From the Departments of Obstetrics and Gynecology,
¶ Urologic Surgery,
Biochemistry, and ** Cell Biology, and
the
Center for Reproductive Biology
Research, Vanderbilt University, School of Medicine,
Nashville, Tennessee 37232-2633
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ABSTRACT |
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The murine epididymis synthesizes and secretes a
retinoic acid-binding protein (mE-RABP) that belongs to the lipocalin
superfamily. The gene encoding mE-RABP is specifically expressed in the
mouse mid/distal caput epididymidis under androgen control. In
transgenic mice, a 5-kilobase pair (kb) promoter fragment, but not a
0.6-kb fragment, of the mE-RABP gene driving the chloramphenicol
acetyltransferase (CAT) reporter gene restricted high level of
transgene expression to the caput epididymidis. No transgene expression
was detected in any other male or female tissues. Immunolocalization of
the CAT protein and in situ hybridization of the
corresponding CAT mRNA indicated that transgene expression occurred
in the principal cells of the mid/distal caput epididymidis, thereby
mimicking the spatial endogenous mE-RABP gene expression. Transgene and mE-RABP gene expression was detected from 30 days and progressively increased until 60 days of age. Castration, efferent duct ligation, and
hormone replacement studies demonstrated that transgene expression was
specifically regulated by androgen but not by any other testicular factors. Altogether, our results demonstrate that the 5-kb promoter fragment of the mE-RABP gene contains all of the information required for the hormonal regulation and the spatial and temporal expression of
the mE-RABP gene in the epididymis.
During their transit through the epididymis, spermatozoa undergo
biochemical and morphological changes to acquire motility and the
ability to fertilize an oocyte in vivo (1, 2). The maturation process progresses along the epididymal duct and is believed
to be dependent on epididymal secretory proteins.
The epididymis displays a highly region-specific pattern of gene
expression. However, little is known regarding the molecular mechanisms
that are involved in the regulation of tissue- and region-specific gene
expression. Gene expression in the epididymis is mainly under androgen
control (for review, see Ref. 3). Indeed, androgen withdrawal, either
by orchiectomy or by hypophysectomy, prevents the sperm maturation
process (4, 5). However, testicular factors (6), estrogen (7), growth
factors (8), and retinoic acid (9) have also been demonstrated to be
involved in epididymis-specific gene expression. Whether these
endocrine and paracrine signal pathways cooperate to restrict gene
expression to a narrow segment of the epididymal duct is unclear.
We previously described two proteins (major and minor forms) that are
generated by the differential cleavage of a unique precursor, initially
named mouse epididymal protein 10 (10). This protein, recently renamed
murine epididymal retinoic acid-binding protein (mE-RABP)1 (11), is
specifically synthesized and secreted by the principal cells of the
distal caput epididymidis. The mE-RABP protein binds active retinoids
(9-cis- and all-trans-retinoic acid) but not retinol (12) and is the orthologue of two other retinoic acid-binding proteins described in the rat epididymis. These rat proteins were successively named B/C (13), EBP 1 and EBP 2 (14), E-RABP (15), and ESP
I (16). Analysis of the amino acid sequence and putative
three-dimensional structure show that mE-RABP belongs to the lipocalin
superfamily (11).
The mE-RABP protein is encoded by a single-copy gene localized to the
[A3-B] region of mouse chromosome 2, a region rich in genes encoding
lipocalin and displaying a similar genomic structure to that of the
mE-RABP gene (17).
The mE-RABP gene expression is androgen-regulated in vivo
(11). In addition, transient transfection studies have shown that a
functional androgen-specific response region (ARR) is localized within
the first 600 bp of the mE-RABP gene promoter (18). The androgen
dependence and the strong tissue- and region-specific expression make
the mE-RABP gene a good candidate to study the molecular mechanisms
that restrict gene expression to a narrow segment of the epididymis
under androgen control. In the absence of appropriate epididymal cell
lines, cis-DNA regulatory elements involved in the
tissue-specific and androgen-regulated expression of the mE-RABP gene
can only be identified in transgenic mice.
In the present study, we demonstrate that 5 kb of the 5'-flanking
region of the mE-RABP gene can drive the specific expression of the
chloramphenicol acetyltransferase (CAT) reporter gene to the principal
cells of the mid/distal caput epididymidis, thereby mimicking the
expression of the endogenous gene. We also demonstrate that the 5-kb
DNA fragment contains most, if not all, of the information required for
the temporal and hormonal regulation of the mE-RABP gene in
vivo.
Animals--
All experiments were conducted in accordance with
the National Institutes of Health Guidelines for Care and Use of
Animals in the Laboratory. Castration or efferent duct ligation was
performed by the abdominal route under light methoxyflurane
(Mallinckrodt, Mundelein, IL) anesthesia. When required, hormone
replacement was begun 30 days after castration with daily subcutaneous
injections of testosterone propionate (2 µg/g),
Chimeric Constructs--
DNA fragments encompassing the mE-RABP
gene promoter were generated from the pHindIII genomic clone (17) using
appropriate restriction enzymes. DNA fragments were purified on a 1%
(w/v) agarose gel and then ligated into the promoterless pBLCAT3
plasmid (19) using standard methods (20) in such a way that the mE-RABP promoter DNA fragment would drive the CAT reporter gene.
Transgenic Mice--
The m0.6Kb-CAT and m5Kb-CAT DNA fragments,
containing 0.6 or 5 kb, respectively, of the mE-RABP gene promoter
driving the CAT reporter gene were excised from the pUC18 vector by
restriction enzyme digest. DNA fragments were purified on a 0.8% (w/v)
agarose gel using the AgarACE® enzyme (Promega,
Madison, WI). Transgenic mice (strain B6D2; Harlan Sprague-Dawley) were
generated by microinjection of the DNA into the male pronucleus of a
fertilized oocyte using standard techniques (21). Transgenic animals
were identified by PCR-based screening assay using isolated tail DNA.
Approximately 1 cm of the tail was digested overnight at 55 °C in a
Proteinase K digestion mix (10 mM Tris-Cl, pH 7.5, 75 mM NaCl, 25 mM EDTA, 1% SDS, 0.5 mg/ml
Proteinase K). Then DNA was extracted with 1 volume of
phenol/chloroform/isoamyl alcohol (25/24/1) and precipitated at room
temperature with 2 volumes of absolute ethanol. Samples were
centrifuged at 10,000 × g at 4 °C for 15 min,
washed with 70% ethanol, centrifuged at 10,000 × g at
4 °C for 15 min, and dried for 2 h at room temperature. 500 ng
of genomic DNA were mixed with 1× PCR buffer II (Perkin Elmer, Foster
City, CA), 2 units of Taq DNA polymerase (Promega), 1.5 mM MgCl2, 1 µM concentration of
each primer (primer 1, 5'-TGGATGGATAGATGCATACATGAG-3'; primer 2, 5'-CAACGGTGGTATATCCAGTG-3'; casein forward, GATGTGCTCCAGGCTAAAGTT-3'; and casein reverse, AGAAACGGAATGTTGTGGAGT-3') and 0.2 mM
dNTP. DNA fragments were amplified for 30 cycles (95 °C, 1 min;
50 °C, 45 s; 72 °C, 45 s) and one cycle (95 °C, 1 min; 50 °C, 45 s; 72 °C, 10 min). PCR products were analyzed
on a 2% (w/v) agarose gel. To monitor CAT activity, organs were
homogenized with 20 strokes with a B pestle in a glass Dounce
homogenizer in 200 µl of 0.1 M Tris-HCl, 0.1% Triton
X-100, pH 7.8. Insoluble material was removed by centrifugation
(14,500 × g, 15 min, 4 °C), and CAT assays were
performed as described previously (22).
Southern Blot of Genomic DNA--
Genomic DNA was extracted from
the liver of adult male mice as described previously (23). Aliquots (25 µg) were digested with 40 units of HindIII restriction
enzyme (Promega), electrophoresed on a 0.8% (w/v) agarose gel, and
then incubated in 0.25 N HCl for 10 min, in 0.5 N NaOH, 1.5 M NaCl for 30 min and twice in 0.5 M Tris-HCl, pH 7.5, 1 mM EDTA, 1.5 M NaCl for 15 min. DNA fragments were transferred overnight
to a Hybond N+ nylon membrane (Amersham Pharmacia Biotech)
by blotting (24). The membrane was baked 2 h at 80 °C and
prehybridized for 3 h at 42 °C in 6× SSC, 1% (w/v) SDS, 100 µg/ml salmon sperm DNA, 50% (v/v) formamide, 5% (v/v) dextran
sulfate. The random-primed 32P-radiolabeled probe was
synthesized using the Rediprime DNA labeling system (Amersham Pharmacia
Biotech) and incubated overnight (106 cpm/ml). The filter
was washed once in 2× SSC for 15 min; once in 2× SSC, 0.1% (w/v) SDS
for 15 min; once in 2× SSC, 0.1% (w/v) SDS for 30 min; once in 0.2×
SSC, 0.1% (w/v) SDS for 15 min at 65 °C; and once in 0.1× SSC,
0.1% (w/v) SDS for 15 min at 65 °C before being autoradiographed
for 0.5-4 days at Immunohistochemistry--
Tissues were fixed in 4%
paraformaldehyde, 1× PBS (pH 7.4) overnight at room temperature,
dehydrated, and embedded in paraplast. Tissues were sectioned at 7 µm
and rehydrated. The slides were washed in H2O for 5 min,
and endogenous peroxidase activity was quenched in the presence of 3%
H2O2 for 30 min. Then the slides were washed with 1× PBS
for 5 min and incubated with 1% blocking reagent (Boehringer Manheim)
for 30 min. Sections were incubated at 4 °C overnight with
polyclonal rabbit IgG anti-CAT (5 Prime Western Blotting--
Tissues were homogenized in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl in the
presence of protease inhibitors (1 µg/ml leupeptin, 1 µg/ml
chymostatin, 1 µg/ml aprotinin, 2 µg/ml antipain, 10 µg/ml benzamidine). Samples were centrifuged at 40,000 × g
for 30 min and the supernatants were stored at In Situ Hybridization--
The epididymis was fixed overnight in
freshly prepared 4% (v/v) paraformaldehyde/phosphate-buffered saline
(pH 7.4), rinsed with PBS, and then dehydrated through a series of
increasing concentrations of ethanol for a period of 3-5 h prior to
embedding in paraplast. Tissues were sectioned at 7 µm. The CAT
reporter gene was excised from the pBLCAT3 vector (19) and ligated into
the pGEM7Zf( Generation of Transgenic Mice Carrying the CAT Reporter Gene Driven
by mE-RABP Gene Promoter DNA Fragments--
Two DNA fragments
containing either 0.6 or 5 kb of the mE-RABP gene promoter were
subcloned from the genomic clone pHindIII (17) and ligated into the
promoterless pBLCAT3 vector (19) so that the mE-RABP gene promoter
fragments would drive CAT reporter gene expression. The CAT reporter
gene was preferred to the lacZ reporter gene due to the high
endogenous
The chimeric genes, named m0.6Kb-CAT and m5Kb-CAT, respectively, were
excised from the pUC18 plasmids using the appropriate restriction
enzymes (Fig. 1A). After
purification, the fusion genes were injected into the male pronuclei of
B6D2 mouse embryos to generate transgenic mice. Two m5Kb-CAT (one male
and one female) and four m0.6Kb-CAT (two males and two females) founder
animals were detected by PCR using DNA extracted from the tail and
primers 1 and 2 (Fig. 1B). All founder animals passed on the
transgene to their offspring in a Mendelian fashion. Analysis of the
genomic DNA on Southern blot was carried out to confirm random
insertion of both transgenes into the mouse genome and to determine the number of transgene copies in each transgenic line (Fig.
1C). A probe encompassing the first 600 bp of the mE-RABP
gene promoter was used to detect both the endogenous mE-RABP gene as
well as the transgenes (Fig. 1A). In the nontransgenic
control animal (wild type), a 9-kb HindIII restriction
fragment corresponding to the endogenous mE-RABP gene was detected as
described previously (17). In addition to the endogenous 9-kb DNA
fragment, other DNA fragments of different lengths were detected for
each transgenic mouse line, indicating that the transgenes were
inserted in a random fashion. Transgene copy number was determined by
calculating the ratio between the hybridization signals of the
transgenes and the endogenous mE-RABP gene (Fig. 1C). The
presence of 18 copies and 1 copy of the m5Kb-CAT transgene was detected
in mouse lines 4 and 19, respectively. In transgenic mouse lines 143, 148, 149, and 391, 9, 6, 2, and 4 copies of the m0.6Kb-CAT transgene were inserted, respectively. The mouse line 4 carrying the m5Kb-CAT transgene exhibited a smaller 5.5-kb band in addition to the full size
of the m5Kb-CAT transgene of interest (6.7 kb). The 5.5-kb band
probably corresponds to the insertion of a truncated transgene and was
not taken into account for determination of copy number. Another probe,
specifically designed to detect only the CAT reporter gene (Fig.
1A), was also used (Fig. 1D). The CAT probe
detected the same restriction fragments described above with the
exception of the endogenous mE-RABP gene. Only in mouse line 4, a 9-kb
restriction fragment was still detectable, suggesting again that a
complete or truncated m5Kb-CAT transgene had been inserted at multiple sites.
The 5-kb Promoter Fragment, but Not the 0.6-kb Fragment, of the
mE-RABP Gene Targets High Level of Transgene Expression to the Caput
Epididymidis--
We have previously shown that the mE-RABP gene is
specifically expressed in the principal cells of the mid/distal caput
epididymidis (11). Therefore, to detect the expression of the
m0.6Kb-CAT and m5Kb-CAT transgenes, adult male mice were killed, and
the CAT activity was first measured in cell extracts of the caput epididymidis (Fig. 2). When compared with
the wild type control animals, no significant CAT activity was
detectable in mouse lines 143, 148, 149, and 391 carrying the
m0.6Kb-CAT transgene. However, high and moderate levels of CAT activity
were found in the caput epididymidis from lines 4 and 19 carrying the
m5Kb-CAT transgene. The m5Kb-CAT transgene expression appeared to be
correlated with transgene copy number, since line 4 expressed 11-fold
greater CAT activity than line 19, and carried the highest copy number (18 versus 1, respectively). In addition, homozygous
transgenic mice expressed on average a 2-fold higher level of CAT
activity than the heterozygous transgenic males (Table
I).
The pattern of tissue-specific expression of the m5Kb-CAT and m0.6Kb
CAT transgenes was then determined. No CAT activity was detected in the
cauda epididymidis and in 12 other tissues, including testis, vas
deferens, seminal vesicles, prostate, spleen, kidney, heart, lung,
brain, small intestine, liver, and muscle, of adult male mice carrying
the m5Kb-CAT transgene (Fig. 3) or the
m0.6Kb-CAT transgene (data not shown). In addition, CAT reporter gene
expression was undetectable in adult female tissues, including ovary,
oviduct, uterus, spleen, kidney, liver, heart, lung, brain, small
intestine, and muscle (data not shown). This demonstrates that 5 kb of
the 5'-flanking region of the mE-RABP gene is required to target high levels of gene expression to the caput epididymidis.
The m5Kb-CAT Transgene Expression Is Restricted to the Principal
Cells of the Mid/Distal Caput Epididymidis--
We have previously
shown that the mE-RABP gene expression is limited to the caput
epididymidis with a highly region- and cell-specific pattern (11). To
address the question of whether the m5Kb-CAT transgene contained all of
the information required for region- and cell-specific gene expression,
in situ hybridization of CAT mRNA was carried out (Fig.
4). Serial tissue sections of the
epididymis from homozygous m5Kb-CAT transgenic mouse line 4 were
incubated either with a CAT antisense or mE-RABP antisense probe. Both
CAT (Fig. 4C) and mE-RABP mRNA (Fig. 4E) were
detected only in the principal cells of the mid/distal caput
epididymidis. No CAT mRNA was detected in the efferent ducts, the
initial segment, the corpus, or cauda epididymidis of m5Kb-CAT
transgenic mice. In addition, no hybridization signal was detected in
the epididymis of wild type mice using the antisense CAT probe (Fig.
4A) or line 4 mice using the sense CAT probe (Fig.
4B).
