Article Effect of protamine-2 deficiency on ICSI outcome

Transcription

1 RBMOnline - Vol 9. No Reproductive BioMedicine Online; on web 1 November 2004 Article Effect of protamine-2 deficiency on ICSI outcome Mohammad Hossein Nasr-Esfahani received his PhD from the University of Cambridge, UK, in 1991 and is presently an academic member of the Royan Institute in Tehran, Iran. He has been working as laboratory director of the Isfahan Fertility and Infertility centre since The main research areas of the group with which he is working are stem cells and sperm chromatin. He has in excess of 15 publications in international journals and over 30 publications in the Iranian medical journal. Dr MH Nasr-Esfahani MH Nasr-Esfahani 1,2,4, M Salehi 1, S Razavi 3, M Mardani 3, H Bahramian 3, K Steger 4, F Oreizi 3 1 Royan Institute, Tehran, Iran; 2 Isfahan Fertility and Infertility Centre, Isfahan, Iran; 3 Isfahan Medical University, Isfahan, Iran; 4 Department of Urology and Pediatric Urology, University of Giessen, Germany 4 Correspondence: Department of Clinical and Experimental Embryology and Andrology, Royan Institute, Tehran, Iran. Tel: ; Fax: ; info@royaninstitute.org Abstract During spermiogenesis, histones are replaced by protamines (P1 and P2), resulting in sperm chromatin condensation followed by a halt to gene expression in haploid spermatids and spermatozoa. As a consequence, protamine deficiency and aberrant P1/P2 ratio have a profound effect on both fertilization and embryo development. However, reports on the effect of the P1/P2 ratio on fertilization and embryo development after intracytoplasmic sperm injection (ICSI) are contradictory between human and animal studies. The question that still remains to be elucidated is which type of protamine deficiency is most common among protamine deficient samples. The present study has a direct bearing on this issue investigating the correlation of the P1/P2 ratio with protamine deficiency, fertilization, embryo quality and embryo development in ICSI patients. This study was carried out on 71 patients. Chromomycin A3 (CMA3) staining was used to determine protamine deficiency. Since this procedure does not indicate the type of protamine deficiency, the P1/P2 ratio was evaluated by nuclear protein extraction, acetic acid urea polyacrylamide gel electrophoresis and analysis of protein bands with software. Polyclonal anti-p1 and anti-p2 antibodies were used to confirm P1 and P2 presence. Results show a negative significant correlation of fertilization rate with protamine deficiency and P1/P2 ratio. No significant correlation was observed between protamine deficiency and P1/P2 ratio. Therefore, it can be concluded that altered P1/P2 ratio effects fertilization rate and embryo quality which subsequently may affect implantation and pregnancy outcome. Keywords: CMA3 (chromomycin A3), embryo quality, fertilization, protamine 652 Introduction During human spermiogenesis, 85% of histones are replaced by protamines which results in sperm DNA packaging and formation of highly stable structure due to formation of covalent (-S-S-) and non-covalent (-SH...Zn SH-) bonds (Barone et al., 1994; Carrell and Liu, 2001). 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 six-fold increase in sperm chromatin compaction (Ward and Coffey, 1991). Sperm DNA packaging results in: (i) facilitation of sperm transport, (ii) protection of DNA from chemical and physical damage, (iii) proper gene reprogramming postfertilization, and (iv) synchronization of cell cycle between the oocyte in MII phase and sperm in G1 (Braun, 2001). In humans, unlike salmon and bulls, there exist two families of protamines, called P1 (protamine 1) and P2 (protamine 2). P1, which is present in all mammalian spermatozoa, contains 50 amino acids and is rich in arginine and cysteine. P2 belongs to a family of three proteins (P2, P3 and P4) that all derive from a single gene but vary in length due to a variable amino terminal cleavage (De Yebra et al., 1993; Carrell and Liu, 2001). P2 protamines are zinc finger proteins with one CYS2/HIS2 motif that are rich in histidine and cysteine and believed to inhibit gene expression in late spermatids (Bianchi et al., 1992). The mechanism by which protamines interact with DNA is still under debate. P1 and P2 may bind to the major groove of the DNA (Pogany et al., 1981), attach to both minor and major grooves (D Auria et al., 1993), or electrostatically bind to the surface of the DNA by interacting with

