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Gonadal regulation of PrRP mRNA expression in the nucleus tractus solitarius and ventral and lateral reticular nuclei of the rat
Molecular Brain Research 87 (2001) 42–47 www.elsevier.com / locate / bres
Research report
Gonadal regulation of PrRP mRNA expression in the nucleus tractus solitarius and ventral and lateral reticular nuclei of the rat Yuko Kataoka a ,1 , Norio Iijima b,1 , Takahiko Yano c , Kenshi Kakihara d , Seiji Hayashi b , e a f b b, Shuji Hinuma , Hideo Honjo , Shinji Hayashi , Masaki Tanaka , Yasuhiko Ibata * a b
Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602 -8566, Japan Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602 -8566, Japan c Discovery Research Laboratories III, Pharmaceutical Discovery Research Division, Takeda Chemical Industries Ltd., Osaka 532 -8686, Japan d Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602 -8566, Japan e Discovery Research Laboratories I, Pharmaceutical Discovery Research Division, Takeda Chemical Industries Ltd., Ibaraki 300 -4293, Japan f Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183 -8526, Japan Accepted 8 November 2000
Abstract We investigated the prolactin-releasing peptide (PrRP) gene expression quantitatively in the rat brain and the involvement of estrogen and progesterone using in situ hybridization. The strongest signals were observed in the nucleus tractus solitarius (NTS), which showed approximately 70% of total PrRP mRNA in the brain. Moderate expression was observed in the ventral and lateral reticular nuclei (VLRN) of the medulla oblongata. PrRP mRNA signals in the hypothalamic ventromedial- and dorsomedial nuclei showed only 5% of total signals. The PrRP mRNA expression among female rats showing normal gonadal cycle and male rats showed that the highest levels were in female rats in proestrus. Administration of estrogen or progesterone after ovariectomy induced an increase in PrRP mRNA expression in the NTS. PrRP mRNA content in the NTS increased with the progress of the pregnancy and reached a peak on the 14th day, the mid-period of pregnancy, when plasma progesterone increases. We also observed the colocalization of PrRP and estrogen receptor alpha in the neurons distributed in the NTS by double labeling immunocytochemistry. These findings indicate that PrRP gene expression is regulated by gonadal steroid hormones in the medulla oblongata, and parts of PrRP synthesizing neurons are considered to be directly influenced by estrogen in the NTS. 2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Other neurotransmitters Keywords: Prolactin-releasing peptide (PrRP); Estrogen; Progesterone; In situ Hybridization; Nucleus tractus solitarius (NTS); Ventral and lateral reticular nuclei (VLRN); Pregnancy; Ovariectomy; Rat
1. Introduction Prolactin-releasing peptide (PrRP) was originally isolated from the bovine hypothalamus on the basis of its ability to bind an orphan G protein-coupled receptor, hGR3, which is expressed in the human pituitary gland [8]. We previously reported that PrRP and its mRNA are expressed in neurons of the ventromedial- and dorsomedial *Corresponding author. Tel.: 181-75-251-5302; fax: 181-75-2515304. E-mail address: [email protected] (Y. Ibata). 1 First and second authors contributed equally to this work.
nuclei of the hypothalamus (HT), and in neurons of the nucleus tractus solitarius (NTS) and ventral and lateral reticular nuclei (VLRN) of the medulla oblongata [9]. Recently, PrRP was reported to increase plasma prolactin levels in a dose-dependent manner in female rats showing normal cycle when applied intravenously [14]. Another study also reported that prolactin secretion in response to PrRP was observed in the female rats during estrus and PrRP gene expression in the medulla oblongata was estrogen dependent [19]. These findings suggest that production of PrRP is under the control of gonadal steroid hormones. In the present study, we investigated the expression of
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00280-1
Y. Kataoka et al. / Molecular Brain Research 87 (2001) 42 – 47
PrRP mRNA in three distinct areas, HT, NTS and VLRN using in situ hybridization histochemistry. We quantified PrRP mRNA levels under various gonadal hormone conditions in vivo such as during proestrus, diestrus, ovariectomized and pregnant stages using a computer-assisted image-analyzing system and found that PrRP gene expression was regulated by both estrogen and progesterone. Moreover, we studied whether estrogen may regulate it directly or indirectly by examining the coexistence of PrRP and estrogen receptor (ER) in neurons of the NTS.
2. Materials and methods
2.1. Animals Adult female (200–230 g) and male (250–280 g) Sprague–Dawley (SD) rats were used in this study (Japan Charles River Co., Kanagawa, Japan). They were maintained in individual cages of the Animal Research Center of our university under 12 h light: 12 h dark conditions with free access to food and water. All experiments in the present study were carried out following the National Institute of Health guidelines for the Care and Use of Laboratory Animals. The Committee of Animal Research in Kyoto Prefectural University of Medicine also approved our experiments.
