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Endocrinology Vol. 139, No. 8 3503-3511
Copyright © 1998 by The Endocrine Society

ARTICLES

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 Acid1

Viqar Syed and Norman B. Hecht

Center for Research on Reproduction and Women’s Health and Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Norman B. Hecht, Center for Research on Reproduction and Women’s Health, 752b Clinical Research Building/6142, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: nhecht{at}mail.med.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
2-Methoxyethanol (ME) and its metabolite, methoxyacetic acid (MAA), produce testicular lesions characterized by pachytene spermatocyte degeneration. To understand the molecular basis of this action on meiotic prophase cells, mRNA differential display was used to identify gene expression changes in control and treated cells. When pachytene spermatocytes were cultured with 5 mM ME or 5 mM MAA for 24 h, two complementary DNAs (cDNAs), of 557 nucleotides (clone 5) and 388 nucleotides (clone 6), were up-regulated; and a cDNA of 648 nucleotides (clone 1) was down-regulated. The altered expression pattern shown by differential display was confirmed by Northern blotting. Sequence analyses indicate that clones 1 and 6 have 83% and 79% homology at the nucleotide level to a polo-like kinase and a thiol-specific antioxidant, respectively. Clone 5 shows no homology to any known gene in the database. Messenger RNAs (mRNAs) encoding the thiol-specific antioxidant and clone 5 are up-regulated within 30 min of the addition of MAA, whereas the polo-like kinase mRNA decreased to undetectable levels after 6 h. Changes in Sertoli cell gene expression were also detected when Sertoli cells were cultured with 5 mM ME or MAA for 24 h. Two cDNAs, of 367 nucleotides (clone 2) and 676 nucleotides (clone 3), were up-regulated; and a cDNA of 538 nucleotides (clone 4) was down-regulated. Homology searches revealed that clones 3 and 4 have 90 and 91% homology at the nucleotide level to an oxidative stress protein and a phosphodiesterase (PDE), respectively. Northern blotting confirmed the differential display expression pattern for the PDE and oxidative stress protein. mRNAs for the latter were induced within 30 min, and PDE mRNAs were down-regulated within one h, after the addition of MAA. To determine whether the changes in gene expression seen with cells in culture also occur in vivo, rats were given a single oral dose of 250 mg/kg ME or MAA. After 24 h, total testis RNAs from control and treated rats were purified and hybridized. The expression patterns seen in vivo for the differentially expressed cDNAs were identical to those seen in vitro. We conclude that, although pachytene spermatocytes seem to be selectively affected by ME and MAA, changes in gene expression are also detected in Sertoli cells, suggesting that the action(s) of ME or MAA on pachytene spermatocytes could be mediated through Sertoli cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
METHOXYETHANOL (ME) is a glycol ether extensively used in paints, textile dyes, printing inks, and brake fluids. Animal studies have revealed a considerable reproductive toxicity for ME in males leading to testicular atrophy and impaired fertility (1). In rats, exposure to 300 ppm ME for 10 days leads to a selective degeneration of primary spermatocytes, with no visible effects on spermatogonia, Sertoli cells, or Leydig cells (2). When rats are exposed for 4 days to 150 mg/kg ME, spermatocytes and round spermatids seem necrotic (3). Foster et al. (4) reported that, 24 h after a single dose of 100 mg/kg ME in rats, the initial testicular lesion seemed to be depletion of primary spermatocytes. In addition, 16 h after a single dose of 500 mg/kg ME in spermatocytes, swelling and disruption of mitochondria, cytoplasmic vacuolization, and early condensation of nuclear chromatin were observed (4).

The toxicity of ME is mediated through its primary metabolite, methoxyacetic acid (MAA) (5). Oral administration of MAA to rats produces testicular lesions characterized by changes in the pachytene spermatocytes in stages XIII-II of the seminiferous epithelium within 12 h of exposure and degeneration often by 24 h (3, 6, 7, 8, 9, 10). Exposure of cocultures of Sertoli cells and germ cells to MAA produces selective detachment of pachytene spermatocytes from the Sertoli cell monolayer and morphological degeneration of the germ cells (9, 11). MAA may also directly affect the viability of isolated pachytene spermatocytes (12).

During spermatogenesis, Sertoli cells function, in part, as nurse cells, synthesizing and secreting proteins and metabolites into the tubule lumen for the differentiating germ cells (13, 14). The Sertoli cell metabolizes glucose, primarily to lactate (15), a primary metabolic substrate of cells early in the spermatogenic cycle (16, 17). ME and MAA induce changes in Sertoli cell lactate concentration and in cyclic protein-2, raising the possibility that the degradation of pachytene spermatocytes results from alterations in Sertoli cell function (18, 19).

The objective of this study was to identify genes that are up-regulated or down-regulated in pachytene spermatocytes, as a result of exposure to ME or MAA. We also investigated the influence of these agents on Sertoli cell gene expression, to evaluate any potential involvement of Sertoli cells in the selective toxicity of ME and MAA to spermatocytes. To identify genes that are differentially expressed in pachytene spermatocytes and in Sertoli cells after exposure to the glycol ethers, we have used messenger RNA (mRNA) differential display. We report here the isolation and identification of a group of complementary DNAs (cDNAs) encoding proteins that show identical up-regulation or down-regulation in spermatocytes and Sertoli cells in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparation and culture
Sertoli cells were isolated from the testes of 20-day-old Sprague-Dawley rats (Charles River, Kingston, MA) by sequential enzymatic digestion, as previously described (20, 21). Decapsulated fragments were digested with trypsin (1 mg/ml; Sigma, St. Louis, MO) for 30 min at 37 C to remove interstitial cells and then with collagenase (1 mg/ml; Sigma) for 25 min at 37 C. The cell suspensions were centrifuged at 800 rpm for 2 min; and the supernatants, containing peritubular cells, were discarded. The pellets were washed twice and incubated in PBS containing hyaluronidase (1 mg/ml; Sigma) for 30 min at 37 C. After incubation, the cell suspensions were centrifuged at 800 rpm for 2 min, and the pellets were washed twice with PBS. The cells were resuspended in DMEM and Ham’s F-12 medium (Gibco, Bethesda, MD) containing gentamycin (4 µg/ml), insulin (10 µg/ml), and transferrin (5 µg/ml). The Sertoli cells were plated at a density of 2 x 106/cm2 in polystyrene {gamma}-irradiated plates (Falcon, Oxnard, CA) under serum-free conditions and maintained in a 5% CO2 atmosphere. After 2–3 days of culture, the Sertoli cells were exposed to a hypotonic wash (20 mM Tris-HCl at pH 7.4) for 2–5 min to remove contaminating germ cells (22). As determined by DNA flow cytometry, Sertoli cells of greater than 98% purity were obtained. Contaminating peritubular cells were monitored by alkaline phosphatase staining at the start of culture and estimated to be 2% (23, 24). A 500 mM stock solution of 2-MAA (99% pure, Aldrich Chemical Company, Milwaukee, WI) was freshly prepared by dilution in culture medium, and the pH was adjusted to 7.0 with 1 N sodium hydroxide. This MAA stock was then diluted to a final concentration of 5 mM in culture medium. Twenty-four hours after the hypotonic wash, ME or MAA was added to the cells from freshly prepared stock solutions. After 24 h, control and treated monolayers were washed with PBS, and guanidine isothiocyanate buffer was added. The cells were scraped from the plate, and RNA was purified (25). Trypan blue exclusion revealed greater than 96% cell viability. To study the time-dependent up- or down-regulation of genes, Sertoli cells were cultured with MAA for 0.5, 1, 3, 6, 18, and 24 h.

