Mol. Hum. Reprod. Advance Access originally published online on July 22, 2005
Molecular Human Reproduction 2005 11(7):503-505; doi:10.1093/molehr/gah192
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Specialized pacemaking cells in the human Fallopian tube
1Department of Surgery and Experimental Research, Cairo University, Cairo and 2Department of Surgery, Menoufia University, Shebin El-Kom, Egypt
3 To whom correspondence should be addressed at: Department of Surgery and Experimental Research, Cairo University, 2 Talaat Harb Street, Cairo 11121, Egypt. E-mail: shafik{at}ahmedshafik.com
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Abstract |
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It is postulated that the propagated electric activity of the gut is generated by the interstitial cells of Cajal (ICCs). We investigated the hypothesis of the presence of ICCs in the Fallopian tube (FT) as initiators of the tubal electric activity. Specimens from various parts of the FT were obtained from 21 female cadavers (mean age 38.2 ±10.9 years) from the PostMortem Department of the Cairo University, Faculty of Medicine. Fixed sections were prepared and stained for c-kit. Controls for antisera specificity consisted of tissue incubated with normal rabbit serum substituted for the primary antiserum. Dendritic, c-kit-positive, ICC-like cells were detected in the tubal musculature of the studied specimens. They were distinguishable from the c-kit-positive nonbranching mast cells and from the c-kit-negative smooth muscle cells (SMC). Immunoreactivity was absent in the negative controls. We have for the first time identified cells in the FT with morphologic and immunologic phenotypes similar to the ICCs of the gut. These cells may be responsible for initiating the slow waves (SWs) recorded from the SMC of the FT. This is a preliminary study, and further studies are needed to investigate the functional role of these cells.
Key words: action potentials/motility/oviduct/slow waves
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Introduction |
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The Fallopian tube (FT) has major functions in reproduction which comprise enabling the transport of ova and spermatozoa, providing favourable environment for fertilization and embryo cleavage and transporting the zygote towards the normal site of implantation in the uterine endometrium (Brosens, 1991
). It is usually described as having four anatomical segments: the fibriae, ampulla, isthmus and the intramural or interstitial segment. The fimbrial segment is poor in muscle fibres. However, its contractions play a role in approximating the fimbriae to the ovary at the time of ovulation (Ahlgren, 1975). The ampullary muscle coats are arranged in three layers comprising an outer longitudinal layer, a middle circular and an incomplete inner longitudinal layer. Meanwhile, the isthmus has a well-developed muscle layer consisting of outer and inner longitudinal layers and a circular layer between them. The uterine musculature is arranged in a loop around the intramural portion of the tube. Muscular contractions are mainly responsible for ovum transport in the isthmic portion of the FT.
A recent study has demonstrated that the FT exhibits electric activity in the form of slow waves (SWs) and fast-activity spikes or action potentials (APs) (Shafik, 1996). The SWs showed a regular rhythm, whereas the APs occurred randomly on top of or following the SWs. The APs were associated with the increase of the intraoviductal pressure (Shafik, 1996). The source of these electric waves has not been exactly defined yet. Previous studies of the rectum have revealed that similar waves are transmitted through the smooth rectal muscles and are partially controlled by the intrinsic and extrinsic rectal innervation (Shafik, 1998, 1999). It was postulated that the electric waves from both the gut and genitourinary system are generated by specialized cells called the interstitial cells of Cajal (ICCs) (Thuneberg, 1982; Rumessen et al., 1992; Liu and Huizinga, 1993; Vanderwinden et al., 1996; Daniel et al., 2001; Ward and Sanders, 2001; McCloskey and Gurney, 2002; Pezzone et al., 2003; Shafik et al., 2004a,b). These cells appear to act as intermediate coordinating factors in the neuronal modulations of gastrointestinal muscular activity (Daniel et al., 2001; Ward and Sanders, 2001). Other investigators suggested that the ICCs act as gut pacemakers, generating electric waves to the gut (Rumessen et al., 1992; Liu and Huizinga, 1993). These cells are located in the gut at the level of the myenteric plexus and within the circular muscle layer (Hagger et al., 1998).
As the FT is a motile organ and exhibits electric activity (Shafik, 1996), we hypothesized that it contains ICCs in its wall. This hypothesis was investigated in this study.
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Materials and methods |
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Specimens from the FT were taken from 21 female cadavers [mean age of 38.2 ± 10.9 SD years (range 16–52)] within 24 h of death. We obtained them at the PostMortem Department of the Cairo University, Faculty of Medicine. The causes of death were motor-car–accident trauma or cardiac infarction. The study was approved by the Review Board and Ethics Committee of the Cairo University Faculty of Medicine.
Specimens of 0.5 x 0.5 cm were obtained from the various FT segments: the infundibulum, ampulla, isthmus and the intramural part. They were immediately placed in 2% buffered paraformaldehyde for 1 h, cryoprotected in 30% sucrose overnight and flash frozen in liquid nitrogen-cooled isopentane. Tissues were cut in to 5 µm frozen sections, mounted on polylysine-coated slides and kept frozen until immunocytochemical processing.
