stability prior to commencing research and may contribute to experimental design in the field of animal reproductive physiology as reference data.

Keywords

Acknowledgement

This work was supported by a grant from the National Research Foundation (NRF) of Korea, funded by the government of the Republic of Korea (NRF-2020R1F1A1076723).

References

  1. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 2002;29:23-39. https://doi.org/10.1677/jme.0.0290023
  2. Lee WJ, Jang SJ, Lee SC, et al. Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos. Reprod Fertil Dev 2017;29:357-67. https://doi.org/10.1071/RD14393
  3. Niu G, Yang Y, Zhang YY, et al. Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs. Peer J 2016;4:e2428. https://doi.org/10.7717/peerj.2428
  4. Cinar MU, Islam MA, Uddin MJ, et al. Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA. BMC Res Notes 2012;5:107. https://doi.org/10.1186/1756-0500-5-107
  5. Lee WJ, Jeon RH, Jang SJ, et al. Selection of reference genes for quantitative gene expression in porcine mesenchymal stem cells derived from various sources along with differentiation into multilineages. Stem Cells Int 2015;2015:235192. https://doi.org/10.1155/2015/235192
  6. Oczkowicz M, Rozycki M, Piorkowska K, PiestrzynskaKajtoch A, Rejduch B. A new set of endogenous reference genes for gene expression studies of porcine stomach. J Anim Feed Sci 2010;19:570-6. https://doi.org/10.22358/jafs/66323/2010
  7. Nygard AB, Jorgensen CB, Cirera S, Fredholm M. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol Biol 2007;8:67. https://doi.org/10.1186/1471-2199-8-67
  8. Uddin MJ, Cinar MU, Tesfaye D, Looft C, Tholen E, Schellander K. Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues. BMC Res Notes 2011;4:441. https://doi.org/10.1186/1756-0500-4-441
  9. Jang SJ, Jeon RH, Kim HD, et al. TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells. Asian-Australas J Anim Sci 2020;33:2021-30. https://doi.org/10.5713/ajas.20.0238
  10. Kim D, Kim HD, Son Y, et al. Establishment of normal reference intervals in serum biochemical parameters of domestic sows in Korea. J Anim Reprod Biotechnol 2021;36:261-9. https://doi.org/10.12750/JARB.36.4.261
  11. Hwang JC, Park BJ, Kim HD, et al. Immunohistological expression of cytochrome P450 1A2 (CYP1A2) in the ovarian follicles of prepubertal and pubertal rat. J Anim Reprod Biotechnol 2020;35:328-37. https://doi.org/10.12750/JARB.35.4.329
  12. Sathagopam S, Ullewar MP, Harne R, Velmurugan S. Chronic kisspeptin delays puberty and reduces feed intake and body weight in female rats. J Anim Reprod Biotechnol 2021;36:25-34. https://doi.org/10.12750/JARB.36.1.25
  13. Tomikawa J, Homma T, Tajima S, et al. Molecular characterization and estrogen regulation of hypothalamic KISS1 gene in the pig. Biol Reprod 2010;82:313-9. https://doi.org/10.1095/biolreprod.109.079863
  14. Ciechanowska M, Lapot M, Mateusiak K, Przekop F. Neuroendocrine regulation of GnRH release and expression of GnRH and GnRH receptor genes in the hypothalamuspituitary unit in different physiological states. Reprod Biol 2010;10:85-124. https://doi.org/10.1016/s1642-431x(12)60054-0
  15. Skovgaard K, Mortensen S, Poulsen KT, Angen O, Heegaard PMH. Validation of putative reference genes for qRT-PCR normalization in tissues and blood from pigs infected with Actinobacillus pleuropneumoniae. Vet Immunol Immunopathol 2007;118:140-6. https://doi.org/10.1016/j.vetimm.2007.04.010
  16. Martinez-Giner M, Noguera JL, Balcells I, Fernandez-Rodriguez A, Pena RN. Selection of internal control genes for real-time quantitative PCR in ovary and uterus of sows across pregnancy. PLoS One 2013;8:e66023. https://doi.org/10.1371/journal.pone.0066023
  17. Herbison AE. A simple model of estrous cycle negative and positive feedback regulation of GnRH secretion. Front Neuroendocrinol 2020;57:100837. https://doi.org/10.1016/j.yfrne.2020.100837
  18. Soedea NM, Langendijk P, Kemp B. Reproductive cycles in pigs. Anim Reprod Sci 2011;124:251-8. https://doi.org/10.1016/j.anireprosci.2011.02.025
  19. Cinar MU, Islam MA, Proll M, et al. Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA. BMC Res Notes 2013;6:56. https://doi.org/10.1186/1756-0500-6-56
  20. Wang J, Wang Y, Wang H, Hao X, Wu Y, Guo J. Selection of reference genes for gene expression studies in porcine whole blood and peripheral blood mononuclear cells under polyinosinic:polycytidylic acid stimulation. Asian-Australas J Anim Sci 2014;27:471-8. https://doi.org/10.5713/ajas.2013.13471
  21. Bahmed K, Henry C, Holliday M, et al. Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B. Cancer Cell Int 2012;12:19. https://doi.org/10.1186/1475-2867-12-19
  22. Wang S, Wang B, He H, Sun A, Guo C. A new set of reference housekeeping genes for the normalization RT-qPCR data from the intestine of piglets during weaning. PLoS One 2018;13:e0204583. https://doi.org/10.1371/journal.pone.0204583
  23. Gan Y, Ye F, He XX. The role of YWHAZ in cancer: A maze of opportunities and challenges. J Cancer 2020;11:2252-64. https://doi.org/10.7150/jca.41316
  24. Gu YR, Li MZ, Zhang K, et al. Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig. J Anim Breed Genet 2011;128:319-25. https://doi.org/10.1111/j.1439-0388.2011.00920.x
  25. Xiang-Hong J, Yan-Hong Y, Han-Jin X, et al. Selection of reference genes for gene expression studies in PBMC from Bama miniature pig under heat stress. Vet Immunol Immunopathol 2011;144:160-6. https://doi.org/10.1016/j.vetimm.2011.07.004
  26. Zinzow-Kramer WM, Horton BM, Maney DL. Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds. Horm Behav 2014;66:267-75. https://doi.org/10.1016/j.yhbeh.2014.04.011
  27. Gholami K, Loh SY, Salleh N, Lam SK, Hoe SZ. Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence. PLoS One 2017;12:e0176368. https://doi.org/10.1371/journal.pone.0176368
  28. van Rijn SJ, Riemers FM, van den Heuvel D, et al. Expression stability of reference genes for quantitative RT-PCR of healthy and diseased pituitary tissue samples varies between humans, mice, and dogs. Mol Neurobiol 2014;49:893-9. https://doi.org/10.1007/s12035-013-8567-7
  29. Lv Y, Zhao SG, Lu G, et al. Identification of reference genes for qRT-PCR in granulosa cells of healthy women and polycystic ovarian syndrome patients. Sci Rep 2017;7:6961. https://doi.org/10.1038/s41598-017-07346-x
  30. Simon A, Javor A, Bai P, Olah J, Czegledi L. Reference gene selection for reverse transcription quantitative polymerase chain reaction in chicken hypothalamus under different feeding status. J Anim Physiol Anim Nutr (Berl) 2018;102:286-96. https://doi.org/10.1111/jpn.12690