Deficiency of leucine-rich repeat kinase 2 aggravates thioacetamide-induced acute liver failure and hepatic encephalopathy in mice.

BACKGROUND: Hepatic encephalopathy (HE) is closely associated with inflammatory responses. However, as a crucial regulator of the immune and inflammatory responses, the role of leucine-rich repeat kinase 2 (LRRK2) in the pathogenesis of HE remains unraveled. Herein, we investigated this issue in thioacetamide (TAA)-induced HE following acute liver failure (ALF). METHODS: TAA-induced HE mouse models of LRRK2 wild type (WT), LRRK2 G2019S mutation (Lrrk2G2019S) and LRRK2 knockout (Lrrk2-/-) were established. A battery of neurobehavioral experiments was conducted. The biochemical indexes and pro-inflammatory cytokines were detected. The prefrontal cortex (PFC), striatum (STR), hippocampus (HIP), and liver were examined by pathology and electron microscopy. The changes of autophagy-lysosomal pathway and activity of critical Rab GTPases were analyzed. RESULTS: The Lrrk2-/--HE model reported a significantly lower survival rate than the other two models (24% vs. 48%, respectively, p < 0.05), with no difference found between the WT-HE and Lrrk2G2019S-HE groups. Compared with the other groups, after the TAA injection, the Lrrk2-/- group displayed a significant increase in ammonium and pro-inflammatory cytokines, aggravated hepatic inflammation/necrosis, decreased autophagy, and abnormal phosphorylation of lysosomal Rab10. All three models reported microglial activation, neuronal loss, disordered vesicle transmission, and damaged myelin structure. The Lrrk2-/--HE mice presented no severer neuronal injury than the other genotypes. CONCLUSIONS: LRRK2 deficiency may exacerbate TAA-induced ALF and HE in mice, in which inflammatory response is evident in the brain and aggravated in the liver. These novel findings indicate a need of sufficient clinical awareness of the adverse effects of LRRK2 inhibitors on the liver.

tRNA-derived fragment tRF-1001: A novel anti-angiogenic factor in pathological ocular angiogenesis.

Transfer RNA-derived fragments (tRFs) are a novel class of non-coding RNA transcripts and play important roles in several physiological/pathological processes. However, the role of tRFs in ocular angiogenesis remains elusive. Herein, we investigate whether the intervention of tRF-1001 expression could suppress pathological ocular angiogenesis. The results show that the levels of tRF-1001 expression were reduced in the retinas of an oxygen-induced retinopathy (OIR) model, choroidal neovascularization model, and endothelial sprouting model in vitro. Increased tRF-1001 expression could suppress ocular angiogenesis and endothelial sprouting in vivo and reduce endothelial migration, specification, and sprouting in vitro. Mechanistically, tRF-1001 regulated endothelial angiogenic effects via tRF-1001/METTL3/RBPJ-MAML1 signaling. The levels of tRF-1001 expression were downregulated in the aqueous humor of age-related macular degeneration (AMD) patients. tRF-1001 upregulation could suppress AMD aqueous humor-induced endothelial sprouting and pathological angiogenesis. Collectively, tRF-1001 acts as an anti-angiogenic factor during ocular angiogenesis. Targeting tRF-1001-mediated signaling is a therapeutic option for ocular neovascular diseases.

METTL3-mediated N6-methyladenosine modification governs pericyte dysfunction during diabetes-induced retinal vascular complication.

Rationale: Microvascular complication is a major cause of morbidity and mortality among the patients with diabetes. Pericyte dysfunction is the predominant pathological manifestation of microvascular complication. N6-methyladenosine (m6A) serves as the most prevalent modification in eukaryotic mRNAs. However, the role of m6A RNA modification in pericyte dysfunction is still unclear. Methods: Quantitative polymerase chain reactions and western blots were conducted to detect the change of m6A RNA modification in pericytes and mouse retinas following diabetic stress. MTT assay, transwell migration assay, caspase 3/7 activity assay, calcein-AM/propidium iodide (PI) staining, and TUNEL staining were conducted to determine the role of METTL3 in pericyte biology in vitro. Retinal trypsin digestion, vascular permeability assay, and IB4-NG2 double immunofluorescent staining were conducted to determine the role of METTL3 in retinal pericyte dysfunction and vascular complication. RNA sequencing, RNA pull-down assays and immunoblots were conducted to clarify the mechanism of METTL3-mediated pericyte dysfunction and vascular complication. Results: The levels of m6A RNA methylation were significantly up-regulated in pericytes and mouse retinas following diabetic stress, which were caused by increased expression of METTL3. METTL3 regulated the viability, proliferation, and differentiation of pericytes in vitro. Specific depletion of METTL3 in pericytes suppressed diabetes-induced pericyte dysfunction and vascular complication in vivo. METTL3 overexpression impaired pericyte function by repressing PKC-η, FAT4, and PDGFRA expression, which was mediated by YTHDF2-dependent mRNA decay. Conclusion: METTL3-mediated m6A methylation epigenetically regulates diabetes-induced pericyte dysfunction. METTL3-YTHDF2-PKC-η/FAT4/PDGFRA signaling axis could be therapeutically targeted for treating microvascular complications.

Targeting circular RNA-MET for anti-angiogenesis treatment via inhibiting endothelial tip cell specialization.

Endothelial tip cell specialization plays an essential role in angiogenesis, which is tightly regulated by the complicated gene regulatory network. Circular RNA (circRNA) is a type of covalently closed non-coding RNA that regulates gene expression in eukaryotes. Here, we report that the levels of circMET expression are significantly upregulated in the retinas of mice with oxygen-induced retinopathy, choroidal neovascularization, and diabetic retinopathy. circMET silencing significantly reduces pathological angiogenesis and inhibits tip cell specialization in vivo. circMET silencing also decreases endothelial migration and sprouting in vitro. Mechanistically, circMET regulates endothelial sprouting and pathological angiogenesis by acting as a scaffold to enhance the interaction between IGF2BP2 and NRARP/ESM1. Clinically, circMET is significantly upregulated in the clinical samples of the patients of diabetic retinopathy. circMET silencing could reduce diabetic vitreous-induced endothelial sprouting and retinal angiogenesis in vivo. Collectively, these data identify a circRNA-mediated mechanism that coordinates tip cell specialization and pathological angiogenesis. circMET silencing is an exploitable therapeutic approach for the treatment of neovascular diseases.

CircRNA expression profile and functional analysis in retinal ischemia-reperfusion injury.

Retinal ischemia-reperfusion (I/R) is involved in the pathogenesis of many vision-threatening diseases. circRNAs act as key players in gene regulation and human diseases. However, the global circRNA expression profile in retinal I/R injury has not been fully uncovered. Herein, we established a murine model of retinal I/R injury and performed circRNA microarrays to identify I/R-related circRNAs. 1265 differentially expressed circRNAs were identified between I/R retinas and normal retinas. Notably, the detection of cWDR37 level in aqueous humor could discriminate glaucoma patients from cataract patients (AUC = 0.9367). cWdr37 silencing protected against hypoxic stress- or oxidative stress-induced retinal ganglion cell (RGC) injury. cWdr37 silencing alleviated IR-induced retinal neurodegeneration as shown by increased NeuN staining, reduced retinal reactive gliosis, and decreased retinal apoptosis. Collectively, this study provides a novel insight into the pathogenesis of retinal I/R injury. cWdr37 is a promising target for the diagnosis or treatment of I/R-related ocular diseases.

Role of METTL3-Dependent N6-Methyladenosine mRNA Modification in the Promotion of Angiogenesis.

Epigenetic alterations occur in many physiological and pathological processes. N6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic mRNAs. However, the role of m6A modification in pathological angiogenesis remains elusive. In this study, we showed that the level of m6A modification was significantly upregulated in endothelial cells and mouse retinas following hypoxic stress, which was caused by increased METTL3 levels. METTL3 silencing or METTL3 overexpression altered endothelial cell viability, proliferation, migration, and tube formation in vitro. METTL3 knockout in vivo decreased avascular area and pathological neovascular tufts in an oxygen-induced retinopathy model and inhibited alkali burn-induced corneal neovascularization. Mechanistically, METTL3 exerted its angiogenic role by regulating Wnt signaling through the m6A modification of target genes (e.g., LRP6 and dishevelled 1 [DVL1]). METTL3 enhanced the translation of LRP6 and DVL1 in an YTH m6A RNA-binding protein 1 (YTHDF1)-dependent manner. Collectively, this study suggests that METTL3-mediated m6A modification is an important hypoxic stress-response mechanism. The targeting of m6A through its writer enzyme METTL3 is a promising strategy for the treatment of angiogenic diseases.

