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BOR - Papers in Press, published online ahead of print May 2, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.060244
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BIOLOGY OF REPRODUCTION 77, 303–311 (2007)
DOI: 10.1095/biolreprod.107.060244
© 2007 by the Society for the Study of Reproduction, Inc.

Chronic Cyclophosphamide Exposure Alters the Profile of Rat Sperm Nuclear Matrix Proteins1

Alexis M. Codrington 3, Barbara F. Hales 2 3, and Bernard Robaire 3 4

Departments of Pharmacology and Therapeutics3 and Obstetrics and Gynecology,4 McGill University, Montreal, Quebec, Canada H3G 1Y6

ABSTRACT

Chronic exposure of male rats to the alkylating agent cyclophosphamide, a well-known male-mediated developmental toxicant, alters gene expression in male germ cells as well as in early preimplantation embryos sired by cyclophosphamide-exposed males. Sperm DNA is organized by the nuclear matrix into loop-domains in a sequence-specific manner. In somatic cells, loop-domain organization is involved in gene regulation. Various structural and functional components of the nuclear matrix are targets for chemotherapeutic agents. Consequently, we hypothesized that cyclophosphamide treatment would alter the expression of sperm nuclear matrix proteins. Adult male rats were treated for 4 wk with saline or cyclophosphamide (6.0 mg kg–1 day–1), and the nuclear matrix was extracted from cauda epididymal sperm. Proteins were analyzed by two-dimensional gel electrophoresis. Identified proteins within the nuclear matrix proteome were mainly involved in cell structure, transcription, translation, DNA binding, protein processing, signal transduction, metabolism, cell defense, or detoxification. Interestingly, cyclophosphamide selectively induced numerous changes in cell defense and detoxification proteins, most notably, in all known forms of the antioxidant enzyme glutathione peroxidase 4, in addition to an uncharacterized 54-kDa form; an overall increase in glutathione peroxidase 4 immunoreactivity was observed in the nuclear matrix extracts from cyclophosphamide-exposed spermatozoa. An increase in glutathione peroxidase 4 expression suggests a role for this enzyme in maintaining nuclear matrix stability and function. These results led us to propose that a change in composition of the nuclear matrix in response to drug exposure was a factor in altered sperm function and embryo development.

chemotherapeutic agent,, glutathione peroxidase 4,, infertility,, nuclear matrix,, proteomics,, sperm,, toxicology

INTRODUCTION

Chromatin structural organization has a significant impact on cell function. Two levels of organization within somatic nuclei, nucleosomal and DNA loop-domain organization, play a role in gene regulation [1]. The formation of DNA loop-domains is of particular interest, as DNA is attached in a sequence-specific manner to the nuclear matrix, the nonchromatin structure of the nucleus [2]. The nuclear matrix consists of an internal ribonucleic protein network and residual nucleoli bounded by peripheral lamins [3, 4]; its interaction with DNA has been implicated in many essential nuclear functions, including DNA replication and repair, transcription, and RNA processing and transport [5–8].

The relationship between nuclear function and organization has been well established in somatic cells [9]; however, the functional significance of sperm structural organization remains elusive. Spermatozoal chromatin is not organized into nucleosomes; protamines replace histones during spermiogenesis, resulting in highly condensed toroids [10]. Interestingly, sperm do maintain the organization of DNA by the nuclear matrix into loop-domains [11]. In the mouse sperm nucleus, the nuclear matrix forms part of the perinuclear matrix, which consists of the surrounding perinuclear theca and a filamentous internal network [12].

Proteins that make up the nuclear matrix vary in a cell type- and tissue-specific manner and change as cells differentiate [13–16]. Changes also occur in the organization and general protein composition of the nuclear matrix during spermatogenesis [17, 18]. Altering the composition of the nuclear matrix could be associated with DNA disorganization; disrupting the association of loop-domains with the nuclear matrix may alter nuclear function [19]. Structural and functional components of the nuclear matrix are targets in somatic cells for chemotherapeutic agents. In somatic cells, alkylating agents interact with nuclear matrix proteins and with DNA close to matrix-bound replication and transcription sites [20–24].

Abnormal DNA organization or an unstable sperm nuclear matrix may play a role in male factor infertility; under these conditions, embryo development is affected [25, 26]. We have shown previously that chronic exposure of male rats to cyclophosphamide (CPA), a bifunctional alkylating agent, results in pre- and postimplantation embryo loss and malformed and growth-retarded progeny [27–29]. Exposure to CPA during spermiogenesis and epididymal transit, crucial times during male germ cell development as the genome is being remodeled and packaged, creates DNA single-strand breaks and cross-links [30–32] and, notably, results in altered gene expression in male germ cells as well as in early preimplantation embryos sired by CPA-exposed males [33–35]. We hypothesized that an action of CPA was to affect germ cell function by targeting components of the sperm nuclear matrix.

Many somatic cell nuclear matrix proteins have been characterized [36]. However, little is known about the components of the sperm nuclear matrix or the precise roles of these proteins in sperm function or embryo development. The aim of this study was to use proteomic strategies to identify proteins of the nuclear matrix and to elucidate the effects of chronic CPA exposure on the expression of matrix proteins.

MATERIALS AND METHODS

Animal Treatments

Adult male Sprague-Dawley rats (400–450 g) were obtained from Charles River Canada (St. Constant, QC, Canada), maintained on a 14L:10D light cycle, and provided with food and water ad libitum. Rats were gavaged with saline or CPA (6 mg kg–1 day–1, CAS 6055-19-2; Sigma-Aldrich, Oakville, ON, Canada). To capture cauda epididymal spermatozoa exposed to CPA throughout spermiogenesis and epididymal transit, animals were killed by decapitation 4 wk after initiation of treatment [37]. Animal handling and care were done in accordance with the guidelines established by the Canadian Council on Animal Care.

Sperm Collection

Sperm collection was done according to Calvin [38] with modifications. Epididymides were first removed, trimmed free of fat, and washed in prechilled phosphate buffer (PB, 20 mM, pH 6.0, containing 1 mM EDTA). The cauda region was removed, transferred to 8 ml of fresh buffer on ice, and thoroughly minced with sterile scalpels. The tissue was left for 5 min on ice to allow the spermatozoa to disperse and was then strained through a BD Falcon 70-µm nylon cell strainer (VWR International, Mississauga, ON, Canada) and washed with 2 ml of fresh PB; the total cell suspension was centrifuged at 1000 x g for 10 min at 4°C. The pellet was washed once and then resuspended in 4 ml of PB containing 40 µl of protease inhibitor cocktail (P8340; Sigma-Aldrich). Sperm were sonicated on ice, and sperm heads were isolated with discontinuous sucrose gradients made with PB. Twelve milliliters of sonicated sperm in 1.80 M sucrose was layered over 13 ml each of cold 2.05 M and 2.20 M sucrose and centrifuged at 91 400 x g in a Beckman SW 28 rotor for 70 min at 4°C. The pellet was resuspended in PB containing protease inhibitor cocktail (1:100 dilution) and stored at –80°C.

Nuclear Matrix Extraction

Sperm heads were resuspended in 500 µl of solution containing 1% SDS, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 5 µl of protease inhibitor cocktail and shaken with a Fisher Vortex Genie 2 mixer fitted with a TurboMix attachment (VWR International) for 10 min at room temperature. This treatment has been shown to remove the acrosome, all membranes, basal striations, the posterior nuclear ring, and the ventral spur of the postacrosomal sheath, leaving the condensed nucleus and partially attached perinuclear theca perforatorium, as well as less prominent remnants of perinuclear material in the midlateral and posterior regions of the sperm head [38, 39]. The samples were then washed three times with 50 mM Tris-HCl, pH 7.5, and resuspended in 500 µl of decondensation buffer (40 mM 1,4-dithiothreitol [DTT], 0.25 M (NH4)2SO4, 25 mM Tris-HCl, pH 7.5, and 5 µl of protease inhibitor cocktail) for 40 min at room temperature. A 261/2-gauge needle was used to gently break up any clumps, and 4000 U of RNase-free deoxyribonuclease I (Sigma-Aldrich) was added for 60 min at room temperature. Samples used for gel electrophoresis were pelleted, air dried, and stored at –20°C or used immediately for immunofluorescence studies.

Two-Dimensional Gel Electrophoresis

Protein separation and gel image analyses were conducted by the McGill University and Genome Quebec Innovation Centre (Montreal, QC, Canada) with material from Invitrogen (Burlington, ON, Canada), except where noted, and by the Invitrogen ZOOM IPGRunner System protocol. Fifty micrograms of protein was resuspended in 155 µl of rehydration buffer (9.8 M urea, 10 mM 1,4-dithioerythritol, 4% CHAPS [3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid], and 20 mM Tris) supplemented with 2% IPG (immobilized pH gradient) Buffer, pH 3–10 normal limits (NL) (Amersham Biosciences, Baie D'Urfe, QC, Canada). Seven-centimeter ZOOM Dry Strips, pH 3–10NL, were rehydrated for 16–18 h, and isoelectric focusing (IEF) was done with a voltage gradient (200-2000 V) applied as recommended by the manufacturer. After the IEF was complete, strips were equilibrated with 1x NuPAGE LDS Sample Buffer containing 2% DTT and then alkylated with iodoacetamide. Both steps were done at room temperature for 15 min. Electrophoresis in the second dimension was done on 4%–12% Bis-Tris precast mini-gels in XCell SureLock Mini-Cells filled with MOPS (morpholinepropanesulfonic acid) SDS Running Buffer. Broad-range protein molecular weight markers (0.9 µg/gel; Amersham) were used, and 200 V was applied for 50 min. Gels (n = 3 each for control and treated) were fixed overnight in 50% methanol:10% acetic acid, silver stained, scanned, and analyzed by Phoretix 2004 Image Analysis software (Amersham). Following background subtraction and normalization, intensities of the spots were calculated. One gel was then chosen as a reference, and the other gels were compared with the reference gel to create an average control and CPA gel, which were then compared. Spots were considered if present in at least two of three gels, and protein expression was considered changed only if the difference was at least 2-fold; this is equivalent to an increase of 100% or a decrease of 50%.

Mass Spectrometry

Spots were excised from the gel and subjected to trypsin digestion on a robotic MassPREP Station (Waters-Micromass, Milford, MA), per the manufacturer's instructions. Gel pieces were first washed twice with water for 20 min, destained twice in a 120-µl solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate mixed 1:1 for 15 min, and then dehydrated with 75 µl of 100% acetonitrile. Samples were reduced, in the dark, with 50 µl of 10 mM DTT for 30 min and then alkylated with 50 µl of 55 mM iodoacetamide for 20 min and 100 µl of 100% acetonitrile for 5 min. After washing and dehydration in 100 mM ammonium bicarbonate and 100% acetonitrile, respectively, gel pieces were covered and digested for 4.5 h with trypsin gold at 6 ng/µl (Promega, Madison, WI) in 100 mM ammonium bicarbonate. Peptides were extracted once with 30 µl of formic acid (FA) solution (1% FA in 2% acetonitrile) for 30 min, twice with 12 µl of FA solution, and then once with 12 µl of 100% acetonitrile for 30 min.

Nanoflow chromatography of digested peptides was done on an Agilent 1100 series nanopump (Agilent Technologies, Mississauga, ON, Canada) at a flow rate of 200 nl/min. Sample injection and desalting were performed with an Isocratic Agilent 1100 series pump at 15 µl/min for 5 min. A trapping column (Agilent) packed with Zorbax 300SB-C18 (5 x 0.3 mm) was used for sample desalting. Peptide separation was done with a Biobasic C18 (10 x 0.075 mm) picofrit column (New Objective, Woburn, MA). Peptides were eluted with a 20-min gradient with solvent A (0.1% FA) and solvent B (95% acetonitrile:0.1% FA) from 90%A:10%B to 100%B.

Electrospray mass spectrometry (MS) was done with a 4000 Q TRAP System (Applied Biosystems/MDS Sciex, Foster City, CA). Enhanced MS scans were acquired between 350 and 1600 mass:charge ratio (m/z) with a scan speed of 4000 atomic mass units (amu)/sec and active dynamic fill time. Information-dependent MS/MS analysis was performed on the three most intense multiply charged ions; a dynamic exclusion of 90 sec was used to limit the resampling of previously selected ions to two events. Three averaged MS/MS scans were acquired between 70 and 1700 m/z at a scan speed of 4000 amu/sec. Fixed fill time was set at 20 msec with Q0 trapping and rolling collision energy of ±3 eV. Peaklists for peptide mapping searches were generated by Mascot script 1.6 for Analyst 1.4.1 software (Applied Biosystems/MDS Sciex). Spectral processing included peak smoothing and centroiding without de-isotoping. Database searches were done by Mascot 1.9 (Applied Biosystems/MDS Sciex) with carbamidomethyl cysteine as a fixed modification, methionine oxidation as a variable modification, and 1.5 Da of precursor mass and 0.8 Da of fragment mass tolerances.

GPX4 Immunoblotting

Immediately after electrophoresis in the second dimension, unstained gels were transferred to Hybond-P polyvinylidene difluoride membranes (Amersham) by means of the Invitrogen XCell II Blot Module and protocol. Briefly, the blot module, gel, and membrane were assembled and inserted into the XCell SureLock Mini-Cell. The module was filled with NuPAGE Transfer Buffer (Invitrogen) supplemented with 10% methanol, and protein transfer was done at 30 V for 1 h. Efficiency of protein transfer was confirmed by staining blots with Ponceau S (Sigma-Aldrich). Following destaining in deionized water, membranes were air dried and stored at room temperature. When ready to use, the membrane was washed in 100% methanol for 2 sec, followed by 10 min in 20 mM Tris-HCl, pH 7.6, containing 0.8% NaCl and 0.1% Tween-20 (TBS-T). Membranes were then blocked for 1 h at room temperature in 5% nonfat milk in TBS-T, washed for 2 min in TBS-T, and then incubated overnight at 4°C with rabbit polyclonal anti-glutathione peroxidase 4 (GPX4) (Abcam, Cambridge, MA) diluted 1:20 000 in TBS-T containing 3% nonfat milk. After two brief washes with TBS-T, membranes were washed once in 40 ml for 15 min and twice in 20 ml for 10 min with TBS-T. Membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated rabbit immunoglobulin G (IgG) antibody (Amersham) diluted 1:15 000 in TBS-T containing 5% nonfat milk and washed. Antibody detection was done with the ECL Plus Western Blotting system (Amersham).

GPX4 Immunofluorescence

GPX4 immunoreactivity was determined in sperm collected after incubation in 1% SDS or after nuclear matrix extraction. Ten-microliter droplets were placed on slides and left on ice for 20 min. Slides were then washed in PBS (3 x 2 min), fixed in 2% paraformaldehyde for 20 min at room temperature, and washed and blocked with PBS containing 5% normal goat serum (Vector Laboratories, Burlington, ON, Canada) and 1% BSA (Sigma-Aldrich) for 30 min at room temperature. Subsequently, cells were covered overnight at 4°C with primary antibody solution containing 1% BSA and rabbit polyclonal GPX4 antibody (1:20 dilution) in PBS, washed with PBS (3 x 5 min), covered for 1.5 h in the dark at room temperature with secondary antibody solution containing Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:200 dilution; Invitrogen) in PBS, and finally washed in PBS. Slides were covered with Vectashield mounting medium containing 4',6'-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and kept at 4°C in the dark. Pictures were taken with a Dage-MTI CCD300-RC camera (Dage-MTI, Michigan City, IN) attached to an Olympus BX51 epifluorescence microscope.

RESULTS

2D Gel Analysis of Sperm Nuclear Matrix Proteins

Extraction of sperm nuclear matrices following DNA digestion was confirmed by negative DAPI staining (Fig. 1). Sperm nuclear matrix proteins analyzed by two-dimensional (2D) gel electrophoresis resulted in reproducible protein patterns. Three gels each were run for control and chronic CPA-treated sperm protein samples; protein profiles differed with treatment (Fig. 2). The average control gel (Fig. 2A) consisted of 290 protein spots that appeared in at least two of the three gels analyzed and corresponded to 90%–96% of the total number of spots detected on individual gels. In comparison, 309 protein spots were found on the average CPA gel (Fig. 2B), corresponding to 94%–98% of all spots appearing on individual gels. Overall changes in protein expression are illustrated in Figure 3. The expression of 7 (2%) protein spots was unique to control samples, and the expression of 26 (8%) was unique to CPA samples. Two hundred eighty-three protein spots were expressed in both groups; analysis of protein expression changes >2-fold revealed 34 (11%) spots that increased and 38 (12%) that decreased following CPA exposure.


