Sexually Dimorphic Roles of Steroid Hormone Receptor Signaling in Gonadal Tumorigenesis
Kathleen H. Burns,
Julio E. Agno,
Lei Chen,
Bisong Haupt,
Samuel C. Ogbonna,
Kenneth S. Korach and
Martin M. Matzuk
Department of Pathology (K.H.B., J.E.A., L.C., B.H., S.C.O., M.M.M.), Department of Molecular and Human Genetics (K.H.B., M.M.M.), and Department of Molecular and Cellular Biology (M.M.M.), Baylor College of Medicine, Houston, Texas 77030; and National Institute of Environmental Health Sciences (K.S.K.), National Institutes of Health, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Martin M. Matzuk, M.D., Ph.D. Stuart A. Wallace Chair and Professor, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu.
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ABSTRACT
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Sex steroids control cellular phenotypes by binding to receptor proteins that in turn regulate downstream gene expression. They are important tropic factors in hormonally responsive tissues and have been implicated in the pathogenesis of both benign proliferations and malignancies at some of these sites. Knockout mice lacking inhibins,
:ß heterodimeric peptide hormones of the TGFß superfamily, develop gonadal tumors that produce sex steroids and depend on pituitary gonadotropin hormones. To better appreciate how sex steroid receptor signaling pathways contribute to the loss of granulosa/Sertoli cell proliferation in the ovary and testis of inhibin
(Inh
) knockout mice, we are using both pharmacologic and genetic approaches. Roles of androgens in testicular tumor development have been investigated in our previous studies using double-mutant mice lacking inhibins and carrying the null testicular feminization (tfm) mutation of the androgen receptor. Herein, we report that androgens also participate in the development of ovarian tumors, as tumor development is forestalled in mice treated with flutamide, a nonsteroidal inhibitor of androgen actions. Additionally, we generated double-mutant mice lacking estrogen receptor
(ER
) and Inh
or ERß and Inh
, as well as triple-mutant mice lacking ER
, ERß, and Inh
to determine the effects of individual and combined ER signaling pathways on tumor development. Although estrogens may have proliferative effects during follicle development and are important in specifying the granulosa cell phenotype, ER
and ERß signaling are not essential for timely granulosa cell tumor development or granulosa cell-like morphological features in ovarian tumors. However, redundant ER signaling through ER
and ERß in males is critical for testicular tumor formation, as triple-knockout, but not double-knockout, males are protected from early Sertoli cell tumorigenesis and death. Together, these studies indicate important and sexually dimorphic functions of estrogens and androgens in tumor development in this mouse model and indicate, for the first time, overlapping functions of ER
and ERß in Sertoli cell pathophysiology.
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INTRODUCTION
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STEROIDS, RETINOIDS, AND other lipid-soluble hormones can directly enter cells to complex with nuclear receptors, which in turn regulate gene expression and silencing. These hormones are key mediators of cellular function and differentiation in many tissues (1). Insights into these functions have been gained in part by studies in tfm mutant mice, which carry a naturally occurring mutation of the androgen receptor (considered below) (2, 3), and knockout mice lacking one or both estrogen receptor (ER) subtypes (ER
and ERß) (4, 5, 6). ER
knockout females are infertile due to a block in ovarian folliculogenesis and the development of cystic ovaries in response to gonadotropin hyperstimulation (7, 8). ER
knockout males are also infertile and show a progressive atrophy in the seminiferous epithelium caused by fluid reabsorption defects in the efferent ductules. ERß knockout mice display more subtle reproductive deficits, including female subfertility owing to developing follicle atresia and decreased responsiveness to the gonadotropins (5). Although partial fertility is preserved in both sexes in the absence of ERß, this ER is highly expressed in ovarian granulosa cells and testicular Sertoli cells, indicating to us that ERß signaling may be especially pertinent to pathologies in this lineage. Interestingly, double-mutant females lacking ER
and ERß develop a postnatal sex reversal wherein granulosa cells express genes and take on morphological features characteristic of Sertoli cells (6). In contrast, ERß knockout males are fertile, and the phenotype of ER
, ERß double-knockout males is not distinct from that of the ER
knockout. These phenotypes are reviewed in Ref. 9 . In addition to their roles in reproductive physiology, androgens and estrogens are important trophic factors contributing to the development of cancers in hormonally responsive tissues. For example, associations between androgen and estrogen signaling and malignancies of the prostate, breast, and ovarian surface epithelium have been well documented, and antagonists of steroid production and response pathways have been employed therapeutically (10).
