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Academic Unit of Reproductive and Developmental Medicine (B.R., B.O., N.C., W.L.), Royal Hallamshire Hospital, Sheffield S10 2SF, United Kingdom; and Derby City General Hospital (S.A.), University of Nottingham, The Medical School, Derby DE22 3NE, United Kingdom
Address all correspondence and requests for reprints to: Professor William Ledger, Academic Unit of Reproductive and Developmental Medicine, Level 4, The Jessop Wing, Royal Hallamshire Hospital, Sheffield S10 2SF, United Kingdom. E-mail: w.ledger{at}sheffield.ac.uk.
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Abstract |
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Objective: We investigated whether epithelia from Fallopian tubes (FTs) bearing an ectopic pregnancy differ from normal tubes in expression of TGF-β family and related proteins and their receptors.
Methodology: Because it is not possible to collect FTs from women carrying a healthy pregnancy, we studied tissue collected at the time of hysterectomy for benign disease. Women were injected with human chorionic gonadotropin in the days leading up to hysterectomy to produce a state of pseudopregnancy. Pseudopregnancy status was confirmed by the presence of high serum progesterone levels and the decidualization of the endometrium. Fifteen FTs bearing ectopic pregnancy and six pseudopregnant tubes were collected and examined using immunohistochemistry and quantitative RT-PCR.
Results: Immunohistochemistry demonstrated clear staining for the βA- and βB-subunits, type II receptor group comprising the activin type IIA and type IIB receptors, and follistatin, which increased in intensity from the isthmus to the ampulla in both models. However, the intensity of expression of these molecules was stronger in the ectopic pregnancy group when compared with the pseudopregnant group. Quantitative RT-PCR showed significant decrease in mRNA levels of βA-subunit, activin type IIA and IIB receptors, and follistatin in ectopic group (P < 0.05) but no changes in βB-subunit (P > 0.05). Overall, there was an apparent paradox of high concentration of protein but low mRNA expression.
Conclusion: Activin-A may stimulate tubal decidualization and trophoblast invasion. A better understanding of the mechanism by which an embryo implants in the tubal epithelium may lead to improved methods for early diagnosis and/or management of ectopic pregnancy.
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Introduction |
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Activins and inhibins are members of the TGF-β (4). Activins and inhibins were identified initially as gonadal proteins that have stimulating (activins) and suppressing (inhibins) effects on FSH production in cultured rat anterior pituitary cells (5, 6, 7, 8). Activins are homodimers of the β-subunits (βA and βB), and the different dimerization of subunits gives rise to three glycoproteins: activin-A (βA-βA), activin-B (βB-βB), and activin-AB (βA-βB) (9). As for all TGF-β members, activins are originally synthesized as larger precursor proteins that are subsequently cleaved to release the mature C-terminal protein.
Activins are secreted proteins, and, as for any external stimulus, responsive cells must possess the proper mechanism to transduce the activin signal intracellularly (10). Like most members of the TGF-β family, activins mediate their actions by binding to a complex of transmembrane serine and threonine kinase receptors (11). These activin receptors can be classified into two main categories, namely the type I receptor group, comprising the activin receptor-like kinase (2 and 4), and the type II receptor group comprising the activin type IIA and type IIB receptors (ActRIIA and ActRIIB) (12, 13).
The coordinated synthesis of follistatin with activin is the main regulator of the local bioactivity of activin because binding of activin to follistatin is almost irreversible (14). The activin-follistatin complex is generally regarded as consisting of one activin dimer and two follistatin molecules. Circulating activin is commonly detected bound with the long-form follistatin (follistatin-315) (15), whereas the short form (follistatin-288) exhibits high affinity for proteoglycans, which are commonly associated with cell membrane (16). Generally, activin A, AB, and B bind to follistatin with similar affinity.
The uterine tubes are biologically active, providing an environment that enhances and supports fertilization and early embryonic development as the embryo travels toward the uterine cavity (17, 18). To complete these events, there is an embryonic-maternal dialog in which the embryo and the maternal reproductive tract induce changes in each other to promote embryonic and endometrial maturation (19). Recently we demonstrated the expression of activin subunits, type II receptors, and follistatin by the premenopausal Fallopian tube. We suggested that activin has a local paracrine or autocrine role in early embryonic development and transport because it is coexpressed with follistatin, which would mitigate against tubal activin having a more distant site of action (20, 21).
Little is known of the mechanism by which the process of embryo transport is coordinated within the tube. Both cilial activity and tubal peristalsis are believed to be necessary for successful transport of the embryo along the tube and to ensure delivery of the embryo at the endometrial cavity at the optimum time for adhesion and implantation (22). This is critical for the successful establishment of pregnancy and avoidance of ectopic pregnancy. Therefore, we investigated whether epithelium from Fallopian tubes bearing an ectopic pregnancy differs from a normal tube in expression of TGF-β family and related proteins and their receptors.
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Subjects and Methods |
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Case group
Fifteen Fallopian tubes were obtained from women diagnosed with ectopic pregnancy for whom salpingectomy was performed on clinical management grounds. All participants in this group conceived spontaneously and were not taking exogenous progesterone.
Control group
Because it is not possible to collect Fallopian tubal tissue from women carrying a healthy pregnancy, we studied tissue collected at the time of hysterectomy performed for benign diseases not affecting the tubes. Women were asked to have treatment with human chorionic gonadotropin (hCG) in the days leading to hysterectomy. This regimen produces a state of pseudopregnancy, is harmless, and has been previously used within U.K. research studies (23, 24). Six Fallopian tubes were obtained from women who were undergoing routine total abdominal hysterectomy for benign disease not affecting the Fallopian tubes, and they were pretreated with sc hCG (5000 IU) every 3 d beginning in the midluteal phase for at least 12 d before total abdominal hysterectomy. The three included patients had had nine, six, and 10 doses, respectively. The menses delayed in the patients by 24, 15, and 30 d, respectively. To confirm the pseudopregnancy status, an endometrial biopsy and a blood sample were collected at the time of hysterectomy to examine decidualization of the endometrium and progesterone levels. All women who donated Fallopian tubes had regular menstrual cycles and were of proven fertility with no evidence of tubal disease.
Sampling and processing
For the case group, the Fallopian tubes were excised at least 1 cm away from the implantation site to avoid collecting any embryonic or trophoblastic tissue and assure the integrity of tubal morphology and function.
Routine histological analysis of the ectopic pregnancy was undertaken. The isthmic and ampullary regions of the excised tubes from the two groups were identified and a small section immediately cut from each region using RNase-free equipment (baked at 200 C for 4 h). These samples were then divided into three equal pieces, with one piece being immediately fixed in 10% buffered formalin for immunohistochemistry and the other parts in 5 ml RNAlater solution (Ambion, Warrington, UK) for the RT-PCR. All the tissues used in the RT-PCR were snap frozen in RNAlater solution (Ambion) and stored at –80 C until fixed in 10% buffered formalin to preserve the RNA stability.
Antibodies
Monoclonal antibodies to detect activin-βA- (E4) and -βB- (C5) subunits were obtained from Serotec Ltd. (Oxford, UK) and follistatin from R&D Systems (Abingdon, UK). Polyclonal antibodies to detect the ActRIIA (N-17) and -IIB (N-16) were obtained from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Blocking peptides for the N-16 and N-17 polyclonal antibodies were obtained from Santa Cruz Biotechnology.
Immunohistochemistry
An avidin-biotin horseradish peroxidase technique was used to localize the activin βA- and βB-subunits, ActRIIA and ActRIIB and follistatin following the protocol described previously (21). Briefly, sections were dewaxed, dehydrated in alcohol, and treated with 2% (vol/vol) hydrogen peroxide for 20 min in methanol to block endogenous peroxidase (25). The sections used for the detection of βB-subunit and ActRIIA were pretreated in an 850-W domestic microwave oven in 0.01 M citrate buffer for 10 min. However, for the activin βA-subunit, follistatin, and ActRIIB, no pretreatment was required. The sections were incubated for 30 min with normal horse serum and then incubated with the primary antibodies (the antibody concentration was 1:200 for the C5, follistatin, and the N-16; 1:400 for E4 and N-17) overnight at 4 C. The following day the sections were washed with 20 mM PBS (pH 7.3) and then incubated with 1:200 biotinylated antimouse (for the E4 and C5) and antigoat (for the N-17 and N-16) secondary antibody for 30 min. After a further wash step, the sections were incubated with the avidin-biotin peroxidase complex ELITE system (Vector Laboratories, Inc., Burlingame, CA) for 30 min and then subsequently with 3,3'-diaminobenzidine (Vector Laboratories) for 10 min. Sections were washed in tap water, counterstained with Gill’s hematoxylin, and then dehydrated in a series of graded ethanol, cleared in xylene, and mounted in DPX (BDH/Merck, Leicestershire, UK). Regarding the N-16 and N-17 polyclonal antibodies, negative controls were designed by blocking each antibody by the corresponding blocking peptide. The same protocol was applied for the negative controls.
The sections were observed on a Labor Lux microscope (Leitz, Wetzlar, Germany), at a magnification of x100, x250 and x400. A positive reaction was characterized by the presence of brown staining. Each section was examined by two observers who were blinded to the source of tissue, and they agreed on the intensity of staining according to the following semiquantitative scale: 0, negative; 1, equivocally positive; 2–4, weakly positive; 5–7, positive; and 8–10, strongly positive. Representative sections were photographed using an digital camera at x250 and x400 magnification (Olympus UK Ltd, Hertfordshire, UK).
Laser capture microscopy
For investigation of mRNA expression levels, epithelial cells from the different segments of the Fallopian tube were microdissected using a laser capture microdissector (Arcturus, Hertfordshire, UK). One of the snap-frozen sections in RNAlater was prepared for microdissection by being fixed in 10% buffered formalin for 24 h. Before microdissection, 10-µm sections of paraffin blocks from the Fallopian tube were dewaxed, dehydrated in alcohol, and then stained with toludine blue (1 min) and destained and dehydrated through graded ethanol concentrations and xylene (2 x 5 min).
cDNA synthesis
RNA was extracted from microdissected Fallopian tube epithelial cells using the RNeasy FFPE kit (QIAGEN Ltd., Crawley, UK) following the manufacturer’s instructions. RNA was treated with RNase-free DNase during the extraction protocol. The first-strand cDNA was carried out using the RETROscript kit (Applied Biosystems, Warrington, UK) and following the manufacturer’s protocol. Briefly, reverse transcription was performed with heat denaturation of the RNA (75 C, 3 min) by adding 2 ng total RNA from each group under investigation and using random decamers in the presence of RNase inhibitor (50 C, 1 h).
Quantitative PCR
PCR was performed with the cDNAs, power SYBR green master mix (Applied Biosystems), and primers from Metabion (Table 1
). Each well of the PCR plate contained 10 µl SYBR green, 7 µl water, 1 µl of each primer (20 pmol), and 1 µl cDNA. The amplification was performed under the following conditions: 50 cycles (95 C 30 sec, 65 C 30 sec, 72 C 30 sec). Universal human RNA (Stratagene, Amsterdam, The Netherlands) was used as a positive control, and two negative controls were included, one with minus-reverse transcription control from the previous step, and a minus-template PCR, which contained all the PCR components but water, was used as a template. All experiments were performed in triplicate.
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Results |
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Immunohistochemistry
Fifteen Fallopian tubes bearing an ectopic pregnancy from 15 patients (median age 29 yr, range 24–36 yr, and mean gestational age calculated from stated light microscope 45.67 ± 7.15 d). Six tubes from three pseudopregnant patients (median age 38 yr, range 36–41 yr) were collected. Positive staining was observed in all tissue samples for the activin-βA (Fig. 1, panel 1.1, A–D) and -βB subunits (Fig. 1, panel 1.2, E–H) as well as ActRIIA (Fig. 1, panel 1.4, M–P), ActRIIB (Fig. 1, panel 1.5, Q–T), and follistatin (Fig. 1, panel 1.3 I, J–L, and Table 2). In the present study, all antibodies demonstrated immunoreactivity in both the ampulla and isthmus. The antibodies clearly labeled the cytoplasm of tubal epithelial cells from the ectopic and pseudopregnant tube. All the epithelial cells were stained, and the intensity of the immunoreaction decreased toward the isthmic region, showing a gradient of expression along the Fallopian tube. The intensity of expression was higher in the ectopic pregnancy group than the pseudopregnant group in all parts of the Fallopian tube. The immunoreactivity for the activin-βB subunits and ActRIIA showed a similar localization to the other antibodies. Of particular interest was that the intensity of expression was concentrated in the apical area in the pseudopregnant group (Fig. 1, panel 1, F, H, J, and L). The immunoreaction intensity again decreased toward the isthmic region with a gradient of expression along the tube. No immunoreactivity was observed by using blocking peptides against the N-16 and N-17 polyclonal antibodies (data not shown).
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We carried out quantitative real-time PCR experiments on Fallopian tube specimens collected from the ectopic and pseudopregnant groups. Loading of RNA from the different samples was similar as shown by β-actin results (data not shown). Our results demonstrated that the expression of activin-βA subunit (Fig. 2, part 1), ActRIIA and ActRIIB (Fig. 2, parts 4 and 5), and follistatin (Fig. 2, part 3) genes (P < 0.05) changed between the two groups, being significantly higher in the pseudopregnant group, compared with that of the ectopic pregnancy group. However, the activin-βB subunit failed to show any significant changes between both groups (P > 0.05).
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Discussion |
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The present study is the first to report an increase in the expression of activin-βA subunit, follistatin, and ActRIIA and ActRIIB within the Fallopian tube bearing an ectopic pregnancy. This increase in protein expression is seen within tissues with lower mRNA than in the controls. These suggest that pathological secretion of activin-A and its receptors may be a key factor in the development of ectopic pregnancy.
There are drawbacks to this study. The gestational age at which the tubes bearing ectopic pregnancies were collected was variable, being dictated by clinical management of this surgical emergency. However, the duration of the pregnancy in all cases was longer than the duration of the state of pseudopregnancy that we were able to achieve with repeated hCG injection. We did not believe that it was ethical to prolong the pseudopregnant state and hence delay elective surgery for more than 21 d. Not surprisingly, recruitment to the pseudopregnant arm was difficult, and hence, we present control data on only six tubes, although all findings were consistent between tubes and patients. Because of the discrepancy in duration of amenorrhea between the pregnant and pseudopregnant groups, it may be that the observed differences in protein and gene expression are related to the length of time that the tissues were exposed to hCG rather than to a difference between healthy and pathological. Although accepting this criticism, it is difficult to derive an ethically acceptable means of addressing this question.
Several studies demonstrated that activin-A promotes and facilitates endometrial decidualization, which is essential for successful implantation. Activins and their type IIA and IIB receptors are expressed by the decidualized cells (29). It has been suggested that activin-A, which is secreted by newly decidualized cells, promotes the decidualization of neighboring cells and thus facilitates the spread of decidualization all through the endometrium (27). Activin-A also up-regulates the matrix metalloproteinases, which promote tissue remodeling and decidualization (27). Therefore, the authors suggested that activin-A induces endometrial decidualization through a downstream mechanism. Moreover, the expression of activin by the cytotrophoblast is low, which suggests that trophoblast invasion is induced by the maternally derived activin (27). A recent study suggested that insufficient secretion of activin-A from the endometrium may lead the placenta to implant in an extrauterine site (28).
The expression of activin-A is dynamic, and it is up- and down-regulated during the process of decidualization (27). Follistatin is expressed by the decidualized cells (29) and it has shown a similar pattern of expression to activin (29).
The present work agrees with the previous observations because it demonstrates an increase in the intensity of expression of activin-βA subunit, ActRIIA and ActRIIB, and follistatin by the epithelial cells of human Fallopian tubes bearing an ectopic pregnancy (Fig. 1
). However, the up-regulation of the proteins has been accompanied by a down-regulation of the mRNA of these molecules (Figs. 1 and 2). An explanation for this paradoxical observation could be that the mRNA of these molecules is rapidly translated and degraded, resulting in rapid turnover of the mRNA, with depletion of these mRNAs due to the prolonged synthesis of large amounts of activin-A, its receptors, and follistatin. Investigation of the time course of changes in mRNA and these proteins will be necessary to address the relationship between mRNA turnover and protein synthesis in this system.
Nitric oxide (NO) and NO synthase have been reported to be expressed by the Fallopian tube epithelial cells and tubal smooth muscle cells (30, 31). The NO is associated with mRNA instability and degradation in the mammalian cells (32, 33, 34). Thus, the observed decrease in mRNA level could be due to NO-dependent mRNA instabilities and degradation.
The NO production is induced by activin-A in a concentration-dependent manner in a variety of tissues and cells (35, 36, 37). It has been reported that NO is involved in a variety of female reproductive functions (31), and it has a potent relaxing effect on Fallopian tube smooth muscles (30, 38, 39). Additionally, a significant increase in tubal transport of ova has been documented after the local administration of NO synthase inhibitors in rat oviduct (40).
Therefore, we suggest that increased activin-A expression by the Fallopian tube epithelial cells may stimulate tubal decidualization and trophoblast invasion within the tube. Furthermore, an increase in activin-A expression by the Fallopian tube epithelial cells may increase the production of NO in a concentration-dependent manner, which will result in pathological relaxation of the tubal smooth muscles, failure of propulsion of the early embryo along the Fallopian tube, and the development of ectopic pregnancy. Further studies are needed to explore this hypothesis.
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Footnotes |
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First Published Online October 30, 2007
Abbreviations: ActRII, Type II receptor group comprising the activin type II receptor; hCG, human chorionic gonadotropin; NO, nitric oxide.
Received July 24, 2007.
Accepted October 24, 2007.
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