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Departments of Obstetrics and Gynecology (J.-J.L., M.-C.O.-C.), Biochemistry (D.E.O.), Cell Biology (V.P.W., S.K., R.J.M., G.E.O., M.-C.O.-C.), and Urologic Surgery (S.K., R.J.M.) and Center for Reproductive Biology Research (S.K., D.E.O., R.J.M., G.E.O., M.-C.O.-C.), Vanderbilt University, Nashville, Tennessee 37232
Address all correspondence and requests for reprints to: Dr. Marie-Claire Orgebin-Crist, Center for Reproductive Biology Research, Vanderbilt University School of Medicine, Medical Center North, Room C-3306, Nashville, Tennessee 37232-2633.
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Abstract |
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Introduction |
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in the mouse cauda epididymidis
(3) results in the degeneration of the epithelium and male
infertility. Therefore, epididymal function is also dependent on the
delivery of retinoids.
We previously identified a mouse epididymal secretory protein (MEP 10)
(4), now named murine epididymal retinoic acid-binding
protein (mE-RABP), that is specifically synthesized by principal cells
of the mouse mid/distal caput epididymidis and is secreted into the
tubule (4). Once in the luminal fluid, mE-RABP is in
contact with spermatozoa, but does not bind tightly to them, as it was
not detected after washing (4). The deduced amino acid
sequence of mE-RABP is highly homologous (75% identity, 90% homology)
to two rat epididymal retinoic acid-binding proteins successively named
proteins B/C (5, 6), epididymal-binding protein-I
and -2 (7), epididymal secretory protein I (ESPI)
(8), and E-RABP (9). These proteins are not
restricted to rodent epididymis, because the N-terminal sequences of
two boar epididymal secretory proteins are highly similar to mE-RABP
and ESP-I (10). The mE-RABP and ESP-I proteins belong to
the lipocalin superfamily (11, 12). The three-dimensional
structure analyses reported to date show that lipocalins have an eight
up and down stranded 
-barrel closed at one end by a single turn of
-helix forming a hydrophobic binding cavity. This hydrophobic pocket
is well adapted for noncovalent binding and transport of small
lipophilic ligands. The mE-RABP binds active retinoids
(9-cis and all-trans-retinoic acid)
(13) and therefore may function as a carrier protein and
mediate a retinoic acid signal pathway in the mouse epididymis
(14).
The gene encoding mE-RABP was isolated and mapped to the locus [A3,B] of mouse chromosome 2 (15). We have recently shown that 5 kb, but not 0.6 kb, of the 5'-flanking region of the mE-RABP gene targeted high levels of chloramphenicol acetyl transferase reporter gene expression to the principal cells of the mid/distal caput epididymidis in transgenic mice (16). Transgene expression was consistent with the temporal and spatial expression pattern of the endogenous mE-RABP messenger RNA (mRNA) and was regulated by androgens. This demonstrates that cis-DNA regulatory elements important for epididymis-, region-, and cellspecific gene expression and regulation are located within the 5 kb upstream from the mE-RABP gene.
In this study we identified within 5 kb of the 5'-flanking region of the mE-RABP gene a new gene encoding a 17-kDa lipocalin. This protein was named mEP17 for mouse epididymal protein of 17 kDa. Northern blot and in situ hybridization analyses were carried out to study the tissue-, region-, and cell-specific expression as well as regulation of the mEP17 mRNA. Further, we demonstrate that a mEP17-like gene is conserved in the rat and hamster genome. The conservation of mEP17-like gene expression during evolution suggests that it may play an important function in male fertility.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation and sequencing of genomic DNA fragments
DNA fragments from the Bac clone 10983, containing 5 kb of the
5'-flanking region of the mE-RABP gene (15), were
subcloned in pBluescript SK+ using appropriate
enzymes (Promega Corp., Madison, WI). DNA templates for
sequencing were purified using the Plasmid Midi kit
(QIAGEN, Santa Clarita, CA). Sequencing reactions were
performed as described in the Thermo Sequenase fluorescent-labeled
primer cycle sequencing kit (Perkin-Elmer Corp., Foster
City, CA). DNA fragments were separated in a denaturing PAGE (6%
acrylamide gel) and analyzed using an ABI 373A automated sequencer
(PE Applied Biosystems, Foster City, CA). Nucleotide
sequences were analyzed using the GeneJockey sequence processor
(Biosoft, Milltown, NJ).
Primer extension analysis
Total RNA was extracted from the mouse epididymis using the
method described previously (17). The mEP17PE2 primer
(5'-CTTCTCTGGTACAAGCTCCACCCTGGT-3') specific for mEP17 mRNA was
radiolabeled using T4 polynucleotide kinase in the presence of 100
µCi [
-32P]ATP (3000 Ci/mmol;
Amersham Pharmacia Biotech, Arlington Heights, IL)
according to the manufacturer’s instructions (New England Biolabs, Inc.). For each reaction, 10 µg epididymal total RNA
or transfer RNA were hybridized to 1 pmol (105
dpm) mEP17PE2 primer for 12 h at 35 C in 10 µl of a solution
containing 0.04 M PIPES (pH 6.4), 1 µM EDTA,
and 80% (vol/vol) formamide. RT was performed in 20 µl containing 50
µM Tris-HCl (pH 8.3), 30 µM KCl, 8
µM MgCl2, 6 µM
dithiothreitol, 0.5 mM of each deoxy-NTP, and 50 U avian
myeloblastosis virus (AMV) reverse transcriptase (Promega Corp.). Samples were incubated for 30 min at 42 C, and then 50 U
AMV reverse transcriptase were added again and incubated for 1 h
more. Elongated radiolabeled fragments were loaded on denaturing PAGE
(7% acrylamide gel) next to sequencing reactions carried out using the
Sequenase sequencing kit (Amersham Pharmacia Biotech). The
clone pHindIII (15) and the mEP17PE2 primer
were used as template and primer, respectively.
Isolation of a complementary DNA (cDNA) encoding mEP17
For RT, 2 µg epididymal total RNA were incubated in 50
mM Tris-HCI (pH 8.3); 75 mM KCl; 10
mM dithiothreitol; 3 mM
MgCl2; 0.5 mM each of deoxy (d)-GTP,
dATP, dCTP, and dTTP; 2 U/µl RNasin (Promega Corp.); 10
µg/ml oligonucleotide RACEIII
(5'-CAGCTGCAGGTACCGGATCCTCGAGAAGC(T)18-3'); and
200 U Moloney murine leukemia virus reverse transcriptase for 1 h
at 42 C. The DNA/RNA hybrids were denatured for 5 min at 90 C and
stored at -80 C. PCR was performed using 100 ng cDNA incubated in a
mixture containing 0.2 mM each of dGTP, dATP, dTTP, and
dCTP; 1 µM of each primer (FwmEP17cDNA,
5'-GGCCCTGAGATGGAAGCTAGG-3'; RiIII,
5'-GCAGGTACCGGATCCTCGAGAAG-3'); 1 x reaction buffer II
(Perkin-Elmer Corp.); and 1 U AmpliTaq (Perkin-Elmer Corp.). DNA was amplified for 29 cycles, consisting of 1 min at
94 C, 2 min at 55 C, and 2 min at 72 C. PCR products were purified on
2% (wt/vol) agarose gels, ligated into pGEM-T easy plasmid
(Promega Corp.), and sequenced.
Southern blot analyses of genomic DNA
Genomic DNA was extracted from the livers of adult male
mice (strain 129 SvJ), adult male rats (Sprague Dawley), and adult male
golden hamster as described previously (18). DNA (15 µg)
was digested with 40 U HindIII (Promega Corp.),
electrophoresed on a 0.8% (wt/vol) agarose gel, and then incubated in
0.25 N HCl for 10 min, in 0.5
N NaOH and 1.5 M NaCl for
30 min, and twice in 0.5 M Tris-HCl (pH 7.5), 1
mM EDTA, and 1.5 M NaCl for
15 min each time. DNA fragments were transferred overnight to a Hybond
N+ nylon membrane (Amersham Pharmacia Biotech) by blotting (19). The membrane was baked
2 h at 80 C and prehybridized for 3 h at 42 C in 6 x
SSC (standard saline citrate), 1% (wt/vol) SDS, 100 µg/ml salmon
sperm DNA, 50% (vol/vol) formamide, and 5% (wt/vol) dextran sulfate.
The random primed 32P-radiolabeled probe was
synthesized using the Rediprime DNA labeling system (Amersham Pharmacia Biotech) and incubated overnight
(106 dpm/ml). The filter was washed once in
2 x SSC for 15 min, once in 2 x SSC and 0.1% (wt/vol) SDS
for 15 min, once in 2 x SSC and 0.1% (wt/vol) SDS for 30 min,
once in 0.2 x SSC and 0.1% (wt/vol) SDS for 15 min at 65 C, and
once in 0.1 x SSC and 0.1% (wt/vol) SDS for 15 min at 65 C
before being autoradiographed for 0.5–4 days at -80 C with Hyperfilm
MP film (Amersham Pharmacia Biotech).
Northern blotting
Total RNA (10 µg) was denatured for 15 min at 65 C and
cooled on ice. RNA samples were loaded on a 1% (wt/vol) agarose gel
containing 20 mM MOPS (pH 7), 5 mM
sodium acetate, 1 mM EDTA, and 6% (vol/vol) formaldehyde
and then transferred to a Hybond N+ nylon
membrane (Amersham Pharmacia Biotech) by blotting
overnight in 20 x SSC. The membrane was washed once in 2 x
SSC, dried, and baked 2 h at 80 C. The prehybridization was
carried out for 3 h at 42 C in 50% (vol/vol) formamide, 6 x
SSC, 5 x Denhardt’s solution, 100 µg/ml salmon sperm DNA, and
0.1% (wt/vol) SDS and then random primed
32P-labeled mE-RABP cDNA prepared with the Random
Prime DNA labeling kit (Amersham Pharmacia Biotech) was
added and incubated overnight. The filter was washed once in 2 x
SSC for 15 min, once in 2 x SSC and 0.1% (wt/vol) SDS for 15
min, once in 2 x SSC and 0.1% (w/vol) SDS for 30 min, once in
0.2 x SSC and 0.1% (wt/vol) SDS for 15 min, and once in 0.l
x SSC and 0.1% (wt/vol) SDS for 15 min at 65 C before being
autoradiographed with Hyperfilm MP (Amersham Pharmacia Biotech). Northern blots were reprobed with a cloned 18S cDNA to
standardize the loaded RNA samples. The relative absorbance of the
mEP17 and 18S RNA was determined using an imaging densitometer (model
GS-670, Bio-Rad Laboratories, Inc., Richmond, CA) and the
application Molecular Analyst.
In situ hybridization
Nonisotopic in situ hybridization
(20, 21) was performed on 4- to 6-µm thick cryosections
of fresh-frozen mouse epididymides. Sections were fixed in 4%
formaldehyde in 0.1 M sodium phosphate buffer, pH
7.2, and then incubated for 10 min in PBS containing 5 µg/ml
proteinase K. After two rinses in PBS, sections were incubated in
0.25% acetic anhydride in 0.1 M triethanolamine,
pH 8.0, for 15 min. Sense and antisense riboprobes were prepared in
20-µl transcription reactions containing SP6 (Promega Corp.) or T7 (New England Biolabs, Inc., Beverly,
MA) polymerase; 1 x transcription buffer; 1
mM each of ATP, CTP, and GTP; 0.65
mM UTP; 0.35 mM
digoxigenin-UTP (Roche, Indianapolis, IN); and 1 µg
linearized F3 plasmid carrying the mEP17 cDNA. Unincorporated
nucleotides were removed on a Chroma Spin-100 500
mM NaCl, 20 mM Tris-HCl (pH
7.5), and 1 mM EDTA (STE) column (CLONTECH Laboratories, Inc., Palo Alto, CA). Digoxigenin
riboprobes were denatured for 5 min at 80 C; diluted in hybridization
buffer composed of 50% (vol/vol) formamide, 10% (wt/vol) dextran
sulfate, 4 x SSC, 1 x Denhardt’s reagent, and 0.5 mg/ml
yeast transfer RNA; and incubated with the sections overnight at 55 C.
The slides were washed at room temperature for 5 min in 2 x SSC,
rinsed in STE, and then incubated for 30 min in STE containing 40
µg/ml ribonuclease A. The sections were washed sequentially for 5 min
each in 2 x SSC and 50% formamide at 50 C, then at room
temperature with 1 x SSC, and finally with 0.5 x SSC. To
detect hybridized probes slides were rinsed in 100
mM Tris-HCl (pH 7.5) and 150
mM NaCl, blocked for 1 h in 100
mM Tris-HCl (pH 7.5) and 150
mM NaCl containing 2% horse serum and 0.1%
Triton X-100 (blocking solution), and incubated for 1 h in 1:500
diluted alkaline phosphatase-conjugated antidigoxigenin
(Roche) in blocking solution. Slides were rinsed three
times in blocking solution and then in a substrate buffer of 100
mM Tris-HCl (pH 9.5), 100
mM NaCl, and 50 mM
MgCl2. Color development was performed in
substrate buffer containing 0.17 mM
5-bromo-4-chloro-3-indolyl phosphate, 10 mM
N-ethyl-maleimide, and 1 mM levamisole
as an inhibitor of endogenous alkaline phosphatase. Color development
was stopped with 10 mM Tris-HCl (pH 8.0) and 1
mM EDTA. Sections were examined and photographed
with a Carl Zeiss Axiophot (New York, NY) using both
brightfield and phase contrast optics.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The DNA sequence of the predicted
exons allowed us to design a DNA primer (FwmEP17cDNA), which was used
to clone the corresponding cDNA by rapid amplification of 3'-cDNA ends
(3'-RACE; Fig. 1). As the 5-kb genomic DNA sequence restricted gene
expression to the epididymis, epididymal total RNA was reverse
transcribed using the RACE III primer. A full-length cDNA (694 bp) was
generated by PCR using the FwmEP17cDNA and RiIII primers (Fig. 2). The exon/intron boundaries were
confirmed by comparison of sequences between the cDNA and the genomic
DNA. The genomic organization, constituted by seven exons interrupted
by six introns, was identical to that of the mE-RABP gene. Moreover,
our results indicated that the exon/intron boundaries were also
strictly conserved between the genes. We also noticed that a short
18-bp pyrimidine-rich nucleotide sequence within the
3'-untranslated region was conserved between mEP17 and mE-RABP cDNA
(Fig. 2).
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). The analysis of the
hydropathic pattern of the precursor indicated that the first 21 amino
acid residues constituted a transmembrane domain (Fig. 3). This 2.4-kDa domain is probably a
signal peptide, because it is 1) highly hydrophobic, 2) similar to the
signal peptide of the mE-RABP protein, and 3) in agreement with the
sliding window/matrix scoring method and -1, -3 rule for predicting
signal peptide cleavage (22). This observation implies
that the new gene encodes a putative 17-kDa mature protein
(17.39582–17.61202 kDa; pI 5.69–5.94) that may be secreted into the
luminal fluid. Therefore, we named the new gene, mEP17, for mouse
epididymal protein of 17 kDa.
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). It is well documented that
members of the lipocalin superfamily share a few highly conserved amino
acid sequence motifs believed to be important for the tertiary
structure of the proteins, but have low overall sequence identity
(
20%) and similarity (50%) (23, 24). As expected,
low, but significant, identity was found with members of the lipocalin
superfamily, including horse lactoglobulin (24.9% identity)
(25), chicken quiescencespecific protein (24.3%)
(26), mE-RABP (23.9%) (12), human placenta
protein of 14 kDa (23.4%) (27), ESP I (22.9%)
(5), murine urinary protein IV (21%) (28),
and lizard epididymal secretory protein IV (15.5%) (29).
The mEP17 protein possesses the three structurally conserved regions
(T-D-Y, G-X-W and two cysteine residues) that have been proposed to be
a prerequisite for a protein to be considered a lipocalin
(30). These motifs are encoded by exons 4, 2, 3, and 6,
respectively, as reported previously for other genes encoding
lipocalins.
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). Two major transcription initiation
sites were localized 22 and 18 nucleotides from the putative
translation initiation site and were numbered +1 and +5, respectively.
Two minor transcription initiation sites were also detected at
positions +2 and +4. A putative TATA box (5'-TATAAG-3') and a CAAT box
(5'-CCAAT-3') are present at positions -26 and -73, respectively
(Fig. 6). A putative SP 1 transcription
factor-binding site is located at position -240. A putative retinoic
acid response element (RARE) constituted by a direct repeat separated
by 1 bp (DR1) is present at position -259.
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). A computer-base analysis of the DNA sequence
revealed the presence of a putative androgen receptor-binding site at
position -342. The presence of several putative activator protein-1
and c-Ets cis-DNA regulatory elements as well as several
potential sex-determining region Y gene product-related transcription
factor-binding sites was also noted.
Another gene related to the mEP17 gene or a pseudogene may be
present in the mouse genome
The 694-bp 32P-radiolabeled mEP17 cDNA was
used to probe the mouse strain 129 genomic DNA in Southern blot
experiments (Fig. 7). A complex
hybridization pattern was obtained with most of the restriction enzymes
used. In particular, two bands (3.8 and 9 kb in size) were detected
when the HindIII restriction enzyme was used. The 9-kb
HindIII restriction fragment, but not the 3.8-kb DNA
fragment, was in agreement with the restriction map of the mEP17 gene
locus. The 3.8-kb HindIII restriction fragment could not be
explained by the presence of a different allele of the mEP17 gene,
because this DNA fragment was not detected when the mE-RABP cDNA or the
mE-RABP promoter was used as probe (15). In addition,
hybridization of the plasmid Bac 10983, containing 175 kb of the mEP17
gene locus, with the mEP17 cDNA used as probe revealed the presence of
a 9-kb HindIII restriction fragment only (not shown).
Therefore, it is likely that the HindIII 3.8-kb restriction
fragment belongs to a pseudogene or to another gene encoding a protein
related to mEP17.
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). Hybridization of the Northern
blots with a 32P-radiolabeled mEP17 cDNA probe
revealed two RNA species of about 3.1 and 1 kb only in the epididymis
(Fig. 8). The total length of the mEP17 gene, including exons and
introns, is 3.1 kb. To determine whether the 3.1-kb RNA could be the
precursor RNA, two epididymal RNA samples were run side by side and
hybridized individually with the cDNA probe or with a probe
encompassing intron 1 of the mEP17 gene (Fig. 8B). The intron 1 probe
hybridized with the 3.1-kb RNA, but not the 1-kb RNA, indicating that
the 3.1-kb RNA may be an unspliced precursor.
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and 10). No hybridization was seen when the
sense riboprobe was used (Fig. 10). mEP17 gene expression was high
throughout the initial segment and decreased progressively at the
beginning of segment 2. A checkerboard pattern was observed in segment
2, i.e. some epithelial cells expressed high levels of mEP17
transcripts, and other did not. No mEP17 mRNA was detected in the
efferent ducts, mid and distal caput, or corpus and cauda epididymidis
using sense or antisense riboprobes.
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Hormonal regulation of the mEP17 gene
The effect of castration on mEP17 mRNA expression was
studied by Northern blot analysis (Fig. 11A). Total RNA extracted from the
epididymis at 5, 10, 20, and 30 days postcastration was hybridized with
the 32P-radiolabeled mEP17 cDNA probe. High
levels of mEP17 transcripts were detected in intact animals as
described above. Castration led to a rapid decrease in mEP17 gene
expression, as no mEP17 transcript could be detected at 5 days
postcastration.
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). Northern
blot analyses showed that mEP17 mRNA expression levels strongly
decreased in the epididymis from the castrated side, but not in that
from the intact side. In addition, testosterone replacement for 10 days
to 35-day-castrated mice did not restore mEP17 gene expression (Fig. 11C). Together our results demonstrate that mEP17 gene expression is
not dependent on circulating androgens, but is dependent on a
testicular factor(s) (that may include androgens) available from
testicular fluid.
mEP17 gene is conserved during evolution from rodents to
hamster
To determine whether the mEP17 gene may be conserved during
evolution, genomic DNA, extracted from mouse (129 SvJ), rat (Sprague
Dawley), and hamster, was analyzed by Southern blots using the
32P-labeled mEP17 cDNA as a probe (Fig. 12). As described previously with the
HindIII restriction digest, two DNA fragments (3.8 and 9 kb
in size) were easily detected in the mouse genome. A 9-kb
HindIII restriction fragment was also observed in the rat
genome. A larger DNA fragment (>15 kb) was detected in the hamster
genome as well. Our result suggests that an mEP17-like gene has been
conserved among the rodents and therefore suggests that mEP17 may play
an important function in male fertility.
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Discussion |
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Gene duplication gives rise to an epididymis-specific lipocalin
multigene family
The genomic organization of the mEP17 gene, consisting of seven
exons and six introns, is similar to that of genes encoding other
lipocalins, including the complement component 8 (31),
PGD2 synthase (32), and mE-RABP
(15). Interestingly, these genes were also mapped to the
proximal region of mouse chromosome 2 (15, 33). This
suggests that they result from the duplication of a common ancestral
gene within this chromosomal region. Furthermore, a phylogenetic
comparison of the ORF and conservation of the exon/intron boundaries
between mEP17 and mE-RABP genes indicates that both genes may result
from a more recent duplication. The conservation of a 18-bp
pyrimidine-rich DNA region within the 3'-untranslated region of the
mEP17 and mouse and rat E-RABP genes supports this assumption. However,
one can also hypothesize that this DNA motif is conserved because it
may be important for mRNA stability and/or translation. The
3'-untranslated region of mRNA contains an important
cis-regulatory element(s) that binds specific stabilizing or
destabilizing proteins. For instance, binding of poly(C)-binding
protein to a pyrimidine-rich sequence, located within the
3'-untranslated region, has been associated with stability of mRNA
encoding the human tyrosine hydroxylase or -globin proteins
(34, 35). Further studies will be required to determine
whether the pyrimidine-rich sequence found in the 3'-untranslated
region of the epididymis-specific lipocalins has a specific
function.
The presence of several lipocalins expressed specifically in the epididymis has previously been described in a lower vertebrate. The lizard epididymal secretory proteins (LESP) family is composed of nine androgen-dependent polypeptides (LESP II–IX) that are immunologically related, with an apparent molecular mass of approximately 18 kDa (36). These lizard proteins, synthesized in the epithelial cells of the caput epididymidis, are secreted into the luminal fluid. Some of these LESP proteins bind tightly to the head region of spermatozoa. N-Terminal microsequencing of the LESP proteins suggests that they are encoded by at least five distinct genes. Interestingly, the recent molecular characterization of LESP IV indicates that the L protein family belongs to the lipocalin superfamily (29). LESP IV is similar to mEP17 and, to a lesser extent, to mE-RABP.
Taken together, these results suggest that mEP17 and mE-RABP genes are the first identified members of a multigene subfamily encoding epididymal lipocalins in the mouse. Although the PGD2 synthase (PGD2-S) gene is not specifically expressed in the caput epididymidis, it may be considered a member of this subfamily. The PGD2-S gene, also localized in the proximal segment of mouse chromosome 2, encodes another related lipocalin with multiple function. The PGD2-S protein catalyzes the conversion of the cyclooxygenase-derived intermediate PGH2 to PGD2 in the presence of sulfhydryl compounds. In addition to its enzymatic activity, PGD2-S binds retinal and retinoic acid, and therefore should be considered a putative retinoid transporter (37). More members of this multigene subfamily may remain to be identified.
Expressions of mEP17 and mE-RABP genes are differently
regulated
The mEP17 and mE-RABP genes are both expressed in the caput
epididymidis and are placed in tandem on mouse chromosome 2. In
situ hybridization experiments have shown that mEP17 gene
expression was restricted to the principal cells of the initial segment
of the caput epididymidis. Interestingly, this region-specific
expression is complementary to that of the mE-RABP gene, as mE-RABP
mRNA are detected only from the mid/distal caput epididymidis
(12). Although mEP17 and mE-RABP genes may share common
cis-DNA regulatory sequences responsible for the
tissue-specific gene expression, our observations suggest that distinct
regulatory elements are responsible for the region-specific gene
expression.
Unlike mE-RABP, mEP17 gene expression almost disappeared 4 days after
hemicastration from the castrated side, suggesting that gene expression
was dependent on factors circulating within the testicular fluid.
Previous studies have described histological modifications in the
initial segment after efferent duct ligation (38). These
changes are probably the consequence of altered gene expression.
Indeed, numerous genes expressed in the initial segment and dependent
on testicular factors other than androgens have been reported,
including the -glutamyl transpeptidase mRNA IV (39),
polyomavirus enhancer activator 3 (40), A-raf
(41), 5reductase (42), and
CRES (43). Regulation of epididymal function by
testicular factors remains poorly understood (44). Soluble
components of rete testis fluid may regulate gene expression. For
instance, a 43-kDa protein found within testicular fluid and
immunologically related to basic fibroblast growth factor has been
reported to regulate the GGT activity in the rat epididymis
(45). In addition, expression of the proenkephalin mRNA
may be regulated by spermassociated factors (46).