The region- and cell-specific expression of the m5Kb-CAT transgene was
further studied by the immunohistochemical localization of the CAT
protein (Fig. 5). Sections of the
epididymis, from wild type and heterozygous transgenic mouse lines,
were incubated in the presence of a rabbit polyclonal antibody directed
against CAT (5 Prime
In agreement with our previous results, no CAT staining was detected in
the caput epididymidis of wild type mice and transgenic mice carrying
the m0.6Kb-CAT transgene (Fig. 5, C and D).
Corpus and cauda epididymidis of wild type or heterozygous mouse line 4 were also negative (not shown).
Altogether, these results demonstrate that the 5-kb promoter fragment
contains all of the cis DNA regulatory elements required for
tissue-, region-, and cell-specific mE-RABP gene expression.
Temporal Expression of the Endogenous mE-RABP Gene and m5Kb-CAT
Transgene Is Identical during Postnatal Development--
The m5Kb-CAT
transgene expression was further studied by monitoring CAT activity in
epididymal cell extracts during development (Fig.
7). No CAT activity was found in the
epididymis of heterozygous mice from 1 to 20 days of age. The CAT
activity was first detected at 30 days and continued to increase until
50 days of age. Finally, a 3-fold increase of CAT activity occurred
between day 50 and 60. This temporal expression pattern of the m5Kb-CAT
transgene was identical to that of the endogenous mE-RABP gene as
demonstrated by Western blot analyses (Fig. 7).
Our results demonstrate that the endogenous mE-RABP gene expression is
developmentally regulated and that the 5-kb DNA promoter fragment
contains all of the information required for correct temporal
expression of the mE-RABP gene.
The m5Kb-CAT Transgene Expression Is Specifically Regulated by
Androgens--
The appearance of the m5Kb-CAT transgene expression at
puberty suggested that androgens might be involved in its regulation. To confirm this hypothesis, adult male mice carrying the m5Kb-CAT transgene were castrated for 5, 10, 20, and 30 days (Fig.
8A). The m5Kb-CAT transgene
expression dramatically decreased during the first 10 days following
castration (47% and 5.8% of the intact heterozygous mice, 5 and 10 days after castration, respectively) and then decreased progressively
to reach background level 30 days after castration (5.7 and 3.4% of
the intact heterozygous mice, 20 and 30 days after castration). Western
blot analyses showed that endogenous mE-RABP protein expression
appeared to decrease more rapidly after castration than the CAT
activity, since the mE-RABP protein was no longer detectable 10 days
after castration as reported previously (11).
To study whether transgene expression was regulated by testicular
factors present in the luminal fluid, efferent duct ligation was
carried out (Fig. 8B). No difference of the m5Kb-CAT
transgene (90% of the heterozygous intact mice) and endogenous mE-RABP
protein expression (98.7% of the intact heterozygous mice) was
observed 30 days after efferent duct ligation.
Interestingly, testosterone replacement for 10 days to heterozygous
transgenic mice that had been castrated for 30 days restored the
expression of the m5Kb-CAT transgene (85% of the intact heterozygous mice) and that of the endogenous mE-RABP protein but to a lesser extent
(30% of the intact heterozygous mice) (Fig. 8B). Estrogen and glucocorticoid replacement failed to restore m5Kb-CAT transgene and
mE-RABP gene expression in heterozygous transgenic mice 30 days
postcastration (not shown).
Altogether, our results demonstrate that the expression of the m5Kb-CAT
transgene is not dependent on testicular factors present in luminal
fluid but is specifically dependent on androgens present in the
circulation as described previously for the mE-RABP gene (11).
Tissue-, Region-, and Cell-specific Expression of the m5Kb-CAT
Transgene--
In the present study, we have shown that the 5-kb, but
not the 0.6-kb, promoter fragment of the mE-RABP gene was able to
direct high levels of CAT reporter gene expression to the epididymis. The m5Kb-CAT transgene expression was not detected in any other tissues
examined from male or female transgenic mice, indicating a degree of
tissue specificity identical to the native gene. This occurred in the
two independent transgenic mouse lines that were established. In
contrast, the m0.6Kb-CAT transgene was not expressed in any of the four
founder transgenic mouse lines. This strongly suggests that the 5-kb
mE-RABP promoter, but not the 0.6-kb fragment, contains all of the
information needed for the epididymis-specific expression of the
mE-RABP gene.
Numerous studies have reported that promoter fragments are able to
direct tissue-specific expression of a reporter gene. For instance, the
0.4-kb promoter fragment of the probasin gene (30, 31) and the 6-kb
promoter region of the prostate-specific antigen gene (28) are able to
target transgene expression to the mouse prostate. However, gene
expression was not correlated with transgene copy number, suggesting
that, although these promoter fragments contain tissue-specific
cis-DNA regulatory elements, they may lack important
elements such as matrix attachment regions or locus control regions.
Matrix attachment regions are believed to facilitate gene expression
and may serve as specific sequence landmarks as they anchor DNA to the
nuclear scaffold (32-34). On the other hand, locus control regions are
strong enhancers that bind multiple transcription factors that function
in a cooperative manner (for review, see Ref. 34). Both of these DNA
elements are believed to ensure a position-independent and copy
number-dependent expression of a gene present in their
vicinity. Although we generated only two different m5Kb-CAT transgenic
mouse lines, transgene expression was independent of site of
integration as shown by Southern blot analysis of the genomic DNA (Fig.
1C) but was well correlated with transgene copy number. This
suggests that matrix attachment regions and/or locus control
region-like elements required for the epididymis-specific expression of
the mE-RABP gene are contained within 5 kb of the 5'-flanking region.
Within the epididymis, the 5-kb mE-RABP promoter directed CAT gene
expression to a narrow region of the epididymis, the mid/distal caput.
This mimicked the localization of the expression of the endogenous
mE-RABP gene. Region specificity is a prominent feature for gene
expression in the epididymis. Although the epididymis is a long
convoluted duct lined primarily by the same cell type, a polarized
columnar principal cell, genes encoding epididymal proteins display a
highly region-specific pattern of expression (for review, see Ref. 3).
As examples, the gpx5 gene is expressed only in the caput
epididymidis (35), while expression of the superoxide dismutase gene is
restricted to the cauda epididymidis (36). Our results show that all of
the cis-DNA regulatory elements necessary to confer
mid/distal caput-specific expression reside within the 5 kb of the
5'-flanking region of the mE-RABP gene. Therefore, we feel that this
5-kb DNA fragment provides a unique tool to identify region-specific
transcription factors that may regulate epididymal genes.
Within the caput epididymidis, the m5Kb-CAT transgene was expressed
specifically in the cytoplasm and nuclei of the principal cells as
shown by in situ hybridization and by immunohistochemistry. No staining was observed in other cell types either in the epithelium or in the connective tissue between the tubules. The nuclear
immunolocalization of the CAT protein may be due to a cryptic nuclear
localization signal. Indeed, the CAT protein contains the motif KKNK in
its primary amino acid sequence, which is homologous to the nuclear localization signal (KKRK) of the DNA helicase Q1 (37). This cell-specific expression of the m5Kb-CAT transgene mimicked that previously reported for the endogenous mE-RABP gene (11). In addition,
as with mE-RABP, there was a checkerboard pattern of expression at the
proximal and distal borders of the distal caput epididymidis such that
some principal cells exhibited strong expression, whereas adjacent
principal cells displayed no or very low levels of expression. A
similar pattern has also been observed for other epididymal genes (for
review, see Ref. 3). These observations imply that the 5-kb mE-RABP
promoter contains all of the information necessary for this highly
cell-specific gene expression. A single transcription factor can
function as a trigger to determine tissue-specific gene expression. For
instance, the critical role of the Pit-1 transcription factor in cell
differentiation of the pituitary somatotropes, lactotropes, and
thyrotropes is well documented (for review, see Ref. 38). However, to
achieve cell-specific gene expression, the tissue-specific
transcription factor may combine with other regulatory proteins to
control gene expression (39, 40). On the basis of a such model, it is
reasonable to anticipate that the m5Kb-CAT transgene contains several
cis-DNA elements that bind ubiquitous, tissue-, and/or
cell-specific transcription factors.
The m5Kb-CAT Transgene Expression Is Specifically Regulated by
Androgens--
Our study clearly demonstrated that the m5Kb-CAT
transgene expression was developmentally regulated and mimicked that of
the endogenous mE-RABP gene. The m5Kb-CAT gene expression was well correlated with the increase of DHT and androgen receptor content that
occurs in the mouse epididymis during development (41, 42). Castration,
efferent duct ligation, and hormonal replacement studies confirmed that
androgens, but not other testicular factors, were required to maintain
m5Kb-CAT transgene expression in adult animals. Moreover, in
vitro transient transfection assays had shown that the 5-kb
promoter fragment of the mE-RABP gene directing the CAT reporter gene
was highly androgen-responsive, indicating that androgens may control
mE-RABP gene expression at the transcriptional level (18). In addition,
only androgens, but not glucocorticoids or estradiol, were able to
increase reporter gene expression in vitro. Therefore, the
m5Kb-CAT transgene expression during development and its
androgen-specific regulation are consistent with our previous in
vitro studies, demonstrating that the 5-kb promoter fragment of
the mE-RABP gene confers androgen-specific responsiveness and that
androgens may act directly at the transcriptional level to modulate
mE-RABP gene expression in vivo.
We have demonstrated in transient transfection studies using HeLa cells
that a functional ARR was localized within the first 600 bp of the
mE-RABP gene promoter (18). A similar androgen response region was also
identified within the 400-bp promoter region of the probasin gene,
which encodes another androgen-regulated lipocalin in the rat prostate
(43). In transgenic mice, this short 400-bp promoter fragment was
sufficient 1) to restrict gene expression to the prostate and 2) to
confer androgen control (30, 31). In the present study, the m0.6Kb-CAT
transgene was not sufficient to direct detectable levels of the CAT
reporter gene expression in four independent transgenic mouse lines. A
similar result has been reported for the human prostate-specific
antigen gene encoding an androgen-regulated prostatic secretory protein and belonging to the kallikrein gene family. The first 600 bp of the
prostate-specific antigen gene promoter did not target gene expression
to the prostate, although two distinct functional androgen receptor
binding sites that cooperate were localized within the first 600 bp of
the prostate-specific antigen gene promoter (44). However, a 6-kb
promoter fragment containing an upstream enhancer was able to place
reporter gene expression under androgen control in the prostate of
transgenic mice (28). Therefore, it is probable that the ARR localized
within the first 600 bp of the mE-RABP gene promoter requires an
upstream enhancer present in the m5Kb-CAT transgene to drive gene
expression in vivo. Further deletions between 0.6 and 5 kb
upstream from the transcription initiation site of the mE-RABP gene
will be required to identify important cis-DNA elements required for
promoter activity in vivo. This enhancer may bind to
tissue-specific transcription factors, but our results do not exclude
the possibility that the 600-bp promoter fragment of the mE-RABP gene
may also be required for androgen-regulated and tissue-specific gene expression.
