Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
AUCES
IN UTERO AND LACTATIONAL EXPOSURE TO DIOXIN ALTERED
THE REPRODUCTIVE FUNCTION IN FEMALE RAT OFFSPRING
BEFORE AND AFTER PUBERTY
*EMAN E. ELSHARKAWY AND **NEVEEN A.El-NISR
* Department of Forensic Medicine and Toxicology, Faculty Of Veterinary Medicine,
Assuit University, Egypt
**Animal Health Institute of Research- Egypt
ABSTRACT:
To evaluate effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-rhodioxin (TCDD) exposure on the reproductive function in female rat offspring, before
and after puberty. The pregnant Sprague Dawely rat administered 0, or 1.0 mg
TCDD/kg on Gestation Day (GD) 8 and 15. Female offspring were examined at the
post-weanling before puberty on posnatal day (PND) 21 and in young adult stage of
development on PND42. Ovulation assessment, radioimmunoassay for serum
gonadotropins, steroids and histo-morphmetric analysis to the ovaries were
evaluated. The analysis included a count, measurement and classification of
preantral and antral follicles throughout the entire ovary on PND 21. The results
indicate that TCDD treatment significantly reduced the ovulation rate, serum
gonadotropins, steroids levels and the number of antral and preantral follicles of
certain size classes. The histopathological examination revealed small preovulatory
follicles displaying an atretic morphologic difference among the ovaries of rats
exposed to TCDD treatments. These data support the hypothesis that TCDD results
in adverse effects on female reproductive function. However, the age of animals
before or after puberty play an important role in the difference between results.
Moreover, TCDD exposure on the GD 8 or 15 has a great concern in the results
observed.
Keywords: Dioxin; Ovary; LH; FSH; Estrogen; Ovarian Follicles.
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Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
INTRODUCTION:
2,3,7,8-Tetrachlorodibenzo
Monly occurs through the consump-
p-dioxin (TCDD) is a persistent envi-
tion of contaminated meats and dairy
ronmental contaminant inadvertently
products (Travis and Hattemer-Frey,
produced as a by-product of herbicide
1991). Toxic effects of TCDD include
and pesticide manufacturing (Kul-
teratogenesis,
karni et al., 2008; Hites, 2011). TCDD
carcinogenesis, and developmental and
is also released during the bleaching
reproductive dysfunction (Pohjanvirta
process at tree pulp and paper mills,
et al., 1994).
immune
impairment,
and during the burning of municipal
The dioxin/aryl hydrocarbon
solid waste (Frakes et al., 1993; Tup-
receptor (AhR) mediates most, if not
purainen et al., 2003). TCDD is the
all, effects of dioxins. TCDD, the most
most toxic member of a class of chemi-
well characterized ligand of AhR, has
cals
like
for a long time been regarded as hav-
TCDD, have a long environmental
ing antiestrogenic properties such as
half-life, bioaccumulate in the food
inhibition
chain, and can be found in human fat
induced uterine weight increase and
tissue, blood serum, breast milk and
decreased
ovarian follicular fluid Schecter et al.,
progesterone receptors in uterus of
2006; Tsutsumi et al., 2011; (Humblet
rats and mice (Safe, 1995). In Ah-
et al., 2011; Ulaszewska et al., 2011).
responsive
The resistance of TCDD to metabo-
cancer cells, TCDD inhibits 17b-
lism, as well as its lipophilic chemical
estradiol-induced cell proliferation and
nature, contributes to bioaccumulation
decreases the mRNA levels of Cathep-
in animals and humans, as well as
sin D (Romkes et al., 1987; Gierthy et
biomagnification within the ecosystem.
al., 1988). One of the suggested
Its half-life can be more than 10 years
mechanisms
in humans (Aylward et al., 2005). Hu-
estrogen
man TCDD exposure at low levels com
involves direct interaction of the AhR
called
dioxins.
Dioxins,
-14-
of
17b-estradiol
levels
of
MCF-7
of
receptor
(E2)-
estrogen and
human
inhibitory
(ER)
breast
AhR-
crosstalk
Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
complex with functional DREs within
;activity and expression of several
the regulatory part of estrogen respon-
ovarian steroidogenic enzymes either
sive genes, such as Cathepsin D (Safe,
directly or indirectly (Dasmahapatra
1995). Interestingly, ligand activated
et al., 2000; Gregoraszczuk et al.,
AhR was more recently shown to be a
2001; Myllym¨aki et al., 2005). Repro-
member of an ubiquitin ligase complex
ductive organs and endocrine system
promoting proteosomal degradation of
are sensitive to TCDD during devel-
ERa (Ohtake et al., 2007). On the con-
opment and sexual maturation Mably
trary, estrogenic effects of TCDD have
et al., 1992) (Gray and Ostby, 1995;
also been reported (Abdelrahim et al.,
Compared to placental transfer, expo-
2003; Watanabe et al., 2004).
sure via lactation plays a major role in
There is an increasing evidence of
offspring receiving maternal PCDD/Fs
TCDD’s endocrine disrupting effects
and dioxin-like PCBs (Chen et al.,
on
reproductive
2001). Female Sprague–Dawley rats
system. In the hypothalamus, TCDD
appear to be more sensitive to dioxin-
has been shown to decrease the re-
like PCBs than males (Chu et al.,
sponsiveness to estradiol (Gao et al.,
1995). It has been proposed, that the
2001). A direct stimulatory effect of
increased susceptibility of females to
TCDD on pituitary gonadotropin se-
TCDD toxicity may be due to the sex-
cretion in immature female rats was
specific kinetics of TCDD (Petroff et
demonstrated by Li et al. (1997). In
al., 2000). The sensitivity to TCDD has
utero and lactational exposure to
been demonstrated to vary with the
TCDD has been shown to disrupt
alterations in ovarian hormonal status
estrus cycles and inhibit ovulation rate
(Petroff et al., 2001), and estrogens
(Salisbury and Marcinkiewicz, 2002).
have been shown to modulate the ef-
Furthermore, maternal exposure to
fects exerted by TCDD (Petroff et al.,
TCDD has potency to directly disturb
2000; Wyde et al., 2001).
immature
female
ovarian steroidogenesis of
female
TCDD exposure causes com-
progeny (Chaffin et al., 1997) and
promised ovarian function through
TCDD has been shown to alter the
alteration
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of
follicle development,
Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
alteration of steroidogenesis and inhi-
Animals:
bition of ovulation (Petroff et al.,
Nine pregnant, eight- week-old, female
2001b). To date, the majority of stud-
Sprague Dawely rats 200- 250g were
ies conducted with the purpose of un-
purchased from laboratory animal's
derstanding the effect of in utero and
house, Assiut University, Egypt. The
lactation TCDD exposure on ovarian
day following overnight mating with
development and function have been
the evidence of the vaginal plug is
conducted using animals before or
designated as day zero of pregnancy
after puberty. Additionally, the results
GD 0; animals were housed individu-
reported vary widely and sometimes
ally in clean plastic cages with constant
are conflicting depending on animal
temperature (22 ± I °C), humidity (55
model, dose of TCDD, length of expo-
+ 5%), and light schedule (12 L: 12 D).
sure, and age of the animals being
The animals were provided with com-
used. Thus, in this study, we set out to
mercial food (Rat Chow) and water ad
determine the effect of in utero and
libitum. All rats were handled in
lactation single oral dose of TCDD on
accordance with the standard guide
ovarian development and function in
for the care and use of laboratory
female rat offspring before and after
animals.
puberty. Moreover, we objected to test
Experimental design:
The pregnant females were
whether the perinatal exposure to
divided into three groups, 3 dams
TCDD on GD8 or on GD 15 can
each, the first group received a single
develop different response.
oral dose of 1.0 µg TCDD in vehicle/kg
Material and methods:
Chemical:
of maternal body mass by gavage on
2,3,7,8-teterachlorodibenzo-p-
gestation day eight (GD8). The second
dioxin(TCDD)solution(99%purity)was
group received the same previous dose
purchased from Grey Hound company
of TCDD on gestation day fifteen
(England).TCDD was dissolved in a
(GD15). The third group was served as
vehicle (corn oil: acetone; 19: 1 [v:v].
a control and received a dose of vehicle
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Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
(corn oil: acetone; 19: 1 [v: v] 2
primordial follicles enter the growing
mL/kg).
pool
during
this
time
period
The dose used in the present
(Hirshfield, 1991). PND 21 was chosen
study was based on extensive pub-
because it is prior to rat vaginal open-
lished work by previous studies involv-
ing, and thus provides a time to
ing in utero and lactational exposure of
determine
rats to TCDD Mably et al., 1992).
occur before puberty. Another nine
(Chen et al., 2001; Gregoraszczuk et
offspring from each group were termi-
al., 2001; GD 8 was chosen because it
nated on PND42 for measurement of
is an important time in fetal ovarian
serum estradiol, progesterone, FSH
development and major fetal organo-
and LH. PND 42 was chosen because it
genesis. During this time, primordial
is just after normal puberty and vagi-
germ cells migrate to the genital ridge
nal opening in the rat (Flaws et al.,
and begin to colonize the indifferent
1997). Moreover, postnatal days 25
gonad (Hirshfield, 1991). GD 15 was
and 42 were chosen in order to follow
used because TCDD exposure at this
the pups’ exposure through lactation.
time has been shown to induce a num-
All female offspring were visually
ber of unusual reproductive altera-
inspected and their body weight was
tions, as delayed vaginal opening and
recorded immediately before termina-
vaginal threads (Gray and Ostby,
tion.
1995). At birth, postnatal day (PND)
whether
abnormalities
Sample collections:
0, live offspring were counted and
on postnatal days 21 and 42,
sexed. On PND 1, litters were adjusted
individual body weight was recorded
to sex females for each dam to allow
and 54 female pups, 18 from each
for a similar nutritional environment
treated group and 18 from the control
and lactational exposure to TCDD.
group were anesthetized with CO, and
Nine female offspring from each group
decapitated. Two pups were randomly
were terminated on PND 21, before
picked from each litter. Trunk blood
puberty, for ovarian histology. Ovaries
was collected after decapitation and
were analyzed on PND 21 because rat
allowed to clot at 4°C. Sera were
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Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
collected and stored at -80°C until
taining the germinal vesicle (Heimler
determination
et al.,1998).
of
radioimmunoassay.
hormones
Ovaries
by
Ovulation assessment:
were
carefully removed from the body
Oviducts were dissected and
cavity, cleaned of adhering tissue and
ova flushed from the oviduct using
weighed. Ovaries from the control group
normal saline containing 0.1% hyalu-
and from the treated groups were
ronidase according to Li et al. (1995).
prepared for the histological analyses.
The number of ova/rat was counted
Ovarian histology and microscopy:
and the ovulation rate was calculated
(%) at PND42.
Ovaries were collected upon
Radioimmunoassay:
sacrifice, weighed, and fixed in Bouin’s
Serum Gonadotropins
Serum
concentrations
solution. Fixed tissues were embedded
of
in paraffin, sectioned at 6 Mm and
lutinzing hormone (LH) and follicular
stained with hematoxylin and eosin for
stimulating
hormone
examination of ovarian morphology by
determined
by
light microscopy.
(Taya et al., 1981).
Morphmetric analysis of the ovar-
Serum Steroid
ian follicles:
Serum concentrations of progesterone
(FSH)
were
radioimmunoassay
Follicles were analyzed in a
(P4) and estradiol (E2) were measured
double-blind paradigm. The analysis
as previously described by Terranova
included a count
and Garza (1983).
classification and
Statistical analysis:
measurement of area (µm2) of preanserially
Data are expressed as means ± SD.
throughout the entire ovary at PND
Statistical analysis was performed to
25. The greatest cross sectional area of
compare treated groups with respec-
each follicle was calculated using the
tive control groups using one-way
equation for an ellipse, rr (A) (B), in
analysis
which A and B are one-half the great-
followed by the Duncan’s multiple
est length and width of the section con-
range test when appropriate. Values of
tral
and
antral
follicles
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of
variance
(ANOVA),
Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
P < 0.05 were considered statistically
Ovarian weights:
significant.
A significant reduction in the
Results:
Body weight:
ovarian weight was obtained in TCDD
treated group in comparison with the
TCDD treatment significantly
control animals. There was not a
retarded the increase in body weight
significant difference between GD 8
seen in the control animals in this
and GD15 on PND 21 or 42 (Table 1).
experiment. There is no significant
difference between GD 8 and GD15 on
PND 21 or 42 (Table 1).
Table 1: Effects of in utero and lactational exposure to TCDD (1.0 µg/kg) or vehicle
control on the body and ovarian weights in female rat offspring.
Body Weight
Ovarian Weight
(g)
(mg/rat)
Control
PND21
PND42
36.2 ± 8.9
65.5 ±3.5┼
43.96±2.3
65.8±3.7┼
GD8
PND21
PND42
28.7±5.4*
51.4±4.7* ┼
38.16±1.3*
53.50±1.6*┼
GD15
PND21
PND42
29.3±4.3*
50.7±4.0* ┼
39.2 ±1.5*
50.5±1.7*┼
*Significantly different from controls (P < 0.05)
┼ Significantly different between the PND21 and PND42 (P < 0.05).
Serum gonadotrophins:
The serum LH and FSH levels
TCDD reduced the LH and FSH
in immature female rats were found to
surges significantly reduced (by 75–
be higher at 21 day of age than later at
80%) than the control in GD8 and 15
day 42 in control and exposed rats.
on PND21 and 42. There was not a
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Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
significant difference between GD 8
Serum 17b-estradiol concentrations
and GD15 on PND 21 or 42 (Table 2).
In female rat offspring treated with
Serum steroid:
Serum progesterone
TCDD, serum E2 concentrations were
significantly reduced when compared
ANOVA confirmed a signifi-
with control in GD 8 and 15 on PND
cant inhibitory effect of TCDD on
21 and 42. Moreover, there was
concentrations of serum P4. This
significant decrease in estrogen level in
inhibition was obtained in female rat
GD8 on PND21and 42 than in GD 15
offspring on PND 42 in both GD8 and
on PND 21 and 42. In addition, there
15. Moreover, there was a significant
difference
in
P4
was a significant difference in E2 levels
concentrations
between female rat offspring sacrificed
between female rat offspring sacrificed
on PND 21 and 42.
on PND 21 and 42 (Table 2).
The inhibitory
effect was more obvious on PND 42
than PND21 (Table 2).
Table 2: Effects of in utero and lactational exposure to TCDD (1.0 µg/kg) or vehicle
control on the serum gonadotrophine and steroids in female rats offspring on
PND25and PND42.
FSH
LH
E2
P4
IU/L
IU/L
pg/mL
ng\ml
Control
PND21
PND42
GD8
PND21
PND42
GD15
PND21
PND42
0.660.03
0.430.04┼
0.590.02
0.490.04┼
0.450.04*
0.29 0.02*┼
0.490.02*
0.440.04*┼
0.470.04*
0.30 0.02*┼
0.460.02*
0.400.04*┼
25.50.4
55.5 0.2┼
21.5 0.2*b
39.2 0.2*d ┼
26.2 0.7a
47.40.2c ┼
8.80±0.12
19.89±0.12┼
6.30+0.10*
14.30+0.10*d┼
7.30+0.10
16.30+0.10*c┼
*Significantly different from controls (P < 0.05)
Different letters indicate significance between the GD8 and GD15 (P < 0.05)
┼ Significantly different between the PND21 and PND42 (P < 0.05)
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Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
Morphmetric
analysis
of
and greater than 100,000 µm2 and
the
preantral follicles less than 50,000 µm2
ovarian follicles:
in female rat offspring in GD 8 and 15
Classification, count and measurement
on PND21. There was a significant dif-
of the ovarian follicles
ference between GD 8 and 15 in the
As observed from morphmetric
number of antral and prantral follicles
analysis of the ovarian sections of
less than 50,000, 50,000-75,000, greater
TCDD(versus vehicle-treated controls)
than 100,000 µm2 and less than 50,000,
reduced significantly the number of
respectively (Table 3).
antral follicles less than 75,000 µm2
Ovulation assessment:
There was a significant inhibition of ovulation by TCDD, hence it alter the
number of ova shed in comparison with control group. This effect on the ovulation
and the decrease in the ova number is more obvious in GD8 than GD 15 on PND 42
(Table 4).
Table 3: Ovarian follicle number in various size categories: effects of in-utero and
lactational exposure to TCDD or vehicle control on PND21.
