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From the Center for Reproductive Biology, School of Molecular
Biosciences, Washington State University, Pullman, WA 99164-4231. *
Present Address: Department of Animal Science,
University of Nebraska, Lincoln, NE, 68583-0908.
Present Address: Nippon Veterinary and Animal
Science University, Department of Veterinary Physiology, 1-7-1, Kyonan-Cho,
Musashino-Shi, Tokyo, 180-8602, Japan.
| Correspondence to: Dr. 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). |
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Abstract |
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Key words: Sertoli, mesenchymal-epithelial, growth, gametogenesis
Methoxychlor is a chlorinated hydrocarbon pesticide currently used in the
United States as a replacement for DDT
(Kapoor et al, 1970). Methoxychlor can be metabolized by the liver into two demethylated compounds
(ie, mono-OH-M and bis-OH-M). The most active estrogenic metabolite is 2,
2-bis-(p-hydroxyphenyl)-1,1,1-trichloro-ethane, (HPTE)
(Dehal and Kupfer, 1994;
Cummings, 1997;
Kupfer et al, 1990). Other
methoxychlor metabolites appear to have antiandrogenic activity
(Kelce et al, 1997). HPTE is
weakly estrogenic (Lamoureux and Feil,
1980; West et al,
1982; Bulger and Kupfer,
1985), and it stimulates the expression of estrogen receptor (ER)
(Eroschenko et al, 1996). Recently it has been found that the estrogenic metabolite of methoxychlor HPTE
has differential effects on ER-
and ER-ß, being an ER-
agonist and ER-ß antagonist (Gaido et
al, 1999; Gaido et al,
2000). Other methoxychlor metabolites also have differential
effects on ER- and ER-ß (Gaido
et al, 2000). Therefore in examining the actions of methoxychlor
or HPTE, the ER agonist and antagonist activities need to be considered as
well as antiandrogenic activities. Consideration of these differential
activities is critical in elucidating the mechanisms of action of endocrine
disruptors such as methoxychlor. Previously, methoxychlor metabolites have
been shown to act differentially on the ER of different species
(Mathews et al, 2000). The
effects of methoxychlor at an embryonic or early postnatal period can
influence reproductive functions at later adult periods
(Cooke and Eroschenko, 1990;
Chapin et al, 1997). Neonatal
exposure to methoxychlor can influence pregnancy
(Swartz and Eroschenko, 1998),
ovarian and hypothalamic function
(Eroschenko et al, 1995),
reproductive behavior (Palanza et al,
1999), and prostate development
(Stoker et al, 1999).
Therefore, transient embryonic exposure to an endocrine disruptor can
reprogram or imprint effects that manifest in the adult on reproductive
physiology. A study has shown that its effects on a gestating mother may
influence subsequent pregnancies as well
(Swartz and Corkern, 1992).
The current study is designed to investigate the reproductive toxicology of
MXC.
The potential environmental exposure concentration to MXC can be based on its recommended use as a pesticide (ACSCEQ, 1983; Cummings, 1997). For the control of mosquitoes, MXC has been used at a final surface water concentration of 1µM (ACSCEQ, 1983). In vivo exposures to 50 to 250 mg/kg/day have been reported (Gray et al, 1989; Facemire et al, 1995; Eroschenko et al, 1997; Swartz and Eroschenko, 1998; Atanassova et al, 1999). Estimating the body volume, the concentrations used in the current study, 50 to 150 mg/kg/day are similar to potential environmental exposures. Previous in vivo studies have used either intraperitoneal (IP) (Facemire et al, 1995; Eroschenko et al, 1997; Swartz and Eroschenko, 1998; Atanassova et al, 1999) or oral gavage (Gray et al, 1989). The IP route of administration controls the exposure dose more efficiently; it was therefore, used in the current study.
The current study was conducted to determine if transient administration of methoxychlor prior to morphological testis development (seminiferous cord formation, embryonic day 13.5 of rat gestation; E0 = plug date) could have detrimental effects on testis morphology that result in impaired adult testis function. Previous studies have primarily examined effects of late embryonic and early postnatal exposure to MXC (Carlsen et al, 1992; Linder et al, 1992; Cummings and Laskey, 1993; Facemire et al, 1995; Swan et al, 1997; Crain and Guillette, 1998; Crisp et al, 1998; Laws et al, 2000). The hypothesis tested is that transient early embryonic exposure to the endocrine disruptor methoxychlor will alter testis morphogenesis and proliferative events during embryonic testis development and will subsequently influence adult male fertility.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Embedding, Histology, and Immunohistochemistry
Tissues were fixed in Histochoice (Amresco, Solon, Ohio) and embedded in
paraffin according to procedures previously described
(Cupp et al, 1999). The tissue
sections (3-5 µm) were deparaffinized, rehydrated, microwaved, and blocked
in 10% goat serum for 30 minutes at room temperature. Immunohistochemistry was
performed as described previously
(Martinez and Swartz, 1991; Itoh et al, 1998). The germ
cell nuclear antigen (GCNA1) antibody was a monoclonal antipeptide antibody
generously provided by George Enders, University of Kansas, Kansas City. The
GCNA1 antibody was diluted 1:50 in 10% goat serum. The antibody stains all
germ cells in the mouse and mitotic germ cells in the rat. As a negative
control, serial sections were put through the same procedure without any
primary antibody. The biotinylated goat antirabbit (Vector Laboratories,
Burlington, Calif) was diluted 1:300. The secondary antibody was detected by
using the Histo stain-SP kit (Zymed Laboratories, South San Francisco, Calif),
and immunohistochemical images were digitized with a slide scanner. All
sections utilized for negative controls had no positive staining. This
demonstrated that the positive staining detected with the GCNA1 antibodies was
not due to nonspecific staining or artifacts of tissue fixation and
processing. Three different experiments were conducted for GCNA1 antibodies
for each developmental time. In each experiment, 3 serial sections of 4 or 5
testes for each developmental age were analyzed. One serial section was used
for the nonimmune control for each time period. There was uniform and
repeatable staining at each developmental age for GCNA1 in all 3
experiments.
Testis Cellular Composition Analysis
Sections from each treatment group at each developmental age were analyzed
at a 200x magnification in the NIH image program for: area of
seminiferous cords, area of interstitium, number of germ cells (through GCNA
staining), and number of seminiferous cords. The total area represented for
each testis section was approximately 308000 pixels. To determine the area of
seminiferous cords or tubules per section, the cords/tubules were outlined and
the area (in pixels) was calculated with the NIH image program. The area of
interstitium was calculated by subtracting the area of the seminiferous
cords/tubules from the total area within the section (308000 pixels). The
number of seminiferous cords/tubules was determined by counting the number of
cords or tubules within the designated testis section. This included any
partial cords or tubules that were present. The total number of germ cells per
section was determined by counting the GCNA positively stained cells within
the section. For each parameter, 3 different experiments were done in
replicate with approximately 8 to 12 sections analyzed per treatment group for
each development age. Two independent measurements were taken for each section
analyzed. These measurements were averaged for each parameter and
statistically analyzed for differences between treatments. The data for each
averaged area are depicted as number of pixels per designated testis area (ie,
total pixels).
Apoptosis Analysis
TUNEL analysis was conducted utilizing the Promega Apoptosis Detection
System, fluorescein kit (Madison, Wisc). Briefly, histochoice-fixed and
paraffin-embedded histological sections
(ACSCEQ, 1983) were
deparafinized and washed in phosphate-buffered saline (PBS) and then fixed in
4% methanol-free formaldehyde solution. The sections were then treated with a
20 ug/ml solution of Proteinase K for 8 minutes. Subsequently, they were
washed in PBS, fixed in 4% methanol-free formaldehyde, and washed again with
PBS. The sections were then placed in an equilibrating solution with a
fluorescein-tagged TdT enzyme and nucleotide (fluorescein-12-dUTP) mix and
incubated at 37°C for 1 hour in a humidified chamber. The reaction was
terminated and washed with PBS to remove unincorporated fluorescein-12-dUTP.
