IMMUNIZATION OF MALE RABBITS WITH SHEEP LUTEAL RECEPTOR TO LH RESULTSIN PRODUCTION OF ANTIBODIES EXHIBITING HORMONE-AGONISTIC AND HORMONE-ANTAGONISTIC ACTIVITIES

Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAG E and ligand blotting) from sheep luteal membrane using human CG (hCG) -Sepharose affinity chromatography, were raised in three adult male ra bbits (R-I, R-II and R-III). Each of the rabbits received 20-30 mu g o i the purified receptor in Freund's complete adjuvant at a time. Prima ry immunization was followed by booster injection at intervals. Produc tion of receptor antibodies was monitored by (1) determining the dilut ion of the serum (IgG fraction) that could specifically bind 50% of I- 125-LH/CG-R added and (2) analysing sera for any chance in testosteron e levels. Following primary immunization and the first booster, all th ree rabbits exhibited a 2.5- to 6.0-fold increase in serum testosteron e over basal levels and this effect was spread over a period of time ( similar to 40 days) coinciding with the rise and fall of receptor anti bodies. The maximal antibody titre (ED(50)) produced at this time rang ed from 1:350 to 1:100 to below detectable limits for R-I, R-II and R- III respectively. Subsequent immunizations followed by the second boos ter resulted in a substantial increase in antibody titre (ED(50) of 1: 5000) in R-I, but this was not accompanied by any change in serum test osterone over preimmune levels, suggesting that with the progress of i mmunization the character of the antibody produced had also changed. T wo pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further expe riments. IgG isolated from bleed I but not from bleed II antiserum sho wed a dose-dependent stimulation of testosterone production by mouse L eydig cells in vitro, thus confirming the in vivo hormone-mimicking ac tivity antibodies generated during the early immunization phase. The I gG fractions from both bleeds were, however, capable of inhibiting (1) I-125-hCG binding to crude sheep luteal membrane (EC(50) of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimu lated testosterone production by mouse Leydig cells in vitro, indicati ng the presence oi antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The: fact that bleed I-stimulated testosterone production could be inhibited ill a dose-dep endent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intr insic to the bleed I antibody. The receptor antibody (bleed II) was al so capable of blocking LH action ill vivo, as rabbits passively (for 2 4 h with LH/CG-R antiserum) as well as actively (for 130 days) immuniz ed against LH/CG-R failed to respond to a bolus injection of LH (50 mu g). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to im munization with hole LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities.