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MRC Human Reproductive Sciences Unit (S.B., K.M., R.P.M., H.N.J.) and Department of Reproductive and Developmental Sciences (H.O.D.C.), Centre for Reproductive Biology, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom
Address all correspondence and requests for reprints to: Dr. H. N. Jabbour, MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, United Kingdom. E-mail: h.jabbour{at}hrsu.mrc.ac.uk.
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Abstract |
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PK1 and PK2 and their receptors were localized to multiple cellular compartments, including glandular epithelial, stromal, and endothelial cells in the endometrium and endothelial and smooth muscle cells in the myometrium. The elevation in PK1 expression in the secretory phase of the menstrual cycle indicated potential regulation of PK1 by progesterone. To investigate this, endometrial tissue was treated with 1 µM (µmol/liter–1) progesterone for 24 h, and PK1 expression was assessed by quantitative RT-PCR. Treatment with 1 µM (µmol/liter–1) progesterone resulted in 2.91 ± 0.75-fold elevation in PK1 expression, compared with controls (P < 0.05). These data identify a paracrine role for the PKs and their receptors in endometrial vascular function.
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Introduction |
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Initial studies examining the functions of PK1 and PK2 have demonstrated diverse physiological roles for these factors. PK1 and PK2 can promote angiogenesis by causing endothelial cell proliferation and chemotaxis in an organ-specific manner (1, 3). In addition, there is evidence that they are secreted in response to hypoxia and involved in the circadian rhythm of the suprachiasmatic nucleus (1, 6).
The human endometrium undergoes cyclical phases of tissue regeneration and shedding during every menstrual cycle, which is orchestrated to prepare the uterus for implantation in the event of pregnancy. Angiogenesis is of crucial importance during the cyclical tissue regeneration and growth of the normal endometrium. Moreover, regulation of angiogenesis and vascular function has been implicated in various endometrial disorders including menorrhagia and endometriosis (7, 8, 9). A role in the control of endometrial angiogenesis has been established for a number of factors including the vascular endothelial growth factor (VEGF) family of genes, angiopoietins, and the fibroblast growth factors (FGFs) (8, 10, 11). The recent discovery of the proangiogenic actions of the PKs has highlighted their potential role in the regulation of endometrial angiogenesis.
The objectives of the current study were to investigate the temporal expression and regulation of the PKs and their receptors in the normal human endometrium across the menstrual cycle. The data confirm the expression of PKs and their receptors in various cellular compartments within the endometrium and myometrium including endothelial cells. PK1 expression demonstrated temporal regulation with levels being elevated during the secretory phase of the menstrual cycle and induced by treatment with progesterone. Taken together, the data outline a potential paracrine role for this novel family of angiogenic factors in endometrial vascular function.
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Patients and Methods |
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Endometrial biopsies (n = 50) at different stages of the menstrual cycle were obtained from women with regular menstrual cycles (25–35 d), who had not received a hormonal preparation in the 3 months preceding biopsy collection. Samples were collected either with an endometrial suction curette (Pipelle, Laboratoire CCD, Paris, France) or as full-thickness endometrial biopsies (including the functional layer and basal-myometrial junction) from women undergoing hysterectomy for benign gynecological indications. Shortly after collection, tissue was snap frozen in dry ice and stored at –70 C (for RNA extraction), fixed in neutral buffered formalin and wax embedded (for immunohistochemical analyses or in situ hybridization), or placed in RPMI 1640 [containing 2 mM (mmol/liter–1) L-glutamine, 100 U penicillin, and 100 µg/ml (µg/ml–1) streptomycin] and transported to the laboratory for in vitro culture.
Biopsies were dated according to stated last menstrual period and confirmed by histological assessment according to criteria of Noyes et al. (12). Furthermore, circulating estradiol and progesterone concentrations at the time of biopsy were consistent for both stated last menstrual period and histological assignment of menstrual cycle stage. Samples were divided according to phase of menstrual cycle as menstrual (d 1–4), early to midproliferative (d 5–10), late proliferative to ovulatory (d 11–14), early secretory (d 15–18), midsecretory (d 19–24), and late secretory (d 25–28). Ethical approval was obtained from Lothian Research Ethics Committee, and written informed consent was obtained from all subjects before tissue collection.
Gene analysis
The DNA sequences of the genes encoding the PKs and their receptors were obtained in complete form from the GenBank human genome database and were located as given in Table 1
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RNA for RT-PCR was extracted from endometrial suction curette biopsies (consisting predominantly of functional layer endometrium), obtained from across the menstrual cycle (n = 33) using Tri Reagent (Sigma, Poole, UK) following the manufacturer’s instructions. RNA samples were quantified and reverse transcribed using 5.5 mM (mmol/liter–1) MgCl2, 0.5 mM (mmol/liter–1) of each deoxynucleotide triphosphate, 2.5 µM (µmol/liter–1) random hexamers, and ribonuclease inhibitor both at 0.4 U/µl (U/µl–1) and 1.25 U/µl (U/µl–1) multiscribe reverse transcriptase (all from Applied Biosystems, Warrington, UK). RNA (400 ng) was added to each reverse transcription reaction and samples were incubated for 90 min at 25 C, 45 min at 48 C, and 5 min at 95 C. The reaction mix for the PCR consisted of 1x mastermix, ribosomal 18S forward and reverse primers, ribosomal 18S probe [50 nM (nmol/liter–1)]; all from Applied Biosystems], forward and reverse primers for PK1, PK2, PKR1, and PKR2 [300 nM (nmol/liter–1)], and their probes [200 nM (nmol/liter–1)] (all from Biosource UK, Nivelles, Belgium). The reaction mix (48 µl) was aliquoted into tubes and 2 µl cDNA were added. Duplicate 24-µl samples plus positive and negative controls were placed in a PCR plate and wells were sealed with optical caps. The PCRs were carried out using an ABI Prism 7700 (Applied Biosystems). All primers and probes were designed using the PRIMER express program (Applied Biosystems). The sequences of primers and probe are given in Table 2. Data were analyzed and processed using Sequence Detector version 1.6.3 (Applied Biosystems) according to the manufacturer’s instructions. Primers and probes were optimized and the linearity of the results validated in serial dilution of a cDNA pool. Results were expressed relative to an internal positive standard (cDNA obtained from a single sample of endometrial tissue) included in all reactions.
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Custom synthesized oligonucleotide double-fluoroscein isothiocyanate (FITC)-labeled cDNA probes for PK2, PKR1, and PKR2 were obtained from Biognostik (Göttingen, Germany). Sections (5 µM) from full-thickness human endometrial biopsies collected across the menstrual cycle (n = 12; two each from the early, mid-, and late proliferative and early, mid-, and late secretory phases) were cut onto gelatin-coated slides. Sections were dewaxed and rehydrated and then treated with proteinase K [20 µg/ml (µg/ml–1) in 100 mM (mmol/liter–1) Tris-HCl (pH 7.6), containing 50 mM (mmol/liter–1) EDTA] for 15 min at 37 C to enhance cDNA probe access. Sections were washed in diethylpyrocarbonate-treated water and prehybridized for 4 h at 30 C with 25 µl of the hybridization buffer supplied with the probe, which had been previously heated to 95 C. The sections were then hybridized overnight at 30 C with the cDNA probe at 6 U/ml (U/µl–1) for PKR1 and 12 U/ml (U/µl–1) for PK2 and PKR2 in hybridization buffer. After hybridization, sections were washed for 2 x 5 min in 1x standard saline citrate at room temperature and 2 x 15 min in 0.1x standard saline citrate at 39 C. After rinsing in Tris-buffered saline, endogenous peroxidase activity was quenched with 10% (vol/vol) H2O2 in methanol at room temperature. The FITC-labeled probes were detected using standard immunohistochemical reagents with an additional amplification step (TSA biotin system, NEN Life Science Products, Hounslow, UK). Sections were incubated with blocking buffer for 30 min. Conjugated anti-FITC-horseradish peroxidase (Roche Diagnostics Ltd., Lewes, UK) was added at a dilution of 1:200 in blocking buffer and the sections incubated for 30 min. After washing, biotinyl tyramide amplification reagent (1:50 dilution) was applied to each slide and incubated for 15 min. Streptavidin-horseradish peroxidase (1:100 dilution) was applied after washing and incubated for 30 min and probe localization visualized with 3,3'-diaminobenzidine substrate. To assess background hybridization, control sections were treated with a double FITC-labeled oligonucleotide probe containing the same proportion of cysteine and guanine bases as each of the PK2, PKR1, and PKR2 probes. All treatments were carried out at room temperature unless otherwise specified.
Immunohistochemistry
Immunohistochemistry was performed as described previously (13). Briefly, sections (5 µM), obtained from full-thickness human endometrial biopsies from across the menstrual cycle (n = 12; two each from the early, mid-, and late proliferative and early, mid-, and late secretory phases), were dewaxed in xylene and rehydrated using decreasing grades of ethanol. Antigen retrieval was performed by treating sections for 5 min in a pressure cooker in boiling 0.1% citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 10% (vol/vol) H2O2 in methanol at room temperature. Nonimmune swine serum (20% serum in Tris-buffered saline) was applied for 1 h before overnight incubation at 4 C with rabbit antihuman PK1 (Phoenix Pharmaceuticals Inc., Belmont, CA) at a dilution of 1:2000. An avidin-biotin peroxidase detection system was then applied (Dako Ltd., Cambridge, UK) with 3,3'-diaminobenzidine as the chromagen.
Tissue culture and treatment
Endometrial tissue from suction curette biopsies (n = 5) collected from across the menstrual cycle was minced finely with scissors and divided into two portions. The tissue was incubated overnight at 37 C in a humidified 5% CO2 incubator in 2 ml Phenol Red Free D-MEM/F-12 medium with L-glutamine (Invitrogen Ltd., Paisley, UK) containing 100 IU penicillin and 100 µg streptomycin. The tissue was then treated for 24 h in the same medium either in the presence of 1 µM (µmol/liter–1) progesterone in ethanol (Sigma) or an equivalent volume of ethanol. Subsequently, RNA was extracted from all samples and PK1 expression was determined by quantitative RT-PCR as described above.
Statistics
Where appropriate, data were subjected to statistical analysis with ANOVA and Fishers protected least significant difference tests (Statview 4.0; Abacus Concepts Inc., Piscataway, NJ) and statistical significance accepted when P < 0.05.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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). Expression of PK1 in the menstrual phase of the cycle was highly variable (80.57 ± 57.05; n = 3) and did not differ significantly from the proliferative or secretory stages. PK2 was also expressed in all samples examined (Fig. 1B) but did not show significant variation with the phase of the menstrual cycle (menstrual: 5.42 ± 3.57; proliferative: 3.17 ± 0.61; secretory: 2.70 ± 0.46). PKR1 and PKR2 were also expressed during all phases of the menstrual cycle (PKR1, menstrual: 0.95 ± 0.51; proliferative: 1.38 ± 0.16; secretory: 1.07 ± 0.13 and PKR2, menstrual: 4.40 ± 3.39; proliferative: 3.08 ± 0.52; secretory: 3.01 ± 0.45).
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, A–C). Greater intensity of staining was detected in the functional layer (Fig. 2C), compared with the basal layer of the endometrium (Fig. 2B). Endothelial cells lining blood vessels in the myometrium and myometrial smooth muscle cells also displayed immunoreactivity for PK1 (Fig. 2A, inset).
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, D–F) was detected in glandular epithelial cells, stromal and endothelial cells predominantly in the functional layer (Fig. 2F), with stronger staining toward the luminal surface of the epithelium. Weak vascular staining was present in the basal layer (Fig. 2E) of the endometrium and in the myometrium. There was also infrequent weak staining of myometrial smooth muscle cells. PKR1 expression was detected in glandular epithelial and endothelial cells throughout the endometrium (Fig. 3, A–C) and in stromal cells, especially in the functional layer (Fig. 3C). In addition, PKR1 reactivity was detected in the blood vessels and smooth muscle cells in the myometrium (Fig. 3A, inset). Very weak PKR2 expression was detected by in situ hybridization, and expression was confined to glandular epithelial cells and endothelial cells, predominantly in the functional layer of the endometrium (Fig. 3, D–F).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The data presented here also confirm expression of both PKR1 and PKR2 in the human endometrium. Both receptors were detected in all samples examined by RT-PCR but without significant temporal variation between the proliferative and secretory phases of the menstrual cycle. This suggests that the major control of prokineticin function in the uterus is mediated through changes in ligand expression and not receptor expression. The coexpression of the G proteincoupled receptors, PKR1 and PKR2, with their ligands has been demonstrated previously in a range of tissues, including endocrine organs such as the adrenal and pituitary glands and ovary and the gastrointestinal tract (4, 5). In the present study, localization of the site of expression of PKR1 and PKR2 was investigated by in situ hybridization. Both receptors were localized in multicellular compartments as observed for PK1 and PK2. This implicates paracrine mechanisms of action for PKs in the human endometrium and myometrium.
The temporal variation in expression of PK1 suggests that it may be regulated by steroid hormones, particularly progesterone. This notion is supported by the in vitro data, demonstrating elevation of PK1 mRNA expression in response to progesterone. The increase in PK1 expression may result from a direct effect of progesterone on PK1 gene transcription via a progesterone receptor response element in the promoter region. This possibility is supported by analysis of the promoter region of the PK1 gene in which two putative progesterone receptor binding sites can be identified. Regulation of expression of uterine growth factors by steroid hormones is well established and has been described for a number of other angiogenic factors, including FGF and VEGF (14) and the type 2 VEGF receptor (15). For example, there is evidence for increased synthesis of FGF and VEGF in response to estrogen and progesterone in uterine tissue in vitro, and there are putative estrogen and progesterone response elements in the promoter regions of both the FGF and VEGF genes (14, 16, 17, 18, 19). It is also plausible that progesterone may mediate its effect on PK1 expression indirectly via paracrine mechanisms. This is supported by recent data from tissue recombination studies using uteri from progesterone receptor null mice (20).
PK1 and PK2 have been linked to a diverse range of physiological functions in vitro, including angiogenic functions such as the proliferation, migration, and fenestration of endothelial cells. These data would indicate a particular role for PK1 and possibly PK2 in uterine angiogenesis, possibly in concert with the other angiogenic factors already described in this tissue. The endometrium is one of the few sites in the adult in which rapid angiogenesis is a normal physiological event as part of regeneration and growth during the menstrual cycle (10). Regeneration commences in the basal layer of the endometrium during menstruation in the functional layer and subepithelial capillary plexus in the proliferative to early secretory phases and in the spiral arterioles during the secretory phase (21). Several growth factors, including the angiopoietins and members of the FGF family, have been identified as having a regulatory role in endometrial angiogenesis (7, 8, 11, 22). Furthermore, there is evidence demonstrating that the VEGF family of growth factors and their receptors are regulatory factors in angiogenesis in the uterus, although the precise role of VEGF and its receptors in the angiogenic process at different stages of the menstrual cycle remains unclear (10, 11, 17, 23, 24). The data presented herein strongly suggest that the newly identified PK family also plays a role in the angiogenic and vascular function of the human endometrium. Moreover, the regulation of PK1 expression by progesterone outlines a potential role for this angiogenic factor in vascular differentiation and spiral arteriole formation during the secretory phase of the menstrual cycle.
The expression of PKR1 and PKR2 in the glandular epithelial cells and smooth muscle cells suggest that the PKs may also have a nonangiogenic function in the human uterus. PK1 may play a role in the regulation of the endometrial environment by progesterone, including the edema and hyperpermeability required for implantation (25). The expression of PK1 in myometrial smooth muscle suggests it may also play a role in myometrial contraction by analogy with the effects of PK1 on the contraction of gastrointestinal smooth muscle in vitro (2).
The demonstration of a marked change in the expression of PK1 in the human endometrium across the menstrual cycle provides a background for a delineation of its significance in normal uterine function and uterine pathologies.
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Acknowledgments |
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Footnotes |
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Received November 18, 2003.
Accepted February 16, 2004.
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