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BOR - Papers in Press, published online ahead of print December 1, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.036053
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BIOLOGY OF REPRODUCTION 72, 805–813 (2005)
DOI: 10.1095/biolreprod.104.036053
© 2005 by the Society for the Study of Reproduction, Inc.

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 Deformities1

Ryota Suganuma3,4,, Charlotte M. Walden5, Terry D. Butters5, Frances M. Platt5, Raymond A. Dwek5, Ryuzo Yanagimachi3, and Aarnoud C. van der Spoel2,5

Institute for Biogenesis Research,3 University of Hawaii School of Medicine, Honolulu, Hawaii 96822 Department of Obstetrics and Gynecology,4 Fukushima Medical University, Fukushima, 960-1295, Japan The Glycobiology Institute,5 Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, Sr2+. Oocytes injected with the misshapen spermatozoa from NB-DNJ- and NB-DGJ-treated mice developed (with or without Sr2+ 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.

assisted reproduction technology, calcium, fertilization, gamete biology, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Administration of N-butyldeoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) renders male mice infertile [1]. Male mice that have been fed with these drugs produce morphologically abnormal spermatozoa that are poorly motile and display abnormal nuclear and acrosomal shapes. Severe structural and functional defects deprive spermatozoa of their fertilizing capacity (for review, see [2, 3]).

Both NB-DNJ and NB-DGJ are alkylated imino sugars. The biochemical properties of these alkylated compounds have been compared with those of deoxynojirimycin (DNJ), an unalkylated imino sugar. These three compounds inhibit enzymes that are involved in the metabolism of various glycoconjugates, and they share some, but not all, of their enzyme targets [4–6]. Both DNJ and NB-DNJ are inhibitors of {alpha}-glucosidases I and II [7], which are located in the lumen of the endoplasmic reticulum and are involved in the processing of asparagine-linked glycans on glycoproteins. However, because NB-DNJ and NB-DGJ, but not DNJ, affect the fertility of male mice, it was inferred that the biochemical basis of the contraceptive effect resides in a property that is common to the alkylated imino sugars [1]. To date, NB-DNJ and NB-DGJ are known as the inhibitors of two distinct enzymes, nonlysosomal glucosylceramidase [8] and ceramide-specific glucosyltransferase (UDP-glucose:N-acylsphingosine glucosyltransferase) [4, 5]. Nonlysosomal glucosylceramidase has been identified as the conduritol-ß-epoxide-resistant ß-glucosidase that can hydrolyze the glycosphingolipid glucosylceramide [9]. The ceramide-specific glucosyltransferase catalyzes the transfer of glucose to ceramide to produce glucosylceramide; this reaction is the first step in the biosynthetic pathway of glucosylceramide-based glycosphingolipids (GSLs), which are a family of approximately 400 structurally distinct glycolipids [10]. To our knowledge, whether the interaction of alkylated imino sugars with either of these two enzymes is responsible for their effects on spermatogenesis remains to be established.

In vivo, the inhibition of the ceramide-specific glucosyltransferase by the alkylated imino sugars results in the reduction of GSL levels [6, 11]. Because of this, NB-DNJ and NB-DGJ have been evaluated as therapeutic agents in mouse models of pathologic conditions that are caused by a diminished capacity to degrade GSLs, such as the lysosomal storage disorders Tay-Sachs disease [12] and Sandhoff disease [13]. In fact, both NB-DNJ and NB-DGJ delay the onset of symptoms and increase the life span of the mice with Sandhoff disease [13, 14]. NB-DNJ has been extensively tested in various mammals, including humans [15], and recently has been approved for clinical use in patients with type 1 Gaucher disease (for review, see [16– 19]).

To evaluate their suitability as male contraceptives, NB-DNJ and related drugs must be investigated carefully for their effects on male reproduction in animals. To distinguish between the distinct biochemical properties of NB-DNJ, we compared the effects of this compound with those of NB-DGJ and DNJ, both because NB-DGJ is a more specific inhibitor than NB-DNJ for enzymes that are involved in sphingolipid metabolism and because DNJ does not interfere with sphingolipid metabolism. We examined the consequences of administration of these three imino sugars for sperm morphology and fertility, and we investigated whether any of these compounds affects the genetic competence of spermatozoa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

The C57BL/6 male mice and B6D2F1 female mice (C57BL/6 x DBA/ 2) were sperm and oocyte donors, respectively. Their ages were 6–10 and 8–12 wk, respectively, when the experiment began. Mature CD-1 females (age, 3–4 mo) were used as surrogate mothers. All animals were kept in a temperature- and light-controlled room with a 14L:10D photoperiod in accordance with the U.K. Animals (Scientific Procedures) Act 1986 (Program License 30/1892) and the guidelines of the Laboratory Animal Services at the University of Hawaii. The protocol of our animal handling and treatment was reviewed and approved by the Animal Care and Use Committee at the University of Hawaii. All mice were housed under standard, nonsterile conditions and were provided with water and either pelleted or powdered chow ad libitum.

Treatment of C57BL/6 Males with Imino Sugars

DNJ was purchased from Toronto Research Chemicals (Downsview, ON, Canada); the NB-DNJ and NB-DGJ were provided by Oxford GlycoSciences (Abingdon, Oxfordshire, U.K.). The NB-DNJ, NB-DGJ, and DNJ were mixed thoroughly with powdered mouse chow (expanded Rat and Mouse Chow 1, ground; SDS, Witham, Essex, U.K.) and stored at room temperature for less than 3 mo. Imino sugars are completely stable for several months at room temperature. Males were fed with the feed containing DNJ, NB-DNJ, or NB-DGJ for 5–7 wk (Fig. 1). Unless stated otherwise, male mice were treated with 15 mg kg–1 day–1 of NB-DNJ and 150 mg kg–1 day–1 of NB-DGJ, the lowest doses of these compounds that result in the production of very high proportions of abnormal spermatozoa. The DNJ was used in a dose that is equimolar to 15 mg kg–1 day–1 of NB-DNJ, which is more than 50-fold lower than the dose of DNJ that was previously found not to induce male infertility [1]. The imino sugar-containing powdered chow was formulated assuming that mice consume 5 g of feed per day and have a body weight of 20 g.



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  		FIG. 1. Experimental protocols. A) In the previous study [1], males were administered an imino sugar for 5 wk, starting from 6 wk after birth. Animals became infertile in 3 wk, and fertility was regained during the fourth week after the end of imino sugar administration. B and C) In the present study, males were given an imino sugar for 5–7 wk, starting from 6 to 10 weeks after birth. B) The fertility of NB-DGJ-administered males was assessed after 5 wk of drug treatment and again after 5 wk after termination of drug administration. C) Sperm fertility, chromosomal constitution, and genetic potential of spermatozoa were determined between 5 and 7 wk after start of imino sugar administration

Mating

Fertility of NB-DGJ-treated males was assessed by caging each male with four C57BL/6 female mice for 9 days, during which time the females were checked daily for postcoital vaginal plugs. Males were then removed, and the females were monitored for signs of pregnancy and litter sizes, if any.

Reagents and Media

Polyvinyl alcohol (PVA; cold water-soluble; molecular weight, ~10 000) was purchased from Sigma Chemical Co. (St. Louis, MO). Polyvinyl pyrrolidone (PVP; molecular weight, ~360 000) and bovine testicular hyaluronidase (200 UPS U/mg) were from ICN Pharmaceuticals (Costa Mesa, CA), and BSA (fraction V) was from Calbiochem (La Jolla, CA). Mineral oil was from Squibb and Sons (Princeton, NJ). All other reagents were obtained from Sigma unless specifically stated. The CZB medium [20], supplemented with 5.56 mM glucose and 4 mg/ml of BSA, was used for the culture of mouse oocytes after microsurgery. The medium for collection of oocytes from oviducts and subsequent oocyte treatments, including micromanipulation, was a modified CZB (Hepes-CZB) [21] containing 20 mM Hepes-HCl, a reduced amount of NaHCO3 (5 mM), and 0.1 mg/ml of PVA instead of BSA. The CZB was used under 5% CO2 in air, and Hepes-CZB was used under air.

Preparation of Oocytes, Spermatozoa, and Spermatids

Oocytes and spermatozoa were collected and prepared for micromanipulation according to the method described by Kimura and Yanagimachi [21, 22]. Briefly, oocytes were collected from oviducts of superovulated females soon after ovulation (13–15 h after injection of hCG), freed from cumulus cells by hyaluronidase treatment, and kept in CZB medium at 37°C for less than 2 h. Spermatozoa from the cauda epididymis were allowed to disperse in Hepes-CZB for 5–10 min. A droplet (~2 µl) of this sperm suspension was transferred into 10 µl of Hepes-CZB containing 10% PVP and thoroughly mixed. This sperm suspension was kept under mineral oil in a micromanipulator dish for less than 1 h. Testicular spermatozoa were collected from the testes and kept in Hepes-CZB with 1% PVP for no more than 1 h at room temperature before injection into oocytes. From epididymides as well as from testes, only motile spermatozoa were used.

In Vitro Fertilization and Intracytoplasmic Sperm Injection

Conventional in vitro fertilization (IVF) was performed according to the method described by Toyoda et al. [23] using cauda epididymal spermatozoa without separating morphologically normal from abnormal spermatozoa. Intracytoplasmic sperm injection (ICSI) was performed according to the method described by Kimura and Yanagimachi [21, 22] except that it was performed at room temperature (~25°C). The ICSI was performed in Hepes-CZB within 60 min after spermatozoa were suspended in PVP-containing Hepes-CZB. The ICSI oocytes were rinsed and cultured in CZB at 37°C. Some ICSI oocytes were activated by incubation in Ca2+-free CZB medium containing 10 mM SrCl2 [24] for 5–6 h at 37°C. Activated oocytes were rinsed and kept in CZB at 37°C under 5% CO2 in air. Between 5 and 6 h after ICSI or IVF, oocytes were examined with a dissecting microscope, and those with distinct signs of cytolysis were discarded. An oocyte with two distinct pronuclei and the second polar body was recorded as being normally fertilized. Some ICSI oocytes were fixed and stained [25] to visualize clearly the decondensation of sperm nuclei and the transformation of decondensed sperm chromatins into male pronuclei.

Embryo Development and Embryo Transfer to Surrogate Mothers

Normally fertilized oocytes were allowed to develop in CZB at 37°C. Embryos reaching the morula/blastocyst stage were transferred to the oviducts of pseudopregnant CD-1 females that had been mated during the previous night with vasectomized males of the same strain. Pregnant females were allowed to deliver and raise their foster pups.

Examination of Acrosomes, Acridine Orange Staining, and Chromosomes of Spermatozoa

Epididymal spermatozoa were treated with antiacrosomal monoclonal Mab18.6 [26], counterstained with propidium iodide (or Hoechst 33342), and examined with a fluorescence microscope as described previously [1]. At least 250 spermatozoa were examined per mouse (n ≥ 3).

Acridine orange (AO) staining was performed according to the method described by Tejada et al. [27] and Kosower et al. [28] except that samples were examined with a fluorescence microscope immediately after washing and mounting in distilled water following AO staining. At least 300 spermatozoa were examined for each group of experiments.

Sperm chromosomes were examined as described previously [29–31]. Approximately 7–9 h after ICSI, oocytes were transferred into CZB medium containing 0.006 µg/ml of vinblastine. Those arrested at the first cleavage metaphase after 19–21 h of culture in the vinblastine solution were treated for a few minutes with 0.5% pronase (1000 tyrosine units/ mg) in PBS (pH 6.8) to remove zonae pellucidae. They were then treated with a hypotonic solution (1:1 mixture of 1% sodium citrate and 30% fetal bovine serum) for a few minutes at room temperature. Chromosomes were spread on clean glass slides by the gradual-fixation/air-dying method [32]. The preparations were stained with 2% Giemsa (Merck, Darmstadt, Germany) in PBS (pH 6.8) for 8 min. It was not always possible to distinguish between chromosomes of paternal and maternal origin. However, because oocyte chromosomes seldom show structural aberrations during the first metaphase cleavage following parthenogenetic activation [33, 34], any abnormal chromosomes within fertilized oocytes were considered to be of sperm origin.

Statistical Analysis

The data were compared with the chi-square test using Yates correction for continuity. A value of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Imino Sugars and Sperm Morphology

Previously, we reported that the minimum dose of NB-DNJ that causes male mice to produce very high proportions of severely misshapen spermatozoa was 15 mg kg–1 day–1 and that NB-DGJ had a very similar effect when used at 1200 mg kg–1 day–1 [1] (Fig. 1A). To determine the minimal dose of NB-DGJ that disturbs spermiogenesis, we treated male C57BL/6 mice for 5 wk with various doses of NB-DGJ, ranging from 5 to 600 mg kg–1 day–1. Figure 2 shows that 15 mg kg–1 day–1 of NB-DGJ had no obvious effect on spermatozoa. At this dose, the proportion of epididymal spermatozoa with a normal nuclear morphology was the same as that for untreated mice. In contrast, 150 mg kg–1 day–1 of NB-DGJ dramatically reduced the incidence of spermatozoa with normal nuclei and normal acrosomes (Figs. 2 and 3, B and E). This effect was more dramatic at higher doses (Fig. 2). Spermiogenesis thus is less sensitive to NB-DGJ than it is to NB-DNJ.



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  		FIG. 2. Dose dependence of the effects of NB-DGJ on cauda epididymal spermatozoa. Sperm nuclei and acrosomes of spermatozoa were stained with propidium iodide and the antiacrosome monoclonal antibody Mab18.6, respectively. The data are presented as the mean + SD



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  		FIG. 3. Fluorescence microscopic images of nuclear and acrosomal morphology of cauda epididymal spermatozoa from control mice (A and D), mice treated with 150 mg kg–1 day–1 of NB-DGJ (B and E), and 15 mg kg–1 day–1 of NB-DNJ (C and F). A–C) Nuclei stained with propidium iodide. D–F) Acrosomes stained with monoclonal antibody Mab18.6 (green) and nuclei (red). Bar = 10 µm

The spermatozoa with abnormal nuclei also had irregular head shapes when examined immediately after suspension in CZB medium. The vast majority of live spermatozoa from the cauda epididymis and testis of NB-DNJ- and NB-DGJ-administered males (15 and 150 mg kg–1 day–1, respectively) had an abnormal head shape (Fig. 4, B–D, and data not shown), whereas the spermatozoa from DNJ-treated males were similar to those of control, nontreated mice (data not shown).



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  		FIG. 4. Differential interference contrast images of spermatozoa of NB-DNJ-treated and control mice. A) A spermatozoon with a normal head from a control male. B–D) Spermatozoa with abnormal heads from NB-DNJ-treated males. E) An injection pipette and sperm heads ready to inject after their separation from tails using piezo-pulses. Larger, normal heads (arrows) and smaller, abnormal heads (arrowheads) are shown. Bar = 5 µm

Male Fertility Assessed by Natural Mating

When male C57BL/6 mice are administered NB-DNJ at doses of at least 15 mg kg–1 day–1, they mate naturally with female mice at frequencies similar to those of control males as judged by the appearance of postcoital vaginal plugs [1]. The drug-treated mice, however, do not impregnate the females [1]. It also has been established that male mice are infertile in natural mating tests after administration of 1200 mg kg–1 day–1 of NB-DGJ [1]. In the present study, we assessed the fertility of male mice treated with low doses of NB-DGJ in a natural mating assay (Fig. 1B). Males treated with 15 mg kg–1 day–1 of NB-DGJ for 5 wk were normally fertile (data not shown), but those administered 150 and 600 mg kg–1 day–1 of NB-DGJ were infertile (Table 1). When these males were maintained for five more weeks without any drug administration, they regained their fertility and sired normal-size litters (Table 1). The NB-DGJ-induced infertility thus was reversible, as was previously found for NB-DNJ [1].


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  		TABLE 1. Mating assays with male mice treated with NB-DGJ and those recovered for 5 wk from NB-DGJ treatment.a

Male Fertility Assessed by IVF

Having established by mating tests that the NB-DNJ- and the NB-DGJ-administered males were infertile, we investigated whether their spermatozoa were capable of fertilizing oocytes in vitro. To this end, we used nonselected, whole populations of cauda epididymal spermatozoa, of which the majority has an abnormal nuclear morphology (for 15 mg kg–1 day–1 of NB-DNJ and 150 mg kg–1 day–1 of NB-DGJ, only 5.7% ± SD 2.2% and 12.1% ± SD 10%, respectively, of the cauda epididymal spermatozoa have normal nuclear shapes [1]) (Fig. 2). We found that the cauda epididymal spermatozoa from the alkylated imino sugar-treated males did not fertilize any cumulus-intact oocyte by IVF (out of 250 and 61 oocytes for spermatozoa from NB-DNJ- and NB-DGJ-treated males, respectively). In contrast, spermatozoa from DNJ-treated males and those from males without imino sugar administration fertilized 39 (68%) of 57 oocytes and 137 (77%) of 179 oocytes, respectively, under the same conditions. Furthermore, many normal spermatozoa (from control males) bound tightly to the zona pellucida so that they were not even removed by hard pipetting, whereas some spermatozoa from the NB-DNJ-administered males made contact with the zona. However, most of them were removed from zona surfaces by pipetting (data not shown). None of the spermatozoa from mice treated with an alkylated imino sugar penetrated the zona (data not shown).

Sperm Protamine Disulfide Status

The nuclear protamines of murine spermatozoa contain sulfhydryl groups, which are in the reduced state when these cells are in the testis. During the transit of spermatozoa through the epididymis, the protamine-SH groups undergo extensive oxidation to intra- and intermolecular disulfide bonds, which confer resistance to chemical and physical factors that can denature nuclear DNA [25, 28, 31]. Thus, the stability of nuclear DNA of epididymal spermatozoa depends on the presence of protamine disulfide bonds. We therefore investigated the sulfhydryl(-SH)/disulfide(-SS-) status of nuclear protamines of spermatozoa from NB-DNJ-treated mice using acridine orange (AO).

Figure 5 shows that in the caput epididymis of NB-DNJ-treated mice the proportion of sperm cells containing -SS- protamines is significantly lower than in the caput of normal mice (P < 0.0001), with the balance predominantly being cells with an intermediate protamine oxidation status. In the cauda epididymis of drug-treated mice, a small fraction (16%) of spermatozoa carries protamines with underoxidized sulfhydryl groups, whereas none of the spermatozoa from control mice are in this state. Thus, after NB-DNJ administration, the sperm protamine disulfide bonds were formed in a delayed fashion and to a slightly lesser extent than in control cells.



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  		FIG. 5. Sulfhydryl/disulfide status of testicular, caput epididymal, and cauda epididymal sperm nuclear protamines as assessed by AO fluorescence. When sperm nuclei are treated with acid (or heat) while the DNA-associated protamines contain relatively few disulfide bonds, the DNA denatures. The AO aggregated on denatured (single-stranded) DNA fluoresces red. In contrast, when protamines are rich in disulfide bonds, protamine-associated DNA is resistant to denaturation. The AO intercalated as a monomer on native (double-stranded) DNA fluoresces green [28]. In normal male mice, nearly 100% of testicular sperm nuclei fluoresce red after AO staining, whereas 100% of cauda sperm nuclei fluoresce green. The formation of protamine disulfide bonds in sperm nuclei occurred in NB-DNJ-treated males, but an efficiency slightly less than that in control males. Average values are presented for n = 3. *Significantly different from the corresponding category of spermatozoa from untreated mice (chi-square test)

Genetic Competence of Dysmorphic Spermatozoa

The genetic competence of testicular and epididymal spermatozoa from the males treated with DNJ, NB-DNJ, or NB-DGJ was assessed by ICSI of sperm heads into oocytes (Fig. 1C). Before ICSI, the spermatozoa from the mice treated with the alkylated imino sugars were separated on the basis of their head shape as assessed by differential interference contrast microscopy: Those with normal head morphology were completely separated from spermatozoa with abnormal head morphology. The morphology of spermatozoa used for each ICSI experiment is indicated in Tables 2–4. We found that the abnormal sperm heads from NB-DNJ- and NB-DGJ-administered mice (as indicated by arrowheads in Fig. 4E) were capable of fertilizing the majority of oocytes by ICSI (Table 2, experiments A, C, D, and F). It should be noted that deformed testicular spermatozoa fertilized 78–82% of oocytes, whereas those from the cauda epididymis fertilized only 20–27% of oocytes (Table 2, experiments A and D vs. experiments B and E). The majority (75–80%) of oocytes injected with deformed epididymal spermatozoa remained unactivated for 5–6 h after ICSI as evidenced by the presence of metaphase II chromosomes and condensed sperm chromatin (data not shown). However, when ICSI oocytes were treated with Sr2+ after injection of misshapen epididymal spermatozoa, 93–99% of them were activated and normally fertilized (Table 2, experiments C and F).


