Oral administration of a retinoic Acid receptor antagonist reversibly inhibits spermatogenesis in mice

Abstract

Here we investigated a pharmacological approach to inhibit spermatogenesis in the mouse model by manipulating retinoid signaling using low doses of the pan-retinoic acid receptor (RAR) antagonist BMS-189453. Spermatogenesis was disrupted, with a failure of spermatid alignment and sperm release and loss of germ cells into lumen, abnormalities that resembled those in vitamin A-deficient and RARα-knockout testes. Importantly, the induced sterility was reversible. Enhanced efficacy and a lengthened infertility period with full recovery of spermatogenesis were observed using systematically modified dosing regimens. Hematology, serum chemistry, and hormonal and pathological evaluations revealed no detectable abnormalities or adverse side effects except the distinct testicular pathology. Our results suggest that testes are exquisitely sensitive to disruption of retinoid signaling and that RAR antagonists may represent new lead molecules in developing nonsteroidal male contraceptives.

Fig. 1.

Characterization of a retinoid receptor antagonist. A, Chemical structure of the synthetic retinoid. BMS-189453. B, Characterization of the synthetic retinoid, BMS-189453, using transactivation. Increasing ATRA concentrations (open circles) stimulated transactivation of the reporter construct as indicated. Closed circles represent the normalized transactivation for each test compound in the same concentration range. Each data point represents the mean of three independent measurements. C, Transactivation competition assay for assessment of antagonist activity. CAT reporter activity was measured in the presence of 10⁻⁷ m ATRA and increasing concentrations of each retinoid. BMS-189453 (C) competes the activity of ATRA for RARα (open circle), RARβ (closed circle), and RARγ (open square) and is thus a pan-antagonist. RA, Retinoic acid; RTA, retinoid-induced transcriptional activation.

Fig. 2.

Acute disruptive effect of BMS-189453 on spermatid alignment and release in testicular tubules immediately after dosing. A–F, Histological sections of mice treated with vehicle alone (A) or 0.1 (B), 1.0 (C–E), and 5.0 (F–H) mg/kg of BMS-189453. Magnification: A–G, ×60; H, ×40. pl, Preleptotene spermatocytes; l, leptotene spermatocytes; p, pachytene spermatocytes; rs, round spermatids. Arabic numerals indicate the step of spermatid differentiation; Roman numerals indicate the stage of the tubules. Although abnormal cell associations complicate staging, an attempt was made to stage the drug-treated tubules using acrosomal system, and tubules are labeled with a Roman numeral followed by an asterisk (e.g. stage IX*). Scale bar, 50 μm.

Fig. 3.

Temporal, cell-specific, and dose-responsive disruption of spermatogenesis in testes at 1 month after treatment. A–F, Histological sections of mice treated with 0 (A and B), 1.0 (C and D), and 5.0 (E–l) mg/kg of BMS-189453. A–G, The testes; H-I, the epididymides. Magnification: A, C–D, F, and G, ×40; B, E, H, and I, ×60. in, Intermediate spermatogonia; z, zygotene spermatocytes; d, diplotene spermatocytes; p, pachytene spermatocytes; MI/II, meiosis I/II; rs, round spermatids. At stage XII* (G, right bottom tubule): MI/MII spermatocytes with chromosomes aligned at metaphase plate, and zygotene and diplotene spermatocytes were readily seen. However, step 1 or 12 spermatids were missing. In stage XII* tubule (G, left upper tubule), zygotene spermatocytes were missing. The missing layer of specific cell types is indicated with an arrow and question mark. Scale bar, 50 μm.

Fig. 4.

Gonad weight and serum testosterone. A, The testicular weight of BMS-189453-treated male mice at different post-dose time points. The bars represent the mean ± sd of at least seven mice for each time point. ***, Significant differences within the same age group as assessed by paired Student's t test. P < 0.0005. B, Male mice were treated with BMS-189453 at three different doses, and sera were obtained for determination of testosterone level. Data points from individual mice are presented.

Fig. 5.

Assessment of spermatogenesis in testes 3 months after treatment with BMS-189453. A–D, Histological sections of mice treated with 1 (A and C) and 5 (B and D) mg/kg of BMS-189453. Magnification: A and B, ×20; C and D, ×40. sg, Spermatogonia; pl/liter, preleptotene/leptotene spermatocytes; ps, pachytene spermatocytes; z, zygotene spermatocytes; rs, round spermatids; es, elongated spermatids. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the tubules. Scale bar, 50 μm.

Fig. 6.

Full recovery of spermatogenesis in testes 6 months after treatment. A–F, Histological sections of mice treated with vehicle alone (A), 0.1 (B), 1 (C and D), and 5 (E and F) mg/kg of BMS-189453. Magnification: A–F: ×40. ps, Pachytene spermatocytes; z, zygotene spermatocytes; rs, round spermatids; es, elongated spermatids. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the tubules. Scale bar, 50 μm.

Fig. 7.

Effects on spermatogenesis and fertility upon systematic modification of dosing regimens to fine tune the inhibition of spermatogenesis. A–D, Induction and reversibility of sterility by BMS-189453. The average numbers of embryos obtained at each assessed time point during the mating period of 14 wk in mice treated with 5 mg/kg of BMS-189453 for 7 d (B, n = 9), compared with control (A, n = 5). Histological sections of 5-mg group (C and D) showed full recovery of spermatogenesis in testes and epididymides from six of nine mice at 3.5 months after treatment. Magnification: C and D, ×40. Roman numerals indicate the stage of the tubules. T=0 is the time the administration stopped, i.e. end of dosing period. E and F, Timely induction and lengthened period of infertility with a full recovery of spermatogenesis with modified dosing regimens. The average numbers of embryos obtained after each assessment time point during the mating period in group I mice treated with 5 mg/kg of BMS-189453 for 2 wk (E), compared with group II mice (F) treated with an even lower dose of 2.5 mg/kg for 4 wk. Two other experiments using different groups of mice and similar dosing regimens (n = 20) yielded similar results.