Concluding Remarks--
Despite the fact that the epididymis is
the site of an important physiological event, the maturation of the
male gamete, little is known about the molecular mechanisms regulating
its function. The highly region-specific pattern of gene expression
observed in the epididymis may be required to coordinate the functions of the different epididymal regions to ensure the efficient
post-testicular maturation of spermatozoa. In the present study, we
demonstrate that the 5-kb promoter fragment of the mE-RABP gene
restricts high levels of gene expression to the principal cells of the
mid/distal caput epididymidis in transgenic mice. To our knowledge,
this is the first demonstration of targeted expression by an epididymal gene promoter. It is now possible to further dissect and identify the
cis-DNA regulatory elements and their associated proteins (steroid
receptor coactivators, tissue- and/or cell-specific transcription factors) that are involved in the regulation of the tissue region- and
cell-specific expression of epididymal genes.
The identification of an androgen-regulated and epididymis-specific
gene promoter is not only relevant to identifying individual regions of
the gene involved in cell specificity and hormonal regulation but also
enables one to study the function of epididymal secretory proteins that
are involved in the sperm maturation process. It is possible to disrupt
gene function by targeted mutation ("knockout"), but it is also
possible to use the antisense RNA or protein engineering technologies
as alternative strategies to inhibit gene function. Due to the high
level of gene expression achieved with the m5Kb-CAT transgene, the
mE-RABP gene promoter will be an appropriate tool to overexpress an
antisense-ribozyme RNA or a dominant negative protein to inhibit the
expression and/or the function of target proteins in
vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
-estradiol-3-benzoate (2 µg/g), hydrocortisone 17-butyrate (2 µg/g) dissolved in sesame oil. Treatments were conducted for 9 days,
and mice were killed 1 day after the last injection. Organs were
excised, immediately frozen in liquid nitrogen, and stored at
80 °C.
80 °C with Hyperfilm MP film (Amersham
Pharmacia Biotech). The relative density of the transgenes and
endogenous mE-RABP gene was determined using an imaging densitometer
(Bio-Rad model GS-670) and Molecular Analyst software.
3 Prime, Inc., Boulder, CO)
(1:1000). The slides were subsequently washed in 1× PBS, incubated at
room temperature for 2 h with anti-rabbit IgG antibody (DAKO,
Carpinteria, CA), washed in 1× PBS for 5 min, and incubated at room
temperature with rabbit PAP (DAKO) for 1 h. The slides were washed
again in 1× PBS for 5 min and equilibrated in 50 mM
Tris-Cl, pH 7.5. Peroxidase activity was revealed using the DAKO liquid
DAB substrate kit. The staining was monitored under the microscope and
stopped in tap water. Sections were dehydrated and mounted with
Permount (Fisher) for photography.
80 °C before use.
10 µg of total protein were separated by SDS-polyacrylamide gel
electrophoresis (17% polyacrylamide gel) and transferred to a
ProtranTM nitrocellulose membrane as described previously
(25). Nitrocellulose membranes were incubated overnight at 4 °C in
PBS, 1% (w/v) bovine serum albumin, washed five times in PBS, 0.1%
(w/v) bovine serum albumin, 0.1% (v/v) Tween 20, prior to incubation
for 1 h with the immune rabbit IgG anti-mE-RABP (10). The
membranes were washed five times in PBS, 0.1% (w/v) bovine serum
albumin, 0.1% (v/v) Tween 20; incubated for 1 h at room
temperature with biotinylated anti-rabbit IgG (Vector Laboratories
Inc., Burlingame, CA); washed again; and incubated for 1 h at room
temperature with ABC-peroxidase reagent (Vector Laboratories). After
washing five times in PBS, 0.1% (v/v) Tween 20 and once in 1× PBS,
reactive bands were visualized using a solution containing 0.5 mg/ml
diaminobenzidine, 0.02% H2O2, 0.04%
NiCl2 in 0.05 M Tris-HCl, pH 7.5. The reaction
was stopped with H2O, and the membrane was air-dried. The
relative density of the mE-RABP bands was determined using densitometry (Bio-Rad model GS-670) and Molecular Analyst software.
) vector (Promega) to obtain the pGEM-CAT construct. CAT
RNA probes were transcribed from pGEM-CAT and labeled with
[35S]UTP to a specific activity of l.2·l09
cpm/µg using the in vitro transcription kit
MAXIscriptTM (Ambion Inc., Austin, TX). Hybridization was
carried out at 55 °C with 2·104 cpm/ml riboprobe
overnight in 50% (v/v) formamide, 300 mM NaCl, 10 mM Tris (pH 7.4), 10 mM
NaH2PO4 (pH 6.8), 5 mM EDTA (pH 8), 0.2% (w/v) Ficoll 400, 0.2% (w/v) polyvinyl pyrrolidone, 10% (w/v) dextran sulfate, 200 µg/ml yeast transfer RNA, and 50 µM dithiothreitol (DTT). Excess of riboprobe was removed
by washing in 2× SSC, 20 mM
-mercaptoethanol for 15 min
at 50 °C, once followed by two washes in 4× SSC, 50% (v/v)
formamide, 20 mM
-mercaptoethanol for 30 min each at
55 °C, and two washes in 4× SSC, 20 mM Tris-Cl, pH 7.5, 2 mM EDTA for 10 min each at 37 °C. Single-stranded RNA was digested in 4× SSC, 20 mM Tris-Cl, pH 7.5, 2 mM EDTA, and 20 µg/ml RNase A for 30 min at 37 °C. The
reaction was stopped by two washes in 4× SSC, 20 mM
Tris-Cl, pH 7.5, 2 mM EDTA for 10 min each at 37 °C and
two washes in 4× SSC, 50% (v/v) formamide, 20 mM
-mercaptoethanol for 30 min each at 55 °C. Slides were quickly
rinsed twice in H2O and air-dried. Slides were dipped in
NTB-2 Kodak emulsion and exposed for 23 days at 4 °C, developed, fixed, and mounted with Permount (Fisher) for photography.
RESULTS
-galactosidase activity present in the epididymis (26,
27), which cannot be suppressed by the usual modification of the
standard protocols of staining for 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (28).
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Fig. 1.
Schematic map of the chimeric constructs and
characterization of the transgenic mouse lines. A,
genomic organization of the mE-RABP gene and schematic maps of the
m5Kb-CAT and m0.6Kb-CAT transgenes. Coding and noncoding regions of the
mE-RABP gene are represented with black and white
boxes, respectively. The initiation start site is indicated
by a broken arrow. The size (bp) of the exons and
introns is indicated below. The nucleotide sequence is
numbered according to the major initiation start site of the mE-RABP
gene. Positions of the primers used to identify the transgenic mice are
represented by horizontal arrows. Finally, m0.6Kb
and CAT DNA probes used to determine transgene copies number are also
illustrated. B, characterization of the transgenic mouse
lines by PCR of tail DNA. Primers 1 and 2 were used to amplify a 320-bp
DNA fragment corresponding to the transgene. The casein forward and
casein reverse primers were also used to amplify a DNA fragment (590 bp) of the casein gene as positive control of the PCR. C,
southern blot analysis of genomic DNA extracted from wild type and
transgenic mouse lines. 20 µg of genomic DNA was digested with
HindIII, and resulting DNA fragments were separated on a
0.8% (w/v) agarose gel. DNA fragments were blotted on a nylon membrane
and hybridized with the 32P-labeled m0.6Kb probe
(left panel). Hybridization signals, observed
after autoradiography, were quantified using an imaging densitometer
(model GS-670, BIORAD). Transgene copy number was determined by
calculating the ratio between the intensity of DNA fragments
corresponding to transgenes (asterisks) and the
9Kb-HindIII DNA fragment corresponding to the endogenous
mE-RABP gene (arrow) multiplied by 2. D, the
identity of DNA bands corresponding to the transgenes was also
confirmed using the CAT probe.
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Fig. 2.
m5Kb-CAT and m0.6Kb-CAT transgene expression
levels in the caput epididymidis of transgenic mouse lines. Three
adult male mice (60 days old) from each transgenic mouse line and from
wild type mice (WT) were killed. The caput from each
epididymis was dissected and stored individually at 80 °C. CAT
activity was determined as described under "Experimental
Procedures." To enhance the sensitivity of the CAT assays, different
concentrations of total protein were used (line 4, 10 µg; line 19, 100 µg; wild type and lines 143, 148, 149, and 391, 200 µg), but
final results are expressed per mg of protein. Values of the CAT
activity are presented as the mean ± S.E.
CAT activity in the epididymis of heterozygous and homozygous m5Kb-CAT
transgenic mice
80 °C. CAT activity was measured
as described under "Experimental Procedures." Different amounts of
total protein were used to determine the CAT activity in the caput
epididymidis as follows: wild type, 200 µg; heterozygous transgenic
mice, 10 and 100 µg for line 4 and 19, respectively; homozygous
transgenic mice, 5 and 50 µg for line 4 and 19, respectively. Values
are presented as the mean ± S.E. Note that CAT activity increases
twice on average between heterozygous and homozygous transgenic mice.
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Fig. 3.
Tissue-specific expression of the m5Kb-CAT
transgene in the caput epididymidis. Tissues were dissected from
three adult male mice (60 days old) belonging to B6D2 wild type or
transgenic mouse lines and then stored individually at 80 °C. CAT
activity was measured as described under "Experimental Procedures."
10 and 100 µg of total protein were used to determine the CAT
activity in the caput epididymidis of mouse lines 4 and 19, respectively. 200 µg of total protein was used when the CAT activity
was assayed in the other tissues. Values are presented as the mean ± S.E. per mg of protein.
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Fig. 4.
In situ hybridization of the
m5Kb-CAT transgene mRNA. The regions of the epididymis are
classified according to Abou-Haila and Fain-Maurel (29). A,
epididymis from wild type mouse incubated with CAT antisense probe. No
CAT mRNA was detected. B, epididymis from homozygous
mouse line 4 incubated with CAT sense probe. No CAT mRNA was
detected. C and D, epididymis from homozygous
mouse line 4 incubated with CAT antisense probe. ED,
efferent ducts; 1, initial segment; 4 and
5, distal caput; 6, corpus; 7,
proximal cauda; 8, distal cauda. Note that the plan of
section is such that segments 2 and 3 are not visible. High levels of
CAT mRNA are detected in the distal caput epididymidis.
E, epididymis from homozygous mouse line 4 incubated with
mE-RABP antisense probe. Note the following: 1) the plan of the section
is identical to that treated with the CAT antisense probe, and 2) the
localization of the m5Kb-CAT transgene mRNA is identical to that of
the endogenous mE-RABP mRNA.
