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Some Effects of advancing age on the histology and reactivity of the mouse ovary.

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SOME EFFECTS O F ADVANCING AGE ON
THE HISTOLOGY AND REACTIVITY
O F THE MOUSE OVARY1
JAMES A. GREEN'
Department of Anatomy, University of North Carolina
Chapel Hill, North Carolina
TWELVE FIQTJBES
The response of the rodent ovary to exogenous gonadotrophin changes with age during prepubertal life; Ortiz ( '47)
studied this in the hamster and her paper should be consulted
for a review of the literature. Surprisingly little information
is available on the reactivity of the ovary of any species
throughout adult life. Ortiz ( ' 5 5 ) observed that the hamster
ovary became less responsive to injected gonadotrophin with
advancing age. Watson, Smith and Kurzrok ('38) reported
that in women the ovarian response t o equine serum gonadotrophin declined with age, and that after the menopause no
response was obtained. On the other hand Westman ('34)
stated that an ovarian response was found in two postmenopausal women given transfusions of blood from pregnant
women. Hoffman ( '31) obtained ovarian stimulation after
implants of bovine anterior pituitary tissue into old mice, but
no data are available as to the ages of these animals. Two
year old, infertile albino rats did not show compensatory hypertrophy of the remaining ovary after unilateral ovariectomy, although ovaJriesof rats of the same age were said to be
responsive to chorionic gonadotrophin ( Wiesner, '32).
This investigation was begun to study the histology and rcactivity of the mouse ovary throughout adult life.
This investigation was supported by research grant C-2246 M & G from
the National Cancer Institute, Public Health Service.
Present address : Department of Anatomy, University of Indiana, Bloomington, Indiana.
333
334
JAMES A. GREEN
MATEEIALS AND METHOD
Adult, virgin, C57BL/6JAX mice were used. At intervals
ranging from two and one half to 19 months of age groups of
10 mice were withdrawn from the colony for study. In order
to conserve mice a technique was followed that utilized each
animal as its own control. A vaginal smear was made and recorded for each mouse; however, it was shown earlier that
the stage of the estrous cycle had no influence on the ovarian
response to gonadotrophin in mice handled in the manner to
be described (Green, ’54). The mice were anesthetized with
nembutal; the right ovary and uterine horn were exteriorized
through a ventral midline incision ; the right uterine horn was
cut at its junction with the cervix; then the entire right side
of the reproductive tract was removed by blunt dissection of
its mesenteries. The excised ovary and uterus were trimmed
free of fat, weighed separately on a torsion balance, and transferred to Bouin’s solution. The left side of the reproductive
tract was inspected for evidence of gross pathological change
before the body wall was sutured. Next, the mouse was given
a subcutaneous injection in the nape of neck of 20 i.u. of equine
serum gonadotrophin (Equinex 3, contained in 0.25 ml. of
distilled water. Seventy two hours later the mice were sacrificed and the left ovary and uterine horn were removed,
weighed and fixed. The tissues obtained were subjected to
routine histological embedding procedure ; serial sections were
made of all of the ovaries, and sections were cut from the
midposrtion of each uterine horn.
OBSERVATION8
Ovarian a d uterine weight data. A plot of the average
weights of the organs (fig. 1) showed that the ovarian weight
of the control ovary tended to increase until about one year
of age, and then it began a gnadual decline that persisted
throughout the rest of the experiment. The ovarian response
t o PMS was pronounced until 14 months of age; then this re= The PMS (“Equinex’’) was obtained in generous amounts through the courtesy
of Dr. John B. Jewell of Ayerst Laboratories, Inc.
335
AGING MOUSE OVARY
sponse fell off abruptly and in two of the last three months of
the experiment only a limited weight increase was observed.
The low response at 17 and 19 months of age are of interest
because the average age at the end of fertility was 16 months
in mice of this strain (Boot and Muhlbock, '54).
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AGE (MONTHS)
Fig. 1 Average ovarian and uterine weights of mice of various ages before and
after the administration of 20 i.u. of PMS.
The average weight of the control uteri showed a rather
large increase toward the end of active reproductive life.
After 15 months of age it fell off, but it seemed to show a
tendency to increase again. The uterine weight increase after
PMS injection demonstrated that the exogenous gonadotrophin stimulated ovarian secretion until 17 months of age. The
lack of uterine weight increase during the last 3 months of the
336
JAMES A. GREEN
experiment suggested strongly that ovarian secretion was
not evoked by the dose level of gonadotrophin employed here.
