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The impairment of metrial gland development in tamoxifen exposed rats
Experimental and Toxicologic Pathology 64 (2012) 121–126
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Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
The impairment of metrial gland development in tamoxifen exposed rats Satoshi Furukawa ∗ , Seigo Hayashi, Koji Usuda, Masayoshi Abe, Izumi Ogawa Biological Research Laboratories, Nissan Chemical Industries, Ltd., 1470 Shiraoka, Minamisaitama, Saitama 349-0294, Japan
a r t i c l e
i n f o
Article history: Received 17 February 2010 Accepted 5 July 2010 Keywords: Metrial gland hypoplasia Placenta Rat Tamoxifen uNK cell
a b s t r a c t We examined the sequential histopathological changes in the placenta from rats exposed to tamoxifen. Tamoxifen was administered intraperitoneally at doses of 0 and 2 mg/kg/day on gestation days (GDs) 8, 9 and 10, and the placentas were sampled on GDs 11, 13, 15, 17, and 21. The fetal mortality rates in the tamoxifen group were increased up to 56%. However, there were no effects on the weights of live embryos/fetuses and their placentas. Histopathologically, the size of metrial gland in the tamoxifen group was reduced on all sampling times. The spiral arteries appeared less well developed in the hypoplastic metrial gland. A decrease in uterine natural killer (uNK) cells and mitotic uNK cells around the spiral arteries in the metrial gland was detected from GD 13 onward and on GDs 11 and 13, respectively. There were no obvious changes in the labyrinth zone or basal zone. We consider that the anti-estrogen effect of tamoxifen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland and leads to inhibition of the proliferative activity of uNK cells, followed by defective development of spiral arteries, and metrial gland hypoplasia. It is assumed that the metrial gland hypoplasia might be involved in the tamoxifen-induced embryo/fetus-toxicity. © 2010 Published by Elsevier GmbH.
The placenta is the interface between dam and developing embryo/fetus and is a specialized organ of O2 /CO2 exchange and nutrient/metabolite requirements during embryonic developments. It also secures an embryo/fetus to the endometrium as a protective barrier of xenobiotics and releases a variety of steroids, hormones and cytokines. Although a placenta is a transient organ, its growth and function play important roles in the maintenance of pregnancy and the influence on fetal growth and development. The placenta is histologically composed of a fetal part and a maternal part. The maternal part of the placenta differentiates from uterine stromal cells after decidualization, and consists of the decidua and the metrial gland. The decidua includes newly developed blood vessels which play essential roles in the development of vascularized decidual–placental interface (Adamson et al., 2002). The metrial gland is the nodular aggregates of heterogeneous tissue that develops in the mesometrial triangle in the uterine wall (Peel, 1989). Uterine natural killer (uNK) cells are the main structural component of the metrial gland. They may play an important immunological role in their tolerogenic form and be necessary for normal arterial
∗ Corresponding author at: Toxicology & Environmental Science Department, Biological Research Laboratories, Nissan Chemical Industries, Ltd., 1470 Shiraoka, Minamisaitama, Saitama 349-0294, Japan. Tel.: +81 480 92 2513; fax: +81 480 92 2516. E-mail address: [email protected] (S. Furukawa). 0940-2993/$ – see front matter © 2010 Published by Elsevier GmbH. doi:10.1016/j.etp.2010.07.004
remodeling (Bilinski et al., 2008; Wooding and Burton, 2008). Thus, the maternal part of the placenta is important in fetal growth and successful placentation. However, there is little attention given to the toxicity for the maternal part of the placenta, despite that it is known that the fetal part of the placenta is a major toxicological target organ for some chemicals. Tamoxifen is a non-steroid selective estrogen-receptor modulator that has mixed agonist/antagonist actions at the estrogen receptor, and has been widely used for therapy of estrogen-receptor positive breast cancer (Martel et al., 1998). Clitoromegaly and labial fusion are reported in human females born from the mothers who are treated with tamoxifen during pregnancy (Tewari et al., 1997). Tamoxifen affects germ cell mutation during spermatogenesis and induces a decrease in fertility index in male rats (Balasinor et al., 2001; Gill-Sharma et al., 2001). Uterine atrophy and a retardation of uterine gland genesis are common abnormalities in rats and mice exposed neonatally to tamoxifen (Chamness et al., 1979; Iguchi et al., 1986). Furthermore, tamoxifen inhibits the decidual cell response of uterus in rats (Barkai et al., 1992; Ohta et al., 1989) and induces failure of the transformation of the placental bed blood vessels in the maternal part of the placenta (Sadek and Bell, 1996). Although tamoxifen is considered to affect the maternal part of the placenta, there have been few reports on the detailed histopathology of the placenta exposed to it. In the present report, we examined the sequential histopathological changes in the placenta from the rats exposed to tamoxifen during gestation day (GD) 8 to GD 10.
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S. Furukawa et al. / Experimental and Toxicologic Pathology 64 (2012) 121–126
1. Materials and methods 1.1. Animals Non-pregnant specific pathogen-free Wistar Hannover GALAS rats (CLEA Japan, Inc., Japan) were purchased at approximately 10–14 weeks of age. A female rat was housed together with a male rat of the same strain and source for mating. The occurrence of copulation was established by daily inspection for a vaginal plug. These mated female rats were assigned in this study. GD 0 was designated as the day when the presence of vaginal plug was identified. The animals were single-housed in plastic cages on softwood chip bedding in an air-conditioned room (22 ± 2 ◦ C; humidity, 55 ± 10%; light cycle, 12 h/day). Feed (CRF-1:Oriental Yeast Co., Ltd., Japan) and water were available ad libitum. 1.2. Experimental design Tamoxifen (Sigma, USA) was made into a stable suspension of the desired drug concentration in aqueous solution of 0.25% carboxymethyl cellulose (Maruishi Pharmaceutical Co., Ltd., Japan) and 0.2% polysorbate 80 (Koso Chemical Co., Ltd., Japan). The 40 pregnant rats were randomly allocated to 2 groups of 20 rats each. Tamoxifen was intraperitoneally administered to the groups at doses of 0 (control) and 2 mg/kg/day with a volume of 0.5 mL/100 g body weight on GDs 8, 9 and 10. The dose level and treatment period in this study were previously reported to induce an impairment of decidual development (Sadek and Bell, 1996). All treatments were made between 9 and 11 am. Maternal body weight was recorded on GDs 0, 8–17, 21. The dams each from the control and tamoxifen groups were sampled on GDs 11, 13, 15, 17, and 21. The dams were euthanized by exsanguination under anesthesia, and necropsied. All embryos/fetuses were removed from the placentas. Half of the placentas were separated between the basal zone and decidua basalis, and removed from the uterus wall. The embryos/fetuses and removed placentas in each dam were weighed, and the fetal–placental weight ratio was calculated individually. The fetuses on GD 21 were macroscopically examined for external malformations. All placentas were fixed in 10% neutral buffered formalin. These experiments were conducted according to the Guidelines for Animal Experimentation, Japanese Association for Laboratory Animal Science, 1987. 1.3. Histopathological examination A total of 40 placentas of the control and tamoxifen groups were obtained randomly from the live embryos/fetuses in the dams at each sampling time (4 placentas/dam). These tissues were embedded in paraffin, sectioned at 4-m thickness, and stained routinely
with hematoxylin and eosin (H&E), and periodic acid Schiff (PAS) for histopathological examination. Immunohistochemical staining, Factor VIII related antigen (DAKO, Japan), were performed on all selected placentas. The thickness of labyrinth zone, basal zone, decidua basalis and metrial gland close to the central portion were measured in placentas from each dam with the aid of an image analyzer (IPAP; Processor for Analytical Pathology, Sumika Technoservice Co., Japan). For the incidence of mitotic cells in the metrial gland, mitotic figures in HE stain sections were counted in 2.5 mm2 areas by an image analyzer with a 40× objective. 1.4. Statistical analysis Means and standard error (SE) of the individual litter values were calculated. Continuous data were analyzed with the F test. When variances were homogeneous, the Student t-test was performed. The Aspin–Welch t-test was performed when variances were not homogeneous. The level of significance was set at P < 0.05, 0.01 and 0.001. 2. Results 2.1. Effects on dams The dam’s body weight gains in the tamoxifen group were reduced from GD 9 onward. However, dams in neither the control nor tamoxifen group showed any other clinical signs during the experimental period. 2.2. Effects on embryos/fetuses and placentas The fetal mortality rates in the tamoxifen group were significantly increased from 27% on GD 13 to 56% on GD 15, and reached the plateau from GD 15 onward (Table 1). However, there were no effects on the weights of live embryos/fetuses and their placentas, or these fetal/placental weight ratios on each sampling time. No macroscopic abnormalities were detected in the fetuses and placentas on GD 21 in both groups (Table 1). 2.3. Histopathological observation of placenta Histopathologically, the size of metrial gland in the tamoxifen group was reduced on all sampling times compared to the control group (Fig. 1). The spiral arteries appeared less well developed in the hypoplastic metrial gland (Fig. 2). The uNK cells around the spiral arteries were decreased from GD 13 onward in the tamoxifen group, and instead the stromal cells were closely arranged in the
Table 1 Effect of tamoxifen on fetus and placenta. Autopsy
GD 11 GD 13 GD 15 GD 17 GD 21
Treatment
No. of dams
Total No. of live fetuses
Fetal mortality (%)a
Fetus weight (g)a
Placenta weight (g)a
Fetus/placenta weight ratio (g/g)a
External malformation ratio (%)a
Control Tamoxifen Control Tamoxifen Control Tamoxifen Control Tamoxifen Control Tamoxifen
4 4 4 4 4 4 4 4 4 4
53 56 52 36 52 20 40 26 37 21
NA NA 1.4 ± 1.4 26.8 ± 11.4 6.9 ± 2.5 55.6 ± 12.5* 7.1 ± 7.1 56.3 ± 0.7** 4.2 ± 4.2 51.3 ± 14.7*
NA NA 0.081 ± 0.014 0.089 ± 0.012 0.242 ± 0.002 0.235 ± 0.009 0.772 ± 0.028 0.688 ± 0.041 5.114 ± 0.137 5.126 ± 0.387
NA NA 0.118 ± 0.012 0.110 ± 0.010 0.192 ± 0.015 0.207 ± 0.016 0.333 ± 0.032 0.312 ± 0.018 0.449 ± 0.013 0.458 ± 0.012
NA NA 0.69 ± 0.06 0.84 ± 0.09 1.29 ± 0.11 1.13 ± 0.06 2.34 ± 0.14 2.17 ± 0.22 11.44 ± 0.35 11.72 ± 1.04
NA NA NA NA NA NA NA NA 0.0 ± 0.0 0.0 ± 0.0
Mean ± SE. NA, not available. a Mean of individual litter values. * Significantly different from control at P < 0.05 (Student’s t/Aspin–Welch test). ** Significantly different from control at P < 0.001 (Student’ t/Aspin–Welch test). ***Significantly different from control at P < 0.0001 (Student’ t/Aspin–Welch test).
S. Furukawa et al. / Experimental and Toxicologic Pathology 64 (2012) 121–126
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Fig. 1. Histological sequential changes in metrial gland. Marked metrial gland hypoplasia is detected with less well development of spiral arteries in the tamoxifen group. (a and b) Metrial gland on GD 11. (a) Control group. (b) Tamoxifen group. (c and d) Metrial gland on GD 13. (c) Control group. (d) Tamoxifen group. (e and f) Metrial gland on GD 17. (e) Control group. (f) Tamoxifen group. (g and h) Metrial gland on GD 21. (g) Control group. (h) Tamoxifen group. H&E stain, Bar = 1 mm. BZ, basal zone; DB, decidua basalis; LZ, labyrinth zone; MG, metrial gland.
Fig. 2. Histological changes in metrial gland and labyrinth zone. (a and b) Marked defective development of spiral arteries in metrial gland is detected on GD 11 in the tamoxifen group. (a) Control group. (b) Tamoxifen group. Factor VIII stain, Bar = 500 m. (c and d) uNK cells with clear cytoplasm and PAS positive granules are decreased around the spiral arteries on GD 13 in the tamoxifen group. The stromal cells are closely arranged in the metrial gland. (c) Control group. (d) Tamoxifen group. PAS stain, Bar = 100 m. (e and f) The number of mitotic cells, likely uNK cells (↑) are decreased on GD 11 in the tamoxifen group. (e) Control group. (f) Tamoxifen group. HE stain, Bar = 50 m. (g and h) Pyknosis or karyorrhexis (↑) is detected in the embryonic blood vessels without any changes of the trophoblastic septa and maternal sinusoid on GD 13 in the tamoxifen group. (g) Control group. (h) Tamoxifen group. HE stain, Bar = 50 m. DB, decidua basalis; MG, metrial gland; SA, spiral artery.
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Table 2 Distribution of uNK cells in metrial part of placenta. Autopsy
Metrial glands Control Tamoxifen Decidua basalis Control Tamoxifen
GD 11
GD 13
GD 15
GD 17
GD 21
+/− +/−
+++ ++
++ +
+ −
+/− −
+/− +/−
− −
− −
− −
− −
Codes: −, almost absent; +/−, slight; +, mild; ++, moderate; +++, severe.
Fig. 3. Mitotic cells in metrial gland. () Control; () Tamoxifen. Each value represents mean ± SE. Significantly different from control at *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s test). The number of mitotic cells, likely uNK cells, is lower on GDs 11 and 13 in the tamoxifen group.
metrial gland (Table 2 and Fig. 2). In addition, the number of mitotic cells which appeared to be uNK cells, was statistically significantly lower on GDs 11 and 13 in the tamoxifen group than that in the control group (Figs. 2 and 3). In the labyrinth zone, there were no obvious changes in almost all examined placentas. However, pyknosis and karyorrhexis were detected in the embryonic blood vessels without any changes of the trophoblastic septa and maternal sinusoid in a few tamoxifen-treated placentas on GD 13 (Fig. 2). These cell debris were likely to be derived from the embryos. There were no changes in the basal zone and decidua basalis.
A significant decrease in the thickness of metrial gland was observed on all sampling times and that in the decidua basalis on GD 13 in the tamoxifen group (Fig. 4). There were no changes in the thickness of labyrinth zone or basal zone on each sampling time.
3. Discussion These results indicated that the administration of tamoxifen at 2 mg/kg/day on GDs 8, 9 and 10 in pregnant rats induced an increased fetal mortality rate (up to 56%). However, there were no effects on the weights of live embryos/fetuses and their placentas on each sampling time. No macroscopic abnormalities were detected in the fetuses and placentas on GD 21. Histopathologically, metrial gland hypoplasia was observed with a decrease in uNK cells and defective development of spiral arteries. Metrial gland is composed of a dynamic mixed cell population of uNK cells, spindle to decidualized endometrial stromal cells, trophoblasts, blood vessels, and fibroblasts (Picut et al., 2009). The metrial gland is normal structure located in the mesometrial triangle of the pregnant rat’s uterus from GD 8 through termination of pregnancy and is known to be fully developed during GD 13–15. Development of metrial gland is a part of decidualization, which is a sequential process of growth and differentiation of uterine stromal cells and uNK cells, and remodeling of extracellular matrix and maternal vasculature (Greenwood et al., 2000). uNK cells appear in the mesometrial triangle on GD 8 and continue to increase in number and reach maximum until GD 12, and the disappearance occurs after GD 15 (Fukazawa et al., 1998; Kusakabe et al., 1999). They belong to a member of natural killer (NK) cells and originate from the bone marrow. They are recruited after conception, rapidly divided and differentiated to a different phenotype from the circulation NK cells in the metrial gland (Croy et al., 2002). The stromal cells produce the signals (hormones, cytokines and chemokines) which trigger the recruitment, guide the uNK cells differentiation to express a different phenotype from the circulating NK cells (Stewart, 1998; Wooding and Burton, 2008). Ovariectomy in rats and mice induces a metrial gland weight loss, and an impairment of the development of metrial gland seems to be attributed to reduction in the proliferative activity of uNK cells (Peel and Bulmer, 1975). Conversely, in NK gene knock-out mice (TgE26 mice) there was no development of mesometrial triangle area into the metrial gland, suggesting that the absence of uNK cells causes small placenta (Guimond et al., 1997). Thus, it assumes that the development of metrial gland and the proliferative activity of uNK cells have a relationship with each other. Human uNK cells express estrogen-1
Fig. 4. Thickness of labyrinth zone, basal zone, decidua basalis and metrial gland. () Control; () Tamoxifen. Each value represents mean ± SE. Significantly different from control at *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s test). A significant decrease in the thickness of metrial gland and decidua basalis in the tamoxifen group is noted on all sampling times and on GD 13, respectively.
