**Models Assessing Direct Effects of Dioxins and Related Compounds on
the Ovary**

Project Scope

Previous studies in rats have indicated that
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has inhibitory effects on
ovulation due to either the direct effects of TCDD on ovarian function
or a change in the pituitary gonadotropins that regulate ovarian
follicular development, steroidogenesis, and ovulation. The objectives
of this study were to investigate if TCDD, other dioxins, furans and
polychlorinated biphenyls (PCBs) directly affect ovarian function using
the hypophysectomized rat as an *in vivo* model.

The main objectives of this research were to determine:

-   The effects of TCDD on physiological responses of the ovarian
    theca-interstitial cells of immature hypophysectomized rats exposed
    *in vivo* to TCDD - specifically, changes in gonadotropin receptor
    binding, second messengers, and steroid production;

-   The effects of TCDD *in vitro* on gonadotropin receptor binding,
    second messengers, and steroid production in the ovarian
    theca-interstitial cells;

-   The *in vivo* effects of TCDD on follicular development in immature
    hypo-physectomized rats exposed to physiologic doses of
    gonadotropin, the steroidogenic responses of the ovary to
    gonadotropin, and ovulation induction and corpora luteal function
    controlled by gonadotropin;

-   If other dioxins, furans, and PCBs have the same mode of direct
    action on ovarian function as TCDD, using ovulation and
    steroidogenesis as biomarkers, and to demonstrate that these models
    can be used for the assessment of toxic equivalency (TEQ) of dioxins
    and related compounds.

# 

# Project Results and Implications 

[Ovulation Blockage by TCDD: Direct and Indirect Effects on
Steroidogenesis in the Immature Rat]{.underline} The main purpose of
this study was to investigate the direct effect of TCDD on ovulation and
steroidogenesis in the ovary. *In vivo* effects of TCDD on ovulation and
on ovarian and circulating steroid hormones were investigated in
immature hypophysectomized rats (IHR) primed with equine chorionic
gonadotropin (eCG) and human chorionic gonadotropin (hCG). The *in
vitro* effects of TCDD on the steroidogenesis of granulosa cells (GC),
theca-interstitial cells (TIC), and whole ovarian dispersates derived
from the ovaries of IHR were also investigated. In the ovulation model,
rats were hypophysectomized at the age of 23 days. At 26 days, the IHR
were given 20 µg/kg TCDD by gavage. On the next day, eCG (10 IU) was
injected subcutaneously to stimulate follicular development, and after
52 hours 10 IU hCG was given to induce ovulation. TCDD was found to
block ovulation and reduc ovarian weight in the IHR. Concentra-tions of
progesterone (P~4~), androstenedione (A~4~), and estradiol (E~2~) in
sera and ovaries were not altered by TCDD at 12, 24, 48, and 72 hours
after eCG injection. In isolated GC from untreated IHR, TCDD (0.1-100
nM) had no significant effect on P~4~ and E~2~ after stimulation by
luteinizing hormone (LH) or follicle stimulating hormone (FSH). In TIC
and whole ovarian dispersates containing GC, TIC, and other ovarian
cells, TCDD (0.1- 800 nM) also had no effect on A~4~ and P~4~ secretion
stimulated by LH. Reverse transcriptase polymerase chain reaction
(RT-PCR) results showed that aryl hydrocarbon receptor (AhR) mRNA was
expressed constitutively in the whole ovaries of IHR, with maximum
down-regulation at 6 hours after TCDD (20 g/kg) administration. Ovarian
*CYP1A1* were induced maximally at 6 hours after TCDD administration,
whereas *CYP1B1* could not be detected. The selective induction of
AhR-related genes by TCDD in the ovary that TCDD may act through
AhR-mediated signal transduction pathways. In summary, these results
indicate that TCDD does not block ovulation in IHR by altering ovarian
steroidogenesis. It appears that TCDD's inhibition of ovulation by
direct action on the ovary is due to processes related to follicular
rupture.

[Direct Effects of TCDD on Ovarian Epithelial Cells: Regulation by
Estradiol Congeners]{.underline} Derivatives of 17β-estradiol (E~2~)
increased the TCDD induction of 7-ethoxyresorufin-*O*-deethylase (EROD)
in a dose-dependent fashion. Potency of the derivatives varied in the
order : estrone (E~1~) \> E~2~ \> 4-hydroxyestradiol (4-OHE~2~) and
2-hydroxyestradiol (2-OHE~2~). In contrast, estriol (E~3~) decreased
EROD activity and *CYP1A1* gene expression. The AhR antagonists
alpha-naphthoflavone and phenanthroline inhibited induction of EROD by
TCDD. Progesterone and gonadotropins (FSH and LH) had no effect on the
induction of EROD by TCDD. E~2~ increased TCDD-induced *CYP1A1* protein
and mRNA in a dose-dependent fashion, whereas E~3~ in combination with
TCDD decreased CYP1A1 expression and activity. E~2~ did not alter the
luciferase activity induced by TCDD in cells transfected with a dioxin
response element (DRE)-containing luciferase reporter, whereas E~3~
decreased its activity. The actions of E~2~ were blocked by a specific
estradiol receptor (ER) antagonist, indicating the involvement of the ER
in modifying TCDD effects. Although *CYP1A1* mRNA was induced by TCDD,
*CYP1B1* mRNA was constitutively expressed and unaffected by TCDD. ER-α
mRNA was constitutively expressed in ID8 cells, but ER-β mRNA and
progesterone receptor mRNAs were not present. These results indicate
that E~2~ derivatives differentially regulate TCDD-inducible *CYP1A1*
expression and provide new insight into different mechanisms by which
estrogen derivatives regulate ovarian responses to TCDD.

[Estradiol Enhancement of the Action of TCDD on Ovulation
Blockade.]{.underline} Immature rats were treated subcutaneously with
0-2 mg/kg estradiol cypionate (ECP), followed by TCDD (0 or 10 µg/kg
orally) 24 hours later. Follicular development was induced by injecting
subcutaneously 5 or 10 IU of eCG followed by an ovulatory dose of hCG
(10 IU, subcutaneously). In another experiment, intact
(non-hypopysectomized) rats were given ECP subcutaneously (0 or 2 mg/kg)
and TCDD into the ovarian bursa (0 or 250 ng), while another group
received TCDD orally (10 µg/kg) and ECP into the ovarian bursa (0 or 1.5
µg). Inhibition of ovulation by TCDD was potentiated by ECP in
hypophysectomized but not intact rats. Only hypophysectomized rats
exposed systemically to TCDD and ECP exhibited weight loss. Pair feeding
mimicked the combined effects of TCDD and ECP in hypophysectomized rats.
Blockade of ovulation by systemic or local TCDD in the intact rats was
alleviated by the ECP pretreatment. These results indicate that estrogen
increased the systemic toxicity of TCDD in hypophysectomized rats while
antagonizing its direct effects on the ovaries.

[Toxic Equivalency, TCDD, and Ovulation]{.underline} For these
experiments, the gonadotropin-primed immature female rat model was used
to study the effect of TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin
(PeCDD), and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) on
ovulation. Single doses of different of single congeners and mixtures
were given orally to 23-day-old rats. Gonadotropin from pregnant mare\'s
serum (PMSG) was injected (5 IU) 24 hours later to induce follicular
maturation. PCDDs were found to decrease the number of ova per ovary and
reduce ovarian weight gain induced by PMSG in a dose-dependent fashion.
The slopes of the dose-response curves generated by individual PCDDs
and/or their mixture were similar. PMSG-induced increases in serum E~2~
on the day of expected ovulation were enhanced by PCDDs. In contrast,
serum P~4~ and FSH were decreased at ovulation. PCDDs also altered the
temporal pattern of serum E~2~, FSH, and LH variations, but not that of
PrL. Histologically, all three PCDDs caused ova to become trapped in
preovulatory follicles, and reduced the number of corpora lutea. These
results indicate that the PCDDs tested in the present model all have the
same mode of action on ovulation and produce similar changes in l
reproductive hormone levels (*e.g*., LH, FSH, P~4~, and E~2~). Further,
the dose- response relationships of the individual congeners are
parallel, and parallel to that of their equipotent mixture. This
confirms the validity of the toxic equivalency (TEQ) concept for these
chemicals for one aspect of endocrine disruption, that is, inhibition of
ovulation.

[PCBs, Furans, and Their Effects on Ovulation: Comparison with
TCDD]{.underline} The objective of another experiment was to investigate
the effect of other structurally related compounds, such as chlorinated
furans and biphenyls, on ovulation and related hormonal endpoints. The
gonadotropin-primed immature female rat model was used to study the
effect of 2,3,4,7,8-pentachlorodibenzofuran (PeCDF),
3,3\',4,4\',5-pentachlorobiphenyl (PeCB), 2,2\',5,5\'
tetrachlorobiphenyl (TCB) and their mixtures with PCDDs on ovulation.
Rats were dosed on post-natal day 23 with individual congeners (PeCDF,
PeCB, and TCB) or a mixtures of TCDD, PeCDD, HxCDD, PeCDF, and PeCB.
Twenty-four hours after dosing, eCG (5 IU) was injected to induce
follicular development. The slopes of the dose--response relationships
for inhibition of ovulation for the individual PeCDF, PeCB, and/or their
mixtures with PCDDs were similar. PeCDF, PeCB, and the mixture increased
serum concentrations of E~2~ at 72 hours after eCG injection.,.In
contrast, serum P~4~ and FSH were decreased at the same time point. Only
the high doses of TCDD, PeCDF, and PeCB blocked LH and FSH surges 58
hours after eCG injection. Examination of ovarian histology revealed
that the effects of PeCDF, PeCB, and the mixture,consisting of ova
trapped in preovulatory follicles and a lack, or reduced number, of
corpora lutea were very similar to that of PCDDs alone. Parallel
dose-response relationships for the individual congeners (PeCDF and
PeCB) and their equipotent mixture with PCDDs support the toxic
equivalency (TEQ) concept for the blockage of ovulation by these
chemicals. PCDDs, PCDFs, and PeCBs appear to block ovulation by the same
or a very similar mechanism of action.

[Gonadotropin Releasing Hormone Partially Overcomes the Blockade of
Ovulation Induced by TCDD]{.underline} The inhibitory effect of TCDD on
ovulation may result from either direct effect(s) of TCDD on ovarian
function or via altered pituitary secretionof LH and FSH. To further
investigate the effects of TCDD on pituitary gonadotropins and their
regulation, the potential role of gonadotropin releasing hormone (GnRH)
was investigated. Immature female Sprague-Dawley rats were dosed with 32
µg/kg of TCDD in corn oil. eCG (5 IU) was injected subcutaneously 24
hours later to induce follicular development. Immediately prior to the
expected time of the LH/FSH surge, 54 hours after eCG injection, half of
the TCDD-treated and corn oil-treated rats were subcutaneously injected
with GnRH (2 µg/ rat) to stimulate pituitary hormone release. TCDD
exposure alone was found to inhibit ovulation by 70-80% (2-3 ova/rat).
GnRH partially restored ovulation (6-7 ova/rat) in TCDD-treated rats,
without reversing its effect on ovarian weight reduction. The LH and FSH
surges triggered by eCG injection in controls were completely abolished
by TCDD at 56-60 hours after eCG injection. However, in rats treated
with TCDD and GnRH, an abnormally increased LH/FSH surge occurred at
56-60 hours after eCG injection. GnRH alone enhanced E~2~ and P~4~ serum
levels at 56-58 hours after eCG injection, but in rats treated with both
TCDD and GnRH, E~2~ secretion was significantly inhibited at 58, 60, and
72 hours. Serum P~4~ was only decreased at 72 hours after eCG injection.
The results indicate that exogenous GnRH given to TCDD treated-rats
induces LH or FSH surges, but only partially overcomes the inhibitory
effects of TCDD on ovulation.

Investigators

Paul F. Terranova - University of Kansas Medical Center, Kansas City, KS

For More Information

Laboratory web page:

<http://www.kumc.edu/physiology/terranova.html>

<http://www2.kumc.edu/crs/people_pages/terranova_pubs.html>

NCER Project Abstract and Reports:

<http://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/165/report/0>