Gonadotropin hormones (FSH and LH) produced by the anterior pituitary stimulate steroidogenesis in the ovary and testes, producing estrogens and androgens, which regulate gametogenesis and signal to distant target tissues. In addition to steroid hormone feedback, gonadotropin production is subject to regulation by inhibins and activins, peptide hormones produced in the gonads and the pituitary (reviewed in Refs. 11 and 12). Inhibins are heterodimeric members of the TGFß superfamily made up of a unique
-subunit and a ß-subunit that is shared with the activins. Inhibins normally suppress FSH production and gonadal tumorigenesis. Both male and female mice homozygous for a targeted null mutation at the
-subunit-encoding locus demonstrate elevated levels of FSH and develop gonadotropin-dependent tumors of the granulosa/Sertoli cell lineage (13, 14). These tumors occur in mixed C57BL/6J/129S6/SvEv genetic background, as well as in C57BL/6J congenic inbred lines (Matzuk, M. M., unpublished data). The gonadal tumors secrete sex steroids and activin dimers, and the activin production causes a cachexia-like wasting syndrome, which ultimately kills the knockout mice (11, 15, 16). In several respects, granulosa/Sertoli cell tumors in the inhibin
(Inh
) knockout mouse model phenocopy naturally occurring human sex cord-stromal cancers. Granulosa/Sertoli cell tumors comprise a small portion of ovarian and testicular neoplasms, but are clinically important because of their potential for invasiveness, recurrence, and endocrinological sequelae. Patients with sex cord-stromal tumors of granulosa/Sertoli cell origin commonly present with symptoms of steroid hormone secretion, and tumor inhibin expression has been used as a marker of disease progression (17). Roles of gonadotropin hormones, sex steroids, and their antagonists in the pathogenesis and clinical management of these malignancies are being explored.
Our laboratory previously demonstrated that androgens are modifiers of testicular tumorigenesis in Inh
knockout mice (18). For these studies, Inh
mutant mice were crossed with mice carrying the loss-of-function testicular feminization mutation (tfm) of the androgen receptor. Males lacking inhibins and functional androgen receptor lived significantly longer than their Inh
knockout (Inh
-/-) littermates and developed tumors that were less hemorrhagic. Although hemorrhage is normally a prominent feature of all testicular tumors in Inh
knockouts, 65% of tumors from Inh
-/-, tfm males had only minimal or focal sites of hemorrhage. Because the androgen receptor locus is X-linked and tfm mutant males are completely sterile with defects in both masculinization and testicular function, it was not possible to generate female double-mutant mice and test the roles of androgens in ovarian tumorigenesis by the same strategy. Investigations of Beamer and colleagues (19, 20, 21), however, have linked androgens and androgen responsiveness to the pathophysiology of naturally occurring granulosa cell ovarian tumors in SWR mice.
In this manuscript, we describe studies that test the roles of androgens in ovarian tumor development by treating Inh
knockout mice with a pharmacological antiandrogen. In addition, we have investigated the roles of estrogens in tumor development in both sexes by intercrossing the Inh
mutant mice with mice harboring targeted mutations of the ERs to produce double- and triple-mutant mice. Together, these studies provide insights into the sexually dimorphic functions of estrogens and androgens in tumor development in the absence of inhibins.
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RESULTS
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Flutamide Treatment Delays, But Does Not Avert, Ovarian Tumor Development
To define the roles of androgens in ovarian tumor development in the absence of inhibins, we treated Inh
knockout females with flutamide, a nonsteroidal androgen antagonist. Time-released flutamide pellets were implanted in 14 experimental mice at 23 wk of age, and the mice were monitored weekly thereafter for signs of tumor development. The flutamide treatment had a subtle, but statistically significant, effect on the survival of Inh
-/- mice (Fig. 1A
). Females in the treatment group had a 50% survival at 1617 wk of age, as opposed to the Inh
-/- untreated mice, which show a 50% survival at 1314 wk of age. By 24 wk of age, 95% of the flutamide-treated Inh
-/- mice succumbed to ovarian tumor development, forestalled modestly from 18 wk of age in the untreated Inh
-/- control mice.
We previously reported that androgens are trophic factors in testicular tumor development and that androgen receptor signaling may contribute to hemorrhage within these tumors (18). In a majority of the ovarian tumors recovered from flutamide-treated mice, the tumors were solid, well-circumscribed masses with little evidence of gross hemorrhage (Fig. 1B
). Histologically, these tumors demonstrated typical features of the sex cord stromal tumors previously described for this mouse model, with a characteristic mix of granulosa cell-like and Sertoli cell-like elements (Fig. 1C
).
To better appreciate how flutamide treatment might influence tumor aggressiveness in Inh
knockouts, we analyzed circulating FSH levels in dying females and found that those receiving flutamide treatment had lower FSH (15.4 ± 3.5 ng/ml) as compared with controls (24.9 ± 7.4 ng/ml). Serum LH and expression of activin ßA and activin ßB subunit mRNAs in tumor samples as assessed by Northern blot analysis were not influenced by flutamide treatment (data not shown).
ER
-/-, Inh
-/- Double Knockouts of Both Sexes Develop Gonadal Tumors
ER
signaling is critical for fertility in both sexes and plays an important role in females in suppressing gonadotropin hormone production at the level of the pituitary (8). To assess the contributions of ER
signaling pathways to the pathology of tumorigenesis in this model, we bred the Inh
knockout line to mice carrying a targeted mutation of the ER
locus (4). We found that both ovarian and testicular tumors developed and progressed in double knockouts in the absence of inhibins and ER
. Although the survival of double-mutant males was comparable to single knockouts lacking Inh
alone (Fig. 2A
), the survival of double-knockout females was compromised (Fig. 2B
). Half of double-knockout females survived to between 9 and 10 wk of age, as compared with Inh
-/- females, in which 50% survival is between 12 and 13 wk. All ER
-/-, Inh
-/- females followed for this study died by 11 wk of age, a time point in which the majority of Inh
-/- females are still alive. At 10 wk of age, the few surviving ER
-/-, Inh
-/- double-knockout females were all showing overt signs of a wasting syndrome, which we associate with tumor progression (see below). These females had an average total body weight of 11.5 ± 1.0 g, as compared with surviving Inh
-/- mice, which average 18.0 ± 0.7 g, and wild-type and ER
-/- mice, which average in excess of 21 g at 10 wk of age (21.6 ± 0.6 g and 22.5 ± 0.5 g, respectively).

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Fig. 2. Tumor Development in ER -/-, Inh -/- Double-Knockout Mice
A and B, Survival curves for ER -/- single-knockout, Inh -/- single-knockout, and double-knockout mice of each sex (n = 10 and 9 for ER -/-, Inh -/- males and females, respectively). The leftward shift in the survival curve of females lacking both ER and inhibins is significant by Students t test (P < 0.01). C, Histology of a testicular tumor from an 8-wk-old ER -/-, Inh -/- male. This portion of the tumor is filled with diffuse granulosa-like cells with pale oval nuclei without grooves. These cells are arranged haphazardly within well delineated insular nests. A point of hemorrhage is marked (*). Magnification, x20. D, The boundary of an ovarian tumor from a 10-wk-old ER -/-, Inh -/- mouse. Several abnormal tubule-like follicle-like structures like the one shown (arrow) are confined to the periphery of the ovarian tumor. This field is largely filled with a solid tubule pattern of tumor cells with off-centered nuclei and cytoplasm marked by lipid vacuolization (**). Magnification, x20. E, Serum estrogen levels for wild-type (WT); Inh -/- knockout; ER -/- knockout; and ER -/-, Inh -/- double-knockout females (n = 10, 14, 9, and 9, respectively) and males (n = 5, 13, 13, and 9, respectively). Inh -/- mice were killed for serum collection with signs of advanced tumor development, and age-matched ER -/- and wild-type mice were used as controls. Values are given as mean ± SEM of the estrogen measurements (pg/ml). Notice the different y-axis scales in these two graphs.
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In all cases, gonadal tumors occurred bilaterally and disrupted essentially the entirety of the testes or ovaries by the time of death. Testicular and ovarian tumors occur in both Inh
-/- mice and ER
-/-, Inh
-/- mice with 100% penetrance and, in this study, were the only cause of death in these populations. Histologically, tumors of ER
-/-, Inh
-/- males (Fig. 2C
) and females (Fig. 2D
) phenocopy features reported for Inh
single knockouts. Tumors in both sexes were found to be mixtures of solid tumor foci, sites of hemorrhage, and Sertoli cell-like cells arranged in tubules (Fig. 2
, C and D).
We measured serum estradiol in double-knockout and Inh
-/- mice with advanced tumors, as well as adult wild-type or ER
-/- mice to begin to evaluate effects of the null ER mutation on the endocrine axis. We found marked elevation of serum estrogens in ER
-/- females, as well as in Inh
-/- single knockouts and ER
-/-, Inh
-/- double knockouts of both sexes with terminal disease progression as compared with wild-type mice (Fig. 2E
). In contrast to females, elevations in male serum estradiol levels seemed to reflect only effects of the Inh
mutation rather than combined effects of this and the ER
mutation (Fig. 2E
).
We suspected that lack of ER
would contribute to ovarian tumorigenesis through elevations in serum gonadotropins, but found lower levels of FSH (9.3 ± 2.0 ng/ml) and LH (0.07 ng/ml) in terminal double-knockout females than in Inh
single knockouts (24.9 ± 7.4 ng/ml and 0.36 ± 0.18 for FSH and LH, respectively). To assess the levels of gonadotropins at earlier ages, we killed 4- to 6-wk-old females of each genotype. At this age, ER
-/-, Inh
-/- double knockouts had already developed large, bilateral ovarian tumors grossly larger than Inh
-/- females. Their FSH and LH levels were 21.9 ± 1.5 ng/ml and 0.31 ± 0.05 ng/ml, respectively, as compared with Inh
knockout mice (35.2 ± 7.5 ng/ml FSH and 0.67 ± 0.18 ng/ml LH). Thus, levels of both gonadotropin hormones decline at the conclusion of the natural course of disease, and they are suppressed earlier and to a greater extent than Inh
knockout females also lacking ER
.
Advanced Disease in ER
-/-, Inh
-/- Double Knockouts Is Characterized by a Wasting Syndrome
Tumor development in ER
-/-, Inh
-/- double knockouts was associated with a cachexia-like wasting syndrome like that previously described for Inh
-/- mice (15). The progression of weight loss and development of kyphoscoliosis in both male and female double-knockout mice were indicative of advanced disease and short survival. Average weights for female wild type, ER
-/-, Inh
-/-, and double-knockout mice followed as part of this study are depicted in Fig. 3A
as a function of their age. Mice lacking inhibins achieve lower total body weights and do not maintain weight gain as compared with controls that are wild type or heterozygous at the Inh
locus. In females, this wasting syndrome was more pronounced in double knockouts lacking both ER
and Inh
.

