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From the Medical Research Council Toxicology Unit, Leicester, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, preadipocyte
factor-1, and insulin-like growth factor-2 were key genes
differentially modified by tamoxifen or toremifene treatment,
relative to the controls. As these genes may play an important role in
regulating differentiation and development of the myometrium,
these data suggest that adenomyosis may be caused primarily by defects
in the formation of the myometrium.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although several theories for the development of adenomyosis have been proposed, its precise etiology remains uncertain and the trigger for this aberrant growth remains unexplained. The conventional view is that adenomyosis results from the abnormal down-growth and invagination of the endometrium into the myometrium, because continuity between the basal endometrium and underlying adenomyosis can often be seen in tissue sections. It has been suggested that this occurs because of weakness in the uterine smooth muscle or increased uterine pressure. The focal smooth muscle hypertrophy and hyperplasia, observed in association with zones of adenomyosis, is explained as a response of the myometrium to the presence of ectopic endometrial cells. Traditionally, adenomyosis is seen as separate from endometriosis, the condition in which endometrial tissue is found in extrauterine locations. It is argued that endometrium reaches these extrauterine locations by seeding of endometrial cells via the fallopian tubes.
However, it has been postulated that both adenomyosis and endometriosis arises by a process of metaplasia. Mai and colleagues3 have proposed that both conditions result from induction of endometrial epithelial cells by the presence of endometrial stroma formed by multipotent pericytes situated in the uterine body and extrauterine locations. More recently, magnetic resonance imaging and vaginal sonography has highlighted anatomical defects in the myometrium in adenomyosis and endometriosis in women. Instead of smooth muscle changes representing a response to invading endometrial tissue, this imaging data has suggested that the primary defect in these conditions is myometrial dysfunction.4,5
A number of primate and rodent models have been used in the study of adenomyosis. Adenomyosis and endometriosis occurs naturally in non-human primates notably Macaca mulata but useful numbers of cases can only be obtained from large breeding colonies or after surgical implantation of autologous endometrium.6 Several mouse strains also develop adenomyosis spontaneously, where it can be found in a small percentage of adult animals.7,8 Adenomyosis has also been induced in mice by hormonal manipulation such as produced by the implantation of a single anterior pituitary gland into the uterine lumen.9,10 The effects of surgical manipulation and tissue repair as well as diverse local hormonal alterations may confound interpretations of the findings in this latter model.
In this article we report on the development of adenomyosis in mice after the administration of the SERMs, tamoxifen or toremifene, for 5 days during the neonatal period. This model, which produces a high incidence of adenomyosis by 3 months of age, allowed examination of the early events in the myometrium and endometrium that might be responsible for the development of adenomyosis. Histological findings, estrogen receptor (ER) status, and gene expression data after tamoxifen or toremifene compared with raloxifene or estradiol treatment during this neonatal period suggested that the predisposition to adenomyosis may be primarily related to alterations in stromal and myometrial differentiation that alter myometrium structure and function in adulthood.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pregnant CD-1 mice were from Charles River, Kent, UK. Groups of 10 female neonatal mice were dosed orally via capillary tubing on days 2 to 5 after birth (day of birth is day 1), for 4 days consecutively. Mice were dosed with 5.3 nmol/kg estradiol, 2.7 µmol/kg tamoxifen base, toremifene citrate, or raloxifene hydrochloride suspended in peanut oil/lecithin/condensed milk mixture (2:0.2:3 v/v) at a dose volume of 5 µl/g body weight. Controls received vehicle only. Oral dosing was used in preference to subcutaneous on animal welfare grounds. On day 6, mice were sacrificed by decapitation and uteri were removed, weighed, and fixed in 3.7% neutral-buffered formalin, Carnoy’s medium, or snap-frozen in liquid nitrogen. Four controls and groups of four mice, also dosed on days 2 to 5 after birth, for 4 days consecutively with tamoxifen, toremifene, raloxifene, and estradiol were examined at 42 days. In addition, 10 controls and groups of 10 mice treated with a similar regimen of tamoxifen, toremifene, and raloxifene and controls were examined at 90 days.
