Characterization of human sperm protamine deficiency and the implications for human male infertility

During spermiogenesis, sperm chromatin undergoes dramatic remodeling. The testis-specific protamine proteins facilitate these nuclear changes by replacing the somatic cell histones, a process that produces highly condense, transcriptionally silent chromatin. In humans there are two forms of sperm protamine, protamine-1 (P1) and protamine-2 (P2), which occur in a strictly regulated one-to-one ratio. Sperm protamine-deficiency and P1/P2 ratio deregulation have been implicated in male infertility. The studies comprising this dissertation aimed to investigate the hypothesis that human sperm protamine deficiency: (1) occurs due to abnormal expression of the P1 and P2 proteins, (2) is associated with diminished semen quality, compromised sperm functional ability, and reduced sperm DNA integrity, (3) arises due to genetic aberrations in the protamine genes and unfaithful translation of the protamine transcripts, and (4) is negatively associated with fertility potential. In order to evaluate the hypothesis, studies were focused on three specific objectives: (1) to better characterize protamine-deficiency in infertile males, (2) to evaluate the molecular basis of protamine-deficiency, and (3) to evaluate the clinical significance and fertility implications of human sperm protamine deficiency. Quantitative analysis of sperm nuclear protein extracts revealed a large population of infertile men with abnormal sperm P1/P2 stoichiometry. Included in this group, were patients with abnormally elevated P1/P2 ratios, as well as a newly identified infertile population with significantly reduced P1/P2 ratios. Abnormal expression of the P1 and P2 proteins were shown to underlie these abnormally reduced and elevated ratios, respectively. Nuclear protein gene sequencing and protamine mRNA quantification subsequently revealed deregulated protamine expression is not a primary result of nuclear protein gene mutations, but instead may arise due to novel mechanisms of aberrant translation regulation or post-translational processing defects. Semen analysis, sperm penetration evaluation, and DNA fragmentation testing revealed aberrations in P1/P2 stoichiometry are associated with diminished semen quality, compromised sperm functional ability, and reduced sperm DNA integrity. Finally, an analysis of in vitro fertilization (IVF) outcomes revealed sperm protamine-deficiency is related to reduced fertilization ability and diminished IVF pregnancy rates. Taken together, these studies provide a comprehensive characterization of sperm protamine deficiency, elucidate the biochemical and molecular basis of abnormal protamine expression, and establish the clinical significance of the protamines in human male infertility.

CHARACTERIZATION OF HUMAN SPERM PROTAMINE DEFICIENCY AND THE IMPLICATIONS FOR HUMAN MALE INFERTILITY by Vincent Wayne Aoki A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Physiology The University of Utah December 2005 Copyright © Vincent Wayne Aoki 2005 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Vincent Wayne Aoki This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. Chair: 1$ouglas T. Carrell 1 \\Jo \J 2.lX)S C. Matthew Peterson 1/1, )£~- Michael =inetti THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Vincent Wayne Aoki in its final fonn and have found that (1) its fonnat, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Y NO\} OS Date Douglas T. ~arrell Chair: Supervisory Committee Approved for the Major Department F. Edward Dudek Chair/Dean Approved for the Graduate Council David S. Chapman Dean of The Graduate School ABSTRACT During spermiogenesis, sperm chromatin undergoes dramatic remodeling. The testis-specific protamine proteins facilitate these nuclear changes by replacing the somatic cell histones, a process that produces highly condense, transcriptionally silent chromatin. In humans there are two forms of sperm protamine, protan1ine-I (PI) and protamine-2 (P2), which occur in a strictly regulated one-to-one ratio. Sperm protamine­deficiency and PlIP2 ratio deregulation have been implicated in male infertility. The studies comprising this dissertation aimed to investigate the hypothesis that human sperm protamine deficiency: (1) occurs due to abnormal expression of the PI and P2 proteins, (2) is associated with diminished semen quality, compromised sperm functional ability, and reduced sperm DNA integrity, (3) arises due to genetic aberrations in the protamine genes and unfaithful translation of the protamine transcripts, and (4) is negatively associated with fertility potential. In order to evaluate the hypothesis, studies were focused on three specific objectives: (1) to better characterize protamine-deficiency in infertile males, (2) to evaluate the molecular basis of protamine-deficiency, and (3) to evaluate the clinical significance and fertility implications of human sperm protamine deficiency. Quantitative analysis of sperm nuclear protein extracts revealed a large population of infertile men with abnormal sperm PlIP2 stoichiometry. Included in this group, were patients with abnormally elevated PlIP2 ratios, as well as a newly identified infertile population with significantly reduced PlIP2 ratios. Abnormal expression of the PI and P2 proteins were shown to underlie these abnormally reduced and elevated ratios, respectively. Nuclear protein gene sequencing and protamine mRNA quantification subsequently revealed deregulated protamine expression is not a primary result of nuclear protein gene mutations, but instead may arise due to novel mechanisms of aberrant translation regulation or post-translational processing defects. Semen analysis, sperm penetration evaluation, and DNA fragmentation testing revealed aberrations in PlIP2 stoichiometry are associated with diminished semen quality, compromised sperm functional ability, and reduced sperm DNA integrity. Finally, an analysis of in vitro fertilization (IVF) outcomes revealed sperm protamine-deficiency is related to reduced fertilization ability and diminished IVF pregnancy rates. Taken together, these studies provide a comprehensive characterization of sperm protamine deficiency, elucidate the biochemical and molecular basis of abnormal protamine expression, and establish the clinical significance of the protamines in human male infertility. v TABLE OF CONTENTS ABSTRACT................................................................................................................ iv LIST OF TABLES ...................................................................................................... IX LIST OF FIGURES..................................................................................................... xi CHAPTER 1. HUMAN PROTAMINES AND THE DEVELOPING SPERMATID: THEIR STRUCTURE, FUNCTION, EXPRESSION AND RELATIONSHIP WITH MALE INFERTILITy....................................................................................... 1 Abstract ........................................................................................................ 2 Introduction ................................................................................................. . Structure and function ofP1 and P2 ............................................................. . Protamine expression regulation .................................................................. . Protamines and infertility ............................................................................ .. Future directions .......................................................................................... . References .................................................................................................. .. 2. IDENTIFICATION AND EVALUATION OF A NOVEL SPERM PROTAMINE ABNORMALITY IN A POPULATION OF 2 3 4 8 8 9 INFERTILE MALES.......................................................................................... 12 Introduction.................................... ................ ....... ..... . ......... . ........... . ..... .... .. 13 Materials and methods.................................................................................. 13 Results.......................................................................................................... 16 Discussion................................................ .................................................... 19 References.. ................................................................................................ .. 21 3. DNA INTEGRITY IS COMPROMISED IN PROTAMINE-DEFICIENT HUMAN SPERM .............................................................................................. 22 Abstract....... .. ............. .. ... ................. ... ............. ....... ........... ........... ............... 23 Materials and Methods.................................................................................. 24 Results .......................................................................................................... 26 Discussion... ...... .............. ........................ ... .. ........... . . ........... ........... ............. 28 References .................................................................................................... 29 4. PROTAMINE LEVELS VARY BETWEEN INDIVIDUAL SPERM CELLS OF INFERTILE HUMAN MALES AND CORRELATE WITH VIABILITY AND DNA INTEGRITy ............................................................................................. 31 Abstract ........................................................................................................ 32 Introduction .................................................................................................. 33 Materials and Methods.................................................................................. 34 Results .......................................................................................................... 42 Discussion.................................................................................................... 45 References....................................... ............................................................. 50 5. IDENTIFICATION OF NOVEL POL YMORPHISMS IN THE NUCLEAR PROTEIN GENES AND THEIR RELATIONSHIP WITH HUMAN SPERM PROTAMINE DEFICIENCY AND SEVERE MALE INFERTILITY .. 53 Abstract.. . . ....................... .. .. .... ....... ......... .. .. ..... ....... .. .. ..... .... ........... ..... ..... . .. 54 Introduction....... .............. .. ....... ............... ..... .. .. ....... .... . . ... .. .. ........... .......... . .. 54 Materials and Methods.................................................................................. 55 Results .......................................................................................................... 59 Discussion.................................................................................................... 62 Acknowledgements........ .... ..... ..... .... ............ ......... ... ........... ..... ........... ... ....... 66 References.................................................................................. .................. 67 6. A NOVEL MECHANISM OF PROTAMINE EXPRESSION DEREGULATION HIGHLIGHTED BY ABNORMAL PROTAMINE TRANSCRIPT RETENTION IN INFERTILE HUMAN MALES WITH SPERM PROTAMINE DEFICIENCY ......................................................................... '" 69 Abstract ........................................................................................................ 70 Introduction................................................................... ................ ............... 71 Materials and Methods.............. .................................................................... 72 Results .......................................................................................................... 82 Discussion. .. ...... ..... .... ....... ............. ... ......... .................................................. 89 Acknowledgements ....................................................................................... 95 References....... .... .. . .... . . . . ....... ..... ...... . . ... ............... .. ....... ..... ...... ..... ............... 96 7. SPERM PROTAMINE lIPROTAMINE 2 RATIOS ARE RELATED TO IVF PREGNANCY RATES AND PREDICTIVE OF FERTILIZATION ABILITY. 99 Abstract ........................................................................................................ 100 Introduction .................................................................................................. 100 Materials and Methods .................................................................................. 102 Results .......................................................................................................... 107 Discussion .................................................................................................... 111 References .................................................................................................... 117 Vll 8. GLOBAL SPERM DNA METHYLATION IS UNAFFECTED IN PROTAMINE-DEFICIENT INFERTILE MALES ............................................. 120 Abstract ........................................................................................................ 121 Introduction .................................................................................................. 121 Materials and Methods .................................................................................. 122 Results .......................................................................................................... 124 Discussion .................................................................................................... 125 References .................................................................................................... 128 9. SUMMARY AND CONCLUSIONS .................................................................. 130 Sperm Protamine Ratios in Infertile Men ...................................................... 132 Characteristics of Protamine-Deficient Human Sperm ................................... 133 The Molecular Basis of Human Sperm Protamine Deficiency ....................... 137 The Clinical Significance of the Hunlan Sperm Protamines ........................... 140 Summary ...................................................................................................... 143 References .................................................................................................... 144 viii LIST OF TABLES 1.1 Testis-specific regulators of PI and P2 transcription and translation........... 5 2.1 Comparison of semen analysis parameters and IVF outcomes between PI/P2 ratio groups...................................................................................... 15 2.II. Protamine quantification summary .... ...... ................ ........... ............... ......... 16 2.III. Contingency table comparing incidence of P 1 and P2 deregulation in patients with an abnormal P lIP2 ratio........................................................ 1 7 2.1V. Contingency table comparing incidence of PI and P2 deregulation in patients with low and high P lIP2 ratios...................................................... 18 2.V. Composition ofPlIP2 ratio groups with respect to male diagnosis ............. 19 3.1 Reproducibility of protamine measurements validated via multiple extractions of aliquots from identical samples............................................ 25 3.2 Semen quality parameters within DNA fragmentation index (DFI) and P I/P2 ratio categories................................................................................. 26 3.3 Frequency of PI and P2 underexpression within DNA fragmentation index (DFI) categories ............................................................................... 27 4.1 Protamine Fluorescence Characteristics among PlIP2 Ratio Groups .......... 43 5.1 PCR Primers for the PI, P2, TP 1, and TP2 Genes...................................... 58 5.2 Identified Single Nucleotide Polymorphisms in the Human Sperm Nuclear Protein Genes............................................................................................. 61 6.1 Real-time PCR primer sequences ............................................................... 77 6.2 Incidence of decreased, normal, and high protamine mRNA levels in patients with low, normal, and high PlIP2 ratios ........................................ 87 6.3 Incidence of decreased, normal, and high protamine mRNA levels in patients with low, normal, and high protamine protein concentrations........ 87 7.1 Comparison of Semen Quality Between P lIP2 Ratio Groups ..................... 105 7.2 Comparison of IVF Outcomes Between P lIP2 Ratio Groups ..................... 108 7.3 Comparison ofIVF Outcomes Between PI and P2 Concentration Groups. 108 7.4 Value of the SPA and PlIP2 Ratio for Predicting Abnormal Standard IVF Fertilization...... .. ......................... ........... ............... ......................... . .......... 110 8.1 Global DNA Methylation Assessed by 5MC Fluorescence Microscopy ..... 124 x LIST OF FIGURES 1.1 (A) Human protamine amino acid sequences and identity. (B) Mammalian protamine 1 amino acid sequence alignment and homology. (C) Mammalian protamine 2 amino acid sequence alignment and homology............................. 3 1.2 Diagram of protamine gene expression and regulation..................................... 7 2.1 (A) Acid gel electrophoresis of purified PI and P2 proteins. (B) Representative standard curves used to calculate PI and P2 quantity....................................... 14 2.2 Distribution of P lIP2 ratios in fertile donors and infertility patients....... .......... 16 2.3 Mean PI and P2 content in the sperm of fertile donors and infertility patients.. 17 2.4 PI and P2 deregulation frequency in patients with abnormal sperm P lIP2 ratios ................................................................................................................ 17 2.5 Frequency of protamine deregulation groups using fertile donors (A) and infertility patients with a normal PI/P2 ratio (B) as comparison groups ........... 18 3.1 DNA Integrity Assay fluorescence cytograms for samples with (A) low and (B) high DNA fragmentation index (DFI) values ............................................. 25 3.2 Mean DNA fragmentation index (DFI) levels among patients with low, normal, and high PI/P2 ratios .......................................................................... 26 3.2 Correlations between DNA fragmentation index (DFI) and (A) the PI/P2 ratio, (B) total protamine concentration, (C) PI concentration, and (D) P2 concentration ................................................................................................... 27 4.1 Immunofluorescence micrographs showing simultaneous evaluation of Individual sperm cell PI levels and DNA damage via the TUNEL assay .......... 37 4.2 Immunofluorescence micrographs showing PI and TPI expression in round and elongating spermatids ................................................................................ 38 4.3 MitoTracker and P2 immuno-flourescence in (A) fertile donor sperm with normal protamine content and (B) patient sperm with globally assessed P2-deficiency ...... 40 4.4 Population-based DNA damage assessments within P l/P2 ratio groups................. 44 4.5 Relationship between DNA damage and protamine levels in individual sperm cells ....................................................................................................................... 46 5.1 Genomic DNA sequences and identified polymorphisms in the protamine-I (P 1) and protamine-2(P2) genes .................................................................................... 60 5.2 Genomic DNA sequences and identified polymorphisms in the transition protein-I (TP 1) and transition protein-2 (TP2) genes............................................. 63 6.1 Semi-quantitative real-time PCR............................................................................ 79 6.2 Comparison of mean PI and P2 mRNA quantity within PI and P2 protein groups ................................................................................................................... 85 6.3 Comparison of mean PI, P2 and GAPDH mRNA quantity within fertile men and patients with low, normal, and high Pl/P2 ratios ............................................. 86 6.4 Relationship between protamine mRNA and protein levels .................................... 88 6.5 Relationship between the Pl/P2 protein ratio and protamine mRNA levels ............ 90 xu CHAPTER! HUMAN PROTAMINES AND THE DEVELOPING SPERMATID: THEIR STRUCTURE, FUNCTION, EXPRESSION AND RELATIONSHIP WITH MALE INFERTILITY Published in the Asian Journal of Andrology in 2003 Authors Vincent W. Aoki Douglas T. Carrell © Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Reproduced by permission of The Science Press/Asian Journal of Andrology. 2 Aoki &: Carrell/Asian J Androl 2003 Dec; 5 (4): 315·324 ·315· © 2003. Asian Journal of Andrology ISSN lOOS-682X Shanghai Institute of Materia Medica Chinese Academy of Sciences http://www.AsiaAndro.com AJA Human protamines and the developing spermatid: their structure, function, expression and relationship with male infertility Vincent W. Aoki, Douglas T. Carrell Andrology and IVF Laboratories, University of Utah School of Medicine, Salt Lake City, UT 84108, USA Keywords: prolamines; spennatids; male infertility; spennatid transition proteins Abstract DUring spermiogenesis. the protamine proteins play an integral role in spermatid chromatin compaction. Recent research has focused on many facets of protamine biology. including protamine gene and protein structure! function relationships, mechanisms of protamine expression regulation and involvement of the prolamines in male fertility. In this paper, we review our current understanding of the structure and function of the protamine-l (PI) and protamine-2 (P2) proteins and genes, the expression and regulation of these genes and the relationship between the protamines and male fertility. In addition, we offer a brief outlook on future investigation into protamine proteins. ( Asian J Andro12003 Dec; 5: 315-324) 1 Introduction During spermiogenesis, spermatid chromatin under­goes substantial compaction. Testis-specific nuclear proteins. the transition proteins and protamines, are re­sponsible for this chromatin condensation [1-5], The first step in this process occurs in haploid round sper­matids and involves replacement of somatic histones with the transition proteins (TPI and TP2). Subsequently, in elongating spermatids, the prolamines (PI and P2) re­place TPI and TP2. The resulting chromatin is highly condensed and transcriptionally silent. In the two decades following the elucidation of the protamine protein sequences by McKay et al [6, 7] nu- Correspondence to: Dr. Douglas T. Carrell. Andrology and IVF Laboratories, University of Utah School of Medicine. 675 Arapeen Dr. Suite 205, Salt Lake Cjty. UT 84108, USA. Tel: [phone] E-mail: [e-mail] Received 2003-03-22 Accepted 2003-09-01 merous studies have focused on these sperm nuclear proteins. The structure of the Pl/P2 proteins and the genes encoding them have been well described. In addition, recent studies have uncovered the details of the unique expression of PI and P2, including the storage of protamine transcripts until translation occurs during sper­matid elongation [5]. Recent research has focused on the regulatory mechanisms controlling this temporal regu­lation of PI and P2 expression during spermatogenesis [5]. Prolamine research has also entered the clinic and we are beginning to understand the link between the prOlamines and male fertility, Meanwhile, there is evi­dence that TPI plays a role in DNA repair processes and, together with TP2, may be required for complete processing of the P2 protein [8, 9]. In this paper, we review our current knowledge of the structure and func­tion of the PIIP2 proteins and genes encoding them. the expression and regulation of the PI and P2 genes, and the relationship between the protamines and male fertility. In addition, we offer a brief outlook on future investiga- 3 ·316· Prolamines and sperm development tion into protamine proteins. 2 Structure and function of PI and P2 The P11P2 genes and proteins are highly conserved in the sperm of all mammalian species. The haploid ge­nome encodes a single copy of the human PI and P2 genes mapped to chromosome 16p13.3 [10]. In addition. TP2 is mapped to the same locus on chromosome l6p 13.3 [11], The PI-P2-TP2locus spans a 28.5-kb region and is organized in a linear array, a structural feature affording concurrent expression of the PI, P2 and TP2 genes [12]. This multigenic locus, therefore, represents a single coordinately expressed chromatin domain. Additionally, the mammalian PI and P2 genes contain only one single intron [13, 14]. The structure of the protamine genes plays a major role in their transcriptional regulation. First, the PI and P2 genes are located in a large methylated domain in round spermatids, which facilitates nuclear matrix attachment and potentiation of the PI-P2-TP2 gene locus [15]. In fact, this P1-P2-TP2 multigenic locus is flanked by ma­trix attachment regions (MAR) that contain repetitive ala­nine (Alu) elements, which serve as sites of methylation [16]. In general, methylation silences gene expression while hypomethylation serves to derepress these genes by allowing chromatin to bind to the nuclear matrix, thereby maintaining a potentiated state [16]. In the case A Human PI ([>2 identil} PI 1'2 H Mammalian P I Homology ItAT IIUMA1\( ST.u,uON BOAR IIAIIIlIT CJIICIJ.'i It,vol (",O.\T Ill"". (,'()I<,'Sl."NSllS :: : : :: ~: r~ ~ :t~~l:: .. It V It, (. c ,'S Q •• a S;;j}l Y \' ... ,v.ae CJ;S Qtl.Q'If,"'t,C ... y .• {' c •• '!' II I .~; .. C \' ltV • C c,: l\ Q •• ~i •. C "....v ya.H {' s aT RI.a "t"ill ~ C I.~.H ••• ;:~:,c. f:;: ~: ~:~:; ::: ~'~t~:~ ::' .,. y • < c .. .:t>~ I r tI,';"/< :r C Mammalian (>2 Homology IIA'r \lOllil: Ul'lU." ':T.u,ut)l<, (i<lIlIU ..... ~lJUI ... \.>IUJ! U'\\I!iTl1.lt U)"'-.., .... ~lS "~'f:t II~:II II, II • IL., II •• , • 'f lI .. k 0 II D II 11.·11 '.'R .. a 'f"lI;G'q,;S II Y Rfl .• \11 Afl,.TAi9;'S Y Ila 'i1t'R It (' a ,.'G II S DYRiIJ- ,:}( •• aY·:lI'G II , D Y II fl - 11: H· • k "',:0 D II H fl 'll II It '(.i", ,JJ" Dr r .:, .at of the P I-P2-TP2 locus, gene potentiation occurs after binding of the chromatin to the nuclear matrix, this asso­ciation being mediated by the MARs [16]. Interestingly, Schmid et al recently reported that MAR binding in the P1-P2-TP2 locus was independent of Alu methylation status, which indicated that the MAR attachment site was important for transcription but was not regulated by the state of methylation [16], Second, all protamine genes contain a TATA-box, which facilitates binding of tran­scription factors to their promoters, thereby playing a major role in transcription initiation. Third, a cAMP­response element (CRE) is present in all protamine genes and is highJy conserved in sequence and location resid­ing from position -57 to -48 [17]. The CRE regulates transcription by binding various CRE proteins to this regu­latory region [18-21], Fourth, upstream regulatory se­quences in the individual PI and P2 promoters bind other trans-acting proteins, thereby directing transcriptional activation or suppression [22-25]. The human P I and P2 genes encode a 50 amino-acid protein and a final processed protein of 57 amino-acids, respectively (Figure 1). Overall, there is approximately 50 % identity between human PIIP2. The ratio of P1:P2 varies in each species and in fertile humans is approxi­mately 0.9 to 1.0 [26-28]. Corzett et al recently re­ported the P2 content to vary from 0 % to 80 % in a selected group of mammalian species [29]. However, within a given genus the PI :P2 stoichiometry is tightly "",11\' \ 111111 Il :·,Jall,.l Figure 1. (A) Human protamine amino acid sequences and identity. The sequences are from McKay et al. [6.7] and show approximately 50 % identity. (B) Mammalian protamine 1 amino acid sequence alignment and homology. (C) Mammalian protamine 2 amino add sequence alignment and homology. Amino acid sequences were obtained in PubMed Protein and shaded areas indicate homology. 4 Aoki &: Carrell IAsian J Androl 2003 Dec; 5 (4): 315·324 ·317· regulated [29]. Both the PI and P2 proteins are highly basic and contain copious amounts of arginine (-50 % of the total amino acid compliment) and cysteine (-10 %). Although the arginine content of both proteins is similar, P2 is slightly more basic due to its higher his­tidine and lysine content. In light of this sequence infonnation. the structure/function relationship is readily apparent. Indeed, the basic character of the protamines facilitates DNA binding due to the strong intennolecular attraction between the positively charged protamine and the negatively charged DNA backbone. Furthennore, the presence of cysteine residues allows the fonnation of disulfide bridges. affording stability in the protamine molecular structure. With respect to synthesis and processing. PI is syn­thesized as a mature protein product. P2 is synthesized as a precursor protein of 103 amino acids and undergoes proteolytic cleavage of its amino-tenninus [30]. Like many other proteins. phosphorylation of the protamines is required for their DNA binding. PI protein is rapidly phosphorylated after translation [31]. Serine/arginine protein-specific kinase I is largely responsible for the phosphorylation of PI [32]. An intennediate form of P2, one derived by proteolysis of the precursor P2, is also rapidly phosphorylated [31]. One important regula­tor of P2 phosphorylation is the Ca2+/calmodulin-depen­dent protein kinase N (Camk4). Camk4 phosphorylates P2 in vitro and targeted mutations in the Camk4 gene have produced infertile mice with a specific loss of P2 and retention ofTP2 [33]. The phosphorylation of P2 is essential for its binding to chromatin which, in tum, is requisite for proteolytic cleavage and processing of the P2 protein. There are two major proposals for the physi­ological significance of protamine phosphorylation. Flrst, it is thought that P IIP2 phosphorylation is required for correct binding to DNA and their subsequent dephos­phorylation induces chromatin condensation in the spenn nucleus [3, 34]. Second, in addition to facilitating proper protamine-DNA binding. phosphorylation (not dephos­phorylation) may also promote correct chromatin con­densation [32]. Although this second hypothesis is some­what counterintuitive, giving that phosphorylation would destabilize a highly condensed chromatin and the phos­phate groups attached to these highly basic proteins may reduce repelling forces and serve to promote their inter­actions and consequent chromatin condensation. Recent studies using electron microscopy and scan­ning probe microscopy have elucidated protamine me­diation of DNA condensation. Binding of protamines to the DNA induces coiling of the DNA into a toroidal or doughnut-shaped structure. These loop domains are very compact, only half the size of somatic cell histone loops, accounting for the 6-fold increase in spenn chromatin compaction [35], The mechanism by which the prota­mines mediate this interaction is currently under debate [36]. PI and P2 may bind to the major groove of the DNA [30], attach to both the minor and major grooves [37] or electrostatically bind to the surface of the DNA by interacting with phosphate residues [38]. PI is present in all mammalian spermatozoa while P2 has been detected in spermatozoa of mouse hamster. vole, rat, stallion and man. Although the P2 protein is not present in some other mammals, such as the bull and boar. the gene encoding P2 is present and transcribed in these species. The absence of the protein is explained by translational regulatory mechanisms [39]. From an evolutionary standpoint, it is likely that mammals have inherited the PI gene from a common ancestor since it is present in all species studied thus far. There are two possible explanations for the origin of the P2 gene. First. P2 may be derived from Pl followed by the divergent evolution of the two genes [40]. Second, it may be pos­sible that P I and P2 were inherited from a single com­mon ancestor and that successive species have lost the ability to express P2 protein [13], 3 Protamine expression regulation The protamines are expressed in the post-meiotic haploid spennatid. An interesting aspect of this expres­sion is that transcription and translation of the proteins are temporarily uncoupled in the developing spennatid [41]. Because protamine-DNA binding results in chro­matin condensation and consequent transcriptional cessation, the protein cannot be transcribed concomi­tantly at the time it is needed during spenniogenesis. Thus, aU transcription must precede translation of the pro­tamines. Indeed, Pl and P2 mRNA have been identified in round spennatids while the proteins remain conspicu­ously absent at this stage [42, 43]. Translation of these mRNAs is delayed until the elongating stage of spenna­tid differentiation [44-46]. We should note that a small amount of mRNA is present in the mature human spennatozoa, which presumably represents remnant message that was never translated [47], The temporal uncoupling of transcription and trans­lation of the PI and P2 genes is due to transcriptional and translational regulatory mechanisms [48, 49]. Tran­scriptional regulation occurs on three fronts: (1) DNA methylation, (2) binding of trans-acting factors to TATA box. CRE-box or other specific promoter DNA sequences of the protamine genes and (3) potentiation via chroma­tin association with the nuclear matrix (Table I), The PIIP2 genes are associated with regions of increased 5 ·318· Prolamines and sperm development Table 1. Testis-specific regulators of P1 and P2 tmnscription and translation. *( +) Activation; (-) Repression. Regulator Target Response' Reference Transcription Methylation/Potentiation TBP PIIP2 gene region (MAR sequences) TATAbox + + 16,48 50 51,52 51.52 51,52 51,52 CREB CREM PAP-I Y -box protein 48/p52 Tet-l cAMP-response element cAMP-response element Trans-acting factor (Binding sites-61 (PI) and-48 (P2) + + + + Nuclear Factor I Translation MRNPs Prbp PABP Trans-acting factor {Binding sites -84 (PI) and -72 (P2) PI Promoter + + 53 54 Pl Promoter mRNA storage in chromatoid bodies + 58,59 74 61 Y -Box Proteins Contrin Translin Unknown - directJindirect action on PI &. P2 mRNA 63-73 67 70 48/50 kDa RNA-BP 72 PI &. P2 poly-A tail and 3'UTR sequences 3' UTR sequences methylation in round spennatids as compared with elon­gating spennatids, accounting for the increased transcrip­tiona] activity at that stage [43]. Transcriptiona1 regula­tion via the TATA box may be mediated by a TATA-bind­ing protein (TBP) recently identified in rodents [50]. TBP binding to the TATA box results in transcriptional activa­tion of the PIIP2 genes [50]. CRE nuclear factors medi­ate transcriptional regulation via the adeny]yl-cyclase pathway [48]. These nuclear factors include cAMP­response element binding protein (CREB) and cAMP­response element modulator protein (CREM) [48]. Fol­lowing the typical cAMP-protein kinase A pathway, CREB and CREM are phosphorylated allowing them to bind to the CRE. This binding, in tum, activates transcription of the PIIP2 genes. Other trans-acting factors include protamine activat­ing factor (PAF-l) and a testis-specific Y -box protein (Y­box protein p48/p52), which activate haploid cell P2 tran­scription by binding to specific regulatory sites located at positions -64/-48 and -84/-72, respectively [51, 52]. This P2 transcriptional activation was exemplified by studies, which demonstrated a 5-fold increase in P2 tran· scription when PAF-l and Y -box protein p48/p52 bound to these regulatory sites [51,52]. Furthermore, alter­ations to the gene sequences of these binding sites re" sulted in a substantial reduction in P2 transcription [51, 52]. In addition, there are a number of ubiquitous and testis·specific proteins. such as Tet-l and nuclear factor I. which bind with the PI promoter to activate PI tran­scription [53,54]. Thus far, no regulatory proteins have been identified which specifically function to inhibit tran-scription of the protamine genes. Although there are general modifiers of gene activity such as the Y -box pro­teins in which interactions with other tissue specific regu­latory proteins determine their role in gene regulation [55]. Lastly, the potentiation of the PI-P2-TP210cus may be mediated through association with the nuclear matrix [56]. Lee et al demonstrated aberrant premature ex­pression of the PI-P2-TP2 locus causes developmental arrest and cell death [57]. It makes sense. therefore, that in non-expressing cells the chromatin is maintained in a transcriptionally silent closed confonnation. Then, during spermatogenesis, the chromatin is selectively opened in specific domains to express those genes needed during differentiation [56]. As mentioned above, in the PI-P2-TP2 locus, gene potentiation occurs after bind­ing of the chromatin to the nuclear matrix. this associa­tion being mediated by the MARs [16]. Although these nuclear matrix attachment regions do not appear to be regulated by methylation status, their attachment to the nuclear matrix does afford potentiation of the PI-P2- TP2 locus [16]. Thus, protamine gene regulation can be integrated into a three-step process consistent with the recently emerging mode] for gene expression [56]. First. poten­tiation of the protamine gene cluster occurs either by hypomethylation of the region or MAR binding to the nuclear matrix thereby rendering the gene cluster in an open chromatin structure affording transcription factor binding. Second, initiation of transcription via binding of trans-acting factors to TATA box, eRE-box or other specific promoter DNA sequences of the protamine genes. 6 Aoki & Carrell/Asian J Androl 2003 Dec; j (4): 315-324 ·319· Lastly, an elongation phase in which the PI, P2, and TP2 transcripts are produced paving the way for translation. Indeed, translational regulation is one of the most important aspects of protamine biology. In fact, if prota­mine transcription and translation is allowed to occur concurrently, the DNA undergoes precocious compac­tion and sperm development is arrested [57]. It is the mechanism of translational regulation that is responsible for the delay in PIIP2 protein appearance until the elon­gation phase of spermiogenesis. One mechanism to ac­complish this temporal regulation of mRNAs is to keep the transcripts physically separated from the cellular trans­lation machinery. In fact, translationally repressed mRNAs are sequestered in messenger ribonucleoprotein (mRNP) particles in chromatoid bodies near the sperm nucleus [58, 59]. Translational repression is evident dur­ing the round spermatid stage by the increased presence of these mRNPs [60]. Before being sequestered, however, the mRNAs un­dergo processing in the nucleus, which contributes to translation suppression. This processing includes a cleav­age of the hnRNA primary transcript followed by the addition of a longpolyadenylated (poly-A) tail, which serves to repress the mRNA from translation [5], The poly-A mRNA is then stored in the mRNPs until deadenylation and shortening of the poly-A tail, at which time it is released from the mRNP and subsequently, undergoes translation [5). Steger has proposed the regu­lation of adenylation may be under the control of cellular signaling cascades related to pituitary hormone control [5]. In addition, a poly-A binding protein (PABP) may have a dual role in the adenylation and consequent trans­lation regulation of the protamines. First, by binding to the poly-A tail, PABP initially protects the mRNA from degradation thereby preserving the transcript until trans­lation repression is removed [61]. Second, PABP may act as a repressor protein when it is bound to the end of the poly-A tail (61]. This repressor action is an indirect consequence to its protecting and maintaining the long poly-A tail. However. PABP then migrates upstream to the 3'-UTR segment leaving the poly-A tail subject to degradation and subsequent translational derepression [61]. Sequence-specific RNA binding proteins also target the 3'UTR sequence of PI and P2 mRNAs to mediate their translational regulation [62]. In general. it is thought that binding of these regulatory proteins to the protamine mRNA induces translational repression while modifica­tion of the mRNP complex results in release of translat­able mRNA and subsequent translation derepression. Translational repression involving the 3'UTR sequences is accomplished mainly by the Y-box protein family [63]. These Y-box proteins contain a central cold shock do­main that facilitates binding with nucleic acids. In general, phosphorylation of the Y -box protein is required for this binding to occur. Identified Y -box proteins which re­press translation of the PIIP2 transcripts include the 481 52 illa Y-box proteins (murine-MSY2, human-contrin and rat-YB2IRYB) , the 18 illa RNA-binding protein. the 26 IDa testislbrain RNA-binding protein (murine-TB­RBP, human-translin) and the 48150 IDa RNA binding proteins [64-73]. A related protein in the rat, Prbp, has been shown to be required for translational activation of the PI mRNA [74]. Prbp contains two copies of a double-stranded­RNA- binding domain and, in vitro, binds to the 3' -UTR of Pl mRNA. The function of the protein in translation regulation was recently elucidated by Zhong et al [74]. This investigation showed that disruption of the gene encoding Prbp results in retention of translational repres­sion of PI mRNA. Consequently, the delayed replace­ment of the transition proteins by the protamines resulted in sterility and severely oligospermic mice [74]. Thus, the authors concluded that Prbp is required for the proper activation of repressed PI mRNA. Curiously, this pro­tein has been shown to bind to the 3'-UTR of PI mRNA, a well-defined characteristic of translational repressors noted above. In fact, a recombinant form of Prbp has been shown to repress translation of multiple mRNAs in wheat germ lysate (57]. Given this apparent discrepancy, a well-defined mechanism of Prbp action is very much needed but remains elusive. Indeed, such a study would be of great value in the light of a recently identified protein, TAR RNA binding protein (TRBP), which is the human homologue to Prbp and is expressed in spermatids at steps 3-4 of spermiogenesis, suggesting a potential role in protamine translation regulation [75]. The temporal expression of the protamine genes is indeed unique and extremely complex. Transcription occurs in round spermatids and involves potentiation of the PI-P2-TP2 chromatin domain, initiation via binding of trans-acting factors and elongation of the primary transcript. Subsequently. translational regulation ac­counts for the delay in protamine protein production and begins immediately during RNA processing with cleav­age and po\yadenylation of the mRNA. As the protamine mRNA enters the cytoplasm, it is translationally repressed via storage in mRNPs, binding by PABP and other RNA binding proteins, which target the 3' -UTR sequence. Translation is then derepressed by covalent modification of the mRNPs, release of translatable mRNA and migra­tion of the PABP to the 3'-UTR, leaving the po1y-A tail susceptible to degradation (Figure 2), 7 ·320· Prolamines and sperm development I POTENTIATION I Z MAR FaclUtated ChromatinlNuclear Matrix Binding ~ + INITIATION Ur:J'} TATA Box Bloding CREBinding Other Trans-Acting Factors tIJ ~ 1BP CREB PAF-I 8 CREM Y -Box Protein p48/52 ~ ~ + Nuclear Factor I E-ELONGATION tg~IJ: hnRNA P+ro duction ~ POST .. TRANSCRIPTION PROCESSING RNA Cleavage 0 ~ Polyadenylation + TRANSLATION REPRESSION mRNA Storage in mRNPs Translation ReRressor Binlllwl PoIy·A Binding PABP Z 3' .. UfR BinW 0 Contrin 5 Translin 40150 kDA RNA-BP tI.l ~ + I r:J'} Z TRANSLATION ACTIVATION ~ E- Covalent Modification of mRNP Release ofmRNA from mRNP tI.l PABP Migration to 3' .. UfR Q Poly-A TaD Degradation ~ Prbp Action Z + 0 ~ PI & P2 Protein ~ Figure 2. Diagram of protamine gene expression and regulation. Transcription occurs in round spermatids and involves potentiation of the PI-P2-TNP2 chromatin domain [16,48], initiation via binding ofTBP [SO), CREB. CREMt PAP-I. Y-box protein p48152 and Nuclear Factor 1 [51-52] and, lastly, elongation of the primary transcript [561. Transcription is followed immediately by post-transcriptional processing of protamine mRNA [5]. Subsequently, translational regulation accounts for the delay in protamine protein production. As the protamine mRNA enters the cytoplasm it is translationally repressed via storage in mRNPs [58, 59), binding by PABP [61] and other RNA binding proteins. which target the 3'-UTR sequence [63-73]. Translation is then derepressed by covalent modification of the mRNPs [5], release of translatable mRNA [5] and migration of the PABP to the 3' -UTR, leaving the poly-A tail susceptible to degradation [61]. 8 Aoki & Carrell/Asian J Androl 2003 Dec; 5 {4}: 315·324 ·321· 4 Protamines and infertility Because of the critical role oonna! protamine replace­ment plays in spermatid differentiation, one might ex­pect aberrations in protamine expression or structure to lead to male infertility. Indeed, numerous reports have emerged in the last decade establishing a relationship be­tween abnormal protamine expression and infertility. Premature translation of PI has been reported to cause precocious nuclear condensation, which resulted in the arrest of spermatid differentiation in mice [57]. Abnor­mal PI :P2 ratios have been reported in the sperm of in­fertile human males, which indicate the relative amounts of each protamine is important for proper spermatid dif­ferentiation [27, 76-77J. In fact, there is evidence that the Pl:P2 ratio is more important for male fertility than the absolute amount of protamines [58, 76, 78]. A re­cent report found P2 precursors to be present in the sperm of infertile males who had reduction in P2 levels [79]. Presumably, this indicates incomplete processing of the P2 protein. The importance of the PI1P2 ratio in spermatogenesis has recently been emphasized by a study, which showed that haploinsufficiency of PIIP2 causes severe infertility in mice' [80]. In addition, the haploin­sufficiency of P2 has recently been reported to lead to sperm DNA damage and embryo death in mice [81]. Consistent with this data. Yebra et al reported the com­plete loss of P2 protein in a small proportion of infertile males [82]. Carrell and Liu later found that P2 was un­detectable in 13 of 75 severely infertile patients analyzed prior to in vitro fertilization. Conversely, P2 was seen,in all 50 donors of known fertility analyzed [27J. That study also showed that low P2 levels were generally associ­ated with low sperm counts, motility and morphology. Additionally, Mengual et al recently reported marked in­creases in the PlIP2 ratios of oligozoospennic and asthenozoospermic patients as compared with fertile control patients [83]. Taken together these studies may indicate that the abnormal protamine content in sperm is a reflection of abnormal transcriptional or translational regulation of PIIP2 expression. Functionally, it appears as if the protamines are re­quired to impart zona pelucida binding and penetration abilities. This was exemplified by a study that showed destruction of the protamines inhibited sperm binding and penetration in the hamster egg penetration test [84]. Recent reports have shown that abnormal P2 is associ­ated with diminished fertilization ability [85]. However, it does not appear as if normal protamine replacement is required for pronulcear formation, because ICSI with round spermatids has been shown to produce chromatin decondensation and pronucleus formation [84]. Additionally, patients without P2 have good success with ICSI during IVF cycles [85]. Furthermore, it has been demonstrated that destruction of the protamines actually increases sperm decondensation [84-85]. Taken together, these studies demonstrate that protamines are important components of spermatid differentiation and aberrations in the protamines are related to infertility and may reflect defects in spenniogenesis. Thus far, the underlying causes of PIIP2 ratio de­regulation in infertile males remains elusive. However, a number of attractive hypotheses may account for the induction of aberrant PIIP2 stoichiometry. First, muta­tions in either the Pl or n, genes may play a role in P II P2 deregulation. Although there have not been any sub­stantial reports on mutations of the protamine genes in patients with specifically identified aberrations in the P 11 P2 ratio, a recent study has identified single nucleotide polymorphisms in the Pl and P2 genes in a large group of both fertile and sterile male patients [86]. Second. gene mutations in any of the accessory proteins includ­ing TPl, TP2, Carnk4 and Serine/arginine protein-spe­cific kinase 1 may result in a breakdown in the normal histone-protamine replacement process and render an abnormal PI1P2 ratio. Third. in the absence of any gene mutations. an irregular PI1P2 ratio may reflect defects in translation regulation and, to a lesser extent transcription regulation through unfaithful function of accessory regu­lator proteins. Such defects could potentially disrupt the temporal expression of PI and P2 and result in irregular PIIP2 ratios. Aberrant translation regulation is more likely to be involved in protamine expression regulation. Furthennore, the data showing disruption of regulatory proteins such as Prbp, which results in retention of trans­lational repression and subsequent severe infertility. also support this hypothesis. Fourth, incomplete processing of the P2 protein could result in aberrant P I1P2 ratios. Indeed, there is evidence supporting this hypothesis, as mentioned above. a group of infertile males with reduced P2levels also displayed an increase in P2 precursors [79]. Considering the importance of TPI and TP2 in the pro­cessing of P2, this may be a reflection of TP deficiency or abnormal function. Finally. disruption of normal PII n phosphorylation would result in incomplete protamine processing and unsuccessful binding to the DNA. Thus, improper signal transduction or malfunction in the ap­propriate kinases could potentially playa role in PIIP2 ratio deregulation. 5 Future directions As explained above, protamines are uniquely regu­lated during spermatogenesis. Proper transcription, 9 ·322· PrOlamines and sperm thvetopmens translation and post-translational modifications are im­perative to requisite function of chromatip compaction and transcriptional silencing during spermiogenesis. Abnormal P2 expression has been shown in both mice and humans to lead to severe infertility. The identifica­tion of infertility patients with abnormal levels of P2 of­fers some interesting opportunities for future studies. First, it will be important to ascertain if an increase in the PIIP2 ratio of the proteins extracted from a pool of ejacu­lated spermatozoa reflects a general decrease in P2 in all sperm in the population or rather if P2 is diminished pref­erably in apoptotic or non-viable sperm. Second, the underlying causes of P2 deficiency can be evaluated, in­cluding the sequencing of relevanr genes in affected patients. In addition to studying the PI, P2 and TP genes, it will be important to evaluate those genes that encode translation regulators, such as Prbp, which has been shown to lead to oligozoospermia when disrupted in mice. Third, temporal regulators may be responsible for ab­normal P2 expression and should be further evaluated in patients with low P2 levels. Finally, we suspect protamine production is a pos­sible regulator or checkpoint of spermatogenesis. There are a number of studies supporting this hypothesis. First, haploinsufficiency of PI and P2 leads to severely de­creased spermatogenesis in mice [80]. Additionally, mice lacking P2 also show increased sperm DNA damage [81 J. Interestingly, in the human, moderately diminished lev­els of P2 are associated with severely diminished sperm counts, motility and normal morphology [27]. Disrup­tion of PI and P2 expression, exemplified by a knockout of the translational regulator Prbp, also leads to severe infertility [74J. 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J BioI Chem 1993; 268: 10553-7. 83 Mengual L. BaUesca JL, Ascaso C. Oliva R. Marked differ­ences in protamine content and PIIP2 ratios in sperm cells from percoll fractions between patients and controls. J Androl 2003; 24: 438-47. 84 Ahmadi A. Ng Sc. Destruction of protamine in human sperm inhibits spenn binding and penetration in the zona-free ham­ster penetration test but increases spenn head decondensation and male pronuclear formation in the hamster-ICSl assay. 1 Assist Reprod Genet 1999; 16: 128-32. 86 Carrell DT. Uu L. Heparin binding sites are present at a higher concentration on spenD of subfertile men than donors of known fertility. Arch Androl2oo2; 48: 147-54. 87 Tanaka H, Miyagawa Y. Tsujimura A. Matsumiya K, Okuyama A. Nishimune Y. Single nucleotide polymotphisms in the prota­mine- I and -2 genes of fertile and infertile human male populations. Mol Hum Reprod 2003; 9: 69-73. CHAPTER 2 IDENTIFICATION AND EVALUATION OF A NOVEL SPERM PROTAMINE ABNORMALITY IN A POPULATION OF INFERTILE MALES Published in Human Reproduction in 2005 Authors Vincent W. Aoki Lihua Liu Douglas T. Carrell © European Society of Human Reproduction and Embryology. Reproduced by permission of Oxford University Press/Human Reproduction. Human R.eproduction VoI.20. No.5 pp.129S-1306, 2005 Advance Access publication February 10, 2005 13 Identification and evaluation of a novel sperm protamine abnormality in a population of infertile males Vincent W.Aoki1.2.3, Lihua Liu1,2 and Douglas T.Carrell1•2,3,4,5 lAndrology and IVF Laboratories. 2Department of Surgery. 3Department of Physiology and 4Department of Obstetrics and Gynecology, University of Utah School of Medicine, Salt Lake City, UT 84108, USA 5To whom correspondence should be addressed at the Andrology and IVF Laboratories. University of Utah School of Medicine. 675 Arapeen Dr. Ste 205. Salt Lake City. UT 84117. USA. E-mail: [e-mail] BACKGROUND: A significant relationship exists between an abnormally high sperm protamine-l (PI)/protamine-2 (P2) ratio and male infertility. In this study we investigate whether a decreased PIIP2 ratio is also linked to male infertility and we attempt to describe, at the protein expression level, the underlying cause of sperm P1JP2 deregu­lation. METHODS: PI and P2 protein concentrations were quantified in sperm from 272 infertility patients and 87 fertile donors. PI1P2 ratios and protamine quantity were correlated with fertility status using semen analysis, sperm penetration capacity, and IVF data. RESULTS: We identified four distinct groups in the study: normal P1JP2 fertDe donors, normal PIIP2 patients, low PIIP2 patients, and high PIIP2 patients. PI and P2 were both under-expressed in patients with a normal PI1P2 ratio, but not in fertile donors. In patients with a low P1JP2 ratio, PI was under-expressed while P2 was over-expressed; in patients with a high PIIP2 ratio, PI was normally expressed and P2 was under-expressed. Patients with abnormal PIIP2 ratios displayed significantly reduced semen quality and sperm penetration ability. CONCLUSIONS: We have identified a novel population of infertile males with a reduced PIIP2 ratio. Aberrant PIIP2 ratios arise from an abnormal concentration of PI and/or P2, either of which is associated with male infertility. Key words: expressionllVFlhuman sperm/protamine/semen quality Introduction During spermiogenesis, sperm protamines replace somatic cell histones in a multi-step process (Oliva and Dixon, 1991). The first step in this process occurs in round spermatids and involves replacement of the histones with the transition nuclear proteins (TPI and TP2). Subsequently, in elongating spermatids, the protamines replace TP1 and TP2. This histone-protamine replacement results in a highly con­densed. transcriptionally silent chromatin (Dadoune, 1995). The importance of the protamines is highlighted by the high level of conservation found within mammalian genera (Corzett et al.. 2002). In human males there are two forms of sperm protamine, protamine-l (Pl) and protamine-2 (P2), which occur in a strictly regulated 111 ratio (Corzett et al .. 2002). It now appears that strict regulation of this protamine-II protamine-2 ratio (PIIP2) is critical for the fertility status of human sperm. A number of studies have established a relationship between deregulated protamine expression and human male infertility (ChevaiUier et ai .. 1987; Balhorn et ai., 1988; Belokopytova et al .. 1993; de Yebra et al., 1993, 1998; Carrell and Liu. 2001). Each of these studies demonstrated that an elevated PI/P2 ratio is highly correlated [0 malc infertility. In addition, the studies conducted by de Yebra el al. (1998) and Carrell and Liu (2001) described a popu­lation of infertile males with undetectable P2 in their sperm. Taken together, these data have led to the assumption that decreased expression of P2 is responsible for the increased PIIP2 ratio observed in inferti1e males. However, studies have failed to elucidate a population of infertile males with a diminished PIIP2 ratio. Such a finding would question the assumption that P2 deregulation is always responsible for aberrant PlIP2 ratios. In this study, we aimed to quantify the spenn PI and P2 concentrations in infertility patients and men of known ferti­lity; to evaluate semen quality and IVF outcome in patients with a reduced PIIP2 ratio; and to assess which of the prota­mine proteins is abnormally expressed in infertile males with an abnormal PIIP2 ratio. Materials and methods Mo1eriols Unless otherwise noted, all chemicals were obtained from Sigma Chemical Company (USA). Reagents for gel electrophoresis were purchased from BioRad Laboratories (USA). 1298 © The Author 2005. Publij'hed by Oxford University Press on bdlG/f of 1m: European Society (~f HUnIG/J Repmduclum and Elnbryology. All rights reserved. For Permission:1. please email: joumaL~.permis.iions@{}upjoumals.org Study population and semen quality evaluation Institutional Review Board approval was obtained prior to initiation of this study. Semen was collected and evaluated from 87 fertile donors and 272 patients presenting for infertility assessment andlor preparing to undergo IVF. A single semen sample was used for diagnostic assays, including protamine protein extraction and quanti­fication. A second sample wa<; used for the IVF procedure. Patients were excluded from the study if the spenn concentration was <] X 106/ml or if they were presenting for post·vase(..'tOmy analysis. Semen quality was evaluated by following WHO (World Health Organization. 2000) standards for semen analysis. including spenn concentration. motility and morphology. In addition, a spenn pen· etration a<;say (SPA) wa~ perfonned on the semen sample as pre· viously described (Carrell and Urry. 1996: Carrell et al.. 1998). The percentage of hamster ova penetrated by one or more sperm is reported. Purification of nuclear proteins Spenn nuclear proteins were extracted a<; previously described from all 87 fertile donors and 272 patients enrolled in the study to deter· mine PI/P2 mtios (Carrell and Liu. 2001). PI and P2 concentrations were subsequently quantified in all 87 fertile donor samples and 139 of the 272 patients. Prior to extraction, sperm concentrations were detennined in samples being quantified for P I and PZ concentrations using WHO criteria. All samples were run in duplicate and the aver­age P I and P2 concentrations and P I/PZ ratio from the two runs were reported. Semen aliquots with a known number of spenn (in samples being quantified for PI and P2 concentrations) were centrifuged at 500 g for 5 min at 4°C, The pellet was wa"hed in I mmolll phenyhnethylsulfonyl fluoride (PMSF) in distilled water, centrifuged at 500 g for 5 mi n at 4°C, and the pellet was resus· pended in 100 Il-I of 100 mmol/l Tris buffer containing 20 mmol/l EDT A and I mmolll PMSF (pH 8.0). One hundred microlitres of () molll guanidine and 575 mmolll dithiothreitol was added and mixed, followed by mixing of 200 Il-I 522 mmolll sodium iodo­acetate. The suspension was protected from light and kept at room temperature for 30min. The suspension was mixed with l.Oml of 100% ethanol at 4°C for 1 min and centrifuged at 12000 g for 10 min at 4°C. The ethanol wash was repeated and the pellet was resuspended in 0.8 ml of 0.5 molll HCl and incubated for 15 min at 37°C and centrifuged at lO000g for 10 min. The supernatant was kept and the nuclear proteins were precipitated by the addition of 100% trichloroacetic acid (TCA) to a final concentration of 20% TCA. The solution was incubated at 4°C for 5 min and centrifuged at l2000g for Wmin. The pellet was wa'ihed twice in 5001l-11% 2· mercaptoethano) in acetone. The final pellet was dried and stored at - 20°C until analysed using gel electrophoresis. Preparation of the human protomine smndard A human protamine standard was prepared as previously described (Mengual et al .. 2003). A pool of 20 semen samples was made in order to extract and quantify speml protamines. Briefly, sperm were washed twice with 0.5 molll HCI before protamine extraction to remove other acid·extracted proteins. After acid treatment. the protanlines were extracted as described above. The protein extract contained highly purified protamine and the final protamine concen­tration was detennined using the RC DC protein a'isay kit (SioRad Laboratories, USA). The protamine extract was run using acid­acrylamide gel electrophoresis to detemline the ratio of PI to P2 (see below). 14 Sperm protamine deregulation in infertile males The final concentration of P) and P2 was calculated from the per­centage composition of each of the protamines in the total protamine standard. Subsequently, 1.52, 0.76, 038 and 0.19 f.l.g of human sperm protamine standard were loaded in each gel and a standard regression curve was made to calculate the amount of protamine in each of the patient samples (Figure I). The r 2 value of the regression curve was 2: 0.96 for each gel run. PIIP2 quantification Acetic acid-urea gel electrophoresis was performed as previously described by Carrell and Liu (2001). The separating gel contained 20% acrylamide. 0.1% bisacrylamide. O.9moUl acetic acid, and 2.5 molll urea. The stacking gel was comprised of 7.5% acrylamide. 0.2% bisacrylamide. 2.5 molll urea, and 0.375 molll potassium acet· ate at pH 4.0. Gels were stained with Coomassie Blue using stan· dard techniques. The gels were scanned using a Umax-SE scanner with the Silver Fast scanning software package (Umax Technologies, USA). The intensity of bands corresponding to PI and P2 wa~ quan­tified using National Institutes of Health Image-J software. PI and P2 quantities were calculated against the standard curve genemted from the human protamine standard as described above (Figure 1). Protein quantity is reported as ng protein/106 spenn. Identity of protamine hands were established using western blot analysis as reported in a previous study (Carrell and Liu, 2001). Protamine quantification quality control We employed two distinct quality controls to ensure our protamine quantification protocol could produce valid and reproducible results A 2 3 4 s 6 8 It9 OJI .ns-dC- ·'Is-dC_ 0.1 ! 0.6 f O.S J 1t4 113 O.l 0.1 0 0.00 O.~I !.Ill} I.$) 2.00 BII IlIlWiI) I<'igure I. (A) Acid gel electrophoresis of purified PI (arrow) and P2 (arrowhead) proteins. Shown are representative banding patterns for a patient with a high PIIP2 ratio (lane I), a low PIIP2 ratio (lane 2), and four human protamine standards u.~ to generate standard curves (lane 3: 0.1895Il-g; lane 4: 037891l-g; lane5: 0.7578Il-g; lane 6: L5156Il-g). (B) Representative standard curves used to calculate PI and P2 quan· tity. Linear regression resolved a P2 standard curve fitting the equation fP2] 0.3919(Intensity) + 0.066 with an R 2 value of 0.9999. The PI standard curve fit the equation [PI} 0.3547(Intensity) + 0.0259 with an R 2 value of 0.9988. 1.299 V.W.Aoki and L.Liu and D.T.Carrell with respect to evaluation of the PIIP2 ratio. [PI] and [P2]. First, aliquots of 20 X 106 sperm were made from a common semen sample taken from a pool of 20 semen samples. One of these aliquots was run with each round of extractions (n 15). The resulting mean PlIP2 ratio (0.85 0.01), PI concen­tration (441.1 ± 3.7 ng/I06 sperm), and P2 concentration (522.1 ± 4.5 ngll06 sperm) showed little between-sample variation and ensured reproducible results within individual samples. Second, to evaluate variations in the PI1P2 ratio, [PI] and [P2) between eja­culates from the same individual. we analyzed the semen from two different ejaculates (6 months apart) in 42 individuals. Results indi­cated no significant differences between ejaculates with respect to the PIIP2 ratio (1.03 ± 0.04 versus 1.11 0.08), PI concentration (560.4 ± 42.2 versus 571.9 49.6 ng/106 sperm). or P2 concen­tration (535.5 ± 30.9 versus 527.1 ± 37.2 ng/106 sperm) as assessed by a paired t-test. IVF A total of 175 patients subsequently underwent lVF. Ovarian stimu­lation was performed using standard techniques of GnRH agonist down-regulation and gonadotrophin stimulation with ultrasound­guided transvaginal oocyte retrieval performed 36 h after heG administration. Pertilization was achieved by standard IVF (11 71 cases). ICSI (n 73). or a combination of standard IVF/ICSI (n 31) depending on the sperm penetration score (Table I). Resulting embryos were cultured in RTF medium supplemented with 15% heat-deactivated maternal serum for 72 h post-oocyte retrieval. at which time embryos were transferred to the uterus. Embryo grade was assessed based on the degree of cellular frag­mentation and the regularity of blastomere morphology, with zero being the highest quality and three being the worst quality. A pre­viously reported embryo scoring system was used which represent.'! the number of blaslomeres minus the embryo grade (Carrell et al., 1999). 15 Stntistical evaluation Ba~ed on the PlIP2 ratio, study I.;ubjects were stratified into four groups: normal PI/P2 fertile donors, normal PIIP2 patients, low PIIP2 patients, and high PIIP2 patients. Low «0.8) and high PIIP2 ratios (> 1.2) were defined by the critical values calculated from the two-tailed nornlal distribution for the PIIP2 ratios of fertile donors with 90% confidence limits (Figure 2). Protamine protein quantity was compared between these groups using the Kruskal-Wallis test. Deviation in PI andlor P2 expression in patients with abnormal PIIP2 ratios is referred to as deregulation and was assessed by comparing their protein concentrations against those of the fertile donors and patients with nonnal PIIP2 ratios. Pl and P2 concentrations falling outside the critical values for PI and P2 concentration in the comparison groups were classified as being deregulated. The critical values for PI and P2 quantity define the protein cOllcentration range within a two-tailed normal distribution at a confidence of 95%. The occurrence of PI deregulation was compared to that of P2 deregulation using "i-analysis. Semen quality parameters including sperm concentration, motility and morphology were compared between groups statistically using Kruskal- Wallis analysis. The fertilization rate, embryo quality and pregnancy rate of those subjects subsequently undergoing IVF was compared between groups. Kruskal- Wallis was used to compare fertilization rate and embryo quality whereas x2-analysis was used to compare pregnancy rates between groups. The relationships between the PIIP2 ratio/protamine measure­ments (PI. P2 and total protamine quantity) and semen quality/IVF outcome measures were evaluated using Spearman's correlation. I.n order to use this test effectively we split the analysis between two groups for correlation analysis with the PIIP2 ratio: 0) patients with low and normal ratios (those with PIIP2 ratios of 0-1.2); and (ii) patients with normal and high ratios (those with PIIP2 ratios >0.8). The critical values define the PIIP2 ratio range within a two-tailed normal distribution of fertile donors with 90% confidence limits, as described above. For individual correlation analysis of PI and P2 concentration with the outcome measures, we used a similar approach by evaluating two groups: (i) those with low and normal PI or P2 Table L COllnnalri"ln and IVF outcomes between PIIP2 n COUnI ( X 106 spennlml) Progressive motility (%) Nonnal heads (Ik) Tapered heads (%) Amorphous heads elk) Spenn penetration assay IVF cycles (n) Fertilization rate (%) Fertilization rate by type ('to) ICSI IVF IVFIICSI FertiIizntion type used (%) ICSI IVF IVFIICSI ES/embryo transferred ESlembryo total Pregnancy rate overall (%) Spclnlllnenus abortion rale (%) PIIP2 <0.8 37 64.8 10.6 25.4 3.0 28.2 2.9 41.2 3.2 22.5 ± 2.2 5 ± 0.94 25 75.4 ± 4.0 75.2 ± 4.0 75.8 ± 13.6 NA 73 (18/25) 27 (7125) o 4.8 ± OA 3.5 ± 0.3 36 (9125) II "Significant difference between columns 2 and columns I or 3. "Significant difference between all columns. 'Significant difference between columns I and columns 2 or 3. NS not significant: NA = nol applicable; ES = embryo score. 1300 PIIP2 = 0.8-1.2 127 108.5 9.56 44.7 ± 2.1 52.8 ± 2.1 26.6 ± 2.4 13.0 ± 1.5 18.5 ± 1.1 89 84.4 ± 2.0 80.5 5.1 86.0 2.7 83.7 ± 3.9 17 (15/89) 50 (45/89) 33 (29/89) 5.2 ± 0.2 4.1 0.2 41 (37189) 3 (Ll37) PIIP2> 1.2 108 79.0 S.I 32.4 1.2 23.9 1.9 57.6 ± 2.0 13.4 ± 1.0 13.7 ± 1.2 61 86.5 ± 2.0 83.5 ± \.9 86.1 2.2 75.0 5 65 (40161) 31 (19/61) 4 (2161) 4.8 ± 0.2 3.5 ± 0.3 44 (27/61) 4 ([124) p < 0.005- < O.OOSo < 0.001- < O.OOlb < 0.05" < O.OOlb < 0.05" NS NS NS < 0.001" NS NS NS NS 16 Sperm protamine deregulation in infertile males »~----------------------------------------------~ i »~--------------~I--~I~------------------~ i I .,.~-----------------4·~~I~~r-------------------~~ .1 J • ......, " lit ~ ~ ~ ~ Ie i ! lit ! ~ 1 ~ ~ ! ! ! 0 <:> .. <:> .. .. 0- 1/ II 1/ " II " II 1/ a " ! " 1/ II II 1/ II .II A : .., .. .. ~ ~ : ; ;; ;; ii ::! 5 ! 5 ! 5 5 5 c co <:> <:> 0- PlIP2 Rllio Figure 2. Distribution of PlIP2 ratios in fertile donors and infertility patients. The histogram shows the relative frequency of fertile donors (solid boxes) and infertility patients (meshed boxes) within bins of 0.10 PllP2 ratio units. A much broader PIIP2 ratio distribution is observed in the patients (range: 0.0-2.82) versus the fertile donors (range: 0.75-1.26). concentrations (0.0-594.4 ng Pl/106 sperm, 0.0-556.6 ng P21lif sperm respectively) and (ii) those with normal and high Pl or P2 concentrations (>483.0ng Pl!106 sperm. > 474.2ng P2I106 sperm re.~peclively). The critical values for PI and P2 quantity define the protein concentration range within a two-tailed normal distribution of fertile donors at a confidence of 95%. as described above. Results P 1IP2 ratio quantification Gel electrophoresis revealed that the mean PIIP2 ratio for fertile donors was 1.06:!: om with a range of 0.75-1.26 (Figure 2). We found a similar mean PIIP2 ratio in the patients (1.09:!: 0.10) but the range was much wider (0.0-2.82). In the patient group. we identified 37 individuals with a significantly reduced PIIP2 ratio «0.8) and 108 with a significantly elevated P1IP2 ratio (> 1.2; Table I). In the fertile donor group, there were five individuals with a reduced PlIP2 ratio and seven individuals with an elevated PlIP2 ratio. However, the mean PlIP2 ratios for these individuals were 0.77 :!: 0,01 and 1.23:!: 0.01 respectively compared to 0.50 :!: 0.4 and 1.55 :!: 0.06 in the patients with low and high PIIP2 ratios respectively (P < 0.01). Table IL Protamine Protamine quantification revealed that the mean PI and P2 concentrations in the sperm of fertile donors were 538.7:!: 28.6 and 515.4:!: 21.1 ng/106 sperm respectively (Table n. Figure 3). Kruslcal-Wallis analysis revealed that the mean PI and P2 concentrations in the patient group with a normal P IIP2 ratio were significantly reduced versus the donors (438.8:!: 14.0 and 427.0:!: 20.8 ngl106 sperm. P < 0.05). The mean PI concentrations of the patients with low and high P11P2 ratios (459.3 :!: 32.9. 453.7 :!: 25.6ngl106 sperm) were significantly reduced versus the fertile donors (P < 0.05) but not different from the patients with a normal PIIP2 ratio (P = 0.434). The mean P2 concentration in patients with a low PIIP2 ratio (580.6 :!: 29.5ng/106 sperm) was significantly increased over that of all other groups (P < 0.001). Conversely, the mean P2 concentration in patients with a high PIIP2 ratio (304.2 :!: 19.9ngl106 sperm) was significantly decreased versus each of the other groups (P < 0.001). PI and P2 deregulation Based on protamine quantification. the boundaries defined by the two-tailed normal distribution in the fertile Fenile donors Patient group p Normal PllP2 11 87 57 Mean PlIP2 ratio 1.06 z 0.01 1.01 z O.oI PI concentr'cltion 538.7 :t 28.6" 438.81 :!: 14.0 P2 concentration 515.4 ± 21.1" 427.0 ± 20.S· ·Significantly different from aU other columns. LowPllP2 30 0.50:!: 0.09" 459.3 z 32.9 580.6 ± 29.5" High PIIP2 52 1.55 z OJ}(5/1 453.7 5.6 304.2 z 19.9' < 0.001 < 0.05 < 0.001 1301 V.W.Aoki and L.Liu and D.T.Carrell 1- r. i I. J-PI Figure 3. Mean P I and P2 content in the sperm of fertile donors and infertility patients. The mean concentration of PI (left) and P2 (right) is shown with standard error hars for the fertile donors and three groups of infertility patients. Patients with normal PIIP2 ratios under-expressed both PI and P2. Meanwhile, P2 deregulation accounts for aberrations in the PIIP2 ratio of the other infertility patients with an over-expression leading to a low PIIP2 ratio and severe under-expression leading to a high PtIP2 ratio. +Significant differences (P < O.OS) from each of the other groups within the PI category. *Significant differences (P < 0.001) from each of the other groups within the P2 category. donor group were 483.0-594.4ng/106 sperm for PI and 474.2-556.6ng/J06 sperm for P2. The boundaries defined by the two-tailed normal distribution in the patient group with normal P11P2 were 411.6-466.0nglJ06 sperm for PI and 395.9-458.1 ng/l06 sperm for P2. x2-Analysis revealed that P2 is subject to deregulation much more frequently than PI in patients with abnormal PIIP2 ratios (73/82 versus 56/82 against fertile donors: P < 0.01; 26/82 versus 9/82 against patients with a normal PlIP2 ratio: P < 0.001; Table III; Figure 4). The frequency of PI and P2 deregulation was compared within the low and high PIIP2 ratio groups (Table IV, Figure 5). P2 under-expression accounts for the majority of cases with elevated PIIP2 ratios. However, both PI under­expression and P2 over-expression are involved in cases where the PIIP2 ratio is diminished. When using fertile donors as a standard. 63% (19/30) of the patient group with a low PlIP2 ratio displayed under-expression of PI, while 50% (15/30) showed over-expression of P2 (not significant). There were four patients within this group that showed low PI con­current with high P2, which accounts for the discrepancy in the overall percentage shown above. Table III. LOlltlnli1;em-:y table comparing incidence of PI and P2 PI"(%) P2"(%) in with an abnormal PIIP2 ratio Parienls with abnormal PIIP2 ratiol> Deregulation 68 (56/82) 89 (73182) No deregulation 32 (26182) II (9/82) 'Protamine deregulation assessed using fertile donors as a standard. 1302 p < 0.01 17 In the high PlIP2 ratio group there were 13% (7/52) with over-expression of PI and 90% (47/52) with under­expression of P2, a significant difference (P < 0.001, Table IV). Two patients in this group showed high PI con­current with low P2. When using the patients with a normal PIIP2 ratio as a standard, 10% (3/30) of the low PIIP2 ratio group exhibited PI under-expression, while 30% (9/30) demonstrated P2 over-expression (P O.ll). In the high P llP2 ratio group 8% (4/52) had over-expressed PI and 17% (9/52) exhibited P2 under-expression (P = 0.19). Reilltionship between sperm PIIP2 content and semen qlUllityRVF outcome Sperm concentrations were reduced in patients with low PIIP2 (64.8 ± 10.6 x lOu/mn as well as high PtlP2 (79.0 ± 5.1 x lOu/mt) versus patients with normal PlIP2 (108.5 ± 9.56 x lOu/ml: P < 0.005; Table I). Spearman's correlation analysis indicates that the PIIP2 ratio is signifi­cantly correlated with count (rs 0.22. P < 0.05: rs = - 0.18, P < 0.0 I) in patients with low and normal PIIP2 ratios (0-1.2) as well as normal and high PIIP2 ratios (>0.8), respectively. Progressive motility was significantly different in patients with low, normal and high PIIP2 (25.4 ± 3.0, 44.7 ± 2.1, 32.4 ± 1.2% respectively; P < 0.005). Spearman's corre­lation indicates that the PIIP2 ratio is significantly correlated with progressive motility in patients with low and normal PIIP2 ratios (0-1.2; rll 0.17. P < 0.(5) but only moder­ately correlated in patients with normal and high PIIP2 ratios (>0.8; rs -0.11, P O.lO). Normal head morphology was significantly reduced in the low and high PIIP2 groups (28.2 ± 2.9 and 23.9 ± 1.9% respectively) versus those with normal PIIP2 (44.7 ± 2.1 %: P < 0.(01). A significant increase in tapered heads was observed in patients with low P1IP2 (41.2 ± 3.2%) compared with the normal PIIP2 group (26.6 ± 2.4%; P < 0.001). In patients with high P 1IP2, there was a concomitant increase in tapered beads (57.6 2.0%; P < 0.001). Furthermore. Spearman's correlation analysis indicates that the PIIP2 ratio .r-------------------------------------~ 1Oi------- 10+---""::"'- 10 It +---'-----' Figure 4. PI and P2 deregulation frequency in patients with abnor­mal sperm PIIP2 ratios. P2 is subject to deregulation much more frequently than PI in patients with abnormal PIIP2 ratios when using both the fertile donors (+ P < 0.0 I) and patienl..~ with normal PIIP2 ratios (*P < 0.001) as comparison groups. 18 Sperm protamine dereguladon in lnfertile males Table IV. Contingency table comparing incidence of PI and deregulati4Jn in patients with low and higb Pl!P2 ratios PI deregulation" P2 deregulation" Patients Witll low PIIP2 ratios Deregulation 63 (19/30) 50 (15/30) No deregulation 37 (11130) 50 (15/30) ·Protamine deregulation assessed using fertile donors as a standard. NS not significanL is significantly correlated with nonna) head morphology (rs = 0.25, P < 0.005; rs = - 0.39, P < 0.005) and tapered head morphology (rs = -0.18, P < 0.05; rs 0.32, P < 0.005) in patients with low and normal PIIP2 ratios (0- L2) as well as normal and high PI/P2 ratios (> 0.8), respect­ively. Amorphous heads were increased in patients with low PIIP2 (22.5 ± 2.2%) versus those with nonnal and high PIIP2 (13.0 ± 1.5 and 13.4 ± 1.0% respectively; P < 0.05). A 35 30 2S i 20 1 IS 10 S () 8 45 40 35 r 30 cr 2S d! 20 IS 10 S 0 .H i1 n n... Prolamine o-,llIatioa OMips Figure S. Frequency of protamine deregulation groups using fertile donors (A) and infertility patients with a normal PIIP2 ratio (B) as comparison groups. Study subjects were grouped according to PI and P2 concentration. Equal numbers of patients with a low PIIP2 ratio displayed over-expression of P2 or under-expression of PI. The majority of patients with an elevated PUP2 ratio were found to have under-expressed P2, whereas a small percentage displayed over-expression of PI. p NS Patients with high PIIP2 ratios Deregulation 13 (7152) 90 (47/52) No deregulation 87 (45152) 10 (5152) p < O.OOJ Spearman's correlation analysis indicates the PIIP2 ratio is significantly correlated with amorphous head morphology in patients with low and normal PIIP2 ratios (0-1.2; fs = - 0.16; P < 0.05) but not in patients with normal and high PIIP2 ratios (>0.8; fs = 0.02; P 0.73). The mean SPA scores differed significantly between patients with decreased (n 37), nonnal (n 127) and ele­vated (n = 108) PIIP2 ratios (5.0 ± 0.94, 18.5 ± 1.1 and 13.7 ± 1.2 respectively; P < 0.001; Table J). Spearman's correlation analysis indicates that the PIIP2 ratio is signifi­cantly correlated with SPA (r. = 0.37, P < 0.00 1; r s = - 0.17, P < 0.05) in patients with low and nonnaJ P I/P2 ratios (0-1.2) as well as nonnal and high PlIP2 ratios (> 0.8) respectively. Evaluation of male infertility diagnosis for patients within PIIP2 ratio groups revealed a significantly increased pro­portion of subfertile males within the low PIIP2 rdtio group (95%, 35/37) and high PIIP2 ratio group (90%, 97/108) versus the normal PIIP2 ratio group (36%, 461127) using l-analysis (P < 0.001, Table V). Conversely, the proportion of fertile males was significantly higher in the normal PIIP2 ratio group (64%, 81/127) versus the low PIIP2 ratio group (5%, 2/37) and high PIIP2 ratio group (10%, 111108, P < 0.005; Table V). Twenty-five of the 37 patients with a diminished PIIP2 ratio, 89 of the 127 patients with a normal PlIP2 ratio. and 61 of the 108 patients with an elevated PIIP2 ratio underwent IVF (Table O. Overall fertilization rate (lVF and ICSI combined) was significantly decreased in patients with a low PtlP2 ratio (75.4 ± 4.0%) compared with patients with normal and elevated Pl/P2 (84.4 ± 2.0 and 86.5 ± 2.0% respectively; P < 0.05). Spearman's correlation analysis revealed that IVF fertilization is not significantly correlated to the PlIP2 ratio in patients with normal to high PIIP2 ratios (r. = 0.03, P = 0.71) but is moderately correlated in patients with low to normal PlIP2 ratios (r. 0.16, P = 0.12). How­ever, no significant differences between PIIP2 ratio groups or correlations with the P11P2 ratio were observed in any other IVF outcome measure. The relationship between semen qualitylIVF outcome and Pl. P2, and total protamine quantity was also evaluated. Spearman's correlation revealed a significant relationship between PI concentration and SPA (r, = - 0.26, P < 0.05), sperm concentration (r. -0.31, P < 0.05), progressive motility (rg - 0.30, P < 0.05) and IVF fertilization rate (rs = - 0.35, P < 0.05) in patients with normal and high PI levels. However, no significant correlations with PI concentration were observed in patients with low and normal 1303 19 V.W.Aoki and L.Liu and D.T.Carrell Table V. of P IIP2 ratio Male infertility diagnosis PIIP2 < 0.8 P11P2 = 0.8-1.2 PI1P2 > 1.2 P Fertile 5 (2137) 64 (811127) 10 (11/108) < 0.001 Subfertlle 95 (35137) 36 (461127) 90 (97/108) Clinical diagnosis Normozoospennic 5 (2/37) 64 (811127) 10 (11/1OS) Astbenozoospennk 3 (1/37) 2 (3/127) 2 (2/\OS) Oligozoospermic 5 (2137) 1(11127) 0(01108) Teratozoospennic 0(0137) 5 (6/127) 17 (181108) Nonnozoospennic with reduced penetration ability 14 (5/37) 7 (91127) 7 (81108) Astbenol.oospennic with reduced penetration ability 11(4137) 9 (111127) 6 (7/10S) Oligoz(lospermic witll reduced penetration ability 3 (1137) 0(0/127) 2 (21108) Teratol.Oospennic witb reduced penetration ability 22 (8/37) 9 (111127) 24 (26110S) Asthenoteratoz()()spennic 3 (1/37) 2 (3/127) 5 (51108) Oligoasthenozoospennic 0(0137) 0(01127) I (llIOS) A~then()terdtozoospennic with reduced penetration ability 22 (8137) I (11127) 19 (21/1OS) Oligoasthenozoospennic with reduced penetration ability Oligoteratozoospennic with reduced penetmtion ability Oligoastbenoteratozoospennic Oligoasthenoter'dtozoospennic with reduced penetration ability Values are percentages (numbers in parentheses). PI levels. P2 and total protamine concentrations showed no correlation with any of the outcome measures. Multiple regression analysis failed to resolve any significant complex relationships between the PIIP2 ratio, PI, P2, and total prota­mine quantity and semen qualitylIVF outcome. Discussion In this study we provide the first description of a population of infertile males with markedly diminished PIIP2 ratios. A number of studies have reported a relationship between abnormally high PIIP2 ratios and male infertility (Balhorn et ai .• 1988; de Yebra et ai .. 1993, 1998; Khara et ai., 1997; Carrell and Liu, 2001; Mengual et al., 2003). Based on these studies it has been assumed that a reduction in P2 expression is responsible for aberrant P IIP2 ratios in infertile males. Support for this conclusion was solidified by two studies that identified infertile males with complete selective absence of P2 (de Yebra et ai., ]993; Carrell and Liu, 2(01), Previous studies have failed to quantify the sperm prota· mines in patients with aberrant PIIP2 ratios, and it has been assumed, without direct evidence. that P2 under-expression is responsible. The identification of infertile patients with abnormally low PIIP2 ratios and the possibility that PI over­expression. rather than P2 under-expression, is responsible for the high PIIP2 ratios observed in infertile patients high­light the need for actual protein quantification. as performed in this study. In the sperm of infertile patients, P2 deregulation occurs much more frequently than does PI deregulation. supporting the conclusion that P2 deregulation is responsible for the majority of cases involving an aberrant P IIP2 ratio. Studies of protamine evolution have revealed that the P2 gene is more recently derived than Pl and highly variable within the mammalian genera (Lewis et al.. 2003). Consistent with this data is the notion that the regulatory mechanisms governing P2 ex.pression are more susceptible to variation than those for 1304 3 (1137) 0(0/127) I (1/108) 3 (1137) I (11127) 2 (21108) 3 (1/37) 0(01127) 2 (21IOS) 5 (2/37) o (0/127) 2 (21108) PI expression. Indeed, 90% of patients with abnormally high PI!P2 ratios exhibited P2 under-expression. However. PI deregulation is also implicated in aberrant human sperm PIIP2 ratios. A small proportion (13%) of patients with high PlIP2 exhibited Plover-expression. Mean­while, PI under-expression and P2 over-expression were shown to be equally involved within the low P 1!P2 group. There was only a small percentage of patients exhibiting low PI levels concurrent with high P2 levels (13%) and high PI levels concurrent with low P2 levels (4%). Therefore, both Pl and P2 deregulation are implicated in aberrant PIIP2 ratio cases in infertile human males and there do not appear to be well·defined mechanisms for compensation of under­expression of one of the protamines by over-expression of the other. The mechanisms underlying the uncoupling of PI and P2 expression remain elusive. An interesting aspect of protamine expression is that transcription and translation are temporally uncoupled in the developing spermatid (Steger, 1999). Studies should focus on four targets in the expression path­way to elucidate the underlying etiology of protamine dereg­ulation: the protamine genes themselves, transcription regulation, translation regulation, and downstream protein processing. Attractive hypotheses have emerged in each of these areas to account for the induction of aberrant PI!P2 stoichiometry . Mutations in the genes encoding PI, P2, or any of the accessory proteins including transition nuclear protein I and 2 (TPI and TP2), serine/arginine protein-specific kinase I (SRPKl). and Ca2+/calmodulin-dependent protein kinase IV (Camk4) may play a role in PI and P2 deregulation. For each of these genes, functional studies and animal knockouts have demonstrated the critical involvement of these proteins in faithful protamine expression, processing, and function (Papoutsopou[ou et ai .• 1999; Wu et aI., 2000; Cho et al., 2001, 2003; Zhao eJ ai., 2004). Although genetic screens have not yet been performed on these genes in patients with specifical1y identified deregulations in the PIIP2 ratio, a recent study ha.o;; identified single nucleotide polymorphisms in the P I and P2 genes of a large group of fertile and infertile patients (Tanaka et aI., 2(03). Future investigations should be undertaken to elucidate the involvement of gene mutations in patients with deregulated PIfP2 ratios. In the absence of any gene mutations, an irregular PI/P2 ratio may reflect defects in transcription and translation regu­lation. The haploid expressed PI and P2 genes exist in a single chromatin domain in human sperm and their transcrip­tion is regulated by the same upstream regulatory elements. thus making transcriptional regulation an unlikely but poss­ible cause of aberrant PIIP2 stoichiometry (Johnson el al., 1988; De Jonckheere et ai., 1994; Nelson and Krawetz. 1994). Aberrant translation regulation is more likely to be involved in protamine expression regulation. A number of regulatory proteins have now been identified which are involved in repression or activation of protamine translation (Aoki and Carrell. 2(03). Future investigations should focus on aberrant expression, activation and function of these trans­lational regulators in patients with deregulated PI1P2 ratios. Finally, incomplete post-translational processing of the protamines may result in deregulated P11P2 ratios. Two reports provide strong evidence for this argument, as a group of infertile males with aberrant P1/P2 ratios also displayed an jncrease in P2 precursors (de Yebra el ai., 1998; Carrell and Liu. 2(X))). Considering the importance of TP1, TP2, Camk4, and SRPK 1 in the processing of PI and P2, deregu­lated protamine ratios may also reflect deficiency or abnor­mal function of these accessory proteins. The majority of these effects would be evidenced by disruption of PIIP2 phosphorylation: thus improper signal transduction or mal­function in the appropriate kinases could lead to incomplete protein processing and unsuccessful DNA binding. Numerous reports have now established a relationship between abnonnal protamine expression and male infertility (Balhorn et af.. 1988: de Yebra el al.. ]993. 1998; Khara er 01 .• 1997; Carrell and Liu. 2001; Mengual el aI., 2003; Steger el al .• 2(03). Our data are consistent with these reports and, on three levels, provide convincing evidence for the relationship between abnormal P11P2 ratios and male inferti­lity. First, the sperm of infertile patients with aberrant P11P2 ratios have reduced motility, sperm concentration, morphology. penetration capacity and, in low P1fP2 ratio patients. IVF fertilization rates. Second, we have now shown that the sperm P IIP2 ratio directly correlates with sperm motility. concentration. morphology, and sperm penetration ability. Third. most of the patients with a PIIP2 ratio were clinically diagnosed with some form of male subfertility. Moreover. the newly discovered group of infertile males with a reduced PllP2 ratio has markedly diminished sperm quality and IVF fertilization versus those with increased PIIP2 ratios. This difference may reflect inefficient sperm chroma­tin packaging when P2 is more abundant than Pl. Clearly. there is a ratio-dependent interaction between PI and P2 critical for proper chromatin packaging and the sub­sequent events in spermiogenesis. The precise nature of this interaction, however, has not yet been elucidated. Recent reports of protamine knockouts indicate that haploinsuffi- 20 Sperm protamine deregulation in infertile males ciency of PI or P2 causes infertility in mice and that mouse P2 deficiency leads to sperm DNA damage and embryo death (Cho et aI.. 2001. 2(03). Taken together, these data demon­strate that protamines are important components of spermatid differentiation and that aberrations in protamine stoichi­ometry are related to infertility and may confer defects during spermiogenesis. An alternative hypothesis is that abnormal protamine expression may not be an independent cause of infertility. but rather a result of generalized abnormal spermiogenesis. For example, the protamines may be regulated in conjunction with other key genes during spermiogenesis that affect their expression. Although the animal models argue against this hypothesis by demonstrating that P I and P2 deficiency are direct causes of infertility, no such studies exist in the human (Cho et al.. 2001. 2(03). rCSI appears to overcome defective sperm function associated with an abnormal PlIP2 ratio in light of our data indicating little effect of the PlfP2 ratio on IVF preg­nancy rates. However, evaluation of the P11P2 ratio may still serve as a valuable clinical diagnostic test for three reasons. First, we have shown that the PIIP2 ratio corre­lates highly with sperm penetration ability, count, mor­phology and motility. Second, recent studies have emphasized the importance of sperm DNA damage for proper embryogenesis. Protamine content appears to be critical for proper chromatin integrity evidenced by increased susceptibility of protamine deficient sperm to DNA damage (Manicardi et ai., 1998: Agarwal and Allamaneni, 2004). Third, in light of recent concerns about imprinting disea.<;es associated with ICSI, human sperm pro­tamines may be of utmost clinical significance even though they do not impair IVF/ICSI pregnancy rates. It is now clear that proper chromatin structure is critical for faithful methylation of imprinted genes (Paldi, 2(03). Since the pri­mary role of the sperm protamines is to impart proper sperm chromatin structure, it is possible that protamine and chromatin structural defects may render sperm susceptible to improper imprinting patterns in critical genes. It will be important to evaluate the relationship between abnormal PIIP2 ratios and sperm DNA damage and imprinting pat­terns, the underlying etiology of PIIP2 defects, and why the P11P2 ratio is linked to sperm penetration and capacitation. To summarize, we have now identified a new classification of infertile patients with diminished PlIP2 ratios. The identi­fication of this group raised the question: which of the prota­mines is deregulated in infertile patients with aberrant Pl/P2 stoichiometry? Quantification data indicate that the majority of these cases are due to P2 deregulation. However. PI deregulation also plays a role, especially in infertile patients with a low PllP2 ratio. Finally, we have shown that the PIIP2 ratio is associated with sperm quality. Moreover, patients with a reduced PIIP2 ratio have severely affected sperm quality and reduced IVF fertilization, greater than the previously described class of patients with an elevated PUP2 ratio. These data highlight the clinical importance of sperm protamines in fertility diagnosis and prognosis. Additionally, 1305 V.W.Aoki and I,.Liu and D.T.Carreli identification of the nature of protamine deregulation at the protei.n expres..'lion level serves as a necessary first step to elucidating the underlying causes of abnormal protamine expression in infertile males. References Agarwal A and Allamaneni SS (2004) The effect of sperm DNA damage on aSsIsted reproducl1on outcomes. A review. Minerva Ginecol 56,235-245. Anki VW and Carrell DT (2003) Human prolamines and the developing Spemlatid: their structure, function. expression and relationship with male infertility. Asian 1 AndroI5,315-324. Balhonl R, Reed S and Tanphaichitr N (1988) Aberrant protamine IIprota­mine 2 ratIOS in sperm of infertile human males. Experientia 44,52-55. Belokopytova lA, Kostyleva EI, 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. Carrell DT and Liu L (2001) Altered protamine 2 expression is uncommon in donors of known fertility. but common among men with pour fertilizing ~i~~t~6r~ may reflect other abnormalities of spermiogenesis. J Androl Carrell DT and Urry RL () 996) Sperm penetration assay modifications for improved prediction of sperm feni1ization capacity. Assist Reprod Rev 6,170-174. Carrell DT. Udoff LC. Hata~aka HH. Jones KP and Peterson CM (1998) Pre­dictability/ variability of a modified sperm penetration a&~ay (SPA): review of 1033 patientll undergoing zona-free hamster oocyte sperm penetration analyseslIVF. Adv Reprod 1,45-56. C.arrell OT, Peterson CM, Jones KP. Hatasaka HH. Udoff LC, Cornwell CE. Thorp C, Kuneck P. Erickson L and Campbell B (1999) A simplified coculture system using homologous. attached cumulus tissue results in improved human embryo morphology and pregnancy rates during in vitro tertilization. J As.~ist Reprod Genet 16,344-349. ChevailIier P. Mauro N, Fenellx D. Jouannet P and David G (1987) Anoma­lous protein complement of sperm nuclei in some infertile men. Lancet 2.806-807. Cho C, Willis WD. Goulding EH. Jung-Ha H, Choi YC. Hecht NB and FAldy EM (200 I) Haploinsufficiency of protamine-lor -2 causes infertilitv in mICe. Nat Genet 28.82-86. • Cho C, lung-Ha H. Willis WD. Goulding EH, Stein P. Xu Z, Schultz RM, Hecht NB and Eddy EM (2003) Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice. Bioi Reprod 69,211-217. Cor.lett M, Mazrimas J and Balhom R (2002) Protamine l:protamine 2 stoi­chiometry in !he sperm of elltherian mammals. Mol Reprod Dev 61.519-527. Dadoune JP (1995) The nuclear status of human sperm cells. Micron 26.323-3~5. De 10nckheere J. Nelson JE. Ginsburg KA, Martin Land Krawetz SA (1994) GA repeat polymorphism at !he PRM2 male fertility locus. Hum Mol Genet 3,1915. 1306 21 de Yebr~ L. Ballesca JL, Vanrell J~. Bussa., L and Oliva R (1993) Complete selecllve absence of protamme P2 in humans. J Bioi Chern 268.10553-10557. de Yebra 1., Ballesca n.., Vanrell IA. Corzett M. Balhorn R a.nd Oliva R (1998) Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels. Ferti! SteriI69.755-759. Johnson PA. Peschon 11, Yelick PC. Palmiter RD and Hecht NB (1988) Sequence homologies in the mouse protamine I and 2 genes. Biochim Biophys Acta 950,45-53. Khara KK, Vlad M. Griffiths M and Kennedy CR (1997) Human protamines and male infertility, J Assist Reprod Genet 14.282-290. Lewis 10, Song Y, de Jong ME. Bagha SM and Ausio J (2003) A walk though vertebrate and invertebrate protamines. Chromosoma 111,473-482. Manicardi GC. Tombacco A, Bizzaro D, Bianchi U. Bianchi PG and Sakkas D (1998) D~A. ~nd break~ in ejaculated human ~'»ermatozoa: comparison of SUsceptJbIhty to the nick translation and terminal transferase assays. Histochem J 30,33-39. Mengual L. Ballesca n... Ascaso C and Oliva R (2003) Marked differences in protamine content and PIIP2 ratios in sperm cells from percoll fractions between patients and controls. J Andml24,438-447. Nelson JE and Krawetz SA (1994) Characterization of a human locus in tran~ sition. J BioI Chern 269.31067-31073, Oliva R and Dixon GH (l99\) Vertebrate protamine genes and the histone· to-protamine replacement reaction. Prog Nucleic Acid Res Mol BioI 40,25-94. Paldi A (2003) Genomic imprinting: could the chromatin structure be the driving force? Curr Top Dev BioI 53.115-138. Papout~opoulou S, Nikolakaki E, Chalepakis G, Kruft V. Chevaillier P and Gi~nakouros T (1999) SR prolein-specilic kinase I is highly expressed in te~IIS and phosphorylates protamine L Nucleic Acids Res 27,2972-2980. Steger K (1999) Transcriptional and translational regulation of gene expression in haploid spermatid!!. Anat Embryo! (Berl) 199.471-487. Steger K, Fink L. Failing K, Bohle RM. IGiesch S. Weidner W and Bergmann M (2003) Decreased protamine-l transcript levels in testes from infertile lnen. Mol Hum Reprod 9,331-336. Tanaka H, Miyagawa Y. Tsujimura A, Matsumiya K, Okuyama A and Nishimune Y (2003) Single nucleotide polymorph isms in the protamine·/ and ·2 genes of fertile and infertile human male populations. Mol Hum Reprod 9.69-73. World Health Organisation (2000) WHO manual for the standardized investi· gation, diagnosis and management of the infertile male. Cambridge University Press. Cambridge. UK. Wu JY, Ribar TI, Cummings DE, Burton KA, McKnight GS and Means AR ~2(00) Spermiogenesis and exchange of basic nuclear proteins are ImpaIred In male germ cells lacking Camk4. Nat Genet 25.448-452. Zhao M. Shirley CR, Hayashi S. Marcon L. Mohapatra. B, Suganuma R Behringer RR. Boissonneault O. Yanagbnachi R and Meistrich ML (2004) Transition nuclear proteins are required for normal chromatin condensation and functional sperm development. Genesis 38,200-213. Submilred on September 24, 2004; resulmlitted on December 17. 2004; accepted 011 January 19, 200'; CHAPTER 3 DNA INTEGRITY IS COMPROMISED IN PROTAMINE-DEFICIENT HUMAN SPERM Published in the Journal of Andrology in 2005 Authors Vincent W. Aoki Sergey 1. Moskovtsev Jennifer Willis Lihua Liu Brendan M. Mullen Douglas T. Carrell © American Society of Andrology, Inc. Reproduced by permission of the American Society of Andrology/Joumal of Andrology. Journal of Andrology, VoL 26, No.6. NovcmberJDecC'lllber 2005 Copyrighl g American SodelY of Andnllogy 23 DNA Integrity Is Compromised in Protamine-Deficient Human Sperm VINCENT W. AOKI,*t:J: SERGEY 1. MOSKOVTSEV,§ JENNIFER WILLIS,§ LIHUA LIU,*:j: J. BRENDAN M. MULLEN,§ AND DOUGLAS T. CARRELL*trI From the *Andrology and lVF Laboratories and the Departments of fSurgery and tPhysiology. University of Utah School of Medicine, Salt Lake City, Utah; the §Alldro!ogy Laborat01Y, Department of Pathology and Laboratory Medicine. Mount Sinai Hospital, Toronto, Ontario. Canada; and the fDepartment of Obstetrics and Gynecology, University of Utah Sclwol of Medicine. Salt Lake City, Utah. ABSTRACT: The objective of this study was to examine the relation· ship between DNA integrity and protamines in human sperm. One hundred forty-nine male infertility patients were included in an Insti­tutional Review Board-approved study. Sperm were evaluated for DNA fragmentation using the DNA Integrity Assay, a test equivalent to the sperm chromatin structure assay (SCSA). Additionally, nuclear proteins were extracted and the protamine-1/protamine-2 ratio (P1! P2). protamine-1 (P1). protamine-2 (P2). and total protamineooncen· trations were evaluated. We identified 37 patients with abnormally low P1!P2 ratiOS, 99 patients with normal P1/P2 ratios, and 13 patients with abnormally high P1/P2 ratios. DNA fragmentation was signifi­cantly elevated in patients with low P1/P2 ratios (37.1 6.02) vs those with normal and high P1/P2 ratios (26.7 ::t: 1.9 and 23.8 3.2, re­spectively; P < .05) and was inversely correlated with the P1/P2 ratio During spermiogenesis. sperm chromatin undergoes substantial compaction. Sperm chromatin packaging occurs in a 2-step process (Oliva and Dixon, 1991). In the first step, the transition nuclear proteins (TPI and TP2) replace the somatic cell histones. In the second step, during the elongating spermatid stage, the sperm prot­amine proteins replace the transition proteins. The result is a highly compact sperm chromatin, which fosters DNA stability and transcriptional quiescence. In humans there are 2 forms of sperm protamine: prot­amine- I (PI) and protamine-2 (P2), which occur in a strictly regulated I-to-l ratio (Corzett et a1, 2002). Sperm protamine deficiency has been implicated in male infer­tility (Chevaillier et at, 1987; Balhorn et aI, 1988; Be]o­kopytova et aI. 1993; de Yebra et aI, ]993. 1998; Carrell and Liu, 2001 ~ Aoki and Carrell, 2003; Aoki et ai, 2oo5). In particular. aberrant PI fP2 ratios significantly relate to fertility status. The studies conducted by Yebra et al Correspondence to: Douglas T. Carrell, PhD, University of Utah IVF and AndroJogy Laboratories. 675 Arapecn Drive. Suite 205, Salt Lake City, UT 84108 (e-mail: [e-mail]). Received for publication March 21, 2005: accepted for publication May 31,2005. DOl: J 0.2 J 64/jandroI.05063 741 (R. -0.18, P < .05), P1 concentration (Rs -0.29. P < .OD1). P2 concentration (R. -0.24. P < .OD5). and total protamine concentration (R. -0.28, P < .001). Furthermore, X2 analysis revealed a significant increase in the incidence of marked DNA fragmentation in patients with diminished levels of either P1 or P2. The present study is the first to report that human sperm protamine content is significantly related to DNA fragmentation. In particular, sperm P1 and P2 concentrations inversely correlate with DNA fragmentation, indicating a protective role of the protamines against sperm DNA damage. In light of recent stud­ies highlighting the negative effect of sperm DNA damage on ART outcomes. these findings indicate a possible clinical significance for human sperm protamine levels. Key words: Chromatin. DFI. DNA damage. J Androl 2005;26:741-748 (1998) and Carrell and Liu (2001) describe a population of infertile males with undetectable sperm P2. Recently, Pl deficiency has also been identified in a population of subfertile males (Aoki et aI, 20(5). It has been postulated that protamine defic.iency is re­lated to DNA damage in human sperm. A number of re­cent studies have focused on the relationship between sperm DNA damage and male infertility (Evenson et a1, 2oo2; Tomsu et ai, 2002; Virant-Klun et ai, 2002; Seli et al. 2004). Although the biological significance of sperm DNA damage remains unclear, it appears to be detrimen­tal to fertility in humans and has been linked to lower embryo quality (Tomsu et al, 2oo2; Virant-KJun et ai, 2002), blastulation rates (Seli et aI, 2004), and in vitro fertilization (IVF) pregnancy rates (Evenson et al, 2oo2~ Bungum et ai, 2004; Henke1 et ai, 2004; Virro et aI, 2oo4). Mice that are haplo-insufficient for either PI or P2 are sterile and have increased levels of sperm cell apo­ptosis, DNA damage, and embryonic arrest (Cho et aI. 2001, 2(03). However, relatively little is known about sperm DNA integrity in protamine-deficient human males. The objective of this study was to examine the rela­tionship between DNA integrity and protamines in human 742 sperm. Specifically, the DNA fragmentation index (DFI) was used as a measure of DNA integrity. Protamine levels (Pl/P2 ratio. PI, P2, and total protamine concentrations) are compared to DFI levels in the sperm of male infertility patients. Materials and Methods Unless otherwise noted. all chemicals were obtained from Sigma Chemical Company (St Louis. Mo). Reagents for gel electro­phoresis were purchased from Bio-Rad Laboratories (Hercules. Calif>. Acridine omnge was purchased from Polysciences Inc (Warrington. Pal. Institutional Review Board approval was obtained before ini­tiation of this study. Semen was collected and evaluated from 149 randomly selected male patients presenting for infertility assessment. A single semen sample was used for all assays, in­cluding the DNA Integrity Assay and protamine protein extrac­tion and quantification. Samples with a sperm concentration of Jess than 3 X 106/mL were excluded because they offered in­sufficient materiaL After semen analysis and within I hour of the time of ejaculation. aliquots of raw semen were frozen at -80°C for later analysis. DNA Integrity Assay The DNA Integrity Assay was used to measure the DNA frag­mentation index (DFl) and was performed as previously de­scribed (Evenson et aI, 2002; Fischer et al. 2003). At the time of analysis. semen samples were thawed on ice and diluted with TNE buffer (0.01 M Tris-HCI. 0.15 M NaCI. and 1 mM ethyl­enediaminetetraacetic acid [EDT A], pH 7.4) to 1-2 x 10<' cells! mL Two hundred-microliter aliquots of diluted sample were mixed with 400 ILL of a low-pH (pH 1.2) detergent solution containing 0.1% Triton X-100, 0.15 M NaCI, and 0.08 N HCI for 30 seconds; this was followed by staining with 1.2 mL of 6 ttglmL chromatographically purified acridine orange (AO) in a phosphate citrate buffer (pH 6.0). Three minutes after the staining procedure started. the cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif) equipped with an air-cooled argon laser. Measurements were collected in duplicate on 5000 cells per sample, and 2 aJiquots were analyz.ed for each semen spec­imen. Under these conditions, AO intercalated with double­stranded DNA emits green fluorescence, and AO associated with single-stranded DNA emits red fluorescence. To avoid instru­ment drift, reference samples were used to set the red and green photomultiplier tube voltages. A new reference sample was run every 6 to 10 samples. FCS Express Version 2 (De Novo Soft­ware, Thornhill. Canada) was used for off-line analysis of the flow cytometric data. DNA denaturation was expressed as the DFI, which represents the ratio of red to red plus green fluorescence intensity (Figure 1). This is similar to the SCSA delinition of DFI as red (F > 630 nm)/red + green (F515-530 band pass). Based on a previ­ously published categorization system (Evenson et ai, 2002), 3 levels of DNA fragmentation were reported: low « 15% DFI). moderate (15%-30% DFI). and high (>30% DFI). These levels 24 Journal of Andrology . NovemberlDecember 2005 correspond to excellent, good, and fair-to-poor fertility potential, respectively. Purification of Nuclear Proteins Sperm nuclear proteins were extr'acted from the semen aliquots of all 149 patients. The PIIP2 mtio, PI, P2, and total protamine concentrations were subsequently quantified in aU 149 patients (Aoki et aI, 2005), Prior to extraction, sperm concentrations and white blood cell counts were determined using World Health Organization (WHO) criteria. AU samples were run in duplicate, and the average PIIP2 ratio, PI, and P2 concentrations from the 2 runs were reported. Semen aliquols with a known number of sperm (5.0-20 X I ()6 cells) were centrifuged at 500 x g for 5 minutes at 4°C. The pellet was washed in I mM phenylmethylsulfonylfluoride (PMSF) in distilled water. centrifuged at 500 X g for 5 minutes at 4°C, and the pellet was resuspended in 100 ILL of 100 mM Tris buffer containing 20 mM EDTA and I mM PMSF (pH 8.0). One hundred microliters of 6 M guanidine and 575 mM dithio­threitol were added and mixed, followed by addition of 200 ILL 522 mM sodium iodoacetate. The suspension was maintained at room temperature for 30 minutes while being protected from light. To this suspension, 1.0 mL of 100% ethanol at 4°C was added and maintained for 1 minute before centrifugation at 12000 X g for 10 minutes at 4°C, The ethanol wash was repeated and the pellet was resus­pended in 0.5 M HCI (0.8 mL), incubated for 15 minutes at 37°C. and centrifuged at 10000 X g for lO minutes. The supernatant was retained and the nuclear proteins were precipitated by the addition of lOO% trichloroacetic acid (TCA) to a final concentration of 20% TCA. The solution was incubated at 4°C for 5 minutes and centrifuged at 12000 X g for lO min­utes. The pellet was washed twice in 1% 2-mercaptoethanol in acetone (500 ILL). The final pellet was dried and stored at -20°C until gel electrophoresis analysis. Preparation of the Human Protamine Standard A human protamine standard was prepared as previously de­scribed (Mengual et ai, 2003). Twenty semen samples were pooled to extract and quantitate a highly purified sperm prol­amine sample. Briefly, sperm were washed twice with 0.5 M HCl before protamine extraction to remove other acid-extracted proteins. After acid treatment. the protamines were extracted as described above. The protein extract contained highly purified protamine. as verified by gel electrophoresis and Western blot. The final protamine concentration was determined using the RC DC protein assay kit (Bio-Rad). The protamine extract was run using acid-acrylamide gel electrophoresis to determine the ratio of PI to P2. The final concentrations of PI and P2 were calculated from the percent composition of each of the protamines in the total protamine standard. Subsequently, 1.52,0.76,0.38, and 0.19 ILg of human sperm protamine standard were loaded in each gel and a standard regression curve was made to calculate the amount of protamine in each of the patient samples. The r value of the regression curve was 0.98 or better for each gel run in this study. Aoki et al Sperm Protamines and DNA Damage A J0231r:=:::::;:~============:=; 256 512 768 1023 Red Fluorescence (Fragmented DNA) 90 r----r---------~~~-~-., 80 70 '" 60 § 8 50 40 ";) 20 1 U 30 l 10 I ()L.Lj~~==- n 256 512 768 1023 DNA Fragmentation Index (DF!) B 25 743 1023 rr=~~~===5======--::;) 4 o o. ~-~-.r-2-56: :'--~-5-1-2 -:;;;;;76;8; ;;----10~2 3 70 60 50 40. 30 20~ 10 Red Fluorescence (Fragmented DNA) o~~~~"8!II""IE=l () 256 512 768 1023 DNA Fragmentation Index (OFf) Figure 1. DNA Integrity Assay fluorescence cytograms for samples with (A) low and (8) high DNA fragmentation index (DFI) values. The top traces for both A and B show fragmented DNA staining vs native DNA staining. The numbered regions represent (1) cells with normal DNA. (2) cells with fragmented DNA, (3) seminal debris, (4) "high-green" stained cells that are not full