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Center for Reproductive Sciences and Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160
Address all correspondence and requests for reprints to: Katherine F. Roby, Ph.D., Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160. E-mail: kroby{at}kumc.edu
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Abstract |
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Introduction |
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TCDD is the prototype for a class of environmental contaminants that includes chlorinated benzenes, phenols, polychlorinated biphenyls, furans, and dibenzo-p-dioxins (6, 7). TCDD is persistent and ubiquitous in the environment and is capable of causing a wide spectrum of toxic effects. Historically, industrial accidents have been a major source of TCDD contamination of the environment; however, TCDD and related compounds were commonly used as herbicides until 1978. The most important direct source of TCDD for humans appears to be food, especially dairy products, meat, and fish (8, 9, 10, 11). This is not surprising in view of the known ability of TCDD to accumulate in the food chain (12, 13, 14, 15, 16, 17, 18). Due to its extreme potency and widespread low level environmental contamination, the effects of TCDD on the reproductive system are of interest.
Mechanisms initiating the early phases of follicle development are
unknown; however, the process is likely to be supported by intraovarian
factors. The appearance of thecal LH receptors and granulosal FSH
receptors in the late preantral to early antral stages of development
chronicle the dependence on gonadotropic support. FSH stimulates
granulosal cell aromatization of estrogens. In turn, estrogens support
further follicle development in part by increasing FSH receptors
(19), inducing granulosa cell proliferation
(20), and stimulating further estrogen production.
Estrogen together with FSH regulate the expression of LH receptors on
the granulosa late in antral follicle development (21, 22). As estrogen production increases, positive feedback results
in the release of a surge of LH, initiating the process of
ovulation (23). LH regulates the expression of
several genes important in the rupture of the follicle, including
progesterone receptor (PR) (24), cyclooxygenase
(25), and the family of plasminogen activators and
inhibitors (26, 27). Studies using knockout technologies
have demonstrated the importance of multiple factors, including
estrogen receptor (ER; both ER
and ERß) (28, 29), PR
(30), FSH receptor (31), and FSH
(32), in the normal process of follicle development and
ovulation.
The objectives of the current study were to investigate potential ovarian-specific mechanisms of TCDD-mediated inhibition of ovulation.
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Materials and Methods |
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10). The dose of TCDD (32 µg/kg) was found to most
completely block ovulation (to 1 ova). Animals were decapitated at
various times as indicated, and trunk blood was collected. Ovaries were
collected, cleaned, weighed, and prepared for histological analysis,
topical autoradiography, or membrane binding studies as described
below. Ovulation was determined by oviductal irrigation 20 h after
hCG as previously described (33). All animal handling and
procedures conformed to the guidelines set forth by the institutional
animal care and use committee of the University of Kansas Medical
Center.
Histological analysis
Ovaries were fixed in Bouin’s solution, and serial sections (8
µm) of the entire ovary were prepared, mounted on glass slides,
stained with hematoxylin and eosin, and evaluated under the light
microscope. Each healthy antral follicle was measured and counted in
the section containing the nucleolus of the oocyte. The diameter at two
perpendicular orientations was measured with a ruled ocular; the final
diameter was the mean of the two measurements. Follicle counts were
obtained using a single ovary from six control and seven TCDD-treated
rats collected 52 h after PMSG administration.
Membrane receptor binding
Receptor binding analysis was performed as previously described
(34). Each ovary was homogenized in 300 µl
homogenization buffer (50 mM Tris-HCl containing 10
mM CaCl2 and 20 mM
MgCl2, pH 7.2). The homogenate (100 µg protein)
was transferred to a 12 x 75-mm tube, and
[125I]hCG or [125I]FSH
(100 µl, 23 µCi/µg) was added. hCG and rat FSH (CR-127 and rat
FSH-I-9 from the National Hormone and Pituitary Program) were iodinated
using lactoperoxidase (35). The mixture of homogenate and
[125I]gonadotropin was incubated overnight at
room temperature; then 1 ml ice-cold receptor buffer (50 mM
Tris-HCl and 5 mg albumin/ml, pH 7.0) was added, the mixture was
centrifuged, and the supernatant was aspirated. The
[125I]gonadotropin bound was determined by
-spectroscopy. Nonspecific binding was determined by incubating the
homogenate with [125I]hCG or
[125I]FSH in the presence of 20 IU hCG or 25
µg ovine FSH (oFSH-19), respectively. Specific binding was determined
as counts per min bound minus nonspecific counts per min. Receptor
binding was performed on ovaries from three different experiments with
between six and eight animals in each treatment group and at each time
point.
Topical autoradiography
Topical autoradiography was performed according to the method of
Oxberry and Greenwald (36). The tissue was frozen in
liquid nitrogen, embedded in OCT (Miles Laboratories, Elkhart, IN), and
cut in a cryostat at 10 µm. The frozen sections were placed on
poly-L-lysine-coated slides, air-dried for 30 min, and
stored in a dessicator box at -20 C until subjected to topical
autoradiography of hCG or FSH receptors. After bringing the slides to
room temperature, the tissue sections were fixed in 4%
paraformaldehyde at 4 C for 30 min. Fixation was followed by rinsing in
two changes of ice-cold PBS, pH 7.0. 125I-Labeled
hormone (hCG CR-127 or rat FSH-I-9; 25,000 cpm of 20 µCi/µg) was
added to the tissue section in 0.2 ml PBS containing 0.1% BSA and
incubated for 2 h at 37 C. Control nonspecific binding sections
had 10 IU unlabeled homologous hormone in addition to the radiolabeled
hormone. The tissue sections were rinsed thoroughly in PBS to remove
excess unbound hormone, and then the sections were postfixed in 4%
glutaraldehyde and rinsed in PBS. The slides were dipped in
autoradiographic emulsion, dried, stored for 5 days at 4 C, and then
developed through routine photographic steps of Dektol, Stop, and Fix.
Then the slides were washed and stained with hematoxylin. Grain
counting was assessed using Optomis Bioscan (Media Cybernetics, Silver
Spring, MD). This program measures the grain density per unit
area. Grain density was assessed under total and nonspecific binding
conditions in the same follicle in adjacent sections. Specific binding
was determined as total binding minus nonspecific binding. Ovaries from
five control and seven TCDD-treated animals were assessed.
RIAs
Serum progesterone and estradiol levels were measured by RIA,
and ovarian cAMP levels were determined with a commercially available
kit according to the manufacturer’s (Biomedical Technologies
Inc., Stoughton, MA) directions as previously described
(33).
Statistical analysis
Experiments were repeated at least three times with between six
and eight animals in each treatment and time point. Data were subjected
to ANOVA, with differences between means detected by Newman-Keuls test.
Differences were considered significant at P 
0.05.
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Results |
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)
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)). Ovaries from
TCDD-treated rats contained scant evidence of corpora lutea. In a group
of eight animals treated with TCDD (Table 1
), four did not ovulate,
three ovulated one ova each, and one ovulated two ova; thus, occasional
corpora lutea were observed histologically. The number of corpora lutea
observed histologically correlated with the number of ova recovered
from the oviduct. TCDD-treated ovaries contained multiple large
unruptured antral follicles and numerous small antral follicles (Fig. 1B).
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). Serum
progesterone levels in TCDD-treated animals also increased after hCG;
however, all peak levels were significantly lower than those measured
in control animals. Also, the morning after ovulation (at 20 h)
serum levels of progesterone were significantly lower in TCDD-treated
animals compared with controls (Fig. 3 and Table 1).
Ovary weight (Fig. 4)
The increase in ovary weight stimulated by PMSG and hCG treatment
in control animals was reduced in TCDD-treated animals. Ovary weight
was increased significantly 52 h after PMSG compared with that
at various time points before PMSG treatment, and ovary weight
continued to increase after hCG administration in control animals.
Compared with controls, ovary weight was significantly lower in
TCDD-treated animals 52 h after PMSG and remained lower throughout
all subsequent time points examined during treatment.
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). The data in Fig. 6 are representative
of follicles 350 µm or more in diameter.
Additional membrane binding studies were carried out on ovaries
collected at multiple times throughout the treatment schedule. FSH
binding to ovarian membrane preparations from control animals was
increased 24 h after the administration of PMSG compared with
times before PMSG administration (Fig. 7). FSH binding increased further at
52 h, remained elevated after hCG administration, and then
increased again near the time of ovulation. FSH binding to ovarian
membrane preparations from TCDD-treated rats also increased after PMSG
(PMSG vs. pre-PMSG time points); however, binding decreased
thereafter when binding to controls remained elevated. Closer to the
time of ovulation, FSH binding in the TCDD samples increased to levels
similar to those in controls (Fig. 7).
hCG binding to membranes in control and TCDD-treated groups was very
similar throughout the treatment schedule with the exception of two
time points. hCG binding was lower at 24 h after treatment with
TCDD and at 52 h after PMSG compared with control binding (Fig. 8). This was observed consistently in
three different experiments.
Ovarian cAMP (Fig. 9)
The ability of the ovary to respond to PMSG and hCG with increased
cAMP was altered in TCDD-treated animals. In control animals, ovarian
cAMP levels increased 24 h after PMSG, increased again at 52
h after PMSG, and then increased further in response to hCG. In animals
treated with TCDD ovarian cAMP increased 24 h after PMSG and was
not different from control levels. However, ovarian cAMP did not
increase further at 52 h post-PMSG. In addition, no further
increase in cAMP occurred in TCDD-treated animals in response to hCG.
cAMP levels in ovaries from TCDD-treated rats were significantly lower
compared with control values 52 h after PMSG and 5 h after
hCG.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data in the present study identify a potential critical period
during PMSG-stimulated follicular development that is altered by TCDD.
Estradiol production, cAMP levels, ovary weight, and gonadotropin
receptor binding were reduced in TCDD-treated animals by 52 h
after PMSG treatment. Thus, critical events occurring between 24 and
52 h after PMSG may be altered by TCDD, resulting in inhibition of
full maturation of the follicles and a reduction in the number of large
antral follicles (those 
350 µm). Several recent studies have
identified genes important in maturation of the follicle. For example,
gene knockout studies in the mouse have illustrated critical
intraovarian actions of estradiol on follicular development. Disruption
of the ER gene (ER) results in blockade of ovulation and the
development of cystic follicles (28). Immature ER
knockout (ERKO) mice stimulated with PMSG and hCG can ovulate.
However, only about 46% of animals ovulate, and those ovulating
release fewer ova than controls (44% of control value)
(29). Although not specifically addressed in that study,
the researchers suggested that the reduced response in the KO mice may
be due to reduced follicular development and maturation, such that
there are fewer antral follicles available to undergo ovulation.
Similar findings were made in the ERßKO mouse. The ability of exogenous gonadotropins to stimulate follicle development and ovulation was reduced in the KO mouse compared with controls (37). Together these data indicate that the presence of the ER is not essential for early stages of follicle development and ovulation; however, the receptors are necessary for the development of fully mature follicles that are capable of responding to the ovulatory stimulus (38). TCDD is an antiestrogen, and the results of TCDD treatment in the present system are similar to deletion of the ER. In the presence of TCDD, follicle development in response to PMSG is initiated; however, maturation is limited, as evidenced by reduced numbers of large antral follicles, reduced serum estradiol levels, and reduced FSH and hCG binding.
Effects of TCDD on follicle development have been demonstrated in other model systems. Immature rats (21 days of age) had altered follicle numbers after being exposed to a single dose of TCDD on day 15 during gestation and the continued lactational exposure until weaning on day 21 (39). The number of small preantral follicles and the number of small and large antral follicles were reduced compared with control values (39). Interestingly, the number of primordial follicles present in the ovary on day 3 of age was reduced in aryl hydrocarbon receptor (AhR) KO mice compared with controls (40). The same study found reduced numbers of antral follicles in AhRKO mature mice compared with controls (40). Although the model systems differ, collectively these studies and the present study indicate the ability of TCDD and the AhR to alter follicle development.
TCDD mediates function after first binding to the AhR. Antiestrogenic effects of TCDD have been described in several different systems, and TCDD has been shown to reduce cancer incidence in hormonally responsive tissues (41, 42, 43, 44, 45). Using MCF-7 breast cancer cells, Kharat and Saatcioglu (46) demonstrated that the TCDD-AhR complex interferes with ER binding to the ERE and the ability of liganded ER to activate transcription (46). Additional studies indicate that TCDD can reduce expression of the ER (47, 48). Using competitive RT-PCR techniques, ER messenger RNA was decreased in several organs, including the ovary, 4 days after a single dose of TCDD in CD-1 mice. Thus, TCDD-mediated altered follicle development may occur due to TCDDs antiestrogenic action. This hypothesis is currently under investigation. The antiestrogenic effects of TCDD may provide a unique model to further identify the role of estrogen in follicle development in the rat. The present studies might indicate a critical action during the developmental transition from small antral follicles to fully mature preovulatory follicles, as the number of small antral follicles was not altered by TCDD, but the number of large antral follicles was significantly reduced.
Estradiol has been shown to play a role in several processes important for follicle development and ovulation, including increasing granulosa cell growth and number (20), increasing granulosal insulin-like growth factor I synthesis (49), maintaining FSH receptor (19), induction of the LH receptor (21, 22), augmentation of aromatase and estradiol production, and attenuation of granulosal apoptosis (50).
Antiestrogenic effects of TCDD might account for reduced gonadotropin receptor expression. Furthermore, reduced gonadotropin receptor expression might account for reduced estradiol production. Rats treated with TCDD responded to PMSG with follicles entering the growing pool (i.e. the numbers of small antral follicles 100–349 µm were similar in control and TCDD-treated groups). However, during the early phases of follicle development (24–52 h after PMSG) when estradiol levels increased in controls, estradiol levels in TCDD-treated rats did not. This in itself might limit further follicle development. Additionally, the effects of TCDD on follicular ER expression are unknown, but ER expression is likely to be decreased based on knockout studies. Follicle counts assessed 52 h after PMSG represent healthy follicles. At this time point the number of atretic follicles was not different in control and TCDD-treated animals. However, assessment of follicular atresia 14 h after the administration of hCG indicated that the majority of the large antral follicles in the TCDD-treated animals were atretic (Roby, K. F., unpublished observations). Thus, it appears that TCDD does not directly induce follicular atresia, but that follicular atresia is probably a result of reduced responsiveness to gonadotropins.
Antiestrogenic effects of TCDD might further limit follicle development through several secondary mechanisms. For example, cyclin D2 is necessary for granulosa cell proliferation in response to FSH. Cyclin D2 KO mice exhibit reduced granulosal proliferation; follicles remain small, developing only up to four layers of granulosa cells; and ovulation does not occur (51, 24). Although follicles do not reach a developmental maturity to ovulate in cyclin D2 KO mice, stimulation with PMSG and hCG does induce changes in gene expression reflecting further differentiation of granulosa cells. For example, follicles develop antra, aromatase expression is induced, and in response to LH, PR and PG synthase messenger RNA are up-regulated (24). Although the follicle can respond at least in part to LH, ovulation does not occur. Thus, in the present experiments, antiestrogenic effects of TCDD resulting in reduced FSH receptor expression might, in turn, reduce cyclin D2 expression.
TCDD-treated animals did not ovulate in response to hCG. Rupture of the follicle occurs after hCG-induced changes in the expression of several genes, including a transient increase in granulosal PR expression (52). KO studies clearly demonstrate the necessity of PR expression for ovulation (30). In addition, changes in specific enzyme expression and activity have been mapped to the time of ovulation and are thought to play critical roles in the breakdown of the tissues necessary for ovulation (53, 54). The ability of TCDD to directly alter these genes is not clear. Using the model described in the present study, expression of PR in granulosa cells was reduced 10 h after hCG administration in TCDD-treated animals compared with controls (Roby, K. F., unpublished observations). In addition, plasminogen activator expression and activity 15 h after hCG administration were reduced in ovaries from TCDD-treated rats compared with controls (Roby, K. F., unpublished observations). These results are difficult to interpret given the data indicating that maturation of the follicles was inhibited in TCDD-treated animals. A previous study indicated that administration of TCDD into the ovarian bursal cavity of intact rats before follicle development/ovulation induction with PMSG/hCG resulted in reduced ovulation (5). Direct effects of TCDD on hCG- induced genes around the time of ovulation might be best addressed using the technique of intrabursal injection. TCDD administered after the completion of follicle development, at the time of hCG treatment, would more directly address this question.
In summary, a single dose of TCDD administered before the initiation of follicle development alters the responsiveness of the ovary to PMSG, limiting the development of fully mature follicles capable of responding to an hCG ovulatory stimulus.
Received August 2, 2000.
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knockout mice.
Biol Reprod 62:599–605 induces clustering in ovarian theca-interstitial
cells in vitro. Endocrinology 131:2503–2513This article has been cited by other articles:
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