Investigations on the triiodothyronine (T3)-specificity of thyrotropic (TSH) and gonadotropic (HCG) hormone in the unicellular Tetrahymena

In a previous experiment thyrotropin (TSH) increased the triiodothyronine (T3) production of Tetrahymena and chorionic gonadotropin (HCG) moderately overlapped the effect. At present the production of three amino acid type (histamine, serotonin, epinephrine) and one peptide (endorphin) hormones were studied under the effect of TSH or HCG, in tryptone-yeast (TY) or salt (Losina-Losinsky) medium. The duration of the effect was 10 min. TSH significantly (with almost 20%) decreased epinephrine production in TY medium and HCG similarly decreased epinephrine and increased histamine level. In salt solution TSH as well as HCG decreased the level of serotonin. The results show that at this low level of phylogeny TSH effect is not completely thyroxine-specific, however it is not general. HCG overlaps TSH effect on epinephrine and serotonin production, however its effect is broader. The experiments also demonstrate that the effect of pituitary trop-hormones can be bidirectional in Tetrahymena, as histamine level was increased and epinephrine level was decreased by HCG, in the same cells.     

Presence (hPL, prostaglandin) and absence (triiodothyronine, thyroxine) of hormones in Tetrahymena: experimental facts and open questions

The RIA technique detected prostaglandin (PGF2) and human placetal lactogen (hPL) in Tetrahymena cultures grown in bacto tryptone + yeast extract medium which, however, itself contained these hormones. About one to two per cent of the total hormone content of the medium was demonstrated intracellularly. Treatment with diiodotyrosine (T2), which is known to stimulate the growth of Tetrahymena, was followed by a decrease in the intracellular prostaglandin level. Triiodothyronine and thyroxine were not detected in Tetrahymena or in the medium, and did not appear in it on induction with TSH either. In the light of these observations it might well be doubted that prostaglandin was native in Tetrahymena: the use of synthetic media, and/or a reliable demonstration of the hormone content of the growth medium is recommended for evidence of hormone biosynthesis by unicellular organisms.     

Effect of polypeptide hormones (insulin, thyrotropin, gonadotropin, adrenocorticotropin) on RNA synthesis in Tetrahymena, as assessed from incorporation of 3H-uridine

Incorporation of 3H-uridine by RNA in Tetrahymena was differently influenced by insulin, glucagon, follicle-stimulating hormone (FSH), thyrotropic hormone (TSH), adrenocorticotropic hormone (ACTH) and chorion-gonadotropic hormone (PMSG). TSH caused it to increase considerably and durably after an initial depression, while glucagon caused it to rise over the control throughout. Insulin, and especially PMSG, depressed the incorporation of label considerably, the latter to 3-6% of the control value by 120 min. ACTH and FSH accounted for an initial depression of RNA synthesis which, however, returned to normal 30 min after treatment. Remarkably, while the chemically similar hormones acted differently, insulin and glucagon showed the same trend of positive and negative influence, respectively.     

Hormonal interactions in Tetrahymena: effect of hormones on levels of epidermal growth factor (EGF)

Tetrahymena pyriformiswas treated with insulin, histamine or serotonin for 30 min and epidermal growth factor (EGF) level was studied inside the cells using specific antibodies and flow cytometry as well as confocal microscopy. The EGF concentration was highly significantly elevated after hormone treatment, regardless of the hormone used. EGF was localized mainly in the cortical region (mucocysts) and in vesicles and this localization did not differ in untreated and treated cells. The results call attention to the possibility of interactions between hormones at unicellular level and points to the presence of a hormonal system in Tetrahymena that includes receptors, hormones and signal transduction pathways as well as hormonal interactions. This could be the basis of further evolution to the hormonal system of multicellulars.     

Effect of gonadotropin (FSH + LH) and thyrotropin (TSH) administered with or without endotoxin at the age of three weeks on the response capacity of the thyroid gland in adult rats

At the age of three weeks the experimental animals received either thyrotropin (TSH), or gonadotropin (FSH + LH), or endotoxin (LPS) alone or in combination. The effectivity of the treatments was evaluated at the age of two months (with or without further hormone treatment). Contrastingly to neonatal TSH treatment, TSH treatment at the age of three weeks did not give rise to imprinting. In female animals, however, TSH treatment increased the sensitivity to the related gonadotropin hormone. At the age of three weeks gonadotropin treatment--on its own--did not cause damages to the TSH receptors of the thyroid gland. While in previous experiments neonatal endotoxin treatment damaged considerably the thyroxin production of the adult thyroid gland, after treatments at the age of three weeks no similar effect could be observed. The treatment, however, decreased the sensitivity of the receptors to TSH. In female animals simultaneous administration of endotoxin and TSH led, even without further hormone treatment, to constant increase in T4 level (the increase could also be detected in the adult animal). Imprinting, however, did not develop. In male animals simultaneous administration of endotoxin and gonadotroph hormone decreased considerably the T4 baseline level, and further TSH or gonadotropin treatment was unable to enhance T4 production.     

The role of Ca2+ in hormonal imprinting of the Tetrahymena

It is known from model experiments on Tetrahymena that primary exposure to a hormone induces receptor formation or amplification, in other words a hormonal imprinting. Substances acting on the intracellular Ca2+ level of the Tetrahymena, such as TMB-8, EDTA, EGTA, NiCl2 and La(NO3)3, interfered with hormonal imprinting of the unicellular to different degrees, and some of them influenced hormone (insulin, TSH) binding also independently of imprinting. Interference with the intracellular Ca-metabolism generally influenced imprinting by insulin and TSH, which were mediated by different mechanisms, to dissimilar degrees, or in opposite directions. On combined application of the agents acting on Ca-metabolism, their effects were additive. It appears that intact Ca-mediation is an essential prerequisite for normal hormonal imprinting.     

Increased hormone levels in Tetrahymena after long-lasting starvation

Tetrahymena contains vertebrate hormone-like materials. The level of one of these, insulin increased during starvation in a previous experiment. We hypothesized that other hormones are also influenced by starvation. To prove the hypothesis Tetrahymena pyriformis cultures were (1) starved for 24h; (2) starved for 24h and re-fed for 30min or (3) starved for 30min. Amount and localization of vertebrate-like hormones, produced by Tetrahymena, beta-endorphin, adrenocorticotropin (ACTH), serotonin, histamine, insulin and triiodothyronine (T(3)) were studied by immunocytochemical methods using flow cytometry and confocal microscopy. Long starvation elevated with 50% the hormone levels, while short starvation moderately elevated only the serotonin level in the cells. After short re-feeding endorphin and histamine returned to the basal level, ACTH and serotonin approached the basal level, however, remained significantly higher, while insulin and T(3) stood at the starvation level. The results show that such a stress as long starvation provokes the enhanced production of hormones which likely needed for tolerating the life-threatening effect of stress.     

Effects of inorganic iodide, epidermal growth factor and phorbol ester on hormone synthesis by porcine thyroid follicles cultured in suspension.

Porcine thyroid follicles cultured in suspension for 96 h synthesized and secreted thyroid hormones in the presence of thyrotropin (TSH). The secretion of newly synthesized hormones was assessed by determining the contents of thyroxine (T4) and triiodothyronine (T3) in the media and by paperchromatographic analysis of 125I-labelled hormones in the media where the follicles were cultured in the presence and absence of inhibitors of hormone synthesis. The hormone synthesis and secretion was modified by exogenously added NaI (0.1-100 microM). The maximal response was obtained at 1 microM. Thyroid peroxidase (TPO) activity in the cultured follicles with TSH for 96 h was dose-dependently inhibited by NaI. One hundred microM of NaI completely inhibited TSH-induced TPO activity. Moreover, both epidermal growth factor (EGF: 10(-9) and 10(-8) M) and phorbol 12-myristate 13-acetate (PMA: 10(-8) and 10(-7) M) inhibited de novo hormone synthesis. An induction of TPO activity by TSH was also inhibited by either agent. These data provide direct evidences that thyroid hormone synthesis is regulated by NaI as well as TSH at least in part via regulation of TPO activity and also that both EGF and PMA are inhibitory on thyroid hormone formation.     

Is there a hormonal network in Tetrahymena? A systematic investigation of hormonal effects on the hormone content

The effect of six hormones (histamine, serotonin, insulin, epidermal growth factor (EGF), oxytocin and gonadotropin) was studied on the hormone (histamine, serotonin, adrenocorticotropic hormone [ACTH], endorphin and triiodothyronine [T(3)]) content of Tetrahymena. The hormones were given in 10(-9) or 10(-12) M concentrations or as 0.1 and 0.001 I.U. ml(-1) (in the case of oxytocin and gonadotropin) for 1 h. The hormones in picomolar concentration, i.e. at levels which can be present also in natural conditions, influence the amount of other hormones inside the cell. Their effect is not a general one: it is individual, the level of one of the hormones was elevated, while that of the others diminished under the effect of the same hormonal stimulus. Insulin was the only hormone, which influenced the concentration of other hormones in one direction, elevating them. This effect could have a role in the life-saving property of this hormone in Tetrahymena, but the hormones were not studied from this point of view. Usually there is no difference between the effect of the two concentrations used, but there are situations when the effect of the two concentrations is opposite. This means that there is a possible concentration dependence and this could influence differently the cells which are far from or near to the secretor cell. Considering earlier observations, the duration of the treatment can also influence the result. The results give new data to the hormonal regulation at unicellular level (which can be the base of regulation at higher evolutionary levels) and point to the possibility of a hormonal network.     

Durable effect of heat-stress on the hormone production of Tetrahymena. Effect of insulin on the consequences of stress

The unicellular Tetrahymena pyriformis was stressed by 37°C heat for 1 h and its hormone (serotonin, histamine, triiodothyronine) content was measured by immunocytochemical flow cytometry in different time points (immediately after treatment and after 1, 2, 8, 16 weeks). The treatment increased each hormone level for two weeks, however, after 8 weeks the hormone concentration inside the cells decreased and in case of serotonin this was similar in the 16th week, while the other two hormones' level was similar to the control. Insulin further increased the hormone production during treatment, but this effect was not durable. After one week the cells behave similar to those, subjected to heath shock only. The results show that a single stress causes deep and durable changes in the hormone household of Tetrahymena which is influenced by exogenously given insulin only in the acute phase.     

Effect of the beta-adrenergic substances propranolol, alprenolol and isoprenaline on cAMP production in bovine thyroid slices

The effect of the beta-adrenergic blockers L-alprenolol and DL-propranolol and of the beta-adrenergic agonist L-isoprenaline on the basal and thyrotropic hormone(TSH)-stimulated cyclic adenosine-monophosphate (cAMP) level in bovine thyroid slices was studied. The main basal cAMP level in bovine thyroid slices was 3 pmol/mg tissue. TSH stimulated cAMP production in correlation to the concentration. Maximum stimulation was achieved with a TSH concentration of 10 mU/ml. The beta-blockers DL-propranolol and L-alprenolol caused 74 and 77% inhibition of TSH-stimulated cAMP synthesis respectively. The beta-adrenergic agonist L-isoprenaline did not significantly affect either the basal or the TSH-stimulated cAMP level. The role of the beta-adrenergic receptor system in the regulation of TSH-stimulated cAMP synthesis is discussed.     

Induction of steroid binding sites (receptors) and presence of steroid hormones in the unicellular Tetrahymena pyriformis

The unicellular Tetrahymena does not normally possess a steroid hormone (dehydroepiandrosterone, DHEA) or a glucocorticoid (dexamethasone) receptor, but both kinds of receptor can be induced in it by pretreatment (imprinting) with the adequate hormone. The specific receptors which arise are demonstrable experimentally. Examination of Tetrahymena cells for endogenous steroids by the radioimmunoassay (RIA) technique detected an appreciable concentration of DHEA and DHEA sulphate, and lesser concentrations of testosterone and estradiol in this unicellular organism.     

Effects of extremely low concentrations of hormones on the insulin binding of Tetrahymena

FITC-insulin binding to previously hormone-treated Tetrahymena was studied by flow cytometry and confocal microscopy. Hormones produced by Tetrahymena were chosen for study and the hormone concentrations were administered between 10(-6) and 10(-21)M for 30 min. Endorphin, serotonin and insulin significantly reduced the hormone binding however histamine did not influence it at all. Endorphin, serotonin and insulin were significantly effective down to 10(-18)M and the effect of insulin and endorphin suggest a similar mechanism. The results call attention to the efficacy of very low hormone concentrations, which can influence the hormone content (earlier experiments) and receptor binding capacity (present study) of a unicellular organism. This seems to be very important, as in wild (natural) conditions the dilution of signaling materials secreted by a water-living protozoan is very high. In addition, the results point to the selectivity of response, as not all of the hormones that deeply influence other physiological indices (e.g. histamine) have an effect on insulin content or insulin receptors.     

Involvement of selection and amplification mechanisms in hormone receptor development in a unicellular model system

It was demonstrated earlier, that long lasting exposure of Tetrahymena to a hormone (histamine) resulted in an increased responsiveness to a later re-exposure. However, it was difficult to establish whether selection or amplification plays a role in receptor differentiation. As diiodotyrosine (T2) enhances the growth of Tetrahymena, in the present experiment the effect of T2-treatment on a long-term culture of Tetrahymena pyriformis was analysed by mathematical-statistical methods to differentiate the effects of selection and amplification mechanisms on hormone receptor development. Although continuous and periodic treatment with T2 enhanced cell division equally, the resulting populations differed in structure. On continuous treatment the population tended to become inhomogenous. The variance tended to increase for 9 days and decreased afterwards without, however, returning to the control level. On periodic treatment the variance was the same as in the control group, but the second and third exposure were significantly more effective than the first treatment, suggesting that the primary encounter with the hormone had given rise to lasting alterations (hormonal imprinting). It follows that continuous exposure involves a selection process which does not, however, account for a steady increase of the growth rate; for initial amplification, taking place also in this condition, and selection which takes effect later, compensate one another's effects. Regarding the unicellular experimental system as a phylo- and ontogenetic model, the conclusion lies close at hand that the selection and amplication mechanisms promote hormone receptor development by joint rather than alternate action.     

