T2DM-elicited oxidative stress represses MTA3 expression in mouse Leydig cells.

Metastasis-associated protein 3 (MTA3) functions as a versatile coregulator in cancers and in physiological contexts. A predominant expression of MTA3 in interstitial Leydig cells (LCs) and its role as a local modulator of testicular steroidogenesis have recently emerged. Incubation with insulin decreased MTA3 expression in a concentration- and exposure time-dependent manner in LCs. This raises the possibility of additional endocrine actions of insulin in the direct control of MTA3 expression, which remains so far unexplored. Herein, we reported that type 2 diabetes mellitus (T2DM)-mediated inhibition of MTA3 was associated with an increase in testicular oxidative stress. In contrast, a gavage of the strong antioxidant melatonin effectively ameliorated oxidative stress and restored the expression of MTA3, but failed to change serum insulin levels in the diabetic mice with testosterone deficiency (TD). Using multiple biochemical approaches, we demonstrated that oxidative stress suppressed MTA3 expression via repression of nuclear receptor subfamily 4 group A member 1 (NR4A1)-mediated transactivation of MTA3 in mouse LCs. By contrast, ectopic expression of NR4A1 ameliorated oxidative stress-impaired MTA3 expression in LCs. By employing an effective in vivo gene transfer method with microinjection of lentiviral plasmids, we showed that replenishment of MTA3 expression in vivo partially restored testicular steroidogenesis and improved male fertility in diabetic mice with TD. Thus, we have unveiled a central regulatory hub, involving oxidative stress-impaired NR4A1-driven transactivation of MTA3 in stimulated LCs, as a potential mechanism regulating crosstalk between hyperinsulinemia and male infertility associated with TD.

N-myc downstream-regulated gene 4, up-regulated by tumor necrosis factor-α and nuclear factor kappa B, aggravates cardiac ischemia/reperfusion injury by inhibiting reperfusion injury salvage kinase pathway.

N-myc downstream-regulated gene 4 (NDRG4) is expressed weakly in heart and has been reported to modulate cardiac development and QT interval duration, but the role of NDRG4 in myocardial ischemia/reperfusion (I/R) injury remains unknown. In the present study, we analyzed the expression as well as potential function of cardiac NDRG4 and investigated how NDRG4 expression is regulated by inflammation. We found that NDRG4 was weakly expressed in cardiomyocytes and that its expression increased significantly both in I/R injured heart and in hypoxia-reoxygenation (H/R) injured neonatal rat ventricular myocytes (NRVMs). The increased NDRG4 expression aggravated myocardial I/R injury by inhibiting the activation of the reperfusion injury salvage kinase (RISK) pathway. Forced over-expression of NDRG4 inhibited RISK activation and exacerbated injury not only in I/R injured heart, but also in H/R treated NRVMs, whereas short hairpin RNA (shRNA)-mediated knock-down of NDRG4 enhanced RISK activation and attenuated injury. Upon injury, myocardial NDRG4 expression was induced by tumor necrosis factor-α (TNF-α) through nuclear factor kappa B (NF-κB), and we found that pre-treatment with inhibitors of either TNF-α or NF-κB blocked NDRG4 expression as well as I/R injury in vivo and H/R injury in vitro. Our study indicates that up-regulation of NDRG4 aggravates myocardial I/R injury by inhibiting activation of the RISK pathway, thereby identifying NDRG4 as a potential therapeutic target in I/R injury.

IL-17 mediates inflammatory reactions via p38/c-Fos and JNK/c-Jun activation in an AP-1-dependent manner in human nucleus pulposus cells.

BACKGROUND: Low back pain and sciatica caused by intervertebral disc (IVD) disease are associated with inflammatory responses. The cytokine interleukin 17 (IL-17) is elevated in herniated and degenerated IVD tissues and acts as a regulator of disc inflammation. The objective of this study was to investigate the involvement of IL-17A in IVD inflammatory response and to explore the mechanisms underlying this response. METHODS: Cells were isolated from nucleus pulposus (NP) tissues collected from patients undergoing surgeries for IVD degeneration. The concentrations of COX2 and PGE2, as well as of select proteins involved in the mitogen-activated protein kinase (MAPK)/activating protein-1 (AP-1) pathway, were quantified in NP cells after exposure to IL-17 with or without pretreatment with MAPK or AP-1 inhibitors. RESULTS: Our results showed that IL-17A increased COX2 expression and PGE2 production via the activation of MAPKs, including p38 kinase and Jun N-terminal kinase (JNK). Moreover, IL-17A-induced COX2 and PGE2 production was shown to rely on p38/c-Fos and JNK/c-Jun activation in an AP-1-dependent manner. CONCLUSION: In summary, our results indicate that IL-17A enhances COX2 expression and PGE2 production via the p38/c-Fos and JNK/c-Jun signalling pathways in NP cells to mediate IVD inflammation.

Different locations of RANTES and its receptors on mouse epididymal spermatozoa.

Our previous study showed that the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) originating from the mouse epididymis bound to the midpiece of luminal spermatozoa. The present study was undertaken to investigate the association between RANTES and epididymal spermatozoa and to determine whether the association is mediated by the RANTES receptors CCR1, CCR3 or CCR5. The use of reverse transcription polymerase chain reaction (RT-PCR), immunohistochemical staining and immunofluorescent staining demonstrated that RANTES secreted by apical and narrow cells of mouse epididymal ducts was associated with luminal spermatozoa. Flow cytometric analysis and immunofluorescent labelling revealed that the association between RANTES and spermatozoa of different regions weakened gradually as the spermatozoa moved along the epididymis. Moreover, CCR1, CCR3 and CCR5 were expressed in epididymal spermatozoa and located on the head of epididymal spermatozoa, while RANTES was generally located at the midpiece. In conclusion, RANTES and its receptors were not in the same sperm location, suggesting that RANTES binding to mouse epididymal spermatozoa is independent of CCR1, CCR3 and CCR5.

ADAMTS-7 exhibits elevated expression in cartilage of osteonecrosis of femoral head and has a positive correlation with TNF- α and NF- κ B P65.

ADAMTS-7 has been reported to exaggerate cartilage degeneration and to be associated with TNF-α and NF-κB signaling pathway. In this study we compared the expression of ADAMTS-7, TNF-α, and Phospho-NF-κB in patients with femoral neck fracture (FNF) and osteonecrosis of femoral head (ONFH) at different stages. We found that expression of ADAMTS-7, TNF-α, and Phospho-NF-κB was significantly upregulated in ONFH patients' articular cartilage and related to the pathogenesis of ONFH. Thus we conclude that ADAMTS-7 level appears to be positively associated with expression of TNF-α and Phospho-NF-κB P65 in cartilage, which may imply its association with cartilage destruction of ONFH.

Up-regulation of NDRG2 through nuclear factor-kappa B is required for Leydig cell apoptosis in both human and murine infertile testes.

Many pro-apoptotic factors, such as nuclear factor-kappa B (NF-κB) and Fas, play crucial roles in the process of Leydig cell apoptosis, ultimately leading to male sterility, such as in Sertoli cell only syndrome (SCO) and hypospermatogenesis. However, the molecular mechanism of such apoptosis is unclear. Recent reports on N-myc downstream-regulated gene 2 (ndrg2) have suggested that it is involved in cellular differentiation, development, and apoptosis. The unique expression of NDRG2 in SCO and hypospermatogenic testis suggests its pivotal role in those diseases. In this study, we analyzed NDRG2 expression profiles in the testes of normal spermatogenesis patients, hypospermatogenesis patients, and SCO patients, as well as in vivo and in vitro models, which were Sprague-Dawley rats and the Leydig cell line TM3 treated with the Leydig cell-specific toxicant ethane-dimethanesulfonate (EDS). Our data confirm that NDRG2 is normally exclusively located in the cytoplasm of Leydig cells and is up-regulated and translocates into the nucleus under apoptotic stimulations in human and murine testis. Meanwhile, transcription factor NF-κB was activated by EDS administration, bound to the ndrg2 promoter, and further increased in expression, effects that were abolished by NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC). Furthermore, siRNA knock-down of ndrg2 led to increased proliferative or decreased apoptotic TM3 cells, while over-expression of ndrg2 had the reverse effect. This study reveals that ndrg2 is a novel gene that participates in Leydig cell apoptosis, with essential functions in testicular cells, and suggests its possible role in apoptotic Leydig cells and male fertility.

Demethylenetetrahydroberberine protects dopaminergic neurons in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a multifactorial disorder of the nervous system where a progressive loss of dopaminergic neurons exist. However, the pathogenesis of PD remains undefined, which becomes the main limitation for the development of clinical PD treatment. Demethylenetetrahydroberberine (DMTHB) is a novel derivative of natural product berberine. This study was aimed to explore the neuroprotective effects and pharmacological mechanism of DMTHB on Parkinson's disease using C57BL/6 mice. A PD model of mice was induced by administration of MPTP (20 mg·kg-1) and probenecid (200 mg·kg-1) twice per week for five weeks. The mice were administered with DMTHB daily by gavage at the dose of 5 and 50 mg·kg-1 for one- week prophylactic treatment and five-week theraputic treatment. The therapeutic effects of DMTHB were evaluated by behavior tests (the open field, rotarod and pole tests), immunohistochemical staining of tyrosine hydroxylase (TH), Nissl staining and biochemical assays. The molecular mechanisms of DMTHB on the key biomarkers of PD pathological states were analyzed by Western blot (WB) and qRT-PCR. DMTHB treatment alleviated the behavioral disorder induced by MPTP-probenecid. Nissl staining and TH staining showed that the damage of dopaminergic neurons in the substantia nigra was remarkably suppressed by DMTHB treatment. Western blot results showed that the ratio of Bcl-2/Bax and TH increased, but the level of α-synuclein (α-syn) was remarkably reduced, which indicated that the apoptosis of dopaminergic neurons in mice was significantly reduced. The protein phosphorylation of p-PI3K, p-AKT and p-mTOR also increased about 2-fold, compared with the model group. Furthermore, qRT-PCR results demonstrated that the mRNA levels of pro-inflammatory cytokines, IL-1ß and TNF-α, were reduced, but the level of anti-inflammatory cytokine IL-10 increased after DMTHB treatment. Finally, the cellular assay displayed that DMTHB was also a strong antioxidant to protect neuron cell line PC12 by scavenging ROS. In this study, we demonstrated DMTHB alleviates the behavioral disorder and protects dopaminergic neurons through multiple-target effects includubg anti-apoptotic, anti-inflammatory and antioxidant effects.

Biocompatible Nano-Hydroxyapatites Regulate Macrophage Polarization.

Research on regulation of the immune microenvironment based on bioactive materials is important to osteogenic regeneration. Hydroxyapatite (HAP) is believed to be a promising scaffold material for dental and orthopedic implantation due to its ideal biocompatibility and high osteoconductivity. However, any severe inflammation response can lead to loosening and fall of implantation, which cause implant failures in the clinic. Morphology modification has been widely studied to regulate the host immune environment and to further promote bone regeneration. Here, we report the preparation of nHAPs, which have uniform rod-like shape and different size (200 nm and 400 nm in length). The morphology, biocompatibility, and anti-inflammatory properties were evaluated. The results showed that the 400 nm nHAPs exhibited excellent biocompatibility and osteoimmunomodulation, which can not only induce M2-phenotype macrophages (M2) polarization to decrease the production of inflammatory cytokines, but also promote the production of osteogenic factor. The reported 400 nm nHAPs are promising for osteoimmunomodulation in bone regeneration, which is beneficial for clinical application of bone defects.

Differential expression of N-Myc downstream regulated gene 2 (NDRG2) in the rat testis during postnatal development.

N-Myc downstream regulated gene 2 (NDRG2) is expressed in the testis of adult animals and is involved in cell differentiation and development. However, little is known about the expression pattern of NDRG2 in the testis during postnatal development. Here, we show that NDRG2 is consistently expressed in Leydig cells in the rat testis during postnatal development. However, its expression has also been detected at a high frequency in spermatogenic cells of the seminiferous tubules in young rats but at a much lower frequency in adult rats. Furthermore, high levels of NDRG2 expression have been found in methoxyacetic-acid-induced apoptotic germ cells, particularly at stages X-XIII of the seminiferous epithelium cycle of adult rats. Interestingly, high levels of NDRG2 expression have also been observed in spontaneously apoptotic germ cells in the seminiferous tubules of young and adult rats. Thus, the expression of NDRG2 in germ cells seems to alter during spermatogenesis. These findings suggest that NDRG2 regulates testicular development and spermatogenesis in rats and is involved in the physiological and pathological apoptosis of germ cells.

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells.

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2'-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation.

Interference with lactate metabolism by mmu-miR-320-3p via negatively regulating GLUT3 signaling in mouse Sertoli cells.

Disruption of the nursery function in Sertoli cells (SCs) by reducing lactate production, a preferred energy substrate for developed germ cells (spermatocytes and spermatids), is tightly associated with spermatogenic failure such as SC-only syndrome (SCOS). However, whether this complicated pathogenesis is regulated by certain miRNAs at the post-transcriptional level remain fascinating but largely unknown. Here we show for the first time that mmu-miR-320-3p was exclusively expressed in murine SCs and this expression was significantly induced in busulphan-treated murine testis. The most efficient stimulatory germ cell types for the induction of apoptosis-elicited mmu-miR-320-3p expression were meiotic spermatocytes and haploid spermatids. Functionally, forced expression of the exogenous mmu-miR-320-3p in SCs compromises male fertility by causing oligozoospermia and defection of sperm mobility. Mechanistically, mmu-miR-320-3p negatively regulates lactate production of SCs by directly inhibiting glucose transporter 3 (GLUT3) expression. Thus, dysregulation of mmu-miR-320-3p/GLUT3 cascade and consequently of lactate deficiency may be a key molecular event contributing the germ cell loss by SC dysfunction. Future endeavor in the continuous investigation of this important circulating miRNA may shed novel insights into epigenetic regulation of SCs nursery function and the etiology of azoospermia, and offers novel therapeutic and prognostic targets for SCOS.

Immunolocalization assessment of metastasis-associated protein 1 in human and mouse mature testes and its association with spermatogenesis.

AIM: To investigate the stage-specific localization of metastasis-associated protein 1 (MTA1) during spermatogenesis in adult human and mouse testis. METHODS: The immunolocalization of MTA1 was studied by immunohistochemistry and Western blot analysis. The distribution pattern of MTA1 in mouse testis was confirmed by using quantitative analysis of purified spermatogenic cells. RESULTS: The specificity of polyclonal antibody was confirmed by Western blot analysis. MTA1 was found expressed in the nucleus of germ cells, except elongate spermatids, and in the cytoplasm of Sertoli cells; Leydig cells did not show any specific reactivity. MTA1 possessed different distribution patterns in the two species: in humans, the most intensive staining was found in the nucleus of round spermatids and of primary spermatocytes while in mice, the most intense MTA1 staining was in the nucleus of leptotene, zygotene and pachytene spermatocytes. In both species the staining exhibited a cyclic pattern. CONCLUSION: The present communication initially provides new evidence for the potential role of MTA1 in mature testis. In addition, its distinctive expression in germ cells suggests a regulatory role of the peptide during spermatogenesis.

Histone deacetylase inhibitor trichostatin A induced caspase-independent apoptosis in human gastric cancer cell.

BACKGROUND: Histone deacetylase inhibitors (HDACIs) have been reported to induce apoptosis in cancer cells. The effects of trichostatin A (TSA) on gastric cancer cells have not been well characterized. This study was aimed to explore the effects and mechanisms of TSA on human gastric cancer SGC-7901 cells. METHODS: The cells were treated with TSA and analyzed by cell proliferation assay, Western blot, TUNEL assay, flow cytometry by fluorescein isothiocyanate (FITC) conjugated with Annexin V and PI staining, immunofluorescence analysis, analysis of subcellular fractionation, gene chips and real time polymerase chain reaction (PCR). RESULTS: TSA could inhibit cell growth and induced apoptosis in gastric cancer SGC-7901 cells through the regulation of apoptosis-related genes, such as Bcl-2, Bax and survivin. Further study indicated that the pan-caspase inhibitor z-VAD-fmk did not inhibit the apoptosis induced by TSA, and we did not observe the cleavage of poly ADP ribose polymerase (PARP) after TSA treatment too. In addition, apoptosis inducing factor (AIF) and EndoG were found to translocate from mitochondria to nucleus in the immunofluorescence assay and the Western analysis of subcellular fractionation confirmed the result of immunofluorescence assay. CONCLUSIONS: The apoptosis induced by TSA in gastric cancer SGC-7901 cells involves a caspase-independent pathway.

[Distribution characteristic of NDRG2 expression in human fetal tissues.].

NDRG2, one of the new N-Myc downstream-regulated gene (NDRG) gene families, is believed to be involved in cell growth event. However, the exact function is still unknown. Identification of the tissue or cell types expressing this gene in vivo will provide clues in clarifying its physiological roles. Using RT-PCR and Western blot, we analyzed the expression level of NDRG2 mRNA and protein in human fetal tissues from different gestational ages. The anti-NDRG2 monoclonal antibody, which has been proved to react specifically with NDRG2 protein, was further used to analyze the cellular location of NDRG2 protein in various human fetal tissues by immunohistochemistry. We found that NDRG2 expression was developmentally dynamic, being generally lower in the early stages of development and markedly increasing during the later stages. NDRG2 mRNA and protein distribution were generally consistent in heart and lung. One of the differences was that NDRG2 protein appeared later than mRNA in kidney. Another unmatched expression was found in liver. NDRG2 mRNA appeared later than protein in liver. In human fetal tissues at sixteen and twenty-eight weeks of gestation, NDRG2 protein immunoreactions could be seen in epithelium of small intestine, epithelium of large intestine, superficial layer of epidermis, whisker follicles, epithelium of small bronchus, hepatocytes, cardiac myocytes, thymus corpuscles and epithelium of renal tubule, and the immunoreactions in those tissues from twenty-eight weeks of gestation was stronger than that from sixteen weeks of gestation. In the present study, we demonstrate the expression pattern and cellular location of NDRG2 protein in a large set of human fetal tissues. This is the first demonstration of NDRG2 protein expression in human fetal tissues. Taken together, the results suggest that NDRG2 protein found in a variety of tissues is not a tissue-specific protein, and may play important roles in histogenesis and organogenesis.

Regular expression of discoidin domain receptor 2 in the improved adjuvant-induced animal model for rheumatoid arthritis.

OBJECTIVE: To investigate the expression of discoidin domain receptor 2 (DDR2) of fibroblast-like synovial cells in improved adjuvant-induced animal (AIA) model for rheumatoid arthritis (RA) and to provide evidence for DDR2's antagonist use clinically. METHODS: AIA was modified by administrating 0.1 mL of complete Freund's adjuvant (CFA, mixed with 5 mg Bacillus Calmette-Guerin vaccine/mL) into rats' right hind paws and 0.125 mL tumor necrosis factor-alpha (2 U/mL) into right ankles and subpatellar fatty tissue. The expression of DDR2 in fibroblast-like synovial cells was assessed using immunohistochemistry, immunofluorescence histochemistry, and in situ hybridization methods. Levels of anti-collagen II antibody were measured using enzyme-linked immunosorbent assay. RESULTS: Given the terms mentioned above, we found a more practical rat model, apparently decreasing immunization time (average 3-5 days). DDR2 can be detected upon the 15th day of immunization; expression gradually increased with time going on, and reaching a peak 35 days after immunization before gradually decreasing. Serum anti-collagen II antibody showed similar expression patterns as DDR2, but reached peak later than DDR2, about 40 days after immunization. CONCLUSION: Regular expression of DDR2 in animal models infers its important role in the pathological process of RA.

[Expression of Smad1 and Smad5 in the testis of infertile rats with kidney-yang deficiency].

OBJECTIVES: To study the expression of Smad1 and Smad5 in the testis of infertile rats with adenine-modeled kidney-yang deficiency and the pathological mechanism of infertility with kidney-yang deficiency, attempting to obtain experimental evidence for the prevention and treatment of male infertility. METHODS: Forty-eight 60 d male SD rats were divided randomly into 6 groups with 8 in each: 7 d, 14 d and 21 d kidney-yang deficiency groups, and 7 d, 14 d and 21 d control groups. The experimental rats had been fed with adenine (300 mg/kg) and the expression levels of Smad1 and Smad5 were measured with immunohistochemical SABC method at the 7th, 14th and 21st day. RESULTS: Smad1 immunoreactivity was mainly located in the spermatogonia, spermatocytes and spermatids, and the reactive substance distributed in cytoplasm with negative nuclei. Sertoli cells and Leydig cells were negative. Compared with the control, the expression level of Smad1 was decreased significantly at the 21st day (P < 0.05), but with no significant difference at the 7th and 14th day (P > 0.05). Smad5 immunoreactivity was mainly located in the spermatogonia and spermatocytes, and the reactive substance distributed in cytoplasm with negative nuclei. Compared with the control, the expression level of Smad5 was not significantly different at the 7th day (P > 0.05). The expression of Smad5 was negative at the 14th and the 21st day. CONCLUSION: The weaker expression of Smad1 and no expression of Smad5 may be one of the pathological mechanisms of infertility with adenine-modeled kidney-yang deficiency.

IL-17A enhances ADAMTS-7 expression through regulation of TNF-α in human nucleus pulposus cells.

ADMATS-7 is known to play an important role in the pathogenesis of various diseases, including cartilaginous diseases. IL-17A is an inflammatory cytokine detected in degenerative disc tissues. However, the interplay between IL-17A and ADMATS-7 in human disc degeneration is still unknown. Samples collected from 50 patients were divided into three groups according to MRI degeneration grading system score. Immunohistochemistry, RT-PCR and western Blotting were used to investigate the expression of ADAMTS-7 in NP tissues. Furthermore, a rat disc degeneration model was established, and the expression level of ADAMTS-7 was assayed using immunohistochemistry, RT-PCR and western Blotting. The human NP cells were cultured in the presence and absence of IL-17A stimulation. RNA extracts were collected, and real-time PCR was performed to determine the expression of ADAMTS-7. Moreover, ADAMTS-7 concentrations were detected in human NP cell culture supernatants by ELISA. After culturing NP cells with IL-17A (with or without Etanercept), ADAMTS-7 levels were detected in each group. ADAMTS-7 expression was dramatically elevated in both human and rat degenerative NP tissues compared with normal controls. The RT-PCR and ELISA results revealed that IL-17A could enhance the production of ADAMTS-7, while ADAMTS-7 expression dramatically decreased in the IL-17A + Etanercept group in comparison to the IL-17A alone group. Our results indicate the presence of ADAMTS-7 in human NP cells and imply its potential role in disc degeneration. Additionally, our results indicate that IL-17A induced ADAMTS-7 expression via TNF-α, which may form a molecular axis in human NP cells.

Abnormal Accumulation of Collagen Type I Due to the Loss of Discoidin Domain Receptor 2 (Ddr2) Promotes Testicular Interstitial Dysfunction.

BACKGROUND: Loss of functional allele for discoidin domain receptor 2 (Ddr2) results in impaired Leydig cell response to luteinizing hormone (LH), low testosterone production and arrested spermatogenesis in older male Ddr2slie/slie mice. However, the underlying mechanism responsible for this phenotype remains unknown. Herein, we reported for the first time that the deregulated expression of Ddr2 cognate ligand, namely collagen type I (COL1), may account for the disruption of the testicular steroidogenesis in Ddr2slie/slie mutant testes. METHODOLOGY/PRINCIPAL FINDINGS: Expression of Ddr2 increased gradually along postnatal development, whereas COL1 expression became negligible from adulthood onwards. In Ddr2slie/slie mutant testis, however, in contrast to the undetectable staining of Ddr2, COL1 expression was constantly detected, with the highest values detected during adulthood. In the experimental vasectomy model, Ddr2slie/slie mutant mice exhibited an early androgen deficiency than wild-type mice, along with the accumulation of fibrotic tissue in the interstitium. Functionally, ablation of endogenous Ddr2 resulted in a significant decrease of testosterone (T) level in TM3 cells in the presence of higher concentration of COL1 treatment. Conversely, overexpression of Ddr2 could help TM3 cells to maintain a normal testicular steroidogenesis even in the presence of high concentration of COL1. Additionally, attenuated expression of Ddr2 correlates to the deregulated level of serum T levels in human pathological testes. CONCLUSIONS: Abnormal accumulation of interstitial COL1 may be responsible for the steroidogenic dysfunction in Ddr2slie/slie mutant testes.

Localization of neurokinin B receptor in mouse gastrointestinal tract.

AIM: To observe the location of neurokinin receptor (NK3r) in the mouse gastrointestinal tract. METHODS: The abdomens of 8 male Kunming mice were opened under anaesthesia with sodium pentobarbital. The exposed gut organs were kept moisture and temperature at the same time. Then the esophagus,jejulum, ileum, and colon, etc were respectively cut and the segments from the stomach to the distal colon were opened along the mesenteric border. A circular 4mm-6mm enteric part(pieces of 1 cm(2) were to be prepared) and mucosa and submucosa were removed, then the longitudinal muscle layer was pulled off from the circular muscle layer under microphotograph. They were rinsed in 50 nmol x L(-1) potassium phosphate-buffered saline(PBS). Immunohistochemistry and immunoreactive fluorescence were used in the staining procedures. RESULTS: There was NK3r-Like(-Li) positive material on the smooth muscle cells of the esophagus, stomach, and intestines and other regions. The nerve cell bodies with immunoreactivity for NK3r were mainly distributed in the submucousal nerve plexus or myenteric nerve plexus of the gastrointestinal tract except for the esophagus, stomach and rectum. The reaction product was located on the surface of the nerve cell plasma. It was occasionally observed in the cell plasma endosomes, but was very weakly stained. Among the NK3-like positive neurons in the plexus,the morphological type in many neurons appeared like Dogiel II type cells. Some neuron cell bodies were big, having many profiles, some were long ones or having grading structure. Cell body diameter was about 10 microm-46 microm and 8 microm-42 microm in myenteric plexus and submucous plexus. CONCLUSION: This study not only described the distribution of neurokinin B receptor in the mouse gut, but also provided a morphological basis for deducing the functional identity of the NK3r-LI immunoreactivity neurons, suggesting the possibility that these neurons were closely related to gastrointestinal tract contraction and relaxing activity.