Doklady Biological Sciences, Vol. 387, 2002, pp. 505–507. Translated from Doklady Akademii Nauk, Vol. 387, No.

4, 2002, pp. 562–564. Original Russian Text Copyright © 2002 by Novikov, Troitskaya, Gladysheva, Churakov.

PHYSIOLOGY

Specific Anosmia to Isovaleric Acid in Laboratory C57BL/6 Mice: Recessive Inheritance
S. N. Novikov, V. T. Troitskaya, O. S. Gladysheva, and G. A. Churakov
Presented by Academician Yu.V. Natochin December 25, 2001 Received August 12, 2002

Specific anosmia, a phenomenon of a strong decrease in sensitivity to certain odorants, was first described in humans by the American researcher Blakeslee [1] at the beginning of the 20th century and provided the basis for Amoore’s highly original theory of odor discrimination [2, 3]. Although the theory seemed promising, the physiological and biochemical processes underlying the phenomenon itself has not been studied sufficiently. Adequate experimental models, primarily genetic ones, are required for further attack on the problem. Researches have great expectations for the natural chemical compounds, pheromones, that are used as stimuli [3]. Isovaleric acid (IVA) is one of them. In some mammalian species [4], it is one of the major components of the vaginal excretion with signaling function. In 1977, when testing nine inbred mouse strains for relative olfactory sensitivity to amyl acetate, pentadecalactone, and IVA by using the conditioned aversion technique, a group of researches [5] identified two contrasting strains, C57BL/6 and AKR, which differed in the olfactory threshold for IVA concentrations by one to two orders of magnitude. At the same time, these two strains did not differ in their sensitivity to the other two compounds. Hence, the conclusion was inferred that a specific anosmia to IVA was characteristic of C57BL/6 mice. Afterwards, using various experimental approaches, the phenomenon was shown to be caused by abnormal functioning of the peripheral part of the olfactory system [6–9]. In this work, the role of the initial link of the olfactory system in the mechanisms of specific anosmia to IVA was studied in the two mouse strains contrasting with respect to this trait (AKR and C57BL/6) and in their hybrids (AKB6F1 and B6AKF1) by recording electroolfactograms (EOGs) from the olfactory layer preparation.

Pavlov Institute of Physiology, Russian Academy of Sciences, nab. Makarova 6, St. Petersburg, 199034 Russia Nizhni Novgorod State University, Nizhni Novgorod, Russia

Sixty-three seven- to eight-month-old mice, both males and females, were used in this study. EOG was recorded using chlorine–silver electrodes with agar passage and a tip diameter of 0.1–0.2 mm [6]. The olfactory stimulation was induced by 0.1 ml of vapor above a paper filter on which either IVA or its freshly prepared solution at a concentration of 10–2, 10–3, 10–4, or 10–5 M was applied. Isoamyl acetate (IA) at a concentration of 10–2, 10–3, 10–4, and 10–5 M and its saturated vapor served as reference stimuli. The EOG was recorded from an isolated olfactory layer of the bone helices at t = 19°C. Under these conditions, the EOG recorded from the preparations displayed an invariant amplitude throughout the experiment in response to the standard stimulus. From 5 to 19 areas were tested in each animal. In C57BL/6 mice, no response to the IVA concentrations from 10–3 to 10–5 M was observed (Fig. 1a). At the same time, all AKR mice responded to 10–4 M IVA, and most preparations responded to the concentration of 10–5 M (Fig. 1b). The two mouse strains studied did not differ in their responses to low IA concentrations (103−104 M). Thus, C57BL/6 mice with a comparatively normal sensitivity to IA exhibited a considerably reduced sensitivity to IVA; i.e., they were characterized by a specific anosmia to IVA [6]. The drop in olfactory sensitivity (an increase in the threshold of the response by one to two orders of magnitude) was only observed in males (Fig. 1a). In the olfactory-lining preparations from mice of both strains, the areas with a complete anosmia to IVA and those displaying a normal response to IA were present simultaneously; no areas insensitive to IA were found. The EOG analysis of direct and reciprocal hybrids showed that the anosmia to IVA was inherited as a recessive trait (Figs. 1c, 1d). Thus, the sharp interstrainar differences in the sensitivity to the IVA effect were revealed in the preparations of the olfactory layer by the electrophysiological method; the recessive inheritance of this trait was also shown. This suggests that the specific anosmia to IVA in male C57BL/6 mice is related to hereditary morphofunctional characteristics of the peripheral part of the olfactory system.

0012-4966/02/1112-0505$27.00 © 2002 MAIK “Nauka /Interperiodica”

506
(a) C57BL/6 strain Males

NOVIKOV et al.
(c) (AKR × C57BL/6) F1 hybrids Females Males Females

log X 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 4.0 3.5 3.0 2.5 2.0

(b) AKR strain

(d) (C57BL/6 × AKR)F1 hybrids 3.5 3.0 2.5 2.0 1.5

1.5 1.0 0.5 0 SV 10–2 10–3 10–4 10–5 SV 10–2 10–3 10–4 10–5 SV 10–2 10–3 10–4 10–5 1.0 0.5 SV 10–2 10–3 10–4 10–5 M

Fig 1. The EOG amplitude dependence on the concentrations of isoamyl acetate (IA) and isovaleric acid (IVA) in male and female C57BL/6 and AKR mice and their hybrids. Abscissa shows the concentrations, M (SV, saturated vapor); ordinate shows the logarithm of the EOG amplitude measured in mV. Dark bars, experiment (IVA); light bars, control (IA).

