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Original Studies |
Hospital for Children and Adolescents, University of Helsinki (V.P., K.E., L.S. L.D.), FIN-00029 Helsinki, Finland; and Department of Anatomy, University of Turku (M.P.), FIN-20520 Turku, Finland
Address all correspondence and requests for reprints to: Virve Pentikäinen, M.D., Hospital for Children and Adolescents, University of Helsinki, P.O. Box 281, FIN-00029 Helsinki, Finland. E-mail: virve.pentikainen{at}hus.fi
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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knockout and aromatase
(cyp 19 gene) knockout mice. However, direct testicular
effects of estrogens in male reproduction have remained unclear. Here
we studied the protein expression of ER and the recently described
estrogen receptor ß in the human seminiferous epithelium and
evaluated the role of 17ß-estradiol, the main physiological estrogen,
in male germ cell survival. Interestingly, both estrogen receptors
and ß were found in early meiotic spermatocytes and elongating
spermatids of the human testis. Furthermore, low concentrations of
17ß-estradiol (10-9 and 10-10 mol/L)
effectively inhibited male germ cell apoptosis, which was induced
in vitro by incubating segments of human seminiferous
tubules without survival factors (i.e. serum and
hormones). Dihydrotestosterone, which, in addition to estradiol, is an
end metabolite of testosterone, was also capable of inhibiting
testicular apoptosis, but at a far higher concentration
(10-7 mol/L) than estradiol. Thus, estradiol appears to be
a potent germ cell survival factor in the human testis. The novel
findings of the present study together with the previously reported
indirect effects of estrogens on male germ cells indicate the
importance of estrogens for the normal function of the testis. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Estrogens exert their cellular effects through estrogen receptors (ER)
that exist in at least two subtypes, ER (11, 12) and the recently
described ERß (13, 14), which differ in the C-terminal ligand-binding
domain and in the N-terminal trans-activation domain. These
two subtypes of ER have similar high affinities for 17ß-estradiol,
but some synthetic and naturally occurring ligands have different
relative affinities for ER and ERß (15). In the male reproductive
tract, ER has been shown to be strongly expressed in the epididymis
and efferent ductules (16, 17, 18, 19, 20). It has also been found in the Leydig
cells of the rat testis (16), whereas the seminiferous epithelium has
been thought to be negative for ER. ERß messenger ribonucleic acid
(mRNA), in turn, has been described in germ cells, particularly in
primary spermatocytes and round spermatids, of the human testis (21).
In addition, ERß mRNA and protein have been shown to be expressed by
Leydig cells and elongated spermatids of the mouse testis (22) as well
as by Sertoli cells and type A spermatogonia of the developing rat
testis and by Sertoli cells, pachytene spermatocytes and round
spermatids of the adult rat testis (23, 24). Thus, the expression
patterns of ERs seem to be rather complicated and to show at least some
species variation. Moreover, a recent study revealed a novel functional
estrogen receptor on the human sperm membrane that is clearly smaller
than the ER and ERß (25). The physiological significance of this
ER remains to be elucidated.
Recent investigations of mice deficient in ER (ER knockout,
ERKO) (26, 27) or aromatase (cyp 19 gene knockout, ArKO)
(28) have provided direct evidence for a physiological role of
estrogens in male reproductive organs. The ERKO males were infertile
(26). Their testes appeared normal until puberty, but then began to
degenerate, with disruption of spermatogenesis (26). The infertility
was suggested to be caused by impaired fluid reabsorption in the
efferent ductules, resulting in diluted sperm, increased back-pressure
in the seminiferous tubules, and related atrophy of the seminiferous
epithelium (27). Thus, estrogens have been thought to have only an
indirect effect on the developing germ cells of the seminiferous
epithelium. However, ArKO mice were also shown to possess progressive
disruption of spermatogenesis and infertility, but there was no
evidence of abnormal fluid reabsorption by the efferent ductules (28).
These results from ArKO mice and the recently reported expression of
ERß in the human (21), mouse (22), and rat (23, 24) seminiferous
epithelium suggest additional direct effects of estrogens on
spermatogenesis.
Spermatogenesis is a unique process of germ cell proliferation and
maturation from diploid spermatogonia through meiosis to mature haploid
spermatozoa. Before reaching maturity, a number of germ cells undergo
physiological apoptotic death, which has been shown to be controlled by
FSH and testosterone (reviewed in Ref. 29). In this context, we have
recently shown that testosterone inhibits apoptosis of human testicular
germ cells in vitro (30). The role of estrogens, however,
has been completely unknown. The aim of the present study was to
evaluate the direct effects of the main physiological estrogen,
17ß-estradiol, on germ cell apoptosis in the human testis. As no
reports concerning the protein expression of ER and ERß in the
human testis were available, we first used immunohistochemistry and
Western blotting to study the expression of these receptors in the
adult human testis. We then induced germ cell apoptosis in our recently
described in vitro model (30) and studied the effects of
17ß-estradiol and dihydrotestosterone (DHT), the two end metabolites
of testosterone, on the process of cell death.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Testis tissue was obtained from men, aged 60–80 yr, undergoing orchidectomy as treatment for prostate cancer. They had not received hormonal or chemotherapeutic medication or radiotherapeutic treatment for the cancer before the operation. They had no endocrinological disease, and none of them had suffered from cryptorchidism. The operations were performed between November 1996 and December 1999 at the Department of Urology, University of Helsinki, and at the Helsinki City Health Department, Surgical Unit (Helsinki, Finland). The ethics committees of the Hospital for Children and Adolescents and the Department of Urology, University of Helsinki, approved the study protocol.
Immunohistochemical staining of ER and ERß
Small segments of human seminiferous tubules (
1 mm in length)
were squashed under coverslips and fixed as previously described (31).
These squash preparations were rehydrated, washed twice for 5 min each
time in phosphate-buffered saline (PBS), and blocked with blocking
solution (PBS containing 5% normal serum, 3% BSA, and 0.1% Tween 20)
for at least 1 h at room temperature. In our preliminary studies
we found that the negative controls, in which the primary antibody was
replaced with PBS, stayed negative regardless of whether the endogenous
peroxidases were blocked in methanol containing 1%
H2O2. Therefore, it appears
that in the human testis, endogenous peroxidases are not present in
amounts that would affect the immunohistochemical staining of the ERs.
The ER protein in the preparations was detected using a rabbit
polyclonal antibody to human ER (HC-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 0.2 µg/mL or mouse
monoclonal antibodies to human ER (NCL-ER-6F11, Novocastra
Laboratories Ltd., Newcastle, UK; or ER-1D5, DAKO Corp.
A/S, Glostrup, Denmark) at dilutions of 1:10 and 1:100,
respectively. The ERß protein was detected using rabbit polyclonal
antibody to human ERß (PAI-313, Affinity BioReagents, Inc., Golden, CO) at 10 µg/mL. The primary antibodies were
added to the preparations in blocking solution, and incubation was
performed overnight at 4 C. After incubation, the slides were washed
three times for 5 min each time in PBS. The primary antibody was
detected using biotin-conjugated goat antirabbit IgG or horse antimouse
IgG secondary antibodies from the corresponding ABC-Elite Kits
(Vector Laboratories, Inc., Burlingame, CA) followed by
incubation with ABC solution. For location of the antibody, 0.05%
diaminobenzidine substrate (Sigma, St. Louis, MO) was
added. Light counterstaining was performed with hematoxylin. A blocking
peptide was used to verify the specificity of the polyclonal antibody
to ER.
Western blot analysis
Small tissue sections were homogenized on ice in homogenization
buffer [1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris (pH 7.4), 1
mmol/L ethylenediamine tetraacetate, 1 mmol/L
ethyleneglycol-bis-(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, 0.2 mmol/L phenylmethylsulfonylfluoride, and 1 µg/mL
leupeptin]. After centrifugation at 17,000 x g for 30
min, the supernatants were collected, and their protein concentrations
were determined by DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Proteins (25–50 µg) were loaded onto
SDS-polyacrylamide gels, and electrophoresis was performed at 180 V.
The proteins were transferred to polyvinylidene difluoride membranes
(Immobilon-P, Millipore Corp., Bedford, MA) by
electrophoresis for 2 h at 4 C in transfer buffer (26 mmol/L Tris,
192 mmol/L glycine, and 10% methanol) at 100 V. The transfer was
checked by staining with 0.2% Ponceau S in 3% trichloroacetic acid.
ER and ERß proteins on the membranes were detected using rabbit
polyclonal antibodies to human ERs. The ER antibodies HC-20
(Santa Cruz Biotechnology, Inc.) and H-184 (Santa Cruz Biotechnology, Inc.) recognize amino acids 576–595 at the
carboxyl-terminus and 2–185 at the amino-terminus of human ER,
respectively, and they were used at 0.1 and 0.4 µg/mL. The ERß
antibody PAI-313 (Affinity BioReagents, Inc.) was used at
1 µg/mL. The primary antibodies were followed by horseradish
peroxidase-conjugated secondary antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA). The bound secondary antibody was located
with the ECL detection kit (Amersham Pharmacia Biotech,
Arlington Heights, IL). The specificity of the HC-20 antibody to ER
was confirmed using the corresponding blocking peptide.
Tissue culture
Apoptosis of the human testicular germ cells was induced
in vitro by incubating segments of seminiferous tubules
under serum- and hormone-free culture conditions (i.e.
without survival factors). Segments of seminiferous tubules were
cultured, instead of isolated germ cells, to maintain an environment as
physiological as possible for the germ cells. Testis tissue was
microdissected under a transillumination stereomicroscope in a petri
dish containing PBS supplemented with 0.1% BSA (Sigma).
Segments of seminiferous tubules (3 mm in length) were isolated and
transferred to culture plates. Each plate contained tissue culture
medium (nutrient mixture Ham’s F-10, Life Technologies, Inc., Europe, Paisley, UK) supplemented with 0.1% human albumin
(Sigma) and 10 µg/mL gentamicin (Life Technologies, Inc.). The samples were incubated for 2–24 h
under serum- and hormone-free conditions (i.e. without
survival factors) at 34 C in a humidified atmosphere containing 5%
CO2.
Inhibition of germ cell apoptosis in the testis tissue culture
To study the effects of estrogens on male germ cell apoptosis,
17ß-estradiol (Sigma) was added to the tissue cultures
at final concentrations of 10-7,
10-9, and 10-10 mol/L (in
preliminary studies, estradiol concentrations from
10-6–10-10 mol/L were
tested). To test whether the effects of 17ß-estradiol were mediated
by the ERs, an ER antagonist, ICI 182,780 (Tocris Cookson Ltd.,
Bristol, UK), was added at 10-7 mol/L
simultaneously with estradiol. The effects of DHT on germ cell death
were also studied by adding DHT (5-androstan-17ß-ol-3-one, Fluka
Chemie Ag, Buchs, Switzerland) to the culture medium at final
concentrations of 10-7,
10-8, and 10-9 mol/L.
Apoptosis was detected by Southern blot analysis of low molecular
weight DNA fragmentation, by in situ end labeling (ISEL)
analysis of apoptotic cells in the squash preparations of seminiferous
tubules, and by electron microscopy using the established morphological
criteria of apoptosis.
Southern blot analysis of apoptotic DNA fragmentation
Tissue samples were snap-frozen in liquid nitrogen and stored at -70 C until DNA isolation. Genomic DNA was extracted using the Apoptotic DNA Ladder Kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions, with some modifications. Briefly, the testis tissue samples were homogenized and incubated for 10 min at room temperature in binding/lysis buffer (6 mol/L guanidine-HCl, 10 mmol/L urea, 10 mmol/L Tris-HCl, and 20% TritonX-100, pH 4.4). The samples were then mixed with isopropanol (final proportion of isopropanol, 25%), loaded into polypropylene tubes, and centrifuged for 1 min at 8000 rpm. The tubes were washed twice with washing buffer (20 mmol/L NaCl and 2 mmol/L Tris-HCl, pH 7.5), and the bound DNA was eluted from the tubes with 10 mmol/L Tris, pH 8.5. Finally, the samples were incubated with ribonuclease (2.5 µg/mL; deoxyribonuclease-free ribonuclease, Roche Molecular Biochemicals) for 20 min at room temperature. After quantification, the DNA samples were 3'-end labeled with digoxigenin-dideoxy-UTP (Dig-dd-UTP; Roche Molecular Biochemicals) by the terminal transferase (Roche Molecular Biochemicals) reaction, subjected to electrophoresis on 2% agarose gels, and blotted onto nylon membranes overnight. The next day the DNA was cross-linked to the membranes by UV irradiation. The membranes were then washed and blocked for 30 min at room temperature. Apoptotic, 3'-end labeled, DNA on the membranes was detected by the antibody reaction (anti-Digoxigenin-AP, alkaline phosphatase-conjugated; Roche Molecular Biochemicals), as recently described (30). For the luminescence reaction, the membranes were incubated in disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}4-yl)phenyl phosphate solution (Roche Molecular Biochemicals) for 5 min at room temperature. The membranes were then enclosed in hybridization bags, incubated for 15 min at 37 C, and exposed to x-ray films. The films were scanned with a tabletop scanner (Microtek ScanMaker, Microtec International, Inc., Taiwan), and the digital image was analyzed with NIH-Image (1.61) analysis software. The digitized quantification of the low molecular weight DNA fragments (185-bp multiples) of the sample cultured for 4 h without survival factors was set at 1.0 (100% apoptosis), and the amounts of low molecular weight DNA fragments in the other samples were expressed in relation to it.
ISEL of apoptotic DNA
Squash preparations from human seminiferous tubules were rehydrated and washed twice for 5 min each time in distilled water. After incubation for 10 min with terminal transferase reaction buffer (1 mol/L potassium cacodylate, 125 mmol/L Tris-HCl, and 1.25 mg/mL BSA, pH 6.6), the apoptotic DNA was 3'-end labeled with Dig-dd-UTP (Roche Molecular Biochemicals) by the terminal transferase reaction for 1 h at 37 C. For the negative controls, the terminal transferase enzyme was replaced with the same volume of distilled water. Dig-dd-UTP was detected with the antidigoxigenin antibody conjugated to horseradish peroxidase (Anti-Digoxigenin-POD, Roche Molecular Biochemicals). For location of the antibody, 0.05% diaminobenzidine substrate (Sigma) was added. Light counterstaining was performed with hematoxylin, and the samples were dehydrated and mounted.
Electron microscopy
Segments of seminiferous tubules were cultured as described above. They were then fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, dehydrated, and embedded in epoxy resin. The samples were sectioned at 50 nm with an E Ultramicrotome (Reichert Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate. Observations were made with a JEM 1200 EX transmission electron microscope (JEOL USA, Inc., Tokyo, Japan). Germ cells were identified according to their characteristic morphology (32). Apoptosis was recognized by typical ultrastructural changes, including condensation of nuclear chromatin and degeneration of cytoplasmic organelles.
Statistical analysis
The experiments for Southern blot analysis of DNA fragmentation were repeated on at least three independent occasions. Quantitative data represent low mol wt DNA (optical density from x-ray films). The data obtained from the samples incubated for 4 h without survival factors were set at 1.0 (100% apoptosis), and the data from the samples treated with 17ß-estradiol or DHT were compared to it. Data obtained from three to seven replicate experiments (mean ± SEM) were analyzed by one-sample t test. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and ERß in the adult human seminiferous
epithelium
Localization of the ER and ERß proteins in the adult human
testis was studied immunohistochemically using a rabbit polyclonal
antibody (HC-20) and mouse monoclonal antibodies (ER-6F11 and ER-1D5)
to human ER and a rabbit polyclonal antibody to human ERß.
Representative samples of cells were obtained from human seminiferous
epithelium by squashing segments of the seminiferous tubules under
coverslips. With this technique, cells from the seminiferous epithelium
migrate under the coverslip to produce a monolayer. The cells maintain
their morphological characteristics, allowing identification of
individual cell types.
Strong positive staining for the ER protein was observed in early
meiotic germ cells (zygotene and early pachytene primary spermatocytes)
and in early elongating spermatids (Fig. 1A
). Whether we used the polyclonal
(HC-20) or the monoclonal (ER-6F11 or ER-1D5) antibodies to ER, the
result was the same. The specificity of the staining with the HC-20
antibody was confirmed by incubating the primary antibody with an
excess of the corresponding peptide before use. When this blocking
peptide was used, there was only faint background staining, which was
not located in the cells. There was no staining when the primary
antibodies were replaced with PBS (negative controls).
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) as the ER. The strong positive staining of the
early primary spermatocytes and the early elongating spermatids for the
ERß protein accords with the previously reported expression of ERß
mRNA in primary spermatocytes and spermatids of the human testis. No
staining was observed in the negative controls, i.e. when
the primary antibody was replaced with an equal volume of PBS.
Western blot analysis of human seminiferous tubules showed strong
expression of both ER and ERß (Fig. 2). Both polyclonal ER antibodies
HC-20 and H-184 recognized a band corresponding to a molecular mass of
about 80 kDa, which is clearly higher than the usually reported
molecular mass of human ER (66–70 kDa). The 80-kDa band was also
detected in the samples of human ovary and endo/myometrium with both
antibodies, although, with H-184, extended ECL exposure time was needed
to detect the band in endo/myometrium. With HC-20, we observed two
additional faint bands of about 50–55 kDa in both the male and female
tissue samples. The 80-kDa band and the upper of the additional bands
disappeared when the HC-20 antibody was incubated with the
corresponding blocking peptide before use, indicating specificity of
these bands. With H-184, in turn, an additional faint band of
approximately 60 kDa was detected in all of the male and female tissue
samples studied. To further confirm the specificity of the polyclonal
antibodies to ER, we performed the Blast sequence similarity search
with the peptide sequences corresponding to HC-20 and H-184. The only
proteins for which similarity with these sequences was found were ER
with its different splice variants. Thus, in Western blot analysis of
human seminiferous tubules, ovary, and endo/myometrium, we observed a
band representing the ER protein and migrating at a mobility of 80
kDa. The other specific bands observed may represent different splice
variants of the ER. Western blot analysis of the same tissues using
the polyclonal antibody to ERß revealed a protein of approximately 60
kDa, corresponding to the size of human ERß. The expression of both
receptors remained constant during the culture of the seminiferous
tubules in serum- and hormone-free conditions for various lengths of
time.
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In the present in vitro model, human germ cells were cultured in their natural surroundings, i.e. in the seminiferous tubules, to maintain as physiological an environment as possible. Germ cell apoptosis was induced in this model by incubating segments of seminiferous tubules under serum- and hormone-free conditions (i.e. without survival factors). Apoptotic cells were identified by electron microscopy, using the characteristic morphology of the different germ cell types (32) and the established morphological criteria of apoptosis. Typical signs of the apoptotic cells were, for example, condensation of nuclear chromatin, degeneration of cytoplasmic organelles, and, in the later stages of apoptosis, dispersion of the nuclear envelope. The morphological signs of apoptosis were seen most often in spermatocytes and spermatids. Some of the apoptotic spermatids showed a ring-like condensation of chromatin around the nuclear periphery, which is characteristic for apoptosis of this type of germ cell. Late apoptotic cells were impossible to identify.
Inhibition of human testicular apoptosis by 17ß-estradiol
To evaluate the direct effects of estrogens on male germ cells, we
studied the role of the natural estrogen, 17ß-estradiol, in germ cell
apoptosis in the present in vitro model. Interestingly,
17ß-estradiol effectively inhibited the germ cell apoptosis that was
induced in vitro by withdrawal of serum and hormones (Fig. 3). The most effective concentrations
were 10-10 and 10-9
mol/L, which are in range of the previously reported physiological
estradiol concentrations in human spermatic vein and testis tissue. In
Southern blot analysis, the total amount of apoptotic low molecular
mass DNA fragmentation was suppressed by 47% (P <
0.001) and 41% (P = 0.003) at estradiol concentrations
of 10-10 and 10-9 mol/L,
respectively. Estradiol concentration of 10-7
mol/L did not significantly inhibit germ cell death. The suppressive
effect of estradiol on germ cell apoptosis was blocked by the ER
antagonist ICI 182,780 at 10-7 mol/L. This
result was obtained from three independent experiments.
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), whereas in the samples cultured for
4 h in the absence of serum and hormones, their amount was greatly
increased (Fig. 4B). As in the electron microscopy, the apoptotic cells
were most often identified as spermatocytes and spermatids. When
17ß-estradiol was added to the culture medium, the number of
positively staining cells was effectively reduced (Fig. 4C). When the
terminal transferase enzyme was replaced with distilled water, there
was no staining (negative control, data not shown).
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In addition to 17ß-estradiol, testosterone can be metabolized
in vivo to DHT. Therefore, we further studied the effects of
this other end metabolite of testosterone on the in vitro
induced apoptotic death of testicular germ cells. In the present
in vitro model, DHT was also capable of inhibiting
testicular apoptosis (Fig. 5). However,
the lowest concentrations of DHT needed for effective inhibition of
germ cell death were 100-1000 times the effective concentrations of
17ß-estradiol, indicating the relatively high potency of estrogens as
germ cell survival factors compared with that of androgens. Apoptotic
low molecular mass DNA fragmentation was suppressed by 38% at DHT
concentration of 10-7 mol/L (P
< 0.05). Lower concentrations of DHT did not significantly inhibit
germ cell death.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We observed strong expression of both the ER and the ERß proteins
in the developing germ cells of the human testis. Expression of ER
in the testicular germ cells is a novel finding. Previous studies have
shown ER expression in the epididymis and the efferent ductules of
various species (16, 17, 18, 19, 20) as well as in the Leydig cells of the rat
testis (16). Testicular germ cells, however, have not been shown to
express ER in any species, and therefore, direct ER-mediated
effects of estrogens on spermatogenesis have not been suggested. In the
present study the cells staining positively for ER by
immunohistochemistry were identified mainly among early meiotic germ
cells in stages VI and I to II of the human seminiferous epithelium
cycle (zygotene and early pachytene primary spermatocytes) and as
elongating spermatids in stages IV to V of the cycle (Fig. 6). When the seminiferous tubules were
cultured in serum-free conditions for increasing lengths of time, the
expression remained constant regardless of the amount of germ cell
apoptosis. The expression pattern of the ERß protein in the human
seminiferous epithelium was similar to that of the ER protein (Fig. 6). Although the ERß mRNA has recently been found in the germ cells
of the human testis (21), this is the first report confirming the
expression of ERß in the human testis at the protein level.
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The suppressive effect of 17ß-estradiol on germ cell apoptosis was
blocked by an ER antagonist, ICI 182,780, indicating that estradiol was
functioning through its receptors. The intracellular mechanisms of ER
action in the present study and the possible involvement of both ER
and ERß in male germ cell survival are not known. The classic ER
signaling pathway involves binding of the ligand-bound ER (either
or ß) to the estrogen-responsive element that regulates transcription
of target genes. However, ERs may also mediate gene transcription by
binding to an activating protein-1 (AP1) element together with the
transcription factors Fos and Jun. ER and ERß have been shown to
signal in opposite ways from the AP1 site; when bound to
17ß-estradiol, ER activates and ERß inhibits transcription (40).
In contrast to the natural estrogen, antiestrogens, including
tamoxifen, raloxifene, and ICI 164,384, have been shown to effectively
activate transcription from an AP1 site when bound to ERß (40). The
effects of AP1-mediated regulation of transcription on germ cell death
are not known. Thus, the possible involvement of AP1 activation in
estrogen prevention of germ cell apoptosis remains unknown. Moreover,
recent studies have revealed a novel nongenomic signaling pathway
through ERs located on the plasma membrane (reviewed in Ref. 41).
Binding of 17ß-estradiol to the membrane receptors results in rapid
(within minutes) activation of the mitogen-activated protein kinase
signaling cascade. In endothelial cells this has been associated with
activation of endothelial nitric oxide synthase and production of
nitric oxide, which, at physiologically relevant levels, has been shown
to suppress apoptotic pathways in a variety of cell types (reviewed in
Ref. 42). As in the present study the antiapoptotic effect of
17ß-estradiol on germ cells was seen after 4-h incubation, it is
possible that the survival of germ cells is at least partly mediated by
this rapid nongenomic ER signaling pathway.
In our previous studies, testosterone has also been shown to be an effective inhibitor of germ cell apoptosis in the human testis in vitro (30). However, the concentrations of testosterone (10-7 mol/L) required for this apoptosis inhibiting effect were 100-1000 times the effective concentrations of estradiol (10-9–10-10 mol/L). Of note, the relative potencies of testosterone and estradiol are in the range of their relative physiological concentrations in the spermatic vein (43, 44, 45, 46, 47) and testis tissue (48, 49). In vivo, testosterone can be metabolized to either estrogens or DHT. We found that DHT was also capable of inhibiting germ cell death in our in vitro model, but, as with testosterone, the lowest effective concentrations of DHT were strikingly higher than the effective concentrations of estradiol. Thus, in vitro estradiol appears to be a more potent inhibitor of male germ cell death than the androgens testosterone and DHT. The essential role of androgens in completing normal spermatogenesis is well established. However, the mechanism by which androgens regulate spermatogenesis has not been resolved, and even the site of androgen action within the testis has remained unclear. Some reports have shown immunoreactive androgen receptor in developing germ cells (50, 51, 52, 53), and others have suggested that only testicular somatic cells, namely Leydig, Sertoli, peritubular myoid, and smooth muscle cells surrounding the walls of the blood vessels, express AR (54, 55, 56, 57, 58). If the germ cells lack functional AR, the effects of androgens on spermatogenesis may be forwarded to germ cells through paracrine regulation of germ cells by the neighboring somatic cells, especially by the Sertoli cells providing structural and nutritional support to the developing germ cells. The results of the present study suggest another interesting mechanism for testosterone-mediated survival of germ cells. Taken the present finding that estradiol is much more potent than androgens in inhibiting germ cell apoptosis and the previously reported expression of P450 aromatase by adult Leydig and germ cells in several species (5, 6, 7, 8, 9, 10), it is possible that testosterone should at least partly be metabolized to estrogen to mediate its protective effects on germ cells.
The importance of local conversion of testosterone to estrogen in the
testis was recently demonstrated in ArKO mice lacking the functional
aromatase (cyp 19) gene (28). These mice were initially
fertile, but developed progressive infertility with disruptions to
spermatogenesis between 4.5 months and 1 yr despite any decrease in the
levels of circulating gonadotropins or androgens. Spermatogenesis was
arrested at early spermiogenic stages and was characterized by
increased germ cell apoptosis and reduction in the number of round and
elongated spermatids. Interestingly, in the ArKO mice there was no
evidence of abnormal fluid reabsorption by the efferent ductules, which
was reported to be the primary defect leading to infertility in ERKO
mice (27). Thus, the mechanism of defective spermatogenesis in the ArKO
animals appears to be a direct effect of estrogen withdrawal on germ
cell development, rather than an indirect effect as in ERKO mice.
Another ArKO mouse with a different mutation site in the aromatase gene
was recently described (59). These animals at the age of 10–18 weeks
had sperm present in their epididymis, but they were infertile. Further
studies of older animals are needed to show whether disruption of
spermatogenesis also develops in these animals.
The relevance of estrogen formation for human male reproduction was
shown by case reports of two men with homozygous inactivating mutation
in the ER gene (60) or in the P-450 aromatase gene (61). The patient
with mutation of the ER gene had normal male genitalia and sperm
density, but sperm viability was severely decreased. The mutation in
the aromatase gene resulted in infertility with a decreased sperm count
and 100% immotile spermatozoa. Thus, studies of both human and mouse
have shown that male infertility results from blocking either the ER
or aromatase gene. Somewhat surprisingly, the recently described male
ERßKO mice lacking ERß were fertile (62). Fertility was, however,
assessed between 6 and 12 weeks of age, at which age no change in
testicular morphology in ArKO mice was observed (28, 63). Therefore,
the evaluation of fertility in older ERßKO animals will be very
interesting.
In the present study the effects of estrogens in the human testis have been evaluated in an in vitro culture model that may naturally have some limitations as to approximation of germ cell physiology in vivo. However, we believe that in the present in vitro model, conditions were sufficiently close to the situation in vivo, firstly because in this model the germ cells were allowed to stay in their natural environment (i.e. in the seminiferous tubules), and secondly because germ cell death can be blocked by physiological concentrations of testicular hormones, such as testosterone and estradiol. In vivo, the situation may, of course, be complicated by the effects of locally produced compounds on interstitial cells. In the case of estrogens, the locally produced hormones could theoretically inhibit Leydig cell androgen production, leading to unpredictable effects on spermatogenesis. However, the in vivo significance of our results indicating direct protective effects of estrogens on male germ cells expressing ERs is strongly supported by the previously shown production of estrogens by testicular germ cells in several species (6, 7, 9, 10) and by the recently reported infertility and germ cell apoptosis without accompanying alterations of efferent ductules in ArKO mice (28).
In conclusion, our results describe a novel function of estradiol in
the human testis. As receptors for estrogens (both and ß) are
strongly expressed by the differentiating and proliferating germ cells
of the human testis, and as the in vitro induced apoptosis
of these cells can be blocked by the natural estrogen 17ß-estradiol,
it appears very likely that estrogens act as germ cell survival factors
in the human testis. The present results together with the results of
recent studies of ERKO (26, 27) and ArKO (28) mice indicate the
importance of estrogens for the normal function of the adult testis.
Therefore, the effects of physiological estrogens on the male
reproductive system should be carefully studied when considering the
potential effects of environmental estrogens on male reproductive
health.
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Acknowledgments |
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Footnotes |
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Received August 18, 1999.
Revised January 11, 2000.
Accepted January 17, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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5-androstane-3ß,17ß-diol and their precursor in human testicular
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