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Departments of Obstetrics and Gynecology, Oncology, Biochemistry, Pharmacology, and Toxicology Medical Research Council Group in Fetal and Neonatal Health and Development University of Western Ontario and London Regional Cancer Center London, Ontario, Canada N6A 4L6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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11-kilobase (kb;
shbg11) or 4.3-kb (shbg4) human
shbg genomic fragments that comprise all eight exons
encoding SHBG as well as 6 kb or 0.9 kb of 5'-flanking DNA,
respectively. Northern blots indicated that human shbg
transcripts were most abundant in liver, kidney, and testis of the
shbg11 mice. The 4.3-kb shbg transgenes were
expressed at similar levels in liver and kidney, but the abundance of
human shbg transcripts in their testes was much lower than
that in shbg11 mice. Primer extension analysis indicated
that transcription starts 60 bp from the translation initiation codon
for SHBG in liver and kidney of shbg11 mice, and that the
shbg transcripts in their testis are derived from a
separate promoter flanking an alternative exon that replaces the exon
containing the translation initiation codon for SHBG or
androgen-binding protein. At the cellular level, the human
shbg transgenes are expressed in clusters of hepatocytes
located mainly within the periportal region of hepatic lobules and in
the epithelial cells lining the proximal convoluted tubules of the
kidney. This results in high levels of human SHBG in serum (1.45–1.72
nmol/ml) and urine (6–16 pmol/ml) of mature male shbg
mice. The abundance and distribution of human shbg
transcripts in the Sertoli cells of shbg11 mice vary
throughout the spermatogenic cycle, with levels increasing in the
Sertoli cell cytoplasm until stage VII of spermatogenesis and declining
after stage IX. At stages X–XII of spermatogenesis, these transcripts
concentrate at the adluminal compartment of the Sertoli cells, and this
suggests that they have a role in the elongation phase of
spermiogenesis. The presence of human SHBG in the blood of
shbg transgenic mice may result in serum levels of
testosterone that are 10–100 times higher than those in wild-type
littermates. Despite this, their reproductive performance is normal,
and there is no obvious phenotypic abnormalities even in animals
homozygous for the transgenes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In addition to regulating the bioavailability of testosterone and estradiol, human SHBG interacts with the plasma membranes of several sex steroid-dependent cells and tissues (13, 14, 15, 16). This appears to involve the binding of unliganded SHBG to a specific membrane receptor protein (17), which may promote its cellular internalization (16, 18) or cause an increase in intracellular cAMP levels when the membrane-bound SHBG becomes occupied by steroid ligand (19, 20). The biological consequences of these activities are not known, but they may enhance the responses of some cells to sex steroids.
Compared with other mammals, rodents are unusual because their livers
do not produce SHBG postnatally, and rat shbg is expressed
in the liver for only a few days during late fetal development (11). As
a result, the amounts of SHBG in the blood of rats and mice are much
lower than those in other species (21, 22). Why rodent livers do not
produce SHBG postnatally and what impact this has on the biological
activities of sex steroids are questions we sought to address by
producing mice expressing human shbg transgenes. We also
wished to define the transcription units responsible for
shbg expression in different tissues and to develop an
animal model for assessing the relative abundance and biological
significance of its various gene products. To accomplish this, we have
introduced two different portions of the human shbg locus
into the mouse genome by a conventional transgenic approach. One of the
transgenes comprises the eight exons that encode SHBG and only 0.9
kilobase (kb) of 5'-flanking DNA, and the other contains an additional
5 kb of 5'-flanking DNA that includes sequences found in several
alternative shbg transcripts identified from a human testis
complementary DNA (cDNA) library (3). In addition to revealing the
requirements for shbg expression in different cell types, it
was anticipated that the presence of human SHBG in these transgenic
mice might cause an imbalance of androgen and estrogen bioavailability
and affect the growth and function of sex steroid-dependent
tissues.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. The shbg11-a line was
derived from a founder in which two separate integrations segregated in
the F1 generation, but only one of these lines was maintained for
further analysis. Lines shbg11-b and shbg4-a were
derived from separate founder animals. Lines shbg4-b and
shbg4-c were derived from a single founder animal and were
maintained as separate lines.
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) indicate that the transgenes
integrated as head to tail multimers in shbg11-a,
shbg11-b, and shbg4-a mice. The estimated
transgene copy numbers in these lines are 6, 6, and 11, respectively.
Southern blots (Fig. 1B and data not shown) indicate that three copies
of the transgene are present in shbg4-b mice and that two of
them integrated in a head to head orientation. In shbg4-c
mice, there also appears to be one head to head integration of the
transgene, and the intensity of signals obtained on the Southern blot
(Fig. 1B) indicates that this line contains eight copies of the
transgene.
Tissue Distribution of Human SHBG mRNA in Transgenic Mice
A Northern blot of RNA extracted from shbg transgenic
and wild-type mouse tissues is shown in Fig. 2. Samples of spleen RNA were included as
negative controls, and the absence of a signal in wild-type tissues
indicates that endogenous murine shbg transcripts are not
recognized by the human SHBG cDNA. A signal that corresponds to SHBG
mRNA was found in the livers of male mice from each transgenic line,
but the relative abundance of SHBG mRNA in the livers of these animals
varied. A similarly sized (1.8 kb) shbg transcript was found
in the kidneys of mice from four of the five transgenic lines. Its
absence in the kidney of shbg4-b mice was confirmed by
overexposing the blot and by analyzing kidney RNA from other animals in
this line (data not shown). Although a transcript of similar size to
SHBG mRNA in liver was clearly detectable in the testis of both lines
of mice carrying the 11-kb shbg transgene (Fig. 2), it could
only be seen in the testicular RNA extracts from shbg4-a and
shbg4-c mice after prolonged exposure of the Northern blot
(not shown).
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). By contrast, the SHBG cDNA for exons
6–8 hybridized with a transcript in the testis that is similar in size
to the SHBG mRNA (defined by the presence of exon 1 sequences) in the
liver and kidney, but failed to detect any shbg transcripts
in whole brain polyadenylated [poly(A)+] RNA extracts.
This provided the first indication that the shbg transcripts
in the testes of shbg11 mice originate from a transcription
start site different from that used in the liver and kidney and
comprise an alternative exon 1 sequence.
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). Poly(A)+
RNA from the brain and spleen of transgenic mice was used as a control,
and poly(A)+ RNA from corresponding wild-type mouse tissues
was analyzed in parallel to confirm that primer extension products
originate from human shbg transcripts rather than from
endogenous mouse shbg transcripts. In this experiment,
primer extension products were obtained only with mRNA from the liver
and kidney of transgenic mice (Fig. 4). These products were all the
same size, and the most abundant product (69 nucleotides) places the
major transcription start site 60 bp 5' from the translation initiation
codon in exon 1 (3).
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) in which testicular mRNA from
wild-type and shbg11-a transgenic mice was tested with the
same exon 1-specific primer (lanes 3 and 4) as well as with an exon
2-specific primer (lanes 5 and 6). Primer extension of actin mRNA was
also used as a control, and this gave an appropriately sized major
product (109 nucleotides) of similar abundance from both wild-type and
transgenic mouse testicular mRNA extracts (lanes 1 and 2). Again, no
specific primer extension products were observed from the transgenic
testicular mRNA extracts when the exon 1-specific primer was used (lane
4). By contrast, multiple primer extension products between 210–235
nucleotides in length were observed from the same mRNA when an exon
2-specific primer was used (lane 6), and these products were not
obtained in a parallel reaction in which wild-type mouse testicular
poly(A)+ RNA was used as the template (lane 5). The lengths
of these products indicate that the size(s) of the alternative exon 1,
which is used in the testis of shbg11-a (data not shown) and
shbg11-b (Fig. 5) mice, would be between 161–186 bp.
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, A and B,
respectively). Furthermore, the relative amounts of SHBG and its mRNA
were generally higher in the cytoplasm of hepatocytes in the periportal
zones of the hepatic lobules compared with those located near the
central veins. Similar results were obtained when a liver from a
shbg4-a mouse was analyzed, whereas livers from wild-type
mice contained no immunoreactive SHBG or SHBG mRNA (data not shown). In
serial sections of a shbg11-a male mouse kidney,
immunoreactive SHBG and SHBG mRNA were both confined to the cytoplasm
of epithelial cells lining the proximal convoluted tubules (Fig. 6, C
and D, respectively), with the most intense staining of immunoreactive
SHBG seen at the luminal aspect of the cells (Fig. 6C). Immunoreactive
SHBG and its mRNA also colocalized to these same cells in the kidneys
of shbg4-a mice, but were undetectable in kidneys of
wild-type mice or transgenic mice (shbg4-b) that do not
express the 4.3-kb shbg transgene in the kidney (data not
shown).
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, E and G), but it could also be
detected in a few Sertoli cells (Fig. 6G). The most intense
immunostaining was associated with the boundary tissue of the
seminiferous tubules as well as the interstitial fluid surrounding
Leydig cells that contain no immunoreactive SHBG. By contrast,
shbg transcripts could only be detected in testes of
shbg11-a (Fig. 6F) and shbg11-b (not shown) mice,
and their abundance in the Sertoli cells of individual seminiferous
tubules differed remarkably. We, therefore, compared the abundance of
shbg transcripts in the Sertoli cells of these mice with the
stage of spermatogenesis in individual seminiferous tubules (Fig. 7). This demonstrated that expression of
the 11-kb shbg transgene in the testis varies in a
stage-specific manner throughout the spermatogenic cycle, with low
levels of transcripts in the seminiferous tubules at stages I–III
followed by a progressive increase until stage VII. Although the
abundance of these transcripts in the Sertoli cells declines steadily
after stage IX, they invariably accumulate in the adluminal regions of
these cells at stages X–XII of spermatogenesis (Fig. 6F). This
apparent cellular partitioning of the shbg transcripts was
quite striking and was very distinct from their relatively uniform
distribution in Sertoli cells at stages V–VIII of the spermatogenic
cycle.
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) suggest that SHBG is produced by these cells and is secreted
into the renal tubules. We, therefore, assessed the amount and
integrity of SHBG in the urine of male mice that either express
(shbg4-a) or do not express (shbg4-b) the 4.3-kb
shbg transgene in the kidney. When a Western blot was used
to detect human SHBG in serum and urine samples from these transgenic
mice and control wild-type mice (Fig. 8),
immunoreactive SHBG was only found in the urine of mice that express
the transgene in the kidney. The SHBG in the urine of these animals
exhibited the typical heavy (50-kDa) and light (48-kDa)
protomeric isoforms seen in human (23) and transgenic mouse serum
samples (Fig. 8, lane 3), and the fact that it can be quantified by a
steroid binding capacity assay (6 pmol/ml) indicates that it has a
functional steroid-binding site. The amounts of SHBG in the urine of
male shbg4-a (n = 3) and shbg11-a
mice were also very similar (6–16 pmol/ml).
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. These
analyses were performed on serum samples from animals hemizygous for
the transgenes and their wild-type littermates, and illustrate the
variability in serum SHBG levels between lines. Although there is
obviously no clear relationship between SHBG and testosterone levels in
these animals, serum testosterone levels in the transgenic animals are
remarkably high compared with those in wild-type mice, and this was
most evident in the shbg11-a mice (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The 11- and 4.3-kb human shbg transgenes were expressed most abundantly in the mouse liver and kidney and to a lesser extent in the testis. This pattern of expression is different from that reported for mature male transgenic mice containing a rat shbg transgene, in which transcripts that encode SHBG or ABP are essentially confined to the testis (24, 25). These differences might imply that gene-specific sequences play a major role in determining shbg expression in a given tissue, but this is difficult to reconcile with the fact that rat shbg is expressed in fetal, but not adult, livers (11). This paradox might, however, be explained by the specific utilization of an alternative promoter in fetal livers or the presence of trans-acting factors at specific developmental stages.
