Influence of Zeranol treatment on the tissue specific co-expression of
estrogen receptors (ER) in various tissues: 
Quantification of ERα and ERβ mRNA with a real-time RT-PCR 

Experimental and Clinical Endocrinology 108, Suppl. 1/00, p 176 (2000)

Michael W. Pfaffl, Iris G. Lange, Andreas Daxenberger & Heinrich H.D. Meyer
Institute of Physiology, FML-Weihenstephan, TU-München (pfaffl@weihenstephan.de)

INTRODUCTION

Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogen active preparation RALGRO on the tissue specific co-
expression and regulation of both ER subtypes. RALGRO contains Zeranol (1x = 36 mg Zeranol), a derivative of the mycotoxin Zearalenon, and shows strong 
estrogenic and anabolic effects. It exhibits all symptoms of hyper-estrogenism in particular reproductive and developmental disorders. It is well known that 
steroids lead to an increased synthesis of specific proteins and it is proposed that estradiol can stimulate via ER its own receptor expression at least in 
the uterus. To quantify these possible transcripts also in low abundant tissues, sensitive and reliable real-time RT-PCR quantification methods were developed 
and validated on the LightCycler.

MATERIAL & METHODS

Eight heifers were treated over 8 weeks with multiple pellet implantations (0x, 1x, 3x, 10x) and after section Zeranol concentrations were measured by 
enzyme-immuno-assay (Zeranol EIA). In plasma and 4 representative edible tissues (longissimus dorsi, kidney, liver, peri-renal fat) concentration were 
elevated corresponding to the multiple treatment. In totally 15 different tissue ER expression profiles were quantified via real-time RT-PCR. ER α/ER β assay 
sensitivities and reliabilities were confirmed by detection limits down to 10 ssDNA molecules and linear quantification ranges between 10² to 10⁹ molecules (
r>0.955) with intra-and inter-assay variations of <19% to <30% respectively.

RESULTS

Real-time RT-PCRs were ERα and ERβ product specific and in all tissues both transcripts were found in different expression ratios (Table 1).High ERα/ERβ
ratios were examined in some muscle parts, liver, udder and uterus; except in kidney and jejunum, the ERα/ERβ ratios were <1. To make the individual tissue 
expression pattern evident all ERα and ERβ expression rates were compared and shown with bi-directional error bars (Figure 1). Each tissue possesses an ER 
subtype specific expression pattern which stays relatively stable even under zeranol treatment and resulted in an ER α/ER β expression cluster.

With increasing Zeranol concentrations a significant down-regulation of ER��mRNA expression could be observed in jejunum (p<0.001) and kidney medulla (p<0.05) 
(Figure 2)

A positive high correlated (r = +0.674; p<0.001) co-expression of both ER subtypes was shown in uterus, liver, jejunum, abomasum, udder, spleen, lung, neck 
and hind leg muscularity.

Table 1: ERα and ERβ mRNA expression levels and expression ratio (ERα/ERβ) in 25 ng cattle total RNA. Mean expression (in mRNA molecules) and variation coefficient 
(VQ) of 8 animals.

		ERα 	   VQ 	ERβ 	 VQ    ratio
uterus 		980,000    53% 	80,000 	140%    12 
udder 		205,000   105% 	 7,250 	 64%    28
liver 		200,000    87% 	 6,250 	179%    32
lung 		  5,400   167% 	   900 	 94%     6
spleen 		 12,000    65% 	10,400 	104%     1.2
heart muscle 	  6,000    82% 	 4,200 	 56%     1.4
kidney medulla	 10,200    52% 	35,100 	 65%     0.3
kidney cortex 	  4,200    57% 	29,300 	 51%     0.14
rumen 		  4,060    53% 	 1,350 	109%     3 
abomasum 	  4,600    84% 	   650 	157%     7 
jejunum 	  1,550    72% 	 2,150 	152%     0.7 
long. dorsi 	 79,400    61% 	 8,650 	 22%     9
hind leg m. 	100,000    47% 	 5,850 	 76%    17
shoulder m. 	 60,500    83% 	 2,600   93% 	23
neck muscles 	145,000    79% 	 2,250 	133% 	64

Figure1: ERα and ERβ expression cluster in 15 bovine tissues.

Figure 2: Relation between multiple RALGRO treatment and ERα expression.

DISCUSSION & CONCLUSION

In view of the data provided for sensitivity, linearity and reproducibility, the developed RT-PCR assay developed herein allows for the absolute and accurate 
quantification of ERα and ERβ mRNA molecules with a sufficiently high sensitivity even for tissues with low abundancies down to a few molecules.

Our expression results indicate the existence of two ER subtypes in various bovine tissues, their different expression pattern and co-expression as well as 
their tissue specific regulation under estrogen treatment. These different expression patterns of ERα and ERβ could be regarded as support for the hypothesis 
that the ER subtype proteins may have different biological functions, especially in kidney and the jejunum where ERβ expression ratio is vice versa in 
comparison to the other investigated tissues.

In future more detailed study of ERα and ERβ must be investigated in all kidney cell types and in all parts of the gastrointestinal system to continue 
investigations of the ER regulation and its subtype physiological function.

source: http://www.wzw.tum.de/gene-quantification/pfaffl-erab-dge-2000.pdf 28dec03
            http://www.wzw.tum.de/gene-quantification/