Removal of the vomeronasal organ disrupts the activation of reproduction in female voles

Physiology & Behavior, Vol. 40, pp. 349-355. Copyright©Pergamon Journals Ltd., 1987. Printed in the U.S.A.

0031-9384/87 $3.00 + .00

Removal of the Vomeronasal Organ Disrupts the Activation of Reproduction in Female Voles J O H N J. L E P R I * A N D C H A R L E S

J. W Y S O C K I * ?

*Monell Chemical Senses Center, 3500 Market Street Philadelphia, PA 19104 and tDepartment o f Animal Biology, School o f Veterinary Medicine University o f Pennsylvania, Philadelphia, PA 19104 R e c e i v e d 6 F e b r u a r y 1987 LEPRI, J. J. AND C. J. WYSOCKI. Removal o.f the vomeronasal organ disrupts the activation of reproduction in female voles. PHYSIOL BEHAV 40(3) 349--355, 1987.--The reproductive system of female prairie voles remains quiescent in the absence of stimulation from males; however, chemosignals from males are capable of at least partially activating female reproduction. In other species, the vomeronasal system mediates some of the reproductive responses of females to males. We found that surgical removal of the vomeronasal organ (VNX) from adult female prairie voles impeded reproductive activation in response to pairing with stud males: ovarian and uterine weights of VNX females paired with stud males for 24 or 60 hours were significantly less than those of normal (NORMAL) or sham-operated (SHAM) females. Furthermore, 8 of 9 NORMAL, 10 of 13 SHAM, but only 4 of 9 VNX females paired with stud males for 60 hours mated. VNX females, however, were still able to use chemosensory cues to locate food. Behavioral observations of females encountering stud males were similar for VNX, SHAM and NORMAL females. We conclude that vomeronasal chemoreception may be a primary component of reproductive activation in female prairie voles, presumably by mediating neuroendocrine responses to chemosignals. Vomeronasal organ Prairie vole Microtusochrogaster Uterus Ovaries Reflex ovulator Chemosignals Vomeronasal chemoreception Reproductive activation Accessory olfactory bulbs

FREE-living populations of prairie voles, Microtus ochrogaster, are found across the central midwestern United

creases in uterine weight, indicative of increased ovarian activity [7], although physical contact with males is necessary to complete the induction of behavioral estrus [8]. These observations leave unspecified the chemosensory mechanisms that mediate the activation of reproduction. The vomeronasal system is a likely candidate for the mediation of chemosignals which modulate neuroendocrine and reproductive status. It is anatomically and functionally distinct from the main olfactory system. Its sensory afferent neurons originate in the vomeronasal organ located in the rostral nasal cavity and project to the accessory (but not main) olfactory bulbs. Subsequent neural projections from the accessory olfactory bulbs terminate in portions of the brain that regulate hypothalamic function (for review see Meredith [25]). The vomeronasal system has been implicated in many chemosignal-induced neuroendocrine responses (reviewed by Wysocki and Meredith [41]). F o r example, female Norway rats reared under continuous light do not express spontaneous estrous cycles, but about half of these acyclic females do respond to brief (30 rain) exposure to the odors of males by ovulating 19-22 hours later. Following

States and exhibit large population fluctuations [13]. Many other arvicoline rodents show these fluctuations [36], stimulating interest in the reproductive physiology of voles and lemmings. Although photoperiod and diet regulate seasonal reproduction in several arvicoline rodents (see contributions in Tamarin [37]), social factors, particularly chemosignals, influence reproduction across seasons. From laboratory investigations on prairie voles it is clear that the reproductive system of mature females generally remains quiescent in the absence of stimulation from males [5,6]. In contrast to the predictable ovarian cycles that result in estrus and ovulation in house mice and Norway rats, the ovaries of isolated female voles do not undergo spontaneous cycles. Rather, upon pairing, unfamiliar male and female prairie voles engage in extensive naso-nasal and naso-anal investigation [12], resulting in the exchange of chemosignals that stimulate gonadotropin secretion [11]. Exposure of female prairie voles to urinary chemosignals from males in the absence of tactile stimulation produces significant in-

~This research was supported by NIH training grant 5T32NS07176-07, NSF grant BNS83-16437 and BRSG 2 SO7 RR05825-07. 2A preliminary report of this work was presented at the IXth International Symposium on Olfaction and Taste, 20--24 July 1986, Snowmass, CO.

