THE IMPACT OF THE CHEMOTHERAPEUTIC DRUG
CYCLOPHOSPHAMIDE
ON 'RAT SPERMIOGENIC CHROMATIN REMODELING
Alexis Codrington
Department of Pharmacology and Therapeutics
McGiII University
Montreal, Quebec
February 2007
A thesis submitted to the Faculty of Graduate Studies and Research in partial
fulfillment of the requirements for the degree of Doctor of Philosophy
© Copyright by Alexis Codrington (2007) Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition
395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada
Your file Votre référence ISBN: 978-0-494-32344-1 Our file Notre référence ISBN: 978-0-494-32344-1
NOTICE: AVIS: The author has granted a non L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell th es es le monde, à des fins commerciales ou autres, worldwide, for commercial or non sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats.
The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission.
ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse.
While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada JkÙj the:!i:! i:! dedicated 10 mlj parent:! Cuve/la and ngelCodrington,
who wilhoul he:!ilalion jacrificed eller'lltng /or me 10 make il lkij Jar .
.J tltank them /or their guidance, :!upport and uncondilional love.
Jhe'l galle me the /oundafion necmarlj 10 :!tep up and tkvelop info the woman !) am todalJ'
Jo my :!i:!ler :J)ionne, who !) tru4 value and who
wa:! alwalj:! there when!) neetkd her mo:!t...withoul me kaving to a:!k. mwana lJa mlumu, JUdIUd --4rukol mampa:Mi hafUja, lenediclion lJa 11zamli.
J lhank him /0,. hi" immea"u,.alle palience ad "pi,.itual guidance.
J.Ii:l limifUj coulJ nol have leen lette,.. 'Wlien l was aown, 'You were tliere; wlien tlie 6urdén was too lieavy, 'You Cigfitenea my foad; ana wfien l fort CiR.! l couca no fonger go on, 'You picR.!a me up anacarriea me tfie rest oftfie way.
%anf0ou, Patfier
"... CEe strong anaof a gooa courage; 6e not afrait!, neitfier 6e tfiougfi aismayea: for tlie Lora tfiy qoais witfi tliee witliersoever tfiou goest. " }osfiua 1: 9 ABSTRACT
Male reproductive health is of growing concern, as male toxicant exposure can affect progeny outco.me; sperm chromatin structure integrity may be a contributing factor. The formation of mature sperm involves the expression of numerous proteins involved in organizing and packaging the chromatin in a specific manner; this ensures transmission and participation of the paternal genome in embryogenesis. Exposure of male rats to cyclophosphamide as spermatid chromatin is remodeled has an adverse effect on embryo development. The hypothesis of this thesis is that cyclophosphamide exposure causes genetic damage and alters the sperm proteome, thus disrupting components of chromatin condensation and organization during spermiogenesis.
The first objective was to assess the phase specificity of the susceptibility of
spermiogenic germ cells to cyclophosphamide-induced DNA damage.
Spermatozoa were analyzed for DNA strand breaks using the comet assay.
Cyclophosphamide-induced DNA damage was dose-related and accumulated
over time. Germ ce Il phase-specific damage was maximal during mid
spermiogenesis; this reflects an increased susceptibility of step 9-14 spermatids
at a key point in sperm chromatin remodeling, the histone-protamine exchange.
The second objective was to examine the sperm chromatin structure and basic
proteome. Multiple assays demonstrated that the effects of cyclophosphamide
on sperm chromatin structure were also germ cell-phase specific; mid
spermiogenic spermatids were most sensitive. Sperm were less condensed with
reduced total thiol and protamine contents. The sperm basic proteome was also
altered; identified proteins are involved in a variety of spermiogenic and fertilization events. The nuclear matrix organizes chromatin into loop domains, and various components of somatic cel! matrices are targets for chemotherapeutic agents. Therefore the last objective of this study was to assess the effect of cyclophosphamide exposure on the protein profile of the sperm nuclear matrix. The expression of several nuclear matrix protein components, a number of which were identified for the first time, was altered fol!owing drug exposure. Together these results show that cyclophosphamide alters male germ cel! chromatin remodeling at both the DNA and protein level; this could alter sperm function and thus explain the adverse effects on early embryo development.
ii ABRÉGÉ
la reproduction masculine devient un enjeu important, sachant que l'exposition d'un homme à des composés toxiques peut affecter sa descendance; l'intégrité de la structure de la chromatine du spermatozoïde pouvant être un des facteurs en jeu. la formation des spermatozoïdes matures nécessite l'expression de diverses protéines impliquées dans l'organisation et la compaction spécifique de la chromatine, assurant ainsi la participation et la transmission du génome paternel lors de l'embryogenèse. l'exposition de rats mâles à la cyclophosphamide lors du remodelage de la chromatine dans les spermatides induit des anomalies du développement embryonnaire. l'hypothèse de ce travail de thèse est que l'exposition à la cyclophosphamide induit des anomalies génétiques et protéiques dans les spermatozoïdes, altérant ainsi les acteurs de la compaction et de l'organisation de la chromatine lors de la spermiogenèse. le premier objectif a été d'établir les périodes de sensibilité lors de la maturation des cellules germinales mâles, en terme d'altération de l'ADN, à l'exposition à la cyclophosphamide. les coupures d'ADN dans les spermatozoïdes ont été analysées par la technique du COMET. la cyclophosphamide induit des altérations de l'ADN de façon dose dépendante et s'accumulant avec le temps d'exposition. les cellules germinales les plus sensibles sont les spermatides aux stades 9 à 14, ce qui correspond à une étape clé du remodelage de la
chromatine: le remplacement des histones par les protamines. le deuxième
objectif a été d'observer la structure de la chromatine ainsi que le protéome des
spermatozoïdes après traitement. Différentes techniques ont confirmé la période
de sensibilité en mi-spermiogenèse, des cellules germinales à la
iii cyclophosphamide. L'analyse de l'expression des protéines basiques contenues dans les spermatozoïdes a aussi révélé des anomalies, certaines protéines étant impliquées dans la spermiogenèse et la fertilisation. La matrice nucléaire
organise la chromatine en boucle, et l'on sait que les agents de chimiothérapie
peuvent altérer certains composants de cette matrice dans les cellules
somatiques. C'est pourquoi, le troisième objectif de cette thèse a été d'étudier
l'effet de la cyclophosphamide sur la matrice nucléaire des spermatozoïdes.
Nous avons montré que l'expression de nombreux composants étaient affectée,
certains d'entre eux étant identifiés pour la première fois. L'ensemble de ces
résultats montre que la cyclophosphamide altère le remodelage de la chromatine
dans les cellules germinales mâles tant au niveau génomique que protéique. Ces
effets peuvent modifier la fonction de ces cellules et expliquer ainsi en partie les
anomalies observer au niveau du développement embryonnaire.
iv TABLE OF CONTENTS
Abstract...... i Abrégé ...... iii List of Figures ...... viii List of Tables ...... , ...... , ...... ix List of Abbreviations ...... x Acknowledgements ...... , ...... , ...... '" ... '" ...... xii Preface ...... xiv
CHAPTER 1. Introduction ...... 1
1.1. Male Reproductive System ...... 2 1 .1.1. Male Gamete Development...... 3 1.1.1.1 Spermacytogenesis ...... 3 1.1.1.2 Spermatidogenesis ...... '" ...... 3 1.1.1.3 Spermiogenesis and Maturation ...... 4 1.1.1.4 Classification and Timing of Spermatogenesis ...... 6 1.1.2. Sperm Chromatin Structure ...... , ...... , ...... 8 1.1.2.1. Nucleoproteins: Solenoids to Toroids ...... 9 1.1.2.1.1. Histones ...... 9 1.1.2.1.2. Transition Proteins ...... '" ...... '" ..... , ..... , ...... 11 1.1.2.1.3. Protamines ...... 12 1.1.2.2. The Nuclear Matrix ...... 15 1.1.2.2.1. DNA Loop Domains and Gene Regulation ...... 17 1.1.2.2.2. Protein Composition ...... 19 1.1.2.2.3. Nuclear Matrix Stability ...... 22 1.2. Assessment of Sperm Quality and Function ...... 23 1.3. Male-Mediated Developmental Toxicity...... 27 1.3.1. Toxicant Exposure and Male Germ Cells ...... 27 1.3.2. Male Germ Cell Susceptibility ...... 29
v 1.4. Cyclophosphamide ...... 31 1.4.1. Cyclophosphamide as a Male-Mediated Developmental Toxicant. .... 33 1.5. Rationale and Formulation of the Question ...... '" ... 36
References ...... 40
CHAPTER 2. Spermiogenic Germ Cell Phase-Specifie DNA Damage Following Cyclophosphamide Exposure ...... 74
2.1. Abstract...... , ...... , ...... 75 2.2. Introduction ...... , ...... '" ...... , ...... 76 2.3. Materials and Methods ...... 79 2.4. Results ...... 82 2.5. Discussion ..... , ...... 86
References ...... 92 Figures and Legends ...... 101
CHAPTER 3. Exposure of Male Rats to Cyclophosphamide Alters the Chromatin Structure and Basic Proteome in Spermatozoa ...... 113
3.1. Abstract. .'...... 114 3.2. Introduction ...... , ...... '" ...... , ...... 115 3.3. Materials and Methods ...... , ... , .. , ...... , ...... 117 3.4. Results ...... '" ...... , ...... , .. , ...... ,. '" ..... , ...... '" ...... 126 3.5. Discussion ...... 133
References ...... 140 Figures and Legends ...... 152 Tables and Legends ...... 163
vi CHAPTER 4. Chronic Cyclophosphamide Exposure Alters the Profile of Rat Sperm Nuclear Matrix Proteins ...... 167
4.1. Abstract...... 168 4.2. Introduction ...... 169 4.3. Materials and Methods ...... 171 4.4. Results ...... 177 4.5. Discussion ...... 182
References ...... 188 Figures and Legends ...... 197 Tables and Legends ...... 206
CHAPTER 5. Discussion ...... 209
5.1. Sperm Genetic Integrity...... 211 5.2. Sperm Chromatin Structure and Proteome ...... 213 5.2.1. Sperm Head Basic Proteome ...... 215 5.2.2. Nuclear Matrix Proteome ...... 216 5.3. DNA Organization ...... 219
List of Original Contributions ...... 221 References ...... 224 Appendix ...... 231
vii LIST OF FIGURES
CHAPTER 1
Figure 1-1. The rat spermatid and spermatozoon ...... 5 Figure 1-2. The rat spermatogenic cycle ...... 7 Figure 1-3. Nucleoprotein exchange and nuclear matrix loop domain model. .. 13 Figure 1-4. Cyclophosphamide metabolism ...... 32
CHAPTER 2
Figure 2-1...... , ...... , ...... '" ...... , ...... 102 Figure 2-2...... : ...... 104 Figure 2-3...... 106 Figure 2-4...... , ...... , .... 108 Figure 2-5...... 11 0
CHAPTER 3
Figure 3-1...... 153 Figure 3-2...... 155 Figure 3-3...... , ...... 157 Figure 3-4...... , ...... 159 Figure 3-5. '" '" ...... , ...... 161
CHAPTER 4
Figure 4-1...... , ...... 198 Figure 4-2...... , .. 200 Figure 4-3...... , ...... 202 Figure 4-4...... , ..... 204
viii LIST OF TABLES
CHAPTER 1
Table 1-1. World Health Organisation threshold values for normal semen parameters ...... '" .. , ...... , ...... 24 Table 1-2. Methods to assess sperm DNA and chromatin structure integrity ...... 26 Table 1-3. Time of toxicant exposure and the germ cell type first exposed in rats ...... 30
CHAPTER 3
Table 3-1...... 16.4
CHAPTER 4
Table 4-1...... '" ...... 207
ix LIST OF ABBREVIATIONS
8-0HdG: 8-hydroxy-2-deoxyguanosin 4-0HCP: 4- hydroxycyclophosphamide BrUTP: Bromouridine-triphosphate CAMK4: Calcium/calmodulin-dependent kinase 4 CAPZB: Capping protein (actin filament) muscle Z-line, beta CASA: Computer assisted semen analysis CMA3: Chromomycin A3 CPA: Cyclophosphamide OBCP: Oibromochloropropane OBO-FISH: ONA breakage detection-fluorescent in situ hybridization OFI: ONA fragmentation index on: Oithiothreitol FSH: Follicle-stimulating hormone GAS2: Growth-arrest-specific 2 GnRH: Gonadotropin-releasing hormone GPX4: Glutathione peroxidase 4 GSTM5: Glutathione-S-transferase mu 5 GST02: Glutathione-S-transferase omega 2 hnRNP: Heterogeneous nuclear ribonucleoprotein ICSI: Intracytoplasmic sperm injection IEF: Isoelectric focusing LH: Luteinizing hormone LPP: LlM domain containing preferred translocation partner in lipoma MALOI-MS: Matrix assisted laser desorption/ionization mass spectrometry MAR: Matrix-associated regions MBBr: Monobromobimane PIP2: Phosphatidylinositol 4,5-bisphosphate PRM: Protamine
x PRKRIP1: Protein kinase R interacting protein 1 PT: Perinuclear theca PVDF: Polyvinylidene difluoride RRM: RNA recognition motif RNP: Ribonucleoprotein SCSA: Sperm chromatin structure assay SSTK: Small serine/threonine protein kinase TNP: Transition protein TUNEL: Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end labelling
xi Acknowledgements
1 would first like to thank my supervisors Dr. Bernard Robaire and Dr. Barbara Hales for giving me the opportunity to be a part of a great research team. Their guidance and support was critical to my success as a graduate student. 1am weil prepared to succeed in any path 1choose to pursue.
1would like to thank my advisor, Dr. Radan Capek for his support over the years.
To the administrative staff, Helene Duplessis, Pam Moore, Tina Tremblay and Mona Azar, thank you for being there for me on more than one occasion.
Many thanks to Chunwei Huang for always coming through and keeping everything under control; her presence definitely helped the years pass by with greater ease.
To the survivin survivors, Amynah, Katia and Sophie, a heartfelt thank you for their friendship and moral support.
xii "Ali-Khat", Kate and Adriana, 1 could not have asked for a better group of friends to start this journey. 1thank them for their support from day one.
Many thanks to Tara for sharing this MMDT experience.
To the aspiring Pharmacology graduate student, and my dear friend, Saif Husain, who was always there for me without question ... thank you.
T 0 friends, past and present, this experience would not have been as enjoyable if 1 didn't have them around.
To those that Ileave behind, 1wish you ail the best this experience hasto give.
xiii PREFACE Format of the Thesis
This manuscript-based thesis consists of five chapters, prepared according to the McGiII University Graduate and Postdoctoral Studies "Guidelines for Thesis Preparation". The introduction in chapter one presents an overview of the male reproductive system including the different test available to assess sperm quality and function, as weil as a literature review of the effects of male toxicant exposure, in particular cyclophosphamide on reproduction. Chapter two is the duplicated text of a paper published in the Journal of Andrology; 25:354-
362, 2004. Chapter three includes the text of a paper accepted for publication in
Human Reproduction; 2007, doi:10.1093/humrep/dem002. Chapter four is comprised of text submitted for publication to Biology of Reproduction, 2007.
Chapter five contains a summary and discussion of the results of this thesis and a list of original contributions. The appendix contains the ethics certificate for work on animais and the copyright permissions from the American Society of
Andrology and Oxford University Press. The copyright agreements of Biology of
Reproduction by the Society for the Study of Reproduction permit the insertion of the manuscript in this thesis.
Contribution of Authors
The candidate performed the majority of experiments presented in this thesis with the exception of the two-dimensional electrophoresis gels which were done at the McGiII University and Genome Quebec Innovation Centre.
xiv CHAPTER 1
Introduction
1 1.1. Male Reproductive System
The male reproductive system is composed of the testes, the excurrent duct system, the accessory sex glands (prostate, seminal vesicles, bulbourethral gland and Cowper's gland), and the external genitalia. The testes have two functions: male gamete and androgen production. The testes contain convoluted seminiferous tubules surrounded by interstitial tissue; the interstitium contains
Leydig cells which synthesize and secrete testosterone. The seminiferous tubules, responsible for gametogenesis, are lined by a stratified epithelium consisting of spermatogenic cells and the supporting Sertoli cells, and conta in a fluid-filled lumen into which fully formed spermatozoa are released (Russell et al.,
1990). Sertoli cells extend from the basement membrane to the luminal surface
of the seminiferous epithelium; they establish the blood-testis barrier and support
the developing germ cells throughout spermatogenesis (Byers et al., 1993).
Successful and complete male gamete development is dependent on the
endocrine system of the hypothalamic-pituitary-testicular axis. The hypothalamus
releases gonadotropin-releasing hormone (GnRH) which acts on the anterior
pituitary gland to stimulate the release of gonadotropins, luteinizing hormone (LH)
and follicle-stimulating hormone (FSH). LH acts on the Leydig cells to trigger the
synthesis and secretion of testosterone. FSH acts on the Sertoli cells to produce
factors that are essential for normal testicular function (Weinbauer and Nieschlag,
1995).
2 1.1.1. Male Gamete Development
Spermatogenesis is a highly synchronized and regulated process by which immature male germ cells develop into mature spermatozoa. It can be divided into three phases: spermacytogenesis (proliferation), spermatidogenesis
(meiosis) and spermiogenesis (differentiation). Each phase is also associated with specifie germ cell types: spermatogonia, spermatocytes and spermatids, respectively.
1.1.1.1 Spermacytogenesis
Spermatogonia are the most immature cells located along the base of the seminiferous epithelium. They proliferate by mitotic division (3-6 depending on the species, 5 in the rat) and multiply repeatedly to continually replenish the seminiferous epithelium. In the rat, there are three types of spermatogonia: stem
cells (Ais), undifferentiating proliferative cells (Apaired and Aaligned) and
differentiating cells (A1, A2, A3, ~, Intermediate and Type B). It is the Aaligned cells
that leave the pool of undifferentiated Type A spermatogonia and commit to
differentiation by undergoing several mitotic divisions. The final mitotic division of
Type B spermatogonia results in the formation of primary spermatocytes (de
Rooij and Russell, 2000).
1.1.1.2 Spermatidogenesis
Spermatocytes undergo two meiotic divisions to form the haploid
spermatid. Primary spermatocytes immediately enter prophase of the first
meiotic division to produce two diploid secondary spermatocytes. The process of
3 meiosis is extended over a long period of time; prophase of the first meiotic division lasts for nearly three weeks in the rat (Hess, 1998). To increase the genetîc variability of the gamete, recombination or chromosomal crossover occurs between paired chromosomes. Secondary spermatocytes rapidly enter meiosis /1 and divide to produce four haploid round spermatids (Russell et al.,
1990).
1.1.1.3 Spermiogenesis and Maturation
Spermiogenesis is the final stage of spermatogenesis in which haploid round spermatids undergo dramatic changes to give rise to spermatozoa; this process occurs without cell division. In the rat, over a span of about three weeks, the nucleus goes through profound alterations. Depending on the species, the sperm nuclear shape varies; spermatozoa in most mammals, including humans, have a spatulate head; however, in rodents, they have a falciform or sickle shaped head (Lalli and Clermont, 1981; Yanagimachi, 1981). Nuclear shaping may be determined or maintained by the formation of the acrosome, manchette or perinuclear theca (subacrosomal perforatorium and postacrosomal sheath) as spermiogenesis progresses (Bellve and O'Brien, 1983; Longo et al., 1987; Longo and Cook, 1991; Russell et al., 1991). Acrosome formation is a slow process that is not complete until late spermiogenesis (Lalli and Clermont, 1981; Leblond and
Clermont, 1952b). The acrosome is of Iysosomal origin and contains a number of enzymes responsible for sperm penetration through the layers surrounding the egg and sperm-egg fusion (Clermont and Tang, 1985; Yoshinaga and Toshimori,
2003). The nuclear shape is also a result of chromatin remodeling; round
4 elongate and the chromatin condenses as the DNA is repackaged (Ward and
Coffey, 1991). Spermatids also develop a flagellum made up of microtubules.
The axoneme, derived from one of the two centrioles, is the core scaffold of the flagellum (Fawcett, 1975) (Figure 1-1).
Nucleus
Postacrosomal ) sheath
Postacrosomal sheath
Earlv Spermatid Spermatozoon
Figure 1-1. The rat spermatid and spermatozoon
Adapted from de Kretser and Kerr, 1994 and Calvin, 1976
Spermatozoa are released from the Sertoli cells into the lumen of the seminiferous tubules ina process called spermiation. However, prior to spermiation the spermatid sheds most of its excess cytoplasm and organelles in the residual body, which is phagocytosed by the Sertoli cells (Kerr and de
Kretser, 1974). Following spermiation the spermatozoa are not motile. They are
5 transported in testicular fluid by peristaltic contractions through the efferent ducts, after which they enter the caput (head) epididymidis, progress to the corpus
(body) and finally reach the cauda (tail) region where they are stored. While they travel down the epididymis they undergo maturation; spermatozoal chromatin is further condensed and stabilized, and the cells acquire motility and fertilizing potential. It takes spermatozoa approximately 8 days to pass through the rat epididymis compared to approximately 6 days in man and 15 days in the mouse
(Hess, 1998; Robaire and Hermo, 1988) .
1.1.1.4 Classification and Timing of Spermatogenesis
Spermatogenesis is highly synchronized as evidenced in seriai cross sections of seminiferous tubules where germ cells develop in distinct associations at a particular point in time. Each association is classified as a stage and each stage varies in duration. Spermiogenesis can be divided into developmental steps based on the shape of the acrosome, and to a lesser extent on spermatid head shape and degree of chromatin condensation. There are 19 steps of spermiogenesis in the rat; steps 1-8 are round, steps 9-14 are elongating, and steps 15-19 are elongated spermatids. Consequently, each stage can be classified based on the steps of spermiogenesis (Leblond and Clermont, 1952a), such that steps 1 to 14 occur in stages 1 to XIV of the cycle, respectively. Steps
15-19 overlap the early round spermatids (Figure 1-2).
6 (nen a> c a> o0> ·Ë L- a.a> en -o
II-III IV V VI VII VIII IX X XI XII XIII XIV
( Stages of the spermatogenic cycle )
Figure 1-2. The rat spermatogenic cycle
Adapted from Russell et al., 1990
A spermatogenic cycle is defined as the time it takes for the reappearance
of the same stage within a given segment of the tubule. Each stage of the cycle
follows in an orderly sequence along the tubule. The number of stages within a
spermatogenic cycle varies between and within species; there are 14 different
stages in the Sprague-Dawley rat lasting 12.9 days, 12 in the mouse lasting 8.6
days and 6 in man lasting 16 days (Clermont, 1972; Clermont and Leblond, 1955;
Leblond and Clermont, 1952b). In the rat, the duration of spermatogenesis
(about four cycles) starting from the stage where proliferating spermatogonia are
7 called differentiating spermatogonia (Aaligned to A1) is 48-52 days, depending on the strain. In mice it takes 34.5 days and in man, spermatogenesis is completed in 64 days (Clermont et al., 1959).
1.1.2. Sperm Chromatin Structure
Ward and Coffey (1991) proposed four levels of organization for spermatozoal packaging: (1) chromosomal anchoring, which refers to the atlachment of the DNA to the nuclear annulus; (2) replacement of histones by protamines to condense the DNA; (3) DNA loop domain formation on the nuclear matrix; and (4) chromosomal positioning. The relationship between nuclear function and organization has been weil established in somatic cells (Cremer et al., 2000); however, the functional significance of sperm structural organization remains elusive. Two levels which determine higher chromatin organization within somatic nuclei and which play an essential role in gene regulation are nucleosomal and DNA loop-domain organization (Demeret et al., 2001). DNA organized into nucleosomes has been proposed to be further coiled into bulky solenoids with six nucleosomes per turn (Ward, 1993), which then make up
DNA/soienoid loop domains. In the case of mature spermatozoa, however, the
DNA is not organized into nucleosomes. Instead of histones, the chromatin is packaged with protamines in such a way that the DNA becomes at least six times more condensed than that of somatic cell nuclei and occupies almost the entire nuclear volume (Ward and Coffey, 1991). It is believed that this compact structure serves to protect and allow delivery of the paternal genome to the egg; in fa ct , haploinsufficiency caused by a mutation in one allele of the protamine
8 gene prevents the production of structurally and functionally intact sperm (Cho et al., 2001). There is increasing interest in sperm chromatin organization as more evidence is reported regarding the greater role it may play in embryo development. In the following sections, each of these two levels will be discussed in greater detail.
1.1.2.1. Nucleoproteins: Solenoids to Toroids
1.1.2.1.1. Histones
Round spermatids contain a complement of somatic-type histones (H 1,
H2A, H2B, H3, H4) and testis-specific histones (H1t, H1t2, TH2A, TH2B, TH3)
(Martianov et al., 2005; Meistrich, 1989). The testis-specific variants replace a significant fraction of the somatic-types during mitosis and meiosis (Meistrich et \ al., 1985). The histones undergo post-translational modifications in order to alter their association with DNA (Poccia, 1986); ubiquitination, acetylation and phosphorylation are thought to facilitate chromatin transformation from a nucleosomal structure to a nucleoprotamine structure during spermatid differentiation. Homozygous mutation of the mouse HR6 ubiquitin-conjugating enzyme causes abnormal chromatin condensation (Roest et al., 1996). Histone
H4 is highly acetylated in rat elongating spermatids when histone displacement commences, in order to relax the chromatin for interaction of the DNA with more basic proteins (Grimes, Jr. and Henderson, 1984; Meistrich et al., 1992).
Additionally, small serine/threonine protein kinase (SSTK), expressed exclusively in elongating spermatids, specifically phosphorylates H1, H2A, and H3 as weil as the H2A variant, H2AX (Spiridonov et al., 2005). In many species, including
9 humans, 85-90% of histones are removed (Gatewood et al., 1990; Tanphaichitr et al., 1978), while in others however, including rats, nearly ail of the histone complement is replaced (Meistrich et al., 1994).
During the histone-protamine exchange, endogenous nicks in DNA are created in step 12-13 rat elongating spermatids which are then ligated before the completion of protamination (McPherson and Longo, 1993a; McPherson and
Longo, 1992; McPherson and Longo, 1993b; Sakkas et al., 1995). These nicks may be present to facilitate protamination by providing relief of tortional stress
(McPherson and Longo, 1992). Interestingly, high levels of poly (ADP-ribose) formation, mediated by DNA damage-dependent poly (ADP-ribose) polymerases
(PARP), occur in step 12-13 spermatids (Meyer-Ficca et al., 2005). In PARP-Z' mice, spermiogenesis is affected as nuclear elongation is delayed (Dantzer et al.,
2006). Additionally, phosphorylated H2AX is present in rat spermatids at the same time that the nicks occur (Meyer-Ficca et al., 2005). Phosphorylated H2AX is an indicator of DNA double strand breaks; in fact H2AX phosphorylation even occurs in human spermatozoa following hydrogen peroxide induction of DNA double strand breaks (Li et al., 2006). Several DNA repair factors have been shown to co-Iocalize with phosphorylated H2AX and seem to be implicated in the repair of DNA damage (Mladenov et al., 2006; Pauli et al., 2000). The presence of both poly (ADP-ribose) and phosphorylated H2AX in rat spermatids may play a role in DNA strand break repair during the exchange between nucleoproteins.
10 1.1.2.1.2. Transition Proteins
The exchange between histones and protamines is not direct; there are other players involved in the transition. After meiosis, the beginning of spermiogenesis is characterized by a wave of transcriptional activity which results in the activation of numerous essential post-meiotic genes in early spermatids
(Schmidt and Schibler, 1995). In particular, transition protein and protamine genes are transcribed and stored in the cytoplasm of round spermatids until they are translated days later (Dadoune, 2003; Morales et al., 1991; Steger, 1999).
Transition proteins are more basic than histones and less basic than protamines.
Four transition proteins (TNP 1-4) have been identified in the rat (Kistler et al.,
1975); TNP1 and TNP2 are the best characterized forms. TNP2 is the first nucleoprotein that replaces the histones. Its appearance is associated with the onset of nuclear elongation. In the rat, immunocytochemical analysis shows that
TNP2 first appears in step 9-11 spermatids followed by high levels in steps 12-
14. It disappears by step 16 (Oko et al., 1996). As the nucleus begins to condense, TNP1 is lowly expressed in step 12 and th en peaks in steps 13-15. It disappears by step 17 (Heidaran et al., 1988; Kistler et al., 1996; Oko et al.,
1996).
Functionally, TNP1 and TNP2 knockouts suggest that these two proteins
1 1 are related to each other; in both TNP1- - and TNP2- - mice, germ cells are able to initiate chromatin condensation (Adham et al., 2001; Yu et al., 2000; Zhao et al.,
2001). Although the absence of either protein is compensated by increased levels of the remaining transition protein, epididymal sperm chromatin condensation is not complete and is accompanied by abnormal condensation
11 · patterns (Yu et al., 2000; Zhao et al., 2001). Both TNP1 and TNP2 may be
necessary for maintaining the normal processing of protamine 2 (PRM2)
precursors (Yu et al., 2000; Zhao et al., 2001); high levels of PRM2 precursors
have been detected in sperm of infertile men (De Yebra et al., 1998). Targeted
deletion of the Tnp2 gene results in abnormal sperm head morphology (Adham et
al., 2001) and altered sperm chromatin structure (Zhao et al., 2001), possibly due
to increased DNA strand breaks. Interestingly, it has been hypothesized that,
apart from their role in DNA compaction, TNP1 and TNP2 mayalso be involved
in the repair of the DNA breaks that occur in elongating spermatids (Caron et al.,
2001; Kierszenbaum, 2001).
1.1.2.1.3. Protamines
Two different types of protamines have been identified in mammals,
protamine 1 (PRM1) and protamine 2 (PRM2). Protamines are very small (-6500
Da) and highly basic arginine- and cysteine-rich proteins. In many mammalian
species, including the rat, only PRM1 is present in spermatozoa, although the
protamine 2 gene sequence exists (Tanhauser and Hecht, 1989); it is the last
nucleoprotein to appear in mature spermatid nuclei (Balhorn, 1989). PRM1 first
appears in step 11 elongating spermatids "and persists throughout the rest of
spermiogenesis; protamine deposition is complete by step 19. The accumulation
of PRM1 displaces the transition proteins as they preferentiallybind to the minor
groove of the DNA helix and neutralize the phosphodiester backbone of DNA to
induce progressive chromatin condensation (Marushige and Marushige, 1975).
The protamine-DNA complex coils into concentric circles, which collapse onto
12 · each other. The proposed end result, in place of bulky histone solenoids is a highly condensed doughnut-shaped toroid (Hud et al., 1993) (Figure 1-3).
SOMATIC CELLS SPERM
DNA Double Helix JJ JJ rrotamine ~~r...... = Nucleosome ~r
Histone Transition Protamines Displacement Proteins
Doughnut ~ Solenoid Loop / T Loop IfMft
Nuclear Matrix
_~"><.J!..._J'"_A_~_tiV~ DNA Loop ------...... :::::~~ ...... Inactive Gene
Figure 1-3. Nucleoprotein exchange and nuclear matrixloop domain model
Adapted from Ward, 1993
There are two processes involved in sperm chromatin condensation: phosphorylation-dephosphorylation of the serine and threonine residues of protamines and the formation of disulfide bonds between trie protamine cysteinyl
13 residues (Balhom et al., 1991; Green et al., 1994; Mayer, Jr. et al., 1981; Pfeifer et al., 2001; Ward and Coffey, 1991). Soon after their synthesis, protamines are highly phosphorylated; this is required for the correct positioning and binding of protamines to DNA (Green et al., 1994). Serine/arginine protein-specific kinase 1 phosphorylates PRM1 (Papoutsopoulou et al., 1999). Calcium/calmodulin dependent kinase 4 (CAMK4) phosphorylates PRM2 and disruption of the Camk4 gene results in male sterility, spermiogenesis impairment and abnormal sperm head morphology (Wu et al., 2000). Prior to spermiation or release of the sperm into the seminiferous tubule lumen and transfer to the epididymis, dephosphorylation of the protamines occurs, which precipitates chromatin condensation and stabilization (Oliva and Dixon, 1991); inter- and intra-molecular disulfide bonds form between adjacent thiol groups. The number of disulfide bonds created progressively increases as spermatozoa travel from the head
(caput) to the tail (cauda) region of the epididymis during the maturation phase of germ cell development. These disulfide bonds have been shown to be created by the 34 kDa selenoprotein, glutathione peroxidase 4, also known as phospholipid hydroperoxide glutathione peroxidase. This enzyme, bound to sperm chromatin, uses the thiol groups of the protamines instead of glutathione and acts as a protamine thiol peroxidase rather than an antioxidant that reduces lipid peroxides (Conrad et al., 2005; Pfeifer et al., 2001).
A higher order of organization exists in mammalian sperm nuclei in addition to protamine DNA condensation. Each doughnut-shaped toroid created represents oneDNA loop domain formed in association with the nuclear matrix; this is discussed in the following section.
14 1.1.2.2. The Nuclear Matrix
Fort Y years ago, Don Fawcett first introduced the terrn "nuclear matrix" to describe the non-chromatin -structure of the nucleus (Fawcetl, 1966). The isolated, mainly proteinaceous, residual structure retains the ove ra Il shape and size of the nucleus (Berezney and Coffey, 1974). The original nuclear matrix isolation protocol, using nuclease and high salt concentrations, was developed by
Berezneyand Coffey (1974) to extract the chromatin, histones and other soluble or loosely bound nuclear proteins from rat liver nuclei; however, many variations of the original protocol have been described depending on the cell type being used. Most isolation protocols include sequential treatments of isolated nuclei with ionic and non-ionic detergents, nucleases, and low and high ionic strength buffers (Martelli et al., 1996). The initial use of high ionic strength 2M NaCI to extract nuclear proteins greatly alters the appearance of the matrix, which may reflect selective removal of matrix components. The use of lower salt concentràtions, such as O.25M (NH4hS04, betler preserves the morphology without significantly affecting the efficacy of protein extraction (Belgrader et al.,
1991; Fey et al., 1986).
The nuclear matrix consists of two domainS: an internai ribonucleic protein network and a peripheral lamina (Berezney and Coffey, 1974; Maraldi et al.,
1986). The internai nuclear matrix is composed of thick, polymorphie fibers, the composition of which is ce II-type specifie; it is constructed on an underlying three dimensional network of branched core filaments that connect to the nuclear lamina. Dense bodies resembling structural remnants of nucleoli are evident in this network. Ribonucleoproteins (RNPs) are atlached directly or indirectly to the
15 core filaments. RNA has been highlighted for its role in maintaining structural integrity; the inclusion of RNase inhibitors, vanadyl ribonucleoside complexes, results in the isolation of intact structures, whereas the use of Rnase A destroys the inner network, leaving behind nuclear shells (He et al., 1990; Nickerson et al.,
1989). The lamina is a continuous thin fibrous layer under the nuclear membrane, mainly composed of three proteins, lamin A, Band C, as weil as nucleokeratins (Franke, 1987; Gerace et al., 1984). Lamins are members of the intermediate filament protein family playing important roles in maintaining nuclear shape and controlling chromatin organization (Mattout et al., 2006).
Similar to somatic cells, components exist in sperm that are resistant to solubilization. In the mouse sperm nucleus, a nuclear matrix has been described consisting of parts of a surrounding rigid perinuclear theca (PT) and a connected filamentous internai network. After the removal of protamines and DNA, the structure still retains the shape of the nutleus (Bellvé et al., 1992). In rodents, the PT surrounding the nucleus is composed of the subacrosomal perforatorium and postacrosomal sheath (Lalli and Clermont, 1981). During spermatogenesis a nuclear lamina as such does not appear to be present. The sperm nucleus either does not contain (Longo et al., 1987) or barely has detectable levels (Goldman et al., 1998; Schatten et al., 1985) of lamins A, Band C; in mouse sperm only lamin
B is expressed beneath the PT in a punctuate pattern surrounding the nucleus
(Moss et al., 1993). The PT resembles, in structure and perhaps function, the lamina of somatic cells even though it is located externally to the nuclear membrane. The protein constituents of the PT have not been completely defined. The perforatorium is composed of several proteins, the main one being
16 PERF 15 orfatty acid binding protein 9 in both mice and rats (Korley et al., 1997;
Oko and Morales, 1994; Pouresmaeili etaI., 1997). The postacrosomal sheath contains actin capping proteins responsible for actin biogenesis and actin binding proteins (Howes et al., 2001; Hurst et al., 1998; Lecuyer et al., 2000; von Bulow et al., 1997); the main component is the keratin-like protein, calicin (Lecuyer et al., 2000; von Bulow et al., 1995). Bovine spermatozoa also contain somatic histones localized to the postacrosomal sheath (Aul and Oko, 2002; Tovich et al.,
2004; Tovich and Oko, 2003).
Analysis of the mouse and human sperm internai nuclear matrix reveals scattered dense globular patches assembled on a network of thick and thin filaments. Immunoelectron microscope localization of RNPs during mouse spermatogenesis detects labeling only in primary spermatocytes up to elongating spermatids, concomitant with the cessation of RNA synthesis and the disappearance of nucleoli (Biggiogera et al., 1993; Biggiogera et al., 1994). In mature sperm nuclear vacuoles containing fibrils remain in place of nucleoli; interestingly, RNPs have been detected associated with vacuole fibrils (Sousa and Carvalheiro, 1994). Whether they play as great a role as in somatic cells is not clear.
1.1.2.2.1. DNA Loop Domains and Gene Regulation
The nuclear matrix in somatic cells has come under intense study over the last ten to fifteen years, as it is no longer considered to act just as structural support. Many studies revolve around the suggestion that the nuclear matrix may play a key role in genome organization and gene expression and have shown
17 that it is involved in many essential nuclear functions, including DNA replication and repair, transcription, and RNA processing and transport (Anachkova et al.,
2005; Mladenov et al., 2006; Mortillaro et al., 1996; van Driel et al., 1991). There are also indications as to a role of the matrix in nuclear signal transduction
(Alessenko and Burlakova, 2002; Maraldi et al., 1992; Struchkov et al., 2002).
The function of the sperm nuclear matrix is comparatively not as weil understood. As in the nucleus of somatic cells, chromatin within the male gamete is organized into loop domains (Klaus et al., 2001; Kramer and Krawetz, 1996;
Nadel et al., 1995; Ward and Coffey, 1991), bound at the base in a non-random, sequence-specifie manner to the matrix (Kramer and Krawetz, 1996; Ward and
Coffey, 1990) (Figure 1-3). These loops differ, however, with respect to their average size; loops within the sperm nucleus are .... 46 kb in size in hamsters
(Ward et al., 1989) and .... 27 kb in humans (Barone et al., 1994), compared with
.... 70 kb in other cell types (Vogelstein et al., 1980). Even during the early stages of spermatogenesis, the loop size decreases; it does not, however, change during spermiogenesis (Klaus et al., 2001). Adeninelthymine-rich matrix associated regions (MARs) anchor DNA loops to the matrix (Heng et al., 2004).
These regions are either constitutive (for structural purposes) or transient
(expression-related), with the latter depending on the functional state of the cell
(Farache et al., 1990; larovaia et al., 2005; Razin, 1987). MARs delimit a domain considered to be an autonomously regulated unit within the genome. Somatic
MARs are also common in or near origins of replication, introns, enhancers and transcription sites (Dijkwel and Hamlin, 1988; Gasser and Laemmli, 1986;
Jarman and Higgs, 1988; Kalandadze et al., 1990). These regulatory sequences
18 relate to the domain-contained gene and regulatory elements act only within the boundaries of the domain. MARs permit regulatory elements to associate with each other on the matrix, thereby providing a platform for the assembly of protein machinery targeted to these specifie nuclear sites, such as replication and transcription factories, or those involved in chromatin modification and RNA spicing (Boulikas, 1994; Boulikas, 1995; Dworetzky et al., 1992).
ln mature human sperm, analysis of a -40 kb region containing the genes for the sperm-specific Prm1, Prm2, and Tnp2, has shown that this region of the genome is demarcated by two MARs which attach it to the nuclear matrix along with other internai MARs which transiently associate with the matrix (Nelson and
Krawetz, 1994). This subset of haploid-specific, as weil as other constitutively (a globin and p-actin) expressed genes, assumes an altered structural conformation as evidenced by their increased sensitivity to DNase 1 (Kramer and Krawetz,
1996). Boundaries defined by MARs have been identified at the ends of DNase
I-sensitive domains in numerous loci (Levy-Wilson and Fortier, 1989) and,
correspondingly, transcriptionally active genes are found in these domains
associated with the nuclear matrix (Ciejek et al., 1983). Interestingly, this
haploid-specific region also contains ail the elements necessary for the correct
spatial and temporal expression of this gene cluster (Choudhary et al., 1995).
1.1.2.2.2. Protein Composition
The nuclear matrix is a dynamic structure whose morphology and protein
composition vary with the functional state of the nucleus (Penman, 1995). The
use of two-dimensional gel electrophoresis to separate proteins based on their
19 isoelectric points (first dimension) and molecular weight (second dimension) has demonstrated that the protein profile of the matrix is exceptionally complicated, revealing several hundred polypeptides; specifie cell and tissue types can be identified by their nuclear matrix proteomic patterns (Fey and Penman, 1988;
Getzenberg, 1994). The expression of several proteins change in differentiating cells (Zhao et al., 2006) and with neoplastic transformation (Getzenberg et al.,
1991). Changes also occur in the organization and general protein composition of the nuclear matrix in spermatocytes, round spermatids and elongating spermatids (Chen et al., 2001); protein expression appears to be either germ cell type-specifie or common to multiple germ cell types. Progressive and systematic changes in sperm matrix protein composition reflect the morphological and functional transitions occurring during spermatogenesis.
Nakayasu and 8erezney (1991) have identified several abundant proteins that are ubiquitous in matrix preparations obtained from different somatic cells and tissues, the matrins. Other components include cell cycle regulators, tissue specifie transcription factors, RNA splicing factors, heterogeneous nuclear RNPs
(hnRNPs), apoptotic proteins, tumor suppressor genes, phospholipases, protein kinases and many others. Residual components of the ribosome biogenesis center, the nucleolus, also make up part of the matrix; fibrillarin, C23/nucleolin and 823/nucleophosmin have been identified. 823/nucleophosmin, involved in
RNA processing and transport and acting as a shuttle protein between the nucleus and cytoplasm, is one of the main constituents, along with RNPs (Martelli et al., 1996). Very little, however, is known about the components of the sperm nuclear matrix, especially since, unlike somatic cells, replication and transcription
20 do not occur in the mature sperm. To date, structural proteins, actin, myosin, and keratin, as weil as CAMK4 and the enzyme topoisomerase Il, have been identified in nuclear matrix fractions (Chen and Longo, 1996; Ocampo et al.,
2005; Wu and Means, 2000). CAMK4 has been implicated in transcriptional regulation and the histone-protamine exchange during spermiogenesis (Sun et al., 1995; Wu et al., 2000). Topoisomerase Il is an enzyme that catalyzes different DNA isomerization reactions, including DNA relaxation by double-strand breakage and rejoining mechanisms (Wang, 1985). It also plays a role in DNA replication and repair, transcription, as weil as the histone protamine exchange, and chromatin loop formation and atlachment to the nuclear matrix (Fernandes et al., 1995; Laberge and Boissonneault, 2005; Pu and Bezwoda, 2000; Wang,
2002). The enzyme exhibits germ cell phase-specifie variations in expression up to the elongating spermatids, and maintains its activity in the matrix of spermatogenic cells (Chen and Longo, 1996). As a major component of both somatic and male germ cell matrices, topoisomerase Il may serve both catalytic and structural functions.
Interestingly, phospholipids and their metabolizing enzymes are also components of nuclear matrices in numerous somatic cell types localized within the nucleolar remnant and inner filamentous matrix (Lubocka et al., 1995; Maraldi et al., 1992). Their presence has been reported to play both structural and functional roles; they represent specifie sites of DNA loop and RNP attachment to the nuclear matrix, and also regulate replication and transcription (Alessenko and
Burlakova, 2002; Maraldi et al., 1992; Struchkov et al., 2002). Additionally, evidence accumulated over the past twenty years has highlighted the presence of
21 lipid-dependent nuclear signaling pathways (Alessenko and Burlakova, 2002;
Maraldi et al., 1992; Martelli et al., 2002; Martelli et al., 2003). Both cell proliferation and differentiation are associated with defined metabolic changes of nuclear phospholipid components, which appear to be independent of phospholipids present in the rest of the Gall. In particular, phosphatidylinositol
4,5-bisphosphate (PIP2), a key substrate for a number of important signaling proteins, has been identified in the nuclear matrix (Mazzotti et al., 1995). In addition to its role as a signal-generating lipid, PIP2 also binds to and regulates the function of actin-binding proteins (Fukami et al., 1992; Janmey and Stossel,
1987; Lassing and Lindberg, 1985), and has the capability to reverse RNA transcription inhibition (Yu et al., 1998). During spermatogenesis, PIP2 is present at the nuclear level in ail cell types and specifically accumulates in the nuclei of late spermatids and spermatozoa (Caramelli et al., 1996); however, there is no evidence to show that this lipid is actually present in the nuclear matrix of the sperm.
1.1.2.2.3. Nuclear Matrix Stability
Given the complexity of sperm DNA structural organization, questions regarding its role in embryo development have arisen and research has focused on the stability of the sperm nuclear matrix with the thought that one factor associated with decreased fertility may be unstable matrices. Barone et al.
(2000) developed the halo assay to assess nuclear matrix stability and have found unstable matrices in infertile cryptorchid patients. Even if sperm DNA is structurally intact, an unstable nuclear matrix will affect embryogenesis (Ward et
22 al., 1999); therefore, apart from the DNA itself, protein components of the matrix may be important. As in somatic cells, the attachment sites of DNA to the matrix may act as initiation sites for transcription and replication. DNA loop domain organization by the matrix may template the male genome for use following fertilization; alterations in structural organization may affect paternal genome expression and contribute to aberrations in early post-fertilization events, including protamine replacement by histones and pronuclear formation (Kramer and Krawetz, 1996). Controversially, Mohar et el (2002) suggest that ail the information needed for the construction of the paternal chromosomes in the zygote is contained within the sperm nuclear halos devoid of protamines and left with just chromatin attached to the nuclear matrix. Even in spermatozoa that appear normal, an unstable matrix would cause chromatin to be organized in such a way that normal functioning of the paternal DNA would be prevented and embryo development would not occur normally.
1.2. Assessment of Sperm Quality and Function
A variety of tests have been and continue to be developed in clinical andrology to examine spermatozoa and to assess individual fertility potential, and in reproductive toxicology to monitor effects from chemical exposures.
Conventional semen analysis measures parameters such as sperm cou nt, motility, and morphology. The World Health Organization (WHO, 1999) has established threshold values of specific sperm parameters associated with the fertilizing potential of normal human semen (Table 1-1). Many of these attributes
23 are measured with greater ease since the development of computer assisted semen analysis (CASA).
Table 1-1. World Health Organization threshold values for normal semen parameters
Volume ~2ml
Sperm concentration ~ 20 million/ml
Total sperm count ~ 40 million per ejaculate
Morphology ~ 30% normal
Motility ~ 50% motile
Sperm viability ~ 75% viable PH 7.2-7.8
Going beyond these basic sperm parameters, a variety of assays have been developed to monitor sperm function and to predict their fertilizing capacity, including sperm cervical mucus penetration, capacitation, zona recognition, acrosome reaction, sperm-egg fusion, and in vitro egg penetration (Aitken, 2006).
Using flow cytometry, multiple sperm features are now rapidly measured simultaneously on an individual cell basis; these include sperm viability and mitochondrial function (Gravance et al., 2001). Numerous fluorescent staining techniques have been developed for the evaluation of specifie sperm characteristics and functions, including plasma membrane integrity (Gillan et al.,
2005). Hypo-osmotic swelling is also performed to assess sperm membrane structural integrity (Oosterhuis et al., 1996).
24 With the abundance of available tests, what was lacking was the assessment of nuclear DNA integrity. Some men with normal sperm measurements, such as sperm count and motility, may have defects within the nucleus which would not be detected with the tests originally used for sperm assessment. A research area that has been studied intensely during the past decade is DNA integrity in mature spermatozoa and more recently, chromosome organization defects. Fertilization with DNA-damaged spermatozoa could lead to paternal transmission of defective genetic material with adverse consequences on embryonic development (Ahmadi and Ng, 1999; Alvarez et al., 2004; Harrouk
et al., 2000a; Lewis and Aitken, 2005). The advantage of the development of assays to address this issue, compared to the aforementioQed tests, is that they
not only yield information on the fertilizing capacity of spermatozoa, but also on their ability to support normal embryonic development. Sorne of the currently
available tests evaluate the integrity of sperm DNA and chromatin structure
(Table 1-2). The cornet assay measures DNA fragmentation (single and double
strand breaks) in individual sperm nuclei based upon gel electrophoretic patterns
(Tice et al., 2000). The sperm chromatin structure assay (SCSA) measures
abnormal chromatin structure based on the susceptibility of sperm nuclear DNA
to acid-induced denaturation in situ; a review of data on hundreds of semen
samples shows that patients with a DNA fragmentation index greater than 30%
are likely to have significantly-reduced fertility potential as weil as a greater risk of
embryo loss (Virro et al., 2004). Other techniques which also identify sperm
chromatin packaging defects include: 1) aniline blue staining that reveals
differences in the basic nuclear protein composition. This dye positively stains
25 sperm when bound to lysine; immature spermatozoa that are rich in histones are also rich in lysine; 2) toluidine blue staining, which binds to chromatin and therefore becomes heavily incorporated in poorly packaged sperm; 3) monobromobimane staining, which binds to reactive protamine thiol groups; poorly condensed and stabilized chromatin is positively stained; and 4) chromomycin A3 staining, which indirectly reveals protamine- deficient chromatin
(Agarwal and Allamaneni, 2005; Agarwal and Said, 2004).
Table 1-2. Methods to assess sperm DNA and chromatin structure integrity
Parameter measured Technique
DNA denaturation SCSA Acridine orange DNA fragmentation Cornet (neutral - DSB) (alkaline - SSB/DSB) ln situ nick translation (SSB) TUNEL DBD-FISH (SSB) Sperm chromatin dispersion Oxidative DNA damage 8-0HdG DNA adduct assay Chromatin packaging Chromomycin A3 (DNA protein composition) Aniline blue (DNA protein composition) MBBr (Thiol-disulfide status) Toluidine blue
8-0HdG, 8-hydroxy-2-deoxyguanosine; OBO-FISH, ONA breakage detection fluorescent in situ hybridization; TUNEL, terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling; OSB, double strand break; SSB, single strand break.
26 Chromatin abnormalities in sperm could present as condensation/packaging defects, ONA breaks, or chromosomal aneuploidies and as discussed above, various tests have been developed to target these chromatin abnormalities; the cause of such anomalies could be exposure to a variety of environmental, occupational, recreational or therapeutic compounds that have the potential to affect male germ cells.
1.3. Male-Mediated Developmental Toxicity
1.3.1. Toxicant Exposure and Male Germ Cells
Investigation into the mechanisms of action of the pesticide, dibromochloropropane (OSCP) as a testicular toxicant prompted great interest in the effects of environmental and occupational chemicals on male reproductive health. OSCP is the classical compound that indisputably impairs fertility in males; exposure causes reduced sperm counts characterized as oligozoospermia, or partial reduction in sperm numbers, and azoospermia, which is the complete absence of sperm (Whorton et al., 1977; Whorton et al., 1979).
Epidemiological studies suggest that paternal occupations, such as welding, auto mechanics, painting, farming, and firefighting that expose men to certain chemicals may be associated with adverse reproductive outcomes, including spontaneous abortions, birth defects, low birth weight and childhood cancer (Chia and Shi, 2002; Savitz, 1994). Exposure to pesticides other than OSCP correlates with altered morphology, decreased sperm counts, sperm aneuploidy and altered sperm chromatin structure (Harkonen, 2005; Kumar, 2004; Sanchez-Pena et al.,
27 2004). Other industrial chemicals associated with reproductive dysfunction in human males include lead, mercury, ethylene glycol ethers, carbon disulfide and ethylene dibromide (Lan cran jan et al., 1975; Schrag and Dixon, 1985; Winker and Rudiger, 2006). For thousands of other chemicals, the association between exposure and male reproductive dysfunction is only suspected and requires further evaluation before conclusions can be drawn.
There are a number of chemicals to which men may be exposed based on their lifestyle, such as alcohol, caffeine, cigarettes, and iIIicit drugs, yet inadequate epidemiological or toxicological evidence exists to clearly and definitively indicate effects on reproductive function or progeny outcome.
Cigarette smoke may be the only exception, as some evidence suggests that there is an association with increased risk for sperm aneuploidy (Robbins, 2003;
Rubes et al., 1998), along with decreased sperm counts, abnormal morphology, and oxidative DNA damage (Robbins, 2003). Exposure to testicular toxicants most often involves complex mixtures of hazardous chemicals; some contain many active ingredients, making it challenging to identify which agent is responsible for toxic effects and to draw any decisive conclusions about the consequences on progeny.
Cancer patients treated with chemotherapy or radiation provide a considerable amount of information on the effects of male preconceptional exposure to toxicants on human reproduction. Chemotherapeutic agents have been shown to be gonadotoxic in a dose-dependent and time-specific manner, rendering a proportion of cancer survivors with persistent oligozoospermia or azoospermia (Magelssen et al., 2006). Germinal cell genetic damage has also
28 been shown in men undergoing cytotoxic therapy (Chatterjee et al., 2000;
Robbins et al., 1997); damaged sperm are still capable of fertilization (Ahmadi and Ng, 1999). Presently, however, there are no data that show any increase in the incidence of birth defects or genetic disease in the children of cancer survivors (Byrne et al., 1988; Green and Hall, 1988; Nygaard et al., 1991). One must keep in mind that epidemiological evidence is clouded by the fact that the number of patients followed in most studies is low and therefore it is difficult to assess the impact of cancer treatment on the offspring of exposed men.
1.3.2. Male Germ Cell Susceptibility
Contrary to the limitations of human studies, animal studies offer the advantage of defining the chemical, dose and duration; specifie windows of toxicant exposure can be assessed to deduce the susceptibility of specifie germ cell types to potential developmental toxicants and to elucidate the mechanisms of action of gonadotoxicants during spermatogenesis. Knowledge of the kinetics of spermatogenesis allows one to ascertain germ ce II-specifie susceptibilities by timing toxicant exposure; by calculating back from the time of sperm collection or mating, it is possible to determine which germ cell type was affected and responsible for effects observed later in the sperm (diminished quantity and/or quality) or progeny (pre- and post-implantation loss, and congenital and behavioural malformations) (Adler, 2000; Clermont, 1972; Russell, 1994). Table
1-3 shows the germ cell types first exposed to an administered toxicant depending on the time between initial exposure and mating or sperm collection in the rat.
29 Table 1-3. Time of toxicant exposure and the germ cell type first exposed in rats
Time of exposure Germ cell type first exposed (weeks)
1 Epididymal spermatozoa 2 Late elongated spermatids 3 Mid elongating spermatids 4 Early round spermatids 5-7 Spermatocytes 8+ Spermatogonia
The differential susceptibility of germ cells to certain toxic compounds has been identified. For example, bleomycin and procarbazine induce specific locus mutations in spermatogonial stem cells of mice (Russell et al., 2000; Witt and
Bishop, 1996). These cells are also particularly susceptible to X-ray exposure in both mice and men (Clifton and Bremner, 1983). Mitomycin C exerts its effects primarily on spermatocytes (Adler et al., 1996). Chlorambucil and melphalan also have reproductive effects attributable to cytotoxicity in specific male germ cell types; both induce dominant lethal mutations and heritable translocations in post- meiotic germ cells, particularly in early to mid spermatids (Generoso et al., 1995).
As for epididymal spermatozoa, it has been shown that they are maximally sensitive to acrylamide and ethylnitrosourea, which induce specific locus
mutations and dominant lethality (embryo death), respectively (Russell, 1994).
Certain agents, such as methyl methanesulfonate and acrylamide, alkylate the
cysteine sulfhydrl groups of sperm protamines (Sega et al., 1989; Sega and
Owens, 1983). Interestingly, the primary mechanism causing dominant lethality
30 has been proposed to be inhibition of normal chromatin condensation in late spermatids and early spermatozoa, which would render chromosomes at risk of breakage (Sega, 1991; Sega and Owens, 1983).
Animal studies have provided clear evidence that paternal exposure to cancer therapeutic agents induces adverse effects on progeny outcome
(Brinkworlh and Nieschlag, 2000; Robaire and Hales, 2003; Russell, 1994;
Trasler and Doerksen, 1999). The most widely studied male-mediated developmental toxicant is the commonly used anti-cancer drug, cyclophosphamide.
1.4. Cyclophosphamide
Clinically used for over forly years, cyclophosphamide is a bifunctional alkylating agent related to nitrogen mustard. It is used extensively in acute regimens for childhood (Kenney et al., 2001; Thomson et al., 2002) and adult malignancies (Colvin, 1999), including breast, ovarian and lung cancer, as weil as
Hodgkin's and non-Hodgkin's Iymphoma, and acute and chronic Iymphocytic leukemia. The drug, in chronic regimens is also an immunosuppressant for disorders such as systemic lupus erythematosus, Wegener's granulomatosis,
rheumatoid arlhritis, and graft-versus-host disease (Colvin, 1999).
Cyclophosphamide requires hepatic metabolic activation in order to be
effective; cytochrome P450 enzymes transform cyclophosphamide into 4-
hydroxycyclophosphamide (4-0HCP), the main active metabolite. 4-0HCP
interconverls with its tautomer, aldophosphamide and both of these metabolites
31 diffuse out of hepatic cells, circulate, and enter other cells. Aldophosphamide is oxidized to form the inactive metabolite carboxyphosphamide by aldehyde reductase (ALDH), or it undergoes a spontaneous elimination reaction to form phosphoramide mustard and acrolein (Boddy and Yule, 2000; Fleming, 1997)
(Figure 1-4). Phosphoramide mustard is a bifunctional alkylating agent with two reactive chloroethyl side chains capable of forming covalent bonds with nucleophilic sites on DNA or protein, such as the N-7 position of guanine, resulting in inter- and intra-strand DNA cross-links, DNA-protein cross links and
DNA strand breaks (Anderson et al., 1995). Cross-lin king inhibits DNA transcription and replication, thereby preventing cancerous cells from multiplying and causing cell death. Acrolein also alkylates DNA and proteins to exert cytotoxic effects (Crook et al., 1986). It is primarily responsible for bladder toxicity leading to hemorrhagic cystitis (Fraiser et al., 1991).
Cyclophosphamide 4-OHCP Aldophosphamide Phosphoramide Mustard
l ALDH Hj:.CH_C~H o Acrolein
Carboxyphosphamide
Figure 1-4. Cyclophosphamide metabolism
32 1.4.1. Cyclophosphamide as a Male-Mediated Developmental Toxicant
Numerous reports indicate that treatment with acute high doses or chronic low doses of cyclophosphamide alters male fertility, resulting in oligozoospermia or azoo~permia in adults (Fairley et al., 1972; Qureshi et al., 1972). Animal experiments have been and continue to be done to elucidate how cyclophosphamide affects the male germ cell and the impact this has on embryo development (Anderson et al., 1995; Robaire et al., 2006; Trasler et al., 1985;
Trasler et al., 1986; Trasler et al., 1987). Different germ ce Il types
(spermatogonia, spermatocytes, spermatids and epididymal spermatozoa) are clearly differentially sensitive to cyclophosphamide and the effects on progeny outcome (different rates of preimplantaion loss, postimplantation loss and congenital malformations) are dependent on the phase of spermatogenesis when germ cells were tirst exposed to the drug (Trasler et al., 1986; Trasler et al.,
1987).
The exposure to cyclophosphamide throughout germ cell development and maturation i.e. from spermatogonia to spermatozoa, results,in an increased incidence of growth retardation and malformations, including hydrocephaly, edema and micrognathia (Trasler et al., 1986); these malformations are also observed in the F2 generation (Hales et al., 1992). Chromosomal aberrations
could be involved in mechanisms leading to potentially heritable defects;
spermatogonia, however, have been reported to be at low risk for the induction of
heritable translocations by cyclophosphamide (Generoso et al., 1981; Sotomayor
and Cumming, 1975). Interestingly, other chromosomal abnormalities associated
with spermatogonial exposure to cyclophosphamide have been reported.
33 Significant increases in the frequency of spermatozoa with chromosome 4 disomy (Y44) and nullisomy (YO) are associated with germ cell drug exposure initiated during mitosis (9 weeks) though not during meiosis· (6 weeks), suggesting that cyclophosphamide disrupts meiotic events before they begin
(Barton et al., 2003).
Interestingly, chronic exposure to low doses of cyclophosphamide during mitosis leads to significant levels of DNA damage in spermatocytes; however, it does not impair meiotic progression (Aguilar-Mahecha et al., 2005). Only acute exposures increase DNA damage in spermatocytes and impair meiotic progression (Aguilar-Mahecha et al., 2005), along with the induction of gene conVersions and mutations (Schimenti et al., 1997; Zhang et al., 2002), synaptonemal complex damage (Allen et al., 1988; Backer et al., 1988) and micronuclei formation (Lahdetie, 1983). Correspondingly, chronic administration of cyclophosphamide, targeting germ cells first exposed to the drug as spermatocytes, results in increased preimplantation loss (Trasler et al., 1986;
Trasler et al., 1987). Ali things considered, these results signify a defect in germ cell DNA checkpoint mechanisms after repeat exposure to low doses of a genotoxic agent, allowing damage to be transmitled throughout spermatogenesis. ln fa ct , reduced ability to respond to stress wasevident, as chronic low dose treatment dramatically decreased the expression of stress response genes
(oxidative stress-related, heat shock and DNA repair) in pachytene spermatocytes and round spermatids (Aguilar-Mahecha et al., 2002).
Post-meiotic germ cells are the most susceptible to cyclophosphamide effects (Ehling and Neuhauser-Klaus, 1988; Generoso et al., 1981; Sotomayor
34 and Cumming, 1975). This may be partly due to their inability to repair DNA or to undergo apoptosis, thus resulting in an accumulation of damage in spermatozoa; studies show that late spermiogenic germ cells have lost many of the enzymes associated with DNA repair (Sotomayor et al., 1978) and drug-induced apoptosis was most prominent in spermatogonia and spermatocytes (Brinkworth and
Nieschlag, 2000; Cai et al., 1997; Sinha Hikim and Swerdloff, 1999). Using the alkaline elution assay, overall assessment of DNA damage in male germ cells after cyclophosphamide administration revealed an increase in DNA single-strand
breaks one week post-treatment (Qiu et al., 1995a), whereas cyclophosphamide given for six weeks significantly increased DNA single-strand breaks as weil as
DNA-DNA cross-links in spermatozoa of drug-exposed rats (Qiu et al., 1995a);
the duration of this treatment exposes germ cells from the meiotic phase
throughout spermiogenesis and epididymal spermatozoa maturation. The
exposure of either epididymal spermatozoa or testicular spermatids to
cyclophosphamide results in an adverse effect on progeny outcome; primarily,
dose-dependent increases in post-implantation loss occur (Qiu et al., 1992;
Trasler et al., 1986). In particular, there are time specifie inereases in post
implantation loss associated with the phase of spermiogenesis exposed to the
drug; a progressive increase in postimplantation loss started at 2 weeks, reaehing
to about 75% post-implantation loss by 4 weeks. Interestingly, this effect is
reversed within 4 weeks post-treatment (Hales and Robaire, 1990), thus
highlighting the importance of spermiogenic chromatin remodeling and sperm
maturation on the suceess of embryo development.
35 1.5. Rationale and Formulation of the Question
Infertility is the failure to achieve a pregnancy after regular unprotected intercourse for at least one year or to carry a pregnancy to full term. Adverse reproductive outcomes include diverse endpoints such as early pregnancy loss or spontaneous abortions, stillbirths and preterm delivery, as weil as growth retardation and congenital malformations. Approximately 15% of couples are infertile (Evers, 2002) with at least 50% of ail conceptions estimated to result in fetal loss before the end of the first trimester (Goldstein, 1994; Wilcox et al.,
1999). In about 40% of cases male infertility is the primary cause or contributing factor (Centers for disease control and prevention' (COC), 2003). The
consequences of exposure of males to drugs, radiation and environmental toxicants on reproduction and development are a growing concern as it becomes
increasingly clear that exposure to such toxicants impairs sperm quality; the
mechanisms involved, however, are not as evident.
To date, assisted reproductive technology, in particular intracytoplasmic
sperm injection (ICSI) is the only effective treatment for most cases of male
infertility. ICSI, first introduced to help oligozoospermic men to become fathers
(Palermo et al., 1992), quickly became a technique to achieve pregnancies in
couples with extreme male factor infertility, using sub-optimal immature sperm
from the epididymis (Oevroey et al., 1995; Schlegel et al., 1995), and testis
(Bourne et al., 1995; Silber et al., 1995), as weil as injecting spermatids (Fishel et
al., 1995; Tesarik et al., 1995) or sperm with abnormal morphology (Harari et al.,
1995; Rybouchkin et al., 1997) into eggs. Whether fertilization occurs naturally or
36 with assistance, there are valid concerns about the consequences to the progeny if male germ cells are altered or defective.
Chromatin abnormalities in male germ cells pose a risk to the offspring if transmitted to the developing embryo; either spontaneous abortion and/or congenital malformations may result. Increasing evidence suggests that these risks are high in embryos generated from assisted reproductive techniques
(Bonduelle et al., 1999; Hansen et al., 2002). There are multiple mechanisms by which xenobiotics may induce adverse embryo outcomes. A few which have been proposed are: 1) direct effects on chromosomes (aneuploidy or clastogenicity) or genes; 2) epigenetic effects affecting gene expression; and 3) indirect effects on chromosomes or genes, via alteration of DNA repair capacity or sperm chromatin packaging/condensation (Ratcliffe, 1994).
Functional maternai and paternal genomes are fundamental to the complex events involved in early embryogenesis. Changes in the DNA of either parent as a result of toxicant exposure may lead to changes in the progression and coordination of development. Previous studies of parental contributions to embryonic development indicate that absence of the paternal genome results in
aberrant development (Barton et al., 1984; McGrath and Solter, 1984). To
ensure the genetic integrity of the zygote and the success of ensuing embryonic
development there are essential features required that include, in addition to
oocyte-specific events, the exchange of proteins in the sperm nucleus (protamine
to histones) and remodeling of the sperm nucleus into a functional pronucleus.
Transcriptional activation of specifie genes is initiated after fertilization and
remodeling of the sperm chromatin after fertilization is fundamental for the
37 expression of the paternal genome (Schultz, 2002). Above ail, gene expression is regulated by chromatin structure (Schultz and Worrad, 1995), thus the sperm chromatin structure is key to ensure that embryogenesis proceeds normally. The unique structure of sperm chromatin depends on a sequence of events that involves the expression of numerous proteins, precise organization of the DNA and condensation of the chromatin during spermiogenesis. Any perturbations to the integrity of sperm DNA or structure could be detrimental; in fact, a significant association between male subfertility and abnormal nuclear condensation has beensuggested (Hammadeh et al., 1999) .
. Cyclophosphamide is widely used as a model drug to ascertain the molecular mechanisms underlying the developmental toxicity induced following paternal exposure. Spermatids and spermatozoa do not have the ability to repair
DNA or undergo apoptosis, rendering them most susceptible to genotoxic damage; the refore , any insult incurred remains. Consequently, DNA damage found in cyclophosphamide-exposed spermatozoa is imparted to the newly fertilized zygote (Harrouk et al., 2000a). As weil, decondensation of drug exposed spermatozoa is disturbed; sperm decondense more rapidly than control spermatozoa in denuded hamster oocytes and male pronuclear formation is earlier in rat oocytes sired by drug-treated males (Harrouk et al., 2000b; Qiu et al., 1995b). Cyclophosphamide damages male germ cells in such a way that altered zygotic gene activation and embryo death result (Robaire et al., 2006).
The aforementioned evidence suggests that altered sperm chromatin remodeling may be a factor in the effects of cyclophosphamide on progeny outcome.
38 Therefore, 1 focused the studies in this thesis on the spermiogenic and maturation events of post-meiotic male germ cells and the effects of cyclophosphamide on the cells during this time. The hypothesis of this thesis is that: Cyclophosphamide exposure causes genetic damage and alters the sperm proteome, thus disrupting components of chromatin condensation and organization during the spermiogenic phase of spermatogenesis.
To address this hypothesis, 1set forth the following objectives:
1. Assess the phase specificity of the susceptibility of spermiogenic germ
cells to genetic damage induced by cyclophosphamide.
2. Assess the effects of cyclophosphamide on the sperm chromatin structure
and basic proteome.
3. Analyze the effect of cyclophosphamide exposure on the protein profile of
the sperm nuclear matrix.
39 REFERENCES
Adham lM, Nayernia K, Burkhardt-Gottges E, Topaloglu 0, Dixkens C, Holstein AF and Engel W (2001) Teratozoospermia in mice lacking the transition protein 2 (Tnp2). Mol Hum Reprod 7,513-520.
Adler ID (2000) Spermatogenesis and mutagenicity of environmental hazards: extrapolation of genetic risk from mouse to man. Andrologia 32,233-237.
Adler ID, Anderson D, Benigni R, Ehling UH, Laehdetie J, Pacchierotti F, Russo A and Tates AD (1996) Synthesis report of the step project detection of germ cell mutagens. Mutat Res 353,65-84.
Agarwal A and Allamaneni SS (2005) Sperm DNA damage assessment: a test whose time has come. Fertil Steril 84,850-853.
Agarwal A and Said T (2004) Sperm chromatin assessment. In Gardner DK, Weissman A, Howles CM and Shoham Z (eds) Textbook of assisted reproductive techniques: laboratory and clinical perspectives. 2nd edn, Taylor & Francis, Abingdon, 93-106.
Aguilar-Mahecha A, Hales BF and Robaire B (2002) Chronic cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells. Biol Reprod 66,1024-1032.
Aguilar-Mahecha A, Hales BF and Robaire B (2005) Effects of acute and chronic cyclophosphamide treatment on meiotic progression and the induction of DNA double-strand breaks in rat spermatocytes. Biol Reprod 72,1297- 1304.
40 Ahmadi A and Ng SC (1999) Developmental capacity of damaged spermatozoa. Hum Reprod 14,2279-2285.
Aitken RJ (2006) Sperm function tests and fertility. Int J AndroI29,69-75.
Alessenko AV and Burlakova EB (2002) Functional role of phospholipids in the nuclear events. Bioelectrochemistry 58,13-21.
Allen JW, Poorman PA, Backer LC, Gibson JB, Westbrook-Collins B and Moses MJ (1988) Synaptonemal complex damage as a measure of genotoxicity at meiosis. Cell Biol ToxicoI4,487-494.
Alvarez JG, Ollero M, Larson-Cook KL and Evenson DP (2004) Selecting cryopreserved semen for assisted reproductive techniques based on the level of sperm nuclear DNA fragmentation resulted in pregnancy.Fertil Steril81,712-713.
Anachkova B, Djeliava V and Russev G (2005) Nuclear matrix support of DNA replication. J Cell Biochem 96,951-961.
Anderson D, Bishop JB, Garner RC, Ostrosky-Wegman P and Selby PB (1995) Cyclophosphamide: review of its mutagenicity for an assessment of potential germ cell risks. Mutat Res 330,115-181.
Aul RB and Oko RJ (2002) The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis. Dev Biol 242,376-387.
41 Backer LC, Gibson JB, Moses MJ and Allen JW (1988) Synaptonemal complex damage in relation to meiotic chromosome aberrations after exposure of male mice to cyclophosphamide. Mutat Res 203,317-330.
Balhorn R (1989) Mammalian protamines: structure and molecular interactions. In Adolph KW (ed) Molecular biology of chromosome function. Springer, New York, 366-395.
Balhorn R, Corzett M, Mazrimas J and Watkins B (1991) Identification of bull protamine disulfides. Biochemistry 30,175-181.
Barone JG, Christiano AP and Ward WS (2000) DNA organization in patients with a history of cryptorchidism. Urology 56,1068-1070.
Barone JG', de Lara J, Cummings KB and Ward WS(1994) DNA organization in human spermatozoa. J AndroI15,139-144.
Barton SC, Surani MA and Norris ML (1984) Role of paternal and maternai genomes in mouse development. Nature 311,374-376.
Barton TS, Wyrobek AJ, Hill FS, Robaire B and Hales BF (2003) Numerical chromosomal abnormalities in rat epididymal spermatozoa following chronic cyclophosphamide exposure. Biol Reprod 69,1150-1157.
Belgrader P, Siegel AJ and Berezney R (1991) A comprehensive study on the isolation and eharaeterization of the Hela S3 nuclear matrix. J Ce Il Sei 98 ( Pt 3),281-291.
42 Bellvé AR, Chandrika R, Martinova YS and Barth AH (1992) The perinuclear matrix as a structural element of the mouse sperm nucleus. Biol Reprod 47,451-465.
Bellvé AR and O'Brien DA (1983) The mammalian spermatozoon: structure and temporal assembly. In Hartman JF (ed) Mechanisms and control of fertilization. Academie Press, New York, 55-137.
Berezney Rand Coffey DS (1974) Identification of a nuclear protein matrix. Biochem Biophys Res Commun 60,1410-1417.
Biggiogera M, Martin TE, Gordon J, Amalric F and Fakan S (1994) Physiologically inactive nucleoli contain nucleoplasmic ribonucleoproteins: immunoelectron microscopy of mouse spermatids and early embryos. Exp Cell Res 213,55-63.
Biggiogera M, Von Schack ML, Martin TE, Gordon J, Muller M and Fakan S (1993) Immunoelectron microscope localization of snRNP, hnRNP, and ribosomal proteins in mouse spermatogenesis. Mol Reprod Dev 35,261- 271.
Boddy AV and Yule SM (2000) Metabolism and pharmacokinetics of oxazaphosphorines. Clin Pharmacokinet 38,291-304.
Bonduelle M, Camus M, De Vos A, Staessen C, Toumaye H, Van Assche E, Verheyen G, Devroey P, Liebaers 1 and Van Steirteghem A (1999) Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum Reprod 14 Suppl 1,243-264.
43 Boulikas T (1994) Transcription factor binding sites in the matrix atlachment region (MAR) of the chicken alpha-globin gene. J Cell Biochem 55,513- 529.
Boulikas T (1995) Chromatin domains and prediction of MAR sequences. Int Rev Cytol 162A,279-388.
Boume H, Richings N, Harari 0, Watkins W, Speirs AL, Johnston WI and Baker HW (1995) The use of intracytoplasmic sperm injection for the treatment of severe and extreme male infertility. Reprod Fertil Dev 7,237-245.
Brinkworth MHand Nieschlag E (2000) Association of cyclophosphamide induced male-mediated, foetal abnormalities with reduced patemal germ cell apoptosis. Mutat Res 447,149-154.
Byers S, Suarez-Quiam C and Pelletier RM (1993) Sertoli cell junctions and the seminiferous epithelium barrier. In Russell LD and Griswold MD (eds) The Sertoli Cell. Cache River Press, Clearwater, 432-446.
Byme J, Mulvihill JJ, Connelly RR, Austin DA, Holmes GE, Holmes FF, Latouretle HB, Meigs JW, Strong LC and Myers MH (1988) Reproductive problems and birth defects in survivors of Wilms' tumor and their relatives. '~ Med Pediatr Oncol 16,233-240.
Cai L, Hales BF and Robaire B (1997) Induction of apoptosis in the germ cells of adult male rats after exposure to cyclophosphamide. Biol Reprod 56,1490- 1497.
Caramelli E, Matleucci A, Zini N, Carini C, Guidotti L, Ricci D, Maraldi NM and Capitani S (1996) Nuclearphosphoinositide-specific phospholipase C,
44 phosphatidylinositol 4,5-bisphosphate and protein kinase C during rat spermatogenesis. Eur J CeU Biol 71,154-164.
Caron N, Veilleux Sand Boissonneault G (2001) Stimulation of DNA repair by the spermatidal TP1 protein. Mol Reprod Dev 58,437-443.
Centers for disease control and prevention (CDC) (2003) 2000 Assisted reproductive technology success rates: national summary and fertility clinic report. MMWR Morbidity and Mo rta lity Weekly Report 52,1-14.
Chatterjee R, Haines GA, Perera DM, Goldstone A and Morris ID (2000) Testicular and sperm DNA damage after treatment with fludarabine for chronic Iymphocytic leukaemia. Hum Reprod 15,762-766.
Chen JL, Guo SH and Gao FH (2001) Nuclear matrix in developing rat spermatogenic cells. Mol Reprod Dev 59,314-321.
Chen JL and Longo FJ (1996) Expression and localization of DNA topoisomerase " during rat spermatogenesis. Mol Reprod Dev 45,61-71.
Chia SE and Shi LM (2002) Review of recent epidemiological studies on paternal occupations and birth defects. Occup Environ Med 59,149-155.
Cho C, Willis WD, Goulding EH, Jung-Ha H, Choi YC, Hecht NB and Eddy EM (2001) Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet 28,82-86.
Choudhary SK, Wykes SM, Kramer JA, Mohamed AN, Koppitch F, Nelson JE and Krawetz SA (1995) A haploid expressed gene cluster exists as a single chromatin domain in human sperm. J Biol Chem 270,8755-8762.
45 Ciejek EM, Tsai MJ and O'Maliey BW (1983) Actively transcribed genes are associated with the nuclear matrix. Nature 306,607-609.
Clermont Y (1972) Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 52,198-236.
Clermont Y and Leblond CP (1955) Spermiogenesis of man, monkey, ram and other mammals as shown by the periodic acid-Schiff technique. Am J Anat 96,229-253.
Clermont Y, Leblond CP and MESSIER B (1959) [Duration of the cycle of the seminal epithelium of the rat.]. Arch Anat Microsc Morphol Exp 48(Suppl),37-55.
Clermont Y and Tang XM (1985) Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat. Anat Rec 213,33-43.
Clitton OK and Bremner W J (1983) The effect of testicular x-irradiation on spermatogenesis in man. A comparison with the mouse. J And roi 4,387- 392.
Colvin OM (1999) An overview of cyclophosphamide development and clinical applications. Curr Pharm Des 5,555-560.
Conrad M, Moreno SG, Sinowatz F, Ursini F, Kolle S, Roveri A, Brielmeier M, Wurst W, Maiorino M and Bornkamm GW (2005) The nuclear form of phospholipid hydroperoxide glutathione peroxidase is a protein thiol peroxidase contributing to sperm chromatin stability. Mol Cell Biol 25,7637-7644.
46 Cremer T, Kreth G, Koester H, Fink RH, Heintzmann R, Cremer M, Solovei l, Zink D and Cremer C (2000) Chromosome territories, interchromatin domain compartment, and nuclear matrix: an integrated view of the functional nuclear architecture. Crit Rev Eukaryot Gene Expr 10,179-212.
Crook TR, Souhami RL and McLean AE (1986) Cytotoxicity, DNA cross-linking, and single strand breaks induced by activated cyclophosphamide and acrolein in human leukemia cells. Cancer Res 46,5029-5034.
Dadoune JP (2003) Expression of mammalian spermatozoal nucleoproteins. Microsc Res Tech 61,56-75.
Dantzer F, Mark M, Quenet D, Scherthan H, Huber A, Liebe B, Monaco L, Chicheportiche A, Sassone-Corsi P, de Murcia G et al. (2006) Poly(ADP ribose) polymerase-2 contributes to the fidelity of male meiosis 1 and spermiogenesis. Proc Natl Acad Sci USA 103,14854-14859. de Rooij DG and Russell LD (2000) Ali you wanted to know about spermatogonia but were afraid to ask. J Androl21 ,776-798.
De Yebra L, Ballesca JL, Vanrell JA, Corzett M, Balhorn R and Oliva R (1998) Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels. Fertil Steril69,755-759.
Demeret C, Vassetzky y and Mechali M (2001) Chromatin remodelling and DNA replication: from nucleosomes to loop domains. Oncogene 20,3086-3093.
Devroey Pt Silber S, Nagy Z, Liu J, Tournaye H, Joris H, Verheyen G and Van Steirteghem A (1995) Ongoing pregnancies and birth after
47 intracytoplasmic sperm injection with frozen-thawed epididymal spermatozoa. Hum Reprod 10,903-906.
Dijkwel PA and Hamlin JL (1988) Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells. Mol Cell Biol 8,5398-5409.
Dworetzky SI, Wright KL, Fey EG, Penman S, Lian JB, Stein JL and Stein GS (1992) Sequence-specific DNA-binding proteins are components of a nuclear matrix-attachment site. Proc Nat! Acad Sci USA 89,4178-4182.
Ehling UH and Neuhauser-Klaus A (1988) Induction of specifie-locus and dominant-Iethal mutations by cyclophosphamide and combined cyclophosphamide-radiation treatment in male mice. Mutat Res 199,21-30.
Evers JL (2002) Female subfertility. Lancet 360,151-159.
Fairley KF, Barrie JU and Johnson W (1972) Sterility and testicular atrophy related to cyclophosphamide therapy. Lancet 1,568-569.
Farache G, Razin SV, Rzeszowska-Wolny J, Moreau J, Targa FR and Scherrer K (1990) Mapping of structural and transcription-related matrix attachment sites in the alpha-globin gene domain of avian erythroblasts and erythrocytes. Mol Cell Biol 10,5349-5358.
Fawcett DW (1966) On the occurrence of a fibrous lamina on the inner aspect of the nuclear envelope in certain cells of vertebrates. Am J Anat 119,129- 145.
48 Fawcett DW (1975) The mammalian spermatozoon. Dev Biol 44,394-436.
Fernandes DJ, Qiu J and Catapano CV (1995) DNA topoisomerase Il isozymes involved in anticancer drug action and resistance. Adv Enzyme Regul 35,265-281.
Fey EG, Krochmalnic Gand Penman S (1986) The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy. J Cell Biol 102,1654-1665.
Fey EG and Penman S (1988) Nuclear matrix proteins reflect cell type of origin in cultured human cells. Proc Nat! Acad Sci USA 85,121-125.
Fishel S, Green S, Bishop M, Thornton S, Hunter A, Fleming S and al Hassan S (1995) Pregnancy after intracytoplasmic injection of spermatid. Lancet 345,1641-1642.
Fleming RA (1997) An overview of cyclophosphamide and ifosfamide pharmacology. Pharmacotherapy 17, 146S-154S.
Fraiser LH, Kanekal Sand Kehrer JP (1991) Cyclophosphamide toxicity. Characterising and avoiding the problem. Drugs 42,781-795.
Franke WW (1987) Nuclear lamins and cytoplasmic intermediate filament proteins: a growing multigene family. Cell 48,3-4.
Fukami K, Furuhashi K, Inagaki M, Endo T, Hatano Sand Takenawa T (1992) Requirement of phosphatidylinositol 4,5-bisphosphate for alpha-actinin function. Nature 359,150-152.
49 Gasser SM and Laemmli UK (1986) Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell 46,521-530.
Gatewood JM, Cook GR, Balhom R, Schmid CW and Bradbury EM (1990) Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones. J Biol Chem 265,20662-20666.
Generoso WM, Cattanach Band Malashenko AM (1981) Mutagenicity of selected chemicals in mammals. In de Serres J and Shelby MD (eds) Comparative chemical·mutagenesis. Plenum Press, New York, 681-701.
Generoso WM, Witt KL, Cain KT, Hughes L, Cacheiro NL, Lockhart AM and Shelby MD (1995) Dominant lethal and heritable translocation tests with chlorambucil and melphalan in male mice. Mutat Res 345,167-180.
Gerace L, Comeau C and Benson M (1984) Organization and modulation of nuclear lamina structure. J Cell Sei Suppl 1 ,137-160.
Getzenberg RH (1994) Nuclear matrix and the regulation of gene expression: tissue specificity. J Cell Biochem 55,22-31.
Getzenberg RH, Pienta KJ, Huang EY and Coffey DS (1991) Identification of nucléar matrix proteins in the cancer and normal ràt prostate. Cancer Res 51,6514-6520.
Gillan L, Evans G and Maxwell WM (2005) Flow cytometric evaluation of sperm parameters in relation to fertility potential. Theriogenology 63,445-457.
50 Goldman RD, Baccetti B, Collodel G, Gambera L, Moretti E and Piomboni P (1998) Localization of lamins in mammalian spermatozoa. J Submicrosc Cytol Pathol 30,573-580.
Goldstein SR (1994) Embryonic death in early pregnancy: a new look at the first trimester. Obstet Gynecol 84,294-297.
Gravance CG, Garner DL, Miller MG and Berger T (2001) Fluorescent probes and flow cytometry to assess rat sperm integrity and mitochondrial function. Reprod ToxicoI15,5-10.
Green DM and Hall B (1988) Pregnancy outcome following treatment during childhood or adolescence for Hodgkin's disease. Pediatr Hematol Oncol 5,269-277.
Green GR, Balhorn R, Poccia DL and Hecht NB (1994) Synthesis and processing of mammalian protamines and transition proteins. Mol Reprod Dev 37,255- 263.
Grimes SR, Jr. and Henderson N (1984) Hyperacetylation of histone H4 in rat testis spermatids. Exp Cell Res 152,91-97.
Hales BF, Crosman K and Robaire B (1992) Increased postimplantation loss and malformations among the F2 progeny of male· rats chronically treated with cyclophosphamide. Teratology 45,671-678.
Hales BF and Robaire B (1990) Reversibility of the effects of chronic paternal exposure to cyclophosphamide on pregnancy outcome in rats. Mutat Res 229,129-134.
51 Hammadeh ME, al Hasani S, Doerr S, Stieber M, Rosenbaum P, Schmidt W and Diedrich K (1999) Comparison between chromatin condensation and morphology from testis biopsy extracted and ejaculated spermatozoa and their relationship to ICSI outcome. Hum Reprod 14,363-367.
Hansen M, Kurinczuk JJ, Bower C and Webb S (2002) The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 346,725-730.
Harari 0, Boume H, Baker G, Gronow M and Johnston 1(1995) High fertilization rate with intracytoplasmic sperm injection in mosaic Klinefelter's syndrome. Fertil Steril 63,182-184.
Harkonen K (2005) Pesticides and the induction of aneuploidy in human sperm. Cytogenet Genome Res 111,378-383.
Harrouk W, Codrington A, Vinson R, Robaire B and Hales BF (2000a) Patemal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo. Mutat Res 461,229-241.
Harrouk W, Khatabaksh S, Robaire B and Hales BF (2000b) Paternal exposure to cyclophosphamide dysregulates the gene activation program in rat preimplantation embryos. Mol Reprod Dev 57,214-223.
He DC, Nickerson JA and Penman S (1990) Core filaments of the nuclear matrix. J Cell Biol 110,569-580.
Heidaran MA, Showman RM and Kistler WS (1988) A cytochemical study of the transcriptional and translational regulation of nuclear transition protein 1
52 (TP1), a major chromosomal protein of mammalian spermatids. J Cell Biol 106,1427-1433.
Heng HH, Goetze S, Ve CJ, Liu G, Stevens JB, Bremer SW, Wykes SM, Bode J and Krawetz SA (2004) Chromatin loops are selectively anchored using scaffold/matrix-attachment regions. J Cell Sci 117,999-1008.
Hess RA (1998) Spermatogenesis Overview. In Knobil E and Neill JO (eds) Encyclopedia of Reproduction. Academic Press, San Diego, 539-545.
Howes EA, Hurst SM and Jones R (2001) Actin and actin-binding proteins in povine spermatozoa: potential role in membrane remodeling and intracellular signaling during epididymal maturation and the acrosome reaction. J Androl 22,62-72.
Hud NV, Allen MJ, Downing KH, Lee J and Balhom R (1993) Identification of the elemental packing unit of ONA in mammalian sperm cells by atomic force microscopy. Biochem Biophys Res Commun 193,1347-1354.
Hurst S, Howes EA, Coadwell J and Jones R (1998) Expression of a testis specific putative actin-capping protein associated with the developing acrosome during rat spermiogenesis. Mol Reprod Dev 49,81-91. larovaia av, Akopov SB, Nikolaev LG, Sverdlov ED and Razin SV (2005) Induction of transcription within chromosomal ONA loops flanked by MAR elements causes an association of loop DNA with the nuclear matrix. Nucleic Acids Res 33,4157-4163.
Janmey PA and Stossel TP (1987) Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate; Nature 325,362-364.
53 Jarman AP and Higgs DR (1988) Nuclear scaffold attachment sites in the human globin gene complexes. EMBO J 7,3337-3344.
Kalandadze AG, Bushara SA, Vassetzky YS, Jr. and Razin SV (1990) Characterization of DNA pattern in the site of permanent attachment to the nuclear matrix located in the vicinity of replication origin. Biochem Biophys Res Commun 168,9-15.
Kenney LB, Laufer MR, Grant FO, Grier H and Oiller L (2001) High risk of infertility and long term gonadal damage in males treated with high dose cyclophosphamide for sarcoma during childhood. Cancer 91 ,613-621.
Kerr JB and de Kretser DM (1974) Proceedings: The role of the Sertoli cell in phagocytosis of the residual bodies of spermatids. J Reprod Fertil 36,439- 440.
Kierszenbaum AL (2001) Transition nuclear proteins during spermiogenesis: unrepaired ONA breaks not allowed. Mol Reprod Oev 58,357-358.
Kistler WS, Henriksen K, Mali P and Parvinen M (1996) Sequential expression of nucleoproteins during rat spermiogenesis. Exp CeliRes 225,374-381.
Kistler WS, Noyes C, Hsu Rand Heinrikson RL (1975) The amino acid sequence of a testis-specific basic protein that is associated with spermatogenesis. J Biol Chem 250,1847-1853.
f' Klaus AV, McCarrey JR, Farkas A and Ward WS (2001) Changesin ONA loop domain structure during spermatogenesis ahd embryogenesis in the Syrian golden hamster. Biol Reprod 64,1297-1306.
54 Korley R, Pouresmaeili F and Oko R (1997) Analysis of the protein composition of the mouse sperm perinuclear theca and characterization of its major protein constituent. Biol Reprod 57,1426-1432.
Kramer JA and Krawetz SA (1996) Nuclear matrix interactions within the sperm genome. J Biol Chem 271,11619-11622.
Kumar S (2004) Occupational exposure associated with reproductive dysfunction. J Occup Health 46,1-19.
Laberge RM and Boissonneault G (2005) On the nature and origin of DNA strand breaks in elongating spermatids. Biol Reprod 73,289-296.
Lahdetie J (1983) Micronuclei induced during meiosis by ethyl methanesulfonate, cyclophosphamide and dimethylbenzanthracene in male rats. Mutat Res 120,257-260.
Lalli M and Clermont Y (1981) Structural changes of the head components of the rat spermatid during late spermiogenesis. Am J Anat 160,419-434.
Lancranjan l, Popescu HI, GAvanescu 0, Klepsch 1and Serbanescu M (1975) Reproductive ability of workmen occupationally exposed to lead. Arch Environ Health 30,396-401.
Lassing 1and Lindberg U (1985) Specificinteraction between phosphatidylinositol 4,5-bisphosphate and profilactin. Nature 314,472-474.
Leblond CP and Clermont Y (1952a) Definition of the stages ofthe cycle of the seminiferous epithelium in the rat. Ann N Y Acad Sci 55,548-573.
55 Leblond CP and Clermont Y (1952b) Spermiogenesis of rat, mouse, hamster and guinea pig as revealed by the periodic acid-fuchsin sulfurous acid technique. Am J Anat 90,167-215.
Lecuyer C, Dacheux JL, Hermand E, Mazeman E, Rousseaux J and Rousseaux Prevost R (2000) Actin-binding properties and colocalization with actin during spermiogenesis of mammalian sperm calicin. Biol Reprod.63,1801- 1810.
Levy-Wilson Band Fortier C (1989) The limits of the DNase I-sensitive do main of .the human apolipoprotein B gene coincide with the locations of chromosomal anchorage loops and define the 5' and 3' boundaries of the gene. J Biol Chem 264,21196-21204.
Lewis SE and Aitken RJ (2005) DNA damage to spermatozoa has impacts on fertilization and pregnancy. Cell Tissue Res 322,33-41.
Li Z, Yang J and Huang H (2006) Oxidative stress induces H2AX phosphorylation in human spermatozoa. FEBS Lett 580,6161-6168.
Longo FJ and Cook S (1991) Formation ofthe perinucleartheca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides. Mol Reprod Dev 28,380-393.
Longo FJ, Krohne Gand Franke WW (1987) Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements. J Cell Biol 105,1105-1120.
56 Lubocka J, Szopa J and Kozubek A (1995) Modification of the lipid component modulates nuclear matrix nucleolytic activity. Acta Biochim Pol 42,211- 215.
Magelssen H, Brydoy M and Fossa SD (2006) The effects of cancer and cancer treatments on male reproductive function. Nat Clin Pract UroI3,312-322.
Maraldi NM, Marinelli F, Coceo L, Papa S, Santi P and Manzoli FA (1986) Morphometric analysis and topological organization of nuclear matrix in freeze-fractured eleetron microscopy. Exp Cell Res 163,349-362.
Maraldi NM, Mazzotti G, Capitani S, Rizzoli R, Zini N, Squarzoni Sand Manzoli FA (1992) Morphological evidence offunction-related localization of phospholipids in the cell nucleus. Adv Enzyme Regul 32,73-90.
Martelli AM, Cocco L,Riederer BM and Neri LM (1996) The nuclear matrix: a critical appraisal. Histol Histopathol 11,1035-1048.
Martelli AM, Manzoli L, Faenza l, Bortul R, Billi A and Cocco L (2002) Nuclear inositol lipid signaling and its potential involvement in malignant transformation. Biochim Biophys Acta 1603,11-17.
Martelli AM, Tabellini G, Borgatti P, Bortul R, Capitani Sand Neri LM (2003) Nuclear lipids: new functions for old molecules? JCeli Biochem 88,455- 461.
Martianov l, Brancorsini S, Catena R, Gansmuller A, Kotaja N, Parvinen M, Sassone-Corsi P and Davidson 1 (2005) Polar nuclear localization of H1T2, a histone H1 variant, required for spermatid elongation and DNA
57 condensation during spermiogenesis. Proc Nat! Acad Sci USA 102,2808- 2813.
Marushige Yand Marushige K (1975) Transformation of sperm histone during formation and maturation of rat spermatozoa. J Biol Chem 250,39-45.
Mattout A, Dechat T, Adam SA, Goldman RD and Gruenbaum Y (2006) Nuclear lamins, diseases and aging. Curr Opin CeU Biol 18,335-341.
Mayer JF, Jr., Chang TS and Zirkin BR (1981) Spermatogenesis in the mouse 2. Amino acid incorporation into basic nucleoproteins of mouse spermatids and spermatozoa. Biol Reprod 25,1041-1051.
Mazzotti G, Zini N, Rizzi E, Rizzoli R, Galanzi A, Ognibene A, Santi S, Matteucci A, Martelli AM and Maraldi NM (1995) Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus. J Histochem Cytochem 43,181-191.
McGrath J and Solter 0 (1984) Completion of mouse embryogenesis requires both the maternai and paternal genomes. CeU37,179-183.
McPherson S andLongo FJ (1993a) Chromatin structure-function alterations during mammalian spermatogenesis: DNA nicking and repair ln elongating spermatids. Eur J Histochem 37,109-128.
McPherson SM and Longo FJ (1992) Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev 31,268-279.
58 McPherson SM and Longo FJ (1993b) Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II. Dev Biol 158,122- 130.
Meistrich ML (1989) Histones and basic nuclear proteintransitions in mammalian spermatogenesis. In Hnilica LS, Stein GS and Stein JL (eds) Histones and other basic nuclear proteins. CRC Press, Boca Raton, 165-182.
Meistrich ML, Bucci LR, Trostle-Weige PK and Brock WA (1985) Histone variants in rat spermatogonia and primary spermatocytes. Dev Biol 112,230-240.
Meistrich ML, Trostle-Weige PK, Lin R, Bhatnagar YM and Allis CD (1992) Highly acetylated H4 is associated with histone displacement in rat spermatids. Mol Reprod Dev 31,170-181.
Meistrich ML, Trostle-Weige PK and Van Beek ME (1994) Separation of specifie stages of spermatids from vitamin A-synchronized rat testés for assessment of nucleoprotein changes during spermiogenesis. Biol Reprod 51,334-344.
Meyer-Ficca ML, Scherthan H, Burkle A and Meyer RG (2005) Poly(ADP ribosyl)ation during chromatin remodeling steps in rat spermiogenesis. Chromosoma 114,67-74.
Mladenov E, Anachkova Band Tsaneva 1 (2006) Sub-nuclear localization of Rad51 in response to DNA damage. Genes Cells 11,513-524.
Mohar l, Szczygiel MA, Yanagimachi R and Ward WS (2002) Sperm nuclear halos can transform into normal chromosomes after injection into oocytes. Mol Reprod Dev 62,416-420.
59 Morales CR, Kwon YK and Hecht NB (1991) Cytoplasmic localization during storage and translation of the mRNAs of transition protein 1 and protamine 1 , two translationally regulated transcripts of the mammalian testis. J Cell Sci 100 (Pt 1),119-131.
Mortillaro MJ, Blencowe BJ, Wei X, Nakayasu H, Du L, Warren SL, Sharp PA and Berezney R (1996) A hyperphosphorylated form of the large subunit of RNA polymerase Il is associated with splicing complexes and the nuclear matrix. Proc Natl Acad Sci USA 93,8253-8257.
Moss SB, Burnham BL and Bellvé AR (1993) The differential expression of lamin epitopes during mouse spermatogenesis. Mol Reprod Dev 34,164-174.
Nadel B, de Lara J, Finkernagel SW and Ward WS (1995) Cell-specific organization of the 5S ribosomal RNA gene cluster DNA loop domains in spermatozoa and somatic cells. Biol Reprod 53,1222-1228.
Nakayasu H and Berezney R (1991) Nuclear matrins: identification of the major nuclear matrix proteins. Proc Natl Acad Sci USA 88,10312-10316.
Nelson JE and Krawetz SA(1994) Characterization of a human locus in transition. J Biol Chem 269,31067-31073.
Nickerson JA, Krochmalnic G, Wan KM and Penman S (1989) Chromatin architecture and nuclear RNA. Proc Natl Acad Sci USA 86,177-181.
Nygaard R, Clausen N, Siimes MA, Marky l, Skjeldestad FE, Kristinsson JR, Vuoristo A, Wegelius Rand Moe PJ (1991) Reproduction following treatment for childhood leukemia: a population-based prospective cohort study of fertility and offspring. Med Pediatr Oncol 19,459-466.
60 Ocampo J, Mondragon R, Roa-Espitia AL, Chiquete-Felix N, Salgado ZO and Mujica A (2005) Actin, myosin, cytokeratins and spectrin are components of the guinea pig sperm nuclear matrix. Tissue Cell 37,293-308.
Oko R, Jando V, Wagner CL, KistlerWS and Hermo LS (1996) Chromatin reorganization in rat spermatids during the disappearance of testis-specific histone, H1t, and the appearance of transition proteins TP1 and TP2. Biol Reprod 54,1141-1157.
Oko R and Morales CR (1994) A novel testicular prote in, with sequence similarities to a family of lipid binding proteins, is a major component of the rat sperm perinuclear theca. Dev Biol 166,235-245.
Oliva Rand Dixon GH (1991) Vertebrate protamine genes and the histone-to- . protamine replacement reaction. Prog Nucleic Acid Res Mol Biol 40,25-94.
Oosterhuis GJ, Hampsink RM, Michgelsen HW and Vermes 1(1996) Hypo osmotic swelling test: a reliable screening assay for routine semen specimen quality screening. J Clin Lab Anal 10,209-212.
~ Palermo G, Joris H, Devroey P and Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340,17-18.
Papoutsopoulou S, Nikolakaki E, Chalepakis G, Kruft V, Chevaillier P and Giannakouros T (1999) SR protein-specific kinase 1 is highly expressed in testis and phosphorylates protamine 1. Nucleic Acids Res 27,2972-2980.
61 Pauli TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M and Bonner WM (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10,886-895.
Penman S (1995) Rethinking cell structure. Proc Natl Acad Sci USA 92,5251- 5257.
Pfeifer H, Conrad M, Roethlein D, Kyriakopoulos A, Brielmeier M, Bornkamm GW and Behne D (2001) Identification of a specific sperm nuclei selenoenzyme necessary for protamine thiol cross-lin king during sperm maturation. FASEB J 15,1236-1238.
Poccia D (1986) Remodeling of nucleoproteins during gametogenesis, fertilization, and early development. Int Rev Cytol 105,1-65.
Pouresmaeili F, Morales CR and Oko R (1997) Molecular cJoning and structural analysis of the gene encoding PERF 15 protein present in the perinucJear theca of the rat spermatozoa. Biol Reprod 57,655-659.
Pu QQ and Bezwoda WR (2000) Alkylator resistance in human B Iymphoid cell lines: (2). Increased levels of topoisomerase 1/ expression and function in a melphalan-resistant B-CLL cell line. Anticancer Res 20,2569-2578.
Qiu J, Hales BF and Robaire B (1992) Adverse effects of cyclophosphamide on progeny outcome can be mediated through post-testicular mechanisms in the rat. Biol Reprod 46,926-931.
Qiu J, Hales BF and Robaire B (1995a) Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure. Biol Reprod 53,1465-1473.
62 Qiu J, Hales BF and Robaire B (1995b) Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa. Biol Reprod 52,33-40.
Qureshi MS, Pennington JH, Goldsmith HJ and Cox PE (1972). Cyclophosphamide therapy and sterility. Lancet 2,1290-1291.
Ratcliffe JM (1994) Paternal exposures and embryonic orfetalloss: the toxicologie and epidemiologic evidence. In Dishan AF and Mattison DR (eds) Male-mediated developmental toxicity. Plenum Press, New York, 185-196.
Razin SV (1987) DNA interactions with the nuclear matrix and spatial organization of replication and transcription. Bioessays 6,19-23.
Robaire B, Codrington AM and Hales BF (2006) Molecular changes in sperm and early embryos afterpaternal exposure to a chemotherapeutic agent. In Anderson D and Brinkworth MH (eds) Male-mediated developmental toxicity. Royal Society of Chemistry, London, ln press.
Robaire B and Hales BF (2003) Mechanisms of action of cyclophosphamide as a male-mediated devèlopmental toxicant. Adv Exp Med Biol 518, 169-180.
Robaire Band Hermo L (1988) Efferent ducts, epididymis, and vas deferens: structure, functions, and their regulation. In Knobil E and Neill JD (eds) The Physiology of Reproduction. Raven Press Ltd., New York, 999-1080.
Robbins WA (2003) FISH (fluorescence in situ hybridi~ation) to detect effects of smoking, caffeine, and alcohol on human sperm chromosomes. Adv Exp Med Biol 518,59-72.
63 Robbins WA, Meistrich ML, Moore D, Hagemeister FB, Weier HU, Cassel MJ, Wilson G,Eskenazi Band Wyrobek AJ (1997) Chemotherapy induces transient sex chromosomal and autosomal aneuploidy in human sperm. Nat Genet 16,74-78.
Roest HP, van Klaveren J, de Wit J, van Gurp CG, Koken MH, Vermey M, van Roijen JH, Hoogerbrugge JW, Vreeburg JT, Baarends WM et al. (1996) Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modification. Cell 86,799- 810.
Rubes J, Lowe X, Moore D, Perreault S, Siott V, Evenson D, Selevan SG and Wyrobek AJ (1998) Smoking cigarettes is associated with increased sperm disomy in teenage men. Fertil Steril70,715-723.
Russell LB (1994) Effects of spermatogenic cell type on quantity and quality of mutations. In Olshan AF and Mattison DR (eds) Male-mediated developmental toxicity. Plenum Press, New York, 37-48.
Russell LB, Hunsicker PR, Kerley MK, Johnson DK and Shelby MD (2000) Bleomycin, unlike other male-mouse mutagens, is most effective in spermatogonia, inducing primarily deletions. Mutat Res 469,95-105.
Russell LD, Ettlin RA, Sinha Hikim AP and Clegg ED (1990) Mammalian .Spermatogenesis. In Russell LD, Ettlin RA, Sinha Hikim AP and Clegg ED (eds) Histological and Histopathological Evaluation of the Testis. Cache River Press, Clearwater, 1-40.
Russell LD, Russell JA, MacGregor GR and Meistrich ML (1991) Linkage of manchette microtubules to the nuclear envelope and observations of the
64 role of the manchette in nuclear shaping during spermiogenesis in rodents. Am J Anat 192,97-120.
Rybouchkin AV, Van der SF, Quatacker J, De Sutter P and Dhont M (1997) Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity. Fertil Steril 68,1144- 1147.
Sakkas D, Manicardi G, Bianchi PG, Bizzaro D and Bianchi U (1995) Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa. Biol Reprod 52,1149-1155.
Sanchez-Pena LC, Reyes BE, Lopez-Carrillo L, Recio R, Moran-Martinez J, Cebrian ME and Quintanilla-Vega B (2004) Organophosphorous pesticide exposure alters sperm chromatin structure in Mexican agricultural workers. Toxicol Appl PharmacoJ 196,108-113.
Savitz DA (1994) Paternal exposures and pregnancy outcome: miscarriage, stillbirth, low birth weight, preterm delivery. In Olshan AF and Mattison DR (eds) Male-mediated developmental toxicity. Plenum Press, New York, 177-184.
Schatten G, Maul GG, Schatten H, Chaly N, Simerly C, Balczon R and Brown DL (1985) Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins. Proc Natl Acad Sci USA 82,4727-4731.
65 Schimenti KJ, Hanneman WH and Schimenti JC (1997) Evidence for cyclophosphamide-induced gene conversion and mutation in mouse germ cells. Toxicol Appl PharmacoI147,343-350.
Schlegel PN, Palermo GD, Alikani M, Adler A, Reing AM, Cohen J and Rosenwaks Z (1995) Micropuncture retrieval of epididymal sperm with in
vitro fertilization: importance of in vitro micromanipulation technique~. Urology 46,238-241.
Schmidt EE and Schibler U (1995) High accumulation of components of the RNA polymerase Il transcription machinery in rodent spermatids. Development 121,2373-2383.
Schrag SD and Dixon RL (1985) Occupational exposures associated with maJe reproductive dysfunction.Annu Rev Pharmacol ToxicoI25,567-592.
Schultz RM (2002) The molecular foundations of the maternai to zygotic transition in the preimplantation embryo. Hum Reprod Update 8,323-331 .
. Schultz RM and Worrad DM (1995) Role of chromatin structure in zygotic gene
activation in the mammalian embryo. Sernin Cell Biol 6,201 ~208.
Sega GA (1991) Adducts in sperm protamine and DNA vs. mutation frequency. Prog Clin Biol Res 372,521-530.
Sega GA, Alcota RP, Tancongco CP and Brimer PA (1989) Acrylamide binding to the DNA and protamine of spermiogenic stages in the mouse and its
relationship to genetic damage. Mutat Res 216,221~230.
66 Sega GA and Owens JG (1983) Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice. Mutat Res 111,227-244.
Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H and Devroey P (1995) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum Reprod 10,148-152.
Sinha Hikim AP and Swerdloff RS (1999) Hormonal and genetic control of germ cell apoptosis in the testis. Rev Reprod 4,38-47.
Sotomayor RE and Cumming RB (1975) Induction of translocations by cyclophosphamide in different germ cell stages of male mice: cytological characterization and transmission. Mutat Res 27,375-388.
Sotomayor RE, Sega GA and Cumming RB (1978) Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen. Mutat Res 50,229-240.
Sousa M and Carvalheiro J (1994) A cytochemical study of the nucleolus and nucleolus-related structures during human spermatogenesis. Anat Embryol (Berl) 190,479-487.
Spiridonov NA, Wong L, Zerfas PM, Starost MF, Pack SD, Paweletz CP and Johnson GR (2005) Identification and characterization of SSTK, a /' serine/threonine protein kinase essential for male fertility. Mol Ce" Biol 25,4250-4261.
Steger K (1999) Transcriptional and translational regulation of gene expression in haploid spermatids. Anat Embryol (Berl) 199,471-487.
67 Struchkov VA, Strazhevskaya NB and Zhdanov RI (2002) Specific natural DNA bound lipids in post-genome era. The lipid conception of chromatin organization. Bioelectrochemistry 56,195-198.
Sun Z, Sassone-Corsi P and Means AR (1995) Calspermin gene transcription is regulated by two cyclic AMP response elements contained in an alternative promoter in the calmodulin kinase IV gene. Mol Cell Biol 15,561-571.
Tanhauser SM and Hecht NB (1989) Nucleotide sequence of the rat protamine 2 gene. Nucleic Aèids Res 17,4395-
Tanphaichitr N, Sobhon P, Taluppeth N and Chalermisarachai P (1978) Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man. Exp Cell Res 117,347-356.
Tesarik J, Mendoza C and Testart J (1995) Viable embryos from injection of round spermatids into oocytes. N Engl J Med 333,525-
Thomson AB, Critchley HO, Kelnar CJ and Wallace WH (2002) Late reproductive sequelae following treatment of childhood cancer and options for fertility preservation. Best Pract Res Clin Endocrinol Metab 16,311-334.
Tice HR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu Je and Sasaki YF (2000) Single cell gel/cornet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35,206-221.
Tovich PR and Oko RJ (2003) Somatic histonl3s are components of the perinuclear theca in bovine spermatozoa. J Biol Chem 278,32431-32438. Tovich PR, Sutovsky P and Oko RJ (2004) Novel aspect of perinucleartheca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis. Biol Reprod 71,1182-1194.
Trasler JM and Ooerksen T (1999) Teratogen update: paternal exposures reproductive risks. Teratology 60,161-172.
Trasler JM, Hales BF and Robaire B (1985) Paternal cyclophosphamide treatment of rats causes fetal loss and malformations without affecting male fertility. Nature 316,144-146.
Trasler JM, Hales BF and Robaire B (1986) Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny. Biol Reprod 34,275-283.
Trasler JM, Hales BF and Robaire B (1987) Atime-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems. Biol Reprod 37,317-326. van Oriel R, Humbel B and de Jong L (1991) The nucleus: a black box being opened. J Cell Biochem 47,311-316.
Virro MR, Larson-Cook KL and Evenson OP (2004) Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperrn injection cycles. Fertil Steril 81,1289-1295.
Vogelstein B, Pardoll OM and Coffey OS (1980) Supercoiled loops and eucaryotic ONA replicaton. Cell 22,79-85.