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  		TABLE 2. Preimplantation development of the oocytes injected with spermatozoa from males treated with imino sugars


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  		TABLE 4. Postimplantation development of the fertilized oocytes injected with spermatozoa from male mice treated with iminosugars

Chromosome analysis of ICSI-fertilized oocytes revealed that the majority of deformed spermatozoa were karyologically normal, irrespective of the site of sperm collection and the treatment of post-ICSI oocytes (Table 3).


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  		TABLE 3. Chromosome analysis of the oocytes injected with testicular and epididymal spermatozoa

In a series of experiments, oocytes were injected with abnormal testicular or cauda epididymal spermatozoa from NB-DNJ- or NB-DGJ-treated males. These post-ICSI oocytes, with or without Sr2+ treatment, were cultured, and the resultant embryos were transferred to surrogate mothers. The results, which are summarized in Table 4, showed that many of these embryos developed normally and were born as normal pups. Embryos grown out of the oocytes injected with the malformed spermatozoa collected from the testis developed into normal live offspring (Table 4, experiments A and D) with a slightly, but not significantly, lower efficiency than embryos arising from oocytes injected with normal testicular spermatozoa from control and DNJ-treated mice (Table 4, experiments G and I). The embryos grown out of the oocytes injected with the misshapen cauda epididymal spermatozoa also developed into live offspring (Table 4, experiments B and E), but at significantly lower rates than embryos stemming from oocytes injected with normal cauda epididymal spermatozoa from control and DNJ-treated mice (Table 4, experiments H and J). However, when the oocytes injected with the misshapen cauda epididymal spermatozoa were incubated in the presence of Sr2+ (Table 4, experiments C and F), the resultant embryos developed with an efficiency comparable to that of the corresponding control embryos (Table 4, experiments H and J). Thus, the proportion of the embryos that gave rise to live pups was, to some extent, dependent on the morphology of the spermatozoa used and on the presence of Sr2+ in the post-ICSI culture medium.

The postnatal development of ICSI-born pups was monitored by weighing these animals at regular intervals until they were 8 wk of age. Figure 6 shows that the ICSI-offspring of control (nontreated) males and those of NB-DNJ- and NB-DGJ-treated males grew at similar rates in a gender-specific manner.



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  		FIG. 6. Growth curves of male and female offspring born via ICSI with spermatozoa from males that had been treated with NB-DNJ or NB-DGJ. Control was offspring of control males. Data are presented as the mean ± SD

Finally, we assessed whether ICSI-derived progeny from the infertile males were fertile. Ten males and 10 females of the offspring from NB-DNJ-treated males as well as 10 males and 10 females of the offspring from NB-DGJ-treated males were randomly selected and mated with untreated B6D2F1 mice. These mating pairs were kept together until evidence of mating was clear, which was 2.2 ± SD 1.3 days (range, 1–5 days) for NB-DNJ-males, 2.2 ± SD 1.5 days (range, 1–6 days) for NB-DNJ-females, 2.8 ± SD 1.4 days (range, 1–6 days) for NB-DGJ-males, and 2.0 ± SD 1.3 days (range, 1–5 days) for NB-DGJ-females. The females in these mating tests delivered normal pups in litters of normal size (8.3 ± SD 2.1 pups for NB-DNJ-derived offspring and 8.0 ± SD 2.2 pups for NB-DGJ-derived offspring). Thus, both the male and female progeny from the infertile sperm donors proved to be fully fertile.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We administered NB-DNJ and NB-DGJ to male mice for 5–7 wk at the minimal dose levels required to induce infertility, and we performed ISCI experiments with the morphologically abnormal spermatozoa from these mice. The misshapen spermatozoa, obtained from the testis as well as the cauda epididymis, were evaluated for their abilities to fertilize post-ICSI oocytes and to support the development of fertilized post-ICSI oocytes into embryos and live pups. Some of the oocytes injected with the malformed cauda epididymal spermatozoa were incubated in the presence of Sr2+, and the effects of this procedure on oocyte fertilization and embryonal development were also studied. Sr2+, a parthenogenetic agent, causes Ca2+ oscillations in oocytes that have frequencies similar to those of the Ca2+ oscillations induced by normal fertilization [35–37]. Sperm-induced Ca2+ oscillations are instrumental in releasing oocytes from their metaphase II arrest, and they enable oocytes to resume the meiotic division and form pronuclei (for review, see [38]).

We observed a difference in the post-ICSI oocyte-fertilizing ability between malformed spermatozoa obtained from the testis and those obtained from the cauda epididymis of NB-DNJ- and NB-DGJ-treated mice. The abnormal testicular spermatozoa had a fertilization rate similar to that of control spermatozoa, but the abnormal germ cells obtained from the cauda epididymis often failed to fertilize oocytes. However, this fertilization deficit of the epididymal spermatozoa could be overcome by incubating the post-ICSI oocytes cells in the presence of Sr2+, which suggests that most of the abnormal spermatozoa from the cauda epididymis cannot activate oocytes.

We also found that the misshapen spermatozoa from the testis as well as those from the cauda epididymis of NB-DNJ- and NB-DGJ-treated mice are capable of supporting post-ICSI embryonal development, resulting in the birth of normal live pups. The competence of abnormal testicular spermatozoa was, in this respect, comparable to that of control spermatozoa. In contrast, the embryos grown out of spontaneously activated oocytes injected with abnormal cauda epididymal spermatozoa developed into live pups with significantly less efficiency than the embryos that arose from oocytes injected with control spermatozoa. However, when the oocytes that had been injected with abnormal cauda epididymal spermatozoa were artificially activated with Sr2+, the resultant embryos developed into live pups as efficiently as control post-ICSI embryos. The question is why a significant proportion of spontaneously activated oocytes injected with misshapen epididymal sperm heads develops to the morula/blastocyst stage but then fails to mature after reimplantation in the female reproductive tract. We have found that this relative developmental deficit can be overcome by incubating the post-ICSI oocytes in the presence of Sr2+, which suggests that the developmental deficiency results from incomplete oocyte activation. It has been discussed before that the formation of male and female pronuclei and early embryo development is not necessarily indicative of complete oocyte activation [39]. The increase in live births from the deformed cauda epididymal spermatozoa by post-ICSI Sr2+ treatment gives further support to the notion that sperm- or Sr2+-induced Ca2+ oscillations may have longer-term consequences for embryonal development that go beyond release from metaphase II arrest [40].

The capacity of spermatozoa to activate oocytes resides in multiple elements of the perinuclear theca that collectively are referred to as the sperm-borne oocyte-activating factors (SOAFs) and are located predominantly in the postacrosomal sheath (for review, see [41]). Therefore, our observations suggest that the testicular spermatozoa from NB-DNJ and NB-DGJ-treated mice contain sufficient equivalents of SOAFs to activate oocytes despite being grossly misshapen. Our results also indicate that the malformed testicular spermatozoa lose their oocyte-activating abilities during the migration through the epididymis, presumably by inactivation or loss of SOAFs. The extent of inactivation or loss of SOAFs seems to be highly variable among the misshapen cauda epididymal spermatozoa. Therefore, after ICSI without Sr2+ treatment, some of the oocytes are fully activated and develop into live pups, others are only partially activated to undergo only early embryonal development, and the majority of oocytes is not activated at all. The reasons for the inactivation or loss of SOAFs from the deformed sperm cells during their testis-cauda epididymis transport are currently unknown.

Finally, we have seen that the live pups fathered via ICSI by the infertile NB-DNJ- and NB-DGJ-treated mice grow at the same rate as the corresponding offspring from control mice. We also have seen that all of these ICSI-derived progeny are equally fertile, irrespective of the source of spermatozoa used for ICSI and the presence or absence of Sr2+ in the post-ICSI culture medium.

Taken together, our results demonstrate that oral administration of NB-DNJ or NB-DGJ does not affect the genetic integrity of sperm nuclei during spermatogenesis. Thus, the drug-induced, severe disturbance of the phenotypic development of spermatozoa is not associated with an impairment of their genetic potential. These findings are in agreement with those of earlier studies regarding sperm dysmorphia despite the underlying cause of the dysmorphia in this case being very different from those in previously described cases. Malformed spermatozoa from mice and men of proven fertility are not necessarily abnormal karyologically [42, 43]. This also has been found in cases of reduced fertility, in which the majority of the misshapen spermatozoa did not have chromosomal aberrations [29, 44, 45]. Moreover, abnormal spermatozoa from BALB/c and quaking-sterile mice as well as from humans can support the development of live and normal offspring after ICSI [46, 47] (for review, see [48]). Therefore, the result of our studies further support the view that the maintenance of genetic integrity of spermatozoa is not necessarily dependent on the morphological development of these cells.

