title: Tamoxifen impairs prepubertal mammary development and alters expression of estrogen receptor α (ESR1) and progesterone receptors (PGR).
abstract: Research has shown that prepubertal heifers experience allometric mammary growth that is influenced by the ovaries. Our purpose was to determine the role of estrogen in prepubertal mammary gland development. Sixteen Holstein calves were randomly assigned to 1 of 2 treatment groups: tamoxifen-injected (TAM) or control (CON). Calves were administered the antiestrogen tamoxifen (0.3 mg kg(1) d(1)) or placebo from 28 to 120 d of age. At 120 d, calves were euthanized and udders removed. Weight and DNA content of trimmed parenchymal tissue were halved (P ≤ 0.0001) in TAM compared with CON calves. Parenchymal samples from 3 zones of the left rear mammary gland (lower, middle, and outer regions) were processed for immunohistochemical staining for estrogen receptor α (ESR1) and progesterone receptor (PGR), Ki67-positive cells, and 5-bromo-2'-deoxyuridine label retaining cells (LRCs). Overall, neither the percentage nor location within the epithelial tissue layer of either ESR1- or PGR-positive cells was impacted by TAM treatment. However, image analysis indicated a 6.2-fold lower (P = 0.0001) level of ESR1 protein expression in TAM calves. Similarly, messenger RNA expression of ESR1 was also reduced (P = 0.0001) in TAM heifers. In contrast, expression of PGR protein was greater by 43% (P = 0.03) in TAM calves, but messenger RNA expression did not differ between treatments. Overall, TAM calves had a higher (P ≤ 0.03) percentage and density (cells per tissue area) of Ki67-positive cells. Irrespective of treatment, there were also more Ki67-labeled cells in the outer zones of the mammary gland (P ≤ 0.001). We were able to effectively use multispectral imaging to identify positive cells and quantify the expression of ESR1 and PGR protein. We also identified and counted the proportion of label retaining cells (LCR) (putative epithelial stem cells). We noted an overall 2.9-fold greater number of LRCs in TAM heifers and more LRCs in the outer sampling zones. This suggests that a cohort of LCR cells in TAM remained inactivated in comparison with CON heifers, which exhibited markedly increased growth of the mammary parenchymal tissue over the treatment period. These results suggest that the impacts of ovariectomy are partially explained by loss of ESR1 expression and/or estrogen receptor signaling in the prepubertal bovine mammary gland. The significance of mammary expression of PGR in control of prepubertal bovine mammary development remains unresolved.
authors.affiliation: Department of Dairy Science, Virginia Tech, Blacksburg, VA, 24061, USA.
authors.forename: H L M
authors.initials: HL
authors.lastname: Tucker
authors.affiliation: Department of Dairy Science, Virginia Tech, Blacksburg, VA, 24061, USA.
authors.forename: C L M
authors.initials: CL
authors.lastname: Parsons
authors.affiliation: Biological Sciences Directorate, National Science Foundation, Arlington, VA 22230, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ellis
authors.affiliation: Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA, 24061, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Rhoads
authors.affiliation: Department of Dairy Science, Virginia Tech, Blacksburg, VA, 24061, USA. Electronic address: rma@vt.edu.
authors.forename: R M
authors.initials: RM
authors.lastname: Akers
mesh_terms: D000818:Animals; D002417:Cattle / Q000254:growth & development* / Q000378:metabolism; D004847:Epithelial Cells / Q000737:chemistry; D004965:Estrogen Antagonists / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000032:analysis / Q000235:genetics*; D004967:Estrogens / Q000502:physiology; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000032:analysis; D008321:Mammary Glands, Animal / Q000737:chemistry / Q000187:drug effects* / Q000254:growth & development*; D012333:RNA, Messenger / Q000032:analysis; D011980:Receptors, Progesterone / Q000032:analysis / Q000235:genetics*; D012741:Sexual Maturation; D013629:Tamoxifen / Q000494:pharmacology
chemical_list: D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D004967:Estrogens; D019394:Ki-67 Antigen; D012333:RNA, Messenger; D011980:Receptors, Progesterone; D013629:Tamoxifen
keywords: Mammary; Stem cell; Steroid receptor; Tamoxifen