The Effect Of BK_Ca On The Endometrial Receptivity And The Mechanism Involved

Biomolecular mechanisms that are involved in the embryo implantation and development process are still unknown. This event, like all complex biological mechanisms, depends on the activity of several factors of both embryonic and endometrial origin. Endometrium is a unique tissue that sustains numerous changes during the menstrual cycle under control of the ovarian hormones, estradiol and progesterone. It shifts from a proliferative state to a secretory state to prepare for establishing receptivity for the embryo implantation. During a short period named the window of implantation (WOI), endometrial cells play a prominent role in the signaling between maternal and fetal tissues. They synthesize and secrete many molecules which modulate uterine function for receipting embryo.Ion channel activity participates in many cellular functions such as synthesis and secretion of endogenous factors including hormones and neurotransmitters. Although some ion channels have been studied in uterus, most of these researches were focused on the roles of ion channels in controlling uterine muscle constriction and dilation. The roles of ion channels in regulation of endometrial WOI factors and embryo implantation are needed to be elucidated. Large-conductance calcium-activated potassium channels (BKCa channels, also known as Kcal.1, KCNMA1, Slol or maxi-K) are important contributors to excitable cells by regulating the duration of calcium action potential trains, shaping the after-hyperpolarization, and curtailing calcium entry cross membrane. channels are activated both by elevated cytosolic Ca2+ levels and by membrane depolarization. They were mainly studied in smooth muscle cells (vascular, airway, uterine, gastrointestinal, and urinary bladder) where they are the key players in setting the contractile tone. However, they also play important roles in other physiological processes such as action potential repolarization; neuronal excitability; neurotransmitter release; hormone secretion; tuning of cochlear hair cells; innate immunity; cell proliferation, apoptosis and oncogenesis. BKCa channels are present in many members of the animal kingdom such as nematodes (Caenorhabditis), insects (Drosophila), and mammals. The human BKCa gene was first cloned from the brain. Although they are discovered widely distributing in many different tissues in human such as brain (cerebellum, habenula, striatum, olfactory bulb, neocortex, granule and pyramidal cells of the hippocampus), skeletal muscle, smooth muscle (vascular, airway, uterine, gastric, bladder), adrenal cortex, cochlear hair cells, odontoblasts, pancreatic islet cells, colonic and kidney epithelium, and in our previous study we found that BKCa expressed in endometriam, it has been still unclear whether BKCa channels contribute endometrial receptivity.In the present study we confirm that BKCa channels were expressed in human uterine endometrium and the discipline in which BKCa channels were expressed temporospatially. Using Iberiotoxin (IbTX, a specific BKCa blocker) and siRNA targeting for BKCa gene, we examined the role of BKCa channels in the regulation of WOI factors synthesis in human endometrium. We also examined the possible effect of BKCa channels on embryo implantation. At last, we try to demonstrate the possible machenism。Part One:The expression of BKCa in human endometrium and its significanceObjective:To confirm whether BKCa channels were expressed in human uterine endometrium as we previous found. To analyse the differential expression of BKCa in infertile women due to tubule pathology.Methods:qRT-PCR and Western blot assay were used to analyse the expression of BKCa channels in human endometrium derived from 80 patients(44 cases, proliferative phase and 36 cases, mid-secretory phase). Immunohistochemical staining was used to locate BKCa in human endometrium. And Patch-Clamping was used to analyze K+ current in human endometrium.Results:1. qRT-PCR and Western blotting analysis shows the expression of BKCa in human endometrium at the mid-secretory phase was significantly higher than that at the proliferative phase.2. The levels of BKCa in the failed subgroup were significantly lower than those in the successful subgroup3. BKCa Immunohistochemical staining was present in both luminal and glandular epithelial cells in human endometrium. Weak staining was also observed in the stromal endometrium.4. Patch-clamp experiment demonstrated that a voltage dependent current was detected in human endometrial cell. This current could be significantly inhibited by treatment of cells with 0.1μM IbTX.Conclusion:BKCa expressed in human endometrium. The differential expression at different time point in menstrual cycle and in different infertile patients may associate with establishment of endometrial receptivity.Part Two:BKca affect endometrial receptivity via altering the production of WOI factorsObjective:To investigate the effect of BKCa on the attachment of the floating blastocyst to the receptive endometrial epithelium. To examine the potential involvement of BKCa channel in EP-induced alteration of WOI factors in human endometrium. Methods:1. In Vitro attachment model was employed with simulated embryos to analyze the association between BKCa channel and embryos attachment.2. qRT-PCR and Western blot assay were used to analyse the alteration of WOI factors in human primary endometrial cells treated with or without IbTX.3. qRT-PCR, ELISA and Western blot assay were used to analyse the alteration of WOI factors in Ishikawa cells treated with or without IbTX. To confirm the results, siRNA targeting for BKCa channel was employed also.Results:1. Attachment rate analysis demonstrated that knock-down BKCa by siRNA significantly decreased JAr speroids attachment rate (p<0.05). However supplement LIF partially rescued the decrease of attachment rate induced by suppression of BKCa expression; Supplement LIF alone did not increase attachment rate in srambled siRNA group.2. qRT-PCR assay showed LIF, integrin p3, claudin-4 and DKK-1 gene expression were significantly increased in EP-stimulating cells compared with control, whereas IbTX could attenuate this effect.3. qRT-PCR, ELISA and western blot analysis showed that EP could induced increase WOI factors, whereas IbTX significantly attenuated this effect induced by EP in Ishikawa cells. LIF, integrinβ3, claudin-4 and DKK-1 mRNA transcription and protein expression were significantly decreased in Ishikawa cells transfected with BKCa siRNA compared with cells transfected with scrambled siRNA (P<0.5 respectively).Conclusion:BKCa channels may affect embryo implantation via altering the production of WOI factors in human endometrium.Part Three:The mechanism involved in regulating WOI factors by BKCa in human endometrium Objective:To investigate the possible machenism of regulating WOI factors by BKca.Methods:1. Single-cell microfluorimetry was used to monitor agonist-evoked cytosolic Ca2+ transients in real time to investigate the contribution of BKCa to the increase of Ca2+ in cytoplasm in human endometrial cells.2.EMS A assay was used to examine whether BKca interfered with the nuclear translocation and activation of NF-κB by EMSA.3. Western blotting was used to detect the level of IKB-a in human endometrial cells to confirm whether the nuclear translocation of NF-κB was happened.Results:1. Single-cell microfluorimetry assay indicated elevations in cytosolic Ca2+ were evoked by ATP. However, IbTX attenuated evoked increase of Ca2+ in cytoplasm significantly.2. EMSA assay showed the levels of the NF-κB activation were significantly decreased in the presence of IbTX, Compared with control.3. Western blotting indicated IKB-awas degraded significantly 30 min after treatment of cells with EP and reappearing at 60 min, however the effect of IκB-degradation at 30 min after treatment by EP was significantly attenuated in the presence of IbTXConclusion:BKCa channels may affect WOI factors expression, regulate endometrial receptivity via altering intracellular Ca2+ level and activation of NF-κB.