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Fig. 3. ER -/-, Inh -/- Double Knockouts Develop a Rapid Cancer-Associated Wasting Syndrome
A, The line graph illustrates changes in average weekly body weight measurements for females of different genotypes (wild type, ER -/-, Inh -/-, and ER , Inh double knockout; n = 9, 9, 18, and 10 for these genotypes, respectively). B, Northern blot analysis of activin ßA (top) and activin ßB (middle) subunit mRNAs in pooled wild-type (WT) ovaries and unpooled ovarian tumors from Inh -/- and ER -/-, Inh -/- mice. The bottom panel shows a Gapd loading control. C, Liver histology from a 22-wk-old ER -/- female. Large, regularly shaped hepatocytes are the predominant cell type with pale cytoplasm lightened by glycogen content. Central venous structures have smooth luminal surfaces, and red blood cells are seen within these spaces. D, Liver histology recovered from a 10-wk-old ER -/-, Inh -/- male. Darkened irregularly shaped and rounding hepatocytes surround the central vein, the boundaries of which are marked with lymphocytic infiltration (arrow). E, The junction of the squamous forestomach and glandular stomach in an 8-wk-old ER -/- male. Stratified squamous epithelium of the forestomach (arrow) confronts the epithelium of the glandular stomach (*) at the limiting ridge. The majority of epithelial cells (70%) within this portion of the fundic glandular stomach are large, eosinophilc parietal cells. F, The limiting ridge of a 6-wk-old ER -/-, Inh -/- male mouse. The squamous epithelium is marked with an arrow. There is striking mucosal atrophy within the glandular epithelium (**), and there is marked depletion of the parietal cell population. For comparison, this picture is taken at the same magnification as panel E.
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High levels of circulating activins in Inh
-/- mice are key to the pathogenesis of the cachexia-like wasting syndrome. Northern blot analysis of activin ßA and ßB subunit mRNAs in ovarian tumors of ER
-/-, Inh
-/- double knockouts reveals expression levels comparable to those seen in Inh
-/- single-knockout tumors (Fig. 3B
). Several histological hallmarks of disease progression have been identified; characteristic findings include hepatocellular necrosis in the liver and mucosal atrophy and depletion of parietal cells in the glandular stomach (15, 22). We found these features in livers and stomachs of ER
-/-, Inh
-/- double-knockout mice succumbing to gonadal tumor development, but not in ER
-/- mice that were heterozygous or wild type at the Inh
locus (Fig. 3
, CF).
ERß-/-, Inh
-/- Double Knockouts of Both Sexes Develop Gonadal Tumors
To investigate the functions of ERß signaling pathways in tumor development, we produced double-mutant mice lacking Inh
and ERß. Like Inh
-/- controls, double-knockout ERß-/-, Inh
-/- mice exhibit severe wasting and succumb to gonadal tumors in all cases. We found no indication that ERß alone is a genetic modifier of tumor development, as survival curves for double-knockout mice overlay those for Inh
-/- mice in both sexes (Fig. 4
, A and B).
There were no differences detected in serum estrogen levels or gonadotropin concentrations in either sex as a function of the loss of ERß in the absence of inhibins at the final stages of gonadal tumor development. Both gross and histological analyses of tumor morphology in ERß-/-, Inh
-/- double knockouts reveal features essentially identical to those previously described for Inh
-/- mice and described above for ER
-/-, Inh
-/- mice. In addition, histological hallmarks of disease progression in the stomachs and livers are evident in ERß-/-, Inh
-/- double knockouts (data not shown).
ER
-/-, ERß-/-, Inh
-/- Triple-Knockout Females Succumb to Early Onset, Aggressive Ovarian Tumors
Female ER
-/-, ERß-/- double knockouts exhibit an ovarian phenotype that is unique when compared with single knockouts lacking either ER, indicating that some separate but contributory functions exist between the two receptors (6). To study the propensity for ovarian tumor development in the absence of inhibins and ERs, we crossed triple heterozygous ER
+/-, ERß+/-, Inh
+/- mice to generate seven triple-knockout females. Triple-knockout females were recovered at approximately the expected Mendelian ratio of 1:128. We followed six of these mice through the natural progression of disease and found that they succumbed to aggressive bilateral ovarian tumors, with the majority of mice dying between 4 and 6 wk of age (Fig. 5A
). All of the triple-knockout female mice were dead by 9 wk of age. Average body weights of triple-knockout females never surpassed 12.2 g, whereas double-knockout females lacking both ER
and ERß weighed an average of 30 g by 9 wk of age.

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Fig. 5. Triple-Knockout Females Succumb Early to Ovarian Tumors with Granulosa Cell-Like Features
A, We followed six ER -/-, ERß-/-, Inh -/- triple-knockout (TKO) females until the final stages of advanced ovarian tumor development. This graph depicts their percent survival over time as compared with Inh -/- single-knockout females and females lacking ER and ERß, but wild type or heterozygous at the Inh locus. The 50% survival of triple-knockout females is at 5 wk, and all triple-knockout females included in this study were dead by 9 wk of age. Incidentally, large bilateral ovarian tumors were found in one ER -/-, ERß-/-, Inh +/- female near death at 28 wk of age. B, Gross tumor specimen recovered from a triple-knockout female killed at between 4 and 5 wk of age. The tumors are solid masses of cells with well delineated sites of hemorrhage (arrow). Below the tumor specimens, two ovaries from an age-matched wild-type mouse are shown for comparison. C, Histology of an ER -/-, ERß-/-, Inh +/- at 20 wk of age. Some multilayered, secondary follicles are seen with rounded granulosa-like cells (open arrow). Within the central stromal portion of the ovary, nests of Sertoli-like cells with lipid-filled vacuolated cytoplasm can be seen (solid arrow). DF, Histology of the triple-knockout ovarian tumor photographed in panel B above. D, At the tumor periphery, a degenerating oocyte can be seen (open arrow); adjacent is a tubule-like structure filled with Sertoli-like cells (solid arrow). The majority of the tumor sample is comprised of less well-differentiated cells like those seen at the rightmost edge of the micrograph. E, A blood-filled space near the tumor surface is indicated (*); it is lined by tubule-like cell formations (solid arrow). F, Within the central region of the tumor, haphazardly arranged, mitotically active granulosa-like cells predominate. Occasionally, these circumscribe small cavities to form microfollicular structures (arrows), characteristic of granulosa cell tumors.
