Development of specific antibodies to rat insulin-like growth factor-binding proteins (IGFBP-2 to -6): analysis of IGFBP production by rat granulosa cells

• Published in: Endocrinology (Philadelphia) Vol. 132; no. 3; p. 1176
• Main Authors: Liu, X J, Malkowski, M, Guo, Y, Erickson, G F, Shimasaki, S, Ling, N
• Format: Journal Article
• Language: English
• Published: United States 01-03-1993
• Subjects: Amino Acid Sequence; Animals; Antibodies; Antibody Specificity; Blotting, Northern; Blotting, Western; Carrier Proteins - analysis; Carrier Proteins - biosynthesis; Carrier Proteins - immunology; Cells, Cultured; Diethylstilbestrol - pharmacology; Female; Granulosa Cells - drug effects; Granulosa Cells - metabolism; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor Binding Protein 6; Insulin-Like Growth Factor Binding Proteins; Molecular Sequence Data; Molecular Weight; Rats; Rats, Wistar; RNA - genetics; RNA - isolation & purification; RNA Probes; Silicone Elastomers; Somatomedins - metabolism; Steroids - biosynthesis
• ISSN: 0013-7227
• DOI: 10.1210/en.132.3.1176

Six insulin-like growth factor-binding proteins (IGFBPs) have been isolated and their cDNAs cloned in the rat and human species. The next step is to develop antibodies to each IGFBP. Toward this goal, we generated rabbit polyclonal antibodies to rat IGFBP-2, -4, -5, and -6, using synthetic peptide fragments of the IGFBPs. A rat IGFBP-3 polyclonal antibody was prepared in a previous study using the native protein. Western immunoblotting demonstrated that the IGFBP-2, -3, -4, -5, and -6 antibodies were highly specific for their respective antigens. The elicited antisera were used to study IGFBP production in primary cultures of rat granulosa cells grown in serum-free medium for 72 h. Ligand blotting with 125I-labeled IGF-I and IGF-II revealed two bands migrating at 29 and 24 kilodaltons (kDa) in the medium of untreated control cells, whereas no bands were detectable in medium from cells incubated with 100 ng/ml FSH. Western blotting of control medium with all of the IGFBP antibodies revealed that the IGFBP-4 antibody stained two bands at 28 and 24 kDa, and the IGFBP-5 antibody stained two bands at 30 and 29 kDa. By contrast, these bands were absent in medium from FSH-stimulated cells; instead, two lower molecular mass bands of 21.5 and 17.5 kDa were detected with the IGFBP-4 antibody, and a 21-kDa band was seen with the IGFBP-5 antibody. The same 21.5- and 17.5-kDa bands were seen when exogenously added IGFBP-4 was incubated with the FSH-conditioned medium, whereas untreated medium did not degrade the added IGFBP-4. Incubation with the conditioned medium also degraded exogenous IGFBP-5 to yield a 21-kDa band. By contrast, medium from control cells did not degrade the exogenous IGFBP-5. This finding indicates that FSH induced the production of a protease from granulosa cells that degraded IGFBP-4 and -5 in the culture medium. No IGFBPs were detectable in medium from control or FSH-treated cells using the IGFBP-2, -3, and -6 antibodies. Northern blotting analysis of the same control granulosa cell cultures revealed a 2.6-kilobase and a 6.0-kilobase transcript for IGFBP-4 and -5, respectively; however, the IGFBP-4 and -5 mRNAs were essentially undetectable in FSH-treated cell cultures. To determine the effects of the IGFBPs on steroidogenesis, dose-response experiments were performed with IGFBP-4 and -5.