Increased dopamine after mating impairs olfaction andprevents odor interference with pregnancy

Che Serguera, Viviana Triaca, Jakki Kelly-Barrett, Mumna Al Banchaabouchi & Liliana Minichiello

In rodents, social odor sensing influences female reproductive status by affecting neuroendocrine cascades. The odor of male

mouse urine can induce ovulation or block pregnancy within 3 d post coitus. Females avoid the action of such olfactory stimuli

after embryonic implantation. The mechanisms underlying these changes are unknown. Here we report that shortly after mating,

a surge in dopamine in the mouse main olfactory bulb impairs the perception of social odors contained in male urine. Treatment

of females at 6.5 d post coitus with a dopamine D2 receptor antagonist restores social odor sensing and favors disruption of

pregnancy by inhibition of prolactin release, when administered in the presence of alien male urine odors. These results show

that an active sensory barrier blocks social olfactory cues detrimental to pregnancy, consistent with the main olfactory bulb

being a major relay through which social odor modulates reproductive status.

The length of the female rodent ovarian cycle is altered by environ-

mental cues, such as urine odors. These cuesmodulate the release of the

pituitary hormones, such as luteinizing hormone and prolactin (PRL),

that are required for ovulation and pregnancy1. In rodents, the

vaginocervical stimulations produced during copulation block cycling

and trigger a biphasic day and night surge of PRL, the primary

luteotrophic factor2. The luteotrophic action of PRL until midpreg-

nancy is characterized by increased progesterone secretion, which is

essential for implantation of the fertilized ovum, maintenance of

pregnancy3 and inhibition of ovulation2,4. The scent of the urine of

an alien male, but not that of the stud male, blocks embryo implanta-

tion between days 0 and 3 of pregnancy (the Bruce effect)5 by inhibiting

PRL secretion6, which results in a wane in progesterone and a return to

ovulation. This effect is mediated by odorants conveyed through the

vomeronasal organ (VNO)7, a sensory structure located in the nasal

cavity specialized in the detection of pheromones1. In contrast, on days

4 to 6 of pregnancy (the implantation stage), females become refractory

to the Bruce effect8. At this stage, the implanted embryos are still

dependent on progesterone production by the corpus luteum3,9,10.

Thus, it remains elusive why the odor of alien male urine can induce

luteolysis before implantation capability but becomes unable to com-

promise pregnancy soon after implantation.

In the nasal cavity of rodents, the VNO and the main olfactory

epithelium (MOE) contain two sets of neuronal populations with

exclusive chemosensory functions, such as detection of pheromones by

the VNO and detection of odorants by the MOE. Recent evidence

suggests, however, that both structures are essential for pheromone-

mediated responses through specific and nonredundant mechanisms11,

which suggest a higher level of complexity in the integration of

chemical information in rodents. The MOE and the VNO project

parallel fibers that innervate the main olfactory bulb (MOB) and the

accessory olfactory bulb (AOB), respectively. Although the MOB and

the AOB share substantial cellular organization, they are characterized

by divergent projecting areas11 and other specific differences. One such

difference is the absence from the AOB of a prominent dopaminergic

neuronal population, the juxtaglomerular dopaminergic interneurons

(JGD), that is characteristic of the MOB12,13.

MOB JGD cells have been shown to be involved in processing

odorant signaling14, and dopamine is known to regulate odor dis-

crimination and some form of olfactory learning15–17. Tyrosine hydro-

xylase, the rate-limiting enzyme in the synthesis of dopamine18, is

modulated in the mouse MOB by estrogen19, and sex hormones and

vaginocervical stimulations have been shown to modulate tyrosine

hydroxylase synthesis and dopamine release in several nuclei of the

hypothalamus, the limbic system and the midbrain20–22. Given these

previous findings, we asked whether JGD cells influence social

odor perception after mating and whether this, in turn, protects

pregnancy. Here we show that shortly after coitus, tyrosine hydroxylase

increases in femalemouseMOB JGD cells and reaches a plateau at 3.5 d

post coitus (dpc), which coincides with the end of the Bruce effect.

This rise in tyrosine hydroxylase expression is associated with an

impairment in social odor discrimination. Treatment of females at

6.5 dpc with a dopamine D2 receptor (D2R) antagonist restores

olfactory perception abilities and allows alien male urine odors to

disrupt the pregnancy by inhibiting PRL secretion. Our results uncover

an important function of JGD cells in the modulation of social odors

perception detrimental to pregnancy. In addition, these results

strengthen recent evidence and emphasize the role of the MOB in

gating stimuli that affect female reproductive status11 together with the

vomeronasal system1,11.

Received 1 April; accepted 3 June; published online 20 July 2008; doi:10.1038/nn.2154

European Molecular Biology Laboratory, Mouse Biology Unit, Via Ramarini 32, 00015 Monterotondo, Italy. Correspondence should be addressed to L.M.

(minichiello@embl.it).

