GENDER BINDING

Effect of Gender Binding Chemicals to Human Reproduction

Effect of Gender Binding Chemicals to Human Reproduction

Introduction

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds androgens and estrogens with high affinity. It is produced by hepatocytes and secreted into the blood, where it regulates the biological activities of male and female sex steroids. Unlike the human gene, shbg in rodents, such as rats and mice, is silent in the liver postnatally, and the gene expression is mainly restricted to the testes.

The testicular gene product is referred to as the androgen binding protein (ABP), as it binds androgens with high affinity in the male reproductive tract. Human testis produces alternative shbg products, the function(s) of which is largely unknown. To understand why shbg is expressed so differently in humans and rodents and to learn more about the function of the gene product, several lines of transgenic mice carrying human shbg in their genome were produced (Diamond, 1985, 359).

These mice express the human gene in the liver and have appreciable amounts of circulating SHBG in the blood. Although it was anticipated that the presence of this high-affinity steroid binding protein would result in imbalances in free and bound sex hormone levels, the reproductive performance of these mice was not compromised in any way.

The comparisons of the expression patterns of two human shbg transgenes with different amounts of 5'-flanking sequences (~6 kb or ~0.85 kb) provided information about the regulatory mechanisms that govern human shbg expression and regulation in vivo. Specifically, upstream sequences present only in the larger human shbg transgene contributed to sexual dimorphism of SHBG levels in mouse plasma, while both transgenes were subject to the regulation by androgens in the kidney.

Materials and Methods

Experimental design

To introduce human shbg sequence from +7 to +60 into -803/+7shbg and -299/+60shbg fragments, a region spanning nucleotides from -41 to +60 was PCR-amplified using an 11 kbp genomic DNA fragment as the template (III). The forward primer spanned human shbg nts -45/-22 and the reverse primer was complementary to nts +43/+60 with a sequence (GAATGAAGCTT) for a HindIII site at its 5'-end. The PCR-product was digested with SmaI and HindIII and linked to nts -803/-41 of the shbg promoter in pSP72 (III). The -803/+60shbg and -299/+60shbg fragments were then subcloned into the XhoI/HindIII and XbaI/HindIII sites of pGL2 Basic (Promega), respectively. Several 5'-deletion fragments of the shbg promoter were also PCR-amplified from the 11 kbp DNA fragment. The forward primers for these constructs corresponded to the following sequences relative to the shbg transcription start site in the liver: nts -696/-679, -670/-650, -617/-596, -552/-531, -509/-489 and -458/-438.

In each case, a sequence (TGAACTCGAG) for an XhoI site was added to these primers for subcloning. The reverse primer spanned human shbg nts -317/-290. PCR-products were digested with XhoI and XbaI and subcloned into pBluescript KS- (Stratagene) for sequencing. Human shbg region -803/-299 in -803/+60pSP72 was replaced with each deletion fragment followed by subcloning into pGL2 Basic as XhoI-HindIII ...