-2851$/ 2) 9H W H U L Q D U \  6FLHQFH J. Vet. Sci. (2005), / 6 (1), 1–5 Expression of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in human placenta Phil-Ok Koh 1 , Chung-Kil Won 1 , Hae-Sook Noh 2 , Gyeong-Jae Cho 2 , Wan-Sung Choi 2, * 1 Department of Anatomy, College of Veterinary Medicine and Institute of Animal Medicine, Gyeongsang National University, Jinju 660-701, Korea 2 Department of Anatomy and Neurobiology, College of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-751, Korea Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was known to stimulate the release of growth factor in various cells. Recently, we reported the cellular localization of PACAP and its type I (PAC 1 ) receptor in rat placenta during pregnancy. Placenta is a critical organ that synthesizes several growth factors and angiogenic factors for the fetal development and its own growth. However, there is little information regarding the cellular localization of PACAP and its receptor in human placenta at various gestations. The aim of the present study was to define the expression and distribution of PACAP and PAC 1 receptor mRNAs in the human placenta during the pregnancy period. PACAP and PAC 1 receptor mRNAs were expressed in stroma cells of stem villi and terminal villi. At the early stage, on 7 and 14 weeks, PACAP and PAC 1 receptor genes were moderately expressed in stroma cells surrounding the blood vessels within stem villi. These genes were strongly expressed in stroma cells of stem villi and terminal villi on 24 and 38 weeks. The expression of these genes was increased as gestation advanced, and localized in the same areas. Localization of PACAP and PAC 1 receptor demonstrate the evidence that PACAP may play an important role, as an autoregulator or pararegulator via its PAC 1 receptor. In conclusion, our findings strongly suggest that PACAP may have a critical role in physiological function of the placenta for gestational maintenance and fetal growth. Key words: PACAP, receptor, placenta, human Introduction The placenta is an essential organ for the fetal development and the maintenance of pregnancy. It is known that placenta synthesis the growth hormone [21] and several growth factors, such as basic fibroblast growth factor and insulin like growth factor [6,29]. Also, placenta produces placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) [6,29], which are critical factors for the placental growth and fetal development. As an important regulator of angiogenesis, VEGF contributes to the development and growth of the endothelium during the tissue growth [5,32]. Also, another member of the VEGF family, PlGF, promotes endothelial cell proliferation in vitro [16]. The previous study showed pituitary adenylate cyclase activating polypeptide (PACAP) stimulates the release of VEGF and acts as a trophic factor in various cells [7,8,17,31, 33]. PACAP has considerable homology with vasoactive interstinal peptide (VIP) and growth hormone releasing hormone [17,31,33]. Recently, it was reported that PACAP and PACAP receptor are present in both the human and rat placenta at term [23]. Also, even more recently, we reported the cellular localization of PACAP and PACAP type I (PAC 1 ) receptor in the rat placenta during pregnancy [14]. Therefore, the existence of PACAP in placenta suggests that PACAP affects placental function. PACAP was originally isolated from ovine hypothalamus and was known to stimulate the production of cAMP in anterior pituitary cells [18]. PACAP exists in two biologically active forms, PACAP 38 and PACAP 27, sharing the same N-terminal 27 amino acids [19]. PACAP binds to three type Ireceptors. Among these receptors, PAC 1 receptor has high affinity with PACAP 38 and PACAP 27, very low affinity with VIP [12,28]. But, VIP1 and VIP2 receptors have approximately equal high affinity for PACAP 38, PACAP 27, and VIP [11,12,28]. PACAP and its receptor have been found in the central nervous system and its peripheral tissues, including the hypothalamus, pituitary gland, adrenal *Corresponding author Tel: 82-55-751-8716; Fax: 82-55-759-0779 E-mail: choiws@nongae.gsnu.ac.kr 2 Phil-Ok Koh medullar, testis, and ovary [2,3,10,25,27]. The presence of PACAP in the hypothalamus, pituitary, and gonads suggests its roles in the reproductive system. However, the existence, localization of PACAP and PAC 1 receptor genes in human placenta at various gestations has been unknown. Thus, the present study was performed to determine the distribution of PACAP and PAC 1 receptor mRNAs in human placenta. Materials