Reproductive
Toxicology,
Vol.
12, No. 0
Printed
I, pp.
in the USA.
1998
Science
Inc.
All rights mewed
0890-623X/98 ELSEVIER
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PI1 SOS90-6238(97)00098-l
NEW AND TRADITIONAL APPROACHES FOR THE ASSESSMENT TESTICULAR TOXICITY
OF
L. SUTER,* N. CLEMANN,~ E. KOCH,? M. BOBADILLA,~and R. BECHTER? *Pharma Non Clinical R&D, Toxicology, F. Hoffman-La Roche Ltd., 4070-Basle, Switzerland; and tPreclinica1 Safety, Experimental
Toxicology,
Nova&
Pharma AC., Bask, Switzerland
Abstract - In this study, the suitability of several methods for the assessment of testicular damage, including histopathology, flow cytometry (FCM), testicular sperm head counts, and secretion of androgen binding protein (ABP), has been evaluated. Testicular toxicity after acute exposure of adult rats to different doses of the known toxicant 1,3-dinitrobenzene (DNB) was analyzed. The effects showed dose dependence, in spite of the large variability within each dose group. Histopathology and FCM showed germ cell depletion, particularly of round spermatids; testicular sperm head counts were reduced and ABP production was increased. All evaluated methods showed similar sensitivities. The increased testicular ABP levels support the theory that the Sertoli cell is the likely target of DNB induced testicular toxicity, producing subsequent germ cell depletion. The presented results show the suitability of FCM for the analysis of testicular damage and also support the usefulness of including a metabolic marker for Sertoli cell function. 0 1998 Elsevier Science Inc. Kq
Words:
spermatogenesis; rat; I $dinitrobenzene;
histopathology; flow cytometry; sperm head counts: androgen binding protein.
tal animal, and that a male rat can still produce normal progeny after having its sperm production reduced to 10% of the normal level (8), it becomes of major importance to develop new and sensitive parameters to assess impairment of spermatogenesis. Histopathology is the traditional method used for the detection of testicular damage produced by physical or chemical insult (9). This method shows the disadvantages of being subjective and time consuming. A variety of methods have been suggested as possible alternatives to histopathology, among others metabolic and endocrinologic markers (IO), in vitro fertilization (1 l), and flow cytometric analysis of testicular tissue or sperm (12,13). Recently, flow cytometry (FCM) has become a useful tool for objective quantitation of the cell types involved in spermatogenesis and supplies, therefore, very valuable information for the detection of testicular toxicity (14,1.5). The advantages of flow cytometry compared to traditional histopathology are the rapidity with which the results can be obtained, the objectivity of the analysis, and the large number of cells that can be analyzed. Nevertheless, both histopathology and the staining utilized in this study for FCM are methods based on the analysis of fixed tissue and do not, therefore, provide information about the functional integrity of the testicular epithelium. In order to investigate the functionality of the germinal epithelium, androgen binding protein (ABP) was measured in testicular and epididymal tissue. This protein is synthesized by the Sertoli cells and is, therefore, an ideal physiologic indicator of Sertoli cell func-
INTRODUCTION
Assessment of potential health risks associated with exposure to chemical or physical agents is of paramount importance when these agents may interfere with the ability of individuals to produce normal progeny. A survey of the literature on male reproductive toxicants indicates that the process of spermatogenesis is the one affected by the overwhelming majority of chemicals so far studied (1). Among these compounds, 1,3-dinitrobenzene (DNB), a chemical intermediate used in various organic syntheses including the manufacture of dyes, plastics and explosives, is a potent testicular toxicant in laboratory animals, as well as in men (2). The effectiveness of DNB as a testicular poison in the rat is illustrated by marked testicular changes following a single oral dose (3-6). Possible approaches to the evaluation of testicular damage after treatment with a noxious substance include traditional mating studies and pregnancy outcome, sperm production and motility, and histopathology (7). Increasing concern about the sensitivity of the male reproductive system to chemical or physical insult has been paralleled by re-evaluation of these end points and their usefulness in reproductive risk assessment. Bearing in mind that the rat is the most commonly used experimenAddress correspondence to Dr. Rudolf Bechter, Novartis Pharma AG, S-2OO.P,P.O. Box, 4002 Basel, Switzerland. Received 24 Frhruury 1997; Revision received 3 June 1997; Awyted
28 June 1997. 39
40
Reproductive
Toxicology
tion (16). Sertoli cells play a key role in the spermatogenie process, since they constitute the structural framework of the seminiferous tubule, participate in the formation of the blood-testis barrier that isolates in a specific microenvironment the part of the seminiferous epithelium where meiosis occurs, and are targets for FSH and testosterone, which are responsible for the initiation and maintenance of spermatogenesis (17). Morphologically, Sertoli cells have numerous cytoplasmic processes that completely enclose various generations of developing germ cells, arranged in defined cell associations along the length of the tubule in precise order (18). The close physical contact between Sertoli and germ cells led to the concept that Sertoli cells provide a nurse function within the germinal epithelium (1). This possible nutritive role of Sertoli cells in germ cell development is supported by observations that Sertoli cells have a high glycolytic rate and secrete lactate and pyruvate. Additionally, studies on isolated germ cells have indicated that germ cells cannot maintain their ATP levels if supplied with glucose, but can do so on addition of lactate and to a certain extent pyruvate (19). Thus, Sertoli cells supply the energy substrate needed for normal germ cell function. On the other hand, Sertoli cell function is at least partly regulated by the surrounding germ cells, with total or partial germ cell depletion leading to impaired Sertoli cell function (20,21). Morphologic and functional integrity of the Sertoli cells is also of major importance since the blood-testis barrier consists of Sertoli cells forming a layer of cells in the basal region of the testicular tubules, joined to each other through tight junctions. This barrier, while intact, prevents macromolecules, including some toxicants, from reaching the germinal epithelium. In this paper we evaluate testicular damage produced after a single oral dose of DNB utilizing testicular histopathology, testicular sperm head counts, FCM, and ABP determination. The results of this work clearly show a good correlation of the results obtained by the different methods. We could observe testicular damage to the germinal epithelium, identifying the affected cell types. Histopathology and FCM results show treatment related depletion of round spermatids, the analysis of testicular sperm head counts shows the subsequent depletion of elongated spermatids and ABP measurements show Sertoli cell dysfunction, which is the likely reason for the observed germ cell depletion.
MATERIALS
AND METHODS
Animals and treatment Adult male Wistar rats (HanIbm), at least 12 weeks old, were purchased from BRL, Ftillinsdorf, Switzerland and kept singly in Macrolon type III cages on sawdust
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bedding. Food (Kliba 343, Switzerland) and tap water were provided ad libitum. Twenty-five animals were randomly numbered 1 to 25 and then assigned to 5 groups of equal size that were treated with a single oral dose of 1,3_dinitrobenzene (DNB, Sigma Chemical Co. St. Louis, MO). The dose groups (5 animals/group) were: 0 (control); 10 (low dose); 20 (low-mid dose); 30 (high-mid dose); and 40 (high dose) mg/kg body weight, respectively. Immediately before application, DNB was first dissolved in acetone and then diluted 1: 10 in corn oil for oral gavage. The dosage volume was 1 mL/kg body weight. Animals were sacrificed by asphyxiation with CO, after completion of a cycle of the seminal epithelium (13 d). Body weights were recorded on the days of treatment and sacrifice. Immediately after sacrifice, both testes and epididymes were excised and organ weights were recorded. One testis and its correspondent epididymis were fixed in Bouin’s solution for histopathology. The contralateral testicle was decapsulated and cut in two approximately equal pieces; one half was divided in two pieces that were separately frozen at -20°C until further preparation for either sperm head counts or ABP-determination. The other half was immediately used for cell isolation for flow cytometry (FCM). The remaining epididymis was frozen at -20°C until ABP determination. Histopathology Testes and epididymes were fixed in Bouin’s solution for approximately 24 h and afterwards washed several times with ethanol (70%) before embedding in paraffin. Embedded tissue was sectioned at 4 pm and tissue sections were stained with PAS (18). Stained slides were analyzed by light microscopy (Leica, DMRB, Germany). Transversal testicular sections were evaluated for the presence and integrity of different cell types and cell associations (stages), utilizing the description of the rat spermatogenesis by Russell et al. (22). For semiquantitative analysis, testicular damage was arbitrarily ranked from 0 (control) to 3 (severe damage). Longitudinal epididymal sections were evaluated for the presence of spermatozoa and degenerative cells. Microphotographs were made on 64 ASA color film. Sperm head counts Testicular tissue was processed following a modification of the method described by Blazak et al. (23). Briefly, frozen tissue in 15 mL NaCl (0.9%) containing 0.1% Triton X-100 (Sigma) was homogenized for approximately 60 s in a Potter S homogenizer (B. Braun, Switzerland) at a speed of 1 lOO/min. Homogenized tissue was then sonicated for a further 60 s in a Sonyprep
Evaluation
of testicular