Isolation and culture of germ cells
To obtain germ cells, testes from 24-day-old rats were decapsulated and incubated with collagenase (1 mg/ml) for 15 min at 37 C. The tubules were allowed to settle, and interstitial cells were removed by decanting the supernatant. The tubules were rinsed twice with PBS and then incubated with trypsin (1 mg/ml) for 20 min at 37 C. The tubules were pipetted up and down several times to produce a single cell suspension and filtered through 30-µm nylon mesh. Germ cells in medium supplemented with 2 mM sodium pyruvate and 6 mM sodium DL-lactate were plated at a density of 8 x 106/ml with 5 mM ME or MAA for 24 h at 32 C. To study the time-dependent up or down-regulation of genes, germ cells were cultured at 32 C with 5 mM MAA for 0.5, 1, 3, 6, 18, and 24 h. Enriched populations of germ cells were obtained from adult rat testes by Staput sedimentation, as previously described (26). The cell separations were performed in BSA gradients (2–4%) in culture media adjusted to pH 7.4. The cells were allowed to sediment for about 31/2 h. Fractions were collected and cells were identified by microscopy and incubated with either ME or MAA for 24 h before RNA was isolated. Throughout the incubations, cell viability, monitored by trypan blue exclusion, revealed greater than 97% viability.

In vivo studies
Male rats (24 days old) were housed in a cage under controlled light conditions (12-h light, 12-h dark cycles). Groups of three rats were given a single dose of 250 mg/kg BW of ME or MAA by gavage (1.5 ml/kg). All control animals received PBS. Rats were killed by CO2 asphyxiation 24 h later, both testes were removed and were snap-frozen in liquid nitrogen and stored at -70 C for subsequent isolation of RNA.

Differential display RT-PCR and isolation of clones
Total RNAs from treated and control Sertoli cells, germ cells, or total testes were isolated as described previously (25). The mRNA differential display was performed, as previously described, on RNAs obtained from Sertoli or on germ cells treated with ME or MAA (21, 22).

DNA fragments, showing reproducibly unique expression patterns, were cut from the dried gels and were reamplified by PCR using the same set of primers. The amplified PCR fragments were gel-purified using a Sephaglas BandPrep kit (Pharmacia Biotech, Piscataway, NJ) and were subcloned into a TA cloning vector system (Invitrogen, San Diego, CA), according to the manufacturer’s instructions. Subcloned fragments were used as probes for Northern analysis. Both strands of clones showing differential expression were sequenced using a CircumVent kit and both M13 and T7 primers (Bio Labs, Beverly, MA). The sequences of the isolated clones were compared with the GenBank and EMBL DNA databases.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rat germ cells and Sertoli cells were cultured in the presence of 5 mM ME or MAA, and differentially expressed mRNAs were detected using a combination of T11GT with RH-1 (5'-AGTGAATGGC-3') and RH-2 (5'-GAGGATCAGC-3') (Fig. 1Go).



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  		Figure 1. mRNA differential display of RNAs from rat Sertoli cells and germ cells. Total RNA was isolated from a 24-h culture of Sertoli cells from 20-day-old rats (SC), Sertoli cells cultured with 5 mM ME (SC + ME) or with MAA (SC + MAA), control germ cells (GC), and germ cells cultured with 5 mM ME (GC + ME) or with MAA (GC + MAA). The RNAs were reverse-transcribed, followed by PCR. The primers used were T11GT and RH-1 (5'-AGTGAATGGC-3') for A and T11GT and RH-2 (5'-GAGGATCAGC-3') for B. The differentially expressed cDNAs are indicated with arrowheads, and nucleotide size markers are on the right. Three independent experiments were performed, and a representative display is shown here.

 
ME or MAA up-regulate a thiol-specific antioxidant in cultured germ cells
A cDNA of 388 nucleotides (clone 6 in Fig. 1Go) with 79% nucleotide similarity to a thiol-specific antioxidant (from nucleotide 482 to 870) and 71% amino acid similarity (from amino acid 180 to 220) was up-regulated in a population of germ cells cultured with ME or MAA (Fig. 2BGo). This thiol-specific antioxidant is encoded by a transcript of 3.6 kb in RNA from germ cells treated with ME or MAA but not in RNAs from control testis, kidney, or liver, confirming the selective expression of this cDNA seen by mRNA differential display (Fig. 3CGo).



