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Animal Model |
From the Department of Gynecology and Obstetrics*
and the Winship Cancer Center,
Division of
Animal Resources, Departments of Pathology and Laboratory Animal
Medicine, Emory University School of Medicine, Atlanta, Georgia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous reports of Leydig cell tumors in rodents include either spontaneously occurring tumors or induction with chronic estrogen supplementation.13 Leydig cell tumors can be induced by pre- and postnatal estrogen treatment in mice, depending on the strain used, and in adult hamsters, but not rats.14 Each of the following hormonal exposures has been shown to cause testicular tumor formation in rodents: chronic exposure to estrogenic compounds of adult mice (inbred strains A and BALB/c) and hamsters; prenatal exposure to estrogenic compounds of mice and humans; and any treatment or condition that induces cryptorchidism in mice and humans.14 To obtain a better understanding of the biological significance of testicular tumorigenesis, specifically hormone-induced tumorigenesis, a reliable in vivo model is necessary to clarify mechanisms and correlations associated with human cancers. Here we describe such a model, which shows that overexpression of aromatase results in increased estrogen production and a changed hormone milieu, leading to the induction of testicular cancer (Leydig cell tumors).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of aromatase transgenic mice (previously referred as MMTV-int-5/aromatase transgenic mice) and their characterization have been described previously.9 Briefly, aromatase cDNA was expressed under the control of mouse mammary tumor virus promoter (MMTV-LTR), which is active in male reproductive organs as well as in mammary tissues. Male transgenic mice overexpressing aromatase were maintained in a standard colony and were used as breeders. Mice were housed in a centralized animal facility accredited by the American Association for Accreditation of Laboratory Animal Care and the United States Department of Agriculture and maintained according to the recommendations established in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Serum Sample Collection
Blood was collected from mice via retro-orbital sinus every other week or from the cardiac puncture at the time of sacrifice. To collect blood from the retro-orbital sinus, mice were sedated with 100 µl of 0.1 ml/10 grams of a mixture of fentanyl (0.011 ng/ml), midazolam (0.1 mg/ml), and droperidol (0.5 mg/ml) by intraperitoneal injection. A drop of piperocaine ophthalmic solution was instilled in the eye using a heparinized microhematocrit capillary tube to provide local anesthesia. Pooled serum samples from retro-orbital collection or samples collected using cardiac puncture were used to measure the serum estradiol (E2) levels.
Serum Estradiol Levels
Serum concentrations of estradiol were measured by double antibody radioimmunoassay using commercially available reagents (Diagnostic Products Corp., Los Angeles, CA). Using an equivalent of 200 µl of serum in duplicate, the assay had a sensitivity of 2.5 pg/ml and an upper limit of 500 pg/ml. Assaying increasing volumes of serum from 50 to 200 µl produced a displacement line parallel to the standard curve. Intra- and interassay coefficients of variation averaged <10.% and 6.7%, respectively. Differences in mean estradiol levels were compared, and significance was determined using paired Student’s t-test.
Histology and Immunohistochemistry
For routine histology, tissues were rapidly removed and fixed in 10% formalin overnight, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin (H&E). For immunostaining of testicular tissue, unstained tissue sections were quenched in 3% hydrogen peroxide in phosphate buffered saline (PBS). After washing with PBS the sections were incubated in PBS containing 1% goat serum at room temperature followed by incubation with primary antibody (1:200 dilution) overnight at 4°C. Aromatase polyclonal antibody15 was a gift from Dr. Evan Simpson of the University of Texas Southwest Medical Center (Dallas, TX). Secondary antibodies (goat anti-rabbit secondary antibody and biotinylated goat anti-rabbit secondary antibody) were obtained from Vector Laboratories (Burlingame, CA) and staining with diamino-benzidine tetrahydrochloride was carried out per the manufacturer’s instructions. Sections were rinsed with deionized water, counterstained in hematoxylin, and mounted with coverslips for evaluation by light microscopy.
Biochemical Analysis of Testicular Tissue
Total RNA from testicular tissues of aromatase transgenic mice and
control nontransgenic litter mates was isolated as described
before.16
Equal amounts of total RNA (exact amounts of
total RNA used are indicated in figure legends) from both aromatase
transgenic animals and nontransgenic animals were analyzed for both
aromatase mRNA and estrogen receptor (ER
) mRNA levels by RT-PCR as
described before.9,17
Initially, the quality of the total
RNA from each sample was confirmed using agarose gels and ethidium
bromide staining for 28 and 18S RNA. To further demonstrate
that an equal amount of total RNA was used from each sample to
determine aromatase and ER expression, we examined the expression of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping
gene, as an invariant control by RT-PCR. After initial
equalization of the each sample based on GAPDH concentration, a
fraction (1 µl) of the reverse-transcribed mixture, the first step in
reverse-transcriptase-polymerase chain reaction (RT-PCR), was used for
PCR amplification of GAPDH; the remaining mixture (19 µl) was used
for the amplification of either aromatase or ER PCR product
in the same cycle of amplification. The densitometric data from
ethidium bromide staining of RT-PCR products on agarose gels were used
for calculating the differences in the expression of various mRNA
levels in different tissue samples. Data were equalized to GAPDH if
required when calculating the differences in the expression of either
aromatase or ER between the samples. All of the analyses were
carried out using at least three separate samples. The representative
data are presented.
