Brain masculinization requires androgen receptor function

Takashi Sato

Takahiro Matsumoto

Hirotaka Kawano

Tomoyuki Watanabe

Andrée Krust+9 authors

^{Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan;}^{Solution Oriented Research for Science and Technology, Japan Science and Technology, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan;}^{Department of Orthopedic Surgery, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;}^{Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université Louis Pasteur, Collége de France, 67404 Illkirch, Strasbourg, France; and}^{Medical Research Laboratories, Taisho Pharmaceutical Co., Ltd., 403, Yoshino-cho 1-chome, Saitama-shi, Saitama, 330-8530, Japan}

^{To whom correspondence should be addressed. E-mail:}pj.ca.oykot-u.cce.liam@otaksu.

^{T.S. and T.M. contributed equally to this work}

Edited by Bert W. O'Malley, Baylor College of Medicine, Houston, TX

Received 2003 Aug 19; Accepted 2003 Nov 24.

Abstract

Testicular testosterone produced during a critical perinatal period is thought to masculinize and defeminize the male brain from the inherent feminization program and induce male-typical behaviors in the adult. These actions of testosterone appear to be exerted not through its androgenic activity, but rather through its conversion by brain aromatase into estrogen, with the consequent activation of estrogen receptor (ER)-mediated signaling. Thus, the role of androgen receptor (AR) in perinatal brain masculinization underlying the expression of male-typical behaviors remains unclear because of the conversion of testosterone into estrogen in the brain. Here, we report a null AR mutation in mice generated by the Cre-loxP system. The AR-null mutation in males (AR^L-/Y) resulted in the ablation of male-typical sexual and aggressive behaviors, whereas female AR-null homozygote (AR^L-/L-) mice exhibited normal female sexual behaviors. Treatment with nonaromatizable androgen (5α-dihydrotestosterone, DHT) was ineffective in restoring the impaired male sexual behaviors, but it partially rescued impaired male aggressive behaviors in AR^L-/Y mice. Impaired male-typical behaviors in ERα^{mice were restored on DHT treatment. The role of AR function in brain masculinization at a limited perinatal stage was studied in}AR^L-/L- mice. Perinatal DHT treatment of females led to adult females sensitive to both 17β-estradiol and DHT in the induction of male-typical behaviors. However, this female brain masculinization was abolished by AR inactivation. Our results suggested that perinatal brain masculinization requires AR function and that expression of male-typical behaviors in adults is mediated by both AR-dependent and -independent androgen signaling.

Abstract

It is thought that local production of estradiol, converted from testicular testosterone by brain aromatase, and the subsequent activation of estrogen receptor (ER)-mediated signaling is sufficient to induce brain masculinizaton in the male fetus and eventual expression of male typical behaviors in the adult (1–3). This hypothesis is supported by findings that mice deficient in either ER or aromatase display severely reduced male-typical behaviors (2–4). However, studies have indicated that when androgens are given to the female fetus, male-typical behaviors can be induced on further androgen treatment in adulthood (5, 6). Thus, although androgen receptor (AR)-mediated androgen actions are thought to be important in the induction of male-typical behaviors, the activity of locally converted estrogen from aromatizable androgens in the brain has prevented the assessment of the role of AR in perinatal brain masculinization and expression of male-typical behaviors.

Naturally occurring mutations in mammalian AR genes, located on the X chromosome and therefore present as a single copy in males, result in AR dysfunction that can lead to androgen-insensitive testicular feminization mutation (Tfm) (7, 8). Tfm is characterized by a variety of phenotypic abnormalities along with species-specific effects (8). However, Tfm mice appear to express a truncated AR protein (9). Most male animals with severe androgen insensitivity exhibit female-like external sexual organs and are infertile (8). This fact makes it impossible to generate female animals homozygous for AR deficiency and precludes the establishment of AR-null mutant (AR knockout, ARKO) lines either in nature or by conventional gene disruption techniques. Therefore, to define AR function we used the Cre-loxP system to allow the AR-null mutation (10) to be passed to both male and female offspring (11, 12). We report here that perinatal brain masculinization requires AR function and that expression of male-typical behaviors in adults is mediated by both receptor-dependent and -independent androgen signaling.

Click here to view.

Acknowledgments

We thank G. K Schütz and K. S. Korach for helpful discussions, S. Aizawa for TT2 embryonic stem cells, J. Miyazaki for CAG-tester mice, and H. Higuchi and R. Nakamura for manuscript preparation. This work was supported in part by funding from the Human Frontier Science Program (to P.C. and S.K.) and a Grant-in-Aid for Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan (to S.K.).

Acknowledgments

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: AR, androgen receptor; ARKO, AR knockout; DHT, 5α-dihydrotestosterone; ER, estrogen receptor; Tfm, testicular feminization mutation; PLA, placebo; E₂, 17β-estradiol; nNOS, NO synthase.

