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Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Dr. Hidetaka Okada, Department of Obstetrics and Gynecology, Kansai Medical University, Moriguchi 570-8507, Japan. E-mail: hodaka{at}takii.kmu.ac.jp.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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A wide variety of proteins are initially synthesized as inactive precursors that undergo posttranslational processing into one or more biologically active polypeptide(s). Proprotein convertases (PCs) recognize various precursors and cleave at the general consensus motif (K/R)Xn (K/R)
, where n = 0, 2, 4, or 6, and X is any amino acid and usually is not a cysteine (6). PCs are structurally related to bacterial subtilisins and yeast kexin (7). Seven PCs have been identified (furin, PC1/3, PC2, PC4, PACE4, PC5/6, and PC7/LPC), and some have isoforms generated via alternative splicing (8). Their substrates include propeptide hormones, proneuropeptides, precursors of growth factors, cell surface receptors, and viral surface glycoproteins (6, 7, 9). A number of these have previously been shown to be expressed in the endometrium and with likely roles at implantation (1, 2). Therefore, the PC family of proteases has the potential to contribute to decidualization (4).
PC5/6 has recently been described as being of importance at implantation sites in the mouse (10, 11). Although PC5/6 mRNA is expressed in many endocrine and nonendocrine tissues, the level of PC5/6 mRNA at implantation sites in mouse uterus is substantially higher than in other organs where its expression has previously been studied (11). PC5/6 mRNA level is low in the nonpregnant and early pregnant uterus before embryo implantation commences (11). The expression of this gene is dramatically up-regulated at implantation sites in the mouse at d 4.5–5.5 of pregnancy when the uterus shows the first morphological changes associated with implantation and pregnancy. In situ hybridization analysis localized the mRNA expression predominantly in the decidual cells immediately surrounding the implanting embryo at the antimesometrial pole (11). Inhibition of uterine PC5/6 during early pregnancy in vivo blocked decidualization and prevented embryo implantation.1 These data indicate that PC5/6 plays an important role in stromal cell decidualization in the mouse. In addition, strong PC5/6 expression was detected in decidual cells during early pregnancy in human (see Footnote 1). We postulated that PC5/6 may play a key role in the preparation of the endometrium for pregnancy in the human.
Although the importance of the steroid hormones 17ß-estradiol (E2) and progesterone for decidualization in vivo is unquestioned, their precise mechanism of action in the endometrium is not known. At a cell biological level, decidualization of ESCs can be induced in vitro in the presence of progestin after estradiol priming. The decidual cells are typically characterized by morphological differentiation and by their secretory products, particularly prolactin (PRL) and insulin-like growth factor binding protein-1 (12). Using an in vitro model of decidualization, we previously demonstrated that activin A and IL-11 were produced by decidualizing stromal cells and enhanced the process of decidualization (13, 14). In the present study, we investigated the regulation of PC5/6 mRNA and protein expression in ESCs during decidualization in vitro and established a function for PC5/6 in decidualization using an antisense approach.
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Patients and Methods |
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Endometrial tissues (n = 12) were obtained by dilatation and curettage between d 7–20 of the menstrual cycle from women with regular menstrual cycles and no apparent endometrial dysfunction. Written informed consent was obtained from patients, and protocols were approved by the institutional Human Ethics Committee at Monash Medical Centre (Clayton, Victoria, Australia). Samples for cell culture were collected into DMEM (Trace Biosciences, Sydney, Australia).
Isolation and culture of ESCs
ESCs were isolated from tissue by enzymatic digestion and filtration as described previously (13, 14, 15). ESCs were cultured with a 1:1 mixture of DMEM-Ham’s F12 medium (Trace Biosciences) supplemented with 10% charcoal-treated fetal calf serum (Trace Biosciences) and 1% antibiotics (penicillin, streptomycin, and Fungizone; Commonwealth Serum Laboratories, Melbourne, Australia) at 37 C in a humidified atmosphere of 5% CO2 in air. The culture medium was replaced 60 min after plating to reduce epithelial cell contamination. The purity of ESCs was determined by morphology and by immunohistochemical staining as described previously (13, 14, 15) with markers specific to ESCs (vimentin). The ratio of vimentin-positive cells in ESCs was more than 97% by immunohistochemical staining.
