ns
eel
three ways: (i) it is necessary for the
initiation of DNA synthesis by epithelial cells of mature tissue as a prelude
to functional differentiation (5—7); (ii)
it must be present during the post-
.
mitotic action of prolactin {8) when
phenotypic differentiation is expressed;
4
48-72 HRS
7
Fig. 1. Effect of culture on synthesis of
the major casein components. Explants
weighing 0.5 to 1 mg were prepared and
cultured in sterile “Medium 199” (2). The
medium contained 5 yg/ml each of
crystalline beef insulin (Lilly), ovine
prolactin (NIH Endocrinology Study Sec-
tion), and hydrocortisone. Explants were
exposed
to
P-labeled
inorganic
phos-
phate (75 wc/ml) during the times indicat-
ed and then were homogenized in the presence of mouse carrier casein. Total casein
was isolated by precipitation with calcium
and rennin and was further characterized
with vertical starch-urea gel electrophoresis
at pH 8.6 (/3). Bars represent counts in
gel sections corresponding to the four
major casein bands with appropriate background subtracted (/0@). Ordinate refers
to radioactivity in terms of counts per
minute per centimeter per 10 mg of tissue.
in the
absence
of insulin,
did
not
synthesize casein. Furthermore, when
explants were cultured in such a hydrocortisone-prolactin system for 96 hours
and insulin was then added during the
final 24 hours, no effect on casein syn-
thesis was observed. This suggests that
actions of insulin other than postmitotic
synergism with prolactin (8) and initiation of DNA synthesis (7) must occur
before functional differentiation can be
of DNAsynthesis which, atone, cannot
comparedto tissue from unprimed mice.
Thus, it is possible to dissociate struc-
differs from the mature in displaying
sue from unprimed immature mice.
required
during the
proliferative phases of immature cells
in some capacity other than initiation
lead to differentiation.
Thus, even though immature tissue
insulin-independent DNA syathesis and
mitosis,
such proliferation apparently
does not lead to functional differentiation unless it occurs in the presence
of insulin.
Although casein bands 2, 3, and 4
are not detectably synthesized by explants of immature mammary gland
during the Ist day of culture in the presence of all three hormones, such explants synthesize q-lactalbumin and ~lactoglobulin. Tissue was exposed to
Cl4-labeled algal hydrolyzate (10 yc/
ml). Explants were homogenized in
mouse skim-milk carrier, and then q-
lactalbumin and -lactoglobulin were
isolated by ammonium sulfate fractionation and electrophoresis on polyacetate strips as previously described
(2). When the culture was exposed to
the hydrolyzate from 0 to 24 hours, the
rate of synthesis of q-lactalbumin was
310 count/min and of #-lactoglobulin
295 count/min per milligram of tissue.
When the period of exposure was 72
milligram of tissue. Thus, the emergence
of the capacity to synthesize the various
milk proteins is asynchronous in this
immature tissue.
Ichinose and Nandi reported (9) that
232
AH
to
6A
FP
se.
I
43241
Fig. 2. Effect of culture on synthesis of
casein components in incomplete hormone
systems. Explants were cultured as in Fig.
1 but in the incomplete hormone system
shown. Pulse labeling with P*-labeled inorganic phosphate was from 96 to 120
hours in all cases. Because these experi-
ments involve systems lacking insulin, the
series was repeated with glucose replaced
in the medium by D-fructose (100 mg/ml).
No
difference was
noted.
NH, no hor-
mones; 7, insulin; F, hydrocortisone; P,
prolactin. Ordinate refers to radioactivity
in terms of counts per minute per centiMeter per 10 mg of tissue.
8 DECEMBER 1967
thymidine-H? incorporation into DNA
was approximately doubled [perhaps
due to a shortened S-phase (DNA synthesis) as suggested by Banerjee (/2)],
and the development of casein produc-
it is
to 96 hours, the rate of synthesis of
BANG
alveolar structures develop in vitro. Un-
der these primed conditions, the rate of
early
(iii)
a-lactalbumin was 880 count/min and
of 8-lactoglobulin 510 count/min per
HOIMONE S*STEM
mary tissue into medium containing insulin, hydrocortisone, and prolactin,
lobuloalveolar development in| mammary explants from immature mice
rarely occurs unless the mice have previously been primed with injections of
estrogen, progesterone, prolactin, and
growth hormone. Our work confirms
this observation. Culture of unprimed
explants
in
the
presence of insulin,
hydrocortisone, and prolactin elicits the
biochemical differentiation described
above, but there is little or no formation of alveolar structures, If, however,
the animals are primed on each of 7
days with estradiol-178 (1 gg), progesterone (I mg), and growth hormone
tion was accelerated about 24 hours,
tural development from biochemical development in vitro with mammary tis-
Our studies in vitro with mammary
gland explants from immature mice
revealed that: (1) the epithelium differs from that of the mature gland in
that DNA synthesis and mitosis occur
in the absence of exogenous insulin;
(ii) this insulin-independent proliferation does not result in the appearance
of differentiated daughter cells; (iii)
addition of insulin to the cultures does
not quantitatively affect DNA synthesis or mitosis but allows functional differentiation to occur when hydrocortisone and prolactin are present; (iv)
emergence of the ability to synthesize
casein bands 2, 3, and 4 is not synchronized with the appearance of wheyprotein synthesis; and (v) it is pos-
sible in vitro to dissociate the capacity
to synthesize secretory proteins from
structural development of this tissue.
ANTHONYE. VoyTOvICcH
YALE J, TOPPER
National Institute of Arthritis
and Metabolic Diseases,
Bethesda, Maryland 20014
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