Taxon-dependence of receptor level cell-to-cell communication in Tetrahymena: possible explanation for the transmission of hormonal imprinting

Insulin treatment induced in Tetrahymena pyriformis a positive hormonal imprinting, and in Tetrahymena thermophila a negative imprinting, resulting in increased and decreased binding capacity, respectively, at re-exposure to the hormone. The imprinting, or the information associated with it, is transferred by the nutrient medium of the insulin-treated cells to those not treated. The issue of transfer depends on the nature of the receiver taxon, leading always to a positive imprinting in Tetrahymena pyriformis, and to a negative imprinting in Tetrahymena thermophila, regardless of the nature of the 'imprinted' transmitter taxon. The findings substantiate the transferability of hormonal imprinting by the nutrient medium at the unicellular level, the key role of the postreceptorial mechanism in determining the trend of imprinting and may explain the persistence of imprinting in the progeny generations.     

An attempt at transmission of hormonal imprinting between foreign cell lines

Hormonal imprinting is transmitted from imprinted to virgin cells of the same cell line. No transmission of imprinting occurred between Chinese hamster ovary (CHO) and human Chang liver cells, and the presence of the latter reduced rather than enhanced the thyrotropic hormone (TSH) binding capacity of the CHO cells. While hormone binding capacity was relatively homogeneous in the control and the mixed cell cultures, it was not homogeneous in the homologous imprinted plus virgin cell population, indicating a continuous transmission of imprinting from the former to the latter.     

Impact of serum concentration of the medium and fasting on the imprintability of the insulin receptors of Chang liver cells.

When the cells of the Chang cell line came into interaction with a hormone (insulin) an imprinting-like phenomenon took place. The binding capacity of the receptors strengthened and this feature was transmitted to the descendant generations. The quality of the nutrient medium influenced the development of imprinting, when the cells were maintained in a medium containing 2% serum it was more difficult to evoke imprinting than in case the cells were kept in a medium containing 10% serum. If the cells were cultured kept in Tyrode (physiological) solution for 24 hours the possibility to evoke imprinting was lost. Difference could be observed between the behaviour of receptors in nuclear membrane and that of receptors in the plasma membrane; i.e. changes were more dynamic in the plasma membrane.     

Impact of 5-azacytidine on insulin binding and insulin-induced receptor formation in tetrahymena.

The unicellular Tetrahymena is able to bind the vertebrate hormone insulin, and the binding sites presented by it become amplified under hormonal influence. The increased binding capacity for insulin reappears in many offspring generations. 5-azacytidine inhibits insulin binding and the insulin-induced formation of binding sites as well in the cell generation directly involved in interaction, but enhances insulin binding in the daughter cell generations. The nutrient medium of the cells whose binding capacity was enhanced by azacytidine treatment transmitted the information accounting for increased binding to "virgin" cells not previously treated with azacytidine.     

Specificity of the cell-to-cell transmission of hormonal imprinting in cell cultures

Chang liver cells and Chinese hamster ovary (CHO) cells were imprinted either with insulin or with thyrotropin (TSH). Chang liver cells responded to insulin but not to TSH. As an effect of imprinting evoked by insulin administration the binding of insulin administered for the second time was enhanced. In the mixed culture of imprinted and intact cells the extent of the binding was similar to that seen in the cultures of the cells having received imprintatory treatment alone. CHO cells also responded to TSH, imprinting developed and was transmitted to the cells which were not in interaction with the hormone (intact cells). In CHO cells also insulin gave rise to imprinting for insulin, whereas TSH gave rise to moderate binding imprinting for insulin. On the other hand, insulin imprinting did not enhance the binding of TSH. The obtained results indicate that both the imprinting itself and the specificity of the transmission of imprinting depend on the characteristics of the cell-type in question. The extent of the transmission, however, is always proportional to the extent of imprinting.     