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receptors but it is not known if this estrogen-receptor functions in uNK cells (Eriksson et al., 2006). Estrogen is not essential for attracting uNK cells precursors in a chimeric mice study (Peel and Stewart, 1986). Meanwhile, estrogen is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. Tamoxifen has the capability of inhibiting the decidual cell response in pregnant or pseudopregnant rats when administered during early pregnancy period (Barkai et al., 1992). Collectively, these results suggest that the anti-estrogen effect of tamoxifen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland after decidualization. This leads to inhibition of the proliferative activity and differentiation of uNK cells, followed by metrial gland hypoplasia. Conversely, there were no obvious abnormalities in the decidua basalis. It has been assumed that the decidual cells become less sensitive to the anti-estrogen effect of tamoxifen, because the administration to the pregnant rats was performed after decidualization. The uNK cells are involved in a role of regulation and restructuring of spiral arteries in the metrial gland. They produce protease that destroys basement membranes and vascular endothelial growth factor to stimulate endothelial cell proliferation in order to promote angiogenesis (Charnock-Jones et al., 2004; Kaloglu and Bulut, 2007; Wang et al., 2003). Interferon-gamma produced by uNK cells promotes a correct development of the maternal part of the placenta, and contributes to vascular modifications that include dilation, elongation and branching (Ashkar et al., 2000). Therefore, it appears that defective development of spiral arteries is caused by a decrease in uNK cells and contributes to the metrial gland hypoplasia. In the present study, it is not clear whether the poor reproductive performance is primarily by a direct effect of tamoxifen or a secondary effect of the placental damages. It is known that the fetal part of the placenta plays an important role of the interface between dam and embryo/fetus, but there were no obvious effects on the development of labyrinth and basal zone in the placentas derived from the live fetuses. In addition, pyknotic cells were detected in only the embryonal blood vessels in a few placentas on GD 13, supporting that the embryos died without damages of the fetal part of the placenta. Thus, we consider that the fetal part of the placenta contributes little to the tamoxifen-induced embryo/fetustoxicity. On the other hand, TgE26 mice can be pregnant, but their placental size is reduced (45% of the size of control) and the reproductive performance is very poor (mortality: 40%) (Guimond et al., 1997). After the reconstitution of uNK cells in TgE 26 mice (T cell, −; NK cell, −; B cell, +) by the engraftment of bone marrow from SCID mice (T cell, −; NK cell, +; B cell, −), the placental size and the fetal viability were recovered (Guimond et al., 1998). There might be maternal allorecognition mediated by uNK cells that recognize unusual fetal trophoblast MHC (Major Histocompatibility Complex) ligands (Moffett-King, 2002). Then, uNK cells play critical roles in maternal immune tolerance form toward invading trophoblast cells at the maternal–fetal interface caused by facilitating the pairing of fetal HLA (Human Leukocyte Antigen) and maternal KIBs (Killer Inhibitory Receptors) (Riley and Yokoyama, 2008). In addition, preeclampsia is a serious complication of pregnancy and leads to high maternal blood pressure, elevated concentrations of urinary protein and poor fetal growth. Preeclampsia is believed to result in part from inadequate maternal blood flow to the implantation site in humans. Then, alterations of uNK cell function and inadequate remodeling of spiral arteries play an important role in preeclampsia (Cross et al., 2002; Eriksson et al., 2006). Therefore, we consider that the tamoxifen-induced embryo/fetustoxicity might be associated with the immune tolerance deficiency caused by the decreased uNK cells in the hypoplastic metrial gland and/or the preeclampsia caused by defective development of spiral arteries.
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In conclusion, we consider that tamoxifen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland and leads to inhibition of the proliferative activity of uNK cells, followed by defective development of spiral arteries, and metrial gland hypoplasia. It is assumed that the metrial gland hypoplasia might be involved in the tamoxifen-induced embryo/fetus-toxicity. Acknowledgements The authors would like to thank Mr. Kiyoshi Kobayashi, Ms. Kaori Maejima, Ms. Hiromi Asako, Mr. Atsushi Funakoshi, Mr. Yoshinori Tanaka, Ms. Yuko Shimizu, and Mr. Shigeru Iimura for their excellent technical assistance. References Adamson SL, Lu Y, Whiteley KJ, Holmyard D, Hemberger M, Pfarrer C, et al. Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta. Dev Biol 2002;250:358–73. Ashkar AA, Di Santo JP, Croy BA. Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. J Exp Med 2000;192:259–70. Balasinor N, Parte P, Gill-Sharma MK, Juneja HS. Effect of tamoxifen on sperm fertilising ability and preimplantation embryo development. Mol Cell Endocrinol 2001;178:199–206. Barkai U, Kidron T, Kraicer PF. Inhibition of decidual induction in rats by clomiphene and tamoxifen. Biol Reprod 1992;46:733–9. Bilinski MJ, Thorne JG, Oh MJ, Leonard S, Murrant C, Tayade C, et al. Uterine NK cells in murine pregnancy. Reprod Biomed Online 2008;16:218–26. Chamness GC, Bannayan GA, Landry Jr LA, Sheridan PJ, McGuire WL. Abnormal reproductive development in rats after neonatally administered antiestrogen (tamoxifen). Biol Reprod 1979;21:1087–90. Charnock-Jones DS, Kaufmann P, Mayhew TM. Aspects of human fetoplacental vasculogenesis and angiogenesis. I. Molecular regulation. Placenta 2004;25:103–13. Cross JC, Hemberger M, Lu Y, Nozaki T, Whiteley K, Masutani M, et al. Trophoblast functions, angiogenesis and remodeling of the maternal vasculature in the placenta. Mol Cell Endocrinol 2002;187:207–12. Croy BA, Chantakru S, Esadeg S, Ashkar AA, Wei Q. Decidual natural killer cells: key regulators of placental development (a review). J Reprod Immunol 2002;57:151–68. Eriksson M, Basu S, Sentman CL. NK cells and pregnancy. In: Mor G, editor. Immunology of pregnancy. Springer-Verlag; 2006. p. 84–95. Fukazawa Y, Yamamura Y, Sato T, Iguchi T, Ohta Y. Mode of cell death in the rat metrial gland during peripartum regression. Anat Rec 1998;252:369–77. Gill-Sharma MK, Balasinor N, Parte P. Effect of intermittent treatment with tamoxifen on reproduction in male rats. Asian J Androl 2001;3:115–9. Greenwood JD, Minhas K, di Santo JP, Makita M, Kiso Y, Croy BA. Ultrastructural studies of implantation sites from mice deficient in uterine natural killer cells. Placenta 2000;21:693–702. Guimond MJ, Luross JA, Wang B, Terhorst C, Danial S, Croy BA. Absence of natural killer cells during murine pregnancy is associated with reproductive compromise in TgE26 mice. Biol Reprod 1997;56:169–79. Guimond MJ, Wang B, Croy BA. Engraftment of bone marrow from severe combined immunodeficient (SCID) mice reverses the reproductive deficits in natural killer cell-deficient tg epsilon 26 mice. J Exp Med 1998;187:217–23. Iguchi T, Hirokawa M, Takasugi N. Occurrence of genital tract abnormalities and bladder hernia in female mice exposed neonatally to tamoxifen. Toxicology 1986;42:1–11. Kaloglu C, Bulut HE. Vascular endothelial growth factor production by rat granulated metrial gland cells and their morphological features in normal and pathological conditions. Reprod Fertil Dev 2007;19:341–50. Kusakabe K, Okada T, Sasaki F, Kiso Y. Cell death of uterine natural killer cells in murine placenta during placentation and preterm periods. J Vet Med Sci 1999;61:1093–100. Martel C, Provencher L, Li X, St Pierre A, Leblanc G, Gauthier S, et al. Binding characteristics of novel nonsteroidal antiestrogens to the rat uterine estrogen receptors. J Steroid Biochem Mol Biol 1998;64:199–205. Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol 2002;2:656–63. Ohta Y, Iguchi T, Takasugi N. Deciduoma formation in rats treated neonatally with the anti-estrogens, tamoxifen and MER-25. Reprod Toxicol 1989;3:207–12. Peel S. Granulated metrial gland cells. Adv Anat Embryol Cell Biol 1989;115:1–112. Peel S, Bulmer D. The effects of late ovariectomy on the proliferation and differentiation of the uterus of the pregnant rat. J Anat 1975;119:569–78. Picut CA, Swanson CL, Parker RF, Scully KL, Parker GA. The metrial gland in the rat and its similarities to granular cell tumors. Toxicol Pathol 2009;37:474–80. Peel S, Stewart J. Oestrogen and the differentiation of granulated metrial gland cells in chimeric mice. J Anat 1986;144:181–7. Riley JK, Yokoyama WM. NK cell tolerance and the maternal–fetal interface. Am J Reprod Immunol 2008;59:371–87.
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Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
The impairment of metrial gland development in tamoxifen exposed rats Satoshi Furukawa ∗ , Seigo Hayashi, Koji Usuda, Masayoshi Abe, Izumi Ogawa Biological Research Laboratories, Nissan Chemical Industries, Ltd., 1470 Shiraoka, Minamisaitama, Saitama 349-0294, Japan
a r t i c l e
i n f o
Article history: Received 17 February 2010 Accepted 5 July 2010 Keywords: Metrial gland hypoplasia Placenta Rat Tamoxifen uNK cell
a b s t r a c t We examined the sequential histopathological changes in the placenta from rats exposed to tamoxifen. Tamoxifen was administered intraperitoneally at doses of 0 and 2 mg/kg/day on gestation days (GDs) 8, 9 and 10, and the placentas were sampled on GDs 11, 13, 15, 17, and 21. The fetal mortality rates in the tamoxifen group were increased up to 56%. However, there were no effects on the weights of live embryos/fetuses and their placentas. Histopathologically, the size of metrial gland in the tamoxifen group was reduced on all sampling times. The spiral arteries appeared less well developed in the hypoplastic metrial gland. A decrease in uterine natural killer (uNK) cells and mitotic uNK cells around the spiral arteries in the metrial gland was detected from GD 13 onward and on GDs 11 and 13, respectively. There were no obvious changes in the labyrinth zone or basal zone. We consider that the anti-estrogen effect of tamoxifen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland and leads to inhibition of the proliferative activity of uNK cells, followed by defective development of spiral arteries, and metrial gland hypoplasia. It is assumed that the metrial gland hypoplasia might be involved in the tamoxifen-induced embryo/fetus-toxicity. © 2010 Published by Elsevier GmbH.
The placenta is the interface between dam and developing embryo/fetus and is a specialized organ of O2 /CO2 exchange and nutrient/metabolite requirements during embryonic developments. It also secures an embryo/fetus to the endometrium as a protective barrier of xenobiotics and releases a variety of steroids, hormones and cytokines. Although a placenta is a transient organ, its growth and function play important roles in the maintenance of pregnancy and the influence on fetal growth and development. The placenta is histologically composed of a fetal part and a maternal part. The maternal part of the placenta differentiates from uterine stromal cells after decidualization, and consists of the decidua and the metrial gland. The decidua includes newly developed blood vessels which play essential roles in the development of vascularized decidual–placental interface (Adamson et al., 2002). The metrial gland is the nodular aggregates of heterogeneous tissue that develops in the mesometrial triangle in the uterine wall (Peel, 1989). Uterine natural killer (uNK) cells are the main structural component of the metrial gland. They may play an important immunological role in their tolerogenic form and be necessary for normal arterial
∗ Corresponding author at: Toxicology & Environmental Science Department, Biological Research Laboratories, Nissan Chemical Industries, Ltd., 1470 Shiraoka, Minamisaitama, Saitama 349-0294, Japan. Tel.: +81 480 92 2513; fax: +81 480 92 2516. E-mail address: [email protected] (S. Furukawa). 0940-2993/$ – see front matter © 2010 Published by Elsevier GmbH. doi:10.1016/j.etp.2010.07.004
remodeling (Bilinski et al., 2008; Wooding and Burton, 2008). Thus, the maternal part of the placenta is important in fetal growth and successful placentation. However, there is little attention given to the toxicity for the maternal part of the placenta, despite that it is known that the fetal part of the placenta is a major toxicological target organ for some chemicals. Tamoxifen is a non-steroid selective estrogen-receptor modulator that has mixed agonist/antagonist actions at the estrogen receptor, and has been widely used for therapy of estrogen-receptor positive breast cancer (Martel et al., 1998). Clitoromegaly and labial fusion are reported in human females born from the mothers who are treated with tamoxifen during pregnancy (Tewari et al., 1997). Tamoxifen affects germ cell mutation during spermatogenesis and induces a decrease in fertility index in male rats (Balasinor et al., 2001; Gill-Sharma et al., 2001). Uterine atrophy and a retardation of uterine gland genesis are common abnormalities in rats and mice exposed neonatally to tamoxifen (Chamness et al., 1979; Iguchi et al., 1986). Furthermore, tamoxifen inhibits the decidual cell response of uterus in rats (Barkai et al., 1992; Ohta et al., 1989) and induces failure of the transformation of the placental bed blood vessels in the maternal part of the placenta (Sadek and Bell, 1996). Although tamoxifen is considered to affect the maternal part of the placenta, there have been few reports on the detailed histopathology of the placenta exposed to it. In the present report, we examined the sequential histopathological changes in the placenta from the rats exposed to tamoxifen during gestation day (GD) 8 to GD 10.
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1. Materials and methods 1.1. Animals Non-pregnant specific pathogen-free Wistar Hannover GALAS rats (CLEA Japan, Inc., Japan) were purchased at approximately 10–14 weeks of age. A female rat was housed together with a male rat of the same strain and source for mating. The occurrence of copulation was established by daily inspection for a vaginal plug. These mated female rats were assigned in this study. GD 0 was designated as the day when the presence of vaginal plug was identified. The animals were single-housed in plastic cages on softwood chip bedding in an air-conditioned room (22 ± 2 ◦ C; humidity, 55 ± 10%; light cycle, 12 h/day). Feed (CRF-1:Oriental Yeast Co., Ltd., Japan) and water were available ad libitum. 1.2. Experimental design Tamoxifen (Sigma, USA) was made into a stable suspension of the desired drug concentration in aqueous solution of 0.25% carboxymethyl cellulose (Maruishi Pharmaceutical Co., Ltd., Japan) and 0.2% polysorbate 80 (Koso Chemical Co., Ltd., Japan). The 40 pregnant rats were randomly allocated to 2 groups of 20 rats each. Tamoxifen was intraperitoneally administered to the groups at doses of 0 (control) and 2 mg/kg/day with a volume of 0.5 mL/100 g body weight on GDs 8, 9 and 10. The dose level and treatment period in this study were previously reported to induce an impairment of decidual development (Sadek and Bell, 1996). All treatments were made between 9 and 11 am. Maternal body weight was recorded on GDs 0, 8–17, 21. The dams each from the control and tamoxifen groups were sampled on GDs 11, 13, 15, 17, and 21. The dams were euthanized by exsanguination under anesthesia, and necropsied. All embryos/fetuses were removed from the placentas. Half of the placentas were separated between the basal zone and decidua basalis, and removed from the uterus wall. The embryos/fetuses and removed placentas in each dam were weighed, and the fetal–placental weight ratio was calculated individually. The fetuses on GD 21 were macroscopically examined for external malformations. All placentas were fixed in 10% neutral buffered formalin. These experiments were conducted according to the Guidelines for Animal Experimentation, Japanese Association for Laboratory Animal Science, 1987. 1.3. Histopathological examination A total of 40 placentas of the control and tamoxifen groups were obtained randomly from the live embryos/fetuses in the dams at each sampling time (4 placentas/dam). These tissues were embedded in paraffin, sectioned at 4-m thickness, and stained routinely
with hematoxylin and eosin (H&E), and periodic acid Schiff (PAS) for histopathological examination. Immunohistochemical staining, Factor VIII related antigen (DAKO, Japan), were performed on all selected placentas. The thickness of labyrinth zone, basal zone, decidua basalis and metrial gland close to the central portion were measured in placentas from each dam with the aid of an image analyzer (IPAP; Processor for Analytical Pathology, Sumika Technoservice Co., Japan). For the incidence of mitotic cells in the metrial gland, mitotic figures in HE stain sections were counted in 2.5 mm2 areas by an image analyzer with a 40× objective. 1.4. Statistical analysis Means and standard error (SE) of the individual litter values were calculated. Continuous data were analyzed with the F test. When variances were homogeneous, the Student t-test was performed. The Aspin–Welch t-test was performed when variances were not homogeneous. The level of significance was set at P < 0.05, 0.01 and 0.001. 2. Results 2.1. Effects on dams The dam’s body weight gains in the tamoxifen group were reduced from GD 9 onward. However, dams in neither the control nor tamoxifen group showed any other clinical signs during the experimental period. 2.2. Effects on embryos/fetuses and placentas The fetal mortality rates in the tamoxifen group were significantly increased from 27% on GD 13 to 56% on GD 15, and reached the plateau from GD 15 onward (Table 1). However, there were no effects on the weights of live embryos/fetuses and their placentas, or these fetal/placental weight ratios on each sampling time. No macroscopic abnormalities were detected in the fetuses and placentas on GD 21 in both groups (Table 1). 2.3. Histopathological observation of placenta Histopathologically, the size of metrial gland in the tamoxifen group was reduced on all sampling times compared to the control group (Fig. 1). The spiral arteries appeared less well developed in the hypoplastic metrial gland (Fig. 2). The uNK cells around the spiral arteries were decreased from GD 13 onward in the tamoxifen group, and instead the stromal cells were closely arranged in the
Table 1 Effect of tamoxifen on fetus and placenta. Autopsy
GD 11 GD 13 GD 15 GD 17 GD 21
Treatment
No. of dams
Total No. of live fetuses
Fetal mortality (%)a
Fetus weight (g)a
Placenta weight (g)a
Fetus/placenta weight ratio (g/g)a
External malformation ratio (%)a
Control Tamoxifen Control Tamoxifen Control Tamoxifen Control Tamoxifen Control Tamoxifen
4 4 4 4 4 4 4 4 4 4
53 56 52 36 52 20 40 26 37 21
NA NA 1.4 ± 1.4 26.8 ± 11.4 6.9 ± 2.5 55.6 ± 12.5* 7.1 ± 7.1 56.3 ± 0.7** 4.2 ± 4.2 51.3 ± 14.7*
NA NA 0.081 ± 0.014 0.089 ± 0.012 0.242 ± 0.002 0.235 ± 0.009 0.772 ± 0.028 0.688 ± 0.041 5.114 ± 0.137 5.126 ± 0.387
NA NA 0.118 ± 0.012 0.110 ± 0.010 0.192 ± 0.015 0.207 ± 0.016 0.333 ± 0.032 0.312 ± 0.018 0.449 ± 0.013 0.458 ± 0.012
NA NA 0.69 ± 0.06 0.84 ± 0.09 1.29 ± 0.11 1.13 ± 0.06 2.34 ± 0.14 2.17 ± 0.22 11.44 ± 0.35 11.72 ± 1.04
NA NA NA NA NA NA NA NA 0.0 ± 0.0 0.0 ± 0.0
Mean ± SE. NA, not available. a Mean of individual litter values. * Significantly different from control at P < 0.05 (Student’s t/Aspin–Welch test). ** Significantly different from control at P < 0.001 (Student’ t/Aspin–Welch test). ***Significantly different from control at P < 0.0001 (Student’ t/Aspin–Welch test).
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Fig. 1. Histological sequential changes in metrial gland. Marked metrial gland hypoplasia is detected with less well development of spiral arteries in the tamoxifen group. (a and b) Metrial gland on GD 11. (a) Control group. (b) Tamoxifen group. (c and d) Metrial gland on GD 13. (c) Control group. (d) Tamoxifen group. (e and f) Metrial gland on GD 17. (e) Control group. (f) Tamoxifen group. (g and h) Metrial gland on GD 21. (g) Control group. (h) Tamoxifen group. H&E stain, Bar = 1 mm. BZ, basal zone; DB, decidua basalis; LZ, labyrinth zone; MG, metrial gland.
Fig. 2. Histological changes in metrial gland and labyrinth zone. (a and b) Marked defective development of spiral arteries in metrial gland is detected on GD 11 in the tamoxifen group. (a) Control group. (b) Tamoxifen group. Factor VIII stain, Bar = 500 m. (c and d) uNK cells with clear cytoplasm and PAS positive granules are decreased around the spiral arteries on GD 13 in the tamoxifen group. The stromal cells are closely arranged in the metrial gland. (c) Control group. (d) Tamoxifen group. PAS stain, Bar = 100 m. (e and f) The number of mitotic cells, likely uNK cells (↑) are decreased on GD 11 in the tamoxifen group. (e) Control group. (f) Tamoxifen group. HE stain, Bar = 50 m. (g and h) Pyknosis or karyorrhexis (↑) is detected in the embryonic blood vessels without any changes of the trophoblastic septa and maternal sinusoid on GD 13 in the tamoxifen group. (g) Control group. (h) Tamoxifen group. HE stain, Bar = 50 m. DB, decidua basalis; MG, metrial gland; SA, spiral artery.
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Table 2 Distribution of uNK cells in metrial part of placenta. Autopsy
Metrial glands Control Tamoxifen Decidua basalis Control Tamoxifen
GD 11
GD 13
GD 15
GD 17
GD 21
+/− +/−
+++ ++
++ +
+ −
+/− −
+/− +/−
− −
− −
− −
− −
Codes: −, almost absent; +/−, slight; +, mild; ++, moderate; +++, severe.
Fig. 3. Mitotic cells in metrial gland. () Control; () Tamoxifen. Each value represents mean ± SE. Significantly different from control at *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s test). The number of mitotic cells, likely uNK cells, is lower on GDs 11 and 13 in the tamoxifen group.
metrial gland (Table 2 and Fig. 2). In addition, the number of mitotic cells which appeared to be uNK cells, was statistically significantly lower on GDs 11 and 13 in the tamoxifen group than that in the control group (Figs. 2 and 3). In the labyrinth zone, there were no obvious changes in almost all examined placentas. However, pyknosis and karyorrhexis were detected in the embryonic blood vessels without any changes of the trophoblastic septa and maternal sinusoid in a few tamoxifen-treated placentas on GD 13 (Fig. 2). These cell debris were likely to be derived from the embryos. There were no changes in the basal zone and decidua basalis.
A significant decrease in the thickness of metrial gland was observed on all sampling times and that in the decidua basalis on GD 13 in the tamoxifen group (Fig. 4). There were no changes in the thickness of labyrinth zone or basal zone on each sampling time.
3. Discussion These results indicated that the administration of tamoxifen at 2 mg/kg/day on GDs 8, 9 and 10 in pregnant rats induced an increased fetal mortality rate (up to 56%). However, there were no effects on the weights of live embryos/fetuses and their placentas on each sampling time. No macroscopic abnormalities were detected in the fetuses and placentas on GD 21. Histopathologically, metrial gland hypoplasia was observed with a decrease in uNK cells and defective development of spiral arteries. Metrial gland is composed of a dynamic mixed cell population of uNK cells, spindle to decidualized endometrial stromal cells, trophoblasts, blood vessels, and fibroblasts (Picut et al., 2009). The metrial gland is normal structure located in the mesometrial triangle of the pregnant rat’s uterus from GD 8 through termination of pregnancy and is known to be fully developed during GD 13–15. Development of metrial gland is a part of decidualization, which is a sequential process of growth and differentiation of uterine stromal cells and uNK cells, and remodeling of extracellular matrix and maternal vasculature (Greenwood et al., 2000). uNK cells appear in the mesometrial triangle on GD 8 and continue to increase in number and reach maximum until GD 12, and the disappearance occurs after GD 15 (Fukazawa et al., 1998; Kusakabe et al., 1999). They belong to a member of natural killer (NK) cells and originate from the bone marrow. They are recruited after conception, rapidly divided and differentiated to a different phenotype from the circulation NK cells in the metrial gland (Croy et al., 2002). The stromal cells produce the signals (hormones, cytokines and chemokines) which trigger the recruitment, guide the uNK cells differentiation to express a different phenotype from the circulating NK cells (Stewart, 1998; Wooding and Burton, 2008). Ovariectomy in rats and mice induces a metrial gland weight loss, and an impairment of the development of metrial gland seems to be attributed to reduction in the proliferative activity of uNK cells (Peel and Bulmer, 1975). Conversely, in NK gene knock-out mice (TgE26 mice) there was no development of mesometrial triangle area into the metrial gland, suggesting that the absence of uNK cells causes small placenta (Guimond et al., 1997). Thus, it assumes that the development of metrial gland and the proliferative activity of uNK cells have a relationship with each other. Human uNK cells express estrogen-1
Fig. 4. Thickness of labyrinth zone, basal zone, decidua basalis and metrial gland. () Control; () Tamoxifen. Each value represents mean ± SE. Significantly different from control at *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s test). A significant decrease in the thickness of metrial gland and decidua basalis in the tamoxifen group is noted on all sampling times and on GD 13, respectively.
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receptors but it is not known if this estrogen-receptor functions in uNK cells (Eriksson et al., 2006). Estrogen is not essential for attracting uNK cells precursors in a chimeric mice study (Peel and Stewart, 1986). Meanwhile, estrogen is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. Tamoxifen has the capability of inhibiting the decidual cell response in pregnant or pseudopregnant rats when administered during early pregnancy period (Barkai et al., 1992). Collectively, these results suggest that the anti-estrogen effect of tamoxifen inhibits the proliferation of decidualized endometrial stromal cells in the metrial gland after decidualization. This leads to inhibition of the proliferative activity and differentiation of uNK cells, followed by metrial gland hypoplasia. Conversely, there were no obvious abnormalities in the decidua basalis. It has been assumed that the decidual cells become less sensitive to the anti-estrogen effect of tamoxifen, because the administration to the pregnant rats was performed after decidualization. The uNK cells are involved in a role of regulation and restructuring of spiral arteries in the metrial gland. They produce protease that destroys basement membranes and vascular endothelial growth factor to stimulate endothelial cell proliferation in order to promote angiogenesis (Charnock-Jones et al., 2004; Kaloglu and Bulut, 2007; Wang et al., 2003). Interferon-gamma produced by uNK cells promotes a correct development of the maternal part of the placenta, and contributes to vascular modifications that include dilation, elongation and branching (Ashkar et al., 2000). Therefore, it appears that defective development of spiral arteries is caused by a decrease in uNK cells and contributes to the metrial gland hypoplasia. In the present study, it is not clear whether the poor reproductive performance is primarily by a direct effect of tamoxifen or a secondary effect of the placental damages. It is known that the fetal part of the placenta plays an important role of the interface between dam and embryo/fetus, but there were no obvious effects on the development of labyrinth and basal zone in the placentas derived from the live fetuses. In addition, pyknotic cells were detected in only the embryonal blood vessels in a few placentas on GD 13, supporting that the embryos died without damages of the fetal part of the placenta. Thus, we consider that the fetal part of the placenta contributes little to the tamoxifen-induced embryo/fetustoxicity. On the other hand, TgE26 mice can be pregnant, but their placental size is reduced (45% of the size of control) and the reproductive performance is very poor (mortality: 40%) (Guimond et al., 1997). After the reconstitution of uNK cells in TgE 26 mice (T cell, −; NK cell, −; B cell, +) by the engraftment of bone marrow from SCID mice (T cell, −; NK cell, +; B cell, −), the placental size and the fetal viability were recovered (Guimond et al., 1998). There might be maternal allorecognition mediated by uNK cells that recognize unusual fetal trophoblast MHC (Major Histocompatibility Complex) ligands (Moffett-King, 2002). Then, uNK cells play critical roles in maternal immune tolerance form toward invading trophoblast cells at the maternal–fetal interface caused by facilitating the pairing of fetal HLA (Human Leukocyte Antigen) and maternal KIBs (Killer Inhibitory Receptors) (Riley and Yokoyama, 2008). In addition, preeclampsia is a serious complication of pregnancy and leads to high maternal blood pressure, elevated concentrations of urinary protein and poor fetal growth. Preeclampsia is believed to result in part from inadequate maternal blood flow to the implantation site in humans. Then, alterations of uNK cell function and inadequate remodeling of spiral arteries play an important role in preeclampsia (Cross et al., 2002; Eriksson et al., 2006). Therefore, we consider that the tamoxifen-induced embryo/fetustoxicity might be associated with the immune tolerance deficiency caused by the decreased uNK cells in the hypoplastic metrial gland and/or the preeclampsia caused by defective development of spiral arteries.
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