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Grossly, ovarian tumors in triple-knockout females were very similar; all tumors were solid, nodular pale-colored masses with scattered hemorrhagic foci of limited extent (Fig. 5B
). Northern blot analysis of activin subunit mRNAs demonstrated expression levels comparable to Inh
-/- single-knockout ovarian tumors (data not shown). Tumor development was associated with elevated serum FSH levels near death (31.5 ng/ml ± 7.6 ng/ml), as was found with tumorigenesis in Inh
-/- single-knockout mice (24.9 ng/ml ± 7.4 ng/ml, compared with 7.9 ng/ml ± 2.5 ng/ml for wild-type females). One triple-knockout female was killed at 3 wk of age, and ovarian tissue was already completely replaced by advanced bilateral tumors at this early time point (data not shown).
ER Signaling Is Not Required for the Granulosa Cell Tumor Phenotype
It has been reported that double-knockout females lacking ER
and ERß exhibit a transdifferentiation of ovarian follicles to tubule-like structures lined by cells reminiscent of Sertoli cells of the testis, both by morphological and gene expression criteria (6). We found these tubule-like structures in ovaries of adult ER
-/-, ERß-/-, Inh
+/- females (Fig. 5C
). Questioning whether ER signaling pathways would be important in establishing the granulosa cell phenotype in the context of ovarian tumors, we examined tumor samples from triple-knockout females for their histological features. We found essentially the same mix of granulosa cell/Sertoli cell characteristics in these specimens as previously described for the sex cord-stromal tumors that develop in mice lacking inhibins alone (Fig. 5
, DF). Thus, despite the critical physiological functions of ERs in specifying the morphology of ovarian granulosa cells, granulosa cell tumors maintain their histological hallmarks even in the absence of both ER
and ERß.
ER
-/-, ERß-/-, Inh
-/- Triple-Knockout Males Are Protected from Early Tumor Development
Like ER
-/-, ERß-/-, Inh
-/- females, triple-knockout males were viable and were represented at approximately the expected Mendelian ratio. To evaluate the functions of estrogens in Sertoli cell tumorigenesis, we monitored ER
-/-, ERß-/-, Inh
-/- males for testicular tumor development. Surprisingly, the most severely affected triple-knockout male had a survival of 19 wk, as compared with Inh
-/- males, which all die of testicular tumors by this age. The majority of triple-knockout males lived longer than 27 wk of age (Fig. 6A
), representing a striking extension over the Inh
-/- lifespan in which 95% are dead by 12 wk of age. These findings indicated to us that overlapping or redundant actions of ER
and ERß are important in promoting advanced tumorigenesis in males in the absence of inhibins.

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Fig. 6. Triple-Knockout Males Are Protected from Testicular Tumor Development
A, Survival curve of ER -/-, ERß-/-, Inh -/- males. The survival of triple knockouts is plotted in comparison with Inh -/- single knockouts (n = 20) and ER -/-, ERß-/- mice wild type or heterozygous at the Inh locus (n = 10). B, An intratubular testicular lesion from a 7-wk-old triple knockout is shown (arrow). Magnification, x10. At this early age, there is relatively modest disruption of the seminiferous epithelium in adjacent tubules, and there is evidence of spermatogenesis. C, Testicular tumors (right) forming in a 26-wk-old cachexic ER -/-, ERß-/-, Inh -/- male. Testes of an age-matched, wild-type male are pictured for comparison (left). The upper testis has several tumor foci visible grossly, as well as numerous sites of hemorrhage. In contrast, the tumor mass in the contralateral testis (arrow) does not extend completely throughout the specimen, and the testis is not enlarged compared with wild type. DF, Histology of the testicular tumors shown in panel C. D, A solid mass of tumor cells can be seen (solid arrow) surrounded by reactive stroma. Several seminiferous tubules can be seen adjacent to this tumor focus (open arrow). These are dilated like those seen in ER -/- knockout males and show evidence of germ cell depletion and degeneration, but not Sertoli cell hyperplasia. E, Well-delineated nests of neoplastic cells are seen against the capsule of the testicular tumor (solid arrow) near large sites of circumscribed hemorrhage (*). F, Within the central region of the tumor, granulosa-like cells form the mass of the tumor; several mitotic figures are seen among them (arrows). Pools of hemorrhage are also visible (*).
