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Differential Localization of Decidual Stathmin During Pregnancy in Rats
Placenta (2004), 25, 449–455 doi:10.1016/j.placenta.2003.10.007
Differential Localization of Decidual Stathmin During Pregnancy in Rats M. Yoshie, K. Tamura* and H. Kogo Department of Pharmacology, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan Paper accepted 14 October 2003
The present study was undertaken to determine the precise localization of stathmin, a protein associated with microtubule dynamics, during decidualization in rat uterus and to compare it with that of cyclin D3. Immunohistochemical analysis revealed that stathmin is exclusively localized in decidual cells, especially in the primary decidual zone surrounding the embryo, on days 7 and 9 of pregnancy. The intensity of staining was much higher on day 9 than day 7. On day 14, when the endometrial stromal cells had completely differentiated into decidual cells, the staining of decidual cells was faint. Cyclin D3 was expressed in decidual cells of the secondary but not the primary decidual zone on days 7 and 9. On day 14, cyclin D3 levels were low in decidua. Proliferating cell nuclear antigen (PCNA) was broadly detected in the uterus on days 7 and 9, and in the placenta and fetus on day 14. In an artificial decidualization model, cyclin D3 expression was stimulated as deciduoma was formed after an artificial stimulus. Stathmin mRNA levels also increased within 24 h and peaked at 48 h. The specific spatio-temporal uterine expression of stathmin and cyclin D3 suggest that they have a specific role in decidualization in rats. Placenta (2004), 25, 449–455 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Embryo implantation and the subsequent extensive endometrial stromal cell proliferation and differentiation (decidualization) are essential processes for the establishment of pregnancy in mammals. The decidua and the decidual cells of which it is composed are thought to play an important role in providing nutrition to the developing embryo, protecting the embryo from the immunologic responses of the mother, and regulating trophoblast invasion into the uterine stroma [1]. In rodents, decidualization occurs in response to either blastocyst implantation or artificial stimuli, such as the intrauterine infusion of oil in pseudopregnant animals during the receptive period or preparation of the uterus by exogenous ovarian steroid hormones. The stromal cells in decidual tissue proliferate, increase in size and establish tight junctional complexes with their neighbors. The decidualization response is prevented by treatment with antiprogestins or a neutralizing antibody that blocks the actions of progesterone, demonstrating the need for progesterone in decidualization [2,3]. Further, knockout mice lacking two types of progesterone receptor (PR), PR-A and PR-B, are defective in decidualization [4]. A recent report showing that estrogen receptor knockout mice are normal in terms of decidual response supports the important role of progesterone, not estrogen, in decidualization [5]. *
To whom correspondence should be addressed. Tel.: +81-426-764536; Fax: +81-426-76-4529; E-mail: [email protected] 0143-4004/$–see front matter
Numerous factors that are important for decidualization have been isolated and investigated. For example, a member of the homeobox gene family, Hoxa-10, is important for implantation and decidualization, and these responses are defective in mice lacking the Hoxa-10 gene [6,7]. Das et al. [8] reported that cyclin D3, which regulates cell proliferation and G1/S phase transition in the cell cycle, is highly expressed in decidual cells in mice. The expression of cyclin D3 in response to a decidualization stimulus is aberrant in the uteri of Hoxa-10 knockout mice. These results suggest that cyclin D3 may also be important for decidualization in mice. We previously found that uterine stathmin expression is up-regulated at the site of embryo implantation on day 6 of pregnancy in rats [9]. Stathmin, also referred to as Op18 [10], is a ubiquitous cytosolic 19 kDa phosphoprotein that is highly expressed in leukemia and solid tumours, including breast and ovarian cancers [11–13]. This protein is associated with microtubule dynamics during cell cycle progression, especially the promotion of microtubule disassembly in vitro and in vivo [14]. Stathmin levels are shown to be regulated by E2F which is a transcriptional regulator that causes cell cycle progression from G1 to S phases [15]. Inhibition of stathmin expression results in growth arrest and accumulation of cells in G2/M phases of the cell cycles [16]. Cyclin D promotes transcriptional regulation of E2F by releasing active E2F from the complex of retinoblastoma (RB) and inactive E2F [17]. Because both cyclin D and stathmin are crucial mitotic regulators, we analysed the localization and expression of 2003 Elsevier Ltd. All rights reserved.
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stathmin and cyclin D3 from the initiation to the termination of decidualization in the present study. MATERIALS AND METHODS Animals and experimental schedules Animals were maintained in the animal care facility at Tokyo University of Pharmacy and Life Science according to the institutional guidelines for the care of experimental animals. Adult female rats (8 weeks old) of the Wistar–Imamichi strain (Imamichi Institute for Animal Reproduction, Ibaraki, Japan) were mated with fertile males (10 weeks old) of the same strain. The morning of finding sperm was designated day 1 of pregnancy. To induce pseudopregnancy, female rats were mated with vasectomized males of the same strain. On day 5 of pseudopregnancy, when the uteri were optimally sensitized for a deciduogenic stimulus, 100 µl of sesame oil was infused into the lumen of one uterine horn to induce artificial decidualization. The contralateral uterine horn, which was not infused with the oil, served as a control. At 0–120 h after the intraluminal oil infusion, the rats were killed and the uterine horns were isolated.
Immunostaining for stathmin, cyclin D3, and PCNA After isolation, the pregnant uteri were immediately fixed in 4 per cent paraformaldehyde dissolved in PBS, dehydrated, and embedded in paraffin. Sections cut at 4 µm were placed on glass slides coated with poly--lysine (Matunami, Tokyo, Japan). Uterine sections were deparaffinized in xylene and then rehydrated. Sections for proliferating cell nuclear antigen (PCNA) immunostaining were autoclaved in 10 m citrate buffer (pH 6.0) to induce epitope retrieval. To quench endogenous peroxidase, sections were incubated in 0.3 per cent H2O2 in methanol. Sections were blocked in 10 per cent normal goat serum in PBS, followed by incubation with anti-stathmin serum (1 : 100 dilution) (kindly donated by Dr. A. Sobel of the Institut du Fer a Moulin, Paris, France), a monoclonal anticyclin D3 antibody (50 µg/ml) (Sigma, St Louis, MO, USA), or a monoclonal anti-PCNA antibody (1 : 100) (DAKO, Glostrup, Denmark) in PBS containing 2 per cent normal goat serum. Slides were washed with PBS and incubated with Histofine Simple Stain Rat MAX-PO (R) (Nichirei, Tokyo, Japan) and then developed with Histofine Simple Stain DAB solution (Nichirei). After the immunostaining, the slides were counterstained with methyl green.
