Fiz_zh 3_2004___12

A.G. Reznikov, N.D. Nosenko, L.V. Tarasenko, P.V. Sinitsyn, A.A. Lymareva Prenatal dexamethasone prevents early and long-lasting neuroendocrine and behavioral effects of maternal stress on male offspring  ðîáîò³ ïåðåâ³ðåíî ã³ïîòåçó ïðî ðîëü ãîðìîí³â ã³ïîòàëàìî-ã³ïîô³çàðíî-àäðåíîêîðòèêàëüíî¿ ñèñòåìè (ÃÃÀÑ) â îïîñåðåäêóâàíí³ ³íäóêîâàíèõ ìàòåðèíñüêèì ñòðåñîì íåéðîåíäîêðèííèõ, íåéðîõ³ì³÷íèõ ³ ïîâåä³íêîâèõ çì³í ó íàùàäê³â ÷îëîâ³÷î¿ ñòàò³. Äëÿ ãàëüìóâàííÿ ñòðåñîâî¿ ðåàêö³¿ ÃÃÀÑ áóëî âèêîðèñòàíî äåêñàìåòàçîí, ÿêèé ââîäèëè â äîç³ 0,1 ìã/êã çà 30 õâ äî ïî÷àòêó îäíîãîäèííî¿ ùîäåííî¿ ³ììîá³ë³çàö³¿ ñàìèöÿì ùóð³â ïðîòÿãîì îñòàííüîãî òèæíÿ âàã³òíîñò³.
Ó ïðåíàòàëüíî ñòðåñîâàíèõ ñàìö³â ðàííüîãî ïîñòíàòàëüíîãî â³êó ñïîñòåð³ãàëîñÿ çíèêíåííÿ çàëåæíèõ â³ä ñòàò³ ðîçá³æíîñòåé ó ðîçïîä³ë³ á³ëê³â òà àðîìàòàçí³é àêòèâíîñò³ â ïðåîïòè÷í³é ä³ëÿíö³ ã³ïîòàëàìóñà. Ïðè äîñÿãíåíí³ ñòàòåâîçð³ëîãî â³êó ó öèõ òâàðèí âèÿâëåíî ïîñëàáëåííÿ êîïóëÿòèâíî¿ ïîâåä³íêè, çíèæåííÿ ñòðåñîâî¿ òà íîðàäðåíåðã³÷íî¿ ðåàêòèâíîñò³ ÃÃÀÑ. Ââåäåííÿ äåêñàìåòàçîíó â ïðåíàòàëüíîìó ïåð³îä³ çàïîá³ãàëî ðîçâèòêîâ³ çàçíà÷åíèõ âèùå çì³í, ñïðè÷èíåíèõ ñòðåñîì ìàòåðèíñüêîãî îðãàí³çìó. Îòðèìàí³ ðåçóëüòàòè ñâ³ä÷àòü ïðî âàæëèâó ðîëü ãîðìîí³â ÃÃÀÑ â îïîñåðåäêóâàíí³ ³íäóêîâàíèõ ïðåíàòàëüíèì ñòðåñîì ïîðóøåíü ïðîöåñ³â tion and might contribute to sexual behavioral and reproductive disorders in adulthood [20, A lot of early environmental influences on the 22, 23, 29]. In human offspring, the adverse developing mammalian brain like prenatal behavioral and cognitive consequences of stress, exposure to excessive amount of glu- stress experienced by the woman during natu- cocorticoids, sex steroids, nutrient restriction, ral environmental disaster have been de- maternal deprivation etc. were shown to be scribed in ice-storm study [14].
capable of modifying neuroendocrine func- Maternal stress during the last week of tions and behavior in adulthood. Obviously, pregnancy generates numerous endocrine, altered programming of the hypothalamic-pi- neurotransmitter, cardiovascular and other tuitary-adrenal (HPA) axis that has resulted homeostatic turbulences in the mother and from maternal stress or glucocorticoid treat- fetus. Although HPA axis responses to stres- ment in humans and animals might be associ- sors in late pregnancy seem to be attenuated ated with an impairment of HPA function, [5], many studies focused at the possible key anxiety and depression-like behavior, high risk role of the HPA activation in the pathogene- of hypertension, glucose intolerance, obesity sis of long-term consequences of prenatal and other metabolic disorders in adult offspring stress in primates, guinea pigs, rats, sheep, both in males and females [12, 26, 34]. Pre- pigs and other species (for review see Kapoor natal stress or glucocorticoid treatment of rats et al., 2006). In rats, maternal stress leads to during the third week of pregnancy modifies significant elevation of corticosterone levels the basic neurochemical and neurophysiologi- in maternal and fetal plasma [10, 15, 31], and cal mechanisms of sexual brain differentia- presumably to increased access of maternal A.G. Reznikov, N.D. Nosenko, L.V. Tarasenko, P.V. Sinitsyn, A.A. Lymareva ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 A.G. Reznikov, N.D. Nosenko, L.V. Tarasenko, P.V. Sinitsyn, A.A. Lymareva endogenous glucocorticoid to the fetus due to protocols approved by the Animal Care Com- stress-induced reduction in placental 11β- mission at the Institute, in accordance with hydroxysteroid dehydrogenase type 2 (11β-HSD- European Convention for the Protection of 2) activity (for review see Seckl et al., 2004).
Vertebrate Animals used for Experimental and However, there are a lot of controversies with other Scientific Purposes (Strasbourg, 1986).
regard to mediating role of HPA hormones and Virgin Wistar female rats (200±10 g) were mechanisms by which fetus exposure to stress mated overnight and pregnancy was confirmed in utero modifies the brain development by presence of spermatozoa in vaginal smears on following morning, which was considered It is still not clear whether stress-induced gestational day 1. Time-mated dams (n=40) HPA activation generates multiple neuro- were subjected to 1 h strict immobilization in endocrine and behavioral changes in prenatally supine position during days 15 to 21 of gesta- stressed animals. Some prenatal stress effects, tion. Some of them (n=20) were injected i.m.
for example, changes in the hypothalamic with Dex (KRKA, Slovenia) in a dose of 0.1 noradrenaline content or sex-related HPA mg/kg b.w. 30 min prior to each stress ses- responses to noradrenergic stimulation in rats, sion. Control mothers (n=35) were injected could not be mimicked by fetal exposure to glu- with physiological saline according to the cocorticoids [23, 30]. In the experiments with above protocol and housed in the vivarium with maternal bilateral adrenalectomy it was shown no handling.
that maternal adrenal hormone mediates the HPA Litter size averaged 6 pups. A total of 75 dysregulation and the alterations of anxiogenic litters with 403 males were allocated into and spatial learning behaviors in rat offspring ex- groups. 47 intact females were taken for perienced prenatal stress [36]. However, accord- comparison with control males. 5- and 10-day- ing to some research, prenatal ACTH or old pups and mature descendants aged 3 corticosterone treatments do not impair sexual months (weight range 140-160 g), 6 months behavior in male rats [10].
(weight range 180 – 200 g) or 8 months Bilateral adrenalectomy eliminates an in- (weight range 220 – 250 g) were taken to the crease in adrenal hormone release during study. The difference in the descendant body stress. In the meantime, stressful stimuli pro- mass values within each animal group did not voke a wide range of homeostatic distur- exceed 15 %.
bances. Within HPA system, they involve Brain protein profile surges of CRH, ACTH, vasopressin, β-endor- Soluble cytosolic proteins isolated from the phin secretion, hypothalamic noradrenaline preoptic area (POA) and medial basal hypo- depletion, glutamate excitation and many other thalamus (MBH) were studied in 5-day-old events in neuroendocrine machinery.
offspring (n=140). The male pups from all In order to clarify the role of HPA function experimental groups and both male and fe- in pathogenesis of adverse neuroendocrine and male control pups were quickly decapitated.
behavioral consequences of prenatal stress in POA and MBH were isolated by brain dis- rats, dexamethasone (Dex) blockade of the HPA section, then combined by 5 - 7 tissue sam- axis was used in this study followed by evalua- ples and frozen at –20 oC until being assayed.
tion of the brain physiology and chemistry in pre- The spectrum of soluble proteins from discrete pubertal and mature male offspring.
brain structures was determined by modified Laemmli method [16].
EXPERIMENTAL PROCEDURES The tissues were homogenized in 10 vol- umes of ice-cold buffer, containing 0.25 M of Animals and experimental protocol sucrose, 0.025 M potassium chloride, 0.005 Experiments were performed according to Ì magnesium chloride and 0.05 M tris-HCl, ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 Prenatal dexamethasone prevents pH 7.4, and centrifuged during 60 min at generating system (2 mM NADP, 10 mM glu- 100,000 g. Supernatant was taken for the cose-6-phosphate and 2 IU/ml glucose-6- elctrophoretic analysis. Protein concentration phosphate dehydrogenase) [18]. [4-14C]Estra- was measured by Lowry method [17].
diol was used as an internal standard. Tritiat- Samples of soluble proteins containing 5 % ed estradiol and 5α-reduced androgen metab- SDS, 10 % mercaptoethanol and 40 % sucrose olites which have been derived from testoste- were heated at 100 oC for 5 min. Protein frac- rone were isolated by two-dimensional thin tionation was carried out by plate disc-elec- layer chromatography on Silica gel 60/Kiesel- trophoresis using 5 % polyacrylamide gel guhr F plates (“Merck”, Germany). Radio- (PAAG) as concentrating and 10 % PAAG activity of isolated steroids was recorded in a as dividing ones. Running buffer, ðÍ 8.4, con- two-channel β-spectrometer (Beckman LS tained 0.025 Ì Tris-HCl, 0.1 % sodium 500TA, USA). Aromatase activity was cal- dodecyl sulfate and 0.193 Ì glycine. Elec- culated as an amount of estradiol, and 5α-re- trophoresis was carried out consecutively at ductase activity as a sum of 5α-dihydrotesto- 20 mA (in concentrating gel) and 30 mA (in sterone and 3α-androstandiol produced for 1 h dividing gel) for 40 – 60 min. Gels were per 1 g tissue.
stained overnight at room temperature in the Sexual behavior evaluation mixture containing 9.0 % acetic acid, 45 % Male sexual behavior tests were run in two methanol and 0.025 % Coomassie Brilliant trials. The male rats (n=19) were 87-90 days Blue R250, and then destained in several old in the first trial and 93-96 days old in the changes of 7.5 % acetic acid. LKB MW70 second one. The control, prenatally stressed kit was used as molecular weight protein and Dex-pretreated prenatally stressed males, markers. Slab gels were scanned using Epson were tested during the second half of the dark perfection 1670 scanner. Densitometry was cycle with ovariectomized female rats made carried out by Scion Image computer program.
receptive by hormonal injection (0.1 mg of The results were expressed as relative den- estradiol benzoate per rat, i.m. in oil, 48 h pri- sity value under curve for each protein frac- or to the test day followed by 0.5 mg of tion with regard to total density.
progesterone in oil i.m., 4 h prior to the test).
Brain enzyme activities The test consisted of placing each male to Aromatase (estrogen sythetase, EC 1.14.14.1) mating chamber 5 min before a receptive fe- and 5α-reductase (3-oxo-5a-steroid: NADP+4- male was introduced. Mating chambers were ene oxydoreductase, EC 1.3.1.22) activities illuminated with dim red lights. Each test last- were determined in POA and MBH of 10-day- ed for 30 min. The frequency of mounts, in- old offspring (n=50). The male pups from all tromissions and ejaculations, and the timing animal experimental groups and both male and of each response as well as time to first female control pups were quickly decapitat- mount after the first ejaculation (postejacula- ed. POA and MBH were isolated by brain dis- tory interval) were recorded. This procedure section, then combined by 2 - 3 tissue sam- was repeated in one week. Results were as- ples and frozen at –20 îC until being assayed.
sessed separately for the first and the second For aromatase and 5α-reductase activities determination, the aliquots of 1000 g super- Stress-test procedure natant of the 10 % tissue homogenates were 6-Month-old male offspring (groups of con- incubated during 1 h in Tris-HCl buffer (pH trol, prenatally stressed and Dex-pretreated 7.4) containing [1,2,6,7-3H]testosterone (s.a.
prenatally stressed males, each consisted of 3.74 TBq/mmol, “Amersham”, UK) as the 5-6 animals) was subjected to 1 h strict re- enzyme substrate in the presence of NADP.H straining. The rats were immobilized in supine ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 A.G. Reznikov, N.D. Nosenko, L.V. Tarasenko, P.V. Sinitsyn, A.A. Lymareva position with the extremities being attached to jugular vein before and then 30 and 60 min a small animal desk. Non-stressed animals after noradrenaline infusion. Every sample were used as controls. The animals were was substituted immediately by an equal vol- quickly decapitated immediately after 1 h re- ume of saline containing 50 IU/ml heparin.
striction. Taking into consideration possible Plasma was separated and the aliquots were impact of general anaesthesia on the cate- stored at -20 oC until measurement of corti- cholamine content in the brain tissues eutha- costerone concentrations. As it was revealed nasia was performed without anaesthetic in the previous studies, intracerebroventricu- drugs. The hypothalamus was isolated by brain lar infusions of 2 µl saline do not affect plas- tissue dissection at +4 oC. Only fresh brain ma corticosterone levels.
tissues were used for catecholamine assay.
Catecholamine assay Trunk heparinized blood samples were taken, Noradrenaline and dopamine (3-hydroxy- and the plasma was separated and stored at tyramine) contents were determined in the –20 oC until measurement of corticosterone hypothalamus of 6-month-old male offspring.
Immediately after decapitation, hypothalamus Study of baclofen effects on the HPA stress was excised as rapidly as possible, then weighed and frozen on dry ice. Hypothalamic The experiments were carried out in 6-month- tissue samples were homogenized on ice in old male offspring (n=48). Baclofen, GABA - 0.01 N HCl (1:50 w/v). Catecholamines were receptors agonist (“Sigma”, USA), dissolved extracted with 5 ml of n-butanol. After cen- in apyrogenic isotonic NaCl solution was in- trifugation at 1500 g for 10 min, the superna- jected i.p. in a dose of 10 mg/kg b.w. 30 min tants were placed to the tubes containing pH prior to an acute stress (1 h restriction). The 6.5 phosphate buffer in order to transfer cate- animals were decapitated immediately after cholamines to aqueous phase. All of these 1 h restriction. Trunk heparinized blood sam- procedures were performed at 4 oC. Norad- ples were taken, and the plasma was sepa- renaline and dopamine contents were deter- rated and stored at -20 oC until measurement mined by spectrofluorimetric assay [11] in 0.5 of corticosterone concentrations.
ml aliquotes of supernatant after centrifuga- Study of the HPA responses to noradrener- tion at 3000 g. Catecholamine fluorophores were produced by oxidation of noradrenaline The experiments were carried out in 8-month- and dopamine by adding 125 µl of Versen so- old male offspring (groups of control, prena- lution, 100 µl 0.3 M KI and 125 µl 0.5 M tally stressed and Dex-pretreated prenatally Na SO in 2 min intervals. The reaction was stressed males, each consisted of 6 animals).
stopped by adding 150 µl 5 N acetic acid. The Noradrenergic reactivity of the HPA axis was test tubes were placed into boiling water for tested in conscious unrestrained animals.
5 min followed by 1 min cooling in ice-bath.
Noradrenaline bitartrate in a dose of 10 µg Fluorescence of noradrenaline and dopamine dissolved in 2 µl of apyrogenic isotonic NaCl was measured immediately using 385/485 nm solution (saline) was infused into the 3rd brain and 320/385 nm optical filters, corresponding- ventricle via a needle inserted into a stainless ly. Noradrenaline bitartrate and 3-hydroxy- steel guide cannula that had been implanted 8 tyramine hydrochloride (“Sigma”, USA) were to 9 days before the experiment using stereo- used as standards. The concentrations of the taxic coordinates [2]. Twenty four hours pri- monoamines were expressed as nmol/g of tissue.
or to the experiment, a Silastic catheter was Plasma corticosterone assay inserted into the right external jugular vein [9].
Blood plasma corticosterone concentrations Surgery was performed under ether anesthe- were measured by spectrofluorimetric mi- sia. Blood samples (0.5 ml) were taken from croassay [3]. 100 µl blood plasma samples ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 Prenatal dexamethasone prevents were extracted with 1.5 ml methylene chlo- denser in females (respectively, 10 % and 15 %, ride for 3 min. Aqueous phase containing cho- p<0.05) while the proteins with 34.7 and 66.0 lesterol and other lipids was frozen at –40 oC.
kDa molecular weight – in males (respectively, Organic extracts were poured into the test 30 % and 10 %, P<0.05). There were no gen- tubes, and 0.5 ml of fluorescent reagent con- der-related differences in the electrophoretic taining research grade H SO and dehydrated profiles of proteins studied in the MBH.
ethanol (7 : 3, v/v) was added. The content of As a result of maternal stress, diminution the tubes was mixed and the upper methylene of the density of 66.0 kDa protein was found chloride layer was discarded. Corticosterone in the POA of males (Fig. 1). This modifying fluorescence was measured in 1.5 h using 470/ effect of prenatal stress on the protein was 524 nm optical filters.
prevented by Dex. Prenatal stress alone or Statistical analysis combined with Dex pretreatment did not All data are presented as mean ± SEM. Stu- change the densities of any soluble proteins dent’s t criterion or Wilcoxon’s U test were in the MBH of male pups.
used for evaluation of the differences between Brain enzyme activities experimental groups. P < 0.05 was considered In the POA of prenatally stressed 10-day-old as the borderline of statistic significance.
pups, aromatase activity declined on average by 41.1 % (Fig. 2) and reached the normal female level (for comparison, 0.352 ± 0.072 nmol estradiol.h-1.g tissue-1 in females vs.
Brain protein profile 0.598 ± 0.082 nmol estradiol.h-1.g tissue-1 in In the POA of 5-day-old normal control pups, males, P<0.05). Dex pretreatment completely a significant sexual dimorphism in the density restored the enzyme activity to normal level in of soluble proteins with molecular weights of prenatally stressed males.
14.3 – 66.0 kDa was observed. The 14.3 kDa Prenatal stress did not affect aromatase and 24.0 kDa molecular weight proteins were activity in the MBH tissue samples in males, % of total area under protein density curve Aromatase activity (pmol estradiol/h/g tissue Figure 2. Aromatase activity in the brain preoptic area Figure 1. Changes in 66.0 kDa protein profile in the brain (POA) and medial basal hypothalamus (MBH) in control preoptic area (POA) in prenatally stressed (dark bar), Dex- (light bars), prenatally stressed (dark bars) and Dex-pre- pretreated prenatally stressed (gray bar) 5-day-old males treated prenatally stressed (gray bars) 10-day-old males.
and control 5-day-old females (light bar) related to control Data are means ± SEM. *P<0.05 compared to control 5-day-old males (accepted as 100 %). *P<0.05 compared group; #P<0.05 compared to prenatally stressed males to control 5-day-old males (Wilcoxon’s U-test) (Student’s t-test) ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 A.G. Reznikov, N.D. Nosenko, L.V. Tarasenko, P.V. Sinitsyn, A.A. Lymareva Table 1. Effects of prenatal exposure to Dex on brain 5α-reductase activity in prenatally stressed 10-day-old male rats 5α-reductase activity, pmol 5α-reduced metabolites.h-1.g tissue-1 Prenatally stressed (n=5) Dex-pretreated prenatally stressed (n=6) * P<0.05 compared to controls while in Dex-pretreated ones significant in- the POA. As for 5α-reductase activity, Dex- crease of enzyme activity compared to pre- pretreated prenatally stressed males did not natally stressed males without Dex premedi- significantly differ either from control or from cation was observed.
prenatally stressed pups.
The data of measuring 5α-reductase ac- tivities are presented in Table 1. In the POA When male offsprings were tested for sexual of control males it was twice lower, and in performance in adulthood, some variables of the MBH it was almost 3.5 times less than in male sexual behavior improved over 2 weeks female pups (POA: 11.61 ± 2.06 pmol 5α-re- of testing due to sexual experience acquisi- duced metabolites.h-1.g tissue-1, P<0.05; tion and development of stereotype reactions.
MBH: 20.37 ± 2.30 pmol 5α-reduced metabo- Therefore, the results are presented separate- lites.h-1.g tissue-1, P<0.001). Prenatal stress ly for the first and the second week (Table 2).
resulted in significant rise of testosterone Actually, latencies to first mount and ejacu- reduction in the male MBH up to values which lation in all animal groups, as well as ejacula- did not statistically differ from those in nor- tion frequency, in contol males significantly mal females, while there were no changes in improved in time course. Prenatal stress re- Table 2. Effects of prenatal exposure to Dex on male sexual behavior in prenatally stressed 3-month-old male rats Prenatally stressed prenatally stressed to first intromission to first ejaculation Postejaculatory interval (sec) Number of ejaculations to first intromission to first ejaculation Postejaculatory interval (sec) Number of ejaculations * P<0.05, ** P<0.01, *** P<0.001 compared to controls; # P<0.05, ## P<0.01 compared to the 1st trial; P<0.05 compared to the prenatally stressed males ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 Prenatal dexamethasone prevents sulted in significant prolongation of the first Prenatal Dex treatment rendered protective mount latency period and decrease in ejacu- effect with regard to diminishing adrenocorti- lation frequency both in the 1st and the 2nd cal and hypothalamic noradrenaline responses trials. Furthermore, in the 2nd trial the time to an acute stress in those animals. Dex-pre- prior to the first intromission was significant- treated prenatally stressed males showed de- ly longer than that of control males. Prenatal creased basal plasma corticosterone level (31,6 exposure to Dex has led to male sexual be- %, P<0.05) but corticosterone response to an havior normalization in the 2nd trial on the acute stress was similar to that of control rats whole. The only exception was the number of (Fig. 3). There were no significant differences ejaculations during the 2nd mating session that in stress-induced declines of hypothalamic was still significantly fewer versus control noradrenaline contents between the control (24.2 indices but greater compared to that of pre- %, P<0.01) and Dex-pretreated prenatally stressed natally stressed rats.
males (20 %, P<0.05) (Table 3, experiment 2).
HPA responses to an acute stress As well no changes were found in resting hypo- Prenatally stressed mature males exhibited thalamic catecholamine contents in the Dex-pre- decreased corticosterone responses to 1 h treated prenatally stressed males compared to restraint with no changes in basal corticos- normal control.
terone levels (Fig. 3). Basal dopamine concen- Baclofen effects on the HPA stress responses tration in the hypothalamus was 15.7 % higher Pretreatment with baclofen diminished stress- than those of normal control (P<0.05), while stimulated secretion of corticosterone in con- noradrenaline maintained at normal levels. HPA trol males but failed to alter this parameter in stress-activation was not followed by a decrease prenatally stressed rats (Fig. 3). Dex-pretreat- in hypothalamic noradrenaline content, although ed prenatally stressed males, as well as con- control males exhibited its depletion (24.2 %, trol ones, showed significant reduction in the P<0.001) (Table 3, experiment 1).
adrenocortical response to an acute stress following treatment with baclofen but the amplitude of blood plasma corticosterone level drop was less than that of control.
Cortocosterone (nmol/L) 500 Cortocosterone (nmol/L) Figure 3. Effects of baclofen pretreatment on blood plasma corticosterone response to 1h restriction in control (Ñ), prenatally stressed (PS) and Dex-pretreated prenatally stressed (Dex + PS) 6-month-old male offspring. Values are means ± SEM of 5-6 animals per group. Light bars - Figure 4. HPA response to intracerebroventricular basal levels; dark bars - after 1 h restriction; gray bars - noradrenaline (NA) infusion in control (continuous line), after 1 h restriction with baclofen pretreatment. *P<0.01 prenatally stressed (dotted line) and Dex-pretreated pre- compared to basal level; # P<0.05 compared to level after natally stressed (pointed line) 8-month-old males. Data 1 h restriction; P<0.05 compared to resting level in control are means ± SEM. *P<0.05 compared to basal level, group (Student’s t-test).
#P<0.05 compared to control group (Student’s t-test).
ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 A.G. Reznikov, N.D. Nosenko, L.V. Tarasenko, P.V. Sinitsyn, A.A. Lymareva Table 3. Hypothalamic catecholamine response to 1 h restriction in control, prenatally stressed and Dex-pretreated prenatally stressed 6-month-old males Catecholamine (nmol/g tissue)Noradrenaline After 1 h restriction Prenatally stressed: After 1 h restriction After 1 h restriction Dexamethasone-pretreated prenatally stressed: After 1 h restriction Data are means + SEM of 5-6 animals per group. *P<0.05; ** P<0.01; ***P<0.001 compared to resting levels; # P<0.05 compared to resting level in control group (Student’s t test).
HPA responses to noradrenergic stimuation monly used for suppressing the HPA axis in Noradrenaline infusion into the 3rd brain ven- the rat [1]. High glucocorticoid activity of Dex tricle resulted in almost 3-fold blood plasma is connected with its high affinity to tissue corticosterone rise in control males at the 30th glucocorticoid receptors, as well as low bind- min (Fig. 4). In the sequel, it lowered by the ing to plasma proteins [7]. Due to binding to 60th min but was still significantly higher as hippocampal glucocorticoid and, to less extent, compared to basal level. Prenatal stress to mineralocorticoid receptors [12], which evoked hyperactivation of the HPA axis fol- mediate corticosteroid feedback control of lowing noradrenaline application in male off- basal and stress-activated adrenal hormone spring: blood plasma corticosterone continued secretion, Dex attenuates the HPA axis re- to elevate by the 60th min. Though the extent sponse to stress.
of response in those animals was less pro- In this study Dex was used for suppres- nounced than in controls (rise above basal lev- sion of stress-stimulated HPA hormone secre- el by 77 and 175 %, respectively, P<0.05).
tion in order to examine whether the hormones The pattern of adrenocortical response to of the HPA axis mediate early and long-term noradrenaline central stimulation in Dex-pre- neurochemical, neuroendocrine and behavioral treated prenatally stressed males was similar effects of prenatal stress in male rats. Dex is to normal one with the only exception of a poor substrate for 11β-HSD-2, which pre- reaction amplitude, which was somewhat vents the fetus from excess of maternal glu- lower at the 30th min in comparison with con- cocorticoids, and therefore it readily passes trol male rats (rise by 147 vs. 175 % in the placenta [29]. For this reason it should attenuate stress-stimulated HPA function both in pregnant mother and the fetus.
Here it has been shown for the first time that supression of HPA hormone secretion Dex, synthetic glucocorticoid, administered in with Dex during restraining rats in late gesta- this study in a dose of 0.1 mg/kg b.w, is com- tion prevents numerous early and long-term ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 Prenatal dexamethasone prevents alterations including brain tissue protein pat- treatment with baclofen were observed both tern, brain testosterone metabolism, male in normal and Dex-pretreated prenatally sexual behavior, hippocampal GABA recep- stressed male rats, which suggests the ability tor, hypothalamic noradrenaline and plasma of GABA receptor to additional activation.
corticosterone responses to an acute stressor Taken together, these findings reflect media- or noradrenergic stimulation of HPA function.
ting role of stimulated HPA hormone secre- Our findings on maintenance of normal tion in maternal stress-induced alteration of hypothalamic noradrenaline and adrenocorti- GABA receptor function in male offspring.
cal responses to an acute stress in adult rats Maternal stress during the last week of exposed in utero to stress with Dex pretreat- gestation leads to demasculinization and/or ment support the concept on the mediating feminization of sexual behavior in adult male role of adrenal glucocorticoid excess in modu- offspring resulted from disorder of androgen- lating effect of prenatal stress on developmen- dependent sexual brain differentiation. This tal programming of HPA hormone activity [12, generally recognized phenomenon (prenatal stress syndrome) has been partially repro- Previously we revealed that adult male rats duced in this study by appearance of insuffi- exposed prenatally to exogenous glucocorti- cient male copulatory behavior.
coid demonstrate a diminution in plasma In male rodents, the POA is known to play corticosterone response to noradrenaline a key role in neuroendocrine regulation of male infused into the 3rd brain ventricle [30], sexual behavior. Decrease in male sex-asso- meanwhile, in prenatally stressed males a hy- ciated 66.0 kDa protein density in the POA persensitivity of the HPA axis to noradrenergic of prenatally stressed 5-day-old animals can stimulation was found [25]. This unexpected be considered as an early sign of impaired effect of prenatal glucocorticoid treatment sexual brain differentiation, though we cannot could appear for its direct influence on de- identify its function. As far as steroid veloping neurotransmitter systems, in particu- aromatase activity in the POA of 10-day-old lar on tyrosine hydroxylase gene expression pups is concerned, it must be taken into ac- [6]. Given preservation of noradrenergic sen- count, that this enzyme is one of the main sitivity of the hypothalamus, the main trigger determinants of androgen-dependent brain dif- of the HPA axis, in Dex-pretreated and pre- ferentiation [18, 23] and it plays crucial role natally stressed male offspring, we hypoth- in maintaining all aspects of sexual behavior esize that neurotransmitter disturbances in the in male rats [28]. Therefore, decrease in the stressed fetal brain could contribute to alter- enzyme activity resulted from prenatal stress ation of programming of noradrenergic control reflects the process of demasculinization of of HPA function resulted from prenatal stress.
male sexual behavior, which was confirmed Neuroendocrine control of HPA hormone by its testing in adult offsprings.
secretion under stress condition involves We have not investigated the lordosis GABA-ergic limitation mechanism mediated reaction in males in this study. Given location by GABA and GABA receptors. This of neuroendocrine regulation of female sexual mechanism tested with baclofen, GABA behavior which in rodents is associated with receptor agonist, was found to be impaired in the MBH, one can suggest that 5β-reductase male rats following prenatal stress. Prenatal activity elevation in this brain structure in pre- exposure of males to exogenous cortisol (hy- pubertal prenatally stressed males might result drocortisone acetate) exerts similar effect not only in demasculinization but presumably [19]. On the contrary, decreased adreno- in feminization of their sexual behavior in cortical responses to an acute stress after pre- ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5 A.G. Reznikov, N.D. Nosenko, L.V. Tarasenko, P.V. Sinitsyn, A.A. Lymareva Since steroid aromatase in the POA of male mediates alterations of programming of brain rats is induced by testosterone [27], the de- development induced by prenatal stress.
cline of the enzyme activity in the critical pe- riod of sexual brain differentiation presumably A.G. Reznikov, N.D. Nosenko, L.V. Tarasenko, results from androgen insufficiency in the fe- P.V. Sinitsyn, A.A. Lymareva tus under stress condition. It is generally be- lieved that prenatal stress syndrome is gene- PRENATAL DEXAMETHASONE PREVENTS rated by an increase of maternal adrenal gluco- EARLY AND LONG-LASTING NEUROENDO- corticoids in blood circulation with concomitant CRINE AND BEHAVIORAL EFFECTS OF MATER- NAL STRESS ON MALE OFFSPRING transitory testicular androgen deficit in male fe- tuses. Our recent manipulations with testoster- The hypothesis on the mediating role of hypothalamic-pitu- one replacement or naltrexone, opioid receptor itary-adrenocortical (HPA) hormone secretion in neuroendo- crine, neurochemical and behavioral alterations generated by antagonist, treatment of stressed pregnant rats prenatal stress in male rat offspring was tested in this study have demonstrated the essential role of andro- with dexamethasone (Dex) used for suppression of HPA stress gen shortage and endogenic opioids in early responses. Pregnant dams were being restrained daily for 1 h neurochemical feminization of the brain in pre- over the last week of gestation. In male offspring this resulted natally stressed male rats [21, 24] which is in in attenuation of sex-specific pattern of the protein fractions (on the 5th postnatal day), steroid aromatase activity (on the line with the results of studying of adult male 10th postnatal day) in the brain preoptic area, and in a decrease sexual behavior in similar experiments [4, 13, 33].
of male copulatory behavior, hypothalamic noradrenaline and Catecholamines (both noradrenaline and plasma corticosterone responses to an acute stress, an increase dopamine) are known to play primarily stimu- in HPA responses to noradrenergic stimulation and other ef- latory or permissive role in sexual behavioral fects in adulthood. All those changes were prevented with prenatal Dex in a dose of 0.1 mg/kg b.w. injected 30 min prior reactions [32]. Our findings on dopamine in- to restraining pregnant dams. As such, HPA hormone secre- crease in the MBH of prenatally stressed adult tion mediates alterations of programming of brain develop- male rats are quite matched with the disor- ment induced by prenatal stress.
ders in male sexual behavior. Moreover, these V.P.Komisarenko Institute of Endocrinology and Metabolism, observations are in concordance with the re- sults reported by Gerardin et al. [8]. Dex pre- treatment of prenatally stressed males was shown to restore both normal dopamine level in the MBH and consequently normal mani- 1. Almeida O.F.X., Canoine V., Ali S. et al. Activational effects of gonadal steroids on hypothalamo-pituitary- festation of motivational and copulatory adrenal regulation in the rat disclosed by response to components of male sexual behavior.
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V.P.Komisarenko Institute of Endocrinology and Metabolism, Kyiv Received 23.06.2008 ISSN 0201-8489 Ô³ç³îë. æóðí., 2008, Ò. 54, ¹ 5

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