Quantification of growth hormone receptor extra- and intracellular domain gene expression in chicken liver by quantitative competitive RT-PCR.
The very sensitive competitive reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate the expression of the extracellular (GHRe) and intracellular (GHRi) parts of the growth hormone receptor (GHR) in the liver tissue of chickens. Two competitors (internal standards), pGHRi MUT and pGHRe MUT, specific to the GHRi and GHRe genes, respectively, were constructed by site-specific mutagenesis. The internal standards defined PCR products of 394 bp for the pGHRi MUT and 330 bp for the GHRe MUT. These were used as competitors to the wild-type GHRi or GHRe which defined PCR products of 382 and 328 bp, respectively. Coamplification, under standardized conditions, of the native RNA in competition with serial dilutions of the mutant RNA in the same PCR reaction followed by enzymatic digestion produced the expected sizes of internal standard cDNA and predicted target cDNA. Expression levels of GHRe and GHRi were determined from standard curves generated. The method was sensitive enough to detect expressions down to picogram levels. Applying this method, the effect of GH and T(3) injection on GHRe and GHRi mRNA expression was determined in the liver of adult female Hisex birds and 1-day-old normal and dwarf chickens. Intravenous GH injection (25 microg/kg body weight) increased plasma levels of GH in Hisex birds after 10 min but rapidly decreased at 60 min followed by an increase in T(3). GH injection significantly increased the expression of the GHRe 60 min after injection but not at 10 min, when the GH level in plasma was high. In the liver of saline-treated dwarf (dw) and nondwarf (Dw) chicks, the level of expression of GHRe was similar in both strains despite disparate levels of basal GH and T(3). However, the level of GHRi was higher in Dw than in dw chicks. Although GH levels increased in both strains after intravenous GH injection (250 microg/kg body wt), the expression of GHRe in both strains was unaffected. However, the mRNA for the GHRi was significantly depressed by injection in the Dw but unaffected in dw chicks. Intravenous injection of T(3) (0.5 and 5 microg/kg body wt) increased plasma levels in both strains but caused depression of GHRi in Dw but not in dw chicks. T(3) injections had no effect on GHRe in either Dw or dw chicks. It is concluded that the expression of the GHRe in adult chickens is GH regulated either directly or indirectly. In contrast, in 1-day-old chicks, GH or T(3) had no effects on the GHRe but regulated the expression of GHRi in Dw chicks, whereas in dwarf chicks both had no effect on GHRe or GHRi expression. It is postulated that GHRe and GHRi gene expression may be regulated by different agonists/antagonists in different strains and depending on the age of the chicken.
Van As
P
P
Leuven Poultry Research Group, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, Leuven, B-3001, Belgium.
Janssens
W
W
Onagbesan
O M
OM
Bruggeman
V
V
Buys
N
N
Sanders
J
J
Van Der Geyten
S
S
Darras
V M
VM
Decuypere
E
E
D000375:Aging / Q000378:metabolism; D000818:Animals; D002645:Chickens / Q000378:metabolism*; D004392:Dwarfism / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000502:physiology*; D013006:Growth Hormone / Q000097:blood; D008099:Liver / Q000378:metabolism*; D008297:Male; D009154:Mutation / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011986:Receptors, Somatotropin / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014284:Triiodothyronine / Q000097:blood
D012333:RNA, Messenger; D011986:Receptors, Somatotropin; D014284:Triiodothyronine; D013006:Growth Hormone