Female Reproductive Tract

A Single-Chain Tetradomain Glycoprotein Hormone Analog Elicits Multiple Hormone Activities In Vivo

Abstract
We previously demonstrated that genetically linking one or more of the glycoprotein hormone-specific subunit genes to the common subunit resulted in single chain analogs that were bioactive in vitro. The ability of such large structures to bind their cognate receptors with high affinity supported the hypothesis that extensive flexibility exists between the ligand and receptor to establish a functional complex. To further characterize the extent of this conformational flexibility, we engineered a single chain analog that consists of sequentially linked TSH, FSH and CG subunits to the subunit and expressed this chimera in transfected CHO cells. Because the four subunits are genetically linked and expressed as a single chain, this analog presumably lacks significant native structural features of the individual heterodimers. However, it exhibited FSH, CG and TSH activities in vitro. Here, we test whether this non-native structure would be stable in vivo, and thus biologically active. Using a variety of bioassay protocols, we demonstrate that the analog elicits multi-hormone activities when injected in vivo. First, treatment with the analog caused increases in ovarian and uterine weights and resulted in elevated serum estradiol. Second, the analog stimulated ovarian follicle growth and pharmacologically rescued in vivo FSH deficiency similar to recombinant hFSH or PMSG as confirmed by induction of aromatase in the ovaries of FSH knockout mice. Third, in a superovulation protocol, when primed with PMSG, the analog elicited a dose-dependent ovulatory response comparable to that by native heterodimeric hCG. Finally, the analog stimulated thyroxin production in hypothyroid mice similar to the pituitary--derived human TSH standard. Based on these data, we conclude that a single chain tetradomain glycoprotein hormone analog, despite its presumed altered conformation is stable and biologically active in vivo. Our results establish the permissiveness and conformational plasticity with which the glycoprotein hormones are recognized in vivo by their target cell receptors.

Key words: Pituitary • Follicle-stimulating hormone • Luteinizing hormone • Ovulation • Thyroid-stimulating hormone

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