A Growth Hormone Binding Factor in the Serum of Pregnant Mice

Abstract

Using a radioreceptor assay (RRA) an apparent growth hormone-like activity (GH-LA) was first detected in the serum of Swiss Webster mice at mid-pregnancy with peak concentrations appearing 1-2 days before parturition. Hysterectomy at day 17 of pregnancy was followed by a transient increase in apparent GH-LA. In the RRA, displacement of human [h] [125I]iodoGH from the receptor by serial dilutions of serum from pregnant mice paralleled that of purified hGH standards. After gel filtration of a sample of pregnant serum on a Sephadex G-100 column the apparent GHLA determined by RRA was found in fractions with a relative elution volume (Ve/Vo) of 1.2. Moreover, when a sample of serum from pregnant mice and h[125I]iodoGH were separated on a similar column, more than 75% of the labeled hormone also eluted with a relative elution volume (Ve/Vo) of 1.2 rather than 2.0 which was found when h[125I]iodoGH alone or with serum from nonpregnant mice was fractionated on the same column. In these fractions more than 65% of the h[125I]iodoGH was precipitable by excess antiserum to hGH. Upon addition of increasing amounts of unlabeled hGH to the mixture of h[125I]iodoGH and serum from pregnant mice a progressive decrease in the amount of h[125I]iodoGH recovered in fractions with a Ve/Vo of 1.2 was found. Bovine GH, but not ovine prolactin (oPRL) caused a similar displacement. When a plasma sample was obtained from pregnant mice after injection of 10 .mu.g of unlabeled hGH and was fractionated in a similar manner, immunoreactive hGH was recovered in eluates corresponding to a Ve/Vo of 1.2. After injection of hGH into nonpregnant mice, immunoreactive hGH was recovered in fractions eluting with a Ve/Vo of 2.0. Following the same procedure, both unlabeled oPRL and [125I]iodoPRL consistently eluted in fractions with Ve/Vo = 2.0. In vivo experiments showed that the half-time disappearance rate of h[125I]iodoGH was significantly longer in the pregnant than in the non-pregnant mouse. When fresh serum obtained from mice on day 18 of pregnancy was fractionated, the endogenous immunoreactive mouse GH was recovered in fractions which also contained the GH-binding factor. Immunoreactive mouse GH from pituitary extracts was shown to bind to this factor on mixing with serum from pregnant mice. Serum concentration of immunoreactive mouse GH appeared to increase during pregnancy. In vitro studies indicated that the GH binding factor competitively inhibited the binding of h[125I]iodoGH to rabbit liver in the standard RRA leading to falsely high estimates of GH-LA in the serum of pregnant mice. Assuming a monovalent interaction between GH and this binding factor, the MW of the latter is estimated to be 60,000 daltons. Aqueous extracts of 11 tissues, including the placenta, failed to reveal the tissue source of the GH-binding factor.