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.     

Caspase-like activity in programmed nuclear death during conjugation of Tetrahymena thermophila

Apoptosis, or programmed cell death, is common in a variety of eucaryotes, from unicellular protozoa to vertebrates. The ciliated protozoan Tetrahymena thermophila has a unique apoptosis-like nuclear death during conjugation, called programmed nuclear death. This death program involves nuclear condensation (pyknosis) and oligonucleosomal DNA fragmentation in the parental macronucleus. Subsequently, the condensed nucleus is entirely resorbed in the autophagosome. Here we demonstrate that caspase-8- and -9-like activity was detected, but no caspase-3-like activity, by in vitro assay during the nuclear resorption process, suggesting that caspase-like activity is associated with both programmed cell death and apoptosis-like nuclear death in Tetrahymena. The use of indicator dye to detect the loss of mitochondrial membrane potential suggested the uptake of mitochondria and the degenerating macronucleus by the autophagosome. An involvement of mitochondria in the programmed nuclear death is discussed.     

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.     

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.     

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.     

The hormonal system of the unicellular Tetrahymena: a review with evolutionary aspects

The unicellular ciliate, Tetrahymena has receptors for hormones of the higher ranked animals, these hormones (e.g. insulin, triiodothyronine, ACTH, histamine, etc.) are also produced by it and it has signal pathways and second messengers for signal transmission. These components are chemically and functionally very similar to that of mammalian ones. The exogenously given hormones regulate different functions, as movement, phagocytosis, chemotaxis, cell growth, secretion, excretion and the cells' own hormone production. The receptors are extremely sensitive, certain hormones are sensed (and response is provoked) at 10-21 M concentration, which makes likely that the function could work by the effect of hormones produced by the Tetrahymena itself. The signal reception is selective, it can differentiate between closely related hormones. The review is listing the hormones produced by the Tetrahymena, the receptors which can receive signals and the signal pathways and second messengers as well, as the known effects of mammalian hormones to the life functions of Tetrahymena. The possible and justified role of hormonal system in the Tetrahymena as a single cell and inside the Tetrahymena population, as a community is discussed. The unicellular hormonal system and mammalian endocrine system are compared and evolutionary conclusions are drawn.     

Enhancement of the sensitivity of Tetrahymena to a second hormonal influence by hormone pretreatment

Diiodotyrosine and serotonin enhance the growth of Tetrahymena. A second exposure of the unicellular to these hormones accounts for a still greater increase of its growth rate, probably due to the amplification of the receptor induced by the first exposure.     

Influence of membrane fluidity changes upon the imprinting of polypeptide hormones in Tetrahymena

Hormonal imprinting is a physiological phenomenon, in which after the first encounter the receptorial and functional responses of a cell change for future occasions. The present experiments demonstrate (using Tetrahymena as a model cell) that the imprinting is very sensitive to the changes in membrane physical state. Cultivation of Tetrahymena cells in 28 or 15 degrees C or in ergosterol-supplemented media caused only quantitative differences in the imprinting; however, the process of cooling (shift-down) or reheating (shift-up) resulted in a false reaction. The combined treatment by ergosterol and cooling completely abolished the imprinting. These results indicate that hormonal imprinting is a membrane-dependent process.     

Preparation,morphology and drug metabolism of isolated hepatocyte and liver organ cultures from the human fetus

Isolated hepatocytes of human fetuses (week 9–20 of gestation) were prepared by digestion of liver slices with Dispase l and repeated centrifugations. These cells metabolized drugs by first order kinetics for at least 4 h and retained their drug metabolizing enzyme activities for more than 24 h. Using cultures of human fetal liver slices (week 5–20 of gestation) first order kinetics of drug metabolism could be measured for up to 3 days. These findings correlate well with concomitant morphological studies. Electron microscopy revealed that the liver cells of human fetuses (week 9–20 of gestation) contained numerous cavities of the rough endoplasmic reticulum (ER) and only single membranes of the smooth ER. The isolated liver cells maintained their morphological integrity for up to 24 h in culture. Sections derived from liver slices cultured for up to 3 days did not show any significant morphological alterations, except for some swellings of mitochondria, vacuolisations and fat inclusions that occurred in the sinus endothelia and blood cells. A gradual disintegration began on day 4, and after 7 days intact cells could no longer be demonstrated. The metabolism of the benzodiazepine drugs prazepam, diazepam and medazepam as well as diphenylhydantoin, hexobarbital and halothane were measured in these systems. Both the isolated hepatocyte and liver organ cultures should be useful to study whether a relationship exists between the emryotoxicity of certain drugs and their metabolism in the human fetus.     