In vitro decidualization
When ESCs were nearly confluent, they were trypsinized and replated in six-well plates (1 x 106 cells/well) for real-time PCR, RT-PCR analyses, and immunoprecipitation, and 24-well plates (2.5 x 105 cells/well) for immunocytochemistry and for treatment with morpholino antisense oligonucleotides (MOs). ESCs were cultured until confluent and treated with E2 (10–8 mol/liter) (Sigma, St. Louis, MO) in the presence or absence of medroxy-progesterone acetate (P; 10–7 mol/liter) (Sigma) for 12 d. Some ESCs were treated with E2 plus P plus ZK 98299 (onapristone; 10–5 mol/liter), a progesterone receptor (PR) antagonist. The culture media were changed every 3 d, and centrifuged supernatants were stored at –70 C until assayed. Each experiment was repeated at least three times with different cell preparations.
PRL assay by ELISA
The production of PRL by cultured ESCs was measured in conditioned medium using ELISA kits (Bioclone Aust. Pty. Ltd., Marrickville, Australia) in duplicate as previously described (13, 14). The lower detection limit of the assays was 10 mIU/liter. The concentration of PRL in the culture medium without cells was below the detection level (data not shown). The inter- and intraassay variabilities were 4.9 and 3.9%, respectively.
RNA extraction and cDNA synthesis
Total RNA was isolated from cultured ESCs using an RNeasy Minikit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. RNA was then treated with ribonuclease-free deoxyribonuclease (Ambion, Austin, TX) to remove contaminating genomic DNA. Total RNA (2 µg) was reverse transcribed at 46 C for 1.5 h in 20 µl reaction mixture using 100 ng random hexanucleotide primers and 6 IU avian myeloblastosis virus reverse transcriptase (Roche, Mannheim, Germany) in the presence of cDNA synthesis buffer (Roche), 1 mmol/liter dNTPs (Roche), 10 mmol/liter dithiothreitol (Roche), and 10 IU ribonuclease inhibitor (RNasin, Promega, Annandale, Australia). The resultant cDNA mixtures were heated at 95 C for 3 min before storage at –20 C. Negative controls were performed by omission of reverse transcriptase.
PCR for PC5/6
A 1.5-µl aliquot of RT product was amplified in a total volume of 25 µl using 12.5 µl PCR master mix [50 U/ml Taq DNA polymerase supplied in a proprietary reaction buffer (pH 8.5), 400 µmol/liter of each dNTP, and 3 mmol/liter MgCl2] (Promega) and 10 pmol sense and antisense primers for PC6 (Table 1
). The PCR was performed in three stages as follows: the first stage involved one cycle of 95 C for 5 min, 58 C for 1.5 min, and 72 C for 1.5 min; the second stage involved 30 cycles of 94 C for 50 sec, 58 C for 1 min, and 72 C for 1 min; and the final stage was 72 C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% agarose gel (Roche) and stained with ethidium bromide. Bands of interest were excised from the gel, purified, and directly sequenced to confirm their identity.
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Quantitative real-time PCR analysis was performed using a Roche LightCycler (Roche) and the FastStart DNA Master SYBR-green 1 system (Roche) as described previously (16). Expression of 18S rRNA was used to correct for differences in the amount of total RNA added to a reaction and to compensate for different levels of inhibition during RT of RNA and PCR. For PCR analysis, sample cDNA was diluted 1:5-fold (PC5/6) and 1:200-fold (18S rRNA). The diluted cDNA template (4 µl) was added to sterile capillaries to a total volume of 20 µl, containing PCR mastermix (Roche), including SYBR Green I, dNTPs, Taq enzyme, and reaction buffer, supplemented with optimal concentrations of MgCl2 and specific primers (5 pmol) (Table 1
). An initial denaturing step was performed for 10 min at 95 C, before 40 cycles of 95 C for 15 sec, 58–60 C for 5 sec, and 72 C for 10–22s (elongation time specific to primer pair; Table 1). The standard curve method was used to quantify the expression of the PC5/6 mRNA and 18S rRNA in each sample. PCR of all standards and samples was performed using duplicate reactions for 40 cycles, after which a melting curve analysis was performed to monitor PCR product purity (Table 1). In initial experiments, PCR product identities were verified by agarose gel electrophoresis and DNA sequencing. The relative-quantitative data were expressed as the ratio of the level of PC5/6 mRNA to that of 18S rRNA in arbitrary units.
Immunocytochemistry for PC5/6
Trypsinized ESCs were cytocentrifuged on poly-L-lysine-treated glass slides. Cells were fixed with 70% ethanol and allowed to air dry. The cells were rehydrated in distilled water and treated with hydrogen peroxide (3%) to block endogenous peroxide activity. They were then washed with Tris-buffered saline (TBS; pH 7.6) for 10 min before incubation with a blocking solution of 10% normal swine serum/10% fetal calf serum in TBS for 30 min followed by incubation with rabbit anti-PC5/6 (1:200, Alexis, San Diego, CA), which was generated against residues 27–540 of recombinant mouse PC5/6, diluted in the blocking solution at 37 C for 1 h. Normal rabbit serum replaced the primary antibody as negative control. The cells were washed in TBS for 5 min, then TBS-Tween 20 (0.6%) for 10 min, followed by TBS for 5 min before incubation with biotinylated swine antirabbit Ig (1:300, Dako, Glostrup, Denmark) for 30 min at room temperature, followed by an avidin-biotin-complex conjugated to horseradish peroxidase (Dako) according to the manufacturer’s instructions. Peroxidase activity was visualized after the application of diaminobenzidine tetrahydrochloride substrate (Dako) for 3 min. Cells were counterstained with Harris hematoxylin, dehydrated, and mounted. Microscopy was performed using an Olympus BX50 microscope fitted with a Fujix HC-2000 high-resolution digital camera.
Immunoprecipitation and Western blotting
To confirm the specificity of the antibody used for immunocytochemistry, immunoprecipitation and Western blotting were performed as described previously (17, 18). In brief, E2 plus P-treated ESCs on d 12 of culture (decidualized ESCs) were solubilized in lysis buffer (50 mmol/liter Tris-HCl, 150 mmol/liter NaCl, 2 mmol/liter EDTA, 2 mmol/liter EGTA, and 25 mmol/liter NaF) with a protease inhibitor cocktail (Calbiochem, San Diego, CA). Precleared lysates were sequentially incubated with rabbit anti-PC5/6 (1:200, Alexis) at 4 C for 1 h and protein G-agarose (Roche) at 4 C for 1 h. Mouse uterine proteins (15 µg/lane) of implantation sites on d 5.5 of pregnancy, in which PC5/6 was highly expressed (11), were extracted for the positive control as previously described (18). Immunoprecipitates and lysates were separated by 8% SDS-PAGE with molecular weight markers and then transferred to a nitrocellulose membrane (Amersham, Sydney, Australia). After blocking for 1 h in blocking buffer of TBS-Tween 20 (0.1%) with 5% skim milk, the filters were incubated for 1 h in blocking buffer with sheep anti-PC5/6 (25 µg/ml), which was generated against a peptide DYDLSHAQSTY, corresponding to residues 117–127 of mouse PC 5/6 (19). Western blots were then incubated with a horseradish peroxidase-conjugated donkey antisheep IgG (1:20,000, Silenus Laboratories, Hawthorn, Australia) and developed by chemiluminescence (ECL Plus system, Amersham).
MO treatment
PC5/6 MOs (5'-GCAGCAGCGGCTCTCCCAATCCATG-3') were designed to target at the initiation site for PC5/6 translation and synthesized by Gene Tools (Philomath, OR). The specificity of the MOs was validated by employing two control MOs: the invert of the PC5/6 antisense sequence (PC5/6-Inv), 5'-GTACCTAACCCTCTCGGCGACGACG-3', and a standard control of irrelevant sequence (control), 5'-CCTCTTACCTCAGTTACAATTTATA-3'. ESCs were initially treated with 1.4 µmol/liter MOs and 0.56 µmol/liter ethoxylated polyethylenimine (EPEI) delivery solution according to the manufacturer’s instructions. However, this treatment resulted in significant cell death (data not shown), presumably because of excessive EPEI, which can be toxic (20). Subsequently, ESCs were treated with 2.5 µmol/liter MOs and 0.38 µmol/liter EPEI because it had previously been reported that increasing the MO concentration and decreasing the EPEI concentration can reduce toxicity while retaining measurable delivery (20). MOs were transfected into ESCs with EPEI on d 9 after E2 plus P treatment in 24-well plates and the cultures maintained until d 12. Studies were performed with three separate cultures, each in triplicate wells. The supernatants were collected at d 9 and 12 and stored at –70 C until assayed. At the end of the experiment, cell number and viability were assessed by trypan blue exclusion.
