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Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats
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Short communication
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Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats
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Toshiya Matsuzaki ∗ , Takeshi Iwasa, Munkhsaikhan Munkhzaya, Altankhuu Tungalagsuvd
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, Takako Kawami, Masahiro Murakami, Mikio Yamasaki, Yuri Yamamoto, Takeshi Kato, 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
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Article history: Received 29 November 2014 Received in revised form 8 January 2015 Accepted 9 January 2015 Available online xxx
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Keywords: Oxytocin Estrogen receptor-␣ Hypothalamus
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1. Introduction
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Oxytocin (OT) affects the central nervous system and is involved in a variety of social and non-social behaviors. Recently, the role played by OT in energy metabolism and its organizational effects on estrogen receptor alpha (ER-␣) during the neonatal period have gained attention. In this study, the developmental changes in the hypothalamic mRNA levels of OT, the OT receptor (OTR), and ER-␣ were evaluated in male and female rats. In addition, the fasting-induced changes in the hypothalamic mRNA levels of OT and the OTR were evaluated. Hypothalamic explants were taken from postnatal day (PND) 10, 20, and 30 rats, and the mRNA level of each molecule was measured. Hypothalamic OT mRNA expression increased throughout the developmental period in both sexes. The rats’ hypothalamic OTR mRNA levels were highest on PND 10 and decreased throughout the developmental period. In the male rats, the hypothalamic mRNA levels of ER-␣ were higher on PND 30 than on PND 10. On the other hand, no significant differences in hypothalamic ER-␣ mRNA expression were detected among the examined time points in the female rats, although hypothalamic ER-␣ mRNA expression tended to be higher on PND 30 than on PND 10. Significant positive correlations were detected between hypothalamic OT and ER-␣ mRNA expression in both the male and female rats. Hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points in either sex. These results indicate that hypothalamic OT expression is not sensitive to fasting during the developmental period. In addition, as a positive correlation was detected between hypothalamic OT and ER-␣ mRNA expression, these two molecules might interact with each other to induce appropriate neuronal development. © 2015 Elsevier Ltd. All rights reserved.
Oxytocin (OT) is a hypothalamic neuropeptide and plays an essential role in labor and lactation in mammals (Kiss and Mikkelsen, 2005). OT also acts on the central nervous system and is involved in a variety of social and non-social behaviors (Yang et al., 2013). Recently, the role of OT in energy metabolism (Tung et al., 2008; Maejima et al., 2009, 2011; Zhang and Cai, 2011) and its organizational effects on estrogen receptor alpha (ER-␣) during the
∗ Corresponding author. Tel.: +81 88 633 7177. E-mail address: [email protected] (T. Matsuzaki).
neonatal period have been examined in several studies (Yamamoto et al., 2006; Perry et al., 2009). OT mRNA expression in the paraventricular nucleus, one of the hypothalamic nuclei, is decreased by fasting and can be restored by re-feeding in mice (Tung et al., 2008). In rodent studies, central and peripheral OT injections were demonstrated to reduce food intake, whereas the injection of a central OT antagonist increased food intake, suggesting that OT acts to suppress appetite and feeding behavior (Maejima et al., 2009, 2011; Zhang and Cai, 2011). Furthermore, chronic peripheral OT injections caused reductions in body weight, visceral fat mass, and obesity in diet-induced obese mice (Maejima et al., 2011). The effects of OT on energy metabolism might be mediated by hypothalamic neuronal systems (Maejima et al., 2011).
http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001 0736-5748/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Matsuzaki, T., et al., Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats. Int. J. Dev. Neurosci. (2015), http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001
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Fig. 1. Hypothalamic mRNA levels of oxytocin (OT), the oxytocin receptor (OTR), and estrogen receptor alpha (ER-␣) on postnatal days (PND) 10, 20, and 30 in male and female rats. The values obtained on PND 10 were defined as 1.0. Hypothalamic OT mRNA expression increased throughout the developmental period. Hypothalamic OTR mRNA expression peaked on PND 10 and then decreased throughout the rest of the developmental period. In the male rats, hypothalamic ER-␣ mRNA expression was significantly higher on PND 30 than on PND 10. On the other hand, in the female rats no significant differences in hypothalamic ER-␣ mRNA expression were detected between any of the examined time points, although it tended to be higher on PND 30 than on PND 10. Data are expressed as mean ± SE values. Different letters (a–c) indicate significant differences (P < 0.05) according to one-way ANOVA followed by the Tukey–Kramer post-hoc test.
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In adulthood, ER-␣ plays important roles in reproduction and social behavior in both sexes. For example, it is heavily involved in sexual receptivity in female rats (Perry et al., 2009) and is associated with social interactions in male mice (Murakami et al., 2011). Some studies have shown that OT, the OT receptor (OTR), and ER-␣ interact with each other in adulthood. Estrogen and ER regulate social behaviors through OT in male mice (Murakami et al., 2011). On the other hand, OT has been shown to have a number of effects on ER-␣ in vitro (Cassoni et al., 2002), and treating adult female prairie voles with OT increased their behavioral sensitivity to estrogen (Cushing and Carter, 1999). Although OT can have short-term consequences in adulthood, neonatal OT treatment has profound and long-lasting effects on ER-␣ and ER-␣-related behaviors. Neonatal OT treatment reduces hypothalamic ER-␣ expression, and neonatal OTR treatment also downregulated hypothalamic ER-␣ expression in female rats (Perry et al., 2009). In addition, both molecules delay sexual maturation and decrease estradiol-induced sexual receptivity, suggesting that they reduce estrogen sensitivity (Withuhn et al., 2003; Perry et al., 2009). As noted above, hypothalamic OT plays important roles in the regulation of the energy balance in adulthood and has organizational effects on ER-␣ during the developmental period. However, as far as we know, the developmental changes in hypothalamic OT and OTR expression and their sensitivity to under-nutrition in the pre-pubertal period have not been examined. In addition, the interaction between OT and ER-␣ during the developmental period has not been evaluated. Examinations of these topics are important for clarifying the mechanisms by which neonatal OT has long-lasting effects on physiological and behavioral functions. In this study, the hypothalamic OT and OTR mRNA levels of male and female rats were measured during the neonatal and pre-pubertal periods. In addition, the changes in the hypothalamic concentra-
tions of these molecules induced by fasting were also evaluated. The rats’ ER-␣ mRNA levels were also measured in order to elucidate how hypothalamic OT and ER-␣ mRNA expression change during the developmental period.
2. Materials and methods The animals were housed under controlled lighting (12 h light: 12 h darkness cycle with light on at 0800 h) and temperature (24 ◦ C) conditions. Pregnant Sprague–Dawley rats (Charles River Japan Inc., Tokyo, Japan) were purchased and housed individually. The day on which the litters were born was defined as postnatal day (PND) 1. On PND 2, 12 pups were randomly assigned to each dam. To adjust the litter size to 10–12 per dam, pups were culled or moved to other dams and were fostered until weaning. The rats used on PND 30 were weaned at PND 21 and housed at three to four per cage. All animal experiments were conducted in accordance with the ethical standards of the animal care and use committee of the University of Tokushima. On PND 10, 20, and 30, rats of both sexes were randomly selected from each dam, weighed, and divided into the fed and fasting groups (n = 7–8 per group). The rats in the fasting groups were subjected to 24 h maternal (PND 10 and 20) or food (PND 30) deprivation. Twenty-four hours later, the rats’ brains were collected by decapitation between 0900 and 1000 h and stored at −80 ◦ C after being snap frozen. Whole hypothalamic explants were dissected from the frozen brains, as described previously (Iwasa et al., 2014a,b). Total RNA was isolated using a TRIzol® reagent kit (Invitrogen Co., Carlsbad, CA, USA) and an RNeasy® mini kit (Qiagen Gmbh, Hilden, Germany). cDNA was synthesized with oligo (deoxythymidine) primers at 50 ◦ C using the SuperScript III first-strand synthesis system for the real-time polymerase chain
Please cite this article in press as: Matsuzaki, T., et al., Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats. Int. J. Dev. Neurosci. (2015), http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001
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Fig. 2. Correlation between the hypothalamic mRNA levels of oxytocin (OT) and estrogen receptor alpha (ER-␣) in male and female rats. Significant positive correlations were detected between hypothalamic OT and ER-␣ mRNA expression in both the male and female rats. The data obtained on postnatal days (PND) 10, 20, and 30 under the fed conditions are shown. The values obtained on PND 10 were defined as 1.0.
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reaction (RT-PCR; Invitrogen Co.). The PCR analysis was performed using the StepOnePlusTM RT-PCR system (PE Applied Biosystems, Foster City, CA, USA) and FAST SYBR® green. The mRNA expression levels of OT, the OTR, and ER-␣ were normalized to that of GAPDH. The following forward and reverse primers were used: OT: F: 5 GAA CAC CAA CGC CAT GGC CTG CCC-3 , R: 5 -TCG GTG CGG CAG CCA TCC GGG CTA-3 ; OTR: F: 5 -CGA TTG CTG GGC GGT CTT-3 , R: 5 -CCG CCG CTG CCG TCT TGA-3 ; ER-␣: F: 5 -GGC TAC GTC AAG TCG ATT CC-3 , R: 5 -ATC TTG TCC AGG ACT CGG TG-3 ; GAPDH: F: 5 -ATG GCA CAG TCA AGG CTG AGA-3 , R: 5 -CGC TCC TGG AAG ATG GTG AT-3 . The PCR conditions were as follows: initial denaturation and enzyme activation were performed at 95 ◦ C for 20 s, followed by 45 cycles of denaturation at 95 ◦ C for 3 s, and annealing and extension at 62 ◦ C for 30 s (OT and ER-␣), 64 ◦ C for 30 s (GAPDH), or 67 ◦ C for 30 s (OTR). All data are presented as mean ± SEM values. Statistical analyses were performed using one-way analysis of variance (ANOVA) together with the Tukey–Kramer post-hoc test or Student’s t test. Correlation analyses were performed using Pearson’s correlation co-efficient as appropriate. Statistical significance was defined as P < 0.05.