2
Area (µm )
GD8
Antral
<50,000
364
50~75,000
Control
GD15
TCDD
GD8
GD15
377
216*a
299*b
177
165
115 *a
140*b
75 ~100,000
70
68
48*
54*
> I00,000
85
82
75*a
55*b
Preantral
<50,000
2100
2110
1760*a
1880*b
50-75,000
>75,000
19
4
20
20
25
6
2*
4*
*Significantly different from controls (P < 0.05)
Different letters indicate significance between the GD8 and GD15 at (P < 0.05)
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Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
Table 4: Effects of in utero and lactational exposure to TCDD on ovulation in female
rat offspring on PND 42.
Ovulation (ova/rat)
Ovulation rate (%)
Control
PND 42
8.3 ± 1.3
100 (18\18)
GD8
PND 42
2.2 ±2.0’*a
33 (6\18)*a
6.2±1.7*b
70 (12\18)*b
GD 15
PND 42
*Significantly different from controls (P < 0.05)
Different letters indicate significance between the GD8 and GD15 (P < 0.05).
Ovarian morphology:
There were marked morphologic differences among the ovaries of
different treatments regarding macroscopic and microscopic appearance.
Treatment with TCDD (1.0 µg/kg) on PND 21 of GD8 resulted in an aberrant
morphology consisting of small ovaries (approximately 1.5 to 2.5 mm) bearing
numerous small preovulatory follicles displaying an atretic morphology (i.e.,
pyknotic nuclei, scattered granulosa) at the ovarian periphery in the rat (Fig.
1a,b). On PND 42 of GD8, TCDD exposure was associated with smaller
ovaries (approximately 2.3-3 mm) bearing fewer corpora lutea (2 to 4) and a
noticeable cohort (15 to 20) of small preovulatory follicles in rats. There were
some degenerated ovum accompanied with an atresia in the secondary
follicles (Fig. 2. a). There were few, if any, corpora lutea in these ovaries and
the hilar region appeared edematous (Fig. 2. b). The morphological alteration
of the ovary was more obvious in GD8 on PND 21 and 42 than in GD 15 on
PND 21and 42. Moreover, TCDD exposure induced pronounced atretic effect
in the ovaries on PND 42 in both GD 8 and 15; it was more obvious on PND42
than on PND21.
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Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
Fig.1: Ovaries from TCDD- exposed female rat offspring on PND21 of GD8 showed a) Atresia in the
secondary and vesicular follicles along with congestion of the ovarian blood vessels X25. b) Numerous
small preovulatory follicles displaying an atretic morphology (i.e., pyknotic nuclei, scattered granulosa) at the ovarian periphery X 25.
Fig.2: Ovaries from TCDD- exposed female rat offspring on PND 42 of GD8 showed a) Degeneration
in the ovum accompanied with an atresia in the secondary follicle X 40. b) An increase in the thickness
of the ovarian epithelium. There are few corpora lutea in these ovaries and the hilar region appeared
edematous X 40.
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Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
DISCUSSION:
Much attention has been paid
on PND21 and 42. Exposure to PCDDs
to the effects of dioxin in offspring
was associated with significantly lower
exposed in- utero and lactationally,
ovarian weights vs. controls in adult
since the dose level required eliciting
Sprague-Dawley rats exposed acutely
these effects is very low (Yonemoto,
just prior to PMSG-induced follicular
2000). TCDD exposures have been
development (Gao et al., 1999a; Son et
linked to delayed puberty and early
al., 1999).
onset of menopause in women (Eske-
The results of the present study
nazi et al., 2005; Warner et al., 2007).
are in accordance with previous stud-
Similarly, TCDD exposures lead to
ies on dioxin-exposed rodents in that
early puberty, irregular estrous cycles,
an activated AhR (here CA-AhR) leads
reduced
ovulation,
to antiestrogenic effects in the presence
decreased circulating E2 levels, and
of estrogen, but to estrogenic effects in
early
in
the absence of estrogen. (Brunnberg et
female rodents Gray and Ostby, 1995;
al., 2006). This finding is consistent
Li et al., 1995; (Chaffin et al., 1996;
with the hypothesis that the activated
Myllymäki et al., 2005; Franczak et al.,
AhR may play an estrogen like role in
2006; Shi et al., 2007) Jablonska et al.,
the absence of estrogen (Ohtake et al.,
2010;.
2003; Boverhof et al., 2006). Activation
In the present manuscript, an in- utero
of the aryl hydrocarbon receptor
and lactational TCDD exposure at a
(AhR) by the highly toxic, prototypical
dose of 1.0 µg/ kg resulted in over
ligand, TCDD
toxicity in female rat's offspring as
compounds
shown by the decreased rate of body
function by altering follicle maturation
weight gain during the experimental
and steroid synthesis (Tischkau et al .,
period. And also our results of perina-
2011). In both an earlier and the
tal TCDD exposure suggest that TCDD
present study, we had shown that an
reduced ovarian weights vs. controls
in-utero and lactational exposure of
or
blocked
reproductive
senescence
-24-
or other dioxin-like
compromises
ovarian
Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
female rats to TCDD results in a
attributed to a significant reduction in
significant reduction in serum estrogen
certain classes of ovarian follicles
concentrations on PND 21 (Chaffin et
which considered the major source of
al., 1996). Where they observed tissues
estrogen (the antral follicles in particu-
specific regulation of estrogen recep-
lar) (Heimler et al., 1998). This expla-
tors (ER) and decreased circulating
nation is agreed with our findings of
estrogen levels in female offspring of
the decrease in the number of antral
Holtzman rats given 1 µg/kg of TCDD
and preantral follicles of certain size-
on GD15. In contrast, Gray et al.
classes. MacLuskyet al. (1998) sug-
(1997) stated that in female rats
gested that the mechanism that under-
exposed to TCDD perinatally, no
lies the alteration of reproductive func-
effects on estrus cyclicity, ovary func-
tion in offspring exposed perinatally to
tion or serum estradiol concentrations
TCDD may be due to incomplete
were observed.
When the pregnant
sexual differentiation of the CNS. Such
Long Evans rats exposed to TCDD on
alteration is probably not mediated by
GD8, female offspring had enhanced
blocking the estrogen response, but
incidence of constant estrus cycle and
may instead involve subtle develop-
reduced fertility from second to fourth
mental changes in other parts of the
litter when monitored by continuous
endocrine system, perhaps also affect-
breeding.
This decrease in estrogen
ing the feedback control of the adeno-
levels resulted in altered ER mRNA
cortical function. More recent studies
levels. Proposed mechanisms for the
confirmed this criterion; Effects of
antiestrogenic
TCDD
TCDD on ovarian function are likely
include oxidative metabolism of estro-
mediated indirectly through the hypo-
gens via products of the AhR gene bat-
thalamic–pituitary axis, as well as
tery, inhibition of expression and: or
directly at the level of the ovary Gao
efficacy of estrogen receptors and sup-
et al., 2001; Mizuyachi et al., 2002;
pression of estradiol-induced gene
Petroff et al., 2001a, 2003). Miller et
expression (Safe, 1995). Moreover, the
al., 2004; Hernandez-Ochoa et al.,
decrease in estrogen levels may be
2009; (Cao et al., 2011; The herein
action(s)
of
-25-
Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
presented results are also compatible
process of folliculogenesis, follicles
with the assumption that TCDD alters
increase in size mainly due to prolif-
reproductive function by a direct
eration of granulose cells. It has been
action at the level of the ovary as well
proposed that TCDD decreases circu-
as an indirect action via the hypotha-
lating E2 levels in part by inhibiting
lamic-pituitary
results
the growth of follicles from the prean-
showed a marked reduction in antral
tral to antral stage and by disrupting
follicles less than 75,000 µm2 and
the synthesis and metabolism of sex
axis.
Our
2
greater than 100,000 µm , and prean-
steroid hormones Heimler et al., 1998;
2
tral follicles less than 50,000 µm , fol-
(Dasmahapatra
lowing TCDD exposure. TCDD causes
Gregoraszczuk et al., 2001; Grochow-
a slowing of the movement of follicles
alski et al., 2001; Morán et al., 2000,
from the preantral stage that are
2003a, 2003b; Myllymäki et al., 2005;
greater than 50,000 µm2 into the antral
Pesonen et al., 2006). In the ovary, E2
stage, and from small primordial or
synthesis requires both the thecal and
primary follicles to preantral follicles
granulosa cell compartments. In the
2
et
al.,
2000;
smaller than 50,000 µm (Heimler et
rodent, the thecal cells are responsible
al.,1998). TCDD attenuated follicular
for producing P4 and androstenedione
maturation as well, reducing the num-
(A4). A4 then diffuses to nearby granu-
ber of antral and preantral follicles
losa cells where it is converted to tes-
without inducing apoptotic cell death
tosterone (T) and E2. In antral follicles,
in rats exposed to TCDD in utero and
the rate limiting steps in steroid hor-
during lactation (Heimler et al., 1998).
mone production are the mobilization
In this regard, antral follicles are the
of cholesterol from the cytoplasm to
main functional unit of the ovary,
the inner mitochondrial membrane via
housing the gametes and serving as the
steroidogenic acute regulatory protein
primary source of sex steroid hor-
(StAR) and the conversion of choles-
mones, such as E2. Follicles grow from
terol to pregnenolone by the mito-
the primordial to the antral stage in a
chondrial enzyme cytochrome P450,
process called folliculogenesis. In the
family 11, subfamily a, polypeptide 1
-26-
Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
(P450scc).Pregnenolone is then con-
TCDD (Li et al., 1995; Gao et al.,
verted to P4, and then by a multistep
1999b). TCDD blocked the LH and
process to the androgens, and finally to
FSH surges (Gao et al., 1999b).
E2 (Edson et al., 2009).
Chaffin et al. (1997) documented that a
Moreover,
our
results
showed
a
significant reduction of pituitary FSH
significant inhibition of ovulation by
mRNA was observed. Despite the
TCDD exposure, hence it alter the
reduced E2 levels, neither serum FSH
number of ova shed in comparison
nor
with control group. This an ovulatory
significantly.
effect of PCDDs is seen in hypophysec-
TCDD exposure suggest that it does
tomized Sprague-Dawley rats induced
not
to ovulate with exogenous gonadotro-
Reduced serum E2 levels appear to
pins (Gao et al., 1999a; Petroff et al.,
result from direct or indirect action on
2000) and direct application of TCDD
the ovary at some point following
to the ovary blocks ovulation as well
androstenedione production. In con-
(Petroff et al., 2000). Thus PCDDs
trast, previous studies suggested that
have direct effects on the ovulatory fol-
TCDD augmented the feedback action
licle that are sufficient to block ovula-
of steroids on the hypothalamic-
tion.
pituitary axis (Bookstaff, 1990). Ovula-
LH
act
concentration
on
Results
serum
of
increased
perinatal
gonadotropin.
The significant decrease in the
tion was reduced when TCDD was
serum level of gonadotropin was
administered on the morning of estrus,
observed in female offspring in the
which is right after the surge in FSH
present study, after perinatal TCDD
inducing the next cohort of follicles to
exposure suggest that TCDD act
ovulate in the next proestrus-estrus
indirectly on the ovary through the
(Smith et al., 1975). This suggests that
hypothalamic–pituitary axis. Previous
TCDD had a direct effect on the ovary
studies indicated that the patterns of
in preventing ovulation. On the other
preovulatory LH and FSH secretion
hand the extension of diestrus indicates
were altered in female rats receiving
that gonadotropin release was insuffi-
-27-
Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
cient
to
produce
estrogen
for
remodeling as evidenced by the failure
subsequent cycles (Li et al., 1995).
of ovulation seen following acute expo-
In the present study, in- utero
sure to these compounds (Gao et al.,
and lactational TCDD exposure alters
1999a,b;Petroff et al., 2000). The
the
mechanisms of this
morphological
appearance
of
interruption of
ovaries of female offspring on PND21
degradation of the
follicular wall are
and 42. The TCDD exposure resulted
as yet unknown and may involve
in an aberrant morphology consisting
modulation of steroid action or direct
of small ovaries bearing numerous
alteration of gene transcription by the
small preovulatory follicles displaying
PCDDs.
an atretic morphology, pyknotic nuclei
The results of the present study
and scattered granulose at the ovarian
are in accordance with Gray et al.
periphery. There were few corpora
(1997) in that TCDD treatment on GD
lutea in these ovaries and the hilar re-
8 has more toxic effect on the ovula-
gion appeared edematous on PND 42.
tion, ovary foliculogenisis, and ovary
Diminution in the number of antral
morphology and steroids levels in
and preantral follicles of certain
female offspring than on GD 15.
size-classes was observed on PND 21.
Administration of 1 mg TCDD/kg on
These ovarian effects of PCDDs are
GD 15 of pregnancy induces a high in-
evident
smaller
cidence of external genitalia malfor-
ovaries with fewer corpora lutea and a
mations in Holtzman female rat prog-
cohort of unruptured antral follicles
eny. Treatment with TCDD earlier in
still containing their oocytes (Gao et
gestation on GD 8 is generally less
al., 1999a,b; Petroff et al., 2000).
effective in inducing malformations of
While the combined direct and indi-
the genitalia of the female offspring,
rect effects of PCDDs on ovarian
but it does induce functional reproduc-
steroidogenesis
tive alterations (Gray and Ostby,
histologically
appear
as
somewhat
equivocal, PCDD treatment clearly
1995).
induces aberrations in periovulatory
proteolysis
and
follicular
The examination of the ovarian
tissue
follicles on PND 21 (before puberty)
-28-
Ass. Univ. Bull. Environ. Res. Vol. 16 No. 1 March 2013
or assessment of the ovulation rat by
body, ovaries weight and serum levels
counting the fully mature shed ova on
of E2, P4, LH and FSH. Moreover, the
PND 42 (after puberty) give the same
obtained alterations in the ovaries
indication of in-utero and lactional
more pronounced on PND 42 than on
exposure to TCDD induce inhibition
PND 21.
in the ovulation rate in both times
These findings may be due to differ-
related to TCDD effect on floculogene-
ence in the age of the animals and
sis. The results of serum steroid hor-
related to the puberty stage. In conclu-
mones (E2 and P4) and serum gonad-
sion, in-utero and lactional exposure to
otrophen (LH and FSH) revealed a
TCDD induce disturbance in the
significant decrease in both times than
female offspring reproductive function
control. Alterations in the morphologi-
through direct and indirect effect on
cal appearance of ovaries of female
the ovaries. TCDD exerts its toxic
offspring on PND 21 and 42 were
effect on both GD 8 and 15 but more
observed. In comparison between the
pronounced in GD 8 than 15. In addi-
effects of TCDD exposure in female rat
tion, TCDD toxic effect is more obvi-
offspring on PND 21 and PND 42,
ous on PND 42 than PND 21. This may
there were significant differences in
be related to time of puberty.
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‫ﺍﻟﺘﻌﺮﺽ ﺇﱃ ﺍﻟﺪﻳﻮﻛﺴﲔ ﻋﻦ ﻃﺮﻳﻖ ﺍﻟﺮﺣﻢ ﻭ ﺍﻟﺮﺿﺎﻋﺔ ﻳﻐﲑ ﺍﻟﻮﻇﻴﻔﺔ ﺍﻹﳒﺎﺑﻴﺔ ﰲ‬
‫ﺫﺭﻳﺔ ﺍﻟﻔﺌﺮﺍﻥ ﺍﻹﻧﺎﺙ ﻗﺒﻞ ﻭﺑﻌﺪ ﺳﻦ ﺍﻟﺒﻠﻮﻍ‬
‫*‪  **‬‬
‫* ﻗﺴﻡ ﺍﻟﻁﺏ ﺍﻟﺸﺭﻋﻲ ﻭﺍﻟﺴﻤﻭﻡ ﺍﻟﺒﻴﻁﺭﻴﺔ – ﻜﻠﻴﺔ ﺍﻟﻁﺏ ﺍﻟﺒﻴﻁﺭﻱ – ﺠﺎﻤﻌﺔ ﺃﺴﻴﻭﻁ‪.‬‬
‫** ﻤﻌﻬﺩ ﺼﺤﺔ ﺍﻟﺤﻴﻭﺍﻥ – ﻓﺭﻉ ﺃﺴﻴﻭﻁ‪.‬‬
‫ﻗﺩ ﺃﺠﺭﻴﺕ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻟﻠﺘﻌﺭﻑ ﻋﻠﻲ ﺇﻤﻜﺎﻨﻴﺔ ﺘﻘﻴﻴﻡ ﺁﺜﺎﺭ ﺍﻟﺘﻌﺭﺽ ﻟﻠﺩﻴﻭﻜﺴﻴﻥ ﻋﻥ ﻁﺭﻴﻕ ﺍﻟﺭﺤﻡ ﻭﺍﻟﺭﻀﺎﻋﺔ‬
‫ﻋﻠﻰ ﺍﻟﻭﻅﻴﻔﺔ ﺍﻹﻨﺠﺎﺒﻴﺔ ﻓﻲ ﺫﺭﻴﺔ ﺍﻟﻔﺌﺭﺍﻥ ﺍﻹﻨﺎﺙ‪ ،‬ﻗﺒل ﻭﺒﻌﺩ ﺴﻥ ﺍﻟﺒﻠﻭﻍ‪ .‬ﻗﺩ ﺘﻡ ﺇﻋﻁﺎﺀ ﺍﻟﺩﻴﻭﻜﺴﻴﻥ ﻟﻠﺠﺭﺫﺍﻥ ﺍﻟﺤﻭﺍﻤل‬
‫ﺒﺠﺭﻋﺎﺕ ‪ ٠‬ﺃﻭ ‪ ١،٠‬ﻤﻴﻜﺭﻭﻏﺭﺍﻡ ‪ /‬ﻜﻎ ﻓﻲ ﻴﻭﻡ ﺍﻟﺤﻤل ﺍﻟﺜﺎﻤﻥ ﻭ ﺍﻟﺨﺎﻤﺱ ﻋﺸﺭ‪ .‬ﻭﻗﺩ ﺒﺩﺍ ﻓﺤﺹ ﺫﺭﻴﺔ ﺍﻹﻨﺎﺙ ﻓﻲ‬
‫ﻓﺘﺭﺓ ﻤﺎ ﺒﻌﺩ ﺍﻟﻔﻁﺎﻡ ﻗﺒل ﺴﻥ ﺍﻟﺒﻠﻭﻍ ﻓﻲ ﺍﻟﻴﻭﻡ ﺍﻟﻭﺍﺤﺩ ﻭﺍﻟﻌﺸﺭﻭﻥ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ ﻭﺃﻴﻀﺎ ﺘﻡ ﻓﺤﺹ ﺫﺭﻴﺔ ﺍﻹﻨﺎﺙ ﻓﻲ‬
‫ﻤﺭﺤﻠﺔ ﻤﺎ ﺒﻌﺩ ﺍﻟﺒﻠﻭﻍ ﻓﻲ ﺍﻟﻴﻭﻡ ﺍﻟﺜﺎﻨﻲ ﻭ ﺍﻷﺭﺒﻌﻴﻥ ﺒﻌﺩ ﺍﻟﻭﻻﺩﺓ‪ .‬ﻭﻗﺩ ﺘﻡ ﺘﻘﻴﻴﻡ ﻟﻤﺴﺘﻭﻱ ﺍﻹﺒﺎﻀﺔ‪ ،‬ﻭ ﺃﻴﻀﺎ ﻭﻗﺩ ﺘﻡ ﻋﻤل‬
‫ﺘﺤﻠﻴل ﻜﻴﻤﻴﺎﺌﻲ ﺤﻴﻭﻱ ﻟﻘﻴﺎﺱ ﻨﺸﺎﻁ ﻜل ﻤﻥ ﺍﻟﻬﺭﻤﻭﻨﺎﺕ ﺍﻟﺨﺎﺼﺔ ﺒﺎﻟﺠﻭﻨﺎﺩﻭﺘﺭﻭﺒﻴﻥ ﻭﺃﻴﻀﺎ ﻗﻴﺎﺱ ﻤﺴﺘﻭﻱ ﻫﺭﻤﻭﻥ‬
‫ﺍﻻﺴﺘﺭﻭﺠﻴﻥ ﻭﺍﻟﺒﺭﻭﺠﺴﺘﻴﺭﻭﻥ ﻓﻲ ﺍﻟﻤﺼل ﻭﺫﻟﻙ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻟﻤﻘﺎﻴﺴﺔ ﺍﻟﻤﻨﺎﻋﻴﺔ ﺍﻟﺸﻌﺎﻋﻴﺔ‪ ،‬ﻜﻤﺎ ﺘﻡ ﻓﺤﺹ‬
‫ﻫﺴﺘﻭﻤﻭﺭﻓﻭﻟﻭﺠﻲ ﻟﻠﻤﺒﻴﻀﻴﻥ‪ .‬ﻭﺸﻤل ﺍﻟﺘﺤﻠﻴل ﺍﻟﻌﺩ ﻭﺍﻟﻘﻴﺎﺱ ﻭﺍﻟﺘﺼﻨﻴﻑ ﻟﻜل ﺃﻨﻭﻉ ﺍﻟﺒﺼﻴﻼﺕ ﻭﺍﻟﺠﺭﻴﺒﺎﺕ ﺍﻟﻐﺎﺭﻴﺔ ﻓﻲ‬
‫ﺠﻤﻴﻊ ﺃﻨﺤﺎﺀ ﺍﻟﻤﺒﻴﺽ ﺒﺎﻟﻜﺎﻤل‪ .‬ﻭﺘﺸﻴﺭ ﺍﻟﻨﺘﺎﺌﺞ ﺇﻟﻰ ﺃﻥ ﺍﻟﺘﻌﺭﺽ ﺇﻟﻰ ﺍﻟﺩﻴﻭﻜﺴﻴﻥ ﻗﺩ ﺃﺩﻱ ﺇﻟﻲ ﺨﻔﺽ ﻤﻌﻨﻭﻱ ﻤﻠﺤﻭﻅ‬
‫ﻓﻰ ﻤﻌﺩل ﺍﻟﺘﺒﻭﻴﺽ‪ ،‬ﻭﺃﻴﻀﺎ ﻓﻲ ﻤﺴﺘﻭﻱ ﺍﻟﻬﺭﻤﻭﻨﺎﺕ ﺍﻟﺨﺎﺼﺔ ﺒﺎﻟﺠﻭﻨﺎﺩﻭﺘﺭﻭﺒﻴﻥ ﻓﻲ ﺍﻟﻤﺼل‪ ،‬ﻭﺃﻴﻀﺎ ﻓﻲ ﻤﺴﺘﻭﻴﺎﺕ‬
‫ﻫﺭﻤﻭﻥ ﺍﻻﺴﺘﺭﻭﺠﻴﻥ ﻭﺍﻟﺒﺭﻭﺠﺴﺘﻴﺭﻭﻥ ﻓﻲ ﺍﻟﻤﺼل‪ ،‬ﻭﻗﺩ ﺃﺩﻱ ﺍﻟﺘﻌﺭﺽ ﻟﻠﺩﻴﻭﻜﺴﻴﻥ ﺇﻟﻲ ﻨﻘﺼﺎﹰ ﻤﻌﻨﻭﻴﺎﹰ ﻤﻠﺤﻭﻅﺎﹰ ﻓﻲ ﻋﺩﺩ‬
‫ﺍﻟﺠﺭﻴﺒﺎﺕ ﺍﻟﻐﺎﺭﻴﺔ ﻭﺍﻟﺒﺼﻴﻼﺕ ﻤﻥ ﻓﺌﺎﺕ ﻤﻌﻴﻨﺔ ﺤﺠﻡ ﻋﻨﺩﻤﺎ ﺘﻡ ﺍﻟﻔﺤﺹ ﺍﻟﻬﺴﺘﻭﻤﻭﺭﻓﻭﻟﻭﺠﻲ ﻟﻠﻤﺒﻴﺽ‪ .‬ﻜﻤﺎ ﺃﻅﻬﺭ‬
‫ﺍﻟﻔﺤﺹ ﺍﻟﻤﺠﻬﺭﻯ ﻟﻠﻤﺒﻴﺽ ﻤﻥ ﺫﺭﻴﺔ ﺍﻟﻔﺌﺭﺍﻥ ﺍﻟﻤﻌﺭﻀﺔ ﻟﻠﺩﻴﻭﻜﺴﻴﻥ‪ ،‬ﺘﺤﻠل ﻭﻓﻘﺩ ﻭ ﺘﻐﻴﺭ ﻓﻰ ﺸﻜل ﺍﻟﺨﻼﻴﺎ ﺍﻟﺘﻲ ﺘﺘﻜﻭﻥ‬
‫ﻤﻨﻬﺎ ﺃﻨﺴﺠﺔ ﺍﻟﺒﺼﻴﻼﺕ ﺍﻟﻤﺴﺌﻭﻟﺔ ﻋﻥ ﺍﻹﺒﺎﻀﺔ‪ .‬ﺒﺎﻟﻨﻅﺭ ﻟﻬﺫﺓ ﺍﻟﻨﺘﺎﺌﺞ ﻴﻤﻜﻥ ﺃﻥ ﻨﺴﺘﻨﺘﺞ ﺃﻥ ﻫﺫﻩ ﺍﻟﺒﻴﺎﻨﺎﺕ ﺘﺩﻋﻡ‬
‫ﺍﻟﻔﺭﻀﻴﺔ ﺍﻟﻘﺎﺌﻠﺔ ﺒﺄﻥ ﺍﻟﺘﻌﺭﺽ ﻟﻠﺩﻴﻭﻜﺴﻴﻥ ﻗﺩ ﻴﺅﺩﻱ ﺍﻟﻲ ﺁﺜﺎﺭ ﻀﺎﺭﺓ ﻋﻠﻰ ﺍﻟﻭﻅﻴﻔﺔ ﺍﻟﺘﻨﺎﺴﻠﻴﺔ ﻟﻺﻨﺎﺙ‪ .‬ﻤﻊ ﺍﻟﻭﻀﻊ ﻓﻲ‬
‫ﺍﻻﻋﺘﺒﺎﺭ‪ ،‬ﺇﻥ ﻋﻤﺭ ﺍﻟﺤﻴﻭﺍﻨﺎﺕ ﻗﺒل ﺃﻭ ﺒﻌﺩ ﺴﻥ ﺍﻟﺒﻠﻭﻍ ﺘﻠﻌﺏ ﺩﻭﺭﺍﹰ ﻫﺎﻤﺎﹰ ﻓﻲ ﺍﻟﻔﺭﻕ ﺒﻴﻥ ﺍﻟﻨﺘﺎﺌﺞ‪ .‬ﻭﻋﻼﻭﺓ ﻋﻠﻰ ﺫﻟﻙ‪،‬‬
‫ﻗﺩ ﺘﻡ ﻤﻼﺤﻅﺔ ﺃﻥ ﻭﻗﺕ ﺍﻟﺘﻌﺭﺽ ﻟﻠﺩﻴﻭﻜﺴﻴﻥ ﺃﺜﻨﺎﺀ ﺍﻟﺤﻤل ﻓﻲ ﺍﻟﻴﻭﻡ ﺍﻟﺜﺎﻤﻥ ﺃﻭ ﺍﻟﺨﺎﻤﺱ ﻋﺸﺭ ﻜﺎﻥ ﻟﻪ ﺘﺄﺜﻴﺭ ﻭﺍﻀﺢ‬
‫ﻟﻠﻔﺭﻕ ﺒﻴﻥ ﺍﻟﻨﺘﺎﺌﺞ‪.‬‬
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