effect of nutritional stress on the hypothalamo-pituitary-gonadal axis in the growing male rat

Original Paper

Neuroimmunomodulation 2002–03;10:153–162DOI: 10.1159/000067177

Effect of Nutritional Stress on theHypothalamo-Pituitary-Gonadal Axis inthe Growing Male Rat

Cecilia V. Compagnuccia Gabriela E. Compagnuccia Alejandro Lomniczia

Claudia Mohnc Irene Vacasb Elisa Cebralc Juan C.Elverdina

Silvia M. Friedmanb Valeria Rettoric Patricia M. Boyera

Departments of aPhysiology, and bGeneral and Oral Biochemistry, School of Dentistry, University of Buenos Aires,and cCenter of Pharmacological and Botanical Studies (CONICET), Buenos Aires, Argentina

Received: December 18, 2001Accepted: May 3, 2002

Dr. Patricia Monica Boyer, PhD, Associate ProfessorDepartment of Physiology, School of Dentistry, University of Buenos AiresMarcelo T de Alvear 2142, 3er piso, sector A.1122, Buenos Aires (Argentina)Fax +54 11 4508 3958, E-Mail [email protected]

ABCFax + 41 61 306 12 34E-Mail [email protected]

© 2002 S. Karger AG, Basel1021–7401/02/0103–0153$18.50/0

Accessible online at:www.karger.com/nim

Key WordsWeanling rat W Food restriction W LHRH W LH W

Testosterone W Leptin W Sperm morphology

AbstractBackground/Objective: Nutritional dwarfing (ND) con-sists of a decrease in weight and height gain and delayedonset of puberty. The aim of the present investigationwas to study the modifications induced in male rats bythe nutritional stress of a mere 20% reduction in foodintake which, however, started immediately after wean-ing. Materials and Methods: At weaning, male Wistarrats were divided into two groups: Control (C) and ND. Crats were fed ad libitum with a balanced rodent diet. NDreceived 80% of the diet consumed by C for 4 weeks (T4);then they were fed ad libitum for another 4 (T8) and 8weeks (T12). The rats were studied at T0, T4, T8 and T12for the effects of nutritional stress and refeeding on nutri-tional status, body composition, hypothalamic-pituitary-gonadal axis, and sperm morphology and concentration.Results: ND body weight and length diminished vs. C(p ! 0.001). ND body fat percentage decreased 40% (p !0.001) without change in the percentage of body proteincontent. The hypothalamic content of LHRH did not

change. However, FSH, LH and testosterone serum lev-els had significantly decreased (p ! 0.001) at T4 in NDrats. A 48.4 % decrease in serum leptin in the ND groupwas observed at T4 (p ! 0.05). The absolute testicular andseminal vesicle weight was significantly decreased byND at T4 (p ! 0.001). At T4 the percentage of anomalies ofcaudal spermatozoa increased in about 64% (p ! 0.001)of ND vs. C rats, despite the unchanged sperm concen-trations. All parameters normalized during refeeding.Conclusion: In this model, a decrease in leptin due tonutritional stress could be responsible, at least in part,for the inhibition of reproductive function. Refeedingnormalized all parameters studied.

Copyright © 2002 S. Karger AG, Basel

Introduction

It is well known that different kinds of neurogenicstress produce very similar hormonal alterations, al-though the original causes of these changes are different.All present an activation of the hypothalamic-pituitary-adrenal axis and an inhibition of the gonadal axis [1].Chronic undernutrition is one of the most importantcauses of metabolic and neuroendocrine alterations. It is

154 Neuroimmunomodulation 2002–03;10:153–162 Compagnucci et al.

also well known that there are links between nutrition andsexual maturation [2]. How nutrition regulates reproduc-tive activity remains a major unsolved question of repro-ductive biology.

In the literature, different animal models were used tostudy the effect of undernutrition on different parametersof the hypothalamic-pituitary-gonadal (HPG) axis by em-ploying diets deficient in or totally devoid of protein, orunder conditions where animals were acutely starved orseverely and chronically underfed [3–6]. In addition, pre-vious investigators have focused their endocrine nutritionstudies almost entirely on adult animal models [7]. Inadult male rats and rhesus monkeys, short-term starva-tion reduces circulating gonadotropin and testosteronelevels and the pulsatile frequency of their secretion [8, 9].Studies in adult rhesus monkeys showed that calories,regardless of their source, regulated LH secretion and thatfasting can produce changes in LH secretion very rapidly[10]. In male rats, whose food was restricted to 50%, asignificant reduction in testis weight and in LH and tes-tosterone concentrations without alterations in LHRHgene expression was observed [11]. However, in situhybridization studies showed inconsistent results regard-ing the effect of food restriction on LHRH gene expres-sion [12]. It seems that the contradictory results reportedmay be due to differences between species and to theseverity and extent of the food restriction. In addition tothe effects of undernutrition on reproductive hormonalprofiles in adulthood, dietary manipulations can delay theonset of puberty [13] and affect reproductive behavior[14].

Since fasting is one of the most severe nutritionalstresses, it is used in experiments to identify the majoreffects on the reproductive axis. In humans, certain par-ental health practices and beliefs can restrict a child’snutrition to the point of inducing a nutritional diseaseknown as nutritional dwarfing (ND) [15–18]. It is impor-tant to investigate whether this prevalent type of chronicmalnutrition has permanent consequences on sexual de-velopment. A previous study evaluated the onset of pu-berty in female rats subjected to different dietary manipu-lations at different ages [19], but the present study wasconducted to elucidate the onset of puberty and sexualmaturation in postweaning ND male rats. Although theaddition of micronutrients is not taken into account inweight control proceedings, in our model the restrictionwas imposed without compensation of mineral and vita-min intake, unlike other models of underfed animals [20,21]. This resembles human ND most closely; ND is aform of malnutrition manifested by a deficit in the height-

for-age index, but with weight-for-height and biochemicalnutritional indices within normal limits. The aim of thepresent investigation was to study the modifications in-duced by the nutritional stress of a mere 20% reduction offood intake in male rats if food restriction starts imme-diately after weaning. The reason for the age at which theanimals were food restricted was based on clinical pediat-ric findings related to infant feeding practices, like hypo-caloric foods that are given to infants after weaning [22,23]. The nutritional status, body composition, HPG axisand sperm morphology and concentration were assessedafter 4 weeks of nutritional stress and during the refeedingperiod.

Materials and Methods

AnimalsWeanling male Wistar rats of 21–22 days (initial body weight:

37.6 B 2.16 g, mean B SEM) were provided by the AnimalResources of the Department of Biochemistry, School of Dentistry,University of Buenos Aires, Argentina. Animals were housed in gal-vanized cages with meshed floors in order to maintain hygienic con-ditions and avoid coprophagia. They were exposed to 12-hour light-dark cycles. Room temperature was maintained at 21 B 1 ° C with ahumidity of 50–60%. All experiments were performed in agreementwith the UFAW Handbook on the Care and Management of Labora-tory Animals, and approved by the University of Buenos Aires EthicsCommittee.

DietAnimals were fed a standard diet (Purina chow) of the following

composition per 100 g: protein 24 g, lipids 5.1 g, ashes 7.0 g, water 9.6g, dextrin up to 100 g.

Experimental DesignOne hundred and twenty rats were randomly assigned to two

groups of 60 animals each, control (C) and ND. C rats were fed freelywith a standard diet. For the first 4 weeks, ND received 80% of theamount of food consumed by C rats on the previous day, correctedfor body weight (food intake in g/100 g body weight/day). For thefollowing 8 weeks, the ND group was freely fed with the same stan-dard diet eaten by C rats. All rats had free access to water. Bodyweight was recorded every day in the morning before food distribu-tion; length was measured every 4 days. Growth data were comparedover time. Dietary intake was registered every day. Twenty rats fromeach group were randomly selected every 4 weeks, of which 10 weresacrificed under anesthesia for chemical body composition or bydecapitation for hormone assays and evaluation of sperm morpholo-gy. After decapitation, trunk blood was collected, allowed to clot, andthe serum was stored at –20 ° C until hormone measurements. Afterremoval of the brain, the medium basal hypothalamus (MBH) wasdissected and homogenized to determine LHRH content. Testes andseminal vesicles were removed and weighed. Evaluation of epididy-mal sperm morphology was performed. Additionally, 20 rats weresacrificed for initial measurements on the day the experiment began(day 0).

Pituitary-Gonadal Dysfunction and FoodRestriction

Neuroimmunomodulation 2002–03;10:153–162 155

Nutritional StatusAnthropometry. Body weight and length were recorded serially

during the experimental period (every day and every 4 days, respec-tively, after a fasting period of 2–4 h). A Mettler PC 4000 scale withan accuracy of B 1 mg was used to measure body weight. For lengthmeasurements, animals were lightly anesthetized. Body length wasdetermined with a scaled ruler in millimeters and measured from thenose tip to the last hairs of the tail base.

Diet Intake. Food consumption was measured by using specialfeeders that allowed the recovery of spilled food. Food intake wasweighed every day with a Mettler scale (accuracy B 1 mg). It wasexpressed as g/100 g rat/day, and the percentage of the control valuewas calculated.

Body CompositionChemical carcass analysis of 10 animals/group sacrificed at 0, 4, 8

and 12 weeks was performed as described previously [24, 25]. Bodyfat content was determined by the Soxhlet extraction method [24].Body protein quantification was performed by the Kjeldahl method[25].

