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 forms of human chorionic gonadotropin (hCG). The 33 kD molecular mass of rat recLH, as determined by SDS-polyacrylamide gel electrophoresis (SDS-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 recLH 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 physiologic salt concentration. Neither rat recLH nor pitLH stimulated cAMP production or bound specifically to HEK 293 cells expressing the rec human LH receptor. When injected subcutaneously on four consecutive days to male rats (8.4-33.7 microg/rat/day) rat recLH did not induce seminal vesicle growth in comparison with a significant effect of human menopausal 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 3.75 and 7.5 microg of rat recLH, respectively, after pretreatment with 10 microg/kg of a GnRH-antagonist (Org 30850). In conclusion, rat recLH displays clearly lower in vivo and in vitro bioactivity than hCG. Nevertheless, it binds effectively to the rat LH receptor (with affinity dependent on salt concentration) and is bioactive in the mouse Leydig cell bioassay. This newly synthesized recombinant hormone provides a useful tool for further studies on the physiology of LH action in the rat, the most common animal model in reproduction research.
Hakola
K
K
Department of Physiology, University of Turku, Finland. kati.hakola@utu.fi
Boogaart
P V
PV
Mulders
J
J
de Leeuw
R
R
Schoonen
W
W
Heyst
J V
JV
Swolfs
A
A
Casteren
J V
JV
Huhtaniemi
I
I
Kloosterboer
H
H
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003001:Cloning, Molecular; D006224:Cricetinae; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000494:pharmacology; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D007986:Luteinizing Hormone / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology*; D008297:Male; D051379:Mice; D010060:Ovulation / Q000187:drug effects; D010062:Ovulation Induction; D010902:Pituitary Gland / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011974:Receptors, LH / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D013737:Testis / Q000378:metabolism; D013739:Testosterone / Q000096:biosynthesis; D014162:Transfection
D006063:Chorionic Gonadotropin; D006727:Hormone Antagonists; D011974:Receptors, LH; D011994:Recombinant Proteins; C067622:Org 30850; D007987:Gonadotropin-Releasing Hormone; D013739:Testosterone; D007986:Luteinizing Hormone