Cyclic Treatment With WIN 18,446, a Potential Male Contraceptive, is as Effective as Continuous Treatment for Suppression of Spermatogenesis in Mice

FIGURE 1. Male mice were treated with a diet containing WIN 18,446 for up to 4 weeks. Testis weight and morphology, and epididymal sperm counts were determined. (A) Epididymal sperm counts were significantly decreased after 2 weeks of WIN 18,446 treatment and remained suppressed at 3 and 4 weeks. (B) Testicular weights were significantly decreased after 2 weeks of WIN 18,446 and further reduced after 4 weeks of treatment. One‐way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (all groups were compared to each other). *, p < 0.05; **, p < 0.01; ****, and p < 0.0001.

FIGURE 2. Male mice were administered a diet containing WIN 18,446 cyclically or continuously and then recovered from the drug treatment for 8 or 16 weeks. At predetermined time points, a subset of mice was euthanized to determine testis weight (A) and sperm counts from the epididymal cauda (B). The testis was histologically evaluated using the Modified Johnsen score (C). Cycle denotes 2 weeks of no treatment followed by 2 weeks of WIN 18,446 treatment. One‐way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (all groups compared to control) was performed, and all groups were significantly different from control (p < 0.0001) in all three categories. Statistical significance is not shown on the graph to avoid clutter.

FIGURE 3. Histological analyses of testicular tubules. (A–E) Testicular tubules at different time points during cyclic treatment. The testis was fixed in modified Davidson fixative and stained with PAS‐H. (F–K) Morphology (left column) and A spermatogonia identified by PLZF immunohistochemistry (right column). Cycle = 2‐week drug cessation followed by 2 weeks WIN 18,446 treatment.

FIGURE 4. Mice were treated with a diet containing WIN 18,446 for 4 weeks, followed by a 2‐week drug cessation, WIN 18,446, and then a drug cessation. A subset of mice was analyzed at predetermined time points for testes weight (A), sperm counts (B), and histology (C). In (A) and (B): Red box, WIN 18,446 treatment; Green box, no treatment, i.e., drug cessation. Numbers in boxes denote treatment duration in weeks. Treatment duration is additive if they are the same color. In (C), cycle = 2‐week drug cessation followed by 2 weeks WIN 18,446 treatment. An asterisk indicates epididymis tubules containing spermatozoa.