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RESULTS

Expression of tyrosine hydroxylase during pregnancy

To determine whether tyrosine hydroxylase is modulated in JGD cells

of the mouse MOB during pseudopregnancy, C57BL/6J females were

mated to vasectomized males and killed at various time points. As early

as 0.5 dpc, the pseudopregnant females hadmore tyrosine hydroxylase–

positive JGD cells in the periglomerular area of the MOB than did

females in estrous (day 0; Fig. 1a). By 4.5 dpc, the number of tyrosine

hydroxylase–positive JGD cells plateaued at 40% more than day 0

(Fig. 1a). The plateau lasted until 8.5 dpc and returned to control value

by 12.5 dpc (end of pseudopregnancy; Fig. 1). Consistent with previous

reports12,13, tyrosine hydroxylase–expressing cells were not found in the

AOB at any time point analyzed (data not shown), indicating that

dopaminergic cells are absent from this region. Females exposed to

male bedding overnight and killed 4.5 d later showed control numbers

of tyrosine hydroxylase–positive cells (22 ± 8; n ¼ 3) in the MOB,

indicating that the increase in tyrosine hydroxylase in the female MOB

is linked to coitus and is not dependent on male odors per se.

We next investigated tyrosine hydroxylase modulation in JGD cells

during pregnancy in other mouse strains. Tyrosine hydroxylase

increased in females of both C57BL/6J and BALB/cJ strains after mating

(Fig. 2a), reaching a plateau at 3.5 dpc in both strains. A similar pattern

was observed with tyrosine hydroxylase immunostaining intensity

(Fig. 2b,c). Given that these two inbred mouse strains behaved

similarly with respect to postcoital tyrosine hydroxylase levels in the

MOB and that the same trend has been described in the ferret, a

reflex ovulator23, this effect might represent a more general postcoital

phenomenon shared by other mammals independent of how

they ovulate.

To examine whether increases in tyrosine hydroxylase alter dopa-

mine levels, we measured dopamine in the MOB of pregnant C57BL/6J

females and found a significant elevation starting at 2.5 dpc compared

to females in estrous (Fig. 2d). Moreover, females in estrous exposed

for 3 d to CD1 male bedding showed dopamine levels equivalent to

C57BL/6J* *

90

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7 8 9 10 11 12 130

a

b

Figure 1 Kinetics of tyrosine hydroxylase (TH) expression in the MOB of

pseudopregnant females. (a) Numbers of TH-positive cells in pseudopregnant

females, as assessed by immunohistochemistry, increased by 40% in the

periglomerular layer of the MOB after mating. A plateau was reached within

4.5 dpc (ANOVA F4 ¼ 54.5, *Po 0.0001). TH levels returned to estrous

(day 0) values at the end of pseudopregnancy (12.5 dpc). n ¼ 3 for each

time point. (b) MOB sections stained with antibody to TH at various stages of

pseudopregnancy. Intensity of TH staining increased from 0.5 to 8.5 dpc and

decreased at the end of pseudopregnancy. Scale bar ¼ 100 mm.

100 300

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0Num

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ng p

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BALB/cJ C57BL/6J

80

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0 1.5 2.5 3.5 0 1.5 2.5 3.5

Time (dpc) Time (dpc)

Time (dpc)

0

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0 1.5 2.5 3.5C57BL/6J

C57BL/6J

C57B/6J

0 0+ Alien male bedding

Time (dpc)

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***

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******

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dFigure 2 Tyrosine hydroxylase (TH) modulation in JGD cells of two mouse strains during pregnancy.

(a) In pregnant females of either strain, numbers of TH-positive cells increased during the first

4 d after mating (ANOVA F3 ¼ 278.7, Po 0.0001 for C57BL/6J; F3 ¼ 62, Po 0.0001 for

BALB/cJ). Comparisons of C57BL/6J mice: ***P ¼ 0.0008 for 1.5 dpc versus 0, ***Po 0.0001

for 2.5 dpc versus 0, ***Po 0.0001 for 3.5 dpc versus 0. Comparisons of BALB/cJ mice:

**P ¼ 0.001 for 1.5 dpc versus 0, ***Po 0.0001 for 2.5 dpc versus 0, ***Po 0.0001 for

3.5 dpc versus 0. n ¼ 3 per time point. (b) Intensity of glomerular TH staining, expressed as

relative density (ANOVA F3 ¼ 22, Po 0.0001 for C57BL/6J; F3 ¼ 21.6, Po 0.0001 for

BALB/cJ). Comparisons of C57BL/6J mice: **P ¼ 0.005 for 1.5 dpc versus 0, ***Po 0.0001

for 2.5 dpc versus 0, ***Po 0.0001 for 3.5 dpc versus 0. Comparisons of BALB/cJ mice:

***Po 0.0001 for 1.5 dpc versus 0, ***Po 0.0001 for 2.5 dpc versus 0, ***Po 0.0001 for

3.5 dpc versus 0. (c) TH-labeled MOB sections show increased staining intensity from estrous

(day 0) to 3.5 dpc in both strains. Scale bar ¼ 100 mm. (d) Dopamine expression in MOB of virgin

C57BL/6J females in estrous (day 0), virgin females in estrous housed for 3 d with CD1 male bedding, and pregnant females at 1.5, 2.5 and 3.5 dpc (ANOVA

F4 ¼ 12, Po 0.0001). Comparisons: **P ¼ 0.001 for 2.5 dpc versus 0, ***Po 0.0001 for 3.5 dpc versus 0. n ¼ 6 bulbs per group.

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those of unexposed females in estrous. This result indicates that the

postcoital tyrosine hydroxylase increase in female mouse MOB is

translated into a local elevation in dopamine.

