Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herath, C. B.
Right arrow Articles by Taya, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herath, C. B.
Right arrow Articles by Taya, K.
Agricola
Right arrow Articles by Herath, C. B.
Right arrow Articles by Taya, K.
Biology of Reproduction 64, 1216-1224 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Exposure of Neonatal Female Rats to p-tert-Octylphenol Disrupts Afternoon Surges of Luteinizing Hormone, Follicle-Stimulating Hormone and Prolactin Secretion, and Interferes with Sexual Receptive Behavior in Adulthood1

Chandana B. Heratha, Gen Watanabea, Shin-ichi Katsudab, Midori Yoshidab, Akira K. Suzukic, and Kazuyoshi Taya2,,a

a Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan b Department of Pathology, Sasaki Institute, Tokyo 101-0062, Japan c Air Pollutants Health Effects Research Team, Environmental Risk Assessment Project, National Institute of Environmental Studies, Ibaraki 305-0053, Japan

ABSTRACT

The present study investigated the effects of exposure of neonatal female rats to p-tert-octylphenol (OP) on estrogen-induced afternoon surges of LH, FSH, and prolactin (PRL) secretion, and on sexual behavior in adulthood. After birth, one group of female Wistar rat pups received s.c. injections of OP (100 mg/kg body weight [BW]; OP group) dissolved in DMSO, while the control group received DMSO only (DMSO group). In order to make a qualitative comparison, a third group was injected with estradiol-17ß (500 µg/kg BW; estradiol group) dissolved in DMSO. Injections were given on Days 1, 3, 5, 7, 9, 11, 13, and 15 of age. The rats from the OP and estradiol groups that were used for subsequent experiments were in persistent vaginal estrus. Spontaneous LH surge measured at Postnatal Days (PND) 78–81 was observed only in the DMSO group on the afternoon of the day of proestrus. At PND 115, randomly selected rats from each of three treatment groups were bilaterally ovariectomized (ovx), and 8 days later, Silastic capsules containing estradiol-17ß were implanted under the skin. Estrogen implants stimulated afternoon surges of LH, FSH, and PRL for two consecutive days in the DMSO group, but not in the OP and estradiol groups. Rats from the OP and DMSO groups underwent ovx at PND 186, and 6 days later they were treated with a combination of estradiol benzoate s.c. (15 µg/kg BW) and progesterone s.c. (2 mg/kg BW) to test the lordosis reflex. In response to this hormone treatment and mounting stimulus delivered by the stud male rats, the OP-treated rats were less receptive compared with control DMSO-treated rats, and thus the lordosis quotient and lordosis rating were significantly (P < 0.05) reduced in the OP group compared with the DMSO group. Analysis of the area of the sexually dimorphic nucleus of the preoptic area of the brain revealed that the area of this nucleus was larger in the OP group than it was in control DMSO rats. We conclude that the exposure of neonatal female rats to higher doses of OP disrupts the cyclic release of LH, FSH, and PRL, and interferes with the display of sexual receptive behavior in adulthood.

environment, follicle-stimulating hormone, hypothalamus, luteinizing hormone, toxicology

INTRODUCTION

An environmental pollutant, p-tert-octylphenol (OP) [p-(1,1,3,3-tetramethylbutyl)-phenol], an endocrine-disrupting chemical, is an alkylphenolic compound derived as one of the major biodegradation products of nonionic surfactants, alkylphenol polyethoxylates (APEOs). These APEOs are released to the environment from the use of many chemicals such as detergents, paints, plastics, herbicides, and pesticides, and have become a major component in wastewater systems [1, 2]. Because of large quantities of alkylphenols are manufactured every year to provide substrate for the production of APEOs, they continue to contaminate the environment along with their respective alkylphenols.

OP shows marked similarities to that of natural estrogens [2, 3] and has been known to have a higher estrogenic potency compared with other alkylphenols of the same class of chemicals [4], and is 10-3 to 10-4 less estrogenic than that of the most potent natural estrogen, estradiol-17ß [2]. The possible link between these environmental pollutants and poor health of humans and animals has been a contentious issue among scientists in recent years, and the issue has become of wide public concern [5, 6]. In male rats, oral ingestion of high concentrations of OP can lead to an increased accumulation of the chemical in various tissues [4], suggesting that these alkylphenolic compounds are capable of bioaccumulation in mammalian cells, and thus, they contribute substantially to the environmental estrogen pool.

In the normal cyclic rat, spontaneous surges of LH, FSH, and prolactin (PRL) occur in the afternoon of the day of proestrus, and these afternoon surges can also be induced by administration of exogenous estrogen or implantation of estrogen-containing canula in certain brain nuclei in ovariectomized rats [7–13]. Lordosis reflex is one component of sexual receptivity in female rats, and on being mounted by a male, the receptive female arches her back, thus elevating her head and perineum for possible intromission. These sexually dimorphic brain functions in the female rat are not determined by genetic factors directly, but are established during the early postnatal life in the absence of significant levels of gonadal steroids [14, 15]. In male neonates, testosterone secreted by the testis is lightly bound by fetal estrogen-binding protein; this allows free testosterone to pass into the brain, where it is aromatized to estradiol and subsequently masculinizes the brain [15, 16]. On the other hand, in female neonates, fetal estrogen-binding protein prevents plasma estrogen from entering the brain [17] so that the female is able to retain her feminine characteristics.

When female neonates are administered with testosterone or estrogen during the first few days of life, the animals, as adults, enter persistent vaginal estrus with loss of estrous cyclicity and the cyclic release of pituitary gonadotropin [18–20]. These androgenized (defeminized) female rats or hamsters, ovariectomized in adulthood, fail to respond with an afternoon surge of LH, FSH, and PRL following exogenous steroid administration [8, 21–24]. Similarly, an adult female rat does not exhibit lordosis reflex if she had been treated with testosterone in the first few days after birth [25–26].

Previous reports indicate that administration of OP into adult male rats can cause deleterious effects on the release of pituitary and gonadal hormones, and on sperm production [27–29]. Similarly, direct or indirect exposure of neonatal male rats to nonylphenol, another alkylphenolic compound with weak estrogenic activity, has been reported to adversely affect various reproductive parameters, including a decrease in the size of the testes and male accessory glands that were associated with a reduced sperm count and sperm motility [30, 31]. However, to our knowledge, the effects of administration of OP into neonatal female rats on estrogen-induced daily surges of pituitary gonadotropins and PRL in rats ovariectomized as adults, and also on the sexual receptive behavior during adulthood, have not been investigated. Therefore, in the present study, we investigated estradiol-17ß-induced afternoon surges of LH, FSH, and PRL, and estrogen/progesterone combination-induced sexual behavior in ovariectomized adult female Wistar rats, which had been exposed to OP neonatally during the critical period of brain sexual differentiation. In order to make a qualitative comparison, a group of neonatal rat pups was exposed to estradiol-17ß using the same protocol. Stereotaxic coordinates of the brain were also obtained to examine the sexually dimorphic nucleus of the preoptic area (SDN-POA) because the size of this nucleus of the rat brain has been shown to be influenced by perinatal hormonal environment [14].

MATERIALS AND METHODS

Animals

Three-mo-old female Wistar rats were purchased from Imamichi Institute for Animal Reproduction, Ibaraki, Japan. They were housed in metal cages and maintained in a room with controlled illumination (14L:10D, lights-on at 0500 h) and temperature (22°C to 24°C), with free access to commercial pellets and given tap water ad libitum. They were checked daily for estrous cyclicity by examining vaginal smears and rats with regular 4-day estrous cycles were selected for mating. The selected female rats were mated with stud male rats that were selected from our own colony in our animal facility. After mating females were housed in groups (4–5 rats per cage) until Day 15 of pregnancy, and then they were housed individually until the day of parturition. After birth, female rat pups were separated from male rat pups and the female pups were mixed together. Eight to nine pups, randomly chosen, were placed with a mother rat so that the pups may or may not have been placed with their genetic mother. All procedures were carried out in accordance with the guidelines established by the Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, for use of laboratory animals.

Chemicals

The OP (minimum 95% pure) and dimethyl sulfoxide (DMSO) were purchased from Wako (Wako Pure Chemical Industries, Osaka, Japan, lot ACK4415). Estradiol-17ß was purchased from Sigma Chemical Co. (St. Louis, MO; lot 10H0065). Known amounts of OP and estradiol-17ß were dissolved in DMSO and these stock vials were then sealed and kept at room temperature during the experiments. Protective clothing such as gloves and apron were used during handling of these chemicals.

Experimental Protocol

All the pups that were used for the experiments were born within a period of 14 h. The morning the female rat pups were first seen was designated as Day 1 after birth. The dosage of OP and estradiol-17ß used for injections in the present experiments was 100 mg/kg body weight (BW) and 500 µg/kg BW, respectively. Fourteen to 20 pups were assigned per treatment group. The rat pups were given a total of eight injections starting at Day 1 after birth. This means that all the pups received their first injection within the first 24 h after they were born. The next seven injections were carried out on Days 3, 5, 7, 9, 11, 13, and 15 after birth. The pups in each treatment group received s.c. injections of either 0.025 ml DMSO alone (DMSO group = control group) or 0.025 ml DMSO containing appropriate amount of OP (OP group) or estradiol-17ß (estradiol group), and the injection site was covered with a drop of collodion to prevent the chemicals from leaking out. All the pups were weighed daily during the injection period and the amount of OP or estradiol-17ß to be injected into each pup was calculated accordingly. The dosage of OP was chosen on the basis of our following preliminary observations [32]. Briefly, different doses of OP ranging from 12.5 mg up to 50 mg/kg BW, employed for the same duration as in the present study, did not induce persistent vaginal estrus in any of the rats during adulthood. A single injection of 100 mg/kg BW given on Day 1 after birth neither induced a state of persistent estrus, although about 65% of the rats entered persistent vaginal estrus when they were given the same dose for 3 days (i.e., on Days 1, 3, and 5 after birth). However, injections of this dose (100 mg/kg BW) from Day 1 to Day 15 after birth, which comprised eight injections as in the present study, consistently induced persistent vaginal estrus in all of the rats during adulthood. We interpreted these data to suggest that this regimen of OP was effective in defeminizing the brain and the effect or effects may probably be same as that obtained by administration of steroids [15]. Although the estrogenic potency of OP is about 1000 times less than that of estradiol-17ß, the molarity of estradiol-17ß that we used in the present study was approximately 260 times less than that of the OP. A comparatively higher dose of estradiol-17ß was chosen on the basis that all of estradiol-17ß injected may not be available due to binding to fetal estrogen-binding protein [17]. After completion of the treatment period the rat pups were weaned at 26 days after birth. They were then housed in separate cages with 5–6 rats per cage and given commercial pellets and tap water ad libitum.

Sampling Procedure

Vaginal opening and vaginal cytology After completion of the injection period, the rats were checked daily for vaginal opening. Following the attainment of puberty, vaginal smears were taken daily to check the cyclicity until the age of 110 days, and thereafter, the smears were checked a few days before the commencement of a particular experiment.

Spontaneous LH Surge

Starting at Postnatal Day (PND) 78, four rats from each treatment group were used to collect blood samples (1 ml) at 1700 h for four consecutive days to examine the spontaneous preovulatory LH surge. The rats from the DMSO group were synchronous in their estrous cyclicity and Day 1 of sampling represented diestrus Day 1, judged by vaginal cytology. The rats from the OP and estradiol groups were in persistent vaginal estrus as exhibited by the presence of epithelial cells, cornified cells, or both. Blood was drawn from the jugular vein under light ether anesthesia, and following the withdrawal of first blood sample on Day 1, blood was replaced with an equal volume of sterile heparinized saline (100 IU/ml 0.9% NaCl) injected through the same vein. For subsequent replacements, after harvesting plasma blood cells were reconstituted with sterile heparinized saline to the original volume and kept at 4°C, and mixed and warmed to body temperature before they were injected back into the same rat after the next blood sample was taken.

Estradiol-17ß-Induced LH, FSH, and PRL Surges in Ovariectomized Rats

At PND 107, a randomly selected group of OP (n = 7)-, estradiol-17ß (n = 5)-, and DMSO (n = 5)-treated rats underwent bilateral ovariectomy (ovx) under light ether anesthesia. After completion of the surgeries, the rats were taken back into the animal room and housed 4–5 rats per cage. Two-centimeter segments of Silastic tubing (inside diameter, 1.57 mm; outside diameter, 3.18 mm; Dow Corning Corp., Midland, MI) were filled with a solution of sesame oil containing estradiol-17ß dissolved at a concentration of 1 mg/ml, and the ends of the tubing were sealed with GC EXAFLEX adhesive. Eight days after ovariectomy (i.e., at PND 115), two Silastic capsules each 2 cm in length were inserted under the skin through a small dorsal midline incision made in the neck while the animals were lightly anesthetized with ether, and pushed toward the tail. The skin incision was closed with synthetic suture material (Matuda Medical Industries, Tokyo, Japan). Implantation of capsules containing estradiol-17ß was carried out at 0900–1000 h (designated as Day 0).

Blood sampling was performed at 1100 h and 1700 h for three consecutive days (i.e., Day 1, Day 2, and Day 3), with the first sample (i.e., at 1100 h on Day 1) being collected 25–26 h after implantation of capsules containing estradiol-17ß. On Day 3, however, the number of rats of the OP group available for blood sampling was four and three at 1100 h and 1700 h, respectively. Blood (1 ml) was drawn from the jugular vein under light ether anesthesia. Blood replacement was carried out as described in the previous section.

Behavioral Testing

At PND 186, bilateral ovx was performed under light ether anesthesia in DMSO- (n = 4) and OP- (n = 5) treated rats. Six days after ovx, all the rats received s.c. injections of estradiol benzoate (Sigma) 15 µg/kg BW dissolved in DMSO. Twenty-four h later they were given s.c. injections of progesterone (Sigma) at a dose of 2 mg/kg BW dissolved in DMSO, and the behavioral testing began 4 h after progesterone treatment. This treatment regime is shown to induce receptive behavior in ovariectomized females in response to stimulus males [33].

A dim red light (60 W) provided illumination during the testing period (i.e., between 1400 h and 1700 h) and the cage used was specially designed for testing the sexual behavior of rats, with wood shavings on the floor. Two sexually experienced stud male rats were used during the testing period, which consisted of 10–15 mounts. Sexual receptivity was quantified by lordosis quotient (LQ, lordosis responses/number of mounts x 100%) and lordosis rating (LR, average degree of arching of the back using the following criteria: 1, slight; 2, moderate; 3, strong dorsiflexion) [33]. Proceptive behavior was also recorded by noting ear-wiggling and hopping.

Brain Tissue Preparation

Ovariectomized rats that had been used for testing sexual behavior were used for this experiment. Around PND 200, rats from the DMSO and OP groups were anesthetized with ether and perfused with NaCl (0.9% w/v) solution to remove blood, and then with 4% paraformaldehyde (PFA) to fix the brains. Brains were immediately removed from the skulls and left in the same fixative for a further 3–4 days. They were then transferred to 15% sucrose (in 0.05 M PBS pH 7.4), incubated for 24 h at 4°C, followed by a further 24-h incubation in 30% sucrose. After incubation in 30% sucrose, stereotaxic coordinates (50 µm) in the Paxinos and Watson plane [34] were obtained by sectioning the frozen brains at -20°C using Microtome Cryostat (DAMON, International Equipment Company, Needham Heights, MA). Sections were mounted onto gelatin-coated slides (0.5% gelatin type A, Sigma; with 0.05% chromium potassium sulfate, Kanto Chemical Co. Inc., Tokyo, Japan), dried overnight at 32°C, and stained with 0.1% thionine, and coverslips were placed over them. Tracings of the area of the SDN-POA were made by an independent investigator who had no knowledge of treatment, using the National Institutes of Health (NIH) Image computer package (version 1.55, NIH, Bethesda, MD). The NIH Image values were obtained for two brain sections for each animal and averaged before the statistical analyses were performed.

Blood Processing

All blood samples were collected either into plastic tubes containing heparin (15 IU/ml blood) or into heparinized syringes as anticoagulant to prevent clotting. The samples were stored in ice and centrifuged at 3300 x g for 15 min at 4°C immediately after completing the experiment. The resulting plasma was decanted and stored at -20°C until assayed for plasma LH, FSH, and PRL concentrations.

Radioimmunoassays

Plasma concentrations of LH, FSH, and PRL were measured using National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) radioimmunoassay kits (Baltimore, MD) for rat LH, FSH, and PRL. The antiserum used was anti-rat LH (S-10), anti-rat FSH (S-11), and anti-rat PRL (S-9). Intraassay and interassay coefficients of variation were 5.4% and 6.9% for LH, 4.8% and 11.4% for FSH, and 5.7% and 12.2 for PRL, respectively.

Statistics

Statistical significance between the means of treatment groups was determined using ANOVA and Duncan multiple range test. Mann-Whitney U-test was used to evaluate the sexual receptivity data [35]. Data are presented as least square means ± SEM. A P value < 0.05 was considered significant. All statistical analyses except that for sexual receptivity data were carried out using the SAS computer package [36].

RESULTS

Vaginal Opening and Vaginal Cytology

Age at vaginal opening was significantly (P < 0.001) shorter in the rats of the OP and estradiol groups than that in the DMSO group and the values were 17.1 ± 0.2, 17 ± 0.3, and 32.9 ± 0.2 days for OP, estradiol, and DMSO groups, respectively. Vaginal smear checking revealed that all of the rats in the estradiol group entered persistent vaginal estrus at around PND 38, whereas the rats in the OP group had irregular cycles until around PND 65, at which time a majority of them (10 out of 14) entered persistent vaginal estrus. Two more rats in this group entered persistent vaginal estrus each at PND 77 and 79. In comparison, the rats in the DMSO group showed normal cyclicity with regularly 4-day cycles following puberty.

Spontaneous LH Surge

Because rats in our colony have 4-day regular estrous cycles, blood sampling at 1700 h was performed only for four consecutive days to examine the spontaneous LH surge, starting on Day 1 of diestrus through to the day of estrus (Day 4) of the DMSO-treated rats. The overall effects due to treatment, day, and hour were all significant (P < 0.001). Plasma LH concentrations in all treatment groups during 4-day sampling period are shown in Figure 1. At Day 1 (diestrus Day 1 for the DMSO-treated rats), plasma LH concentrations were below 4 ng/ml in all treatment groups, though the OP group had small but significantly (P < 0.05) elevated LH levels compared with that of the DMSO group. At Day 2 (diestrus Day 2 for the DMSO-treated rats), LH concentrations were not different between groups and the levels were below 0.5 ng/ml in all treatment groups. However, at Day 3, which was also the day of proestrus in rats in the DMSO-treatment group, LH concentrations in the DMSO group rose to high levels, which was more than a 40-fold increase from the levels observed in the preceding day. This proestrus afternoon LH surge occurred only in the DMSO group, but not in the OP or estradiol groups. The increases in LH levels over that seen in the preceding day were less than twofold and fourfold in the rats in the OP and estradiol groups, respectively. The levels in the OP and estradiol groups were significantly (P < 0.001) lower compared with the surge levels in the DMSO group. At Day 4, which was also the day of estrus in the rats of the DMSO group, LH levels fell below 0.5 ng/ml in the DMSO group, whereas there were no changes in plasma LH levels in the OP and estradiol groups.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 1. Spontaneous preovulatory LH surge measured in female Wistar rats at 1700 h during a 4-day period between PND 78 and 81. Day 1 in the figure corresponds to the day of diestrus 1 of DMSO-treated control rats (DMSO group). The LH surge occurred on the afternoon of the day of proestrus (Day 3) in rats in the DMSO, but not in the OP or estradiol group that were treated with p-tert-octylphenol (100 mg/kg BW) or estradiol-17ß (500 µg/kg BW), respectively, on Days 1, 3, 5, 7, 9, 11, 13, and 15 after birth. Each bar represents the mean ± SEM concentration of plasma LH from four rats per treatment group. ***P < 0.001, Significance of differences between the DMSO and OP or estradiol groups

Estradiol-17ß-Induced LH, FSH, and PRL Surges in Ovariectomized Rats

The experiment was performed 8 days after ovx. At 0900–1000 h on the 8th day after ovx, Silastic capsules containing estradiol-17ß were implanted s.c. and blood sampling commenced from the following day and continued for 3 consecutive days with two samples per day (i.e., at 1100 h and 1700 h).

Plasma LH

The overall effects due to treatment, hour, and the interaction effects between treatment and day, treatment and hour, and treatment, day, and hour were all significant (P < 0.001). Furthermore, the overall effects due to day, and the interaction between day and hour were also significant (P < 0.01). At 1100 h on Day 1, plasma LH concentrations were below 1 ng/ml in all treatment groups and there was no difference in LH levels between the groups (Fig. 2A). At 1700 h there was a small increase in LH levels in the DMSO group, although it was not significant. The afternoon LH levels in the OP and estradiol groups were similar to that in the morning. At 1100 h on Day 2, LH levels in the DMSO group decreased to the levels that were observed in both OP and estradiol groups. However, at 1700 h of that day, LH surge occurred in the DMSO group. A more than 10-fold increase in LH levels from preceding levels at 1100 h in rats of the DMSO group was significantly (P < 0.001) higher than that in the OP and estradiol groups in which the levels were approximately the same as those observed in the preceding sampling time (i.e., at 1100 h). The LH levels were below 0.5 ng/ml at 1100 h on Day 3 and were approximately the same in the three groups. Again at 1700 h, plasma LH in the DMSO group rose to significantly (P < 0.05) high levels from its morning levels at 1100 h, but the magnitude of this second surge was smaller (P < 0.001) than that of the first LH surge, which had occurred at 1700 h on Day 2. The elevated LH levels in the DMSO group were significantly (P < 0.05) higher than those in the OP and estradiol groups in which afternoon LH levels were approximately similar to those observed at 1100 h.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2. Estrogen implant-induced afternoon surges of LH (A), FSH (B), and PRL (C) secretion in ovariectomized female Wistar rats treated neonatally with p-tert-octylphenol (OP group), estradiol-17ß (estradiol group), and DMSO alone (DMSO group = control rats). For details of neonatal treatment schedule, see Figure 1. The rats were ovariectomized at PND 107 and 8 days later, Silastic capsules containing estradiol-17ß were implanted under the skin. Blood samples were collected at 1100 h and 1700 h for three consecutive days, starting from the day following estrogen implantation. Each bar represents the mean ± SEM concentration of plasma LH (A), FSH (B), and PRL (C) from three to seven rats per treatment group. In A–C, ***P < 0.001; in A, *P < 0.05; in B, acP < 0.001, abP < 0.05, significance of differences between the DMSO and OP or estradiol groups. In C, aP < 0.10, tended to be different from the estradiol group

Plasma FSH

The overall effects due to treatment (P < 0.001) and day, hour, and the interaction effects between treatment and day, treatment and hour, and treatment, day, and hour (P < 0.05) were all significant. At 1100 h on Day 1, plasma FSH concentrations in the DMSO group were not different from those of the OP group, but were significantly (P < 0.05) higher than those of the estradiol group (Fig. 2B). There were no significant changes in FSH levels in either of the treatment groups measured at 1700 h on the same day, but FSH levels were significantly (P < 0.05) lower in the estradiol group than in the DMSO and OP groups. However, FSH fell significantly (P < 0.05) to low levels in the OP group and tended to be decreased (P < 0.10) in the DMSO and estradiol groups when measured at 1100 h on Day 2, and the levels were significantly (P < 0.05) higher in the DMSO group than in the estradiol group. At 1700 h on Day 2, however, plasma FSH in the DMSO group rose significantly (P < 0.001) to high levels from the levels observed at 1100 h, and the levels were significantly (P < 0.001) higher than those of the OP and estradiol groups at this time. This FSH surge occurred only in the DMSO group, but not in the OP or estradiol groups. There was no change in FSH levels of the OP group, although a small and insignificant increase was seen in the estradiol group. Plasma FSH in the DMSO group fell significantly (P < 0.001) to low levels the next morning at 1100 h (Day 3). But the levels in the OP and estradiol groups were not changed from their preceding levels. The rats in the DMSO group had significantly higher FSH levels compared with those in the OP (P < 0.05) and estradiol (P < 0.001) groups. A second FSH surge occurred at 1700 h on Day 3, which was significantly (P < 0.05) higher than the levels observed at 1100 h. The FSH levels achieved by this second surge in the DMSO group were significantly (P < 0.001) higher than the levels in the OP and estradiol groups, although the magnitude of this surge in the DMSO group was smaller (P < 0.05) than that of the first surge, which occurred at 1700 h on Day 2.

Plasma PRL

The overall effects due to day, hour, and the interaction between treatment and hour were significant (P < 0.01), although the overall treatment effect was not significant. At 1100 h on Day 1, plasma PRL concentrations were below 20 ng/ml in all treatment groups, and the levels were not different between the three groups (Fig. 2C). There were no significant changes in PRL levels in either of the treatment groups measured at 1700 h on the same day, although small increases in PRL levels were seen in all groups. The PRL levels were not changed when measured at 1100 h on Day 2. At 1700 h on Day 2, however, plasma PRL in the DMSO group rose significantly (P < 0.05) to high levels from its preceding levels observed at 1100 h, although the levels achieved by this surge in the DMSO group tended (P < 0.10) to be higher than those in the estradiol group only. Although a significant PRL surge had occurred in the rats of the DMSO group, because of relatively high levels of PRL in the rats of the OP and estradiol groups, the differences between groups at 1700 h did not reach significant levels. However, this PRL surge occurred only in the DMSO group, but not in the OP or estradiol groups. Thereafter, PRL in the DMSO group fell significantly (P < 0.05) to low levels on the next morning at 1100 h (Day 3). But PRL in the OP and estradiol groups were not changed from their preceding levels. A second and relatively the largest PRL surge occurred in the DMSO group at 1700 h on Day 3, and the levels were significantly (P < 0.001) higher than those observed at 1100 h. The PRL levels achieved by this second surge in the DMSO group were significantly (P < 0.001) higher than the levels in the OP and estradiol groups, and unlike LH and FSH, the magnitude of the second PRL surge in the DMSO group was larger (P < 0.01) than that of the first surge that occurred at 1700 h on Day 2.

Behavioral Testing

One of the four female rats in the DMSO group was restricted to only seven mounts because of the time limit, whereas one of the five female rats in the OP group had 19 mounts due to a continuous mounting by both stud rats within a very short time.

All the rats in the DMSO group exhibited a highly positive receptive behavior, and thus, they displayed lordosis reflex in response to mounting by the stud male rats following estradiol benzoate/progesterone treatment. Because these rats exhibited lordosis response to every single mounting, the LQ was 100% for the DMSO group. In contrast, the rats in the OP group were less receptive and one of the rats did not display a single lordosis reflex in response to 10 mounts by the stud male rats. The LQ in the OP group was significantly (P < 0.05) reduced compared with the DMSO group (Fig. 3A). The OP rats displayed lordosis reflex; however, these reflexes were either slight or moderate and rarely achieved rating "3", which was assigned for strong dorsiflexion, and thus the mean LR was significantly (P < 0.05) reduced in the OP group than in the DMSO group (Fig. 3B). Proceptive behavior such as ear-wiggling and hopping were frequently observed in rats in the DMSO group, whereas the frequency of these proceptive behaviors were comparatively low in the OP group (data not shown).



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 3. Sexual receptive behavior (A and B), and the area of the sexually dimorphic nucleus of the preoptic area (SDN-POA) (C) in female Wistar rats treated neonatally with either OP or DMSO alone (DMSO group = control rats). For details of neonatal treatment schedule, see Figure 1. The rats were ovariectomized at PND 186 and 6 days later they were injected with estradiol benzoate s.c. (15 µg/kg BW), followed by progesterone s.c. (2 mg/kg BW) 24 h later. Four h after progesterone injection sexual receptivity was examined using two male stud rats. Each bar represents the mean ± SEM lordosis quotient (A), lordosis rating (B), or relative area of the SDN-POA (C) from four to five rats per treatment group. The area of the SDN-POA was examined in rats aged approximately 200 days. Note that the relative values are given for comparison between the two groups. *P < 0.05, significance of difference between the DMSO and the OP group

SDN-POA

The area of the SDN-POA was significantly (P < 0.05) larger in the OP group than in the DMSO group (Fig. 3C). The photographs shown in Figure 4 are representative sections of the brains obtained from a female rat of the DMSO (Fig. 4A) and OP (Fig. 4B) groups.



View larger version (114K):
[in this window]
[in a new window]
  		FIG. 4. Photomicrographs of thionine-stained histological sections (50 µm) through the rat brain at the SDN-POA of a female control rat treated with DMSO (A) and a female treated with p-tert-octylphenol (B) neonatally. For details of neonatal treatment schedule, see Figure 1. Brains were obtained from rats aged around 200 days. Note: the enlargement of the SDN-POA of the females treated with p-tert-octylphenol. Magnification x25. f, Fornix; oc, optic chiasm; so, supraoptic nucleus; v, third ventricle

DISCUSSION

This is the first report to show that exposure of neonatal rats to OP can adversely affect the surge secretion of LH, FSH, and PRL, and the display of sexual receptive behavior in adulthood. In line with this, it appears that like steroids, OP is capable of interfering with sexual differentiation of the brain, which normally occurs within the first 2 wk of life in rats.

The organization of the sexually dimorphic brain areas during early postnatal life has been shown to be a prerequisite in order to regulate afternoon surges of LH, FSH, and PRL in female rats during adulthood [8, 14, 15, 18–24]. In female neonates, this process of brain sexual differentiation occurs in the absence of significant levels of steroids. In the normal cyclic rat, spontaneous surges of LH, FSH, and PRL occur in the afternoon of the day of proestrus, and the characteristic circadian rhythmicity of these surges are dependent on the rise of estrogen secreted between diestrus Day 2 and proestrus [7, 9, 10]. This rising estrogen in conjunction with a circadian neural signal originating from the suprachiasmatic nucleus (SCN) of the hypothalamus [37] facilitates an increased release of GnRH from its neurons in the preoptic area (POA), causing gonadotropin surges [38, 39]. If female neonates were administered with steroid hormones during the first 2 wk of life, then as adults, the defeminized female rats do not show cyclic release of pituitary gonadotropins, nor do they respond with gonadotropin surges to exogenous estrogen following ovariectomy [21–23]. In the present study, the spontaneous preovulatory LH surge and daily surges of LH secretion following implantation of estradiol capsules were absent in the rats treated neonatally with either OP or estradiol-17ß. These data are consistent with those obtained by Harlan and Gorski [21], who demonstrated that female rats androgenized using testosterone propionate and ovariectomized as adults showed a significantly lowered afternoon LH surge compared with control rats in response to administration of exogenous estrogen alone or coupled with progesterone. Furthermore, proportionate differences in the magnitude of LH responses between the DMSO and OP or estradiol groups were about the same as those reported in ovariectomized hamsters that were androgenized neonatally using testosterone propionate [24].

In the present study, control rats treated neonatally with DMSO showed the first significant afternoon LH surge 56 h after, and a second surge with reduced magnitude 80 h after implantation of estrogen capsules. Others have also reported estradiol-induced LH surge in response to implantation for 12 days or a single injection immediately following or 15 days after ovariectomy [9, 10]. However, in the present study, a lack of afternoon LH surge on Day 1 after the capsule implantation may probably be due either to low levels of plasma estradiol-17ß because other investigators have found the best surge response on Day 1 after implantation of estradiol-17ß capsules [9, 10] or insufficient estrogen priming effect [40]. Similarly, a relatively low magnitude of LH response on Day 2 following implantation of ovx DMSO rats compared with that in proestrus ovary-intact rats is probably due to lower estradiol-17ß levels, to a lack of progesterone in ovx rats, or both [11]. A comparison of FSH levels between the DMSO and OP or estradiol groups reveals that FSH levels in the OP or estradiol groups were about one-third to one-fourth of the levels of the FSH surge found in the DMSO group. Donham and Stetson [24] reported about the same ratios of the responses of estrogen-induced afternoon FSH surges between controls and androgenized hamsters ovariectomized as adults.

Rats treated with DMSO neonatally and ovariectomized as adults exhibited afternoon surges of PRL in response to estrogen implantation. However, afternoon surges of PRL secretion were not produced in response to estrogen implantation in the rats treated neonatally with either OP or estradiol. The present data are comparable with those obtained by Neill [8], who showed that female rat pups injected with testosterone propionate on the fifth day after birth and ovariectomized as adults did not produce a surge of PRL secretion in response to estradiol benzoate treatment. Although the mechanism involved in estrogen-induced surge of PRL secretion appears to differ from that of the pituitary gonadotropin secretion, the brain regions that concentrate estrogen receptors probably serve as common feedback sites for estrogen-induced surges of pituitary gonadotropins and PRL secretion [13, 15, 41, 42]. The present experiments do not provide evidence to suggest the specific brain regions that may have affected by neonatal OP treatment, causing a suppression of the afternoon PRL surge in the adults rats. But the previous reports have suggested that the POA and the ventromedial hypothalamus (VMH) serve as major sites that accumulate estrogen receptors in the brain [41]. It was also reported that the effects of OP appear to mediate through estrogen receptors [2, 43]. It is therefore possible to assume that in the present study, neonatal OP treatment may have affected early sexual differentiation of the brain regions that are implicated in the regulation of afternoon PRL surge in female rats [13, 42, 44]. Furthermore, the present finding that a relatively higher magnitude of PRL surge on Day 3 in the DMSO group, in contrast to that of gonadotropin surges, may support the previous findings that the mechanism involved in the surge secretion of PRL is different from that of the pituitary gonadotropins [45, 46].

The regulation of sexual receptive behavior of female rats serves as another excellent model of the sexually differentiated functions of the brain. In the present study, the control female rats treated with DMSO and ovx as adults exhibited lordosis reflex with increased lordosis rating in response to estradiol benzoate/progesterone treatment followed by tactile stimulation of the male rats. However, this receptive behavior following the same hormonal treatment and mounting stimulus by male rats was significantly reduced in ovx rats that had been treated with OP neonatally. It has been adequately demonstrated that a single injection of testosterone propionate into a neonatal female rat within a few days after birth suppresses her ability to display lordosis reflex as an adult [25, 26]. Although the present study does not provide any evidence as to which brain regions might have adversely affected by neonatal OP treatment and subsequently were responsible for reduced receptivity, other reports indicate that the VMH is the estrogen and progesterone feedback center for induction of female sexual behavior [47–49]. This may be further supported by the present finding that a surge of PRL was not produced in OP-treated ovx rats following estradiol capsule implantation, and that the VMH is considered to be one of the major sites that regulates afternoon PRL surge [13].

The sexual dimorphism in the size of the SDN-POA can be well-recognized in the adult rat brain in which the size of the nucleus is usually several times larger in males than in females. Because it is endogenous testosterone that by conversion into estrogen is responsible for the increased size of the nucleus in adult males, administration of exogenous testosterone into neonatal female rats increases the size of this nucleus [14, 50]. In the present study, administration of OP into neonatal female rats increased the area of the SDN-POA of the brain. Although the number of animals used to investigate this anatomical difference was relatively small, the present data indicate that like steroids, OP can also influence the size of the SDN-POA and perhaps other brain regions in female rats.

The present study was designed to investigate estrogenic effects of OP administered into neonatal female rats on some of the well-known sexually dimorphic female characteristics in adulthood. Although it was not our primary intention to relate or compare the higher OP levels used in the present study to the levels of environmental OP to which humans or animals might be exposed, it has been reported that concentrations of an OP-related alkylphenolic compound 2,4-di-tert-pentylphenol were in the 10-3 M range in fat tissue in carp in the Detroit River's Trenton Channel [51]. This level is higher than that reported for other derivatives of alkylphenol polyethoxylates in drinking water, sewage sludge, and river water sediment [2, 52, 53], indicating that these alkyphenolic compounds have the ability to bioaccumulate in mammalian tissues. Furthermore, it has also been reported that environmental estrogens have the potential to act additively [54]. It should be noted, however, that control of reproductive endocrine function in humans is less-affected by events in postnatal life, and thus it may not be possible to identify direct human health risks from the mechanisms tested in the present study.

In conclusion, it is suggested that exposure of neonatal female rats to higher doses of OP disrupts the spontaneous preovulatory LH surge, and estradiol-induced afternoon surges of LH, FSH, and PRL in ovx rats as adults. The present data also provide evidence to suggest that the exposure of female neonates to OP can interfere with the display of sexual receptive behavior in adulthood. Based on the present SDN-POA data and previous reports, it may be possible to conclude that the OP administered into neonatal female rats might have adversely affected the specific brain regions that are implicated in the regulation of the cyclic release of pituitary gonadotropins and PRL, and the display of sexual behavior.

ACKNOWLEDGMENTS

We are grateful to the National Hormone and Pituitary Program, NIDDK, and Dr. A.F. Parlow for rat LH, FSH, and PRL radioimmunoassay kits.

FOOTNOTES

First decision: 24 October 2000.

1 Supported by a grant-in-aid from the Japan Society for the Promotion of Science. Back

2 Correspondence: Kazuyoshi Taya, Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, 3–5–8 Saiwai-cho, Fuchu, Tokyo 183–8509, Japan. FAX: 0081 42 367 5767; taya{at}cc.tuat.ac.jp Back

Accepted: November 28, 2000.

Received: September 22, 2000.

REFERENCES

  1. Giger W, Brunner PH, Schaffner C. 4-Nonylphenol in sewage sludge: accumulation of toxic metabolites from nonionic surfactants. Science 1984; 225:623–625.[Abstract/Free Full Text]
  2. White R, Joblin S, Hoare SA, Sumpter JP, Parker MG. Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 1994; 135:175–182.[Abstract]
  3. Blake CA, Ashiru OA. Disruption of rat estrous cyclicity by the environmental estrogen 4-tert-octylphenol. Proc Soc Exp Biol Med 1997; 216:446–451.[Abstract]
  4. Certa H, Fedtke N, Wiegand H-T, Muller AMF. Toxicokinetics of p-tert-octylphenol in male Wistar rats. Arch Toxicol 1996; 71:112–122.[CrossRef][Medline]
  5. Saegusa A. Japanese media fuel fears of ‘endocrine disrupters’. Nature 1998; 393:613.
  6. Toppari J, Skakkebaek NE. Sexual differentiation and environmental endocrine disrupters. Clin Endocrinol Metab 1998; 12:143–156.
  7. Neill JD, Freeman ME, Tillson SA. Control of the proestrus surge of prolactin and luteinizing hormone secretion by estrogens in the rat. Endocrinology 1971; 89:1448–1453.[Medline]
  8. Neill JD. Sexual differences in the hypothalamic regulation of prolactin secretion. Endocrinology 1972; 90:1154–1159.[Medline]
  9. Legan SJ, Karsch FJ. A daily signal for the LH surge in the rat. Endocrinology 1975; 96:57–62.[Abstract]
  10. Legan SJ, Coon GA, Karsch FJ. Role of estrogen as initiator of daily LH surges in the ovariectomized rat. Endocrinology 1975; 96:50–56.[Medline]
  11. Caligaris L, Astrada JJ, Taleisnik S. Biphasic effect of progesterone on the release gonadotropin in rats. Endocrinology 1971; 89:331–337.[Medline]
  12. Caligaris L, Astrada JJ, Taleisnik S. Oestrogen and progesterone influence on the release of prolactin in ovariectomized rats. J Endocrinol 1974; 60:205–215.[Abstract/Free Full Text]
  13. Pan J-T, Gala RR. Central nervous system regions involved in the estrogen-induced afternoon prolactin surges. II. Implantation studies. Endocrinology 1985; 117:388–395.[Abstract]
  14. Arnold AP, Gorski RA. Gonadal steroid induction of structural sex differences in the central nervous system. Annu Rev Neurosci 1984; 7:413–442.[CrossRef][Medline]
  15. Gorski RA, Harlan RE, Christensen LW. Perinatal hormonal exposure and the development of neuroendocrine regulatory processes. J Toxicol Environ Health 1977; 3:97–121.[Medline]
  16. McEwen BS, Plapinger L, Chaptal C, Gerlach J, Wallach G. Role of fetoneonatal estrogen binding proteins in the association of estrogen with neonatal brain cell nuclear receptors. Brain Res 1975; 96:400–406.[CrossRef][Medline]
  17. Raynaud JP, Mercier-Bodard C, Baulieu EE. Rat estradiol binding plasma protein (EBP). Steroids 1971; 18:767–788.[CrossRef][Medline]
  18. Sutherland SD, Gorski RA. An evaluation of the inhibition of androgenization of the neonatal female rat brain by barbiturate. Neuroendocrinology 1972; 10:94–108.[Medline]
  19. Barraclough CA. Production of anovulatory, sterile rats, by single injections of testosterone propionate. Endocrinology 1961; 68:62–67.
  20. Barraclough CA, Gorski RA. Evidence that the hypothalamus is responsible for androgen-induced sterility in the female rat. Endocrinology 1961; 68:68–79.
  21. Harlan RE, Gorski RA. Steroid regulation of luteinizing hormone secretion in normal and androgenized rats at different ages. Endocrinology 1977; 101:741–749.[Medline]
  22. Harlan RE, Gorski RA. Correlations between ovarian sensitivity, vaginal cyclicity and luteinizing hormone and prolactin secretion in lightly androgenized rats. Endocrinology 1977; 101:750–759.[Medline]
  23. Mennin SP, Gorski RA. Effects of ovarian steroids on plasma LH in normal and persistent estrous adult female rats. Endocrinology 1975; 96:486–491.[Abstract]
  24. Donham RS, Stetson MH. Neonatal androgen abolishes clock-timed gonadotropin release in prepubertal and adult female hamsters. J Reprod Fertil 1985; 73:215–221.[Abstract/Free Full Text]
  25. Barraclough CA, Gorski RA. Studies on mating behavior in the androgen-sterilized rat and their relation to the hypothalamic regulation of sexual behavior in the female rat. Endocrinology 1962; 25:175–182.
  26. Harris GW, Levine S. Sexual differentiation of the brain and its experimental control. J Physiol 1965; 181:379–400.[Free Full Text]
  27. Sharpe RM, Fisher JS, Millar MM, Joblin S, Sumpter JP. Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production. Environ Health Perspect 1995; 103:1136–1143.[Medline]
  28. Blake CA, Boockfor FR. Chronic administration of the environmental pollutant 4-tert-octylphenol to adult rats interferes with the secretion of luteinizing hormone, follicle-stimulating hormone, prolactin, and testosterone. Biol Reprod 1997; 57:255–266.[Abstract]
  29. Boockfor FR, Blake CA. Chronic administration of 4-tert-octylphenol to adult male rats causes shrinkage of the testes and male accessory sex organs, disrupts spermatogenesis, and increases the incidence of sperm deformities. Biol Reprod 1997; 57:267–277.[Abstract]
  30. Lee PC. Disruption of male reproductive tract development by administration of the xenoestrogen, nonylphenol, to male newborn rats. Endocrine 1998; 9:105–111.[CrossRef][Medline]
  31. Lee PC, Arndt P, Nickels KC. Testicular abnormalities in male rats after lactational exposure to nonylphenols. Endocrine 1999; 11:61–68.[CrossRef][Medline]
  32. Katsuda S, Yoshida M, Watanabe G, Taya K, Maekawa A. Irreversible effects of neonatal exposure to p-tert-octylphenol on the reproductive tract in female rats. Toxicol Appl Pharmacol 2000; 165:217–226.[CrossRef][Medline]
  33. Bivens CLM, Olster DH. Abnormal estrous cyclicity and behavioral hyporesponsiveness to ovarian hormones in genetically obese zucker female rats. Endocrinology 1997; 138:143–148.[Abstract/Free Full Text]
  34. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates, 2nd ed. San Diego, CA: Academic Press; 1986.
  35. Jerrold HZ. Biostatistical Analysis. Chapter 9. Englewood Cliffs, NJ: Prentice-Hall; 1984.
  36. SAS. Statistics, Version 6.11. Cary, NC: Statistical Analysis System Institute Inc.; 1987.
  37. Everett J, Sawyer CH. A 24h periodicity in the ‘LH release apparatus' of female rats, disclosed by barbiturate sedation. Endocrinology 1950; 46:198–216.
  38. Park OK, Ramirez VD. Spontaneous changes in GnRH release during the rat estrous cycle as measured with repetitive push-pull perfusions of the pituitary gland in the same female rats. Neuroendocrinology 1989; 50:66–72.[Medline]
  39. Levine JE. New concepts of the neuroendocrine regulation of gonadotropin surges in rats. Biol Reprod 1997; 56:293–302.[Abstract]
  40. Wise PM. Estradiol-induced daily luteinizing hormone and prolactin surges in young and middle-aged rats: correlations with age-related changes in pituitary responsiveness and catecholamine turnover rates in microdissected brain areas. Endocrinology 1984; 115:801–809.[Abstract]
  41. Pfaff DW, Keiner M. Atlas of estradiol-concentrating cells in the central nervous system of the female rat. J Comp Neurol 1973; 151:121–158.[CrossRef][Medline]
  42. Pan J-T, Gala RR. Central nervous system regions involved in the estrogen-induced afternoon prolactin surge. I. Lesion studies. Endocrinology 1985; 117:382–387.[Abstract]
  43. Mueller GC, Kim U-H. Displacements of estradiol from estrogen receptors by simple alkylphenols. Endocrinology 1978; 102:1429–1435.[Abstract]
  44. van der Beek EM, Swarts HJ, Wiegant VM. Central administration of antiserum to vasoactive intestinal peptide delays and reduces luteinizing hormone and prolactin surges in ovariectomized, estrogen-treated rats. Neuroendocrinology 1999; 22:227–237.
  45. Arey BJ, Freeman ME. Hypothalamic factors involved in the endogenous stimulatory rhythm regulating prolactin secretion. Endocrinology 1989; 124:878–883.[Abstract]
  46. Huang SK, Pan JT. Stimulatory effects of vasoactive intestinal peptides and pituitary adenylate cyclase-activating peptide on tuberoinfundibular dopaminergic neuron activity in estrogen-treated ovariectomized rats and their correlation with prolactin secretion. Neuroendocrinology 1996; 64:208–214.[Medline]
  47. Davis PG, McEwen BS, Pfaff DW. Localized behavioral effects of tritiated estradiol implants in the ventromedial hypothalamus of female rats. Endocrinology 1979; 104:898–903.[Medline]
  48. Davis PG, Kieger MS, Barfield RJ, McEwen BS, Pfaff DW. The site of intrahypothalamic estrogen implants in feminine sexual behavior: an autoradiographic analysis. Endocrinology 1982; 111:1581–1586.[Medline]
  49. Rubin BS, Barfield RJ. Priming of estrous responsiveness by implants of 17ß-estradiol in the ventromedial hypothalamic nucleus of female rats. Endocrinology 1980; 106:504–509.[Abstract]
  50. Gorski RA, Gordon JH, Shryne JE, Southam AM. Evidence for a morphological sex difference within the medial preoptic area of the rat brain. Brain Res 1978; 148:333–346.[CrossRef][Medline]
  51. Shiraishi H, Carter DS, Hites RA. Identification and determination of tert-alkylphenols in carp from the Trenton Channel of the Detroit River, Michigan, USA. Biomed Environ Mass Spectrom 1989; 18:478–483.[CrossRef][Medline]
  52. Clark LB, Rosen RT, Hartman TG, Louis JB, Suffet IH, Lippincott RL, Rosen JD. Determination of alkylphenol ethoxylates and their acetic acid derivatives in drinking water by particle beam liquid chromatography/mass spectometry. Int J Environ Anal Chem 1992; 47:167–180.
  53. Giger W, Brunner PH, Schaffner C. 4-Nonylphenol in sewage sludge: accumulation of toxic metabolites from nonionic surfactants. Science 1984; 225:623–625.
  54. Ramamoorthy K, Wang F, Chen I-C, Safe S, Norris JD, McDonnell DP, Gaido KW, Bocchinfuso WP, Korach KS. Potency of combined estrogenic pesticides. Science 1997; 275:405.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Toxicol SciHome page
S. A. Myllymaki, T. E. Haavisto, L. J. S. Brokken, M. Viluksela, J. Toppari, and J. Paranko
In Utero and Lactational Exposure to TCDD; Steroidogenic Outcomes Differ in Male and Female Rat Pups
Toxicol. Sci., December 1, 2005; 88(2): 534 - 544.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol PatholHome page
M. Yoshida, A. Takenaka, S.-I. Katsuda, Y. Kurokawa, and A. Maekawa
Neonatal Exposure to p-tert-octylphenol Causes Abnormal Expression of Estrogen Receptor {alpha} and Subsequent Alteration of Cell Proliferating Activity in the Developing Donryu Rat Uterus
Toxicol Pathol, April 1, 2002; 30(3): 357 - 364.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herath, C. B.
Right arrow Articles by Taya, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herath, C. B.
Right arrow Articles by Taya, K.
Agricola
Right arrow Articles by Herath, C. B.
Right arrow Articles by Taya, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS