Hum. Reprod. Advance Access originally published online on August 24, 2007
Human Reproduction 2007 22(10):2615-2622; doi:10.1093/humrep/dem263
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Histone deacetylase inhibitor-induced glycodelin enhances the initial step of implantation
Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan
1 Correspondence address. Tel: +81-3-3353-1211 (ext. 63401); Fax: +81-3-3226-1667; E-Mail: uchida{at}sc.itc.keio.ac.jp
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Abstract |
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BACKGROUND: The complex molecular pathways governing implantation are unclear and ethical limitations limit studies in humans. Reversible histone acetylation regulates gene transcription and histone deacetylase inhibitors (HDACI) induce specific genes. Suberoylanilide hydroxamic acid (SAHA), a HDACI recently approved as an anti-cancer drug, induces the morphological and functional differentiation of human endometrial gland cells through up-regulation of glycodelin, a secretory phase dominant protein.
METHODS: We investigated whether SAHA improves implantation in an in vitro implantation assay using the human endometrial adenocarcinoma cell line, Ishikawa and the choriocarcinoma cell line, JAR.
RESULTS: In an in vitro implantation assay, JAR spheroids attached and adhered to Ishikawa cells in a time dependent manner. Glycodelin induction, following treatment with ovarian steroid hormones or SAHA, enhanced implantation. The improvement in implantation was also obtained when glycodelin was overexpressed without stimulation and was almost completely abrogated by glycodelin gene silencing.
CONCLUSIONS: This study demonstrates that glycodelin is a key regulatory protein of implantation and suggests that SAHA may have a capacity to supplant steroid derivatives in the treatment of infertility.
Key words: glycodelin/histone deacetylase inhibitor/implantation/SAHA
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Introduction |
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Implantation begins with the attachment of the floating blastocyst onto the endometrial glandular epithelium, followed by broad adhesion and penetration (Lessey, 2002
). For implantation to progress, both transdifferentiation of the endometrial epithelium and a change in embryo characteristics are required (Thie et al., 1997; Heneweer et al., 2002). The human endometrium is first estrogen primed prior to being differentiated by ovarian progesterone. Endometrial changes are effected by a steroid mediated pathway and a non-steroid signalling pathway, the molecular events of which are only partially understood. Thus identification of the molecular events regulating endometrial differentiation would enable the identification of new drugs and reagents useful in the treatment of recurrent implantation failure.
Reversible nucleosomal histone acetylation, a histone modification i.e. regulated by histone acetyltransferases and histone deacetylases, regulates gene transcription (Berger, 2002). Histone deacetylase inhibitors (HDACI) are a novel class of anticancer drugs that induce cell cycle arrest, differentiation and apoptosis in cancer cells and transformed cells (Thiagalingam et al., 2003). Recently, one HDACI, suberoylanilide hydroxamic acid (SAHA), has received Food and Drug Administration (FDA) approval for the treatment of advanced cutaneous T-cell-lymphoma. SAHA also has anti-proliferative activity and acts as a transdifferentiation reagent in the breast cancer cell line, MCF7 (Munster et al., 2001).
Glycodelin is a 28 kDa secretory protein that has a unique temporo-spacial expression pattern. It is primarily expressed in reproductive organs such as the uterine endometrium, cervical glands, fallopian tubes, ovaries and mammary glands (Seppälä et al., 2002). Glycodelin has several tissue-specific glycoforms, each bearing a different glycosylation pattern. For example, glycodelin-A, glycodelin-S, glycodelin-F and glycodelin-C are expressed in the endometrium, sperm, follicular fluid and cumulus cells, respectively (Yeung et al., 2006; Seppälä et al., 2007). In human endometrial glands, glycodelin (glycodelin-A) is scantily expressed during the estradiol (E2)-based proliferative phase. Its expression is induced by progesterone exposure in the early secretory phase, peaks 10 days after ovulation, and persists until menstruation begins or pregnancy is established. The period of glycodelin expression has been referred to as the 'implantation window' (Seppälä et al., 2002). Glycodelin-A inhibits human sperm–oocyte binding (Oehninger et al., 1995) and has an immunosuppressive activity mediated by the inactivation of natural killer cells (Okamoto et al., 1991). Glycodelin-S in sperm affects capacitation and acrosomal reactivity (Chiu et al., 2003, 2005). Thus, glycodelin regulation of the key events in fertilization and implantation, such as sperm capacitance and maternal immunosuppression, is a function of both the specific glycoform and the time at which it is expressed.
We previously reported that trichostatin A, another HDACI, differentiates human endometrial stromal cells (Sakai et al., 2003). Additionally, a human endometrial adenocarcinoma cell line, Ishikawa (of epithelial origin) is morphologically and functionally differentiated through up-regulation of glycodelin by both trichostatin A and SAHA (Uchida et al., 2005, 2007). Both of these agents belong to the hydroxamic acid group of HDACI. These observations prompted the question of whether SAHA could be used in the treatment of infertility arising from an insufficiently differentiated endometrial epithelium. We explored this possibility using ovarian steroids or SAHA in an in vitro implantation assay using simulated endometrium and embryos. An in vitro implantation assay using cell lines, which approximated the normal endometrium and embryo, can simulate in vivo implantation (Thie et al., 1997; Hohn et al., 2000; Nishida, 2002; Kosaka et al., 2003; Li et al., 2003). This model circumvents the ethical limitations associated with obtaining human endometrium and fertilized oocytes for primary culture and provides sufficient materials for a quantitative analysis. We found that SAHA enhanced the number of attaching and adhering simulated embryos and that SAHA-induced glycodelin mediated this improvement in implantation.
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Materials and Methods |
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Reagents
Phenol red-free minimum essential medium (MEM), Dulbecco’s modified essential medium (DMEM), RPMI1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Tokyo, Japan). SAHA was obtained from BIOMOL (Plymouth Meeting, PA, USA). Antibodies against glycodelin [(N-20, sc-12289 and Q-13, sc-12290 (epitopes mapping near the N-terminus and C-terminus of Glycodelin of human origin, respectively)] (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), enhanced green fluorescent protein (EGFP) (BD Biosciences, Bedford, MA, USA), mitogen-activated kinase (MAPK) (Upstate Biotechnology, Inc., Lake Placid, NY, USA), and horse-radish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were purchased from commercial sources. Unless indicated otherwise, all other chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA) and Wako (Osaka, Japan).
Cell culture
Ishikawa (clone 3-H-12) (Nishida, 2002), a human endometrial adenocarcinoma cell line of epithelial origin was a kind gift from Dr M. Nishida (National Kasumigaura Hospital, Ibarak, Japan). JAR cells, a human choriocarcinoma cell line, were kindly provided by Dr N. Suzuki (St Marianna University, Kanagawa, Japan). HEK293 cells (human embryo kidney cells), HeLa cells (human cervical cancer cell line) and MCF7 cells (human breast cancer cell line) were purchased from American Type Cell Culture (Rockville, MD, USA). Ishikawa cells and JAR cells were cultured in phenol-red free MEM and RPMI1640 medium, respectively, supplemented with 10% heat-inactivated FBS, 100 U/ml of penicillin and 100 mg/ml of streptomycin. HEK293 cells, HeLa cells and MCF7 cells were cultured in DMEM medium, supplemented with 10% heat-inactivated FBS, 100 U/ml of penicillin and 100 mg/ml of streptomycin. Ishikawa cells were used within 10 passages according to the provider’s recommendation to avoid changes in cell characteristics including down-regulation of estrogen receptor and progesterone receptor expression.
Plasmids
Total RNA was extracted from cultured Ishikawa cells using the RNeasy mini kit (QIAGEN, Hilden, Germany). Glycodelin cDNA without the signal sequence and stop codon was amplified by RT–PCR using the One-Step RT–PCR kit (QIAGEN). The forward primer (5'-CGGAATTCCACCATGGACATCCCCCAGA-3') has an EcoR I site and a Kozak sequence, and the reverse primer has a BamH I site, but no stop codon (5'-CGGGATCCGAAACGGCACGGCTCTTCCA -3'). The various PCR products containing the full length and splicing variants of the glycodelin gene and the pEGFP-N3 plasmid (CLONTECH, Palo Alto, CA, USA) were both digested with EcoR I and BamH I and ligated to generate an expression plasmid harboring EGFP-fused glycodelin genes (pcGd1-EGFP, pcGd2-EGFP, pcGd3-EGFP and pcGd4-EGFP). Gd2 is missing half of exon 2 (nt 162–236). Gd4 lacks exon 2, exon 3 and half of exon 4 (nt 379–421). Gd2 and Gd4 are isoforms (splicing variants) -2 and -3 (Swiss-Prot primary accession number P09466
[GenBank]
-2 and P09466
[GenBank]
-3), respectively. Gd3 lacks only exon 4. DNA sequences were verified using an automated Applied Biosystems sequencer and the BigDye Terminator Kit (Perkin-Elmer Life and Analytical Sciences, Boston, MA, USA).
In vitro implantation assay
To generate spheroids of JAR cells for use as blastocyst models (Hohn et al., 2000; Li et al., 2003), the JAR cell concentration was adjusted to 2 x 105 cells/ml, and the cell suspension was cultured with shaking at a speed of 70 rpm. Spheroids of 50–200 µm in diameter, which were similar in size to an implanting blastocyst, were generated after 24 h shaking culture. Ishikawa cells (2–4 x 104 cells) were prepared in 96-well plates and grown with or without stimulation (ovarian steroid hormones or SAHA) for 3 days or were used for transfection (small interference RNAs or glycodelin expression vector). After Ishikawa cells had reached confluence, co-culture of Ishikawa cells and JAR spheroids (approximately 100 spheroids/well) for the indicated period was performed with refreshed medium containing no stimulants, followed by fixation with 3.7% paraformaldehyde. To visualize and count the attached or adhered lipophilic-dye (Vybrant DiO cell-labelling solution, purchased from Invitrogen [Carlsband, CA, USA]) coated spheroids, confocal images were acquired using a Leica TCS SP2 confocal microscopy system with a Leica DMIRE2 inverted microscope (Leica Microsystems). Relative attachment/adhesion ratio was calculated as the number of attached or adhered spheroids divided by one of control cells. The relative attachment/adhesion ratio of control cells was set at 100.
Transfection
Glycodelin coding plasmids for transient transfection (pcGd-EGFP), as well as small interference RNAs: GAPDH siRNA (Ambion, Austin, TX, USA) or glycodelin siRNA designed by QIAGEN (Uchida et al., 2005, 2007), were transfected using LipofectAmine (Invitrogen, Carlsbad, CA, USA). To establish permanent clones expressing EGFP-fused glycodelin, semi-confluent Ishikawa cells were transfected with pEGFP-N3 or pcGd-EGFP using LipofectAmine. Transfected Ishikawa cells were cultured in medium containing 500 µ/ml geneticin for more than 2 weeks to select cells stably expressing EGFP or EGFP-tagged glycodelin. The expression levels of the exogenous proteins in geneticin-resistant subcloned Ishikawa cells were analysed with immunoblotting using anti-EGFP and anti-glycodelin antibodies.
Immunoprecipitation and immunoblotting
Confluent Ishikawa cells transfected without or with siRNA were cultured in the indicated medium for 3 days and lysed on ice with RIPA buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Na-deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 mM Na3VO4, 50 mM NaF, 1 mM Na2MoO4) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were determined using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as a standard. Each 250 µg of protein was subjected to immunoprecipitation with anti-glycodelin antibody (N-20, sc-12 289) and protein G-sepharose beads (Amersham Biosciences, Piscataway, NJ, USA) for 4 h at 4°C. Immunoprecipitates were separated by electrophoresis on a 15% SDS–polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene difluoride membrane. After incubation with the anti-glycodelin antibody (Q-13, sc-12 290), followed by a HRP-conjugated secondary antibody, the immunoreactive proteins were detected by the enhanced chemiluminescence method (Amersham Biosciences). An appropriate combination of anti-glycodelin antibody for the immunoprecipitation and immunoblotting was determined by our preliminary study.
Statistical analysis
All experimental data from the bioassays represent the results obtained from three independent experiments each with triplicate assays, expressed as the mean and SEM. Statistical analysis was performed utilizing the ANOVA, followed by a Bonferonni test. Immunoprecipitation and immunoblotting studies were repeated three times and the representative images are shown.
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JAR spheroids attach and adher to Ishikawa cells (in vitro implantation model)
To investigate the effect of ovarian steroid hormones and SAHA on the attachment and adhesion of the floating blastocyst to the receptive endometrial epithelium, we employed an in vitro implantation assay with simulated embryos (Thie et al., 1997
; Hohn et al., 2000; Kosaka et al., 2003; Li et al., 2003). Overnight shaking culture of the human choriocarcinoma cell line, JAR results in spheroid formation (Fig. 1A, left panel). To simulate blastocysts, we cultured JAR cells on a shaker in the presence of a fluorescent lipophilic-dye and generated spheroids similar in size to blastocysts (50–200 µm) (Fig. 1A, right panel). We co-cultured the spheroids with a monolayer of Ishikawa cells (Nishida, 2002) and timed their attachment and adherence to them. The JAR spheroids attached after a short co-culture period of 10 min (Fig. 1B, left panel) and required 24 h to adhere and spread onto the Ishikawa cells (Fig. 1B, right panel). The initial attachment by co-culture for 10 min was loose and the unattached portions of the spheroids broke off during the washing and fixation process. The attached remnants were subsequently visualized with fluorescent microscopy (Fig. 1B, left panel). Therefore, the visible fluorescent signal areas of the attached part of spheroids (10 min co-culture) were significantly smaller than that of adhered and spread spheroids (24 h co-culture) (Fig. 1B).
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Implantation of JAR spheroids on Ishikawa cells is enhanced in a time-dependent fashion
Using the in vitro implantation assay, we investigated whether JAR spheroids attached and adhered to other cell lines. In this assay, we tested Ishikawa cells, HEK293 cells, HeLa cells and MCF7 cells. All of these lines except HEK293 cells permitted the attachment (10 min co-culture) and adhesion (24 h co-culture) of JAR spheroids to varying degrees (Fig. 2A). Ishikawa cells had the highest level of implantation (attachment and adhesion) of JAR spheroids. Following co-culture for 10 min, approximately half of the JAR spheroids attached to the Ishikawa monolayer (Fig. 2A and B). We used the percentage attaching after 10 min to estimate the implantation rate. When the co-culture time was extended, the JAR spheroids adhered and spread as shown in the right panel of Fig. 1B. The numbers of attached and adhered spheroids were gradually increased in a time-dependent fashion (Fig. 2B). HEK293 cells had the lowest number of spheroids attaching after 10 min, compared with the other cell lines, which are derived from glycodelin-expressing tissues (Fig. 2A) (Seppälä et al., 2002
).
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Ovarian steroids and SAHA up-regulate the implantation activity in Ishikawa cells
We next examined whether stimulation with ovarian steroid hormones or HDACI affected the implantation activity of Ishikawa, HeLa and MCF7 cells. In all cells tested, glycodelin expression was up-regulated by stimulation with ovarian steroid hormones or SAHA (Fig. 3A). The attachment activity to JAR spheroids was significantly up-regulated to varying degrees following treatment with either ovarian steroid hormones or SAHA (Fig. 3B). Steroid hormones had a more pronounced effect on MCF7 than did the HDACI. The optimal concentration of SAHA differed among the cell types. In Ishikawa cells, a relatively low concentration of SAHA (0.25 µM) enhanced the attachment of JAR spheroids to a level that was similar to the level induced by the steroid hormones (Fig. 3B). The treatment of Ishikawa cells with ovarian steroid hormones or HDACI induced glycodelin expression, indicating that these cells are sensitive to SAHA (Uchida et al., 2005
, 2007). The augmentation of attachment activity paralleled the induction of glycodelin protein expression in all three cell lines (Fig. 3A and B). Adhesion activity was increased to a lesser extent than attachment following treatment with steroids or SAHA. A weak enhancement of adhesion activity was obtained in Ishikawa cells and HeLa cells, but not in MCF7 cells (Fig. 3C).
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-E2 in combination with 1 µM progesterone (EP)] or the indicated concentrations (µM) of SAHA for 3 days. Cell lysates were extracted, immunoprecipitated and immunoblotted by anti-glycodelin antibody (A, upper panel, Gd). Each input cell lysate (1/25 volume) was also subjected to immunoblotting with anti-MAPK antibody (A, lower panel). Each value (A) represents the mean + SEM of the relative ratio of the immunoblot staining intensity with anti-Gd to that with anti-MAPK, as determined by densitometric image analysis with control cells set at 1.0. After refreshing the culture medium without stimulants, cells were subjected to the in vitro implantation assay. Each bar indicates the mean + SEM of the relative attachment ratio (B) and relative adhesion ratio (C) in the in vitro implantation assay incubated for 10 min (B) or 24 h (C). The attachment or adhesion of spheroids to each control cell type was set to 100%. Asterisks show significant differences compared with each control (P < 0.05)Glycodelin gene silencing abrogates the up-regulation of attachment activity
The synchronicity between the induction of glycodelin expression and the up-regulation of implantation activity prompted us to investigate whether glycodelin regulates implantation activity. We performed the in vitro implantation assay in the presence of glycodelin siRNA. An immunoprecipitation assay confirmed that glycodelin siRNA suppressed glycodelin induction following stimulation by ovarian steroid hormones or SAHA (Fig. 4A). In this assay, suppression of glycodelin expression significantly abrogated the augmentation of attachment and adhesion activity to JAR spheroids caused by steroids or SAHA (Fig. 4B and C).
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Overexpression of glycodelin enhances implantation activity
Finally, we tested whether glycodelin gene transfection, in the absence of ovarian steroid hormones or SAHA stimulation, could up-regulate the implantation activity of Ishikawa cells towards JAR spheroids. We constructed the full length-coding (cGd1) and splicing variants (cGd2, cGd3 and cGd4) of the glycodelin gene tagged with EGFP (Fig. 5A and B) and established four types of glycodelin expressing permanent clones (Fig. 5C). Using these clones in an in vitro implantation assay demonstrated that only those cells expressing full length glycodelin, and not those expressing either the splice variants or EGFP alone, enhanced attachment and adhesion activity (Fig. 5D and E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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Reversible histone acetylation, which is regulated by histone deacetylase and HDACI, plays an important role in gene transcription. HDACI result in the continuous acetylation of histone proteins and induce functional differentiation by up-regulating the transcription of specific genes. In the human breast cancer cell line, MCF7, SAHA treatment increases the production of lipids and milk proteins (Munster et al., 2001
). Additionally, both HDACI and ovarian steroid hormones, through up-regulation of glycodelin, induce morphological and functional differentiation in Ishikawa cells (Uchida et al., 2005, 2007). These results suggest that SAHA treatment could enhance the implantation activity of human endometrial gland cells through induction of differentiation.
To investigate implantation activity, we employed an in vitro implantation assay system (Thie et al., 1997; Hohn et al., 2000; Kosaka et al., 2003; Li et al., 2003) using cell lines. Given that Ishikawa cells and JAR cells have phenotypes similar to normal endometrial glandular cells and trophoblastic cells, respectively (Hohn et al., 2000; Nishida, 2002), they are suitable for simulating the interaction between the human endometrial glandular epithelium and the trophoblast. This circumvents the ethical restrictions associated with obtaining human endometrium and fertilized oocytes for primary culture. Additionally, a cell line-based in vitro implantation assay provides the large quantity of cells needed to study signalling pathways.
In an in vitro implantation assay, the attachment and adhesion between cell lines depends on the lines used (Fig. 2A). The phenomenon of two distinct cell types attaching, adhering and ultimately fusing is unique to reproductive biology. With the exception of implantation and leukocyte extravasation, cells of differing types generally do not adhere under flow (Alon and Feigelson, 2002; Genbacev et al., 2003). In addition to human endometrial gland derived Ishikawa cells, HeLa cells (uterine cervical gland) and MCF7 cells (mammary gland) also have the potential to attach to JAR spheroids. However, it is not unexpected that simulated blastocysts can adhere onto non-endometrial gland cells. Blastocysts have the ability to attach and adhere onto a variety of tissues including the fallopian tube, ovary, omentum and peritoneum, resulting in ectopic pregnancy. In contrast HEK293 cells (human embryo kidney) do not allow attachment and adherence (Fig. 2A). Ishikawa cells, in particular, had a high adhesion rate to JAR spheroids, compared with HeLa and MCF7 cells (Fig. 2B). Thus, attachment and adhesion of the simulated embryo varies depending on factors intrinsic to the cell line. The sensitivity of attachment and the adhesion to the embryo is probably affected by the quantity and type of adhesion molecules on the cell surface.
In the cell lines demonstrating attachment in the embryo model, glycodelin is scantily expressed but can be induced by stimulation with either ovarian steroid hormones or SAHA (Fig. 3A). The progesterone receptor is expressed and administration of progesterone significantly up-regulates glycodelin expression and/or glycodelin promoter activation in HeLa cells (Konishi et al., 1991; Taylor et al., 1998). Progesterone also enhances the glycodelin expression and/or glycodelin promoter activation of Ishikawa cells and human endometrium (Taylor et al., 1998; Mueller et al., 2000; Uchida et al., 2005, 2007). It is reported that progesterone increases the expression of Sp1 in human endometrial stromal cells (Krikun et al., 2000) and that SAHA stimulates Sp1 sites of promoter regions, including that of glycodelin (Huang et al., 2000; Uchida et al., 2005).
Interestingly, both glycodelin protein expression and attachment activity against JAR spheroids showed parallel up-regulation in all three cell lines tested (Fig. 3A), but notably in the Ishikawa and HeLa cells. Adhesion activity also paralleled glycodelin expression but to a lesser extent compared with attachment. In MCF-7 cells, SAHA stimulated glycodelin expression to a lesser degree (Fig. 3A). This weakness of stimulation on glycodelin expression probably resulted in the lack of significant changes of adhesion activity on MCF-7 cells (Fig. 3B). Glycodelin gene silencing with siRNA abrogated the enhancement of attachment and adhesion caused by ovarian steroids or SAHA (Fig. 4). This suggests that the up-regulation of glycodelin affects the steps of implantation including attachment, adhesion and finally invasion.
Glycodelin is a secretory protein, but it is not found in conditioned media from Ishikawa cells (Chatzaki et al., 1994). Although CD45 and fucosyl transferase serve as receptors for the secretory form of glycodelin in T cells and human sperm (Rachmilewitz et al., 2003; Chiu et al., 2007), it is unclear whether they exist and function in endometrial glandular cells. We have always focused on the intracellular function of glycodelin in Ishikawa cells (Uchida et al., 2005, 2007). In this study, to overexpress glycodelin in Ishikawa cells, we used an EGFP-tagged fragment of the glycodelin gene named cytoplasmic glycodelin (cGd), lacking the signal peptide necessary for secretion (Mukhopadhyay et al., 2004). Several splicing variants of glycodelin have also been reported (Garde et al., 1991; Koistinen et al., 1997; Seppälä et al., 2002), and we used the splicing variants of glycodelin available by RT–PCR from Ishikawa cell total RNA. Only permanent clones expressing full-length glycodelin demonstrated a dramatic enhancement of both attachment and adhesion to JAR spheroids (Fig. 5D and E). Furthermore, the enhancement of adhesion depended on the degree of glycodelin overexpression (Fig. 5D). Clones expressing the splicing variant lacking glycodelin exon 4 (cGd3) showed down-regulation of implantation activity. Exon 4 does not contain amino acid residues that anchor carbohydrates (Asp28 and Asp63 in exon 2 and exon 3, respectively) (Seppälä et al., 2002) or affect cell motility (from Lys69 to Ile84) (Song et al., 2001). Exon 4 of glycodelin is located on the surface of the three-dimensional protein (Seppälä et al., 2002) and is a possible binding site for still to be discovered ligands that could regulate implantation activity. There was no significant difference in implantation activity of clones expressing the shortest variant which lacked exons 2, 3 and half of exon 4 (cGd4). The shortest variant (half the length of glycodelin) has a markedly different three-dimensional structure and is probably a non-functional protein. These results suggest that both exons 2 and 4 may be involved in the implantation activity of glycodelin.
An intracellular binding partner or receptor for glycodelin has not been found. Implantation is a complex process that begins with the simple attachment of cells of different tissue types. Dynamic interactions between the membranous receptors of both trophoblast and endometrial gland cells mediate the process. This study demonstrates that glycodelin is a key regulatory protein of the attachment between the embryo and the recipient endometrial gland. The HDACI, SAHA, may promote the initial step of implantation through up-regulation of glycodelin. Thus, SAHA could possibly supplant progesterone derivatives in the treatment of infertility.
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This work was supported in part by grants-in-aid for scientific research C(2) 16591683, C 18591812 (to H.U.) from the the Japan Society for the Promotion of Science (JSPS).
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Acknowledgements |
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We thank Drs Masato Nishida (National Kasumigaura Hospital, Ibarak, Japan) and Nao Suzuki (St Marianna University, Kanagawa, Japan) for gifts of Ishikawa cells and JAR cells, respectively. We are also grateful to Dr Hiroshi Fujiwara (Kyoto University, Kyoto, Japan) for technical advice of in vitro implantation assay.
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Submitted on May 9, 2007; resubmitted on June 8, 2007; accepted on June 19, 2007.
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