Maiko Kawaguchi

Exposure to bisphenol A during gestation and lactation causes loss of sex difference in corticotropin-releasing hormone-immunoreactive neurons in the bed nucleus of the stria terminalis of rats.

It has been suspected that endocrine disrupters induce abnormal differentiation and development of reproductive organs. In the present study, we examined whether exposure to bisphenol A (BPA), a known endocrine disrupter, during gestation and lactation affects sex difference in the number of corticotropin-releasing hormone-immunoreactive neurons (CRH neurons) in the preoptic area (POA) and the bed nucleus of the stria terminalis (BST). For that purpose, pregnant female Wistar rats (n=8-11 per treatment group) were treated with either 0.1% ethanol (control group) or 10 mg/l BPA (BPA group) dissolved in their drinking water until their offspring were weaned. In the control group, we confirmed a previous report that the POA of female rats contained significantly more CRH neurons than that of male rats (p<0.05). This significant sex difference was also evident in the BPA group, indicating that BPA exposure used in the present study had no effect on the sex difference in CRH neurons in the POA. We also found in the control group that the BST of female rats contained significantly more CRH neurons (p<0.05) than that of male rats. However, this significant sex difference was not observed in the BPA group (p>0.05), suggesting that BPA exposure affected the sex difference in CRH neurons in the BST. Since there was no statistically significant difference in the number of CRH neurons between the control and the BPA group, irrespective of the sex, the results suggested that a loss of sex difference in CRH neurons was due to both an increase in CRH neurons in male rats and a decrease in CRH neurons in female rats. The present study indicates that there is a significant sex difference in the number of CRH neurons in the BST as well as in the POA and that exposure to BPA during gestation and lactation causes a loss of this sex difference in the rat BST, but not in the POA. We suggest that CRH neurons in the BST are more susceptible to endocrine disrupters than those in the POA, irrespective of the sex.

The Endocrine Disrupters Butyl Benzyl Phthalate and Bisphenol A Increase the Expression of Progesterone Receptor Messenger Ribonucleic Acid in the Preoptic Area of Adult Ovariectomized Rats

Butyl benzyl phthalate (BBP) and bisphenol A (BPA), so-called endocrine disrupters, are known to mimic the action of estrogens: they are thus liable to influence reproductive functions. Since little is known about their action on gene expression in the adult hypothalamus, we examined the effects of these chemicals on the expression of estrogen-regulated mRNAs, i.e., progesterone receptor (PR) mRNA, preproenkephalin (PPE) mRNA and neurotensin (NT) mRNA, in the hypothalamus and pituitary of adult female rats. Two weeks after ovariectomy, rats were subcutaneously injected with 10 mg BBP, 10 mg BPA, or 10 µg 17β-estradiol (E2) in sesame oil, or with sesame oil alone as a control. Twenty-four hours after the injection, tissues including the preoptic area (POA), mediobasal hypothalamus (MBH) and anterior pituitary were collected. Northern blot revealed that injection of E2 resulted in expected changes, i.e., significant increases in PR mRNA in the POA, MBH and anterior pituitary, and in PPE mRNA in the MBH. We also found that injection of BPA significantly increased PR mRNA in the POA and anterior pituitary, while injection of BBP increased PR mRNA in the POA and anterior pituitary, although the increase in the anterior pituitary was not significant. No significant effect of E2, BPA, or BBP on NT mRNA in the POA was detected. The present study demonstrates that the two endocrine disrupters BPA and BBP can increase the expression of PR mRNA in the POA of adult ovariectomized rats.

Menstrual changes in sleep, rectal temperature and melatonin rhythms in a subject with premenstrual syndrome

We studied a sighted woman with premenstrual syndrome who showed menstrual changes in circadian rhythms. She showed alternative phase shifts in the sleep rhythm in the menstrual cycle: progressive phase advances in the follicular phase and phase delays in the luteal phase. Rectal temperature rhythm also showed similar menstrual changes, but the phase advance and delay started a few days earlier than changes in sleep-wake rhythm so that the two rhythms were dissociated around ovulation and menstruation. These results suggest that her circadian rhythms in sleep and temperature are under the control of ovarian steroid hormones and that these two rhythms have different sensitivity to the hormones.

Expression of Nerve Growth Factor (NGF), and Its Receptors TrkA and p75 in the Reproductive Organs of the Adult Male Rats

Abstract Immunolocalization of nerve growth factor (NGF) and its receptors, TrkA and p75 in the reproductive organs of adult male rats was investigated. Sections of the testis, efferent duct, epididymis, deferent duct, seminal vesicle, coagulating gland and prostate of adult male rats were immunostained by the avidin-biotin-peroxidase complex methods (ABC). NGF was expressed in Leydig cells, primary spermatocytes and pachytene spermatocytes in the testis. TrkA only immunoreacted to elongate spermatids and p75 showed positive immunostaining in the Sertoli cells, Leydig cells, the pachytene spermatocytes and elongate spermatids. Immunoreactions for NGF and its two receptors were detected in epithelial cells of efferent duct, deferent duct and epididymis. In addition, immunoreactions for NGF and its two receptors were also observed in columnar secretory epithelium lines of the seminal vesicles, prostate and coagulating gland. These results suggest that NGF is an important growth factor in gonadal function of adult male rats.

Intravenous injections of nicotine decrease the pulsatile secretion of LH by inhibiting the gonadotropin-releasing hormone (GnRH) pulse generator activity in female rats

Whether nicotine inhibits the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator to suppress pulsatile LH secretion, and whether this suppression of LH secretion by nicotine is mediated by opioid neurons, were studied in ovariectomized rats by examining changes in LH secretion and the multiunit activity (MUA) of the medial basal hypothalamus. Intravenous (i.v.) injection of nicotine (nicotine bitartrate, 100 micrograms) significantly increased the interval between characteristic increases (volleys) in MUA and LH pulses. This inhibitory effect of nicotine on the GnRH pulse generator activity was not blocked by the prior injection of an opiate receptor antagonist naloxone (naloxone hydrochlolide, 2 mg/kg bw), which was effective in significantly decreasing the interval between MUA volleys. The results suggest that nicotine alters the activity of the GnRH pulse generator, and that cholinergic neurons appear to be directly involved in suppressing pulsatile secretion of LH.

Maternal isobutyl-paraben exposure alters anxiety and passive avoidance test performance in adult male rats.

Isobutyl-paraben (IBP), one of the most widely used preservatives, exhibits estrogenic activity. In this study, we analyzed the effects of maternal IBP treatment on the emotional behavior and learning performance in mature offspring. Pregnant female Sprague-Dawley rats were treated with IBP via a subcutaneous Silastic capsule. Consequently, the offspring were exposed to IBP during gestation through the placentae, and before weaning through the milk. Male and female offspring were tested for emotional behavior in an open field and in an elevated plus maze at five and six weeks old, respectively. IBP-exposed male (but not female) rats spent less time in the open arms of the elevated plus maze. At 11 weeks old, all females were gonadectomized and treated chronically with 17beta-estradiol or cholesterol by Silastic capsules; all males were kept intact. They were tested for learning performance in a passive avoidance test and a Morris water maze. IBP exposure impaired the performance of males in the passive avoidance test. These findings suggest that male rats are more affected by early exposure to IBP than female rats. IBP affects their adult behavior including anxiety and learning abilities.

Effects of p-Nonylphenol and 4-tert-Octylphenol on the Anterior Pituitary Functions in Adult Ovariectomized Rats

p-Nonylphenol (NP) and 4-tert-octylphenol (OP) are known to mimic the action of estrogens as endocrine disruptors. However, their acute effects on the pituitary and the hypothalamus functions in vivo have been uncertain. We therefore determined their effects on the anterior pituitary, in particular, gonadotropin secretion. Two weeks after ovariectomy, the rats were given a subcutaneous injection of 10 mg NP, 10 mg OP, 10 mg bisphenol A, 1 µg 17β-estradiol, or sesame oil alone as control. Twenty-four hours after the treatment, the expression of progesterone receptor mRNA in the anterior pituitary and the level of luteinizing hormone (LH), follicle-stimulating hormone, and prolactin were determined. The expression of progesterone receptor mRNA in the anterior pituitary was significantly increased by either NP, OP, bisphenol A, or estradiol, but bisphenol A was less effective. The level of LH was significantly decreased by either NP or OP, but not by bisphenol A and estradiol. Only estradiol significantly increased the level of prolactin. The level of follicle-stimulating hormone was unchanged by any of the treatments. To check the effects of NP and OP on pulsatile LH secretion, blood samplings were done at 6-min intervals for 3 h. Twenty-four hours after treatment in ovariectomized adult rats, we found that the injection of NP significantly decreased the amplitude of LH pulses and the mean LH concentrations, but not the frequency of LH pulses. The injection of OP significantly decreased the mean LH concentrations without affecting the frequency and amplitude of the LH pulses. Finally, the rats given an injection of NP or sesame oil were intravenously injected with 50 ng of gonadotropin-releasing hormone (GnRH) to check whether NP affected the LH secretory responsiveness of the anterior pituitary to GnRH. We found that the responsiveness to GnRH in NP-injected rats was significantly attenuated compared to the sesame oil-injected rats. The present study suggests that NP, even with a single injection, suppresses the pulsatile LH secretion in adult ovariectomized rats, probably by affecting the anterior pituitary level.