2.1.1. PrRP in sex and cycling differences Stage of proestrus or diestrus was determined by examining vaginal cytology for 2 weeks before all experiments to confirm that all female rats showed regular cycles. In the study to examine whether sex differences exist in PrRP mRNA signals, intact adult female (in proestrus / diestrus) and male rats were used (four rats in each group). 2.1.2. Ovariectomy and hormonal supplementation To examine whether sex steroids regulate PrRP mRNA expression, adult female rats were bilaterally ovariectomized (OVX) or sham operated under sodium pentobarbital anesthesia (40 mg / kg) via an abdominal approach. From 2 weeks after surgery, the rats were subcutaneously (s.c.) injected daily for 2 weeks with estrogen (17-b-estradiol, 20 mg / kg in sesame oil), or progesterone (4-pregnene-3, 20-dione, 0.1 mg / kg in sesame oil) or vehicle (sesame oil). The animals were divided into four groups (4 rats for each group): sham-operated control rats in diestrus injected with vehicle s.c. (sham), OVX rats injected with vehicle s.c. (OVX1V), OVX rats injected with estrogen s.c. (OVX1E) and OVX rats injected with progesterone s.c. (OVX1P). 2.1.3. PrRP mRNA expression during pregnancy We also examined PrRP mRNA expression in the NTS during pregnancy and lactation. Twenty female SD rats
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were mated during proestrus with male rats, and the day vaginal plugs were observed was designated day 0 of pregnancy. The day of delivery was designated day 0 of lactation. Animals were killed on days 7, 14 and 20 of pregnancy and on day 7 of lactation (five rats for each day).
2.2. In situ hybridization histochemistry Rats were perfused through the cardiac ventricle with a fixative containing 4% paraformaldehyde in 150 ml of 0.1 M phosphate buffer (PB) under deep anesthesia with sodium pentobarbital (70 mg / kg). The brains were immediately removed, post-fixed in the same fixative for 4 h at 43, and transferred into 20% sucrose in 0.1 M PB for 60 h. Serial coronal sections (40 mm in thickness) were cut along the rostral to caudal axis using a cryostat, collected into 48C standard saline citrate (SSC) and then processed for in situ hybridization as described previously [9,18]. The sections were treated with 1 mg / ml proteinase K (Sigma) in 10 mM Tris buffer, pH 7.4, and 10 mM EDTA for 7 min at 378C and 0.25% acetic anhydride in 0.1 M triethanolamine for 9 min at room temperature. Then, sections were hybridized with an 35 S-labeled cRNA probe, which was synthesized from full-length PrRP cDNA in plasmid (Bluescript). Hybridization buffer contained 10 mM Tris (pH 7.4) containing 60% formamide, 13 Denhardt’s solution, 10% dextran sulfate, 10 mM dithiothreitol, 200 mM tRNA, 1 mM EDTA and 0.25% sodium dodecyl sulfate (10 h at 558C). After hybridization, the sections were rinsed in 23SSC / 50% formamide (2 times at 558C) and treated with RNase. Then, they were mounted onto gelatin-coated slides, dried and dehydrated through a graded alcohol series. The slides were exposed to an imaging plate (radiosensitive plates coated with BaFBr:Eu 21 ; Fuji Film) for 24 h.
2.3. Quantitative analysis of PrRP mRNA signals To quantify the PrRP mRNA signals, the radioactivity of each nuclei (HT, NTS, VLRN) on the imaging plate was measured as photo-stimulated luminescence (PSL) when scanned with a laser beam, and analyzed using a microcomputer interfaced to an imaging analyzing system (BAS 2000; Fuji Film) [18]. The total radioactivity count from all sections in which positive signals were detected, was considered proportional to the amount of PrRP mRNA present in these nuclei. When PSL of HT and that of NTS / VLRN were calculated, the signal intensity in the cerebral cortex and in the hypoglossal nucleus were used as the background levels, respectively. In the examination of PrRP mRNA expression in the NTS during pregnancy and lactation, we used ‘relative RNA contents,’ which referred to the value of virgin females in diestrus which was adjusted to 100. All values obtained in this study were presented as
Y. Kataoka et al. / Molecular Brain Research 87 (2001) 42 – 47
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mean6S.E.M. A P-value of ,0.05 was considered significant.
2.4. Immunocytochemistry For this experiment, three adult female rats were ovariectomized 1 week before perfusion. After perfusion with 150 ml 0.1 M PB containing 4% paraformaldehyde and 0.25% glutaraldehyde, the brains were immediately removed, post-fixed in the same fixative for 24 h at 48C and transferred into 20% sucrose in 0.1 M PB for 72 h. Frontal sections (15 mm in thickness) were cut using a cryostat. Following washing in 0.1 M PBS, free-floating sections were immersed in 0.1 M PB containing 5% calf serum for 1 h at room temperature for blocking nonspecific reaction. They were then incubated with primary antibodies consisting of anti-PrRP (P2L-1T, 10 mg / ml) and anti-ERa (AS409, 1:2000). Positive immunostaining was detected using FITC- or rhodamine-labeled secondary antibody. The specificity of anti-PrRP and anti-ERa was described previously [11,20]. In order to estimate the rate of colocalization of PrRP and ERa in the NTS, we used 3 rat’s brains and selected 20 slices containing the NTS at random per each brain.
