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Knock-Out and Wild-Type Mice1
Departments of Animal Sciences (C.S.R., D.B.L.), Veterinary Biomedical Sciences (V.K.G.), Biochemistry and Child Health (J.A.T., X.-H.Y., D.B.L.), University of Missouri, Columbia, Missouri 65211; Affinity BioReagents (J.S.), Golden, Colorado 80401; and The Population Council (M.P.H.), New York, New York 10021
Address all correspondence and requests for reprints to: Dr. Dennis B. Lubahn, University of Missouri, 163 ASRC East Campus Drive, Columbia, Missouri 65211. E-mail: asld{at}muccmail.missouri.edu
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Abstract |
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(ER) has been identified in the male
reproductive tract, but the role of estrogen in the male has not been
well characterized. In vivo mutations in ER genes
have demonstrated the necessity for ER-mediated action in male
fertility. We asked whether both ERß messenger RNA and protein were
present in the male reproductive tract of wild-type and ER knock-out
(ER KO) mice, and whether ERß could compensate for the lack of
ER in infertile male ER KO mice. Immunohistochemical localization
with both N- and C-terminal anti-ERß antibodies demonstrated that
ERß is present in the Leydig cells of the testes and in the
epithelium of both the efferent ductules and the initial segment of the
epididymis. RT-PCR amplification was used to confirm ERß
transcription in these tissues. In conclusion, we observed that ERß
messenger RNA and protein continue to be expressed in the Leydig cells,
elongated spermatids, efferent ductules, and the initial segment of the
epididymides of ER KO mice, but the presence of ERß is
not able to compensate for the absence of ER in male reproductive
function. |
Introduction |
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In men, mutations in either the aromatase enzyme or the estrogen
receptor- (ER) gene result in infertility problems (1, 2, 3), and
targeted disruption of the ER gene causes sterility in both male and
female mice (4, 5). Fluid reabsorption in the efferent ductules and, to
a lesser extent, in the initial segment of the epididymis was recently
discovered to be under estrogen regulation (6). These findings suggest
an absolute prerequisite for estrogen/ER in normal male reproductive
function.
Histopathological examination of testes from young ER knock-out
(ER KO) mice reveals that there is normal development of the
seminiferous tubules. As the animals reach puberty, however, the
seminiferous epithelium begins to exfoliate, and marked amounts of
fluid accumulate within the seminiferous tubules, rete testis, and
efferent ductules (6, 7). The efferent ductules become severely ectatic
(dilated), and metaplasia of the lining epithelium subsequently
develops as the epithelium degenerates from simple columnar to simple
cuboidal epithelium. The dilation and metaplasia of the efferent
ductules are consistent with their inability to reabsorb the rete
testis fluid.
Past work has demonstrated that ER is present within the male
reproductive tract. Specifically, ER has been localized to Leydig
cells and Leydig cell precursors (8). In addition, ER immunostaining
is observed in the rete testis, the epithelium of the efferent
ductules, and sporadically within the epididymis (9, 10, 11).
A novel ER (ERß) has been localized to the testis, ovary,
prostate, hypothalamus, bone, and various other internal organs by
messenger RNA (mRNA) analyses (12, 13, 14, 15, 16). The specific physiological
actions of ERß and its functional interaction with ER have not yet
been resolved, although in vitro functional
heterodimerization of ER and ERß have been reported (17, 18, 19).
Recently, using a rabbit polyclonal antibody generated against an
18-amino acid stretch within exon 5 of rat ERß, ERß was
immunohistochemically localized to the Sertoli cells within the rat
seminiferous epithelium (20). To more fully understand ERß and its
potential role in male fertility, we used RT-PCR amplification and
immunohistochemistry to localize ERß in Leydig cells, elongated
spermatids, efferent ductules, and the initial segment of the
epididymis of wild-type (WT) and ER KO mice. Neither method revealed
any qualitative differences in expression of ERß in ER KO
vs. WT tissue.
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Materials and Methods |
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KO and WT sibling male mice in a mixed
C57BL/6J/129 background were anesthetized with carbon dioxide and
killed by cervical dislocation. Institutional animal care protocols
were followed. Five WT and ER KO 100- to 110-day-old male mice were
used for immunohistochemistry and RT-PCR. The testes, efferent
ductules, and epididymides were fixed for histology in Bouin’s
solution for 12 h. The tissue was then dehydrated in increasing
concentrations of ethanol and embedded in paraffin. Sections of 5–6
µm were cut and mounted on poly-L-lysine-coated
microscope slides. The tissue was rehydrated in decreasing
concentrations of ethanol. The slides were then incubated in 0.05
M glycine-HCl and 0.1 M Tris-buffered saline
(TBS; pH 3.5) and heated in a microwave oven at high temperature for 5
min. After washing in 0.1 M TBS, endogenous peroxidase
activity was blocked by placing the slides in a 1:10 dilution of 30%
hydrogen peroxide-methanol for 30 min. The slides were washed with 0.1
M TBS and incubated in normal goat serum for 30 min to
block any nonspecific binding.
The two primary epitope-specific rabbit polyclonal antibodies
against ERß were obtained from Affinity BioReagents (Golden, CO). The
N-terminal antibody was generated against a peptide from amino acids
55–70 of the rat ERß sequence (AEPQKSPWCEARSLEH), and the C-terminal
antibody was generated against a peptide from the last 19 amino acids
of the rat ERß sequence (CSSTEDSKNKESSQNLQSQ). The region of rat
ERß from which the N-terminal antibody was generated has the same
amino acid sequence as mouse ERß. There are only three amino acid
differences within the C-terminal peptide between mouse and rat that
still permit cross-reactivity of the antibody in murine tissues.
Western blots and gel mobility shift assays were performed for the both
the N- and C-terminal ERß antibodies (Jurutka, P. W., and
M. R. Haussler, manuscript in preparation). Using transfected
COS-7 monkey kidney epithelial cells, the C-terminal ERß antibody
detected the protein via Western blots in transfected extracts, but not
in untransfected extracts. Gel mobility shift assay revealed that C-
and N-terminal ERß antibody specifically shifted an ERß-containing
complex; preimmune sera and an ER monoclonal antibody were not able
to shift the ERß-containing complex.
The N-terminal antibody was used at a dilution of 1:500, and the C-terminal antibody was used at a dilution of 1:50. These dilutions were chosen based on multiple preliminary trials in which dilutions spanning 1:50 to 1:1000 for both antibodies were used. The respective antibodies were placed on the tissue and incubated overnight at 4 C in a humidified chamber. Unbound primary antibody was washed off the tissue with 0.1 M TBS buffer (pH 7.4). The tissue was incubated with antirabbit IgG secondary antibody (Vectostain kit, Vector Laboratories, Burlingame, CA) for 30 min. The secondary antibody was washed off the tissue with 0.1 M TBS buffer (pH 7.4). Avidin and biotin from the Vectostain kit were mixed and incubated on the tissues for 30 min. Peroxidase was detected by a mixture of 3,3'-diaminobenzidine (DAB; Dako Corp., Carpinteria, CA) and 0.03% hydrogen peroxide. The slides were counterstained for 1 min with Gill’s hematoxylin, dehydrated, and coverslipped with Permount (Fisher, Fairlawn, NJ). Photomicrographs were digitized using a Nikon microscope (Nikon Corp., Melville, NY) attached to a Sony ccd iris RGB camera (Sony, Tokyo, Japan). Images were digitalized using Image I software (NIH, Bethesda, MD); they were compiled using Adobe Photoshop 3.0 for Macintosh and printed with a Mitsubishi Codotonic dye sublimation printer (Mitsubishi, Tokyo Japan).
Leydig cell purification
Mouse Leydig cells from the testes of groups of 10 ER KO and
WT mice were dispersed enzymatically with collagenase and dispase (21).
Seminiferous tubule elements were removed by filtration through two
layers of 100-µm pore size nylon mesh, and the preparation was
further purified by centrifugal elutriation at 2000 rpm and a flow rate
of 16 ml/min to eliminate sperm and other germ cells. The final
fraction of the purified Leydig cells was obtained after centrifugation
through Percoll, collecting cells with a buoyant density of 1.070 g/ml
or greater. Assessment of purity was performed by histochemical
staining for 3ß-hydroxysteroid dehydrogenase (an enzyme that is
specific to the Leydig cells), which was typically 95% or greater.
Total RNA isolation
Testes, epididymides, and purified Leydig cells from ER KO
and WT sibling mice were rapidly frozen in liquid nitrogen and then
stored at -80 C. RNA was isolated using guanidine thiocyanate and
phenol/chloroform extraction (Tri-Reagent, Sigma Chemical Co., St.
Louis, MO). RNA was reconstituted in 50 µl
diethylpyrocarbonate-treated water and then stored at -80 C. The
quality of the RNA was checked by agarose gel electrophoresis and
quantitated spectrophotometrically.
RT-PCR amplification protocol
One microgram of total isolated RNA was reverse
transcribed to complementary DNA. RT-PCR amplification was carried out
using the Titan one-tube RT-PCR system kit (Boehringer Mannheim,
Indianapolis, IN). Each reaction tube contained (final concentrations)
0.2 mM deoxy-NTPs (Promega), 5 mM
dithiothreitol (Boehringer Mannheim), 5 U RNasin (Promega), 1 x
RT-PCR buffer with 1.5 mM Mg2+ mix (Boehringer
Mannheim), and enzyme mix, AMV, and Expand High Fidelity PCR-system
(Boehringer Mannheim). The RT reaction was carried out at 42 C for 30
min. Touchdown PCR, which spanned from 68–50 C for 40 cycles, was used
(22). A second generation of PCR was performed that had a predwell at
94 C for 1 min, followed by 15 cycles of 94 C for 30 sec, 58 C for 30
sec, and 72 C for 1 min, and ending with a postdwell at 72 C for 5 min.
A heminested reverse primer in both the N- and C-terminal regions was
used in combination with the same forward primer used in the first
generation. The amplified DNA was fractionated electrophoretically on a
2% agarose gel, then stained with ethidium bromide and visualized
under UV light. The gels were digitized using a Mitsubishi image
capture system.
Primers
Primers for both the N- and C-terminal regions of ERß primers
were designed based on the mouse ERß sequence (17, 23). ERß gene
intron/exon splice sites and exon numbers were predicted based on the
conserved gene structures of ER and other steroid receptors
(24, 25, 26). The splice sites between exons 2 and 3 as well as those
between exons 8 and 9 were confirmed by PCR amplification of
complementary DNA and genomic DNA and by sequencing (data not shown).
The location and sequence of the primers used are listed in Table 1
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Results |
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KO sibling mice
(Fig. 1, A and C).
Within these cells, immunoreactive staining was detected within the
cytoplasmic, perinuclear, and nuclear regions. The Leydig cells in the
ER KO appeared to be hypertrophied, which is compatible with the
2-fold increase in testosterone that has been previously reported (5).
Using the N-terminal antibodies, staining was also present in the
elongated spermatids (Fig. 1I) in WT, but not ER, KO mice. Within
the efferent ductules, moderate amounts of immunoreactive staining for
ERß were present in the cytoplasm of the nonciliated and ciliated
epithelial cells (Fig. 1, E, G, M, and O). Staining was also present in
the cytoplasm of the pseudostratified simple columnar epithelial lining
of the epididymis in both WT (Fig. 1, E and M) and ER KO (Fig. 1, G
and O). In the WT efferent ductules and initial segment of the
epididymis, the epithelial cells of the efferent ductules and initial
segment had more nuclear staining than the ER KO tissues. The
immunoreactive staining was judged to be specific based on the lack of
significant DAB staining after incubations of the primary antibody with
competing amounts of N- or C-terminal peptides (Fig. 1, B, D, F, H, J,
L, N, and P). Additionally, DAB staining was not detected in sections
that were incubated with rabbit preimmune serum alone or in sections
that were not exposed to the primary antibody (data not shown). The
urinary bladder, in which copious ERß mRNA and protein expression
have been identified (13, 14, 20), was used as a positive control
tissue. The transitional epithelium of the urinary bladder was more
intensely stained than the submucosa or muscularis externa using the
N-terminal antibody in WT and ER KO males (Fig. 1, Q and S). This
staining was not present in peptide-competed sections (Fig. 1, R
and T). Similar staining of the transitional epithelium of WT and ER
KO urinary bladders was seen using the C-terminal antibody (data not
shown).
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KO and WT sibling mice revealed the expected PCR product of
approximately 230 bp for the N-terminal primers (Fig. 2A, lanes 1–4). The C-terminal primers
resulted in the expected approximately 186-bp band in the WT testes,
ER KO testes, WT epididymis, and ER KO epididymis (Fig. 2B, lanes
1–4). Signal intensities were not measured, but no qualitative
differences could be detected in the band intensities between WT and
ER KO. No amplification was observed in samples that lacked RNA
(Fig. 2, A and B, lane 7). No amplified product was present in RNA
samples that were RT-PCR amplified without AMV reverse transcriptase
(data not shown). Additionally, to further eliminate the possibility of
contaminating DNA, the samples were pretreated with ribonuclease-free
deoxyribonuclease before the RT enzyme was added. To verify the
identity of the amplified PCR product, the bands were sequenced and
confirmed to be mouse ERß.
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KO Leydig cells were purified, and total RNA was isolated for
subsequent RT-PCR. The RT-PCR conditions were the same as those used
for the testis and epididymides. Primers from both the N- and
C-terminal regions were used as before. As demonstrated in Fig. 2A
, lanes 5 and 6, the expected 230-bp band (using the N-terminal primers)
was present in both the WT and ER KO Leydig cells. Using primers in
the C-terminal region, an approximately 186-bp band product was
detected in both the WT and ER KO Leydig cells (Fig. 2B, lanes 5 and
6). This is consistent with the immunohistochemical data, which
localized ERß to the Leydig cells of the testes. These bands were
excised, sequenced, and verified to be mouse ERß. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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It has been demonstrated using immunohistochemical and/or
immunogold staining that ER can be present in both the cytoplasm and
nucleus of human mammary carcinoma cells, rabbit endometrial epithelial
cells, and MCF-7 cells (28, 29, 30). Different fixation techniques may also
play a role in the apparent subcellular immunohistochemical staining of
ER (31).
A recent study using a rabbit antirat ERß antibody generated from
exon 5 of the rat demonstrated positive staining in Sertoli cell nuclei
(20). However, in contrast with the present data in the mouse, these
researchers stated that although there was interstitial staining, it
appeared to be nonspecific. Thus, they could not determine whether the
Leydig cells or peritubular myoid cells were positive for ERß. Using
the N- and C-terminal ERß antibodies that were generated from exons 2
and 9, respectively, we found Leydig cell staining in both WT and ER
KO mouse testes, which was confirmed by RT-PCR analysis.
One potential reason for these different findings may be species
variation between mouse and rat in the cellular localization of ERß,
as has been observed in ER immunolocalization in rat and marmoset
monkey male reproductive tracts (10). Neonatal Leydig cells from both
rat and marmoset were immunopositive for ER. Adult rat Leydig cells
were strongly positive for ER, whereas adult marmoset Leydig cells
were only weakly positive for ER. Additionally, ER was
immunolocalized to rat rete testis and efferent ductules, but in the
marmoset only the efferent ducts were positive.
Similar to the rat (20), the transitional epithelium of the urinary bladder in the mouse stained positively for ERß. In the rat, the muscularis externa of the urinary bladder also stained strongly positive for ERß (20). However, when we used both the N- and C-terminal ERß antibodies, little or no staining of the muscularis externa was detected in the mouse urinary bladder.
The elongated spermatids demonstrated ERß staining with the N-terminal ERß antibody, but not the C-terminal antibody. There are several possible reasons for this difference in antibody binding. The first is that there may be novel alternate spliced forms of ERß within the testes in addition to those previously identified in the ovary, pituitary, and various other tissues (23, 32, 33, 34).
Alternatively, this difference in spermatid staining using the N- vs. the C-terminal ERß antibodies may be due to differences in affinities of the N- and C-terminal ERß antibodies. This affinity difference may exist because of sequence variation within the peptide antigen between rat and mouse. This possible difference in affinities combined with potentially lower concentrations of ERß in the elongated spermatids may also account for the staining differences. Finally, the N-terminal ERß spermatid staining may simply be the result of binding to another unrelated protein with a common epitope. Further developmental studies are underway to confirm the presence of ERß protein in elongated spermatids.
No qualitative difference was detected in either ERß mRNA or protein
concentration in ER KO vs. WT tissues. No difference was
detected using ribonuclease protection assay for ERß mRNA expression
in WT vs. ER KO mice (35). This suggests that ERß alone
cannot maintain normal male reproductive function. As it has been shown
that ERß preferentially heterodimerizes with ER (17, 18, 19), the
possibility thus exists that ERß may not exert significant
physiological action without ER.
Although ERß concentrations are similar in both genotypes, there
seems to be a difference in the subcellular localization of ERß in
the efferent ductules and epididymis of WT vs. ER KO
mice. In ER KO animals, cytoplasmic staining is present, but nuclear
staining is scant to absent, whereas in the epithelium of the WT
efferent ductules and epididymis, ERß staining is present in the
nuclei and cytoplasm. This differential subcellular localization is not
observed in the testes and urinary bladder. We do not have an
explanation for this finding. It is interesting to speculate that
perhaps ER may be needed, either directly or indirectly, for ERß
localization or retention in the nucleus in certain cell types.
Estrogen is produced in the male reproductive tract and may exert local effects in the male reproductive system (36). In the prepubescent animal, the Sertoli cells are the main source of estrogen via aromatase conversion of testosterone (37). As the animal matures, the Leydig cells and spermatozoa become the main sources of estrogen within the testes (38, 39). As the sperm traverse the excurrent duct system, there is a decrease in their P450 aromatase activity (40). This suggests that the estrogens synthesized by the sperm act within the efferent ductules and/or the epididymis.
It has been demonstrated that prenatal exposure to estrogen in mice
results in Leydig cell tumors and/or adenoma formation (41, 42). As
most tumors are derived from premature or primordial cells, estrogen
may potentially prevent differentiation of the progenitor Leydig cells
and/or result in the Leydig cells regressing back to a dedifferentiated
state, which would make them more susceptible to uncontrolled division
and subsequent neoplastic transformation. This work, performed on
normal and neoplastic Leydig cells, suggests that estrogen may act in
an autocrine and/or paracrine manner via ER and/or ERß within the
Leydig cells.
In this report we have positively identified, via
immunohistochemistry and RT-PCR amplification, ERß in some of the
same male reproductive tissues and cells as ER. The exact function
and regulators of ERß need to be further determined, although it is
now clear from in vitro studies that ERß may regulate
specific genes differently from ER (43). Based on our findings, it
does not appear that ER regulates ERß concentrations in the murine
male reproductive tract. As ERß has a proclivity to heterodimerize
with ER, additional work needs to be performed to resolve the roles
of ER and ERß in conjunction with one another and the individual
functions of the receptors. Potentially, each receptor may play
different roles at various stages of life and in various tissues. Taken
together, the immunohistochemical and RT-PCR amplification data
indicate that there is qualitatively no difference in the mRNA and
protein expression of ERß in WT vs. ER KO mice. Thus,
ERß alone does not appear to be capable of maintaining normal
reproductive function in ER KO mice.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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Received October 3, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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within the testis and excurrent ducts of the rat and marmoset monkey
from perinatal life to adulthood. J Endocrinol 153:485–495 and ß)
expression in the excurrent ducts of the adult male rat reproductive
tract. J Androl 18:602–611 and ß. Endocrinology 138:863–870 knockout mouse. Endocrinology 138:5649–5652. Mol Endocrinol 11:1486–1496 and ß form heterodimers on DNA. J Biol
Chem 272:19858–19862. J Biol Chem 272:25832–25838 (ER-) and ß
(ER-ß) mRNA in the midgestational human fetus. J Clin Endocrinol
Metab 82:3509–3512 (ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER- knockout mouse.
Endocrinology 138:4613–4621 and ERß at AP1 sites. Science 277:1508–1510This article has been cited by other articles:
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N. Atanassova, C. McKinnell, K. Williams, K. J. Turner, J. S. Fisher, P. T. K. Saunders, M. R. Millar, and R. M. Sharpe Age-, Cell- and Region-Specific Immunoexpression of Estrogen Receptor {{alpha}} (But Not Estrogen Receptor {beta}) during Postnatal Development of the Epididymis and Vas Deferens of the Rat and Disruption of This Pattern by Neonatal Treatment with Diethylstilbestrol Endocrinology, February 1, 2001; 142(2): 874 - 886. [Abstract] [Full Text] [PDF]
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C. S. Rosenfeld, P. S. Cooke, T. H. Welsh Jr., G. Simmer, M. G. Hufford, J.-A. Gustafsson, R. A. Hess, and D. B. Lubahn The Differential Fate of Mesonephric Tubular-Derived Efferent Ductules in Estrogen Receptor-{alpha} Knockout Versus Wild-Type Female Mice Endocrinology, October 1, 2000; 141(10): 3792 - 3798. [Abstract] [Full Text] [PDF]
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 V. Pentikäinen, K. Erkkilä, L. Suomalainen, M. Parvinen, and L. Dunkel Estradiol Acts as a Germ Cell Survival Factor in the Human Testis in Vitro J. Clin. Endocrinol. Metab., May 1, 2000; 85(5): 2057 - 2067. [Abstract] [Full Text]
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K. Kinuta, H. Tanaka, T. Moriwake, K. Aya, S. Kato, and Y. Seino Vitamin D Is an Important Factor in Estrogen Biosynthesis of Both Female and Male Gonads Endocrinology, April 1, 2000; 141(4): 1317 - 1324. [Abstract] [Full Text] [PDF]
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W. N. Jefferson, J. F. Couse, E. P. Banks, K. S. Korach, and R. R. Newbold Expression of Estrogen Receptor {beta} Is Developmentally Regulated in Reproductive Tissues of Male and Female Mice Biol Reprod, February 1, 2000; 62(2): 310 - 317. [Abstract] [Full Text]
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 S Dupont, A Krust, A Gansmuller, A Dierich, P Chambon, and M Mark Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes Development, January 10, 2000; 127(19): 4277 - 4291. [Abstract] [PDF]
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 K. M. Robertson, L. O'Donnell, M. E. E. Jones, S. J. Meachem, W. C. Boon, C. R. Fisher, K. H. Graves, R. I. McLachlan, and E. R. Simpson Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene PNAS, July 6, 1999; 96(14): 7986 - 7991. [Abstract] [Full Text] [PDF]
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J. F. Couse and K. S. Korach Estrogen Receptor Null Mice: What Have We Learned and Where Will They Lead Us? Endocr. Rev., June 1, 1999; 20(3): 358 - 417. [Abstract] [Full Text]
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C. S. Rosenfeld, X. Yuan, M. Manikkam, M. D. Calder, H. A. Garverick, and D. B. Lubahn Cloning, Sequencing, and Localization of Bovine Estrogen Receptor-ß within the Ovarian Follicle Biol Reprod, March 1, 1999; 60(3): 691 - 697. [Abstract] [Full Text]
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