Western blot analysis Uterine tissues were homogenized in cold homogenization buffer (50 m Tris–HCl [pH 7.5] containing 150 m NaCl, 10 m EDTA, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 per
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cent Tween-20, 0.1 per cent [v/v] -mercaptoethanol, and 0.1 m PMSF). The homogenates were centrifuged to remove insoluble materials. The supernatants (30 µg of protein) were subjected to 15–25 per cent gradient SDS-polyacrylamide gel electrophoresis and then electrotransferred on to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Western blot analysis was performed as previously described [9]. After the detection of stathmin, the same membrane was incubated in stripping solution (75.3 m Tris–HCl [pH 6.7] containing 2 per cent SDS and 0.007 per cent [v/v] -mercaptoethanol) and re-probed with the anti-cyclin D3 antibody (20 µg/ml). The same analysis was repeated at least twice and the representative data are shown (Figures 2, 4 and 5).
RT–PCR analysis Uterine tissues were homogenized in EASYPrep RNA (TaKaRa, Siga, Japan) to extract total RNA. Isolation of poly (A)+ RNA from total RNA was performed using the QuickPrep Micro mRNA Purification Kit (Amersham Biosciences, UK Ltd, Buckinghamshire, UK) according to the manufacturer’s instructions. The poly (A) + RNA (0.2 µg) was reversetranscribed using a One Step RNA kit (AMV) (TaKaRa). Oligonucleotide primers specific to rat stathmin (5#GTGCGGAAGAACA AAGAATCC-3# and 5#-GAATTG GGATCGCAAAGTGA-3#) were used for PCR amplification. The PCR conditions were as follows: 50 (C for 60 min and 94 (C for 2 min, followed by 21 cycles of 95 (C for 1 min, 55 (C for 45 sec, and 72 (C for 2 min, and a 5 min extension at 72 (C on the final cycle. As an internal control, specific primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (5#-TGAAGGTCG GGTGTCAACGGATTTG GC-3# and 5#-CATGTAGGCCATGAGGTCCACCAC-3#) were also used. Amplified PCR products were separated on a 1.2 per cent agarose gel containing 0.45 µg/ml ethidium bromide. The bands were analysed using NIH Image, and each value was normalized against that of G3PDH.
RESULTS Immunolocalization of stathmin and cyclin D3 in the uterus, placenta, and fetus during the process of decidualization The distribution and expression level of stathmin in the uterus, placenta, and fetus during the process of decidualization was examined using an immunohistochemical analysis. On day 7 of pregnancy, when implantation was completed and active decidualization was underway, high levels of stathmin signals were detected exclusively at the primary decidual zone (pdz), closely surrounding the implanting embryo (Figure 1A). The signals were stronger in the decidual zone on day 9 than on day 7 (Figure 1B). On day 14, when the endometrial
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Figure 1. Immunostaining of uterine stathmin (A, B and C), cyclin D3 (D, E and F), and proliferating cell nuclear antigen (PCNA) (G, H and I) in decidual and placental cells in rats. Longitudinal sections on Day 7 (A, D and G) and Day 9 (B, E and H), and cross sections on Day 14 (C, F and I) are shown. Original magnification: 25 (A, D and G), 20 (B, C, E, F, H and I). Bar represents 500 µm. ge: glandular epithelium, le: luminal epithelium, myo: myometrium, pdz: primary decidual zone, sdz: secondary decidual zone, fe: fetus, am: amnion, pl: placenta, tg: trophoblast giant cells, dc: decidual cells.
stromal cells had completely differentiated into decidual cells (dc), the stathmin staining in decidual zone was faint but the staining was also seen in the placenta (pl) and fetus (fe) (Figure 1C). Intense immunostaining for cyclin D3 was mainly detected in decidual cells at the secondary decidual zone (sdz) on day 7, and was not detected in the primary decidual zone (Figure 1D). The intensity of cyclin D3 expression was further increased in the secondary decidual zone on day 9 (Figure 1E). On day 14, the signals of cyclin D3 were low in decidua but high in the area of the placenta close to decidual tissues (Figure 1F). To examine the association of stathmin or cyclin D3 with stromal cell proliferation, we compared the distribution of PCNA with that of stathmin or cyclin D3. The signals for PCNA were detected in all areas of uterine sections on days 7 (Figure 1G) and 9 (Figure 1H), although decidual cells were stained much more strongly than other cell types. On day 14, PCNA was widely localized in the placenta and fetus. Intense
signals were obtained in the spinal cord (sc) and the umbilical cord (uc). Staining was weaker in endometrial tissues than in placenta (Figure 1I).
Expression patterns of stathmin and cyclin D3 in the uterus during pregnancy Western blot analysis was carried out to examine the expression patterns of stathmin and cyclin D3 in the uterus during the process of decidualization and placentation, and to confirm semi-quantitatively the signal intensity of the immunohistochemical data in Figure 1. As previously reported [9], stathmin was highly expressed during days 6 to 12 and decreased during days 14 to 16 (Figure 2). A similar pattern of cyclin D3 expression was detected. The expression of cyclin D3 was low on day 1, but gradually increased during days 3 to 6.
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Figure 2. Changes in uterine stathmin and cyclin D3 expression during pregnancy in rats. Each pregnant uterine sample, without the embryo and placenta, was subjected to Western blot analysis. Stathmin protein expression was detected using a polyclonal anti-stathmin antibody (upper panel). After the immunodetection of stathmin, the same membrane was re-probed with an anti-cyclin D3 antibody (lower panel). Each reaction was performed using homogenate pooled from 2 to 3 animals.
Figure 3. Effect of artificial decidualization on stathmin mRNA expression in the pseudopregnant rats. Uterine stathmin levels were measured 0–120 h after sesame oil infusion into one uterine horn on Day 5 of pseudopregnancy [oil (+)]. As a control, the intact horn was subjected to the same analysis [oil ()]. A: Stathmin mRNA levels were detected by RT–PCR analysis. B: Quantitation of stathmin mRNA levels was conducted using NIH Image and the values obtained were normalized against GAPDH mRNA levels. The levels are expressed as a ratio to the density of the band in the oil () lane at 0 h. Each reaction was performed using RNA sample pooled from 2 to 3 animals.
Remarkable up-regulation of cyclin D3 levels was detected on days 7 to 10, and the levels then decreased slowly between days 10 and 16 until they reached basal levels.
Changes in uterine expression of stathmin and cyclin D3 in an artificial decidualization model In addition to the analysis of stathmin expression at the protein level, we used RT–PCR analysis to determine changes in uterine stathmin mRNA expression during artificial decidualization (Figure 3). There were no differences in stathmin
mRNA levels between the intact horn and the oil-infused horn at 0 h. The expression of stathmin dramatically increased within 24 h, peaked at 48 h, and then decreased between 72 h and 120 h in the oil-infused horn. The relationship between the uterine expressions of cyclin D3 and decidualization was examined in this model by Western blot (Figure 4). Cyclin D3 levels increased in the oil-infused uterine horn within 24 h, peaked at 72 h, and decreased slightly at 96 h and 120 h. This up-regulation of cyclin D3 was concomitant with the formation of the deciduoma; cyclin D3 levels in the intact horn were low at all times. Thus, the pattern of cyclin D3 expression was basically the same as that of stathmin.
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Figure 4. Effect of artificial decidualization on cyclin D3 expression in pseudopregnant rats. Uterine cyclin D3 levels were measured 0–120 h after sesame oil infusion into one uterine horn on Day 5 of pseudopregnancy [oil (+)] by Western blot analysis. The intact horn [oil ()] served as a control. Each reaction was performed using homogenate pooled from 2 to 3 animals.
Figure 5. Tissue distribution of stathmin protein and uterine expression of the protein during the oestrous cycle in rats. A: Tissue samples pooled from 2 to 3 adult rats were subjected to Western blot analysis. B: Uterine samples obtained at prooestrus (P), oestrus (E), and dioestrus (dioestrus-1: D1, dioestrus-2: D2) were also subjected to Western blot analysis. Each blot represents an individual animal (n=3).
Comparison of stathmin protein levels in various tissues and in non-pregnant uteri during the oestrous cycle We also examined the distribution of stathmin protein in various rat tissues (Figure 5A). Stathmin protein was abundant in brain, testis, and ovary. Distinct expression was also found in lung and day 6 pregnant uteri. Low or no stathmin expression was seen in heart, stomach, liver, kidney, spleen, jejunum, pancreas, and adrenal gland. We then compared stathmin levels in the uterus throughout the oestrous cycle (Figure 5B). No significant changes in the levels were detected due to the stage of the oestrous cycle (prooestrus, oestrus, and dioestrus).
DISCUSSION We have previously demonstrated that uterine stathmin levels are up-regulated during embryo implantation and decidualization in rats [9]. Previous results have shown localization of its mRNA and protein in the primary decidual zone at day 7 of pregnancy. However, the precise localization of uterine stathmin expression from the initiation to termination of decidual-
ization was still unknown. The main contribution of the present study, therefore, is the understanding of the spatiotemporal expression of uterine stathmin from the initiation of decidualization until placentation, as well as that of cyclin D3, which is reported to be associated with decidualization in mice [8,18]. Both stathmin and cyclin D3 expression increased in the decidual zone during the early phase of decidualization, and decreased when the stromal cells terminally differentiated into decidual cells. Although the pattern of uterine stathmin expression analysed by Western blot was similar to that of cyclin D3 during the process of decidualization, the localization of stathmin was different from that of cyclin D3 in decidual cells. The major site of stathmin expression was the primary decidual zone, whereas cyclin D3 expression was localized to the secondary decidual zone. During the formation of the decidua, PCNA, a proliferating cell marker, was localized mainly in decidual cells, overlapping the localization of stathmin and cyclin D3, but was also widely expressed in uterine cells. Stathmin is highly expressed in various tumours, and it is expressed at high levels in proliferating cells than in non-proliferating cells [19]. These differences of localization among PCNA, stathmin, and cyclin D3 suggest that the expression of stathmin and cyclin D3 is not merely associated with cell proliferation.
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In an artificial decidualization model, transient upregulation of stathmin mRNA expression was accompanied by the formation of the deciduoma. This result is consistent with a previous observation showing that stathmin protein increases 24 h after a decidual stimulus [9]. In the same model, protein levels of cyclin D3 were also up-regulated with the initiation of decidualization and gradually decreased later. Das et al. [8] reported that cyclin D3 mRNA accumulated in the decidual bed of artificially induced deciduoma in mice. Thus, the up-regulation of cyclin D3 in deciduoma is a characteristic common to rodents. Using immunohistochemistry, we observed high levels of placental stathmin expression in rats, in concordance with a report demonstrating the presence of stathmin mRNA in mouse placenta [20]. In contrast, we detected cyclin D3 only close to the decidua. However, the precise roles of stathmin and cyclin D3 in the placenta are not yet known. The intense and restricted stathmin and cyclin D3 signals in the placenta suggest that these proteins are also involved in placental function. Intense staining of stathmin in fetal tissues is consistent with a previous report showing abundant expression of stathmin in the nervous system [21] and neonatal tissues [22]. We have previously reported that the mRNA levels of stathmin during dioestrus and oestrus were not different [9]. The lack of significant changes in stathmin protein during the oestrous cycle indicates that stathmin protein levels are not simply regulated by ovarian steroids. Furthermore, the levels of uterine stathmin expression during early pregnancy are relatively high compared with other organs. These data imply that dynamic up-regulation of stathmin expression depends upon embryo implantation and decidualization. It has been suggested that stathmin acts to integrate the activation of diverse intracellular signaling pathways involved in the control of cell proliferation and differentiation [23]. For example, antisense depletion of stathmin prevents nerve growth factor-stimulated differentiation of PC12 cells into sympathetic-like neurons [24]. Stathmin is a critical regulator of microtubule dynamics during cell cycle progression, because the inhibition of stathmin results in growth arrest and accumulation of cells in G2/M phases of cell cycle [16]. The stathmin promoter contains three E2F-binding sites, and a recent study indicates that stathmin expression is regulated by E2F [15] whose activity is inhibited by its binding to nonphosphorylated RB protein. On the other hand, the main function of cyclin D is thought to promote transcriptional regulation of E2F by releasing active E2F from the inactive form of complex of RB [17]. Thus, both cyclin D3 and stathmin are critical mitotic regulators and stathmin expression might be regulated by cyclin D3. Stathmin undergoes serine phosphorylation in response to a diverse group of extracellular factors. The level of phosphorylated stathmin increases as cells enter the G2/M phases of the cell cycle [16], and its phosphorylation on multiple sites is required for orderly progression through the cell cycle [25]. The unphosphorylated form of stathmin promotes depolymerization of microtubules and phosphorylation of stathmin eliminates the ability of
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microtubules to depolymerize, allowing the mitotic spindle to form [26]. However, stathmin levels increase during hepatectomic regeneration [27] without detectable changes in its phosphorylation state. The phosphorylation-dependent activity of stathmin might play an important role in the control of the dynamic instability of microtubules during the decidualization of endometrial cells. The analyses of phosphorylated form of stathmin will be required to confirm this question. During decidualization, the stromal cells show large monoor bi-nucleated cells consisting of DNA with multiples of the haploid complement. The expression of cyclin D3 during stromal cell decidualization has been shown to be related to polyploidization caused by an unusual cell cycle called an endocycle [18]. During an endocycle (endomitosis), nuclear replication occurs repeatedly without cell division [28]. Furthermore, the coordinate expression and functional interaction of cyclin D3 with cyclin-dependent kinase (cdk) 4 are important for stromal cell proliferation, and the interaction of cyclin D3 with p21, an inhibitor of cdk, and cdk6 is important for development of polyploidy during decidualization [18]. In mammals, megakaryocytes, a blood cell type specialized to produce platelets, enter this unusual cell cycle during differentiation and become polyploid [29]. There are some reports suggesting that stathmin is involved in this megakaryocyte polyploidization as well. Stathmin may reduce megakaryocyte survival during the early stage of differentiation, but enhance polyploidization efficiency [30]. Furthermore, stathmin is necessary for the proliferation and differentiation of early megakaryoblasts and for polyploidization via its suppression in the later stages of megakaryocytic maturation [31]. These reports seem to support the possibility that stathmin expression in decidual cells is important for stromal cell polyploidization during decidualization. Remarkable up-regulation of stathmin expression probably reflects the initial stromal cell proliferation during decidualization, and the maintenance of the high expression of stathmin and its down-regulation in decidual cells during the later phase of decidualization might promote stromal cell polyploidization. However, there is no evidence to indicate a direct relationship between expressions of stathmin and cyclin D3 in this process. The present study described the localization of uterine stathmin and cyclin D3 during the process of decidualization, and indicated that the spatio-temporal expression of stathmin and cyclin D3 in decidual cells is tightly regulated during decidualization in rats. REFERENCES [1] Dey SK. Implantation. In: Adashi EY, Rock JA, Rosenwaks Z, editors. Reproductive endocrinology, surgery and technology. New York: Lippincott-Raven Publishers; 1996, p. 421–31. [2] Crombie DL, Mukherjee R, McDonnell DP, Hayes JS, Wang MW. Creatine kinase activity as an indicator of unopposed estrogen action in the mouse uterus associated with anti-progesterone treatment. J Steroid Biochem Mol Biol 1994;49:123–9. [3] Zhang Z, Funk C, Glasser SR, Mulholland J. Progesterone regulation of heparin-binding epidermal growth factor-like growth factor gene expression during sensitization and decidualization in the rat uterus: effects of antiprogestin, ZK98,299. Endocrinology 1994;135:1256–63.
Yoshie et al.: Uterine Stathmin and Rat Decidualization [4] Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery CA Jr et al. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 1995;9:2266–78. [5] Paria BC, Tan J, Lubahn DB, Dey SK, Das SK. Uterine decidual response occurs in estrogen receptor deficient mice. Endocrinology 1999;140:2704–10. [6] Benson GV, Lim H, Paria BC, Satonaka I, Dey SK, Maas RL. Mechanisms of reduced fertility in Hoxa-10 mutant mice: Uterine homeosis and loss of maternal Hoxa-10 expression. Development 1996; 122:2687–96. [7] Lim H, Ma L, Ma WG, Maas RL, Dey SK. Hoxa-10 regulates uterine stromal cell responsiveness to progesterone during implantation and decidualization in the mouse. Mol Endocrinol 1999;13:1005–17. [8] Das SK, Lim H, Paria BC, Dey SK. Cyclin D3 in the mouse uterus is associated with the decidualization process during early pregnancy. J Mol Endocrinol 1999;22:91–101. [9] Tamura K, Hara T, Yoshie M, Irie S, Sobel A, Kogo H. Enhanced expression of uterine stathmin during the process of implantation and decidualization in rats. Endocrinology 2003;144:1464–73. [10] Hailat N, Strahler JR, Melhem R, Zhu XX, Brodeur G, Seeger RC et al. N-myc gene amplification in neuroblastoma is associated with altered phosphorylation of a proliferation related polypeptide (Op 18). Oncogene 1990;5:1615–8. [11] Hanash SM, Strahler JR, Kuick R, Chu EH, Nichols D. Identification of a polypeptide associated with the malignant phenotype in acute leukemia. J Biol Chem 1988;263:12813–5. [12] Curmi PA, Nogues C, Lachkar S, Carelle N, Gonthier MP, Sobel A et al. Overexpression of stathmin in breast carcinomas points out to highly proliferative tumors. Br J Cancer 2000;82:142–50. [13] Price DK, Ball JR, Bahrani-Mostafavi Z, Vachris JC, Kaufman JS, Naumann RW et al. The phosphoprotein Op18/stathmin is differentially expressed in ovarian cancer. Cancer Invest 2000;18:722–30. [14] Belmont LD, Mitchison TJ. Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell 1996;84:623–31. [15] Ishida S, Huang E, Zuzan H, Spang R, Leone G, West M et al. Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol 2001;21:4684–99. [16] Luo XN, Mookerjee B, Ferrari A, Mistry S, Atweh GF. Regulation of phosphoprotein p18 in leukemic cells. Cell cycle regulated phosphorylation by p34cdc2 kinase. J Biol Chem 1994;269:10312–8. [17] Bartek J, Bartkova J, Lukas J. The retinoblastoma protein pathway and the restriction point. Curr Opin Cell Biol 1996;8:805–14. [18] Tan J, Raja S, Davis MK, Tawfik O, Dey SK, Das SK. Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the
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Differential Localization of Decidual Stathmin During Pregnancy in Rats M. Yoshie, K. Tamura* and H. Kogo Department of Pharmacology, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan Paper accepted 14 October 2003
The present study was undertaken to determine the precise localization of stathmin, a protein associated with microtubule dynamics, during decidualization in rat uterus and to compare it with that of cyclin D3. Immunohistochemical analysis revealed that stathmin is exclusively localized in decidual cells, especially in the primary decidual zone surrounding the embryo, on days 7 and 9 of pregnancy. The intensity of staining was much higher on day 9 than day 7. On day 14, when the endometrial stromal cells had completely differentiated into decidual cells, the staining of decidual cells was faint. Cyclin D3 was expressed in decidual cells of the secondary but not the primary decidual zone on days 7 and 9. On day 14, cyclin D3 levels were low in decidua. Proliferating cell nuclear antigen (PCNA) was broadly detected in the uterus on days 7 and 9, and in the placenta and fetus on day 14. In an artificial decidualization model, cyclin D3 expression was stimulated as deciduoma was formed after an artificial stimulus. Stathmin mRNA levels also increased within 24 h and peaked at 48 h. The specific spatio-temporal uterine expression of stathmin and cyclin D3 suggest that they have a specific role in decidualization in rats. Placenta (2004), 25, 449–455 2003 Elsevier Ltd. All rights reserved.
INTRODUCTION Embryo implantation and the subsequent extensive endometrial stromal cell proliferation and differentiation (decidualization) are essential processes for the establishment of pregnancy in mammals. The decidua and the decidual cells of which it is composed are thought to play an important role in providing nutrition to the developing embryo, protecting the embryo from the immunologic responses of the mother, and regulating trophoblast invasion into the uterine stroma [1]. In rodents, decidualization occurs in response to either blastocyst implantation or artificial stimuli, such as the intrauterine infusion of oil in pseudopregnant animals during the receptive period or preparation of the uterus by exogenous ovarian steroid hormones. The stromal cells in decidual tissue proliferate, increase in size and establish tight junctional complexes with their neighbors. The decidualization response is prevented by treatment with antiprogestins or a neutralizing antibody that blocks the actions of progesterone, demonstrating the need for progesterone in decidualization [2,3]. Further, knockout mice lacking two types of progesterone receptor (PR), PR-A and PR-B, are defective in decidualization [4]. A recent report showing that estrogen receptor knockout mice are normal in terms of decidual response supports the important role of progesterone, not estrogen, in decidualization [5]. *
To whom correspondence should be addressed. Tel.: +81-426-764536; Fax: +81-426-76-4529; E-mail: [email protected] 0143-4004/$–see front matter
Numerous factors that are important for decidualization have been isolated and investigated. For example, a member of the homeobox gene family, Hoxa-10, is important for implantation and decidualization, and these responses are defective in mice lacking the Hoxa-10 gene [6,7]. Das et al. [8] reported that cyclin D3, which regulates cell proliferation and G1/S phase transition in the cell cycle, is highly expressed in decidual cells in mice. The expression of cyclin D3 in response to a decidualization stimulus is aberrant in the uteri of Hoxa-10 knockout mice. These results suggest that cyclin D3 may also be important for decidualization in mice. We previously found that uterine stathmin expression is up-regulated at the site of embryo implantation on day 6 of pregnancy in rats [9]. Stathmin, also referred to as Op18 [10], is a ubiquitous cytosolic 19 kDa phosphoprotein that is highly expressed in leukemia and solid tumours, including breast and ovarian cancers [11–13]. This protein is associated with microtubule dynamics during cell cycle progression, especially the promotion of microtubule disassembly in vitro and in vivo [14]. Stathmin levels are shown to be regulated by E2F which is a transcriptional regulator that causes cell cycle progression from G1 to S phases [15]. Inhibition of stathmin expression results in growth arrest and accumulation of cells in G2/M phases of the cell cycles [16]. Cyclin D promotes transcriptional regulation of E2F by releasing active E2F from the complex of retinoblastoma (RB) and inactive E2F [17]. Because both cyclin D and stathmin are crucial mitotic regulators, we analysed the localization and expression of 2003 Elsevier Ltd. All rights reserved.
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stathmin and cyclin D3 from the initiation to the termination of decidualization in the present study. MATERIALS AND METHODS Animals and experimental schedules Animals were maintained in the animal care facility at Tokyo University of Pharmacy and Life Science according to the institutional guidelines for the care of experimental animals. Adult female rats (8 weeks old) of the Wistar–Imamichi strain (Imamichi Institute for Animal Reproduction, Ibaraki, Japan) were mated with fertile males (10 weeks old) of the same strain. The morning of finding sperm was designated day 1 of pregnancy. To induce pseudopregnancy, female rats were mated with vasectomized males of the same strain. On day 5 of pseudopregnancy, when the uteri were optimally sensitized for a deciduogenic stimulus, 100 µl of sesame oil was infused into the lumen of one uterine horn to induce artificial decidualization. The contralateral uterine horn, which was not infused with the oil, served as a control. At 0–120 h after the intraluminal oil infusion, the rats were killed and the uterine horns were isolated.
Immunostaining for stathmin, cyclin D3, and PCNA After isolation, the pregnant uteri were immediately fixed in 4 per cent paraformaldehyde dissolved in PBS, dehydrated, and embedded in paraffin. Sections cut at 4 µm were placed on glass slides coated with poly--lysine (Matunami, Tokyo, Japan). Uterine sections were deparaffinized in xylene and then rehydrated. Sections for proliferating cell nuclear antigen (PCNA) immunostaining were autoclaved in 10 m citrate buffer (pH 6.0) to induce epitope retrieval. To quench endogenous peroxidase, sections were incubated in 0.3 per cent H2O2 in methanol. Sections were blocked in 10 per cent normal goat serum in PBS, followed by incubation with anti-stathmin serum (1 : 100 dilution) (kindly donated by Dr. A. Sobel of the Institut du Fer a Moulin, Paris, France), a monoclonal anticyclin D3 antibody (50 µg/ml) (Sigma, St Louis, MO, USA), or a monoclonal anti-PCNA antibody (1 : 100) (DAKO, Glostrup, Denmark) in PBS containing 2 per cent normal goat serum. Slides were washed with PBS and incubated with Histofine Simple Stain Rat MAX-PO (R) (Nichirei, Tokyo, Japan) and then developed with Histofine Simple Stain DAB solution (Nichirei). After the immunostaining, the slides were counterstained with methyl green.
Western blot analysis Uterine tissues were homogenized in cold homogenization buffer (50 m Tris–HCl [pH 7.5] containing 150 m NaCl, 10 m EDTA, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 per
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cent Tween-20, 0.1 per cent [v/v] -mercaptoethanol, and 0.1 m PMSF). The homogenates were centrifuged to remove insoluble materials. The supernatants (30 µg of protein) were subjected to 15–25 per cent gradient SDS-polyacrylamide gel electrophoresis and then electrotransferred on to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Western blot analysis was performed as previously described [9]. After the detection of stathmin, the same membrane was incubated in stripping solution (75.3 m Tris–HCl [pH 6.7] containing 2 per cent SDS and 0.007 per cent [v/v] -mercaptoethanol) and re-probed with the anti-cyclin D3 antibody (20 µg/ml). The same analysis was repeated at least twice and the representative data are shown (Figures 2, 4 and 5).
RT–PCR analysis Uterine tissues were homogenized in EASYPrep RNA (TaKaRa, Siga, Japan) to extract total RNA. Isolation of poly (A)+ RNA from total RNA was performed using the QuickPrep Micro mRNA Purification Kit (Amersham Biosciences, UK Ltd, Buckinghamshire, UK) according to the manufacturer’s instructions. The poly (A) + RNA (0.2 µg) was reversetranscribed using a One Step RNA kit (AMV) (TaKaRa). Oligonucleotide primers specific to rat stathmin (5#GTGCGGAAGAACA AAGAATCC-3# and 5#-GAATTG GGATCGCAAAGTGA-3#) were used for PCR amplification. The PCR conditions were as follows: 50 (C for 60 min and 94 (C for 2 min, followed by 21 cycles of 95 (C for 1 min, 55 (C for 45 sec, and 72 (C for 2 min, and a 5 min extension at 72 (C on the final cycle. As an internal control, specific primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (5#-TGAAGGTCG GGTGTCAACGGATTTG GC-3# and 5#-CATGTAGGCCATGAGGTCCACCAC-3#) were also used. Amplified PCR products were separated on a 1.2 per cent agarose gel containing 0.45 µg/ml ethidium bromide. The bands were analysed using NIH Image, and each value was normalized against that of G3PDH.
RESULTS Immunolocalization of stathmin and cyclin D3 in the uterus, placenta, and fetus during the process of decidualization The distribution and expression level of stathmin in the uterus, placenta, and fetus during the process of decidualization was examined using an immunohistochemical analysis. On day 7 of pregnancy, when implantation was completed and active decidualization was underway, high levels of stathmin signals were detected exclusively at the primary decidual zone (pdz), closely surrounding the implanting embryo (Figure 1A). The signals were stronger in the decidual zone on day 9 than on day 7 (Figure 1B). On day 14, when the endometrial
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Figure 1. Immunostaining of uterine stathmin (A, B and C), cyclin D3 (D, E and F), and proliferating cell nuclear antigen (PCNA) (G, H and I) in decidual and placental cells in rats. Longitudinal sections on Day 7 (A, D and G) and Day 9 (B, E and H), and cross sections on Day 14 (C, F and I) are shown. Original magnification: 25 (A, D and G), 20 (B, C, E, F, H and I). Bar represents 500 µm. ge: glandular epithelium, le: luminal epithelium, myo: myometrium, pdz: primary decidual zone, sdz: secondary decidual zone, fe: fetus, am: amnion, pl: placenta, tg: trophoblast giant cells, dc: decidual cells.
stromal cells had completely differentiated into decidual cells (dc), the stathmin staining in decidual zone was faint but the staining was also seen in the placenta (pl) and fetus (fe) (Figure 1C). Intense immunostaining for cyclin D3 was mainly detected in decidual cells at the secondary decidual zone (sdz) on day 7, and was not detected in the primary decidual zone (Figure 1D). The intensity of cyclin D3 expression was further increased in the secondary decidual zone on day 9 (Figure 1E). On day 14, the signals of cyclin D3 were low in decidua but high in the area of the placenta close to decidual tissues (Figure 1F). To examine the association of stathmin or cyclin D3 with stromal cell proliferation, we compared the distribution of PCNA with that of stathmin or cyclin D3. The signals for PCNA were detected in all areas of uterine sections on days 7 (Figure 1G) and 9 (Figure 1H), although decidual cells were stained much more strongly than other cell types. On day 14, PCNA was widely localized in the placenta and fetus. Intense
signals were obtained in the spinal cord (sc) and the umbilical cord (uc). Staining was weaker in endometrial tissues than in placenta (Figure 1I).
Expression patterns of stathmin and cyclin D3 in the uterus during pregnancy Western blot analysis was carried out to examine the expression patterns of stathmin and cyclin D3 in the uterus during the process of decidualization and placentation, and to confirm semi-quantitatively the signal intensity of the immunohistochemical data in Figure 1. As previously reported [9], stathmin was highly expressed during days 6 to 12 and decreased during days 14 to 16 (Figure 2). A similar pattern of cyclin D3 expression was detected. The expression of cyclin D3 was low on day 1, but gradually increased during days 3 to 6.
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Figure 2. Changes in uterine stathmin and cyclin D3 expression during pregnancy in rats. Each pregnant uterine sample, without the embryo and placenta, was subjected to Western blot analysis. Stathmin protein expression was detected using a polyclonal anti-stathmin antibody (upper panel). After the immunodetection of stathmin, the same membrane was re-probed with an anti-cyclin D3 antibody (lower panel). Each reaction was performed using homogenate pooled from 2 to 3 animals.
Figure 3. Effect of artificial decidualization on stathmin mRNA expression in the pseudopregnant rats. Uterine stathmin levels were measured 0–120 h after sesame oil infusion into one uterine horn on Day 5 of pseudopregnancy [oil (+)]. As a control, the intact horn was subjected to the same analysis [oil ()]. A: Stathmin mRNA levels were detected by RT–PCR analysis. B: Quantitation of stathmin mRNA levels was conducted using NIH Image and the values obtained were normalized against GAPDH mRNA levels. The levels are expressed as a ratio to the density of the band in the oil () lane at 0 h. Each reaction was performed using RNA sample pooled from 2 to 3 animals.
Remarkable up-regulation of cyclin D3 levels was detected on days 7 to 10, and the levels then decreased slowly between days 10 and 16 until they reached basal levels.
Changes in uterine expression of stathmin and cyclin D3 in an artificial decidualization model In addition to the analysis of stathmin expression at the protein level, we used RT–PCR analysis to determine changes in uterine stathmin mRNA expression during artificial decidualization (Figure 3). There were no differences in stathmin
mRNA levels between the intact horn and the oil-infused horn at 0 h. The expression of stathmin dramatically increased within 24 h, peaked at 48 h, and then decreased between 72 h and 120 h in the oil-infused horn. The relationship between the uterine expressions of cyclin D3 and decidualization was examined in this model by Western blot (Figure 4). Cyclin D3 levels increased in the oil-infused uterine horn within 24 h, peaked at 72 h, and decreased slightly at 96 h and 120 h. This up-regulation of cyclin D3 was concomitant with the formation of the deciduoma; cyclin D3 levels in the intact horn were low at all times. Thus, the pattern of cyclin D3 expression was basically the same as that of stathmin.
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Figure 4. Effect of artificial decidualization on cyclin D3 expression in pseudopregnant rats. Uterine cyclin D3 levels were measured 0–120 h after sesame oil infusion into one uterine horn on Day 5 of pseudopregnancy [oil (+)] by Western blot analysis. The intact horn [oil ()] served as a control. Each reaction was performed using homogenate pooled from 2 to 3 animals.
Figure 5. Tissue distribution of stathmin protein and uterine expression of the protein during the oestrous cycle in rats. A: Tissue samples pooled from 2 to 3 adult rats were subjected to Western blot analysis. B: Uterine samples obtained at prooestrus (P), oestrus (E), and dioestrus (dioestrus-1: D1, dioestrus-2: D2) were also subjected to Western blot analysis. Each blot represents an individual animal (n=3).
Comparison of stathmin protein levels in various tissues and in non-pregnant uteri during the oestrous cycle We also examined the distribution of stathmin protein in various rat tissues (Figure 5A). Stathmin protein was abundant in brain, testis, and ovary. Distinct expression was also found in lung and day 6 pregnant uteri. Low or no stathmin expression was seen in heart, stomach, liver, kidney, spleen, jejunum, pancreas, and adrenal gland. We then compared stathmin levels in the uterus throughout the oestrous cycle (Figure 5B). No significant changes in the levels were detected due to the stage of the oestrous cycle (prooestrus, oestrus, and dioestrus).
DISCUSSION We have previously demonstrated that uterine stathmin levels are up-regulated during embryo implantation and decidualization in rats [9]. Previous results have shown localization of its mRNA and protein in the primary decidual zone at day 7 of pregnancy. However, the precise localization of uterine stathmin expression from the initiation to termination of decidual-
ization was still unknown. The main contribution of the present study, therefore, is the understanding of the spatiotemporal expression of uterine stathmin from the initiation of decidualization until placentation, as well as that of cyclin D3, which is reported to be associated with decidualization in mice [8,18]. Both stathmin and cyclin D3 expression increased in the decidual zone during the early phase of decidualization, and decreased when the stromal cells terminally differentiated into decidual cells. Although the pattern of uterine stathmin expression analysed by Western blot was similar to that of cyclin D3 during the process of decidualization, the localization of stathmin was different from that of cyclin D3 in decidual cells. The major site of stathmin expression was the primary decidual zone, whereas cyclin D3 expression was localized to the secondary decidual zone. During the formation of the decidua, PCNA, a proliferating cell marker, was localized mainly in decidual cells, overlapping the localization of stathmin and cyclin D3, but was also widely expressed in uterine cells. Stathmin is highly expressed in various tumours, and it is expressed at high levels in proliferating cells than in non-proliferating cells [19]. These differences of localization among PCNA, stathmin, and cyclin D3 suggest that the expression of stathmin and cyclin D3 is not merely associated with cell proliferation.
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In an artificial decidualization model, transient upregulation of stathmin mRNA expression was accompanied by the formation of the deciduoma. This result is consistent with a previous observation showing that stathmin protein increases 24 h after a decidual stimulus [9]. In the same model, protein levels of cyclin D3 were also up-regulated with the initiation of decidualization and gradually decreased later. Das et al. [8] reported that cyclin D3 mRNA accumulated in the decidual bed of artificially induced deciduoma in mice. Thus, the up-regulation of cyclin D3 in deciduoma is a characteristic common to rodents. Using immunohistochemistry, we observed high levels of placental stathmin expression in rats, in concordance with a report demonstrating the presence of stathmin mRNA in mouse placenta [20]. In contrast, we detected cyclin D3 only close to the decidua. However, the precise roles of stathmin and cyclin D3 in the placenta are not yet known. The intense and restricted stathmin and cyclin D3 signals in the placenta suggest that these proteins are also involved in placental function. Intense staining of stathmin in fetal tissues is consistent with a previous report showing abundant expression of stathmin in the nervous system [21] and neonatal tissues [22]. We have previously reported that the mRNA levels of stathmin during dioestrus and oestrus were not different [9]. The lack of significant changes in stathmin protein during the oestrous cycle indicates that stathmin protein levels are not simply regulated by ovarian steroids. Furthermore, the levels of uterine stathmin expression during early pregnancy are relatively high compared with other organs. These data imply that dynamic up-regulation of stathmin expression depends upon embryo implantation and decidualization. It has been suggested that stathmin acts to integrate the activation of diverse intracellular signaling pathways involved in the control of cell proliferation and differentiation [23]. For example, antisense depletion of stathmin prevents nerve growth factor-stimulated differentiation of PC12 cells into sympathetic-like neurons [24]. Stathmin is a critical regulator of microtubule dynamics during cell cycle progression, because the inhibition of stathmin results in growth arrest and accumulation of cells in G2/M phases of cell cycle [16]. The stathmin promoter contains three E2F-binding sites, and a recent study indicates that stathmin expression is regulated by E2F [15] whose activity is inhibited by its binding to nonphosphorylated RB protein. On the other hand, the main function of cyclin D is thought to promote transcriptional regulation of E2F by releasing active E2F from the inactive form of complex of RB [17]. Thus, both cyclin D3 and stathmin are critical mitotic regulators and stathmin expression might be regulated by cyclin D3. Stathmin undergoes serine phosphorylation in response to a diverse group of extracellular factors. The level of phosphorylated stathmin increases as cells enter the G2/M phases of the cell cycle [16], and its phosphorylation on multiple sites is required for orderly progression through the cell cycle [25]. The unphosphorylated form of stathmin promotes depolymerization of microtubules and phosphorylation of stathmin eliminates the ability of
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microtubules to depolymerize, allowing the mitotic spindle to form [26]. However, stathmin levels increase during hepatectomic regeneration [27] without detectable changes in its phosphorylation state. The phosphorylation-dependent activity of stathmin might play an important role in the control of the dynamic instability of microtubules during the decidualization of endometrial cells. The analyses of phosphorylated form of stathmin will be required to confirm this question. During decidualization, the stromal cells show large monoor bi-nucleated cells consisting of DNA with multiples of the haploid complement. The expression of cyclin D3 during stromal cell decidualization has been shown to be related to polyploidization caused by an unusual cell cycle called an endocycle [18]. During an endocycle (endomitosis), nuclear replication occurs repeatedly without cell division [28]. Furthermore, the coordinate expression and functional interaction of cyclin D3 with cyclin-dependent kinase (cdk) 4 are important for stromal cell proliferation, and the interaction of cyclin D3 with p21, an inhibitor of cdk, and cdk6 is important for development of polyploidy during decidualization [18]. In mammals, megakaryocytes, a blood cell type specialized to produce platelets, enter this unusual cell cycle during differentiation and become polyploid [29]. There are some reports suggesting that stathmin is involved in this megakaryocyte polyploidization as well. Stathmin may reduce megakaryocyte survival during the early stage of differentiation, but enhance polyploidization efficiency [30]. Furthermore, stathmin is necessary for the proliferation and differentiation of early megakaryoblasts and for polyploidization via its suppression in the later stages of megakaryocytic maturation [31]. These reports seem to support the possibility that stathmin expression in decidual cells is important for stromal cell polyploidization during decidualization. Remarkable up-regulation of stathmin expression probably reflects the initial stromal cell proliferation during decidualization, and the maintenance of the high expression of stathmin and its down-regulation in decidual cells during the later phase of decidualization might promote stromal cell polyploidization. However, there is no evidence to indicate a direct relationship between expressions of stathmin and cyclin D3 in this process. The present study described the localization of uterine stathmin and cyclin D3 during the process of decidualization, and indicated that the spatio-temporal expression of stathmin and cyclin D3 in decidual cells is tightly regulated during decidualization in rats. REFERENCES [1] Dey SK. Implantation. In: Adashi EY, Rock JA, Rosenwaks Z, editors. Reproductive endocrinology, surgery and technology. New York: Lippincott-Raven Publishers; 1996, p. 421–31. [2] Crombie DL, Mukherjee R, McDonnell DP, Hayes JS, Wang MW. Creatine kinase activity as an indicator of unopposed estrogen action in the mouse uterus associated with anti-progesterone treatment. J Steroid Biochem Mol Biol 1994;49:123–9. [3] Zhang Z, Funk C, Glasser SR, Mulholland J. Progesterone regulation of heparin-binding epidermal growth factor-like growth factor gene expression during sensitization and decidualization in the rat uterus: effects of antiprogestin, ZK98,299. Endocrinology 1994;135:1256–63.
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