150 (N. Zivy & Cie. S.A., Switzerland) at 75% of its maximal power. After homogenization and sonication, an aliquot of the cell suspension was loaded on a hemocytometer (Neubauer) and sonication resistant sperm heads, belonging to spertnatids steps 17 to 19 and spermatozoa, were counted under a light microscope equipped with a green light filter (IF 550, Olympus, Japan). Sperm head counts were expressed per gram of testicular tissue. Crll isolation Testicular cell suspensions were prepared as described by Suter et al. (24). Briefly, tissue was first incubated for 30 min in medium containing 0.25% trypsin (Gibco), 0.1% glucose (Merck), and 25 pg/mL deoxyribonuclease 1 (Sigma) and for a further 30 min in PBS containing 1 mg/mL collagenase (Type IA; Sigma) supplemented with glucose and DNase. Obtained cell suspensions were filtered through a 200 pm nylon mesh in order to discard tissue debris. One aliquot of each cell suspension was stained with 0.2% trypan blue for the determination of cell number and viability. Cells were then fixed in ethanol (70%) and stored at -20°C for at least 24 h before fluorescent staining. Cell staining fkjlow cytometry Cell staining was carried out following the description of Suter et al. (24). Approximately 2 X lo6 fixed cells were incubated with a monoclonal mouse antivimentin antibody (DAKO, 1:20) followed by incubation with the second antibody, 7-amino-4-methylcoumarin-3acetic acid (AMCA)-conjugated goat antimouse-IgG (Molecular Probes, 1:20). After immunostaining, DNA staining was carried out by adding ribonuclease A (Sigma) and propidium iodide (PI; Sigma) to final concentrations of 100 pg/mL and 50 pg/mL, respectively. Mitochondrial staining was then carried out by addition of nonyl-acridine orange (NAO; Molecular Probes) to a final concentration of 0.42 PM. Immediately before flow cytometric analysis, stained cells were fitered through a 45-pm nylon mesh. Flow cytometric analysis The fluorescence emission of the stained cells was measured on a FACS Vantage flow cytometer and cell sorter interfaced to a Hewlett Packard computer equipped with the LYSYS 11 software (Becton & Dickinson). The excitation sources consisted of two argon-ion lasers (lnnova 305) operated on the 488-nm (300 mW) and 35 1 to 364 nm ( 100 mW) lines, respectively. PI and NAO fluorescent emissions were monitored using 630 and 530 nm band-pass filters, respectively, along with a 610SP and a 560SP DC. Blue fluorescence was collected with a 424 nm band-pass filter together with a 630 LP
41
toxicity 0 L. SUTER ET AL.
dichroic mirror. The threshold was located on red fluorescence and at least 10,000 events were evaluated for each sample. Determination of androgen binding protein For the androgen binding protein (ABP) assay a portion of testicular tissue and one epididymis from each animal were processed to obtain cytosol. Tissue was homogenized with a Poter S homogenizer (B. Braun, Switzerland) in ice-cold buffer containing 10 mM Tris-HCl, 2 mu EDTA, and 10% glycerol (pH 7.4). The tissue:buffer ratios were I:4 for testis and 1:9 for epididymis. The homogenates were centrifuged at 106,000 X g for I h at 4°C and the supernatants (cytosol) collected and stored at -20°C until assayed. ABP was determined by steady state polyacrylamide gel electrophoresis (SS-PAGE) as described by Ritzen et al. (25). Briefly, ‘H-dihydrotestosterone was polymerized in 6% acrylamide:bisacrylamide slab gels on which aliquots of cytosol were run under constant current (15 mA/gel). ABP bound to ‘H-dihydrotestosterone is directly proportional to the ABP present in the probe and was quantified in a counter (LS 180 I, Beckman Instruments Inc., Fullerton, USA). The results were expressed as dpm/g tissue and the intra-assay coefficient of variation was below 7%. Statistical ana1ysi.r The results of body and organ weights, as well as testicular sperm head counts and FCM obtained for the treatment groups were compared utilizing variance analysis (ANOVA, one way) with P 5 0.05. For parameters showing significant differences between the groups, the Scheffk test a posteriori was carried out. Incidental anomalies of the seminiferous epithelium in the animals of the control and low-dose groups were compared by means of the unpaired Student t-test. Due to the large standard deviations, no statistical analysis could be carried out for the ABP determination. RESULTS Body and organ weights Body weight gain between Days 0 and 13 was not significantly affected by the treatment. Nevertheless, in the two high dose groups (30 and 40 mg/kg body weight), a reduction of body weight gain accompanied by an increase in the standard deviations was observed (Table 1). This finding was due to the large intragroup variability in the response of the animals in these two dose groups. While control animals showed a mean body weight gain of 27 g animals 18 (30 mg/kg body weight) and 22, 23, 24 (40 mg/kg body weight) showed much smaller body weight gain, ranging from 2 g to 12 g for
42
Reproductive
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Table I. Effect of DNB treatment on body weight gain and testicular and epididymal weights 13 d after a single oral dose DNB dose
(mgk weight) 0 10 20 30 40
body
Body weight gain
between Days 0 and 13 mean t SD (g) 21 22 24 18 15
? 8 i- 7 +- 5 t 10 * 11
Absolute testicular weights mean -C SD (g) 1.72 1.66 1.47 1.15 1.06
Absolute epididymal weights mean ? SD (g)
+- 0.15 t 0.08 +- 0.12 ? 0.26; r 0.21”
0.65 0.58 0.54 0.49 0.49
2 0.06 -+ 0.04 2 0.05 +- 0.03” * 0.06;
Relative (to body weight) testicular weights mean ? SD (%) 0.48 0.48 0.43 0.35 0.31
-t ? ? i+
0.04 0.04 0.3 0.08” 0.06”
Relative (to body weight) epididymal weights mean + SD (%) 0.18 0.17 0.16 0.15 0.14
t + t t *
0.01 0.02 0.01 0.02” 0.01”
Body weight gain (mean 2 SD of 5 animals per treatment group) between day of treatment (Day 0) and the day of killing (Day 13). Testicular and epididymal weights (mean IT SD of 5 animals per treatment group) as absolute (expressed in grams) and relative to body weight (as percentages) values. For each animal, the mean value of left and right organs were considered. “Significantly different from the control value after ANOVA (one way) and Scheffe test a posteriori.
the same period of time. Regarding testicular and epididymal weights, the values obtained for the two high dose groups (30 and 40 mg/kg body weight) showed a significant reduction of both absolute and relative (to body weight) organ weights (Table 1). For these parameters too, large differences in the response shown by different individuals were observed. Sperm head counts Quantification of sonication resistant sperm heads in testicular tissue showed a dose dependent reduction in testicular sperm head counts at doses of 30 and 40 mg/kg
10
body weight to approximately 10% of the control value in the high dose group (Figure 1). Histopathology Testicular histopathology of the treated animals showed a dose dependent effect of DNB on spermatogenesis in the rat (Figure 2). The results are summarized in Table 2. Control animals showed normal spermatogenesis, e.g. all known cell types were present and formed normal cell associations (Figure 2A). Animals treated with the low dose of DNB (10 mg/kg body weight) did not show significant testicular damage (Fig-
20
30
DNB dose (mgkg body weight) Fig. 1. Effects of DNB on testicular sperm head counts 13 d after a single oral dose. Values given relate to millions/g testicular tissue and represent the means t SD of 5 animals per treatment group. *Significantly different from the control value after ANOVA (one way) and Scheffe test a posteriori.
Evaluation
of testicular
toxicity
l
L. SUTEK ET
AL.
43
Fig. 2. Photomicrographs of testicular tissue sections from animals treated with different DNB doses and stained with PAS. A: control, stage IV; B: 10 mglkg, stage I; C: 20 mg/kg, stage II-III; D: 30 mg/kg; E: 40 mg/kg. For the animals treated with the high doses of DNB (D and E) the stage could not be determined. Magnification, X400. V, vacuoles: d, degenerative cells: :k, germ cell depletion.
ure 2B). For this low-dose group as well as for the control group, tubules presenting slight vacuolation and/or few degenerative cells were quantified. Control animals showed an average of 3.38% rt 1.14% affected tubules and the low dose group showed 5.4% t 1.14% affected tubules. These values did not significantly differ. In animals treated with higher DNB doses (20, 30, and 40 mg/kg body weight) testicular toxicity was evident (Figure 2C-E). In these groups, the response to treatment strongly differed between individuals, nevertheless the severity of the testicular impairment showed, to some extent, dose dependency. Round spermatids were the most strongly depleted cells. This cell type was partially affected in the animals treated with 20 mg/kg body weight DNB (Figure 2C), especially in Stages VII Table 2. Histopathologic DNB dose (m&/kg body weight)
n
Affected animals
evaluation
and VIII. With increasing dose, at 30 mglkg body weight DNB, elongated spermatids also appeared strongly depleted and a slight depletion of pachytene spermatocytes was observed (Figure 2D). For this dose group, the most affected stages were VII through XIV. Animals treated with the highest dose showed severe depletion of germ cells and some tubules showed Sertoli cells only (Figure 2E). The severity grade of the vacuolation of the Sertoli cells also increased in a dose dependent manner and the diameter of the testicular tubules was decreased for the mid-high and high dose groups (data not shown). Regarding epididymal histopathology, mainly spermatozoa were seen in the epididymides of the animals treated with vehicle and low-dose DNB. In the males treated with 20 mg/kg body weight DNB, some degen-
of the effect of DNB on rat spermatogenesis
Depleted cell types
Affected stages
0
5
-
-
IO
5
-
-
20
5
Degenerative
Vacuolation
13 d after a single oral dose Sperm retention
Severity Summary
Epidiymal
findings
IOnly Spz
30
40
5
5
4
3
2
Round Spd (grade 2)
Round Spd (grade 3), Elongated Spd (grade 2). Pachhytene Spc (grade I) All types of Spd (grade 3) Pachytene Spc (grade 2) Some Sertoli cell only tubules
VII-VIII
VII-XIV
All
I-t
2
3
I
2
3
I
-
-
I+
2+
3p
Only Spz Sp7 Some degenerative Fewer Spa, Degenerative
cells
cells
Very few Spz Very few Spz Degenerative cells
Effects of DNB on spermatogenesis I3 d after a single oral dose. Histopathologic evaluation of PAS-stained testicular tissue sections. Sections were examined by light microscopy at a magnification of 400X, and the presence of degenerative cells, vacuolation of the germinal epithelium, sperm retention, and germ cell depletion were evaluated. Damage was ranked utilizing an arbitrary severity scale ranging from 0 (normal) to 3 (severe damage). Spc, spermatocyte; Spd, spermatid: Spz, spermatozoon.
