effects of increasing gestation, cortisol and maternal undernutrition on hypothalamic neuropeptide y...

Journal of Neuroendocrinology, 1998, Vol. 10, 51–57

Effects of Increasing Gestation, Cortisol and MaternalUndernutrition on Hypothalamic Neuropeptide YExpression in the Sheep Fetus

K. E. Warnes*, M. J. Morris†, M. E. Symonds‡, I. D. Phillips*, I. J. Clarke§, J. A. Owens* and I. C. McMillen**Department of Physiology, University of Adelaide, Adelaide, SA 5005, Australia.†Department of Pharmacology, University of Melbourne, Parkville, Vic 3052, Australia.‡Department of Child Health, Queens Medical Centre, University Hospital, Nottingham, UK.§Prince Henrys Medical Research Institute, Monash Medical Centre, Clayton, Vic 3168, Australia.

Key words: NPY, hypothalamus, fetus.

Abstract

We have characterized the localization and the ontogenetic changes in Neuropeptide tyrosine (NPY)before birth and investigated the regulation of NPY expression by cortisol and undernutrition in thefetal sheep hypothalamus during late gestation. Using immunohistochemistry, we have identified NPY-containing neurons in the infundibular nucleus and the internal layer of the median eminence in fetalhypothalami collected between 110 and 147 days gestation. NPY projections were also present in theparaventricular nucleus (PVN) of fetal hypothalami at all ages between 110 days gestation and term.There was a significant increase in the amount of immunoreactive NPY/g hypothalamus between 87and 113 days and 131–140 days gestation and a further significant increase after 141 days gestation.The total hypothalamic content of immunoreactive NPY increased significantly between 87 and 113days and 141–145 days gestation. The levels of NPY mRNA: 18S rRNA in the mediobasal region of thefetal hypothalamus were significantly higher at 145–146 days gestation than at any earlier gestationalage between 116 and 141 days gestation. Cortisol (2.5–3.0 mg/24 h) was infused intrafetally between109 and 116 days gestation. The ratio of NPY mRNA: 18s rRNA in the mediobasal region of the fetalhypothalamus was significantly higher in the cortisol-infused group when compared with the saline-infused control group at 116 days gestation. Maternal, and hence fetal undernutrition, was inducedbetween 110 and 146 days gestation. At 145–146 days gestation the ratio of NPY mRNA: 18S rRNA inthe mediobasal region of the fetal hypothalamus was significantly higher in the undernutrition groupwhen compared with control animals. We have therefore demonstrated that NPY is present in thehypothalamus of the sheep fetus before birth and that hypothalamic NPY content and NPY mRNAincrease before delivery. We have also found that glucocorticoids and undernutrition stimulateincreases in NPY mRNA levels in the hypothalamus before birth.

Neuropeptide Y (NPY ) is a 36 amino acid peptide which is are food restriction (2–9), decreased circulating insulin levels(10) and increased glucocorticoid concentrations (11–13).synthesized in the hypothalamic arcuate nuclei and secreted

by nerve terminals in the paraventricular nucleus (PVN ) (1). NPY mRNA levels in the arcuate nucleus (2–4), NPYconcentrations in the PVN (5–7) and NPY secretion into theEndogenous hypothalamic NPY appears to play a major role

in adult life in the coordination of energy metabolism through PVN (8, 9) all increase in response to food deprivation and,subsequently, return to normal when food is provided (2, 5,regulation of food intake and the pituitary-adrenal axis. NPY

is the most potent appetite stimulant known, and central 7). NPY concentration and mRNA in the hypothalamus arealso increased in diabetes mellitus and this effect is reversedadministration of NPY into the PVN significantly increases

feeding and can lead to obesity (1). The major stimuli for by insulin, probably via an action at the insulin receptorswithin the arcuate nucleus (14–17). Type II glucocorticoidNPY synthesis and secretion in the adult rat hypothalamus

Correspondence to: Professor I. C. McMillen, Department of Physiology, University of Adelaide, Adelaide, SA 5005, Australia.

© 1998 Blackwell Science Ltd

52 NPY in the fetal sheep hypothalamus

receptors (GR) are present in the NPY-containing cell bodies were scattered throughout the infundibular nuclei and theinternal and external layers of the median eminence (Fig. 1,).of the arcuate nucleus in the rat hypothalamus (18), and

prepro-NPY mRNA levels and the NPY content of the NPY-containing fibres were also observed in the periven-tricular hypothalamus, extending between the mid region ofhypothalamus, are increased by dexamethasone administra-

tion in vivo and in rat fetal hypothalamic neurons in culture the infundibular nucleus and the paraventricular nuclei. Therewere positively stained NPY-containing fibres throughout the(11–13, 19). Administration of NPY either directly into the

PVN or into the cerebral ventricles in the rat, dog or sheep rostrocaudal extent of the PVN.also results in the stimulation of CRF and AVP secretioninto the hypophysial portal circulation (20, 21) and anincrease in the plasma concentrations of ACTH and glucocor- Hypothalamic NPY content in late gestationticoids (22, 23).

There are key differences, however, between the regulation The hypothalamic content of immunoreactive NPY(expressed as either mg/g hypothalamus or as total hypothal-of energy balance in fetal and adult life. The fetus cannot

alter feed intake to maintain nutrient availability, and in all amic content) increased significantly (P<0.005) with increas-ing gestational age. There was an increase (P<0.05) in thespecies the fetus is hypoglycaemic and hypoinsulinaemic

relative to the mother because a maternal-fetal glucose gradi- amount of immunoreactive NPY/g hypothalamus between 87and 113 days (0.52±0.06 mg/g; n=4) and 131–140 daysent is essential to drive the placental delivery of glucose from

the mother to the fetus (24). In the pregnant sheep, for gestation (0.89±0.05 mg/g; n=4) and a further increase(P<0.05) after 141 days gestation (1.30±0.11 mg/g; n=6)example, circulating glucose concentrations are lower on

the fetal side (0.7–1.5 mmol/l ) than on the maternal side (Fig. 2). The total hypothalamic content of immunoreactiveNPY increased significantly (P<0.05) between 87 and(2.5–4.0 mmol/l ) of the placenta throughout pregnancy (24).

Information on the role of hypothalamic NPY in the coor- 113 days (1.09±0.51 mg) and 141–145 days gestation(4.34±0.55 mg) (Fig. 2).dination of energy balance before birth and in the regulation

of the fetal pituitary-adrenal axis is limited. NPY is presentin the fetal rat hypothalamus (25), where its expression orabundance is reduced by maternal hyperglycaemia or by Hypothalamic NPY mRNA levels in late gestationintracerebroventricular infusion of insulin into the fetal brain(26). This suggests that the NPY axis is present before birth The level of NPY mRNA: 18S rRNA in the mediobasal

region of the fetal hypothalamus was significantly higherand may be active, but the influence of the major postnatalmodulators of hypothalamic NPY, glucocorticoids and (P<0.05) at 145–146 days gestation (157±13; n=6) than at

any earlier gestational age (116–141 days) (Figs 3 and 4).undernutrition, on hypothalamic NPY expression in the fetusare unknown. There was no difference, however, in the abundance of NPY

mRNA: 18S rRNA in the fetal hypothalamus between 116The present study describes the localization of NPY-containing cell bodies within the infundibular nuclei of the days and 141 days (116–117 days, 80±3; n=6: 124–126

days, 63±8; n=6: 138–141 days, 65±10) (Fig. 3).fetal sheep hypothalamus (corresponding structures to thearcuate nuclei of the rat hypothalamus) and the ontogeneticchanges in hypothalamic NPY content and mRNA levels inthe sheep fetus during late gestation. We have also investi- Effect of cortisol on hypothalamic NPY mRNA levelsgated whether hypothalamic NPY mRNA expression isstimulated by administration of cortisol or fetal under- The mean fetal plasma concentrations of cortisol between

109 and 116 days gestation were significantly higher (P<0.05)nutrition before birth.in the cortisol infused group (2.5–3.0 mg cortisol/24 h for 7days) when compared with saline infused control animals(31.8±4.8 nmol/l vs 1.4±0.2 nmol/l ). The ratio of NPYResultsmRNA: 18s rRNA in the mediobasal region of the fetalhypothalamus was significantly higher (P<0.05) in the corti-Localization of NPY in the fetal sheep hypothalamussol infused group (310±60) when compared with the salineinfused control group (150±20) (Fig. 5).There were positively stained nerve cell bodies and fibres for

