From INSERM Unit 317, Institut Louis Bugnard,
Université Paul Sabatier, Hôpital Rangueil, 31403 Toulouse
Cedex 4, France, the § GERM-INSERM Unit 435, Campus de
Beaulieu, Université de Rennes I, 35042 Rennes Cedex, Bretagne,
France, the
INSERM Unit 129, Institut Cochin de
Génétique Moléculaire, Faculté de
Médecine, 24 rue du Faubourg Saint Jacques, 75014 Paris Cedex,
France, and the ** Section for Molecular Signaling, Department of Cell
and Molecular Biology, Lund University, 22100 Lund, Sweden
Received for publication, October 5, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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A testicular form of hormone-sensitive lipase
(HSLtes), a triacylglycerol lipase, and cholesterol
esterase, is expressed in male germ cells. Northern blot analysis
showed HSLtes mRNA expression in early spermatids.
Immunolocalization of the protein in human and rodent seminiferous
tubules indicated that the highest level of expression occurred in
elongated spermatids. We have previously shown that 0.5 kilobase
pairs of the human HSLtes promoter directs testis-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice and determined regions binding nuclear
proteins expressed in testis but not in liver (Blaise, R., Grober, J.,
Rouet, P., Tavernier, G., Daegelen, D., and Langin, D. (1999)
J. Biol. Chem. 274, 9327-9334). Mutation of a
SRY/Sox-binding site in one of the regions did not impair in
vivo testis-specific expression of the reporter gene. Further
transgenic analyses established that 95 base pairs upstream of the
transcription start site were sufficient for correct testis expression.
In gel retardation assays using early spermatid nuclear extracts, a
germ cell-specific DNA-protein interaction was mapped between Hormone-sensitive lipase
(HSL)1 hydrolyzes
triacylglycerol and, cholesterol and retinyl esters (1, 2). In adipose
tissue, HSL catalyzes the rate-limiting step in lipolysis, the
catabolic pathway that mobilizes fatty acids from triacylglycerol
stored in the lipid droplet. HSL is also expressed in rodent and human testis (3-5). In situ hybridization experiments performed
in rat testis showed strong labeling of cells in the adluminal parts of
the seminiferous tubules at stages X-XIV and sparsely distributed grains in the basal parts (6). Immunohistochemistry experiments showed
an HSL-like immunoreactivity in the adluminal part of the rat
seminiferous tubules at stages XIII-VIII (5). The data suggested that
rodent HSL mRNA and protein are expressed in haploid germ cells
with a lag between mRNA and protein appearance. However, the
precise germ cell type(s) expressing HSL was not determined in rat and
it was not possible to rule out HSL expression in Sertoli cells.
Moreover, the expression of HSL in germ cells of other mammalian
species, including man, has not been documented. The effects of HSL
gene disruption in mice have recently been reported (7). The most
striking feature of the phenotype is male sterility due to
oligospermia. Degenerated spermatocytes and spermatids were observed in
HSL-deficient testis with a lack of mature spermatozoa. The data
clearly demonstrate that HSL is required for spermatogenesis.
The human adipose tissue form of HSL is encoded by 9 exons spanning 11 kb (8). The transcription start site was mapped in a short 5'-noncoding
exon located 1.5 kb upstream of the first coding exon (9). The 2.8-kb
mRNA encodes a 775-amino acid protein. A specific form of HSL,
named HSLtes, is expressed in human and rat testis (5). The
3.9-kb human HSLtes mRNA encodes a 1076-amino acid
protein. HSLtes contains a unique NH2-terminal
domain in addition to the 775 amino acids common to adipocyte and
testis HSL. This additional domain is encoded by a testis-specific exon located 15 kb upstream of the first of the 9 exons encoding adipocyte HSL.
The genomic organization suggested, as is often seen when a gene is
expressed in somatic tissues and in germ cells, the use of different
promoters to govern tissue-specific expression. We recently
investigated the molecular mechanisms that control the testis-specific
expression of HSLtes (10). Transgenic mice were generated
with 1.4 and 0.5 kb of the 5'-flanking region of the human
HSLtes-specific exon linked to the chloramphenicol
acetyltransferase (CAT) gene. High levels of CAT activity were measured
in testis from different lines of sexually mature transgenic mice. No
reporter gene activity was observed in nongonadal tissues in males and in all tissues studied in the females. Therefore, the sequences present
in the first 0.5 kb of the human HSLtes promoter confer germ cell-specific expression. To characterize nuclear protein-DNA interactions in the HSLtes promoter, a series of gel
retardation assays was performed with oligonucleotides spanning the
0.5-kb region. Four regions bound nuclear proteins expressed in testis but not in liver, an organ that does not express HSL. The most proximal
region contained a sequence AACAAAG that bound recombinant Sox
proteins. Sox proteins contain a high mobility group DNA-binding domain and are related to the testis-determining factor SRY (11).
