Human Sex Hormone-Binding Globulin Gene Expression in Transgenic Mice

Abstract

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 approximately 11-kilobase (kb; shbg11) or 4.3-kb (shbg4) human shbg genomic fragments that comprise all eight exons encoding SHBG as well as approximately 6 kb or approximately 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.

Monosaccharide-induced lipogenesis regulates the human hepatic sex hormone–binding globulin gene

“…We first manipulated the diets of mice expressing different human SHBG transgenes (21): one line contains the 4.3-kb transcription unit expressed in liver and kidney (22), while the other contains a larger, 11-kb region of human SHBG that is also expressed in male germ cells (23). When male mice expressing an 11-kb human SHBG transgene were fed a highsucrose diet, human SHBG levels in the blood were reduced by about 50% after 1 week ( Figure 1A).…”

Unique Approaches to Androgen Effects on Prostate Cancer

“…10. Quantitated amounts of SHBG transcripts present in prostate, breast, liver and testis, and in the LNCaP, MCF-7, and HepG2 cell lines.…”

Human sex hormone–binding globulin variants associated with hyperandrogenism and ovarian dysfunction

“…These results were even more remarkable because the proband was being treated at the time of blood sampling with an oral estrogen and antiandrogen formulation that would normally be expected to increase serum SHBG levels (see Methods). Overlapping genomic fragments spanning the entire transcription unit encoding SHBG (9,12) were therefore PCR-amplified using the proband's DNA (Figure 1a). Sequence analysis of the PCR products revealed two alleles based on the presence or absence of an SNP (P 1 ) within exon 4 ( Figure 1b).…”

“…Apart from the MspI polymorphism (P 1 ), the allele encoding the SHBG P156L variant showed no other deviations from the consensus SHBG sequence that might contribute to a reduction in its expression. This sequence analysis also included the 800-bp proximal promoter responsible for human SHBG expression in the liver (12). Since the MspI polymorphism is located close to the exon 4/intron 4 boundary, we performed an experiment to determine whether it influences the splicing or relative abundance of SHBG transcripts, or their translation in hepatocytederived cell lines.…”

“…This was done by introducing the sequences of the P 1 variant or consensus SHBG into BW-1 mouse hepatoma cells and human HepG2 hepatoblastoma cells. These genomic fragments span the translation start site for the SHBG precursor polypeptide and the polyadenylation sequence within exon 8 of human SHBG (9,12) and were expressed constitutively under the control of a CMV promoter. RT-PCR and Northern blot analysis of SHBG mRNA from stable transformants obtained after neomycin selection indicated that cells producing SHBG P156L contained only appropriately spliced SHBG transcripts, as did cells expressing the consensus SHBG sequences (Figure 6a).…”