Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm

Available online at www.sciencedirect.com

Theriogenology 76 (2011) 1450 –1464 www.theriojournal.com

Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm E. Floresa, L. Ramió-Llucha, D. Buccib, J.M. Fernández-Novellc, A. Peñaa, J.E. Rodríguez-Gila,* a

Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra, Spain b DIMORFIPA, University of Bologna, Ozzano Emilia I-40064, Bologna, Italy c Department of Biochemistry and Molecular Biology and IRRB, Barcelona Science Park, University of Barcelona, E-08028 Barcelona, Spain Received 11 November 2010; received in revised form 4 May 2011; accepted 4 May 2011

Abstract The main aim of this work is to gain insight into the mechanisms by which freezing-thawing alters the nucleoprotein structure of boar sperm. For this purpose, the freezing-thawing-related changes of structure and location of histones-DNA domains in the boar sperm head were analyzed through Western blot and immunocytochemistry. Afterwards, it was analyzed whether freezing-thawing induced changes in tyrosine phosphorylation levels of both protamine 1 and histone H1, through Western blot analyses in samples previously subjected to immunoprecipitation. This analysis was completed with the determination of the changes induced by freezing-thawing on the overall levels of sperm-head disulfide bonds through analysis of free-cysteine radicals levels. Freezing-thawing induced significant changes in the histones-DNA structures, which were manifested in the appearance of a freezing-thawing-linked histone H1-DNA aggregate of about a 35-kDa band and in the spreading of histone H1-positive markings from the caudal area of the sperm head to more cranial zones. Freezing-thawing did not have any significant effect on the tyrosine phosphorylation levels of either protamine 1 or histone H1. However, thawed samples showed a significant (P ⬍ 0.05) increase in the free cysteine radical content (from 3.1 ⫾ 0.5nmol/␮g protein in fresh samples to 6.7 ⫾ 0.8 nmol/␮g protein). In summary, our results suggest that freezing-thawing causes significant alterations in the nucleoprotein structure of boar sperm head by mechanism/s linked with the rupture of disulfide bonds among the DNA. These mechanisms seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Furthermore, results suggest that the boar-sperm nuclear structure is heterogeneous suggesting the existence of a zonated pattern, differing in their total DNA density and the compactness of the precise nucleoprotein structures present in each zone. © 2011 Elsevier Inc. All rights reserved. Keywords: Boar sperm; Freezing-Thawing; Histone H1; Disulfide bonds

1. Introduction In the last few years there has been an increasing amount of information regarding freezing-thawing-related alteration of sperm nuclear structure. These alter-

* Corresponding author. Tel.: 34-935811045; fax: 34-935812006. E-mail address: [email protected] (J.E. RodríguezGil). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.05.039

ations can strongly affect the fertilizing ability of the sperm without modifying other aspects of sperm function, such as motility or oocyte penetration ability [1]. In the majority of cases and species, the most-studied nuclear sperm alteration linked to freezing-thawing has been DNA fragmentation. In this way, freezing-thawing-induced DNA fragmentation has been observed in several species, such as human and horse [1–2]. Nevertheless, in other species reports regarding DNA frag-

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

mentation during freezing-thawing are conflicting. In this regard, no effect was observed in frozen-thawed ram sperm [3]. Centering on boar, reports vary from the presence of a clear increase in freezing-linked DNA damage to the absence of any effect on DNA fragmentation [4 –7]. It is noteworthy that these discrepancies have been observed even after the utilization of similar techniques to detect DNA fragmentation, such as Neutral Comet Assay and SCSA [3–7]. This opens doubts about the importance and mechanisms of freezingthawing-induced alterations of sperm head-DNA structure. However, although DNA fragmentation would be the clearest effect related to changes in nuclear sperm structure during freezing-thawing, it is not the only possible effect. In this way, our laboratory has shown that freezing-thawing of boar sperm in conditions in which there was not any significant increase of DNA fragmentation induces alterations in the protamine 1-DNA structural interaction instead [7]. This is of importance, since this alteration causes significant changes in the overall boar-sperm nuclear structure after thawing, with the possible consequences of an overall loss of sperm fertilizing ability. Notwithstanding, the exact nature that causes the alteration of the protamine 1-DNA structure is not known. In this respect, it should be remembered that nuclear proteins can modulate their union to DNA through two main mechanisms. The first is through changes in the phosphorylation levels of the nuclear proteins. It is well known that both protamines and histones are present in different degrees of phosphorylated forms, from unphosphorylated forms to highly phosphorylated ones, in mature mammalian spermatozoa, although there are many differences among species. In this sense, protamines are in a non-phosphorylated form in mature sperm [8]. On the contrary, histones are present in eucaryotic cells in various degrees of phosphorylation, with maximal phosphorylation levels during the interphase and mitotic phases of the cell cycle [9]. Changes in the phosphorylation levels of nuclear proteins induce variations in the strength of the proteins-DNA bonds of the nuclear structure. In this way, it has been described that a decrease in the protamine phosphorylation levels induces a loosening in their capacity to link DNA, thus inducing an increased flexibility in the protaminesDNA structure [10]. On the contrary, an increase in histone phosphorylation induces a loosening of the histones-DNA unions [11]. These changes lead histones to play an important role as regulatory elements of gene activity during the cell cycle [11]. Thus, it is possible

1451

that the observed changes in the nucleoprotein structure of boar sperm during freezing-thawing are related to changes in the phosphorylation levels of the present nucleoproteins. Nevertheless, there is another mechanism that can account for the alterations observed. This would be alterations of the disulfide bonds, which are one of the most important union mechanisms between nucleoproteins and DNA [8,12,13]. It is well known that mechanisms such as osmotic oxidative stress induced by osmotic disturbances are able to destroy disulfide bonds in a whole series of cellular structures [14,15]. This is important, since osmotic stress is one of the most important mechanisms, together with oxidative stress, responsible for the main freezing-thawing-induced alterations in sperm, including structural alterations in cellular membranes and mitochondria volume and shape, as well as the rupture of the peri-mitochondrial and head actin network [1,16 –25]. Hence, these mechanisms could be at the basis of the already observed freezing-thawing-linked alterations of the boar-sperm nuclear structure. The study of DNA-proteins structural changes in the sperm head is further complicated, however, by the existence of a heterogeneous structure. It is well known that, although the majority of nuclear sperm proteins are protamines, there are several specific domains in the sperm nucleus in which DNA is associated with histones [26,27]. In this way, the percentage of human sperm DNA that is structured around histones is about 15% [27]. This is important, since the structure associated with the histones-linked DNA domains are much less compact and rigid than that related to the protamines-linked DNA domains [26,27]. Taking into account that the histones-linked domains are located at the telomeric sequences [27], it is reasonable to assume that the freezing-thawing-related alterations of the sperm head-structure would of different intensity in both the histones-linked nuclear domains and the protamines-linked DNA ones. However, there is not any direct evidence that supports this hypothesis, since there are no published data regarding freezing-thawingrelated alterations of the histones-DNA structures in sperm. Taking into account all of the data described above, the aim of this manuscript is centered on two objectives. The first objective is the study of the freezingthawing-related alterations of the histones-DNA domains in the boar sperm head. This was performed through both Western blot analysis and immunocytochemistry. The second objective is to study the freez-

1452

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

ing-thawing-related changes in both the phosphorylation levels of boar-sperm protamine 1 and histone H1 and the overall destruction of disulfide bonds in the boar sperm head. This objective was studied by Western blot against tyrosine phosphorylation of immunoprecipitation-isolated protamine 1 and histone H1 and through the analysis of sperm head free cysteine radicals through spectrophotometric analysis. Finally, the combined analysis of the results observed here and others previously published elsewhere led us to launch a general hypothesis regarding the specific nuclear structure of boar sperm. 2. Materials and methods 2.1. Animals and samples collection Eight healthy boars of 2–3 years of age from a commercial farm were used in this study. The boars were from three separate lines (three Landrace, two Large White and three Pietrain). All boars had proven fertility after AI using extended, liquid semen. The total number of ejaculates utilized was 24, utilizing three ejaculates from each boar obtained in different days. Eight ejaculates (one per boar) were utilized for Western blotting and immunocytochemistry of histone H1. Another eight ejaculates, again one per boar, were utilized for immunoprecipitation techniques. Finally, the other eight ejaculates, one per boar, were utilized for the determination of free-cysteine radicals. The spermrich fraction of each ejaculate utilized in this study was manually collected twice weekly using the gloved-hand method and analyzed to ensure the quality and the homogeneity of the ejaculates. Immediately after collection, the ejaculated semen was suspended (1:2; v/v) in a commercial extender (MR-A; Kubus SA; Majadahonda, Spain). The extended semen samples were cooled and maintained at 17 °C for shipment to the laboratory of the Autonomous University of Barcelona within 24 h post-collection, for further processing and analyses. 2.2. Semen cryopreservation Immediately after receiving the shipped semen samples, an aliquot was taken to perform the appropriate semen assessments and studies (extended semen sample). Only those samples displaying a minimum of 70% progressive motile and 80% of morphologically normal spermatozoa were further processed for freezing-thawing after the application of a proven protocol [28,29]. Progressive motility was defined as the percentage of

sperm that showed a mean velocity (VAP, defined as the mean velocity of the sperm head along a straight line from its first to its last position) greater than 35 ␮m/sec together with a straightness coefficient (STR, defined as the coefficient between the linear velocity and VAP) above 80%, whereas morphologically normal spermatozoa were those that did not show either any evident morphological abnormality or any acrosome alteration after counting 200 spermatozoa in a sample extension stained by the Eosin-Nigrosin technique. Ejaculates were considered as being valid with a percentage of morphologically normal sperm above 80%. The extended semen was centrifuged in a programmable refrigerated centrifuge (Medifriger BL-S; JP Selecta; Barcelona, Spain) set at 17 °C, at 800 g for 10 min. After centrifugation, the supernatant was discarded. The remaining pellets were re-extended with a lactose-egg yolk (LEY) extender composed of 20% (v:v) egg yolk, 248 mM ␤-lactose and 0.1 g/L kanamicin (pH 6, osmolarity 350 mOsm/Kg), at a ratio that led to a final concentration of 1.5 ⫻ 109 sperm/mL. The sperm concentration was manually assessed in a Thoma haemocytometer. At this point, and after thorough mixing, the semen was further cooled to 5 °C for 2 h in a programmable freezer (Ice-Cube 14S; Minitüb) in which was applied a cooling ramp of 0.1 °C/min. Then, an aliquot of the refrigerated semen was taken to carry out the appropriate analysis (refrigerated or 5 °C semen sample) and then the semen was slowly mixed with a third extender consisting of 89.5 mL LEY extender, 9 mL glycerol and 1.5 mL of Equex STM (Nova Chemicals Sales Inc.; Scituate, MA, USA), which is equivalent to Orvus Es Paste [30], at a ratio of two parts of semen to one part of extender, yielding a final concentration of glycerol of 3% and a concentration of 1 ⫻ 109 sperm/mL at 5 °C, which was verified by counting in a Thoma haemocytometer. Spermatozoa were packaged at 5 °C in a cool cabinet (IMV; L’Aigle, France) in 0.5-mL polyvinyl chloride (PVC) plastic straws (Minitüb; Tiefenbach, Germany), which were sealed with PVC powder and placed on racks for freezing. The racks were transferred to the chamber of a programmable freezer (Ice-Cube 14S; Minitüb) set at 5 °C. The cooling-freezing rate used was: 3 °C/min from 5 °C to ⫺5 °C, 1 min for crystallization, and thereafter 50 °C/min from ⫺5 °C to ⫺140 °C. The samples were then plunged into liquid N2 (⫺196 °C) for storage. When stated, samples were thawed by plunging them into a water bath at 37 °C for 20 sec. Straws were then carefully dried and opened, and samples were immedi-

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

ately processed in the appropriate analysis (frozenthawed semen sample). 2.3. Analysis of semen-quality parameters Percentages of viability and altered acrosomes were determined by using the bis-benzamidine-propidium iodide-Mitotracker Green FM-Alexa Fluor 1488-conjugated lectin trypsin-inhibitor from soybean (SBTI) quadruple staining as described in [31]. This technique is based on the simultaneous utilization of four different stains, namely, bis-benzamidine, propidium iodide, Mitotracker Green FM and Alexa Fluor 1488-conjugated SBTI, which allow us the simultaneous determination of both the percentages of viability and that of morphologically intact acrosomes. For this purpose, an aliquot of sperm suspension was firstly incubated with a solution of 15 mM bis-benzamidine (Hoechst 33258; Boehringer Mannheim; Mannheim, Germany; proportion 1:1000, v:v) for 10 min at 37 °C. Afterwards, a propidium iodide solution was added (Sigma Aldrich; Saint Louis, MO, USA; proportion 6:1000, v:v) and the sperm was subjected to further incubation for 10 min at 37 °C. After this incubation, the sperm suspension was centrifuged at 1500 g for 10 min and the supernatant discarded. The obtained sperm pellet was resuspended in 1 mL of a solution of 100 nM Mitotracker Green FM (Molecular Probes; Paisley, United Kingdom) and FMAlexa Fluor1488-conjugated SBTI (Molecular Probes) in phosphate buffered medium (PBS).The sperm suspension was incubated in this solution for 20 min at 37 °C and then was immediately centrifuged at 1500 g for 12 min. The resultant supernatant was discarded and the sperm pellet was resuspended in 100 mL of PBS at 37 °C. The sperm suspension was spread onto slides and fluorescence was immediately determined in a Zeiss Axioskop-40 fluorescence microscope (Karl Zeiss Gmbh; Jena, Germany) with the appropriate filters for green, red and orange fluorescence. Intact, unaltered acrosomes were exclusively considered to be those which showed a uniform STBI lectin stain. Acrosomes that showed any other aspect (saturation of the color intensity of satin, non-uniform acrosomal marking, lack of staining) were considered to be altered structures. Furthermore, viable sperm showed a blue stain of the sperm head, whereas non-viable cells showed an intense red stain of the head. Following these criteria, viability and altered acrosome percentages were simultaneously determined after counting 200 –300 spermatozoa per experiment, from one slide obtained per each utilized ejaculate, at 1000x.

1453

The osmotic resistance test (ORT) was carried out as described in [32], whereas the hyperosmotic resistance test (HRT) was carried out as in [33]. Briefly, the ORT was performed through simultaneous incubation of two aliquots of a semen sample in both an isoosmotic solution (osmolarity of 305 ⫾ 4 mOsm/Kg) of 3.2% (w:v) sodium citrate adjusted to pH 7.4 and a hypoosmotic solution (oslmolarity of 102 ⫾ 6 mOsm/Kg) of 1% (w:v) sodium citrate adjusted to pH 7.4. Incubations were maintained for 10 min at 37 °C and the percentage of altered acrosomes in sperm subjected to both isoosmotic and hypoosmotic media were determined by counting 200-300 sperm per experiment, from one slide obtained per each utilized ejaculate, at 1000x. Acrosomes were visualized without staining by observation through a phase-contrast lens. The ORT values were calculated following the formula: ORT (%) ⫽

(AIM ⫹ AHM) 2

,

in which AIM is the percentage of morphologically intact acrosomes in isoosmotic conditions and AHM is the percentage of morphologically intact acrosomes in hypoosmotic conditions. Intact acrosomes were considered to be those that showed a uniform and visible acrosomal ridge, whereas any other aspect of the apical head zone was considered as being morphologically altered acrosomes. The HRT was carried out by placing an aliquot of a semen sample in a hyperosmotic medium (osmolarity of 1169 ⫾ 10 mOsm/Kg) of 0.9 mol/Kg glucose adjusted to pH 7.4. Incubation was maintained for 5 min at 37 °C. After this time, an aliquot was extracted and placed into an isoosmotic Krebs-Henseleit-Ringer solution at 37 °C (osmolarity of 296 ⫾ 2 mOsm/Kg, pH of 7.4). Both the hyperosmotic and the isoosmotic solutions were further incubated for another 5 min at 37 °C. Afterwards, the percentage of altered acrosomes in sperm subjected to both hyperosomotic and hyperosmotic-to-isoosmotic media were determined by counting 200 –300 sperm per experiment, from one slide obtained per ejaculate utilized, at 1000x. Acrosomes were visualized without staining by observation through a phase-contrast lens, following the pattern described for the ORT. The HRT values were calculated following the formula: HRT ⫽

AD AU

,

in which AD is the percentage of morphologically altered acrosomes in the final isosmotic medium and AU

1454

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

is the percentage of morphologically altered acrosomes in the initial hyperosmotic medium. Total motility was evaluated through analysis by using a commercial computer-assisted analysis of sperm motility (CASA system; Integrated Sperm Analysis System V1.0; Proiser; Valencia, Spain). In this procedure, samples were warmed at 37 °C for 5 min in a water bath and afterwards 5 ␮L aliquots of these samples were placed onto a warmed (37 °C) slide and covered with a 625 mm2 coverslip. Total motility was defined as the percentage of spermatozoa which showed a VAP above 10 ␮m/sec. The analysis of putative DNA fragmentation was analyzed through the Sperm–Sus Halomax® stain (Chromacell; Madrid, Spain). This kit is specifically designed for boar sperm. It is based on the different response that intact and fragmented DNA show after a de-proteinization treatment. It has been described that the results obtained with this technique strongly correlated with those obtained with other previously utilized, but much more difficult to apply, tests like the neutral comet assay [34]. Following the application of the protocol of the commercial kit, boar sperm cells that present DNA fragmentation show a stained halo around their heads. These stained halos had a width of at least 2 ␮m, following the criteria established in the instructions of the kit. The percentage of DNA fragmentation were determined after counting 200 –300 spermatozoa per experiment, from one slide obtained per utilized ejaculate utilized, at 1000x. 2.4. Western blot analyses of boar-sperm histone H1 Both Western blot analyses and immunocytochemical detection of boar-sperm histone H1 were performed by using the same commercial monoclonal antimouse histone H1 antibody (Abcam; Cambridge, United Kingdom). To perform Western blot analyses, boar spermatozoa were homogenized by sonication in ice-cold 10 mM Tris–HCl buffer (pH 7.4) containing 1% (w/v) sodium dodecyl sulfate (SDS), 15 mM ethylene diamine tetraacetic acid (EDTA), 150 mM KF, 0.6 M sacarose, 14 mM ␤-mercaptoethanol, 10 ␮g/mL leupeptin, 1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF). The homogenates were then centrifuged at 10,000 g for 5 min at 4 °C and the resultant pellets were discarded. Western blot was only then performed on soluble sperm fractions of homogenates. Western blot was carried out in samples subjected to SDS gel electrophoresis [35], followed by transfer of electrophoreted samples to nitrocellulose [36]. To carry out the SDS gel electrophoresis, 20 ␮g of

total protein per sample were loaded in each lane. Total protein content of samples was determined by the Bradford method [37], after applying a commercial kit (BioRad; Hercules, CA, USA). The transferred samples were tested with the anti-histone H1 antibody at a dilution (v/v) of 1:1000. Immunoreactivity was tested using peroxidase-conjugated goat anti-mouse secondary antibody (Santa Cruz Technologies; Santa Cruz, CA, USA) and the reaction was developed with an ECL-Plus detection system (Amersham; Buckinghamshire, Great Britain). Moreover, the specificity of the observed immunoreactivity was tested after subjecting mouse-liver extracts processed in the same manner as that described for boar sperm subjected to Western blot in the presence of the anti-histone H1 antibody This was utilized as positive controls, since mouse liver extracts contain great quantities of histone H1. Additionally, a negative control of the observed immunoreactivity was also tested after subjecting experiments to Western blot in the presence of the anti-histone H1 antibody previously pre-adsorbed with a commercial, specific peptide (Santa Cruz Technologies) to a final concentration of 15 mg/mL. This pre-adsorption prevents the union of the anti-histone H1 antibody to the specific protein and, in this way, this is an adequate negative control for any putative methodological mistake. For this purpose, we divided each sample into two aliquots. One was subjected to Western blot against the antibody without any manipulation, while the other was concomitantly subjected to Western blot, but in the presence of the previously preadsorbed antibody. Lastly, the first two experiments that we carried out indicated that the specific reaction obtained in samples could be due to the presence of histone H1–DNA low molecular-weight complexes similar to those previously described for the protamine-1 in boar sperm [7]. To elucidate upon this question, Western blot analyses were carried out in samples previously incubated with 40 mg/mL DNAase-1 for 2 h at 25 °C. This incubation was performed prior to the application of samples to the SDS gel electrophoresis that started the Western blot process. 2.5. Detection of histone H1 in boar sperm through Immunocytochemistry Immunocytochemistry was carried out on sperm sections of samples subjected to cryofixation. The samples utilized came from 5 mL of commercial IA refrigerated doses in extended samples, from 0.5 mL of samples after the cooling phase of the freezing-thawing procedure or from a 0.5 mL straw for frozen-thawed

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

semen. The procedure started by the washing of sperm samples three times with PBS and subsequent fixation with 500 ␮L of a 2% (w/v) paraformaldehyde solution in PBS for 15 min at 25 °C. Fixed samples were centrifuged at 600 g for 3 min, and the supernatants were discarded. The cellular pellet was resuspended in 500 ␮L of PBS and centrifuged again at 600 g for 3 min. Supernatants were again discarded, and the pellets obtained were embedded in 40 ␮L of the OCT1 inclusion medium (Leica Instruments; Wetzlar, Germany). Samples were immediately frozen with liquid N2 and stored until their processing at -80 °C. When stated, the included samples were sectioned in slices of 1 mm of thickness by using a cryostat. Sections were subsequently placed onto gelatin-coated slides (76 mm ⫻ 26 mm). Immediately, the slides were covered with a PBS solution containing 0.1 (v/v) commercial Hoechst 33258 solution (Boehringer Mannheim). This stain allowed for the determination of an exact co-localization between the histone H1 signal obtained with the specific antibody and the sperm nuclear DNA. Incubation with Hoechst 33258 was maintained for 15 min at 38.5 °C, preventing any light source from reaching the slides. From this moment on, all of the further steps were carried out preventing any direct incidence of any light source on the samples. After this, the excess liquid on the slides was eliminated by decantation, and slides were thoroughly washed three times with PBS. Histone H1 immunocytochemistry was started by incubation with 1 mg/mL NaBH4 for 15 min to prevent autofluorescence. This step was followed by a permeabilization with 0.2% (v/v) Triton X-100 in PBS for 30 min and a blocking step with 3% (w/v) bovine serum albumin (BSA) for 30 min. The sperm sections were then incubated with the monoclonal anti-mouse histone H1 antibody (dilution 1/100 v/v) described above for 1–2 h at 15 °C, washed with PBS, and treated with an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Santa Cruz Technologies). Fluorescent images were obtained with a Leica TCS 4D confocal scanning microscope (Leica Lasertechnik; Heidelberg, Germany) adapted to an inverted Leitz DMIRBE microscope and a 63X (NA 1.4 oil) Leitz Plan-Apo Lens (Leitz; Stuttgart, Germany). The light source was an argon/krypton laser (75 mW). Images were captured by the software associated with the microscope. Three-tofour images per sample were captured. The captured images were stored as TIFF format archives. Duplicate images were simultaneously captured and stored under visible light in a phase contrast system. The combination of visible light and laser images allowed for the

1455

exact location of the positive reactions in sperm-head sections, thus permitting a better analysis of the data obtained. Finally, the specificity of the observed immunoreactivity was tested after subjecting several experiments to immunocytochemistry in the presence of the anti-histone H1 antibody previously pre-adsorbed with a commercial, specific peptide (Sigma) to a final concentration of 15 mg/mL. This was done for the same purpose as that described for the Western blot analyses. 2.6. Processing boar-sperm samples for specific immunoprecipitation and subsequent phosphotyrosylation and both protamine-1 detection and histone H1 detection through Western blot analysis Boar sperm samples were immediately diluted to a final volume of 1 mL with an ice-cold 10 mM Tris-HCl buffer (pH 7.4) containing 600 mM sucrose, 10 ␮g/mL leupeptin, 1 mM benzamidine, 1 mM phenyl methyl sulphonyl fluoride and 1M Na2VO4 (lysis buffer). Samples were homogenized by ultrasonication and were then incubated with 40 ␮g/mL DNAase-1 for 2 h at 15 °C and were then centrifuged at 12,000 g for 10 min at 4 °C, in order to eliminate the contaminating DNA that could alter the immunoprecipitation process. Afterwards, samples were divided into two aliquots of 500 ␮L, in order to perform the immunoprecipitation of both protamine-1 and histone H1 per sample. Each aliquot was then added to 12.5 ␮L of a commercial presentation of protein G-sepharose (Protein G Sepharose 4 Fast Flow. GE Healthcare Bio-Sciences AB; Uppsala, Sweden) which was previously diluted in lysis buffer at a dilution rate of 1:1 (v:v). Samples were then incubated for 1h at 4°C in continuous shaking by utilizing an orbital shaker. The same treatment was applied to 500 ␮L of lysis buffer alone as negative control for the immunoprecipitation. Next, samples were centrifuged at 12,000 g for 10 min at 4 °C and the resultant pellet was discarded. Afterwards, the obtained supernatants were incubated with the chosen, specific antibody against either protamine-1 (anti-goat protamine-1 antibody; Santa Cruz Technologies) or histone H1 (anti-mouse histone H1 antibody; Abcam) at a final dilution of 1:150. Samples were incubated with the antibody for 1 h at 4 °C in continuous shaking by using an orbital shaker. Subsequently, samples were added to 30 ␮L of a commercial presentation of protein Gsepharose (Protein G Sepharose 4 Fast Flow. GE Healthcare Bio-Sciences AB; Uppsala, Sweden) which was previously diluted in lysis buffer at a dilution rate of 1:1 (v:v). Incubation with protein G-sepharose was

1456

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

maintained for 1 h at 4 °C in continuous shaking by using an orbital shaker. After this, samples were again centrifuged at 12,000 g for 10 min at 4 °C and the resultant supernatants and pellets were separated, reserving the supernatants obtained as controls for the immunoprecipitation effectiveness. The pellets obtained were resuspended in 1 mL of lysis buffer and were then centrifuged at 12,000 g for 10 min at 4 °C. The supernatants were discarded, and the pellets were subjected to two more steps of resuspension-centrifugation, in order to completely wash the immunoprecipitates. The final pellets were resuspended in 1 mL of a 50 mM Tris buffer (pH 8.0) at 4 °C and a new centrifugation at 12,000 g for 10 min at 4 °C was carried out. The obtained pellets that contained the immunoprecipitated proteins were finally resuspended in 20 ␮L of a 50 mM Tris/HCl buffer (ph 7.5) added to 100 mM dithiotreitol (DTT) and 1% (w:v) SDS. At this step, samples were heated at 95 °C for 3 min and they were subsequently centrifuged at 12,000 g for 10 min at 4 °C. The obtained supernatants that contained the immunoprecipitated proteins were stored at -80 °C until their Western blot analysis. The Western blot analysis was carried out following the standard protocol of transferring the SDS-polyacrylamide gel electrophoresis (polyacrylamide gel percentage of 10%, w:v) to nitrocellulose membranes described above. Transferred samples were tested by applying an antiphosphotyrosine antibody (Chemicon International; Temecula, CA, USA) at a final dilution of 1:1,000 (v/v). Immunoreactive proteins were detected by using peroxidase-conjugated anti-rabbit secondary antibody (Amersham; Buckinghamshire, UK). The reaction was developed with an ECL-Plus detection system (Amersham). After this, the nitrocellulose membranes were stripped off by using a standard protocol and they were subsequently utilized for a new Western blot analysis, this time against either protamine-1 or histone H1 by using the appropriate specific primary antibody described above, all of them to a final dilution of 1:1,000, v/v. Care was taken in order to analyze the samples obtained after precipitation with either the antiprotamine-1 antibody or the anti-histone H1 antibody with the same anti-protamine-1 and anti-histone H1 antibodies. As described in the Western blot procedure, immunoreactive protamine 1 and histone 1 were detected by using the peroxidase-conjugated secondary antibodies specific to each primary antibody utilized (for protamine-1, rabbit anti-goat: Amersham; for histone H1, goat anti-mouse: Santa Cruz Technologies). The reaction was developed with an ECL-Plus detec-

tion system (Amersham). The intensity of the marks obtained was quantified using specific software for image analysis of blots and arrays (Multi Gauge v3.0; Fujifilm Europe; Düsseldorf, Germany), in which the background was previously standardized for all of the samples analyzed. 2.7. Determination of sperm head free cysteine radicals The first step in order to determine the levels of free cysteine radicals in boar sperm heads was the separation of sperm heads from the other spermatozoal structures. For this purpose, sperm samples were first homogenized through sonication. This was made by subjecting samples to 11 consecutive pulses of ultrasounds at maximal intensity, taking care to avoid the overheating of the sample by continuous introduction in ice. The homogenized samples were then centrifuged at 850 g for 20 min at 4 °C and the resultant supernatants, as well as the upper layer of the pellet, were discarded. Afterwards, pellets were then resuspended in 500 ␮L of PBS and the purity of the separation was determined after direct observation at 40x in a phase-contrast microscope. The purity of samples was described as the percentage of loose heads in comparison with the presence of whole, unfractioned sperm and separated tails in the sample. The mean purity percentage was above 95% of loose heads in comparison with other sperm presentations, such as intact sperm or cells with different types of tail rupture without separating the heads from their respective midpieces. The determination of the free-cysteine radical levels was performed by using the 2,2=-dipyrydil disulfide technique, as described in [38]. Briefly, the 10 ␮L aliquots of resuspended, isolated sperm heads obtained as described above were added to 990 ␮L of an aqueous solution of 0.4mM 2-2= dipyrydil disulfide. The mixture was incubated for 1 h at 37 °C and, afterwards, free cysteine levels were determined through spectrophotometric analysis at a wavelength of 343 nm. Normalization of the results was obtained through a parallel determination of the total protein content of samples by utilizing the Bradford technique [37], after applying a commercial kit (BioRad; Hercules, CA, USA). 2.8. Statistical analyses Data were analyzed by using the SAS statistical package for Windows [39]. Only data obtained after the analysis of free-cysteine radicals were subjected to statistical analysis. Thus, the number of experiments subjected to statistical analysis was eight, which came

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

from eight separate ejaculates, one per boar. The determination of putative differences among the studied phases of the freezing-thawing protocol was performed by applying the GLM procedure included in the SAS package. Normality of data was assessed by the Kolmogoroff-Smirnoff Test. This test indicates that data did not follow a normal distribution. The optimal application of the chosen statistical procedure required a previous normalization of data. For this purpose, data were normalized through an arcsin[Hx/100] transformation, “x” being the transformed data. After the transformation, no data were detected as outliers. Differences among data were considered as being significant from P ⬍ 0.05. 2.9. Suppliers All reactives were of analytical grade and came from Sigma, Merck (Darmstadt; Germany) and BioRad. 3. Results 3.1. Freezing-thawing-induced changes in the mean semen-quality parameters of boar spermatozoa The cooling phase of the freezing-thawing protocol induced several significant changes in the majority of the tested semen-quality parameters of boar spermatozoa. As shown in Table 1, cooling induced a significant (P ⬍ 0.05) decrease in the percentages of viability, total motility and ORT, which were concomitant to a significant (P ⬍ 0.05) increase in the percentage of altered acrosomes. Furthermore, freezing-thawing induced greater changes in the tested semen-quality parameters of boar spermatozoa. In this way, the percentages of viability, total motility and ORT underwent a subsequent, significant (P ⬍ 0.05) decrease when compared with samples after cooling, which were also concomitant to a further increase in the percentage of altered acrosomes, which increased from 21.7 ⫾ 1.2% in cooled samples to 48.6 ⫾ 1.9% in thawed sperm (means ⫾ SEM for eight separate experiments, see Table 1). Furthermore, staining of boar spermatozoa through the Sperm–Sus Halomax® specific DNA-fragmentation kit did not show any significant increase in the freezing-thawing-induced DNA fragmentation rate. Thus, sperm from extended samples which present distinct DNA fragmentation was practically absent (Table 1). The cooling phase of the freezingthawing process did not affect sperm DNA fragmentation (Table 1). Furthermore, subsequent freezing-thawing did not induce any significant

1457

Table 1 Effects of freezing-thawing on the mean semen-quality parameters and the percentage of cells with DNA fragmentation of boar spermatozoa.

Viability (%) Altered acrosomes (%) ORT (%) HRT (arbitrary units) Total motility (%) DNA fragmentation (%)

Extended sperm

5 °C

Frozenthawed sperm

90.7 ⫾ 1.1a 6.8 ⫾ 0.2a

78.4 ⫾ 1.3b 21.7 ⫾ 1.2b

50.2 ⫾ 1.1c 48.6 ⫾ 1.9c

90.9 ⫾ 1.3a 1.05 ⫾ 0.04a

48.8 ⫾ 2.9b 1.01 ⫾ 0.04a

32.6 ⫾ 1.7c 1.00 ⫾ 0.03a

88.2 ⫾ 1.2a 1.2 ⫾ 0.7a

64.8 ⫾ 1.6b 1.1 ⫾ 0.7a

53.9 ⫾ 1.9c 1.3 ⫾ 0.8a

The table shows means ⫾ SEM of eight separate experiments separately performed from eight ejaculates, one per boar utilized in the study. Semen quality parameters have been defined in the Materials and methods section. Extended sperm: Samples observed immediately after their arrival at the laboratory previous to their processing for the freezing-thawing protocol. 5 °C: Samples taken at the final stage of the cooling phase of the freezing-thawing protocol. Frozenthawed sperm: samples taken after the thawing of frozen samples. ORT: Results obtained in the Osmotic Resistance Test. HRT: Results obtained through the Hyperosmotic Resistance Test. Total Motility: The percentage of spermatozoa that showed a mean velocity above 10 ␮m/sec. Different superscript letters in a row indicate significant (P ⬍ 0.05) differences among groups after applying the GLM procedure included in the SAS package.

change in the percentage of spermatozoa with detectable DNA fragmentation (Table 1). 3.2. Effects of freezing-thawing on the expression and location of histone H1 of boar sperm The Western blot analysis of the boar sperm head extracts from freshly obtained ejaculates showed the presence of a specific pattern of expression. This pattern consisted of the presence of at least six majoritary bands with an approximate molecular weight of 75 kDa, 60 kDa, 55 kDa, 50 kDa, 40 kDa and 30 kDa (Fig. 1). When samples were pre-digested with DNAase, the pattern was substituted by a single band of about 25 kDa, corresponding to monomeric histone H1 (Fig. 1). This band was accompanied by another band of about 35 kDa only in samples obtained after the cooling phase of the freezing-thawing protocol. The disappearance of the majority of bands after DNAase treatment indicates that these bands were constituted by specific DNA-histones fragments of low molecular weight. Additionally, the frozen-thawed samples showed an additional band of about 35 kDa that also disappeared after DNAase

1458

kDa

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464 - DNAase

MW MW

+ DNAase

C+

C-

75 50 37

25 E

5ºC

F-T

E

5ºC F-T

Fig. 1. Western blot analysis of histone H1 in boar-sperm supernatant extracts subjected to freezing-thawing. Samples were processed as described in the Materials and methods section. Western Blot was performed utilizing samples without (⫺DNAase) and with a previous incubation with DNAase to eliminate the DNA (⫹DNAase). E: Extended samples from freshly collected ejaculates immediately before starting the freezing-thawing protocol. 5 °C: Samples taken after the refrigeration phase included in the standard freezing-thawing protocol. F-T: Samples obtained after thawing of samples subjected to the standard freezingthawing protocol. C⫹: A lane from a representative Western blot against histone H1 in mouse liver extracts after applying the same antibody as that utilised in boar sperm and carried out as a positive control of the immunological reaction. C⫺: A lane from a representative Western blot against histone H1 in a freshly obtained boar-semen sample without previous DNAase treatment and incubated with the anti-histone H1 antibody previously pre-absorbed with a commercial, specific peptide. This was considered as the negative control for the immunological reaction. The figure shows a representative image for eight separate experiments separately performed from eight ejaculates, one per boar utilized in the study. Representativeness of the images was based on the fact that all of the eight performed experiments rendered similar results. In this way we chose the Western blot that showed the minimal amount of background debris for the Figure.

incubation, thus indicating that freezing-thawing induced the appearance of a specific, DNA-histones low-molecular-weight fragment (Fig. 1).

E

5ºC

Immunocytochemistry of histone H1 in freshly obtained boar sperm showed the presence of the histone in the caudal area of the sperm head (Fig. 2). This presence takes the form of a few separate marks with a punctuated aspect. The refrigeration phase of the freezing-thawing process induced the appearance of a greater number of punctuated marks at the caudal area of the sperm head. Finally, frozen-thawed sperm showed a more spread location of histone H1, which, in some spermatozoa, attained the form of punctuated marks located at the apical sperm-head area (Fig. 2). 3.3. Effects of freezing-thawing in the phosphorylation levels of boar sperm-head protamine 1 and histone H1 Specific immunoprecipitation of protamine 1 rendered a specific mark of about 15 kDa in sperm before, during and after the freezing-thawing protocol (Fig. 3A). The analysis of tyrosine phosphorylation levels of protamine 1 in all of the conditions studied showed a lack of phosphorylation in all cases (Fig. 3B). Similar results were obtained after the specific immunoprecipitation of histone H1 (Fig. 3C,D). 3.4. Effects of freezing-thawing on the level of free cysteine radicals in boar sperm heads Sperm heads from freshly extended ejaculates showed levels of free cysteine radicals of 3.1 nmol/␮g protein ⫾ 0.5 nmol/␮g protein (means⫾S.E.M for eight separate experiments). The refrigeration phase of the freezing-thawing process did not induce any significant change of these levels (Fig. 4). On the contrary,

F-T

C-

Fig. 2. Immunocytochemical images of histone H1 location in the boar sperm-head subjected to freezing-thawing. Samples were processed as described in the Materials and methods section. E: A representative sperm head from extended samples of freshly collected ejaculates immediately before starting the freezing-thawing protocol. 5 °C: Sperm after the cooling phase of the standard freezing-thawing protocol. F-T: Sperm head from a thawed sample. C⫺: Sperm head from a fresh sample in which immunocytochemistry was carried out with a previous pre-absorption of the histone H1 antibody with the corresponding, specific synthetic peptide. Arrows indicate the presence of specific signaling for histone H1. Bars indicate a size of 3.5 ␮m. The figure shows representative images obtained from eight separate experiments separately performed from eight ejaculates, one per boar utilized in the study. The representativeness of the images chosen here was based on a careful selection of 700 – 800 heads observed per each experimental point. Election criteria were based on the obtainment of a good sagital section of the head, the obtainment of a good contrast in the staining of both DNA and histone H1 and the presence of the minimal amount of background debris.

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

IgG

E

5ºC

F-T

C-

MW B

Protamine 1 IgG

E

5ºC

F-T

C-

MW

C Histone H1 IgG

E

5ºC

F-T

C-

MW D

Histone H1 IgG

E

5ºC

F-T

MW

C-

Fig. 3. Determination of tyrosine phosphorylation levels of both protamine 1 and histone H1 in fresh and frozen-thawed boar sperm. Both freezing-thawing and specific immunoprecipitation techniques were performed as described in the Materials and methods section. Figures show the detection of both the immunoprecipitated protamine 1 (A) and histone H1 (C) and also the detection of tyrosine phosphorylation in the same immunoprecipitates corresponding to protamine 1 (B) and histone H1 (D). Arrows indicate the placement of the corresponding specific bands. Protamine 1, Histone H1: Placement in which specific signals for both protamine 1 and histone H1 should be located. IgG: Placement in which the signal for the light chain of the immunoglobulin G, utilized in the immunoprecipitation procedure, is placed. E: Extended samples of freshly collected ejaculates immediately before starting the freezing-thawing protocol. 5 °C: Sperm after the cooling phase of the standard freezing-thawing protocol. F-T: Sperm head from a thawed sample. The figure shows representative images obtained from eight separate experiments separately performed from eight ejaculates, one per boar utilized in the study. The representativeness of the images was based on the fact that all of the eight experiments performed rendered similar results. In this way we chose the Western blot that showed the minimal amount of background debris for the Figure.

freezing-thawing induced a significant (P ⬍ 0.05) increase in the sperm-head free cysteine radicals, which reached values of 6.7nmol/␮g protein ⫾ 0.8nmol/␮g protein (Fig. 4). 4. Discussion The interpretation of the results shown in this manuscript clearly shows that the histones-DNA structures that are present in the head of boar sperm undergo

alterations during the freezing-thawing process that were very similar to those previously described for the equivalent protamines-DNA structures in the same cells [7]. In this respect, results concerning Western blot analyses against histone H1 need a careful explanation. We must bear in mind that these analyses were carried out only on supernatants from sperm homogenates obtained after sonication. This indicates that the signal obtained here shows the presence of fragments of histones-DNA complexes of low molecular weight that were bound to the rest of the nuclear structure in a manner such that the mechanical traction originated by sonication was able to detach them. That these lowmolecular-weight fragments were constituted by histone H1-DNA aggregates was demonstrated when Western blot was performed in samples previously incubated with DNAase-1. This incubation separated histones from DNA, and the result was the appearance of a single band of about 20 kDa, which corresponds to monomeric histone H1, which also corresponds to the single band obtained in mouse liver extracts utilized as positive controls. The results are similar to those previously described by protamines-DNA, low-molecularweight aggregates [7]. Additionally, the freezing-thawing process also induced changes in the presence of these aggregates. In this case, the presence of a specific band of about 35 kDa only in thawed sperm, but not in samples before applying the freezing-thawing protocol and those after the refrigeration phase of the freezing-

Free cystein radicals (nmol/µg protein)

A Protamine 1

1459

8

*

6 4 2 0

Extended

5ºC

F-T

Fig. 4. Determination of total free cysteine radicals levels in isolated boar sperm heads during freezing-thawing. Processing of samples and spectrophotometric detection of free cysteine radicals levels were carried out as described in the Materials and methods section. Results are expressed as means ⫾ SEM. for eight separate experiments separately performed from eight ejaculates, one per boar utilized in the study. Extended: Extended samples of freshly collected ejaculates immediately before starting the freezing-thawing protocol. 5 °C: Samples after the cooling phase until 5 °C of the freezing-thawing protocol. F-T: frozen-thawed samples. An asterisk indicates the existence of a significant (P ⬍ 0.05) difference when compared with the other experimental points after applying the GLM procedure included in the SAS package.

1460

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

thawing protocol, is noteworthy (See Fig. 1). The presence of a specific 25-kDa protein-DNA aggregate in cells after the refrigeration step which is not digested by DNAase treatment is also noteworthy (see Fig. 1). This presence suggests a freezing-thawing linked effect on the nuclear structure that becomes evident during the cooling phase of the protocol. Furthermore, our results also indicate that the processes that affect the sperm nuclear structure during the freezing-thawing process act in a similar form on both the protamines-DNA and the histones-DNA complexes. Thus, these processes seem to be unspecific, probably linked to the strong physical changes associated with freezing-thawing. In this sense, we must remember that freezing-thawing induces strong osmotic changes that, in turn, induce a great mechanical cellular stress, based on the fast entry (or releasing) of water from the cell [40]. These fast changes in the intracellular water content would cause changes and subsequent alterations in the form and strength of all internal structures, including the nuclear organization. In this way, the results obtained after the analysis of both the sperm head-free cysteine radicals and protein phosphorylation levels would agree with this interpretation. It is evident that freezing-thawing linked alterations of the boar-sperm nuclear structure are not related to changes in the phosphorylation levels of both protamines and histones, since, in fact, these proteins were not phosphorylated and there were no changes in this lack of phosphorylation after thawing. Meanwhile, it has been described that the strength of disulfide bonds are greatly weakened when osmotic conditions are quickly and greatly modified. This phenomenon, which causes the rupture of disulfide bonds, has been described in different macromolecular systems, including cryopreservation [40,41]. Accordingly, we have observed a significant increase of sperm-head disulfide bond rupture after freezing-thawing. Summarizing, we would suggest that the osmotic-caused alterations would be one of the most important mechanisms underlying the observed structural alterations of the boar sperm nucleus during freezing-thawing. This, of course, does not preclude the existence of other alteration mechanisms. In this sense, oxidative damage induced through a freezing-thawing linked overall increase in the reactive oxygen species (ROS) can also participate as a cause of the observed effects. Taking into account that boar sperm is very sensitive to oxidative damage, in part due to its characteristic cellular membrane composition [42– 44], it would be possible that the freezing-thawing linked ROS increase could contribute to the alterations of the nuclear structure.

Notwithstanding, osmotic stress would be present as an important cause of alteration in our conditions, regardless of the concomitant action of the oxidative damage on its nuclear structure. The results concerning histone H1 immunocytochemistry merit a specific explanation by themselves. First of all, these results confirm the existence of a heterogeneous boar-sperm nuclear structure in which histones are not uniformly distributed along the entire head. The overall loosening of the histones-DNA unions during the freezing-thawing process is also confirmed by the spreading of histone-marking for the sperm nucleus slices, especially in the entire distal zone. We must remember that the lack of positive marking for histones in several nuclear areas does not necessarily imply the total absence of histone in these areas. In fact, immunocytochemistry against protamine 1 in similarly obtained sperm head slices also showed areas in which there was not positive marking against the protamine, despite the fact that some type of nuclear proteins must be present in these areas [7]. These results, then, indicate the existence of nuclear areas with different structural compactness. Nuclear areas with a highly compact structure do not allow for the penetration of the utilized antibodies. In this way, these areas would be negative for both protamine and histones marking, regardless of the technique used to detect them. On the other hand, areas with a more relaxed structure allow for the antibodies penetration, and these areas would exhibit positive marks for the nuclear proteins present there. Following this interpretation, the results obtained here confirm that freezing-thawing induces a relaxation of the nuclear structure. This relaxation will permit the spreading of histone-positive markings to areas in which no marking is observed in freshly obtained cells. Another point of interest regarding histone H1 immunocytochemistry is the observed aspect of markings, especially in freshly obtained sperm. It is noteworthy that positive marks are present as very well defined spots, which become less defined in frozen-thawed cells. This aspect is totally different from the previously published positive markings for protamine 1, which present a much less defined and amorphous aspect (see [7] and Fig. 5). This presentation would agree with the observations of O’Brien and Zini [27], who indicated that sperm nuclear histones are specifically located on telomeric sequences in human sperm. Thus, the areas in which positive, well-defined histone H1 markings are observed would be those in which the maximal presence of telomeric sequences occur. Taking into account that the

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

1461

A

C

B

Medium DNA content; medium DNA density Protamine, histones. APICAL AREA

D

Low DNA content; high DNA density Protamine?, histones? MEDIAL AREA High DNA content; low DNA density Protamine, histones. DISTAL AREA

Fig. 5. Image representation of the working hypothesis regarding a zonated structure of the boar sperm nucleus. A: representative image of a whole boar sperm-head stained through the Hoechst 33258 method, showing different intensity of staining in three separate areas. This image was taken from a negative control of the immunocytochemistry performed in this work. B: Immunocytochemistry against protamine 1 in a boar sperm-head from freshly obtained semen samples immediately. This image was taken from the work previously published in [7]. C: A representative image of the immunocytochemistry against histone H1 in sperm heads from freshly obtained samples. This immunocytochemistry is that which has been performed in this manuscript. D: General scheme of the boar-sperm nucleus zonated structure, as developed in the Discussion section. Bars of the image indicate a size of 3.5 ␮m. The Hoechst 33258 image is representative for different experiments performed in the present work and in others previously published [7,45]. The representativeness of the images chosen here was based on a careful selection of 700 – 800 heads observed per experimental point both in the cases of histone H1 and protamine 1. In the case of the DNA staining alone the number of observed heads was of about 500 – 600. Election criteria were based on the obtainment of a good sagital section of the head, the obtainment of a good contrast in the staining of DNA, histone H1 and protamine 1 in [7] and on the presence of the minimal amount of background debris.

histone-positive markings are mainly present in the caudal area of the sperm head, we would assume that telomeric sequences would be mainly present in this area, thus reinforcing the existence of a heterogeneous structural distribution of the boar sperm-head. Moreover, the results concerning the specific DNA Hoechst 33258 staining reinforce sperm nuclear heterogeneity. Thus, the analysis of photographs, both from this work and from others previously published by us in which Hoechst 33258 staining was utilized [7,45] suggests the presence of three different areas with separate staining intensity. These areas are an apical area, with a medium staining inten-

sity, another medial area, with the lowest staining intensity, and a caudal area, which shows the highest Hoechst 33258 staining intensity (see [7,45] and Figs. 2 and 5). Of course, the results observed with the Hoechst 33258 staining are not totally conclusive, since the intensity of the DNA staining is modified by the precise protocol utilized (specific nuclear staining, type of detergent, etc.). This weakens a more exact explanation of data. In this way, the differences that can be observed in the images shown in this manuscript regarding points like intensity of DNA stain or width of the separated nuclear areas can be explained by the inherent technical

1462

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

variability linked to both the immunocytochemistry technique and observation through the confocal laser microscope. Thus, the width of the observed nuclear areas will vary depending on the exact angle in which the sperm head was localized on the slide. The confocal laser microscope will draw a 2-dimensional projection of the 3-dimensional nucleus image, and the 2-dimensional projection will be slightly different when comparing two nuclei if the longitudinal axles of these nuclei are located in different angles, with respect to the main horizontal plane. Since there is no possibility to place all of the sperm in the same longitudinal axle, the existence of width differences among sperm is unavoidable. Furthermore, it is possible that there were also individual differences in width among spermatozoa, but these putative individual differences will be added to that induced by the methodology utilized. Regarding differences in the intensity of the stain, this can be caused by the combination of two separate technical aspects. First, the unavoidable, slight differences in the exact amount of stain and the incubation time which will yield intensity differences among samples, especially if the observation system is highly sensitive, as the confocal laser microscope is. Second, the confocal laser microscope works with digitally obtained images in which we modulate intensities by taking the obtained luminosity of the background surrounding sperm as reference. The background luminosity changes among samples and even among separate areas of a single sample, depending on factors such as background debris, unspecific background stain, changes in the width of the coating applied to the slides, etc. Our policy is to work with the same background characteristics of both brightness and contrast, avoiding the standardization of images by taking light intensity of cells as reference. Following this policy, we must modulate background light intensity in each image, and this modulation, in turn, induces changes in the luminosity of cell stain, including nuclear stain. Notwithstanding, there are several points that can sustain the hypothesis of a heterogeneous structure of boar sperm nuclear structure, despite the methodological problems expressed. First of all, the differences in the intensity of nuclear stain indicate that the sperm nucleus does not respond uniformly to the treatment of the samples, which includes processes like permeabilization or inclusion in OCT and subsequent freezing. This indicates that the sperm nucleus has subtle differences in its structure that induce non-homogeneous responses to manipulation. These non-homogeneous responses are finally translated into different nuclear zones with separate intensity

to nuclear staining, regardless of the manipulating technique applied. Additionally, these differences have also been strengthened by the existence of separate nucleoprotein structures inside the sperm nucleus. Our results concerning immunolocation of both protamine 1 and histone H1 indicate this, thus reinforcing the hypothesis of separate zones of nuclear sperm. In fact, the nonuniform responses to nuclear staining, even with techniques different from Hoechst 33258, such as the acridine orange stain, have been observed by other authors in species such as bull and human [26,27,46,47]. We have not found data regarding boar sperm other than [7]. However, the sum of DNA stain plus the results shown here regarding histone H1 and those published before on protamine 1 [7] all suggest the expressed hypothesis. In this way, the existence of a non-homogeneous structure of nuclear sperm and, specifically, in boar cells, seems to be clear. However, although all of the data yielded both in this work and in others previously published [7,26,27,45] reinforce the existence of a heterogeneous sperm head structure, we have not found an overall hypothesis that describes this heterogeneity with more precision. Taking this into account, we suggest the following hypothesis concerning the nuclear structure of boar sperm (See Fig. 5). Boar-sperm nuclear structure is mainly formed by three well-defined, separate areas. An apical area that presents a medium DNA content and a nucleoprotein structure of medium compactness. This structure allows for the penetration of at least the anti-protamine 1 antibody. The second area is medial. This area presents the lowest DNA content and the highest compactness in the nuclear structure, which makes the penetration of antibodies greatly difficult. Finally, the third area is caudal. This presents the highest DNA content and, in turn, the lowest structural compactness, which allows for relatively easy antibodies penetration. This hypothesis is provisional and somewhat speculative, especially taking into account the methodological difficulties that the interpretation of DNA staining data poses. However, we think that the importance of the existence of a heterogeneous nucleoprotein structure, at least in species such as boar, human and bull, is important enough to merit the launching of our hypothesis. It is also evident that we can only speculate about the biological significance of this proposed structure and much more work is needed in order to evaluate the strength of the hypothesis, as well as the biological consequences of the presence of a definite structure in the boar-sperm nucleus.

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464

In summary, our results reinforce the idea that freezing-thawing causes significant alterations in the nucleoproteinic structure of the boar sperm-head much before the induction of DNA fragmentation. These alterations imply the rupture of disulfide bonds between the DNA and the nucleoproteins and they seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Finally, boarsperm nuclear structure is heterogeneous, suggesting the existence of a zonated pattern. Following this pattern, the three hypothesized zones differ each other in their total DNA amount and the compactness of the precise nucleoprotein structures present in each zone.

Acknowledgments The first two authors contributed equally to this work. We would like to thank Mr. Chuck Simmons for his accurate revision of the English grammar of this manuscript. This work has been supported by Grant AGL2008-01792GAN (Dirección General de Programas y Transferencia de Conocimiento, Ministerio de Ciencia e Innovación, Spain).

