Effects of Estrogenic Hormones on the Mammary Tissue of Agent-Free and Agent-Bearing
Male Mice I, 2
WILLIAM T. HALL
3
and DAN H. MOORE,' The Rocke-
feller University, New York, New York 10021
SUMMARY-Three-week-old male (C3H X A}F1 X C3H agent.bearing
backcrossed hybrid mice were castrated and given injections of diethylstilbestrol thrice weekly. Agent.free (C3Hf X Ax)Fl X C3Hf backcrossed
hybrids from the same strains were similarly prepared and given injections of
diethylstilbestrol or delestrogen. Mammary adenocarcinomas developed
during the 7th to 11 th months in 90 percent of the agent.bearing mice given
injections. All the mammary glands were enlarged and Ailed with milk.
The epithelial cells of the tumors and the nontumorous glands of the agent·
bearing animals had budding B particles, and the intracellular spaces contained an abundance of mature B particles like those seen in female mice.
None of the agent.free mice produced either a mammary tumor or B particles.
These Andings indicate that mice free of virus particles do not develop
mammary tumors from steroid hormone treatment alone.-J Nat Cancer
Inst 36: 181-188, 1966.
SEVERAL INVESTIGATORS (1-4) have shown
that the mammary tumor agent (MTA) can be
transmitted by the male parent. It is also known
that mammary adenocarcinoma can be induced
in the males of agent.bearing strains by prolonged
inoculation of estrogen or estrogen-like hormones
(5-70). There are conflicting reports in the literature, however, as to whether mammary carcinomas
can be induced by hormone stimulation in MTAfree strains or families of mice. Gardner (77) gave
weekly subcutaneous injections of estradiol benzoate starting at the age of 28 to 56 days and
continuing throughout life to first-generation reciprocal hybrids of agent-bearing CBA and agentfree C57BL mice. When the mother was CBA,
the mammary tumor incidence was 60 percent in
females and 58 percent in males. When the mother
was C57BL, no mammary tumors occurred.
Haagensen and Randall (72) gave estrone benzoate
in oil to the limit of tolerance in both RIll and
Received January 14,1965; revised August 9,1965.
This investigation was supported in part by Public
Health Service research grant CA-04573 from the National
Cancer Institute, and by a grant from the Lillia Babbitt
Hyde Foundation.
I Pr6!ent address: National Cancer Institute, Bethesda,
Md. 20014.
- t We wish to thank Dr. George Miroff for generously
supplying the agent-bearing and agent-free, backcrossed
hybrid mice, Dr. Marcus A. Hairstone for scanning many
of the sections and taking some of the electron micrographs,
and Mrs. J uIiette Crump for her efficient technical
assistance.
1
2
181
792-910-G(}--::
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HALL AND MOORE
C57BL strains. In the RIll mice thus treated,
the incidence of mammary carcinoma in males
increased to a level higher than that of the female
bred controls, but in C57BL mice no mammary
carcinoma was induced in either male or female.
Boot and Muhlbock (13), working with so-called
virus-free C3H animals, concluded that a combination of hormonal stimulation and high susceptibility
to mammary cancer resulted in the production
of mammary tumors. They stated that "the only
logical explanation of this susceptibility seems to
be to consider it as an expression of the genetic
constitution of the agent-free C3H substrain."
Again in 1956 Muhlbock (14) recorded the production of mammary tumors in virus-free mice
of strains C3H, DBA, 020, and C57BL by subcutaneous transplantation of hypophyses.
The validity of these conclusions clearly rests
on the definition of a mammary tumor virus and
the attendant demonstration that nothing falling
within the definition is present. Usually mice are
called agent-free if their tumor incidence is low
and their milk or tissue extract contains no assayable tumor-producing agent. Agent-bearing mice
are defined as those with a high incidence of
mammary tumors and an assayable agent in their
milk and tissues. In addition to these two kinds
of mice there is yet a third category-those having
zero incidence of mammary carcinoma, no assayable agent, and an inability to develop tumors
even under excessive hormonal stimulation. This
paper reports on the third category.
MATERIALS AND METHODS
Agent-bearing hybrids.-Thirty-three (C3H X A)
F 1 X C3HjBi/Miroff 5 agent-bearing male mice
were castrated at 21 to 24 days of age. After 3
days' recovery, 22 were given intraperitoneal injections of 0.25 mg diethylstilbestrol (DES) in
0.05 ml sesame oil thrice weekly. The remaining
11 were kept as controls. When the mice were
4 months old, the injections were discontinued
for 1 month and then resumed at half the rate
until the appearance of tumors could be ascertained macroscopically, at which time an animal
was killed and both tumorous and nontumorous
mammary tissues were studied with the electron
microscope. For comparison, an agent-bearing
control animal of the same age was also killed
and attempts were made to observe its mammary
tissue.
Agent-jree hybrids.-Seventy (C3Hf X Ax)Fl X
C3Hf/Bi/Miroff 6 male mice Were castrated at
21 to 24 days of age. Mter 3 days' recovery,
30 were given injections of DES under the same
conditions described and 30 were given injections
of 0.25 mg delestrogen half as often. Ten were
kept as controls. Injections were continued at
this rate until the mice began losing hair and
several had died, at which time (4 months of age)
injections were temporarily halted. Mter 1 month
the injections were resumed at half the initial
rate until the surviving animals were 1 year old.
For electron microscopy, tumors and mammary
gland tissues were fixed in phosphate-buffered
osmium, dehydrated through a series of alcohols,
embedded in Epon, sectioned on a Porter-Blum
ultramicrotome with glass knives, and stained
with lead hydroxide (75). They were examined
in an RCA EMU3F electron microscope and a
Siemens Elmiskop I.
Specimens from a few tumors were also prepared
for light microscopy. They were fixed in Bouin's
solution and stained with hematoxylin and eosin.
RESULTS
Agent-Bearing Mice
Of the 22 agent-bearing mice given lllJections,
2 died within the first 6 months, but without
palpable tumors. Since all treated mice became
visibly sluggish under hormone administration,
dosage was withheld during the 3d month of the
experiment. By the end of this interval, the
animals appeared to have regained much of their
liveliness. During the 7th month of treatment 4
mice developed large tumors and were killed for
electron microscope studies. By the 11th month,
all but 2 of the remaining mice had developed
tumors, although the injections were also dis6 Miroff obtained both the C3H and the A stocks from
Bittner in 1951. The tumor incidence in the former was
95 percent and in the latter, 97 percent.
8 The agent-free C3Hf and Ax lines were originally
fostered on C57BL by Bittner and have been maintained
by Miroffsince 1951. No mammary tumors have occurred
during this time.
JOURNAL OF THE NATIONAL CANCER INSTITUTE
EFFECTS OF HORMONES ON MTA-FREE MICE
continued during the 8th and 9th months. A
photomicrograph of a typical tumor section is
shown in figure 1.
None of the agent-bearing controls not given
injections developed tumors during the entire
period. At the 9th month, injections were given
to 4 of the controls and continued to the end of
the 11th month (24 injections) but no tumors
appeared, although 2 mice lived beyond the 20th
month.
B particles were found in all the tumors and in
the nontumorous, stimulated mammary glands of
agent-bearing mice. Figures 2 and 3 show not
only mature B particles in acinar spaces of the
tumorous tissue but also new particles budding off
the cell membranes. Figure 4 illustrates a similar
space from a stimulated gland with no visible
tumor. Milk particles were also present.
Agent-Free Mice
In the 60 treated mice used in this part of the
experiment no mammary tumor was observed.
That the hormones were administered to the limit
of tolerance was evidenced by several phenomena.
By the end of the 7th month the 10 castrated controls not given injections were all alive and 33
percent heavier than the 36 surviving animals
that had received injections. Of those given delestrogen, 15 (50%) had died by the 7th month
and, of those receiving DES, 9 (30%) had died.
The delestrogen survivors had lost the hair on
their backs. By the 14th month, all the delestrogen-treated and 60 percent of the DES-treated
animals were dead, and by the end of the 18th
month all the animals had died. Although 12
DES agent-free mice survived for more than 14
months, none developed tumors, whereas 90 percent of the similarly treated agent-bearing animals
had developed tumors by the 11 th month.
DISCUSSION
In these experiments we have used Ax and C3Hf
strains of mice known to be genetically susceptible
to mammary tumors. Their agent-carrying, genetically identical counterparts, A and C3H, have
always had a high tumor incidence, and the castrated males readily respond to hormonal stimulaVOL.
36, NO.2, FEBRUARY 1966
183
tion by developing a high incidence of mammary
tumors. That none of the agent-free animals developed tumors would seem to indicate that the
agent is a necessary factor in the mice we have used.
The question then arises: Why have other investigators obtained tumors in their hormonally stimulated
agent-free mice and concluded that the agent was
unnecessary? In the first place, results of experiments have not permitted unanimous agreement
on this conclusion. Loeb (76), who was the first
to increase tumor incidence by pituitary transplantation, pointed out that in some strains this
expedient had no effect. When steroids were used,
several investigators found that in some strains a
high tumor incidence was achieved, whereas in
others no tumors were elicited. It now seems clear
that some of these mice were carrying a latent
tumor agent and others were not. In recent years,
when mammary tumors have been studied with
the electron microscope, virus particles (B particles)
have been found in almost all, if not all, of them,
whether they occurred in agent-bearing or so-called
agent-free mice. In spite of the high correlation
of milk agent activity with the B particle (77), the
inactivity of the particles in low-cancer strains
(particularly C3Hf) caused some workers to doubt
the identity of the particles. Pitelka and her associates (78), however, have attempted to bridge
the gap by hypothesizing a second virus which,
though indistinguishable in the electron microscope, has different biological properties than the
agent readily transmitted via the milk. The second
virus is transmitted only at conception and by either
parent. It can remain latent in the progeny or
produce an occasional tumor, usually late in life,
under normal conditions, but it can respond to
excessive hormonal stimulation and cause a high
incidence relatively earlier in life. Apparently
neither virus is present in our agent-free strains.
The source of the agent in males has not been
settled. Extensive examination of organs and
tissues other than mammary glands of both sexes
of strain DBA/212 by Feldman (79) did not reveal
budding B particles. However, we have been
able to extract B particles from homogenized
genitalia of DBAf212 and RIll males (20), and
recently Smith (21) has found extracellular B
particles and also intracellular A particles in thin
sections of cauda epididymis and seminal vesicles
184
HALL AND MOORE
of high-cancer strain C3H males, but we have
never found budding particles in any organ except
the mammary gland. From what is known about
other viruses, it is not necessary to have the whole
virus particle to transmit viral activity. Infectivity by virus-free supernatants has been reported
for influenza (22), western equine. encephalitis (23),
poliomyelitis (24), and vaccinia (25), and almost
all the work dealing with the identification and
isolation of MTA has indicated that tumorigenic
activity is associated with a small particle (17, 26,
27) as well as a large one.
The nature of the small particle carrying the
tumorigenic activity in the Bittner virus has not
been clarified, but in other viruses both subviral
or previral elements and viral fragments carrying
the nucleic acid have been shown to be infective.
REFERENCES
(1) ANDERVONT, H. B., and DUNN, T. B.: Mammary
tumors in mice presumably free of the mammarytumor agent. J Nat Cancer Inst 8: 227-233, 1948.
(2) MiiHLBOCK, 0.: Studies on the transmission of the
mouse mammary tumor agent by the male parent.
J Nat Cancer Inst 12: 819-837,1952.
(3) HUMMEL, K. P., and LITTLE. C. C.: Comparative
virulence of the mammary tumor agent from
different sources; qualitative and quantitative differences. J Nat Cancer Inst 30: 593-604, 1963.
(4) BITTNER, J. J.: Transfer of the agent for mammary
cancer in mice by the male. Cancer Res 12: 387398,1952.
(5) GARDNER, W. U.: The effect of ovarian hormones
and ovarian grafts upon the mammary glands of
male mice. Endocrinology 19: 656-667, 1935.
(6) LACASSAGNE, A.: Apparition de cancers de la mamelle
chez la souris mAle, soumise a des injections de
folliculine. C R Acad Sci (Paris) 195: 630-632,
1932.
(7) - - - - : Apparition d'adenocarcinomes mammaires
chez des souris mAles traitees par une substance
oestrogene synthetique. C R Soc BioI (Paris) 129:
641-643,1938.
(8) - - - : Statistique des differents cancers constates
dans des lignees selectionnees de souris, apres
action prolongee d'hormones oestrogenes. Bull
Assoc Franc Cancer 27: 96-116, 1938.
(9) SHlMKlN, M. B., and GRADY, H. G.: Mammary
carcinomas in mice following oral administration
of stilbestrol. Proc Soc Exp BioI Med 45: 246-248,
1940.
(10) HESTON, W. E., and DERINGER, M. K.: Occurrence
of tumors in agent-free strain C3Hf male mice
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
implanted with estrogen-cholesterol pellets. Proc
Soc Exp BioI Med 82: 731-734, 1953.
GARDNER, W. U.: The effect of estrogen on the
incidence of mammary and pituitary tumors in
hybrid mice. Cancer Res 1: 345-358, 1941.
HAAGENSEN, C. D., and RANDALL, H. T.: Production
of mammary carcinoma in mice by estrogens.
Arch Path 33: 411--442,1942.
BOOT, L. M., and MiiHLBOCK, 0.: The mammary
tumour incidence in the C3H mouse-strain with
and without the agent (C3H, C3Hf, C3He). Acta
Un Int Cancr 12: 569-581, 1956.
MiiHLBOCK, 0.: The hormonal genesis of mammary
cancer. Advances Cancer Res 4: 371-391, 1956.
FELDMAN, D. G.: A method of staining thin sections
with lead hydroxide for precipitate-free sections.
J Cell Bioi 15: 592-595,1962.
LOEB, L., and KIRTZ, M. M.: The effects of transplants of anterior lobes of the hypophyses on the
growth of the mammary gland and on the development of mammary gland carcinoma in various
strains of mice. Amer J Cancer 36: 56-82, 1939.
MOORE, D. H., LASFARGUES, E. Y., MURRAY, M. R.,
HAAGENSEN, C. D., and POLLARD, E. C.: Correlation of physical and biological properties of mouse
mammary tumor agent. J Biophys Biochem Cytol
5(1): 85-92,1959.
PITELKA, D. R., BERN, H. A., NANDI, S., and DEOME,
K. B.: On the significance of virus-like particles in
mammary tissues of C3Hf mice. J Nat Cancer
Inst 33: 867-885,1964.
FELDMAN, D. G.: Origin and distribution of viruslike particles associated with mammary tumors in
DBA strain mice. II. Virus-like particles in the
blood and organs. J Nat Cancer Inst 30: 503-515,
1963.
MOORE, D. H., LUNGER, P. D., and LYONS, M. J.:
Unpublished data reported by Moore and Lyons
in VlI'uses, Nucleic Acids, and Cancer. Seventeenth Annual Symposium on Fundamental Cancer
Research at The University of Texas M.D. Anderson
Hospital and Tumor Institute. Baltimore, The
Williams & Wilkins Company, 1963, p 224.
SMITH, G. H.: The role of the milk agent in the
disappearance of mammary tumors in inbred
C3HjStWi mice. Proc Amer Assoc Cancer Res 6:
60(236). Philadelphia, 56th Annual Meeting, April
7-10, 1965.
BOURDILLON, J.: Analytical diffusion of influenza
virus and mouse encephalomyelitis virus. J Gen
Physiol25: 263-273,1941.
RICHTER, A., and WECKER, E.: The reaction of EEE
virus preparations with sodium deoxycholate.
Virology 20: 263-268, 1963.
TOLBERT, 0., and ENGLER, R.: Isolation of infectious
poliovirus ribonucleic acid from supernatant fluids
after ultracentrifugation. Nature (London) 200:
498--499, 1963.
JOURNAL OF THE NATIONAL CANCER INSTITUTE
EFFECTS OF HORMONES ON MTA-FREE MICE
(25)
ABEL,
P., and
TRAUTNER, T. A.:
Formation of an
animal virus within a bacterium.
Z Vererbungsl
95: 66-72, 1964.
(26)
BRYAN,
W.
R., KAHLER, H., SHIMKIN, M. B.,
ANDERVONT,
H.
B.:
and
Extraction and ultracentrif-
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185
ugation of mammary tumor inciter of mice. J Nat
Cancer Inst 2: 451-455, 1942.
(27) GRAFF, S., MOORE, D. H., STANLEY, W. M., RANDALL,
H. T., and HAAGENSEN, C. D.: Isolation of mouse
mammary carcinoma virus. Cancer 2: 755-762,
1949.
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PLATE 49
l.-Photomicrograph of section of tumor from male mouse. Some ductular epithelium shows slight
hyperplasia; other areas are acutely hyperplastic. X 130
FIGURE 2.-Electron micrograph of section from tumor of the mammary gland of hormonally stimulated, castrated
male mouse 216 days of age, showing columnar cells surrounding an acinar space. X 16,000
FIGURE
HALL AND MOORE
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PLATE
50
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 36
3.-A small acinar space showing both budding and mature B particles. X 71,000
4.-Electron micrograph of section from nontumorous male mammary gland showing an acinar space
containing both B particles and milk particles. Many microvilli extend from epithelial cells seen at bottom.
Milk particles are indicated by arrows. X 71,000
FIGURE
FIGURE
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HALL AND MOORE