The Effect of GH and GnRH Hormone
Injections on the Production of IGF-I in
Goldfish Livers
Name
July 18th 2020
Introduction
In vertebrates, growth is a multifactorial characteristic resulting from a very complex genetic
and molecular interactions in which hormones are the major factors. The growth regulator GH
also carries out other physiological process such as having an effect on lipids, protein and
carbohydrate metabolism. (Davidson 1987; Moller and Norrelund, 2003) immune system
maintenance, Jeay et al., 2003) and response behavior to stress (Yoshizato et al.,1998). GH
biological processes are carried are control solely at the somatotrophs in the anterior part of the
pituitary gland and on all of the target organs that interact with certain cell surface receptors
called the GHR. Both the GHR and GH depends on both the environment and biological factors.
(Flores-Morales et al, 2006).
Insulin-like growth factors (IGFS) are polypeptides that are primarily produced in the livers
and are important regulators of growth and differentiation. The nucleotide and amino acids
sequences IGF-I have been determine in a number of mammalian and nonmammalian species. In
mammals, growth hormones (GH) stimulates the production of IGF-1. (Kermouni et al., 1998).
Similar GH stimulatory effects of IGF-I have been reported in several fish species.IGF-I has
been isolated from goldfish liver and ovary with five IGF-I isoforms identified. PCR, a DNA
amplification technique, may be used to quantify the levels of multiple samples of DNA PCR
acts by replicating multiple copies of DNA sample using the appropriate primer and a pool of
nucleotides.( (Kermouni et al.,1998).
IGF-I is mainly produced in liver which is the main source of circulating( endocrine) IGF-I
under the influence of growth hormone( GH). IGF-I released from the liver into the circulation
acts on a variety of target cells. Also IGF-I also expressed, in extrahepatic sites and most likely
stimulates organ –specific functions by paracrine and autocrine processes. There is increasing
evidence that GH stimulates the expression of IGF-I also in extrahepatic sites. (Vong et al., 2003,
Biga et al., 2004).
In this virtual lab, the researcher hypothesized that the goldfish that received either GH or
GnHR injections will result in elevated levels of IGF-I production in the goldfish liver.
Methods
Goldfish specimens were obtain. Carassius auratus, that range in size from fout to five grams.
Maintain the fish in flow-through aquaria and feed them commercial fish food. Then two
experiments were set up, one testing the effects of GH on IGF-I production and a second testing
the effects of GnRH on IGF-l production. (Kermouni et al.,1998)
In the first experiment, the experiment group of fish was set up and the fish received two
intraperitoneal injection (12 hours apart) of 2micrograms/g of carp GH. After that the control
group of fish received no hormone treatment. (Kermouni et al.,1998)
In the second experiment, the experiment group of fish was set up and the fish received two
intraperitoneal injections (12 hours apart) of 2micrograms/g of salmon GnRH. After that the
control group of fish received no hormone treatment. (Kermouni et al.,1998)
After the application of treatments was applied, the fish were sacrificed and all of their livers
were removed. Then RNA was extracted from the liver. After that cDNA for goldfish IGF-
mRNA using primers of carp IGF-I sequences. After synthesizing cDNA was synthesized from
the goldfish IGF-l, then a Competitive Quantitative-PCR system called Q- PCR was set up. This
system involved preparing two separate DNA samples with the same 3’ and 5’ ends so that the
samples compete for the same PCR primers and will result in a higher PCR product. (Kermouni
et al.,1998)
Specifically in these experiments, an internal standard template was set up using a 700-bp
fragment coding for a plant protein, oleosin. After that, a synthetic oligonucleotide was ligated
for a specific goldfish IGF-l to the 3’ and 5’ ends of the oleosin cDNA. The chimeric oleosin
cDNA was then placed into PCR reaction tubes with the 500-bp goldfish IGF-I cDNA and
specific IGF-I primers. The concentration of oleosin was adjusted and the standard template was
added to the PCR tubes from 0.1 to 1 pg/tube in the GH treatment and 0.0001 to 0.04 ng/tube in
the GnRH treatment. Because both the oleosin template and the goldfish IGF-I cDNAs, have
identical 3’ and 5’ ends they complete for the same primers. The PCR products were separated
using gel electrophoresis. Two PCR results, a 500-bp DNA band, corresponding to the IGF-I
cDNA and a DNA band, corresponding to the oleosin template cDNA. (Kermouni et al.,1998).
After that gels images were scanned and the band densities were measured. The relative
quantities of IGF-I and oleosin template c DNA were determine, then the ratio of oleosin internal
standard/ IGF-I was calculated for each concentration of oleosin standard that was added to the
PCR system. Then the ratio was plotted this against the concentration of the oleosin standard.
The effect of hormone treatment was determined by comparing the experimental ratios to the
control ratios. Then the results of the GH experiment were recorded. (Kermouni et al.,1998).
Results
According to the experimental results in the virtual lab, table 1 shows the effect of GH
hormone on IGF-I production in goldfish liver at which the oleosin template added to the PCR
on the ratio of oleosin template on the production of IGF-I c DNA. It also shows the mean of the
control and the GH treatment on IGF-I production. Figure 1 shows GH injections on IGF-1
production in goldfish livers. Table 2 shows the effect of Gnarr hormone on IGF-I production in
goldfish liver at which the oleosin template added to the PCR on the ratio of oleosin template on
the production IGF-I c DNA. It also shows the mean value for the control and Gnarr treatment on
IGF-I production. Figure 2 shows Gnarr injections on IGF-I production in goldfish livers.
A statistical analysis two tailed t-test was performed on GH and Gnarr injections on IGF-I
production in goldfish livers. The mean values for the GH treatment in group 1 was 0.425 and
group two was 2.3. The mean for the control in group 1 was 0.45 and in group 2 it was 8.45. The
p values were .360 for GH treatment and 0.308 for the control. The t values were 0.99 for GH
treatment and 1.11 for the control. The degree of freedom was 6 for both the GH treatment and
the control. The standard deviation for the GH treatment and the control was the same at .40311
in group 1, but in group 2 it was 14.2 for the control and 3.8 for the GH treatment. The p values
were 1.01 for the control and .056 for Gnarr treatment. The t values for the control was 1.8 and
for the Gnarr it was 2.16. The degree of freedom was the same for both at 10. The means for
group 1 were the same at 0.009 for both the control and the Gnarr treatment. The mean for group
2 was 8.34 for the control and Gnarr was 1.06. The standard deviation were the same for group 1
control and Gnarr at 0.01545 and group 2 it was 11.2 and 1.19 respectively. The degree of
freedom was the same at 10.
Table 1
The Effect of GH Hormone on IGF-I Production in Goldfish Liver
GH Injections in Goldfish Causing Increase Production of IGF-I
Oleosin template added to PCR(pg)
Ratio of Oleosin template/IGF-I cDNA
0.1
0.2
0.4
1
Mean 0.425
s.d 0.403
Control
0.3
0.5
3
30
Mean 8.45
P=0.308 t=1.11 s.d 14.1 d.f=6.0
GH Treatment
0.09
0.2
1
8
Mean 2.3
P=.360 t=0.99 s.d= 3.81 d.f=6.0
Figure 1
GH Injections on IGF-I in Production in Goldfish Livers
Ratio of oleosin template/IGF-I cDNA
100
10
GH Treatment
Control
1
0.1
0.1
0.2
0.4
1
Oleosin template added to PCR (pg)
Table 2
The Effect of GnHR Hormone on IGF-I Production in Goldfish Liver
GnHR Injections in Goldfish Causing Increase Production of IGF-I
Oleosin template added to PCR(pg)
Ratio of Oleosin template/IGF-I cDNA
0.0001
0.0004
0.001
0.005
0.01
0.04
Mean 0.09
s.d .015
Control
0.04
1
2
7
10
30
Mean
8.34
P=1.01 t= 1.81 d.f =10 s.d=11.3
GnHR Treatment
0.3
0.04
0.2
0.8
2
3
Mean 1.06
P=.056 t=2.16 d.f.=10 s.d.=1.19
Figure 2
GnHR Injections on IGF-I Production in Goldfish Livers
Ratio of oleosin template/IGF-I cDNA
100
10
Control
1
GnHR Treatment
0.1
0.01
0.0001
0.0004
0.001
0.005
0.01
0.04
Oleosin template added to PCR (ng)
Discussion
In conclusion, growth in vertebrates is controlled by many factors, such as genetics and
environmental factors (Moriyama et al., 2000) The hypothalamus in mammals produces two
antagonistic hormones, growth hormone –releasing hormone (GHRH) and somatostatin (SS)
which act to control the growth hormone (GH) release from the pituitary glands through the
process of stimulation or inhibition of its secretion, respectively (Arvat et al., 2002). This process
is more complicated in fish, because the regulation of growth hormones basically happens
through the use of GHRH and SS a well as though other hormones, which includes dopamine,
GnRH, thyrotropin-releasing hormones (TRH), and pituitary adenylate cyclase- activating
polypeptide ( PACAP) (Riley et al., 2002)
The higher the ratio of oleosin template/ IGF-I the greater the levels of oleosin template
compared to IGF-1. If there were no effect of GH treatment, the ratios for the control and GH
treatment groups would be the same. There was a positive correlation between the concentration
level of the oleosin template added to PCR and the ratio of oleosin template/ IGF-I c DNA.
