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Alterations in Follicle Development, Steroidogenesis,and Gonadotropin Receptor Binding in a Model of

Ovulatory BlockadeKATHERINE F. ROBY

Center for Reproductive Sciences and Department of Anatomy and Cell Biology, University of KansasMedical Center, Kansas City, Kansas 66160

ABSTRACTImmature female rats treated with 2,3,7,8-tetrachlorodibenzo-p-

dioxin (TCDD) before gonadotropin-induced follicle development andovulation ovulate significantly fewer ova compared with controls.This study was designed to investigate potential ovarian-specificmechanisms of TCDD-mediated inhibition of ovulation. Immaturehypophysectomized rats were treated with TCDD (32 g/kg) or corn

oil vehicle. Follicle development was initiated by injection of 10 IUPMSG 24 h after TCDD, and ovulation was induced 52 h after PMSGby injectionof 10 IU hCG. Thenumber of ovaflushed from theoviductwas assessed the morning after hCG injection, and ovaries werecollected at multiple times throughout the treatment schedule forhistological analysis and analysis of FSH and hCG receptor bindingand ovarian cAMP levels. In addition, serum levels of estradiol andprogesterone were determined. Control rats ovulated 9.3 1.5 ova,whereas TCDD-treated rats ovulated 0.6 0.3. Gonadotropin recep-tor binding was evaluated 52 h after PMSG at the usual time of hCG

injection to induce ovulation. Both FSH binding and hCG bindingwere significantly reduced in animals treated with TCDD. Serumestradiol levels in control animals were increased by 52 h after PMSGadministration. In contrast, serum levels of estradiol in TCDD-treated animals remained low. In response to the ovulatory dose ofhCG, serum levels of both estradiol and progesterone increased incontrol animals. Steroid levels also increased in TCDD-treated ani-

mals, but did not reach the peak levels observed in controls. TCDDtreatment further resulted in lower ovarian cAMP levels at 52 hpost-PMSG and at 5 h post-hCG. Together the data indicate thatTCDD treatment altered the ability of the ovary to respond to PMSG,resulting in the development of follicles not comparable to controls(lower gonadotropin binding, lower estradiol production, lower levelsof cAMP). It appears that critical steps in the development and mat-uration of folliclesare disruptedby TCDD. (Endocrinology 142: 23282335, 2001)

IN A RECENT series of studies, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was shown to be a potent reproductivetoxin in females (15). After a single oral dose of TCDD (10

g/kg), the cyclicity of female rats was severely altered,characterized mainly by prolonged periods of diestrus withloss of proestrous and estrous phases of the cycle (1). AmongTCDD-treated rats with a normal first cycle, the number ofova shed was also distinctly reduced. In a gonadotropin-stimulated immature rat model, the number of ova shed wassignificantly reduced in TCDD-treated rats (2). Similar effectswere also observed in gonadotropin-primed hypophysecto-mized rats, indicating the possibility of a direct modulatoryeffect of TCDD on follicular development and ovulation(2, 3).

TCDD is the prototype for a class of environmental con-taminants that includes chlorinated benzenes, phenols, poly-chlorinated biphenyls, furans, and dibenzo-p-dioxins (6, 7).

TCDD is persistent and ubiquitous in the environment andis capable of causing a wide spectrum of toxic effects. His-torically, industrial accidents have been a major source ofTCDD contamination of the environment; however, TCDDand related compounds were commonly used as herbicidesuntil 1978. The most important direct source of TCDD for

humans appears to be food, especially dairy products, meat,and fish (811). This is not surprising in view of the knownability of TCDD to accumulate in the food chain (1218). Due

to its extreme potency and widespread low level environ-mental contamination, the effects of TCDD on the reproduc-tive system are of interest.

Mechanisms initiating the early phases of follicle devel-opment are unknown; however, the process is likely to besupported by intraovarian factors. The appearance of thecalLH receptors and granulosal FSH receptors in the late pre-antral to early antral stages of development chronicle thedependence on gonadotropic support. FSH stimulatesgranulosal cell aromatization of estrogens. In turn, estrogenssupport further follicle development in part by increasingFSH receptors (19), inducing granulosa cell proliferation (20),and stimulating further estrogen production. Estrogen to-gether with FSH regulate the expression of LH receptors onthe granulosa late in antral follicle development (21, 22). Asestrogen production increases, positive feedback results inthe release of a surge of LH, initiating the process of ovulation(23). LH regulates the expression of several genes important inthe rupture of the follicle, including progesterone receptor (PR)(24), cyclooxygenase (25), and the family of plasminogen acti-vators and inhibitors (26, 27). Studies using knockout technol-ogies have demonstrated the importance of multiple factors,including estrogenreceptor(ER;both ERand ER) (28,29), PR(30), FSH receptor (31), and FSH (32), in the normal process offollicle development and ovulation.

Received August 2, 2000.Address all correspondence and requests for reprints to: Katherine F.

Roby, Ph.D., Department of Anatomy and Cell Biology, University ofKansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas66160. E-mail: [email protected].

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The objectives of the current study were to investigatepotential ovarian-specific mechanisms of TCDD-mediatedinhibition of ovulation.

Materials and Methods

Animals

Female Sprague Dawley rats were hypophysectomized by the sup-plier at 23 days of age (Charles River Laboratories, Inc., Portage, MI).Thereafter, the rats were provided food and 5% dextrose water (wt/vol)ad libitum and maintainedin roomswith a temperature of approximately72 F and lighting of 12 h/day (lights on at 0600 h). TCDD (32 g/kg)or corn oil vehicle was given orally at 0900 h on day 26. One day laterthe rats were injected with PMSG (10 IU, sc) to stimulate follicle devel-opment. Fifty-two hours later, all rats were injected with hCG (10 IU, sc)to mimic the LH surge and induce ovulation. In preliminary studiesthese doses of PMSG and hCG were found to result in a physiologicalnumber of ovulations (10). The dose of TCDD (32 g/kg) was foundto most completely block ovulation (to 1 ova). Animals were decap-itated at various times as indicated, and trunk blood was collected.Ovaries were collected, cleaned, weighed, and prepared for histologicalanalysis, topical autoradiography, or membrane binding studies as de-scribed below. Ovulation was determined by oviductal irrigation 20 hafter hCG as previously described (33). All animal handling and pro-

cedures conformedto the guidelines setforthby theinstitutional animalcare and use committee of the University of Kansas Medical Center.

Histological analysis

Ovaries were fixed in Bouins solution, and serial sections (8 m) ofthe entire ovary were prepared, mounted on glass slides, stained withhematoxylin and eosin, and evaluated under the light microscope. Eachhealthy antral follicle was measured and counted in the section con-taining the nucleolus of the oocyte. The diameter at two perpendicularorientations was measured with a ruled ocular; the final diameter wasthe mean of the two measurements. Follicle counts were obtained usinga single ovary from six control and seven TCDD-treated rats collected52 h after PMSG administration.

Membrane receptor binding

Receptor binding analysis was performed as previously described(34). Each ovary was homogenized in 300 l homogenization buffer (50mm Tris-HCl containing 10 mm CaCl2 and 20 mm MgCl2, pH 7.2). Thehomogenate (100 g protein) was transferred to a 12 75-mm tube, and[125I]hCG or [125I]FSH (100 l, 23 Ci/g) was added. hCG and rat FSH(CR-127 and rat FSH-I-9 from the National Hormone and PituitaryProgram) were iodinated using lactoperoxidase (35). The mixture ofhomogenate and [125I]gonadotropin was incubated overnight at roomtemperature; then 1 ml ice-cold receptor buffer (50 mm Tris-HCl and 5mg albumin/ml, pH 7.0) was added, the mixture was centrifuged, andthe supernatant was aspirated. The [125I]gonadotropin bound was de-termined by -spectroscopy. Nonspecific binding was determined byincubating the homogenate with [125I]hCG or [125I]FSH in the presenceof 20 IU hCG or 25 g ovine FSH (oFSH-19), respectively. Specificbinding was determined as counts per min bound minus nonspecificcounts per min. Receptor binding was performed on ovaries from threedifferent experiments with between six and eight animals in each treat-ment group and at each time point.

Topical autoradiography

Topical autoradiography was performed according to the method ofOxberry and Greenwald (36). The tissue was frozen in liquid nitrogen,embedded in OCT(Miles Laboratories, Elkhart, IN), andcut in a cryostatat10m. Thefrozen sections were placedon poly-l-lysine-coated slides,air-dried for 30 min, and stored in a dessicator box at 20 C untilsubjected to topical autoradiography of hCG or FSH receptors. Afterbringing the slides to room temperature, the tissue sections were fixedin 4% paraformaldehyde at 4 C for 30 min. Fixation was followed byrinsing 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 Ci/g) was added to the

tissue section in 0.2 ml PBS containing 0.1% BSA and incubated for 2 hat 37 C. Control nonspecific binding sections had 10 IU unlabeled ho-mologous hormone in addition to the radiolabeled hormone. The tissuesections were rinsed thoroughly in PBS to remove excess unboundhormone, and thenthe sectionswere postfixedin 4% glutaraldehyde andrinsed in PBS. The slides were dipped in autoradiographic emulsion,dried, stored for 5 days at 4 C, and then developed through routinephotographicsteps of Dektol, Stop, andFix.Then theslideswere washedand stained with hematoxylin. Grain counting was assessed using Op-tomis Bioscan (Media Cybernetics, Silver Spring, MD). This programmeasures the grain density per unit area. Grain density was assessedunder total and nonspecific binding conditions in the same follicle inadjacent sections. Specific binding was determined as total bindingminus nonspecific binding. Ovaries from five control and seven TCDD-treated animals were assessed.

RIAs

Serum progesterone and estradiol levels were measured by RIA, andovarian cAMP levelswere determined with a commerciallyavailable kitaccording to the manufacturers (Biomedical Technologies Inc., Stough-ton, MA) directions as previously described (33).

Statistical analysis

Experiments were repeated at least three times with between six andeight animals in each treatment and time point. Data were subjected toANOVA, with differences between means detected by Newman-Keulstest. Differences were considered significant at P 0.05.

Results

Ovulation (Table 1)

Ovulation occurs approximately 16 h after hCG injectionin the rat model used in this study. Therefore, 20 h after hCG,ovulation was assessed by enumerating the ova flushed fromthe oviducts. Control rats ovulated 9.29 1.52 ova comparedwith 0.63 0.26 ova for TCDD-treated rats (P 0.001).Consistent with reduced ovulation, serum progesterone lev-els the morning after ovulation were significantly lower in

TCDD-treated animals compared with controls. Estradiollevels were not different at this time point.

Ovarian morphology (Fig. 1)

Ovaries from control, corn oil-treated animals containednewly formed corpora lutea 20 h after hCG administration,evidence of recent ovulation. In addition, multiple smallantral follicles and a few large, atretic follicles were present(Fig. 1A). Ovaries from TCDD-treated rats contained scantevidence of corpora lutea. In a group of eight animals treatedwith TCDD (Table 1), four did not ovulate, three ovulatedone ova each, and one ovulated two ova; thus, occasionalcorpora lutea were observed histologically. The number of

corpora lutea observed histologically correlated with thenumber of ova recovered from the oviduct. TCDD-treated

TABLE 1. The effects of TCDD treatment in hypophysectomizedrats on the number of ova shed, and serum progesterone andestradiol levels 20 h after administration of hCG

No. of ova(no. ovulating/no. treated)

Progesterone(ng/ml)

Estradiol(pg/ml)

Control 9.29 1.52 (7/7) 10.32 2.29 38.41 4.51TCDD 0.63 0.26a (4/8) 3.13 0.71a 48.63 7.60

a P 0.001 TCDD vs. control.

TCDD ALTERS FOLLICLE DEVELOPMENT 2329



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ovaries contained multiple large unruptured antral folliclesand numerous small antral follicles (Fig. 1B).

Follicular development (Fig. 2)

The numberand size of healthy antral follicles present 52 hafter PMSG, just before hCG was given to induce ovulation,were determined in control and TCDD-treated animals. Ova-ries of TCDD-treated animals contained fewer large antralfollicles compared with controls. The total number of folliclesper ovary with a diameter of 350 m and greater was 5.2 1.2 in TCDD-treated animals compared with 13.2 1.9 incontrols (P 0.004). The number of smaller antral follicles in

each size group between 100349 m in diameter was notdifferent in control and TCDD-treated ovaries. The numberof atretic follicles in ovaries from control and TCDD-treatedanimals did not differ 52 h after PMSG (data not shown).

Serum estradiol and progesterone (Fig. 3)

Serum concentrations of estradiol in control rats remainedlow until 52 h after PMSG treatment; levels then increased topeak 5 h after hCG treatment and thereafter decreased. Incontrast to estradiol levels measured in control animals, es-tradiol levels did not increase in TCDD-treated animals 52 hafter PMSG. In addition, estradiol levels in TCDD-treatedanimals did not reach similar peak levels at 5 h after hCG asobserved in controls.

Serum progesterone levels remained low in control ani-mals until after treatment with hCG, when progesteroneincreased, reached a peak at 510 h after hCG, and decreased

thereafter (Fig. 3). Serum progesterone levels in TCDD-treated animals also increased after hCG; however, all peaklevels were significantly lower than those measured in con-trol animals. Also, the morning after ovulation (at 20 h)serum levels of progesterone were significantly lower inTCDD-treated animals compared with controls (Fig. 3 andTable 1).

Ovary weight (Fig. 4)

The increase in ovary weight stimulated by PMSG andhCG treatment in control animals was reduced in TCDD-

FIG. 2. The size and number of healthy antral follicles present inovaries from control and TCDD-treated rats 52 h after administrationof PMSG. Follicle numbers were obtained from one ovary in six con-trols and seven TCDD-treated animals. Data are the mean SEM. *,P 0.05 TCDD vs. control within the same diameter group.

FIG. 1. Morphology of ovaries from control (A) andTCDD-treated (B) rats collected 20 h after an ovulatory dose of hCG. Control ovaries containmultiple corpora lutea (CL) and small antral follicles. Ovaries from TCDD-treated animals contain large unruptured follicles (UF) and smallantral follicles. Ovaries were photographed at the same magnification. Note that ovaries from TCDD-treated animals are smaller than controls(see also Fig. 4).

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treated animals. Ovary weight was increased significantly52 h after PMSG compared with that at various time pointsbefore PMSG treatment, and ovary weight continued to in-crease after hCG administration in control animals. Com-pared with controls, ovary weight was significantly lower inTCDD-treated animals 52 h after PMSG and remained lowerthroughout all subsequent time points examined duringtreatment.

FSH and hCG receptor binding (Figs. 58)

Gonadotropin receptor binding was assessed by mem-brane receptor binding and topical autoradiography at 52 hafter PMSG administration. FSH and hCG receptor bindingwere significantly reduced in whole ovary membrane prep-arations from TCDD-treated rats compared with controls(Fig. 5).

Topical autoradiography studies revealed results compa-

rable to membrane binding studies. Reduced FSH binding(grain density per m2 membrana granulosa) was observedin granulosa from TCDD-treated animals compared withcontrols. hCG binding was reduced in both thecal and granu-losal compartments in ovaries of TCDD-treated rats com-pared with controls (Fig. 6). The data in Fig. 6 are represen-tative of follicles 350 m or more in diameter.

Additional membrane binding studies were carried out onovaries collected at multiple times throughout the treatment

FIG. 3. Effectof TCDD on PMSG- andhCG-induced estradiol andprogesterone production. Serum concentrations of estradiol andprogesteronein control (oil) and TCDD-treated rats were assessed by RIA before treatment and at multiple time points after PMSG andhCG administration.The times of PMSG and hCG administration are indicated by arrows. n 712/treatment and time point. *, P 0.05 TCDD vs. control withinthe same time point.

FIG. 4. Effect of TCDD on ovarian weight after PMSG and hCGadministration. Increased ovarian weight stimulated by PMSG andhCG was inhibited by TCDD treatment. Ovaries from TCDD-treatedanimals weighed significantly less than control ovaries at 52 h after

PMSG and at all subsequent time points examined. n 6/treatmentand time point. *, P 0.01, control vs. TCDD within the same timepoint.

FIG. 5. Effect of TCDD on PMSG-stimulated FSH and hCG binding.FSH and hCG binding to ovarian membrane preparations from con-trol and TCDD-treated rats was determined 52 h after the adminis-tration of PMSG. n 8/treatment. *, P 0.05.




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schedule. FSH binding to ovarian membrane preparationsfrom control animals was increased 24 h after the adminis-tration of PMSG compared with times before PMSG admin-istration (Fig. 7). FSH binding increased further at 52 h,remained elevated after hCG administration, and then in-creased again near the time of ovulation. FSH binding toovarian membrane preparations from TCDD-treated ratsalso increased after PMSG(PMSG vs. pre-PMSG time points);however, binding decreased thereafter when binding to con-trols remained elevated. Closer to the time of ovulation, FSH

binding in the TCDD samples increased to levels similar tothose in controls (Fig. 7).

hCG binding to membranes in control and TCDD-treatedgroups was very similar throughout the treatment schedulewith the exception of two time points. hCG binding waslower at 24 h after treatment with TCDD and at 52 h afterPMSG 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 withincreased cAMP was altered in TCDD-treated animals. Incontrol animals, ovarian cAMP levels increased 24 h afterPMSG, increased again at 52 h after PMSG, and then in-creased further in response to hCG. In animals treated withTCDD ovarian cAMP increased 24 h after PMSG and was notdifferent from control levels. However, ovarian cAMP didnot increase further at 52 h post-PMSG. In addition, no fur-ther increase in cAMP occurred in TCDD-treated animals inresponse to hCG. cAMP levels in ovaries from TCDD-treated

rats were significantly lower compared with control values52 h after PMSG and 5 h after hCG.

Discussion

Results from this study indicate TCDD-mediated inhibi-tion of ovulation is probably due to the effects of TCDD onfollicular development. The present data indicate that by thetime of hCG treatment to induce ovulation, the folliclespresent in the TCDD-treated ovaries are not comparable tothe follicles in the control ovaries. For example, the numberof large follicles, the production of estradiol, andLH and FSHreceptor binding are all lower in TCDD-treated animals.Thus, it would seem likely that the ability of these follicles tofully respond to the ovulatory surge of LH (hCG injection)

FIG. 6. Specific binding of hCG and FSH to granulosal and thecalcompartments of antral follicles 350 m or less in diameter in TCDD-treated and control ovaries.Insitu binding of [125I]FSH and[125I]hCGto specific receptors was assessed by topical autoradiography, as de-scribed in Materials and Methods, on ovaries (five control and seventreated) collected 52 h after administration of PMSG. *, P 0.05.

FIG. 7. Effects of TCDD treatment on FSH binding. FSH binding towhole ovarian membranes prepared from TCDD-treated and controlrats was assessed at multiple time points throughout treatment. n 6 8/treatment and time point. *, P 0.05 TCDD vs. control withinthe same time point.

FIG. 8. Effects of TCDD treatment on hCG binding. hCG binding toovarian membranes from TCDD-treated and control rats was as-sessed at multiple time points throughout treatment. *, P 0.05TCDD vs. control within the same time point.




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At this time point the number of atretic follicles was notdifferent in control and TCDD-treated animals. However,assessment of follicular atresia 14 h after the administrationof hCG indicated that the majority of the large antral folliclesin the TCDD-treated animals were atretic (Roby, K. F., un-published observations). Thus, it appears that TCDD doesnot 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. Forexample, cyclin D2 is necessary for granulosa cell prolifer-ation in response to FSH. Cyclin D2 KO mice exhibit reducedgranulosal proliferation; follicles remain small, developingonly up to four layers of granulosa cells; and ovulation doesnot occur (51, 24). Although follicles do not reach a devel-opmental maturity to ovulate in cyclin D2 KO mice, stimu-lation with PMSG and hCG does induce changes in geneexpression reflecting further differentiation of granulosacells. For example, follicles develop antra, aromatase expres-sion is induced, and in response to LH, PR and PG synthase

messenger RNA are up-regulated (24). Although the folliclecan respond at least in part to LH, ovulation does not occur.Thus, in the present experiments, antiestrogenic effects ofTCDD resulting in reduced FSH receptor expression might,in turn, reduce cyclin D2 expression.

TCDD-treated animals didnot ovulate in response to hCG.Rupture of the follicle occurs after hCG-induced changes inthe expression of several genes, including a transient increasein granulosal PR expression (52). KO studies clearly dem-onstrate the necessity of PR expression for ovulation (30). Inaddition, changes in specific enzyme expression and activityhave been mapped to the time of ovulation and are thoughtto play critical roles in the breakdown of the tissues necessary

for ovulation (53, 54). The ability of TCDD to directly alterthese genes is not clear. Using the model described in thepresent study, expression of PR in granulosa cells was re-duced 10 h after hCG administration in TCDD-treated ani-mals compared with controls (Roby, K. F., unpublished ob-servations). In addition, plasminogen activator expressionand activity 15 h after hCG administration were reduced inovaries from TCDD-treated rats compared with controls(Roby, K. F., unpublished observations). These results aredifficult to interpret given the data indicating that matura-tion of the follicles was inhibited in TCDD-treated animals.A previous study indicated that administration of TCDD intothe ovarian bursal cavity of intact rats before follicle devel-

opment/ovulation induction with PMSG/hCG resulted inreduced ovulation (5). Direct effects of TCDD on hCG-induced genes around the time of ovulation might be bestaddressedusing the technique of intrabursal injection. TCDDadministered after the completion of follicle development, atthe time of hCG treatment, would more directly address thisquestion.

In summary, a single dose of TCDD administered beforethe initiation of follicle development alters the responsive-ness of the ovary to PMSG, limiting the development of fullymature follicles capable of responding to an hCG ovulatorystimulus.

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