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

Hormones and Behavior 66 (2014) 309–316

Contents lists available at ScienceDirect

Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh

Regular article

Hypothalamic Kiss1 and RFRP gene expressions are changed by a high dose of lipopolysaccharide in female rats Takeshi Iwasa a,⁎, Toshiya Matsuzaki a, Altankhuu Tungalagsuvd a, Munkhsaikhan Munkhzaya a, Takako Kawami a, Hirobumi Niki a, Takeshi Kato a, Akira Kuwahara a, Hirokazu Uemura b, Toshiyuki Yasui c, Minoru Irahara a a b c

Department of Obstetrics and Gynecology, The University of Tokushima Graduate School, Institute of Health Biosciences, 3-18-15 Kuramoto-Cho, Tokushima 770-8503, Japan Department of Preventive Medicine, Institute of Health Biosciences, The University of Tokushima Graduate School, Japan Department of Reproductive Technology, Institute of Health Biosciences, The University of Tokushima Graduate School, Japan

a r t i c l e

i n f o

Article history: Received 26 November 2013 Revised 29 May 2014 Accepted 6 June 2014 Available online 19 June 2014 Keywords: Stress LPS Kisspeptin Kiss1 RFRP GPR147 GnRH

a b s t r a c t 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 (5 mg/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 (500 μg/kg) in ovariectomized rats. Rats with highdose 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. © 2014 Elsevier Inc. All rights reserved.

Introduction Humans and animals have a finite amount of energy for their activities. Therefore, if any activity has to be energetically prioritized, energy for other activities will be suppressed. Such energy insufficiency induces certain behaviors, for example anorexia and lethargy, called ‘sickness behaviors’ (Hart, 1988). In addition, the suppression of reproductive function, which is not essential for individual survival, often occurs simultaneously with sickness behaviors (Bonneaud et al., 2003; Yirmiya et al., 1995). The concept of allostasis has been used to explain these alterations (Goymann and Wingfield, 2004; McEwen, 1988; McEwen and Gianaros, 2011; McEwen and Wingfield, 2003). Allostasis, which means “maintaining stability through change”, is a process that supports homeostasis, which is essential for life, and therefore maintains physiological processes within a limited range in the face of external and ⁎ Corresponding author. E-mail address: [email protected] (T. Iwasa).

http://dx.doi.org/10.1016/j.yhbeh.2014.06.007 0018-506X/© 2014 Elsevier Inc. All rights reserved.

internal demands. Allostasis is produced by triggering changes in some mediators, such as the hypothalamic–pituitary–adrenal axis, autonomic nerves, and metabolic and immune factors (McEwen and Gianaros, 2011). Although allostasis plays pivotal roles in the regulation of homeostasis, prolonged (chronic) imbalance of the mediators of allostasis, which is known as allostatic load, has negative consequences for normal physiological function and accelerates some diseases (Goymann and Wingfield, 2004; McEwen and Gianaros, 2011). Recently, the concepts of allostasis and allostatic load have been applied to stress-induced reproductive dysfunction, such as anovulation and amenorrhea (Pauli and Berga, 2010). One type of allostatic overload occurs when energy demand exceeds supply, and this directs animals away from normal life stages into a survival mode that decreases allostatic load and restores positive energy balance (McEwen and Wingfield, 2003). Infections are thought to be one of the pivotal triggers of these responses in humans and animals. Large amounts of energy will be used for immune responses, while other activities, including reproductive function, will be suppressed for survival in individuals experiencing infection.

310

T. Iwasa et al. / Hormones and Behavior 66 (2014) 309–316

In addition, severe immune stresses induce some chronic pathophysiological changes in the central nervous system, such as brain neuroinflammation, cognitive changes, and disturbance of circadian rhythms (O'Callaghan et al., 2012; Qin et al., 2007; Weberpals et al., 2009). Although the pathophysiology of some sickness behaviors during lifethreatening infection is well established, to the best of our knowledge, changes in reproductive function and their underlying mechanisms during such conditions have never been examined. Reproductive function is regulated by the hypothalamic–pituitary– gonadal (HPG) axis in humans and animals. Physical and psychological stressors suppress HPG activity, mainly through inhibition of gonadotropin-releasing hormone (GnRH) in both males and females (Matsuwaki et al., 2006; Rivier and Rivest, 1991; Rivier et al., 1986). Consequently, these alterations induce the suppression of luteinizing hormone (LH) release from the pituitary in both sexes (Du Ruisseau et al., 1979; Gonzarez-Quijano et al., 1991). When the relationships between stress and reproductive functions are evaluated, lipopolysaccharide (LPS), a Gram-negative bacterial cell wall component, is frequently used to induce immune stresses. Similar to other stressors, LPS induces some sickness behaviors, and it also suppresses HPG activity through the inhibition of GnRH synthesis and secretion in several mammals and birds (Adelman et al., 2010; Ebisui et al., 1992; Harris et al., 2000; He et al., 2003; Lopes et al., 2012; Owen-Ashley et al., 2006; Refojo et al., 1998; Watanobe and Hayakawa, 2003; Xiao et al., 2000). Gonadotropin inhibitory hormone (GnIH)/RFamide-related peptides (RFRPs) and kisspeptin, a newly discovered hypothalamic neuropeptide, have pivotal roles in the regulation of GnRH and gonadotropin in mammals. GnIH, which was discovered in birds, is a hypothalamic RFamide peptide that acts within the hypothalamus and pituitary to suppress the HPG axis (Tsutusi et al., in press; Ubuka et al., 2013). Similarly to GnIH in birds, mammalian GnIH orthologs, known as RFRPs, act to inhibit the HPG axis. The RFRP gene encodes two biologically active GnIH orthologous peptides, RFRP-1 and RFRP-3 (Kriegsfeld et al., 2010; Tsutusi et al., in press; Ubuka et al., 2013). The cognate receptor for RFRP, which is also known as GPR147, is expressed in the hypothalamus, pituitary, and testes of mammals (Clarke et al., 2009; Hinuma et al., 2000; Tsutusi et al., in press). In mammals, RFRP3 acts on GnRH neurons in the hypothalamus in order to inhibit GnRH release and synthesis (Ducret et al., 2009; Kriegsfeld et al., 2006; Smith et al., 2008; Wu et al., 2009). Similarly, RFRP-3 also acts directly on the pituitary in order to inhibit gonadotropin release and synthesis (Murakami et al., 2008; Sari et al., 2009). Such inhibitory actions of RFRP on gonadotropin secretion raise the question of whether RFRP systems are involved in stress-induced reproductive dysfunction. A recent study indicates that LPS injection suppresses GnRH mRNA and peptide expression, but does not affect GnIH in birds (Lopes et al., 2012). On the other hand, although it has been reported that restraint stress, which is a psychological stress, increases RFRP but not GPR147 gene expression in rats (Kirby et al., 2009), the effects of LPS-induced immune stress on the RFRP system have never been examined in mammals. Interestingly, it has been reported that GnIH also acts as an orexigenic factor in birds and mammals (Chowdhury et al., 2012; Murakami et al., 2008; Tachibana et al., 2008). Thus, it is possible that the RFRP system plays some roles in both reproductive dysfunction and in changes of feeding behavior under LPS-induced immune stress conditions. Kisspeptin, a Kiss1 gene product, is a hypothalamic peptide that stimulates GnRH synthesis and release (Roa et al., 2011; Terasaka et al., 2013). We and other groups have demonstrated that a variety of experimental stresses, including LPS injection and undernutrition, down-regulate the expression of Kiss1 and/or its receptor, G proteincoupled receptor 54 (GPR54/Kiss1r), and that such alterations inhibit gonadotropin secretion and delay pubertal onset (Castellano et al., 2010; Iwasa et al., 2008, 2010a, 2010b; Knox et al., 2009). However, alterations of hypothalamic Kiss1 gene expression in life-threatening septic conditions remain poorly understood.

We hypothesized that some specific changes would potently suppress reproductive function during life-threatening immune stress conditions in order to save energy for other important survival responses. To test this hypothesis, we investigated the changes of serum gonadotropin levels and underlying mechanisms during moderate and severe (life-threatening) immune stress conditions. In particular, we focused on changes in kisspeptin and the RFRP system because, as noted above, they have pivotal roles in the regulation of GnRH and gonadotropin in mammals. It has been reported that the gonadal steroid milieu affects Kiss1 and/or RFRP gene expression and gonadotropin secretions mediated through feedback action (Kriegsfeld et al., 2006; Smith, 2013). If the gonadal steroid milieu is altered by immune stress, these hypothalamic factors and serum gonadotropin levels may be secondarily affected. Therefore, both gonadal intact and ovariectomized female rats were used in the present study to distinguish the primary and secondary effects of immune stress on hypothalamic factors. Fever anorectic responses, which are indicators of the degree of immune stress, were also measured. In addition, we measured appetite regulatory factors, i.e. leptin, leptin receptor (OBRb), and NPY, because it has been reported that these factors are involved in both sickness behavior and gonadotropin secretion (Faggioni et al., 1998; Grunfeld et al., 1996; Luheshi et al., 1999; Plata-Salaman, 2001). Materials and methods Animals and treatments Sprague-Dawley rats (Charles River Japan, Tokyo, Japan) were purchased and housed in a room under controlled light (12 h light:12 h darkness; lights turned on at 0800 and turned off at 2000) and temperature (24 °C). In total, 110 rats were used in this study. All animal experiments were conducted in accordance with the ethical standards of the institutional Animal Care and Use Committee of the University of Tokushima. All surgical procedures were carried out under sodium pentobarbital (60–80 mg/kg, intraperitoneal, i.p.) or sevoflurane anesthesia. At 10 weeks of age, 73 rats were ovariectomized bilaterally (OVX), and 37 rats underwent sham surgery (sham). Briefly, small incisions were made on the bilateral sides of the back to expose the ovaries retroperitoneally. The ovaries were clamped and removed (OVX rats), or touched with the surgical instruments (sham rats), and then the skin was sutured (Lam et al., 2002). After 6–7 weeks of recovery, rats were ready for study. Immune stress was induced by i.p. injection of LPS (0111:B4; Sigma, St. Louis, MO). LPS was dissolved in sterile saline; the injection volume did not exceed 0.3 ml. Core BT Core body temperature (BT) was measured using remote radiobiotelemetry. Pre-calibrated temperature-sensitive radio transmitters (TA11TA-F10; Data Sciences International, New Brighton, MN) were implanted into the peritoneal cavity. Rats were given at least 5 days to recover. Frequency data (Hz) from each transmitter were recorded every 15 min by a receiver board antenna placed underneath each rat's cage, and were logged into a peripheral processor. Frequency data were converted to °C using a DATAQUEST software (Data Sciences). Hormone assay Blood was collected at 6 h (in saline, low-dose LPS, and high-dose LPS-injected rats) and at 24 h (in high-dose LPS-injected rats) after saline or LPS injection, and serum was separated by centrifugation and stored at − 20 °C. Serum LH and leptin levels were measured in duplicate using radioimmunoassay (RIA) (rat LH [I-125] RIA kit, Institute of Isotopes Co., Ltd., Tokyo, Japan; rat leptin RIA kit, Linco Research, St. Charles, MO, USA).

T. Iwasa et al. / Hormones and Behavior 66 (2014) 309–316

Quantitative real-time polymerase chain reaction Whole brains were collected at each time-point and snap-frozen. The tissues were stored at − 80 °C. Hypothalamic explants were dissected out from the frozen brains as described elsewhere (Iwasa et al., in press). Briefly, the brain sections were dissected out via an anterior coronal cut at 2 mm anterior from the optic chiasm, a posterior coronal cut at the posterior border of the mammillary bodies, parasagittal cuts along the hypothalamic fissures, and a dorsal cut at 2.5 mm from the ventral surface. Total RNA was isolated using a TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) and an RNeasy mini-kit (Qiagen, Hilden, Germany). cDNA was synthesized with oligo (deoxythymidine) primers at 50 °C using the SuperScript® III first-strand synthesis system (Invitrogen Co., Carlsbad, CA, USA) for real-time polymerase chain reaction (RT-PCR). RT-PCR analysis was performed by using the StepOnePlus™ real-time PCR system (PE Applied Biosystems, Foster City, CA, USA) and SYBR® green. Standard curves, generated from 4 dilution series of a sample, were used for the relative quantifications of Kiss1, Kiss1r, RFRP, GPR147, GnRH, NPY, and OBRb. The expression levels were normalized by dividing by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. Dissociation curve analysis was also performed for each gene at the end of PCR. Each amplicon generated a single peak. Primer sequences, product size, and annealing temperatures are shown in Table 1. The PCR conditions were as follows: initial denaturation and enzyme activation at 95 °C for 20 s, followed by 45 cycles of denaturation at 95 °C for 3 s, and annealing and extension for 30 s. Effects of high-dose LPS injection in OVX rats The effects of high-dose LPS (5 mg/kg) injection on core body temperature (BT), body weight (BW) change, cumulative food intake (FI), serum hormone levels, and hypothalamic gene expressions were measured. This dose of LPS has been commonly used in previous studies, including a study of reproductive function, to induce the septic condition in rats (Reddy et al., 2006). In addition, this dose of LSP triggers chronic changes in the central nervous system (O'Callaghan et al., 2012; Qin et al., 2007). OVX rats were divided into three groups. In the first group, core BT was measured for 3 h before and 36 h after LPS injection. In the second group, BW change and cumulative FI were measured at 6, 24, and 48 h after LPS or saline injection. In the third group, serum LH and leptin levels and hypothalamic mRNA levels of Kiss1, Kiss1r, RFRP, GPR147, GnRH, NPY, and OBRb were measured by real-time PCR after LPS (at 6 and 24 h after injection) or saline injection (at 6 h after injection). These time-points for sampling were determined according to the BT profile, which was initiated 6 h after LPS injection, and then the plateau phase was continued

311

until 36 h after LPS injection. In addition, our previous and preliminary studies indicated that responses of some central and/or peripheral inflammatory factors to high-dose LPS would peak around 6 to 12 h after injection (Iwasa et al., 2011). Effects of high-dose LPS injection in sham rats The effects of high-dose LPS (5 mg/kg) injection on core BT, BW change, cumulative FI and hypothalamic gene expressions were measured. Sham rats were divided into three groups. In the first group, core BT was measured for 3 h before and 36 h after LPS injection. In the second group, BW change and cumulative FI were measured at 6, 24, and 48 h after LPS or saline injection. In the third group, hypothalamic mRNA levels of Kiss1, Kiss1r, RFRP, GPR147, GnRH, and NPY were measured by real-time PCR after LPS (at 6 and 24 h after injection) or saline injection (at 6 h after injection). Because serum LH levels were below the sensitivity level of the measurement kit, we could not evaluate them in sham rats. Effects of moderate-dose LPS injection in OVX rats The effects of moderate-dose LPS (500 μg/kg) injection on core BT, BW change, cumulative FI, serum LH levels, and hypothalamic gene expressions were measured. OVX rats were divided into three groups. In the first group, core BT was measured for 3 h before and 18 h after LPS injection. In the second group, BW change and cumulative FI were measured at 6 and 24 h after LPS or saline injection. In the third group, serum LH levels and hypothalamic mRNA levels of Kiss1, Kiss1r, RFRP, GPR147, and GnRH were measured by real-time PCR at 6 h after LPS or saline injection. These time-points for sampling were determined according to the BT profile, which was elevated and peaked at around 6 h after LPS injection and then dropped thereafter. Because BT recovered to basal levels at 24 h after injection, sampling was not carried out at this time-point. Statistical analyses With regard to temperature data, hourly average values were calculated and used for analysis. Body weight change and food intake data were compared using one-way or two-way analysis of variance (ANOVA). Following identification of significant differences between both groups, values of each time-point were compared using Student's unpaired t-tests. Hormone and mRNA levels were compared using one-way ANOVA followed by Dunnett's test or Student's unpaired t-tests. Cohen's d effect size statistics were calculated for each pairwise comparison (small = 0.2, medium = 0.5, large = 0.8). Eta-squared (η2) was calculated for ANOVA effects as an indicator of effect size

Table 1 Primer sequences, product sizes and annealing temperature. Primer

Sequence

Kiss1 forward Kiss1 reverse Kiss1r forward Kiss1r reverse RFRP forward RFRP reverse GPR147 forward GPR147 reverse GnRH forward GnRH reverse NPY forward NPY reverse OBRb forward OBRb reverse GAPDH forward GAPDH reverse

ATG ATC TCG CTG GCT TCT TGG GGT TCA CCA CAG GTG CCA TTT T TGT GCA AAT TCG TCA ACT AC A TCC AGC ACC GGG GCG G A A ACA GCT GC GAG TCC TGG TCA AG A GCA AC ACT GGC TGG AGG TTT CCTA T GTG TCT GCA TCG GTT TTC AC TTC CG A AGG GTC AGC TTC GCA GAA CCC CAG AAC TTC GA TGC CCA GCT TCC TCT TCA AT GGG GCT GTG TGG ACT G AC CCT GAT GTA GTG TCG CAG AGC GGA G GCA GCT ATG GTC TCA CTT CTT TTG GGT TCC CTG GGT GCT CTG A ATG GCA CAG TCA AGG CTG AG A CGC TCC TGG AAG ATG GTG AT

Product size (bp)

Annealing T (°C)

91

65

193

65

93

60

92

60

101

64

148

66

114

63

64

64

312

T. Iwasa et al. / Hormones and Behavior 66 (2014) 309–316

(small = 0.01, medium = 0.06, large = 0.14). Correlation analyses were performed using Pearson's correlation as appropriate. Differences were considered significant for P b 0.05. Data are expressed as means ± SEM. Results Effects of high-dose LPS injection in OVX rats Fever responses were induced by LPS injection in OVX rats. Fever response was initiated around 6 h and continued until 36 h after injection (Fig. 1A). BW (two-way ANOVA; F (1,28) = 110.2, P b 0.01, η2 = 0.46) and FI (two-way ANOVA; F (1,28) = 173.3, P b 0.01, η 2 = 0.28) were decreased by LPS injection, and BW change and cumulative FI during 6, 24, and 48 h in LPS-injected rats were significantly lower than those in saline-injected rats (Figs. 1B, C). Serum LH levels at 6 h after LPS injection were significantly lower than those for saline injection (Fig. 2A). On the other hand, serum leptin levels were not changed by LPS injection. Hypothalamic Kiss1 mRNA levels were decreased by LPS injection (one-way ANOVA; F (2,20) = 15.5, P b 0.01, η2 = 0.59), and Kiss1 mRNA levels at 6 h and 24 h after LPS injection were significantly lower than those in saline-injected rats (Fig. 2B). Kiss1r mRNA levels were not changed by LPS injection. RFRP mRNA levels at 6 h after LPS injection were significantly higher than those in salineinjected rats. GPR147 mRNA levels were increased by LPS injection (one-way ANOVA; F (2,20) = 4.91, P b 0.01, η2 = 0.33), and GPR147 mRNA levels at 6 h after LPS injection were significantly higher than those in saline-injected rats. GnRH mRNA levels were decreased by LPS injection (one-way ANOVA; F (2,20) = 6.77, P b 0.01, η2 = 0.41), and GnRH mRNA levels at 6 h after LPS injection were significantly higher than those in saline-injected rats. NPY mRNA levels were not changed by LPS injection; however, OBRb mRNA levels at 24 h after LPS injection were significantly lower than those in saline-injected rats. There was a strong positive correlation between hypothalamic Kiss1 and GnRH mRNA levels (Fig. 2C). Similarly, there was a strong positive correlation between hypothalamic Kiss1 mRNA and serum LH levels. On the other hand, there were no correlations between hypothalamic RFRP and GnRH mRNA or serum LH levels. Effects of high-dose LPS injection in sham rats Fever responses were induced by LPS injection in sham rats. Fever responses were initiated at around 6 h and continued until 36 h after injection (Fig. 3A). BW (two-way ANOVA; F (1,32) = 51.0, P b 0.01, η2 = 0.28) and FI (two-way ANOVA; F (1,32) = 100.6, P b 0.01, η 2 = 0.25) were decreased by LPS injection, and BW change and cumulative FI during 6, 24, and 48 h in LPS-injected

rats were significantly lower than those in saline-injected rats (Figs. 3B, C). Hypothalamic Kiss1 mRNA levels were decreased by LPS injection (one-way ANOVA; F (2,20) = 7.70, P b 0.01, η2 = 0.44), and Kiss1 mRNA levels at 24 h after LPS injection were significantly lower than those in saline-injected rats (Fig. 4A). Kiss1r mRNA levels were not changed by LPS injection. RFRP mRNA levels were increased by LPS injection (one-way ANOVA; F (2,20) = 14.60, P b 0.01, η2 = 0.14), and RFRP mRNA levels at 6 h and 24 h after LPS injection were significantly higher than those in saline-injected rats. GPR147 mRNA levels were increased by LPS injection (one-way ANOVA; F (2,20) = 9.40, P b 0.01, η2 = 0.49), and GPR147 mRNA levels at 6 h and 24 h after LPS injection were significantly higher than those in salineinjected rats. The LPS-induced increase of RFRP mRNA levels in sham rats was significantly larger than that in OVX rats (two-way ANOVA; F (1,40) = 11.30, P b 0.01, η2 = 0.12). In sham rats, fold changes of RFRP mRNA at 6 h and 24 h after LPS injection were 1.87 and 1.51, respectively. On the other hand, those in OVX rats were 1.30 and 1.02, respectively. In addition, LPS-induced changes of Kiss1, Kiss1r, and GPR147 mRNA levels were not different between sham rats and OVX rats. GnRH mRNA levels were decreased by LPS injection (one-way ANOVA; F (2,20) = 3.59, P b 0.01, η2 = 0.26), and GnRH mRNA levels at 6 h after LPS injection were significantly higher than those in saline-injected rats. NPY mRNA levels were not changed by LPS injection. There was a strong positive correlation between hypothalamic RFRP and GnRH mRNA levels (Fig. 4B). Similarly, there were inverse correlations between hypothalamic RFRP and GnRH mRNA levels. Effects of moderate-dose LPS injection in OVX rats Fever response was induced by LPS injection in OVX rats. Fever response peaked at around 6 h and was reduced to basal levels by 12 h after injection (Fig. 5A). BW (two-way ANOVA; F (2,33) = 168.7, P b 0.01, η2 = 0.38) and FI (two-way ANOVA; F (2,33) = 199.6, P b 0.01, η2 = 0.16) were decreased by LPS injection, and BW change and cumulative FI during 6 and 24 h in LPS-injected rats were significantly lower than those in saline-injected rats (Figs. 5B, C). Hypothalamic Kiss1, Kiss1r, RFRP, GPR147, and GnRH mRNA levels were not changed by LPS injection (Fig. 5D). Serum LH levels in LPS-injected rats tended to be lower than those in saline-injected rats; however, the differences did not reach statistical significance (t (11) = 1.93, P = 0.07, d = 1.08) (Fig. 5E). Discussion In the present study, we showed that life-threatening immune stress, induced by a high dose of LPS (5 mg/kg), decreased hypothalamic Kiss1 mRNA levels, while it increased hypothalamic RFRP and GPR147 mRNA

Fig. 1. Body temperature after LPS (5 mg/kg) injection (n = 5), and (B) body weight change and (C) cumulative food intake after saline or LPS (5 mg/kg) injection (n = 5 per group) in OVX rats. Data are presented as means ± SEM. *P b 0.05 and **P b 0.01.

T. Iwasa et al. / Hormones and Behavior 66 (2014) 309–316

313

Fig. 2. (A) Serum LH and leptin levels, and (B) hypothalamic mRNA levels before, at 6 h, and at 24 h after LPS injection (n = 6–9). (C) Correlation between Kiss1/RFRP and LH/GnRH levels. Data are presented as means ± SEM. *P b 0.05 and **P b 0.01.

levels in both OVX and sham rats. Because kisspeptin and RFRP act as pivotal regulators of GnRH and gonadotropin in mammals, changes in their action may be involved, at least in part, in the suppression of GnRH and serum LH levels observed in the present study. On the other hand, although serum LH levels tended to be decreased, hypothalamic Kiss1, Kiss1r, RFRP, and GPR147 mRNA levels were not changed by a moderate dose of LPS (500 μg/kg). These results suggest that the underlying mechanisms for the dysfunction of gonadotropin secretion change according to the severity of stress, and that changes in some reserved factors, such as kisspeptin and RFRP, begin to participate in the suppression of GnRH and gonadotropin under severe immune stress conditions. 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 by reducing other activities when under the high-dose condition. Thus, we assume that reproductive function, which needs a large amount of energy, might be strongly suppressed to maintain immune responses under such severe conditions. Usually, although GnRH and

serum LH levels are suppressed during several hours after a small dose of LPS (around 100 μg/kg), they recover 2 h later (Li et al., 2007). On the other hand, GnRH expression and serum LH levels were suppressed at 6 or 24 h after a high dose of LPS injection in the present study. In addition, changes in Kiss1, RFRP, and GPR147 mRNA levels were also observed at the same time-points, supporting our hypothesis that these alterations may be involved in the suppression of GnRH and LH. To the best of our knowledge, this is the first report that has evaluated the mechanisms of dysfunction in gonadotropin secretion under life-threatening immune stress conditions. Similarly, this is the first study that has shown up-regulation of the RFRP system under immune stress conditions. Because severe immune stresses bring about some chronic changes in the central nervous system (O'Callaghan et al., 2012; Qin et al., 2007; Weberpals et al., 2009), it is possible that dysfunction of the gonadotropin regulation system as observed in this study may also continue over a long period. However, further experiments focused on the functional and morphological changes of neuronal systems are needed to fully evaluate this hypothesis.

Fig. 3. (A) Body temperature after LPS (5 mg/kg) injection (n = 4), and (B) body weight change and (C) cumulative food intake after saline or LPS (5 mg/kg) injection (n = 5 per group) in sham rats. Data are presented as means ± SEM. *P b 0.05 and **P b 0.01.

314

T. Iwasa et al. / Hormones and Behavior 66 (2014) 309–316

Fig. 4. (A) Hypothalamic mRNA levels before, at 6 h, and at 24 h after LPS (5 mg/kg) injection (n = 5–10 per each group). (B) Correlation between Kiss1/RFRP and GnRH levels. Data are presented as means ± SEM. *P b 0.05 and **P b 0.01.

As noted above, GnIH/RFRP has inhibitory actions at several levels of the reproductive axis (Tsutusi et al., in press; Ubuka et al., 2013). Such actions raise the question of whether the GnIH/RFRP system is involved in stress-induced dysfunction of gonadotropin secretion. Because gonadotropin plays important roles in follicular growth and ovulation in the ovaries, dysfunction of its action might induce anovulation and

subsequently prevent pregnancy. Recently, Lopes et al. have shown that LPS injection decreases hypothalamic GnRH mRNA, but does not affect the GnIH system in birds (Lopes et al., 2012). In their study, although a high dose of LPS (2 mg/kg) was used to induce immune stress, it altered neither GnIH immunoreactivity nor gene expression. Their data support our result that a moderate dose of LPS did not affect

Fig. 5. (A) Body temperature after LPS (500 μg/kg) injection (n = 4), and (B) body weight change and (C) cumulative food intake after saline or LPS (500 μg/kg) injection (n = 7–8 per group) in OVX rats. (D) Hypothalamic mRNA levels, and (E) serum LH levels before and 6 h after LPS injection (n = 8 per group). Data are presented as means ± SEM. *P b 0.05 and **P b 0.01.

T. Iwasa et al. / Hormones and Behavior 66 (2014) 309–316

RFRP or GPR147 mRNA levels. In contrast, Kirby et al. have shown that acute and chronic immobilization stresses, common psychological stress models, lead to up-regulation of RFRP mRNA levels, and that changes of RFRP mRNA levels negatively correlate with serum LH levels in male rats (Kirby et al., 2009). Taken together, the underlying mechanisms of dysfunction in gonadotropin secretion under immune and psychological stresses may be partially different, and the GnIH/RFRP system might be more susceptible to psychological stress. However, we cannot directly compare the effects of these two kinds of stresses on physiological function and cannot entirely exclude the possibility that psychological stress leads to more serious damage than LPS-induced immune stress on the gonadotropin regulation system. It has been reported that GnIH/RFRP plays a role in energy control in birds and mammals (Chowdhury et al., 2012; Murakami et al., 2008; Tachibana et al., 2008). Under non-stress conditions, intracerebroventricular injection of GnIH/RFRP increases food intake. In addition, a reduction of food intake increases GnIH mRNA levels in chicks. These data indicate that GnIH/RFRP acts as an orexigenic factor in undernourished conditions. In the present study, a high dose of LPS induced marked anorectic responses and body weight loss. It has been established that upregulation of hypothalamic pro-inflammatory cytokines and the adipose leptin gene is strongly involved in anorectic responses under immune stress conditions (Faggioni et al., 1998; Grunfeld et al., 1996; Luheshi et al., 1999; Plata-Salaman, 2001). We assume that up-regulation of the RFRP system may also act as an opponent of those anorectic factors to preserve the feeding behavior under such conditions. In the present study, food intake in sham rats was lower than that in OVX rats under both saline-injected and LPS-injected conditions. It has been reported that endogenous and exogenous estrogen attenuates appetite and body weight gain throughout the modulations of hypothalamic factors and with a reduction of energy expenditure (Meli et al., 2004; Rogers et al., 2009). It has also been reported that estrogen increases the anorectic response to immune stress via up-regulation of pro-inflammatory cytokines (Geary et al., 2004; Straub, 2007). We assume that sensitivities of hypothalamic proinflammatory cytokines to high-dose LPS might be higher in sham rats than in OVX rats, resulting in the differences of anorectic responses. Kisspeptin, which is a potent stimulatory factor of GnRH, is also affected by some kinds of stress. We and Castellano et al. have reported that acute inflammatory stress induced by LPS injection reduces Kiss1 mRNA levels and kisspeptin immunoreactivity in female and male rats (Castellano et al., 2010; Iwasa et al., 2008). In these studies, a high dose of LPS (1 mg/kg) or repeated LPS injection (three consecutive injections of LPS (250 μg/kg)) was used to induce immune stress, indicating that severe immune stress may be needed to induce alterations of kisspeptin and Kiss1 mRNA levels. These data support our present result showing that only a high dose of LPS, but not a moderate dose, affected Kiss1 mRNA levels. On the other hand, it has been reported that the kisspeptin system may be more susceptible to undernutrition. The activity of the kisspeptin system is easily reduced by food restriction or food deprivation in animals at several developmental stages, including the juvenile and prepubertal periods (Iwasa et al., 2008, 2010a, 2010b; Matsuwaki et al., 2006). Consequently, it is possible that stress- and fasting-induced suppression of the kisspeptin system may be caused by different mechanisms. In the present study, we first evaluated LPS-induced changes in OVX rats in order to focus on the hypothalamic responses to immune stress. Subsequently, we evaluated sham rats to clarify whether the existence of gonadal steroids would lead to different results. As a result, the change in hypothalamic factors was similar in both OVX and sham rats, indicating that these changes were independent of gonadal steroid changes. However, the correlations between hypothalamic RFRP and GnRH mRNA levels were slightly different in OVX and sham rats; a significant correlation was observed only in the latter. In addition, the LPS-induced increase of RFRP mRNA was more evident in sham rats than in OVX rats. It has been reported that the Kiss1 mRNA level is

315

increased after OVX because the negative feedback action of estrogen is excluded (Smith, 2013). Thereafter, up-regulation of Kiss1 mRNA induces an increase in GnRH and serum gonadotropin levels. Actually, in the present study, Kiss1 mRNA levels in OVX rats were increased by about five times compared with sham rats (data not shown). We speculate that GnRH is mainly regulated by kisspeptin, and that the effect of RFRP on GnRH is relatively attenuated in ovariectomized animals. Past studies have indicated that leptin positively regulates kisspeptin systems, whereas NPY negatively regulates them (Kalamatianos et al., 2008; Sanchez-Garrido and Tena-Sempere, 2013). However, because serum leptin levels and hypothalamic NPY mRNA levels were not changed by LPS injection in this study, these factors may not be involved in the suppression of kisspeptin under immune stress conditions. There were some limitations in the present study. First, because only changes of mRNA levels in hypothalamic factors were measured, it is unclear whether such alterations led to changes in DNA and functional protein levels. In addition, because mRNA was quantified in whole hypothalamic tissue, changes of mRNA levels at each nucleus could not be examined in this study. Second, because reproductive abilities, for example sexual behavior, pregnancy rate, and nursing, were not tested in the present study, it remains unclear whether changes in hypothalamic factors and gonadotropin levels observed in this study actually affected individual reproductive function. Further comprehensive studies, for example with in situ hybridization and immunohistochemistry, are needed to clarify the mechanisms by which stress induces reproductive dysfunctions. In the third, there was temporal variability in peak core body temperature when comparing the treatment conditions. However, we cannot explain the causes of such variability. In summary, our data add to the understanding of how immune stress affects gonadotropin secretion by evaluating the effects of LPS injection on hypothalamic factors and serum hormone levels. Hypothalamic Kiss1 and RFRP mRNA levels were changed by high-dose LPS, but not by moderate-dose LPS, suggesting that alterations of these factors may be involved in the prolonged suppression of GnRH and LH under severe immune stress conditions. Because large amounts of energy are needed for reproduction, e.g. pregnancy, delivery, and nursing, dysfunction of gonadotropin secretion, which might possibly prevent pregnancy by the induction of anovulation after LPS injection, may be a biophylatic mechanism by which more energy is saved for the immune response.

References Adelman, J.S., Bentley, G.E., Wingfield, J.C., Martin, L.B., Hau, M., 2010. Population differences in fever and sickness behaviors in a wild passerine: a role for cytokines. J. Exp. Biol. 213, 4099–4109. Bonneaud, C., Mazuc, J., Gonzalez, G., Haussy, C., Chastel, O., Faivre, B., Sorci, G., 2003. Assessing the cost of mounting an immune response. Am. Nat. 161, 367–379. Castellano, J.M., Bentsen, A.H., Romero, M., Pineda, R., Ruiz-Pino, F., Garcia-Galiano, D., Sanchez-Garrido, M.A., Pinilla, L., Mikkelsen, J.D., Tena-Sempere, M., 2010. Acute inflammation reduces kisspeptin immunoreactivity at the arcuate nucleus and decreases responsiveness to kisspeptin independently of its anorectic effects. Am. J. Physiol. Endocrinol. Metab. 299, 54–61. Chowdhury, V.S., Tomonaga, S., Nishimura, S., Tabata, S., Cockrem, J.F., Tsutsui, K., Furuse, M., 2012. Hypothalamic gonadotropin-inhibitory hormone precursor mRNA is increased during depressed food intake in heat-exposed chicks. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 162, 227–233. Clarke, I.J., Qi, Y., Sari, I.P., Smith, J.T., 2009. Evidence that RF-amide related peptides are inhibitors of reproduction in mammals. Front. Neuroendocrinol. 30, 371–378. Du Ruisseau, P., Tache, Y., Brazeau, P., Collu, R., 1979. Effects of chronic immobilization stress on pituitary hormone secretion, on hypothalamic factor levels, and on pituitary responsiveness to LHRH and TRH in female rats. Neuroendocrinology 29, 90–99. Ducret, E., Anderson, G.M., Herbison, A.E., 2009. RFamide-related peptide-3, a mammalian gonadotropininhibitory hormone ortholog, regulates gonadotropin-releasing hormone neuron firing in the mouse. Endocrinology 150, 2799–2804. Ebisui, O., Fukata, J., Tominaga, T., Murakami, N., Kobayashi, H., Segawa, H., Muro, S., Naito, Y., Nakai, Y., Masui, Y., Nishida, T., Imura, H., 1992. Roles of interleukin-1a and -1b in endotoxin-induced suppression of plasma gonadotropin levels in rats. Endocrinology 130, 3307–3313. Faggioni, R., Fantuzzi, G., Fuller, J., Dinarello, C.A., Feingold, K.R., Grunfeld, C., 1998. IL-1b mediates leptin induction during inflammation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 274, 204–208.

316

T. Iwasa et al. / Hormones and Behavior 66 (2014) 309–316

Geary, N., Asarian, L., Sheahan, J., Langhans, W., 2004. Estradiol-mediated increases in the anorexia induced by intraperitoneal injection of bacterial lipopolysaccharide in female rats. Physiol. Behav. 82, 251–261. Gonzarez-Quijano, M.I., Ariznavarreta, C., Martin, A.I., Treguerres, J.A., Lopez-Calderon, A., 1991. Naltrexone does not reverse the inhibitory effect of chronic restraint on gonadotropin secretion in the intact male rat. Neuroendocrinology 54, 447–453. Goymann, W., Wingfield, J.C., 2004. Allostatic load, social status and stress hormones: the costs of social status matter. Anim. Behav. 67, 591–602. Grunfeld, C., Zhao, C., Fuller, J., Pollock, A., Moser, A., Friedman, J., Feingold, R., 1996. Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters. J. Clin. Invest. 97, 2152–2157. Harris, T.G., Battaglia, D.F., Brown, M.E., Brown, M.B., Carlson, N.E., Viguie, C., Williams, C.Y., Karsch, F.J., 2000. Prostaglandins mediate the endotoxin-induced suppression of pulsatile gonadotropin-releasing hormone and luteinizing hormone secretion in the ewe. Endocrinology 141, 1050–1058. Hart, B.L., 1988. Biological basis of the behavior of sick animals. Neurosci. Biobehav. Rev. 12, 123–137. He, D., Sato, I., Kimura, F., Akema, T., 2003. Lipopolysaccharide inhibits luteinizing hormone release through interaction with opioid and excitatory amino acid inputs to gonadotropin-releasing hormone neurones in female rats: possible evidence for a common mechanism involved in infection and immobilization stress. J. Neuroendocrinol. 15, 559–563. Hinuma, S., Shintani, Y., Fukusumi, S., Iijima, N., Matsumoto, Y., Hosoya, M., Fujii, R., Watanabe, T., Kikuchi, K., Terao, Y., Yano, T., Yamamoto, T., Kawamura, Y., Habata, Y., Asada, M., Kitada, C., Kurokawa, T., Onda, H., Nishimura, O., Tanaka, M., Ibata, Y., Fujino, M., 2000. New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals. Nat. Cell Biol. 2, 703–708. Iwasa, T., Matsuzaki, T., Murakami, M., Shimizu, F., Kuwahara, A., Yausi, T., Irahara, M., 2008. Decreased expression of kisspeptin mediates acute immune/inflammatory stress-induced suppression of gonadotropin secretion in female rat. J. Endocrinol. Invest. 31, 656–659. Iwasa, T., Matsuzaki, T., Murakami, M., Fujisawa, S., Kinouchi, R., Gereltsetseg, G., Kuwahara, A., Yasui, T., Irahara, M., 2010a. Effects of intrauterine undernutrition on hypothalamic Kiss1 expression and the timing of puberty in female rats. J. Physiol. 588, 821–829. Iwasa, T., Matsuzaki, T., Murakami, M., Kinouchi, R., Gereltsetseg, G., Fujisawa, S., Kuwahara, A., Yasui, T., Irahara, M., 2010b. Sensitivities of mRNA expression levels of Kiss1 and its receptor, Kiss1r, to undernutritional status are changed during the developmental period in female rats. J. Endocrinol. 207, 195–202. Iwasa, T., Matsuzaki, T., Murakami, M., Kinouchi, R., Gereltsetseg, G., Nakazawa, H., Yamamoto, S., Kuwahara, A., Yasui, T., Irahara, M., 2011. Changes in responsiveness of appetite, leptin and hypothalamic IL-1β and TNF-α to lipopolysaccharide in developing rats. J. Neuroimmunol. 236, 10–16. Iwasa, T., Matsuzaki, T., Kinouchi, R., Gereltsetseg, G., Murakami, M., Munkhzaya, M., Althankhuu, T.S., Kuwahara, A., Yasui, T., Irahara, M., 2014. Changes in central and peripheral inflammatory responses to lipopolysaccharide in ovariectomized female rats. Cytokine (in press). Kalamatianos, T., Grimshaw, S.E., Poorun, R., Hahn, J.D., Coen, C.W., 2008. Fasting reduces KiSS-1 expression in the anteroventral periventricular nucleus (AVPV): effects of fasting on the expression of KiSS-1 and neuropeptide Y in the AVPV or arcuate nucleus of female rats. J. Neuroendocrinol. 20, 1089–1097. Kirby, E.D., Geraghty, A.C., Ubuka, T., Bentley, G.E., Kaufer, D., 2009. Stress increases putative gonadotropin inhibitory hormone and luteinizing hormone in male rats. Proc. Natl. Acad. Sci. U. S. A. 106, 11324–11329. Knox, A.M., Li, X.F., Kinsey-Jones, J.S., Wilkinson, E.S., Wu, X.Q., Cheng, Y.S., Milligan, S.R., Lightman, S.L., O'Byrne, K.T., 2009. Neonatal lipopolysaccharide exposure delays puberty and alters hypothalamic Kiss1 and Kiss1r mRNA expression in the female rat. J. Neuroendocrinol. 21, 683–689. Kriegsfeld, L.J., Mei, D.F., Bentley, G.E., Ubuka, T., Mason, A.O., Inoue, K., Ukena, K., Tsutsui, K., Silver, R., 2006. Identification and characterization of a gonadotropin-inhibitory system in the brain of mammals. Proc. Natl. Acad. Sci. U. S. A. 103, 2410–2415. Kriegsfeld, L.J., Gibson, E.M., Williams III, W.P., Zhao, S., Mason, A.O., Bentley, G.E., Tsutsui, K., 2010. The roles of RFamide-related peptide-3 in mammalian reproductive function and behavior. J. Neuroendocrinol. 22, 692–700. Lam, K.K., Hu, C.T., Ou, T.Y., Yen, M.H., Chen, H.I., 2002. Effects of oestrogen replacement on steady and pulsatile haemodynamics in ovariectomized rats. Br. J. Pharmacol. 136, 811–818. Li, X.F., Kinsey-Jones, J.S., Knox, A.M.I., Wu, X.Q., Tahsinsoy, D., Brain, S.D., Lightman, S. L., O'Byrne, K.T., 2007. Neonatal lipopolysaccharide exposure exacerbates stressinduced suppression of LH pulse frequency in adulthood. Endocrinology 148, 5984–5990. Lopes, P.C., Wingfield, J.C., Bentley, G.E., 2012. Lipopolysaccharide injection induces rapid decrease of hypothalamic GnRH mRNA and peptide, but does not affect GnIH in zebra finches. Horm. Behav. 62, 173–179. Luheshi, G.N., Gardner, J.D., Rushforth, D.A., Loudon, A.S., 1999. Leptin actions on food intake and body temperature are mediated by IL-1. Proc. Natl. Acad. Sci. U. S. A. 96, 7047–7052. Matsuwaki, T., Kayasuga, Y., Yamanouchi, K., Nishihara, M., 2006. Maintenance of gonadotropin secretion by glucocorticoids under stress conditions through the inhibition of prostaglandin synthesis in the brain. Endocrinology 147, 1087–1093.

