Impact of single neonatal serotonin treatment (hormonal imprinting) on the brain serotonin content and sexual behavior of adult rats

Hormonal imprinting takes place perinatally at the first encounter between the developing receptor and its target hormone. As a consequence of imprinting the receptor accomplishes its maturation and reaches the binding capacity characteristic to the adult age. In the excess of target hormone or presence of molecules similar to the target hormone, which are able to bind to the unmatured receptors, faulty imprinting develops with life-long consequences. At present, serotonin was given to neonatal rats and their sexual activity, brain serotonin level and steroid receptor's binding capacity was measured in adult age. Brain serotonin level was significantly reduced in male's striatum and parallel with this, male's sexual activity significantly increased. In other regions of the male brain (prefrontal cortex, hypothalamus, hippocampus) there was a statistically non-significant tendency for a decrease in serotonin level. No significant differences were detected in female brain values, and there was only slight change in female's sexual activity. There was also no change in the binding capacity of thymic glucocorticoid and uterine estrogen receptors. The experiments call attention to the possibility of perinatal imprinting by a neurotransmitter causing changes in brain neurotransmitter level for life, which is manifested in altered sexual activity.     

Effect of neonatal beta-endorphin imprinting on sexual behavior and brain serotonin level in adult rats

A single dose (3 microg) beta-endorphin was administered to newborn female and male rats (hormonal imprinting). In adult age (at 5 months) sexual behavior, steroid hormone binding capacity and brain serotonin content was studied. Females' sexual activity (lordosis quotient) significantly decreased and more animals protested against mounting (ratio of kicking and crying 21/24 vs. 8/24; p < 0.001). Males' sexual activity did not change, however more males were aggressive (4/10 vs. 1/10). Uterine estrogen receptor density significantly increased and affinity decreased. There was no change in the binding capacity of thymic glucocorticoid receptors. In the brain, five regions were studied for serotonin content. There was a gender difference in serotonin level and the intragroup differences were also high. In the endorphin treated males the serotonin level was significantly lower than in the controls. In the endorphin treated females the intragroup scattering has been significantly reduced. Nociceptin content of the cerebrospinal fluid was not changed. The experiments call attention to the possibility of adjustment of sexual and behavioral sphere by the individually different endorphin surge during labor.     

Effect of single neonatal treatment with the soy bean phytosteroid,genistein on the sexual behavior of adult rats

Hormonal imprinting develops during the perinatal critical period, when the target hormone meets the yet unmatured receptor. As a consequence of imprinting the receptor accomplishes its maturation reaching the binding capacity characteristic to adults. In this period in the presence of foreign molecules similar to the target hormone faulty imprinting may occur with life-long consequences. Soy bean contains phytosteroids which can mimic estrogen effects. In the present experiments single genistein (20 microg) or combined genistein + benzpyrene (20 microg) treatments were done neonatally and the sexual behavior of male and female adult animals was studied. Genistein significantly increased the lordosis quotient of females, which was compensated by neonatal benzpyrene treatment. Genistein also enhanced the sexual activity of males, and this was significantly not reduced by parallel benzpyrene treatment. The results show that neonatal genistein exposure can imprint sexual activity for life and the presence of a second imprinter can modify genistein's behavioral effect.     

Effect of treatment at weaning with the serotonin antagonist mianserin on the brain serotonin and cerebrospinal fluid nocistatin level of adult female rats: a case of late imprinting

Four weeks old (weanling) female rats were treated with the tricyclic antidepressant and histamine/serotonin receptor blocker mianserin for studying its faulty hormonal imprinting effect. Measurements were done four months later. Brain serotonin levels significantly decreased in four regions (hippocampus, hypothalamus, striatum and brainstem), without any change in the cortex. Sexual activity of the treated and control rats was similar. Cerebrospinal fluid nocistatin level was one magnitude higher in the treated rats, than in the controls. The density of uterine estrogen receptors was significantly reduced, while binding capacity of glucocorticoid receptors of liver and thymus remained at control level. The results call attention to the possibility of 1. a broad spectrum imprinting at the time of weaning by a receptor level acting non-hormone molecule 2. imprinting of the brain in a non-neonatal period of life and 3. a very durable (lifelong?) effect of the late imprinting with an antidepressant.     

Direct and transgenerational effect of benzpyrene treatment at adolescent age on the uterine estrogen receptor and thymic glucocorticoid receptor of the adult rat

Hormonal imprinting develops perinatally at the first encounter between the maturing receptor and the target hormone, helping the normal accomplishment of receptor maturation. In the presence of hormone excess or foreign molecules able to bind to the maturing receptor, faulty imprinting takes place, which disturbs the normal receptor function for life. Earlier experiments demonstrated that the effect of faulty perinatal benzpyrene imprinting of the steroid hormone receptors is transmitted to the progeny generations. In certain organs which are maturing later (such as the uterus) imprinting can be executed at adolescence. In the present experiments pubertal benzpyrene imprinting caused a durable decrease in female's estrogen receptor density. The transgenerational effect of this type of imprinting was also studied. The pubertal imprinting of the parents was transgenerationally transmitted to the offspring generation in which--without further treatment--the density (Bmax) of the uterine estrogen receptors was significantly higher than that in the controls. There were measurable effects neither in the affinity (Kd) of uterine estrogen receptors nor in the Kd and Bmax of the male thymus glucocorticoid receptors. The experiments call attention to the profound and comprehensive imprinting effect of the environmental pollutant benzpyrene.     

Effect of tamoxifen treatment at adolescent age on the sexual behaviour and steroid hormone receptor binding of adult female rats

Hormonal imprinting takes place perinatally, at the first encounter between the target hormone and its developing receptor. However, there is a secondary critical period of imprinting at puberty. In these periods molecules similar to the hormones (members of the same hormone family, antagonists, certain environmental pollutants, etc.) can cause faulty imprinting with lifelong consequences. In the present experiments 5+2 days of tamoxifen treatment (120 microg/day) at adolescent age dramatically (from approx. 40% to 10%) reduced the sexual activity (Meyerson index and lordosis quotient) of female rats, soon after the finishment of the treatment and between four to six weeks after treatment. Similar results were observed in animals neonatally treated with allylestrenol and tamoxifen treated at puberty. Thymic glucocorticoid receptor and uterine estrogen receptor binding capacity were not influenced.     

The effect of perinatal hormonal imprinting with 13-cis-retinoic acid (isotretinoin) on the thymic glucocorticoid receptors of female and testosterone level of male adult rats.

In earlier experiments, the long-term effect of perinatal treatment (hormonal imprinting) with all-trans-retinol and all-trans-retinoic acid on the thymic glucocorticoid and uterine estrogen receptors was studied and was found effective. In the present experiments, the imprinting effect of four retinoids (13-cis-retinaldehyde, 13-cis-retinoic acid, 9-cis-retinaldehyde and 9-cis-retinoic acid) was investigated, using receptor kinetic analysis and sexual hormone (testosterone and progesterone) level determinations. Exclusively 13-cis-retinoic acid (isotretinoin) had an effect, significantly decreasing glucocorticoid receptor affinity and increasing serum testosterone level. Relationships with RAR-RXR receptor binding and teratogenicity is discussed.     

Effect of neonatal treatment with mifepristone or tamoxifen on the binding capacity of the thymic glucocorticoid or uterine estrogen receptor of adult rats: data on the mechanism of hormonal imprinting

For studying the mechanism of perinatal hormonal imprinting newborn rats were treated with a single injection of the antihormones, mifepristone (RU486) or tamoxifen (100 microg each). Glucocorticoid receptors of thymi of 6 weeks old male and female, and uterine estrogen receptors of 2 months old female rats were studied for dexamethasone or estradiol binding, respectively. Tamoxifen caused faulty imprinting both in the thymic and uterine receptors, increasing affinity and density of males, and decreasing females' glucocorticoid receptors as well, as decreasing the density of uterine estradiol receptors. Neonatal mifepristone treatment was indifferent to the thymus, and decreasing to density of uterine estrogen receptors. Males' body weight significantly decreased 6 weeks after tamoxifen treatment. The results suggest that imprinting can not be provoked by a molecule (hormone antagonist) which can bind to the receptor without any postreceptorial events (mifepristone/glucocorticoid receptor), in the presence of some postreceptorial effects the reaction takes place, however the strongest reaction can be observed by the hormone analogue (tamoxifen) with postreceptorial (agonist) effect, not considering that the receptor is the direct target of the molecule or a cross-reaction is present.     

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).     

Single neonatal treatment with beta-endorphin (hormonal imprinting) extremely enhances nocistatin level of cerebrospinal fluid in adult rats

In earlier experiments endorphin treatment of newborn rats caused the decrease of brain serotonin content, increasing aggressivity, enhanced sexual activity of females and changes in the binding capacity of uterine estrogen receptors at adult age, however nociceptin content of the cerebrospinal fluid was not changed. In the present experiment neonatal treatment of male and female rats was done with a single dose of 3 microg beta-endorphin and in five months old rats the level of nociceptin antagonist nocistatin was determined by radioimmunoassay in the cerebrospinal fluid. In both genders the amount of nocistatin was one magnitude higher in the endorphin treated groups. There was also a significant difference between the male and female nocistatin level in the treated and non-treated groups alike, with the advantage of females. The results call attention to the possibility of influencing pain-tolerance for life, by the pain-provoked endorphin levels during delivery.     

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.     

Effect of endorphin exposure at weaning on the endorphin and serotonin content of white blood cells and mast cells in adult rat

Hormonal imprinting takes place perinatally at the first encounter between the developing receptor and its target hormone, resulting in the accomplishment of normal receptor development. In the presence of an excess of target hormone or the absence of it, or an excess of related molecules which can be bound by the receptor, faulty imprinting develops with life-long consequences. In previous experiments neonatal endorphin exposure caused a decrease in endorphin and serotonin content of peritoneal mast cells of adult animals. In the present experiment 25-day-old (weaned) female rats received 2 microg endorphin, and the endorphin as well as serotonin content of adult mast cells and white blood cells was studied by flow cytometry and confocal microscopy. Peritoneal lymphocytes and blood monocytes contained significantly (p<0.01) less endorphin and peritoneal mast cells less serotonin (p<0.07, i.e. of questionable significance) than the untreated control. The results bring attention to the possibility of durable imprinting of differentiating cells later in life and to the durable (possibly life-long) effect of an endorphin excess (perhaps caused by injury) manifested in the change of endorphin and serotonin content of immune cells.     

Subcellular distribution of native estrogen receptor alpha and beta isoforms in rabbit uterus and ovary

The association of estrogen receptors with non-nuclear/cytoplasmic compartments in target tissues has been documented. However, limited information is available on the distribution of estrogen receptor isoforms, specially with regard to the newly described beta isotype. The subcellular localization of estrogen receptor alpha and beta isoforms was investigated in rabbit uterus and ovary. Native alpha and beta subtypes were immunodetected using specific antibodies after subjecting the tissue to fractionation by differential centrifugation. The ovary expressed alpha and beta estrogen receptors in predominant association to cytosolic components. However, in the uterus, a substantial proportion of the total estrogen binding capacity and coexpression of the two isoforms was detected in mitochondria and microsomes. The mitochondrial-enriched subfraction represented an important source of 17beta-estradiol binding, where the steroid was recognized in a stereospecific and high affinity manner. The existence of mitochondrial and membrane estrogen binding sites correlated with the presence of estrogen receptor alpha but mainly with estrogen receptor beta proteins. Using macromolecular 17beta-estradiol derivatives in Ligand Blot studies, we could confirm that both alpha and beta isoforms were expressed as the major estrogen binding proteins in the uterus, while estrogen receptor alpha was clearly the dominant isoform in the ovary. Other low molecular weight estrogen receptor alpha-like proteins were found to represent an independent subpopulation of uterine binding sites, expressed to a lesser extent. This differential cellular partitioning of estrogen receptor alpha and beta forms may contribute to the known diversity of 17beta-estradiol effects in target organs. Both estrogen receptor alpha and beta expression levels and cellular localization patterns among tissues, add complexity to the whole estrogen signaling system, in which membrane and mitochondrial events could also be implicated.     

Influence of a single treatment with vitamin E or K (hormonal imprinting) of neonatal rats on the sexual behavior of adults

The effect of a single neonatal treatment (imprinting) with vitamin E or vitamin K1 on the sexual activity of three-month old rats, was studied. In female animals vitamin E treatment significantly lowered the Meyerson index and lordosis quotient, among males there were significantly more inactive animals and no multiple ejaculations could be observed. Vitamin K1 treatment caused only slight changes in the same direction, in both sexes. Considering also earlier results concerning vitamin A and D neonatal treatments (alterations in receptor binding capacity, sex hormone levels and sexual behavior), and receptorial changes caused by neonatal vitamin E and K1 treatments, the present experiment also calls attention to the lifelong effects of perinatal treatment with lipid soluble vitamins.     

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.     

Interaction of serine protease inhibitors and substrates with human uterine estrogen receptor

The human uterine estrogen receptor has a site which regulates estrogen binding and which structurally resembles the substrate binding site of chymotrypsin. The hormone binding capacity and the affinity of the receptor is decreased in the presence of 4 mM serine protease inhibitors tosyl-lysine chloromethyl ketone and diisopropylfluorophosphate and the protease substrates tryptophan methyl ester and toluene sulphonyl-arginine methyl ester. The protease inhibitors tosylamide-phenylethyl-chloromethyl ketone and phenyl methyl sulphonyl fluoride caused an increase in the binding capacity whereas the affinity was decreased.     

Effect of gonadotropin (FSH-LH) and thyrotropin (TSH) treatment in adolescence on TSH-sensitivity in adult rats

Hormonal imprinting is characteristic of the neonatal age, in which the receptor of the target cell matures, i.e. acquires its adult binding capacity, and cellular response becomes established in presence of the adequate hormone. The normal course of imprinting may be altered by certain molecules (related hormones, hormone analogons) which are able to bind to the receptor of the adequate hormone. The chemically related gonadotropic and thyrotropic hormones may overlap on each other's receptors not only in the perinatal age, but also in the early adulthood, and this overlap of the binding may give rise to an imprinting-like effect. An example of this phenomenon was observed in the present study, in which rats of seven weeks of age treated with gonadotropin showed a significant decrease in thyroidic response to TSH, and exposure to TSH failed to increase their basic thyroxine concentration to the normal (control) level. This depressive effect of gonadotropin was slightly reduced in the presence of LPS (endotoxin), causing membrane perturbation, while pretreatment with LPS and TSH accounted for an increased sensitivity to TSH in later phases of the rat's life. These experimental observations support the possibility of a special form of imprinting in adolescence.     

