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From the Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington.
| Correspondence to: Michael K. Skinner, Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4231 (e-mail: skinner{at}mail.wsu.edu). |
| Received for publication April 13, 2006; accepted for publication July 11, 2006. |
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Abstract |
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Key words: Epigenetic, testis, gametogenesis, male infertility, antiandrogen, apoptosis
A casual factor recently shown to induce a transgenerational phenotype is exposure to environmental endocrine disruptors (Anway et al, 2005). Endocrine disruptors are hormonally active environmental compounds that have been shown to influence both male and female reproductive development and function (Gray et al, 1994; Laws et al, 2000; Sharpe, 2001; Bayley et al, 2002). Endocrine disruptors such as pesticides (eg, DDT and methoxychlor) (Cummings, 1997), fungicides (eg, vinclozolin) (Kelce et al, 1994), insecticides (eg, trichlorfon) (Voccia et al, 1999), herbicides (eg, atrazine) (Cooper et al, 1999), and plastics (eg, phthalates) (Fisher, 2004) affect normal reproductive physiological development and functions by acting as weak estrogenic, antiestrogenic, or antiandrogenic compounds.
Vinclozolin (3-(3-5-dichlorophenyl)-5-methyl-oxazolidine-2, 4-dione) is a systemic fungicide registered for use on fruits and vegetables and commonly used in the wine industry (Kelce et al, 1994). Vinclozolin and metabolites, butenoic acid (M1) and enanilide (M2) derivatives, act as antiandrogens through actions on the androgen receptor (Pothuluri et al, 2000). Transient exposure of neonates to vinclozolin delays puberty and inhibits androgen-dependent male reproductive tract development (Gray et al, 1994). Embryonic exposure to vinclozolin influences male sexual differentiation and development as well as adult spermatogenesis (Wolf et al, 2000; Uzumcu et al, 2004). A previous study (Uzumcu et al, 2004) demonstrated that administrating 100 mg/kg/day of vinclozolin to pregnant rats during embryonic sex determination (embryonic day [E] 8–14 in the rat) reduced the spermatogenic capacity by decreasing germ cell survival in the subsequent F1 adult male offspring. Subsequently, vinclozolin exposure later in embryonic development (E15-postnatal day 0 [P0]) had no affect on adult spermatogenesis (Wolf et al, 2000; Uzumcu et al, 2004). In addition, Omezine and coworkers (Omezzine et al, 2003) demonstrated that embryonic exposure (E6-P0) of the antiandrogen flutamide resulted in a similar reduced spermatogenic capacity in the subsequent F1 male offspring. Combined observations suggest that embryonic testis development is sensitive to androgen receptor signaling and can affect germ cell survival in the adult testis.
Embryonic testis development prior to and during sex determination (ie, E8–14 in the rat) appears to be the sensitive exposure period to antiandrogenic compounds (ie, vinclozolin) in the promotion of the reduced spermatogenic capacity in the adult male offspring (Uzumcu et al, 2004). The mechanism for a toxicant to influence embryonic development and transfer a disease state to an adult phenotype remains to be elucidated. The current study further investigates (Anway et al, 2005) the ability of the spermatogenic cell phenotype to be transferred to subsequent generations in both outbred and inbred strains of rats.
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Materials and Methods |
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Sperm Motility and Concentration Analyses
The sperm motility was determined using caudal epididymal sperm from the
P60 males as previously described (Uzumcu
et al, 2004). Briefly, the epididymis was dissected free of
connective tissue and a small cut made to the cauda. The tissue as placed in 5
mL F12 culture medium containing 0.1% BSA for 10 minutes at 37°C. Fifty
microliters was placed on a warm slide and gently cover-slipped. The specimen
was immediately examined using phase contrast microscopy with 100x
magnification. The sperm motility assays examined rapid progressive, slow
progressive, and nonprogressive motility according to WHO category
(Kvist and Björndahl,
2002). The ratio of motile sperm to the total number of sperm,
including immotile sperm, was calculated. Approximately 50– 100 sperm
were counted per microscopic field. The procedure was repeated at least twice,
with a new specimen from the same epididymis. The average value was considered
as percent motility for that rat and used as one replicate in statistical
analysis. Epididymal sperm count was determined using the same epididymis
according to a previously described method, with some modifications
(Taylor et al, 1985;
Uzumcu et al, 2004). Briefly,
the epididymis that was placed in the 5 mL of culture medium was minced. The
tissue pieces were removed, and the remaining sperm suspension was diluted
with equal volume of 0.2% glutaraldehyde in 1x PBS to immobilize the
sperm. Three independent sperm samples were counted using a hemacytometer. The
counts were averaged and used as a replicate in statistical analysis. The
control and vinclozolin generation analysis for an individual experiment were
done at the same time. All analyses were done blinded, and different
individuals were used for collection and counting.
Testicular Histology and Cellular Apoptosis
Following the weight determination, testes were cannulated with Bouin
fixative (Sigma, St. Louis, Mo), cut in half, and submerged in Bouin for
6–10 hours, then washed with 70% ethanol. Two cross sections from each
testis were embedded in parafilm using standard procedures performed by the
Center for Reproductive Biology Histology Core Laboratory. Paraffin-embedded
tissues were serially sectioned. At least two nonserial sections were stained
with hematoxylin and eosin (H&E) using standard procedures for
morphological analyses. Apoptotic cells were detected on duplicate slides by
TUNEL assay using a Fluorescein In Situ Cell Death Detection Kit (Roche
Applied Science, Indianapolis, Ind). The fluorescent cells in each testis
cross-section were counted at 200x magnification. The average number of
fluorescent germ cells per testis section from stages I–V,
VI–VIII, and IX–XIV from duplicate slides from one animal (2
testis sections per slide) was used as a replicate in statistical analysis.
Approximately the same number of tubules from stages I–V, VI–VIII,
and IX–XIV were present in control and vinclozolin generation testis
sections during the apoptosis analyses. The negative control sections where
the terminal transferase enzyme was excluded from the assay showed no labeling
(data not shown).
Homogenization-Resistant Spermatid Analysis
The number of homogenization-resistant spermatid heads was determined for
F3 control (n = 3) and vinclozolin (n = 3) animals as previously described
(Robb et al, 1978). Briefly,
the right testis from control and vinclozolin generation animals was excised
and tunica albuginea was removed and homogenized in 50 mL of saline-triton
buffer (0.15 M NaCl and 0.05% (v/v) Triton X-100). Each elongated spermatid
head resistant to the homogenization was counted using a hemacytometer. Each
sample was counted 4 times and averaged. Data were represented as average
spermatids per testis (Zirkin et al,
1989).
Radioimmunoassays
Serum and testicular fluid (TF) were collected according to previously
described methods (Turner et al,
1984; Hill et al,
2004). All samples were stored at –80°C until assay for
testosterone. The serum and TF testosterone was determined by radioimmunoassay
with a testosterone double antibody RIA kit (Diagnostic Systems, Webster, Tex)
and was assayed by the Center for Reproductive Biology Assay Core Laboratory.
The sensitivity of the assay was 10 pg/tube. Serum testosterone concentrations
were determined on all samples collected, whereas TF testosterone levels were
measured on 3 control and 4 vinclozolin-treated SD P60 males from the F2
generation.
Statistical Analysis
The data from testis weights, apoptotic cell counts, testosterone assays,
sperm motility, sperm count, and spermatid count were analyzed using SAS. The
values were expressed as the mean ± SEM to account for sample and
animal variation within a data set. Statistical analysis was performed, and
the differences between the means of treatments and respective controls were
determined using a paired Student's t test for single comparison made
between control and vinclozolin generation animals. No multiple comparisons
were made that would require an ANOVA, so the Student's t test was
optimal. Experiments were repeated with 3–17 rats per experimental
group. A statistically significant difference was confirmed at P <
.05.
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Results |
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The testis/body weight ratios between the control and vinclozolin generations were not statistically different (Figure 1). The testes/body weight ratios for the vinclozolin-treated generations F1, F2, F3, F4, VOC and the RVOC generations was 9.3 ± 0.2, 9.9 ± 0.2, 10.2 ± 0.5, 8.2 ± 0.3, 9.9 ± 0.4 and 9.8 ± 0.1, respectively. None of the adult P60 males tested had significantly reduced testis weights in the control or vinclozolin generations in Sprague Dawley rats (Figure 1A). Similar observations were made with Fisher rats, except the F1 generation had a statistically smaller testis/body ratio (Figure 1B). The Fisher inbred strain did show some toxicology in testis weight in the F1, but not in the F2 or F3 generations. Combined observations suggest minimal toxicology of vinclozolin for any generation at the embryonic or early postnatal ages.
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A homogenization resistant spermatid analysis was performed to confirm that the testis spermatogenic cell number was decreased in vinclozolin generation animals and the potential causal factor in alterations in epididymal sperm numbers. The testis from F3 generation control animals had 470 ± 20 million spermatids per testis, while F3 vinclozolin generation animals had 336 ± 17 million spermatids per testis, which was statistically different (P < .008). In contrast, an F3 vinclozolin male that was found to not have a decreased epididymal sperm count also did not have a decreased spermatid per testis count (data not shown). Therefore, the decline in epididymal sperm count corresponded to a reduction in spermatid numbers per testis.
Testes of each male rat were collected, fixed, sectioned, and stained for morphological analysis. The testis morphology of the control SD P60 males was normal in all the generations (Figure 2A). The testis morphology of the vinclozolin-treated SD P60 males was generally normal, with a percentage of the vinclozolin generation animals having morphological abnormalities in the testis. Approximately 15% of the vinclozolin generation animals had testes with more than one large vacuole within seminiferous tubules per testis cross section (Figure 2B). Furthermore, approximately 7% of the vinclozolin generation animals had testes that contained more than one seminiferous tubule with complete spermatogenic failure per testis cross section (Figure 2C and D). These seminiferous tubules were devoid of any advanced germ cell populations, with some tubules containing Sertoli cells only. When present, these abnormal tubules in testis cross sections made up 2%–12% of the total seminiferous tubules in the cross section. Morphological analyses of SD P20 testes from vinclozolin generations were similar to controls and did not show any abnormal morphology (Figure 2E and F). The testis morphology of the VOC rats was similar to the vinclozolin generations in that approximately 20% of the testis cross sections had more than one large vacuole and/or a lack of spermatogenesis within a tubule. In contrast, the RVOC testes were similar to controls and had no abnormalities (data not shown). In contrast to the SD rats, the vinclozolin-treated CDF rats showed no apparent increase in abnormalities in the morphology of the testis. Therefore, the outbred strain had a more dramatic phenotype.
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The serum and testicular fluid testosterone levels were measured in the control and vinclozolin Sprague Dawley (SD) rat generations to determine if the vinclozolin exposure effected androgen production (Figure 7). The serum levels of testosterone in the vinclozolin F1, F2, F3, F4, VOC, and RVOC generations were 4.2 ± 0.8, 4.4 ± 0.6, 3.8 ± 0.8, 6.7 ± 2.0, 6.4 ± 1.2, and 5.8 ± 1.6, respectively. Testosterone levels were not different from their control values (Figure 7A). The testicular fluid was collected and analyzed in the control and vinclozolin F2 generation to measure the testis concentration of testosterone. As with the serum levels of testosterone, the testicular fluid level of testosterone in the vinclozolin F2 generation was 86 ± 16 and did not differ from the control value of 71 ± 12 (Figure 7B). Analysis of Fisher (CDF) rat control and vinclozolin generation males also demonstrated no effects on serum testosterone levels (Figure 8). The basal level of serum testosterone was lower in the CDF rats than SD, but no change in serum levels was observed. Therefore, the reduced spermatogenic capacity observed was not due to endocrine affects and a reduction in testosterone levels.
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Discussion |
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The spermatogenic cell defect was primarily localized to stage IX–XIV seminiferous tubules. This is a stage where spermatogenesis has advanced germ cell maturation and early spermatocyte development. Interestingly, both SD and Fisher rats had the same localization of spermatogenic cell apoptosis. Future studies with procedures such as cell sorting will help identify the most sensitive spermatogenic cell population. Epididymal sperm concentration and motility were also reduced in the vinclozolin generation animals for both strains of rats (Anway et al, 2005). The current study demonstrates that spermatid number per testis was also decreased in vinclozolin F3 generation animals. The inbred Fisher rats did show signs of greater toxicity to vinclozolin in the F1 generation males, with decreased testis/body weight and increased apoptosis in all stage tubules for the F1 vinclozolin males. However, this was lost in the F2 and F3 generations, which were similar to the SD rats.
The serum testosterone and testicular fluid testosterone levels did not change in any of the vinclozolin generation males. Therefore, the transgenerational phenotype and spermatogenic cell defect was not due to an endocrine defect. The transgenerational phenotype is likely more of a local molecular alteration in the germ line. Although the initial action of vinclozolin during embryonic sex determination is through an antiandrogenic mechanism, the adult phenotype and transgenerational transmission is presumed to be hormone-independent.
Transgenerational transmission of such a phenotype requires either an epigenetic phenomenon involving DNA methylation or stable alterations involving a DNA sequence mutation (Rakyan and Whitelaw, 2003). The frequency of the phenotype observed in this study based on the apoptotic TUNEL labeling analyses was approximately 90%. This frequency is substantially higher than the mutational rate (less than 1%) observed in the irradiation-induced mutational analyses previously described (Barber et al, 2002). The DNA sequence mutation rate of a hot spot unstable DNA sequence may reach 1%–5%, but the general DNA sequence mutation rate is less than 0.001% (Barber et al, 2002). This suggests that an epigenetic mechanism involving DNA methylation appears to be responsible for the transgenerational phenotype. Recent reports have led to the idea that an epigenetic reprogramming of the DNA methylation state in the germ line is possible (Rakyan and Whitelaw, 2003; Anway et al, 2005). Recently we have reported evidence for a transgenerational epigenetic effect of endocrine disruptors on specific genes in the male germ line (Anway et al, 2005).
Gonad formation in embryonic development is initiated when primordial germ cells (PGCs) migrate from the hindgut into the genital ridge prior to E12 in the rat. During PGC migration the DNA in the PGCs undergo a demethylation process that is completed prior to colonization of the early gonad (Durcova-Hills et al, 2001; Hajkova et al, 2002). In the rat, sex determination and testis development occurs between E11 and E15 and is initiated by the differentiation of precursor Sertoli cells in response to the testis determining factor Sry. In the gonads during sex determination, E11–E15, the germ cells undergo a remethylation process involving sex-specific determination of the germ cells. This remethylation is dependent on the direct interactions with the somatic cells of the gonad (Reik and Walter, 2001; Hajkova et al, 2002). The aggregation of the precursor Sertoli cells, PGC, and migrating mesonephros cells (precursor peritubular myoid cells) promotes testis morphogenesis and cord formation. Testis morphogenesis (ie, cord formation) is completed prior to E14 in the rat. The androgen receptor (AR) and estrogen receptor-beta (ERß) are present in Sertoli cells, precursor peritubular myoid cells and prespermatogonial cells at the time of cord formation (E14). Although the testis does not produce steroids at this stage of development, estrogenic and androgenic substances appear to have the ability to influence early testis cellular functions. Future studies with defined antiandrogenic substances (ie, flutamide) are now needed to assess the role of the endocrine disruptor versus potential toxicology of the vinclozolin.
Sex steroids, estrogen and androgens, have the ability to influence the methylation state of DNA sequences (Sasaki et al, 2000; Rosinski-Chupin et al, 2001; Kumar and Thakur, 2004; Szabo et al, 2004). Although many reports on sex steroids influencing the methylation state of DNA involve the onset of cancers, such as uterine (Sasaki et al, 2000), prostate (Tekur et al, 2001), and breast (Leu et al, 2004), sex steroids can influence the methylation state of DNA during normal tissue development, including imprinting of genes in the germ line (Rosinski-Chupin et al, 2001; Kumar and Thakur, 2004; Szabo et al, 2004). Imprinted genes, H19 and Igf2, are differentially methylated in the male germ line during gonadal development (Szabo et al, 2004). Szabo et al (2004) proposed that Igf2 and H19 DNA sequence methylation patterns were influenced by steroid receptor binding to the specific sequences. This suggests that an environmental endocrine disruptor could influence the germ line methylation pattern during sex determination and differentiation. This hypothesis is further supported by the actions of diethylstilbestrol (DES), an environmental synthetic estrogen, on uterine tissue (Li et al, 2003a,b). The administration of DES to neonatal mice decreased the methylation state of the c-fos gene approximately 30% in uterine tissue, as well as inducing reproductive tract abnormalities and increased incidents of cancers (Li et al, 2003a,b).
In summary, observations provide evidence that an endocrine disruptor exposure results in a transgenerational effect on spermatogenic capacity and testis function. The embryonic exposure to vinclozolin resulted in a reduced spermatogenetic capacity in the male offspring, and this phenotype was transmitted to the subsequent generations, F2–F4. The potential of such endocrine disruptors to alter the epigenetics and impact evolutionary biology and disease states has been discussed (DeRosa et al, 1998; Walter and Paulsen, 2003; Anway et al, 2005; Guerrero-Bosagna et al, 2005). The observations reported support that fetal exposure to environmental toxicants (ie, endocrine disruptors) increases the potential of adult disease states. The epigenetic transgenerational actions previously shown (Anway et al, 2005) provide a potential mechanism for the fetal basis of adult disease (Basha et al, 2005; Heindel, 2005). For example, environmental toxicants have been speculated as a potential cause for the reported regional specific decline in adult sperm numbers (Swan et al, 1997; Sharpe, 2001). This report suggests that fetal exposure to endocrine disruptors could have the potential to reduce sperm number. Although the dose of vinclozolin used in the current in vivo study was lower than previously used (Gray et al, 1994; Kelce et al, 1994; Wolf et al, 2000), the exposure levels in these studies were higher than anticipated environmental exposures, and thus the impact on human populations remains to be elucidated in further toxicology studies. Independent of the environmental toxicology impacts, the epigenetic transgenerational phenotype observed is anticipated to have a significant impact on the area of andrology and requires further investigation.
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Acknowledgments |
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Footnotes |
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* Present address: Department of Animal Science, Rutgers University, 84
Lipman Dr, New Brunswick, NJ 08901-8525. 
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) represent control and solid bars (
)
represent vinclozolin generations. Statistically significant differences
between control and vinclozolin generation rats are indicated by (*) for
P < .05. Numbers of Sprague Dawley animals analyzed are F1 (n = 7
control, n = 7 vinclozolin); F2 (n = 8 control, n = 17 vinclozolin); F3 (n =
10 control, n = 8 vinclozolin); F4 (n = 5 control, n = 5 vinclozolin); and
outcross VOC (n = 10), RVOC (n = 6) and wild-type as outcross controls (n =
5). Numbers of Fisher animals analyzed are F1 (n = 4 control, n = 10
vinclozolin); F2 (n = 4 control, n = 5 vinclozolin); and F3 (n = 11 control, n
= 13 vinclozolin).
