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. 2011 Jan-Feb;32(1):111-9.
doi: 10.2164/jandrol.110.010751. Epub 2010 Aug 12.

Suppression of spermatogenesis by bisdichloroacetyldiamines is mediated by inhibition of testicular retinoic acid biosynthesis

John K Amory  1 , Charles H Muller, Jakob A Shimshoni, Nina Isoherranen, Jisun Paik, Jan S Moreb, David W Amory Sr, Ryan Evanoff, Alex S Goldstein, Michael D Griswold
Affiliations

Suppression of spermatogenesis by bisdichloroacetyldiamines is mediated by inhibition of testicular retinoic acid biosynthesis

John K Amory et al. J Androl. 2011 Jan-Feb.

Abstract

The bisdichloroacetyldiamine WIN 18,446 reversibly inhibits spermatogenesis in many species, including humans; however, the mechanism by which WIN 18,446 functions is unknown. As retinoic acid is essential for spermatogenesis, we hypothesized that WIN 18,446 might inhibit retinoic acid biosynthesis from retinol (vitamin A) within the testes by inhibiting the enzyme aldehyde dehydrogenase 1a2 (ALDH1a2). We studied the effect of WIN 18,446 on ALDH1a2 enzyme activity in vitro, and on spermatogenesis and fertility in vivo, in mature male rabbits for 16 weeks. WIN 18,446 markedly inhibited ALDH1a2 enzyme activity in vitro with an IC(50) of 0.3 μM. In vivo, the oral administration of 200 mg/kg WIN 18,446 to male rabbits for 16 weeks significantly reduced intratesticular concentrations of retinoic acid, severely impaired spermatogenesis, and caused infertility. Reduced concentrations of intratesticular retinoic acid were apparent after only 4 weeks of treatment and preceded the decrease in sperm counts and the loss of mature germ cells in tissue samples. Sperm counts and fertility recovered after treatment was discontinued. These findings demonstrate that bisdichloroacetyldiamines such as WIN 18,446 reversibly suppress spermatogenesis via inhibition of testicular retinoic acid biosynthesis by ALDH1a2. These findings suggest that ALDH1a2 is a promising target for the development of a reversible, nonhormonal male contraceptive.

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Figures

Figure 1
Figure 1
WIN 18,446 is a potent inhibitor of the testicular retinal dehydrogenase, ALsDH1a2. Lysate from H1229 cells transduced with ALDH1a2 was incubated for 30 minutes with 10 μM all-trans-retinal in the presence of various concentrations of WIN 18,446, and retinoic acid was measured by high-performance liquid chromatography. All experiments were conducted in triplicate. Values are means ± SEM (note that error bars are contained within the symbol in certain instances).
Figure 2
Figure 2
Sperm concentrations over time in New Zealand white rabbits dosed orally with 200 mg/kg WIN 18,446 daily for 16 weeks (N = 3). All animals were azoospermic between week 16 of treatment and week 8 of recovery. Animals underwent a hemiorchiectomy at the end of treatment, which accounts for the 50% reduction in sperm output at the end of recovery. *indicates P < .05 compared with baseline.
Figure 3
Figure 3
Testicular histology in New Zealand white rabbits prior to treatment (A, B) or after daily treatment with 200 mg/kg WIN 18,446 orally for 4 (C, D), 8 (E, F) or 16 weeks (G, H). Se indicates Sertoli cell; Sg, spermatogonia in Figure 3H (arrows). Original magnification ×100 (A, C, E, G); ×400 (B, D); ×200 (F, H).
Figure 4
Figure 4
Testicular tissue retinoic acid concentration (pmol/g tissue) in New Zealand white rabbits dosed orally with 200 mg/kg WIN 18,446 daily (n = 4/group). All values are means ± SEM. *indicates P < .05 compared with placebo; **, P < .01.
Figure 5
Figure 5
Expression of the retinoic acid–induced gene Stra8 in the testes of New Zealand white rabbits dosed orally with WIN 18,446 200 mg/kg daily (n = 4/group). All values are means ± SEM. *indicates P < .05 compared with placebo; **, P < .01.
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