Hum. Reprod. Advance Access originally published online on February 15, 2007
Human Reproduction 2007 22(5):1431-1442; doi:10.1093/humrep/dem002
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Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa
1 Departments of Pharmacology and Therapeutics 2 Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada
3 To whom correspondence should be addressed at: Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, Canada H3G 1Y6. E-mail: barbara.hales{at}mcgill.ca
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Abstract |
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BACKGROUND: The formation of mature sperm involves the expression of numerous proteins during spermiogenesis and the replacement of histones with protamines to package the genome. Exposure to cyclophosphamide (CPA), an anticancer alkylating agent, during spermiogenesis may disrupt chromatin condensation with adverse consequences to the offspring.
METHODS: Adult male rats were given CPA in one of two schedules: (i) subchronic, 4 days—day 1 (100 mg kg–1) and days 2–4 (50 mg kg–1 per day) or (ii) chronic—daily (6.0 mg kg–1 per day). Animals were euthanized on days 14, 21 or 28.
RESULTS: The effects of CPA on epididymal sperm chromatin structure were germ-cell-phase specific; mid-spermiogenic spermatids were most sensitive. The acridine orange DNA denaturation assay showed significant increases in susceptibility to denaturation (P < 0.01). Chromatin packaging assessment revealed 1,4-dithiothreitol-dependent chromomycin A3 DNA binding and less condensed, protamine-deficient sperm; the total thiol (P < 0.001) and protamine contents (P < 0.01), measured using monobromobimane and the HUP1N protamine 1 antibody, respectively, were reduced. The sperm basic proteome was also altered; proteins that were identified are involved in events during spermiogenesis and fertilization.
CONCLUSIONS: Paternal exposure to CPA alters sperm chromatin structure, as well as the composition of sperm head basic proteins. We speculate that these changes underlie effects on fertilization and embryo development.
Key words: chemotherapeutic agents/chromatin packaging/infertility/proteomics/toxicology
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Mature spermatozoa are organized and packaged in a specific manner to ensure transmission of genetic material to the offspring and successful pregnancy. The unique structure of sperm and stability of the DNA are due to a sequence of events that occur to shape, condense and protect the nucleus; the most important of these involves a loss of the bulky nucleosomal structure in elongating spermatids, as transition proteins transiently replace histones. This exchange facilitates the preferential binding of highly basic, cysteine-rich protamines to DNA in late spermatids (Poccia, 1986
; Wouters-Tyrou et al., 1998) Further stabilization is accomplished by the formation of intra- and intermolecular protamine disulphide bonds, as cysteines progressively become oxidized during sperm epididymal transit (Shalgi et al., 1989). Perturbation of events at any point of spermiogenesis or sperm maturation may affect sperm function.
Indeed, human male infertility has been associated with changes in sperm chromatin integrity and packaging (Evenson et al., 1999; Irvine et al., 2000; Spano et al., 2000), as well as with changes in protamine content and affinity to DNA (Belokopytova et al., 1993; Torregrosa et al., 2006). Exposure to chemotherapeutic agents results in increased DNA damage in human spermatozoa (Chatterjee et al., 2000; Morris, 2002); such damage has been associated also with abnormal sperm chromatin packaging (Manicardi et al., 1995; Manicardi et al., 1998).
A commonly used anticancer drug, cyclophosphamide (CPA), is a bifunctional alkylating agent and well-known male-mediated developmental toxicant with clear stage-specific effects on male germ cells (Trasler et al., 1985; Anderson et al., 1995). Despite the appearance of morphologically normal spermatozoa, evidence of hidden anomalies exists (Bianchi et al., 1996) and may be the case in spermatozoa chronically exposed to CPA. Exposure of male germ cells to CPA during spermiogenesis and epididymal transit leads to DNA single-strand breaks, cross-links and altered in vitro spermatozoal decondensation and template function (Qiu et al., 1995a,b) in cauda epididymal sperm and to pre- and post-implantation embryo loss as well as growth retarded progeny (Trasler et al., 1986). The most damaging effects to DNA occurred during mid-spermiogenesis, as the histone–protamine exchange begins (Codrington et al., 2004). Exposure to CPA at this time may alter the binding of protamines to DNA because of increased DNA damage, as well as result in protamine alkylation, given that protamines are known to be especially susceptible to alkylation (Sega and Owens, 1983). If this is the case, the end result may be faulty or incomplete protamine deposition and blockage of normal disulphide bond formation, thus preventing proper chromatin condensation and limiting fertilizing ability.
The formation of mature sperm involves the controlled, sequential expression of a large number of proteins during spermiogenesis. Paternal CPA exposure alters gene expression in male germ cells (Aguilar-Mahecha et al., 2001; Aguilar-Mahecha et al., 2002). These changes in gene expression may be translated into an altered protein expression profile, which could be important for spermiogenic events and fertilization; the characterization of sperm proteins should identify such changes.
