Charlotte Walden

Imino sugars and mouse spermatogenesis

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)

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?

• 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

Fertility -mating assays

10 month 12 month

0.0

0.5

1.0

1.5

2.0

2.5

3.0

untreated treated recovered

Group

pre

gn

an

cie

s/

male

0

2

4

6

8

10

untreatedtreated recovered

Group

litt

er

siz

e

pregnancies litter size

Long-term effects on the testis

Testis and epididymis weights

Test

is w

eig

ht

(%

body w

eig

ht)

Epi d

idym

is weig

ht

(% b

ody w

eig

ht )

untreated treated recovered

6 m

onth

s12 m

onth

sLong-term effects on the testis-

Tubule morphology

Structure of the testis

Nature Reviews Genetics 3 :790-801

Elongated spermatids

Round spermatids

Pachytene spermatocytes

Pre-leptotene spermatocytes

spermatogonium

Basement membrane

lumen

Spermatogenesis

Nature Reviews Genetics 3 :790-801

acrosome

Acrosome development in murine spermatids

Step 2 Step 3 Step 5 Step 7

Fusion of pro-acrosomal vesicles: ONE vesicle attaches to nucleus

Flattening and spread of acrosome: head cap covers anterior nucleus

Spermiogenesis

Long-term effects on fertility- morphology

control treated recovered

6 m

onth

s12 m

onth

s

Long-term effects on fertility - morphology

normal nucleiacrosomal stainingnormal acrosomal staining

0

20

40

60

80

100

120

0 10 20 30 40 50 60

%

40

NB-DNJ treatment Recovery

Time (Days)

Motility analysis - untreated

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Motility analysis - treated

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Long-term effects on fertility- Motility

0

10

2030

40

50

60

7080

90

100

untreated treated recovered

% motile

0

10

2030

40

50

60

7080

90

100

untreated treated recovered

% motile

3 months 12 months

Long-term effects on fertility - sperm counts

* short-term NB-DNJ treatment does not reduce sperm count

Long-term effects on the testis- Apoptosis

TUNEL assay

0.0

0.2

0.4

0.6

0.8

1.0

No.

ap

op

toti

c c

ells/t

ub

ule

10 month12 month

untreated

treated

untreated

reco

vered

treated

reco

vered

un

treate

dtr

eate

d

Non-fertility parameters

0 8 16 24 32 40 4820

25

30

35

40

45

Treatment (weeks)

Long-term effects- general

growth curves endocrinology

Long term effects- Serum Chemistry

Liver Kidney

Behavioural data - rota rod

Accelerating treadmill

Motor coordination

Strength

Behavioural data - open field

Open box

2 matrices of infrared light beams

Spontaneous exploratory behaviour

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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

Acknowledgements

•Dr Aarnoud van der Spoel

•Dr Frances Platt

•Prof Raymond Dwek

•Dr Terry Butters

•Mr David Smith

•Mr Mohan Masih