3. Results Under the fed conditions, hypothalamic OT mRNA expression differed significantly among the examined age groups in both the male (one-way ANOVA; F(2,22) = 33.7, P < 0.01) and female rats (one-way ANOVA; F(2,22) = 53.1, P < 0.01) (Fig. 1). Hypothalamic OT mRNA expression increased throughout the developmental period and was significantly higher on PND 30 than on PND 10 and 20. Hypothalamic OTR mRNA expression also differed significantly among the examined age groups in both the male (one-way ANOVA; F(2,22) = 7.96, P < 0.01) and female rats (one-way ANOVA; F(2,22) = 50.6, P < 0.01) (Fig. 1). However, it peaked on PND 10 and then decreased throughout the rest of the developmental period. The hypothalamic OT mRNA levels of the male and female rats did not differ at PND 10 or 20, whereas the males exhibited significantly higher hypothalamic OT mRNA levels than the females at PND 30 (data not shown). On the other hand, the males demonstrated significantly lower hypothalamic OTR mRNA levels than the females at PND10, but no such differences were seen at PND 30 (data not shown). Hypothalamic ER-␣ mRNA expression differed significantly among the examined age groups in both the male
(one-way ANOVA; F(2,22) = 9.51, P < 0.01) and female rats (one-way ANOVA; F(2,22) = 3.54, P < 0.05). In the male rats, hypothalamic ER␣ mRNA expression was significantly higher on PND 30 than on PND 10. On the other hand, no significant differences in hypothalamic ER-␣ mRNA expression were detected between any of the examined time points in the female rats, although it tended to be higher on PND 30 than on PND 10 (P = 0.08). Significant positive correlations were detected between hypothalamic OT and ER-␣ mRNA expression in both the male and female rats (Fig. 2). In the male rats, hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points (Fig. 3). In the female rats, whilst hypothalamic OTR mRNA expression was not altered by fasting at any of the examined time points, hypothalamic OTR mRNA expression was downregulated at PND 10.
4. Discussion In this study, we found that hypothalamic OT mRNA expression increased from the neonatal to the pre-pubertal period, whereas hypothalamic OTR mRNA expression peaked during the neonatal period and subsequently decreased. We also detected a positive correlation between the hypothalamic mRNA expression levels of OT and ER-␣ during the developmental period. In addition, we demonstrated that hypothalamic OT mRNA expression was not affected by fasting during the neonatal to pre-pubertal period. OT acts on the central nervous system and plays a role in a variety of social and non-social behaviors (Yang et al., 2013). For example, it regulates social memory, attachment, and sexual behavior (social behaviors), as well as the energy balance, reproduction, and brain development (non-social behaviors). Several studies have focused on the effects of OT on the energy balance (Maejima et al., 2009, 2011) and brain development (Yamamoto et al., 2006; Perry et al., 2009). However, most of these studies used adult animal models or evaluated the effects of exogenous OT injections. In previous studies, we found that the sensitivity of the hypothalamic expression patterns of orexigenic and reproductive factors, such as neuropeptide Y (NPY), proopiomelanocortin, and kisspeptin, to a negative energy balance changed markedly from the neonatal to the pre-pubertal period (Delahaye et al., 2008; Iwasa et al., 2011). In addition, some studies have shown that severe fasting or fasting-related hormonal conditions during the prenatal to neonatal period has long-lasting effects on the functions of
Please cite this article in press as: Matsuzaki, T., et al., Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats. Int. J. Dev. Neurosci. (2015), http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001
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Fig. 3. Effects of fasting on the hypothalamic mRNA levels of oxytocin (OT) and the oxytocin receptor (OTR) on postnatal days (PND) 10, 20, and 30 in male and female rats. Hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points in the male rats. In the female rats, whilst hypothalamic OT mRNA expression was not altered by fasting at any of the examined time points, hypothalamic OTR mRNA expression was downregulated at PND 10. The values obtained on PND 10 in the fed rats were defined as 1.0. Data are expressed as mean ± SE values. *P < 0.05.
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these factors (Delahaye et al., 2008; Iwasa et al., 2010, 2014c). These studies indicated that fasting stress has long-lasting effects on the functions of the abovementioned molecules in the immature neuronal system. As noted above, hypothalamic OT expression is not sensitive to fasting during the neonatal to pre-pubertal period. Because previous our studies have shown that other hypothalamic factors already have sensitivities to 24 h fating at postnatal day 10 and/or 20 (Matsuzaki et al., 2015), OT insensitivity, observed in this study, may be caused by the immaturity of OT system, but not caused by the insensitivity of hypothalamus to fasting. Therefore, it is possible that severe fasting during this period might induce longlasting changes in the effects of hypothalamic OT expression on the energy balance. If this hypothesis is true, long-lasting alterations in hypothalamic OT expression might be responsible for certain metabolic disorders because OT has been shown to play pivotal roles in preventing obesity in adulthood (Tung et al., 2008; Maejima et al., 2009, 2011; Zhang and Cai, 2011). However, further studies are needed to confirm these hypotheses. In addition, it is necessary to clarify when hypothalamic OT expression first becomes sensitive to