BIOLOGY OF REPRODUCTION 80, 1146–1151 (2009)Published online before print 28 January 2009.DOI 10.1095/biolreprod.108.075341

Rapid Induction of Cell Proliferation in the Adult Female Ungulate Brain (Ovis aries)Associated with Activation of the Reproductive Axis by Exposure to Unfamiliar Males1

Penelope A.R. Hawken,2 Trina Jorre de St. Jorre, Jennifer Rodger, Tammy Esmaili, Dominique Blache,and Graeme B. Martin

School of Animal Biology, University of Western Australia, Perth, Western Australia, Australia

ABSTRACT

In many species, the reproductive centers of the brain areprofoundly affected by sociosexual stimuli. This is particularlyevident in female ungulates such as sheep, in which exposure tomales switches them from reproductively quiescent to fertile. Intwo experiments with female sheep, we tested whether the braincenters that control gonadotropin-releasing hormone (GnRH)neuronal activity respond differentially to ‘‘novel’’ vs. familiarmales and whether the neuroendocrine response is associatedwith increased cell proliferation in the hippocampus, a siteassociated with memory formation. In experiment 1, groups of10 female sheep that had previously been habituated to malesfor 3 mo were re-exposed to familiar males or were exposed tonovel males. Only the novel males increased luteinizinghormone (LH) pulse frequency, indicating stimulation of GnRHneuronal activity. In experiment 2, groups of six female sheepwere injected with bromodeoxyuridine (BrdU) and thenmaintained in isolation from males or exposed to novel males.Two days later, the hippocampus and hypothalamus wereremoved and processed for fluorescence immunohistochemistry.Again, exposure to males increased LH pulse frequency. Mostimportant, male exposure also doubled the number of BrdU-positive cells in the dentate gyrus of the hippocampus. No BrdU-positive cells were detected in the hypothalamus. We concludethat the stimulus from novel males switches on the reproductivecenters of the brain of female sheep and rapidly doubles the rateof cell proliferation in the hippocampus. The rapidity of thisresponse contrasts with rodents, in which several days ofexposure to male pheromones seem necessary for an effect onneurogenesis.

cell proliferation, female reproductive tract, gonadotropin-releasing hormone, hippocampus, LH, luteinizing hormone,neurogenesis, novelty, pheromones

INTRODUCTION

The brain centers that control reproduction integrateinformation from a variety of sources to formulate thereproductive strategy of the animal. This information can stemfrom the animals’ internal environment (e.g., metabolic factors)and external environment (e.g., photoperiod and sociosexual

milieu). Sociosexual information can include auditory, visual,behavioral, and olfactory signals. Olfactory signals, orpheromones, are perhaps the most important, and they caninhibit (e.g., block pregnancy [1]) or facilitate (e.g., synchro-nization of estrus [2–4]) the reproductive process. Arguably,the most profound facilitatory effect of male sociosexualstimuli identified to date is observed in ungulates such assheep, in which exposure to males leads within minutes to acomplete change in the female’s reproductive status fromquiescent to fertile [5–7]. Moreover, the epidermal pheromoneof sheep and goats seems to encode specific information aboutmale identity, as only unfamiliar (‘‘novel’’) males seem able toinduce ovulation [8, 9]. However, in these studies, changes inpulsatile luteinizing hormone (LH) secretion were not mea-sured. Pulsatile LH secretion directly reflects GnRH cellularactivity, so it is still not clear whether the brain centers thatemit the major neuroendocrine signal that controls reproductioncan discriminate between individual males.

Clues to the brain mechanisms underlying these hormonaland behavioral changes in female ungulates have been obtainedfrom studies in mice, in which male pheromones inducepuberty [10] and synchronize proestrus [2–4]. Most important,these urinary pheromones also seem to encode the individualidentity of males, enabling females to discriminate amongmales according to familiarity or dominance [11]. Furthermore,7 days after exposure to male pheromones, changes in prolactinand LH secretion appear to increase the rate of neurogenesis inbrain regions critical to the processing of olfactory information(subventricular zone [SVZ]) and learning and memory(hippocampus); this seems to be required for female mice toselect and mate with the dominant male [12].

In contrast to the situation in rodents, exposure of femalesheep to male stimuli induces a dramatic neuroendocrineresponse within minutes and leading to ovulation within a fewdays, raising the question of whether recognition of maleidentity, potentially supported by increased neurogenesis, canoccur within such a short time frame. This process in sheep isdriven by activation of neurons in the preoptic-hypothalamiccontinuum that produce pulses of gonadotropin-releasinghormone (GnRH), leading to pulses of luteinizing hormone(LH) in the peripheral circulation. In the present study, wecompared the hormonal profile of females exposed to familiarand novel males to determine the importance of novelty for theneuroendocrine response that drives male-induced ovulation.We also measured cell proliferation in the hippocampus andhypothalamus within the first few hours of male exposure, asthe cascade of neuronal and neuroendocrine responses that maydrive increased cell proliferation begins within minutes ofexposure to the male stimulus. We targeted the hippocampusbecause the male effect in sheep involves a dramatic increase inthe secretion of LH, the hormone that seems to be responsiblefor pheromone-induced neurogenesis in the hippocampus offemale mice. We also investigated the hypothalamus because it

1Supported by the Australian Research Council’s Discovery fundingscheme (Project DP0558952) and Meat & Livestock Australia (ProjectB.MGS.0027).2Correspondence: Penelope A.R. Hawken, School of Animal Biology,University of Western Australia, Crawley, Perth, WA 6009, Australia.FAX: 61 8 6488 1029; e-mail: phawken@animals.uwa.edu.au

Received: 14 December 2008.First decision: 29 December 2008.Accepted: 22 January 2009.� 2009 by the Society for the Study of Reproduction, Inc.eISSN: 1259-7268 http://www.biolreprod.orgISSN: 0006-3363

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is an important site of pheromone-induced neurogenesis infemale prairie voles [13] and has a critical role in mediating theneuroendocrine response of female sheep to male stimuli.

MATERIALS AND METHODS

Both experiments were performed in accord with the Australian Code ofPractice for the Care and Use of Animals for Scientific Purposes (seventhedition, 2004) and were approved by the Animal Ethics Committee of theUniversity of Western Australia (RA05/100/483). Both experiments werecarried out during the anovulatory season (July to December in the southernhemisphere [318 58 min south]).

Experiment 1

During August, adult multiparous female Merino sheep (n ¼ 20) that hadbeen previously isolated from males (i.e., .500 m away from males for aminimum of 2 mo) were habituated to adult male Merino sheep (n ¼ 4) atpasture for approximately 3 mo. Two weeks before the experiment, all animalswere moved into a facility at the University of Western Australia, Perth, andwere allocated to one of the following two groups: familiar (n¼ 10) or novel (n¼ 10), balanced for age (mean 6 SEM, 2.7 6 0.3 y for familiar and 2.6 6 0.2y for novel) and live weight (55.0 6 2.2 kg for familiar and 53.0 6 1.9 kg fornovel). The female sheep were maintained in group pens under natural light(12.5L:11.5D), and each animal received a daily ration of 150 g of lupin grain,750 g of rough-cut chaff, and 25 g of minerals. Progesterone pessaries (CIDR;Pacific Vet, Cheltenham, VIC, Australia) were inserted 11 days before the dayof male exchange (Day 0). Progesterone does not block the neuroendocrineresponse of female sheep to males [14] but ensures a consistent endocrinemilieu within which to monitor LH secretion. The mean 6 SEM concentrationsof progesterone on Day 0 were 1.30 6 0.14 ng/ml (familiar) and 1.20 6 0.09ng/ml (novel). On Day 0, blood was sampled every 15 min from 6 h before to 6h after male exchange to study the effect of male exchange on LH secretion(and thus GnRH activity). At male exchange, the two familiar males wereremoved for 15 min and returned (familiar) or replaced with two novel males(novel).

Experiment 2

Animals and experimental procedures. During November, adultmultiparous female Merino sheep (n ¼ 12) that had been previously isolatedfrom males (i.e., .500 m away from males for a minimum of 2 mo) wereallocated to one of the following two groups: male exposed (n¼ 6) and control(n¼ 6), with each group balanced for age (6 y) and live weight (mean 6 SEM,54.0 6 2.1 vs. 53.0 6 2.1 kg). Two weeks before the experiment, all animalswere moved into a facility at the University of Western Australia, Perth. Thefemales were maintained in group pens under the same regimen for lighting andnutrition as that described for experiment 1. Blood was sampled twice weeklyfor progesterone for 2 wk before the start of the experiment to confirm theanovulatory status of the females. On the day of bromodeoxyuridine (BrdU)injection (Day 0), blood was sampled every 12 min from 6 h before to 6 h afterBrdU injection, to monitor changes in pulsatile LH secretion.