Hypothalamic LHRH ContentHypothalami were homogenized individually in 500 Ìl 2 M acet-

ic acid. Homogenates were boiled at 100 ° C for 5 min and centri-fuged for 10 min at 20,000 g. After centrifugation the supernatantswere separated and the pellets resuspended again in another 500 Ìl2 M acetic acid solution and centrifuged; the second supernatant wasadded to the first. The 1-ml pooled fractions were evaporated in acentri-vap evaporator and stored at –20 ° C until assayed for LHRH.

Hormone AssaysSerum LH and FSH were measured by RIA using kits provided

by the NIDKK National Pituitary Agency (Baltimore, Md., USA),standard LH LP-3 and FSH I-6. Testosterone was measured by RIAusing a kit of DSL-4000 (Active TM Testosterone) with a sensitivityof 0.08 ng/ml. Leptin was assessed in duplicate by enzyme-linkedimmunosorbent assay using a DSL-10-24100 Mouse Leptin ELISAKit (Diagnos Med). The sensitivity of this test was 0.04 ng/ml at aserum volume of 100 Ìl. LHRH was measured by RIA with a highlyspecific LHRH antiserum kindly provided by Ayala Barnea (Univer-sity of Texas Southwestern Medical Center, Dallas, Tex., USA). Thesensitivity of the assay was 0.2 pg per tube and the curve was linearup to 100 pg of LHRH.

Evaluation of Sperm Morphology and ConcentrationA portion of each cauda epididymal sperm suspension was fixed

and smeared onto microscopic slides. After airdrying, slides wereadvanced through Lillie-Mayer hematoxylin, sequentially dehy-drated with 50, 70, 80 and 95% ethanol and cleared with xylene.Slides were coated with a liquid coverslip before microscopic exami-nation.

Spermatozoa were examined by bright-field microscopy with anoil immersion lens (! 97). One hundred randomly selected sperma-tozoa from each sample were evaluated. Anomalies were groupedinto five major categories: (1) head anomalies, including microcepha-ly, macrocephaly, duplicates, amorphous, rotated (broken neck);(2) tail anomalies, including coiled, kinked, duplicate; (3) occurrenceof sperm precursors (i.e., spermatids, spermatocytes); (4) immaturespermatozoa (i. e., those spermatozoa to which the cytoplasmic drop-let remained attached), and (5) decapitated spermatozoa. Results

were expressed as percentage of spermatozoa displaying any of theabove anomalies [26].

For the evaluation of sperm concentration, rats were killed andthe caudal epididymides were dissected and placed in a drop of200 Ìl phosphate-buffered saline. Spermatozoa were obtained bymaking small cuts in the cauda. The dense mass of spermatozoa wasallowed to drain, then the tissue was removed and sperm concentra-tion was determined using a Neubauer chamber. The final concentra-tion was expressed as mean number of spermatozoa ! 106/ml BSEM for each animal.

Statistical AnalysisThe results were expressed as mean B SEM. Data were analyzed

by two-way analysis of variance (ANOVA) for the main effects andinteraction of degree of food restriction and time (weeks of experi-mental period). Differences between means were determined by theStudent-Neuman-Keuls multiple-comparison test. Differences wereconsidered significant if p ! 0.05 [27]. Analyses were performedusing a Graphpad Prism (version 3.0) statistical package (GraphpadSoftware, San Diego, Calif., USA).

ChemicalsLHRH for iodination and standards were purchased from Penin-

sula Laboratories Inc. (Belmont, Calif., USA). Iodine-125 for iodin-ation was purchased from DuPont New England Nuclear (NEN; Bos-ton, Mass., USA). NaCl, KCl, MgCl2, NaH2PO4, CaCl2, EDTA-Na,dextrose, NaHCO3, ascorbic acid and acetic acid were purchasedfrom Sigma (St. Louis, Mo., USA).

Results

Food restriction induced a highly significant (p !

0.001) decrease in growth velocity in ND rats. At week 4of food restriction, the reduction in body weight was 59.4and the reduction in length was 24.1%. There was a catch-up growth during refeeding, with ND rats reaching thesame values as C animals after 6 or 7 weeks (fig. 1a, b).

Changes in body fat content as a function of timebetween weeks 0 and 12 of the postweaning period areshown in figure 2a. ND body fat percentage had decreased40% (p ! 0.01) at the end of the restrictive period. Duringrefeeding, no significant differences between ND and Crats were seen at weeks 8 and 12 of the experimental peri-od. However, fat content increased significantly as ani-mals grew when compared with the moment of weaning(considered as week 0).

Food restriction did not modify the percentage of pro-tein content when compared with control animals. How-ever, there was a significant increase in the percentage ofprotein content between week 0 and weeks 4, 8 and 12(fig. 2b).

The hypothalamic content of LHRH increased signifi-cantly (p ! 0.001) in both C and ND rats after the first 4


Fig. 1. Changes with time after weaning(week 0) in body weight (a) and body length(b) in C and ND rats. Values are expressedas mean B SEM. p values from two-wayANOVA were: interaction: p ! 0.001; degreeof food restriction: p ! 0.001; time: p !

