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From the Departments of * Medicine,
Urology,
Pharmaceutics, and
Comparative Medicine, University of Washington
School of Medicine and School of Pharmaceutics, Seattle, Washington; the ||
Department of Medicine, University of Florida,
Gainesville, Florida; the ¶ School of Molecular
Biosciences, Washington State University, Pullman, Washington; and the #
Focused Scientific, Newcastle, Washington.
| Correspondence to: Dr John K. Amory, University of Washington, Box 356429, 1959 NE Pacific St, Seattle, WA 98195 (e-mail: jamory{at}u.washington.edu). |
| Received for publication April 27, 2010; accepted for publication July 12, 2010. |
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 IC50 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.
Key words: Retinol, vitamin A, male contraception, sperm concentration
Disulfiram (Antabuse) is a medication used in the management of alcoholism (Suh et al, 2006). Individuals taking disulfiram feel well as long as they do not drink alcohol. If they do imbibe, they experience a disulfiram reaction, which is characterized by flushing, diaphoresis, dysphoria, nausea, vomiting, and other symptoms. This reaction is mediated by disulfiram's inhibition of the liver enzyme aldehyde dehydrogenase-2 (ALDH2). ALDH2 catalyzes the conversion of acetaldehyde, the main metabolite of ethanol, to acetic acid (Heilig and Egli, 2006). The combination of disulfiram and alcohol therefore leads to increased serum concentrations of acetaldehyde and the symptoms of the disulfiram reaction (Deitrich et al, 2007). The presence of a disulfiram reaction when WIN 18,446 and alcohol are coadministered strongly suggests that WIN 18,446 also inhibits ALDH2.
Nineteen isozymes of aldehyde dehydrogenase have been identified (Marchitti et al, 2008). ALDH2, the isozyme involved in alcohol metabolism, is expressed in many tissues, including the liver, lung, and kidney (Steward et al, 1996). In 1996, a novel aldehyde dehydrogenase called aldehyde dehydrogenase 1a2 (ALDH1a2) was cloned from the rat and found to be expressed almost exclusively in the testes (Wang et al, 1996). ALDH1a2 localizes to developing germ cells within the seminiferous tubules and strongly binds retinal, but does not recognize acetaldehyde as a substrate, suggesting that ALDH1a2 functions in testicular retinoic acid biosynthesis (Wang et al, 1996; Vernet et al, 2006a).
It has been recognized since 1925 that vitamin A is required for spermatogenesis (Wolbach and Howe, 1925). Vitamin A is converted in tissues to its principal biologically active derivative, retinoic acid. In the testes, retinoic acid is necessary both for the initiation of spermatogenesis at puberty and for the maintenance of spermatogenesis in adults (Lufkin et al, 1993; Ghyselinck et al, 2006; Vernet et al, 2006b). Dietary beta-carotene and retinol are transported to the testes, where they are converted to retinoic acid within the developing germ cells by alcohol and aldehyde dehydrogenases (Bishop and Griswold, 1987; Napoli, 2000; Paik et al, 2004). Retinoic acid then binds one of several retinoic acid receptors that regulate gene expression (Chung et al, 2004; Bowles et al, 2006). Among other effects, retinoic acid induces the spermatogonial differentiation protein stimulated by retinoic acid-8 (STRA8) within the developing germ cells. STRA8 appears to play a central role in spermatogonial differentiation (Zhou et al, 2008).
As testicular retinoic acid biosynthesis requires ALDH1a2, and this enzyme is structurally similar to the one inhibited by WIN 18,446 in the disulfiram reaction (ALDH2), we hypothesized that WIN 18,446 suppressed spermatogenesis via inhibition of ALDH1a2. Therefore, we studied the effect of WIN 18,446 on ALDH1a2 enzyme activity in vitro and on spermatogenesis in vivo in adult male New Zealand white rabbits. In this way, we sought to ascertain if suppression of spermatogenesis by WIN 18,446 was mediated by the inhibition of testicular retinoic acid biosynthesis.