For microscopic examination, standard histological sections of the uterus vagina and ovaries were prepared from formalin-fixed material and were stained with hematoxylin and eosin. Oil red O staining for lipid was conducted on unfixed frozen sections. Animal care and procedures were conducted in accordance with the codes and practice of the Animals (Scientific Procedures) Act 1986 of the United Kingdom.
RNA Isolation and Mouse 1.2 Atlas cDNA Microarray Analysis
Total RNA was extracted from the uteri of each group of treated
neonatal mice using a Microisolation kit (Stratagene, La Jolla, CA)
according to manufacturer’s instructions. For each group, uterine
tissues from four to six newborn mice were pooled. DNase-treated total
RNA (5 µg) was used to synthesize
32P-radiolabeled cDNA probes. RNA was transcribed
by reverse transcriptase with (-32P) dATP and
specific primers for genes represented on the Clontech mouse 1.2 atlas
array (Clontech, Palo Alto, CA). Clontech atlas cDNA expression arrays
include 1176 mouse cDNA spotted onto a nylon membrane. Plasmid and
bacteriophage DNAs are included as negative controls to confirm
hybridization specificity along with several housekeeping genes that
act as orientation marks and as positive controls for normalizing RNA
abundance. Labeled probe (pooled fractions, 2 to 20 x
106
cpm) was added to Cot-1
DNA and incubated at 68°C. Prepared probe was hybridized with the
array for 18 hours at 68°C with constant agitation. Arrays were
processed in duplicate for each treatment group. The arrays were washed
according to the manufacturer’s protocol and exposed to a phosphor
imaging plate for up to 72 hours. Hybridization signals were quantified
with a phosphor imager (Bio-Rad, Hercules, CA) and analyzed using image
analysis software (Molecular Dynamics).
Immunocytochemistry Analysis
Formalin-fixed, 5-µm paraffin sections of rodent uterus were
de-waxed by immersing in Histoclear, taken to water, and microwaved in
citrate buffer, pH 6.0, for 20 minutes at 700 W. Sections were placed
in distilled water and endogenous peroxidase activity was blocked with
3% (v/v) H2O2 in water for
20 minutes. ER was detected using a Novocastra mouse monoclonal
antibody (NCL-ER-6F11) raised against a recombinant protein
corresponding to the full-length human ER. The antibody was diluted
1:40. ERß was detected using an Upstate Biotechnology rabbit
polyclonal antibody (06-629) raised against a synthetic peptide
representing amino acids 54 to 71 of mouse ERß. The antibody was
diluted 1:10. Primary antibody was detected with DAKO
StreptABComplex/HP Duet kit, following the manufacturer’s
instructions, and visualized with 3,3'-diaminobenzidine
tetrahydrochloride solution. Sections were lightly
counterstained with hematoxylin before de-hydration though graded
alcohol, clearing, and mounting with DPX. Human breast carcinoma
sections were used as a positive control and omission of primary
antibody was used as a negative control on parallel sections.
Statistical Analysis
Statistical analysis was performed using Minitab version 10 (Minitab Inc., PA). Difference between groups was tested using analysis of variance with Dunnett’s test for significance at the 5% level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A comparison of effects on uterine weights of tamoxifen,
toremifene, and raloxifene at day 6 after dosing and at day 42 and day
90, relative to vehicle-dosed controls, is shown in Table 1
. At day 6, all treatments resulted in a
significant increase in uterine weights relative to controls. In
contrast, after 42 days both tamoxifen and toremifene showed a
significant decrease in weight compared to controls and, after 90 days,
only the uteri of the raloxifene-treated animals showed an increase in
weight, whereas tamoxifen treatment resulted in a significant decrease.
