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Armand Zini, M.D.

Associate professor

McGill University

Division of urology

St. Mary's Hospital
3830 Lacombe
Montreal, Quebec

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Telephone: (514) 345-3511 ext:3282

 

 

Research Interests :

DNA studies

  • Sperm DNA integrity
  • Semen oxidants
  • Varicocele
Sperm function and reactive oxygen species
  • Sperm motility, capacitation, acrosome reaction
  • Reactive oxygen species and their generators
  • Signal transduction cascades
  • Sulfhydryl-disulfide pair in proteins

 

Summary of results:  

DNA studies

Our laboratory is involved in the study of male factor infertility particularly the investigation of sperm DNA damage, its nature and etiology and effects on reproductive outcomes. Evidence shows that spermatozoa of infertile men possess substantially more DNA damage (genetic and epigenetic alterations) than those of fertile men. Our initial studies focused on DNA fragmentation, abnormal chromatin packaging and protamine deficiency. We also looked at gene-specific DNA damage specifically the telomeres using quantitative real time PCR. Recently, we have been evaluating the effects of varicocele repair and vitamin supplementation (the latter is supported by a CIHR grant) on male fertility using conventional semen parameters as well as tests of sperm DNA integrity. 

Sperm protamine deficiency is observed in a subset of infertile men. We extracted sperm nuclear proteins from fertile (n = 10) and infertile men (n = 20). Histones and protamines were separated by acid-urea (AU) polyacrylamide gel electrophoresis. Sperm samples from the infertile men had a higher proportion of histone H2B to protamine (PRM1 + PRM2) than those from the fertile men (0.38 vs 0.08, P < 0.001). H2B was located throughout the sperm head and the labeling was more intense and in a higher proportion of spermatozoa in infertile men. Therefore, infertile men have a higher proportion of spermatozoa with an increased histone to protamine ratio than fertile controls. 

Using quantitative PCR analysis (real-time PCR), we have shown that spermatozoa of infertile men have higher levels of gene-specific DNA damage (telomere sequences) than that of fertile men. The reduced amplification efficiency is indicative of template DNA damage, but the exact nature of this damage is unknown. These first data suggest that sperm DNA damage in infertile men may be focal (gene-specific) rather than global. Further studies are underway to better characterize the nature of the sperm DNA damage.  

It is reported that 35 to 40 % of infertile men have varicocele (dilated testicular veins) and this has an adverse effect on male fertility. We examined the effect of varicocelectomy on sperm chromatin and DNA integrity. We assessed conventional sperm parameters and sperm chromatin structure assay (SCSA) parameters (% DFI - DNA fragmentation index and % HDS - high DNA stainability, an index of chromatin compaction) before, and, 4 and 6 months after microsurgical varicocelectomy. Sperm DNA integrity improved after surgery (% DFI from 18 ± 11% before surgery to 10 ± 5%, and 7 ± 3%, at 4 and 6 months after surgery, respectively). Sperm chromatin compaction also improved after surgery (% HDS from 11 ± 7% before surgery to 8 ± 6%, and 7 ± 5%, at 4 and 6 months after surgery, respectively). Sperm concentration and progressive motility improved after surgery but differences were not statistically significant.

Recently, we also compared aniline blue (AB) staining of sperm cells collected from pre- and post-varicocelectomy patients. Deeply stained samples are indicative of histone retention and lower chromatin compaction. There was a decrease in the percentage of sperms with dark AB staining 4 months after the surgery which was sustained at 6 months. There was no relationship between AB staining and %DFI, motility or concentration. However, there was a notable correlation between AB staining and HDS post-varicocelectomy.

Sperm mitochondrial DNA (mtDNA) integrity and copy number have also been studied in the context of male infertility. We started quantifying the mitochondrial DNA copy number of the pre- and post-varicocelectomy patients using real-time PCR. Our results show that mtDNA copies per spermatozoa were higher in infertile men with a clinical varicocele and poor sperm motility than in fertile donors. Furthermore, we also observed a significant inverse correlation between sperm motility and sperm mtDNA copy number. Most notably, mtDNA copies were reduced significantly 4-month after varicocele repair.

We are also in the process of recruiting patients for the evaluation of sperm DNA integrity and function after folate supplementation. The parameters that we will examine will include the conventional parameters as well as % DFI and % HDS.  

 

Sperm function and reactive oxygen species 

We study the mechanisms controlling sperm capacitation (CAP), hyperactivated motility and acrosome reaction (AR) with the aim to elucidate how reactive oxygen species (ROS), such as the superoxide anion (O2•¯), hydrogen peroxide (H2O2) and nitric oxide (NO•) generated by human spermatozoa regulate these processes and the associated transduction events. We use different approaches. 

