RECOMBINANT RAT LUTEINIZING-HORMONE - PRODUCTION BY CHINESE-HAMSTER OVARY CELLS, PURIFICATION AND FUNCTIONAL-CHARACTERIZATION

Rat recombinant (rec) luteinizing hormone (LH) was produced in Chinese hamster ovary (CHO) cells, to enable studies on LH physiology in this species with homologous hormone. The synthesized hormone was purified , and characterized physico-chemically and biologically in comparison with highly purified preparations of rat pituitary (pit) LH (NIDDK-rLH -I-7 and I-9) and to highly purified urinary (NIH, CR-121) and rec for ms of human chorionic gonadotropin (hCG). The 33 kD molecular mass of rat recLH, as determined by SDS-polyacrylamide gel electrophoresis (SD S-PAGE) and Western blot, was comparable with the 32 kD size of pitLH. In chromatofocusing, the isoforms of rat recLH distributed in the pI range 6.5-7.8, similar to rat pitLH. In receptor binding assays using rat testicular membranes, and physiologic salt concentration, rat recL H displayed a 5-10-fold higher affinity than rat pitLH, but about 100- fold lower affinity than hCG. In contrast, in low salt concentrations the affinities of rat recLH and rechCG to rat LH receptor were rather similar. The differences in potency in the mouse Leydig cell in vitro bioassay were in agreement with the receptor binding data at physiolog ic salt concentration. Neither rat recLH nor pitLH stimulated cAMP pro duction or bound specifically to HEK 293 cells expressing the rec huma n LH receptor. When injected subcutaneously on four consecutive days t o male rats (8.4-33.7 mu g/rat/day) rat recLH did not induce seminal v esicle growth in comparison with a significant effect of human menopau sal gonadotropin (hMG; 12.5-50 IU/rat/day). In contrast, ovulation was induced in 5/6 and 6/6 female rats following single injections of 3.7 5 and 7.5 mu g of rat recLH, respectively, after pretreatment with 10 mu g/kg of a GnRH-antagonist (Org 30850). In conclusion, rat recLH dis plays clearly lower in vivo and in vitro bioactivity than hCG. Neverth eless, it binds effectively to the rat LH receptor (with affinity depe ndent on salt concentration) and is bioactive in the mouse Leydig cell bioassay. This newly synthesized recombinant hormone provides a usefu l tool for further studies on the physiology of LH action in the rat, the most common animal model in reproduction research. (C) 1997 Elsevi er Science Ireland Ltd.