REPRODUCTIVE MEDICINE. FACULTY OF MEDICINE OF THE ... conferred by the Faculty of Medicine of the University of Saarland, Germany. Submitted by.
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DEPARTMENT OF OBSTETRICS, GYNAECOLOGY & REPRODUCTIVE MEDICINE FACULTY OF MEDICINE OF THE UNIVERSITY OF SAARLAND HOMBURG/SAAR
Protamines and DNA Integrity as A Biomarkers of Sperm Quality of Smokers and Non-Smokers Patients Undergoing Assisted Reproduction Therapy
Dissertation in partial fulfillment of the requirement for the award the degree of Doctor Rerum Medicinae (Dr. rer. med.) conferred by the Faculty of Medicine of the University of Saarland, Germany
Submitted by Mohammed Faiz Khaleel Hamad Born in Amman-Jordan in 24.11.1968
2009
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“For anything worth having one must pay the price; and price is always work, patience, love, and self-sacrifice”
Dedication
This work is dedicated to the persons whom supported me all through my life and never saved an effort to lead me to what I am now, my father and mother. My wife and our children as a sign of love and appreciation for enduring my absence during the long stay abroad, their patience and moral support to complish this piece of work
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Contents
1- Summary 1-1- Summary 1-2- Zusammenfassung
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2- Introduction 13 2-1- Human sperm chromatin structure 13 2-2- Sperm Nuclear Proteins 16 2-2-1- Histones 16 2-2-2- Transition nuclear proteins (TPs) 17 2-2-3- Replacement of Histones by Protamine 18 2-2-4- Protamines 20 2-2-4-1- Genes of protamines 21 2-2-4-2- Synthesis of protamines 22 2-2-4-3- Characteristic structural features 25 2-2-4-3-1- Protamine P1 25 2-2-4-3-2- Protamine P2 25 2-2-4-4- Protamines Functions 26 2-2-4-5- Structural – functional relationships 26 2-2-4-6-Sperm DNA integrity and Protamines 27 2-2-4-7-Protamines and infertility 27 2-2-4-8- Determination of protamines 28 2-2-4-8-1- Direct determination 28 2-2-4-8-2- Indirect assessment of sperm chromatin structure by histochemical procedures 30 2-3- Reactive Oxygen Species (ROS) 30 2-3-1- Physiologic role of ROS 31 2-3-2- Consequences of excessive generation of ROS 31 2-3-3- (8-hydroxy-2'-deoxyguanosine (8-OHdG)) level 32 2-3-4- Lipid peroxidation (Malondialdehyde, MDA) 32 2-4- Smoking and sperm parameters 33 2-4-1- Introduction 33 2-4-2-Smoking and semen quality 33 2-4-3- Effects of Smoking on Semen Volume 34 2-4-4- Effect of Smoking on Sperm Concentration 34 2-4-5- Effect of Smoking on Sperm Motility 34 2-4-6- Effect of smoking on sperm Morphology 35 2-4-7- Effect of smoking on sperm chromatin integrity (DNA) 35 2-4-8- Cotinine level in seminal plasma 36 2-5- Factors that influence sperm chromatin structure 36 2-5-1- Temperature 36 2-5-2- Chemicals and toxins 37 2-5-3- Disease 37 2-5-4- Molecular disturbances 37 2-5-5- Age and spermatozoal DNA damage 37 2-6- Origin of sperm DNA damage 37 2-6-1- Defects in recombination 39 2-6-2- Spermatid maturation abnormalities 39 2-6-3- Oxidative stress 39 2-6-4- Abortive apoptosis 41 2-7- Clinical significance of sperm DNA damage 42 2-7-1- DNA damage and semen parameters 42 2-7-2- The relationship between sperm DNA integrity and fertilization 43
