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
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Abstract |
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Key words: PAF receptor/PAF acetylhydrolase/Fallopian tube/endosalpinx/embryo transport
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Introduction |
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Since human oviductal stage embryos produce PAF (O'Neill et al., 1985) 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., 1999
) and increases intracellular free calcium concentration in bovine oviduct epithelial cells in culture (Tiemann et al.1996
), 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, 1989
; Bito et al.1994
). 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., 1993
). 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.1996
). This enzymatic activity has been detected in plasma and in the cytosolic fraction of some cells and tissues (Stafforini et al., 1991
; Hattori et al., 1993
). These, so called, plasma and cytosolic forms of this enzyme are encoded by different genes (Hattori et al., 1995
; Tjoelker et al., 1995
). PAF inactivation by PAF-AH regulates its overall biological function; indeed PAF-AH has been considered a signal terminator (Stafforini et al., 1997
). 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.
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Materials and methods |
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Tissue samples |
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RNA preparation
Total RNA was prepared from Fallopian tubes by the acid-phenol extraction method (Chomczynski and Sacchi, 1987) as modified (Ojeda et al., 1990
). 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, 1994). Specificity of the amplification was determined by restriction-enzyme digestion analysis.
Preparation of tissue homogenate
All procedures were carried out at 04°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, 1976). 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., 1992), 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, 46 µm thick, were placed onto gelatin-coated slides and were fixed in cold 4% paraformaldehyde in PBS pH 7.47.6 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.
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Results |
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Discussion |
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We previously showed that PAF is the embryonic signal that times oviductal embryo transport to the uterus in the hamster (Velasquez et al., 1995). PAF is also secreted by mouse, human and sheep embryos (O'Neill et al., 1985
; Batty et al., 1991
; Velasquez et al., 1995
). 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, 1997
) and PAF-AH activity in the uterus decreases at the time when the embryo and endometrium are producing PAF (O`Neill, 1995
), 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, 1995). 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., 1999
). 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, 1988). 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., 1999
).
PAF production by human embryos has been linked with the systemic thrombocytopenia seen in early pregnancy (O'Neill et al., 1985). 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, 1993
; Imaizumi et al., 1995
), 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., 1992
). 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.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 4, 2001; accepted on April 26, 2001.