Differential Gene Expression Detected by Suppression Subtractive ...

56, 165–174 (2000) Copyright © 2000 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Differential Gene Expression Detected by Suppression Subtractive Hybridization in the Ethylene Glycol Monomethyl Ether-Induced Testicular Lesion Wei Wang and Robert E. Chapin 1 Laboratory of Toxicology, National Toxicology Program, National Institute of Environmental Health Sciences, Mail Drop B3-05, P. O. Box 12233, Research Triangle Park, North Carolina 27709 Received November 22, 1999; accepted March 6, 2000

The solvent ethylene glycol monomethyl ether (EGME) produces the same testicular lesions in rodents and human testis cultures, whose onset is characterized by apoptosis of pachytene spermatocytes. To identify gene changes early in the lesion and determine the possible involvement of cells other than the spermatocytes, we employed a suppression subtractive hybridization technique using whole testes from mice treated 8 h previously with 500 mg/kg EGME to generate two subtracted mouse testis cDNA libraries enriched for gene populations either up-regulated or down-regulated by EGME. A total of 70 clones were screened, and 6 of them were shown by Northern blotting to be differentially expressed in the EGME lesion. The three clones with increased expression after EGME treatment were identical to t-complex testis expressed gene 1 (tctex1), a gene encoding ribosomal protein S25, and a heretofore uncharacterized mouse testis expressed sequence tag. Three other genes suppressed by EGME were tctex2, alpha-2,6-sialyltransferase gene, and another uncharacterized mouse testis expressed sequence tag. Predicted peptide sequences of these clones contain multiple motifs for phosphorylation, glycosylation, and myristoylation. In situ hybridization with the antisense RNA probes further supported the expression changes of these six clones and localized the changes in multiple germ cell stages as well as other cell types (Sertoli, interstitial and peritubular cells). These data at the gene expression level are the first to demonstrate the early involvement in this lesion of cell types other than the dying spermatocytes. Key Words: gene expression; suppression subtractive hybridization; ethylene glycol monomethyl ether; apoptosis; testis; mouse.

Ethylene glycol monomethyl ether (EGME, also known as 2-methoxyethanol) is an important organic solvent in paints, printing inks, thinners, and photoresists. Significant hematologic and central nervous system disturbances have been observed following occupational exposure to EGME (reviewed by Hardin, 1983), and adverse effects on the male reproductive system were reported more than 60 years ago (Wiley et al., 1936). Animal studies showed the cytotoxic effects of EGME 1

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leading to testicular atrophy and impaired fertility (Anderson et al., 1987; Chapin et al., 1984, 1985; Foster et al., 1983). The most sensitive target cell appears to be the primary spermatocyte in the dividing and early and late pachytene stages (Chapin et al., 1984), with heavily stained grainy cytoplasm and nuclei with uniformly condensed chromatin or dispersed chromatin masses; neighboring Sertoli cells were also visibly affected (Creasy et al., 1986). EGME-induced cell death was recently shown to be an apoptotic process (Ku et al., 1995). The early steps in this lesion are unknown, and the mechanism of this apoptosis is poorly understood. However, there are data to support the involvement of calcium movement (Ghanayem and Chapin, 1990) and protein kinases in this event (Jindo et al., 1999; Wang et al., 2000). Using differential display, Syed and Hecht (1998) found several gene products might be involved in the EGME-induced germ cell death in Sertoli and germ cell cocultures. Because of the requirement for intact tubules in the EGME lesion (Ku and Chapin, 1994), it is reasonable to postulate that this apoptosis is not an isolated process in the pachytene spermatocyte only. The hypothetical involvement of other cell types has been raised and discussed several times (Jindo et al., 1999; Li et al., 1997; Syed and Hecht, 1998), but there are still few direct data in support of this, and only Syed and Hecht addressed changes in the gene expression level. The current studies were aimed at identifying and characterizing the gene changes early in the EGME lesion by the suppression subtractive hybridization (SSH) technique (Diatchenko et al., 1996). This method is based on the suppressive PCR that selectively suppresses amplification of undesirable sequences in PCR procedures (Siebert et al., 1995), and also equalizes for relative abundance of cDNAs within a target population by incorporating a normalization step. Thus, this approach should enhance the probability of identifying increased expression of low abundance transcripts, and represents a potential advantage over other methods for identifying differentially regulated genes such as differential display-PCR (Liang and Pardee, 1992) and cDNA representation difference analysis (Hubank and Schatz, 1994). We used Northern blots to

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verify changes in these cDNA clones, as well as mRNA in situ hybridization to localize the gene changes within the tissue. MATERIALS AND METHODS Animals. Adult male mice (Crl:CD-1[ICR]BR, 65–75 days of age) were purchased from Charles River Laboratories (Raleigh, NC) and acclimated 4 days to the NIEHS animal facility. All the animals were housed in polycarbonate cages with 12:12 h light/dark cycles, 50 ⫾ 10% humidity, ambient temperature of 20 ⫾ 1°C, and were given NIH-31 diet and water ad libitum, all in accordance with the NIEHS Guidelines for the Humane Use of Animals in Research. These animals (n ⫽ 5/group) were treated by gavage with a single dose of 500 mg/kg EGME (Aldrich Chemical Co., Milwaukee, WI) in distilled water; control animals received vehicle alone. The animals were killed by CO 2 asphyxiation 8 h after treatment, and the testes were removed and handled as described below. Northern blot analysis. Total RNA from 10 pooled treated or 10 pooled control mouse testes was isolated as described previously (Ausubel, 1995). 20 ␮g total RNA was fractionated in a 1% agarose-formaldehyde gel and transferred onto a Nytran membrane (Schleicher & Schuell, Keene, NH) in the NorthernMax buffer (Ambion, Austin, TX) via the TurboBlotter System (Schleicher&Schuell) for 1 h. The RNA was cross-linked to the membrane with a UV Stratalinker 2400 (Stratagene, La Jolla, CA). Probes were generated by random priming with Klenow fragments in the presence of [32P]dCTP (Ausubel, 1995). The membrane was hybridized in ULTRAhyb solution (Ambion) according to the manufacturer’s protocol, followed by autoradiography for 0.5–16 h at – 80°C. Quantification of Northern blots was carried out by using a Kodak Digital Science Image Station 440CF (NEN Life Sciences, Boston, MA), in which the relative expression values were determined by normalizing against ␤-actin. Each Northern blot was done at least thrice, using three independently-generated probes and three different pools of murine testis RNA. Suppressive subtractive hybridization. The RNA pools generated above were used for subtractive hybridization. A PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA) was employed following the manufacturer’s protocol with modifications. In addition to the forward subtraction to detect the gene expression up-regulated by EGME treatment, we also performed a reverse subtraction to identify those genes down-regulated by EGME exposure. In the forward subtraction, using the conventions of the Subtraction Kit, testicular cDNA from EGME-treated mice were referred to as tester, and the cDNA from control mice testes were called driver. In the reverse subtraction, as indicated from its name, the forward tester cDNA from EGME-treated mice served as the reverse driver, and the forward driver as the reverse tester. Each tester or driver cDNA sample was generated from 2 ␮g polyadenylated RNA, isolated using the Oligotex mRNA Midi Kit (QIAGEN, Valencia, CA). To minimize the nonspecific messages, two rounds of subtraction were carried out in our study, in which the subtracted cDNA from the first round subtraction was used as the tester in the second round to hybridize against fresh driver cDNA, following the same subtraction protocol. cDNA cloning and sequence analysis. PCR products generated by the second round SSH were inserted into pCRII-TOPO vector using the TOPO TA cloning technique (InVitrogen, Carlsbad, CA), followed by the transformation into E. coli host and subsequent culture. Plasmid DNAs extracted from these cultured E. coli colonies would be used as probe templates for Northern blots of mRNA from testes of control and EGME-treated mice. Automated sequencing primed by T7 or M13 primers was performed using the ABI Prism Dye Terminator Kit (Perkin-Elmer, Foster City, CA), when clones were confirmed by Northern blotting as being differentially expressed after EGME treatment. The sequences obtained were analyzed by programs from the Wisconsin Genetic Computer Group (Madison, WI) to search for nucleotide sequence homology, peptide sequence prediction, and conserved protein motifs. In situ hybridization. The presence and position of the T7 promoter upstream of the cDNA reverse strand was confirmed by nucleotide sequencing