Increased tyrosine hydroxylase impairs odor perception

JGD cells in the MOB modulate sensory input from olfactory sensory

neurons (OSNs) to mitral cells (the output neurons of the MOB)

through D2R-mediated presynaptic inhibition14. Functionally, JGD

cells modulate the threshold of odor detection and the discrimination

between odors. D2R antagonists increase odor discrimination and

reduce odor detection threshold, whereas D2R agonists have an

opposite effect24. We examined whether the increase in tyrosine

hydroxylase expression in the MOB during pregnancy is associated

with an impairment of social odorant perception.

The interest of pregnant females in male or female urine odors was

observed at 0.5 and 6.5 dpc, stages at which tyrosine hydroxylase levels

in the MOB are respectively low and high (Fig. 1). Urine can be

separated into two fractions: the low-molecular-weight (LMW) frac-

tion, which causes OSN- and MOB-dependent exploratory behavior

for which the VNO is dispensable25,26; and the high-molecular-weight

(HMW) fraction, the exploration of which requires an intact VNO25.

Female mice prefer to explore male over female urine odors of either

fraction25. Accordingly, at 0.5 dpc, females spent significantly more

time sniffing male urine of either fraction (Fig. 3a,b). At 6.5 dpc,

females explored the HMW fraction of male

urine more than that of females (Fig. 3c) but

were no longer interested in the LMW frac-

tion of male urine (Fig. 3d), indicating that

the processing of inputs through the MOB,

but not the AOB, is altered during pregnancy.

Because tyrosine hydroxylase levels in the

JGD cells at 6.5 dpc were high (Fig. 1a), which

is linked to increased levels of dopamine

(Fig. 2d), we hypothesized that the lack of

social odor discrimination at this stage of

pregnancy is caused by increased dopamine

levels in the MOB. To verify this hypothesis,

we treated pregnant females with the D2R

antagonist spiperone. In rats, spiperone has

been used to increase olfactory discrimination

at 0.6 mg per kg of body weight17. This dose

in mice caused pronounced cataleptic

motor side effects that impaired exploratory

behavior. A lower dosage (0.03 mg per kg

intraperitoneally) did not cause motor side effects, and instead allowed

exploratory behavior and restored in pregnant mice the interest for the

LMWmale urine fraction when administered at 6.5 dpc (Fig. 3e).

To determine whether increased dopamine in the MOB can modify

olfactory sensitivity, we measured the threshold of odor detection

during early gestation. C57BL/6J females, either in estrous or at

6.5 dpc, were given the choice to explore cotton buds soaked with

water or increasing concentrations of the LMW fraction of CD1 male

urine. Females in estrous spent the same amount of time smelling water

and urine, up to a 10–3 dilution of urine (Fig. 4a). In contrast,

females at 6.5 dpc needed at least a tenfold greater concentration

(10–2 dilution) of urine to start exploring (Fig. 4b). Spiperone (0.03 mg

per kg) restored and even improved odor detection in pregnant females

to a lower threshold (10–4 dilution of urine) compared to females

in estrous (Fig. 4c). Moreover, spiperone did not affect tyrosine

hydroxylase levels in the MOB of pregnant females at 4.5 dpc (Supple-

mentary Fig. 1 online), although it did antagonize the action of

dopamine. Because the MOB receives no centrifugal dopaminergic

innervations, the effect of spiperone on olfactory perception is likely to

have been induced by attenuating dopaminergic inhibition of

OSNs. These results indicate that olfaction is altered in pregnant

mice because of a local increase in dopamine in the MOB. The

findings also strongly suggest that the lack of preference for LMW

male urine at 6.5 dpc is a consequence of lower discrimination

30

25

20

15

10

Seconds s

pent

sniffing

HMW urine fraction, 0.5 dpc (n = 9)

***

5

0

HMW urine fraction, 6.5 dpc (n = 13)

**

30

25

20

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pent

sniffing

Seconds s

pent

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5

0

LMW urine fraction, 6.5 dpc (n = 13)

30

25

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pent

sniffing

5

0

LMW urine fraction, 6.5 dpc + spiperone (n = 13)

30

25

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pent

sniffing

5

0

***

LMW urine fraction, 0.5 dpc (n = 9)

30

25

20

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10

5

0

a b c d e

*

Figure 3 Progression of pregnancy is associated with loss of social olfactory discrimination. (a�d) Time spent by females at 0.5 dpc (a,b) and 6.5 dpc (c,d)

sniffing HMW (a,c) or LMW (b,d) odor cues of urine of fertile males or females in estrous. (a), ***P ¼ 0.0006; (b), ***P ¼ 0.0002; (c), **P ¼ 0.002;

(d), P ¼ 0.2 for male versus female urine. (e) Females at 6.5 dpc treated with spiperone 15 min before the social odor discrimination test gained interest

for the LMW male urine fraction (*P ¼ 0.01). Male and female urine fractions are indicated by gender symbols. n ¼ number of females analyzed per group.

C57BL/6 females in estrous C57BL/6 females at 6.5 dpc C57BL/6 females at 6.5 dpc + spiperone

Urine dilutions 10–7

10–5

10–4

10–3

10–2

**

10–5

10–4

10–5

10–4

10–3

10–2

Ratio o

f tim

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pent sniffing

uri

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us w

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r

3.0

2.0 2.0

1.0

0

3.0

2.0

1.0

0

3.0

1.0

0

***

a b c

Figure 4 Threshold of detection of LMW male urine fraction by C57BL/6J estrous or pregnant females.

Shown are ratios of seconds spent sniffing increasing concentrations of LMW male urine fraction to

seconds spent sniffing water. (a) Females in estrous (n ¼ 5) started detecting the LMW male urine

fraction at 10–3 dilution (ANOVA F4 ¼ 3.4, P ¼ 0.02; *P ¼ 0.04 for 10–3 versus 10–5 or 10–7).

(b) Females at 6.5 dpc (n ¼ 6) needed at least tenfold (10–2) more concentrated solution to start

exploring the LMW male urine fraction (ANOVA F3 ¼ 1.8, P ¼ 0.2; P ¼ 0.07 for 10–2 versus 10–3).

(c) Females at 6.5 dpc treated with spiperone (0.03 mg per kg; n ¼ 5) detected the LMW male urine

fraction at 10–4 dilution (ANOVA F1 ¼ 31, P ¼ 0.0005; ***P ¼ 0.0005 for 10–4 versus 10–5).

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and sensitivity to odors rather than of pregnancy-related motivational

changes or habituation to male urine odors.

Pregnancy reduces neuronal activation in the MOB

We next examined whether increased presynaptic inhibition of OSNs

by dopamine hampers neuronal activation in theMOB. To that end, we

evaluated neuronal activation through c-Fos immunoreactivity in the

MOB of C57BL/6J females, either in estrous or pregnant (4.5 dpc),

stimulated or not with CD1 male bedding and treated or not treated

with spiperone. As expected, c-Fos immunoreactivity was significantly

higher in the glomerular, mitral and granular MOB layers of females in

estrous stimulated withmale bedding (Fig. 5a–d). Notably, the number

of c-Fos–immunoreactive neurons in the MOB layers of 4.5-dpc

females stimulated with male bedding was significantly lower than

that of stimulated estrous females. However, in females at 4.5 dpc

treated with spiperone and stimulated with male bedding, c-Fos

immunoreactivity was restored to levels similar to those of females in

estrous stimulated with male bedding. In contrast, spiperone did not

modify c-Fos immunoreactivity in theMOB of unstimulated females in

estrous. These results indicate that neuronal activation is significantly

reduced in all MOB layers of pregnant mice as a consequence of local

increases in dopamine acting through D2R activation. It seems that a

sensory barrier occurs at early stages of pregnancy in the mouse MOB,

presumably linked to the process of pregnancy itself.

Mitral cells of the MOB project to several cortical structures and

limbic nuclei, known as the olfactory cortex and the olfactory amyg-

dala, that in turn extend projections to many other areas of the brain,

including hypothalamic structures that regulate sexual behavior and

reproductive neuroendocrine cascades27–30. We examined whether

decreased neuronal activation in the MOB during pregnancy would

equally affect c-Fos immunoreactivity in the main brain targets of the

MOB in the presence of olfactory cues. c-Fos immunoreactivity was

significantly higher in the anterior olfactory nucleus of C57BL/6J

females in estrous stimulated with CD1 male bedding compared to

control mice, but remained at low levels in pregnant females (4.5 dpc)

stimulated with CD1 male bedding (Fig. 5e). However, in females at

4.5-dpc treated with spiperone and stimulated with CD1male bedding,

c-Fos immunoreactivity was restored to levels similar to that of females

in estrous stimulated with CD1 male bedding (Fig. 5e). This is

consistent with the observation that the MOB hardly responds to

urine odorants at this stage of pregnancy, thus allowing limited output.

Moreover, spiperone in the absence of male urine odorants did not

affect c-Fos immunoreactivity in the anterior olfactory cortex of

females in estrous (Fig. 5e), indicating that c-Fos immunoreactivity

in those mice was induced by odors. In the arcuate nucleus, a structure

pivotal in regulating neuroendocrine cascades associated with cycling

and pregnancy1,4,27, CD1 male odors did not induce significant c-Fos

immunoreactivity in females in estrous or pregnant mice (Fig. 5f), as

observed previously31. c-Fos immunoreactivity in the arcuate nucleus

increased significantly in females at 4.5-dpc treated with spiperone and

stimulated with CD1 male bedding, whereas the drug alone had no

effect on c-Fos immunoreactivity in females in estrous.

The mitral cells of the AOB, in contrast to those of the MOB, avoid

cortical structures and instead send projections to nuclei of the limbic

Figure 5 c-Fos immunoreactivity (c-Fos–ir) is a

marker of neuronal activation. (a�c) Bars from

left to right indicate average number of c-Fos–

positive cells in C57BL/6J females in estrous not

exposed to male bedding (1), in estrous

stimulated with CD1 male bedding (2), in estrous

stimulated with CD1 male bedding and treated

with spiperone (3), pregnant (4.5 dpc) and

stimulated with CD1 male bedding (4) and

4.5 dpc stimulated with CD1 male bedding and

treated with spiperone (5). (a), Glomerular layer of

the MOB (ANOVA F4 ¼ 42.3, Po 0.0001).

(b), Mitral layer (ANOVA F4 ¼ 28, Po 0.0001).

(c), Granular layer (ANOVA F4 ¼ 37, Po

0.0001). Comparisons of stimulated versus

unstimulated: (a), ***Po 0.0001; (b), ***P ¼

0.0002; (c), ***Po 0.0001. Comparisons of

estrous stimulated versus 4.5 dpc stimulated:

(a), **P ¼ 0.002; (b), **P ¼ 0.005;

(c), ***Po 0.0001. Comparisons of 4.5 dpc

treated with spiperone and stimulated versus

estrous stimulated: (a), P ¼ 0.3; (b), *P ¼ 0.05;

(c), *P ¼ 0.04. NS, not significant. (d) Repre-

sentative c-Fos–labeled MOB sections showing

part of the granular (gr) and mitral (mi) layers of

groups 1–5. Scale bar ¼ 100 mm. (e�g) Average

numbers of c-Fos–positive cells in cortical and

hypothalamic nuclei of groups 1–5. (e), Anterior

olfactory nucleus (ANOVA F4 ¼ 13, P ¼ 0.0006).

**P ¼ 0.002 for estrous stimulated versus

estrous unstimulated; **P ¼ 0.003 for estrous

stimulated versus 4.5 dpc stimulated; P ¼ 0.5 for

4.5 dpc treated with spiperone and stimulated versus estrous stimulated; P ¼ 0.7 for estrous unstimulated versus estrous unstimulated and treated with

spiperone. (f), Arcuate nucleus (ANOVA F4 ¼ 44, Po 0.0001). ***Po 0.0001 for 4.5 dpc treated with spiperone and stimulated versus estrous or 4.5 dpc

stimulated; P ¼ 0.2 for estrous unstimulated versus estrous unstimulated and treated with spiperone. (g), Ventromedial hypothalamus (ANOVA F4 ¼ 77,

Po 0.0001). ***Po 0.0001 for estrous stimulated versus estrous unstimulated; ***P ¼ 0.0002 for estrous stimulated versus 4.5 dpc stimulated;

*P ¼ 0.02 for 4.5 dpc treated with spiperone and stimulated versus estrous stimulated; P ¼ 0.2 for estrous unstimulated versus estrous unstimulated and

treated with spiperone. n ¼ 3 per group.

Glo

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MOB layers MOBns

*****

***

*** ***

*** ****

*

*****

*

Different brain regionsns

****

1 2 3 4 5 1 2 3 4 5

+ + + – –

– – – + +

– – + + +

–

Estrous

4.5 dpc

Alien male bedding

Spiperone + – – +

+ + + – –

– – – + +

– – + + +

–

Estrous

4.5 dpc

Alien male bedding

Spiperone + – – +

a

b

c

ed

f

g

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system, including the medial amygdaloid nucleus. Neurons from these

nuclei project to hypothalamic nuclei such as the ventromedial

hypothalamus11,32,33. CD1 male bedding significantly increased c-Fos

immunoreactivity in the AOB of females in estrous compared to

unstimulated females in estrous (Supplementary Fig. 2 online).

c-Fos immunoreactivity was also significantly increased by male

bedding in females at 4.5 dpc, although to a lesser extent than in

females in estrous; this effect was not significantly modulated by

spiperone (Supplementary Fig. 2). c-Fos immunoreactivity was

similarly increased in the medial amygdaloid nucleus of females in

estrous or at 4.5 dpc stimulated with CD1 male bedding, and as in the

AOB, this effect was not modulated by spiperone (Supple-

mentary Fig. 2). These results indicate that spiperone does not alter

social odor signaling through the VNO-AOB system in pregnant

females at 4.5 dpc

Notably, after stimulation with CD1 male bedding, c-Fos immuno-

reactivity was significantly lower in the ventromedial hypothalamus of

females at 4.5 dpc compared to females in estrous (Fig. 5g). Moreover,

spiperone restored c-Fos immunoreactivity in pregnant mice stimu-

lated with CD1 male bedding to levels observed in stimulated females

in estrous (Fig. 5g). In contrast, spiperone had no effect on unstimu-

lated females in estrous, indicating a direct response of the ventrome-

dial hypothalamus to MOB stimuli. These results indicate that during

pregnancy, the activation of many brain structures by social odors

undergoes changes similar to those measured in the MOB, implying

that the postcoital dopaminergic block of olfaction modifies the central

influence of social odors.

Male urine odor blocks PRL secretion after implantation

Because male odors can induce ovulation and preclude pregnancy6,34,

we hypothesized that the sensory block of olfaction experienced by a

female mouse soon after coitus is a physiological feature that protects

gestation from male odor stimuli. In rodents, both surges of PRL—the

diurnal surge that peaks around 5 p.m. and the nocturnal surge that

peaks around 3 a.m.—are necessary to maintain pregnancy, and

blocking one of the surges compromises gestation2,4,6. We thus sought

to determine whether detection of male urine odors at 7 dpc by a

pregnant female treated with spiperone would hamper the day and

night surges of PRL necessary at that stage for progesterone production

and embryonic development2–4,9,10.

We first assessed whether spiperone restores olfaction throughout

the time of both surges of PRL. C57BL/6J females at 6.5 dpc were

treated with spiperone (0.3 mg per kg) or control vehicle at 7 a.m., and

their threshold detection for the LMW male urine fraction was

measured 12 h later. Pregnant females treated with spiperone could

still detect the LMWmale urine fraction, starting at a 10–4 dilution, in

contrast to females treated with vehicle (Supplementary Fig. 3 online).

Thus, spiperone has long-term effects on olfaction.

