Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development, function and involution.
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)-alpha and ERbeta, 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, 96-108 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 ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha 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 ERbeta was about 1000-fold lower (ag/ micro g 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 ERalpha 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. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta 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.
Institute of Physiology, Technical University Munich-Weihenstephan, D-85350 Freising, Germany.
D
D
Schams
S
S
Kohlenberg
W
W
Amselgruber
B
B
Berisha
M W
MW
Pfaffl
F
F
Sinowatz
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002417:Cattle / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004847:Epithelial Cells / Q000737:chemistry; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D005260:Female; D007150:Immunohistochemistry / Q000379:methods; D007774:Lactation / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000737:chemistry / Q000378:metabolism*; D011247:Pregnancy; D020033:Protein Isoforms / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis* / Q000235:genetics; D011980:Receptors, Progesterone / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D020033:Protein Isoforms; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone