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Molecular Reproduction Research Laboratory, Clinical Research Institute of Montréal (N.D., M.R.S.), Montréal, Québec, Canada H2W 1R7; Department of Medicine, Division of Experimental Medicine, McGill University (N.D., M.R.S.), Montréal, Canada H3A IA3; and Department of Pathology, St Mary’s Hospital of McGill University (I.R.) H3T 1M5, and Department of Medicine, Université de Montréal (M.R.S.), Montréal, Québec, Canada H3T 1J4
Address all correspondence and requests for reprints to: Dr. M. Ram Sairam, Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montréal, Québec, Canada H2W 1R7. E-mail: sairamm{at}ircm.qc.ca
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In general, the only risk factor to be clearly recognized for ovarian
cancer is the inherited germline mutations in the BRCA1 or BRCA2 genes,
but uninterrupted ovulation has been postulated to be a contributing
factor (4). The molecular changes that induce various
ovarian tumors are ill defined at present. There is also no reliable
and general diagnostic screening method available for ovarian cancer.
Some studies have linked granulosa cell tumors of the ovary to the
actions of pituitary gonadotropins, FSH, and LH. A causal connection is
often suspected, because as the concentrations of gonadotropins rise in
the early menopausal years the incidence of ovarian cancer also
increases, to become quite prominent during postmenopause
(5). Some investigations in animals have shown a relation
between chronic and abnormal gonadotropin exposure and the development
of granulosa cell tumors. For instance, targeted overexpression of the
LH ß-subunit in transgenic mice causing high circulating levels of LH
at an early age results in polycystic ovaries and ovarian tumors
inducing infertility in female mice (6). Mice deficient in
the inhibin 
-subunit gene, a partner in the heterodimeric ovarian
protein inhibin belonging to the TGFß family, also develop granulosa
and/or Sertoli cell tumors (7). Besides a possible tumor
suppressor role for inhibin , the accompanying rise in activin
levels that elevate FSH in the circulation may contribute to tumor
formation. Whether the propensity of the ovary to progress toward a
cancerous state increases after stimulation by gonadotropins remains
highly controversial. Thus, arguments that incessant ovulation as
induced during infertility treatments may or may not predispose the
ovaries to cancer are mired in extreme controversy (8, 9).
Some reports that found no abnormal elevation of serum LH and FSH
levels of granulosa tumor patients (10, 11, 12) at the time of
diagnosis suggest that the hypothesis of excess gonadotropins driving
tumorigenesis might be simplistic.
Upon binding to structurally related, but distinct, receptors in the ovary, the two gonadotropins FSH and LH cause a cascade of events leading to stimulation of gonadal growth, differentiation, and steroidogenesis (13, 14). As ovarian function requires a perfect interaction between FSH and its receptor, disruption in any manner is likely to cause various reproductive deficits. For instance, an inactivating point mutation in extracellular domain of the FSH receptor gene converting Ala189 to Val causes absolute infertility due to primary amenorrhea in Finnish women (15). This mutation, however, is not present in infertile women of other backgrounds. Recently a case of compound heterozygotic mutation of the FSH receptor gene provoking a partial loss of function of the receptor has been described in a woman with secondary amenorrhea (16).
Given these considerations, it was logical to assume that the FSH receptor might be a good candidate for activating mutations that could alter the growth and function of ovarian tumors. Reports of finding binding sites for FSH, albeit low in cells derived from human granulosa cell and thecal cell tumors (17, 18), have been interpreted as suggesting that FSH may influence the growth and activity of sex cord-stromal tumors. Although several studies have looked for mutations of the FSH receptor that may lead to tumor initiation, none has been found (19). The single report by Gromoll et al. (20) that the Asp567Gly substitution in a man led to constitutive activation also remains in question (21).
To understand the biology of FSH receptor-dependent processes in the ovary, we have produced mice lacking FSH receptor(s) (22) by using homologous recombination. The resulting mutant female or FORKO mice are sterile despite very high levels of FSH (22). In continuing our investigations on these mutants, we have now found for the first time that essentially all mutant females analyzed after 12 months of age demonstrated macro- or microscopic evidence of ovarian tumors with various pathologies. This strongly suggests that removal of the FSH receptor induces major perturbations in the gonadotropin signal transduction pathway(s) and internal milieu of the ovary leading to the development of tumors in mutants.
Based on the high incidence of tumor appearance in the ovary of aging FORKO mice, we propose that the lack of the beneficial effects of FSH receptor signaling in association with other confounding factors might predispose the ovarian structures to assume a variety of pathologies. In fact, in the dysfunctional ovary after menopause in women there is a natural loss of functional FSH receptor. We believe that a better understanding of the molecular changes that underlie ovarian pathogenesis during aging could be gained by studying models such as the FORKO mouse.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histological analysis
Animals were exsanguinated during ether anesthesia, and all
internal organs were examined for visual signs of abnormalities. The
ovaries were cleaned of extraneous tissue for weighing and then fixed
in 10% formalin at room temperature for 16 h. All tissues were
embedded in paraffin, sectioned, and stained with hematoxylin and eosin
according to standard histological procedures that we have used in
previous studies (24). Classification of ovarian pathology
including tumor type was performed according to the descriptions
provided in the atlas on basic histopathology (25) and
pathology of the female genital tract (26).
Immunohistochemistry
Immunohistochemistry of the following antigens was performed
according to established procedure (24) using antibodies
from different sources. Thin sections were processed for immunostaining
using the ImmunoCruz Staining System (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) according to the supplier’s instructions.
The 3ß-hydroxysteroid dehydrogenase (3ßHSD) antibody was provided
by Dr. A. H. Payne (Stanford University, Palo Alto, CA). This
rabbit antiserum produced against the recombinant mouse 3ßHSD1
protein was used at a dilution of 1:750. Dr. Payne also supplied the
antibody to porcine P450c17 enzyme that was used at 1:600 dilution.
Purified antibody against human placental aromatase (used at 1:500
dilution) was a gift from Dr. N. Harada (Toyoake, Japan). An antibody
to the N-terminal peptide of inhibin -subunit was given to us by Dr.
B. D. Schanbacher (formerly of USDA, Clay Center, NE). This reacts
with all forms of inhibin and was used at a dilution of 1:1000. Goat
antibodies to the transcription factor GATA 4 and Müllerian
inhibiting substance (MIS) obtained from Santa Cruz Biotechnology, Inc., were used at 1:200 dilution. The
corresponding rabbit second antibody was used for subsequent
processing. Antigen retrieval procedure was performed for localization
of inhibin, GATA 4, and MIS.
Plasma and pituitary gonadotropins
Plasma LH and FSH were measured by RIA using kits provided by
the National Hormone and Pituitary Program (courtesy of Dr. A. F.
Parlow, University of California-Los Angeles, Torrance, CA). As none of
the FORKO females show cycles, plasma samples were collected at random,
but usually in the morning before noon. Although the wild-type mice
continue to cycle, we did not make any distinction with regard to the
stage of the cycle for collection of samples. These were also obtained
randomly. For estimating pituitary hormone content by the same RIA,
each frozen tissue was homogenized with an extraction buffer (0.2
M glycine-NaOH, pH 9.0) containing 5 mM
benzamidine hydrochloride. The supernatant obtained after
centrifugation at 13,000 x g was frozen until used for
RIA. The inter- and intraassay variations were 5–8% for both RIAs.
The hormone contents are expressed in terms of the respective rat
pituitary hormone standards provided in the kit.
Steroid hormone profile
The two major ovarian steroid hormones (estradiol and
testosterone) in serum samples of 12-month-old females (wild-type and
FORKO) were estimated by solid phase RIAs (24) using
commercially available kits (Diagnostic Products, Los
Angeles, CA). Where pertinent, statistical significance was calculated
for all comparisons by performing one-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Circulating testosterone levels were higher in the 1-yr-old mutants
(
2-fold) compared with those in the +/+ females of comparable age;
however, they were not as exaggerated as in the 3-month-old (Table 1)
or 4- to 5-month-old FORKO mice (24).
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). At 12+
months of age the plasma levels (per ml) of both FSH and LH in FORKO
mice had increased about 3- to 4-fold compared to those in age-matched
wild-type females. Although plasma LH in 12- to 15-month-old FORKO
females declined significantly (P < 0.01) from the 7
month level, it was still higher (P < 0.05) than that
in the corresponding age group +/+ mice. The pituitary FSH content was
higher at 3 months and continuously increased with age in FORKO mice as
well as in +/+ animals. The highest FSH levels were found in pituitary
of 12- to 15-month-old mice. The pituitary content of LH that became
different in FORKO only at the seventh month remained high at 1 yr.
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Between 3–5 months of age all null female mutants examined
demonstrated the characteristic underdeveloped phenotype, with
follicular development blocked before antrum formation, without any
corpora lutea. Thus, there was no ovulation in FORKO mice, in agreement
with our previous reports (22, 24). In those few follicles
that had any semblance of antrum there was atresia (not shown).
Hyperplasia was noted in the interstitial tissue in the middle of the
FORKO ovary (Fig. 2A). Another
interesting characteristic of the 3- to 5-month-old FORKO ovary was the
presence of large islands of polygonal cells with a round central
nucleus and abundant cytoplasm-containing lipid droplets (Fig. 2B).
These cells with hyperplastic appearance have the features of
luteinized cells that are steroidogenically active, consistent with the
high testosterone values in the circulation at 3–5 months
(24) (Table 1
). In contrast to the FORKO mice, 3- to
5-month-old wild-type mice do not have such exaggerated luteinization
(Fig. 2, C and D).
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Morphological examination also revealed that ovaries containing tumors had solid or solid and cystic consistency and yellowish-white color. Two FORKO females had huge balloon-like right ovaries filled with a large amount of liquid. Ovaries from two null mutants remained atrophic and did not have any macroscopic signs of tumorigenesis or cysts.
Pathology and histological appearance
The histological examination of FORKO ovaries revealed the
presence of tumors in 23 mutants. Among these we were able to identify
sex cord-stromal tumors of the Sertoli-Leydig cell type based on their
appearance (Fig. 2, E–H). In most cases, as shown in the example (Fig. 2F), the small ovarian component was overshadowed by the tumor. These
tumors showed a variable proportion of Sertoli and Leydig type cells.
Some were characterized by an equal admixture of tubular structures
(Sertoli cells) and vacuolated plump stromal cells (Leydig cells; Fig. 2G). Others were almost devoid of the tubular element, consisting
almost entirely of clusters of Leydig type cells (Fig. 2H).
Occasionally there was a focal microfollicular change in the neoplastic
cells, reminiscent of granulosa cells, a feature sometimes described in
Sertoli-Leydig cell tumors of the human ovary (26).
The cysts encountered in a few of the ovaries were lined by a columnar
epithelium with occasional vacuoles, with subjacent stroma containing
vacuolated plump cells (Fig. 3, A and B).
The epithelial cells ranged from a single cell-lining population to a
stratified cell population thrown into papillary folds. One cyst showed
a transition from a simple columnar epithelium to a stratified
epithelium with a papillary configuration (Fig. 3, C and D). It is
interesting to note that these cysts resemble the retiform type of
Sertoli-Leydig cell tumors used in describing the pathology of some
ovarian cancer (2).
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, E and
F). All of these structures had disappeared. However, there was
widespread luteinization of the interstitial tissue with the
characteristic indication of steroidogenically active cells. We also
observed multiple areas of calcification within the nonaffected
ovaries, possibly caused by destruction of follicular structures by
this age (Fig. 3F).
Body weight and cachexia
As reported recently (24) all FORKO females develop
obese tendencies at 3 months of age. This condition increased
progressively (Fig. 4) up to about 10
months, after which there was a steady decline. At the time of death,
the tumor-bearing animals were definitely smaller, with a reduction in
body weight by about 50% compared with their peak at 10 months. By
about 15 months they were leaner than wild-type littermates, exhibiting
signs of cachexia that is normally associated with advanced stages of
neoplasia. In addition, there were signs of splenomegaly and anemia, as
indicated by the pale extremities of the mutants. These changes were
not evident in any of the control wild-type mice.
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) many months after ovulation had
ceased, indicating that loss of one FSH receptor allele might also
create imbalances sufficient to produce pathological changes upon
aging.
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peptide antiserum), the
transcription factor GATA 4, and MIS. Representative staining patterns
of ovaries of aging FORKO and wild-type mice are shown in Figs. 6 and 7. There was an intense staining
for 3ßHSD in cells resembling Leydig cells in neoplastic ovaries
containing Sertoli-Leydig cell tumors (arrows in Fig. 6B)
compared with that in +/+ ovary of the same age (Fig. 6A). Similarly,
P450c17 was localized in these cells (Fig. 6D). In contrast to this, it
may be noted that the tubular structures lined with Sertoli-like cells
did not express 3ßHSD (Fig. 6B, asterisks) or the P450c17
(Fig. 6D, asterisks), indicating differences in
steroidogenic activities of these two types of cells in the tumors.
Aromatase staining was weak in all FORKO ovaries (Fig. 6F) compared
with that in wild-type mice (Fig. 6E), suggesting that expression of
this enzyme had decreased in the aging FORKO ovary. Interestingly, this
is in contrast to the situation previously reported for the 3- to
5-month-old FORKO ovary. At this age there was no difference in mRNA
(RT-PCR) or protein, as verified by Western blot analysis of the
ovaries of FORKO and wild-type mice (24). In ovaries
collected at random from 1-yr-old wild-type females there were many
antral follicles with granulosa cells that intensely stained for the
inhibin peptide that was tested in our study (Fig. 6G). In older
FORKO ovaries (Fig. 6H) there were no such structures, although some
weakly stained cells were present. When we compared the total content
of aromatase and inhibin by Western blots, both were drastically
reduced in the aging FORKO ovaries (data not shown).
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, A and B), whereas the expression of
this protein was much stronger in the aging FORKO ovary. In the
1-yr-old atrophic FORKO ovary that had not yet developed tumors there
were multiple foci of MIS expression. From the example shown (Fig. 7, C
and D), intense and localized expression of MIS in what was previously
a degenerating follicle is clearly evident. In the FORKO ovary with
tumor (Fig. 7, E and F) there was a more generalized expression of MIS.
Like MIS, the transcription factor GATA 4 was also selectively
expressed only in the granulosa cells of a few large follicles (Fig. 7, G and H) of the 1-yr-old +/+ ovary. In Fig. 7, I and J, are depicted
two examples of the altered expression of this factor in the aging
FORKO ovaries with tumors. In both cases expression was confined to
Sertoli-type cells that were present in the tubular-like
structures. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the consequences of the disruption of the FSH receptor signaling on reproductive function as well as long-term implications for changes in gonadal structures, we generated homozygous mutant mice that lack the FSH receptor. As noted previously (22), these animals are infertile and have no reproductive cycles (24, 27). Our results recorded here clearly suggest that ablation of the FSH receptor causes development of gonadal tumors in aging female mutants. Considering the normal life span of about 3 yr for mice, the ovarian tumors were discovered quite early, by about 1 yr. The exact stage at which the tumors begin to appear has not been pinpointed, as this requires more detailed examination of the mutants between 5 and 12 months. It is quite likely that the hormonal imbalances may have altered the expression of regulatory genes in a manner that led to the appearance of large tumors. It was interesting to find that the loss of FSH receptor caused various types of Sertoli-Leydig cell tumors and cysts. The retiform type tumors described in women (2) also occur in FORKO mice. As these tumors are apparently very aggressive in women compared with nonretiform Sertoli-Leydig cell types, the prognosis becomes very poor (2). Similarly, FORKO mice with such tumors were also among those that became anemic and lost body weight.
Confinement of the tumors to the right ovary in a majority of the aging FORKO females is interesting and worthy of note. In a minority of the mutants tumors were also induced in the left or both ovaries. We are not aware of reports of such selective localization of ovarian tumors in other types of transgenic mice (6, 7, 28, 29) or in women with ovarian cancer (4). Although we have no rational explanation at the present time for this intriguing anatomical confinement of the tumor in our mutant mice, attention might be drawn to reports that note differences in the sympathetic innervation of the right and left ovary (30). Whether these had any bearing on tumor localization in the mutants can only be a matter of speculation. Further studies are needed to explore this question and other possibilities.
The potential involvement of pituitary gonadotropins, LH and FSH, in
ovarian tumorigenesis has been under investigation for a long time. It
has been noted that the incidence of ovarian neoplasms rises around the
onset of menopause, accompanied by high levels of plasma gonadotropins
(4, 31). Although a causal link between ovarian
stimulation in women undergoing in vitro fertilization and
tumor development remains controversial (4, 8, 9, 32, 33, 34),
the precise mechanisms remain unknown. Experimental studies in
transgenic animals tend to draw an apparent correlation between serum
gonadotropins and tumor development due to overexpression of LH
(6). That this does not occur in mice of all genotypes
further suggests that there are other modifying genetic factors that
could be important in particular backgrounds (29),
situations that might also occur in women of different races. The 2- or
3-fold elevation of FSH levels in inhibin -deficient mice that
causes gonadal stromal tumors (7, 35) has been suggested
to stimulate the development and/or progression of the tumors. On the
contrary, attempts to induce granulosa cell tumors in BALB/c mice with
exogenous PMSG, a biological surrogate hormone for pituitary FSH,
have been unsuccessful (36). Exogenous gonadotropin
treatment for 180 d of hypogonadal (hpg/hpg) mice,
deficient in GnRH and lacking serum FSH and LH, also did not cause
ovarian tumors (37). In addition, the recent report in
transgenic mice overexpressing FSH has shown that elevated FSH levels
alone do not directly cause gonadal tumors (38).
As sex cord-stromal tumors in women are known to have steroidogenic capabilities and apparent binding sites for LH and FSH (17, 18), several different groups have actually searched for mutations in the FSH receptor gene in granulosa cell tumors to test the implications of such changes in ovarian tumor induction. As these attempts have not been successful (19, 39), other possibilities must be considered to explain the findings. Our observations reported here in the FORKO mouse model suggest that it may be the loss of receptor function that triggers the abnormality leading to certain ovarian tumors. As nearly all (92%) of our aging FORKO mice developed ovarian pathology, it is reasonable for us to propose a beneficial role of the FSH receptor’s signaling function in protecting the ovary from developing tumors. Therefore, it would be instructive to identify these protective (tumor suppressors?) genes that are influenced by FSH receptor signaling. A lack of such effects, as happens in the postmenopausal state in women, may predispose the ovary to tumors at later stages in life, especially if other confounding factors are also present. Indeed, infertility has been suggested as a significant risk factor for various gynecological cancers (40). It should be noted again that in the FORKO mice ovulation is completely suppressed (22, 24, 27). Other findings, such as increased angiogenesis after loss of ovarian function (41) in menopause, may also support our proposition of the FSH receptor system’s protective effect on the ovary. The appearance of ovarian pathology in our +/- female mice that follows early reproductive senescence is noteworthy and requires more detailed investigations, as this experimental paradigm may indeed duplicate menopausal conditions in some manner. Whether there was a complete loss of FSH receptor function at this stage in the +/- female has not yet been ascertained. Nevertheless, these mice could provide an interesting model to examine many issues related to menopause and the appearance of ovarian tumors.