4 2-7-3- Sperm DNA damage and preimplantation development 2-7-4- Intrauterine insemination (IUI) 2-7-5- In vitro fertilization (IVF) and Intracytoplasmic sperm injection (ICSI) 2-7-6- Embryonal loss 2-7-7- Effect of sperm DNA quality on offspring 2-8- Assessment of sperm DNA damage 2-8-1- Factors that influence the predictive value of DNA damage tests 2-8-2- Assessment Methods 2-8-2-1- Chromatin structural probes using nuclear dyes 2-8-2-1-1- Acridine Orange Assay (AOA) 2-8-2-1-2- Chromomycin–A3 Staining (CMA3) 2-8-2-1-3- Sperm chromatin structure assay (SCSA) 2-8-2-1-4- DNA breakage detection–fluorescence in situ hybridization test (DBD-FISH) 2-8-2-1- 5- Acidic Aniline Blue Assay (AB) 2-8-2-1- 6- Toluidine blue Assay (TB) 2-8-2-2- Tests for direct assessment of sperm DNA fragmentation 2-8-2-2-1- In situ nick translation assay (ISNT) 2-8-2-2-2- Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick-end abelling assay (TUNEL) 2-8-2-2-3- COMET assay (Single Cell Gel Electophoresis) Assay 2-8-2-3- Sperm nuclear matrix assays 2-8-2-3-1- Sperm chromatin dispersion assay (SCDA) 2-9- Aims of the study
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3- Materials and Methods 3-1- Materials 3-1-1- Semen samples 3-1-2- Chemicals and laboratory materials 3-1-3- Buffers and Solutions 3-1-4- Apparatus and Instruments 3-1-5- Antibodies 3-1-5-1- Primary antibodies 3-1-5-2- Secondary antibody 3-1-6- ELISA Kits 3-2- Methods 3-2-1- Ejaculates and Sperm Preparation 3-2-2- Assessment of Sperm Morphology 3-2-3- Assessment of Sperm Vitality (Eosin Test) 3-2-4- Assessment of the Sperm Membrane Integrity (Hypo-Osmotic Test; HOS) 3-2-5- Assessment of Chromatin Condensation (Chromomycin, CMA3) of Spermatozoa 3-2-6- DNA Fragmentation Analysis (Terminal deoxynucleotidyltransferasemediated dUTP nick end labeling, TUNEL Test) 3-2-6- Protamine Quantification 3-2-7-1- Determination of protein concentration according to Bradford 3-2-7-2- Total Nuclear Protein Extraction: 3-2-7-2-1- Tail Dissociation 3-2-7-2-2- Protein Extraction 3-2-7-3- Production of a Protamine Standard 3-2-7-4- Protein Extraction Control Sample 3-2-7-5- Acid-Urea Polyacrylamide Gel Electrophoresis (AU-PAGE) 3-2-7-5-1- AU-PAG preparation 3-2-7-5-2-Electrophoresis 3-2-7-5-3- Staining 3-2-7-6- Western Blot
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5 3-2-8- Reactive oxygen species measurement 3-2-9- 8-hydroxy-2-deoxyguanosine (8-OH-dG) measurement 3-2-10- Lipid peroxidation assay (Measurment of Malondialdehyde, MDA) In Seminal Plasma 3-2-11- Cotinine measurement 3-2-12- Statistical analysis
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4- Results 4-1- Semen and Sperm Parameters for all Participants 4-2- Normal volunteers versus Patients 4-2-1- Normal Volunteers 4-2-2- Patients 4-3- Non-smokers versus Smokers 4-3-1- All participants 4-3-2- Normal volunteers 4-3-3- Patients 4-3-4- Normal smokers versus patient smokers 4-4- Protamines Quantification 4-4-1- Volunteers versus Patients 4-4-2- Non-smokers versus smokers 4-4-2-1-Volunteers non-smokers versus smokers 4-4-2-2-Patients non-smokers versus smokers 4-4-2-3- Patients smokers versus volunteers smokers 4-4-3- P1/P2 ratio grouping 4-4-3-1- Relationship between P1/P2 ratios and seminal parameters 4-4-3-1-1- All participants 4-4-3-1-2- Smokers 4-5- Clinical Data
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5- Discussion 5-1- Sperm DNA Integrity and Semen Parameters (CMA3 and TUNEL) 5-2- Oxidative stress 5-2-1- Oxidative Stress and Semen Parameters 5-3- Smoking and infertility 5-3-1- Smoking and oxidative damage 5-3-1-1- Smoking and ROS 5-3-1-2- Cotinine and sperm parameters 5-3-1-3- Smoking cigarette and MDA levels 5-3-1-4 Smoking cigarette and 8-OHdG levels 5-3-2- Smoking and Semen Parameters 5-3-3- Smoking and Semen volume 5-3-4- Smoking and Sperm motility 5-3-5- Smoking and Sperm Morphology 5-3-6- Smoking and DNA damage 5-4- Protamines Quantifications 5-4-1- P1 and P2 Concentrations 5-4-2- Protamines and DNA integrity 5-4-3- P1/P2 Content and Sperm Quality 5-4-4- P1/P2 Content and Semen Parameters 5-4-5- P1/P2 Content and Fertilization and Pregnancy Rates 5-4-6- P1/P2 ratio Grouping 5-4-7- P1/P2 ratios and seminal characteristics 5-4-8- Relationship between Protamines and Smoking Conclusion
