Recombinant rat follicle-stimulating hormone; production by Chinese hamster ovary cells, purification and functional characterization.
In order to obtain homologous follicle-stimulating hormone (FSH) for in vivo and in vitro studies in the rat, rat recombinant (rec) FSH was produced in Chinese Hamster Ovary (CHO) cells. The synthesized rat recFSH was purified and subjected to physico-chemical and biological characterization. including a comparison with two rat pituitary (pit) and reference preparations (NIDDK-rFSH I-8 and NIDDK-rFSH-RP2) as well as with human recFSH (Org 32489). The molecular masses of rat recFSH and human recFSH were determined by SDS-polyacrylamide (SDS-PAGE) and were found to be similar, about 40 kD. The pI distribution of rat recFSH is similar to rat pitFSH, and slightly more acidic than human recFSH (3.6-5.6 vs 3.9-5.5, respectively) as determined by isoelectric focussing in immobilized pH gradients. Rat recFSH displayed dose-response curves parallel and in the same dose range as the rat pitFSH in receptor binding and in vitro bioassays. However, the in vivo activities of rat recFSH and rat pitFSH were 8824 and 3051 IU/mg, respectively, determined by the Steelman Pohley assay. Rat (pit and rec) and human FSH are very different. Human recFSH bound to both calf testicular membranes and CHO cells expressing the human FSH receptor (CHO hFSH-R) with about 10-fold higher affinity (Ka) than pituitary and recombinant rat FSH. In in vitro bioassays with immature rat Sertoli cells and CHO hFSH-R cells human recFSH was also about 10-fold more potent than the rat FSH preparations. In the in vitro bioassays with immature rat granulosa cells the difference was about 5-10-fold. These studies indicate that the receptor binding and in vitro activities of rat pitFSH and rat recFSH are similar. The differences in in vivo activity are probably due to the differences in glycosylation. The biological behaviour of rat FSH (pit and rec) is different from that of human FSH. Therefore, if the rat is used as a model for physiology of gonadotropic action, the results may be greatly influenced by the type (species) of hormone preparation used. The availability of homologous hormone preparations is therefore crucial.
Hakola
K
K
Department of Physiology, University of Turku, Finland. kati.hakola@utu.fi
Van der Boogaart
P
P
Mulders
J
J
de Leeuw
R
R
Schoonen
W
W
Van Heyst
J
J
Swolfs
A
A
Van Casteren
J
J
Huhtaniemi
I
I
Kloosterboer
H
H
D000818:Animals; D001681:Biological Assay; D016466:CHO Cells / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D005640:Follicle Stimulating Hormone / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D006801:Humans; D007525:Isoelectric Focusing; D051379:Mice; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone