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Vaginal cycles in the rat after x-ray or other damage to one or both ovaries.

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Vaginal Cycles in the Rat after X-ray or Other
Damage to One or Both Ovaries'
DepaTtment of Anatomy, University of Illinois, College of Medicine,
Chicago, Illinois
Damage to the rat ovary at 60 days of age causes irregularities in the
estrous cycle and leads to continuous comification of the vagina. These vaginal changes
occurred within two to three months after bilateral irradiation (800 or 2,000 r ) or after
bilateral castration and autotransplantation of a portion of one ovary. Unilateral
irradiation or ligation of the ovarian arteries at their junction with the uterine artery
produces similar changes within five to seven months. Comparable vaginal changes
did not occur in control animals until they reached the age of ten months.
The estrous cycle of rats exhibits periods
of irregularity during the last two thirds of
life in which extended periods of continuous estrus are characteristic (Asdell and
Crowell, '35; Bloch, '57; Everett, '39; Ingram, '59 and others). Bloch and Flury
('59) found continuous estrus in the rat to
last for varying periods up to the age of
30 months. Thereafter, their animals
showed an anestrus. Comparable irregularities have been noted after various insults to the ovary. In both the mouse
(Parkes, '27) and the rat (Drips and Ford,
'32) x-ray irradiation of the ovaries may
be followed by regular cycles or, after a
latent interval, by periods of continuous
estrus (Mandl and Zuckerman, '56; Ingram, '58; and Slate and Bradbury, '62).
The continuous estrus lasts for varying
periods and is followed by diestrus. Continuous corniiication is also found after
transplantation of the ovary or interference with its normal innervation and blood
supply (Fels, '44; Hernandez, '43; Kullander, '59, and '60; Lipschutz, Mena and
Vinals, '34 and others). The time at which
constant estrus appears, and its extent,
may be dependent upon the degree of
ovarian damage (Fels, '35 and Slate and
Bradbury, '62) and may also be an indication of an acceleration of the processes
which are seen in the untreated animals.
In most observations all the ovarian tissue of the animal was subjected to the
injury. Ingram ('58) and others suggest
that the alteration may be due in part to
the loss of ova in the damaged ovary. Pencharz and Long ('32) found that when
only one ovary was irradiated the injured
ovary was inactive in the presence of an
untreated ovary. There is some question
of whether the control of the estrous cycle
by the normal ovary is affected by the
presence of a damaged ovary.. The age of
the animal at the time of ovarian damage
may also be a significant factor. This investigation compares the effects of various
doses of x-ray or other damaging agents
administered to one or both ovaries of the
rat at 60 days of age.
Vaginal cycles were followed in 169 rats.
Smears were started at 45 to 50 days of
age and irradiations, transplantations and
other procedures were done on day 60.
Irradiations with 800,2,000or 3,000 roentgens were applied to the exteriorized ovary
or ovaries with the animal under Nembutal
anesthesia. The female was placed in a
lead compartment and the gonad or gonads
brought through both the dorsal body wall
and an opening in the lead shield. Lead
plates were placed around the mesovanum
with care to avoid trauma to the ovary or
its blood vessels. The entire unit was then
placed under the x-ray tube to irradiate
(140 KV and a current of 5 mA at a dose
of 520 r/min.) the gonad or gonads. A
dosimeter in the lead container during
irradiation had an average reading of 0.6 r
of scatter rays in an 800 r exposure. Sham
1Aided by grants from National Institutes of
Health United States Public Health Service and the
Univerhty of Illinois Research Board, The Graduate
irradiation, irradiation of the left ear or
only anesthesia with Nembutal, as well as
unilateral and bilateral castrates and untreated animals served as controls.
Ovarian transplantations were made to
the subcutaneous tissue of the dorsal body
wall. Both ovaries were removed and onehalf of the left ovary was transplanted.
In another series the blood supply to both
ovaries was disturbed to the extent of
placing a ligature on each ovarian artery
at its junction with the uterine artery.
The most evident effects of irradiation
on the vaginal cycle as reported in the
literature and in our observations was the
appearance of a continuous cornification
(CC will be used to designate this type of
smear). The first change was usually an
irregularity in the cycle in which extended
periods of cornification occurred. The time
at which the irregularity of the cycle appeared varied and will be considered with
specific groups. As the interval following
irradiation became longer, the periods of
cornification increased in length to replace
the cycle with CC. The length of a given
period of CC varied. At times a typical
diestrous smear would appear for one to
several days with a return to the CC state.