To examine intracellular MOs localization and transfection efficiency, ESCs were seeded on coverslips in 24-well plates, and the treatments were carried out when the cells were nearly confluent. ESCs were treated with either fluorescein isothiocyanate (FITC) control MOs or unlabeled control MOs as described above. The coverslips were washed twice with PBS after 24 h MOs treatment, and the cells were then stained with 100 nmol/liter 4',6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene, OR) for 5 min at room temperature in the dark. The coverslips were mounted onto slides using FluorSave reagent (Calbiochem) and examined at 488 nm using a fluorescence microscope, fitted with the Analytical Imaging Station (Imaging Research Inc., St. Catherine, Ontario, Canada) software.
Statistical analysis
Data are expressed as mean ± SEM. Results were analyzed with a statistical software package, StatView II version 4.0 (Abacus Concepts, Inc., Berkeley, CA). Differences in the measured parameters across the different groups were statistically assessed using ANOVA with repeated measurements, followed by Fisher’s protected least significant difference multiple range test. A level of P < 0.05 was considered statistically significant.
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Results |
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Cultured human ESCs undergo differentiation in response to P after E2 priming. The induction of PRL production, a typical marker for decidualization, was detected after 6 d culture (Fig. 1A), confirming published data. The presence of PC5/6 mRNA was determined in nondecidualized and decidualized ESCs using RT-PCR. A PC5/6 transcript corresponding to the expected 545-bp size was present in the nondecidualized and decidualized ESCs on d 12 (Fig. 1B). The bands were excised from the gel, purified, and directly sequenced to confirm their identity (data not shown).
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PC5/6 mRNA levels in ESCs during decidualization were determined using quantitative real-time RT-PCR (Fig. 2). Addition of E2 to the culture medium had no significant effect on the levels of PC5/6 mRNA expression. In contrast, E2 and P caused a significant increase in PC5/6 mRNA after 9 d of culture, and this continued to increase until the end of these studies at 12 d.
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ESCs treated with E2 plus P for 12 d underwent characteristic morphological changes, from elongated spindle-shaped cells to characteristic enlarged polygonal cells. Decidualized and cytocentrifuged ESCs retained enlarged nuclei and an increased amount of cytoplasm compared with nondecidualized ESCs (Fig. 3, A–C). Immunocytochemistry showed the localization of PC5/6 in the cultured ESCs on d 12 of the culture period (Fig. 3, A–C). Consistent with the results of real-time PCR, intense PC5/6 immunoreactivity was observed in the cytoplasm of E2 plus P-treated ESCs on d 12 of culture (decidualized cells) (Fig. 3B). In contrast, weak PC5/6 immunoreactivity was observed in E2-treated ESCs on d 12 of culture (nondecidualized cells) (Fig. 3A) and in E2 plus P-treated ESCs on 3 d of culture (data not shown). To determine whether PR is necessary for induction of PC5/6 protein during decidualization, ESCs were cultured for 12 d with E2 plus P plus ZK 98299, a PR antagonist. Cotreatment of cells with ZK 98299 blocked both the change in morphology and the increase in PC5/6 protein (Fig. 3C). Western blot analysis detected PC5/6 protein in mouse implantation sites on d 5.5 of pregnancy, as the positive control, and in immunoprecipitates of decidualized ESCs (Fig. 3L). The results showed bands around 120 kDa, representing pro- (126 kDa) and mature (117 kDa) forms, and a 65-kDa band that represents a C-terminally truncated form as previously reported (19). These data also show that use of the sheep antibody raised against the mouse N-terminal antigen, which has two amino acids different from the human sequence, allows detection by Western blot after immunoprecipitation.
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To investigate whether the induction of PC5/6 was necessary for decidualization in vitro, MOs were used to block PC5/6 synthesis in cultured ESCs. Transfection efficiency was monitored by examining immunofluorescence in cells treated with FITC control MOs (Fig. 3, E–H). The FITC label was located in the cytoplasm of ESCs after 24 h of MO treatment (Fig. 3E), whereas no fluorescence was observed in cells treated with unlabeled MOs (Fig. 3F). To determine the proportion of cells transfected, DAPI was applied to stain the nuclei (Fig. 3G); virtually all DAPI-positive cells also showed FITC labeling in the cytoplasm (Fig. 3H). This indicates successful delivery into the cytoplasmic site of antisense action.
Effects of PC5/6 MOs on PRL production from decidualized ESCs
Treatment with MOs was limited to 3 d because extending the period of treatment to >4 d resulted in significant cell death (data not shown). Because PC5/6 mRNA expression rose in parallel with PRL secretion and significantly increased after 9 d of culture in the presence of E2 plus P (Fig. 2), MOs were transfected into ESCs with EPEI at d 9 after the start of E2 plus P treatment (Fig. 4A). Immunocytochemistry demonstrated a depletion of PC5/6 in ESCs treated with PC5/6 MOs compared with control or Inv-PC5/6 MOs on d 12 (Fig. 3, I–K). PRL production at d 12 was significantly inhibited in ESCs treated with PC5/6 MOs from d 9 of culture compared with those treated with control or Inv-PC5/6 MOs (Fig. 4B). However, PRL production for these treatment groups was not significantly different during d 6–9, before the addition of MOs (Fig. 4C). The number of viable cells was not significantly different between different treatment groups at d 12 of culture (PC5/6 MOs, 100.8 ± 1.8%; Inv-PC5/6 MOs, 100.6 ± 3.1% compared with control MOs 100%, across three separate experiments). Taken together, these results suggest that PC5/6 action is necessary for PRL production from decidualized ESCs.
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Discussion |
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Decidualization is an essential preparative event for blastocyst implantation and establishment of pregnancy and represents differentiation of stromal cells to a distinct morphological appearance accompanied by a unique biosynthetic and secretory phenotype (4, 21). Understanding the cellular and functional changes in ESCs will provide clues to the mechanisms underlying this process as well as potential pathological changes preventing implantation. Gene-targeting studies have identified several genes with possible roles in decidualization. These models include mice lacking leukemia inhibitory factor, prostaglandin synthase 2, epidermal growth factor (EGF) receptor, IL-11 receptor 
, and Hoxa-10 (22, 23, 24, 25, 26).
Recent studies have also aimed at identifying differentially expressed genes associated with the decidualization process in human endometrium (3, 5). During the process of decidualization, up-regulation of IGF family members, vascular endothelial growth factor (VEGF) family members, EGF family members, transforming growth factor-ß family members, and their receptors were found (3, 5). In addition, up-regulation of neurotransmitter receptors, neuromodulators, renin/angiotensin family, integrins, and matrix metalloproteinases were observed. These up-regulated factors are believed to participate in autocrine/paracrine signaling in the endometrium during normal cycles and in early pregnancy (1, 2). Furthermore, many of these proteins are synthesized as inactive precursors that are subsequently converted to their mature active forms by proteolytic enzymes known as PCs (6, 7, 9). According to the known functions of PCs, PC5/6 expression in decidualized ESCs may determine the cascade of events leading to the tissue-specific production of biologically active peptides and proteins essential for decidualization.
In this study, we examined the function of PC5/6 on PRL production from decidualized ESCs using an antisense morpholino approach. MOs have been reported to be a successful tool in reducing levels of their specific target proteins by blocking translation of mRNA in zebra fish embryos, in cell culture, and during implantation in vivo (20, 27, 28). Our results showed that PRL production was significantly attenuated in decidualized ESCs treated with PC5/6 MOs compared with those of control or Inv-PC5/6 MOs (Fig. 4B). This inhibition was not due to cytotoxicity for MOs treatment because ESCs cultured in the presence of MOs showed no evidence of significant change in viable cell number. These results suggest that the role of PC5/6 on PRL production from decidualized ESCs involves autocrine or paracrine mechanisms.
What is the mechanism underlying the inhibition of PRL production with depletion of PC5/6? It is suggested that PC5/6 is involved in proteolytic processing and hence activation of a number of growth factors and receptors previously associated with decidualization, including EGF and activin A (9, 29), whose up-regulation has been reported in microarray studies comparing nondecidualized with decidualized ESCs in vitro (3, 5). This is consistent with immunoreactivity of those growth factors in decidualizing ESCs and in the decidua of early pregnancy in vivo (16, 30). Moreover, using the same in vitro model of decidualization as used in the present study, EGF and activin A promote decidualization of ESCs in the presence of progestin (14, 31). Thus, the decrease of PRL production when PC5/6 is inhibited in the present study may be caused via effects on EGF and activin A whose bioactivity is dependent on posttranslational modification. However, other as yet unidentified factors with appropriate spatial and temporal expression during decidualization are also potential substrates for PC5/6.
PC5/6 may also be involved in the establishment of pregnancy, because its substrates include precursors to VEGF and heparin-binding EGF (9). The ESC-derived heparin-binding-EGF stimulates epithelial expression of the key endometrial proteins for uterine receptivity, including LIF, HOXA-10, and the ß3 integrin subunit (32). The level of VEGF mRNA expression also increases in decidualized ESCs in comparison with nondecidualized ESCs (3). VEGF is expressed in the endometrium and probably participates in the increased angiogenesis and vascular permeability necessary for successful implantation (1). In addition, PC5/6 can potentially cleave a variety of other precursors, including pro-mullerian-inhibiting substance, prorenin, proneurotensin, pro-PTPµ receptor, pro-cholecystokinin, integrin pro- subunits, human immunodeficiency virus gp160, ß-amyloid-converting enzyme, and transforming growth factor-ß like Lefty (9, 33). Although we have carried out Western blot analysis for activin A in the decidualized cells and shown that both the pro- and the mature-forms are present, at this time we have no conclusive evidence to support its processing by PC5/6 (Okada, H., G. Nie, and L. A. Salamonsen, unpublished data). Such possibilities are a focus of ongoing studies to define the mechanisms of PC5/6 action in the endometrium; however, potential redundancy with other PCs such as furin in the processing of pro-proteins make this a challenging venture.
In summary, we revealed that PC5/6 mRNA and protein expression was up-regulated during decidualization of human ESCs in vitro and that this induction of PC5/6 was necessary for decidualization. These findings provide evidence not only for an integral role for PC5/6 in decidualized ESCs but also insights into important functions for PC5/6 in the endometrium. Further investigation is required to establish the specific substrates and the precise role of PC5/6 during decidualization and implantation. The knowledge will be benefit our understanding of key reproductive processes such as decidualization and the establishment and maintenance of early pregnancy and those of endometrial dysfunction such as infertility and menstrual disorders.
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Acknowledgments |
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Footnotes |
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Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole; E2, 17ß-estradiol; EGF, epidermal growth factor; EPEI, ethoxylated polyethylenimine; ESC, endometrial stromal cell; FITC, fluorescein isothiocyanate; MO, morpholino antisense oligonucleotide; P, medroxy-progesterone acetate; PC, proprotein convertase; PR, progesterone receptor; PRL, prolactin; TBS, Tris-buffered saline; VEGF, vascular endothelial growth factor.
1 Nie G, Li Y, Wang M, Liu YX, Findlay JK, Salamonsen LA 2004 Inhibiting uterine PC6 blocks embryo implantation: an obligatory role for a proprotein convertase in fertility. Biol Reprod 2004 Dec 15 (Epub ahead of print) 
This work was supported by Grants 143798 (to L.A.S.) and 241000 (to G.N.) from the National Health and Medical Research Council of Australia and by the World Health Organization/Rockefeller Foundation Initiative on Implantation.
Received May 12, 2004.
Accepted October 27, 2004.
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