For immunocytochemical processing, tissue sections were rehydrated in potassium phosphate buffered saline (KPBS) at room temperature for 20 min, blocked with 10% normal goat serum for 20 min and incubated overnight at 4°C with the primary antiserum, a rabbit polyclonal IgG antibody to the human C-kit protein (Oncogene Research Products, Cambridge, MA, USA), diluted 1:100 in KPBS, 0.05% goat serum and 0.1% Triton X-100. The next day, slides were rinsed with KPBS three times (each for 10 min) and then incubated with a Cy3-conjugated goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature for 2 h at a dilution of 1:800 in KPBS, 0.05% goat serum and 0.1% Triton X-100. As mast cells also contain the C-kit receptor and thus stain positive with C-kit antibodies, dual staining with fluoroscein-avidin DCS (Vector Laboratories, Burlingame, CA, USA) diluted 1:200 for 2 h was used to specifically identify mast cell staining. Slides were again washed three times with KPBS and overslipped. They were then imaged using an Olympus Fluoview 500 scanning confocal microscope.
Controls for the specificity of the antisera consisted of tissue incubated with normal rabbit serum substituted for the primary antiserum.
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Results |
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The C-kit-positive cells were detected in the musculature of the FT (Figure 1). They were elongated with dendritic processes which were located commonly on the cell sides but occurred also at both poles of the cell (Figures 1 and 2). The cytoplasm of the cell was granular, and the nucleus was large and oval (Figure 2). The cells were variably small, medium and large, ranging in size from 52 to 168 µm (mean 118.7 ± 37). They occurred either separately or in groups. Because the cells were c-kit positive, branched and had oval nuclei, they were identified as ICCs.
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The C-kit-positive mast cells were also detected in the section. However, these cells were rounded, unbranched and had round nuclei (Figure 3). Furthermore, the mast cells, in contrast to ICCs, stained positively for fluoroscein-avidin DCS. The ICCs and mast cells were surrounded by the smooth muscle cells (SMC) in the wall of the FT (Figure 4). The SMC had a typical spindle shape with more regular dimensions compared with the morphologic heterogeneity of the ICCs; they were c-kit negative and not branched.
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The ICCs were detected in all segments of the FT: the infundibulum, ampulla, isthmus and intramural part. They existed among the SMC of the circular muscle layer; in the ampulla and isthmus, the ICCs also occurred in the area between the longitudinal and circular muscle layers.
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Discussion |
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The FT is a muscular organ which mainly functions to transport the zygote to the uterus to be implanted into the uterine endometrium (Brosens, 1991
). This zygote transport is mediated by the FT motile activity which is presumably produced by electric waves that are discharged from the FT musculature (Shafik, 1996) and are claimed to be generated by the ICCs (Thuneberg, 1982; Rumessen et al., 1992; Liu and Huizinga, 1993; Vanderwinden et al., 1996; Daniel et al., 2001; Ward and Sanders, 2001; McCloskey and Gurney, 2002; Pezzone et al., 2003; Shafik et al., 2004a,b).
This study could demonstrate, for the first time, the presence of c-kit-positive, ICC-like cells in all segments of the FT. The cellular morphology of these c-kit-positive cells is characteristic of cells that were earlier identified in the gastrointestinal tract as ICCs or pacemaker cells (Thuneberg, 1982; Rumessen et al., 1992; Liu and Huizinga, 1993; Vanderwinden et al., 1996; Daniel et al., 2001; Shafik et al., 2004a). We postulate that these cells discharge electric waves that effect tubal motility. They appear to act as pacemaker cells, which are probably responsible for the generation of the FT motile activity and, consequently, the transport of the zygote from the FT to the uterus.
The resting electric activity of the FT seems to be controlled by the constant firing of pacemaker cells, which are most likely the ICCs, the activity of which is conducted to the smooth muscle. This is in accord with the postulation of other investigators who hold that the electric waves from the gut and the genitourinary system are generated by the ICCs (Thuneberg, 1982; Rumessen et al., 1992; Liu and Huizinga, 1993; Vanderwinden et al., 1996; Daniel et al., 2001; Ward and Sanders, 2001; McCloskey and Gurney, 2002; Pezzone et al., 2003; Shafik et al., 2004a,b). It appears that the release of excitatory transmitters acts either directly on the tubal smooth muscle fibres or indirectly through stimulation of the pacemakers’ firing with a resulting tubal smooth muscle contraction. Furthermore, a pattern similar to the ICCs apparently mediating the response to nerves in the gastrointestinal tract may also exist in the FT (Thuneberg, 1982; Rumessen et al., 1992; Liu and Huizinga, 1993; Vanderwinden et al., 1996; Daniel et al., 2001; Ward and Sanders, 2001; Shafik et al., 2004a).
The ICCs could be morphologically differentiated from the c-kit-positive mast cells and the c-kit-negative SMC. Recognition of abnormalities of the ICC-like system in the FT may offer insight into a variety of motility disorders of the FT which may be congenital or acquired and thought to result from a deficiency in tubal innervation. Therefore, absence or deficiency of ICCs in the FT may interfere with ovum and/or sperm transportation through the tube and can hence induce infertility of a kind that may very likely be diagnosed as idiopathic. So it seems recommendable in examinations related to female infertility that the patient tests for the existence of these cells in her FT. The development of an appropriate method to use the potential of the ICC-like cells as investigative tool seems indicated.
In conclusion, we have identified cells in the FT with morphologic and immunologic phenotypes similar to the ICCs of the gut. These cells appear responsible for the generation of the SWs recorded from the FT. The role of these cells in the pathologic conditions of the FT needs to be studied.
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Acknowledgements |
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Margot Yehia assisted in preparing the manuscript.
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References |
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