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Systems Biology in Reproductive Medicine

Volume 54, 2008 - Issue 6
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Original

Presence and Release of Bovine Sperm Histone H1 During Chromatin Decondensation by Heparin-Glutathione

María Luisa Sánchez-Vázquez Laboratorio de Biología de la Reproducción, Centro de Investigación Biomédica de Oriente, Hospital General de Zona #5, Metepec, Instituto Mexicano del Seguro Social, Puebla, México
,
Juan Carlos Flores-Alonso Laboratorio de Biología de la Reproducción, Centro de Investigación Biomédica de Oriente, Hospital General de Zona #5, Metepec, Instituto Mexicano del Seguro Social, Puebla, México
,
Horacio Merchant-Larios Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, México
&
Rosalina Reyes Laboratorio de Biología de la Reproducción, Centro de Investigación Biomédica de Oriente, Hospital General de Zona #5, Metepec, Instituto Mexicano del Seguro Social, Puebla, MéxicoCorrespondencerreyesluna@hotmail.com
Pages 221-230
Received 08 Feb 2008
Accepted 17 Jun 2008
Published online: 09 Jul 2009

Original

Presence and Release of Bovine Sperm Histone H1 During Chromatin Decondensation by Heparin-Glutathione

Abstract

During spermatogenesis, changes in sperm nuclear morphology are associated with the replacement of core somatic histones by protamines. Although protamines are the major nucleoproteins of mature sperm, not all species totally replace the histones. Histone H1, along with protamines, mediates chromatin condensation into an insoluble complex that is transcriptionally inactive. In vitro, heparin-reduced glutathione causes sperm decondensation, and the structures formed are morphologically similar to the in vivo male pronucleus. To study the participation of histone H1 in bovine sperm chromatin remodelling, we measured the presence and release of histone H1 by immunofluorescence, acetic acid-urea-triton-polyacrylamide gel electrophoresis, and immunoblotting. Nuclear decondensation was induced by 80 μM heparin and 15.0 mM reduced glutathione (GSH) for 7, 14, and 21 h at 37°C. Additionally, nucleons, composed of nuclei isolated from the sperm, were decondensed with 20.0 μM heparin and 5.0 mM GSH for 4.0 h at 37°C. Controls were incubated in buffer for similar periods of time. Immunofluorescent localization of histone H1 was carried out with mouse monoclonal antibody, and DNA localization was visualized by 0.001% quinacrine staining. Chromatin decondensation was accompanied by increased sperm nuclei and nucleon surface area. We observed that histone H1 was localized exclusively in the nuclei of intact sperm and nucleons. Histone H1 immunofluorescent intensity did not change in control samples but decreased over time in samples treated with heparin-GSH. There was a negative correlation between the surface area of sperm nuclei and immunohistochemical intensity of histone H1 (P < 0.05). Nucleon decondensation showed a similar relationship. By electrophoresis and immunoblotting, we verified the loss of histone H1 from the sperm and nucleons and its release into the incubation media. Based on these results, we propose that histone H1 is present in the bovine sperm nuclei. H1 depletion may participate in chromatin decondensation and nuclear swelling induced by heparin-GSH.

Introduction

During spermatogenesis, the haploid genome undergoes extensive reorganization through meiosis and DNA compaction in which it is reduced to a volume of about 5% of the somatic cell nucleus. This notable repackaging event, which takes place as a multi-step process during the final stages of spermatogenesis, is achieved in part by replacing histones with protamines rich in arginine and cysteine residues [Wouters-Tyrou et al. [1998]].

Although protamines are the major nucleoproteins of mature sperm, not all species totally replace the histones during spermatogenesis. Some species, such as carp [Christensen et al. [1984]], retain histones as typical proteins in the sperm nuclei. In human and other mammals some persistence of histones occurs in mature ejaculated sperm [Fuentes-Mascorro et al. [2000]; Silvestroni et al. [1976]]. Human sperm may contain up to 15% histones [Gatewood et al. [1987]] including a telomere-binding protein complex isolated that contains a variant of histone H2B [Gineitis et al. [2000]]. The retained histones may be located within specific regions of chromatin [Li et al. [2008]] where it identifies genes that may be preferentially activated during early embryonic development [Evenson [1999]].

At fertilization, the highly condensed and transcriptionally inert chromatin of the sperm becomes remodelled into the decondensed and transcriptionally competent chromatin of the male pronucleus. Nuclear decondensation requires disulphide bond reduction [Perreault et al. [1984]; Zirkin et al. [1989]], and the process correlates with the presence of free thiol glutathione in the ooplasma [Perreault et al. [1988]; Zirkin et al. [1989]]. Based on the affinity for histones, heparin and other poly-anions have been used to study the structure of histone-DNA complexes and functions such as DNA template availability, transcription, and replication [Brotherton et al. [1989]; Delgado et al. [1984]; Villeponteau [1992]]. Courvalin et al. [[1982]] reported that heparin optimally solubilizes rat liver nuclear DNA, histone H1, and 70% of the core histone oligomers at a heparin-to-DNA ratio of 1:1. Heparin binds tightly to intact nucleosomes, predominantly through interactions with trypsin-sensitive lysine and arginine residues in histone H1 and with the N-terminal segments of the core histones [Villeponteau [1992]]. Under in vitro conditions, heparin-reduced glutathione induces sperm nuclei decondensation in six different mammalian species [Reyes et al. [1989], [1996]; Sánchez-Vázquez et al. [1996], [1998]; Delgado et al. [2001]]. The structures formed are morphologically similar to the in vivo male pronucleus [Yanagimachi [2005]; Ajduk et al. [2006]]. However, there is limited information about the presence of histone H1 in bovine sperm during in vitro decondensation. In this report we demonstrate by direct immunofluorescence, acetic acid-urea-triton-polyacrylamide gel electrophoresis (AUT PAGE) and immunoblotting, the presence of histone H1 in bovine sperm nuclei. We also show that histone H1 is released during in vitro decondensation induced by heparin-reduced glutathione.

Results

All of the control sperm cells remain condensed after 21 h in 100 mM Tris, pH 8.0, alone (Fig. 1A). Similarly, sperm cells treated with heparin alone or GSH after 21 h remain condensed. In contrast, treatment with heparin plus GSH induced decondensation of sperm nuclei (Fig. 1B). After treatment with N-cetyl-N,N,N-trimethyl ammonium bromide/DL-dithiothreitol (CTAB/DTT), the recovered nucleons preserved their original nuclear shape (Fig. 2A). The acrosome, mid piece and tail were removed (Fig. 2B). Like control sperm cells, nucleons remain condensed after 4 h in 100 mM Tris (Fig. 2C). They also remained condensed when treated with heparin or GSH alone. However treatment for 4 h with the combination of heparin plus GSH induced nucleon decondensation (Fig. 2D).

FIGURE 1 Phase-contrast microscopy of bovine sperm intact and decondensed. (A) Bovine sperm controls incubated for 21 h at 37°C in 100 mM Tris, pH 8.0, retained the structural organization of freshly isolated sperm. (B) Sperm treated for 21 h at 37°C in 100 mM Tris, pH 8.0, containing 80 μM heparin and 15 mM GSH contained cells in Phases I, II, and III of nuclear decondensation.

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FIGURE 2 Bovine sperm nucleon. (A) As viewed by phase-contrast microscopy, the original nuclear shape was retained after isolation in CTAB/DTT. (B) By electron microscopy, there was a complete absence of acrosome, perinuclear theca, mid piece, and tails from each nucleon. (C) Sperm nucleon controls incubated for 4 h at 37°C in 100 mM, Tris, pH 8.0, showed no evidence of decondensation. (D) Nucleons underwent similar phase changes as when sperm cells treated with 20 μM heparin and 5 mM GSH for 4 h.

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We identified three nuclear decondensation phases based on surface area of the sperm heads and nucleons. The percent of sperm nuclei in phase I was highest at 7 h. Between 7 and 21 h, there was a progression of nuclei decondensation through phases I–III (Fig. 3A). At 21 h of incubation, 98% of the sperm were in phase I, II, or III of decondensation and 80% of these were in phase III (Fig. 3A). Nucleon decondensation took less time than sperm decondensation. By 4 h, 95% of the nucleons were decondensed, with 64% being in phase III (Fig. 3B).

FIGURE 3 Heparin-GSH time-dependent sperm and nucleon decondensation. Each bar represents the percent of (A) sperm or (B) nucleons in decondensation phases I, II and III after each incubation period. The data represent the mean±SD of 7 different experiments. P < 0.05.

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Bovine oocytes served as positive controls for the immunohistochemical localization of histone H1. Nuclear immunofluorescence was clearly detected (Fig. 4A inset). Immunohistochemical staining of intact bovine sperm and nucleons showed the nuclear localization of histone H1 (Fig. 4A, B). The immunofluorescence of histone H1 was essentially the same when the cells were treated 24 h and 4 h, respectively, with heparin or GSH (data not shown). After a 21 h treatment with heparin and GSH, the sperm in phase I decondensation lost 20% of their fluorescence intensity (Fig. 4C, Table 1). For sperm cells in phase II, 42% of the staining intensity was lost (Fig. 4C, Table 1), and 75% was lost in phase III (Fig. 4C, Table 1). Similar results were obtained for nucleons incubated for 4 h at 37°C in 100 mM Tris, pH 8.0, containing 20 μM heparin and 5 mM GSH (Fig. 4D, Table 2). There was a negative correlation between the area of the decondensed nuclei and the histone H1 immunohistochemical staining intensity (r2=0.97, p < 0.05; Fig. 5A). Similar results were obtained for nucleons after 4 h of treatment with heparin-GSH (r2=0.91, p < 0.05; Table 2).

FIGURE 4 Immunofluorescent localization of histone H1. Confocal images show that the somatic histone H1 antibody is localized to sperm nuclei and nucleons. (A inset) Bovine oocytes, used as positive controls, showed histone H1 antibody localized to the nucleus. Similarly, histone H1 antibody in intact sperm (A) and nucleon (B) was localized to the nucleus. (C) The immunofluorescent intensity of histone H1 decreased as sperm nuclei decondensed from phase I, condensed II and finally III decondensed. (D) Nucleon decondensation exhibited similar decreased histone H1 immunofluorescence.

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FIGURE 5 Sperm decondensation and immunofluorescent localization of H1. (A) There was a significant negative correlation between the mean nuclear area and the mean histone H1 antibody fluorescence intensity for cells in each of the phases of decondensation (white line, r2=0.97). The DNA fluorescence intensity (quinacrine mustard) remained constant during each phase of decondensation (yellow line, r2=0.11). A similar correlation was observed between area and histone H1 antibody immunofluorescence intensity in nucleons (r2=0.91, p < 0.05). (B) Control sperm cells were incubated in 100 mM Tris, pH 8.0, for 21 h while others (C) were decondensed for 21 h. Both were treated with histone H1 antibody and counter-stained with 0.001% quinacrine to visualize DNA. Loss of histone H1 is marked by the diminished immunofluorescence (red) in cells designated as phases I, II and III of decondensation.

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Decondensation-Dependent Changes in Surface Area, Histone H1 and DNA Localization in Bovine Spermatozoa

Decondensation-Dependent Changes in Surface Area. Localization of Histone H1 and DNA in Nucleons

When histone H1 immunostaining was followed by quinacrine staining, the nuclei of control cells (Fig. 5B) and cells treated with heparin-GSH for 21 h (Fig. 5C) were orange. Thus quinacrine staining remained constant (Table 1, Fig. 5A), indicating that while histone H1 was lost during nuclear decondensation, DNA remained constant.

Acetic acid-urea-triton-polyacrylamide gel electrophoresis (AUT PAGE) analysis from intact and decondensed bovine sperm and nucleons revealed one 20.8 kDa histone (Fig. 6A). The band was present in samples from intact whole sperm after 21 h of incubation in control medium (Fig. 6A, lane 3). In comparison, it was absent in the incubation medium (Fig. 6A, lane 2). After sperm decondensation for 21 h, histone H1 was absent from the sperm (Fig. 6A, lane 5); however it was present in the incubation medium (Fig. 6A, lane 4) suggesting that histone H1 was released into the medium. Histone H1 was distributed in a similar manner with intact and decondensed nucleons (data not shown). Western blot analysis of both sperm and nucleons (Fig. 6B) confirmed these observations.

FIGURE 6 Detection of histone H1 in bovine sperm chromatin. (A) Acid-Urea-Triton-Polyacrylamide gel electrophoresis (AUT-PAGE) of acid extracted histone H1: lane 1, histone H1; lane 2, incubation medium of whole intact sperm; lane 3, whole intact sperm; lane 4, incubation medium of decondensed sperm; lane 5, decondensed sperm. (B) Western blot analysis of histone H1 transferred to a nitrocellulose membrane. The antibodies showed identical immunoreactivity with the histone H1 standard (lane 1), intact sperm (lane 3) and the incubation medium of decondensed sperm (lane 4). The same histone profile was obtained from intact and decondensed nucleons and incubation medium (data not shown).

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Protein quantitation of the histone extracts from the intact sperm yielded 0.43 pg/sperm. After decondensation for 21 h, the histone content was 0.07 pg/sperm and 0.35 pg in the incubation medium. Thus treatment with heparin-GSH released 83% of histone H1.

Discussion

Several reports concerning the presence of histones in mammalian sperm have been published. Based on high performance liquid chromatography, Gatewood et al. [[1990]] identified H2A variants in human sperm as H2A.X and H2A.Z. They proposed that this chromatin component facilitates the programming of genes that are active in early development. Tovich and Oko [[2003]] presented biochemical and ultrastructural evidence for the presence of stable, non-nuclear somatic-type core histones H3, H2B, H3A, and H4 in mature bovine sperm. The synthesis of these histones occurs in the late stage spermatid [Tovich et al. [2004]] and they are confined to the post-acrosomal sheath of the perinuclear theca. The localization of somatic histones in the perinuclear theca mechanistically favors a role in sperm-mediated gene transfer [Lavitrano et al. [1989]]. In our study, we observed that histone H1, as detected by specific histone H1 antibody immunofluorescence was confined to the nucleus. Thus, these histones, which are somatic in nature, may be synthesized de novo as are other histones in the late stage of spermatid formation. We speculate that histone H1 stabilizes the sperm DNA structure contributing to chromosomal condensation.

The remodelling of sperm chromatin occurs after several interdependent events during spermatogenesis. The nuclei become fully condensed due to the partial replacement of somatic histones by protamines [Dadoune [2003]; Palmer et al. [1991]]. The retention of nuclear histone H1 is consistent with the in vitro observation that decondensed bull sperm retain 54.0% of the original haploid level of histones [Zakhidov et al. [1985]]. Even though the exact role of histones is well known, the temporary accumulation of histone H1 in mature bovine sperm might indicate its potential involvement in fertilization.

Sulphated glycosaminoglycans (GAGs), including heparin, play important roles during fertilization. They are present in both male and female reproductive tracts, in reproductive secretions, and in both gametes [Reyes et al. [2004]; Binette et al [1996]; Boushehri et al. [1996]; Sloan et al. [1996]]. Under physiological conditions, GAGs bind to specific receptors in the sperm plasma membrane [Peluso et al. [1992]; Sánchez-Prieto et al. [1996]], induce capacitation [Medeiros and Parrish [1996]; Miller et al. [1990]; Reyes et al. [1989]] and the acrosome reaction [Reyes et al. [1984]; Delgado et al. [1988]]. Moreover, they induce the release of basic nucleoproteins [Reyes et al. [1991]] and facilitate nuclear swelling [Delgado et al. [1999], Romanato et al. [2003], [2005]]. The observations reported in this study are consistent with these known roles of GAGs in fertilization.

Solubilization of sperm nuclei due to the release of histone H1 by heparin is a prerequisite for chromatin decondensation after the oocyte fertilization [Courvalin et al. [1982]]. This is consistent with and is likely facilitated by the GAGs, including heparin, in mammalian oocytes [Reyes et al. [2004]; Romanato et al. [2008]]. In this study we showed that histone H1 is present in the bull sperm nucleus and is released during nuclear decondensation mediated by the heparin-GSH. After decondensation, the amount of histone H1 that remains in sperm represents 17% of the original.

Our previous results [Delgado et al. [2001]; Reyes et al. [1996]; Sánchez-Vázquez et al. [1996]] and those presented here strengthen the hypothesis that GSH induces disulphide bound reduction between protamines, while the heparin induces sperm nuclei swelling by mediating the release of histone H1. Therefore, we propose that the depletion of histone H1 in bull sperm nuclei is part of nuclear decondensation and swelling that occurs during the remodelling of paternal chromatin after the oocyte fertilization.

Materials and methods

All reagents were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise noted.

Bovine Sperm Cells

Bovine epididymides were obtained at the local abattoir at Atlixco, Puebla, México and transported to the laboratory in 0.9% NaCl at 35–37°C. Sperm cells were obtained by flushing the lumen of Cauda epididymis with TALP medium (114.0 mM NaCl, 3.2 mM KCl, 25.0 mM NaHCO3, 0.4 mM NaH2PO4, 10.0 mM Na-Lactate, 0.5 mM Na-Pyruvate, 2.0 mM CaCl2, 0.5 mM MgCl2, pH 7.4) at 37°C. To diminish inter-donor variability, sperm cells from five different animals were pooled and used for individual experiments. These samples were washed two times by centrifugation at 600×g for 10 min and then resuspended and diluted if necessary to obtain a final concentration 1.67×108 cells/ml in 100 mM Tris, pH 8.0.

Bovine Sperm Nuclei Preparation

To isolate nucleons from sperm nuclei [Delgado et al. [1999]], aliquots of 108 cells/0.6 ml sperm suspension were treated with 0.2 ml of 9.0 mM freshly prepared DL-dithiothreitol (DTT, Merck, Darmstadt, Germany) in 50 mM Tris-HCl, pH 8.0, and allowed to stand for 18 min at room temperature. At the end of this period, 0.2 ml of 1% N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB, Merck, Darmstadt, Germany) was added and the mixture was allowed to stand for 15 min [Delgado et al. [2001]]. The CTAB is a cationic detergent that induces micelle formation with the membrane lipids while maintaining the nuclei intact. The nucleons were immediately centrifuged at 600×g for 10 min then washed twice by resuspension and centrifugation with 50.0 mM Tris-HCl, pH 8.0. Nucleon purity was observed by phase contrast microscopy using a Nikon microscope (E-600, Técnica en Laboratorios, S. A., Distrito Federal, México).

Electron Microscopy

The nucleons were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 1 h [Zetterqvist [1956]]. The samples were washed twice by centrifugation and resuspension with 0.1 M cacodylate buffer alone and post-fixed in 1% OsO4 for 2 h. This was followed by dehydration in ascending ethanol concentrations and embedding in epon [Luft [1981]]. Thin sections were stained with uranyl acetate/lead citrate [Reynolds [1963]] and observed using an electron microscope (JEM 1010, JEOL Ltd, Tokyo, Japan).

Sperm Cell and Nucleon Decondensation by Heparin-Glutathione (GSH)

Sperm decondensation was achieved by incubation of 108 cells/ml with 80.0 μM heparin and 15.0 mM GSH dissolved in 100 mM Tris, pH 8.0, for either 7, 14 or 21 h at 37°C. Controls included incubation in (a) buffer, (b) 80.0 μM heparin without GSH, and (c) 15.0 mM GSH without heparin. Nucleon decondensation was achieved in a similar manner with 108 nucleons/ml with 20.0 μM heparin and 5.0 mM GSH dissolved in 100 mM Tris, pH 8.0, buffer for 4 h at 37°C. Controls included (a) buffer alone, (b) 20.0 μM heparin without GSH, and (c) 5.0 mM GSH without heparin.

Evaluation of Decondensed Sperm Nuclei

From seven different experiments, sample smears of control and decondensed sperm nuclei at each time and treatment point were prepared and dried on glass microscope slides. The DNA was stained with 50.0 μl 0.001% quinacrine mustard solution for 20 min in a dark humid chamber. Nuclear decondensation was observed using a Nikon confocal microscope as described above. Fluorescence was detected upon excitation with a helio-neon laser at 488 nm. Using the confocal system imaging software, the total surface area (μm2) and the quinacrine fluorescence intensity were measured for control and experimental sperm and nucleons. Fluorescence intensity was calculated as the mean pixel intensity after subtracting the background. For each incubation period, decondensation of sperm nuclei and nucleons was evaluated and classified by the surface area as follows: Phase I, 31.0–50.0 μm2, Phase II, 51.0–100.0 μm2, and Phase III, >100.0 μm2. Nuclei and nucleons with surface areas less than 31 μm2 were classified as condensed.

Direct Immunofluorescence Localization of H1

The anti-H1 histone antibody specificity was assessed using mature bovine oocytes. The oocytes were obtained as described by Flores-Alonso et al. [[2008]] then permeabilized with 1% Triton X-100 in PBS for 10 min. They were washed and incubated for 30 min in 10 μl (2 μg) undiluted mouse monoclonal H1 antibody (Histone H1 Ab-1, Clone 1415-1, Fisher Scientific, Fremont, CA. USA), using the manufacturer's instructions (Neomarkers, Fremont, CA, USA).

Slides with control or treated bovine sperm were washed with phosphate buffered saline (PBS), fixed for 5 min with ice-cold methanol, washed, then allowed to air dry. The specimens were incubated at room temperature for 45 min with 10 μl anti-H1 histone antibody and washed with PBS. Additionally, in some samples the DNA was counter-stained with 0.001% quinacrine mustard solution. The quinacrine and histone H1 antibody fluorescence intensity were analyzed by confocal microscopy as described above. Fluorescence was detected upon excitation with a helio-neon laser at 488 nm for the quinacrine-labelled DNA and 633 nm for the antibody-labelled H1.

Histone Extraction, Electrophoresis and Western Blotting

Aliquots of 108 bovine intact sperm, nucleons and sperm or nucleons treated 21 h and 4 h respectively with heparin-GSH were centrifuged at 5,000 rpm for 15 min at 4°C. The cell pellets were resuspended in 1 ml of 7 M urea, 3 M NaCl and 0.35 M 2-mercaptoethanol, then gently shaken. To extract histones from the sperm and nucleons, 1/4 volume of cold 1.0 N H2SO4 was slowly added and the resulting mixture allowed to stand for 30 min on ice with stirring. The cells and incubation media were centrifuge at 12,000 rpm for 20 min at 4°C.

To precipitate the histones, four volumes of cold absolute ethanol were added at each extraction and stored for 24 h at −20°C. The precipitates were collected by centrifugation at 2,000 rpm for 30 min, and the pellets were dissolved in 50 μl PBS added with 2 μl 2-mercaptoethanol and 12.5 μl sample buffer (0.9N acetic acid, 1.0 M urea, 0.1 M 2-mercaptoethanol, 15% sucrose, 0.02% pyronine Y). The total protein concentration of all samples was determined by the Bradford method [[1976]] and analyzed by AUT PAGE as previously described [Bonner et al. [1980]]. All gels were stained with 0.1% Coomassie brilliant blue R in 7.5% acetic acid/40% methanol for protein band detection. The gels were destained in 7.5% acetic acid/30% methanol/62.5% H2O.

Migration was compared with 10 μg of histone H1 (Histone type I, Sigma H4524). For Western blotting, the proteins from intact and decondensed whole sperm and nucleons were immobilized on nitrocellulose membrane then probed with affinity purified histone H1 antibody. Nonspecific sites were blocked with 5% nonfat dried milk in 0.3% Tween-20 in PBS for 2 h at room temperature. Undiluted primary antibody (histone H1 Ab-1, 200 μg/ml) was incubated overnight at 4°C. The membrane was washed twice with 0.3% Tween-20 in PBS. Alkaline phosphatase-conjugated anti-mouse IgG, (Jackson ImmunoReserch West Grove, PA, USA) was used for secondary incubations. Finally, the second antibody was washed and a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate was added to color development [Sambrook et al. [1989]].

Statistical Analysis

Morphometric and fluorescence data were analyzed by Sigma Stat 3.1 (SYSTAT Software, Inc., Chicago, IL, USA). Values were expressed as means±standard deviations, and Student's t-test (two tailed) was applied. Differences of p < 0.05 were significant. The correlation coefficient between the area of the decondensed sperm or nucleons and the H1 immunohistochemical staining intensity was assessed by linear regression analysis.

Acknowledgments

We are grateful to M. V. Z. Juan Manuel Ayestaran Nava and M. V. Z. Eduardo Cabrera Bautista of the Rastro Municipal de Atlixco, Puebla, México, for the bovine epididymis and to Dr. Gerardo Santos for his assistance on the western blot technique.

