JOURNAL OF EXPERIMENTAL ZOOLOGY 283:573–579 (1999)

© 1999 WILEY-LISS, INC.

Agouti Locus May Influence ReproductionUnder Food Deprivation in the Water Vole(Arvicola terrestris)

N.M. BAZHAN,* T.V. YAKOVLEVA, AND E.N. MAKAROVAInstitute of Cytology and Genetics, Novosibirsk, Russia, 630090

ABSTRACT The effect of 16-hr food deprivation on day 3 and again on day 5 of pregnancy onthe fecundity of female water voles homozygous (ae/ae) or heterozygous (A/ae) for, an allele at theAgouti (A) locus, non agouti extreme (ae) was studied. 63 A/ae females (mated to ae/ae males)produced 115 food-deprived and 115 control pregnancies, and 52 ae/ae females (mated to A/ae

males) produced 55 food-deprived and 57 control pregnancies. Regardless of the experimental group,pregnant ae/ae females weighed less than A/ae females. The effect of food deprivation on fecun-dity depended on the Agouti-locus genotype of the female. In food-deprived A/ae females, fecun-dity was diminished due to fewer successful pregnancies (P < 0.001) and lower survival of theyoung (P < 0.05). In food-deprived ae/ae females, reproductive performance was not changed; asomewhat reduced rate of successful pregnancies was compensated for by significantly increased(P < 0.002) postnatal survival of the young. In progeny weaned from food-deprived mothers, thefrequency of A/ae females was diminished. Resistance of ae/ae females to the negative effect ofnutritional stress, and predominance of ae/ae young in progeny produced by food-deprived moth-ers, may favour the maintenance of polymorphism for the Agouti-locus in natural populations ofthe water vole. J. Exp. Zool. 283:573–579, 1999. © 1999 Wiley-Liss, Inc.

Natural water vole (Arvicola terrestris) popula-tions are polymorphic for coat color. There is aspectrum in which three main coat colors can bedistinguished: brown, black-brown, and black(Nikolaeva, ’78). Laboratory studies have revealedthat brown individuals are homozygous for thedominant wild-type allele Agouti (A); black, mela-nic individuals are homozygous for extreme non-agouti (ae); and black-brown individuals are A/ae

heterozygotes (Nasledova et al., ’80; Prasolova etal., ’91). In western Siberia, populations of thewater vole exhibit 7–8 year population cycles(Moshkin et al., ’90). A sharp decrease in popula-tion size usually is coincident with drought, de-creased availability of green vegetation, andincreased frequency of black voles from 4 to 10%(Nikolaeva, ’78; Moshkin et al., ’90). We suspectthat malnutrition and short-term food deprivationoccur in nature in the beginning of the breedingseason as a consequence of food stores beingexausted, in the years of high density. Studies byGerlinskaya et al. (’94) indicate that malnutritioncan inhibit the reproduction of the water vole.They found that in the course of the populationcycle there is a positive correlation between bloodlevels of fatty free acids (index of undernutrition)and the number of embryos lost. Under labora-

tory conditions, food deprivation in late pregnancyreduces fecundity in the water vole (Bazhan etal., ’96a). Accordingly, we hypothesize that the in-creased frequency of black (ae/ae) voles that oc-curs in years of low population density resultsfrom the fact that ae/ae females are better ablethan A/ae females to reproduce successfully whenfood is scarce.

The Agouti locus is one of the major coat colorloci in mammals. The Agouti gene of mice hasbeen cloned and shown to encode a 131-amino acidprotein-signaling factor that dictates melanocytepigment production within individual hair follicles(Bultman et al., ’92; Miller et al., ’93). The mouseagouti protein also has been shown to be an an-tagonist of α-MSH on melanocortin-1 receptors(MC1R) in skin and ACTH and α-MSH on MC4Rin brain tissue (Lu et al., ’94). It also regulatesthe concentration of intracellular calcium (Zemel

Grant sponsor: Program of Russian Ministry of Education “Fun-damental Researches in Natural Sciences”; Grant number: 3H-200-98; Grant sponsor: Russian Foundation for Fundamental Researches;Grant number: 98-04-49502.

*Correspondence to: Nadezhda M. Bazhan, Institute of Cytologyand Genetics, Lavrentjev Av., 10, Novosibirsk, 630090, Russia. E-mail:bazhan@bionet.nsc.ru

Received 9 April 1998; Accepted 11 August 1998.

574 N.M. BAZHAN ET AL.

et al., ’95). These findings suggest possible rolesfor the agouti protein in a variety of cellular andphysiological functions. In mice, the recessive mu-tation ae results in a lack of translation initiation,and hence no agouti protein (Hustad et al., ’95).Although little is known about the pleiotropic ef-fects of the ae allele in mice, the absence of thefunctional agouti protein does not appear to affecteither viability or fertility (reviewed in Manne etal., ’95). In other species, the ae allele was shownto effect neuronal and hormonal functions. A pleio-tropic effect of the ae allele on brain catecholamines(Hayssen et al., ’94) and ability to reproduce un-der stressful conditions (Hayssen, ’98) have beendemonstrated in deer mice (Peromyscus mani-culatus). Our earlier studies have revealed a pleio-tropic effect of the ae allele on secretion of adrenalcorticosterone and progesterone in immature fe-male water voles (Bazhan and Ivanova, ’89) andadrenal response to osmotic stress in adults(Bahzan, ’91). The object of this study was to testour hypothesis that the increased frequency ofae/ae animals that occurs during periods of fooddeprivation stems from their enhanced ability toreproduce successfully when nutritionally deprived.Accordingly, we have compared the reproductiveperformances of A/ae and ae/ae females when ex-perimentally deprived of food.

MATERIALS AND METHODSAnimals

Wild water voles were introduced into the labo-ratory in 1974 from the subtaiga region of WestSiberia (Ubinsk). A colony has since been success-fully bred at the vivarium of the Institute of Cy-tology and Genetics (Siberian Division, RussianAcademy of Science), at an ambient temperatureof 21°C under natural light conditions. Judgingfrom the frequency of litters and their size, thereappears to have been no diminution in reproduc-tive ability in captivity. Details on the laboratorybreeding of water voles have been described else-where (Bazhan et al., ’96a). The breeding seasonof the water vole under natural and laboratory con-ditions lasts from April to August (Panteleev, ’68).Ovulation is induced and implantation occurs onthe 7th day of pregnancy (Nasledova et al., ’87).The gestation period is 21 days, animals areweaned at about three weeks of age (Blake, ’82).

BreedingFemales (150–170 g) and males (170–190 g),

aged 8–9 months, were housed individually before

the beginning of the breeding season. Femaleswere paired with males, and vaginal smears werechecked daily for spermatozoa. The day that sper-matozoa were detected was designated day 0 ofpregnancy. After mating, males were removedfrom females. Cages with pregnant females werechecked daily at time of expected parturition. Theday young were found was designated day 1 ofage. Young were separated from mothers on day21 postpartum, and females were paired for thesecond time with another male. Under those ex-perimental conditions each female can produce3–4 litters during the breeding season. Litter sizeis not related to the number of pregnancies.

CrossesPolymorphism for Agouti locus alleles in our

laboratory population has been maintained by anoutbreeding regime. Inbreeding coefficient does notexceed 12.5%. The fecundity of heterozygous andhomozygous females has been shown to be thesame in reciprocal crosses A/Ae × ae/ae and ae/ae

× A/ae (Bazhan et al., ’96b). We worked only withthese reciprocal crosses in this study, so litters weregenetically similar (only the mothers differed).

Food deprivationFemales were denied access to food for 16 hours

(from 1700 hr to 0900 hr) on day 3 and again onday 5 of pregnancy. Paper shavings were substi-tuted for hay, and fresh water was freely avail-able. Females were not food deprived for twopregnancies in succession. During control preg-nancies females received the standard laboratorydiet and food and water ad libitum (Bazhan etal., ’96a). Sixty-three A/ae females produced 115pregnancies that were food deprived (experimen-tal group) and 115 pregnancies that were treatednormally (control group). Similarly, 52 ae/ae fe-males produced 55 food-deprived and 57 controlpregnancies. Both A/ae and ae/ae females weremated 3–4 times in each breeding season. Therewere fewer pregnancies in ae/ae females (both con-trol and food-deprived) because it was necessaryto use some of these females for test crosses inother experiments.

Reproductive measuresFor each female we recorded the total number

of pregnancies, the number of successful pregnan-cies (i.e., that resulted in birth), the number ofyoung (litter size) at parturition, at 8 days, andat weaning (day 21). Since mortality is signifi-cantly lower in litters that are not disturbed post-

FOOD DEPRIVATION AND AGOUTI LOCUS 575

partum (Blake, ’82) animals were not handled(sexed) until they were 8 days old, the youngestage at which their genotype can be ascertained.In each litter the number of young homozygousand heterozygous females and males also was re-corded at weaning. The litters in which all younglived to weaning were designated successful, lit-ters where some did not live, partially successful,and litters where none lived to weaning, unsuc-cessful. The total index of observed fecundity, ex-pressed as the mean number of weaned young perpregnancy, was calculated for each pregnancy. Inthe case of pregnancy failure the total index offecundity was set equal to zero. Daily weightswere measured during 56 control and 17 food-de-prived pregnancies in A/ae females and during 34control and 12 food-deprived pregnancies in ae/ae

females. The day that female ceased gainingweight was designated the day of pregnancy fail-ure. The weights of females in which pregnancyfailed were excluded from the consideration.

Statistical analysisWeight data were analyzed by two-way ANOVA.

The ANOVA includes two factors: female agoutigenotype (A/ae and ae/ae) and group (control andfood-deprived). To compare the rates of successfulpregnancies, weaned litters, weaned young, malesand melanics (ae/ae genotype) in progeny, the χ2-test was applied with Yates correction for conti-nuity. Litter size at birth and number of survivingyoung at weaning were compared with a t-test.Nonparametric variances such as relative frequen-cies of young A/ae and ae/ae females and malesin successful litters and mean number of youngweaned per one pregnancy were analysed with useof Mann-Whitney U-test. Statistical significancewas defined as P < 0.05. Data are presented asmean ± 1 S.E.

RESULTSFood deprivation resulted in failed pregnancies.

Among control pregnancies in which female

weights were measured daily 30% (27 of 90) failed,whereas in food-deprived groups, 48% (14 of 29)failed (P < 0.05). The risk of pregnancy failureincreased in the second week (Table 1). The rateof pregnancy failure in this period was signifi-cantly greater in food-deprived than in control fe-males. In week two, in food-deprived groups, allpregnancies failed from 8 to 12 days postcoitumand most (62%) failed on days 8 and 9 (Table 1),i.e., just after implantation.

Regardless of experimental group, ae/ae femalesweighed less than A/ae females throughout ges-tation (ANOVA, P < 0.001). Food deprivation for16 hours on days 3 and 5 of pregnancy inducedweight loss on days 4 and 6. In aa/aa females,loss (6.9%) was less than in A/ae females (8.2%,ANOVA, P < 0.001). After the second week of preg-nancy the weight was regained. At parturitionweights in food-deprived and control females werenot differed (in A/ae: control 214 ± 6 g, food dep-rivation 215 ± 10 g; in ae/ae: control 201 ± 5 g,food deprivation 187 ± 10 g).

There were no significant differences in littersize by experimental conditions or genotype (over-all mean in control groups 4.5 ± 0.2, n = 121; infood-deprived groups 4.6 ± 0.2, n = 76).

