petitive inhibitor of concanavalin A
such as 0.2M methyl a-D-glucoside must
be added to block recombination of
concanavalin A with serum glycopro-
teins in the column.
Myron A. LEON
Pathology Research Department,
St. Luke’s Hospital,
‘Cleveland, Ohio 44104
References and Notes
1. 8. Nakamura, K. Tanaka, S. Murakawa, Nature 188, 144 (1960).
2. H. Harris and E. B. Robson, Vox Sang. 8,
348 (1963).
3. I. J. Goldstein and R. N. Iyer, Biochim.
Biophys, Acta 121, 197 (1966).
4.B. B. L. Agrawal and I. J. Goldstein,
Biochem. J, 96, 23C (1965).
3. The serum inhibitor of C’l (first component of
complement) esterase was measured by Dr,
J. Pensky.
6. I thank Miss Janet Wilson, in Dr. A. Steinberg’s laboratory, for performing the Gm
titrations.
7. J. R. Clamp, G. Dawson, L. Hough, Biochem.
J. 100, 35C (1966). References to earlier
papers on IgG carbohydrate are given here.
8. Dr. G. Bernier, Dr. W. D. Terry, and Dr.
G. A. Spengler provided the typed myeloma
serums. Supported by NIH grant 5-ROIA103104-07.
18 October 1967
sulin; however, proliferation does not
lead to differentiation unless hydrocorti-
sone and prolactin are present in the
culture medium. The sequence of action of the hormones in relation to the
cell cycle in explants of mammary gland
from pregnant mice has been described
(8).
Although the relationships between
hormone-dependent proliferation and
differentiation have been characterized
in the mature gland, the biochemical
behavior of the immature gland in
vitro has been less clear. We examined
the capacity of the immature tissue to
differentiate in vitro and compared the
requirements for and responses to the
hormones with those of the mature
tissue.
Although the onset of “maturity” is
variable, 21-day-old female weanling
mice of the C3H/HeN strain are
termed immature in our report. The
glands of the 21-day-old mouse consist principally of branching ducts several cell layers thick with terminal
buds. No alveoli are seen in serial sections, nor have any been seen in whole
Hormone-Dependent Differentiation
of Immature Mouse
Mammary Gland in vitro
Abstract. Explants from the mammary glands of 3-week-old mice can
be induced to synthesize casein in vitro
in the absence of lobuloalveolar development, Maximum biochemical differentiation requires the presence of insulin, hydrocortisone, and prolactin in
the culture medium. In contrast to explants from adult mice, the mammary
epithelium of immature animals undergoes DNA synthesis and mitosis in
vitro in the absence of exogenous insu-
lin; however, such proliferation does not
lead to the formation of differentiated
daughter cells. Insulin acts in at least
two ways during the proliferative
phases of the cell cycle of differentiating mammary epithelial cells,
Mammary gland epithelial cells in
explants derived from pregnant or non-
pregnant mature mice proliferate, assume a secretory appearance, and be-
gin to synthesize certain milk proteins
when cultured in the presence of in-
sulin,
hydrocortisone,
and
prolactin
(/-4). This functiona! differentiation of
mounts in other laboratories (9).
Mammary epithelial cells in immature animals proliferate into the mam-
mary fat; yet, as the animal matures,
proliferation virtually ceases and does
not resume until pregnancy. We con-
firmed this by injecting thymidine-H?
(1 pe per gram of body weight) intraperitoneally and preparing autoradiographs of the mammary gland 24
hours later. Only about 1.5 percent of
the epithelial cells from 3-month-old
mice were labeled, whereas 18 percent of the epithelial cells from
3-
week-old animals were labeled. It is
unlikely that the results reflect a
difference in pool size, because almost
all the labeled nuclei had approximately 35 to 40 grains.
Synthesis of DNA in vitro by mammary epithelial cells from mature mice
is completely dependent on the pres-
ence of insulin in the culture medium
(5-7). In contrast, our studies indicate
that DNA synthesis in vitro by mammary gland explants derived from im-
mature mice [as reflected by the
amount of thymidine-H? incorporated
during 4-hour periods as previously de-
scribed (5)] is quantitatively independent of the addition of insulin. Since
these explants contain large numbers of
the epithelial cells has been found to
be necessarily coupled to their prolifera-
sulin-independent. data on total incor-
sequent cell division require only in-
terpreted with caution. To determine
tion (5-7). Synthesis of DNA and sub1326
fibroblasts whose DNA synthesis is in-
poration of thymidine-H? must be in-
whether such incorporation of thymidine-H? into DNA reflected epithelial
cell activity, we made autoradiographs
of explants exposed to thymidine-H?
(1
ype/ml) for 72 hours. Similar ex-
plants were cultured in the presence of
colchicine (0.05 »g/ml) for 72 hours
and were examined for mitotic activity.
The duct epithelium, especially the
epithelium of smaller terminal branches,
was heavily labeled, and mitotic figures
were numerous in either the presence
or absence of insulin in the cultures.
In explants cultured without the addition of hormones, 12 percent of the
cells were in metaphase (5000 epithelial
cells counted); in those cultured in the
presence of insulin, 9 percent were in
metaphase
(5000
epithelial
cells
counted); and in those cultured in the
presence of hydrocortisone and prolactin, 11.5 percent were in metaphase
(1000 epithelial cells counted). In contrast to tissue from mature mice (5-7)
then, proliferation of mammary epithelial cells from immature animals occurs in vitro in the absence of exogenous insulin. However, proliferation
in the absence of exogenous insulin
does not lead to functional differentia-
tion.
Figure 1 shows that explants of mam-
mary glands of immature mice can be
induced to synthesize the major casein
components when cultured in the presence of insulin, hydrocortisone, and
prolactin. During the first 24 hours,
the synthesis of casein bands 2, 3,
and 4 is virtually undetectable, but by
the 4th or 5th day these proteins are
synthesized at greatly accelerated rates
in ratios similar to those observed in
tissue from mature animals (10). Casein
band 1, a more heavily phosphorylated
component (7/7), is present from the
start and is least affected by culture.
This component persists longest in ma-
ture tissue cultured in incomplete hor-
mone systems (J0).
Figure 2 shows patterns of casein
production during the 5th day of cuiture in several media. The full differentiative response is elicited only when
the three hormones are present. Of
the incomplete systems, only those con-
taining insulin are even partially effective, and insulin alone is as effective
as it ts in combination with hydrocortisone or prolactin. In the absence of
insulin,
not
even
band
1
is
sythe-
sized.
The hydrocortisone-prolactin system
(Fig. 2), in which proliferation occurs
SCIENCE, VOL. 158