In the present study, we determined the germ cell types expressing HSL
mRNA and protein and further delimited the region conferring testis
specificity in the human HSLtes promoter.
Immunohistochemistry experiments performed on rat, mouse, and human
testis showed that, in the three species, HSL-like immunoreactivity
exhibits a biphasic pattern with a first wave of expression in
spermatogonia and primary spermatocytes and, a second wave of
expression in elongating and elongated spermatids. The highest
expression levels were observed in the latter cells. Northern blot
analyses showed that HSLtes mRNA is abundant in early
spermatids and in the cytoplasmic fragments of late spermatids and
residual bodies. Studies performed on the human HSLtes
promoter showed that, although a Sox-like protein present in nuclear
extracts from pachytene spermatocytes binds to the promoter, mutation
of the corresponding AACAAAG sequence does not abolish testis-specific
CAT expression in transgenic mice. Additional transgenic analyses
established that 95 bp upstream of the transcription start site were
sufficient for testis-specific expression of the CAT reporter gene.
Using early spermatid nuclear extracts and transgenesis, a GT-rich
binding region was identified in the 95-bp sequence and shown to be
involved in the testis-specific expression of HSL.
Tissue Preparation and Immunohistochemistry--
Mice (8, 16, 24, and 48 days old) and rats (9, 20, 35, 45, and 90 days old) were
purchased from Elevage Janvier (Le Genest Saint Isle, France). Human
testes were obtained from patients undergoing therapeutic orchidectomy
for metastatic prostate carcinoma (protocol approved by the Ethics
Committee of the city of Rennes, France). Testes were stored in
Bouin's solution for 24 h (mouse), 72 h (rat), and 48 h
(human). The fixed tissues were embedded in paraffin wax. Sections (5 µm thick) were dried overnight at 37 °C, deparaffined, and
rehydrated through decreasing grades of alcohol. Sections were
microwaved three times (5 min each) in 0.01 M sodium
citrate (pH 6) buffer. Endogenous peroxidase was quenched with 3%
H2O2 for 5 min. Sections were incubated with affinity-purified polyclonal anti-rat HSL antibodies (rat and mouse
sections) and afffinity-purified polyclonal anti-human HSL antibodies
(human sections) at 0.5 and 2 µg/ml, respectively. Complexes were
revealed using biotinylated antibodies (Dako) at a working dilution of
1:500 coupled with streptavidin-peroxidase amplification. Preimmune
sera were used as negative controls. Sections were counterstained with
0.2% hematoxylin, dehydrated, and mounted in Eukitt (Polylabo,
Strasbourg, France) for microscopic observation.
Rat Testis Cell Isolation and RNA Extraction--
Leydig,
Sertoli, and peritubular cells and, spermatogonia were isolated as
described (12). For germ cell elutriation, testes of 8 adult rats were
decapsulated in PBS buffer and tubules were dissociated mechanically
using scalpels. Tubules were washed twice in PBS buffer and digested
with 0.025% trypsin for 30 min at 30 °C. Digestion was stopped by
addition of 3.2 mg of soybean trypsin inhibitor (Sigma). Tubules were
filtered through a 100-µm filter, then through glass wool and
centrifuged at 100 × g for 10 min. The cell pellet was
resuspended in PBS+ buffer (PBS buffer with 800 µM CaCl2 and 500 µM
MgCl2) and then centrifuged at 100 × g for
10 min. The resulting pellet was resuspended in PBS+c
buffer (PBS+ buffer with 5 g/liter bovine serum albumin and
300 mg/liter glucose). The solution was filtered through a 20-µm
nylon filter. Pachytene spermatocytes, early spermatids, and fragments
of late spermatids plus residual bodies that are shed by late
spermatids at the time of spermiation were purified by centrifugal
elutriation with respective purity of 90, 90, and 75-85% (13). Total
RNA of elutriated cells was extracted using the RNeasy kit (Qiagen).
Northern blot analysis was performed as previously described (10).
Nuclear Extracts from Elutriated Cells--
Elutriated germ
cells (107 to 108 cells) were resuspended into
0.8 ml of buffer A (10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 2 µg/ml aprotinin, and 0.05% Nonidet P-40), homogenized
with 10-20 strokes of a glass tissue grinder with a Teflon pestle and
incubated 15 min at 4 °C. The solution was centrifuged at
11,000 × g for 10 min at 4 °C. Nuclear pellets were
washed twice in buffer A and resuspended into 0.4 ml of buffer B (20 mM HEPES, pH 7.9, 0.14 M NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
0.1 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 2 µg/ml aprotinin, and 6.25% glycerol) with an all-glass
Dounce pestle B. An equal volume of buffer C (20 mM HEPES,
pH 7.9, 0.7 M NaCl, 1.5 mM MgCl2,
0.5 mM DTT, 0.2 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, and 6.25%
glycerol) was added dropwise. The suspension was incubated for 30 min
at 4 °C and then centrifuged at 20,000 × g for 30 min at 4 °C. An equal volume of buffer D (20 mM HEPES,
pH 7.9, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol) was added to the supernatant and
small aliquots were frozen into liquid nitrogen. The protein
concentrations of early spermatid and pachytene spermatocyte nuclear
extracts ranged between 0.5 and 1 mg/ml (14). Liver and whole testis
nuclear extracts were prepared as previously described (10).
Gel Retardation Assays--
Oligonucleotide purification and
labeling were performed as described (10). The positions from the
transcription start site and sequences of oligonucleotides were as
follows: Transgenic Mice--
All constructs were derived from the
p0.5HSLtesCAT vector (renamed p-515HSLtesCAT in the present study)
(10). Microinjection fragments with 316 and 95 bp of the human
HSLtes 5'-flanking region (
Fragments were microinjected into mouse oocytes and transgenic mice
were produced as described (10). Screening of the positive transgenic
animals was performed with DNA prepared from tails by Southern blot and
PCR using as sense primer, an oligonucleotide located in the human
HSLtes promoter and as antisense primer, an oligonucleotide
located in the CAT gene (14). Copy number was determined by Southern
blot analysis. Protein extracts for CAT assays were prepared from the
following tissues of hemizygous transgenic mice: testis, epididymis,
kidney, spleen, liver, small intestine, heart, lung, brain, skeletal
muscle, and adipose tissue. Briefly, tissues were rapidly frozen in
liquid nitrogen and homogenized in 0.5 ml of 250 mM Tris
(pH 7.6), 5 mM EDTA, and 1 mM DTT. Homogenates were heated 7.5 min at 65 °C and centrifuged at 4 °C 15 min at 18,000 × g. Supernatants were kept for CAT and protein
analyses (14).
HSL Is Expressed in Different Germ Cell Types--
To precisely
determine the localization of HSL in testis, we performed light
immunohistochemistry on adult mouse, rat, and human testes. Serial
adjacent testis sections were labeled with antibodies directed against
rat or human HSL. Fig. 1 shows
representative sections and a schematic summary of HSL expression in
germ cells during the cycle of the seminiferous epithelium. In mouse
and human, HSL-like immunoreactivity was first observed in the
cytoplasms of B spermatogonia and primary spermatocytes. In rat,
primary spermatocytes were labeled. In the three species, a second wave of expression was observed in the elongating and elongated spermatids. The staining was intense in the latest steps of spermiogenesis. After
spermiation, strong labeling was found in residual bodies in the mouse
and rat and residual staining was visible in spermatozoa. Residual
bodies are difficult to observe in human testis sections (16). In human
testis, weak labeling was also observed in Sertoli cells. The staining
of Sertoli cells was clearly visible in tubules devoid of germ cells.
To investigate the ontogeny of HSL, immunohistochemistry experiments
were also performed in testis from mice (8-, 16-, 24- and 48-day-old)
and rats (9-, 20-, 35-, 45-, and 90-day-old) at different ages (data
not shown). In rodents, the first wave of spermatogenesis in
prepubertal animals is synchronized and the appearance of novel germ
cell type can be associated with protein expression. In the rat,
HSL-like immunoreactivity appeared at day 20 with weak labeling in
pachytene spermatocytes. At day 35, a strong labeling was observed in
elongated spermatids. The results at day 45 were similar to the data in
90-day-old animals (Fig. 1). In the mouse, the appearance of pachytene
spermatocytes was associated with a strong labeling at day 16. At day
24, the first early spermatids were not labeled. In 48-day-old animals, high HSL-like immunoreactivity was observed in elongated spermatids. Northern blot analyses using total RNA prepared from elutriated rat
germ cells showed that the 3.9-kb HSLtes mRNA is
strongly expressed in early spermatids (Fig.