In the liver, the shbg transgenes exhibited some variation in expression between lines, with the lowest levels of SHBG mRNA observed in the line (shbg4-b) with only three copies of the 4.3-kb transgene. In the other four lines, this variability was less pronounced and suggests that the transgenes are essentially unaffected by differences in their position within the mouse genome, and that the additional sequences in the 11-kb transgenes exert little effect on the expression of shbg in this tissue. More importantly, these results indicate that all of the information required for transcription of human shbg in the liver is contained within the 4.3-kb shbg transgene, which comprises only 0.85 kb of 5'-DNA flanking exon 1 of the human shbg gene. Our primer extension analysis indicates that human shbg transcription in the liver initiates 60 bp 5' of the translation initiation codon for SHBG in exon 1, and when this is compared with the major transcription start site for ABP mRNA in the rat testis (26), it places the proximal promoters responsible for shbg transcription in the liver and testis in a very similar context.
At the cellular level, human SHBG mRNA was confined to hepatocytes in
transgenic mouse livers, and this was expected because the human
hepatoma cell line (HepG2) produces and secretes SHBG (2). However,
immunoreactive SHBG and its mRNA generally colocalized in hepatocyte
clusters within the periportal region of the hepatic lobules, and a
subset of hepatocytes in this location, therefore, appears to be
largely responsible for producing plasma SHBG. This is consistent with
the concept that gene expression in hepatocytes is often influenced by
differentiation events determined by their anatomical location as well
as developmental stage, as reported recently for the mouse
-fetoprotein gene (27).
Both 11- and 4.3-kb shbg transgenes are expressed in the mouse kidney, and our primer extension experiments indicate that human shbg is under the control of the same promoter in the liver and kidney. Immunoreactive SHBG and its mRNA also colocalize in epithelial cells lining the proximal convoluted tubules, and the SHBG in these cells appears to concentrate at their luminal surface. This suggests that SHBG is produced and secreted by these cells into the renal tubules, and we confirmed this by showing that SHBG is present only in the urine of mice expressing shbg transgenes in their kidneys. The shbg4-b mice that express human shbg in their livers, but not in their kidneys, represent a perfect control for this experiment and are of particular interest to us because the transgenes in this line may have lost sequences that act in concert with the proximal promoter flanking exon 1 to direct the kidney-specific expression of shbg or may have integrated adjacent to a host sequence that acts as a tissue-specific repressor.
The relatively high levels of SHBG mRNA in the kidneys of shbg transgenic mice were unexpected, even though we were aware that shbg transcripts have been identified in hamster kidney (28). Thus, the kidney may represent an important and largely overlooked site of shbg expression in some species, and the local production of SHBG in the kidney could influence the activities of sex steroids within the renal tubules, perturbations of which are associated with the development of lupus in both mice and humans (29). We have detected mouse shbg transcripts in kidney RNA extracts from wild-type mice by Northern blotting (our unpublished data), and it will therefore be important to determine whether shbg is also expressed in the adult human kidney, and if it influences the way sex steroids impact on renal physiology in mice and humans.
Human and rat shbg transgenes are obviously expressed quite differently in the mouse testis. In Sertoli cells, the rat transgene gives rise to appreciable amounts of ABP in the testis and epididymis, and this is associated with impaired spermatogenesis and infertility (25). By contrast, we were unable to identify immunoreactive human SHBG in the seminiferous tubules or epithelial cells of the epididymis from any of our transgenic mice, and this may explain why their testes appear to function normally. These differences obviously reflect a much greater transcriptional activity at the promoter flanking exon 1 of the rat shbg transgene compared with the human shbg transgenes in the testis, and this was confirmed by our failure to detect human shbg transcripts that originate from the promoter flanking exon 1 in testes of shbg11 mice by primer extension as well as the very low levels of shbg transcripts in testes of the mice containing the 4.3-kb transgene.