Figure 01
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  		FIG. 1. Phase-contrast images of whole spermatozoa and nuclear matrix (NM) extracts (top panels). DNA stained with DAPI (bottom panels). Original magnification x400.


Figure 02
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  		FIG. 2. Two-dimensional electrophoretic separation of cauda epididymal spermatozoal nuclear matrix proteins from (A) control and (B) 4-wk chronic cyclophosphamide (CPA)-treated rats. Spots unique to each treatment group are indicated with blue circles. Red and yellow circles indicate spots showing increased or decreased expression following CPA exposure, respectively. Spots with <2-fold change in expression are indicated with green circles. Protein identification was achieved for spots 1–24 (see Table 1). MW, Molecular weight; pI, isoelectric point.


Figure 03
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  		FIG. 3. Overall changes in the expression of 316 nuclear matrix protein spots following a 4-wk chronic cyclophosphamide (CPA) treatment; >100% increase (horizontal lines), >50% decrease (black), control-specific (white), CPA-specific (cross-hatch), and no change (<2-fold change in spot intensity, diagonal lines). Numbers in parenthesis = the number of spots in each category.

Thirty-two spots were chosen for MS from each expression group (increased, decreased, control-specific, CPA-specific, and no change). Twenty-four spots were identified, as labeled in Figure 2 and summarized in Table 1. Selection was based on the following: 1) spots present in charge-trains, such as spot 5; 2) spots located in the acidic region where other somatic nuclear matrix proteins have been previously identified, such as spots 7–12; and 3) spots that consistently appeared on all three gels, such as spots 4 and 24. The majority of identified spots were either increased in expression or unique to CPA samples; none was unique to control sperm, and spot 2 was the only one that decreased (by 67%). Ten of the identified proteins were represented by at least two distinct spots on the gels, suggesting that these proteins are modified or exist in different isoforms (for example, the LIM [for Lin-11 Isl-1 Mec-3] domain containing preferred translocation partner in lipoma [LPP], in spots 3 and 4, or GPX4, found in spots 5, 16, 17, 18, 19, 20, 21, and 23). Thirteen spots matched more than one protein (for example, analysis of spot 24 gave significant results for phosphatidylethanolamine binding protein [PEBP]; and proteosome [prosome, macropain] subunit, beta type 6 [PSMB6]) and similar to Ran-interacting protein MOG1 (predicted). Of note, protein fragments were identified from significant MS analysis results for peptides that only partially covered a protein sequence. Despite the use of protease inhibitors during sample preparation, these protein fragments may be proteolytic cleavage products. Analyzed spots for three proteins (heterogeneous nuclear ribonucleoprotein K [HNRPK]; regulator of telomere elongation helicase 1 [RTEL1]; and spermidine/spermine N1-acetyl transferase [mapped]) contained only a fragment of the identified proteins. The mass of the proteins calculated from the 2D gel was below the expected mass of the proteins, calculated from their amino acid compositions.


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  		TABLE 1. Expression of proteins identified by mass spectrometry.

On the basis of information in the nuclear matrix NMPdb database (Database of Nuclear Matrix Proteins) [40] and in the literature, 11 of the identified proteins are known nuclear matrix components (HNRPK, GPX4, PSMB6, and protein phosphatase 1, catalytic subunit, gamma isoform [PPP1CC]) or are related to previously identified nuclear matrix proteins (DnaJ [Hsp40] homolog, subfamily B, member 6 [DNAJB6]; glutathione-S-transferase omega 2 [GSTO2]; glutathione-S-transferase mu 5 [GSTM5]; heat shock 70-kDa protein 2 [HSPA2]; RTEL1; proteosome [prosome, macropain] subunit, alpha type 5 [PSMA5] and beta type 4 [PSMB4]). Along with GPX4, HNRPK, and PPP1CC, glutathione-S-transferases and DnaJ (Hsp40) proteins are expressed in nucleoli; all except for PPP1CC have been identified in spots that mainly increased in expression following CPA exposure or were specific to CPA-exposed sperm. These proteins may be a part of the nuclear matrix residual nucleoli; these are known to be present in somatic cells [3, 41].

Functional Analysis of Identified Proteins

Surprisingly, only 7 of the 24 identified proteins were previously identified as components of spermatozoa heads (GPX4, HSPA2, calicin, DNAJB6, PEBP, capping protein [actin filament] muscle Z-line, beta [CAPZB], and testis-specific serine kinase 2 [TSSK2]), while 4 others have other known roles in spermatogenesis in the testis (GSTO2, GSTM5, PPP1CC, and RTEL1). Information concerning the putative functions of the proteins was found in the National Center for Biotechnology Information (Bethesda, MD) nonredundant and SWISS-PROT protein sequence databases or in the literature. In general, the proteins are involved in cell structure, transcription and translation regulation, DNA binding, protein processing, signal transduction, metabolism, cell defense, and detoxification (Table 1). The unknown proteins have not yet been characterized.

Of the three structural proteins identified, calicin is a known component of the sperm head perinuclear theca [42, 43]. Spot 4, containing calicin and growth-arrest-specific 2 (GAS2), increased by 1850% after CPA exposure compared with the control. There was no change in the expression of CAPZB; however, it migrated higher than its calculated molecular mass of 32 kDa. Despite this discrepancy, CAPZB was accepted as a positive identification because multiple significant peptide matches were found following MS analysis.

GPX4 Expression

Surprisingly, cell defense and detoxification proteins were present in abundance on the 2D gels. CPA induced changes in the amount of all known forms of the antioxidant enzyme GPX4; in addition, an uncharacterized 54-kDa form was identified (Table 1). The MS results were confirmed by Western blotting and showed the full effect of CPA on GPX4 expression (Fig. 4). Charge-trains between 20 and 31 kDa were observed, with an apparent overall increase in expression after CPA treatment compared with controls. Distinct spots were clearly different between control and CPA blots (Fig. 4, open arrows). In particular, a form of GPX4 at ~54 kDa is shown in the CPA blot (Fig. 4B, black arrow), and an increased amount of the 34-kDa posttranslationally modified form of GPX4 was detected (Fig. 4, arrowhead); this form has been shown to be localized to the nucleoli of spermatogonia, spermatocytes, and spermatids [44].


Figure 04
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  		FIG. 4. Two-dimensional Western blots probed with an antibody to glutathione peroxidase 4 (GPX4). A) Control. B) 4-wk chronic cyclophosphamide (CPA)-treated nuclear matrix samples. Open arrows: immunoreactive spots that clearly differ with treatment. Black arrow: uncharacterized 54-kDa GPX4 form. Arrowhead: posttranslationally modified and increased expression of 34-kDa form.

Figure 5 further validates the results obtained by MS and confirms the presence of GPX4 in the nuclear matrix. For comparison, membrane-free sperm heads treated with 1% SDS were immunostained (Fig. 5A). Not only was there increased GPX4 immunoreactivity in CPA-exposed samples compared with controls, but also, this increased immunoreactivity was found in the sperm nuclear matrix extract. Interestingly, immunoreactivity was greater in the SDS-treated samples than in the respective nuclear matrix extracts (Fig. 5B). Thus, these results indicate that CPA treatment resulted in an increase in chromatin-bound GPX4 as well as matrix-specific increased expression. Omission of the primary antibody resulted in an absence of immunoreactivity (data not shown).


Figure 05
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  		FIG. 5. Expression of glutathione peroxidase 4 (GPX4) in (A) SDS-treated membrane-free spermatozoa and (B) sperm nuclear matrix extracts. Left panels: control. Right panels: 4-wk chronic cyclophosphamide-treated sperm. Samples (n = 3) were immunostained with anti-GPX4. Original magnification x400.

