The Endometrial Polarity Paradox: Differential Regulation of Polarity Within Secretory-Phase Human Endometrium

The Endometrial Polarity Paradox: Differential Regulation of Polarity Within Secretory-Phase... Abstract A major cause of infertility in normal and assisted reproduction cycles is failure of the endometrium to undergo appropriate changes during the secretory phase of the menstrual cycle as it acquires receptivity for an implanting blastocyst. Current dogma states that loss of epithelial polarity in the luminal epithelial cells, the point of first contact between maternal endometrium and blastocyst, may facilitate embryo implantation. Loss of polarity is likely an important change during the secretory phase to overcome mutual repulsion between otherwise polarized epithelial surfaces. Although “plasma membrane transformation” describes morphological/molecular alterations associated with loss of polarity, direct measures of polarity have not been investigated. Transepithelial resistance, a proxy measure of polarity, was downregulated in endometrial epithelial (ECC-1) cells by combined estrogen/progestin, mimicking the hormonal milieu of the secretory phase. Examination of defined polarity markers within human endometrium throughout the menstrual cycle identified downregulation of atypical protein kinase C, Stardust, Crumbs, and Scribble within the luminal-epithelial layer, with upregulation of Scribble within the stromal compartment as the menstrual cycle progressed from the estrogen-dominated proliferative to progesterone-dominated secretory phase. Epithelial (ECC-1) Scribble expression was downregulated in vitro by combined estrogen/progestin and estrogen/progestin/human chorionic gonadotropin treatment, whereas knockdown of Scribble in these cells enhanced “embryo” (trophectodermal spheroid) adhesion. In contrast, Scribble was upregulated within decidualized primary human endometrial stromal cells, with decidualization downregulated upon Scribble knockdown. These data highlight an important contribution of polarity modulation within the human endometrium, likely important for receptivity. Clinical investigations examining how polarity may be modulated in the infertile endometrium may facilitate fertility. The processes and mechanisms underlying human embryo implantation remain enigmatic despite intense research effort directed at understanding this complex process. This is partially due to the unique and transient nature of endometrial preparation for embryo implantation, which is controlled by cyclic variations in ovarian steroid hormones and their impact on local endometrial factors. Although embryos can attach and implant with ease into other tissues within the human body, their attachment to the human uterine luminal epithelium and subsequent implantation is limited to ∼4 days during the secretory phase of each menstrual cycle when the endometrium becomes “receptive,” a span of time also termed the “window of implantation” (1). Before this specific window, the endometrium is hostile, and afterward it remains permissive to implantation but less likely to result in live birth, as observed during the late secretory phase of the menstrual cycle (2). Within an idealized 28-day menstrual cycle, changes for receptivity occur under the influence of rising progesterone (in the presence of estrogen) following ovulation, and the receptive window encompasses approximately days 19 to 23 of the menstrual cycle (luteinizing hormone+7-10). However, with variation in timing of menstrual cycles between women and even between different cycles within the same woman, there is clearly considerable scope for variation in the timing of endometrial receptivity. Nowhere is this more clear than in assisted reproductive technology cycles, in which, despite the use of genetically normal embryos, implantation failure is commonly attributed to inadequate endometrial receptivity (3–5). Indeed, it is thought that up to 75% of pregnancy failures represent a failure of implantation (6, 7). Recent advances have been made in attempts to characterize changes during the secretory phase of the cycle leading toward and encompassing the implantation window by genetic analysis of endometrial tissue. Despite gene array analyses of the endometrium demonstrating little concordance in up- or downregulated genes at the time of receptivity [reviewed in Altmäe, et al. (8)], an endometrial receptivity array provides a genetic signature of receptivity that can determine if the endometrium is “out of phase” and therefore nonreceptive (9). However, although the endometrial receptivity array test can be used to “diagnose” a receptive endometrium, it does not provide enlightenment on the mechanisms underlying attainment of receptivity and particularly the intricate changes that occur within different endometrial cellular compartments. This is an important issue when we consider that the point of first contact between the maternal endometrium and the embryo, the luminal epithelial layer, must achieve adhesion competence during the secretory phase of the cycle under the influence of progesterone to facilitate attachment, whereas the underlying stroma must undergo an appropriate degree of decidual differentiation to act as a “quality control” mechanism ensuring subsequent development of only high-quality embryos (10–12). Understanding of how these processes may be modulated is critical for both enhancing receptivity and therefore fertility, and for maintaining the endometrium in a state refractory to implantation in the development of novel contraceptive strategies. Cellular transformation during the process of embryo implantation presents a cell biological paradox in a number of aspects. Both the trophectodermal layer of the preimplantation embryo (the blastocyst) and the uterine luminal epithelium present with a polarized phenotype. Generally, epithelial layers are polarized to maintain a barrier function and, in specialized cells, directional secretion of proteins. Throughout most of the cycle, the endometrial luminal epithelium is specialized to resist adhesion. However, occurring progressively throughout the secretory phase under the influence of progesterone, this epithelial layer must develop adhesion competence at its apical pole, which is related to cellular rearrangements that result in modulation of their apico-basal polarity as part of the plasma membrane transformation (13). Indeed, a number of “polarity-related” changes have been observed within this luminal epithelium. In marmosets, the electronegativity of the uterine epithelium decreases progressively from the preovulatory stage in preparation for implantation with an associated increase in its adhesive capacity, due to enhanced adhesion as repulsive charges on opposing membranes are decreased (14). In association with these charge changes in the luminal epithelium, a partial epithelial-to-mesenchymal transition (EMT) is observed, associated with a downregulation of E-cadherin (15) and, paradoxically, a reduction in paracellular permeability, while the cells of the epithelial layer become less firmly adhered to each other (16). Downregulation of E-cadherin is a common phenomenon in EMT; however, endometrial epithelial cells do not downregulate their complete epithelial program, but only selected parameters, such that epithelial identity is maintained (17). Conversely, upon initiation of hormone-directed differentiation, the endometrial stroma undergoes a mesenchymal-to-epithelial transition (MET) during the decidualization process. Therefore, the opposing processes of EMT and MET are occurring within adjacent endometrial cellular compartments under the influence of the same endocrine and local milieu. Dogma within this area consistently states that apico-basal polarity is altered within the luminal epithelium during the mid to late secretory phases, with loss of polarity typically associated with EMT. However, to date, specific intracellular determinants of epithelial polarity have not been investigated within the human endometrium in the transition from a nonreceptive state during the proliferative phase of the cycle to a prereceptive and subsequently receptive/permissive state during the secretory phase of the cycle. Cellular polarity is governed by an asymmetrical arrangement of intracellular polarity molecules within specific regions of the cell to specify apical and basal cellular identity of the cell and thus present a polarized epithelial-sheet phenotype (Fig. 1). Maintenance of this apico-basal polarity is dependent on appropriate intracellular localization of polarity complexes. The Crumbs and Par complexes specify the apical domain, whereas the basolateral domain is specified by the Scribble complex (Fig. 1). A complex interplay of positive feedback, resulting in recruitment of polarity determinants to their cellular poles, and negative feedback, resulting in mutually antagonistic interactions between the complexes, within each domain, is required to maintain cellular polarity (Fig. 1). Figure 1. View largeDownload slide Localization and mutual antagonism of polarity determinants within epithelial cells. Much of the background on interactions of polarity determinants has been derived from examination of interactions within Drosophila epithelium; it is presumed that many of these interactions translate to mammalian species. The major plasma membrane domains comprise the apical and basolateral domains. The main apical complexes are the Crumbs (Crb; orange) and Par (green) complexes, whereas the main basolateral complex is the Scribble (Scrib; pink) complex. Subconstituents of each of these complexes are indicated. Mutually antagonistic interactions (“T” bars) between apical and basolateral polarity determinants form and maintain the apical-basolateral axis. Some apical polarity determinants interact with tight junctions (TJ; yellow) and adherens junctions (AJ; purple) in a supportive manner (double-headed arrows). Specific subconstituents of the polarity complexes are involved in these interactions, including Bazooka (Baz) and PALS1-associated tight junction protein (Patj; tight junctions) and atypical PKC (αPKC) and Bazooka (adherens junctions). Desmosomes (D) are also shown (blue); however, the manner in which polarity determinants may interact with these is unclear. This schematic represents a “best working knowledge” model; however, specifically how these polarity determinants interact with each other and with intercellular junction proteins is still being determined in mammalian cells. Data from Lamouille et al. (18). Dlg, discs large; Lgl, lethal giant larvae; Par6, partition defective gene-6; Std, Stardust. Figure 1. View largeDownload slide Localization and mutual antagonism of polarity determinants within epithelial cells. Much of the background on interactions of polarity determinants has been derived from examination of interactions within Drosophila epithelium; it is presumed that many of these interactions translate to mammalian species. The major plasma membrane domains comprise the apical and basolateral domains. The main apical complexes are the Crumbs (Crb; orange) and Par (green) complexes, whereas the main basolateral complex is the Scribble (Scrib; pink) complex. Subconstituents of each of these complexes are indicated. Mutually antagonistic interactions (“T” bars) between apical and basolateral polarity determinants form and maintain the apical-basolateral axis. Some apical polarity determinants interact with tight junctions (TJ; yellow) and adherens junctions (AJ; purple) in a supportive manner (double-headed arrows). Specific subconstituents of the polarity complexes are involved in these interactions, including Bazooka (Baz) and PALS1-associated tight junction protein (Patj; tight junctions) and atypical PKC (αPKC) and Bazooka (adherens junctions). Desmosomes (D) are also shown (blue); however, the manner in which polarity determinants may interact with these is unclear. This schematic represents a “best working knowledge” model; however, specifically how these polarity determinants interact with each other and with intercellular junction proteins is still being determined in mammalian cells. Data from Lamouille et al. (18). Dlg, discs large; Lgl, lethal giant larvae; Par6, partition defective gene-6; Std, Stardust. This study investigated the expression and localization of polarity proteins within the human endometrium across the menstrual cycle, including the proliferative phase, when the endometrium is restored following menstruation, the early secretory phase, when epithelial differentiation for receptivity is initiated, and the receptive mid secretory phase and permissive late secretory phase of the cycle, to determine if apico-basal polarity is altered via modulation of polarity proteins. We identified cyclic changes in both apical [atypical protein kinase C (PKC); Stardust and Crumbs] and basolateral determinants (Scribble) within the epithelial and stromal compartments. We further investigated the hormonal regulation and functional role of the basolateral determinant, Scribble, in endometrial function and demonstrated: (1) its differential regulation by ovarian steroid hormones in epithelial (ECC-1 endometrial epithelial cell line) and primary human stromal cells; (2) its functional role in embryo adhesion; and (3) its contribution to decidual transformation of endometrial stromal cells. This study confirms that apico-basal polarity is altered within both the luminal epithelial and stromal compartments of the endometrium during the secretory phase of the cycle, encompassing the window of receptivity, and that these changes are functionally important for embryo implantation and stromal cell decidualization. Materials and Methods Ethics and tissue collection Ethical approval was obtained for all tissue collections from the Institutional Ethics Committees at Monash Health and Monash Surgical Private Hospital. Written informed consent was obtained from all subjects prior to tissue collection. Endometrial tissue collection and patient details Endometrial biopsies for immunohistochemistry were collected by curettage from normally cycling women throughout the menstrual cycle, covering the proliferative (n ≥ 10), early secretory (n ≥ 10), mid secretory (n ≥ 10), and late secretory (n ≥ 10) phases