The influence of psychological stress in early life on sexual maturation and sexual behavior in male and female rats.

PURPOSE: We studied the influence of psychological stress during the early neonatal period on sexual maturation and sexual behavior in rats. METHODS: Neonatal male and female rats were divided into control (C) and maternal separation (MS) groups (n = 20-24 per group). The pups in the MS groups were placed in isolation cages for 240 minutes/d from postnatal days 2-11. Vaginal opening (VO) in females and preputial separation (PS) in males (indicators of sexual maturation) were monitored, as was the estrous cycle in females. Thereafter, sexual behavior was monitored twice at 13 and 15 weeks of age. RESULTS: As for sexual maturation, the onset of PS occurred significantly earlier in the MS group than in the C group, whereas the onset of VO did not differ between the groups. The length of the estrous cycle did not differ between the groups. The frequencies of sexual behaviors did not differ between the groups in either sex. CONCLUSIONS: In conclusion, early-life psychological stress induced by MS advanced sexual maturation in male rats, whereas it did not affect sexual maturation in female rats. On the other hand, early-life psychological stress might not affect sexual behavior in adulthood in either sex.

The reduction in sexual behavior of adult female rats exposed to immune stress in the neonatal period is associated with reduced hypothalamic progesterone receptor expression.

PURPOSE: We examined the mechanism by which neonatal immune stress reduces the sexual behavior of female rats in adulthood. METHODS: Neonatal female rats were randomly divided into 3 groups: control (n = 11), postnatal day 10 lipopolysaccharide (PND10LPS) (n = 23), and PND25LPS (n = 11) groups, which received intraperitoneal injections of LPS (100 µg/kg) or saline on PND10 and 25. Daily inspections of the vaginal opening (VO) were performed from PND27 to PND37. Thereafter, the frequency of estrus was assessed for 15 days. Female rats (at 11-12 weeks of age) were placed in a cage with male rats, and their sexual behavior was monitored for 30 min. The hypothalamic mRNA expression levels of factors related to sexual behavior were examined via real-time PCR. RESULTS: VO occurred later and the frequency of estrus was lower in the PND10LPS group compared to the control group. The number of lordosis behaviors and the total number of mounts performed by male partners were lower in the PND10LPS and PND25LPS groups than in the control group. Acceptability: The lordosis quotient and lordosis rating were lower in the PND10LPS group than in the control group. Proceptive behavior: the number of ear wiggling events was lower in the PND10LPS group than in the other groups, and the number of hops/darts was lower in the PND10LPS group than in the control group. The hypothalamic mRNA expression level of progesterone receptors (PR)A + B was lower in the PND10LPS group than in the control group, and the hypothalamic PRB mRNA expression level was lower in the PND10LPS and PND25LPS groups than in the control group. CONCLUSION: Neonatal immune stress impeded sexual behavior and hypothalamic PR mRNA expression in female rats. Decreased progesterone activity in the hypothalamus might explain the reduction in sexual behavior seen in these rats.

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.

Neurokinin B receptor agonist and Dynorphin receptor antagonist stimulated luteinizing hormone secretion in fasted male rodents.

Kisspeptin/neurokinin B (NKB)/dynorphin (Dyn) (KNDy) neuron in hypothalamic arcuate nucleus plays a key role in GnRH/LH pulsatile secretion. We aimed to determine whether stimulation of NKB/neurokinin 3 receptor (NK3R) signaling and inhibition of Dyn/kappa-opioid receptor (KOR) signaling recover LH secretion that is suppressed by acute fasting in male rats. Furthermore, we determined dose dependent effect of NKB/NK3R signaling on serum LH level under acute fasting condition in male mice. Mature male rats were injected saline (0.1 mL) and senktide (20 µg/kg), a NK3R agonist, or nor-BNI (800 µg/kg), a KOR antagonist intraperitoneally (ip) after 72 h fasting. And mature male mice were injected multiple doses of senktide, ip after 48 h fasting. Blood and brain sample were collected 90 min after injections for LH measurement and hypothalamic mRNA expressions. All three studies showed significantly lower LH concentration in fasted groups than non-fasted groups. Senktide did not recover LH suppressed by acute fasting in male rats, whereas nor-BNI injected male rats showed significantly higher LH than 72 h fasted male rats (p < 0.05). Mice study showed significantly higher LH concentration in higher doses senktide groups than 48 h fasted group and one of lower doses senktide group. These results suggest that stimulation of NKB/NK3R signaling and attenuation of Dyn/KOR signaling could recover suppressed LH secretion under acute fasting condition in male rodents.

