Document Type : Original Article
Authors
Abstract
Keywords
The female reproductive system and, therefore human
fertility may be affected by exposure to environmental
toxicants. In this regard, most attention
has been paid to toxic environmental factors that
cause ovarian toxicity (
Lead is a ubiquitous environmental pollutant widely
dispersed in the environment and remains in the biotope.
Exposure to lead may be via contaminated
food or water and fuel additives (
Golmohammadi et al. found an association between
mean concentrations in blood lead of mothers and
newborns (
Lead can be concentrated in the cell nucleus, thus
perturbing cell proliferation and DNA synthesis
(
The Ethics Committee of Shahid Chamran University of Ahwaz approved this research project. Forty female Wistar rats were obtained from the animal house of the Jundishapour Medical Sciences University of Ahwaz and kept under specific conditions on a constant 12-hour light/dark cycle and at a controlled temperature of 22 ± 2°C. All rats had unlimited access to standard pellet food (Pars Co.) and distilled water. After acclimatizing to the laboratory conditions for one week, female Wistar rats (100 ± 10 days old) were mated overnight at a proportion of three females per male. After childbirth, mothers and their pups were randomly divided into four equal groups: control and three treatment groups that received 20, 100 and 300 mg/L/day lead acetate (Merk Co.) in drinking water from day 1 to day 21 of the lactational period. Doses were established from related studies of reproductive toxicity. Then, at 30, 60, 90 and 120 days of age five pups were randomly selected, weighed and under chloroform (Merk Co.) inhalation anesthesia, their left and right ovaries were removed, trimmed of fat and extraneous tissue, weighed and fixed by immersion in Bouin’s solution for 24 hours.
Following tissue processing, 5 μm serial paraffin
sections were prepared and stained with hematoxylin-
eosin. For microscopic analysis, sections
were selected using a non-random 10% sampling.
Numbers of ovarian follicles and corpora lutea
were counted in each 10th section of the ovary
(
For measuring the diameter of ovarian follicles in each developmental stage, 45 microscopic fields were randomly chosen in each rat. Then, using an ocular micrometer of light microscopy (Olympus EH), at a magnification of ×10, the largest and smallest diameters of each ovarian follicle were measured and the mean was calculated. To avoid counting the same follicle more than once, only individual follicles having an oocyte with a nucleus were evaluated, and we measured the size of the follicles in which the oocyte was present with an ocular micrometer.
All data were analyzed using SPSS version 10.0 for Windows. The data in different groups were compared by one-way analysis of variance (ANOVA) and Tukey’s test was used as a post hoc test. Differences were considered to be significant when p<0.05, p<0.01 and p<0.001.
Mean body weight showed significant decreases
in the highest dose group at 30 (p<0.001), 60
(p<0.01) and 90 and 120 (p<0.05) days of age
in comparison with control group. Significant
(p<0.05) decreases were observed in the moderate
dose group at 30 days of age in comparison with
the control group (
There were significant differences between mean
relative ovary weight in the 300 mg/L/day dose
group and control group at 30, 60 and 90 (p<0.05)
days of postnatal development (
Mean number of primordial follicles was higher
significantly at 30 days of age in 100 (p<0.01) and
300 (p<0.001) mg/L/day dose groups and at 60
(p<0.01) and 90 (p<0.05) days of age in the 300
mg/L/day dose group in comparison with the control
group (
Mean ± SEM body weight (g) and relative ovary weight (%) in control and neonatal lead-treated offspring Wistar rats during different stages of postnatal development
Groups | Days of age | Body weight | Relativeovary weight |
---|---|---|---|
30 | 28.37 ± 0.71cd | 0.055 ± 0.003d | |
60 | 79.96 ± 1.62d | 0.046 ± 0.001d | |
90 | 84.38 ± 2.13d | 0.054 ± 0.002d | |
120 | 95.12 ± 2.16d | 0.050 ± 0.002 | |
30 | 24.54 ± 0.18d | 0.053 ± 0.003 | |
60 | 77.91 ± 1.27d | 0.045 ± 0.003 | |
90 | 81.65 ± 2.38d | 0.051 ± 0.002 | |
120 | 91.65 ± 2.01 | 0.050 ± 0.001 | |
30 | 20.47 ± 0.35a** | 0.052 ± 0.002 | |
60 | 74.66 ± 2.80 | 0.043 ± 0.001 | |
90 | 79.25 ± 2.81 | 0.053 ± 0.001 | |
120 | 91.05 ± 2.11 | 0.048 ± 0.003 | |
30 | 17.26 ± 1.50ab*** | 0.050 ± 0.002a* | |
60 | 69.29 ± 1.24ab** | 0.040 ± 0.002a* | |
90 | 75.82 ± 1.16ab* | 0.047 ± 0.003a* | |
120 | 85.42 ± 2.36a* | 0.048 ± 0.001 | |
Different letters indicates significant (p<0.05) differences between groups.
*Significant difference between control and treatment groups. *p<0.05, **p<0.01 and ***p<0.001
Mean ± SEM number of ovarian follicles in control and neonatal lead-treated offspring Wistar rats during different stages of postnatal development
Groups | Days of age | Primordial F. | Primary F. | Secondary F. | Antral F. |
---|---|---|---|---|---|
30 | 12.30 ± 0.27cd | 17.30 ± 0.25cd | 17.20 ± 0.35cd | 6.63 ± 0.41cd | |
60 | 12.07 ± 0.23c | 16.97 ± 0.21d | 15.80 ± 0.48d | 6.93 ± 0.47d | |
90 | 11.97 ± 0.15d | 16.55 ± 0.38d | 15.87 ± 0.25bd | 6.97 ± 0.46d | |
120 | 11.32 ± 0.20 | 15.43 ± 0.23 | 16.64 ± 0.45 | 7.65 ± 0.50 | |
30 | 12.36 ± 0.25c | 17.11 ± 0.38d | 16.92 ± 0.22d | 5.39 ± 0.15d | |
60 | 12.63 ± 0.21 | 16.56 ± 0.27 | 15.44 ± 0.28 | 6.58 ± 0.23d | |
90 | 12.08 ± 0.18 | 16.35 ± 0.31 | 15.49 ± 0.33 | 6.17 ± 0.21d | |
120 | 11.47 ± 0.20 | 15.30 ± 0.24 | 15.55 ± 0.28 | 6.71 ± 0.20 | |
30 | 14.87 ± 0.36a** | 13.03 ± 0.21a* | 13.79 ± 0.42a* | 4.67 ± 0.22a* | |
60 | 12.79 ± 0.28 | 15.34 ± 0.32 | 14.13 ± 0.45 | 4.03 ± 0.31a* | |
90 | 12.10 ± 0.16 | 16.20 ± 0.62 | 15.23 ± 0.60 | 5.83 ± 0.26 | |
120 | 11.65 ± 0.24 | 15.05 ± 0.51 | 15.84 ± 0.46 | 6.46 ± 0.19 | |
30 | 15.63 ± 0.26a*** | 12.53 ± 0.30ab** | 11.80 ± 0.38ab** | 3.4 ± 0.20ab** | |
60 | 15.52 ± 0.42a** | 13.12 ± 0.31a* | 12.31 ± 0.43a* | 4.86 ± 0.31ab* | |
90 | 13.07 ± 0.32a* | 15.01 ± 0.55 | 14.73 ± 0.54 | 5.50 ± 0.28 | |
120 | 12.11 ± 0.29 | 15.21 ± 0.34 | 15.33 ± 0.50 | 6.00 ± 0.25 | |
Different letters indicates significant (p<0.05) differences between groups.
*Significant difference between control and treatment groups. *p<0.05, **p<0.01 and ***p<0.001
Mean (±SEM) number of ovarian follicles in control and neonatal lead-treated offspring Wistar rats during different stages of postnatal development
Groups | Days of age | Primordial F. | Primary F. | Secondary F. | Antral F. |
---|---|---|---|---|---|
30 | 21.40 ± 0.66 | 52.61 ± 0.55 | 99.40 ± 4.60cd | 207.17 ± 4.05cd | |
60 | 22.93 ± 0.75 | 52.57 ± 0.75 | 103.00 ± 2.26d | 232.00 ± 3.34cd | |
90 | 22.50 ± 0.74 | 52.57 ± 0.69 | 106.87 ± 3.27d | 237.00 ± 5.23d | |
120 | 21.42 ± 0.80 | 52.56 ± 0.54 | 108.33 ± 3.30 | 244.54 ± 4.14 | |
30 | 21.30 ± 0.37 | 52.30 ± 0.51 | 98.10 ± 2.25d | 205.36 ± 3.14d | |
60 | 22.57 ± 0.46 | 51.92 ± 0.63 | 100.33 ± 1.88 | 225.97 ± 2.20d | |
90 | 21.74 ± 0.39 | 51.87 ± 0.41 | 102.71 ± 2.38 | 231.57 ± 3.32d | |
120 | 21.56 ± 0.60 | 52.66 ± 0.49 | 107.22 ± 1.80 | 240.54 ± 2.77 | |
30 | 21.13 ± 0.64 | 52.34 ± 0.46 | 90.16 ± 4.53a* | 193.50 ± 4.41a* | |
60 | 22.90 ± 0.74 | 52.11 ± 0.71 | 97.67 ± 1.82 | 217.33 ± 6.63a* | |
90 | 22.45 ± 0.73 | 51.76 ± 0.52 | 100.67 ± 3.70 | 229.67 ± 9.83 | |
120 | 22.31 ± 0.61 | 52.08 ± 0.48 | 104.77 ± 2.47 | 237.38 ± 5.11 | |
30 | 20.27 ± 0.44 | 52.18 ± 0.54 | 87.83 ± 2.90ab** | 188.15 ± 2.89ab** | |
60 | 22.68 ± 0.52 | 51.69 ± 0.33 | 96.03 ± 2.50a* | 209.63 ± 2.10ab** | |
90 | 21.86 ± 0.65 | 51.55 ± 0.52 | 98.33 ± 3.29ab* | 222.33 ± 2.34ab* | |
120 | 22.05 ± 0.58 | 51.81 ± 0.50 | 102.36 ± 2.44 | 235.81 ± 2.02 | |
Different letters indicates significant (p<0.05) differences between groups.
