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To demonstrate expression and regulation of prokineticins (PROKs) and their receptors (PROKRs) in Fallopian tube (FT) from non-pregnant women and women with ectopic pregnancy (EP).
Large UK teaching hospital
Women undergoing hysterectomy for benign gynecological conditions (n=15) and surgery for EP (n=16).
Quantitative RT-PCR and immunohistochemistry were used to determine FT PROK/PROKR mRNA expression and protein localization, respectively. PROK/PROKR levels were measured in tubal explant cultures stimulated with estrogen and progestogen.
Differential expression of PROK and PROKR.
FT PROK2 and PROKR1 mRNA levels were upregulated during the progesterone-dominant mid-luteal phase of the menstrual cycle. Increased PROKR1 expression was observed in tubal explant cultures treated with medroxyprogesterone acetate. PROK and PROKR proteins were localized to the epithelium and smooth muscle layers of the FT. PROKR1 and PROKR2 mRNA levels were lower in FT from women with EP compared to non-pregnant FT from the mid-luteal phase.
These data suggest a potential role for PROKs in FT function. PROKs are known to affect smooth muscle contraction in the gut. Dysregulated PROK expression in FT could affect FT smooth muscle contractility and embryo-tubal transport, providing a potential cause for EP.
Ectopic pregnancy remains a considerable cause of morbidity and occasional mortality (1). One in 80 pregnancies is ectopic and over 98% implant in the Fallopian tube (1;2). The exact mechanism leading to tubal implantation is uknown. However, given that the human embryo appears to have the ability to implant on any given epithelial surface (3), it is likely that ectopic pregnancy is the result of embryo retention in the Fallopian tube due to Fallopian tube dysfunction. Transport of the pre-implantation embryo through the Fallopian tube is accomplished, in part, by smooth muscle contraction (4;5).
The prokineticins, PROK1 and PROK2, have angiogenic actions but are primarily known for their function as regulators of specific and potent contractions of smooth muscle (6). They are the cognate ligands for two closely homologous G protein-coupled receptors, PROK receptor (PROKR) 1 and PROKR2, through which either PROK can signal (6). PROKs were initially reported to be expressed in the gastrointestinal tract, where they were shown directly to stimulate contraction of the ileum longitudinal muscle of guinea pigs (7). However, the opposing effect of relaxation through a nitric oxide-mediated mechanism has also been reported recently in the proximal colon in mice (8). In addition, it has been reported that PROK2 has no effect on fore-stomach or colon contraction (9), suggesting that the intracellular milieu in different tissues results in differential coupling and different phenotypic effects.
PROKs and their receptors are expressed in the ovary, uterus and in various tissues of pregnancy (10;11). PROK1 is expressed in the uterine epithelium, as well as the smooth muscle cells of the myometrium, with maximum expression at the time of implantation in the progesterone dominant mid-secretory phase (11). PROK1 is expressed in much greater levels than PROK2. PROK2 and the PROK receptors are also expressed in the various cellular compartments of the uterus but they do not show a temporal variation across the menstrual cycle (11), suggesting a regulatory role for PROK1 and a more permissive role for PROK2 in uterine function. Furthermore, it has been hypothesized that PROK1 facilitates successful intrauterine embryo implantation through induction of leukemia inducible factor (LIF) expression (12), which has been shown to promote adhesion of trophoblast cells to extracellular matrix proteins, in vitro.
It is therefore plausible that the PROKs and their receptors could regulate smooth muscle contraction in the human Fallopian tube facilitating controlled and timely embryo transport into the uterine cavity, and that attenuated expression could lead to embryo retention and ectopic pregnancy. However, PROK/PROKR expression has to our knowledge never been demonstrated in human Fallopian tube. In this study, we report expression and tissue localization of the PROKs and PROKRs in human Fallopian tube, their regulation by progesterone and show that PROKR expression is dysregulated in ectopic pregnancy.
Ethical approval for this study was obtained from the Lothian Research Ethics Committee (04/S1103/20) and informed written consent was obtained from all patients. Fallopian tube tissues from the ampullary region of the Fallopian tube were collected at the time of hysterectomy (n = 15) or during surgical management of tubal pregnancy (n = 16). Women were between 18 and 45 years of age (mean age for hysterectomy patients was 40 ± 4 and mean age for tubal ectopic pregnancy patients was 31 ± 5). Samples were prepared by either a) short-term storage in phosphate-buffered saline (PBS) prior to explant culture, b) immersion in RNAlater ™ (Ambion, Texas, USA) at 4 °C overnight and then flash frozen at −80 ° for RNA extraction or c) fixation in 4% neutral-buffered formalin overnight at 4 °C followed by storage in 70% ethanol, and embedding in paraffin wax for for immunohistochemical staining. The menstrual cycle phase of each patient at the time of hysterectomy was determined using the following parameters: 1) last menstrual period confirmed by patient, b) histological examination and staging of an endometrial biopsy taken with the Fallopian tube by a gynecological pathologist and c) measurement of estradiol and progesterone levels in serum of patients. The clinical data corresponding to each sample are listed in Table 1. None of the gynaecological conditions listed have been reported to date to affect PROK/PROKR expression in the endometrium so it was assumed that they would have no effect on expression in the Fallopian tube. Fallopian tubes removed during surgical management for ectopic pregnancy were all from the ampullary region of the Fallopian tube. Gestational age, human chorionic gonadotropin (hCG) and progesterone levels of each ectopic pregnancy are listed in Table 1.
Fallopian tube tissue culture was performed, in triplicate, using tissue from three different patients. Two of the Fallopian tubes were from the follicular phase of the menstrual cycle and one was from the late-luteal phase. Each tissue was cut into small pieces (2-3 mm) which were placed in each well of a 12-well dish and cultured in RPMI containing 10% charcoal-stripped fetal-bovine serum at 37 °C in 5% CO2. PROK expression in the endometrium has previously been demonstrated to be upregulated by progesterone after 24 hours (11). Explants were therefore treated with 10 nM estradiol (Sigma-Aldrich, Dorset, UK), 1 μM medroxyprogesterone acetate (MPA) (Sigma-Aldrich, Dorset, UK) (both in ethanol) and a combination of the two steroids for 24 hours according to this protocol. Explants were also treated with ethanol to control for the steroid diluent. After 24 hours, media was removed and tissues were collected for RNA extraction.
Total RNA was extracted from tissues using the RNAeasy kit (Qiagen, West Sussex, UK) according to the manufacturer’s protocol. RNA concentrations were determined using a Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). 200 ng of RNA was reverse transcribed into cDNA in a 10 μL reaction using random hexamers (Applied Biosystems, Foster City, CA, USA).
Taqman RT-PCR was used to quantify transcript levels. Reactions were performed using an ABI Prism 7900 (Applied Biosystems, Foster City, CA, USA) using standard conditions. Previously validated primer and FAM (6-carboxyfluorescein) labeled probe sequences are listed in Supplementary Table 1. 1 μL of cDNA (from above) was used per 25 μL qRT-PCR reaction and all reactions were performed in triplicate. Gene expression was normalized to RNA loading using primers and VIC ™ labeled probe (Applied Biosystems, Foster City, CA, USA) for ribosomal 18S as an internal standard. Using the 2-ΔΔCt method, mRNA expression results were normalized against 18S and expressed as relative to a positive RNA standard (cDNA from a single mid-secretory endometrial tissue) which was included in all reactions.
Standard immunohistochemistry protocols were used (11). All antibodies were diluted in normal goat serum: PROK1 rabbit polyclonal serum antibody (Phoenix Pharmaceuticals, Belmont CA, USA), 1000-fold; PROKR1 rabbit polyclonal antibody (IgG purified) (MBL International Corporation, Woburn, MA, USA), 250-fold; PROK2 rabbit polyclonal antibody (IgG purified) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 50-fold; PROKR2 rabbit polyclonal antibody (IgG purified) (MBL International corporation, Woburn, MA, USA), 67-fold and smooth muscle actin mouse monoclonal antibody (Sigma-Aldrich, Dorset, UK), 5000-fold. Rabbit serum diluted 1000-fold in normal goat serum was used as a negative control for PROK1 staining. Rabbit IgG diluted 50-fold in normal goat serum was used as a negative control for PROK2, PROKR1 and PROKR2 staining. Mouse IgG diluted 5000-fold in normal goat serum was used as a negative control for smooth muscle actin staining. Staining was reviewed by an expert gynecological pathologist.
Statistical analyses were performed with GraphPad Prism (version 5.0, GraphPad Software, La Jolla, CA, USA). Analysis of the differences in PROK and PROKR levels in mid-luteal phase Fallopian tubes and in Fallopian tubes from ectopic pregnancies was performed using a Mann-Whitney test. Samples for which q-RT-PCR found undetermined levels of transcript were removed from the analysis. For the analysis of PROK and PROKR levels in hormonally treated explants, a One-Way ANOVA was performed followed by Dunnett’s post-hoc analysis.
PROK1, PROK2 PROKR1, and PROKR2 mRNA levels were measured in Fallopian tubes from the follicular and mid-luteal phases of the menstrual cycle and in Fallopian tubes from women with ectopic pregnancies using Taqman q-RT-PCR. PROK1 and PROKR1 levels were relatively lower compared with PROK2 and PROKR2 levels in all Fallopian tube tissue samples compared (Figure 1). PROK1 levels did not vary across the menstrual cycle (Figure 1a). PROK2 levels were higher in the mid-luteal phase compared to the follicular phase of the menstrual cycle (Figure 1c; p<0.05). PROKR1 levels were significantly higher in the mid-luteal phase of the menstrual cycle compared to the follicular phase (Figure 1e; p<0.05). PROKR1 levels were also significantly lower in Fallopian tubes from ectopic pregnancies compared to levels in Fallopian tubes from the mid-luteal phase of the menstrual cycle (Figure 1f; p<0.05). PROKR2 levels did not vary across the menstrual cycle but were significantly lower in Fallopian tubes from ectopic pregnancies compared to levels in Fallopian tubes from the mid-luteal phase of the menstrual cycle (Figure 1h; p<0.01). Due to ethical constraints, it is not possible to collect Fallopian tube from women with intra-uterine pregnancies and so Fallopian tube collected from the mid - luteal phase, when circulating progesterone levels are raised, provides the most appropriate control.
PROK1, PROK2 and PROKR2 mRNA levels were not influenced by hormonal treatment of human Fallopian tube explants (Figure 2). PROKR1 levels were significantly upregulated by MPA treatment (Figure 2c; p<0.01) and combined estrogen and MPA treatment (Figure 2c; p<0.05). Estrogen treatment alone did not alter the expression of any of the genes analyzed. We also compared PROKR levels in Fallopian tube from women with low serum hCG levels (< 1000 IU/L) with PROK levels in Fallopian tube from women with high serum hCG levels (> 1000 IU/L) and found no statistically significant differences in expression between these two groups (data not shown).
PROK1, PROK2, PROKR1 and PROKR2 proteins were found to immunolocalize to the epithelium of the Fallopian tube (Figure 3 a, c, e, g). These proteins were also found in the smooth muscle layer as confirmed by colocalization of PROKs and PROKRs with smooth muscle actin in serial tissue sections (Figure 3 b, d, f, h). In Fallopian tubes collected from women with ectopic pregnancies, the PROKs and PROKRs showed similar localization to that in sections from non-pregnant women (Figure 4).
To our knowledge this is the first comprehensive description of PROK and PROKR expression in human Fallopian tube at different phases of the menstrual cycle. We have also used an in vitro model system to investigate the impact of acute exposure to estradiol and/or MPA on expression of PROK and PROKR expression in this tissue. In addition, we report on altered PROKR expression in Fallopian tube from women with ectopic pregnancy compared to non-pregnant Fallopian tube.
PROK2 and PROKR1 mRNA are more highly expressed in Fallopian tube from the progesterone-dominant, mid-luteal phase of the menstrual cycle compared with the follicular phase, suggesting that tubal PROK2 and PROKR1 are regulated by progesterone. This is also supported by our in vitro data which shows that tubal PROKR1 mRNA is significantly upregulated by treatment with MPA and a combination of estradiol and MPA. PROK2 mRNA expression was not significantly upregulated by MPA treatment in vitro, suggesting that factors, in addition to progesterone, may control PROK2 expression in vivo.
In contrast to the endometrium we have shown that tubal PROK2 and PROKR2 mRNA levels are relatively higher compared to PROK1 and PROKR1 levels, suggesting that PROK2 may the dominant PROK active in the Fallopian tube in the mid-luteal phase. PROK2 has previously been shown to signal through PROKR1 (13). Thus, it is possible that, in the Fallopian tube, PROK2 may be signaling through PROKR1 whereas in the endometrium, PROK1 has been shown to signal through PROKR1 (14).
We have also demonstrated that PROKR mRNA levels are lower in Fallopian tube from ectopic pregnancies than in mid-luteal phase Fallopian tube collected from non-pregnant women, and that PROK/PROKR protein is localized to the smooth muscle of the Fallopian tube. This adds support to our hypothesis that the control of Fallopian tube smooth muscle contractility and embryo transport could be regulated by PROK and PROKR expression.
It is plausible that reduced PROK expression in Fallopian tube from women with ectopic pregnancy could contribute to impaired smooth muscle contractility and dysregulated embryo tubal transport through the cyclo-oxygenases, COX-1/COX-2, and subsequent production of prostaglandins. PROK1 has been shown to upregulate cyclo-oxygenase-2 (COX-2) levels in the endometrium (14). COX-2 is a member of the cyclo-oxygenase family of enzymes responsible for the conversion of arachidonic acid into prostaglandin, and has been shown to play a role in implantation in mice (15). The human Fallopian tube is known to express COX-1 and COX-2 (16) and prostaglandins have recently been shown to increase the contractility of smooth muscle in the Fallopian tube (17).
Nevertheless, the reduced PROK1 levels we observed in Fallopian tubes from women with ectopic pregnancies may simply be the result of lower maternal serum beta-human chorionic gonadotropin (hCG) levels observed in ectopic pregnancy. hCG increases PROK1 expression in endometrium (12), and generally, ectopic pregnancies have lower hCG levels than normal intrauterine pregnancies (18;19). However, when we compared PROKR levels in Fallopian tube from women with low serum hCG levels (< 1000 IU/L) with PROK levels in Fallopian tube from women with high serum hCG levels (> 1000 IU/L), we found no statistically significant differences in expression between these two groups. The signaling downstream of PROKR1 in the Fallopian tube therefore warrants further investigation.
In summary, we show that PROK and PROKR are expressed in the epithelium and smooth muscle of human Fallopian tube and have demonstrated that PROK and PROKR expression is regulated by the hormonal changes that occur during the menstrual cycle. Our finding of altered PROKR expression in Fallopian tube from women with ectopic pregnancy suggests that PROKs may be important in tubal wall smooth muscle contractility and successful embryo transport.
Embryo retention in human Fallopian tube is thought to lead to ectopic pregnancy. Attenuated prokineticin expression may affect embryo-tubal transport and lead to retention of embryos in the Fallopian tube.
The authors would like to thank Paula Lourenco and Sheila Wright for technical support; Catherine Cairns, Sharon McPherson and Catherine Murray for patient recruitment; and Ronnie Grant for graphical assistance.
This work was supported by a Wellbeing of Women Project Grant (R40608) and by MRC Core funding to HN Jabbour (U.1276.00.004.00002.02).