Why the human and rat shbg transgenes are expressed so differently in the mouse testis is unclear, but mouse shbg is also expressed at relatively low levels in the testis compared with the rat gene (4). One obvious difference between these transgenic mice, however, is that the concentration of SHBG in the plasma of mice expressing the human transgenes, as determined by its steroid binding capacity, is at least 300-fold greater than that in mice expressing the rat shbg transgene (25) and might influence the intratesticular bioavailability of testosterone if it gains access to the interstitial compartment of the testis. This may be relevant because the expression of the rat shbg gene in the testis is increased by androgen treatment in vivo (30). It is also possible that androgens do not exert this effect directly and function through an intermediary androgen-responsive paracrine factor produced by peritubular cells, which, in turn, influences the transcriptional control of ABP production in Sertoli cells. In support of this, the interstitial lymphatic spaces of all shbg transgenic mouse testes contained appreciable amounts of immunoreactive SHBG. The most intense staining was observed in the vicinity of blood vessels of the perfused-fixed testes, and this supports our assumption that the SHBG in the interstitial compartment of the testes is largely derived from the blood circulation. In addition to being distributed throughout the interstitial lymphatic space and surrounding the Leydig cells, immunoreactive SHBG appeared to concentrate in the boundary tissue of the seminiferous tubules, and this is consistent with the idea that it might restrict testosterone bioavailability at this location. However, any effect this may have on testicular function must be subtle and certainly does not compromise the reproductive performance of these animals.
Although shbg transcripts in shbg4 mouse testes most likely comprise the exon 1 sequences that encode the signal polypeptide required for plasma SHBG or testicular ABP secretion (3), this exon appears to be replaced by an alternative sequence in the majority of testicular transcripts that originate from the 11-kb shbg transgene. The observation that the alternative human shbg transcripts predominate in the testes of transgenic mice is intriguing and raises the question of how abundant they might be in the human testis. This is an important issue because although several cDNAs representing shbg transcripts in human testis have been identified, none of them comprises exon 1 sequences (3, 9).
The alternative shbg exon 1 sequence in the transcripts extracted from shbg11 mouse testes is obviously located within the unique 5'-region of the transgene and is identical (our unpublished data) to that used in the human testis (3). We do not know whether these transcripts are translated, but if they are, their products are not recognized by antiserum against human SHBG. However, their accumulation in Sertoli cells varies in a spermatogenic stage-specific manner, and it appears that the transgene is activated at stages IV–V of the spermatogenic cycle and that the resulting transcripts are most abundant at stages VII and VIII. At stages IV–VIII of the spermatogenic cycle, the shbg transcripts are distributed uniformly in the Sertoli cell cytoplasm, but after spermiation they accumulate in the adluminal compartment of these cells at stages X–XII. This is indicative of their possible localization in the penetrating processes that reside in the cytoplasmic lobules of step 10–12 spermatids. These penetrating processes are involved in the transfer of cytoplasmic constituents between Sertoli cells and germ cells (31, 32), and it is possible that shbg transcripts containing an alternative exon 1 sequence and are transferred to the germ cell, where they might have some function during the elongation phase of spermiogenesis. After stage XII of spermatogenesis, there is a very rapid loss of shbg transcripts in the Sertoli cell cytoplasm, and residual transcripts remain associated primarily with step 13 spermatids. During spermatid condensation and spermiation, shbg transcripts are no longer associated with step 15 and 16 spermatids, and this is consistent with a transient function for these transcripts in spermatids just before condensation.
Differences in the metabolic clearance of sex steroids in human blood are related directly to their relative affinities for SHBG and its plasma concentration (33). The presence of human SHBG in the blood of the transgenic mice we have produced, therefore, probably accounts for the extraordinarily high (>1 µM) plasma concentrations of testosterone in some of these animals compared with those in wild-type mice. Despite this, their reproductive performance does not appear to be affected in any way, and we have been able to breed all five lines of human shbg transgenic mice to homozygosity. Male mice homozygous for a rat shbg transgene also have ABP in their blood that presumably originates from the testis (25), but its plasma concentrations are much lower than those of human SHBG in any of the transgenic mice we have produced. Furthermore, the plasma levels of testosterone in rat shbg transgenic mice are not different from those in wild-type mice (25), and this might be due to the fact that the affinity of rat ABP for sex steroids is much lower than that for human SHBG (22). However, mice expressing the rat shbg transgene not only suffer from impaired fertility, but are also characterized by a hind limb motor dysfunction (25) that was not observed in any of the mice expressing human shbg transgenes. Only one line of rat shbg transgenic mice has been characterized, and the neuromuscular defect observed in these animals might be the result of disruption of an endogenous gene during transgene insertion. This underscores the importance of generating and characterizing more than one transgenic line.
