Uterine EMG activity in the non-pregnant sow during estrous cycle

Uterine EMG activity in the non-pregnant sow during estrous cycle Background: Uterine myoactivity is crucial for successful reproductive performance of the sow. Spontaneous contractions of the uterus are strictly controlled and coordinated. Uterine electromyographic (EMG) activity undergoes hormonal regulation with rapid and long-term effects. What is more, interstitial Cajal-like Cells (ICLC) appear essential for smooth muscle contractility in the reproductive tract where they are suspected to be playing a major role in generating, coordinating, modulating and synchronizing slow triggering waves. The aim of this study was to investigate the myoelectrical activity of sow’s uterus during estrus cycle. Results: Study was conducted on 10 Polish Landrace sows. Propagation mechanisms and their connection with the uterine EMG activity were considered in correlation with expression of c-kit, progesterone and oxytocin receptors of the non-pregnant sow. ICLC were labeled with antibody directed against c-kit receptor and visualized by confocal microscopy and scanning cytometer for positive cells percentage assessment. EMG signal was recorded directly from the myometrium with telemetry transmitters and electrodes located in different topographic regions of reproductive tracts. The stages of estrus cycle were determined by monitoring levels of luteinizing hormone, progesterone and estrogen with radioimmunoassays. Significant differences of the EMG signal parameters between diestrus and estrus and the correlations with density of labelled receptors were demonstrated. Moreover, the electrophysiological studies indicated that ICLC in the myometrium in the tip of uterine horn may participate in the regulation of slow waves duration and frequency. Conclusions: The pattern of EMG signal propagation in the wall of the non-pregnant porcine uterus occurs in an orderly, bidirectional fashion and at distinctive speed, with no differences between diestrus and estrus. Keywords: EMG activity, Direction, Speed, Estrous cycle, Sow, Myometrium Background relaxation are based on a number of intracellular molecu- Reproductive performance of the sow is a critical compo- lar processes (i.e. rise in intracellular calcium increase in nent of profitable production. Spontaneous contractions myosin phosphorylation; increase in myosin light-chain of uterus must be controlled and coordinated for the suc- kinase activity) [1]. In myometrium, an increase in cess of various reproductive functions. Suitable uterine myosine phosphorylation accompanies spontaneous contractility is involved in the transport of gametes and and hormone-induced contractions. Spontaneous and embryo implantation. Unsuitable uterine contractility may agonist-induced relaxation is accompanied by decrease in lead to ectopic pregnancies, miscarriages, embryonic loss myosin light-chain phosphorylation [2]. The long-term and abnormalities of puerperium. effects concern hormonal regulation (including Uterine electromyographic (EMG) activity comes under reproductive hormones) of components of intracellular systems hormonal regulation due to acute and long-term effects. at the level of the plasma membrane (i.e. receptor-operated, The acute effects promoting uterine contraction and voltage-operated, second-messenger-operated, GTP-binding protein (Guanosine-5′-triphosphate-binding protein), gated ion channels) [1]. * Correspondence: zgajewski@supermedia.pl Department of Large Animal Diseases with Clinic, Veterinary Research Centre It is generally accepted that progesterone, estrogens and Center for Biomedical Research, Faculty of Veterinary Medicine, Warsaw and oxytocin are key regulators of uterine contraction University of Life Sciences (WULS – SGGW), Nowoursynowska 100, 02-797 [3, 4]. Progesterone promotes sustained myometrial Warsaw, Poland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Domino et al. BMC Veterinary Research (2018) 14:176 Page 2 of 9 relaxation, estrogens and oxytocin favor myometrial con- generated at cellular and organ level, propagation mech- tractility and excitability [5]. Recent studies have sug- anisms and their correlation with the uterine EMG gested that contractility in spontaneously active organs signal recorded internally from the myometrium and ex- occurs as an intrinsic property of the muscle [6]. At the pression of c-kit, PR and OXTR of the non-pregnant sow. plasma membrane level, progesterone interacts with Although a precise role for myometrial Cajal-like intersti- membrane-associated receptors (progesterone receptors tial cells has not yet been identified, this study presents re- - PR) to directly modulate intracellular calcium and sults, that may support understanding of functional role cyclic adenosine monophosphate levels. Progesterone of ICLC in regulating uterine contractile activity. also indirectly inhibits estrogen-induced oxytocin recep- tors (OXTR) expression. At the plasma membrane level Methods oxytocin also interacts with membrane-associated recep- Animals tors and induces inositol triphosphate production and The experiment has been conducted on 10 mature Ca2+ mobilization [5]. Polish Landrace sows (n = 10) according to protocol ap- Based on their gastrointestinal (GI) tract investiga- proved by the III Local Ethical Committee on Animal tions, Torihashi et al. (1999) have proposed a population Testing in Warsaw (Permit Number: 71/2009, from of independent interstitial cells as pacemakers of con- 19.11.2009) on behalf of the National Ethical Committees tractile rhythm. on Animal Testing. Spontaneous uterine activity in In GI the interstitial cells of Cajal (ICC) create a net- non-pregnant state was recorded by the combination of work which initiates and propagates the slow waves. three electrodes connected to 3-channel transmitter used Moreover, cells with morphology and antigenicity similar in large animals [15]. The experiment started with surgery to ICC have been found outside the gastrointestinal tract in the diestrus and the estrus began on average between and named interstitial Cajal-like cells (ICLC). The ICLC day 7 and 12 after surgery when the highest quality EMG appear essential for smooth muscle contractility in the signal was collected. One registration period lasted 5 to urinary and reproductive tract where they generate, co- 6 weeks with 1.87 ± 0.40 estrus cycles occurring during ordinate, modulate and synchronize slow triggering this time. Surgery was carried out under general waves [7]. The discovery of the c-kit receptor (type III anesthesia and the telemetry EMG recording method was tyrosine kinase receptor; CD117) as a marker of ICLC performed. According to the standard protocol [16] allows for the recognition of these cells under light and animals were premedicated with an intramuscular injec- confocal microscopes [8]. The presence of ICLC located tion of azaperone (Stresnil, 3 [mg/kg b.wt.], IM, Janssen among smooth muscle cells (SMC) were demonstrated Pharmaceutica) and then catheter was inserted into the in non-pregnant human [9] and porcine [10] myome- auricular vein. General anesthesia was achieved with trium. Moreover, the presence of estrogen, progesterone combined administration of medetomidine (Cepetor, 1 and oxytocin receptors was demonstrated in the nuclei [mg/kg b.wt.], IV, CP- Pharma Handelsges), butorphanol of ICLC in human myometrium [11, 12]. (Butomidor, 0,2 [mg/kgb.wt.], IV, Ricgter Pharma AG), Anatomical structure of uterus with billions of SMC ketamine (Bioketan, 3[mg/kg b.wt.], IV, Vetoquinol comprising myometrium interacting in a complex man- Biowet) and propofol (Propofol, 2 ± 4 [mg/kg b.wt.], IV, ner in longitudinal and circular muscle layers inter- Pfizer). The telemetry transmitter TL10M3-D70-EEE twined with the network of pacemaker cells (ICLC) (DSI, USA) was surgically positioned between abdominal must be considered in myoactivity signal propagation muscles and electrodes sutured into different topographic analysis. Individual electrical activities within the myo- regions of reproductive tract. In experiment 1: right uter- metrial tissue may differ in speed and direction. A single ine horn (RUH-channel 1), corpus uteri (CU-channel 2) electrical activity (burst) can initiate a myometrial con- and the left uterine horn (LUH-channel 3) surfaces with traction but multiple, coordinated activities (bungle) are 17 cm distance between electrodes and in experiment 2: needed for powerful and sustained contractions [1, 13]. isthmus of oviducts (IO-channel 1), right uterine horn tip SMC contract when the action potentials reaches a (RUHT-channel 2), right uterine horn (RUH-channel 3) depolarization threshold and generates an electromag- surfaces with 17 cm distance between electrodes 2 and 3. netic field, possible to measure as voltage. Therefore, When pigs recovered from surgery analgesic - meloxicam electromyography (EMG) is the most accurate method (Metacam 0.4 [mg/kg BWT], IM, Boehringer Ingelheim) to measure action potential changes leading to syn- and anti-microbial - cefquinom (Cobactan, 2.0 [mg/kg chronous contraction on the organ level [14]. BWT], IM, Intervet) had been administered for 5 days. The primary objective of this paper is to discuss the Directly after surgery, one day before the start of registra- reproductive phenomenon, which is the myoelectrical tion, the EMG signal quality was inspected. At day 3 to 5 activity of sow uterus during estrus cycle. We considered after surgery the disturbance-free signal was obtained and physiological modeling of uterine electrical activity the regular registrations was carried during next 5 to Domino et al. BMC Veterinary Research (2018) 14:176 Page 3 of 9 6 weeks. The EMG signals were collected 10 h daily al- bungle propagation was evaluated basing on the time ways in the same time periods. Obtained analog signal elapsed for highly synchronized signals to move from was digitalized and sent to the telemetric receiver (DL10 the first electrode to the second one. (DSI, USA)). The signal was acquired with a 3-channel transmitter PowerLab (ADInstruments, Australia) with Microscopy analysis sampling frequency 100 Hz and archived for off-line ana- Samples were fixed in paraformaldehyde and embedded lysis. Pigs were euthanized at the end of the experiment, in paraffin using standard histology procedures. by Sodium Pentobarbital (Morbital 100.0 [mg/kg BWT], IV, Afterwards, samples were cut to 5 μm thin sections and Biowet Pulawy) in different stages of estrus cycle (half in es- stained with hematoxilin-eosine (H-E) using a standard trus (n = 5) and half in diestrus (n = 5)). Full-thickness protocol. Immunofluorescent labeling against CD117 tissue samples of muscle layer were obtained from uterus (c-kit), progesterone receptor (PR) and oxytocine recep- (corpus-CU, middle of the horn-UH, horn tip-UHT) tor (OXTR) was performed. For the assessment of and oviducts (isthmus-IO, infundibulum-IFO) of all immunofluorescent reaction (CD117) specificity, porcine pigs (n =10). ileum paraffin blocks were used as positive control (PC), while staining protocols, with the primary antibody EMG data analysis omitted, were used as negative control (NC). The EMG signals were digitally filtered with a band-pass Immunofluorescent labeling against CD117 (c-kit), PR filter [5–50 Hz]. Mean and linear trends were removed and OXTR was performed using a published staining [17]. Uterine contractions were defined as series of elec- protocol [8, 15]. Antigens were retrieved through citrate trical potentials with amplitude above 5 μV and a dur- buffer heating, washing with PBS-buffer and blocked with ation longer than 3 s, separated from each other by the BSA to minimize non-specific antibody binding. Slides time period no shorter than 5 s. Any new electrical ac- were then incubated with antibodies: anti-CD117 (goat tivity after 5 s was interpreted as a subsequent contrac- polyclonal anti-human CD117; RD System, USA; AF332; tion [18]. Contraction was described using burst and 120 min, room temperature; dilution 1:200), anti-PR bungle parameters. The EMG signal spectral content (mouse monoclonal anti-human Progesterone Receptor was analyzed in time and frequency domain features. In 1A6; PROGEN, Germany, no16077; 120 min, room time domain, the bursts represented single action poten- temperature; dilution 1:250), anti-OXTR (mouse mono- tials, while the bungles comprised of multiple action po- clonal anti-human hOXTR; R&D Systems, USA, tentials. Mean amplitude [mV], mean RMS (root mean MAB6616; 180 min, room temperature; concentration 15 square) [mV], duration of electrical activity [s], duration μg/ml). Cross-reactivity for above-mentioned antibodies of pauses [s], and number of bursts forming a bungle has been confirmed in previous studies [15, 22]. Polyclonal were analyzed. In frequency domain, means of Fourier Alexa Fluor 405 (chicken anti-mouse), Alexa Fluor 488 analysis (FFT–Fast Fourier transform), dominant fre- (chicken anti-mouse, chicken anti-goat), Alexa Fluor 660 quency (DF) [Hz] (the frequency at which most signal (chicken anti-mouse, chicken anti-goat) (Abcam, UK) energy was transmitted) was assessed for each data labeled antibodies (dilution 1:500, 120 min, room series. We used the Hamming window for the Fourier temperature) were used respectively to detect the analysis [19, 20]. The similarity measures were used to primary antibodies. Nuclei were counterstained with describe synchronization between EMG signals. Degree 7-Aminoactinomycin D (Sigma Aldrich, Poland) or of synchronization between three simultaneously re- HOECHST33342 (Sigma Aldrich, Poland). After labeling, corded data series (channels 1(x), 2(y), 3(z) in experi- the coverslips were mounted using mounting medium for ment 1) was estimated for two signal pairs (xy and yz) fluorescence microscopy. Immunofluorescence labeled using linear measures: the cross-correlation function cells were examined with confocal microscope (FV-500, (ƒx,y(l), ƒy,z(l)) and the cross-coherence function Olympus, Poland). Quantitative evaluation was performed (Cxy(ƒ), Cyz(ƒ)) [19, 21]. Similarity measures, based on using a scanning cytometer (SCAN^R, Olympus, Poland, the concept of time series data synchronization, allows magnification × 200). The positive cells density was signal propagation detecting in different topographic re- defined as a number of cells with appropriate immuno- gions. It was used to “identify” the frequency fingerprint phenotype and morphology in 20 fields of view (mean of the spatial bungle and determine, if the bungle density ± SD). Afterwards the relative density (%) was reached the other location during propagation. Locations calculated as percentage of positive cells relative to the of the first and second electrodes (on the x/y or y/z axis) highest density region. were used to measure highly synchronized signals, and the direction of bungle propagation was evaluated. The Hormone concentrations analysis second signal location in the time function pointed to During the surgery, a silicone cannula was inserted into cervico-tubal and tubo-cervical directions. The speed of the brachial vein to enable collection of blood samples. Domino et al. BMC Veterinary Research (2018) 14:176 Page 4 of 9 Each day during the experiment (from 7 days before to Table 1 The EMG signal parameters in different topographic regions of reproductive tract during diestrus and estrus 42 days after surgery) 7 mL sample of blood was collected every 4 h. All samples were used to determine EMG Diestrus Estrus stages of estrus based on luteinizing hormone (LH), Region parameter mean SEM mean SEM 1 a b 17β-estradiol (E2) and pregn-4-ene3,20-dione (P4) con- A [mV] 0.64 ±0.04 0.81 ±0.02 centrations. The serum LH concentrations were mea- 2 c d IO RMS [mV] 0.05 ±0.003 0.12 ±0.001 sured with noncommercial radioimmunoassay test [21]. 3 e ef D [s] 153.10 ±9.64 111.10 ±14.48 The sensitivity was 0.08 ng/mL (intra-assay CV < 6.7%, 1 a b A [mV] 0.71 ±0.03 0.99 ±0.04 interassay CV < 12.5%). The serum levels of E2 and P4 2 c d UHT RMS [mV] 0.09 ±0.002 0.12 ±0.002 were determined with RIA. The sensitivity for estradiol was 3 e f 5 pg/mL (intra-assay CV < 8.0%, interassay CV < 11.1%; D [s] 194.70 ±9.25 99.97 ±20.97 1 a g ESTR-US-CT; Cisbio assays, France) and for progesterone UH A [mV] 0.74 ±0.02 2.22 ±0.15 0.15 ng/mL (intra-assay CV < 5.6%, inter-assay CV < 8.8%; 2 c h RMS [mV] 0.10 ±0.001 0.26 ±0.02 KIP1458; DIAsource ImmunoAssays SA, Belgium). The 3 e i D [s] 221.60 ±25.53 33.30 ±2.73 stages of estrus cycle were determined by monitoring 1 a j CU A [mV] 0.57 ±0.02 4.24 ±0.19 hormone levels and divided into estrus (mean concen- 2 c k RMS [mV] 0.07 ±0.001 0.47 ±0.02 tration: E2 > 8 pg/ml, P4 < 1 ng/ml, LH > 4 ng/ml) and 3 e l diestrus (mean concentration: E2 < 2 pg/ml, P4 > 4 ng/ml, D [s] 209.70 ±22.92 42.60 ±3.23 1 2 3 LH < 1 ng/ml). A-amplitude, RMS-root mean square, D-duration of electrical activity. The different letters in superscript indicate the statistical significant differences (P < 0.05): diestrus/estrus and regions of reproductive tract Statistical analysis All statistical evaluation was performed by Graph-Pad InStat software (San Diego, USA), the level of statistical percentages (mean% ± SEM) of bungle propagated significance was set to P < 0.05, using specific tests: CU→ UH during diestrus (45.30 ± 3.27) and estrus Mann-Whitney test, Kruskal-Wallis test with Dunn’s (50.40 ± 1.95) and UH→ CU during diestrus (54.73 ± 3.25) multiple comparisons, one-way ANOVA test with Tukey’s and estrus (49.74 ± 1.95) in relation to the total number of multiple comparisons. The Pearson correlation coefficient highly synchronized uterine contraction signals. (r) and Spearman’s rank correlation coefficient (Sr) were The EMG signals propagated along the UH with three used to determine correlation between receptors (c-kit, significantly different (P < 0.0001), independent speeds: PR, OXTR) distribution and for selected EMG parameters SBMR (slow basic migration rhythm), RBMR (rapid in different regions of reproductive tract. basic migration rhythm) and RAMR (rapid accessory migration rhythm). There were no significant differences Results (P > 0.05) between EMG signal propagation speeds The bungle parameters were characterized in details in values along the UH and percentage of each speeds dur- three different topographic regions of the reproductive ing diestrus compared to estrus (Table 3). tract, with significant differences between diestrus and The cells with morphology similar to ICLC were estrus (Table 1). We demonstrate significantly higher shown by H-E staining, however the reliable amplitude and RMS (P < 0.0001) and lower duration of electrical activity (P < 0.0001) in estrus compared to di- Table 2 EMG signal propagation along uterus during diestrus and estrus in all uterine regions (UHT, UH, CU). We also estrus demonstrate significantly higher amplitude (P = 0.0003) The directions of EMG signal propagation 1 2 3 4 5 and RMS (P < 0.0001) in estrus compared to diestrus in LUH-CU RUH-CU UH(L,R)-CU CU-LUH CU-RUH CU-UH IO with no differences in duration of electrical activity. (L,R) There were no differences (P > 0.05) in amplitude, RMS Diestrus and duration of electrical activity in IO and uterus a a b a a b mean 24.51 30.20 54.73 24.51 20.82 45.30 (UHT, UH, CU) in diestrus, but in estrus, amplitude and RMS were significantly higher (P < 0.05) and duration of SEM ±2.27 ±1.07 ±3.25 ±0.53 ±3.75 ±3.27 electrical activity was significant lower (P < 0.05) in CU Estrus and UH compared to the oviductal region (IO, UHT). a a b a a b mean 26.60 23.13 49.74 27,32 23.11 50.40 The EMG signals were propagated along the UH in both cervico-tubal (CU→ UH) and tubo-cervical (UH→ CU) SEM ±1.55 ±0.35 ±1.95 ±0.70 ±1.15 ±1.95 directions during diestrus and estrus with no signifi- 1 2 3 4 5 6 LUH➔CU; RUH➔CU; UH(L,R)➔CU; CU➔LUH; CU➔RUH; CU➔UH(L,R). cant differences (P >0.05) (Table 2). Therewereno The different letters in superscript indicate the statistical significant differences significant differences (P > 0.05) between the (P < 0.05): diestrus/estrus and the directions of EMG signal propagation Domino et al. BMC Veterinary Research (2018) 14:176 Page 5 of 9 Table 3 EMG signal propagation speeds along uterus during in the cytoplasm for PR and intense at membranes and diestrus and estrus weak in the cytoplasm for OXTR (Fig. 2). The speeds of EMG signal propagation In recent study we demonstrated that the ICLC dens- 1 2 3 ity differed among different parts of the porcine repro- SBMR RBMR RAMR ductive tract during diestrus [15]. In present study we Diestrus assessed corresponding differences in estrus and com- a b c mean speed [mm/min] 1.25 2.47 7.07 pared our current results with previous data in one table SEM ±0.04 ±0.08 ±0.60 in order to test the dependency of density differences on d d d %of speeds 46.01 26.30 27.30 stages of estrus cycle (Table 4). Current results Estrus demonstrate that in estrus there is significantly higher a b c (P < 0.01) density of ICLC in the IO compared to UH mean speed [mm/min] 1.16 2.53 8.70 with no differences (P > 0.05) between UHT and UH. SEM ±0.05 ±0.10 ±0.67 We also demonstrate significantly lower density of ICLC d d d %of speeds 33.03 29.01 38.01 in IFO (P < 0.01) and in CU (P < 0.01) when compared 1 2 SBMR-slow basic migration rhythm; RBMR-rapid basic migration rhythm; 3 to IO. There were no significant differences (P > 0.05) RAMR-rapid accessory migration rhythm. The different letters in superscript indicate the statistical significant differences (P < 0.05): diestrus/estrus and the between density of ICLC in different topographic regions speeds of EMG signal propagation of the reproductive tract during diestrus [15] and estrus (Table 4). differentiation of m-ICLC from fibroblasts according to The correlations (Pearson’s r or Spearman’s rank) be- Hutchings et al. [12] criteria was possible in porcine spe- tween ICLC, PR, OXTR density (mean ± SD) and the cimen after immunofluorescence labeling. Therefore, the following EMG signal parameters: dominant frequency, histology approach was used only for initial screening amplitude, RMS, duration of bungles and bursts, dur- and to examine tissue architecture and the cells with an- ation of pauses between bungles and bursts, number of tigenicity similar to ICLC were demonstrated in all in bursts forming bungle (mean ± SEM) were calculated for examined parts of the porcine reproductive tract. The different topographic regions of the reproductive tract c-Kit-immunopositive nucleated cells that present char- during diestrus and described below. acteristic cell morphology (triangular, spindle-shaped, Strong positive correlation has been confirmed in CU star-like body with two or more, very long, moniliform (r = 0.86; P = 0.03) between ICLC density (1.22 ± 0.38) processes) were demonstrated in each topographic re- and OXTR density (0.53 ± 0.02) and strong, negative cor- gions of the reproductive tract. The presence of similar relation (Sr = − 0.71; P < 0.0001) between PR (0.20 ± 0.02) cells was confirmed in positive control-porcine ileum and number of bursts forming bungle (3.40 ± 0.40). used as reference material and so the c-kit positive cells In UH there was strong negative correlation (Sr = − 0.87; were considered as ICLC (Fig. 1). The lack of such cells P < 0.0001) between ICLC density (3.17 ± 1.04) and number was demonstrated in negative control. We demonstrated of bursts forming bungle (3.40 ± 0.24); strong positive cor- the presence of ICLC that were single-positive for c-kit relation (r =0.82; P = 0.04) between PR density (0.31 ± and double positive for c-kit/PR as well as c-kit/OXTR 0.02) and EMG signal dominant frequency (4.01 ± in all examined topographic regions. Double immuno- 0.21); strong negative correlation (r = − 0.86; P=0.03) be- fluorescence confirmed the same distribution of PR and tween OXTR density (0.61 ± 0.01) and EMG signal OXTR on c-kit positive cells: intense in nuclei and weak amplitude (1.18 ± 0.17); strong positive correlation Fig. 1 The representative images of presence of Cajal cells in uterine tissues (ICLC) and ileum (ICC). Fusiform cells with long processes and oval nucleus show morphology typical for Cajal cells (arrows). Legend: A. Muscular layer of UHT. Widefield microscopy of a H-E stained tissue section, lens magnification 40×; B. Muscular layer of UHT. Confocal microscopy following IF labeling, lens magnification 40×; C. Muscular layer of porcine ileum (PC - positive control). Confocal microscopy following IF labeling, lens magnification 63× Domino et al. BMC Veterinary Research (2018) 14:176 Page 6 of 9 Fig. 2 The representative images of IF labelling of c-kit (CD117), PR and OXTR. Legend: A. Muscular layer of UHT - Triangular ICLC positive for c-kit only; emission spectra of AF 488/Hoechst; B. Muscular layer of UHT - Star-like shape ICLC double positive for c-kit and PR intense at nuclear level; emission spectra of AF 488/AF 660/Hoechst; C. Muscular layer of UHT - Fusiform ICLC double positive for c-kit and OXTR intense at membrane level; emission spectra of AF 488/AF 660 /Hoechst. Confocal microscopy, lens magnification 63×, digital zoom 0.75–3.00 (r =0.85; P = 0.03) between OXTR density (0.61 ± 0.01) Discussion and EMG signal dominant frequency (4.40 ± 0.31) and In this study, we have thoroughly and systematically very strong positive correlation between OXTR density characterized the electrical activity of the porcine uterus (0.61 ± 0.01) duration of bungles (3.77 ± 0.46) (r = 0.99; throughout the estrus and diestrus. Our complex experi- P = 0.01) and duration of bursts (9.21 ± 1.54) (r = 0.97; ment combines parallel studies: on cellular level in situ P = 0.03). Strong negative correlations between OXTR and in vivo on organ level, in living organism, encom- density and duration of pauses between bungles (7.23 ± 1.54) passed by natural biological processes. In pigs, an activ- (r = − 0.94; P = 0.01) and duration of pauses between bursts ity of the myometrium seems to be myogenic in origin, (13.20 ± 2.73) (r = − 0.97; P = 0.03) were observed. similarly to described in Langendijk et al. (2002) reports, In UHT there was strong negative correlation between and to be driven by pacemaker activity of not only the ICLC density (3.10 ± 0.92) and duration of bungles uterine smooth muscle cells [23] but also ICLC similar (2.31 ± 0.36) (r = − 0.85; P = 0.03) as well as duration to these found in humans [12]. The anatomical arrange- of bursts (9.59 ± 0.88) (r = − 0.86; P = 0.03) and very ment of SMC in uterus with ICLC located as a network strong positive correlation (r = 0.95; P = 0.01) between between SMC is consistent with the immunohistochemi- OXTR density (0.52 ± 0.03) and EMG signal RMS cal observations in humans [9] and pigs [10]. (0.11 ± 0.01). Popescu et al. (2005) showed that ICLC establish close Last but not least, in IO there was strong negative contacts (gap junctions) with each other, SMC, nerve fi- correlation between ICLC density (4.12 ± 1.01) and bers and capillaries. These authors also stated that re- duration of pauses between bungles (2.72 ± 1.26) (r = productive hormones could influence ICLC function via − 0.81; P = 0.04) as well as between OXTR density Cx43-mediated mechanism [23, 24] and via OXTR and (0.90 ± 0.02) and duration of pauses between bursts PR [25]. Cretoiu et al. (2009) suggested that ICLC could (5.08 ± 0.10) (r = − 0.82; P = 0.04). act as steroid hormone sensors [12, 25]. In the current manuscript we present the data showing that ICLC, OXTR and PR are functionally connected in porcine Table 4 ICLC density (mean ± SD) in topographic regions of uterus and oviduct. This relation is particularly visible in reproductive tract during diestrus [15] and estrus CU, where significant correlations between ICLC and IFO IO UHT UH CU those receptors were demonstrated. Diestrus Furthermore, Hutchings et al. (2009) described the a b c c d mean density 2.16 4.12 3.10 3.17 1.22 stimulatory function of ICLC on neighbouring SMC as SD ±0.82 ±1.01 ±0.92 ±1.04 ±0.38 an acute effect of promoting uterine contraction. They a b c c d suggested that ICLC generate the subsequent calcium relative density 52.4% 100.0% 75.3% 77.1% 29.6% wave travelling at a relatively lower velocity towards the Estrus center of the smooth muscle fibers. [12]. In all porcine a b c c d mean density 2.31 5.35 4.23 3.96 1.09 specimens the ICLC were seen to be located mainly on SD ±0.03 ±0.53 ±0.50 ±0.12 ±0.68 the boundaries of smooth muscle bundles throughout a b c c d relative density 43.1% 100.0% 79.0% 74.0% 20.4% the myometrium and formed the cell network similar to Immunofluorescent labeling; Density defined as cells number in 20 fields of view that found in human tissues [26]. The presence of high and relative density as percentage of positive cells relative to the highest density correlations between steroid hormone receptors, ICLC region. The different letters in superscript indicate groups with statistically significant differences (P < 0.05): diestrus/estrus and regions of reproductive tract and parameters of EMG signal suggests that in vivo Domino et al. BMC Veterinary Research (2018) 14:176 Page 7 of 9 ICLC could behave as hormone sensors controlling the positive cells, were predominantly negative for ER and uterine contraction and playing the role of major pace- PR at the fourth passage [11]. It has to be noted, how- maker, in a similar way to that found in humans [27]. In ever, that ER and PR expression on human uterine SMC porcine specimens this network can be observed on in culture significantly decreases with every passage and structural and cellular level; moreover, it seems to play a is much lower compared to cells in situ [34]. functional role, but for this conclusion further research Recent studies have stated that the propagation of is necessary. The results presented in current manu- myoelectrical activity in uterus is linear and that speed script support the functional role of ICLC in regulating of this propagation can be measured but have not pre- uterine contractile activity. sented any measurements showing the speed of EMG Spontaneous myoactivity, as well as contractility of the signal propagation along porcine reproductive tract [35, cycling, nonpregnant uterus in pigs has been studied 36]. We propose to divide the propagation speed in por- using nonsurgical, open-end catheter technique [28] and cine non-pregnant uterus into one of three distinctive long-term electromyography combined with the telem- categories: SBMR, RBMR and RAMR; there is, however, etry recording system [18, 29, 30]. The induced myome- no evidence for any differences in any of those categor- trial activity during estrus in sow has also been studied ies between estrus and diestrus. due to clenbuterol, cloprostenol [28], oxytocin [4] and Recent studies show that in mice [37] and rats [38] semen effect [31]. Similarly, to recent studies, we sup- contractile activity is predominantly initiated from IO posed that in porcine uterus and oviduct there are cer- and UHT and it is the dominant peacemaking site of re- tain motility patterns that are more prevalent at each productive tract. Gajewski et al. (2001) suggested that phase of the cycle. Signal parameters and its propagation also in pigs both UHT act as tentative anatomical re- depend on properties of wave propagation medium. gions with the strong pacemaker activity [30]. Our find- Current report is, to our knowledge, the first to ap- ings support this hypothesis in porcine specimen in proach the problem of complex signal propagation estrus state. In both estrus and diestrus the highest medium, such as syncytium of myometrium [32], by par- density of ICLC was found in the IO and UHT. At allel indirect research (in vivo EMG signal analyzing) as the same time the EMG signal parameters were sig- well as direct research (in situ cellular structure analyz- nificantly “sharper” (higher amplitude and RMS with ing) in pigs. Significant difference in properties of the lower duration of electrical activity) in estrus com- propagation medium in estrus and diestrus was reported pared to diestrus in oviductal region, similar to recent [3]. Bower (1974) suggested, that in sows uterine con- studies [18, 39]. tractions occur along uterine horns in two opposite di- According to Radhakrishanan et al. (2000), stimuli rections, tubo-cervical and cervico-tubal, although the for SMC originates from ICLC, or neighboring myo- tubo-cervical direction appeared to be predominant [33]. cytes and is affecting surrounding and distant (via Our results suggest that EMG signals in porcine uterus processes creating network) cells organizing syncyt- are propagated relatively uniformly in both cervico-tubal ium [40]. Frequency, amplitude and duration of con- and tubo-cervical directions, with no differences be- tractile wave depend upon number of stimulated tween estrus and diestrus. In estrus, when EMG signal simultaneously cells [41]. Strong correlations stated was propagating in tubo-cervical direction, there was in IO and UHT between time-dependent variables strong trend for increase of signal energy, amplitude and (duration and pause between bungles and bursts, as duration of electrical activity. However, in cervico-tubal well as RMS) suggest regulating function of duration direction these values were rather decreasing. These ten- of myoelectric activity of those parts. It may be in- dencies were not observed in diestrus, which may sug- ferred from that findings that IO and UHT deter- gest participation of specific modulators, dependent on mines the rhythm of uterus contractility. This theory hormonal status, that are controlling and connecting is confirmed with strong correlation between ICLC myometrium activity. In a recent study there was specu- density and duration of bursts and pauses between lation about ICLC involvement in myometrial contractil- bungles. Region IO/UHT may have a pacemaker ity regulation as steroid hormone ‘sensors’ [11, 12, 25, function, however this thesis requires further investi- 34]. This is consistent with our results and the hypoth- gation. In human studies Andersen and Barclay esis that there may be a functional connection between (1995) suggested that action potential may be gener- ICLC and endocrine regulation of reproductive system. ated in random pacemaker cells in reproductive sys- This hypothesis is based on structural relationships, dir- tem, however Popescu et al. (2007) stated that IO is ect contact of ICLC processes with blood vessels and possibly responsible for contractility stimulation in nerve terminals observed in microscopic studies [23, 24] rats and humans [24, 42]. In our research we sup- and the distribution of ER and PR [11]. Under in vitro port hypothesis that analogous mechanism is func- conditions the uterine myocytes, in contrast to c-kit tional in sows. Domino et al. BMC Veterinary Research (2018) 14:176 Page 8 of 9 ICLC density in UH and CU was relatively lower than Acknowledgements This work was conducted in the Veterinary Research Centre WULS (VRC) and in IO and UHT, nonetheless EMG signal parameters dif- the Center for Biomedical Research (CBR) supported by EFRR RPO WM 2007- ferences between estrus stage are similar and even more expressed. Those findings are convergent with results Funding obtained in vitro from mouse and rat specimens [36, This work was supported by the State Committee for Research Investigation 37]. Data obtained from rodent models suggested that grant NN308 077439, Narodowe Centrum Nauki, https://www.ncn.gov.pl/. contractions in UH and CU persisted following blockade Availability of data and materials of activity at the oviductal region [37]. Strong correlation All data generated or analyzed during this study are included in this published between amplitude, dominant frequency and OXTR article. If any additional material used and/or analysed during the current study is density, as well as between PR density and dominant fre- required, these are available from the corresponding author on reasonable request. quency indicates possibility for strengthening or weaken- Authors’ contributions ing signal generated in UHT during its propagation. ZG, MD planned and conducted the experiments. MD, BP performed signal Both force of contraction (amplitude, RMS) and contrac- and data analysis. MD, MG, MS wrote the draft of the paper. ZG, MD, TJ, MS wrote the paper. All authors read and approved the final manuscript. tion duration (duration and pauses between bungles and bursts) are probably regulated via receptors for repro- Ethics approval and consent to participate ductive system hormones. Oxytocin demonstrates higher The protocol was approved by the III Local Ethical Committee on Animal Testing in Warsaw (Permit Number: 71/2009, from 19.11.2009) on behalf of activity than progesterone, which is consistent with bio- the National Ethical Committees on Animal Testing. logical outcome of those hormones. It is thought that ef- fect of oxytocin on contractility is stronger than Competing interests The authors declare that they have no competing interests. progesterone [43, 44], which is compatible with our findings. Publisher’sNote In gastrointestinal tract frequency of slow waves in Springer Nature remains neutral with regard to jurisdictional claims in published smooth muscles is regulated by ICC, while amplitude maps and institutional affiliations. depends on neuroendocrine activity [45]. 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Uterine EMG activity in the non-pregnant sow during estrous cycle

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Medicine & Public Health; Veterinary Medicine/Veterinary Science; Zoology; Transgenics
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Abstract

Background: Uterine myoactivity is crucial for successful reproductive performance of the sow. Spontaneous contractions of the uterus are strictly controlled and coordinated. Uterine electromyographic (EMG) activity undergoes hormonal regulation with rapid and long-term effects. What is more, interstitial Cajal-like Cells (ICLC) appear essential for smooth muscle contractility in the reproductive tract where they are suspected to be playing a major role in generating, coordinating, modulating and synchronizing slow triggering waves. The aim of this study was to investigate the myoelectrical activity of sow’s uterus during estrus cycle. Results: Study was conducted on 10 Polish Landrace sows. Propagation mechanisms and their connection with the uterine EMG activity were considered in correlation with expression of c-kit, progesterone and oxytocin receptors of the non-pregnant sow. ICLC were labeled with antibody directed against c-kit receptor and visualized by confocal microscopy and scanning cytometer for positive cells percentage assessment. EMG signal was recorded directly from the myometrium with telemetry transmitters and electrodes located in different topographic regions of reproductive tracts. The stages of estrus cycle were determined by monitoring levels of luteinizing hormone, progesterone and estrogen with radioimmunoassays. Significant differences of the EMG signal parameters between diestrus and estrus and the correlations with density of labelled receptors were demonstrated. Moreover, the electrophysiological studies indicated that ICLC in the myometrium in the tip of uterine horn may participate in the regulation of slow waves duration and frequency. Conclusions: The pattern of EMG signal propagation in the wall of the non-pregnant porcine uterus occurs in an orderly, bidirectional fashion and at distinctive speed, with no differences between diestrus and estrus. Keywords: EMG activity, Direction, Speed, Estrous cycle, Sow, Myometrium Background relaxation are based on a number of intracellular molecu- Reproductive performance of the sow is a critical compo- lar processes (i.e. rise in intracellular calcium increase in nent of profitable production. Spontaneous contractions myosin phosphorylation; increase in myosin light-chain of uterus must be controlled and coordinated for the suc- kinase activity) [1]. In myometrium, an increase in cess of various reproductive functions. Suitable uterine myosine phosphorylation accompanies spontaneous contractility is involved in the transport of gametes and and hormone-induced contractions. Spontaneous and embryo implantation. Unsuitable uterine contractility may agonist-induced relaxation is accompanied by decrease in lead to ectopic pregnancies, miscarriages, embryonic loss myosin light-chain phosphorylation [2]. The long-term and abnormalities of puerperium. effects concern hormonal regulation (including Uterine electromyographic (EMG) activity comes under reproductive hormones) of components of intracellular systems hormonal regulation due to acute and long-term effects. at the level of the plasma membrane (i.e. receptor-operated, The acute effects promoting uterine contraction and voltage-operated, second-messenger-operated, GTP-binding protein (Guanosine-5′-triphosphate-binding protein), gated ion channels) [1]. * Correspondence: zgajewski@supermedia.pl Department of Large Animal Diseases with Clinic, Veterinary Research Centre It is generally accepted that progesterone, estrogens and Center for Biomedical Research, Faculty of Veterinary Medicine, Warsaw and oxytocin are key regulators of uterine contraction University of Life Sciences (WULS – SGGW), Nowoursynowska 100, 02-797 [3, 4]. Progesterone promotes sustained myometrial Warsaw, Poland © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Domino et al. BMC Veterinary Research (2018) 14:176 Page 2 of 9 relaxation, estrogens and oxytocin favor myometrial con- generated at cellular and organ level, propagation mech- tractility and excitability [5]. Recent studies have sug- anisms and their correlation with the uterine EMG gested that contractility in spontaneously active organs signal recorded internally from the myometrium and ex- occurs as an intrinsic property of the muscle [6]. At the pression of c-kit, PR and OXTR of the non-pregnant sow. plasma membrane level, progesterone interacts with Although a precise role for myometrial Cajal-like intersti- membrane-associated receptors (progesterone receptors tial cells has not yet been identified, this study presents re- - PR) to directly modulate intracellular calcium and sults, that may support understanding of functional role cyclic adenosine monophosphate levels. Progesterone of ICLC in regulating uterine contractile activity. also indirectly inhibits estrogen-induced oxytocin recep- tors (OXTR) expression. At the plasma membrane level Methods oxytocin also interacts with membrane-associated recep- Animals tors and induces inositol triphosphate production and The experiment has been conducted on 10 mature Ca2+ mobilization [5]. Polish Landrace sows (n = 10) according to protocol ap- Based on their gastrointestinal (GI) tract investiga- proved by the III Local Ethical Committee on Animal tions, Torihashi et al. (1999) have proposed a population Testing in Warsaw (Permit Number: 71/2009, from of independent interstitial cells as pacemakers of con- 19.11.2009) on behalf of the National Ethical Committees tractile rhythm. on Animal Testing. Spontaneous uterine activity in In GI the interstitial cells of Cajal (ICC) create a net- non-pregnant state was recorded by the combination of work which initiates and propagates the slow waves. three electrodes connected to 3-channel transmitter used Moreover, cells with morphology and antigenicity similar in large animals [15]. The experiment started with surgery to ICC have been found outside the gastrointestinal tract in the diestrus and the estrus began on average between and named interstitial Cajal-like cells (ICLC). The ICLC day 7 and 12 after surgery when the highest quality EMG appear essential for smooth muscle contractility in the signal was collected. One registration period lasted 5 to urinary and reproductive tract where they generate, co- 6 weeks with 1.87 ± 0.40 estrus cycles occurring during ordinate, modulate and synchronize slow triggering this time. Surgery was carried out under general waves [7]. The discovery of the c-kit receptor (type III anesthesia and the telemetry EMG recording method was tyrosine kinase receptor; CD117) as a marker of ICLC performed. According to the standard protocol [16] allows for the recognition of these cells under light and animals were premedicated with an intramuscular injec- confocal microscopes [8]. The presence of ICLC located tion of azaperone (Stresnil, 3 [mg/kg b.wt.], IM, Janssen among smooth muscle cells (SMC) were demonstrated Pharmaceutica) and then catheter was inserted into the in non-pregnant human [9] and porcine [10] myome- auricular vein. General anesthesia was achieved with trium. Moreover, the presence of estrogen, progesterone combined administration of medetomidine (Cepetor, 1 and oxytocin receptors was demonstrated in the nuclei [mg/kg b.wt.], IV, CP- Pharma Handelsges), butorphanol of ICLC in human myometrium [11, 12]. (Butomidor, 0,2 [mg/kgb.wt.], IV, Ricgter Pharma AG), Anatomical structure of uterus with billions of SMC ketamine (Bioketan, 3[mg/kg b.wt.], IV, Vetoquinol comprising myometrium interacting in a complex man- Biowet) and propofol (Propofol, 2 ± 4 [mg/kg b.wt.], IV, ner in longitudinal and circular muscle layers inter- Pfizer). The telemetry transmitter TL10M3-D70-EEE twined with the network of pacemaker cells (ICLC) (DSI, USA) was surgically positioned between abdominal must be considered in myoactivity signal propagation muscles and electrodes sutured into different topographic analysis. Individual electrical activities within the myo- regions of reproductive tract. In experiment 1: right uter- metrial tissue may differ in speed and direction. A single ine horn (RUH-channel 1), corpus uteri (CU-channel 2) electrical activity (burst) can initiate a myometrial con- and the left uterine horn (LUH-channel 3) surfaces with traction but multiple, coordinated activities (bungle) are 17 cm distance between electrodes and in experiment 2: needed for powerful and sustained contractions [1, 13]. isthmus of oviducts (IO-channel 1), right uterine horn tip SMC contract when the action potentials reaches a (RUHT-channel 2), right uterine horn (RUH-channel 3) depolarization threshold and generates an electromag- surfaces with 17 cm distance between electrodes 2 and 3. netic field, possible to measure as voltage. Therefore, When pigs recovered from surgery analgesic - meloxicam electromyography (EMG) is the most accurate method (Metacam 0.4 [mg/kg BWT], IM, Boehringer Ingelheim) to measure action potential changes leading to syn- and anti-microbial - cefquinom (Cobactan, 2.0 [mg/kg chronous contraction on the organ level [14]. BWT], IM, Intervet) had been administered for 5 days. The primary objective of this paper is to discuss the Directly after surgery, one day before the start of registra- reproductive phenomenon, which is the myoelectrical tion, the EMG signal quality was inspected. At day 3 to 5 activity of sow uterus during estrus cycle. We considered after surgery the disturbance-free signal was obtained and physiological modeling of uterine electrical activity the regular registrations was carried during next 5 to Domino et al. BMC Veterinary Research (2018) 14:176 Page 3 of 9 6 weeks. The EMG signals were collected 10 h daily al- bungle propagation was evaluated basing on the time ways in the same time periods. Obtained analog signal elapsed for highly synchronized signals to move from was digitalized and sent to the telemetric receiver (DL10 the first electrode to the second one. (DSI, USA)). The signal was acquired with a 3-channel transmitter PowerLab (ADInstruments, Australia) with Microscopy analysis sampling frequency 100 Hz and archived for off-line ana- Samples were fixed in paraformaldehyde and embedded lysis. Pigs were euthanized at the end of the experiment, in paraffin using standard histology procedures. by Sodium Pentobarbital (Morbital 100.0 [mg/kg BWT], IV, Afterwards, samples were cut to 5 μm thin sections and Biowet Pulawy) in different stages of estrus cycle (half in es- stained with hematoxilin-eosine (H-E) using a standard trus (n = 5) and half in diestrus (n = 5)). Full-thickness protocol. Immunofluorescent labeling against CD117 tissue samples of muscle layer were obtained from uterus (c-kit), progesterone receptor (PR) and oxytocine recep- (corpus-CU, middle of the horn-UH, horn tip-UHT) tor (OXTR) was performed. For the assessment of and oviducts (isthmus-IO, infundibulum-IFO) of all immunofluorescent reaction (CD117) specificity, porcine pigs (n =10). ileum paraffin blocks were used as positive control (PC), while staining protocols, with the primary antibody EMG data analysis omitted, were used as negative control (NC). The EMG signals were digitally filtered with a band-pass Immunofluorescent labeling against CD117 (c-kit), PR filter [5–50 Hz]. Mean and linear trends were removed and OXTR was performed using a published staining [17]. Uterine contractions were defined as series of elec- protocol [8, 15]. Antigens were retrieved through citrate trical potentials with amplitude above 5 μV and a dur- buffer heating, washing with PBS-buffer and blocked with ation longer than 3 s, separated from each other by the BSA to minimize non-specific antibody binding. Slides time period no shorter than 5 s. Any new electrical ac- were then incubated with antibodies: anti-CD117 (goat tivity after 5 s was interpreted as a subsequent contrac- polyclonal anti-human CD117; RD System, USA; AF332; tion [18]. Contraction was described using burst and 120 min, room temperature; dilution 1:200), anti-PR bungle parameters. The EMG signal spectral content (mouse monoclonal anti-human Progesterone Receptor was analyzed in time and frequency domain features. In 1A6; PROGEN, Germany, no16077; 120 min, room time domain, the bursts represented single action poten- temperature; dilution 1:250), anti-OXTR (mouse mono- tials, while the bungles comprised of multiple action po- clonal anti-human hOXTR; R&D Systems, USA, tentials. Mean amplitude [mV], mean RMS (root mean MAB6616; 180 min, room temperature; concentration 15 square) [mV], duration of electrical activity [s], duration μg/ml). Cross-reactivity for above-mentioned antibodies of pauses [s], and number of bursts forming a bungle has been confirmed in previous studies [15, 22]. Polyclonal were analyzed. In frequency domain, means of Fourier Alexa Fluor 405 (chicken anti-mouse), Alexa Fluor 488 analysis (FFT–Fast Fourier transform), dominant fre- (chicken anti-mouse, chicken anti-goat), Alexa Fluor 660 quency (DF) [Hz] (the frequency at which most signal (chicken anti-mouse, chicken anti-goat) (Abcam, UK) energy was transmitted) was assessed for each data labeled antibodies (dilution 1:500, 120 min, room series. We used the Hamming window for the Fourier temperature) were used respectively to detect the analysis [19, 20]. The similarity measures were used to primary antibodies. Nuclei were counterstained with describe synchronization between EMG signals. Degree 7-Aminoactinomycin D (Sigma Aldrich, Poland) or of synchronization between three simultaneously re- HOECHST33342 (Sigma Aldrich, Poland). After labeling, corded data series (channels 1(x), 2(y), 3(z) in experi- the coverslips were mounted using mounting medium for ment 1) was estimated for two signal pairs (xy and yz) fluorescence microscopy. Immunofluorescence labeled using linear measures: the cross-correlation function cells were examined with confocal microscope (FV-500, (ƒx,y(l), ƒy,z(l)) and the cross-coherence function Olympus, Poland). Quantitative evaluation was performed (Cxy(ƒ), Cyz(ƒ)) [19, 21]. Similarity measures, based on using a scanning cytometer (SCAN^R, Olympus, Poland, the concept of time series data synchronization, allows magnification × 200). The positive cells density was signal propagation detecting in different topographic re- defined as a number of cells with appropriate immuno- gions. It was used to “identify” the frequency fingerprint phenotype and morphology in 20 fields of view (mean of the spatial bungle and determine, if the bungle density ± SD). Afterwards the relative density (%) was reached the other location during propagation. Locations calculated as percentage of positive cells relative to the of the first and second electrodes (on the x/y or y/z axis) highest density region. were used to measure highly synchronized signals, and the direction of bungle propagation was evaluated. The Hormone concentrations analysis second signal location in the time function pointed to During the surgery, a silicone cannula was inserted into cervico-tubal and tubo-cervical directions. The speed of the brachial vein to enable collection of blood samples. Domino et al. BMC Veterinary Research (2018) 14:176 Page 4 of 9 Each day during the experiment (from 7 days before to Table 1 The EMG signal parameters in different topographic regions of reproductive tract during diestrus and estrus 42 days after surgery) 7 mL sample of blood was collected every 4 h. All samples were used to determine EMG Diestrus Estrus stages of estrus based on luteinizing hormone (LH), Region parameter mean SEM mean SEM 1 a b 17β-estradiol (E2) and pregn-4-ene3,20-dione (P4) con- A [mV] 0.64 ±0.04 0.81 ±0.02 centrations. The serum LH concentrations were mea- 2 c d IO RMS [mV] 0.05 ±0.003 0.12 ±0.001 sured with noncommercial radioimmunoassay test [21]. 3 e ef D [s] 153.10 ±9.64 111.10 ±14.48 The sensitivity was 0.08 ng/mL (intra-assay CV < 6.7%, 1 a b A [mV] 0.71 ±0.03 0.99 ±0.04 interassay CV < 12.5%). The serum levels of E2 and P4 2 c d UHT RMS [mV] 0.09 ±0.002 0.12 ±0.002 were determined with RIA. The sensitivity for estradiol was 3 e f 5 pg/mL (intra-assay CV < 8.0%, interassay CV < 11.1%; D [s] 194.70 ±9.25 99.97 ±20.97 1 a g ESTR-US-CT; Cisbio assays, France) and for progesterone UH A [mV] 0.74 ±0.02 2.22 ±0.15 0.15 ng/mL (intra-assay CV < 5.6%, inter-assay CV < 8.8%; 2 c h RMS [mV] 0.10 ±0.001 0.26 ±0.02 KIP1458; DIAsource ImmunoAssays SA, Belgium). The 3 e i D [s] 221.60 ±25.53 33.30 ±2.73 stages of estrus cycle were determined by monitoring 1 a j CU A [mV] 0.57 ±0.02 4.24 ±0.19 hormone levels and divided into estrus (mean concen- 2 c k RMS [mV] 0.07 ±0.001 0.47 ±0.02 tration: E2 > 8 pg/ml, P4 < 1 ng/ml, LH > 4 ng/ml) and 3 e l diestrus (mean concentration: E2 < 2 pg/ml, P4 > 4 ng/ml, D [s] 209.70 ±22.92 42.60 ±3.23 1 2 3 LH < 1 ng/ml). A-amplitude, RMS-root mean square, D-duration of electrical activity. The different letters in superscript indicate the statistical significant differences (P < 0.05): diestrus/estrus and regions of reproductive tract Statistical analysis All statistical evaluation was performed by Graph-Pad InStat software (San Diego, USA), the level of statistical percentages (mean% ± SEM) of bungle propagated significance was set to P < 0.05, using specific tests: CU→ UH during diestrus (45.30 ± 3.27) and estrus Mann-Whitney test, Kruskal-Wallis test with Dunn’s (50.40 ± 1.95) and UH→ CU during diestrus (54.73 ± 3.25) multiple comparisons, one-way ANOVA test with Tukey’s and estrus (49.74 ± 1.95) in relation to the total number of multiple comparisons. The Pearson correlation coefficient highly synchronized uterine contraction signals. (r) and Spearman’s rank correlation coefficient (Sr) were The EMG signals propagated along the UH with three used to determine correlation between receptors (c-kit, significantly different (P < 0.0001), independent speeds: PR, OXTR) distribution and for selected EMG parameters SBMR (slow basic migration rhythm), RBMR (rapid in different regions of reproductive tract. basic migration rhythm) and RAMR (rapid accessory migration rhythm). There were no significant differences Results (P > 0.05) between EMG signal propagation speeds The bungle parameters were characterized in details in values along the UH and percentage of each speeds dur- three different topographic regions of the reproductive ing diestrus compared to estrus (Table 3). tract, with significant differences between diestrus and The cells with morphology similar to ICLC were estrus (Table 1). We demonstrate significantly higher shown by H-E staining, however the reliable amplitude and RMS (P < 0.0001) and lower duration of electrical activity (P < 0.0001) in estrus compared to di- Table 2 EMG signal propagation along uterus during diestrus and estrus in all uterine regions (UHT, UH, CU). We also estrus demonstrate significantly higher amplitude (P = 0.0003) The directions of EMG signal propagation 1 2 3 4 5 and RMS (P < 0.0001) in estrus compared to diestrus in LUH-CU RUH-CU UH(L,R)-CU CU-LUH CU-RUH CU-UH IO with no differences in duration of electrical activity. (L,R) There were no differences (P > 0.05) in amplitude, RMS Diestrus and duration of electrical activity in IO and uterus a a b a a b mean 24.51 30.20 54.73 24.51 20.82 45.30 (UHT, UH, CU) in diestrus, but in estrus, amplitude and RMS were significantly higher (P < 0.05) and duration of SEM ±2.27 ±1.07 ±3.25 ±0.53 ±3.75 ±3.27 electrical activity was significant lower (P < 0.05) in CU Estrus and UH compared to the oviductal region (IO, UHT). a a b a a b mean 26.60 23.13 49.74 27,32 23.11 50.40 The EMG signals were propagated along the UH in both cervico-tubal (CU→ UH) and tubo-cervical (UH→ CU) SEM ±1.55 ±0.35 ±1.95 ±0.70 ±1.15 ±1.95 directions during diestrus and estrus with no signifi- 1 2 3 4 5 6 LUH➔CU; RUH➔CU; UH(L,R)➔CU; CU➔LUH; CU➔RUH; CU➔UH(L,R). cant differences (P >0.05) (Table 2). Therewereno The different letters in superscript indicate the statistical significant differences significant differences (P > 0.05) between the (P < 0.05): diestrus/estrus and the directions of EMG signal propagation Domino et al. BMC Veterinary Research (2018) 14:176 Page 5 of 9 Table 3 EMG signal propagation speeds along uterus during in the cytoplasm for PR and intense at membranes and diestrus and estrus weak in the cytoplasm for OXTR (Fig. 2). The speeds of EMG signal propagation In recent study we demonstrated that the ICLC dens- 1 2 3 ity differed among different parts of the porcine repro- SBMR RBMR RAMR ductive tract during diestrus [15]. In present study we Diestrus assessed corresponding differences in estrus and com- a b c mean speed [mm/min] 1.25 2.47 7.07 pared our current results with previous data in one table SEM ±0.04 ±0.08 ±0.60 in order to test the dependency of density differences on d d d %of speeds 46.01 26.30 27.30 stages of estrus cycle (Table 4). Current results Estrus demonstrate that in estrus there is significantly higher a b c (P < 0.01) density of ICLC in the IO compared to UH mean speed [mm/min] 1.16 2.53 8.70 with no differences (P > 0.05) between UHT and UH. SEM ±0.05 ±0.10 ±0.67 We also demonstrate significantly lower density of ICLC d d d %of speeds 33.03 29.01 38.01 in IFO (P < 0.01) and in CU (P < 0.01) when compared 1 2 SBMR-slow basic migration rhythm; RBMR-rapid basic migration rhythm; 3 to IO. There were no significant differences (P > 0.05) RAMR-rapid accessory migration rhythm. The different letters in superscript indicate the statistical significant differences (P < 0.05): diestrus/estrus and the between density of ICLC in different topographic regions speeds of EMG signal propagation of the reproductive tract during diestrus [15] and estrus (Table 4). differentiation of m-ICLC from fibroblasts according to The correlations (Pearson’s r or Spearman’s rank) be- Hutchings et al. [12] criteria was possible in porcine spe- tween ICLC, PR, OXTR density (mean ± SD) and the cimen after immunofluorescence labeling. Therefore, the following EMG signal parameters: dominant frequency, histology approach was used only for initial screening amplitude, RMS, duration of bungles and bursts, dur- and to examine tissue architecture and the cells with an- ation of pauses between bungles and bursts, number of tigenicity similar to ICLC were demonstrated in all in bursts forming bungle (mean ± SEM) were calculated for examined parts of the porcine reproductive tract. The different topographic regions of the reproductive tract c-Kit-immunopositive nucleated cells that present char- during diestrus and described below. acteristic cell morphology (triangular, spindle-shaped, Strong positive correlation has been confirmed in CU star-like body with two or more, very long, moniliform (r = 0.86; P = 0.03) between ICLC density (1.22 ± 0.38) processes) were demonstrated in each topographic re- and OXTR density (0.53 ± 0.02) and strong, negative cor- gions of the reproductive tract. The presence of similar relation (Sr = − 0.71; P < 0.0001) between PR (0.20 ± 0.02) cells was confirmed in positive control-porcine ileum and number of bursts forming bungle (3.40 ± 0.40). used as reference material and so the c-kit positive cells In UH there was strong negative correlation (Sr = − 0.87; were considered as ICLC (Fig. 1). The lack of such cells P < 0.0001) between ICLC density (3.17 ± 1.04) and number was demonstrated in negative control. We demonstrated of bursts forming bungle (3.40 ± 0.24); strong positive cor- the presence of ICLC that were single-positive for c-kit relation (r =0.82; P = 0.04) between PR density (0.31 ± and double positive for c-kit/PR as well as c-kit/OXTR 0.02) and EMG signal dominant frequency (4.01 ± in all examined topographic regions. Double immuno- 0.21); strong negative correlation (r = − 0.86; P=0.03) be- fluorescence confirmed the same distribution of PR and tween OXTR density (0.61 ± 0.01) and EMG signal OXTR on c-kit positive cells: intense in nuclei and weak amplitude (1.18 ± 0.17); strong positive correlation Fig. 1 The representative images of presence of Cajal cells in uterine tissues (ICLC) and ileum (ICC). Fusiform cells with long processes and oval nucleus show morphology typical for Cajal cells (arrows). Legend: A. Muscular layer of UHT. Widefield microscopy of a H-E stained tissue section, lens magnification 40×; B. Muscular layer of UHT. Confocal microscopy following IF labeling, lens magnification 40×; C. Muscular layer of porcine ileum (PC - positive control). Confocal microscopy following IF labeling, lens magnification 63× Domino et al. BMC Veterinary Research (2018) 14:176 Page 6 of 9 Fig. 2 The representative images of IF labelling of c-kit (CD117), PR and OXTR. Legend: A. Muscular layer of UHT - Triangular ICLC positive for c-kit only; emission spectra of AF 488/Hoechst; B. Muscular layer of UHT - Star-like shape ICLC double positive for c-kit and PR intense at nuclear level; emission spectra of AF 488/AF 660/Hoechst; C. Muscular layer of UHT - Fusiform ICLC double positive for c-kit and OXTR intense at membrane level; emission spectra of AF 488/AF 660 /Hoechst. Confocal microscopy, lens magnification 63×, digital zoom 0.75–3.00 (r =0.85; P = 0.03) between OXTR density (0.61 ± 0.01) Discussion and EMG signal dominant frequency (4.40 ± 0.31) and In this study, we have thoroughly and systematically very strong positive correlation between OXTR density characterized the electrical activity of the porcine uterus (0.61 ± 0.01) duration of bungles (3.77 ± 0.46) (r = 0.99; throughout the estrus and diestrus. Our complex experi- P = 0.01) and duration of bursts (9.21 ± 1.54) (r = 0.97; ment combines parallel studies: on cellular level in situ P = 0.03). Strong negative correlations between OXTR and in vivo on organ level, in living organism, encom- density and duration of pauses between bungles (7.23 ± 1.54) passed by natural biological processes. In pigs, an activ- (r = − 0.94; P = 0.01) and duration of pauses between bursts ity of the myometrium seems to be myogenic in origin, (13.20 ± 2.73) (r = − 0.97; P = 0.03) were observed. similarly to described in Langendijk et al. (2002) reports, In UHT there was strong negative correlation between and to be driven by pacemaker activity of not only the ICLC density (3.10 ± 0.92) and duration of bungles uterine smooth muscle cells [23] but also ICLC similar (2.31 ± 0.36) (r = − 0.85; P = 0.03) as well as duration to these found in humans [12]. The anatomical arrange- of bursts (9.59 ± 0.88) (r = − 0.86; P = 0.03) and very ment of SMC in uterus with ICLC located as a network strong positive correlation (r = 0.95; P = 0.01) between between SMC is consistent with the immunohistochemi- OXTR density (0.52 ± 0.03) and EMG signal RMS cal observations in humans [9] and pigs [10]. (0.11 ± 0.01). Popescu et al. (2005) showed that ICLC establish close Last but not least, in IO there was strong negative contacts (gap junctions) with each other, SMC, nerve fi- correlation between ICLC density (4.12 ± 1.01) and bers and capillaries. These authors also stated that re- duration of pauses between bungles (2.72 ± 1.26) (r = productive hormones could influence ICLC function via − 0.81; P = 0.04) as well as between OXTR density Cx43-mediated mechanism [23, 24] and via OXTR and (0.90 ± 0.02) and duration of pauses between bursts PR [25]. Cretoiu et al. (2009) suggested that ICLC could (5.08 ± 0.10) (r = − 0.82; P = 0.04). act as steroid hormone sensors [12, 25]. In the current manuscript we present the data showing that ICLC, OXTR and PR are functionally connected in porcine Table 4 ICLC density (mean ± SD) in topographic regions of uterus and oviduct. This relation is particularly visible in reproductive tract during diestrus [15] and estrus CU, where significant correlations between ICLC and IFO IO UHT UH CU those receptors were demonstrated. Diestrus Furthermore, Hutchings et al. (2009) described the a b c c d mean density 2.16 4.12 3.10 3.17 1.22 stimulatory function of ICLC on neighbouring SMC as SD ±0.82 ±1.01 ±0.92 ±1.04 ±0.38 an acute effect of promoting uterine contraction. They a b c c d suggested that ICLC generate the subsequent calcium relative density 52.4% 100.0% 75.3% 77.1% 29.6% wave travelling at a relatively lower velocity towards the Estrus center of the smooth muscle fibers. [12]. In all porcine a b c c d mean density 2.31 5.35 4.23 3.96 1.09 specimens the ICLC were seen to be located mainly on SD ±0.03 ±0.53 ±0.50 ±0.12 ±0.68 the boundaries of smooth muscle bundles throughout a b c c d relative density 43.1% 100.0% 79.0% 74.0% 20.4% the myometrium and formed the cell network similar to Immunofluorescent labeling; Density defined as cells number in 20 fields of view that found in human tissues [26]. The presence of high and relative density as percentage of positive cells relative to the highest density correlations between steroid hormone receptors, ICLC region. The different letters in superscript indicate groups with statistically significant differences (P < 0.05): diestrus/estrus and regions of reproductive tract and parameters of EMG signal suggests that in vivo Domino et al. BMC Veterinary Research (2018) 14:176 Page 7 of 9 ICLC could behave as hormone sensors controlling the positive cells, were predominantly negative for ER and uterine contraction and playing the role of major pace- PR at the fourth passage [11]. It has to be noted, how- maker, in a similar way to that found in humans [27]. In ever, that ER and PR expression on human uterine SMC porcine specimens this network can be observed on in culture significantly decreases with every passage and structural and cellular level; moreover, it seems to play a is much lower compared to cells in situ [34]. functional role, but for this conclusion further research Recent studies have stated that the propagation of is necessary. The results presented in current manu- myoelectrical activity in uterus is linear and that speed script support the functional role of ICLC in regulating of this propagation can be measured but have not pre- uterine contractile activity. sented any measurements showing the speed of EMG Spontaneous myoactivity, as well as contractility of the signal propagation along porcine reproductive tract [35, cycling, nonpregnant uterus in pigs has been studied 36]. We propose to divide the propagation speed in por- using nonsurgical, open-end catheter technique [28] and cine non-pregnant uterus into one of three distinctive long-term electromyography combined with the telem- categories: SBMR, RBMR and RAMR; there is, however, etry recording system [18, 29, 30]. The induced myome- no evidence for any differences in any of those categor- trial activity during estrus in sow has also been studied ies between estrus and diestrus. due to clenbuterol, cloprostenol [28], oxytocin [4] and Recent studies show that in mice [37] and rats [38] semen effect [31]. Similarly, to recent studies, we sup- contractile activity is predominantly initiated from IO posed that in porcine uterus and oviduct there are cer- and UHT and it is the dominant peacemaking site of re- tain motility patterns that are more prevalent at each productive tract. Gajewski et al. (2001) suggested that phase of the cycle. Signal parameters and its propagation also in pigs both UHT act as tentative anatomical re- depend on properties of wave propagation medium. gions with the strong pacemaker activity [30]. Our find- Current report is, to our knowledge, the first to ap- ings support this hypothesis in porcine specimen in proach the problem of complex signal propagation estrus state. In both estrus and diestrus the highest medium, such as syncytium of myometrium [32], by par- density of ICLC was found in the IO and UHT. At allel indirect research (in vivo EMG signal analyzing) as the same time the EMG signal parameters were sig- well as direct research (in situ cellular structure analyz- nificantly “sharper” (higher amplitude and RMS with ing) in pigs. Significant difference in properties of the lower duration of electrical activity) in estrus com- propagation medium in estrus and diestrus was reported pared to diestrus in oviductal region, similar to recent [3]. Bower (1974) suggested, that in sows uterine con- studies [18, 39]. tractions occur along uterine horns in two opposite di- According to Radhakrishanan et al. (2000), stimuli rections, tubo-cervical and cervico-tubal, although the for SMC originates from ICLC, or neighboring myo- tubo-cervical direction appeared to be predominant [33]. cytes and is affecting surrounding and distant (via Our results suggest that EMG signals in porcine uterus processes creating network) cells organizing syncyt- are propagated relatively uniformly in both cervico-tubal ium [40]. Frequency, amplitude and duration of con- and tubo-cervical directions, with no differences be- tractile wave depend upon number of stimulated tween estrus and diestrus. In estrus, when EMG signal simultaneously cells [41]. Strong correlations stated was propagating in tubo-cervical direction, there was in IO and UHT between time-dependent variables strong trend for increase of signal energy, amplitude and (duration and pause between bungles and bursts, as duration of electrical activity. However, in cervico-tubal well as RMS) suggest regulating function of duration direction these values were rather decreasing. These ten- of myoelectric activity of those parts. It may be in- dencies were not observed in diestrus, which may sug- ferred from that findings that IO and UHT deter- gest participation of specific modulators, dependent on mines the rhythm of uterus contractility. This theory hormonal status, that are controlling and connecting is confirmed with strong correlation between ICLC myometrium activity. In a recent study there was specu- density and duration of bursts and pauses between lation about ICLC involvement in myometrial contractil- bungles. Region IO/UHT may have a pacemaker ity regulation as steroid hormone ‘sensors’ [11, 12, 25, function, however this thesis requires further investi- 34]. This is consistent with our results and the hypoth- gation. In human studies Andersen and Barclay esis that there may be a functional connection between (1995) suggested that action potential may be gener- ICLC and endocrine regulation of reproductive system. ated in random pacemaker cells in reproductive sys- This hypothesis is based on structural relationships, dir- tem, however Popescu et al. (2007) stated that IO is ect contact of ICLC processes with blood vessels and possibly responsible for contractility stimulation in nerve terminals observed in microscopic studies [23, 24] rats and humans [24, 42]. In our research we sup- and the distribution of ER and PR [11]. Under in vitro port hypothesis that analogous mechanism is func- conditions the uterine myocytes, in contrast to c-kit tional in sows. Domino et al. BMC Veterinary Research (2018) 14:176 Page 8 of 9 ICLC density in UH and CU was relatively lower than Acknowledgements This work was conducted in the Veterinary Research Centre WULS (VRC) and in IO and UHT, nonetheless EMG signal parameters dif- the Center for Biomedical Research (CBR) supported by EFRR RPO WM 2007- ferences between estrus stage are similar and even more expressed. Those findings are convergent with results Funding obtained in vitro from mouse and rat specimens [36, This work was supported by the State Committee for Research Investigation 37]. Data obtained from rodent models suggested that grant NN308 077439, Narodowe Centrum Nauki, https://www.ncn.gov.pl/. contractions in UH and CU persisted following blockade Availability of data and materials of activity at the oviductal region [37]. Strong correlation All data generated or analyzed during this study are included in this published between amplitude, dominant frequency and OXTR article. If any additional material used and/or analysed during the current study is density, as well as between PR density and dominant fre- required, these are available from the corresponding author on reasonable request. quency indicates possibility for strengthening or weaken- Authors’ contributions ing signal generated in UHT during its propagation. ZG, MD planned and conducted the experiments. MD, BP performed signal Both force of contraction (amplitude, RMS) and contrac- and data analysis. MD, MG, MS wrote the draft of the paper. ZG, MD, TJ, MS wrote the paper. All authors read and approved the final manuscript. tion duration (duration and pauses between bungles and bursts) are probably regulated via receptors for repro- Ethics approval and consent to participate ductive system hormones. Oxytocin demonstrates higher The protocol was approved by the III Local Ethical Committee on Animal Testing in Warsaw (Permit Number: 71/2009, from 19.11.2009) on behalf of activity than progesterone, which is consistent with bio- the National Ethical Committees on Animal Testing. logical outcome of those hormones. It is thought that ef- fect of oxytocin on contractility is stronger than Competing interests The authors declare that they have no competing interests. progesterone [43, 44], which is compatible with our findings. Publisher’sNote In gastrointestinal tract frequency of slow waves in Springer Nature remains neutral with regard to jurisdictional claims in published smooth muscles is regulated by ICC, while amplitude maps and institutional affiliations. depends on neuroendocrine activity [45]. 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BMC Veterinary ResearchSpringer Journals

Published: Jun 5, 2018

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