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J. Biol. Chem., Vol. 277, Issue 47, 45291-45298, November 22, 2002
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From the Departments of Obstetrics & Gynecology and Pharmacology & Toxicology, and the Canadian Institutes of Health Research Group in
Fetal and Neonatal Health and Development, University of Western
Ontario, London, Ontario N6A 4L6, Canada
Received for publication, June 13, 2002, and in revised form, September 12, 2002
Human sex hormone-binding globulin (SHBG) binds
estradiol and testosterone with high affinity. Plasma SHBG is produced
by hepatocytes, but the human SHBG gene is also expressed
in the testis. Little is known about SHBG gene expression
in the human testis, but human SHBG transcripts accumulate
in a spermatogenic stage-dependent manner in the testes of
mice containing an 11-kb human SHBG transgene. We have now
found that human SHBG transcripts containing an alternative
exon 1 sequence are located specifically in the testicular germ cells
of these transgenic mice, whereas murine SHBG transcripts
are confined to Sertoli cells. In addition, we have detected
immunoreactive human SHBG in the acrosome during all stages of
spermiogenesis in mice containing an 11-kb human SHBG
transgene. Western blots of germ cell extracts from these transgenic
mice and from human sperm indicate that the immunoreactive human SHBG
in the acrosome composes electrophoretic variants, which are 3-5 kDa
smaller than the major electrophoretic isoforms of human SHBG in the
blood. This apparent size difference is due in part to differences in
glycosylation of plasma and acrosomal SHBG isoforms. The function of
the human SHBG isoform in the acrosome is unknown, but it binds steroid
ligands with high affinity. This is the first demonstration that human
SHBG transcripts encode an SHBG isoform that remains within
a cellular compartment.
Mammalian genes encoding sex hormone-binding globulin
(SHBG)1 contain at
least two transcription units (1, 2). In humans, the transcription unit
responsible for the production of plasma SHBG by hepatocytes consists
of eight exons that span ~3.2 kb on chromosome 17 (1, 3) and is under
the control of a promoter sequence that contains several well defined
binding sites for liver-enriched transcription factors (4, 5). The
human SHBG gene contains a second transcription unit that
consists of an alternative exon 1 sequence that replaces the exon 1 sequence present in the SHBG mRNA found in the liver. As a
consequence, these differentially spliced SHBG transcripts
lack the secretion signal sequence associated with the plasma SHBG
precursor polypeptide.
The expression of the SHBG gene in the testis has been
studied extensively in the rat (2). In this species, the
SHBG gene is expressed in Sertoli cells (6, 7) and encodes
the SHBG homologue that is generally known as the testicular
androgen-binding protein (ABP). The ABP produced by rat Sertoli cells
is secreted into the lumen of seminiferous tubules where it is thought
to serve primarily as a carrier of testosterone throughout the male reproductive tract (2). Although SHBG transcripts are
present in the human testis (1), virtually nothing is known about their function or how they are regulated, and evidence that they encode a
precursor polypeptide containing a leader sequence for secretion is
lacking. In fact, all the available evidence suggests that the human
testis contains several alternative SHBG transcripts comprising a non-coding alternative exon 1 sequence and some of them
also lack exon 7 sequences (1, 8). Differentially spliced human
SHBG transcripts lacking exon 7 sequences have also been identified in several other tissues (9, 10), but their 5'-sequences have not been characterized. Like the human SHBG gene, the
rat SHBG gene produces transcripts that consist
of alternative exon 1 sequences, and these have been identified in the
rat brain (11) and the fetal rat liver (12). There is no obvious
sequence similarity between the alternative exon 1 sequences associated
with various SHBG transcripts in different species, but
there is evidence they encode SHBG isoforms comprising subcellular
localization signals within a unique amino-terminal sequence (13).
To study the tissue-specific expression of various human
SHBG transcripts, we have produced several lines of
transgenic mice (14) containing either a 4- or an 11-kb human
SHBG transgene. The 4-kb transgene consists of the eight
exons encoding plasma SHBG (1) and 0.9 kb of 5'-flanking DNA that
includes the promoter utilized in the liver (4), whereas the 11-kb
human SHBG transgene contains an additional 5'-flanking DNA
sequence that includes an alternative exon 1 sequence associated with
the SHBG transcripts present in the human testis (1, 8). We
have shown previously that only the 11-kb human SHBG
transgene is expressed in the mouse testis, as evidenced by the
presence of human SHBG transcripts in the seminiferous
epithelium (14). These studies also indicated that the human
SHBG transcripts accumulate in a spermatogenic cycle
stage-dependent manner in this location, but the cell type in which they were located could not be clearly identified, and their
protein products eluded detection (14). We have therefore re-examined
this issue, and we have found that the majority of human
SHBG transcripts in the testis of these mice consist of the
alternative exon 1 sequence associated with SHBG cDNAs from a human
testis library (1). Furthermore, these alternatively spliced human
SHBG transcripts are confined to testicular germ cells and
an immunoreactive human SHBG isoform accumulates in the acrosome of
developing spermatids and immature sperm in the transgenic mice. We
have also obtained direct evidence that this acrosomal SHBG isoform
binds steroids and is also present in human sperm.
Animals--
Transgenic mice containing 11-kb (lines
shbg 11-a and shbg 11-b) or 4-kb (lines
shbg 4-a and shbg 4-b) regions of human
SHBG gene have been characterized previously (14, 15).
Animals were housed under standard conditions and provided with food
and water ad libitum. At ~10 weeks of age, mice were
sacrificed for the isolation of Sertoli cells and/or germ cells for
protein and RNA analysis (see below). For immunohistochemistry, mice
were perfused with phosphate-buffered saline (PBS) followed by 4%
paraformaldehyde (14). All procedures were approved by the Animal Use
Subcommittee of the University Council on Animal Care (University of
Western Ontario, Canada).
Immunohistochemistry--
The anti-human SHBG antibodies for
immunohistochemistry were purified from a rabbit antiserum by
immunoaffinity chromatography using an
N-hydroxysuccinimide-activated HiTrap column coupled to
purified human SHBG (16), according to instructions provided by
Amersham Biosciences. Testes from perfused mice (see above) were
further fixed in 4% paraformaldehyde at 4 °C for 24 h,
subsequently dehydrated with a series of ethanol solutions, and
embedded in paraffin. The paraffin sections were de-waxed and incubated
at high power in a microwave oven for 10 min in citrate buffer, pH 9.9. The sections were then cooled at room temperature for 20 min and
treated with a 0.03% hydrogen peroxide solution for 7 min, prior to
incubation (overnight at 4 °C) with affinity-purified rabbit
antibodies against human SHBG. The immunoreactive human SHBG was
detected using the EnVisionTM + System, HRP (DAB)
from DAKO (Carpinteria, CA).
Sperm from transgenic mice (14) were spread on slides, fixed for 5 min
in 4% paraformaldehyde, and washed with PBS. After incubation in
citrate buffer, pH 9.9, for 30 min at room temperature, the sections
were treated with 0.03% hydrogen peroxide solution for 7 min at room
temperature. The sections were incubated with the affinity-purified
antibodies against human SHBG overnight at 4 °C, and
immunoreactivity was detected using the EnVisionTM + System (DAKO).
Sertoli Cell and Germ Cell Isolation--
A mixed population of
Sertoli cells and germ cells was isolated from the testes of wild-type
and transgenic mice (17). Briefly, the testes were excised and washed
in Dulbecco's modified Eagle's medium/NUT mix F-12 culture medium
(Invitrogen Canada Inc., Burlington, Ontario, Canada) supplemented with
penicillin, streptomycin, and amphotericin (17). The testes were then
de-capsulated and incubated in a collagenase solution (0.9 mg/ml) at
33 °C for 10 min with agitation. After centrifugation, the pellets
were resuspended in 50 ml of the same culture medium and allowed to
sediment for 15 min. This was repeated three times. A second incubation
with collagenase (0.9 mg/ml) was then performed at 33 °C for 10 min. After centrifugation, the pellets were washed twice with PBS and frozen
for RNA and protein extraction (see below).
Germ cells were isolated separately from the mouse testes using an
established method (17). Briefly, testes were excised and washed with
PBS supplemented with penicillin, streptomycin, and amphotericin (17),
de-capsulated, and minced for 5 min in the PBS solution. The medium was
removed, and the remaining testicular fragments were digested in PBS
containing trypsin (80 mg/ml) at 33 °C for 10 min. The reaction was
stopped by adding 25 mg/ml trypsin inhibitor, and the resulting
solution was treated with deoxyribonuclease I (0.4 mg/ml) at room
temperature for 5 min. The isolated tubules were subjected to several
rounds of mincing and filtration, as described previously (17, 18).
After centrifugation, the pellet was resuspended in 15 ml of
Dulbecco's modified Eagle's medium/NUT mix F-12 culture medium
(Invitrogen) supplemented with 10% fetal bovine serum and incubated in
a tissue culture flask for 5 h. The supernatant containing germ
cells without Sertoli cells was recovered and centrifuged. After two
washes with PBS, the pellet was frozen in aliquots for protein or RNA extraction.
Western Blot Analysis--
Soluble protein was extracted from
mixed populations of Sertoli cells and germ cells and from isolated
germ cells (see above) with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS at 4 °C for 12 h. Human donor sperm samples were
centrifuged (350 × g for 10 min) to fractionate
seminal plasma and sperm, and sperm samples were either washed in HTF culture medium (Irvine Scientific, Santa Ana, CA) or purified by
Percoll® density gradient centrifugation prior to extraction with 0.25 M Tris-HCl, pH 8.0, by sonication in a water bath and three
freeze-thaw cycles. Samples were heat-denatured in loading buffer and
subjected to discontinuous SDS-PAGE with 4 and 10% polyacrylamide in
the stacking and resolving gels, respectively. Proteins in the gel were
transferred (19) to Hybond ECL nitrocellulose membranes (Amersham
Biosciences). The membranes were first blocked for 1 h in PBS
containing 0.01% Tween 20 and 5% skim milk and were then incubated
overnight at 4 °C with primary antibodies against human SHBG (DAKO;
kindly provided by Dr. Francina Munell) in the same buffer. The blots
were then washed three times in PBS containing 0.01% Tween 20 for 15 min to remove excess antibody, and specific antibody-antigen complexes
were identified using a horseradish peroxidase-labeled donkey
anti-rabbit IgG and chemiluminescent substrates (Pierce) by exposure to
x-ray film.