Unless the break was longer than a few
days the smear was recorded as CC. There
were, however, periods of diestrus, periods
of irregularity or even periods of regular
cycling after an extended period of CC.
As described by others, the cell type in
the smear was not always typically that of
estrus. There were periods of large nucleated epithelial cells, with or without
white blood cells. Another occasional variation was the presence of dark fluid in
the vagina resembling that found at the
time of the placental sign. This type of
smear often lasted for a considerable number of days and the blood was thought to
be uterine in origin.
In some of the females which had had
extended periods of CC the uterus was
greatly distended. Volumes of 15 to 30 ml
of fluid were obtained from the uterus of
a single rat. It was either clear estrous
fluid or contained blood. Not all uteri contained fluid but all showed stimulation.
The mammary glands in many of the fe-
males in CC were stimulated and contained
The vaginal cycles in ten of the 36 females in which both ovaries received 800 r
were normal prior to irradiation and for
approximately the first two months after
irradiation (fig. 1 ) . In the other 26 females of this series smearing was not
started until one and one-half, two and onehalf, three and one-half or four and onehalf months after the irradiation date. It
is seen that after two months post irradiation the cycles became either irregular or
continuously cornified. Once the CC had
been established, it generally continued
during the period of observation. Normal
cycling did not occur during interruptions
of the CC. After a long period of diestrus
female 868 returned to CC, as did several
animals in the 2,000 r series (fig. 3).
The mechanical stimulus of smearing
(noted by Wade and Doisey, '35) was not
the cause of cornification since CC was
present in the animals when smearing was
started three and one-half or four and onehalf months after irradiation.
One ovary was removed from 13 of these
animals. In seven (662, 733, 748, 725,
775, 776 and 676 as well as one animal in
the 2,000 r series, 811) ovariectomy was
done during a period of CC. With the exception of 776 there was no effect, or only
a short break in the CC due to the removal
of one ovary. Removal of both ovaries
(807, 808, 809) resulted in an immediate
interruption of cornification.
The effects of mating are to be reported
elsewhere but it may be mentioned that in
the early post irradiation period fertilization occurred (867). On the other hand,
seven females placed with males two and
one-half months after irradiation did not
become pregnant even though sperm were
found occasionally in the smear.
Irradiation of the left ovary by 800 r
and removal of the right ovary did not
cause a marked change in the vaginal
state from that seen in the animals with
800 r to both ovaries. The smear records
of five of 12 animals so treated are shown
in figure 2.
After 2,000 r to both ovaries ( 8 femdes)
disturbances in the cycle appeared somewhat earlier than in the 800 r animals
(fig. 3). Irregularities and CC were preva-
Figs. 1 to 9.
Graphic representation of vaginal cycles of female rats which received
various treatment at 60 days of age.
Fig. 1
Fig. 2
Fig. 3
800 r to both ovaries.
800 r to left ovary; right ovary removed.
2,000 r to both ovaries.
lent during the second month. Thereafter
extended periods of diestrus were seen in
all animals but one (811). Prolonged CC
appeared in three of these animals after
months of diestrus (896, 897,898).
Three females which received 3,000 r
to both ovaries had irregular cycles during
the first 50 days after irradiation and then
had diestrous smears throughout the period
of observation ( 6 months).
Vaginal records of 18 of the 30 females
in which only the left ovary was exposed
to 800 r are shown in figure 4. In general
the cycles were normal during the first
three to five months. The 12 animals
which were followed for only five months
are not included in the figure. It is evident
that at six to seven months post irradiation (8-9 months of age) irregularities
and CC became prevalent. The onset of
CC was later than in the bilaterally irradiated females and there were extended
periods of irregular cycles which appeared
at about the time CC appeared in the bilateral group. Periods of regular cycles
followed CC and were observed up to ten
months after irradiation. When the normal (right) ovary was removed the animal
showed a period of diestrus. Two of these
(547, 797) returned to normal cycling.
Continuous comification occured in the
others about a month after the ovariectomy. Removal of the irradiated ovary
(798,799) caused no change. Removal of
both ovaries (799,547) produced anestrus.
Unilateral irradiation with 2,000 r had
much the same effect as with 800 r (fig. 5).
The greater damage produced by the 2,000
r to the single ovary of these seven animals
did not seem to change the time at which
the irregular and CC vaginal smears appeared.
Irregular cycles or CC were observed
seven to ten months after irradiation in
36 females which were unilaterally irradiated and the irradiated ovary removed
six to 90 days later. Four typical cases
are shown in figure 6.
In four females the left ear was irradiated as one of the control procedures.
Rats 873 and 874 received 800 r and numbers 875 and 876 received 2,000 r (fig. 7).
Continuous cornification appeared in two
of these animals at 8 months post irradiation.
The cycles of five untreated control animals (889, 891, 892, 899 and 924) were
essentially normal (fig. 8). Exceptions
were found in that 899 had some CC at
380 days of age and 924 showed an extended period of irregularity between 9
and 11 months of age.
Eight females were unilaterally castrated. Their cycles were essentially normal and three are shown in figure 8 (836,
918, 920). The one exception (920)
showed cornification between 10 and 11
months of age. The one sham irradiated
animal (834) cycled normally whereas the
animal with Nembutal anesthesia (840)
showed irregularity and CC (fig. 8).
Five females were castrated bilaterally
at 60 days of age and their smears were
recorded at intervals for periods of 14
days. These females showed no vaginal
cornification through the ten months of
Transplantation of half an ovary to the
dorsal body wall and the removal of the
remainder of the ovarian tissue produced
marked changes in the vaginal picture.
Four of seven animals are shown in figure
9 (942, 943, 945, 947). Vaginal changes
in these animals began at a time comparable to, and were similar to, those in the
bilaterally irradiated series (800 r).
Continuous cornification also was found
in six animals after ligatures had been
placed on the ovarian arteries at their
junction with the uterine arteries. Two of
these (948, 951) are shown in figure 9.
Cornification appeared during the sixth
month after surgery, and their uteri showed
distention beyond the limits of normal
Fig. 4 800 r to left ovary only.
Fig. 5 2,000 r to left ovary only.
Fig. 6 800 r to left ovary; left ovary removed
6 to 24 days post irradiation.
Fig. 7 800 r (873,874) or 2,000 r (875,876)
to left ear.
Fig. 8 Untreated (889, 891, 892, 899, 924);
unilaterally castrated (836, 918, 920); sham irradiated (834); Nembutal anesthesia (840).
Fig. 9 Subcutaneous autotransplantation of
an ovarian fragment after bilateral castration
(942, 943,945, 947); bilateral ligation at junction
of ovarian and uterine vessels (948, 951).
Figures 4-9.
The continuous cornification found in
the vaginal smears of animals after ovarian irradiation was not due to the effects
of continuous smearing noted by Wade
and Doisey ('35). The changes in the
vaginal cycle produced by damage to the
ovary appeared at an earlier age than comparable changes which occur in the aging
female rat. Normal cycles continued for
a period which is, in general, inversely proportional to the degree of damage to the
ovary (Fels, '35; Ingram, '59 and Slate and
Bradbury, '62). Unilateral ovariectomy
did not hasten the appearance of the vaginal changes. Vaginal changes appeared
at the same time as in untreated animals
and as in those of Bloch and Flury ('59).
The effects of aging may be the determining factor in animals which received irradiation to the ear or nembutal anesthesia.
There is no apparent explanation of why
irradiation of one ovary, followed by its removal a short time later, resulted in a
higher percentage of cornification than was
seen in the untreated or unilaterally ovariectomized female. Again, this may have
been an aging process.
From our observations there is no evidence why alterations occurred in the
vaginal cycle after unilateral irradiation.
Damage to one ovary may introduce some
new factor. The same hypothesis may also
be applied to bilateral irradiation and to
the other forms of ovarian damage. It is
clear that the damaged ovaries are responsible for the observed changes, because in
every case anestrus followed bilateral ovariectomy. This would support the observations of Mandl and Zuckerman ('56) that
the suprarenals do not maintain the cornification.
Whatever the changes may be, the endocrine balance is disturbed. This leads to
a masking of the cyclic activity, resulting
in a continuous cornification of the vagina
and an excessive stimulation of the uterus
and mammary gland. There may be an
altered estrogen effect, as suggested by
Fels ('55) or Ingram and Mandl ('58),
which may activate the accessory reproductive organs but have no effect on the
pituitary. Or there may be a difference in
the threshold at which the various end
organs will respond to estrogen stimula-
tion. Ingram and Mandl ('58) have shown
that gonadotrophins are necessary for the
damaged ovary to continue its activity.
Their evidence suggests that the damaged
ovary may not be able to respond fully to
the gonadotrophins.
It is not possible to determine if the
effects are due to interference of the nerve
(Hill, '48) or blood supply (Fels, '44) or to
a direct effect on the ovarian tissue. Since
the ovary is capable of maintaining an
estrogenic influence on the vagina, uterus
and mammary gland, it would seem that
the damage leads to an endocrine imbalance which begins in, and is mediated
through, the ovary.
Asdell, S. A., and Mary F. Crowell 1935 The
effect of retarded growth upon the sexual development of rats. J. Nutrition, 10: 13-24.
Bloch, Suzanne 1957 Untersuchungen iiber das
funktionelle Altern tierischer Genitalorgane.
Gynaecologia, 144: 313-316.
Bloch, Suzanne, and E. Flury 1959 Untersuchungen iiber Klimakterium und Menopause
an Albino Ratten. Ibid., 147: 414-438.
Drips, Della G., and Frances A. Ford 1932 The
study of the effects of roentgen rays on the
estrual cycle and the ovaries of the white rat.
Surg. Gynec. and Obst., 55: 596-606.
Everett, J. W. 1939 Spontaneous persistent
estrus in a strain of albino rats. Endocrinology,
25: 123-127.
Fels, E. 1935 Ergebnisse expenmenteller Eierstocks und Nierenbestrahlung bei der weissen
Ratte. Strahlentherapie, 54: 279-293.
1944 El efecto de la inyecci6n de
hormona estrdgeno sobre la hipdfisis de rata con
ovarios ligados. Rev. Assoc. Med. Argentina,
58: 778.
1955 Transformations tumorales des
greffes ovariennes dans le rein de la Ratte
castree. Compt. Rend. SOC.Biol., 149: 16661667.
Hernandez, T. 1943 Hormonal ambisexuality
of ovarian grafts in female rats. Amer. J. Anat.,
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artery and nerves. Anat. Rec., 100: 42.
Ingram, D. L. 1958 Fertility and oocyte numbers after x-irradiation of the ovary. J. Endocrin., 17: 81-90.
1959 The vaginal smear of senile laboratory rats. Ibid., 19: 182-188.
Ingram, D. L., and Anita M. Mandl 1958 The
hypophyseal control of x-ray sterilized ovary.
Ibid., 17: 1-12.
Kullander, S. 1959 Studies on the histogenesis
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1960 On tumor formation in gonadal
and hypophyseal transplants into the anterior
eye chamber of gonadectomized rats. Cancer
Research, 20: 1079-1082.
Levine, W. T., and E. Witschi 1933 Endocrine
reactions in female rats after x-ray treatment
of the ovaries. Proc. SOC.Exp. Biol. and Med.,
30: 1152-1153.
Lipschutz, A., J. Mena and E. Vinals 1934 Intervention ovarienne et loi de la puberte.
Compt. Rend. SOC.Biol. Paris, 116: 136S1367.
Mandl, Anita M.,and S. Zuckerman 1956 The
reactivity of the x-irradiated ovary of the rat.
J. Endocrin., 13: 243-261.
Parkes, A. S. 1927 On the occurrence of the
estrous cycle after x-ray sterilization. Part 111.
The periodicity of estrus after sterilization of
the adult. Proc. Roy. SOC.London B, 101 : 421449.
1956 Survival time of ovarian homographs in two strains of rats. J. Endocrin., 13:
Pencharz, R. I., and J. A. Long 1932 The effect
on a subsequent pregnancy after x-raying one
ovary of a rat. Am. J. Anat., 50: 1-8.
Slate, W.G.,and J. T. Bradbury 1962 Ovarian
function and histology after x-ray irradiation
in albino rats. Endocrinology, 70: 1-6.
Wade, N. J., and E. A. Doisey 1935 Cornification of vaginal epithelium of ovariectomized
rat produced by smearing. Proc. SOC.Exp. Biol.
and Med., 32: 707-709.
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