Estrogen and progesterone are critical regulators of Stat5a expression in the mouse mammary gland

Abstract

Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the prepubertal gland and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution, and then increased again after weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double-labeling experiments in animals treated with E plus P for 3 d demonstrated that Stat5a was localized exclusively to cells containing both E and P receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland and suggest that these hormones act at the cellular level through their cognate receptors.

Figure 1

Immunofluorescent staining of Stat5a during mouse mammary gland development. A, Sections from 4-wk-old prepubertal, 5-wk-old pubertal, 19-wk-old mature adult, 7-d pregnant, 10-d lactating, and 8- and 28-d involuting mammary glands were stained with α-Stat5a antibody (green), and nuclei were labeled with DAPI (blue) as described in Materials and Methods. Control sections from 4-wk-old glands without primary antibody are also shown. Green arrowheads indicate Stat5a-positive cells and white arrowheads indicate Stat5a-negative cells. B, Mammary glands from mature (19-wk-old) mice were stained for SMA (red) and Stat5a (green). Scale bar for all images, 5 μm. C, Quantitation of nuclear Stat5a expression at different stages of mouse mammary gland development. Immunofluorescent staining using an α-Stat5a antibody was carried out as described in A on tissue sections from 4-, 5-, or 19-wk-old virgin, 7-d pregnant (P), 10-d lactating (L), and 8- or 28-d involuting (I) mice. The values represent the mean ± sem from three animals per group, with a minimum of 1000 cells per mouse analyzed, and significance was determined by Student’s t test. No nuclear staining was detected (ND) at 4 wk of age or 8 d of involution.

Figure 2

Stat5a expression in hormone-treated mice. A, Virgin 18-wk-old mice were ovariectomized and then treated with vehicle control, E, P, PRL, E+P, or PRL+P for 3 d as described in Materials and Methods. Representative photomicrographs of Stat5a staining are shown. B, The 3-d E+P-treated mammary glands were stained with an α-pY694 Stat5 antibody. All images show Stat5a staining (green) in the left panel and the merged image with DAPI-stained nuclei in the right panel. Scale bar for all images, 20 μm. C, Quantitation of Stat5a staining intensity in E+P-treated mice. The total pixel intensity of every cell (nucleus plus cytoplasm) was measured from a single representative structure from the 3-d control (white bars) and E+P-treated (black bars) mice (each bar represents one cell).

Figure 3

Cellular localization of Stat5a in BC-treated mice. The 18-wk-old virgin mice were ovariectomized and treated with E+P+vehicle control (oil) or E+P+BC for 3 d. Staining for Stat5a (green) and nuclei (red) was visualized with an Olympus laser scanning confocal microscope. White arrowheads indicate nuclear Stat5a staining, and red arrowheads indicate cytoplasmic Stat5a staining. Scale bar, 20 μm.

Figure 4

Colocalization of Stat5a with PRA or PRB. A, Staining for Stat5a and PRA was carried out on sections from 3- and 10-d E+P-treated ovariectomized mice and was visualized with a Nikon Eclipse fluorescent microscope. Staining for Stat5a and PRB was carried out on sections from 10-d E+P-treated mice only and was visualized with a Pascal laser scanning confocal microscope. Yellow arrowheads indicate Stat5a and PRA or Stat5a and PRB colocalization (yellow nuclei). White arrowhead indicates Stat5a-negative, PRA-positive cell (red nucleus). Green arrowheads indicate Stat5a-positive, PRA- or PRB-negative cells (green nuclei). Scale bar, 20 μm. B, Quantitation of Stat5a and PR colocalization in 3- and 10-d E+P-treated animals. The values represent the mean ± sem from three animals per group, with a minimum of 800 cells per mouse analyzed. The percentage of PRA-positive cells and Stat5a/PRA-positive cells were both significantly lower in 10-d E+P alveolar structures than 3-d E+P ducts (P < 0.001) as determined by Student’s t test.

Figure 5

Colocalization of Stat5a with ERα. A, Staining for Stat5a and ERα was carried out on sections from 3-d E+P-treated ovariectomized mice. White arrowheads indicate Stat5a- and ERα-positive cells. Scale bar, 20 μm. B, Quantitation of Stat5a and ERα colocalization in 3-d E+P-treated animals. The values represent the mean ± sem from three animals per group, with a minimum of 800 cells per mouse analyzed.

Figure 6

Immunodetection of Stat5a and BrdU in mammary glands of hormone-treated mice. Ovariectomized 19-wk-old mice were treated with E+P for 3 or 10 d, and animals were injected with BrdU 2 h before being euthanized as described in Materials and Methods. A, Representative structures from mammary glands showing Stat5a (green), BrdU (red), DAPI-stained nuclei (blue), and merged images. Green arrowheads indicate Stat5a-positive, BrdU-negative cells. Red arrowheads indicate BrdU-positive, Stat5a-negative cells. White arrowheads indicate BrdU-positive, Stat5a-positive cells. Scale bar, 25 μm. B, Quantitation of Stat5a and BrdU colocalization in 3- and 10-d E+P-treated animals. The values represent the mean ± sem from three animals per group, with a minimum of 800 cells per mouse analyzed.

Figure 7

Immunodetection of Stat5a and WAP or Stat5a and RANKL. Tissue sections from 3-d E+P-treated ovariectomized mice were stained for Stat5a and either WAP or RANKL. A, Representative structures showing merged images of Stat5a (red), WAP (green), and DAPI-stained nuclei (blue). B, Representative structures of merged images of Stat5a (red), RANKL (green), and DAPI-stained nuclei (blue). Scale bar, 25 μm.