McEwen, B.S., 1998. Stress, adaptation, and disease. Allostasis and allostatic load. Ann. N. Y. Acad. Sci. 840, 33–44. McEwen, B.S., Gianaros, P.J., 2011. Stress- and allostasis-induced brain plasticity. Annu. Rev. Med. 62, 431–445. McEwen, B.S., Wingfield, J.C., 2003. The concept of allostasis in biology and biomedicine. Horm. Behav. 43, 2–15. Meli, R., Pacilio, M., Raso, G.M., Esposito, E., Coppola, A., Nasti, A., Di Carlo, C., Nappi, C., Di Carlo, R., 2004. Estrogen and raloxifene modulate leptin and its receptor in hypothalamus and adipose tissue from ovariectomized rats. Endocrinology 145, 3115–3121. Murakami, M., Matsuzaki, T., Iwasa, T., Yasui, T., Irahara, M., Osugi, T., Tsutsui, K., 2008. Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats. J. Endocrinol. 199, 105–112. O'Callaghan, E.K., Anderson, S.T., Moynagh, P.N., Coogan, A.N., 2012. Long-lasting effects of sepsis on circadian rhythms in the mouse. PLoS One 7, e47087. Owen-Ashley, N.T., Turner, M., Hahn, T.P., Wingfield, J.C., 2006. Hormonal, behavioral, and thermoregulatory responses to bacterial lipopolysaccharide in captive and free-living white-crowned sparrows (Zonotrichia leucophrys gambelii). Horm. Behav. 49, 15–29. Pauli, S.A., Berga, S.L., 2010. Athletic amenorrhea: energy deficit or psychogenic challenge? Ann. N.Y. Acad. Sci. 1205, 33–38. Plata-Salaman, C.R., 2001. Cytokines and feeding. Int. J. Obes. 25, 48–52. Qin, L., Wu, X., Block, M.L., Liu, Y., Breese, G.R., Hong, J.S., Knapp, D.J., Crews, F.T., 2007. Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia 55, 453–462. Reddy, M.M., Mahipal, S.V., Subhashini, J., Reddy, M.C., Roy, K.R., Reddy, G.V., Reddy, P.R., Reddanna, P., 2006. Bacterial lipopolysaccharide-induced oxidative stress in the impairment of steroidogenesis and spermatogenesis in rats. Reprod. Toxicol. 22, 493–500. Refojo, D., Arias, P., Moguilevsky, J.A., Feleder, C., 1998. Effect of bacterial endotoxin on in vivo pulsatile gonadotropin secretion in adult male rats. Neuroendocrinology 67, 275–281. Rivier, C., Rivest, S., 1991. Effect of stress on the activity of the hypothalamic–pituitary– gonadal axis: peripheral and central mechanisms. Biol. Reprod. 45, 523–532. Rivier, C., Rivier, J., Vale, W., 1986. Stress-induced inhibition of reproductive functions: role of endogenous corticotropin-releasing factor. Science 231, 607–609. Roa, J., Navarro, V.M., Tena-Sempere, M., 2011. Kisspeptins in reproductive biology: consensus knowledge and recent developments. Biol. Reprod. 85, 650–660. Rogers, N.H., Perfield II, J.W., Strissel, J., Obin, M.S., Greenberg, A.S., 2009. Reduced energy expenditure and increased inflammation are early events in the development of ovariectomy-induced obesity. Endocrinology 150, 2161–2168. Sanchez-Garrido, M.A., Tena-Sempere, M., 2013. Metabolic control of puberty: roles of leptin and kisspeptins. Horm. Behav. 64, 187–194. Sari, I.P., Rao, A., Smith, J.T., Tilbrook, A.J., Clarke, I.J., 2009. Effect of RF-amide-related peptide-3 on luteinizing hormone and follicle-stimulating hormone synthesis and secretion in ovine pituitary gonadotropes. Endocrinology 150, 5549–5556. Smith, J.T., 2013. Sex steroid regulation of kisspeptin circuits. Adv. Exp. Med. Biol. 784, 275–295. Smith, J.T., Coolen, L.M., Kriegsfeld, L.J., Sari, I.P., Jaafarzadehshirazi, M.R., Maltby, M., Bateman, K., Goodman, R.L., Tilbrook, A.J., Ubuka, T., Bentley, G.E., Clarke, I.J., Lehman, M.N., 2008. Variation in kisspeptin and RFamide-related peptide (RFRP) expression and terminal connections to gonadotropin-releasing hormone neurons in the brain: a novel medium for seasonal breeding in the sheep. Endocrinology 149, 5770–5782. Straub, R.H., 2007. The complex role of estrogens in inflammation. Endocr. Rev. 28, 521–574. Tachibana, T., Masuda, N., Tsutsui, K., Ukena, K., Ueda, H., 2008. The orexigenic effect of GnIH is mediated by central opioid receptors in chicks. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 150, 21–25. Terasaka, T., Otsuka, F., Tsukamoto, N., Nakamura, E., Inagaki, K., Toma, K., Ogura-Ochi, K., Glidewell-Kenney, C., Lawson, M.A., Makino, H., 2013. Mutual interaction of kisspeptin, estrogen and bone morphogenetic protein-4 activity in GnRH regulation by GT1-7 cells. Mol. Cell. Endocrinol. 381, 8–15. Tsutusi, K., Ubuka, T., Bentley, G.E., Kriegsfeld, L.J., 2014. Review: regulatory mechanisms of gonadotropin-inhibitory hormone (GnIH) synthesis and release in photoperiodic animals. Front. Neurosci. (in press). Ubuka, T., Son, Y.L., Bentley, G.E., Millar, R.P., Tsutsui, K., 2013. Gonadotropin-inhibitory hormone (GnIH), GnIH receptor and cell signaling. Gen. Comp. Endocrinol. 190, 10–17. Watanobe, H., Hayakawa, Y., 2003. Hypothalamic interleukin-1b and tumor necrosis factor-a, but not interleukin-6, mediate the endotoxin-induced suppression of the reproductive axis in rats. Endocrinology 144, 4868–4875. Weberpals, M., Hermes, M., Hermann, S., Kummer, M.P., Terwel, D., Semmler, A., Berger, M., Schäfers, M., Heneka, M.T., 2009. NOS2 gene deficiency protects from sepsisinduced long-term cognitive deficits. J. Neurosci. 29, 14117–14184. Wu, M., Dumalska, I., Morozova, E., van den Pol, A.N., Alreja, M., 2009. Gonadotropin inhibitory hormone inhibits basal forebrain vGlu T2-gonadotropin-releasing hormone neurons via a direct postsynaptic mechanism. J. Physiol. 587, 1401–1411. Xiao, E., Xia-Zhang, L., Ferin, M., 2000. Inhibitory effects of endotoxin on LH secretion in the ovariectomized monkey are prevented by naloxone but not by an interleukin-1 receptor antagonist. Neuroimmunomodulation 7, 6–15. Yirmiya, R., Avitsur, R., Donchin, O., Cohen, E., 1995. Interleukin-1 inhibits sexual behavior in female but not in male-rats. Brain Behav. Immun. 9, 220–223.