Although a putative androgen response element is present within the mEP17 gene promoter, testosterone replacement to hemicastrated male mice failed to restore mEP17 gene expression. Testosterone may have no effect on mEP17 gene transcription, but one cannot exclude that the androgen receptor requires cooperation with other transcription factors induced by an extracellular signal present within the luminal fluid.
A putative RARE is present at position -259 nt within the mEP17 gene promoter. Most genes encoding a retinoid-binding protein show the presence of a functional RARE in the promoter region. For instance, RAREs have been identified 0.2, 0.6, 1, and 5.6 kb upstream the promoter of the human retinol-binding protein (47), human cellular retinol binding protein type II (CRBP-II) (48), rat CRBP-I (49), and human CRABP-II (50) genes, respectively. Thus, mEP17 gene expression may be regulated by retinoic acid.
Putative function of the mEP17 protein
Because we still do not know what ligands bind to mEP17, all
discussion of function, although based upon the amino acid sequence
homology and the region-specific expression pattern, remains
hypothetical. Lipocalins are primarily extracellular transport protein
found in most body fluids and involved in the delivery of small
lipophilic ligand (30). As mEP17 presents high homology
with other lipocalins that bind to retinoids, the most plausible
function for mEP17 is as a transport protein for retinoids within the
epididymis.
There is evidence that retinoids play an important role in regulating
gene expression in the epididymis (51). Moreover, a
vitamin A-free diet leads to pathological changes in the epididymal
epithelium (52). The retinoids required for the epididymal
function may have different sources. The retinol binding protein binds
retinol that circulates in the plasma and is recognized by a
membrane-bound receptor found on target cells (53, 54).
Once retinol is internalized into the cells, it is bound to an
intracellular binding protein termed CRBP-I. Within the mouse
epididymis, CRBP-I is expressed in the initial segment (Zheng, W.
L., unpublished data), suggesting that the uptake of retinol is likely
in this segment. The CRBP-I expression pattern overlaps that of mEP17,
suggesting that CRBP-I may deliver retinol or retinol-derived
metabolites to mEP17. Upon binding to mEP17, these retinoids may be
exported out of the cells and delivered to downstream epididymal
epithelial cells lining the duct or to spermatozoa. Indeed, retinoic
acid and retinyl esters are present in rat epididymal spermatozoa
(55). However, the former hypothesis appears more
plausible, as the overexpression of a dominant negative retinoic acid
receptor in transgenic mice results in a loss of the cauda
epithelium integrity (3). This demonstrates that this
segment is highly dependent on retinoic acid.
In addition, mEP17 may capture retinoids from the spermatozoa and release it to the epididymal epithelial cells lining the duct. This would imply that sperm-derived retinoids may play the role of a sperm-associated signal that regulates epididymal gene expression. Such a model has been proposed for regulation of proenkephalin gene expression in the rat caput epididymidis (46).
Finally, other functions have been assigned to lipocalins, including maintenance of blood-organ barriers, regulation of the immune response, and mediation of cellular homeostasis (30).
In conclusion, mEP17 may play an important role in the sperm maturation process or in the paracrine regulation of the epididymal function. Such roles have also been proposed for mE-RABP (14, 56). However, it is unlikely that mEP17 and mE-RABP functions are redundant, because of the different region-specific expression patterns. This, then, implies that epididymal lipocalins bind to different ligands or to the same ligand, but localize/transfer that ligand to different cellular compartments.
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Acknowledgments |
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Footnotes |
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2 Present address: Institut National de la Recherche Agronomique,
Station Commune de Recherches en Ichtyophysiologie, Biodiversité
et Environnement, Campus de Beaulieu, 35042 Rennes Cedex, France.
Received August 17, 2000.
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. Biol Reprod 56:985–990[Abstract]
2u-globulin superfamily. Biochem Int 14:235–240[Medline]
-lactoglobulin II from horse
colostrum (Equus caballus, Perissodactyla):
-lactoglobulins are retinol-binding proteins. Biol Chem Hoppe Seyler 366:601–608[Medline]
-lactoglobulins from various species.
Endocrinology 120:2620–2622: homology to the lipocalin gene
family. Biochemistry 33:5162–5166[CrossRef][Medline]
chain, prostaglandin-D-synthase,
oncogene- 24p3, and progestagen-associated endometrial protein map to
HSA9 and MMU2. Genomics 23:145–150[CrossRef][Medline]
-globin mRNA
stability [published erratum appears in Mol Cell Biol 1995 Apr;
15(4):2331]. Mol Cell Biol 15:1769–1777[Abstract]
-trace) is a newly recognized type of retinoid transporter. J
Biol Chem 272:15789–15795-glutamyl transpeptidase messenger ribonucleic acid transcripts in
the adult rat epididymis is differentially regulated by androgens and
testicular factors in a region-specific manner. Endocrinology 135:1146–1156[Abstract]
-glutamyl
transpeptidase mRNA-IV in the rat epididymis. Biol Reprod 57:186–193[Abstract]
-reductase type 1 along the rat
epididymis during postnatal development. Endocrinology 134:2298–2306[Abstract]
-glutamyl transpeptidase catalytic activity and protein level in the
initial segment of the rat epididymis by testicular factors: role of
basic fibroblast growth factor. Biol Reprod 58:197–206This article has been cited by other articles:
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T. G. Cooper, A. Wagenfeld, G. A. Cornwall, N. Hsia, S. T. Chu, M.-C. Orgebin-Crist, J. Drevet, P. Vernet, C. Avram, E. Nieschlag, et al. Gene and Protein Expression in the Epididymis of Infertile c-ros Receptor Tyrosine Kinase-Deficient Mice Biol Reprod, November 1, 2003; 69(5): 1750 - 1762. [Abstract] [Full Text] [PDF]
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S. Fouchecourt, J.-J. Lareyre, P. Chaurand, B. B. DaGue, K. Suzuki, D. E. Ong, G. E. Olson, R. J. Matusik, R. M. Caprioli, and M.-C. Orgebin-Crist Identification, Immunolocalization, Regulation, and Postnatal Development of the Lipocalin EP17 (Epididymal Protein of 17 Kilodaltons) in the Mouse and Rat Epididymis Endocrinology, March 1, 2003; 144(3): 887 - 900. [Abstract] [Full Text] [PDF]
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N. Ezer and B. Robaire Gene Expression Is Differentially Regulated in the Epididymis after Orchidectomy Endocrinology, March 1, 2003; 144(3): 975 - 988. [Abstract] [Full Text] [PDF]
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Y. Araki, K. Suzuki, R. J. Matusik, M. Obinata, and M.-C. Orgebin-Crist Immortalized Epididymal Cell Lines From Transgenic Mice Overexpressing Temperature-Sensitive Simian Virus 40 Large T-Antigen Gene J Androl, November 1, 2002; 23(6): 854 - 869. [Abstract] [Full Text] [PDF]
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P. Sipila, T. G. Cooper, C.-H. Yeung, M. Mustonen, J. Penttinen, J. Drevet, I. Huhtaniemi, and M. Poutanen Epididymal Dysfunction Initiated by the Expression of Simian Virus 40 T-Antigen Leads to Angulated Sperm Flagella and Infertility in Transgenic Mice Mol. Endocrinol., November 1, 2002; 16(11): 2603 - 2617. [Abstract] [Full Text] [PDF]
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S. Fouchecourt, P. Chaurand, B. B. DaGue, J.-J. Lareyre, R. J. Matusik, R. M. Caprioli, and M.-C. Orgebin-Crist Epididymal Lipocalin-Type Prostaglandin D2 Synthase: Identification Using Mass Spectrometry, Messenger RNA Localization, and Immunodetection in Mouse, Rat, Hamster, and Monkey Biol Reprod, February 1, 2002; 66(2): 524 - 533. [Abstract] [Full Text] [PDF]
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N. Hsia and G. A. Cornwall CCAAT/Enhancer Binding Protein {beta} Regulates Expression of the Cystatin-Related Epididymal Spermatogenic (Cres) Gene Biol Reprod, November 1, 2001; 65(5): 1452 - 1461. [Abstract] [Full Text] [PDF]
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Q. Liu, K. G. Hamil, P. Sivashanmugam, G. Grossman, R. Soundararajan, A. J. Rao, R. T. Richardson, Y.-L. Zhang, M. G. O'Rand, P. Petrusz, et al. Primate Epididymis-Specific Proteins: Characterization of ESC42, a Novel Protein Containing a Trefoil-Like Motif in Monkey and Human Endocrinology, October 1, 2001; 142(10): 4529 - 4539. [Abstract] [Full Text] [PDF]
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