Delayed puberty in prenatally glucocorticoid administered female rats occurs independently of the hypothalamic Kiss1–Kiss1r–GnRH system

Int. J. Devl Neuroscience 29 (2011) 183–188

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Delayed puberty in prenatally glucocorticoid administered female rats occurs independently of the hypothalamic Kiss1–Kiss1r–GnRH system Takeshi Iwasa ∗ , Toshiya Matsuzaki, Masahiro Murakami, Riyo Kinouchi, Ganbat Gereltsetseg, Satoshi Yamamoto, Akira Kuwahara, Toshiyuki Yasui, Minoru Irahara Department of Obstetrics and Gynecology, The University of Tokushima Graduate School, Institute of Health Biosciences, 3-18-15 Kuramoto-Cho, Tokushima 770-8503, Japan

a r t i c l e

i n f o

Article history: Received 28 August 2010 Received in revised form 22 October 2010 Accepted 3 November 2010 Keywords: IUGR Kisspeptin CRH Glucocorticoid

a b s t r a c t Intrauterine growth retardation (IUGR) is an important risk factor for the pathogenesis of diseases after birth. Long-lasting alterations in the structure and function of tissues and the neuroendocrine system, which are known as ‘programming effects’, increase the risks of these diseases. To investigate the pathophysiology of programming effects, several kinds of IUGR rodent models have been used in experiments. Sexual maturation and the onset of puberty are delayed in these models. We have previously reported that decreased action of hypothalamic kisspeptin, which is a positive regulator of GnRH, contributes to the delayed onset of puberty in undernutrition-induced IUGR rats. The aim of this study was to evaluate whether the hypothalamic Kiss1–Kiss1r–GnRH system is also altered in dexamethasone-induced IUGR rats. Compared with offspring from an untreated mother (control), offspring from a dexamethasone administered mother (DEX) showed a significant reduction in body weight throughout the experimental period (from birth to the prepubertal period) and the delayed onset of puberty. There were no significant differences between the control and DEX groups with regard to their hypothalamic Kiss1, Kiss1r, GnRH, CRH, NPY and POMC mRNA levels during the experimental period or their serum LH, FSH, or leptin concentrations at postnatal day 28 or vaginal opening (VO). Compared with the control, DEX showed significantly lower ovarian weight at postnatal day 28, but not at VO. These results suggested that the delayed onset of puberty induced by maternal dexamethasone administration would occur independently of hypothalamic Kiss1–Kiss1r–GnRH system. © 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

1. Introduction Recently, epidemiological and experimental evidence has suggested that intrauterine growth retardation (IUGR) is an important risk factor for the pathogenesis of diseases after birth (Ravelli et al., 1976; Godfrey and Barker, 2000; Breier et al., 2001), which has given rise to the concept of developmental origins of health and disease (Breier et al., 2001; Gluckman and Hanson, 2004). In humans, IUGR and low birth weight are associated with increased rates of insulin resistance, type 2 diabetes, hypertension, and ischemic heart disease in adulthood (Barker et al., 1993a,b; Phillips et al., 1998; Phillips, 1998). It has been supposed that long-lasting alterations in the structure and function of tissues and the neuroendocrine system, which are known as ‘programming effects’, lead to higher risks of these diseases. To investigate the mechanisms by which IUGR induces disease development, several kinds of animal models, for example,

∗ Corresponding author. E-mail address: [email protected] (T. Iwasa). 0736-5748/$36.00 © 2010 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2010.11.001

uterine artery ligation (Engelbregt et al., 2000), prenatal food or protein restriction (Zambrano et al., 2005; Guzman et al., 2006; Yura et al., 2005; Iwasa et al., 2010a), and overexposure to dexamethasone (Benediktsson et al., 1993; Nyirenda et al., 1998, 2001; Smith and Waddell, 2000; de Vries et al., 2007), have been widely used in experiments. These animal models should be used appropriately according to the purpose of the experiment. In general, prenatal food or protein restriction is used as a maternal undernutrition-induced IUGR model, and overexposure to dexamethasone is used to represent maternal chronic stress or a therapeutic dexamethasone-induced IUGR model. These models of IUGR show similar neuroendocrine and clinical characteristics, for example, disturbance of the hypothalamic-pituitary-adrenal (HPA) axis (Levitt et al., 1996; Lesage et al., 2001; de Vries et al., 2007), fewer nephrons and hypertension (O’Regan et al., 2004; Kawamura et al., 2007; Woods and Weeks, 2005), and glucose intolerance in rodents and non-human primates (Nyirenda et al., 1998; de Vries et al., 2007; Yura et al., 2005; Shahkhalili et al., 2010). Although, some studies have shown that the undernutrition-induced IUGR model and dexamethasone-induced IUGR model may induce programming effects, at least in part, by a common mechanism (Lesage

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T. Iwasa et al. / Int. J. Devl Neuroscience 29 (2011) 183–188

et al., 2001; Woods and Weeks, 2005), another study has shown that the programming effects observed in these two models are mediated by different pathophysiological mechanisms (Shahkhalili et al., 2010). It has been reported that sexual maturation and the onset of VO, which is an indicator of puberty in females, are delayed in both undernutrition-induced and dexamethasone-induced IUGR rodent models (Politch and Herrenkohl, 1984; Smith and Waddell, 2000; Iwasa et al., 2010a). Several studies have suggested mechanical hypotheses to explain the relationship between IUGR and delayed sexual maturation (Deligeorgis et al., 1996; Faria et al., 2008; Ristic et al., 2008). Recently, we have reported that decreased action of hypothalamic kisspeptin contributes to the delayed onset of puberty in undernutrition-induced IUGR rats (Iwasa et al., 2010a). Kisspeptin, a Kiss1 gene product (Kotani et al., 2001; Ohtaki et al., 2001), stimulates gonadotropin releasing hormone (GnRH) release via its receptor Kiss1r (Irwig et al., 2004; Matsui et al., 2004; Shahab et al., 2005; Jayasena et al., 2009; Roseweir and Millar, 2009) and is a key factor for pubertal onset in humans, rodents, and monkeys (de Roux et al., 2003; Funes et al., 2003; Seminara et al., 2003; Navarro et al., 2004; Shahab et al., 2005). To the best of our knowledge, there have been no studies about the alterations in hypothalamic reproductive function, including the kisspeptin system, in dexamethasone-induced IUGR rats. The aim of this study was to evaluate the effects of prenatal dexamethasone exposure-induced IUGR on the development of the hypothalamic Kiss1–Kiss1r–GnRH systems, serum gonadotropin concentrations and the timing of onset of puberty. We have evaluated the hypothalamic mRNA level of corticotrophin releasing hormone (CRH), a central factor in the HPA axis, because it may play an important role in modulating Kiss1 and Kiss1r mRNA expression and the timing of puberty (Kinsey-Jones et al., 2009, 2010). In addition, we also evaluated the hypothalamic mRNA levels of neuropeptide Y (NPY) and proopiomelanocortin (POMC), central components of the appetite regulation system, because it was reported that they may play roles in controlling GnRH release and the timing of the onset of puberty (Wiemann et al., 1989; Lebrethon et al., 2000) and that their mRNA expression levels and neuronal activities might be altered by kisspeptin (Backholer et al., 2010; Fu and van den Pol, 2010; Kim et al., 2010).

2. Materials and methods 2.1. Animals Pregnant Sprague–Dawley rats were purchased (Charles River Japan, Inc., Tokyo, Japan) and housed individually (female rats were mated with males overnight at 8–10 weeks of age and the day on which spermatozoa were present in a vaginal smear was designated day 1 of pregnancy). The animal rooms were maintained under controlled lighting (14 h light, 10 h dark cycle) and temperature (24 ◦ C) conditions. All animal experiments were conducted in accordance with the ethical standards of the Animal Care and Use Committee of the University of Tokushima. The animals were humanely killed by decapitation at the end of the experiments. In total, 18 pregnant rats and their offspring were used in this study. The pregnant rats were divided into two groups. In the untreated group (n = 9), the dams were allowed normal drinking water during the gestation and lactation period. In the dexamethasone administration group (n = 9), dexamethasone (1 ␮g/mL, Sigma, St Louis, MO, USA) was administered in drinking water from day 13 of pregnancy until delivery, and then normal drinking water was provided during the lactation period. Litter size was examined 1 day after delivery, and the pups were randomized among all mothers (untreated and dexamethasone administered dams). To adjust the litter size to 10–12 per dam, pups were culled or moved to other dams and were fostered until weaning. The pups were weighed at various postnatal ages and weaned at 21 days of age. After weaning, the pups were housed one per cage. Only female offspring were used in this study. To obviate any litter effects, the animals used for each experiment were randomly chosen from different litters. Some factors that affect sexual maturation and the onset of puberty were evaluated and compared between the offspring from the untreated dams (control) and those from the dexamethasone administered dams (DEX).

2.2. Assessment of serum hormone concentrations, mRNA levels of hypothalamic factors, and the onset of puberty At various postnatal stages (from day 5 of age to VO), several pups (n = 5–8) from the control and DEX groups were killed by decapitation between 09.00 and 11.00 h, and whole brains and trunk blood were obtained to measure the serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), and leptin and the mRNA levels of hypothalamic factors (Kiss1, Kiss1r, GnRH, CRH, NPY and POMC). The ovaries of the rats were dissected and weighed. Eighteen pups from the control group and 15 pups from the DEX group were used to assess the onset of puberty. From postnatal day 28 onwards, the pups were inspected daily for VO. The onset of puberty was defined as day on which VO was first noticed. 2.3. Hormone assays The serum LH concentrations were measured using an 125 I-radioimmunoassay (RIA) kit (Rat LH [I-125] RIA kit, Institute of Isotopes Co., Ltd. Tokyo, Japan). The sensitivity of the assay was 0.2 ng mL−1 , and the inter- and intra-assay coefficients of variation (CV) were 6.6% and 6.5%, respectively. The serum FSH concentrations were measured using an 125 I-RIA kit (Rat FSH [I-125] RIA kit, Institute of Isotopes Co., Ltd. Tokyo, Japan). The sensitivity of the assay was 0.09 ng mL−1 , and the interand intra-assay CV were 8.1% and 4.2%, respectively. The serum leptin concentration was measured using an 125 I-RIA kit (Rat leptin RIA kit, Linco Research Inc., MO, USA). The sensitivity of the assay was 0.5 ng mL−1 , and the inter- and intra-assay CV were 4.8% and 2.4%, respectively. Hormone assays were performed in duplicate. 2.4. Quantitative real time polymerase chain reaction Hypothalamic explants, including of the median preoptic area (POA), anteroventral periventricular nucleus (AVPV), arcuate nucleus (ARC) and paraventricular nucleus (PVN) were dissected out. Kisspeptin containing neurons are mainly in AVPV and ARC and GnRH containing neurons are in POA. Similarly, NPY and POMC containing neurons are mainly in ARC and CRH containing neurons are in PVN. The brain sections were dissected out by making an anterior coronal cut at 1 mm anterior from the optic chiasm and a posterior coronal cut at the posterior border of the mammillary bodies. Then, two parasagittal cuts were made along the hypothalamic fissures and another dorsal cut was made at 2.0 mm from the ventral surface. Total RNA was isolated from the hypothalamus using a TRIzol reagent kit (Invitrogen Co., Carlsbad, CA, USA) and an RNeasy Mini kit (Qiagen Gmgh, Hilden, Germany). cDNA was synthesized with oligo (deoxythymidine) primers at 50 ◦ C using the SuperScript III First-Standard Synthesis System for RT-PCR (Invitrogen Co., USA). Real-time PCR analysis was performed using the PCR System 7500 (PE Applied Biosystems, Foster City, CA, USA) with SYBR green (Applied Biosystems). The sequences of the selected forward and reverse primers were as follows: Kiss1: F: 5 -AGC TGC TGC TGC TTC TCC TCT GT-3 , R: 5 -AGG CTT GCT CTC TGC ATA CC-3 ; Kiss1r: F: 5 -GCA GAC CGT CAC CAA TTT CT-3 , R: 5 -GGG AAC ACA GTC ACG TAC CA-3 ; GnRH: F: 5 -GCA GAA CCC CAG AAC TTG GA-3 , R: 5 -TGC CCA GCT TCC TCT TCA AT-3 ; CRH: F: 5 -TCT CTC TGG ATC TCA CCT TCC ACC-3 , R: 5 -AGC TTG CTG AGC TAA CTG CTC TGC-3 ; NPY: F: 5 -GGG GCT GTG TGG ACT GAC CCT-3 , R: 5 -GAT GTA GTG TCG CAG AGC GGAG-3 ; POMC: F: 5 -CCC GAG AAA CAG CAG CAG TG-3 , R: 5 -AGG GGG CCT TGG AGT GAG AA-3 and ˇ-actin: F: 5 -TCA TGA AGT GTG ACG TTG ACA TCC GT-3 , R: 5 -CTT AGA AGC ATT TGC GGT GCA CG-3 . The PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95 ◦ C for 10 min, followed by 45 cycles of denaturation at 95 ◦ C for 15 s; annealing at 63 ◦ C for 30 s (Kiss1, Kiss1r), 58 ◦ C for 30 s (GnRH, CRH), or 65 ◦ C for 30 s (ˇ-actin); and extension at 72 ◦ C for 1 min. The copy numbers of the transcripts were normalized against those of ˇ-actin transcripts for each sample. 2.5. Statistical analyses All data are presented as means ± SEM. Statistical analyses were performed using two-way analysis of variance (ANOVA) with Fisher’s least significant difference test or the Student’s t test. Statistical significance was defined as P < 0.05.

3. Results There were no significant differences in body weight between the untreated and dexamethasone treated dams on day 13. Compared with the untreated dams, the dexamethasone treated dams showed significantly less body weight gain during pregnancy (twoway ANOVA; P < 0.01, F(3,92) = 164.9) (Fig. 1), and litter size did not differ between the two groups (litter size: 12.9 ± 0.9 in control vs. 14.7 ± 0.4 in DEX, P = 0.08). Compared with the control (offspring from the untreated dams), the DEX group (offspring from the dexamethasone administered dams) showed a significant reduction in body weight from birth to postnatal day 28 (two-way ANOVA; P < 0.01, F(3,85) = 913.5) (Fig. 2).

body weight change (% of day 13)

T. Iwasa et al. / Int. J. Devl Neuroscience 29 (2011) 183–188

140

**

130

** 120

**

4. Discussion

**

** 100 90 80

day13

day14

day15

day16

day17

day18

day19

day20

untreated dams dexamethasone administered dams Fig. 1. Body weight change in untreated dams and dexamethasone treated dams (n = 9 per group). In the dexamethasone treated dams, dexamethasone was administered in drinking water from day 13 of pregnancy until delivery. There were no significant differences in body weight between the two groups at day 13. Data are presented as means ± SEM. **P < 0.01 vs. dexamethasone treated dams.

120 **

body weight (g)

100 80 60

control DEX

** **

40 **

20 **

0

day0

**

day5

day10

day15

ANOVA; P = 0.15, F(4,00) = 2.17) or POMC (two-way ANOVA; P = 0.26, F(4,01) = 1.44) mRNA levels during any experimental period (Fig. 4).

**

**

110

185

day20

day28

Fig. 2. Body weight changes in the offspring of the untreated dams (control) and those of the dexamethasone administered dams (DEX) (n = 23–103 per group per age). Both male and female data were included at day 0, and only female data were included from day 5 to day 28. Data are presented as means ± SEM. **P < 0.01 vs. DEX.

Compared with the control, the DEX group demonstrated delayed onset of puberty (age at VO: 31.1 ± 0.3 days in control vs. 34.4 ± 0.4 days in DEX, P < 0.01) (Fig. 3). Body weight at VO was not different between the two groups (body weight at VO: 122.8 ± 2.4 days in control vs. 119.0 ± 2.4 days in DEX, P = 0.27). There were no significant differences between the control and DEX groups with regard to serum LH, FSH, or leptin concentrations (Table 1). Compared with control, the DEX group showed a significantly lower ovarian weight at postnatal day 28. However, ovarian weight at VO was not different between the two groups. There were no significant differences between the control and DEX groups with respect to hypothalamic Kiss1 (two-way ANOVA; P = 0.20, F(4,01) = 1.63), Kiss1r (two-way ANOVA; P = 0.22, F(4,01) = 1.54), GnRH (two-way ANOVA; P = 0.22, F(4,01) = 1.52), CRH (two-way ANOVA; P = 0.12, F(4,01) = 2.54), NPY (two-way

This study demonstrates that the hypothalamic mRNA levels of Kiss1, Kiss1r, GnRH, CRH, NPY and POMC and the serum LH and FSH concentrations are not altered by prenatal exposure to dexamethasone, suggesting that delayed onset of puberty might occur in dexamethasone-induced IUGR rats independently of these factors. While glucocorticoid plays an important role in the final maturation of fetal organ systems, overexposure to it retards fetal growth and induces the development of adult disease (Benediktsson et al., 1993; Nyirenda et al., 1998, 2001; Smith and Waddell, 2000; de Vries et al., 2007). In general, exposure of the fetus to endogenous maternal glucocorticoid is limited by 11␤-hydroxysteroid dehydrogenase type 2 (11␤-HSD2), the placental glucocorticoid barrier molecule (Burton and Waddell, 1999). Placental 11␤-HSD activity may positively correlate with birth weight (Benediktsson et al., 1993), and treatment of adrenal intact pregnant rats with 11␤HSD2 inhibitor reduces birth weight and leads to hypertension (Lindsay et al., 1996a) and hyperglycemia (Lindsay et al., 1996b) in their offspring. It has been reported that both maternal chronic stress, which attenuates placental 11␤-HSD2 function, and treatment with dexamethasone, which can freely crosses the placenta, induce fetal exposure to glucocorticoid and are linked to IUGR and subsequent pathophysiology (Drake et al., 2007). Some programming effects in undernutrition-induced IUGR rats are induced by the same mechanism as in dexamethasone-induced IUGR rats. Maternal food restricted rats during the last week of gestation show decreased placental 11␤-HSD2 expression, which overexposes the fetus to glucocorticoid and disturbs the development of the HPA axis (Lesage et al., 2001). In addition, maternal body weight loss is a common cause of impaired development and hypertension in these two models (Woods and Weeks, 2005). On the other hand, another study has suggested that these two models may have different mechanisms for some of their programming effects. Recently, Shahkhalili et al. (2010) directly compared the early catch up growth and subsequent glucose intolerance of these two rat models of IUGR. As a result, the undernutrition-induced IUGR rats showed more rapid catch up growth and development of glucose intolerance. Similarly, while the undernutrition-induced IUGR rats used in our previous study showed rapid catch up growth by postnatal day 12 (Iwasa et al., 2010a), the dexamethasoneinduced IUGR rats used in present study did not catch up with the control at any point during the experimental period (Fig. 2). It has been reported that IUGR and the consequent rapid catch up growth increase the risk of severe glucose intolerance (Jimenez-Chillaron et al., 2005). In addition, it has been shown that IUGR rats that demonstrated rapid catch up growth in the early developmental period showed hypothalamic leptin resistance and increased body weight and body fat (Desai et al., 2005). Leptin, an anorexigenic factor, plays a crucial role in the sexual maturation and fertility of rodents (Ahima et al., 1997; Cheung et al., 1997; Cunningham et al.,

Table 1 Serum hormone and ovarian weigh profiles of the control and DEX. Day 28

Day at VO

Control LH (ng/mL) FSH (ng/mL) Ovarian weight (mg) Leptin (pg/mL)

2.2 9.8 28.6 2.1

± ± ± ±

DEX 0.10 2.2 2.9 0.4

2.2 10.7 22.0 2.8

Control ± ± ± ±

0.2 1.4 5.9* 0.6

2.2 17.4 47.3 1.5

± ± ± ±

DEX 0.3 8.5 10.5 0.5

Control: offspring from untreated mother, DEX: offspring from dexamethasone administered mother. n = 8 per group. Values are presented as means ± SEM. * P < 0.01.

2.7 15.5 42.8 1.6

± ± ± ±

0.7 6.1 6.7 0.3

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T. Iwasa et al. / Int. J. Devl Neuroscience 29 (2011) 183–188

Cumulative percentage of VO (%)

A

120 100 80 60 40

DEX 0

B

control

20

day29

day30

day31

day32

day33

day34

day35

day36

day37

130

38

body weight at VO (g)

Age at VO (days)

** 36 34 32 30

125

120

115

110

28

Fig. 3. Cumulative percentage of vaginal opening (VO) (A) and mean age and body weight at VO in the offspring of the untreated dams (control, n = 18) and those of the dexamethasone administered dams (DEX, n = 15) (B). Data are presented as means ± SEM. **P < 0.01 vs. each other.

1999), and leptin resistance induces hypothalamic infertility, which is caused by the suppression of GnRH mRNA expression (Tortoriello et al., 2004). It has been shown that Kiss1 mRNA expression is regulated by leptin, as well as estrogen, and that the decreased action of leptin suppresses Kiss1 mRNA expression in the hypotha-

1.0 0.5

3.0

day5

day10

day15

day20

day28 days at VO

1.5

1.0

0.5

0 3.0

B

2.5 2.0 1.5 1.0 0.5

NPYmRNA / β-actin

1.5

1.0

C

day5

day10

day15

day20

day28 days at VO

2.5 2.0 1.5 1.0 0.5 0

0 day5

day10

day15

day20 day28 days at VO

day5

day10

day15 day20

day28 days at VO

E

0.8 0.6 0.4 0.2 0

1.2

D POMC mRNA / β-actin

GnRHmRNA / β-actin

2.0

0

Kiss1r mRNA / β-actin

2.0

A

CRHmRNA / β-actin

Kiss1 mRNA / β-actin

2.5

lamus (Smith et al., 2006; Castellano et al., 2009). In our previous study, undernutrition-induced IUGR rats began to show a higher serum leptin concentration and lower Kiss1 mRNA expression after catch up growth, suggesting that leptin resistance occurs in the hypothalamic kisspeptin system and delays the onset of puberty

day5

day10 day15 day20 day28

VO

day10 day15 day20 day28

VO

F

1.0 0.8 0.6 0.4 0.2 0

day5

control DEX Fig. 4. Hypothalamic Kiss1, Kiss1r, GnRH, CRH, NPY and POMC mRNA levels from day 5 until vaginal opening (VO) in the offspring of the untreated dams (control) and those of the dexamethasone administered dams (DEX) (n = 5–8 per group per age) (A–F). Only female data were included. The relative expression levels were calculated by dividing by ˇ-actin mRNA expression. There were no significant differences between the control and DEX groups in any experimental period. Data are presented as means ± SEM.

T. Iwasa et al. / Int. J. Devl Neuroscience 29 (2011) 183–188

(Iwasa et al., 2010a). No such alterations were detected in this study, and we speculate that leptin resistance of the kisspeptin system was not induced and that kisspeptin action was preserved in the dexamethasone-induced IUGR rats. These results indicate that the dexamethasone-induced IUGR rats and the undernutritioninduced IUGR rats have different programming mechanisms in sexual maturation. On the other hand, it is also unclear why the hypothalamic expression of Kiss1 mRNA was not decreased despite the low body weight of the dexamethasone-induced IUGR rats. When body weight is decreased by food restriction in the prepubertal and adult period, the hypothalamic expression of Kiss1 mRNA is decreased, and consequently, the serum LH concentration is reduced (Castellano et al., 2009; Iwasa et al., 2010b). In these conditions, the actions of orexigenic and stress related factors are increased and that of anorexigenic factors is decreased. While these alterations stimulate feeding behavior, some of them; i.e. increased CRH activity and/or a decreased serum leptin concentration, might suppress the hypothalamic expression of Kiss1 mRNA. On the other hand, no such alterations were found in the dexamethasoneinduced IUGR rats used in this study, although their body weights were lower than those of the controls throughout the experimental period. We suppose that when food is restricted and body weight is acutely decreased, the action of the kisspeptin system is suppressed, regardless of basal body weight. In other words, the dexamethasone-induced IUGR rats used in this study were able to consume sufficient levels of nutrition for their body weight after birth, and therefore, they might have been able to preserve normal Kiss1 mRNA expression levels. We also evaluated the hypothalamic CRH mRNA level. It has been reported that prenatal stress and dexamethasone disturb the HPA axis, in which CRH acts as a central regulator (Levitt et al., 1996; Lesage et al., 2001; de Vries et al., 2007). As CRH is a negative regulator of the kisspeptin system and the onset of puberty (Kinsey-Jones et al., 2009, 2010), changes in its hypothalamic level could alter the action of the kisspeptin system and affect sexual maturation. However, as a result, CRH mRNA level was not altered in the present model. In addition, we measured hypothalamic NPY and POMC mRNA levels because these factors might mediate the action of kisspeptin on GnRH. It has been reported that kisspeptin increases NPY expression and reduces POMC gene expression (Backholer et al., 2010; Kim et al., 2010) and that both NPY and POMC affect GnRH release and the onset of puberty (Wiemann et al., 1989; Lebrethon et al., 2000). However, as for CRH, the mRNA levels of these molecules were not altered in the present model. In this study, we could not clarify the cause of the delayed onset of puberty observed in the dexamethasone-induced IUGR rats. As the levels of gonadotrophin were changed, the cause might not have been related to hypothalamic or pituitary mRNA levels. However, it has been reported that the serum LH concentration is increased in the mid-afternoon in the prepubertal period and is accompanied by the establishment of a diurnal pattern of pulsatile LH secretion. As we used single blood samples, which were taken in the morning, to evaluate serum LH concentrations in the present study, we were unable to evaluate the diurnal pattern of LH secretion. If the establishment of the diurnal LH secretion pattern is disturbed in dexamethasone-induced IUGR rats, this might have caused the observed delays in sexual maturation and puberty onset. Further experiments using multiple blood samples are therefore needed to compare the diurnal pattern of LH secretion between dexamethasone-induced IUGR rats and control rats (Urbanski and Ojeda, 1985). On the other hand, some studies focusing on alterations in ovarian factors have shown that follicle number and/or the expression level of the gonadotrophin receptor are decreased in undernutrition and/or dexamethasone-induced IUGR rats, and we speculate that these alterations affect sexual maturation (Deligeorgis et al., 1996; Faria et al., 2008; Ristic et al.,

187

2008). As ovarian weight in the dexamathasone-induced IUGR rats were lower during the prepubertal period (postnatal day 28), but not at VO, than in the control, the retardation of ovarian function development might be involved in the delayed onset of puberty. In summary, the present study shows that hypothalamic mRNA levels of Kiss1, Kiss1r, GnRH, CRH, NPY and POMC and the serum LH and FSH concentrations in the morning were not altered in dexamethasone-induced IUGR rats, suggesting that the delayed onset of puberty observed in this model might occur independently of hypothalamic Kiss1–Kiss1r–GnRH system. Conflict of interest The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific works. References Ahima, R.S., Dushay, J., Flier, S.N., Prabakaran, D., Flier, J.S., 1997. Leptin accelerates the onset of puberty in normal female mice. J. Clin. Invest. 99, 391–395. 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