2). No band was visible in other cell
types. An abundant expression was also observed in late spermatid
cytoplasmic fragments and residual bodies where The AACAAAG SRY/Sox Consensus Binding Site Is Dispensable for
Testis-specific Expression in Transgenic Mice--
We recently showed
that 0.5 kb of the human HSLtes promoter conferred
testis-specific expression in transgenic mice (10). In this region, we
characterized an AACAAAG site for Sox proteins that bound a protein
expressed in testis but not in liver. Using purified germ cell nuclear
extracts, we observed using gel retardation assays that the
testis-specific protein binding to the
We showed previously that mutation of the AACAAAG sequence abolished
Sox protein binding (10). To determine whether the binding site was
critical for testis-specific expression of HSL, a CAT construct
containing the 0.5-kb 5'-flanking region with a mutation of the AACAAAG
sequence was microinjected into mouse oocytes (Fig.
4). Eight independent transgenic lines
were obtained (Table I). In 5 out of 8 transgenic lines, high levels of CAT activity were measured in testis
from adult mice. Nongonadal tissues showed very low levels of CAT
activity (<10 cpm/min/mg of protein). The testis of mice from 3 transgenic lines exhibited background levels of CAT activity,
presumably due to the insertion of the transgene at a chromosomal
location that suppresses expression.
A 95-bp Promoter Region Is Sufficient to Direct Testis-specific
Expression--
In the first 0.5 kb of the HSLtes
promoter, four regions bound nuclear proteins expressed in testis but
not in liver. To determine whether these regions played a role in the
testis specific activity of the promoter, CAT constructs with
progressive deletions of the 5'-flanking region were used to generate
transgenic mice (Fig. 4). Deletion of the first 3 testis-specific
binding regions did not abolish CAT activity in transgenic testis
(Table II). In agreement with the data
obtained with the fragment containing the mutated Sox-binding site,
high level of CAT activity was found in testis from transgenic mice
produced with a 209-bp 5'-flanking region that does not contain the
AACAAAG sequence. Mice harboring a transgenic construct containing 95 bp upstream of the transcription start site still showed strong
testicular CAT activity. In all the lines, CAT activity associated to
transgene expression in spermatozoa was detected in epididymis. None of
the transgenic lines showed CAT activity above background levels in
nongonadal tissues (<10 cpm/min/mg of protein). The different lines
were tested for testis expression of the transgenes at day 21 which in
prepubertal mice corresponds to the accumulation of pachytene
spermatocytes and the appearance of the first early spermatids in the
seminiferous tubules. In agreement with an activity of the human
HSLtes promoter in haploid germ cells, background levels of
CAT activity were measured (data not shown).
The In this paper, we establish the precise cellular localization of
HSL in rodent and human seminiferous tubules and show that a short
region of the human HSLtes promoter confers testis-specific expression. Study of HSL expression in male germ cells revealed that
the peak of expression occurs during spermatogenesis in elongated spermatids. A similar pattern was found in rat, mouse, and man. In
combination with Northern blot analyses of total RNA from isolated rat
germ cells (Fig. 2) and the ontogeny of HSLtes mRNA
expression in rat and mouse testes (6, 10), the data reveal that
HSLtes transcription and translation occur in early
spermatids and elongated spermatids, respectively. This feature is
characteristic of many genes expressed in haploid germ cells (23). The
determination of HSLtes stage-specific expression shown in
our immunohistochemistry experiments help to understand the testicular
alterations observed in HSL-deficient mice (7). The mice were
characterized by severe oligospermia and a reduction from 12 to 5-7
epithelium layers in seminiferous tubules. The lack of elongated
spermatids and spermatozoa could be the consequence of the absence of
HSL in the preceding germ-cell types, i.e. during elongation
of spermatids. The data strongly suggest that the testicular isoform of
HSL is necessary for germ cell differentiation during spermiogenesis.
The immunohistochemistry data also show HSL-like immunoreactivity in
human spermatogonia B, early spermatocytes, and Sertoli cells. It is
possible that the primary spermatocyte and Sertoli cell protein is
translated from a HSL mRNA different from the HSLtes
mRNA that encodes the 1076-amino acid protein. In support of this
hypothesis, two HSL mRNAs are expressed in human testis, the 3.9-kb
HSLtes mRNA and an ~3-kb mRNA (5). The
5'-noncoding region of the shorter mRNA differs from previously
characterized HSL mRNAs and corresponds to a novel
exon.2 The origin of HSL-like
immunoreactivity in rodent pachytene spermatocytes is more elusive. A
single 3.9-kb HSLtes mRNA is expressed in rat and mouse
testes (6, 10). The rodent HSLtes promoter may govern two
waves of expression, the first in spermatocytes and the second in spermatids.