Although we have focused here on studying the expression of human
shbg transgenes in male mice, their expression in female
mice also results in high concentrations of SHBG and increased sex
steroid hormone levels in their blood (unpublished data). The actual
amounts of nonprotein-bound, or free, sex steroids in these animals is
not known, and will be difficult to determine because the percentage of
free hormone in serum samples will be very much less than 1% in
animals with greater than 1 nM SHBG in their blood. It is
remarkable, however, that there is no clear correlation between serum
SHBG and testosterone concentrations in different animals, and this is
most obvious when different lines of mice are compared. For instance,
the concentrations of testosterone are approximately 10-fold lower in
male shbg4-a mice than in male shbg11-a mice of
similar age despite the fact that these animals have very similar serum
levels (1 µM) of SHBG. The reason for this is unclear,
but this will result in marked differences in the relative amounts of
bioavailable sex steroids in the blood of these different lines.
Mice homozygous for human shbg transgenes are fertile, and the litter sizes and sex ratios of their progeny are within normal limits. Despite the extraordinarily high serum testosterone levels in some of the male shbg transgenic mice, we have not observed any obvious phenotypic differences in these animals to date, but it is possible that abnormalities in sex steroid-sensitive tissues will become evident as the animals age. However, the expression of human shbg transgenes in mice has already provided us with some insight into the tissue-specific expression of this gene in vivo, and these animals now represent a model system for dissecting the molecular basis for its regulation in different tissues during development and after treatment with various hormones.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Production of Transgenic Mice
An 11-kb HindIII fragment comprising all eight exons
that encode SHBG together with 6 and 2 kb of 5'- and 3'-flanking
sequences, respectively (Fig. 1), was excised from a human
shbg cosmid clone (3) and subcloned into pBluescript
(Stratagene, La Jolla, CA). This genomic fragment includes sequences
that contribute to shbg transcripts found in human testis
(Fig. 1A) and are located 1.5 kb 5' to the exon (exon 1) containing the
initiation codon for the SHBG precursor polypeptide (3). Digestion of
the 11-kb shbg HindIII fragment with XhoI
releases a 4.3-kb portion of human shbg that also comprises
all eight exons encoding SHBG plus 0.9 kb 5' of the translation
initiation codon in exon 1 and 0.5 kb 3' from the polyadenylation
sequence in exon 8 (Fig. 1A).
The pBluescript containing the 11-kb shbg sequence was purified using a Qiagen Tip-500 column (Qiagen, Chatsworth, CA), and the shbg fragments obtained by HindIII (11 kb) and XhoI (4.3 kb) digestion were purified twice by agarose gel electrophoresis and electroelution. They were then passed through an Elutip-D column (Schleicher and Schuell, Keene, NH) before microinjection into the pronuclei of one-cell mouse embryos (34). Injected embryos were implanted into pseudopregnant recipient mice using a standard protocol (34) approved by the animal care committee of the University of Western Ontario.
Founder Identification and Transgene Analysis
Genomic DNA (5 µg) from tail biopsies was digested with
EcoRI and subjected to agarose gel electrophoresis (34). The
DNA fragments in the gel were transferred to a ZetaProbe (Bio-Rad,
Mississauga, Canada) nylon membrane and subsequently hybridized with a
32P-labeled SHBG cDNA (3'-EcoRI fragment) that
recognizes exons 6–8 of human shbg (3, 35). The blotting,
hybridization, and washing conditions were recommended by Bio-Rad.
Radioactivity on the blots was detected by exposure to x-ray film
(DuPont Canada, Mississauga, Canada) against an intensifying
screen.