Reproductive Toxicology
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12,
Number 1, 1998
n Control q 10 mgkg El20 mgkg I9 30 mg/kg n 40 mgkg
oPrL Spcl
LIZ spc
PSpc
Set Spc
Spg
RSpd (steps 1-5)
Spd (steps 6-16)
Spd (steps 17-18)
Spd (step 19)/Spz
Testicular germ cells Fig. 3. Effects of DNB on spermatogenesis 13 d after a single oral dose. Bar graphs representing the proportions of each germ cell subpopulation quantified by three-parameter FCM (mean -t SD of 5 animals per treatment group). Spc, spermatocyte; Spd, spermatid; Spg, spermatogonia; PrL, preleptotene; L/Z, leptotene/zygotene; P, pachytene; Set, secondary; R, round; Spz, spermatozoon. *, significantly different from the control value after ANOVA (one way) and Scheffe test a posteriori.
cells were detected and in the animals treated with 30 and 40 mg/kg body weight, the number of degenerative cells in the epididymal tubules increased, whilst the number of spermatozoa was markedly reduced.
erative
Flow cytometric analysis Flow cytometric analysis of testicular cell suspensions of the treated animals compared with the control animals showed, according to vimentin immunoreactivity, a dose-dependent decrease in the proportion of germ cells, indicated by an increase of vimentin-positive somatic cells. In the control animals, only 4.4 -t 0.9% of the cells present in the testicle were somatic cells. The proportion of somatic cells increased in a dose dependent manner and was 5.78 f 1.61%; 6.69 -+ 2.48%; 18.53 + 14.67%; and 30.6 2 13.06% for the increasing DNB doses, respectively. This increase means that the germ cells present in the testicular tissue decreased from a control value of around 95% down to 70% for the highest dose (40 mg/kg body weight). As has previously been described (13), three-param-
eter FCM allows 10 germ cell subpopulations to be identified and quantified. Analysis of these germ cell subpopulations showed that the most affected cell type was the round spermatid, the percentages of which were dose-dependently decreased after treatment with DNB (Figure 3). Concomitantly with the reduction of the proportion of haploid cells, a relative increase in the proportion of other cell subpopulations, especially the spermatogonia and early primary spermatocytes (preleptotene to zygotene) was observed. Determination of androgen binding protein ABP concentrations were determined in cytosol obtained from epididymal and testicular tissues and the results are summarized in Table 3. The ABP concentration (expressed as dpmlg tissue) showed a dose-dependent increase in the testis, reaching a three-fold increase for the highest DNB dose. Conversely, the amount of ABP measured in epididymes showed a dose-dependent decrease. The decrease observed for the epididymal ABP content (25% of the control value for the high dose group) was not as marked as the increase observed in
Evaluation
Table
3. ABP
DNB dose (mg/kg body weight) 0
I0 20 30 40
measured in testicular and epididymal 13 d after a single oral dose
of testicular
tissue
ABP mean 2 SD (dpm/g tissue) Epididymis
Testis 7905 7482 8820 15426 25718
t 2145 2 4708 L 1631 I?r 10281 2 12919
34492 31038 37463 25387 26106
+ 2 t k 2
15905 8825 207 IO 15077 21505
Effects of DNB on ABP content (mean i: SD of 5 animals per treatment group) in testicular and epididymal tissue 13 d after a single oral dose, measured by steady-state electrophoresis and expressed as dpm/g tissue. The large SD made statistical analysis impossible.
testicular tissue. Both testicular values showed large interanimal statistical analysis impossible.
and epididymal ABP variability, rendering
DISCUSSION This work attempted to detect testicular injury in male rats treated with the known testicular toxicant DNB and to compare the results obtained evaluating different endpoints. The utilization of histopathology and FCM, in conjunction with the quantification of testicular sperm head counts and ABP, provides information concerning several aspects of the testicular toxicity induced by DNB. Testicular histopathology has been widely used to assess disruption of spermatogenesis. Histopathology supplies information about the cell associations present in the germinal epithelium and allows for a semiquantitative evaluation of testicular damage. This method shows the disadvantages of being subjective, time consuming, and only allowing a very limited number of testicular tubules to be evaluated. Flow cytometric analysis of cell suspensions is a relatively new method and has already been utilized to detect testicular damage after treatment with a range of toxicants, including DNB (26-28). The results obtained in this study by means of FCM for the control animals are consistent with the control pattern defined by the same method in a previous paper (13). As described elsewhere (6), the low DNB dose did not produce any toxic effect in the rat. In this study, for all analyzed parameters including body and testicular weights no statistically significant differences between the 20 mgikg body weight dose group and the control were detected, although a clear trend was observed. That was partly due to the moderate effect of this DNB dose on the spermatogenesis and partly to the interanimal variability in response found for each dose group generating large standard deviations and resulting in no clear-cut dose response. For the rat, a similar variability within dose groups after a single dose of DNB has already been reported (10,28,29). The results of histopathology and FCM obtained in
toxicity
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L. SUTER ET AL
45
the present study show a dose dependent effect of DNB on the rat testis. This effect appeared to be moderate for animals treated with 20 mg/kg body weight and marked to severe for animals belonging to the 30 and 40 mg/kg body weight dose groups. The histopathologic evaluation as well as the FCM analysis are in agreement with a previous report, in which a dose-response study showed no effects with 10 mg/kg body weight, minimal effects with 20, and extensive damage with 30 mg/kg body weight. (10). Both methods showed that the effects caused by the treatment produced partial to marked depletion of round spermatids and, to some extent, of elongated spermatids, accompanied by a relative increase of spermatogonia and early primary spermatocytes (e.g. preleptotene to zygotene spermatocytes). For the less affected animals, the effects caused by the treatment were limited to the stages VII and VIII, more stages becoming affected as the damage grew more severe. Similar results have been reported by other investigators (6,30,3 1). The significant reduction down to approximately 10% of the control value at 40 mg/kg body weight of testicular sperm head counts in the high dose groups correlates well with the above mentioned findings, indicating a disruption of the later steps of spermatogenesis. This is also in agreement with the results of Linder et al. (30), who described a decay in testicular sperm head counts after treatment with 48 mg/kg body weight DNB, reaching the lowest level of ca. 20% of the control value 16 d after treatment. Evaluation of testicular sperm head counts seems to be a good indicator of spermatogenic damage (32). Transient increases in testicular sperm head counts are consistent with selective retention of late spermatids and subsequent decreases rellect maturation depletion due to loss of earlier cell types (30). Testicular sperm head counts can, however, overlook effects affecting earlier stages if the affected cells have not yet reached the stage of mature spermatids. ABP is a reliable marker for Sertoli cell function and can therefore provide information about whether or not the mechanism of action of a given chemical affects this particular cell type (10). ABP determination in testicular tissue showed for the high dose groups a twoto three-fold increase in the testicular ABP content accompanied by large standard deviations, which prevented statistical analysis from being carried out. Nevertheless, such an increase should be considered as biologically significant. It has already been described that the Sertoli cell is the main target of DNB induced testicular toxicity (33) and the modified production of ABP supports this theory. Histopathology results also confirm the site of the testicular lesion as being the Sertoli cell, since vacuolation of this cell type was observed. Furthermore, the stages particularly affected in
46
Reproductive
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the animals treated with DNB at 20 mg/kg body weight, which displayed limited testicular damage, were mainly VII and VIII. These two stages are also the ones in which the peak of secretion of ABP and plasminogen activator were observed (34). Whether the effect on the Sertoli cell is due to a direct action of DNB or to an indirect effect caused by depletion of germ cells producing an impaired modulation of Sertoli cell function could not be determined. The onset of both Sertoli cell effects and degenerative changes in germ cells (primary spermatocytes) after treatment with 25 mg/kg body weight DNB has been reported to be simultaneous (6,lO). For the same DNB dose, plasma ABP levels reached a maximum value 48 h after treatment, but returned to control values 14 d after treatment (10). To our knowledge, this is the first study to investigate testicular and epididymal ABP content after treatment with DNB. The higher (compared to the control) levels of testicular ABP 13 d after treatment with 30 and 40 mg/kg body weight DNB indicate a persistent impairment of Sertoli cell function. The increased testicular ABP levels found in this investigation 13 d after DNB treatment and the normal plasma ABP levels reported by Reader et al. (10) 14 d after treatment probably indicate a modification of the ABP secretion ratio. Under normal conditions, Sertoli cells secrete 20% of their ABP production into the plasma and 80% into the tubular lumen (10). Treatment with a toxicant like DNB could induce the Sertoli cell to secret its products preferentially through its apical membrane toward the testis, leading to a higher concentration of the protein in the testicular tissue, even after the normalization of the systemic level. A similar effect was reported by Morris et al. (35), who found that the bidirectional secretion of ABP was modulated by the selective impairment of spermatogenesis. Indeed, lower ABP epididymal values also suggest an organ specific effect generating a higher ABP concentration in the testis, but the results obtained in the present study do not supply sufficient information to hypothesize on a physiologic explanation. Further studies should be carried out to clarify this point. Summarizing, the results from the histopathologic studies, FCM, and testicular sperm head counts suggest that the early stages of primary spermatocytes were not affected 13 d after DNB treatment. Bearing in mind that DNB affects in the first instance Sertoli cell function (1,33) and primary spermatocytes and round spermatids (6) by 48 h after treatment and that this acute effect is followed by a recovery phase (30,31), we conclude that our data represent the beginning of this recovery phase. The early stages of primary spermatocytes were partly recovered in some animals and the more mature stages were still missing. On the other hand, the recovery seemed to be dose-dependent, since some of the animals treated with the highest DNB dose were still seriously
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affected. This is in agreement with the results of Linder et al. and Hess et al. (30,31) who described a partial recovery of the germinal epithelium, starting 16 d after DNB treatment. There was good correlation of all the evaluated parameters. Although large differences were observed between animals, the males showing the most damaged testes in histopathology were also massively affected according to the FCM results and also showed the lower values for testicular sperm head counts and the highest levels of testicular ABP. We conclude, therefore, that DNB, as has been described before, produced reversible damage of the Sertoli cells, which was reflected in degeneration and depletion of primary spermatocytes. This effect produced a sequential depletion of other germ cells and at the same time a slow recovery of the affected cells. This process was partly masked by the large variations of sensitivity to the treatment found for different animals. The results presented in this paper support results presented in a previous paper (13) concerning the suitability of FCM after triple fluorescent staining of testicular cell suspensions for the evaluation of spermatogenesis. Three-parameter FCM is, therefore, a useful tool to detect testicular damage and should be considered as an alternative to histopathologic testicular staging. Additionally, testicular ABP measurements provide information for detecting dysfunction of the Sertoli cells, which play a major role during the spermatogenic process. Future studies should validate these alternative methods utilizing a larger number of animals and a variety of toxicants. wish to thank Dr. Patricia Brander for her advice concerning histopathologic evaluation and Ms. Brigitte Greiner and Mr. Christian Elsaesser for their technical assistence and advice. We would also like to thank Mr. Julian Dick for the critical reading of this manuscript and his constant support.
Acknowledgments-We
REFERENCES 1. Foster PMD. Testicular
2.
3.
4.
5.
6.
7.
organization and biochemical function. In: Lamb JC 4th, Foster PMD, eds. Physiology and toxicology of male reproduction. San Diego, CA: Academic Press Inc.; 19887-3 I. Ishihara N, Kanaya A, Ikeda M. m-Dinitrobenzene intoxication due to skin absorption, Int Arch Occup Environ Hlth. 1976;36: 161-8. Linder RE, Strader LF, Barbee RR, Rehnberg GL, Perreault SD. Reproductive toxicity of a single dose of 1,3_dinitrobenzene in two ages of young adult male rats. Fundam Appl Toxicol. 1990;14: 284-98. Obasaju MF, Katz DF, Miller MG. Species differences in suceptibility to 1,3-dinitrobenzene-induced testicular toxicity and methemoglobinemia. Fundam Appl Toxicol. 1991;16:257-66. Brown CD, Forman CL, McEuen SF, Miller MC. Metabolism and testicular toxicity of 1,3-dinitrobenzene in rats of different ages. Fundam Appl Toxicol. 1994;23:439-46. Blackburn DM, Gray AJ, Lloyd SC, Sheard CM, Foster PMD. A comparison of the effects of the three isomers of dinitrobenzene on the testis in the rat. Toxicol Appl Pharmacol. 1988;92:54-64. Lamb JC 4th. Fundamentals of’ male reproductive toxicity testing.
Evaluation
8. 9.
IO.
I I.
12.
13.
14.
IS.
16.
17. 18.
19.
20.
21. 22.
of testicular
In: Lamb JC 4th, Foster PMD, eds. Physiology and toxicology of male reproduction. San Diego, CA: Academic Press Inc.; 137-53. Aafjes JH. Vels JM, Schenk, E. Fertility of rats with artificial oligozoospermia. J Reprod Fertil. 1980;58:345-5 I. Russell LD, Ettlin RA, SinhaHikim AP, Clegg ED. Staging foi laboratory species. In: Evaluation of the testis, First Edition. Clearwater: Cache River Press; 1990:62-194. Reader SCJ, Shingles C, Stonard MD. Acute testicular toxicity of 1,3-dinitrobenzene and ethylene glycol monomethyl ether in the rat: Evaluation of biochemical effect markers and hormonal responses. Fundam Appl Toxicol. 1991;16:61-70. Holloway AJ, Moore HDM, Foster PMD. The use of in vitro fertilization to detect reductions in the fertility of malerats exposed to I &dinitrobenzene. Fundam Appl Toxicol. 1990; 14: I 13-22. Hittmair A, Rogatsch H, Offner F, Feichtinger H, dfner D, Mikuz G. Deoxyribonucleic acid flow cytometry and semiquantitative histology of spermatogenesis: a comparative study. Fertil Steril. 1992;58:1040-45. Suter L, Bobadilla M, Koch E. Bechter R. Flow cytometric evaluation of the effects of doxorubicin on rat spermatogenesis. Reprod Toxicol. 1997; I I :in press. Petit JM, Ratinaud MH, Cordelli E, Spano M, Julien R. Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis. Cytometry. 1995;19:304-12. Spano M, Bartoleschi C, Cordelli E, Leter G, Tiveron C, Pacchierotti F. Flow cytometric assessment of trophosophamide toxicity on mouse spermatogenesis. Cytometry. 1996;24: 174-80. Gunsalus CL, Larrea F, Musto NA, becker RR, Mathier JP, Bardin CW. Androgen binding protein as a marker for Sertoli cell function. J Steroid Biochem. 1981;15:99-106. Ritzen EM. Hansson V, French FS. The Sertoli cell. Burger H, de Kretser DM. eds. New York: Raven Press; 1981:171-94. Leblond CP. Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann NY Acad Sci. 1952: 55:548-73. Jutte NHPM, Jansen R, Grootegoed JA, Rommerts FFG, van der Molen HJ. FSH stimulation of the production of pyruvate and lactate by rat Sertoli cells may be involved in hormonal regulation of spermatogenesis. J Reprod Fertil. 1983;68:219-26. Galdieri M. Monaco L, Stefanini M. Secretion of androgen binding protein by Sertoli cells is influenced by contact with germ cells. J Androl. 1984;5:409-15. Sharpe RM. lntratesticular factors controlling testicular function. Biol Reprod. 1984:30:29-49. Russell LD. Ettlin RA. SinhaHikim AP, Clegg ED Mammalian
toxicity 0 L. SUTER ET
23.
24.
25.
26.
27. 28.
29.
30.
31.
32.
33. 34. 35.
AL.
47
Spermatogenesis In: Evaluation of the testis, first edition. Clearwater: Cache River Press; 1990:1-40. Blazak WF, Ernst TL, Stewart BE. Potential indicators of reproductive toxicity: Testicular sperm production and epididymal sperm number, transit time, and motility in Fischer 344 rats. Fundam Appl Toxicol. 1985;5:1097-102. Suter L, Bechter R, Koch E, Bobadilla M. Three parameter flow cytometric analysis of rat spermatogenesis. Cytometry. 1997;27: 161-68. Ritzen EM, French FS, Weddington SC, Nayfeh SN, Hanson V. Steroid binding in polyacrylamide gels. J Biol Chem. 1974:249: 6597-6604. Hacker U, Schumann J, Goehde W. Effects of acute gamma irradiation on spermatogenesis as revealed by Bow cytometry. Acta Radiologica Oncologa. 1980;19:36 l-8. Spano M. Evenson DP. Flow cytometric studies in reproductive toxicology. New horizons in biologic dositometry. 199 I :497-5 I I. Evenaon DP, Janca FC, Jost LK, Baer RK, Karabinus DS. Flow cytometric analysis of effects of 1.3.dinitrobenzene on rat spermatogenesis. J Toxicol Environ Health. 1989;28:X l-98. McEuen SF, Jacobson CF, Brown CD. and Miller MG. Metabolism and testicular toxicity of I ,3-dinitrobenzene in the rat: Effect of route of administration. Fundam Appl Toxicol. 1995;28:94-9. Linder PE, Hess RA, Perreault SD, Strader LF, Barbee RR. Acute effects and long-term sequelae of 1.3.dinitrobenzene on male reproduction in the rat. I. Sperm quality. quantity and fertilizing ability. J Androl. I988;9:3 17-26. Hess RA, Linder RE, Strader LF. Perreault SD. Acute effects and long-term sequelae of I $dinitrobenzene on male reproduction in the rat. II. Qualitative and quantitative hi\topathology of the testis. J Androl. 1988;9:327-42. Cassidy SL, Dix K,,, Jenkins T. Evaluation of a testicular sperm head counting technique using rats exposed to dimethoxy-ethyl phthalate (DMEP), glycerol a-monochlorohydrin (GMCH), epichlorohydrin (ECH), formaldehyde (FA), or methyl metanesulphonate (MMS). Arch Toxicol. 1983;53:7 I-8. Foster PMD, Sheard CM, Lloyd SC. I $dinitrobenzene: a Sertoli cell toxicant? Excerpta Med Int Congr Ser. 1986:7 16:28 l-88. Parvinen M. Regulation of the seminiferous epithelium. Endocrine Rev. 1982;3:404-17. Morris lD, Bardin CW, Musto NA. Thau RB, Gunsalus GL. Evidence suggestmg that germ cells intluence the bidirectional secretion of androgen binding protein by the seminiferous epithelium demonstrated by selective impairment of spermatogenesis with busulfan. Int J Androl. 1987;10:691-7.