D. Grouselle

Differentiation of Fetal Mouse Hypothalamic Cells in Serum-Free Medium

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 beta-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.

Release of immunoreactive TRH in serum-free cultures of mouse hypothalamic cells

Serum-free cultures of mouse hypothalamic cells were used as a model for studying TRH (thyroliberin) secretion in vitro. Supplementation of the culture medium with triiodothyronine, corticosterone and polyunsaturated fatty acids is necessary to obtain a substantial release capacity of TRH neurons. Under these conditions depolarization of the cells with 60 mM K+ results in a calcium-dependent release of immunoreactive TRH.

An Enzyme Immunoassay for Rat Prolactin: Application to the Determination of Plasma Levels

Pure acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus has been covalently coupled to rat prolactin using the heterobifunctional reagent: N-succinimidyl-4 (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). This conjugate was used as a tracer in a competitive enzyme immunoassay using a rabbit antiserum, raised against rat prolactin, as first antibody. The assay was performed in 96-well microtiter plates coated with a mouse monoclonal anti-rabbit immunoglobulin antibody. This second antibody solid phase ensured separation of bound and free moieties of the tracer during the specific immunoreaction. The total reaction volume was 150 microliters. Each component (tracer, antiserum and standard) was added in a volume of 50 microliters. The sensitivity of the assay was good since calculation indicated a detection threshold of 25 pg (0.5 ng/ml) and a B/Bo 50% value of 220 pg (4.4 ng/ml). Intra-assay variation was better than 10% over a wide range (135 to 2500 pg) with an optimum of 4% at 300 pg. The inter-assay coefficient of variation was less than 15% for rat plasma samples in the concentration range of 8 to 1000 ng/ml. The good parallelism observed between the standard curve and sample dilution curves, and recovery experiments, indicated that direct assay is possible. This was confirmed by molecular sieve fractionation of plasma samples. The reliability of the assay was confirmed by good correlation with conventional radioimmunoassay (r = 0.996, slope = 0.978).

Effect of thyreotrope-releasing hormone (TRH) on prolactin turnover in culture

A kinetic study of the influence of thyreotrope-releasing hormone (TRH) on prolactin turnover and synthesis by a new rat pituitary prolactin cell line (SD1) has been performed by means of pulse-chase experiments. After a 10-min [3H]leucine pulse, the chase was carried out in the presence or absence of TRH (54 nM), cycloheximide (3.6 X 10(-5)M) and/or [14C]-proline. The prolactin content of the cells in the medium was estimated using a radioimmunoassay technique. The specific radioactivity of prolactin in the medium was estimated after its isolation by disc gel electrophoresis. This kinetic study demonstrated, firstly, a rapid intracellular transit of newly synthesized prolactin (15 + 10 min or less); secondly, the existence of at least two intracellular prolactin pools; thirdly, a rapid effect of TRH on release of stored prolactin, which is independent of de novo protein synthesis, and fourthly, a delayed stimulating effect of TRH on prolactin synthesis.

A radioimmunoassay for thyroliberin (TRH). comparison with a TRH radioreceptor assay

A radioimmunoassay (RIA) for thyroliberin (TRH) is described which differs on several points from the previously published ones. A TRH antiserum was raised in rabbits against TRH coupled to a seed-globulin from the sun-flower; [(3)H]TRH was used as radioligand and precipitation of the antibody-antigen complex was achieved with polyethylene glycol (PEG). The RIA sensitivity is around 4 pg. It is compared to a radioreceptor assay (RRA) which has been previously settled [5] using a pituitary cell line in culture (GH3 clone) (sensitivity: 200 pg). No cross-reactivity is seen in either assay with various hormones or TRH analogues, except with some methylated derivatives. Both techniques led to comparable values when biological extracts were assayed, provided they were partially purified.

Axons Containing a Prolactin-Like Peptide Project into the Perivascular Layer of the Median Eminence: An Immunocytochemical Light and Electron Microscope Study in Adult and Infant Rats

A light and electron microscopic immunocytochemical study was undertaken to explore the fine structural organization of prolactin-immunoreactive axons in the rat median eminence. In adult intact males and females and in hypophysectomized females, light microscopic immunocytochemical labeling of the mediobasal hypothalamus revealed a marked concentration of prolactin-like immunoreactive fibers in the perivascular layer throughout the median eminence and the hypophysial stalk. At the electron microscopic level, immunostaining was associated with typical neurosecretory axons located either in the palisade layer where they displayed numerous contacts with tanycyte processes, or in the perivascular layer where they frequently contacted the perivascular space. Within the labeled axonal profiles, immunostaining was essentially located on secretory granules, 90-120 nm in diameter, whereas the microvesicles accumulated in some perivascular profiles constantly remained unlabeled. These data strongly suggest that most prolactin-immunoreactive axons of the median eminence release their content into the hypophysial portal vessels. In 1-day-old infant rats, intensely prolactin-like immunoreactive fibers were similarly localized in the most external layer of the median eminence, in which, contrary to adult animals, very slight if any tyrosine-hydroxylase-immunoreactive fibers were detected. Since earlier studies have provided evidence for a nondopaminergic prolactin-release-inhibiting factor in the hypothalamus of infant rats, and for an inhibitory effect of prolactin on pituitary mammotrophs, we propose that hypothalamic prolactin may contribute, as an additional prolactin-release-inhibiting factor, to the multifactorial control of pituitary mammotrophs.