Tissue RNA Analysis
TRIzol reagent (Life Technologies, Burlington, Canada) was used
to extract RNA from mouse tissues, and poly(A)+ RNA was
isolated using a PolyATtract mRNA Isolation System IV (Promega,
Madison, WI). Total RNA (10 µg) or poly(A)+ RNA
(0.5–2.5 µg) was electophoresed in a formaldehyde/agarose gel and
transferred to a ZetaProbe nylon membrane, as recommended by Bio-Rad.
The RNA was first hybridized with 32P-labeled human SHBG
cDNA (3' EcoRI fragment). After washing at high stringency
and exposure to x-ray film to detect shbg transcripts, the
hybridized SHBG cDNA probe was stripped from the blot, which was then
rehybridized with a cDNA of mouse 18S ribosomal RNA as a loading and
transfer control. A Northern blot of poly(A)+ RNAs was
first hybridized with a 32P-labeled
SmaI-ApaI fragment of human shbg that
contains a 183-bp sequence within exon 1 spanning the translation start
site (3). After detection on an x-ray film, the radioactive probe was
stripped from the blot for sequential rehybridization with a human SHBG
cDNA (3'-EcoRI fragment) and a mouse ß-actin cDNA.
Primer Extension of shbg Transcripts
Poly(A)+ RNA (2.5 µg) from shbg11 and
wild-type mouse tissues was hybridized with a 32P-labeled
oligonucleotide complementary to human shbg exon 1
(5'-GCTCTCCATAATCAGCCACTGTCC-3') or exon 2
(5'-CTTGTCCTGGGCCATTGCTGAGGTG-3') sequences, and primer extended with
Superscript reverse transcriptase (Life Technologies) using a standard
method (36). A 32P-labeled oligonucleotide
(5'-ACCAGCGCAGCGATAATCGCCATCCAT-3') that recognizes mouse actin mRNA
(37) was also used as a control. The primer-extended products were
analyzed by electrophoresis on an 8% acrylamide DNA sequencing gel and
autoradiography, and their sizes were determined by comparison with the
products of a DNA sequencing reaction.
Immunohistochemistry
Tissues were fixed in situ by transcardial perfusion
of anesthetized animals with ice-cold Bouin’s fluid or 70
mM phosphate buffer, pH 7.0, containing 4%
paraformaldehyde, followed by immersion in the same fixatives at 4 C
for 20 h. Tissues were also removed and immersed directly in these
fixatives for routine histology. Fixed tissues were embedded in
paraffin wax, and 5-µm thick sections were cut and mounted onto
SuperFrost-coated slides (VWR, Westchester, PA). After dewaxing and
rehydration, tissue sections were incubated sequentially in 0.3%
hydrogen peroxide to quench endogenous peroxidase activity, in 0.1%
trypsin for 10 min, and then in nonimmune serum for 20 min. They were
then incubated with diluted (1:500 to 1:2000) rabbit antihuman SHBG
antiserum (38) at 4 C for 16 h. After washing in PBS, antibody
complexes were detected by incubation with a biotinylated secondary
antibody and the avidin-biotin-peroxidase complex (Vectastain, Vector
Laboratories, Burlingame, CA) with 3,3'-diaminobenzidine tetrachloride
as the chromagen. Slides were counterstained with Harris’s hematoxylin
and mounted with Permount (Fisher Scientific, Unionville, Canada).
In Situ Hybridization
Sense and antisense human SHBG riboprobes were transcribed in
the presence of digoxigenin-11-UTP (Boehringer Mannheim, Laval, Canada)
or [35S]UTP (DuPont Canada, Mississauga, Canada) from a
0.7-kb 5'-EcoRI fragment of a human SHBG cDNA in a
pT3/T7mp18 vector (35) using commercially available reagents
(Promega).
For hybridization with digoxigenin (DIG)-labeled riboprobes, rehydrated tissue sections were first treated sequentially with 0.2 M HCl and PBS containing 0.2% Triton-X 100, and then incubated with 40 µg proteinase K/ml PBS at room temperature for 10 min, followed by a wash in PBS. After a postfixation in PBS containing 4% paraformaldehyde for 30 min, sections were washed in PBS and treated with 0.1 M triethanolamine containing 2.6 mM acetic anhydride at room temperature for 10 min. Sections were then dehydrated through ascending ethanols (70–100%) and air-dried.
