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Development and maintenance of a polycystic condition in ovaries autotransplanted under the kidney capsule.

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THE ANATOMICAL RECORD 225118-123 (1989)
Development and Maintenance of a Polycystic
Condition in Ovaries Autotransplanted Under the
Kidney Capsule
Department of Anatomy, McGill Uniuersity, Montreal, P.Q. H3A 2B2, Canada
A single injection of estradiol valerate (EV) produces a polycystic
ovarian (PCO) condition in the rat. The development of the PCO condition coincides with alterations in the endogenous plasma gonadotropin patterns, suggesting that PCO may be a response to abnormal gonadotropin stimulation. Other
factors, however, such a s direct autonomic innervation, also contribute significantly to the regulation of the ovary and could be important in generating and/or
maintaining PCO. We have, therefore, removed and autotransplanted one ovary in
each of eight rats under the capsule of the ipsilateral kidney, thus totally disrupting its innervation. The animals were injected with EV and both ovaries of each
animal were examined 8 weeks later. In a second group of animals, we induced the
PCO condition, autotransplanted one polycystic ovary in each animal under the
kidney capsule, and examined the ovaries 2 weeks later. In both groups the autotransplanted ovaries exhibited the full range of polycystic morphology, a s did the
intact ovary in each animal. We conclude that since a major perturbation in innervation affects neither the development nor the maintenance of PCO, autonomic
innervation does not play a crucial role in this disorder.
Anovulatory acyclicity characterized by ovaries containing multiple cystic follicles is a reproductive anomaly that occurs in a wide range of mammalian species
(including the human) under a variety of circumstances. It occurs in the laboratory rat as a result of age
(Aschheim, 1976), neonatal androgen treatment (Gorski, 1971), and treatment in adulthood with dihydroepiandrosterone (DHEA), (Parker and Mahesh, 19761,
estradiol valerate (EV) (Brawer et al., 19781, antibody
to LHRH (Fraser and Baker, 1978; Popkin et al., 19831,
or thiouracil and hCG (Copmann and Adams, 1981).
The condition can also be produced by anterior hypothalamic deafferentation (Halasz, 19691, constant light
exposure (Daane and Parlow, 1971), and implantation
of estradiol benzoate into the anterior hypothalamus
(Kawakami and Vissuvan, 1979).
The histologically definitive polycystic condition
(PCO) does not appear to reflect intrinsic ovarian pathology. The polycystic ovaries in both the neonatally
androgenized and EV-induced models ovulate in response to a n LHRH challenge (Hemmings et al., 1983).
In the DHEA-treated and constant-light-induced models, the ovaries revert to normal function and histologic
appearance following the cessation of treatment. Moreover, ovulatory cyclicity and normal ovarian histology
can be restored in EV-induced PCO by the removal of
one polycystic ovary (i.e., hemiovariectomy) (Farookhi
et al., 1985).
Since we have identified hypothalamic and pituitary
impairments in animals with PCO (Brawer e t al., 1986;
Simard et al., 1987; Carrier et al., 19891, it seemed
most likely that the polycystic condition reflects the
response of a normal ovary to abnormal gonadotropic
stimulation. This hypothesis was strengthened by the
observation that the development of polycystic ovaries
in the EV-treated model is preceded by the emergence
of a specific abnormal episodic plasma pattern of LH
(McCarthy et al., 1986). This pattern is invariably
present in animals with established PCO (Grosser et
al., 1987).
Although it seems likely t h a t the plasma LH pattern
plays a role in PCO, there are other factors, such as
direct autonomic innervation, which contribute to the
regulation of ovarian function and that may also be
significant in generating and maintaining PCO.
The rat ovary is richly supplied with both sympathetic (Lawrence and Burden, 1980) and parasympathetic (Burden et al., 1978) innervation. In addition, a
beta adrenergic mechanism is involved in steroid-induced increases in ovarian blood flow (Varga et al.,
1985) a s well as in the facilitation of LH-induced steroidogenesis in hypertrophied thecal and secondary interstitial cells (Erickson et al., 1985). Moreover, these
cells have been shown to receive direct adrenergic innervation (Erickson et al., 1985).
