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Ovulation of ovarian implants in unilaterally ovariectomized rats.

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THE ANATOMICAL RECORD 209:331-336 (1984)
Ovulation of Ovarian Implants in Unilaterally
Ovariectomized Rats
Department of Anatomy, Virginia Commonwealth University, Medical
College of Virginia, Richmond, VA 23298
Subcutaneous autografts of ovarian tissue were made in unilaterally ovariectomized adult rats and were examined by light microscopy a t
various times after implantation. The implants were surrounded by a dense
connective tissue capsule. They were well vascularized and contained follicles
in varying stages of development as well as in different stages of atresia.
Oocytes and fresh corpora lutea indicated that grafts ovulated in the presence
of the in situ ovary but that the number of ovulations and their frequency
were reduced when compared to normal ovaries or ovaries grafted in bilaterally castrated animals. Ovulation results in the formation of a cyst that
contains follicular fluid, the oocyte, and cumulus in the ovarian stroma. Macrophages are associated with the oocyte-cumulus complex but are not prominent in association with the fluid in the cyst. It is suggested that follicular
fluid is retained owing to inefficient resorption mechanisms and that this
coupled with occasional ovulations results in the formation and maintenance
of the large cysts.
For many years grafts or implants of ovarian tissue have been made under a variety of
conditions in efforts to understand the normal function of the ovary and reproductive
system in general (Krohn, 1977). Because of
variations in the age and hormonal condition
of the donor and host, the site of the implant
and other conditions, the results of these procedures, in terms of the ability of the implant
to exhibit normal morphogenesis, have not
always been comparable.
It is generally agreed that unless the implant has vascular drainage to the hepatic
portal circulation, ovaries implanted in castrated females will ovulate and support normal vaginal cyclicity. However, it also has
been stated that implants are unable to function normally in the presence of a n in situ
ovary (Deanesly, 1956; Lane and Markee,
1941). In all ovarian implants, both vasculature and innervation of the tissue are altered
and either of these components may affect
the function of the organ (Burden, 1978; Ellinwood et al., 1978).The failure of the tissue
to respond normally in hemicastrates has
been attributed to either a lack of gonadotrophin stimulation or a failure of the grafted
tissue to reorganize sufficiently to compete
0 1984 ALAN R. LISS. INC.
with the in situ ovary for that stimulation
(Deanesly, 1956; Welschen, 1971). The present study reexamines subcutaneous autografts of ovarian tissue in hemicastrate rats.
Cycling Sprague-Dawley rats approximately 60 days of age were used in this study.
Autografts were made on the day following
estrus by removing the right ovary and implanting one-fourth to one-third of that ovary
into a subcutaneous pocket in the midscapular region. Vaginal smears were examined
daily, and animals were killed at various
times up to 50 days after implantation. In
additional animals, implants were placed in
the kidney capsule. Also for comparison, intact animals were exposed to constant light,
and after 10 days of estrous smears, ovaries
were removed for examination. Such ovaries
are known to contain follicular cysts and be
devoid of corpora lutea (Lawton and
Schwartz, 1967). Implants from 24 animals
were fixed in picric acid-formaldehyde,
embedded in paraffin, and serial-sectioned.
Additionally, in order to visualize erythroReceived November 22, 1983; accepted January 31,1984
cyte-filled vascular channels, tissues of three
animals were fixed in situ by injection of
fixative into the abdominal cavity and the
subcutaneous region containing the implant.
Vibratome sections (50 or 100 pm) of these
tissues were incubated with a diaminobenzidine-hydrogen peroxide solution (Karnovsky,
1967) and examined by light microscopy. In
two additional animals horseradish peroxidase was used as a n indicator of vascular
permeability. Forty-five seconds after intracardiac infusion of 250 m g k g body weight
horseradish peroxidase in 0.2 ml of 0.9% saline, ovarian implants and in situ ovaries
were fixed in place as described above. Peroxidase was visualized by incubation of vibratome sections with diaminobendizinehydrogen peroxide.
In each of the grafted animals compensatory hypertrophy occurred in the in situ
ovary. Ovarian implants were found in the
subcutaneous connective tissue, which
formed a loose adventitia around the implant. There was no mesothelium or bursa
associated with any of the implants. However, each was surrounded by a dense connective tissue capsule (Fig. 1).Such a capsule
was not seen in the in situ ovaries. Figure 2
shows the periphery of a n ovary from a n
animal in persistent estrus induced by constant light. Ovulation did not occur in these
animals, the tunica albuginea did not appear
hypertrophied. In implants, normal-appearing follicles were present, but there was considerable variation in the number and
distribution of different types between implants. (Figs. 1,3). Similar variation also was
seen in the number and types of atretic follicle observed. While theca and interstitial tissue did develop, it did not appear as abundant
Fig. 1. Periphery of implant showing primordial follicle (F), a vascularized stroma (S), the capsule of dense
connective tissue (C), and the subcutaneous connective
tissue (El in which it is imbedded. ~ 4 0 0 .
Fig. 2. Periphery of in situ ovary of a persistent-estrus animal. Macrophages (M) are seen in the antrum of
a n atretic follicle. The tunica albuginea (T)and a portion
of a Graafian follicle (F)also are present. ~ 4 0 0 .
Fig. 3. Section through an implant showing the connection of two locules (L)of a cyst. ~ 3 5 .
Fig. 4. Portion of a cyst containing an oocyte-cumulus
complex (0)and erythrocytes. Wedges mark inner surface of cyst. ~ 4 0 0 .
as it is in nongrafted animals or the in situ
control. The implants appeared to be well
Whether implants were examined a t 15 or
40 days after grafting, the majority of implants contained cystic structures. Six out of
nine implants were cystic a t 15 days, and 11
of 13 were cystic a t 40 days. The cyst (Fig. 3)
was generally a single, thin-walled, fluidfilled structure. Although some ovaries exhibited multilocular structures, examination
of the serial sections proved these cysts to be
continuous. Unilocular cysts 3-4 mm in diameter were found in six implants. The cyst
was generally lined with what appeared to
be a connective tissue layer (Fig. 4). Occasional phagocytic cells were seen a t the periphery, but these were not abundant. They
were frequently associated with the remains
of a n oocyte or oocyte-cumulus complex. In
contrast, within the antra of atretic Graafian
follicles of persistent-estrus animals, numerous macrophages filled with large vacuoles
were found (Fig. 2). The material filling the
cyst in the implant was eosinophilic, homogeneous, and of the same appearance as follicular fluid. In many cysts red blood cells
could be seen, and these frequently were concentrated in one region of the cyst.
In several implants a n oocyte with accompanying cumulus cells was found in the periphery of the cyst (Fig. 4).Out of six animals
killed on estrus 15 days after the implant
was made, four implants had cysts, and two
of these exhibited a single oocyte and newly
formed corpus luteum. When examining animals 40-50 days after implants were made,
nine of 13 had implants with cysts greater
than 1 mm in diameter, of the five cystic
implants examined on estrus, two had a n
oocyte within the cyst and a fresh corpus
luteum associated with it. At no time was
more than one oocyte seen in a cyst. In implants where oocytes were present, the corpus luteum was seen connected with or in
close proximity to the cyst. In Figure 5 the
lumen of a new corpus luteum is seen in
continuity with a newly formed cyst in the
connective tissue. Examination of grafts to
the kidney capsule showed identical results.
All of the implants exhibited a well-vascularized connective tissue stroma. The vascular network was demonstrated by examination of 100-pmvibratome sections that had
been incubated with diaminobenzidine. This
procedure resulted in visualization of a brown
reaction product in red blood cells. In addi-
tion to exhibiting a well-vascularizedstroma,
a wreath of vessels was seen in the theca
surrounding antral follicles (Fig. 6), and the
corpus luteum exhibited its characteristic
capillary bed (Fig. 7). In animals given the
tracer horseradish peroxidase, reaction product was present in all tissue compartments
in both the in situ and implanted ovary
within 45 seconds of infusion. Primary and
secondary follicles exhibited reaction product
adjacent to the oocyte (Fig. 8).
It has been generally stated that ovarian
implants in hemispayed hosts are dormant
until the in situ ovary is removed (Krohn,
1977). The present study, however, indicates
that ovulation does take place within 15 days
of implantation and as late as 50 days after
implantation, although at a much reduced
frequency at both times. Additionally the
present study suggests that follicular fluid
expelled from the follicle is not eliminated
but is retained in the connective tissue
stroma forming a cyst-like structure.
These findings were not a contradiction of
previous studies. In one of the studies most
frequently cited (Deanesly, 1955)there was a
failure to ovulate. However, the donor tissue
for those implants was from animals 7-10
days old; adult tissue was used in the present
study. Welschen (1971) indicated that ovulation occurred in kidney capsule implants of
adult ovaries. He based his conclusion on the
presence of “fresh” corpora lutea and noted
that the numbers of these corpora were significantly less than that found in normal
ovaries and decreased further as time between implantation and sampling decreased.
In that study corpora lutea frequently contained trapped ova. This was considered as
Fig. 5 . Lumen of new corpus luteum (X) in continuity
with forming cyst (C), which contains cumulus cells.
x 100.
Fig. 6 . Vibratome section treated to show erythrocytes in vessels (wedges) surrounding a secondary follicle. Follicle borders a cyst (C) containing free
erythrocytes. x 200.
Fig. 7. Implant corpus luteum treated to demonstrate
erythrocytes filling vascular bed. Center of corpus luteum is marked “C”; periphery is at wedges. X 100.
Fig. 8. An unstained vibratome section of an antral
follicle in an implant following vascular infusion with
horseradish peroxidase. Reaction product seen in all extracellular spaces. x 170.
an indication of inadequate gonadotropin
stimulation; such a condition was not noted
in the present study. Welschen (1971) made
no mention of the fate of the oocyte or follicular fluid. In their study of kidney capsule
grafts, Peckham and Randak (1952)did make
note of cysts, but they identified them as
being of tubal origin. The cysts described in
the present study of both subcutaneous and
kidney capsule grafts were definitely not
tubal in origin.