of the menstrual cycle. By this method, it is anticipated that the functionalis and a small amount of basalis endometrium would be sampled. The women had no known endometrial pathologies, regular menstrual cycles (28 to 34 days), and at least one parous pregnancy. All women were under 40 years of age and had not received steroid hormone therapy in the last 6 months. The biopsies were fixed in 10% formalin for 24 hours prior to processing to paraffin wax. All women were determined to have morphologically normal endometrium. Menstrual cycle stage was assessed by standard histological dating by a highly experienced gynecological pathologist. Immunohistochemistry Five-micrometer-thick tissue sections were placed onto superfrost slides and dried overnight at 37°C. Sections were dewaxed in Histosol and rehydrated with decreasing concentrations of ethanol (100% to 70%) to distilled water. Citrate antigen retrieval was performed, as determined by prior optimization, for each antibody (Table 1) in citrate buffer (10 mM citric acid, pH 6.0), followed by incubation in hot buffer for 20 minutes. Endogenous peroxidase activity was blocked by incubation of tissue sections in 3% hydrogen peroxide for 10 minutes at room temperature. Nonspecific binding was subsequently blocked with nonimmune serum [10% nonimmune serum (Table 1), 2% human serum, Tris-buffered saline (TBS)] for 60 minutes at room temperature. Sections were then incubated overnight in a humidity chamber at 4°C with the appropriate primary antibody or immunoglobulin G negative control, as determined by prior optimization (Table 1). Subsequently, sections were thoroughly washed in TBS 0.2% Tween 20 (TBST) and incubated at room temperature for 60 minutes with a biotinylated secondary antibody (Table 1). Sections were rewashed with TBS 0.2% Tween 20 and an avidin/biotin-peroxidase detection system [Vectastain Elite ABC Kit (Standard; catalog no. PK-6100; Vector Laboratories, Inc., Burlingame, CA)] applied for 30 minutes at room temperature. Immunostaining was then performed by addition of the peroxidase substrate 3,3′-diaminobenzidine (catalog no. K3468; Dako, Agilent, Santa Clara, CA). Sections were counterstained with hematoxylin and dehydrated in increasing concentrations of ethanol (70% to 100%) and Histosol, with coverslips mounted using DPX. Imaging used an Olympus BX53 microscope at ×20 magnification. Immunohistochemical staining was assessed within the glandular epithelium, luminal epithelium, and stroma using Image J. Average immunostaining and standard error of the mean (SEM) were calculated for each protein, within each cellular compartment (glandular epithelium, luminal epithelium, and stroma) across each phase of the menstrual cycle. Table 1. Antibodies Used 1° Ab, Catalog No. (RRID)  Nonimmune Serum  1° Antibody Species  Concn 1° Antibody  Negative Control  2° Antibody, Batch  α-PKCζ (C-20), SC-216 (RRID: AB_2300359)  Goat  Rabbit  1 µg/mL  Rabbit IgG  Goat anti-rabbit, BA-1000  Crumbs (N-20), SC-27901 (RRID: AB_2261069)  Horse  Goat  1 µg/mL  Goat IgG  Horse anti-goat, BA-9500  Hugl-1 (B-6), SC-136993 (RRID: AB_2135864)  Horse  Mouse  2 µg/mL  Mouse IgG  Horse anti-mouse, BA-2000  Scribble, ab115240 (RRID: AB_11155336)  Goat  Rabbit  1 µg/mL  Rabbit IgG  Goat anti-rabbit, BA-1000  Stardust, LS-C152791 (RRID: AB_2687873)  Goat  Rabbit  1:100  Rabbit IgG  Goat anti-rabbit, BA-1000  PAR3, HPA030443 (RRID: AB_10600926)  Goat  Rabbit  1:5000  Rabbit IgG  Goat anti-rabbit, BA-1000  1° Ab, Catalog No. (RRID)  Nonimmune Serum  1° Antibody Species  Concn 1° Antibody  Negative Control  2° Antibody, Batch  α-PKCζ (C-20), SC-216 (RRID: AB_2300359)  Goat  Rabbit  1 µg/mL  Rabbit IgG  Goat anti-rabbit, BA-1000  Crumbs (N-20), SC-27901 (RRID: AB_2261069)  Horse  Goat  1 µg/mL  Goat IgG  Horse anti-goat, BA-9500  Hugl-1 (B-6), SC-136993 (RRID: AB_2135864)  Horse  Mouse  2 µg/mL  Mouse IgG  Horse anti-mouse, BA-2000  Scribble, ab115240 (RRID: AB_11155336)  Goat  Rabbit  1 µg/mL  Rabbit IgG  Goat anti-rabbit, BA-1000  Stardust, LS-C152791 (RRID: AB_2687873)  Goat  Rabbit  1:100  Rabbit IgG  Goat anti-rabbit, BA-1000  PAR3, HPA030443 (RRID: AB_10600926)  Goat  Rabbit  1:5000  Rabbit IgG  Goat anti-rabbit, BA-1000  Abbreviations: Ab, antibody; BA, batch; Concn, concentration; IgG, immunoglobulin G; RRID, Research Resource Identifier. View Large Cell culture ECC-1 endometrial epithelial cells are an endometrial cancer cell line with characteristics of the endometrial luminal epithelial layer. These cells were obtained from the American Type Culture Collection and their validity independently validated via short tandem repeat DNA profiling of human cell lines per American Type Culture Collection guidelines (19). They were routinely maintained in a 1:1 mix of Dulbecco’s modified Eagle medium (DMEM)/F12 Glutamax (Gibco, Invitrogen, Mt Waverley, VIC, Australia) supplemented with 1% volume-to-volume ratio (v/v) penicillin/streptomycin and 10% v/v fetal calf serum (FCS; Gibco, Invitrogen). These cells were seeded and used for experimental purposes as described later. Trophectodermal cells [kind gift of Susan Fisher (20)] are derived from trophoblast stem cells. They were routinely maintained in a 1:1 mix of DMEM:F12 Glutamax (Gibco, Invitrogen) supplemented with 1% v/v penicillin/streptomycin and 10% v/v FCS with addition of 10 ng/mL bovine fibroblast growth factor and 10 uM SB431542 (R&D Systems, In Vitro Technologies, Melbourne, Australia). Cells were grown on flasks coated with 0.5% gelatin (Sigma-Aldrich, NSW, Australia) prior to experimental seeding. Primary cell culture Endometrial biopsies for cell isolations were collected as described previously, from women meeting the same inclusion criteria, from the proliferative and early secretory phases of the menstrual cycle. Tissue biopsies were finely scissor minced and incubated in phosphate-buffered saline containing 7.5 IU/mL collagenase III (Sigma-Aldrich) and 100 mg/mL DNase I (Worthington, Lakewood, NJ) at 37°C with shaking at 130 rpm for 40 minutes. Digestion was stopped by addition of excess DMEM/F12 (Invitrogen). Digested samples were sequentially vacuum filtered through 45- and 11-μm filters to remove debris and epithelial clusters, before collection of the stromal cell pellet by centrifugation. Cells were sequentially seeded in DMEM/F12 media containing 10% charcoal-stripped FCS (csFCS; Invitrogen) and 1% penicillin/streptomycin (Sigma) into sterile cell culture flasks for 25 minutes to allow cell attachment before removal of the blood contamination. Stromal cells were then seeded into six-well plates and used in two different experimental paradigms: (1) Stromal cells (n = 5 separate preparations) were treated with 10–8 M 17β-estradiol (henceforth referred to as estrogen)/10–7 M medroxyprogesterone acetate (MPA; to mimic progesterone, henceforth referred to as progestin; Sigma-Aldrich) in DMEM/F12 with 2% csFCS (decidualization media) for 2 or 12 days, media collected for prolactin (PRL) assay (later), and cells lysed for Western immunoblot analysis (later) and (2) stromal cells (n = 5 separate preparations) were transfected with small interfering RNA (siRNA)–targeting Scribble (or control scramble sequence; protocol outlined later) and then treated with decidualization media for 12 days followed by media collection for PRL assay (later). Transepithelial resistance assays ECC-1 cells (2 × 105) were seeded onto polyester bicameral chambers (12 mm, 0.4-μm pore; Sigma) and coated with fibronectin according to the manufacturer’s protocol (BD Bioscience) prior to cell seeding. Cells were allowed to attach overnight in DMEM/F12 medium containing 10% FCS, confluency checked, and then incubated in DMEM/F12 with 0.5% csFCS in both the basal and apical chambers. Cells were incubated for a further 24 hours followed by assessment of baseline transepithelial resistance (TER). To assess the integrity of interepithelial cell-tight junctions, TER was quantified using a Millipore Millicell-Electrical Resistance System (Millipore), with measurements taken daily. Cells and media were maintained at 37°C; following removal from the incubator, cells were equilibrated on a warming plate within the culture hood for a minimum of 30 minutes before TER measurement. After basal readings, 24 hours post media change to 0.5% csFCS DMEM/F12, cells were treated with estrogen for 24 hours followed by assessment of TER. Cells were subsequently maintained in estrogen or treated with estrogen/progestin for a further 48 hours with TER readings taken every 24 hours. Changes in TER were calculated as a percentage of basal readings for each individual well. Duplicate culture wells were used for each treatment, and the entire experiment was performed four times (n = 4). Cell transfection with Scribble siRNA Both ECC-1 cells and primary stromal cells were transfected with Scribble siRNA (Santa-Cruz Biotechnology, Dallas, TX). ECC-1 cells were seeded at 2 × 105 cells/well in 12-well plates. Primary stromal cells were seeded into six-well plates and grown to 80% confluence. All cells were seeded in media without penicillin/streptomycin. Initially, lyophilized siRNA/scramble sequence was resuspended in RNAse free water to a final concentration of 10 uM. Two microliters of siRNA/scramble was mixed with 100 uL siRNA transfection media and incubated for 5 minutes. Concurrently, 2 uL of the transfection reagent (lipofectamine; Invitrogen) was mixed with 100 uL siRNA transfection media and incubated for 5 minutes. These two solutions were then mixed by pipetting and incubated at room temperature for 30 minutes. After incubation, the transfection solution was mixed with 800 uL of siRNA transfection media, cells washed with siRNA transfection media, and the transfection mix added to the cells for 8 hours. All volumes were doubled for transfection of primary stromal cells in six-well plates. After the incubation period, media were replaced with 10% FCS or csFCS media and cells maintained for 48 hours with daily visual checking of cell viability. ECC-1 cells were then used for spheroid adhesion assays (later), and primary stromal cells were decidualized for 12 days in decidualization media as previously. Western immunoblot ECC-1 cells were seeded at 1 × 106 cells in six-well plates, allowed to adhere overnight, and then deprived of serum for 8 hours. Cells were then treated with a vehicle control (ethanol) or primed with estrogen for 24 hours. After estrogen, priming cells were (1) continued in estrogen (2) treated with estrogen/progestin, and (3) treated with estrogen/progestin/10 IU human chorionic gonadotropin (hCG; to represent peri-implantation blastocyst secretion), each for 24 hours. Cells were then lysed in radioimmunoprecipitation assay buffer with protease inhibitors, lysates were clarified by centrifugation at 14,000 rpm at 4°C for 15 minutes, and supernatants were retained. Twenty-five micrograms of lysate from each treatment condition was loaded onto 4% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and run at 100 V for ∼90 minutes. Proteins were blotted to polyvinylidene difluoride membrane using a transblot turbo and washed thoroughly in TBST. Nonspecific binding was then blocked by incubation in 5% nonfat milk/TBS for 1 hour at room temperature. Membranes were again washed to remove excess block solution and incubated overnight at 4°C with anti-Scribble antibody at 1:500 dilution in TBST. Membranes were washed thoroughly and incubated with a goat anti-rabbit horseradish peroxidase–labeled antibody, 1:5000 in TBST, for 1 hour at room temperature. Membranes were again washed with TBST, developed by application of enhanced chemiluminescence substrate and bands visualized using a ChemiDoc. Membranes were stripped using reblot plus, washed thoroughly in TBST, and blocked in 5% nonfat milk/TBS as previously. Membranes were washed and probed with anti-β-actin horseradish peroxidase for 1 hour at room temperature and developed/imaged as previously. Scribble densitometry was normalized for β-actin, and the experiment was performed four times. Following decidualization, primary human stromal cells were lysed in radioimmunoprecipitation assay buffer, and lysates were prepared and examined for Scribble as described for ECC-1 cells (n = 5 patient samples/cells). PRL assay PRL release from primary stromal cells transfected with a scramble construct or siRNA-targeting Scribble was determined after 12 days of incubation in decidualizing media. PRL assays were performed at Monash Health pathology using the access/DXI PRL assay, which is a simultaneous one-step immunoenzymatic (sandwich) assay carried out on a Beckman Coulter Unicel DXI 800. Briefly, the culture media sample was added to a reaction vessel along with polyclonal goat anti-PRL alkaline phosphatase conjugate and paramagnetic particles coated with mouse monoclonal anti-PRL antibody. The sample PRL binds to the monoclonal anti-PRL on the solid phase, whereas the goat anti-PRL-alkaline phosphatase conjugate reacts with a different antigenic site on the cell culture PRL. After incubation in a reaction vessel, the sample is subjected to separation in a magnetic field and washing to remove materials not bound to the solid phase. A chemiluminescent substrate, Lumi-Phos 530, is added to the reaction vessel, and light generated by the reaction is measured with a luminometer. The light production is directly proportional to the concentration of PRL in the sample. The amount of analyte in the sample is determined from a stored, multipoint calibration curve. The analytical range of the assay is from 5.3 to 4240 mIU/L. The interassay coefficient of variation was 5.11%, and the intra-assay coefficient of variation was 5.3%. Spheroid adhesion assay Trophectodermal cells were seeded at 2.5 × 103 cells per well into round-bottom, 96-well plates in the presence of 20% methylcellulose (Sigma-Aldrich) for 48 hours to facilitate spheroid formation. These spheroids are used as a mimic of human embryos. Twenty spheroids were placed into 15-mL centrifuge tubes and washed three times with serum-free DMEM/F12 to ensure removal of methylcellulose. Spheroids were resuspended in DMEM/F12 media containing 1% FCS and placed onto ECC-1 cells transfected with a scramble construct or siRNA-targeting Scribble for 6 hours. Spheroids were then counted to determine the total number of spheroids per well. Media were removed and the epithelial cell/spheroid cocultures gently washed with phosphate-buffered saline to remove nonadhered spheroids. The number of firmly adhered spheroids was counted, and adhered spheroids were expressed as a percentage of total spheroids. Each experimental condition was assessed in triplicate, and the experiment was performed four times. Statistics GraphPad Prism Version 6 for Windows was used for all statistical analyses. Before analysis, all data were tested for normality. If the data were found to be nonparametric, a Kruskal-Wallis or Mann-Whitney U analysis was performed. If the data were parametric, one-way analysis of variance with a Tukey or Dunnett post hoc test or an unpaired t test was performed. Significance was given as P < 0.05, and all data were presented as the mean ± SEM. Results Progestin alters cellular polarity TER, a proxy indicator of epithelial cellular polarity, was observed to rise gradually over 72 hours in ECC-1 cells treated with estrogen (E, Fig. 2). However, treatment with estrogen/progestin following 24 hours of estrogen priming mediated a significant reduction in TER (E/P, Fig. 2), indicative of a decrease in epithelial polarity. Figure 2. View largeDownload slide Progestin alters endometrial epithelial (ECC-1) cell polarity. Endometrial luminal epithelial (ECC-1) polarity, as assessed by TER, was significantly decreased by treatment with 10–8 M estrogen (E)/10–7 M MPA [progestin (P), E/P] vs treatment with 10–8 M estrogen alone (E). Data presented as mean ± SEM (n = 4). *P < 0.05. Figure 2. View largeDownload slide Progestin alters endometrial epithelial (ECC-1) cell polarity. Endometrial luminal epithelial (ECC-1) polarity, as assessed by TER, was significantly decreased by treatment with 10–8 M estrogen (E)/10–7 M MPA [progestin (P), E/P] vs treatment with 10–8 M estrogen alone (E). Data presented as mean ± SEM (n = 4). *P < 0.05. Expression and localization of polarity molecules within the human endometrium In vitro data suggested that progesterone altered epithelial polarity. To determine if specific regulators of cellular polarity were altered in vivo, expression and localization of established polarity determinants were investigated in the human endometrium across the menstrual cycle with comparisons in expression of polarity determinants made between the estrogen-dominated proliferative (days 5 to 10) phase of the cycle and the progesterone-dominated early secretory (days 14 to 18), mid secretory (days 19 to 23), and late secretory (days 24 to 28) phases of the cycle. Within the apical domain, atypical PKC localized to the luminal and glandular epithelium during all phases of the menstrual cycle, with limited immunostaining within the stromal compartment [Fig. 3(a)–3(d)]. Quantification of staining intensity demonstrated downregulation of atypical PKC within the luminal epithelium during the early secretory (P < 0.05), mid secretory (P = 0.06), and late secretory (P < 0.05) phases of the cycle vs proliferative phase [Fig. 3(e)]. Immunostaining within the glandular epithelium demonstrated a trend toward downregulation [Fig. 3(f)], whereas no significant changes were observed in the stromal compartment [Fig. 3(g)]. Figure 3. View largeDownload slide Localization and cyclic changes in atypical PKC within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, atypical PKC localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for atypical PKC within the (e) luminal epithelium during the secretory phase of the menstrual cycle, with no significant differences in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Figure 3. View largeDownload slide Localization and cyclic changes in atypical PKC within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, atypical PKC localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for atypical PKC within the (e) luminal epithelium during the secretory phase of the menstrual cycle, with no significant differences in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Crumbs localized mainly to the luminal and glandular epithelial cells with limited localization within the stroma [Fig. 4(a)–4(d)]. Quantification demonstrated a progressive decrease in Crumbs immunostaining within the luminal epithelium, which reached significance during the late secretory phase [vs proliferative phase, P = 0.01; Fig. 4(e)]. No patterns in immunostaining changes were observed throughout the menstrual cycle within the glandular epithelial [Fig. 4(f)] or stromal [Fig. 4(g)] compartments. Figure 4. View largeDownload slide Localization and cyclic changes in Crumbs within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Crumbs localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for Crumbs within the (e) luminal epithelium during the late secretory phase of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Figure 4. View largeDownload slide Localization and cyclic changes in Crumbs within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Crumbs localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for Crumbs within the (e) luminal epithelium during the late secretory phase of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Stardust (MPP5) also localized to the luminal and glandular epithelium, with variable staining within the stroma [Fig. 5(a)–5(d)]. As observed with Crumbs, immunostaining for Stardust within the luminal epithelium progressively decreased throughout the secretory phase, with significantly lower levels of immunostaining during the late secretory phase of the cycle [vs proliferative phase, P = 0.01; Fig. 5(e)]. No trends in immunostaining intensity were observed within the glandular epithelial [Fig. 5(f)] or stromal [Fig. 5(g)] compartments. Figure 5. View largeDownload slide Localization and cyclic changes in Stardust within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Stardust localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for Stardust within the (e) luminal epithelium during the late secretory phase of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Figure 5. View largeDownload slide Localization and cyclic changes in Stardust within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Stardust localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for Stardust within the (e) luminal epithelium during the late secretory phase of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Within the basolateral domain, Scribble localized to the luminal and glandular epithelial cells throughout the menstrual cycle, with increased stromal staining evident as the menstrual cycle progressed [Fig. 6(a)–6(d)]. Quantification of immunostaining demonstrated a highly significant downregulation of Scribble immunostaining during the mid (P < 0.05) and late (P < 0.01) secretory phases of the cycle [vs proliferative phase; Fig. 6(e)]. No significant trends in Scribble immunostaining were observed within the glandular epithelium [Fig. 6(f)]. However, staining within the stromal compartment increased as the menstrual cycle progressed and was significantly elevated during the late secretory phase of the cycle [P < 0.01; Fig. 6(g)]. Figure 6. View largeDownload slide Localization and cyclic changes in Scribble within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Scribble localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, immunostaining also observed within the stroma (arrows), particularly in areas exhibiting signs of decidualization. Quantification of staining revealed a significant downregulation in staining intensity for Scribble within the (e) luminal epithelium during the mid- and late secretory phases of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and a significant upregulation in immunostaining within the (g) stroma during the late secretory phase of the menstrual cycle. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Figure 6. View largeDownload slide Localization and cyclic changes in Scribble within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Scribble localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, immunostaining also observed within the stroma (arrows), particularly in areas exhibiting signs of decidualization. Quantification of staining revealed a significant downregulation in staining intensity for Scribble within the (e) luminal epithelium during the mid- and late secretory phases of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and a significant upregulation in immunostaining within the (g) stroma during the late secretory phase of the menstrual cycle. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. The apical polarity determinant Par3 and basolateral polarity determinant Lgl (Hugl-1) displayed little immunostaining within the endometrium and were therefore not quantified (data not shown). Hormonal regulation of Scribble in endometrial epithelial cells Because the basolateral polarity determinant Scribble was the most significantly downregulated polarity factor in luminal epithelium during the secretory phase of the menstrual cycle when progesterone is the dominant influence, it was chosen for further investigation. Levels of Scribble protein were comparable between estrogen-treated ECC-1 cells and vehicle-treated controls. Treatment with a combination of estrogen/progestin mediated a nonsignificant decrease (P = 0.08) in Scribble protein within ECC-1 cells, whereas treatment with estrogen/progestin/hCG (mimicking the hormonal influence of the incoming blastocyst) significantly downregulated Scribble protein (P < 0.05; Fig. 7). Figure 7. View largeDownload slide Hormone regulation of Scribble within human endometrial luminal epithelial (ECC-1) cells. Treatment of endometrial luminal epithelial cells (ECC-1) with 10–8 M estrogen (E)/10–7 M MPA [progestin (P), E/P, P = 0.08] and 10–8 M estrogen/10–7 M MPA (progestin)/10 IU hCG (E/P/hCG *P < 0.05) mediated a downregulation of Scribble protein abundance vs untreated [control (C)] or estrogen-only (E) groups. Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Figure 7. View largeDownload slide Hormone regulation of Scribble within human endometrial luminal epithelial (ECC-1) cells. Treatment of endometrial luminal epithelial cells (ECC-1) with 10–8 M estrogen (E)/10–7 M MPA [progestin (P), E/P, P = 0.08] and 10–8 M estrogen/10–7 M MPA (progestin)/10 IU hCG (E/P/hCG *P < 0.05) mediated a downregulation of Scribble protein abundance vs untreated [control (C)] or estrogen-only (E) groups. Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Downregulation of endometrial epithelial Scribble enhances “embryo” adhesion To investigate the role that luminal epithelial Scribble downregulation may play in preparation of the endometrium for implantation, ECC-1 cells were transfected with siRNA-targeting Scribble or a scramble construct. Trophectodermal spheroids were placed on transfected ECC-1 cell monolayers, and adhesion was determined at 6 hours. ECC-1 cells transfected with the scrambled construct demonstrated 77.5% ± 3.5% adhesion (Fig. 8), whereas ECC-1 cells transfected with siRNA-targeting Scribble (proven by Western immunoblot) demonstrated a significant increase in spheroid adhesion (93.9% ± 2.6%, P < 0.001; Fig. 8). Figure 8. View largeDownload slide Downregulation of Scribble in endometrial luminal epithelial (ECC-1) cells enhances “blastocyst” adhesion. Downregulation of endometrial epithelial (ECC-1) cell Scribble expression, using siRNA-targeting Scribble, mediated a significant increase in trophectodermal cell spheroid adhesion (Scrib siRNA, **P < 0.001) vs cells transfected with a scramble construct (siRNA control). Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Figure 8. View largeDownload slide Downregulation of Scribble in endometrial luminal epithelial (ECC-1) cells enhances “blastocyst” adhesion. Downregulation of endometrial epithelial (ECC-1) cell Scribble expression, using siRNA-targeting Scribble, mediated a significant increase in trophectodermal cell spheroid adhesion (Scrib siRNA, **P < 0.001) vs cells transfected with a scramble construct (siRNA control). Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Scribble is elevated in decidualized endometrial stromal cells Immunohistochemical data demonstrated a progressive increase in Scribble immunostaining in the stromal compartment with progression of the secretory phase, with maximal levels seen after prolonged exposure to progesterone during the late secretory phase of the menstrual cycle, suggestive of an association with decidualization. Scribble protein was examined by Western blot in nondecidualized (2 days of treatment with decidualizing media) vs decidualized (12 days of treatment with decidualizing media) primary human endometrial stromal cells, demonstrating a significant (P = 0.05) increase in Scribble protein with decidualization [Fig. 9(a)]. Figure 9. View largeDownload slide Decidualization is associated with, and partially dependent on, upregulation of Scribble. (a) In vitro decidualization of primary endometrial stromal cells by treatment with 10–8 M estrogen/10–7 M MPA (progestin) for 12 days (day 12 decidualization) mediated a significant increase in Scribble protein abundance vs cells treated for 2 days (day 2 decidualization). (b) SiRNA knockdown of Scribble (Scribble siRNA) inhibited in vitro decidualization of primary human stromal cells (P = 0.06) vs scramble transfected controls (scramble control). Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Figure 9. View largeDownload slide Decidualization is associated with, and partially dependent on, upregulation of Scribble. (a) In vitro decidualization of primary endometrial stromal cells by treatment with 10–8 M estrogen/10–7 M MPA (progestin) for 12 days (day 12 decidualization) mediated a significant increase in Scribble protein abundance vs cells treated for 2 days (day 2 decidualization). (b) SiRNA knockdown of Scribble (Scribble siRNA) inhibited in vitro decidualization of primary human stromal cells (P = 0.06) vs scramble transfected controls (scramble control). Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Stromal cell decidualization is partially dependent on de novo Scribble expression Scribble is elevated upon decidualization in primary human endometrial stromal cells. Therefore, to determine if decidualization is dependent on/directed by acquisition of cellular polarity, endometrial stromal cells were transfected with siRNA-targeting Scribble or a scramble construct prior to decidualization. Decidualization, as determined by PRL secretion, demonstrated a trend toward downregulation in primary human endometrial stromal cells transfected with Scribble siRNA [P = 0.06; Fig. 9(b)] vs scramble transfected cells. Discussion and Conclusions This study clearly demonstrates that molecular determinants mediating cell polarity changes are cyclically, but differently, regulated in endometrial luminal epithelial and stromal cells in the progression from the nonreceptive state during the proliferative phase of the cycle to the gradual progesterone-mediated acquisition of a receptive/permissive state during the mid and late secretory phase of the cycle, respectively (2). In addition, these polarity changes alter cell functions important in implantation and preparation for pregnancy. Inadequate endometrial preparation for receptivity is a major cause of infertility. In both natural and artificially cycling women, inadequate endometrial receptivity is thought to account for approximately 30% of infertility (21). The loss of luminal epithelial polarity, accompanied by a partial EMT, is an important step in the gradual acquisition of progesterone-mediated endometrial preparation for receptivity. This likely contributes to overcoming the mutual repulsion between the otherwise polarized endometrial epithelium and embryonic trophectoderm, enabling attachment to and subsequent invasion of the blastocyst through the luminal epithelium, the first steps in implantation. We provide evidence that ovarian steroid hormones differentially impact endometrial epithelial cell polarity, with combined estrogen/progestin treatment decreasing TER, a proxy measure of cellular polarity, vs treatment with estrogen alone. MPA/progestin was used in place of natural progesterone in the current study, as it is much more stable in solution, and hence data are better replicated. Although MPA may activate androgen receptors, such actions are also involved in acquisition of receptivity (22, 23). We have previously shown that a range of progesterone analogs, including both progesterone and MPA, have similar effects in vitro (24). In vivo, we demonstrated progressive downregulation of polarity determinants predominantly within the luminal epithelium during progression of the menstrual cycle from the estrogen-dominated proliferative phase to the progesterone-dominated secretory phase. Both apical and basolateral determinants of cellular polarity were altered during the window in which receptivity is attained and during the late secretory phase under the prolonged influence of progesterone. Because these apical and basolateral determinants interact to promote mutual antagonism, thus maintaining the determinants in their appropriate intracellular localization, such downregulation of both apical and basolateral determinants suggests removal of the mutual antagonistic regulation, resulting in a significant downregulation in epithelial polarity within the endometrial epithelium during the secretory phase of the cycle. Of the molecules examined, the basolateral polarity determinant, Scribble, exhibited the most profound progressive downregulation in the luminal epithelium during the secretory phase of the menstrual cycle, being significantly downregulated during the mid (receptive) and late (permissive) secretory phases, and was therefore selected for further investigation. hCG was added to the cultures to mimic the hormonal milieu that would be present at the time of embryo implantation when the endometrium is under the influence of estrogen, progesterone, and hCG. The latter is released in close proximity to the endometrium by the incoming blastocyst. As the endometrium is exposed to hCG prior to adhesive contact with the embryo, hCG treatment in this context is important to mimic the immediate preimplantation hormonal milieu. In vitro, combined ovarian steroid hormones and the peri-implantation pregnancy hormone (hCG) decreased Scribble protein levels beyond the reduction seen with ovarian steroids alone. Thus the polarity changes during the secretory phase in the nonpregnant menstrual cycle may be enhanced in the presence of a hCG-secreting blastocyst, further increasing loss of polarity and aiding implantation competence. This expands previous findings that hCG mediates proimplantation changes in endometrial epithelial cells (25, 26). We also demonstrated that functionally, Scribble downregulation enhances implantation competence. In an in vitro spheroid adhesion assay, which utilizes spheroids of trophectodermal cells and ECC-1 cells to mimic initial luminal epithelial-trophectodermal interactions at implantation, knockdown of Scribble in luminal epithelial cells enhanced spheroid adhesion to the monolayers, implying that modulation of apico-basal polarity is an important facilitative step in the complex changes required within the endometrium to achieve embryo implantation. Impaired polarity changes may be one of the factors that contribute to infertility by reducing the likelihood of embryo adhesion. Importantly, we also observed modulation of polarity determinants, particularly Scribble, within the endometrial stromal compartment. In contrast to its downregulation within the luminal epithelium, Scribble was significantly elevated during the late secretory phase of the menstrual cycle when stromal cells are decidualized. Whereas stromal fibroblasts are not conventionally considered to exhibit apico-basal polarity, endometrial stromal fibroblasts undergo an MET during the process of decidualization that occurs progressively during the secretory phase of each menstrual cycle: The decidual cells are epithelioid and highly secretory and develop a basal lamina. We demonstrate here that acquisition of this epithelioid phenotype is accompanied by the development of apico-basal polarity. In vitro decidualized cells expressed elevated levels of Scribble protein. Additionally, decidual transformation appeared to be partially dependent on this acquisition of apico-basal polarity, as stromal cells, when transfected with siRNA-targeting Scribble, did not decidualize as effectively as cells transfected with a scramble construct. It is important to note that specialized changes in the luminal epithelium and stroma that facilitate implantation may be unique to menstrual species that exhibit intrusive penetration of the trophectoderm/early trophoblast through the luminal epithelium and spontaneous decidualization. Endometrial preparation may be different/absent in commonly examined rodent species that exhibit entosis (endocytosis of luminal epithelial by trophoblast cells) as their mode of gaining access to the underlying endometrial stroma during implantation and conceptus-dependent decidualization (27). This highlights the necessity of examining human tissues/menstrual species to understand their specific endometrial changes during the secretory phase of the menstrual cycle, encompassing the receptive and permissive windows when implantation can occur. Early studies of the human endometrium observed a decrease in the area of tight junctions and the overall geometric complexity of the junctional structure as the menstrual cycle progressed (28). Indeed, it was proposed that a less complex junctional structure may facilitate penetration of the luminal epithelium and thus contribute to successful pregnancy (17). In the fertile receptive endometrium downregulation of the adherens junction, proteins E-cadherin and β-catenin are critical to fertility (29). As Scribble stabilizes the E-cadherin/catenin complex to reduce the rate of cadherin internalization, downregulation of Scribble may have been expected as a corollary of adherens junction alterations within the receptive endometrium (30, 31). However, upon disruption of adherens junction complexes (cadherin-catenin interactions), epithelial apico-basal polarity is generally lost (32), leading to disruption of the epithelial structure and EMT. Within the luminal epithelium during the secretory phase of the cycle, we are faced with a cell polarity paradox; epithelial identity is required while partial loss of their polarity is needed for implantation, as structural features of nonpolarized cells strongly correlate with functional features of adhesiveness for the implanting blastocyst (17). The requirement for partial maintenance of the epithelial phenotype may underlie the progressive, but not significant, downregulation of atypical PKC, Crumbs, and Stardust within the human luminal epithelium during the secretory phase of the menstrual cycle in preparation for receptivity. The progressive decidual transformation of the endometrial stroma throughout the secretory phase of the menstrual cycle is associated with a significant increase in the basolateral determinant Scribble and some increase in the apical determinants atypical PKC and Crumbs, indicating acquisition of a partial epithelial identity that may be important in the success of pregnancy. Although an embryo may be able to breach the luminal epithelial layer of the endometrium due to the cycle-dependent changes in polarity and associated partial EMT within this cellular compartment, the decidualizing endometrial stroma appears to act as a more stringent “quality control” mechanism, actively rejecting bad-quality embryos (12). Acquisition of cellular polarity and a more epithelial-like cellular phenotype may play a role in this quality control mechanism. Therefore, if polarity acquisition is compromised as stromal cells decidualize, this may enable implantation of low-quality embryos, as observed in the population of women with recurrent pregnancy loss. This population of women should be investigated for the regulation of stromal/decidual polarity determinants. Should these be dysregulated, they may provide novel uterine targets for modulation to intervene in cases of recurrent pregnancy loss. A cellular paradox exists not just in the adhesion of the endometrial epithelium to the blastocyst trophectodermal epithelium via their apical poles, but in the differential regulation of polarity determinants within the epithelial and stromal compartments under the influence of the same hormonal milieu. Estrogen/progestin treatment of endometrial luminal epithelial (ECC-1) cells decreased Scribble protein, whereas the identical treatment upregulated Scribble protein in primary endometrial stromal cells. This is likely due to the unique properties of the endometrium, whereby the influence of endogenous hormones and local factors differentially transform the epithelium vs the stroma, with the epithelium undergoing a partial EMT, while the stroma undergoes an MET during the same timespan. The associated downregulation of apico-basal polarity within the luminal epithelium upon EMT contributes to the adhesiveness of these cells for the blastocyst. In contrast, the endometrial stroma uniquely undergoes hormone-mediated differentiation; we demonstrate here that this differentiation is partially dependent on de novo development of apico-basal polarity. These data strongly support the plasma membrane transformation theory proposed by Murphy (13). In conclusion, this work demonstrates a downregulation in polarity determinants within the luminal epithelium during the secretory phase of the menstrual cycle encompassing the phase of endometrial receptivity, changes which are likely associated with a partial EMT and which are mediated by the endogenous progesterone-dominated hormonal milieu. Concurrently, polarity determinants are increased within the stromal compartment, associated with decidual transformation, similarly governed by progesterone dominance. These differential changes within the luminal epithelial and stromal endometrial cellular compartments may be functionally important for blastocyst adhesion and decidual transformation, respectively. In vivo, these changes were demonstrated immunohistochemically within normal fertile women who were not under the influence of exogenous steroid hormones. Therefore, given the apparent importance of these changes in receptivity and embryo implantation, it remains to be determined whether these polarity determinants display altered regulation/expression within the endometrium of infertile women, those undergoing hormonal stimulation for assisted reproduction, and those who have experienced recurrent pregnancy loss. Modulation of polarity determinants within the endometrium presents an opportunity to enhance or block development of receptivity in facilitating embryo implantation or development of novel contraceptives, in association with targeting of other aspects of endometrial receptivity. Because the role of polarity determinants has been extensively investigated in organ development and cancer, translation from these fields may provide cues in how to modulate endometrial polarity. Abbreviations: csFCS charcoal-stripped fetal calf serum DMEM Dulbecco’s modified Eagle medium EMT epithelial-to-mesenchymal transition FCS fetal calf serum hCG human chorionic gonadotropin MET mesenchymal-to-epithelial transition MPA medroxyprogesterone acetate PKC protein kinase C PRL prolactin SEM standard error of the mean siRNA small interfering RNA TBS Tris-buffered saline TBST Tris-buffered saline 0.2% Tween 20 TER transepithelial resistance v/v volume-to-volume ratio. Acknowledgments PRL assays were performed by Michael Desakalis at Monash Health pathology department. Judi Hocking collected the endometrial tissue. We thank the women who donated the endometrial tissues used in this study. Financial Support: This work was supported by National Health and Medical Research Council (NHMRC) Fellowship 1002028 (to L.A.S.), NHMRC Project Grant GNT1047756, and the Victorian Government’s Operational Infrastructure funding to the Hudson Institute. 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The Endometrial Polarity Paradox: Differential Regulation of Polarity Within Secretory-Phase Human Endometrium

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2016-1877
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Abstract

Abstract A major cause of infertility in normal and assisted reproduction cycles is failure of the endometrium to undergo appropriate changes during the secretory phase of the menstrual cycle as it acquires receptivity for an implanting blastocyst. Current dogma states that loss of epithelial polarity in the luminal epithelial cells, the point of first contact between maternal endometrium and blastocyst, may facilitate embryo implantation. Loss of polarity is likely an important change during the secretory phase to overcome mutual repulsion between otherwise polarized epithelial surfaces. Although “plasma membrane transformation” describes morphological/molecular alterations associated with loss of polarity, direct measures of polarity have not been investigated. Transepithelial resistance, a proxy measure of polarity, was downregulated in endometrial epithelial (ECC-1) cells by combined estrogen/progestin, mimicking the hormonal milieu of the secretory phase. Examination of defined polarity markers within human endometrium throughout the menstrual cycle identified downregulation of atypical protein kinase C, Stardust, Crumbs, and Scribble within the luminal-epithelial layer, with upregulation of Scribble within the stromal compartment as the menstrual cycle progressed from the estrogen-dominated proliferative to progesterone-dominated secretory phase. Epithelial (ECC-1) Scribble expression was downregulated in vitro by combined estrogen/progestin and estrogen/progestin/human chorionic gonadotropin treatment, whereas knockdown of Scribble in these cells enhanced “embryo” (trophectodermal spheroid) adhesion. In contrast, Scribble was upregulated within decidualized primary human endometrial stromal cells, with decidualization downregulated upon Scribble knockdown. These data highlight an important contribution of polarity modulation within the human endometrium, likely important for receptivity. Clinical investigations examining how polarity may be modulated in the infertile endometrium may facilitate fertility. The processes and mechanisms underlying human embryo implantation remain enigmatic despite intense research effort directed at understanding this complex process. This is partially due to the unique and transient nature of endometrial preparation for embryo implantation, which is controlled by cyclic variations in ovarian steroid hormones and their impact on local endometrial factors. Although embryos can attach and implant with ease into other tissues within the human body, their attachment to the human uterine luminal epithelium and subsequent implantation is limited to ∼4 days during the secretory phase of each menstrual cycle when the endometrium becomes “receptive,” a span of time also termed the “window of implantation” (1). Before this specific window, the endometrium is hostile, and afterward it remains permissive to implantation but less likely to result in live birth, as observed during the late secretory phase of the menstrual cycle (2). Within an idealized 28-day menstrual cycle, changes for receptivity occur under the influence of rising progesterone (in the presence of estrogen) following ovulation, and the receptive window encompasses approximately days 19 to 23 of the menstrual cycle (luteinizing hormone+7-10). However, with variation in timing of menstrual cycles between women and even between different cycles within the same woman, there is clearly considerable scope for variation in the timing of endometrial receptivity. Nowhere is this more clear than in assisted reproductive technology cycles, in which, despite the use of genetically normal embryos, implantation failure is commonly attributed to inadequate endometrial receptivity (3–5). Indeed, it is thought that up to 75% of pregnancy failures represent a failure of implantation (6, 7). Recent advances have been made in attempts to characterize changes during the secretory phase of the cycle leading toward and encompassing the implantation window by genetic analysis of endometrial tissue. Despite gene array analyses of the endometrium demonstrating little concordance in up- or downregulated genes at the time of receptivity [reviewed in Altmäe, et al. (8)], an endometrial receptivity array provides a genetic signature of receptivity that can determine if the endometrium is “out of phase” and therefore nonreceptive (9). However, although the endometrial receptivity array test can be used to “diagnose” a receptive endometrium, it does not provide enlightenment on the mechanisms underlying attainment of receptivity and particularly the intricate changes that occur within different endometrial cellular compartments. This is an important issue when we consider that the point of first contact between the maternal endometrium and the embryo, the luminal epithelial layer, must achieve adhesion competence during the secretory phase of the cycle under the influence of progesterone to facilitate attachment, whereas the underlying stroma must undergo an appropriate degree of decidual differentiation to act as a “quality control” mechanism ensuring subsequent development of only high-quality embryos (10–12). Understanding of how these processes may be modulated is critical for both enhancing receptivity and therefore fertility, and for maintaining the endometrium in a state refractory to implantation in the development of novel contraceptive strategies. Cellular transformation during the process of embryo implantation presents a cell biological paradox in a number of aspects. Both the trophectodermal layer of the preimplantation embryo (the blastocyst) and the uterine luminal epithelium present with a polarized phenotype. Generally, epithelial layers are polarized to maintain a barrier function and, in specialized cells, directional secretion of proteins. Throughout most of the cycle, the endometrial luminal epithelium is specialized to resist adhesion. However, occurring progressively throughout the secretory phase under the influence of progesterone, this epithelial layer must develop adhesion competence at its apical pole, which is related to cellular rearrangements that result in modulation of their apico-basal polarity as part of the plasma membrane transformation (13). Indeed, a number of “polarity-related” changes have been observed within this luminal epithelium. In marmosets, the electronegativity of the uterine epithelium decreases progressively from the preovulatory stage in preparation for implantation with an associated increase in its adhesive capacity, due to enhanced adhesion as repulsive charges on opposing membranes are decreased (14). In association with these charge changes in the luminal epithelium, a partial epithelial-to-mesenchymal transition (EMT) is observed, associated with a downregulation of E-cadherin (15) and, paradoxically, a reduction in paracellular permeability, while the cells of the epithelial layer become less firmly adhered to each other (16). Downregulation of E-cadherin is a common phenomenon in EMT; however, endometrial epithelial cells do not downregulate their complete epithelial program, but only selected parameters, such that epithelial identity is maintained (17). Conversely, upon initiation of hormone-directed differentiation, the endometrial stroma undergoes a mesenchymal-to-epithelial transition (MET) during the decidualization process. Therefore, the opposing processes of EMT and MET are occurring within adjacent endometrial cellular compartments under the influence of the same endocrine and local milieu. Dogma within this area consistently states that apico-basal polarity is altered within the luminal epithelium during the mid to late secretory phases, with loss of polarity typically associated with EMT. However, to date, specific intracellular determinants of epithelial polarity have not been investigated within the human endometrium in the transition from a nonreceptive state during the proliferative phase of the cycle to a prereceptive and subsequently receptive/permissive state during the secretory phase of the cycle. Cellular polarity is governed by an asymmetrical arrangement of intracellular polarity molecules within specific regions of the cell to specify apical and basal cellular identity of the cell and thus present a polarized epithelial-sheet phenotype (Fig. 1). Maintenance of this apico-basal polarity is dependent on appropriate intracellular localization of polarity complexes. The Crumbs and Par complexes specify the apical domain, whereas the basolateral domain is specified by the Scribble complex (Fig. 1). A complex interplay of positive feedback, resulting in recruitment of polarity determinants to their cellular poles, and negative feedback, resulting in mutually antagonistic interactions between the complexes, within each domain, is required to maintain cellular polarity (Fig. 1). Figure 1. View largeDownload slide Localization and mutual antagonism of polarity determinants within epithelial cells. Much of the background on interactions of polarity determinants has been derived from examination of interactions within Drosophila epithelium; it is presumed that many of these interactions translate to mammalian species. The major plasma membrane domains comprise the apical and basolateral domains. The main apical complexes are the Crumbs (Crb; orange) and Par (green) complexes, whereas the main basolateral complex is the Scribble (Scrib; pink) complex. Subconstituents of each of these complexes are indicated. Mutually antagonistic interactions (“T” bars) between apical and basolateral polarity determinants form and maintain the apical-basolateral axis. Some apical polarity determinants interact with tight junctions (TJ; yellow) and adherens junctions (AJ; purple) in a supportive manner (double-headed arrows). Specific subconstituents of the polarity complexes are involved in these interactions, including Bazooka (Baz) and PALS1-associated tight junction protein (Patj; tight junctions) and atypical PKC (αPKC) and Bazooka (adherens junctions). Desmosomes (D) are also shown (blue); however, the manner in which polarity determinants may interact with these is unclear. This schematic represents a “best working knowledge” model; however, specifically how these polarity determinants interact with each other and with intercellular junction proteins is still being determined in mammalian cells. Data from Lamouille et al. (18). Dlg, discs large; Lgl, lethal giant larvae; Par6, partition defective gene-6; Std, Stardust. Figure 1. View largeDownload slide Localization and mutual antagonism of polarity determinants within epithelial cells. Much of the background on interactions of polarity determinants has been derived from examination of interactions within Drosophila epithelium; it is presumed that many of these interactions translate to mammalian species. The major plasma membrane domains comprise the apical and basolateral domains. The main apical complexes are the Crumbs (Crb; orange) and Par (green) complexes, whereas the main basolateral complex is the Scribble (Scrib; pink) complex. Subconstituents of each of these complexes are indicated. Mutually antagonistic interactions (“T” bars) between apical and basolateral polarity determinants form and maintain the apical-basolateral axis. Some apical polarity determinants interact with tight junctions (TJ; yellow) and adherens junctions (AJ; purple) in a supportive manner (double-headed arrows). Specific subconstituents of the polarity complexes are involved in these interactions, including Bazooka (Baz) and PALS1-associated tight junction protein (Patj; tight junctions) and atypical PKC (αPKC) and Bazooka (adherens junctions). Desmosomes (D) are also shown (blue); however, the manner in which polarity determinants may interact with these is unclear. This schematic represents a “best working knowledge” model; however, specifically how these polarity determinants interact with each other and with intercellular junction proteins is still being determined in mammalian cells. Data from Lamouille et al. (18). Dlg, discs large; Lgl, lethal giant larvae; Par6, partition defective gene-6; Std, Stardust. This study investigated the expression and localization of polarity proteins within the human endometrium across the menstrual cycle, including the proliferative phase, when the endometrium is restored following menstruation, the early secretory phase, when epithelial differentiation for receptivity is initiated, and the receptive mid secretory phase and permissive late secretory phase of the cycle, to determine if apico-basal polarity is altered via modulation of polarity proteins. We identified cyclic changes in both apical [atypical protein kinase C (PKC); Stardust and Crumbs] and basolateral determinants (Scribble) within the epithelial and stromal compartments. We further investigated the hormonal regulation and functional role of the basolateral determinant, Scribble, in endometrial function and demonstrated: (1) its differential regulation by ovarian steroid hormones in epithelial (ECC-1 endometrial epithelial cell line) and primary human stromal cells; (2) its functional role in embryo adhesion; and (3) its contribution to decidual transformation of endometrial stromal cells. This study confirms that apico-basal polarity is altered within both the luminal epithelial and stromal compartments of the endometrium during the secretory phase of the cycle, encompassing the window of receptivity, and that these changes are functionally important for embryo implantation and stromal cell decidualization. Materials and Methods Ethics and tissue collection Ethical approval was obtained for all tissue collections from the Institutional Ethics Committees at Monash Health and Monash Surgical Private Hospital. Written informed consent was obtained from all subjects prior to tissue collection. Endometrial tissue collection and patient details Endometrial biopsies for immunohistochemistry were collected by curettage from normally cycling women throughout the menstrual cycle, covering the proliferative (n ≥ 10), early secretory (n ≥ 10), mid secretory (n ≥ 10), and late secretory (n ≥ 10) phases of the menstrual cycle. By this method, it is anticipated that the functionalis and a small amount of basalis endometrium would be sampled. The women had no known endometrial pathologies, regular menstrual cycles (28 to 34 days), and at least one parous pregnancy. All women were under 40 years of age and had not received steroid hormone therapy in the last 6 months. The biopsies were fixed in 10% formalin for 24 hours prior to processing to paraffin wax. All women were determined to have morphologically normal endometrium. Menstrual cycle stage was assessed by standard histological dating by a highly experienced gynecological pathologist. Immunohistochemistry Five-micrometer-thick tissue sections were placed onto superfrost slides and dried overnight at 37°C. Sections were dewaxed in Histosol and rehydrated with decreasing concentrations of ethanol (100% to 70%) to distilled water. Citrate antigen retrieval was performed, as determined by prior optimization, for each antibody (Table 1) in citrate buffer (10 mM citric acid, pH 6.0), followed by incubation in hot buffer for 20 minutes. Endogenous peroxidase activity was blocked by incubation of tissue sections in 3% hydrogen peroxide for 10 minutes at room temperature. Nonspecific binding was subsequently blocked with nonimmune serum [10% nonimmune serum (Table 1), 2% human serum, Tris-buffered saline (TBS)] for 60 minutes at room temperature. Sections were then incubated overnight in a humidity chamber at 4°C with the appropriate primary antibody or immunoglobulin G negative control, as determined by prior optimization (Table 1). Subsequently, sections were thoroughly washed in TBS 0.2% Tween 20 (TBST) and incubated at room temperature for 60 minutes with a biotinylated secondary antibody (Table 1). Sections were rewashed with TBS 0.2% Tween 20 and an avidin/biotin-peroxidase detection system [Vectastain Elite ABC Kit (Standard; catalog no. PK-6100; Vector Laboratories, Inc., Burlingame, CA)] applied for 30 minutes at room temperature. Immunostaining was then performed by addition of the peroxidase substrate 3,3′-diaminobenzidine (catalog no. K3468; Dako, Agilent, Santa Clara, CA). Sections were counterstained with hematoxylin and dehydrated in increasing concentrations of ethanol (70% to 100%) and Histosol, with coverslips mounted using DPX. Imaging used an Olympus BX53 microscope at ×20 magnification. Immunohistochemical staining was assessed within the glandular epithelium, luminal epithelium, and stroma using Image J. Average immunostaining and standard error of the mean (SEM) were calculated for each protein, within each cellular compartment (glandular epithelium, luminal epithelium, and stroma) across each phase of the menstrual cycle. Table 1. Antibodies Used 1° Ab, Catalog No. (RRID)  Nonimmune Serum  1° Antibody Species  Concn 1° Antibody  Negative Control  2° Antibody, Batch  α-PKCζ (C-20), SC-216 (RRID: AB_2300359)  Goat  Rabbit  1 µg/mL  Rabbit IgG  Goat anti-rabbit, BA-1000  Crumbs (N-20), SC-27901 (RRID: AB_2261069)  Horse  Goat  1 µg/mL  Goat IgG  Horse anti-goat, BA-9500  Hugl-1 (B-6), SC-136993 (RRID: AB_2135864)  Horse  Mouse  2 µg/mL  Mouse IgG  Horse anti-mouse, BA-2000  Scribble, ab115240 (RRID: AB_11155336)  Goat  Rabbit  1 µg/mL  Rabbit IgG  Goat anti-rabbit, BA-1000  Stardust, LS-C152791 (RRID: AB_2687873)  Goat  Rabbit  1:100  Rabbit IgG  Goat anti-rabbit, BA-1000  PAR3, HPA030443 (RRID: AB_10600926)  Goat  Rabbit  1:5000  Rabbit IgG  Goat anti-rabbit, BA-1000  1° Ab, Catalog No. (RRID)  Nonimmune Serum  1° Antibody Species  Concn 1° Antibody  Negative Control  2° Antibody, Batch  α-PKCζ (C-20), SC-216 (RRID: AB_2300359)  Goat  Rabbit  1 µg/mL  Rabbit IgG  Goat anti-rabbit, BA-1000  Crumbs (N-20), SC-27901 (RRID: AB_2261069)  Horse  Goat  1 µg/mL  Goat IgG  Horse anti-goat, BA-9500  Hugl-1 (B-6), SC-136993 (RRID: AB_2135864)  Horse  Mouse  2 µg/mL  Mouse IgG  Horse anti-mouse, BA-2000  Scribble, ab115240 (RRID: AB_11155336)  Goat  Rabbit  1 µg/mL  Rabbit IgG  Goat anti-rabbit, BA-1000  Stardust, LS-C152791 (RRID: AB_2687873)  Goat  Rabbit  1:100  Rabbit IgG  Goat anti-rabbit, BA-1000  PAR3, HPA030443 (RRID: AB_10600926)  Goat  Rabbit  1:5000  Rabbit IgG  Goat anti-rabbit, BA-1000  Abbreviations: Ab, antibody; BA, batch; Concn, concentration; IgG, immunoglobulin G; RRID, Research Resource Identifier. View Large Cell culture ECC-1 endometrial epithelial cells are an endometrial cancer cell line with characteristics of the endometrial luminal epithelial layer. These cells were obtained from the American Type Culture Collection and their validity independently validated via short tandem repeat DNA profiling of human cell lines per American Type Culture Collection guidelines (19). They were routinely maintained in a 1:1 mix of Dulbecco’s modified Eagle medium (DMEM)/F12 Glutamax (Gibco, Invitrogen, Mt Waverley, VIC, Australia) supplemented with 1% volume-to-volume ratio (v/v) penicillin/streptomycin and 10% v/v fetal calf serum (FCS; Gibco, Invitrogen). These cells were seeded and used for experimental purposes as described later. Trophectodermal cells [kind gift of Susan Fisher (20)] are derived from trophoblast stem cells. They were routinely maintained in a 1:1 mix of DMEM:F12 Glutamax (Gibco, Invitrogen) supplemented with 1% v/v penicillin/streptomycin and 10% v/v FCS with addition of 10 ng/mL bovine fibroblast growth factor and 10 uM SB431542 (R&D Systems, In Vitro Technologies, Melbourne, Australia). Cells were grown on flasks coated with 0.5% gelatin (Sigma-Aldrich, NSW, Australia) prior to experimental seeding. Primary cell culture Endometrial biopsies for cell isolations were collected as described previously, from women meeting the same inclusion criteria, from the proliferative and early secretory phases of the menstrual cycle. Tissue biopsies were finely scissor minced and incubated in phosphate-buffered saline containing 7.5 IU/mL collagenase III (Sigma-Aldrich) and 100 mg/mL DNase I (Worthington, Lakewood, NJ) at 37°C with shaking at 130 rpm for 40 minutes. Digestion was stopped by addition of excess DMEM/F12 (Invitrogen). Digested samples were sequentially vacuum filtered through 45- and 11-μm filters to remove debris and epithelial clusters, before collection of the stromal cell pellet by centrifugation. Cells were sequentially seeded in DMEM/F12 media containing 10% charcoal-stripped FCS (csFCS; Invitrogen) and 1% penicillin/streptomycin (Sigma) into sterile cell culture flasks for 25 minutes to allow cell attachment before removal of the blood contamination. Stromal cells were then seeded into six-well plates and used in two different experimental paradigms: (1) Stromal cells (n = 5 separate preparations) were treated with 10–8 M 17β-estradiol (henceforth referred to as estrogen)/10–7 M medroxyprogesterone acetate (MPA; to mimic progesterone, henceforth referred to as progestin; Sigma-Aldrich) in DMEM/F12 with 2% csFCS (decidualization media) for 2 or 12 days, media collected for prolactin (PRL) assay (later), and cells lysed for Western immunoblot analysis (later) and (2) stromal cells (n = 5 separate preparations) were transfected with small interfering RNA (siRNA)–targeting Scribble (or control scramble sequence; protocol outlined later) and then treated with decidualization media for 12 days followed by media collection for PRL assay (later). Transepithelial resistance assays ECC-1 cells (2 × 105) were seeded onto polyester bicameral chambers (12 mm, 0.4-μm pore; Sigma) and coated with fibronectin according to the manufacturer’s protocol (BD Bioscience) prior to cell seeding. Cells were allowed to attach overnight in DMEM/F12 medium containing 10% FCS, confluency checked, and then incubated in DMEM/F12 with 0.5% csFCS in both the basal and apical chambers. Cells were incubated for a further 24 hours followed by assessment of baseline transepithelial resistance (TER). To assess the integrity of interepithelial cell-tight junctions, TER was quantified using a Millipore Millicell-Electrical Resistance System (Millipore), with measurements taken daily. Cells and media were maintained at 37°C; following removal from the incubator, cells were equilibrated on a warming plate within the culture hood for a minimum of 30 minutes before TER measurement. After basal readings, 24 hours post media change to 0.5% csFCS DMEM/F12, cells were treated with estrogen for 24 hours followed by assessment of TER. Cells were subsequently maintained in estrogen or treated with estrogen/progestin for a further 48 hours with TER readings taken every 24 hours. Changes in TER were calculated as a percentage of basal readings for each individual well. Duplicate culture wells were used for each treatment, and the entire experiment was performed four times (n = 4). Cell transfection with Scribble siRNA Both ECC-1 cells and primary stromal cells were transfected with Scribble siRNA (Santa-Cruz Biotechnology, Dallas, TX). ECC-1 cells were seeded at 2 × 105 cells/well in 12-well plates. Primary stromal cells were seeded into six-well plates and grown to 80% confluence. All cells were seeded in media without penicillin/streptomycin. Initially, lyophilized siRNA/scramble sequence was resuspended in RNAse free water to a final concentration of 10 uM. Two microliters of siRNA/scramble was mixed with 100 uL siRNA transfection media and incubated for 5 minutes. Concurrently, 2 uL of the transfection reagent (lipofectamine; Invitrogen) was mixed with 100 uL siRNA transfection media and incubated for 5 minutes. These two solutions were then mixed by pipetting and incubated at room temperature for 30 minutes. After incubation, the transfection solution was mixed with 800 uL of siRNA transfection media, cells washed with siRNA transfection media, and the transfection mix added to the cells for 8 hours. All volumes were doubled for transfection of primary stromal cells in six-well plates. After the incubation period, media were replaced with 10% FCS or csFCS media and cells maintained for 48 hours with daily visual checking of cell viability. ECC-1 cells were then used for spheroid adhesion assays (later), and primary stromal cells were decidualized for 12 days in decidualization media as previously. Western immunoblot ECC-1 cells were seeded at 1 × 106 cells in six-well plates, allowed to adhere overnight, and then deprived of serum for 8 hours. Cells were then treated with a vehicle control (ethanol) or primed with estrogen for 24 hours. After estrogen, priming cells were (1) continued in estrogen (2) treated with estrogen/progestin, and (3) treated with estrogen/progestin/10 IU human chorionic gonadotropin (hCG; to represent peri-implantation blastocyst secretion), each for 24 hours. Cells were then lysed in radioimmunoprecipitation assay buffer with protease inhibitors, lysates were clarified by centrifugation at 14,000 rpm at 4°C for 15 minutes, and supernatants were retained. Twenty-five micrograms of lysate from each treatment condition was loaded onto 4% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and run at 100 V for ∼90 minutes. Proteins were blotted to polyvinylidene difluoride membrane using a transblot turbo and washed thoroughly in TBST. Nonspecific binding was then blocked by incubation in 5% nonfat milk/TBS for 1 hour at room temperature. Membranes were again washed to remove excess block solution and incubated overnight at 4°C with anti-Scribble antibody at 1:500 dilution in TBST. Membranes were washed thoroughly and incubated with a goat anti-rabbit horseradish peroxidase–labeled antibody, 1:5000 in TBST, for 1 hour at room temperature. Membranes were again washed with TBST, developed by application of enhanced chemiluminescence substrate and bands visualized using a ChemiDoc. Membranes were stripped using reblot plus, washed thoroughly in TBST, and blocked in 5% nonfat milk/TBS as previously. Membranes were washed and probed with anti-β-actin horseradish peroxidase for 1 hour at room temperature and developed/imaged as previously. Scribble densitometry was normalized for β-actin, and the experiment was performed four times. Following decidualization, primary human stromal cells were lysed in radioimmunoprecipitation assay buffer, and lysates were prepared and examined for Scribble as described for ECC-1 cells (n = 5 patient samples/cells). PRL assay PRL release from primary stromal cells transfected with a scramble construct or siRNA-targeting Scribble was determined after 12 days of incubation in decidualizing media. PRL assays were performed at Monash Health pathology using the access/DXI PRL assay, which is a simultaneous one-step immunoenzymatic (sandwich) assay carried out on a Beckman Coulter Unicel DXI 800. Briefly, the culture media sample was added to a reaction vessel along with polyclonal goat anti-PRL alkaline phosphatase conjugate and paramagnetic particles coated with mouse monoclonal anti-PRL antibody. The sample PRL binds to the monoclonal anti-PRL on the solid phase, whereas the goat anti-PRL-alkaline phosphatase conjugate reacts with a different antigenic site on the cell culture PRL. After incubation in a reaction vessel, the sample is subjected to separation in a magnetic field and washing to remove materials not bound to the solid phase. A chemiluminescent substrate, Lumi-Phos 530, is added to the reaction vessel, and light generated by the reaction is measured with a luminometer. The light production is directly proportional to the concentration of PRL in the sample. The amount of analyte in the sample is determined from a stored, multipoint calibration curve. The analytical range of the assay is from 5.3 to 4240 mIU/L. The interassay coefficient of variation was 5.11%, and the intra-assay coefficient of variation was 5.3%. Spheroid adhesion assay Trophectodermal cells were seeded at 2.5 × 103 cells per well into round-bottom, 96-well plates in the presence of 20% methylcellulose (Sigma-Aldrich) for 48 hours to facilitate spheroid formation. These spheroids are used as a mimic of human embryos. Twenty spheroids were placed into 15-mL centrifuge tubes and washed three times with serum-free DMEM/F12 to ensure removal of methylcellulose. Spheroids were resuspended in DMEM/F12 media containing 1% FCS and placed onto ECC-1 cells transfected with a scramble construct or siRNA-targeting Scribble for 6 hours. Spheroids were then counted to determine the total number of spheroids per well. Media were removed and the epithelial cell/spheroid cocultures gently washed with phosphate-buffered saline to remove nonadhered spheroids. The number of firmly adhered spheroids was counted, and adhered spheroids were expressed as a percentage of total spheroids. Each experimental condition was assessed in triplicate, and the experiment was performed four times. Statistics GraphPad Prism Version 6 for Windows was used for all statistical analyses. Before analysis, all data were tested for normality. If the data were found to be nonparametric, a Kruskal-Wallis or Mann-Whitney U analysis was performed. If the data were parametric, one-way analysis of variance with a Tukey or Dunnett post hoc test or an unpaired t test was performed. Significance was given as P < 0.05, and all data were presented as the mean ± SEM. Results Progestin alters cellular polarity TER, a proxy indicator of epithelial cellular polarity, was observed to rise gradually over 72 hours in ECC-1 cells treated with estrogen (E, Fig. 2). However, treatment with estrogen/progestin following 24 hours of estrogen priming mediated a significant reduction in TER (E/P, Fig. 2), indicative of a decrease in epithelial polarity. Figure 2. View largeDownload slide Progestin alters endometrial epithelial (ECC-1) cell polarity. Endometrial luminal epithelial (ECC-1) polarity, as assessed by TER, was significantly decreased by treatment with 10–8 M estrogen (E)/10–7 M MPA [progestin (P), E/P] vs treatment with 10–8 M estrogen alone (E). Data presented as mean ± SEM (n = 4). *P < 0.05. Figure 2. View largeDownload slide Progestin alters endometrial epithelial (ECC-1) cell polarity. Endometrial luminal epithelial (ECC-1) polarity, as assessed by TER, was significantly decreased by treatment with 10–8 M estrogen (E)/10–7 M MPA [progestin (P), E/P] vs treatment with 10–8 M estrogen alone (E). Data presented as mean ± SEM (n = 4). *P < 0.05. Expression and localization of polarity molecules within the human endometrium In vitro data suggested that progesterone altered epithelial polarity. To determine if specific regulators of cellular polarity were altered in vivo, expression and localization of established polarity determinants were investigated in the human endometrium across the menstrual cycle with comparisons in expression of polarity determinants made between the estrogen-dominated proliferative (days 5 to 10) phase of the cycle and the progesterone-dominated early secretory (days 14 to 18), mid secretory (days 19 to 23), and late secretory (days 24 to 28) phases of the cycle. Within the apical domain, atypical PKC localized to the luminal and glandular epithelium during all phases of the menstrual cycle, with limited immunostaining within the stromal compartment [Fig. 3(a)–3(d)]. Quantification of staining intensity demonstrated downregulation of atypical PKC within the luminal epithelium during the early secretory (P < 0.05), mid secretory (P = 0.06), and late secretory (P < 0.05) phases of the cycle vs proliferative phase [Fig. 3(e)]. Immunostaining within the glandular epithelium demonstrated a trend toward downregulation [Fig. 3(f)], whereas no significant changes were observed in the stromal compartment [Fig. 3(g)]. Figure 3. View largeDownload slide Localization and cyclic changes in atypical PKC within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, atypical PKC localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for atypical PKC within the (e) luminal epithelium during the secretory phase of the menstrual cycle, with no significant differences in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Figure 3. View largeDownload slide Localization and cyclic changes in atypical PKC within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, atypical PKC localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for atypical PKC within the (e) luminal epithelium during the secretory phase of the menstrual cycle, with no significant differences in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Crumbs localized mainly to the luminal and glandular epithelial cells with limited localization within the stroma [Fig. 4(a)–4(d)]. Quantification demonstrated a progressive decrease in Crumbs immunostaining within the luminal epithelium, which reached significance during the late secretory phase [vs proliferative phase, P = 0.01; Fig. 4(e)]. No patterns in immunostaining changes were observed throughout the menstrual cycle within the glandular epithelial [Fig. 4(f)] or stromal [Fig. 4(g)] compartments. Figure 4. View largeDownload slide Localization and cyclic changes in Crumbs within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Crumbs localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for Crumbs within the (e) luminal epithelium during the late secretory phase of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Figure 4. View largeDownload slide Localization and cyclic changes in Crumbs within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Crumbs localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for Crumbs within the (e) luminal epithelium during the late secretory phase of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Stardust (MPP5) also localized to the luminal and glandular epithelium, with variable staining within the stroma [Fig. 5(a)–5(d)]. As observed with Crumbs, immunostaining for Stardust within the luminal epithelium progressively decreased throughout the secretory phase, with significantly lower levels of immunostaining during the late secretory phase of the cycle [vs proliferative phase, P = 0.01; Fig. 5(e)]. No trends in immunostaining intensity were observed within the glandular epithelial [Fig. 5(f)] or stromal [Fig. 5(g)] compartments. Figure 5. View largeDownload slide Localization and cyclic changes in Stardust within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Stardust localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for Stardust within the (e) luminal epithelium during the late secretory phase of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Figure 5. View largeDownload slide Localization and cyclic changes in Stardust within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Stardust localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, with limited immunostaining also observed within the stroma (arrows). Quantification of staining revealed a significant downregulation in staining intensity for Stardust within the (e) luminal epithelium during the late secretory phase of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and (g) stroma. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Within the basolateral domain, Scribble localized to the luminal and glandular epithelial cells throughout the menstrual cycle, with increased stromal staining evident as the menstrual cycle progressed [Fig. 6(a)–6(d)]. Quantification of immunostaining demonstrated a highly significant downregulation of Scribble immunostaining during the mid (P < 0.05) and late (P < 0.01) secretory phases of the cycle [vs proliferative phase; Fig. 6(e)]. No significant trends in Scribble immunostaining were observed within the glandular epithelium [Fig. 6(f)]. However, staining within the stromal compartment increased as the menstrual cycle progressed and was significantly elevated during the late secretory phase of the cycle [P < 0.01; Fig. 6(g)]. Figure 6. View largeDownload slide Localization and cyclic changes in Scribble within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Scribble localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, immunostaining also observed within the stroma (arrows), particularly in areas exhibiting signs of decidualization. Quantification of staining revealed a significant downregulation in staining intensity for Scribble within the (e) luminal epithelium during the mid- and late secretory phases of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and a significant upregulation in immunostaining within the (g) stroma during the late secretory phase of the menstrual cycle. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. Figure 6. View largeDownload slide Localization and cyclic changes in Scribble within the human endometrium. Within the (a) proliferative, (b) early secretory, (c) mid secretory, and (d) late secretory endometrium, Scribble localized to the glandular (black arrowheads) and luminal (white arrowheads) epithelium, immunostaining also observed within the stroma (arrows), particularly in areas exhibiting signs of decidualization. Quantification of staining revealed a significant downregulation in staining intensity for Scribble within the (e) luminal epithelium during the mid- and late secretory phases of the menstrual cycle, with no significant differences noted in immunostaining within the (f) glandular epithelium and a significant upregulation in immunostaining within the (g) stroma during the late secretory phase of the menstrual cycle. Data presented as mean ± SEM (n ≥ 10 tissues per cycle stage). *P < 0.05 vs proliferative phase of cycle. ES, early secretory; LS, late secretory; MS, mid secretory; P, proliferative. The apical polarity determinant Par3 and basolateral polarity determinant Lgl (Hugl-1) displayed little immunostaining within the endometrium and were therefore not quantified (data not shown). Hormonal regulation of Scribble in endometrial epithelial cells Because the basolateral polarity determinant Scribble was the most significantly downregulated polarity factor in luminal epithelium during the secretory phase of the menstrual cycle when progesterone is the dominant influence, it was chosen for further investigation. Levels of Scribble protein were comparable between estrogen-treated ECC-1 cells and vehicle-treated controls. Treatment with a combination of estrogen/progestin mediated a nonsignificant decrease (P = 0.08) in Scribble protein within ECC-1 cells, whereas treatment with estrogen/progestin/hCG (mimicking the hormonal influence of the incoming blastocyst) significantly downregulated Scribble protein (P < 0.05; Fig. 7). Figure 7. View largeDownload slide Hormone regulation of Scribble within human endometrial luminal epithelial (ECC-1) cells. Treatment of endometrial luminal epithelial cells (ECC-1) with 10–8 M estrogen (E)/10–7 M MPA [progestin (P), E/P, P = 0.08] and 10–8 M estrogen/10–7 M MPA (progestin)/10 IU hCG (E/P/hCG *P < 0.05) mediated a downregulation of Scribble protein abundance vs untreated [control (C)] or estrogen-only (E) groups. Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Figure 7. View largeDownload slide Hormone regulation of Scribble within human endometrial luminal epithelial (ECC-1) cells. Treatment of endometrial luminal epithelial cells (ECC-1) with 10–8 M estrogen (E)/10–7 M MPA [progestin (P), E/P, P = 0.08] and 10–8 M estrogen/10–7 M MPA (progestin)/10 IU hCG (E/P/hCG *P < 0.05) mediated a downregulation of Scribble protein abundance vs untreated [control (C)] or estrogen-only (E) groups. Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Downregulation of endometrial epithelial Scribble enhances “embryo” adhesion To investigate the role that luminal epithelial Scribble downregulation may play in preparation of the endometrium for implantation, ECC-1 cells were transfected with siRNA-targeting Scribble or a scramble construct. Trophectodermal spheroids were placed on transfected ECC-1 cell monolayers, and adhesion was determined at 6 hours. ECC-1 cells transfected with the scrambled construct demonstrated 77.5% ± 3.5% adhesion (Fig. 8), whereas ECC-1 cells transfected with siRNA-targeting Scribble (proven by Western immunoblot) demonstrated a significant increase in spheroid adhesion (93.9% ± 2.6%, P < 0.001; Fig. 8). Figure 8. View largeDownload slide Downregulation of Scribble in endometrial luminal epithelial (ECC-1) cells enhances “blastocyst” adhesion. Downregulation of endometrial epithelial (ECC-1) cell Scribble expression, using siRNA-targeting Scribble, mediated a significant increase in trophectodermal cell spheroid adhesion (Scrib siRNA, **P < 0.001) vs cells transfected with a scramble construct (siRNA control). Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Figure 8. View largeDownload slide Downregulation of Scribble in endometrial luminal epithelial (ECC-1) cells enhances “blastocyst” adhesion. Downregulation of endometrial epithelial (ECC-1) cell Scribble expression, using siRNA-targeting Scribble, mediated a significant increase in trophectodermal cell spheroid adhesion (Scrib siRNA, **P < 0.001) vs cells transfected with a scramble construct (siRNA control). Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Scribble is elevated in decidualized endometrial stromal cells Immunohistochemical data demonstrated a progressive increase in Scribble immunostaining in the stromal compartment with progression of the secretory phase, with maximal levels seen after prolonged exposure to progesterone during the late secretory phase of the menstrual cycle, suggestive of an association with decidualization. Scribble protein was examined by Western blot in nondecidualized (2 days of treatment with decidualizing media) vs decidualized (12 days of treatment with decidualizing media) primary human endometrial stromal cells, demonstrating a significant (P = 0.05) increase in Scribble protein with decidualization [Fig. 9(a)]. Figure 9. View largeDownload slide Decidualization is associated with, and partially dependent on, upregulation of Scribble. (a) In vitro decidualization of primary endometrial stromal cells by treatment with 10–8 M estrogen/10–7 M MPA (progestin) for 12 days (day 12 decidualization) mediated a significant increase in Scribble protein abundance vs cells treated for 2 days (day 2 decidualization). (b) SiRNA knockdown of Scribble (Scribble siRNA) inhibited in vitro decidualization of primary human stromal cells (P = 0.06) vs scramble transfected controls (scramble control). Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Figure 9. View largeDownload slide Decidualization is associated with, and partially dependent on, upregulation of Scribble. (a) In vitro decidualization of primary endometrial stromal cells by treatment with 10–8 M estrogen/10–7 M MPA (progestin) for 12 days (day 12 decidualization) mediated a significant increase in Scribble protein abundance vs cells treated for 2 days (day 2 decidualization). (b) SiRNA knockdown of Scribble (Scribble siRNA) inhibited in vitro decidualization of primary human stromal cells (P = 0.06) vs scramble transfected controls (scramble control). Data presented as mean ± SEM. Representative Western immunoblot presented. Scrib, Scribble. Stromal cell decidualization is partially dependent on de novo Scribble expression Scribble is elevated upon decidualization in primary human endometrial stromal cells. Therefore, to determine if decidualization is dependent on/directed by acquisition of cellular polarity, endometrial stromal cells were transfected with siRNA-targeting Scribble or a scramble construct prior to decidualization. Decidualization, as determined by PRL secretion, demonstrated a trend toward downregulation in primary human endometrial stromal cells transfected with Scribble siRNA [P = 0.06; Fig. 9(b)] vs scramble transfected cells. Discussion and Conclusions This study clearly demonstrates that molecular determinants mediating cell polarity changes are cyclically, but differently, regulated in endometrial luminal epithelial and stromal cells in the progression from the nonreceptive state during the proliferative phase of the cycle to the gradual progesterone-mediated acquisition of a receptive/permissive state during the mid and late secretory phase of the cycle, respectively (2). In addition, these polarity changes alter cell functions important in implantation and preparation for pregnancy. Inadequate endometrial preparation for receptivity is a major cause of infertility. In both natural and artificially cycling women, inadequate endometrial receptivity is thought to account for approximately 30% of infertility (21). The loss of luminal epithelial polarity, accompanied by a partial EMT, is an important step in the gradual acquisition of progesterone-mediated endometrial preparation for receptivity. This likely contributes to overcoming the mutual repulsion between the otherwise polarized endometrial epithelium and embryonic trophectoderm, enabling attachment to and subsequent invasion of the blastocyst through the luminal epithelium, the first steps in implantation. We provide evidence that ovarian steroid hormones differentially impact endometrial epithelial cell polarity, with combined estrogen/progestin treatment decreasing TER, a proxy measure of cellular polarity, vs treatment with estrogen alone. MPA/progestin was used in place of natural progesterone in the current study, as it is much more stable in solution, and hence data are better replicated. Although MPA may activate androgen receptors, such actions are also involved in acquisition of receptivity (22, 23). We have previously shown that a range of progesterone analogs, including both progesterone and MPA, have similar effects in vitro (24). In vivo, we demonstrated progressive downregulation of polarity determinants predominantly within the luminal epithelium during progression of the menstrual cycle from the estrogen-dominated proliferative phase to the progesterone-dominated secretory phase. Both apical and basolateral determinants of cellular polarity were altered during the window in which receptivity is attained and during the late secretory phase under the prolonged influence of progesterone. Because these apical and basolateral determinants interact to promote mutual antagonism, thus maintaining the determinants in their appropriate intracellular localization, such downregulation of both apical and basolateral determinants suggests removal of the mutual antagonistic regulation, resulting in a significant downregulation in epithelial polarity within the endometrial epithelium during the secretory phase of the cycle. Of the molecules examined, the basolateral polarity determinant, Scribble, exhibited the most profound progressive downregulation in the luminal epithelium during the secretory phase of the menstrual cycle, being significantly downregulated during the mid (receptive) and late (permissive) secretory phases, and was therefore selected for further investigation. hCG was added to the cultures to mimic the hormonal milieu that would be present at the time of embryo implantation when the endometrium is under the influence of estrogen, progesterone, and hCG. The latter is released in close proximity to the endometrium by the incoming blastocyst. As the endometrium is exposed to hCG prior to adhesive contact with the embryo, hCG treatment in this context is important to mimic the immediate preimplantation hormonal milieu. In vitro, combined ovarian steroid hormones and the peri-implantation pregnancy hormone (hCG) decreased Scribble protein levels beyond the reduction seen with ovarian steroids alone. Thus the polarity changes during the secretory phase in the nonpregnant menstrual cycle may be enhanced in the presence of a hCG-secreting blastocyst, further increasing loss of polarity and aiding implantation competence. This expands previous findings that hCG mediates proimplantation changes in endometrial epithelial cells (25, 26). We also demonstrated that functionally, Scribble downregulation enhances implantation competence. In an in vitro spheroid adhesion assay, which utilizes spheroids of trophectodermal cells and ECC-1 cells to mimic initial luminal epithelial-trophectodermal interactions at implantation, knockdown of Scribble in luminal epithelial cells enhanced spheroid adhesion to the monolayers, implying that modulation of apico-basal polarity is an important facilitative step in the complex changes required within the endometrium to achieve embryo implantation. Impaired polarity changes may be one of the factors that contribute to infertility by reducing the likelihood of embryo adhesion. Importantly, we also observed modulation of polarity determinants, particularly Scribble, within the endometrial stromal compartment. In contrast to its downregulation within the luminal epithelium, Scribble was significantly elevated during the late secretory phase of the menstrual cycle when stromal cells are decidualized. Whereas stromal fibroblasts are not conventionally considered to exhibit apico-basal polarity, endometrial stromal fibroblasts undergo an MET during the process of decidualization that occurs progressively during the secretory phase of each menstrual cycle: The decidual cells are epithelioid and highly secretory and develop a basal lamina. We demonstrate here that acquisition of this epithelioid phenotype is accompanied by the development of apico-basal polarity. In vitro decidualized cells expressed elevated levels of Scribble protein. Additionally, decidual transformation appeared to be partially dependent on this acquisition of apico-basal polarity, as stromal cells, when transfected with siRNA-targeting Scribble, did not decidualize as effectively as cells transfected with a scramble construct. It is important to note that specialized changes in the luminal epithelium and stroma that facilitate implantation may be unique to menstrual species that exhibit intrusive penetration of the trophectoderm/early trophoblast through the luminal epithelium and spontaneous decidualization. Endometrial preparation may be different/absent in commonly examined rodent species that exhibit entosis (endocytosis of luminal epithelial by trophoblast cells) as their mode of gaining access to the underlying endometrial stroma during implantation and conceptus-dependent decidualization (27). This highlights the necessity of examining human tissues/menstrual species to understand their specific endometrial changes during the secretory phase of the menstrual cycle, encompassing the receptive and permissive windows when implantation can occur. Early studies of the human endometrium observed a decrease in the area of tight junctions and the overall geometric complexity of the junctional structure as the menstrual cycle progressed (28). Indeed, it was proposed that a less complex junctional structure may facilitate penetration of the luminal epithelium and thus contribute to successful pregnancy (17). In the fertile receptive endometrium downregulation of the adherens junction, proteins E-cadherin and β-catenin are critical to fertility (29). As Scribble stabilizes the E-cadherin/catenin complex to reduce the rate of cadherin internalization, downregulation of Scribble may have been expected as a corollary of adherens junction alterations within the receptive endometrium (30, 31). However, upon disruption of adherens junction complexes (cadherin-catenin interactions), epithelial apico-basal polarity is generally lost (32), leading to disruption of the epithelial structure and EMT. Within the luminal epithelium during the secretory phase of the cycle, we are faced with a cell polarity paradox; epithelial identity is required while partial loss of their polarity is needed for implantation, as structural features of nonpolarized cells strongly correlate with functional features of adhesiveness for the implanting blastocyst (17). The requirement for partial maintenance of the epithelial phenotype may underlie the progressive, but not significant, downregulation of atypical PKC, Crumbs, and Stardust within the human luminal epithelium during the secretory phase of the menstrual cycle in preparation for receptivity. The progressive decidual transformation of the endometrial stroma throughout the secretory phase of the menstrual cycle is associated with a significant increase in the basolateral determinant Scribble and some increase in the apical determinants atypical PKC and Crumbs, indicating acquisition of a partial epithelial identity that may be important in the success of pregnancy. Although an embryo may be able to breach the luminal epithelial layer of the endometrium due to the cycle-dependent changes in polarity and associated partial EMT within this cellular compartment, the decidualizing endometrial stroma appears to act as a more stringent “quality control” mechanism, actively rejecting bad-quality embryos (12). Acquisition of cellular polarity and a more epithelial-like cellular phenotype may play a role in this quality control mechanism. Therefore, if polarity acquisition is compromised as stromal cells decidualize, this may enable implantation of low-quality embryos, as observed in the population of women with recurrent pregnancy loss. This population of women should be investigated for the regulation of stromal/decidual polarity determinants. Should these be dysregulated, they may provide novel uterine targets for modulation to intervene in cases of recurrent pregnancy loss. A cellular paradox exists not just in the adhesion of the endometrial epithelium to the blastocyst trophectodermal epithelium via their apical poles, but in the differential regulation of polarity determinants within the epithelial and stromal compartments under the influence of the same hormonal milieu. Estrogen/progestin treatment of endometrial luminal epithelial (ECC-1) cells decreased Scribble protein, whereas the identical treatment upregulated Scribble protein in primary endometrial stromal cells. This is likely due to the unique properties of the endometrium, whereby the influence of endogenous hormones and local factors differentially transform the epithelium vs the stroma, with the epithelium undergoing a partial EMT, while the stroma undergoes an MET during the same timespan. The associated downregulation of apico-basal polarity within the luminal epithelium upon EMT contributes to the adhesiveness of these cells for the blastocyst. In contrast, the endometrial stroma uniquely undergoes hormone-mediated differentiation; we demonstrate here that this differentiation is partially dependent on de novo development of apico-basal polarity. These data strongly support the plasma membrane transformation theory proposed by Murphy (13). In conclusion, this work demonstrates a downregulation in polarity determinants within the luminal epithelium during the secretory phase of the menstrual cycle encompassing the phase of endometrial receptivity, changes which are likely associated with a partial EMT and which are mediated by the endogenous progesterone-dominated hormonal milieu. Concurrently, polarity determinants are increased within the stromal compartment, associated with decidual transformation, similarly governed by progesterone dominance. These differential changes within the luminal epithelial and stromal endometrial cellular compartments may be functionally important for blastocyst adhesion and decidual transformation, respectively. In vivo, these changes were demonstrated immunohistochemically within normal fertile women who were not under the influence of exogenous steroid hormones. Therefore, given the apparent importance of these changes in receptivity and embryo implantation, it remains to be determined whether these polarity determinants display altered regulation/expression within the endometrium of infertile women, those undergoing hormonal stimulation for assisted reproduction, and those who have experienced recurrent pregnancy loss. Modulation of polarity determinants within the endometrium presents an opportunity to enhance or block development of receptivity in facilitating embryo implantation or development of novel contraceptives, in association with targeting of other aspects of endometrial receptivity. Because the role of polarity determinants has been extensively investigated in organ development and cancer, translation from these fields may provide cues in how to modulate endometrial polarity. Abbreviations: csFCS charcoal-stripped fetal calf serum DMEM Dulbecco’s modified Eagle medium EMT epithelial-to-mesenchymal transition FCS fetal calf serum hCG human chorionic gonadotropin MET mesenchymal-to-epithelial transition MPA medroxyprogesterone acetate PKC protein kinase C PRL prolactin SEM standard error of the mean siRNA small interfering RNA TBS Tris-buffered saline TBST Tris-buffered saline 0.2% Tween 20 TER transepithelial resistance v/v volume-to-volume ratio. Acknowledgments PRL assays were performed by Michael Desakalis at Monash Health pathology department. Judi Hocking collected the endometrial tissue. We thank the women who donated the endometrial tissues used in this study. Financial Support: This work was supported by National Health and Medical Research Council (NHMRC) Fellowship 1002028 (to L.A.S.), NHMRC Project Grant GNT1047756, and the Victorian Government’s Operational Infrastructure funding to the Hudson Institute. 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EndocrinologyOxford University Press

Published: Jan 1, 2018

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