In this work, for the first time, we elucidate the mechanism by which bisdichloroacetyldiamines such as WIN 18,446 suppress spermatogenesis. Specifically, we conclude that these compounds block spermatogenesis by inhibiting testicular retinoic acid biosynthesis. This conclusion is supported by the demonstration that WIN 18,446 potently inhibits the testes-specific enzyme ALDH1a2, which is involved in testicular retinoic acid biosynthesis. In addition, we have shown that the oral administration of WIN 18,446 to male rabbits significantly reduces intratesticular concentrations of retinoic acid and Stra8 expression and very severely suppresses spermatogenesis and fertility. It is possible that the observed reductions in retinoic acid and Stra8 expression could be due, in part, to the reduction in the number of testicular spermatids present during treatment. However, the observation that the reduction in testicular retinoic acid concentration occurred before any apparent reduction in spermatogenesis strongly supports the hypothesis that the reduction in spermatogenesis was a result of the inhibition of retinoic acid biosynthesis and not due to some other cause. Taken together, these findings suggest that WIN 18,446 reversibly suppresses spermatogenesis via inhibition of testicular retinoic acid biosynthesis by ALDH1a2, and identifies ALDH1a2 as a promising target for the development of novel, nonhormonal male contraceptives. Furthermore, this finding raises the possibility that a defect in the biosynthetic pathway of retinoic acid could underlie some causes of male infertility.
The finding that bisdichloroacetyldiamines suppress spermatogenesis by inhibition of testicular retinoic acid biosynthesis makes sense in light of emerging knowledge about the importance of retinoic acid in spermatogenesis. For example, retinoic acid appears to promote spermatogonia to enter into the meiotic pathway by up-regulating the expression of Kit
in germ cells while also increasing expression of Kit
ligand in Sertoli cells (Pellegrini et al, 2008
). Furthermore, the absence of retinoic acid signaling is markedly deleterious to spermatogenesis. For example, retinoic-acid receptor-α
knockout males are sterile because of degenerating or abnormal spermatogonia (Lufkin et al, 1993
). This appears to be due in part to aberrant coupling between cells within the seminiferous tubules, which may be secondary to reduced expression of the junctional complex constituents connexin-40 and vimentin (Chung et al, 2010
). Similarly, mice deficient in either RARγ or RXRβ are infertile, owing to low sperm counts and spermiation failure, respectively (Lohnes et al, 1993
; Kastner et al, 1996
). Moreover, retinoic acid receptor antagonists such as BMS-189453 potently inhibit spermatogenesis after oral doses as low as 5 mg/kg, with mice becoming temporarily infertile after 4 weeks of treatment (Schulze et al, 2001
Given the importance of retinoic acid in spermatogenesis, one might wonder why WIN 18,446 takes so long to exhibit its contraceptive effect. It is tempting to speculate that tubules either have variable stores of retinoic acid (likely bound to cellular binding proteins) or only require it at a given stage of the spermatogenic cycle. This theory would explain the variability in tubular appearance after 8 weeks of treatment (). Perhaps tubules lacking access to retinoic acid exhibit hypospermatogenesis before tubules that have adequate stores of retinoic acid or had recently completed the stage in the spermatogenic cycle that required retinoic acid before treatment began.
In addition, it is somewhat surprising that WIN 18,446 treatment reduced testicular retinoic acid concentrations only to approximately 40% of baseline values. It is possible that testicular retinoic acid is partially synthesized via non-ALDH1a2 pathways, possibly via ALDH1a1, which is also expressed in testicular tissue (Steward et al, 1996
). Alternatively, retinoic acid from other tissues could be diffusing into the testes; however, less than 1% of testicular retinoic acid is derived from the circulation (Kurlandsky et al, 1995
). Moreover, this finding may imply that spermatogenesis requires a certain threshold concentration of retinoic acid within the testes. Determining the minimal concentration of testicular retinoic acid required for spermatogenesis will be the subject of future research.
Importantly, treatment with WIN 18,446 did not have any toxic effects, as measured by tests of hematopoiesis and liver and kidney function; however, animals did lose 5%–10% of their body weight in the first several weeks of treatment. A slight decrease in the body weights of male cats treated with WIN 18,446 was also observed (Munson et al, 2004
), and humans treated with this compound complained of upset stomachs, without changes in weight (Heller et al, 1961
). Alternatively, the loss in body weight observed in our study could have been due to the stress of daily oral dosing of the medication, which has been observed in other settings with rabbits (Foote and Carney, 2000
Interestingly, and despite a 75% reduction in testicular volume, there was no apparent change in the serum testosterone concentrations during treatment. This observation implies that the change in the volume of the testis was mostly attributable to the absence of maturing sperm in the seminiferous tubules. Reductions in testicular volume are observed in humans during treatment with male hormonal contraceptives (Wu et al, 1996
). In these trials the epididymal volume is also reduced, as the epididymis is an androgen-dependent organ (Goyal et al, 1994
), and hormonal contraceptives markedly reduce intratesticular concentrations of testosterone (Page et al, 2007
). In contrast, epididymal weight did not change during treatment with WIN 18,446, implying that local concentrations of testosterone remained normal during treatment. The lack of change in serum testosterone and epididymal weight demonstrates that WIN 18,446 functions as a contraceptive without impacting the hypothalamic–pituitary–Leydig cell axis. Such a nonhormonal contraceptive could have significant appeal, as men could be reassured that their testosterone levels would be unaffected by treatment.
Importantly, WIN 18,446 exhibited almost no discernable toxic effects in vivo in experimental animals aside from its effect on the testes. The LD50
after oral administration of WIN 18,446 exceeds 10 000 mg/kg, and there were no deaths after the intraperitoneal injection of 750 mg/kg of WIN 18,446 in rats (Coulston et al, 1960
). Although WIN 18,446 is likely safe in adult animals, WIN 18,446 and other bisdichloroacetyldiamines have teratogenic effects on rat and chicken embryos (Tasaka et al, 1991
; Choy et al, 1999
), similar to those observed with retinoids (Nau, 2001
). Therefore, care must be taken to avoid exposure of pregnant women to these compounds.
There is a great need for new approaches to preventing unintended pregnancy. Despite currently available contraceptives, the world's population exceeds 6.9 billion and is increasing by 80 million yearly (Department of Economic and Social Affairs, 2010
). Much of this population growth is unintended (Henshaw, 1998
). Therefore, there is a great need for better access to existing contraceptives, improved contraceptive education, and more contraceptive options. Currently, male-directed contraceptive options are particularly limited, and despite decades of research into hormonal and immunological methods of male contraception, no regimen based on either of these approaches is near clinical approval. Bisdichloroacetyldiamines, such as WIN 18,446, safely and effectively suppress spermatogenesis in men, but cause a disulfiram reaction when coadministered with alcohol, which prevented their introduction for contraceptive purposes. We have now demonstrated that the contraceptive effect of these compounds is mediated by inhibition of testicular retinoic acid biosynthesis via the testes-specific enzyme ALDH1a2. Hopefully, this finding will allow for the development of novel, specific inhibitors of ALDH1a2 that do not cause the disulfiram reaction and can be developed into a safe, effective, and reversible form of male contraception.