fasting. In adulthood, ER-␣ plays important roles in reproduction and social behavior in both sexes (Perry et al., 2009; Murakami et al., 2011). Recently, it has been reported that neonatal OT treatment has long-lasting effects on ER-␣ expression and ER-␣-related behaviors. (Withuhn et al., 2003; Perry et al., 2009), suggesting that the organizational effects of OT on ER-␣ persist into adulthood. In this study, we found that the hypothalamic mRNA expression levels of OT and ER-␣ tended to increase during the developmental period and were positively correlated during the developmental period. These findings indicate that the functions of OT and ER are not in operation in the pre-pubertal period and that these molecules might interact with each other to promote appropriate neuronal development. On the other hand, OTR mRNA expression tended to decrease during the developmental period, suggesting that cells might become less responsive to OT as development progresses. Another possibility is that estrogen affects OT and ER-␣ mRNA
expression during development. Further examinations are needed to evaluate these hypotheses. In this study, the pups in the fasting groups were separated from their mothers. Some studies have reported that the hypothalamic-pituitary-adrenal axis, which is partially regulated by OT, is activated by maternal separation (Levine et al., 1991; Smith et al., 1997; Schmidt et al., 2006). In addition, maternal separation-induced hypothermia might also affect the mRNA level of OT. Therefore, it is possible that the effects of maternal separation and hypothermia (if such effects exist) compensate for fastinginduced changes in OT expression. Thus, it would be necessary to control all of these factors in order to confirm whether food deprivation alone affects OT mRNA levels. It is also possible that the fasting period employed in the present study was not long enough to affect the OT system, which is known to be robust. Experiments performed under more strictly controlled conditions are required to examine these hypotheses. In summary, this study showed that hypothalamic OT mRNA expression was increased, but was not sensitive to fasting, during the developmental period. In addition, as hypothalamic mRNA OT and ER-␣ expression were found to be positively correlated these two factors might interact with each other to induce appropriate neuronal development. References Cassoni, P., Catalano, M.G., Sapino, A., Marrocco, T., Fazzari, A., Bussolati, G., Fortunati, N., 2002. Oxytocin modulates estrogen receptor alpha expression and function in MCF7 human breast cancer cells. Int. J. Oncol. 21, 375–378. Cushing, B.S., Carter, C.S., 1999. Prior exposure to oxytocin mimics social contact and facilitates sexual behaviour in females. J. Neuroendocrinol. 11, 765–769. Delahaye, F., Breton, C., Risold, P.Y., Enache, M., Dutriez-Casteloot, I., Laborie, C., Lesage, J., Vieau, D., 2008. Maternal perinatal undernutrition drastically reduces postnatal leptin surge and affects the development of arcuate nucleus proopiomelanocortin neurons in neonatal male rat pups. Endocrinology 149, 470–475. Iwasa, T., Matsuzaki, T., Murakami, M., Fujisawa, S., Kinouchi, R., Gereltsetseg, G., Kuwahara, A., Yasui, T., Irahara, M., 2010. Effects of intrauterine undernutrition on hypothalamic Kiss1 expression and the timing of puberty in female rats. J. Physiol. 588, 821–829.
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Iwasa, T., Matsuzaki, T., Kinouchi, R., Gereltsetseg, G., Murakami, M., Nakazawa, H., Yasui, T., Irahara, M., 2011. Changes in the responsiveness of serum leptin and hypothalamic neuropeptide Y mRNA levels to food deprivation in developing rats. Int. J. Dev. Neurosci. 29, 377–380. Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kawami, T., Murakami, M., Yamasaki, M., Kato, T., Kuwahara, A., Yasui, T., Irahara, M., 2014a. Pre-pubertal serum leptin levels and sensitivity to central leptin injection of prenatally undernourished female rats. Int. J. Dev. Neurosci. 35, 52–54. Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kawami, T., Murakami, M., Yamasaki, M., Kato, T., Kuwahara, A., Yasui, T., Irahara, M., 2014b. Changes in the responsiveness of hypothalamic prokineticin 2 mRNA expression to food deprivation in developing female rats. Int. J. Dev. Neurosci. 34, 76–78. Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kawami, T., Murakami, M., Yamasaki, M., Kato, T., Kuwahara, A., Yasui, T., Irahara, M., 2014c. Prenatal exposure to glucocorticoids affects body weight serum leptin levels, and hypothalamic neuropeptide-Y expression in pre-pubertal female rat offspring. Int. J. Dev. Neurosci. 36, 1–4. Kiss, A., Mikkelsen, J.D., 2005. Oxytocin anatomy and functional assignments: a minireview. Endocr. Regul. 39, 97–105. Levine, S., Huchton, D.M., Wiener, S.G., Rosenfeld, P., 1991. Time course of the effect of maternal deprivation on the hypothalamic–pituitary–adrenal axis in the infant rat. Dev. Psychobiol. 24, 547–548. Matsuzaki, T., Iwasa, T., Altankhuu, T., Munkhsaikhan, M., Kawami, T., Yamasaki, M., Murakami, M., Kato, T., Kuwahara, A., Yasui, T., Irahara, M., 2015. The responses of hypothalamic NPY and OBRb mRNA expression to food deprivation develop during the neonatal-prepubertal period and exhibit gender differences in rats. Int. J. Dev. Neurosci., in press. Maejima, Y., Sedbazar, U., Suyama, S., Kohno, D., Onaka, T., Takano, E., Yoshida, N., Koike, M., Uchiyama, Y., Fujiwara, K., Yashiro, T., Horvath, T.L., Dietrich, M.O., Tanaka, S., Dezaki, K., Oh, I., Hashimoto, S., Shimizu, K., Nakata, H., Mori, M., Yada, M., 2009. Nesfatin-1-regulated oxytocinergic signaling in the paraventricular nucleus causes anorexia through a leptin-independent melanocortin pathway. Cell Metabol. 10, 355–365.
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Maejima, Y., Iwasaki, Y., Yamahara, Y., Kodaira, M., Sedbazar, U., Yada, T., 2011. Peripheral oxytocin treatment ameliorates obesity by reducing food intake and visceral fat mass. Aging 3, 1169–1177. Murakami, G., Hunter, R.G., Fontaine, C., Ribeiro, A., Pfaff, D., 2011. Relationships among estrogen receptor: oxytocin and vasopressin gene expression and social interaction in male mice. Eur. J. Neurosci. 34, 469–477. Perry, A.N., Paramadilok, A., Cushing, B.S., 2009. Neonatal oxytocin alters subsequent estrogen receptor alpha protein expression and estrogen sensitivity in the female rat. Behav. Brain. Res. 205, 154–161. Schmidt, M.V., Levine, S., Alam, S., Harbich, D., Sterlemann, V., Ganea, K., de Kloet, E.R., Holsboer, F., Muller, M.B., 2006. Metabolic signals modulate hypothalamic–pituitary–adrenal axis activation during maternal separation of the neonatal mouse. J. Neuroendocrinol. 18, 865–874. Smith, M.A., Kim, S.Y., van Oers, H.J., Levine, S., 1997. Maternal deprivation and stress induce immediate early genes in the infant rat brain. Endocrinology 138, 4622–4628. Tung, Y.C., Ma, M., Piper, S., Coll, A., O’Rahilly, S., Yeo, G.S.H., 2008. Novel leptin regulated genes revealed by transcriptional profiling of the hypothalamic paraventricular nucleus. J. Neurosci. 19, 12419–12426. Withuhn, T.F., Kramer, K.M., Cushing, B.S., 2003. Early exposure to oxytocin affects the age of vaginal opening and first estrus in female rats. Physiol. Behav. 80, 135–138. Yamamoto, Y., Carter, C.S., Cushing, B.S., 2006. Neonatal manipulation of oxytocin affects expression of estrogen receptor alpha. Neuroscience 137, 157–164. Yang, H.P., Wang, L., Han, L., Wang, S.C., 2013. Nonsocial functions of hypothalamic Q4 oxytocin. ISRN Neuroscience, 179272. Zhang, G., Cai, D., 2011. Circadian intervention of obesity development via resting stage feeding manipulation or oxytocin treatment. Am. J. Physiol. Endocrinol. Metab. 301, E1004–E1012.
Please cite this article in press as: Matsuzaki, T., et al., Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats. Int. J. Dev. Neurosci. (2015), http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001
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Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Short communication
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Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats
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Toshiya Matsuzaki ∗ , Takeshi Iwasa, Munkhsaikhan Munkhzaya, Altankhuu Tungalagsuvd
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, Takako Kawami, Masahiro Murakami, Mikio Yamasaki, Yuri Yamamoto, Takeshi Kato, 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
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Article history: Received 29 November 2014 Received in revised form 8 January 2015 Accepted 9 January 2015 Available online xxx
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Keywords: Oxytocin Estrogen receptor-␣ Hypothalamus
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1. Introduction
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Oxytocin (OT) affects the central nervous system and is involved in a variety of social and non-social behaviors. Recently, the role played by OT in energy metabolism and its organizational effects on estrogen receptor alpha (ER-␣) during the neonatal period have gained attention. In this study, the developmental changes in the hypothalamic mRNA levels of OT, the OT receptor (OTR), and ER-␣ were evaluated in male and female rats. In addition, the fasting-induced changes in the hypothalamic mRNA levels of OT and the OTR were evaluated. Hypothalamic explants were taken from postnatal day (PND) 10, 20, and 30 rats, and the mRNA level of each molecule was measured. Hypothalamic OT mRNA expression increased throughout the developmental period in both sexes. The rats’ hypothalamic OTR mRNA levels were highest on PND 10 and decreased throughout the developmental period. In the male rats, the hypothalamic mRNA levels of ER-␣ were higher on PND 30 than on PND 10. On the other hand, no significant differences in hypothalamic ER-␣ mRNA expression were detected among the examined time points in the female rats, although hypothalamic ER-␣ mRNA expression tended to be higher on PND 30 than on PND 10. Significant positive correlations were detected between hypothalamic OT and ER-␣ mRNA expression in both the male and female rats. Hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points in either sex. These results indicate that hypothalamic OT expression is not sensitive to fasting during the developmental period. In addition, as a positive correlation was detected between hypothalamic OT and ER-␣ mRNA expression, these two molecules might interact with each other to induce appropriate neuronal development. © 2015 Elsevier Ltd. All rights reserved.
Oxytocin (OT) is a hypothalamic neuropeptide and plays an essential role in labor and lactation in mammals (Kiss and Mikkelsen, 2005). OT also acts on the central nervous system and is involved in a variety of social and non-social behaviors (Yang et al., 2013). Recently, the role of OT in energy metabolism (Tung et al., 2008; Maejima et al., 2009, 2011; Zhang and Cai, 2011) and its organizational effects on estrogen receptor alpha (ER-␣) during the
∗ Corresponding author. Tel.: +81 88 633 7177. E-mail address: [email protected] (T. Matsuzaki).
neonatal period have been examined in several studies (Yamamoto et al., 2006; Perry et al., 2009). OT mRNA expression in the paraventricular nucleus, one of the hypothalamic nuclei, is decreased by fasting and can be restored by re-feeding in mice (Tung et al., 2008). In rodent studies, central and peripheral OT injections were demonstrated to reduce food intake, whereas the injection of a central OT antagonist increased food intake, suggesting that OT acts to suppress appetite and feeding behavior (Maejima et al., 2009, 2011; Zhang and Cai, 2011). Furthermore, chronic peripheral OT injections caused reductions in body weight, visceral fat mass, and obesity in diet-induced obese mice (Maejima et al., 2011). The effects of OT on energy metabolism might be mediated by hypothalamic neuronal systems (Maejima et al., 2011).
http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001 0736-5748/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Matsuzaki, T., et al., Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats. Int. J. Dev. Neurosci. (2015), http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001
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Fig. 1. Hypothalamic mRNA levels of oxytocin (OT), the oxytocin receptor (OTR), and estrogen receptor alpha (ER-␣) on postnatal days (PND) 10, 20, and 30 in male and female rats. The values obtained on PND 10 were defined as 1.0. Hypothalamic OT mRNA expression increased throughout the developmental period. Hypothalamic OTR mRNA expression peaked on PND 10 and then decreased throughout the rest of the developmental period. In the male rats, hypothalamic ER-␣ mRNA expression was significantly higher on PND 30 than on PND 10. On the other hand, in the female rats no significant differences in hypothalamic ER-␣ mRNA expression were detected between any of the examined time points, although it tended to be higher on PND 30 than on PND 10. Data are expressed as mean ± SE values. Different letters (a–c) indicate significant differences (P < 0.05) according to one-way ANOVA followed by the Tukey–Kramer post-hoc test.
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In adulthood, ER-␣ plays important roles in reproduction and social behavior in both sexes. For example, it is heavily involved in sexual receptivity in female rats (Perry et al., 2009) and is associated with social interactions in male mice (Murakami et al., 2011). Some studies have shown that OT, the OT receptor (OTR), and ER-␣ interact with each other in adulthood. Estrogen and ER regulate social behaviors through OT in male mice (Murakami et al., 2011). On the other hand, OT has been shown to have a number of effects on ER-␣ in vitro (Cassoni et al., 2002), and treating adult female prairie voles with OT increased their behavioral sensitivity to estrogen (Cushing and Carter, 1999). Although OT can have short-term consequences in adulthood, neonatal OT treatment has profound and long-lasting effects on ER-␣ and ER-␣-related behaviors. Neonatal OT treatment reduces hypothalamic ER-␣ expression, and neonatal OTR treatment also downregulated hypothalamic ER-␣ expression in female rats (Perry et al., 2009). In addition, both molecules delay sexual maturation and decrease estradiol-induced sexual receptivity, suggesting that they reduce estrogen sensitivity (Withuhn et al., 2003; Perry et al., 2009). As noted above, hypothalamic OT plays important roles in the regulation of the energy balance in adulthood and has organizational effects on ER-␣ during the developmental period. However, as far as we know, the developmental changes in hypothalamic OT and OTR expression and their sensitivity to under-nutrition in the pre-pubertal period have not been examined. In addition, the interaction between OT and ER-␣ during the developmental period has not been evaluated. Examinations of these topics are important for clarifying the mechanisms by which neonatal OT has long-lasting effects on physiological and behavioral functions. In this study, the hypothalamic OT and OTR mRNA levels of male and female rats were measured during the neonatal and pre-pubertal periods. In addition, the changes in the hypothalamic concentra-
tions of these molecules induced by fasting were also evaluated. The rats’ ER-␣ mRNA levels were also measured in order to elucidate how hypothalamic OT and ER-␣ mRNA expression change during the developmental period.
2. Materials and methods The animals were housed under controlled lighting (12 h light: 12 h darkness cycle with light on at 0800 h) and temperature (24 ◦ C) conditions. Pregnant Sprague–Dawley rats (Charles River Japan Inc., Tokyo, Japan) were purchased and housed individually. The day on which the litters were born was defined as postnatal day (PND) 1. On PND 2, 12 pups were randomly assigned to each dam. To adjust the litter size to 10–12 per dam, pups were culled or moved to other dams and were fostered until weaning. The rats used on PND 30 were weaned at PND 21 and housed at three to four per cage. All animal experiments were conducted in accordance with the ethical standards of the animal care and use committee of the University of Tokushima. On PND 10, 20, and 30, rats of both sexes were randomly selected from each dam, weighed, and divided into the fed and fasting groups (n = 7–8 per group). The rats in the fasting groups were subjected to 24 h maternal (PND 10 and 20) or food (PND 30) deprivation. Twenty-four hours later, the rats’ brains were collected by decapitation between 0900 and 1000 h and stored at −80 ◦ C after being snap frozen. Whole hypothalamic explants were dissected from the frozen brains, as described previously (Iwasa et al., 2014a,b). Total RNA was isolated using a TRIzol® reagent kit (Invitrogen Co., Carlsbad, CA, USA) and an RNeasy® mini kit (Qiagen Gmbh, Hilden, Germany). cDNA was synthesized with oligo (deoxythymidine) primers at 50 ◦ C using the SuperScript III first-strand synthesis system for the real-time polymerase chain
Please cite this article in press as: Matsuzaki, T., et al., Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats. Int. J. Dev. Neurosci. (2015), http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001
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Fig. 2. Correlation between the hypothalamic mRNA levels of oxytocin (OT) and estrogen receptor alpha (ER-␣) in male and female rats. Significant positive correlations were detected between hypothalamic OT and ER-␣ mRNA expression in both the male and female rats. The data obtained on postnatal days (PND) 10, 20, and 30 under the fed conditions are shown. The values obtained on PND 10 were defined as 1.0.
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reaction (RT-PCR; Invitrogen Co.). The PCR analysis was performed using the StepOnePlusTM RT-PCR system (PE Applied Biosystems, Foster City, CA, USA) and FAST SYBR® green. The mRNA expression levels of OT, the OTR, and ER-␣ were normalized to that of GAPDH. The following forward and reverse primers were used: OT: F: 5 GAA CAC CAA CGC CAT GGC CTG CCC-3 , R: 5 -TCG GTG CGG CAG CCA TCC GGG CTA-3 ; OTR: F: 5 -CGA TTG CTG GGC GGT CTT-3 , R: 5 -CCG CCG CTG CCG TCT TGA-3 ; ER-␣: F: 5 -GGC TAC GTC AAG TCG ATT CC-3 , R: 5 -ATC TTG TCC AGG ACT CGG TG-3 ; GAPDH: F: 5 -ATG GCA CAG TCA AGG CTG AGA-3 , R: 5 -CGC TCC TGG AAG ATG GTG AT-3 . The PCR conditions were as follows: initial denaturation and enzyme activation were performed at 95 ◦ C for 20 s, followed by 45 cycles of denaturation at 95 ◦ C for 3 s, and annealing and extension at 62 ◦ C for 30 s (OT and ER-␣), 64 ◦ C for 30 s (GAPDH), or 67 ◦ C for 30 s (OTR). All data are presented as mean ± SEM values. Statistical analyses were performed using one-way analysis of variance (ANOVA) together with the Tukey–Kramer post-hoc test or Student’s t test. Correlation analyses were performed using Pearson’s correlation co-efficient as appropriate. Statistical significance was defined as P < 0.05.
3. Results Under the fed conditions, hypothalamic OT mRNA expression differed significantly among the examined age groups in both the male (one-way ANOVA; F(2,22) = 33.7, P < 0.01) and female rats (one-way ANOVA; F(2,22) = 53.1, P < 0.01) (Fig. 1). Hypothalamic OT mRNA expression increased throughout the developmental period and was significantly higher on PND 30 than on PND 10 and 20. Hypothalamic OTR mRNA expression also differed significantly among the examined age groups in both the male (one-way ANOVA; F(2,22) = 7.96, P < 0.01) and female rats (one-way ANOVA; F(2,22) = 50.6, P < 0.01) (Fig. 1). However, it peaked on PND 10 and then decreased throughout the rest of the developmental period. The hypothalamic OT mRNA levels of the male and female rats did not differ at PND 10 or 20, whereas the males exhibited significantly higher hypothalamic OT mRNA levels than the females at PND 30 (data not shown). On the other hand, the males demonstrated significantly lower hypothalamic OTR mRNA levels than the females at PND10, but no such differences were seen at PND 30 (data not shown). Hypothalamic ER-␣ mRNA expression differed significantly among the examined age groups in both the male
(one-way ANOVA; F(2,22) = 9.51, P < 0.01) and female rats (one-way ANOVA; F(2,22) = 3.54, P < 0.05). In the male rats, hypothalamic ER␣ mRNA expression was significantly higher on PND 30 than on PND 10. On the other hand, no significant differences in hypothalamic ER-␣ mRNA expression were detected between any of the examined time points in the female rats, although it tended to be higher on PND 30 than on PND 10 (P = 0.08). Significant positive correlations were detected between hypothalamic OT and ER-␣ mRNA expression in both the male and female rats (Fig. 2). In the male rats, hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points (Fig. 3). In the female rats, whilst hypothalamic OTR mRNA expression was not altered by fasting at any of the examined time points, hypothalamic OTR mRNA expression was downregulated at PND 10.
4. Discussion In this study, we found that hypothalamic OT mRNA expression increased from the neonatal to the pre-pubertal period, whereas hypothalamic OTR mRNA expression peaked during the neonatal period and subsequently decreased. We also detected a positive correlation between the hypothalamic mRNA expression levels of OT and ER-␣ during the developmental period. In addition, we demonstrated that hypothalamic OT mRNA expression was not affected by fasting during the neonatal to pre-pubertal period. OT acts on the central nervous system and plays a role in a variety of social and non-social behaviors (Yang et al., 2013). For example, it regulates social memory, attachment, and sexual behavior (social behaviors), as well as the energy balance, reproduction, and brain development (non-social behaviors). Several studies have focused on the effects of OT on the energy balance (Maejima et al., 2009, 2011) and brain development (Yamamoto et al., 2006; Perry et al., 2009). However, most of these studies used adult animal models or evaluated the effects of exogenous OT injections. In previous studies, we found that the sensitivity of the hypothalamic expression patterns of orexigenic and reproductive factors, such as neuropeptide Y (NPY), proopiomelanocortin, and kisspeptin, to a negative energy balance changed markedly from the neonatal to the pre-pubertal period (Delahaye et al., 2008; Iwasa et al., 2011). In addition, some studies have shown that severe fasting or fasting-related hormonal conditions during the prenatal to neonatal period has long-lasting effects on the functions of
Please cite this article in press as: Matsuzaki, T., et al., Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats. Int. J. Dev. Neurosci. (2015), http://dx.doi.org/10.1016/j.ijdevneu.2015.01.001
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Fig. 3. Effects of fasting on the hypothalamic mRNA levels of oxytocin (OT) and the oxytocin receptor (OTR) on postnatal days (PND) 10, 20, and 30 in male and female rats. Hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points in the male rats. In the female rats, whilst hypothalamic OT mRNA expression was not altered by fasting at any of the examined time points, hypothalamic OTR mRNA expression was downregulated at PND 10. The values obtained on PND 10 in the fed rats were defined as 1.0. Data are expressed as mean ± SE values. *P < 0.05.
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these factors (Delahaye et al., 2008; Iwasa et al., 2010, 2014c). These studies indicated that fasting stress has long-lasting effects on the functions of the abovementioned molecules in the immature neuronal system. As noted above, hypothalamic OT expression is not sensitive to fasting during the neonatal to pre-pubertal period. Because previous our studies have shown that other hypothalamic factors already have sensitivities to 24 h fating at postnatal day 10 and/or 20 (Matsuzaki et al., 2015), OT insensitivity, observed in this study, may be caused by the immaturity of OT system, but not caused by the insensitivity of hypothalamus to fasting. Therefore, it is possible that severe fasting during this period might induce longlasting changes in the effects of hypothalamic OT expression on the energy balance. If this hypothesis is true, long-lasting alterations in hypothalamic OT expression might be responsible for certain metabolic disorders because OT has been shown to play pivotal roles in preventing obesity in adulthood (Tung et al., 2008; Maejima et al., 2009, 2011; Zhang and Cai, 2011). However, further studies are needed to confirm these hypotheses. In addition, it is necessary to clarify when hypothalamic OT expression first becomes sensitive to fasting. In adulthood, ER-␣ plays important roles in reproduction and social behavior in both sexes (Perry et al., 2009; Murakami et al., 2011). Recently, it has been reported that neonatal OT treatment has long-lasting effects on ER-␣ expression and ER-␣-related behaviors. (Withuhn et al., 2003; Perry et al., 2009), suggesting that the organizational effects of OT on ER-␣ persist into adulthood. In this study, we found that the hypothalamic mRNA expression levels of OT and ER-␣ tended to increase during the developmental period and were positively correlated during the developmental period. These findings indicate that the functions of OT and ER are not in operation in the pre-pubertal period and that these molecules might interact with each other to promote appropriate neuronal development. On the other hand, OTR mRNA expression tended to decrease during the developmental period, suggesting that cells might become less responsive to OT as development progresses. Another possibility is that estrogen affects OT and ER-␣ mRNA
expression during development. Further examinations are needed to evaluate these hypotheses. In this study, the pups in the fasting groups were separated from their mothers. Some studies have reported that the hypothalamic-pituitary-adrenal axis, which is partially regulated by OT, is activated by maternal separation (Levine et al., 1991; Smith et al., 1997; Schmidt et al., 2006). In addition, maternal separation-induced hypothermia might also affect the mRNA level of OT. Therefore, it is possible that the effects of maternal separation and hypothermia (if such effects exist) compensate for fastinginduced changes in OT expression. Thus, it would be necessary to control all of these factors in order to confirm whether food deprivation alone affects OT mRNA levels. It is also possible that the fasting period employed in the present study was not long enough to affect the OT system, which is known to be robust. Experiments performed under more strictly controlled conditions are required to examine these hypotheses. In summary, this study showed that hypothalamic OT mRNA expression was increased, but was not sensitive to fasting, during the developmental period. In addition, as hypothalamic mRNA OT and ER-␣ expression were found to be positively correlated these two factors might interact with each other to induce appropriate neuronal development. References Cassoni, P., Catalano, M.G., Sapino, A., Marrocco, T., Fazzari, A., Bussolati, G., Fortunati, N., 2002. Oxytocin modulates estrogen receptor alpha expression and function in MCF7 human breast cancer cells. Int. J. Oncol. 21, 375–378. Cushing, B.S., Carter, C.S., 1999. Prior exposure to oxytocin mimics social contact and facilitates sexual behaviour in females. J. Neuroendocrinol. 11, 765–769. Delahaye, F., Breton, C., Risold, P.Y., Enache, M., Dutriez-Casteloot, I., Laborie, C., Lesage, J., Vieau, D., 2008. Maternal perinatal undernutrition drastically reduces postnatal leptin surge and affects the development of arcuate nucleus proopiomelanocortin neurons in neonatal male rat pups. Endocrinology 149, 470–475. Iwasa, T., Matsuzaki, T., Murakami, M., Fujisawa, S., Kinouchi, R., Gereltsetseg, G., Kuwahara, A., Yasui, T., Irahara, M., 2010. Effects of intrauterine undernutrition on hypothalamic Kiss1 expression and the timing of puberty in female rats. J. Physiol. 588, 821–829.
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