Notes

This paper was submitted directly (Track II) to the PNAS office.

References

1. Couse, J. F. & Korach, K. S. (1999) Endocr. Rev.20, 358-417. [[PubMed]
2. Ogawa, S., Chester, A. E., Hewitt, S. C., Walker, V. R., Gustafsson, J. A., Smithies, O., Korach, K. S. & Pfaff, D. W. (2000) Proc. Natl. Acad. Sci. USA97, 14737-14741.
3. Matsumoto, T., Honda, S. & Harada, N. (2003) Neuroendocrinology77, 416-424. [[PubMed]
4. Ogawa, S., Lubahn, D. B., Korach, K. S. & Pfaff, D. W. (1997) Proc. Natl. Acad. Sci. USA94, 1476-1481.
5. Plapinger, L. & McEwen, B. S. (1978) in Biological Determinants of Sexual Behaviour, ed. Hutchison, J. B. (Wiley, New York).
6. Olsen, K. L. (1985) in Neurobiology, eds. Gilles, R. & Balthazart, J. (Springer, Heidelberg), pp. xv, 149-164.
7. Takeyama, K., Ito, S., Yamamoto, A., Tanimoto, H., Furutani, T., Kanuka, H., Miura, M., Tabata, T. & Kato, S. (2002) Neuron35, 855-864. [[PubMed]
8. Quigley, C. A., De Bellis, A., Marschke, K. B., el-Awady, M. K., Wilson, E. M. & French, F. S. (1995) Endocr. Rev.16, 271-321. [[PubMed]
9. Gaspar, M. L., Meo, T., Bourgarel, P., Guenet, J. L. & Tosi, M. (1991) Proc. Natl. Acad. Sci. USA88, 8606-8610.
10. Li, M., Indra, A. K., Warot, X., Brocard, J., Messaddeq, N., Kato, S., Metzger, D. & Chambon, P. (2000) Nature407, 633-636. [[PubMed]
11. Sato, T., Matsumoto, T., Yamada, T., Watanabe, T., Kawano, H. & Kato, S. (2003) Biochem. Biophys. Res. Commun.300, 167-171. [[PubMed]
12. Kato, S(2002) Clin. Pediatr. Endocrinol.11, 1-7. [PubMed][Google Scholar]
13. Ohtake, F., Takeyama, K., Matsumoto, T., Kitagawa, H., Yamamoto, Y., Nohara, K., Tohyama, C., Krust, A., Mimura, J., Chambon, P., et al. (2003) Nature423, 545-550. [[PubMed]
14. Suzawa, M., Takada, I., Yanagisawa, J., Ohtake, F., Ogawa, S., Yamauchi, T., Kadowaki, T., Takeuchi, Y., Shibuya, H., Gotoh, Y., et al. (2003) Nat. Cell Biol.5, 224-230. [[PubMed]
15. Cologer-Clifford, A., Simon, N. G., Richter, M. L., Smoluk, S. A. & Lu, S. (1999) Physiol. Behav.65, 823-828. [[PubMed]
16. Matsumoto, T. & Yamanouchi, K. (2000) Neurosci. Lett.291, 143-146. [[PubMed]
17. Ogawa, S., Chan, J., Chester, A. E., Gustafsson, J. A., Korach, K. S. & Pfaff, D. W. (1999) Proc. Natl. Acad. Sci. USA96, 12887-12892.
18. Yeh, S., Tsai, M. Y., Xu, Q., Mu, X. M., Lardy, H., Huang, K. E., Lin, H., Yeh, S. D., Altuwaijri, S., Zhou, X., et al. (2002) Proc. Natl. Acad. Sci. USA99, 13498-13503.
19. Meisel, R. L. & Sachs, B. D. (1994) in The Physiology of Reproduction, eds. Knobil, E. & Neill, J. D. (Raven, New York), pp. 3-105.
20. Dupont, S., Krust, A., Gansmuller, A., Dierich, A., Chambon, P. & Mark, M. (2000) Development (Cambridge, U.K.) 127, 4277-4291. [[PubMed]
21. Simon, N. G. & Whalen, R. E. (1987) Horm. Behav.21, 493-500. [[PubMed]
22. Thornton, J. & Goy, R. W. (1986) Horm. Behav.20, 129-147. [[PubMed]
23. Revelli, A., Massobrio, M. & Tesarik, J. (1998) Endocr. Rev.19, 3-17. [[PubMed]
24. Zhu, Y., Rice, C. D., Pang, Y., Pace, M. & Thomas, P. (2003) Proc. Natl. Acad. Sci. USA100, 2231-2236.
25. Mangelsdorf, DJ., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., et al. (1995) Cell83, 835-839. [Google Scholar]
26. Glass, C. K. & Rosenfeld, M. G. (2000) Genes Dev.14, 121-141. [[PubMed]