Sperm count, viability, membrane fluidity, capacitation status, acrosomal integrity and mitochondrial function, as well as DNA integrity and chromatin structure and packaging can be measured rapidly in a large number of spermatozoa by using flow cytometry (Gillan et al., 2005). The value of flow cytometry to study mammalian sperm has been recognized in the areas of reproductive toxicology (to monitor effects from environmental, occupational and therapeutic exposures) and clinical andrology (to assess individual fertility potential) (Spano and Evenson, 1993). A multiplex approach, combining flow cytometry assays with proteomic and genomic methods, should improve our ability to predict fertility status and help identify susceptible subpopulations at risk for infertility. This study uses such an approach to elucidate the effects of CPA on chromatin structure and on the expression of sperm head basic proteins in the rat.
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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 (CAS 6055-19-2, Sigma-Aldrich Ltd, Oakville, ON, Canada) in one of two schedules: (i) high dose/subchronic, 4 days—day 1 (100 mg kg–1) and days 2–4 (50 mg kg–1 per day); (ii) low dose/chronic—daily (6 mg kg–1 per day). To capture cauda epididymal spermatozoa first exposed to CPA during late-, mid- and early spermiogenesis, animals were euthanized by decapitation on days 14, 21 or 28 after initiation of treatment (Clermont, 1972
). Animal handling and care were done in accordance with the guidelines established by the Canadian Council on Animal Care.
Sperm collection
Epididymides were first removed, trimmed free of fat and washed in 6 ml of pre-chilled 1x phosphate buffered saline (PBS), pH 7.4 (Roche Diagnostics, Laval, QC, Canada). The caput and cauda regions were removed and transferred to separate sterile Petri dishes containing 8 ml of fresh PBS on ice. Each region was 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 Co., Mississauga, ON, Canada), washed with 2 ml of fresh PBS and the total cell suspension was centrifuged at 1000 g for 10 min at 4°C. The pellet was washed twice in 10 ml of 0.45% NaCl, once in PBS and then resuspended in 4 ml of PBS containing 40 µl of protease inhibitor cocktail (Sigma-Aldrich, Ltd). Aliquots were stored at –80°C.
Cauda epididymal sperm used for protein extraction were further processed according to the method of Yu et al. (2000), with a few modifications. Following the final wash in PBS, sperm were resuspended in 4 ml of water containing 40 µl of protease inhibitor cocktail. After sonication on ice, sperm heads were isolated using discontinuous sucrose gradients (Calvin, 1976) made with a 0.2x concentration of MP buffer (5 mM MgCl2, 5 mM sodium phosphate, pH 6.5) containing 0.25% Triton X-100. Twelve millilitres of sonicated sperm in 1.80 M sucrose were layered over 13 ml each of cold 2.05 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 washed in MP buffer and then frozen at –80°C.
SCSA®/acridine orange DNA denaturation assay
Altered chromatin structure measured by the susceptibility of sperm DNA to acid-induced denaturation was assessed with the metachromatic dye, acridine orange (AO) (Sigma-Aldrich Ltd), on the basis of a method previously described (Evenson and Jost, 2000). The dye fluoresces green when bound to double-stranded DNA and red when bound to single-stranded or denatured DNA. Samples of spermatozoa from the cauda epididymidis were placed in a 37°C water bath until thawed, immediately placed on ice and then sonicated to separate tails from heads. Two hundred microlitres of the 1–2 x 106 sperm ml–1 samples were mixed with 400 µl of a solution containing 0.08 N HCl, 0.1% Triton X-100 and 150 mM NaCl, pH 1.4, to denature sperm DNA. After 30 s, 1.2 ml of AO staining solution (0.2 M Na2HPO4, 0.1 M citric acid buffer, 1 mM EDTA, 150 mM NaCl, pH 6.0 and 6.0 µg ml–1 AO) was added.
Exactly 2.5 min after the addition of the staining solution, the samples were vortexed and analysed using a BD FACScan Analyser System (BD Bioscience, San Jose, CA, USA) fitted with a 488 nm argon-ion laser. Green fluorescence was detected with 502LP and 530/30BP filters. Red fluorescence was detected with 670LP and 660/20BP filters. A total of 10 000 sperm were analysed per sample (n = 6–8) and each sample was measured twice.
Raw data were processed using WinList Software (Verity Software, Topsham, ME, USA). The extent of DNA denaturation was determined by calculating the DNA fragmentation index (DFI), which represents the shift from green to red fluorescence and is the ratio of denatured DNA (red intensity) to total DNA (red + green intensity). For each sample, the mean DFI, indicating shifts within a population of cells, and percentage of abnormal sperm with denatured DNA, defined as %DFI, were calculated.
Chromomycin A3 staining
The state of sperm chromatin packaging was assessed on the basis of the accessibility of the fluorochrome, chromomycin A3 (CMA3) (Sigma-Aldrich Ltd) (Bianchi et al., 1993). This assay was done with the following modifications. Aliquots of spermatozoa from the caput and cauda epididymidis were first thawed and sonicated on ice. Following one wash in cold PBS, spermatozoa were incubated in 0, 0.1, 0.2, 0.5, 1, 2, 5, 10 or 20 mM of 1,4-dithiothreitol (DTT) in PBS for 10 min at 37°C and then washed in PBS. Five hundred microlitres of CMA3 solution (0.25 mg ml–1 CMA3 in McIlvaine buffer, 0.1 M citric acid, 0.2 M Na2HPO4, pH 7.0, containing 10 mM MgCl2) was added to each sample and incubated for 20 min at 25°C in the dark. The samples were finally resuspended in 1 ml McIlvaine buffer and stored at 4°C in the dark until analysis the next day.
Spermatozoal heads were analysed using a MoFlo High Performance Cell Sorter (Cytomation Inc., Fort Collins, CO, USA) equipped with an argon laser (457 nm line excitation) and a 460/10 filter. The fluorescence emitted was detected with a 580/30 bandpass filter and quantified using Summit v3.1 software (Cytomation Inc.). A total of 10 000 sperm were analysed for each sample (n = 4).
Monobromobimane thiol labelling
Thiol labelling was done according to Shalgi et al. (1989), with minor modifications. Aliquots of spermatozoa from the caput and cauda epididymidis (4 x 106 cells ml–1) were sonicated on ice and incubated with or without 1 mM DTT in PBS at 37°C for 10 min. Samples were washed twice and resuspended in PBS. A 50 mM monobromobimane (mBBr) (Calbiochem, San Diego, CA, USA) stock solution was prepared in acetonitrile and added to the sperm suspensions to a final concentration of 0.5 mM in the dark for 10 min. Sperm were washed twice with PBS and stored in the dark at 4°C until analysis the next day.
Flow cytometric analysis was done using a MoFlo High Performance Cell Sorter (Cytomation Inc.) equipped with an argon laser (UV excitation). MBBr fluorescence emission was detected with 450/65 and dichroic 510LP filters. Quantification was done using Summit v3.1 software (Cytomation Inc.). A total of 10 000 sperm were analysed for each sample (n = 4).
Protamine immunostaining
Aliquots of cauda epididymal spermatozoa (3 x 107 cells ml–1) were resuspended in 1 ml of solution containing 1% sodium dodecyl sulphate (SDS), 50 mM Tris–HCl pH 7.5, 1 mM EDTA and protease inhibitor cocktail (1:100 dilution) for 10 min at room temperature to remove peri-nuclear material. The samples were then cooled on ice for 5 min, sonicated for 5 s and washed three times with 50 mM Tris–HCl, pH 7.5. Before the last wash, samples were divided into three groups and then resuspended in 1 ml decondensation buffer (25 mM Tris–HCl, pH 7.5, and 10 µl protease inhibitor cocktail) containing 0, 1 or 20 mM DTT for 1 h at room temperature. Ten microlitre droplets were placed on slides (two sample areas per slide) and left on ice for 20 min to allow cells to settle and adhere to the slides. Slides were then washed 3 x 2 min in PBS, fixed in 2% paraformaldehyde for 20 min at room temperature and washed again.
Slides were first blocked with 2% normal goat serum (Vector Laboratories Inc., Burlington, ON, Canada) and 1% bovine serum albumin (BSA, Sigma-Aldrich, Ltd) in PBS (200 µl per sample area) for 30 min at room temperature. Subsequently, cells were covered with 200 µl of primary antibody solution containing 1% BSA and Hup1N monoclonal human protamine 1 (P1) antibody (1:100 dilution, kindly provided by Dr Rod Balhorn, Lawrence Livermore National Laboratory) in PBS, overnight at 4°C, washed with PBS (3 x 2 min), covered for 1.5 h in the dark with 200 µl of secondary antibody solution containing fluorescein-conjugated mouse immunoglobulin antibody (1:150 dilution, Amersham Biosciences, Baie D'Urfe, QC, Canada) in PBS and finally washed three times in PBS. Slides were covered with Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories Inc.) and kept at 4°C in the dark.
Pictures were taken using a DAGE-MTI CCD300-RC camera (DAGE-MTI Inc., Michigan City, IN, USA) attached to an Olympus BX51 epifluorescence microscope. Fluorescence intensity and sperm head area were measured using the MCID Elite 6.0 image analysis system (Imaging Research Inc., St Catharines, ON, Canada). Fifty cells were randomly chosen for analysis from each sample area for a total of 100 cells per group (n = 3).
Protein extraction
Cauda epididymal sperm head proteins were extracted according to the method of Balhorn et al. (1977) with the following modifications. Every step was done at 4°C. Pellets were dissolved in 5 M guanidine-HCl, 10 mM Tris pH 8, EDTA pH 8, 100 mM DTT and protease inhibitor cocktail (1:100 dilution) for 30 min. After sonication for 1 min, urea, 2-mercaptoethanol and NaCl were added to give a final concentration of 0.5 M guanidine-HCl, 3 M urea, 0.5 M 2-mercaptoethanol and 2 M NaCl for 1 h. HCl was added to a concentration of 0.5 M for 1 h to precipitate the DNA, and the DNA pelleted by centrifugation at 18 300g for 10 min. The supernatant was dialyzed against 0.01 N HCl and 10 mM DTT with two changes of solution using 3500 Da dialysis cassettes (Pierce, Rockford, IL, USA). Proteins were precipitated with 25% trichloroacetic acid for 1 h and the precipitate pelleted at 21 000g for 10 min, washed once with cold acid-acetone (one drop 5 N HCl in 10 ml acetone), once with cold acetone and then air-dried. Samples were stored at 4°C.
2D basic gel electrophoresis
Protein separation and analyses were conducted by the McGill University and Genome Quebec Innovation Centre (Montreal, QC, Canada) using material from Invitrogen Inc. (Burlington, ON, Canada), except where noted, and the Invitrogen ZOOM IPGRunner System protocol. Fifty micrograms of protein were resuspended in 155 µl of rehydration buffer (9.8 M urea, 10 mM 1,4-dithioerythritol, 4% CHAPS, 20 mM Tris) supplemented with 1% ZOOM® Focusing Buffer, pH 7–12. ZOOM Dry Strips, pH 9–12, were rehydrated for 16–18 h and isoelectric focusing (IEF) done with a voltage gradient (200–2000 V) applied, as recommended by the manufacturer. After 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 4–20% Tris-glycine precast gels in XCell SureLockTM Mini-Cells filled with Tris-glycine SDS Running Buffer. Broad range protein molecular weight markers (0.9 µg per gel, Amersham Biosciences) were used and 125 V were applied for 1.5 h. Gels (n = 5) were silver stained, scanned and analysed with Phoretix 2004 Image Analysis software (Amersham Biosciences). Following background subtraction and normalization, intensities of the spots were calculated. One gel was then chosen as a reference and the other gels 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 three out of five gels, and protein expression was considered changed only if the difference was at least 1.5-fold; this is equivalent to an increase of 50% or decrease of 33%.
Mass spectrometry
Selected spots were excised from the gel and subjected to trypsin digestion on a robotic MassPREP Station (Waters-Micromass, Milford, MA, USA), as per the manufacturer's instructions. Gel pieces were first washed twice with water for 20 min, destained twice in 120 µl solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulphate 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 followed by alkylation 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 6 ng µl–1 of trypsin gold (Promega, Madison, WI, USA) in 100 mM ammonium bicarbonate. Peptides were extracted with 30 µl formic acid (FA) solution (1% FA in 2% acetonitrile) for 30 min, twice with 12 µl FA solution and then 12 µl of 100% acetonitrile for 30 min.
Nanoflow chromatography of digested peptides was performed on an Agilent 1100 series nanopump (Agilent Technologies Inc., Mississauga, ON, Canada) at a flow rate of 200 nl min–1. Sample injection and desalting were performed with an Isocratic Agilent 1100 series pump at 15 µl min–1 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, USA). Peptides were eluted using 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.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was done with a MALDI Q-Time-of-Flight (ToF) Ultima instrument (Waters-Micromass). Samples were spotted along with a saturated solution of alpha-hydroxycinnamic acid in 50% acetonitrile. Peak lists for peptide-mapping searches were generated with Proteinlynx v.1.5 (Waters-Micromass). Searches were performed with Mascot 1.9 using carbamidomethyl cysteine as a fixed modification, methionine oxidation as a variable modification and a precursor mass tolerance of 0.3 Da.
Statistical analysis
Significant differences were determined using two- and three-way analysis of variance followed by the Holm–Sidak post hoc test (P < 0.05). The percentage of abnormal sperm with denatured DNA among populations of spermatozoa from control and CPA-treated animals was compared with 
2 analysis. Statistical analyses were done using the SigmaStat 3.0 software package (SPSS Inc., Chicago, IL, USA).
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Sperm chromatin structure
Altered sperm chromatin structure has been attributed to changes in protamine content and the thiol-disulphide status of proteins (Kosower et al., 1992
; Sailer et al., 1995; Love and Kenney, 1999; Zini et al., 2001; Aoki et al., 2005; Pina-Guzman et al., 2005). The SCSA®/AO DNA denaturation assay was used for a general assessment of the effects of CPA exposure on sperm chromatin structure. DNA in sperm with abnormal chromatin structure showed increased red fluorescence yielding a broader distribution of DFI values with an increase in mean DFI and more cells with denatured DNA (%DFI). Figure 1A shows that chronic CPA treatment for 28 days, exposing spermatids throughout spermiogenesis and epididymal maturation, resulted in a 1.2-fold increase in the mean DFI of treated spermatozoa when compared with controls. Subchronic exposure resulted in significant increases in mean DFI values in spermatozoa exposed to the drug only at the end of spermiogenesis (14 days), when they were elongated spermatids, and during mid-spermiogenesis, as elongating spermatids (21 days). Cells collected after 28 days, and therefore first exposed as round spermatids, exhibited no increase in mean DFI.
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The percentage of sperm with denatured DNA was dramatically higher in drug-exposed samples (Figure 1B). Chronic exposure resulted in a 6.9-fold increase in %DFI. Subchronic treatment revealed significant increases in the %DFI in spermatozoa collected after 14 days (3.8-fold) and 21 days (6.9-fold). In contrast, there was no significant difference in %DFI in cells targeted as round spermatids. Mid-spermiogenic elongating spermatids were most sensitive to the effects of CPA; exposure at this time appears to account for most of the abnormalities seen following chronic exposure.
Protamination and condensation status of sperm
Chronic CPA exposure showed no CMA3 staining above background levels in either control or drug-exposed cauda epididymal spermatozoa, suggesting no change in protamine content (Figure 2A, 0 mM DTT); however, sperm condensation by protamine disulphide bond formation, in addition to the level of protamination, could be a limiting factor controlling fluorochrome accessibility. To determine whether thiol bonds do play a role in CMA3 binding, caput spermatozoa were examined. Indeed, less condensed, immature caput spermatozoa were CMA3 positive. Not only was fluorescence intensity 7-fold higher in control caput sperm compared with control cauda sperm, but also the fluorescence intensity of CPA-exposed caput sperm was 1.6-fold higher than that of control caput sperm and 9-fold higher than that of CPA cauda sperm.
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, significantly different from control cauda epididymal spermatozoa; ¶, significantly different from CPA cauda epididymal spermatozoa (P < 0.001).The relationship between CMA3 DNA binding and chromatin condensation was further assessed by in vitro decondensation induced by the disulphide reducing agent, DTT (Figure 2A, inset). With increasing amounts of DTT, a concentration-dependent increase in staining was observed, suggesting that the thiol status or decondensation status of sperm does indeed affect CMA3 accessibility and binding. In caput spermatozoa, with just 0.1 mM DTT, CMA3 binding increased to levels significantly higher than in cells not treated with the reducing agent (control: 4-fold; chronic CPA: 3.8-fold). More importantly, the CPA-exposed cell population exhibited unique susceptibility at this concentration of DTT, as fluorescence intensity was 1.6-fold higher than in controls. Maximum intensity in both groups was observed with 1 mM DTT (Figure 2A).
In cauda spermatozoa, higher concentrations of DTT were required to observe significant differences in CMA3 fluorescence intensity. One millimolar of DTT was needed to achieve a 9.2-fold increase in control cauda spermatozoa and 17.5-fold increase in CPA-exposed cauda spermatozoa when compared with those that were not treated with DTT. Notably, the only difference in CMA3 binding in cauda sperm due to drug treatment was seen when using 1 mM DTT (1.9-fold increase, Figure 2A). Therefore, chromatin packaging, whether due to protamine deficiency or not, was affected by CPA exposure; however, such an affect was only evident after decondensing the cells with 1 mM DTT. It appears that a certain thiol-disulphide status threshold had to be passed before CMA3 binding was achieved. These results suggest that CPA-exposed spermatozoa were less condensed and/or were protamine deficient when compared with control sperm. Cauda spermatozoa collected following subchronic exposure to CPA did not exhibit any changes in CMA3 DNA binding at any time point in the absence of DTT (Figure 2B). However, in the presence of 1 mM DTT, significant differences (>2-fold) due to CPA treatment were observed at all time points, with no cell type being more sensitive than the other (Figure 2B).
Thiol-disulphide status
Results obtained using CMA3 led us to assess the thiol-disulphide status of spermatozoa by incubating cells with and without DTT and then labelling them with mBBr. In Figure 3A, as expected, the proportion of reactive thiols [SH/(SH + SS)], estimated from the mean fluorescence of sperm incubated without DTT (free thiols, SH) and the fluorescence of DTT pretreated cells (total thiols, SH + SS), decreases as sperm mature from the caput to cauda epididymal regions. The effects of CPA were assessed in cauda sperm as well as caput sperm, since the thiol status changes during epididymal transit as protamine disulphide bonds form. Using both cell types, therefore, also highlights any epididymal effects of CPA. Following chronic exposure to CPA, the fluorescence intensities of caput and cauda sperm not incubated with DTT were similar to controls. In contrast, total thiol levels significantly decreased by 5.7% in caput and 10% in cauda spermatozoa pretreated with DTT when compared with controls, thus indicating that not only were thiol groups affected during epididymal transit, but also that the effect occurred before cells reached the caput epididymal region.
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Subchronic CPA exposure revealed a decrease in total thiols (7.4%) only in cauda sperm collected after 21 days, representing an effect on mid-spermiogenic spermatids (Figure 3B). No other cell type exposed to CPA and incubated with or without DTT showed an effect different from that of controls.
Protamine content
Protamines are the main basic protein present in spermatozoal heads. To follow-up on results obtained using CMA3 and mBBr, which point to the possibility of protamine deficiency in mature spermatozoa following CPA exposure, the level of protamination was assessed in cauda epididymal sperm. By the time spermatozoa enter the epididymis, protamination is complete; therefore, only cauda sperm were required for this assessment. Protamine 1 (P1) detection in cauda spermatozoa was dependent on the extent of decondensation induced by DTT; differences due to chronic drug treatment were only evident if cells were pretreated with DTT (Figure 4A). Minimal fluorescence was seen in both control and CPA-exposed sperm in the absence of DTT (Figure 4B). With increasing concentrations of DTT, the amount of protamines detected significantly increased in control sperm. The fluorescence intensity of CPA-exposed sperm was significantly different from control if DTT was used; compared with controls, less protamine was detected in CPA-exposed sperm. Differences in fluorescence between control and CPA-exposed sperm were not due to disproportionate decondensation because although sperm head areas increased with increasing concentrations of DTT, they were not affected by the CPA treatment (Figure 4C). Thus, sperm head fluorescence intensity results show that protamine content was indeed lower following chronic CPA exposure (Figure 4D).
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Sperm head basic protein expression
Sperm head proteins were analysed by 2D basic gel electrophoresis. Five gels each were run for control and chronic CPA-treated sperm protein samples. The overall pattern of proteins was reproducible between experiments; protein profiles differed with treatment (Figure 5). The average control gel (Figure 5A) consisted of 68 protein spots that appeared in at least 3 out of 5 gels analysed, corresponding to 73–98% of the total number of spots detected on individual gels. In comparison, 59 protein spots were found on the average CPA gel (Figure 5B), corresponding to 70–99% of all spots appearing on individual gels. Eleven protein spots were uniquely expressed in control samples and two in CPA samples. Fifty-seven protein spots were expressed in both groups; analysis of protein-expression changes > 1.5-fold revealed nine spots that increased and six that decreased following CPA exposure.
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Twenty-two spots were chosen for identification; however, we were only successful in confidently identifying 12; these spots are labelled in Figure 5 and summarized in Table I. Three of the identified proteins (histone 4, HIST2H4; fatty-acid-binding protein 9, FABP9; zona-pellucida-binding protein, ZPBP) were represented by at least two distinct spots on the gels, suggesting that these proteins are modified or exist in different isoforms. ZPBP was found as the precursor (46.3 kDa) and mature protein (39.7 kDa). Interestingly, the expression of the precursor increased by 68%, whereas that of the mature protein was not changed (
1.5-fold).
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Of note, protein fragments were identified from significant MALDI Q-ToF MS analysis results for peptides that only partially covered a protein sequence. Protein fragments may be proteolytic cleavage products, despite the use of protease inhibitors during sample preparation. Analysed spots for two proteins (heterogeneous nuclear ribonucleoprotein A1, HNRPA1; chromodomain helicase DNA-binding protein 4, CHD4) contained only a fragment of the identified protein; the masses of the proteins calculated from the 2D gel (11.8 and 21.0 kDa, respectively) were below the expected masses of these proteins, calculated from their amino acid compositions (38.9 and 205.5 kDa, respectively). Peptides for HNRPA1 covered the amino acid sequence containing the RNA recognition motif 1 (RRM1), RRM2 and a phosphothreonine phosphorylation site. Those for CHD4 contained the helicase superfamily C-terminal (HELICc) domain; the two chromatin organization modifier (CHROMO) domains, which play a role in the functional organization of the nucleus, were not present (data not shown). We do not know whether the expression of the HNRPA1 and CHD4 protein fragments (64% increase with CPA and no change, respectively) reflects that of the full-length proteins.
The majority of proteins identified, with the exception of HNRPA1, protein kinase R interacting protein (PRKRIP1) and CHD4, are known components of spermatozoal heads expressed in the nucleus, perinuclear theca (PT) subacrosomal layer and sheath or on the surface of the sperm. Information concerning the putative functions of these proteins was found in the National Centre for Biotechnology Information non-redundant and SWISS-PROT protein sequence databases or in the literature. In general, these proteins are involved in transcription and translation regulation (HNRPA1, PRKRIP1 and CHD4), chromatin organization [CHD4, HIST2H4 and histone H2B (HIST1H2BL)], sperm structure and stability (FABP9) and fertilization (HIST2H4, HIST1H2BL, ZPBP and beta-defensin 20).