69 von Bulow M, Heid H, Hess H and Franke WW (1995) Molecular nature of calicin, a major basic protein of the mammalian sperm head cytoskeleton. Exp Cell Res 219,407-413. von Bulow M, Rackwitz HR, Zimbelmann Rand Franke WW (1997) CP beta3, a novel isoform of an actin-binding protein, is a component of the cytoskeletal calyx of the mammalian sperm head. Exp Cell Res 233,216- 224.
Wang JC (1985) DNA topoisomerases. Annu Rev Biochem 54,665-697.
Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3,430-440.
Ward WS (1993) Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa. Biol Heprod 48,1193-1201.
Ward WS and Coffey DS (1990) Specifie organization of genes in relation to the sperm nuclear matrix. Biochem Biophys Res Commun 173,20-25.
Ward WS and Coffey DS (1991) DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells: Biol Reprod 44,569-574.
Ward WS, Kimura Y and Yanagimachi R (1999) An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development. Biol Reprod 60,702-706.
Ward WS, Partin AW and Coffey DS (1989) DNA loop domains in mammalian spermatozoa. Chromosoma 98,153-159.
70 Weinbauer GF and Nieschlag E (1995) Gonadotrophin control of testicular germ cell development. Adv Exp Med Biol 377,55-65.
WHO (1999) WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 4th edn, Cambridge University Press, Cambridge.
Whorton D, Krauss RM, Marshall Sand Milby TH (1977) Infertility in male pesticide workers. Lancet 2,1259-1261.
Whorton D, Milby TH, Krauss RM and Stubbs HA (1979) Testicular function in DBCP exposed pesticide workers. J Occup Med 21,161-166.
Wilcox AJ, Baird DD and Weinberg CR (1999) Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 340,1796-1799.
Winker Rand Rudiger HW (2006) Reproductive toxicology in occupational settings: an update. Int Arch Occup Environ Health 79,1-10.
Witt KL and Bishop JB (1996) Mutagenicity of anticancer drugs in mammalian germ cells. Mutat Res 355,209-234.
Wu JY and Means AR (2000) Ca(2+)/calmodulin-dependent protein kinase IV is expressed in spermatids and targeted to chromatin and the nuclear matrix. J Biol Chem 275,7994-7999.
Wu JY, RibarTJ, Cummings DE, Burton KA, McKnight GS·and Means AR (2000) Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4. Nat Genet 25,448-452.
71 Yanagimachi R (1981) Mechanisms offertilisation in mammals. In Mastroianni L and Biggers JD (eds) Fertilization and embryonic development in vitro. Plenum Press, New York, 81-182.
Yoshinaga K and Toshimori K (2003) Organization and modifications of sperm acrosomal molecules during spermatogenesis and epididymal maturation. Microsc Res Tech 61,39-45.
Yu H, Fukami K, Watanabe Y, Ozaki C and Takenawa T (1998) Phosphatidylinositol 4,5-bisphosphate reverses the inhibition of RNA transcription caused by histone H1. Eur J Biochem 251,281-287.
Yu YE, Zhang Y, Unni E, Shirley CR, Deng JM, Russell LD, Weil MM, Behringer RR and Meistrich ML (2000) Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice. Proc Natl Acad Sci U SA 97,4683-4688.
Zhang Y, Monckton DG, Siciliano MJ, Connor TH and Meistrich ML (2002) Detection of radiation and cyclophosphamide-induced mutations in individual mouse sperm at a human expanded trinucleotide repeat locus transgene. Mutat Res 516,121-138.
Zhao CH, Li QF, Zhao Y, Niu JW, Li ZX and Chen JA (2006) Changes ofnuclear matrix proteins following the differentiation of human osteosarcoma MG-63 cells. Genomics Proteomics Bioinformatics 4,10-17.
Zhao M, Shirley CR, Yu YE, Mohapatra B, Zhang Y, Unni E, Deng JM, Arango NA, Terry NH, Weil MM et al. (2001) Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice. Mol Cell Biol 21,7243-7255.
72 Connecting Text
Chronic cyclophosphamide dosing regimens targeting post-meiotic germ cells result in increased drug-induced DNA damage, as weil as time-specific increases in adverse progeny outcomes. Of interest is whether spermiogenic germ cells are differentially susceptible to damage, therefore accounting for the time-specific effects observed. We used the cornet assay to determine among the different types of spermatids, if the extent of DNA damage incurred depends on when during spermiogenesis spermatids acquire damage. The first study was designed using acute, subchronic and chronic dosing regimens with cyclophosphamide to assess spermiogenic germ cell phase-specifie DNA damage.
73 CHAPTER 2
Spermiogenic Germ Cell Phase-Specifie DNA Damage
following Cyclophosphamide Exposure
Alexis M. Codrington, Barbara F. Hales and Bernard Robaire
Published in Journal of Andrology J Andro/; 25(3):354-362, 2004
74 2.1 ABSTRACT
The production of genetically competent spermatozoa is essential for normal embryo development. The chemotherapeutic drug cyclophosphamide creates cross-links and DNA strand breaks in many ceU types, including germ cells. This study assessed the phase specificity of the susceptibility of spermiogenic germ cells to genetic damage induced by cyclophosphamide. Adult male rats were given cyclophosphamide using one of four schedules: 1) high dose/acute- day 1,100 mg/kg; 2) low dose/subchronic, 4 days-days 1--4, 6.0 mg/kg/d; 3) high dose/subchronic, 4 days-day 1, 100 mg/kg, and days 2--4, 50 mg/kg/d; and 4) low dose/chronic-daily, 6.0 mg/kg/d for 14-28 days. To capture cauda epididymal spermatozoa exposed to cyclophosphamide during late, mid-, and early spermiogenesis, animais were sacrificed on days 14, 21, and 28, respectively. Spermatozoa were analyzed for DNA strand breaks using the cornet assay. No dramatic increases in damage were seen after high-dose/acute exposure to cyclophosphamide. Subchronic exposure showed a dose-related increase in DNA damage; maximal damage, as demonstrated by cornet tail parameters, was seen after 21 days, reflecting an increased susceptibility of step
9-14 spermatids. Low-dose chronic exposure to cyclophosphamide induced DNA damage, which reached a plateau by day 21. The magnitude of damage at ail time points after low-dose chronic exposure was much greater than that following low-dose exposure for 4 days, indicating an accumulation of damage over time.
Thus, the DNA damage induced by cyclophosphamide is germ cell phase specific. The most damaging effects of cyclophosphamide occurred during a key point of sperm chromatin remodeling (histone hyperacetylation and transition
75 protein deposition). We speculate that strand breaks disrupt chromatin remodeling, hence affecting chromatin structure and embryo development.
2.2 INTRODUCTION
Society has growing concerns about the consequences of exposure to drugs and toxicants on the reproductive system, germ ceUs, fertilization, and development. Preconceptional exposure to drugs, radiation, or chemicals results in embryo loss, birth defects, and predisposition to cancer (Dlshan and Matlison,
1994; Brinkworth, 2000; Robaire and Hales, 2003). Paternal exposure to toxicants may cause changes in sperm quality, thereby contributing to infertility and adverse progeny outcomes. Evidenceis now accumulating about the importance to embryogenesis of genetically competent spermatozoa during both natural and assisted fertilization. Several authors have reported significant correlations between sperm DNA damage and effects on fertilization as weil as embryo
cleavage and pregnancy rates (Sun et al, 1997; Lopes et al, 1998b; Larson et al,
2000; Morris et al, 2002).
Among the commonly used anticancer drugs, alkylating agents have been
associated most often with the development of infertility (Schilsky, 1980; Pont,
1997). Cyclophosphamide is a cytotoxic alkylating agent widely used in
chemotherapeutic regimens. Its cytotoxic effects are the result of chemically
reactive metabolites that create DNA adducts, DNA-DNA and DNA-protein
crosslinks, sister chromatid exchanges, chromosomal aberrations, and DNA
strand breaks (Sotomayor and Cumming, 1975; Bishop et al, 1997). Exposure of
male germ ceUs to cyclophosphamide leads to single-strand breaks, crosslinks,
76 and altered in vitro spermatozoal decondensation and template function (Qiu et al, 1995a,b). With little effect on the male reproductive system, chronic paternal treatment of rats with cyclophosphamide results in increases in embryo death and growth-retarded and malformed fetuses (Trasler et al, 1985, 1986, 1987;
Jenkinson and Anderson, 1990). Although proliferating premeiotic germ cells are sensitive to alkylating agents, nondividing postmeiotic spermatids and spermatozoa are the most susceptible to DNA damage, leading to dominant lethality, heritable translocations, specifie locus mutations, and malformations
(Jackson, 1964; Ehling et al, 1968). Certain monofunctional alkylating agents, such as methyl methanesulfonate and ethylene oxide, produce more dominant lethal mutations and translocations in late spermatids and early spermatozoa than in any other germ cell stage (Ehling, 1980; Generoso et al, 1980). In contrast, other chemicals, such as eth yi nitrosourea and methyl nitrosourea, show no germ cell stage-specific selection, with a wide range of effects on both pre- and postmeiotic germ cells (Russell et al, 1979; Sega et al, 1981). Cross-linking agents are also active throughout spermatogenesis; however, maximal genetic damage has been observed in early round spermatids (Russell, 1989, 1992).
Dramatic increases inpostimplantation loss are dose-dependent and maximal
after a 3-week treatment of male rats with a low dose of cyclophosphamide
(Trasler et al, 1985, 1986, 1987), which thereby exposes germ cells during
spermiogenesis and sperm maturation.
Abnormal sperm chromatin packaging, identified as poorly protaminated
spermatozoa or a high susceptibility of DNA to acid-induced denaturation using
the sperm chromatin structure assay, has been correlated with the presence of
77 DNA strand breaks (Gorczyca et al, 1993; Manicardi et al, 1995, 1998; Sai 1er et al, 1995). Mature spermatozoa contain highly packaged chromatin organized in a specific manner to allow access to required genetic information during embryogenesis. The proposed model for sperm DNA packaging consists of several levels of organization, including DNA loop domains attached to the nuclear matrix in a sequence-specific manner (Ward and Coffey, 1991; Ward,
1993) and protamine DNA binding (Balhom, 1982). With such an intricate organization of the sperm nucleus, the possibility exists that damage to the DNA will, in tum, affect overall nuclear organization. Continuous treatment with cyclophosphamide for 4 weeks targets the male germ cell through ail stages of spermiogenesis and sperm maturation. The adverse progeny outcomes associated with patemal exposure to this drug may be due to the accumulation of
DNA damage in male germ ce Ils as round spermatids develop into spermatozoa or due to spermiogenic germ cell phase-specific susceptibility to damage.
Genotoxic damage in the form of single- and double-stranded breaks can be measured using the cornet assay. This assay is used extensively to assess
DNA damage in somatic cells and has been applied to human and murine spermatozoa (Singh et al, 1989; Haines et al, 1998; Tice et al, 2000). Increased
DNA damage in human spermatozoa measured by the cornet assay is associated with infertility (Irvine et al, 2000) and exposure to chemotherapeutic agents
(Chatterjee et al, 2000). In this study, we have developed the cornet assay under alkaline conditions for rodent spermatozoa. We have used this assay to determine to what extent and at which point during spermiogenesis sperm acquire
78 DNA damage after acute, subchronic, and chronic dosing regimens with cyclophosphamide.
2.3 MATE RIALS AND METHODs
Animal Treatments
Adult male Sprague-Dawley rats (400-450 g) were obtained from Charles
River Canada (St Constant, Canada), maintained on a 14:10 light-dark cycle, and provided with food and water ad libitum. Rats were gavaged with saline or cyclophosphamide (CAS 6055-19-2; Sigma-Aldrich, Oakville, Canada) using one offour schedules: 1) high dose/acute-day 1, 100 mg/kg; 2) low dose/subchronic,
4 days-days 1-4, 6.0 mg/kg/d; 3) high dose/subchronic, 4 days-day 1, 100 mg/kg, and days 2-4,50 mg/kg/d; and 4) low dose/chronic-daily, 6.0 mg/kg/d for
14-28 days. To capture spermatozoa tirst exposed to cyclophosphamide during late, mid-, and early spermiogenesis, animais were sacrificed by decapitation on days 14, 21, and 28 after the initiation of treatment (Clermont, 1972). Animal handling and care were performed in accordance with the guidelines established by the Canadian Council on Animal Care.
Sperm Collection
To isolate spermatozoa from the cauda epididymides, the epididymides were first removed, trimmed free of fat, and washed in 2 ml of prewarmed (37°C) 10 mM
Tris-HCI buffer containing 50 mM NaCI and 50 mM EGTA, pH 8.2-8.4. This medium maintains the genetic integrity of frozen spermatozoa (Kusakabe et al,
2001). The corpus-cauda epididymal junction was then clamped with a hemostat,
79 and an incision was made in the distal cauda epididymidis. Spermatozoa released from the site of incision were collected in 2 ml of media at 3rC, incubated for 5 minutes to allow the spermatozoa to disperse, and then diluted
1: 10 in fresh media. Aliquots of 100 JJl were stored at -80°C until comet analysis was performed.
Detection of Damage in Individual Gells Using the Gomet Assay
DNA damage in spermatozoa was evaluated using the comet assay as previously described (Singh et al, 1989; Haines et al, 1998) with the following modifications. Frozen sperm samples were thawed on ice and resuspended in
Tris-HCI-buffered EGTA medium to a concentration of 1 x 105 ceUs/mL. Fifty microliters of the cell suspension was added to 500 JJl of molten aga rose (0.5% low-melting-point grade in Mg2+ and Ca2+-free phosphate-buffered saline, pH
7.4, at 42°C). Sixt Y microliters was immediately pipetted and evenly spread onto slides (Trevigen Inc, Gaithersburg, Md), and the gel was allowed to solidify at 4°C in the dark for 10 minutes. Ali subsequent steps were performed under yellow light or in the dark to prevent any additional DNA damage.
Siides were immersed in prechilled (4°C) Iysis buffer (2.5 M NaCI, 100 mM
EDTA, and 10 mM Tris-HCI; final pH, 10) containing 10% dimethylsulfoxide, 1%
Triton X-100, and 40 mM dithiothreitol for 1 hour on ice. Following initial Iysis, slides were washed in distilled water for 5 minutes. Proteinase K, at a concentration of 0.1 mg/ml, was added to fresh prewarmed Iysis buffer, and additionallysis was performed for 3 hours at 3rC. Siides were washed in distilled water, placed fiat at 4°C for 10 minutes to reset the agarose, and then immersed
80 in freshly prepared alkaline solution (1 mM EDTA and 0.05 M NaOH, pH 12.1)for
45 minutes in the dark. Siides were washed twice in 1x Tris-Borate-EDTA buffer
, (TBE, pH 7.4) for 5 minutes and th en placed equidistant from the electrodes on a
gel tray submerged in TBE in a horizontal electrophoresis apparatus (Mini-Sub
Cell GT; Bio-Rad Laboratories, Inc, Mississauga, Canada). The level of TBE was
2-4 mm above the slides. Electrophoresis was carried out at 14 V (0.7 V/cm) for
10 minutes. Siides were drained and fixed in ice-cold 70% ethanol for 5 minutes
and left to air dry prior to storage with desiccant at room temperature. Similar to
the standard alkaline cornet assay defined by Singh et al (1988), treatment with
alkali to unwind and denature the DNA followed by electrophoresis in neutral
buffer permits the detection of single-strand breaks. These conditions, however,
tend to give lower background levels of DNA damage, result in betler dose
responses, and avoid saturating the assay so that estimation of damage is over a
wider range (Koppen and Angelis, 1998; Angelis et al, 1999).
DNA was stained with 50 !-IL of SYBR Green solution (Trevigen) diluted
1:10 000 in Tris-EDTA buffer, pH 7.5, and immediately analyzed at 200x
magnification using a DAGE-MTI CCD300-RC camera (DAGE-MTI Inc, Michigan
City, Ind) atlached to an Olympus BX51 epifluorescence microscope. Fifty cells
were randomly analyzed per slide for a total of 100 cells per animal, and
fluorescent images were scored for cornet parameters. Tail length, percent tail
DNA, and tail moment (tail length x fraction of tail DNA) were measured using the
KOMET 5.0 image analysis system (Kinetic Imaging Ltd, Liverpool, United
Kingdom).
81 Slalistical Analysis
Significant differences due to drug treatment or time of drug exposure were determined using a 2-way analysis of variance followed by the Bonferroni post hoc test (P < .05). The percent abnormal sperm among populations of spermatozoa from saline- and cyclophosphamide-treated animais was compared by chisquare analysis. Statistical analyses were performed by the SigmaStat 2.03 software package (SPSS Inc, Chicago, III).
2.4 RESULTS
ln the alkaline comet assay, an increase in DNA strand breaks leads to greater DNA migration out of the nucleus and into the tait of the comet. The majority of control spermatozoa had little if any cornet tail (Figure 2-1A).
Differences in tait length and tail intensity were observed. among cyclophosphamide-exposed sperm populations (Figure 2-1 B through D). The extent of damage was dependent on dose as weil as on time and length of exposure to cyclophosphamide.
Acufe 1-Day Exposure
Exposure of male germ cells to a single high dose of cyclophosphamide resulted
in time-specific increases in DNA damage (Figure 2-2). Small yet significant differences were observed only in the percentage of tail DNA values 14 days after
drug administration (1.4-fold increase) and in taillength values after 14 days (1.2-
fold increase) and 21 days (1.7-fold increase). On the integration of these 2
comet parameters to obtain the tail moment, significant differences between drug-
82 exposed and time-matched controls were observed after 14 days (1.6-fold) and
21 days (2.2-fold). Acute exposure to cyclophosphamide did not alter sperm DNA integrity after 28 days; this may indicate either a decrease in susceptibility to damage or the presence of repair in early spermiogenic germ cells.
Subchronic 4-0ay Exposure
Short-term repeat exposure to cyclophosphamide resulted in dose- and time-dependent increases in DNA damage (Figure 2-3). Sy day 14, no significant differences were seen between sperm exposed to low doses of cyclophosphamide and control sperm for any cornet parameters. Administration of a higher dose, however, resulted in significant differences in tait length (1.6- fold) and tail moment (2.3-fold). Drug-exposed spermatozoa collected on day 21 showed the highest levels of DNA strand breaks detected at any time point.
Measurements of cornet tail moment after exposure to low or high cyclophosphamide doses were 3.3- and 9.4-fold higher than for the control, respectively (Figure 2-3C). Similar changes were observed for the percentage of tait DNA and tait length; however, the magnitudes were not as great (Iow dose:
2.4- and 2.2-fold increases, respectively; high dose: 3.8-fold increase for both
parameters). Spermatozoa collected on day 28 had relatively lower levels of DNA
strand breaks than spermatozoa collected on day 21.
Chronic Exposure
Significant increases in the percentage of tait DNA, tait length, and tail
moment were detected at ail time points after chronic exposure to low doses of
83 cyclophosphamide (Figure 2-4). There were significant yet very small differences in the percent tail DNA for cyclophosphamide-exposed sperm populations at the 3 time points. In contrast, the mean tail length showed a significant increase in damage from day 14 (80.1 J,Jm) to day 21 (102.8 J,Jm) and then a slight decrease by day 28 (94.2 J,Jm). Tail moments showed a 1.9-fold increase in DNA damage after 14 days; damage was maximal by 21 days of chronic cyclophosphamide exposure (3.4-fold increase). In comparison to the mean tail moments observed after low-dose subchronic exposure (Figure 2-3C), the mean tail moment values for each time point were much higher after chronic drug exposure, indicating an accumulation of cyclophosphamide-induced damage during the entire course of
. drug administration. This increase in damage appeared to reach a plateau by day
21, as there was no difference between the fold increases in damage for days 21 and 28 (3.4- and 3.5-fold increases, respectively).
Distribution of Sperm from Low to High Damage Groups
Frequency histograms of tail moment represent the distribution of sperm damage. Tail moments for drug-exposed spermatozoa had a heterogeneous
distribution pattern, ranging from 0 to 100, rather than discrete populations of low
and high DNA damage. Differences in the percentage of sperm with increasing
amounts of damage were, however, still noticeable. An overwhelming majority
(~94%) of tail moments for control animais ranged from 0 to 15; therefore, in
comparison, the percentage of abnormal sperm with tail moments greater than 15
in drug-treated populations was calculated (Figure 2-5).
84 Signiticant increases in the percentage of abnormal sperm were seen at ail time points among the cyclophosphamide-treated group in the high-doselacute study (Figure 2-5A). By day 14, 12% of sperm were damaged, while by day 21,
10% of sperm had tail moments above 15. Note that although no significant difference was found between tail moment means of drug-exposed and control sperm at day 28 (Figure 2-2C), there was still a higher number (14%) of sperm from cyclophosphamide..:treated animais with abnormal DNA. For the subchronic cyclophosphamide study, the highest percentage of abnormal sperm was found within the population of cells collected after 21 days for both low and high cyclophosphamide doses (12% and 32%, respectively; Figure 2-5B). In the day
28 high-dose cyclophosphamide-exposed sperm, signiticant differences were found, even though the tail moment mean was not significantly different from controls. At days 14 and 28, only sperm collected after high-dose cyclophosphamide administration had a significantly higher than control percentage of sperm with increased DNA damage.
Chronic low-dose administration of cyclophosphamide resulted in a
signiticant increase in the percentage of abnormal sperm compared to controls
(Figure 2-5C). Only 13% of sperm presented with ab normal DNA (2.6-fold
increase) by day 14. Cyclophosphamide-induced damage accumulated in 22% of the germ cells that were tirst exposed during mid-spermiogenesis and collected
21 days later (4.1-fold increase). The percentage of abnormal sperm did not
further increase by day 28.
85 2.5 DISCUSSION
Using alkaline elution, previous studies (Qiu et al, 1995b) have shown that
6 weeks of chronic cyclophosphamide treatment significantly increases both DNA single-strand breaks and cross-links in spermatozoa. Chronic exposure to cyclophosphamide, targeting spermatids and epididymal spermatozoa, resulted in increases in peri-implantation embryo loss and fetal growth retardation and malformations (Trasler et al, 1985, 1986, 1987). The goal of this study was to elucidate the extent of DNA damage in spermatozoa induced by cyclophosphamide at specific spermiogenic time points. The cornet assay is a sensitive measure· of DNA damage in individual sperm nuclei, thus making it possible to determine whether or not ail cells within a population demonstrate the same degree of damage. Only a few studies have used the cornet assay to assess genotoxic damage to male germ cells after in vivo exposure to a testicular genotoxicant (Anderson et al, 1996; Haines et al, 2001,2002).
The germ cell phase most sensitive to the induction of mutations by cyclophosphamide was the early spermatid phase (Sotomayor and Cumming,
1975). As measured by the cornet assay, the mid-spermiogenic germ cells were most susceptible to DNA damage after cyclophosphamide exposure. These data, and those of Haines et al (2002), on germ ceU phase-specific susceptibility to radiation damage suggest that the cornet assay is not predictive of subtle mutational changes. However, high amounts of DNA damage as detected by the cornet assay have been associated with the failure of embryo development after
intracytoplasmic sperm injection (Morris et al, 2002) and with infertility in humans
(Irvine et al, 2000). The DNA damage measured by the cornet assay may also
86 provide an accu rate reflection of adverse reproductive outcome after chronic low dose administration of cyclophosphamide to male rats (Trasler et al, 1985, 1986,
1987).
Cyclophosphamide exposure resulted in dose-dependent and time-specific increases in the levels of DNA damage; as dose and exposure time increased, so did the fraction of cells with DNA damage. Acute exposure to a high dose of cyclophosphamide did not create any major elevations in damage, and the damage that was noted was readily reversibJe. Step 9 and 15 spermatids collected as spermatozoa on days 21 and 14, respectively, incurred only low levels of damage. Acute cycJophosphamide exposure affected gene expression most dramatically in round spermatids; the expression of DNA repair genes was induced (Aguilar-Mahecha et al, 2001). These data suggest that round spermatids attempt to compensate for DNA damage induced by the drug. Neither acute nor subchronic cyclophosphamide exposure resulted in a significant increase in damage in germ cells exposed to the drug as round spermatids
(acute: step 1 spermatids, subchronic: steps 1-5 spermatids). Postmeiotic spermatids do not undergo apoptosis (Cai et al, 1997; Sinha Hikim and Swerdloff,
1999; Brinkworth and Nieschlag, 2000); however, early to mid-spermatids do have the ability to repair DNA lesions (Sega, 1976; Sotomayor et al, 1978) that may give ri se to DNA strand breaks. The ultimate effect of cyclophosphamide on germ cens will depend on both the extent of damage and their ability to undergo
DNA repair. While there was no significant increase in ove ra Il damage at this stage, there was still a discrete population of ab normal sperm presenting increased levels of damage, and these cells may be capable of affecting progeny.
87 Repeat exposure to cyclophosphamide was required for higher levels of damage to occur. Subchronic treatment of male rats produced maximal damage in elongating spermatids exposed to the drug from stages 9 to 14. Chronic administration of cyclophosphamide resulted in the greatest accumulation of damage over time; the damage reached a plateau after 21 days of drug treatment, at a time point when germ cells exposed to cyclophosphamide are developing from elongating spermatids (steps 9-19) to mature spermatozoa.
Cyclophosphamide may exert its maximal effect on elongating spermatids and spermatozoa, since these cells have lost the ability to repair DNA and undergo apoptosis.
Differences in susceptibility to genetic damage between mid- (steps 9-14) and late (steps 15-19) spermatids may be due to chromatin structural changes during spermiogenesis. The structural organization of DNA in the sperm may determine the participation of the paternal genome in embryo development.
During spermiogenesis, mammalian sperm DNA is reorganized into loop domains attached at specific sites to the nucJear matrix (Ward and Coffey, 1991; Ward,
1993). The unique chromatin architecture that results may be required to facilitate scheduled transcription after fertilization. DNase-l-hypersensitivity regions have been localized to transcriptionally active or potentially active genes near nucJear matrix attachment sites (Gross and Garrard, 1988). The pattern of DNase-l
hypersensitivity regions changes during spermiogenesis; step 12-13 elongating
spermatids are the most sensitive to enzymatic action (McPherson and Longo,
1992). This stage of spermiogenesis may represent a point at which the male
genome is more accessible to insult. In comparison, alkylating agents
88 preferentially damage DNA regions that are in close proximity to matrix-bound replication and transcription sites (Muenchen and Pienta, 1999). The template function of spermatozoal DNA was markedly affected after 6 weeks of treatment with cyclophosphamide (Qiu et al, 1995b). During spermiogenesis, certain gene loci may be more susceptible to cyclophosphamide alkylation; we hypothesize that these genes play an important role in regulating early embryo development.
Final packaging of the sperm DNA occurs as histone acetylation and ubiquitination increase in mid-spermiogenic spermatids to allow transition proteins to bind to the DNA. These transition proteins then facilitate the preferential binding of protamines in late spermatids to condense the chromatin (Poccia,
1986; Wouters-Tyrou et al, 1998). Endogenous nicks in DNA, present normally in step 12-13 rat spermatids, are ligated before the completion of protamination
(McPherson and Longo 1992, 1993a,b; Sakkas et al, 1995). These nicks may be present to facilitate protamination by providing relief of tortional stress
(McPherson and Longo, 1992). Improper ligation of these breaks may result in altered chromatin structure and residual DNA strand breaks in spermatozoa
(Sailer et al, 1995; Caron et al, 2001; Kierszenbaum, 2001). The presence of
DNA damage in mature spermatozoa has been correlated with poor chromatin packaging (Gorczyca et al, 1993; Sailer, 1995; Manicardi et al, 1998), possibly due to underprotamination (Manicardi et al, 1995; Sakkas et al, 1996).
Cyclophosphamide may affect the relationship between protamination and endogenous nick creation and ligation. Thus, cyclophosphamide maximally damages DNA at its most vulnerable state, during mid-spermiogenesis, when nucleoproteins are involved in the histone-protamine exchange.
89 Decreased iodoacetamide binding, indicating decreased reducible sulfhydryl content of sperm nuclei, was obser\ted in cyclophosphamide-exposed germ ceUs, suggesting that protamine is affected in these cells (Qiu et al, 1995a).
Effects on reducible sulfhydryl content may be due to either incomplete protamine deposition or an increase in alkylation of protamine sulfhydryl groups. Protamines in the testis may be especially susceptible to alkylation; if so, the end result would be blockage of normal disulfide bond formation, thus preventing proper chromatin condensation. This could lead to stress in the chromatin structure and result in
DNA strand breaks (Sega and Owens, 1983). Fertilization with damaged or poor quality sperm may le ad to aberrant pronuclear development (Lopes et al, 1998a).
Indeed, male pronulear formation was early in rat oocytes sired by
cyclophosphamide-treated males (Harrouk et al, 2000b). Sega and Owens (1983)
have paralleled patterns of alkylation of protamine in late spermatids and early
spermatozoa to the occurrence of dominant lethal mutations.
ln summary, cyclophosphamide-induced DNA damage accumulates over
time, with the most damaging effects occurring during mid-spermiogenesis. The
step 9-14 spermatids appear to be most susceptible to DNA damage; this
represents a key time point in sperm chromatin remodeling (histone acetylation
and transition protein deposition). Fertilization can be achieved using damaged
spermatozoa; however, proper embryo development depends on spermatozoal
genetic integrity (Ahmadi and Ng, 1999). In previous studies, using the comet
assay, we have shown that cyclophosphamide-exposed spermatozoa imparted
significantly greater DNA damage to fertilized eggs at the single-cell stage than
did control spermatozoa (Harrouk et al, 2000a). Given the results of the present
90 study, we speculate that cyclophosphamide disrup~s chromatin remodeling, hence affecting sperm chromatin structure and embryo development.
91 REFERENCES
Aguilar-Mahecha A, Hales BF, Robaire B. Acute cyclophosphamide exposure has germ cell specifie effects on the expression of stress response genes during rat spermatogenesis. Mol Reprod Dev. 2001 ;60:302-311.
Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool. 1999;284:696-704.
Anderson D, Dhawan A, Yu TW, Plewa MJ. An investigation of bone marrow and testicular cells in vivo using the cornet assay. Mutat Res. 1996;370: 159-174.
Angelis KJ, Dusinska M, Collins AR. Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity. Electrophoresis. 1999;20:2133- 2138.
Balhom R. A model for the structure of chromatin in mammalian sperm. J Cell Biol. 1982;93:298-305.
Bishop JB, Witt KL, Sioane RA. Genetic toxicities of human teratogens. Mutat Res. 1997;396:9-43.
Brinkworth MH. Patemal transmission of genetic damage: findings in animais and humans. Int J Androl. 2000;23:123-135.
Brinkworth MH, Nieschlag E. Association of cyclophosphamide-induced male mediated, foetal abnormalities with reduced patemal germ-cell apoptosis. Mulal Res. 2000;447:149-154.
92 Cai L, Hales BF, Robaire B. Induction of apoptosis in the germ cells of adult male rats after exposure to cyclophosphamide. Biol Reprod. 1997;56:1490-1497.
Caron N, Veilleux S, Boissonneault G. Stimulation of DNA repair by the spermatidal TP1 protein. Mol Reprod Dev. 2001 ;58:437-443.
Chatterjee R, Haines GA, Perera DM, Goldstone A, Morris ID. Testicular and sperm DNA damage after treatment with tludarabine for chronic Iymphocytic leukaemia. Hum Reprod. 2000;15:762-766.
Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev. 1972;52: 198-236.
Ehling UH. Induction of gene mutations in germ cells of the mouse. Arch Toxicol. 1980;46:123-138.
Ehling UH, Cumming RB, Malling HV. Induction of dominant lethal mutations by alkylating agents in male mice. Mutat Res. 1968;5:417-428.
Generoso WM, Cain KT, Krishna M, Sheu CW, Gryder RM. Heritable translocation and dominant-Iethal mutation induction with ethylene oxide in mice. Mutat Res. 1980;73:133-142.
Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z. Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis of somatie cells. Exp Cell Res. 1993;207:202-205.
Gross OS, Garrard WT. Nuclease hypersensitive sites in chromatin. Annu Rev Biochem. 1988;57:159-197.
93 Haines GA, Hendry JH, Daniel CP, Morris ID. Increased levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or X-irradiation of spermatogonia. Mutat Res. 2001;495:21-32.
Haines GA, Hendry JH, Daniel CP, Morris ID. Germ cell and dose-dependent DNA damage measured by the cornet assay in murine spermatozoa after testicular X-irradiation. Biol Reprod. 2002;67:854-861.
Haines G, Marples 8, Daniel P, Morris 1. DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the cornet assay. Adv Exp Mê'd Biol. 1998;444:79-91.
Harrouk W, Codrington A, Vinson R, Robaire 8, Hales 8F. Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo. Mutat Res. 2000a;461 :229- 241.
Harrouk W, Khatabaksh S, Robaire 8, Hales 8F. Paternal exposure to cyclophosphamide dysregulates the gene activation program in rat preimplantation embryos. Mol Reprod Dev. 2000b;57:214-223.
Irvine OS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ. DNA integrity in human spermatozoa: relationships with semen quality. J Androl. 2000;21 :33- 44.
Jackson H. The Effects of Alkylating Agents on Fertility. Br Med Bull. 1964;20:107-114.
Jenkinson PC, Anderson D. Malformed foetuses and karyotype abnormalities in the offspring of cyclophosphamide and allyl alcohol-treated male rats. Mutat Res. 1990;229: 173-184.
94 Kierszenbaum AL. Transition nuclear proteins during spermiogenesis: unrepaired DNA breaks not allowed. Mol Reprod Dev. 2001 ;58:357-358.
Koppen G, Angelis KJ. Repair of X-ray induced DNA damage measured by the cornet assay in roots of Vicia faba. Environ Mol Mutagen. 1998;32:281-285.
Kusakabe H, Szczygiel MA, Whittingham DG, Yanagimachi R. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc Nat! Acad Sei USA. 2001;98:13501-13506.
Larson KL, DeJonge CJ, Bames AM, Jost LK, Evenson OP. Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod. 2000;15:1717-1722.
Lopes S, Jurisicova A, Casper RF. Gamete-specific DNA fragmentation in unfertilized human oocytes after intracytoplasmic sperm injection. Hum Reprod. 1998a;13:703-708.
Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Feriil Steril. 1998b;69:528-532.
Manicardi GC, Bianchi PG, Pantano S, Azzoni P, Bizzaro D, Bianchi U, Sakkas D. Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biol Reprod. 1995;52:864-867.
95 Manicardi GC, Tombacco A, Bizzaro D, Bianchi U, Bianchi PG, Sakkas D. DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays. Hislochem J. 1998;30:33-39.
McPherson SM, Longo FJ. Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev. 1992;31 :268-279.
McPherson SM, Longo FJ. Chromatin structure-function alterations during mammalian spermatogenesis: DNA nicking and repair in elongating spermatids. Eur J Hislochem. 1993a;37:109-128.
McPherson SM, Longo FJ. Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II. Dev Biol. 1993b;158:122-130.
Morris ID, 1I0tt S, Dixon L, Brison DR. The spectrum of DNA damage in human sperm assessed by single cel! gel electrophoresis (Cornet assay) and its relationship to fertilization and embryo development. Hum Reprod. 2002; 17:990-998.
Muenchen HJ, Pienta KJ. The role of the nuclear matrix in cancer chemotherapy. Gril Rev Eukaryol Gene Expr. 1999;9:337-343.
Olshan AF, Mattison DR, eds. Male Medialed Deve/opmenlal Toxicily. New York: Plenum Press; 1994.
Poccia D. Remodeling of nucleoproteins during gametogenesis, fertilization, and early development. Inl Rev Gy/o/. 1986; 105: 1-65.
96 Pont J, Albrecht W. Fertility after chemotherapy for testicular germ cell cancer. Fertil Stenl. 1997;68: 1-5.
Qiu J, Hales BF, Robaire B. Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure. Biol Reprod. 1995a;53:1465-1473.
Qiu J, Hales BF, Robaire B. Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa. Biol Reprod. 1995b;52:33-40.
Robaire B, Hales BF, eds. Advances in Male-Mediated Developmental Toxieity. New York: Kluwer AcademiclPlenum Press; 2003.
Russell LB, Hunsicker PR, Cacheiro NL, Bangham JW, Russell WL, Shelby MD. Chlorambucil effectively induces deletion mutations in mouse germ cells. Proe Natl Aead Sei USA. 1989;86:3704-3708.
Russell LB, Hunsicker PR, Shelby MD. Melphalan, a second chemical for which specific-Iocus mutation induction in the mouse is maximum in early spermatids. Mutat Res. 1992;282:151-158.
Russell WL, Kelly EM, Hunsicker PR, Bangham JW, Maddux SC, Phipps EL. Specific-Iocus test shows ethylnitrosourea to be the most pote nt mutagen in the mouse. Proe Natl Aead Sei USA. 1979;76:5818-5819.
Sailer BL, Jost LK, Evenson OP. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. J Androl. 1995;16:80-87.
97 Sakkas D, Manicardi G, Bianchi PG, Bizzaro D, Bianchi U. Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa. Biol Reprod. 1995;52:1149- 1155.
Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner l, Jaquenoud N, Manicardi G, Campana A. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod. 1996;11 :837-843.
Schilsky RL, Lewis BJ, Sherins RJ, Young RC. Gonadal dysfunction in patients receiving chemotherapy for cancer. Ann Intem Med. 1980;93: 109-114.
Sega GA. Molecular dosimetry of chemical mutagens. Measurement of molecular dose and ONA repair in mammalian germ cells. Mutat Res. 1976;38:317-326.
Sega GA, Owens JG. Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice. Mutat Res. 1983; 111 :227- 244.
Sega GA, Wolfe KW, Owens JG. A comparison of the molecular action of an SN1-type methylating agent, methyl nitrosourea and an SN2-type methylating agent, methyl methanesulfonate, in the germ cells of male mice. Chem Biol Interact. 1981 ;33:253-269.
Singh NP, OannerOB, Tice RR, McCoy MT, Collins GD, Schneider EL. Abundant alkali-sensitive sites in ONA of human and mou se sperm. Exp Cell Res. 1989;184:461-470.
Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of ONA damage in individual cells. Exp Cell Res. 1988; 175: 184-91.
98 Sin ha Hikim AP, Swerdloff RS. Hormonal and genetic control of germ cel! apoptosis in the testis. Rev Reprod. 1999;4:38-47.
Sotomayor RE, Cumming RB. Induction of translocations by cyclophosphamide in different germ cel! stages of male mice: cytological characterization and transmission. Mutat Res. 1975;27:375-388.
Sotomayor RE, Sega GA, Cumming RB. Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen.Mutat Res. 1978;50:229-240.
Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod. 1997;56:602-607.
Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/cornet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35:206-221.
Trasler JM, Hales BF, Robaire B. Paternal cyclophosphamide treatment of rats causes fetal loss and malformations without affecting male fertility. Nature. 1985;316: 144-146.
Trasler JM, Hales BF, Robaire B. Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny. Biol Reprod. 1986;34:275-283.
Trasler JM, Hales BF, Robaire B. A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems. Biol Reprod. 1987;37:317-326.
99 Ward WS. Oeoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa. Biol Reprod. 1993;48:1193-1201.
Ward WS, Coffey OS. ONA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod. 1991;44:569-574.
Wouters-Tyrou D, Martinage A, Chevaillier P, Sautiere P. Nuclear basic proteins in spermiogenesis. Biochimie. 1998;80: 117 -128.
100 FIGURES and LEGENDS
101 Figure 2-1. Images of spermatozoal nuclei following the cornet assay. (A) Control sperm; (8-0) cyclophosphamide-exposed sperm. Ali images are at 200x magnification. Scale bar = 40lJm.
102
Figure 2-2. DNA damage assessed by the cornet assay in epididymal spermatozoa on days 14, 21 and 28 after acute 1-day cyclophosphamide exposure. Effects measured as (A) % tail DNA, (8) tai! length and (C) tail moment are shown for saline (open bars) and cyclophosphamide-exposed (crosshatched bars) sperm populations. The data shown represent means ± SEM (n= 3). *Significantly different from time-matched controls (P<0.05).
104 ACUTE EXPOSURE A 25 « 20 5 15 * ~ 10 5
o ~----~~~~~--~~--~~~~----- Day 14 Day 21 Day 28 Days Post-Treatment
B 120 Ê 100 3; 80 .c..... * 0)c: 60 Q) -' 40 cu ~ 20
o ~----~~~~~--~~--~~~~----- Day 14 Day 21 Day 28 Days Post-Treatment
C 25 ..... 20 c: Q) E 15 o ~ 10 5
o ~----~~~~~--~~--~~~~----- Day 14 Day 21 Day 28 Days Post-Treatment Figure 2-3. DNA damage assessed by the cornet assay in epididymal spermatozoa on days 14, 21 and 28 after sub-chronic 4-day cyclophosphamide exposure. Effects measured as (A) % tail DNA, (8) tai! length and (C) tail moment are shown tor saline (open bars), low-dose cyclophosphamide (crosshatched bars) and high-dose cyclophosphamide (black bars) sperm populations. Data shown represent means ± SEM (n= 3). * Significantly different trom time-matched controls; § significantly different trom time-matched low-dose cyclophosphamide group; 1f significantly different trom dose-matched day 21 cyclophosphamide group (P<0.05).
106 SUB-CHRO'NIC EXPOSURE A 25 20 *§ «z 0 15 ëil J- 10 '*' 5 0 Day 14 Day 21 Day 28 Days Post-Treatment B 120 *§ -100 E 2: 80 ..c:...... C> c:: 60 Q) ....J 40 Oro J- 20 O Day 14 Day21 Day 28 Days Post-Treatment
25 C *§ ...... 20 c:: Q) E 15 0 :2: 10 o-ro J- 5
0 Day 14 Day21 Day 28 Days Post-Treatment Figure 2-4. DNA damage assessed by the cornet assay in epididymal spermatozoa on days 14, 21 and 28 after chronic cyclophosphamïde exposure. Effects measured as (A) % taïl DNA, (8) taïl length and (C) taïl moment are shown for saline (open bars), and cyclophosphamide-exposed (crosshatched bars) sperm populations. Data shown represent means ± SEM (n= 4). * Significantly different from time-matched controls; 11 significantly different from dose-matched day 21 cyclophosphamide group (P<0.05).
108 CHRONIC EXPOSURE A 25 « 20 5 15 * ~ 10 5
o ~----~~~~~--~~--~~~~----- Day 14 Day 21 Day 28 Days Post-Treatment
8 120 * Ê 100 2; 80 .r:; -c:0> 60 (l) .....J 40 ~ 20
o~----~~~~~--~~--~~~~----- Day 14 Day21 Day 28 Days Post-Treatment
C 25 _ 20 c: * (l) E 15 o ~ 10 ~ 5
O~----~~~~-L--~~--~~~~----- Day 14 Day21 Day 28 Days Post-Treatment Figure 2-5. Percentage of epididymal spermatozoa with tail moments greater than 15. CeUs were coUected on days 14, 21 and 28 after (A) acute 1-day, (8) sub chronic 4-day and (C) chronic cyclophosphamide exposure and then analysed for DNA damage measured as cornet tait moment. The percentage of abnormal sperm presenting increased DNA damage is shown for saline (open bars), low dose cyclophosphamide (crosshatched bars) and high-dose cyclophosphamide (black bars). TM indicates tait moment. * Significantly different from time-matched controls; § significantly different from time-matched low-dose cyclophosphamide group (P<0.05).
110 A 35 E ACUTE "- 30 ID a. 25 en-l{) 20 mA * E::2: 15 * 0t-c ...... 0« 10 0~ 5 0 Day 14 Day 21 Day 28 Days Post-Treatment 8 35 *§ E SUB-CHRONIC "- 30 a.ID en 10 25 mA 20 E::2: 15 * 0t-c ...... 0« 10 ~0 5 0 Day 14 Day 21 Day 28 Days Post-Treatment C 35 E CHRONIC "- 30 ID a. 25 * en-l{) mA 20 E::2: 15 c0t- ...... 0« 10 0~ 5 0 Day 14 Day21 Day 28 Days Post-Treatment Connecting Text
ln the previous study we found that cyclophosphamide induced DNA damage in a spermiogenic germ cell-phase specifie manner, thus highlighting the susceptibilities of the spermatids to genetic damage. This time of exposure targets the germ cell as it remodels its chromatin to condense the nucleus and form the mature spermatozoon, a process that requires the involvement of diverse proteins. This unique event could be altered by drug-induced damage. The second objective of this thesis assessed the effects of cyclophosphamide exposure on two aspects which establish spermatozoal chromatin structure, chromatin condensation and basic proteome expression.
112 CHAPTER 3
Exposure of Male Rats to Cyclophosphamide Alters the
Chromatin Structure and Basic Proteome in Spermatozoa
Alexis M. Codrington, Barbara F. Hales and Bernard Robaire
Accepted for publication in Human Reproduction Hum Reprod; 2007 doi:10.1093/humrep/dem002
113 3.1 ABSTRACT
BACKGROUND: The formation of mature sperm involves the expression of numerous proteins during spermiogenesis and the replacement of histones with protamines to package the genome. Exposure to cyclophosphamide (CPA), an anticancer alkylating agent, during spermiogenesis may disrupt chromatin condensation with adverse consequences to the. offspring. METHODS: Adult male rats were given CPA in one of two schedules: 1) sub-chronic, 4 days - day
1, 100mg/kg and days 2 - 4, 50mg/kg/day or 2) chronic - daily, 6.0mg/kg/day.
Animais were euthanized on days 14, 21 or 28. RESUL TS: The effects of CPA on epididymal sperm chromatin structure were germ cel/-phase specific; mid spermiogenic spermatids were most sensitive. The SCSA® 1 acridine orange
DNA denaturation assay showed significant increases in susceptibility to
denaturation (P dithiothreitol-dependent chromomycin A3 DNA binding, and less condensed, protamine-deficient sperm; the total thiol (P (P antibody, respectively, were reduced. The sperm basic proteome was also altered; proteins that were identified are involved in events during spermiogenesis and fertilization. CONCLUSIONS: Paternal exposure to CPA alters sperm chromatin structure, as weI/ as the composition of sperm head basic proteins. We speculate that these changes underlie effects on fertilization and embryo development. 114 3.2 INTRODUCTION Mature spermatozoa are organized and packaged in a specific manner to ensure transmission of genetic material to the offspring and successful pregnancy. The unique structure of sperm and stability of the DNA is due to a sequence of events that occur to shape, condense and protect the nucleus; the most important of these involves a loss of the bulky nucleosomal structure in elongating spermatids as transition proteins transiently replace histones. This exchange facilitates the preferential binding to DNA of highly basic, cysteine-rich protamines in late spermatids (Poccia, 1986; Wouters-Tyrou et al., 1998) Further stabilization is accomplished by the formation of intra- and intermolecular protamine disulfide bonds as cysteines progressively become oxidized during sperm epididymal transit (Shalgi et al., 1989). Perturbation of events at any point of spermiogenesis or sperm maturation may affect sperm function. Indeed, human male infertility has been associated with changes in sperm chromatin integrity and packaging (Evenson et al., 1999; Irvine et al., 2000; Spano et al., 2000), as weil as with changes in protamine content and affinity to DNA (Belokopytova et al., 1993; Torregrosa et al., 2006). Exposure to chemotherapeutic agents results in increased DNA damage in human spermatozoa (Chatterjee et al., 2000; Morris, 2002); such damage has been associated also with abnormal sperm chromatin packaging (Manicardi et al., 1995; Manicardi et al., 1998). A commonly used anticancer drug, cyclophosphamide (CPA), is a bifunctional alkylating agent and well-known male mediated developmental toxicant with clear stage-specific effects on male germ cells (Anderson et al., 115 1995; Trasler et al., 1985). Despite the appearance of morphologically normal spermatozoa, evidence of hidden anomalies exists (Bianchi et al., 1996), and may be the case in spermatozoa chronicallY exposed to CPA. Exposure of male germ cells to CPA during spermiogenesis and epididymal transit leads to DNA single-strand breaks, crosslinks, and altered in vitro spermatozoal decondensation and template function (Qiu et al., 1995a; Qiu et al., 1995b) in cauda epididymal sperm, and to pre- and post-implantation embryo loss as weil as growth retarded progeny (Trasler et al., 1986). The most damaging effects to DNA occurred during mid-spermiogenesis, as the histone-protamine exchange begins (Codrington et al., 2004). Exposure to CPA at this time may alter the binding of protamines to DNA due to increased DNA damage, as weil as result in protamine alkylation, given that protamines are known to be especially susceptible to alkylation (Sega and Owens, 1983). If this is the case, the end result may be faulty or incomplete protamine deposition and blockage of normal disulfide bond formation, thus preventing proper chromatin condensation and limiting fertilizing ability. The formation of mature sperm involves the controlled, sequential expression of a large number of proteins during spermiogenesis. Paternal CPA exposure alters gene expression in male germ cells (Aguilar-Mahecha et al., 2001; Aguilar-Mahecha et al., 2002). These changes in gene expression may be translated into an altered protein expression profile, which could be important for spermiogenic events and fertilization; the characterization of sperm proteins should identify such changes. 116 Sperm count, viability, membrane fluidity, capacitation status, acrosomal integrity, and mitochondrial function, as weil as DNA integrity, and chromatin structure and packaging can be measured rapidly in a large number of spermatozoa by using flow cytometry (Gillan et al., 2005). The value of flow cytometry to study mammalian sperm has been recognized in the areas of reproductive toxicology (to monitor effects from environmental, occupational and therapeutic exposures), and clinical andrology (to assess individual fertility potential) (Spano and Evenson, 1993). A multiplex approach, combining flow cytometry assays with proteomic and genomic methods, should improve our ability to predict fertility status and help identify susceptible subpopulations at risk for infertility. This study uses such an approach to elucidate the effects of CPA on chromatin structure and on the expression of sperm head basic proteins in the rat. 3.3 MATERIALS AND METHODS Animal Treatments Adult male Sprague-Dawley rats (400-450 g) were obtained from Charles River Canada (St. Constant, OC, Canada), maintained on a 14L:10D light cycle and provided with food and water ad libitum. Rats were gavaged with saline or CPA (CAS 6055-19-2, Sigma-Aldrich Ltd., Oakville, ON, Canada) in one of two schedules: 1) high dose/sub-chronic, 4 days - day 1, 100 mg/kg and days 2 - 4, 50 mg/kg/day; 2) low dose/chronic - daily, 6 mg/kg/day. To capture cauda epididymal spermatozoa first exposed to CPA during late, mid and early spermiogenesis, animais were euthanized by decapitation on days 14, 21 or 28 117 after initiation of treatment (Clermont, 1972). Animal handling and care were done in accordance with the guidelines established by the Canadian Council on Animal Care. Sperm Collection Epididymides were first removed, trimmed free of fat and washed in 6ml of pre-chilled 1X phosphate buffered saline (PBS), pH 7.4 (Roche Diagnostics, Laval, QC, Canada). The caput and cauda regions were removed and transferred to separate sterile Petri dishes containing 8ml of fresh PBS on ice. Each region was thoroughly minced with sterile scalpels. The tissue was left for 5 min on ice to a"ow the spermatozoa to disperse and was then strained through a BD Falcon 70J..lm nylon ce" strainer 0JWR International Co., Mississauga, ON, Canada), washed with 2ml of fresh PBS, and the total ce" suspension was centrifuged at 1000 x 9 for 10 min at 4°C. The pellet was washed twice in 10ml of 0.45% NaCI, once in PBS, then resuspended in 4ml of PBS containing 40J..lI of protease inhibitor cocktail (Sigma-Aldrich, Ltd.). Aliquots were stored at -80°C. Cauda epididymal sperm used for protein extraction were further processed according to the method of Yu and co"eagues (Yu et al., 2000), with a few modifications. Fo"owing the final wash in PBS, sperm were resuspended in 4ml of water containing 40J..lI of protease inhibitor cocktail. After sonication on ice, sperm heads were isolated using discontinuous sucrose gradients (Calvin, 1976) made with a 0.2X concentration of MP buffer (5mM MgCI2, 5mM sodium phosphate, pH 6.5) containing 0.25% Triton X-100. Twelve milliliters of sonicated 118 sperm in 1.80M sucrose were layered over 13ml each of cold 2.05M and 2.20M sucrose and centrifuged at 91,400 x gin a Beckman SW 28 rotor for 70 min at 4°C. The pellet was washed in MP buffer and then frozen at -80°C. SCSA ® / Acridine Orange DNA Denaturation Assay Altered chromatin structure measured by the susceptibility of sperm DNA to acid-induced denaturation was assessed with the metachromatic dye, acridine orange (AO) (Sigma':'Aldrich Ltd.), based on a method previously described (Evenson and Jost, 2000). The dye f1uoresces green when bound to double stranded DNA and red when bound to single stranded or denatured DNA. Samples of spermatozoa from the cauda epididymidis were placed in a 37°C water bath until thawed, immediately placed on ice and then sonicated to separate tails from heads. Two hundred microliters of the 1-2 x 106 sperm/ml samples were mixed with 400~1 of a solution containing 0.08N HCI, 0.1 % Triton X-100, and 150mM NaCI, pH 1.4 to denature sperm DNA. After 30 seconds, 1.2ml of AO staining solution (O.2M Na2HP04, 0.1 M citric acid buffer, 1mM EDTA, 150mM NaCI, pH 6.0 and 6.0~g/ml AO) was added. Exactly 2.5 min after the addition of the staining solution, the samples were vortexed and analyzed using a BD FACScan Analyzer System (BD Bioscience, San Jose, CA) fitted with a 488nm argon-ion laser. Green fluorescence was detected with 502LP and 530/30BP filters. Red fluorescence was detected with 670LP and 660/20BP filters. A total of 10,000 sperm were analyzed per sam pie (n=6-8) and each sample was measured twice. 119 Raw data were processed using Win List Software (Verity Software, Topsham, ME, USA). The extent of ONA denaturation was determined by calculating the ONA fragmentation index (OFI), which represents the shift from green to red fluorescence and is the ratio of denatured ONA (red intensity) to total ONA (red + green intensity). For each sample, the mean OFI, indicating shifts within a population of cells and percent abnormal sperm with denatured ONA, defined as %OFI, were calculated. Chromomycin A3 Staining The state of sperm chromatin packaging was assessed based on the accessibility of the f1uorochrome, chromomycin A3 (CMA3) (Sigma-Aldrich Ltd.) (Bianchi et al., 1993). This assay was done with the following modifications. Aliquots of spermatozoa from the caput and cauda epididymidis were first thawed and sonicated on ice. Following one wash in cold PBS, spermatozoa were incubated in 0, 0.1, 0.2, 0.5, 1,2, 5, 10 or 20mM of 1,4-dithiothreitol (OTT) in PBS for 10 min at 37°C, and then washed in PBS. Five hundred microliters of CMA3 solution (0.25mg/ml CMA3 in Mcllvaine buffer, 0.1 M citric acid, 0.2M Na2HP04, pH 7.0, containing 10mM MgCb) was added to each sample and incubated for 20 min at 25°C in the dark. The samples were finally resuspended in 1ml Mcllvaine buffer and stored at 4°C in the dark until analysis the next day. Spermatozoal heads were analyzed using a MoFlo High Performance Cell Sorter (Cytomation Inc., Fort Collins, CO, USA) equipped with an argon laser (457nm line excitation) and a 460/10 filter. The fluorescence emitted was 120 detected with a 580/30 bandpass tilter and quantified using Summit v3.1 software (Cytomation Inc.). A total of 10,000 sperm were analyzed for each sam pie (n=4). Monobromobimane Thiot Labeling Thiol labeling was done according to Shalgi et al. (1989), with minor modifications. Aliquots of spermatozoa from the caput and cauda epididymidis (4 x 106 ceUs/ml) were sonicated on ice and incubated with or without 1mM OTT in PBS at 37°C for 10 min. Samples were washed twice and resuspended in PBS. A 50mM monobromobimane (mBBr) (Calbiochem, San Diego, CA, USA) stock solution was prepared in acetonitrile and added to the sperm suspensions to a final concentration of 0.5mM in the dark for 10 min. Sperm were washed twice with PBS and stored in the dark at 4°C until analysis the next day. Flow cytometric analysis was done using a MoFlo High Performance CeU Sorter (Cytomation Inc.) equipped with an argon laser (UV excitation). MBBr fluorescence emission was detected with 450/65 and dichroic 510LP filters. Quantification was done using Summit v3.1 software (Cytomation Inc.). A total of 10,000 sperm were analyzed for each sample (n=4). Protamine tmmunostaining Aliquots of cauda epididymal spermatozoa (3 x 107 ceUs/ml) were resuspended in 1ml of solution containing 1% SOS, 50mM Tris-HCI pH 7.5, 1mM EDTA, and protease inhibitor cocktail (1:100 dilution) for 10 min at room temperature to remove peri-nuclear material. The samples were then cooled on ice for 5 min, sonicated for 5 sec and washed three times with 50mM Tris-HCI pH 121 7.5. Before the last wash, samples were divided into three groups and then resuspended in 1ml decondensation buffer (25mM Tris-HCI, pH 7.5 and 10Jjl protease inhibitor cocktail) containing 0, 1 or 20mM DIT for 1 hr at room temperature. Ten microliter droplets were placed on slides (two sam pie areas per si ide) and left on ice for 20 min to allow cells to setlle and adhere to the slides. Slides were then washed 3 x 2 min in PBS, fixedin 2% paraformaldehyde for 20 min at room temperature and washed again. Siides were first blocked with 2% normal goat serum (Vector Laboratories Inc., Burlington, ON, Canada) and 1% bovine serum album in (BSA, Sigma Aldrich, Ltd.) in PBS (200Jjl per sample area) for 30 min at room temperature. Subsequently, cells were covered with 200Jjl of primary antibody solution containing 1% BSA and HUP1N monoclonal human protamine 1 antibody (1:100 dilution, kindly provided by Dr. Rod Balhom, Lawrence Livermore National Laboratory) in PBS, ovemight at 4°C, washed with PBS (3 x 2 min), covered for 1.5 hrs in the dark with 200Jjl of secondary antibody solution containing fluorescein-conjugated mouse Ig antibody (1:150 dilution, Amersham Biosciences, Baie D'Urfe, QC, Canada) in PBS, and finally washed three times in PBS. Siides were covered with Vectashield mounting medium containing DAPI (Vector Laboratories Inc.) and kept at 4°C in the dark. Pictures were taken using a DAGE-MTI CCD300-RC camera (DAGE-MTI Inc., Michigan City, IN, USA) atlached to an Olympus BX51 epifluorescence microscope. Fluorescence intensity and sperm head area were measured using the MCID Elite 6.0 image analysis system (Imaging Research Inc., St. 122 Catharines, ON, Canada). Fifty cells were randomly chosen for analysis from each sample area for a total of one hundred cells per group (n=3). Protein Extraction Cauda epididymal sperm head proteins were extracted according to the method of Balhorn et al (1977) with the following modifications. Every step was done at 4°C. Pellets were dissolved in 5M guanidine-HCI, 10mM Tris pH 8, EOTA pH 8, 100mM OTT and protease inhibitor cocktail (1 :100 dilution) for 30 min. After sonication for 1 min, urea, 2-mercaptoethanol and NaCI were added to give a final concentration of 0.5M guanidine-HCI, 3M urea, 0.5M 2- mercaptoethanol and 2M NaCI for 1 hr. HCI was added to a concentration of 0.5M for 1 hr to precipitate the DNA, and the DNA pelleted by centrifugation at 18,300 x 9 for 10 min. The supernatant was dialyzed against 0.01N HCI and 10mM OTT with two changes of solution using 3500 Da dialysis cassettes (Pierce, Rockford, IL, USA). Proteins were precipitated with 25% trichloroacetic acid for 1 hr, the precipitate pelleted at 21,000 x 9 for 10 min, washed once with cold acid-acetone (1drop 5N HCI in 10ml acetone), once with cold acetone, and then air-dried. Samples were stored at 4°C. 20 Basic Gel Electrophoresis Protein separation and analyses were conducted by the McGiII University and Genome Ouebec Innovation Centre (Montreal, OC, Canada) using material from Invitrogen Inc. (Burlington, ON, Canada), except where noted, and the Invitrogen ZOOM IPGRunner System protocol. Fifty micrograms of protein were 123 resuspended in 155J.11 of rehydration buffer (9.8M urea, 10mM 1,4-dithioerythritol, 4% CHAPS, 20mM Tris) supplemented with 1% ZOOM® Focusing Buffer pH 7- 12. ZOOM® Dry Strips, pH 9-12 were rehydrated for 16-18 hrs and isoelectric focusing (IEF) done with a voltage gradient (200V- 2000V) applied as recommended by the manufacturer. After IEF was complete, strips were equilibrated with 1X NuPAGE® LOS Sample Buffer containing 2% OTT and then alkylated with iodoacetamide. Both steps were done at room temperature for 15 min. Electrophoresis in the second dimension was done on 4-20% Tris-Glycine precast gels in XCell SureLock™ Mini-Cells filled with Tris-Glycine SOS Running Buffer. Broad range protein molecular weight markers (0.9 J.lg/gel, Amersham Biosciences) were used and 125V were applied for 1.5 hrs. Gels (n=5) were silver stained, scanned and analyzed with Phoretix 2004 Image Analysis software (Amersham Biosciences). Following background subtraction and normalization, intensities of the spots were calculated. One gel was then chosen as a reference and the other gels compared to the reference gel to create an average control and CPA gel, which were then compared. Spots were considered if present in at least three out of five gels and protein expression was considered changed only if the difference was at least 1.5-fold; this is equivalent to an increase of 50% or decrease of 33%. Mass Spectrometry Selected spots were excised from the gel and subjected to trypsin digestion on a robotic MassPREP Station (Waters-Micromass, Milford, MA, USA), as per the manufacturer's instructions. Gel pieces were tirst washed twice with 124 water for 20 min, destained twice in a 120fJI solution of 30mM potassium ferricyanide and 100mM sodium thiosulfate mixed 1: 1 for 15 min and then dehydrated with 75fJI of 100% acetonitrile. Samples were reduced, in the dark, with 50fJI of 10mM OTT for 30 min followed by alkylation with 50fJI of 55mM iodoacetamide for 20 min and 100fJI of 100% acetonitrile for 5 min. After washing and dehydration in 100mM ammonium bicarbonate and 100% acetonitrile, respectively, gel pieces were covered and digested for 4.5 hours with 6 ng/fJl of trypsin gold (Promega, Madison, WI, USA) in 100mM ammonium bicarbonate. Peptides were extracted with 30fJI formic acid (FA) solution (1 % FA in 2% acetonitrile) for 30 min, twice with 12fJI FA solution and then 12fJI 100% acetonitrile for 30 min. Nanoflow chromatography of digested peptides was performed on an Agilent 1100 series nanopump (Agilent Technologies Inc., Mississauga, ON, Canada) at a flow-rate of 200nl/min. Sam pie injection and desalting were performed with an Isocratic Agilent 1100 series pump at 15 fJl/min for 5 min. A trapping column (Agilent) packed with Zorbax 300SB-C18 (5 x 0.3 mm) was used for sample desalting. Peptide separation was done with a Biobasic C18 (10 x 0.075 mm) picofrit column (New Objective, Woburn, MA, USA). Peptides were eluted using a 20 min gradient with solvent A (0.1 % FA) and solvent B (95% acetonitrile:0.1%FA) from 90%Al10%B to 100%B. Matrix assisted laser desorption/ionization mass spectrometry (MALOI MS) was done with a MALOI Q-Time of Flight (ToF) Ultima instrument (Waters Micromass). Samples were spotted along with a saturated solution of alpha hydroxycinnamic acid in 50% acetonitrile. Peaklists for peptide mapping 125 searches were generated with Proteinlynx v.1.5 (Waters-Micromass). Searches were performed with Mascot 1.9 using carbamidomethyl cysteine as a fixed modification, methionine oxidation as a variable modification and a precursor mass tolerance of 0.3 Da. Statistical Analysis Significant differences were determined using two- and three-way analysis of variance foUowed by the Holm-Sidak post-hoc test (p < 0.05). The percent abnormal sperm with denatured DNA among populations of spermatozoa from control and CPA-treated animais was compared by Chi-square analysis. Statistical analyses were done using the SigmaStat 3.0 software package (SPSS Inc., Chicago, IL). 3.4 RESULTS Sperm Chromatin Structure Altered sperm chromatin structure has been attributed to changes in protamine content and the thiol-disulfide status of proteins (Aoki et al., 2005; Kosower et al., 19Q2; Love and Kenney, 1999; Pina-Guzman et al., 2005; Sailer et al., 1995; Zini et al., 2001). The SCSA® tAO DNA denaturation assay was used for a general assessment of the effects of CPA exposure on sperm chromatin structure. DNA in sperm with abnormal chromatin structure showed increased red fluorescence yielding a broader distribution of DFI values with an increase in mean DFI and more ceUs with denatured DNA (%DFI). Figure 3-1A shows that chronic CPA treatment for 28 days, exposing spermatids throughout 126 spermiogenesis and epididymal maturation, resulted in a 1.2-fold increase in the mean OFf of treated spermatozoa, compared to controls. Sub-chronic exposure resulted in significant increases in mean OFI values in spermatozoa exposed to the drug only at the end of spermiogenesis (14 days), when they were elongated spermatids, and during mid-spermiogenesis, as efongating spermatids (21 days). Cells collected after 28 days, and therefore first exposed as round spermatids, exhibited no increase in mean OFI. The percentage of sperm with denatured ONA was dramatically higher in drug-exposed samples (Figure 3-18). Chronic exposure resulted in a 6.9-fold increase in %OFI. Sub-chronic treatment revealed significant increases in the %OFI in spermatozoa collected after 14 days (3.8-fofd) and 21 days (6.9-fold). In contrast, there was no significant difference in %OFI in cells targeted as round spermatids. Mid-spermiogenic elongating spermatids were most sensitive to the effects of CPA; exposure at this time appears to account for most of the abnormalities seen following chronic exposure. Protamination and Condensation Status of Sperm Chronic CPA exposure showed no CMA3 staining above background levels in either control or drug-exposed cauda epididymal spermatozoa, suggesting no change in protamine content (Figure 3-2A, OmM OTT); however, sperm condensation by protamine disulfide bond formation, in addition to the level of protamination, could be a limiting factor controlling fluorochrome accessibility. To determine whether thiol bonds do play a role in CMA3 binding, caput spermatozoa were examined. Indeed, less condensed immature caput 127 spermatozoa were CMA3 positive. Not only was fluorescence intensity 7 -fold higher in control caput sperm compared to control cauda sperm, but fluorescence intensity of CPA-exposed caput sperm was 1.6-fold higher then control cap ut sperm and 9-fold higher then CPA cauda sperm. The relationship between CMA3 ONA binding and chromatin condensation was further assessed by in vitro decondensation induced by the disulfide reducing agent, on (Figure 3-2A, inset). With increasing amounts of on, a concentration-dependent increase in staining was observed, suggesting that the thiol status or decondensation status of sperm does indeed affect CMA3 accessibility and binding. In caput spermatozoa, with just 0.1 mM on, CMA3 binding increased to levels significantly higher then in cells not treated with the reducing agent (control: 4-fold, chronic CPA: 3.8-fold). More importantly, the CPA-exposed cell population exhibited unique susceptibility at this concentration of on as fluorescence intensity was 1.6-fold higher than in controls. Maximum intensity in both groups was observed with 1mM on (Figure 3-2A). ln cauda spermatozoa, higher concentrations of OTT were required in order to observe significant differences in CMA3 fluorescence intensity. One millimolar of on was needed to achieve a 9.2-fold increase in control cauda spermatozoa and 17.5-fold increase in CPA-exposed cauda spermatozoa, compared to those that were not treated with on. Notably, the only difference in CMA3 binding in cauda sperm due to drug treatment was seen using 1mM on (1.9-fold increase, Figure 3-2A). Therefore, chromatin packaging, whether due to protamine deficiency or not, was affected by CPA exposure; however, such an affect was only evident after decondensing the cells with 1mM on. lt appears 128 that a certain thiol-disulfide status threshold had to be passed before CMA3 binding was achieved. These results suggest that CPA-exposed spermatozoa were less condensed and/or were protamine deficient compared to control sperm. Cauda spermatozoa collected following sub-chronic exposure to CPA did not exhibit any changes in CMA3 ONA binding at any time point in the absence of on (Figure 3-28). However, in the presence of 1 mM on, significant differences (> 2-fold) due to CPA treatment were observed at ail time points, with no cell type being more sensitive than the other (Figure 3-28). Thiol-Disulfide Status Results·obtained using CMA31ead us to assess the thiol-disulfide status of spermatozoa by incubating cells with and without on, and then labeling them with m8Br. In Figure 3-3A, as expected the proportion of reactive thiols (SH 1 (SH+SS», estimated from the mean fluorescence of sperm incubated without on (free thiols, SH) and the fluorescence of on pre-treated cells (total thiols, SH+SS), decreases as sperm mature from the caput to cauda epididymal regions. The effects of CPA were assessed in cauda sperm as weil as caput sperm, since the thiol status changes during epididymal transit as protamine disulfide bonds form. Using both cell types therefore also highlights any epididymal effects of CPA. Following chronic exposure to CPA, the fluorescence intensities of caput and cauda sperm not incubated with OTT were similar to controls. In contrast, total thiol levels significantly decreased by 5.7% in caput and 10% in cauda spermatozoa pre-treated with OTT compared to controls, thus 129 indicating that not only were thiol groups affected during epididymal transit, but also that the effect occurred before cells reached the caput epididymal region. Sub-chronic CPA exposure revealed a decrease in total thiols (7.4%) only in cauda sperm collected after 21 days, representing an effect on mid spermiogenic spermatids (Figure 3-3B). No other ceU type exposed to CPA, and incubated with or without on showed an effect different from that of controls. Protamine Content Protamines are the main basic protein present in spermatozoal heads. To follow-up on results obtained using CMA3 and mBBr, which point to the possibility of protamine deficiency in mature spermatozoa following CPA exposure, the level of protamination was assessed in cauda epididymal sperm. By the time spermatozoa enter the epididymis protamination is complete, therefore only cauda sperm were required for this assessment. Protamine 1 (P1) detection in cauda spermatozoa was dependent on the extent of decondensation induced by OTT; differences due to chronic drug treatment were only evident if cells were pre-treated with on (Figure 3-4A). Minimal fluorescence was seen in both control and CPA-exposed sperm in the absence of OTT (Figure 3-4B). With increasing concentrations of OTT, the amount of protamines detected significantly increased in control sperm. The fluorescence intensity of CPA exposed sperm was significantly different from control if on was used; compared to controls, less protamine was detected in CPA-exposed sperm. Differences in fluorescence between control and CPA-exposed sperm were not due to disproportionate decondensation because although sperm head areas 130 increased with increasing concentrations of DIT, they were not affected by CPA treatment (Figure 3-4C). Thus, sperm head fluorescence intensity results show that protamine content was indeed lower following chronic CPA exposure (Figure 3-40). Sperm Head Basic Profein Expression Sperm head proteins were analyzed by 20 basic gel electrophoresis. Five gels each were run for control and chronic CPA-treated sperm protein samples. The ove ra Il pattern of proteins was reproducible between experiments; protein profiles differed with treatment (Figure 3-5). The avèrage control gel (Figure 3- 5A) consisted of 68 protein spots that appeared in at least three out of five gels analyzed, corresponding to 73-98% of the total number of spots detected on individual gels. In comparison, 59 protein spots were found on the average CPA gel (Figure 3-5B), corresponding to 70-99% of ail spots appearing on individual gels. Eleven protein spots were uniquely expressed in control samples and two in CPA samples. Fifty-seven protein spots were expressed in both groups; analysis of protein expression changes > 1.5-fold revealed nine spots that increased and six that decreased following CPA exposure. Twenty-two spots were chosen for identification; however, we were only successful in confidently identifying 12; these spots are labeled in Figure 3-5 and summarized in Table 3-1. Three of the identified proteins (histone 4, HIST2H4; fatty acid binding protein 9, FABP9; and zona pellucida binding protein, ZPBP) were represented by at least two distinct spots on the gels, suggesting that these proteins are modified or exist in different isoforms. ZPBP was found as the 131 precursor (46.3 kOa) and mature protein (39.7kOa). Interestingly, the expression of the precursor increased by 68%, while that of the mature protein was not changed (:S 1.5-fold). Of note, protein fragments were identified from significant MALOI Q-ToF MS analysis results for peptides that only partially covered a protein sequence. Protein fragments may be proteolytic cleavage products, despite the use of protease inhibitors during sample preparation. Analyzed spots for two proteins (heterogeneous nuclear ribonucleoprotein A 1, HNRPA 1; and chromodomain helicase DNA binding protein 4, CH04) contained only a fragment of the identified protein; the masses of the proteins calculated from the 20 gel (11.8 and 21.0 kOa, respectively) were below the expected masses of these proteins, calculated from their amino acid compositions (38.9 and 205.5 kOa, respectively. Peptides for HNRPA1 covered theamino acid sequence containing the RNA recognition motif 1 (RRM1), RRM2 and a phosphothreonine phosphorylation site. Those for CH04 contained the helicase superfamily C-terminal (HELICe) domain; the two chromatin organization modifier (CHROMO) domains, which play a role in the functional organization of the nucleus, were not present (data not shown). We do not know if the expression of the HNRPA1 and CH04 protein fragments (64% increase with CPA and no change, respectively) reflect the expression of the full-Iength proteins. The majority of proteins identified, with the exception of HNRPA 1, protein kinase R interacting protein 1 (PRKRIP1) and CH04, are known components of spermatozoal heads expressed in the nucleus, perinuclear theca (PT) subacrosomal layer and sheath, or on the surface of the sperm. Information 132 concerning the putative functions of these proteins was found in the NCBI non redundant and SWISS-PROT protein sequence databases or in the literature. ln general, these proteins are involved in transcription and translation regulation (HNRPA1, PRKRIP1 and CHD4), chromatin organization (CHD4, HIST2H4 and histone H2B (HIST1 H2BL), sperm structure and stability (FABP9), and fertilization (HIST2H4, HIST1 H2BL, ZPBP and beta-defensin 20). 