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Proenkephalin A and the γ-aminobutyric acid A receptor π subunit: expression, localization, and dynamic changes in human secretory endometrium

      Objective

      To compare mRNA and protein levels of proenkephalin A (PEA) and γ-aminobutyric acid A receptor π subunit (πGABA-R) in human secretory endometrium before and during receptivity and to determine the cell phenotypes where they are expressed.

      Design

      Prospective and observational, comparing prereceptive vs. receptive stages of secretory endometrium within the same nonconceptional menstrual cycle.

      Setting

      University and non-governmental organization (NGO)–based academic and clinical-research facilities.

      Patient(s)

      Seven healthy, multiparous, surgically sterilized women with spontaneous regular menstrual cycles.

      Intervention(s)

      Endometrial biopsies were obtained on LH+3 and LH+7 within the same cycle.

      Main Outcome Measure(s)

      Levels of PEA and πGABA-R mRNA were determined by real-time PCR, and protein presence, by immunofluorescence.

      Result(s)

      The mRNA level of PEA fell, whereas that of πGABA-R increased, during endometrial receptivity. Positive immunostaining of PEA was found in the luminal and glandular epithelium, whereas that of πGABA-R was in luminal epithelium and stromal cells.

      Conclusion(s)

      The discrete cell-phenotype localization and timing of the changes in the level of PEA and of πGABA-R mRNA and protein suggest an important role for these molecules in switching the human endometrium from a refractory to a receptive state.