We study protein phosphorylation and cross-talks related to human sperm CAP (time course, regulation by kinases and ROS, cellular distribution). We reported that ROS regulate several of the transduction events associated with CAP. ROS increase sperm cAMP. The peak of sperm PKA activity (30 min of CAP) corresponds to phosphorylation of the Arg-X-X-Ser/Thr motif (PKA substrates) in Triton-insoluble sperm proteins (80 and 105 kDa); PKA, PTK and ROS regulate this process. The ERK pathway (Grb2 to ERK) is present in human spermatozoa and plays a role in CAP and related events. The double phosphorylation of the Thr-Glu-Tyr motif on Triton-insoluble sperm proteins (80 and 105 kDa) during CAP is regulated by MEK-like kinase, PTK and NO• (but not O2•¯ or H2O2). On the other hand, the increased phosphorylation of MEK-like proteins during CAP is controlled by PKA and ROS. The PI3K/Akt axis is also modulated by ROS but appears to be independent from PKA during CAP; one of its downstream effectors may be NO• synthase (NOS). Collectively, results stress that ROS play a role at early, intermediate and late steps of CAP by regulating several phosphorylation events and that parallel pathways are needed that lead to the late protein Tyr phosphorylation.

Sulfhydryl groups (-SH) are often first targets for ROS in regulating protein functions. Induction of CAP modofies the sulfhydryl level on 10 of the 14 Triton-soluble proteins chosen for analysis (2D gel electrophoresis). The increases (by 200-400%, 5 proteins) and the decreases (by 45-95%, 5 proteins) observed are prevented by superoxide dismutase and/or catalase. These alterations occur within 5-15 min but are reversed within 30-120 min. Therefore, CAP is associated with rapid and reversible changes in protein sulfhydryl groups that appear to be redox regulated and could be the first targets for the ROS generated by spermatozoa.

We also study the interactions between ROS (NO• and O2•¯) and their generators (NOS, and a still elusive oxidase) as well as interactions and modulation by zinc (Zn2+) and semenogelin (Sg, the main protein of the semen coagulum). The several inducers tested (albumin, fetal cord serum ultrafiltrate, zinc chelators, L-arginine, etc) trigger CAP, ROS (NO• and O2•¯) generation and tyrosine phosphorylation. CAP due to exogenous NO• (or O2•¯) is also blocked by SOD (or NOS inhibitors). CAP causes a time-dependent raise in protein tyrosine nitration that is prevented by SOD and NOS inhibitors, suggesting a likely formation of peroxinitrite (ONOO–). Spermatozoa treated with NO• (or O2•¯) initiate a dose-dependent O2•¯ (or NO•) production, providing, for the first time in cells, an evidence for two-sided ROS-induced ROS generation.

Inhibitors of kinases (PKC, PI3K, Akt, ERK, PTK) that block CAP have no effect on the generation of O2•¯ but prevent that of NO•. Also, NADPH-induced capacitation is blocked by L-NMMA but not by SOD, which agrees with the observed increase in NO• (but not O2•¯) generation.

Spermatozoa quickly internalize Sg upon incubation and Sg is then rapidly degraded in a Zn2+-inhibitable manner. Triton-soluble and -insoluble sperm fractions contain Sg and Sg peptides, the levels of which decrease with initiation of CAP. This drop is prevented by SOD, NOS inhibitor or Zn2+ and is reproduced by addition of O2•¯, NO• or Zn2+ chelator. In conclusion, Sg and Zn2+ block CAP mainly via inhibition of ROS generation and appear as natural factors that prevent premature CAP.

The studies proposed above are important because they cover many aspects of ROS regulation of human sperm CAP and will allow us to elucidate mechanisms, and to characterize specific elements involved in this process. These studies will improve our understanding of the regulation of sperm CAP while forming the basis on which a rational approach can be developed to diagnose and treat male infertility.   

 

Lab Members: 

Name

Position

Email Address

Eve de Lamirande

Research Associate

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Maria Conception San Gabriel

Research Associate

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Krista Zeidan

Student

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labo

Left to right: Armand, Cristian (voisin et collaborateur), Naif, Eve, Krista (à l'arrière) et Connie

 

Keywords:

Human spermatozoa; sperm DNA; sperm DNA damage; DNA oxidation; male infertility; varicocele, protamines and histones. 
Sperm capacitation; acrosome reaction; hyperactivation; sperm motility; signal transduction; protein phosphorylation; reactive oxygen species; superoxide anion; nitric oxide; hydrogen peroxide; sulfhydryl/disulfide couple; semenogelin.

 

Publications: 

BOOK: Sperm Chromatin: Biological and Clinical Applications in Male Infertility and Assisted Reproduction. Armand Zini and Ashok Agarwal Editors; Springer, New York, 2011; 512 pages

 

Baazeem A, Belzile E, Ciampi A, Dohle G, Jarvi K, Salonia A, Weidner W, Zini A. Varicocele and Male Factor Infertility Treatment: A New Meta-analysis and Review of the Role of Varicocele Repair. Eur Urol. 2011;60:796-808

Zini A, Al-Hathal N. Antioxidant therapy in male infertility: fact or fiction? Asian J Androl. 2011;13:374-381.

Zini A, Jamal W, Cowan L, Al-Hathal N. Is sperm DNA damage associated with IVF embryo quality? A systematic review. J Assist Reprod Genet. 2011;28:391-397.

Zini A. Are sperm chromatin and DNA defects relevant in the clinic? Syst Biol Reprod Med. 2011;57:78-85

Jarvi K, Lo K, Fischer A, Grantmyre J, Zini A, Chow V, Mak V. CUA Guideline: The workup of azoospermic males. Can Urol Assoc J. 2010;4:163-167.

Barratt CL, Aitken RJ, Björndahl L, Carrell DT, de Boer P, Kvist U, Lewis SE, Perreault SD, Perry MJ, Ramos L, Robaire B, Ward S, Zini A. Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications--a position report. Hum Reprod. 2010;25:824-38.

Zini A, Phillips S, Courchesne A, Boman JM, Baazeem A, Bissonnette F, Kadoch IJ, San Gabriel M. Sperm head morphology is related to high deoxyribonucleic acid stainability assessed by sperm chromatin structure assay. Fertil Steril. 2009;9:2495-500.

Zhang X, San Gabriel M, Libman J, Phillips S, Courchesne A, Zini A. Localization of single-stranded DNA in human sperm nuclei. Fertil Steril. 2007;88:1334-8.

Zini A, Gabriel MS, Zhang X. The histone to protamine ratio in human spermatozoa: comparative study of whole and processed semen. Fertil Steril. 2007;87:217-9.

San Gabriel M, Zhang X, Zini A. Estimation of human sperm gene-specific deoxyribonucleic acid damage by real-time polymerase chain reaction analysis. Fertil Steril. 2006;85:797-9.

Zhang X, San Gabriel M, Zini A. Sperm nuclear histone to protamine ratio in fertile and infertile men: evidence of heterogeneous subpopulations of spermatozoa in the ejaculate. J Androl. 2006;27:414-20.

 

Concernant les fonctions des spermatozoïdes (mobilité, capacitation, réaction d'acrosome) et le rôle des dérivés actifs de l'oxygène:

 

de Lamirande E, O'Flaherty CM. Sperm capacitation as a mild oxidative event. Chapter 4. In: Studies on Men's Health and Fertility. Ashok Agarwal, John Aitken, and Juan Alvarez Editors; Springer, New York, 2010; in press

de Lamirande E, Lamothe G. Levels of semenogelin in human spermatozoa decrease during capacitation: involvement of reactive oxygen species and zinc. Hum Reprod. 2010;25:1619-30.

de Lamirande E, Lamothe G and Villemure M, 2009. Controls of superoxide and nitric oxide formation during human sperm capacitation. Free Radic Biol Med, 46:1420–1427.

de Lamirande E, Lamothe G, 2009. Free radical-induced free radical production during human sperm activation. Free Radic Biol Med, 46: 502-510.

de Lamirande E, O'Flaherty C, 2008. Sperm activation: Role of reactive oxygen species and kinases. BBA - Proteins and Proteomics, 1784: 106-115.

O’Flaherty C, de Lamirande E, Gagnon C, 2006. Positive role of reactive oxygen species in mammalian sperm capacitation triggering and modulation of phosphorylation events. Free Radic Biol Med, 41: 528-540.

O’Flaherty C, de Lamirande E, Gagnon C, 2006. Reactive oxygen species modulate independent protein phosphorylation pathways during human sperm capacitation. Free Radic Biol Med, 40: 1045-1055.

O’Flaherty C, de Lamirande E, Gagnon C, 2005. Reactive oxygen species and protein kinases modulate the level of phospho-MEK-like proteins during human sperm capacitation. Biol Reprod, 73: 94-105.

O’Flaherty C, de Lamirande E, Gagnon C, 2004. Protein kinase A-dependent phosphorylation in human sperm capacitation is modulated by reactive oxygen species and protein tyrosine kinase. Mol Hum Reprod, 10: 355-363.

de Lamirande E, Gagnon, C. 2003. Redox control of changes in protein sulfhydryl levels during human sperm capacitation. Free Radic Biol Med, 35: 1271-1285

Thundathil J, de Lamirande E, Gagnon C, 2002. Nitric oxide regulates the phosphorylation of the threonine-glutamine-tyrosine motif in proteins of human spermatozoa during capacitation. Biol Reprod, 68: 1290-1298  

de Lamirande E, Gagnon C, 2002. Involvement of the extracellular signal regulated protein kinase pathway in human sperm function; Involvement of the superoxide anion. Mol Hum Reprod, 8:124-135.

Chatterjee S, de Lamirande E, Gagnon C, 2001. Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione. Mol Reprod Dev, 60:498-506.

de Lamirande E, Tsai C, Harakat A, Gagnon C, 1998. Involvement of reactive oxygen species in human sperm acrosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. J Androl, 19:585-594.

de Lamirande E, Gagnon C, 1998. Paradoxical effect of reagents for sulfhydryl and disulfide groups on human capacitation and superoxide production. Free Radic Biol Med, 25:803-817.