Human serum LH inhibitor(s): behaviour and contribution to in vitro bioassay of LH using dispersed mouse Leydig cells

Abstract

The present study aimed at investigating the nature and causes of non-parallelism in testosterone responses to serial dilutions of peripheral serum and standard LH preparations in the mouse Leydig cell in vitro bioassay of LH. Immunoadsorption with monoclonal antibody to the beta-subunit of LH was used to obtain LH-free serum; the procedure removed more than 98% of the immunoassayable LH. When a constant amount of the LH-free serum was added to standard dilutions, the bioassay dose-response curves to serum dilutions, the standards became parallel, i.e. the well-known source of error of this assay system was eliminated. When standard curves prepared in medium and LH-free serum (final concentration 10%) were compared, no effect of the serum was found on basal cAMP and testosterone production. However, the LH-stimulated testosterone and cAMP production were suppressed by serum by a rather constant factor of 40%. Mild heating (60 degrees C, 15 min) or treatment with dextran-coated charcoal, but not other extraction, was able to eliminate the inhibitory activity of the LH-free serum. Binding studies demonstrated that [125I]HCG interaction with mouse Leydig cell homogenates was inhibited by LH-free serum in a fashion indicative of reduced LH receptor number, but not of reduced binding affinity. In conclusion, these data show that human serum contains LH inhibitor(s) which affect the LH-receptor interaction and LH stimulated testosterone production in mouse Leydig cell in vitro. The effect is marked in serum concentration over 1.5% and it shows only minor variation between individual sera. This source of error can be effectively removed from the LH in vitro bioassay by using LH-free serum for preparation of dilutions of LH standards.