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Departments of Obstetrics and Gynecology, and Pharmacology and Toxicology, and the Medical Research Council Group in Fetal and Neonatal Health and Development, University of Western Ontario, London, Ontario, Canada N6A 4L6
Address all correspondence and requests for reprints to: Geoffrey L. Hammond, Ph.D., London Regional Cancer Center, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6. E-mail: ghammond{at}julian.uwo.ca
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Abstract |
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-dihydrotestosterone treatment. Moreover, exogenous androgen
increased human SHBG mRNA levels in the kidneys of female mice. In
summary, comparisons of how different human shbg
transgenes are expressed in vivo provides information
about the positions of potential regulatory sequences that may control
the hormonal regulation and tissue-specific expression of this gene
during development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of shbg in Sertoli cells gives rise to the testicular form of SHBG known as the androgen-binding protein (ABP), which is secreted into the lumen of seminiferous tubules and is believed to promote sperm maturation in the male reproductive tract (12). In addition to a messenger RNA (mRNA) for ABP, several differentially spliced shbg transcripts, originating from an alternative upstream promoter, have been identified in human testis (13). These alternative shbg transcripts lack the coding sequence for the signal polypeptide required for SHBG or ABP secretion, and they accumulate in a spermatogenic stage-dependent manner within Sertoli cells of mice carrying and expressing human 11-kb shbg transgenes (3).
Hepatocytes and Sertoli cells are major sites of shbg expression, but shbg transcripts have been identified in several other tissues (14, 15, 16), including hamster kidneys (17) and the kidneys of mice expressing human shbg transgenes (3). In these mice, expression of the transgenes results in the production and secretion of appreciable amounts of immunoreactive human SHBG, which concentrates at the luminal surface of epithelial cells lining the proximal convoluted tubules (3). The SHBG excreted in their urine retains its steroid-binding properties (3), but the physiological role of shbg expression in the mouse and hamster kidney and whether it is expressed in human kidney remain to be determined.
To learn more about the tissue-specific expression and regulation of
human shbg, two regions of the human shbg locus
were introduced as transgenes into the mouse genome, and their
activities have been monitored during fetal and postnatal development.
The smallest of these transgenes spans 4.3 kb of human shbg
and comprises the entire coding sequence for the SHBG precursor
polypeptide as well as approximately 0.9 kb of promoter sequence
containing a number of cis-active elements that may regulate
liver- and kidney-specific expression (18). Several other lines of
shbg transgenic mice that carry a much larger (
11-kb)
region of human shbg were also studied because these
transgenes include an alternative promoter sequence used in the testis
of mature animals (3). Moreover, we reasoned that any differences in
the spatial and temporal patterns of expression or hormonal regulation
of these two different human shbg transcription units might
reveal the locations of important regulatory sequences.
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Materials and Methods |
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In situ hybridization
Sense and antisense human SHBG riboprobes were transcribed using
commercially available reagents (Promega Corp., Madison,
WI) in the presence of [35S]UTP (DuPont Canada,
Mississauga, Canada) from a 0.7-kb 5'-EcoRI fragment of a
human SHBG complementary DNA (cDNA) in a pT3/T7mp18 vector (19). The
protocol for in situ hybridization with
35S-labeled riboprobes was described previously (20). To
detect hybridized probes, slides were first subjected to
autoradiography by overnight exposure to x-ray film (DuPont) and then
coated with NTB-2 emulsion (Eastman Kodak Co., Rochester,
NY), stored for 1 week at 4 C, and developed in D19 developer
(Eastman Kodak Co.). Sections were counterstained with
Harris’s hematoxylin to identify the cellular location of silver
grains.
Western blot analysis
Diluted mouse serum 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 (21) to Hybond ECL nitrocellulose
membranes (Amersham Pharmacia Biotech, Mississauga,
Canada). The membranes were preincubated in a 5% skim milk solution
and then incubated overnight at 4 C with primary antisera diluted in
TBS-T [10 mM Tris-HCl (pH 7.5), 150 mM NaCl,
and 0.05% Tween-20] containing 1% skim milk powder. The blots were
then washed several times in TBS-T 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, Inc.,
Burlington, Canada) by exposure to x-ray film. The primary antisera
used were raised in rabbits against pure human SHBG (22) or against
mouse SHBG expressed as a glutathione-S-transferase fusion
protein in Escherichia coli using a pGex2T expression vector
(Pharmacia Biotech, Baie d’Urfe, Canada). The mouse SHBG
was released from the glutathione-S-transferase fusion
protein by thrombin cleavage and purified for use as an immunogen, as
recommended by Pharmacia Biotech.