References [1] Silva PFN, Gadella BM. Detection of damage in mammalian sperm cells. Theriogenology 2006;65:958 –78. [2] Baumber J, Ball BA, Linfor JJ, Meyers SA. Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. J Androl 2003;24:621– 8. [3] Martínez-Pastor F, Johanisson A, Gil J, Kaabi M, Anel L, Paz P, et al. Use of chromatin stability assay, mitochondrial stain JC-1 and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen. Anim Reprod Sci 2004;84:121–33. [4] Evenson DP, Thompson L, Jost L. Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. Theriogenology 1994;41:637–51. [5] Fraser L, Strezezek J. Effects of freezing-thawing on DNA integrity of boar spermatozoa assessed by the Neutral Comet Assay. Reprod Domest Anim 2005;40:530 – 6. [6] Hernández M, Roca J, Ballester J, Vázquez JM, Martínez EA, Johanisson A, et al. Differences in SCSA outcome among boars with different sperm freezability. J Androl 2006;29:583–91. [7] Flores E, Cifuentes D, Fernández-Novell JM, Medrano A, Bonet S, Briz MD et al. Freeze-thawing induces alterations in the protamine-1/DNA overall structure in boar sperm. Theriogenology 2008;69:1083–94. [8] Balhorn R. The protamine family of sperm nuclear proteins. Genome Biol 2007;8:227–38. [9] Mueller RD, Yasuda H, Bradbury EM. Phosphorylation of histone H1 through the cell cycle of “Physarum polycephalum”. J Biol Chem 1985;260:5081– 6.

1463

[10] Willmitzer L, Bode J, Wagner KG. Phosphorylated protamines I: Binding stoichiometry and thermal stability of complexes with DNA. Nucl Acid Res 1977;4:149 – 62. [11] Zheng Y, John S, Pesavento JJ, Schultz-Norton JR, Schiltz RL, Baek S, et al. Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II. J Cell Biol 2010; 189:407–10. [12] Brewer L, Corzett M, Lau EY, Balhorn R. Dynamics of protamine-1 binding to single DNA molecules. J Biol Chem 2003; 278:42403– 8. [13] Dorigo B, Schalch T, Kulangara A, Duda S, Schroeder RR, Richmond TJ. Nucleosome arrays reveal the two-start organization of the chromatin fiber. Science 2004;306:1571–3. [14] Cumming RC, Andon NL, Haynes PA, Park M, Fischer WH, Schubert D. Protein disulfide bond formation in the cytoplasm during oxidative stress. J Biol Chem 2004;279:21749 –58. [15] Yang Y, Song Y, Loscalzo J. Regulation of the protein disulfide proteome by mitochondria in mammalian cells. Proc Nat Am Soc 2007;104:10813–7. [16] Hinshaw DB, Sklar LA, Bohl B, Schraufstatter IU, Hyslop PA,Rossi MW, et al. Cytoskeletal and morphologic impact of cellular oxidant injury. Am J Pathol 1986;123:454 – 64. [17] Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biol Reprod 1989;41:183–97. [18] Alvarez JG, Storey BT. Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a model of sublethal cryodamage to human sperm during cryopreservation. J Androl 1992;13:232– 41. [19] De Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa: I. Effects on the motility of intact spermatozoa and sperm axonemes. J Androl 1992;13:368 –78. [20] Holt WV, North RD. Effects of temperature and restoration of osmotic equilibrium during thawing on the induction of plasmamembrane damage in cryopreserved ram spermatozoa. Biol Reprod 1994;51:414 –24. [21] O’Flaherty C, Beconi M, Beorlegui N. Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa. Andrologia 1997;29: 269 –75. [22] Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998;13:896 –900. [23] Mazur P, Katkov I, Katkova N, Critser JK. The enhancement of the ability of mouse sperm to survive freezing and thawing by the use of high concentrations of glycerol and the presence of an Escherichia coli membrane preparation (Oxyrase) to lower the oxygen concentration. Cryobiology 2000;40:187–209. [24] Chatterjee S, Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing and thawing. Mol Reprod Develop 2001;59:451– 8. [25] Correa LM, Thomas A, Meyers SA. The macaque sperm actin cytoskeleton reorganizes in response to osmotic stress and contributes to morphological defects and decreased motility. Biol Reprod 2007;77:942–53. [26] Wykes SM, Krawetz A. The structural organization of sperm chromatin. J Biol Chem 2003;278:29471– 4. [27] O’Brien J, Zini A. Sperm DNA integrity and male infertility. Urology 2005;65:16 –22. [28] Eriksson BM, Rodríguez-Martinez H. Effect of freezing and thawing rates on the post-thaw viability of boar spermatozoa

1464

[29]

[30]

[31]

[32]

[33]

[34]

[35] [36]

[37]

[38]

E. Flores et al. / Theriogenology 76 (2011) 1450 –1464 frozen in FlatPacks and Maxi-straws. Anim Reprod Sci 2000;63:205–20. Saravia F, Wallgren M, Nagy S, Johannisson A, RodríguezMartínez H. Deep freezing of concentrated boar semen for intrauterine insemination: effects on sperm viability. Theriogenology 2005;63:1320 –33. Graham EF, Rajamannan AHJ, Schmehl MKL, Maki-Laurila M, Bower RE. Fertility studies with frozen boar spermatozoa. AI Digest 1972,19:61. Bussalleu E, Pinart E, Yeste M, Briz M, Sancho S, García-Gil N, et al. Development of a protocol for multiple staining with fluorochromes to assess the functional status of boar spermatozoa. Microsc Res Tech 2005;68:277– 83. Rodríguez-Gil JE, Rigau T. Effects of slight agitation on the quality of refrigerated boar sperm. Anim Reprod Sci 1995;39: 141– 6. Quintero-Moreno A, Rigau T, Rodríguez-Gil JE. Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis. Theriogenology 2004;61:673–90. Enciso M, López-Fernández C, Fernández JL, García P, Gosálbez AI, Gosálvez J. A new method to analyze boar sperm DNA fragmentation under bright-field or fluoresecence microscopy. Theriogenology 2006;65:308 –16. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;15:680 –5. Burnett WN. “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radiodinated protein. A. Anal Biochem 1981;112: 195–203. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 –54. Brocklehurst K, Stuchbury T, Malthouse JPG. Reactivities of neutral and cationic forms of 2,2=-dipyridyl disulphide towards

[39] [40] [41]

[42]

[43]

[44]

[45]

[46]

[47]

thiolate anions: detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe. Biochem J 1979;183:233– 8. SAS. SAS/SATC Software. Cary, NC, USA: SAS Institute Inc.;1996. Meryman HT. Osmotic stress as a mechanism of freezing injury. Cryobiology 1971;8:489 –500. Oganesyan N, Ankoudinova I, Kim SH, Kimb R. Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization. Prot Express Purif 2007;52:280 –5. Flesch M, Brouwers JF, Nievelstein PF, Verkleij AJ, van Golde LM, Colenbrander B, et al. Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane. J Cell Sci 2001;114:3543–55. Breininger E, Beorlegui NB, O’Flaherty CM, Beconi MT. Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen. Theriogenology 2005;63: 2126 –35. Holt WV, Medrano A, Thurston LM, Watson PF. The significance of cooling rates and animal variability for boar sperm cryopreservation: insights from the cryomicroscope. Theriogenology 2005;63:370 – 82. Flores E, Fernández-Novell JM, Peña A, Rigau T, RodríguezGil JE. Cryopreservation-induced alterations in boar spermatozoa mitochondrial function are related to changes in the expression and location of midpiece mitofusin-2 and actin network. Theriogenology 2010;74:354 – 63. Martic G, Karetsou Z, Kefala K, Politou AS, Clapier CR, Straub T, Papamarcaki T. Parathymosin affects the binding of linker histone H1 to nucleosomes and remodels chromatin structure. J Biol Chem 2005;16142–50. Martins CF, Dode MN, Báo SN, Rumpf R. The use of acridine orange test and the TUNEL assay to assess the integrity of freeze-dried bovine spermatozoa DNA. Genet Mol Res 2007; 6:94 –104.