Cellular mechanism underlying leptin-induced anion secretion of rat epididymal epithelial cells.

BACKGROUND: A large number of studies have shown that leptin plays an important role in the regulation of fertility via the hypothalamus-pituitary-gonad axis. However, its peripheral function in epididymis was still elusive. OBJECTIVE: The purpose of this study was to determine the pro-secretion effect of leptin on the rat epididymal epithelium. MATERIALS AND METHODS: In the present study, real-time quantitative polymerase chain reaction, western blot, and immunohistochemical analysis were employed to detect the expression pattern of leptin receptors in rat epididymis. The pro-secretion effect of leptin on epididymal epithelial cells was measured by short-circuit current, and the prostaglandin E2 and cyclic adenosine monophosphate level was evaluated by enzyme-linked immunosorbent assay. RESULTS: We verified that the leptin receptor was located on the epididymal epithelium, with a relatively high expression level in corpus and cauda epididymis. Ussing chamber experiments showed that leptin stimulated a significant rise of the short-circuit current in rat epididymal epithelial cells, which could be abolished by the specific leptin receptor antagonist peptide Allo-aca, or by removing the ambient Cl- and HCO3 -. Furthermore, the leptin-stimulated short-circuit current response could be abrogated by blocking the apical cystic fibrosis transmembrane regulator or the basolateral Na+-K+-2Cl- cotransporter. Our pharmacological experiments manifested that interfering with the prostaglandin H synthase-2-prostaglandin E2-EP2/EP4-adenylate cyclase pathways could significantly blunt the cystic fibrosis transmembrane regulator-mediated anion secretion induced by leptin. The enzyme-linked immunosorbent assay demonstrated that leptin could induce a substantial increase in prostaglandin E2 release and cyclic adenosine monophosphate synthesis of primary cultured rat cauda epididymal epithelial cells. Our data also suggested that JAK2, ERK, and PI3K-dependent phosphorylation may be involved in the activation of prostaglandin H synthase-2 and the subsequent prostaglandin E2 production. CONCLUSIONS: The present study demonstrated the pro-secretion function of leptin in rat epididymal epithelium via the activation of cystic fibrosis transmembrane regulator and Na+-K+-2Cl- cotransporter, which was dependent on the paracrine/autocrine prostaglandin E2 stimulated EP2/EP4-adenylate cyclase pathways, and thus contributed to the formation of an appropriate microenvironment essential for sperm maturation.

Aerobic exercises regulate the epididymal anion homeostasis of high-fat diet-induced obese rats through TRPA1-mediated Cl- and HCO3- secretion†.

Aerobic exercises could improve the sperm motility of obese individuals. However, the underlying mechanism has not been fully elucidated, especially the possible involvement of the epididymis in which sperm acquire their fertilizing capacity. This study aims to investigate the benefit effect of aerobic exercises on the epididymal luminal milieu of obese rats. Sprague-Dawley male rats were fed on a normal or high-fat diet (HFD) for 10 weeks and then subjected to aerobic exercises for 12 weeks. We verified that TRPA1 was located in the epididymal epithelium. Notably, aerobic exercises reversed the downregulated TRPA1 in the epididymis of HFD-induced obese rats, thus improving sperm fertilizing capacity and Cl- concentration in epididymal milieu. Ussing chamber experiments showed that cinnamaldehyd (CIN), agonist of TRPA1, stimulated an increase of the short-circuit current (ISC) in rat cauda epididymal epithelium, which was subsequently abolished by removing the ambient Cl- and HCO3-. In vivo data revealed that aerobic exercises increased the CIN-stimulated Cl- secretion rate of epididymal epithelium in obese rats. Pharmacological experiments revealed that blocking cystic fibrosis transmembrane regulator (CFTR) and Ca2+-activated Cl- channel (CaCC) suppressed the CIN-stimulated anion secretion. Moreover, CIN application in rat cauda epididymal epithelial cells elevated intracellular Ca2+ level, and thus activate CACC. Interfering with the PGHS2-PGE2-EP2/EP4-cAMP pathway suppressed CFTR-mediated anion secretion. This study demonstrates that TRPA1 activation can stimulate anion secretion via CFTR and CaCC, which potentially forming an appropriate microenvironment essential for sperm maturation, and aerobic exercises can reverse the downregulation of TRPA1 in the epididymal epithelium of obese rats.

Mechanosensitive Piezo1 channel in rat epididymal epithelial cells promotes transepithelial K+ secretion.

The Piezo1 channel, a mechanosensitive channel that exhibit a preference for Ca2+, play multifarious physiological and pathological roles in the endothelium and epithelium of various tissues. However, the functional expression of Piezo1 channel in the epithelium of the male reproductive tract remains unknown. In the present study, the expression of Piezo1 channel in the rat epididymis was determined by real-time quantitative PCR, western blot and immunohistochemical analysis. Our data revealed that Piezo1 channel was located in the epithelial layer of the rat epididymis, with higher expression levels in the corpus and cauda regions. The pro-secretion function of Piezo1 channel was then investigated using short circuit current (ISC) and intracellular Ca2+ imaging techniques. Application of Yoda1, a selective Piezo1 channel activator, stimulated a remarkable decrease in the ISC of the epididymal epithelium. Pharmacological experiments revealed that the ISC response induced by Piezo1 channel activation was abolished by pretreating epithelial cells with the Yoda1 analogue, Dooku1, the selective mechanosensitive cation channel blocker, GsMTx4, or removal of basolateral K+. Meanwhile, we demonstrated that activation of Piezo1 channel triggered a robust Ca2+ influx in epididymal epithelial cells. The possible involvement of Ca2+- activated K+channels (KCa) in transepithelial K+ secretion was then evaluated. And that big conductance KCa (BK), but not small conductance or intermediate conductance KCa, mediated Piezo1-elicited transepithelial K+ secretion. Moreover, we demonstrated that NKCC and NKA were responsible for supplying substrate K+ during transepithelial K+ secretion. These data demonstrate that the activation of Piezo1 channel promotes BK-mediated transepithelial K+ secretion, and thus may plays an important role in the formation of a high K+ concentration in epididymal intraluminal fluid.

Activation of TRPV4 stimulates transepithelial K+ secretion in rat epididymal epithelium.

The maturation of sperms is dependent on the coordinated interactions between sperm and the unique epididymal luminal milieu, which is characterized by high K+ content. This study investigated the involvement of transient receptor potential vanilloid 4 (TRPV4) in the K+ secretion of epididymal epithelium. The expression level and cellular localization of TRPV4 and Ca2+-activated K+ channels (KCa) were analyzed via RT-PCR, real-time quantitative PCR, western blot and immunofluorescence. The functional role of TRPV4 was investigated using short-circuit current (ISC) and intracellular Ca2+ imaging techniques. We found a predominant expression of TRPV4 in the corpus and cauda epididymal epithelium. Activation of TRPV4 with a selective agonist, GSK1016790A, stimulated a transient decrease in the ISC of the epididymal epithelium. The ISC response was abolished by either the TRPV4 antagonists, HC067047 and RN-1734, or the removal of basolateral K+. Simultaneously, the application of GSK1016790A triggered Ca2+ influx in epididymal epithelial cells. Our data also indicated that the big conductance KCa (BK), small conductance KCa (SK) and intermediate conductance KCa (IK) were all expressed in rat epididymis. Pharmacological studies revealed that BK, but not SK and IK, mediated TRPV4-elicited transepithelial K+ secretion. Finally, we demonstrated that TRPV4 and BK were localized in the epididymal epithelium, which showed an increased expression level from caput to cauda regions of rat epididymis. This study implicates that TRPV4 plays an important role in the formation of high K+ concentration in epididymal intraluminal fluid via promoting transepithelial K+ secretion mediated by BK.

[Assessment of enhancement release of Oncorhynchus keta in Suifen River, Northeast China]. / 绥芬河大麻哈鱼增殖放流效果评估.

To evaluate the effects of enhancement release of Chum salmon (Oncorhynchus keta) in Suifen River, the homing Chum salmon was monitored in Dongning section of Suifen River from 2012 to 2017. A total of 462 samples were collected, 41 samples out of which were tagged indivi-duals which were cut off adipose fin before they were released. The recapture rate and the effects of Chum salmon enhancement release were analyzed and evaluated based on the releasing information. The results showed that the entire recapture rate of Chum salmon from 2010 to 2012 was 0.295%, and the input-output ratio of enhancement release was 1:2.87. Both the tagged and non-tagged groups were composed of 1+ to 5+ age individuals, with the average age being 3.93 and 3.63 years, respectively. The fork length at 50% individuals reached sexual maturity (L50) was estimated using a logistic moderating function, which was 53.13 cm and 49.89 cm for tagged and non-tagged groups, respectively. Results from ARSS analysis showed that there was no significant difference in fork length growth between tagged and non-tagged groups, but a significant difference in sexual maturity ratio. Our results confirmed the positive effects of enhancement release on recovery of Chum salmon resource and provided suggestions for the enhancement release efforts.

Influence of the three-dimensional printing technique and printing layer thickness on model accuracy.

OBJECTIVE: The accuracies of three-dimensional (3D) printed dental models using various digital light processing (DLP) and stereolithography (SLA) printers at different thicknesses were compared. MATERIALS AND METHODS: Based on digital dental models (originally digitized using R700; 3Shape, Copenhagen, Denmark), physical dental models were printed using three types of DLP printers: (1) EvoDent (UnionTec, Shanghai, China) with layer thicknesses of 50 µm and 100 µm; (2) EncaDent (Encashape, WuXi, China) with layer thicknesses of 20, 30, 50 and 100 µm; (3) Vida HD (Envisioned, Dearborn, MI, USA) with layer thicknesses of 50 and 100 µm. Models with the SLA printer Form 2 (Formlabs, Somerville, MA, USA) were printed with layer thicknesses of 25, 50 and 100 µm. All 22 printed models were converted to digital dental models using a D2000 model scanner (3 Shape, Copenhagen, Denmark) and compared three-dimensionally to the source files using Geomagic Qualify 12.0 (3D Systems, Rock Hill, SC, USA). RESULTS: The printing accuracy of all printers was higher at 50 µm. When the layer thickness was set at 100 µm, the printing speed and printing accuracy of DLP printer were both superior to those of the SLA printer. In all groups, the EvoDent 50 µm group had the highest consistency with the source files (mean absolute deviation of 0.0233 mm in the maxilla and 0.0301 mm in the mandible). While the accuracy of Form 2 100 µm group was the lowest (mean absolute deviation of 0.0511 mm in the maxilla and 0.0570 mm in the mandible). CONCLUSION: For the 3D printers studied, 50 µm was the optimum layer thickness for DLP technology, and the printing accuracy using SLA technology increased with decreasing layer thickness. The DLP technology also had higher printing accuracy at a layer thickness of 100 µm. EvoDent 50 µm had the highest and Form 2 100 µm the lowest printing accuracy.

[Tongjingling reduces ROS level and improves mitochondrial function in spermatogenic cells of varicocele rats with liver-qi stagnation].

OBJECTIVE: To explore the effects of the Chinese herbal compound Tongjingling (TJL) on the ROS level, mitochondrial membrane potential (MMP) and mitochondrial ultrastructure in spermatogenic cells in varicocele (VC) rats with liver-qi stagnation (LQS). METHODS: Totally, 72 male SPF Wistar rats were randomly divided into six groups of equal number, sham operation, VC+LQS model, low-, medium- and high-dose TJL, and L-carnitine (LC) control. The VC+LQS model was established by the Saypol method combined with tail-clamping. At 4 weeks after modeling, the rats in the low-, medium- and high-dose TJL groups were treated intragastrically with TJL at 0.6, 1.2 and 2.4 g/ml respectively, those in the sham operation and VC+LQS model groups with 0.9% saline, and those in the LC control group with LC oral liquid at 2.1 ml/kg, qd, for 8 weeks. Then the ROS level and MMP in the spermatogenic cells were measured by flow cytometry and the mitochondrial ultrastructure observed under the transmission electron microscope. RESULTS: The ROS level in the spermatogenic cells was significantly higher in the VC+LQS model and low-dose TJL groups than in the sham operation group (ï¼»62.72 ± 9.90ï¼½% and ï¼»52.25 ± 8.15ï¼½% vs ï¼»36.53 ± 5.25ï¼½%, P < 0.01), but remarkably lower in the medium-dose TJL (ï¼»41.65 ± 4.89ï¼½%), high-dose TJL (ï¼»39.60 ± 5.19ï¼½%) and LC control (ï¼»38.33 ± 7.46ï¼½%) than in the VC+LQS model group (P < 0.01). MMP in the spermatogenic cells was significantly lower in the VC+LQS model and low-dose TJL than in the sham operation group (ï¼»34.80 ± 20.18ï¼½% and ï¼»44.39 ± 19.60ï¼½% vs ï¼»60.24 ± 22.25ï¼½%, P < 0.01 and P < 0.05), but markedly higher in the medium-dose TJL (ï¼»50.62 ± 14.83ï¼½%), high-dose TJL (ï¼»52.42 ± 7.55ï¼½%) and LC control (ï¼»50.75 ± 15.65ï¼½%) than in the VC+LQS model group (P < 0.05). The mitochondria in the spermatogenic cells of the VC+LQS model rats exhibited swelling, vacuolization, rupture or disappearance of the mitochondrial cristae, and breakdown of the internal structure. The pathological injury of the mitochondrial structure was improved in different degrees in the TJL and LC groups. CONCLUSIONS: TJL can reduce the ROS level, increase MMP and improve pathological injury of the mitochondrial ultrastructure in the spermatogenic cells of VC rats with LQS, which may be the underlying molecular mechanism of TJL improving fertility in infertile VC patients.

[Soft tissue angle evaluation of fixed Twin-Block appliance treatment and tooth extraction treatment in skeletal Class II malocclusion].

PURPOSE: This cephalometric study was designed to evaluate the soft tissue profile effects of Twin-Block combined with straight wire appliances in the treatment of skeletal Class II malocclusion with mandibular retrognathia in growing individuals by angulation. METHODS: The sample comprised 31 growing individuals with skeletal Class II and mandibular retrognathia, which were divided into 2 groups. One group (18 patients) was treated with fixed Twin-Block combined with fixed appliance， the other group (13 patients) was only treated with fixed appliance after tooth extraction. Pretreatment and posttreatment lateral cephalograms were taken before and after Twin-Block treatment and after all the fixed appliance treatment. Angular cephalometric measurements of soft tissue were analyzed statistically. Intragroup and intergroup changes of the 2 groups were evaluated by Student's t test using SPSS 15.0 software package. RESULTS: Significant difference of FH-N'Pg', G-Pn-Pg', LiSi-SN and SnLs-SiLi were discovered in both groups (P<0.05). With Twin-Block combined with fixed appliance, N'-Sn-Pg', Sn-N'-Si and Si-N'-S changed significantly, while LsSn-SN and Z angle differed significantly before and after treatment with extraction and fixed appliance. After treatment of Twin-Block combined with fixed appliance, Sn-N'-Si decreased significantly, and the difference between the change of the 2 groups was significant. Meanwhile LsSn-SN was stable in combined treatment group while it decreased greatly after extraction. CONCLUSIONS: Compared with extraction with fixed appliance treatment, Twin-Block combined with fixed appliance treatment is more favorable in promoting lower facial profile in growing skeletal Class II individuals with mandibular retrognathia.

[Soft tissue linear evaluation of fixed Twin-block appliance treatment and tooth extraction in skeletal Class II malocclusion].

PURPOSE: This cephalometric study was designed to evaluate the soft tissue profile effects of Twin-block combined with straight wire appliances to treat growing adolescents with skeletal Class II malocclusion and mandibular retrognathia. METHODS: The samples comprised 31 growing individuals with skeletal Class II and mandibular retrognathia. They were divided into 2 groups. The study group (18 patients) was treated with fixed Twin-block combined with fixed appliance, while the control group (13 patients) was only treated with fixed appliance after tooth extraction. Pretreatment and posttreatment lateral cephalograms were taken before and after Twin-block treatment and after all the fixed appliance treatment. Linear cephalometric measurements of soft tissue were analyzed statistically. Intragroup and intergroup changes of the 2 groups were evaluated using SPSS 15.0 software package. RESULTS: Significant changes of G'-Pg', Li-1, Si-B and Ls-E line between before and after treatment were found in the study group, while Li-1, Ls-E line and Li-E line changed significantly in control group. The change of Li-E line was different between the 2 groups. CONCLUSIONS: Twin-block combined with fixed appliance treatment is better than extraction with fixed appliance treatment in promoting lower facial profile in cases of skeletal Class II malocclusion and mandibular retrognathia with growth potentials.

[Exogenous Sr2+ sedimentation on otolith of chum salmon embryos].

To explore the exogenous Sr2+ sedimentation on otolith of chum salmon embryos, chum salmon embryos were exposed to culture water contained Sr2+ at Sr2+ concentration of 50, 100, 200 or 400 mg . L-1 for 48 h to imitate Sr2+ sedimentation. After a culturing period of 12 d and 100 d, the otoliths of the chum salmon were taken to detect exogenous Sr2+ sedimentation with electro-probe microanalyzer (EPMA). The results showed that obvious deep red strontium signatures were produced in the otolith of chum salmon at different concentrations of Sr2+. The mean and extreme values of peak strontium area were not stable for the same Sr2+ dose, but the lowest of all the peak values was 35.1 times as much as that of control. Overall, the strontium value increased with the increase of Sr2+concentration. The strontium peak had no signs of abating after a culture period of 100 d. The results also showed that strontium was gradually deposited in the otolith, and had obvious hysteresis to immersion. Strontium sedimentation could also return to a normal level after the peak. These characteristics accorded exactly with the requirement of discharge tag technology, which indicated that exogenous Sr2+ was suitable in the marking of salmon otolith.

[Efficacy evaluation of fixed Twin-block appliance and tooth extraction in skeletal Class II malocclusion].

PURPOSE: To evaluate the skeletal and dentoalveolar effects of Twin-Block combined with straight wire appliances and tooth extraction in the treatment of skeletal Class II malocclusion with mandibular retrognathia in growing individuals. METHODS: The sample comprised 31 growing individuals with skeletal Class II and mandibular retrognathia, divided into 2 groups. One group (18 patients) was treated with fixed Twin-block combined with fixed appliance; the other group (13 patients) was only treated with fixed appliance after tooth extraction. Lateral cephalograms pretreatment and posttreatment were taken before and after Twin-block treatment and after all fixed appliance treatment. Pancherz's cephalometric measurements were analyzed statistically. Intragroup and intergroup changes of the two groups were evaluated by test using SPSS 15.0 software package. RESULTS: According to Pancherz's analysis, mandibular length increased, point Pg, mandibular incisors and first molars moved forward, maxillary incisors moved backward, overjet decreased (P<0.05) after treatment with Twin-block and fixed appliance. Within the rectification of anterior overjet, proportion of dentofacial factor was 39.5%, skeletal factor was 60.5%. Within the rectification of posterior relationship, proportion of dentofacial factor was 44.9%, skeletal factor was 55.1%. CONCLUSIONS: Twin-block combined with fixed appliance treatment can efficiently promote mandibular growth, restrict forward growth of maxilla to some extent, correct incisor and molar relationship and improve skeletal profile in growing skeletal Class II individuals with mandibular retrognathia.