References

  • Ajduk A., Yamauchi Y., Ward M. A. Sperm chromatin remodeling after intracytoplasmic sperm injection differs from that of in vitro fertilization. Biol Reprod 2006; 75: 442–451 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Binette J. P., Ohishi H., Burgi W., Kimura A., Suyemitsu T., Seno N., Schmid K. The content and distribution of glycosaminoglycans in the ejaculates of normal and vasectomized men. Andrologia 1996; 28: 145–149 [Google Scholar]
  • Bonner W. M., West M. H., Stedman J. D. Two-dimensional gel analysis of histones in acid extracts of nuclei, cells, and tissues. Eur J Biochem 1980; 109: 17–23 [Google Scholar]
  • Boushehri I., Yadav M. C., Meur S. K. Characteristic of proteoglycans of buffalo ovarian follicular fluid during maturation of follicles. Indian J Biochem Biophys 1996; 33: 213–217 [Google Scholar]
  • Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Brotherton T. W., Jagannadham M. V., Ginder G. D. Heparin binds to intact mononucleosomes and induces a novel unfolded structure. Biochemistry 1989; 28: 3518–3525 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Christensen M. E., Rattner J. B., Dixon G. H. Hyperacetylation of histone H4 promotes chromatin decondensation prior to histone replacement by protamines during spermatogenesis in rainbow trout. Nucleic Acids Res 1984; 12: 4575–4592 [Google Scholar]
  • Courvalin J. C., Dumontier M., Bornens M. Solubilization of nuclear structures by the polyanion heparin. J Biol Chem 1982; 257: 456–463 [Google Scholar]
  • Dadoune J. P. Expression of mammalian spermatozoal nucleoproteins. Microsc Res Tech 2003; 61: 56–75 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Delgado N. M., Flores-Alonso J. C., Rodríguez-Hernández H. M., Merchant-Larios H., Reyes R. Heparin and glutathione II: Correlation between decondensation of bull sperm cells and its nucleons. Arch Androl 2001; 47: 47–58 [Taylor & Francis Online], [Google Scholar]
  • Delgado N. M., Magdaleno V. M., Merchant H., Rosado A., Reyes R. Heparin-induced release of DNA template restrictions in human sperm zinc-depleted nuclei. Arch Androl 1984; 12: 211–216 [Taylor & Francis Online], [Google Scholar]
  • Delgado N. M., Reyes R., Mora-Galindo J., Rosado A. Size-uniform heparin fragments as nuclear decondensation and acrosome reaction inducers in human spermatozoa. Life Sci 1988; 42: 2177–2183 [Google Scholar]
  • Delgado N. M., Sánchez-Vázquez M. L., Reyes R., Merchant-Larios H. Nucleons I: A model for studying the mechanism of sperm nucleus swelling in vitro. Arch Androl 1999; 43: 85–95 [Taylor & Francis Online], [Google Scholar]
  • Evenson D. P. Alterations and damage of sperm chromatin structure and early embryonic failure. Towards Reproductive Certainty: Fertility and Genetics Beyond 1999 The Plenary Proceedings of the 11th World Congress, R. Jansen, D. Mortimer. Taylor & Francis, New York 1999; 313–329 [Google Scholar]
  • Flores-Alonso J. C., Lezama-Monfil L., Sánchez-Vázquez M. L., Reyes R., Delgado N. M. Heparin effect on in vitro nuclear maturation of bovine oocytes. Zygote 2008; 16: 1–8 [Google Scholar]
  • Fuentes-Mascorro G., Serrano H., Rosado A. Sperm chromatin. Arch Androl 2000; 45: 215–225 [Taylor & Francis Online], [Google Scholar]
  • Gatewood J. M., Cook G. R., Balhorn R., Bradbury E. M., Schmid C. W. Sequence-specific packaging of DNA in human sperm chromatin. Science 1987; 236: 962–964 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Gatewood J. M., Cook G. R., Balhorn R., Schmid C. W., Bradbury E. M. Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones. J Biol Chem 1990; 265: 20662–20666 [Google Scholar]
  • Gineitis A. A., Zalenskaya I. A., Yau P. M., Bradbury E. M., Zalensky A. O. Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment. J Cell Biol 2000; 151: 1591–1598 [Google Scholar]
  • Lavitrano M., Camaioni A., Fazio V. M., Dolci S., Farace M. G., Spadafora C. Sperm cells as vectors for introducing foreign DNA into eggs: Genetic transformation of mice. Cell 1989; 57: 717–723 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Li Y., Lalancette C., Miller D., Krawetz S. A. Characterization of nucleohistone and nucleoprotamine components in the mature human sperm nucleus. Asian J Androl 2008; 10: 535–541 [Google Scholar]
  • Luft J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 1981; 9: 409–414 [Google Scholar]
  • Medeiros C. M., Parrish J. J. Changes in lectin binding to bovine sperm during heparin-induced capacitation. Mol Reprod Dev 1996; 44: 525–532 [Google Scholar]
  • Miller D. J., Winer M. A., Ax R. L. Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol Reprod 1990; 42: 899–915 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Palmer D. K., O'Day K., Trong H. L., Charbonneau H., Margolis R. L. Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc Natl Acad Sci USA 1991; 88: 3734–3738 [Google Scholar]
  • Peluso J. J., Luciano A. A., Nulsen J. C. The relationship between alterations in spermatozoa deoxiribonucleic acid, heparin binding sites and semen quality. Fertil Steril 1992; 57: 665–670 [Google Scholar]