Testicular tissues from both aromatase and nontransgenic control
animals were also used to determine the ER protein levels using
Western blot analysis. Briefly, testicular tissue was homogenized in
lysis buffer, 60 µg total protein from each sample was separated on
polyacrylamide gels and transferred to nylon membrane. Nonspecific
binding of antibodies were blocked by incubation for at least 4 hours
at room temperature with Tris-buffered saline (TBS) containing 0.05%
Triton X-100 (TBST) and 5% nonfat dry milk. Filters were incubated
with respective primary antibodies in TBST-milk overnight at 4°C, and
specific binding was visualized by using anti-mouse IgG followed by
enhanced chemiluminescent detection (ECL kit; Amersham). The
densitometric data from Western blots (X-ray image of chemiluminescent
proteins) were used for calculating the differences in the expression
of receptor protein levels in various samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Originally, select male breeders from this colony were suspected
of being infertile because of poor reproductive performance. The ages
of these mice ranged from 6 to 12 months. About half of the aromatase
male breeders (n = 30) were determined to
be infertile. On close examination some of these mice were found to
contain larger than normal testicles (Figure 1, A and B)
. Grossly, the testicular
tumors from aromatase transgenic mice ranged in size from 1 to 3 cm in
diameter. Some tumors were bilateral and well circumscribed, having a
soft to firm consistency. The cut surface of the tumor was homogeneous
and yellow-tan (data not shown). Only the larger tumors were associated
with necrosis. Histologically, these tumors were made up of large
interstitial cells that were polygonal in shape, with round nuclei and
abundant clear to granular eosinophilic cytoplasm. The supporting
stroma was highly vascular with multiple cystic areas containing
numerous red blood cells (Figure 1, D
-I). We have observed tumors in
various human Leydig cell tumors; 30 to 40% of cases show
intracytoplasmic, eosinophilic rod-shaped crystals of
Reinke.18
However, crystals of Reinke were not observed to
be present in any of the mouse tissues we examined. No cellular atypia
or mitoses were observed. Gross and histological evaluation of other
tissues were normal. These results clearly suggest that the
pathological changes were restricted to testicular tissue.
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Our previous studies, as expected, showed the overexpression of
aromatase in transgenic mammary glands, so we examined whether
transgene is similarly overexpressed in testicular tissue. Our results
using RT-PCR analysis (Figure 2)
show
that, compared to aromatase mRNA levels in testicular tissue of control
animals, the levels of aromatase mRNA in testicular tissue of aromatase
animals are almost fourfold higher. These results suggest that
increased aromatase expression may play a role in change in the
hormonal milieu of testicular tissue and may also affect circulating
hormonal levels.
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To determine whether the overexpression of aromatase in testicular tissue changes the hormonal milieu in these animals, serum estradiol levels were estimated. Results clearly indicate that estradiol levels are significantly higher for transgenic mice in comparison to nontransgenic mice. The serum estradiol levels for transgenic mice were at least twice as high as those for nontransgenic mice. Mean estradiol levels for transgenic and nontransgenic mice were 5.71 and 2.55 pg/ml, respectively. Differences in mean estradiol levels were compared, and significance was determined by paired Student’s t-test (P < 0.001).
Leydig Cells in Tumor Express Aromatase
To characterize the histological features of Leydig cell tumors
and to verify the cellular localization of aromatase overexpression, we
carried out immunohistochemical staining for the aromatase protein
expression. In general, the testes from transgenic mice were found to
contain immunoreactive aromatase within the cytoplasm of interstitial
Leydig cells (Figure 3C)
. The pattern of
staining within the testicular tissue was not uniform and ranged from
focal to locally extensive in distribution. Immunoreactive aromatase
was present within the interstitial Leydig cells and absent from other
interstitial cells such as Sertoli cells, fibroblasts, and cells within
the seminiferous tubules. Areas of intense staining were noted in the
periphery of the tumor tissue, whereas areas of light to no staining
were observed in the more central areas. In addition, the degree of
immunostaining appeared to be strongest in testes with more advanced
stages of neoplasia. Nontransgenic tissue showed very weakly positive
reactivity with aromatase antibody, indicating very low expression of
aromatase (data not shown). The negative controls (without primary
antibody) of testicular sections from nontransgenic, nonsyngeneic mice
were negative for aromatase (Figure 3, A and B)
.