Serum SHBG and testosterone measurements
The concentrations of human SHBG in transgenic mouse serum were
determined using a saturation ligand binding assay (23). In this assay,
endogenous steroids were first removed from serum samples by dilution
(1:100) in a dextran-coated charcoal suspension and incubation (30 min)
at room temperature. Samples were then further diluted (1:10 to 1:20)
and incubated (1 h) at room temperature with 10 nM
[3H]5-dihydrotestosterone (Amersham Pharmacia Biotech) followed by an additional incubation (30 min) at 0 C.
Nonspecific binding was estimated in the presence of a 400-fold molar
excess of nonradioactive 5-dihydrotestosterone. Free ligand was
removed by incubation (10 min) with an ice-cold dextran-coated charcoal
slurry and separation by centrifugation. Supernatants containing
SHBG-bound ligand were taken for radioactivity measurements to
determine serum SHBG concentrations by assuming one steroid ligand
bound per molecule of SHBG (23).
Serum testosterone concentrations were determined using a RIA kit (Orion Diagnostica, Oulunsalo, Finland).
Tissue RNA analysis
Total RNA isolated from mouse tissues using TRIzol reagent
(Life Technologies, Inc.) was separated by electrophoresis
on a 1% agarose gel in the presence of formaldehyde and transferred to
a Zeta-Probe nylon membrane (Bio-Rad Laboratories, Inc.
Mississauga, Canada). Membranes were hybridized with either a
32P-labeled human SHBG cDNA (556-bp 3'-EcoRI
fragment) or a 32P-labeled mouse SHBG cDNA (535-bp
3'-EcoRI fragment) followed by high stringency washing and
exposure to x-ray film. The blots were then stripped of hybridized
32P-labeled SHBG probes. After overnight exposure to x-ray
film to ensure complete removal of probe, membranes were reprobed with
a 32P-labeled cDNA for 18S ribosomal RNA as a control for
loading and transfer. The relative intensities of radiographic signals
obtained for human shbg and 18S transcripts were compared by
densitometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Examination of the in situ
hybridization of the antisense complementary RNA probe under high power
(x63) revealed that the signal was distributed throughout the liver,
whereas in the gut it was confined to the epithelial cells of the
duodenum (not shown).
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) and was
compared with the levels of mouse and human SHBG in serum samples taken
from late fetal mice at the same gestational ages (Fig. 2B). In liver
extracts of shbg transgenic mice, the major shbg
transcript is approximately 1.6 kb in size (Fig. 2A). An additional
transcript of approximately 2.5 kb is also detected using human and
mouse SHBG cDNA probes, and this is believed to represent a partially
processed transcript (13). These data indicate that expression of
murine shbg declines in the fetal liver from day 16 of
gestation to birth together with a corresponding decline in amounts of
SHBG in the blood of the wild-type neonatal mice. Differences were
observed in the relative amounts of mouse SHBG present in serum from
wild-type and shbg transgenic mice at 17 dpc (Fig. 2B), and
this was confirmed in a repeat experiment. It is therefore possible
that expression of shbg transgenes influences the expression
of mouse shbg in the liver. More importantly, the expression
of both the shbg11 and shbg4 transgenes in the
fetal liver increases between day 17 of gestation and term (Fig. 2A),
with greater amounts of both human shbg transcripts in the
liver and human SHBG in the blood of neonatal mice expressing the 11-kb
shbg transgene (Fig. 2B). Interestingly, human SHBG is
present in the serum of shbg4-a fetal mice at 17 dpc (Fig. 2B) whereas human SHBG mRNA is undetectable in the liver at this stage
(Fig. 2A), indicating that the transgene is probably expressed in the
fetus at some earlier stage of gestation. Although the antiserum
against mouse SHBG appears to cross-react partially with human SHBG
(see Fig. 2B, shbg11-b d0 sample), there is a clear size
difference between human and mouse SHBG that allows the specific
recognition of mouse SHBG in serum samples from fetal shbg
transgenic mice (Fig. 2B). This size difference can be attributed to
the fact that mouse SHBG has three sites for
N-glycosylation, whereas human SHBG has only two
N-linked carbohydrate chains (24).
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-fetoprotein) that may have been present at
different stages of mouse development. Moreover, as the liver is the
major site of plasma SHBG biosynthesis (3), measurements of human SHBG
concentrations in blood samples provides a convenient means of
monitoring the hepatic expression of different shbg
transgenes in relatively large numbers of transgenic animals.
The serum levels of human SHBG in male and female
shbg11-a transgenic mice increased markedly within the first
10 days after birth. In the male mice, the levels increased
progressively thereafter until they reached a plateau at about day 40
(Fig. 3A). By contrast, serum SHBG levels
in the female mice did not change appreciably after 10 days of age.
However, a 3-fold difference was apparent in human SHBG serum levels in
male and female shbg11-a transgenic mice by the time they
reached sexual maturity on day 40 (Fig. 3A). This sex difference was
confirmed in another line (shbg11-b) of mice carrying an
approximately 11-kb shbg transgene, and the serum
concentrations of human SHBG in sexually mature male
shbg11-b mice were about 2.5 times greater than those in
their female counterparts (data not shown).
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). There was also no sex difference in the
serum levels of human SHBG in a separate line (shbg4-b) of
mice carrying a 4.3-kb human shbg transgene (data not
shown). Thus, additional sequences present in the approximately 11-kb
human shbg transgenes must account for their sexually
dimorphic expression. These sequences also appear to repress the
expression of the transgenes in the livers of shbg11-a
female mice at about the time they reach sexual maturity (Fig. 3A),
because this does not occur in female mice carrying a 4.3-kb
shbg transgene (Fig. 3B). The ontogenic changes and gender difference in serum SHBG levels observed in these mice were also reflected in corresponding differences in the relative abundance of human SHBG mRNA in the liver (data not shown).
Relationship between serum SHBG and testosterone levels in human
shbg transgenic mice
The presence of human SHBG in the blood of shbg
transgenic mice results in levels of serum testosterone that are
10–100 times higher than those in wild-type mice of the same age (25).
At birth, serum testosterone levels were much higher in
shbg11-a and shbg4-a male transgenic mice (Fig. 4, A and C, respectively) compared with
those in their female littermates (Fig. 4, B and D, respectively). In
the male mice, the serum testosterone levels decreased to very low
levels after birth and remained low until the animals were 30 days of
age, after which they fluctuated considerably from 35 nmol/liter to
1.75 µmol/liter. Serum testosterone levels in shbg11-a and
shbg4-a female transgenic mice were generally very low in
animals before weening, but increased by 20–30 days of age days with
the onset of sexual maturation. However, there was considerable
variation in serum testosterone levels between sexually mature animals
(Fig. 4, B and D, respectively). Despite the wide variations in serum
testosterone levels in mature male and female mice, these levels were
generally much higher than those measured in their wild-type
counterparts at 90 days (3). As expected, correlations were observed
between serum SHBG and testosterone levels in male (r = 0.52;
P < 0.001) and female (r = 0.49;
P < 0.001) shbg transgenic mice.
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). Compared with
the changes in serum testosterone levels in the same animals during the
first 20 days of life (Fig. 4A), it is apparent that the human
shbg transcripts in the testes accumulate (Fig. 5) well
before any appreciable increases in serum testosterone.
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20 days of age) and reached mature levels earlier (at 40 days of
age) in shbg11-a mice (Fig. 6B) compared with shbg4-a mice
(Fig. 6A). In the shbg4-a mice, SHBG mRNA increased in
abundance at 30 days of age and did not reach peak levels until at
least 60 days of age. The expression of both shbg11-a and
shbg4-a transgenes in the kidney was also clearly sexually
dimorphic, with much higher SHBG mRNA levels in the kidneys of male
mice (Fig. 6, A and B).
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, A and C). When this was considered
together with the differences in human SHBG mRNA abundance in kidneys
of male and female shbg transgenic mice (Fig. 6), we
reasoned that the expression of human shbg transgenes in the
mouse kidney might be androgen dependent. This was confirmed by first
demonstrating a clear reduction in the abundance of SHBG mRNA in the
kidneys of male shbg4-a (Fig. 7, A and C, panel i) and
shbg-11a (Fig. 7B, panel i) mice after castration.
Densitometric analysis of these Northern blots revealed that after
castration the relative abundance of SHBG mRNA was 19–56% of the
level in intact control mice depending on their ages (Fig. 7A). By
contrast, castration had no effect on the relative abundance of human
SHBG mRNA in the livers of the transgenic mice (Fig. 7A) or on SHBG
levels in their serum (data not shown). Additional evidence that human
shbg expression in the mouse kidney is regulated by
androgens was obtained by treating castrated male (Fig. 7, B, panel ii,
and C, panel i) and female (Fig. 7C, panel ii) shbg
transgenic mice daily with 100 µg 5-dihydrotestosterone given sc
in sesame oil (vehicle) for up to 5 days. In castrated male mice, the
relative abundance of human SHBG mRNA in the kidneys increased after
only 2 days of treatment (Fig. 7B, panel ii) and increased further
after 5 days of treatment (Fig. 7, B, panel ii, and C, panel i).
Furthermore, in ovariectomized female shbg4-a mice, the same
treatment with 5-dihydrotestosterone for 5 days resulted in a marked
increase the amount of SHBG mRNA in the kidney (Fig. 7C, panel ii).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse and rat shbg genes are transcriptionally silent in the liver postnatally (4, 8), but the hepatic production of human SHBG in transgenic mice increases steadily after birth. What is remarkable, however, is that the hepatic biosynthesis of SHBG in female mice expressing the 11-kb shbg transgene shows no increase after the onset of sexual maturation. By contrast, serum concentrations of human SHBG continue to increase in mice containing the 4.3-kb shbg transgene until about 60 days regardless of gender. Thus, ovarian steroids probably act through upstream regulatory sequences present in the 11-kb shbg transgenes to repress a postpubertal increase in hepatic SHBG production. This was unexpected, because the plasma levels of SHBG in humans are also sexually dimorphic after puberty, with levels falling much more markedly in males compared with females (10, 27). Although this decrease in young men has been attributed to an androgen-dependent decrease in the biosynthesis of plasma SHBG (28), this explanation remains questionable, because orchidectomy has no effect on serum SHBG levels (29). Castration of male shbg transgenic mice also had no effect on SHBG mRNA levels in the liver, and this excludes the possibility that the sexual dimorphism in 11-kb shbg transgene expression is due to the much higher plasma levels of testosterone in male mice.
It is widely accepted that SHBG reduces the MCR of sex steroids that interact with its binding site (30), and this probably contributes to the much higher serum concentrations of testosterone in SHBG mice compared with those in wild-type mice (25). It also explains why there are good correlations between plasma SHBG and testosterone levels in both male and female SHBG transgenic animals. This was not observed previously in transgenic mice that overexpress a rat shbg transgene in the testis and have detectable levels of rat ABP in their blood (31), but this is probably due to the much lower levels of serum rat ABP in these mice and its much lower affinity for sex steroids compared with human SHBG (32). Increased serum testosterone levels could result in part from an up-regulation of testicular androgen biosynthesis in response to decreased sex steroid bioavailability at the level of the hypothalamic-pituitary axis. In support of this, the steroid-binding capacity of SHBG is much higher than the concentration of testosterone in serum, and this would tend to maintain a low free testosterone concentration in blood. Surprisingly, the presence of very high concentrations of human SHBG in the blood of these mice throughout postnatal development does not have appreciable effects on their sexual development or fertility.
Human SHBG transcripts cannot be detected by Northern blotting in the testis of mice carrying a 4.3-kb human shbg transgene (3). In the latter study we also found that a majority of transcripts in the testis of shbg11 transgenic mice originate from an alternative promoter that is not present in the 4.3-kb shbg transgenes. These transcripts in the testis of shbg11-a transgenic mice contain an alternative exon 1 sequence (13), and their presence in Sertoli cells is tightly regulated throughout the spermatogenic cycle (3). Although the biological significance of these transcripts is not known, they may encode an N-terminally truncated form of SHBG that remains within cells (33). These transcripts within the mouse testis increased in abundance starting on about day 10 and reached a plateau by day 30. It will be of interest to determine how their expression is regulated. From the present study it appears that androgens may not be involved in this process, because these alternative human shbg transcripts begin to accumulate within the mouse testis well before any significant increases in serum testosterone levels.
Expression of human shbg transgenes in the mouse kidney was
unexpected, but we have recently obtained evidence that the mouse
shbg gene is also expressed in the kidney (unpublished
data). It was, therefore, of interest to examine how the human
shbg transgenes are regulated in the kidney, and our present
results indicate that the levels of human SHBG mRNA in the kidney do
not increase until animals undergo sexual maturation. At this time,
there is a marked increase in human SHBG mRNA abundance in the kidney,
particularly in male animals, and this appears to occur earlier in
animals expressing the 11-kb shbg transgenes. The sexual
dimorphism in human SHBG mRNA abundance in the kidneys indicated that
gonadal steroids, and in particular androgens, might be responsible for
increasing the expression of shbg in this tissue at puberty.
This was confirmed by castrating male mice and observing a marked
decrease in kidney SHBG mRNA abundance, which could be reversed by
treatment with 5-dihydrotestosterone. This androgen dependence was
also demonstrated in the kidneys of female mice. Furthermore, this
effect of androgens was observed in both male and female
shbg4 mice, and the cis-elements that mediate
this must, therefore, be located within this region of the human
shbg gene. We do not know what role SHBG gene expression
plays in the mouse kidney, and whether our observations in transgenic
mice recapitulate the situation in the human kidney, but the kidney is
very sensitive to sex steroids, and some renal diseases are known to be
sex hormone dependent (34).
In summary, an analysis of mice expressing human shbg transgenes has provided new insight into the spatial and temporal expression of the human shbg in an in vivo context and has revealed the locations of elements within the gene that might be involved in its hormonal regulation. These mice, therefore, not only provide a model system for studying the hormonal control of human shbg, but may provide insight into the function of this protein.
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Acknowledgments |
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Footnotes |
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Received January 8, 1999.
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) mice are shown. At each time point,
three or four animals were studied.
