JOURNAL OF EXPERIMENTAL ZOOLOGY 283:573–579 (1999) Agouti Locus May Influence Reproduction Under Food Deprivation in the Water Vole (Arvicola terrestris) N.M. BAZHAN,* T.V. YAKOVLEVA, AND E.N. MAKAROVA Institute 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 on the fecundity of female water voles homozygous (ae/ae) or heterozygous (A/ae) for, an allele at the Agouti (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 fecundity depended on the Agouti-locus genotype of the female. In food-deprived A/ae females, fecundity was diminished due to fewer successful pregnancies (P < 0.001) and lower survival of the young (P < 0.05). In food-deprived ae/ae females, reproductive performance was not changed; a somewhat 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, the frequency of A/ae females was diminished. Resistance of ae/ae females to the negative effect of nutritional stress, and predominance of ae/ae young in progeny produced by food-deprived mothers, may favour the maintenance of polymorphism for the Agouti-locus in natural populations of the water vole. J. Exp. Zool. 283:573–579, 1999. © 1999 Wiley-Liss, Inc. Natural water vole (Arvicola terrestris) populations are polymorphic for coat color. There is a spectrum in which three main coat colors can be distinguished: brown, black-brown, and black (Nikolaeva, ’78). Laboratory studies have revealed that brown individuals are homozygous for the dominant wild-type allele Agouti (A); black, melanic individuals are homozygous for extreme nonagouti (ae); and black-brown individuals are A/ae heterozygotes (Nasledova et al., ’80; Prasolova et al., ’91). In western Siberia, populations of the water vole exhibit 7–8 year population cycles (Moshkin et al., ’90). A sharp decrease in population size usually is coincident with drought, decreased availability of green vegetation, and increased frequency of black voles from 4 to 10% (Nikolaeva, ’78; Moshkin et al., ’90). We suspect that malnutrition and short-term food deprivation occur in nature in the beginning of the breeding season as a consequence of food stores being exausted, in the years of high density. Studies by Gerlinskaya et al. (’94) indicate that malnutrition can inhibit the reproduction of the water vole. They found that in the course of the population cycle there is a positive correlation between blood levels of fatty free acids (index of undernutrition) and the number of embryos lost. Under labora© 1999 WILEY-LISS, INC. tory conditions, food deprivation in late pregnancy reduces fecundity in the water vole (Bazhan et al., ’96a). Accordingly, we hypothesize that the increased frequency of black (ae/ae) voles that occurs in years of low population density results from the fact that ae/ae females are better able than A/ae females to reproduce successfully when food is scarce. The Agouti locus is one of the major coat color loci in mammals. The Agouti gene of mice has been cloned and shown to encode a 131-amino acid protein-signaling factor that dictates melanocyte pigment production within individual hair follicles (Bultman et al., ’92; Miller et al., ’93). The mouse agouti protein also has been shown to be an antagonist of α-MSH on melanocortin-1 receptors (MC1R) in skin and ACTH and α-MSH on MC4R in brain tissue (Lu et al., ’94). It also regulates the concentration of intracellular calcium (Zemel Grant sponsor: Program of Russian Ministry of Education “Fundamental Researches in Natural Sciences”; Grant number: 3H-20098; Grant sponsor: Russian Foundation for Fundamental Researches; Grant number: 98-04-49502. *Correspondence to: Nadezhda M. Bazhan, Institute of Cytology and Genetics, Lavrentjev Av., 10, Novosibirsk, 630090, Russia. E-mail: email@example.com Received 9 April 1998; Accepted 11 August 1998. 574 N.M. BAZHAN ET AL. et al., ’95). These findings suggest possible roles for the agouti protein in a variety of cellular and physiological functions. In mice, the recessive mutation ae results in a lack of translation initiation, and hence no agouti protein (Hustad et al., ’95). Although little is known about the pleiotropic effects of the ae allele in mice, the absence of the functional agouti protein does not appear to affect either viability or fertility (reviewed in Manne et al., ’95). In other species, the ae allele was shown to effect neuronal and hormonal functions. A pleiotropic effect of the ae allele on brain catecholamines (Hayssen et al., ’94) and ability to reproduce under stressful conditions (Hayssen, ’98) have