Human Sex Hormone-Binding Globulin Gene Expression in Transgenic Mice

Marja Jänne, Harminder K. Deol, Stephen G. A. Power, Siu-Pok Yee and Geoffrey L. Hammond

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The sex hormone-binding globulin gene (shbg) is expressed in the liver and testis as well as in several other tissues that play important roles in reproduction. Expression of shbg in the human liver results in the production of plasma sex hormone-binding globulin (SHBG), which regulates the bioavailability of sex steroids in the blood. Although shbg is not expressed in rodent livers postnatally, it gives rise to the androgen-binding protein in their testes upon sexual maturation. Human shbg is also expressed in the testis, but its products and their function are less well characterized. To study the expression of human shbg in different tissues and the consequences of overexpressing this gene in vivo, we have produced several lines of mice containing ~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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sex hormone-binding globulin (SHBG) transports sex steroids in the blood and regulates their access to target cells (1). Hepatocytes are probably the major site of plasma SHBG biosynthesis (2), but the SHBG gene (shbg) is also expressed in the testis (3, 4) and several other tissues (5, 6, 7). Expression of shbg in the rat testis gives rise to the androgen-binding protein (ABP), which is secreted by Sertoli cells into the seminiferous tubules where it is thought to influence sperm maturation (8). Several differentially spliced shbg transcripts are also present in human (3, 9) and rat testis (10) as well as in fetal rat liver and adult rat brain (10, 11). Their protein products have never been identified or shown to have any biological function, but one alternative shbg transcript in rat tissues (10) encodes a protein that is capable of translocating to the cell nucleus and is also recognized by an antiserum against rat ABP (12).

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Analysis of Human shbg Transgenic Mice
Two founder animals were used to establish lines for each of the two human shbg fragments injected into mouse embryos, and a Southern blot of DNA from their progeny is shown in Fig. 1BGo. 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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Figure 1. Human shbg Transgenes and Identification of Transgenic Mice

A, Partial restriction maps of the human shbg genomic DNA fragments, shbg11 and shbg4, used for microinjection of one-cell mouse embryos. Both fragments contained the entire coding region for human SHBG (exons 1–8) and different amounts of 5'- and 3'-flanking sequences. The shbg11 also contained sequences for alternative exons (*) that replace exon 1 in human testicular cDNAs (3). B, Southern blot representing the genotypes of the five different human shbg transgenic mouse lines. Human shbg sequences were identified among EcoRI-digested genomic DNA using a cDNA probe encoding human shbg exons 6–8. Wild-type (wt) mouse DNA was analyzed as a control in lane 1. The positions of DNA size markers are shown on the left.

 
The number and sizes of EcoRI restriction fragments that hybridized with the SHBG cDNA (Fig. 1BGo) 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. 1BGo 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. 1BGo) 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. 2Go. 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. 2Go), 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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Figure 2. Northern Blot of Human shbg Transcripts in Transgenic Mouse Tissues

Total RNA from male transgenic (five independent lines shown in Fig. 1Go) and wild-type (wt) mouse spleen (lanes 1–2), liver (lanes 3–8), testis (lanes 9–14), and kidney (lanes 15–20) was subjected to formaldehyde/agarose gel electrophoresis and blotted onto a nylon membrane. Human shbg transcripts were detected using a SHBG 3'-cDNA probe (exons 6–8), and RNA loading and transfer were controlled by reprobing with a cDNA encoding mouse 18S ribosomal RNA.

 
To further characterize the shbg transcripts in the testis, a Northern blot of poly(A)+RNA from several shbg11-b mouse tissues was prepared and first hybridized with a cDNA for shbg exon 1 sequences, which span the translation initiation codon for the SHBG precursor polypeptide, and then rehybridized with a cDNA that corresponds to sequences encoded by shbg exons 6–8. Despite the obvious presence of ß-actin transcripts in testis and brain, the shbg exon 1-specific probe failed to hybridize with any transcripts in these tissues (Fig. 3Go). 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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Figure 3. Relative Abundance of Exon 1-Specific Sequences in shbg Transcripts Extracted from Different Transgenic Mouse Tissues

Poly(A)+ RNA (0.5–2.5 µg) from shbg11-b mouse tissues was subjected to formaldehyde/agarose gel electrophoresis and transferred to a nylon membrane. The RNAs on the Northern blot were hybridized sequentially with human shbg exon 1 and SHBG 3'-cDNA (exons 6–8) probes. Mouse ß-actin was used to demonstrate the presence and integrity of mRNA in extracts from different tissues.

 
Mapping the Tissue-Specific shbg Transcription Start Sites
To map the shbg transcription start sites in shbg11 transgenic mouse tissues, primer extension of poly(A)+ RNA from shbg11-b mouse liver, kidney, and testis was performed using an oligonucleotide complementary to a region in exon 1 spanning the translation initiation codon for the SHBG precursor (Fig. 4Go). 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. 4Go). 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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Figure 4. Mapping the Human shbg Transcription Start Site in Transgenic Mouse Tissues by Primer Extension Analysis

Poly(A)+ RNA (2.5 µg) from wild-type (wt; lanes 1, 3, 5, 7, and 9) and shbg11-b (lanes 2, 4, 6, 8, and 10) mouse tissues was hybridized with a 32P-labeled human shbg exon 1 oligomer and primer-extended with Superscript reverse transcriptase. The products were analyzed by PAGE and autoradiography. The sizes of the major extension products (inclusive of the primer) were determined from a DNA sequencing ladder and are indicated beside the arrowheads.

 
The absence of a primer extension product from the testicular mRNA of the transgenic mice provides additional evidence that the shbg transcript in this tissue lacks this exon 1 sequence. This was confirmed by an additional primer extension experiment (Fig. 5Go) 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. 5Go) mice, would be between 161–186 bp.



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Figure 5. Utilization of Alternative Human shbg Transcription Start Sites in Transgenic Mouse Testis

Poly(A)+ RNA from shbg11-a transgenic and wild-type (wt) mouse testes was hybridized with 32P-labeled actin or human shbg exon 1 or exon 2 oligomers, and primer-extended with Superscript reverse transcriptase. The products were analyzed by PAGE and autoradiography. The sizes of the major extension products (inclusive of the primer) were determined from a DNA sequencing ladder and are indicated beside the arrowheads.

 
Cellular Localization of SHBG and shbg Transcripts in Transgenic Mice
The hepatocytes of a shbg11-a male mouse contain immunoreactive SHBG and SHBG mRNA (Fig. 6Go, 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. 6Go, C and D, respectively), with the most intense staining of immunoreactive SHBG seen at the luminal aspect of the cells (Fig. 6CGo). 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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Figure 6. Cellular Localization of Human SHBG and shbg Transcripts in the Liver, Kidney, and Testis of Mature Male Transgenic Mice Brightfield micrographs show the cellular localization of immunoreactive human SHBG (A, C, E, and G) and human shbg transcripts using DIG-labeled SHBG antisense riboprobes (B and D), and the darkfield micrographs (monochrome threshold image) are mouse testes sections probed for shbg transcripts with an 35S-labeled SHBG antisense riboprobe (F and H). Serial cross-sections of a hepatic lobule from a 65-day-old shbg11-a mouse liver illustrating the presence of human SHBG (A) and its mRNA (B) in the same hepatocyte populations. Central (cv) and portal (pv) veins are shown, with arrows indicating the periportal regions of a hepatic lobule. Serial longitudinal sections of the S2 segment (inner zone of the cortex) of a 43-day-old shbg11-a mouse kidney showing immunoreactive human SHBG (C) and its mRNA (D) in the same epithelial cells lining the proximal convoluted tubules. The most intense immunoreactivity was present at the luminal aspects of these cells (D). The position of a glomerulus (g) is marked. Cross-sections of testes from shbg11 (E and F) and shbg4 (G and H) mice. Immunoreactive human SHBG is most abundant at the boundary tissue of the seminiferous tubules and in interstitial compartments of testes taken from 54-day-old shbg11-b (E) and 42-day-old shbg4-a (G) mice, but can also be detected (arrows) in a few Sertoli cells (G). A monochrome threshold image (F) shows the intensity of silver grains that mark the shbg transcripts in a 43-day-old shbg11-a mouse testis, which are distributed uniformly throughout the Sertoli cells of seminiferous tubules at stages VII and VIII and are concentrated in the adluminal region of the Sertoli cells at stages X–XII of spermatogenesis. No signals marking shbg transcripts could be detected in the Sertoli cells of a 42-day-old shbg4-a mouse testis (H). Bar = 100 µm.

 
Most of the immunoreactive SHBG was restricted to the interstitial compartment of testes from both shbg11-b and shbg4-a mice (Fig. 6Go, E and G), but it could also be detected in a few Sertoli cells (Fig. 6GGo). 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. 6FGo) 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. 7Go). 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. 6FGo). 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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Figure 7. Quantitative in situ Hybridization of shbg Transcripts in the Seminiferous Tubules of a 43-day-old shbg11-a Mouse Testis with Respect to the Stage of Spermatogenesis

Relative abundance of human shbg transcripts (pixel gray values/seminiferous tubule) in Sertoli cells plotted with respect to the stage of spermatogenesis. The intensity of signals is obtained by measuring the pixel gray values on a monochrome threshold image (Northern Eclipse Image Analysis Software) in the Sertoli cells of individual seminiferous tubules, and each measurement was divided by the area of the seminiferous tubule excluding the lumen. Values are the mean (columns) ± SD (bars) for at least four seminiferous tubules per stage (I–XII) of the spermatogenic cycle.

 
Human SHBG in the Urine of Transgenic Mice
The presence of shbg transcripts in the epithelial cells lining the proximal convoluted tubules of the kidney and the accumulation of immunoreactive human SHBG at their luminal surface (Fig. 6CGo) 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. 8Go), 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. 8Go, 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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Figure 8. Western Blot of Human SHBG in the Transgenic Mouse Serum and Urine

Serum (1:500 in PBS) and urine samples from wild-type (wt) mice and transgenic mice that either express (shbg4-a) or do not express (shbg4-b) a human shbg4 transgene in kidney were subjected to SDS-PAGE and transferred to a nitrocellulose membrane for Western blot analysis with an antiserum specific for human SHBG. The relative positions of protein size markers are shown on the left.

 
Blood Levels of SHBG and Testosterone in shbg Transgenic Mice
The serum concentrations of SHBG and testosterone in 90-day-old shbg11-a, shbg4-a and shbg4-c male mice are presented in Table 1Go. 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 1Go).


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Table 1. Serum Levels of SHBG and Testosterone in Male shbg Transgenic Mice and Their Wild-Type Littermates

 
Phenotype Analysis
The body weights, and testis and kidney weights of mature male shbg11 and shbg4 transgenic mice were similar to those in age-matched wild-type mice, and no differences were observed in their epididymal sperm counts or sperm morphology at the light microscopic level. Routine histology of male accessory sex organs revealed no obvious differences between shbg transgenic and wild-type mice. Male shbg transgenic mice from all five lines were fertile up to at least 9 months of age. The litter sizes of all five lines of homozygous shbg transgenic mice were normal, as were the sex ratios of homozygous shbg transgenic pups within each litter.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The introduction of human genomic DNA fragments encoding SHBG into the mouse genome has allowed us to identify sites of human shbg expression and to define the transcription units expressed in different tissues. At least two independent lines of transgenic mice were generated per DNA sequence, and this was done to control for inappropriate transcription caused by positional effects at the transgene integration sites. In most lines, the transgenes integrated as head to tail multimers, exhibited remarkable consistency with respect to where they were expressed, and remained stable over several generations. In one founder male, they did not appear to have integrated as strictly head to tail multimers, and segregated as two separate genotypes that were maintained for further comparisons because one of them lacked expression in the kidney.

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 {alpha}-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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Mice were obtained from in-house breeding stocks and housed in a pathogen-free facility. The one-cell embryos for microinjections were obtained by superovulating CBA x C57BL6 hybrid females with pregnant mare serum and hCG (Sigma Chemical Co. Mississauga, Canada) and mating them with CBA x C57BL6 males (34). The same hybrid cross was used to propagate transgenic mouse lines. Female CD-1 mice were used to produce pseudopregnant recipients by mating with vasectomized CD-1 males.

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. 1Go), 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. 1AGo) 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. 1AGo).

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{alpha}-[3H]dihydrotestosterone (Amersham) in the presence or absence of a 400-fold molar excess of 5{alpha}-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).


    ACKNOWLEDGMENTS
 
The authors thank Denise Power and Gail Howard for secretarial assistance.


    FOOTNOTES
 
Address requests for reprints to: Geoffrey L. Hammond, Ph.D., Cancer Research Laboratories, London Regional Cancer Center, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6. E-mail: ghammond{at}julian.uwo.ca

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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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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