3.5 DISCUSSION Several alkylating agents, including ethylnitrosourea, methyl methanesulfonate, thiotepa and triethylenemelamine, have been reported to result in sperm chromatin structural changes, as measured by flow cytometry (Evenson et al., 1985; Evenson et aL, 1986; Evenson et aL, 1989; Evenson et aL, 1993). In the present study, the SCSA® tAO DNA denaturation assay revealed significant epididymal sperm nuclear structural effects of CPA exposure; maximal changes occurred in mid-spermiogenic elongating spermatids. These findings are in accordance with our results using the cornet assay, which showed increased DNA damage after chronic exposure to CPA and a similar maximal sensitivity to the drug in elongating spermatids (Codrington et aL, 2004). Protamine content and thiol-disulfide status are sources of chromatin stability examined in this study and reduced chromatin stability can affect genetic integrity. CPA effects on chromatin condensation may lead to stress in the chromatin structure and result in DNA strand breaks. The fluorochrome CMA3 has been used as an indirect tool to detect abnormal sperm chromatin packaging, as increased CMA3 staining is indicative 133 of decondensed, protamine-depleted spermatozoa (Bianchi et al., 1993; Bianchi et al., 1996; Manicardi et al., 1995). It is thought that either CMA3 accesses DNA in the minor groove of the DNA helix (Behr et aL, 1969; Berman et al., 1985), making it a protamine competitor, or protamines bind within the major groove (Fita et al., 1983; Hud et aL, 1994; Prieto et aL, 1997) thereby possibly obstructing CMA3 access to the minor groove (Bizzaro et al., 1998). Classical CMA3 analysis, as documented in the literature and first developed using human and murine samples (Bianchi et al., 1993), failed to reveal an effect following drug exposure in rats. Mouse caput, corpus and cauda epididymal spermatozoa do not positively stain with CMA3 (Sakkas et al., 1995); however, in rats the thiol disulfide status of epididymal spermatozoal nuclei influences the binding capacity of CMA3. This and other studies (Shalgi et al., 1989) show that the proportion of reactive thiols decreases from -85% in caput spermatozoa to ..... 22% in cauda spermatozoa. Interestingly, CPA affected male germ cells exposed as round spermatids, altering both sperm chromatin packaging, as assessed with the CMA3 binding assay, and genetic integrity, as assessed with the cornet assay (Codrington et al., 2004). While the reasons for this are unclear, it is possible that CPA-induced DNA damage in early-spermiogenic spermatids affects mRNA transcripts synthesized at this time that are required for spermatid differentiation, such as protamine 1 (P1). Decreased template function has been observed in sperm chronicallyexposed to this alkylating agent (Qiu et al., 1995a); this may result in transcription termination and truncated RNA molecules (Gray et al., 1991; Pieper et al., 1989; Pieper and Erickson, 1990). Indeed, studies on infertile men report 134 decreased P1 transcript levels, thereby affecting the P1 :P2 ratio and sperm chromatin structure (Aoki et al., 2005). P1, the only protamine expressed in rats, appears to be the most critical factor, in comparison to P2, for male fertili~y (Cho et al., 2001; Steger et al., 2003). Metabolites of CPA (phosphoramide mustard and acrolein) can alkylate nucleophilic sites of ONA, RNA and protein (Murthy et al., 1973; Sanderson and Shield, 1996). In addition to consequences of DNA dam~ge on mRNA transcription, CPA could also bind directly with transcripts and affect protein synthesis. Protamines are synthesized during mid- and late-spermiogenesis and bind to late elongated spermatid ONA (Kistler et al., 1996). CMA3 staining was at its highest in spermatozoa exposed to CPA as elongating mid-spermiogenic spermatids; this is best explained by decreased P1 expression, which in turn could account for the noted reduction in thiol content. These results substantiate previous findings from our laboratory that report decreased 14C-iodoacetamide binding in spermatozoal nuclei chronicaUy exposed to CPA; however, an effect was also seen in ceUs not pre-treated with OTT (Qiu et al., 1995b). Oiscrepancies may be accounted for by differences in the level of sensitivity of the methods used. In addition to decreased P1 content, an effect on available reactive thiols could be due also to increased alkylation of protamine thiol groups; protamines in the testis are especially susceptible to alkylation, thus blocking normal disulfide bond formation (Sega and Owens, 1983), preventing proper chromatin condensation, and altering sperm structure. Indeed, the thiol-disulfide status of protamines determines the AO fluorescence of spermatozoal nuclei, such that increased susceptibility to acid-induced ONA denaturation occurs if 135 ONA-associated protamines are poor in disulfides (Kosower et aL, 1992; Zini et aL, 2001). Previously, we reported earlier male pronuclear formation in rat oocytes fertilized by CPA-treated males, as weil as altered sperm chromatin decondensation, both in vitro and in denuded hamster oocytes (Harrouk et aL, 2000; Qiu et al., 1995b). It would be of interest to examine modifications to protamines following CPA exposures that affect sperm chromatin structure, namely alkylation and phosphorylation. Protamine phosphorylation/dephosphorylation maybe required for proper chromatin condensation; shortly after their synthesis, protamines are phosphorylated, thereby facilitating their correct binding to ONA. Once the ONA protamine complex is formed, protamines are dephosphorylated and sperm then enter the epididymis where final chromatin condensation occurs (Oliva and Oixon, 1991; Pirhonen et aL, 1994). Standard 20 systems provide more information about a protein sample, but they do not resolve ail proteins present in a sample, especially basic proteins with isoelectric points above 9. The 20 basic protein system used in this study did not resolve and identify modified protamines because they are extremely basic and rich in disulfide bridges; however, histones were present on the gels. It is tempting to speculate that these may be residual nuclear histones, although somatic histones are known also to be present in the subacrosomal sheath of the perinuclear theca (PT) (Tovich and Oko, 2003). The PT, a specialized cytoskeletal structure under the acrosome and surrounding sperm nuclei, has been implicated in acrosome-nuclear docking· and nuclear shaping during spermiogenesis (Au 1 and Oko, 2002; Oko, 1995), as weil as in egg activation, and pronuclear formation (Kimura et al., 1998; Manandhar and 136 Toshimori, 2003; Sutovsky et aL, 1997; Sutovsky et aL, 2003); the potential exists for PT -derived histones to contribute to male pronuctear development. The histone-protamine exchange occurs along with other functionally important spermiogenic morphological and biochemical events. If protamine expression is altered, this could reflect a wide range of spermiogenic defects. Indeed, in this study we have identified proteins involved in various aspects of spermatid differentiation, sperm maturation and fertilization, sorne of which had altered expression following CPA exposure. As weil, sorne of the proteins appear to be post-translationally modified, which altered their isoelectric point, and, therefore, the 2D gel protein expression profile. A few proteins with altered expression were identified that have unknown functions during spermiogenesis. PRKRIP1, highly expressed in the testis, is a nuclear protein with high nucleolar expression. It acts as a negative regulator of PRKR (Yin et al., 2003). Decreased expression the refore, may relieve PRKR from inhibition. PRKR, expressed in spermatogonia (Melaine et al., 2003), inhibits protein synthesis by phosphorylating the initiation factor, elF-2a (Hovanessian, 1989); it has been implicated also in apoptosis (Der et al., 1997), cellular transformation (Donze et al., 1995; Meurs et al., 1993), differentiation (Samuel et al., 1997) and transcription (Deb et al., 2001; Wong et al., 1997). It has been demonstrated that PRKR is involved in the cellular response to genotoxic stress, possibly by modulating DNA repair mechanisms to rem ove bulky DNA adducts (Bergeron et al., 2000). HNRPA1 is a member of a group of core mammalian HNRP proteins that bind pre-mRNA and are involved in RNA processing (Krecic and Swanson, 137 1999). Many HNRPs are expressed in germ cells from the spermatogonial to the round spermatid phase of development, when RNA synthesis is known to cease (Biggiogera et al., 1993); however, the expression of HNRPA1 is restricted to spermatogonia (Matsui et al., 2000). Interestingly, the N-terminal end of HNRPA 1 is cleaved to produce unwinding protein 1 (UP1), which contains the two RNA recognition motifs required for efficient RNA binding (Myers and Shamoo, 2004). It is possible that the proteolytic cleavage product present on our 2D gels is UP1. Both HNRPA 1 and UP1 are also helix-destabilizing, single- stranded DNA binding proteins (Nadler et al., 1991). More importantly they are involved in telomere biogenesis (LaBranche et al., 1998). Reduced telomere length in sperm can affect embryogenesis; if not re-Iengthened, germ cells containing shortened telomeres may limit the replication of cells derived from the zygote (Baird et al., 2006; Bekaert et al., 2004). Chemotherapeutic agents, including drugs such as bleomycin, mitomycin C and CPA, can cause telomere shortening (Arutyunyan et al., 2004; Kiyozuka et al., 2000). Increased expression of HNRPA1/UP1 may be in response to reduced telomeres in sperm following chronic CPA exposure. This study used multiple assays to demonstrate that CPA exposure alters ~ sperm chromatin structure and protein expression. Changes in proteins involved in sperm function, fertilization, and post-fertilization events important for proper embryo development have been shown. An increasing number of groups are attempting to identify proteins of the sperm proteome (Chu et al., 2006; Martinez- Heredia et al., 2006; Pixton et al., 2004). We have used proteomic techniques to identify components of rat spermatozoa heads. The results of this study 138 encourage further investigation into changes of the sperm proteome in response to exposure to CPA and other male mediated developmental toxicants. The clinical significance of these analyses rests in their role in both natural and assisted reproduction success rates, and the possibly high prognostic value in assessing fertility in cancer patients. Acknowledgements We thank Martine Dupuis and Eric Masicotle at L'Institut de Researches Cliniques de Montreal for their enthusiastic assistance with the flow cytometer, and Dr. Rod Balhom at Lawrence Livermore National Laboratory for his generous gift of the HUP1 N protamine 1 antibody. We greatly appreciate the assistance of Leonid Kriazhev from the McGiII University and Genome Quebec Innovation Center with the 2D gel electrophoresis and mass spectrometry. This work was supported by a grant from the Canadian Institutes of Health Research. 139 REFERENCES Aguilar-Mahecha A, Hales BF and Robaire B (2001) Acute cyclophosphamide exposure has germ cell specifie effects on the expression of stress response genes during rat spermatogenesis. Mol Reprod Dev 60,302-311. Aguilar-Mahecha A, Hales BF and Robaire B (2002) Chronic cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells. Biol Reprod 66,1024-1032. Anderson D, Bishop JB, Garner RC, Ostrosky-Wegman P and Selby PB (1995) CycJophosphamide: review of its mutagenicity for an assessment of potential germ cell risks. M utat Res 330,115-181 . Aoki VW, Moskovtsev SI, Willis J, Liu L, Mullen JB and Carrell DT (2005) DNA integ rit y is compromised in protamine-deficient human sperm. J Androl 26,741-748. Arutyunyan R, Gebhart E, Hovhannisyan G, Greulich KO and Rapp A (2004) Comet-FISH using peptide nucleic acid probes detects telomeric repeats in DNA damaged by bleomycin and mitomycin C proportional to general DNA damage. Mutagenesis 19,403-408. Aul RB and Oko RJ (2002) The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis. Dev Biol 242,376-387. Baird DM, Britt-Compton B, Rowson J, Amso NN, Gregory L and Kipling 0 (2006) Telomere instability in the male germline. Hum Mol Genet 15,45-51. 140 Balhom R, Gledhill Bl and Wyrobek AJ (1977) Mouse sperm chromatin proteins: quantitative isolation and partial characterization. Biochemistry 16,4074- 4080. Behr W, Honikel K and Hartmann G (1969) Interaction of the RNA polymerase inhibitor chromomycin with DNA. Eur J Biochem 9,82-92 .. Bekaert S, Derradji H and Baatout S (2004) Telomere biology in mammalian germ cells and during development. Dev Biol 274,15-30. Belokopytova lA, Kostyleva El, Tomilin AN and Vorob'ev VI (1993) Human male infertility may be due to a decrease of the protamine P2 content in sperm chromatin. Mol Reprod Dev 34,53-57. Bergeron J, Benlimame N, Zeng-Rong N, Xiao D, Scrivens PJ, Koromilas AE and Alaoui-Jamali MA (2000) Identification of the interferon-inducible double stranded RNA-dependent protein kinase as a regulator of cellular response to bulky adducts. Cancer Res 60,6800-6804. Berman E, Brown SC, James Tl and Shafer RH (1985) NMR studies of chromomycin A3 interaction with DNA. Biochemistry 24,6887-6893. Bianchi PG, Manicardi GC, Bizzaro D, Bianchi U and Sakkas D (1993) Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa. Biol Reprod 49,1083-1088. Bianchi PG, Manicardi GC, Umer F, Campana A and Sakkas D (1996) Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa. Mol Hum Reprod 2,139-144. 141 Biggiogera M, Von Schack ML, Martin TE, Gordon J, Muller M and Fakan S (1993) Immunoelectron microscope localization of snRNP, hnRNP, and ribosomal proteins in mouse spermatogenesis. Mol ReprodDev 35,261- 271. Bizzaro D, Manicardi GC, Bianchi PG, Bianchi U, Mariethoz E and Sakkas 0 (1998) ln-situ competition between protamine and fluorochromes for sperm DNA. Mol Hum Reprod 4,127-132. Calvin HI (1976) Isolation and subfractionation of mammalian sperm heads and tails. Methods Cell Biol 13,85-104. Chatterjee R, Haines GA, Perera DM, Goldstone A and Morris ID (2000) Testicular and sperm DNA damage after treatment with fludarabine for chronic Iymphocytic leukaemia. Hum Reprod 15,762-766. Cho C, Willis WD, Goulding EH, Jung-Ha H, Choi YC, Hecht NB and Eddy EM (2001) Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet 28,82-86. Chu OS, Liu H, Nix P, Wu TF, Ralston EJ, Yates JR, III and Meyer BJ (2006) Sperm chromatin proteomics identifies evolutionarily conserved fertility factors. Nature 443,101-105. Clermont Y (1972) Kinetics of spermatogenesis· in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 52,198-236. Codrington AM, Hales BF and Robaire B (2004) Spermiogenic germ cel! phase specifie DNA damage following cyclophosphamide exposure. J Androl 25,354-362. 142 Deb A, Haque SJ, Mogensen T, Silverman RH and Williams BR (2001) RNA dependent protein kinase PKR is required for activation of NF-kappa B by IFN-gamma in a STAT1-independent pathway. J ImmunoI166,6170-6180. Der SD, Yang YL, Weissmann C and Williams BR (1997) A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-· induced apoptosis. Proc Natl Acad Sci USA 94,3279-3283. Donze 0, Jagus R, Koromilas AE, Hershey JW and Sonenberg N (1995) Abrogation of translation initiation factor e1F-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J 14,3828-3834. Evenson D and Jost L (2000) Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sei 22,169-189. Evenson DP, Baer RK and Jost LK (1989) Long-term effects of triethylenemelamine exposure on mouse testis cells and sperm chromatin structure assayed by flow cytometry. Environ Mol Mutagen 14,79-89. Evenson DP, Baer RK, Jost LK and Gesch RW (1986) Toxicity of thiotepa on mouse spermatogenesis as determined by dual-parameter flow cytometry. Toxicol Appl PharmacoI82,151-163. Evenson DP, Higgins PJ, Grueneberg D and Ballachey BE (1985) Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea. Cytometry 6,238-253. Evenson OP, Jost LK and Baer RK (1993) Effects of methyl methanesulfonate on mouse sperm chromatin structure and testicular cell kinetics. Environ Mol Mutagen 21,144-153. 143 Evenson OP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P and Claussen OP (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility cJinic. Hum Reprod 14,1039-1049. Fita l, Campos JL, Puigjaner LC and Subirana JA (1983) X-ray diffraction study of ONA complexes with arginine peptides and their relation to nucJeoprotamine structure. J Mol Biol 167,157-177. Gillan L, Evans G and Maxwell WM (2005) Flow cytometric evaluation of sperm parameters in relation to fertility potential. Theriogenology 63,445-457. Gray PJ, Cullinane C and Phillips DR (1991) ln vitro transcription analysis of ONA alkylation by nitrogen mustard. Biochemistry 30,8036-8040. Harrouk W, Khatabaksh S, Robaire B and Hales BF (2000) Patemal exposure to cyclophosphamide dysregulates the gene activation program in rat preimplantation embryos. Mol Reprod Oev 57,214-223. Hovanessian AG (1989) The double stranded RNA-activated protein kinase induced by interferon: dsRNA-PK. J Interferon Res 9,641-647. Hud NV, Milanovich FP and Balhom R (1994) Evidence of novel secondary structure in ONA-bound protamine is revealed by Raman spectroscopy. Biochemistry 33,7528-7535. Irvine OS, Twigg JP, Gordon EL, Fulton N, Milne PA and Aitken RJ (2000) ONA integrity in human spermatozoa: relationships with semen quality. J Androl 21,33-44. 144 Kimura Y, Yanagimachi R, Kuretake S, Bortkiewicz H, Perry AC and Yanagimachi H (1998) Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biol Reprod 58,1407-1415. Kistler WS, Henriksen K, Mali P and Parvinen M (1996) Sequential expression of nucleoproteins during rat spermiogenesis. Exp CeU Res 225,374-381. Kiyozuka Y, Yamamoto D, Yang J, Uemura Y, Senzaki H, Adachi Sand Tsubura A (2000) Correlation of chemosensitivity to anticancer drugs and telomere length, telomerase activity and telomerase RNA expression in human ovarian cancer ceUs. Anticancer Res 20,203-212. Kosower NS, Katayose H and Yanagimachi R (1992) Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei. J Androl 13,342-348. Krecic AM and Swanson MS (1999) hnRNP complexes: composition, structure, and function. Curr Opin Ce" Biol 11,363-371. laBranche H, Dupuis S, Ben David Y, Bani MR, Wellinger RJ and Chabot B (1998) Telomere elongation by hnRNP A1 and a derivative that interacts with telomeric repeats and telomerase. Nat Genet 19,199-202. love CC and Kenney RM (1999) Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biol Reprod 60,615-620. Manandhar Gand Toshimori K (2003) Fate of postacrosomal perinuclear theca recognized by monoclonal antibody MN13 after spermhead microinjection and its role in oocyte activation in mice. Biol Reprod 68,655-663. 145 Manicardi GC, Bianchi PG, Pantano S, Azzoni P, Bizzaro D, Bianchi U and Sakkas D (1995) Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to chromomycin A3 accessibility. Biol Reprod 52,864-867. Manicardi GC, Tombacco A, Bizzaro D, Bianchi U, Bianchi PG and Sakkas D (1998) DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays. Histochem J 30,33-39. Martinez-Heredia J, Estanyol JM, Ballesca JL and Oliva R (2006) Proteomic identification of human sperm proteins. Proteomics 6,4356-4369. Matsui M, Horiguchi H, Kamma H, Fujiwara M, Ohtsubo R and Ogata T (2000) Testis- and developmental stage-specific expression of hnRNP A2/B1 splicing isoforms, BOa/b. Biochim Biophys Acta 1493,33-40. Melaine N, Lienard MO, Guillaume E, Ruffault A, Dejucq-Rainsford N and Jegou B (2003) Production of the antiviral proteins 2'5'oligoadenylate synthetase, PKR and Mx in interstitial cells and spermatogonia. J Reprod Immunol 59,53-60. Meurs EF, Galabru J, Barber GN, Katze MG and Hovanessian AG (1993) Tumor suppressor function of the interferon-induced double-stranded RNA activated protein kinase. Proc Natl Acad Sci USA 90,232-236. Morris ID (2002) Sperm DNA damage and cancer treatment. Int J Androl 25,255- 261. 146 Murthy W, Becker BA and Steele WJ (1973) Effects of dosage, phenobarbital, and 2-diethylaminoethyl-2,2-diphenylvalerate on the binding of cyclophosphamide and-or its metabolites to the DNA, RNA, and protein of the embryo and liver in pregnant mice. Cancer Res 33,664-670. Myers JC and Shamoo y (2004) Human UP1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (RRM). J Mol Biol 342,743-756. Nadler SG, Merrill BM, Roberts WJ, Keating KM, Lisbin MJ, Barnett SF, Wilson SH and Williams KR (1991) Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions. Biochemistry 30,2968-2976. Oko R (1995) Developmental expression and possible role of perinuclear theca proteins in mammalian spermatozoa. Reprod Fertil Dev 7,777-797. Oliva Rand Dixon GH (1991) Vertebrate protamine genes and the histone-to protamine replacement reaction. Prog Nucleic Acid Res Mol Biol 40,25-94. Pieper RO and Erickson LC (1990) DNA adenine adducts induced by nitrogen mustards and their role in transcription termination in vitro. Carcinogenesis 11,1739-1746. Pieper RO, Futscher BW and Erickson LC (1989) Transcriplion-terminating lesions induced by bifunctional alkylating agents in vitro. Carcinogenesis 10,1307-1314. 147 Pina-Guzman B, Solis-Heredia MJ and Ouintanilla-Vega B (2005) Diazinon alters sperm chromatin structure in mice by phosphorylating nuclear protamines. Toxicol Appl PharmacoI202,189-198. Pirhonen A, Linnala-Kankkunen A and Maenpaa PH (1994) Identification of phosphoseryl residues in protamines from mature mammalian spermatozoa. Biol Reprod 50,981-986. Pixton KL, Deeks ED, Flesch FM, Moseley FL, Bjomdahl L, Ashton PR, Barratt CL and Brewis lA (2004) Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report. Hum Reprod 19,1438-1447. Poccia 0 (1986) Remodeling of nucleoproteins during gametogenesis, fertilization, and early development. Int Rev CytoI105,1~65. Prieto MC, Maki AH and Balhom R (1997) Analysis of DNA-protamine interactions by optical detection of magnetic resonance. Biochemistry 36,11944-11951. Oiu J, Hales BF and Robaire B (1995a) Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure. Biol Reprod 53,1465-1473. Oiu J, Hales BF and Robaire B (1995b) Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa. Biol Reprod 52,33-40. Sailer BL, Jost LK and Evenson OP (1995) Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks 148 as measured by the terminal,deoxynucleotidyl transferase assay. J And roi 16,80-87. Sakkas D, Manicardi G, Bianchi PG, Bizzaro 0 and Bianchi U (1995) Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa. Biol Reprod 52,1149-1155. Samuel CE, Kuhen KL, George CX, Ortega LG, Rende-Fournier R and Tanaka H (1997) The PKR protein kinase--an interferon-inducible regulator of cell growth and differentiation. Int J HematoI65,227-237. Sanderson BJ and Shield AJ (1996) Mutagenic damage to mammalian cells by therapeutic alkylating agents. Mutat Res 355,41-57. Sega GA and Owens JG (1983) Methylation of DNA and protamine by methyl methanesulfonate in the germ cells of male mice. Mutat Res 111,227-244. Shalgi R, Seligman J and Kosower NS (1989) Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane. Biol Reprod 40,1037-1045. Spano M, Bonde JP, Hjollund HI, Koistad HA, Cordelli E and Leter G (2000) Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertil Steril 73,43-50. Spano M and Evenson OP (1993) Flow cytometric analysis for reproductive biology. Biol Cell 78,53-62. 149 Steger K, Fink l, Failing K, Bohle RM, Kliesch S, Weidner W and Bergmann M (2003) Decreased protamine-1 transcript levels in testes from infertile men. Mol Hum Reprod 9,331-336. Sutovsky P, Manandhar G, Wu A and Oko R (2003) Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti polyspermy defense, and assisted reproduction. Microsc Res Tech 61,362-378. Sutovsky P, Oko R, Hewitson land Schatten G (1997) The removal of the sperm perinuclear theca and its association with the bovine oocyte surface during fertilization. Dev Biol 188,75-84. Torregrosa N, Dominguez-Fandos D, Camejo MI, Shirley CR, Meistrich Ml, Ballesca Jl and Oliva R (2006) Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients. Hum Reprod 21,2084-2089. Tovich PR and Oko RJ (2003) Somatic histones are components of the perinuclear theca in bovine spermatozoa. J Biol Chem 278,32431-32438. Trasler JM, Hales BF and Robaire B (1985) Paternal cyclophosphamide treatment of rats causes fetal loss and malformations without affecting male fertility. Nature 316,144-146. Trasler JM, Hales BF and Robaire B (1986) Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny. Biol Reprod 34,275-283. 150 Wong AH, Tarn NW, Yang YL, Cuddihy AR, Li S, Kirchhoff S, Hauser H, Decker T and Koromilas AE (1997) Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and double-stranded RNA signaling pathways. EMBO J 16,1291-1304. Wouters-Tyrou D, Martinage A, Chevaillier P and Sautiere P (1998) Nuclear basic proteins in spermiogenesis. Biochimie 80,117-128. Yin Z, Haynie J, Williams BR and Yang YC (2003) C114 is a novel IL-11- inducible nuclear double-stranded RNA-binding protein that inhibits protein kinase R. J Biol Chem 278,22838-22845. Yu YE, Zhang Y, Unni E, Shirley CR, Deng JM, Russell LD, Weil MM, Behringer RR and Meistrich ML (2000) Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice. Proc Natl Acad Sci U S A 97,4683-4688. Zini A, Kamal KM and Phang D (2001) Free thiols in human spermatozoa: correlation with sperm DNA integrity. Urology 58,80-84. 151 FIGURES and LEGENDS 152 Figure 3-1. Susceptibility to acid-induced DNA denaturation in cauda epididymal spermatozoa assessed with acridine orange. Cells were collected after 28 days of chronic cyclophosphamide exposure, and on days 14, 21 and 28 after sub chronic exposure. (A) Extent of DNA denaturation represented by the mean DNA fragmentation index (DFI) of cell populations. (B) The percentage of abnormal spermatozoa with denatured DNA (%DFI). Black bars, control; gray bars, cyclophosphamide. Data shown represent means ± SEM (n=6-8). *Significantly different from time-matched controls; # significantly different from sub-chronic cyclophosphamide day 14 and 28 groups (p 153 300 A Germ Cells Targeted 14 Days = Round Spermatids 21 Days =Elongating Spermatids 280 28 Days =Elongated Spermatids ü: *# 0 * c tU Q) 260 ~ 1 * E..... Q) a. (j) 240 tU "'C :J tU ü 220 28 Days 14 Days 21 Days 28 Days Chronic Exposure Sub-Chronic Exposure B 50 40 * ü: o '* 30 E ~ CI) tU 20 "'C :J tU Ü 10 0-'---- 28 Days 14 Days 21 Days 28 Days Chronic Exposure Sub-Chronic Exposure Figure.3-2. Chromatin protamination and condensation status of epididymal spermatozoa assessed by chromomycin A3 (CMA3) ONA binding. (A) 28-day chronic cyclophosphamide-exposed caput and cauda epididymal spermatozoa pre-treated with 0, 0.1 and 1mM dithiothreitol (OTT). (A, inset) OTT-dependent CMA3 binding in 28-day chronic cyclophosphamide-exposed spermatozoa pre treated with 0-20mM OTT. (B) Sub-chronic cyclophosphamide-treated spermatozoa collected on days 14, 21 and 28 and pre-treated with or without 1mM OTT. Black bars, control; gray bars, cyclophosphamide. Data shown represent means ± SEM (n=4). *Significantly different from matched controls; t significantly different from control cauda epididymal spermatozoa; ff significantly differentfrom cyclophosphamide cauda epididymal spermatozoa (p<0.001). 155 A 1100 1200 Chronic Exposure ~ ~1000 1000 E Q; 800 0 900 c ~ ~ 600 "iii ~ -- Control - Caput c: 800 ~ 400 (]) IL ---0- Control - Cauda c: (") -T- CPA - Caput - 700 ~ 200 (]) ü --- CPA- Cauda * 'li 0 0 c: 0.1 0.2 0.5 1 2 5 (J) 600 o 0 OTT (mM) Cf) ~ 500 0 :::1 iL 400 (") 100 0 Caput Cauda Caput . Cauda Caput Cauda OmM OTT 0.1mM OTT 1mM OTT B Sub-Chronic Exposure 600 Germ CeUs Targeled 14 Days = Round Spermatids 21 Days = Elongating Spermatids * 500 28 Days = Elongated Spermatids ~ "ii> * c: * 2 c: 400 (]) ü c: (]) 0 Ul 300 ~ 0 :::1 iL 200 «(") :2: u 100 0 OmM 1mM OmM 1mM OmM 1mM 140ays 210ays 280ays Cauda Sperm Figure 3-3. Thiol content in heads of caput and cauda epididymal spermatozoa after (A) 28-day chronic cyclophosphamide treatment and (B) sub-chronic CPA treated spermatozoa collected on days 14, 21 and 28. Measurements were taken without dithiothreitol (DIT; free thiols, SH) or with 1mM DIT pre-treatment (total thiols, SS+SH). Black bars, control; gray bars, cyclophosphamide. Data shown represent means ± SEM (n=4). *Significantly different from matched controls (p 157 A 1000 Chronic Exposure Z. 900 "iii c: Q) .....c: c:~ 800 ~ VJ ~o ::l LL 700 0::m m E 600 200 0...1.--- OmM OTT 1mM OTT OmM OTT 1mM OTT Caput Sperm Cauda Sperm Sub-Chronic Exoosure B 400 Germ CeUs Targeted 14 Days = Round Spermatids 21 Days =Elongating Spermatids 375 28 Days =ElongatedSpermatids Z. "iiic: .....Q) c: 350 ~ c: ~ VJ ~ 325 o ::l LL mL- m 300 E 100 o OmM 1mM OmM 1mM OmM 1mM 14 Days 21 Days 28 Days Cauda Sperm Figure 3-4. Immunofluorescence assessment of protamine 1 content in cauda epididymal spermatozoa pre-treated with or without 1 or 20mM dithiothreitol, .(DTT). (A) Images of spermatozoa labeled with HUP1N protamine 1 antibody, x400. Parameters measured were (8) fluorescence intensity of spermatozoa, (C) spermatozoal head area and (D) fluorescence intensity of spermatozoal heads 2 per IJm . (8-D) 81ack bars, control; gray bars, cyclophosphamide. Data shown represent means ± SEM (n=3). *Significantly different from OmM DTT group (p 159 A Control CPA B 110 100 ~ OmM 'iii c: 90 DlT CI> Cl>ë c: - "Ë ~ 80 s ~ e 0 70 a.. ~ 0 ::J 60 20mM u: OlT 50 0 OmM 1mM 20mM Cauda Epididymal Sperm on C D 23 6 N 22 *# *# '"~ 5 *# E 2- ~ "t:l eii 21 c: 4 '"~ '"CI> $ « * I E "t:l 20 3 E CI> CI> CI> <> '" c. c: I en CI> E 19 0 2 CI> ~ a. '"0 en ::J 18 u: 0 0 OmM lmM 20mM OmM lmM 20mM on on Figure 3-5. Two-dimensional electrophoretic separation of cauda epididymal spermatozoal head basic proteins from (A) control and (8) 28-day chronic cyclophosphamide (CPA)-treated rats. Spots unique to each treatment group are indicated with blue circles. Red and yellow circles indicate spots showing increased or decreased expression compared to the other treatment, respectively. Spots with <1.5-fold change in expression are indicated with green circles. Protein identification was achieved for spots 1-12 (see Table 3-1). MW, molecular weight; pl, isoelectric point. (n=5). 161 12 r- MW (kDa) A pl9 Control ~ 116 ~97 ... "66 /11 • r ./10 .'45 _ .....,,.31 _21 _____ 14 __---6.5 12 r--" MW (kDa) B 1~19 CPA 1 ...... 116 1 ...... 97 1 1 .-...- 66 11 .111.45 = • 31 21 7 r 14 f\a • r 6.5 TABLES and LEGENDS 163 Table 3-1 Expression of proteins identified by mass spectrometry CPA, cyclophosphamide; HNRPA 1, heterogeneous nuclear ribonucleoprotein A 1; PRKRIP1j)redicted, protein kinase R interacting protein 1 (interleukin 11 inducible) (predicted); CHD4, chromodomain helicase DNA binding protein 4; HIST2H4, germinal histone H4; HIST1 H2BL, histone H2B; FABP9, fatty acid binding protein 9; ZPBP, zona pellucida binding protein. a Change in expression is < 1.5-fold. 164 Protein Spot No. MW (kDa) pl Control CPA % Change Function Localization HNRPA1 1 11.8 10.8 0.11 0.18 64 RNAIDNA binding Nucleus/Cytoplasm/Nucleolus PRKRIP1_predicted 2 21.5 11.8 2.18 0.76 -65 RNA bindingrrranslationai regulator Nucleolus CHD4 3 21.0 11.5 6.63 7.48 13" DNA bindingrrranscriptionai regulator Nucleus HIST2H4 4 12.3 11.4 0.86 0.56 _35" DNA binding/Male pronuclear formation Nucleus/Perlnuclear Theca 5 12.1 11.6 4.26 4.02 -68 HIST1H2BL 6 14.3 10.8 6.35 5.06 -20· DNA bindlng/Male pronuclear formation Nucleus/Perinuclear Theca FABP9 7 15.1 9.1 0.53 1.15 117 Lipid binding/Sperm structure and stability Perinuclear Theca 8 15.7 9.2 1.50 1.90 27· 9 15.6 11.5 2.35 1.29 -45 ZPBP (Sp38) 10 39.7 10.1 1.16 1.39 20· Egg zona pelluclda blndlng Sperm Surface 11 46.3 11.2 0.37 0.62 68 Beta-Defensin 20 12 11.8 10.7 0.06 0.13 117 Anti-mlcroblal sperm bindlng protein/ Sperm maturation and capacitation Sperm Surface Connecting Text On one level, chromatin remodelling during spermiogenesis was affected following cyclophosphamide exposure; effects on chromatin condensation were . germ cell-phase specific. Oh another level, spermatozoal chromatin structure depends on the precise organisation of the DNA into loop domains by mainly proteinaceous, nuclear matrix; this structure may serve to regulate gene expression. Previously reported altered gene expression in male germ cells and early preimplantation embryos following chronic cyclophosphamide exposure prompted the development of the last objective of this thesis. T 0 assess whether cyclophosphamide affects the sperm nuclear matrix, the next chapter reveals the effects chronic cyclophosphamide treatment on the expression of sperm nuclear matrix proteins. 166 CHAPTER4 Chronic Cyclophosphamide Exposure Alters the Profile of Rat Sperm Nuclear Matrix Proteins Alexis M. Codrington, Barbara F. Hales and Bernard Robaire Submitled to Bi%gy of Reproduction 167 4.1 ABSTRACT Chronic exposure of male rats to the alkylating agent cyclophosphamide, a well-known male-mediated developmental toxicant, alters gene expression in male germ cells, as weil as in early preimplantation embryos sired by cyclophosphamide-exposed males. Sperm DNA is organized by the nuclear matrix into loop domains in a sequence specifie manner. In somatic cells, loop domain organization is involved in gene regulation. Various structural and functional components of the nuclear matrix are targets for chemotherapeutic agents. Consequently, we hypothesize that cyclophosphamide treatment alters the expression of sperm nuclear matrix proteins. Adult male rats were treated for 4 weeks with saline or cyclophosphamide (6.0 mg/kg/day) and the nuclear matrix was extracted from cauda epididymal sperm. Proteins were analyzed by two dimensional gel electrophoresis. Identified proteins withinthe nuclear matrix proteome were mainly involved in cell structure, transcription, translation, DNA binding, protein processing, signal transduction, metabolism, cell defense or detoxification. Interestingly, cyclophosphamide selectively induced numerous changes in cell defense and detoxification proteins, most notably, in ail known forms of the antioxidant enzyme, glutathione peroxidase 4, in addition to an uncharacterized 54 kDa form; an ove ra Il increase in glutathione peroxidase 4 immunoreactivity was observed in the nuclear matrix extracts from cyclophosphamide-exposed spermatozoa. An increase in glutathione peroxidase 4 expression suggests a role for this enzyme in maintaining nuclear matrix stability and function. These results lead us to propose that a change in 168 composition of the nuclear matrix in response to drug exposure may be a factor in altered sperm function and embryo development. 4.2 INTRODUCTION Chromatin structural organization has a significant impact on cel! function. Two levels of organization within somatic nuclei, nucleosomal and DNA loop domain organization, play a role in gene regulation [1]. The formation of DNA loop-domains is of particular interest as DNA is atlached in a sequence-specifie manner to the nuclear matrix, the non-chromatin structure of the nucleus [2]. The nuclear matrix consists of an internai ribonucleic protein network and residual nucleoli bounded by peripheral lamins [3,4]; its interaction with DNA has been implicated in many essential nuclear functions, including DNA replication and repair, transcription, and RNA processing and transport [5-8]. The relationship between nuclear function and organization has been weil established in somatic cells [9]; however, the functional significance of sperm structural organization remains elusive. Spermatozoal chromatin is not organized into nucleosomes; protamines replace histones during spermiogenesis, resulting in highly condensed toroids [10]. Interestingly, sperm do maintain the organization of DNA by the nuclear matrix into loop-domains [11]. In the mouse sperm nucleus, the nuclear matrix forms part of the perinuclear matrix, which consists of the surrounding perinuclear theca and a filamentous internai network [12]. Proteins that make up the nuclear matrix vary in a cel! type- and tissue specifie manner and change as cells differentiate [13-16]. Changes also occur in 169 the organization and general protein composition of the nuclear matrix dûring spermatogenesis [17,18]. Altering the composition of the nuclear matrix could be associated with DNA disorganization; disrupting the association of loop-domains with the nuclear matrix may alter nuclear function [19]. Structural and functional components of the nuclear matrix are targets in somatic ce Ils for chemotherapeutic agents. In somatic cells, alkylating agentsinteract with nuclear matrix proteins and with DNA close to matrix-bound replication and transcription sites [20-24]. Abnormal DNA organization or an unstable sperm nuclear matrix may play a role in male factor infertility; under these conditions, embryO development is affected [25,26]. We have shown previously that chronic exposure of male rats to cyclophosphamide (CPA), a bifunctional alkylating agent, results in pre- and post implantation embryo loss, and malformed and growth retarded progeny [27-29]. CPA exposure during spermiogenesis and epididymal transit, crucial times during male germ cell development as the genome is being remodeled and packaged, creates DNA single-strand breaks and crosslinks [30-32] and, notably, results in altered gene expression in male germ cells as weil as in early preimplantation embryos sired by CPA-exposed males [33-35]. We hypothesize that an action of CPA is to affect germ cell function by targeting components of the sperm nuclear matrix. Many somatic cell nuclear matrix proteins have been characterized [36]. However, little is known about the components of the sperm nuclear matrix or the precise roles of these proteins in sperm function or embryo development. The aim of this study was to use proteomic strategies to identify proteins of the 170 nuclear matrix and to elucidate the effects of chronic CPA exposure on the expression of matrix proteins. 4.3 MATERIALS AND METHODS Animal Treatments Adult male Sprague-Dawley rats (400-450 g) were obtained from Charles River Canada (St. Constant, OC, Canada), maintained on a 14L:10D light cycle and provided with food and water ad libitum. Rats were gavaged with saline or CPA (6mg/kg/day, CAS 6055-19-2, Sigma-Aldrich Ltd., Oakville, ON, Canada). To capture cauda epididymal spermatozoa exposed to CPA throughout spermiogenesis and epididymaltransit, animais were euthanized by decapitation 4 weeks after initiation of treatment [37]. Animal handling and care were done in accordance with the guidelines established by the Canadian Council on Animal Care. Sperm Collection Sperm collection was done according to Calvin [38] with modifications. Epididymides were first removed, trimmed free of fat and washed in pre-chilled phosphate buffer (PB, 20mM, pH 6.0, containing 1mM EDTA). The cauda region was removed, transferred to 8ml of fresh buffer on ice and thoroughly minced with sterile scalpels. The tissue was left for 5 min on ice to allow the spermatozoa to disperse and was then strained through a BD Falcon 70J,Jm nylon cell strainer (VWR International Co., Mississauga, ON, Canada), washed with 2ml of fresh PB, and the total cell suspension centrifuged at 1000 x 9 for 10 min 171 at 4°C. The pellet was washed once and then resuspended in 4ml of PB containing 40J.J1 of protease inhibitor cocktail (P8340, Sigma-Aldrich, Ltd.). Sperm were sonicated on ice and sperm heads isolated using discontinuous sucrose gradients made with PB. Twelve milliliters of sonicated sperm in 1.80M sucrose were layered over 13ml each of cold 2.05M and 2.20M sucrose and centrifuged at 91,400 x 9 in a Beckman SW 28 rotor for 70 min at 4°C. The pellet was resuspended in PB containing protease inhibitor cocktail (1 :100 dilution) and stored at -BOoC. Nuclear Matrix Extraction Sperm heads were resuspended in 500J.J1 of solution containing 1% SOS, 50mM Tris-HCI pH 7.5, 1mM EDTA, and 5J.J1 protease inhibitor cocktail and shaken using a Fisher Vortex Genie 2 mixer fitted with a TurboMix attachment (VWR International Co.) for 10 min at room temperature. This treatment has been shown to remove the acrosome, ail membranes, basal striations, the posterior nuclear ring, and the ventral spur of the postacrosomal sheath, leaving the condensed nucleus and partially attached perinuclear theca perforatorium, as weil as less prominent remnants of perinuclear material in the midlateral and posterior regions of the sperm head [38,39]. The samples were then washed three times with 50mM Tris-HCI pH 7.5 and resuspended in 500J.J1 of decondensation buffer (40mM 1,4-dithiothreitol, 0.25M (NH4)2S04, 25mM Tris HCI, pH 7.5, 5J.J1 protease inhibitor cocktail) for 40 min at room temperature. A 26- 1/2G needle was used to gently break up any clumps and 4000U of RNase-free deoxyribonuclease 1 (Sigma-Aldrich Ltd.) was added for 60 min at room 172 temperature. Samples used for gel electrophoresis were pelleted, air-dried and stored at -20°C or were used immediately for immunofluorescence studies. Two-Oimensional (20) Gel Electrophoresis Protein separation and gel image analyses were conducted by the McGiII University and Genome Quebec Innovation Centre (Montreal, QC, Canada) using material from Invitrogen Inc. (Burlington, ON, Canada), except where noted, and the Invitrogen ZOOM IPGRunner System protocol. Fifty micrograms of protein were resuspended in 155~1 of rehydration buffer (9.8M urea, 10mM 1,4- dithioerythritol, 4% CHAPS, 20mM Tris) supplemented with 2% IPG Buffer pH 3- 10NL (Amersham Biosciences, Baie D'Urfe, QC, Canada). Seven centimeter ZOOM Dry Strips, pH 3-10NL were rehydrated for 16-18 hrs andisoelectric focusing (IEF) done with a voltage gradient (200V- 2000V) applied as recommended by the manufacturer. After IEF was complete, strips were equilibrated with 1X NuPAGE LOS Sam pie Buffer, containing 2% OTT and then alkylated with iodoacetamide. Both steps were done at room temperature for 15 min. Electrophoresis in the second dimension was done on 4-12% Bis-Tris precast mini-gels in XCeli SureLock Mini-Cells filled with MOPS SOS Running Buffer. Broad range protein molecular weight markers (0.9 J,Jg/gel, Amersham Biosciences) were used and 200V were applied for 50min. Gels (n=3 each for control and treated) were fixed overnight in 50% methanol/10% acetic acid, silver stained, scanned and analyzed using Phoretix 2004 Image Analysis software (Amersham Biosciences). Following background subtraction and normalization, intensities of the spots were calculated. One gel was then chosen as a reference 173 and the other gels compared to the reference gel to create an average control and CPA gel, which were then compared. Spots were considered if present in at least two out of three gels and protein expression was considered changed only if the difference was at least 2-fold; this is equivalent to an increase of 100% or decrease of 50%. Mass Spectrometry Spots were excised from the gel and subjected to trypsin digestion on a robotic MassPREP Station (Waters-Micromass, Milford, MA, USA), as per the manufacturer's instructions. Gel pieces were first washed twice with water for 20 min, destained twice in a 120".11 solution of 30mM potassium ferricyanide and 100mM sodium thiosulfate mixed 1: 1 for 15 min and then dehydrated with 751-11 of 100% acetonitrile. Samples were reduced, in the dark, with 501-11 of 10mM OTT for 30 min followed by alkylation with 501-11 of 55mM iodoacetamide for 20 min and 1001-11 of 100% acetonitrile for 5 min. After washing and dehydration in 100mM ammonium bicarbonate and 100% acetonitrile, respectively, gel pieces were covered and digested for 4.5 hours with 6 ng/I-II of trypsin go Id (Promega, Madison, WI, USA) in 100mM ammonium bicarbonate. Peptides were extracted with 301-11 formic acid (FA) solution (1% FA in 2% acetonitrile) for 30 min, twice with 121..J1 FA solution and then 121-11100% acetonitrile for 30 min. Nanoflow chromatography of digested peptides was done on an Agilent 1100 series nanopump (Agilent Technologies Inc., Mississauga, ON, Canada) at a flow-rate of 200nl/min. Sam pie injection and desalting were performed with an Isocratic Agilent 1100 series pump at 151..JI/min for 5 min. A trapping column 174 (Agilent) packed with Zorbax 300SB-C18 (5 x 0.3 mm) was used for sam pie desalting. Peptide separation was done with a Biobasic C18 (10 x 0.075 mm) picofrit column (New Objective, Woburn, MA, USA). Peptides were eluted using a 20 min gradient with solvent A (0.1 % FA) and solvent B (95% acetonitrile:0.1%FA) from 90%Al10%B to 100%B. Electrospray mass spectrometry was done with a 4000 Q TRAP System (Applied Biosystems/MDS Sciex, Foster City, CA, USA). Enhanced MS scans were acquired between 350-1600 m/z using a scan speed of 4000 amu/sec and active dynamic filt time. Information-dependent MS/MS analysis was performed on the three most intense multiply charged ions; a dynamic exclusion of 90 sec was used to limit resampling of previously selected ions to two events. Three averaged MS/MS scans were acquired between 70-1700 m/z at a scan speed of 4000 amu/sec. Fixed fill time was set at 20 ms with QO trapping and rolling collision energy of .±. 3 eV. Peaklists for peptide mapping searches were generated with Mascot script 1.6 for Analyst 1.4.1 software (Applied Biosystems/MDS Sciex). Spectral processing included peak smoothing and centroiding without de-isotoping. Database searches were done with Mascot 1.9 (Applied Biosystems/MDS Sciex) using carbamidomethyl cysteine as a fixed modification, methionine oxidation as a variable modification, and 1.5 Da precursor mass and 0.8 Da fragment mass tolerances. Glutathione Peroxidase 4 (GPX4) Immunoblotfing Immediately after electrophoresis in the second dimension, unstained gels were transferred to Hybond-P polyvinylidene difluoride (PVDF) membrane 175 (Amersham Biosciences), using the Invitrogen XCeli1i Blot Module and protocol. Briefly, the blot module, gel and membrane were assembled and inserted into the XCeli SureLock Mini-Cell. The module was filled with NuPAGE Transfer Buffer (Invitrogen) supplemented with 10% methanol and protein transfer was done at 30V for 1 hr. Efficiency of protein transfer was confirmed by staining blots with Ponceau S (Sigma-Aldrich Ltd.). Following destaining in deionized water, membranes were air dried and stored at room temperature. When ready to use, the membrane was washed in 100% methanol for a 2 seconds, followed by 10 min in 20mM Tris-HCI pH 7.6, containing 0.8% NaCI and 0.1 % Tween-20 (TBS T). Membranes were then blocked for 1 hr at room temperature in 5% non-fat milk in TBS-T, washed for 2 min in TBS-T and then incubated overnight at 4°C with rabbit polyclonal anti-GPX4 (Abcam Inc., Cambridge,MA, USA) diluted 1:20000 in TBS-T containing 3% non-fat milk. After two brief washes with TBS-T, membranes were washed once in 40ml for 15 min and twice in 20ml for 10 min with TBS-T. Membranes were then incubated for 1 hour at room temperature with horseradish peroxidase-conjugated rab bit IgG antibody (Amersham Biosciences) diluted 1:15000 in TBS-T containing 5% non-fat milk and washed. Antibody detection was done using the ECL Plus Western Blotting system (Amersham Biosciences). GPX41mmunofluorescence GPX4 immunoreactivity was determined in sperm collected after incubation in 1% SOS or after nuclear matrix extraction. Ten microliter droplets were placed on slides and left on ice for 20 min. Siides were then washed in 176 PBS (3 x 2 min), fixed in 2% paraformaldehyde for 20 min at room temperature, washed and blocked with PBS containing 5% normal goat serum (Vector Laboratories Inc., Burlington, ON, Canada) and 1% bovine serum album in (BSA, Sigma-Aldrich, Ltd.) for 30 min at room temperature. Subsequently, cells were covered overnight at 4°C with primary antibody solution containing 1% BSA and rabbit polyclonal GPX4 antibody (1 :20 dilution) in PBS, washed with PBS (3 x 5 min), covered for 1.5 hrs in the dark at room temperature with secondary antibody solution containing Alexa Fluor 488 conjugated goat anti-rabbit IgG antibody (1 :200 dilution, Invitrogen) in PBS, and finally washed in PBS. Slides were covered with Vectashield mounting medium containing DAPI (Vector Laboratories Inc.) and kept at 4°C in the dark. Pictures were taken using a DAGE-MTI CCD300-RC camera (DAGE-MTI Inc., Michigan City, IN, USA) atlached to an Olympus BX51 epifluorescence microscope. 4.4 RESULTS 20 Gel Analysis of Sperm Nuclear Matrix Proteins Sperm nuclear matrix proteins analyzed by 2D gel electrophoresis resulted in reproducible protein patterns. Three gels each were run for control and chronic CPA-treated sperm protein samples; protein profiles differed with treatment (Figure 4-1). The average control gel (Figure 4-1A) consisted of 290 protein spots that appeared in at least two out of the three gels analyzed and corresponded to 90-96% of the total number of spots detected on individual gels. ln comparison, 309 protein spots were found on the average CPA gel (Figure 4- 1 B), corresponding to 94-98% of ail spots appearing on individual gels. Overall 177 changes in protein expression are iIIustrated in Figure 4-2A. The expression of 7 protein spots (2%) was unique to control samples and 26 (8%) to CPA samples. Two hundred and eighty-three protein spots were expressed in both groups; analysis of protein expression changes> 2-fold revealed 34 spots (11 %) that increased and 38 (12%) that decreased following CPA exposure. Thirty-two spots were chosen for mass spectrometry from each expression group (increased, decreased, control-specifie, CPA-specific and no change). Twenty-four spots were identified, as labeled in Figure 4-1 and summarized in Table 4-1. Selection was based on: 1) spots present in charge trains, such as spot 5; 2) spots located in the acidic region where other somatic nuclear matrix proteins have been previously identified, such as spots 7-12; and 3) spots that consistently appeared on ail three gels, such as spots 4 and 24. The majority of identified spots was either increased in expression or unique to CPA samples; none were unique to control sperm and spot 2 was the only one that decreased (by 67%). Ten of the identified proteins were represented byat least two distinct spots on the gels, suggesting that these proteins are modified or exist in different isoforms (for example L1M domain containing preferred translocation partner in lipoma (LPP), in spots 3 and 4, or GPX4, found in spots 5, 16, 17, 18, 19, 20, 21 and 23). Thirteen spots matched to more than one protein (for example, analysis of spot 24 gave significant results for phosphatidylethanolamine binding protein, PEBP; proteosome (prosome, macropain) subunit, beta type 6, PSMB6; and similar to Ran-interacting protein MOG-1 (predicted». Of note, protein fragments were identified from significant mass spectrometry analysis results for peptides that only partially covered a 178 protein sequence. Despite the use of protease inhibitors during sample preparation, these protein fragments may be proteolytic cleavage products. Analyzed spots for three proteins (heterogeneous nuclear ribonucleoprotein K, HNRPK; regulator of telomere elongation helicase 1, RTEL 1; and spermidine/spermine N1-acetyl transferase (mapped» contained only a fragment of the identified proteins; the mass of the proteins calculated from the 2D gel was below the expected mass of the proteins, calculated from their amino acid compositions. Based on information in the nuclear matrix NMP-db database [40] and in the literature, 11 of the identified proteins are known nuclear matrix components (HNRPK, GPX4, PSMB6 and protein phosphatase 1, catalytic subunit, gamma isoform; PPP1 CC) or are related to previously identified nuclear matrix proteins (DnaJ (Hsp40) homolog, subfamily B, member 6, DNAJB6; glutathione-S transferase omega 2, GST02; glutathione-S-transferase mu 5, GSTM5; heat shock 70kDa protein 2, HSPA2; RTEL 1; and proteosome (prosome, macropain) subunit, alpha type 5, PSMA5 and beta type 4, PSMB4). Along with GPX4, HNRPK, and PPP1 CC, glutathione-S-transferases and DnaJ (Hsp40) proteins, are expressed in nucleoli; ail except for PPP1 CC have been identified in spots that mainly increased in expression following CPA exposure or were specifie to CPA-exposed sperm. These proteins may be a part of nuclear matrix residual . nucleoli; these are known to be present in somatic cells [3,41]. 179 Functional Analysis of Identified Proteins Surprisingly, only 7 of the 24 identified proteins were previously identified as components of spermatozoa heads (GPX4, HSPA2, calicin, DNAJB6, PEBP, capping protein (actin filament) muscle Z-line, beta (CAPZB), and testis-specific serine kinase 2 (TSSK2», while 4 others have other known roles in spermatogenesis in the testis (GST02, GSTM5, PPP1 CC and RTEL 1). Information concerning the putative functions of the proteins was found in the NCBI non-redundant and SWISS-PROT protein sequence databases or in the literature. In general, the proteins are involved in cell structure, transcription and translation regulation, DNA binding, protein processing, signal transduction, metabolism, cell defense and detoxification (Table 4-1). The unknown proteins have not yet been characterized. Out of the three structural proteins identified, calicin is a known component of the sperm head perinuclear theca [42,43]. Spot number 4, containing calicin and growth-arrest-specific 2 (GAS2), increased by 1850% after CPA exposure compared to control. There was no change in the expression of CAPZB; however, it migrated higher then its calculated molecular weight of 32 kDa. Despite this discrepancy, CAPZB was accepted as a positive identification since multiple significant peptide matches were found following mass spectrometry analysis. Morphological assessment of whole mature spermatozoa following chronic CPA exposure showed that cells were normal and that such treatment did not appear to affect the stability of the nuclear matrix, as the structure after extraction (confirmed by negative DAPI staining) was intact (Figure 4-2B). 180 GPX4 Expression Surprisingly, cell defense and detoxification proteins were present in abundance on the 2D gels. CPA induced changes in the amount of ail known forms of the antioxidant enzyme, GPX4; in addition, an uncharacterized 54 kDa form was identified (Table 4-1). Mass spectrometry results were confirmed by western blotting and revealed the full effect of CPA on GPX4 expression (Figure 4-3). Charge-trains between 20 and 31 kDa were observed with an apparent ove ra Il increase in expression after CPA treatment compared to controls. Distinct spots were clearly different between control and CPA blots (Figure 4-3, open arrows). In particular, a form of GPX4 at -54 kDa is shown in the CPA blot (Figure 4-38, black arrow) and an increased am ou nt of the 34 kDa post translationally modified form of GPX4 was detected (Figure 4-3, arrowhead); this form has been shown to be localized to the nucleoli of spermatogonia, spermatocytes and spermatids [44]. Figure 4-4 further validates the results obtained by mass spectrometry and confirms the presence of GPX4 in the nuclear matrix. For comparison, membrane-free sperm heads treated with 1% SDS were immunostained (Figure 4-4A). Not only was there increased GPX4 immunoreactivity in CPA-exposed samples compared to controls but also this increased immunoreactivity was found in the sperm nuclear matrix extract. Interestingly, immunoreactivity was greater in SDS-treated samples compared to the respective nuclear matrix extracts (Figure 4-48). Thus, these results indicate that CPA-treatment resulted in an increase in chromatin-bound GPX4, as weil as matrix-specific increased 181 expression. Omission of the primary antibody resulted in an absence of immunoreactivity (data not shown). 4.5 DISCUSSION The nuclear matrix is a dynamic structure with a morphology and protein composition that varies with the functional state of the nucleus [45]. It has been possible to identify specifie cell and tissue types by the electrophoretic pattern of their nuclear matrix proteins [14-16]. Both normal and neoplastic samples can be identified by differences in nuclear matrix protein patterns [46]. The present study is the first to go beyond structural evaluation and extensively examine the rat spermatozoal nuclear matrix by identifying sorne of the protein components. Significantly, we have demonstrated that exposure to an alkylating agent and male-mediated developmental toxicant alters the spermatozoal nuclear matrix protein profile. A major transition in sperm chromatin structure and function occurs during spermiogenesis, resulting in the formation of mature spermatozoa competent to fertilize. Changes reported in the nuclear matrix structure and protein profile of round and elongating spermatids reflect the morphological changes and remodeling of chromatin that occur during spermatid differentiation [17]. Our results show that targeting germ cells throughout spermiogenesis with CPA modifies the make-up of the nuclear matrix. Such an effect may alter chromatin reorganization by the nuclear matrix both during spermatogenesis and in the zygote after fertilization. Interestingly, there was no apparent effect of CPA exposure on the structure of the nuclear matrix. Calicin and F-actin capping proteins like CAPZB 182 are known components of the sperm head perinuclear theca, possibly involved in sperm morphogenesis and stability, and F-actin organization and biogenesis, respectively [42,47]. GAS2 has been implicated also in actin filament organization [48]; hyperphosphorylation and proteolytic c1eavage at its C-terminal end result in an irregular cell shape and actin rearrangement, processes which are triggered as part of a series of apoptotic events [49]. Overexpression of other cytoskeletal proteins results in a change in actin organization; however, this is not the case with overexpressed GAS2 [48], as we see in this study. We did not identify any of the previously described classical cytoskeletal proteins found in somatic cell matrices, such as vimentin, keratin or actin, or the perinuclear lamins A, 8 and C [50,51]; interestingly, actin, myosin, cytokeratins and spectrin have been described in the guinea pig sperm nuclear matrix [52]. Previous studies report differential expression of stress response genes in male germ cells after chronic exposure of male rats to CPA [33]. Factors other than matrix instability may alter DNA organization and, subsequently, affect gene function. Alkylating agents preferentially' bind to matrix proteins and matrix associated DNA [23,24]. Proteins are most likely to give rise to multiple spots as a consequence of multiple post-translational modifications; most eukaryotic proteins are modified and these modifications are often essential for their function [53]. In this study, some differences in expression are probably due to modification of the nuclear matrix proteins following drug exposure. Tew and colleagues [21] have shown that fibrillar components of the matrix and ribonucleoproteins are alkylated following exposure to 1-(2-chloroethyl-3- 183 cyclohexyl)-1-nitosourea (CCNU) and chlorozotocin, but the effects of these modifications on protein function are not known. Reduced binding of DNA to the matrix may be a function of interference with the DNA recognition sites by alkylation at specifie bases; in vitro alkylated DNA has a reduced interaction with matrix proteins [21]. Chromatin loops associate with the nuclear matrix at specifie regions called matrix attachment regions (MARs) [54]; these MARs are involved in DNA replication and repair and in various aspects of gene regulation [55], including mRNA transcription and processing via their involvement in attachment and/or association with newly transcribed mRNA, pre-mRNA splicing machinery and ribonucleoprotein particles [56]. In light of this,two proteins, HNRPK and LPP that were present at elevated levels in the nuclear matrix extracted from CPA-exposed sperm are of particular interest. HNRPK is a unique member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that preferentially binds single-stranded DNA [57]. It acts as a docking site to integrate signaling cascades between anchored protein complexes and, as such, is a multifunctional molecule implicated in transcription activation and chromatin remodeling, in addition to the more typical hnRNP functions of mRNA splicing, transport and translation [58]. Studies on LPP demonstrate that it has the capa city to activate transcription and suggest that, like HNRPK, it may serve as a scaffold upon which protein complexes are assembled [59]. Increased expression of GPX4 in the nuclear matrix was intriguing, given its role not only as an intracellular antioxidant enzyme, directly reducing lipid 184 hydroperoxides [60], but also in apoptosis inhibition [61], œil cycle regulation [62], and embryo development [63]. Most interestingly, in spermatozoa GPX4 appears to have two functions: 1) protamine disulfide crosslinking, where it uses the protamine cysteine residues instead of glutathione as reductants and acts as a thiol peroxidase when bound to DNA, and 2) protection of sperm against oxidative damage [64]. Sperm nuclear GPX4 has a molecular weight of 34 kDa and is bound to DNA. However, once spermatozoa reach the caput epididymal region, about two-thirds of the 34 kDa enzyme is processed into smaller proteins, with molecular masses between 22 and 29 kDa, that do not bind to DNA; their enzymatic properties are not affected [64,65]. A 20 kDa form, identical to cytosolic GPX4, is also present in heads of spermatozoa [65]. Each of these forms was present in our extracts. The nucleoli in mature spermatozoa are inactive; only nuclear vacuoles containing fibrils remain [66]. Work done by Puglisi [67] showed that GPX4 localizes to fibrous material in electron-Iucent spots in condensed epididymal sperm; these could be areas of residual nucleoli. A nucleolar GPX4 with a molecular mass of 34 kDa has also been identified in the nucleoli of spermatogonia, spermatocytes and spermatids [44]. Under normal conditions, GPX4 appears to be present within the head of spermatozoa both bound and unbound to DNA and, to a lesser extent, in the nuclear matrix; GPX4 expression increases following CPA exposure. Exposure to CPA or acrolein, a metabolite of CPA, induces the formation of reactive oxygen species (ROS) (68,69] and lipid peroxidation [70]. Sperm are highly susceptible to lipid peroxidation, and the induction of ROS is correlated with decreased 185 capacity to undergo the acrosome reaction [71] and DNA damage [72]. Increased nuclear expression of GPX4 may contribute to antioxidant defense mechanisms; however, functions served by localization of GPX4 to the nuclear matrix are less evident. In somatic ce Ils , overexpression of nucleolar GPX4 protects nucleoli from oxidative stress-induced damage [44]. Additionally, lipids are not only membrane components, but also represent important components of chromosomes, chromatin and the nuclear matrix [73]; sorne studies suggest that they are involved in DNA loop atlachment to the nuclear matrix, replication, transcription as weil as nuclear signal transduction [73-75]. If this is the case in sperm nuclei, CPA may induce oxidative stress and lipid peroxidation that may be reduced by GPX4. Changes in protein composition, or modifications thereof, may correlate with alterations in DNA organization, leading to changes in DNA function and protein expression. Changes in protein expression could be important in the regulation of sperm function. The impact on the post-fertilization early embryo remains to be determined; however, one effect may be ectopic protein expression in fertilized eggs. Studies on preimplantation embryos sired by CPA-treated males report temporal and spatial disruption of the rat zygotic genome activation; total RNA synthesis was higher and gene expression profiles were altered as early as at the 1-cell stage in comparison to controls [34,35,76]. This study provides further insight into the mechanisms by which CPA may exert its male-mediated effects on embryo development. The identification of sperm nuclear matrix components and their functions brings us closer to unraveling the mysteries of the matrix. 186 Acknowledgements We greatly appreciate the assistance of Leonid Kriazhev from the McGiII University and Genome Quebec Innovation Center with the 2D gel electrophoresis and mass spectrometry. 187 REFERENCES 1. Demeret C, Vassetzky Y, Mechali M. Chromatin remodelling and DNA replication: from nucleosomes to loop domains. Oncogene 2001; 20:3086- 3093. 2. Kramer JA,· Krawetz SA. Nuclear matrix interactions within the sperm genome. J Biol Chem 1996; 271 :11619-11622. 3. Berezney R, Coffey DS. Identification of a nuclear protein matrix. Biochem Biophys Res Commun 1974; 60:1410-1417. 4. Getzenberg RH, Pienta KJ, Ward WS, Coffey DS. Nuclear structure and the three-dimensional organization of DNA. J Ce" Biochem 1991; 47:289-299. 5. Mladenov E, Anachkova B, Tsaneva 1. Sub-nuclear localization of Rad51 in response to DNA damage. Genes Ce Us 2006; 11 :513-524. 6. Anachkova B, Djeliova V, Russev G. Nuclear matrix support of DNA replication. J Ce" Biochem 2005; 96:951-961. 7. Mortillaro MJ, Blencowe BJ, Wei X, Nakayasu H, Du L, Warren SL, Sharp PA, Berezney R. A hyperphosphorylated form of the large subunit of RNA polymerase " is assoeiated with splicing complexes and the nuclear matrix. Proc Natl Acad Sei USA 1996; 93:8253-8257. 8. van Driel R, Humbel B, de Jong L. The nucleus: a black box being opened. J Ce" Biochem 1991; 47:311-316. 9. Cremer T, Kreth G, Koester H, Fink RH, Heintzmann R, Cremer M, Solovei l, Zink D, Cremer C. Chromosome territories, interchromatin domain compartment, and nuclear matrix: an integrated view of the functional nuclear architecture. Crit Rev Eukaryot Gene Expr 2000; 10:179-212. 188 10. Hud NV, Allen MJ, Downing KH, Lee J, Balhorn R. Identification of the elemental packing unit of DNA in mammalian sperm cells by atomic force microscopy. Biochem Biophys Res Commun 1993; 193:1347-1354. 11. Ward WS, Coffey DS. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 1991; 44:569-574. 12. Bellve AR, Chandrika R, Martinova YS, Barth AH. The perinuclear matrix as a structural element of the mouse sperm nucleus. Biol Reprod 1992; 47:451- 465. 13. Zhao CH, Li QF, Zhao Y, Niu JW, Li ZX, Chen JA. Changes of nuclear matrix proteins following the differentiation of human osteosarcoma MG-63 cells. Genomics Proteomics Bioinformatics 2006; 4:10-17. 14. Getzenberg RH. Nuclear matrix and the regulation ofgene expression: tissue specificity. J Cell Biochem 1994; 55:22-31. 15. Kallajoki M, Osborn M. Gel electrophoretic analysis of nuclear matrix fractions isolated from different human cell lines. Electrophoresis 1994; 15:520-528. 16. Fey EG, Penman S. Nuclear matrix proteins reflect cell type of origin in cultured human ceUs. Proc Natl Acad Sci USA 1988; 85:121-125. 17. Chen JL, Guo SH, Gao FH. Nuclear matrix in developing rat spermatogenic cells. Mol Reprod Dev 2001; 59:314-321. 18. Klaus AV, McCarrey JR, Farkas A, Ward WS. Changes in DNA loop domain structure during spermatogenesis· and embryogenesis in the Syrian golden hamster. Biol Reprod 2001; 64:1297-1306. 19. Pienta KJ, Getzenberg RH, Coffey DS. Cell structure and DNA organization. Crit Rev Eukaryot Gene Expr 1991; 1:355-385. 189 20. Muenchen HJ, Pienta KJ. The role of the nuclear matrix in cancer chemotherapy. Crit Rev Eukaryot Gene Expr 1999; 9:337-343. 21. Tew KD, Wang AL, Schein PS. Alkylating agent interactions with the nuclear matrix. Biochem Pharmacol1983; 32:3509-3516. 22. Pienta KJ, Lehr JE. Inhibition of prostate cancer growth by estramustine and etoposide: evidence for interaction at the nuclear matrix. J Urol 1993; 149:1622-1625. 23. Fernandes DJ, Catapano CV. Nuclear matrix targets for anticancer agents. Cancer Cells 1991; 3:134-140. 24. Fernandes DJ, Catapano CV. The nuclear matrix as a site of anticancer drug action. Int Rev Cytol 1995; 162A:539-576. 25. Ward WS, Kimura Y, Yanagimachi R. An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development. Biol Reprod 1999; 60:702-706. 26. Barone JG, Christiano AP, Ward WS. DNA organization in patients with a history of cryptorchidism. Urology 2000; 56: 1068-1 070. 27. Trasler JM, Hales BF, Robaire B. Paternal cyclophosphamide treatment of rats causes fetal loss and malformations without affecting male fertility. Nature 1985; 316:144-146. 28. Trasler JM, Hales BF, Robaire B. Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny. Biol Reprod 1986; 34:275-283. 29. Trasler JM,- Hales BF, Robaire B. A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems. Biol Reprod 1987; 37:317-326. 190 30. Qiu J, Hales BF, Robaire B. Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa. Biol Reprod 1995; 52:33-40. 31. Qiu J, Hales BF, Robaire B. Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure. Biol Reprod 1995; 53:1465-1473. 32. Codrington AM, Hales BF, Robaire B. Spermiogenic germ cell phase specific DNA damage following cyclophosphamide exposure. J Androl 2004; 25:354-362. 33. Aguilar-Mahecha A, Hales BF, Robaire B. Chronic· cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells. Biol Reprod 2002; 66:1024-1032. 34. Harrouk W, Codrington A, Vinson R, Robaire B, Hales BF. Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo. Mutat Res 2000; 461 :229-241. 35. Harrouk W, Robaire B, Hales BF. Paternal exposure to cyclophosphamide alters ce II-ce Il contacts and activation of embryonic transcription in the preimplantation rat embryo. Biol Reprod 2000; 63:74-81. 36. Jackson DA. The principles of nuclear structure. Chromosome Res 2003; 11:387-401. 37. Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 1972; 52:198- 236. 38. Calvin HI. Isolation and subfractionation of mammalian sperm heads and tails. Methods Cell Biol 1976; 13:85-104. 39. O'Brien DA, Bellve AR. Protein constituents of the mouse spermatozoon. 1. An electrophoretic characterization. Dev Biol 1980; 75:386-404. 191 40. Mika S, Rost B. NMPdb: Oatabase of Nuclear Matrix Proteins. Nucleic Acids Res 2005; 33:0160-0163. 41. Nickerson JA, Krockmalnic G, Wan KM, Penman S. The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus. Proc Natl Acad Sci USA 1997; 94:4446-4450. 42. Lecuyer C, Oacheux JL, Hermand E, Mazeman E, Rousseaux J, Rousseaux-Prevost R. Actin-binding properties and colocalization with actin during spermiogenesis of mammalian sperm caliein. Biol Reprod 2000; 63:1801-1810. 43. von Bulow M, Heid H, Hess H, Franke WW. Molecular nature of caliein, a major basic protein of the mammalian sperm head cytoskeleton. Exp Cel! Res 1995; 219:407-413. 44. Nakamura T, Imai H, Tsunashima N, Nakagawa Y. Molecular cloning and functional expression of nucleolar phospholipid hydroperoxide glutathione peroxidase in mammalian cells. Biochem Biophys Res Commun 2003; 311 :139-148. 45. Penman S. Rethinking cell structure. Proc Natl Acad Sei USA 1995; 92:5251-5257. 46. Getzenberg RH, Pienta KJ, Huang EY, Coffey OS. Identification of nuclear matrix proteins in the cancer and normal rat prostate. Cancer Res 1991; 51 :6514-6520. 47. Hurst S, Howes EA, Coadwell J, Jones R. Expression of a testis-speeific putative actin-capping protein assoeiated with the developingacrosome during rat spermiogenesis. Mol Reprod Oev 1998; 49:81-91. 48. Brancolini C, Benedetti M, Schneider C. Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases. EMBO J 1995; 14:5179-5190. 192 49. Brancolini C, Schneider C. Phosphorylation of the growth arrest-specific protein Gas2 is coupled to actin rearrangements during Go-->G1 transition in NIH 3T3 cells. J Cell Biol 1994; 124:743-756. 50. Fey EG, Wan KM, Penman S. Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition. J Cel! Biol 1984; 98:1973-1984. 51. Matlern KA, Humbel BM, Muijsers AO, de Jong l, van Oriel R. hnRNP proteins and B23 are the major proteins of the internai nuclear matrix of Hela S3 cells. J Cell Biochem 1996; 62:275-289. 52. Ocampo J, Mondragon R, Roa-Espitia Al, Chiquete-Felix N, Salgado ZO, Mujica A. Actin, myosin, cytokeratins and spectrin are components of the guinea pig sperm nuclear matrix. Tissue CeU 2005; 37:293-308. 53. Larsen MR, Roepstorff P. Mass spectrometrie identification of proteins and characterization of their post-translational modifications in proteome analysis. Fresenius J Anal Chem 2000; 366:677-690. 54. Heng HH, Goetze S, Ye CJ, Liu G, Stevens JB, Bremer SW, Wykes SM, Bode J, Krawetz SA. Chromatin loops are selectively anchored using scaffold/matrix-atlachment regions. J Cell Sci 2004; 117:999-1008. 55. Boulikas T. Chromatin domains and prediction of MAR sequences. Int Rev Cytol 1995; 162A:279-388. 56. Oworetzky SI, Wright Kl, Fey EG, Penman S, Lian JB, Stein Jl, Stein GS. Sequence-specifie ONA-binding proteins are components of a nuclear matrix-atlachment site. Proc Natl Acad Sei USA 1992; 89:4178-4182. 57. Tomonaga T, Levens O. Heterogeneous nuclear ribonucleoprotein K is a DNA-binding transactivator. J Biol Chem 1995; 270:4875-4881. 193 58. Bomsztyk K, Denisenko 0, Ostrowski J. hnRNP K: one protein multiple processes. Bioessays 2004; 26:629-638. 59. Petit MM, Fradelizi J, Goisteyn RM, Ayoubi TA, Menichi B, Louvard D, Van de Ven WJ, Friederich E. LPP, an actin cytoskeleton protein related to zyxin, harbors a nuclear export signal and transcriptional activation capacity. Mol Biol Ce1l2000; 11 :117-129. 60. Ursini F, Maiorino M, Valente M, Ferri L, Gregolin C. Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides. Biochim Biophys Acta 1982; 710:197- 211. 61. Imai H, Sumi D, Sakamoto H, Hanamoto A, Arai M, Chiba N, Nakagawa Y. Overexpression of phospholipid hydroperoxide glutathione peroxidase suppressed cell death due to oxidative damage in rat basophile leukemia cells (RBL-2H3). Biochem Biophys Res Commun 1996; 222:432-438. 62. Wang HP, Schafer FQ, Goswami PC, Oberley LW, Buettner GR. Phospholipid hydroperoxide glutathione peroxidase induces a delay in G1 of the cell cycle. Free Radie Res 2003; 37:621-'630. 63. Imai H, Hirao F, Sakamoto T, Sekine K, Mizukura Y, Saito M, Kitamoto T, Hayasaka M, Hanaoka K, Nakagawa Y. Early embryonic lethality caused by targeted disruption of the mouse PHGPx gene. Biochem Biophys Res Commun 2003; 305:278-286. 