A possible cyclic AMP-mediated regulation of microsomal fatty acyl-CoA desaturation system in Tetrahymena microsomes

Profound alterations in the microsomal fatty acyl-CoA desaturase activities and cyclic AMP production of a unicellular eukaryote, Tetrahymena pyriformis NT-1, originally grown in the glucose-deficient medium, were observed, following the administration of glucose or beta-adrenergic agonists such as epinephrine and isoproterenol. There was a great increase of stearoyl-CoA (delta 8) desaturase activity coincident with a 2-fold decrease of oleoyl-CoA (delta 12) desaturase activity over the first 2 h after administration of these compounds. During this period of time, it was found that the production in vivo of labeled oleic acid from [14C]acetic or [3H]palmitic acid increases 2-fold and the formation in vivo of each labeled linoleic and gamma-linolenic acids drastically decreases. Glucose or beta-adrenergic agonists caused an increase of stearoyl-CoA-stimulated reoxidation rate of NADH-reduced cytochrome b5 but depressed oleoyl-CoA-stimulated reoxidation rate of b5, indicating that both desaturase activities are controlled by the respective terminal components of the desaturase system. A significant and reproducible increase of adenylate cyclase activity and a slight decrease of cyclic AMP phosphodiesterase activity were observed to occur within the first 2 h after the addition of these compounds, when cyclic AMP content in Tetrahymena cell rose by 3-4-fold. Propranolol, a beta-adrenergic blocker, abolished the effects of glucose or beta-adrenergic agonists on the activities of fatty acyl-CoA desaturases and the terminal components as well as cyclic AMP production of cells. These results suggest that glucose and beta-adrenergic agonists may modulate the microsomal fatty acyl-CoA desaturase system in Tetrahymena by acting through the increase of intracellular cyclic AMP content.     

Prolonged elevation of insulin content in the unicellular tetrahymena after insulin treatment: induction of insulin production or storage?

In the unicellular organism, Tetrahymena, the first encounter with an exogeneously given hormone results in hormonal imprinting. This causes an increase of the binding capacity of receptors and the production of the appropriate hormone in the progeny generations of the treated cell. In the present experiments the quantity (using radioimmunoassay) and localization (using confocal laser scanning microscopy) of the immunologically insulin‐like material (hereafter insulin) were studied for 10 days after 4 h or 24 h 10⁻⁶ m insulin treatment (hormonal imprinting). Forty‐eight hours after both insulin treatments a high quantity of insulin was present in the cells. This value was also significantly increased after 96 h. After 8 days the difference to the control was significant only in the 24 h treated group. Confocal microscopy (using antibody to pig insulin) localized insulin in the cell body. The oral field contained extremely high quantities of the endogeneous hormone. Insulin treatment (after 48 and 96 h) caused an elevation of insulin content in general, and specific accumulation in the posterior sections of the cell, around the nucleus and in the periphery were observed. Ten days after both treatments only the peripheral region of the cell body and the ciliary row contained more insulin than the control. This means that after insulin treatment the quantity of insulin increases for a lengthy time period which is followed by the expression of insulin in the peripheral region. Insulin contained by Tetrahymena 48 h after imprinting stimulated glucose uptake of rat diaphragm. Copyright © 1999 John Wiley & Sons, Ltd.     

Immunocytochemical verification of the insulin receptor's specificity in the nuclear envelope of Tetrahymena. Comparison with receptors of the plasma membrane

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.     

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 hexamethylene bisacetamide (HMBA) on the microtubular system of Tetrahymena pyriformis

HMBA (10-[3-hydroxy-4-methoxy benzylidene)]-9(10H)-anthracenone) is an inhibitor of tubulin polymerization and a developmental inducer in mammalian cells. The effect of HMBA on the microtubular system of Tetrahymena was investigated. This is the first case when its effect was studied in an unicellular animal, by using immunocytochemistry, confocal microscopy, Flutax-1 staining and flow cytometry. In Tetrahymena, HMBA (20 nM.; 10 and 45 min) significantly decreased the label of transversal microtubules (without affecting longitudinal ones) and also decreased the diffuse cytoplasmic fluorescence (tubulin-dimer pool). However, it increased the gross amount of alpha-tubulin and acetylated tubulin. Cilia showed an extraordinary strong labeling. Longer treatments (45 min) were toxic. There is a possibility, that the extremely rich tubulin content of cilia was due to the inducer effect of HMBA or to the self-defense of the cell.     

Analysis of expressed sequence tags (ESTs) in the ciliated protozoan Tetrahymena thermophila

To assess the utility of expressed sequence tag (EST) sequencing as a method of gene discovery in the ciliated protozoan Tetrahymena thermophila, we have sequenced either the 5' or 3' ends of 157 clones chosen at random from two cDNA libraries constructed from the mRNA of vegetatively growing cultures. Of 116 total non-redundant clones, 8.6% represented genes previously cloned in Tetrahymena. Fifty-two percent had significant identity to genes from other organisms represented in GenBank, of which 92% matched human proteins. Intriguing matches include an opioid-regulated protein, a glutamate-binding protein for an NMDA-receptor, and a stem-cell maintenance protein. Eleven-percent of the non-Tetrahymena specific matches were to genes present in humans and other mammals but not found in other model unicellular eukaryotes, including the completely sequenced Saccharomyces cerevisiae. Our data reinforce the fact that Tetrahymena is an excellent unicellular model system for studying many aspects of animal biology and is poised to become an important model system for genome-scale gene discovery and functional analysis.     

