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