0.001. Letters express significant differencesbetween groups: a p ! 0.05; b p ! 0.01; c p !0.001; n = 10 animals/group.

0 1 2 3 4 5 6 7 8 9 10 11 120

a

b

100

200

300

400CND

CND

WeeksFood restriction

a

aa

c c c c cc

c

cc

cc

cc

c

b

b

Body

weig

ht (

g)

0 1 2 3 4 5 6 7 8 9 10 11 128

12

16

20

24

Food restriction Weeks

aa

a aa

b

b

c

cc

c

Body

length

(cm

)

weeks of the experimental period, without significant dif-ferences between groups. From this time until the end ofthe study, hypothalamic LHRH content appeared to havestabilized (fig. 3). Since hypothalamic LHRH content re-mained unchanged in both groups at week 8 of the study,it was not measured at week 12.

Serum FSH levels decreased 64.9% (p ! 0.001) in NDrats after 4 weeks of food restriction when compared withthe C group (fig. 4a). At week 8, ND rat serum FSH levelshad achieved C values.

There was a significant increase in serum LH duringthe experimental period in the C group. Serum LH levels

decreased significantly (p ! 0.001) after 4 weeks of foodrestriction. During refeeding, serum LH levels had alrea-dy increased at week 8, being slightly lower than in C (butnot statistically significant), and at week 12 the valueswere equal to the C group (fig. 4b).

Serum testosterone levels were also significantly de-creased after 4 weeks of food restriction, but reached thesame levels as C rats after refeeding (fig. 5a). There wasalso a significant increase in testosterone levels at week 4in C animals when compared with week 0, and at week 8compared with week 4. There was no difference between 8and 12 weeks.



Fig. 2. a Body fat content in C and ND ratsat different times after weaning (week 0).Values are expressed as mean B SEM. p val-ues from two-way ANOVA were: interac-tion: p ! 0.001; degree of food restriction:p ! 0.05; time: p ! 0.001. Different lettersabove the bars indicate significant differ-ences between groups: p ! 0.05; n = 8–10animals/group. b Body protein content in Cand ND rats at different times after weaning(week 0). Values are expressed as mean BSEM. p values from two-way ANOVA were:interaction: p = 0.2253; degree of food re-striction: p ! 0.4782; time: p ! 0.001. Differ-ent letters above the bars indicate significantdifferences between groups: p ! 0.05; n = 8–10 animals/group.

0 4 8 12

2

4

6

8

10

12C

ND


a/ba/b

a/c

a/c

b

c

cc

Bod

y fa

t (%

)

0 4 8 126

10

b

a

14

18

22

ND

C


a a

bb b b

b

b

Body

pro

tein

(%

)

Fig. 3. LHRH content from the medial basalhypothalamus (MBH) in C and ND rats atdifferent times after weaning (week 0). Val-ues are expressed as mean B SEM. p valuesfrom two-way ANOVA were: interaction:p = 0.7642; degree of food restriction: p =0.6336; time: p ! 0.001. Different lettersabove the bars indicate significant differ-ences between groups: p ! 0.05; n = 9–10animals/group.

0 4 80

2

4

6

8

ND

C

Food restrictionWeeks

LH

RH

conte

nt (�

g/M

BH

)

a a

b b

bb


Fig. 4. a Serum FSH in C and ND rats atdifferent times after weaning (week 0). Val-ues are expressed as mean B SEM. p valuesfrom two-way ANOVA were: interaction:p ! 0.001; degree of food restriction: p =0.0421; time: p ! 0.001. Different lettersabove the bars indicate significant differ-ences between groups: p ! 0.05; n = 9–10animals/group. b Serum LH in C and NDrats at different times after weaning (week0). Values are expressed as mean B SEM.p values from two-way ANOVA were: inter-action: p = 0.9881; degree of food restriction:p ! 0.01; time: p ! 0.001. Different lettersabove the bars indicate significant differ-ences between groups: p ! 0.05; n = 9–10animals/group.

0 4 8 120

a

2

4

6

8 ND

C


a a

a

a

a

a

a

b Seru

m F

SH

(ng/m

l)

0 4 8 120

0.1

0.2

0.3

C

ND

0.5

1.0

1.5

2.0

2.5

3.0


a a

a

b

c

cc

c

Seru

m L

H (pg/m

l)

b

Circulating leptin levels during the experimental pe-riod are shown in figure 5b. Food restriction resultedin a 48.4% decrease (p ! 0.05) in serum leptin in theND group. This difference disappeared after refeeding(fig. 5b). The pattern of leptin levels in C and ND animalswas similar to those of LH and testosterone serum levels.

