The developmental neuroendocrinology of reproduction and adaptation: lessons from animal research

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The developmental neuroendocrinology of reproduction and adaptation: lessons from animal research

A.G. Reznikov

V.P. Komisarenko Institute of Endocrinology and Metabolism of National Academy of Medical Sciences of Ukraine, 04114 Kyiv

In order to commemorate the 30th anniversary of the establishing Department of Endocrinology of Reproduction and Adaptation at the V.P. Komisarenko Institute of Endocrinology and Metabolism (Kyiv, Ukraine), the results of animal research in the field of developmental neuroendocrinology of reproduction and adaptation in early ontogenesis are reviewed in the article. Special attitude is paid to sex differentiation of the brain and developmental programming of hypothalamic-pituitary-adrenal axis. Presented are reprogramming effects of perinatal steroids, stress, some drugs, and chemical endocrine disruptors on the developing brain. Phenomenology and neurochemical mechanisms underlying hormone-neurotransmitter imprinting of morphology of the hypothalamus, sexual behavior, reproductive and endocrine functions, and stress reactivity are under discussion. The results of the studies could contribute to prenatal prevention of neuroendocrine and behavioral disorders.

Key words: brain; sexual differentiation; androgens; estrogens; glucocorticoids; neurotransmitters; prenatal stress; sexual behavior; stress reactivity; endocrine disruptors; rat.

INTRODUCTION

animal neuroendocrinology reproduction programming hypothalamic pituitary adrenal

Androgen-dependent sex brain differentiation

In both sexes, an immature brain is potentially capable of developing along with the female type. In adulthood, its manifestations are female sexual behavior and the ability for pre-ovulation estrogen-induced surge of pituitary LH. Fetal testicular (in males) or exogenous (in females) androgens program hypothalamic and other neuroendocrine centers of sexual behavior and regulation of gonadotropin secretion towards male type. In rodents (rats, mice, hamsters), the critical period of SDB covers the last days before birth and a few postnatal days (PND). Early research by our team aimed at clarification of pathogenesis of early life androgen-induced endocrine and behavioral abnormalities in female rats [8-12].

Newborn Wistar female pups were given testosterone propionate (TP) oil solution subcutaneously in daily doses of 150 mcg to 1.25 mg per animal once or repeatedly. Subsequently, they developed permanent estrus or a dramatic violation of the estrous cycles (persistent or irregular estrus), as well as polycystic ovaries. The contents of progesterone and 20a-hydroxyprogesterone in the ovaries, isolated with two-dimensional thin-layer chromatography in Silicagel, reduced significantly. Blood plasma progesterone levels measured by radioimmunoassay in females with persistent estrus decreased significantly as well. With moderate androgenization, estradiol-17p levels decreased compared with those in normal animals and were increased in animals after the highest dose of TP. Surprisingly, LH levels in plasma that have been determined with the rat LH radioimmunoassay did not change, while contents of bioactive LH in the adenohypophysis and LH-RH in the hypothalamus decreased. In neonatally androgen-sterilized females, which were ovariectomized at age of 4-5 months, the hypothalamus, adenohypophysis, and uterus tissue binding of 6,7-3H-estradiol-17p with high specific activity (10 Ki/mmole) were significantly reduced.

In parallel with disorders of neuroendocrine control of reproductive functions, in neonatally androgenized females (NAF), the programming of HPA axis function is modified. It was suggested that the sex differences of HPA axis function in adults might be the result of sex hormone- dependent organization of the brain [13]. In rats, there is a coincidence in time between the organizing effect of sex hormones on the brain and the maturation of HPA axis [14]. Investigation of the mechanisms that cause long-term disorders of HPA axis in NAF has shown that they are accompanied by changes in the expression of corticoliberin mRNA and the content of this neurohormone in the hypothalamus, as well as an increase in the content of glucocorticoid receptor in paraventricular nuclei [15].

Photo. From left to right: P.V. Sinitsin, L.I. Polyakova, N.D. Nosenko, A.G. Reznikov, L.V Chaykovskaya, L.V.Tarasenko

We studied the condition of HPA axis in 6-month-old female rats with persistent estrus who received 250 mcg TP on PND 3. Blood plasma corticosterone levels were measured before and after 1 h strict immobilization. In control females, immobilization induced a 2.2- fold increase in corticosterone level on average, whereas, in NAF, the response to an acute stress was completely absent. One possible reason for this might be estrogen deficiency in females with polycystic ovaries. However, a more important pathogenetic mechanism is probably a violation of the hypothalamic neurotransmitter regulation that was confirmed in further experiments.

