Histological Expression of Metallothionein in the Developing Rat Placenta

J Toxicol Pathol 2008; 21: 223–227

Original Histological Expression of Metallothionein in the Developing Rat Placenta

Satoshi Furukawa1, Koji Usuda1, Masayoshi Abe1, Seigo Hayashi1, and Izumi Ogawa1

1Biological Research Laboratories, Nissan Chemical Industries, Ltd., 1470 Shiraoka, Minamisaitama, Saitama 349–0294, Japan

Abstract: In order to clarify the metallothionein (MT) localization in the developing placenta, we histologically investigated the sequential MT expression in placentas and fetal livers using pregnant rats during gestation days (GDs) 9 – 21. The placentas were sampled and weighed on GDs 9, 11, 13, 15, 17, 19 and 21. In the early post implantation period, the expression of MT was slightly detected in the yolk sac and the primary decidual zone around the embryo. MT was then mainly present in the deciduas parietalis and yolk sac. After the deciduas parietalis ruptured, MT was subsequently detected in the yolk sac and deciduas basalis. MT continued to be detected in the yolk sac until GD 21, but it was reduced in the deciduas basalis in accordance with development of the fetal liver with elevated MT expression. In conclusion, the main expression site of MT changes from the maternal placenta to the fetal placenta, and then to the fetal liver in accordance with the fetal development. However, we speculate that the MT-positive cells in the placenta are positioned between the maternal and embryonic environments throughout the gestation period and always surround the embryo/fetus. (J Toxicol Pathol 2008; 21: 223–227) Key words: fetus, liver, metallothionein, placenta, rat

Introduction fetal blood2. On the other hand, cadmium is known to accumulate in the placenta and induces cellular damage, Metallothionein (MT) is a family of cysteine-rich, low which can result in teratogenic or embryotoxic effects3. The molecular weight (MW: 3500 to 14000 Da) metalloproteins placental transfer of cadmium has been studied in the rat4, that have high affinity for several divalent metals, such as mouse5, rabbit6 and human7. The placenta is thought to cadmium, zinc, mercury and copper. MT is expressed to a protect the developing embryo/fetus from this toxic metal certain extent in almost all mammalian tissues. MT plays a mechanism by which placental MT restricts entry of role in the homeostasis of zinc and copper, and also provides cadmium into the embryonic environment. However, there protection from the toxic effects of cadmium and mercury. have been no reports on the sequential morphological details MT has been extensively studied in relation to cadmium of MT expression in the rat placenta. In order to clarify the toxicity and has been shown to play an important role in the MT localization in the developing placenta, we toxicokinetics of cadmium1. histologically investigated the sequential MT expression in The placenta plays a major role in the transfer of O2/ placentas and fetal livers in normal rats during gestation days CO2 and nutrient/metabolite requirements from the dam to (GDs) 9 – 21. the embryo and also in protecting the embryo from toxic compounds during mammalian fetal development. Both Materials and Methods zinc and copper are essential metals for growth and development of the fetus. Placental MT can be considered to Animals regulate metal transport of these metals from maternal to A total of 28 pregnant, specific-pathogen-free Wistar Hannover GALAS rats (CLEA Japan, Inc., Japan) Received: 19 May 2008, Accepted: 14 July 2008 approximately 10 – 14 weeks of age were purchased for use Mailing address: Satoshi Furukawa, Toxicology Group, Toxicology & in the present study. The animals were housed individually Environmental Science Department, Biological Research Laboratories, in wire mesh cages in an air-conditioned room (22 ± 2°C; Nissan Chemical Industries, Ltd., 1470 Shiraoka, Minamisaitama, humidity, 55 ± 10%; light cycle, 12 hr/day). Feed (Oriental Saitama 349–0294, Japan Yeast Co., Ltd., Japan) and water were available ad libitum. TEL: 81-480-92-2513 FAX: 81-480-92-2516 E-mail: [email protected] 224 Metallothionein in the Rat Placenta Furukawa, Usuda, Abe et al. 225

Experimental design Labyrinth and basal zones GD 0 was designated as the day when the presence of a A few MT-positive spongiotrophoblasts in the basal vaginal plug was identified. Four maternal rats were zone were detected during GDs 15 – 21, although there was sampled on GDs 9, 11, 13, 15, 17, 19 and 21. Dams were no MT staining in the glycogen cells or giant cells in the euthanized by exsanguination under anesthesia and basal zone or in the trophoblasts in the labyrinth zone necropsied. All embryos/fetuses and half of the placentas throughout the gestation period (Figs. 1b1, c1, d1, e1). were removed from each uterus and weighed. The placentas and embryos/fetuses were fixed in 10% neutral buffered Decidua formalin. This experiment was conducted according to the On GD 9, MT was detected in the cytoplasm and nuclei Guidelines for Animal Experimentation, Japanese of decidual cells in the primary decidual zone around the Association for Laboratory Animal Science, 1987. embryo (Fig. 1a1). In addition, there were a few MT- positive cells scattered in the secondary decidual zone. On Histopathological examination GD 11, MT was more prominent in the deciduas parietalis Four placentas that were not removed from the uterus of than in the deciduas basalis (Fig. 1b1). After GD 13, MT each dam at each sampling, and four fetal livers from each staining was observed in the decidual cells in the deciduas dam during GDs 13 – 21 were embedded in paraffin, basalis beneath the basal zone (Figs. 1c1, c3). The intensity sectioned at a 4-µm thickness and stained routinely with of positive staining with MT in the deciduas basalis hematoxylin and eosin (H&E). Immunohistochemical increased until GD 15 and then decreased with the progress staining using an antibody against rat MT-1 and MT-2 (MT of placental development (Figs. 1d1, d3, e1, e3). E9, Dako Cytomation, USA) was performed on these tissues according to the avidin-biotin complex (ABC) method Fetal liver (VECTSTAIN ABC Kit, Vector Laboratories Inc., Canada). MT was detected mainly in the cytoplasm and nuclei of hepatocytes beginning on GD 15 (Figs. 1c4, d4). The Results intensity of positive staining with MT gradually increased with the progress of fetal development, and reached the Table 1 shows the total numbers and weights of the maximum level on GD 21 (Fig. 1e4). live embryos/fetuses, the dead embryo/fetus ratio and the placenta weights at each sampling time point. Table Uterus 2 and Fig. 1 show the sequential changes in the Strong expression of MT was detected in the uterine immunohistochemical MT expression in the placenta, fetal glandular epithelium throughout the gestation period. liver and uterus. Discussion Yolk sac On GD 9, minimal staining of MT was detected in the MTs consist of four major groups (MT-1, MT-2, MT-3 cytoplasm of epithelial in the visceral yolk sac (Fig. 1a2). and MT-4), and the most well-studied MTs are mammalian After the embryo turned and the yolk sac covered the entire MT-1 and MT-2. MT-1 is present with MT-2 and is embryo, the intensity of positive staining with MT in the expressed in almost all tissues1. In the present study, yolk sac gradually increased until GD 19 (Figs. 1b2, c2, d2) immnohistochemical sequential MT-1 and MT-2 expression and then slightly decreased on GD 21 (Fig. 1e2). MT was cell-specifically examined in placentas and fetal livers staining was prominent at the free surface of the epithelial using normal pregnant rats. We found that MT was mainly cells. present in the decidua and yolk sac in the placenta and in the

Fig. 1. Immnohistochemical metallothionein expression in the placenta and fetal liver. a: Gestation day 9. a1) Low magnification. A few metallothionein-positive decidual cells are scattered in the primary decidual zone around embryo (↑). EPC: Ectoplacental cone. E: Embryo. Bar=1 mm. a2) Visceral yolk sac (Y) and primary decidual zone (PDZ). Very slight MT-positive staining in the epithelial cells of the yolk sac. Bar=100 µm. b: Gestation day 11. b1) Low magnification. MT-positive staining in the decidual cells of the deciduas parietalis (↑). CAP: Chrioallantoic placenta. E: Embryo. Y: Yolk sac. Bar=1 mm. b2) Visceral yolk sac (Y) and decidual cells (D). Slight MT-positive staining in the epithelial cells of the visceral yolk sac. Bar=100 µm. c: Gestation day 13. c1) Low magnification. MT-positive staining in the epithelial cells of the visceral yolk sac and decidual cells (↑). L: Labyrinth zone. B: Basal zone. D: Deciduas basalis. Y: Yolk sac. Bar=1 mm. c2) Visceral yolk sac. Bar=100 µm. c3) Basal zone (B) and deciduas basalis (D). Bar=100 µm. c4) Fetal liver. No MT-positive cells in the fetal liver. Bar=100 µm. d: Gestation day 17. d1) Low magnification. MT-positive staining in the decidual cells (↑). L: Labyrinth zone. B: Basal zone. D: Deciduas basalis. Bar=1 mm. d2) Visceral yolk sac. MT-positive staining in the epithelial cells. Bar=100 µm. d3) Basal zone (B) and deciduas basalis (D). Bar=100 µm. d4) Fetal liver. MT-positive staining in some hepatocytes. Bar=100 µm. e: Gestation day 21. e1) Low magnification. Slight MT-positive staining in the deciduas basalis (↑). Bar=1 mm. L: Labyrinth zone. B: Basal zone. D: Deciduas basalis. e2) Visceral yolk sac. MT-positive staining in the epithelial cells. Bar=100 µm. e3) Basal zone (B) and deciduas basalis (D). Bar=100 µm. e4) Fetal liver. MT-positive staining in almost all hepatocytes. Bar=100 µm. 226 Metallothionein in the Rat Placenta

Table 1. Fetal and Placental Weight in the Normal Pregnant Rat

Gestation day No. of dams Total No. of live Dead embryo Embryo/fetus Placenta weight embryos/fetuses ratio (%)a weight (mg)a (mg)a

9 4 54 0.0 ± 0.0 NE NE 11 4 53 0.0 ± 0.0 NE NE 13 4 54 0.0 ± 0.0 65.7 ± 3.7 105.5 ± 7.5 15 4 54 1.7 ± 1.4 257.3 ± 4.0 193.7 ± 9.7 17 4 50 5.5 ± 1.6 777.9 ± 12.6 291.5 ± 15.1 19 4 44 2.5 ± 2.2 2062.2 ± 80.0 448.7 ± 13.0 21 4 52 0.0 ± 0.0 4799.4 ± 44.8 452.9 ± 13.9

NE: Not examined. Mean ± SE. a: Mean values for individual litters.

Table 2. Immunohistochemical Metallothionein Expression in the Rat Placenta and Fetal Liver Gestation day Tissue / organ Cell type 9 111315171921

Yolk sac (visceral) Epithelial cells – to ± ± + ++ +++ +++ ++ Labyrinth zone Trophoblasts NE –––––– Basal zone Spongiotrophoblasts – – – ±±±± Glycogen cells––––––– Giant cells––––––– Decidua Deciduas parietalis ± ++ NE NE NE NE NE Deciduas basalis ± ± to + ++ +++ ++ + + Fetal liver Hepatocytes NE NE – ± to + ++ ++ +++ Uterus Glandular epithelium +++ +++ +++ +++ +++ +++ +++

NE: Not examined. –: Negative, ±: Minimal, +: Mild, ++: Moderate, +++: Severe.

fetal liver. bilaminar, and is composed of trophoblast and a single layer MT can avidly sequester intracellular essential metals of hypoblast (endodermal cells) in the early post such as zinc and copper and also provide protection from the implantation period. When the yolk sac surrounds the early toxic effects of cadmium and mercury during pregnancy8,9. embryo as a double-layer sac, the yolk sac placenta begins to Normal pregnant rats have higher plasma cadmium levels transfer substances and nutrition from the dam to the and MT levels compared with non-pregnant rats10, and the embryo. In this period, MT was slightly detected in the yolk basal levels of MT already increased on GD 8 in pregnant sac and primary decidual zone around the embryo. The rats11. It is likely that maternal plasma MT originates in the expression of MT then mainly increased in the yolk sac and liver12. On the other hand, MT is also detected in the fetal deciduas parietalis. After the chorioallantoic placenta began liver and placenta during pregnancy in rats4,9. Both zinc and to function and deciduas parietalis ruptured, MT was copper are found at high concentrations in the human and subsequently detected in the yolk sac and the deciduas rodent fetal liver bound to MT during the perinatal basalis. The rodent yolk sac is largely responsible for development2. In the rat fetal liver, MT is present on GD 16, providing amino acid nutrition to the early post-implantation and the MT level continues to increase into the neonatal embryo and possibly also in the late fetal stage of gestation, period as determined by an enzyme-linked immunosorbent unlike other mammalian placentas15. In the present study, assay13. In the human placenta obtained from full-term MT continued to be detected in the yolk sac until GD 21, and healthy infants, MT has been immunohistochemically it seems that the yolk sac in rats might also be involved in identified in amniotic cells, syncytial trophoblasts, villous homeostasis of essential and toxic metals until birth. In interstitial cells and decidual cells2. In the mouse placenta, contrast, the expression of MT gradually decreased in the cell-specific expression of MT genes occurs in the yolk sac, deciduas basalis, but increased in the fetal liver in spongiotrophoblasts and decidual cells throughout the accordance with the fetal development. Therefore, the main gestation period14. expression site of MT changes from the maternal placenta to In the present study, the MT location in the normal the fetal placenta and then to the fetal liver in accordance developing rat placenta was almost the same as shown in with the fetal development. However, we speculate that the mice. In development of the rat placenta, the yolk sac is MT-positive cells in the placenta are positioned between the Furukawa, Usuda, Abe et al. 227 maternal and embryonic environments throughout the Cadmium-metallothioneins in mother animal, placenta and gestation period and always surround the embryo/fetus for fetus. Proceedings of the 2nd International Conference on homeostasis of essential metals and protection from metal Metallothionein. Zürich. 1985. toxic effects. We suppose that MT may play an important 7. Waalkes MP, Poisner AM, Wood GW, and Klaassen CD. role in embryonic development and maintenance of normal Metallothionein-like proteins in human placenta and fetal membranes. Toxicol Appl Pharmacol. 74: 179–184. 1984. pregnancy. 8. Goyer RA and Cherian MG. Role of metallothionein in human placenta and rats exposed to cadmium. IARC Sci Acknowledgements: The authors would like to thank Mr. Publ. 118: 239–247. 1992. Kiyoshi Kobayashi, Ms. Kaori Maejima, Ms. Hiromi Asako, 9. Webb M. Metallothionein in regeneration, reproduction and Mr. Atsushi Funakoshi, Mr. Yoshinori Tanaka, Ms. Mayumi development. In: Metallothionein II. JHR Kagi and Y Seta and Mr. Shigeru Iimura for their excellent technical Kojima (eds). Birkhauser-Verlag, Basel. 483–498. 1987. assistance. 10. Chan HM, Tamura Y, Cherian MG, and Goyer RA. Pregnancy-associated changes in plasma metallothionein concentration and renal cadmium accumulation in rats. Proc References Soc Exp Biol Med. 202: 420–427. 1993. 11. Nordberg M and Nordberg GF. Toxicological aspects of 1. Nordberg M and Nordberg GF. Toxicological aspects of metallothionein. Cell Mol Biol. 46: 451–463. 2000. metallothionein. Cell Mol Biol. 46: 451–463. 2000. 12. Chan HM and Cherian MG. Mobilization of hepatic 2. Goyer RA, Haust MD, and Cherian MG. Cellular cadmium in pregnant rats. Toxicol Appl Pharmacol. 120: localization of metallothionein in human term placenta. 308–314. 1993. Placenta. 13: 349–355. 1992. 13. Chan HM and Cherian MG. Ontogenic changes in hepatic 3. Webb M, Holt D, Brown N, and Hard GC. The metallothionein isoforms in prenatal and newborn rats. teratogenicity of cadmium-metallothionein in the rat. Arch Biochem Cell Biol. 71: 133–140. 1993. Toxicol. 61: 457–467. 1988. 14. De SK, McMaster MT, Dey SK, and Andrews GK. Cell- 4. Arizono K, Ota S, and Ariyoshi T. Purification of specific metallothionein gene expression in mouse decidua metallothionein-like protein in rat placenta. Bull Environ and placentae. Development. 107: 611–621. 1989. Contam Toxicol. 27: 671–677. 1981. 15. Maranghi F, Macri C, Ricciardi C, Stazi AV, and Mantovani 5. Hanlon DP, Specht C, and Ferm VH. The chemical status of A. Evaluation of the placenta: Suggestions for a greater role cadmium ion in the placenta. Environ Res. 27: 89–94. 1982. in developmental toxicology. Adv Exp Med Biol. 444: 129– 6. Nakabayashi-Kodama J, Kojima Y, and Itokawa Y. 136. 1998.