Reversible infertility in male mice after oral administration of alkylated imino sugars: a nonhormonal approach to male contraception

Abstract

During mammalian spermatogenesis, male germ cells undergo a dramatic transformation, which includes a change of shape, nuclear condensation, and development of specialised structures, such as an acrosome, and a flagellum with a mitochondrial sheath. We have found a previously undescribed pharmacological approach to intervene in these events. After oral administration of the alkylated imino sugar N-butyldeoxynojirimycin (NB-DNJ) to mice, epididymal spermatozoa displayed a spectrum of abnormal head shapes, and acrosomal antigens were mostly absent or displayed irregular patterns. In addition, the mitochondria of these cells often had an aberrant morphology, and were arranged in relatively short and wide mitochondrial sheaths. The motility of the affected spermatozoa was severely impaired. After 3 weeks of administration of NB-DNJ, male mice became sterile, and regained their fertility during the fourth week off drug. The NB-DNJ-induced infertility was not associated with a reduction in the serum testosterone level. Biochemically, the capacity of imino sugars to impair spermatogenesis was associated with their potential to attenuate the biosynthesis of glucosylceramide-based sphingolipids. Our study reveals that male fertility is affected by partial glycosphingolipid depletion, or, alternatively, by a distinct as yet unidentified property that is shared by alkylated imino sugars that inhibit glucosylceramide biosynthesis. These compounds therefore may be new leads in the development of a male contraceptive, especially because NB-DNJ has already been through extensive evaluation in various mammals, including man.

Figures

Fig 1.

Mating tests. After drug treatment, fertility of male mice was assessed by caging each of them with four female mice for 7 (A) or 9 (B) days, during which the females were monitored for vaginal mucous plugs (see text), and subsequently for pregnancies and litter sizes. (A) Effects of oral administration and withdrawal of NB-DNJ on fertility of male mice. ON, Animals were given the compound for 1–5 weeks. OFF, Recovery of fertility was studied by using males that had received the compound for 5 weeks. These animals were further assayed for 5 weeks without drug by caging each of them each week with a new set of four female mice. (B) Comparison of effects of imino sugars with different biochemical activities on reproductive capacity of male mice. The data are presented as the mean ± SD for 3 male and 12 female mice per data point.

Fig 2.

Dose-effect study. Males were given NB-DNJ in a range of concentrations for 5 weeks, and then caged with four female mice each for 9 days (n = 6 males in A, n = 3 males in B). The females were monitored for pregnancies and litter sizes (A) and vaginal mucous plugs (B). Alternatively, after a 5-week treatment, epididymal spermatozoa were obtained and incubated with either monoclonal antibody Mab18.6 or anti-sp56, and the nuclear dye Hoechst 33342. By examining >100 spermatozoa per mouse, we determined the percentage of nuclei with grossly abnormal morphology (n ≥ 4; C) and the percentage of spermatozoa that stain with monoclonal antibodies Mab18.6 (n = 3) or anti-sp56 (n = 2) (D). The data are presented as the mean ± SD.

Fig 3.

Reproductive endocrinology. Male mice were treated with NB-DNJ at four different doses, and sera and were obtained for determination of luteinizing hormone (A), follicle-stimulating hormone (B), and testosterone (C). (D) Testes were also assayed for testosterone. Data points from individual mice are presented.

Fig 4.

Histology of the testis and immunocytochemistry of epididymal spermatozoa. Sections from stage I seminiferous tubules from a control mouse (A) and a mouse treated with 50 mg/kg/day NB-DNJ (B). Insets show close-ups of step 13 spermatid heads. Epididymal spermatozoa from normal mice (C), 15 mg/kg/day NB-DNJ-treated mice (D), and mice recovered from a 15 mg/kg/day NB-DNJ regimen (E) were incubated with anti-acrosomal monoclonal Mab18.6 and counterstained with the nuclear dye propidium iodide. (Original magnification of A and B = ×1,000; bar in C–E = 10 μm.)

Fig 5.

Morphological features of epididymal spermatozoa from normal mice (A and D), NB-DNJ-treated mice (B and E, 15 mg/kg/day), and mice recovered from a 15 mg/kg/day NB-DNJ regimen (C). (A–C) Scanning electronmicrographs, showing examples of a normal head and midpiece (A), a spatulate sperm head with a disorganized squat mitochondrial sheath (B), and a normal head and midpiece after recovery from drug treatment (C). (D and E) Transmission electronmicrographs, displaying a normal spermatozoon (D) and a spermatozoon with alterations in nuclear shape, tail orientation, and morphology and arrangement of mitochondria (E). (Bar = 2 μm.)