Scent of receptive females as signal to reproduction stimulate male mice to olfactory search of a potential breeding partner^1, 2^. This searching behavior is coupled with infection risk due to bacterial contamination of the fecal and urine scent marks^4^. The theoretical consideration of host evolution under inevitable parasitic pressures, including helminthes, bacteria, virus etc., predicts adaptations that help protect against parasites associated with breeding^7^. In this study, we propose that acceptation of female signals by male mice leads to adaptive redistribution of immune defense directed to protection against respiratory infection risks. Our results reveal migration of macrophages and neutrophils to upper airways upon exposure to female odor stimulus resulting in increased resistance to influenza virus in male mice. Contrary to widely accepted immunosuppressive function of female sexual signals, our data provide the first demonstration of the adaptive immunological response to female odor stimulus through induction of nonspecific immune response in upper airways

Alla M. Zaidman

Ekaterina A. Litvinova

Elena P. Goncharova

Marina A. Zenkova

Mikhail P. Moshkin

English

Nature Precedings

FEMALE SCENT SIGNALS ENHANCES MALE RESISTANCE TO 
INFLUENZA 
Ekaterina A. Litvinova1, Elena P. Goncharova2, Alla M. Zaidman3, Marina A. Zenkova2, 
Mikhail P. Moshkin1,4 
1Institute of Systematics and Ecology of Animals, SB RAS, Frunze 11, Novosibirsk, 
630091, Russia. 
2Institute of Chemical Biology and Fundamental Medicine, SB RAS, Lavrentyeva 8, 
Novosibirsk, 630090, Russia. 
3Novosibirsk Research Institute of Traumatology and Orthopaedics, Frunze 17, 
Novosibirsk, 630091, Russia. 
4Institute of Cytology and Genetics, SB RAS, Lavrentyeva 10, Novosibirsk, 630090, 
Russia. 
Summary paragraph 
Scent of receptive females as signal to reproduction stimulate male mice to olfactory 
search of a potential breeding partner1, 2. This searching behavior is coupled with infection risk 
due to bacterial contamination of the fecal and urine scent marks4. The theoretical consideration 
of host evolution under inevitable parasitic pressures, including helminthes, bacteria, virus etc., 
predicts adaptations that help protect against parasites associated with breeding7. In this study, 
we propose that acceptation of female signals by male mice leads to adaptive redistribution of 
immune defense directed to protection against respiratory infection risks. Our results reveal 
migration of macrophages and neutrophils to upper airways upon exposure to female odor 
stimulus resulting in increased resistance to influenza virus in male mice. Contrary to widely 
accepted immunosuppressive function of female sexual signals, our data provide the first 
demonstration of the adaptive immunological response to female odor stimulus through 
induction of nonspecific immune response in upper airways. 
The scent of female mice as a signal for reproduction stimulates male mice to search for a 
breeding partner1-3. In turn, searching behavior increases risk of respiratory infections in male 
mice due to their sniffing of the contaminated female fecal and urinary marks, which harbor 
many infection agents4-6. The theoretical consideration of host evolution under inevitable 
parasitic pressures, including helminthes, bacteria, virus etc., predicts adaptations that help 
protect against parasites associated with breeding7. We hypothesized that the scent of female 
soiled bedding as most naturalistic sexual signal8-10 induces integrative neuroendocrine response, 
which activated migration of immunocompetent cells according to breeding related change of the 
infection risks. Here we studied leukocyte trafficking into upper air-ways and resistance to 
respiratory infection (influenza as an example) in male mice kept with or without female soiled 
bedding during 1-3 weeks. In all experiments BALB/c male and females, 12-16 weeks old, were 
maintained in unisex groups of 4-5 animals under standard temperature, light-dark period (12:12 
h) and food and water ad libitum. 
Resistance to influenza. Mature BALB/c males were kept in two odor environments: with 
(n=41) or without the exposure (n=41) to the female soiled bedding as most naturalistic sexual 
signals in the mouse. One week later, they received an intranasal application (while under ether 
anesthesia) of the mice adapted influenza virus A/WSN/33 (H1N1) in 50 μl phosphate-buffered 
saline (PBS). We used 5 serially diluted doses that contain from 2.5*101 to 2.5*105 focus-
forming units (FFU). The mice were observed daily for the following 3 weeks. The total 
mortality rate was lower in the males exposed to the female bedding (8 from 41), in comparison 
with male mice not exposed (19 from 41 mice, χ2 = 5.52, P= 0.019). The males exposed to the 
female bedding had a higher LD50 than the males isolated from females (Fig. 1).  
Male mice were challenged with mouse-adapted influenza virus A/WSN/33 (H1N1) by 
intranasal inoculation. The total mortality rate was lower in the males exposed to the female 
bedding (8 from 41), in comparison with not exposed male mice (19 from 41 mice, χ2 = 5.52, P= 
0.019). The males exposed to the female bedding had a higher LD50 than the males isolated from 
females (Fig. 1). 
Lung histology and broncho-alveolar lavage (BAL). A possible mechanism of scent 
induced resistance to respiratory infection may be the migration of immune cells to body 
compartments where they are likely to be more effective, for example to upper airways. 
Therefore we have made histological preparations of lung tissue harvested from the additional 
groups of non-infected male mice kept either exposed (n=3) or non-exposed (n=3) to female 
bedding. We found a significantly increased number of leukocytes in the perivenous and 
peribronchial areas in male mice that were exposed to the female bedding (Fig. 2). A deferential 
count of the leukocytes in the lung tissue from the male mice revealed a statistically significant 
difference between the female soiled bedding treated (n=5) and non-treated (n=5) groups (Fig. 
3a). The prevalence of macrophages and neutrophils was higher in the BAL of male mice 
exposed to the female bedding; whereas, male mice kept in isolation from the female bedding 
showed a higher percent of lymphocytes in their BAL (Fig. 3b). Since neutrophils and 
macrophages are major effectors of the nonspecific immune defense against respiratory 
infections11-13 and its migration to upper airway increase resistance of mice to experimental 
pneumonia14. That may help explain the observed enhancement of resistance to influenza in male 
mice exposed to the female soiled bedding.  
Aerobic performance. Leukocyte migration into lung tissue is a common manifestation of 
respiratory airway inflammation, which entails a decline in the efficiency of both lung 
ventilation and aerobic performance15, 16. But in our study, the average size of the leukocyte 
aggregations was smaller (11 – 1029 cells per aggregation) than has been reported for typical 
inflammatory infiltrations 17, 18. Also we determined the aerobic performance in the new set of 
male mice exposed (n=5) and not exposed (n=5) to female bedding. The cold-induced maximum 
oxygen consumption (MOC) was used as criteria of the aerobic performance. There were no 
statistically significant differences between the male mice exposed to female soiled bedding and 
those not exposed (Fig. 4). Thus, the female scent induced increases in the number of small sized 
aggregations of leukocytes does not reduce the aerobic performance in male mice.  
 
Thus, besides the well known induction of the neuroendocrine regulation of 
spermatogenesis and breeding behavior by female signals2, 3, 19, this work demonstrates that such 
signals have additional role in increasing the leukocyte migration into the upper airways of male 
mice. Moving of the immunocompetent cells into the upper airways compensates the increased 
risk of respiratory infections, which accompanies the olfactory searching of a breeding partner. 
The proximate explanation of this phenomenon could be related with neuroendocrine effects of 
the female pheromones and neuroendocrine and immune links. Also we can not refuse non-
pheromonal explanations such as an effect of the pathogen inhalation from contaminated soiled 
bedding. How it was found recently some bacterial compound might be detected by formil 
peptide receptors (FPR-rs) in vomeronasal olfactory epithelium20. Our data is the first to 
demonstrate the migration of leukocytes in response to the naturalistic breeding signals that 
provided an anticipatory adaptation of male mice to potential risk of the respiratory infections. 
 
METHODS 
Female bedding treatment 
Bedding (wooden flakes) from cages with mature BALB/c females were used for female 
scent treatment of males. Using 5 cages with 5 females per cage let as collected every day 
bedding that was soiled during the previous 5 days. The examination of vaginal smears revealed 
at least 30 % female in estrus. The soiled beddings or fresh wooden flakers were placed on the 
lid of male cages in wire mesh containers (about 5 ml) and were changed daily just before lights 
off. Four experiments have been done on BALB/c males that were kept one week before study 
with or without female scent. 
Influenza A challenge 
Mouse-adapted influenza virus A/WSN/33 (H1N1) was grown in embryonated chicken 
eggs. Virus containing allantoic fluid was harvested, and stored in aliquots at -80°C. Stock 
(5×107 FFU/ml) was serially ten-fold diluted by cold PBS.  
Leukocyte trafficking into upper airwas 
Lungs samples were fixed in 10% buffered formalin. Histologic sections (4 µm) of lung 
were stained with hematoxylin and eosin (HE). The number of leukocyte aggregations, their size, 
and their optical density in grey light were estimated in each section. Using regression equation, 
described previously we calculated number leukocytes per section21. Total leukocyte count in 
BAL was determined with a hemacytometer. For differential count of the leukocytes in BAL 
each samples (5 ml) were cytocentrifuged 5 min at 3g and 4°C. Then slices were stained by 
Wright-Giemsa. 
Lung capacity 
The maximum oxygen consumption (MOC) was assayed in male mice, which were treated 
either by female bedding or by fresh wooden flakes during week. Mice were exposed to HELOX 
(20 % O2: 80 % He, +10°C) to stimulate the energy metabolism22. 
 
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Acknowledgements We thank Professor R. Nagatomi and members of the Department of 
Medicine & Science in Sports & Exercise, Graduate School of Medicine Tohoku 
University (Sendai, Japan) for teaching BAL methodology and assistance in study of 
leukocyte in BAL. This project was supported by the Russian Foundation for Basic 
Research and Heiwa Nakajima. Fist draft of manuscript was dramatically improved by 
Professor J. Burns (Bethany College, USA). We thank Professors M. Ferkin, D. Penn and 
Dr. Sara Zala for the useful comments to manuscript. 
 
Author Contributions  
E.A.L. measured the maximum cold-induced oxygen consumption; E.P.G. passaged the 
virus and infected mice; A.M.Z. made histological preparations of lung tissue; M.M.P. 
analyzed cells in BAL fluid. M.A.Z., M.M.P. and E.A.L. designed the study. E.A.L. and 
M.M.P. analyzed data and wrote the paper. All authors discussed the results and 
commented on the manuscript. 
 
Author Information 
Reprints and permissions information is available at www.nature.com/reprints. The 
authors declare competing financial interests: details accompany the full-text HTML 
version of the paper at www.nature.com/nature. Correspondence and requests for 
materials should be addressed to E.A.L. (dimkit@ngs.ru) or M.M.P. 
(mmp@bionet.nsc.ru). 
 
 
Figure 1 Cumulative mortality after challenge with influenza virus A/WSN/33 
(H1N1).  Non-exposed (white dots) and exposed to the female bedding (black dots) 
BALB/c male mice. LD50 in males exposed to the female bedding (4.73±0.30 LogFFU) 
was significantly higher compare to males isolated from female bedding (3.28±0.35 
LogFFU, t=2.84, p<0.01). Mean mortality±s.e.m. are shown. 
Figure 2 Leukocyte aggregations in lung tissue. a, Non-exposed to the female bedding 
BALB/c male mice. b, Exposed to the female bedding BALB/c male mice. Light 
microscopy (×10 and ×20 magnifications). 
Figure 3 Number of leukocytes. a, Average number of leukocytes per histological slide 
in BALB/c male mice non-exposed (white) and exposed to the female bedding (black). 
Mean leukocytes±s.e.m. are shown; asterisks indicated P=0.02, n=9, Z=2.32. b, Percent 
of different types of leucocytes in broncho-alveolar lavage in BALB/c male mice non-
exposed (right) and exposed to the female bedding (left). * indicated P<0.01, n=10, 
Z=2.56, † indicated P<0.02, n=10, Z=2.45, ‡ indicated P<0.02, n=10, Z=2.45. 
Figure 4 Aerobic performance. Oxygen consumption during a 13 min exposure in 
HELOX by male mice that had been non-exposed (white dots) and exposed to the female 
bedding (black dots) (repeated measures ANOVA F2,13=1.92, p=0.39). MOC was similar 
in males exposed (n=8, 9.73±0.39) and not exposed (n=8, 9.00±0.29) to the female 
bedding. Mean oxygen consumption±s.e.m. are shown. 
 






Female Scent Signals Enhances Male Resistance to Influenza

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Female Scent Signals Enhances Male Resistance to Influenza

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