Abstract
Regucalcin (RGN) is a mammalian Ca2+-binding protein that plays an important role in intracellular Ca2+ homeostasis. Recently, RGN has been identified as a target gene for sex steroid hormones in the prostate glands and testis of rats and humans, but no studies have focused on RGN expression in bovine tissues. Thus, in the present study, we examined RGN mRNA and protein expression in the different tissues and organs of veal calves and beef cattle. Moreover, we investigated whether RGN expression is controlled through sex steroid hormones in bovine target tissues, namely the bulbo-urethral and prostate glands and the testis. Sex steroid hormones are still illegally used in bovine husbandry to increase muscle mass. The screening of the regulation and function of anabolic sex steroids via modified gene expression levels in various tissues represents a new approach for the detection of illicit drug treatments. Herein, we used quantitative PCR, western blot and immunohistochemistry analyses to demonstrate RGN mRNA and protein expression in bovine tissues. In addition, estrogen administration down-regulated RGN gene expression in the accessory sex glands of veal calves and beef cattle, while androgen treatment reduced RGN gene expression only in the testis. The confirmation of the regulation of RGN gene expression through sex steroid hormones might facilitate the potential detection of hormone abuse in bovine husbandry. Particularly, the specific response in the testis suggests that this tissue is ideal for the detection of illicit androgen administration in veal calves and beef cattle.
Highlights
Regucalcin (RGN) was first identified in 1978 as a calcium (Ca2+)-binding protein [1], which does not contain the typical EF-hand Ca2+-binding motif [2]
The presence of RGN protein in the same tissues was further confirmed through western blot (WB) analysis (Figure 1C and D), showing a immunoreactive band of 33 kDa
RGN expression was down-regulated in the testis of testosterone-treated veal calves (7.14E-03 ¡ 1.16E-03) compared with the control group K1 (2.85E-02 ¡ 3.35E-03) (P,0.01) (Figure 2C)
Summary
Regucalcin (RGN) was first identified in 1978 as a calcium (Ca2+)-binding protein [1], which does not contain the typical EF-hand Ca2+-binding motif [2]. RGN regulates intracellular Ca2+ homeostasis through the modulation of the activity of Ca2+ channels, Ca2+-ATPase in the membrane of mitochondria and endoplasmic reticulum [4, 5] and (Ca2+-Mg2+)ATPase in the plasma membrane [6, 7]. RGN plays an important role in the regulation of Ca2+-dependent enzymes, such as protein kinases, tyrosine kinases, phosphatases, phosphodiesterase, nitric oxide synthase and proteases [8,9,10,11,12,13]. RGN is widely expressed in a variety of tissues and cell lines [25,26,27] and was first identified in the liver, where this protein is highly expressed [1]. RGN protein has been shown to be secreted to biological fluids, namely plasma [31, 40,41] and seminiferous tubules fluid [29]
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