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Effects of knockout of antioxidant genes on spermatogenesis

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  • The Issue of infertilityInfertility – as defined by the CDC, not being able to get pregnant after a year of trying - it is estimated that worldwide, 1 in 7 couples have problems conceiving - a 2002 study by the CDC found that 12% of couples in the US are affected by infertility - infertility is often thought of as only a woman’s condition. It’s not just a woman’s concern. - The same CDC study found that 18% of males were diagnosed with male-related infertility problems - in fact, a male-related infertility problem has been found to be the sole, or a contributing cause, of 40% of infertile couplesCauses – abnormal sperm – abnormalities in shape and number of heads, tails, kinks and curls in tails etc. 60% and even as low as 30% normal sperm and motility Low sperm count (oligospermia) – the normal number of sperm is 20+ mil/mLAzoospermia (no sperm) – usually caused by blockagesVaricoceles – enlarged varicose veins of the spermatic cord Abnormal semen – blood in ejaculate, thick/watery, low volume of semen etc.
  • Environmental factors:These causes of infertility stem from different physiological, genetic and environmental factors. physiological like varicoceles, cancer, infections many are caused by environmental factors like smoking, pollution all of these factors lead to oxidative damage in the cells - this oxidative damage is caused by reactive oxygen species (peroxides and oxygen free radicals) - reactive molecules that contain oxygen - formed as natural byproduct of normal metabolism of oxygen and have an important role in cell signaling: apoptosis, host defense and ion transport - During environmental stress (like UV, heat, pollutants), ROS levels increase dramatically, which can result in significant damage to cell structures due to oxidative stress, eventually leading to infertility - cell structures like protein, biomembranes and DNA damageBiological systems have an ability to readily detoxify reactive intermediates - antioxidant enzyme mechanisms through the use of superoxide dismutases, catalases and glutathione peroxidases - small moleule antioxidants such as Glutathione, Vitamin E, and Vitamin C
  • The main focus of this study is centered around Glutathione (GSH) Antioxidant tripeptide synthesized de novo from three different amino acids taken from our diet (Glutamate, Cysteine and Glycine) Synthesis happens in two ATP-dependent steps, directed by two different enzymes, Glutamate CysteineLigase and GSH Synthase The first step of the reaction is performed by GCLThis enzyme is a heterodimer composed of a catalytic (GCLc) and a modulatory (GCLm) subunits. glutathione gets conjugated to cytotoxins (ROS) and is then transported outside the cell together with the ROS. Ablation of gclm leads to decreased levels of glutathione in various tissues, including testes and epid.
  • There is another important factor in the antioxidant response: Nrf2 (nuclear factor erythroid-2 related factor 2)Transcription factor that regulates the antioxidant response. This factor induces genes important in combating oxidative stress (genes like GCL) Nrf2 is retained in the cytosol by association with the cytoskeletal protein Keap1. Upon oxidative stress from environment, ROS etc, Nrf2 is phosphorylated and translocates to the nucleus in response to Protein Kinase C activation and MAPK pathways. In the nucleus, Nrf2 activates genes through binding to AREs (antioxidant response elements) and promoting transcription. mice lacking Nrf2 have reduced expression of numerous antioxidant genes, including Gclm and Gclc previous studies from our lab have shown that Nrf2 KO male mice have an accelerated decline in sperm counts and fertility with age.
  • GCLM: - used 10 month old male mice, KO and WT - testes were analyzed and sperm morphology was scored -epid. Sperm - looking at abnormalities in sperm head, tail and cytoplasmic droplet (indicative of immature sperm)NRF2: - KO and WT male mice were randomly assigned (n=7/group) - fed either a low Vitamin E diet (minimum daily requirement) or a high Vitamin E diet (10 fold minimum) - looked at sperm counts using testicular sperm - scored for sperm morphology using epid. sperm
  • Figure 4 shows the average # of sperm/testis in millions.The Y axis shows the average number of sperm/testis in millionsThe X axis shows the High and Low Vitamin E dietBlack means WT, white means KOWe can see that WT had about 20 mil. Sperm/testis while KO had about 15 milThis is a significant reduction in sperm count of about 25% between the two genotypesHowever the Vitamin E diet did not have an effect on actual sperm counts as both KO groups had relatively similar numbers
  • Figure 5 shows the average number of sperm per caudaepidydimis. The Y axis tells the average number of sperm in millionsThe X axis shows the two treatment groups (high and low) with black being WT and white being KO we again see a decrease in sperm from WT to KO with about a 40% decrease in total sperm in the low diet group.
  • Figure 6 is a reference picture of sperm morphology. It shows different sperm abnormalities so you can see what we were looking for while scoring. This picture is also relevant for the GCLM study.A shows normal sperm, with a characteristic “hook” that mouse sperm has in comparison with human sperm, which lacks this hook. B is an example of an abnormal head. We can see that its shape is different from normal sperm and its also missing the hookC is an example of a cytoplasmic droplet. The small circle in the tail is that droplet. Its an indication that the sperm is still immature and has not reached maturityD shows abnormal tail. Really, there is no tail which determines the abnormality. These sperm heads were missing tails. Other examples would include kinks in the tail or twisted tails.
  • Figure 7 shows average percent of abnormal sperm scored from Nrf2 mice. The Y axis shows average percent abnormal while the X axis shows the different treatment groups. Black means WT and white is KOThere is no significant difference between the two genotypes or between the diet groupsWe do however see a trend: higher abnormalities in WT and in high diet groups, a trend that is consistent across head and tail abnormalitiesWe also see consistent abnormalities of over 20%
  • Figure 9 shows average percent of abnormal tail and head in the GCLM genotype of the line. The Y axis gives average percent of abnormalitiesThe X axis shows the WT and KO groupsThere is a slight trend of lower abnormalities in WT than in KO but nothing is significantWe only had 3 WT animals as opposed to 6 KO animals
  • Figure 10 shows average percent of total abnormal sperm (in A). The trend of higher abnormalities in KO translates and remains consistent. There is high variability leading to insignificance.Figure 10 B shows percent immature sperm and we can see a decrease in immaturity in WT animals, however again the high variability leads to non-significance
  • We observed a significant decrease in testicular and epidydimal sperm counts in the KO groupsWe did not observe any significant effects of diet or genotype on sperm morphology but we come back to the interesting trend of increased abnormalities in the high treatment groupA possible cause to these abnormalities could be the dose size. Maybe a 10-fold increase in Vitamin E was too much and actually became destructive rather than beneficial. According to literature, C57BL/6 mice, at 4 months of age, have between 11 and 13% abnormalities in sperm morphology but in our study, we observed abnormalities of over 20%, indicating a possible difference in the strain.
  • We did not observe any significant effects of genotype on sperm morphology, with averages between 8-15% abnormal, consistent with literature that indicates about 12-13% abnormalities in 10 month old C57BL/6 miceWe did observe that KO showed a higher percentage of immature sperm but again, the high variability within the groups rendered the results to not be significantThis variability is most likely coming from the small sample size of the study. The WT group only had 3 males while the KO group had 6 males. Further scoring of WT and KO samples could provide further insight into this trend and reduce variability.
  • From the Nrf2 data, it seems that Vitamin E alone is not a suitable antioxidant to protect Nrf2 knock-out mice from declines in spermatogenesis. Perhaps a lower dose, or the use of a different antioxidant, like Vitamin C or Glutathione, or a combination of these would provide the protection needed.Furthermore, differences in Gclm genotypes do not seem to have an effect on sperm morphology and further scoring could provide more insight into the trend of immature sperm.
  • Transcript

    • 1. Effects of knockout of antioxidant genes on spermatogenesis
      Bogdan Rau
      Department of Medicine
      PI: Dr. Ulrike Luderer
    • 2. BACKGROUND
      The issue of infertility
      12% of couples are infertile
      Male factor ~40-50%
      Major causes of male infertility:
      Abnormal sperm
      Low sperm count (oligospermia)
      Azoospermia
      Varicoceles
      Abnormal semen
    • 3. Figure 1. Association of ROS production with infertility. (International Braz J Urol. 2007)
    • 4. A
      B
      Figure 2. Glutathione Production and Pathway. A. de novo glutathione synthesis. B. Glutathione antioxidant activity and recycling pathway (LP Institute)
    • 5. 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)
    • 6. Objective
      Determine fertility and sperm counts of Gclmknock-out and wild type mice
      Determine if vitamin E (known antioxidant) will protect Nrf2 -/- males from testicular oxidative damage
    • 7. Methods
      GCLM – C57BL/6J
      Mouse testes analyzed at 10 mos.
      Sperm morphology
      Abnormalities in head and tail
      NRF2 – C57BL/6NCrl
      KO and WT randomly assigned (n=7/group)
      Low (normal) & High (10 fold increase)
      Sperm counts – hemacytometer
      Sperm morphology
    • 8. 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
    • 9. 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
    • 10. 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
    • 11. 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
    • 12. Results – Nrf2
      A
      B
      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
    • 13. Results – Gclm
      A
      B
      Figure 9. Average percent abnormal sperm tail (A) or head (B). Samples taken from 10 month old GclmKO & WT male mice. Scored using light microscopy. (n=3 for WT and n=6 for KO)
    • 14. Results – Gclm
      A
      B
      Figure 10. Average percent total abnormal sperm tail (A) and average percent immature (B). Samples taken from 10 month old GclmKO & WT male mice. Scored using light microscopy. (n=3 for WT and n=6 for KO)
    • 15. Discussion – Nrf2
      KO mice had a significant decrease in testicular and epidydimal sperm counts.
      No significant effects of diet or genotype on sperm morphology
      Interesting trend: increased abnormalities in WT and in high diet groups
      dose = cause?
      Vitamin E is not a suitable antioxidant
      Regular strain
      11-13% abnormal
    • 16. Discussion – Gclm
      No significant effects of genotype on sperm morphology
      Trend: KO showed higher percentage of immature sperm
      High variability and low sample size
      Regular strain
      12-13% abnormal
    • 17. Conclusion
      Vitamin E alone is not a suitable antioxidant to protect Nrf2 -/- from declines in spermatogenesis.
      Differences in Gclmgenotypes do not have an effect on sperm morphology
    • 18. 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