first. The plasmids were then linearized by HindIII and BamHI (Roche Molecular Biochemicals, Indianapolis, IN) and purified by QIAquick Gel Extraction (QIAGEN). Biotinylated anti-sense RNA transcripts were generated by incubation at 37°C for 3 h with AmpliScribe T7 reagents from Epicentre, Madison, WI (7.0 ␮l dH 2O, 2.5 ␮l 10X reaction buffer, 1.5 ␮l ATP, 1.5 ␮l CTP, 1.5 ␮l GTP, 1.0 ␮l UTP, 2.0 ␮l [1 ␮g] linearized template with T7 promoter, 2.0 ␮l AmpliScribe T7 enzyme solution) and 6.0 ␮l biotin-UTP (10 mM, Roche). One volume of 5 M ammonium acetate was added at the end of each reaction to precipitate and purify the anti-sense RNA probe. Pellets were resuspended in RNase-free dH 2O and quantified by absorbance at 260 nm. To perform the in situ hybridization, animals were treated with EGME or water and killed after 8 h. Testes were removed immediately and fixed with 10% neutral buffered formalin for 8 h at 4°C, then embedded in paraffin, sectioned at 5 ␮m by standard methods, and hybridized, as described in the mRNAlocator-Hyb in situ Hybridization kit (Ambion). In brief, tissue sections were deparaffinized, then treated with 4 ␮g/ml proteinase K for 10 min at 37°C. Fifty microliters per slide of the Ambion in situ Hyb Buffer-diluted RNA probe (0.6 ␮g/ml) was applied to the tissue section, to denature the tissue RNA and prehybridize at 68°C for 10 min in a water-saturated chamber. Hybridization at 55°C was performed for 16 –20 h. Other slides used Ambion Biotinylated GAPDH RNA probe as the positive control, and blank in situ hybridization buffer only as the negative control. Following three 4-min washes in 1:20 diluted in situ wash solution, the sections were incubated under 50 ␮l 1:400 Streptavidin-Alkaline Phosphatase Conjugate at 37°C for 60 min. Fifty microliters NBT/BCIP was then added at 37°C for 60 –90 min to generate the blue/purple color at the sites where biotinylated probe had hybridized to target sequences. Color development was terminated once the desired color intensity was achieved, followed by washing in dH 2O, dehydration, and mounting.

RESULTS

Involvement of Six Transcripts in the EGME Lesion Two subtracted cDNA libraries were generated at the end of the second round of the forward and reverse SSH, with the size ranging from 0.1 to 4 kb. The subtracted cDNAs were then transformed into E. coli for amplification. Seventy colonies were picked up from the transformants for screening: 36 (F1F36) from the forward SSH library, 34 (R1-R34) from the reverse SSH. Six of them (F27, F28, F31, R22, R27, R33) were confirmed by Northern analysis to differ in expression from controls by 2- to 4-fold (Fig. 1). The size of their mRNA from mouse testis were 1.0 ⬃ 2.5 kb, corresponding to cDNAs of 0.52 ⬃ 0.83 kb. The DNAs extracted from the other 64 colonies either were nondifferentially expressed or contained no cDNA inserts. Characterization of the Differentially Expressed Clones with EGME Treatment Sequencing was performed on the double-stranded DNA templates of the six clones, followed by GCG FastA searching against GenBank/EMBL database for sequence homology (Table 1). Clone F27 showed 98.2% homology to 40S ribosomal protein S25. F28 was similar to mouse testis expressed sequence tag (EST) GB#AA183 774 with a percentage of 94.6%. Another gene up-regulated after EGME treatment (F31) was found to encode mouse t-complex testis-expressed protein Tctex1 (99%). For the EGME down-regulated genes, database

SSH-DETECTED GENE CHANGES IN EGME LESION

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FIG. 1. Northern blots of mouse testicular genes differentially expressed with EGME treatment. Of 70 randomly selected clones (36 forward subtracted, 34 reverse subtracted), 6 (3 forward, 3 reverse) were demonstrated by Northern blotting to be differentially expressed and their size was estimated as shown. ␤-actin was hybridized with the same blots to confirm similar loading and transfer of the mRNA, which also served to normalize the phosphorimaging values. Differential expression (-fold) is the degree of expression relative to that found in control. The size of PCR-generated cDNA clone was estimated by electrophoresis in 2% agarose gel.

searching indicated R22 was 97.7% homologous to the uncharacterized mouse testis EST GB#AA183 842, R27 was 99.7% homologous to mouse Gel beta-1,3-GalNAc-specific GalNAc alpha-2,6-sialyltransferase gene (STF), and R33 was 98.5% identical to tctex2. Peptide sequences of 117–203 AA in length were predicted using the GCG Translate program from the longest open reading frames of the six cDNA clones. Motif analysis of the predicted peptide sequences found protein kinase C phosphorylation sites in all six clones. N-glycosylation sites were found in the three forward subtracted clones that were up-regulated by EGME. Casein kinase II phosphorylation sites were found in the reverse subtracted clones and in F28 (EST 774). Nmyristoylation sites were found in the predicted peptide sequences of clones F28 (EST 774), F31(tctex1), R22 (EST 842), and R33 (tctex2). A cAMP-dependent protein kinase (PKA) phosphorylation site (amino acid sequence KKWS) and a tyrosine kinase phosphorylation site (KLCKEVPNY) were also detected from the ribosomal protein S25. Localization of EGME-Induced Gene Expression Changes in Mouse Testis In situ hybridization (Fig. 2) was employed to confirm the Northern blot data of these gene changes in their normal milieu. Although the nonradioactive ISH provides better resolution than the isotope labeling strategy, our ability to discern specific cellular compartments was limited by the protease treatments required to enable hybridization. Despite the suboptimal structural preservation, hybridizations in cellular loca-

tions within and outside the tubules were detectable using these anti-sense RNA probes. The negative control (Figs. 2A and 2B), which was blank hybridization buffer lacking RNA probes, showed no background signal. The housekeeping gene GAPDH served as the positive control (Figs. 2C and 2D). In tissue from control mice, low expression of clone F27 (S25) was detected in spermatogonia and both early and late spermatocytes (Fig. 3A). EGME treatment induced an overall increase of signal intensity, especially in the dying spermatocytes and Sertoli cells (Fig. 3B). The EST 774 (F28) was faintly discernable in control pachytene spermatocytes and in some interstitial cells (Fig. 3C). EGME treatment up-regulated the expression of this clone in the dying spermatocytes, Sertoli cells, and spermatogonia (Fig. 3D), as expected from SSH and Northern blot data. Tctex1 (F31) gene expression could be localized in spermatocytes and probably spermatogonia in control tissue (Fig. 4A), but even greater staining intensity was seen in dying germ cells, early pachytene cells, and interstitial cells after EGME treatment (Fig. 4B). After EGME treatment, the intense staining of clone EST842 (R22) in early spermatocytes in control tissue (Fig. 4C) was diminished, but Sertoli cells were still clearly stained (Fig. 4D). The STF (R27) gene was highly expressed in the basallylocated early spermatocytes in control tubules, and the neighboring interstitial cells also showed a high expression level

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TABLE 1 Sequence Analysis of Differentially Expressed Clones in EGME Lesion Predicted peptide sequence analysis 2 cDNA sequence analysis 1

Motif Length (AA)

Clone

Reference

Identity

F27

Human ribosomal protein S25 mRNA (mRNA 497bp, GenBank#M64716)

98.2% in 440 nt overlap

117

Stratagene mouse testis (#937308) Mus musculus cDNA clone (mRNA 609bp, GenBank#AA183774)