Testicular and seminal vesicle absolute weights de-creased by food restriction. It was more evident in semi-nal vesicle weight (34.0 B 2.45 vs. 250.0 B 29.44 mg; p !0.001) than in testicular weight (1.08 B 0.11 vs. 2.03 B0.11 g; p ! 0.001) when compared with the freely fed ani-mals. After refeeding, at week 12 of the experimental peri-od, no differences were observed in both testicular (2.97



Fig. 5. a Serum testosterone in C and NDrats at different times after weaning (week0). Values are expressed as mean B SEM.p values from two-way ANOVA were: inter-action: p = 0.9881; degree of food restriction:p ! 0.05; time: p ! 0.001. Different lettersabove the bars indicate significant differ-ences between groups: p ! 0.05; n = 9–10animals/group. b Serum leptin in C and NDrats at different times after weaning (week0). Values are expressed as mean B SEM.p values from two-way ANOVA were: inter-action: p = 0.2948; degree of food restriction:p ! 0.05; time: p ! 0.001. Different lettersabove the bars indicate significant differ-ences between groups: p ! 0.05; n = 9–10animals/group.

0 4 8 120

a

b

0.1

0.2

0.3

0.4

0.5

C

ND

1.0

2.0

3.0

4.0


a aSeru

m test

ost

ero

ne (ng/m

l)

b

a

cc c

c

0 4 8 120

0.4

0.8

1.2

1.6

2.0

2.4

2.8C

ND


a a

b

c

b/db/d

dd

Ser

um

leptin (ng/m

l)

B 0.09 vs. 2.78 B 0.07 g) and seminal vesicle (1,134 B100 vs. 1,232 B 40 mg) weights between the C and NDgroups.

Morphological characteristics of spermatozoa andsperm concentrations between C and ND rats after foodrestriction and at the end of the experimental period aredescribed in table 1A, B, respectively. After food restric-tion, the percentage of anomalies of spermatozoa withinthe caudal epididymis increased in about 64% of ND vs.

C rats. Significantly greater (p ! 0.001) numbers of sper-matozoa with head anomalies were observed, without evi-dent differences in the tail. There were no differences insperm morphology between C and ND groups after nutri-tional rehabilitation (table 1A). Since no significant dif-ferences in sperm morphology between groups were ob-served at week 8 of the experimental period, this evalua-tion was not performed at week 12.


Table 1. Morphology and concentration ofspermatozoa of cauda epididymis in C andND rats at different times

A Morphology

Anomalies Week 4

C ND

Week 8

C ND

Head 22.04B2.09 36.92B2.34* 2.82B0.74 4.30B2.10Tail 0.78B0.53 0.46B0.28 0.21B0.15 0.50B0.50Total 22.84B1.96 37.42B2.17* 5.30B1.90 4.75B1.98Total number of

spermatozoa counted 533 586 604 452

Values are expressed as mean percentages B SEM. p values from two-way ANOVA were:interaction p ! 0.001; degree of food restriction p ! 0.003; time p ! 0.001. Significantlydifferent from C at the same time point: * p ! 0.001; n = 5 animals/group.

B Sperm concentration (!106 spermatozoa/ml)

Group Week 4 Week 8 Week 12

C 3.56B0.41 90.0B11.8 176.0B0.75ND 3.84B0.71 63.6B6.3* 191.0B2.50

Values are expressed as mean percentages B SEM. p values from two-way ANOVA were:interaction p ! 0.01; degree of food restriction p = 0.4234; time: p ! 0.001. Significantlydifferent from C at the same time point: * p ! 0.05; n = 5 animals/group.

At week 4 the sperm concentration, being very low incomparison with the levels found in adult animals, wasnot significantly different between ND and C (table 1B).At week 8, however, we found significantly decreased (p !0.05) sperm concentrations in ND males compared withC males. ND sperm concentration achieved C values atweek 12 (table 1B).

Discussion

The nutritional stress model that we used in this studywas developed in weanling male rats in our laboratory[28] and resembles a suboptimal nutrition observed inchildren who consume inappropriate diets with insuffi-cient total energy to sustain normal growth and weightgain.

Growth failure and delayed sexual development arewell-recognized complications of ND patients [18, 29]. Inthe present study we found a dramatic decrease in growthrate in ND animals, which could be due to the combinedeffect of mild suboptimal intake and the duration of therestriction. It was more evident in body weight, consid-ered as an expression of overall body growth, than in body

length, which gives information of longitudinal bonegrowth. The important reduction in weight gain observedin the ND group could be attributed to a decrease in bodyfat mass, since body protein percentage was not modified,even though the absolute content diminished. Despite thenegative effect of the nutritional stress on body weightand length in these postweaning male rats, a completecatch-up and a normalization of body fat content wereobserved with refeeding, allowing ND rats to reach theirnormal adult body size [30]. These results are in agree-ment with those of other authors, who also reported catch-up growth in another model of nutritional stress [31].

In the present study we found an increase in anomaliesof sperm head morphology when compared with the per-centage of anomalies observed in normal animals of thesame age, even though the sperm concentrations weresimilar. At approximately 50 days of age, male rats are inthe peripubertal phase, where the process of spermatogen-esis and sperm production is being completed [32, 33].Thus, at this phase, spermatozoa levels are not yet equalto those in the adult phase. Therefore C males presentedincreased percentages of morphological sperm abnormali-ties that will be reduced after reaching sexual maturity,while the ND males presented significantly increased per-



centages of abnormal spermatozoa compared with the Cmales. Despite these results, we found similar, reducedpercentages of abnormal spermatozoa at week 8 in C andND males, which can be indicative of a sperm morpholo-gy recovery. At week 4 the sperm concentrations, despitebeing very low in comparison with the levels found inadults, were similar in ND and C males. However, atweek 8 we found significantly decreased sperm concentra-tions in the ND males compared with C males, indicatinga slow catch-up since there were no differences betweenND and C groups at week 12.

Ronnekliev et al. [19] reported in 1978 that basalserum gonadotropin levels were within normal limits intheir model of restrictive diet in female rats. In our studywe found a decrease in serum FSH (64.9%), LH (64.7%)and testosterone (70%) levels that could be due to the gen-der difference and model of undernutrition.

We found no differences in hypothalamic LHRH con-tent between C and ND rats after 4 weeks of nutritionalstress. This does not imply that the pulsatile release ofLHRH was not inhibited. The decreased serum FSH, LHand testosterone levels after 4 weeks of food restrictioncould be due to a decrease in LHRH pulses, as suggestedby Goto et al. [34] and supported by the finding that exog-enous application of LHRH to food-restricted adult maleand female rats could prevent the inhibitory effect ofundernutrition on reproduction. Others studies showedinconsistent results regarding the effect of suboptimalfood intake on LHRH gene expression and/or release [12,35]. The conflicting results related to LHRH levels, geneexpression and secretion might be due to differencesbetween species, age of the experimental animal model,pattern, degree and extent of undernutrition, and in vitroor in vivo LHRH release measurements, among other rea-sons. However, there are few reports that demonstrate theinhibitory action of food restriction on the in vivo releaseof LHRH from the hypothalamic LHRH neuronal net-work [36–39].

The increase in LHRH content 4 weeks after weaningin C and ND animals is in agreement with other authorswho reported that hypothalamic LHRH content, pituitarynumber of LHRH receptors and pituitary content of LHbegin to show a clear increase early in life in the male rat,probably determined by a variety of signals routedthrough the brain to the neuroendocrine mechanisms con-trolling LHRH release [40].

The influence of metabolic cues on the timing ofpuberty has been suspected for many years. The earlywork of Kennedy [41] indicated that vaginal opening wasmore related to body weight than to chronological age. It

was postulated that the state of somatic growth is assessedby the hypothalamus through some metabolic cues thatact as signals to initiate puberty once a critical bodyweight is achieved [42]. This hypothesis was subsequentlyextended from ‘critical weight’ to ‘critical ratio of lean tofat tissue’ to ‘critical level of metabolic signal’ necessaryfor the occurrence of puberty [39, 43]. Leptin wouldappear to play a role in relaying metabolic information tothe reproductive axis for the modulation of its activity ateither the hypothalamus, the pituitary or the gonads sincethere is evidence of a functional ob-receptor in these tis-sues [44]. In the present study it is evident that weightgain was negatively affected during food restriction com-pared with freely fed animals, with a considerable reduc-tion in fat mass/body weight ratio and circulating leptinlevels. Since plasma levels of leptin are linked both tometabolism and reproduction, it seems that the normalonset of puberty may reflect, in part, the activation of theneuroendocrine reproductive axis by this adipocyte-de-rived hormone, possibly signaling the state of energystores at different levels of the HPG axis. In our model ofnutritional stress there was a decrease of about 48% inserum leptin levels. Recent studies suggest that leptin isone of several permissive factors necessary to initiatepuberty [45, 46]. Yu et al. [47] have shown that leptinstimulates LHRH release from the hypothalami in vitro,as well as the release of LH from the anterior pituitary invitro and in vivo. Since there was a decrease of about 50%in serum leptin levels, we hypothesize that this decrease inthe adipocyte-derived hormone could be responsible, atleast in part, for the inhibition of the reproductive axis inthis nutritional stress model in postweaning male rats.Furthermore, refeeding normalized the leptin serum lev-els already at week 8, and all other parameters betweenweeks 8 and 12.

Acknowledgements

This work was supported by research grants from the Universityof Buenos Aires (UBACyT TO20). The authors thank Dr. PatriciaLouzan (Instituto de Neurobiologia-CONICET) for helping withRIA of LH.


References

1 Chrousos GP: The stress response and immunefunction: Clinical implications. The 1999 No-vera H. Spectator Lecture. Ann NY Acad Sci2000;917:38–67.

