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EFFECTS OF KNOCKOUT OF
ANTIOXIDANT GENES ON
SPERMATOGENESISBogdan Rau
Department of Medicine
PI: Dr. Ulrike Luderer
BACKGROUND
The issue of infertility12% of couples are infertile
○ Male factor ~40-50%
Major causes of male infertility:Abnormal spermLow sperm count (oligospermia) Azoospermia Varicoceles Abnormal semen
Figure 1. Association of ROS production with infertility. (International Braz J Urol. 2007)
Figure 2. Glutathione Production and Pathway. A. de novo glutathione synthesis. B. Glutathione antioxidant activity and recycling pathway (LP Institute)
A
B
Figure 3. Nrf2 activation pathway. After being phosphorylated, Nrf2 travels to the nucleus to bind to ARE and promote the production of Phase II enzymes (Nature 2003)
Objective
Determine fertility and sperm counts of Gclm knock-out and wild type mice
Determine if vitamin E (known antioxidant) will protect Nrf2 -/- males from testicular oxidative damage
Methods
GCLM – C57BL/6JMouse testes analyzed at 10 mos. Sperm morphology
○ Abnormalities in head and tail
NRF2 – C57BL/6NCrlKO and WT randomly assigned (n=7/group)
○ Low (normal) & High (10 fold increase) Sperm counts – hemacytometerSperm morphology
Results – Nrf2
Figure 4. Average # of sperm/testis. Samples taken from 4 month old Nrf2 KO and WT male mice. Scored using light microscopy. *P<0.01. n=7/group
Results – Nrf2
Figure 5. Average # of sperm/epidydimis. Samples taken from 4 month old Nrf2 KO and WT male mice. Scored using light microscopy. n=7/group
Results – Nrf2
Figure 6. Photograph of normal/abnormal/immature sperm. Samples taken from 4 month old Nrf2 WT/KO male mice; A. Normal, B. Abnormal head, C. Cytoplasmic Droplet, D. Abnormal tail. Magnification: 400x
Results – Nrf2
Figure 7. Average percent abnormal sperm. Samples taken from 4 month old Nrf2 KO & WT male mice. Scored using light microscopy. n=7/group
Results – Nrf2
Figure 8. Average percent abnormal sperm tail (A) and head (B) Samples taken from 4 month old Nrf2 KO & WT male mice. Scored using light microscopy. n=7/group
A B
Results – Gclm
Figure 9. Average percent abnormal sperm tail (A) or head (B). Samples taken from 10 month old Gclm KO & WT male mice. Scored using light microscopy. (n=3 for WT and n=6 for KO)
A B
Results – Gclm
Figure 10. Average percent total abnormal sperm tail (A) and average percent immature (B). Samples taken from 10 month old Gclm KO & WT male mice. Scored using light microscopy. (n=3 for WT and n=6 for KO)
A B
Discussion – Nrf2
KO mice had a significant decrease in testicular and epidydimal sperm counts.
No significant effects of diet or genotype on sperm morphologyInteresting trend: increased abnormalities in WT
and in high diet groups○ dose = cause?
Vitamin E is not a suitable antioxidant Regular strain
11-13% abnormal
Discussion – Gclm
No significant effects of genotype on sperm morphology
Trend: KO showed higher percentage of immature spermHigh variability and low sample size
Regular strain12-13% abnormal
Conclusion
Vitamin E alone is not a suitable antioxidant to protect Nrf2 -/- from declines in spermatogenesis.
Differences in Gclm genotypes do not have an effect on sperm morphology
Acknowledgements
I would like to thank Dr. Ulrike Luderer for her mentoring and assistance
Brooke Nakamura for her assistance in lab UROP for funding and support