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To evaluate earlier stages of tumor development, we killed one triple knockout at 7 wk, and one at 12 wk of age, and found no gross evidence of tumor formation in the former, and modest tumor foci in the latter. Histologically, the testicular tissue of the 7-wk-old triple knockout demonstrated a single site of intratubular Sertoli cell proliferation where cells filled the tubule lumen (Fig. 6B
). These two time points, 7 and 12 wk of age, represent periods in which Inh
-/- males already uniformly demonstrate extensive testicular tumors (13). Another indication of the delayed nature of tumor development in ER
-/-, ERß-/-, Inh
-/- males is their ability to gain and maintain body weight. Whereas average weights of ER
-/-, ERß-/-, Inh
-/- and Inh
-/- males are comparable at 7 wk of age (20.7 g and 20.1 g, respectively), by 10 wk of age these measurements have diverged such that ER
-/-, ERß-/-, Inh
-/- males weigh considerably more than surviving Inh
-/- males (24.1 g and 15.4 g, respectively).
When triple-knockout males did develop wasting syndrome signs and were killed, we found that tumor foci did not encompass the entirety of both testes, as is always the case in Inh
knockouts alone (Fig. 6C
). In these ER
-/-, ERß-/-, Inh
-/- males, large regions of the seminiferous epithelium were observed with minimal Sertoli cell hyperplasia; however, tubules were dilated and there was a loss of germ cells consistent with the loss of ER
. Within tumor regions, typical histological features of sex cord stromal tumors can be appreciated (Fig. 6
, E and F).
Gonadal Tumors Express Cyclin D2 mRNA Independent of ER Status
Estrogen responsiveness in breast cancers has been associated with amplification and overexpression of cyclin D1 (23). Because up-regulation of cyclin D2 mRNA accompanies tumorigenesis, and cyclin D2 and ERs are both critical for early and aggressive tumor development in males (24 24A ), we hypothesized that ER
/ERß signaling pathways may direct Sertoli cell cyclin D2 expression. To test this, we performed Northern blot analysis to quantify the accumulation of cyclin D2 mRNA in testicular tissue from mice that were either wild type, ER
-/-, ERß-/-, or ER
-/-, ERß-/- for the ER loci and either wild type or null at the Inh
locus. In all cases, Inh
-/- mice were killed with advanced testicular tumors, and Inh
wild-type littermates were killed as age-matched controls. We found that cyclin D2 mRNA is 8- to 12-fold higher in testicular tumor samples as compared with testicular samples from mice with functional inhibins, irrespective of the ER genotype (Fig. 7
). Thus, although there may be a period of time early in tumor development when ER signaling contributes to the formation of tumors cells with high levels of cyclin D2, ultimately Sertoli cells in ER
-/-, ERß-/-, Inh
-/- mice retain the ability to up-regulate this cyclin during the course of tumorigenesis without functional ERs.
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DISCUSSION
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Steroid hormones and other aspects of the endocrine environment have been implicated in the progression of diverse types of cancer. In the present study, we focused on androgen and estrogen receptor signaling pathways in gonadal tumorigenesis. Previous studies from our laboratory showed that the androgen receptor can function as a genetic modifier of testicular tumor development, as males lacking inhibins and carrying the loss-of-function tfm mutation at the androgen receptor locus lived longer and had less hemorrhagic tumors as compared with their Inh
-/- counterparts with normal androgen receptor function (18). Breeding females null for the androgen receptor was not feasible because the tfm mutation is carried on the X chromosome and cannot be inherited paternally, although recently created Cre/LoxP conditional androgen receptor mutations promise to remedy this (25). We chose instead to evaluate the role of androgens in ovarian tumorigenesis using a pharmacological approach. We found that treatment with a nonsteroidal androgen antagonist, flutamide, prolonged the lifespan of Inh
-/- females and that a number of these tumors had minimal hemorrhage. In these ways, flutamide treatment in females was able to affect (but not prevent) tumor development, much like the loss-of-function androgen receptor mutation in males. Affymetrix (Santa Clara, CA) GeneChip expression profiling studies in our laboratory indicate that androgen receptor is expressed in ovarian tumors that develop in Inh
knockout females (average difference values of 1907 and 1482, respectively, for two ovarian tumor samples) (Burns, K. H., G. E. Owens, J. H. Nilson, and M. M. Matzuk, unpublished data). Based on this observation, we postulate that effects of flutamide treatment are partially, if not principally, due to direct action on tumor cells (see below). Some flutamide effects, however, may be mediated through effects on the central endocrine axis suppressing gonadotropin production (26).
Although androgens seem to have trophic effects on ovarian tumor development, ER signaling pathways may have protective effects on gonadal tumor development in females. This is based on the finding that the ovarian tumor phenotype in the absence of inhibins is exacerbated with homozygous mutations of the ER
locus, or the loss of both ER
and ERß. GeneChip studies to evaluate the transcriptomes of Inh
-/- ovarian tumors indicate that the tumors express ERß mRNA (average difference values of 4080 and 4178, respectively, for two ovarian tumor samples), but not ER
, suggesting that ER
effects on tumor progression (although perhaps not initiation) are mediated centrally (Burns, K. H., G. E., Owens, J. H., Nilson, and M. M. Matzuk, unpublished data). Although we were unable to demonstrate augmented increases in gonadotropin hormones in the terminal stages of tumorigenesis when these hormone levels are falling, it seems plausible that in the absence of ER
, disturbances in feedback mechanisms that normally suppress anterior pituitary production of FSH and LH result in a hypergonadotropism that exacerbates tumor progression. Anterior pituitary hormones are crucial to tumor development in the absence of inhibins and promote this pathology, in part, by elevating steroid and activin production (14), as well as enhancing cyclin D2 expression within tumor cells (27). Indeed, high levels of LH alone are sufficient as a primary cause of granulosa cell tumors in some genetic backgrounds (28). There is compelling evidence to connect elevated gonadotropin levels with loss of estrogen signaling. Absence of ER
alone, for example, leads to elevated serum gonadotropins causing a cystic ovarian pathology without targeted mutation of the Inh
locus (9). Presumably, such a scenario contributes to elevations in circulating estrogens in the absence of ER
, which is more pronounced in ER
-/-, Inh
-/- double-knockout females than Inh
-/- counterparts. Currently, it is unclear whether elevated serum estrogens can directly affect biological functions without ER
and ERß and contribute to pathology in these mice. Interestingly, combined actions of ER
and ERß and local effects of inhibins are critical for specifying the granulosa cell phenotype and precluding Sertoli cell transdifferentiation and tubule formation within the ovary (6, 11). Nevertheless, granulosa cell features are evident in gonadal tumors of males and females in the absence of ER function. Why these receptors are not required for establishing granulosa cell-like features within the tumors that develop in the absence of inhibins remains unclear. Finally, it is noteworthy that 40% of ER
-/-, ERß-/- double-knockout females develop sex cord stromal ovarian tumors by 1520 months of age (9). What similarities these tumors have to those that develop in the Inh
-/- mice, their dependence on gonadotropin hormones, and associated aberrations in inhibin-mediated pathways, if these exist, remain to be evaluated.
The studies reported here reveal an intriguing sexual dimorphism related to the roles of estrogens in gonadal tumors. Triple-knockout males, unlike females, are protected from early weight loss and tumor development. Even when delayed testicular tumors develop in these males, they do not encompass the entire gonad, and large regions retain histological features reminiscent of ER
knockout mice and are spared Sertoli cell hyperplasia. Such limited tumor development is never observed in knockout males lacking inhibins and either ER
or ERß. These findings enhance our appreciation for the roles of estrogens in the testis and in the context of testicular tumorigenesis. Notably, some evidence suggests that estrogens mediate the proliferation of Sertoli cells during development (29, 30, 31) and that exposure to exogenous estrogens inhibits Sertoli cell differentiation programs (32). However, despite high levels of aromatase and ERß expression in dividing Sertoli cells and their relative lack of ER
, defects in Sertoli cell proliferation are not recapitulated in either aromatase or ERß knockout models. This suggests that other targets of FSH, such as cyclin D2, produce its principal mitogenic effects normally in males. Even if evidence that estrogens mediate Sertoli cell proliferation during testis development is equivocal, it seems clear that ER functions are key to loss of Sertoli cell proliferation control in the absence of inhibins. Interestingly, only triple-knockout males are afforded a measure of protection from testicular tumorigenesis, in contrast to double knockouts lacking ER
or ERß and inhibins. Although redundancy of ER
and ERß has been demonstrated in granulosa cells by the unique ovarian phenotype of ER
-/-, ERß-/- double-knockout females (6), this is the first study to reveal overlapping ER
and ERß functions in males where consequences of loss of ERs are only manifest in double-mutant mice. The molecular mechanisms underlying compensation pathways in single-knockout mice in this regard will be of great interest.
The male-specific rescue of the Inh
knockout phenotype we observe in triple knockouts is reminiscent of that seen in two double-mutant models, Inh
knockouts also lacking cyclin D2 (24A ) or FSHß (33). To investigate the possibility that ER signaling was important for up-regulation of cyclin D2 expression in the course of tumorigenesis, we performed Northern blot analysis on tumors from double- and triple-knockout males, but found that cyclin D2 transcript is markedly up-regulated with testicular tumorigenesis regardless of ER expression. Thus, it seems that loss of inhibins and high circulating levels of FSH promote testicular tumor development by enhancing steroidogenesis and ER signaling pathways, as well as up-regulating cyclin D2 mRNA, the latter being separable in vivo from the former.
In conclusion, this study underscores differences between males and females in how estrogens feed back to regulate the central endocrine axis, and in how endocrine factors influence proliferation pathways intrinsic to granulosa/Sertoli cells (Fig. 8
). In female Inh
knockouts, estrogens may have dominant functions in controlling pituitary gonadotropin hormone production, whereas in males, they are critical trophic factors accelerating tumor development. Although we ascribe this dichotomy largely to different estrogen effects within the central endocrine axis, there are also likely to be differences in estrogen signaling pathways and cellular thresholds of proliferation control in granulosa cells vs. Sertoli cells. The utility of the Inh
knockout mice extends to a third tumor-prone cell type, as castrate knockouts demonstrate critical functions of inhibins in tumor suppression in the adrenal cortex (15). In addition, we have shown that these adrenal tumors are sex steroidogenic (11) and that they express high levels of LH receptor and aromatase mRNAs (Burns, K. H., and M. M. Matzuk, unpublished data). Thus, there is potential for estrogen pathways to influence adrenal tumor development in this model, and future studies may be directed at defining these functions.

