Multiple structural and functional abnormalities in the p450 aromatase expressing transgenic male mice are ameliorated by a p450 aromatase inhibitor

Abstract

The present study was undertaken to analyze the effect of a P450 aromatase inhibitor (finrozole) on 4-month-old transgenic mice expressing human P450 aromatase (P450arom) under the human ubiquitin C promoter (AROM+). AROM+ mice present several dysfunctions, such as adrenal and pituitary hyperplasia, cryptorchidism, Leydig cell hypertrophy and hyperplasia, and gynecomastia. The present study demonstrates that these abnormalities were efficiently treated by administration of a P450arom inhibitor, finrozole. The treatment normalized the reduced intratesticular and serum testosterone levels, while those of estradiol were decreased. The body weight and several affected organ weights were normalized with the treatment. Histological analysis revealed that both the pituitary and adrenal hyperplasia were diminished. Furthermore, the cryptorchid testes present in the untreated AROM+ males descended to scrotum, 4 to 15 days after inhibitor treatment. In addition, the disrupted spermatogenesis was recovered and qualitatively complete spermatogenesis appeared with the inhibitor treatment. This was associated with normalized structure of the interstitial tissue, as analyzed by immunohistochemical staining for Leydig cells and macrophages. One of the features was that the Leydig cell hypertrophy was markedly diminished in the treated mice. AROM+ mice also present with severe gynecomastia, while the development and differentiation of the mammary gland in AROM+ males was markedly diminished with the inhibitor treatment. Interestingly, the mammary gland involution was associated with the induction of androgen receptor in the epithelial cells, while estrogen receptors were still detectable in the epithelium. The data show that AROM+ mouse model is a novel tool to further analyze the use of P450arom inhibitors in the treatment of the dysfunctions in males associated with misbalanced estrogen to androgen ratio, such as pituitary adenoma, testicular dysfunction, and gynecomastia.

Figure 1

Intratesticular T and E2, serum LH concentrations in control and finrozole-treated AROM+ male mice and in WT males. Asterisks indicate a significant difference (***, P < 0.001).

Figure 2

A–E: Localization of Prl-producing cells in the pituitary gland. Pituitary sections from WT mice (A and B), AROM+-placebo (C and D), and finrozole-treated AROM+ (E) male mice were stained with an antibody to rat Prl. Higher magnifications (B and D) indicate specific brown staining in the cytoplasm (arrows). F–K: Histology of the adrenal gland in WT (F and I), AROM+-placebo (G and J), and finrozole-treated AROM+ (H and K) male mice. In AROM+-placebo mice, large vacuole-filled cells appeared in the innermost cortex (J, arrow). The thickness of the cell layer was reduced after finrozole treatment (H and K). Scale bars: 50 μm (A, C, E), 100 μm (F–K).

Figure 3

A: Testis descent in WT mice, and in AROM+-placebo and finrozole-treated AROM+ mice. B and C: Identical intraabdominal descent of testes in WT and AROM+ mice. D: Intratesticular T concentration in embryonic day 17.5 is identical in WT and AROM+ males.

Figure 4

A–D: Testis histology in WT (A), AROM+-placebo (B), and finrozole-treated AROM+ (C and D) mice. In AROM+ males, the Leydig cell hyperplasia and the present of giant cells were evident, and no germ cells were present in the seminiferous tubules (B). The testes phenotype is largely reversible as indicated by the normalized Leydig cell structure (C), and by the qualitatively full spermatogenesis in finrozole-treated AROM+ males (D). E–G: Immunostaining with 3βHSD1 demonstrates the number and size of Leydig cells in WT (E), AROM+-placebo (F), and finrozole-treated AROM+ (G) mice. H–K: Immunostaining of macrophages with F4/80 macrophage marker antibody in WT (H), AROM+-placebo (I and J), and finrozole-treated AROM+ (K) mice. The pericellular staining is indicated with higher magnification (J). L–O: Immunostaining with cell proliferation marker (PCNA) demonstrates the positive nuclear staining in WT testis (L), AROM+-placebo testis (M and N) and inhibitor-treated AROM+ testis (O) in Leydig cells (arrows). Scale bars: 200 μm (A–C), 100 μm (E–O).

Figure 5

Wholemount staining of the mammary gland structure in placebo-treated (A1) and inhibitor-treated (A2) AROM+ males demonstrated the less differentiated phenotype of the inhibitor-treated AROM+ males, with less alveolar structures (arrow). B1–D2: Immunohistochemical staining of steroid receptors in AROM+ male mammary glands. Sections from placebo- and inhibitor-treated AROM+ male mice were stained with an antibody against ERα (B), ERβ (C), and AR (D). Data indicate the presence of ERs in the mammary gland epithelium in both placebo-treated and inhibitor-treated mice, while AR is markedly induced by the inhibitor treatment. E1–E2: Immunostaining for phosphotyrosine-Stat5 (D1–D2) reveals that the inhibitor treatment reduces Stat5 activation. Scale bars, 50 μm.