and Methods Tissue preparation Human placental tissue from legal abortions aged between 6-7 weeks post menstruation (pm) were collected from normal pregnancies by curettages. Second trimester placenta from 14-24 weeks of gestation were obtained from induced abortion of healthy pregnancies and term placenta (38-41 weeks pm) by caesarian section or normal delivery. For in situ hybridization studies, tissues were fixed with 4% paraformaldehyde in 0.1M phosphate buffered saline (PBS) and cryoprotected with 20% sucrose phosphate buffer for 24 hr. Placental sections were cut in a cryomicrotome at a thickness of 15 µ m, mounted on the Probe-on slides (Fisher Scientific, USA), and stored at − 70 o C. Slides from each placenta were stained with hematoxylin and eosin for general morphological observation. In situ Hybridization All solutions were made with sterile water and glassware was autoclaved to prevent contamination by RNase. In situ hybridization histochemistry was carried out, as described by Angerer et al . [1]. Briefly, the slides were dried, washed with 0.1 M PBS, and treated proteinase K, TE buffer, and an acetylation solution. Sections were covered with prehybridization buffer containing 50% deionized formamide and incubated at 37 o C for 1 hr. After removal of the prehybridization buffer, the slides were covered with the mixture containing the prehybridization buffer, 50 µ g/ml yeast tRNA, 10 mM dithiothreitol, and 35 S-labeled PACAP cRNA probe or PAC 1 receptor cRNA probe [13]. The slides were then covered with cover glasses and incubated at 60 o C for 24 hr. 35 S-UTP-labeled probes were prepared using in vitro transcription kit (Promega, USA). Antisense and sense cRNA probes were purified with a Sephadex G-50 nick column (Pharmacia Biotech, Sweden) and eluted with SET buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris, and 10 mM DTT). Tissue slides were posthybridized in a posthybridization buffer. Following a wash in 4 × SSC for 30 min, the sections were then treated with ribonuclease A (50 µ g/ ml) at 37 o C for 10 min, washed twice in 2 × SSC and 1 × SSC, transferred to a wash buffer containing 0.1 × SSC at 65 o C for 30 mins, and dehydrated in alcohol solutions with ascending concentrations. Slides were exposed to β -max hyperfilm (Amersham, Sweden) for 4 days in light-tight cassettes at − 70 o C, and were dipped into NTB2 emulsion (1 : 1 dilution, Eastman Kodak, USA), exposed at 4 for 2 weeks, developed in Kodak D19 developer (1 : 1 dilution, Eastman Kodak, USA) at 15 o C, and counterstained with hematoxylin. The slides were observed under a dark and a bright field microscope, and photographed. Results The present study showed the expression and distribution of PACAP and PAC 1 receptor mRNAs in the human placenta at various gestations. In situ hybridization revealed the expression of PACAP and PAC 1 receptor mRNAs in stem villi and terminal villi. Positive cells of PACAP mRNA were detected in stroma cells surrounding the blood vessels within stem villi on 7 weeks (Figs. 1A & 2A). PACAP mRNA was expressed in stroma cells of stem villi on 14 week (Figs. 1B & 2B). Furthermore, positive signals of PACAP mRNA were strongly observed in the stroma cells of stem villi and terminal villi on 21 and 38 weeks (Figs. 1C, 1D, 2C & 2D). Signals for PACAP mRNA in these cells were gradually increased as gestation advanced. However, PACAP mRNA was very weakly expressed in cytotrophoblast cells and syncytiotrophoblast cells. There were no detectable signals in negative control with a sense probe (Fig. 1E). The hybridization of adjacent sections with PACAP and PAC 1 receptor cRNA probes showed that PACAP and PAC 1 receptor mRNAs were expressed in the same areas. Positive signals of PAC 1 receptor mRNA were detected in stroma cells of stem villi on 7 and 14 weeks (Figs. 3A, 3B, 4A & 4B). PAC 1 receptor mRNA was strongly expressed in stroma cells of stem villi and terminal villi on 21 and 38 weeks F ig. 1. Dark-field photomicrographs of PACAP mRNA expressi on i n the human placenta from 7 (A), 14 (B), 24 (C), and 38 (D ) w eeks gestation by in situ hybridization. A and B: Positive signa ls w ere moderately observed in stroma cells of stem villi. C and D: P ositive cells were strongly detected in stroma cells of stem vi lli a nd terminal villi. E: No positive signals were detected in negati ve c ontrol with a sense probe. Bar = 200 µm. PACAP and its type I receptor in human placenta 3 (Figs. 3C, 3D, 4C & 4D). As similar like to the expression pattern of PACAP, positive signals for PAC 1 receptor in these cells became strong as gestation advanced. But, PAC 1 receptor mRNA was very weakly expressed in cytotrophoblast cells and syncytiotrophoblast cells. No positive signals of PAC 1 receptor mRNA was detected in negative control with a sense probe (Fig. 3E). Discussion In the present study, we showed the cellular localization of PACAP and PAC 1 receptor mRNAs in human placenta on 7, 14, 24, and 38 weeks. Recently, we reported evidence that PACAP and PAC 1 receptor mRNAs were expressed in decidual cells, chorionic vessels, and stroma cells of chorionic villi in the rat placenta [14]. Also, Scaldaferri et al. [23] demonstrated the presence of PACAP and PAC 1 receptor in both the human and the rat placenta at term, using RT-PCR and immunohistochemistry techniques. They showed the expression of PACAP at term placenta, but they did not offer any information as to the distribution of PACAP and PAC 1 receptor in human placenta at various gestations, during pregnancy. In this study, we utilized in situ hybridization to determine the existence of PACAP and PAC 1 receptor mRNAs in human placenta. Our data showed the expression of PACAP and PAC 1 receptor mRNAs in stroma cells of stem villi and terminal villi. As gestation advanced, the expression of PACAP and PAC 1 receptor mRNAs was increased in these cells. It is known that placenta produces several growth factors such as insulin like growth factor and basic fibroblast growth factor. Especially, VEGF and placenta growth factor (PlGF), which are essential factor for the placental growth and fetal development [5,32]. VEGF was expressed in stroma cells within villi in human placenta [26]. Localization of VEGF in stroma cells demonstrates that VEGF play an important role in the physiological growth and function of the vascular system in the villous stroma. Furthermore, PACAP that acts as a growth factor in various cells, stimulates VEGF release F ig. 2. Bright-field photomicrographs of PACAP mRN A e xpression in the human placenta from 7 (A), 14 (B), 24 (C), a nd 3 8 (D) weeks gestation by in situ hybridization. A and B: PACA P m RNA was strongly expressed in stroma cells of stem villi. C a nd D: Positive signals were strongly detected in stroma cells of s tem villi. Arrows indicate the positive cells. Bar = 20 µ m. F ig. 3. Expression of PAC 1 receptor mRNA in the hum an p lacenta from 7 (A), 14 (B), 24 (C), and 38 (D) weeks gestati on b y in situ hybridization. A and B: Positive signals we re m oderately observed in stroma cells of stem villi. C and D: P ositive cells were strongly detected in stroma cells of stem vi lli a nd terminal villi. E: No positive signals were detected in n egative control with a sense probe. Bar = 200 µ m. F ig. 4. Localization of PAC 1 receptor mRNAs expression in t he h uman placenta from 7 (A), 14 (B), 24 (C), and 38 (D) wee ks g estation by in situ hybridization. A and B: PAC 1 receptor mRN A w as strongly expressed in stroma cells of stem villi. C and D: P ositive signals were strongly detected in stroma cells of ste m v illi. Arrows indicate the positive cells. Bar = 20 µ m. 4 Phil-Ok Koh [7]. Also, VEGF has been known to act directly on vascular endothelial cells by promoting cell proliferation and permeability. In this study, PACAP was strongly expressed in stroma cells of stem villi and terminal villi. PACAP mRNA was localized in the stroma cell surrounding the blood vessel on 14 and 24 weeks. Also, PACAP mRNA was expressed in the whole stroma cells on 38 weeks. Furthermore, PACAP 38 immunostaining was detected in stroma cells of stem and terminal placental villi [23]. Both PACAP and VEGF were expressed in stroma cells of terminal villi. Thus, our data suggest that PACAP stimulates the release of VEGF and promotes the growth of placenta. In the previous studies, VIP/PACAP neuropeptide family regulates the blood flow and hormone secretion in human placenta [4,9,30]. Steenstrup et al. [30] reported that PACAP were expressed in the uteroplacental unit, where it causes a concentration dependent relaxation on stem villous and umbilical cord arteries. These results suggest that PACAP mediates the placental growth and fetal development during the pregnancy period. In the previous studies, many researchers demonstrated that PACAP acts an autocrine and/or paracrine regulator in various tissues, including ovarian granulose cells, testicular Leydig cells, and placental tissue [15,20,22,24]. Also, in our results, PACAP and PAC 1 receptor mRNAs were expressed in the same areas. 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