Amniotic fluid (AF) collected from ewes and goats at mid gestation displayed mitogenic activity in mouse fibroblasts. Upon fractionation of this material by size exclusion chromatography, the mitogenic activity was resolved into two peaks, whose activity was inhibited by an anti-IGF type 1 receptor blocking antibody. One of the peaks contained IGF-I and IGF-II (mature form), whereas the other contained high M r precursor forms of IGF-II. The presence in this latter fraction of IGF-binding proteins (IGFBP) suggests that the AF IGFBPs do not efficiently inhibit the mitogenic activity of the high M r forms of IGF-II. In agreement with this conclusion, exogenous IGFBP-1 failed to affect this activity. Analysis of IGF-II in sheep AF showed that the AF concentrations of both forms of IGF-II increased dramatically from mid pregnancy until 106-120 days of gestation, and fell thereafter. The amniotic IGFBPs followed a similar evolution. High M r forms of IGF-II were also found in human AF, with a pattern of electrophoretic migration different from that of sheep. We suggest that the precursor forms of IGF-II may play an important role in foetal development.

Growth hormone releasing factor (GHRH) has been described in the rat, mouse and human placentae. This study reports the presence of an immunoreactive GHRH activity (IR-GHRH) in the ovine placenta. This activity was detected by radioimmunoassay from day 50 (D50) until the end of pregnancy. Higher IR-GHRH concentration in placental tissue was observed on days 100 (543 +/- 123 pg/g) and 140 (550 +/- 62 pg/g) and, when compared with the GHRH content of the ovine hypothalamus (1.2 ng/hypothalamus), represents a considerable amount of GHRH per placenta (a mean of 200 ng). Perifused placenta explants released IR-GHRH in vitro at a mean rate of 200 pg/g/h. Depolarization by 55 mM KCl increased the IR-GHRH concentration of the perifusion media 1.7 times over basal values. The elution position of GHRH immunoreactivity in the gel filtration chromatography profiles was the same for placenta and hypothalamus extracts and lay very near to the molecular weight of bovine GHRH. Northern blot hybridization analysis revealed the existence of a placental transcript whose size (0.75 kb) was comparable to the size of the ovine hypothalamus and rat placenta GHRH transcripts. Hybridization signal was observed at each stage studied from D50 until D120 of pregnancy. This study demonstrated the existence of a IR-GHRH peptide in the ovine placenta.

Amniotic fluid collected from ewes on various days of gestation was examined for the presence of insulin-like growth factor (IGF) binding proteins. IGF-binding proteins with a molecular mass of 40-45 kDa appeared at day 41 of gestation. The level of these major IGF-binding proteins increased during pregnancy and reached a maximum at day 106. Smaller IGF-binding molecules with an approximate molecular mass of 35 kDa and 25 kDa appeared at day 90, also reaching a concentration peak at day 106. The mitogenic activity of sheep amniotic fluid after chromatography on Sephadex G-50 was separated into two peaks. The peak having lower molecular mass corresponded to an elution profile of 125I-IGF-I. The first peak, having higher molecular mass, was eluted immediately after the void volume of column. Electrophoresis and ligand blotting showed that proteins in the first peak had similar properties as IGF-binding proteins.