We show here that cells dissociated from fetal mouse hypothalamus and cerebral hemispheres can be grown in primary culture in a serum-free medium (SFM). We describe several properties of these cultures and compare them to those in serum-supplemented medium (SSM). The SFM used is a modification of that described for neuroblastoma cells: neuronal survival is improved when 17 β-estradiol is added. Initial events in culture development were similar to those observed in SSM. However, after 1 week, several differences could be noted: in SFM, the proportion of neuron-like cells was increased while the basal glial layer was noticeably reduced, and the neurite network remained less developed than in SSM. These findings demonstrate that the use of SFM permits manipulation of the types and proportions of cells in these primary cultures. This point has been already made. Several neuronal activities were studied. In cultures from both hypothalamus and cerebral hemispheres, thyroliberin (TRH)-immunoreactive cells were visualized by immunohistochemistry, and TRH was radioimmunoassayed in cell extracts and in the medium. In hypothalamic cultures, tyrosine hydroxylase was shown to remain stable for 1 week, and then declined. Glutamic acid decarboxylase disappeared very quickly in vitro, whereas choline acetyltransferase activity increased more rapidly in SFM than in SSM. It is concluded that the use of an SFM for growing normal fetal hypothalamic cells offers a promising model for studying neuroendocrine regulatory mechanisms in culture.

PRL compartments have been studied in normal rat pituitary cells cultured for 6 days. The cells were pulse-labeled for 15 min with 35S-methionine and then chased for 24 h in the absence or presence of cycloheximide (3.6 X 10(-5) M). TRH (30 nM) was introduced into the medium either at the beginning or after increasing durations of chase. The findings were compared with those obtained with GH3B6 cells in similar experimental conditions. Despite the fact that normal PRL cells differ from GH3B6 cells by a large intracellular PRL store, several similarities were found between the two systems: newly synthesized PRL was rapidly and preferentially released in basal conditions, the pattern of the decay of the specific radioactivity of PRL released into the medium suggested the existence of at least two PRL pools with different half-lives: 2.5 h and 22 h, respectively, TRH induced the preferential release of stored PRL synthesized before the pulse, only 20% of the pulse-labeled PRL was released into the medium after 24 h of chase. However, normal PRL cells differed in several respects from GH3B6 cells: the turnover time of the two PRL pools is 8 times greater in normal PRL cells, an asynchrony in the time of appearance of labeled PRL in the medium was observed, suggesting a functional heterogeneity of these cells, at the end of the chase, 40% of the pulse-labeled PRL was lost in the case of normal cells, but not of GH3B6 cells, and this was prevented by cycloheximide, polyacrylamide gel electrophoresis analysis of this labeled immunoprecipitated intracellular material revealed the existence, in addition to the mol wt of 23,000 PRL and the large PRL-like forms (mol wt, 45,000 and 50,000), as observed with GH3B6 cells, of smaller proteins (mol wts, 39,000, 36,000, 20,000, 18,000, 15,000), which might represent degradation products.

PRL compartments were studied in a clonal strain of rat pituitary tumor cells (GH3B6). The cells were pulse-labeled for 10 min with 35S-methionine and then chased for 20 h in the absence or presence of TRH (30 nM) or cycloheximide (3.6 X 10(-5) M), or both. The specific radioactivity (SA) of PRL was followed in the cells and chase medium as a function of chase time and treatments. The transit of labeled and unlabeled PRL has been investigated in cells treated with monensin (1 microM), a drug which is known to perturb the Golgi zone. Newly synthesized PRL was rapidly (15 min of chase) and preferentially released in basal conditions. The pattern of the decay of the SA of PRL released in the medium suggested the existence of at least two PRL pools with different half-lives: 15 min and 3 h, respectively. TRH induced the preferential release of a PRL pool synthesized before the labeling pulse. Monensin decreased the basal release of total radioimmunoassayable PRL without affecting that of the newly synthesized PRL. In contrast, it did not affect the stimulating effect of TRH on the release of unlabeled PRL. These results are in favor of the existence of different intracellular routes for the basal release of PRL (mostly newly synthesized) and the TRH-stimulated release of PRL (mostly stored). Moreover, after 20 h of chase a large fraction (approximately 80%) of the labeled immunoprecipitated material remained intracellularly located and not degraded. This material was not mobilizable by TRH even in the presence of cycloheximide. Polyacrylamide gel electrophoresis analysis revealed that it consisted of large immunoreactive proteins (mol wt, 45,000 and 50,000) instead of mol wt 23,000 PRL which was found in the medium.

The effects of reduced temperatures (20, 15 or 10 degrees C) and brefeldin A (BFA) on prolactin (PRL) secretion in the GH3 rat pituitary cell line have been compared. Both treatments inhibit PRL release to different extents. Ultrastructural immunocytochemistry reveals that, depending on the treatment, PRL is blocked at different steps during its intracellular transit. The temperatures of 20 and 15 degrees C block the PRL transport at one face of the Golgi stacks whereas both the temperature of 10 degrees C and BFA treatment induce an arrest of PRL at the level of the rough endoplasmic reticulum (RER) cisternae. Moreover, exposure to 10 degrees C or BFA induces an accumulation of a specific Golgi membrane antigen in the dilated RER structures. However, although disorganized and no longer definable under BFA treatment, the Golgi apparatus remains visible at 10 degrees C. These two last treatments cause also an increase in the number of partly rough, partly smooth tubular structures tentatively called 'paired cisternae'.

First isolated in porcine pituitary glands the protein 7B2 subsequently proved to be a specific biochemical marker of the secretory granules. Likewise 7B2 was detected in almost all normal and tumoral endocrine tissues. Unexpectedly, several authors failed to demonstrate its presence in rat and human adrenocorticotrophin (ACTH)-secreting cells. In order to definitely establish whether this cell type also produces 7B2 we chose the mouse pituitary corticotroph tumour cell line AtT-20 as a model. Serial dilutions of the mouse culture medium generated displacement curves parallel to that of the standard in a specific 7B2 RIA directed against the human 7B2(23-39) fragment. Under basal secretory conditions immunoreactive 7B2 accumulated in the culture medium in parallel with proopiomelanocortin (POMC) and its fragments N-terminal-joining peptide (NT-JP), joining peptide (JP), beta-lipotropin (beta-LPH), and beta-endorphin (beta-end), although at a much lower (approximately 100-fold) molar concentration. As expected mouse corticotroph cells responded to the stimulatory action of cyclic AMP (3.5 mM) with a preferential increase in the release of POMC end-products, JP and beta-end, which was accompanied by a parallel increase in immunoreactive 7B2 release.

The cytoskeletal organization of the rat pituitary tumor cell line GH3B6 was analysed using immunofluorescence, in basal conditions and after stimulation by thyroliberin (TRH). Under basal conditions, a dense and entangled cytoplasmic microtubule network, a perinuclear cage of cytokeratin fibers, and a diffuse distribution of F-actin were revealed. Short-term stimulation of these cells by TRH induces a first early phase of PRL release (0–2 min), concomitant with a rarefaction of cytoplasmic PRL-containing granules, followed by a second plateau phase (5–30 min), concomitant with modifications of the Golgi zone. We show that TRH induced early and transient modifications in the cytoskeletal distribution during these short periods of stimulation. First, after 2 min of stimulation, small fluorescent tubulin blebs appeared under the plasma membrane. Then, after 5 min they disappeared, and a thin actin network, accentuated by thicker fibers, organized transiently in the cytoplasm. After 30 min, the microtubules and cytokeratin networks had extended throughout the cytoplasm and the actin distribution was diffuse again. So, in this study, we have shown the existence of a parallelism between the redistribution of intracellular PRL compartments and the reorganization of cytoskeletal elements, during exposure to TRH. We could not clearly correlate these modifications with transduction mechanisms involved in TRH action.

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