Prenatal undernutrition suppresses sexual behavior in female rats.

Infectious, psychological and metabolic stresses in the prenatal and early neonatal period induce long-lasting effects in physiological function and increase the risk of metabolic disorders later in life. We examined the sexual behavior of female rats that were subjected to undernutrition in the prenatal period. Eight pregnant rats were divided into two groups: a maternal normal nutrition group (mNN; n = 4) and a maternal undernutrition group (mUN; n = 4), which received 50% of the daily food intake amount of the mNN group from gestation day 13 to delivery. Nine and seven female offspring were randomly selected from the mNN and mUN groups, respectively. Vaginal opening (VO), estrous cycle length, sexual behavior and mRNA expression levels of the factors that regulate sexual behavior were observed. In the mUN group, VO day was later, the estrous cycle was longer, and the lordosis quotient and lordosis rating were lower than in the mNN group; such differences were not seen in other sexual performances, such as ear wiggles, darts, kick bouts and box. The hypothalamic mRNA expression level of progesterone receptor (PR) A + B and oxytocin (OT) were significantly lower in the mUN group than in the mNN group. These findings indicated that prenatal undernutrition disrupted puberty onset, the estrous cycle, sexual behavior and hypothalamic mRNA expression of PR and OT in female rat pups.

The reduction in sexual behavior induced by neonatal immune stress is not related to androgen levels in male rats.

PURPOSE: It is known that various types of stress in early life increase the incidence of diabetes, myocardial infarctions, and psychiatric disorders in adulthood. We examined the mechanism by which neonatal immune stress reduces sexual behavior in adult male rats. METHODS: Male rats were randomly divided into 3 groups: the control (n = 17), postnatal day 10 lipopolysaccharide (PND10LPS) (n = 31), and PND25LPS (n = 16) groups, which received intraperitoneal injections of LPS (100 µg/kg) or saline (injection volume: ≤0.1 ml/g) on postnatal days 10 and 25. In experiment 1, male rats (age: 11 to 12 weeks) were put together with female rats in a one-to-one setting for mating, and sexual behavior (mounting, intromission, and ejaculation) was monitored for 30 minutes. The serum levels of luteinizing hormone (LH) and testosterone (T) and the hypothalamic mRNA expression levels of factors related to sexual behavior were examined. After experiment 1 finished, the remaining 37 male rats were used for experiment 2: the control group (n = 8), PND10 LPS group (n = 21) and PND25LPS group (n = 8) these rats had been given an i.p. injection of the saline during the expriment1. All of the rats were orchidectomized at 14 weeks of age. After a 3-week recovery period, a silastic tube containing crystalline T was subcutaneously implanted into the back of each rat. The rats' sexual behavior, serum hormone concentrations, and hypothalamic mRNA expression levels were assessed. RESULTS: In experiment 1, preputial separation occurred significantly later in the PND10LPS group than in the control group. The frequency of sexual behavior was significantly lower in the PND10LPS group than in the control group. The serum T concentrations of the PND10LPS and PND25LPS groups were significantly lower than that of the control group, but the serum LH concentrations of the 3 groups did not differ significantly. The hypothalamic mRNA expression levels of progesterone receptor B (PRB) and gonadotropin-releasing hormone (GnRH) were significantly lower in the PND10LPS and PND25LPS groups than in the control group, whereas the hypothalamic PRA + B mRNA expression levels of the 3 groups did not differ significantly. In experiment 2, after T supplementation the frequency of sexual behavior was significantly lower in the PND10LPS and PND25LPS groups than in the control group, although there were no significant differences in the serum T or LH concentrations or the hypothalamic PRB, PRA + B, or GnRH mRNA expression levels of the 3 groups. CONCLUSION: In male rats, immune stress in the early neonatal period delayed sexual maturation, reduced sexual behavior, suppressed the serum T concentration, and downregulated the hypothalamic mRNA expression levels of GnRH and the PR in adulthood. The delayed sexual maturation was presumed to have been caused by the reduction in the serum T concentration. However, the rats that experienced neonatal stress exhibited reduced sexual behavior irrespective of their serum T concentrations.

Effects of chronic testosterone administration on the degree of preference for a high-fat diet and body weight in gonadal-intact and ovariectomized female rats.

Energy balance and reproductive functions are closely linked in some species. The sex hormones (estrogens and androgens) are involved in the regulation of appetite, metabolism, body weight (BW), and body composition in mammals. Previously, we showed that the effects of testosterone on BW, appetite, and fat weight were markedly affected by alterations to the gonadal hormonal milieu. In this study, we examined whether testosterone administration changes food preferences and whether these effects of testosterone depend on gonadal status in female rats. We also evaluated the underlying mechanisms responsible for these effects, focusing on hypothalamic inflammation and endoplasmic reticulum (ER) stress. In gonadal-intact (sham) female rats, chronic testosterone administration promoted a preference for a high-fat diet (HFD) and increased BW gain, fat weight, and adipocyte size, whereas no such effects were observed in ovariectomized (OVX) rats. Testosterone administration increased hypothalamic interleukin-1 mRNA expression in the sham rats, but not the OVX rats. On the contrary, testosterone administration decreased the hypothalamic mRNA levels of ER stress-response genes in the OVX rats, but not the sham rats. These testosterone-induced alterations in OVX rats might represent a regulatory mechanism for preventing hypothalamic inflammation and the overconsumption of a HFD. In conclusion, testosterone's effects on food preferences and the subsequent changes were affected by gonadal status. Testosterone-induced changes in hypothalamic inflammatory cytokine production and ER stress might be related to these findings.

The effects of chronic testosterone administration on body weight, food intake, and adipose tissue are changed by estrogen treatment in female rats.

In females, estrogens play pivotal roles in preventing excess body weight (BW) gain. On the other hand, the roles of androgens in female BW, appetite, and energy metabolism have not been fully examined. We hypothesized that androgens' effects on food intake (FI) and BW regulation change according to the estrogens' levels. To evaluate this hypothesis, the effects of chronic testosterone administration in ovariectomized (OVX) female rats with or without estradiol supplementation were examined in this study. Chronic testosterone administration decreased BW, FI, white adipose tissue (WAT) weight, and adipocyte size in OVX rats, whereas it increased BW, WAT weight, and adipocyte size in OVX with estradiol-administered rats. In addition, chronic testosterone administration increased hypothalamic CYP19a1 mRNA levels in OVX rats, whereas it did not alter CYP19a1 mRNA levels in OVX with estradiol-administered rats, indicating that conversion of testosterone to estrogens in the hypothalamus may be activated in testosterone-administered OVX rats. Furthermore, chronic testosterone administration decreased hypothalamic TNF-α mRNA levels in OVX rats, whereas it increased hypothalamic IL-1ß mRNA levels in OVX with estradiol-administered rats. On the other hand, IL-1ß and TNF-α mRNA levels in visceral and subcutaneous WAT and liver were not changed by chronic testosterone administration in both groups. These data indicate that the effects of chronic testosterone administration on BW, FI, WAT weight, and adipocyte size were changed by estradiol treatment in female rats. Testosterone has facilitative effects on BW gain, FI, and adiposity under the estradiol-supplemented condition, whereas it has inhibitory effects in the non-supplemented condition. Differences in the responses of hypothalamic factors, such as aromatase and inflammatory cytokines, to testosterone might underlie these opposite effects.

The effects of prenatal undernutrition and a high-fat postnatal diet on central and peripheral orexigenic and anorexigenic factors in female rats.