349

350

LEPRI AND WYSOCKI

lesion of the vomeronasal system, however, only IlY~. of such female rats ovulated [18]. Thus, vomeronasal chemoreception has been implicated in reproductive responses of female rats that have been artificially sensitized to stimulation by chemosignals from males. Female prairie voles are naturally sensitive to male-induced activation, and preliminary evidence suggests that chemosensation mediates reponse to males: olfactory bulbectomy, which removes both main and accessory bulbs, blocks estrus induction [16]. In this experiment, we tested the hypothesis that female prairie voles utilize vomeronasal chemoreception for reproductive responses to males.

TABLE 1 SAMPLE SIZES FOR T H E P H Y S I O L O G I C A L V A R I A B L E S

Surgical History Hours With Stud Males 0 12 24 60

NORMAL

SHAM

VNX

8 13 3 9

9 12 6 13

8 9 9 9

METHOD

Subjects The virgin female and stud male prairie voles (Microtus ochrogaster) used in this research were drawn from a laboratory colony descended from wild voles trapped in Missouri during the late 1950's by Dr. M. E. Richmond [30], and subsequently bred at the UDSI Patuxent Wildlife Center (Laurel, MD) by Dr. G. Linder before transfer to the Monell Center. Experimental subjects were sexually mature females of 3 to 11 months of age. The voles were housed on pine shavings in polypropylene cages fitted with wire lids. All animals were transferred to clean cages weekly. All animals were housed and tested in the same colony room after weaning. Colony room photoperiod was 14L: 10D (lights on at 0300 EST) and temperature was maintained at 23+-3°C. Water and Agway Rabbit Chow were available ad lib. Surgical Procedures Removal of the vomeronasal organs of females followed procedures similar to those described for house mice by Wysocki et al. [42]. The animals designated for vomeronasal organ removal (VNX) were anesthetized with ketamineacepromazine (100 mg/kg and 1 mg/kg, respectively) and then placed in a head-holder in a supine position. Anesthesia was augmented with halothane in oxygen when necessary. The lower jaw was retracted and approximately 0.05 ml of 2% lidocaine (to enhance local anesthesia) and 0.1% norepinephrine (to cause vasoconstriction and limit bleeding) were applied to the skin covering the palate before a midline incision was made with a scalpel. The epithelial tissue was retracted and, using a dental drill, portions of the incisive bones rosral to the incisive foramina were cleared bilaterally to gain unrestricted access to the vomeronasal organ. At this point the vomeronasal organ was removed, usually in its entirety, by grasping it with fine forceps and applying a twisting upward pull. If the vomeronasal capsule was damaged in the process of removal, electrocautery was used to destroy residual vomeronasal neuroepithelium. Blood was continuously aspirated from the nasal cavity. Following surgery, the incision was closed with cyanoacrylate glue. The animals were allowed to recover from anesthesia under a heat lamp. All subjects were individually housed after surgery. Females designated for sham surgery (SHAM) were anesthetized, placed in the head holder, given topical lidocaine and norepinephrine on the palate, received the midline incision and the palate was retracted as in the VNX procedure. However, the incision was closed after a small amount of surface drilling on the incisive bones. Recovery from anesthesia was also permitted under a heat lamp.

