Toshiya Matsuzaki

Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats

Gonadotropin-inhibitory hormone (GnIH), a newly discovered hypothalamic RFamide peptide, inhibits reproductive activity by decreasing gonadotropin synthesis and release in birds. The gene of the mammalian RFamide-related peptides (RFRP) is orthologous to the GnIH gene. This Rfrp gene gives rise to the two biologically active peptides RFRP-1 (NPSF) and RFRP-3 (NPVF), and i.c.v. injections of RFRP-3 suppress LH secretion in several mammalian species. In this study, we show whether RFRP-3 affects LH secretion at the pituitary level and/or via the release of GnRH at the hypothalamus in mammals. To investigate the suppressive effects of RFRP-3 on the mean level of LH secretion and the frequency of pulsatile LH secretion in vivo, ovariectomized (OVX) mature rats were administered RFRP-3 using either i.c.v. or i.v. injections. Furthermore, the effect of RFRP-3 on LH secretion was also investigated using cultured female rat pituitary cells. With i.v. administrations, RFRP-3 significantly reduced plasma LH concentrations when compared with the physiological saline group. However, after i.c.v. RFRP-3 injections, neither the mean level of LH concentrations nor the frequency of the pulsatile LH secretion was affected. When using cultured pituitary cells, in the absence of GnRH, the suppressive effect of RFRP-3 on LH secretion was not clear, but when GnRH was present, RFRP-3 significantly suppressed LH secretion. These results suggest that RFRP-3 does not affect LH secretion via the release of GnRH, and that RFRP-3 directly acts upon the pituitary to suppress GnRH-stimulated LH secretion in female rats.

Effects of intrauterine undernutrition on hypothalamic Kiss1 expression and the timing of puberty in female rats

Recent studies have suggested that intrauterine undernutrition is closely associated with the pathogenesis of diseases after birth. Perinatal undernutrition is known to disturb the development of reproductive function and delay the onset of puberty in some species. Using a rat model, we determined the effects of prenatal undernutrition on the development of the hypothalamic kisspeptin system and evaluated whether the alteration of the kisspeptin system contributes to the delayed onset of puberty induced by prenatal undernutrition. We also evaluated the effects of prenatal undernutrition on the developmental changes in serum leptin levels because leptin was a putative positive regulator of the hypothalamic kisspeptin system. We compared the timing of vaginal opening (VO) and the developmental changes in body weight, hypothalamic Kiss1 mRNA levels, and serum leptin concentrations between offspring with prenatal undernutrition (UN offspring) and normal nutrition (NN offspring). After birth, the UN offspring showed rapid growth and had caught up to body weight of the NN offspring by postnatal day 12. After postnatal day 16, the UN offspring showed significantly lower Kiss1 mRNA levels than the NN offspring, despite their significantly higher serum leptin levels (at days 20 and 28). The timing of VO in the UN offspring was delayed compared with that in the NN offspring, and chronic central injection of kisspeptin normalized the timing of VO in the UN offspring. These results suggest that decreased hypothalamic kisspeptin action contributes to the delayed onset of puberty in prenatally undernourished female rats. Increased leptin resistance in the kisspeptin system might be involved in these alterations.

Developmental changes in the mammalian gonadotropin-inhibitory hormone (GnIH) ortholog RFamide-related peptide (RFRP) and its cognate receptor GPR147 in the rat hypothalamus.

The mammalian gonadotropin‐inhibitory hormone (GnIH) ortholog RFamide‐related peptide (RFRP) is considered to act on gonadotropin‐releasing hormone (GnRH) neurons and on the pituitary to inhibit gonadotropin release and synthesis. To understand the functional significance of this neuropeptide, we investigated the physiological changes in RFRP at mRNA and peptide levels, as well as at the mRNA level of its cognate receptor, G protein‐coupled receptor 147 (GPR147), in the rat hypothalamus during development. We also investigated the effects of gonadal steroids on mRNA expression levels of these molecules. In male rats, mRNA expressions of both RFRP and GPR147 increased from postnatal days 12 and 16, peaking at postnatal days 35 and 42, respectively. However, their expressions fell at postnatal day 49. In female rats, mRNA expression of RFRP continued to increase throughout development; mRNA expression of GPR147 in female rats increased from postnatal day 16, peaking at postnatal day 28, but decreased from postnatal day 35. The hypothalamic contents of RFRP on postnatal days 28 and 42 were significantly higher than on postnatal day 4 in male rats, and those on postnatal day 42 were significantly higher than those on postnatal days 4 and 28 in females. Neither orchidectomy nor ovariectomy influenced mRNA expression levels of RFRP or GPR147 in the prepubertal period when endogenous sex steroid levels were low in males and females. Administration of estradiol‐17β (E2) increased mRNA expression of RFRP in prepubertal females. These results suggest that the hypothalamic RFRP system changes during development. An ovarian sex steroid, E2, may stimulate mRNA expression of RFRP in the prepubertal period when the basal E2 concentration is low.

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.

Decreased expression of kisspeptin mediates acute immune/inflammatory stress-induced suppression of gonadotropin secretion in female rat

Kisspeptin and its corresponding receptor, the G protein-coupled receptor 54, play an important role in reproductive systems. It has been suggested that reproductive disorders in metabolically disrupted animals are caused by the alteration of hypothalamic KiSS-1 systems. Immune/inflammatory challenge is also known to disrupt reproductive function. However, the effects of immune/inflammatory challenge on KiSS-1 systems have not been investigated. In this study, we showed that lipopolysaccharide (LPS) injection decreased hypothalamic KiSS-1 mRNA expression as well as plasma LH levels in ovariectomized rats. Indomethacin completely blocked the suppressive effects of LPS on LH secretion and KiSS-1 mRNA level. Furthermore, we showed that iv injection of kisspeptin increased plasma LH levels in LPS-administrated rats to the same degree as in saline-injected rats. These results suggest that KiSS-1 systems are sensitive to immune/inflammatory challenge conditions and transmit these signals into the central reproductive system.

Neonatal LPS injection alters the body weight regulation systems of rats under non-stress and immune stress conditions

It has been reported that prenatal immune stress induced by lipopolysaccharides or cytokines increases food intake and leads to obesity and other features of metabolic syndrome in adulthood. Using Sprague–Dawley rats, we evaluated whether neonatal LPS injection altered their body weight regulation systems under non‐stress and immune stress conditions. On Day 10 after birth, all pups were injected with LPS (100 μg/kg, i.p.) (PND10LPS) or saline (PND10Saline). After weaning, body weight was significantly elevated in PND10LPS compared with PND10Saline. Thereafter, the rats were injected with LPS (100 μg/kg, i.p.) or saline (used as a basal condition) from 7 to 8 weeks of age. Under basal conditions, cumulative food intake were significantly higher, serum leptin concentration was significantly increased, and hypothalamic NPY mRNA expression was significantly decreased in PND10LPS compared with PND10Saline. Under adult LPS injected conditions, body weight gain and cumulative food intake were suppressed in both the PND10LPS and PND10Saline groups compared with those observed under basal adult saline‐injected conditions. The suppressive effects induced by adult LPS injection were less evident in the PND10LPS group than in the PND10Saline group. Adult LPS injection increased the serum leptin concentration in the PND10Saline rats, but not in the PND10LPS rats. In addition, adult LPS injection increased the mRNA expression of anorexinergic factors (IL‐1β, and TNF‐α), and decreased that of the orexinergic factor NPY in both groups. However, the influence of adult LPS injection upon these factors was less evident in the PND10LPS group than in the PND10Saline group. These results suggest that neonatal LPS injection alters body weight regulation under both non‐stress and immune stress conditions in male rats. Changes in the endocrine, neuropeptide, and cytokine regulation systems might be involved in these alterations.

Hypothalamic ghrelin suppresses pulsatile secretion of luteinizing hormone via β-endorphin in ovariectomized rats.

Objectives: Ghrelin, an endogenous growth hormone secretagogue, is a known accelerator of feeding behavior and suppresses pulsatile secretion of luteinizing hormone (LH) in ovariectomized rats. However, the mechanisms underlying this action remain unclear. We examined the effects of naloxone (NAL), a specific opioid antagonist, on the suppression of pulsatile LH secretion by ghrelin to determine whether β-endorphin (β-END) is involved in this suppressive effect. Methods: Ghrelin was administered intracerebroventricularly, and NAL was injected intravenously in ovariectomized rats; then, serum LH concentrations were measured by radioimmunoassay in blood samples drawn every 6 min for 2 h to analyze pulsatile secretion. Results: Administration of ghrelin significantly reduced mean LH concentration and pulse frequency. Coadministration of NAL with ghrelin significantly restored mean LH concentration and pulse frequency. Conclusion: Suppressive effect of intracerebroventricular injection of ghrelin on pulsatile LH secretion was mediated by β-END, suggesting that hypothalamic ghrelin suppressed pulsatile gonadotropin-releasing hormone secretion via β-END in female rats.

Neonatal immune challenge affects the regulation of estrus cyclicity and feeding behavior in female rats

A single immune challenge with lipopolysaccharide (LPS) in the neonatal period has a long‐lasting influence on immune response. Using female Sprague–Dawley rats, we examined whether neonatal LPS challenge influences the life‐long neuroendocrine sensitivity of reproductive function and feeding behavior to LPS, and whether stress‐related neuropeptides and their receptors are involved in neonatal LPS‐induced physiological change. On day 10 after birth, all pups were injected with LPS (100 μg/kg, i.p.) or saline. Then, in Experiment 1, LPS (100 μg/kg, i.p.) or saline was injected at diestrous in adulthood, and the length of the estrous cycle, 24 h food intake and body weight change were recorded. In Experiment 2, the mRNA expression levels of corticotropin‐releasing hormone (CRH), urocortin (UCN), urocortin 2 (UCN2), CRH receptor type 1 (CRH‐R1) and CRH receptor type 2 (CRH‐R2) in the hypothalamus were measured using real‐time PCR. LPS injection in adulthood prolonged the estrous cycle in neonatal LPS‐injected rats. LPS injection in adulthood decreased food intake and body weight in both neonatal LPS‐ and saline‐injected rats, more so in the latter. Basal expressions of UCN2 and CRH‐R2 mRNA were higher in neonatal LPS‐injected rats than in saline‐injected rats. These findings indicate that neonatal immune challenge influences the anti‐stress regulation of the estrous cycle and feeding behavior in adulthood. Increased expression of UCN2 and CRH‐R2 might enhance the sensitivity of the estrous cycle in suppressing the effects of LPS.

Neonatal lipopolysaccharide exposure attenuates the homotypic stress-induced suppression of LH secretion in adulthood in male rat

Neonatal immune challenges have a long‐lasting influence on immune response. Using male Sprague–Dawley rats, we examined whether neonatal lipopolysaccharide (LPS) challenge alters the sensitivity of male reproductive function to adult LPS challenge and at which level (central or testes) the alteration occurs. We also examined the mRNA expression of proinflammatory cytokines in the hypothalamus and testes because they have a pivotal role in immune stress‐induced suppression of gonadotropin secretion and testosterone synthesis. On day 10 after birth, all the pups were injected with LPS (100 μg/kg, i.p.) or saline. Thereafter, LPS (100 μg/kg, i.p.) or saline was injected in adulthood at 8 weeks of age. The serum LH concentration was decreased by LPS injection during adulthood in the neonatal saline‐injected rats. This suppressive effect was not seen in the neonatal LPS‐injected rats. The serum testosterone concentration was decreased by adult LPS injection in both the neonatal LPS‐injected and neonatal saline‐injected rats. The expression levels of KiSS‐1, which encodes kisspeptin, known to have a crucial role in the regulation of gonadotropin secretion, and GnRH mRNA in the hypothalamus and LHβ mRNA in the pituitary were not influenced by neonatal or adult LPS injection. On the other hand, the expression levels of tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) mRNA in the hypothalamus and testes were increased by adult LPS injection in both the neonatal LPS‐injected and neonatal saline‐injected rats. Furthermore, the expression levels of these factors in the hypothalamus after adult LPS injection were significantly lower in the neonatal LPS‐injected rats than in the neonatal saline‐injected rats. These findings indicate that neonatal LPS challenge reduces the sensitivity of male reproductive function to the suppressive effects of LPS, mainly at the central level. Attenuation of proinflammatory cytokine synthesis in the hypothalamus might be involved in this alteration.

Associations of circulating adiponectin with estradiol and monocyte chemotactic protein-1 in postmenopausal women.

Objective: The aim of the present study was to clarify the association of serum adiponectin concentrations with serum 17&bgr;-estradiol concentrations in pre-, peri-, and postmenopausal women. In addition, the associations of serum adiponectin with serum concentrations of proinflammatory and anti-inflammatory cytokines were examined in women during the menopausal transition. Design: A total of 197 women were enrolled in this study: 33 premenopausal women, 80 perimenopausal women, and 84 postmenopausal women. Serum adiponectin concentration was measured by an enzyme-linked immunosorbent assay. Serum concentrations of the proinflammatory cytokines interleukin (IL)-1&bgr;, IL-6, and tumor necrosis factor &agr;, anti-inflammatory cytokine IL-10, and the chemokines IL-8, macrophage inflammatory protein-1&bgr; and monocyte chemotactic protein-1 were measured by using a multiplexed human cytokine assay. Results: Serum adiponectin concentration showed a significant negative correlation with serum estradiol concentration (r = −0.400, P = 0.001) in postmenopausal women but not in pre- and perimenopausal women, and this correlation was significant after adjustment for age and body mass index. Serum adiponectin concentration also showed a significant negative correlation with serum monocyte chemotactic protein-1 concentration (r = −0.244, P = 0.05) in postmenopausal women. Conclusion: An increase in adiponectin level due to a decrease in estradiol results in a reduction in monocyte chemotactic protein-1 level in postmenopausal women, suggesting that adiponectin may be associated with a protective role against insulin resistance and atherosclerosis, which occur in the postmenopausal stage.