Hum. Reprod. Advance Access originally published online on January 5, 2006
Human Reproduction 2006 21(5):1309-1315; doi:10.1093/humrep/dei463
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Long-term non-hormonal male contraception in mice using N-butyldeoxynojirimycin
The Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, UK
1 To whom correspondence should be addressed: The Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. E-mail: aarnoud.vanderspoel{at}bioch.ox.ac.uk
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Abstract |
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BACKGROUND: The imino sugar N-butyldeoxynojirimycin (NB-DNJ) causes reversible infertility in male mice. This compound may have promise as a male contraceptive, because it is already in clinical use, for a non-reproductive condition. As contraceptives need to be taken for extended periods of time, it was essential to evaluate NB-DNJ for its reproductive effects over a long period of administration. METHODS: We have assessed the imino sugar for its long-term effects on the fertility of male C57BL/6 mice, reversibility and potential cumulative toxicity by monitoring various reproductive and systemic parameters over 12 months. RESULTS: Long-term low-dose (15 mg/kg/day) administration of NB-DNJ was sufficient to maintain infertility in male mice. In contrast to short-term drug treatment, imino sugar exposure for more than 3 months resulted in reduced sperm counts. Male mice that had been administered imino sugar for 6, 10 or 12 months and were then maintained without drug administration regained their fertility within 9 weeks after withdrawal of the drug. Prolonged NB-DNJ intake did not affect reproductive hormone levels, serum biochemistry or animal behaviour. CONCLUSION: Low-dose treatment with NB-DNJ over a long period is an effective approach for the regulation of fertility in a male mammal by non-hormonal means, without causing overt adverse effects.
Key words: acrosome/behaviour/imino sugar/male contraception/sperm morphology
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Introduction |
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The imino sugar N-butyldeoxynojirimycin (NB-DNJ) is a glucose mimetic and has been licensed for clinical use in type 1 Gaucher disease, a genetic disorder of glycosphingolipid metabolism (Butters et al., 2003
; Cox et al., 2003; Lachmann, 2003; Pastores and Barnett, 2003). In addition, this compound and its galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ) cause infertility in male C57BL/6 mice (van der Spoel et al., 2002; Suganuma et al., 2005). Mice become infertile after at least 3 weeks of oral administration of NB-DNJ, and the minimal dose to induce infertility is 15 mg/kg/day (van der Spoel et al., 2002). This effect of short-term treatment is fully reversible following the withdrawal of the drug for a further 3 weeks (van der Spoel et al., 2002). Mice treated with this dose of NB-DNJ produce morphologically abnormal spermatozoa that display a wide variety of different head shapes and have severely deformed or no acrosomes. Furthermore, the tails of some of the spermatozoa from drug-treated mice have disorganised mitochondrial sheaths and poor motility. Short-term treatment with 15 mg/kg/day NB-DNJ does not lead to alterations in mating behaviour, sperm counts, reproductive hormone levels, body weight or testis weight (van der Spoel et al., 2002).
Mature spermatozoa from NB-DNJ-treated mice have a reduced ability to bind to the zona pellucida and are incapable of fertilizing oocytes in vitro (Suganuma et al., 2005). The majority of epididymal spermatozoa from NB-DNJ-treated mice cannot activate oocytes following ICSI (Suganuma et al., 2005). However, when the oocytes injected with the abnormal epididymal spermatozoa from drug-treated mice are incubated in the presence of Sr2+, most oocytes (>90%) are fertilized (Suganuma et al., 2005). These Sr2+-activated post-ICSI oocytes develop into live pups at similar rates as oocytes injected with control epididymal spermatozoa (Suganuma et al., 2005). The progeny obtained by ICSI with spermatozoa from NB-DNJ-treated mice develop normally and are fertile (Suganuma et al., 2005). Thus, it has been concluded that the genetic competence of spermatozoa is unaffected by NB-DNJ administration (Suganuma et al., 2005)
The alkylated imino sugars NB-DNJ and NB-DGJ are two of a small number of non-hormonal compounds that can induce reversible infertility in male mice (Cheng et al., 2002; Lyttle and Kopf, 2003; Lee and Cheng, 2004). Considering that NB-DNJ has been licensed for therapeutic use in humans for an unrelated indication, and that NB-DGJ is currently being evaluated in man, it seems that alkylated imino sugars may hold promise as candidate compounds for the development of a non-hormonal male contraceptive. As male contraceptives need to be taken for long periods of time, we have now addressed the following questions. Is a low-dose NB-DNJ regimen sufficient to maintain infertility for several months? If so, is the prolonged infertility reversible? And finally, does long-term low-dose imino sugar administration have any adverse effects?