Sexual differentiation of gonadotrophin secretion, sexual orientation and gender role behavior

The positive estrogen feedback was found to be a relatively sex-specific reaction of the hypothalamo-hypophyseal system in rats as well as in human beings. It is dependent--most of all--on the estrogen convertible androgen level during sexual brain differentiation, but also on an estrogen priming effect in adulthood. The lower the estrogen convertible androgen or primary estrogen level during brain differentiation, the higher is the evocability of a positive estrogen action on LH secretion in later life. In clinical studies, we were able to induce a positive estrogen feedback on LH secretion in most intact homosexual men in clear-cut contrast to intact hetero- or bisexual men. These findings were strongly confirmed by Gladue and associates. In addition, the evocability of a positive estrogen feedback was also demonstrable in most homosexual male-to-female transsexuals in significant contrast to hetero- or bisexual male-to-female transsexuals. These findings suggest that homosexual males possess, at least in part, a predominantly female-differentiated brain, which may be caused by a low estrogen convertible androgen level during brain organization. Recently, the following relations were found between sex hormone levels during brain differentiation and sex-specific responses in adulthood: (1) estrogens--which are mostly converted, however, from androgens--are responsible for the sex-specific organization of gonadotrophin secretion and hence the evocability of a positive estrogen feedback in later life; (2) estrogens and androgens, occurring during brain differentiation, predetermine synergistically sexual orientation and (3) androgens--without conversion to estrogens--are responsible for the sex-specific organization of gender role behaviour in later life. Furthermore, the organization periods for sex-specific gonadotrophin secretion, sexual orientation and gender role behaviour are not identical but overlapping. Thus, combinations as well as dissociations between deviations of the neuroendocrine organization of sex-specific gonadotrophin secretion, sexual orientation and gender role behaviour are conceivable. Most recently, female-type sexual orientation could be converted to male-type sexual orientation in adult rats by administration of the dopamine agonist and serotonin antagonist lisuride.     

Single treatment (hormonal imprinting) of newborn rats with serotonin increases the serotonin content of cells in adults

Hormonal imprinting takes place perinatally at the first encounter between the hormone and its target receptor, causing the finishment of the maturation of receptor-signal transduction system. In the presence of an excess of the target hormone or related molecules faulty imprinting develops with life-long consequences. In earlier experiments single neonatal treatment with minute dose of IL-6 caused also prolonged stimulation of IL-6 production. In the present experiment newborn female and male rats were treated with 20 microg serotonin (hormonal imprinting) and were studied for serotonin content of different cell types in adult age. Serotonin content was measured by flow cytometry and its localization was determined by confocal microscopy. Serotonin content was detected in white blood cells (lymphocytes, monocytes and granulocytes); in lymphocytes, monocytes (macrophages), granulocytes and mast cells of peritoneal fluid and thymic lymphocytes. Serotonin was present in all cell types of control animals studied. Serotonin content extremely elevated in the white blood cells and also increased in the peritoneal cells of neonatally treated female animals. There was no elevation in thymic lymphocytes. The mean values of male animals remained at the control level. The experiments call attention to the life-long effect of the perinatal hormonal imprinting manifested presently in the elevation of serotonin content and point to the gender differences of serotonin imprinting. Considering the role of serotonin in mood and psychiatric diseases, the observations could have some clinical importance.     

Prolonged impact of five imprinters on the serotonin content of white blood cells and mast cells of weanling rats: outstanding effect of benzpyrene and chlorpheniramine

In previous experiments, treatment at weaning or adult age with endorphin, serotonin or an antihistamine (late hormonal imprinting) durably influenced the serotonin content of white blood cells and mast cells of rat. In the present experiments, five molecule (approved imprinters in other indexes) were studied for imprinting effect of immune cells, 3 weeks after a single treatment at weaning. Three steroid hormone-like molecule (vitamin D3, mifepristone and dexamethasone) were ineffective (except dexamethasone in 1/4 indexes), while benzpyrene (aromatic hydrocarbon) and chlorpheniramine (H1-receptor blocker antihistamine) were highly effective (5/6 and 4/4 respectively). The results indicate: (1) a prolonged (late imprinting) effect of a single treatment with certain molecules acting at receptor level; (2) non-generality of late imprinting, and (3) the very extensive effects of benzpyrene, which in earlier experiments was one of the strongest imprinter at receptorial and behavioral level at any periods of life studied.