MATERIALS AND METHODS
Time course study
The number of experimental (CERM D1 -/-) and control (non-transgenic, CERM, CERM
D1 +/-, cyclin D1 +/- and cyclin D1 -/-) mice included in the study was as follows:
CERM D1 -/- (n=10), non-transgenic (n=16), CERM (n=11), CERM cyclin D1 +/- (n=9),
cyclin D1 +/- (n=15), cyclin D1 -/- (n=9). The following time points throughout
mammary gland development were studied: 1-, 3-, 6-, 8- and 12-weeks. It is important to
note that CERM D1 -/- mice showed relatively normal development at one week of age
and that all CERM D1 -/- mice showed abnormal development at 3, 6, 8 and 12 weeks of
age. Abnormal mammary gland development was defined in whole mounts as absence of
a ductal tree and/or abnormal stroma, and in H&E sections as significantly reduced
number of epithelial cells composing the TEBs and/or disorganization of the
myoepithelial cell layer of the TEB and/or absence of fat cells and/or increased number
of fibroblasts and collagen deposition in the stroma. A contingency table was created
with the number of control (n=0) and CERM D1 -/- (n=8) mice showing abnormal
development and the number of control (n=60) and CERM D1 -/- (n=2) mice without
abnormal development.
Morphological and histological analyses of mammary glands
The number of TEBs per gland was counted on whole mounts at 1X and then the mean
and standard error of the mean (SEM) were calculated. Four H&E slides, 6 slides apart
from each other, from 6-week-old CERM (n=4) and CERM D1 -/- (n=4) were randomly
selected from the middle part of the gland (estimated by lymph node diameter); the
number of MECs composing the TEB was determined by counting epithelial cells in all
TEB present in those four slides and then calculating the mean number of epithelial cells
and SEM per TEB.
Sirius Red Staining
Tissue sections were deparaffinized, rehydrated and a small volume of Picro-Sirius Red
(American Master Tech Scientific, Lodi, CA) solution was added onto the slides. Slides
were incubated overnight at room temperature, and washed twice with acidified water
(5ml of glacial acetic acid glacial in 1 liter of distiled water) the next day. Slides were
then dehydrated in butanol and xylene, mounted using a xylene-based permount solution,
and examined with polarized light microscopy. Sirius Red staining was performed on
mammary glands from experimental CERM D1 -/- (n=10) and control mice (n=10).
Immunohistochemistry
Primary antibodies used for immunohistochistry on mouse mammary gland tissue: mouse
cyclin D1, rabbit cyclin D2, rabbit cyclin D3, and rabbit cyclin E (Santa Cruz
Biotechnology Inc., Santa Cruz, CA); rabbit polyclonal P-cadherin (Santa Cruz
Biotechnology Inc., Santa Cruz, CA); mouse monoclonal Ki67 antibody clone MM1
(Novocastra, United Kingdom); mouse polyclonal E-cadherin (BD Transduction Labs);
mouse monoclonal p63 (Neomarkers/Lab Vision, Fremont, CA); mouse monoclonal
SMA (Neomarkers/Lab Vision, Fremont, CA); rabbit monoclonal H2AX (Cell
Signaling, Danvers, MA); rabbit polyclonal pS-p53 (Abcam Inc., Cambridge, MA);
rabbit polyclonal pT-Chk2 (Abcam Inc., Cambridge, MA). Detection of apoptosis by
terminal deoxytransferase-mediated deoxy uridine nick end-labelling (TUNEL) assay was
carried out utilizing the Apoptag®peroxidase in situ apoptosis detection kit (Chemicon
International,
Temecula,
CA)
according
to
manufacturer’s
instructions.
All
inmunohistochemical procedures were performed in 6-week-old CERM (n=4) and
CERM D1 -/- (n=4) mammary glands and in transplanted CERM and CERM D1 -/mammary epithelium. For cyclin D2, D3 and E immunohistochemistry cell counting: a
total of 1000 ductal MECs per section were counted to determine the percentage of ductal
MECs demonstrating nuclear localized cyclin D2, D3 and E. For Ki67 and TUNEL
immunohistochemistry cell counting: the number of MECs within each TEB
demonstrating positively-stained nuclei was counted and proliferative/apoptotic rates
were calculated. All the TEBs present in each section were counted. For H2AX and pTChk2 immunohistochemistry cell counting: the number of MECs within the TEBs
demonstrating positively-stained nuclei was counted and the percentage of cells showing
positive staining was calculated. For pS-p53 immunohistochemistry cell counting: a
contingency table was created with the number of CERM TEBs with no positive cells
(n=8) or 1 to 10% positive cells (n=0) and the number of CERM D1-/- TEB with no
positive cells (n=7) or 1 to 10% positive cells (n=4). All the TEBs present in each
section were counted. In all cases, negative control slides in which the primary antibody
was omitted were analyzed in parallel; no nuclear specific staining was observed in the
absence of primary antibody.
Cyclicity Measurements
Estrous cycles were monitored in 6-week-old CERM D1 -/- mice (n=4) by analysis of
vaginal cytology beginning on postnatal day 35. A plastic pipette was inserted into the
vagina, with care not to stimulate the cervix, and the area was gently flushed with PBS.
Vaginal cells were analyzed by light microscopy according to standard morphological
criteria.
Hormonal supplementation and whole organ culture
Twenty-one-day-release 17-estradiol (0.01 mg) + progesterone (10 mg) pellets
(Innovative Research of America) were inserted in 3-week-old CERM D1 -/- and control
mice (n=3). After 7 days of priming with estradiol and progesterone pellet, the abdominal
number 4 mammary glands were removed aseptically, temporarily placed in a sterile dish
with a small amount of sterile media without hormones in order to keep the glands moist,
stretched on a dry fluorcarbon spectrum mesh, and then placed on a 6 well culture dish
containing Waymouth’s MB 752/1 media (Invitrogen, Carlsbad, CA) plus all the
necessary hormones: 0.1 g/ml hydrocortisone , 5 g/ml bovine insulin,
1 g/ml
prolactin, 0.1 g/ml aldosterone (HIPA media). Glands were maintained in a tri-gas
incubator in an atmosphere of 50% O2 and 5% CO2 in air. The medium was replaced
every 24 hours initially and every other day for a total of seven days. After 7 days, glands
were whole mounted for morphological examination.
Mammary gland transplantation
For clearing of the endogenous mammary epithelium, the tissue between the nipple and
the lymph node was excised from 3-week-old female nude mice and mammary anlagen
from E13.5 embryos were implanted into the center of the remaining fat pad. The
transplants were harvested six weeks after the surgery. The number of transplants per
genotype was as follows: D1 -/- (n=1), MMTV-rtTA D1 +/- (n=2), tet-op-ERD1 -/(n=2), tet-op-ERD1 +/- (n=2), CERMD1 -/- (n=2), CERMD1 +/- (n=1), CERM (n=1).
SUPPLEMENTARY EXPERIMENTS
Experiments performed to test whether alterations in ovarian function were
responsible for the phenotype
CERM D1 -/- mice showed vaginal opening at day 30 on average, which is within normal
range for C57BL/6 mice, and completed at least one documented complete estrous cycle
by day 46 on average indicating that loss of cyclin D1 in CERM mice did neither retard
the onset of puberty nor prevent normal estrous cyclicity. In addition, normal antral
follicles, responsible for estrogen production, were found in 12-week-old CERM D1 -/mice. The phenotype was not rescued by estrogen and progesterone supplementation of
intact CERM D1-/- mice. CERM D1-/- mammary glands failed to undergo alveolar
differentiation following exposure to prolactin in vitro in whole mammary gland organ
culture. These results suggest that the phenotype was not due to alterations in ovarian
function or to estrogen, progesterone or prolactin deficiency.
TITLES AND LEGENDS TO SUPPLEMENTARY FIGURES
Figure 1: CERM D1 -/- mice showed abnormally increased collagen I/III deposition
Sirius Red Staining of collagen I and III in mammary glands from 12-week-old
nulliparous female CERM (a) and CERM D1 -/- (b) mice.
Figure 2: CERM D1 -/- mice demonstrated normal ovarian function
H&E stained section of ovary from a 12-week-old nulliparous female CERM D1 -/mouse (a). Mammary gland whole mounts from 4-week-old nulliparous female CERM
D1 -/- mice treated with Estrogen and Progesterone pellets for 7 days (b) and then
cultured in media containing prolactin, insulin, aldosterone and hydrocortisone (c). Black
arrow indicates the presence of antral follicles (a). CERM D1 -/- mammary gland failed
to develop normally even after estrogen and progesterone (b) and prolactin (c)
supplementation.