SKLB1002, a potent inhibitor of VEGF receptor 2 signaling, inhibits endothelial angiogenic function in vitro and ocular angiogenesis in vivo.

Ocular angiogenesis is a major cause of severe vision loss, which can affect several parts of the eye, including the retina, choroid and cornea. Vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors have demonstrated great potential for treating ocular angiogenesis and SKLB1002 is a potent inhibitor of VEGF receptor 2 signaling. The present study investigated the effects of SKLB1002 administration on ocular angiogenesis. SKLB1002 administration did not show obvious cytotoxicity and tissue toxicity at the tested concentrations. In an alkali­burn corneal model, SKLB1002 administration significantly decreased the mean length and number of new corneal blood vessels. SKLB1002 administration significantly reduced endothelial cell proliferation, migration and tube formation in vitro. Mechanistically, SKLB1002 inhibited endothelial angiogenic functions by blocking the phosphorylation of ERK1/2, JNK and p38. Thus, selective inhibition of VEGFR­2 through SKLB1002 administration is a promising therapy for ocular angiogenesis.

Osthole: A Traditional Chinese Medicine for Ocular Anti-Angiogenic Therapy.

PURPOSE: Osthole is an agent isolated from Cnidium monnieri (L.) Cusson and has been used to treat several disorders. Corneal neovascularization is a sight-threatening condition associated with several inflammatory or infectious ocular disorders. In this study, we investigated the anti-angiogenic effects of osthole on corneal neovascularization and the underlying mechanism. METHODS: MTT assay, HE staining, and calcein-AM/propidium iodide staining was conducted to detect the toxicity of osthole in vitro and in vivo. Corneal neovascularization of ICR mice was induced by alkali burn and observed by a slit lamp microscopy on day 7 after alkali injury. EdU assay, Ki67 immunofluorescence assay, Transwell migration assay, and Matrigel assay were conducted to investigate the role of osthole in endothelial angiogenic effects in vitro. Western blots were conducted to investigate the anti-angiogenic mechanism of osthole in corneal neovascularization. RESULTS: Administration of osthole ranging from 0.05 to 25 µM had no detectable cytotoxicity or tissue toxicity in vivo and in vitro. Topical administration of osthole inhibited corneal neovascularization induced by alkali burn. Osthole decreased the proliferation, migration, and tube-formation of endothelial cells induced by VEGF. Osthole inhibited endothelial angiogenic functions through blocking the phosphorylation of ERK1/2, JNK, and p38. CONCLUSION: Our study provides evidence that osthole is a promising drug for the treatment of corneal neovascularization.

Hepatic Cyp1a2 Expression Reduction during Inflammation Elicited in a Rat Model of Intermittent Hypoxia.

BACKGROUND: Intermittent hypoxia (IH) is a key element of obstructive sleep apnea (OSA) that can lead to disorders in the liver. In this study, IH was established in a rat model to examine its effects on the expression of hepatic cytochrome P450 (CYP) and CYP regulators, including nuclear receptors. METHODS: Hematoxylin and eosin staining was conducted to analyze the general pathology of the liver of rats exposed to IH. The messenger RNA (mRNA) expression levels of inflammatory cytokines, CYPs, nuclear factor-κB (NF-κB), and nuclear factors in the liver were measured by quantitative reverse transcription polymerase chain reaction. RESULTS: We found inflammatory infiltrates in the liver of rats exposed to IH. The mRNA expression level of interleukin-1beta was increased in the liver of the IH-exposed rats (0.005 ± 0.001 vs. 0.038 ± 0.008, P = 0.042), whereas the mRNA expression level of Cyp1a2 was downregulated (0.022 ± 0.002 vs. 0.0050 ± 0.0002, P = 0.029). The hepatic level of transcription factor NF-κB was also reduced in the IH group relative to that in the control group, but the difference was not statistically significant and was parallel to the expression of the pregnane X receptor and constitutive androstane receptor. However, the decreased expression of the glucocorticoid receptor upon IH treatment was statistically significant (0.056 ± 0.012 vs. 0.032 ± 0.005, P = 0.035). CONCLUSIONS: These results indicate a decrease in expression of hepatic CYPs and their regulator GR in rats exposed to IH. Therefore, this should be noted for patients on medication, especially those on drugs metabolized via the hepatic system, and close attention should be paid to the liver function of patients with OSA-associated IH.

