Fertility and Sterility
Volume 95, Issue 1 , Pages 105-109, January 2011

Protamine 1 to protamine 2 ratio correlates with dynamic aspects of DNA fragmentation in human sperm

  • Agustín García-Peiró, B.S.

      Affiliations

    • Departament de Biologia Cel·lular, Fisiologia i Immunologia, Bellaterra, Spain
    • Càtedra de Recerca Eugin-UAB, Universitat Autònoma de Barcelona, Bellaterra, Spain
    • Corresponding Author InformationReprint requests: Jordi Benet, Ph.D., and Agustín García-Peiró, B.S., Departament de Biologia Cel·lular, Fisiologia i Immunologia, Unitat de Biologia Cel·lular i Genètica Mèdica, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain (TEL: 34-93-581-1773; FAX: 34-93-581-1025).
    • ,
  • Juan Martínez-Heredia, Ph.D.

      Affiliations

    • Departament de Biologia Cel·lular, Fisiologia i Immunologia, Bellaterra, Spain
    • ,
  • María Oliver-Bonet, Ph.D.

      Affiliations

    • Departament de Biologia Cel·lular, Fisiologia i Immunologia, Bellaterra, Spain
    • ,
  • Carlos Abad, M.D.

      Affiliations

    • Servei d'Urologia, Sabadell, Spain
    • ,
  • María José Amengual, Ph.D.

      Affiliations

    • UDIAT, Consorci Hospitalari Parc Taulí, Sabadell, Spain
    • ,
  • Joaquima Navarro, Ph.D.

      Affiliations

    • Departament de Biologia Cel·lular, Fisiologia i Immunologia, Bellaterra, Spain
    • ,
  • Celine Jones, B.S.

      Affiliations

    • Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
    • ,
  • Kevin Coward, Ph.D.

      Affiliations

    • Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
    • ,
  • Jaime Gosálvez, Ph.D.

      Affiliations

    • Departamento de Biología, Unidad de Genética, Universidad Autónoma de Madrid, Madrid, Spain
    • ,
  • Jordi Benet, Ph.D.

      Affiliations

    • Departament de Biologia Cel·lular, Fisiologia i Immunologia, Bellaterra, Spain
    • Corresponding Author InformationReprint requests: Jordi Benet, Ph.D., and Agustín García-Peiró, B.S., Departament de Biologia Cel·lular, Fisiologia i Immunologia, Unitat de Biologia Cel·lular i Genètica Mèdica, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain (TEL: 34-93-581-1773; FAX: 34-93-581-1025).

Received 22 April 2010; received in revised form 8 June 2010; accepted 16 June 2010. published online 29 July 2010.

Article Outline

Objective

To investigate the relationship between the protamine 1 to protamine 2 (P1/P2) ratio and the rate of sperm DNA fragmentation in sperm samples from human males with proven fertility and three different cohorts of male patients.

Design

P1/P2 ratio was analyzed using acid-urea polyacrylamide acid-urea gels electrophoresis (PAGE). Sperm DNA fragmentation using sperm chromatin dispersion methodology was analyzed after 0, 4, 8, and 24 hours of incubation at 37°C.

Setting

University medical school and hospital.

Patient(s)

A total of 32 human males: six with proven fertility, seven carriers of chromosome reorganizations, nine clinical varicocele patients, and ten subclinical varicocele patients.

Intervention(s)

None.

Main Outcome Measure(s)

P1/P2 ratio, sperm DNA fragmentation (SDF) and the rate of sperm DNA fragmentation (rSDF).

Result(s)

P1/P2 ratio correlated with SDF and rSDF. Statistical differences were detected between fertile controls and patients for the three pathologies studied. rSDF yielded information that differed from baseline SDF. No differences were detected for P1/P2 ratio among patient groups, in reference to the three pathologies studied.

Conclusion(s)

SDF and rSDF correlates with P1/P2 ratio in human sperm, and statistical differences were detected when fertile controls were compared with three different cohorts of patients.

Key Words: Spermatozoa, protamine, P1/P2 ratio, DNA integrity, infertility, DNA damage

 

The protamines are the most abundant nuclear proteins present in mammalian sperm. Human sperm express two types of these key nucleoproteins: protamine 1 (P1) and the protamine 2 (P2) family of proteins (P2, P3, and P4). Under normal conditions, fertile and healthy human sperm express P1 and P2 in almost identical amounts (1). However, studies of infertile human males have shown that the P1/P2 ratio deviates from that seen in normal fertile controls (2). One of the predominant functions proposed for these important sperm proteins is preserving DNA integrity in the sperm head by preventing attack from exogenous or endogenous agents 3, 4. Of particular note is that a correlation exists between P1/P2 ratio and the incidence of sperm DNA fragmentation (SDF), suggesting that this observation may be of use for clinical diagnosis in infertility clinics 5, 6.

Previous studies have established that the integrity of sperm DNA is a highly limiting factor for the correct transmission of paternal genetic information to the developing embryo (7). Because it has been further demonstrated that sperm samples from different human males can exhibit differing dynamic properties of SDF, evaluation of the rate of SDF (rSDF; SDF measured between two consecutive time points) could provide accurate information pertaining to nuclear sperm vulnerability and differential susceptibility to DNA damage. This observation has already been demonstrated in a variety of mammalian species 8, 9, 10.

The present study aimed to determine the dynamics underlying sperm DNA fragmentation and, in particular, its potential relationship with protamine content. To achieve these objectives, P1/P2 ratio was compared with baseline SDF and rSDF in sperm from healthy fertile human males and in groups of males diagnosed with three differing pathologies of the reproductive system. In addition, we assessed the possibility that further understanding of the dynamic aspects of sperm DNA damage could provide key additional information about sperm health and subsequent potential embryo quality that is not apparent from analyses of baseline SDF alone, and such knowledge could be of value to clinical diagnosis.

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Materials and methods 

Sample Selection 

Semen samples from 32 individuals (six control donors of proven fertility, seven patients who were carriers of structural chromosome reorganization, nine patients with clinical varicocele, and 10 patients with subclinical varicocele) were obtained by masturbation after 3 days of sexual abstinence. Fresh ejaculate was allowed to liquefy and was then cryopreserved. Written informed consent was given by all patients, and the study was approved by the institutional ethics committee.

Extraction of Sperm Proteins 

An aliquot of semen sample containing 14 × 106 spermatozoa was washed three times with Ham's F10 1x (GibCo, Grand Island, NY). Sperm cells were then resuspended in 200 μL of 1 mM phenylmethylsulphonyl fluoride (Sigma-Aldrich, St. Louis, MO). Sediment was then resuspended in 200 μL of 20 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, and 100 mM Tris–HCl (pH 8) and processed as described previously (6). Finally, each sample was resuspended in 20-μL buffer of 5.5 M urea, 20% β-mercaptoethanol, and 5% acetic acid.

Separation and Analysis of Sperm Proteins 

Acid-urea polyacrylamide gels electrophoresis was performed on a Miniprotean System (Bio-Rad, Hercules, CA) using gels containing (final concentrations) 0.9 M acetic acid, 2.5 M urea, 15% acrylamide, 0.09% bis-acrylamide, 0.53% ammonium persulfate, and 0.53% TEMED (N,N,N,N-tetramethyl-ethylenediamine; Amersham Biosciences, Uppsala, Sweden). Gels were prerun for 1 hour at 150 V before loading. Samples were then loaded onto the gel (2.5 μL per sample) and separated by electrophoresis for one additional hour at 150 V in 0.9 M acetic acid buffer. Gels were stained with a solution of BioSafe Coomassie Blue G-250 (Bio-Rad) following the manufacturer's instructions. Finally, gels were scanned using a GS-800 scanner (Bio-Rad), and band intensity quantified with Quantity One software (Bio-Rad).

Sperm DNA Fragmentation and the Dynamics Underlying Fragmentation 

An aliquot of each sperm sample was incubated at a physiologic temperature of 37°C in phosphate-buffered saline buffer. SDF, defined as the proportion (%) of fragmented sperm present among the total number of spermatozoa in the analyzed sample, was assessed after 0, 4, 8, and 24 hours of incubation (t0, t4, t8, and t24). Samples were assessed for SDF immediately after thawing (t0), and the corresponding value was considered as the baseline for each sample—SDF (t0). Values for rSDF were determined according to the following equation: rSDF = (SDFt8 – SDFt0) ÷ 8 hours.

The Halosperm Kit (Halotech DNA SL, Madrid, Spain), which is based on the sperm chromatin dispersion test (SCDt), was used to determine sperm DNA fragmentation following commercial instructions (11). Slides were stained for fluorescence microscopy using propidium iodide (2.5 μg/mL in Vectashield; Vector Laboratories, Burlingame, CA). For this study, 200 spermatozoa were scored per experimental point.

Statistical Analysis 

Data analyses were performed using the Statistics Package for the Social Sciences software, version 15 (SPSS, Inc., Chicago, IL). Values were compared using the Kruskal-Wallis test. Correlations were studied using the Pearson test. The level of significance was established at 95% of the confidence interval to be considered as statistically significant.

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Results 

Determination of P1/P2 Ratio 

Figure 1 shows analysis of P1 and P2 in human sperm samples. P1/P2 ratio was 1.2 ± 0.48 (average ± standard deviation) and ranged from 0.5 to 1.9 in fertile control sperm (Table 1), compared with 1.85 ± 0.68 (range, 1–4.68) in sperm obtained from a group of patients suffering pathologies of the reproductive system (Fig. 2A). P1/P2 ratio was significantly higher in the group of patients than in the controls (P=0.023). There were no significant differences in sperm P1/P2 ratio when compared between the three pathologic conditions. P1/P2 ratio was 1.7 ± 0.46 (range, 1–2.41), 2.12 ± 1.19 (range, 1.41–4.68), and 1.72 ± 0.34 (range, 1.25–2.34) in patient groups with rearranged genome, clinical varicocele, and subclinical varicocele, respectively (Fig. 2B).

  • View full-size image.
  • Figure 1. 

    Analysis of protamine 1 (P1) and protamine 2 (P2) ratio. (A) Proteins extracted from spermatozoa, separated on a polyacrylamide-acetic-urea gel and stained with Coomassie blue. Lanes 1–5 and 6–10 correspond to different sperm samples from clinical and subclinical varicocele patients, respectively. (B) Example from a fertile donor sample with normal P1/P2 ratio (left) and a patient sample with a highly altered P1/P2 ratio (right).

Table 1. P1/P2r, rSDF and SDF (t0) outcomes.
P1/P2rrSDFSDF (t0)
Control (n = 6)1.23 ± 0.480.93 ± 0.3013.00 ± 4.97
All patients (n = 26)1.85 ± 0.68a1.84 ± 1.10a30.42 ± 9.29b
Rearranged genome (n = 7)1.70 ± 0.462.57 ± 1.34a19.85 ± 6.98
Clinical varicocele (n = 9)2.12 ± 1.192.10 ± 1.00b34.77 ± 7.91b, c
Subclinical varicocele (n = 10)1.72 ± 0.341.09 ± 0.44d, e33.90 ± 5.68b, c

Note: Values are expressed as mean ± SD. Comparisons were performed using the Kruskal-Wallis test.

aStatistical differences versus control group; P<0.05.

bStatistical differences versus control group; P<0.01.

cStatistical differences versus rearranged genome group; P<0.05.

dStatistical differences versus rearranged genome group; P<0.01.

eStatistical differences versus clinical varicocele group; P<0.05.

  • View full-size image.
  • Figure 2. 

    Box-and-whisker plots showing (A) differences between control and patients groups for P1/P2 ratio, rSDF, and SDF (t0) and (B) differences between control, rearranged genome, and clinical and subclinical varicocele groups for P1/P2 ratio, rSDF, and SDF (t0).

Determination of SDF (t0) and rSDF 

Data pertaining to SDF (t0) and rSDF are also given in Table 1. SDF (t0) was significantly higher in sperm from patients (30.42 ± 9.29; range, 11–48) than in sperm from the control group (13.0 ± 4.97; range, 4–19; Fig. 2A). Statistical differences were found between both varicocele groups (clinical and subclinical) when compared with the rearranged genome group (P < 0.01; Fig. 2B). rSDF was significantly higher (P=0.033) in sperm from the group of patients (1.84 ± 1.10; range, 0.62–4) than in sperm from the fertile control group (0.93 ± 0.30; range, 0.5–1.37; Fig. 2A). rSDF in the rearranged genomes and clinical and subclinical varicocele patients was 2.57 ± 1.34 (range, 0.75–4), 2.10 ± 1.10 (range, 1.25–4), and 1.09 ± 0.44 (range, 0.62–1.87), respectively. Statistical differences were detected between the subclinical varicocele patients and both the clinical varicocele and rearranged genome groups (P < 0.05). No statistical differences were found between patients with a rearranged genome and those diagnosed with clinical varicocele (Fig. 2B).

Correlation of P1/P2 Ratio with rSDF and SDF (t0) 

Analyses showed that the P1/P2 ratio was correlated with SDF at t0 (r = 0.424; P=0.015). However, a stronger correlation was detected between the P1/P2 ratio and rSDF (r = 0.525; P=0.002), suggesting that the latter relationship was of more use to the development of new clinical diagnostic assessments.

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Discussion 

The results of the present study strongly indicate that the dynamic assessment of sperm DNA fragmentation may provide new insight to our understanding of the loss of sperm DNA quality, a sperm characteristic that remains hidden when only baseline SDF is considered. In the present experimental approach, we have demonstrated that both SDF and rSDF correlate with P1/P2 ratio. Correlation between baseline DNA damage and protamine content in sperm has been determined previously by Manicardi et al. (12) using chromomycin A3 staining. This correlation was confirmed using similar experimental approaches by several other authors 13, 14, 15. Direct correlations between P1/P2 ratio and baseline SDF were reported by Aoki et al. (5) and Torregrosa et al. (6) using different experimental approaches such as the sperm chromatin structure assay and the TUNEL assay. The use of the SCDt also demonstrates that when the P1/P2 ratio is altered, SDF is increased. Using this methodology, we were able to demonstrate that rSDF is also correlated with P1/P2 ratio, and more importantly, that this correlation was higher than that obtained for SDF when assessed at t0. This finding is of great interest because these results indicate that any imbalance in P1/P2 ratio may render the sperm more susceptible to stressors. It is evident that the level of DNA damage produced when spermatozoa are confronted by a multistep and potentially aggressive methodology, such as sperm selection for assisted reproductive technologies (ART), may be the result of a synergistic process, whereby the level of damage in each step is reinforcing the damage produced in the next. Consequently, the immediate hypothesis to test would be that the highest imbalance in P1/P2 ratio will render the highest levels of rSDF, thus yielding sperm with compromised potential to create a viable embryo. The confirmation of this observation may be of great significance in common and established assisted reproduction technique protocols when using ejaculated sperm samples for controlled clinical procedures. Future studies should critically analyze sperm selected via such protocols for downstream clinical application and relate such sperm to P1/P2 ratio, SDF, and rSDF. Moreover, even under natural conditions devoid of clinical intervention, the sperm that survive the stressful passage in the first part of the female tract may have fewer opportunities for deleterious effects on DNA integrity. Sperm must reach and make contact with the cervical mucus and enter the cervix; only then can the sperm fraction exhibiting adequate biologic characteristics reach the fallopian tubes with the physiologic potential to fertilize an oocyte. In such competitive and potentially aggressive environments, the fraction of sperm that is maintained in a fully functional state by connecting with endosalpingeal epithelium must maintain an orthodox DNA molecule with fully physiologic capacity for fertilization 16, 17, and this could depend on the P1/P2 ratio.

P1/P2 Ratio in Sperm From Fertile Controls and Patient Groups Exhibiting Pathology of the Reproductive System 

The present study detected a statistically elevated P1/P2 ratio and SDF in patients exhibiting pathologies of the reproductive system compared with healthy fertile controls. These results concur with earlier studies by other authors 1, 18. Studies have attempted to establish the prognostic values of these variables for pregnancy outcome 2, 7, 19, 20. However, the conclusions of these investigative studies remain controversial, because other authors report conflicting results 21, 22, 23. The present results show that statistical differences in rSDF are detectable between control and patient cohorts. However, until recently, studies have failed to incorporate patients exhibiting reproductive anomalies in their analyses. It has been reported that in human males of proven fertility, sperm DNA experience significant elevations in SDF, which can be variable in intensity when compared with different individuals (9). Interestingly, the tendencies for increased elevations in SDF can be logarithmic, linear, or exponential (10). This finding indicates that some individuals may exhibit a more intense rSDF than others, and conversely some sperm samples may be more resistant to DNA fragmentation than others (9).

P1/P2 ratio has not been determined previously in the three patient subgroup types included in the present study. However, our results are in agreement with formerly published values for fertile male and infertile patients 2, 19. We did not detect statistical differences for P1/P2 ratio when the three patient subgroups were analyzed. However, the highest values of P1/P2 ratio were recorded in the clinical varicocele group. This finding can be explained if we consider that a drastically altered P1/P2 ratio is normally associated with a reduction in the levels of P2 concurrent with elevated levels of the pre-P2 precursor form (1). A common feature of varicocele is increased testicular temperature and elevated levels of oxidative agents 24, 25. Evenson et al. (26), however, reported that thermal stress does not influence P1/P2 ratio. However, De Iuliis et al. (14) described a direct relationship between oxidative stress and chromatin remodeling. Therefore, it is reasonable to postulate that oxidative stress may be a factor that affects P1/P2 ratio in a manner yet to be described. However, this interpretation needs to be confirmed by future work addressing a larger number of appropriate patient samples.

Rearranged Genomes 

Carriers of a rearranged genome are characterized by an increased risk of unbalanced sperm production, which in some cases could lead to infertility 27, 28. A number of different reports have reported that carriers of a rearranged genome exhibit increased levels of sperm DNA fragmentation 29, 30. However, we failed to detect any statistically differences, although the P value was close to statistical significance (P=0.06). We speculate that this effect is not apparent in our study, owing mainly to a relatively low sample size. We need to highlight that in some of the patients of this study, the level of SDF at t0 would be considered as being normal by some authors 7, 31. The situation is different when the rSDF was analyzed; the rSDF average for the rearranged genome group was the highest of all patient subgroups (Table 1).

Varicocele 

Varicocele is defined as the dilation of the pampiniform plexus of the internal spermatic veins, and it is one of the main causes of male infertility (32). Depending on its severity, and although its diagnosis remains controversial, we have distinguished a subclinical varicocele condition, which is less intense and diagnosed by ultrasound examination. Clinical varicocele, however, is more severe and can be diagnosed by physical examination. The results obtained in regard to the level of SDF at t0 in the present study are also consistent with published data 33, 34, 35. To our knowledge, there are no earlier reports concerning DNA fragmentation in the subclinical varicocele condition. Our current data demonstrate that both varicocele patient groups (clinical and subclinical) exhibit an increased and statistically significant level of SDF when compared with the control group. No differences were detected between the subclinical and clinical varicocele patients. Nevertheless, when dynamic aspects of SDF were considered by determining rSDF, we detected differences between the two varicocele groups. No differences in rSDF were found between the control group and the subclinical varicocele cohort, whereas the clinical varicocele group differed statistically when compared with the control and subclinical groups. Moreover, it is interesting to note that basal SDF levels for clinical and subclinical groups were similar, but the increasing rate of sperm DNA damage was higher in patients with clinical varicocele. This finding could be related to the higher levels of ROS associated with the clinical varicocele condition (36).

Baseline levels of SDF evaluated at t0, along with rSDF, correlates with the P1/P2 ratio determined from individual patients. Evaluation of dynamic aspects of SDF offers significant new insight into our understanding of the potential capacity of a sperm sample to achieve fertilization when used in assisted reproductive technique treatments such as intrauterine insemination or in vitro fecundation (IVF). Furthermore, detailed evaluation of rSDF may provide important information concerning the specific use and temporal windows in which we must handle sperm samples for ICSI, intracytoplasmic morphologically selected sperm injection (IMSI), or alternative procedures for sperm selection.

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Acknowledgment 

We thank Sara de Mateo for technical assistance.

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References 

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 A.G-P. has nothing to disclose. J.M-H. has nothing to disclose. M.O-B. has nothing to disclose. C.A. has nothing to disclose. M.J.A. has nothing to disclose. J.N. has nothing to disclose. C.J. has nothing to disclose. K.C. has nothing to disclose. J.G. has nothing to disclose. J.B. has nothing to disclose.

 A.G-P. and J.M-H. contributed equally to this work.

 Supported by the Fondo de Investigación Sanitaria (FIS) (PI051834, PI080623), Generalitat de Catalunya (2009 SGR 1107), Ministerio de Ciencia y Tecnología (MCYT) (BFU 2007-66340/BFI), and Càtedra de Recerca Eugin-UAB.

PII: S0015-0282(10)01006-X

doi:10.1016/j.fertnstert.2010.06.053

Fertility and Sterility
Volume 95, Issue 1 , Pages 105-109, January 2011

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