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Adult Animals
At the 90-day time point, all 10 mice treated with tamoxifen and 9
of 10 treated with toremifene in the neonatal period showed
adenomyosis, only 1 of 10 animals treated with raloxifene, and none of
the three groups of 10 untreated controls. In affected animals
adenomyosis was characterized histologically by the presence of simple
endometrial glands, associated with variable but small amounts of
endometrial stroma that infiltrated all layers of the myometrium,
reaching but not penetrating the uterine peritoneal surface (Figure 1)
. The loose mesometrial aspects of the
myometrium were preferentially involved. Another feature associated
with affected animals was the disruption to the concentric and
longitudinal smooth muscle bands. Instead of regular concentric layers
of smooth muscle cells seen in control mice at 90 days (Figure 1)
,
there were interwoven, thickened bands of smooth muscle cells
interspersed with prominent layers of collagen and prominent blood
vessels. In many of these altered zones there was a scattering of
polymorphonuclear cells, particularly eosinophils that was not evident
among controls. These stromal changes were frequently, but not always,
associated with the presence of ectopic endometrial glands. The
endometrial glands and the vagina epithelium were unaltered in treated
groups and both control and treated mice showed typical cyclical
alterations. Likewise, ovaries were similar in treated mice and
controls. At the 42-day time point adenomyosis was also observed in all
mice treated with tamoxifen, three of four treated with toremifene, but
none of controls or mice treated with raloxifene or estradiol. In
treated animals the vagina, oviducts, and ovaries showed no significant
histological differences from controls.
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At 6 days, in mice treated with raloxifene or estradiol the uterus
showed similar histological appearances to controls. The endometrial
lumen was a simple slit lined by hyperchromatic epithelial cells. The
lumen was surrounded by a layer of cellular stromal tissue in which
occasional mitoses were evident. A uniform layer of young concentric
smooth muscle fibers also containing mitotic figures surrounded the
endometrial tissue. A thin external layer of longitudinal smooth muscle
cells was also present (Figure 2)
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. Within this stroma small blood vessels were prominent. Another
striking feature of the outer aspect of the altered myometrium was the
presence of vacuolated cells that special stains showed were lipocytes
(Figure 2)
. The endometrial lumen retained its slit-like character with
some infolding but it was lined by glandular tissue that was moderately
hyperplastic with enlarged cells containing pseudostratified nuclei
compared with controls or mice given raloxifene or estradiol. Immunocytochemistry
ER and ERß labeling of control neonatal uterine tissue was
most dense in the cells of the endometrial stroma, with little or no
staining in smooth muscle or endometrial glandular cells. In
tamoxifen-treated mice, labeling of the disordered stroma was similar
to that of endometrial stroma in controls. Labeling of nuclei from the
hyperplastic glandular tissue was also more prominent than in controls
(Figure 3)
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Changes in gene expression in uterine tissues of neonatal mice
were assessed 6 days after birth. Figure 4
shows log-log scatterplots of the cDNA
microarray data. Comparisons were made between cDNA hybridizations of
pooled uterine samples of controls and either tamoxifen (Figure 4a)
,
toremifene (Figure 4b)
, raloxifene (Figure 4c)
, or estradiol (Figure 4d)
. Each point on the scatterplots represents 1 of the 1176 genes on
the Atlas array. The density of each point has been normalized to the
sum of the density of all of the genes. There was good reproducibility
between duplicate assays performed on the pooled RNA extracts. Changes
in gene expression resulting from tamoxifen or toremifene treatment
were broadly similar, whereas raloxifene had a much less marked effect.
Comparing expression between tamoxifen and control: 22 genes were
up-regulated and 22 were down-regulated greater than twofold.
Toremifene treatment resulted in the up-regulation of 45 genes and the
down-regulation of 31 genes greater than twofold when compared with
control. Comparison of estradiol to control showed a greater than
twofold up-regulation of 19 genes and the down-regulation of 47 genes.
In contrast, treatment with raloxifene resulted in a greater than
twofold up-regulation of just four genes and down-regulation of eight
genes.
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. The 7S
nerve growth factor- subunit (Ngfa) was strongly
up-regulated by tamoxifen and toremifene treatment but in contrast, was
strongly down-regulated by estradiol (Table 2)
. The paternally
imprinted insulin-like growth factor 2 (Igf-2) and
preadipocyte factor-1 (Pref-1) were down-regulated by
tamoxifen, toremifene, raloxifene, and estradiol.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Why tamoxifen and toremifene but not raloxifene should have this effect remains unclear, although different SERMs are known to possess diverse effects in various tissues and organs.
The histological features of the adenomyosis in this present study using this 5-day treatment regimen of SERMs possessed the histological features described previously both in animal models and in humans.1,11,12 Moreover, this was produced without the confounding effects of surgical trauma of the mouse pituitary implantation model. Nodules of endometrial glands and stroma were present deep within the myometrium, sometimes extending to, but not penetrating, the serosa. Notable was the disordered arrangement of the smooth muscle of the myometrium. In contrast to controls where smooth muscle fibers were arranged in uniform concentric layers around the endometrium, in tamoxifen- and toremifene-treated mice, this zone comprised irregular smooth muscle bundles interspersed with collagen and often penetrated by prominent blood vessels. The smooth muscle changes were more extensive than the zones of abnormal glands. This suggests that the penetration of endometrial glands might follow the changes to myometrium rather than precede them. These areas of altered smooth muscle organization were associated with a scattering of polymorphonuclear leukocytes, notably eosinophils. The presence of eosinophils has been reported in both human endometriosis and carcinoma where it has been postulated that they are involved in general tissue-remodeling.13 It is of note that there was neither evidence of vaginal adenosis nor the extension of the oviduct epithelium through the muscularis described in stilbestrol-treated CD-1 mice by Newbold and colleagues14 in the treated mice.
The histological alterations and principle genetic changes, which occurred in the neonatal uterus immediately after treatment with tamoxifen and toremifene, provide additional support for the concept that the primary derangement of the uterine mesenchymal tissue differentiation is the basis for the development of adenomyosis. At 6 days the uterine body in mice treated with tamoxifen and toremifene, but not raloxifene or estradiol, was devoid of the normal developing layers of smooth muscle but retained histological appearances and ER status of endometrial stroma. In addition, some unusual patterns of differentiation were noted, particularly the presence of lipocytes in the outermost parts of the uterus, features resembling adipose tissue of the developing mesenteric tissue in the surrounding mesenchyme.
In contrast to the major changes in mesenchymal differentiation, alterations to histological and receptor status in endometrial glandular tissue, produced by treatment, were modest. This was reflected by the lack of major alteration in the ER gene in endometrial glandular tissues. Not only did endometrial glands show no major differences in ER status, the endometrium itself continued to show regular cyclical alterations in treated groups similar to those in controls. These data suggest that the functional status of the endometrial glandular tissue is not greatly altered in adenomyosis. In the present study, no cystic ovaries were observed and corpora lutea were seen in all of the treatment groups. This contrasts to previous findings at 14 to 17 months after dosing where corpora lutea were absent when tamoxifen was given subcutaneously on days 1 to 5 to newborn CD-1 mice.15
Although the conventional view is that adenomyosis represents down-growth of endometrial glands and stroma into the myometrium with secondary local tissue response,1 other recent evidence also underlines the importance of mesenchymal tissue in the development of adenomyosis. It has been postulated, based on histopathological and immunocytochemical analysis of the human uterus, that the abnormal smooth muscle in adenomyosis results from a similar metaplastic process proposed for endometriosis where smooth muscle has been found associated with endometrium in extrauterine locations. It has been suggested that aberrant endometrial stroma may be produced by metaplasia from primitive pericytes. It is postulated that this is capable of inducing the formation of endometrial glands through local autocrine or paracrine mechanisms controlled by genetic, hormonal, and immunological factors.3,16 It has also been suggested that excessive myocyte proliferation may be the cause of adenomyosis rather than its consequence based on T2-weighted magnetic resonance imaging and vaginal sonography in women with adenomyosis. These techniques show more extensive derangement of the myometrium compared with the patchy and focal nature of adenomyosis.4,5
