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Uterine metaplasia and plasma levels of vitamin A.

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Uterine Metaplasia and Plasma Levels of Vitamin A '
Department of Anatomy, Bowman Gray School of Medicine,
Winston-Salem, North Carolina
The relationship between vitamin A deficiency and estrogen as etiological agents of uterine stratified squamous metaplasia (keratinizing metaplasia)
was studied using the rat. This is the initial attempt to correlate plasma levels of
vitamin A with the uterine lesions. From the results the following conclusions can
be drawn. Uterine metaplastic lesions which develop in estrogen-treated rats are not
a result of generalized hypovitaminosis A, since plasma levels of vitamin A are not
altered in animals treated with estrogen. Estrogen per se can cause uterine keratinizing metaplasia. The histopathologic lesions occur more frequently in vitamin Adeficient estrogenized rats having decreased plasma vitamin A levels as compared to
rats with normal vitamin A intake. The two insults are complementary in producing
epithelial metaplasia of the endometrium of the rat.
Uterine stratified squamous metaplasia
(keratinizing metaplasia) occurs both in
vitamin A-deficient and in estrogen-treated
rats. Wolbach and Howe ('25) initially
described the characteristic alterations of
the endometrium in avitaminotic A rats.
These investigators concluded that the primary effect of vitamin A deficiency was the
withdrawal of factors necessary for the
differentiation and maintenance of the epithelia concerned. In 1935, Selye et a1 noted
that the normal columnar epithelium was
partially replaced by stratified squamous
cells when rats were chronically treated
with estrone. The definitive lesions which
are found in the uteri of avitaminotic A
rats and those which appear in uteri of rats
stimulated excessively with estrogen have
been reported to be morphologically identical (Bern, '52).
McCollough and Dalldorf ('37) theorized
that uterine metaplasia induced by estrogen may be a manifestation of vitamin A
deficiency. From their studies, they concluded that the causative agent of uterine
metaplasia in rats was vitamin A deficiency
and that estrogen was acting as a growth
stimulant. Likewise, Cramer ('42) found
that Progynon (an estrogen compound)
induced metaplasia of the uterus and
cervix of virgin female rats. This change
could be inhibited by the injection of vitamin A into the uterine horns. Cramer believed, like McCollough and Dalldorf ('37),
that the epidermalization process induced
by estrogen may, in fact, be a manifestation of hypovitaminosis A.
ANAT. REC., 152: 1-8.
More recently, Gilten ('54), Fluhmann
('55), and Bo ('57) independently presented
comprehensive discussions on the origin
and development of estrogen-induced uterine metaplasia in the rat. Although these
investigators differ on the description of
the mode of development of the histopathologic lesions, because of their results
they concluded that estrogen per se can
cause keratinizing metaplasia of the uterine epithelium.
The difference of opinion which exists
as to the causative agent of endometial
keratinizing metaplasia prompted the present investigation. In the investigations discussed in the previous paragraphs, no
attempt was made to correlate blood levels
of vitamin A with the metaplastic changes
seen in the uterus. A low level of vitamin
A in the blood is known to be an absolute
criterion of generalized hypovitaminosis A
(Dowling and Wald, '58). Thus, correlation of the plasma levels of vitamin A with
uterine lesions seen in estrogen-treated
rats fed normal or vitamin A-free diets
would help to elucidate the role of estrogen
and hypovitaminosis A as causative agents
of uterine keratinizing metaplasia.
Twenty-eight to 30-day-old ovariectomized rats were divided into three groups of
1 In part from a thesis prepared by the author as a
partial requirement for the Doctor of Philosophy
degree at the Bowman Gray School of Medicine, Winston-Salem. N. C. Suuuorted bv a erant from the
2 Present address : Physiology Division, Directorate
of Medical Research, Edgewood Arsenal, Maryland.
ten rats each. The rats of groups I and I11
were fed a vitamin A-free diet. The animals of group I1 were fed a normal Purina
Chow diet and each was paired with a n
animal of group I.
The animals of group K began to develop
the regular signs of vitamin A deficiency
after approximately 13 weeks. When the
weight of these rats reached a plateau, the
animals of group I1 were increasing in
weight normally. During the subsequent
14 days, each rat of groups I and I1 received 2 clg of estrogen (depoestradiol, Upjohn Co.) subcutaneously per diem. At the
end of the two-week period, all rats of
group I and their group I1 mates were sacrificed. Rats of group I11 were treated
similarly to those of grciup I. When the
weights of the vitamin A-deficient rats of
group 111 began to decrease, 2 clg of estrogen were given daily for 14 days. In
addition, 200 International Units (I.U.) of
vitamin A (diluted from vitamin A ester
concentrate) per day in cottonseed oil was
administered orally for the same period
of time, after which the animals were
At the time of sacrifice, blood was taken
from the thoracic aorta according to the
technique of Lushbough and Moline ('61)
for plasma vitamin A determinations. The
blood from each animal was collected
directly into a graduated centrifuge tube
and centrifuged immediately. No anticoagulant was used. Plasma determinations of vitamin A were conducted using
the technique of Kimble ('39). Uteri were
excised and after processing, serial sections
were stained with Harris' hematoxylin and
counterstained with eosin.
In the hypovitaminotic A rats of group I,
the endometria were hyperplastic and the
columnar cells appeared hypertrophic. The
cylindrical epithelial cells became multilayered in many cases (fig. 2), and not
uncommonly epithelial polyps of the endometrium were observed. Focal absence of
the endometrial epithelium was noted and
occasionally, columns of epithelial cells extended into the lamina propria. These
structures were occasionally associated
with small metaplastic lesions (fig. 3 ) . Of
the ten animals in this group, nine developed partial keratinizing metaplasia
and six of these showed extensive lesions
(table 1). Both the luminal and glandular
epithelial were involved. The endometrial
epithelium of one uterus was nearly replaced by stratified squamous metaplasia
and keratinized lamellae were being desquamated into the uterine lumen (fig. 4).
Frequently the lesions originated in the
necks of glands or occasionally in association with polypoid structures. The stratified squamous lesions usually appeared to
originate beneath the columnar epithelium
(fig. 2) and supplant it by peripheral
growth .
The uterine columnar epithelium of the
rats from group I1 was also hyperplastic,
often becoming stratified into several
layers and epithelial polyps were infrequently noted. Only three of ten animals
had small areas of uterine metaplasia
(table 1).
Seven of the uteri of rats of group I11
had keratinizing metaplastic lesions, however, the changes involved only small areas
of the endometrial surface. Not uncommonly the focus or origin appeared to be
Uterine m.etaplasia i n rats injected w i t h estrogen f o r 14 days
No. rats
Estrogen and vit. A
Extent of
lesions 1.2
+(3); ++(5); + + + ( I )
+ ( 6 ) ; ++(I)
indicates one to severd rnetaplastic foci of the glandular or luminal epithelium;
indicates very extensive metaplastic lesions involvmore numerous small metaplastic lesions;
ing the entire or nearly the entire endometrium.
2 Number in parentheses iindicates number of animals with a lesion of this extent.
A deficiency, leads to a rapid replacement
of the columnar epithelium by stratified
squamous metaplasia. Recently, Aydelotte
( ' 6 3 ) studied chick tracheal epithelium in
tissue culture and noted that the basal
cells had a low mitotic rate. However, if
the cells were subjected to a vitamin A
deficiency, they divided more rapidly and
became stratified. She speculated that the
mitotic rate of the basal cells is inhibited
by the normal concentration of vitamin A
in the blood, but when a deficiency develops, the cells are released from this inhibition. Also, the in vivo production of a
hypovitaminosis A leads to a proliferation
of mucous membranes culminating in a
stratified squamous metaplasia. It is theorized that the stimulation of mitotic activity by vitamin A deficiency coupled with
the proliferative action of estrogen in the
uterus results in the rapid replacement of
the uterine mucosa with epidermoid heteroplasia, as seen in the present investigation.
This would explain the high index of
squamous metaplasia in group I, even
estrogen was administered for only
14 days.
Only three of ten animals fed a normal
Fig. 1 Estimations of the plasma levels of
vitamin A (vertical lines indicate range of val- chow diet (containing adequate vitamin
A) prior to estrogen injections exhibited
ues) in rats injected with estrogen for 14 days.
Area between broken lines represents the average
histopathologic endometrial changes. Sevnormal plasma levels of vitamin A for the albino
eral workers (Hohlweg, '51, '52; Kahn and
rat (Moore, '57).
Bern, '50) have demonstrated an antagin the necks of the uterine glands (fig. 5 ) . onism between vitamin A and estrogen.
The plasma levels of vitamin A were within
Epithelial polyps were rarely observed.
The mean plasma vitamin A level of the the normal limits in rats fed a normal chow
ten rats of group I was 35 I.U. per 100 ml. diet prior to the estrogen injections. This
This average is significantly lower than the tends to preclude the idea that the pathnormal range of plasma vitamin A in the ologic changes were an indirect manifestaalbino rat (fig. 1). The levels of vitamin tion of vitamin A deficiency. Rather, the
A in the blood of rats of groups I1 and I11 results can be attributed to excess exogenous estrogen stimulation. Since estrogen
were within normal limits.
stimulation did not alter blood vitamin A
levels, the uterine keratinizing metaplastic
In the present investigation, nine of ten lesions induced by estrogen are not a result
uteri of hypovitaminotic A rats had meta- of generalized hypovitaminosis A. Furtherplastic changes when stimulated with estro- more, the other common signs of vitamin
gen for 14 days. In this group of animals, A deficiency (e.g., unkempt fur, dyspnea)
the plasma levels of vitamin A were greatly were not obvious, although the time interreduced from normal. On the other hand, val was so short they may not have become
if the rats were fed a standard laboratory manifested.
Most animals treated simultaneously
chow diet prior to the estrogen injections,
rarely did metaplasia appear. This demon- with oral vitamin A and subcutaneous
strates that the combined effect of the two estrogen after being fed a diet deficient in
insults, estrogen stimulation and vitamin vitamin A developed endometrial changes.
Analyses of vitamin A in the blood of these relative avitaminotic A condition within
animals showed the levels to be within the endometrial cells. The findings of the
normal limits, and yet seven of ten uteri present investigation show that treatment
exhibited changes. However, the animals with exogenous estrogen for two weeks
became hypovitaminotic A before estrogen does not alter the plasma vitamin A level.
and vitamin A were administered. At this Therefore, the uterine changes noted in
time, the uterine endometrial cells were these animals cannot be attributed to genalso subjected to the deficit of vitamin A. eralized hypovitaminosis A.
Since Jensen ('60) has shown that carbonACKNOWLEDGMENTS
14-labeledestrogens can be detected within
The author expresses his gratitude to
the endometrial cells very shortly after injection, the endometrial epithelial cells Dr. Walter J. Bo for his help in preparing
may have responded while still in the dys- this manuscript.
vitaminotic A condition to produce squaLITERATURE CITED
mous metaplasia. Had the animals conAydelotte,
B. 1963 The effects of vitamin
tinued for a greater period of time to
A and citral on epithelial differentiation in
receive both vitamin A and estrogen, the
vitro. 1. The chick tracheal epithelium. J.
incidence of stratified squamous lesions
Embryol. Exp. Morph., 11: 279-291.
may have decreased. The concentration of Beaver, D. L. 1961 Vitamin A deficiency i n the
germ-free rat. Am. J. Path., 38: 335-357.
vitamin A used in the present investigaBern, H. A. 1952 Alkaline phosphatase activity
tion lowers the incidence of uterine kerain epithelial metaplasia. Cancer Res., 12: 85tinizing metaplasia induced by estrogen
Bo, W. J. 1957 Relation of vitamin A defici(unpublished observations).
ency and estrogen to induction of keratinizing
That chronic or acute septic states acmetaplasia i n the uterus of the rat. Amer. J.
companying vitamin A deficiency and/or
Clin. Path., 5: 666-673.
chronic estrogen treatment may have Cramer, V. H. 1942 Wachstumssteuerung Gefahrdeter Epithelial gebiete. Deut. Med. Wschr.,
caused the metaplastic lesions must be
68: 809-814.
considered a possibility. Vitamin A deJ. E., and G. Wald 1958 On the
ficiency in experimental animals is often Dowling,
mechanism of vitamin A deficiency and nightassociated with infection (Tyson and
blindness. Fourth International Congress of
Biochemistry, Vol. XI - Vitamin Metabolism,
Smith, '29). However, it has never been
Vienna, pp. 185-197.
established conclusively that the septic
C. F. 1955 Squamous metaplasia in
state is always the cause of epithelial metathe rat uterus. Arch. Path., 59: 238-256.
plasia in these hypovitaminotic A animals. Gilten, G. 1954 On the mode of development
Also, it is known that vitamin A deficiency
of stratified squamous epithelium in the rat's
uterus following prolonged estrogen adminiscan cause metaplastic lesions in many
tration. Anat. Rec., 120: 637-662.
organs of germ-free rats precluding the
W. 1951 ifber die Hemmung der
possibility that they are invariably a result HoNweg,
Oestrusreaktion durch Vitamin-A-ifberdosierof secondary infection (Beaver, '61). If
ung. Klin. Wschr., 29: 193-195.
the uterine metaplastic changes seen in
1952 Die Testierung von Vitamin APreparation mit Hilfe der Antioestrusreaktion.
the uteri of animals of the present investiDie Pharmazie, 7: 280-284.
gation resulted from septic states produced
E. V. 1960 Fate of steroid estrogens in
by estrogen administration, the number Jensen,
target organs. First International Congress of
and extent of the lesions should have been
Endocrinology, Copenhagen, p. 733.
similar in the three groups since all Kahn, R. H., and H. A. Bern 1950 Antifolliculoid activity of vitamin A. Science, 111: 516the rats were given identical estrogen
M. S. 1939 The photo-colorimetric deIf the postulation, propounded by pre- Kimble,
termination of vitamin A and carotene in huvious investigators (McC!ollough and Dallman plasma. J. Lab. Clin. Med., 24: 1055dorf, '37; Cramer, '42) - that the etiology
of estrogen-induced uterine metaplasia is Lushbough, C. H., and S. W. Moline 1961 Improved terminal bleeding method. Proc. Animal
vitamin A deficiency - is correct, then it
Care Panel, 11: 315-318.
would be necessary to explain how excess McCollough,
K., and G. Dalldorf 1937 Epitheestrogen stimulation causes an absolute or
lial metaplasia. Arch. Path., 24: 4 8 6 4 9 6 .
Moore, T. 1957 Vitamin A. Elsevier Publishing Company, Amsterdam.
Selye, H., D. L. Thompson and J. B. Collip 1935
Metaplasia of the uterine epithelium produced
by chronic oestrin administration. Nature, 135:
Tyson, M. D., and A. H. Smith 1929 Tissue
changes associated with vitamin A deficiency
in the rat. Am. J. Path., 5: 57-70.
Wolbach, S. B., and P. R. Howe 1925 Tissue
changes following deprivation of fat-soluble A
vitamin. J. Exp. Med., 42: 753-778.
Uterine section of a hypovitaminotic A rat treated with estrogen for 14 days. Note
the pronounced stratification of the columnar epithelium and the metaplastic
lesion. x 100.
Uterine section of a hypovitaminotic A rat treated with estrogen for 14 days.
Note the column of epithelial cells extending into the lamina propria and the
underlying squamous metaplastic lesion. X 100.
Uterine section of a hypovitaminotic A rat treated with estrogen for 14 days.
The columnar epithelium is nearly replaced by stratified squamous metaplasia.
x 100.
Uterine section of a rat treated with vitamin A and estrogen showing small lesion
originating i n the neck of the gland. X 100.
Russel J. Reiter
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level, vitamins, metaplasia, plasma, uterine
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