To study the human HSLtes promoter, we used a combination
of transgenic mouse analyses and in vitro DNA-protein
binding assays. The use of transgenic mouse technology to assess the
activity of testis promoters is necessary because of the lack of
suitable male germ cell lines. Furthermore, this approach allows a
determination of regulatory elements involved in tissue-specific
expression. In a previous study, we showed that 0.5 kb of the
5'-flanking region of the HSLtes promoter governed
testis-specific expression of the transgene (10). A systematic analysis
of the region using gel retardation assay revealed 4 binding sites for
nuclear proteins expressed in testis but not in liver. One of the
binding sites located between Analyses of transgenic lines revealed that the first 95 bp of the human
HSLtes promoter mediates the in vivo expression
of a reporter gene in post-meiotic male germ cells. In all the lines examined, no transgene expression was observed in somatic tissues. In vivo analyses of male germ cell promoters show that
testis-specific expression is often conferred by ~100 bp 5'-flanking
regions (26-30). However, the transcriptional mechanisms differ
between promoters. A subset of them are under the direct control of
cAMP-responsive element modulator To conclude, we have shown that HSL is highly expressed in late
spermatids in humans and rodents. A short genomic region of the human
HSLtes promoter confers testis-specific expression in transgenic mice and contains an essential cis-acting element binding an
early spermatid-specific nuclear protein.
46 and
29 base pairs. The DNA binding nuclear protein showed properties of
zinc finger transcription factors. Mutation of the region abolished
reporter gene activity in transgenic mice, showing that it is necessary
for testis expression of HSLtes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
250/
216, 5'-CAACCATTTGTAGGAATGAACAAAGAGGGAAATAA-3';
96/
47, 5'-GCCTAAATTGGGATGCTTGCCTTATGAGAAGAAACATTTTAACGGAGTGG-3';
61/
12, 5'-ATTTTAACGGAGTGGTGGGTGGGGTGGGGCCCTATTTATGACACAAGAGA-3';
69/
25(mut
49/
36),
5'-GAAGAAACATTTTAACGGAGgaattctgttctgtGGGCCCTATTT-3';
28/+22,
5'-ATTTATGACACAAGAGAGCAAGCCCCTCCCTTCTTGTAAGAGAGTGCTAG-3'. 32P-Labeled DNA (1 ng at ~100,000 cpm/ng) was
incubated in binding buffer (10 mM HEPES, pH 7.9, 60 mM KCl, 1 mM EDTA, 5 mM DTT, 4 mM spermidine, 5 mM MgCl2, 10%
glycerol, and 1 µg of poly(dI-dC) (Amersham Pharmacia Biotech)) on
ice for 30 min with testis (10 µg), liver (8 µg), and germ cell (10 µg) nuclear extracts in a total reaction volume of 25 µl. Binding
buffer for
250/
216 oligonucleotide included 0.5 µg of poly(dG-dC)
(Amersham Pharmacia Biotech) instead of 1 µg of poly(dI-dC). In some
experiments with the
61/
12 oligonucleotide, ZnCl2 was
added in the binding buffer and 4% Ficoll 400 was used instead of 10%
glycerol. DNA-protein complexes were resolved on 6% nondenaturing
polyacrylamide/bisacrylamide (29:1) gels at 10 V/cm for 3 h in a
23 mM Tris borate (pH 8), 0.5 mM EDTA migration buffer. Polyacrylamide gels were dried under vacuum and subjected to
digital imaging (Molecular Dynamics).
316HSLtesCAT and
95HSLtesCAT) were generated by KpnI/BamHI and
StuI/BamHI enzymatic digestions, respectively.
Deletions of the 0.5-kb HSLtes 5'-flanking region to 460 and 209 bp (
460HSLtesCAT and
209HSLtesCAT) were generated by PCR
with high fidelity pfu DNA polymerase (Stratagene). Mutation
of the 5'-AACAAAG-3' SRY/Sox consensus site in the p-515HSLtesCAT
vector into the sequence 5'-CCGCGGT-3' (
515mutSoxHSLtesCAT) was
obtained by a two-step overlap PCR extension method (15) with high
fidelity pfu DNA polymerase. Using the same method, the
49/
31 bp region in
209HSLtesCAT was mutated into
5'-GAATTCTGTTCTGTG-3' (
209mutHSLtesCAT). All PCR constructs were
sequenced on ABI PRISM 310 Genetic Analyser (PerkinElmer Life Sciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin mRNA was
not detected. Reverse transcription-polymerase chain reaction assay
showed that HSL transcripts were weakly expressed in pachytene
spermatocytes (data not shown).
