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The effect of theelin and theelol on the growth of the mammary gland.

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THE E F F E C T O F THEELIN AND THEELOL ON THE
GROWTH O F THE MAMMARY GLAND1
C. W. TURNER, A. H. FRANK, W. U. GARDNER, A. B. SCHULTZE, AND
E. T. GOMEZ
Department of Dairy Rusbandry, University of Missouri, Columbia
FOUR PLATES (FOURTEEN FIGURES)
Since the introduction of the rat unit test of estrogenic
hormones by Allen and Doisy ( '23), the physiological effect
of various crude extracts upon the growth of the mammary
gland has been reported. Allen et al. ( '24) noted changes in
the mammary glands of rats and mice following injections of
ovarian follicular fluid extracts. Vintemberger ( '24) produced growth of the ducts of the mammary gland in normal
virgin female rabbits as well as in young castrate males and
females with follicular fluid.
The effect of estrogenic hormones of ovarian and placental
origin on the growth of the mammary gland of the monkey
was demonstrated by Allen ('27). Laqueur et al. ('27-28)
reported an increase in the size of the mammary glands of
rats and guinea-pigs following subcutaneous or intraperitoneal injections of Menformon (extract of ovary, placenta, or
amniotic fluid). In the guinea-pig similar results have been
observed by Champy and Keller ( '27), Haterius ( '281, Loeb
and Kountz ('28), and Steinach et al. ( '28)' using ovarian or
placent a1 extracts.
With the finding of large amounts of an estrogenic hormone
in the urine of pregnant cattle by Turner, Frank, Lomas, and
This study was aided in part by a grant from the Committee on Grants-in Aid
of the National Research Council. From the Dairy Husbandry Department,
Agricultural Experiment Station, University of Missouri, Journal Series paper
no. 325.
227
T H E ANATOMICAL RECORD, VOL.
53, NO. 2
228
C. W. TURNER A N D OTHERS
Nibler ( '30), a series of studies were initiated in this laboratory to determine its effect on the growth of the mammary
glands of experimental animals. Up to the present time
studies on the growth of the mammary glands of gonadectomized male and female and normal male rabbits (Turner
and Frank, '30), rats (Turner and Schultze, '31), mice and
guinea-pigs (Turner and Gomez, unpublished), have shown
that the initial growth of the mammary gland complex, including the teats and duct systems, is stimulated by the crude
extracts of estrogenic hormones whether obtained from the
ovary, placenta, amniotic fluid, or urine of pregnancy. The
continued stimulation of the mammary glands of these animals eventually causes the growth of an extensive duct
system.
I n addition there is, in some species, a slight stimulus for
the growth of the lobules of the gland. I n large amounts and
over long periods of time the estrogenic hormones have been
found ineffective in producing lobules in the mammary glands
of the mouse. I n the rabbit and rat there is a slight lobule
growth, while in the guinea-pig there appears to be great
lobule development. An explanation of these specie differences in regard to the effect of the estrus-producing hormones
on the duct and lobule growth must await further study.
I n all of these studies it will be noted that estrogenic extracts from various sources and of varying degrees of purity
have been used. With the crystallization of theelin by Doisy,
Veler, and Thayer ( '30), and the more recent discovery of a
second estrogenic hormone, theelol, in the urine of pregnancy
(Doisy and Thayer, '31), a number of questions arise as to
their relation to' the growth of the duct system of the mammary gland. Does pure theelin stimulate the growth of the
mammary gland or is theelol the effective agent? If both
are effective, do they cause the same type of duct development 41
Through the kindness of Dr. Oliver Kamm, of the Research
Laboratory of Parke, Davis & Company, a supply of highly
purified theelin and theelol was made available for our use,
GROWTH O F THE MAMMARY GLAND
229
which permitted us to obtain an answer to the above questions.
The solution of theelin used was made up to contain fifty
rat units per cubic centimeter, while the theelol solution contained % mg. of pure crystalline material per cubic centimeter
dissolved in just sufficient alkali (.003/N) t o maintain it in
solution.
I n the study of the growth of the duct system of the mammary gland of the rabbit, rat, and mouse, it has been found
that gross mounts of the entire gland show the extent of
development very satisfactorily. The method of fixation and
staining which has given us best results is described below.
Except in the male rat and mouse it has been found possible
to remove check glands at the beginning and at intervals
during the experiment by placing the animal under ether
anesthesia. The glands are spread out and tacked on pieces
of flat cork, then fixed in Bouin’s fluid for twenty-four hours.