2 phosphate residues (Bianchi et al., 1994). Due to the critical role of correct histone-to-protamine exchange for spermatid differentiation, one might expect aberrations in protamine expression or structure leading to male infertility. Indeed, abnormal P1/P2 ratios have been reported in spermatozoa of infertile human males (Silvestroni et al., 1976; Balhorn et al., 1988; De Yebra et al., 1993, 1998; Mengual et al., 2003). In addition, Steger et al. (2003) reported an aberrant P1/P2 mrna ratio in round spermatids of testicular biopsies. In-vitro studies by Carrell and Liu (2001) suggest that an increased P1/P2 ratio is associated with decreased sperm penetration and fertilization rates. Complementary studies in mice suggest that haploinsufficiency of P2 leads to sperm DNA damage and early embryo death (Cho et al., 2003), while premature translation of P1 causes precocious nuclear condensation, which resulted in an arrest of spermatid differentiation (Lee et al., 1995). During in-vivo and in-vitro fertilization, the zona pellucida acts as a selective barrier for penetration of abnormal sperm. Indeed, previous studies have shown that abnormal sperm morphology, protamine deficiency and aberrant P1/P2 ratio are negatively related to in-vitro fertilization rates (Carrell and Liu, 2001; Nasr-Esfahani et al., 2001). However, unlike IVF, applying intracytoplasmic sperm injection (ICSI) spermatozoa with abnormal morphology, protamine deficiency and aberrant P1/P2 ratio find the chance to initiate fertilization and subsequent development. Therefore, the present study aimed at evaluating the effect of the aforementioned parameters on fertilization and subsequent embryo development post-icsi and, in addition, the P1/P2 ratio in protamine deficient samples. Materials and methods Samples Semen samples were obtained from 71 couples referred to the Isfahan Fertility and Infertility Centre. Forty-five of these samples belonged to patients who were ICSI candidates. Mean age of males and females was 36 ± 5 and 31 ± 5 years respectively. Semen samples were collected by masturbation after 3 4 days of abstinence on the day of oocyte recovery. A major portion of the semen samples were prepared for routine ICSI using discontinuous pure sperm gradients (80:40) (Nidacon, Gothenburg, Sweden), while the remaining part was used for semen analysis, assessment of sperm chromatin status using CMA3 staining and cryopreservation for nuclear protein extraction. Assessment of protamine deficiency (chromomycin A3 staining) Semen samples were washed in Ca 2+ Mg 2+ -free PBS and were fixed in Carnoy s solution [methanol:glacial acetic acid 3:1 (Merck, Darmstadt, Germany)] at 4 C for 5 min. Smears were prepared and each slide was treated for 20 min with 100 μl of CMA3 (Sigma, St Louis, MO, USA) solution [0.25 mg/ml in McIlvaine buffer (7 ml citric acid 0.1 M ml Na 2 HPO 4.7H 2 O, 0.2 mol/l, ph 7.0, containing 10 mmol/1 MgCl 2 )]. The slides were then rinsed in buffer and mounted with buffered glycerol (1:1). Microscopic analysis of the slides was performed on a Nikon Eclipse 600 fluorescence microscope (Tokyo, Japan) with the appropriate filters ( nm). On each slide, 200 sperm cells were evaluated. Evaluation of CMA3 staining was carried out by distinguishing between spermatozoa with bright yellow staining (CMA3 positive) and spermatozoa with dull yellow staining (CMA3 negative). Patients were grouped as normal or protamine-deficient, according to CMA3 cut-off value of 30% (Sakkas et al., 1996; Razavi et al., 2003). ICSI After oocyte collection, the oocytes were treated in hyaloronidase (Hyase; Vitrolife, Gothenburg, Sweden) in G-MOPS medium (Vitrolife Fertility System, GIII-Series; Vitrolife). Oocytes were then washed in fresh G-MOPS and transferred to G-MOPS under oil in a Falcon 1006 dish for microinjection. The pure sperm-processed semen sample was also introduced into ICSI100 (a viscous sperm handling solution containing sterile filtered PVP and human serum albumin from Vitrolife) in the same dish. An Eppendorf micromanipulator mounted on a Nikon inverted microscope was used to perform ICSI. Motile spermatozoa with the best morphology were selected for each oocyte insemination. The injected oocytes were then washed and incubated in G1.III medium. All media used for ICSI procedures were obtained from Vitrolife. Fertilization was assessed for presence of pronuclei h after injection. Percentage of fertilization was calculated by multiplying the ratio of fertilized oocytes to total number of injected MII oocytes by 100. In order to reduce female factors, any patients with fewer than four matured MII oocytes that had survived the ICSI procedure were excluded from this study. Furthermore, immature, deformed and post-mature oocytes, or any oocyte with certain types of abnormality, were also excluded from this study. Therefore, 26 patients had the inclusion criteria. Evaluation of embryo quality score and cleavage score Twenty-four to 48 h after sperm injection, embryos were scored for their cleavage and quality. The cell cleavage stages of embryos were recorded and the embryos were graded as A, B and C. Grade A embryos with even-sized blastomeres and less than 10% fragmentation were given score 3. Grade B embryos with even-sized blastomeres and between 10 50% fragmentation were given score 2. Grade C embryos with uneven-sized blastomeres and with >50% fragmentation were given score 1. The cumulative quality scores of the embryos in each group for each patient were calculated as follows: sum of scores of embryos/total number of embryos. Each embryo was also given a cleavage score, which was calculated as follow: 2 3, 4 5, 6 8 and 9 16 cell embryos were given score 1, 2, 3, and 4 respectively. The cleavage scores of each group for each patient were calculated as follows: sum of cleavage scores of embryos in that group/total number of embryos. 653

3 654 Semen cryopreservation Each semen sample was diluted with sperm freeze cryoprotectant medium (made up according to Elder et al., 1990) according to sperm concentration and allowed to stay at room temperature for 10 min. The diluted sample was then drawn into 0.25 ml straw and suspended in liquid nitrogen vapour (1 cm above the level of liquid nitrogen) for 30 min. The straws were then plunged into liquid nitrogen and stored until required. All the material used for cryopreservation, nuclear protein extraction and gel electrophoresis were obtained from Merck, Germany. Nuclear protein extraction Sperm nuclear proteins were extracted using slight modifications of the previously described procedure (De Yebra and Oliva, 1993). Approximately 10 7 sperm cells from each semen sample were thawed and washed three times (5 min at 800 g) with Ham F-10 (Sigma). The pellet was washed in 1 mmol/l phenylmethylsulphonyl fluoride (PMSF) in distilled water to lyse the sperm membranes by osmotic shock, then resuspend in 100 μl of 100 mmol/l Tris buffer containing 20 mmol/l ethylenediamine tetraacetic acid (EDTA) and 1 mmol/l PMSF (ph 8). One hundred microlitres of 6 mol/l guanidine and 575 mmol/l dithiothreitol was added to break hydrogen and disulphide bonds. To allow chromatin expansion, the suspension was protected from light and kept at 4 C for 30 min. The suspension was then mixed with 1 ml of cold ethanol ( 20 C) for 1 min, then centrifuged for 14 min at 10,800 g at 4 C. The supernatant was decanted and the ethanol wash was repeated. Following the second wash, the pellet was resuspended in 0.8 ml of 0.5 mol/l HCl and incubated for 10 min at 37 C, then centrifuged for 5 min at 2240 g. Trichloroacetic acid (TCA) 100% was added for 5 min at 4 C to the supernatant (to a final concentration of 20% TCA) in order to induce protein precipitation. This solution was centrifuged for 10 min at 10,800 g and the pellet was washed twice in 500 μl of 1% 2-mercaptoethanol acetone for 5 min at 10,800 g each time. The final pellet was dried and stored at 20 C. Separation and analysis of proteins Acetic acid urea polyacrylamide gel electrophoresis was performed on 20 cm long gel composed of stacking and running gels. The running gel was made of 20% acrylamide, 0.29% bisacrylamide, 2.5 mol/l urea, 0.9 mol/l acetic acid, 0.33% TEMED and 0.18% ammonium persulphate. The stacking gel was made of 7.5% acrylamide, 0.11% bisacrylamide, 2.5 mol/l urea, 0.27 mol/l acetic acid, 0.5% TEMED and 0.48% ammonium persulphate. The gel was pre-run at 180 V at room temperature until the current falls to a steady level. The extracted protein was dissolved in 0.9 mol/l glacial acetic acid, 30% sucrose and 0.4% methyl green and then electrophoresed (0.9 mol/l acetic acid, 180 V, 270 min). After electrophoresis, gels were fixed with fixing solution (methanol 50% and acetic acid 10%) and then stained with 1.1 g of Coomassie brilliant blue G-250 dissolved in 250 ml methanol, 250 ml H 2 O and 50 ml acetic acid overnight. Destaining was carried out for 5 min in fixing solution, and then overnight in 10% methanol and 10% acetic acid. Acidic acid urea gels were scanned using Adobe Photoshop 7 software and resulting images were analysed with UVP-Lab works software. Unstained gels were analysed by western blot using polyclonal antibody (rabbit-anti-human) protamine 1 and protamine 2, secondary antibody goat-anti-rabbit, streptavidin-hrp and diaminobenzidine (DAB) according to a previously described procedure with modification (De Yebra et al., 1998) in order to confirm that the proteins being analysed were P1 and P2. Standardization of results In order to compare results within and between experiments, inter- and intra-assay coefficient of variation were obtained as follow: eight semen samples were mixed and diluted to get 40 straws for freezing. Initially, 10 straws were thawed, protein extraction and gel electrophoresis were carried out and the ratios of P1/P2 were evaluated. Coefficient of variation for intra assay was calculated by multiplying the ratio of standard deviation over the mean value times 100. For inter-assay variation, during each experiment two of the aforementioned straws were thawed and protein extraction and gel electrophoresis were carried out simultaneously with the patient s samples and then the ratios of P1/P2 were evaluated. Inter-assay variation was calculated by multiplying the ratio of standard deviation over the mean value times 100. Statistical evaluation Comparison between parameters was performed using correlation coefficient, χ 2 and Student s t-test analysis. All statistical analysis was performed using SPSS software. Results Extractions of sperm nuclear proteins from 71 patients were carried out. Sperm nuclear proteins were analysed by acetic acid urea polyacrylamide gel electrophoresis and visualized by Coomassie blue staining. Subsequent western blot analysis confirmed the presence of P1 and P2 bands. Both protamine family and low mobility bands possibly representing protamine precursor molecules were observed in all samples (Figure 1). The mean P1/P2 ratio in CMA3 positive (>30%) and negative (<30%) samples was 0.77 ± 0.3 and 0.79 ± 0.4 respectively, revealing no significant difference (P > 0.05). Since the normal P1/P2 ratio has been reported to be 0.98 ± 0.12 (Balhorn et al., 1988), samples were divided into three groups: (i) patients with P1/P2 ratio of lower than 0.86, (ii) patients with normal P1/P2 ratio between 0.87 and 1.1, and (iii) patients with P1/P2 ratio of higher than However, no difference in frequency of patients with P1/P2 ratio in samples with less or more than 30% CMA 3 positivity was observed (Table 1). Both the P1/P2 ratio and the protamine deficiency show a significant negative correlation with the fertilization rate of ICSI patients (Figure 2). Only on day 3, could a significant negative correlation be demonstrated between P1/P2 ratios and embryo quality score. However, no significant relation was observed between P1/P2 ratio and embryo cleavage score (Table 2).

4 Figure 1. Sperm nuclear proteins analysed by acetic acid urea polyacrylamide gel electrophoresis and visualized by Coomassie blue staining and western blot. Lane 1: sperm proteins from pooled infertile men containing histones, transitional proteins (possibly HPS1 and HPS2 which are believed to be precursor of protamine 2 family) and protamines. Lane 2: western blot analysis of sperm proteins separated by PAGE and detected using the anti-protamine 1 and 2 antibodies. Table 1. Frequency of P1/P2 ratio in cases with low and high protamine deficiency. Chi-squared analysis between frequency of patients in the low and high CMA3 groups showed no significant difference in the frequency of patients in the normal, low and high P1/ P2 ratio. Figures in parentheses are percentages. P1/P2 ratio < >1.11 CMA3 >30% 10 (58.8) 5 (29.4) 2 (11.8) CMA3 <30% 26 (53.1) 13 (26.5) 10 (20.4) Total 36 (54.5) 18 (27.3) 12 (18.2) a b Figure 2. Correlation between fertilization rate with (a) P1/P2 ratio (r = 0.464, P = 0.017) and (b) protamine deficiency (r = 0.412, P = 0.002). Table 2. Correlation between P1/P2 ratio with embryo cleavage and quality scores. r P-value Embryo cleavage score day Embryo quality score day Embryo cleavage score day Embryo quality score day a

5 656 Discussion Testis-specific nuclear proteins, namely transition proteins and protamines, are responsible for substantial chromatin condensation in haploid spermatid. The first step in this process occurs in round spermatids and involves replacement of somatic histones by transitional proteins (TP1, TP2, HPS1 and HPS2). Subsequently, in elongating spermatid, transitional proteins should be replaced by protamines (P1 and P2). The resulting chromatin is highly condensed and transcriptionally silent (Ward and Coffey, 1991; Barone et al., 1994; Steger, 1999). Numerous studies have used chromomycin A3, in order to evaluate protamine deficiency on male reproductive outcome (Bianchi et al., 1993; Lolis et al., 1996; Iranpour et al., 2000). These studies suggest that there is correlation between protamine deficiency and fertilization rate following IVF and ICSI (Esterhuizen et al., 2000; Nasr-Esfahani et al., 2001; Razavi et al., 2003). Furthermore, protamine deficiency affects embryo development and implantation rate (Sakkas et al., 1998). However, what remains to be elucidated is which type of protamine deficiency was most common among the protamine deficient samples. The results of this study showed no correlation between protamine deficiency and P1/P2 ratio suggesting that chromomycin A3 does not distinguish between P1 and P2 deficiency. Furthermore, the frequencies of patients with normal, low and high P1/P2 ratio were similar in patients with low and high CMA3 positivity (Table 1). The fact that no correlation was observed between these two parameters is probably due to the fact that both P1 and P2 bind electrostatically to the surface of the DNA by interacting with phosphate residues (Bianchi et al., 1994) or bind to the major groove, while CMA3 binds to the minor groove of the DNA (Gao and Patel, 1990) when either P1 or P2 is absent from the major groove or from the surface of the DNA, suggesting that there is possibly no difference in the site of attachment between P1 and P2. The results of Table 1 also suggest that the majority of patients have a low P1/P2 ratio, suggesting that they either have low P1 or high P2. These results are in concordance with pervious reports (Steger et al., 2003) suggesting that P1 deficiency has important implication in azoospermic male infertility and this is possibly true for other ICSI patients. Majority of the patients with normal amount of CMA3 have an altered P1/P2 ratio, suggesting that spermatozoa in these patients may have adequate amount of protamine; however, they may have an altered expression of P1 or P2. Looking at the raw data, there were two patients with normal P1/P2 ratio which had protamine deficiency, one with CMA3 of 32% and the other with CMA3 of 62%, with fertilization rates of 75 and 50% respectively. These data suggest that patients with high CMA3 and normal P1/P2 ratio have fairly good fertilization rates; however, to further verify this statement, further study with a high number of patients is required. The results of this study, in addition, showed a negative significant correlation between the P1/P2 ratio and protamine deficiency with fertilization rate post-icsi indicating that not only protamine deficiency, but also the type of protamine deficiency has a profound impact on fertilization outcome post-icsi. Previous studies using CMA 3 also suggest that the overall protamine deficiency decreases fertilization rate and implantation rate in ICSI patients (Esterhuizen et al., 2000; Razavi et al., 2003) The negative significant correlation between P1/P2 ratio and fertilization post-icsi suggests that decrease in P2 or possibly overexpression of P1 is associated with a lower fertilization rate. This finding is contradictory to the results reported by Carrell and Liu (2001), which showed patients without P2 have normal fertilization, development and pregnancy rate. One of the differences between this study and of Carrell and Liu (2001) is that in the present study, all of the patients had at least some level of P2; however, in the report by Carrell and Liu (2001), 12 out of 21 patients had no measurable P2. The results of present study are complementary to the study by Cho et al. (2003) showing that P2 deficiency affects the reproductive outcome of the embryos derived from P2-deficient spermatozoa (produced from knock-out protamine-deficient embryonic stem cells). These authors concluded that only 11% of P2 deficient embryos developed to the blastocyst stage and 86% were arrested in the 2 6 cell stages. These results are in agreement with the negative correlation observed between embryo quality score and P1/P2 ratio on day 3.The results of this study show patients with good quality embryos have low, normal or high P1/P2 ratio; however, the majority of patients with poor embryo quality had a P1/P2 ratio >1. This suggests that over-expression P1 or P2 deficiency can lead to poor embryo quality. However, embryo quality showed no correlation with protamine deficiency, thereby suggesting that the type of protamine deficiency has a more profound effect on embryo quality. Lower fertilization observed in cases with P2 deficiency could be due to other anomalies. Carrell and Liu (2001) stated that it is unlikely that the altered protamine expression is directly related to, or causative of, diminished fertilization ability. Rather, it is probably indicative of abnormal, late stage spermiogenesis in general. Histone-to-protamine exchange is a late spermiogenesis event, along with acrosome formation, membrane remoulding and other significant morphological and biochemical events that are necessary for normal sperm function (Sakkas et al., 2003; Kimmins et al., 2004). Furthermore, such anomalies might result in failure of oocyte activation that, in turn, may prone protamine-deficient spermatozoa to premature chromatin condensation (Nasr- Esfahani et al., 2004). Another possible explanation for lower fertilization of P2-deficient spermatozoa could be greater DNA damage in P2-deficient spermatozoa (Cho et al., 2003). This conclusion is contradictory to results obtained by Twigg et al. (1998), who showed that exposure of spermatozoa to high concentrations of H 2 O 2 does not prevent human sperm pronucleus formation in zona-free hamster oocytes. However, higher levels of DNA damage in P2-deficient sperm could provide an explanation for the lower embryo quality observed in this study around day 3 (Sakkas et al., 2003), which is concomitant with genomic activation and in agreement with the report of Cho et al. (2003) who used P2-deficient spermatozoa to perform ICSI in mouse. Another point that needs to be stated with P2-deficient spermatozoa is that P2 is considered to be a zinc finger protein that may inhibit transcription of genes during late spermiogenesis and influence the developmental outcome of the embryos injected with P2-deficient spermatozoa.

6 Imprinting anomalies has been associated with male infertility, and imprinting disorders have been suggested as the underlying cause of oligozoospermia (Marques et al., 2004). Proper histone-to-protamine exchange is associated with maintenance of imprinted genes. Imprinted genes are generally believed to be packed in the form of nucleohistone, which protects the imprinted genes from wide demethylation postfertilization, while non-imprinted genes are packed in the form of nucleoprotamine, which undergoes wide demethylation post-fertilization (Reik and Walter, 2001). Therefore, genes that are required for normal early embryonic development might remain methylated in protamine-deficient or P2- deficient spermatozoa, and therefore may affect fertilization and especially the developmental capacity of the embryo. In conclusion, the results of this study suggest that protamine deficiency and especially P2 deficiency affects ICSI fertilization outcome. Furthermore, P2 deficiency appears to effect embryo quality, while P1 deficiency may affect spermatogenesis. Therefore, procedures that may reduce or elevate these conditions, and elimination of spermatozoa lacking in protamine from insemination samples, may influence ICSI outcome. Acknowledgements The authors wish to express their gratitude to the Royan Institute for their financial support (grant no ), and the members and staff of Isfahan Fertility and Infertility Centre and Isfahan Medical School of Sciences for their kind collaboration. References Balhorn R, Reed S, Tanphaichitr N 1988 Aberrant protamine 1/protamine 2 ratios in sperm of infertile human males. Experientia 44, Barone J, De Lara J, Cummings K, Ward S 1994 DNA organization in human spermatozoa. Journal of Andrology 15, Bianchi F, Rousseaux-Prevost R, Sautiere P, Rousseaux J 1992 P2 Protamines from human sperm are zinc-finger proteins with one CYS2/HIS2 motif. Biochemical and Biophysical Research Communications 182, Bianchi PG, Manicardi GC, Bizzaro D et al Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human mature spermatozoa. Biology of Reproduction 49, Bianchi F, Rousseaux-Prevost R, Bailly C, Rousseaux J 1994 Interaction of human P1 and P2 protamines with DNA. Biochemical and Biophysical Research Communications 201, Braun RE 2001 Packaging paternal chromosomes with protamine. Nature Genetics 28, Carrell DT, Liu L 2001 Altered protamine 2 expression is uncommon in donors of known fertility, but common among men with poor fertilizing capacity, and may reflect other abnormalities of spermiogenesis. Journal of Andrology 22, Cho C, Jung-Ha H, Willis WD et al Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice. Biology of Reproduction 69, D Auria G, Paolillo L, Sartorio R, Wurzburger S 1993 Structure and function of protamines: a 1H nuclear magnetic resonance investigation of the interaction of clupeines with mononucleotides. Biochimica et Biophysica Acta 1162, De Yebra L, Oliva R 1993 Rapid analysis of mammalian sperm nuclear proteins. Analytical Biochemistry 209, De Yebra L, Ballesca JL, Vanrell JA et al Complete selective absence of protamine P2 in humans. Journal of Biological Chemistry 268, De Yebra L, Ballesca JL, Vanrell JA et al Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels. Fertility and Sterility 69, Elder KT, Avery S, Mills C 1990 IVF Laboratory Procedures. Broadwater Press, Welwyn Garden City, Hertfordshire, UK, pp Esterhuizen AD, Franken DR, Lourens JG et al Chromatin packaging as an indicator of human sperm dysfunction. Journal of Assisted Reproduction and Genetics 17, Gao XL, Patel DJ 1990 Chromomycin dimer-dna oligomer complexes. Sequence selectivity and divalent cation specificity. Biochemistry 29, Iranpour FG, Nasr-Esfahani MH, Valojerdi MR, al-taraihi TM 2000 Chromomycin A3 staining as a useful tool for evaluation of male fertility. Journal of Assisted Reproduction and Genetics 17, Kimmins S, Kotaja N, Fienga G et al A specific programme of gene transcription in male germ cells. Reproductive BioMedicine Online 8, Lee K, Haugen HS, Clegg CH, Braun RE 1995 Premature translation of protamine 1 mrna causes precocious nuclear condensation and arrests spermatid differentiation in mice. Proceedings of National Academy of Sciences of the USA 92, Lolis D, Georgiou I, Syrrou M et al Chromomycin A3-staining as an indicator of protamine deficiency and fertilization. International Journal of Andrology 19, Marques CJ, Carvalho F, Sousa M, Barros A 2004 Genomic imprinting in disruptive spermatogenesis. Lancet 22, Mengual L, Ballesca JL, Ascaso C, Oliva R 2003 Marked differences in protamine content and P1/P2 ratios in sperm cells from Percoll fractions between patients and controls. Journal of Andrology 24, Nasr-Esfahani MH, Razavi S, Mardani M 2001 Relation between different human sperm nuclear maturity tests and in vitro fertilization. Journal of Assisted Reproduction and Genetics 18, Nasr-Esfahani MH, Razavi S, Mozdarani H et al Relationship between protamine deficiency with fertilization rate and incidence of sperm premature chromosomal condensation post- ICSI. Andrologia 36, Pogany GC, Corzett M, Weston S, Balhorn R 1981 DNA and protein content of mouse sperm. Implications regarding sperm chromatin structure. Experimental Cell Research 136, Razavi S, Nasr-Esfahani MH, Mardani M et al Effect of human sperm chromatin anomalies on fertilization outcome post- ICSI. Andrologia 35, Reik W, Walter J 2001 Genomic imprinting: paternal influence on the genome. Nature Reviews Genetics 2, Sakkas D, Urner F, Bianchi PG 1996 Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Human Reproduction 11, Sakkas D, Urner F, Bizzaro D et al Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development. Human Reproduction (suppl.) 13, Sakkas D, Seli E, Bizzaro D, Tarozzi N et al Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reproductive BioMedicine Online 7, Silvestroni L, Frajese G, Fabrizio M 1976 Histones instead of protamines in terminal germ cells of infertile, oligospermic men. Fertility and Sterility 27, Steger K 1999 Transcriptional and translational regulation of gene expression in haploid spermatids. Anatomy and Embryology (Berlin) 199, Steger K, Fink L, Failing K et al Decreased protamine-1 transcript levels in testes from infertile men. Molecular Human Reproduction 9,

7 Twigg JP, Irvine DS, Aitken RJ 1998 Oxidative damage to DNA in human sperm does not preclude pronucleus formation at intracytoplasmic sperm injection. Human Reproduction 13, Ward WS, Coffey DS 1991 DNA packaging and organization in mammalian spermatozoa: comparison with somatic cell. Biology of Reproduction 44, Received 17 August 2004; refereed 20 September 2004; accepted 22 October