Bisphenol A induces transforming growth factor-β3 mRNA in the preoptic area: A cDNA expression array and Northern blot study

To gain better understanding of the effects of bisphenol A (BPA) in the adult brain, a cDNA expression array was used to screen possible candidates for BPA-inducible genes in the medial preoptic area (MPOA). Adult ovariectomized rats were given a subcutaneous injection of 10 mg BPA or sesame oil alone as a control. Twenty-four hours after the injection, the MPOAs were dissected and total RNAs were extracted. When expression levels of cDNAs derived from pooled samples were compared to controls, the expression levels of some genes in BPA-injected rats appeared to be different from those in sesame oil-injected rats. Among the candidate genes, we focused on an increase in the expression of transforming growth factor (TGF)-beta3 mRNA. To quantify the change in TGF-beta3 mRNA by BPA, we examined the effects of 10 mg BPA (n=6), 1 microg 17beta-estradiol (n=6), or oil injection (n=6) on the expression of TGF-beta3 mRNA in the MPOA of ovariectomized rats by Northern blot. The TGF-beta3 mRNA level in the MPOA of BPA-injected rats was significantly increased compared to the level in oil-injected rats (p<0.05). Injection of 1 microg 17beta-estradiol did not have any significant effect. The results suggest that, in the adult female rat, BPA acts on the MPOA by altering the expression of the TGF-beta3 gene in a manner distinct from that of estrogen.

Dietary deprivation of each essential amino acid induces differential systemic adaptive responses in mice

SCOPE Dietary deprivation of essential amino acids (EAAs) in mammals is known to cause reductions in food intake and body weight. The aim of this study was to determine whether and how mice respond to deprivation of individual EAA species. METHODS AND RESULTS Dietary deprivation of any single EAA (not non-EAA) in mice led to progressive weight loss in the order of Ile(-) > Val(-) > Thr(-) > Leu(-) > Trp(-) > His(-) > Phe(-) > Met(-) > Lys(-) , which correlated with the reduction in food intake. Decreased levels of the deprived EAAs as well as increased levels of all or some of the other amino acids were detected in the serum, although these levels differed among the diets examined. Serum biochemistry identified significant increases in creatine phosphokinase, blood urea nitrogen, alanine aminotransferase, and aspartate aminotransferase, and decreases in glucose and triglycerides; computed tomography revealed a marked reduction in abdominal/femoral fat and muscle depots; histology identified diffuse myofiber atrophy in the rectus femoris muscle, all in that approximate order. In contrast, amino acid response, autophagy, and ubiquitination marker genes as well as amino acid transporter genes were induced in both deprived EAA-specific and tissue-specific manners. CONCLUSION Dietary deprivation of individual EAAs induced systemic adaptive responses that differed in magnitude and molecular machinery.

A Single Neonatal Injection of Ethinyl Estradiol Impairs Passive Avoidance Learning and Reduces Expression of Estrogen Receptor α in the Hippocampus and Cortex of Adult Female Rats.

Although perinatal exposure of female rats to estrogenic compounds produces irreversible changes in brain function, it is still unclear how the amount and timing of exposure to those substances affect learning function, or if exposure alters estrogen receptor α (ERα) expression in the hippocampus and cortex. In adult female rats, we investigated the effects of neonatal exposure to a model estrogenic compound, ethinyl estradiol (EE), on passive avoidance learning and ERα expression. Female Wistar-Imamichi rats were subcutaneously injected with oil, 0.02 mg/kg EE, 2 mg/kg EE, or 20 mg/kg 17β-estradiol within 24 h after birth. All females were tested for passive avoidance learning at the age of 6 weeks. Neonatal 0.02 mg/kg EE administration significantly disrupted passive avoidance compared with oil treatment in gonadally intact females. In a second experiment, another set of experimental females, treated as described above, was ovariectomized under pentobarbital anesthesia at 10 weeks of age. At 15–17 weeks of age, half of each group received a subcutaneous injection of 5 μg estradiol benzoate a day before the passive avoidance learning test. Passive avoidance learning behavior was impaired by the 0.02 mg/kg EE dose, but notably only in the estradiol benzoate-injected group. At 17–19 weeks of age, hippocampal and cortical samples were collected from rats with or without the 5 μg estradiol benzoate injection, and western blots used to determine ERα expression. A significant decrease in ERα expression was observed in the hippocampus of the estradiol-injected, neonatal EE-treated females. The results demonstrated that exposure to EE immediately after birth decreased learning ability in adult female rats, and that this may be at least partly mediated by the decreased expression of ERα in the hippocampus.