Anovulatory status caused by the injections of 150 meg TP during PND 2-4, was characterized by a statistically significant decrease in the hypothalamic concentration of noradrenaline to 1.53 ± 0.15 x 10^-9 g/mg tissue (control - 2.46 ± 0.13x 10^-9 g/mg tissue) and dopamine up to 1.46 ± 0.37 x 10^-9 g/mg tissue (control - 3.35 ± 0.31 x 10^-9 g/mg tissue). Serotonin concentration did not change. The histamine content increased to 1.25 ± 0.10 x 10^-9 g/mg from 0.91 ± 0.07 x 10^-9 g/mg in the control group, the acetylcholine concentration decreased to 0.68 ± 0.07 x 10^-9 g/ mg from 0.97 ± 0.08 x 10^-9 g/mg in the control group.

Response of HPA axis to noradrenergic stimulation was studied in females aged 8 months. The experiments were carried out on unanaestetized, freely moving in the cage animals with a pre-installed metal cannula in the third brain ventricle and a Sylastic catheter in the external jugular vein. 10 mcg of noradrenaline bitartrate in 2 mcl of apyrogenic saline were infused for 1 min into the brain ventricle. Blood samples for spectrofluorometric assay of plasma corticosterone were collected before and after noradrenaline bitartrate infusion at intervals of 30 min, followed by replacement with an equal volume of heparin solution.

In control females, in response to noradrenergic stimulation corticosterone level grew 1.6 times on average after 30 min., then starting from 60 min it approached the baseline. In NAF, noradrenergic stimulation did not cause an increase in corticosterone levels at all.

A decrease in hypothalamic concentrations of noradrenaline and acetylcholine correlates with decrease in the content of hypophyseal gonadotropins and hypothalamic LH-RH in NAF, and an increase in histamine concentration correlates with an increased prolactin in adenohypophysis (apparently due to inhibition of secretion). Supposedly, catecholamine deficiency in the hypothalamus and HPA axis refractoriness to noradrenergic stimulation of the circumventricular structures are relevant pathogenetic mechanisms of impaired HPA response to an acute stress.

Our studies, similarly to ones by other researchers, have shown significant weakening of female sexual behavior and the presence of homo- and bisexual behavior induced by neonatal TP. In particular, the lordosis index in NAF who were ovariectomized and primed with estradiol and progesterone decreased ten times.

In search of early markers of SDB, we turned to proteins of neuroendocrine structures. Two proteins were found in the preoptic region, the contents of which differ in individuals of different sex. According to some data [16-17], the content of brain proteins with molecular masses of 53-56 kDa in neonatal rats can reflect sexual differences in the level of microtubulin, which is a marker of testosterone-induced neuronal growth. However, it remained unknown if the sexual features of the protein spectrum are due to genetic inheritance or whether hormones program them during perinatal development. Using disc electrophoresis in agar gel and densitometry, the spectrum of soluble proteins in the discrete brain areas involved in the neuroendocrine regulation of reproduction and HPA function was explored in our laboratory in normal males, females and NAF [18]. In normal rats, sex differences were found in a wide range of proteins mostly in the preoptic area (POA) of the hypothalamus, to a lesser extent in the mediobasal hypothalamus (MBH) at PND 5-10. In particular, the ratio of protein with a molecular weight of 66 kDa in the POA was 71% higher in males. Neonatal androgenization eliminated sex differences in the protein spectrum in this area by increasing the percentage optical density of the protein bands to a level that of normal males.

The most reliable early marker of impaired SDB in NAF is the changes in the steroid aromatase activity (AA) in the hypothalamus. This enzyme catalyzes the conversion of testosterone to estradiol and androstenedione to estrone. Testosterone induces aromatase synthesis in the hypothalamus. AA differs in male and female rats, especially in the POA, where it is higher in males [12, 19].

We studied in vitro conversion of [1,2,6,7- 3H] testosterone to radiolabeled estradiol-17p and 5a-reduced metabolites in the supernatant (1000 g) of 10% brain tissue homogenate in the presence of a NADP-H-generating system in NAF and normal rats after isolation of steroids with two-dimensional thin-layer chromatography in Silicagel followed by P-radiometry [6]. In normal males, AA in the POA was three- five times higher than that of females, and the 5a-reductase activity, on the contrary, was significantly lower. Sex differences of 5a-reductase activity were not detected in either POA or MBA. Neonatally administered TP induced in females elevation of AA in the POA measured on 10th postnatal day with no significant changes in 5a-reductase activity in both areas studied.

According to some early reports from Japan and the USA, 5a-dihydrotestosterone and other non-aromatizable androgens being introduced parenterally or intracerebrally to newborn females were unable to program masculinization of developing brain. On the other hand, inhibition of aromatase prevented the brain defeminization in male rats [20]. We contributed to the hypothesis of mediating the role of estrogenic testosterone metabolites in SDB by studying the long-term effects of androgen receptor antagonists, flutamide and hydroxyflutamide, and steroid aromataze inhibitors, androst-1,4,6- trien-3,17-dion and androst-4-en-3,6,17-trion, on NAF. Both enzyme inhibitors reduced by 6 times the frequency of anovulation and persistent estrus caused by the injection of 50 mcg of TP on the PND 5 [21]. The blockade of androgen receptor signaling did not have a preventive effect that confirms the role of the conversion of testosterone to estrogens, which does not require the participation of the androgen receptor.

The next step in our research of the neurochemical mechanisms of androgen-dependent SDB was elucidation of the participation of catechol estrogens, which are formed in the hypothalamus from monophenolic estrogens (estradiol, estrone) by hydroxylation at positions 2 or 4 of ring A in a steroid molecule. They are capable of binding to estrogen receptors and initiating signaling pathways. The content of catechol estrogens in the hypothalamus is several times higher than that of estradiol and estrone; as ovulation inducers, they are superior to estradiol.

It has been reported that the administration of 2-hydroxy- or 4-hydroxyestradiol-17p to newborn female rats reproduces the effects of neonatal androgenization on the development of anovulation [22]. We investigated the ability of 2-hydroxyestrone, as well as the isomers of catechol estradiol, 2- and 4-hydroxylated estradiol-17p, and 4-hydroxylated estradiol-17a, with systemic (subcutaneous) or intracerebral administration in doses of 5-25 mcg to newborn female rats to cause the brain defeminization. In contrast to the above authors, there was no disturbance of the estrous cycle and ovulation in adulthood in animals subjected to 2-hydroxyl- ated estradiol-17p. Similar negative results were obtained in experiments with 2-hydroxyestrone and 4-hydroxylated estradiol-17a. Instead, 4-hydroxyestradiol-17p caused the same effect as TP. Probably the difference in the defeminiz- ing activity of steroids is due to the known lower metabolic clearance and the much greater affinity of 4-hydroxyestradiol-17p to estrogen receptor as compared to other catechol estrogens.

One of the interesting phenomena is an increase in the content of noradrenaline in the whole brain of newborn female rats under the influence of testosterone [23]. We have found out a twofold increase in noradrenaline content in the hypothalamus of seven-day-old female rats to the level of normal males as a result of the administration of 250 mcg TP on PND 3 (7.3 ± 2.1 x 10^-9 g/mg tissue vs 2.6 ± 0.1 x 10^-9 g/mg in controls, P < 0.05). Nevertheless, it remained unknown whether this phenomenon is relevant to SDB or to another testosterone effects. Dopamine content remained unchanged, and serotonin decreased slightly.

According to our research, the introduction of a-methyl-p-tyrosine, an inhibitor of catecholamine synthesis, prevented in newborn male rats the programming of hypothalamus refractoriness for the stimulating effect of estrogen on the regulation of LH secretion in adulthood. This evidenced by the appearance of luteal bodies in the fragments of ovaries of prepuberal females transplanted into the anterior chamber of experimental males [24]. Therefore, hypothalamic noradrenaline is critically important for the normal differentiation of the male hypothalamus, and its deficiency leads to the genetically determined brain programming by the female type.

In experiments on NAF, there were used alpha- and beta-blockers, presynaptic blockers of catecholamine and serotonin synthesis, cate- chol-O-methyltransferase and steroid aromatase inhibitors and other agents. TP at a dose of 25 mcg was injected on PND 3 or 4. At the same time, one of the neurotropic drugs was administered, and the treatment continued for a total of 5 days. Cyclic morphological changes in vaginal smears, ovarian morphology, concentrations of estradiol, progesterone, and LH in blood plasma were investigated at the age of 3 months.

a-Methyl-p-tyrosine was the most effective protector of anovulation syndrome. It preserved the proestrus surge of LH secretion and prevented cystic transformation of the ovaries. Another argument for the participation of noradrenaline in SDB was that an additional increase in the concentration of noradrenaline in the hypothalamus of NAF by giving them tropolone, a catechol-O-methyltransferase inhibitor (100 mcg from 4th to 10th day), increased the number of animals with anovulation syndrome from 70 to 100%, that is augmented defeminizing effect of TP [25]. However, tropolone-induced increase in noradrenaline content in the hypothalamus of non-androgenized neonatal females did not cause anovulation in adulthood. Therefore, noradrenaline alone is unable to direct the development of the neuroendocrine system along with the male type; it is only effective in cooperation with steroids.