In view of the prominence of hypertrophied thecal
and secondary interstitial cells in the polycystic ovary
(Brawer et al., 1989) we have asked whether direct
innervation andlor any other local factor is essential to
the production or maintenance of polycystic ovaries.
Our approach has been to disrupt the innervation to
the ovary and to alter the local environment by remov-
Received December 6, 1988; accepted February 21, 1989.
ing the ovaries and implanting them beneath the kidney capsule. We can then determine whether EV
treatment will produce the PCO morphology in transplanted normal ovaries and whether the PCO morphology is maintained in transplanted polycystic ovaries.
TABLE 1. Variety of follicular structures occurring in
initially normal ovaries autotransplanted under the
kidney capsule and examined 8 weeks after an
injection of estradiol valeratel
Rat No. Precystic Cystic Atretic 2" Healthy 2" luteum
Female Wistar rats (Charles River, Quebec) were
maintained under conditions of controlled light (lights
on between 0500 and 1900 hours) and temperature
(22°C). They were fed Purina rat chow and permitted to
drink water freely. Estrous cycles prior to and after
treatment were monitored by daily examination of vaginal smears.
Experimental Groups
Two groups of animals exhibiting at least two consecutive 4-day estrous cycles were used in this study.
One group of eight animals underwent unilateral excision and reimplantation of the ovary under the kidney capsule on the left side only. The contralateral
ovary was left in place. Immediately following surgery,
each animal was injected intramuscularly with 2 mg of
EV (Delestrogen, E.R. Squibb and Sons, Princeton,
NJ). The ovaries were fixed by perfusion 8 weeks later
to allow for the complete development of the PCO condition (Brawer et al., 1978, 1986).
Each of seven animals in the second group was injected with EV and left for 8 weeks time, during which
the ovaries became polycystic. One cystic ovary of each
animal was then implanted under the capsule of the
ipsilateral kidney, while the remaining ovary was left
in place. Two weeks after transplantation, the ovaries
were fixed by perfusion and examined.
Rats were anesthetized with ether, and a 1 cm-wide
incision was made approximately 2 cm below the last
rib in the midclavicular region. The ovary was removed, stripped of adherent connective tissue, and
washed in saline. The kidney was then localized
through the same incision, and a small aperture was
made in the kidney capsule into which the ovary was
then inserted.
Perfusion and Preparation of Tissues
Animals were anesthetized with 0.9 ml of urethane
per 100 g body weight, and the descending aorta was
exposed through a ventral midline incision. The descending aorta was clamped immediately above the bifurcation. An 18-gauge needle was introduced into the
aorta, above the clamp, and perfusion with lactated
Ringer's solution was begun. A second clamp was
placed on the aorta just below the level of the liver and
the renal vein of the kidney containing the ovarian
transplant was cut. When the kidney appeared uniformly blanched, the perfusate was switched to fixative. This fixative consisted of 1% formaldehyde
and 1% glutaraldehyde solution in 0.12 M phosphate
Both ovaries were removed and cleaned of adherent
connective tissue. Each ovary was then cut into halves
or thirds and these blocks of tissue were left in fixative
Follicle tvDe
'The numbers in the column on the left each designate an individual
animal. The categories across the top each indicate a type of follicular
structure. The occurrence of a specific type of follicular structure in
the ovary of an individual animal is designated by + ; the absence of
that structure is indicated by -. The first symbol refers to the control
(intact) ovary and the second to the autotransplanted ovary. + / would, therefore, indicate that the intact ovary, but not the autotransplanted ovary in a particular rat, has the designated structure.
overnight. The following day, the tissues were postfixed in a solution of 1% Os04, and 1.5% ferrous cyanide for 4 hours. The tissues were then dehydrated in
graduated concentrations of methanol, left in a 1:l
mixture of propylene oxide overnight, and embedded in
Epon; 2-3 pm-thick sections were cut on a Reichert
microtome and stained with toluidine blue.
The transplanted ovaries in all animals of both experimental groups exhibited the typical polycystic morphology that characterized the nontransplanted (control) ovary. Both the transplanted and control ovary of
each animal expressed the full complement of morphologic features that exemplify the polycystic condition in
the EV-treated model (Hemmings et al., 1983; Brawer
et al., 1986,1989).These included the presence of large
precystic and cystic follicles, numerous atretic secondary follicles (as well a s a few normal secondary follicles), absence of corpora lutea, and a n extensive stroma
composed of hypertrophied secondary interstitial cells.
In addition, occasional very large healthy follicles described previously as type I11 large follicular structures
(Brawer et al., 1989) were also seen in both control and
transplanted ovaries. These results are summarized in
Tables 1 and 2.
The histological appearance of the cysts and precystic follicles was archetypic of the EV-induced PCO condition. The cystic follicle consisted of a large antrum
surrounded by a highly attenuated membrana granulosa (Figs. 1-3). This layer was often no more than one
cell thick. Occasional large macrophagelike cells appeared on the antral surface of the membrana granulosa (Fig. 3). Precystic follicles resembled cysts except
for irregular, occasionally extensive patches of degenerate granulosa cells occurring at variable intervals
along the perimeter of the antrum (Figs. 4, 5).
The thecal cells in cystic, precystic, and atretic secondary follicles were generally large, polygonal in
shape, and filled with lipid ghosts (Figs. 2, 3). Occasional small fusiform thecal cells typical of normal fol-
TABLE 2. Polycystic ovaries autotransplanted under
the kidney capsule and examined 2 weeks later'
Follicle type
Rat No. Precvstic Cvstic Atretic 2" Healthy 2" luteum
'Results are expressed as the presence ( + 1 or absence (-) of the indicated follicular structure in the intact/autotransplanted ovaries of
each animal.
licles were also occasionally seen in cystic and precystic
Large clusters of hypertrophied, lipid-containing interstitial cells occurred throughout the ovaries (Fig. 2).
In addition to the cystic, precystic, and atretic follicles,
a unique follicular structure which we have called the
type I11 large follicular structure (Brawer et al., 1989)
was occasionally observed. Cystic and precystic follicles have been designated as type I and I1 large follicular structures (Brawer et al., 1989). The type I11 large
follicular structure was characterized by a very large
antrum surrounded by an extremely thick layer of
healthy granulosa cells (Figs. 3, 6). The theca is composed of a diffuse layer of small fusiform cells, characteristic of healthy follicles. Occasional large polygonal
Fig. 2. Cyst wall and secondary interstitial cells in 2-week ovarian
transplant. The membrana granulosa (arrowheads) is one to two cells
thick. Immediately beneath the membrana granulosa is a basement
membrane beneath which is the hypertrophied thecal cell layer. The
thecal cells are large, polygonal, and often filled with lipid ghosts. The
left quarter of the field is occupied by secondary interstitial cells (arrows) similar in appearance to the cystic thecal cells. x 800.
cells appeared scattered throughout the thecal cell
layer (Fig. 3). The type I11 large follicular structure
differs from a preovulatory follicle in that it is larger
and in that occasional mitotic figures occur in the perimural granulosa region. Furthermore, the absence of
corpora lutea in these ovaries indicates that the type
I11 large follicular structure is not ovulatory.
Fig. 1. Cystic follicle in 8-week ovarian transplant. The large antrum is surrounded by a single layer of granulosa cells over most of
the perimeter. In the upper right quadrant of the cyst the membrana
granulosa is slightly thicker (arrowheads). x 130.
The polycystic ovary in the estradiol-treated rat exhibits a unique constellation of morphologic features.
This includes absence of corpora lutea, a dearth of
healthy secondary follicles, although atretic secondary
follicles are commonly encountered, and an abundance
of secondary interstitial tissue (Brawer et al., 1986,
1989). In addition, there are three types of follicular
structures unique to the polycystic ovary. These are the
cystic and precystic follicles and the type I11 large follicular structure. This latter structure is of Darticular
interest because of its size and the healthy nbrmal appearance Of the granulosa and
Moreover, it
is the only follicular structure in the PolYcYstic ovary to
exhibit LH-binding sites on the perimural granulosa
planted under the kidney capsule sustain normal estrous cyclicity and exhibit the typical range of follicular structures as well as normally appearing corpora
lutea (Del Castillo, 1928; Vivien, 1948; Harris and
Eakin, 1949; Krohn, 1959, 1962). Although some cyst
formation as well as other expressions of pathology
have been reported following transplant to spleen or
subcutaneous connective tissue at very long postimplantation intervals (months) (Krohn, 1962; Parkes,
19561, no abnormal structures occur in ovaries implanted beneath the kidney capsule (see Krohn, 1962,
for review). The appearance of pathologic changes in
old ovarian grafts in spleen or connective tissue is attributed to inadequate vascularization.
