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3, 2001, pp. 407–409. Original Russian Text Copyright © 2001 by Babalyan, Novikov.
The Pheromone Inhibiting Spermatogenesis in Laboratory Mice Is Associated with the Urinary Protein Fraction
V. V. Babalyan and S. N. Novikov
Presented by Academician A.D. Nozdrachev May 10, 2000 Received October 4, 2000
Physiological proteinuria reaching sometimes 20 mg of protein per 1 ml of urine a day in mature male house mice is one of mysteries of rodent living activity . The phenomenology and molecular and genetic basis of proteinuria have been studied in detail; nevertheless, the biological implication of an increased protein excretion in this large systematic group is still an enigma. In the early 1990s, ﬁrst reports appeared in which close functional association was demonstrated between urinary protein and physiological activity of pheromones controlling the rate of sexual maturation in female laboratory mice . In this study, we analyzed physiological effects of the volatile compounds contained in both protein and nonprotein urine fractions of six- to eight-month-old male CBA/LacSto mice. To evaluate the physiological activity of the fractions, a biotest was used, namely, the count of morphologically abnormal spermia . Low-molecular-weight compounds were separated from the urinary proteins by salting-out with a saturated solution of ammonium sulfate followed by centrifugation. To obtain the lipophilic fraction, the supernatant was four times extracted with diethyl ether, and the solvent was removed with a nitrogen ﬂow under vacuum. The solid residual was dissolved in distilled water to restore the initial volume. Physiological activity of the fractions was evaluated from their effect on spermatogenesis in 30-day-old male CBAB6F1 mice after a 2-h exposure to a ﬂow of air containing the odorant in an olfactometer operating in a deﬁnite mode . The results obtained (table) indicate the following. (1) In native urine of mature CBA/LacSto mice, low-molecular-weight (volatile) compounds were found. Their effect on the 30-day-old recipients led to an increased frequency of abnormal spermia in the epididymis.
Pavlov Institute of Physiology, Russian Academy of Sciences, nab. Makarova 6, St. Petersburg, 199034 Russia
(2) Physiologically active compounds are represented by substances of different polarity, which are present in both lypophilic and hydrophilic urinary fractions. (3) After separation of the low-molecular-weight and protein urinary fractions, the latter retains a high pheromone activity. Thus, we have demonstrated that, after multiple extraction, the protein urinary fraction of mature male CBA mice retains pheromone physiological activity interfering with spermatogenesis in young CBAB6F1 mice. As early as in the mid-1970s, it was suggested that the structure and function of urinary proteins (peptides) of mature male mice were related to the physiological activity of the androgen-dependent pheromones that inﬂuence sexual maturation in females. . This suggestion was experimentally conﬁrmed later: two compounds accelerating sexual maturation were chemically identiﬁed as 2-(sec-buthyl)thiazoline and 2,3-dehydro-exo-brevicomin and afterwards synthesized. They were also found to be associated with the so-called major urinary protein (MUP) with a molecular weight of 18–19 kDa, which forms the basis of the protein fraction . Proteins of the MUP complex belong to a family of phylogenetically ancient proteins that serve several important functions in an organism, such as binding and transport of hydrophobic physiologically active compounds (hormones, vitamins, pheromones), interaction with membrane receptors of the target cells, formation of functional complexes with various substances, etc. . To date, at least 20 members of this family have been identiﬁed; the best studied proteins are fetoprotein, lactoglobulin, retinol-binding protein, and apolipoprotein D. Despite a high variation in the primary structure, lipocalins are characterized by a conserved conformation of the protein molecule, which is barrel-shaped with an open cuplike pocket and a ligand-binding site situated within the “barrel” . In the MUP complex, as many as 15 protein fractions are distinguished. They differ in molecular weight, amino-acid composition , and, probably, in the afﬁnity for the volatile pheromone-like physiologically active compounds. Both qualitative and quantita-
