The Effects of Erectile Dysfunction Drugs on Mouse (Mus musculus) Spermatozoa Heather Stultz Mentor : Dr. Deborah Ricker Introduction ● Erectile dysfunction (ED) is a condition that affects about 10% of the adult population, including young males (Plessis et al. 2004). Drugs such as Levitra, Cialis, and Viagra are frequently prescribed to relieve ED symptoms (Glenn et al. 2007), each with a unique active ingredient. ●Penile erection requires cGMP-mediated vasodilation within the penis. Increased blood flow causes penile pressure to increase, causing the erection. To stop the erection, phosphodiesterase (PDE) 5 enzyme degrades the cGMP. ED drugs are PDE 5 inhibitors. This means they stop the enzyme from degrading cGMP by binding to its catalytic sites (Figure 1). This will cause the cGMP to accumulate, therefore promoting an erection (Plessis et al. 2004). ● Since these drugs are being prescribed to younger men, research has been conducted to study their effect on sperm. All three ED drugs have shown no effect on sperm morphology but there is controversy over how they affect motility (Pomara et al. 2007). A recent study of Viagra, found that it induces premature acrosome reaction (Figure 2), thus decreasing sperm fertility (Glenn et al. 2007). ● Each ED drug has been investigated individually for their effect on sperm motility and morphology, but a comparative study has yet to be conducted. Furthermore, the effects of Cialis and Levitra on the acrosome reaction have not been determined. MethodsResults Overall Conclusions 1)Both tadalafil and sildenafil decreased the overall percent of motile sperm, while vardenafil had no significant effect. This could be because tadalafil and sildenafil were at higher concentrations causing them to be poisonous to the sperm. It could also be because the drugs are from the PDE-inhibitor family. This means that they can inhibit other secondary PDEs which could affect the motility of the sperm (Pomara et al. 2007). 2) Vardenafil, tadalafil, and sildenafil had no significant effects on the tail and head morphology of mouse sperm. The morphology of the sperm at 1000X was found to have no differences which would suggest the drugs don’t have a structural effect on the sperm. However, a closer examination of the sperm may show a morphological impact. Also, sperm in the epididymis have already gone through meiosis which could be why the morphology wasn’t effected 3) The effect of the erectile dysfunction drugs on the acrosome could not be determined due to technical difficulties with the CTC staining procedure. Future Studies Repeat CTC staining or reassess acrosome status Complete a dose-response curve for each drug Perform closer microscopic evaluation of sperm structure Assess drug impact on sperm fertility Objectives 1.To determine whether mouse sperm motility is affected differently by the active ingredients in Cialis, Levitra, and Viagra 2.To determine whether mouse sperm morphology is affected differently by the active ingredients in Cialis, Levitra, and Viagra. 3.To determine if the active ingredients in Cialis, Levitra, and Viagra cause a premature acrosome reaction in mouse sperm. Figure 2: The Sperm Acrosome Reaction. Acrosome intact sperm (left) contact the egg, which induces fusion of the plasma membrane and outer acrosomal membrane. Enzymes within the acrosome are released (right) and facilitate penetration of the egg. ansci.wisc.edu Sperm dissected from cauda epididymis and diluted in medium Sildenafil citrate, tadalafil, and vardenafil dissolved in ethanol and diluted in medium Motility and Morphology Methods Acrosome Methods Sperm were diluted to approx. 1,000,000 sperm/mL and incubated 1 hour in 5% CO 2 at 37°C in equal amounts of medium (negative control) calcium ionophore A23187 (positive control), tadalafil (270ng/mL), vardenafil (18.125ng/mL), or sildenafil (450ng/mL) Sperm sample was placed on a slide and stained with chlortetracycline (CTC) 100 randomly selected sperm were assessed using Fluorescence microscopy Equal numbers of sperm mixed with: - sildenafil citrate (450ng/mL), - tadalafil (270ng/mL), - vardenafil (18.125ng/mL), or - medium (control) Sperm incubated 5 min in 5% CO 2 incubator at 37°C Samples placed on a pre-warmed slide 100 sperm assessed (5 fields/slide) Effect on Percent Motility Effect on Sperm Tail Morphology CurledNormal Effect on Sperm Head Morphology Normal Abnormal Absent Bi et al Literature Cited Bi, Y., Xu, W., Wong, H., Zhu, H., Zhou, Z., Chan, H., Sha, J NYD-SP27, a novel intrinsic deacapacitation factor in sperm. Asian Journal of Andrology [serial online] 11: Available from: Glenn, D.R., McVicar, C.M., McClure, N., Lewis, S Sildenafil citrate improves motility but causes premature acrosome reaction in vitro. Fertility and Sterility [serial online] 87: Available from: Academic Search Premier. Plessis, S., Jongh, P., Hons, B., Franken, D Effects of acute in vivo sildenafil citrate and in vitro 8-bromo-cGMP treatments on semen parameters and sperm function. Fertility and Sterility [serial online] 81: Available from: Academic Search Premier. Pomara, G., Morelli, G., Canale, D., Turchi, P., Caglieresi, C., Moschini, C., Liguori, G., Selli, C., Macchia, E., Martino, E., and Francesca, F Alterations in sperm motility after acute oral administration of sildenafil or tadalfil in young, infertilie men. Fertility and Sterility [serial online] 88: Available from: Pubmed Central. Acknowledgments I thank Dr. Deborah Ricker for assisting me with taking on this project, Dr. Karl Kleiner for directing me in the statistical analysis, Ashley Swift for blinding my experiment results, and the York College Biology Department for funding my research. Figure 1: The cGMP/PDE5 Pathway. Accumulation of cGMP in penile smooth muscle and endothelial cells facilitates relaxation and vasodilation. PDE5 prevents cGMP breakdown thus promoting penile erection. cvpharmacology.com Statistical Analysis performed Acrosome intact mouse sperm Acrosome reacted mouse sperm Effect on Acrosome Reaction CTC staining of mouse sperm acrosome reaction was inconclusive.