203

F28

ASN GLYCOSYL

CAMP PHOSPHO

CK2 PHOSPHO

MYRISTYL

24NKSG

33KKWS

—

—

166NKTL

—

194NSSL 99NKTM

—

PKC PHOSPHO

TYR PHOSPHO

15SAK

57KLCKEV

PNY

96SWED

15GAPGSR

74SER 19SRR

195SSLE

127GAFYAA 184GMLFAA 76GLHSAS

106TLR 168TLK 94TVR

—

—

—

F31

Mouse tetex-1 mRNA (mRNA 680bp, GenBank#M25824)


113

R22

Stratagene mouse testis (#937308 Mus musculus cDNA clone 567456 5⬘ (mRNA 603bp, GenBank#AA183842)


200

—

—

67SGLD

76GLGNC

71SIK

—

R27

Mus musculus Gal beta-1,3-GalNAcspecific GalNAc alpha-2,6sialyltransferase gene (mRNA 1995bp, GeneBank#X93999)


117

—

—

8SGPE

79GNCPTN —

29TER

—

R33

Mus musculus t complex testisspecific protein (Tctex2) mRNA (mRNA 761bp, GeneBank#U21673)


130

—

—

10SIAD

101GQAINI

30SYR

—

55SLKD

55SLK

1

Based on the GCG FastA searching against GenBank/EMBL database. Based on the GCG Translate program to predict peptide sequences from the longest open reading frames, and the Motif program to analyze the conserved peptide motifs. 2

(Fig. 5A). Figure 5B shows that EGME reduced STF expression to near background in all cell types. In contrast to tctex1, the staining pattern of tctex2 (R33) was broadly strong in the control section, particularly remarkable in the spermatocytes and Sertoli cells (Fig. 5C). EGME treatment markedly reduced the staining intensity in Sertoli cells and pachytene spermatocytes, but staining was still present in dying germ cells (Fig. 5D). DISCUSSION

The molecular basis for EGME-induced germ cell apoptosis is poorly understood. Apoptosis, as a programmed process, is thought to involve orderly changes in gene expression (rev. in Schwartzman and Cidlowski, 1993). To shed light on the genes whose expression might be changed by EGME exposure, we have compared the pattern of gene expression in the testes of control and treated mice. Suppression subtractive hybridization was used to generate two cDNA libraries that contained genes relatively overexpressed in either the control or treated testes.

A panel of 70 clones was isolated from these two libraries, with six being finally confirmed as showing the greatest treatmentrelated changes and reported here. Based on sequence analysis, transcript size, differential expression, and EGME responsiveness, we estimate that each of the six SSH clones represents a different mouse gene. Database analysis suggests that several of these (tctex1 and 2, EST 774, 842) are likely to be preferentially expressed in the testis. Predicted peptide sequences of the six genes revealed several conserved motifs such as phosphorylation sites for protein kinase C, tyrosine kinase, casein kinase II, cAMP-dependent protein kinase (PKA), and sites of glycosylation and myristoylation. Of course, the mere presence of the consensus peptide motifs is not sufficient to conclude that the residues are phosphorylated, glycosylated, or myristoylated (Pless and Lennarz, 1977). However, motif analysis may provide some clues about the possible involvement of phosphorylation, myristoylation, or glycosylation in the EGME lesion. Membrane changes were reported to be an early event in the


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FIG. 2. In situ hybridization of the differentially expressed genes in EGME lesion on the mouse testis sections. Biotin-labeled antisense RNA probes were applied in the reactions, except for the negative controls with blank hybridization buffer on testicular sections from normal (a) and EGME-treated (b) mice. The housekeeping gene GAPDH was used as a positive control of the distribution patterns of the gene expression in normal (c) and EGME treated (d) sections. Bar ⫽ 20 ␮m.

pathogenesis of the lesion (Creasy et al., 1986). N-myristoylation is an acylation process suggested as a first step by which a protein associates with the membrane (Boutin, 1997). The N-myristoylation motifs found in the EGME-related clones are consistent with membrane association, although immunohistochemistry will be necessary to confirm a membrane location for the proteins. Several indirect lines of evidence hint at a possible involvement of glycosylation changes in the EGME-induced lesion. First, there are glycosylation sites in all three up-regulated

clones. Additionally, the EGME-suppressed Gal beta-1,3-GalNAc-specific GalNAc alpha-2, 6-sialytransferase (R27, STF) catalyzes the transfer of sialic acids to the terminal positions of the carbohydrate groups of glycoproteins and glycolipids to extend their biologic functions (Kurosawa et al., 1996). Moreover, this STF was reported to be highly expressed in testis and to be involved in apoptosis (e.g., in the induction of apoptosis, the phagocytosis of apoptotic cells, or the detachment of apoptotic cells from adjacent cells and/or extracellular matrices) by altering glycans with glycoconjugates (Kimura et al., 1999;

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FIG. 3. Low basal expression of clone F27 (ribosomal protein S25) was detected in spermatogonia and early and late spermatocytes in control sections (a); EGME treatment induced an overall increase of signal intensity, especially in the dying spermatocytes, late spermatocytes, and Sertoli cells (b). Compared to controls (c), EGME treatment also up-regulated expression of clone F28 (EST774) in dying spermatocytes, Sertoli cells, and spermatogonia (d).

Kurosawa et al., 1996). One might speculate a potential involvement of altered glycosylation in EGME-induced germ cell death. Clone F27 is identical to 40S ribosomal protein S25 and was up-regulated after EGME. S25 was found to play a growthsuppressive role in the nonproliferating liver and was reduced during liver regeneration (Sun et al., 1995). Collectively, the preexisting data show reduced expression of S25 during growth and increased expression during cell death, consistent with our up-regulation noted in apoptosis. However, it remains

to be determined if this increase seen in mRNA expression in the EGME lesion also occurs at the protein level. It is interesting to find two members (tctex1, F31; tctex2, R33) of t-complex testis expressed (tctex) gene family associated with the EGME lesion, especially given the opposite directions of their change. The t-complex is a large region of mouse chromosome 17 that contains four inversions that suppress recombination, which represents the most extreme vertebrate example of meiotic drive or transmission ratio distortion (Silver, 1993). Based on mapping and expression studies,


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FIG. 4. Tctex1 (F31) gene expression could be localized in the pachytene spermatocytes as well as other tubular cell types on the control sections (a), but greater staining intensity was seen in dying germ cells, early pachytene cells, and selected interstitial cells in EGME-treated mice (b). After EGME treatment, the intense staining for clone R22 (EST842) in the early spermatocytes (c) was diminished, but Sertoli cell staining was apparently unchanged (d).

members of the tctex gene family have been implicated in the multigene phenomena of tail length, embryonic lethality, sperm cell transmission ratio distortion, and possibly also in male sterility. The tctex-1 gene maps to the extreme proximal end of the t-complex and is overexpressed 8-fold in t/t testis of the homozygous males, compared to wild-type ⫹/⫹ males (Lader et al., 1989). The tctex-2 message is encoded in the distal inversion. However, in contrast to tctex-1, tctex-2 is underexpressed in t/t testis 6-fold less than that in wild-type mice (Ha et al., 1991). Analysis of the tctex genes has been

limited until King et al. (1996) demonstrated that Tctex-1 protein is a light chain component of the cytoplasmic dynein complex. Later on, the presence of Tctex-1 was also identified in the inner arm I1 of flagellar dynein with the location of Tctex-2 in the outer arm dynein light chain (Harrison et al., 1998; Patel-King et al., 1997). Dyneins are complex, microtubule-dependent molecular motors, classified into four structurally and functionally distinct groups: cytoplasmic, outer arm, inner arm I1, inner arms I2/3 (Witman et al., 1994). Cytoplasmic dynein plays important roles in intracellular retrograde

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FIG. 5. STF (R27) expression was high in the early spermatocytes in controls, while the neighboring interstitial cells also showed high expression level (a). This was reduced by EGME treatment (b). The staining pattern of tctex2 (R33) was strong overall in controls, particularly so in the spermatocytes and Sertoli cell (c). EGME treatment reduced the staining intensity, but still visible was staining in dying germ cells and nuclei of Sertoli cells and pachytene spermatocytes (d). Bar ⫽ 20 ␮m.

organelle transport, membrane trafficking, mitotic spindle localization, centrosome separation during mitosis (Schroer, 1994), and preventing apoptotic cell death (Dick et al., 1996; Puthalakath et al., 1999), while the light chain of flagellar outer arm dyneins participate in cAMP-dependent phosphorylation, Ca 2⫹ binding, and sulfhydryl oxidoreductase activity (PatelKing et al., 1996). These background references, as well as the recent report on interaction between Src tyrosine kinase family member Fyn and dynein light chain Tctex-1 (Campbell et al., 1998), are congruent not only with the kinase motif data shown

in this paper but also with our prior study of kinase involvement in the EGME lesion discussed above. Although it has always been considered possible that cell types other than the pachytene spermatocyte might be involved in EGME-induced apoptosis, no evidence has emerged to date. However, 8 h after dosing, which is the very beginning of histologic change in the testis, our in situ hybridization studies detected changes in the expression of a group of genes not only in the dying spermatocytes, but also in other germ cells, Sertoli cells, peritubular cells, and interstitial cells. These early


changes of gene expression, not restricted only to the dying spermatocytes, help to explain the requirement for intact seminiferous tubules for the in vitro replication of the in vivo lesion (Ku and Chapin, 1994). Importantly, the current data suggest that the EGME-induced lesion is a complex network of changes involving both structural and functional proteins. Ongoing research aims to define more precisely the critical players in this lesion, with a shorter duration of exposure and employing a cDNA microarray of ⬃9K mouse genes. In the only other report of which we are aware using a similar strategy, Syed and Hecht (1998) showed a decrease in polo-like kinase-1 gene expression in testis after EGME treatment in rats, using differential display. With this exception we could find no reports of studies that looked broadly at changes in gene expression. In summary, we have identified six genes that are differentially expressed in mouse testis after EGME treatment. Early changes of gene expression were detected not only in the apoptotic germ cells, but also in other testicular cell types. The current data demonstrate an influence of EGME on more testis cells than was thought previously and provide clues for further studies in this field. REFERENCES Anderson, D., Brinkworth, M. H., Jenkinson, P. C., Clode, S. A., Creasy, D. M., and Gangolli, S. D. (1987). 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. 7, 141–158. Ausubel, F. M. (1995). Short protocols in molecular biology: a compendium of methods from current protocols in molecular biology. Wiley, New York. Boutin, J. A. (1997). Myristoylation. Cell Signal. 9, 15–35. Campbell, K. S., Cooper, S., Dessing, M., Yates, S., and Buder, A. (1998). Interaction of p59 fyn kinase with the dynein light chain, Tctex-1, and colocalization during cytokinesis. J. Immunol. 161, 1728 –1737. Chapin, R. E., Dutton, S. L., Ross, M. D., and Lamb, J. C. IV (1985). Effects of ethylene glycol monomethyl ether (EGME) on mating performance and epididymal sperm parameters in F344 rats. Fundam. Appl. Toxicol. 5, 182–189. Chapin, R. E., Dutton, S. L., Ross, M. D., Sumrell, B. M., and Lamb, J. C. IV (1984). The effects of ethylene glycol monomethyl ether on testicular histology in F344 rats. J. Androl. 5, 369 –380. Creasy, D. M., Beech, L. M., Gray, T. J., and Butler, W. H. (1986). An ultrastructural study of ethylene glycol monomethyl ether-induced spermatocyte injury in the rat. Exp. Mol. Pathol. 45, 311–322. Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D., and Siebert, P. D. (1996). Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. U S A 93, 6025– 6030. Dick, T., Ray, K., Salz, H. K., and Chia, W. (1996). Cytoplasmic dynein (ddlc1) mutations cause morphogenetic defects and apoptotic cell death in Drosophila melanogaster. Mol. Cell. Biol. 16, 1966 –1977. Foster, P. M., Creasy, D. M., Foster, J. R., Thomas, L. V., Cook, M. W., and Gangolli, S. D. (1983). Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat. Toxicol. Appl. Pharmacol. 69, 385–399. Ghanayem, B. I., and Chapin, R. E. (1990). Calcium channel blockers protect

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