Clinical outcome of various metformin treatments for women with polycystic ovary syndrome.

Aim: Polycystic ovary syndrome (PCOS) is an ovulatory disorder and insulin resistance and diabetes are involved in its pathophysiology. Metformin, an anti-diabetic agent, has been reported to be useful to induce ovulation. Methods: Metformin treatment was classified into four types: (1) clomiphene-metformin combination treatment for clomiphene-resistant patients; (2) clomiphene-metformin combination for clomiphene-sensitive patients; (3) clomiphene-metformin combination for naïve patients; and (4) metformin monotherapy. The patients underwent physical, endocrinological, and clinical examinations for their ovulation rates, pregnancy rates, and follicular development. Results: The ovulation rates, pregnancy rates, and single follicular development were not significantly different among the clomiphene-metformin combination treatment groups. In the Body Mass Index (BMI) subanalysis, the pregnancy rate was higher in the BMI≥30 kg/m2 group than in the other three groups with a BMI of ≤30 kg/m2 in both cycles and cases. The ovulation rates and pregnancy rates were significantly higher in the group with a fasting insulin of ≥15 µU/mL than in the groups with a fasting insulin of <15 µU/mL in both cycles and cases. Conclusion: Clomiphene-metformin combination treatment appears to be useful, at least for clomiphene-resistant patients, and a BMI of >30 kg/m2 and a fasting insulin of ≥15 µU/mL appear to be predictors of a good result with this treatment.

Prenatal undernutrition disrupted the sexual maturation, but not the sexual behavior, in male rats.

Purpose: Exposure to various stressors, including psychological, metabolic, and immune, in the perinatal period induces long-lasting effects in physiological function and increase the risk of metabolic disorders in later life. In the present study, sexual maturation and sexual behavior were assessed in prenatally undernourished mature male rats. Methods: All the pregnant rats were divided into the maternal normal nutrition (mNN) group and the maternal undernutrition (mUN) group. The mUN mothers received 50% of the amount of the daily food intake of the mNN mothers. Preputial separation and sexual behavior were observed in randomly selected pups of the mNN and mUN groups. Results: The body weight of the mothers was significantly lighter in the mUN group than in the mNN group. Similarly, the pups in the mUN group showed a significantly lower body weight than those in the mNN group from postnatal day (PND) 1 to PND 15. The preputial separation day was significantly delayed in the mUN group, compared to the mNN group. Sexual behavior did not show any significant difference between the two groups. Conclusion: These findings indicated that prenatal undernutrition delayed sexual maturation, but did not suppress sexual behavior, in mature male rats.

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.

Relationship between serum anti-Mullerian hormone and clinical parameters in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an ovulatory disorder that affects 6-10% of women of reproductive age. Serum AMH level may be an additional factor, or surrogate of PCOM, in the diagnostic criteria of PCOS. We evaluated the correlations between the serum AMH level and various endocrine and metabolic features in PCOS using the latest fully automated assay. Serum AMH level was compared between 114 PCOS patient (PCOS group) and 95 normal menstrual cycle women (Control group). Correlations between serum AMH level and various endocrine and metabolic factors were analysed in PCOS group. The serum AMH level was significantly higher in the PCOS group (8.35±8.19 ng/mL) than in the Control group (4.99±3.23 ng/mL). The serum AMH level was independently affected by age and the presence of PCOS on multiple regression analysis. Ovarian volume per ovary (OPVO) showed the strongest positive correlation (r=0.62) with the serum AMH level among related factors. On receiver operating characteristic (ROC) curve analysis, the cut-off value of AMH for the diagnosis of PCOS was 7.33 ng/mL, but this value did not have high efficacy (sensitivity 44.7%, specificity 76.8%). A cut-off value of 10 ng/mL had a high specificity of 92.6%, although the sensitivity was low (24.6%). The serum AMH level was elevated and reflected ovarian size in PCOS patients. The serum AMH level could be a surrogate for ultrasound findings of the ovaries in PCOS and might be useful for estimating ovarian findings without transvaginal ultrasound in the diagnosis of PCOS.

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.

Kisspeptin mRNA expression is increased in the posterior hypothalamus in the rat model of polycystic ovary syndrome.

Hypersecretion of luteinizing hormone (LH) is a common endocrinological finding of polycystic ovary syndrome (PCOS). This derangement might have a close relationship with hypothalamic kisspeptin expression that is thought to be a key regulator of gonadotropin-releasing hormone (GnRH). We evaluated the relationship between the hypothalamic-pituitary-gonadal axis (HPG axis) and kisspeptin using a rat model of PCOS induced by letrozole. Letrozole pellets (0.4 mg/day) and control pellets were placed subcutaneously onto the backs of 3-week-old female Wistar rats. Body weight, vaginal opening and vaginal smear were checked daily. Blood and tissues of ovary, uterus and brain were collected at 12-weeks of age. An hypothalamic block was cut into anterior and posterior blocks, which included the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), respectively, in order to estimate hypothalamic kisspeptin expression in each area. The letrozole group showed a similar phenotype to human PCOS such as heavier body weight, heavier ovary, persistent anovulatory state, multiple enlarged follicles with no corpus luteum and higher LH and testosterone (T) levels compared to the control group. Kisspeptin mRNA expression in the posterior hypothalamic block including ARC was higher in the letrozole group than in the control group although its expression in the anterior hypothalamic block was similar between groups. These results suggest that enhanced KNDy neuron activity in ARC contributes to hypersecretion of LH in PCOS and might be a therapeutic target to rescue ovulatory disorder of PCOS in the future.

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.

The sensitivity of adipose tissue visfatin mRNA expression to lipopolysaccharide-induced endotoxemia is increased by ovariectomy in female rats.

Visfatin plays an important role in inflammatory and metabolic conditions. In this study, the effects of septic stress on the serum, white-adipose-tissue (WAT), and liver visfatin levels of male and female rats were examined. Both gonadally intact (sham) and ovariectomized (OVX) female rats were used in order to evaluate the effects of the gonadal hormonal milieu on visfatin responses. Under the saline-injected conditions, the serum visfatin levels and the hepatic, subcutaneous, and visceral WAT visfatin mRNA levels of the OVX and sham rats did not differ. The serum visfatin levels and the subcutaneous, visceral WAT, and hepatic visfatin mRNA levels of both male and female rats were increased by the injection of a septic dose (5mg/kg) of LPS. At 6h after the injection of LPS, the WAT visfatin mRNA levels of the OVX rats were higher than those of the sham rats, whereas the serum visfatin levels and hepatic visfatin mRNA levels of the two groups did not differ. In the cultured visceral WAT, visfatin antagonist (FK-866) attenuated the LPS-induced up-regulations of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α). The pathophysiological roles of visfatin under septic conditions remain to be clarified. In addition, the precise mechanisms responsible for the increased WAT visfatin expression seen after ovariectomy and the effects of such changes should also 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.

Prenatal undernutrition results in greater lipopolysaccharide-induced changes in hypothalamic TNF-α expression, but does not affect the equivalent changes in the serum levels of luteinizing hormone and testosterone, in adult male rats.

Immune stress can cause reproductive dysfunction. Some hypothalamic factors such as pro-inflammatory cytokines play pivotal roles in reproductive disorders under immune stress conditions. Recently, it has been reported that prenatal undernutrition affects not only metabolic functions, but also the responses of physiological functions to immune stress in adulthood. In this study, the long-term effects of prenatal undernutrition on the responses of hypothalamic pro-inflammatory cytokine (interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6) expression; reproductive endocrine factors; i.e., the serum levels of gonadotropins and testosterone; and hypothalamic kisspeptin expression to lipopolysaccharide (LPS) were examined in male rats. Pregnant rats were divided into two groups; i.e., the normally nourished group and the undernourished (50% food restricted) group. The offspring of the normally nourished mothers (control) and undernourished mothers (the intrauterine growth restriction [IUGR] group) were sub-divided into saline-injected and LPS (500 µg, i.p.)-injected groups at 10 weeks of age. The rats' hypothalamic pro-inflammatory cytokine levels and serum luteinizing hormone (LH) and testosterone levels were measured and compared between the control and IUGR groups. The hypothalamic pro-inflammatory cytokine mRNA levels of the LPS-injected rats were significantly higher than those of the saline-injected rats in both the control and IUGR groups. The changes in the hypothalamic expression level of TNF-α, but not those of the other cytokines, induced in response to LPS were more marked in the IUGR group than in the control group. On the other hand, although the serum LH and testosterone levels of the LPS-injected rats were significantly lower than those of the saline-injected rats in both the control and IUGR groups, their levels did not differ between the control and IUGR groups under the LPS-injected conditions. These results suggest that prenatal undernutrition results in more marked LPS-induced changes in hypothalamic TNF-α expression, but does not alter the effects of LPS on the serum levels of LH or testosterone, in adult male rats.

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.