BrdU labeling. BrdU is an analogue of thymidine and incorporates into theDNA of dividing cells during the S phase of the cell cycle. The population ofcells susceptible to BrdU labeling is dictated by many aspects of theexperimental protocol, particularly the dose of BrdU and the time of injectionrelative to stimulus exposure and to euthanasia [15, 16]. In this study, BrdU(100 mg/kg in 0.9% saline) was injected intravenously into the female sheepimmediately before male exposure and at a parallel time point in the controlgroup. BrdU had not previously been used in adult sheep, so the dose wasselected based on a rodent study [17]. The short half-life of 2 h [18], incombination with injection immediately before male exposure, ensures that theonly cells labeled with BrdU were those proliferating within hours of maleexposure. Approximately 48 h after BrdU injection, all females wereheparinized (25 000 IU i.v.), killed (100 mg/kg of sodium pentobarbitonei.v.), and swiftly decapitated, and the heads were perfused with 2 L of 0.9%saline and 3 L of 4% paraformaldehyde (pH 7.4). The 48-h time point wasselected because newborn cells are typically small, irregular in shape, anddistributed in clusters and thus are easier to quantify accurately several daysafter BrdU injection [15]. Blocks of tissue containing the hippocampus andhypothalamus were dissected out and stored in 4% paraformaldehyde for afurther 24 h (48C) followed by 24–48 h in 15% sucrose (48C). We also intendedto study the SVZ and rostral migratory stream, but for technical reasons it wasnot feasible to collect these regions for the present study. The blocks of tissuewere then embedded (OCT; ProSciTech, Thuringowa, QLD, Australia) andstored at�808C. A sample of small intestine was also removed and processed

as already described to act as a positive control. Coronal sections of thehippocampus, hypothalamus, and small intestine were cut at 20 lm on acryostat (CM3050; Leica, Wetzlar, Germany), mounted onto charged slides,and stored at �808C.

Immunohistochemistry and BrdU-positive quantification. One in 10sections (20 lm at 200-lm intervals) through the hippocampus was processedfor fluorescence immunohistochemistry to detect BrdU-immunoreactive (BrdUpositive) cells. Briefly, on Day 1 of the protocol, sections were air dried,rehydrated (0.1 M PBS [pH 7.4]), and incubated in 50% hydrochloric aciddiluted in 1% Triton X-100 for 20 min to denature the DNA. They were thenrinsed (0.1 M PBS) and incubated overnight (48C) with anti-mouse BrdU(1:2000; Chemicon International, Temecula, CA) diluted in 0.2% Triton X-0.1M PBS. Sections were double labeled with neuron-specific enolase (1:500;Chemicon International) for identification and measurement of relevantstructures. On Day 2, sections were rinsed (0.1 M PBS) and incubated (2 h)with Alexa 546 anti-mouse and Alexa 488 anti-rabbit (both 1:400; Invitrogen,San Diego, CA) diluted in 0.2% Triton X-0.1 M PBS. They were then rinsed(0.1 M PBS) and stained with 0.3% Sudan Black B for 2–3 min to removelipofuscin autofluorescence [19]. Sections of small intestine were included ineach run to validate the BrdU technique for sheep. BrdU-positive cells werevisualized at 403 magnification on an Axioskop II fluorescent microscope andphotographed using an Axiocam digital camera (Carl Zeiss, Thornburg, NY).BrdU-positive cells in the hippocampus were counted in the dentate gyrus andhippocampus proper by an operator blind to the identity and treatment group ofthe animals. The area of each structure was calculated using photographs ofeach section (e.g., see Figure 3d) and an image analysis program (ImageJ 1.38;http://rsb.info.nih.gov/ij/download.html). The numbers of BrdU-positive cellswere then adjusted for the area of each structure to give BrdU-positive cells permm�2. The numbers of BrdU cells per mm�2 in each structure were normallydistributed per Anderson Darling test (Minitab 13.1; Minitab Ltd., Coventry,England) and were thus compared between male-exposed and control ewesusing one-way ANOVA (GENSTAT 5 for Windows, second edition;Numerical Algorithms Group, Oxford, England). Three representative sectionsof the anterior, medial, and posterior ventromedial hypothalamus of eachanimal were also processed for BrdU immunohistochemistry as alreadydescribed. No BrdU-positive cells were observed in the ventromedialhypothalamus, so we processed one in 20 sections (20 lm at 400-lm intervals)through the entire hypothalamic block (mammillary bodies to diagonal band ofbroca [20]) of one control female and one male-exposed female. However, wefound no BrdU-positive cells in any structures within the hypothalamus.

General Studies for Experiments 1 and 2

Radioimmunoassay. Plasma LH was measured in duplicate by a double-antibody radioimmunoassay [21] using ovine LH (NIDDK-oLH-1–4; AFP-8614B) for iodination and standards kindly supplied by A. Parlow, NationalHormone and Pituitary Program, National Institute of Diabetes and Digestiveand Kidney Diseases, Torrance, CA. The limit of detection was 0.06 ng/ml(experiments 1 and 2). Quality control samples for experiment 1 (0.41, 0.74,and 1.83 ng/ml) and experiment 2 (0.60, 1.11, and 2.05 ng/ml) were used toestimate intraassay coefficients of variation (13.8%, 5.3%, and 5.9% forexperiment 1 and 6.6%, 5.1%, and 8.2% for experiment 2). Plasmaprogesterone was measured in duplicate using an active progesteroneradioimmunoassay kit (Diagnostic Systems Laboratories Inc., Webster, TX)as previously described [22]. The limit of detection of the assay was 0.1 ng/ml.Quality control samples (1.40 and 9.43 ng/ml for experiment 1 and 1.21 and8.95 ng/ml for experiment 2) were used to estimate intraassay coefficients ofvariation (4.4% and 9.43% for experiment 1 and 5.6% and 7.8% for experiment2). Plasma estradiol was measured in duplicate using an Adaltis MAIAestradiol radioimmunoassay kit (Diagnostic Technology, Belrose, NSW,Australia). The limit of detection was 0.4 pg/ml. Quality control samples(2.73 and 8.28 pg/ml) were used to estimate intraassay coefficients of variation(3.0% and 2.0%).

LH data analysis. Luteinizing hormone pulses were detected using theMunro algorithm, a modified version of the Pulsar algorithm [23]. Data for LHpulse frequency and mean concentrations of LH before and after male exchange(experiment 1) or male exposure (experiment 2) were subjected to repeated-measures ANOVA in GENSTAT 5 for Windows. Progesterone concentrationson Day 0 were included in the repeated-measures analysis as a covariate buthad no effect on any parameters of LH secretion in either experiment (P . 0.1).If a significant effect of male exchange or exposure was detected, data beforeand after male exchange (experiment 1) or male exposure (experiment 2) werecompared within treatment by paired t-test or between treatments by Student t-test (GENSTAT 5 for Windows for both comparisons). Before analysis, thenormality of the data was assessed using Anderson Darling test for normality(Minitab 13.1), and log10 transformation was used to overcome skewness inthe mean concentration of LH and progesterone (experiments 1 and 2).

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RESULTS

Experiment 1

The differences between the neuroendocrine responses to

novel vs. familiar males are shown in Figure 1. Novel maleswere clearly able to elicit an immediate robust change in the

pulsatile pattern of LH secretion (Fig. 1, b and c), whereasreintroduction of familiar males had no detectable effect (Fig.1, a and c). Detailed quantitative analysis of the data confirms

this, with novel males inducing a highly significant (P ,

0.001) increase in LH pulse frequency (the mean concentra-tions of LH are given in Table 1). In contrast, reintroduction of

familiar males did not affect (P . 0.1) any of the threevariables (Table 1).

Experiment 2

Neuroendocrine response. The neuroendocrine responsesof female sheep to novel males are shown in Figure 2. Noteagain the immediate robust increase (P , 0.001) in thesecretion of LH (Fig. 2a) that was accompanied by increased (P, 0.01) circulating concentrations of estradiol-17b (Fig. 2b).Detailed quantitative analysis of the data confirms this, withnovel males inducing highly significant (P , 0.001) increasesin LH pulse frequency and thus in the mean concentrations ofLH (Table 1). Plasma estradiol concentrations increased from amean 6 SEM of 0.58 6 0.14 to 0.98 6 0.15 pg/ml (Fig. 2b).In contrast, in the females that remained isolated from males,there were no significant changes in LH secretion (Table 1) orestradiol concentration (mean 6 SEM, 0.55 6 0.20 vs. 0.60 60.20 pg/ml) (Fig. 2b).

BrdU-labeled cells. Exposure to males doubled the numberof BrdU-positive cells in the dentate gyrus of the hippocampusfrom a mean 6 SEM of 4.68 6 1.19 to 9.60 6 1.09 cellsmm�2 (P , 0.05). Representative images of BrdU-positivecells located in this region are shown in Figure 3. There was aparallel trend in the hippocampus proper (mean 6 SEM, 0.786 0.18 vs. 1.68 6 0.18 cells mm�2), but the difference justfailed to reach significance (P ¼ 0.06). In contrast to thehippocampus, there was no evidence of cell proliferation in theventromedial region or any other region of the hypothalamus.

DISCUSSION

The systems that control the reproductive centers in thebrain of female sheep and ultimately control the secretion ofGnRH are activated by exposure to unfamiliar (novel) males,and this response is associated with increased cell proliferation,perhaps neurogenesis, in the dentate gyrus of the hippocampus,a key structure involved in learning and memory [24]. Thefollowing two aspects of the increase in hippocampal cellproliferation are markedly different from that recently reportedfor mice [12]. First the response is very robust, effectivelydoubling the constitutive rate of cell proliferation. Second, theresponse is very rapid, suggesting that if these cells areinvolved in the formation of a memory of the new male, thisprocess occurs within only a few hours of initial exposure.

The differences between rodents and ungulates in hippo-campal cell proliferation might be attributed to differences inthe biological action of pheromones in these animals. In adultfemale mice, male pheromones do not directly induceovulation but subtly alter the hormonal patterns that controlthe cycle so that there is a synchronization of proestrus among

FIG. 1. Representative profiles of LH secretion in female sheep whosefamiliar males were removed for 15 min and returned (a) or replaced withnovel males (b). c) Mean 6 SEM concentrations of LH in female sheepwhose familiar males were removed for 15 min and returned (graydiamonds) or replaced with novel males (black diamonds). The malesymbol and associated arrow represent the time of male exchange.

TABLE 1. Mean characteristics of LH secretion (6 SEM) in female sheepreintroduced to familiar or novel males, or isolated from or introduced tonovel males midway through a blood sampling regimen.

Experiment typeMale

exchange

LH pulsefrequency(pulses/h)

Meanconcentration

(ng/ml)

Experiment 1Familiar Before 0.26 6 0.04 0.17 6 0.03

After 0.16 6 0.06 0.17 6 0.04Novel Before 0.35 6 0.04 0.23 6 0.05

After 0.87 6 0.06* 0.60 6 0.10*Experiment 2

Control Before 0.39 6 0.15 0.27 6 0.07After 0.17 6 0.43 0.15 6 0.02

Male–exposed Before 0.19 6 0.11 0.32 6 0.06After 1.14 6 0.12* 1.34 6 0.10*

* Values differ within treatment (P , 0.001).

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females. Two key hormones involved in this response, pituitaryLH and prolactin, are thought to elicit the increase inneurogenesis [12]. Similarly, in prairie voles, exposure tomales raises the concentration of estrogen and alters theexpression of behavioral estrus [13]. In each case, theresponses are not observed until after days of intense exposureto male pheromones [12, 13]. In sheep, the concentrations ofLH and estradiol increase within minutes of exposure to themale stimulus. It seems likely, therefore, that differences in thetiming of the hippocampal response to the male stimulus arerelated to species-specific differences in the neural andneuroendocrine response to pheromonal signals. However,the short half-life of BrdU [18] means that the frequency, dose,and timing of injection, relative to the time of tissue sampling,dictate the populations of cells susceptible to BrdU labeling.Previous studies [12, 13, 25, 26] in rodents focused theirinjection regimen several days after exposure to malepheromones, and this strategy may hide a more immediateand robust effect of pheromones on neurogenesis in thesespecies.

To our knowledge, this study is the first to reportconstitutive proliferation of cells in the dentate gyrus of thehippocampus of adult sheep, at a rate of about 2500 cells perday. The increase in cell proliferation induced by exposure offemale sheep to novel males is associated with a dramaticchange in the endocrine milieu. From observations in rodents,

it would seem that this phenomenon may be driven by changesin one or more reproductive hormones [12, 13, 25, 26].However, in contrast to rodents, exposure to males has littleeffect on the concentration of prolactin in sheep [27]. Rather,male exposure increases GnRH/LH pulse frequency withinminutes [21], and each LH pulse induces a pulse of estradiol[28]. The concentrations of follicle-stimulating hormone do notincrease until the preovulatory surge [29]. Therefore, LH andestradiol are potential drivers of male-induced cell proliferationin the hippocampus of female sheep. This hypothesis is yet tobe tested, and a role for LH is potentially problematical becausethis large glycoprotein molecule seems unlikely to cross theblood-brain barrier. However, there is evidence of LH receptorsin the hippocampus of rodents [12, 30], and LH is thought tohave a role in Alzheimer disease in humans [31] and thus mayhave a role in the brain of ungulates.

In contrast to the hippocampus, we did not detect anyproliferating cells in the hypothalamus of female sheep. This isperhaps surprising, as neural activity in the hypothalamusdrives the rapid neuroendocrine response of female sheep tomales and it is a key site of pheromone-induced neurogenesisin female prairie voles [13]. However, our injection regimentargeted only cells proliferating during the first few hours ofmale exposure, and it might be that cell proliferation in thisregion is associated with the preovulatory surge and/or

FIG. 2. Mean 6 SEM of LH (a) andestradiol (b) of female sheep exposed tomales (black diamonds) or isolated frommales (gray diamonds). Mean concentra-tions are significantly different from thosebefore male exposure (**P , 0.01 and ***P, 0.001). The male symbol and associatedarrow represent the time of male exposure.

FIG. 3. Images of BrdU-positive cells inthe dentate gyrus of the hippocampus (a andb) and small intestine (c). d) Compositeimage of the hippocampus used to calculatethe area of the dentate gyrus (solid line) andthe hippocampus proper (area bound bysolid line).

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ovulation, processes that occur 1–3 days after initial maleexposure [6]. This hypothesis requires further investigation.

In mice, prolactin seems to drive pheromone-inducedneurogenesis in the SVZ [12]. We did not study this regionbut may include it in future studies for two reasons. First, adultneurogenesis has been reported in the SVZ of many species,and cells born in this region migrate along the rostral migratorystream to become integrated into the functional circuitry of theolfactory bulb [15]. Second, olfactory stimuli are a vitalcomponent of sociosexual communication in sheep [5, 32].However, in sheep, prolactin seems to have no role in male-induced ovulation [27]; if it occurs, male-induced cellproliferation in the SVZ of female sheep is unlikely to bedriven by prolactin.

The ability of novel but not familiar males to induce aneuroendocrine response in female sheep suggests that thebrain centers controlling reproduction respond differentially toindividual males, at least in terms of their social familiarity. Inmice, volatile compounds in male urine encode specificinformation that enables females to distinguish betweenindividual males [33]. In sheep, the male pheromone seemsto be a mix of long-chain fatty acids [34] that might besufficiently complex to encode the identity of individual males[5]. However, visual stimuli are also important for socialcommunication in sheep [35–38], and we recently found thatprojected images of male sheep elicit a small but significantneuroendocrine response in female sheep [39]. Therefore,further studies are required to elucidate the mechanism bywhich female sheep can discriminate between novel andfamiliar males.

In conclusion, there is constitutive proliferation of cells inthe hippocampus but not the hypothalamus of female sheep.Cell proliferation in the hippocampus increases rapidly inassociation with an increase in the neuroendocrine activity ofthe brain centers that control reproduction following theintroduction of unfamiliar (novel) males. We propose that thisphenomenon is driven by rapid increases in LH and/or estradiolsecretion within minutes of exposure to males and that the newcells may be involved in forming memories of individual malesthat dictate the responsiveness of females to future male-femaleinteractions.

ACKNOWLEDGMENTS

We would like to acknowledge the facilities and scientific andtechnical assistance of the Australian Microscopy and MicroanalysisResearch Facility at the Centre for Microscopy, Characterisation andAnalysis, University of Western Australia, a facility funded by theuniversity, state, and commonwealth governments. We also thank M.Blackberry for her assistance with the LH assays and C. Blyth, L. Host, andS. Seeber for their assistance in data collection.

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