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Overexpressed in Transgenic Testicular Tissue
Our previous studies have shown that overexpression of aromatase
in transgenic mammary glands leads to up-regulation of ER as well as
induction of various histopathological changes mammary epithelial
cells.9,19
Therefore, we have examined whether
overexpression of aromatase in testicular tissue that results in
increased circulating estrogen levels also leads to up-regulation of
ER. Our results (Figure 4
, top panel)
show that, compared to ER protein levels in testicular tissue of
control animals that are too low to be detected, the levels of ER
protein in testicular tissue of aromatase transgenic animals is very
high. Similarly, the expression of ER mRNA is fourfold higher in
aromatase transgenic mice testicular tissue compared to nontransgenic
testicular tissue. These results suggest that increased biosynthesis of
estrogen as a result of aromatase overexpression leads to up-regulation
of ER in testicular tissue.
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To determine whether overexpression of aromatase in testicular
tissue leads to changes in estrogen-dependent cell cycle proteins, we
estimated the levels of cyclin D1 in testicular tissues from transgenic
and control nontransgenic mice. The data presented in Figure 5
clearly show that the testicular
tissue cyclin D1 protein level in transgenic mice is about threefold
higher than that in control animals. No difference was observed
in the levels of PCNA between nontransgenic and transgenic tissue.
These results suggest that estrogen-mediated epigenetic changes may
play a significant role in testicular tumorigenesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in human testicular Leydig cell tumors is
consistent with the Leydig cells being the source of estrogen in both
rodent and human testis.21,22
Recent studies by
Berensztein et al23
and Valenski et al24
with
human tissue samples have shown that aromatase activity and estrogen
synthesis is higher in Leydig cell tumor fraction than in normal tissue
surrounding the tumor of the same patient. Studies by Kerlam et
al25
have shown further that estradiol-producing tumors in
men responded well to human chorionic gonadotropin (hCG)
administration in regulating aromatase activity and estradiol
production. Our studies clearly show the level of expression of ER
and aromatase are higher in the testicular tissue of these mice. In
contrast, the expression of both aromatase and ER was lower in
testicular tissue of nontransgenic mice (Figures 2–4)
. This study and
a number of previous animal and human studies clearly suggest that
aromatase may play a critical role in Leydig cell tumorigenesis.
Estrogen plays a critical role in regulating the G1 cyclins in a number
of target tissues. Our previous studies with mammary and testicular
tissue and other studies with breast cancer cells26,27
have shown that estrogen also plays direct role in activation of genes
involved in the G1 phase of the cell cycle. These studies have shown
that estrogen induces cyclin D1 levels significantly in mammary
tissues, whereas the levels of cyclin E, cdk2, and the p21 and p27 cdk
inhibitors are relatively constant. However, the p21 cdk inhibitor
shifts from its association with cyclin E-cdk2 to cyclin D1-cdk4, thus
suggesting that cyclin D1 has an important role in steroid-dependent
cell proliferation and that estrogen, by regulating the activities of
G1 cyclin-dependent kinases, can control the proliferation of breast
cancer and other hormone-dependent cells.26,27
Cyclin D1
is a very important cell cycle protein that has been demonstrated to
play a significant role in mammary gland development and human mammary
carcinomas. Recent work has postulated that cyclin D1 binds directly to
ERs and potentiates transcription of ER-regulated
genes.27
Our results not only confirm increased ER
expression in testicular tissue, but also show that serum estradiol
levels are significantly higher in transgenic mice than in
nontransgenic litter mates. This finding indicates that enhanced
circulating estrogen production does occur. The increased estradiol
levels may be due to the increased aromatization and direct secretion
from the Leydig cells. Our expression studies directly support this
notion. Based on our observations we suggest that an enhanced ER
phenotype of Leydig cells and increased peripheral aromatization may
contribute to the Leydig cell tumorigenesis.
One advantage of using this transgenic mouse model, in contrast to
previous animal models, is that this is a non-invasive model that does
not use exogenous estrogen administration. We believe the spontaneous
development of testicular tumors in these transgenic mice better
characterizes the natural physiological and molecular progression of
events that occur in humans who develop testicular tumors, specifically
Leydig cell tumors. In conclusion, the results from this study provide
the first qualitative and quantitative relationship between testicular
aromatase, ER expression, serum estradiol levels, and Leydig cell
tumor development in a transgenic animal model. We have demonstrated
that the aromatase transgenic mouse model mimics the human Leydig cell
tumor and should provide future insight into the molecular
pathogenesis of Leydig cell tumors and possibly other testicular
tumors.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health/National Cancer Institute grants CA 57559 and 75018 (to R. R. T.).
Accepted for publication September 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, McPhaul MJ, Kozlowski G, Conley AJ, Lephart ED: Immunohistochemical distribution of aromatase cytochrome P450 in the rat brain using peptide generated polyclonal antibodies. Endocrinology 1991, 129:2834–2844
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