Altankhuu Tungalagsuvd

Hypothalamic Kiss1 and RFRP gene expressions are changed by a high dose of lipopolysaccharide in female rats

Reproductive function is suppressed by several types of stress. Hypothalamic kisspeptin, which is a product of the Kiss1 gene, and GnIH/RFRP have pivotal roles in the regulation of GnRH and gonadotropins through their receptors Kiss1r and GPR147 in many species. However, alterations of these factors under stress conditions have not been fully evaluated. This study investigated the mechanisms of immune stress-induced reproductive dysfunction, especially focusing on the changes of Kiss1 and RFRP gene expression. Serum LH levels and hypothalamic Kiss1 and GnRH mRNA levels were decreased, while hypothalamic RFRP and GPR147 mRNA levels were increased by administration of a high dose of LPS (5mg/kg) in both ovariectomized and gonadal intact female rats. In this condition, Kiss1 and/or RFRP mRNA levels were positively and negatively correlated with GnRH expression, respectively. In contrast, hypothalamic Kiss1, RFRP, and GPR147 mRNA levels were not changed by administration of a moderate dose of LPS (500μg/kg) in ovariectomized rats. Rats with high-dose LPS injection showed more prolonged fever responses and severe anorexia compared with rats with moderate-dose LPS injection, indicating that more energy was used for the immune response in the former. These results suggest that the underlying mechanisms of dysfunction of gonadotropin secretion are changed according to the severity of immune stress, and that changes of some reserved factors, such as kisspeptin and RFRP, begin to participate in the suppression of GnRH and gonadotropin in severe conditions. As reproduction needs a large amount of energy, dysfunction of gonadotropin secretion under immune stress may be a biophylatic mechanism by which more energy is saved for the immune response.

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.

Changes in the responsiveness of hypothalamic prokineticin 2 mRNA expression to food deprivation in developing female rats

Prokineticin 2 (PK2) is highly expressed in several regions of the central nervous system, including the hypothalamus. Recently, it has been suggested that PK2 plays a role in appetite regulation. In adult male rodents, the administration of PK2 decreased food intake, and PK2 mRNA expression was reduced by food deprivation. Usually, the changes in the expression levels of appetite‐regulating factors induced in response to fasting are not fully established during the neonatal period. Thus, we investigated the developmental changes in hypothalamic PK2 mRNA expression and the alterations in hypothalamic PK2 mRNA expression induced by fasting during the pre‐pubertal period in female rats. The changes in hypothalamic neuropeptide Y (NPY) mRNA expression were also examined because NPY is a potent appetite regulatory factor. Hypothalamic PK2 mRNA expression was extremely high during the early neonatal period (postnatal day (PND) 5) compared with that observed during subsequent periods (PND15, 25, and 42), while hypothalamic NPY mRNA expression did not differ among any of the examined periods. A fasting‐induced reduction in hypothalamic PK2 mRNA expression was observed on PND5, but no fasting‐induced increase in hypothalamic NPY mRNA expression was seen during the same period. In addition, the fasting‐induced reduction in hypothalamic PK2 mRNA expression observed on PND5 was more marked than that seen on PND25. These results suggest that the sensitivity of hypothalamic PK2 expression to undernutrition develops during the early neonatal period, when the responses of other appetite regulatory factors to such pressures remain immature.

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 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 (100 μg/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 2 h 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‐1β 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.

LH and testosterone production are more sensitive to the suppressive effects of food deprivation in prenatally undernourished male rats

Although prenatal undernutrition affects the development of metabolic, physiological, and reproductive functions, it remains unclear whether it also affects physiological responses to undernutrition in adulthood. Therefore, in this study we examined whether prenatal undernutrition alters the sensitivity of the hypothalamic–pituitary–gonadal (HPG) axis to fasting in adult male rats. The offspring of ad libitum fed dams (control) and ∼50% food‐restricted (during the late gestational period) dams (IUGR) were sub‐divided into ad libitum fed (fed) and 48 h food deprivation (FD) groups at 10 weeks of age. In each group, the serum levels of luteinizing hormone (LH), testosterone, and leptin and the hypothalamic mRNA expression levels of gonadotropin‐releasing hormone (GnRH) regulatory factors were measured. The serum LH and testosterone levels of the IUGR‐fed rats were significantly or tend to be higher than those of the control‐fed rats, respectively. The serum LH levels of the IUGR‐FD rats were lower than those of the IUGR‐fed rats. Similarly, the serum testosterone levels of the IUGR‐FD rats tended to be lower than those of the IUGR‐fed rats. On the other hand, the serum LH and testosterone levels of the control‐fed and control‐FD rats did not differ. The serum leptin levels of the IUGR fed rats were higher than those of the control‐fed rats. The serum leptin levels of the control‐FD and IUGR‐FD rats were lower than those of the control‐fed and IUGR‐fed rats, respectively. The hypothalamic neuropeptide Y (NPY) mRNA levels of the IUGR‐FD rats were higher than those of the IUGR‐fed rats. Similarly, hypothalamic NPY mRNA levels of control‐FD rats were higher than those of the control‐fed rats. The hypothalamic kisspeptin, kisspeptin receptor, RFamide‐related peptide, GPR147, and OBRb mRNA levels of control fed rats did not differ between control‐fed and IUGR‐fed rats. Their mRNA levels of the fed and FD rats did not differ in the control or IUGR groups. These results suggested that prenatal undernutrition increased the basal LH and testosterone production, whereas they are easily reduced by food deprivation in male rats. Changes of serum leptin level, but not of hypothalamic reproductive related factors, might be involved in these alterations. However, the precise mechanisms responsible for these effects remain unclear.

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.

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.

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.

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)‐1β, 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.