2 Wade GN, Schneider JE, Li HY: Control of fer-tility by metabolic cues. Am J Physiol 1996;270:E1–E19.

3 Cagampang FRA, Maeda K, Yokoyama A, ÔtaK: Effect of food deprivation on the pulsatileLH release in the cycling and ovariectomizedfemale rat. Horm Metab Res 1990;22:269–272.

4 Thomas GB, Mercer JE, Karalis T, Rao A,Cummins JT, Clarke IJ: Effect of restrictedfeeding on the concentrations of growth hor-mone (GH), gonadotropins, and prolactin(PRL) in plasma, and on the amounts of mes-senger ribonucleic acid for GH, gonadotropinsubunits, and PRL in the pituitary glands ofadult ovariectomized ewes. Endocrinology1990;126:1361–1367.

5 Cameron JL, Nosbisch C: Suppression of pul-satile luteinizing hormone and testosterone se-cretion during short term food restriction in theadult male rhesus monkey (Macaca mulatta).Endocrinology 1991;128:1532–1540.

6 Cameron JL, Weltzin TE, McConaha C, Hem-reich DL, Kaye WH: Slowing of pulsatile lu-teinizing hormone secretion in men after forty-eight hours of fasting. J Clin Endocrinol Metab1991;73:35–41.

7 Campbell GA, Kurcz S, Marshall S, Meites J:Effects of starvation in rats on serum levels offollicle stimulating hormone, luteinizing hor-mone, thyrotropin, growth hormone, and pro-lactin; response to LH-releasing hormone andthyrotropin-releasing hormone. Endocrinology1977;100:580–587.

8 Bergendahl M, Wiemann JN, Clifton DK,Huhtaniemi I, Steiner RA: Short-term starva-tion decreases POMC mRNA but does notalter GnRH mRNA in the brain of adut malerats. Neuroendocrinology 1992;56:913–920.

9 Cameron JL, Helmreich DL, Schreihofer DA:Modulation of reproductive hormone secretionby nutritional intake: Stress signals versus met-abolic signals. Hum Reprod 1993, 8:162–167.

10 Cameron JL: Search for the signal that conveysmetabolic status to the reproductive axis. CurrOpin Endocrinol Diabetes 1997;4:158–163.

11 Leonhardt S, Shahab M, Luft H, Wuttke W,Jarry H: Reduction of luteinizing hormone se-cretion induced by long-term feed restriction inmale rats is associated with increased expres-sion of GABA-synthesizing enzymes withoutalterations of GnRH gene expression. J Neu-roendocrinol 1999;11:613–619.

12 Nappi RE, Rivest S: Effect of immune andmetabolic challenges on the luteinizing hor-mone-releasing hormone neuronal system incycling female rats: An evaluation at the tran-scriptional level. Endocrinology 1997;138:1374–1384.

13 Foster DL, Olster DH: Effect of restricted nu-trition on puberty in the lamb: Patterns of tonicluteinizing hormone (LH) secretion and com-petency of the LH surge system. Endocrinology1985;116:375–381.

14 Gill CJ, Rissman EF: Female sexual behavior isinhibited by short- and long-term food restric-tion. Physiol Behav 1997;61:387–394.

15 Pugliese MT, Weyman-Daum M, Moses N,Lifshitz F: Parental health beliefs as a cause ofnonorganic failure to thrive. Pediatrics 1987;80:175–182.

16 Lifshitz F, Moses N, Cervantes C, Ginsberg L:Nutritional dwarfing in adolescents. Semin Ad-olesc Med 1987;3:255–266.

17 Lifshitz F, Moses N: Nutritional dwarfing:Growth, dieting and fear of obesity. J Am CollNutr 1988;7:367–376.

18 Lifshitz F, Moses N: Growth failure. A compli-cation of dietary treatment of hypercholesterol-emia. Am J Dis Child 1989;143:537–542.

19 Ronnekleiv OK, Ojeda SR, McCann SM: Un-dernutrition, puberty and the development ofestrogen positive feedback in the female rat.Biol Reprod 1978;19:414–424.

20 Tarim O, Chasalow FI, Murphy J, Rising R,Carrillo A, Lifshitz F: Evaluation of differentialeffects of carbohydrate and fat intake on weightgain, serum IGF-1 and erythrocyte Na+K+

ATPase activity in suboptimal nutrition inrats. J Am Coll Nutr 1997;16:159–165.

21 Carrillo A, Rising R, Tverskaya R, Lifshitz F:Effects of exogenous recombinant humangrowth hormone on an animal model of subop-timal nutrition. J Am Coll Nutr 1998;17:276–281.

22 Kaplan RM, Toshima MT: Does a reduced fatdiet cause retardation in child growth? PrevMed 1992;21:33–52.

23 Akeson PK, Axelsson IEM, Raiha NCR, WarmA, Minoli I, Moro G: Fat intake and metabo-lism in Swedish and Italian infants. Acta Pae-diatr 2000;89:28–33.

