Figure 1.
Analysis of morphology, histology and epithelial content of control, aged -nulliparous and aged-multiparous FVB/N-RC mammary glands.
(A) Photomicrographs of aged-nulliparous (panel a, b and c), aged-multiparous (panel d, e and f) and 14-wk-old (panels g, h and i) control mice whole-mounts and histological secions. Whole-mounts of the aged-nulliparous (panel a compare to panel g) and aged-multiparous (panels d compare to panel g) mice had the appearance of mammary glands from pregnant mice. Similary sections of mammary glands of the aged-nulliparous (panel b and c) and aged-multiparous (panel e and f) mice had the appearance of those from pregnant mice. Mammary glands of the aged-nulliparous (Panel b and c) mice displayed few budding alveoli (arrows, panel c) emanating from primary ducts; the aged-multiparous glands consisted of higher order of branching containing extensive numbers of fully developed alveoli (arrows, panel e and f). Also, the ducts of the aged-nulliparous and multiparous mice are dilated and filled with secretory material (arrow head in panel c and f). The control 14-wk-old nulliparous female mammary glands were composed of primary ducts (Panel g, h and i). Magnification, Panels a, d and g are at 5×, panels b, e and h are at 10× and panels c, f and i are at 20×. (B) Quantification of epithelial content of mammary glands from 14-wk-old control, aged-nulliparous and aged-multiparous mice, was conducted on the whole-mounts using AxioVision 4 Module AutoMeasure Program. Aged-multiparous mice had the highest epithelial contents followed by the aged-nulliparous and the control mice. Data represent mean±SEM from ten mice per group. In Figure 1B * indicates a statistically significant difference of p<0.05.
Figure 2.
Mammary gland, ovary, pituitary and uterine anomalies.
Representative H&E stained sections of mammary gland (A), ovary (B), and uterus (C & D). Focal nodules of squamous metaplasia were rarely observed in mammary sections of aged-multiparous mice (A), 10× magnification. Ovary anomalies occur frequently in the aged-multiparous mice, ovarian atrophy (panel B, arrow) was common in aged-multiparous mice, 5× magnification. Large fluid containing cysts suggestive of hydrometra (panel C arrow) and multiple smaller cysts were common in the uterus of aged-nulliparous females (panel D arrow), 5× magnification.
Figure 3.
Pars distalis abnormalities and immunostaining.
(A) Histological features of the pars distalis. H&E stained sections of pars distalis were examined in the 14-wk-old control (a), aged-nulliparous (b) and aged-multiparous (c) mice. Note the presence of cysts in the aged- nulliuparous (panel b) and aged-multiparous (panel c) females (arrow). Cysts were undetectable in the control 14-weeks-old (panel a) mice. (B) prolactin Immuno-histochemistry analysis of the pars distalis of 14-wk-old (a), aged-nulliparous (b) and aged-multiparous (c) mice showed uniform positive staining for prolactin in the control and experimental mice. Stained images illustrate the location of prolactin positive cells (arrows). Magnification 5×.
Table 1.
Effects of age and parity on serum hormone levels in the FVB/N-RC mice.
Figure 4.
Effect of age and parity on proliferation, progesterone receptor and estrogen receptor-alpha (ER-α) levels in FVB/N-RC mice mammary glands.
(A) Representative immuno-histochemistry sections of mammary tissue collected from 14-wk-old (panels a, d and g), aged-nulliparous (panels b, e and h) and aged-multiparous (panels c, f and i) female mice. Tissue was fixed and processed for age and parity associated proliferation marked by PCNA expression (Fig. 4A panels, a–c), progesterone receptor (Fig. 4A panels, d–f) and ER-α (Fig. 4A panels, g–i). Magnification 100×. (B) Proliferation, progesterone receptor and ER-α percent positive cells were scored and labeling index expressed as a percentage of positive nuclei of at least 3000 counted cells in mammary ducts (Fig. 4B panel a) and alveoli (Fig. 4B panel b). In the ducts, proliferation was significantly lower in the aged-nulliparous (p<0.05) and the aged-multiparous (p<0.05) mice than the 14-wk-old nulliparous mice. Also, proliferation was significantly lower (p<0.05) in the aged-multiparous than in the aged nulliparous mice mammary ducts. Progesterone receptor positive cells were significantly less in the ducts of the aged-nulliparous (p<0.05) and the aged-multiparous (p<0.05) mice than in the ducts of the 14-wk-old nulliparous mice. In the alveoli, proliferation was significantly higher in the aged-multiparous mice than in the aged-nulliparous (p<0.05). Also, progesterone receptor was more prevalent in the alveoli of the aged-nulliparous than in the aged-multiparous (p<0.05) mice mammary glands. In Figure 4B * indicates a statistically significant difference of p<0.05.
Figure 5.
Transplantation results from implantation of dispersed, aged-nulliparous and aged-multiparous mammary epithelial cells into mammary fat pads of 3-wk old female mice.
Representative whole-mounts of FVB/N-RC cleared fat pads transplanted with 250 (panel a and b) or 100 cells (panel c and d), collected from aged-nulliparous (panel a and c) and aged-multiparous (panel b and d), showing outgrowth filling of the fat pad. There was no difference in the results obtained with tissues from aged versus young donors regardless of parity.
Figure 6.
Transplantation results from implantation of mammary tissue fragments of, aged-nulliparous and aged-multiparous mammary epithelial cells into mammary fat pads of 3-wk old female mice.
Hosts were maintained as virgins for 8 weeks after the transplant. At the end of the 8 weeks, mammary glands were collected for morphology analysis and fragments were collected for the serial transplantation. All fragments contributed to complete outgrowth of the mammary gland when implanted into cleared mammary fat pad. (A) Portrays a representative whole-mount image of the fifth generation transplant (panel a) of an outgrowth of aged-nulliparous mammary tissue fragment into the cleared mammary fat pad (CFP), (panel b) shows the comparable outgrowth of aged-multiparous mammary tissue fragment into the CFP. (B) Serial transplantation results from transplant of paired aged-nulliparous and aged-multiparous mammary donor fragments into cleared mammary fat pads. Tissue fragments were collected from 20-month old mice (20-Mo-Donor), 22 month old mice (22-Mo-Donor) or 23-month old mice (23-Mo-Donor) as donors for the first transplant generation. Each donor mouse represented an independent experiment. In all cases fragments from the matched donors produced comparable mammary outgrowths.
Table 2.
Number of positive outgrowths (takes) originating from the implant of mammary gland cells into cleared fat pads.