Developmental changes of thyroliberin (TRH) in the rat brain

Levels of thyroliberin (TRH) were measured by radioimmunoassay in the hypothalamus and in the extrahypothalamic brain tissue of rats from fetal day 12 up to day 47 of postnatal life. TRH was detectable as early as on day 12 of intrauterine life in both the hypothalamic primordia and the extrahypothalamic brain tissue. The increase of hypothalamic TRH content was marked between fetal days 12 and 15, then up to the end of gestation it exhibited a slower tendency of increase. From fetal day 15 up to postnatal day 2 the TRH content of the hypothalamus was always higher than that of the extrahypothalamic brain. Both hypothalamic and extrahypothalamic TRH content increased up to day 35 of postnatal life.

Immunocytochemical evidence for in vivo internalization of thyroliberin into rat pituitary target cells

The in vivo internalization of thyrotropin-releasing hormone (TRH) was studied by using a semiquantitative immunoelectron microscopic method. Pituitary glands of normal male rats intravenously injected with 100 ng TRH and sacrificed after 5-60 min were used. Ultrathin sections were obtained by cryoultramicrotomy of fixed pituitary glands. Pituitary cellular types were identified by appropriate antiserums. An antiserum specifically directed against TRH was used. TRH-like immunoreactivity due to endogenous TRH was observed in thyrotropes and prolactin cells, but never in somatotropes, gonadotropes or corticotropes. At the subcellular level, the reaction was detected within the cytoplasmic matrix, the secretory granules, and the nucleus but only occasionally at the plasma membrane. After in vivo injection of TRH, the immunocytochemical reaction was still restricted to thyrotropes and prolactin cells, increased with time elapsed after injection up to 15-30 min and then returned to basal intensity in cytoplasm, secretory granules, and nucleus, and became very frequent at the plasma membrane. These data provide evidence for endogenous TRH within thyrotropes and prolactin cells, i.e., in physiological target cells for TRH, and support the hypothesis that normal TRH target cells can, in vivo, internalize exogenous as well as endogenous TRH into several subcellular compartments including the nucleus.

Binding of thyrotropin releasing hormone and prolactin release by synthronized GH3 rat pituitary cell line

Abstract GH3 cells were synchronized by growing them in a low serum concentration (1%). They were thereafter put back in normal medium (17.5% serum) (time 0 of synchronization). Four parameters were then examined every two hours for up to 40 hours : rate of [ 3 H] thymidine incorporation, cell number, binding of [ 3 H] Thyrotropin Releasing Hormone (TRH) after a 30 min exposure, and prolactin (PRL) content of culture medium and cell extract. The rate of thymidine incorporation presented a 10–20 fold increase in S phase, beginning on 12–16 hours and lasting at 26 hours. The cell population was doubled at 28 hours. [ 3 H] TRH binding to attached cells was observed throughout the cell cycle, but presented a significant increase (40–80%) during the S phase. In contrast, the % increase of PRL release in response to TRH was optimum (300% of control) in G 1 phase. Variations of the PRL cell content as well as of the PRL spontaneous release ability of the cell do not account for the variations of TRH responsiveness. The discrepancy between the two parameters of the TRH-GH3 cells interaction strongly suggest a morphological or functional heterogeneity of the TRH-binding sites.

Enzyme immunoassays for Thyroliberin (TRH) and TRH-elongated peptides in mouse and rat hypothalamus

Enzyme immunoassays (EIAs) for Thyroliberin (TRH) and TRH-elongated peptides were developed. Three haptens less than E-H-P-NH2 (TRH). Less than E-H-P-OH (TRH-OH), and S-K-R-Q-H-P-G-K-R-F (P10) were conjugated by the use of different heterobifunctional cross-linking agents either to sun-flower globulin as carrier or to acetylcholinesterase as tracer. For a same hapten, the same chemical group in the peptide was used to prepare the immunogen and the enzyme conjugate. These EIAs were performed with a second antibody solid phase technique using acetylcholinesterase as label. They permitted the measurement of TRH and TRH-elongated peptides with a sensitivity threshold of 10 fmol/well for TRH and 2 fmol/well for P10. TRH EIA only detected authentic TRH whereas TRH-OH EIA detected TRH and TRH peptides elongated on C terminal part. Anti-P10 serum was specific of TRH peptides elongated both on C and N terminal parts and no cross reactivity was observed with TRH. Using these assays, TRH and TRH-elongated peptides were determined in crude or chromatographed mouse and rat hypothalamus tissular extracts. Several TRH extended forms were identified by P10 EIA, whereas TRH-OH EIA permitted detection of both TRH and TRH-elongated peptides in chromatographed extracts. Authentic TRH was measured by TRH EIA both in crude and chromatographed hypothalamic extracts. These assays can permit the study of the processing and maturation of TRH.