* Significant difference between control and treatment groups. *p<0.5, **p<0.01, ***p<0.001
Comparison of mean ± SEM number of atretic follicles and corpus luteum in control and neonatal lead-treated offspring Wistar rats during different stages of postnatal development. *p<0.05, **p<0.01 and ***p<0.001.
Histological sections of ovarian follicles in ovary of offspring Wistar rats at 60 days of age in control group (hematoxyline & eosin); primordial (A) (scale bar: 20 μm), primary (B) (scale bar: 100 μm), secondary (C) (scale bar: 100 μm) and antral (D) (scale bar: 100 μm) follicles.
Histological sections of ovarian follicles in ovary of offspring Wistar rats at 60 days of age in control group (hematoxyline & eosin); primordial (A) (scale bar: 20 μm), primary (B) (scale bar: 100 μm), secondary (C) (scale bar: 100 μm) and antral (D) (scale bar: 100 μm) follicles.
Significant decreases were observed in the mean
numbers of primary, secondary and antral follicles
at 30 days of age in 100 (p<0.05) and 300 (p<0.01)
mg/L/day dose groups and at 60 (p<0.05) days of
age in the 300 mg/L/day dose group in comparison
with the control group (
In addition, the means of secondary and antral follicle
diameters decreased significantly (p<0.05) in
the 100 mg/L/day dose group at 30 (p<0.05) days
of age and in the 300 mg/L/day dose group at 30
(p<0.01), 60 and 90 (p<0.05) days of age in comparison
with the control group (
There were significant increases in the mean
number of atretic follicles at 30 days of age in
100 (p<0.01) and 300 (p<0.001) mg/L/day dose
groups and at 60 (p<0.01) and 90 (p<0.05) days
of postnatal development in the 300 mg/L/day
dose group in comparison with the control group
(Figes
Significant decreases were seen in the mean
number of corpora lutea in 100 (p<0.05) and 300
(p<0.01) mg/L/day dose groups at 60 days of age
in comparison with the control group (
During recent decades concerns have been raised
about human infertility that might stem from exposure
to environmental contamination. Exposure
to environmental contamination prior and after the
initiation of pregnancy, and during the early period
of postnatal development could affect reproductive
efficacy of offspring (
Mean body weight of the offspring decreased significantly
in neonatal lead treatment of Wistar rats,
particularly in the 300 mg/L/day dose group. Ronis
et al. observed that lead exposure during pregnancy
and lactation resulted in significant dose-responsive
decreases in birth weight and crown-to-rump
length in all litters of the treatment group (
Neonatal lead treatment caused dose-related reductions
of ovaries’ relative weights in offspring rats.
It seems that these reductions may be due to dosedependent
increases of atretic follicles, as well as
decreases of secondary and antral follicular diameters
in the ovaries of rats’ offspring. McGivern
et al. and El Feki et al. have shown that ovarian
weight reduces in offspring exposed maternally to
low levels of lead (
The present study showed that neonatal lead treatment
reduced the number of primary, secondary
and antral follicles in the ovaries of offspring rats,
particularly in the 300 mg/L/day dose group. Junaid
et al. showed that low lead acetate levels reduced
small and medium follicle numbers and high
levels resulted in fewer large follicles numbers in
mice (
McGivern et al. and El-Feki et al. found that maternal
exposure to low lead levels causes fewer
corpora lutea and abnormal estrous cycles in offspring
(
Overall, these data suggest that neonatal lead treatment
inhibits follicular development in the ovaries
of offspring in a dose-related manner. Ercal et al.
observed that chronic exposure to lead damaged
primordial and medium follicles and arrested follicular
development in Rhesus monkeys (
However, the present study showed no significant
differences in numbers of growing follicles and
corpora lutea at 90 and 120 days of age in the treatment
groups. Additionally, the mean numbers of
secondary and antral follicles, and ovarian weight
in the treatment groups normalized until 120 days
of age in comparison with 30 days of age. In this
regard, Mansouri and Abdennour have shown that
increase of exposure time to lead caused more toxic
effects to gametes (
Consequently, the present study shows that maternal lead acetate exposure during lactation affects prepubertal ovarian follicle development in a dose dependent manner, but ovarian parameters become better gradually during the postpubertal period.