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Several alkylating agents, including ethylnitrosourea, methyl methanesulphonate, thiotepa and triethylenemelamine, have been reported to result in sperm chromatin structural changes, as measured by flow cytometry (Evenson et al., 1985
; Evenson et al., 1986; Evenson et al., 1989; Evenson et al., 1993). In the present study, the SCSA®/AO DNA denaturation assay revealed significant epididymal sperm nuclear structural effects of CPA exposure; maximal changes occurred in mid-spermiogenic elongating spermatids. These findings are in accordance with our results using the comet assay, which showed increased DNA damage after chronic exposure to CPA and a similar maximal sensitivity to the drug in elongating spermatids (Codrington et al., 2004). Protamine content and thiol-disulphide status are sources of chromatin stability examined in this study, and reduced chromatin stability can affect genetic integrity. CPA effects on chromatin condensation may lead to stress in the chromatin structure and result in DNA strand breaks.
The fluorochrome CMA3 has been used as an indirect tool to detect abnormal sperm chromatin packaging, as increased CMA3 staining is indicative of decondensed, protamine-depleted spermatozoa (Bianchi et al., 1993; Manicardi et al., 1995; Bianchi et al., 1996). It is thought that either CMA3 accesses DNA in the minor groove of the DNA helix (Behr et al., 1969; Berman et al., 1985), making it a protamine competitor, or protamines bind within the major groove (Fita et al., 1983; Hud et al., 1994; Prieto et al., 1997), thereby possibly obstructing CMA3 access to the minor groove (Bizzaro et al., 1998). Classical CMA3 analysis, as documented in the literature and first developed using human and murine samples (Bianchi et al., 1993), failed to reveal an effect following drug exposure in rats. Mouse caput, corpus and cauda epididymal spermatozoa do not positively stain with CMA3 (Sakkas et al., 1995); however, in rats, the thiol-disulphide status of epididymal spermatozoal nuclei influences the binding capacity of CMA3. This and other studies (Shalgi et al., 1989) show that the proportion of reactive thiols decreases from 
85% in caput spermatozoa to 22% in cauda spermatozoa.
Interestingly, CPA affected male germ cells exposed as round spermatids, altering both sperm chromatin packaging, as assessed with the CMA3-binding assay, and genetic integrity, as assessed with the comet assay (Codrington et al., 2004). Although the reasons for this are unclear, it is possible that CPA-induced DNA damage in early-spermiogenic spermatids affects mRNA transcripts synthesized at this time, such as P1, which are required for spermatid differentiation. Decreased template function has been observed in sperm chronically exposed to this alkylating agent (Qiu et al., 1995a); this may result in transcription termination and truncated RNA molecules (Pieper et al., 1989; Pieper and Erickson, 1990; Gray et al., 1991). Indeed, studies on infertile men report decreased P1 transcript levels, thereby affecting the P1:P2 ratio and sperm chromatin structure (Aoki et al., 2005). P1, the only protamine expressed in rats, appears to be the most critical factor, in comparison to P2, for male fertility (Cho et al., 2001; Steger et al., 2003). Metabolites of CPA (phosphoramide mustard and acrolein) can alkylate nucleophilic sites of DNA, RNA and protein (Murthy et al., 1973; Sanderson and Shield, 1996). In addition to consequences of DNA damage on mRNA transcription, CPA could also bind directly with transcripts and affect protein synthesis.
Protamines are synthesized during mid- and late spermiogenesis and bind to late elongated spermatid DNA (Kistler et al., 1996). CMA3 staining was at its highest in spermatozoa exposed to CPA as elongating mid-spermiogenic spermatids; this is best explained by decreased P1 expression, which in turn could account for the noted reduction in thiol content. These results substantiate previous findings from our laboratory, which report decreased 14C-iodoacetamide binding in spermatozoal nuclei chronically exposed to CPA; however, an effect was also seen in cells not pretreated with DTT (Qiu et al., 1995b). Discrepancies may be accounted for by differences in the level of sensitivity of the methods used. In addition to decreased P1 content, an effect on available reactive thiols could be due also to increased alkylation of protamine thiol groups; protamines in the testis are especially susceptible to alkylation, thus blocking normal disulphide bond formation (Sega and Owens, 1983), preventing proper chromatin condensation and altering sperm structure. Indeed, the thiol-disulphide status of protamines determines the AO fluorescence of spermatozoal nuclei, such that increased susceptibility to acid-induced DNA denaturation occurs if DNA-associated protamines are poor in disulphides (Kosower et al., 1992; Zini et al., 2001). Previously, we reported earlier male pronuclear formation in rat oocytes fertilized by CPA-treated males, as well as altered sperm chromatin decondensation, both in vitro and in denuded hamster oocytes (Qiu et al., 1995b; Harrouk et al., 2000).
It would be of interest to examine modifications to protamines following CPA exposures that affect sperm chromatin structure, namely alkylation and phosphorylation. Protamine phosphorylation/dephosphorylation may be required for proper chromatin condensation; shortly after their synthesis, protamines are phosphorylated, thereby facilitating their correct binding to DNA. Once the DNA–protamine complex is formed, protamines are dephosphorylated and sperm then enter the epididymis in which final chromatin condensation occurs (Oliva and Dixon, 1991; Pirhonen et al., 1994). Standard 2D systems provide more information about a protein sample, but they do not resolve all proteins present in a sample, especially basic proteins with isoelectric points above 9. The 2D basic protein system used in this study did not resolve and identify modified protamines because they are extremely basic and rich in disulphide bridges; however, histones were present on the gels. It is tempting to speculate that these may be residual nuclear histones, although somatic histones are known also to be present in the subacrosomal sheath of the perinuclear theca (PT) (Tovich and Oko, 2003). The PT, a specialized cytoskeletal structure under the acrosome and surrounding sperm nuclei, has been implicated in acrosome-nuclear docking and nuclear shaping during spermiogenesis (Aul and Oko, 2002; Oko, 1995), as well as in oocyte activation and pronuclear formation (Sutovsky et al., 1997; Kimura et al., 1998; Manandhar and Toshimori, 2003; Sutovsky et al., 2003); the potential exists for PT-derived histones to contribute to male pronuclear development.
The histone–protamine exchange occurs along with other functionally important spermiogenic morphological and biochemical events. If protamine expression is altered, this could reflect a wide range of spermiogenic defects. Indeed, in this study, we have identified proteins involved in various aspects of spermatid differentiation, sperm maturation and fertilization, some of which had altered expression following CPA exposure. In addition, some of the proteins appear to be post-translationally modified, which altered their isoelectric point and, therefore, the 2D gel protein expression profile.
A few proteins with altered expression were identified, which have unknown functions during spermiogenesis. PRKRIP1, highly expressed in the testis, is a nuclear protein with high nucleolar expression. It acts as a negative regulator of PRKR (Yin et al., 2003). Decreased expression therefore, may relieve PRKR from inhibition. PRKR, expressed in spermatogonia (Melaine et al., 2003), inhibits protein synthesis by phosphorylating the initiation factor, eIF-2
(Hovanessian, 1989); it has been implicated also in apoptosis (Der et al., 1997), cellular transformation (Meurs et al., 1993; Donze et al., 1995), differentiation (Samuel et al., 1997) and transcription (Wong et al., 1997; Deb et al., 2001). It has been demonstrated that PRKR is involved in the cellular response to genotoxic stress, possibly by modulating DNA repair mechanisms to remove bulky DNA adducts (Bergeron et al., 2000).
HNRPA1 is a member of a group of core mammalian HNRP proteins that bind pre-mRNA and are involved in RNA processing (Krecic and Swanson, 1999). Many HNRPs are expressed in germ cells from the spermatogonial to the round spermatid phase of development, when RNA synthesis is known to cease (Biggiogera et al., 1993); however, the expression of HNRPA1 is restricted to spermatogonia (Matsui et al., 2000). Interestingly, the N-terminal end of HNRPA1 is cleaved to produce unwinding protein 1 (UP1), which contains the two RRM required for efficient RNA binding (Myers and Shamoo, 2004). It is possible that the proteolytic cleavage product present in our 2D gels is UP1. Both HNRPA1 and UP1 are also helix-destabilizing, single-stranded DNA-binding proteins (Nadler et al., 1991). More importantly, they are involved in telomere biogenesis (LaBranche et al., 1998). Reduced telomere length in sperm can affect embryogenesis; if not re-lengthened, germ cells containing shortened telomeres may limit the replication of cells derived from the zygote (Bekaert et al., 2004; Baird et al., 2006). Chemotherapeutic agents, including drugs such as bleomycin, mitomycin C and CPA, can cause telomere shortening (Kiyozuka et al., 2000; Arutyunyan et al., 2004). Increased expression of HNRPA1/UP1 may be in response to reduced telomeres in sperm following chronic CPA exposure.
This study used multiple assays to demonstrate that CPA exposure alters sperm chromatin structure and protein expression. Changes in proteins involved in sperm function, fertilization and post-fertilization events important for proper embryo development have been shown. An increasing number of groups are attempting to identify proteins of the sperm proteome (Pixton et al., 2004; Chu et al., 2006; Martinez-Heredia et al., 2006). We have used proteomic techniques to identify components of rat spermatozoa heads. The results of this study encourage further investigation into changes of the sperm proteome in response to exposure to CPA and other male-mediated developmental toxicants. The clinical significance of these analyses rests in their role in both natural and assisted reproduction success rates and the possibly high prognostic value in assessing fertility in cancer patients.
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We thank Martine Dupuis and Eric Masicotte at L'Institut de Researches Cliniques de Montreal for their enthusiastic assistance with the flow cytometer and Dr Rod Balhorn at Lawrence Livermore National Laboratory for his generous gift of the HUP1N protamine 1 antibody. We greatly appreciate the assistance of Leonid Kriazhev from the McGill University and Genome Quebec Innovation Centre with the 2D gel electrophoresis and mass spectrometry. This work was supported by a grant from the Canadian Institutes of Health Research.
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