To assess the influence of glycosylation on the electrophoretic
mobility of immunoreactive human SHBG in different samples (i.e. diluted serum and protein extracts from isolated
testicular cells), we performed a similar Western blotting experiment.
For this purpose, protein was extracted from isolated testicular cells with 0.25 M Tris-HCl, pH 8.0, by sonication in a water bath
and three freeze-thaw cycles, and cell debris was removed by
centrifugation. Samples were then treated with
N-glycosidase F (Roche Diagnostics) at 37 °C
overnight, as recommended by the enzyme supplier, prior to analysis by
SDS-PAGE and Western blotting, as described above.
RNA Analysis--
Total RNA was extracted from mouse testicular
cells and liver using TRIzol reagent (Invitrogen), separated by
electrophoresis on a 1% agarose gel in the presence of formaldehyde,
and transfered to a Zeta-Probe nylon membrane (Bio-Rad). The membrane
was hybridized with various 32P-labeled human SHBG
cDNAs, i.e. the 3' EcoRI fragment spanning exons 6-8 (20), the SHBG exon 1 sequence encoding the
leader sequence for secretion of SHBG, and the SHBG
alternative exon 1 sequence (1). In addition, cDNAs for mouse
vimentin and transition protein 1 were used as markers for somatic
cells and germ cells, respectively. In some experiments, a mouse SHBG
cDNA corresponding to exon 6-8 sequences was used as a probe, and
a cDNA for 18 S ribosomal RNA was also used as an additional
control for RNA loading and transfer (15).
We also used the total RNA from isolated germ cells to further analyze
the human SHBG transcripts. To accomplish this, reverse transcription (RT) was performed at 42 °C for 50 min using 3 µg of
total RNA and 200 units of Superscript II together with an oligo(dT)
primer and reagents provided by Invitrogen. An aliquot (1 µl) of the
RT product was amplified in a 20-µl reaction in the presence of 1 unit of Taq polymerase, 0.05 mM
MgCl2, 1.25 µM of each dNTP, and 0.2 µM of each oligonucleotide primer. For this purpose, we
used an oligonucleotide corresponding to a 5'-sequence (5'-GCGGTTCAAAGGCTCCC) in the SHBG alternative exon 1 and a
reverse primer complementary to a sequence (5'-TGGCTTCTGTTCAGGGCC)
within exon 8 of the human SHBG gene (1). The PCR was
performed for 40 cycles at 94 °C for 30 min, 65 °C for 30 s,
and 72 °C for 1 min. A mouse transition protein 1 cDNA was
amplified by RT-PCR under the same conditions using as two specific
primers (5'-CCAGCCGCAAGCTAAAGACTCATGC and 5'-AGCTCATTGCCGCATCACAAGTGGG)
to control for the integrity and relative amounts of germ cell mRNA
in the samples. The PCR products were resolved by electrophoresis in a
1% agarose gel and purified using the GenElute Gel Extraction kit
(Sigma). They were then cloned using the Zero Blunt TOPO PCR Cloning
kit (Invitrogen), and plasmids containing PCR products were sequenced.
SHBG Steroid Binding Assays--
To determine whether the
immunoreactive SHBG extracted from testicular cells binds steroids, we
used a saturation ligand binding assay (23). In this assay, the
endogenous steroids were first removed from protein extracts (25 µg/µl) by dilution (1:3) in a dextran-coated charcoal (DCC)
suspension and incubation for 30 min at room temperature. After
centrifugation to remove the DCC, aliquots of the supernatants were
further diluted (1:3) and incubated at room temperature for 1 h
with 10 nM 5 The 11-kb Human SHBG Transgene Is Expressed in Germ Cells of the
Mouse Testis--
A single Northern blot was used to measure the
relative abundance of human SHBG transcripts in mixed
populations of Sertoli cells and germ cells, as well as a pure
population of germ cells isolated from the testes of wild-type mice and
mice containing 4- or 11-kb human SHBG transgenes (Fig.
1). When a human SHBG cDNA that
recognizes exon 6-8 sequences was used as probe, human SHBG
transcripts were only detected in RNA extracts of testicular cells from
mice containing the 11-kb human SHBG transgene. The use of
mouse vimentin (a marker of somatic cells) and transition protein 1 (a
germ cell specific marker) cDNAs allowed us to demonstrate the
purity of the germ cells isolated from the testes of transgenic mice
(Fig. 1, lanes 7 and 8). These data indicate that
similar numbers of germ cells were present in all samples, as evidenced by the presence of similar amounts of transition protein 1 mRNA. Therefore, because the relative abundance of human SHBG
transcripts in the germ cell-only preparation was similar to that in
the mixed population of Sertoli cells and germ cells, this suggested
that the SHBG transcripts in the testis of 11-kb human
SHBG transgenic mice cannot be derived from Sertoli
cells.
