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Center for Experimental Therapeutics and Department of Pharmacology (Y.M.Q., X.J.S., M.H.T., X.P.L., W.C.S.) and Center for Research on Reproduction and Women’s Health, Department of Genetics (J.R.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: W.-C. Song, Ph.D., University of Pennsylvania School of Medicine, Room 1351, Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: song{at}spirit.gcrc.upenn.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the various metabolic routes, sulfation has been recognized for many years as a prominent pathway for estrogen transformation in vivo (5, 6). Sulfated estrogen has diminished receptor-binding affinity and is more water-soluble. Although sulfation is generally regarded as a metabolic process, the observation that, in some species, estrogen sulfate actually circulates in the blood at levels higher than that of the parent hormone suggests that estrogen sulfate may not be produced merely for the purpose of excretion (5, 6). Because estrogen sulfate can be hydrolyzed to release the active hormone, it may exist in the blood as a reservoir for expeditious production of active estrogen in target tissues (5, 6). Irrespective of its role in systemic estrogen homeostasis, in vitro experiments have suggested that sulfation may represent a physiologic mechanism to regulate local estrogen activity in target tissues (7, 8, 9).
To test this hypothesis, we have chosen to study the male reproductive system of the mouse as a model. In earlier studies, we have shown that an estrogen-specific sulfotransferase (EST) is expressed abundantly in the Leydig cells of the mouse testis (10, 11, 12). Because testis is a major source of estrogen biosynthesis in the males (13, 14, 15, 16, 17, 18), and several tissues within the male reproductive system are established estrogen target sites (19, 20, 21, 22), we speculated that Leydig cell EST may act as a molecular switch to modulate the intracrine and paracrine activity of the locally produced estrogen. In this study, we have generated an EST-deficient mouse through targeted disruption of the EST gene and characterized the phenotype of the male reproductive system of the mutant mouse. We found that EST knockout male mice developed age-dependent structural and functional lesions in their reproductive systems because of increased local estrogen activity. This finding provides direct evidence for a physiological role of estrogen metabolism in intracrine and paracrine estrogen regulation. It also implies that inhibition of estrogen transformation enzymes by environmental chemicals, as has been shown recently for the human EST (23, 24), may constitute a novel mechanism of endocrine disruption in vivo that is independent of their direct interaction with the estrogen receptors (25).
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Materials and Methods |
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Construction of targeting vector
To clone the mouse EST gene, a 129J/Sv 
FixII murine genomic
library (Stratagene, La Jolla, CA) was screened,
with the full-length mouse EST cDNA as a probe (10). The
mouse EST gene was found to consist of eight exons (unpublished
results). A 13-kb genomic clone, containing exon II to VII, was chosen
for targeting vector construction. In the first step, a cassette
containing the herpes simplex virus-thymidine kinase gene under the
control of the mouse phosphoglycerate kinase-1 gene promoter was
excised from the pPNT vector (26) at the unique
HindIII and EcoRI sites and was subcloned into
pCDNA3 vector (Invitrogen, Carlsbad, CA). This was
followed by insertion of the 13-kb EST gene fragment at the
NotI site of pCDNA3. Finally, a neo gene
cassette (NEO) containing the SV40 gene promoter was amplified by PCR
from the pKO vector (Stratagene) using primers containing
a KpnI site, cloned into the TA vector
(Invitrogen), and then inserted into exon III of the
EST gene fragment at a unique KpnI site (Fig.
1A).
Generation of EST-deficient mice
The targeting vector was linearized by XhoI digestion
and transfected into TL-1 embryonic stem (ES) cells (kindly provided by
Dr. P. Laboski, University of Pennsylvania) by electroporation. This
procedure and the subsequent colony screening were carried out as
previously described (27). Genomic DNAs of ES cells were
digested with XbaI and screened by Southern blot analysis
using a 300-bp probe 5' to the targeting sequence (in the 5' flanking
region of the gene, Fig. 1A
). In this screening scheme, the wild-type
(WT) EST gene produced a 6-kb XbaI band, whereas the
targeted allele was expected to show a 7-kb band attributable to
incorporation of the neo gene (Fig. 1A). Confirmation
hybridization on positive clones was subsequently carried out by using
the neo cDNA as a probe. Targeted ES cell clones were
injected into C57BL/6 mouse blastocyst to produce chimeric mice
(28). Germline transmission of the targeted gene was
established by coat color (agouti) of the progeny from the matings
between male chimeric mice and normal C57BL/6 female mice. Heterozygous
EST knockout mice (F1 generation) were intercrossed, and the genotypes
of their offspring were determined by Southern blot analysis of
XbaI digested tail DNA (26, 27). Phenotypic
characterization was carried out by using mice of the F2 (first
generation of homozygous knockout) or F3 generation that had a mixed
129/B6 background.
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Steroid measurement and fertility test
Blood was obtained by tail vein bleeding or through cardiac
puncture. Serum T and E2 levels were measured with RIA kits by
following the manufacture’s instructions (Diagnostic Systems Laboratories, Inc., Webster, TX). To assess the knockout
mouse fertility, 4 mating experiments were performed. In the first
experiment, 5 mating pairs of 2-month-old WT and knockout mice were set
up, and their litter and pup numbers were recorded for 6 months. In the
second experiment, 6 WT and 6 knockout males were mated for 2 months
with 8-wk-old WT C57BL/6 females (The Jackson Laboratory,
Bar Harbor, ME). For this experiment, each male was housed with
four females in a cage. In the third mating experiment, 15 WT and 15
knockout females were mated for 2 months with 8-wk-old WT B6/D2 F1
males. The females were divided into 4 mating cages (4, 4, 4, 3), with each cage containing 1 male mouse. In the fourth mating
experiment, 5 WT and 5 knockout males, between 17–18 months old, were
mated with 8-wk-old WT C57BL/6 females (The Jackson Laboratory) for 2 months. In this experiment, each male was
housed with 2 females in a cage.
Histology and immunohistochemistry
Testes were harvested from age-matched WT and knockout mice. In
some experiments, 2-month-old WT and knockout mice were treated with
exogenous estrogen for 3 wk (10 µg, 3-wk release E2 pellet, sc;
Innovative Research of America, Sarasota, FL), and
testes were collected. Tissues were fixed in cold Bouin’s solution for
24–72 h. They were then dehydrated and paraffin-embedded. Sections of
the embedded testes were made at 5 µm, and the slides were processed
either for conventional hematoxylin and eosin staining or for
immunohistochemistry. The later procedure was carried out using the
Vectastain ABC Elite kit from Vector Laboratories, Inc. (Burlingame, CA) and a rabbit polyclonal antimouse EST
antibody, as previously described. (11).
Sperm motility assays
Sperm were collected from caudal epididymides and placed in
Hanks’ medium containing 2 mg/ml BSA. They were kept at 37 C until
being examined microscopically. Sperm total and forward motility were
determined visually on a computer screen by phase contrast microscopy.
For each sperm sample, at least 10 viewing fields, each containing
20–40 sperm, were counted. Total sperm motility was defined as clear
head and flagellar movement, whereas only progressive sperm movement
was counted as forward motility.
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Results |
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. Insertion of the neo gene cassette into exon III was
expected to disrupt the coding frame of the EST gene and yield a
nonfunctional protein. The successful generation of homozygous
EST-deficient mice was shown first by Southern blot analysis of tail
DNAs. As can be seen in Fig. 1B, WT (+/+) mouse DNA produced a single
6-kb XbaI band; whereas in heterozygous EST knockout mice
(+/-), a 7-kb band (attributable to 1-kb neo gene cassette
insertion), as well as the 6-kb band, was observed. In homozygous EST
knockout mice (-/-), only the 7-kb band was detected, indicating that
both alleles of the EST gene had been mutated (Fig. 1B). The genotypes
of offspring from heterozygous mating displayed a characteristic
Mendelian distribution (of 105 mice examined, 26 were WT, 52 were
heterozygous, and 27 were nullizygous). There was also no gender bias,
with 13 of the 27 nullizygotes being males.
To confirm that the EST gene had been functionally inactivated and to
investigate whether anomalous EST mRNA and protein (attributable to
added neo sequence in exon III) had been produced, Northern
and Western blot analyses and EST enzyme activity assays of testicular
homogenate were performed. As shown in Fig. 2, A and B, whereas both EST mRNA and
protein were detected in the testis of WT mice as expected, no mRNA or
protein of either normal or aberrant size could be detected in the
knockout mice. Similarly, E2-sulfating activity was detected in the
testis homogenate of WT but not that of nullizygous mice (Fig. 2C).
Immunohistochemical analysis also confirmed that EST expression in
Leydig cells is disrupted (Fig. 2, D and E).
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). However,
subsequent cross-breeding experiments suggested that the reduced
fertility is most likely caused by an uncharacterized impairment in the
female reproductive function (Table 1).