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  		Figure 2. A, Amino acid sequence comparison of the coding region of clone 1 (GenBank accession no. AF053092) with a Rattus norvegicus polo-like kinase (GenBank accession no. U10188). One hundred and eleven amino acids of the open reading frame of clone 1 were compared with amino acids 489 to 600 of a polo-like kinase. B, Amino acid sequence comparison of clone 6 (GenBank accession no. AF053093) with a Rattus norvegicus thiol-specific antioxidant (GenBank accession no. U06099). Forty amino acids of open reading frame of clone 6 share homology with amino acids 180–220 of the thiol-specific antioxidant. The shaded area indicates identity, and boxes show homology. C, Nucleotide sequence of clone 5 (GenBank accession number AF053094). A computer search against GenBank showed no homology to any known genes. Both strands of the clones were sequenced twice using M13 and T7 primers

 


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  		Figure 3. Northern blot of RNAs differentially expressed in rat germ cells. Total RNAs (10 µg) from control germ cells (GC), germ cells treated with 5 mM ME (GC + ME) or with 5 mM MAA (GC + MAA), testis, kidney, and liver were subjected to Northern blotting, as described in Materials and Methods. The germ cell cDNAs encoding the polo-like kinase (A), clone 5 (B), and the thiol specific antioxidant (C) were used as probes for hybridization. Actin (2.1 kb) was used as a control to monitor for equal RNA loading. The experiment was done twice.

 
ME or MAA down-regulate a polo-like kinase gene in cultured germ cells
A cDNA of 648 nucleotides (clone 1 in Fig. 1Go) was present in a population of germ cells but absent from germ cell RNA after treatment with ME or MAA. Clone 1 shows 83% similarity at the nucleotide level (from nucleotide 1561 to 2209) and 79% similarity at the amino acid level (from amino acid 489 to 600) to a polo-like kinase (Fig. 2AGo). When the cDNA encoding this polo-like kinase was used to probe Northern blots of control and treated RNAs, it hybridized to a transcript of 2.2 kb in RNA from control germ cells and total testis but not from treated germ cells, kidney, or liver RNA preparations (Fig. 3AGo).

In addition to the differentially expressed thiol-specific antioxidant and the polo-like kinase, we have detected a third cDNA of 557 nucleotides (clone 5 in Fig. 1Go). Although the cDNA for clone 5 does not show homology to any known genes in the database (Fig. 2CGo), its mRNA differential expression pattern was confirmed by Northern blotting. Clone 5 hybridizes to RNA of 3.8 kb from germ cells cultured with ME or MAA but not to RNAs from total testis, kidney, or liver (Fig. 3BGo).

To determine the germ cell types that express the thiol-specific antioxidant, the polo-like kinase, and clone 5, we have isolated and cultured enriched populations of pachytene spermatocytes, round spermatids, and elongated spermatids with ME or MAA for 24 h. When purified RNAs from each of these cell types were hybridized with these three cDNAs, the polo-like kinase transcripts were only detected in RNA preparations from untreated pachytene spermatocytes or total testis RNA (Fig. 4AGo). mRNAs encoding the thiol-specific antioxidant and clone 5 were seen in RNA from pachytene spermatocytes exposed to ME or MAA but not in RNAs from round or elongated spermatids (Fig. 4Go, B and C).



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  		Figure 4. Northern blot of RNAs from control and treated enriched germ cell populations. Total RNAs (10 µg) were isolated from 24-h cultures of pachytene spermatocytes (Pach), pachytene spermatocytes cultured with 5 mM ME (Pach + ME), pachytene spermatocytes cultured with 5 mM MAA (Pach + MAA), round spermatids (RS), round spermatids cultured with ME (RS + ME), round spermatids cultured with MAA (RS + MAA), elongated spermatids (ES), elongated spermatids cultured with ME (ES + ME), elongated spermatids cultured with MAA (ES + MAA), and from testis. RNAs were electrophoresed and hybridized individually with cDNAs encoding the polo-like kinase (A), clone 5 (B), and the thiol-specific antioxidant (C). For A, the blot was rehybridized with an actin cDNA. For B and C, an actin cDNA was added at the time of hybridization. The experiment was performed once.

 
The up-regulated pachytene spermatocyte transcripts are rapidly induced, whereas down-regulation of polo-like kinase occurs more slowly
To study the time-dependent expression of the up-regulated and down-regulated pachytene spermatocyte genes, RNAs were isolated from germ cells cultured with 5 mM MAA for increasing time periods and hybridized to cDNAs encoding the polo-like kinase, clone 5, and the thiol-specific antioxidant. Transcripts of clone 5 and the thiol-specific antioxidant were detected within 30 min of incubation (Fig. 5Go, B and C), whereas polo-like kinase transcripts were detected up to 6 h after addition of MAA (Fig. 5AGo).



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  		Figure 5. Time-dependent expression of germ cell mRNAs up-regulated or down-regulated by MAA. Total RNA (10 µg) was isolated from germ cells cultured with 5 mM MAA for 0.5, 1, 3, 6, 18, and 24 h and analyzed by Northern blotting using cDNAs encoding the polo-like kinase (A), clone 5 (B), and the thiol-specific antioxidant (C) as probes. The experiment was performed twice.

 
ME or MAA induce identical up- or down-regulation of pachytene spermatocyte genes in vivo
To determine whether ME and MAA induce the same changes in gene expression in vivo that we observe in cultured cells, RNAs were isolated from testes of animals given one dose of either ME or MAA 24 h earlier. By Northern blotting, the 2.2-kb polo-like kinase mRNA was detected in RNA preparations from control testes but not from treated testes (Fig. 6AGo), whereas the expected 3.8-kb mRNA of clone 5 (Fig. 6BGo) and the 3.6-kb mRNA of the thiol-specific antioxidant (Fig. 6CGo) were detected in RNAs from testes of treated rats. We conclude that the changes in gene expression that we detect in cultured primary spermatocytes are also seen in vivo.



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  		Figure 6. Expression of germ cell clones 1, 5, and 6 in rat testis. Total RNAs (10 µg) from testes of control rats, rats treated with 250 mg/kg ME for 24 h, or rats treated with 250 mg/kg MAA for 24 h were electrophoresed and hybridized with cDNAs encoding the polo-like kinase (A), clone 5 (B), and the thiol-specific antioxidant (C). The experiment was performed once.

 
ME or MAA up-regulate an oxidative stress-induced gene in cultured Sertoli cells
Changes in gene expression were also detected in Sertoli cells exposed to ME or MAA. Three differentially expressed cDNAs were detected by mRNA differential display using RNAs prepared from cultured Sertoli cells. A cDNA of 676 nucleotides (clone 3 of Fig. 1Go) was up-regulated in Sertoli cells treated with ME or MAA. Sequence analysis revealed a 91% similarity to the oxidative stress-induced protein A-170 at the nucleotide level (between nucleotides 1090 and 1775) and an 84% similarity at the amino acid level (between amino acids 520 and 605) (Fig. 7AGo). A transcript of 3.2 kb, encoding the oxidative stress-induced protein A-170, was detected in treated Sertoli cells but not in control Sertoli cells, total testes, kidney, or liver (confirming the differential expression pattern of this oxidative stress induced protein by Northern blotting) (Fig. 8CGo). As previously seen for the two up-regulated pachytene spermatocyte genes, the 3.2-kb transcript was induced within 30 min after exposure to MAA (Fig. 9BGo).



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  		Figure 7. A, Amino acid sequence comparison of clone 3 (GenBank accession no. AF053095) with Mus musculus stress-induced protein (GenBank accession no. U40930). Eighty-five amino acids of the open reading frame of clone 3 were compared with amino acids 520 to 605 of a stress-induced protein. The shaded area indicates identity, and boxes show homology. B, Amino acid sequence comparison of clone 4 (GenBank accession no. AF053097) with Rattus norvegicus PDE (GenBank accession no. M25349). One-hundred-eleven amino acids of the open reading frame of clone 4 were compared with amino acids 415 to 526 of a PDE. C, Nucleotide sequence of clone 2 (GenBank accession no. AF053096). A computer search against GenBank showed no homology to any known genes. Both strands of the clones were sequenced twice using M13 and T7 primers

 


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  		Figure 8. Northern blot of RNAs from control and treated Sertoli cells. RNAs (10 µg) were isolated from a 24-h culture of Sertoli cells (SC), Sertoli cells cultured with 5 mM ME (SC + ME), Sertoli cells cultured with MAA (SC + MAA), testis, kidney, and liver. RNAs were electrophoresed and hybridized with cDNAs encoding PDE (A), clone 2 (B), and the oxidative stress induced protein (C). The blots were rehybridized with an actin cDNA. The experiment was done twice.

 


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  		Figure 9. Time-dependent expression of up-regulated or down-regulated Sertoli cell mRNAs. Total RNA (10 µg) was isolated from Sertoli cells cultured with 5 mM MAA for 0.5, 1, 3, 6, 18, and 24 h and analyzed by Northern blotting using cDNAs encoding phosphodiesterase (A) and oxidative stress-induced protein (B) as probes. The experiment was performed twice.

 
A phosphodiesterase (PDE) is down-regulated in cultured Sertoli cells after exposure to ME or MAA
A cDNA of 538 nucleotides (clone 4 in Fig. 1Go), showing 91% similarity to a PDE (from nucleotide 1425 to 1963) and 80% amino acid similarity (from amino acid 415 to 526), was down-regulated in Sertoli cells after exposure to ME or MAA (Fig. 7BGo). The cDNA encoding this PDE hybridized to a 3.2-kb transcript in RNA from control Sertoli cells and total testis RNAs but not from treated Sertoli cells, kidney, or liver (Fig. 8AGo). The expression pattern seen by differential display was confirmed by Northern blotting. Unlike the more slowly down-regulated polo-like kinase, no PDE transcript was detected after 60 min of Sertoli cells exposure to MAA (Fig. 9AGo). A third cDNA of 367 nucleotides (clone 2 of Fig. 1Go) was up-regulated in treated Sertoli cells. Computer analysis of the clone 2 sequence did not reveal homology to any known genes in the database (Fig. 7CGo). The clone 2 cDNA hybridized to a 1.6-kb mRNA from control and treated Sertoli cells (Fig. 8BGo).

ME or MAA induce a similar up- or down-regulation of Sertoli cell genes in vivo
To determine whether the changes in gene expression that we detect in cultured Sertoli cells also occur in vivo, rats were given a single oral dose of MAA and, after 24 h, total testis RNA was isolated. RNAs from control and treated rats were hybridized with each of the differentially expressed Sertoli cell cDNAs. The PDE was down-regulated in the treated rat testis, whereas the oxidative stress-induced protein was induced after MAA treatment, exactly matching the results obtained with cultured Sertoli cells (Fig. 10Go, A and C). Clone 2 failed to hybridize to any RNA (Fig. 10BGo).



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  		Figure 10. Expression of Sertoli cell clones 2, 3, and 4 in rat testis. Total RNAs (10 µg) were isolated from testes of control rats or rats treated with 250 mg/kg BW ME or with 250 mg/kg BW MAA, and were hybridized with cDNAs encoding phosphodiesterase (A), clone 2 (B), and the oxidative stress induced protein (C). An actin cDNA was used to monitor RNA loading. The blot was hybridized twice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have demonstrated that administration of MAA to rats results in a cell- and stage-specific loss of pachytene spermatocytes (4, 6, 7, 9, 29, 30). However, the mechanisms by which MAA exerts this apparently selective effect are unknown. The primary objective of this study was to begin to define the molecular basis of this cell loss by examining changes in gene expression in the cells of the testis. To detect changes in gene expression induced by the toxicants, we have used the technique of mRNA differential display. Although unanticipated, we find both down- and up-regulation of specific genes in both pachytene spermatocytes and Sertoli cells after exposure to ME or MAA for 24 h. To avoid artifact and to relate in vitro findings to the animal, we have analyzed RNAs from cells in culture and from in vivo sources.

In pachytene spermatocytes, after exposure to ME or MAA, a polo-like kinase-1 (Plk-1) is down-regulated and a thiol-antioxidant is up-regulated. Protein kinases are integral components of many signal transduction pathways; not only do they serve to phosphorylate their physiological substrates, their enzyme activities are often regulated through interactions with other protein kinases and phosphatases (31, 32, 33). Kinases may be involved in a signaling pathway from receptors expressed on the surface of meiotic germ cells. An intriguing observation to emerge from this study is that the cells most sensitive to ME or MAA are the spermatocytes entering diplotene and the first meiotic division (stages XIII and XIV). A polo-like kinase-1 gene has been shown to be specifically expressed in the diplotene and diakinesis stages of meiotic prophase (34, 35). Like the Drosophila polo-like kinase, the Plk-1 enzyme is believed to play an important role in meiosis, with an involvement in the regulation of microtubule organization and consequent spindle formation. Polo-like kinases may also function in cell cycle progression, facilitating the completion of meiosis (36). The time-dependent decrease in mRNA expression of the polo-like kinase within 6 h of exposure to MAA is in agreement with the observation of Ku and Chapin (9), who report DNA damage and disassembly of mitotic spindles in pachytene spermatocytes after 8 h of exposure to MAA. Taken together, the expression of a polo-like kinase in meiotic cells of the testis and the sensitivity of spermatocytes to ME or MAA suggest that down-regulation of this crucial meiotic gene product could trigger a cascade of genes, leading to apoptosis.

At the time a polo-like kinase is down-regulated in pachytene spermatocytes after exposure to ME or MAA, a thiol-specific antioxidant is up-regulated. Organisms living in aerobic environments must prevent or limit cellular damage caused by reactive forms of oxygen and sulfur to prevent protein oxidation, lipid peroxidation and DNA base modification, and strand breaks (37, 38). Alternatively, oxidative stress can be induced by decreasing the ability of cells to scavenge or detoxify reactive oxygen intermediates. To counteract these destructive processes, cells have evolved protective enzyme systems that prevent and repair radical-linked damage (37, 38). It has been shown that testicular cells contain mRNA encoding several enzymes, glutathione peroxidase, catalase, and superoxide dismutase 1 and 2, which protect them from oxidative damage (39, 40). Because ethers such as MAA are highly susceptible to peroxide formation, pachytene spermatocytes may defensively synthesize antioxidants when exposed to ME or MAA. We find that the thiol-specific antioxidant is produced in spermatocytes in culture and in vivo within 30 min of cellular exposure to MAA, a finding consistent with the rapid induction of other antioxidants (41, 42). From the work of Chapin and colleagues (3), we know that spermatocytes start to degenerate within 12 h of exposure, suggesting that the rapid increase of the thiol-specific antioxidant mRNA, after exposure to MAA, cannot maintain the needed oxidation-antioxidation balance. A third cDNA, clone 5, is also up-regulated in pachytene spermatocytes after treatment of cells with ME or MAA. Based upon its lack of similarity to database sequences, we conclude that it is a transcript from a novel gene.

ME and MAA also induce changes in gene expression in cultured Sertoli cells and in Sertoli cells in vivo. We have detected one cDNA, clone 4, that is down-regulated and two cDNAs, clones 2 and 3, that are up-regulated in Sertoli cells after exposure to ME or MAA. The down-regulated cDNA shares 80% similarity at the protein level to a PDE. The synthesis of intracellular cAMP is regulated by adenylate cyclase and the degradation of cAMP by PDEs. Multiple PDEs have been identified (43), and at least four genes encoding different isoforms of PDEs are differentially expressed in somatic and germ cells of the testis (44). Localization studies of PDE in rat seminiferous tubules indicate that PDE1 and PDE2 are predominantly expressed in germ cells, whereas PDE3 and PDE4 are mainly restricted to Sertoli cells (45, 46). Three rat PDE3 mRNAs with divergent 5' untranslated regions are present in Sertoli cells (47). The PDE of Sertoli cells may be a testis-specific enzyme, because its cDNA only hybridizes to a 3.2-kb mRNA in Sertoli cells and in testicular RNA. The size differences between the mRNA encoding the down-regulated PDE and the other testicular PDEs (45, 46) further argues that this PDE may be a new member of the PDE family.

A second cDNA, clone 3, is up-regulated in Sertoli cells after exposure to ME or MAA. This cDNA shares a 84% similarity, at the protein level, to a 60-kDa protein (A-170), originally cloned and characterized from murine peritoneal macrophages in response to stress, and it has a structural similarity to a tyrosine kinase p56 lck gene (48). The A-170 protein is 90% identical to a human protein that binds to the Src homology 2 domain of the T-cell-specific tyrosine kinase p56lck, believed to play a role in oxidative stress-responsive signal transduction in macrophages (48). Many specific protein-protein interactions are modulated through common structural domains, such as the Src homology regions 2 and 3 (SH2 and SH 3) (49). The SH2 domain is highly conserved in signaling molecules, and it mediates protein-protein interactions by binding to proteins containing phosphotyrosine. The similarity between the up-regulated Sertoli cell protein and the A-170 stress-inducible protein suggests a similar role for this protein in Sertoli cells, leading us to propose that it functions as a modulator of signal transduction, inducing cellular responses to oxidative stress. The A-170 protein is rapidly induced in macrophages exposed to diethyl maleate, whereas the Sertoli cell A-170 is induced within 30 min of exposure to MAA (48).

A third Sertoli cell clone, clone 2, is also up-regulated in Sertoli cells cultured with ME or MAA. No sequences matching clone 2 are detected in the database. Interestingly, we do not detect a similar up-regulation of clone 2 in vivo, suggesting it may only be expressed in isolated cells. The lack of clone 2 expression in vivo could be explained by the fact that, when cell-cell contact is intact, the expression of this gene is masked; and when the contact is disrupted, the gene is unmasked and expressed in isolated cells. It has been shown that breaking of cell contacts, and subsequent culture of hepatocytes, lead to an increase in jun B (50). Furthermore, a significant increase in jun B expression, after collagenase treatment of liver cells, has been reported (51). The up-regulation of clone 2 may be induced by the mechanics of Sertoli cell isolation similar to the up-regulation of jun proto-oncogenes after germ cell dissociation (52). Clone 2 is not expressed in vivo, suggesting that it may not have significance in testicular physiology.

In summary, our data indicate that ME and MAA induce changes in the gene expression of both pachytene spermatocytes and Sertoli cells. Changes in Sertoli cell metabolism may mediate the apparent toxicity of these agents to a specific population of meiotic germ cells. The identical response of Sertoli cells and pachytene spermatocytes to ME and MAA in vitro and in vivo suggests that the effects seen in Sertoli cells in culture are not an artifact of culture. We propose that cells exist in a state of oxidative siege in which survival requires an appropriate balance of oxidants and antioxidants. ME or MAA perturb this balance, resulting in the up-regulation in both Sertoli cells and pachytene spermatocytes of oxidative stress proteins, in an effort to prevent the resulting deaths of pachytene spermatocytes.


   Acknowledgments
 
We are indebted to Dr. Marianthi Kiriakidou for help in GenBank analysis and to Ms. Judith Wood for her excellent secretarial assistance.


   Footnotes
 
1 The sequences reported in this paper have been deposited in the GenBank database (accession no. AF053092-AF053097). This study was supported by NICHD Grant HD-11878. Back

Received December 5, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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