Cysts similar to those described herein
have been identified in grafts as follicular
cysts (Parkes, 1956).The multilocular nature
of some cysts encourages this assumption.
However, the presence on the days of estrus
of fresh corpora lutea whose hyalin center is
in continuity with the cyst, or corpora which
protrude into the cyst argues against this
being an atretic follicle. One also is less likely
t o find a follicle cyst on the day of estrus with
no follicle cells in its wall than an unfragmented oocyte with a cumulus.
These findings suggest that the normal
surface associations are not necessary for
ovulation or corpus luteum formation. While
intraovarian ovulation has been noted in
normal rats, these involved only preantral
follicles, and no cyst was formed (Spanel-Borowski et al., 1982). The present study suggests that in the graft, the basal lamina, and
follicle cell layer ruptures, spilling the oocyte
and antral fluid into the stroma. The aggregation of follicle lutein cells that follows rupture may be a property of those cells rather
than the result of action of the surrounding
tissue. It is not known whether once a cyst
forms, future ovulations occur at the surface
of that cyst or whether cysts fuse. The dense
connective tissue capsule apparently prevents the loss of this fluid in the loose adventitia surrounding the implant. It is of
additional interest that a thickened connective tissue capsule is characteristic of polycystic ovaries in humans (Blaustein, 1982).
The present study offers no explanation of
the diminished number of follicles that ovulate in a given cycle in the presence of an in
situ ovary. The grafts are well vascularized,
although the origin of these vessels is not
that which normally serves the tissue. Both
corpora lutea and developing follicles have
vascular patterns comparable in appearance
to those of similar structures in normal ovaries. The tracer horseradish peroxidase penetrates t o the oocyte within 45 seconds of
cardiac infusion, suggesting that all com-
partments in the graft have access to hor- umented, our observations of the persistentmone. The innervation is a possible cause for estrus animal suggest a continuous turnover
the impaired function of the graft. While one of follicles, and the presence of vacuole-filled
might assume that the tissue is reinnervated macrophages in the absence of granulosa
as it becomes revascularized, there is no di- cells suggests that macrophages play a role
rect evidence for this in the present study. in resorption. In the implant, macrophages
However, even if reinnervation takes place, are observed, but they are associated with
as is the case with the vasculature, the source the oocyte and cumulus, not the follicular
of the reinnervation would not be that which fluid. If they are responsible for elimination
normally serves the ovary.
of fluid in normal ovaries their failure to
The presence of the cysts, particularly in function in the implant is of interest. The
grafts where very few follicles remain, sug- reasons for the failure of antral fluid resorpgests that the implanted ovary is incapable tion also may be significant to studies of
of disposing of this follicular fluid. There are pathological conditions in humans, dairy catseveral factors that support the assumption tle, and swine, where follicle cysts also are
that fluid is retained for more than one cycle. retained and related to sterility (Nalbandov,
Based on the number of corpora lutea and 1976).
ovulated oocytes in a given graft, it can be
stated that it is most unlikely that more than
one follicle ovulates in a given cycle. Also Blaustein, A. (1982) Non-neoplastic cysts of the ovary.
In: Pathology of the Female Genital Tract. A. Blaubased on the infrequency of finding oocytes
stein, ed. Springer-Verlag, New York, pp. 449-463.
in a cyst and the small number of old corpora Burden,
H.W. (1978) Ovarian innervation. In: The Verlutea in a graft, it is concluded that the imtebrate Ovary. R.E. Jones, ed. Plenum Press, New York,
plant does not ovulate during each vaginal
pp. 615-638.
cycle. A final reason to suspect retention of Deanesly, R. (1956) Cyclic function in ovarian grafts. J.
Endocrinol., 13t211-220.
the follicular fluid is that a cyst 2-4 mm in Ellinwood,
W.E., T.M. Nett, and G.D. Niswender (1978)
diameter would require contribution from
Ovarian vasculature: Structure and function. In: The
several follicles to form it.
Vertebrate Ovary. R.E. Jones, ed. Plenum Press, New
York, pp. 583-614.
It is obvious that the thick capsule of conJ.W., and C.H. Sawyer (1950) A 24-hour periodnective tissue confines the antral fluid to the Everett,
icity in the “LH-release apparatus” of female rats,
implant, but that does not explain the failure
disclosed by barbiturate sedation. Endocrinology,
of phagocytic elements to remove this mate47~98-218.
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tic ovary may prevent ovulation. While such Krohn, P.L. (1977)Transplantation of the ovary. In: The
follicles do not rupture, the resultant ovary
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contains numerous follicle cysts (Blaustein,
C.E., and J.E. Markee (1941) Responses of ovarian
1982).There are additional conditions in lab- Lane,
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oratory animals where ovulation fails and
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method of their resorption has not been doc-
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ovarian, implants, ovulation, unilateral, rats, ovariectomized
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