Porcine ovarian granulosa cells secrete insulin-like growth factor-binding proteins-4 and -5 and express their messenger ribonucleic acids: regulation by follicle-stimulating hormone and insulin-like growth factor-1.
Using ligand blotting, Western immunoblotting, and Northern analysis, we have characterized the insulin-like growth factor (IGF)-binding proteins (IGFBPs) produced by cultures of porcine granulosa cells. Ligand blot analysis of conditioned medium from untreated cultures of moderately differentiated granulosa cells (MDGCs; from 4-6-mm follicles) revealed mainly IGF-binding activity associated with IGFBP-2 (34 kDa) and IGFBP-3 (40/44-kDa doublet), which have previously been identified and characterized. In addition, these cultures secreted 30- and 22-kDa forms under some circumstances. The identification and regulation of these IGFBPs of lower molecular mass were the focus of the current studies. Treatment of these MDGCs with IGF-I dramatically stimulated the production (to a detectable level) of a 30-kDa IGFBP that was identified by immunoblotting with antiserum to IGFBP-5 but not antisera to IGFBP-1, -2, -3, -4, or -6. Production of IGFBP-5 was attenuated by concurrent treatment with FSH. IGFBP-5 mRNA in these cultures was correspondingly stimulated by IGF-I but unaffected by FSH. FSH increased the level of a minor 22-kDa IGFBP. Messenger RNAs for IGFBP-1, -4, and -6 were also examined but only IGFBP-4 mRNA was detectable, suggesting that the 22-kDa band was IGFBP-4. These results were compared to those in cultures of immature granulosa cells from 1-3-mm follicles, in which 22- and 30-kDa IGFBPs were readily detectable. An antiserum to IGFBP-4 precipitated the 22- and 30-kDa bands whereas deglycosylation shifted the 30-kDa IGFBP to 22 kDa, suggesting that both these bands represent glycosylation variants of IGFBP-4.(ABSTRACT TRUNCATED AT 250 WORDS)
Grimes
R W
RW
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Barber
J A
JA
Shimasaki
S
S
Ling
N
N
Hammond
J M
JM
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D015870:Gene Expression; D006031:Glycosylation; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008970:Molecular Weight; D012333:RNA, Messenger / Q000378:metabolism*; D013552:Swine
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone