Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print January 16, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.065151
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/5/822    most recent
biolreprod.107.065151v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wade, M. G.
Right arrow Articles by Yauk, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wade, M. G.
Right arrow Articles by Yauk, C.
Agricola
Right arrow Articles by Wade, M. G.
Right arrow Articles by Yauk, C.
BIOLOGY OF REPRODUCTION 78, 822–831 (2008)
DOI: 10.1095/biolreprod.107.065151
© 2008 by the Society for the Study of Reproduction, Inc.

Toxicology; 

Methoxyacetic Acid-Induced Spermatocyte Death Is Associated with Histone Hyperacetylation in Rats1

Michael G. Wade 2 3, Alice Kawata 3, Andrew Williams 5, and Carole Yauk 4

Systemic Toxicology and Pharmacokinetics Section,3 Biostatistics & Epidemiology Division,5 Environmental Health Sciences Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario, Canada K1A 0L2 Mutagenesis Section,4 Environmental & Occupational Toxicology Division,

ABSTRACT

We used high-density microarrays to evaluate the possible mechanisms by which 2-methoxyacetic acid (MAA) disrupts spermatogenesis. Levels of mRNA transcripts were determined in total RNA isolated from testes of MAA-treated (650 mg/kg i.p.) or concurrent control rats killed 4, 8, 12, or 24 h postexposure (PE). Germ cell death was examined in testis sections using in situ staining for DNA fragmentation. MAA treatment caused increased death of pachytene spermatocytes starting 8 h PE and increasing dramatically at 12 and 24 h PE. Microarray results indicated that at 4 h PE the transcript levels of seven different genes were significantly overrepresented in the testes of MAA-exposed animals. One gene (histone H1 zero [H1f0]) was significantly overrepresented in MAA-treated samples at 4, 8, and 12 h PE. Because expression of this gene has been associated with increased acetylation of core histones, we examined MAA-induced changes in the acetylation of histones H4 (HISTH4) and H3 (HISTH3) in testis nuclear protein. Western blots of acid-extracted testis nuclei indicated that the levels of tetraacetyl histone H4 (4acHIST1H4) and of diacetyl histone H3 (2acHIST1H3) were elevated by MAA treatment at 4, 8, and 12 h PE. Using the same antibodies, 4acHIST1H4 and 2acHIST1H3 were localized primarily to elongating spermatids in testis sections from control animals. At 4 h PE, staining for either histone modification was dramatically increased in spermatogonia and all primary spermatocyte populations except for dividing spermatocytes. MAA treatment of testis nuclear protein extracts from unexposed animals caused both a significant increase in histone acetyltransferase activity and a significant inhibition of histone deacetylase activity, suggesting that increased core histone acetylation results from a combination of these complementary modes of action. Our results indicate that exposure to MAA causes increased acetylation of core histones in several testis germ cell populations, including those in prophase of meiosis, a large proportion of which die rapidly following this treatment.

histone acetylation, meiosis, methoxyacetic acid, microarray, spermatogenesis, testis, toxicology

INTRODUCTION

Mammalian spermatogenesis is a complex process of division of diploid germ cells followed by meiosis and dramatic differentiation to highly modified haploid gametes. The transition from spermatogonia to spermatocyte coincides with the initiation of meiosis, during which homologous recombination occurs, DNA integrity is assessed and repaired, and the genome is halved through two sequential, highly ordered divisions. In addition, epigenetic programming of the sperm genome, encoded as methylation of DNA, is partially established during this phase. Meiotic prophase also corresponds to a peak in the rate of spontaneous death [1–3] through processes that are not fully understood. In addition, hormonal manipulation (e.g., [1, 3]), some physical agents (e.g., testis hyperthermia resulting from crytorchidism [4]), or exposure to chemical agents (e.g., [5–7]) all cause a dramatic increase in the rate of cell death in meiotic prophase. Impairment of DNA methylation, whether consequent to genetic deficiencies in the enzymes responsible for cytosine methylation [8–10] or due to treatment with drugs that impair this reaction [11–14], also causes increased death of cells in meiotic prophase. Investigation of the molecular basis of rapid death of spermatocytes induced by chemical agents will very likely uncover molecular or signaling processes that are critically important to meiosis and/or germ cell differentiation.

One agent that causes spermatocyte death is methoxyacetic acid (MAA). MAA is an in vivo metabolite of ethylene glycol monomethoxy ether (EGME), a solvent used in a variety of industrial and household applications, including inks, paints, and water-based cleaners, and as an anti-icing additive in fuels, hydraulic brake fluids, and aircraft de-icing fluid [15]. Exposure to either EGME or MAA results in impaired fertility consequent to a reversible disruption of spermatogenesis in a variety of mammalian species (e.g., rats [16], mice [17], guinea pigs [18], and rabbits [19]). Recent studies in our laboratory have also implicated MAA as the active metabolite in disruption of spermatogenesis resulting from 1,6-dimethoxyhexane exposure [20]. The primary testicular effect of MAA is the rapid (<12 h) induction of cell death in pachytene spermatocytes [7, 21, 22]. Dying spermatocytes in EGME-treated rats appear necrotic, suggesting that toxicant-induced damage impairs cell integrity [21]. However, the pattern of chromatin fragmentation in these cells indicates that affected cells are undergoing programmed cell death, suggesting that MAA impairs some physiological factor or signal required to maintain cell viability [23].

Despite considerable effort directed toward understanding the cellular responses to EGME or MAA in inducing testis toxicity, the precise mechanism of toxicity is poorly understood. A number of studies using pharmacological agents have shown that oxidative stressors [24], various protein kinases [25] and, in particular, protein tyrosine kinase src [26], voltage-gated ion channels [27, 28], and calcium channels [29, 30] all seem to mediate MAA-induced germ cell toxicity. Upon further investigation of the role of calcium channels, this latter group [31] failed to detect, in rat seminiferous tubules, immunoreactivity or functional activity of the calcium channels sensitive to the pharmacological agents tested. These results suggest that blockade of MAA-induced spermatocyte death by these pharmacological agents is due to the agents' effects on some mechanism(s) other than impairment of function of voltage-operated calcium channels. Other investigators have suggested that MAA alteration of steroid hormone receptor signaling (either estrogens [32] or androgens [33]) underlies MAA-induced spermatocyte toxicity. In addition, several studies have attempted to determine the mechanism of EGME- or MAA-induced spermatocyte toxicity by evaluating genome-wide patterns of gene expression, using either differential display hybridization [34], suppression-subtractive hybridization [35] or microarrays [36], or by examining changes in protein expression levels using proteomic analyses [37]. However, all of these studies have examined the response at only a single time point postexposure (PE) and none have led to a clear hypothesis of how MAA causes death in spermatocytes.

In the current study, we have investigated the response of the testis transcriptome over time after a single exposure to MAA using high-density microarrays and have correlated changes to steady-state mRNA levels with morphological and biochemical evidence of germ cell responses. By determining the temporal changes in both gene transcript levels and cell death we have been able to focus on the relatively few tissue responses that precede the frank expression of toxicity, as these are most likely to be related to the mechanism of toxic action. These studies provide evidence that MAA-induced death of meiotic germ cells is preceded by the hyperacetylation of core histones in these cells.

MATERIALS AND METHODS

Chemicals and Reagents

MAA (CAS# 625-45-6; 98% purity), sodium butyrate (But; 99%), sodium valproate (VPA; 98%),and trichostatin (TSA; 98%) were purchased from Sigma Aldrich Chemical Canada (Oakville, ON). Rabbit anti-acetyl histone antibodies were obtained from Millipore (Charlottesville, VA). They included anti-acetyl H4 (4acHIST1H4; Cat #06-866), specific for acetyl-lysines K5, K8, K12, and K16, and anti-acetyl H3 (2acHIST1H3; Cat #06-599), specific for acetyl-lysines K9and K14. Goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP; 12-348) and non-immune rabbit serum (S20-100ML) were also purchased from Millipore. Vectastain Elite ABC kit, HRP label, and diaminobenzidine (DAB) were purchased from Vector Laboratories (Burlington, ON).

Animals

Sexually mature Sprague-Dawley rats (8–9 wk of age) were purchased from Charles River Canada (St. Constant, QC) and housed in pairs. For studies of testis histone deacetylase (HDAC) activity, male pups were purchased as 13-day-old pups with lactating dams. All animals were housed in hanging polycarbonate cages on hardwood chip bedding with free access to water and laboratory rat chow (Purina 5008, Agribrand Purina; St-Hubert, QC) under controlled photoperiod (12L:12D), humidity (40%–70%), and temperature (20–24°C). All males were acclimatized to the study facilities for at least 12 days prior to exposure to test substances. All animal handling procedures adhered to Canadian Council on Animal Care guidelines and were approved by the Health Canada Animal Care Committee prior to the initiation of the study.

For microarray studies of MAA effects, animals were randomly assigned to vehicle or MAA groups of 24 animals each, with four subgroups (n = 6) to be killed at 4, 8, 12, or 24 h PE. On the morning of the experiment, all animals were injected i.p with 10 ml/kg of sterile saline (vehicle) or 650 mg/kg of MAA (in deionized water, pH adjusted to 7.2 with 10 M NaOH). This dose and route of exposure have previously been reported to cause rapid death of spermatocytes in a similar model [32]. Six males from each group were killed by CO2 asphyxiation at 4, 8, 12, and 24 h PE, and the testes were rapidly removed. The right testis was decapsulated and the parenchyma was divided into four roughly equal pieces that were individually snap-frozen in liquid nitrogen and stored at –80°C until analysis. The left testis was fixed for histological processing and analyses.

Microarray Analysis

Total RNA was extracted from one of the portions of the right testis from each animal using the RNaqueous-4PCR kit (Ambion, Austin, TX) and quantified using the Ultraspec 3100 UV/visible spectrophotometer (Biochom, Cambridge, England). Total RNA quality was assessed using the RNA 6000 Nano LabChips with the 2100 Bioanalyzer system (Agilent Technologies Inc., Mississauga, ON, Canada). Only samples with the ratios of 28S/18S ribosomal RNA intensity between 1.65 and 2.0 were used for microarray analyses.

Individual RNA samples were labeled with Cyanine 3-CTP (Perkin Elmer Life Sciences, Woodbridge, ON, Canada) and rat universal reference total RNA (BD Biosciences Clontech, Palo Alto, CA) were labeled with Cyanine 5-CTP (Perkin Elmer Life Sciences) using Agilent low RNA input fluorescent linear amplification kits (Agilent Technologies Inc.). Briefly, double-stranded cDNA was synthesized using MMLV-RT with T7 promoter primer, starting with 2 µg total RNA. Cyanine-labeled cRNA targets were transcribed in vitro using T7 RNA polymerase. The synthesized cRNA was purified using Qiagen's RNeasy mini kit (Qiagen, Mississauga, ON, Canada). Labeled cRNA (0.75 µg) was fragmented at 60°C for 30 min with fragmentation solution. Cy3- sample cRNA and Cy5- universal reference cRNA were hybridized to Agilent rat oligo microarrays (containing ~20 000 unique 60 mer oligonucleotides; Cat #G4130A; Agilent Technologies Inc.) at 60°C overnight with Agilent hybridization solution and washed according to the manufacturer's instructions. Arrays were scanned on a ScanArray Express (Perkin Elmer Life Sciences), and data were acquired with ImaGene 5.5 (BioDiscovery Inc., El Segundo, CA). Probes with fluorescent intensity greater than mean background plus 3 SD of background intensity were identified as being expressed (called present).

Real-Time RT-PCR Analysis

RT-PCR was used to confirm differential gene expression of transcripts from microarray analyses. Reverse transcription was carried out in a 20-µl reaction mix with the QuantiTect Reverse Transcription kit (Qiagen) using 1 µg of total RNA per animal. Quantitative PCR was performed with an iCycler iQ real-time detection system using iQ SYBR green supermix (BioRad, Mississauga, ON, Canada) and gene-specific primers (QuantiTect, Qiagen). PCR reactions were performed in duplicate, and the values of threshold cycle were averaged. Gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase. PCR efficiency was estimated using the standard curve for each gene. The primer specificity was determined by the melting curve for each gene. Relative expression software tool was used for statistical and fold change evaluation of each gene as described elsewhere [38].

Statistical Analysis of Microarray Data

A partially confounded block design [39] for the control and exposed rats at four time points was used to analyze the data. The block design was based on the date of hybridization of the slides, as four slides were hybridized per day. Data from one 4-h control sample were excluded from the analyses based on concerns that differences in sample collection may have led to differences in sample results.

The data were normalized by LOWESS curve [40] using the SAS/STAT software [41]. Ratio intensity plots for the raw and normalized data, boxplots and heatmaps, were constructed for each array using R language [42] and used to evaluate data quality.

To detect differentially expressed genes between the control and treated groups at any of the four time points, an ANOVA model was applied using the MAANOVA library [43] in R. The model included the date of hybridization as a block effect and fixed effects of time and treatment with a treatment by time interaction. This model was applied to the log 2 relative intensities. The Fs statistic [44], a shrinkage estimator for the gene-specific variance components, was used, and the P-values for all the statistical tests were estimated using the permutation method (2000 permutations with residual shuffling). These P-values were then adjusted for multiple comparisons by using the Benjamini-Hochberg false discovery rate approach [45]. Estimated marginal means also known as least square means [46, 47] were estimated for each group. These means are a function of the model parameters and are adjusted for the other factors in the model such as date of hybridization. The least square means were then used to estimate the fold change for each contrast that was tested.

Effect of MAA on HDAC and Histone Acetyltransferase Activity Assays

The influence of MAA on rat testis nuclear or cytosolic HDAC activities was determined using the HDAC fluorimetric assay/drug discovery kit (BioMol International, Plymouth Meeting, PA). Approximately 30 mg of testis tissue from an untreated juvenile rat (27 days old) was homogenized in 400 µl of buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 1X Complete mini protease inhibitor [Roche Diagnostics Canada, Laval, QC]; pH 7.9). The cell lysate was incubated on ice for 10 min, vortexed vigorously for 10 sec, then centrifuged at 14 000 rpm for 30 sec, and supernatant (cytosolic extract) was retained. The remaining cell pellet was resuspended in 100 µl of Buffer B (20 mM HEPES, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM DTT, 1X Complete mini protease inhibitor; pH 7.9) and incubated on ice for 20 min. After centrifuging at 14 000 rpm for 3 min, the second supernatant (nuclear extract) was transferred into a new tube. Total protein was assayed using Bradford Reagent (Sigma Aldrich Chemical).

To assay HDAC activity, 0.3 mg protein per ml of HeLa cell extract (supplied with the kit), the testicular cytosolic or nuclear extracts were incubated with various test compounds and 50 µM of the HDAC kit Fluor de Lys substrate for 45 min. The compounds tested were MAA (0.5, 2, or 5 mM), trichostatin (TSA; 0.1 µM: a potent HDAC inhibitor) sodium butyrate (But; 5 mM: a weak inhibitor) and sodium valproate (VPA; 0.5, 2, or 5 mM: a weak inhibitor). The reaction was stopped by adding Fluor de Lys developer and incubated for 15 min. The fluorescence generated by the deacetylated substrate was measured using the SpectraMax M2 multidetection microplate reader (Molecular Devices). The assay was repeated on four separate occasions using freshly prepared tissue extracts, with triplicate incubations for each preparation and test substance combination.

The influence of MAA on rat testis nuclear histone acetyltransferase (HAT) activity was also examined. Nuclear extracts were prepared from adult rat testes using the Nuclear/Cytosol Extraction kit (K266–100; Biovision Inc.) according to the manufacturer's protocol, with the exception that DTT was not added to any of the extraction buffers. HAT activity was determined in incubations containing 50 µg of nuclear protein in the presence of various MAA concentrations (HAT activity assay kit; K332–100; Biovision Inc.). Reaction progression was monitored by changes in NADH absorbance (440 nm) over a 2-h period using SpectraMax Plus384 spectrophotometer (Molecular Devices). Enzyme activity was estimated from the linear part of the activity curve and expressed as a percent of activity in the absence of MAA.

Western Blot Analyses

Frozen testis (about 30 mg) was homogenized in 200 µl of ice-cold homogenization buffer (Dulbecco PBS [Invitrogen, Burlington, ON] containing 0.5% TritonX 100 [Sigma Aldrich Chemical], 1X Complete mini protease inhibitor, and 5 mM of But; pH 7.9) and incubated for 10 min on ice. The homogenate was then centrifuged at 2000 rpm for 10 min at 4°C, and the pellet, containing nuclei, was rehomogenized in 100 µl of ice-cold homogenization buffer and centrifuged as above. The pellet was then resuspended in 0.2 N HCl using a micropestel. After overnight incubation at 4°C, homogenates were centrifuged as above, and supernatant aliquots were stored at –80°C until analyzed by Western blot. Protein was determined using Bradford reagent (Sigma).

Nuclear extracts were resolved on a 15% polyacrylamide gel, transferred to nitrocellulose membranes (Bio-Rad Laboratories), and probed with anti-4acHIST1H4 (1:2000) or anti-2acHIST1H3 (1:5000) and with secondary HRP-conjugated goat anti-rabbit (1:5000; Upstate). Blots were visualized using ECL plus Western blotting detection reagent (GE Healthcare Life Sciences Canada, Baie d'Urfe, QC) and imaged using a Typhoon Trio+ variable mode Imager (GE Healthcare). The intensity of staining was quantified on digital images of blots using ImageProPlus (MediaCybernetics, Bethesda, MD).

Testis Histology and Histochemistry

Testes were fixed in modified Davidson fix [48] for 48 h, postfixed for 24 h in 10% neutral buffered formalin, and embedded in paraffin. Germ cell death was visualized in testis sections by TUNEL staining using a commercial kit (R&D Systems, Minneapolis, MN). Testis sections were also stained for acetylation of histones H3 and H4 following antigen retrieval. Briefly, 5-µm paraffin sections were rehydrated and immersed in retrieval solution (1 mM EDTA, 0.05% Tween 20; pH 8.0) at 95°C for 40 min then washed twice (5 min) in PBS. After background peroxidase quenching (0.3% H2O2 in methanol for 30 min, room temperature), slides were probed for 4acHIST1H4 or 2acHIST1H3, and staining was visualized using the anti-rabbit Vectastain Elite ABC kit (Vector Laboratories). Adjacent sections were stained with PAS to allow accurate staging of seminiferous tubules [49].

To determine the testis cell types in which MAA caused acetylation of core histones, testis sections were stained with the same antibodies against 4acHIST1H4 and 2acHIST1H3 that were used in immunoblot above. As it has been shown that hyperacetylation of core histones occurs in elongating spermatids (spermatid steps 9–12) and a previous study reported that 4acHIST1H4 was not detected in pachytene spermatocytes [50], we titrated the primary antibody to replicate this staining pattern. In the absence of EDTA treatment, no staining was seen in testis sections with either antibody at a concentration of 1:200. However, EDTA pretreatment dramatically improved antigen detection and intense staining of condensing spermatids, and significant staining of virtually all testis cell nuclei was seen for both antibodies at this dilution. Incubation of EDTA-pretreated testis sections from control animals with serial dilutions of anti-4acHIST1H4 revealed that a dilution of 1:10 000 resulted in negligible staining of spermatocyte, spermatogonia, most spermatids, and all somatic nuclei in all seminiferous tubule stages, but caused intense staining of condensed spermatids (e.g., step 12 spermatids; see Fig. 6E). Serial dilutions of anti-2acHIST1H3 incubated with similar sections revealed that a dilution of 1:2000 resulted in negligible staining in most germ cell nuclei but barely visible staining in condensing spermatids (not shown). Increasing the antibody concentration to 1:1500 resulted in intense staining in these latter nuclei and light staining in various germ cell nuclei in control testes.


Figure 06
View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6. Changes in the acetylation of histones H4 (A, C) and H3 (B, D) in acid extracts of whole testis nuclear fraction with time after MAA treatment. A representative of three replicate blots for 4acHIST1H4 (A) and 2acHIST1H3 (B) are depicted. Densitometry quantitation from all blots examined, normalized to the mean of control bands, is provided for 4acHIST1H4 (C) and 2acHIST1H3 (D). Asterisk (*) indicates significant increase relative to pooled control (P < 0.05).

Statistical Analyses

The incidence of TUNEL-positive cells was expressed as the number of positive cells per tubule examined for one entire testis section per animal. These data were log transformed and analyzed by two-way ANOVA, with time PE and treatment being the factors examined. Data from each repeat experiment for HDAC activity assay were analyzed separately due to interassay variability. All activity data for a given enzyme preparation were converted to percent of the mean for their respective control. Converted data were tested for normality (Kolmogorov-Smirnov test) and for homogeneity of variance (Levene Median test) and analyzed via parametric or nonparametric (Kruskal-Wallis) ANOVA depending on the results. Specific effects were identified using Student-Newman-Keuls multiple comparisons. Statistical analyses for nonmicroarray data were performed using SigmaStat 3.1 (Systat Software Inc., San Jose, CA).

RESULTS

Progression of Germ Cell Death

Treatment of male rats with MAA resulted in a marked increase in spermatocyte death, as indicated by positive TUNEL staining in testis sections of animals killed 12 and 24 h PE. There was no significant difference in the number of TUNEL-positive cells between MAA-treated and control rats at 4 h (Fig. 1). At 8 h PE, the number of TUNEL-positive cells was slightly but significantly increased in testes from MAA-treated animals, whereas at later time points this effect was markedly increased (P < 0.01; Fig. 1). The cell types identified as TUNEL positive in sections from MAA-treated animals were primary spermatocytes mainly at the pachytene or diplotene stages, with a few zygotene spermatocytes observed (Fig. 2). In control animals, apoptotic cells were either dividing meiotic cells in stage XIV or early pachytene spermatocytes in stage I tubules.


Figure 01
View larger version (7K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1. Changes over time in the number of TUNEL-positive germ cells present in rat seminiferous tubule cross sections after exposure of rats to saline (control) or MAA. Data are expressed as the mean ± SD of all positively stained cells divided by the number of tubules examined for three control or four MAA-treated animals per time point. Asterisk (*) indicates significant difference (P < 0.05) from concurrent control value.


Figure 02
View larger version (98K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2. The timing and pattern of germ cell death after MAA exposure. A representative TUNEL-stained cross section of a stage XIII seminiferous tubule from a section of testis from control (A, concurrent to 24 h PE) and MAA-treated animal at 4 (B), 8 (C), 12 (D), and 24 (E) h PE. St, Condensed spermatids; Di, diplotene spermatocytes; z, TUNEL-negative zygotene spermatocytes; z', TUNEL-positive zygotene spermatocytes; Sg, spermatogonia; s, Sertoli cells. All micrographs are the same magnification and the bar in A = 40 µm for A–E.

Microarray Analysis

The data from microarray analyses are publicly available at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/GEO Accession Number GSE10032). Using Benjamini-Hochberg False discovery rate [34] adjusted P-values of the F-test, we found that the transcript levels of 814 genes were significantly altered (P < 0.05) by MAA treatment at one or more sampling times PE (data not shown). Only seven and six genes exhibited significantly altered transcript levels at 4 and 8 h, respectively, whereas the transcript levels of 500 and 513 genes were found to be altered at 12 and 24 h PE (data not shown).

As the initial focus of this research was on the immediate mechanism of toxicity of MAA, we will focus only on genes observed to be altered at 4 h PE in the current manuscript. Longer-term response (i.e., at 8, 12, and 24 h PE) will be examined in detail in a subsequent publication. All gene expression changes observed at 4 h PE were the result of transcript overrepresentation in testes from MAA-treated animals (Table 1). Of these, three of the seven genes were significantly different from control exclusively at the 4 h time point (Fig. 3). Of the genes that were differentially expressed at 4 h, histone H1 zero (H1f0) was the only gene for which transcripts remained significantly elevated in MAA-treated samples at both 8 and 12 h PE (Fig. 3). Real-time PCR quantification of H1f0 transcripts confirmed the pattern of expression revealed by microarray data (Table 2).


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 1. The identity and major function of all seven genes whose transcript levels were found to be significantly altered by MAA at 4 h PE (Benjamini-Hochberg adjusted; P < 0.05).a


Figure 03
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3. Changes in relative transcript levels over time for each of the seven genes whose transcript levels were significantly altered 4 h PE to MAA. The identity and description of the seven genes are provided in Table 1. Asterisk (*) denotes that the transcript levels were significantly altered by MAA treatment at the indicated time PE (Benjamini-Hochberg corrected P < 0.05).


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 2. Verification of microarray results by RT-PCR quantitation of mRNA levels of H1f0.

Effect of MAA on HDAC/HAT Activities and Acetylated Histones in Testis

As elevated transcript levels of histone H1f0 have been reported from cells or tissues in which the enzyme HDAC has been inhibited or in which core histones have been found to be highly acetylated [51–55], we examined the effects of MAA on HDAC activity in vitro and the degree of histone acetylation in the testes of MAA-treated animals. To determine if MAA inhibits HDAC activity in the testis, juvenile rat testes (27 days old) were used as a source of enzyme activity because these contain a much higher proportion of meiotic germ cells relative to testes from sexually mature rats due to the reduced proportion of spermatids at this age. Control data indicated that both cytosolic and nuclear testis fractions demonstrated considerable HDAC activity; however, the nuclear fraction routinely demonstrated much higher activity than the cytosolic (3.3 ± 0.8-fold higher; mean and SD of four replicate assays). Like HeLa cell extract HDAC activity, the activity in both nuclear and cytosolic extracts was inhibited by both strong (TSA) and weak (But and VPA) HDAC inhibitors (Fig. 4). Addition of MAA to the reaction mixture caused a concentration-dependent inhibition of HDAC activity in both testis fractions and in HeLa cell nuclear fraction (Fig. 4).


Figure 04
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4. The influence of MAA, VPA, TSA, and But on HDAC activity in HELA cell nuclear extract (A), testis cytosolic (B), and nuclear extract (C). Activity depicted is the mean of pooled data from four separate replicate assays each using freshly extracted testis and a fresh aliquot of commercial HELA cell nuclear extract. All data points were normalized to the mean of their respective control. Asterisk (*) denotes significant difference from control (P < 0.05).

As histone acetylation occurs through the action of HATs, we examined the effects of MAA on HAT activity in nuclear protein extracted from whole testis from sexually mature rats. HAT activity was significantly increased by addition of 0.5 mM MAA (Fig. 5), and this effect was enhanced with increasing MAA concentration.


Figure 05
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5. The effects of MAA on cell-free HAT activity in testis nuclear protein. Activity of HAT is expressed as the percent of control values. Values represent mean activity from three separate extracts (each from different animals; assayed in triplicate). Asterisk (*) indicates significant difference (P < 0.05) from control.

To determine the effect of MAA exposure on testis histone acetylation, testis nuclear proteins were fractionated by Western blot and probed with anti-4acHIST1H4 or anti-2acHIST1H3. 4acHIST1H4 was detected in all samples as an intensely stained band at 14 kDa and a second, fainter band at approximately 16 kD (Fig. 6A). This antibody has previously been reported to detect fainter, larger bands in addition to acetylated HIST4H4 [56], suggesting, as the manufacturer indicates, some cross-reactivity with other acetylated nuclear proteins. Treatment with MAA led to a sharp increase in intensity of both bands, particularly the 14-kDa band, over concurrent control (Fig. 6, A and C; P < 0.05), and these bands remained significantly more intensely stained than control in MAA-treated animals at all periods (Fig. 6C). 2acHIST1H3 was detected in all samples as a band of approximately 17 kDa (Fig. 6B). As with 4acHIST1H4, the intensity of 2acHIST1H3 staining was markedly increased 4 h after MAA treatment and remained significantly more intensely stained up to 24 h PE (Fig. 6D). These results clearly demonstrate that MAA causes hyperacetylation of these core histones in the testes.

Testis sections from all animals (control and MAA treated) killed at 4 h PE were stained for either 4acHIST1H4 or 2acHIST1H3 using the optimum concentration of the respective antibodies. As expected, the intensity of both 4acHIST1H4 and 2acHIST1H3 staining was markedly increased in many cell types in MAA-treated animals (Fig. 7, B, D, and F, and Fig. 8, B, D, and F). To determine the pattern of MAA-induced increase in histone acetylation, the intensity of staining in germ cell types was evaluated semiquantitatively in a stage-specific manner by an observer who was unaware of animal treatment. MAA clearly increased the acetylation of histone H4 in germ cells at stages of differentiation from spermatogonia through to pachytene spermatocytes. Of these, spermatogonia and early spermatocytes (preleptotene and leptotene) appeared to be the most markedly effected, while the intensity of MAA-induced staining declined with progression through meiosis, with late pachytene and diplotene spermatocytes having minimal staining, and dividing spermatocytes showing no acH4 staining (Table 3). Immediately, postmeiotic round spermatids showed moderate staining (Fig. 7B) in response to MAA, but the degree of MAA-induced staining declined with progression and was absent in round spermatids from stages IV through VIII. There was no apparent effect of MAA on the intensity of 4acHIST1H4 staining in condensing spermatids in tubule stages IX–XII (Table 3; Fig. 7F). Notably, there was no 4acHIST1H4 staining seen in any Sertoli cells observed in any tubules in any of the control or MAA-treated animals (Fig. 7, A, C, and E).


Figure 07
View larger version (143K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 7. The distribution of 4acHIST1H4 immunoreactivity in seminiferous tubules at stages II (A, B), VIII (C, D), and XII (E, F) 4 h after saline (A, C, E) or MAA (B, D, F) treatment. St, Condensing spermatids; r, round spermatids; Di, diplotene spermatocytes; p, pachytene spermatocytes; z, zygotene spermatocytes; pl, preleptotene spermatocytes; Sg, spermatogonia; s, Sertoli cells. All micrographs are at the same scale and the bar in A = 40 µm for A–F.


Figure 08
View larger version (165K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 8. The distribution of 2acHIST1H3 immunoreactivity in seminifierous tubules at stages II (A, B), VIII (C, D), and XII (E, F) 4 h after saline (A, C, E) or MAA (B, D, F) treatment. St, Condensing spermatids; r, round spermatids; Di, diplotene spermatocytes; p, pachytene spermatocytes; z, zygotene spermatocytes; pl, preleptotene spermatocytes; Sg, spermatogonia; s, Sertoli cells. All micrographs are at the same scale and the bar in A = 40 µm for A–F.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 3. Relative intensity of immunostaining of various spermatogenic cell populations for acetylation of histones H3 and H4 in control and MAA-treated testes.a

As described above, 2acHIST1H3 staining was observed in all premeiotic (spermatogonia) and early meiotic germ cells (preleptotene, leptotene, and zygotene spermatocytes) in testis sections from 4 h PE concurrent control animals. Beyond slight staining seen in some early pachytene spermatocytes, there was no staining observed in other meiotic spermatocytes in control testes (late pachytene, diplotene, and dividing spermatocytes) and only slight staining in some early round spermatids (Fig. 8, A, C, and E). In testes from MAA-treated animals, there was increased staining intensity in spermatogonia, preleptotene, leptotene, zygotene, pachytene, and diplotene spermatocytes (Fig. 8, B, D, and F). As with 4acHIST1H4, 2acHIST1H3 staining intensity in MAA-treated spermatocytes declined from zygotene, which were very intensely labelled, through to diplotene, which were only faintly stained. Dividing spermatocytes showed no staining in either control or MAA-treated testes. There was no obvious MAA-induced increase in 2acHIST1H3 staining in condensing spermatids. As with 4acHIST1H4, Sertoli cells showed no staining for 2acHIST1H3 in any tubule in control or MAA-treated sections. These results demonstrate that MAA induces histone hyperacetylation in all premeiotic and meiotic prophase germ cells and some postmeiotic germ cells, but not in Sertoli cells.

DISCUSSION

In the current study we provide evidence suggesting that rapid spermatocyte death following MAA exposure is associated with hyperacetylation of the core histones H3 and H4 in these cells. The data suggest that hyperacetylation of histones results from MAA-induced inhibition of testis HDAC activity and activation of HAT activity.

The use of high-density microarrays, in combination with an experimental design that incorporates concurrent controls and that allows for the neutralization of significant confounding factors (especially day of hybridization), has yielded a high-quality data set that permits the identification of genes with modestly changed expression levels. Statistical analyses of this data set identified changes in the transcript levels of very few genes at 4 h PE, and only one of these, H1f0, showed a sustained increase over time. Therefore, investigation of the factors that modulate histone H1f0 transcription may provide insight into the molecular mechanisms of MAA toxicity. The mRNA of H1f0 is frequently measured as an indicator of cell response to But [51, 52, 54] and TSA [53, 55] and is strongly correlated with increased acetylation of core histones [55], possibly as a compensatory response of the cell to reduce histone hyperacetylation [57]. Analysis of the temporal and cell/stage-specific pattern of histone H3/H4 hyperacetylation in response to MAA demonstrates that the increased acetylation of the core histones of the cell types vulnerable to MAA toxicity either precedes or occurs concurrently with the onset of cell death, providing support to the hypothesis that hyperacetylation leads to spermatocyte death.

The reversible acetylation of histones has been suggested to play a very significant role in freeing DNA from its tight association with nucleosomes, likely due to acetylation-induced neutralization of the charged lysine residues on the amino terminal tail regions of all core histone subtypes [58]. Acetylation of core histones is essential to a) allow histone insertion into newly synthesized DNA [59], b) facilitate access of cis acting gene regulatory elements to transcriptional machinery [60], and c) permit the replacement of nucleosomes (core histone hetero-octamers) with transition proteins and protamines during spermatid nuclear restructuring and DNA condensation [50, 61]. As germ cells progress normally through spermatogenesis, the patterns of acetylation of core histones have been shown to change in a consistent fashion. As noted above, hyperacetylation of core histones occurs in maturing spermatids (step 9–12 spermatids in rats) in association with nuclear condensation [50, 61–63]. Other testis germ cell populations have also been observed with acetylated core histones, particularly spermatogonia, preleptotene spermatocytes, and round spermatids [50, 61, 62]. Histones from these cell types were reported to have predominantly monacetylated forms [62], although a study in mouse testes reported polyacetylation of histone H4 in spermatogonia and preleptotene spermatocytes [50]. In situ analysis of histone acetylation in meiotic prophase germ cells indicates a complete absence of acetylated histone H4 in any primary spermatocytes [50, 62, 64]. Our current results in control animals showing a complete lack of staining of nuclei of any germ cell other than step 9–12 spermatids for acetylated histone H4 agree with these previous studies. Moreover, staining of adjacent sections with an antibody that recognizes 2acHIST1H3 revealed acetylation in spermatogonia, while staining was absent in all other germ cell nuclei except condensing spermatids. Our results also indicate that 4 h after MAA treatment, staining for either of these modifications was markedly increased in all premeiotic (spermatogonia, preleptotene spermatocytes), most meiotic (leptotene, zygotene, pachytene, diplotene) and at least some postmeiotic (early round spermatids) germ cell stages (Figs. 7 and 8). Although we did not evaluate in situ staining for histone acetylation at other times PE, Western blot analyses indicate that levels of acetylation of both of these histones remained elevated at least until 12 h PE. The changing pattern of hyperacetylation of both histone targets revealed by Western blot (Fig. 6) closely mirrors the changing pattern of H1f0 transcript levels, which were highest at 4 and 8 h PE (Fig. 3). Given that no staining of either acetyl-histone modification was seen in Sertoli cells in control or MAA-treated animals, the increased H1f0 expression is most likely due to the direct effects of MAA on germ cell nuclei.

Our data show that MAA treatment causes acetylation of histones H3 and H4 in the nuclei of several germ cell populations. Moreover, we have also demonstrated that MAA both inhibits the activity of HDAC and increases activity of HAT in protein extracts from testis tissue. The dose dependencies of both of these effects are comparable to that reported for in vitro inhibition of human type 1 HDAC (HDAC 1, 2, and 3 and HELA cell nuclear extract) by MAA [65]. In the rat testis nuclear fraction, which we have determined contains HDAC1 immunoreactivity (Wade et al., unpublished observation), HDAC activity was reduced to 62% of control by 5 mM MAA (Fig. 4C). This is very similar to previously published data showing a 50% reduction in HDAC1 activity induced by the same MAA concentration [65]. The testis has also been shown to be the major site of Hdac6 expression [66]; we have observed that immunoreactivity of this type II HDAC was primarily associated with the cytosolic fraction (Wade et al., unpublished results). The activity of HDAC in the cytosolic fraction was equally sensitive to MAA, as was the nuclear fraction. Although we did not evaluate the concentration of MAA in the testes of treated animals, previous studies on MAA report serum levels of MAA around 5 mM 4 h after a single oral exposure to MAA precursor methoxyethanol at a dose that caused spermatocyte death [67]. The close match between the median effective concentration for in vitro inhibition of HDAC1 activity and the reported in vivo serum concentration of MAA that precedes spermatocyte death suggests a causal role for histone hyperacetylation in this lesion. Moreover, the observed effects of MAA in enhancing HAT activity and inhibiting HDAC activity, with comparable dose dependency, suggests that MAA-induced increase in histone acetylation is the result of these combined activities.

It is not clear what role, if any, histone hyperacetylation plays in the MAA-induced apoptotic deletion of pachytene spermatocytes. However, other substances with HDAC inhibitory activity have also been reported to disrupt spermatogenesis. Valproic acid is a widely prescribed anticonvulsant and mood stabilizer that has recently been recognized to have HDAC-inhibiting activity [68]. Chronic or subchronic administration of VPA to rats or beagle dogs has been shown to cause reduced testis size, atrophy of seminiferous epithelium, and reduced fertility [69–72]. In addition, repeated exposure of mice to potent HDAC inhibitor TSA has been shown to dramatically disrupt spermatogenesis [64]. This latter study identified pachytene spermatocytes as the target of TSA toxicity, because a quantitative evaluation of germ cells in testis sections revealed a sharp reduction in the ratio of pachytene to zygotene spermatocytes in TSA-exposed animals. These cells are also the primary target of MAA (Table 3) in the testis, which suggests a similar mechanism of action for these substances.

Although these observations suggest an association between histone hyperacetylation and spermatocyte death, further investigation will be necessary to confirm a causal role for this modification. Also, the germ cell stages most vulnerable to the killing action of MAA, pachytene and diplotene (Table 3), are not the cells that are most heavily labeled for polyacetylated histones. However, consideration of the nuclear events that are occurring in these cells as they progress through prophase 1 of meiosis, in particular the dynamic changes in DNA methylation that occur during this transition and the importance of histone modifications in mediating this process, may provide clues to the vulnerability of these cells. Recent reports have demonstrated that the transition from spermatogonia to meiosis coincides with marked changes in methylation of DNA at diverse sites throughout the genome ([73, 74]) and that inhibition of DNA methylation results in disruption of spermatogenesis [11–13]. Moreover, male transgenic mice that lack a functional gene for Dnmt3l, the enzyme largely responsible for creating novel CpG methyl sites during meiosis in mammalian germ cells, are sterile due to meiotic failure [8]. Intriguingly, germ cells in the testes of sexually mature dnmt3l knockout mice show intense acetylation of histones H4 and H3 [10] similar to the pattern seen in MAA-treated testes, suggesting that core histone acetylation is intimately related to meiotic failure and spermatocyte apoptosis.

The current results are the first observation that MAA causes covalent modifications of chromatin proteins in spermatogenic cells that precede the onset of cell death. Although our data do not provide proof that these changes contribute to spermatocyte death, the recognized role of chromatin structure in regulating meiotic events and DNA methylation suggest at least a role for MAA-induced increased histone acetylation in spermatocyte death.

ACKNOWLEDGMENTS

The authors would like to thank Lynn Berndt for her assistance in performing microarray hybridization and Lorraine Casavant for her assistance with immunohistochemical procedures. We would also like to thank Drs. Vern Seligy, George Douglas, and Guillaume Pelletier for their valuable critiques of this manuscript.

FOOTNOTES

1Supported in part by a grant from the Health Canada Office of the Chief Scientist. The data from microarray analyses are publicly available at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/GEO Accession Number GSE10032). Back

Correspondence: 2M. Wade, P.L. 0803D, Environmental Health Centre, 50 Columbine Driveway, Tunney's Pasture, Ottawa, ON, Canada K1A 0L2. FAX: 613 957 8800; e-mail: Mike_Wade{at}hc-sc.gc.ca

Received: 31 August 2007.

First decision: 20 September 2007.

Accepted: 8 January 2008.

REFERENCES

  1. Russell LD, Clermont Y. Degeneration of germ cells in normal, hypophysectomized and hormone treated hypophysectomized rats. Anat Rec 1977; 187:347–366.[CrossRef][Medline]
  2. Huckins C. The morphology and kinetics of spermatogonial degeneration in normal adult rats: an analysis using a simplified classification of the germinal epithelium. Anat Rec 1978; 190:905–926.[CrossRef][Medline]
  3. Bartlett JM, Kerr JB, Sharpe RM. The effect of selective destruction and regeneration of rat Leydig cells on the intratesticular distribution of testosterone and morphology of the seminiferous epithelium. J Androl 1986; 7:240–253.[Abstract/Free Full Text]
  4. Shikone T, Billig H, Hsueh AJ. Experimentally induced cryptorchidism increases apoptosis in rat testis. Biol Reprod 1994; 51:865–872.[Abstract]
  5. Sjoblom T, West A, Lahdetie J. Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide, adriamycin, and diepoxybutane. Environ Mol Mutagen 1998; 31:133–148.[CrossRef][Medline]
  6. Strandgaard C, Miller MG. Germ cell apoptosis in rat testis after administration of 1,3-dinitrobenzene. Reprod Toxicol 1998; 12:97–103.[CrossRef][Medline]
  7. Creasy DM, Flynn JC, Gray TJ, Butler WH. A quantitative study of stage-specific spermatocyte damage following administration of ethylene glycol monomethyl ether in the rat. Exp Mol Pathol 1985; 43:321–336.[CrossRef][Medline]
  8. Bourc'his D, Bestor TH. Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 2004; 431:96–99.[CrossRef][Medline]
  9. Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, Sasaki H. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 2004; 429:900–903.[CrossRef][Medline]
  10. Webster KE, O'Bryan MK, Fletcher S, Crewther PE, Aapola U, Craig J, Harrison DK, Aung H, Phutikanit N, Lyle R, Meachem SJ, Antonarakis SE, et al. Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis. Proc Natl Acad Sci U S A 2005; 102:4068–4073.[Abstract/Free Full Text]
  11. Doerksen T, Benoit G, Trasler JM. Deoxyribonucleic acid hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is associated with altered testicular histology. Endocrinology 2000; 141:3235–3244.[Abstract/Free Full Text]
  12. Doerksen T, Trasler JM. Developmental exposure of male germ cells to 5-azacytidine results in abnormal preimplantation development in rats. Biol Reprod 1996; 55:1155–1162.[Abstract]
  13. Kelly TL, Li E, Trasler JM. 5-aza-2'-deoxycytidine induces alterations in murine spermatogenesis and pregnancy outcome. J Androl 2003; 24:822–830.[Abstract/Free Full Text]
  14. Oakes CC, Kelly TL, Robaire B, Trasler JM. Adverse effects of 5-aza-2'-deoxycytidine on spermatogenesis include reduced sperm function and selective inhibition of de novo DNA methylation. J Pharmacol Exp Ther 2007; 322:1171–1180.[Abstract/Free Full Text]
  15. Johanson G. Toxicity review of ethylene glycol monomethyl ether and its acetate ester. Crit Rev Toxicol 2000; 30:307–345.[CrossRef][Medline]
  16. Foster PM, Creasy DM, Foster JR, Thomas LV, Cook MW, Gangolli SD. Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat. Toxicol Appl Pharmacol 1983; 69:385–399.[CrossRef][Medline]
  17. Anderson D, Brinkworth MH, Jenkinson PC, Clode SA, Creasy DM, Gangolli SD. Effect of ethylene glycol monomethyl ether on spermatogenesis, dominant lethality, and F1 abnormalities in the rat and the mouse after treatment of F0 males. Teratog Carcinog Mutagen 1987; 7:141–158.[CrossRef][Medline]
  18. Ku WW, Ghanayem BI, Chapin RE, Wine RN. Comparison of the testicular effects of 2-methoxyethanol (ME) in rats and guinea pigs. Exp Mol Pathol 1994; 61:119–133.[CrossRef][Medline]
  19. Foote RH, Farrell PB, Schlafer DH, McArdle MM, Trouern-Trend V, Simkin ME, Brockett CC, Giles JR, Li J. Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits. Reprod Toxicol 1995; 9:527–539.[CrossRef][Medline]
  20. Wade MG, Poon R, Li N, Lee A, McMahon A, Chu I. Testicular toxicity of candidate fuel additive 1,6-dimethoxyhexane: comparison with several similar aliphatic ethers. Toxicol Sci 2006; 89:304–313.[Abstract/Free Full Text]
  21. Creasy DM, Beech LM, Gray TJ, Butler WH. An ultrastructural study of ethylene glycol monomethyl ether-induced spermatocyte injury in the rat. Exp Mol Pathol 1986; 45:311–322.[CrossRef][Medline]
  22. Creasy DM, Foster PM. The morphological development of glycol ether-induced testicular atrophy in the rat. Exp Mol Pathol 1984; 40:169–176.[CrossRef][Medline]
  23. Ku WW, Wine RN, Chae BY, Ghanayem BI, Chapin RE. Spermatocyte toxicity of 2-methoxyethanol (ME) in rats and guinea pigs: evidence for the induction of apoptosis. Toxicol Appl Pharmacol 1995; 134:100–110.[CrossRef][Medline]
  24. Rao AV, Shaha C. N-acetylcysteine prevents MAA induced male germ cell apoptosis: role of glutathione and cytochrome c. FEBS Lett 2002; 527:133–137.[CrossRef][Medline]
  25. Jindo T, Wine RN, Li LH, Chapin RE. Protein kinase activity is central to rat germ cell apoptosis induced by methoxyacetic acid. Toxicol Pathol 2001; 29:607–616.[Abstract/Free Full Text]
  26. Wang W, Wine RN, Chapin RE. Rat testicular Src: normal distribution and involvement in ethylene glycol monomethyl ether-induced apoptosis. Toxicol Appl Pharmacol 2000; 163:125–134.[CrossRef][Medline]
  27. Barone F, Aguanno S, D'Alessio A, D'Agostino A. Sertoli cell modulates MAA-induced apoptosis of germ cells throughout voltage-operated calcium channels. FASEB J 2004; 18:353–354.[Abstract/Free Full Text]
  28. Barone F, Aguanno S, D'Agostino A. Modulation of MAA-induced apoptosis in male germ cells: role of Sertoli cell P/Q-type calcium channels. Reprod Biol Endocrinol 2005; 3:13.[CrossRef][Medline]
  29. Ghanayem BI, Chapin RE. Calcium channel blockers protect against ethylene glycol monomethyl ether (2-methoxyethanol)-induced testicular toxicity. Exp Mol Pathol 1990; 52:279–290.[CrossRef][Medline]
  30. Li LH, Wine RN, Chapin RE. 2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testes: an in vitro comparison. J Androl 1996; 17:538–549.[Abstract/Free Full Text]
  31. Li LH, Wine RN, Miller DS, Reece JM, Smith M, Chapin RE. Protection against methoxyacetic-acid-induced spermatocyte apoptosis with calcium channel blockers in cultured rat seminiferous tubules: possible mechanisms. Toxicol Appl Pharmacol 1997; 144:105–119.[CrossRef][Medline]
  32. Tirado OM, Selva DM, Toran N, Suarez-Quian CA, Jansen M, McDonnell DP, Reventos J, Munell F. Increased expression of estrogen receptor beta in pachytene spermatocytes after short-term methoxyacetic acid administration. J Androl 2004; 25:84–94.[Abstract/Free Full Text]
  33. Tirado OM, Martinez ED, Rodriguez OC, Danielsen M, Selva DM, Reventos J, Munell F, Suarez-Quian CA. Methoxyacetic acid disregulation of androgen receptor and androgen-binding protein expression in adult rat testis. Biol Reprod 2003; 68:1437–1446.[Abstract/Free Full Text]
  34. Syed V, Hecht NB. Rat pachytene spermatocytes down-regulate a polo-like kinase and up- regulate a thiol-specific antioxidant protein, whereas sertoli cells down-regulate a phosphodiesterase and up-regulate an oxidative stress protein after exposure to methoxyethanol and methoxyacetic acid. Endocrinology 1998; 139:3503–3511.[Abstract/Free Full Text]
  35. Wang W, Chapin RE. Differential gene expression detected by suppression subtractive hybridization in the ethylene glycol monomethyl ether-induced testicular lesion. Toxicol Sci 2000; 56:165–174.[Abstract/Free Full Text]
  36. Fukushima T, Yamamoto T, Kikkawa R, Hamada Y, Komiyama M, Mori C, Horii I. Effects of male reproductive toxicants on gene expression in rat testes. J Toxicol Sci 2005; 30:195–206.[CrossRef][Medline]