We then tested whether spiperone affects PRL secretion at 7 dpc

when administered in the presence of alien male urine odors. The

LMWconstituents of alienmale urine are sufficient to block pregnancy,

but the HMW constituents improve their effectiveness by concentrat-

ing and fixing them30. We therefore used CD1 male soiled bedding

containing both urine fractions. C57BL/6J females were treated at 7 dpc

with spiperone (0.3 mg per kg) at 3 p.m., 2 h before the peak of the day

surge but 12 h before the peak of the night surge. Serum levels of PRL

were measured at both the day surge peak (5 p.m.) and the night surge

peak (3 a.m.). PRL levels in C57BL/6J females treated with spiperone

and housed with alien (CD1) male bedding were not affected at the

peak of the day surge compared to control females housed with CD1

male bedding, control females treated with spiperone or control

females left undisturbed (Fig. 6a). In contrast, the combination of

spiperone and alien male bedding induced a significant drop in PRL

serum concentration during the night surge compared to control

females left undisturbed, housed with CD1 male bedding or treated

with spiperone only (Fig. 6b). We confirmed pregnancy in each animal

by uterine dissection. These results indicate that spiperone alone is not

able to modify the amplitude of the PRL surges but, presumably by

improving olfactory perception, allows the alien male odors to disrupt

the night surge of PRL. Together, these findings suggest that a maternal

MOB dopaminergic block prevents environmental modulation of PRL

during pregnancy.

Olfactory dopaminergic barrier protects pregnancy

Given the above results, we determined whether restoring olfaction

at 7 and 8 dpc would compromise pregnancy in the presence of

alien male bedding containing both the LMW and HMW urine

fractions. C57BL/6J and BALB/cJ females were treated with spiperone

(0.3 mg per kg) for 2 successive days (6.5 and 7.5 dpc) at 3 p.m. to

restore their ability to discriminate urine odors during this period.

Females of either strain only exposed to the male bedding for 48 h

(from 6.5 to 8.5 dpc) carried their pregnancies to term at a rate of

90–100%, similar to pregnant females left undisturbed (Fig. 7). In

contrast, only 50% of females treated with spiperone at 6.5 and 7.5 dpc

and exposed to alien male bedding for 48 h (from 6.5 to 8.5 dpc)

carried their pregnancies to term. However, females either treated

with spiperone at 6.5 and 7.5 dpc and exposed to original stud male

bedding for 48 h (from 6.5 to 8.5 dpc) or treated with spiperone at

6.5 and 7.5 dpc with no exposure to soiled bedding carried their

pregnancies to term at a rate of 90–100%. This experiment indicates

that odors contained in the bedding of an alien male can still

induce pregnancy failure after embryos are implanted if the dopa-

minergic barrier in the mother’s MOB is concomitantly disrupted.

Moreover, given the results of the previous experiment (Fig. 6b), it is

conceivable that this effect occurs through a drop of the night surge of

PRL at 7 and 8 dpc.

400 Day surge PRL Night surge PRL

350

300

250

200

150

100

Seru

m P

RL (

ng m

l–1)

Seru

m P

RL (

ng m

l–1)

50

Spiperone Spiperone

CD1 soiledbedding

CD1 soiledbedding

0– –

– – – –

––

400

350

300

250

200

150

100

50

0

+ + +

+ +

+

**

++

a bFigure 6 After embryonic implantation, the night surge of PRL is hampered

by alien male odor in females treated with the D2R antagonist spiperone.

Shown are serum PRL levels during day and night surge peaks in C57BL/6J

females 1 week after fecundation (7 dpc). (a) PRL levels during the day surge

peak (5 p.m.) were similarly high in females that were left undisturbed,

treated with spiperone, housed with alien CD1 male bedding or treated with

spiperone and housed with CD1 male bedding (n ¼ 3 for all groups). (b) In

contrast, PRL levels during the night surge peak (3 a.m.) were significantly

lower in females treated with spiperone and housed with CD1 male bedding

compared to control females left undisturbed (ANOVA F3 ¼ 5.5, P ¼ 0.01;

**P ¼ 0.006; n ¼ 6), treated with spiperone (P ¼ 0.01; n ¼ 3) or housed

with CD1 male bedding (P ¼ 0.01; n ¼ 3).

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DISCUSSION

In mammals, exchange of body odors favors the establishment of social

and sexual interactions and contributes to the regulation of reproduc-

tion. In rodents, these odorants are mainly contained in urine, affecting

neuroendocrine status, ovulation, opposite-sex tethering and

mating1,27,35,36. Urine constituents activate the VNO-AOB and the

MOE-MOB olfactory systems30, which, through their respective out-

puts11, modulate innate responses such as sexual receptivity (lordo-

sis)25 and ovulation1 and adaptive responses such as sex discrimination

and mate choice35,37. An effective mating therefore requires both

systems to be functional25,37,38. Because odors favor fecundity, they

might harm pregnancy, a condition that is not compatible with cycling.