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). Unlike normal Leydig
cell clusters, which were composed of 10–20 cells of similar size that
stained prominently with eosin (Fig. 3, A and C), masses of
hypertrophic Leydig cells with volumes 2–3 times larger than those of
normal Leydig cells were detected in the mutant mouse testes (Fig. 3; B
and E). Compared with that of the WT cells, the cytoplasm of the
knockout Leydig cells stained less uniformly with eosin and contained
vacuoles (Fig. 3D). Hyperplasia occurred more frequently (although not
exclusively) in the peripheral space under the testicular capsule (Fig. 3, B and E), but hypertrophy of Leydig cells was commonly observed
within inner tubular junctions (Fig. 3D). The second abnormality in the
knockout testes was the presence of numerous so-called giant yellow
cells (Fig. 3, E and F). These cells contained multiple dark stained
nuclei and were detected in the hyperplastic lesions found in the
peripheral space (Fig. 3E) as well as in Leydig cell clusters located
at inner tubular junctions (Fig. 3F). In many of the sections, we also
noticed a thickening of the capsule of the knockout testis, but this
putative phenotype remains to be verified with more objective
morphometric analysis. Finally, seminiferous tubule damage was observed
in the majority of testis sections of 12-months and older knockout
mice. These abnormal tubules were filled with vacuoles and had
disrupted spermatogenic epithelium (Fig. 4, A and B). They were usually localized
adjacent to hypertrophic and hyperplastic Leydig cell lesions (Fig. 4, A and B).
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, it did not cause appreciable Leydig cell damage in the WT
mouse. In contrast, severe Leydig cell hypertrophy/hyperplasia and
seminiferous tubule damage were observed in the estrogen-treated
knockout mouse testis (Fig. 4D). Thus, exogenous estrogen accelerated
the development of structural lesions in the knockout mouse testis.
Reduced sperm motility and litter size, and other reproductive
tract abnormalities in older knockout mice
To further evaluate the long-term consequence of EST deficiency in
the male reproductive system, we examined, in addition to testis
histology, the sperm motility and general morphology of the male
reproductive tracts of 18- to 22-month-old mice and compared them with
that of 3-month-old mice. In addition to Leydig cell
hypertrophy/hyperplasia and seminiferous tubule damage, as described
above, there was also a significant reduction in the relative weights
of the testis and epididymis of 18- to 22-month-old knockout mice
(organ weight/body weight x 100: testis, 0.63 ± 0.05 for
WT, 0.51 ± 0.04 for knockout; epididymis, 0.43 ± 0.06 for
WT, 0.31 ± 0.07 for knockout, mean ± SD,
P < 0.05, t test, Fig. 5E). A most striking abnormality was
found with the knockout mouse seminal vesicle/coagulating gland, which
had significantly increased wet weight because of tissue swelling
[organ weight/body weight x 100: 0.85 ± 0.15 for WT, 1.44
±0.47 for knockout, mean ± SD, P < 0.05,
t test (Fig. 5; A, B, and E)]. Furthermore, although caudal
sperm number was not significantly decreased, total and forward
motility of sperm isolated from caudal epididymis of 18- to
22-month-old knockout mice were reduced by 60% and 80%, respectively
(Fig. 5F). In contrast, the reproductive tracts of 3-month-old knockout
mice were largely normal (organ weight/body weight x 100: testis,
0.81 ± 0.03 for WT, 0.70 ± 0.05 for knockout; epididymis,
0.45 ± 0.05 for WT, 0.41 ± 0.07 for knockout; seminal
vesicle/coagulating gland, 1.04 ± 0.15 for WT, 1.09 ± 0.11
for knockout; mean ± SD, Fig. 5C), and there was no
reduction in sperm total or forward motility in these younger knockout
mice (Fig. 5D). To assess whether the reduced motility of caudal sperm
from the older knockout males is associated with reduced fertility, a
controlled mating experiment was conducted in which five 17- to
18-month-old WT or knockout males were mated with 8-wk-old WT females.
During a 2-month period, the knockout males sired a number of litters
similar to that of the WT mice. However, the litter size in the
knockout group was significantly smaller (Table 2).
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Discussion |
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Except for a small decrease in testicular weights (Fig. 5C), the
reproductive system of younger (3–6 months) knockout male mice
appeared normal, both structurally and functionally. Serum T and E2
levels in the knockout males were in the normal range. Although we
initially found a reduction in the fertility of the knockout mice,
subsequent cross-breeding experiments suggested that the impairment
most likely lay with the female knockout mice. The conclusion that
younger knockout males had normal fertility is consistent with the
result of sperm motility assays, which detected no abnormality in the
knockout males of this age group (Fig. 5D). The nature of the female
reproductive defect in the EST knockout mice remains to be
characterized; but we recently have discovered that, in addition to
Leydig cells of the testis, EST is also expressed in late stages of the
mouse placenta and uterus (our unpublished observations).
