Different molecular and messenger ribonucleic acid forms of insulin-like growth factor-binding protein-3 in the pregnant baboon (Papio anubis)
Authors: Gargosky S E , Giudice L C , Rosenfeld R G , Fazleabas A T 
Published: December 2008

Abstract
The ratio of the serum concentrations of insulin-like growth factors (IGF) to IGF-binding protein (IGFBP)-3 is highly correlated (Baxter & Martin 1986). During pregnancy in the baboon, this ratio is perturbed; serum IGFBP-3 concentrations increase 10-fold, yet IGF-I levels are unaltered and IGF-II is increased only 2-fold (Giudice et al. 1993). The aims of this study were to determine the molecular distribution of IGFBP-3 and to identify the tissue source and form(s) of IGFBP-3 during pregnancy in the baboon. Serum of non-pregnant and pregnant baboons, and conditioned media of decidua and placental explant cultures were characterized using neutral size-exclusion chromatography in combination with Western ligand blot, Western immunoblot, an IGFBP-3 radioimmunoassay (RIA) and an IGFBP-3 protease assay. Localization of immunoreactive IGFBP-3 was determined by immunocytochemistry, and expression of IGFBP-3 mRNA in the placental and decidual explants was examined by Northern blot analysis.
RIA confirmed that immunoreactive IGFBP-3 is increased 10-fold in pregnancy serum compared with non-pregnancy serum. Size-exclusion chromatography combined with an IGFBP-3 RIA revealed that, unlike non-pregnancy serum where 70% of the immunoreactive IGFBP-3 elutes in the 150 kDa ternary complex, equal amounts of immunoreactive IGFBP-3 were measured in pregnancy serum in the ≤150 and 60 kDa IGFBP regions. Western analysis revealed that non-pregnancy serum contained predominantly a 45–40 kDa IGFBP-3 doublet and a 28 kDa immunoreactive form of IGFBP-3, while in pregnancy serum IGFBP-3 existed as a 45–40 kDa doublet, as well as 26–28 kDa and 18 kDa immunoreactive forms. These alternative forms of IGFBP-3 were not attributable to detectable IGFBP-3 protease activity.
To identify the source of the increased serum levels of IGFBP-3 during pregnancy, we examined explant culture media of baboon decidua and placenta. Size-exclusion chromatography combined with RIA and Western analysis revealed that: (1) more immunoreactive IGFBP-3 was produced by decidual cultures than by placental explants, but less 45–40 kDa IGFBP-3 was present in decidua; (2) the immunoreactive forms of IGFBP-3 detected in decidua were similar to those found in maternal serum; (3) placental explants secreted only 45–40 kDa IGFBP-3 in culture. IGFBP-3 was immunohistochemically localized to the cells of placental villi, and to the perinuclear region of the decidual cells and staining for IGFBP-3 was more intense in the decidua than in the placenta.
Northern analysis of the explant cultures revealed two IGFBP-3 mRNA transcripts of 2·4 and 1·7 kb in both decidua and placenta which may account for the different immunoreactive forms of IGFBP-3 detected in the baboon. However analysis of non-pregnancy liver also revealed two IGFBP-3 transcripts of 2·4 and 1·7 kb. These data suggest that the two transcripts are not solely pregnancy-associated and levels of protein may be the reason for detection of multiple immunoreactive IGFBP-3 fragments in pregnancy.
Journal of Endocrinology (1995) 147, 449–461