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6 References Acknowledgment Curriculum Vitae (CV)
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1- Summary 1-1- Summary Unlike the chromatin structure of somatic cells, sperm chromatin is very tightly compacted by virtue of the unique associations among the DNA, the nuclear matrix, and sperm nuclear proteins. Protamines (P1 and P2 family) are the most abundant proteins in human sperm nucleus and are important for condensation of the paternal genome of the spermatozoa that produce a more compact and hydrodynamic nucleus. This compact structure protects the genetic materials during transport of the paternal genome through the male and female reproductive tracts, also, allow proper fusion of male and female gametes and correct expression of the genetic information by the developing embryo. Human sperm protamine deficiency has been correlated significantly with reduced semen quality parameters (counts, motility and morphology), sperm penetration ability, sperm DNA integrity, and male infertility. Sperm DNA damage may occur at testicular, epididymal or post-ejaculatory levels. DNA damage in the male germ line has been associated with poor semen quality, low fertilization rates, impaired embryonal preimplantation development, increased abortion and an elevated incidence of disease in the offspring, including childhood cancer. Oxidative stress resulting from an imbalance in the productions of reactive oxygen species (ROS) and the antioxidant capacity appears to be the major cause of DNA damage in the male germ line. Spermatozoa are particularly susceptible to damage induced by excessive ROS; their plasma membrane contains large quantities of polyunsaturated fatty acids and their cytoplasm contains low concentrations of scavenging enzymes. Smoking was associated with an increase in ROS levels and a decrease in ROS-Total antioxidant capacity (TAC) scores. Many studies revealed that semen samples from smokers have a significantly higher ratio of single-strand-to double-strand breaks of DNA spermatozoa. The current study evaluates the relationship between the oxidative stress, smoking and human sperm DNA integrity. Other evaluations include the effect of smoking and sperm parameters, cotinine and oxidative stress biomarkers (ROS, MDA, and 8-OHdG) levels in seminal plasma, the effect of smoking on protamines, and the ratios of protamine 1 to protamine 2 in human spermatozoa and their effect on intracytoplasmic sperm injection (ICSI) and standard semen parameters. Semen samples of patients who underwent ICSI therapy (n=116) and healthy volunteers (n=50) were included in this study. The patients group and volunteers group were divided into two groups: smokers (where n=53 patients and n=19 volunteers) and non-smokers (n=63 patients and n= 31 volunteers). Each semen sample was evaluated according to the WHO guideline (1999) except the morphology, which was evaluated undergo strict criteria. Chromatin condensation, DNA integrity, and protamines concentrations were investigated. In addition, the concentrations of ROS, MDA, 8OHdG and cotinine in seminal plasma were measured. Smears were prepared for evaluation of: sperm vitality (Eosin test) and morphology by normal staining; chromatin condensation (CMA3 assay) and DNA integrity (TUNEL assay) by fluorescence technique. Aliquots of each sperms sample were used for protamine extraction and evaluation using acid-urea polyacrylamide gel electrophoresis. ROS, cotinine and 8-OHdG were evaluated by ELISA, while MDA were evaluated by chemical reactions. The results from this study showed significant differences (p