24 Method 963.15. Official Methods of Analysisof the Association of Official Analytical Chem-ists, ed 15. Arlington, Association of OfficialAnalytical Chemists, 1990.

25 Method 920.105. Official Methods of Analysisof the Association of Official Analytical Chem-ists, ed 15. Arlington, Association of OfficialAnalytical Chemists, 1990.

26 Zaneveld LJD, Polakoski K: Collection andphysical examination of the ejaculate; in HafezESE (ed): Techniques of Human Andrology.Amsterdam, Elsevier, 1977, pp 147–172.

27 Sokal R, Rohlf J: Biometry: The Principles andPractice of Statistics in Biological Research, ed3. New York, Freeman, 1995.

28 Friedman SM, Rodriguez PN, Olivera MI,Bozzini C, Norese F, Gamba CA, Boyer PM:Enanismo por desnutricion: Cronodinamia delos procesos metabolicos en ratas. Medicina(Buenos Aires) 1998;58:282–286.

29 Pugliese M, Lifshitz F, Grad G, Fort P, Gins-berg L: Fear of obesity: A cause of short statureand delayed puberty. N Engl J Med 1983;309:513–518.

30 Iossa S, Lionetti L, Mollica MP, Barletta A,Liverini G: Energy intake and utilization varyduring devepment in rats. J Nutr 1999;129:1593–1596.

31 Dulloo AG, Girardier L: Adaptive role of ener-gy expenditure in modulating body fat and pro-

tein deposition during catch-up growth afterearly undernutrition. Am J Clin Nutr 1993;58:614–621.

32 Malkov M, Fisher Y, Don J: Developmentalschedule of the postnatal rat testis determined byflow cytometry. Biol Reprod 1998;59:84–92.

33 Yang ZW, Wreford N, de Kretser DM: A quan-titative study of spermatogenesis in the devel-oping rat testis. Biol Reprod 1990;43:629–635.

34 Goto K, Kotsuji F, Tominaga T: Divergenteffects of gonadotrophin-releasing hormone(GnRH) analogue and authentic GnRH on theanterior pituitary gland of rats with restrictedfeeding. J Endocrinol 1995;145:501–511.

35 Gruenewald DA, Matsumoto AM: Reduced go-nadotrophin-releasing hormone gene expres-sion with fasting in the male rat brain. Endo-crinology 1993;132:480–482.

36 Prasad BM, Conover CD, Sarkar DK, Rabii J,Advis JP: Short-term feed restriction decreasesin vivo release of beta-endorphin (but not ofLHRH and NPY) from median eminence andincreases NPY release from paraventricular nu-cleus in ewes. Soc Neurosci 1991;17:1362.

37 Prasad B, Gore A, Roberts J, Sarkar D, Rabii J,Advis JP: Feed restriction decreases arcuatemRNA and in vivo median eminence release ofß-endorphin in ewe lambs. Soc Neurosci 1992;18:18–111.

38 Advis JP, Prasad B, Conover C, Sarkar D, RabiiJ: Delayed puberty induced by feed restrictionin ewe lambs is associated with decreased invivo release of LHRH and ß-endorphin (but notNPY) from median eminence (ME). Biol Re-prod 1992;46:176.

39 Foster DL, Nagatani S: Physiological perspec-tives on leptin as a regulator of reproduction:Role in timing puberty. Biol Reprod 1999;60:205–215.

40 Ojeda SR, Urbanski HF: Puberty in the rat; inKnobil E, Neill J (ed): The Physiology of Repro-duction. New York, Raven, 1988, pp 1699–1737.

41 Kennedy GC: The development with age ofhypothalamic restraint upon the appetite of therat. J Endocrinol 1957;16:9–17.

42 Kennedy GC, Mitra J: Body weight and foodintake as initiating factors for puberty in the rat.J Physiol 1963;166:408–418.

43 Matthew J, Cunningham, Clifton DK, SteinerRA: Leptin’s action on the reproductive axis:Perspectives and mechanisms. Biol Reprod1999;60:216–222.

44 Yarnell DO, Knight DS, Hamilton K, Tulp O,Tso P: Localization of leptin receptor immuno-reactivity in the lean and obese Zucker ratbrain. Brain Res 1998;785:80–90.

45 Cheung CC, Thornton JE, Nurani SD, CliftonDK, Steiner RA: A reassessment of leptin’s rolein triggering the onset of puberty in the rat andmouse. Neuroendocrinology 2001;74:12–21.

46 Urbanski HF: Leptin and puberty. Trends En-docrinol Metab 2001;12:428–429.

47 Yu WH, Kimura M, Walczewska A, Karanth S,McCann SM: Role of leptin in hypothalamic-pituitary function. Proc Natl Acad Sci USA1997;94:1023–1028.

effect of nutritional stress on the hypothalamo-pituitary-gonadal axis in the growing male rat

Documents