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

Abstract

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.

Figure 1

Sperm motility (%) and morphology in rabbits. A. Spermatozoa were released in medium and percentage motile spermatozoa was estimated by counting flagellating or non flagellating spermatozoa. B. For assessment of morphology, a smear was air-dried on a slide and stained with PNA and DAPI and counted manually for abnormal sperm heads. Data are presented as the mean ± SD of percentage motile spermatozoa for 5 males and as the mean ± SD of percentage abnormal spermatozoa. Nuclear morphology and acrosomal staining were assessed by examining 100 spermatozoa per rabbit.

Figure 2

Morphology of rabbit spermatozoa. Smears of ejaculated spermatozoa were air-dried and stained with PNA/DAPI. A. Spermatozoa from control rabbits. B. Spermatozoa from rabbits treated with 50 mg/kg/day miglustat for 8 weeks. Bar = 20 μm.

Figure 3

Effects of miglustat on morphology of spermatozoa from various mouse strains. Acrosomal and nuclear morphology of cauda epididymal spermatozoa from (A, B, C, G, H and I) control and (D, E, F, J, K and L) miglustat-treated mice from inbred mouse strains, (A and D) C57BR, (B and E) FVB/N, (C and F) BALB/c, (G and J) DBA/2, (H and K) AKR/J and (I and L) MA/MyJ. Acrosomes were stained with the monoclonal antibody Mab18.6 (green) and nuclei with propidium iodide (red). Drug administration was at 150 mg/kg/day. Bar = 10 μm.

Figure 4

Quantitation of effects of miglustat on morphology of spermatozoa from inbred mouse strains and interstrain hybrid mice. Effects of miglustat administration on morphological features of cauda epididymal spermatozoa from (A and B) mice of various inbred strains (n = 2–3 per strain) and from (C and D) fourth-generation C57BL/6 × FVB/N hybrid mice. In (A and C) spermatozoa were scored for possession of an acrosome (present/absent), irrespective of acrosomal staining pattern, and for possession of a grossly abnormal non-falciform nucleus. In (B and D) spermatozoa were scored for acrosomal staining pattern (normal/abnormal), and for the normality of their nuclear morphology. Falciform nuclei (flat and curved) that deviated from the typical shape of spermatozoa from untreated mice were score as abnormal. In (C and D) data are presented from the hybrid mice that were most affected by miglustat, and from a representative number of hybrid mice that showed a lesser response. Each datapoint in (A and B) expresses the difference between the average score of drug-treated mice from one inbred strain and the average score of control mice of the same strain. Each datapoint in (C and D) expresses the difference between the score of one drug-treated hybrid mouse and the average score of untreated FVB/N mice. Data points were fitted by linear regression; the corresponding trend lines are displayed in grey (correlation coefficients in A, B, C and D were 0.97, 0.79, 0.98 and 0.84, respectively). Miglustat administration was at 150 mg/kg/day, except SM/J mice (15 mg/kg/day). At least 200 spermatozoa per mouse were scored for nuclear morphology and acrosomal staining.

Figure 5

Effects of miglustat on acrosomal proteins as detected by western blotting. The impact of miglustat administration on the levels of the acrosomal proteins sp56 and IAM38 was assessed both in C57BL/6 and in FVB/N mice. Replicate western blots were prepared with lysates of epididymal spermatozoa and probed with antibodies against sp56, IAM38, or cytochrome C oxidase subunit I. Miglustat treatment was at 15 mg/kg/day.

Figure 6

Effects of miglustat on morphology of spermatozoa from interstrain hybrid mice. Acrosomal and nuclear morphology of cauda epididymal spermatozoa from miglustat-treated (A) C57BL/6 (15 mg/kg/day) and (B) FVB/N mice (600 mg/kg/day), and from (C, D, E and F) fourth-generation C57BL/6 × FVB/N hybrid mice (15 mg/kg/day). These drug-treated hybrid mice differed in their proportion of non-falciform acrosome-less spermatozoa. This proportion increases from C to F. Acrosomes were stained with the monoclonal antibody Mab18.6 (green), and nuclei with propidium iodide (red). Bar = 10 μm.