DISCUSSION

The nuclear matrix is a dynamic structure with a morphology and protein composition that varies with the functional state of the nucleus [45]. It has been possible to identify specific cell and tissue types by the electrophoretic pattern of their nuclear matrix proteins [14–16]. Both normal and neoplastic samples can be identified by differences in nuclear matrix protein patterns [46]. To our knowledge, the present study is the first to go beyond structural evaluation and extensively examine the rat spermatozoal nuclear matrix by identifying some of the protein components. Significantly, we have demonstrated that exposure to an alkylating agent and male-mediated developmental toxicant alters the spermatozoal nuclear matrix protein profile. A major transition in sperm chromatin structure and function occurs during spermiogenesis, resulting in the formation of mature spermatozoa competent to fertilize. Changes reported in the nuclear matrix structure and protein profile of round and elongating spermatids reflect the morphologic changes and remodeling of chromatin that occur during spermatid differentiation [17]. Our results show that targeting germ cells throughout spermiogenesis with CPA modifies the makeup of the nuclear matrix. Such an effect may alter chromatin reorganization by the nuclear matrix, both during spermatogenesis and after fertilization in the zygote.

Interestingly, there was no apparent effect of CPA exposure on the structure of the nuclear matrix. Calicin and F-actin capping proteins like CAPZB are known components of the sperm head perinuclear theca and are possibly involved in sperm morphogenesis and stability and F-actin organization and biogenesis, respectively [42, 47]. GAS2 has also been implicated in actin filament organization [48]; hyperphosphorylation and proteolytic cleavage at its C-terminal end result in an irregular cell shape and actin rearrangement, processes that are triggered as part of a series of apoptotic events [49]. Overexpression of other cytoskeletal proteins results in a change in actin organization; however, this is not the case with overexpressed GAS2 [48], as we see in the present study. We did not identify any of the previously described classical cytoskeletal proteins found in somatic cell matrices, such as vimentin, keratin, or actin, or the perinuclear lamins A, B, and C [50, 51]; interestingly, actin, myosin, cytokeratins, and spectrin have been described in the guinea pig sperm nuclear matrix [52].

Protein expression specific to CPA-exposed sperm may be due to effects on RNA transcripts, altering their amounts, localization, or translation. Previous studies report differential expression of stress response genes in male germ cells after chronic exposure of male rats to CPA [33]. Factors other than matrix instability may alter DNA organization and, subsequently, affect gene function. Alkylating agents preferentially bind to matrix proteins and matrix-associated DNA [23, 24]. Proteins are most likely to give rise to multiple spots as a consequence of multiple posttranslational modifications; most eukaryotic proteins are modified, and these modifications are often essential for their function [53]. In this study, some differences in expression are probably due to modification of the nuclear matrix proteins following drug exposure. Indeed, the majority of the CPA-specific proteins are expressed within charge-trains; charge-trains created by protein spots with the same molecular mass but different isoelectric points indicate modified proteins. Tew et al. [21] have shown that fibrillar components of the matrix and ribonucleoproteins are alkylated following exposure to 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea and chlorozotocin, but the effects of these modifications on protein function are not known.

Reduced binding of DNA to the matrix may be a function of interference with the DNA recognition sites by alkylation at specific bases; in vitro alkylated DNA has a reduced interaction with matrix proteins [21]. Chromatin loops associate with the nuclear matrix at specific regions called matrix attachment regions (MARs) [54]; these MARs are involved in DNA replication and repair and in various aspects of gene regulation [55], including mRNA transcription and processing via their involvement in attachment or association with newly transcribed mRNA, pre-mRNA splicing machinery, and ribonucleoprotein particles [56]. In light of this, two proteins, HNRPK and LPP, which were present at elevated levels in the nuclear matrix extracted from CPA-exposed sperm, are of particular interest. HNRPK is a unique member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that preferentially binds single-stranded DNA [57]. It acts as a docking site to integrate signaling cascades between anchored protein complexes and, as such, is a multifunctional molecule implicated in transcription activation and chromatin remodeling, in addition to the more typical hnRNP functions of mRNA splicing, transport, and translation [58]. Studies on LPP demonstrate that it has the capacity to activate transcription and suggest that, like HNRPK, it serves as a scaffold upon which protein complexes are assembled [59].