been demonstrated in deer mice (Peromyscus maniculatus). Our earlier studies have revealed a pleiotropic effect of the ae allele on secretion of adrenal corticosterone and progesterone in immature female water voles (Bazhan and Ivanova, ’89) and adrenal response to osmotic stress in adults (Bahzan, ’91). The object of this study was to test our hypothesis that the increased frequency of ae/ae animals that occurs during periods of food deprivation stems from their enhanced ability to reproduce successfully when nutritionally deprived. Accordingly, we have compared the reproductive performances of A/ae and ae/ae females when experimentally deprived of food. MATERIALS AND METHODS Animals Wild water voles were introduced into the laboratory in 1974 from the subtaiga region of West Siberia (Ubinsk). A colony has since been successfully bred at the vivarium of the Institute of Cytology and Genetics (Siberian Division, Russian Academy of Science), at an ambient temperature of 21°C under natural light conditions. Judging from the frequency of litters and their size, there appears to have been no diminution in reproductive ability in captivity. Details on the laboratory breeding of water voles have been described elsewhere (Bazhan et al., ’96a). The breeding season of the water vole under natural and laboratory conditions lasts from April to August (Panteleev, ’68). Ovulation is induced and implantation occurs on the 7th day of pregnancy (Nasledova et al., ’87). The gestation period is 21 days, animals are weaned at about three weeks of age (Blake, ’82). Breeding Females (150–170 g) and males (170–190 g), aged 8–9 months, were housed individually before the beginning of the breeding season. Females were paired with males, and vaginal smears were checked daily for spermatozoa. The day that spermatozoa were detected was designated day 0 of pregnancy. After mating, males were removed from females. Cages with pregnant females were checked daily at time of expected parturition. The day young were found was designated day 1 of age. Young were separated from mothers on day 21 postpartum, and females were paired for the second time with another male. Under those experimental conditions each female can produce 3–4 litters during the breeding season. Litter size is not related to the number of pregnancies. Crosses Polymorphism for Agouti locus alleles in our laboratory population has been maintained by an outbreeding regime. Inbreeding coefficient does not exceed 12.5%. The fecundity of heterozygous and homozygous females has been shown to be the same in reciprocal crosses A/Ae × ae/ae and ae/ae × A/ae (Bazhan et al., ’96b). We worked only with these reciprocal crosses in this study, so litters were genetically similar (only the mothers differed). Food deprivation Females were denied access to food for 16 hours (from 1700 hr to 0900 hr) on day 3 and again on day 5 of pregnancy. Paper shavings were substituted for hay, and fresh water was freely available. Females were not food deprived for two pregnancies in succession. During control pregnancies females received the standard laboratory diet and food and water ad libitum (Bazhan et al., ’96a). Sixty-three A/ae females produced 115 pregnancies that were food deprived (experimental group) and 115 pregnancies that were treated normally (control group). Similarly, 52 ae/ae females produced 55 food-deprived and 57 control pregnancies. Both A/ae and ae/ae females were mated 3–4 times in each breeding season. There were fewer pregnancies in ae/ae females (both control and food-deprived) because it was necessary to use some of these females for test crosses in other experiments. Reproductive measures For each female we recorded the total number of pregnancies, the number of successful pregnancies (i.e., that resulted in birth), the number of young (litter size) at parturition, at 8 days, and at weaning (day 21). Since mortality is significantly lower in litters that are not disturbed post- FOOD DEPRIVATION AND AGOUTI LOCUS partum (Blake, ’82) animals were not handled (sexed) until they were 8 days old, the youngest age at which their genotype can be ascertained. In each litter the number of young homozygous and heterozygous females and males also was recorded at weaning. The litters in which all young lived to weaning were designated successful, litters where some did not live, partially successful, and litters where none lived to weaning, unsuccessful. The total index of observed fecundity, expressed as the mean number of weaned young per pregnancy, was calculated for each pregnancy. In the case of pregnancy failure the total index of fecundity was set equal to zero. Daily weights were measured during 56 control and 17 food-deprived pregnancies in A/ae females and during 34 control and 12 food-deprived pregnancies in ae/ae females. The day that female ceased gaining weight was designated the day of pregnancy failure. The weights of females in which pregnancy failed were excluded from the consideration. Statistical analysis Weight data were analyzed by two-way ANOVA. The ANOVA includes two factors: female agouti genotype (A/ae and ae/ae) and group (control and food-deprived). To compare the rates of successful pregnancies, weaned litters, weaned young, males and melanics (ae/ae genotype) in progeny, the χ2test was applied with Yates correction for continuity. Litter size at birth and number of surviving young at weaning were compared with a t-test. Nonparametric variances such as relative frequencies of young A/ae and ae/ae females and males in successful litters and mean number of young weaned per one pregnancy were analysed with use of Mann-Whitney U-test. Statistical significance was defined as P < 0.05. Data are presented as mean ± 1 S.E. RESULTS Food deprivation resulted in failed pregnancies. Among control pregnancies in which female 575 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 failure increased in the second week (Table 1). The rate of pregnancy failure in this period was significantly greater in food-deprived than in control females. In week two, in food-deprived groups, all pregnancies failed from 8 to 12 days postcoitum and most (62%) failed on days 8 and 9 (Table 1), i.e., just after implantation. Regardless of experimental group, ae/ae females weighed less than A/ae females throughout gestation (ANOVA, P < 0.001). Food deprivation for 16 hours on days 3 and 5 of pregnancy induced weight 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 pregnancy the weight was regained. At parturition weights in food-deprived and control females were not differed (in A/ae: control 214 ± 6 g, food deprivation 215 ± 10 g; in ae/ae: control 201 ± 5 g, food deprivation 187 ± 10 g). There were no significant differences in litter size by experimental conditions or genotype (overall mean in control groups 4.5 ± 0.2, n = 121; in food-deprived groups 4.6 ± 0.2, n = 76). A/ae and ae/ae females differed in the index of fecundity—the mean number of young weaned per pregnancy—when they were deprived of food (Table 2). In the control, there was no difference, but after food deprivation, the index of fecundity for A/ae females dropped to 0.55 times that of the controls (P < 0.001). By contrast, food deprivation in early pregnancy did not alter the index of fecundity ae/ae females. There were somewhat fewer successful pregnancies in this group, but survival of young actually increased after food deprivation (P < 0.002; Table 2). Breeding under food deprivation, ae/ae females had some advantages over A/ae females; the rates of litters weaned and of young surviving were much greater (P < 0.001, Table 2), and as a result the index of fecundity tended 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 week of pregnancy and by distribution of failures in second week of pregnancy (when failures were greatest) Distribution of failures throughout pregnancy Group Control Food-deprived P1 1 Total no. of failures 27 14 Week of pregnancy Days in second week 1 2 3 8–9 10–12 13–14 4 (15%) 0 n.s. 18 (67%) 13 (93%) <0.003 5 (18%) 1 (7%) n.s. 9 (50%) 8 (62%) n.s. 5 (28%) 5 (38%) n.s. 4 (22%) 0 n.s. Probability levels are given for differences between control and food-deprived females. 576 N.M. BAZHAN ET AL. TABLE 2. Effect of food deprivation during pregnancy (16 hr on days 3 and 5) on reproduction in water voles of two genotypes, A/ae (n = 65) and ae/ae (n = 52)1 Parental genotype (female × male) A/ae × ae/ae ae/ae × A/ae Survival of young Experimental group Reproductive success (litters/ pregnancy) (litters weaned/litters born) (young weaned/young born) Weaning rate (number of young per pregnancy mean ± 1 SE) Control Food-deprived P Control Food-deprived 85/115 (74%) 49/115 (43%) <0.001 36/57 (63%) 27/55 (49%) 71/85 (84%) 35/49 (71%) <0.1 31/36 (86%) 26/27 (96%) 311/371 (84%) 170/220 (77%) <0.05 143/179 (80%) 118/127 (93%) 2.7 ± 0.2 1.5 ± 0.2 <0.001 2.5 ± 0.4 2.2 ± 0.3 P n.s. n.s. <0.002 n.s. P (food-deprived A/ae vs. ae/ae) n.s. <0.001 <0.001 <0.1 1 Probability levels for differences between control and experimental females shown in body of table; probabilities for differences between two female genotypes shown in last row of table. Some litters were entirely lost before weaning, when in other litters a few young were lost. Nevertheless, there were no significant differences in the losses of partial litters in any categories examined (Table 3). By contrast, the losses of whole litters varied with time (most of them occurred in the first 8 days postpartum), experimental conditions and genotype. Food deprivation caused more losses 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% of lost litters (6/14) consisted of only one infant; therefore, although fewer litters survived, their mean size at weaning was significantly greater than 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 of gender and genotype of young weaned. There was a tendency for more males (P < 0.1) and more ae/ae offspring (P < 0.06) to be weaned from A/ae mothers following food deprivation (Table 4). There also were more (55%) ae/ae offspring weaned from both food-deprived ae/ae and A/ae mothers (P < 0.05). Since we could not distinguish the agouti genotype before 8 days of age, any differential elimination of offspring between birth and 8 days would contribute to, or might totally account for, the observed frequency differences. Therefore, in order to estimate the effect of prenatal elimination on litter composition, we compared only successful litters in which all young survived weaning. In the successful litters born to fooddeprived mothers, there were more males of both genotypes (28% A/ae and 29% ae/ae) and a decrease (to 19%) in A/ae females. DISCUSSION e e In A/a and a /ae food-deprived females, gestation was an all-or-none phenomenon, in that either complete litters developed or all implants were resorbed. Most pregnancy failures (93%) occurred on days 8–12 postcoitum, i.e., just after implantation (Nasledova et al., ’87). In rats (Berg, ’65) and mice (Archunan, et al., ’94), fasting and food restriction in early pregnancy also caused the death of whole litters at the time of implantation. 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 genotype (female × male) A/ae × ae/ae ae/ae × A/ae Experimental group Control Food-deprived P Control Food-deprived P P (food-deprived A/ae vs. ae/ae) No. of litters born 85 49 36 27 No. of litters in which young died Partially successful litters Unsuccessful litters Days 1–8 Days 9–21 Days 1–8 Days 9–21 9 (11%) 5 (10%) n.s. 3 (8%) 1 (4%) n.s. n.s. 1 (1%) 1 (2%) n.s. 2 (6%) 2 (7%) n.s. n.s. 10 (12%) 14 (29%) <0.02 5 (14%) 1 (4%) n.s. <0.001 4 (5%) 0 n.s. 0 0 n.s. n.s. Young weaned in completely successful litters Young weaned in all litters Parental genotype (female × male) A/ae × ae/ae ae/ae × A/ae Total a Experimental group Sex ratio (males/total) (%) Agouti ratio (ae/ae out of total) (%) Control Food-deprived P Control Food-deprived P Control Food-deprived P 147/311 (47)a 92/170 (54) <0.1 80/143 (56)a 67/118 (57) n.s. 227/454 (50) 159/288 (55) n.s. 142/311 (46) 93/170 (55) <0.06 74/143 (52) 65/118 (55) n.s. 216/454 (48) 158/288 (55) <0.05 Relative frequencies No. of litters Male A/ae Male ae/ae 61 29 0.26 ± 0.03 0.28 ± 0.04 n.s. 0.26 ± 0.05 0.29 ± 0.06 n.s. 0.26 ± 0.03 0.28 ± 0.03b n.s. 0.25 ± 0.03 0.28 ± 0.03 n.s. 0.29 ± 0.05 0.31 ± 0.06 n.s. 0.26 ± 0.03 0.29 ± 0.03c n.s. 26 23 87 52 Female A/ae 0.27 ± 0.03 0.20 ± 0.03 <0.07 0.22 ± 0.05 0.17 ± 0.04 n.s. 0.25 ± 0.02 0.19 ± 0.02bc <0.05 P < 0.05, differences between ae/ae and A/ae control females. P < 0.05, differences between relative fequencies of A/ae young females and males, in litters weaned by food-deprived females. c P < 0.05, differences between relative frequencies of A/ae young females and ae/ae young males, in litters weaned by food-deprived females. b Female ae/ae 0.23 ± 0.03 0.25 ± 0.04 n.s. 0.23 ± 0.04 0.23 ± 0.04 n.s. 0.23 ± 0.02 0.24 ± 0.03 n.s. FOOD DEPRIVATION AND AGOUTI LOCUS TABLE 4. Effect of 16 hr maternal food deprivation during pregnancy (days 3 and 5) on relative frequencies of male and female young of different agouti genotypes 577 578 N.M. BAZHAN ET AL. It may be assumed that food deprivation induced pregnancy failures by disturbance of progesterone and corticosterone levels before implantation. In water voles, 24-hr fasting on day 3 and on day 5 of pregnancy significantly increased blood corticosterone and decreased urinary progesterone concentrations on day 6, (our unpublished data). We assume that in pregnant voles fasting-increased corticosterone level may reduce progesterone level. Corticosterone was shown to inhibit progesterone secretion in pregnant rats by affecting the corpus luteum directly, (Sugino et al., ’91). The lack of progesterone at implantation blocks pregnancies in the ferret (Rider and Heap, ’86) and in the mouse (Rider et al., ’87; Heap et al.,’92). Food deprivation affected the survival of nestlings both in A/ae and ae/ae females. Nevertheless, the effect differed strikingly: in A/ae females, postnatal survival decreased compared to controls, whereas in ae/ae females it increased. Nutritional stress apparently has different effects on maternal behaviour in A/ae and ae/ae females. We believe so because in A/ae females, food deprivation increased postnatal death of whole litters (rather than individual pups) within the first week of lactation, when maternal care is of primary importance to neonatal survival. Postpartum maternal behaviour is regulated by endocrines, including progesterone (Bridges ’84). In mice, short-term depletion of active progesterone, induced by a single administration of anti-progesterone antibody on day 2 of pregnancy, blocks embryo development and implantation (Vinijsanun et al., ’90) and it also causes aberrant maternal behaviour towards the neonate within the first 5 days of lactation (Wang et al., ’95). We suggest that in food-deprived A/ae females the same hormonal mechanism—a decreased level of progesterone before implantation—inhibited survival of offspring both prenatally and postnatally. It is not clear why the opposite effect—the increased survival of young of ae/ae mothers—occurred. Doubtless this is related to pleiotrophic effects of the nonagouti allelle ae on the endocrine system. Adult females of the two genotypes differ in their adrenal response to stress, for ae/ae females do not increase their level of serum corticosterone 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 there are differences in fertility associated with the Agouti locus. In deer mice (Peromyscys maniculatus), reproduction after the stress of transport was sup- pressed to a different extent in nonagouti and agouti females, with nonagouti deer mice having more failures after transatlantic transportation (Hayssen, ’98). It is not clear how the Agouti locus influences reproduction under stressful conditions, obviously its pleiotropic effect depends on the nature of the stress and on the species. In additional to changes in fertility, there were also differences in gender and genotype of progeny born after food deprivation with fewer A/ae females born than would be expected. It suggests that fasting-induced prenatal elimination of embryos was not random; litters with predominance of A/ae females were more vulnerable and more likely were eliminated. It indicates that the Agouti locus is expressed at an early stage of development in the water vole. This suggestion agrees with observation in mice, in which homozygosity for lethal Agouti locus alleles (Ay and a(x)) results in embryo death at about the time of implantation (Duhl et al., ’94; Miller et al., ’94). In summary, the ae allele in homozygote has a positive pleiotropic effect on female fecundity and on embryo viability under nutritional stress. Our earlier report has demonstrated that maintenance of polymorphism for the Agouti locus in natural populations of the water vole might be associated with positive pleiotropic effect of the ae allele on fecundity of homozygous and heterozygous females (Bazhan et al., ’96b). In nature, in years of high population density, food deprivation may occur in populations of the water vole if winter food stores are exhausted (Evsikov et al., ’97). Present findings show that resistance of ae/ae females to the negative effect of nutritional stress, and predominance of ae/ae young in progeny produced by food-deprived mothers, may also favour the maintenance of polymorphism for the Agouti locus in natural populations of the water vole. ACKNOWLEDGMENTS We thank Dr. Tatiana Aksenovich who advised on statistical analyses. We are grateful to Dr. Barbara Blake, Bennett College, who read various versions of the manuscript and made many useful suggestions. We thank her also for help with English. LITERATURE CITED Archunan G, Aruldhas MM, Govindarajulu P. 1994. Postimplantation fasting does not induce pregnancy failure in newly inseminated mice. Acta Physiol Hung 82:377–381. Bazhan NM. 1991. 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