1. Charlotte Walden Imino sugars and mouse spermatogenesis

2. Short-term NB-DNJ treatment causes infertility in male mice Oral intake Minimal dosage of 15mg/kg/day Minimal induction and recovery time of 3 weeks Structurally abnormal spermatozoa Poor motility No effect on endocrinology (LH, FSH or testosterone) No effect on sperm counts or organ weights Van der Spoel et al., PNAS 99:17173-17178 (2002)

3. Long-term NB-DNJ treatment Is long-term, low dose administration of NB-DNJ sufficient to impair spermatogenesis? Can normal spermatogenesis be regained after long-term treatment? Do we observe the same abnormal germ cell morphology in long-term treated mice? Characterize development of NB-DNJ treated spermatozoa Are there any other effects when mice are treated for long periods?

4. 1 year study, 4 time points (3, 6, 9, 12 months) Control, treated and recovered groups 6 and 9 week recovery periods Variety of fertility, biochemical and toxicity parameters measured Long-term study

5. Fertility -mating assays 10 month12 month 0.0 0.5 1.0 1.5 2.0 2.5 3.0 untreatedtreatedrecovered Group pregnancies/male 0 2 4 6 8 10 untreatedtreatedrecovered Group litter size pregnancieslitter size

6. Long-term effects on the testis Testis and epididymis weights Testis weight (% body weight) Epididymis weight (% body weight)

7. untreatedtreatedrecovered 6 months 12 months Long-term effects on the testis- Tubule morphology

8. Structure of the testis Nature Reviews Genetics 3 :790-801

9. Elongated spermatids Round spermatids Pachytene spermatocytes Pre-leptotene spermatocytes spermatogonium Basement membrane lumen Spermatogenesis Nature Reviews Genetics 3 :790-801 acrosome

10. Acrosome development in murine spermatids Step 2Step 3Step 5 Step 7 Fusion of pro-acrosomal vesicles: ONE vesicle attaches to nucleus Flattening and spread of acrosome: head cap covers anterior nucleus

11. Spermiogenesis

12. Long-term effects on fertility- morphology controltreated recovered 6 months 12 months

13. Long-term effects on fertility - morphology

14. Motility analysis - untreated

15. Motility analysis - treated

16. Long-term effects on fertility- Motility 3 months12 months

17. Long-term effects on fertility - sperm counts * short-term NB-DNJ treatment does not reduce sperm count

18. Long-term effects on the testis- Apoptosis TUNEL assay 0.0 0.2 0.4 0.6 0.8 1.0 No. apoptotic cells/tubule 10 month 12 month untreated treated untreated recovered treated recovered untreated treated

19. Non-fertility parameters

20. Long-term effects- general growth curves endocrinology

21. Long term effects- Serum Chemistry LiverKidney

22. Behavioural data - rota rod Accelerating treadmill Motor coordination Strength

23. Behavioural data - open field Open box 2 matrices of infrared light beams Spontaneous exploratory behaviour

24. Summary of long term study Low-dose administration of NB-DNJ is an effective method to induce and maintain infertility in male mice for long periods of time Normal fertility is fully regained after long-term treatment Infertility during long-term treatment :abnormal morphology reduced motility reduced sperm count Prolonged administration of low-dose NB-DNJ does not cause overt adverse effects

25. Acknowledgements Dr Aarnoud van der Spoel Dr Frances Platt Prof Raymond Dwek Dr Terry Butters Mr David Smith Mr Mohan Masih