NPY present in fetal sheep hypothalami at all ages between110 and 147 days gestation (n=11). Positive staining for NPYwas predominantly present in the nerve cell bodies of Undernutrition and fetal hypothalamic NPY mRNAthe hypothalamic infundibular nuclei (Fig. 1–). NPY-containing nerve cell bodies were observed throughout the The mean plasma glucose concentration between 116 and

145 days gestation was significantly lower (P<0.05) in therostrocaudal extent of the infundibular nuclei and had oval orfusiform perikarya with maximal diameters of around undernutrition group (0.93±0.08 mmol/l ) than in the control

group (1.42±0.05 mmol/l ). At 145–146 days gestation. the15–20 mm (Fig. 1). Positively stained NPY-containing cellswere found mainly in the ventromedial part of the infundibular ratio of NPY mRNA: 18S rRNA in the mediobasal region

of the fetal hypothalamus was significantly higher (P<0.05)nuclei, around the wall of the third ventricle (Fig. 1,). NPY-containing cells were also present in the internal layer of the in the undernutrition group (590±190) when compared with

the control animals (250±30) (Fig. 6).median eminence and immunoreactive NPY-containing fibres

© 1998 Blackwell Science Ltd, Journal of Neuroendocrinology, 10, 51–57

NPY in the fetal sheep hypothalamus 53

F. 1. Immunohistochemical staining for NPY in the hypothalami offetal sheep between 125 and 141 days gestation. () Cell bodies, nervesand nerve terminals in the mid region of the infundibular nucleus andin the internal layer of the median eminence in a sheep fetus at 132 daysgestation. Scale bar=200 mm. () The highlighted region of () is shownat a higher magnification to demonstrate NPY-containing cell bodiesand nerve fibres in the internal layer of the median eminence at 132 daysgestation. Scale bar=100 mm. () NPY-containing cell bodies and nerveterminals in the internal layer of the median eminence in a sheep fetusat 141 days gestation. The orientation of the NPY projections is Gestational age (days)

87–113 131–140 141–145

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predominantly towards the external layer of the median eminence. ScaleF. 2. Mean NPY content of the fetal sheep hypothalamus, expressedbar=100 mm. ( ) NPY-containing neurons in the periventricular regionas either mg/g hypothalamus (top panel ) or as total hypothalamic NPYof the infundibular nucleus in a sheep fetus at 141 days gestation. Thecontent (bottom panel ) in three gestational age groups. Different super-NPY cell bodies are oval and fusiform in shape. Scale bar=25 mm. ()script letters within a panel represent mean values which are significantlyNPY-containing cell bodies in the internal layer of the median eminencedifferent (P<0.05).in a sheep fetus at 125 days gestation. Scale bar=50 mm.

Discussionpresent in the ventromedial portion of the infundibularnucleus and in the internal layer of the median eminenceWe have demonstrated that NPY is present in the cell bodies

of the infundibular nuclei of the fetal sheep hypothalamus (28). In the rat, the arcuate nucleus, which corresponds tothe infundibular nucleus in the sheep, contains the highestand that NPY-containing nerve terminals are present in the

fetal PVN throughout late gestation. There is an increase in number of cell bodies in the central nervous system thatexpress NPY mRNA (29). It has also been demonstrated inthe hypothalamic content of NPY during the last 10–15 days

of gestation and in NPY mRNA levels during the last week the rat, that there is a projection from the arcuate nucleus tothe PVN and that there are NPY-containing fibres in contactbefore delivery. Intrafetal infusion of cortisol and fetal under-

nutrition each stimulated an increase in NPY mRNA levels with each major class of output neuron from the PVN (31).The NPY-containing terminals within the rat PVN are derivedin the fetal hypothalamus.

The pattern of localization of NPY and the size and shape from two sources: the arcuate nucleus, and catecholamine-containing neurons in the brainstem. In the rat brainstem,of the cell bodies containing NPY in the fetal hypothalamus

are similar to those described in the adult sheep hypothalamus NPY is extensively colocalized within adrenergic neurons ofthe C1, C2 and C3 cell groups, while the correspondence in(28). McShane et al. (28) have demonstrated that NPY

mRNA is present in the infundibular nucleus of the lamb noradrenergic cell groups is less complete and generallylimited to a subset of neurons in the A1 cell group (31). Ithypothalamus and immunohistochemical studies in the adult

sheep have also localized NPY in oval or fusiform cell bodies appears, however, from lesion studies that the arcuato-



paraventricular projection represents the more importantNPY input to the PVN (31).

