Impaired expansion of trophoblast spheroids cocultured with endometrial cells overexpressing cellular retinoic acid-binding protein 2

Impaired expansion of trophoblast spheroids cocultured with endometrial cells overexpressing cellular retinoic acid-binding protein 2

Impaired expansion of trophoblast spheroids cocultured with endometrial cells overexpressing cellular retinoic acid-binding protein 2 We previously sh...

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Impaired expansion of trophoblast spheroids cocultured with endometrial cells overexpressing cellular retinoic acid-binding protein 2 We previously showed that the cellular retinoic acid-binding protein 2 (CRABP2) gene was up-regulated during the implantation window period in the endometrium of patients with unexplained recurrent spontaneous abortion. Here, we report that trophoblast spheroids cocultured with a human endometrial cell line stably overexpressing CRABP2 are defective in expansion and exhibit apoptosis, suggesting that the altered expression level of endometrial CRABP2 is involved in abnormal endometrium–trophoblast interaction, which leads to implantation failure. (Fertil Steril 2011;95:2599–601. 2011 by American Society for Reproductive Medicine.) Key Words: Cellular retinoic acid-binding protein 2, endometrium, in vitro implantation model, trophoblast spheroid, recurrent spontaneous abortion

Recurrent spontaneous abortion (RSA) is defined as three or more clinically consecutive miscarriages during the first trimester and affects approximately 3% of women with diagnosed pregnancies (1, 2). Unexplained RSA is potentially due to alterations in endometrial components essential for implantation and continuing pregnancy. The process of implantation involves interaction between the developing embryo and the receptive maternal endometrium during the implantation window (3). This process begins with apposition, attachment, and invasion. Although the molecular mechanism of implantation is poorly understood, any defects in each step may lead to implantation failure. In fact it has been suggested that RSA is associated with limited trophoblast invasion (4–6). We previously identified endometrial genes that were differentially expressed during the implantation window in patients with unexplained RSA (7). One of the identified genes encodes cellular retinoic acid-binding protein 2 Jiae Lee, Ph.D. Jeong Su Oh, Ph.D. Chunghee Cho, Ph.D. School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, South Korea Received March 1, 2011; accepted April 18, 2011; published online May 24, 2011. J.L. has nothing to disclose. J.S.O. has nothing to disclose. C.C. has nothing to disclose. Supported by a Korea Science and Engineering Foundation Grant (2010-0028776), a Korea Research Foundation Grant (KRF-2008313-C00736), and a GIST Systems Biology Infrastructure Establishment Grant. Present addresses: Jiae Lee, Ph.D., Hajin Diagnotics, Seoul 157-033, South Korea; Jeong Su Oh, Ph.D., Department of Obstetrics, Gynecology and Reproductive Sciences, University of California-San Francisco, San Francisco, CA 94143. Reprint requests: Chunghee Cho, Ph.D., School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea (E-mail: [email protected]).

0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.04.065

(CRABP2). The CRABP2 expression was significantly higher in the endometrium of patients with unexplained RSA at the transcript and protein levels when compared with healthy fertile subjects (7). In the present study, we investigated the function of CRABP2 using an in vitro implantation assay in which human trophoblast spheroids expand on human endometrial cells. The human endometrium-derived epithelial cell line, RL95-2, is known to serve as a suitable in vitro model for receptive uterine epithelium because the cell line is highly adhesive to trophoblast-derived cells, allowing expansion of the cells (8–10). A stable cell line overexpressing CRABP2 was established using RL95-2, as previously described (7). The RL95-2 cells were maintained in a 1:1 mixture of Dulbecco’s modified Eagle medium (DMEM) and Ham’s F12 supplemented with 10% fetal bovine serum, 10 mM HEPES, 5 mg/mL of bovine insulin, and 1% penicillin/streptomycin. Full-length human CRABP2 complementary DNA (cDNA) was generated from human endometrium and cloned into pcDNA3.1-myc/his vector (Invitrogen). The CRABP2 full-length cDNA tagged with myc/his to detect recombinant protein was subcloned into the bicistronic expression vector pIRESneo3 (Clontech). The RL95-2 cells were stably transfected with pIRES-CRABP2 or pIRES vector using Fugene HD reagent (Roche Applied Science) according to the manufacturer’s protocol. At 48 hours after transfection, cells were treated with culture medium containing 1 mg/mL geneticin (GIBCO; Invitrogen cell culture), which had been predetermined to kill cells completely within 2 weeks. Cells were grown in the presence of 1 mg/mL geneticin for 3 weeks to select stably transfected cells (7). Human BeWo choriocarcinoma cells (ATCC) were used to produce trophoblast spheroids. These cells were cultured in Ham F-12K nutrient mixture supplemented with 15% fetal bovine serum. For preparation of spheroids, 10 mL of BeWo cells at a density of 2  105 cells/mL were plated on a 100-mm plastic Petri dish and incubated on a gyratory shaker at 110 rpm for 24 hours. Spheroids were selected under a microscope. Stable cells expressing CRABP2 were seeded to each well of Lab-Tek Tissue Culture

Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.


FIGURE 1 Expansion and survival of trophoblast spheroids cocultured with a stable endometrial cell line. (A) Morphological changes in trophoblast spheroids at 0, 12, and 24 hours during coculture with control cells (parental RL cell line and mock cell line) or a stable cell line expressing CRABP2. The arrows indicate the margins of the spheroid-endometrial cell interface. Bar ¼ 100 mm. (B) Fold expansion of trophoblast spheroids interacting with the endometrial cells, as described in (A). The numbers of trophoblast spheroids examined are indicated in the parentheses. Data shown are the means  SEM. *P< .05 when compared with control cells. (C) Detection of apoptotic cells in trophoblast spheroids at 24 hours after coculture with the endometrial cells. Arrows indicate apoptotic cells. Bar ¼ 100 mm. (D) Apoptotic index of trophoblast spheroids cocultured with the endometrial cells, as described in (C). The apoptotic index was calculated as the percentage of the number of positive cells in the spheroid expansion area. The numbers of trophoblast spheroids examined are indicated in the parentheses. Data shown are the means  SEM. *P< .01 compared with control cells.

Lee. Correspondence. Fertil Steril 2011.

Chamber Slides II (Nunc International) and cultured to 90% confluence. The slides were then washed twice with medium, after which spheroids were loaded onto each well. Spheroid expansion was observed at different times (1, 6, 9, 12, or 24 hours) during cultivation under an inverted microscope and photographed using a cooled charge-coupled device camera system. Fold expansion of spheroids at 6, 9, 12, and 24 hours was calculated by measuring the expanded area. After 24 hours of coculture, the slides were washed twice and subjected to TUNEL staining to detect apoptotic cells according to the ApopTag Plus (Chemicon) protocol. The statistical significance of spheroid expansion and degree of apoptosis was calculated using the Student’s t-test. A P value of less than .05 was considered to be statistically significant. In our in vitro implantation assay, parental human endometrial cells and mock cells (cells stably transfected with empty vector) were used as control cells. We found that trophoblast spheroids on parental RL95-2 cells expanded 2.48-fold at 24 hours. Spheroids on mock cells showed a similar extent of expansion at 24 hours during cocultivation (2.59-fold). In contrast, the degree of expansion of spheroids on the stable cell line overexpressing CRABP2 was significantly lower (1.92-fold) than that of spheroids on the control cells (Fig. 1A and B), revealing a link between endometrial CRABP2 and trophoblast expansion. To further investigate the effect of CRABP2 on trophoblast– endometrium interaction, we examined apoptosis in trophoblast spheroids at 24 hours of cocultivation. As shown in Figure 1C


Lee et al.


and D, we found a small but significant increase in the percentage of apototic cells in spheroids on cells overexpressing CRABP2 (4.25  0.39) when compared with those on parental RL95-2 cells and mock cells (2.71  0.26 and 2.50  0.32, respectively). CRABP2 is a 15-kDa protein containing a lipocalin domain involved in retinoic acid (RA) binding. CRABP2 regulates the access of RA to specific nuclear receptors, such as the retinoic acid receptor (RAR) and the retinoid X receptor (RXR). The RAR-RXR heterodimer activates gene transcription, regulating multiple biological processes. Previous studies have suggested that CRABP2 is an important component of the antiproliferative effects of RA. For instance, CRABP2 overexpression in mammary carcinoma dramatically sensitizes cells to RA-induced growth inhibition (11). During the implantation window, the endometrium proliferates and differentiates to facilitate implantation of the developing embryo, which is very tightly controlled. Our previous study showed that the growth of a RL95-2 stable cell line expressing CRABP2 was markedly inhibited, leading to accumulation in the S and G2–M phase fractions (7). In the present study, we found that trophoblast spheroids are defective in expansion on the endometrial cells overexpressing CRABP2. Thus, the impaired proliferation of endometrial cells by CRABP2 overexpression could be related to altered adhesion or invasion of trophoblast spheroids, which leads to reduced spheroid expansion. It should be noted that the initial process of implantation begins with trophoblast attachment

Vol. 95, No. 8, June 30, 2011

and expansion to the uterine epithelial cells. In addition to the abnormal spheroid expansion, we observed a small but significant increase in the rate of apoptosis in spheroids on the cell line expressing CRABP2. It has previously been demonstrated that, in contrast to normal pregnancy, apoptotic cells are dominant in the syncytiotrophoblast layer in cases of spontaneous abortion (12), and that changes in apoptosis in the placenta are associated with abnormal pregnancy outcome (13). Thus, our findings suggest the presence of a relationship of endometrial CRABP2 with trophoblast outgrowth and apoptosis.

In conclusion, we demonstrated the decreased expansion of trophoblast spheroids cocultured with the stable cell line overexpressing CRABP2 previously identified as an up-regulated gene in patients with unexplained RSA. Furthermore, a higher rate of apoptosis occurred in spheroids cocultured with these cells. The results of the present study suggest that CRABP2 may play a role in pathogenesis underlying implantation failure with defective endometrial cell growth and altered trophoblast behavior. Thus, this study provides novel information regarding the role of CRABP2 in unexplained RSA.

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