64. Pfeifer H, Conrad M, Roethlein D, Kyriakopoulos A, Brielmeier M, Bornkamm GW, Behne p. Identification of a specifie sperm nuclei selenoenzyme necessary for protamine thiol cross-lin king during sperm maturation. FASEB J 2001; 15:1236-1238. 194 65. Maiorino M, Mauri P, Roveri A, Benazzi L, Toppo S, Bosello V, Ursini F. Primary structure of the nuclear forms of phospholipid hydroperoxide glutathione peroxidase (PHGPx)· in rat spermatozoa. FEBS Lett 2005; 579:667-670. 66. Sousa M, Carvalheiro J. A cytochemical study of the nucleolus and nucleolus-related structures during human spermatogenesis. Anat Embryol (Berl) 1994; 190:479-487. 67. Puglisi R, Tramer F, Panfili E, Micali F, Sandri G, Boitani C. Differentiai splicing of the phospholipid hydroperoxide glutathione peroxidase gene in diploid and haploid male germ cells in the rat. Biol Reprod 2003; 68:405- 411. 68. Sulkowska M, Sulkowski S, Skrzydlewska E, Farbiszewski R. Cyclophosphamide-induced generation of reactive oxygen species. Comparison with morphological changes in type Il alveolar epithelial cells and lung capillaries. Exp Toxicol Patho11998; 50:209-220. 69. Adams JO, Jr., Klaidman LK. Acrolein-induced oxygen radical formation. Free Radic Biol Med 1993; 15: 187-193. 70. Berrigan MJ, Struck RF, Gurtoo HL. Lipid peroxidation induced by cyclophosphamide. Cancer Biochem Biophys 1987; 9:265-270. 71. Griveau JF, Dumont E, Renard P, Callegari JP, Le Lannou D. Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa. J Reprod Fertil 1995; 103: 17-26. 72. Twigg JP, Irvine OS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod 1998; 13:1864-1871. 195 73. Struchkov VA, Strazhevskaya NB, Zhdanov RI. Specifie natural DNA-bound lipids in post-genome era. The lipid conception of chromatin organization. Bioelectrochemistry 2002; 56: 195-198. 74. Maraldi NM, Mazzotti G, Capitani S, Rizzoli R, Zini N, Squarzoni S, Manzoli FA. Morphological evidence of function-related localization of phospholipids in the cell nucleus. Adv Enzyme Regu11992; 32:73-90. 75. Alessenko AV, Burlakova EB. Functional role of phospholipids in the nuclear events. Bioelectrochemistry 2002; 58:13-21. 76. Harrouk W, Khatabaksh S, Robaire B, Hales BF. Paternal exposure to cyclophosphamide dysregulates the gene activation program in rat preimplantation embryos. Mol Reprod Dev 2000; 57:214-223. 196 FIGURES and LEGENDS 197 Figure 4-1. Two-dimensional electrophoretic separation of cauda epididymal spermatozoal nuclear matrix proteins from (A) control and (8) 4-week chronic cyclophosphamide (CPA)-treated rats. Spots unique to each treatment group are indicated with blue circles. Red and yellow circles indicate spots showing increased or decreased expression following CPA exposure, respectively. Spots with <2-fold change in expression are indicated with green circles. Protein identification was achieved for spots 1-24 (see Table 4-1). MW, molecular weight; pl, isoelectric point. 198 A 3 21 3 ~ • 6 , o D Figure 4-2. (A) Overall changes in the expression of 316 nuclear matrix proteins spots following 4-week chronic cyclophosphamide (CPA) treatment. >100% increase (horizontallines), >50% decrease (black), control-specifie (white), CPA specifie (cross-hatch), and no change «2-fold change in spot intensity, diagonal lines). Numbers in parenthesis = the number of spots in each category. (8) Phase contrast images of whole spermatozoa and nuclear matrix extracts (top panels). DNA stained with DAPI (bottom panels). Original magnification X400. 200 A Increase 11 % (34) Decrease 12% (38) Control-only 2% (7) CPA-only 8% (26) No change 67% (211) B , Whole Sperm NM Extract Figure 4-3. Two-dimensional western blots probed with an antibody to glutathione peroxidase 4 (GPX4). (A) Control and (B) 4-week chronic 'cyclophosphamide (CPA)-treated nuclear matrix samples. Open arrows: Immunoreactive spots that clearly differ with treatment. Black arrow: Uncharacterized 54 kOa GPX4 form. Arrowhead: Post-translationally modified and increased expression of 34 kOa form. 202 A X 3 M r 10- 66 45 ,.>,,; 31 21 B X 3 M r 10- 66 45 31 21 Figure 4-4. Expression of glutathione peroxidase 4 (GPX4) in (A) SDS-treated membrane-free spermatozoa and (B) sperm nuclear matrix extracts. Left panels: Control. Right panels: 4-week chronic cyclophosphamide-treated sperm. Samples (n=3) were immunostained with anti-GPX4. Original magnification X400. 204 A. SDS-treated Control CPA B. NM Extract TABLES and LEGENDS 206 Table 4-1. Expression of proteins identified by mass spectrometry. a Values represent spot intensities; b % Change in expression; * Change in expression is < 2-fold; CAPZB, capping protein (actin filament) muscle Z-line, beta; DNAJB6, DnaJ (Hsp 40) homolog, subfamily B, member 6; GAS2, growth arrest-specific 2; GPX4, glutathione peroxidase 4; GSTM5,glutathione-S transferase mu 5; GST02, glutathione-S-transferase omega 2; HNRPK, heterogeneous nuclear ribonucleoprotein K; HSPA2, heat shock 70kDa protein 2; LPP, UM domain containing preferred translocation partner in lipoma; MGC156832, similar to BTB/POZ domain containing protein 6; PCBP1, poly(rC) binding protein 1; PEBP, phosphatidylethanolamine binding protein; PPP1 CC, protein phosphatase 1, catalytic subunit, gamma isoform; PSMA5/B4/B6, proteosome (prosome, macropain) subunit, alpha type 5/beta type 4/beta type 6; RTEL 1, regulator of telomere elongation helicase 1; SAT_MAPPED, spermidine/spermine N1-acetyl transferase (mapped); TSSK2, testis-specific serine kinase 2; TPI1, triosephosphate isomerase 1. 207 3 a b Function Protein Spot No. M,x 10. pl Control CPA a % Change Cell structure CAPZB 7 45.8 6.0 0.54 0.67 24* 8 47.8 7.1 0.36 0.49 36* 10 40.2 7.0 1.35 1.41 4* 12 37.4 6.1 1.09 1.67 53* Calicin 4 74.1 9.3 0.12 2.34 1850 GAS2 4 74.1 9.3 0.12 2.34 1850 TranscriptionITranslation LPP 3 74.1 7.2 0.39 0.71 82* 4 74.1 9.3 0.12 2.34 1850 HNRPK 23 20.4 8.3 0.02 0.18 800 PCBPI 8 47.8 7.1 0.36 0.49 36* DNAbinding RTELl 16 25.4 7.5 0.10 0.45 350 17 25.4 7.6 0.56 CPA-specifie Protein processing DNAJB6 5 53.9 8.9 0.15 0.60 300 6 47.8 9.2 0.80 1.05 31* HSPA2 3 74.1 7.2 0.39 0.71 82* PSMA5 13 27.1 4.4 0.09 0.20 122 PSMB4 22 23.3 7.2 0.04 CPA-specific PSMB6 24 21.1 5.3 0.51 0.69 35* Signal transduction PPP 1CC 7 45.8 6.0 0.54 0.67 24* 8 47.8 7.1 0.36 0.49 36* 9 42.7 6.0 0.71 0.93 31* TSSK2 6 47.8 9.2 0.80 1.05 31* PEBP 24 21.1 5.3 0.51 0.69 35* Metabolism SAT_MAPPED 3 74.1 7.2 0.39 0.71 82* TPIl Il 37.4 5.8 0.69 1.13 64* 12 37.4 6.1 1.09 1.67 53* Cell defenselDetoxification GPX4 5 53.9 8.9 0.15 0.60 300 16 25.4 7.5 0.10 0.45 350 17 25.4 7.6 0.56 CPA-specifie 18 26.5 8.6 0.26 CPA-specific 19 24.1 6.0 0.10 0.23 130 20 24.3 6.1 0.09 0.23 155 21 22.4 6.5 0.65 0.80 23* 23 20.4 8.3 0.02 0.18 800 GST02 14 29.3 8.4 2.31 3.86 67* GSTMS 15 26.6 6.8 0.47 0.79 68* 16 25.4 7.5 0.10 0.45 350 17 25.4 7.6 0.56 CPA-specific 18 26.5 8.6 0.26 CPA-specific 21 22.4 6.5 0.65 0.80 23* Unknown Sirnilar to Ran-interacting 24 21.1 5.3 0.51 0.69 35* protein MOG 1 (predicted) MGCI56832 7 45.8 6.0 0.54 0.67 24* 8 47.8 7.1 0.36 0.49 36* Similar to KlAAI683 113.5 9.2 0.43 0.90 109 2 115.9 9.4 0.43 0.14 -67 Similar to hypothetical protein 6 47.8 9.2 0.80 l.05 31* DKFZp434H2215 (predicted) CHAPTER 5 Discussion 209 At least 50% of ail conceptions are estimated to result in fetal loss before the end of the first trimester (Goldstein, 1994; Wilcox et al., 1999). This figure is most likely underestimated since many spontaneous abortions happen very early in a pregnancy, before a woman may even know she is pregnant (Jauniaux et al., 1996). Evidence from animal studies clearly indicates that male-mediated effects on reproduction, particularly following paternal exposure to a drug, may include an increased incidence of early spontaneous abortions; in humans, such an effect would likely go unnoticed and be diagnosed as idiopathic infertility. Cyclophosphamide is a prime example of a male-mediated developmental toxicant with >80% early post-implantation loss reported in embryos sired by male rats exposed to the drug during post-meiotic germ cell development (Trasler et al., 1987). How or why this occurs was the basis for the studies of this thesis. The W.H.O. uses seven parameters for semen analysis (WHO, 1999); however, these have been proven in many circumstances to be non-predictive of fertility potential or more importantly, ability to support embryonic development. There is now a battery of assays that extend beyond the scope of the W.H.O. normal semen criteria to evaluate sperm quality and function (Agarwal and Allamaneni, 2005; Aitken, 2006). The studies presented in this thesis highlight a few of these which contribute to our understanding of the mechanisms of cyclophosphamide action in the male germ cell. The paternal genome is key to successful embryo development and, collectively, the results of the present studies show that cyclophosphamide alters male germ cell chromatin structure at both the DNA and protein javel. In this final chapter, the significance of this work in terms of its relevance in and advancement of the field will be discussed. 210 5.1. Sperm Genetic Integrity The results of chapter two clearly show that chronic cyclophosphamide exposure has a great impact on the genetic integrity of sperm nuclei. Most striking, are the differential susceptibilities of spermiogenic germ cells to cyclophosphamide-induced DNA damage; the consequences of such effects on gene expression, in particular on the transcriptional state of the male gamete, are intriguing. The beginning of spermiogenesis is characterized by a wave of transcriptional activity which results in the activation of numerous essential post meiotic genes in early spermatids (Dadoune et al., 2004). Alkylating agents, including cyclophosphamide, induce DNA structural modifications by creating strand breaks and DNA-DNA or DNA-protein crossHnks; these modifications can delay or stop RNA polymerases, resulting in truncated RNA molecules (Masta et al., 1995; Tornaletti and Hanawalt, 1999). Interestingly, there is no detectable damage in epididymal spermatozoa after early round spermatids were exposed to acute or repeat doses of cyclophosphamide. Assuming that damage is induced by this treatment, the results imply that round spermatids have the ability to repair any damage incurred. If this were the case, then the synthesis of transcripts would not be affected. In chapter three, however, we showed that the expression of proteins critical to sperm function, fertilization and embryo development was still altered in the mature sperm. Another mechanism affecting gene expression may be at the level of translation; cyclophosphamide may alter transcript quality by RNA alkylation. Previous in vitro studies have shown that nitrogèn mustards have the capability to inhibit translation, resulting in truncated 211 polypeptides (Masta et al., 1995). This is interesting considering that protein fragments were identified in the 2D gels used in our experiments in chapters three and four. Due to the highly condensed architecture of mammalian sperm chromatin, the male gamete is rendered transcriptionally silent late in spermiogenesis (Kierszenbaum and Tres, 1975). Interestingly, several studies have reported the presence of RNA transcripts in human ejaculated spermatozoa (Ostermeier et al., 2002; Ostermeier et al., 2004; Wykes et al., 1997) and mouse mature spermatozoa (Wykes et al., 2000). One study clearly proves that mature human spermatozoa cannot transcribe novel RNA (Grunewald et al., 2005). It has been suggested that these sperm transcripts may be remnants of stored testicular transcribed RNAs that were not translated and that were retained as a consequence of nuclear shutdown or that they may play a more active role in post-fertilization eventsas essential paternal RNAs required for embryo development (Ostermeier et al., 2004). The effect of xenobiotic exposure on the quality of the transcripts is of concern if the possibility exists that they may participate in embryogenesis. Cyclophosphamide maximally damaged DNA at its most vulnerable state during mid-spermiogenesis, when nucleoproteins are in the process of exchanging histones for protamines and therefore leave the genome temporarily open to cyclophosphamide atlack. Both transcription and translation are ongoing in elongating spermatids (Steger, 1999). Most importantly, DNase-1 hypersensitivity regions have been located within step 12-13 elongating spermatids; these regions have been associated also with transcriptionally active 212 genes near nuclear matrix sites (Gross and Garrard, 1988; McPherson and Longo, 1992). In somatic cells, alkylating agents preferentially bind to DNA near matrix-bound transcription sites (Muenchen and Pienta, 1999). It is tempting to speculate that cyclophosphamide alkylation may also target specifie gene loci within these regions; these genes may be required for early embryo development. Analysis of RNAs from spermatozoa of infertile men is being used as a diagnostic tool for identifying germline mutations in candidate infertility associated genes (Yatsenko et al., 2006). Interestingly, previous studies from our laboratory report increased transcript levels in elongating spermatids following acute and chronic dosing regimes using cyclophosphamide (Aguilar Mahecha et al., 2001; Aguilar-Mahecha et al., 2002). It remains to be determined whether these transcripts persist in the mature rat spermatozoon; nevertheless, this approach could be used to characterize the RNA of rat spermatozoa, should any be present, following cyclophosphamide exposure. By altering the integrity of genomic DNA, the drug may subsequently affect the expression and quality of the RNA synthesized during mid-spermiogenesis and still present in mature spermatozoa. These transcripts may, in fact, be required for post-meiotic events including sperm condensation, maturation, fertilization or embryogenesis. 5.2. Sperm Chromatin Structure and Proteome ln chapter three, the use of multiple assays demonstrated that cyclophosphamide exposure alters sperm chromatin structure, with effects specifically on thiol and protamine content. The results of this study tie in very weil with previous reports which have shown that the male pronucleus forms 213 earlier in embryos sired by cyclophosphamide-treated male rats than in those sired by controls (Harrouk et al., 2000). Transcriptional activation of specifie genes is initiated after fertilization and remodelling of the sperm chromatin after fertilization is fundamental for the expression of the paternal genome (Schultz, 2002). Interestingly, earlier male pronuclear formation was also followed by alterations in the gene activation program; bromouridine-triphosphate (BrUTP) incorporation into RNA and Sp1 transcription factor immunostaining increased in 2-cell embryos sired by cyclophosphamide-treated males compared to controls (Harrouk et al., 2000). Our findings are relevant since less condensed sperm present in newly fertilized eggs could be the reason for more rapid decondensation, pronuclear formation and zygotic gene activation with detrimental effects on embryo development. Proteomic tools can be used to analyze sperm proteins important for sperm development and proper embryo development. Recently, there have been a few reports using the present proteomic technology to identify proteins present in the mature germ cell (Chu et al., 2006; Martinez-Heredia et al., 2006; Pixton et al., 2004). Chapters three and four used 20 gel electrophoresis and subsequent mass spectrometry to assess two different fractions of sperm, the sperm head basic proteome and nuclear matrix. To my knowledge, these studies are the first to analyze sperm in this manner, as weil as to determine the effects of xenobiotic exposure on protein expression. 214 5.2.1. Sperm Head Basic Proteome , The exchange between histones and protamines is a carefully regulated and complex process. Studies have demonstrated that the expression of protamines 1 and 2 is uniquely regulated by transcription and translation factors, including storage of the mRNA in RNP particles in the cytoplasm until translation initiation (Dadoune, 2003; Steger, 2001). Recent studies have indicated that infertile men with abnormal protamine levels have either decreased protamine transeript levels, thereby affecting the P1/P2 ratio (Steger et al., 2003), or elevated levels of protamine transcripts in mature sperm (Aoki et al., 2006), indicating a possible defect in transcription or translation. There are several possible mechanisms which may be responsible for the dysregulation of protamine expression. Whatever the meehanism may be, the problem could be a global issue and not just protamine expression-specifie. For example, such abnormalities may be indicative of a defeet in mRNA storage and/or translation overall which, in turn, may affect other mRNA transeripts. Sperm chromatin condensation defects in general may be indicative of a wide range of spermiogenic anomalies. Following ehronic eyclophosphamide exposure, we saw that the sperm basic proteome was indeed affected. Identified proteins were involved in spermatid differentiation, sperm maturation and fertilization. The results of this study incite further investigation into the proteins of the sperm head, not just with 2D gel protein identification, but also with immunofluorescence and functionality assays in order to completely characterize proteins of interest involved in germ cell and embryo development. This approach would allow for the potential identification of biomarkers specific to the male-mediated effects of 215 drug exposure. For example, PRKRIP1, is highly expressed in the testis yet has no known spermiogenic function (Yin et al., 2003). Its novelty is exciting. It is a negative regulator of the protein kinase, PRKR which has been implicated in many cellular functions, including translation (Hovanessian, 1989), transcription (Oeb et al., 2001) and differentiation (Samuel et al., 1997), as weil as ONA repair (Bergeron et al., 2000); any one of these roles could be critical to the development and function of the male germ cell. Cyclophosphamide exposure resulted in decreased expression of PRKRIP1 compared to control spermatozoa. Given the multiple roles played by PRKR, future studies focused on either protein could prove them to be important factors in the mechanisms of cyclophosphamide action. 5.2.2. Nuclear Matrix Proteome Research into the relationship between the development of cancer and nuclear matrix aberrations has been fundamental in establishing biomarkers used as prognostic tools for neoplastic diseases (Leman et al., 2003). Whether biomarkers of sperm defects can be established and used to elucidate the reasons behind male-mediated developmental toxicant effects on progeny outcome remains to be seen with great anticipation. The nuclear matrix is known to be important for normal cellular functions such as transcriptional regulation and ONA replication (Anachkova et al., 2005; Mortillaro et al., 1996; van Oriel et al., 1991). Clearly the sperm nuclear matrix is important for embryo development since studies have reported that sperm nuclei with disrupted nuclear matrix structures cannot participate in embryogenesis (Ward et al., 1999). Sperm 216 nuclear DNA is apparently organized in such a way that it contains heritable information required for development (Sotolongo and Ward, 2000); this organization may template the paternal genome for use in the early post fertilization embryo (Kramer and Krawetz, 1996). If this is indeed the function of the sperm nuclear matrix th en questions naturally emerge concerning the proteins that make up the nuclear matrix. What is it that makes the nuclear matrix so important? To date, the proteins of the spermatozoa nuclear matrix have not been examined extensively nor has the impact of exposure to drugs on their structure or function been determined. Chapter four serves to answer this question. The expression of several matrix proteins was altered following chronic cyclophosphamide exposure and, for the first time, a number of the proteins were identified. Of utmost interest is the increased or cyclophosphamide-specific expression of nucleolar proteins following drug exposure. The nucleolus is the ribosomal biogenesis center where ribosomal RNA (rRNA) genes are transcribed. Nucleolar inactivation commences in early round spermatids; rRNA synthesis gradually decreases as spermiogenesis progresses to the point where nucleoli disappear in parallel with the cessation of RNA synthesis in elongating spermatids (Schultz and Leblond, 1990). The use of specifie antibodies directed against nucleolar proteins, including ribosomal proteins, hnRNPs and fibrillarin, only results in positive immunolabeling of fibrillarin in spermatid residual nucleoli (Biggiogera et al., 1994). The increased expression of nucleolar-specific proteins in our experiments leads me to speculate that nucleolar inactivation may be disrupted. 217 It would be fascinating to examine sperm nucleolar inactivation during spermiogenesis following cyclophosphamide exposure. Important end points to consider would be the persistent expression of the nucleolar proteins mentioned above, including the prime nucleolar marker, nucleolin. As weil, ultrastructural evaluation of spermatid nucleoli would be helpful. Preliminary experiments in our laboratory not only indicate increased expression of nucleolin in mature spermatozoa following chronic cyclophosphamide exposure, but also differential localization of the protein from what appears to be the acrosomal or subacrosomal region in control sperm to the nucleus in cyclophosphamide exposed sperm. Further experiments are necessary to confirm these findings. Additionally, increased expression of the 34 kDa form of glutathione peroxidase 4 (GPX4) was observed. Interestingly, this protein is expressed in the nucleoli of spermatids; these findings spur great curiosity into their function in this organelle. Overexpression of GPX4 in somatic cells has a protective role in that nucleoli are protected from oxidative-stress induced damage (Imai et al., 1996). It is tempting to speculate that the spermatid nucleolus may be prevented from degrading due to the increased expression of GPX4 following cyclophosphamide exposure. Additional experimentation is necessary to resolve this mystery. Correspondingly, should the nucleolus still be active in mature sperm, one might expect dysregulated nucleogenesis and, consequently, altered gene expression in the early embryo which is exactly what our laboratory has discovered using similar chronic cyclophosphamide dosing regimes (Harrouk et al., 2000); the formation of the nucleolus after fertilization is temporally regulated and is essential for embryonic protein synthesis and viability. 218 5.3. DNA Organization A plethora of information can be gained from examination of the nuclear matrix protein profile. While further work can be done to identify additional proteins within the proteome, future directions of this work should include investigation of the actual organization of DNA within mammalian sperm nuclei, with particular emphasis on whether drug treatment affects the organization of DNA loops by the matrix. One route which can be taken to accomplish this goal, is visualizing, by fluorescence in situ hybridization (FI8H), the structural organization of specific DNA segments. Changes in individual DNA loop domains can be visualized in nuclear halo preparations by FI8H using probes to specific genes of interest. Changes in DNA loop organization have been shown to occur based on the functional state of the cell in question (Gerdes et al., 1994; larovaia et al., 2005). Probes to 58 rDNA have been used successfully for studying changes in complete loop domain structures during spermatogenesis (Klaus et al., 2001; Nadel et al., 1995). The beauty of this technique is that it is possible to distinguish between actively transcribing genes, which are more likely to be associated with the nuclear matrix as a single focus, and inactive genes, which would extend into the nuclear halo (Gerdes et al., 1994; larovaia et al., 2005). The same can then be true in sperm; even if they are not transcriptionally active, changes in DNA loop attachment due to drug exposure may be visible. Probes for 58 rDNA and other candidates, such as paternally imprinted genes, would be likely choices to start. Interestingly, the expression of imprinted genes was altered in embryos sired by cyclophosphamide treated males; expression peaked in 2-cell embryos 219 compared to a peak in expression of these genes not observed until the 8-cell stage in control embryos (Harrouk et al., 2000). Previous reports suggest that DNA loop domain organization is specifically regulated during spermatogenesis and embryogenesis (Klaus et al., 2001). Using 5S rDNA probes, it has been shown that DNA organization changes during spermacytogenesis and spermatidogenesis; however, it does not appear to change during spermiogenesis (Klaus et al., 2001). At this stage, the loop domains may be set in place, thus suggesting that they may be important in the function of the paternal genome in the newly fertilized zygote, for DNA replication and/or RNA transcription. If this is the case and this structure is altered following drug exposure then the consequences to the embryo would be detrimental. Collectively, the results of this thesis contribute to our understanding of the susceptibility of male germ cells to damage at the DNA and protein level and the consequent diminished quality of the male germ cell following cyclophosphamide exposure. The necessity of an intact and stable paternal genome to support embryo development is also highlighted in the studies presented. These analyses are indeed also clinically significant for the diagnosis and treatment of male infertility. Finally, the data collected cou Id potentially be used towards the discovery of future therapeutic interventions and male contraceptive targets. The findings presented in this thesis should act as a catalyst to delve deeper into unraveling the mechanisms of male-mediated developmental toxicity. 220 List of Original Contributions 1- 1 have developed the cornet assay under alkaline conditions for rodent spermatozoa. In particular, by using this assay, 1 have identified the consequences of cyclophosphamide exposure on the genetic integrity of individual sperm nuclei, thus revealing that there are heterogeneous populations with varying degrees of DNA damage. 2- Cyclophosphamide exposure results in dose-dependent and time-specific increases in the levels of DNA damage measured in epididymal spermatozoa, evidenced by acute, subchronic and chronic dosing regimens. 3- The extent of DNA damage incurred in spermatid nuclei is germ cell phase specifie; mid-spermiogenic elongating spermatids are most susceptible to DNA damage following cyclophosphamide exposure. 4- Chronic exposure to cyclophosphamide alters the epididymal sperm chromatin structure; sperm have limited disulfide and protamine content and reduced chromatin stability. 221 5- Sy using subchronic doses of cyclophosphamide 1 was able to expose the germ cell specifie effects of the drug on chromatin structure. Altered chromatin structure, determined by increased susceptibility to in situ denaturation and reduced thiol content in sperm, was most evident in epididymal spermatozoa exposed to the drug during mid spermiogenesis. 6- Classical use of chromomycin A3 analysis, used to detect abnormal chromatin packaging in protamine-depleted spermatozoa, failed to reveal an affect in rat spermatozoa exposed to cyclophosphamide. However the method 1 have developed shows that, in addition to protamine deficiency, chromomycin A3 binding in the rat depends on the thiol or decondensation status of the ce Il , thus this fluorochrome can be used to measure the effect of drug exposure on chromatin packaging providing spermatozoa are first decondensed. 7- 1 have uniquely designed a method using two-dimensional basic gels to characterize the basic proteome of spermatozoal heads which shows that chronic cyclophosphamide exposure alters the proteome make-up. Proteins identified are involved in transcription and translation regulation, chromatin organization, sperm structure and stability and fertmzation and thus could be essential for normal sperm function and embryo development 222 8- My studies were the first to demonstrate that xenobiotic agents can alter the sperm nuclear matrix; chronic cyclophosphamide exposure affects the expression of the nuclear matrix proteome in epididymal spermatozoa and therefore could affect the potential role of the matrix in gene regulation. 9- 1 have expanded the current knowledge of sperm nuclear matrix proteins by identifying a number of the components. From the proteins identified 1 have shown that the functions of sorne of the sperm nuclear matrix proteins include involvement in cell structure, transcription, translation, DNA binding, protein processing, signal transduction, metabolism, cell defense and detoxification. 223 REFERENCES Agarwal A and AUamaneni SS (2005) Sperm DNA damage assessment: a test whose time has come. Fertil SteriI84,850-853. Aguilar-Mahecha A, Hales BF and Robaire B (2001) Acute cycJophosphamide exposure has germ ceU specifie effects on the expression of stress response genes during rat spermatogenesis. Mol Reprod Dev 60,302-311. Aguilar-Mahecha A, Hales BF and Robaire B (2002) Chronic cycJophosphamide treatment alters the expression of stress response genes in rat male germ ceUs. Biol Reprod 66,1024-1032. Aitken RJ (2006) Sperm function tests and fertility. Int J Androl 29,69-75. Anachkova B, Djeliova V and Russev G (2005) Nuclear matrix support of DNA replication. J CeU Biochem 96,951-961. Aoki VW, Liu Land Carrell DT (2006) A novel mechanism of protamine expression deregulation highlighted by abnormal protamine transcript retenti on in infertile human males with sperm protamine deficiency. Mol Hum Reprod 12,41-50. Bergeron J, Benlimame N, Zeng-Rong N, Xiao D, Scrivens PJ, Koromilas AE and Alaoui-Jamali MA (2000) Identification of the interferon-inducible double stranded RNA-dependent protein kinase as a regulator of cellular response to bulky adducts. Cancer Res 60,6800-6804. 224 Biggiogera M, Martin TE, Gordon J, Amalric F and Fakan S (1994) Physiologically inactive nucleoli contain nucleoplasmic ribonucleoproteins: immunoelectron microscopy of mouse spermatids and early embryos. Exp Cell Res 213,55-63. Chu OS, Liu H, Nix P, Wu TF, Ralston EJ, Yates JR, III and Meyer BJ (2006) Sperm chromatin proteomics identifies evolutionarily conserved fertility factors. Nature 443,101-105. Oadoune JP (2003) Expression of mammalian spermatozoal nucleoproteins. Microsc Res Tech 61,56-75. Oadoune JP, Siffroi JP and Alfonsi MF (2004) Transcription in haploid male germ cells. Int Rev Cytol 237,1-56. Oeb A, Haque SJ, Mogensen T, Silverman RH and Williams BR (2001) RNA dependent protein kinase PKR is required for activation of NF-kappa B by IFN-gamma in a STAT1-independent pathway. J ImmunoI166,6170-6180. Gerdes MG, Carter KC, Moen PT, Jr. and Lawrence JB (1994) Oynamic changes in the higher-Ievel chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos. J Cell Biol 126,289-304. Goldstein SR (1994) Embryonic death in early pregnancy: a new look at the first trimester. Obstet Gynecol 84,294-297. Gross OS and Garrard WT (1988) Nuclease hypersensitive sites in chromatin. Annu Rev Biochem 57,159-197. 225 Grunewald S, Paasch U, Glander HJ and Anderegg U (2005) Mature human spermatozoa do not transcribe novel RNA. Andrologia 37,69-71. Harrouk W, Khatabaksh S, Robaire B and Hales BF (2000) Paternal exposure to cyclophosphamide dysregulates the gene activation program in rat preimplantation embryos. Mol Reprod Dev 57,214-223. Hovanessian AG (1989) The double stranded RNA-activated protein kinase induced by interferon: dsRNA-PK. J Interferon Res 9,641-647. larovaia OV, Akopov SB, Nikolaev LG, Sverdlov ED and Razin SV (2005) Induction of transcription within chromosomal DNA loops t1anked by MAR elements causes an association of loop DNA with the nuclear matrix. Nucleic Acids Res 33,4157-4163. Imai H, Sumi D, Sakamoto H, Hanamoto A, Arai M, Chiba N and Nakagawa Y (1996) Overexpression of phospholipid hydroperoxide glutathione peroxidase suppressed cell death due to oxidative damage in rat basophile leukemia cells (RBL-2H3). Biochem Biophys Res Commun 222,432-438. Jauniaux E, Gavriil P and Nicolaides KH (1996) Ultrasonographic assessment of early pregnancy complications. In Jurkovic D and Jauniaux E (eds) Ultrasound and Early Pregnancy. Parthenon Publishing, Carnforth, 53. Kierszenbaum AL and Tres LL (1975) Structural and transcriptional features of the mouse spermatid genome. J Cel! Biol 65,258-270. 226 Klaus AV, McCarrey JR, Farkas A and Ward WS (2001) Changes in DNA loop domain structure during spermatogenesis and embryogenesis in the Syrian golden hamster. Biol Reprod 64,1297-1306. Kramer JA and Krawetz SA (1996) Nuclear matrix interactions within the sperm genome. J Biol Chem 271,11619-11622. Leman ES, Madigan MC, Brunagel G, Takaha N, Coffey OS and Getzenberg RH (2003) Nuclear matrix localization of high mobility group protein I(Y) in a transgenic mouse model for prostate cancer. J Cell Biochem 88,599-608. Martinez-Heredia J, Estanyol JM, Ballesca JL and Oliva R (2006) Proteomic identification of human sperm proteins. Proteomics 6,4356-4369. Masta A, Gray PJ and Phillips DR (1995) Nitrogen mustard inhibits transcription and translation in a œil free system. NucieicAcids Res 23,3508-3515. McPherson SM and Longo FJ (1992) Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev 31,268-279. Mortillaro MJ, Blencowe BJ, Wei X, Nakayasu H, Du L, Warren SL, Sharp PA and Berezney R (1996) A hyperphosphorylated form of the large subunit of RNA polymerase " is associated with splicing complexes and the nuclear matrix. Proc Natl Acad Sci USA 93,8253-8257. Muenchen HJ and Pienta KJ (1999) The role of the nuclear matrix in cancer chemotherapy. Crit Rev Eukaryot Gene Expr 9,337-343. 227 Nadel B, de Lara J, Finkernagel SW and Ward WS (1995) Cell-specific organization of the 5S ribosomal RNA gene cluster DNA loop domains in spermatozoa and somatic cells. Biol Reprod 53,1222-1228. Ostermeier GC, Dix DJ, Miller D, Khatri P and Krawetz SA (2002) Spermatozoal RNA profiles of normal fertile men. Lancet 360,772-777. Ostermeier GC, Miller D, Huntriss JO, Diamond MP and Krawetz SA (2004) Reproductive biology: delivering spermatozoan RNA to the oocyte. Nature 429,154- Pixton KL, Deeks ED, Flesch FM, Moseley FL, Bjorndahl L, Ashton PR, Barratl CL and Brewis lA (2004) Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report. Hum Reprod 19,1438-1447. Samuel CE, Kuhen KL, George CX, Ortega LG, Rende-Fournier R and Tanaka H (1997) The PKR protein kinase--an interferon-induciblè regulator of cell growth and differentiation. Int J Hematol 65,227-237. Schultz MC and Leblond CP (1990) Nucleolar structure and synthetic activity during meiotic prophase and spermiogenesis in the rat. Am J Anat 189,1- 10. Schultz RM (2002) The molecular foundations of the maternai to zygotic transition in the preimplantation embryo. Hum Reprod Update 8,323-331. Sotolongo B and Ward WS (2000) DNA loop domain organization: the three dimensional genomic code. J Cell Biochem Suppl Suppl 35,23-26. 228 Steger K (1999) Transcriptional and translational regulation of gene expression in haploid spermatids. Anat Embryol (Berl) 199,471-487. Steger K (2001) Haploid spermatids exhibit translationally repressed mRNAs. Anat Embryol (Berl) 203,323-334. Steger K, Fink L, Failing K, Bohle RM, Kliesch S, Weidner W and Bergmann M (2003) Oecreased protamine-1 transcript levels in testes from infertile men. Mol Hum Reprod 9,331-336. Tornaletti Sand Hanawalt PC (1999) Effect of ONA lesions on transcription elongation. Biochimie 81,139-146. Trasler JM, Hales BF and Robaire B (1987) A time-course study of chronic paternal cyclophosphamide treatment in rats: effects on pregnancy outcome and the male reproductive and hematologic systems. Biol Reprod 37,317-326. van Oriel R, Humbel B and de Jong L (1991) The nucleus: a black box being opened. J Cell Biochem 47,311-316. Ward WS, Kimura Y and Yanagimachi R (1999) An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development. Biol Reprod 60,702-706. WHO (1999) WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 4th edn, Cambridge University Press, Cambridge. 229 Wilcox AJ, Baird DO and Weinberg CR (1999) Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 340,1796-1799. Wykes SM, Miller 0 and Krawetz SA (2000) Mammalian spermatozoal mRNAs: tools for the functional analysis of male gametes. J Submicrosc Cytol PathoI32,77-81. Wykes SM, Visscher OW and Krawetz SA (1997) Haploid transcripts persist in mature human spermatozoa. Mol Hum Reprod 3,15-19. Yatsenko AN, Roy A, Chen R, Ma L, Murthy LJ, Yan W, Lamb DJ and Matzuk MM (2006) Non-invasive genetic diagnosis of male infertility using spermatozoal RNA: KLHL 10 mutations in oligozoospermic patients impair homodimerization. Hum Mol Genet 15,3411-3419. Yin Z, Haynie J, Williams BR and Yang YC (2003) C114 is a novel IL-11- inducible nuclear double-stranded RNA-binding protein that inhibits protein kinase R. J Biol Chem 278,22838-22845. 230 APPENDIX 231