Presence and localization of histidine decarboxylase enzyme (HDC) and histamine in Tetrahymena pyriformis.

Histidine decarboxylase (HDC) enzyme and its function under hormonal influences were studied in a low level of phylogeny. HDC protein is present in the unicellular ciliate Tetrahymena and its expression was not altered by insulin or histamine treatment. Starvation for 24 h enormously decreased the quantity of histamine in the cells. However, insulin influenced the activity of the HDC enzyme, demonstrated by the seven-fold quantity of histamine in the starved cells after insulin treatment. Insulin also increased the uptake of histamine from the tryptone-yeast extract medium. HDC was found in different parts of the cytoplasm, mainly in the periphery (epiplasm) of the cells. The experiments demonstrated the uptake and synthesis of histamine by Tetrahymena as well as the possibility of hormonal regulation of HDC activity.     

Regulation of thyrotropin biosynthesis. Discordant effect of thyroid hormone on alpha and beta subunit mRNA levels

We have studied the regulation of the biosynthesis of thyrotropin (TSH) and its alpha and beta subunits by thyroid hormone in thyrotropic tumors carried in hypothyroid mice. Treatment with 3,5,3'-triiodo-L-thyronine (T3) (20 micrograms/100 g, body weight) daily for 4 or 10 days reduced serum TSH to 3 and 0.3% of control, respectively. Serum levels of free alpha subunit were reduced to 60 and 11% of control at 4 days and 10 days, respectively, and serum free TSH-beta was undetectable at both time points. There was no significant decrease in tumor TSH content after 4 days of treatment and, after 10 days, TSH content was reduced to 15% of control levels. There was no significant effect of T3 on tumor alpha subunit levels at either 4 or 10 days. In contrast, tumor TSH-beta content was markedly reduced after 4 days and 10 days of T3 treatment, to 29 and 10% of control levels, respectively. Translation of tumor poly(A) mRNA in a rabbit reticulocyte lysate system showed that thyroid hormone decreased translatable TSH-beta mRNA to undetectable levels at both 4 and 10 days, whereas translatable alpha mRNA was reduced strikingly only at 10 days in one of two tumors. RNA blot hybridization with 32P-labeled plasmid probes containing alpha or TSH-beta cDNAs showed that TSH-beta mRNA was reduced to less than 10% of control after both 4 and 10 days of T3 treatment, whereas, again, alpha mRNA was only reduced in one of two tumors at 10 days. Our data thus show that thyroid hormone affects alpha and TSH-beta mRNA and protein levels discordantly and suggest that regulation of TSH biosynthesis may occur predominantly at the level of TSH-beta mRNA.     

Verification of epigenetic inheritance in a unicellular model system: multigenerational effects of hormonal imprinting

The unicellular Tetrahymena has receptors for hormones of higher vertebrates, produces these hormones, and their signal pathways are similar. The first encounter with a hormone in higher dose provokes the phenomenon of hormonal imprinting, by which the reaction of the cell is quantitatively modified. This modification is transmitted to the progeny generations. The duration of the single imprinter effect of two representative signal molecules, insulin and 5-HT (5-hydroxytryptamine), in two concentrations (10-6 and 10-15 M) were studied. The effects of imprinting were followed in 5 physiological indices: (i) insulin binding, (ii) 5-HT synthesis, (iii) swimming behaviour, (iv) cell growth and (v) chemotaxis in progeny generations 500 and 1000. The result of each index was different from the non-imprinted control functions, growth rate, swimming behaviour and chemotactic activity to insulin being enhanced, while others, e.g. synthesis and chemotactic responsiveness of 5-HT and the binding of insulin were reduced. This means that a function-specific heritable epigenetic change during imprinting occurs, and generally a single encounter with a femtomolar hormone concentration is enough for provoking durable and heritable imprinting in Tetrahymena. The experiments demonstrate the possibility of epigenetic effects at a unicellular level and call attention to the possibility that the character of unicellular organisms has changed through to the present day due to an enormous amount of non-physiological imprinter substances in their environment. The results - together with results obtained earlier in mammals - point to the validity of epigenetic imprinting effects throughout the animal world.