Materials and Methods
In Vitro Inhibition of ALDH1a2 by WIN 18,446
To produce cells expressing high levels of ALDH1a2 activity, a cDNA for
ALDH1a2 was cloned into lentiviral plasmids under the control of EF1a promoter
and virally transduced into the lung cancer cell line H1229, which expresses
little ALDH activity at baseline (Zaiss et
al, 2002). Expression of ALDH1a2 was then verified by Western blot
analysis as described previously (Moreb et
al, 2002). Inhibition of ALDH1a2 by WIN 18,446
(N,N'-bis(dichloroacetyl)-1,8-octanediamine; Acros Organics, Geel,
Belgium) was determined using lysate of the transduced H1229 cells. Lysate was
incubated with 10 µM all-trans-retinal in the presence of varying
concentrations of WIN 18,446 for 30 minutes at 37°C. The reaction was
stopped by adding 100% ethanol containing 0.025 M potassium hydroxide.
Retinoic acid was extracted and measured by high-performance liquid
chromatography as described previously
(Kane et al, 2005;
Paik et al, 2005). Loss during
extraction was accounted for by adjusting the recovery to that of known
concentrations of the internal standard, acitretin (Sigma-Aldrich, St Louis,
Missouri).
Spermatogenesis in Rabbits
All procedures in rabbits were approved in advance by the Institutional
Animal Use and Care Committee at the University of Washington. Six-month-old
male New Zealand white rabbits were dosed orally for 4, 8, and 16 weeks (n =
4/group) with 200 mg/kg WIN 18,446 daily, as this dose had previously been
shown to be effective at suppressing spermatogenesis in a variety of other
species (Coulston et al, 1960;
Beyler et al, 1961;
Heller et al, 1963;
Singh and Dominic, 1980;
Asa et al, 1996;
Munson et al, 2004), and could
be used to determine the effect of WIN 18,446 administration on
intratesticular retinoic acid biosynthesis and function during suppression of
spermatogenesis. The WIN 18,446 was compounded in a simple syrup base at a
concentration of 100 mg/mL with banana crème flavoring and dosed orally
using a syringe. Spermatogenesis was assessed by having the animals ejaculate
sperm into an artificial vagina at baseline and weekly during treatment and
recovery (Naughton et al,
2003). Sperm count, concentration, and motility were quantified
manually using a hemocytometer (Seed et
al, 1996). Two chambers were counted and averaged for each time
point. At baseline, and after 4, 8, and 16 weeks of treatment, anesthetized
rabbits underwent a unilateral orchiectomy to ascertain the impact of
treatment on spermatogenesis by histology, intratesticular retinoic acid
concentrations, and Stra8 expression. For histological examinations,
approximately 0.1–0.3 g testis was placed in Bouin solution for fixation
and paraffin embedment, sectioned at 5 µm, and stained with
hematoxylin–periodic acid-Schiff stain. For testicular and epididymal
sperm counts, approximately 0.3–0.6 g of the testis and 0.1–0.3 g
of the cauda epididymis were individually weighed and homogenized in 0.1 M
sodium phosphate buffer, pH 7.4, containing 0.1% Triton X-100, using an
all-glass Kontes 15-mL homogenizer. Testes were homogenized with 8 strokes and
the epididymis was homogenized with 15 strokes. Ten microliters of the
homogenate was loaded into each side of a Neubauer phase contrast
hemocytometer and counted. Total homogenization-resistant spermatids or
epididymal sperm per gram or per organ were calculated by correcting for the
number of squares counted, dilution, volume, and weight
(Amann, 1986).
|
For the measurement of testicular Stra8 mRNA expression, total RNA
was isolated from testes homogenized in Trizol (Invitrogen, Carlsbad,
California) according to the manufacturer's protocol. Four testes were used
for each time point. One microgram of total RNA was reverse transcribed using
the iScript kit (BioRad, Hercules, California). Quantitative PCR was performed
by using Fast SYBR Green Mastermix (Applied Biosystems, Foster City,
California) on an ABI7500 Fast Real-Time PCR instrument (Applied Biosystems).
Stra8 primers amplify a 154-bp product (primers: 5'-GATGCTGGGGA
GAAGTTTCA-3' and 5'-AATCGTCGTCATCGAAGGTC-3'); control
Rps2 primers amplify a 112-bp product (primers:
5'-CTGACTCCCGACCTCTGGAAA-3' and 5'-GAGCCTGGG
TCCTCTGAACA-3'). Results were analyzed using the 
- Ct
method with Rps2 as the normalization control
(Zhou et al, 2008).
Fertility was assessed by mating experiments. Six-month-old females were placed with treated animals both after 16 weeks of treatment and after recovery from treatment, and copulation was observed. Fifteen days after mating, the females were sacrificed and the number of embryos counted.
Statistical Analysis
For the analysis of the testicular spermatids, Stra8 expression,
and retinoic acid concentrations, as well as the sperm parameters in the
ejaculates, mean values were calculated at each time point and compared with
baseline by the Wilcoxon signed rank test. No corrections were made for
multiple comparisons. For assessment of fertility, the number of embryos
between treatment and recovery sired by a given male were compared using a
2-sample t-test with unequal variances. STATA (version 8.0; College
Park, Texas) was used for all statistical analysis. For all comparisons, an
of 
.05 was considered significant.
Results
In Vitro Inhibition of ALDH1a2 by WIN 18,446
WIN 18,446 potently inhibited the ability of ALDH1a2 to convert retinal (10
µM) to retinoic acid in vitro with an IC50 of 0.3 µM
(Figure 1). Concentrations of
WIN 18,446 greater than 1 µM suppressed over 75% of the formation from
retinal under these assay conditions, demonstrating that WIN 18,446 is a
strong inhibitor of retinal oxidation. WIN 18,446 also potently inhibited
ALDH2 activity in a similar fashion (data not shown).
Spermatogenesis in Rabbits
Treatment of rabbits with WIN 18,446 at an oral daily dose of 200 mg/kg
resulted in marked suppression of testicular weight over time
(Table 1). After 16 weeks of
treatment, testicular weight was reduced almost 75% compared with untreated
controls. Conversely, treatment with WIN 18,446 had no apparent effect on
epididymal weight compared with baseline.
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Treatment of rabbits with WIN 18,446 dramatically suppressed spermatogenesis, both as measured by testicular and epididymal sperm counts from orchiectomy specimens (Table 1) as well as in ejaculated sperm from the 3 animals that were able to ejaculate into the artificial vagina (Figure 2). Interestingly, there were no significant reductions in any measure of spermatogenesis after 4 weeks of treatment. After 8 weeks of treatment, however, significant reductions in ejaculated sperm concentrations and testicular and epididymal sperm counts were evident. After 16 weeks of treatment, sperm was absent from the ejaculates in all treated animals and very few spermatids were present in either the testes or the epididymides (Table 1). Sperm motility was similarly affected, dropping from greater than 75% at baseline and after 4 weeks of treatment to 14% after 8 weeks of treatment and 0% (epididymal sperm) after 16 weeks of treatment (data not shown). Ejaculated sperm concentrations remained severely suppressed for 8–10 weeks after treatment was discontinued. However, thereafter sperm concentrations rapidly increased to the anticipated 50% of baseline values in all animals. This level takes into account the 50% reduction in spermatogenetic capacity attributable to the hemiorchiectomy performed at the end of the treatment phase.
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Histological examination of testicular tissue from rabbits treated with WIN 18,446 revealed no obvious changes after 4 weeks of treatment (Figure 3C and D), compared with controls (Figure 3A and B). By 8 weeks of treatment, however, roughly one-half of the tubules revealed marked evidence of spermatogenic arrest and hypospermatogenesis (Figure 3E and F). After 16 weeks of treatment, no active spermatogenesis was apparent (Figure 3G and H). Instead, within the seminiferous tubules only spermatogonia and Sertoli cells were identifiable, implying that treatment was blocking the ability of spermatogonia to enter into meiosis.
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Discussion
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 intra-testicular 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- 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 RAR
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 (Figure 3E and F). 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.
Acknowledgments
We thank Ms Constance Pete and Ms Pilar Cordero for assistance with sperm analysis; William Bremner, MD, PhD, and Alvin M. Matsumoto, MD, for critical review of the manuscript; Stu Sommerville for medication compounding; and the University of Washington Veterinary Services for animal care and assistance.
Footnotes
Supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, a division of the National Institute of Health, through cooperative agreement U01 HD060408 and, in part, by NIH grant R01GM081569-02S1.
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