      Key Words

      After ovulation, the endometrium undergoes conspicuous structural and biochemical changes that are driven by increasing levels of P, and it becomes functionally receptive to blastocyst implantation in the midluteal phase (
      • Nikas G.
      Endometrial receptivity: changes in cell-surface morphology.
      ,
      • Adams E.C.
      • Hertig A.T.
      • Rock J.
      A description of 34 human ova within the first 17 days of development.
      ,
      • Nikas G.
      Pinopodes as markers of endometrial receptivity in clinical practice.
      ,
      • Wilcox A.J.
      • Baird D.D.
      • Weinberg C.R.
      Time of implantation of the conceptus and loss of pregnancy.
      ,
      • Tabibzadeh S.
      • Babaknia A.
      The signals and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion.
      ). This structural and functional unfolding intimately is associated with changes in gene-expression profiles, such that endometrial receptivity is likely to be determined by a specific mRNA profile (
      • Martin J.
      • Dominguez F.
      • Avila S.
      • Castrillo J.L.
      • Remohi J.
      • Pellicer A.
      • Simon C.
      Human endometrial receptivity: gene regulation.
      ).
      By using a one-by-one approach, several investigators have identified molecules associated with receptivity, such as adhesion molecules, growth factors, and cytokines (
      • Paria B.C.
      • Reese J.
      • Das S.K.
      • Dey S.K.
      Deciphering the cross-talk of implantation: advances and challenges.
      ,
      • Salamonsen L.A.
      • Nie G.
      Proteases at the endometrial-trophoblast interface: their role in implantation.
      ,
      • Aplin J.D.
      • Meseguer M.
      • Simon C.
      • Ortiz M.E.
      • Croxatto H.
      • Jones C.J.
      MUC1, glycans and the cell-surface barrier to embryo implantation.
      ,
      • Cavagna M.
      • Mantese J.C.
      Biomarkers of endometrial receptivity—a review.
      ). More recently, microarray technology has allowed the characterization of a broader spectrum of genes associated with receptivity (
      • Kao L.C.
      • Tulac S.
      • Lobo S.
      • Imani B.
      • Yang J.P.
      • Germeyer A.
      • et al.
      Global gene profiling in human endometrium during the window of implantation.
      ,
      • Carson D.D.
      • Lagow E.
      • Thathiah A.
      • Al-Shami R.
      • Farach-Carson M.C.
      • Vernon M.
      • et al.
      Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.
      ,
      • Borthwick J.M.
      • Charnock-Jones D.S.
      • Tom B.D.
      • Hull M.L.
      • Teirney R.
      • Phillips S.C.
      • et al.
      Determination of the transcript profile of human endometrium.
      ,
      • Riesewijk A.
      • Martin J.
      • van Os R.
      • Horcajadas J.A.
      • Polman J.
      • Pellicer A.
      • et al.
      Gene expression profiling of human endometrial receptivity on days LH+2 vs. LH+7 by microarray technology.
      ,
      • Mirkin S.
      • Arslan M.
      • Churikov D.
      • Corica A.
      • Diaz J.I.
      • Williams S.
      • et al.
      In search of candidate genes critically expressed in the human endometrium during the window of implantation.
      ). Taken together, these data provide clues about the genes that are likely to be involved in endometrial receptivity. One of the limitations of microarray technology is that to exclude false-positive results, each difference detected demands confirmation by an independent technique such as real-time PCR.
      We chose to submit to closer examination the previously reported differential expression of proenkephalin A (PEA) (
      • Carson D.D.
      • Lagow E.
      • Thathiah A.
      • Al-Shami R.
      • Farach-Carson M.C.
      • Vernon M.
      • et al.
      Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.
      ,
      • Borthwick J.M.
      • Charnock-Jones D.S.
      • Tom B.D.
      • Hull M.L.
      • Teirney R.
      • Phillips S.C.
      • et al.
      Determination of the transcript profile of human endometrium.
      ) and the γ-aminobutyric acid A receptor π subunit (πGABA-R), which was detected with high-throughput microarrays (
      • Kao L.C.
      • Tulac S.
      • Lobo S.
      • Imani B.
      • Yang J.P.
      • Germeyer A.
      • et al.
      Global gene profiling in human endometrium during the window of implantation.
      ,
      • Borthwick J.M.
      • Charnock-Jones D.S.
      • Tom B.D.
      • Hull M.L.
      • Teirney R.
      • Phillips S.C.
      • et al.
      Determination of the transcript profile of human endometrium.
      ). We postulated that these two genes play an important role in endometrial physiology.
