Truncated mutants of beta-glucosidase 2 (GBA2) are localized in the mitochondrial matrix and cause mitochondrial fragmentation.

The enzyme ß-glucosidase 2 (GBA2) is clinically relevant because it is targeted by the drug miglustat (Zavesca®) and because it is involved in inherited diseases. Mutations in the GBA2 gene are associated with two neurological diseases on the ataxia-spasticity spectrum, hereditary spastic paraplegia 46 (SPG46) and Marinesco-Sjögren-like syndrome (MSS). To establish how GBA2 mutations give rise to neurological pathology, we have begun to investigate mutant forms of GBA2 encoded by disease-associated GBA2 alleles. Previously, we found that five GBA2 missense mutants and five C-terminally truncated mutants lacked enzyme activity. Here we have examined the cellular locations of wild-type (WT) and mutant forms of GBA2 by confocal and electron microscopy, using transfected cells. Similar to GBA2-WT, the D594H and M510Vfs*17 GBA2 mutants were located at the plasma membrane, whereas the C-terminally truncated mutants terminating after amino acids 233 and 339 (GBA2-233 and -339) were present in the mitochondrial matrix, induced mitochondrial fragmentation and loss of mitochondrial transmembrane potential. Deletional mutagenesis indicated that residues 161-200 are critical for the mitochondrial fragmentation of GBA2-233 and -339. Considering that the mitochondrial fragmentation induced by GBA2-233 and -339 is consistently accompanied by their localization to the mitochondrial matrix, our deletional analysis raises the possibility that that GBA2 residues 161-200 harbor an internal targeting sequence for transport to the mitochondrial matrix. Altogether, our work provides new insights into the behaviour of GBA2-WT and disease-associated forms of GBA2.

Correction to: Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson's disease.

The original article [1] contains an error in the y-axes of Fig. 8's sub-figures whereby 'CSF' is mistakenly mentioned instead of 'serum'.

Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson's disease.

BACKGROUND: Haploinsufficiency in the Gaucher disease GBA gene, which encodes the lysosomal glucocerebrosidase GBA, and ageing represent major risk factors for developing Parkinson's disease (PD). Recently, more than fifty other lysosomal storage disorder gene variants have been identified in PD, implicating lysosomal dysfunction more broadly as a key risk factor for PD. Despite the evidence of multiple lysosomal genetic risks, it remains unclear how sphingolipid hydrolase activities, other than GBA, are altered with ageing or in PD. Moreover, it is not fully known if levels of glycosphingolipid substrates for these enzymes change in vulnerable brain regions of PD. Finally, little is known about the levels of complex gangliosides in substantia nigra which may play a significant role in ageing and PD. METHODS: To study sphingolipid hydrolase activities and glycosphingolipid expression in ageing and in PD, two independent cohorts of human substantia nigra tissues were obtained. Fluorescent 4-methylumbelliferone assays were used to determine multiple enzyme activities. The lysosomal GBA and non-lysosomal GBA2 activities were distinguished using the inhibitor NB-DGJ. Sensitive and quantitative normal-phase HPLC was performed to study glycosphingolipid levels. In addition, glycosphingolipid levels in cerebrospinal fluid and serum were analysed as possible biomarkers for PD. RESULTS: The present study demonstrates, in two independent cohorts of human post-mortem substantia nigra, that sporadic PD is associated with deficiencies in multiple lysosomal hydrolases (e.g. α-galactosidase and ß-hexosaminidase), in addition to reduced GBA and GBA2 activities and concomitant glycosphingolipid substrate accumulation. Furthermore, the data show significant reductions in levels of complex gangliosides (e.g. GM1a) in substantia nigra, CSF and serum in ageing, PD, and REM sleep behaviour disorder, which is a strong predictor of PD. CONCLUSIONS: These findings conclusively demonstrate reductions in GBA activity in the parkinsonian midbrain, and for the first time, reductions in the activity of several other sphingolipid hydrolases. Furthermore, significant reductions were seen in complex gangliosides in PD and ageing. The diminished activities of these lysosomal hydrolases, the glycosphingolipid substrate accumulation, and the reduced levels of complex gangliosides are likely major contributors to the primary development of the pathology seen in PD and related disorders with age.

19q13.12 microdeletion syndrome fibroblasts display abnormal storage of cholesterol and sphingolipids in the endo-lysosomal system.

Microdeletions in 19q12q13.12 cause a rare and complex haploinsufficiency syndrome characterized by intellectual deficiency, developmental delays, and neurological movement disorders. Variability in the size and interval of the deletions makes it difficult to attribute the complex clinical phenotype of this syndrome to an underlying gene(s). As an alternate approach, we examined the biochemical and metabolic features of fibroblasts from an affected individual to derive clues as to the molecular basis for the syndrome. Immunofluorescence and electron microscopy of affected fibroblasts revealed an abnormal endo-lysosomal compartment that was characterized by rapid accumulation of lysosomotropic dyes, elevated LAMP1 and LAMP2 expression and vacuoles containing membrane whorls, common features of lysosomal lipid storage disorders. The late endosomes-lysosomes (LE/LY) of affected fibroblasts accumulated low-density lipoprotein cholesterol, and displayed reduced cholesterol esterification and increased de novo cholesterol synthesis, indicative of defective cholesterol transport to the endoplasmic reticulum. Affected fibroblasts also had increased ceramide and sphingolipid mass, altered glycosphingolipid species and accumulation of a fluorescent lactosylceramide probe in LE/LY. Autophagosomes also accumulated in affected fibroblasts because of decreased fusion with autolysosomes, a defect associated with other lysosomal storage diseases. Attempts to correct the cholesterol/sphingolipid storage defect in fibroblasts with cyclodextrin, sphingolipid synthesis inhibitors or by altering ion transport were unsuccessful. Our data show that 19q13.12 deletion fibroblasts have abnormal accumulation of cholesterol and sphingolipids in the endo-lysosomal system that compromises organelle function and could be an underlying cause of the clinical features of the syndrome.

Characterization of the Zebrafish Homolog of ß-Glucosidase 2: A Target of the Drug Miglustat.

The small-molecular compound miglustat (N-butyldeoxynojirimycin, Zavesca(®)) has been approved for clinical use in type 1 Gaucher disease and Niemann-Pick type C disease, which are disorders caused by dysfunction of the endosomal-autophagic-lysosomal system. Miglustat inhibits a number of enzymes involved in glycoconjugate and glycan metabolism, including ß-glucosidase 2 (GBA2), which is exceptionally sensitive to inhibition by miglustat. GBA2 is a glucosylceramide-degrading enzyme that is located on the plasma membrane/endoplasmic reticulum, and is distinct from the lysosomal enzyme glucocerebrosidase (GBA). Various strands of evidence suggest that inhibition of GBA2 contributes to the therapeutic benefits of miglustat. To further explore the pharmacology and biology of GBA2, we investigated whether the zebrafish homolog of GBA2 has similar enzymatic properties and pharmacological sensitivities to its human counterpart. We established that zebrafish has endogenous ß-glucosidase activity toward lipid- and water-soluble GBA2 substrates, which can be inhibited by miglustat, N-butyldeoxygalactonojirimycin, and conduritol B epoxide. ß-Glucosidase activities with highly similar characteristics were expressed in cells transfected with the zebrafish gba2 cDNA and in cells transfected with the human GBA2 cDNA. These results provide a foundation for the use of zebrafish in screening GBA2-targeting molecules, and for wider studies investigating GBA2 biology.

Verification and refinement of cellular glycosphingolipid profiles using HPLC.

Glycosphingolipids (GSLs) are hybrid molecules consisting of the sphingolipid ceramide linked to a mono- or oligo-saccharide. In comparison to other membrane lipids, the family of GSLs stands out because of the extensive variation in the carbohydrate headgroup. GSLs are cell surface binding partners, in cis with growth factor receptors, and in trans with bacterial toxins and viruses, and are among the host-derived membrane components of viral particles, including those of HIV. In spite of their biological relevance, GSL profiles of commonly used cell lines have been analyzed to different degrees. Here, we directly compare the GSL complements from CHO-K1, COS-7, HeLa, HEK-293, HEPG2, Jurkat, and SH-SY5Y cells using an HPLC-based method requiring modest amounts of material. Compared to previous studies, the HPLC-based analyses provided more detailed information on the complexity of the cellular GSL complement, qualitatively as well as quantitatively. In particular for cells expressing multiple GSLs, we found higher numbers of GSL species, and different levels of abundance. Our study thus extends our knowledge of biologically relevant lipids in widely used cell lines.

Lack of enzyme activity in GBA2 mutants associated with hereditary spastic paraplegia/cerebellar ataxia (SPG46).

Glucosylceramide is a membrane glycolipid made up of the sphingolipid ceramide and glucose, and has a wide intracellular distribution. Glucosylceramide is degraded to ceramide and glucose by distinct, non-homologous enzymes, including glucocerebrosidase (GBA), localized in the endolysosomal pathway, and ß-glucosidase 2 (GBA2), which is associated with the plasma membrane and/or the endoplasmic reticulum. It is well established that mutations in the GBA gene result in endolysosomal glucosylceramide accumulation, which triggers Gaucher disease. In contrast, the biological significance of GBA2 is less well understood. GBA2-deficient mice present with male infertility, but humans carrying mutations in the GBA2 gene are affected with a combination of cerebellar ataxia and spastic paraplegia, as well as with thin corpus callosum and cognitive impairment (SPastic Gait locus #46, SPG46). To improve our understanding of the biochemical consequences of the GBA2 mutations, we have evaluated five nonsense and five missense GBA2 mutants for their enzyme activity. In transfected cells, the mutant forms of GBA2 were present in widely different amounts, ranging from overabundant to very minor, compared to the wild type enzyme. Nevertheless, none of the GBA2 mutant cDNAs raised the enzyme activity in transfected cells, in contrast to the wild-type enzyme. These results suggest that SPG46 patients have a severe deficit in GBA2 activity, because the GBA2 mutants are intrinsically inactive and/or reduced in amount. This assessment of the expression levels and enzyme activities of mutant forms of GBA2 offers a first insight in the biochemical basis of the complex pathologies seen in SPG46.

ß-Glucosidase 2 (GBA2) activity and imino sugar pharmacology.

ß-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher KI) and a lower rate of enzyme inactivation (k(inact)). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the ß-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5-6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated ß-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.

Glycomimetic affinity-enrichment proteomics identifies partners for a clinically-utilized iminosugar.

Widescale evaluation of interacting partners for carbohydrates is an underexploited area. Probing of the 'glyco-interactome' has particular relevance given the lack of direct genetic control of glycoconjugate biosynthesis. Here we design, create and utilize a natural product-derived glycomimetic iminosugar probe in a Glycomimetic Affinity-enrichment Proteomics (glyco-AeP) strategy to elucidate key interactions directly from mammalian tissue. The binding partners discovered here and the associated genomic analysis implicate a subset of chaperone and junctional proteins as important in male fertility. Such repurposing of existing therapeutics thus creates direct routes to probing in vivo function. The success of this strategy suggests a general approach to discovering 'carbohydrate-active' partners in biology.

The cell biology of disease: lysosomal storage disorders: the cellular impact of lysosomal dysfunction.

Lysosomal storage diseases (LSDs) are a family of disorders that result from inherited gene mutations that perturb lysosomal homeostasis. LSDs mainly stem from deficiencies in lysosomal enzymes, but also in some non-enzymatic lysosomal proteins, which lead to abnormal storage of macromolecular substrates. Valuable insights into lysosome functions have emerged from research into these diseases. In addition to primary lysosomal dysfunction, cellular pathways associated with other membrane-bound organelles are perturbed in these disorders. Through selective examples, we illustrate why the term "cellular storage disorders" may be a more appropriate description of these diseases and discuss therapies that can alleviate storage and restore normal cellular function.

Fusion failure of dense-cored proacrosomal vesicles in an inducible mouse model of male infertility.

The acrosome is a specialized secretory vesicle located in the head of spermatozoa and has an essential role during fertilization. This organelle and the sperm nucleus have aberrant morphologies in forms of male infertility in humans (teratozoospermia), often associated with poor motility (asthenoteratozoospermia). To further our understanding of the aetiology of these conditions, we have performed a pathological investigation of a model of asthenoteratozoospermia that can be induced in mice by N-butyldeoxynojirimycin (NB-DNJ). We have found that, in mice treated with NB-DNJ, instead of an acrosome forming over the round spermatid nucleus, multivesicular bodies (MVB) accumulate in the vicinity of this nucleus. Electron microscopy has revealed that proacrosomic vesicles or granules (PAG) secreted during the Golgi phase of spermiogenesis do not fuse together to form an acrosomic vesicle, but rather attach transiently to the spermatid nucleus. Immunocytochemistry has shown that acrosomal membrane proteins and cytosolic acrosome-associated proteins are redirected to MVB in affected testes, whereas glycoproteins originating in the dense core of the PAG are degraded. Thus, the major effect of NB-DNJ is to inhibit membrane fusion of Golgi-derived secretory vesicles destined for acrosome formation, raising the possibility that these vesicles are critically affected in forms of (astheno)teratozoospermia.

Differential sensitivity of mouse strains to an N-alkylated imino sugar: glycosphingolipid metabolism and acrosome formation.

This review deals with the pharmacological properties of an alkylated monosaccharide mimetic, N-butyldeoxynojirimycin (NB-DNJ). This compound is of pharmacogenetic interest because one of its biological effects in mice - impairment of spermatogenesis, leading to male infertility - depends greatly on the genetic background of the animal. In susceptible mice, administration of NB-DNJ perturbs the formation of an organelle, the acrosome, in early post-meiotic male germ cells. In all recipient mice, irrespective of reproductive phenotype, NB-DNJ has a similar biochemical effect: inhibition of the glucosylceramidase beta-glucosidase 2 and subsequent elevation of glucosylceramide, a glycosphingolipid. The questions that we now need to address are: how can glucosylceramide specifically affect early acrosome formation, and why is this contingent on genetic factors? Here we discuss relevant aspects of reproductive biology, the metabolism and cell biology of sphingolipids, and complex trait analysis; we also present a speculative model that takes our observations into account.

Beneficial effects of substrate reduction therapy in a mouse model of GM1 gangliosidosis.

GM1 gangliosidosis is an inherited neurodegenerative disorder caused by lysosomal beta-galactosidase deficiency, resulting in the storage of GM1 and GA1, primarily in the central nervous system. This disease typically afflicts infants and young children and there is currently no effective therapy. Substrate reduction therapy (SRT) could be of potential benefit. The imino sugars N-butyldeoxynojirimycin (NB-DNJ, miglustat, Zavesca) and N-butyldeoxygalactonojirimycin (NB-DGJ) used for SRT inhibit glucosylceramide synthase (GlcCerS) that catalyses the first committed step in glycosphingolipid biosynthesis. We have compared the efficacy and tolerability of NB-DNJ and NB-DGJ in the beta-galactosidase knockout mouse. NB-DGJ was better tolerated than NB-DNJ, due to intrinsic gastrointestinal tract dysfunction that was exacerbated by NB-DNJ. However, functional improvement was greatest with NB-DNJ treatment which may potentially be caused by novel anti-inflammatory properties of NB-DNJ.

Male germ cells require polyenoic sphingolipids with complex glycosylation for completion of meiosis: a link to ceramide synthase-3.

Previously, it was found that a novel class of neutral fucosylated glycosphingolipids (GSLs) is required for male fertility. These lipids contain very long-chain (C26-C32) polyunsaturated (4-6 double bonds) fatty acid residues (VLC-PUFAs). To assess the role of these complex GSLs in spermatogenesis, we have now investigated with which of the testicular cell types these lipids are associated. During postnatal development, complex glycosylated and simple VLC-PUFA sphingolipids were first detectable at day 15, when the most advanced germ cells are pachytene spermatocytes. Their synthesis is most likely driven by ceramide synthase-3. This enzyme is encoded by the Cers3/Lass3 gene (longevity assurance genes), and out of six members of this gene family, only Cers3 mRNA expression was limited to germ cells, where it was up-regulated more than 700-fold during postnatal testicular maturation. Increasing levels of neutral complex VLC-PUFA GSLs also correlated with the progression of spermatogenesis in a series of male sterile mutants with arrests at different stages of spermatogenesis. Remarkably, fucosylation of the complex VLC-PUFA GSLs was not essential for spermatogenesis, as fucosylation-deficient mice produced nonfucosylated versions of the complex testicular VLC-PUFA GSLs, had complete spermatogenesis, and were fertile. Nevertheless, sterile Galgt1(-/-) mice, with a defective meiotic cytokinesis and a subsequent block in spermiogenesis, lacked complex but contained simple VLC-PUFA GSLs, as well as VLC-PUFA ceramides and sphingomyelins, indicating that the latter lipids are not sufficient for completion of spermatogenesis. Thus, our data imply that both glycans and the particular acyl chains of germinal sphingolipids are relevant for proper completion of meiosis.

The postacrosomal assembly of sperm head protein, PAWP, is independent of acrosome formation and dependent on microtubular manchette transport.

PAWP (postacrosomal sheath WW domain-binding protein) exclusively resides in the postacrosomal sheath (PAS) of the sperm perinuclear theca (PT). Because of the importance of this region in initiating oocyte activation during mammalian fertilization [Sutovsky, P., Manandhar, G., Wu, A., Oko, R., 2003. Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction. Microsc. Res. Tech. 61, 362-378; Wu, A., Sutovsky, P., Manandhar, G., Xu, W., Katayama, M., Day, B.N., Park, K.W., Yi, Y.J., Xi, Y.W., Prather, R.S., Oko, R., 2007. PAWP, A sperm specific ww-domain binding protein, promotes meiotic resumption and pronuclear development during fertilization. J. Biol. Chem. 282, 12164-12175], we were interested in resolving the origin and assembly of its proteins during spermatogenesis, utilizing PAWP as a model. Based on previous PT developmental studies, we predicted that the assembly of PAWP is dependent on microtubule-manchette protein transport and manchette descent and independent of subacrosomal PT formation. Consequently, we hypothesized that PAWP will colocalize with manchette microtubules during spermiogenesis. Utilizing specific antibodies, PAWP was first detected in the cytoplasmic lobe of spermatids beginning to undergo elongation and became most prominent in this region just prior to and during manchette descent. During this peak period, PAWP was concentrated over the manchette and colocalized with alpha- and beta-tubulin. It was then assembled as part of the PAS in the wake of manchette descent over the caudal half of the elongated spermatid nucleus. PAWP mRNA, on the other hand, was first detected in mid-pachytene spermatocytes, peaked by early round spermatids, and declined during spermatid elongation. In order to confirm that PAWP-PAS assembly was independent of subacrosomal PT development, PAWP immunolocalization was performed on the testes of NB-DNJ-treated mice which fail to form an acrosome and subacrosomal layer during spermiogenesis [van der Spoel, A.C., Jeyakumar, M., Butters, T.D., Charlton, H.M., Moore, H.D., Dwek, R.A., Platt, F.M., 2002. Reversible infertility in male mice after oral administration of alkylated imino sugars: a nonhormonal approach to male contraception. Proc. Natl. Acad. Sci. U.S.A. 99, 17173-17178] but whose elongated spermatids still retain egg-activating ability [Suganuma, R., Walden, C.M., Butters, T.D., Platt, F.M., Dwek, R.A., Yanagimachi, R., and van der Spoel, A.C., 2005. Alkylated imino sugars, reversible male infertility-inducing agents, do not affect the genetic integrity of male mouse germ cells during short-term treatment despite induction of sperm deformities. Biol. Reprod. 72, 805-813]. The same temporal and manchette-based pattern of PAWP-PAS assembly during spermiogenesis was evident as in controls supporting our hypothesis that PAS assembly is independent of subacrosomal PT formation and that egg-activating ability resides within the PAS.

Accumulation of glucosylceramide in murine testis, caused by inhibition of beta-glucosidase 2: implications for spermatogenesis.

One of the hallmarks of male germ cell development is the formation of a specialized secretory organelle, the acrosome. This process can be pharmacologically disturbed in C57BL/6 mice, and thus infertility can be induced, by small molecular sugar-like compounds (alkylated imino sugars). Here the biochemical basis of this effect has been investigated. Our findings suggest that in vivo alkylated imino sugars primarily interact with the non-lysosomal glucosylceramidase. This enzyme cleaves glucosylceramide into glucose and ceramide, is sensitive to imino sugars in vitro, and has been characterized as beta-glucosidase 2 (GBA2). Imino sugars raised the level of glucosylceramide in brain, spleen, and testis, in a dose-dependent fashion. In testis, multiple species of glucosylceramide were similarly elevated, those having long acyl chains (C16-24), as well as those with very long polyunsaturated acyl chains (C28-30:5). Both of these GlcCer species were also increased in the testes from GBA2-deficient mice. When considering that the very long polyunsaturated sphingolipids are restricted to germ cells, these results indicate that in the testis GBA2 is present in both somatic and germ cells. Furthermore, in all mouse strains tested imino sugar treatment caused a rise in testicular glucosylceramide, even in a number of strains, of which the males remain fertile after drug administration. Therefore, it appears that acrosome formation can be derailed by accumulation of glucosylceramide in an extralysosomal localization, and that the sensitivity of male germ cells to glucosylceramide is genetically determined.

Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.

The sensitivity of murine spermiogenesis to miglustat is a quantitative trait: a pharmacogenetic study.

BACKGROUND: A major event in the post-meiotic development of male germ cells is the formation of the acrosome. This process can be perturbed in C57BL/6 mice by administration of the small molecule miglustat (N-butyldeoxynojirimycin, NB-DNJ). The miglustat-treated mice produce morphologically abnormal spermatozoa that lack acrosomes and are poorly motile. In C57BL/6 mice, miglustat can be used to maintain long-term reversible infertility. In contrast, when miglustat was evaluated in normal men, it did not affect spermatogenesis. To gain more insight into this species difference we have now evaluated the reproductive effects of miglustat in rabbits, in multiple mouse strains and in interstrain hybrid mice. METHODS: Male mice of 18 inbred strains were administered miglustat orally or via miniosmotic pumps. Rabbits were given the compound in their food. Fourth-generation interstrain hybrid mice, bred from C57BL/6 and FVB/N mice (which differ in their response to miglustat), also received the drug. Data on fertility (natural mating), sperm motility and morphology, acrosome status, and serum drug levels were collected. RESULTS: In rabbits the drug did not induce aberrations of sperm shape or motility, although the serum level of miglustat in rabbits far exceeded the level in C57BL/6 mice (8.4 microM and 0.5 microM, respectively). In some strains of the Swiss and Castle lineages of inbred mice miglustat did not cause infertility, severe morphological sperm aberrations or reduced sperm motility. In these strains miglustat only had milder effects. However, miglustat strongly disturbed acrosome and sperm nucleus development in AKR/J and BALB/c mice and in a number of C57BL/6-related strains. The consequences of drug administration in the interstrain hybrid mice were highly variable. Judging by the number of grossly abnormal spermatozoa, these genetically heterogeneous mice displayed a continuous range of intermediate responses, distinct from either of their parental strains. CONCLUSION: The effects of miglustat on spermatogenesis in mice are strain-dependent, while in rabbits the drug is ineffective. Evaluation of interstrain hybrid mice indicated that the sensitivity of spermatogenesis to miglustat is a quantitative trait. These studies pave the way for identifying the genetic factors underlying the strain/species differences in the effect of miglustat.

Long-term non-hormonal male contraception in mice using N-butyldeoxynojirimycin.

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.

Alkylated imino sugars, reversible male infertility-inducing agents, do not affect the genetic integrity of male mouse germ cells during short-term treatment despite induction of sperm deformities.

Reversible infertility can be induced in male mice by oral administration of the alkylated imino sugars N-butyldeoxynojirimycin (NB-DNJ) and N-butyldeoxygalactonojirimycin (NB-DGJ). Spermatozoa of these mice have grossly misshapen heads and reduced motility. Because NB-DNJ and related compounds may hold promise as nonhormonal male contraceptives, a comprehensive examination of their effects on male reproduction is necessary. To this end, we further examined reproductive properties of the dysmorphic spermatozoa that are produced after short-term imino sugar administration at the minimal dose that completely abolishes the ability of male C57BL/6 mice to produce offspring by natural mating. Here, we report that, in vitro, the abnormal spermatozoa from the NB-DNJ- and NB-DGJ-treated mice were unable to fertilize oocytes. In addition, we investigated whether the imino sugars damage the genetic integrity of spermatozoa. To test this, we microsurgically injected deformed spermatozoa from imino sugar-treated males into oocytes. The deformed spermatozoa from the testis were able to activate oocytes very efficiently, but those from the cauda epididymis often failed to do so. This problem was overcome when the sperm-injected oocytes were treated with a parthenogenetic agent, Sr(2+). Oocytes injected with the misshapen spermatozoa from NB-DNJ- and NB-DGJ-treated mice developed (with or without Sr(2+) treatment) into live offspring that grew normally and were normally fertile. This indicates that during short-term administration, alkylated imino sugars alter sperm morphology and physiology but do not diminish the genetic potential of spermatozoa.