Before hybridization with DIG-labeled riboprobes, sections were prehybridized in the presence of 50% formamide in 2 x SSC (1 x = 0.15 M NaCl and 0.015 M sodium citrate) at 42 C for 2 h. Hybridization with DIG-labeled riboprobes was performed at 55 C for 20 h in the presence of 50% formamide in a buffer containing 2 x SSC, 1 x Denhardt’s reagent, 0.4 mg/ml salmon sperm DNA, and 10% dextran sulfate. Nonhybridized riboprobes were removed by washing in 2 x SSC and 1 x SSC at 50 C, and sections were then incubated with 20 µg ribonuclease A/ml 10 mM PIPES (pH 7.2), 0.5 M NaCl, and 0.1% Tween-20 at 37 C for 30 min. After washing in 100 mM Tris-HCl, pH 7.5, and 150 mM NaCl, hybridized DIG-labeled riboprobes were detected using alkaline phosphatase-labeled anti-DIG antibody, as recommended by Boehringer Mannheim. Sections were counterstained with methyl green.
The protocol for in situ hybridization with 35S-labeled riboprobes was described previously (39). Slides were coated with NTB-2 emulsion (Eastman Kodak, Rochester, NY), stored for 1 week at 4 C, and developed in D19 developer (Eastman Kodak). Sections were counterstained with Harris’s hematoxylin, and silver grains were quantified under darkfield illumination using a Northern Eclipse image analysis system (Empix Imaging, Inc., Mississauga, Canada). Serial sections of testes were stained with periodic acid-Schiff’s stain to identify the stages of spermatogenesis.
Western Blot Analysis
Proteins in mouse urine and diluted serum (1:500 in PBS) were
heat denatured in loading buffer and subjected to discontinuous
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 4% and 10%
polyacrylamide in the stacking and resolving gels, respectively.
Proteins in the gel were transferred electrophoretically (40) to a
Hybond ECL nitrocellulose membrane (Amersham, Mississauga, Canada). The
membrane was preincubated in a 5% skim milk solution and then
incubated overnight at 4 C with rabbit antihuman SHBG antiserum (38)
diluted 1:500 in TBS [10 mM Tris-HCl (pH 7.5), 150
mM NaCl, and 0.05% Tween-20] containing 0.5% skim milk
powder. The blot was then washed several times in TBS to remove excess
antibody, and specific antibody-antigen complexes were identified using
a second antibody (horseradish peroxidase-labeled donkey antirabbit
IgG) and chemiluminescent substrates (Life Technologies) by exposure to
x-ray film.
Serum SHBG and Testosterone Assays
The amounts of SHBG in transgenic mouse serum were determined
using a steroid binding capacity assay (41). In brief, serum samples
were diluted 1:100 in a slurry of dextran-coated charcoal at room
temperature to remove endogenous steroids and then further diluted 1:10
to 1:20 in PBS containing 0.01% gelatin. Aliquots of the diluted serum
samples containing about 0.1 pmol SHBG were incubated (1 h at room
temperature and 30 min at 0 C) with 1 pmol
5-[3H]dihydrotestosterone (Amersham) in the presence
or absence of a 400-fold molar excess of 5-dihydrotestosterone to
measure nonspecific binding and total binding, respectively. A
dextran-coated charcoal slurry was then added at 0 C for 10 min to
separate free ligand, and the SHBG-bound fraction was recovered by
centrifugation and decantation of supernatants into scintillation vials
for counting radioactivity. Serum concentrations of SHBG were
determined from the steroid binding capacity measurements after
correction for dilution and assuming one steroid-binding site per mol
SHBG (41). Serum testosterone concentrations were determined by RIA
(Endocrine Sciences, Calabasas Hills, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the Medical Research Council of Canada.
Received for publication July 28, 1997. Revision received September 18, 1997. Accepted for publication October 16, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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