It seems likely that removing the FSH receptor from the scene of action
causes alterations in several important genes. For example, the plasma
level of inhibin that showed some tendency to decrease at 4 months
as reported in one study (27) could be further reduced
upon aging, as shown by our immunohistochemical analysis of the FORKO
ovary at 1 yr. Although we have not measured the dimeric inhibin in the
present study, it is reasonable to assume that levels would be low in
the circulation, as total expression itself is extremely weak in the
FORKO ovary. This would in some way be consistent with the report of
loss of inhibin -subunit gene in transgenic mice, strongly
predisposing the ovary to granulosa cell tumors (7).
Interestingly, as double homozygous mutant mice that lack both FSHß
and inhibin -subunit develop slow-growing ovarian neoplasm
(38), tumor suppressors of the TGFß family may also be
important.
The mechanisms underlying tumor development in aging FORKO females are
not clear at present. The elevated LH levels in the FORKO mice could
contribute to ovarian pathology, as was apparently the case in
LH-overexpressing transgenic mice (6). Even though
circulating FSH remained high at all times, this hormone could not
function in FORKO mice, as all receptors had been ablated. Other
changes that include parameters such as increased concentration of
plasma testosterone and the presence of large islands of polygonal
cells with abundant cytoplasm containing lipid droplets and high
intensity of 3ßHSD and P450c17 (features of steroidogenic cells) in
the ovarian stroma may also have played a part in the induction of
pathology. Ovarian androgens synthesized in response to LH action may
be among the signals for induction of tumors (42), and
FORKO mice have high levels of circulating testosterone at 3–5 months
(24) and also at the time of tumor detection. However,
because of the fact that the +/- mouse that does not show such high
levels of testosterone (24) also develops ovarian
pathology later in life after reproductive senescence, we may infer
that other mechanisms must also be involved. Regardless of the
underlying mechanisms it is remarkable that the granulosa cells of the
mutant ovary acquire the capacity to reorganize themselves into
tubular-like structures. The immunohistochemical evidence of expression
of two Sertoli cell markers, namely MIS and GATA 4, supports these
conclusions. In this context it is interesting to point out that a
similar redifferentiation of the granulosa cells into Sertoli cells has
been reported in female mice that lacked both ER and ERß
(43). Although these animals have high estrogen levels,
the hormone is nonfunctional due to lack of the two deleted nuclear
receptors. However, in our FORKO mutants the circulating estrogen
ligand level became extremely low (or absent), but the expression of
both ER and ERß was unaltered at the age of 3–5 months.
In conclusion, we observed that the loss of FSH receptor signaling results in ovarian tumor development of aging mutant mice. Our results provide the first in vivo evidence that the complete elimination of FSH receptor is involved in the induction of gonadal tumorigenesis. We believe that further studies in this animal model may provide valuable insights into the molecular mechanisms of this insidious disease in women. The induction of such varied pathology caused by the loss of a single receptor might provide a new perspective in understanding why ovarian tumors are precipitated during menopause in women. Based on the present studies we hypothesize that the loss of FSH receptor expression/signaling that occurs naturally in all women at the time of menopause predisposes the ovary to neoplasia, a condition that might be exacerbated in combination with other confounding (genetic or environmental?) factors.
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Acknowledgments |
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Footnotes |
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Abbreviations: FORKO, Follitropin receptor knockout; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; MIS, Müllerian inhibiting substance.
Received March 16, 2001.
Accepted for publication April 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Inhibin is a tumour-suppressor gene with
gonadal specificity in mice. Nature 360:313–319[CrossRef][Medline]
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