1. Transgenerational (TG) Effects of Endocrine-Disrupting Chemicals
on Female Reproduction in Rats
Li Ching Sheng1, Arpita Bhurke, Aparna Zama, and Mehmet Uzumcu
Department of Animal Sciences, School of Environmental and Biological Sciences, and 1School of Pharmacy, Rutgers, The State University of New Jersey
Methods ResultsAbstract
Developmental exposure to endocrine-disrupting chemicals (EDCs) such as
bisphenol a (BPA), phthalates, and methoxychlor (MXC) can have long lasting effects
on reproductive health. The current study examined potential transgenerational (TG)
effects of such EDC exposures. Diethylhexyl phthalate (DEHP; 500 mg/kg), BPA (50
mg/kg), or MXC (75 mg/kg) were administered to timed-pregnant female rats (F0)
daily between embryonic day (E) 11 and 21. Once born, both male and female
offspring (F1) continued to be treated between postnatal day (PND) 0 and 7.
Synthetic estrogen diethylstilbestrol (DES; 0.1 μg/kg) was used as positive control.
Negative control rats received 1 ml/kg ethanol:oil (1:9) or dimethyl sulfoxide
(DMSO):oil (1:2) as vehicle. F1 females were bred with non-sibling treated males to
produce F2. In a similar manner, F2 females were mated with F2 males to produce
F3. Of note is that only the F1 generation was directly exposed to the EDCs.
Examination of various reproductive parameters suggests that MXC and DES caused
accelerated puberty in the F1 rats while phthalates caused late puberty in the F1. In
addition, DES caused a significantly reduced litter size. Lastly, MXC resulted in
irregular estrous cycle in the F1 rats. These effects were only observed in F1
generation and were absent in both F2 and F3. Further follicular composition and
gene expression analyses will be conducted on ovaries from adult littermates to
further examine potential TG effects. This study is important because it helps
explore the etiology of human health disorders linked to environmental causes.
Background
• EDCs are toxins in the environment that mimic certain essential hormones, such as
estrogen, and disrupt the endocrine functions in mammals. Studies have shown that
beyond a certain dosage, EDCs can cause cancers, infertility, pregnancy
complications, birth defects, and other disorders.
• Phthalate is a common plasticizer that contributes to the flexibility of plastics. It
can be found as a component in a variety of products including common household
products, medical devices, and personal care products. Most people can be exposed
to low levels of phthalate through air, water, or food that has been in contact with
phthalate-consisting containers. Recent studies have shown that phthalate exposure
disrupts the reproductive functions in male testis, and such effect is
transgenerational (Doyle et al., 2013 Biology of Reproduction).
• Diethylstilbestrol (DES) is a synthetic form of estrogen that was prescribed to
pregnant women in the 1940s to 1970s to prevent pregnancy complications such as
miscarriage and premature labor. However, women who were exposed to DES
showed increased risk of breast cancer. Their children also suffered from fertility
problems and pregnancy complications. Studies of their grandchildren are ongoing.
• Bisphenol A (BPA) is an EDC found in epoxy resins and polycarbohydrate
plastics. BPA has a variety of applications: the coating of food and beverage
packaging and of other metal products. Humans are exposed to BPA primarily
through diet since BPA can be leached into the food when in contact with high
temperature. Other sources of exposure such as air, dust, and water are possible.
• Methoxychlor (MXC) is an insecticide used against common pests. It is also
used as pesticides in agricultural farms. Humans can be exposed to MXC from
consuming food or water that has been contaminated with MXC. Farmers and other
agricultural workers are exposed primarily through inhaling or direct contact with
products that contain MXC. According to the Agency for Toxic Substances and
Diseases Registry, high level of exposure to MXC during pregnancy may increase
the likelihood of a miscarriage and cause other pregnancy complications.
• DES has been shown to cause pathologies in F2 males and females, an observation
was noted with MXC as well.
Objective
The purpose of this experiment is to examine whether there is a TG effect of the
following EDCs on the female reproductive functions in rats.
• Chemicals analyzed: diethylhexyl phthalate (DEHP; 500 mg/kg), diethylstilbestrol
(DES; 0.1 µg/kg), bisphenol A (BPA; 50 mg/kg), and methoxychlor (MXC; 75 mg/kg)
• Control groups: ethanol:oil (1:9) and dimethyl sulfoxide (DMSO):oil (1:2)
• Pregnant Fischer CDF rats (F0) were treated with phthalate, DES, BPA or MXC
between embryonic day (E) 11 and 21. Once born, the F1 pups were further treated
from postnatal day (PND) 0 to PND 7, as illustrated in figure 1. The control groups
received either the ethanol:corn oil (1:9) mixture or the DMSO:corn oil (1:2) mixture.
• The female rats are followed for their pubertal age and the regularity of their
estrous cycle. Pubertal age was determined by the appearance of vaginal opening,
and once opened, the estrous cycles were observed through vaginal cytology and
recorded. For every two female rats, one was kept for breeding while the other was
dissected between PND 50 and 60 to collect its ovaries, uteri, liver, and sera for
histology and measurement of hormones. The F1 female rats were bred to treated
male rats (also F1) to produce F2, and similarly, F2 rats were bred to produce F3.
However, only pregnant F0 rats and their F1 pups received the EDC treatment.
• Reproductive parameters analyzed:
 Litter size was determined by counting the pups on the day of birth and by
sexing the litters.
 The rats were weaned at PND 28, and their vaginal opening determines their
pubertal age.
 Estrous cycle was determined through vaginal cytology, which was performed
every morning in the duration of the experiment. As shown in figure 2, depending
on the cells present, the rats are classified as proestrus (P), estrus (E), or diestrus
(D). Proestrus is characterized by nucleated epithelial cells. Estrus mainly
consists of keratinized cells in the smear. Diestrus is determined by the presence
of white blood cells, or leukocytes. A normal estrous cycle lasts for 4-5 days. It
consists of one day of E followed by three days of D and then one day of P
(EDDDP), or sometimes without P (EDDD). Cycles that are shorter or longer than
that are abnormal.
Results
Hubscher et al. (2005)
Figure 2: Vaginal cytology.
Classification of different
stages of the estrous cycle
as observed under the
microscope.
Figure 4: Litter size. The offspring from each generation was counted at the respective date of
birth. To analyze the results, one-way ANOVA and t-test were performed. Data reveals that the
litter size of DES-treated F1 rats was significantly reduced. ** - P<0.01.
Figure 5: Abnormal cyclicity. The above graphs show the percent of abnormal cycle based on the
first three estrous cycles of the rats. A normal estrous cycle lasts for 4-5 days, with one day of
estrus followed by three days of diestrus and one day of proestrus (sometimes without proestrus).
Any cycle that is longer or shorter than that was considered abnormal. One-way ANOVA and t-test
were conducted. As shown, there was a significantly high percent of abnormal cyclicity in MXC-
treated F1 rats. ** - P<0.01.
Conclusions
• F1 rats were directly exposed to EDCs while F2 and F3 animals were not.
• Disruption of arrival of puberty has long-lasting effects on the reproductive life
span. Data show that MXC and DES resulted in accelerated puberty in the F1 rats
while phthalate caused delayed puberty. However, these effects were not seen in
either F2 or F3 generation.
• DES had a significant effect on F1 litter size while other EDCs did not. Once again,
this effect was absent in F2 and F3 generations. This means that MXC, BPA, and
phthalate do not affect litter size, and that DES, though affects the first generation,
has no transgenerational effect on litter size.
• Estrous cyclicity was disrupted only in the F1 generation. The same effect was not
found in F2 and F3 generations.
Future Directions/ Studies
The effects of EDCs on the female reproductive system are not only on the
hypothalamus-pituitary gonadal axis but also on the uteri and ovaries. These
reproductive tissues have been collected from the littermates of the animals
examined in the above study. They are currently being processed and prepared for
multiple analyses of protein, DNA and RNA. Histology to examine ovarian follicular
composition and immunohistochemistry to study gene expression patterns will be
conducted.
Reference
• Agency for Toxic Substances and Diseases Registry, (2002). Public health statement for methoxychlor. Retrieved from
website: http://www.atsdr.cdc.gov
• Environmental Protection Agency, (2007). Methoxychlor. Retrieved from website: http://www.epa.gov
• National Cancer Institute, (2011). Diethylstilbestrol (DES) and cancer. Retrieved from website: http://www.cancer.gov
• National Institute of Environmental and Health Sciences, (2013). Bisphenol A (BPA). Retrieved from website:
http://www.niehs.nih.gov
• National Library of Medicines, (2013). Phthalates. Retrieved from website: http://toxtown.nlm.nih.gov
• Zama, A. M., & Uzumcu, M. (2010). Epigenetic effects of endocrine-disrupting chemicals on female reproduction: an ovarian
perspective. Frontiers in neuroendocrinology, 31(4), 420-439.
Embryonic Day Postnatal Day
7.5 11 21 1 3 7 21 Adult 50-60
Germ cell lineage
specification
F0 dam and F1 fetus
exposure F1 exposure
Figure 1: Outline of experimental method
Critical ovarian
developmental processes
Reproductive
parameter analysis
Number of Female Rats
Control Groups Experimental Groups
DMSO Eth:Oil MXC DES BPA Phthalate
F1 13 15 16 20 23 18
F2 14 13 8 12 29 20
F3 12 12 8 10 29 14
Figure 3: Pubertal Age. Vaginal opening served as the sign of puberty. After weaning on the 28th
day, the offspring was observed daily for the appearance of vaginal opening. One-way ANOVA and
t-test were performed on the data. The results show that there was accelerated puberty in F1 MXC
and DES groups, and delayed puberty in F1 phthalate group. ** - P<0.01.