Significance of carbohydrate residues in human chorionic gonadotropin (hCG) for expression of biological activity

Testosterone is produced in male rats by the action of luteinizing hormone (LH), an anterior pituitary hormone which interacts with receptors on Leydig cells (testes) to activate adenylate cyclase. A most frequently used agonist for LH is human chorionic gonadotropin (hCG), a hormone synthesized by the placenta during the early stages of pregnancy. Like LH, it is a heterodimer consisting of two non-covalently bound subunits designated as alpha and beta. Each subunit of LH/hCG contains oligosaccharide chains of varying sizes and intersugar linkages. In order to determine the significance of carbohydrate residues of hCG in receptor interaction and signal transduction, deglycosylated hCG (DGhCG), free of m r e than 80% of its sugar residues, was prepared by treatment of hCG with anhydrous HF. The effect of this derivative was tested on two different hCG responsive purified testicular interstitial cell fractions as described (Bhalla, V., Flasch, M., Browne, E., Sohal, G., and Sharawy, M. (1987) J. Biol. Chem. =:5322). Fraction I nonsteroidogenic light cells, previously found to bind 1251-labeled hCG with high affinity (Kd 3.0 x lO-lOM), also bound 1251-labeled DG-hCG with a similar high affinity (Kd 6.3 x 1O-l0M) without stimulating testosterone production. The binding of 125I-labeled hCG to light cells was also inhibited by DG-hCG. Fraction IV steroidogenic Leydig cells, which produce CAMP and testosterone in response to hCG ( 2 ng/2 million cells/250 ul) without detectable high affinity binding sites for that hormone, neither bound DG-hCG with high affinity nor sufficiently produced CAMP and testosterone in the presence of DG-hCG alone. With addition of intact hCG (2 ng/2 million cells/250 ul), DG-hCG inhibited hCG stimulated CAMP levels by 70-90 %, although this was insufficient to inhibit testosterone production even at higher DG-hCG concentrations. This observation was contrary to previous studies in which DG-hCG was shown to be an antagonist to hCG action in vitro. From this study we conclude that DG-hCG (a) retains its receptor binding activity in the non-steroidogenic light cells and this high affinity binding is unrelated to steroidogenesis, (b) like hCG, shows an extremely small number of high affinity binding sites on Leydig cells, (c) loses its biological activity as a result of deglycosylation, (d) actions in this study support the concept of two different hCG responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high affinity binding and biological response in the same cell and (e) partially antagonizes the activation of adenylate cyclase but not enough to block testosterone production thus questioning the usefulness of DG-hCG in developing means to control and/or regulate fertility.