3 Prime, Inc.). No CAT expression was detected in the efferent ducts and initial segment of the epididymis from heterozygous mouse line 4 (Fig. 5A). A faint staining was
first detected in the cytoplasm and nucleus of principal cells in
segment 2 (Fig. 5E) (classification of Abou-Haila and
Fain-Maurel (29)). Levels of CAT gene expression progressively
increased in segment 3, reaching a maximum in the distal portion of
segment 3 and segment 4 (Fig. 5F). Finally, expression
decreased in segment 5 and ended with a checkerboard pattern,
i.e. some principal cells expressed CAT and others did not,
at the boundary between the distal caput and the proximal corpus (Fig.
5G). No staining was observed in the apical cells, the clear
cells, the basal cells of the epithelium, the myoid cells surrounding
the tubule, or cells of the connective tissue between the tubules (Fig.
6). No transgene expression was seen in
the corpus, the cauda or the vas deferens (data not shown). Thus, the
pattern of transgene expression was identical to that described
previously for the endogenous mE-RABP gene (11).
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Fig. 5.
Immunolocalization of the CAT and mE-RABP
proteins in the m5Kb-CAT transgenic mice. A, the CAT
protein is expressed in both the cytoplasm and nuclei of principal
cells from segments 2 to 5 of the epididymis in heterozygous m5Kb-CAT
transgenic mice (line 4). E, note that the principal cells
of the initial segment are negative (large arrow)
and that the intensity of staining increases more distally with some
cells displaying a more intense staining (magnification × 125).
F, principal cells of region 4 are uniformly stained but
clear cells (small arrows) are negative
(magnification × 125). G, principal cells in region 5 display a checkerboard pattern with some cells stained and others not
stained (magnification × 125). B, the mE-RABP protein
is secreted and is visible in the lumen of the duct from region 4 to
more distal regions. C and D, immunolocalization
of the CAT protein in the epididymis of wild type (C) and
heterozygous m0.6Kb-CAT transgenic (line 149) mice (D). No
CAT protein was detected.
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Fig. 6.
Immunolocalization of the CAT protein in the
distal caput epididymidis of the m5Kb-CAT transgenic mice.
A, principal cells display uniform levels of CAT expression.
However, apical cells (small arrows), clear cell
(long arrow), and peritubular cells (basal cells
and myoid cells) (short arrow) are not stained
(magnification × 600). B, matched phase contrast
photograph (magnification × 600).
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Fig. 7.
Developmental expression of the m5Kb-CAT
transgene in the epididymis of mouse line 19. The CAT activity was
determined, as described under "Experimental Procedures," at day 1 from a pool containing 40 epididymides, at day 10 from two pools
containing 20 epididymides, and at days 20, 30, 40, 50, and 60 from
three animals. 200 µg of total protein was used in CAT assays at days
1, 10, and 20, whereas 100 µg of total protein was used at days 30, 40, 50, and 60. Values are presented as the mean ± S.E.
Lower panel, Western blot analysis of mE-RABP
during postnatal development. Since mE-RABP is a major epididymal
secretory protein, only 20 µg of total epididymal proteins was
separated on a SDS-polyacrylamide gel electrophoresis (15%
polyacrylamide gel) and transferred to a nitrocellulose membrane. The
mE-RABP expression was detected using a purified IgG raised against
mE-RABP (10).
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Fig. 8.
Hormonal regulation of the m5Kb-CAT transgene
in the epididymis of mouse line 4. A, m5Kb-CAT
transgene expression after castration. CAT activity was determined in
the epididymis of intact (I) and 5-, 10-, 20-, and 30 day-castrated heterozygous (+/ ) adult male mice (Line 4)
and compared with that of intact wild type adult male mice
(WT). Values are presented as the mean ± S.E. of three
individual animals. B, m5Kb-CAT expression after efferent
duct ligation or androgen replacement. Three 30-day castrated adult
heterozygous male mice were injected with heptylate testosterone
(testosterone propionate (TP); 200 µg/day) or sesame oil
(SO; 100 µl/day) for 10 days. Three mice had their
efferent ducts ligated for 30 days (Lig). Lower
panel, Western blot analysis of mE-RABP expression in intact
(I), castrated (5, 10, 20, and 30 days postcastration), and
30-day castrated male mice supplemented with testosterone heptylate
(testosterone propionate; 200 µg/day for 10 days) or sesame oil (100 µl/day for 10 days). Total epididymal proteins (10 µg/lane) were
separated on a SDS-polyacrylamide gel electrophoresis (15%
polyacrylamide gel) and transferred to a nitrocellulose membrane. The
mE-RABP expression was detected using a purified IgG raised against
mE-RABP (10).
DISCUSSION
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the Vanderbilt Transgenic/ES cell Shared Resource for generating the transgenic mouse lines. We thank Drs. C. Pettepher and J. Wright for helpful advice throughout the course of these studies. We thank Dr. B. J. Danzo for critical comments on the manuscript. The DNA sequencing was performed by the Cancer Center DNA Sequencing Core, directed by Dr. K. Bhat.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by National Institutes of Health Grants HD03820, HD05797, HD36900, and HD25206.
§ Present address: Station Commune de Recherches en Ichtyophysiologie Biodiversité et Environnement, Institut National de la Recherche Agronomique, Campus de Beaulieu, 35042 Rennes Cedex, France.
§§ To whom correspondence should be addressed: Center for Reproductive Biology Research, Vanderbilt University, School of Medicine, Medical Center North, Room D2303, Nashville, TN, 37232-2633. Tel.: 615-322-7484; Fax: 615-343-7797; E-mail: m-c.orgebin-crist{at}mcmail.vanderbilt.edu.
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ABBREVIATIONS |
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The abbreviations used are: mE-RABP, murine epididymal retinoic acid-binding protein; ARR, androgen-specific response region; bp, base pair(s); kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
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REFERENCES |
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