The Advantage of Absolute Quantification in ...

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“somatomedin” hypothesis, IGF-I is mainly produced in the liver under the control of GH from the anterior pituitary. After its release into the circulation, IGF-I acts ...

The Advantage of Absolute Quantification in Comparative Hormone Research as Indicated by a Newly Established Real-Time RT-PCR GH, IGF-I, and IGF-II Gene Expression in the Tilapia, Oreochromis niloticus ELISABETH EPPLER, ANTJE CAELERS, GIORGI BERISHVILI, AND MANFRED REINECKE Division of Neuroendocrinology, Institute of Anatomy, University of Zürich, Zürich, Switzerland

ABSTRACT: We have developed a real-time RT-PCR that absolutely quantifies the gene expression of hormones using the standard curve method. The method avoids cloning procedures by using primer extension to create templates containing a T7 promoter gene sequence. It is rapid since neither separate reverse transcriptions nor postamplification steps are necessary, and its low detection level (2 pg/␮g total RNA) allows precise absolute quantification. Using the method, we have quantified the gene expression of GH, IGF-I, and IGF-II in the tilapia. KEYWORDS: growth hormone; insulin-like growth factors; mRNA; absolute quantification

Knowledge on the principles of gene regulation during bony fish growth, differentiation, and development is important in basic and environmental research as well as for aquaculture industry.1 Several semiquantitative methods (RT-PCR, RNase protection assay, Northern blot) are established tools to determine changes in gene expression that occur during ontogeny or after experimental treatment.2–4 However, it gets increasingly important to deal with absolute amounts. This is especially true in clinical studies aimed to compare virus load, cytokines, and tumor marker expression,5,6 and in endocrine disruptor research. Especially in the latter case, species differences in hormone and cytokine gene expression necessitate higher considerations to allow new comparative approaches. To date, very few studies have used PCR for absolute quantification7,8 in fish hormone research.

Address for correspondence: M. Reinecke, Division of Neuroendocrinology, Institute of Anatomy, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Voice: +41-1-6355370; fax: +41-1-635-5702. [email protected] Ann. N.Y. Acad. Sci. 1040: 301–304 (2005). © 2005 New York Academy of Sciences. doi: 10.1196/annals.1327.047 301

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We mainly focus on the expression of the insulin-like growth factors (IGFs) and their regulation by growth hormone (GH) in bony fish.2,8,9 According to the classical “somatomedin” hypothesis, IGF-I is mainly produced in the liver under the control of GH from the anterior pituitary. After its release into the circulation, IGF-I acts on various target cells. Several in vivo studies revealed that GH regulates the production of liver IGF-I also in teleosts. Injection of GH increased IGF-I mRNA and/or IGF-I serum level.10,11 In agreement, GH raised IGF-I expression also in hepatocyte cultures.2,4,12 As in mammals, there is evidence that GH stimulates the expression of IGF-I also in extrahepatic sites of bony fish.3,13,14 There are few studies on the potential GH dependence of IGF-II expression in bony fish. While early studies yielded controversial results,10,13 a recent investigation in juvenile common carp showed that injection of GH significantly increased the amount of IGF-II mRNA in liver and extrahepatic sites.3 The controversial results might well be due to species differences, but also could be caused by different sensitivities of the detection methods because the earlier studies used RNase protection assay and the latter used the more sensitive real-time PCR. Therefore, it is reasonable to assume that not only the IGF-I gene, but also the IGF-II gene is regulated by GH in bony fish liver and extrahepatic sites. This gives bony fish a quite unique state in phylogeny since, in mammals, GH most likely regulates only the expression of the IGF-I gene. However, species differences remain to be clarified as only limited data on absolute quantification of GH, IGF-I, and IGF-II in bony fish exist and, thus, limited conclusions can be drawn on species diversities (or similarities), simplifying comparisons. Therefore, we have established a real-time RT-PCR8 using the standard curve method to measure the absolute amounts of GH, IGF-I, and IGF-II gene expression in the tilapia (Oreochromis niloticus). The method avoids labor-intense cloning procedures6 because it uses primer extension to create templates containing a T7 promoter gene sequence for in vitro transcription of standardized cRNAs. The onetube technology avoids contamination. The method is rapid as there are neither separate reverse transcriptions nor postamplification steps, and its low detection level (2 pg/µg total RNA) allows precise absolute quantification. Using this method in a preliminary study on 5 adult individuals, we detected GH mRNA exclusively in the pituitary (242 ± 45 pg/µg total RNA). In contrast, in developing rainbow trout, GH mRNA was found in pituitary and in several other organs.15 In coho salmon, GH mRNA was expressed in pituitary and intestine, but was detectable in the latter only in small fish.16 Thus, during fish development, GH seems to be expressed also in extrapituitary sites. This suggestion is supported by a study on adult four-spine sculpin17 that also detected GH mRNA only in pituitary. We have further used our real-time PCR method to measure the absolute amounts of IGF-I and IGF-II mRNA in several organs of tilapia.8 In liver, the levels of IGF-I and IGF-II mRNA were determined as 8.90 ± 1.90 pg/µg total RNA (22.5 × 107 copies/µg total RNA) for IGF-I and 3.59 ± 0.98 pg/µg total RNA (8.7 × 107 copies/µg total RNA) for IGF-II. The level of IGF-II mRNA was significantly (P < 0.001) lower than that of IGF-I mRNA. In agreement, a relatively lower content of IGF-II mRNA than of IGF-I mRNA was determined in the liver of common carp.3 Comparable absolute data are available only for rainbow trout liver. By the use of RNase protection assay and real-time PCR, 1.68 pg IGF-I/µg 18S rRNA10 and 7.6 × 106 copies/µg total RNA7 were detected, which are less than our data. On the one hand, the lower IGF-I liver mRNA levels in trout may reflect the species difference or the develop-