Boehmenan, a lignan from the Chinese medicinal plant Clematis armandii, induces apoptosis in lung cancer cells through modulation of EGF-dependent pathways.

BACKGROUND: Epidermal growth factor receptor (EGFR) is an effective molecular target for cancer treatment. Boehmenan, a lignan from the dried stems of Clematis armandii, exhibited the potent cytotoxic effects against many cancer cell lines in previous studies. However, the effects and underlying mechanism of boehmenan on non-small cell lung cancer (NSCLC) remains unclear. PURPOSE: The present study was designed to determine the in vitro anti-cancer properties and underlying molecular mechanisms of boehmenan on A549 NSCLC cells. STUDY DESIGN/METHODS: Cellular viability and chemoattractive properties of macrophages were investigated by using MTT and transwell migration assay, respectively. Mitochondrial membrane potential (ΔΨm), apoptotic ratio, and cell cycle were measured by flow cytometry. Protein expression was visualized by Western blot using specific antibodies. RESULTS: Boehmenan concentration-dependently suppressed proliferation and induced G1 phase arrest in A549 NSCLC cells, which were accompanied by reduction of migration, colony formation and increase of apoptosis in A549 cells. In addition, boehmenan treatment markedly modulated apoptosis-related protein (p53, p21, cleaved caspase 3, and cleaved PARP) and cyclin D1 expression and induced ΔΨm collapse in a concentration dependent manner. Furthermore, boehmenan concentration-dependently inhibited EGF-induced activation of EGFR and its downstream signaling molecules, including MEK, Akt, ERK1/2, and STAT3. CONCLUSION: Taken together, our results suggested that boehmenan-mediated anti-tumor property was mediated by modulation of mitochondria and EGFR signaling pathway in A549 NSCLC cells.

(7R,8S)-9-acetyl-dehydrodiconiferyl alcohol inhibits inflammation and migration in lipopolysaccharide-stimulated macrophages.

BACKGROUND: (7R, 8S)-9-Acetyl-dehydrodiconiferyl alcohol (ADDA), a novel lignan compound isolated from Clematis armandii Franch (Ranunculaceae) stems, has been found to exert potential anti-inflammatory activities in vitro. PURPOSE: To investigate the pharmacological effects and molecular mechanisms of ADDA on lipopolysaccharide (LPS)-induced activation and migration of macrophages. STUDY DESIGN/METHODS: Macrophages were stimulated with LPS in the presence or absence of ADDA. Expression of inflammatory mediators, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) were measured by Western blot and commercial NO detection kit. Cellular viability and chemotactic properties of macrophages were investigated using MTT and transwell migration assays. The activation and expression of mitogen activated protein kinases, nuclear factor-κB (NF-κB), protein kinase B (Akt), Src, and focal adhesion kinase (FAK) were analyzed by Western blot. RESULTS: Non-toxic concentrations (12.5-50 µM) of ADDA concentration-dependently inhibited expression/release of inflammatory mediators (COX-2, iNOS, and NO), suppressed Akt and c-jun N-terminal kinase 1/2 (JNK) phosphorylation, and NF-κB activation in LPS-stimulated macrophages. In addition, ADDA blocked LPS-mediated macrophage migration and this was associated with inhibition of LPS-induced Src and FAK phosphorylation as well as Src expression in a concentration dependent manner. Notably, the inhibitory effects of ADDA on iNOS, NO, and Src could be mimicked by a Src inhibitor PP2 or an iNOS inhibitor l-NMMA. CONCLUSION: Our results suggested that ADDA attenuated LPS-induced inflammatory responses in macrophages and cell migration, at least in part, through inhibition of NF-κB activation and modulation of iNOS/Src/FAK axis.