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THE PHEROMONE INHIBITING SPERMATOGENESIS IN LABORATORY MICE Frequency of abnormal spermia in the caudal segment of the epididymis (AC, %) in male CBAB6F1 mice exposed to volatile components of the protein and nonprotein fractions of urine of male CBA/LacSto mice Variant of treatment VUC P Lnf Hyd.Lnf Lip.Lnf CI CO N 3338 3225 3242 2170 2623 2572 4104 AC, % 10.1 ± 0.52 7.0 ± 0.45** 7.5 ± 0.46** 7.8 ± 0.58** 4.7 ± 0.41* 2.8 ± 0.33 2.5 ± 0.24
the MUP-complex proteins have been localized to the same chromosome . Comparison of these data with the results obtained in our study leads to a proposal that the MUP and OBP proteins are involved in a phylogenetically ancient transport chain ensuring chemical communication and reproduction regulation by means of pheromones in the animal world. If so, the question arises as to whether the artiﬁcial structural analogues of the MUP exhibiting high afﬁnity for the physiologically active compounds can be used for directed (vector) regulation of reproduction by means of olfactory stimulation. ACKNOWLEDGMENTS This work was partly supported by the Russian State Program “Frontiers in Genetics” (project no. 2.152) and the program “ASGL-Olfaction Pharmacology.” REFERENCES
1. Parfentjev, J.A., Proc. Soc. Exp. Biol. Med., 1932, vol. 29, pp. 1285–1286. 2. Böcskei, Z., Groom, C.R., Flower, D.R., et al., Nature (London), 1992, vol. 360, pp. 186–188. 3. Novikov, S.N. and Babalyan, V.V., Dokl. Akad. Nauk SSSR, 1988, vol. 302, no. 2, pp. 470–472. 4. Vandenbergh, J.G., Whitsett, J.M., and Lombardi, J.R., J. Reprod. Fert., 1975, vol. 43, pp. 515–523. 5. Flower, D.R., Biochem. J., 1996, vol. 318, pp. 1–14. 6. Robertson, D.H.L., Hurst, J.L., Bolgar, M.S., et al., Rapid Commun. Mass Spectrum., 1997, vol. 11, pp. 786–790. 7. Cavaggioni, A., Sorbi, R.T., Keen, J.N., et al., FEBS Lett., 1987, vol. 212, pp. 225–228. 8. Pelosi, P., J. Neurobiology, 1996, vol. 30, pp. 3–19. 9. Boudjelal, M., Sivaprasadarao, A., and Findlay, J.B.C., Biochem. J., 1996, vol. 317, pp. 23–27. 10. Sabanov, V.S., Plakhova, V.B., Shchegolev, B.F., et al., Dokl. Akad. Nauk, 2000, vol. 370, no. 1, pp. 126–129. 11. Roy, A.K., Byrd, J.G., Biswas, N.M., and Chowdhury, A.K., Nature (London), 1976, vol. 260, pp. 719–721. 12. Ghosh, P.K., Steger, R.W., and Bartke, A., Neoruendocrinology, 1991, vol. 53, pp. 7–11. 13. Antakly, T., Pelletier, G., and Feigelson, P., Proc. Nat. Acad. Sci. USA, 1983, vol. 80, pp. 4000–4002. 14. Stirna, J., Genet. Res., 1991, vol. 57, pp. 135–138. 15. Hainey, S. and Bishop, J.O., Genet. Res., 1982, vol. 39, pp. 31–39.
Notes: VUC, volatile urinary components; P, urinary protein fraction; Lnf, low-molecular-weight nonprotein urinary fraction; Lip.Lnf, and Hyd.Lnf, lipophilic and hydrophilic low-molecular-weight nonprotein urinary fractions, respectively; CO, intact males; CI, exposure of male CBAB6F1 mice to water vapor. * Signiﬁcant differences from the variant CI at p < 0.05. ** Signiﬁcant differences from the variant CI at p < 0.01.
tive compositions of the MUPs is highly polymorphic . We suggest, therefore, that the partial concentration of physiologically active compounds in a solution are determined by genetically determined differences in the MUP expression and variations in ligand (pheromone) binding with one or another fraction, which, in turn, depend on the structural or conformation properties of the protein molecule. In general, these properties of the MUP complex determine the functional activity of the pheromone “bouquet” serving as a vector directed to the recipient organism. The MUP structure is surprisingly similar to that of the odorant-binding protein (OBP) playing a key role in mechanisms of the olfactory reception, because OBP transports the odorant molecules to the receptor cells of the olfactory layer [7, 8]. Therefore, the reports in which the special membrane receptors for OBP are described , as well as the effect of pyrazine compounds characterized by high afﬁnity for OBP on the functioning of slow sodium channels of the sensory neurons  are of particular interest. Note that a MUP homologue, α2u-globulin, isolated from rat urine and injected i.c. has a pronounced antiestrogen effect  and causes changes in the levels of gonadotropin, prolactin, and testosteron . A more surprising fact is that, in rats, a relatively high concentration of α2u-globulin of extrahypophysial origin was determined in cells of the anterior lobe of hypophysis . There is evidence indicating that one of the genes controlling spermatogenesis and formation of the spermatozoon head in laboratory mice is linked to chromosome 4 ; the Mup genes responsible for synthesis of
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