A method has been described for the purification of ovine placental lactogen (oPL) involving the use of freshly obtained sheep foetal cotyledons. Tissue was extracted with 0·1 m-ammonium bicarbonate and the supernatant fraction, adjusted to pH 7, was brought to 60% saturation with ammonium sulphate. The resulting precipitate was then subjected to a sequence of chromatographic steps using columns of Sephadex G-100 and carboxymethylcellulose. During each stage of the purification, the lactogenic activity was monitored with a pregnant rabbit mammary gland radioreceptor assay. The yield of oPL corresponded to 8 mg/kg wet foetal tissue and the oPL possessed lactogenic activity equivalent to 1 mg ovine prolactin/mg protein and GH-like activity equivalent to 0·8 mg human GH/mg protein. The biological activity of oPL was confirmed using a rabbit intraductal mammary gland assay in vivo.

After polyacrylamide gel electrophoresis at pH 8·9, oPL was resolved into one major band (isoelectric point 8·2–8·4) and four minor components, which were thought to be deamidation products of oPL. Microimmunoelectrophoresis and immunodiffusion studies confirmed that the preparation of oPL was free from serum protein contaminants.

SUMMARY

The rate of clearance from the circulation and uptake into tissues of radioactive label was studied after i.v. injection of ¹²⁵I-labelled human placental lactogen (HPL) into rats at various stages of pregnancy. The half-life was obtained for the disappearance of the trichloroacetic acid-precipitable material from the plasma. The half-life, t _½(S), calculated over the first 5 min after injection of the hormone was 5·4 ± 1·1 (s.d.) min, while a half-life, t _½(L), of 27·9 ± 2·3 min was obtained from the decay period of 15–35 min.

In the non-pregnant and pregnant rat the highest ratio of the radioactivity in an organ to that in the blood was 12–14:1 in the kidney. That the kidney is mainly involved in the uptake of exogenous HPL is further confirmed by the application of the histochemical immunoperoxidase technique. Human placental lactogen was localized in the cells of the proximal tubules of the cortex and to a lesser extent in the tubular lumen and the tubules of the medulla region.

Uptake of HPL in vivo also occurs in the mammary gland tissue of the post-partum rat and reaches a maximum uptake between 15 and 30 min after injection of the hormone. Furthermore, specific uptake of HPL was observed on the alveolar cell membranes after the incubation of paraffin-embedded sections of formalin-fixed mammary gland and subsequent treatment by the peroxidase-labelled antibody method.

These findings support the work of others who have demonstrated the presence of specific membrane receptors in the mammary gland for hormones with prolactin-like activity.

Obesity and its related disorders are the most prevalent health problems in the Western world. Using the paradigm of fetal programming we developed a rodent model which displays the phenotype of obesity and metabolic disorders commonly observed in human populations. We apply maternal undernutrition throughout gestation, generating a nutrient-deprived intrauterine environment to induce fetal programming. Maternal undernutrition results in fetal growth retardation and in significantly decreased body weight at birth. Programmed offspring develop hyperphagia, obesity, hypertension, hyperleptinemia and hyperinsulinism during adult life and postnatal hypercaloric nutrition amplifies the metabolic abnormalities induced by fetal programming. The adipoinsular axis has been proposed as a primary candidate for linking the status of body fat mass to the function of the pancreatic beta-cells. We therefore investigated the relationship between circulating plasma concentrations of leptin and insulin and immunoreactivity in the endocrine pancreas for leptin and leptin receptor (OB-R) in genetically normal rats that were programmed to become obese during adult life. Virgin Wistar rats were time mated and randomly assigned to receive food either available ad libitum (AD group) or at 30% of the ad libitum available intake (UN group). Offspring from UN mothers were significantly smaller at birth than AD offspring (AD 6.13+/-0.04 g, UN 4.02+/-0.03 g, P<0.001). At weaning, offspring were assigned to one of two diets (a standard control diet or a hypercaloric diet consisting of 30% fat) for the remainder of the study. At the time of death (125 days of age), UN offspring had elevated (P<0.005) fasting plasma insulin (AD control 1.417+/-0.15 ng/ml, UN control 2.493+/-0.33 ng/ml, AD hypercaloric 1.70+/-0.17 ng/ml, UN hypercaloric 2.608+/-0.41 ng/ml) and leptin (AD control 8.8+/-1.6 ng/ml, UN control 14.32+/-1.9 ng/ml, AD hypercaloric 15.11+/-1.8 ng/ml, UN hypercaloric 30.18+/-5.3 ng/ml) concentrations, which were further increased (P<0.05) by postnatal hypercaloric nutrition. The elevated plasma insulin and leptin concentrations were paralleled by increased immunolabeling for leptin in the peripheral cells of the pancreatic islets. Dual immunofluorescence histochemistry for somatostatin and leptin revealed that leptin was co-localized in the pancreatic delta-cells. OB-R immunoreactivity was evenly distributed throughout the pancreatic islets and was not changed by programming nor hypercaloric nutrition. Our data suggest that reduced substrate supply during fetal development can trigger permanent dysregulation of the adipoinsular feedback system leading to hyperleptinemia, hyperinsulinism and compensatory leptin production by pancreatic delta-cells in a further attempt to reduce insulin hypersecretion in the progression to adipogenic diabetes.

Limited information is available on the role of MAPK phosphatase 1 (MKP1) signaling in osteoblasts. We have recently reported distinct roles for MKP1 during osteoblast proliferation, differentiation, and skeletal responsiveness to parathyroid hormone (PTH). As MKP1 regulates the phosphorylation status of MAPKs, we investigated the involvement of P-ERK and P-p38 MAPKs in MKP1 knockout (KO) early and mature osteoblasts with respect to mineralization and PTH response. Calvarial osteoblasts from 9–14-week-old WT and MKP1 KO male and female mice were examined. Western blot analysis revealed downregulation and sustained expressions of P-ERK and P-p38 with PTH treatment in differentiated osteoblasts derived from KO males and females respectively. Exposure of early osteoblasts to p38 inhibitor, SB203580 (S), markedly inhibited mineralization in WT and KO osteoblasts from both genders as determined by von Kossa assay. In osteoblasts from males, ERK inhibitor U0126 (U), not p38 inhibitor (S), prevented the inhibitory effects of PTH on mineralization in early or mature osteoblasts. In osteoblasts from KO females, PTH sustained mineralization in early osteoblasts and decreased mineralization in mature cells. This effect of PTH was attenuated by S in early osteoblasts and by U in mature KO cells. Changes in matrix Gla protein expression with PTH in KO osteoblasts did not correlate with mineralization, indicative of MKP1-dependent additional mechanisms essential for PTH action on osteoblast mineralization. We conclude that PTH regulation of osteoblast mineralization in female mice is maturation stage specific and involves MKP1 modulation of P-ERK and P-p38 MAPKs.