Interestingly, as mice aged, EST knockout mice were found to develop with high-incidence Leydig cell hypertrophy/hyperplasia and seminiferous tubule damage. The time-dependent nature in the development of these structural lesions suggested that the damaging effect of EST deficiency is chronic and progressive. In theory, these Leydig cell abnormalities could result from changes in the hypothalamus-pituitary-testis axis or from increased intracrine estrogen stimulation in the absence of intracellular EST. Because EST is not expressed in the mouse brain (10) and serum steroid levels were normal in the knockout mice, the most likely explanation for this phenotype is increased intracrine estrogen stimulation, rather than systemic hormonal disturbance. Consistent with this hypothesis, administration of exogenous E2 caused no visible Leydig cell abnormalities in the WT mice but induced highly similar structural lesions in the testes of younger knockout mice. This differential response by the knockout Leydig cells contrasted with spermatogenesis suppression, which occurred, to a similar degree, in the WT and knockout mice and which was likely attributable to the systemic effect of estrogen involving the hypothalamus-pituitary-testis axis (29). However, the possibility cannot be excluded that knockout Leydig cells had altered sensitivity to gonadotropin disturbances caused by E2 administration. On the other hand, it is well known that Leydig cells not only produce estrogen (13, 30) but also are targets of direct estrogen stimulation. For example, estrogen receptors have been detected in both the human and mouse Leydig cells (31, 32), and ectopic exogenous E2 has been shown to cause Leydig cell tumors in susceptible strains of mice (33, 34).
Two conspicuous features of the Leydig cell abnormality in the knockout mice were noticed and remain to be explained mechanistically. The first is that the hypertrophic/hyperplastic Leydig cell masses were more frequently localized in the peripheral space under the testicular capsule. This suggests that either Leydig cells localized in these regions were phenotypically different (i.e. more responsive to estrogen stimulation) from those residing in the inner areas of the testis, or that there were important paracrine factors released from other cell types nearby (e.g. stroma cells under the capsule) that acted in concert with estrogen to cause Leydig cell proliferation. A second conspicuous feature concerning the knockout Leydig cells is the presence of multinucleated giant yellow cells in the interstitial space and the hyperplastic lesions. The identity of these cells is not yet known. It is possible that these cells were estrogen-stimulated Leydig cells that had undergone repeated, but incomplete, mitogenesis. Alternatively, they may represent a subclass of testicular macrophages undergoing active phagocytosis of damaged Leydig cells.
Because EST is not expressed in the seminiferous tubules
(11), the disruption of tubular structure in older EST
knockout mice is likely a result of altered paracrine stimulation.
Changes in paracrine stimulation may be caused by increased paracrine
estrogen activity. Additionally, the hypertrophic/hyperplastic Leydig
cells may also release other nonsteroidal paracrine factors
(35) that could negatively impact the local
microenvironment and ultimately contribute to the development of a
seminiferous tubule defect. This scenario was supported by the fact
that abnormal tubular segments were mostly observed in areas
immediately adjacent to the hyperplastic Leydig cell lesions (Fig. 4A).
A similar paracrine effect of unmetabolized estrogen originating from
the testis may account for the structural and functional abnormalities
observed in the seminal vesicle and epididymis in older knockout mice,
although potential localized expression of EST in various accessory
organs at different developmental stages remains to be examined in
detail. The effect of estrogen on the accessory structures of the male
reproductive tract of the mouse has been demonstrated in other studies
and is known to be mediated by estrogen receptor (20, 21, 22, 36).
In summary, we have described a number of age-dependent abnormalities
in the male reproductive system of EST knockout mice. These
observations provide direct evidence for a physiological role of
estrogen metabolism in intracrine and paracrine regulation of estrogen
activity. They also shed new light on the biology of estrogen in the
male reproductive system of mammals. There is now little doubt that
estrogen is required for normal testicular function, as has been
demonstrated by recent studies of estrogen receptor 
and P450
aromatase gene knockout mice (19, 20, 37, 38, 39). The present
study indicates that, whereas estrogen is required in the testis, there
is also a physiological need to maintain proper intracrine and
paracrine estrogen homeostasis in the male reproductive tract. Our
results further imply that inhibition of estrogen transformation
enzymes, such as EST, as has been demonstrated recently for human EST
with several common environmental chemicals (23, 24), may
constitute a novel mechanism of endocrine disruption by environmental
chemicals that is independent of their direct interaction with the
estrogen receptors.
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Acknowledgments |
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Footnotes |
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Abbreviations: ES, Embryonic stem; EST, estrogen sulfotransferase; WT, wild-type.
Received June 12, 2001.
Accepted for publication August 15, 2001.
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