We found no increase in NPY mRNA levels in the medio-basal hypothalamus between 117 and 141 days gestation,although total hypothalamic content of NPY appeared toincrease between 113 and 140 days gestation. HypothalamicNPY content represents an integral of both NPY synthesisand secretion in the whole hypothalamus and may thereforenot correlate directly with NPY mRNA levels in the medio-basal hypothalamus. There was however, a significantincrease in both hypothalamic NPY mRNA levels and NPYcontent after 141 days gestation, i.e. 7–10 days before deliv-ery. One possibility is that the increase in hypothalamic NPYmRNA and content is related to the exponential increase incirculating cortisol that occurs during the last 20 days ofgestation (32). Type II corticosteroid receptors are presentin the NPY-containing cell bodies of the arcuate nucleus inthe rat hypothalamus (18), and prepro-NPY mRNA levelsand the NPY content of the hypothalamus are increased bydexamethasone administration in vivo and in rat fetal hypo-thalamic neurons in culture (11–13, 19). In the present study,we found that inducing an increase in circulating cortisol Gestational age (days)

116–117 138–141 145–146

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concentrations at 116 days gestation, to levels similar to those F. 4. Ratios of NPY mRNA: 18S rRNA in the mediobasal region ofmeasured at around 125–135 days gestation, stimulated an hypothalami of fetal sheep in four gestational age groups. The asterisk

indicates that the NPY mRNA: 18S rRNA ratio at 145–146 days isincrease in hypothalamic NPY mRNA levels. Liu et al. (22)significantly higher than at any previous gestational age.have also found, in the adult sheep, that intracerebroventricu-

lar administration of NPY caused a subsequent increase inthe levels of CRF and AVP in the hypophysial portal NPY peptide in the arcuate nucleus (2–5). We have demon-

strated that maternal undernutrition, which is associated withcirculation. Similarly, Brooks et al. (32) demonstrated thatcentral NPY administration stimulated ACTH secretion in fetal hypoglycaemia and hypoinsulinaemia (24), stimulates

an increase in NPY mRNA levels in the fetal hypothalamus.the adult and fetal sheep. One intriguing possibility in lategestation, therefore, is that there is a positive feedback effect This indicates that the NPY axis in the sheep hypothalamus

is responsive to changes in energy balance before birth. Foodof cortisol on hypothalamic NPY synthesis and secretion,which in turn could stimulate the fetal pituitary-adrenal axis. restriction in the adult rat (2–5) and sheep (28) consistently

increases hypothalamic levels of NPY mRNA and NPYIt has been demonstrated that food restriction in the adultrat results in a parallel increase in NPY mRNA levels and peptide. Current evidence supports a predominant role for

NPY mRNA

116 – 117 days 124 – 126 days

138 – 141 days 145 – 146 days

18s RNA

NPY mRNA

18s RNA

F. 3. Signals detected by the phosphorimager corresponding to NPY mRNA and 18s RNA in the mediobasal region of hypothalami from individualfetal sheep at 116–117 days (n=5), 124–126 days (n=6), 138–141 days (n=6) and 145–146 days (n=6) gestation.



in the pregnant rat has shown that maternal diabetes associ-ated with fetal hyperglycaemia in the absence of changes infetal insulin concentrations led to a decrease in fetal brainNPY mRNA and peptide levels (26). This suggests that inthe fetus, in contrast to the adult, hyperglycaemia alone cansuppress brain NPY synthesis. In these studies, intracerebraladministration of insulin did decrease NPY peptide concen-trations in the fetal rat brain but did not alter NPY mRNAlevels (26). It is interesting to speculate whether the increasein hypothalamic NPY mRNA levels and content in lategestation is a result of changes in fetal nutrient availabilityat this stage of pregnancy as well as the preparturient surgein cortisol. Between 75 and 145 days gestation, fetal glucoseconcentrations decrease by more than 30% to increase thematernal-fetal glucose gradient (34, 35). This is a necessaryphysiological adaptation, because it increases glucose deliveryto the fetus to meet its rapidly increasing needs and it occursdespite 5-fold increases in placental glucose transfer capacityover this time (35). Further work is clearly required to establishwhether NPY synthesis within the infundibular nucleus of thefetal hypothalamus is stimulated by the prevailing low glucoseCortisol

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and insulin concentrations in late gestation.F. 5. Ratios of NPY mRNA: 18S rRNA in the mediobasal region of Recent studies have clearly identified leptin as an a regu-hypothalami of fetal sheep infused with either saline or cortisol

lator of hypothalamic NPY expression (36, 37) and circulat-(2.5 mg/24 h) between 109 and 116 days gestation. The asterisk indicatesa significant difference (P<0.05) between the groups. ing leptin may also be an important signal and act at the

fetal hypothalamus to alter NPY expression before birth.In summary, we have found that NPY is present in the

cell bodies of the infundibular nuclei of the fetal sheephypothalamus and that there is a significant increase in thehypothalamic levels of NPY mRNA and peptide during thelast week before delivery. Intrafetal infusion of cortisol andfetal undernutrition each stimulated an increase in hypothal-amic NPY mRNA levels. It is possible, therefore, thathypothalamic NPY may play a role in the coordination ofenergy balance and in the regulation of the pituitary-adrenalaxis in the late gestation sheep fetus.

Materials and methods

Immunohistochemistry

Eleven pregnant ewes between 110 and 147 days gestation, carrying singletonor twin fetal lambs of known gestational age, were used in these studies. Allewes were sacrificed with an overdose of sodium pentobarbitone and the fetalsheep were delivered immediately by hysterotomy and decapitated prior todissection of the hypothalamus, which was immersed in Zambonis fixative(2% formaldehyde, 15% picric acid in 0.1 M phosphate buffered saline) forat least 48 h before clearing and permeabilization of the tissue with 80%ethanol (four 10 min washes), followed by Dimethylsulphoxide (four 10 minUndernutrition

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washes). The tissues were washed with several volumes of 0.1 M phosphateF. 6. Ratios of NPY mRNA: 18S rRNA in the mediobasal region of buffered saline (PBS), pH 7.4, then immersed in 30% sucrose in 0.1 M PBShypothalami of fetal sheep in well nourished control animals or under- prior to embedding and storage at −70 °C. Coronal hypothalamic sectionsnourished pregnant ewes. The asterisk indicates a significant difference (30–60 mm) were cut and placed in 0.1 M PBS. The free floating sections were(P<0.05) between the groups. incubated in 0.1 M PBS at 4 °C overnight and all reagents for immunohisto-

chemical staining were also diluted in 0.1 M PBS unless otherwise stated.Hypothalamic sections were incubated with hydrogen peroxide (H2O2, 3%)insulin rather than glucose in regulating NPY synthesis duringfor 10 min and then either bovine serum albumin (1%) or sheep serum (10%)

food restriction. In the adult rat, streptozotocin-induced for 30 min. Sections were then incubated overnight with the primary antiseradiabetes associated with hyperglycaemia and hypoinsulinae- (rabbit antiporcine NPY, 151600 diluted in 1% BSA/0.1 M PBS; Peninsula

Laboratories, Belmont, CA, USA) before addition of the second antibodymia also results in an increase in hypothalamic NPY levels(biotinylated donkey antirabbit IgG, 15100: Vector Laboratories, Bulinghame,similar to that observed in association with hypoglycaemiaCA, USA) for 120–180 min After three 10 min washes in 0.1M PBS, the(15). Furthermore, insulin administration reversed the sections were exposed to the avidin biotin complex (15100, Vectastain Kit,

changes in the NPY concentrations induced by diabetes in Vector Laboratories, Bulinghame, CA, USA) for 60 min. After three 5 minwashes in 0.1 M PBS, diaminobenzidine tetrahydrochloride tartratethe adult rat hypothalamus (16). Recently, however, a study



(0.5 mg/ml ) was added for 10 min and, subsequently, H2O2 was added for Oligonucleotide probes. A 48 bp oligonucleotide antisense probe complement-ary to the nucleotide sequences encoding amino acids 231–278 of human30–60 s The sections were then washed in PBS (three 5 min washes) and twice

in Milli Q water (5 min) before mounting on to slides. NPY mRNA and an antisense 30 bp oligomer to part of the 18S rRNA wereend labelled with c32-P ATP (4000 Ci mmol, Bresatec, Adelaide, SA,

Extraction and radioimmunoassay of NPY Australia) by T4 polynucleotide kinase (Pharmacia, Wintirna South,Victoria, Australia).Fourteen hypothalami from fetal sheep in three separate age groups (87–113

days, n=4; 131–140 days, n=4; 141–145 days gestation, n=6) were collectedNorthern blot analysis. Hypothalamic RNA samples (20 mg/lane) were separ-

for extraction and measurement of NPY. The hypothalamic tissue was minced ated on a 1% agarose gel containing 2.2 M formaldehyde, and subsequentlyfinely on ice, boiled for 10 min in 5 volumes acetic acid (1 mol/l ), then

transferred by capillary blotting to Zetaprobe nylon membranes. Membraneshomogenised for 15 s in glass/teflon homogeniser (six passes) and tissue rinsed

were washed in 10×SSC (1×SSC: 0.15 M NaCl, 15 mM sodium citrate,from the homogeniser with another five volumes acetic acid (1 mol/l ). Tissues

pH 7.0) for 10 min at room temperature before being baked for 30 min atwere left standing on ice for 1 h prior to centrifugation at 7500 r.p.m. at 4 °C 80 °C and stored flat at −20 °C until hybridization. Membranes were pre-for 30 min. The supernatant was decanted, weighed and a known volume

hybridized at 50 °C for 18 h in a solution consisting of 5×SSC, 20 mMlyophilised overnight (Speed Vac, Savant Instruments, Farmingdale, New York,

NaH2PO4, pH 7.2, 7% SDS, 5×Denhardt’s, and 100 mg/ml of heat denaturedUSA) for subsequent measurement of immunoreactive NPY. Immunoreactive- Salmon sperm DNA. Membranes were hybridized with 1×106 cpm/ml ofNPY was determined by radioimmunoassay as previously described (38) using labelled NPY oligonucleotide probe for 20 h at 50 °C. Membranes werean antibody raised in a rabbit immunized with synthetic NPY (39). Dried

washed in a series of steps, 1×SSC, 0.1×SDS at room temperature forextracts were reconstituted in assay buffer and assayed in triplicate in a total

30 min, 1×SSC, 0.1×SDS at 50 °C for 30 min and 0.1×SSC, 0.1×SDS atvolume of 300 ml. Standard curves were constructed using porcine NPY 50 °C for 25 min. Membranes were then exposed to Fuji phosphorimage(10–1280 pg/tube; Auspep, Australia). Following 24 h incubation at 4 °C, 125I— plates. The NPY probe was stripped from the membranes by washing inNPY (Amersham, UK) was added and the incubation continued overnight.

0.1×SSC (1×SSC: 0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 0.5% SDSBound and free radioactivity was separated by incubation with sheep antirabbit

for 10 min at 90 °C. The consistency of lane loading was verified by hybridiza-second antibody (Silenus, Australia) and centrifugation at 4 °C. In all assays, tion of the membrane with radiolabeled 18s rRNA oligomer probe. Thethe non specific binding was calculated for every sample. All reagents were of signals present on the phosphorimage plates were visualized and quantifiedanalytical grade. Inter-and intra-assay coefficient of variation were 13% and

on a Fuji-BAS 1000 phosphorimager.6%, respectively, and the sensitivity of the assay was less than 3 pg.

Plasma glucoseNPY mRNA studiesPlasma glucose concentrations were determined by enzymatic analysis usingOntogeny. Twenty-three pregnant ewes carrying singleton or twin fetal lambshexokinase and glucose-6-phosphate dehydrogenase and measuring the forma-between 116 and 146 days gestation (116–117 days, n=5, 124–126 days, n=tion of NADH photometrically at 340 nm (COBAS MIRA automated6, 138–141 days, n=6, 145–146 days,=6) were used in this protocol. All ewesanalysing system, Rouch Diagnostic, SA, Australia). The intra-assay coeffi-were sacrificed with an overdose of sodium pentobarbitone and the fetal sheepcient of variation was 1.4%.were delivered by hysterotomy and sacrificed by decapitation prior to the

removal of the brain. The fetal hypothalamus was dissected using the rostral Cortisol radioimmunoassayborder of the optic chiasm and the caudal border of the mamillary bodies as

Total cortisol concentrations were measured in fetal sheep plasma by radio-landmarks on the ventral surface and the lateral hypothalamic sulci as theimmunoassay using an Orion Diagnostica kit (Orion Diagnostica, Turku,lateral boundaries. Several hypothalamic blocks were dissected, including aFinland). Prior to assay, cortisol was extracted from the plasma usingblock containing and extending 3 mm vertically from the base of the mediandichloromethane. The efficiency of recovery of [125I ]-cortisol from fetal plasmaeminence. This block contained the mediobasal region of the fetal hypothalamususing this procedure was at least 90%. The sensitivity of the assay wasand included the infundibular nuclei. In preliminary experiments, we found0.39 nmol/l and the cross reactivity of the rabbit anticortisol antibodythat NPY mRNA expression was predominantly present in this block andwas<1% with cortisone and 17-hydroxyprogesterone and<0.01% with preg-therefore this hypothalamic block was routinely collected in this and thenenolone, aldosterone, progesterone and estradiol. The inter and intra assayremaining experimental protocols. The hypothalamic tissue was snap-frozen incoefficents of variation were always less than 10%.liquid nitrogen and stored at −80 °C prior to the extraction of total RNA.

Cortisol infusion. Surgery was performed on twelve pregnant ewes and their Statistical analysisfetal sheep at either 103 or 104 days gestation under general anaesthesia and All data are expressed as mean±standard error of the mean (SEM). The NPYusing aseptic techniques (40). A catheter was implanted into a fetal carotid content data are expressed as mg peptide per g hypothalamic tissue or as totalartery and jugular vein, filled with heparinised saline and exteriorised through hypothalamic NPY-LI content (mg/hypothalamus). NPY content data werean incision in the ewe’s flank. Cortisol (2.5–3.0 mg/5 ml/24 h, n=6; logarithmically transformed where necessary to reduce heterogeneity of vari-Solucortef, Upjohn Pharmaceuticals Ltd, Tullamarine, Victoria, Australia) or ance. NPY mRNA: 18S rRNA ratios were routinely multiplied by 100. Onesaline (n=6) was infused intravenously between 109 and 116 days gestation. way analysis of variance () and the Duncans Multiple Range test wereFetal arterial blood samples (2 ml ) were collected twice daily throughout the used to determine whether there were significant differences in the hypothalamicperiod of the infusion until the ewe and fetus were killed at 116 days gestation. content of NPY or in the ratio of hypothalamic NPY mRNA: 18S rRNAThe mediobasal region of the fetal hypothalamus containing the median levels between the gestational age groups. Student’s unpaired t-test was usedeminence and infundibular nuclei was dissected and snap frozen at −80 °C. to determine whether there were significant differences in the ratio of hypothal-

amic NPY mRNA: 18S rRNA levels between experimental groups (i.e. cortisolUndernutrition. At 115 days gestation, ewes were placed on either a restrictedvs saline infused fetal sheep and undernutrition vs control feeding groups). Afeeding regime (10 g chopped lucerne and 1.5 g oats/kg body weight; under-probability of 5%, i.e. P<0.05 was taken to be significant.nutrition group) or an ad libitum feeding regime (20 g chopped lucerne and

3 g oats/kg body weight; control group). The amount of feed offered wasincreased by 15% in each feeding group every 10 days. Fetal arterial bloodsamples (3 ml ) were collected three times per week for measurement of Acknowledgementsarterial blood gas status and plasma glucose concentrations. Ewes were killedat 141–146 days gestation and the mediobasal region of the fetal hypothalamus We are grateful to the National Health and Medical Research Council ofcontaining the median eminence and infundibular nuclei was dissected from Australia for financial support for this project. We are very grateful to Vickifetal sheep in the undernourished group (n=6) and control group (n=6) and Mau and Helen Rodgers for their expert assistance with the immunohisto-frozen as described above. chemical staining procedures and photography. We are also grateful to

Timothy Butler and Jacob Ross for their assistance with the cortisol infusionExtraction of total RNA. Hypothalamic blocks were homogenised in 4 M experiments and to Anne Jurisevic for her expert assistance with fetalguanidine hydrochloride, and after ultracentrifugation through a 5.7 M sheep surgery.caesium chloride cushion, the purity and concentration of the total RNA wasquantified by spectrophotometric measurement at 260 nm and 280 nm asdescribed previously (40). Accepted 26 September 1997



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effects of increasing gestation, cortisol and maternal undernutrition on hypothalamic neuropeptide y...

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