Here we report that female mice experience a postcoital dopaminergic

surge in the MOB that hampers olfaction. We provide evidence that

male urine odors, channeled through the MOB, decrease PRL release at

7 dpc. This further implicates mouse MOB projections in the regula-

tion of reproductive neuroendocrine status and suggests that in mice,

as well as in hamsters27 and rats39, both sensory systems send inputs to

shared structures such as the amygdala and the hypothalamus and so

interact in eliciting behavioral and neuroendocrine responses. This

suggestion is strengthened by our finding that c-Fos is proportionally

activated in the MOB and the ventromedial hypothalamus by male

odorants during estrous and/or pregnancy. The ventromedial hypotha-

lamus is typically a target of the medial amygdala nucleus32

and contributes, together with other hypothalamic nuclei of the

vomeronasal system, to the integration of olfactory33,35 and vagino-

cervical inputs36 controlling female sexual behavior and postcoital PRL

surges. Thus, odors gated through the MOB might reach the ventro-

medial hypothalamus either directly through projections from the

olfactory cortex or indirectly through MOB efferences that modulate

inputs from the medial amygdala to the ventromedial hypothalamus

and tune mating behaviors35,37 and cycling40.

How odorant cues modulate PRL release is not clear. Because

dopamine release by tuberoinfundibular neurons, located in the

arcuate nucleus, is a major PRL inhibitor through D2R activation in

lactotrophs2, it has been proposed that the

tuberoinfundibular neurons are the final

target of luteolytic male odorants6. We showed

that administration of the D2R antagonist

spiperone concomitant with exposure to

alien male bedding affects the night surge,

but not the day surge, of PRL in females

at 7 dpc, presumably a result of the

PRL-releasing effect of spiperone administered

shortly before the day surge41. In contrast, 12 h

later, because induction of PRL is acute and

brief after spiperone injection41,42, no such

interference occurred; spiperone, which has a

long-term facilitating effect on olfaction,

allowed disruption of the night surge of PRL.

The day and the night surges of PRL in rats are

differently affected by a D2R antagonist42,

indicating that there is variation in the

effects of dopamine and other distinct

factors modulating PRL release at each

surge2. Male odors may have a different impact

on each surge, for example by modu-

lating hypothalamic dopamine during the

day surge and modulating other neuro-

transmitters with effector properties during

the night surge2.

Our observation that MOB inputs can affect PRL release is contrary

to the current view of the MOB being dispensable for blocking

pregnancy in the context of the Bruce effect, with VNO-AOB projec-

tions transmitting the luteolytic stimulus (blocking PRL)6,7,43,44. As

this is true until 3�4 dpc but not later8, the circuits leading to blockade

of PRL surges and return to estrous in response to male odor may be

progressively conditioned by progestational hormonal changes after

mating36. One main effect linked to such changes could be hormonal19

induction of the postcoital dopaminergic surge in the MOB that

effectively obstructs olfaction, which would consequently block

VNO-AOB outputs. In fact, deafferentation of the MOB prevents

c-Fos expression in mouse AOB in response to opposite-sex urine

odorants45, suggesting that themain olfactory pathway indeed provides

modulatory inputs to VNO efferent circuits. This is consistent with our

observation of decreased c-Fos immunoreactivity in the AOB of

females at 4.5 dpc, compared to estrous females, after exposure to

male bedding. It is also notable that the plateau of the dopaminergic

surge in the MOB coincides with the end of the Bruce effect (3�4 dpc)

and the embryonic implantation stage, when maternal investment

prevails over mate choice and estrous-inducing male odors. Thus,

the dopaminergic surge in the MOB may have evolved to prevent

interference of male odors with progestational status from implanta-

tion on. To this extent, the Bruce effect might be an emblematic

manifestation of mate choice that benefits both sexes at periovulation

but becomes deleterious to the species at peri-implantation time.

Our findings emphasize the importance of the MOB in regulating

reproductive processes in mice. VNO ablation, for example, does not

affect estrous or mating46, whereas bulbectomy induces ovarian

atrophy47 and infertility48. In addition, recent studies have challenged

the common view of exclusive functions for the MOB and the AOB,

showing that classical odorants and pheromones can activate either

system49,50. It has also been shown that the mouse MOB not only

projects fibers to neurons expressing luteinizing hormone–releasing

hormone, a key regulator of the ovarian cycle28,29, but also transduces

sensory inputs that regulate sexual and aggressive behaviors38. Finally,

100

a bP

erc

enta

ge o

f pre

gnancie

s t

o t

erm

in C

57B

L/6

J

90

80

***

70

60

50

40

30

20

10

0

100

Perc

enta

ge o

f pre

gnancie

s t

o t

erm

in B

ALB

/cJ

90

80

70

60

50

40

30

20

10

01

+

– –

––

– – – –

–

Plugged Plugged

Spiperone Spiperone

Original studbedding (C57BL/6J)

Original studbedding (BALB/cJ)

Alien(CD1)

Alien(C57BL/6J)

+

+ +

++ +

+

+ + +

2 3 4 5 1

+

– –

––

– – – –

–

+

+ +

++ +

+

+ + +

2

*

3 4 5

bedding bedding

Figure 7 Alien male odors compromise pregnancy. Shown are percentages of pregnancies carried to term

in C57BL/6J (a) and BALB/cJ (b) females. Group 1, females plugged and left undisturbed; group 2,

females plugged and exposed to alien male bedding for 48 h from 6.5 to 8.5 dpc; group 3, females

plugged and injected with spiperone at 6.5 and 7.5 dpc; group 4, females plugged, injected with

spiperone at 6.5 and 7.5 dpc (at 3 p.m.) and exposed to alien male bedding for 48 h from 6.5 to

8.5 dpc (w2 test with 4 degrees of freedom; ***P ¼ 0.0007 for C57BL/6J; *P ¼ 0.01 for BALB/cJ);

group 5, females plugged, injected with spiperone and exposed to original stud male bedding for 48 h

from 6.5 to 8.5 dpc. n ¼ 10 for each group, except n ¼ 12 for group 5 in b.

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our results support the idea that the vomeronasal and the olfactory

systems convey convergent sensory inputs27–29 rather than being relays

of unrelated cognitive and instinctive information.

The postcoital rise of dopamine in the MOB and consequent

decrease of olfactory perception abilities are an unexpected mechanism

by which female mice prevent male odor interference with neuroendo-

crine luteotrophic processes. Our results indicate that stimuli gated

through the MOB affect female reproductive status, strengthening the

view that the main olfactory system, together with the vomeronasal

system, contributes to modulation of reproductive behavior and

physiology in rodents.

METHODSExperimental animals. We used 6- to 10-week-old C57BL/6J and BALB/cJ

males and females and CD1 males (Harlan). The EMBL Monterotondo ethical

committee approved all animal procedures.

Dopamine tissue levels. Levels of endogenous dopamine were measured as

described in Supplementary Methods online.

Social olfactory discrimination test and threshold detection of odors. Urine

was collected from fertile BALB/cJ males and females. The LMWurine fraction

was separated from the HMW fraction using a Microcon YM10 centrifugal

filter for 30 min at 14,000g to retain proteins heavier than 10 kDa. The LMW

fraction was collected in the flow-through and the HMW fraction retained in

the filter and resuspended in a volume of water equivalent to the original

volume of urine. The fractions were stored at �80 1C until use.

To measure discrimination for social odors, mice were kept in their own

cages. Cotton buds were impregnated with either 2 ml of male urine or 2 ml of

female urine (either the LMWor HMW fraction) and placed at opposite ends

of the cage. Mice were subjected to two successive trials of 5 min during which

the time spent sniffing the cotton buds impregnated with the LMW, and

90 min later the HMW, urine was quantified using a chronometer. To restore

social odor discrimination, 6.5-dpc females were treated with spiperone

(0.03 mg per kg intraperitoneally; Sigma) 15 min before the social

odor discrimination test.

To measure the lowest concentration of LMWmale urine fraction that could

be detected by C57BL/6J females, the mice were exposed to increasing

concentrations of urine on one side of their cage and water on the other side.

More details are given in Supplementary Methods.

c-Fos immunoreactivity. The induction of c-Fos in the olfactory bulbs and

other brain areas of 2-month-old C57BL/6J virgin estrous females exposed to

alien male (CD1) urine odor was measured and compared to unstimulated

control estrous females or pregnant (4.5 dpc) females, either treated or not with

spiperone (0.03 mg per kg) and exposed or not to alien male urine odor. The

odor exposure always started at 3:30 p.m., and c-Fos expression was measured

after 90 min (time of the second daily prolactin surge) in the five experimental

groups. More details about the experimental groups are given in Supplemen-

tary Methods.

Histology. Histology, cell counts, and tyrosine hydroxylase staining intensity

were analyzed as described in Supplementary Methods.

Pregnancy block experiment. Virgin females (C57BL/6J and BALB/cJ) in

proestrous or early estrous, as assessed by vaginal smears, were housed in a cage

with syngeneic stud males (one male per female) overnight. Females with

vaginal plugs were considered 0.5-dpc. Plugged females were removed from the

stud male and housed individually. For the pregnancy block experiment, five

experimental groups were used. Group 1 females were left undisturbed until the

end of pregnancy. Group 2 females were exposed at 6.5 dpc, in the morning, to

alien male bedding for 48 h (CD1 males for C57BL/6J females and C57BL/6J

males for BALB/cJ females). Group 3 females were injected with spiperone

(0.3 mg per kg) at 6.5 and 7.5 dpc at 3 p.m. and left undisturbed. Group 4

females were injected with spiperone as for group 3 and exposed at 6.5 dpc to

alien male bedding for 48 h. Group 5 females were injected with spiperone as

for group 3 and returned at 6.5 dpc to the original stud male bedding for 48 h.

Pregnancy block was inferred by rating the number of completed gestations

3 weeks after plug. Females not giving birth at the expected time were deemed

not pregnant.

PRL dosing was done as described in Supplementary Methods.

Statistical analysis. A two-way ANOVA test (Statview 5.0) was used to evaluate

statistical significance, followed by a post hoc Fisher’s probable least-squares

difference test. The unpaired Student t test was used when an experiment had

only two groups to compare. The alpha level for all statistical tests wasr0.05.

Error bars of graphs represent standard error of the mean.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTS

We are grateful to M. Hamon and F. Saurini (INSERM U288) for the tissue

dopamine dosage and C. Sciarretta for comments. C.S. was supported by an

EMBO fellowship.

AUTHOR CONTRIBUTIONS

C.S. planned and performed most of the experiments and, together with L.M.,

drafted the manuscript. V.T. conducted the c-Fos experiment with the help of

J.K.-B. and provided conceptual input. J.K.-B. also contributed to the PRL dosage

experiments. M.A.B. provided the expertise for and helped conduct the behavioral

experiments. L.M. is the PI; she contributed to the experimental plans, supervised

the project, provided theoretical input and wrote the manuscript.

Published online at http://www.nature.com/natureneuroscience/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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