MORPHOLOGICAL OBSERVATIONS
Two a d one-half t o eleverc rvLow%s of age. The untreated
ovaries of these animals contained a copious supply of follicles
in all stages of development, corpora lutea and a large quantity of uniformly distributed interstitial tissue (fig. 2). The
most prominent cell type of the interstitium was a large epithelioid cell with an abundant eosinophilic cytoplasm and a vesicular nucleus with a prominent nucleolus and diffuse, small
chromatin particles (fig. 7). The resemblance of these cells to
luteal cells was striking. In the rat, Rennels ('51) was of the
opinion that these cells produced estrogen. At 4 to 5 months
of age large, often multinucleated cells containing a yellow
pigment appeared in the interstitium ; these became progressively more numerous with age. The appearance and increase
of these lipochrome cells in aging C57BL mice has been reported by Leob ('48) and Fekete ('53) ; Deane and Fawcett
( '52) described their histochemical characteristics in this
strain of mice.
After P M S stimulation the ovaries showed a distinct increase in the number of follicles containing an antrum and
also showed 4 to 8 new corpora lutea (fig. 3). The medulla of
these ovaries had a conspicuous labyrinthine network of dilated sinusoids ; a similar vasodilation after gonadotrophin
was reported for the hamster ovary (Ortiz, '55). The lipochrome cells were unaffected by the gonadotrophin. The uteri
after P M S stimulation showed a well developed myometrium,
a high, often pseudostratified epithelium, an edematous endometrial stroma and vesicular stromal nuclei. This uterine morphology indicated combined stimulation by estrogen and
progestin.
Twelve to sizteen months of a,ge. The untreated ovaries of
these mice contained fewer follicles than the previous group.
This was particularly true for primary and secondary follicles. During this period small anovular follicles made their
AGING MOUSE OVARY
337
appearance at the periphery of the ovary. These structures
had a diameter approximately the same as that of a primordial
follicle. The granulosa cells of these anovular follicles were
identical with those of ovular follicles. The lipochrome cells
had increased further in number and had become a prominent
feature of the ovarian morphology (fig. 4). The interstitial
cells seemed ,more densely packed due to a diminution of cytoplasm. The nuclear membrane of these cells frequently showed
a slight wrinkling during the first half of this period. By 15
months of age nuclei were encountered that were oval to fusiform and had large chromatin particles (fig. 8). These nuclear changes were not uniform throughout the interstitial tissue, but seemed to occur most frequently in the medullary
region.
Follicular growth and corpus luteum formation occurred
after PMS injection, but not on as extensive a scale as in the
younger mice. All of the changes noted in the interstitial cells
of the control ovaries were repaired by PMS and the interstitial cells of the treated ovaries from this group were identical
to those of the young mice.
The uteri taken before PMS injection showed a morphology
that was indicative of stimulation by substantial amounts of
estrogen and progestin. The endometrium and myometrium
were a great deal thicker in these mice than in the younger
series. The glandular development was more extensive in this
series of mice than in the previous one. and cystic dilation of
the glands was frequent. Silver impregnation of the connective tissue fibers revealed a fibrous network in the endometrium that was f a r more extensive than any encountered in the
younger series. The uteri taken after P M S injection showed
no gross change from that just dcscribed for the control uteri.
Seventeen t o nineteen months of age. The ovaries of mice
sacrificed during this period uniformly showed definite
changes (fig. 5). Follicles were present but were not numerous. Around the periphery an occasional primordial follicle
with an oocyte was encountered, but small anovular follicles
were numerous. Medium sized folliclcs were scarce ; the num-
338
JAMES A. GREEN
ber of graafian follicles was small but they were invariably
present. Counts were made of the graafian follicles in the untreated ovary of the 27 mice in this series, and it was determined that there was an average of 2.2 graafian follicles per
ovary. One to three corpora lutea were present in 12 of the
mice, and corpora were absent in 15 mice. The interstitial
cells had very little cytoplasm and their nuclei were small and
contained large chromatin particles (fig. 10). These cells resembled the interstitial cells of the hypophysectomized rat
(Selye, Collip and Thomson, '33). There are no descriptions of
the interstitium of the ovary of the hypophysectomized mice
available, but the author has noted that the interstitial cells in
a series of hypophysectomized mice of the strain used here
were identical to those described here in the old mice. The
lipochrome cells at this time constituted a considerable portion
of the total ovarian volume.
