Brain masculinization requires androgen receptor function - PubMed (original) (raw)

. 2004 Feb 10;101(6):1673-8.

doi: 10.1073/pnas.0305303101. Epub 2004 Jan 27.

Takahiro Matsumoto, Hirotaka Kawano, Tomoyuki Watanabe, Yoshikatsu Uematsu, Keisuke Sekine, Toru Fukuda, Ken-ichi Aihara, Andrée Krust, Takashi Yamada, Yuko Nakamichi, Yoko Yamamoto, Takashi Nakamura, Kimihiro Yoshimura, Tatsuya Yoshizawa, Daniel Metzger, Pierre Chambon, Shigeaki Kato

Affiliations

Brain masculinization requires androgen receptor function

Takashi Sato et al. Proc Natl Acad Sci U S A. 2004.

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 (5alpha-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 ERalpha(-/-) 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 17beta-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.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Establishment of the AR-null mutant mouse line. (a) Diagram of the wild-type AR genomic locus (+), the floxed AR L3 allele (L3), and the AR allele (L-) obtained after Cre-mediated excision of exon 1. K, _Kpn_I; E, _Eco_RI; H, _Hin_dIII; B, _Bam_HI; LoxP sites are indicated as black arrowheads. The targeting vector consisted of a 7.6-kb 5′ homologous region containing exon 1, a 1.3-kb 3′ homologous region, a single loxP site, and the neo cassette with two loxP sites. (b) Southern blot analysis of targeted embryonic stem clones. Genomic DNA from WT TT2 embryonic stem cells (WT) and homologous targeted clones (FB-18 and FC-61) were digested with _Kpn_I for hybridization with probe 1 or with _Eco_RI for hybridization with probe 2. (c) Strategy for the disruption of the floxed AR gene by using the CMV-Cre transgene. ARL3/Y mice were crossed with CMV-Cre(tg/0)/ARL-/+ mice to generate CMV-Cre(0/0)/ARL-/Y and CMVCre(tg/0)/ARL-/L- mice. (d) Detection of the Y chromosome-specific Sry gene in ARL-/Y mice by PCR of mouse-tail DNA. (e) Lack of AR transcripts in male (ARL-/Y) and female (ARL-/L-) AR-null mutant mouse brains as shown by Northern blot analysis with an AR E-domain cDNA probe. (f) Absence of AR protein in ARL-/Y and ARL-/L- mouse brains by Western blot analysis with a specific C-terminal antibody. (g) Female-like external genitalia in ARL-/Y mice (Upper). Atrophic testis without internal male and female reproductive organs in ARL-/Y mice. Ep, epididymis; Pr, prostate; Sv, seminal vesicle; Te, testis; Vd, vas deferens; Od, oviduct, Ov, ovary; Ut, uterus.

Fig. 2.

Fig. 2.

Ablation of AR in male mice resulted in the lack of both male and female sexual behaviors. (a) Loss of all components of male sexual behavior in intact (gonads: +) 10-week-old ARL-/Y mice. E2 treatment of gonadectomized (gonads: -) ARL-/Y mice partially induced mounts and intromissions but not ejaculation. Results of 90-min sexual behavioral tests performed after 30-min tests performed twice every week for 2 weeks are shown and were basically identical with those of the 30-min tests (data not shown). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (b) Percentage of intact (gonads: +) and gonadectomized (gonads: -) 10-week-old mice exhibiting aggressive behaviors (Incidence of male aggressive behavior) toward olfactory bulbectomized (OBX) male intruder mice. Three tests in total were carried out every week, and results of the third test are shown. *, P < 0.05; **, P < 0.01. (c) Number of bouts with attacks. **, P < 0.01; ***, P < 0.001. (d) Latency to the first attack during resident–intruder tests. Aggressive behavioral acts were markedly reduced in intact ARL-/Y mice and were partially recovered after DHT treatment. *, P < 0.05; ***, P < 0.001. (e) Absence of female sexual behaviors in intact and E2- and progesterone-primed gonadectomized 10-week-old ARL-/Y mice. Twenty days after implantation of E2 pellets, progesterone (P; 500 μg) was administered to ARL-/Y and WT mice 4 h before the behavioral test. Normal expression of female sexual behaviors was observed in intact 10-week-old ARL-/L- mice. (f) Lordosis was not induced in gonadectomized ARL-/Y mice after treatment with E2. (g) Absence of AR protein and no reduction in ERα and ERβ protein levels in the medial preoptic area of ARL-/Y mice analyzed by immunohistochemistry.

Fig. 3.

Fig. 3.

Partial recovery of impaired male-typical behaviors in _ER_α-/- mice after DHT treatment. (a) Treatment with DHT, but not E2, restored the impaired mount and intromission but not ejaculation of gonadectomized 10-week-old male _ER_α-/- mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (b) Impaired male aggressive behaviors of male _ER_α-/- mice were restored by DHT treatment. *, P < 0.05. (c) Number of bouts with attacks. *, P < 0.05. (d) Latency to the first attack during resident–intruder tests. *, P < 0.05.

Fig. 4.

Fig. 4.

Failure of ARL-/L- mice to express male-typical behaviors after DHT-induced perinatal brain masculinization. (a) WT female mice, but not ARL-/L- mice, showed male sexual behaviors in response to either DHT or E2 treatment as adults after perinatal exposure to DHT. Behavioral tests were performed as described in Fig. 2_a_. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (b) Percentage of mice exhibiting aggressive behaviors (incidence of male aggressive behavior) toward olfactory bulbectomized (OBX) male intruder mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (c) Number of bouts with attacks. *, P < 0.05. (d) Latency to the first attack during resident–intruder tests. *, P < 0.05; **, P < 0.01. (e) Expression of female sexual behaviors is independent of AR/androgen signaling during the perinatal stage. Behavioral tests were performed after E2 and progesterone treatment (E2/P) as described in Fig. 2_e_. (f) No clear reduction in serum testosterone levels in ARL-/Y neonate mice (1 day after birth). (g) Reduced expression levels of nNOS transcript in ARL-/Y hypothalamus by Northern blot analysis. Densitometric analysis of the relative expression level by semiquantitative RT-PCR, expressed as fold WT after normalization to GAPDH. *, P < 0.05.

References

    1. Couse, J. F. & Korach, K. S. (1999) Endocr. Rev. 20**,** 358-417. - PubMed
    1. 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. USA 97**,** 14737-14741. - PMC - PubMed
    1. Matsumoto, T., Honda, S. & Harada, N. (2003) Neuroendocrinology 77**,** 416-424. - PubMed
    1. Ogawa, S., Lubahn, D. B., Korach, K. S. & Pfaff, D. W. (1997) Proc. Natl. Acad. Sci. USA 94**,** 1476-1481. - PMC - PubMed
    1. Plapinger, L. & McEwen, B. S. (1978) in Biological Determinants of Sexual Behaviour, ed. Hutchison, J. B. (Wiley, New York).

Publication types

MeSH terms

Substances

LinkOut - more resources