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

L.A. Velasquez1,4, K. Maisey1, R. Fernandez1, D. Valdes1, H. Cardenas1, M. Imarai1, J. Delgado2, J. Aguilera2 and H.B. Croxatto3

1 Laboratorio de Inmunología de la Reproducción, Facultad de Química y Biología, Universidad de Santiago de Chile (USACH), 2 Servicio de Obstetricia y Ginecología, Hospital Félix Bulnes, and 3 Unidad de Reproducción y Desarrollo, Facultad de Ciencias Biológicas and MIFAB, Pontificia Universidad Católica de Chile, Santiago, Chile


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Tissue samples
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Prostaglandin-E2 and platelet-activating factor (PAF) are embryonic-derived signals that time embryo passage into the uterus in the mare and hamster respectively. PAF-like activity is detectable in the spent media of preimplantation human embryos and it has been suggested that PAF may be the embryonic signal that controls embryo transport to the uterus in our species. The actions of PAF are regulated at the level of its synthesis and degradation as well as the expression of a specific cell surface receptor (PAFr). The enzyme PAF acetylhydrolase (PAF-AH) degrades PAF. This study was undertaken to examine whether or not PAFr and PAF-AH are expressed in the human Fallopian tube and to identify the cell types in which they are expressed. METHODS: The presence of PAFr mRNA in tissue extracts was investigated using reverse transcription-polymerase chain reaction. We amplified the predicted amplicon for PAFr mRNA from RNA samples extracted from Fallopian tubes. The expression of PAF-AH was detected by Western blot and the localization of PAFr and PAF-AH proteins was detected by immunohistochemistry. RESULTS: Utilizing antibodies against PAFr and PAF-AH, co-localization of the two proteins in the epithelium and stromal cells were demonstrated. CONCLUSIONS: These observations show that the human Fallopian tube expresses PAFr and PAF-AH at a location compatible with the proposed paracrine role of early embryo-derived PAF.

Key words: PAF receptor/PAF acetylhydrolase/Fallopian tube/endosalpinx/embryo transport


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Tissue samples
 Results
 Discussion
 Acknowledgements
 References
 
The passage of embryos into the uterus is timed by a complex signalling system that controls oviductal transport. Ovarian hormones play diverse roles in this process in mammals, since their administration can hasten, delay or have no effect on oocyte transport depending on the nature of the steroid, the dose and time of administration, and the species (Villalon et al., 1999Go). Acute administration of high doses of oestradiol or progesterone to women in the postovulatory period does not alter the recovery of oocytes from the tubes up to the time they would normally pass into the uterus (Croxatto, 1996Go). Thus, these hormones do not seem to have an important role in the short-term control of oviductal transport in the human. Marked differences in the timing of oviductal transport between embryos and oocytes in mares, bats, mice and hamsters have been taken as indirect evidence that, in these species, the embryo is a source of regulatory signals for timing its transport to the uterus (Rasweiler, 1979Go; Ortiz et al., 1986Go; Croxatto and Ortiz, 1991Go; Weber et al., 1991aGo, bGo). Such evidence is lacking for the human since no one has compared oocyte and embryo transport in women. In the mare, the five-day morula secretes prostaglandin E2 which acts on the oviduct to stimulate transport of the oocyte to the uterus (Weber et al., 1991aGo, bGo). In contrast, in the hamster platelet-activating factor (PAF):1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine of embryonic origin triggers the passage of embryos from the oviduct to the uterus (Velasquez et al., 1995Go).

Since human oviductal stage embryos produce PAF (O'Neill et al., 1985Go) we hypothesized that this substance may also play a role in the control of embryo transport in our species. The fact that PAF, applied on the apical surface of human oviductal cells in vitro, increases the potential difference and short-circuit current (Downing et al., 1999Go) and increases intracellular free calcium concentration in bovine oviduct epithelial cells in culture (Tiemann et al.1996Go), suggests that these cells may be responsive to embryonic-derived PAF. PAF is a potent phospholipid mediator with important functions in many physiological and/or pathological situations (Harper, 1989Go; Bito et al.1994Go). Its diverse and potent effects suggest that there are mechanisms for precise regulation of its concentration in tissues and body fluids. These may include synthesis of PAF and rapid clearance and metabolism to an inactive product (Venable et al., 1993Go). A key mechanism for the removal of PAF is hydrolysis catalysed by PAF acetylhydrolase (PAF-AH), which converts PAF to the biologically inactive lyso-PAF (Stafforini et al.1996Go). This enzymatic activity has been detected in plasma and in the cytosolic fraction of some cells and tissues (Stafforini et al., 1991Go; Hattori et al., 1993Go). These, so called, plasma and cytosolic forms of this enzyme are encoded by different genes (Hattori et al., 1995Go; Tjoelker et al., 1995Go). PAF inactivation by PAF-AH regulates its overall biological function; indeed PAF-AH has been considered a signal terminator (Stafforini et al., 1997Go). Thus, the hypothesis proposed above assumes that PAFr and PAF-AH are expressed in human Fallopian tube cells. This was tested in the present study using the highly sensitive technique of reverse transcription-polymerase chain reaction (RT-PCR) to determine the presence of PAFr mRNA. In addition, Western blot was used to determine whether or not PAF-AH expression occurs in the Fallopian tube and immunohistochemistry was used to identify the cell types in which PAFr and PAF-AH are expressed. Our results are consistent with the hypothesis that PAF of embryonic origin could be the signal used by human embryos to time their transport to the uterus.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Tissue samples
 Results
 Discussion
 Acknowledgements
 References
 
Antibodies
Rabbit polyclonal antiserum against a synthetic peptide (MEPHDSSHMDSEFRYTL) from the N-terminus of human PAFr and rabbit polyclonal antiserum against a synthetic peptide (TNINTTNQHMLQNSSGIEKYN) from the C-terminus of human plasma PAF-AH were obtained from Cayman Inc., Ann Arbor, FL, USA. The latter does not cross-react with the other forms of this enzyme.


    Tissue samples
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 Abstract
 Introduction
 Materials and methods
 Tissue samples
 Results
 Discussion
 Acknowledgements
 References
 
Volunteers for this study were four healthy multiparous women with no history of pelvic inflammatory disease and whose request for surgical sterilization had been approved by the hospital ad hoc committee. All subjects were using barrier methods of contraception and none had used hormonal contraception recently. The protocol was approved by the Ethics Committee of USACH, Santiago, Chile, and all volunteers gave informed consent. Fallopian tube samples were obtained at the time of surgical sterilization at the Department of Obstetrics and Gynecology of Felix Bulnes Hospital, Santiago, Chile. Surgical sterilization was done at mid-cycle through minilaparotomy, following the Pomeroy technique for partial tubal resection and ligature (Murray et al., 1992Go). A 1.5 cm tubal segment was available for the study and corresponded in most cases to ampullary segment. Samples were immediately embedded in Tissue-Tek OCT compound (Miles Inc., Elkhart, IN, USA), and were frozen in nitrogen and stored at –80°C.

RNA preparation
Total RNA was prepared from Fallopian tubes by the acid-phenol extraction method (Chomczynski and Sacchi, 1987Go) as modified (Ojeda et al., 1990Go). Each RNA sample was treated with DNase-I, amplification grade (Life Technologies Inc., Gaithersburg, MD, USA), to remove contaminating genomic DNA.

RT-PCR procedures
RT was carried out for 1 h at 42°C in a total volume of 20 µl. Each reaction mixture contained 1 µg of total RNA from Fallopian tube, 1xRT buffer (50 mmol/l Tris-HCl, pH 8.3; 75 mmol/l KCl; 3 mmol/l MgCl2), 0.01 mol/l dithiothreitol, 0.5 mmol of each dNTP, 20 U of RNasin, 25 pmol of oligo (dT) primer and 200 IU of Superscript II reverse transcriptase (Life Technologies Inc.). Reaction tubes were incubated at 42°C for 60 min. At the end of the incubation period, the reaction was stopped by heating at 90°C for 5 min. Then, RT products were treated with Ribonuclease H (Life Technologies Inc.) to remove mRNA for the second-strand cDNA synthesis. Each PCR amplification was performed in 75 µl final reaction volume containing 1/10 of the cDNA mixture diluted with the reaction buffer (10 x) to a final composition of: 10 mmol/l Tris-HCl, pH 8.3, 50 mmol KCl, 1.5 mmol MgCl2, 100 µmol/l dNTPs, 2.5 U of Taq polymerase and 25 pmol of each primer. The tubes were placed in the Programmed Tempcontrol system which was programmed as follows: (a) incubation at 96°C for 1 min (initial melt); (b) 35 cycles of the following sequential steps: 94°C for 60 s (melt); 59°C for 50 s (anneal); 72°C for 50 s (extend); and (c) 72°C for 10 min (final extension).

The following primer pairs were used to amplify the coding region of PAFr gene (Accession Number P25105): Sense 5'-CGGACATGCTCTTCTTGATCA-3', Antisense 5'-GTCTAAGACACAGTTGGTGCTA-3' (Bastien and Mazer, 1994Go). Specificity of the amplification was determined by restriction-enzyme digestion analysis.

Preparation of tissue homogenate
All procedures were carried out at 0–4°C. The tissues were homogenized in a Potter homogenizer in 500 µl of buffer A [10 mmol/l Tris pH 7.4, 150 mmol/l NaCl, 2.5 mmol/l EDTA, 0.05% w/v sodium dodecyl sulphate (SDS)], and then the homogenates were centrifuged at 15 000 g for 30 min to remove the bulk of the solid material. The supernatant was centrifuged at 15 000 g for 5 min. The protein concentration of the homogenates was determined using Bradford protein assay reagent with bovine serum albumin (BSA) as standard (Bradford, 1976Go). Western blot analysis was performed on the tissue homogenate (50 µg of protein) with PAF-AH antibody. PAFr antibody proved to be unsuitable for use in Western blots.

Western blot
The samples were resolved by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) on a 12% acrylamide gel (Ausubel et al., 1992Go), and blotted onto a nitrocellulose membrane. The membrane was blocked overnight at 4°C with phosphate-buffered saline (PBS) containing 5% skim milk and then was incubated with either one of the primary antibodies diluted 1:500 in 1% PBS-BSA-or primary antibody 1:500 preincubated with 10 µg/ml blocking peptide for 2 h at room temperature. The filter was washed three times with PBS and incubated for 60 min with alkaline phosphatase-conjugated goat anti-rabbit Ig polyclonal antibodies, diluted to 1:1000 in PBS containing 5% skim milk. After washing the filters 3 times with PBS, the blots were detected using bromochloroindolyl phosphate/nitro blue tetrazolium (BCIP/NBT) at room temperature with agitation until the stain was suitably dark.

Immunohistochemistry
Cryostat sections, 4–6 µm thick, were placed onto gelatin-coated slides and were fixed in cold 4% paraformaldehyde in PBS pH 7.4–7.6 for 60 min, before sequential transfer to 10% w/v sucrose in PBS for 60 min at 4°C and 30% w/v sucrose in PBS at 4°C overnight.

After blocking with 1% PBS-BSA for 120 min, the primary antibody diluted 1:50 in 1% PBS-BSA was added to the sections. Binding was allowed to occur at 4°C overnight. Three PBS rinses were followed by 60 min incubation at room temperature with secondary antibody biotin-conjugated anti rabbit IgG (Biosource, Nivelles, Belgium) diluted in 1% PBS-BSA. After three PBS rinses, the slides were incubated with Avidin-FITC (Sigma, St Louis, MO, USA.) diluted 1:5000 for 60 min at room temperature. Samples were subsequently washed with PBS, counterstained with 1µg/ml propidium iodide and mounted in DABCO (Sigma). As negative controls (a) the primary antibody was omitted, (b) the primary antibody was replaced by preimmune serum and (c) the primary antibody was preincubated with blocking peptide. The resulting staining was evaluated on a Zeiss confocal laser scanning microscope.


    Results
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 Abstract
 Introduction
 Materials and methods
 Tissue samples
 Results
 Discussion
 Acknowledgements
 References
 
Detection of PAFr gene expression by RT-PCR
RT-PCR was performed on RNA from four Fallopian tubes. Figure 1Go depicts the ethidium-stained gel of these fractions. A band of predicted size (682 bp) was observed in all oviductal samples upon ethidium bromide staining of the RT-PCR products (Lanes 4–7). The control reactions, one containing no RNA (Lane 2) and another containing RNA but no reverse transcriptase (Lane 3), were negative confirming the absence of genomic DNA contamination. Verification that the PCR products corresponded indeed to the PAFr gene was performed by restriction-enzyme digestion analysis. Digestion with AvaII yielded the predicted products (103 and 579 bp) (data not shown).



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Figure 1. PAFr mRNA detected by RT-PCR in human Fallopian tube. PCR products of 682 bp visualized by ethidium bromide staining are shown. Lane 1: DNA ladder, size marker; Lane 2: negative control (without RNA); Lane 3: negative control (with RNA but no reverse transcriptase); Lanes 4–7, human Fallopian tube mRNA transcribed and amplified using PAFr specific primers, as described in materials and methods.

 
Western blotting
As shown in Figure 2Go PAF-AH protein is detectable in human Fallopian tube by Western blot analysis. A band of predicted molecular weight (45 kDa) was observed in all samples.



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Figure 2. Western blot analysis of plasma PAF-AH expression in the human Fallopian tube. Panel A: Lanes 1–2 are Fallopian tube samples. Immunoreactive PAF-AH was detected with a molecular mass of 45 kDa (arrow), the positions of molecular mass markers are indicated in the first lane. Panel B: Primary antibody blockage by means of the same peptide used to generate the antibody. Note the absence of reaction demonstrating the PAF-AH identity.

 
Cell localization of PAFr and PAF-AH in Fallopian tube tissue layers
As shown in Figure 3Go PAFr and PAF-AH exhibited strong staining in the endosalpinx, with apical distribution in the epithelial cells and pericellular distribution in the stromal cells. Orthogonal and phase contrast analysis of the samples showed strong staining in the luminal border and weak staining in the cytoplasm (data not shown). The ampullary segment was examined in all cases and the isthmic segment was examined in two cases. No differences were found between ampullary and isthmic segment with regard to the immunostaining of the two proteins. No staining was observed in myosalpinx.



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Figure 3. Immunostaining of PAFr and PAF-AH in sections of human Fallopian tube ampulla. (A) negative control, original magnification x280; (B) PAFr positive staining at x280; (C) at x720; (D) at x1680. Note the strong staining in the epithelial and stromal cells (green label); (E) PAF-AH positive staining x280 and (F) at x840. Note the similarity in the distribution of PAF-AH and PAFr. Nuclei stained red with propidium iodide.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Tissue samples
 Results
 Discussion
 Acknowledgements
 References
 
The results of this study disclose the presence and spatial pattern of expression of PAFr and PAF-AH in the human Fallopian tube. Both were found to be expressed at the apical surface of epithelial cells and subepithelial stromal cells. In human endometrium PAFr mRNA was localized in both stromal and epithelial cells (Baldi et al., 1994Go). In hamster oviduct, expression of mRNA encoding PAFr is restricted to the endosalpinx and is most prominent in subepithelial cells located in the mucosal folds that protrude into the lumen (Velasquez et al., 1997Go).

We previously showed that PAF is the embryonic signal that times oviductal embryo transport to the uterus in the hamster (Velasquez et al., 1995Go). PAF is also secreted by mouse, human and sheep embryos (O'Neill et al., 1985Go; Batty et al., 1991Go; Velasquez et al., 1995Go). In these species it may play the same function as in hamsters, but there is no evidence as yet. This hypothesis presumes that PAFr is expressed in oviductal cells which are accessible from the lumen. The observations presented here confirm this assumption in the case of the human. PAF released by mouse embryos is highly resistant to the action of PAF-AH (Ammit and O'Neill, 1997Go) and PAF-AH activity in the uterus decreases at the time when the embryo and endometrium are producing PAF (O`Neill, 1995Go), thus these features may be very important for passing the barrier of PAF-AH expressed in the epithelial cells in order to stimulate the stromal cells.

Plasma PAF-AH is generally not found in the cytoplasm of cells, however it has been detected in endometrial tissue and in uterine flushings of mouse uterus during the oestrous cycle (O`Neill, 1995Go). The antiserum used in the present study was raised against a peptide whose sequence is not shared by the cytosolic form of PAF-AH, therefore the epitope recognized must correspond to the so called plasma PAF-AH. Since it was closely associated with the luminal border, we assume that it was either taken up from extracellular fluid or produced by the epithelial cells. In addition, aminoacid sequence analysis in the SOSUI Data Base (http://sosui.proteome.bio.tuat.ac.jp) of plasma PAF-AH shows it has a transmembrane helix in the amino terminal end. Moreover, kinetic studies using recombinant plasma PAF-AH have shown that this enzyme acts as a membrane-bound enzyme (Min et al., 1999Go). In view of these facts, we surmise that the terms plasmatic and cytosolic PAF-AH are misleading misnomers.

In order to explain how embryonic derived PAF may trigger embryo passage to the uterus, some facts need to be reconciled. At least in rats, accelerated embryo transport is associated with increased frequency of myosalpinx contractions (Moore and Croxatto, 1988Go). However the site of action of PAF is at the cells located in the endosalpinx rather than the myosalpinx. Therefore the endosalpinx may act as a relay station between the embryo and the myosalpinx. Possible endosalpinx-derived factors induced by PAF, which may act in turn on smooth muscle cells, are endothelin, nitric oxide and prostaglandin (Villalon et al., 1999Go).

PAF production by human embryos has been linked with the systemic thrombocytopenia seen in early pregnancy (O'Neill et al., 1985Go). However, it is unlikely that a few blastomeres are able to produce large enough amounts of PAF to induce systemic thrombocytopenia. In other systems, cells respond to PAF by producing more PAF in a positive feedback loop (Chao and Olson, 1993Go; Imaizumi et al., 1995Go), therefore an alternative explanation is that the embryonic PAF is targeted to the nearby endosalpinx cells rich in PAFr and these oviductal cells amplify the local PAF signal. PAF produced by stromal cells of the endosalpinx, in response to embryonic PAF, is likely to diffuse into the capillaries. This would allow systemic effects such as thrombocytopenia. The presence of PAF-AH in the epithelial surface may limit the action of stromally produced PAF to the endosalpinx. The fact that rabbit oviductal membrane preparations metabolise PAF quite readily is consistent with this idea (Yang et al., 1992Go). Here we propose that PAF-AH localized in the luminal surface of the epithelium plays a key role limiting spatially the action of non embryonic PAF in early pregnancy.

In conclusion the human Fallopian tube expresses PAFr and PAF-AH at a location compatible with a paracrine role of early embryo-derived PAF.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Tissue samples
 Results
 Discussion
 Acknowledgements
 References
 
We thank Dr John Aplin, of the Department of Obstetrics and Gynaecology and School of Biological Sciences, University of Manchester, for his critical reading of this manuscript. This work was supported by DICYT and SDT from Universidad de Santiago de Chile, Proyecto Lineas complementarias FONDECYT # 8980008, Catedra Presidencial Dr Horacio Croxatto, RF 98024 # 98 and MIFAB. The Millenium Institute for Fundamental and Applied Biology (MIFAB) is financed in part by the Ministerio de Planificación y Cooperación (Chile).


    Notes
 
4 To whom correspondence should be addressed at: Universidad de Santiago de Chile, Alameda 3363, Casilla 40, Correo 33, Santiago, Chile. E-mail: lvelasqu{at}lauca.usach.cl Back


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 Discussion
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 References
 
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Submitted on January 4, 2001; accepted on April 26, 2001.