Increased expression of GPX4 in the nuclear matrix was intriguing, given its role not only as an intracellular antioxidant enzyme, directly reducing lipid hydroperoxides [60], but also as an inhibitor of apoptosis [61], cell cycle regulation [62], and embryo development [63]. Most interestingly, in spermatozoa, GPX4 appears to have two functions: 1) protamine disulfide cross-linking, where it uses the protamine cysteine residues instead of glutathione as reductants and acts as a thiol peroxidase when bound to DNA, and 2) protection of sperm against oxidative damage [64].

Sperm nuclear GPX4 has a molecular mass of 34 kDa and is bound to DNA. However, once spermatozoa reach the caput epididymal region, about two thirds of the 34-kDa enzyme is processed into smaller proteins, with molecular masses between 22 and 29 kDa, that do not bind to DNA; their enzymatic properties are not affected [64, 65]. A 20-kDa form, identical to cytosolic GPX4, is also present in the heads of spermatozoa [65]. Each of these forms was present in our extracts. The nucleoli in mature spermatozoa are inactive; only nuclear vacuoles containing fibrils remain [66]. A study done by Puglisi et al. [67] showed that GPX4 localizes to fibrous material in electron-lucent spots in condensed epididymal sperm; these could be areas of residual nucleoli. A nucleolar GPX4 with a molecular mass of 34 kDa has also been identified in the nucleoli of spermatogonia, spermatocytes, and spermatids [44].

Under normal conditions, GPX4 appears to be present within the head of spermatozoa both bound and unbound to DNA and, to a lesser extent, in the nuclear matrix; GPX4 expression increases following CPA exposure. Exposure to CPA or acrolein, a metabolite of CPA, induces the formation of reactive oxygen species (ROS) [68, 69] and lipid peroxidation [70]. Sperm are highly susceptible to lipid peroxidation, and the induction of ROS is correlated with a decreased capacity to undergo the acrosome reaction [71] and DNA damage [72]. Increased nuclear expression of GPX4 may contribute to antioxidant defense mechanisms; however, functions served by localization of GPX4 to the nuclear matrix are less evident. In somatic cells, overexpression of nucleolar GPX4 protects nucleoli from oxidative stress-induced damage [44]. Additionally, lipids are not only membrane components, but also represent important components of chromosomes, chromatin, and the nuclear matrix [73]; some studies suggest that they are involved in DNA loop attachment to the nuclear matrix, replication, and transcription as well as nuclear signal transduction [73–75]. If this is the case in sperm nuclei, CPA may induce oxidative stress and lipid peroxidation that may be reduced by GPX4.

Changes in protein composition, or modifications thereof, may correlate with alterations in DNA organization, leading to changes in DNA function and protein expression. Changes in protein expression could be important in the regulation of sperm function. Complete analysis of all the spots on the gels may uncover some of the expected components of sperm nuclear matrices, such as the cytoskeletal proteins, and additional unexpected components. Both unknown proteins and CPA-specific proteins are interesting because new proteins discovered by this proteomic approach may be major players in spermiogenesis or sperm function or may serve as possible biomarkers of altered sperm. The impact on the postfertilization early embryo remains to be determined; however, one effect may be ectopic protein expression in fertilized eggs. Studies of preimplantation embryos sired by CPA-treated males report temporal and spatial disruption of the rat zygotic genome activation; total RNA synthesis was higher, and gene expression profiles were altered as early as the single-cell stage in comparison with controls [34, 35, 76].

The present study provides further insight into the mechanisms by which CPA may exert its male-mediated effects on embryo development. The identification of sperm nuclear matrix components and their functions brings us closer to unraveling the mysteries of the matrix.

ACKNOWLEDGMENTS

We greatly appreciate the assistance of Leonid Kriazhev from the McGill University and Genome Quebec Innovation Centre with the 2D gel electrophoresis and MS.

FOOTNOTES

1Supported by a grant from the Canadian Institutes of Health Research. Back

Correspondence: 2Barbara F. Hales, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, Canada H3G 1Y6. FAX: 514 398 7120; e-mail: barbara.hales{at}mcgill.ca

Received: 20 January 2007.

First decision: 14 February 2007.

Accepted: 25 April 2007.

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