      Proenkephalin A belongs to the opioid neuropeptide precursor family, consisting of 2,667 amino acids and containing six interspersed Met-enkephalin sequences and one Leu-enkephalin sequence (
      • Comb M.
      • Seeburg P.H.
      • Adelman J.
      • Eiden L.
      • Herbert E.
      Primary structure of the human Met- and Leu-enkephalin precursor and its mRNA.
      ). Enkephalins play a number of physiological functions, including pain perception, response to stress, and modulation of the immune system (
      • Plotnikoff N.P.
      • Faith R.E.
      • Murgo A.J.
      • Herberman R.B.
      • Good R.A.
      Methionine enkephalin: a new cytokine-human study.
      ,
      • Heagy W.
      • Teng E.
      • Lopez P.
      • Finberg R.W.
      Encephalin receptors and receptor-mediated signal transduction in cultured human lymphocytes.
      ). Progesterone and E2 regulate PEA expression in the rodent (
      • Muffly K.E.
      • Jin D.F.
      • Okulicz W.C.
      • Kilpatrick D.L.
      Gonadal steroids regulate proenkephalin gene expression in a tissue-specific manner within the female reproductive system.
      ,
      • Cheon Y.P.
      • Li Q.
      • Xu X.
      • Demayo F.J.
      • Bagchi I.C.
      • Bagchi M.K.
      A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation.
      ) and primate uterus (
      • Low K.G.
      • Nielsen C.P.
      • West N.B.
      • Douglas J.
      • Brenner R.M.
      • Maslar I.A.
      • et al.
      Proenkephalin gene expression in the primate uterus: regulation by estradiol in the endometrium.
      ).
      The main inhibitory neurotransmitter in the mammalian central nervous system, γ-aminobutyric acid (GABA), also is present in peripheral tissues, including the female reproductive tract (
      • Erdo S.L.
      • Lapis E.
      Presence of GABA receptors in rat oviduct.
      ,
      • Erdo S.L.
      • Laszlo A.
      • Szporny L.
      • Zsolnai B.
      High density of specific GABA binding sites in the human fallopian tube.
      ,
      • Erdo S.L.
      • Laszlo A.
      • Kiss B.
      • Zsolnai B.
      Presence of gamma-aminobutyric acid and its specific receptor binding sites in the human term placenta.
      ,
      • Murashima Y.L.
      • Kato T.
      Distribution of gamma-aminobutyric acid and glutamate decarboxylase in the layers of rat oviduct.
      ,
      • Apud J.A.
      • Tappaz M.L.
      • Celotti F.
      • Negri-Cesi P.
      • Masotto C.
      • Racagni G.
      Biochemical and immunochemical studies on the GABAergic system in the rat fallopian tube and ovary.
      ). In mammals, 14 subunits of GABA A receptor have been categorized within five structural classes that are thought to assemble in different pentameric complexes that together form a transmembrane chloride-ion channel (
      • Macdonald R.L.
      • Olsen R.W.
      GABA A receptor channels.
      ,
      • Whiting P.J.
      • McKernan R.M.
      • Wafford K.A.
      Structure and pharmacology of vertebrate GABA A receptor subtypes.
      ). The πGABA-R is expressed in the hippocampus and in several nonneuronal tissues, and its transcript was found to be abundant in the human endometrium and rat uterus (
      • Hedblom E.
      • Kirkness E.F.
      A novel class of GABA A receptor subunit in tissues of the reproductive system.
      ,
      • Neelands T.R.
      • Macdonald R.L.
      Incorporation of the pi subunit into functional gamma-aminobutyric acid (A) receptors.
      ).
      The GABA A receptor is activated by GABA binding, and in addition, it is modulated by benzodiazepines, barbiturates, convulsants, and neurosteroids, including P metabolites (
      • Lan N.C.
      • Chen J.S.
      • Belelli D.
      • Prtchett D.B.
      • Seeburg P.H.
      • Gee K.W.
      A steroid recognition site is functionally coupled to an expressed GABA(A)-benzodiazepine receptor.
      ,
      • Compagnone N.A.
      • Mellon S.H.
      Neurosteroids: biosynthesis and function of these novel neuromodulators.
      ). The degree to which each ligand modulates GABA A receptor function depends on the subunit composition of the receptor (
      • Scholze P.
      • Ebert V.
      • Sieghart W.
      Affinity of various ligands for GABA A receptors containing alpha 4 beta 3 gamma 2, alpha 4 gamma 2, or alpha 1 beta 3 gamma 2 subunits.
      ).
      The aim of this investigation was to characterize the expression of πGABA-R and PEA in the human endometrium during the prereceptive and receptive stages. Our results confirm that these two molecules are important components of the specific repertory of genes whose expression changes during the transition from a nonreceptive to a receptive endometrium.

      Materials and methods

       Subjects

      Seven proven-fertile healthy women who were aged 28–39 years old, menstruating regularly every 26–35 days, and surgically sterilized and who had no history of endometriosis or pelvic inflammatory disease were chosen to participate in this study. The protocol was approved by the Ethics Review Committees of Instituto Chileno de Medicina Reproductiva and Universidad de Santiago de Chile (Santiago, Chile), and the subjects entered the study after signing the informed consent. Each volunteer contributed to the study with a single spontaneous menstrual cycle while taking no medication or hormones or consuming alcohol, cigarettes, or drugs. No subject had used hormonal contraception or taken drugs able to modify the metabolism of steroid hormones for a minimum of 3 months before the study.

       Tissues

      Once subjects entered the study cycle, follicular growth was monitored by daily transvaginal ultrasound, beginning on day 8 of the menstrual cycle, until follicular rupture was detected. A blood sample of 10 mL was obtained from the antecubital vein every other day or daily, as needed, from the time that the leading follicle reached 12 mm until the first biopsy was taken. Estradiol, P, LH, and FSH were measured in these samples by RIA as described elsewhere (
      • Croxatto H.B.
      • Brache V.
      • Pavez M.
      • Cochon L.
      • Forcelledo M.L.
      • Alvarez F.
      • et al.
      Pituitary-ovarian function following the standard levonorgestrel emergency contraceptive dose or a single 0.75-mg dose given on the days preceding ovulation.
      ). Because we anticipated that interindividual variation in gene-expression levels would be substantial, the two endometrial biopsies from each volunteer were compared within the same menstrual cycle, one on day 3 (LH+3) and the other on day 7 (LH+7) after the LH peak (LH+0). This same strategy to control biological variability in gene expression was used by Riesewijk and colleagues (
      • Riesewijk A.
      • Martin J.
      • van Os R.
      • Horcajadas J.A.
      • Polman J.
      • Pellicer A.
      • et al.
      Gene expression profiling of human endometrial receptivity on days LH+2 vs. LH+7 by microarray technology.
      ). A total of 14 biopsies were obtained at two time points in the secretory phase: 7 in the prereceptive phase (day LH+3) and 7 during the receptive phase (day LH+7). Endometrial biopsies were obtained from the uterine fundus under sterile conditions, with pipelles de Cornier (CCD Laboratories, Paris, France). A portion of each sample immediately was frozen in liquid nitrogen for subsequent RNA isolation, and the remainder was transported in cold phosphate-buffered saline (PBS). The tissues then were placed in 30% sucrose in PBS overnight. Subsequently, they were embedded in optical cutting temperature (OCT) compound for immunohistochemistry and dating according to Noyes et al. (
      • Noyes R.N.
      • Hertig A.T.
      • Rock J.
      Dating the endometrial biopsy.
      ).

       Real-Time PCR

      Total RNA was isolated by using Trizol reagent (Invitrogen, Gaithersburg, MD), and complementary DNA synthesis was performed as described elsewhere (
      • Velasquez L.A.
      • Maisey K.
      • Fernandez R.
      • Valdes D.
      • Cardenas H.
      • Imarai M.
      • et al.
      PAF receptor and PAF acetylhydrolase expression in the endosalpinx of the human Fallopian tube: possible role of embryo-derived PAF in the control of embryo transport to the uterus.
      ). The Light Cycler Instrument (Roche Diagnostics, GmbH, Mannheim, Germany) was used to quantify relative expression of πGABA-R and PEA. We chose glyseraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference housekeeping gene. The SYBR Green I double-strand DNA-binding dye was the reagent of choice for these assays. The Light Cycler instrument, even running SYBR Green, provides a broad linear dynamic range for detecting specific PCR products, provided there are no associated byproducts. The oligonucleotide sequences used are the following: πGABA-R forward: 5′-AGCCACCGAATAAACAGCC-3′, πGABA-R reverse: 5′-AGCTCCAACCATTGTTCTAAGC-3′; and PEA forward: 5′-AAGGCGAAAGTTACTCCAA-3′, and PEA reverse: 5′ TAGTTATCCAGACAATGAAGTCAA-3′. Primers were designed by using the EPRIMER3 (Institute Pasteur, Paris, France). The PCR products were isolated and subjected to automated sequencing by using the ABI Prism310 sequencer as described by Muscillo et al. (
      • Muscillo M.
      • La Rosa G.
      • Marianelli C.
      • Zaniratti S.
      • Capobianchi M.R.
      • Cantiani L.
      • et al.
      A new RT-PCR method for the identification of reoviruses in seawater samples.
      ). The obtained sequences displayed 100% homology for πGABA-R and PEA (data not shown).
      All real-time PCR assays were run by using SYBR Green PCR Master Mix and the thermal-cycling parameters that were indicated by the oligonucleotide manufacturer (57°C and 53°C annealing temperature for πGABA-R and PEA, respectively). Quantification was performed by using the SYBR Green dye. Data are presented as relative median ± upper quartile after normalization to the housekeeping gene (GAPDH). No significant difference in the expression of GAPDH between samples was observed when the same starting amount of template was used, thus confirming the adequacy of this gene as an ad hoc standard of reference. Quantitative analysis was based on the relative quantification of each gene of interest in the samples of each group by using the so-called Delta-delta method developed by PE Applied Biosystems (
      • Soong R.
      • Beyser K.
      • Basten O.
      • Kalbe A.
      • Rueschoff J.
      • Tabiti K.
      Quantitative reverse transcription-polymerase chain reaction detection of cytokeratin 20 in noncolorectal lymph nodes.
      ):
      2[Ct(gene X in  reference gene X in endometrial samples)][Ct(GAPDH in referenceGAPDH in endomentrial  samples)],


      where Ct (cycle thresholds) is the cycle in the amplification reaction in which the fluorescence begins to be exponential above the background base line and gene X corresponds to each gene of interest analyzed. Statistical significance was determined by paired t test analysis.

       Immunohistochemistry

      Frozen endometrial cryostat sections (4–6 μm thick) were transferred to gelatin-coated slides. Sections were incubated with ammonium chloride (100 mM) for 45 minutes, followed by washing in PBS and permeabilization in cold acetone for 10 minutes. After three rinses in PBS, sections were blocked with 10% normal serum in PBS for 120 minutes, and the primary antibodies (anti πGABA-R goat >polyclonal 1:50, Santa Cruz Biotechnology, Santa Cruz, CA; or anti Met-enkephalin rabbit polyclonal 1:100; CHEMIKON, Chemicon International, Temecula, CA) were added. Anti–Met-enkephalin antibody also recognizes Leu-enkephalin according to the manufacturer. Incubation was performed overnight at 4°C. Three rinses in PBS were followed by 60 minutes of incubation at room temperature with the corresponding secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). After three rinses in PBS, the samples were counterstained with 1 μg/mL of propidium iodide and were mounted in 1,4-diazabicyclo-(2,2,2)-octane (DABCO, Sigma, St. Louis, MO). Nonimmune goat IgG (enkephalins) and rabbit IgG (πGABA-R) were used as negative controls. The primary antibody was omitted as additional control. The resulting staining was evaluated on a Carl Zeiss confocal laser scanning microscope (LSM-510, Zeiss, Göttingen, Germany).

      Results

      The endocrine profile and transvaginal ultrasound data were consistent with normal ovulatory cycles in all seven cycles under study (data not shown).

       Messenger RNA Levels of PEA and πGABA-R in Human Endometrium

      The mRNA levels of πGABA-R and PEA was determined in duplicate in all samples. The data confirmed that πGABA-R and PEA are transcribed in human endometrium. Specificity of each RT-PCR product was assessed by sequencing and melting-curve analysis, performed after every real-time PCR run; both analyses resulted in a single product for each target gene.
      Data in Figure 1 are presented as a median value for each gene investigated, normalized to the median value of the housekeeping gene (GAPDH) obtained on the respective day LH+3 or LH+7.
      Figure thumbnail gr1
      FIGURE 1Relative tissue levels of mRNA of PEA and πGABA-R in the human endometrium at LH+3 and LH+7. The cross-points of PEA and πGABA-R were normalized with the corresponding cross-point of GAPDH. (A, C) Individual data. To accommodate large interindividual variations in the basal level of these two transcripts, the y-axis in this panel was drawn in logarithmic scale. (B, D) Median and the upper and lower quartiles of 7 subjects. The levels of PEA mRNA diminished 47-fold, whereas those of πGABA-R increased 12-fold from LH+3 to LH+7, respectively (*P<.05 vs. LH+3).
      Quezada. PEA and the GABA-R in human endometrium. Fertil Steril 2006.
      Figure 1 shows individual and median mRNA levels of PEA and πGABA-R on day LH+3 and LH+7. The basal level of these two transcripts differs among volunteers, which is to be expected because of interindividual variability. In all cases, the same tendency of change was displayed between LH+3 to LH+7. The level of these two transcripts changed several fold from LH+3 to LH+7 in all cases. The level of PEA mRNA decreased 47-fold (P<.05), whereas the level of πGABA-R increased 12-fold (P<.05).

       Immunohistochemical Analysis

      To identify the cell phenotypes that express PEA and πGABA-R, samples from four cases included in the expression analysis were submitted to indirect immunofluorescence. Enkephalins clearly were detected in biopsies taken from subjects on LH+3 (Fig. 2A), whereas there was complete absence of such labeling in biopsies obtained from the same subjects on LH+7 (Fig. 2B). They were detected in the luminal and glandular epithelium in all samples analyzed, with no apparent signal in the stroma (Fig. 2A and B; Table 1). Staining for the γ-aminobutyric acid A receptor π subunit was positive on LH+7 and LH+3 in the four samples (Fig. 3A and B). However, the level of expression clearly was less in biopsies taken on LH+3 (Fig. 3A) than in those taken on LH+7 (Fig. 3B). γ-Aminobutyric acid A receptor π subunit was localized in the cell periphery of both luminal epithelium and stromal cells (Fig. 3A and B; Table 2). In the absence of primary antibodies, secondary antibodies produced no signal. (Figs. 2C and D and 3C and D). As a positive control, we used human prostate for PEA and placenta for πGABA-R. Both gave intense staining (data not shown).
      Figure thumbnail gr2
      FIGURE 2Immunofluorescent staining of enkephalin in sections of human endometrium. Enkephalins abundantly are expressed in the cell periphery of luminal and glandular epithelium and are absent from the surrounding stroma on LH+3 (A) and are undetectable on LH+7 (B). Nonimmune goat IgG (negative control) does not cross-react with the tissue sections (C and D). LE = Luminal epithelium; GE = glandular epithelium; S = stroma. Original magnification, ×400.
      Quezada. PEA and the GABA-R in human endometrium. Fertil Steril 2006.
      TABLE 1Semiquantitative assessment of immunostaining of enkephalins in prereceptive (LH+3) and receptive (LH+7) human endometrium.
      VolunteerLH+3LH+7
      Luminal epitheliumGlandular epitheliumStromaLuminal epitheliumGlandular epitheliumStroma
      1++++++
      2++++++
      3++++
      4+++++
      Quezada. PEA and the GABA-R in human endometrium. Fertil Steril 2006.
      Figure thumbnail gr3
      FIGURE 3Immunofluorescent staining of πGABA-R in sections of human endometrium. πGABA-R is expressed in the cell periphery in luminal epithelium and stroma with less intense green fluorescent stain on LH+3 (A) in comparison with LH+7 (B). Nonimmune rabbit IgG (negative control) did not react with the tissue sections (C and D). LE = Luminal epithelium; GE = glandular epithelium; S = stroma. Original magnification, ×400.
      Quezada. PEA and the GABA-R in human endometrium. Fertil Steril 2006.
      TABLE 2Semiquantitative assessment of immunostaining of πGABA-R in prereceptive (LH+3) and receptive (LH+7) in human endometrium.
      VolunteersLH+3LH+7
      Luminal epitheliumGlandular epitheliumStromaLuminal epitheliumGlandular epitheliumStroma
      1+++++++
      2+++++++
      3++++++
      4+++++++
      Quezada. PEA and the GABA-R in human endometrium. Fertil Steril 2006.
      For both targets, we conducted a double-blind semi-quantitative assessment by using the following scale: +++, intense stain; ++, moderate stain; +, minimal stain; and −, absent stain.
      The results are summarized in Tables 1 (immunofluorescence for PEA) and 2 (immunofluorescence for πGABA-R).

      Discussion

      This study shows remarkable changes in the levels of PEA and πGABA-R during the secretory phase of the human endometrium, in agreement with microarray data reported elsewhere (
      • Kao L.C.
      • Tulac S.
      • Lobo S.
      • Imani B.
      • Yang J.P.
      • Germeyer A.
      • et al.
      Global gene profiling in human endometrium during the window of implantation.
      ,
      • Carson D.D.
      • Lagow E.
      • Thathiah A.
      • Al-Shami R.
      • Farach-Carson M.C.
      • Vernon M.
      • et al.
      Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.
      ,
      • Borthwick J.M.
      • Charnock-Jones D.S.
      • Tom B.D.
      • Hull M.L.
      • Teirney R.
      • Phillips S.C.
      • et al.
      Determination of the transcript profile of human endometrium.
      ,
      • Riesewijk A.
      • Martin J.
      • van Os R.
      • Horcajadas J.A.
      • Polman J.
      • Pellicer A.
      • et al.
      Gene expression profiling of human endometrial receptivity on days LH+2 vs. LH+7 by microarray technology.
      ,
      • Mirkin S.
      • Arslan M.
      • Churikov D.
      • Corica A.
      • Diaz J.I.
      • Williams S.
      • et al.
      In search of candidate genes critically expressed in the human endometrium during the window of implantation.
      ). By semiquantitative assay of the mRNA by real-time PCR and respective proteins by immunohistochemistry, the level of PEA falls, whereas the level of πGABA-R rises from the prereceptive to the receptive stage.
      This procedure, taking two biopsies a few days apart within the same cycle, has the potential to generate a difference in gene expression between the two because of the procedure. However, in microarray assays conducted to characterize the repertory of molecules associated with receptivity, the biopsies compared were taken in the same or in different cycles, and at least the direction of the changes was found to coincide (
      • Kao L.C.
      • Tulac S.
      • Lobo S.
      • Imani B.
      • Yang J.P.
      • Germeyer A.
      • et al.
      Global gene profiling in human endometrium during the window of implantation.
      ,
      • Carson D.D.
      • Lagow E.
      • Thathiah A.
      • Al-Shami R.
      • Farach-Carson M.C.
      • Vernon M.
      • et al.
      Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.
      ,
      • Borthwick J.M.
      • Charnock-Jones D.S.
      • Tom B.D.
      • Hull M.L.
      • Teirney R.
      • Phillips S.C.
      • et al.
      Determination of the transcript profile of human endometrium.
      ). This observation and the regularity of the findings in our material make it highly unlikely that they are a result of artifact rather than of true physiologic changes.
      Real-time PCR showed low levels of mRNA for PEA on LH+7 in comparison with LH+3. This may be caused by rising levels of P because the PEA gene displays a single P-response element (
      • Borthwick J.M.
      • Charnock-Jones D.S.
      • Tom B.D.
      • Hull M.L.
      • Teirney R.
      • Phillips S.C.
      • et al.
      Determination of the transcript profile of human endometrium.
      ), and this hormone directly reduces the transcription of PEA mRNA. In addition, P may induce the expression of other genes that inhibit PEA expression, as in the case of ebaf (
      • Tabibzadeh S.
      • Mason J.M.
      • Shea W.
      • Cai Y.
      • Murray M.J.
      • Lessey B.
      Dysregulated expression of ebaf, a novel molecular defect in the endometria of patients with infertility.
      ).
      Low et al. (
      • Low K.G.
      • Nielsen C.P.
      • West N.B.
      • Douglas J.
      • Brenner R.M.
      • Maslar I.A.
      • et al.
      Proenkephalin gene expression in the primate uterus: regulation by estradiol in the endometrium.
      ) suggested that gene expression of PEA in monkey endometrium is regulated positively by E2 and negatively by P, a relationship that is consistent with the diminution in PEA expression level during receptivity shown in the present work. In contrast, expression of PEA in rats, mice, and hamster is regulated positively by P (
      • Cheon Y.P.
      • Li Q.
      • Xu X.
      • Demayo F.J.
      • Bagchi I.C.
      • Bagchi M.K.
      A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation.
      ,
      • Soong R.
      • Beyser K.
      • Basten O.
      • Kalbe A.
      • Rueschoff J.
      • Tabiti K.
      Quantitative reverse transcription-polymerase chain reaction detection of cytokeratin 20 in noncolorectal lymph nodes.
      ), indicating divergent regulation of PEA in the uterus of primates and rodents.
      With respect to cell localization of enkephalins in human endometrial tissue, immunofluorescence revealed that these molecules are present in the epithelium, as in rodents (
      • Jin D.F.
      • Muffly K.E.
      • Okulicz W.C.
      • Kilpatrick D.L.
      Estrous cycle- and pregnancy-related differences in expression of the proenkephalin and proopiomelanocortin genes in the ovary and uterus.
      ). This cell-type localization is compatible with a role of PEA in endometrial refractoriness to implantation.
      The multiple functions attributed to enkephalins in nonneuronal tissues (
      • Plotnikoff N.P.
      • Faith R.E.
      • Murgo A.J.
      • Herberman R.B.
      • Good R.A.
      Methionine enkephalin: a new cytokine-human study.
      ,
      • Heagy W.
      • Teng E.
      • Lopez P.
      • Finberg R.W.
      Encephalin receptors and receptor-mediated signal transduction in cultured human lymphocytes.
      ,
      • Rosen H.
      • Itin A.
      • Schiff R.
      • Kesshet E.
      Local regulation within the female reproductive system and upon embryonic implantation: identification of cells expressing proenkephalin A.
      ,
      • Blebea J.
      • Mazo J.E.
      • Kihara T.K.
      • Vu J.H.
      • McLaughlin P.J.
      • Atnip R.G.
      • et al.
      Opioid growth factor modulates angiogenesis.
      ) invites speculation on their role in the human endometrium during the prereceptive period. Enkephalin-mediated enhancement of immune responses has been demonstrated (
      • Salzet M.
      • Tasiemski A.
      Involvement of pro-enkephalin-derived peptides in immunity.
      ,
      • Zunich K.M.
      • Kirkpatrick C.H.
      Methionine-enkephalin as immunomodulator therapy in human immunodeficiency virus infections: clinical and immunological effects.
      ,
      • Jankovic B.D.
      • Maric D.
      Enkephalin-induced stimulation of humoral and cellular immune reactions in aged rats.
      ), so our results showing decreased endometrial enkephalin expression during the receptive period are consistent with the simultaneous expression of immunosuppressors such as glycodelin (
      • Seppala M.
      • Koistinen H.
      • Mandelin E.
      • Koistinen R.
      Importance of uterus and sperm glycodelin in the regulation of reproduction.
      ).
      In the vascular system, activation of endothelial δ-opioid receptors decreases the intracellular cyclic adenosine 3′:5′ monophosphate levels and inhibits integrin-mediated lymphocyte adhesion (
      • Zhu Y.
      • Pintar J.E.
      Expression of opioid receptors and ligands in pregnant mouse uterus and placenta.
      ,
      • Rangarajan S.
      • Enserink J.M.
      • Kuiperij H.B.
      • de Rooij J.
      • Price L.S.
      • Schwede F.
      • et al.
      Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor.
      ). If a similar regulatory mechanism operates in the endometrial epithelium, decreased expression of enkephalins could lead to decreased activation of δ receptors and increased intracellular cyclic adenosine 3′:5′ monophosphate levels. This in turn would increase integrin-mediated cell adhesion. Experimental testing of this hypothesis is needed to elucidate the interaction of opioid peptides and integrins in embryo adhesion.
      An explanation for the elevated level of πGABA-R during endometrial receptivity has to take into account that P levels are high in this period. Progesterone receptors can act on P-response elements in the promoter region of this gene (
      • Borthwick J.M.
      • Charnock-Jones D.S.
      • Tom B.D.
      • Hull M.L.
      • Teirney R.
      • Phillips S.C.
      • et al.
      Determination of the transcript profile of human endometrium.
      ).
      Pharmacologic and electrophysiological studies show that GABA channels are expressed in human endometrium (
      • Whiting P.J.
      • McKernan R.M.
      • Wafford K.A.
      Structure and pharmacology of vertebrate GABA A receptor subtypes.
      ). Therefore, increased expression of πGABA-R may augment the affinity of GABA receptor for P metabolites, increasing the entrance of chloride to the cell (
      • Hedblom E.
      • Kirkness E.F.
      A novel class of GABA A receptor subunit in tissues of the reproductive system.
      ).
      The opening of GABA-receptor ion channels may be crucial to transfer water from the endometrial cavity into the epithelium. This could foster the formation of uterodomes at the apical pole of the epithelium, which takes place at the onset of receptivity and allows trophoblast–epithelium attachment to proceed. The expression of πGABA-R in luminal epithelium and stroma suggests that this molecule may participate in early and late stages of implantation.
      The differential expression and cell localization of PEA and πGABA-R in human endometrium are compatible with an important role for these molecules in the acquisition of endometrial receptivity.

      Acknowledgment

      The authors thank Gareth Owen, Ph.D., from Pontificia Universidad Católica de Chile (Santiago, Chile), for his critical reading of this manuscript.

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