Growth Factor/Cytokine Secretion by a Permanent Human Endometrial Cell Line with Embryotrophic Properties

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1 Journal of Assisted Reproduction and Genetics, VoL 13, No. L 1996 CLINICAL ASSISTED REPRODUCTION Growth Factor/Cytokine Secretion by a Permanent Human Endometrial Cell Line with Embryotrophic Properties NINA N. DESAI 2'3 and JAMES M. GOLDFARB 2 Submitted: December 15, 1995 Accepted: Februao' 19,1996 Objective: Our goal was to identif)' and quantitate growth factors and cytokines actively secreted by a permanent human endometrial cell line with emb~otrophic properties and to determine if cell lines enhancing emb~o development secrete common growth factors and/or cytokines. Design: Culture medium conditioned by this human endometrial cell line was screened for platelet-derived growth factor ( PDG F), leukemia inhibitory factor ( LtF), interleukin-6 ( IL- 6). epidermal growth factor ( EGF), and transforming growth factor-~3 (TGF-f3) by enzyme-linked immunoassay (ELISA). Comparisons were made to the African Green monkey kidney epithelial cell line (Vero), which has been well documented to be emb~. otrophic. Setting: This study was conducted in a university-based research laboratory associated with a clinical IVF program. Results: The monkey Vero cell line and our permanent human endometrial cell line produced several common growth factors and cytokines, such as IL-6, PDGE and LIE IL-6 was secreted in substantial amounts by both Vero cells (400 pglml per 100,000 cells) and endometrial cells (100 pg/ml). PDGF levels in culture supernatant were measured to be 72 and 35 pg/ml, respectively, for Vero and endometriat cells. LIF could not be assayed in our human cell line by ELISA but its presence was evidenced by immunocvtochemistry, EGF and active TGF-~ were not detectable in conditioned medium from either cell line. Conclusions: The two emb~otrophic cell lhtes analyzed in this investigation synthesized several common growth factors and c3,tokines. Identification of factors being secreted by cell lines which positively influence emb~o development may aid us in understanding the basic growth requirements of the preimplantation embryo. Presented at the 51 st Annual Meeting of the American Society/'or Reproductive Medicine, October 7-12, 1995, Seattle, Washington. 2 Department of Obstetrics and Gynecology, University MacDonald Women's Hospital, Case Western Reserve University, II 100 Euclid Avenue, Cleveland, Ohio To whom correspondence should be addressed. KEY WORDS: growth factors; cytokine; leukemia inhibitory factor; platelet-derived growth /'actor; interleukin-6; coculture; Vero cells; embryotrophic; secretion; endometrial cell line. INTRODUCTION Early embryonic development, genomic activation, and differentiation of embryonic cells into inner cell mass and trophectoderm at the blastocyst stage can occur in relatively simple media, implying autocrine control of early events by the developing embryo. Growth factor transcripts of both maternal and embryonic origin have been identified in early embryos and blastocysts of several species (1-3). Numerous data point to growth factor involvement in embryonic genome activation and blastulation (reviewed in Ref. 4). it has been suggested that retarded rates of embryo cleavage and blastulation, along with reductions in total cell number observed in blastocysts grown in vitro (5), may be attributed to the absence of oviductand uterine-derived growth factors. Cultivation of human embryos on feeder cell monolayers has been used as a tool to overcome some of these difficulties. Improvements in embryo quality, blastocyst formation, and pregnancy outcome have been reported (6). A variety of cell types has been used effectively for the coculture of human embryos, i.e., epithelial cells from the African Green monkey kidney (Vero) (7), human oviduct (6), and bovine oviduct (8). Secretion of growth factors by coculture cells could account for some of their beneficial effects. Recently, release of leukemia inhibitory factor (LIF) by Vero cell cocultures has been reported (9). In a subsequent study, Kauma et al. have hypothesized that L1F secretion by coculture cells may be responsible for some of their beneficial effects on in vitro embryo development (10). In their study, coculture cell types expressing L1F (i.e., Vero cells and human embryonic fibroblasts) were 1058-(gl68/96/~800-( /0 19q6 Plenum Publishing Cot'potation 546

2 GROWTH FACTOR SECRETION BY EMBRYOTROPHIC CELLS 547 found to be more embryotrophic than the non-lifproducing cell line tested. In this investigation, we further explore the possibility that cell lines promoting embryo development may secrete common growth factors by analyzing a novel human endometrial cell line with embryotrophic properties (11). This human cell line exhibits epithelial cell morphology and like vero cells is easily handled, passaged, and frozen. In an initial comparative trial, this endometrial cell line proved to be superior to both human oviduct and monkey kidney Vero cells in promoting blastocyst development and hatching of in vitro fertilized mouse oocytes (1 i). Blastocyst transfer studies have since confirmed the viability of these endometrial cell cocultured embryos (Desai et al., 1996, abstract). The purpose of this investigation was therefore (a) to identify and quantitate growth factors and cytokines actively secreted by this embryotrophic human endometrial cell line and (b) to determine if cell lines enhancing embryo development secrete common growth factors and/or cytokines. Comparisons were made to the Vero cell coculture system, which has been well documented to be embryotropic to both murine and human embryos. Assay for Growth Factors Culture supematants were assayed for plateletderived growth factor (PDGF), leukemia inhibitory factor (LIF), epidermal growth factor (EGF), transforming growth factor-13 (TGF-[3), and interleukin-6 (IL-6) using enzyme-linked immunoassay kits (ELISA) from R&D Systems (Minneapolis, MN). Assays were run following manufacturer's instructions. Unconditioned medium was used for controls. All measurements were made in duplicate. Secretions are expressed as picograms per milliliter and are standardized for 100,000 cells. lmmunochemistry Endometrial monolayers for immunochemical identification of LIF were grown in chamber slides and fixed with 4% paraformaldehyde after 2 days of culture. Monolayers were permeabilized and blocked with normal donkey serum before application of primary sheep anti-lif antibody (1:100; R&D Systems) for 1 hr. LIF was visualized by indirect immunofluorescence using a secondary donkey anti-sheep antibody (1:100; R&D systems) labeled with fluorescein isothiocyanate (FITC). In negative control slides the primary antibody was omitted. MATERIALS AND METHODS Cell Culture African green monkey kidney epithelial cells (Vero) were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). The human endometrial cell line described in this investigation was initially isolated from benign proliferative endometrium (11). This cell line has since undergone more than 45 passages. Vero and endometrial cells were seeded into Falcon 25-cm 2 tissue culture flasks at a concentration of approximately 100,000 cells in modified a-minimum essential medium (GIBCO, Grand Island, NY) with 5% fetal calf serum. Flasks were incubated in a humidified incubator at 37 C with 5% CO2 for 5 days. At the end of this interval, conditioned culture medium was collected and centrifuged at 400g to remove any dead cells. Supernatants were stored at -20 C until assay. Culture flasks were trypsinized and the cell concentration per millimeter was determined for each cell line. RESULTS Figure 1 summarizes our findings on the secretions by these two cell lines. PDGF was actively secreted by both cell lines. Secretion by our endometrial cell line (35 pg/ml) was approximately half that by the Vero cells (72 pg/ml). IL-6 was secreted in substantial amounts into the culture medium by both cell lines. IL-6 output by Vero cells was 400 pg/ml, compared to 100 pg/ml by endometrial cells. Vero cell supernatant contained approximately 15 pg/ml LIF. Unlike the monkey kidney cells, our human endometrial cell line did not secrete detectable amounts of LIF into culture supernatant. We explored the possibility that LIF secretion by these cells might be below the sensitivity of our ELISA assay (2 pglne). Immunoreactive LIF could in fact be identified in the cytoplasm of cultured endometrial cells using indirect immunoflourescence. The signal was weak but clearly positive when contrasted to negative controls (Fig. 2). EGF and active TGF-13 were not detected in culture supernatant from either cell line (assay sensitivity, 0.2 and 2 pg/ml, respectively). Journal of Assisted Reproduction and Genetics, VoL 13, No. 7, 1996

3 548 DESAI AND GOLDFARB D to O,T.I Q. V -. O o T Vero Cells 35O 30O 250 2O O 0 PDGF IL6 LIF Fig.!. Comparison of growth factor and cytokine secretion by two embryotrophic cell lines, Fig. 2. Identification of immunoreactive LIF in a human endometrial cell line by indirect immunoflourescence. DISCUSSION In this study we have clearly demonstrated several common growth factors and cytokines being secreted by two permanent cell lines with embryotrophic properties. LIF as the putative embryotrophic agent mediating the positive effects observed with several coculture systems is an intriguing possibility. From the work of Stewart et al., it is apparent that maternal uterine production of LIF is critical to the implantation process (12). Knockout mice lacking the LIF gene were unable to implant their blastocysts, yet these blastocysts, when transferred to foster mothers with functional LIF genes, implanted and gave rise to viable offspring (12), Uterine expression of LIF has been shown to coincide with the onset of blastocyst implantation (13,14). LIF secretion by isolated human endometrial glandular epithelial cells has been shown to be menstrual cycle stage specific, with the highest LIF secretion around the window of implantation and low levels in follicular and late luteal phases (15). LIF may also play a role in the developing blastocyst, by maintaining proliferation in cells of the inner cell mass (ICM) and therein indirectly controlling trophoblast proliferation (12). Although LIF expression by murine (16) and human blastocysts (Desai et at., unpublished results) has been documented, one might speculate that the low levels of LIF produced may be inadequate to maintain proliferation of the ICM in vitro. Supplementation of embryo culture medium with recombinant LIF can in fact enhance viability and hatching of ovine embryos (17). Although LIF does not affect early cleavage and blastocyst formation of murine embryos, the proportion of blastocysts that hatch is significantly increased (18). A similar effect was not, however, observed by Juriscova et al. with spare human embryos (19). Of course, if the primary action of LIF is at later stages, observation of positive effects of LIF on human embryos may be obscured by unidentified in vitro factors which compromise the ability of these embryos to overcome the initial hurdle of genomic activation (eight-cell stage) and morulation. To date there has been no mention of coculture cells secreting large amounts of IL-6 into the culture medium; an important outcome from this investiga- Journal ~[Assisted RepnMuction and Genetics, Vol, 13, No. 7, 1996

4 GROWTH FACTOR SECRETION BY EMBRYOTROPHIC CELLS 549 tion was the identification of IL-6 release by both Vero cells and our endometrial cell line. Many of the biological activities attributed to LIF are also shared by IL6 (20). Both IL-6 and LIF share a common signal transducer molecule (gp 130), although they bind to different membrane receptors (21). IL-6 expression has been detected in mouse (16) blastocysts and further confirmed by measurement of low levels of cytokine in blastocyst-conditioned media. Little information is currently available on the impact of IL-6 on embryo development, blastocyst cell number, and differentiation/proliferation of trophectodermal and ICM cells. The desirability of using coculture cell monolayer to improve the in vitro development of human embryos is an area of continued controversy. Bavister has argued against coculture since it represents an additional layer of technology which may mask underlying culture media problems, expose embryos to numerous unknown factors/proteins synthesized by the feeder layer, and potentially introduce disease organisms into the culture system, especially when animal cells are being used for cocutture (22). Moreover, many investigators contend that the beneficial effects observed with coculture might not be the result of specific secretions ("positive conditioning") but, rather, that the coculture cell type simply alters a less than optimal culture medium through its metabolic processes, thus indirectly creating a more favorable environment for embryos ("negative conditioning") (22,23). The arguments supporting embryotrophic factor secretion by coculture cells have been weakened by conflicting results from conditioned medium studies. Whereas oviduct-conditioned medium has been reported to improve in vitro development of bovine embryos (24), other investigators have reported no effect of conditioned medium (25). Leppens and Sakkas examined different molecular weight fractions of Vero cell-conditioned medium and their effect on murine embryo development. They failed to identify any clear association between a particular fraction and embryo enhancement (23). In our laboratory, initial results with a-mem medium conditioned by this human endometrial cell line have indicated an increase in the percentage of murine IVF oocytes reaching the hatched blastocyst stage (Desai et al. unpublished results). Alpha MEM was selected for this study because it supported high blastocyst rates during mouse IVF experiments even without coculture. One explanation for the inability of conditioned medium alone to elicit maximum embryo response could be that embryos need to be presented with growth factors/cytokines in a dynamic fashion, perhaps in minute but steadily increasing amounts. Exposure of embryos to one factor could conceivably alter their response to another growth factor presented simultaneously or at a later interval. Indeed, several in vitro studies have clearly demonstrated that growth factor influence on embryo development is both cell stage and concentration dependent (26-28). Another consideration is that while "negative conditioning" of culture environment by coculture cells may be reflected by gross improvements in early embryo quality and blastocyst transformation rates, the "'positive" effects of so-called embryotrophic factors could be much more subtle. These effects may become evident only by examination of the distribution of trophectoderm and ICM ceils within the cocultured blastocyst (29), metalioproteinase and plasminogen activator secretion by trophoblast cells (30), and overall invasiveness. Many questions remain to be answered as regards the interaction among coculture cells, their products, and the developing embryo. Identification of factors being secreted by cell lines which positively influence in vitro development of embryos may bring us one step closer to understanding the basic growth requirements of the preimplantation embryo. In summary, the two embryotrophic cell lines tested in this study secreted several common growth factors and cytokines: LIE IL-6, and PDGE Although the abundance of data on leukemia inhibitor factor and its function in early implantation events makes it a likely candidate for mediating the embryotrophic effects seen with several coculture cell types, our findings suggest that IL-6 should also be considered in this capacity. Future research should focus on each of these factors and how they influence blastocoel formation, differentiation of inner cell mass and trophectoderm, and ultimately implantation rates. Screening of conditioned medium for additional growth factors and cytokines known to influence embryo development may yield further information on the environment to which cocultured embryos are being exposed. ACKNOWLEDGMENTS The authors are grateful to Donna Kinzer, MS, and Meera Scarrow, BA, JD, for technical assistance and performance of mouse IVF and coculture experiments. 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