  37. Yamamoto T, Fukushima T, Kikkawa R, Yamada H, Horii I. Protein expression analysis of rat testes induced testicular toxicity with several reproductive toxicants. J Toxicol Sci 2005; 30:111–126.[CrossRef][Medline]
  38. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002; 30:e36.[Abstract/Free Full Text]
  39. Kerr MK. Design considerations for efficient and effective microarray studies. Biometrics 2003; 59:822–828.[CrossRef][Medline]
  40. Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 2002; 30:e15.[Abstract/Free Full Text]
  41. SAS System for Windows, version 8.2. Cary, NC:: SAS Institute Inc.; 2001.
  42. R Development Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria; 2004.
  43. Kerr MK, Wu H, Cui X, Churchill GA. MAANOVA: A software package for the analysis of spotted cDNA microarray experiments. In: Parmigiani G, Garrett ES, Irizarry RA, Zeger S (eds.), The Analysis of Gene Expression Data: Methods and Software. Berlin, Germany:: Springer-Verlag; 2003:313–431.
  44. Cui XG, Hwang JT, Oui J, Blades NJ, Churchill GA. Improved statistical tests for differential gene expression by shrinking variance components. Biostatistics 2005; 6:59–75.[Abstract]
  45. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 1995; 57:289–300.
  46. Searle SR, Speed FM, Milliken GA. The population marginal means in the linear model: an alternative to least squares means. Am Statistician 1980; 34:216–221.[CrossRef]
  47. Goodnight JH, Harvey WR. LeastSquares Means in the FixedEffects General Linear Models. SAS Technical Report R103. Cary, NC:: SAS Institute Inc.; 1978.
  48. Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM. Fixation of testes and eyes using a modified Davidson's fluid: comparison with Bouin's fluid and conventional Davidson's fluid. Toxicol Pathol 2002; 30:524–533.[Abstract/Free Full Text]
  49. Hess RA. Quantitative and qualitative characteristics of the stages and transitions in the cycle of the rat seminiferous epithelium: light microscopic observations of perfusion-fixed and plastic-embedded testes. Biol Reprod 1990; 43:525–542.[Abstract]
  50. Hazzouri M, Pivot-Pajot C, Faure AK, Usson Y, Pelletier R, Sele B, Khochbin S, Rousseaux S. Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases. Eur J Cell Biol 2000; 79:950–960.[CrossRef][Medline]
  51. D'Anna JA, Gurley LR, Becker RR, Barham SS, Tobey RA, Walters RA. Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H1(0); IP25) from Chinese hamster cells. Biochemistry 1980; 19:4331–4341.[CrossRef][Medline]
  52. Chabanas A, Khoury E, Goeltz P, Froussard P, Gjerset R, Dod B, Eisen H, Lawrence JJ. Effects of butyric acid on cell cycle regulation and induction of histone H1(0) in mouse cells and tissue culture. Inducibility of H1 (0) in the late S-G2 phase of the cell cycle. J Mol Biol 1985; 183:141–151.[CrossRef][Medline]
  53. Kypreou KP, Sourlingas TG, Sekeri-Pataryas KE. Age-dependent response of lymphocytes in the induction of the linker histone variant, H1 degrees and histone H4 acetylation after treatment with the histone deacetylase inhibitor, trichostatin A. Exp Gerontol 2004; 39:469–479.[CrossRef][Medline]
  54. Hochhuth C, Doenecke D. Differential expression of the histone H1 zero gene in U937 and HL-60 leukemia cell lines. J Cell Biochem 1992; 50:316–323.[CrossRef][Medline]
  55. Sourlingas TG, Kypreou KP, Sekeri-Pataryas KE. The effect of the histone deacetylase inhibitor, trichostatin A, on total histone synthesis, H1(0) synthesis and histone H4 acetylation in peripheral blood lymphocytes increases as a function of increasing age: a model study. Exp Gerontol 2002; 37:341–348.[CrossRef][Medline]
  56. Jing Y, Xia L, Waxman S. Targeted removal of PML-RARalpha protein is required prior to inhibition of histone deacetylase for overcoming all-trans retinoic acid differentiation resistance in acute promyelocytic leukemia. Blood 2002; 100:1008–1013.[Abstract/Free Full Text]
  57. Gunjan A, Sittman DB, Brown DT. Core histone acetylation is regulated by linker histone stoichiometry in vivo. J Biol Chem 2001; 276:3635–3640.[Abstract/Free Full Text]
  58. Grunstein M. Histone acetylation in chromatin structure and transcription. Nature 1997; 389:349–352.[CrossRef][Medline]
  59. Parthun MR, Widom J, Gottschling DE. The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism. Cell 1996; 87:85–94.[CrossRef][Medline]
  60. Eberharter A, Becker PB. Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics. EMBO Rep 2002; 3:224–229.[CrossRef][Medline]
  61. Meistrich ML, Trostle-Weige PK, Lin R, Bhatnagar YM, Allis CD. Highly acetylated H4 is associated with histone displacement in rat spermatids. Mol Reprod Dev 1992; 31:170–181.[CrossRef][Medline]
  62. Grimes SR Jr, Henderson N. Acetylation of histones during spermatogenesis in the rat. Arch Biochem Biophys 1983; 221:108–116.[CrossRef][Medline]
  63. Oliva R, Mezquita C. Histone H4 hyperacetylation and rapid turnover of its acetyl groups in transcriptionally inactive rooster testis spermatids. Nucleic Acids Res 1982; 10:8049–8059.[Abstract/Free Full Text]
  64. Fenic I, Sonnack V, Failing K, Bergmann M, Steger K. In vivo effects of histone-deacetylase inhibitor trichostatin-A on murine spermatogenesis. J Androl 2004; 25:811–818.[Abstract/Free Full Text]
  65. Jansen MS, Nagel SC, Miranda PJ, Lobenhofer EK, Afshari CA, McDonnell DP. Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition. Proc Natl Acad Sci U S A 2004; 101:7199–7204.[Abstract/Free Full Text]
  66. Seigneurin-Berny D, Verdel A, Curtet S, Lemercier C, Garin J, Rousseaux S, Khochbin S. Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways. Mol Cell Biol 2001; 21:8035–8044.[Abstract/Free Full Text]
  67. Moss EJ, Thomas LV, Cook MW, Walters DG, Foster PM, Creasy DM, Gray TJ. The role of metabolism in 2-methoxyethanol-induced testicular toxicity. Toxicol Appl Pharmacol 1985; 79:480–489.[CrossRef][Medline]
  68. Gottlicher M. Valproic acid: an old drug newly discovered as inhibitor of histone deacetylases. Ann Hematol 2004; 83(suppl 1):S91–S92.[Medline]
  69. Sveberg RL, Tauboll E, Isojarvi JI, Pakarinen AJ, Huhtaniemi IT, Knip M, Gjerstad L. Effects of chronic valproate treatment on reproductive endocrine hormones in female and male Wistar rats. Reprod Toxicol 2002; 16:767–773.[CrossRef][Medline]
  70. Sveberg RL, Tauboll E, Berner A, Berg KA, Aleksandersen M, Gjerstad L. Morphological changes in the testis after long-term valproate treatment in male Wistar rats. Seizure 2001; 10:559–565.[CrossRef][Medline]
  71. Walker RM, Smith GS, Barsoum NJ, Macallum GE. Preclinical toxicology of the anticonvulsant calcium valproate. Toxicology 1990; 63:137–155.[CrossRef][Medline]
  72. Nishimura T, Sakai M, Yonezawa H. Effects of valproic acid on fertility and reproductive organs in male rats. J Toxicol Sci 2000; 25:85–93.[Medline]
  73. Oakes CC, La Salle S, Smiraglia DJ, Robaire B, Trasler JM. Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells. Dev Biol 2007; 307:368–379.[CrossRef][Medline]
  74. Oakes CC, La Salle S, Smiraglia DJ, Robaire B, Trasler JM. A unique configuration of genome-wide DNA methylation patterns in the testis. Proc Natl Acad Sci U S A 2007; 104:228–233.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/5/822    most recent
biolreprod.107.065151v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wade, M. G.
Right arrow Articles by Yauk, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wade, M. G.
Right arrow Articles by Yauk, C.
Agricola
Right arrow Articles by Wade, M. G.
Right arrow Articles by Yauk, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS