Chemical Cues Are Necessary but Insufficient for Reproductive Activation of Female Pine Voles

BIOLOGY OF REPRODUCTION 54, 1038-1045 (1996)

Chemical Cues Are Necessary but Insufficient for Reproductive Activationof Female Pine Voles (Microtus pinetorum)'

Nancy G. Solomon,2 '3 John G. Vandenbergh, 4 Kennedy S. Wekesa, 4 and Laura Barghusen3

Department of Zoology,3 Miami University, Oxford, Ohio 45056

Department of Zoology,4 North Carolina State University, Raleigh, North Carolina 27695-7617

ABSTRACT

Among various arvicoline rodents, reproduction is influenced to varying degrees by social factors, including behavioral or chemicalcues. Since previous research suggested that chemosignals from adult males reproductively activate female pirie voles (Microtus pine-torum), we sought to determine specifically what types of stimuli promote the activation response. In these experiments, females wereexposed to unfamiliar adult males, or to some combination of cues from males, or were housed alone. Using uterine mass as a measureof reproductive activation, we found that females were not activated by exposure either to male urine by itself or to male-soiled beddingby itself, but full contact with a male clearly resulted in heavier uteri. Females whose vomeronasal organs were surgically excised failedto undergo reproductive activation when housed with males. Finally, females allowed physical contact by being housed directly underneathmales had heavier uteri than did females whose housing allowed contact only with the chemical cues from males. Among female arvicolinerodents, it appears that there exists a physiological continuum between absolute dependence on both contact and chemical cues frommales vs. absolute independence for reproductive activation. The present results place female pine voles closer to the former extremethan to the latter.

INTRODUCTION

The timing of reproduction is one of the most importantfactors influencing an individual's reproductive success andcan be affected by a number of determinants including ge-netics, ecological parameters such as resource quality orquantity, temperature, photoperiod, and social conditions[1]. Social effects on reproduction appear to be widespreadamong mammals [2, 3] and are not restricted to a particulartaxon, mating system, or social system.

The best-documented and most extensively studied so-cial effects are those that influence sexual maturation in fe-male house mice, Mus musculus (for review see [2]). Ingeneral, the presence of a male, or chemical cues from urineof males or breeding females, accelerate puberty in juvenilefemale house mice [4-6]. Females housed alone achievefirst estrus 12-20 days later than females exposed to cuesfrom males [4, 5]. In contrast, chemical cues from femalesliving in high densities delay puberty [7-10].

In some species of mammals that show spontaneous es-trus, stimuli from males synchronize estrous cycles (e.g.,house mice [11]) or are necessary to organize ovulatory cy-cles (e.g., some Callitrichid monkeys [12-14]). In othermammals, estrus and ovulation do not occur spontaneously.Male-related stimuli appear to be essential for induction ofestrus in a number of arvicoline rodents (voles and lem-mings) [15-17]. Stimuli from males induce rapid changes inneuroendocrine and endocrine functions. These physiolog-

Accepted November 28, 1995.Received September 18, 1995.'This research was supported by grants from the Committee on Faculty Research at

Miami University and NIH MH 52471-01 to N.G.S. and by the North Carolina ResearchService Project NC 06263.

2Correspondence. FAX: (513) 529-4900: e-mail: solomon@msmail muohio.edu

ical changes, in turn, influence female behavior and the en-docrinological processes leading to estrus and subsequentlyto ovulation.

The nature of the stimuli controlling these changes hasreceived considerable attention. Chemical cues were oncethought to be sufficient for reproductive activation in arvi-coline rodents, but more recently this finding has been re-examined [16-18]. It has been hypothesized that there maybe a relationship between the type of mating system dis-played by a rodent species and the type of cues sufficientto induce estrus [16-18]. In socially monogamous prairievoles (Microtus ochrogaster), initial changes are induced bycontact with chemical cues in male urine, but extended co-habitation with a male is necessary for behavioral receptiv-ity [19]. Among California voles (M. californicus), only di-rect contact with a male-not chemical cues from a male--results in increased uterine mass, an indirect index ofestrogen secretion [20]. In contrast, meadow voles (M.pennsylvanicus) and other promiscuous species may beless dependent on cues from males and may even enterestrus spontaneously [21].

Pine voles (M. pinetorum), like prairie voles, are usuallyclassified as monogamous on the basis of laboratory obser-vations and patterns of cohabitation found by means of livetrapping [22-24]. Lepri and Vandenbergh [25] found that aslittle as 12 h of contact with an adult male can result in asignificant increase in uterine mass. When females werehoused in wire-topped cages and males were housed di-rectly overhead in wire-bottomed cages, there was a smallbut statistically significant increase in uterine mass. Lepriand Vandenbergh [25] suggested that exposure to excreta(urine or feces) led to estrogen secretion in young females.

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ACTIVATION IN PINE VOLES

In their experiment [25], females were exposed to a com-bination of cues, i.e., those in the feces and urine as well asauditory and possibly visual cues from males. The purposeof the present experiments was to determine specificallywhich stimuli from males will induce estrogen secretion inpine voles. If the cues that induce uterine changes are as-sociated with the type of mating system and if pine volesare socially monogamous like prairie voles, we expectedthat exposure to chemical cues present in male urine shouldbe capable of activating reproduction in sexually naive fe-male pine voles. Experiments 1, 2, and 5 were conductedat North Carolina State University, and the others were con-ducted at Miami University. The results of each experimentled to each subsequent experiment.

MATERIALS AND METHODS

Experiment 1: Effect of Male Urine on ReproductiveActivation of Females

Pine voles used in this experiment were descendants ofvoles live-trapped in Henderson County, North Carolina.Food (Prolab Diets; Agway Inc., Syracuse, NY) and waterwere provided ad libitum. Animals were maintained on a14L: 1OD schedule in a climate-controlled environment witha temperature of 25 3C. Lights came on at 0600 h.

Breeding pairs were housed in 36 X 30 x 18-cm plasticcages with corncob bedding and shredded paper towel fornest material. Pups were weaned at 28 days of age. Afterweaning, juvenile females were weighed and individuallyhoused in 27 16.5 X 13-cm plastic cages. Some femaleswere left in their home cages until they reached 60-70 daysof age. In these cages, subsequent litters were removedwhen they reached 24-28 days of age.

Sexually inexperienced adult males (- 90 days of age)were isolated from littermates at least 1 wk prior to the be-ginning of the experiment and were housed individuallyunder the same conditions as juvenile females. Male urine,to be used as a stimulus, was collected by placing each maleon a wire screen in a 18 29 X 12.5-cm cage. Urine wascollected from the bottom of the cage at the end of a 4-5-hperiod. Male urine was pooled and frozen until needed. Inaddition, sexually experienced males (- 90 days of age)were housed with adult females. Urine was collected fromthese males after 1 wk of housing with a female but priorto delivery of pups and was pooled and refrigerated for usewithin 2 days.

Sexually naive juvenile (24-28 days of age) and mature(60-70 days of age) females were tested in this experiment.Within each age class, females were randomly assigned toone of three treatments: control, exposure to male urine, orexposure to presence of adult male. One drop (0.3 ml) ofurine or saline was applied to the nasal philtrum of juvenilefemales daily for 7 days beginning on the day after removalfrom the home cage. Lepri and Vandenbergh [25] have ex-

amined reproductive activation by exposing females tomale excreta for a week. Therefore, females in this experi-ment were treated with urine for the same length of time.Mature females were treated in the same way as juvenilesexcept that treatment began when they were 60-70 days ofage. An additional group of adult females (n = 10) weretreated with fresh urine from sexually experienced malesfor 7 days. On the eighth day from the beginning of treat-ment, females were killed by means of a lethal i.p. injectionof pentobarbital.

Females subjected to direct contact with an adult malewere placed individually into 36 X 30 X 18-cm cages eachcontaining an adult male (greater than 90 days of age) andmale-soiled bedding. Lepri and Vandenbergh [25] reporteduterine hypertrophy after 12-72 h of contact with an adultmale and mating after about 48 h. Therefore, females indirect contact with males were killed after 24 h to maximizedetection of uterine hypertrophy but to prevent mating.