To characterize further the human SHBG transcripts that
accumulate in testicular germ cells, a Northern blot of total RNA extracts of germ cells, isolated from transgenic mice containing either
the 4- or 11-kb human SHBG transgenes, was performed using exon 1- and alternative exon 1-specific cDNA probes. As a control, we also included a similar amount of total RNA from an 11-kb human SHBG transgenic mouse liver (Fig.
2). When comparing the ratios of signals
obtained using these exon 1-specific cDNAs with those obtained
using a cDNA corresponding to human SHBG exon 6-8
sequences (Fig. 2), it is again apparent that SHBG
transcripts are only present in germ cells from the 11-kb human
SHBG transgenic mice. As expected, the SHBG
transcript in the liver RNA sample comprises predominantly the exon 1 sequence containing the translation initiation codon for the SHBG
precursor polypeptide and the leader sequence for secretion. By
contrast, the SHBG transcript in the germ cells from mice
containing the 11-kb human SHBG transgene can only be detected using the cDNAs that recognize the alternative exon 1 sequence and sequences corresponding to exons 6-8.
The human alternative SHBG transcripts were also analyzed in
germ cells from transgenic mice by an RT-PCR with specific primers for
human SHBG alternative exon 1 and human SHBG
exon 8, and mouse transition protein 1-specific primers were again used
in an RT-PCR as a positive control for the presence of intact mRNA
species. Human SHBG transcripts were detected only in the
germs cells of the 11-kb human SHBG transgenic mice, whereas
mouse transition protein 1 was amplified in all samples including those
from 4-kb human SHBG transgenic mice and wild-type mice
(Fig. 3). When the two differently sized
RT-PCR products amplified using human SHBG-specific primers
were cloned and sequenced, the ~1.1-kb RT-PCR product (Fig. 3)
was found to contain the alternative exon 1 sequence followed by the
sequences of exons 2-8, whereas the smaller and less abundant
RT-PCR product (~0.9 kb) contained the alternative exon 1 sequence
followed by the sequences of exons 2-6 and 8.
Expression of the endogenous mouse SHBG gene was examined in
the testicular cell preparations from wild-type mice by Northern blotting, and mouse vimentin and transition protein 1 cDNAs were again used to monitor for the presence of somatic cells and germ cells,
respectively (Fig. 4). When we used a
cDNA corresponding to mouse SHBG exon 6-8 sequences,
murine SHBG transcripts were only detected in the mixed
population of Sertoli cells and germ cells. Although similar levels of
transition protein 1 mRNA levels were detected in the two different
isolated testicular cell preparations, murine SHBG transcripts could
not be detected in the purified germ cells (Fig. 4). These data
therefore indicate that the mouse SHBG gene is expressed in
Sertoli cells rather than in germ cells.
Immunoreactive Human SHBG Accumulates in the Acrosome during
Spermiogenesis in Mice Expressing the 11-kb Human SHBG
Transgene--
The immunoaffinity-purified rabbit antibodies against
human SHBG do not detect antigens in the testes of wild-type mice, and this illustrates the specificity of the immunoreactivity observed at
low power magnification (10×) in the sections of testes from transgenic mice containing the 4- or 11-kb human SHBG
transgenes (Fig. 5A). In these
sections, similar amounts of immunoreactive human SHBG are present in
the interstitial compartment of both transgenic mouse lines,
irrespective of the size of the human SHBG transgene.
Because we have been unable to detect any human SHBG transcripts in the
testes of mice containing the 4-kb human SHBG transgene, and
because the plasma levels of human SHBG are similar in these two lines
of transgenic mice (14), the immunoreactivity in the interstitial
compartment reflects the sequestration of SHBG from the plasma. In
contrast to previous studies (14), in which we were not able to detect
any immunoreactive human SHBG within the seminiferous tubules of
mice containing human SHBG transgenes, microwave
pretreatment of histology sections in a high pH buffer most likely
exposed human SHBG epitopes within the seminiferous tubules of the
11-kb human SHBG transgenic mouse testis (Fig.
5A). At higher power magnification, this immunoreactive human SHBG could be detected within the acrosome of spermatids during
spermiogenesis only in the seminiferous tubules of transgenic mice
containing the 11-kb human SHBG transgene (Fig.
5B). Moreover, this immunoreactivity could be detected in
the acrosome as soon as it begins to form on spermatids (stage VII
of spermatogenesis) and persists in the acrosome as it develops
throughout the elongation stages (stages IX-XII) of spermiogenesis
(Fig. 5B).
To investigate whether the immunoreactive human SHBG remains within the
acrosome after sperm are released into the male reproductive tract, we
performed immunohistochemistry on sperm isolated from the epididymis of
transgenic mice containing either the 4- or 11-kb human SHBG
transgenes (Fig. 5C), and this clearly shows that
immunoreactive human SHBG is only present in the acrosome of sperm from
the 11-kb human SHBG transgenic mice (Fig.
5C).
Biochemical Characteristics of the Immunoreactive Human SHBG
Extracted from Testicular Cells of Transgenic Mice and Human
Sperm--
To determine the molecular size of the immunoreactive SHBG
within the acrosome of 11-kb human SHBG transgenic mouse
germ cells, we used Western blotting to examine protein extracts
isolated from testicular cells, i.e. a mixed population of
Sertoli cells and germ cells or isolated germ cells. The lack of any
immunoreactive molecules in the protein extract of testicular cells
from wild-type mice confirms the specificity of the anti-human SHBG
antibodies used for this purpose (Fig.
6). In addition, there was no
immunoreactivity in the protein extract of isolated testicular cells
from mice containing a 4-kb human SHBG transgene (Fig. 6),
despite the fact that the testes of these animals contain appreciable
amounts of immunoreactivity in the interstitial cell compartment (Fig.
5A). Thus, the ~45-kDa immunoreactive protein observed in
the isolated testicular cell extracts of mice containing the 11-kb
transgene cannot be accounted for by contamination of SHBG from blood
or the interstitial cell compartment.
In preliminary Western blotting experiments of the testicular protein
extracts, we noticed that the apparent molecular size of the
immunoreactive human SHBG in these samples was slightly smaller than
that in a serum sample from the same animals and that its
electrophoretic heterogeneity was different from that associated with
human SHBG purified from serum. To explore this further, we compared
the electrophoretic behavior of immunoreactive SHBG in serum and
testicular cell extracts from the 11-kb human SHBG
transgenic mice. For this experiment, aliquots of the samples were also
treated with N-glycosidase F to remove N-linked
oligosaccharides prior to Western blot analysis (Fig.
7). This confirmed that the apparent
molecular size of immunoreactive human SHBG in the testicular protein
extract (44-46 kDa) is smaller and electrophoretically more
heterogeneous than the major electrophoretic isoform of SHBG in serum
(50-51 kDa). However, treatment with N-glycosidase F clearly reduces the electrophoretic heterogeneity of immunoreactive SHBG in both samples and results in similarly sized immunoreactive products of about 43 kDa for deglycosylated serum SHBG and 42 kDa for
deglycosylated acrosomal SHBG (Fig. 7).
The steroid-binding properties of the immunoreactive human SHBG in
the protein extracts from isolated testicular cells were also examined
using a steroid-binding capacity assay. Our experiments indicated that
specific binding could only be detected in the extracts from mice
containing the 11-kb human SHBG transgene and that the
highest levels were present in one particular line (shbg 11-b) of these mice (14). We therefore prepared protein extracts of
mixed populations of Sertoli cells and germ cells from these mice to
study the steroid binding characteristics of the SHBG extracted from
the acrosome. A Scatchard analysis using [3H]DHT as
labeled ligand indicates that the affinity constant of the acrosomal
SHBG is essentially the same as SHBG in serum (Fig. 8). Furthermore, a competition analysis
with other SHBG ligands indicates that testosterone and estradiol
compete equally as effectively for the binding of [3H]DHT
to the acrosomal SHBG, as compared with serum SHBG (Table I).
Our finding that expression of alternative human
SHBG transcripts in the testis of transgenic mice results in
accumulation of an SHBG isoform in the acrosome of epididymal sperm led
us to determine whether SHBG is also present in human sperm. To
accomplish this, we compared the electrophoretic mobility of human SHBG
in a serum sample with human SHBG in seminal plasma, unwashed sperm in
seminal plasma, washed sperm, and sperm that had been purified by
Percoll® density gradient centrifugation (Fig.
9). This Western blot demonstrates that
the apparent molecular size (Mr) of the immunoreactive SHBG in human sperm is about 5 kDa smaller than SHBG in
either blood or seminal plasma. Furthermore, the
Mr of immunoreactive SHBG in human sperm is
similar to that of the immunoreactive SHBG extracted from the
testicular cells of 11-kb human SHBG transgenic mice but is
less heterogeneous with respect to its electrophoretic mobility (Fig.
9).
Based on extensive studies of ABP production by the rat testis
(2), it is generally assumed that the presence of an SHBG-like protein
in the epididymis of other mammalian species is the result of
SHBG gene expression in Sertoli cells (23). In rats, the transcription unit responsible for plasma SHBG production by the fetal
liver is also expressed in the testis of sexually mature animals (2),
with the highest levels of expression occurring in Sertoli cells during
sexual development (6, 24). In contrast, the 4-kb human SHBG
transcription unit that is expressed in the liver of transgenic mice is
not expressed in the testis (14), despite the fact that the
corresponding rat SHBG transcription unit with a similar
5'-regulatory region is expressed strongly as a transgene in the mouse
testis (25). These observations provided an indication that there are
species-specific differences in the way the SHBG gene is
expressed in the testis.
Our results confirm this because they clearly show that the human and
rodent SHBG genes are expressed in different cell types within the testis. Like the rat, expression of the mouse
SHBG gene appears to be confined to Sertoli cells. However,
expression of human SHBG transgenes in the mouse testis
occurs in germ cells, and the resulting transcripts consist of an
alternative exon 1 sequence identical to that present in several SHBG
cDNAs isolated from a human testis library (1). Thus, cell
type-specific differences in SHBG expression in the testis
of different species is likely due to the utilization of different
transcription units under the control of distinct promoter sequences.
In the germ cells, the human SHBG gene appears to be under
the control of a promoter flanking the alternative exon 1 sequence,2 and 4-kb human
SHBG transgenes are not expressed in the mouse testis
because they lack this alternative exon 1 and its flanking regulatory
sequences. Essentially nothing is known about how this promoter is
controlled, but our previous studies (14) have shown that human
SHBG transcripts within the seminiferous epithelium of our
transgenic mice begin to increase in abundance at stage V of
spermatogenesis, and the highest levels are found between stages VII
and IX. This corresponds well with the stage (VII) at which the
proacrosomal vesicles form (26) and when we first detect
immunoreactive human SHBG in the germ cells. The identification of
germ cells as the cell type in which the alternative SHBG
transcripts are located will now allow us to select an appropriate cell
type for studies of how the promoter that controls their expression is regulated.
Differentially spliced SHBG transcripts have been identified
in several human tissues, and in many cases these transcripts lack exon
7 sequences (1, 9, 10). Analysis of human SHBG transcripts
in the germ cells of our transgenic mice by RT-PCR also indicate that
some alternative exon 1 containing human SHBG transcripts
lack exon 7 sequences. This type of alternatively spliced transcript
would result in a premature termination of the open reading frame
encoding an SHBG isoform, and this type of product would most likely
fold abnormally and undergo rapid degradation, as shown recently (27)
for a human SHBG variant encoded by an abnormal allele with a premature
stop codon in exon 8. In addition, if human SHBG transcripts
lacking exon 7 sequences produce a carboxyl-terminally truncated form
of SHBG, this protein would lack consensus sites for
N-glycosylation, and our Western blot analysis indicates
that the immunoreactive human SHBG in the mouse germ cells is
N-glycosylated. We therefore conclude that the human SHBG
isoform we have identified in the acrosome cannot be the product of a
transcript lacking exon 7.
Our data also indicate that the most abundant human SHBG
transcript in the mouse germ cells consists of the alternative exon 1 sequence followed by a sequence corresponding to human SHBG exons 2-8 (1). The 5'-end of this alternative exon 1 sequence has not
yet been identified, but preliminary primer extension analysis2 and comparisons with the published human
SHBG sequence (1) indicate that the complete alternative
exon 1 sequence lacks an in-frame AUG codon. This might imply that the
first conventional translation initiation codon is the AUG codon for
Met-30 in the mature SHBG protein sequence, which is located in exon 2 (1). However, because the size of the acrosomal SHBG isoform after de-glycosylation is within 1-2 kDa of the size of serum SHBG treated in the same way, it is unlikely that its amino terminus corresponds to
Met-30 in SHBG because the deglycosylated acrosomal SHBG isoform would
then be at least 3 kDa smaller than the deglycosylated serum SHBG.
Furthermore, the acrosomal SHBG isoform accumulates in the proacrosomal
vesicle, which forms from granules that originate from the Golgi
apparatus, and it must therefore consist of a leader sequence that is
removed as the nascent protein undergoes translocation through the
rough endoplasmic reticulum. Thus, we speculate that translation of the
major alternative human SHBG transcript in testicular germ
cells might start from a non-conventional translation initiation codon
as part of an internal ribosome entry site (28) and that the precursor
polypeptide it encodes comprises a novel amino-terminal leader sequence.
The electrophoretic mobility of acrosomal SHBG is more heterogeneous
than SHBG in serum when analyzed by SDS-PAGE, and our data indicate
that this is due to a difference in the extent of N-glycosylation. This might also be attributed to the fact
that spermatids at all stages of development were used for the
extraction of acrosomal SHBG, and the fact that the carbohydrate
composition of glycoproteins in the acrosome changes throughout
spermiogenesis in a species-specific manner (29). Although the
functional significance of N-linked carbohydrates within the
carboxyl-terminal domain of SHBG remains obscure, it has been shown
previously (30) that the glycosylation has no influence on its steroid
binding activity. However, one particular N-glycosylation
site is invariably conserved across a wide variety of mammalian species
and is likely to be functionally important (31). It could for instance
influence the ability of SHBG to interact with other proteins on the
surface of specific cell types (32), and this might be relevant to its function in the acrosome.
Given the morphological differences in the testes of humans and
rodents, and the fact that rodents lack SHBG in the blood, it is not
surprising that there are differences in the way the human and rodent
SHBG genes are expressed in the testis. Previous studies
have shown that the overexpression of a rat SHBG transgene in the Sertoli cells of the mouse testis results in an increase in germ
cell apoptosis (18), but this does not occur in 11-kb human
SHBG transgenic mice in which the transgene is expressed within the germ cells. Furthermore, there are marked differences in the
levels of SHBG gene expression in the testis of mice and rats (33), and attempts to demonstrate that human Sertoli cells secrete
a protein with steroid-binding properties similar to SHBG have not been
successful (34). In this context, there is also no reason to assume
that SHBG gene products function similarly in the human and rodent
testis. Based on the observation that human SHBG accumulates in the
acrosome of sperm in our transgenic mice, we examined human sperm
samples and confirmed that an SHBG isoform, which can be distinguished
from plasma SHBG on the basis of its electrophoretic mobility, is also
present in ejaculated human sperm. The SHBG extracted from human sperm
is slightly smaller and less heterogeneous than that extracted from
transgenic mouse testicular cells, and this might be due to more
uniform glycosylation of the SHBG in ejaculated human sperm when
compared with the SHBG extracted from mixed populations of germ cells
at different stages of maturation (29). These data lead us to conclude
that the expression of the human SHBG transgene in mice
mimics the situation in the human testis and that production of an SHBG
isoform in germ cells must also result in its accumulation in the
acrosome of human sperm.
Male mice expressing the 11-kb human SHBG transgene in their
testes are reproductively normal (14), and this suggests the presence
of human SHBG in the acrosome has no deleterious effects on sperm
function in this animal model. Any positive effects that it might have
on either sperm viability or capacity for fertilization would be
difficult to assess in these animals. It may also not be relevant to
the human situation because of species-specific differences in sperm
maturation events, sperm-egg recognition and fertilization. Why human
SHBG might accumulate in the acrosome therefore remains to be
determined, and analysis of the content of SHBG in various normal and
abnormal human sperm samples may shed some light on this. In this
context, the fact that acrosomal SHBG retains the capacity to bind
steroids is of particular interest and raises the question of what
steroid ligands are bound to human SHBG in this location. Thus, in
addition to providing new information about human SHBG gene
expression in the testis, our studies provide the first evidence that
human SHBG transcripts encode an SHBG isoform that is
distinct from plasma SHBG, and which might serve a novel function
unrelated to extracellular steroid transport.
We thank Denise Power for secretarial
assistance, Dr. Gabe DiMattia for helpful comments, Dr. George
Avvakumov for the pure human SHBG, and David Dales and Rana Al-Nouno
for technical help.
*
This work was supported in part by a grant from the Canadian
Institutes of Health Research.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.
§
Holds the Ivey Chair in Environmental Toxicology. To whom
correspondence should be addressed: London Regional Cancer Centre, 790 Commissioners Rd. East, London, Ontario N6A 4L6, Canada. Tel.: 519-685-8637; Fax: 519-685-8616; E-mail: ghammond@uwo.ca.
Published, JBC Papers in Press, September 13, 2002, DOI 10.1074/jbc.M205903200
2
D. M. Selva, K. N. Hogeveen, K. Seguchi, F. Tekpetey, and G. L. Hammond, unpublished data.
The abbreviations used are:
SHBG, sex
hormone-binding globulin;
ABP, androgen-binding protein;
DHT, 5
A Human Sex Hormone-binding Globulin Isoform Accumulates in the
Acrosome during Spermatogenesis*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-[3H]dihydrotestosterone
(PerkinElmer Life Sciences) followed by an additional incubation (30 min) at 0 °C. Nonspecific binding was estimated in the presence of
excess unlabeled 5-dihydrotestosterone (DHT). Free ligand was
removed by incubation (10 min) with an ice-cold DCC suspension, and
following separation of the DCC by centrifugation, the supernatant
containing SHBG-bound ligand was taken for radioactivity measurements
(21). A similar protocol was used to determine the steroid-binding
properties of the immunoreactive SHBG extracted from the acrosome and
involved a Scatchard plot (22) to determine the affinity constant and a
competitive binding assay to determine steroid-binding specificity, as
described previously (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (56K):
[in a new window]
Fig. 1.
Human SHBG transcripts
accumulate in testicular cells from 11-kb human SHBG
transgenic mice. Human SHBG transcripts were only detected
in total RNA extracted from testicular cells of 11-kb human
SHBG transgenic mice on a Northern blot (lanes 5, 6, and 8). Mouse vimentin (marker of somatic cells)
mRNA was detected in the mixed populations of Sertoli cells and
germ cells from wild-type and transgenic mice (lanes 1-6)
but was not detected in isolated germ cells samples from transgenic
mice (lanes 7 and 8). Mouse transition protein 1 (marker of germ cells) expression was detected in all samples from
wild-type and transgenic mice (lanes 1-8). Based on similar
amounts of transition protein 1 mRNA in all samples, it appears
that the relative abundance of SHBG transcripts is greater
in the germ cells (lane 8) of 11-kb human SHBG
transgenic mice than in the mixed population of Sertoli cells and germ
cells (lanes 5 and 6) from these animals.
View larger version (67K):
[in a new window]
Fig. 2.
Human SHBG expression occurs
in the germ cells of 11-kb human SHBG transgenic
mice, and the resulting human SHBG transcripts
contain an alternative exon 1 sequence. Human SHBG
transcripts were only detected with a human SHBG cDNA (exon 6-8
sequences) in total RNA extracts from germ cells (lanes 2 and 5) and liver (lanes 3 and 6) of
11-kb human SHBG transgenic mice. Human SHBG
transcripts containing the exon 1 sequence could only be detected in
the liver RNA extracts (lane 6). When the same samples were
examined using a probe for the alternative human SHBG exon 1 sequence, the intensities of signals obtained for RNA from the germ
cells of 11-kb human SHBG transgenic mice were similar to
those obtained using the human SHBG cDNA that recognizes human
SHBG exon 6-8 sequences (lane 2). By contrast,
when these two probes were used to examine the RNA extract from the
liver (lane 3), the signal obtained using the alternative
human SHBG exon 1 sequence was almost undetectable, and this
is most apparent when the ratios of these signals is compared (see
below the blots). A cDNA for mouse 18 S RNA was used to
demonstrate that similar amounts of total RNA were present in all
samples.
View larger version (57K):
[in a new window]
Fig. 3.
Two human SHBG transcripts
containing alternative exon 1 sequences are present in total RNA
extracts of 11-kb human SHBG transgenic mouse germ
cells. An RT-PCR with oligonucleotide primers corresponding to
human SHBG alternative exon 1 and exon 8 sequences resulted
in two products of 1.13 and 0.93 kb when total RNA extracts from 11-kb
human SHBG transgenic mouse germ were used as template. No
products were obtained when similar amounts of testicular germ cell RNA
extracts (as defined by the RT-PCR of mouse transition protein 1) were
used from wild-type (WT) or 4-kb human SHBG
transgenic mice.
View larger version (31K):
[in a new window]
Fig. 4.
Mouse SHBG transcripts are
present in Sertoli cells but are absent in germ cells. A mouse
SHBG transcript of ~1.6 kb is present only in the total
RNA extract of the mixed population of Sertoli cells and germ cells
isolated from a wild-type (WT) mouse testis. A mouse
vimentin cDNA was used as to monitor somatic cells, and
vimentin mRNA was absent in the isolated germ cell RNA extract.
However, the presence of mouse transition protein 1 mRNA in the
total RNA from both types of testicular cell extract indicated that
similar amounts of germ cells were present in both samples.
View larger version (79K):
[in a new window]
Fig. 5.
Immunoreactive human SHBG is present
in the acrosome of germ cells within the 11-kb human SHBG
transgenic mouse testis. A, immunoreactive human
SHBG (brown stain) is present in the interstitial
compartment of the testis from 4- and 11-kb human SHBG transgenic mice
(×10 magnification), whereas the testis of wild-type (WT)
mice was completely devoid of any immunoreactivity. B, at
higher magnification (×60), immunoreactive human SHBG (brown
stain) can be detected only within the seminiferous tubules of
11-kb human SHBG transgenic mice, and it can be seen to
accumulate in the acrosome (arrowheads) of the germ cells
during stages VII-XII of spermatogenesis. C, immunoreactive
human SHBG (brown stain) is specifically located in the
acrosome (arrowhead) of epididymal spermatozoa taken from
11-kb human SHBG transgenic mice (×100).
View larger version (37K):
[in a new window]
Fig. 6.
Immunoreactive human SHBG is present in
testicular cell extracts from 11-kb human SHBG
transgenic mice. Two immunoreactive proteins of ~47 and 45 kDa were detected by Western blotting in protein extracts from a mixed
population of Sertoli cells and germ cells (S + g) and from
the isolated germ cells (g) of 11-kb human SHBG
transgenic mice. When similar amounts of these cell extracts (50 µg)
from wild-type (WT) and 4-kb human SHBG
transgenic mice were examined in the same way, no immunoreactive human
SHBG was detectable. The positions of protein size markers are shown on
the left.
View larger version (25K):
[in a new window]
Fig. 7.
Electrophoretic variants of plasma and
acrosomal human SHBG from 11-kb human SHBG
transgenic mice reflect differences in glycosylation. The
Western blot shows differences in the electrophoretic mobility of
plasma SHBG and acrosomal SHBG extracted from a mixed population of
Sertoli cells and germ cells before ( 
) and after (+) treatment of
N-glycosidase F to remove N-linked
oligosaccharides. The positions of protein size markers are shown on
the left.
View larger version (9K):
[in a new window]
Fig. 8.
Scatchard plots indicate that the
steroid-binding affinities of plasma SHBG (closed
circles) and acrosomal SHBG (open circles)
from 11-kb human SHBG transgenic mice are very
similar. The steroid-binding affinities of plasma SHBG
(Kd = 1.74 nM) and acrosomal SHBG
(Kd = 2.25 nM) were determined using
[3H]DHT as labeled ligand. The bound over free
[3H]DHT ratio (B/F) was plotted against the
amount of [3H]DHT bound specifically to SHBG.
Comparison of the relative binding affinity of steroid ligands for
serum and acrosomal SHBG isoforms
View larger version (37K):
[in a new window]
Fig. 9.
Western blot demonstrating the presence of an
immunoreactive human SHBG isoform in human sperm samples that differs
in size from SHBG blood plasma or seminal plasma. For comparison,
an extract of Sertoli cells and germ cells (S + g) from
11-kb human SHBG transgenic mice has been analyzed next to
an extract of Percoll®-purified human sperm. The positions of
molecular size markers in kDa are shown on the left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of a Canadian Institutes of Health Research studentship.
ABBREVIATIONS
-dihydrotestosterone;
PBS, phosphate buffered saline;
WT, wild
type;
RT, reverse transcription;
DCC, dextran-coated charcoal.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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