(Kermouni et al.,1998).
The GnRH treatment resulted in decreases in the ratios of oleosin template IGF-I, indicating an
increase in IGF-I production in fish treated with GnRH. There was a positive correlation between
the concentration level of the oleosin template added to PCR and the ratio of oleosin template/
IGF-I c DNA. (Kermouni et al.,1998). In this virtual lab, the hypothesis was supported and
accepted by this data which showed both p values at < 5.
Literature Cited
Arvat, E., Broglio, F., Aimaretti, G., Benso, A., Giordano, R., Deghenghi, R., & Ghigo, E.
(2002). Ghrelin and synthetic GH secretagogues. Best Practice & Research Clinical
Endocrinology & Metabolism, 16(3), 505-517.
Biga, P. R., Schelling, G. T., Hardy, R. W., Cain, K. D., Overturf, K., & Ott, T. L. (2004). The
effects of recombinant bovine somatotropin (rbST) on tissue IGF-I, IGF-I receptor, and GH
mRNA levels in rainbow trout, Oncorhynchus mykiss. General and comparative
endocrinology, 135(3), 324-333.
Davidson, M.B., 1987. Effect of growth hormone on carbohydrate and lipid metabolism. Endocr.
Rev. 8, 115–131.
Flores-Morales, A., Greenhalgh, C.J., Norstedt, G., Rico-Bautista, E., 2006. Negative regulation
of growth hormone receptor signaling. Mol. Endocrinol. 20, 241–253.
Jeay, S., Sonenshein, G.E., Postel-Vinay, M.C., Kelly, P.A., Baixeras, E., 2002. Growth
hormone can act as a cytokine controlling survival and proliferation of immune cells: new
insights into signalling pathways. Mol. Cell. Endocrinol. 188, 1–7.
Kermouni, A., Mahmoud, S. S., Wang, S., Moloney, M., & Habibi, H. R. (1998). Cloning of a
full-length insulin-like growth factor-I complementary DNA in the goldfish liver and ovary and
development of a quantitative PCR method for its measurement. General and comparative
endocrinology, 111(1), 51-60.
Moriyama, S., Ayson, F. G., & Kawauchi, H. (2000). Growth regulation by insulin-like growth
factor-I in fish. Bioscience, biotechnology, and biochemistry,64(8), 1553-1562.
Moller, N., Norrelund, H., 2003. The role of growth hormone in the regulation of protein
metabolism with particular reference to conditions of fasting. Horm. Res. 59 (Suppl. 1), 62–68.
Riley, L. G., Hirano, T., & Grau, E. G. (2002). Rat ghrelin stimulates growth hormone and
prolactin release in the tilapia, Oreochromis mossambicus.Zoological science, 19(7), 797-800.
Vong, Q. P., Chan, K. M., & Cheng, C. H. (2003). Quantification of common carp (Cyprinus
carpio) IGF-I and IGF-II mRNA by real-time PCR: differential regulation of expression by
GH. Journal of Endocrinology, 178(3), 513-521.
Yoshizato, H., Fujikawa, T., Soya, H., Tanaka, M., Nakashima, K., 1998. The growth hormone
(GH) gene is expressed in the lateral hypothalamus: enhancement by GH-releasing hormone and
repression by restraint stress. Endocrinology 139, 2545–2551.