The pathological changes in mice were mirrored by alterations seen at 6
days in genes that are believed to be important in the developmental
regulation of mesenchymal cells. The up-regulation of Ngfa
by tamoxifen and toremifene (Figure 4
, Table 2
) is in marked contrast
to its down-regulation by estradiol treatment. Nerve growth factor and
its low-affinity receptor p75NTR are believed to
have not only a role in synchronizing the developing visceral nervous
system but also on myogenic differentiation. The receptor
p75NTR is highly expressed in the smooth muscle
cells of the mouse uterus.17
Further studies in a mouse
muscle-derived cell line (C2C12) demonstrate down-regulation of nerve
growth factor and p75NTR during myogenic
differentiation.18
We suggest that the up-regulation of
Ngfa observed in the tamoxifen- and toremifene-treated uteri
may contribute to the repression of myometrial differentiation in these
tissues.
Pref-1 is abundant in preadipocytes and is down-regulated during adipocyte differentiation; it is also known that the expression of Pref-1 inhibits differentiation of mouse 3T3-L1 preadipocytes.19 The microarray data presented here demonstrate the down-regulation of Pref-1 in tamoxifen- and toremifene-treated neonatal uteri. We suggest that the down-regulation of Pref-1 contributes to the appearance of adipocytes observed in the abnormally differentiated neonate tissue.
The paternally imprinted Igf-2 precursor is repressed by all of the SERMs tested, although the effect was most marked with toremifene treatment. In contrast, the maternally imprinted gene for the mannose 6-phosphate/insulin-like growth factor-2 receptor showed little treatment-related change in expression (not shown). Insulin-like growth factors are believed to mediate and modulate steroid hormone actions in the endometrium,20 IGF-2 expression is also involved in endometrial differentiation.21 A previous study of patients with endometriosis showed a reduced level of staining for IGF-2 in the eutopic endometria of affected women.22 Elevated expression of IGF-2 is often found in tumors, and loss of imprinting is one mechanism by which its expression is deregulated.23 However, we speculate that the observed reduction in Igf-2 expression may contribute to the altered growth and differentiation of the uterus.
With the present technology, we could only examine changes in gene expression taking RNA from the whole uterus. Clearly, the response of the different endometrial, stromal, and myometrial components will differ. If the molecules are expressed in different cell types the changes in their levels may be larger than when analyzed with the present methods. Techniques such as laser capture microdissection will be needed to examine cell-specific responses to these drugs.
In summary, these experimental data from this study support the hypothesis that adenomyosis represents a condition of the uterine body in which the stromal cells have a primary pathogenetic role although some contribution of accelerated epithelial downgrowth cannot be entirely excluded. Disruption of the mesenchymal layers surrounding the endometrium in the neonatal period can give rise to disordered development of uterine stroma, smooth muscle, blood vessels, and possibly its innervation. This alteration to the normal functional fibromuscular anatomy of the uterine body provides the framework for the abnormal and aberrant growth of endometrial tissue. Most importantly, these data suggest that discrete hormonal derangements, that produce defects in the formation of the myometrium early in neonatal life in humans, may explain the predisposition to adenomyosis in adulthood.
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Acknowledgements |
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Footnotes |
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Accepted for publication April 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This article has been cited by other articles:
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  A. Hever, R.B. Roth, P.A. Hevezi, J. Lee, D. Willhite, E.C. White, E.M. Marin, R. Herrera, H.M. Acosta, A.J. Acosta, et al. Molecular characterization of human adenomyosis Mol. Hum. Reprod., December 1, 2006; 12(12): 737 - 748. [Abstract] [Full Text] [PDF]
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