The slides were then coverslipped using a drop of Anti-Fade mounting solution
(Molecular Probes, Eugene, Ore) and analyzed on fluorescent and confocal
microscopes in the Center for Reproductive Biology, Histology Core Laboratory
at Washington State University. Fluorescently labeled cells were counted per
area of testis for each treatment group at each developmental age. Eight to 10
microscopic fields from each section were digitally imaged and the
fluorescently labeled cells counted. Five to 7 sections were evaluated for
each developmental age from 3 different experiments and averaged to obtain the
data presented. Positive controls used DNAse I instead of Proteinase K to
determine if enzyme was labeling properly, since DNAse I causes DNA strand
breaks. Negative controls without enzyme were also conducted to determine the
background of the fluorescent procedure.
Statistical Analysis
Data were analyzed with the JMP 3.1 statistical analysis program (SAS
Institute, Cary, NC). All values were derived from 3 different experiments
done in replicate and are expressed as the mean ± SEM. Statistical
analysis was performed using one-way ANOVA. Significant differences were
determined using Dunnett's test for comparing with controls and the
Tukey-Kramer significant difference tests for multiple comparisons.
Statistical difference was confirmed at P < .05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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At P4, P10, P17, and P60 (Figures 2, 3, and 4) there were no differences in area of seminiferous cords/tubules, interstitial area, or in number of seminiferous cords between MXC and control testes. In addition, MXC did not appear to influence Sertoli cell or Leydig cell numbers (data not shown). Therefore, any changes in testis cellular composition that occurred at E16 did not influence the cellular composition of testis postnatally. However, there was a trend for a reduction (but not statistically significant) in the numbers of seminiferous tubules in P17 MXC exposed male testes when compared with controls (Figure 4).
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Transient exposure to the low 50 mg/kg/day or higher 150 mg/kg/day methoxychlor had no effect on pubertal P20 (Figure 5) or adult P60 (Figure 6) testis weights. In addition, methoxychlor exposure did not influence the weights of a large number of different organs (Figures 5 and 6). Therefore, the transient embryonic exposure did not have any gross effects on general organ development.
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Methoxychlor exposed males had reduced germ cell numbers at P17 of development as determined by positively stained cells for the germ cell nuclear antigen (GCNA) (Figure 7). No other developmental age demonstrated significant differences in number of germ cells per testis area. However, there were trends for reductions (but not statistically significant) in the number of germ cells present in MXC exposed males at P10 and P60 of development when compared with their counterpart controls.
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In addition to differences in germ cell numbers at P17, there was also an
increase in germ cell apoptosis in P17 MXC exposed testes when compared with
controls (Figures 8 and
9). At P17-20, an increase in
germ cell apoptotis at low MXC exposure is shown in
Figure 8B and D. The high MXC
exposure has a similar effect on germ cell apoptosis
(Figure 9). Testes collected at
earlier developmental timepoints did not have differences in apoptotic cells
between MXC exposed and control testes (data not shown). Although testis
composition was not different in P17, there were alterations in germ cell
number and germ cell viability in MXC treated testes when compared with
controls (Figures 7,
8, and
9). In MXC exposed P60 testes
there appeared to be an increase in germ cell apoptosis in elongating
spermatids associated with stage X-XI of the spermatogenic cycle
(Figure 8E and F). Apoptotic
cell numbers in tubules other than stage X-XI of the treated testis were
higher than control but the difference was statistically not significant
(P > .05). However, in the MXC-treated group, the number of X-XI
stage tubules with 5 or more apoptotic cells was dramatically higher. While
approximately 
of the stage X-XI tubules in control group had 5 or
more apoptotic germ cells, approximately 
of the X-XI stage tubules in
the treated group had 5 or more apoptotic germ cells. The number of tubules
with apoptotic germ cells was doubled by the MXC treatment. Low-level exposure
(50 mg/kg/day) MXC treatment did not have consistent effects on apoptotic germ
cell number at P60 (data not shown).
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Testosterone levels in serum collected from P20 and P60 MXC treated animals and control animals were tested to determine potential endocrine effects of the MXC exposure. Serum levels of testosterone (ng/ml) in MXC treated animals compared with control demonstrated no significant change in testosterone levels (Figure 10). Therefore, the transient embryonic exposure to MXC does not appear to influence the endocrine status of the adult male.
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Postnatal day 60 MXC exposed males (n = 4) and control males (n = 4) were allowed to mate with control 60-day-old females. All females became pregnant and produced normal litter sizes and pup weights with no difference between control and MXC treated animals (data not shown). Therefore, the MXC exposed males were determined to be fertile even though some sperm cells may have been compromised. Preliminary analysis of caudal epididymal sperm showed no change in sperm number, a small (15%) decrease in the percentage of motile sperm and forward movement, and a small increase in sperm with abnormal morphology (data not shown). However, the majority of sperm had normal morphology and motility; this remains to be more thoroughly investigated.
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Discussion |
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The effects of exposure to methoxychlor in utero resulted in an imbalance of seminiferous cords to interstitium at embryonic day 16 (E16) of testis development. Exposure to methoxychlor was at a time when both seminiferous cord formation and somatic and germ cell proliferation were occurring. Different testicular cell types are required to migrate during this period of testis morphogenesis (Magre and Jost, 1991; Karl and Capel, 1998; Levine et al, 2000). Some interstitial cells (eg, preperitubular and endothelial) are derived from the migration of mesonephric cells into the testis to promote formation of the seminiferous cords, while some Sertoli cells migrate from the coelomic epithelium (Karl and Capel, 1998). Previous in vitro studies (Cupp and Skinner, 2001) have shown that both MXC and its metabolite HPTE interfere with or inhibit cord formation. More recent studies (Uzumcu et al, 2002; Cupp et al, 2003) have shown that one of the reasons for abnormal cord formation is the inhibition of mesonephros cell migration.
Sertoli, interstitial, and peritubular cells are rapidly proliferating from E14 to P0 of testis development (Akingbemi et al, 2000; Levine et al, 2000). Germ cells rapidly proliferate until approximately E18, when mitosis arrests until it resumes after birth (Orth, 1982). Since the area of seminiferous cords was reduced, observations suggest that either the germ cell numbers, Sertoli cell numbers, or size may have been affected by methoxychlor exposure. Since there was no apparent change in germ cell numbers at E16, reduction in Sertoli cell number or size appears to at least contribute to the reduced seminiferous cord area. Alternatively, the number or size of the Leydig cells may be affected by the methoxychlor exposure, which subsequently can alter the normal ratio between cord and interstitium. This is possible since some of the MXC metabolites (eg, HPTE) are antiandrogenic (Maness et al, 1998) which may have led to hypertrophy or hyperplasia of the Leydig cell. A similar mechanism was suggested for phthalates. When prepubertal rats were exposed to shorter (14 days) or longer (28 days) exposure to phthalates, the shorter exposure reduced testosterone production but longer exposure increased testosterone and LH levels, potentially leading to Leydig cell hypertrophy (Akingbemi et al, 2001). Thus, the exposure to methoxychlor may have resulted in inefficient migration, differentiation, or proliferation of Sertoli cells or hypertrophy of Leydig cells that would result in a relative reduction of seminiferous cord area. This reduction was transient since no further alterations in seminiferous cord/tubule area or interstitial cell area was observed in subsequent testis collections. Furthermore, the size of the adult testes in the methoxychlor exposed males appears to be similar to controls. Therefore, any alteration in testis cellular composition observed at E16 did not result in differential size or abnormal structures within the testes collected at the postnatal ages. Similar transient effects (eg, reduction in serum testosterone) in rats that were in utero exposed to phthalates were observed (Akingbemi et al, 2001).
A major effect of methoxychlor exposure was the reduction in number of mitotic germ cells detected at P17 through positive germ cell nuclear antigen (GCNA) staining. This reduction in germ cell number was also supported by an increase in germ cell DNA fragmentation in P17-20 in males exposed to methoxychlor during gestation. Therefore, the transient exposure to methoxychlor appears to result in abnormal germ cell survival or development during puberty. The abnormal germ cell development or accelerated germ cell apoptotis may be caused by inappropriate cell differentiation after fetal exposure to methoxychlor. This may result in an insensitivity of cells, such as the Sertoli cell, to respond adequately to FSH or Leydig cells to respond to LH. Several studies have demonstrated that exposure to high doses of estrogen or estrogenic endocrine disruptors reduces serum testosterone levels (Akingbemi et al, 2000; McKinnell et al, 2001) and alters or inhibits fetal Leydig cell differentiation (Abney, 1999); it can also abolish androgen receptor populations (Abney, 1999) within the testis. In a similar manner, methoxychlor has been demonstrated in the female to cause an insensitivity to gonadotropins that would normally cause superovulation (Eroschenko et al, 1997). The metabolite of methoxychlor-HPTE has been demonstrated to reduce Leydig cell function in cell culture (Akingbemi et al, 2000). Therefore, methoxychlor may act in an estrogenic manner to inhibit testosterone function and induce abnormal Leydig cell differentiation or insensitivity to gonadotropin stimulation.
There also appeared to be an effect of methoxychlor exposure on adult animal germ cells that are at stage X-XI of the spermatogenic cycle. In P60, high-dose, methoxychlor exposed testes, the majority of the stage X-XI seminiferous tubules had apoptotic, elongated spermatids. This stage of the seminiferous tubule is especially sensitive to testosterone withdrawal and to antiandrogens such as flutamide that inhibit the development of spermatids. A 5-alpha reductase inhibitor also reduced spermatid development, but not to the extent of flutamide (O'Donnell et al, 1999). Analysis of serum testosterone demonstrated that the transient embryonic methoxychlor exposure did not influence the endocrine status of the pubertal or adult males. The lack of methoxychlor effect on any of the reproductive organ weights examined correlated with the normal testosterone levels. Embryonic testis development does not require endocrine control; however, the effects of methoxychlor treatment on embryonic and early postnatal serum testosterone remains to be elucidated. It is speculated that a transient embryonic exposure to methoxychlor may alter the abilities of Leydig and Sertoli cells to respond to gonadotropins, which promotes a reduction of viable germ cells within the testis. The reduction in germ cells does not appear to inhibit reproductive function because the P60 littermate males were capable of producing offspring with normal litter size and pup weights.
In summary, the current study demonstrates that a transient in vivo embryonic exposure to the endocrine disruptor methoxychlor influences testis development and function. Males exposed to methoxychlor during E7 to E15 of gestation appear to have inappropriate differentiation of cells during the initial stages of embryonic testis differentiation and growth. The altered testis differentiation may subsequently lead to accelerated germ cell apoptosis or altered germ cell development in the adult male. While the reduction in germ cell numbers and increased germ cell apoptosis may not result in infertility, there is the possibility that offspring of matings between individuals subjected to methoxychlor exposure may have reproductive problems. Future studies will investigate the potential transgenerational effects of estrogenic endocrine disruptors (eg, methoxychlor) on male reproduction. Although the current study selected a relatively low-dose exposure of methoxychlor to provide relevance to environmental exposure levels (Atanassova et al, 1999; ACSCEQ, 1983), the possibility that a higher dose exposure would cause more profound effects on testis development or male fertility also will be investigated in future studies.
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Acknowledgments |
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 T. M. Price, S. K. Murphy, and E. V. Younglai Perspectives: The Possible Influence of Assisted Reproductive Technologies on Transgenerational Reproductive Effects of Environmental Endocrine Disruptors Toxicol. Sci., April 1, 2007; 96(2): 218 - 226. [Abstract] [Full Text] [PDF]
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 J. Merlet, C. Racine, E. Moreau, S. G. Moreno, and R. Habert Male fetal germ cells are targets for androgens that physiologically inhibit their proliferation PNAS, February 27, 2007; 104(9): 3615 - 3620. [Abstract] [Full Text] [PDF]
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 M. D. Anway, C. Leathers, and M. K. Skinner Endocrine Disruptor Vinclozolin Induced Epigenetic Transgenerational Adult-Onset Disease Endocrinology, December 1, 2006; 147(12): 5515 - 5523. [Abstract] [Full Text] [PDF]
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M. D. Anway and M. K. Skinner Epigenetic Transgenerational Actions of Endocrine Disruptors Endocrinology, June 1, 2006; 147(6): s43 - s49. [Abstract] [Full Text] [PDF]
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 H. H-C Yao and B. Capel Temperature, Genes, and Sex: a Comparative View of Sex Determination in Trachemys scripta and Mus musculus J. Biochem., July 1, 2005; 138(1): 5 - 12. [Abstract] [Full Text] [PDF]
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 M. D. Anway, A. S. Cupp, M. Uzumcu, and M. K. Skinner Epigenetic Transgenerational Actions of Endocrine Disruptors and Male Fertility Science, June 3, 2005; 308(5727): 1466 - 1469. [Abstract] [Full Text] [PDF]
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