Body mass of females was recorded prior to autopsy.Reproductive activation results in estrogen secretion thatcauses cell proliferation in the uterus [26]. Cell proliferationcan be detected by an increase in the mass of the uterus;this measure has been commonly used as an indirect mea-sure of estrogen secretion accompanying reproductive ac-tivation [19, 26, 271. Fat, connective tissue, and the cervixwere removed from uteri, which were then blotted dry onabsorbent paper before being weighed to the nearest 0.1mg. Body mass was examined with a two-way ANOVA [28]and was found to differ between treatments. Therefore,uterine mass was examined with a two-way ANOVA withbody mass as a covariate. Student-Newman-Keuls testswere used for post hoc comparisons if a significant differ-ence was found in the ANOVA [28]. Only mature femaleswere examined to determine the effectiveness of freshurine. Body mass of mature females treated with saline, fro-zen-stored urine, or fresh urine or housed with a male wastested with a one-way ANOVA and did not differ amongtreatments. Therefore, a one-way ANOVA was used to ex-amine uterine weights for this comparison.

Experiment 2: Effect of Male-Soiled Bedding onReproductive Activation of Females

The purpose of this experiment was to examine the re-sponse to multiple types of olfactory cues by exposing fe-males to male-soiled bedding. The methods followed in thisexperiment were similar to those described for experiment1. Females 60 to 70 days old were randomly assigned to oneof two treatments: exposure to male-soiled or to clean bed-ding (n = 15 per treatment). Beginning when they wereapproximately 60-70 days of age, females received dailyfor 7 days the quantity of bedding that filled a 100-mlbeaker. This quantity of bedding, three times as much asthat used by Vandenbergh [5] with house mice, was chosento ensure a sufficient quantity of chemical cues. Before the

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bedding was sprinkled evenly in the cage, including thenest, an equivalent amount was removed.

Sexually inexperienced adult males provided soiled bed-ding for test females. As in experiment 1, they were isolatedat least one week prior to the start of the experiment andhoused in 27 16.5 x 3 cm cages. Pine voles tend to useone corner of the cage as a latrine [291, so each day, soiledbedding was collected primarily from that area of the cage,and an equivalent quantity of clean bedding was added.Care was taken to make sure that each female received bed-ding from the same male throughout the experiment toeliminate possible confounding effects from exposure tostrange males [30], such as occurred when urine was pooledin experiment 1. On the eighth day after the beginning ofthe treatment, females were killed with pentobarbital, andweight data were collected as previously described. Dataon body and uterine masses were analyzed with Student'st-tests.

Experiment 3: Effect of Housing in a Male-Soiled Cage onReproductive Activation of Females

The purpose of this experiment was to examine the re-sponse to a larger quantity of chemical cues by housingfemales in male-soiled cages. Voles used in this experimentwere descendants of animals from the North Carolina StateUniversity colony; in addition, a small number of animalsfrom an apple orchard in Henderson County, North Caro-lina, were used. Voles were provided with water ad libitumand were fed Purina Rodent Breeder chow (Ralston-Purina,St. Louis, MO), a diet similar in the percentages of fat, pro-tein, fiber, and metabolizable energy to the diet used atNorth Carolina State University. Nestlets (Ancare, Corp.,North Bellmore, NY) were provided for nest material. Thelighting schedule was the same as that used in the NorthCarolina State University colony, and room temperature wasmaintained at 25 + 3C. Animals were removed from theirhome cages at approximately 24-26 days of age and thenhoused with littermates. Each female was isolated from herlittermates at least 1 wk before the beginning of the treat-ment and, in a room separate from the breeding colony,was housed alone in a 27 x 16.5 x 13-cm cage with corn-cob bedding. Males were sexually inexperienced and werehoused as described for experiment 2 and left in their cagesfor 7 days before the start of the experiment.

Females 60 days old were randomly assigned to one oftwo treatments. For these treatments, each female was ei-ther housed alone in a 27 x 16.5 x 13-cm cage for 48 h orplaced for 48 h in a cage that had been soiled by an adultmale. This length of time was selected because Gardner etal. 131] have shown that exposure of female prairie voles tomale-soiled cages for 48 h results in increased uterine mass.At the end of the 48-h period, females were killed with pen-tobarbital, and weight data were collected as previously de-

scribed. Data on body and uterine masses were analyzedwith Student's t-tests.

Experiment 4: Effect of Separation of the Male and Femaleby a Barrier

This experiment was an examination of the effect on re-productive activation of all sensory cues from males excepttactile and contact olfactory cues. Specifically, females inthis experiment were exposed to visual, auditory, and vol-atile olfactory cues. Sixty-day-old females were separatedfrom their families, housed as described for experiment 3,and randomly assigned to one of three treatments (n = 15per treatment). Stimulus males, older than 90 days of ageand sexually inexperienced, were housed as described forexperiment 2.

Each female was placed in a clean 36 x 30 x 18-cm cagewith an adult male, with an adult male housed on the otherside of a double wire-mesh barrier, or with no male on eitherside of the barrier. The barrier was constructed of a doublethickness of 0.5-mm mesh hardware cloth (average distancebetween layers of hardware cloth, 5 cm). A layer of processedpaper bedding (Cell Sorb Plus; A & W Products Inc., NewPhiladelphia, OH) was placed on the bottom of each side ofthe cage. On the eighth day, females were killed and weightdata were collected as previously described. One-way AN-OVAs were used to compare data on body and uterine massesamong the three treatments. Student-Newman-Keuls testswere used for post hoc comparisons if a significant differencewas found in the ANOVA [28].

Experiment 5: Effect of Excision of the VomeronasalOrgan on Reproductive Activation

In prairie voles, chemical cues from the male act throughthe vomeronasal organ system, leading to hormonal changesthat result in estrogen secretion and subsequent increases inuterine mass [19, 32, 33]. If chemical cues are not requiredfor reproductive activation in pine voles, then vomeronasalorgan removal should not prevent uterine mass increases infemales paired with unfamiliar males. If chemical cues areimportant, then such treatment should prevent or reduce in-creases in uterine masses.

Sexually inexperienced females were 60 days of age atthe beginning of the experiment. They had been separatedfrom their families at 24 days of age and were housed aloneuntil surgery, which was conducted when they reached 39days of age. For surgery, females were assigned to one oftwo treatment groups: vomeronasalorganectomy (VNX) orsham-operated (SHAM). Those assigned to the VNX groupwere anesthetized with an i.p. injection of a ketamine (100mg/kg) and acepromazine (1 mg/kg) mixture, and the vo-meronasal organ was surgically removed via the oral cavityusing techniques described by Lepri and Wysocki [32]. TheSHAM procedure was identical to the VNX procedure interms of anesthesia, incision, and drilling of the incisive

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bone, but the vomeronasal organ remained intact. Femaleswere housed individually and undisturbed for a period of 3wk after surgery and then were paired with 60-day-old, un-familiar, sexually inexperienced males. Twenty-four hourslater, females were killed with an overdose of pentobarbital(150 mg/kg), and uteri were removed as described for ex-periment 1. Data on body and uterine mass were comparedamong treatments using Student's t-tests.

Surgical procedures were verified at the termination ofthe experiment. Females were then perfused with 0.1 M PBS(pH 7.3; at least 10 times total blood volume) followed byaldehyde fixatives (2.5% glutaraldehyde, 1% paraformal-dehyde in 0.08 M PBS, pH 7.2; at least 40 times total bloodvolume).

Brains were carefully removed from skulls. The olfactorybulbs and frontal poles of the cerebral cortex were cut awayfrom the rest of the brain, cryoprotected in 30% sucrosesolution for 24 h, and then serial sectioned at 40 glm in thehorizontal plane. VNX was verified in thionin-stained sec-tions (a) by degeneration of the vomeronasal nerve whereit travels from the midline between the two olfactory bulbsto the more laterally positioned accessory olfactory bulbs(AOB) and (b) by the complete absence of glomeruli in theAOBs as described in previous studies [32-34]. SHAMs pro-vided a positive control. For further verification, the nasalcavity of each VNX subject was thoroughly examined forfragments of the vomeronasal organ.

Experiment 6: Effect of Chemical Cues and Contact onReproductive Activation of Females

The chemical cues responsible for reproductive activa-tion of female pine voles may be extremely volatile. Thishypothesis is consistent with the fact that females in Lepriand Vandenbergh's [251 experiment were exposed to a con-tinuous barrage of fresher excreta than females in the cur-rent experiments and, without direct contact, were repro-ductively activated. But this is not the most likelyexplanation, because a number of important rodent pher-omones are not volatile [35-38]. Alternatively, since pinevoles spend time climbing and hanging from cage lids (N.G.Solomon, personal observation), it is possible that the fe-males in Lepri and Vandenbergh's experiment made contactwith males through the cages and that direct contact withmales and olfactory cues are needed for reproductive acti-vation. Reproductive activation through contact betweenmales and females is especially likely since the vomeronasalorgan is thought to be a receptor for molecules of low vol-atility and high molecular weight that are transported to thevomeronasal organ in a liquid medium like urine [39, 40].

The purpose of experiment 6 was to distinguish betweenthe two hypotheses. If contact with males is necessary forreproductive activation, then females in cages directly un-der male cages should be activated but those in cages sep-arated from male cages by a space, therefore incapable of

contact, should not. On the other hand, if the cues necessaryfor reproductive activation are volatile, then females bothin direct contact with the male's cage and those that receivefresh excreta but no actual male contact should show uter-ine hypertrophy. Use of both sexually experienced and in-experienced males allowed us to verify that sexual experi-ence did not alter the ability of males to reproductivelyactivate females.

Animal husbandry was as described for experiment 3.Females were removed from their home cages at approxi-mately 28 days of age and placed individually in 25.4 X 20x 14-cm plastic cages with processed paper bedding.

A random sample of females (n = 10) were killed im-mediately after removal from their home cages at 28 daysof age so that uterine weights could be examined prior totreatment. For the remainder of this experiment, each fe-male was randomly assigned to one of six treatments in a 2X 3 factorial design (sexual experience of males x type ofstimulus: female alone, male contact + chemical, and malechemical cues only). In approximately half of each of these,females were exposed to cues from sexually experiencedmales and the other half to cues from sexually inexperi-enced males. The sexually experienced males had fathereda litter previously and had been housed with a female for3 days immediately prior to the experiment. In the femalealone treatment, females were individually housed in 20 X25.4 X 14-cm cages. In the male contact + chemical treat-ment, females in their individual cages were placed imme-diately underneath a cage containing an unfamiliar, unre-lated adult male. Males were housed in 20 X 25 X 17-cmstainless steel cages with a wire bottom that allowed nestmaterial (paper towel strips) and excreta to fall through tothe female's cage. Males and females also could make min-imal amounts of contact through the bottom of the male'sand the top of the female's cages. In the male chemical cuetreatment, the female's cage was placed underneath the male'scage but was separated from the bottom of the male's cageby 7 cm of airspace. Thus, females in this treatment were ex-posed to similar amounts of chemical cues as in the contact+ chemical treatment but contact between the male and fe-male was impossible.

After 7 days, females were killed with pentobarbital andweight data were collected as previously described. Bodyand uterine masses were compared among treatments withtwo-way ANOVAs. Student-Newman-Keuls tests were usedfor post hoc comparisons if a significant difference wasfound in the ANOVA [28].

RESULTS

Experiment 1: Effect of Male Urine on ReproductiveActivation of Females

There was no main effect of age class; the pattern ofresponse to cues was the same in juvenile and mature fe-

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males that had contact with males (18.8 2.3 mg; F(3 39, =7.68, p = 0.0004).

There was a significant interaction between age andtreatment in terms of body mass (F(2,55 = 3.36, p = 0.04).Even though females were randomly assigned to treatment,juveniles in the saline treatment were significantly heavierthan juvenile females in the other two groups. There wasno statistically significant difference in body mass among

treatment groups of mature females.

Experiment 2. Effect of Male Bedding on ReproductiveActivation of Females

Female voles exposed to bedding soiled by adult malesdid not show an increase in uterine mass as compared tocontrols receiving clean bedding (t(22) = 1.30, p = 0.21).Comparison with the uterine masses of females housed withmales in experiment 1 further confirms that females ex-posed to male-soiled bedding were not reproductively ac-tivated (Table 1). There was no difference in body mass offemales between the two treatments (t(22 = 0.35, p = 0.73).

FIG. 1. Mean + 1 SE uterine mass (mg) of juvenile and mature females exposedto saline, urine, or intact males. Different letters indicate statistically significant dif-ferences found with a Student-Newman-Keuls test. Sample sizes are found withinbars. Groups: male = male present in cage with female; urine = female treatedwith male urine; saline = female treated with saline as a control.

males (F(1 54) = 0.52, p = 0.47). Contact with an adult maleresulted in a significant increase in uterine mass (F(2 54) =

20.36, p = 0.0001; Fig. 1). Surprisingly, there was no statis-tically significant difference between uterine mass of fe-males that had been treated with male urine and that of thecontrol females (Fig. 1). Neither fresh (9.5 + 1.7 mg; mean± SE) nor frozen urine (10.2 + 1.3 mg) led to increases inuterine mass in mature females as compared to mature fe-

TABLE 1. Mean + SE of body mass (g) and uterine mass (mg).

Body mass Uterine mass

Experiment 1:Mature females with males 24.28 + 1.47 18.84 + 2.35

Experiment 2:Clean bedding 20.80 + 0.81 6.02 + 0.52Male bedding 20.38 ± 0.82 7.71 + 1.03

Experiment 3:Female alone 21.17 + 0.97 11.80 + 1.27In male cage 20.57 + 0.58 12.41 + 1.13

Experiment 4:Female alone 23.53 ± 0.89 11.49 + 1.18Male across a barrier 22.15 + 0.97 12.05 + 1.71Female with male 22.07 + 0.56 23.37 + 1.65

Experiment 5:SHAM females 25.76 + 2.22 34.62 + 4.30VNX females 22.42 ± 1.88 10.04 + 1.77

Experiment 3. Effect of Housing in a Male-Soiled Cage onReproductive Activation of Females

Uteri from females housed in male-soiled cages were notsignificantly heavier than those from controls (t(2 9 , = 0.36.p = 0.72; Table 1). There was no difference in body massof females in the two treatments (t( 29, = 0.53, p = 0.60).

Experiment 4: EffJct qf'Separation of the Male and Femaleby a Barrier

Females housed with males had significantly heavieruteri than females housed alone or with males across a dou-ble-wire barrier (F(2 42 ) = 19.12, p = 0.0001; Table 1). Therewas no significant difference in body mass among treat-

ments (F(2 42) = 0.98, p = 0.38).

Experiment 5: EffJct of Excision of the VomeronasalOrgan on Reproductive Activation

Females whose vomeronasal organs had been removeddid not show an increase in uterine mass with male expo-sure as did the sham-operated controls (t(, = 4.90; p =0.0008; Table 1). There was no significant difference in bodymass between treatments (t(9 , = 1.12, p = 0.29).

Experiment 6: Effect of Chemical Cues and Contact onReproductive Activation of Females

Body and uterine masses of females upon removal fromtheir home cages were 19.05 0.79 g and 11.47 1.09mg, respectively (mean + SE). In some cases in whichmales' cages were placed directly on top of cages contain-ing females, males and females were observed to make con-tact by sticking their snouts through the cages. Females

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whose cages were in direct contact with males' cages hadheavier uteri than females in cages with no male housedabove them (F(2,90 ) = 4.53, p = 0.01; Fig. 2). Females whosecages were housed beneath but not in contact with males'cages had uterine weights that were intermediate, but thesedid not differ significantly from uterine weights for either ofthe other two treatment groups. The sexual experience ofthe male had no effect on reproductive activation of females(F( 1,90) = 0.16, p = 0.69). There was no significant differ-ence in body mass of females among the three treatments(F(2,90 ) = 0.38, p = 0.68). Females exposed to sexually ex-perienced males were heavier (22.34 0.46 g) than thoseexposed to cues from inexperienced males (20.19 + 0.30g; F(1,90 ) = 16.72, p = 0.0001).

