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ABSTRACT

Transgenic mice which expressed human IGF-binding protein-3 (hIGFBP-3) were generated by pronuclear injection of an hIGFBP-3 cDNA driven by the mouse metallothionein 1 promoter. Two of the seven founder mice had measurable levels of hIGFBP-3 in the circulation. The serum levels of hIGFBP-3 increased as the mice were bred to homozygosity and were further induced by supplementing the drinking water with 25 mm ZnCl₂. While the birth weight, litter size and body weight of transgenic mice were not significantly different from non-transgenic litter mates or wild-type mice derived from the same genetic background, the transgenic mice demonstrated selective organomegaly. The spleen, liver and heart of mice derived from both founders were significantly heavier compared with organs from non-transgenic mice (P<0·05, P<0·005 and P<0·01 respectively). The weights of the brain and kidney were similar in transgenic and non-transgenic mice. Expression of the transgene was detected in the kidney, small intestine and colon by Northern blot analysis.

Western ligand blotting of serum from transgenic mice did not demonstrate any change in the abundance of the IGFBPs detected by this method. When serum from transgenic mice was incubated with ¹²⁵I-labeled IGF-I and analyzed by Sephacryl S-200 chromatography under neutral conditions a significantly (P<0·05) increased amount of the radioactivity was found in the 140 kDa ternary complex compared with serum from wild-type mice. Immunoreactive hIGFBP-3 was detected in the 140 kDa ternary complex but the majority of immunoreactive hIGFBP-3 present in transgenic mouse serum eluted in later fractions indicating that it was not associated with the acid-labile subunit. These data demonstrate that modest constitutive expression of hIGFBP-3 has a selective effect on organ growth and development. The establishment of these IGFBP-3 transgenic mouse strains may provide useful models to investigate further the physiological role of IGFBP-3.

ABSTRACT

Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common α subunit and a hormone-specific β subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian β subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-β. Using site-directed mutagenesis, each of these two lysines in hCG-β was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine α subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine α subunit bound to hCG-β (Glu²) and to hCG-β (Glu¹⁰⁴), as well as with the control, i.e. the bovine α subunit bound to the hCG-β wild-type subunit. In two in-vitro assays, one a competitive binding assay with ¹²⁵I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant β subunit exhibited bioactivity, but their potencies were lower than that of the bovine α subunit bound to the hCG-β wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-β participate, either directly or indirectly, in receptor binding.

ABSTRACT

IGF-I is an important local regulator of ovarian function, stimulating follicular growth and steroidogenesis in human granulosa cells. However, it is not known whether ovarian IGF-I is derived from the circulating serum pool or from local production. IGF-I peptide has only been detected in human thecal cells and not in granulosa cells. This study has used the sensitive technique of reverse transcription of mRNA followed by PCR amplification (RT/PCR) to examine IGF-I gene expression in human preovulatory granulosa cells.

Granulosa-lutein cell (GLC) samples were obtained by follicular puncture of seven women enrolled in an ovulation induction programme. Treatment had included buserelin acetate, human menopausal gonadotrophin to stimulate follicular growth and human chorionic gonadotrophin to induce ovulation. Total RNA (TRNA), extracted from the GLCs, was amplified by RT/PCR, using combinations of leader and 3′ IGF-I exon-specific primers, to yield four IGF-I gene products: IGF-IA (exons 1, 3, 4, 6), IGF-IB (exons 1, 3, 4, 5), IGF-IA′ (exons 2, 3, 4, 6) and IGF-IB′ (exons 2, 3, 4, 5). As controls from other tissues, an identical procedure was undertaken on TRNA from peripheral blood monocytes and liver. All four mRNAs were expressed in GLCs, monocytes and liver. However the pattern of IGF-I mRNA expression differed between the tissues; in liver and GLCs, the IGF-IA transcript was dominant, but in monocytes the IGF-IA′ species was the most prominent. Quantitative RT/PCR using standardization to the house-keeping gene for glyceraldehyde-3′-phosphate dehydrogenase revealed that IGF-IA mRNA was 300-fold more abundant in liver than GLCs.

This study has indicated that: i) RT/PCR can be used to detect multiple IGF-I mRNA species in small amounts of TRNA from human tissue, ii) the IGF-I gene is expressed at low level in human preovulatory GLCs and iii) there is preferential use of leader exon 1 in human GLCs and leader exon 2 in monocytes. Further studies of IGF-I gene expression by this method in other ovarian cell types and granulosa cells from the developing follicle may help to clarify the local role of IGF-I in the human ovary.

ABSTRACT

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2^b and H-2^d) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22–35 (A10 and All, both IgG2b from H-2^b animals), amino acids 402–415 (A7, IgG2b from H-2^b animals) and amino acids 147–228 (A9, IgG1 from H-2^d animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and All was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and All, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.