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Materials and methods |
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Drug administration and mating tests
All animals were housed and sacrificed in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. NB-DNJ (gift from Oxford GlycoSciences, Abingdon, UK/CellTech UK, Slough, Berkshire, UK), as a dry crystalline solid powder, was mixed thoroughly with powdered mouse chow (expanded rat and mouse chow 1, SDS, Witham, Essex, UK) and stored at room temperature. C57BL/6 male mice were fed powdered mouse chow with or without 15 mg/kg/day NB-DNJ for up to 1 year. The mice were 10 weeks old at the start of imino sugar treatment, unless otherwise stated. Natural mating tests were performed as described by van der Spoel et al. (2002)
. Briefly, each male mouse was caged with four female 10-week-old C57BL/6 mice for 9 days, after which the male was removed, and the females were monitored for pregnancies and litter sizes.
Histology
Mice were fixed by perfusion through the left cardiac ventricle with 10 ml of 15 U/ml heparin in phosphate-buffered saline (PBS) followed by 100 ml of 2% glutaraldehyde and 2% paraformaldehyde in PBS. Testes were excised, transferred to 2% glutaraldehyde, 2% paraformaldehyde in PBS for 4 h and then placed in PBS overnight. Tissues were post-fixed in 1% osmium tetroxide and embedded in LR White (TAAB, Aldermaston, Berkshire, UK). Tissues were sectioned and stained with toluidine blue. Images were acquired using a Zeiss Axioskop 2 Plus microscope fitted with an Axiocam camera. Diameters of seminiferous tubules were measured from microscope images using a Kontron image processing system and Zeiss IBAS 2000 software (Image Associates, Oxfordshire, UK). In each mouse, 20–32 tubules with a circular contour were measured (n = 4).
Sperm counts and motility
One cauda/vas deferens per mouse was homogenized in 1 ml M2 medium (Sigma, Poole, Dorset, UK) using a glass homogenizer with teflon pestle. One testis per mouse was homogenized in 1 ml M2 medium, and Triton X-100 was added to a final concentration of 0.05%. Cells were stained with 1.7 µg/ml Hoechst 33342 and counted in 20 µm Standard Count Analysis Chambers (Leja, Nieuw-Vennep, The Netherlands) using Simple PCI image analysis software (Digital Pixel, Brighton, UK). For the evaluation of sperm motility, spermatozoa from freshly excised caudae epididymides were incubated in either M2 medium or in Modified HEPES-buffered HTF medium (Irvine Scientific, Smiths Medical Int., Hythe, Kent, UK) at 37°C for 1 h. Sperm movements were registered by videomicroscopy in 20 µm Standard Count Analysis Chambers on a 37°C stage. For each mouse, three to four recordings of 2 min each were made in independently filled compartments of an Analysis Chamber. Spermatozoa exhibiting any level of progressive motility were scored as motile.
Fluorescence microscopy
The cauda epididymis was dissociated in M2 medium with forceps. Smears of spermatozoa were prepared as described by van der Spoel (2002) and stained by indirect immunofluorescence with monoclonal antibody Mab18.6 (Moore et al., 1985) and flourescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G (Sigma). Hoechst 33342 (333 ng/ml) was used as a nuclear stain. All antibodies were diluted in PBS containing 0.5% (w/v) bovine serum albumin and 0.15 M glycine. Nuclear morphology and acrosomal staining were assessed by examining at least 250 spermatozoa per mouse.
In situ detection of apoptotic cells
Testes fixed as described above were embedded in paraffin and sectioned (at 5 µm). TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick-end labelling) staining was performed using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Oncor, Gaithersburg, MD, USA) according to the manufacturer’s instructions. In each mouse, at least 80 seminiferous tubules were examined.
Endocrinology and serum chemistry
Serum FSH, LH and testosterone levels and testicular testosterone contents were measured as described (Huhtaniemi et al., 1985; Haavisto et al., 1993; van Casteren et al., 2000). Serum markers (urea, creatinine, alanine amino-transferase, alkaline phosphatase, aspartate amino-transferase, total protein, albumin and globulin) were determined with a Clinical Chemical Analyser IL600 (Instrumentation Laboratory, Cheshire, UK).
Behavioral assays
Motor co-ordination and balance were measured with an accelerating Rota-rod (Rota-rod Treadmill 7650, Ugo Basile, Middlesex, UK) using the method of Jones and Roberts (1968). One training session, 48 h prior to the first test, was sufficient to familiarize the mice with the apparatus. Spontaneous activity and exploration was assessed for 5 min in an automated open-field test and recorded with an AM Activity Monitor with AM Logger software (Ugo Basile).
Statistical analyses
Values of experimental animals were tested for significant differences from values of untreated animals. Statistical analyses were performed using GraphPad InStat, version 3.0a (San Diego, USA). All tests were two-tailed with significance set at P 
0.05. One-way analysis of variance (ANOVA) was used to determine significant differences among treatment groups at each timepoint. Normality of variance was assessed by the Kolmogorov–Smirnov test, and normality of SD was calculated according to the Bartlett method. Parametric ANOVA followed by Tukey’s post-hoc test was used if the data met the test criteria of normality of variances and SD. When required, data were transformed to meet the test criteria. For data that did not meet the criteria, nonparametric Kruskal–Wallis ANOVA followed by Dunn’s post-hoc test was performed. For body weight and behavioural analyses, unpaired t-tests were performed. When the SD did not pass the normality test, t-tests with Welch correction were used.
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Results |
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Study design
To evaluate efficacy, reversibility and systemic effects of a long-term low-dose NB-DNJ regimen, the drug was administered to 10-week old male C57BL/6 mice at 15 mg/kg/day for up to 1 year. At 3–4-month intervals of NB-DNJ treatment, the drug was withdrawn from groups of mice, and they were fed normal mouse chow for a further 6 weeks, to recover from the consequences of drug administration. Following 12 months of NB-DNJ treatment, mice were examined after 6–12 weeks after termination of drug administration. At each timepoint, various fertility parameters of untreated, treated and recovered mice were assessed, as well as serum indicators of liver and kidney function. Fertility of male mice in natural mating was assessed after 10 and 12 months of drug administration. In addition, some aspects of mouse behaviour (spontaneous exploratory activity, motor co-ordination and balance) were monitored throughout the treatment period.
Weights
High doses of NB-DNJ (1200–2400 mg/kg/day) have previously been shown to reduce the body weight of mice (Platt et al., 1997). To see the effect of long-term low-dose drug treatment (15 mg/kg/day) on body weight, treated and untreated mice were weighed at 2-week intervals for 1 year following the commencement of drug treatment. After 30 weeks, the weights of treated mice were significantly reduced (P < 0.05), but at all other timepoints, there was no significant difference between control and treated groups (Supplementary Data 1). Testicular weights were significantly reduced (P < 0.05) in treated and recovered groups at the 6-month timepoint, but at no other point during the study (Supplementary Data 2). Epididymal weights were unaffected throughout the study, with the exception of recovered epididymis weights at 12 months which were significantly increased (P < 0.001) (Supplementary Data 2). Thus, prolonged imino sugar exposure did not have a consistent impact on gonadal weights.