Abnormally shaped epididymal spermatozoa from various mouse strains and humans have been evaluated for their capacity to activate oocytes after ICSI and been found to differ in this respect. For example, the triangular, collapsed, and calyculate spermatozoa from BALB/c mice as well as the club- and crescent-shaped spermatozoa from azh/azh mutant mice are able to activate oocytes after ICSI [46, 49], whereas human round-headed epididymal spermatozoa often cannot do so despite being genetically competent [50, 51].

How NB-DNJ and NB-DGJ cause male mice to produce epididymal spermatozoa that are morphologically and functionally defective is not clear. First, the essential target of these imino sugars that is responsible for their effects on spermiogenesis needs to be identified. Interestingly, NB-DNJ inhibits the ceramide-specific glucosyltransferase as a competitive inhibitor of ceramide [52]. Also, this imino sugar and its galactose analogue can be modeled on ceramide so that both imino sugars can be regarded as ceramide mimics [52]. Therefore, one cannot exclude that NB-DNJ and NB-DGJ not only interact with the ceramide-specific glucosyltransferase and the nonlysosomal glucosylceramidase but also with other ceramide-metabolizing enzymes, many of which have been characterized in recent years (for review, see [53, 54]). In any case, it is not unlikely that these compounds act via modulation of sphingolipid metabolism. Second, it is not clear which cell type is primarily affected by the alkylated imino sugars. They may act directly on their target in round spermatids. Alternatively, these compounds may impact Sertoli cells and, thus, indirectly undermine spermiogenesis. In addition, they might change physiological and biochemical characteristics of the epididymis so that it cannot properly support the maturation of spermatozoa. It remains to be elucidated how any peculiarity of sphingolipid metabolism in germ cells or in testicular nongerm cells is affected by alkylated imino sugars and is responsible for the observed effects on germ cell development.

In summary, it has become clear, both from our previous study and from the present study, that short-term, low-dose administration of NB-DNJ or NB-DGJ affects the fertility of male mice at three levels: sperm motility, entry of sperm into the oocyte, and oocyte-activating ability of spermatozoa. The data presented here strengthen the profile of alkylated imino sugars as male contraceptives in C57BL/6 mice: Both NB-DNJ and NB-DGJ can be administered orally, induce fully reversible infertility, and do not affect the genetic competence of the germ cells when given for 5–7 wk at the lowest dose required to render male mice infertile. In addition, because NB-DNJ does not affect reproductive endocrinology [1], it is highly likely that NB-DGJ also does not do this.

Finally, because the current studies were done with short-term, low-dose imino sugar-treated mice, the present results do not necessarily imply that the reproductive consequences of long-term, high-dose imino sugar treatment also are limited to the fertilizing capacity of male germ cells. At present, we have no information regarding the impact of long-term, high dose imino sugar treatment on the genetic potential of male germ cells. Because contraceptives are to be taken chronically, the effects of long-term, low-dose NB-DNJ administration are currently being studied.


   ACKNOWLEDGMENTS
 
We thank Dave Smith for technical assistance, J. Anton Grootegoed (Erasmus University, Rotterdam, The Netherlands) for helpful suggestions, and Harry D. Moore (University of Sheffield, Sheffield, United Kingdom) for providing the Mab18.6 monoclonal antibody.


   FOOTNOTES
 
1 Supported by the Harold Castle Foundation, the Kosasa Family Foundation, the Victoria Geist Foundation (R.S. and R.Y.), Oxford GlycoSciences/ Celltech UK (C.M.W), and NIH Grant 1 U01 HD45861 (F.M.P. and A.C.S.) Back

2 Correspondence: Aarnoud C. van der Spoel, The Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. FAX: 44 01865 27 5216; aarnoud.vanderspoel{at}bioch.ox.ac.uk Back

Received: 3 September 2004.

First decision: 11 October 2004.

Accepted: 24 November 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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