Discrepancy in hormone binding and information transfer between sister cells of Tetrahymena clones

Tetrahymena cells treated with insulin in mass cultures were separated to single-cell clones or one of the "sister-cells" of dividing Tetrahymena (in single-cell culture) was treated with insulin. In both cases the FITC-insulin binding of sister-cells were compared. The insulin imprinting significantly increased the insulin binding of cells. There was also a significant difference between the imprinted and not imprinted sisters as well as between the not imprinted sisters. This demonstrates the existence of a difference (in hormone binding) between sister-cells and justifies that the information of the first hormone treatment (imprinting) is not equally divided between the sister-cells.     

Permanence of the cell-to-cell transmission of insulin induced hormonal imprinting

When insulin-treated (imprinted) Chang liver cell cultures were mixed with cultures which did not receive insulin treatment the information of imprinting was transmitted to the cultures which were not in direct contact with insulin. The ability of the cells to transmit imprinting was long lasting and could be detected even after four weeks, when it was nearly of the same degree as at the first measurement. Difference was found between the binding capacity of the receptors of the plasma membrane and those of the nuclear membrane.     

Steroid hormone (hydrocortisone, oestradiol and testosterone) uptake, storage or induced synthesis in tetrahymena

After cyclodextrin-coated 10(-6) m steroid hormone treatment for 3 days (hormonal imprinting), Tetrahymena cells and their media were analysed by radioimmunoassay for the same hormone and for the presence of the other two. In the absence of hormone treatment, the cells contained no detectable levels of the three steroids. By 2 days in fresh medium following exposure of cells to a 72 h pretreatment of each specific hormone, correspondingly high quantities of hydrocortisone and oestradiol, but lesser quantities of testosterone, were found in both the media and the cells. One week after treatment only traces of hydrocortisone were found, exclusively within the cells themselves. Oestradiol was present in measurable quantities in both cells and media, whereas testosterone was only present in the medium. The presence of the other two hormones to the one used in the pretreatment were not usually present, except that when testosterone had been given, some oestradiol was also detected at 48 h, suggesting Tetrahymena has a functional cytochrome P(450)aromatase.     

Prolonged effect of an H1-receptor blocker antihistamine on the histamine content of white blood cells and mast cells

Hormonal imprinting usually takes place perinatally at the first encounter between the developing receptor and its target hormone, determining the future binding capacity of the receptor for life. Molecules similar to a hormone can cause faulty imprinting also with life-long consequences. Hormone production of the imprinted cell is also durably influenced. In cytogenic organs imprinting can also be provoked in adulthood. At present the effect of a single terfenadine treatment in adult rats on the histamine content of peritoneal cells (lymphocytes, mast cells and the monocyte-macrophage-granulocyte group), white blood cells (lymphocytes, granulocytes, monocytes) and thymic lymphocytes was studied 3 weeks after treatment to clarify the effect of prolonged treatment with an antihistamine in adulthood.The cells were studied by flow cytometric analysis. Peritoneal mast cells contained significantly more and thymic lymphocytes significantly less histamine than controls. In the other cells the differences were not significant. The results support earlier observations on the effect of antihistamines on mast cell histamine release (inhibition) and call attention to the fact that this effect is durable (hormonal imprinting provoked in adults).     

Prolonged impact of pubertal serotonin treatment (hormonal imprinting) on the later serotonin content of white blood cells

The first encounter between the developing receptor and its target hormone establishes the hormonal imprinting which is needed for the normal function of the cell. In the presence of foreign-however able to bind-molecules, faulty imprinting develops with lifelong consequences. Hormonal imprinting influences not only the receptors, but also the later hormone production of cells. The critical time of hormonal imprinting is the perinatal period, however it can be executed sometimes (in continuously differentiating cells) also at puberty. As in earlier experiments single neonatal serotonin treatment caused a life-long alteration of white blood serotonin content in female rats, the early (10-19 day) and late (8 weeks) effect of single pubertal serotonin treatment was studied presently, by using flow cytometry. In contrast to the earlier (neonatal) results, pubertal treatment caused a radical reduction of serotonin content in male's lymphocytes, monocytes, granulocytes and mast cells, independent on the time of study. The effect in females was rather increasing, however uncertain. The experiments call attention to the possible different effects of neonatal and pubertal hormonal imprinting and to the imprintability of blood cells in adolescence.     