In the recombinant strains of laboratory mice obtained by the parental cross C57BL/6 × DBA/2, the genes responsible for anosmia to IVA were mapped to chromosomes 4 and 6 and shown to display recessive inheritance [9]. Note that one of the genes responsible for spermatogenesis and the high level of sperm abnormality characteristic of animals of the C57 family of strains was also linked to chromosome 4, and the trait itself was a recessive one [10]. It should be emphasized that males from this family display abnormal mating behavior, have low plasma testosterone level and neuroendocrine disturbances [3]. Comparison of these data brings up the question as to whether the specific anosmia to IVA in male C57BL/6 mice is related to the abnormal mating function, and whether the same genetic basis underlies the anosmia at the peripheral region of the olfactory system and the neuroendocrine status of the organism.

Thus, the relationships between the sensitivity to pheromones and reproduction is closely connected with the urgent problem of the correlation between physiological mechanisms of olfaction, neuroendocrine system, and reproduction in general [11]. In our opinion, of special interest is the hormonal control of expression of odorant-binding proteins (OBPs), the key and universal link in the cascade of the perireceptor events of the olfaction process in terrestrial vertebrates, which may be responsible for the primary recognizing of the odorant [12]. In addition, according to [13], in mouse cells of the olfactory layer, a significant amount of mRNA transcribed from the Mup gene complex, which is well-known to be also located on chromosome 4, is expressed along with OBP [14]. The above data and the results obtained in this study, taken together, suggest that the C57BL/6 mouse strain has a genetic linkage group, which, on the one hand, has an effect on the characteristics of male reproducDOKLADY BIOLOGICAL SCIENCES Vol. 387 2002

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tive, neuroendocrine function, and formation of the spermatozoon head shape [3, 10]. On the other hand, the same genetic group controls the spectral composition of isomorphic proteins, such as OBPs and MUPs, which are presumed to differ significantly in the degree of the affinity to ligands important for communication (to IVA, in particular). To provide the basis for the controlled influence on the organism [14], detailed analysis of the OBP and MUP differential binding to a number of physiologically active compounds is required. Note that the discovery of the membrane receptors with high affinity to OBPs on the cells of the olfactory layer and the lungs in cows [15] suggests some selective pathways and specific mechanisms underlying the directed axonal transport of physiologically active ligands within the OBP complex and (or) its polypeptide fragments upon endocytosis. This study provides the basis for development of some new approaches to the experimental correction of olfactory deficiency and the mammalian neuroendocrine function. ACKNOWLEDGMENTS This work was supported by the State Program “Frontiers in Genetics” (project no. 2.152) and the Russian Foundation for Basic Research (project no. 02-0449273).

REFERENCES
1. Blakeslee, A.F., Science, 1918, vol. 48, pp. 298–299. 2. Amoore, J.E., Nature, 1967, vol. 214, pp. 1094–1098. 3. Novikov, S.N., Feromony i Razmnozhenie Mlekopitayushchikh (Pheromones and Reproduction in Mammals), Leningrad: Nauka, 1988. 4. Michael, R.P., Keverne, E.B., and Bonsall, R.W., Science, 1971, vol. 172, pp. 964–966. 5. Wysocki, C.J., Whitney, G., and Tucker, D., Behav. Genet., 1977, vol. 7, pp. 171–188. 6. Troitskaya, V.T., Gladysheva, O.S., and Novikov, S.N., Neirofiziologiya, 1987, vol. 19, pp. 133–135. 7. Sicard, G., Royet, J.-P., and Jourdan, F., Brain Res., 1989, vol. 481, pp. 325–334. 8. Wang, H.-W., Wysocki, C.J., and Gold, G.H., Science, 1993, vol. 260, pp. 998–1000. 9. Griff, I.C. and Reed, R.R., Cell, 1995, vol. 83, pp. 407– 414. 10. Styrna, J., Genet. Res., 1991, vol. 57, pp. 135–138. 11. Vowles, R.H., Bleach, N.R., and Rowe-Jones, J.M., Int. J. Pediatr. Otorhinolaryngol., 1997, vol. 41, pp. 207–214. 12. Tegoni, M., Pelosi, P., Vincent, F., et al., Biochim. Biophys. Acta, 2000, vol. 1482, pp. 229–240. 13. Utsumi, M., Ohno, K., Kawasaki, Y., et al., J. Neurobiol., 1999, vol. 39, pp. 227–236. 14. Babalyan, V.V. and Novikov, S.N., Dokl. Akad. Nauk, 2001, vol. 378, no. 3, pp. 407–409. 15. Boudjelal, M., Sivaprasadarao, A., and Findlay, J.B.S., Biochem. J., 1996, vol. 317, pp. 23–27.

DOKLADY BIOLOGICAL SCIENCES

Vol. 387

2002

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