charlotte walden imino sugars and mouse spermatogenesis
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Charlotte Walden
Imino sugars and mouse spermatogenesis
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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)
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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?
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• 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
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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
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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 )
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untreated treated recovered
6 m
onth
s12 m
onth
sLong-term effects on the testis-
Tubule morphology
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Structure of the testis
Nature Reviews Genetics 3 :790-801
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Elongated spermatids
Round spermatids
Pachytene spermatocytes
Pre-leptotene spermatocytes
spermatogonium
Basement membrane
lumen
Spermatogenesis
Nature Reviews Genetics 3 :790-801
acrosome
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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
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Spermiogenesis
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Long-term effects on fertility- morphology
control treated recovered
6 m
onth
s12 m
onth
s
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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)
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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
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Long-term effects on fertility - sperm counts
* short-term NB-DNJ treatment does not reduce sperm count
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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
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Non-fertility parameters
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0 8 16 24 32 40 4820
25
30
35
40
45
Treatment (weeks)
Long-term effects- general
growth curves endocrinology
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Long term effects- Serum Chemistry
Liver Kidney
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Behavioural data - rota rod
Accelerating treadmill
Motor coordination
Strength
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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
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Acknowledgements
•Dr Aarnoud van der Spoel
•Dr Frances Platt
•Prof Raymond Dwek
•Dr Terry Butters
•Mr David Smith
•Mr Mohan Masih