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In the paraventricular nucleus of the mammalian hypothalamus, hypophysiotropic thyrotropin releasing hormone (TRH) neurons integrate metabolic information and control the activity of the thyroid axis. Additional populations of TRH neurons reside in various hypothalamic areas, with poorly defined connections and functions, albeit there is evidence that some may be related to energy balance. To establish extracellular modulators of TRH hypothalamic neurons activity, we performed a screen of neurotransmitters effects in hypothalamic cultures. Cell culture conditions were chosen to facilitate the full differentiation of the TRH neurons; these conditions had permitted the characterization of the effects of known modulators of hypophysiotropic TRH neurons. The major end-point of the screen was Trh mRNA levels, since they are generally rapidly (0.5-3h) modified by synaptic inputs onto TRH neurons; in some experiments, TRH cell content or release was also analyzed. Various modulators, including histamine, serotonin, β-endorphin, met-enkephalin, and melanin concentrating hormone, had no effect. Glutamate, as well as ionotropic agonists (kainate and N-Methyl-d-aspartic acid), increased Trh mRNA levels. Baclofen, a GABAB receptor agonist, and dopamine enhanced Trh mRNA levels. An endocannabinoid receptor 1 inverse agonist promoted TRH release. Somatostatin increased Trh mRNA levels and TRH cell content. Orexin-A rapidly increased Trh mRNA levels, TRH cell content and release, while orexin-B decreased Trh mRNA levels. These data reveal unaccounted regulators, which exert potent effects on hypothalamic TRH neurons in vitro.

SummaryOocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases.In vitrostudies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesisin vitroby timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized.Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 °C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7 ± 1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0 ± 1.1 h to 9.0–11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.

Primary cultures of dispersed cells from fetal nervous tissue are extensively used for studying multiple neuronal properties. Analyses of the developmental expression of thyrotropin releasing hormone (TRH; pglu-his-pro-NH(2)) biosynthesis in primary cultures of fetal dissociated hypothalamic cells have shown that cellular TRH levels per dish increase with time in culture, after a lag period of a few days, but do not attain the values observed in vivo, hampering its use as a model system for the study of peptide biosynthesis and release. We have demonstrated that homologous conditioned medium (CM) enhances TRH expression in dissociated cell cultures from fetal mice hypothalamus, maintained in presence of serum. We report here experimental conditions that allow the expression, during the second or third week in vitro, of higher cellular TRH levels than previously described in primary cultures of dissociated hypothalamic cells from 17-day rat fetuses. The medium used was Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%), vitamins, glucose, glutamine and insulin (DMEM-S). Cellular levels of TRH/mg protein increased with cell density between 1 and 2.7x10(6) cells per 35-mm dish. Addition of 10(-5) M cytosine arabinoside (CAr) at the 4th day in vitro (DIV) improved TRH cell content per dish compared to addition at 5 DIV; 2.5-5x10(-5) M bromodeoxyuridine added at seeding reduced cell survival and did not enhance TRH levels, in comparison to CAr-treated cultures. Addition of ascorbic acid (0.5-1x10(-4) M) increased TRH levels per dish. Substitution of DMEM by DMEM-F12 (1:1) did not improve TRH levels. Cellular levels of TRH, in Neurobasal plus B27 (a serum-free medium), were similar to levels in serum-supplemented media. In the optimized conditions, a small number of pro-TRH mRNA expressing cells (2% of total cells) was detected by in situ hybridization; 40% coexpressed the pro-protein convertase PC1 mRNA. Conditioning the medium, controlling glial proliferation, and adding ascorbic acid improved the expression of TRH in primary culture of hypothalamic cells in DMEM-S.