Induction of hormone receptor formation in the unicellular tetrahymena

Studies based on treatment with antibodies to thyrotropic hormone, luteotropic hormone, growth hormone or adrenocorticotropic hormone have shown that although the unicellular Tetrahymena does not possesssui generis receptors to all polypeptide hormones, such binding structures may arise, or become established in the membrane of the unicellular Tetrahymena in the presence of exogenous hormone. The Tetrahymena subjected to hormonal imprinting still contained an increased amount of hormone after six generation changes, which suggested that either hormone production had been induced by treatment, or the internalized hormone had not been degraded intracellularly. Thus the role of hormonal imprinting in receptor formation has also been substantiated by the immunocytochemical approach used in the present study.     

Diazepam affects both level and amplitude of rat locomotor activity rhythm but has no effect on core body temperature

The present study investigates the effects of a chronic administration of diazepam, a benzodiazepine widely used as an anxiolytic, on locomotor activity and body core temperature rhythms in male Wistar rats housed under 12∶12 light∶dark (LD) cycle conditions. Diazepam was administered subcutaneously for 3 wks in a dosage of 3 mg/kg body weight/day, 1 h before the onset of darkness. Diazepam increased the level of locomotor activity from the first day until the end of treatment, and also increased the amplitude of the activity circadian rhythm, but only on the third wk of treatment. Diazepam exerted no effects on the length of the period and did not affect the phase of the locomotor activity rhythm. The body temperature rhythm of rats was affected neither by short‐term (a single injection) nor by long‐term (every day for 3 wks) diazepam treatment. Diazepam lacked effect on body core temperature even on the first day of administration, thereby ruling out the possibility of drug tolerance development. The fact that diazepam affects locomotor activity, but not core body temperature, suggests that different mechanisms mediate the actions of diazepam on locomotor activity and on core body temperature.     

Characterization of chemotactic ability of peptides containing N-formyl-methionyl residues in Tetrahymena fMLP as a targeting ligand

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C-terminal f-Met and N-terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f-MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f-Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis-activating processes.     

Diazepam affects both level and amplitude of rat locomotor activity rhythm but has no effect on core body temperature

The present study investigates the effects of a chronic administration of diazepam, a benzodiazepine widely used as an anxiolytic, on locomotor activity and body core temperature rhythms in male Wistar rats housed under 12:12 light:dark (LD) cycle conditions. Diazepam was administered subcutaneously for 3 wks in a dosage of 3 mg/kg body weight/day, 1 h before the onset of darkness. Diazepam increased the level of locomotor activity from the first day until the end of treatment, and also increased the amplitude of the activity circadian rhythm, but only on the third wk of treatment. Diazepam exerted no effects on the length of the period and did not affect the phase of the locomotor activity rhythm. The body temperature rhythm of rats was affected neither by short-term (a single injection) nor by long-term (every day for 3 wks) diazepam treatment. Diazepam lacked effect on body core temperature even on the first day of administration, thereby ruling out the possibility of drug tolerance development. The fact that diazepam affects locomotor activity, but not core body temperature, suggests that different mechanisms mediate the actions of diazepam on locomotor activity and on core body temperature.     

游仆虫信息素Er-1和Er-2对四膜虫的种间作用

信息素是生物体向外释放的化学物质,在细胞及生物体中具有种内信息传递的生理学功能。信息素这一类分子广泛分布于系统发生史中,它们的特异活性在单细胞生物、昆虫以及脊椎动物中均有报道。脊椎动物中信息素的信号传输已被证实是一嗅觉依赖过程,7TM-受体被认为是信号传输过程中的信号转换器。在低等单细胞生物(例如:来可夫游仆虫)的细胞膜上存在有信息素异构体,作为信息素分子的有效结合位点而行使其功能。本研究主要探讨单细胞的信息素(Er-1和Er-2)的基础细胞生理学作用是仅限于产生该信息素的物种,还是对其它的原生动物(例如:四膜虫)或对系统发育中分类地位较高的细胞(例如:MRC5成纤维细胞或J774巨噬细胞)均具有调节活性。研究结果表明,游仆虫的两种信息素对梨形四膜虫GL的生长调节有显著不同的作用:当信息素浓度为10-11M时,Er-1具有正调控作用,而Er-2具有抑制剂的作用。这两种配体的趋化作用也有很不同:Er-1具有一种广范的化学排斥特性,而Er-2具有一个双峰的化学吸引剂的性质。计算机检测发现,与Er-2的作用不同,Er-1可略微降低被测细胞的游动速率。趋化现象的选择特性表明Er-2信息素的受体有一种“短期”的特性;而Er-1是不能选择任何亚种群的,这也支持了我们先前的研究数据,即这两种信息素在四膜虫GL内产生两种不同的信号。四膜虫对信息素特异性的反应表明四膜虫能辨别非常近似但带有微小差异的配体(如Er-1和Er-2的电荷差异)。