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Review

Glucocorticoids, prenatal stress and the programming of disease

Anjanette Harris , Jonathan SecklUniversity of Edinburgh, Endocrinology Unit, Centre for Cardiovascular Science, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK

a b s t r a c ta r t i c l e i n f o

Article history:Received 26 February 2010Revised 26 May 2010Accepted 8 June 2010Available online 19 June 2010

Keywords:GlucocorticoidsPrenatal stress11 hydroxysteroid dehydrogenase type 2:glucocorticoid receptorEpigeneticPTSDFoetal programmingHPA axisAnxiety

An adverse foetal environment is associated with increased risk of cardiovascular, metabolic, neuroendocrineand psychological disorders in adulthood. Exposure to stress and its glucocorticoid hormone mediators mayunderpin this association. In humans and in animal models, prenatal stress, excess exogenous glucocorticoidsor inhibition of 11-hydroxysteroid dehydrogenase type 2 (HSD2; the placental barrier to maternalglucocorticoids) reduces birth weight and causes hyperglycemia, hypertension, increased HPA axis reactivity,and increased anxiety-related behaviour. Molecular mechanisms that underlie the developmental program-ming effects of excess glucocorticoids/prenatal stress include epigenetic changes in target gene promoters. Inthe case of the intracellular glucocorticoid receptor (GR), this alters tissue-specic GR expression levels, whichhas persistent and profound effects on glucocorticoid signalling in certain tissues (e.g. brain, liver, andadipose). Crucially, changes in gene expression persist long after the initial challenge, predisposing theindividual to disease in later life. Intriguingly, the effects of a challenged pregnancy appear to be transmittedpossibly to one or two subsequent generations, suggesting that these epigenetic effects persist.

2010 Elsevier Inc. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280Birth weight and the programming of disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280Mechanisms of programming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

Glucocorticoids during pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280Excess glucocorticoids and foetal development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281Maternal stress in pregnancy and the offspring brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281Role of the placenta and HSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282Regulation of feto-placental HSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282Other stress-related programming mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

Excess glucocorticoids and the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283Cells and structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283Gene targets of programming in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284Programming of the HPA axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284Prenatal stress and PTSD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284Neurochemical changes in HPA axis programming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284Programming brain MR/GR balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284The programming of affective disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285Central nervous system programming mechanisms: hippocampal GR as an exemplary target . . . . . . . . . . . . . . . . . . . . . . . . 285Epigenetics as a mediator of programming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286Transgenerational effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

Hormones and Behavior 59 (2011) 279289

Corresponding author.E-mail address: [email protected] (A. Harris).

0018-506X/$ see front matter 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.yhbeh.2010.06.007

Contents lists available at ScienceDirect

Hormones and Behavior

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Introduction

Early life events have long lasting impacts on tissue structure andfunction. These effects appear to underlie the developmental origins ofvulnerability to chronic degenerative diseases that have been revealedby human epidemiological studies. In addition to the well-recognisedeffects of both genes and adult environment, it is clear thatundernutrition, stress and exposure to excess glucocorticoids (themain hormonal mediator of stress) during foetal development causepermanent cardiometabolic, neuroendocrine and cognitive effects(Meaney et al., 2007; Seckl and Meaney, 2006). The concept ofdevelopmental programmingwas put forward in anattempt to explainthis association between environmental challenge during pregnancy,altered foetal growth and development and later pathophysiology(Barker et al., 1993a; Seckl, 1998). During programming, environmentaladversity is transmitted to the foetus and acts on specic tissues duringsensitive periods in their development to change developmentaltrajectories and thus their organisation and function. Since differentcells and tissues are sensitive to various factors at different times, theeffects of adversity on an animal's biology will be tissue, time andchallenge specic.

It is currently unclear what the biological purpose of early lifeprogramming is. However, since the phenomenon of programmingseems highly conserved through evolution (humans, non-humanprimates and many animal models show evidence of developmentalprogramming) it may be assumed that programming confers adaptiveDarwinian advantages. For example, a mother may transmit a signalto the foetus it is tough out here be it because of reduced foodavailability and/or increased stress (e.g. predation, war, etc.) and theresulting changes in the foetus affect the offspring to promote survivaluntil reproduction is secured. However, in the post-reproductiveperiod these changes may prove to be disadvantageous. This has beensuggested to be particularly pertinent if the adult environment doesnot match that for which the developmental plasticity is aimed, e.g. ifearly life undernutrition programmes a thrifty phenotype and thenfood is plentiful, the adult may be at risk of developing metabolicdiseases such as diabetes and obesity (Gluckman and Hanson, 2004).

Birth weight and the programming of disease

In humans there is a clear association between low birth weightand the development of hypertension, type 2 diabetes and cardio-vascular disease in adulthood (Barker et al., 1993b; Fall et al., 1995;Moore et al., 1996; RichEdwards et al., 1997). This relationship isindependent of other life style-associated risk factors (e.g. smoking,excess alcohol consumption, obesity, and social class) and holds trueover a continuum of birth weights within the normal range (Leon etal., 1996; Levine et al., 1994; Osmond et al., 1993). While it is possiblethat shared genetics explains the above ndings, data from twinstudies show that the twin with the lowest birth weight has higherblood pressure in adulthood (Gluckman and Hanson, 2004) thoughnot always (Baird et al., 2001). Additionally, ndings from apparentlyisogenic rodent models provide further evidence of a link betweenearly life environmental manipulations, reduced birth weight andadult pathophysiology.

Low birth weight is also associated with affective and cognitivedisorders in adulthood (Thompson et al., 2001; Wiles et al., 2005). Forexample, low birth weight has been linked to schizophrenia, attentiondecit/hyperactivity disorder (ADHD), antisocial behaviour, increasedvulnerability to post-traumatic stress disorder (PTSD), anxietydisorders, learning difculties and depression (Cannon et al., 2002;Famularo and Fenton, 1994; Jones et al., 1998; Khashan et al., 2008a;Lahti et al., 2009; Raikkonen et al., 2008; Wust et al., 2005). Andcrucially, rodent models show that early environmental challenges(such as restraint stress in third week of pregnancy) increase anxietyand depressive-like behaviour and impair cognitive ability in adults,

further substantiating the human correlative data (Meaney and Szyf,2005). Consequently, low birth weight has been proposed as anindicator of environmental adversity during foetal development.Though low birth weight per se may not be the cause of disease, itdoes seem to suggest foetal programming processes are at work. Birthweight is, however, an unsophisticatedmeasure and presumably therearemany factors or lesser degrees of challenge thatmay alter offspringbiology without affecting birth weight. Furthermore, humans androdents with reduced birth weight may experience rapid postnatalcatch-up growth, altered fat content and distribution and lowadiponectin levels, which may predispose to cardiovascular disease,obesity and type 2 diabetes in adulthood (Owen andMatthews, 2007).However, postnatal dexamethasone treatment of newborn rat pups,whilst causing immediate and marked growth retardation withsubsequent catch-up growth does not appears to induce cardiometa-bolic sequellae (Nyirenda et al., 1998) suggesting that the window ofsensitivity in prenatal life is key in this species at least.

Mechanisms of programming

Two mechanistic hypotheses have been proposed to explain howfoetal programming may arise: foetal malnutrition and foetaloverexposure to glucocorticoids, which may have effects eitherdirectly or indirectly upon the developing foetus (Barker et al.,1993a; Berney et al., 1997; Edwards et al., 1993; Matthews, 2000;Meaney et al., 2007). However, these hypotheses are not mutuallyexclusive since stress may reduce maternal food intake and reducedfood intake may invoke stress responses in the mother and foetus(Gardner et al., 1997). Moreover, maternal glucocorticoids maymediate the effects of diet on foetus biology. For example, the feedingof low-protein diets to rats during pregnancy results in higher bloodpressure in the offspring from the age of weaning. However, blockingmaternal glucocorticoid synthesis eliminates the impact of proteinrestriction on the offspring (Gardner et al., 1997; Langley and Jackson,1994; Langley-Evans, 1997; Langley-Evans et al., 1996a). And it wouldseem that the relevant physiological systems are glucocorticoid-sensitive from an early age, since infusion of glucocorticoids directlyto the foetus in utero or at birth elevates blood pressure, at least insheep (Berry et al., 1997; Tangalakis et al., 1992). Furthermore, thelow birth weight syndrome rather resembles the endocrinologicaldisorder Cushing's syndrome (glucocorticoid excess) which alsocauses type 2 diabetes, hypertension, dyslipidemia, atherosclerosisand osteoporosis (Anagnostis et al., 2009; Andrew et al., 2002; Seckl etal., 2004; Walker, 2006; Wei et al., 2004). Here we focus predomi-nantly on antenatal glucocorticoid and stress-mediated effects.

Glucocorticoids during pregnancy

Glucocorticoids inuence the developing foetus by binding toglucocorticoid and mineralocorticoid receptors (GR and MR, respec-tively), which subsequently act as transcription factors to alter geneexpression. In addition, MR and GR can alsomediate fast non-genomicactions via membrane-located receptors, at least, in the hippocampus(de Kloet et al., 2008; Karst et al., 2005). Although, any role of thisbiology indevelopmental programming is unexplored. GR is expressedin most foetal tissues, including the placenta, from early embryonicstages and is essential for survival, as indicated by the lethal postnatalphenotype of GR null mice (Cole et al., 1995). Indeed, glucocorticoidsplay a vital role during normal foetal development. Most notable istheir role in promoting maturation of the lung and production of thesurfactant necessary for extra-uterine lung function (Ward, 1994).Glucocorticoids also promote correct brain development by initiatingterminal maturation, remodelling of axons and dendrites, andaffecting cell survival (Meyer, 1983; Yehuda et al., 1989). Expressionof the higher afnity MR is more limited and present only during thelater stages of development in rodents (Brown et al., 1996; Diaz et al.,

280 A. Harris, J. Seckl / Hormones and Behavior 59 (2011) 279289


1998). In vivo, MR (and perhaps GR) may be protected from illicitglucocorticoid signalling if co-localised with the enzyme 11-hydro-xysteroid dehydrogenase type 2 (HSD2) (see below). HSD2 catalysesthe rapid inactivation of glucocorticoids (cortisol and corticosterone)to inert 11-keto forms (cortisone and 11-dehydrocorticosterone). Inthe distal nephron and other aldosteroneselective target cells HSD2,by inactivating glucocorticoids, allows the non-substrate aldosteroneselective access to MR, which are intrinsically non-selective andotherwise bind glucocorticoids and mineralocorticoids with similarafnity. High levels of HSD2 expression are also found in the placenta.Here this enzyme plays an important role in shielding the developingfoetus from the mother's relatively high glucocorticoid levels(Edwards et al., 1993; Meaney et al., 2007).

HSD2 shows a complex cell- and stage-specic ontogeny in thefoetuswhichmay gate glucocorticoid access toMR and/or GR, perhapsminimising their activation despite expression in developing organs(Brown et al., 1996; Speirs et al., 2004). Thus, glucocorticoids canexert genome wide effects from early development, and in a tissuespecic manner (i.e. receptor expression location) which can bemodulated by tissue glucocorticoid metabolism.

Excess glucocorticoids and foetal development

Despite the clear physiological importance of endogenous gluco-corticoids during development, there are a plethora of data to showthat exposure to excess exogenous glucocorticoids during pregnancycorrelates with reduced birth weight and adverse outcomes in theoffspring, especially if glucocorticoids are administered during lategestation when growth is accelerating and presumably thus mostsusceptible to the catabolic effects of steroids (Bloom et al., 2001;French et al., 1999; Nyirenda et al., 2001; Reinisch et al., 1978).Nevertheless, pregnant women at risk of preterm delivery areroutinely given glucocorticoids to speed up foetal lung development(Kay et al., 2000), and glucocorticoid treatment is also used in theantenatal treatment of foetuses at risk of congenital adrenalhyperplasia (CAH) (Lajic et al., 2008). However, birth weight istypically reported as normal in infants at risk of CAH whose mothersreceived low-dose dexamethasone (a synthetic GR ligand that is littlemetabolised by HSD2 and so passes freely across the placenta) in uteroduring the rst trimester (Forest et al., 1989; Mercado et al., 1995).

In pregnancies with complications it is difcult to determine theimpact of glucocorticoid treatment on cognition and adult behaviourin the offspring because these studies are confoundedby subjects oftenborn prematurely and so already at risk of delayed neurologicaldevelopment (e.g. Pesonen et al., 2008). Furthermore, antenatalglucocorticoid treatment only started to be used routinely from themid 1980s, so its long-term impact cannot be fully known until theoffspring reachmiddle or old age in the coming decades. Nevertheless,there are data that suggest prenatal glucocorticoid administration towomen at risk of preterm delivery is associated with higher bloodpressure in their 14 year old offspring (Doyle et al., 2000), higherinsulin levels in adulthood (Dalziel et al., 2005), reduced headcircumference at birth and an increase in distractibility and inattentionin the teenage offspring (French et al., 1999). Treatment of babies withdexamethasone immediately after birth for the prevention of chroniclung disease is also associated with signicantly smaller headcircumference, lower IQ scores, poorer motor skills, coordination andvisualmotor integration as compared with control children whentested at school age (Yeh et al., 2004). However, in children at risk ofCAH and receiving prenatal dexamethasone treatment, albeit in lowdoses, no overall effects on full-scale IQ have been observed, but anegative effect on verbal working memory has been reported (Lajicet al., 2008; Trautman et al., 1995). Administration of betamethasone(a GR specic synthetic glucocorticoid) in preterm risk pregnancieshad no signicant effect on cognitive development or progress atschool when investigated in children under the age of ten (MacArthur

et al., 1982). Also, repeated antenatal betamethasone did not inducealterations in toddler temperament, however a longer duration ofexposure was associated with higher impulsivity scores in two yearold children (Pesonen et al., 2009). In sum, whilst more clinical trialsare needed, it would seem that late gestation, short-term prenatalcorticosteroid treatment prevents respiratory diseases with littleimpact on adult biology, and that repeated treatment regimes orpostnatal therapy are likely to have persisting adverse effects with noclear additional benets to the new born (Kay et al., 2000). The long-term effects of low doses in early gestation remain uncertain.

Maternal stress in pregnancy and the offspring brain

Unlike prenatal glucocorticoid treatment, which is often GRspecic, prenatal stress involves both MR and GR signalling andcatecholamine release. And there is evidence that endogenousmaternal and foetal glucocorticoids (and possibly other stress-relatedhormones) reduce birth weight and have implications for thedeveloping foetal hypothalamicpituitaryadrenal (HPA) axis andaffective behaviour (i.e. emotional behaviour, such as anxiety)(Khashan et al., 2008b). The children from mothers who self reporthigh levels of stress during pregnancy (and salivary cortisol levelsverify elevated glucocorticoids), are reported to have temperamentaland behavioural problems as toddlers (Gutteling et al., 2005b) andimpaired attention and concentration at 7 years of age (Guttelinget al., 2006). There is some evidence that low birth weight coupledwith lower levels of maternal care associate with reduced hippocampalvolume (determined by magnetic resonance imaging) in adult women(Buss et al., 2007).However, the effects of prenatal stress on cognition inoffspring are conicting (Bergman et al., 2007; Gutteling et al., 2006).Other outcomes that have been reported to associate with exposure tohigh levels of glucocorticoids during development include autism,attention decit and hyperactivity disorder, language problems anddepression (O'Donnell et al., 2009). Severe trauma (e.g. death of a closerelative) during rst trimester is associated with an increased risk ofdeveloping schizophrenia in adulthood (Khashan et al., 2008a).

Higher basal HPA axis activity coupled with greater stressreactivity is associated with the development of many depressiveand anxiety-related disorders. And crucially, mothers who self reportanxiety and/or depression during pregnancy give birth to offspringwith higher basal HPA axis activity at 6 months, 5 years and 10 yearsof age (Gutteling et al., 2005a). Therefore, it has been proposed thatthese children may be at greater risk of developing affective disordersin adulthood. In other words, early life events may engender alteredHPA axis activity as a vulnerability factor for later disease. Whether ornot the link is direct or merely co-causation remains unresolved.

In corroboration of the human data, ndings from experimentswith inbred rodent models show that prenatal stress (such asrestraint) and administration of exogenous glucocorticoids reducesbirth weight, impairs cognition, increases anxiety and reactivity tostress and alters brain development (Henry et al., 1994; Igoshevaet al., 2004; Takahashi et al., 1992b; Vallee et al., 1999; Welberg et al.,2000). There is also evidence that prenatal stress increases sensitivityto addictive drugs. For example, the offspring of rats that werestressed in the nal week of pregnancy self-administer higher levelsof intravenous amphetamine in adulthood than non-stressed controlrat offspring (Deminiere et al., 1992).

If maternal glucocorticoid synthesis is prevented by adrenalectomy,stressing the mother appears to have little effect on the offspring and,though a role for other stress-related hormones cannot be ruled outcompletely (catecholamines are secreted from the adrenal medulla),replacement of glucocorticoid hormones to adrenalectomised damsrestores the adverse effects of prenatal stress on offspring physiologyand behaviour (Barbazanges et al., 1996; Ordyan and Pivina, 2003;Zagron andWeinstock, 2006). Although the ndings from these studiessuggest that maternal glucocorticoid levels are the sole mediator of

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foetal programming, observational data from humans implicates a roleof foetal glucocorticoid levels in reducing birth weight, because foetusesin intrauterine growth retardation and in pre-eclampsia have highglucocorticoids levels (Goland et al., 1993, 1995). However, this mightalso be transplacental passage of maternal glucocorticoids, as discussedbelow.

Overall, whilst many reports link maternal stress in pregnancywith altered offspring outcomes, as with many human ndings thesestudies are predominantly associational. They have somewhatincreased cause-and-effect plausibility in the light of the pre-clinicalbiology reviewed here, but more needs to be done to convince thatthis is the case in humans.

Role of the placenta and HSD2

The placenta plays a vital role in moderating foetal exposure tomaternal factors. Despite being able to pass freely across the placenta,glucocorticoid levels are signicantly lower in the foetus than in themother. This is because the placenta expresses high levels of HSD2,which has been suggested to provide a protective barrier between thefoetus and mother (Edwards et al., 1993), shielding glucocorticoid-sensitive tissues in the foetus from inappropriately high glucocorticoidlevels during development (Meaney et al., 2007). This barrier isapparently incomplete though, with 1020% of maternal glucocorti-coids reaching the foetus intact (Benediktsson et al., 1997a). Sincematernal glucocorticoid levels are so much higher than that of thefoetus, modest perturbations in placental HSD2 levels/activity canhave a profound impact on foetal glucocorticoid exposure. In themouse, placental HSD2 mRNA expression drops dramatically towardslate gestation (Brown et al., 1996) with a subsequent loss of activity,perhaps to allow maternal glucocorticoids to stimulate late foetalmaturation. In the rat, the fall in placental HSD2 occurs later ingestation with less loss of activity (Waddell et al., 1998), perhapsbecause the foetal adrenals contributemore to thematurational surge.In the human placenta, HSD2 levels steadily increase throughoutgestation (McTernan et al., 2001). Since GR expression remainsconstant in the placenta throughout gestation, natural uctuations inHSD2 levels may control glucocorticoid action in the placenta as wellas the foetus during development (altered GR signalling may alterplacental function e.g. efcacy of nutrient transfer).

It has been hypothesised that reduced placental HSD2 activityresults in high levels of glucocorticoids reaching the foetus, whichretard growth and programme disease susceptibility (Benediktssonet al., 1997a; Edwards et al., 1993; Seckl, 1998). Compatible with thishypothesis, low levels of placental HSD2 activity are correlated withlow birth weight in humans (McTernan et al., 2001; Stewart et al.,1995) and rodent models (Brown et al., 1996), though this has notalways been reported (Rogerson et al., 1996, 1997). Humanshomozygous for deleterious genetic mutations in the HSD11B2 genehave birth weights, on average, 1.2 kg less than their heterozygote orunaffected siblings (Dave-Sharma et al., 1998). Additionally, foetalmarkers of glucocorticoid exposure such as cord blood levels ofosteocalcin (a GC sensitive osteoblast product that does not cross theplacenta) correlate with HSD2 activity in the placenta in humans(Benediktsson et al., 1995).

Studies in which HSD2 activity is inhibited reveal an importantrole for feto-placental barrier in mediating physiological andpsychological disease. Administration of the liquorice derivativecarbenoxolone, a potent (Ki low nM) inhibitor of HSD2 reducesbirth weight and produces permanent alterations of the HPA axis andincreases anxiety-like behaviour in aversive situations in adult rats(Welberg et al., 2000). Maternal carbenoxolone administrationthroughout pregnancy in rats also leads to the development ofhypertension and predisposes to hyperglycaemia in later life, but onlyin the of presence of maternal adrenal products, since carbenoxolonehas no effect on offspring biology in adrenalectomised pregnant rats

(Lindsay et al., 1996a,b). In humans, excessive maternal consumptionof glycyrrhizin (N500 mg/week), a natural constituent of liquorice,which inhibits placental HSD2 (Monder et al., 1989), leads to offspringwith signicant decrements in verbal and visuo-spatial abilities andnarrative memory, and signicant increases in externalising symp-toms, attention, rule breaking and aggression problems, moreover,the effects on cognitive performance appear dose-related (Rikknenet al., 2009). However, heavy glycyrrhizin exposure during pregnancy(N500 mg/week) did not signicantly affect birth weight or maternalblood pressure, but was signicantly associatedwith shorter gestation(Strandberg et al., 2002, 2001), illustrating that changes in birthweight are not necessary for apparent programming effects to occur,paralleling ndings in non-human primates exposed to low doses ofdexamethasone in the last half of pregnancy (de Vries et al., 2007).Consequently, women are advised against excessive intake ofliquorice-containing foodstuffs in pregnancy (Rikknen et al., 2009).

The production of genetically modied mice allows the feto-placental unit to be investigated under varying genetic conditions, forexample, heterozygote crosses (of HSD2+/ mice) allows a singlemother to bear wild-type, heterozygote and null offspring (whichhave normal, reduced and zero levels of placental HSD2, respectively).Using this approach, HSD2 nullizygosity was found to reduce birthweight and increase anxiety relative to wild-type littermates (Holmeset al., 2006). Crucially, the heterozygotes had intermediate birthweights, suggesting that variation, and not just complete absence, ofHSD2 mediates foetal exposure to glucocorticoid levels, and thusprogramming. In this neat experiment, the effects of maternal stressand nutrition were eliminated and a clear key role of feto-placentalHSD2 in prenatal glucocorticoid programming was established.

It is also possible that reduced placental HSD2 levels allow greaterglucocorticoid signalling within the placenta itself, which canindirectly impact foetal development by altering placental function.Indeed, variation in placental HSD2 levels correlate with alteredexpression levels of various glucose and amino acid transportermolecules and growth factors in the placenta. Null mice generatedfromHSD2+/ heterozygote crosses are able to maintain normal bodyweight up to embryonic day (E) 15 (term is E19) with concomitantincreases in amino acid and glucose transporters (Wyrwoll et al.,2009). However, by E18, both foetal and placental weights aresignicantly reduced in the null mice relative to wild-type littermates,and correspondingly the null placentas have reduced foetal capillarysurface area, plausibly due to lower placental vascular endothelialgrowth factor (VEGF) expression (VEGF is inhibited by glucocorti-coids) and reduced amino acid and glucose transporters, all clearlyessential for foetal growth (Wyrwoll et al., 2009). One implicationfrom these data is that the placenta is able to overcome decreasedHSD2 levels, and thus increased local GR signalling, initially byincreasing nutrient transfer to the foetus. But in the nal stages ofpregnancy, when growth is most rapid the placenta fails to maintainhigh nutrient transfer to the foetus. Furthermore, demonstrating thatreduced nutrient transfer is a cause of, rather than a consequence of,being a smaller foetus, one study showed that placental amino acidtransport is down-regulated before foetal weight is retarded (Janssonet al., 2006). It is currently unclear if variation in placental HSD2 levels(and thus exposure to glucocorticoids) causes changes in the ability ofthe placenta to transport (free) fatty acids to, or waste products from,the developing foetus.

Regulation of feto-placental HSD2

Clearly then, placental HSD2 plays a key role in mediating the owof maternal environmental signals to the foetus. So what alters HSD2?There is evidence that placental HSD2 levels/activity are affected bystress. Somewhat paradoxically, there is some evidence that prenatalstress leads to a reduction in placental HSD2 activity in rats,suggesting that the foetus and placenta are exposed to extra excessive



amounts of glucocorticoids (Mairesse et al., 2007). Perhaps this is toensure that the foetus receives a reliable signal about the environ-mental adversity that it may face after birth. However, the genetics ofthe rodent are important. Firstly, rats bred for low anxiety are able toincrease placental HSD2 and withstand the detrimental effects of highglucocorticoid exposure during pregnancy on hippocampal neuro-genesis relative to rats bred for high anxiety (Lucassen et al., 2009).And secondly, HSD2 null mice on a 129 MF1 background havenormal foetal birth weight (Kotelevtsev et al., 1999) whereas those onC57Bl/6J have reduced birth weight, though the effects of geneticvariation between animals in the earlier study may have reduced thepower to detect small differences (c.f. to Holmes et al., 2006).

Dietary protein restriction during pregnancy also reduces placen-tal HSD2 activity, apparently selectively, suggesting a commonmechanism by which excess glucocorticoids and malnutrition canprogramme adult biology (e.g. Bertram et al., 2001; Langley-Evanset al., 1996b). Indeed, in the maternal protein restriction diet model,antenatal prevention of glucocorticoid synthesis prevents the devel-opment of hypertension in the adult offspring (Langley-Evans, 1997).And there is also evidence to suggest that placental HSD2 expressionand activity levels are oxygen dependent in the rst and thirdtrimesters during human gestation (Alfaidy et al., 2002). It is currentlyunclear what effect various drugs and chemicals (e.g. caffeine, envi-ronmental pollutants, and anti-depressants) or life style factors (e.g.working night shifts) have on placental HSD2 levels and thus foetalprogramming.

In vitro studies using humanplacental cell lines reveal that hypoxia,catecholamines and inammatory cytokines can down-regulate HSD2levels (Sarkar et al., 2001; Tsugita et al., 2008). Conversely,physiologically relevant levels of ethanol and nicotine do not appearto inuence the activity of HSD2 in clonal cell cultures, freshly isolateddually-perfused intact human placentas or placentas from in vivotreated rats (Benediktsson et al., 1997b).

In addition to placental HSD2, foetal tissueHSD2 levelsmay also playa role in programming adult disease. The impact of altered foetal HSD2levels on adult biology are, as yet, not fully elucidated, but there is someevidence that restricted maternal oxygen intake leads to foetalhypoxemia-induced acidosis which selectively down-regulates HSD2mRNAexpression in the preterm foetal sheep kidney (Yang et al., 1997).These data indicate a potential mechanism whereby altered oxygenlevels inuencedevelopmental processes by alteringHSD2 levels,whichultimately regulates the bioavailability of glucocorticoids in specicfoetal organs, altering maturational trajectories and programmingoffspring biology and adult disease risk.

Other stress-related programming mediators

Glucocorticoids clearly inuence development, but other substancesare released by the mother and foetus in response to stress, andrelatively little is known about the impact of these onplacental function,foetal development and disease susceptibility. The catecholamines,adrenaline and noradrenaline, are of particular relevance since they arereleased by stress and the placenta has adrenergic receptors suggestingthat catecholaminesmay inuence placental function (Ganapathy et al.,1993). And there is evidence that catecholamines can be transportedacross the placenta between foetus and mother (Morgan et al., 1972;Thomas et al., 1995). Crucially, in vitro studies have demonstrated thatnoradrenalineandadrenaline rapidly down-regulateHSD2mRNAlevelsin early and late gestation human trophoblast cell lines (Sarkar et al.,2001). The suppression of HSD2 was mediated via both alpha (1)- andalpha (2)-adrenoceptors and was independent of beta-adrenergicstimulation. Therefore, it is plausible that in vivo, high circulating levelsof catecholamines (from mother or foetus) play a role in programmingdisease by altering placental and/or foetal HSD2 levels/activity, nutrienttransport, and/orGR density. This certainlymerits exploration. The feto-placental unit is protected to some extent by placental expression of the

enzyme monoamine oxidase A (MAO-A) which catalyses the oxidationof monoamines, but it is currently unclear how, if at all, stress affectsplacental MOA-A levels/activity, or how other factors inuence MAO-Ain the placenta, and potentially, programming of target tissues andperhaps disease risk.

Intriguingly, excess foetal glucocorticoid exposure programmescatecholamine sensitivity in adulthood. For example, prenataldexamethasone treatment associates with exaggerated hypertensiveresponses to catecholaminergic stimulation in offspring rats (O'Reganet al., 2008). Furthermore, in vitro stimulation of isolated mesentericvasculature with noradrenaline and vasopressin had greater vaso-constricting effects in rats from dexamethasone treated mothers(O'Regan et al., 2008). Therefore, it is plausible that alteredsympathetic responses to noradrenaline underlie the developmentof hypertension in the glucocorticoid programming models.

Excess glucocorticoids and the brain

High levels of glucocorticoids impact on brain structure duringdevelopment, which may alter neurotransmitter activity and synapticplasticity resulting in subtle or drastic changes in subsequent function,notably altering behaviour and cognition, as well as diseasesusceptibility in adulthood (Weinstock 2008).

Cells and structure

During development sufcient glucocorticoid levels are essentialfor normal maturation of many parts of the central nervous system.However, either excess or decient glucocorticoid signalling duringcritical windows of development alters the developmental trajectoryof vulnerable brain structures often with permanent consequences.For example, antenatal betamethasone injections retard foetal braingrowth in sheep (Huang et al., 1999) and are associated with areduced cortex convolutions index and surface area in humanoffspring (Modi et al., 2001). And prenatal stress in rats signicantlydecreases dendritic spine density in the anterior cingulate gyrus andorbitofrontal cortex (Murmu et al., 2006). Additionally, repeatedadministration of synthetic glucocorticoids to pregnant sheep delaysastrocyte and capillary tight junction maturation, and delaysmyelination of the corpus callosum in the brains of the developingfoetuses (Antonow-Schlorke et al., 2009; Huang et al., 2001a,b). Thisdelay in maturation does not appear to be simply due to a lack of CNSglucose, uptake of which is affected by glucocorticoids (Virgin et al.,1991) since the structural changes are not associated with changes inglucose transporter protein GLUT1 in the blood brain barrier or GLUT3in neuronal membranes (Antonow-Schlorke et al., 2006).

The hippocampus highly expresses GR and MR (Reul and de Kloet,1985) and is particularly vulnerable to glucocorticoid manipulations,especially in early life. In rats, prenatal stress signicantly decreasessynaptic spine density of the hippocampus (by 32%) on postnatal day(P) 35, and subsequently reversal learning is impaired (Hayashi et al.,1998). Also, maternal stress reduces offspring neurogenesis and resultsin faster ageing (i.e. decline in cell proliferation associated withlearning) which correlates with decits in cognition (Lemaire et al.,2000). Similar effects are also seen in primates. Thus, in foetal baboons,betamethasone exposure decreases cytoskeletalmicrotubule associatedproteins and the presynaptic marker protein synaptophysin, whichmay cause reduced levels of neuritogenesis and neuronal plasticity(Antonow-Schlorke et al., 2003). And in rhesus monkeys prenataladministration of dexamethasone decreases the number of pyramidalneurons in the hippocampal CA regions and granular neurons in thedentate gyrus and causes pronounced degeneration of the axodendriticsynaptic terminals of the mossy bres in the CA3 hippocampal region(Uno et al., 1990). Moreover, these effects are dose-associated withmultiple injections inducingmore severe damage than single injectionsof the same total dose (Uno et al., 1990). These ndings raise the



intriguing question of whether chronic, but low level, stress is moredetrimental than a short sharp trauma to foetal development. Perhapsdifferent types of stress confer adaptation to specic types of challengein later life, though the mechanisms of such ne-tuning are moot.However, the complexity of hormonal responses to stress (e.g.glucocorticoids, catecholamines etc. versus straight synthetic glucocor-ticoid treatment) and the substantial interindividual differences instress responses in humans make extrapolation to our species fraught.Whatever, if early exposure affects hippocampal structure and altersfunction, myriad studies in animal models and humans illustrate thatsuch effects are likely to alter cognitive ability, behaviour and the risk ofpsychological disorders (e.g. Sheline et al., 1996).

Gene targets of programming in the brain

Exploration of glucocorticoid-responsive genes in the brains ofprenatally stressed rats has provided insight into mechanisms thatmay underlie glucocorticoid-related neurodevelopmental disorders.Fukumoto et al. (2009) demonstrated that defective neuronalmigration, essential for normal network formation, is caused by excessglucocorticoid-activated GR binding to the CALD1 gene whichincreases Caldesmon1 (CaD) protein levels in the developing cerebralcortex in vivo and in vitro. CaD is a cytoskeletal related protein involvedin negative regulation of myosin II function. Increased CaD levels incerebral cortex cause neuronal precursor cells to change shapeimpairing their migration (Fukumoto et al., 2009). These ndingsraise intriguing questions as to which other genes and structures aretargeted by GR andMR during excess glucocorticoid exposure in utero.

Programming of the HPA axis

The HPA axis and its key limbic regulator, the hippocampus, areparticularly sensitive to glucocorticoid levels during development(Gould and Tanapat, 1999; Gould et al., 1991; Welberg and Seckl,2001). High glucocorticoid exposure during foetal development in ratsand primates permanently elevates basal glucocorticoid levels inoffspring (de Vries et al., 2007; Levitt et al., 1996; Welberg et al., 2000,2001), often with sex differences (Weinstock et al., 1992). Maternalstresshas similar effects. Thus restraint stress in pregnant rats during thethirdweek of gestation is associatedwith greater plasma corticosteronelevels in response to novelty in the offspring (Henry et al., 1994).

Intriguingly, the human literature, though far from complete,suggests that antenatal glucocorticoid exposure reduces HPA activityin the neonate (perhaps expectedly), but does not clearly associatewithelevated glucocorticoid levels in later life (Tegethoff et al., 2009). It maybe that following prenatal glucocorticoid overexposure in humans,offspringHPA axis changes occur but that the direction of change varies,perhaps related to developmental timing, genetics or other factors. Insupport of this notion, healthy young adult humans whose mothersexperienced severe stress during their pregnancy have greater cortisolresponses during a psychological challenge, the Trier Social Stress Test(TSST) (Entringer et al., 2009). Salivary cortisol responses to theTSSTarealso signicantly and inversely related to birth weight (Wust et al.,2005). In contrast, pregnant women directly exposed to the 9.11WorldTrade Centre atrocity in 2001 gave birth to babies which showed lowersalivary cortisol aged 1 year, but only if themother developed PTSD andwas exposed in the last trimester of pregnancy (Yehuda et al., 2005).Neither maternal PTSD nor exposure alone were sufcient to producethis effect in the offspring. These data suggest that underlyingvulnerability factors and developmental timing critically determinethe occurrence and vector of HPA axis programming in humans.

Prenatal stress and PTSD

Approximately 1040% of trauma sufferers develop PSTD (Davidsonet al., 2004). HPA axis perturbations are associated with PTSD: typically

reduced urinary, plasma and salivary cortisol levels suggestingenhanced negative feedback on the HPA axis and/or increased tissuesensitivity to glucocorticoids (Yehuda et al., 2009b, 2004). Indeed, PTSDpatients show greater suppression of cortisol in response to dexameth-asone than normal subjects (Yehuda et al., 1993).

Though it was not initially clear if an attenuated HPA axis is a causeor a consequence of PTSD, recent data suggest that low HPA axisreactivity may actually be a vulnerability factor. Data from pregnantwomen that were in or near the World Trade Centre attack on Sept11th 2001 show that womenwho went on to develop PTSD had lowercortisol levels than women who did not develop PTSD (Yehuda et al.,2005). The effect on the offspring's HPA axis (Yehuda et al., 2005),appears corroborated in studies of Nazi Holocaust survivors and theirchildren. Healthy offspring of women with PTSD who survived havesignicantly lower cortisol levels than do offspring of survivorswithout PTSD or control mothers (Yehuda, 2002). To convince, moreprospective studies are needed on vulnerability to PTSD in suchoffspring (and e.g. the grandchildren).

Glucocorticoid metabolism may also play a role. A recent studyshowed that Holocaust survivors with PSTD who underwent extrememalnutrition/stress during early life had lowered glucocorticoidcatabolism by 5alpha-reductase and HSD2 (Yehuda et al., 2009b).These effects are thought to be adaptive, sustaining metabolic andmineralocorticoid actions of glucocorticoids within liver and kidneywithout generalised glucocorticoid excess, which exerts deleteriouseffects on muscle and CNS (Yehuda et al., 2009a). However, recentdata suggests that diminished 5alpha-reductase activity may mark astate of primary vulnerability, perhaps via attenuated peripheralcatabolism of cortisol resulting in the suppression of HPA axisresponsiveness (Yehuda et al., 2009b).

Neurochemical changes in HPA axis programming

Prenatal dexamethasone treatment in rats increases circulatingcorticosterone and ACTH levels in the offspring (e.g. Levitt et al.,1996), which is associated with increased CRH in the paraventricularnucleus of the hypothalamus (PVN), and in the central nucleus of theamygdala, where it is key for the expression of fear and anxiety(Welberg et al., 2000, 2001). Prenatal stress also impacts upon thedevelopment of CRH neurons in the PVN (Fujioka et al., 1999; Shoeneret al., 2006) with greater vulnerability to PVN cell death (Tobe et al.,2005), which has implications for HPA axis programming. Theresulting mild chronic glucocorticoid excess can be further exacer-bated by increased hepatic and visceral adipose tissue glucocorticoidactivity due to increased tissue levels of 11-hydroxysteroid dehy-drogenase type 1 (HSD1) (Cleasby et al., 2003; Nyirenda et al., 2009,2006). HSD1 catalyzes the reverse reaction to HSD2, thus regeneratingactive glucocorticoids from inert 11-keto metabolites (Rajan et al.,1996; Yau et al., 2001) in CNS and peripheral metabolic organs (e.g.liver, adipose tissue). The resulting chronic mild intracellularglucocorticoid excess may lead to the development of hypertensionand hyperglycemia, cognitive decits, affective disorders, immuno-suppression and cardio-metabolic disease, as seen in Cushing'ssyndrome. Antenatal glucocorticoid exposure also up-regulatesHSD1 in the hippocampus (Shoener et al., 2006). Whilst this mightseem at odds with the HPA axis feedback decits observed, increasedlevels of HSD1 is glucocorticoid-induced, has little effect on HPA axisfeedback in most situations (Carter et al., 2009), and may underpinlater neuronal toxicity (e.g. Yau et al., 2007).

Programming brain MR/GR balance

Programmed HPA axis activation has plausibly been ascribed torelative deciency of glucocorticoid feedback sensitivity with reducedGR and MR in the hippocampus and other feedback sites (Muellerand Bale, 2008; Noorlander et al., 2006; Shoener et al., 2006; Welberg



et al., 2000). Thus glucocorticoid excess in the last trimester of ratpregnancy permanently attenuates GR and MR mRNA expressions inspecic hippocampal subelds (Henry et al., 1994; Levitt et al., 1996;Noorlander et al., 2006; Shoener et al., 2006; Welberg et al., 2000).Similarly, in guinea-pigs prenatal dexamethasone exposure results insex and region-specic changes in MR and GR mRNA in foetal brains(Dean andMatthews, 1999). This not only impacts directly on the HPAaxis, but also mediates glucocorticoid actions on behaviour, cognitionand disease susceptibility. Offspring whosemothers received an HSD2inhibitor during pregnancy, have increased GR mRNA expression inthe amygdala and increased anxiety-like behaviour in adulthood(Welberg et al., 2000). It is also plausible that increases in GR levels inthe basolateral, central and medial nuclei of the amygdala increasepositive drive onto the HPA axis which increases stress responsivity(Welberg et al., 2000).

The balance between MR and GR in hippocampus and other CNSregions is an important factor determining individual susceptibility orresilience to disease in adulthood (de Kloet, 2004, 1991; de Kloet andReul, 1987; De Kloet et al., 1998). MR and GR gene variants in thehuman population which associate with altered MR and GRexpression/activity have consequences for stress responsivity, cogni-tive performance and emotional arousal in later life (DeRijk and deKloet, 2005; DeRijk, 2009). Thus, an imbalance in MR/GR caused bygenetic factors and/or early life experience my give rise to a particularphenotype that is vulnerable to later life stressors. Clearly the adultenvironment plays a crucial role here too.

Further, evidence for the importance of MR and GR signalling inemotional behaviour comes from studies using mutant mice withaltered GR levels in the brain. Mice with selective loss of GR in thebrain show impaired behavioural response to stress and displayreduced anxiety relative to littermate controls (Tronche et al., 1999).Although, a global reduction of GR, which more closely models thehuman gene variant condition, has no impact on anxiety-likebehaviour, but impairs cognition in mice (Oitzl et al., 2001; Ridderet al., 2005). Forebrain specic loss of MR leads to impaired learningand memory and increased behavioural perseverance and stereotypy(Berger et al., 2006), whereas forebrain overexpression of MRfacilitates learning and reduces anxiety-like behaviours (Lai et al.,2007; Rozeboom et al., 2007). Undoubtedly, total body and brainmanipulations of MR or GR may cause multiple or compensatoryeffects that either cancel out ormask other subtle effects. One solutionis to use an inducible gene manipulation methodology (e.g. Mitraet al., 2009). Simultaneous manipulation of MR and GR in a region-specic manner using gene inducible methods may prove to be aparticularly fruitful avenue for future research into the role of MR/GRbalance in affective health. Moreover, the impact of early life, prenataland/or chronic stress paradigms on these mutant mice will beespecially illuminating in dissecting out the role of adult environment,genes and development in resilience and susceptibility to chronicdisease.

The programming of affective disease

In humans, maternal stress has been linked with poor copingbehaviour under adversity, aggressive and antisocial behaviour,attention decit and hyperactivity disorders and depression inchildren and adults (Weinstock, 2008). For example, a retrospectivestudy of severely emotionally disturbed children and adolescents,revealed that these children were signicantly more likely to havemothers that had experienced chronic prenatal stress than emotion-ally stable children (Ward, 1991). Maternal anxiety has been linked togreater levels of self-reported anxiety in 8 and 9 year old offspring(Van den Berg and Marcoen, 2004). However, as with all observa-tional human data, it is difcult to imply causation because maternalanxiety may be transmitted to offspring via genes, prenatal and/orpostnatal effects. In other words, babies from anxious mothers may

be genetically anxious, may learn to be anxious or may indeed beprogrammed to be anxious.

Fortunately, there are myriad data from rodentmodels of maternalstress (in which cross-fostering removes confounding maternaleffects) that substantiate the relationship between excess foetalglucocorticoid exposure and unfavourable behavioural traits. Forexample, comparedwith non-stressed control rats, prenatally stressedrats exhibit impaired social behaviour and a reduced propensity toplay (Takahashi et al., 1992a). Depressive-like behaviours are alsoaltered. In rodents, prenatal stress increases immobility time in theforced swim test and tail suspension test and leads to anhedonia(Alonso et al., 2000; Sobrian et al., 2000) though not always (Hauseret al., 2009). And anxiety is increased too. The offspring of prenatallystressed rats spend less time than controls in the open arms of anelevated plus maze (Estanislau andMorato, 2005; Murmu et al., 2006;Vallee et al., 1997). And rats from mothers that were prenatallystressed exhibit higher levels of anxiety (such as defensive freezingbehaviour or thigmotaxis) than control rats in novel environments,such as the open eld (Dickerson et al., 2005; Takahashi et al., 1992b;Ward et al., 2000). While it is tempting to conclude that changes inanxiety-related behaviour are the direct result of an altered HPA axisresponse to novelty, it is possible to see increased anxiety-relatedbehaviour in the absence of HPA axis changes (Fride and Weinstock,1988; Holmes et al., 2006; Takahashi et al., 1992b).

A plausible alternative explanation for the increased hyper-emotional state of prenatally stressed rats is altered functioning ofthe amygdala. The amygdala mediates fear and anxiety-relatedbehaviour, learning and memory and thus, is instrumental in theexpression of fear-conditioned learning (Davis, 1992). The amygdalacontains MR, GR and CRH receptors and CRH producing cells. Indeed,CRH may be the key neurotransmitter that mediates the effect ofprenatal stress on anxiety. Firstly, CRH levels are increased in thecentral nucleus of the amygdala in prenatally stressed or glucocor-ticoid-overexposed rats (Cratty et al., 1995; Welberg et al., 2000,2001). And secondly, CRH injections directly into the amygdalaincrease anxiety-related behaviour in rats (Dunn and Berridge, 1990).

Central nervous system programming mechanisms: hippocampal GR asan exemplary target

How do prenatal environmental factors programme lifespanphysiology and chronic disease? Looking to mechanisms of postnatalprogramming provides some ideas. In rats, for example, high levels ofmaternal care permanently increase GR levels in the hippocampus andprefrontal cortex (Liu et al., 1997). This results in lower plasmaglucocorticoid levels and tighter feedback (Meaney et al., 1989).Increased GR expression is thought to involve increased serotonergic(5HT) release in response to tactile stimulation (Smythe et al., 1994)and increasedhippocampal expressionof the transcription factornerve-growth-factor-inducible factor-A (NGFI-A). In vitro and in vivo studiesshow that increased 5HT signalling induces GR expression in hippo-campalneurons (Mitchell et al., 1990; Yauet al., 1997) andNGFI-Abindsto the GR promoter to induce GR expression (Weaver et al., 2007). It isplausible that this post-natal mechanism may also be responsible forprenatalprogramming, since dexamethasone treatment in the lastweekof pregnancy in rats increases 5HT transporter expression in the brain(Fumagalli et al., 1996), an effect that is predicted to reduce local 5HTlevels in the brain and thus reduce MR and GR in the hippocampus.

How is tissue-specic GR expression regulated by early environ-mental manipulations? Although GR is present in most cells, GR geneexpression is regulated in a tissue-specic manner. This is because theGR promoter has multiple tissue-specic untranslated rst exons,most within a transcriptionally active CpG island (McCormick et al.,2000; Turner and Muller, 2005). All of the GR mRNA species aretranslated into the same receptor protein, which is encoded by exons29. Crucially, perinatal manipulations permanently programme



increased expression of exon 17 which is relatively specic to thehippocampus (McCormick et al., 2000). NGF1-A can bind to thisexon's promoter (Weaver et al., 2005).

Epigenetics as a mediator of programming

How is, for example, GR exon 17 permanently programmed?Emerging data suggest that early life experiences cause selectivemethylation/demethylation of specic cytosine residues, for instancein the GR 17 promoter. DNA hypermethylation and specic histoneresidue hypoactylation are epigenetic modications that are usuallyassociated with reduced gene expression. Offspring that haveincreased GR expression levels in the hippocampus as a result ofhigh levels of maternal care, also have lower levels of DNAmethylation in the NGFI-A binding site of the GR 17 promoter,increased histone H3-K9 acetylation (a marker of transcriptionalactivation) and increased NGFI-A binding to the 17 promoter relativeto offspring from low care mothers (Weaver et al., 2004, 2007).

The GR gene is not the only HPA axis target to undergo epigeneticmodication in early life. Early life stress in mice results in elevatedCRH and AVP transcription in the PVN. Increased AVP gene expressionis associated with DNA hypomethylation of a regulatory region thatserves as a DNA binding site for the methyl CpG-binding protein 2(MeCP2; involved in silencing gene expression). Persistently reducedMeCP2 binding results in permanent increases in AVP gene expressionand thus sustained hyperactivity of the HPA axis (Murgatroyd et al.,2009). Similarly, there is evidence that differential expression levels ofthe HSD2 enzyme correlate with differential levels of methylation ofCpG islands covering the promoter and exon 1 region of the HSD11B2gene in human cells in vitro and in rats in vivo, suggesting thatepigenetic mechanisms may play a role in regulating HSD2 levels(Alikhani-Koopaei et al., 2004). In addition, in the placenta, thisenzyme is regulated by a host of factors including cytokines, hypoxia,glucocorticoids, peptide hormones and maternal nutritional cues,though the precise molecular mechanisms remain to be determined.

Human epidemiological studies also show that early life environ-mental conditions can cause epigenetic changes in humans that canpersist throughout life. For example, individuals that were prenatallyexposed to famine during the Dutch Hunger Winter have less DNAmethylation of the maternally imprinted IGF2 gene compared to theirunexposed same sex siblings some six decades later (Heijmans et al.,2008). At present, the phenotypic consequences of these epigeneticmarks remain to be determined, but prenatal famine is associatedwith schizophrenia and coronary heart disease (Painter et al., 2005;Susser et al., 1996; Susser and Lin, 1992). And examination of thebrains from suicide victims who experienced childhood abuse,revealed reduced GR mRNA and correspondingly increased levels ofmethylation of the NR3C1 exon 1F (the human homolog of the ratexon 17 region) which regulates GR expression in the hippocampus,relative to control humans (McGowan et al., 2009).

Transgenerational effects

Programmingmay not be restricted to the immediate offspring of achallenged pregnancy, but may also affect subsequent generations, socalled transgenerational effects. For this to happen, exposure to thefoetus must induce chromosomal alterations and/or epigeneticmodications that are stable and maintained down the germ line.Indeed, there is evidence that this can occur. In rats, the offspring fromF1 progeny that were prenatally exposed to dexamethasone alsoshow reductions in birth weight and persisting neuroendocrinechanges even though they themselves were not prenatally exposedto dexamethasone (Drake et al., 2005). This transgenerationalprogramming stopped at the third generation, suggesting againsta uterine effect. Moreover, programming effects were transmittedby either maternal or paternal lines, implying an epigenetic and/or

nucleic acid associated mechanism. Similarly, moderate maternalundernutrition was found to modify heart structure and HPA axisfunction in adult male offspring for two generations in a guinea pigmodel of maternal undernutrition (Bertram et al., 2008). In humansepidemiological surveys have linked grand-parental nutrition withdisease rates in the second generation (Kaati et al., 2002, 2007). Thefundamental processes that underpin such effects remain largelyunexplored but have been reviewed recently (Drake and Walker,2004; Gluckman et al., 2007; Matthews and Phillips, 2010).

In summary, prenatal exposure to stress and its glucocorticoidmediators exerts long-term effects on the offspring, altering brain andperipheral biology and changing the lifetime risk of disease. Sucheffects are clear in animal models and supported to an increasingextent by human observations. Major challenges include establishingthe fundamental mechanisms underpinning such effects, dissectingtheir importance versus other factors causing disease and determin-ing the impact or otherwise of intergenerational effects.

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