EPPLER et al.: NEWLY ESTABLISHED REAL-TIME RT-PCR

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mental stage because adult individuals were investigated in the present study and juveniles by Shamblott et al.10 On the other hand, the nutritional status18 or the environmental temperature7 may also account for the differences. ACKNOWLEDGMENTS This work was supported by the SNF (Project 32-061481 and NRP 50, Project 4050-66580). REFERENCES 1. REINECKE, M. & C. COLLET. 1998. The phylogeny of the insulin-like growth factors. Int. Rev. Cytol. 183: 1–94. 2. SCHMID, A.C., M. REINECKE & W. KLOAS. 2000. Primary cultured hepatocytes of the bony fish Oreochromis mossambicus, the tilapia: a valid tool for physiological studies on insulin-like growth factor I (IGF-I) expression in liver. J. Endocrinol. 166: 265–273. 3. VONG, Q.P., K.M. CHAN & C.H. CHENG. 2003. Quantification of common carp (Cyprinus carpio) IGF-I and IGF-II mRNA by real-time PCR: differential regulation of expression by GH. J. Endocrinol. 178: 513–521. 4. PIERCE, A.L., J.T. DICKEY, D.A. LARSEN et al. 2004. A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes. Gen. Comp. Endocrinol. 135: 401–411. 5. NIESTERS, H.G. 2004. Molecular and diagnostic clinical virology in real time. Clin. Microbiol. Infect. 10: 5–11. 6. FRONHOFFS, S., G. TOTZKE, S. STIER et al. 2002. A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction. Mol. Cell. Probes 16: 99–110. 7. GABILLARD, J.C., C. WEIL, P.Y. RESCAN et al. 2003. Effects of environmental temperature on IGF1, IGF2, and IGF type I receptor expression in rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 133: 233–242. 8. CAELERS, A., G. BERISHVILI, M.L. MELI et al. 2004. Establishment of a real-time RT-PCR for the determination of absolute amounts of IGF-I and IGF-II gene expression in liver and extrahepatic sites of the tilapia. Gen. Comp. Endocrinol. 137: 196–204. 9. REINECKE, M., A. SCHMID, R. ERMATINGER et al. 1997. Insulin-like growth factor I in the teleost Oreochromis mossambicus, the tilapia: gene sequence, tissue expression, and cellular localization. Endocrinology 138: 3613–3619. 10. SHAMBLOTT, M.J., C.M. CHENG, D. BOLT et al. 1995. Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone. Proc. Natl. Acad. Sci. USA 92: 6943–6946. 11. KAJIMURA, S., K. UCHIDA, T. YADA et al. 2002. Effects of insulin-like growth factors (IGF-I and -II) on growth hormone and prolactin release and gene expression in euryhaline tilapia, Oreochromis mossambicus. Gen. Comp. Endocrinol. 127: 223–231. 12. DUAN, C., S.J. DUGUAY & E.M. PLISETSKAYA. 1993. Insulin-like growth factor I (IGF-I) mRNA expression in coho salmon, Oncorhynchus kisutch: tissue distribution and effects of growth hormone/prolactin family protein. Fish Physiol. Biochem. 11: 371–379. 13. DUGUAY, S.J., J. LAI-ZHANG, D.F. STEINER et al. 1996. Developmental and tissue-regulated expression of IGF-I and IGF-II mRNAs in Sparus aurata. J. Mol. Endocrinol. 16: 123–132. 14. BIGA, P.R., G.T. SCHELLING, R.W. HARDY et al. 2004. The effects of recombinant bovine somatotropin (rbST) on tissue IGF-I, IGF-I receptor, and GH mRNA levels in rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 135: 324–333. 15. YANG, B.Y., M. GREENE & T.T. CHEN. 1999. Early embryonic expression of the growth hormone family protein genes in the developing rainbow trout, Oncorhynchus mykiss. Mol. Reprod. Dev. 53: 127–134.

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16. MORI, T. & R.H. DEVLIN. 1999. Transgenic and host growth hormone expression in pituitary and nonpituitary tissues of normal and growth hormone transgenic salmon. Mol. Cell. Endocrinol. 149: 129–139. 17. INOUE, K., H. IWATANI & Y. TAKEI. 2003. Growth hormone and insulin-like growth factor I of a euryhaline fish Cottus kazika: cDNA cloning and expression after seawater acclimation. Gen. Comp. Endocrinol. 131: 77–84. 18. LARSEN, D.A., B.R. BECKMAN & W.W. DICKHOFF. 2001. The effect of low temperature and fasting during the winter on metabolic stores and endocrine physiology (insulin, insulin-like growth factor-I, and thyroxine) of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 123: 308–323.

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