The glands are then washed in water until the yellow color
is largely removed; then stained in Mayer’s haemalum overnight; washed with 1 per cent potassium alum, followed by
further washing in water. The glands are now differentiated
in 70 per cent acid alcohol until the color in the connective
tissue has been removed, showing the ducts in sharp contrast
to the light background. The glands are now dehydrated in
a series of alcohols, cleared in xylol and mounted in balsam
on slides of suitable size.
GROWTH O F THE RABBIT GLAND
The theelin and theelol solutions supplied were diluted with
two parts of water and 1 cc. injected daily subcutaneously
into a young male rabbit. The daily injection, therefore, was
16% rat units of theelin, or 1/15 mg. of crystalline theelol. One
animal was injected with each of the preparations. One animal, R17, which received the theelin, had been castrated five
months previously; the other, R20, a non-castrated animal of
approximately the same age, received the theelol injections.
Control glands removed at the time the experiments started
revealed very rudimentary glands extending scarcely beyond
the base of the teat and consisting of unbranched ducts.
230
C. W. TURNER A N D OTHERS
A second gland removed on the twelfth day after the injections started showed a marked duct development in both
animals. The rapidly growing saccular duct ends produced a
thickening distinguishable through the skin. The ducts had
begun to branch and increased several times in length.
Further injection of the theelol and theelin over a ten-day
period resulted in the further development of the duct system
both in total extent and complexity.
Ten days later a third period of injection was started. Both
animals received the usual dose (16% units) of theelin. Glands
removed after fifteen daily injections showed further development. The distal ends of the ducts were still proliferating as
indicated by their enlarged sac-like terminations. At this
time the gland of R17 appeared to be slightly better developed
as indicated by more numerous and more definite lateral
branches and a slightly greater total area.
These glands were similar to the glands of male rabbits
which had been injected with the crude extract of the estrusproducing hormones obtained from the urine of pregnant
cows.
GROWTH O F T H E RAT GLAND
To determine the effect of each of the estrogenic substances
on the growth of the mammary gland of the albino rat, four
females, twenty days of age, of the same litter were selected
for the experiment. Three were castrated and one was
sacrificed at the time of the beginning of the experiment as
a check. The first of the three castrated was treated with
2.5 rat units of the theelin fraction daily for twenty days,
the glands being removed on the twenty-first day. The second
castrated female was treated with 0.05 cc. of the theelol fraction daily for twenty days and the glands removed after this
treatment. The last of the three castrated received no treatment after castration and was sacrificed as a check on the
twenty-first day after castration.
The gland removed from the control twenty-day-old female
was comparatively small with numerous end buds, this growth
GROWTH O F THE MAMMARY GLAND
231
being characteristic of the duct development at this age. The
gland from the animal castrated, but receiving no other treatment, was very similar to the gland from the twenty-day-old
female previously described. The gland from the castrated
female that had received treatment with the theelin fraction
when compared to the control glands showed significantly
greater growth, being approximately two and one-half times
greater in spread than the controls. The character of the
growth, however, was the typical duct development. No
lobule proliferation was apparent. The growth of the gland
from the castrated female treated with the theelol fraction
was apparently similar in extent and character to that of the
theelin treated animal. Its area of growth was somewhat
greater than that of the gland of the theelin treated individual, but probably not significantly so. I n neither of the
individual glands was lobule proliferation .observed as the
result of the treatment.
GROWTH O F THE MOUSE GLAND
In studying the effect of theelin and theelol upon the mammary glands of the mouse, the use of males was given preference, in view of the fact that the gland complex of these
animals is very slightly developed and remains rudimentary
in normal adult life. This condition, coupled with the absence
of the nipples which serve as an external guide in locating
the exact position of the glands, made it extremely difficult
t o remove a gland for the control. For this reason, an animal
was sacrificed at the beginning of the experiment as a control.
Three adult male mice were each injected subcutaneously
with 0.1 cc. (5 rat units) of theelin; and a second group of
three with 0.05 cc. of theelol fractions. These injections were
administered daily, and on the fifteenth and twenty-first days,
respectively, one animal was sacrificed at each time in both
series. The treatments were continued with the remaining
animal with an increase in dosage from 0.1 to 0.2 cc. (10 rat
units) in the case of theelindand from 0.05 to 0.1 cc. in the
case of theelol fraction f o r an extended period of twenty and
ten days, respectively.
232
C. W. TURNER AND OTHERS
An adult castrated female mouse (castrated before weaning
or fifteen days of age) was also injected with 0.1 cc. of the
theelin fraction daily for twelve days. Control gland was
removed on the day the first injection was administered.
Microscopic examinations of the prepared mammary glands
of the male mice which received 0.1 cc. ( 5 rat units) of the
theelin and 0.05 cc. of the theelol fractions daily for fifteen
and twenty days, respectively, reveal no sign of growth. These
glands, except for the slight individual variation in size and
form of the rudimentary ducts, correspond closely with that
of the control gland. However, the glands from the animals
which received a n increased dosage of the theelin and theelol
fractions for an additional period of treatment of twenty and
ten days, both showed extensive growth of the duct system
with prominent end buds.
I n the castrated female, after twelve days of daily injection
with theelin, the mammary glands showed a very significant
growth of the duct system. The spread of the duct system in
this case could roughly be approximated at about five times
that of the control gland. I n all cases, however, no lobular
proliferation was observed.
SUMMARY AND CONCLUSIONS
These experiments indicate that the purified estrogenic
hormones theelin and theelol are capable of stimulating the
growth of the duct system of the mammary glands of the
rabbit, rat, and mouse. No significant difference was observed
in the type of duct growth produced by theelin, theelol, or the
crude extract from the urine of pregnant dairy cattle. The
slight proliferation of lobules which has been observed following the extended injection of the crude extract in the rabbit
and rat was not observed during the course of these experiments. The lack of quantitative measures of mammary duct
growth make difficult the comparison of the relative growth
stimulating effect of theelin and theelol. No conclusions may
be drawn from these observations in this regard.
GROWTH O F THE MAMMARY GLAND
233
LITERATURE CITED
ALLEN, EDGAR1927 The menstrual cycle of the monkey, Macacus rhesus:
Observations on normal animals, the effects of removal of the ovaries
and the effects of injections of ovarian and placental extracts into
spayed animals. Contrib. to Embryol., Carnegie Institute, Washington,
vol. 19, p. 1.
ALLEN, E., AND DOISY, E. A. 1923 An ovarian hormone. Preliminary report
on its localization, extraction, and partial purification, and action in
test animals. J . Am. Med. Assn., vol. 81, p. 819.
ALLEN, E., FIGANCIS,
B. F., ROBERTSON,
L. L., COLGATE,C. E., JOIINSTON,
C . G.,
DOISY, E. A., KOUNTZ,
W. B., A N D GIBSON, H. V. 1924 The hormone
of the ovarian follicle; its localization and action in test animals,
and additional points bearing upon the internal secretion of the
ovary. Am. J. Anat., vol. 34, p. 133.
CHAMPY,C., AND KELLER,T. 1927 DBveloppement uterin et mammaire par
injection d’hormone ovarienne. Compt. rend. Acad. d. sc., T. 85, p. 302.
DOISY, E. A., VELER, C. D., AND THAYER,S. A. 1930 The preparation of the
crystalline ovarian hormone from the urine of pregnant women. J.
Biol. Chem., vol. 86, p. 499.
DOISY, E. A,, AND THAYER,S. A. 1931 The preparation of theelol. J. Biol.
Chem., vol. 91, p. 641.
HATERIUS,H. 0. 1928 Effect of placental extract on mammary glands of male
guinea-pigs. Proc. SOC.Exper. Biol. and Med., vol. 25, p. 471.
LAQUEUR,E., DE JONGH,
S. E., AN^ TAUSK, M. 1927 Uber weibliehes SexualHormone, Menformon. V. Uber den feminisierenden Einfluss des
Menformons auf des unentwickelte Brustdruse. Deutsche med. Wchnschr., Bd. 53, S. 867.
LAQUEUR,E., BORCHARDT,
E., DINGEMANSE,E., AND DE JONQH,
S. E. 1928
Uber weibliches (sexual-) Hormon, Menformon. Deutsche med.
Wchnschr., Bd. 54, S. 465.
LOEB,L., AND KOUNTZ,W. B. 1928 The effect of injection of follicular extract
on the sex organs in the guinea pig and the interaction between the
follicular substances given off by the corpus luteum. Am. J. Physiol.,
voI. 84, p. 283.
STEINACH, E., DOHRN,M., SCHOELLEJL,
W., HOHLWEG,
W., AND FAURE,
W. 1928
h e r die biologischen Wirkungen des weiblichen Sexualhormons.
Arch. f. d. ges. Physiol., Bd. 219, S. 306.
TURNEE, G. W., AND FRANK,
A. H. 1930 The effect of the estrus producing
hormone on the growth of the mammary gland. Mo. Agr. Exper.
Sta. Res. Bul. 145.
TURNER,C. W., A N D SCHULTZE,
A. B. 1931 A study of the causes of the
normal development of the mammary glands of the albino rat. Mo.
Agr. Exper. Sta. Res. Bul. 157.
TURNER, C. W., FRANK,
A. H., LOMAS,C. H., AND NIBLER, C. W. 1930 A
study of the estrus producing hormone i n the urine of cattle during
pregnancy. Mo. Agr. Exper. Sta. Rea. Bul. 150.
V I N T E M B ~ E RP., 1925 Action des injections de liquide folliculaire sur la
gland mammaire. Arch. de biol., T. 35, p. 125.
PLATE 1
EXPLANATION OF FIGURCS
1 Control gland of a normal young male rabbit (R20). X 3%.
2 Control gland of a young male rabbit (R17) that had been castrated five
months before receiving the theelol injection. I n both rabbits the glands consist
of short, unbranched ducts. X 31h.
3 A section of a gland of R20 extending from the region of the teat medially
mg. of theelol. The duct system had
after twenty-two injections containing
increased in extent and complexity. The distal ends of the ducts are enlarged
and in a stage of growth. X 3%.
4 A section of a gland of R20 after fifteen daily injections of 16% rat units
of theelin. These injections were started ten days after the gland shown in
figure 3 had been removed. X 3%.
234
GROWTH O F TIIE AIA3IAIARY GLAND
PLATE 1
C. JY. T E R S E R A N D O T H E R S
235
THP A Y h T O Y I C A L R E C O R D , TOT.. 5 3 , HO.
2
PLATE 2
EXPLANATION OF FIGURES
5 A section of a inamniary gland of R17 after thirty-seven injections of 16%
rat units of tbeelin. A control gland of R17 is shown in figure 2. The extent and
complexity of the duet system was greatly increased. Numerous siriall lateral
ducts aiid a distal growth area are shown. X 3%.
6 A section of a maininary gland of X106, a male rabbit after thirty daily
injections of 20 rat units of a crude extract of the estrus-producing hormone
recovered from the uriiic of pregnant cattle. The ducts are generally increased
in length aiid diameter, with extensive branching and a rapid-growing periphery.
236
GROWTH OF THE AIAJIXARY GLAND
PLATE 2
C W. T U H N > H 9ND OTHERS
237
PLATE 3
EXPLANATION OF FIGURES
7 Mammary gland of a normal feiriale albino rat twenty days of age. Control.
X 3.
8 Mamniary gland of a female albino r a t spayed at t v e n t g days of age
followed by a daily treatinelit with 0.03 ee. of theelol for twenty days. X 3 .
9 Maiiiniary gland of a fcinale albino rat spayed when twenty days of age
followed by daily treatment with 2..5 r a t units of theelin f o r twenty days. X 3.
10 Mammary gland of a female albino rat spayed at twenty days of nge followed by treatment of 1 r a t unit daily f o r tnenty days of the estius-producing
hormone extracted from the urine of pregnant cattle. X 3.
238
PLATE 3
GROWTH OE' THE DZAIlDfARY GLAND
0. W. TURNER A N D OTIIERS
239
PLATE 1
EXPIANATION OF FIGURES
11 The niainniary gland of the normal adult inale iiiousc. The ducts of the
glands remain Tery rudimentary throughout life. Control. X 7 .
12 The nixmmary gland of an adult inalc mouse which received 0.2 cc. (10 rat
units) of tlieelin daily f o r twenty days. Extensive growth of the duct system
has becn stimulated. X 7 .
13 The ntaniinary gland of a n adult male mouse which rcccived 0.05 cc. of
tltcclol daily f o r fifteen days followed by 0.1 cc. of theelol daily f o r ten days. X 7.
1 4 The mammary gland of a n adult male mouse which reccired 0.2 cc. (10 rat
units) of a crude extract of the estrus-producing hornionc recovered from the
urine of prcgnant cattle for thirty days. x 7 .
240
GROWTH O F THE MA3IMARY G L 4 N D
C W
TURNER AHD O T H E R S
241
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