Clinical Effectiveness of Scapulothoracic Joint Control Training Exercises on Shoulder Joint Dysfunction.

The objective of this study was to examine the clinical effectiveness of scapulothoracic joint control training exercises on shoulder joint dysfunction. Forty patients with traumatic shoulder pain and joint dysfunction were randomized into the treatment or control group. Standard rehabilitation interventions included glenohumeral joint mobilization techniques, ultrasound therapy, traditional Chinese medicine, interference current therapy, and other comprehensive interventions. Patients received scapulothoracic joint control training exercises, including active and passive motions of the scapulothoracic joints, peri-joint muscle exercise, and joint stability exercises for 1 month. Patient status was evaluated by Constant-Murley scales before and after the prescribed interventions. The pain conditions, daily activities, range of movement, strength tests and total scores were significantly improved compared to prior treatment. Moreover, improvements in pain, daily activities, scope of activities, and total scores for patients in the treatment group were statistically significant when compared to the control group (P < 0.05). However, there was no inter-group difference in strength testing. The combination of standard rehabilitation interventions and scapulothoracic joint control training exercises are an effective treatment of the shoulder joint dysfunction. Moreover, the pain outcomes, scope of activities, and total scores were better in the treatment group.

Comprehensive study of cationic liposomes composed of DC-Chol and cholesterol with different mole ratios for gene transfection.

The ability of cationic liposomes composed of DC-Chol and cholesterol to carry pDNA into 293 T cells was investigated. Lipoplexes formed between DC-Chol/cholesterol liposomes and pDNA (encoding green fluorescent protein, GFP) were analyzed in terms of morphology observation, turbidity determination, particle size and zeta potential measurement, differential scanning calorimetry (DSC), gel retardation assay, cytotoxicity analysis in 293 T cells and transfection efficiency. The results showed that liposome preparation at or above 66.7 mol% cholesterol in formulation exhibited a calorimetric transition caused by anhydrous cholesterol domain at about 41°C. In comparison with the control, DOPE-containing liposomes, DC-Chol/cholesterol carriers showed more stable particle size, lower turbidity, higher activity for transfecting cells in the presence of high concentration serum (50% FBS), primarily due to the neutral domain formation by increasing mole ratios of cholesterol in formulation, as well as relatively lower cytotoxicity. Based on the results, it is suggested that incorporating high contents of cholesterol might be a potentially applicable method for various kinds of cationic lipids to obtain the gene carriers with high capability for in vivo transfection.

Correlation of Sperm Associated Antigen 11 (SPAG11) and its Isoforms with Varicocele in Rats.

BACKGROUND: We undertook this study to investigate the variation relationship of sperm associated antigen 11 (Spag11) mRNA expression and SPAG11E protein in the epididymis and spermatozoa of experimental left varicocele (ELV) rats. These findings could contribute to the understanding of the role of epididymal proteins in sperm functions and the mechanism of male infertility induced by varicocele. METHODS: The ELV model was established in adolescent male Sprague-Dawley rats. Four weeks after the operation, tissue distribution and changes in the expressions of Spag11 mRNA and SPAG11E protein caused by ELV in the whole of left epididymis and spermatozoa were studied using quantitative reverse transcription-polymerase chain reaction (RT-QPCR), immunohistochemistry and immunofluorescence. Significant differences were identified using one-way ANOVA followed by Student-Newman-Keuls test. Significance level (p) was fixed at 0.05. RESULTS: The expected product of Spag11 was 96 bp that amplified by RT-QPCR was detected in the epididymal tissue and spermatozoa. SPAG11E protein was confined mainly to the supranuclear region of the principal cells and the stereocilium of the epididymal epithelium, it was concentrated on the acrosome and the tail of spermatozoa except the terminal piece. Statistical analyses of the images and the data indicated that Spag11 mRNA and SPAG11E protein expressions in the left epididymis and spermatozoa of ELV rats presented a considerable decrease (p<0.001) compared with that of the corresponding control group. CONCLUSION: The expressions of Spag11 mRNA and SPAG11E protein declined markedly in ELV rats, which suggest that SPAG11E may not only play an important role in sperm maturation, but it may also be influenced by varicocele.

The study of gene variation and phylogenetic analysis of HPV16 E6 and E7 gene in Hubei, China / 病毒学报

To study the gene variation and the distribution of HPV16 variant in Hubei, China, DNA was extracted from cervical cancer tissue samples. The E6 and E7 genes of HPV16 were amplified and the PCR products were sequenced using E6- and E7-specific primers. Fortyseven cases were found mutations at nucleotide position 178 of HPV16 E6 gene in 80 cervical cancer samples. This mutation resulted in amino acid change from Asp to Glu. The rate of mutation at nucleotide position 178 of E6 gene was 58. 75%. Twenty two cases were found mutations at nucleotide position 647 of HPV16 E7 gene in 31 cervical cancer samples. This mutation resulted in amino acid change from Asn to Ser. The rate of mutation was 70.97%. These results showed that mutations at nucleotide position 178 of E6 gene, nucleotide position 647 of E7 gene of HPV16 in cerveical cancer samples were prevalent in Hubei, China. Phylogenetic analysis showed that Asian (As) variants of HPV16 are predominated in Hubei, China. European (Ep) varinats were also found in Samples in Hubei areas. None of Asian American (AA), African-1 (Af-1), African-2 (Af-2) variants of HPV16 was found in this region. Whether Asian (As) variants of HPV16 are more oncogenic and play a much more important role in the progress of cervical cancer than European (Ep) variants is not clear. More sequences of E6 and E7 gene in CIN and normal cervical tissue samples and study of the function of E6 and E7 protein of these HPV16 variants are needed to adress above question.

[Experimental varicocele affects VEGF and Flt-1 protein expressions in the testis and epididymis of adolescent rats].

OBJECTIVE: To investigate the effects of experimental left varicocele (ELV) on the vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase-1 (Flt-1) proteins in the testis and epididymis of adolescent rats, and to find out the correlation of the two proteins with varicocele-induced male infertility. METHODS: We established the ELV model in adolescent male SD rats, and detected the expressions of VEGF and Flt-1 proteins in the testis and epididymis by immunohistochemistry at 2 and 4 weeks after surgery. RESULTS: Cell- and region-specific expressions of VEGF and Flt-1 were observed in the testis and epididymis of the ELV and control groups. Statistical analysis showed that, in comparison with the corresponding control groups, the 2- and 4-week ELV groups exhibited a notable increase in the VEGF protein expression in the hibateral testis and epididymis (P < 0.01, P < 0.05); the Flt-1 expression was obviously upregulated in the hibateral testis and epididymis of the 2-week ELV group (P < 0.01, P < 0.01), but remarkably reduced in the hibateral testis and left epididymis of the 4-week ELV group (P < 0.01, P < 0.05), with no statistic difference in the right epididymis (P > 0.05). CONCLUSION: ELV can cause changes in the expressions of VEGF and Flt-1 proteins in the testis and epididymis of adolescent rats, and consequently affect spermatogenesis and spermiotelcosis, which may be one of the causes of varicocele-induced male infertility or subfertility.

The expression of Usp26 gene in mouse testis and brain.

Deubiquitinating enzymes (DUBs) play an important role in ubiquitin-dependent processes as negative regulators of protein ubiquitination. Ubiquitin-specific protease 26 (USP26) is a member of this family. The expression of Usp26 in mammalian testis and in other tissues has yet to be fully elucidated. To study the expression of Usp26 mRNA and protein in various murine tissues, reverse transcription (RT)-PCR and immunohistochemistry analyses were carried out. The RT-PCR analysis showed that the Usp26 transcript was expressed in all of the tested tissues. USP26 protein localization was examined by immunohistochemistry, and it was shown that USP26 was not detectable at 20 days postpartum, with the expression restricted to the cytoplasm of condensing spermatids (steps 9-16), Leydig cells and nerve fibers in the brain. In addition, the USP26 protein was detected at moderate levels in myocardial cells, the corpus of epidydimis, epithelium of the renal tubules and the seminal gland of postnatal day 35 mice. Its spatial and temporal expression pattern suggests that Usp26 may play an important role in development or function of the testis and brain. Further research into these possibilities is in progress.

[Effects of experimental left varicocele on SPAG11 mRNA and SPAG11E in the testis and epididymis of adolescent rats].

OBJECTIVE: To investigate the effects of experimental left varicocele (ELV) on the expression of sperm associated antigen 11 (SPAG11) mRNA and its protein isomer SPAG11E in the testis and epididymis of adolescent rats, and to explore the mechanism of infertility caused by varicocele. METHODS: The experimental left varicocele model was established in the adolescent male Sprague-Dawley rats. Two and 4 weeks after the operation, the changes of SPAG11 mRNA and SPAG11E expression in the testis and epididymis were detected using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expected product of SPAG11 367 bp amplified by RT-PCR was detected only in the epididymis. SPAG11E protein was observed mainly in the acrosomal vesicles and acrosome of round and elongating spermatids of the seminiferous epithelium, in the cytoplasm of Leydig cells, and in the supranuclear region of principle cells and stereocilia of the epididymal epithelium. Imaging and statistical analysis showed that SPAG11 mRNA and SPAG11E protein expressions in the left epididymis of the 2- and 4-week ELV groups presented a remarkable decrease (P < 0.05 or P < 0.01) compared with the right side and the corresponding control group, and the same decreased change in the left epididymis (P < 0.05 or P < 0.01) and an obvious reduction of SPAG11E immunopositive reaction in the right epididymis (P < 0.01) were noted in the 4-week group as compared with the 2-week group. No statistical difference of SPAG11E expression in the bilateral testes was found (P > 0.05) between the ELV group and the control, as well as between the 2- and 4-week ELV groups. CONCLUSION: SPAG11 is a specific gene expressed in the epididymis. The localization and expression of SPAG11E exhibited a region- and cell-specific pattern in both the testis and epididymis of adolescent rats. The expression levels of both SPAG11 mRNA and SPAG11E protein altered obviously in ELV rats. The results suggest that SPAG11 may not only play an important role in spermatogenesis and sperm maturation, but also be associated with varicocele-induced male infertility or subfertility.

[Expressions of VEGF and Flt-1 in the testis, epididymis and epididymal sperm of adolescent rats].

OBJECTIVE: To study the expressions of the vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase-1 (Flt-1) in the testis, epididymis and epididymal sperm of adolescent rats and explore the functions of both the proteins in the male reproductive system. METHODS: The expressions of VEGF and Flt-1 were detected in 20 adolescent SD rats, immunohistochemical staining used for both the testis and the epididymis and immunofluorescent staining for sperm. RESULTS: VEGF and Flt-1 proteins were specifically present in the testis, epididymis and sperm. In the testis, VEGF immunoreactive particles were localized in the cytoplasm of spermatogenic cells, the developing acrosome of spermatids, Sertoli cells and Leydig cells, while Flt-1 expressed mainly in the developing acrosome of spermatids and Leydig cells. In the epididymis, the cell-specific and region-specific expressions of VEGF and Flt-1 proteins were observed in the principal cells of epididymal epithelia, VEGF in the whole epididymis, while Flt-1 only in the caput and cauda segments. Both VEGF and Flt-1 were localized in the acrosome of the sperm head as well as in the neck, middle and principal segments of the sperm tail. CONCLUSION: The specific expression patterns of VEGF and Flt-1 in the rat testis, epididymis and sperm indicate that they may independently or collectively affect spermatogenesis and spermiotelcosis in either an autocrinological or a

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility.

AIM: To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 (USP26) gene and its involvement in idiopathic male infertility in China. METHODS: Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. RESULTS: Of 41 infertile men, 9 (22.0%, P = 0.01) had changes in USP26 gene on the X chromosome. A compound mutation (364insACA; 460G right triple arrow A) was detected in 8 patients (19.5%, P = 0.01) and a 1044T right triple arrow A substitution was found in 1 patient (2.4%, P > 0.05). All three variations led to changes in the coding amino acids. Two substitutions predict some changes: 460G right triple arrow A changes a valine into an isoleucine, and 1044T right triple arrow A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. CONCLUSION: The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.