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Fig. 8. A Model for the Effects Of Steroid Hormones on Gonadal Tumor Development
In females, we speculate that the chief role of estrogen signaling through ER and ERß receptors serves to inhibit gonadotropin hormones rather than to directly promote cell division within the ovarian tumor. This is based on the finding that tumors develop and progress with rapidity in ER -/-, Inh -/- and ER -/-, ERß-/-, Inh -/- mice. These mice develop elevated serum FSH and the activin-mediated wasting syndrome as do Inh -/- mice, but at early ages, and these tumors produce high levels of ineffectual estrogens. In males, we hypothesize that ER signaling is most critical directly within Sertoli cells, promoting their switch to mitotically active tumor cells with granulosa-like cell characteristics. Thus, ER and ERß loss in triple-knockout males has a protective effect. Androgens serve more analogous roles in males and females, directly promoting tumor development and hemorrhage. There are minimal effects of the absence of androgen receptor on the central endocrine axis.
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MATERIALS AND METHODS
|
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Generation and Genotyping of Mice
Mice were maintained as described in the NIH Guide for the Care and Use of Laboratory Animals. Generation and genotyping of Inh
(Inha), ER
(Ers1, also known as ERKO), and ERß (Esr2, also known as BERKO) mutant mice by Southern blot or PCR genotyping have been described (4, 5, 13). Double-knockout mice were identified in litters from matings of double heterozygotes (ER
+/-, Inh
+/-; ERß +/-, Inh
+/-), and triple-knockout mice were identified in litters from matings of triple mutants (either ER
+/-, ERß+/-, Inh
+/- or ER
+/-, ERß-/-, Inh
+/-). ER
-/-, Inh
-/- and ERß-/-, Inh
-/- double-knockout mice and ER
-/-, ERß-/-, Inh
-/- triple-knockout mice were obtained at approximately the expected Mendelian ratio. All lines of mice were maintained on a mixed C57BL/6J/129S6/SvEv genetic background.
Flutamide Treatment
To study ovarian tumor progression in the presence of flutamide, a nonsteroidal androgen antagonist, mice were given sc time-released drug implants. For the implantation, each mouse was anesthetized by avertin injection, and a flutamide pellet was placed under the skin of the neck behind the ears (Innovative Research of America, Sarasota, FL). Mice without palpable tumors and overt signs of cachexia 60 d later had pellets replaced to ensure continuous flutamide exposure.
Weight Data and Survival Curves
Mice were followed weekly for tumor development by measuring total body weight and monitoring for kyphoscoliosis as described (15). Mice were killed when they developed signs of cachexia. Statistical significance was determined by Students t test comparing weeks of age at death.
Morphological and Histological Analysis
Mice of each sex and genotype were killed and tissues collected immediately into 10% buffered formalin (ovaries) or Bouins fixative (testes). After overnight fixation, tissues were embedded in paraffin blocks, sectioned, and stained with hematoxylin and eosin. Embedding and staining were performed by standard procedures in the Baylor College of Medicine Pathology Core Services laboratory.
Serum Analyses
Mice were anesthetized by isoflurane inhalation (Abbott Laboratories, North Chicago, IL), and blood was recovered by closed cardiac puncture. Serum was separated by centrifugation in Microtainer tubes (Becton Dickinson and Co., Franklin Lakes, NJ) and stored at -80 C before analysis. FSH and LH measurements were made by the University of Virginia Ligand Core Facility (Charlottesville, VA). Methods are provided at http://www.healthsystem.virginia.edu. Serum estrogens were measured by RIA at this Core Facility or in our laboratory using the Coat-A-Count kit (Diagnostic Products Corp., Los Angeles, CA.) according to the manufacturers instructions.
Northern Blot Analyses
Northern blot analyses were performed as previously described (34, 35). Briefly, 15 µg of total RNA were electrophoresed in each gel lane and transferred onto nylon membranes as detailed (36). Tumor RNA samples were not pooled for these studies, but samples from individual mice of the same genotype were run in adjacent lanes. [32P]dATP-labeled probe was synthesized from a template corresponding to the coding sequence of cyclin D2 mRNA (Ccnd2, 115-1244 of NM 009829), or activin ßA (Inhba, 10491474 of X69619), or activin ßB (Inhbb, 1602 of X83376 + 240 bp upstream) using the Strip-EZ kit (Ambion, Inc., Austin, TX). Blots were then hybridized and washed as recommended. Probe hybridization was assessed by autoradiography and PhosphorImager analysis with ImageQuant software (Molecular Dynamics, Sunnyvale, CA) (37). A background level was determined and subtracted, and then each blot was stripped and reprobed for glyceraldehyde 3-phosphate dehydrogenase (Gapd) mRNA to allow for loading corrections when comparing the signal intensities.
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ACKNOWLEDGMENTS
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We thank Steven Elledge for providing of the cyclin D2 cDNA probe; Valerie Long, Sherry Cipriano, and Micheal Klysik for their technical assistance; T. Rajendra Kumar and Wei Yan for insightful discussions; Joanne Richards for critical review of the manuscript; and Shirley Baker for her assistance with manuscript formatting.
 |
FOOTNOTES
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Serum sample analyses were performed by the University of Virginia Ligand Core Facility supported by NICHD/NIH through cooperative agreement (U54 HD28934) as part of the Specialized Cooperative Centers Program in Reproduction Research. K.H.B. is a student in the Medical Scientist Training Program at Baylor College of Medicine supported in part by NIH Grant T32GM07330. M.M.M. was supported by NIH Grant CA60651, which provided funds for research recovery efforts after Tropical Storm Allison.
Abbreviations: ER, Estrogen receptor; Inh
, inhibin
.
Received for publication February 5, 2003.
Accepted for publication July 3, 2003.
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