Although the transplant procedure obviously causes
a total disruption of ovarian innervation, there is little
information on the extent to which the transplanted
ovaries may be reinnervated. Hill (1949), using the
protargol stain, failed to observe any reinnervation of
adult ovaries autotransplanted t o the spleen in mice.
On the other hand, Jacobowitz and Laties (1970),using
induced catecholamine fluorescence, observed reinnervation of ovarian tissue transplanted to the anterior
chamber of the eye in the cat. Although fluorescent
fibers were observed 35 days after transplant, no fibers
were observed at 15 days. Moreover, the extent of
adrenergic innervation in the transplanted ovaries (at
35 or 75 days) was considerably less than that seen in
the normal ovary.
In the present study, it is possible that reinnervation
fig. 3. Segments of a cystic follicle and a type 111 large follicular
structure in 8-week ovarian transplant. The antrum of the cystic follicle (C) is surrounded by a single layer of granulosa cells. Peripheral
to the basement membrane is the thecal layer composed mostly of
large, lipid-filled polygonal cells. The antrum of the type III large
follicular structure (T) is surrounded by multiple layers of healthy
granulosa cells. The theca consists of scattered cells many of which
are small and fusiform. x 400.
cells as well as on thecal cells (Brawer et al., 1989).The
occurrence of the type I11 large follicular structure indicates that the polycystic ovary engages in a unique
form of folliculogenesis in addition to the degenerative
processes that produce atretic secondary follicles, the
abundance of secondary interstitial tissue, and cysts.
This study demonstrates that the complete spectrum
of histological characteristics that define the polycystic
ovary can be produced and maintained in ovaries
transplanted under the kidney capsule. In each animal,
the transplanted ovary exhibited the same morphological features as the intact (control) ovary. This suggests
that neither direct innervation t o the ovary nor any
other local factor need play a significant role in the
generation of PCO. This interpretation is based on two
assumptions. The first is that the transplant procedure
does not itself induce the PolYcYstic condition, and the
second is that the transplantation results in a significant disruption in ovarian innervation.
The first of these assumpt~onsis well supported by
the literature, representing Over 40 Years of experience
with ovarian transplants. Healthy ovaries trans-
Fig. 4. Segments ofa cystic and precystic follicle in 2-week ovarian
transplant. The antrum of the cyst ( C )is surrounded by a n attenuated
irregular membrana granulosa. The thecal cells are typically large
and contain numerous lipid ghosts. Although the theca of the precystic follicle (PI resembles that of cysts, the membrana granulosa contains numerous cells many of which show clear signs of degeneration.
x 4,000.
Fig. 5. Precystic follicle in 8-week nontransplanted (control side)
ovary. In the top of the field is a typical follicle (PI. This particular
section includes the degenerating ovum. To the immediate left is a
segment of a cystic follicle (C).The bottom of the field is occupied by
atretic follicles (a) and clusters of secondary interstitial cells (arrowheads). x 52.
occurs to some extent in the long-term (8-weekI-transplanted ovaries. It is unlikely, however, that significant reinnervation occurs in the l k d a y transplants.
In any event we have shown that even discrete temporary perturbations in the neuroendocrine axis of rats
with PCO rapidly produce major alterations in ovarian
histology (Hemmings et al., 1983; Carriere et al.,
1989). Clearly, a significant disruption in ovarian innervation has no such effect, suggesting that innervation plays a minor, if any, role in PCO.
The authors gratefully acknowledge the technical assistance of Ms. Dalia Chen. This work was supported
by a n operating grant to J. Brawer from the Medical
Research Council of Canada.
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development, autotransplants, maintenance, kidney, capsules, conditions, ovaries, polycystic
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