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Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice

¹ Hannah Research Institute, Ayr KA6 5HL, UK
² Roslin Institute, Roslin, Midlothian EH25 9PS, UK

View larger version (52K): [in a new window]   Fig. 1. 125I-IGF-I western ligand blot analysis of IGFBP-5 expression in milk from transgenic mice. Lane 1: 48 hours involution (positive control); lanes 2-7: transgenic lines, 211, 285, 289, 114 (used in this study), 137 and 292 respectively. Lane 8: from lactating wild-type mice (negative control).

View larger version (52K): [in a new window]   Fig. 2. IGFBP-5, BLG and ß-casein mRNA expression in various tissue of transgenic mice. Blots were probed with (A) cDNA from the 3' UTR of BLG present in both the BLG and IGFBP-5 construct; thus two transcripts are present, the upper band representing IGFBP-5 and the lower band representing BLG; (B) with IGFBP-5 cDNA and (C) ß-casein riboprobe. (D) Ethidium bromide stained RNA as loading control.

View larger version (15K): [in a new window]   Fig. 3. Cumulative litter weight change (A) in pups suckled by wild-type dams (circles) or transgenic dams (squares), and (B) in pups alternately suckled by wild-type dams (circles) and transgenic dams (squares), where litters were exchanged on a daily basis between wild-type and transgenic dams. Note that the weight gains in B do not represent the actual weight gain of individual litters but represent the cumulative weight gain achieved by litters (whether transgenic or wild type) when being nursed by the wild-type or transgenic dam. Values are mean±s.e.m. of 5-6 mice.

View larger version (15K): [in a new window]   Fig. 4. Mammary gland weight and total DNA content on day 10 of lactation in IGFBP-5 transgenic and wild-type mice. Values are means ± s.e.m. of 10-11 mice. *** P<0.001 compared with wild-type mice.

View larger version (12K): [in a new window]   Fig. 5. DNA content in mammary glands of wild-type mice (circles) or IGFBP-5 transgenic mice (squares) at various stages of pregnancy and lactation. Values are means ± s.e.m. of 3-7 mice.

View larger version (82K): [in a new window]   Fig. 6. Whole mounts of mouse mammary glands showing ductal branching on day 10 of pregnancy in a wild-type mouse (A) and two transgenic mice (B,C).

View larger version (147K): [in a new window]   Fig. 7. Sections of wild-type (A,C,E) or IGFBP-5 transgenic (B,D,F) mammary gland on day 10 (A,B) or 15 (C,D) of pregnancy or day 2 of lactation (E.F). M, mammary epithelium, D, duct, A, adipose tissue.

View larger version (72K): [in a new window]   Fig. 8. BrdU-labelled cells in 2 wild-type mice (A,B) and two transgenic mice (C,D). Representative sections are shown for animals with the highest and lowest incorporations for each group. Right hand panel shows quantitative analysis of the data. Wild-type mice, grey bar; transgenic mice, black bar. At least 1500 nuclei were counted from at least three random fields. *P<0.05 compared to wild type.

View larger version (33K): [in a new window]   Fig. 9. (A) Western immunoblots for Bcl-2 and Bcl-xL, in day-1 and day-10 lactating mammary glands from wild-type and transgenic mice. Three representative animals are shown and the bar chart in C illustrates quantitative analysis of the data. (B) Endonucleosomal ladders in mammary gland DNA preparations. Lanes 1-4 show ethidium bromide detection, and lanes 5-10 show [32P]dCTP-end labelling. Lane 1, wild type, day 2 of lactation; lane 2, transgenic, day 2 of lactation; lane 3, wild type, day 10 of lactation; lane 4, day 2 of involution (positive control). Lanes 5-7 are samples from 3 representative day-1 lactating transgenic mice and lanes 8-10 are samples from 3 representative day-1 lactating wild-type mice. (C) Quantitative analyses for values on day 1 of lactation. White bars, transgenics, black bars, wild type. Values are mean ± s.e.m. for 5-6 mice per group. *P<0.05, **P<0.01 compared with wild-type mice.

View larger version (19K): [in a new window]   Fig. 10. Caspase-3 and plasmin activities in mammary homogenates of wild-type or transgenic females on day 2 (L2) or 10 (L10) of lactation. Values are means ± s.e.m. of 3-7 mice. ***P<0.001 compared with wild type on day 2 of lactation for caspase-3 or plasmin activity.

View larger version (52K): [in a new window]   Fig. 11. Western immunoblots for IGF receptor, IGF receptor phosphorylation, Akt and phospho-Akt in mammary tissue from lactating wild-type and transgenic mice. Samples from three representative mice of each type are shown. Quantitative analysis is shown in the bar chart. White bars, transgenic L1; black bars, wild-type L1; grey bars, transgenic L10; cross-hatched bars, wild-type L10. Values are means ± s.e.m. for 5 mice. **P<0.01 compared with wild-type mice.

View larger version (16K): [in a new window]   Fig. 12. Mammary gland weight and DNA content in wild-type mice and in transgenic mice receiving R3-IGF-I or GH. Values are means ± s.e.m. of 4-13 observations. *P<0.05, **P<0.01, ***P<0.001 compared with respective wild-type values.

View larger version (17K): [in a new window]   Fig. 13. Cumulative litter weight gain for wild-type dams (solid circles), transgenic dams (squares) or transgenic dams receiving R3-IGF-1 (triangles) or GH (open circles). Values are means ± s.e.m. of 4-13 observations.