Prenatal undernutrition and postnatal overnutrition increase the risk of some peripheral and central metabolic disorders in adulthood. We speculated that disturbances of appetite/metabolic regulatory factors might already have been established in the early stages of life and contribute to obesity later in life. The effects of a high-fat diet on the levels of peripheral and central appetite/metabolic regulatory factors were compared between the offspring of normally nourished dams and those of undernourished dams in the peri-pubertal period. In the offspring of the normally nourished dams (control), the consumption of the high-fat diet resulted in lower hypothalamic mRNA levels of orexigenic factors (neuropeptide Y (NPY) and prepro-orexin (pporexin)), whereas no such changes were seen in the offspring of the undernourished dams (subjected to intrauterine growth restriction). These results indicate that in high-energy conditions either the adaptive response does not function properly or has not been established in the offspring of undernourished dams. Because NPY and pporexin are negatively regulated by leptin, these findings suggest that in the intrauterine growth restriction group, the leptin resistance of hypothalamic functions, which is usually caused by diet-induced obesity in adulthood, had already been established in the peri-pubertal period.

Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats.

The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. In this study, we examined the developmental changes in the hypothalamic mRNA levels of brain-derived neurotrophic factor (BDNF), which is a potent anorectic factor and the changes in the sensitivity of the hypothalamic expression of this factor to fasting during the neonatal to pre-pubertal period. Under fed conditions, hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum levels of leptin, which is a positive regulator of hypothalamic BDNF expression, also tended to fall during the developmental period. The serum leptin level and the hypothalamic BDNF mRNA level were found to be positively correlated in both sexes under the fed conditions. Hypothalamic BDNF mRNA expression was decreased by 24h fasting (separating the rats from their mothers) in the early neonatal period (postnatal day 10) in both males and females, but no such changes were seen at postnatal day 20. Twenty-four hours' fasting (food deprivation) did not affect hypothalamic BDNF mRNA expression in the pre-pubertal period (postnatal day 30). On the other hand, the rats' serum leptin levels were decreased by 24h fasting (separating the rats from their mothers at postnatal day 10 and 20, and food deprivation at postnatal day 30) throughout the early neonatal to pre-pubertal period. The correlation between serum leptin and hypothalamic BDNF mRNA levels was not significant under the fasted conditions. It can be speculated that leptin partially regulates hypothalamic BDNF mRNA levels, but only in fed conditions. Such changes in hypothalamic BDNF expression might play a role in maintaining appropriate metabolic and nutritional conditions and promoting normal physical development. In addition, because maternal separation induces a negative energy balance and short- and long-term stress responses, it is also possible that reductions in hypothalamic BDNF mRNA levels in the early neonatal period (postnatal day 10) may be partially induced by stress responses of the maternal deprivation.

Effects of chronic testosterone administration on body weight and food intake differ among pre-pubertal, gonadal-intact, and ovariectomized female rats.

In females, estrogens play pivotal roles in preventing excessive body weight gain. On the other hand, the roles of androgen in female appetite and body weight regulation have not been fully studied. In this study, whether the roles of androgen in the regulation of body weight and appetite were different among ages and/or the estrogen milieu in females was evaluated. Body weight gain and food intake were increased by chronic testosterone administration in pre-pubertal and gonadal-intact female rats, but not in ovariectomized female rats. Testosterone administration also affected the serum leptin level and adipose leptin gene expression levels differently in each experimental condition. Hypothalamic mRNA levels of ERα, which plays pivotal roles in regulation of body weight and metabolism, were decreased by chronic testosterone administration in pre-pubertal and gonadal-intact female rats, but not in ovariectomized female rats. These results indicate that the effects of testosterone on body weight and appetite differed among ages and/or estrogen milieu in female rats, and that attenuation of estrogens' actions on the hypothalamus might be partly involved in the androgen-induced increases of body weight gain and food intake in females.

Developmental changes in the hypothalamic mRNA expression levels of PACAP and its receptor PAC1 and their sensitivity to fasting in male and female rats.

The actions and responses of hypothalamic appetite regulatory and factors change markedly during the neonatal to pre-pubertal period. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been found to play pivotal roles in the regulation of metabolic and nutritional status through its specific receptor PAC1. PACAP/PAC1 have anorectic roles, and their functions are regulated by leptin in adulthood. In the present study, we showed that hypothalamic PACAP mRNA expression decreases during the neonatal to pre-pubertal period (from postnatal day 10-30) in both male and female rats. During this period, hypothalamic PACAP mRNA expression was not affected by 24h fasting in either sex, while the serum leptin levels (leptin is a positive regulator of hypothalamic PACAP expression in adulthood) of both sexes were decreased by fasting. On the other hand, hypothalamic PAC1 mRNA expression did not change during the neonatal to pre-pubertal period in either sex; however, its levels were consistently higher in males than in females. Hypothalamic PAC1 mRNA expression was decreased by 24h fasting in males, but no such changes were observed in females. These results indicate while hypothalamic PACAP expression is sensitive to a negative energy state and the serum leptin level in adulthood, no such relationships are seen in the pre-pubertal period. In addition, we speculate that differences in the gonadal steroidal milieu might induce sexual dimorphism in the basal hypothalamic PAC1 mRNA level and its response to fasting. The mechanisms responsible for and the physiological effects of such changes in hypothalamic PACAP and PAC1 expression during the developmental period remain to be clarified.

Effects of chronic DHEA treatment on central and peripheral reproductive parameters, the onset of vaginal opening and the estrous cycle in female rats.

The neonatal and/or prepubertal androgen milieu affects sexual maturation and reproductive function in adulthood. However, the effects of chronic dehydroepiandrosterone (DHEA) treatment on reproductive functions have not been fully elucidated. Therefore, the reproductive phenotypes and parameters of rats that had been subjected to chronic DHEA treatment were evaluated in this study. The chronic DHEA-treated (from postnatal day 23-12 weeks of age) rats exhibited earlier vaginal opening, indicating that DHEA treatment promotes sexual maturation. In addition, the estrus phase lasted longer in the DHEA-treated rats, suggesting that their estrous cycles had been disrupted. As the DHEA-treated rats' serum luteinizing hormone levels and hypothalamic Kiss1 mRNA expression levels were decreased and their uterine weight was increased, DHEA and/or estrogen might directly affect reproductive phenotypes. While DHEA treatment caused changes in body weight and body composition in chronic testosterone-treated models in previous studies, no such changes were seen in the present study.

Developmental changes in the hypothalamic mRNA levels of nucleobindin-2 (NUCB2) and their sensitivity to fasting in male and female rats.

Nesfatin-1 is a central anorectic peptide derived from the precursor protein nucleobindin-2 (NUCB2). In the present study, the changes in hypothalamic NUCB2 mRNA expression and their responses to food deprivation during the neonatal to pre-pubertal period (postnatal days 10, 20, and 30) were evaluated in male and female rats. The rats' serum leptin levels were also measured because NUCB2 mRNA expression is positively regulated by leptin. In both the female and male rats, hypothalamic NUCB2 mRNA expression tended to fall throughout development. Similarly, higher serum leptin levels were detected on postnatal day 10 than on postnatal days 20 and 30 in both sexes. Hypothalamic NUCB2 mRNA expression was positively correlated with the serum leptin level in both the female and male rats; however, the relationship was not significant in males. The hypothalamic NUCB2 mRNA levels of the fed and 24h fasted groups did not differ at any time point in either sex. On the other hand, the serum leptin levels of the 24h fasted group were significantly lower than those of the fed group at all time points in both sexes. It can be speculated that the upregulation of hypothalamic leptin activity might induce a transient increase in hypothalamic NUCB2 mRNA expression during the early postnatal period (postnatal day 10) in both sexes. However, hypothalamic NUCB2 mRNA expression does not become sensitive to a negative energy balance during the neonatal to pre-pubertal period.

The expression of orexigenic and anorexigenic factors in middle-aged female rats that had been subjected to prenatal undernutrition.

Fetal growth retardation, which affects short- and long-term fetal brain development, is associated with metabolic, hematological, and thermal disturbances, which can increase the risk of metabolic syndrome later in life. Orexigenic and anorexigenic factors regulate food intake and energy expenditure. We studied how the expression of these factors was affected by food deprivation (FD) in middle-aged female rats that had been subjected to prenatal undernutrition. Eight pregnant rats were divided into two groups, the normal nutrition (NN) (n=4) group and the undernutrition (UN) (n=4) group, which received 50% (approximately 11 g) of the daily food intake of the normal nutrition rats from day 13 of pregnancy to delivery. The pups from these dams were defined as the maternal NN (mNN) and maternal UN (mUN) groups, respectively. After weaning, all of the pups were housed and allowed ad libitum access to food and water. At the age of 6 months, both groups of pups were sub-divided into three groups. One group was allowed to consume normal amounts of food (Fed), and the other two groups were subjected to 24h or 48 h FD (n=7-8 per group). The rats' serum leptin levels and hypothalamic mRNA expression levels of various orexigenic or anorexigenic factors were measured. In both the mNN and mUN rats, the serum leptin levels of the 24h and 48 h FD groups tended to be lower than those of the Fed group, and the serum leptin levels of the 24h FD mUN rats and the Fed mUN rats differed significantly. The hypothalamic neuropeptide Y (NPY) mRNA expression levels of the 24h and 48 h FD groups were significantly higher in the mUN rats than in the mNN rats. In addition, among the mUN rats the hypothalamic NPY mRNA expression levels of the 48 h FD group were significantly higher than those of the Fed group. In both the mNN and mUN rats, prepro-orexin mRNA expression was lower in the 48 h FD group than in the corresponding Fed group. Among the mUN rats, the 48 h FD group exhibited significantly lower hypothalamic proopiomelanocortin (POMC) mRNA expression than the Fed group, and a similar tendency was seen among the mNN rats. Among the mNN rats, the 24h FD group displayed significantly higher hypothalamic leptin receptor (OBRb) mRNA levels than the Fed group. However, no such differences were seen among the mUN rats. As a result, the hypothalamic OBRb mRNA expression levels of the mUN rats in the 24h and 48 h FD groups were lower than those of the corresponding mNN rat groups. These findings indicate that rats that are subjected to prenatal undernutrition exhibit upregulated expression of orexigenic factors and are more sensitive to FD in middle age, which might increase their risk of developing metabolic disorders in later life.

Effects of LPS injection on the hypothalamic and testicular mRNA expression levels of reproductive factors in male rats.

OBJECTIVES: In the hypothalamus, kisspeptin and RFamide-related peptide (RFRP) regulate gonadotropin-releasing hormone expression. Kisspeptin and RFRP are also found in the testes and might play roles in steroidogenesis and spermatogenesis. DESIGN AND RESULTS: The present study demonstrated that the hypothalamic mRNA expression level of the kisspeptin receptor was decreased by the injection of lipopolysaccharide (LPS) (500 µg/kg) in male rats, and it was suggested that such changes might contribute to reductions in serum luteinizing hormone levels. Contrary to our expectations, hypothalamic RFRP and testicular GPR147 (the RFRP receptor) mRNA expression were also decreased by LPS injection. CONCLUSIONS: We speculate that changes in hypothalamic RFRP expression might represent a protective response aimed at attenuating LPS-induced anorectic responses.

Developmental changes in the hypothalamic mRNA levels of prepro-orexin and orexin receptors and their sensitivity to fasting in male and female rats.

Orexin, which is also called as hypocretin (Hcrt), a product of the prepro-orexin (pp-orexin//Hcrt) gene, affects various physiological and behavioral functions, such as the sleep-wake cycle and appetite. The developmental changes in the hypothalamic mRNA levels of pp-prexin and the orexin receptors OX1R and OX2R and their sensitivity to fasting were evaluated in both male and female rats. During development, hypothalamic pp-orexin/Hcrt mRNA expression increased in both male and female rats, whereas hypothalamic OX1R mRNA expression decreased in both sexes. In addition, hypothalamic OX2R mRNA expression increased in male rats, but did not change in female rats. Fasting did not affect hypothalamic pp-orexin/Hcrt mRNA expression in either sex. Hypothalamic OX1R mRNA expression was increased by fasting in the prepubertal period (postnatal days 20 and 30) in female rats, but was not affected by fasting in males. In male rats, hypothalamic OX2R mRNA expression was decreased by fasting during the neonatal period (postnatal day 10), but not the prepubertal period (postnatal days 20 and 30). In females, hypothalamic OX2R mRNA expression was also decreased by fasting; however, the fasting-induced downregulation of hypothalamic OX2R expression persisted until postnatal day 20. These results indicate that the developmental patterns of components of the orexin system and their sensitivity to fasting during the neonatal and prepubertal periods only differ slightly between the sexes. These differences might be involved in the development of some physiological and behavioral functions.

The suppressive effect of immune stress on LH secretion is absent in the early neonatal period in rats.

Some physiological functions display weak responses to stress in the early neonatal period; i.e., they exhibit stress hyporesponse periods. In this study, we evaluated whether gonadotropin regulatory factors exhibit stress hyporesponsive periods in male and female rats. Rats were intraperitoneally injected with lipopolysaccharide (100µg/kg) (LPS group) or saline (control group) on postnatal day (PND) 5, 10, 15, or 25. Then, their serum luteinizing hormone (LH) concentrations and hypothalamic mRNA levels of gonadotropin regulatory factors; i.e., kisspeptin (Kiss1), the kisspeptin receptor (Kiss1r), and gonadotropin-releasing hormone (GnRH), were measured at 2h after the injection. The hypothalamic mRNA levels of pro-inflammatory cytokines were also measured because they suppress gonadotropin secretion. The serum LH concentration of the LPS group was lower than that of the control group at PND25 in both sexes, but no such difference was seen at PND5, 10, or 15 in either sex. In both sexes, the hypothalamic tumor necrosis factor (TNF)α and interleukin (IL)-6 mRNA expression levels of the LPS group were higher than those of the control group at PND25, but not at PND5 or 10. The hypothalamic IL-1ß mRNA expression level of the LPS group was higher than that of the control group at all time points. The hypothalamic Kiss1, Kiss1r, and GnRH mRNA expression levels of the LPS and control groups did not differ at any time point in either sex. These findings suggest that gonadotropin regulatory factors exhibit stress hyporesponse periods. The hypothalamic-pituitary-gonadal axis (HPG) might become responsive to immune stress between PND15 and 25, which could be related to enhanced hypothalamic cytokine expression. The avoidance of infectious stress during the early neonatal period might be important for normal development of the HPG axis.

Prenatal undernutrition increases the febrile response to lipopolysaccharides in adulthood in male rats.

It has been reported that prenatal undernutrition affects the development of the peripheral immune system. In this study, the effects of prenatal undernutrition on the febrile response and hypothalamic innate immune system were evaluated in male rats. Pregnant rats were divided into normally nourished (NN) and undernourished groups (UN). The febrile and anorectic responses to lipopolysaccharides (LPS) were evaluated in the offspring of NN and UN dams. The hypothalamic expression levels of pro-inflammatory cytokines, toll-like receptor 4 (TLR4), and neuropeptide Y (NPY) were also evaluated. The UN rats exhibited significantly lighter body weights than the NN rats at birth; however, their mean body weight was the same as that of the NN rats by postnatal day 10. In adulthood, the UN rats exhibited significantly stronger febrile responses than the NN rats, and the anorectic responses of the UN rats also tended to be stronger than those of the NN rats. On the other hand, no differences in hypothalamic interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, TLR4, or NPY mRNA expression were detected between the NN and UN rats. These results suggest that prenatal undernutrition has long-lasting effects on the febrile response to LPS. However, the precise mechanism underlying these effects and their pathophysiological significance remain unclear.

The effects of prenatal undernutrition and postnatal high-fat diet on hypothalamic Kiss1 mRNA and serum leptin levels.

Prenatal undernutrition and postnatal overnutrition increase the risk of some metabolic disorders in adulthood, and hypothalamic leptin resistance makes an important contribution to these effects. Leptin plays important roles in the maintenance of reproductive function, and its actions might be partially mediated by kisspeptin, which is a potent positive regulator of gonadotropin-releasing hormone. In this study, the effects of prenatal undernutrition and postnatal overnutrition on reproductive parameters and sexual maturation during the peripubertal period were evaluated. Rats subjected to prenatal undernutrition (IUGR) and fed a postnatal high-fat diet (HFD) (n = 7) exhibited 40% higher serum leptin levels and 30% lower hypothalamic Kiss1 (the gene encoding kisspeptin) mRNA levels than those subjected to prenatal undernutrition (IUGR) and fed a normal diet (n = 7). No such HFD-induced postnatal alterations were observed in the rats fed a normal diet during the prenatal period (control) (n = 7 per group). Although the consumption of the HFD did not affect the serum luteinizing hormone levels or body weight of the IUGR or control rats, it did promote vaginal opening in both groups (evaluated in 14 rats per group). These findings indicate that hypothalamic leptin resistance might occur in IUGR-HFD rats, but these changes do not influence downstream effectors of the reproductive endocrinological system. They also suggest that the relationships between nutritional conditions, body weight, reproductive factors, and sexual maturation are complex.

Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats.

Oxytocin (OT) affects the central nervous system and is involved in a variety of social and non-social behaviors. Recently, the role played by OT in energy metabolism and its organizational effects on estrogen receptor alpha (ER-α) during the neonatal period have gained attention. In this study, the developmental changes in the hypothalamic mRNA levels of OT, the OT receptor (OTR), and ER-α were evaluated in male and female rats. In addition, the fasting-induced changes in the hypothalamic mRNA levels of OT and the OTR were evaluated. Hypothalamic explants were taken from postnatal day (PND) 10, 20, and 30 rats, and the mRNA level of each molecule was measured. Hypothalamic OT mRNA expression increased throughout the developmental period in both sexes. The rats' hypothalamic OTR mRNA levels were highest on PND 10 and decreased throughout the developmental period. In the male rats, the hypothalamic mRNA levels of ER-α were higher on PND 30 than on PND 10. On the other hand, no significant differences in hypothalamic ER-α mRNA expression were detected among the examined time points in the female rats, although hypothalamic ER-α mRNA expression tended to be higher on PND 30 than on PND 10. Significant positive correlations were detected between hypothalamic OT and ER-α mRNA expression in both the male and female rats. Hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points in either sex. These results indicate that hypothalamic OT expression is not sensitive to fasting during the developmental period. In addition, as a positive correlation was detected between hypothalamic OT and ER-α mRNA expression, these two molecules might interact with each other to induce appropriate neuronal development.

The responses of hypothalamic NPY and OBRb mRNA expression to food deprivation develop during the neonatal-prepubertal period and exhibit gender differences in rats.

Neuropeptide Y (NPY) is an important hypothalamic orexigenic neuropeptide that acts in the brain. It has been established that the fasting-induced up-regulation of NPY expression is mainly caused by a reduction in the activity of leptin, which is a hormone secreted by adipose tissue. We have reported that in female rats hypothalamic NPY mRNA expression does not respond to fasting during the early neonatal period, but subsequently becomes sensitive to it later in the neonatal period. In this study, we compared the developmental changes in the responses of NPY and leptin expression to fasting between male and female rats during the neonatal to pre-pubertal period. Fasting was induced by maternal deprivation during the pre-weaning period (postnatal days 10 and 20) and by food deprivation during the post-weaning period (postnatal day 30). Hypothalamic NPY mRNA expression was not affected by fasting on postnatal day 10, whereas it was increased by fasting on postnatal day 20 and 30 in both males and females. On the other hand, the serum leptin level was decreased by fasting at all examined ages in both sexes. Namely, hypothalamic NPY mRNA expression was not correlated with the reduction in the serum leptin level at postnatal day 10 in either sex. Under the fasted conditions, the hypothalamic NPY mRNA levels of the males were higher than those of the females on postnatal days 20 and 30, whereas no such differences were observed under the normal nourishment conditions. The serum leptin levels observed under the fasted conditions did not differ between males and females at any examined age. These results suggest that some hypothalamic NPY functions develop during the neonatal period and that there is no major difference between the sexes with regard to the time when NPY neurons become sensitive to fasting. They also indicate that hypothalamic NPY expression is more sensitive to under-nutrition in male rats than in female rats, at least during the pre-pubertal period.

The advancement of the onset of vaginal opening in female rats subjected to chronic testosterone treatment occurs independently of hypothalamic Kiss1 and RFRP expression.

OBJECTIVE: The neonatal and/or prepubertal androgen milieu affects sexual maturation. In rodents, neonatal chronic testosterone treatment, which is used as a model of polycystic ovary syndrome (PCOS), results in the onset of vaginal opening occurring earlier in the pubertal period. DESIGN: In the present study, the changes in hypothalamic Kiss1 (a gonadotropin-releasing hormone (GnRH)-stimulating factor) and RF-amide related peptide (RFRP; a GnRH inhibitory factor) mRNA expression induced by testosterone treatment were examined in order to clarify whether these factors are involved in the testosterone-induced acceleration of sexual maturation. RESULTS: The onset of vaginal opening occurred earlier and uterine weight was increased in female rats subjected to chronic (from postnatal day 23 to day 31) testosterone treatment. Contrary to our expectations, the rats' hypothalamic Kiss1 and Kiss1 receptor mRNA levels were not changed, and their serum luteinizing hormone (LH) levels were decreased. Although hypothalamic RFRP mRNA expression was decreased in the testosterone-treated rats, this change was not reflected in their serum LH levels. CONCLUSIONS: These results indicate that the advancement of sexual maturation observed in chronic testosterone-treated rats might be caused by a peripheral, rather than a central, mechanism.

Developmental changes in hypothalamic toll-like-receptor 4 mRNA expression and the effects of lipopolysaccharide on such changes in female rats.

Hypothalamic pro-inflammatory cytokine expression exhibits a weaker response to lipopolysaccharides (LPS) during the early neonatal period than during the later developmental period. Although toll-like receptor 4 (TLR4), which recognizes bacterial molecules, activates pro-inflammatory cytokine responses, the developmental changes in hypothalamic TLR4 expression have not been evaluated. In this study, the hypothalamic TLR4 mRNA levels of saline-injected and LPS-injected rats were measured during the neonatal, pre-pubertal, and post-pubertal periods. The rats' hypothalamic TLR4 mRNA levels gradually increased from the neonatal to pubertal period and were altered by the injection of LPS at all examined ages (postnatal day (PND) 5, 15, 25, and 42). LPS injection resulted in decreased hypothalamic TLR4 mRNA expression at PND5, whereas it increased hypothalamic TLR4 mRNA expression at PND15, 25, and 42. After the injection of LPS, the hypothalamic mRNA levels of the pro-inflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor α, and IL-6 were attenuated during the early developmental period and increased acutely on PND42. The expression profiles of these pro-inflammatory cytokines exhibited similar, but not entirely consistent, changes to those displayed by TLR4 during the developmental period. Hypothalamic TLR4 mRNA expression gradually increased throughout the developmental period, whereas the mRNA expression levels of the pro-inflammatory cytokines increased acutely at PND42. Thus, it is assumed that hypothalamic TLR4 hypoactivity contributes to the low sensitivity of pro-inflammatory cytokines to LPS during the early developmental period.