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Fig. 1.
Immunolocalization of HSL in mouse, rat, and
human testes. Left panels show the
immunolocalization of HSL in mouse (A), rat (B),
and human (C) testis sections. Affinity-purified polyclonal
anti-rat HSL (A and B) and anti-human
(C) HSL antibodies were used. Complexes were revealed using
biotinylated antibodies coupled with streptavidin-peroxidase
amplification. Sections were counterstained with Masson's hematoxylin.
Labeled primary spermatocytes and elongated spermatids are shown with
arrowheads in A, B, and C,
(top left). The weak labeling of the human Sertoli
cells is clearly visible in a "Sertoli-cell only" tubule
(arrowheads in C, bottom left). Right
panels show summaries of the HSL immunolocalization data
superimposed on the map of spermatogenesis (A, from Oakberg
(35) modified by Russell et al. (36); B, from
Leblond and Clermont (37) modified by Dym and Clermont (38);
C, from Clermont (39) modified by Sharpe (40)).
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Fig. 2.
HSLtes mRNA expression in rat
testicular cell types. RNA blots were prepared with 20 µg of
total RNA from peritubular cells (PT), spermatogonia
(G), Sertoli cells (S), Leydig cells
(L), pachytene spermatocytes (PS), early
spermatids (ES), and late spermatid cytoplasmic fragments
plus residual bodies (RB). The blot was hybridized with rat
HSL and -actin cDNA probes.
250/
216 oligonucleotide
containing the AACAAAG sequence (10) was more abundant in pachytene
spermatocytes than in early spermatids (Fig. 3). Efficient competition of the band was
observed with an oligonucleotide containing an AACAAT sequence that has
been shown to bind members of the Sry/Sox family (17-20). Proteins
containing a high mobility group DNA-binding domain such as Sox
proteins interact with A-T pairs in the minor groove of the DNA helix
(11). The A-T pair selective minor groove DNA ligand distamycin (21,
22) inhibited the testis-specific binding.
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Fig. 3.
Binding of liver, testis, and germ cell
nuclear extracts on the 250/
216 oligonucleotide probe containing
the AACAAAG sequence. Lane 1, liver (L)
nuclear extracts; lane 2, testis (T) nuclear
extracts; lanes 3-6, early spermatid (ES)
nuclear extracts; lanes 7-14, pachytene spermatocyte
(PS) nuclear extracts. A 100-fold excess of unlabeled
250/
216 oligonucleotide (lanes 4 and 8), of
unrelated unlabeled oligonucleotide from the 0.5-kb HSLtes
promoter (lanes 5 and 9), of an oligonucleotide
that contains the AACAAT SRY/Sox consensus sequence (lanes 6 and 10) were added in competition experiments. The minor
groove DNA binding drug distamycin was added to a final concentration
of 5 (lane 11), 10 (lane 12), 15 (lane
13), and 20 (lane 14) µM. A
testis-specific protein-DNA complex is shown with
arrowheads.
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Fig. 4.
Schematic representation of the CAT
constructs used to generate transgenic mouse lines. The original
515HSLtesCAT construct from Blaise et al. (10) contains
515 bp of the 5'-flanking region upstream of the transcription start
site shown by a broken arrow and 196 bp of the 5'-noncoding
region linked to the CAT gene. The constructs with deletions of the
5'-flanking region were obtained by PCR (
460HSLtesCAT and
209HSLtesCAT) or enzymatic digestion (
316HSLtesCAT and
95HSLtesCAT). Open boxes represent the previously
characterized (10) testis-specific protein-DNA interactions. The
AACAAAG sequence binding Sox proteins in the fourth testis-specific
binding region was mutated using PCR to obtain the
515mutSoxHSLtesCAT construct. The mutated region is shown as a
hatched box. A novel testis-specific zinc finger
protein-like-DNA complex in the 95-bp region is shown as an open
ellipse. The
209mutHSLtesCAT construct was obtained through
PCR-mediated mutation of the
49/
31 bp region shown as an
hatched ellipse.
CAT activity of 515mutSoxHSLtesCAT in transgenic mouse testis
CAT activity of human HSLtes promoter-CAT gene constructs in
transgenic mouse testis
49/
31 bp Region Is Required for Testis Specific Activity of
HSLtes Promoter--
The 95-bp region did not contain
known consensus sequences for transcription factors expressed in
testis. To characterize DNA binding of nuclear proteins expressed in
cells with active HSLtes gene transcription, nuclear
extracts were prepared from a purified preparation of early spermatids
and used in gel retardation assays with 3 overlapping 50-bp
oligonucleotides covering the entire 95-bp region (Fig.
5). The
96/
47 probe revealed weak binding with spermatid nuclear extracts. The
61/
12 and
28/+22 probes bound several proteins present in liver and spermatid nuclear extracts. When ZnCl2 was added in the binding buffer, a
retarded band was observed with the
61/
12 probe using nuclear
extracts prepared from early spermatids but not from liver (Figs. 5 and 6). Competition experiments showed that
the binding site was located between
46 and
29 bp from the
transcription start site. An oligonucleotide with a mutation of the
49/
36 bp region did not compete the testis-specific binding
observed with the
61/
12 probe (Fig. 6) and did not bind testis-specific nuclear proteins (data not shown). To determine the
in vivo importance of this region, we generated eight
independent transgenic lines with a
209HSLtesCAT construct bearing
the mutation of the
49/
31 bp region (
209mutHSLtesCAT). In all the
transgenic lines, very low CAT activities were observed in testis
(Table III) and in nongonadal tissues
(data not shown) of transgenic adult mice. These results showed that
mutation of the
49/
31-bp region abolished testis-specific promoter
activity. The whole data suggest that the binding of a testis-specific
zinc finger transcription factor within the
46/
29 bp region is
required for the testis-specific expression of HSLtes.
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Fig. 5.
Gel retardation analysis of the 96/+22
region. Lanes 1, 5, and 14, liver nuclear
extracts; lanes 2-4, 6-13, and 15-17, early
spermatid nuclear extracts. A 100-fold excess of unlabeled
96/
47
oligonucleotide (lanes 3 and 12), of unlabeled
61/
12 oligonucleotide (lanes 7 and 10), of
unlabeled
28/+22 oligonucleotide (lanes 13 and
16) and of unrelated unlabeled oligonucleotide from the
0.5-kb HSLtes promoter (lanes 4, 8, 11, and
17) were added in competition experiments. Lanes
5 and 9-13, ZnCl2 was added to a final
concentration of 0.5 mM. A testis-specific protein-DNA
complex is shown with an arrowhead.
View larger version (111K):
[in a new window]
Fig. 6.
Binding of early spermatid and liver nuclear
extracts on the 61/
12 oligonucleotide probe containing a
testis-specific protein-DNA binding region. Lanes 1-3,
liver nuclear extracts; lanes 4-11, early spermatid nuclear
extracts. A 100-fold excess of unlabeled
61/
12 oligonucleotide
(lanes 2 and 9), of unrelated unlabeled
oligonucleotide from the 0.5-kb HSLtes promoter
(lanes 3 and 10) and of unlabeled
69/
25
oligonucleotide mutated between nucleotides
49 and
36 (lane
11) were added in competition experiments. ZnCl2 was
added to a final concentration of 0.05 (lane 5), 0.1 (lanes 1-3 and 6), 0.5 (lanes 7 and
9-11), and 1 mM (lane 8). To enhance
the specificity of protein-DNA interaction, binding buffer contained
4% Ficoll 400 instead of 10% glycerol. A testis-specific protein-DNA
complex is shown with an arrowhead.
CAT activity of 209mutHSLtesCAT in transgenic mouse testis
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
232 and
226 bp from the transcription
start site contained the consensus motif AACAAAG for Sox proteins.
Several lines of evidence support that the binding protein is a member of the Sox family. First, the germ cell-specific binding was competed by an oligonucleotide containing an AACAAT sequence with high affinity
for Sox proteins (Fig. 3). Second, mutation of the site abolished
binding of Sox proteins (10). Third, distamycin, a minor groove DNA
ligand (21, 22), competed the binding suggesting that the protein-like
Sox proteins contained an high mobility group-binding domain (Fig. 3).
Two members of the Sox family, a short form of Sox5 and Sox6 are
expressed in early spermatids (18-20, 24). However, the endogenous
Sox-like protein binding to the HSLtes promoter is not
likely to correspond to Sox5 and Sox6 although recombinant Sox5 and
Sox6 readily bind to the HSLtes promoter (10). Antibodies
used to demonstrate the binding of Sox5 and Sox6 to the
chondrocyte-specific enhancer of the type II collagen gene (24) had no
effect on the Sox-like protein binding to the HSLtes
promoter.3 To determine
whether the AACAAAG sequence was important for in vivo
testis expression of a reporter gene, a CAT construct containing the
0.5-kb region with a mutation of the site was used to produce different
lines of transgenic mice. Five of the lines showed strong testis-specific CAT activity (Table I). The data clearly show that, in
transgenic testis, the Sox-binding site is not necessary for
tissue-specific expression of the human HSLtes promoter.
They, however, do not rule out a role for Sox proteins in the
regulation of HSLtes promoter activity. The role of most
Sox proteins expressed in adult tissues is not known. They could
indirectly affect transcription activity by altering chromatin
structure. We showed that the high mobility group domain of Sox5
induces a strong bend in DNA through binding to the AACAAAG sequence
(10). In pachytene spermatocytes, the interaction between Sox protein
and DNA minor groove could promote a reorganization of the local
chromatin structure preceding the HSLtes gene transcription
in early spermatids. Such a pattern of activation has been reported for
the pgk2 gene, DNase I-hypersensitive sites appearing
in spermatogonia whereas transcription starts in preleptotene
spermatocytes (25).
, e.g. the
angiotensin-converting enzyme and protamine 1 promoters (31, 32).
Others are activated by cAMP-responsive element modulator
-independent mechanisms such as the lactate dehydrogenase
c,
1,4-galactosyltransferase I, and HSLtes
promoters (29, 30). In previous gel retardation analyses, testis and liver nuclear extracts revealed similar binding of nuclear proteins to
the
95-bp region (10). To better characterize this region, we used
longer oligonucleotides and nuclear extracts from a purified preparation of early spermatids rather than from the whole testis (Fig.
5). Moreover, we tested different binding buffers (data not shown).
Most of the protein-DNA interactions observed did not differ between
early spermatids and liver. An early spermatid-specific protein-DNA
interaction was observed in the region between
46 and
29 bp from
the transcription start site that was strongly enhanced by the addition
of Zn2+ into the binding buffer. Zinc dependence of the
binding suggests DNA interaction with zinc finger transcription factors
(33). The binding region contains a GT-rich sequence that could bind members of the Sp1 family. It is, however, unlikely that the early spermatid nuclear protein binding to the HSLtes promoter
regulatory element is Sp1 because this protein-DNA interaction (shown
by an arrowhead on Fig. 5) was not found with liver nuclear extracts and was not competed by an oligonucleotide containing a consensus site
GGGGCGGGG for Sp1.3 Of note, an unidentified germ cell
nuclear protein different from Sp1 binds to a GC box in the proximal
promoter region of lactate dehydrogenase c that confers
testis-specific expression (34). Mutation of the GT-rich binding region
in transgenic mice abolished the testis-specific expression of the
reporter gene, showing that, unlike the SRY/Sox consensus binding site,
this region is required for the testis specificity of human
HSLtes promoter activity. Further experiments are necessary
to establish if the early spermatid nuclear protein binding to the
GT-rich sequence in the HSLtes promoter is a novel zinc
finger transcription factor.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Michel Raymondjean (CNRS-UPRESA 7079, Paris, France) for insightful discussion, Maxime Fontanié and Stéphanie Lucas (INSERM U317, Toulouse, France) for help with transgenic mice, Dr. Philippe Rouet (INSERM U317, Toulouse, France) for GC-box oligonucleotides, Dr. Véronique Lefebvre (University of Texas M. D. Anderson Cancer Center, Houston, TX) for anti-Sox6 and anti-Sox5 antibodies, and Nathalie Melaine for purified germ cell and RNA preparations (GERM-INSERM U435, Rennes, France). The contribution of Dr. Yara Barreira and the staff of the Louis Bugnard Institute Animal Care Facility is deeply acknowledged.
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FOOTNOTES |
---|
* The work was supported by INSERM and Swedish Medical Research Council Grant 11284 (to C. H.).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.
¶ Contributed equally to the results of this work.
To whom correspondence should be addressed: INSERM U317,
Institut Louis Bugnard, Bâtiment L3, CHU Rangueil, F-31403
Toulouse Cedex 4, France. Tel.: 33-5-62172950; Fax: 33-5-61331721;
E-mail: langin@rangueil.inserm.fr.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M009103200
2 H. Laurell, L. Holst, J. Grober, C. Holm, and D. Langin, unpublished observations.
3 R. Blaise and D. Langin, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: HSL, hormone-sensitive lipase; CAT, chloramphenicol acetyltransferase; HSLtes, testicular hormone-sensitive lipase; Sox, SRY-type high mobility group box protein; SRY, sex-determining Y chromosome protein; kb, kilobase pair(s); bp, base pair(s); PBS, phosphate-buffered saline; DTT, dithiothreitol; PCR, polymerase chain reaction.
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