We also tested females that were neither anesthetized nor underwent surgery (NORMAL) to ensure that surgical trauma per se did not disrupt reproduction. Assessment o f Chemosensory Ability to Find Food Two weeks after the surgeries, VNX, S H A M and NORM A L females were tested on 3 consecutive days for their ability to find food using odor cues. The females were removed from their cages for 60 seconds, and a small (5-7 g) piece of apple was hidden under the cage bedding in a random location, but at least 2 cm away from the cage walls. The females were then returned to their cages and the latency to find the hidden apple was recorded using a stop watch. This test served as a measure of chemosensory ability, presumably mediated by the main olfactory system, to determine whether the surgical procedures had severely compromised the ability of the females to respond behaviorally to odor cues. Data were analyzed using repeatedmeasures analysis of variance. Behavioral and Physiological Variables Three weeks after the surgeries, the females were assigned to one of four treatment groups in which the amount of time they spent physically paired with stud males was varied: (1) zero hours; (2) 12 hours; (3) 24 hours; and (4) 60 hours. Behavioral interactions between a subset of the subject females in the 12 and 60 hour pairings with stud males were observed during the initial 10 minutes of pairing. Behavioral data were combined between 12 and 60 hour groups of the same surgery treatments since observations could be conducted at the same time of day: in the dark phase of the light cycle, using a dim red light for illumination. Behavioral variables were recorded using a microcomputer and included: temporal durations of female sniffing male's nasal or ano-genital regions; male sniffing female's nasal or anogenital regions; and frequencies of activity (crossing a line across the short axis of the cage). The presence or absence of ultrasonic vocalizations during each of 120 5-sec blocks of the tests also was recorded with the aid of a QMC S-100 bat detector set to 40 kHz, as were male and female aggressive behaviors, such as leap-threats, attacks, and bites (see Garish et al. [ 12]). Behavioral data were analyzed using oneway analysis of variance (ANOVA) followed by Duncan's new multiple range test for comparison of individual means [35]. Vaginal smears were collected from all females in the 60 hour treatment group at 36 and 60 hours after pairing, and at necropsy for the 12 and 24 hour treatment groups. The vaginal contents were microscopically examined for the presence of sperm, which was used as evidence that mating had oc-

VOMERONASAL ACTIVATION OF FEMALE VOLES

351

TABLE 2 BEHAVIORAL OBSERVATIONS OF STUD MALES DURING FIRST l0 MINUTES OF HETEROSEXUAL PAIRING

Female Condition NORMAL n=20 Frequencies* Activity Ultrasonic Vocalizations Aggression Digging

33.1 53.6 6.0 4.2

Sniffing Durations (sec)* Female's nasal region Female's perianal region

22.1 _+ 4.7 33.7 -+ 7.8

- 2.6 -+ 7.2 - 1.3 _+ 0.7

SHAM n= 17

29.9 42.8 3.7 5.1

_+ _+ -+ _+

VNX n= 15

3.2 5.7 0.9 1.3

33.1 47.1 4.7 4.3

24.6 _+ 4.6 32.2 _+ 11.5

_+ 4.5 _+ 6.9 _+ 1.2 _+ 1.5

30.2 __+8.4 39.7 _+ 8.1

*Mean _ SEM.

curred. At the end of the pairing interval all females were given an overdose of sodium pentobarbital and then perfused intracardially with phosphate-buffered saline (PBS) solution followed by gluteraldehyde-paraformaldehyde fixative in PBS at p H 7.3. All females were killed at 1000 EST (-+ 1 hour) to help offset any possible circadian pattern of changes in weights of reproductive organs. The ovaries and uteri were removed from the carcasses, cleaned of fat and connective tissue and weighed to the nearest 0.1 rag. Sample sizes for the physiological variables as shown in Table 1 are variable due to attrition of subjects that died after surgery or failed to meet the criteria for histological verification of VNX surgery (see below). Uterine and ovarian weights in mg were divided by body weight and then multiplied by 40 to give standardized tissue weights in mg per 40 g body weight. The average weight of females in this experiment was 40 g and was statistically equivalent across surgery treatments. The distribution of standardized uterine weights departed from normality in several treatment groups, necessitating a log10 transformation to meet the normality assumption of A N O V A [34]. Organ weight data were analyzed using two-way A N O V A with the main effects of surgery treatment (NORMAL, S H A M and VNX) and time treatment (0, 12, 24, or 60 hours with stud males). Student-Newman-Keuls (SNK) tests were used a posteriori to compare means [34].

80 ..J 0,. 0..

c,, z_ t,l.

NX ,

60

0

I->-

4O '~)VNX

UJ

.................

20

ORM^

z UJ

1

2 DAY

3.

TESTED

FIG. 1. Mean (-+SEM) latency to find a small piece of fresh apple hidden in the cage bedding for NORMAL (n=30), SHAM (n--34) and VNX (n=25) female prairie voles. No statistically significant differences were found.

Histology F o r histological verification of VNX, brains of all VNX and 6 S H A M females were carefully removed from the skulls following perfusion with fixative, and refrigerated overnight in a 30% sucrose solution. The olfactory bulbs and frontal poles of the brains were then serially sectioned in the horizontal plane at 40/zm in a -20°C cryostat. The sections were stained with 0.25% thionin. VNX was verified by the complete lack o f glomeruli in the accessory olfactory bulbs (AOB). Histological assessments were conducted bfindly with respect to treatment groups. A n y data collected from VNX females that had one or more AOB glomeruli were excluded from the behavioral or physiological analyses, but comments on females receiving incomplete surgeries are presented in the discussion.

RESULTS

Food Finding Deafferentation of the vomeronasal system did not affect the ability of females to find apple hidden in their cage bedding (Fig. 1). Although there was a nonsignificant trend for VNX females to take longer to find the apple on the first day of testing than the SHAM and N O R M A L females, their performance matched that of the S H A M and N O R M A L females on days 2 and 3.

Behavioral Observations Stud males treated the females equivalently in the first 10 minutes after pair formation, regardless of the females' sur-

352

LEPRI A N D WYSOCKI TABLE 3 BEHAVIORAL OBSERVATIONSOF FEMALES DURING FIRST l0 MINUTES OF HETEROSEXUAL PAIRING Female Condition

Frequencies* Activity Aggression Digging Sniffing Durations (sec)* Male's nasal region Male's perianal region

NORMAL n=20

SHAM n=17

VNX n=15

27.2 _+ 2.8+ 19.4 _+ 2.2 6.4 + 0.7

19.5 +_ 2.1 15.9 -+ 2.8 6.0 _+ 0.8

15.5 _+ 2.0 18.9 +_ 4.4 4.5 _+ 0.7

16.8 +_ 3.1 16.0 _+ 3.1

15.4 _+ 3.3 21.4 _+ 7.2

21.9 -+ 7.5 11.3 + 3.6

*Mean _+ SEM. *Significantly greater than SHAM and VNX females (p<0.05).

gical histories (Table 2). None of the A N O V A comparisons reached statistical significance. The males engaged in equivalent amounts of naso-nasal and naso-anal investigation, emitted equivalent amounts of ultrasonic vocalizations (when presented with anesthetized conspecifics, male prairie voles emit many ultrasonic vocalizations whereas females emit negligible amounts [22]), and engaged in equivalent levels of locomotor activity. We conclude that the stud males' initial behavioral responses to females were not influenced by the surgical history of the females. Similarly, the initial behavioral responses of N O R M A L , SHAM, and VNX females toward stud males were equivalent (Table 3). Although S H A M and VNX females had significantly lower activity scores than N O R M A L females, F(2,49)=6.1, p<0.01; Duncan's test, p<0.05, perhaps a result of neophobia enhanced by surgical trauma, the 3 groups of females were not signficantly different in any other behavioral measures. We conclude that the surgical histories of these females did not produce important changes in initial behavioral responses to stud males.

Physiological Observations VNX females paired with stud males for 24 or 60 hours had smaller increases in uterine weight than N O R M A L and S H A M females paired with males for the same length of time (Fig. 2), a conclusion supported by a significant interaction between surgery and time treatments, F(6,99)=2.33, p <0.04, and S N K comparison of means (O<0.05). The main effect of surgery treatment approached significance, F(2,99)=2.68, p<0.07, and the main effect of time spent with stud males was highly significant, F(3,99) = 17.48, p <0.0001. VNX females paired with stud males for 24 or 60 hours also had smaller increases in ovarian weight than N O R M A L and S H A M females paired for the same length of time (Fig. 2), a conclusion also supported by a significant interaction between surgery and time treatments, F(6,99) = 2.16, p <0.05, and S N K comparison of means (o<0.05). The main effect of surgery closely approached significance, F(2,99)=2.84, p<0.06, and the main effect of time treatment was highly significant, F(3,99)=21.33, p<0.0001. Unlike the comparisons for uterine weights, the ovaries of S H A M females paired with males for 60 hours were significantly heavier than all other groups (SNK, p<0,05).

The diminished responsiveness of VNX females to stud males was not absolute: in the 60 hour groups, 4 of 9 VNX, compared to 8 of 9 N O R M A L and 10 of 13 SHAM, females had sperm in the vagina or uterus, indicating that mating had occurred. Three of 4 VNX females that mated had enlarged uteri (>40 mg/40 g body weight) and 2 had ovulation sites present on the ovaries. None of the females tested positive for sperm in the vagina at 12, 24 or 36 hours. DISCUSSION These experiments represent the first demonstration that vomeronasal chemoreception contributes to the activation of female reproduction in a reflex-ovulating species. Female prairie voles whose vomeronasal organs had been surgically removed had smaller increases in uterine and ovarian weights following 24 or 60 hours contact with stud males than did normal or sham-operated females. We suggest that the vomeronasal system may facilitate the activation of female reproduction by responding to stimulatory chemosignals from males. In the formation of a potential breeding relationship, unfamiliar male and female prairie voles immediately engage in extensive ano-genital investigation [12], at which time urinary and glandular chemosignals from males can gain access the females' vomeronasal organ [40]. The neuroendocrine response to the stimulatory chemosignals presumably includes the secretion of luteinizing hormone-releasing hormone (LHRH), which stimulates the release of gonadotropins from the anterior pituitary. As there are many steps between peripheral chemoreception and central translation into gonadotropin secretion in this response, many different mechanisms are possible. The simplest mechanism for vomeronasal chemoreception to produce reproductive activation posits the peripheral release of L H R H into the circulation as a direct response to chemosignals. Although no other examples of this type of peripheral response are known, L H R H is indeed a prevalent component of many peripheral and central neurons of chemosensory systems, including prairie voles [29]. Immunoreactive L H R H fibers are clearly a major component of nervus terminalis, a putative chemosensory system which may have efferent neurons [17, 33, 38, 39]. Given the close anatomical relationship between the vomeronasal organ and nervus terminalis, it is inevitable that our VNX technique

V O M E R O N A S A L ACTIVATION OF F E M A L E VOLES

353

y

.....I

SHAM

SO

UTERUS

. . /./.S"

eO

1 VNX

40

~.~ ~o *.~

I

.O

I

I ,

,,

I

r~ cD O) +l 0

v

12

OVARIES

.......(~)SHAM ........'"" ..... . . . . " "

'

10

"•

i 0

I 12

! 24 HOURS

I 60 WITH

STUD

MALE

FIG. 2. Mean (-+SEM) uterine and ovarian weights of female prairie voles having undergone no surgery (NORMAL), sham surgery (SHAM), or surgical removal of the vomeronasal organ (VNX), and having been paired with stud males for the intervals indicated on the x-axis. Uterine and ovarian weights of NORMAL and SHAM females paired with males for 24 or 60 hours were significantly heavier than those of VNX females paired for the same length of time. Uterine weight data were analyzed after log10 transformation. Sample sizes are in Table 1.

damaged some portion of nervus terminalis; thus our VNX prairie voles might have lost L H R H from this system. Jennes [17], however, cautions that the short half-life of L H R H in the circulation may preclude peripherally-released L H R H from ever reaching the anterior pituitary in its active form. Alternatively, L H R H may be a neuromodulator or neurotransmitter, and as such may prime the neural substrate for chemoreception. Processing of chemosignals affecting L H R H may take place at the level of the olfactory bulbs. Witkin et al. [39] described immunoreactivity for L H R H in fibers associated with the glomernlar and plexiform layers of the main and

accessory olfactory bulbs of Norway rats. Reger et al. [29] recently reported that L H R H fibers in the accessory olfactory bulbs are present in prairie voles. Jennes [17] further noted that L H R H fibers in house mice are closely associated with capillaries and raised the possibility that this constitutes a potential mechanism for chemosensory-induced secretion of L H R H into the blood. Using female prairie voles as subjects, Dluzen and associates have demonstrated increased L H R H levels in the posterior halves of the olfactory bulbs (which contain the accessory olfactory bulbs) and increased serum L H levels after exposure to a single drop of male urine [ 111. Similar effects have been noted in house mice following

354

LEPR1 AND WYSOCKI

social interactions [10]. These results demonstrate that changes in bulbar LHRH are a consequence of social behavior, but it is uncertain whether this directly affects LH secretion or produces modulation of neural activity. The central neural targets of the vomeronasal system include the posteromedial cortical nucleus of the amygdala. The amygdala is one of several neural substrates that modulate the activity of hypothalamic loci. The amygdaloid projection from the accessory olfactory bulbs provides a neuroanatomical circuit for hypothalamic-pituitary responses to chemosignals received via the vomeronasal organ [4]. Vomeronasal chemoreception may cause changes in catecholamine turnover in these loci, which can subsequently influence gonadotropin secretion [1, 3, 20, 21]. Studies of chemosensory-induced changes in amygdaloid and hypothalamic catecholamine turnover could shed more light on the neurotransmitter mechanisms of social stimulation of reproduction. The initial behavioral responses of the female prairie voles in these experiments were apparently unchanged by surgical history (Table 3). This supports evidence from other species that the main olfactory system may mediate behavioral attractiveness to conspecifics and that it mediates social interactions [19]. The ability of VNX females to find apples hidden in their bedding also indicates that they were not impaired in the use of chemosensory cues that guide behavior. Non-vomeronasal sources of sensory information are important to reproductive activation in female voles as evidenced by the smaller increases in ovarian and uterine weights of VNX compared to SHAM and NORMAL females (Fig. 2) and in the observation that 4 of 9 VNX females in the 60 hour group mated. Chemosensory information processed by the main olfactory system or nervus terminalis may interact with the stimulation of physical contact with males to cause LH release and reproductive activation. Although chemosignals in male urine activate reproduction in normal female prairie voles, physical contact with males is required to complete reproductive activation into behavioral estrus [8]. Although VNX female house mice and Norway rats given prolonged mating opportunities with males were impregnated and successfully delivered and reared litters [24,32], VNX female rats showed deficits in mating behavior, including lower levels of lordosis, which were ameliorated by estrogen and LHRH therapy [32], This provides further support for the potential impact on reproductive be-

havior of socially-induced release of LHRH from neurons in the vomeronasal system. In reference to our own study, we expect that injections of LHRH or prolonged exposure of VNX female voles to males would result in a higher percentage of matings. Our criterion for histological verification of successful deafferentation of the vomeronasal system requires complete absence of glomeruli in the accessory olfactory bulbs. Data from 6 VNX females paired with stud males for 60 hours had to be removed from analysis because histology identified intact glomeruli in the accessory olfactory bulbs. All 6 of these incomplete-VNX females mated and their mean uterine weight was 75.51 mg/40 g body weight, well into the range that we would consider reproductively activated. This indicates that even a small portion of vomeronasal receptors may facilitate normal activation, suggesting that the entire vomeronasal neuroepithelium might be composed of an even distribution of homogeneous receptor types. Leaving behind even a small subset of such receptors may be sufficient to retain normal responsiveness. Stimulation of these few receptors may be analogous to an all-ornone switch; once activated the appropriate neuroendocrine response is obtained. Although the rigid classification of mammalian species into categories of spontaneous versus induced ovulators is artificial and often unjustified [26], neuroendocrine responsiveness to stimulation provided by the opposite sex is a common characteristic of mammalian reproduction [27]. The sensitivity of female voles to male-induced activation of reproduction is exemplary in this regard. Stimulation of female reproduction can also be accomplished by chemosignals from males in many species of voles [2, 9, 14, 15, 23, 28, 31]. The present results identifying the vomeronasal system as the potential mediator of this phenomena provide support and a physiological mechanism for the general hypothesis that chemosensory information coordinates reproduction.

ACKNOWLEDGEMENTS We wish to extend our appreciation to Dr. Gregory Linder of the United States Fish and Wildlife Service for providing the breeding stock of prairie voles, Willie Whitfield for his expert care of the animals, L. Wysocki for histological assistance and to Drs. G. K. Beauchamp, C. S. Carter, J. A. Cherry, K. J. Darney, Jr., J. B. Labor and J. G. Vandenbergh for constructive comments on this report.

REFERENCES

1. Adler, B. A., M. D. Johnson, C. O. Lynch and W. R. Crowley. Evidence that norepinephrine and epinephrine systems mediate the stimulatory effects of ovarian hormones on luteinizing hormone and luteinizing hormone-releasing hormone. Endocrinology 113: 1431-1438, 1983. 2. Baddaloo, E. G. Y. and F. V. Clulow. Effects of the male on growth, sexual maturation, and ovulation of young female meadow voles, Microtus pennsylvanicus. Can J Zool 59: 415421, 1981. 3. Barraclough, C. A. and P. M. Wise. The role of catecholamines in the regulation of pituitary luteinizing hormone and folliclestimulating hormone secretion. Endocr Rev 3: 91-119, 1982. 4. Beltramino, C. and S. Taleisnik. Facilitatory and inhibitory effects of electrochemical stimulation of the amygdala on the release of luteinizing hormone. Brain Res 144: 95-107, 1978.

5. Carter, C. S. and L. L. Getz. Social and hormonal determinants of reproductive patterns in the prairie vole. In: Comparative Neurobiology. edited by R. Gilles and J. Balthazart. Berlin: Springer-Verlag, 1986, pp. 18-36. 6. Carter, C. S., L. L. Getz and M. Cohen-Parsons. Relationships between social organization and behavioral endocrinology in a monogamous mammal. Adv Stud Behav 16: 10%145, 1986. 7. Carter, C. S., L. L. Getz, L. Garish, J. L. McDermott and P. Arnold. Male-related pheromones and the activation of reproduction in the prairie vole (Microtus ochrogaster). Biol Reprod 23: 1038-1045, 1980. 8. Carter, C. S., D. M. Witt, J. Schneider, Z. L. Harris and D. Volkening. Male stimuli are necessary for female sexual behavior and uterine growth in prairie voles (Microtus ochragaster). Horrn Behav 21: 74-82, 1987.

VOMERONASAL

ACTIVATION OF FEMALE

VOLES

9. Clarke, J. V. and F. V. Clulow. The effect of successive matings upon bank vole (Clethrionomys glareolus) and vole (Microtus agrestis) ovaries. In: The Development of the Ovary and its Functions, edited by H. Peters. Amsterdam: Excerpta Medica, 1973, pp. 160--170. 10. Dluzen, D. E. and V. D. Ramirez. Localized and discrete changes in neuropeptide (LHRH and TRH) and neurotransmitter (NE and DA) concentrations within the olfactory bulbs of male mice as a function of social interaction. Horm Behav 17: 139-145, 1983. 11. Dluzen, D. E., V. D. Ramirez, C. S. Carter and L. L. Getz. Male vole urine changes luteinizing hormone-releasing hormone and norepinephrine in female olfactory bulb. Science 212: 573575, 1981. 12. Gavish, L., C. S. Carter and L. L. Getz. Male-female interactions in prairie voles. Anim Behav 31: 511-517, 1983. 13. Getz, L. L., L. Verner, F. R. Cole, J. E. Hofmann and D. E. Avalos. Comparisons of population demography of Mierotus ochrogaster and M. Pennsyh,anicus. Acta Theriol 24: 319-344, 1979. 14. Gray, G. D., H. N. Davis, M. Zerylnick and D. A. Dewsbury. Oestrus and induced ovulation in montane voles. J Reprod Fertil 38: 193-196, 1974. 15. Hasler, M. J. and A. V. Nalbandov. The effect of weanling and adult males on sexual maturation in female voles (Microtus ochrogaster). Gen Comp Endocrinol 23: 237-238, 1974. 16. Horton, L. W. and B. A. Shepherd. Effects of olfactory bulb ablation on estrus induction and frequency of pregnancy. Physh~l Behav 22: 847-850, 1979. 17. Jennes, L. The olfactory gonadotropin-releasing hormone immunoreactive system in mouse. Brain Res 386: 351-363, 1986. 18. Johns, M. A., H. H. Feder, B. R. Komisurak and A. D. Mayer. Urine-induced reflex ovulation in anovulatory rats may be a vomeronasal effect. Nature 272: 446-447, 1978. 19. Johnston, R. E. and K. Rasmussen. Individual recognition of female hamsters by males: role of chemical cues and the olfactory and vomeronasal systems. Physiol Beha v 33: 95-104, 1984. 20. Kalra, P. S. and S. P. Kalra. Control ofgonadotropin secretion. In: The Pituitary Gland, edited by H. Imura. New York: Raven Press, 1985, pp. 18%220. 21. Keverne, E. B. Pheromonal influences on the endocrine regulation of reproduction. Trends Neurosci g: 381-384, 1983. 22. Lepri, J. J., M. Theodorides and C. J. Wysocki. Ultrasonic vocalizations by adult prairie voles. Submitted. 23. Lepri, J. J. and J. G. Vandenbergh. Puberty in pine voles (Microtus pinetorum) and the effects of chemosignals on female reproduction. Bh~l Reprod 34: 370-377, 1986. 24. Lepri, J. J., C. J. Wysocki and J. G. Vandenbergh. Mouse vomeronasal organ: effects on chemosignal production and maternal behavior. Physiol Behav 35: 809-814, 1985. 25. Meredith, M. Sensory physiology of pheromone communication. In: Pheromones and Reproduction in Mammals. edited by J. G. Vandenbergh. New York: Academic Press, 1983, pp. 199--252.