A/ae and ae/ae females differed in the index offecundity—the mean number of young weaned perpregnancy—when they were deprived of food(Table 2). In the control, there was no difference,but after food deprivation, the index of fecundityfor A/ae females dropped to 0.55 times that of thecontrols (P < 0.001). By contrast, food deprivationin early pregnancy did not alter the index of fe-cundity ae/ae females. There were somewhat fewersuccessful pregnancies in this group, but survivalof young actually increased after food deprivation(P < 0.002; Table 2). Breeding under food depri-vation, ae/ae females had some advantages overA/ae females; the rates of litters weaned and ofyoung surviving were much greater (P < 0.001,Table 2), and as a result the index of fecunditytended to be higher (P < 0.1, Table 2).

TABLE 1. Times of pregnancy failures due to food deprivation (for 16 hr on days 3 and 5), as shown by failures in each weekof pregnancy and by distribution of failures in second week of pregnancy (when failures were greatest)

Distribution of failures throughout pregnancy

Total no. Week of pregnancy Days in second weekGroup of failures 1 2 3 8–9 10–12 13–14

Control 27 4 (15%) 18 (67%) 5 (18%) 9 (50%) 5 (28%) 4 (22%)Food-deprived 14 0 13 (93%) 1 (7%) 8 (62%) 5 (38%) 0P1 n.s. <0.003 n.s. n.s. n.s. n.s.1Probability levels are given for differences between control and food-deprived females.

576 N.M. BAZHAN ET AL.

Some litters were entirely lost before weaning,when in other litters a few young were lost. Nev-ertheless, there were no significant differences inthe losses of partial litters in any categories ex-amined (Table 3). By contrast, the losses of wholelitters varied with time (most of them occurred inthe first 8 days postpartum), experimental condi-tions and genotype. Food deprivation caused morelosses in A/ae females—more than in controls (P< 0.05) and more than in ae/ae females (P < 0.001;Table 3). In food-deprived A/ae mothers, 43% oflost litters (6/14) consisted of only one infant;therefore, although fewer litters survived, theirmean size at weaning was significantly greaterthan the mean size of litters at birth (5.3 ± 0.3 g,vs. 4.5 ± 0.3 g, P < 0.05).

Food deprivation also altered the frequency ofgender and genotype of young weaned. There wasa tendency for more males (P < 0.1) and moreae/ae offspring (P < 0.06) to be weaned from A/ae

mothers following food deprivation (Table 4). Therealso were more (55%) ae/ae offspring weaned fromboth food-deprived ae/ae and A/ae mothers (P <

0.05). Since we could not distinguish the agoutigenotype before 8 days of age, any differentialelimination of offspring between birth and 8 dayswould contribute to, or might totally account for,the observed frequency differences. Therefore, inorder to estimate the effect of prenatal elimina-tion on litter composition, we compared only suc-cessful litters in which all young survivedweaning. In the successful litters born to food-deprived mothers, there were more males of bothgenotypes (28% A/ae and 29% ae/ae) and a de-crease (to 19%) in A/ae females.

DISCUSSIONIn A/ae and ae/ae food-deprived females, gesta-

tion was an all-or-none phenomenon, in that ei-ther complete litters developed or all implantswere resorbed. Most pregnancy failures (93%) oc-curred on days 8–12 postcoitum, i.e., just afterimplantation (Nasledova et al., ’87). In rats (Berg,’65) and mice (Archunan, et al., ’94), fasting andfood restriction in early pregnancy also caused thedeath of whole litters at the time of implantation.

TABLE 2. Effect of food deprivation during pregnancy (16 hr on days 3 and 5) on reproduction in water voles of twogenotypes, A/ae (n = 65) and ae/ae (n = 52)1

Reproductive Survival of young Weaning rateParental success (litters (young (number of younggenotype Experimental (litters/ weaned/litters weaned/young per pregnancy(female × male) group pregnancy) born) born) mean ± 1 SE)

A/ae × ae/ae Control 85/115 (74%) 71/85 (84%) 311/371 (84%) 2.7 ± 0.2Food-deprived 49/115 (43%) 35/49 (71%) 170/220 (77%) 1.5 ± 0.2P <0.001 <0.1 <0.05 <0.001

ae/ae × A/ae Control 36/57 (63%) 31/36 (86%) 143/179 (80%) 2.5 ± 0.4Food-deprived 27/55 (49%) 26/27 (96%) 118/127 (93%) 2.2 ± 0.3P n.s. n.s. <0.002 n.s.P (food-deprived n.s. <0.001 <0.001 <0.1

A/ae vs. ae/ae)1Probability levels for differences between control and experimental females shown in body of table; probabilities for differences between twofemale genotypes shown in last row of table.

TABLE 3. Ages at which neonates died in litters in which some died (“partially successful”) and in litters in which all died(“unsuccessful”), following food deprivation of mothers during pregnancy (for 16 hr on days 3 and 5)

Parental No. ofNo. of litters in which young died

genotype Experimental litters Partially successful litters Unsuccessful litters(female × male) group born Days 1–8 Days 9–21 Days 1–8 Days 9–21

A/ae × ae/ae Control 85 9 (11%) 1 (1%) 10 (12%) 4 (5%)Food-deprived 49 5 (10%) 1 (2%) 14 (29%) 0P n.s. n.s. <0.02 n.s.

ae/ae × A/ae Control 36 3 (8%) 2 (6%) 5 (14%) 0Food-deprived 27 1 (4%) 2 (7%) 1 (4%) 0P n.s. n.s. n.s. n.s.P (food-deprived n.s. n.s. <0.001 n.s.

A/ae vs. ae/ae)

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TABLE 4. Effect of 16 hr maternal food deprivation during pregnancy (days 3 and 5) on relative frequencies of male and female young ofdifferent agouti genotypes

Young weaned in all litters Young weaned in completely successful litters

Parental Sex ratio Agouti ratio Relative frequenciesgenotype Experimental (males/total) (ae/ae out No. of Male Male Female Female(female × male) group (%) of total) (%) litters A/ae ae/ae A/ae ae/ae

A/ae × ae/ae Control 147/311 (47)a 142/311 (46) 61 0.26 ± 0.03 0.25 ± 0.03 0.27 ± 0.03 0.23 ± 0.03Food-deprived 92/170 (54) 93/170 (55) 29 0.28 ± 0.04 0.28 ± 0.03 0.20 ± 0.03 0.25 ± 0.04P <0.1 <0.06 n.s. n.s. <0.07 n.s.

ae/ae × A/ae Control 80/143 (56)a 74/143 (52) 26 0.26 ± 0.05 0.29 ± 0.05 0.22 ± 0.05 0.23 ± 0.04Food-deprived 67/118 (57) 65/118 (55) 23 0.29 ± 0.06 0.31 ± 0.06 0.17 ± 0.04 0.23 ± 0.04P n.s. n.s. n.s. n.s. n.s. n.s.

Total Control 227/454 (50) 216/454 (48) 87 0.26 ± 0.03 0.26 ± 0.03 0.25 ± 0.02 0.23 ± 0.02Food-deprived 159/288 (55) 158/288 (55) 52 0.28 ± 0.03b 0.29 ± 0.03c 0.19 ± 0.02bc 0.24 ± 0.03P n.s. <0.05 n.s. n.s. <0.05 n.s.

aP < 0.05, differences between ae/ae and A/ae control females.bP < 0.05, differences between relative fequencies of A/ae young females and males, in litters weaned by food-deprived females.cP < 0.05, differences between relative frequencies of A/ae young females and ae/ae young males, in litters weaned by food-deprived females.

578 N.M. BAZHAN ET AL.

It may be assumed that food deprivation inducedpregnancy failures by disturbance of progesteroneand corticosterone levels before implantation. Inwater voles, 24-hr fasting on day 3 and on day 5of pregnancy significantly increased blood corti-costerone and decreased urinary progesterone con-centrations on day 6, (our unpublished data). Weassume that in pregnant voles fasting-increasedcorticosterone level may reduce progesterone level.Corticosterone was shown to inhibit progesteronesecretion in pregnant rats by affecting the corpusluteum directly, (Sugino et al., ’91). The lack ofprogesterone at implantation blocks pregnanciesin the ferret (Rider and Heap, ’86) and in themouse (Rider et al., ’87; Heap et al.,’92).

Food deprivation affected the survival of nest-lings both in A/ae and ae/ae females. Neverthe-less, the effect differed strikingly: in A/ae females,postnatal survival decreased compared to controls,whereas in ae/ae females it increased. Nutritionalstress apparently has different effects on mater-nal behaviour in A/ae and ae/ae females. We be-lieve so because in A/ae females, food deprivationincreased postnatal death of whole litters (ratherthan individual pups) within the first week of lac-tation, when maternal care is of primary impor-tance to neonatal survival. Postpartum maternalbehaviour is regulated by endocrines, includingprogesterone (Bridges ’84). In mice, short-termdepletion of active progesterone, induced by asingle administration of anti-progesterone anti-body on day 2 of pregnancy, blocks embryo devel-opment and implantation (Vinijsanun et al., ’90)and it also causes aberrant maternal behaviourtowards the neonate within the first 5 days oflactation (Wang et al., ’95). We suggest that infood-deprived A/ae females the same hormonalmechanism—a decreased level of progesterone be-fore implantation—inhibited survival of offspringboth prenatally and postnatally.

It is not clear why the opposite effect—the in-creased survival of young of ae/ae mothers—oc-curred. Doubtless this is related to pleiotrophiceffects of the nonagouti allelle ae on the endocrinesystem. Adult females of the two genotypes differin their adrenal response to stress, for ae/ae

females do not increase their level of serum cor-ticosterone when subjected to osmotic stress(Bazhan, ’91) or to social stress (Moshkin et al.,’90), in contrast to A/ae females, which do.

Our data support other observations that thereare differences in fertility associated with the Ago-uti locus. In deer mice (Peromyscys maniculatus),reproduction after the stress of transport was sup-

pressed to a different extent in nonagouti andagouti females, with nonagouti deer mice havingmore failures after transatlantic transportation(Hayssen, ’98). It is not clear how the Agouti lo-cus influences reproduction under stressful con-ditions, obviously its pleiotropic effect depends onthe nature of the stress and on the species.

In additional to changes in fertility, there werealso differences in gender and genotype of prog-eny born after food deprivation with fewer A/ae

females born than would be expected. It suggeststhat fasting-induced prenatal elimination of em-bryos was not random; litters with predominanceof A/ae females were more vulnerable and morelikely were eliminated. It indicates that the Agoutilocus is expressed at an early stage of developmentin the water vole. This suggestion agrees with ob-servation in mice, in which homozygosity for lethalAgouti locus alleles (Ay and a(x)) results in embryodeath at about the time of implantation (Duhl etal., ’94; Miller et al., ’94). In summary, the ae allelein homozygote has a positive pleiotropic effect onfemale fecundity and on embryo viability under nu-tritional stress.

Our earlier report has demonstrated that main-tenance of polymorphism for the Agouti locus innatural populations of the water vole might beassociated with positive pleiotropic effect of theae allele on fecundity of homozygous and heterozy-gous females (Bazhan et al., ’96b). In nature, inyears of high population density, food deprivationmay occur in populations of the water vole if win-ter food stores are exhausted (Evsikov et al., ’97).Present findings show that resistance of ae/ae fe-males to the negative effect of nutritional stress,and predominance of ae/ae young in progeny pro-duced by food-deprived mothers, may also favourthe maintenance of polymorphism for the Agoutilocus in natural populations of the water vole.

ACKNOWLEDGMENTSWe thank Dr. Tatiana Aksenovich who advised

on statistical analyses. We are grateful to Dr. Bar-bara Blake, Bennett College, who read variousversions of the manuscript and made many use-ful suggestions. We thank her also for help withEnglish.

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