Testis histology
The overall appearance of seminiferous tubules was comparable between untreated, treated and recovered mice, except for an increased level of vacuolization in the tubules of imino sugar-exposed animals (Supplementary Data 3), as was seen after 5 weeks of NB-DNJ intake at 50 mg/kg/day (van der Spoel et al., 2002). Morphometric analysis revealed that there were no significant differences in tubule diameter between any of the experimental groups in the long-term study. The diameters of the seminiferous tubules in control, treated and recovered mice were 202 ± 33, 200 ± 24 and 197 ± 23 µm (mean ± SD), respectively, averaged over all timepoints.
One of the hallmarks of early post-meiotic differentiation of a male germ cell is the generation of a dense-core proacrosomal vesicle that associates with and spreads over the nucleus in stage II–III and IV seminiferous tubules (Russell et al., 1990) (Figure 1A and D). In contrast, in stage II–III and IV tubules of NB-DNJ-treated mice, a large proportion of round spermatids lacked a proacrosomal granule and instead displayed step 1 morphology (Figure 1B and E). In stage II–III and IV tubules from animals recovered from imino sugar administration, round spermatids contained similar proacrosomal vesicles as in control tubules (Figure 1C and F).
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In stage VIII–IX seminiferous tubules of normal mice, spermatid nuclei begin to elongate and the acrosome forms a wide cap with a large central granule over the anterior nucleus (Russell et al., 1990) (Figure 1G and J). During stages VIII–IX, spermatids of imino sugar-treated mice elongated, but in a less-regimented fashion than in controls, exhibiting a variety of asymmetrical abnormal nuclear morphologies with most of them without an acrosomal cap (Figure 1H and K). Conversely, in corresponding seminiferous tubules of recovered mice, nuclear shapes of elongating spermatids and acrosomal phenotypes were similar to those of control animals (Figure 1I and L).
Sperm counts
Previously, sperm counts were found to be unaltered after 5 weeks of NB-DNJ treatment at doses up to 150 mg/kg/day (van der Spoel et al., 2002). However, in animals that were provided with 15 mg/kg/day NB-DNJ for 6 months or more, both cauda epididymal and testicular sperm counts were significantly lower than in untreated mice (up to 56 and 34% lower, respectively, Figure 2). The epididymal sperm counts of the mice recovered from 3, 6 and 9 months drug exposure were not significantly different from those of untreated control animals (Figure 2A). The epididymal and testicular sperm counts from mice recovered from 12 months of NB-DNJ administration were also at normal levels (Figure 2A and B). Thus, long-term low-dose administration of the imino sugar caused a reversible depression of sperm numbers.
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) P < 0.001; n = 4.To assess whether the reduction in sperm counts was caused by an increased frequency of germ cell apoptosis, testis sections from long-term drug-treated mice were processed for TUNEL staining. Comparable numbers of apoptotic cells were found in the testes of untreated, treated and recovered mice at the 3-month and 12-month timepoints (Supplementary Data 4). Therefore, the reduction in sperm counts appeared not to be caused by an increased frequency of germ cell apoptosis.
Morphology of spermatozoa
We observed previously that over 90% of cauda epididymal spermatozoa had an abnormal nuclear morphology and a lack of acrosomal antigens after administration of 15 mg/kg/day NB-DNJ for 5 weeks (van der Spoel et al., 2002). Here, we have investigated the kinetics of this phenomenon in more detail over a 5-week drug treatment period and a 25-day recovery period. Between 15 and 35 days of drug exposure, the percentage of cauda epididymal spermatozoa that have a normal nuclear morphology decreased sharply to below 5% (Figure 3A). The percentage of cells with any acrosomal staining (irrespective of staining pattern) and the fraction of cells with normal acrosomal staining also decreased over this period, in close parallel with the reduction in the proportion of cells having a normal nuclear shape (Figure 3A). Furthermore, between 15 and 25 days after termination of the 5-week drug treatment, nuclear morphology and acrosomal staining parameters rose in concert to similar levels as those of untreated mice (Figure 3A).
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), acrosomal staining (irrespective of staining pattern, 
) and normal acrosomal staining (x) were scored during (A) 35 days of N-butyldeoxynojirimycin treatment of 6-week old mice, followed by a 25-day recovery period, and (B) 52 weeks of NB-DNJ treatment of 10-week old mice, followed by a 12-week recovery period. The data are presented as the mean ± SD; (A) n = 5; (B) n = 4, except for 12-month treated/12-week recovered; n = 3.The nuclear and acrosomal morphologies of cauda epididymal spermatozoa were also examined after long-term low-dose NB-DNJ administration. The majority of the epididymal spermatozoa from these mice had abnormal nuclear shapes. This was seen after 3, 6, 9 and 12 months of imino sugar administration (Figure 4B, E, H and K). From 3 to 12 months of drug administration, the percentage of spermatozoa with normal falciform nuclei was very low and did not change throughout this period (range 6–5%, Figure 3B). In mice that had been drug-treated for 6, 10, or 12 months and then maintained for 6 weeks without medication, the percentages of epididymal spermatozoa with a normal nuclear shape were much higher than in drug-treated mice (88 ± 2.6, 65 ± 8.0 and 80 ± 12%, respectively) but significantly lower (P < 0.001) than the corresponding values from untreated mice of the same age (90–98% normal spermatozoa). In contrast, in mice that had been drug-treated for 12 months and then maintained for 12 weeks without medication, the percentage of epididymal spermatozoa with a normal nuclear shape was 91 ± 1.2%, which was comparable to that of untreated mice of the same age (92 ± 1.2% normal spermatozoa, Figure 3B).
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In control spermatozoa, the acrosome is a large, crescent-shaped organelle that lies over the anterior/dorsal part of the nucleus (Figure 4A, D, G and J). In long-term NB-DNJ-administered mice, the percentages of sperm cells with any acrosomal staining (irrespective of staining pattern) ranged from 19.1 to 32.4%, which were significantly lower (P < 0.001) than in age-matched untreated mice (94–96%; Figures 3B and 4B, E, H and K). Many of the spermatozoa from the drug-treated mice that showed acrosomal fluorescence displayed an abnormal staining pattern (Figure 4B, E, H and K), so that, in these mice, the proportions of spermatozoa with a normal acrosomal staining pattern were significantly lower (P < 0.001) than in control animals (4–6% versus 85–90%, respectively; Figure 3B). In mice that had been drug-treated for 3, 6, 10 or 12 months and then maintained for at least 6 weeks without medication, the frequencies and patterns of acrosomal staining were similar to those of age-matched untreated mice (Figures 3B and 4C, F, I and L, data not shown).
Sperm motility
In our first study we found that, after 5 weeks of NB-DNJ administration at 15 mg/kg/day, the percentage of motile spermatozoa was considerably reduced (van der Spoel et al., 2002). After 3, 9 and 12 months of exposure to NB-DNJ, the proportions of motile epididymal spermatozoa were also significantly reduced (Figure 5). In turn, after several months of imino sugar administration and 6 or 9 weeks of withdrawal, the percentages of motile spermatozoa were comparable with those of age-matched control mice (Figure 5).
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) P < 0.01; (
2 for the 12-month timepoint.
Fertility in natural mating
The fertility of 6-, 10- and 12-month NB-DNJ-treated male mice was assessed by natural mating. None of the imino sugar-treated males impregnated female mice (Table I) in spite of the fact that matings took place, as evidenced by post-copulatory vaginal mucous plugs (data not shown). In contrast, the males that had been administered the drug for 6 or 10 months, and then maintained for 6 weeks without medication, respectively, impregnated similar numbers of female mice as age-matched untreated males and sired comparable numbers of pups per litter. Similar results were obtained with male mice that had been treated with NB-DNJ for a whole year and then had been kept without the drug for 9 weeks (Table I).
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Endocrinology and serum chemistry
We determined the serum concentrations of LH, FSH and testosterone and the testicular testosterone levels in 3-, 6-, 9- and 12-month NB-DNJ-administered male mice and in males that had been maintained for 6 weeks after cessation of each of these imino sugar treatments. Within each of the timepoints, the LH, FSH and testosterone levels of untreated, treated and recovered mice did not differ significantly (Supplementary Data 5). Thus, the drug administration did not alter the levels of these reproductive hormones.
To monitor potential consequences of imino sugar administration on liver and kidney functions, we also determined the serum levels of alanine amino-transferase (ALT), aspartate amino-transferase, alkaline phosphatase (ALP), protein, creatinine and urea (Supplementary Data 6). The values of these serum markers in NB-DNJ-treated or recovered mice did not significantly differ from control values, except for three measurements. At 3 months, the urea level in recovered mice was significantly decreased (7.53±0.5 mmol/l in comparison to 9.45±0.4 mmol/l in controls, P < 0.01), at 12 months ALT activity was significantly lower in recovered mice (32.3±13.9 U/l in comparison to 97.8±74.1 U/l in controls, P < 0.05) and at 12 months ALP activity in treated mice was significantly reduced (102.5±28.8 U/l in comparison to 235.3±47.0 U/l in controls, P < 0.01). Thus, these serum markers of liver and kidney functions were not consistently affected by long-term NB-DNJ administration.
Mouse behaviour
Rotarod and open field behavioural tests were carried out at 3-month intervals with NB-DNJ-treated mice. Co-ordination and muscle strength (as assessed in the Rota-rod test) and spontaneous exploratory behaviour (as assessed in the open field test) were not significantly affected by drug treatment (Supplementary Data 7). Only at the initial time point, in the Rotarod test, the length of time it took imino sugar-administered mice to lose their footing on the rotating rod was significantly longer than untreated mice (P < 0.05). These results indicate that long-term imino sugar administration did not alter motor co-ordination, muscle strength or exploratory behaviour.
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Discussion |
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Previously, we reported that NB-DNJ causes reversible infertility in male C57BL/6 mice after 5 weeks of treatment (van der Spoel et al., 2002
). In order to determine whether NB-DNJ is suitable for administration over prolonged periods of time, we have investigated the effects of low-dose administration of this imino sugar in mice for up to 1 year. Two aspects have been studied here: the effectiveness of NB-DNJ as a reversible disrupter of spermatogenesis and the potential occurrence of adverse consequences, including both testis-specific and general effects. Firstly, mating assays demonstrated that all 6-, 10- and 12-month NB-DNJ-treated mice were infertile and that these mice had regained their fertility 6–9 weeks after withdrawal of drug administration. None of the drug-treated mice were able to induce pregnancies, whereas all of the recovered mice did. These results show that low-dose administration of NB-DNJ is an effective method to induce and maintain reversible infertility in male mice for a long period of time. Secondly, we have established that long-term low-dose NB-DNJ treatment does not affect body or gonad weights, reproductive endocrinology, serum chemistry or animal behaviour. These data indicate that a low-dose NB-DNJ regimen does not cause overt adverse effects upon prolonged administration and is thus well tolerated.
As with short-term NB-DNJ treatment, the morphology of epididymal spermatozoa was radically altered during long-term drug administration. In long-term treated mice, the proportion of cells with normal nuclear morphology and normal acrosomal staining was greatly reduced, to the same degree as observed in 5-week treated mice. The percentage of spermatozoa with progressive motility was also considerably lower after several months of NB-DNJ treatment. However, the consequences of long-term imino sugar exposure differed in two ways from those of short-term treatment. Firstly, the percentage of epididymal spermatozoa with any anti-acrosomal antibody staining (irrespective of staining pattern) was higher after 3 or more months of NB-DNJ treatment than after 5 weeks. Secondly, we have observed that treatment with low-dose NB-DNJ for more than 3 months leads to a considerable reduction in both testicular and epididymal sperm counts, while it has previously been noted that short-term low-dose treatment does not affect sperm counts (van der Spoel et al., 2002). Thus, compared to short-term imino sugar ingestion, long-term treatment results in the production of fewer spermatozoa, of which a higher proportion carries acrosomal antigens. It must be noted that most of the spermatozoa from long-term drug-treated mice that carry acrosomal antigens have an abnormal acrosomal staining pattern, similar to short-term treated mice. It is unlikely that an incomplete acrosome is capable of performing the acrosome reaction.
It is notable that the sperm counts are reduced only after more than 3 months of NB-DNJ administration. Since this happens much later than the first observed effects of the imino sugar (disruption of spermiogenesis and production of abnormal spermatozoa), it is likely that the reduction in sperm numbers is a secondary (indirect) effect of the drug and is brought about by the prolonged disturbance of spermiogenesis. Having established by TUNEL staining that long-term drug intake does not enhance germ cell apoptosis, it remains to be established how long-term imino sugar administration causes a reduction in sperm counts. In addition, it is unclear at present why the proportion of epididymal spermatozoa that carry acrosomal antigens is higher in long-term dosed mice than in 5-week treated males.
Another difference between long- and short-term NB-DNJ treatment is in the recovery after cessation of drug administration, in particular, the length of time it took the animals to produce normal numbers of spermatozoa with normal nuclear morphology. After a 5-week treatment and a 25-day recovery period, the proportion of cells with normal nuclear morphology was comparable to that of control mice. In contrast, in animals that had taken the drug for 6 months or longer and then had the drug withdrawn for 6 weeks, the number of spermatozoa with normal nuclear morphology was significantly (10%) lower than in control mice. However, 12-month drug-treated mice that were maintained without medication for 12 weeks produced similar numbers of spermatozoa with normal nuclear morphology as untreated control mice. This suggests that following long-term NB-DNJ treatment, a longer period of recovery is required for nuclear morphology to return to normal. We can assume that the 10-month dosed/6-week recovered males also had a relative deficit in normal spermatozoa compared to age-matched control animals. In spite of this, these recovered males were equally successful in natural mating tests as the age-matched controls.
To summarize, we have demonstrated the efficacy and reversibility of long-term low-dose NB-DNJ administration in inducing infertility in male C57BL/6 mice. Our study shows that long-term imino sugar-treated mice are infertile because of the combined consequences of the drug on sperm number, sperm motility, sperm nuclear morphology and acrosomal phenotype. In addition, we have not found any evidence for cumulative toxicity caused by prolonged low-dose NB-DNJ treatment. Having investigated the effects of NB-DNJ on the fertility of male mice, the drug now merits evaluation in other species.
It must be noted that the dose of NB-DNJ used here (15 mg/kg/day) is at least an order of magnitude lower than the dose needed to prevent or delay accumulation of glycosphingolipids in mouse models of glycosphingolipid storage diseases (Jeyakumar et al., 1999; Andersson et al., 2004). Therefore, if spermatogenesis in humans is equally sensitive to the drug as in mice, we estimate that the dose required for use as a non-hormonal contraceptive in men is at least an order of magnitude lower than that prescribed for type 1 Gaucher disease patients, which is 100–300 mg/day (Cox et al., 2000).
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Acknowledgements |
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We thank David Smith and Denise Jelfs and co-workers (Department of Biochemistry, University of Oxford, UK) for expert technical assistance, Mohan Masih for excellent histology services, Lee-Anne Tupper for help with morphometric analysis (Department of Human Anatomy and Genetics, University of Oxford, UK), Prof Harry D. Moore (University of Sheffield, Sheffield, UK) for provision of the Mab 18.6 monoclonal antibody, Dr Richard Callaghan (Nuffield Department of Clinical Biochemistry and Cellular Science, University of Oxford, UK) for assistance with histology, and Prof Ilpo Huhtaniemi, Dr Pirjo Pakarinen and Ms Tarja Laiho (University of Turku, Turku, Finland) for analysis of reproductive endocrinology. This study was supported by The Oxford Glycobiology Institute, Oxford GlycoSciences/Celltech UK (C.M.W.) and NIH Grant 1 U01 HD45861 (F.M.P. and A.C.S.).
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Submitted on October 13, 2005; resubmitted on November 14, 2005; accepted on November 17, 2005.
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