Mechanisms underlying the diminished sensitivity to prolactin negative feedback during lactation: Reduced STAT5 signaling and up-regulation of cytokine-inducible SH2 domain-containing protein (CIS) expression in tuberoinfundibular dopaminergic neurons

Hyperprolactinaemia during lactation is a consequence of the sucking stimulus and in part due to reduced prolactin (PRL) negative feedback. To date, the mechanisms involved in this diminished sensitivity to PRL feedback are unknown but may involve changes in PRL signal transduction within tuberoinfundibular dopaminergic (TIDA) neurons. Therefore, we investigated signal transducers and activators of transcription (STAT) 5 signaling in the TIDA neurons of lactating rats. Dual-label confocal immunofluorescence studies were used to determine the intracellular distribution of STAT5 within TIDA neurons in the dorsomedial arcuate nucleus. In lactating rats with pups removed for 16 h, injection of ovine PRL significantly (P < 0.05) increased the STAT5 nuclear/cytoplasmic ratio compared with vehicle-treated mothers. In contrast, ovine PRL injection did not increase the STAT5 nuclear/cytoplasmic ratio in lactating mothers with pups, demonstrating that PRL signal transduction through STAT5 is reduced in TIDA neurons in the presence of pups. To investigate possible mechanisms involved in reduced PRL signaling, we examined the expression of suppressors of cytokine signaling (SOCS) proteins. Northern analysis on whole hypothalamus showed that CIS (cytokine-inducible SH2 domain-containing protein), but not SOCS1 or SOCS3, mRNA expression was significantly (P < 0.01) up-regulated in suckled lactating rats. Semiquantitative RT-PCR on arcuate nucleus micropunches also showed up-regulation of CIS transcripts. Immunofluorescence studies demonstrated that CIS is expressed in all TIDA neurons in the dorsomedial arcuate nucleus, and the intensity of CIS staining in these neurons is significantly (P < 0.05) increased in lactating rats with sucking pups. Together, these results support the hypothesis that loss of sensitivity to PRL-negative feedback during lactation is a result of increased CIS expression in TIDA neurons

Purification, partial characterization, and radioimmunoassay of prolactin and growth hormone from the Bennett's wallaby

Bennett's wallaby prolactin (wPRL) and growth hormone (wGH) were purified from an aqueous extract of pituitary glands. The extract from 202 glands (6.5 g wet wt) was processed by gel filtration on Sephadex G-100, gel filtration on Sephadex G-100 SF, and then anion-exchange chromatography on DEAE-Sepharose CL-6B. The yields of wPRL and wGH were 5.2 and 15.7 mg, respectively. Since recovery of wPRL from the anion exchange column was 10%, anion exchange was performed in the presence of 20% acetonitrile in a subsequent purification. Recovery from this column was markedly increased to 42%. The purified hormones each gave a single vand on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight under reducing conditions of 21,000 and 23,000 for GH and PRL, respectively. Each hormone was positively identified by its N-terminal amino acid sequence, which showed high sequence identity with the equivalent eutherian hormone. Semianalytical gel filtration of purified hormone was used to demonstrate that each hormone remained as a monomer in aqueous solution. Each purified hormone was tested in the heterologous PRL radioimmunoassay (RIA) which has been used in many earlier studies to measure marsupial PRL. Highly purified wPRL was less potent than ovine prolactin (5.3 compared with 1.5 ng/ml at 50% displacement) and the cross-reaction of wGH wa