Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution

Abstract

It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-and ER , and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28,(96)(97)(98)(99)(100)(101)(102)(103)(104)(105)(106)(107)(108) h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ER , ER and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ER , ER and PR in bovine mammary gland. Both ER and PR are expressed in fg/µg total RNA range. The highest mRNA expression was found for ER and PR in the tissue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ER was about 1000-fold lower (ag/µg total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ER revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tissue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ER and ER protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ER was weaker in the tissue of non-pregnant heifers and during involution (4 weeks). ER protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tissue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.

Chromosomal mapping and quantitative analysis of estrogen-related receptor alpha-1, estrogen receptors alpha and beta and progesterone receptor in the bovine mammary gland

“…In contrast, transcripts for ER were present in exceedingly low concentration and the protein was undetectable by IHC throughout all physiological stages. Similarly, Schams et al (2003) showed low expression levels of ER transcripts and low, but detectable, levels of protein expression by Western blot. These data suggest a relative lack of a role for ER in bovine mammary gland development and lactation.…”

“…Although we demonstrated the presence of PR in lactating bovine mammary gland by ligand binding methodology (Capuco et al 1982), it is commonly stated that lactating tissue does not contain PR. A recent study has evaluated changes in ER and PR expression in the bovine mammary gland (Schams et al 2003). Much remains to be learned about the regulation of ER and PR in the bovine gland, despite their importance to mammary gland function.…”

Molecular aspects of bovine cystic ovarian disease pathogenesis

“…Both PGRA and PGRB may activate different sets of genes, even within the same cell, revealing the enormous complexity of progesterone-dependent activation in the target cell (Graham & Clarke 2002, Alfaro et al 2012. This suggests that changes in the expression of PGR isoforms could regulate the biological activity of progesterone, resulting in functional hormone withdrawal at the ovarian level, under unchanged progesterone serum concentrations (Schams et al 2003, Amrozi et al 2004, Goldman et al 2005.…”

Sex steroid modulation of cortisol secretion in sheep

“…Primers to specifically amplify cDNA of MC2R, ERS1 and STAR, as well as the housekeeping genes hypoxanthine phosphoribosyltranspherase (HPRT) and ribosomal protein L19 (RPL19) were obtained from the literature (Schams et al, 2003;Myers et al, 2005;Chen et al, 2006;Jaquiery et al, 2006;Carriquiry et al, 2009) and further primer information is given in Supplementary Table S1. Primers to amplify cDNA of AR were designed based on ovine nucleotide sequences available from NCBI (http://www.ncbi.…”