In Utero through Lactational Exposure to Ethinyl Estradiol Induces
Cleft Phallus and Delayed Ovarian Dysfunction in the Offspring
Masakuni Sawaki, Shuji Noda, Takako Muroi, Hideo Mitoma, Saori Takakura, Satoko Sakamoto, and Kanji Yamasaki
Chemicals Evaluation and Research Institute, Chemicals Assessment Center, Hita, Oita, Japan
Received March 30, 2003; accepted June 20, 2003
Most of the attention currently focused on endocrine-active
chemicals is directed to their effects on the development of off-
spring exposed to them in utero or during the neonatal period.
Pregnant Crj:CD(SD)IGS rats were given ethinyl estradiol (EE)
orally in doses of 0.5–50
g/kg/day from gestational day 7 to
postnatal day 18, and their offspring were examined for its effects.
Our previous study according to a similar protocol demonstrated
the occurrence of cleft phallus in the female offspring exposed to
50
g/kg of EE in utero and during the lactation period. The
present study was designed to assess (1) the reproducibility of the
induction of cleft phallus, (2) the fertility of female rats with cleft
phallus, and (3) whether any delayed effects, possibly delayed
anovulation, were induced. At 50
g/kg cleft phallus was observed
in almost all of the female offspring, and slight retardation of body
weight gain was detected in both sexes. At 15–17 weeks of age the
animals with cleft phallus could copulate and had fertility com-
parable to the control group. At 6 months of age, on the other
hand, 6/8 of the female offspring at 50
g/kg exhibited abnormal
cyclicity, including persistent estrus, and histological examination
revealed follicular cysts and absence of corpora lutea in the ovaries
of the rats with persistent estrus. These findings are consistent
with delayed anovulation syndrome. The results suggest that ob-
servation of cyclicity at 6 months old is able to detect possible
delayed ovarian dysfunction induced by perinatal exposure to
chemicals.
Key Words: ethinyl estradiol; delayed ovary dysfunction; cleft
phallus.
Numerous chemicals have been demonstrated to modify
endocrine functions of humans and wildlife (Colborn et al.,
1993; Danzo, 1997; McLachlan, 1993), and most of the atten-
tion that is currently focused on endocrine-active chemicals is
being directed at their effects on the development of the off-
spring exposed to such chemicals in utero or during the neo-
natal period. An in utero and lactational exposure method has
been employed to identify the developmental toxicities of
various chemicals (Awoniyi et al., 1998; Moore et al., 2001;
Mylchreest et al., 1998; Odum et al., 2002; You et al., 1998),
and “in utero through lactational exposure” protocols have
been proposed in the draft report by the United States Envi-
ronmental Protection Agency as a method of detecting effects
of prenatal and early postnatal exposure that would not be
detected as a result of pubertal or adult exposure (Tyl and
George, 2003).
In the previous study we conducted a preliminary trial of an
“in utero through lactational exposure” method using ethinyl
estradiol and discovered the occurrence of cleft phallus in only
the female offspring at a dose at which no major adverse
effects were induced (Sawaki et al., 2003). Excessive cleavage
of the urethral slit, occasionally associated with insufficient
raphe formation between the urethral orifice and vagina in
female rats, is referred to as “cleft phallus.” Cleft phallus has
been reported in female rats exposed in utero to 2,3,7,8-
tetrachlorodibenzo-p-dioxin and an androgen, testosterone pro-
pionate (Gray et al., 1997; Wolf et al., 2002), as well to
estrogens such as diethylstilbestrol (Henry and Miller, 1986;
Henry et al., 1984). This means that cleft phallus is induced by
disruption of the complicated process of the development of
the external genitalia during the critical period of their mor-
phogenesis, not simply by an estrogenic action. Although the
mechanisms of the development of the external genitalia have
not been well characterized, cleft phallus is considered to
represent a mild form of female rodent hypospadias (Gray et
al., 1997). In spite of the abnormality of the external genitalia,
the female offspring exhibited normal cyclicity, and no gross
or histopathological changes were detected in their internal
reproductive tracts (Sawaki et al., 2003). This phenomenon led
us to investigate whether the offspring with cleft phallus have
normal reproductive performance.
The study described in this report was similar to the in utero
through lactational study of exposure to ethinyl estradiol re-
ported previously (Sawaki et al., 2003) and was conducted to
confirm the reproducibility of the formation of cleft phallus as
well as to evaluate the reproductive performance of the off-
spring with cleft phallus. In addition, some of the offspring
were sacrificed at weaning or at 6 months of age. Sacrifice at
6 months of age was performed in view of the possibility of the
occurrence of “delayed anovulation syndrome,” a term pro-
1 To whom correspondence should be addressed at Chemicals Evaluation
and Research Institute, Chemicals Assessment Center, 3-822 Ishii-machi, Hita,
Oita 877-0061, Japan. E-mail: sawaki-masakuni@ceri.jp.
Toxicological Sciences 75(2), © Society of Toxicology 2003; all rights reserved.
TOXICOLOGICAL SCIENCES 75, 402–411 (2003)
DOI: 10.1093/toxsci/kfg180
402

posed by Gorski (Gorski, 1968) to describe the condition of
female rats neonatally exposed to low doses of testosterone
propionate that retain the ability to ovulate for a time after
puberty but eventually become anovulatory (Arai, 1971). De-
layed anovulation induced by neonatal exposure to the estro-
genic compound p-t-octylphenol has been documented (Yo-
shida et al., 2002).
MATERIALS AND METHODS
Chemicals. Ethinyl estradiol (EE, lot No. KSF1601, purity 99.0%) was
purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and olive
oil (Fujimi Pharmaceutical Co., Ltd., Osaka, Japan) was used as the vehicle.
The physical stability of EE was assessed with a Fourier transform infrared
spectrophotometer (FTS-135, Nippon Bio-Rad Laboratories K.K., Tokyo, Ja-
pan), and the stability, homogeneity, and concentration of each EE suspension
prepared for administration were confirmed by HPLC.
Animals and experimental design. Timed-pregnant Crj: CD
(SD) IGS
BR rats (Charles River Japan Inc., Shiga, Japan) were used in this study. They
were nulliparous, 11 weeks old, and at gestational day (GD) 1 on arrival
(vaginal plugs noted
GD 0). On GD 6, 10 dams each were randomly
assigned to four experimental groups by a body-weight-stratified randomiza-
tion method to minimize the variation in body weights between the groups, and
EE 0.5, 5, or 50
g/kg/day or vehicle alone, was administered to them by
gavage from GD 7 to postnatal day 18 (day 0
delivery day). Their body
weight at the time they were divided into groups ranged from 237.5 g to 304.7
g. Our previous study according to a similar protocol demonstrated cleft
phallus in the female offspring at 50
g/kg/day (Sawaki et al., 2003).
The pups were kept in polycarbonate pens (280 W  440 D  150 H mm,
Tokiwa Kagaku Kikai Co., Ltd., Tokyo, Japan) with the dams until weaning.
Bedding was SUNFLAKE
supplied by Charles River Japan Inc (Atsugi,
Japan). To maximize the uniformity of growth rates, at 4 days of age the litters
were culled randomly to four male and four female pups per dam. When the
number of pups of either sex was insufficient to obtain four offspring of each
sex, litter size was adjusted to eight by excluding pups of the sex with animals
in surplus but adjustment was not made when the number of pups per dam was
less than eight. Pups were weaned at 19 days of age, that is, on the day after
the final dose. Before weaning the pups in each group were randomly assigned
to three subgroups: a subgroup sacrificed at weaning, a subgroup sacrificed at
18–20 weeks of age subjected to caesarean section to evaluate the reproductive
performance of the offspring (caesarean section subgroup) and a subgroup
sacrificed at 6 months of age to examine the animals for delayed toxicities
(6-month-sacrifice subgroup). The reproductive organs of the subgroup sacri-
ficed at weaning were collected for a gene expression study, but the results
will not be reported here. The numbers of animals in each group are shown in
Table 1.
After weaning, offspring were housed individually in stainless steel wire-
mesh cages (260 mm W  380 mm D  180 mm H, Tokiwa Kagaku Kikai
Co., Ltd., Tokyo, Japan) in a controlled environment of 12 h light/12 h
darkness (lights on at 0700 h), 23  20C and 50  10% humidity with 10–15
air exchanges per h. The rats were fed an MF diet (certified pellet rodent diet,
Oriental Yeast Co., Tokyo, Japan) and given free access to tap water. All
animals were cared for according to the principles outlined in the guide for
animal experimentation prepared by the Japanese Association for Laboratory
Animal Science. At sacrifice, animals were euthanized by exsanguination from
the femoral arteries under deep anesthesia using diethyl ether. The culled pups
were euthanized only by deep anesthesia.
Endpoints. The general and nursing states and body weight of all dams
were monitored daily. The gestational period of each dam was calculated. On
the day of delivery, the numbers of live newborns and stillborns and the sex
ratio of the pups were recorded, and the pups were inspected for external
anomalies. The dams were euthanized and necropsied on the day after wean-
ing. The number of implantation sites in each uterus was counted by immersing
the incised uterus in ammonium sulfate solution, and the birth rate was
calculated (number of newborns/ number of implantation sites). The duration
of pregnancy, numbers of live newborns and of stillborns, and the sex ratios of
the live newborns were also recorded.
The general condition of the pups was observed daily. Body weight was
measured at 0, 4, 7, 12, 15, and 19 days of age and at weekly intervals
thereafter until sacrifice. The females were examined for vaginal opening daily
from 28 days of age, and the males for preputial separation from 35 days of age
until they occurred. Ano-genital distance was measured at 4 days of age. The
estrous cycle of the female offspring was evaluated based on vaginal cytology
performed for at least 14 days starting at 13 weeks in the caesarian section
subgroup and for at least 20 days prior to sacrifice in the 6-month-sacrifice
subgroup. Following criteria were used for determination of cyclicity: cyclicity
is normal when animals show 1–2 days length of estrus stage and cyclicity at
4–6 day intervals; persistent estrus is denoted by predominant appearance of
proestrus or estrus stages and occasionally other stages sporadically occur
without cyclicity.
In the caesarean section subgroup, the female animals were mated with the
males in the corresponding dose groups at 15–17 weeks of age. At gestational
day 20 (18–20 weeks of age), caesarean section was performed under ether
anesthesia. The numbers of females that copulated and conceived, and the
numbers of corpora lutea, implantation sites, and live fetuses of each female
were counted. The sex ratio of the fetuses was recorded, and they were
inspected for external anomalies. The male offspring were sacrificed at the
same time.
In the 6-month-sacrifice subgroup, the animals were killed in the diestrus
stage, and animals with persistent estrus were sacrificed on the 25th day after
start of the vaginal smear evaluation. The male offspring were also sacrificed
at 6 months of age. On the day of necropsy, the animals were weighed, and the
following organs were collected, weighed, and fixed with 10% neutral buffered
formalin: adrenals, hypophysis, testes (fixed with Bouin’s solution), epididy-
mides, seminal vesicles, ventral prostate, uterus, and ovaries. The vagina was
also collected and fixed. The hypophysis was weighed approximately 24 h after
fixation to avoid crushing during weighing because of its fragility. The ovary,
uterus, and vagina were prepared for hematoxylin and eosin staining and
examined under a light microscope.
To quantitate the extent of cleft phallus, the female external genitalia of the
female offspring in both the caesarean section and 6-month-sacrifice subgroups
were measured morphometrically with calipers having a reproducible precision
of 0.01 mm (Digimatic Caliper CD-15CP, Mitsutoyo Corp., Kanagawa, Ja-
pan). Morphometry included the length of the urethral slit across top of the
phallus and the vertical distance between the tip of the phallus and the urethral
orifice.
Statistical analyses. Body weight, organ weight, ano-genital distance, and
the morphometric data were analyzed by the Bartlett’s test for homogeneity of
TABLE 1
Number of Dams and Their Offspring after Weaning
Dose of EE (
g/kg/day)
Number of animals
VC 0.5 5 50
Dams 10 8 10 9
Weanlings (male/female) 40/40 28/28 36/35 31/33
Sacrificed after weaninga 13/13 7/7 9/9 8/9
Caesarian section 19/19 14/14 18/17 16/16
Sacrificed at 6 months of age 8/8 7/7 9/9 7/8
Note. EE, ethinyl estradiol; VC, vehicle control.
aNot reported in this paper.
403DELAYED OVARY DYSFUNCTION BY ESTROGEN IN RATS