3. Results
3.1. Comparisons of PrRP mRNA among NTS, VLRN and HT We examined PrRP mRNA expression throughout the brain from the olfactory bulb to the medulla oblongata and observed its expression only in neurons of four nuclei, that is, ventromedial- and dorsomedial nuclei in the HT and NTS and VLRN in the medulla oblongata. In all groups (intact adult females in proestrus / diestrus and males), expression of PrRP mRNA signals was strongest in the NTS (2000688.6 PSL in female rats in proestrus, 1,683644.1 PSL in female rats in diestrus, 1,536653.1 PSL in male rats); Almost of two-thirds to three-fourths (62.4% in female rats in proestrus, 76.7% in female rats in diestrus, 73.1% in male rats) of total PrRP mRNA in the brain was present in this nucleus. Moderate expression was observed in the VLRN (1,027671.6 PSL (32.0% of total PrRP mRNA in the brain) in female rats in proestrus, 373.4666.2% PSL (17.0%) in female rats in diestrus, 417.4655.9% PSL (19.9%) in male rats). PrRP mRNA signals in the HT were the weakest (180.4612.4 PSL (5.6%) in female rats in proestrus, 138.1637.8 PSL (6.3%) in female rats in diestrus, 147.8636.5% PSL (7.0%) in male rats) (Fig. 1).
3.2. Comparisons of PrRP mRNA among male and stages of gonadal cycle in female rats PrRP mRNA levels of NTS and VLRN were the highest
Fig. 1. The quantified signal intensity of PrRP mRNA levels in the HT, NTS and VLRN. Comparison among female rats in proestrus (solid bars), in diestrus (stippled bars) and male rats (open bars). Autoradiography showing PrRP mRNA signals were shown in the sections of HT, NTS and VLRN on the above of the histogram. The PSL measurements are described in the text. Values are presented as the mean6S.E.M. *P,0.05, one-way ANOVA followed by Fisher’s multiple comparisons test (n54).
in female rats during proestrus (2000688.6 PSL in the NTS, 1027671.6 in the VLRN). During diestrus, the PrRP mRNA level was 1683644.1 PSL in the NTS, 373.4666.2 in the VLRN in the HT. In male rats, PrRP mRNA levels in the NTS and VLRN (1536653.1, 417655.9 PSL, respectively) were lower than those of female rats during proestrus. There were significant differences of PrRP mRNA between proestrus and other groups (diestrus and males) in the NTS and VLRN (P,0.05) (Fig. 1). However, we could not detect a significant difference between male rats and female rats during the diestrus stage.
3.3. The role of gonadal steroid hormones in regulating PrRP mRNA expression The administration of estrogen (OVX1E) and progesterone (OVX1P) induced increases in PrRP mRNA levels in the NTS (27846335, 26066466, respectively) (Fig. 2a). PrRP mRNA content of OVX1E, and that of OVX1P within the NTS were approximately 2-fold higher than that of OVX1V. A similar tendency was observed where estrogen supplementation after ovariectomy increased PrRP mRNA levels in the VLRN. PrRP mRNA content of OVX1E (26006647) was approximately 3-fold larger than that of OVX1V. However, PrRP mRNA of OVX1P (16506435 PSL) did not show a significant increase compared with that of the OVX1V group (Fig. 2b).
Y. Kataoka et al. / Molecular Brain Research 87 (2001) 42 – 47
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Fig. 3. Change in the relative amount of PrRP mRNA during pregnancy and lactation. The value of the virgin controls was adjusted to 100. Differences between virgin controls and other groups were evaluated using one-way ANOVA followed by Scheffe’s multiple comparisons test (*P,0.01, **P,0.05, n55). Values are the mean6S.E.M.
3.5. Colocalization of ERa immunoreactivity in PrRPsynthesizing neurons
Fig. 2. The expression of PrRP mRNA in the NTS (a: n54) and VLRN (b: n54) of the sham-operated, OVX1V, OVX1E and OVX1P groups. Values are the mean6S.E.M. *P,0.05, one-way ANOVA followed by Fisher’s multiple comparisons test.
In the HT, we could not detect a significant change in the PrRP mRNA level in either of the OVX1E and OVX1P groups compared with the OVX1V group (data not shown).
ERa immunoreactivity was observed in the nucleus of neuronal cells in the medulla oblongata. ERa was strongly labeled in neurons of the NTS but only weakly labeled in the VLRN. PrRP immunoreactive neurons were observed only in the NTS and VLRN. Almost all neurons in the NTS were PrRP immunoreactive and they were more densely labeled than the VLRN. Since both PrRP and ERa immunoreactivity in the NTS were much stronger than those in VLRN, we performed a colocalization study in the NTS. Many double-labeled cells displayed green cytoplasmic staining for PrRP and red nuclear staining for ERa in the NTS (Fig. 4). We detected the colocalization as following rate: PrRP and ERa-immunoreactive cells / PrRP-immunoreactive cells574 / 101, 86 / 116, 78 / 111. Therefore 72.661.2% of PrRP-positive neurons within the NTS expressed ERa.
4. Discussion
3.4. PrRP mRNA levels during pregnancy and lactation The PrRP mRNA level in the NTS increased corresponding to the progress of pregnancy and reached a peak on day 14 of pregnancy (370617.3% of basal level), and then decreased towards terms and lactation. On day 7 of lactation, PrRP mRNA content was reduced to near the basal level (127632.2%) in virgin rats (Fig. 3). In the HT and the VLRN, we could not detect significant changes in PrRP mRNA contents during pregnancy and lactation (data not shown).
The study of PrRP mRNA expression in female rats showing normal gonadal cycle and male rats revealed that in the NTS and VLRN, its level was higher in female rats during proestrus than in female rats during diestrus or in male rats. In female rats, sufficient estrogen administration after ovariectomy markedly increased PrRP mRNA levels in the NTS and VLRN. OVX1P also upregulated PrRP mRNA level in the NTS. These findings indicate that ovarian steroid hormones stimulate PrRP gene expression in neurons of the NTS and VLRN of the rat brain.
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Y. Kataoka et al. / Molecular Brain Research 87 (2001) 42 – 47
Fig. 4. Double labeling immunocytochemistry of PrRP and ERa in neurons of the NTS. A higher magnified picture of colocalized neuron is shown in b. Arrows indicate both immunoreactive neurons. Calibration bars, 100 mm (a) and 5 mm (b).
The double labeling immunocytochemistry revealed the presence of estrogen receptor a type (ERa) in PrRP neurons of the NTS. The finding that more than 70% of PrRP immunoreactive neurons showing coexpression of ERa in the NTS suggests that neurons coexpressing PrRP and ERa are directly regulated by estrogen via ERa. With regard to estrogen receptor b type (ERb), its mRNA was reported to be expressed in the rat NTS and VLRN [16]. This implies that PrRP neurons in these nuclei also express ERb and estrogen may directly influence PrRP gene expression via ERb, although we did not confirm the coexpression of PrRP and ERb in the present study. Estrogen is known to upregulate some neuropeptide mRNAs in the brain such as neurotensin (NT), oxytocin and luteinizing hormone-releasing hormone [1,4,15]. NT gene expression within the anterior medial preoptic nucleus (AMPO) exhibits sexual dimorphism, changes across the estrus cycle with fluctuations in endogenous hormone levels, and is stimulated by estradiol treatment in female rats [2]. Estrogen is thought to influence NT neurons directly in the preoptic area, as these neurons express ERa [3]. Intranuclear regulation of PrRP gene transcription by
estrogen in NTS neurons may have a similar mechanism to that of NT in AMPO neurons. We previously reported that PrRP expressing neurons in the NTS and VLRN are also catecholaminergic neurons [9]. The neurons in these two nuclei were termed noradrenergic A1 (VLRN) and A2 (NTS), respectively [5]. Both estrogen and progesterone receptors are expressed in A2 neurons [10,17]. Haywood et al. recently reported that substantial numbers of ERa are expressed, but few progesterone receptors are found, in A1 neurons [7]. Their results apparently support our present findings that ERa was observed to express in PrRP neurons in the NTS and OVX1E increased the PrRP mRNA level in both the NTS and VLRN, but OVX1P increased it significantly only in the NTS. During pregnancy, the PrRP mRNA level in the NTS showed a greater than 3.5-fold increase in virgin rats. Plasma progesterone levels are known to increase and peak during pregnancy (on day 16) and then decrease at term [6]. Such a rise in the plasma progesterone level appears to be correlated with the PrRP mRNA expression in the NTS. However, plasma estrogen levels increase towards term.
Y. Kataoka et al. / Molecular Brain Research 87 (2001) 42 – 47
PrRP gene expression during pregnancy may be mainly regulated by progesterone. Since this 370% increase in PrRP mRNA expression was the largest observed in the present study (larger than in OVX1P rats) and various hormones were released into blood from the placenta during pregnancy, there might be other factors involved in the upregulation of PrRP gene expression. In the present study, we demonstrated that PrRP mRNA of the brain is most intensively expressed in the NTS. Approximately 70% of total brain PrRP mRNA existed in the NTS. This means NTS is the principal site of PrRP production in the brain. The PrRP immunoreactive fibers, which are known to be densely distributed in the forebrain such as the hypothalamus, basolateral amygdaloid nucleus and bed nucleus of the stria terminals [9,11], may considerably originated from the PrRP neurons in the NTS as well as the hypothalamus. A retrograde tracing study is now progress to clarify the origins of those dense PrRP immunoreactive fibers distributed in the forebrain. Recently, plasma oxytocin and ACTH levels were reported to be upregulated by intracerebroventricular injection of PrRP [12,13]. The hypothalamic paraventricular and supraoptic nuclei receive dense innervation of PrRP fibers [11]. Taken together with the above findings, these hypothalamic neuroendocrine neurons may be directly regulated by PrRP neurons distributed in the NTS. Further studies will be required to clarify the gonadal function to control hypothalamic neuroendocrine system through PrRP neurons in the NTS.
Acknowledgements This work was supported in part by a grant (No. 11480240) from the Ministry of Education, Science, Sports and Culture, Japan.
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Research report
Gonadal regulation of PrRP mRNA expression in the nucleus tractus solitarius and ventral and lateral reticular nuclei of the rat Yuko Kataoka a ,1 , Norio Iijima b,1 , Takahiko Yano c , Kenshi Kakihara d , Seiji Hayashi b , e a f b b, Shuji Hinuma , Hideo Honjo , Shinji Hayashi , Masaki Tanaka , Yasuhiko Ibata * a b
Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602 -8566, Japan Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602 -8566, Japan c Discovery Research Laboratories III, Pharmaceutical Discovery Research Division, Takeda Chemical Industries Ltd., Osaka 532 -8686, Japan d Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602 -8566, Japan e Discovery Research Laboratories I, Pharmaceutical Discovery Research Division, Takeda Chemical Industries Ltd., Ibaraki 300 -4293, Japan f Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183 -8526, Japan Accepted 8 November 2000
Abstract We investigated the prolactin-releasing peptide (PrRP) gene expression quantitatively in the rat brain and the involvement of estrogen and progesterone using in situ hybridization. The strongest signals were observed in the nucleus tractus solitarius (NTS), which showed approximately 70% of total PrRP mRNA in the brain. Moderate expression was observed in the ventral and lateral reticular nuclei (VLRN) of the medulla oblongata. PrRP mRNA signals in the hypothalamic ventromedial- and dorsomedial nuclei showed only 5% of total signals. The PrRP mRNA expression among female rats showing normal gonadal cycle and male rats showed that the highest levels were in female rats in proestrus. Administration of estrogen or progesterone after ovariectomy induced an increase in PrRP mRNA expression in the NTS. PrRP mRNA content in the NTS increased with the progress of the pregnancy and reached a peak on the 14th day, the mid-period of pregnancy, when plasma progesterone increases. We also observed the colocalization of PrRP and estrogen receptor alpha in the neurons distributed in the NTS by double labeling immunocytochemistry. These findings indicate that PrRP gene expression is regulated by gonadal steroid hormones in the medulla oblongata, and parts of PrRP synthesizing neurons are considered to be directly influenced by estrogen in the NTS. 2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Other neurotransmitters Keywords: Prolactin-releasing peptide (PrRP); Estrogen; Progesterone; In situ Hybridization; Nucleus tractus solitarius (NTS); Ventral and lateral reticular nuclei (VLRN); Pregnancy; Ovariectomy; Rat
1. Introduction Prolactin-releasing peptide (PrRP) was originally isolated from the bovine hypothalamus on the basis of its ability to bind an orphan G protein-coupled receptor, hGR3, which is expressed in the human pituitary gland [8]. We previously reported that PrRP and its mRNA are expressed in neurons of the ventromedial- and dorsomedial *Corresponding author. Tel.: 181-75-251-5302; fax: 181-75-2515304. E-mail address: [email protected] (Y. Ibata). 1 First and second authors contributed equally to this work.
nuclei of the hypothalamus (HT), and in neurons of the nucleus tractus solitarius (NTS) and ventral and lateral reticular nuclei (VLRN) of the medulla oblongata [9]. Recently, PrRP was reported to increase plasma prolactin levels in a dose-dependent manner in female rats showing normal cycle when applied intravenously [14]. Another study also reported that prolactin secretion in response to PrRP was observed in the female rats during estrus and PrRP gene expression in the medulla oblongata was estrogen dependent [19]. These findings suggest that production of PrRP is under the control of gonadal steroid hormones. In the present study, we investigated the expression of
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00280-1
Y. Kataoka et al. / Molecular Brain Research 87 (2001) 42 – 47
PrRP mRNA in three distinct areas, HT, NTS and VLRN using in situ hybridization histochemistry. We quantified PrRP mRNA levels under various gonadal hormone conditions in vivo such as during proestrus, diestrus, ovariectomized and pregnant stages using a computer-assisted image-analyzing system and found that PrRP gene expression was regulated by both estrogen and progesterone. Moreover, we studied whether estrogen may regulate it directly or indirectly by examining the coexistence of PrRP and estrogen receptor (ER) in neurons of the NTS.
2. Materials and methods
2.1. Animals Adult female (200–230 g) and male (250–280 g) Sprague–Dawley (SD) rats were used in this study (Japan Charles River Co., Kanagawa, Japan). They were maintained in individual cages of the Animal Research Center of our university under 12 h light: 12 h dark conditions with free access to food and water. All experiments in the present study were carried out following the National Institute of Health guidelines for the Care and Use of Laboratory Animals. The Committee of Animal Research in Kyoto Prefectural University of Medicine also approved our experiments.
2.1.1. PrRP in sex and cycling differences Stage of proestrus or diestrus was determined by examining vaginal cytology for 2 weeks before all experiments to confirm that all female rats showed regular cycles. In the study to examine whether sex differences exist in PrRP mRNA signals, intact adult female (in proestrus / diestrus) and male rats were used (four rats in each group). 2.1.2. Ovariectomy and hormonal supplementation To examine whether sex steroids regulate PrRP mRNA expression, adult female rats were bilaterally ovariectomized (OVX) or sham operated under sodium pentobarbital anesthesia (40 mg / kg) via an abdominal approach. From 2 weeks after surgery, the rats were subcutaneously (s.c.) injected daily for 2 weeks with estrogen (17-b-estradiol, 20 mg / kg in sesame oil), or progesterone (4-pregnene-3, 20-dione, 0.1 mg / kg in sesame oil) or vehicle (sesame oil). The animals were divided into four groups (4 rats for each group): sham-operated control rats in diestrus injected with vehicle s.c. (sham), OVX rats injected with vehicle s.c. (OVX1V), OVX rats injected with estrogen s.c. (OVX1E) and OVX rats injected with progesterone s.c. (OVX1P). 2.1.3. PrRP mRNA expression during pregnancy We also examined PrRP mRNA expression in the NTS during pregnancy and lactation. Twenty female SD rats
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were mated during proestrus with male rats, and the day vaginal plugs were observed was designated day 0 of pregnancy. The day of delivery was designated day 0 of lactation. Animals were killed on days 7, 14 and 20 of pregnancy and on day 7 of lactation (five rats for each day).
2.2. In situ hybridization histochemistry Rats were perfused through the cardiac ventricle with a fixative containing 4% paraformaldehyde in 150 ml of 0.1 M phosphate buffer (PB) under deep anesthesia with sodium pentobarbital (70 mg / kg). The brains were immediately removed, post-fixed in the same fixative for 4 h at 43, and transferred into 20% sucrose in 0.1 M PB for 60 h. Serial coronal sections (40 mm in thickness) were cut along the rostral to caudal axis using a cryostat, collected into 48C standard saline citrate (SSC) and then processed for in situ hybridization as described previously [9,18]. The sections were treated with 1 mg / ml proteinase K (Sigma) in 10 mM Tris buffer, pH 7.4, and 10 mM EDTA for 7 min at 378C and 0.25% acetic anhydride in 0.1 M triethanolamine for 9 min at room temperature. Then, sections were hybridized with an 35 S-labeled cRNA probe, which was synthesized from full-length PrRP cDNA in plasmid (Bluescript). Hybridization buffer contained 10 mM Tris (pH 7.4) containing 60% formamide, 13 Denhardt’s solution, 10% dextran sulfate, 10 mM dithiothreitol, 200 mM tRNA, 1 mM EDTA and 0.25% sodium dodecyl sulfate (10 h at 558C). After hybridization, the sections were rinsed in 23SSC / 50% formamide (2 times at 558C) and treated with RNase. Then, they were mounted onto gelatin-coated slides, dried and dehydrated through a graded alcohol series. The slides were exposed to an imaging plate (radiosensitive plates coated with BaFBr:Eu 21 ; Fuji Film) for 24 h.
2.3. Quantitative analysis of PrRP mRNA signals To quantify the PrRP mRNA signals, the radioactivity of each nuclei (HT, NTS, VLRN) on the imaging plate was measured as photo-stimulated luminescence (PSL) when scanned with a laser beam, and analyzed using a microcomputer interfaced to an imaging analyzing system (BAS 2000; Fuji Film) [18]. The total radioactivity count from all sections in which positive signals were detected, was considered proportional to the amount of PrRP mRNA present in these nuclei. When PSL of HT and that of NTS / VLRN were calculated, the signal intensity in the cerebral cortex and in the hypoglossal nucleus were used as the background levels, respectively. In the examination of PrRP mRNA expression in the NTS during pregnancy and lactation, we used ‘relative RNA contents,’ which referred to the value of virgin females in diestrus which was adjusted to 100. All values obtained in this study were presented as
Y. Kataoka et al. / Molecular Brain Research 87 (2001) 42 – 47
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mean6S.E.M. A P-value of ,0.05 was considered significant.
2.4. Immunocytochemistry For this experiment, three adult female rats were ovariectomized 1 week before perfusion. After perfusion with 150 ml 0.1 M PB containing 4% paraformaldehyde and 0.25% glutaraldehyde, the brains were immediately removed, post-fixed in the same fixative for 24 h at 48C and transferred into 20% sucrose in 0.1 M PB for 72 h. Frontal sections (15 mm in thickness) were cut using a cryostat. Following washing in 0.1 M PBS, free-floating sections were immersed in 0.1 M PB containing 5% calf serum for 1 h at room temperature for blocking nonspecific reaction. They were then incubated with primary antibodies consisting of anti-PrRP (P2L-1T, 10 mg / ml) and anti-ERa (AS409, 1:2000). Positive immunostaining was detected using FITC- or rhodamine-labeled secondary antibody. The specificity of anti-PrRP and anti-ERa was described previously [11,20]. In order to estimate the rate of colocalization of PrRP and ERa in the NTS, we used 3 rat’s brains and selected 20 slices containing the NTS at random per each brain.
3. Results
3.1. Comparisons of PrRP mRNA among NTS, VLRN and HT We examined PrRP mRNA expression throughout the brain from the olfactory bulb to the medulla oblongata and observed its expression only in neurons of four nuclei, that is, ventromedial- and dorsomedial nuclei in the HT and NTS and VLRN in the medulla oblongata. In all groups (intact adult females in proestrus / diestrus and males), expression of PrRP mRNA signals was strongest in the NTS (2000688.6 PSL in female rats in proestrus, 1,683644.1 PSL in female rats in diestrus, 1,536653.1 PSL in male rats); Almost of two-thirds to three-fourths (62.4% in female rats in proestrus, 76.7% in female rats in diestrus, 73.1% in male rats) of total PrRP mRNA in the brain was present in this nucleus. Moderate expression was observed in the VLRN (1,027671.6 PSL (32.0% of total PrRP mRNA in the brain) in female rats in proestrus, 373.4666.2% PSL (17.0%) in female rats in diestrus, 417.4655.9% PSL (19.9%) in male rats). PrRP mRNA signals in the HT were the weakest (180.4612.4 PSL (5.6%) in female rats in proestrus, 138.1637.8 PSL (6.3%) in female rats in diestrus, 147.8636.5% PSL (7.0%) in male rats) (Fig. 1).
3.2. Comparisons of PrRP mRNA among male and stages of gonadal cycle in female rats PrRP mRNA levels of NTS and VLRN were the highest
Fig. 1. The quantified signal intensity of PrRP mRNA levels in the HT, NTS and VLRN. Comparison among female rats in proestrus (solid bars), in diestrus (stippled bars) and male rats (open bars). Autoradiography showing PrRP mRNA signals were shown in the sections of HT, NTS and VLRN on the above of the histogram. The PSL measurements are described in the text. Values are presented as the mean6S.E.M. *P,0.05, one-way ANOVA followed by Fisher’s multiple comparisons test (n54).
in female rats during proestrus (2000688.6 PSL in the NTS, 1027671.6 in the VLRN). During diestrus, the PrRP mRNA level was 1683644.1 PSL in the NTS, 373.4666.2 in the VLRN in the HT. In male rats, PrRP mRNA levels in the NTS and VLRN (1536653.1, 417655.9 PSL, respectively) were lower than those of female rats during proestrus. There were significant differences of PrRP mRNA between proestrus and other groups (diestrus and males) in the NTS and VLRN (P,0.05) (Fig. 1). However, we could not detect a significant difference between male rats and female rats during the diestrus stage.
3.3. The role of gonadal steroid hormones in regulating PrRP mRNA expression The administration of estrogen (OVX1E) and progesterone (OVX1P) induced increases in PrRP mRNA levels in the NTS (27846335, 26066466, respectively) (Fig. 2a). PrRP mRNA content of OVX1E, and that of OVX1P within the NTS were approximately 2-fold higher than that of OVX1V. A similar tendency was observed where estrogen supplementation after ovariectomy increased PrRP mRNA levels in the VLRN. PrRP mRNA content of OVX1E (26006647) was approximately 3-fold larger than that of OVX1V. However, PrRP mRNA of OVX1P (16506435 PSL) did not show a significant increase compared with that of the OVX1V group (Fig. 2b).
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Fig. 3. Change in the relative amount of PrRP mRNA during pregnancy and lactation. The value of the virgin controls was adjusted to 100. Differences between virgin controls and other groups were evaluated using one-way ANOVA followed by Scheffe’s multiple comparisons test (*P,0.01, **P,0.05, n55). Values are the mean6S.E.M.
3.5. Colocalization of ERa immunoreactivity in PrRPsynthesizing neurons
Fig. 2. The expression of PrRP mRNA in the NTS (a: n54) and VLRN (b: n54) of the sham-operated, OVX1V, OVX1E and OVX1P groups. Values are the mean6S.E.M. *P,0.05, one-way ANOVA followed by Fisher’s multiple comparisons test.
In the HT, we could not detect a significant change in the PrRP mRNA level in either of the OVX1E and OVX1P groups compared with the OVX1V group (data not shown).
ERa immunoreactivity was observed in the nucleus of neuronal cells in the medulla oblongata. ERa was strongly labeled in neurons of the NTS but only weakly labeled in the VLRN. PrRP immunoreactive neurons were observed only in the NTS and VLRN. Almost all neurons in the NTS were PrRP immunoreactive and they were more densely labeled than the VLRN. Since both PrRP and ERa immunoreactivity in the NTS were much stronger than those in VLRN, we performed a colocalization study in the NTS. Many double-labeled cells displayed green cytoplasmic staining for PrRP and red nuclear staining for ERa in the NTS (Fig. 4). We detected the colocalization as following rate: PrRP and ERa-immunoreactive cells / PrRP-immunoreactive cells574 / 101, 86 / 116, 78 / 111. Therefore 72.661.2% of PrRP-positive neurons within the NTS expressed ERa.
4. Discussion
3.4. PrRP mRNA levels during pregnancy and lactation The PrRP mRNA level in the NTS increased corresponding to the progress of pregnancy and reached a peak on day 14 of pregnancy (370617.3% of basal level), and then decreased towards terms and lactation. On day 7 of lactation, PrRP mRNA content was reduced to near the basal level (127632.2%) in virgin rats (Fig. 3). In the HT and the VLRN, we could not detect significant changes in PrRP mRNA contents during pregnancy and lactation (data not shown).
The study of PrRP mRNA expression in female rats showing normal gonadal cycle and male rats revealed that in the NTS and VLRN, its level was higher in female rats during proestrus than in female rats during diestrus or in male rats. In female rats, sufficient estrogen administration after ovariectomy markedly increased PrRP mRNA levels in the NTS and VLRN. OVX1P also upregulated PrRP mRNA level in the NTS. These findings indicate that ovarian steroid hormones stimulate PrRP gene expression in neurons of the NTS and VLRN of the rat brain.
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Fig. 4. Double labeling immunocytochemistry of PrRP and ERa in neurons of the NTS. A higher magnified picture of colocalized neuron is shown in b. Arrows indicate both immunoreactive neurons. Calibration bars, 100 mm (a) and 5 mm (b).
The double labeling immunocytochemistry revealed the presence of estrogen receptor a type (ERa) in PrRP neurons of the NTS. The finding that more than 70% of PrRP immunoreactive neurons showing coexpression of ERa in the NTS suggests that neurons coexpressing PrRP and ERa are directly regulated by estrogen via ERa. With regard to estrogen receptor b type (ERb), its mRNA was reported to be expressed in the rat NTS and VLRN [16]. This implies that PrRP neurons in these nuclei also express ERb and estrogen may directly influence PrRP gene expression via ERb, although we did not confirm the coexpression of PrRP and ERb in the present study. Estrogen is known to upregulate some neuropeptide mRNAs in the brain such as neurotensin (NT), oxytocin and luteinizing hormone-releasing hormone [1,4,15]. NT gene expression within the anterior medial preoptic nucleus (AMPO) exhibits sexual dimorphism, changes across the estrus cycle with fluctuations in endogenous hormone levels, and is stimulated by estradiol treatment in female rats [2]. Estrogen is thought to influence NT neurons directly in the preoptic area, as these neurons express ERa [3]. Intranuclear regulation of PrRP gene transcription by
estrogen in NTS neurons may have a similar mechanism to that of NT in AMPO neurons. We previously reported that PrRP expressing neurons in the NTS and VLRN are also catecholaminergic neurons [9]. The neurons in these two nuclei were termed noradrenergic A1 (VLRN) and A2 (NTS), respectively [5]. Both estrogen and progesterone receptors are expressed in A2 neurons [10,17]. Haywood et al. recently reported that substantial numbers of ERa are expressed, but few progesterone receptors are found, in A1 neurons [7]. Their results apparently support our present findings that ERa was observed to express in PrRP neurons in the NTS and OVX1E increased the PrRP mRNA level in both the NTS and VLRN, but OVX1P increased it significantly only in the NTS. During pregnancy, the PrRP mRNA level in the NTS showed a greater than 3.5-fold increase in virgin rats. Plasma progesterone levels are known to increase and peak during pregnancy (on day 16) and then decrease at term [6]. Such a rise in the plasma progesterone level appears to be correlated with the PrRP mRNA expression in the NTS. However, plasma estrogen levels increase towards term.
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PrRP gene expression during pregnancy may be mainly regulated by progesterone. Since this 370% increase in PrRP mRNA expression was the largest observed in the present study (larger than in OVX1P rats) and various hormones were released into blood from the placenta during pregnancy, there might be other factors involved in the upregulation of PrRP gene expression. In the present study, we demonstrated that PrRP mRNA of the brain is most intensively expressed in the NTS. Approximately 70% of total brain PrRP mRNA existed in the NTS. This means NTS is the principal site of PrRP production in the brain. The PrRP immunoreactive fibers, which are known to be densely distributed in the forebrain such as the hypothalamus, basolateral amygdaloid nucleus and bed nucleus of the stria terminals [9,11], may considerably originated from the PrRP neurons in the NTS as well as the hypothalamus. A retrograde tracing study is now progress to clarify the origins of those dense PrRP immunoreactive fibers distributed in the forebrain. Recently, plasma oxytocin and ACTH levels were reported to be upregulated by intracerebroventricular injection of PrRP [12,13]. The hypothalamic paraventricular and supraoptic nuclei receive dense innervation of PrRP fibers [11]. Taken together with the above findings, these hypothalamic neuroendocrine neurons may be directly regulated by PrRP neurons distributed in the NTS. Further studies will be required to clarify the gonadal function to control hypothalamic neuroendocrine system through PrRP neurons in the NTS.
Acknowledgements This work was supported in part by a grant (No. 11480240) from the Ministry of Education, Science, Sports and Culture, Japan.
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