355 26. Milligan, S. R. Induced ovulation in mammals. In: Oxford Reviews in Reproductive Biology, Vol 4, edited by C. A. Finn. New York: Oxford University Press, 1982, pp. 1-46. 27. Miiligan, S. R. Pheromones and rodent reproductive physiology. Symp Zool Soc London 45: 251-275, 1980. 28. Milligan, S. R. Social environment and ovulation in the vole, Microtus agrestis. J Reprod Fertil 41: 35-47, 1974. 29. Reger, R. L., C. J. Wysocki, C. S. Carter and A. A. Gerall, Luteinizing hormone releasing hormone system in the accessory olfactory bulb of the prairie vole. Abstract from the 18th Conference on Reproductive Behavior, Concordia University, Montreal, Canada, 8-11 June 1986. 30. Richmond, M. E. and C. H. Conaway. Induced ovulation and estrus in Microtus ochrogaster. J Reprod Fertil Suppl 6: 357376, 1969. 31. Richmond, M. E. and R. Stehn. Olfaction and reproductive behavior in microtine rodents. In: Mammalian Olfaction, Reproductive Processes, and Behavior, edited by R. Doty. New York: Academic Press, 1976, pp. 197-217. 32. Saito, T. R. and H. Moltz. Sexual behavior in the female rat following removal of the vomeronasal organ. Physiol Behav 38: 81-87, 1986. 33. Schwanzel-Fukuda, M., J. I. Morrell and D. W. Pfaff. Ontogenesis of neurons producing luteinizing hormone-releasing hormone (LHRH) in the nervus terminalis of the rat. J Comp Neurol 238: 348--364, 1985. 34. Sokal, R. and F. J. Rohlf. Biometry. San Francisco: W. H. Freeman and Co., 1969. 35. Steel, R. G. D. and J. H. Torrie. Principles and Procedures of Statistics. New York: McGraw-Hill, 1980. 36. Taitt, M. J. and C. J. Krebs. Population dynamics and cycles. In: Bh~logy of New World Microtus, edited by R. H. Tamarin. Lawrence, Kansas: American Society of Mammalogists, Special Publication No. 8, 1985, pp. 567-620. 37. Tamarin, R. H. (editor). Biology of New World Microtus. Lawrence, KS. American Society of Mammalogists, Special Publication No. 8, 1985. 38. Wirsig, C. R. and C. M. Leonard. The terminal nerve projects centrally in the hamster. Neuroscienee 19: 70%717, 1986. 39. Witkin, J. W., C. M. Paden and A. J. Silverman. The luteinizing hormone-releasing hormone systems in the rat brain. Neuroendocrinology 35: 42%438, 1982. 40. Wysocki, C. J., G. K. Beauchamp, R. R. Reidinger and J. L. Wellington. Access of large and nonvolatile molecules to the vomernasal organ of mammals during social and feeding behaviors. J Chem Ecol 11: 1147-1159, 1985. 41. Wysocki, C. J. and M. Meredith. The vomeronasal system. In: Neurobh~logy of Taste and Smell. New York: Wiley and Sons, 1987, pp. 125-150. 42. Wysocki, C. J., J. Nyby, G. Whitney, G. K. Beauchamp and Y. Katz. The vomeronasal organ: Primary role in mouse chemosensory gender identification. Physiol Beha v 29:315-327, 1982.