  • Perreault S. D., Barbee R. R., Slott V. L. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol 1988; 125: 181–186 [Google Scholar]
  • Perreault S. D., Wolff R. A., Zirkin B. R. The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev Biol 1984; 101: 160–167 [Google Scholar]
  • Reyes R., Carranco A., Hernández O., Rosado A., Merchant H., Delgado N. M. Glycosaminoglycan sulfate as acrosomal reaction-inducing factor of follicular fluid. Arch Androl 1984; 12: 203–209 [Taylor & Francis Online], [Google Scholar]
  • Reyes R., Carranco A., Huacuja L., Delgado N. M. Male pronuclei formation release of phosphorylation of histone H-3 during decondensation of human sperm nuclei activated in vitro by heparin. Arch Androl 1991; 26: 53–60 [Taylor & Francis Online], [Google Scholar]
  • Reyes R., Ramírez G., Delgado N. M. Fluorescent berberine binding as a marker of internal glycosaminoglycans sulfate in bovine oocytes and sperm cells. Arch Androl 2004; 50: 327–332 [Taylor & Francis Online], [Google Scholar]
  • Reyes R., Rosado A., Hernández O., Delgado N. M. Heparin and glutathione: Physiological decondensing agents of human sperm nuclei. Gamete Res 1989; 23: 39–47 [Google Scholar]
  • Reyes R., Sánchez-Vázquez M. L., Merchant-Larios H., Rosado A., Delgado N. M. Effect of heparin-reduced glutathione on hamster sperm DNA unpacking and nuclear swelling. Arch Androl 1996; 37: 33–45 [Taylor & Francis Online], [Google Scholar]
  • Reynolds E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 1963; 17: 208–212 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Romanato M., Cameo M. S., Bertolesi G., Baldini C., Calvo J. C., Calvo L. Heparan sulphate: a putative decondensing agent for human spermatozoa in vivo. Hum Reprod 2003; 18: 1868–1873 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Romanato M., Julianelli V., Zappi M., Calvo L., Calvo J. C. The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo. Hum Reprod 2008; 23: 1145–1150 [Google Scholar]
  • Romanato M., Regueira E., Cameo M. S., Baldini C., Calvo L., Calvo J. C. Further evidence on the role of heparan sulfate as protamine acceptor during the decondensation of human spermatozoa. Hum Reprod 2005; 20: 2784–2789 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Sambrook J., Fritsch E. F., Maniatis T. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New York, USA 1989 [Google Scholar]
  • Sánchez-Prieto J., González J., Illera M. J., Lorenzo P. L., Orensanz L. M. [3H]heparin binding in boar spermatozoa: Characterization and correlation with routine semen quality parameters. Biol Reprod 1996; 55: 860–867 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
  • Sánchez-Vázquez M. L., Reyes R., Merchant-Larios H., Rosado A., Delgado N. M. Differential decondensation of class I (rat) and class II (mouse) spermatozoa nuclei by physiological concentrations of heparin and glutathione. Arch Androl 1996; 36: 161–176 [Taylor & Francis Online], [Google Scholar]
  • Sánchez-Vázquez M. L., Reyes R., Ramírez G., Merchant-Larios H., Rosado A., Delgado N. M. DNA unpacking in guinea pig sperm chromatin by heparin and reduced gluthatione. Arch Androl 1998; 40: 15–28 [Taylor & Francis Online], [Google Scholar]
  • Silvestroni L., Frajese G., Fabrizio M. Histones instead of protamines in terminal germ cells of infertile, oligospermic men. Fertil Steril 1976; 27: 428–437 [Google Scholar]
  • Sloan R., Archbold G. P., Lloyd F., Elliott R. J. Glycosaminoglycans-sulfate accumulation in amniotic fluid during early pregnancy. Biochem Soc Trans 1996; 24: 1035 [Google Scholar]
  • Tovich P. R., Oko R. J. Somatic Histones are components of the perinuclear theca in bovine spermatozoa. J Biol Chem 2003; 278: 32431–32438 [Google Scholar]
  • Tovich P. R., Sutovsky P., Oko R. J. Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis. Biol Reprod 2004; 71: 1182–1194 [Google Scholar]
  • Villeponteau B. Heparin increases chromatin accessibility by binding the trypsin-sensitive basic residues in histones. Biochem J 1992; 288: 953–958 [Google Scholar]
  • Wouters-Tyrou D., Martinage A., Chevaillier P., Sautière P. Nuclear basic proteins in spermiogenesis. Biochimie 1998; 80: 117–128 [Crossref], [Web of Science ®], [Google Scholar]
  • Yanagimachi R. Intracytoplasmic injection of spermatozoa and spermatogenic cells: Its biology and applications in humans and animals. Reprod Biomed Online 2005; 10: 247–288 [Google Scholar]
  • Zakhidov S. T., Boronchuk G. V., Nauk V. A., Brodskiiˇ V. Quantitative changes in histones during the formation of structures characteristic of the male pronucleus in mammalian spermatozoa in vitro. Ontogenez 1985; 16: 73–75 [Google Scholar]
  • Zetterqvist, H. (1956). The structural organization of columnar absorbing cells of the mouse jejunum. Thesis, Karolinska, Institute, Stockholm. [Google Scholar]
  • Zirkin B. R., Santulli R., Awoniyi C. A., Ewing L. L. Maintenance of advanced spermatogenic cells in the adult rat testis: Quantitative relationship to testosterone concentration within the testis. Endocrinology 1989; 124: 3043–3049 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
 

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