We were lucky to find out the neurochemical mechanism of increasing the concentration of hypothalamic noradrenaline under the influence of neonatal testosterone. Since the 70s of the last century, it was known that 2-hydroxyestra- diol and 2-hydroxyestrone inhibit catechol-O- methyltransferase activity in the brain [26]. This enzyme metabolizes catecholamines and catechol estrogens by transferring a methyl group from S-adenosylmethionine to one of the phenolic hydroxyl groups. The consequence of the competition between catechol estrogens and noradrenaline for the active sites of catechol-O- methyltransferase is the inhibition of the conversion of noradrenaline to normethanephrine and an increase in the concentration of noradrenaline in tissues.

We hypothesized that 4-hydroxyestradiol- 17p also has a similar property, and as a testosterone mediator in the process of SDB is responsible for the increase in hypothalamic noradrenaline level. This was confirmed by the results of studies, according to which steroid aromatase inhibitors prevented the rise of hypothalamic noradrenaline in NAF [27]. In addition, 24 h after subcutaneous injections of 4-hydroxyestradiol-17p to normal female rats at a daily dose of 10 mcg during the first postnatal days, the concentration of noradrenaline in the hypothalamus increased from 3.04 ± 0.20 x 10^-9 mole/g tissue to 5.50 ± 0.62 x 10^-9 mole/g tissue [11, 28]. The concentration of dopamine remained unchanged. Thus, it was proved that the increase in the concentration of noradrenaline in the hypothalamus under the influence of 4-hydroxyestradiol-17p is associated with defeminizing effect of the latter in females during the critical period of SDB.

Noticeably, the blockade of adrenergic receptors was not capable of interfering with testosterone to disrupt SDB. We have come to the conclusion that the participation of noradrenaline in SDB does not require synaptic transmission, that is, catecholamine in this case acts as a “pre-nerve” inducer of neurocytes differentiation.

The association between changes in noradrenaline levels in the hypothalamus of newborn rats and long-term consequences of SDB is presented in the diagram (Fig. 1). Alteration of the neurochemical phenotype of the brain as a consequence of programming caused by neonatal androgenization reflects the key mechanisms in the pathogenesis of male-type brain differentiation. Androgen-dependent SDB is the result of the cooperative action of catecholamines and neurosteroids, the last ones formed in the nervous tissue are estrogens and catechol estrogens. We have called this phenomenon a hormone-neurotransmitter con-induction that provides differentiation and functional maturation of immature neurocytes by epigenetic imprinting. We believe that estrogens (in particular, 4-hydroxyestradiol-17P) and noradrenaline are the substantial neurochemical determinants of androgen-dependent SDB [10, 29]. Estrogen involvement in SDB is necessary considering that estrogen receptor antagonists prevent testosterone-induced anovulation development as well as steroid aromatase inhibitors. A commonly recognized activity of estrogens is stimulation of synaptogenesis in neuroendocrine structures of the developing brain, which is essential for SDB.

Given the role of calcium ions in neurogenesis (neurocytes differentiation, migration, proliferation, apoptosis), it was considered appropriate to investigate their involvement in SDB. We studied the effects of neonatal administration of verapamil, a calcium L-type channel blocker, on the stress and noradrenergic responses of HPA axis in adult female rats with anovulatory syndrome induced by neonatal androgenization (250 mcg TP on PND 3). Although the pharmacodynamic effects of verapamil occur predominantly in the myocardium and blood vessels, it is able to penetrate the blood-brain barrier and block membrane calcium channels in the brain neurons [30].

There were no HPA axis responses to 1 h immobilization or noradrenaline bitartrate (10 mcg) infusion in the third brain ventricle in females with persistent estrus. Verapamil treatment (50 mcg/100 g b.w., s/c for PND 3-7) preserved the rise in plasma corticosterone in both cases. These finding demonstrates the possible involvement of calcium signaling in the pathogenesis of the HPA disorders in NAF.

The proposed mechanisms of early differentiation of neuroendocrine control of gonadotropin secretion, sexual behavior, and HPA axis function in NAF are shown in the scheme (Fig. 2).

Prenatal glucocorticoid effects on developing brain

Hydrocortisone. The developing brain is characterized by extremely high plasticity and is very sensitive to changes in hormonal balance. Glucocorticoids of maternal or exogenous origin easily cross the placental barrier and play an important role in the differentiation and programming of fetal neurons [31, 32].

According to numerous animal research, prenatal glucocorticoids cause a variety of behavioral disorders in adult life, modification of the basal level of corticosteroids, HPA axis reactions to stress stimuli, violation of reciprocal relationships between the adrenal glands and the hypothalamic-pituitary complex. The latter is associated with a decrease in the expression of corticoliberin mRNA in the hypothalamus, and with a change in the density of corticosteroid receptor distribution in the brain, in particular, in the hippocampus [31]. One of the possible mechanisms of the programming effect of glucocorticoids on the developing brain is inhibition of the neurocytes apoptosis. From a practical point of view, it is of importance that the administration of glucocorticoids in the perinatal period is associated with long-term adverse effects on neurodevelopment [33].

Our studies in this field concerned the effects of prenatal glucocorticoids on Wistar rats. Although corticosterone is the main glucocorticoid in rats, hydrocortisone was used in the experiments, because it is very close to corticosterone in terms of hormonal activities.

Suspension of hydrocortisone acetate (HA) was administered subcutaneously to pregnant females at a daily dose of 50 mg/kg b.w. on gestational days 15-21 or 16-18. Due to the hormone supplementation during the 15-21th day of pregnancy, the level of 11-hydroxycorti- costeroids (hydrocortisone plus corticosterone) in blood plasma on the 21st day increased by an average of 53%. Early and long-term postnatal effects of prenatal HA on sex-related peculiarities of the brain morphology, biogenic monoamine contents and turnover, testosterone metabolism, adrenocortical responses to an acute stress and noradrenergic reactivity of HPA axis were studied [12, 34-36].

Disorders of SDB have been revealed in the next few days after birth. The sex differences of AA in the POA on PND 10 were absent because of an increase in enzyme activity in female offspring. In the MBH, aromatase sexual dimorphism was absent in both control and experimental animals, although AA in the experimental females was significantly reduced compared to the corresponding control.

Prenatal HA impaired the formation of 5a-reduced metabolites of testosterone in the MBH. Under normal conditions, the activity of 5a-reductase (measured as the sum of 5 a-dihydrotestosterone and 5a-androstan-3a,17p- diol, formed in vitro from testosterone for 1 h per g of protein) in the POA of females was higher than that of males (on average, 329 U vs 212 U, P < 0.05 by the nonparametric criterion U). In experimental rats, this difference disappeared (females - 237 U, males - 190 U protein, P > 0.05). The concentration of dopamine in the POA did not change.

Glucocorticoid treatment eliminated the sex difference in noradrenaline concentrations in the POA of 10-day-old offspring due to a decrease in females (control: females, 4.22 ± 0.48 x 10^-9 mole/g tissue; males, 2.74 ± 0.34 x 10^-9 mole/g tissue, P < 0.05; HA: females, 2.64 ± 0.37 x 10^-9 mole/g tissue; males, 2.16 ± 0.31 x 10^-9 mole g tissue, P > 0.05). Probably, this phenomenon relates to the programming inhibitory effect of prenatal HA on the rat hypothalamic activity of tyrosine hydroxylase, a limiting enzyme in catecholamine biosynthesis, in the fetal brain [37].

In the study of the rate of catecholamine turnover in the discrete structures of the hypothalamus of 10-day-old rats there was used tyrosine hydroxylase inhibitor, a-methyl-p-tyrosine, which was injected subcutaneously 1 h or 2 h before decapitation of animals. Catecholamine turnover rates were calculated according to Brodie et al. [38]. It is known that a change in turnover rate during the first hour after injection reflects the velocity of using the functional catecholamine pool.

Prenatal HA led to a significant decrease in noradrenaline turnover in the male POA (0.33 ± 0.09 x 10^-9 mole/g-h vs 1.89 ± 0.45 x 10^-9 mole/g-h in control males, P < 0.05) and an increase in the dopamine turnover rate in females (0.69 ± 0.12 x 10^-9 mole/g-h vst 0.33 ± 0.11 x 10^-9 mole/g-h in control females, P < 0.05). There were no changes in these values in the MBH of the experimental animals.

No sex differences were detected in serotonin and 5-hydroxyindolacetic acid levels assayed by high-performance liquid chromatography in both areas of the hypothalamus of normal rats. Prenatal HA caused the appearance of sex differences in the concentration of 5-hy- droxyindolacetic acid in both POA and MBH and serotonin metabolism - in the POA due to an increase in these parameters in females. The concentration of serotonin in the POA was higher in the experimental males.

Fig. 1. Dependence of the sex brain differentiation on changes in the concentration of noradrenaline in the hypothalamus of female rats on the 7th day after birth (shown with the arrows).

Abbreviations: TP - TP; MPT - a-methyl-p-tyrosine; AI - aromatase inhibitor; 4-OH-E2 - 4-hydroxyestradiol- 17P; Tr - tropolone

Fig. 2. Neurochemical determinants of the sex brain differentiation in neonatally androgenized female rat. Abbreviations: HPA axis - hypothalamic-pituitary-adrenal axis; COMT - catechol-O-methyltransferase

Prenatal HA affected the characteristics of SDB and HPA axis function in adulthood (6-8 months). Samples from experimental and control females were collected in the diestrus stage.

According to our research, a neuromorpho- logical marker of HA-induced SDB disorders was the disappearance of sexual dimorphism of the neurocyte nuclei size in medial preoptic (MPN) and suprachiasmatic nuclei of the hypothalamus. In normal rats, it is 20-25% higher in males compared to females. These differences are attenuated in the suprachiasmatic nuclei because of reducing those values to that of normal females. In MPN, sexual dimorphism of karyo- metric indices also disappeared, but by increasing the size in females. These changes correlate with disorders of sexual behavior and fertility in male rats prenatally exposed to HA [39].

It has been confirmed that HPA response to 1 h immobilization weakened in adult male offspring prenatally exposed to HA. This is probably due to the modification of neurotransmitter regulatory systems. HA-exposed males lacked stress-specific decline in the concentration of noradrenaline in the hypothalamus and increased activity in the hippocampal glutamate decarboxylase, which is a limiting enzyme in the synthesis of GABA. In contrast to the males, the experimental females responded to an acute stress with a more expressed elevation of corticosterone level relative to controls associated with a more pronounced decrease in hypothalamic noradrenaline concentration and an increase in glutamate decarboxylase activity in the hippocampus.

On unanesthetized and unrestricted adult females and males, we have studied the HPA response to corticotropic or central noradrenergic stimulation (10 mcg of noradrenaline bitartrate into the 3rd brain ventricle). In contrast to the control males, the HA-exposed animals did not experience corticosterone rise in blood plasma during the 90-minute observation. This did not related to changes in the adrenal cortex, because of maintaining its sensitivity to P-1- 24-corticotropin intravenous infusion either in males or in females.

Thus, induced by prenatal HA changes in the functional state of neuroendocrine system depend on sex.

Dexamethasone. One of the widely used glucocorticoid drugs is the synthetic steroid, dexamethasone. It binds the glucocorticoid receptor with high affinity and almost not metabolized by placental 11-beta-hydroxysteroid dehydrogenase. It crosses the placental barrier freely into the fetal circulatory system and is relatively easy to overcome the fetal blood-brain barrier.

Prenatal administration of dexamethasone led to sexual behavior abnormalities in adult male rat offspring [40, 41]. This preceded by a decrease in AA (by an average of 45%) in the POA of 10-day-old rats. Under the influence of dexamethasone injections (100 mcg/kg daily during the 15-21st days of pregnancy), sex differences in the rate of metabolic conversion of testosterone to estradiol disappeared; however, there were no changes in AA in the MBH [42]. There was found out an inversion of sex differences in the formation of 5a-reduced metabolites of testosterone in the POA and the MBH due to decreased 5a-reductase activity in females.

Changes in HPA axis response to an acute stress in terms of plasma corticosterone levels and hypothalamic noradrenaline concentrations and turnover in adult males and females were the same as those under prenatal HA [43]. Analysis of the GABA_B receptor as a participant of stress-limiting mechanism in the central nervous system was carried out using this receptor agonist, baclofen, which was introduced 30 min before 1 h immobilization intraperitoneally at a dose of 10 mg/kg BW. In control males, baclofen caused a limitation of corticosterone response to an acute stress. Instead, prenatally exposed to dexamethasone animals did not show any stress-response after baclofen premedication. It seems that prenatal dexamethasone damages in males the programming of the stress-limiting GABA_B signaling. In females exposed prenatally to dexamethasone, the stress-induced HPA reaction on the background of the activation of GABA_B receptor persisted: administration of baclofen prior stress restricted the hormonal response similarly to control animals. Thus, alteration of the HPA axis function with prenatal dexamethasone demonstrates distinct sex differences.

Pharmacological analysis of mechanisms of developing brain programming

Prenatal stress syndrome

Prenatal stress syndrome was discovered and named in 1972 by I. Ward 1972 [49]. PS males exhibited low copulatory activity (21% of animals mated and ejaculated vs 64% in the control group) and high rates of lordosis, that is, female sexual behavior. The author associated the identified changes with a stress- induced reduction in fetal testicular testosterone secretion during the critical stage of SDB. G. Dorner and co-workers were the first to obtain evidences that PS syndrome is present in real human life [50, 51]. Environmental pollution, social turbulence, wars, technogenic accidents, fear of losing a job, physical violence, family disputes can all cause severe stress (distress).

Later in various laboratories, it was shown that similar disorders occur in other species of animals, as well as with other methods of stressing - pain, cold, fasting, emotional factors and so on. The accumulation of facts about the negative impact of gestational stress on offspring continues in various countries, such as Israel, where populations are exposed to rocket attacks,

Canada, where natural disasters occur, and so on. The concept of PS syndrome expanded significantly when it became clear that it causes alteration not of sexual behavior only, but also other numerical abnormalities: disorders of lipid and carbohydrate metabolism, changes in stress reactivity of HPA axis, subfertility of females, oxidative stress, etc. in both sexes. Stressing rats at an early postnatal life has similar consequences, and this is due to the fact that SDB has not yet completed during this period.

For many years, our team studied the pathogenesis of PS syndrome and the possibilities of its prevention [7, 12, 34, 44-47, 52-61]. The experimental model was the daily one- hour immobilization of rats in the last week of pregnancy. The effects of PS were examined in rats aged 3, 6-8, 19-28 months.

Below is a list of phenotypic features of PS syndrome. The results of our research are marked with an asterisk.

Male rats

• Homo- or bisexual behavior, decrease in the number of mounts, intromissions, ejaculations, increase of their latent periods *

• Increasing aggressive behavior

• Reduction of the size of the sex-dimorphic nucleus of the anterior hypothalamic region

• Reduction of the volume of neuron nuclei in the MPN and suprachiasmatic nuclei of the hypothalamus *

• Early changes in the concentrations and metabolic turnover of catecholamines in the brain*

• Early decline of testosterone conversion to estradiol-17p in the POA *

• Early disorders of testosterone conversion to 5-alpha-dihydrotestosterone and other 5-alpha-reduced metabolites in the MBH *

• Puberty delay *

• Suppression of pituitary LH response to LH releasing hormone, aromatase inhibitors and androgen receptor antagonists *

• Suppression of pituitary ACTH response to arginine-vasopressin *

• Acceleration of age-related involution of

spermatogenesis *

• Reduction of the response of the adrenal cortex to an acute stress *

• Changes in the number of glucocorticoid receptors in the hippocampus

• Delayed degranulation of hypophyseal corticotropocytes in response to an acute stress *

• Insufficient release and utilization of catecholamines in the hypothalamus in an acute stress *

• Disruption of serotonergic control of HPA axis response to an acute stress *

• HPA axis nordarenergic hypersensitivity *

• Activation of the brain stress-limiting systems (GABA *, atrial natriuretic peptide, melatonin, beta-endorphin*, PG 2, prostacyclin, etc.)

• Disappearance of early sex differences in certain protein in discrete brain regions*

• Violation of lipid and protein peroxidation in the brain

• Decrease in the activity of microsomal monooxygenases in the liver *

Female rats

• Puberty delay *

• Changes in the structure of estrous cycles *

• Decrease in fertility potential and fecundity

• Moderate increase in adrenal cortex response to an acute stress *

• Reduced physical endurance *

• Reduction of the HPA axis noradrenergic sensitivity *

• Increase in the activity of microsomal monooxygenases in the liver *

From this list, it is obvious that the fetal male brain is much more sensitive than in females to the damaging effect of PS. It is proved that the main factor of demasculinization/feminization of the male brain is transient deficiency of testicular testosterone. This is justified by the preventive effect of perinatal use of testosterone on the demasculinization and feminization of sexual behavior of adult males [62] and on testosterone aromatization and protein pattern in the POA of newborns [59] resulting from PS for the last gestational week. Impairment of brain programming in females is probably due to an increase in the secretion of androstenedione by maternal adrenals and its penetration into the fetus's blood circulation.

Changes in the neurochemical determinants of SDB in PS males have been revealed mainly in the POA, the most important locus of androgen-dependent sexual programming of the developing brain. AA in 10-day-old males was almost halved (0.38 ± 0.05 x 10^-12 mole/g-h tissue vs. 0.62 ± 0,07 x10^-12 mole/g-h tissue in control), while in females it did not change.

Sexual differences in the content of noradrenaline in the rat hypothalamus develop during the maturation of the brain catecholaminergic system on the PND 7 and reach a statistical significance on the PND 10. Therefore, we conducted appropriate studies on 10-day-old rats.

Under normal conditions, the concentration of noradrenaline in the POA of males is almost half that of females. This corresponds to a more accelerated metabolic turnover of noradrenaline in males, which we studied using a-methyl-p- tyrosine for blockage of catecholamine synthesis. Sexual differences in the content and circulation of noradrenaline in the male POA disappeared due to their approximation to these parameters in females. In particular, noradrenaline level grew from 2.9 ± 0.2 x 10^-9 mol/g tissue to 4.7 ± 0.6 x 10^-9 mol/g tissue (P < 0.05), while it did not change in females (control - 4.4 ± 0.6 x 10^-9 mol/g tissue, PS - 3.5 ± 0.4 x 10^-9 mol/g tissue, P > 0.05). PS did not change dopamine and serotonin levels in the POA of males.

Changes in soluble protein spectrum also reflect the disorders of SDB after PS. PS eliminated sexual differences in the relative content of 66.0 kDa protein and at the same time caused them to appear in 18.4 kDa protein in the POA. Although under normal conditions the sex difference of the studied protein spectrum was not observed in the MBH and hippocampus, it appeared as a result of PS.

Sex-dimorphic formations of the hypothalamus also include suprachiasmatic nuclei. The karyometry of the neurocytes of these nuclei in 10-day-old PS males demonstrates a change in SDB toward demasculinization. The mean volume of nuclei of neurons in males averaged 233 ± 7 ^m³ vs 193 ± 10 ^m³ in females (P < 0.05). This difference disappeared in the PS rats due to the nuclei volume reduction in males to 189 ± 7 ^m³, while maintaining the volume of 189 ± 21 ^m³ in females. A similar effect of PS was also seen in 3-month-old rats.

Early neurochemical and microstructural changes in the male hypothalamus are predictors of neuroendocrine and behavioral abnormalities in adulthood. PS prolonged latency of the first mount and intromission, and post-ejaculatory refractory period twice, significantly reduced the number of ejaculations. All PS males who were castrated and primed with estradiol and progesterone displayed lordosis reactions in the presence of normal males.

The weakening of the HPA response to one-hour restriction in 3-month-old males was associated with the absence of a stress-induced decrease in noradrenaline concentration in the hypothalamus. Instead, the response of HPA axis to central noradrenergic stimulation lengthened in time. Plasma corticosterone response to P-1- 24-corticotropin stimulation in non-anesthetized males did not change, as well as in experiments with stimulation of corticosterone secretion with corticotropin in vitro by rat adrenal slices. Alterations in the HPA axis were not detected in 3-month-old PS females, although at the age of 18-21 months, corticosterone response to an acute stress was insufficient when compared with controls of the same age.

The state of GABA receptors as a part of a stress-limiting system was analyzed in adult PS rats using GABA_a receptor agonist, muscimol, and GABA_B receptor agonist, baclofen, which were administered intraperitoneally at a dose of 0.1 and 10 mg/kg, respectively, 30 min before immobilization. Under normal conditions, they respond to an acute stress by reducing the plasma corticosterone response. In prenatally stressed adult males, there is a refractoriness of GABA_a and GABA_B receptors to their activation, possibly as a manifestation of compensation for catecholaminergic deficiency.

In order to address the ways and mechanisms of realization of pathogenic effect of maternal stress on the programming of neuroendocrine functions in intrauterine fetus, various pharmacological preparations were used.

Blockade of the HPA axis stress response with dexamethasone (s/c 100 mcg/kg, 30 min before one-hour immobilization of females during 15-21 days of pregnancy) prevented early and long-term negative neurochemical, behavioral, and other PS effects in male progeny. This indicates initiating role of glucocorticoids and their receptors as mediators in the pathogenesis of PS syndrome.