Placental hormones,genomic imprinting,and maternal—fetal communication

Placental hormones are produced by one genetic individual (the fetus) to act on the receptors of another genetic individual (the mother). Mothers are probably able to extract some information from placental hormones, but this information may be limited to a crude measure of fetal vigor. Placental hormones are most easily interpreted as fetal attempts to manipulate maternal metabolism for fetal benefit. An evolutionary model is presented for a hypothetical hormone that increases the nutrient content of maternal blood. The model predicts that, at an evolutionary equilibrium, the hormone will be produced solely by the mother or solely by the placenta, but not by both. If the gene for the hormone is subject to genomic imprinting, the paternally-derived allele will be active and the maternally-derived allele will be silent. Hormone production benefits the members of the mother's current litter at some cost to future litters. Therefore, paternity changes between litters increase the level of hormone production. On the other hand, offspring that produce less of the hormone than litter-mates share the benefits but have lower costs. Therefore, multiple paternity within litters reduces the level of hormone production.     

Influence of hormone concentration and time factor on development of receptor memory in a unicellular (Tetrahymena) model system

1. Treatment of Tetrahymena pyriformis cells with diiodotyrosine (T2) gave rise to a considerable, concentration-dependent increase of the growth rate within the range of 10(-15) and 10(-9) M, but did not influence it at the level of 10(-18) M. 2. Re-exposure of the cells 1, 2 and 4 weeks later to the hormone concentrations originally used accounted for a marked increase of growth rate at all hormone levels tested, indicating that the extremely low concentration of 10(-18) M, which failed to stimulate growth on first exposure, did nevertheless give rise to hormonal imprinting, which caused the cells to "remember" the hormone, as judged from their increased responsiveness to it on re-exposure. 3. The degree of growth response was concentration-dependent on both first and second exposure: higher levels of treatment gave rise to firmer imprinting, and to greater response on re-exposure. 4. The length of exposure time proved to be more decisive than the level of treatment in respect of the development of hormonal imprinting. 5. Short-term exposures up to 60 min, although they stimulated cell growth by direct effect, gave rise to lasting inhibition of cellular response to re-exposure(s) rather than to hormonal imprinting.     

Study of the imprinting and overlap of insulin and concanavalin-A at the receptor level in a protozoan (Tetrahymena) model system

In a protozoan (Tetrahymena) model system, insulin treatment produced a long-term imprinting which upon re-exposure to the hormone resulted in an enhanced binding of the hormone. Insulin pretreatment produced similar effect with regard to the binding of concanavalin-A. Concanavalin-A could only induce a short-term imprinting for itself and was not capable at all of inducing imprinting for insulin. Based on the results of this study it appears that the binding of the sugar component of the receptor, which can be achieved also by lectin, is not sufficient to induce imprinting but the whole (hormone) molecule is needed.     

The biological basis and clinical significance of hormonal imprinting, an epigenetic process

The biological phenomenon, hormonal imprinting, was named and defined by us (Biol Rev, 1980, 55, 47-63) 30?years ago, after many experimental works and observations. Later, similar phenomena were also named to epigenetic imprinting or metabolic imprinting. In the case of hormonal imprinting, the first encounter between a hormone and its developing target cell receptor-usually at the perinatal period-determines the normal receptor-hormone connection for life. However, in this period, molecules similar to the target hormone (members of the same hormone family, synthetic drugs, environmental pollutants, etc), which are also able to bind to the receptor, provoke faulty imprinting also with lifelong-receptorial, behavioral, etc.,-consequences. Faulty hormonal imprinting could also be provoked later in life in continuously dividing cells and in the brain. Faulty hormonal imprinting is a disturbance of gene methylation pattern, which is epigenenetically inherited to the further generations (transgenerational imprinting). The absence of the normal or the presence of false hormonal imprinting predispose to or manifested in different diseases (e.g., malignant tumors, metabolic syndrome) long after the time of imprinting or in the progenies.     

Experimental observations on the mechanism of hormonal imprinting: influence of actinomycin D, methylamine and colchicine on receptor memory in a unicellular model system

The first interaction between target cell and hormone gives rise to hormonal imprinting, which accounts for greater responsiveness of the cell at later interactions. The mechanism of hormonal imprinting is obscure; we based experimental approach to its closer study on combined treatment of Tetrahymena, as model cells, with diiodotyrosine (T2), which stimulates the division, and cell growth inhibitors, which interfere with different stages of cell reproduction, and methylamine, which inhibits cluster formation in the membrane. Of these, actinomycin D and methylamine inhibited the growth of the Tetrahymena, while colchicine did not, and all three suppressed the division stimulating action of T2, but could not prevent hormonal imprinting, as demonstrated on later re-exposure to T2 of cells preexposed and not preexposed to T2 in combination with the inhibitors. It appears that the underlying mechanism of hormonal imprinting is highly complex, and involves many subcellular mechanisms and structures, but suppression of, or gross interference with, one or another of these cannot delete, only quantitatively reduce, the consequence of the first interaction with the hormone, i.e. hormonal imprinting.     

Influence of hormone treatment applied or begun in different phases of the cell cycle on hormonal imprinting in Tetrahymena

Primary exposure to a hormone (hormonal imprinting) alters--in the case of the Tetrahymena increases--cellular response to re-exposure(s) to the same hormone. The intensity of hormonal imprinting depends on the phase of the cell cycle in which the primary exposure has taken place. The effect of imprinting was greater on the cells exposed to the hormone in phase G1 than on those exposed in phase S or G2. The response pattern of the progeny generations corresponded to that of the primarily exposed (imprinted) ancestor cell, irrespective of their own pre-exposure in phase G1, G2 or S of their cycle.     

Studies into hormonal imprinting in intact and transformed 3T3 fibroblast cell lines

The cells of the NIH 3T3 fibroblast line responded to primary interaction with insulin by a positive imprinting, i.e. by an increased binding capacity for the hormone on re-exposure. Positive imprinting, although to a lesser degree, was also induced by thyrotropin. However, oncogenic transformation by polyoma virus oncogens resulted in decreased imprinting in both the middle-T-antigen (MT3) and small-T-antigen-expressing (N4) cells.     

Influence of hormone concentration and time factor on TSH-FSH imprinting and overlap in CHO cells

Exposure of Chinese hamster ovarian cell cultures (cell line CHO) to TSH of FSH gave rise to hormonal imprinting. In earlier studies re-exposure after 48 h displayed a considerable increase in hormone binding. In the present experiments similar increase was demonstrated with an interval of five days. After 14 days, the increment was of lesser degree or even a decrease was noted in hormone-binding capacity. Although the CHO line originates from the target cells of gonadotropin, long-term positive imprinting was greater for TSH than for FSH, imprinting for FSH being negative rather than positive. The experimental results suggest that even very low concentrations (10(-13) mol) of hormone induce imprinting after an exposure as short as 60 min.     

Studies into secretions of Tetrahymena: enzymes secreted into inorganic medium.

The peptides secreted by Tetrahymena cells into inorganic medium were chromatographed. Six fractions showing a marked enzyme-like activity were examined for influence on certain physiological parameters of Tetrahymena. The enzymatically active fractions increased the phagocytic activity of Tetrahymena and decreased its binding capacity for lectins and hormone (insulin), but enhanced insulin imprinting at primary interaction. It remains to be clarified whether these effects were due to the enzymatic or other components of the fractions investigated, or to lack of the compensatory influence of the fractions not studied.     

Studies into hormonal imprinting in a Tetrahymena thermophila mutant incapable of lysosomal enzyme secretion

Reexposure to insulin after primary interaction (hormonal imprinting) was followed by a binding increase in T. pyriformis and by a binding decrease in T. thermophila. The sec. mutant, MS-1 strain of T. thermophila, which is unable of lysosomal enzyme secretion, also showed a binding increase on a second exposure to insulin, from which it follows that alteration of the enzyme secretion, or other factors associated with mutation, accounted for reversion of the trend of imprinting. Thyrotropic hormone (TSH) also gave rise to a negative imprinting in T. thermophila, but did not alter the binding relations of the MS-1 mutant strain.