The treated ovaries from almost all of these old animals
contained recently formed corpora lutea, and there was little,
if any, increase in follicular development such as that seen in
the younger ovaries after stimulation. Counts of the number
of graafian follicles and corpora lutea gave an average of 1.2
and 2.2 respectively. The interstitial cells of the treated ovaries had undergone a remarkable rejuvenation and possessed
large vesicular nuclei with a prominent nucleolus and small
diffuse chromatin particles. Their cytoplasm had increased,
but it was not as abundant as that seen in young mice (fig. 11).
Vasodilation of the medullary vessels was not marked in the
ovaries of this series.
The uteri before PMS treatment had a uniform morphology
that was not related to the vaginal smear. With few exceptions
the uteri had a thick cellular endometrium (fig. 12) and stroma1 nuclei of the progestational type (Hooker, '45). The epithelium was only moderately developed and did not exhibit
pseudo-stratification in any of the mice ; the glands were long
and well developed. Silver impregnation of these uteri revealed that they did not have as much fibrous material in the
stroma as did the preceding age series. The columnar epi-
AGING MOUSE OVARY
339
thelium and well developed myometrium indicated the presence of estrogen, but the lack of stromal edema and the argyrophilia suggest that it was present in minimal amounts. On
the other hand the cellularity of the stroma and the structure
of the stromal nuclei intimate that substantial amounts of progestin were circulating. After PMS treatment there was little
if any change in the uterine morphology.
Twenty-two mo&hs. One animal survived for 22 months
and was sacrificed at that time. The ovaries were atrophic
and sections of them showed a medulla composed almost entirely of lipochrome cells and a thin cortex (fig. 6) that was
composed of fusiform fibroblast-like cells (fig. 9). The uterus
of this mouse was atrophic and histologically it resembled that
of an ovariectomized mouse. The argyrophilic fibers seemed
to be reduced in number and size and resembled those seen in
young, adult ovariectomized mice.
DISCUSSION
The weight increase response of the ovary to 20 i.u. of PMS
remained fairly uniform until 14 months of age and then it
began to decline steadily. Examination of the ovaries of these
mice showed that several factors seemed to be contributing to
this decreased response. The decline in the follicular population was probably the factor of greatest consequence in this
loss of reactivity. I n connection with this it was interesting
to note that the decrease in follicular population of these mice
was far greater than that reported in 25 months old hamsters
(Ortiz, '55). A slight decline in the volume of reactive interstitial tissue may have played a minor role. However, it was
felt that the limted weight increase obtained in the oldest mice
was due in part to the increase in cell volume by the interstitial
cells. Another contributory factor was the failure of the
medullary vessels to undergo the widespread vasodilation seen
in the younger mice.
The increase in uterine weight after PMS injection during
the period of active breeding life was ample evidence that the
gonadotrophin dosage was an adequate stimulus for ovarian
340
JAMES A. GREEN
secretion. The failure of the uterus to increase in weight after
P M S injection during the last 3 months of the experiment
suggested that ovarian changes had occurred. It was of interest to note in this connection that in the group of oldest hamsters used by Ortiz ('55) 4 of 8 animals had uteri that were
not stimulated after P M S injection.
The presence of large follicles, deficient interstitial tissue,
and scarcity of corpora lutea in the aged ovaries could be interpreted as signs of pituitary gonadotrophin undergoing
qualitative changes with age. Such a structural pattern in the
ovary suggests that the pituitary secretion contained adequate
FSH content and inadequate LH content. It has been reported
that preparations of purified FSH will cause follicle growth
without interstitial cell repair in the hypophysectomized rat
(Evans, Simpson and Pencharz, '37; Greep, van Dyke and
Chow, '42). The rapid repair of the deficient interstitial cells
of aged mice after PMS injection indicated that their pretreatment morphology was a consequence of LH deficiency.
It has been reported that in the human female pituitary the
FSH :LH ratio is 1: 1during reproductive life and 3 :1 after
the menopause (Albert ct al., '56). The appearance of deficiency cells during reproductive life is somewhat puzzling ;
Wolfe ('43) has reported similar findings for the rat.
