The Role of Interstitial Cells of Cajal in Oviduct Pacemaker Activity

Abstract

The experiments described in this dissertation were performed to investigate the spontaneous electrical activity of the oviduct myosalpinx and to determine its cellular origin and ionic basis. We have also examined the impact of chlamydia on this activity, using a murine model of genital chlamydia infection. <italic>In vitro</italic> spatio-temporal mapping and video imaging of myosalpinx contractions and egg and luminal particle movement within the oviduct have revealed that spontaneous myosalpinx contractions are critical for egg transport along the duct, not ciliary beating. Intracellular microelectrode recordings and simultaneous isometric force recordings have shown that slow waves provide the electrical basis for the rhythmic contractions of the myosalpinx. Immunohistochemistry against the KIT protein has revealed an extensive network of ICC-OVI along the length of oviducts. Organotypic culture experiments performed on P0 oviducts incubated with or without the KIT neutralizing antibody ACK2, have shown that slow wave activity is absent from oviducts with severely disrupted ICC-OVI networks, revealing for the first time, that ICC-OVI are the pacemakers of the oviduct. RT-PCR has revealed transcriptional expression of several Ca<super>2+</super> and K<super>+</super> channels as well as Cl_Ca channels (encoded by <italic>Tmem16a</italic>) in the oviduct myosalpinx. The ionic basis of slow wave activity has been thoroughly examined using intracellular electrophysiology and specific ion channel agonists and antagonists. Adequate extracellular Ca<super>2+</super> and intact intracellular Ca<super>2+</super> stores have been found to be critical for pacemaker activity. Cl_Ca channel activity has also been determined to be essential for pacemaker activity, since slow waves are absent from oviducts exposed to the Cl_Ca channel antagonist niflumic acid and from oviducts taken from animals that are homozygotes for the null allele of TMEM16A. TREK, K_ATP and K_v channels are regulators of slow wave frequency. These three channels along with IK, have all been found to contribute to the setting of the RMP in the oviduct. Caffeine has been demonstrated to activate K_ATP channels in a cAMP-dependent manner in the oviduct. This causes membrane hyperpolarization, inhibition of slow wave activity and loss of myosalpinx contractions and may explain why women with high caffeine intakes take longer to conceive than women who do not consume caffeine. In oviducts 2 wk post-infection with <italic>C. muridarum</italic>, slow wave activity has been found to be absent and ICC-OVI populations were severely disrupted or absent. Damage to pacemaker activity is suggested to be a result of the host immune response to infection, more specifically to the upregulation of proinflammatory mediators NOS2 and PTGS2. Pacemaker activity can be protected from LPS induced inflammatory damage in vitro by inhibition of NOS2 with 1400W. ICC-OVI networks begin to recover or re-establish and pacemaker activity returns as the infection is resolved, 4 - 7 weeks post-infection. In conclusion, this dissertation provides compelling evidence that ICC-OVI networks are the pacemakers of the oviduct, responsible for generating spontaneous electrical slow wave activity that underlies the rhythmic contractions of the myosalpinx which are critical to egg transport. These pacemaker cells are damaged by the host immune response to chlamydia infection, leading to oviduct stasis and pseudo-obstruction and providing an explanation as to why women with a history of chlamydia infections have an increased risk for ectopic pregnancies and tubal factor infertility.