SHBG is a homodimeric plasma glycoprotein. It functions as a carrier for sex steroids in blood and regulates their access to target cells. In human and rabbit, SHBG is a single-copy gene comprised of eight exons and is expressed primarily in the liver and testis. In the present study, the ontogeny of rabbit SHBG (rbSHBG) gene expression was examined in both fetus and mothers. Trace amounts of rbSHBG mRNA were detected in fetal liver from d 11 to d 29 gestation. These levels increased dramatically at d 30 and remained high until parturition (d 33). In contrast, high levels of rbSHBG mRNA were detected in the maternal liver early during pregnancy, with maximal levels being attained by d 22 and declining markedly thereafter. A rbSHBG transcript lacking the exon 4 sequences was consistently expressed along with the rbSHBG mRNA. When expressed as a glutathione-S-transferase-fusion protein, this alternatively spliced rbSHBG transcript resulted in a product with almost no steroid binding activity, unlike the full-length rbSHBG-glutathione-S-transferase fusion protein, which bound 5alpha-dihydrotestosterone. Antibody specific to the novel rbSHBG isoform lacking the exon 4-encoding domain was raised, and a single immunoreactive protein of 33-35 kDa was detected by Western blot analysis in both fetal and maternal liver, and this indicates that the rbSHBG transcripts lacking exon 4 sequences are translated in vivo. An RT-PCR analysis further revealed that this alternatively spliced SHBG transcript is present in human HepG2 cells as well as human and mouse testes, indicating that exon 4 splicing in SHBG transcription is conserved among mammalian species. To our knowledge, this is the first report of the identification of a SHBG exon 4 splice variant that is translated. Because the SHBG isoform it encodes lacks appreciable steroid-binding activity, it may function beyond that of the widely accepted role of SHBG as a steroid-transport protein.
Testin is a secretory protein that was initially identified from rat Sertoli cell-enriched cultures and has been suggested to be a sensitive marker to monitor the integrity of Sertoli-germ cell junctions. However, the expression of the testin gene in other species and the molecular mechanisms that govern its transcription are unknown. To address these issues, we cloned and characterized the mouse testin gene. A full-length mouse testin cDNA encoding a polypeptide of 333 amino acid residues was isolated by library screening. Sequence analysis revealed that mouse testin shares 90.1%, 58.9%, 62.2%, and 64.6% identity with rat testin and cathepsin L of mouse, rat, and human, respectively, at the amino acid level. Reverse transcription-polymerase chain reaction and Southern blot analysis demonstrated that mouse testin transcripts were predominantly expressed in the gonads. The mouse testin gene spans over 21 kilobases (kb) and contains eight exons interrupted by seven introns. Primer extension analysis and 5' rapid amplification of cDNA ends identified a major transcription start site located 134 base pairs upstream from the translation initiation codon. Analysis of a 2.3-kb mouse testin 5'-flanking region revealed that it lacked TATA and CAAT boxes, and the region was not GC rich. By the use of deletion analysis, in vitro DNase I footprinting, and site-directed mutagenesis, we identified within the proximal promoter region three closely spaced putative binding sites for GATA, sex-determining factor, and steroidogenic factor 1 that are important for testin gene transcription in mouse Sertoli (MSC-1) cells. These cis-acting elements are also present in the conserved Mullerian-inhibiting substance (MIS) proximal promoters, raising a possibility that the transcriptions of testin and MIS genes are controlled by similar mechanisms.
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