The uteri of these aged mice showed increased connective
tissue fibers toward the end of reproductive life, but this was
reversed in the senile mice. This is not in accordance with
other reports on the effect of advancing age on uterine connective tissue (Loeb, Suntzeff and Burns, '39; Wolfe, et al.,
'42 ;Rolle and Charipper, '49). The uteri of these mice showed
little evidence of estrogenic activity, and since estrogen has
a fibrogenic action on the endometrium it is felt that the uterine connective tissue merely reflects the ovarian secretion, and
thus was another sign of estrogen deficiency.
The aging changes that bring on the cessation of reproduction must be intricate and interrelated. It is probable they
are far more than a derangement of pituitary and ovarian
AGING MOUSE OVARY
341
function but there is little doubt that those organs play a
major role in the process.
ACKNOWLEDGMENTS
The author is deeply grateful to Dr. Jacob F u r t h for his
interest and encouragement a t the outset of this project; also
for his making available the ovaries of hypophysectomized
mice for comparison with the aged ovaries reported on here.
SUMMARY
Weight increase and histological response of ovaries and
uteri from 207 adult, virgin C57BL/&JAX mice to 20 i.u. of
P M S was examined a t ages ranging from 3 to 19 months by a
technique that utilized each animal as its own control. At 14
months o r younger the ovaries responded with a pronounced
and fairly uniform weight increase. With advancing age the
response progressively decreased. I n control ovaries of mice
17 months of age or older a few large graafian folliclels were invariably present, but corpora lutea were absent in over half of
the mice. After PMS injection corpora lutea were formed. At
15 months of age ovarian interstitial cells resembling the “deficiency cells” of hypophysectomized mice were encountered,
and these became progressively more numerous with age.
These cells were rcpaired by PMS in all mice. The uterine
weight increase and histological changes demonstrated that
the PMS injection caused incrcascd ovarian secretion until 17
months of age. a t 17 to 19 months the lack of uterine response
indicated that the exogenous gonadotrophin did not markeclly
influence ovarian secretion.
LTTERATURE CITED
ALBERT,A., R. V. RANDALL,R. A. SnrITH AND C. E. JOHNSON
1956 Urinary
excretion of gonadotrophin as a function of age. Hormones and the
Aging Process. Ed. by E. T. Engle and G . Pincus. Academic Press,
h c . , 49-62.
BOOT,L. M., AND 0. MUIILBOCK 1954 The ovarian function in old mice. Acta
Physiol. et Phsrmscol., 9 : 463-464.
342
JALWES A. GHEEN
1952 Piginented interstitial cclls showing
DEANE,H. W., AND D. W. FAWVRTT
“brown degeneratioii” in the ovariw of old mice. Anat. Rw.,1.73:
239-246.
1937 An aliterior pituitary
EVANS,
H. M., M. E. SIMPSON
AND R. I. PENCHARZ
gonadotrophic fraction ( I C S H ) sprcifically stimulating the interstitial
tissue of testis and ovary. Cold Spring Harbor Symposia on Quantit;itive Biology, 5: 229-240.
E‘EEETE, E. 1953 A inorphological study of the ovaries of virgin mice of right
inbred strains shoiving qriaiititative differences in their hormone producing components. Anat. Rec., 117 : 93-113.
GREEN,J. A. 1954 Ovarian weight and responsiveness t o gonadotrophins during
tlie estrous cycle of mice. Anat Rrc., 120: 714-715.
GREEP, R. O., H. E. VAN DYKE AND B. F. CHOW 1942 Gonadotrophins of tlie
swine pituitary. I. Various biological effects of purified thylakentrin
( F S H ) and pure mrtnkentrin (LH) Endocrinology, 30 : 635-649.
IIOFFYAN,
J. 1931 Tho effect of anterior hypophyseal implants upon senile
ovarics of mice. Am. J. Obstet. and Gynecol., 2%’: 231-238.
HOOKER,
C. W. 1945 A criterion of luteal activity i n the mouse. Antit. Rec.,
93: 333-347.
LEOB,L. 1948 Aging procesxes in the ovaries of mice belonging t o strains differing in the incidence of mamniary carcinoma. Arch. Path., 4 6 : 401-440.
LEOB,L., v. SUNTZEFF
AND E. I,. BURNS 1939 Changes in the nature of the
stroms in vagina, cervix and uterus of the mouse produced by longcontinued injections of estrogen and by advancing age. Am. J. Cancer,
35: 159-174.
Owm, E. 1947 The postnatal development of the reproductive system of the
hamster (Cricetus auratub) and its reactivity t o hormones. Physiol.
ZOO^., 2 0 : 45-67.
ORTIZ,
E. 1955 The relation of advancing age t o reactivity of the reproductive
system i n the female hamster. Anat. Hec., 12%: 517-538.
RENNE:LS,E. G. 1951 Influence of hormones on the histochemistry of ovarian
interstitial tissue in the immature rat. Am. J. ,4nat., 88: 63-108.
ROLLE,G. K., AND H. A. CHARIPPER 1949 The effects of advancing age upon the
histology of the ovary, uterus and vagina of the female golden hamster. Anat. Ree., 106: 281-297.
SELYE,
H., J. B. COLLIPAND D. L. THOMSOX 1933 On the effect of the anterior
pituitary-like hormone on the ovary of the hypophysectomized rat.
Endocrinology, 27 : 494-500.
1956 Seiiile changes i n the oestrous
THUNG,P. J., L.M. BOOTAND 0.YUHLBOCK
cycle and in ovnrinn structure in some inbred strains of mice. Acta.
Endocrinol., 83 : 8-32.
1938 The relation of the pituitary
AND R. KURZROK
WATSON,B. P., P. E. SMITH
gland to the menopause. Am. J. Obstet. and Gynecol., 36: 562-570.
WESTMAN,A. 1934 Rcaktiverung voii setiilcn menxchlirhen Ovarian. Zentralbl.
Gynlkol., 58 : 1090-1096.
WIESNER,B. P. 1932 Experimental study of senescence. Brit. Med. J., 2 :
585-587.
.
AOlNC: M O U S E OVARY
WOLFE,J. M.
WOLF'E,
a.
343
1943 The effcwts of ac1v:uieing age 011 the structure of the anterior
Iiypopliysis a i d ovaries of female rats. Am. J. Anat., 72: 361-383.
M., E. BURACK,
W. LANSING
AND A. \v. WKIGHT 1942 The effscts of
ndvaiiciiig age oii the coiiIiective tissue of the uterus, cervix and vagina
of the rat. Am. J. Aiiat., 7 U : 135-165.
PLATE 1
EXPLANATION OF FIGUElES
2 A section of the m t r c a t e d ovary of a 5-mOllth old mouse. X 42.
3
A section of the treated ovary of a 5-month old mouse; the untreated ovary
of this m o u ~ cis shown in figure 2. There is an abundant folliculsr growth
and corpora lutea formation. X 42.
4 Tlie untreated ovary of a 12-month old mouse. The number of small a i d
medium follicles is less than in the ovary show11 i n figure 2. The lipocbromc
cells are prominent. X 42.
5
Tho untreated ovary of a11 l8-nionth old mouse. A t the bottom of the photograph is a large grwfian follicle. There is very little glandular appearing
interstitial tissue. X 42.
6
The ovary of a 22-1no11th old mouse. The medulla is composed almost eutirely
of lipochrome cells. X 42.
344
PLATE 1
AGING MOUSE OVARY
J A k l E S A. GRl3Y.Y
345
PLATE 2
EXPLANATION OF FIGURES
7
The epitheloid interstitial cells of the u n t r r a t d ovary of a 5-1nont,h old mouse.
X 970.
8
The interstitial cells of the untreated ovary of a 15-month old mouse. A t the
bottom of the photograph are several l i p c h r o m e cells. X 970.
9
The interstitial cells of the cortex of the 22-month old mousc ovary sliowii in
figure 6. X 970.
10
The interstitiid cells of the untrrated ovary of
pare with figure 7. X 970.
13
The interstititd cells of thr treated ovary from the same mouse the ovary slionii
in figure 10 was taken from. Compare with figures 7 arid 10. X 970.
12 A section showing the endometrium of
gestational stroma cells. x 440.
346
R
RII
18-month old iiinusc. Cnm-
19-moiith old mouse. Note the pro-
AGING MOUSE OVARY
PLATE 2
JAMES A. G R E X X
341
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