DISCUSSION

Researchers have shown considerable interest in the cuesthat stimulate estrus in female mammals. Early work sug-gested that, in arvicoline rodents, chemical cues from malesstimulate activation of the hypothalamus-hypophyseal-ovarian axis [41-43] and subsequent increases in uterineweight [19, 25]. In one of the earlier studies that more pre-cisely tested the prevailing wisdom, Rissman [44] found thatfemale California voles did not show a significant increasein uterine weight when exposed to male urine or male-soiled bedding in the absence of an intact male vole unlessfemales were fed lettuce, which contains 6-MBOA (6-meth-oxy-2-benzoxazolinone). Following a series of recent stud-ies that examined reproductive activation in females withdifferent mating systems, the original hypothesis that chem-ical cues can reproductively activate female voles was re-fined to state that only females in social species can be ac-tivated by chemical cues [16, 17]. We tested this hypothesisusing the pine vole, a social arvicoline rodent.

The results from experiment 1 suggest that a cue otherthan male urine is needed to activate female pine voles. Wealso found that female pine voles did not show reproductiveactivation in response to cues present in the bedding fromunfamiliar, intact adult males. The lack of reproductive ac-tivation from exposure to male-soiled bedding was not dueto an insufficient quantity of chemical cues, because hous-ing females in cages previously inhabited by males did notresult in reproductive activation. Additionally, auditory, vi-sual, and volatile olfactory cues did not reproductively ac-tivate a female pine vole housed across a barrier from anunfamiliar, intact adult male. Furthermore, females whosevomeronasal organs were excised did not become repro-ductively activated as would be expected if the pheromonewas extremely volatile.

It is becoming increasing clear that it is difficult to gen-eralize about social effects on reproduction in this group ofrodents. As in the prairie vole, chemical cues are necessaryfor reproductive activation in female pine voles, but in con-

FIG. 2. Mean + 1 SE uterine mass (mg) of juvenile females housed alone, exposedto chemical cues, or exposed to chemical and tactile cues. Different letters indicatestatistically significant differences found with a Student-Newman-Keuls test. Sam-ple sizes are given within bars. Treatments are as follows: female alone = femalehoused underneath an empty cage; suspended cage = male's cage suspended overthe cage of a female so female received chemical cues but no direct contact withthe male; direct contact = male's cage placed directly on top of the female's cage.In the latter treatment, females received chemical cues and also had some contactwith the male.

trast to what occurs with prairie voles, these cues alone arenot sufficient to induce uterine hypertrophy. Rather, a com-bination of chemical cues and contact with an unfamiliarmale is necessary for reproductive activation in female pinevoles. This result may explain the extremely low rate ofmating found by Taylor et al. [171 when they exposed femalepine voles to males for 1 h per day across a wire meshbarrier. The barrier may have prevented sufficient contactthat would have resulted in more females becoming behav-iorally receptive.

Contact alone is ineffective as seen in experiment 5, inwhich the vomeronasal organs were removed from femaletest subjects. It appears that contact acts synergistically withchemical cues from adult males as has been found for ac-celeration of puberty in house mice [6, 45] and behavioralreceptivity in prairie voles [27]. Separation of the male andfemale by a barrier (experiments 4 and 6) suggests that theprimary cue involved in reproductive activation of femalepine voles may be tactile. In contrast to our expectations,chemical cues alone do not result in an increase in uterinemass as seen in house mice and prairie voles. It appears thatpine voles are intermediate between California voles and

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prairie voles in terms of cues that activate reproduction. Par-tial support for this hypothesis stems from observations thatlimited male exposure resulted in mating by three times asmany prairie voles as pine voles [17].

Preliminary observations suggest that female pine volesrequire relatively long exposure to a male before becomingreproductively activated (N.G. Solomon, personal obser-vation); therefore, casual contact while foraging probably isnot effective in reproductively activating female pine voles.This observation again suggests that more than just a chem-ical cue is necessary for reproductive activation in this spe-cies. Thus, in species for which contact with unfamiliarmales may be rare prior to dispersal from the natal nest, itmay be adaptive for activation to occur only after extensivecontact. Pine voles are fossorial and live in undergroundburrows with members of their social group [23]; theamount of time spent above ground or in contact with un-familiar males is unknown.

What male characteristics cause reproductive activationin female pine voles? It does not appear that sexual expe-rience of males affects the chemical cues in their urine. Inthe first and sixth experiments, females were exposed tourine, excreta, or contact with sexually experienced malesthat had been housed with females prior to the birth of theirlitters. Presumably these males had the highest testosteronetiters (they had received stimulation from a female but werenot parental) of any in these experiments. Even under theseconditions there was no significant effect on reproductiveactivation of chemical cues from sexually experiencedmales. Further, uterine weights were not higher in femalesin contact with sexually experienced as compared to sex-ually inexperienced males.

Chemical cues may be a necessary factor involved in re-productive activation because they indicate the familiarityof the male. Female pine and prairie voles remaining in theirfamily groups beyond the age of sexual maturity do notbreed with their father or brothers [46, 47]. It has beenthought that incest avoidance in these species is mediatedby familiarity. In the prairie vole, it was found that separa-tion of brothers and sisters for 8 days resulted in matingupon reunion [48], suggesting that familiarity indeed pre-vents inbreeding.

In addition to signaling a lack of relatedness, the unfa-miliarity of a male may result in an increase in generalarousal in females. In a recent study, Saltzman et al. [491have shown that adrenal activity appears to be closely as-sociated with ovarian status in the common marmoset (Cal-lithrix jacchus). Females that began cycling showed in-creases in levels of plasma cortisol, suggesting increasedarousal, as compared to noncycling females. In addition,female house mice showed increased levels of corticoster-one after exposure to stimuli from an unfamiliar male [50].Glucocorticoid levels in female prairie voles also changerapidly in the presence of an unfamiliar male [51], but cor-

ticosterone levels of female pine voles have yet to be in-vestigated.

It is an oversimplification to group social mammals (e.g.,cooperative breeders) together and to regard them as if theyrespond in the same way to environmental stimuli. In ad-dition to the different types of cues needed for reproductiveactivation, there are other differences in social behavior andreproductive physiology among these cooperatively breed-ing mammals. For example, pine voles also differ from prai-rie voles in the amount of time that the male parent spendsin the nest with pups. Male prairie voles spend as muchtime as females in the nest with pups [52]. Although malepine voles engage in parental care of young, they spendconsiderably less time with pups than do females [53]. Fur-ther, prairie voles do not respond with an increase in uterineweight to 6-MBOA [54], yet this chemical is effective in fe-male pine voles [551.

The idea of a continuum of behavioral responsivenesshas been proposed to describe differences in the degree ofsociality [56, 57] and reproductive suppression [581 in mam-malian cooperative breeders. Females' responsiveness tomale cues also may be viewed as a physiological responsecontinuum instead of discrete categories of responsiveness[17]. At one end of the continuum would be mammals likemeadow voles that may come into estrus spontaneously,and at the other end would be those like prairie voles thatshow an increase in uterine weight in response to maleurine alone. This continuum may be useful with regard toother mammals as well as arvicoline rodents.

ACKNOWLEDGMENTS

We thank Chris Salatti, Inga Van Nynatten, Ryna Rowell, and Andrew Robinson forassistance with data collection. Bruce Steinly designed the wire harrier and Traci Cashmanassisted with data entry. We also thank Christine Brant for her insightful comments duringdiscussion of the results of these experiments and Lee Drickamer, John Lepri, and threeanonymous reviewers for helpful suggestions on a previous version of this manuscript.

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Documents

Chemical Cues Are Necessary but Insufficient for Reproductive Activation of Female Pine Voles