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Objective To determine the pathophysiological progresses following bilateral cavernous nerve crushing injury, as an index for a treatment point and establishment of adequate treatment strategies for neurogenic erectile dysfunction. Materials and Methods Thirty-six rats were assigned to 1 of 6 groups, and bilateral cavernous nerve crushing or sham surgery was performed. Functional testing and ultrastructural analyses were performed immediately and at 7, 14, 21, and 28 days after the cavernous nerve (CN) injury (n = 6). Results Intracavernos pressure lowered progressively from 7 days to 14 days post-injury, and histologic staining revealed that the number of neuronal nitric oxide synthase-positive nerve fibers on the dorsal penile nerve decreased significantly and progressively from 7 days until 21 days post-injury. Furthermore, ultrastructural analyses revealed axon loss and demyelination of the CN at 7 and 14 days post-injury. However, it is followed by partial spontaneous recovery of erectile function and regeneration of the CN at 28 days post-injury, suggesting that these time points may be useful for evaluating the effects of drug treatments. Furthermore, we found that CN injury-induced damage to corporal smooth muscle cells was irreversible; therefore, focusing on protecting the corpus cavernosum from apoptosis may be more important than nerve protection when assessing treatment mechanisms in the CN injury model. Conclusion Our study makes a significant contribution to the human diagnostic pathology literature because it describes characteristics of relevant tissue in the rat and provides information regarding time points that may be useful for future studies of pathologic mechanisms or treatment evaluations.

// Yen-Lin Chen 1, 2, 3 , Yi-Ting Tsai 1 , Ting-Ting Chao 4 , Yi-No Wu 5 , Meng-Chuan Chen 6 , Ying-Hung Lin 3 , Chun-Hou Liao 3, 7 , Shang-Shing P. Chou 2 and Han-Sun Chiang 3, 7 1 Department of Pathology, Cardinal Tien Hospital, New Taipei, Taiwan 2 Department of Chemistry, Fu-Jen Catholic University, New Taipei, Taiwan 3 Graduate Institute of Biomedical and Pharmaceutical Science, Fu-Jen Catholic University, New Taipei, Taiwan 4 Medical Research Center, Cardinal Tien Hospital, New Taipei, Taiwan 5 School of Medicine, Fu-Jen Catholic University, New Taipei, Taiwan 6 Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan 7 Division of Urology, Department of Surgery, Cardinal Tien Hospital, New Taipei, Taiwan Correspondence to: Han-Sun Chiang, email: 05382@mail.fju.edu.tw Keywords: cavernous nerve; Schwann cell; CIP2A; DAPK; GAS6/AXL Received: July 22, 2017 Accepted: October 28, 2017 Published: January 05, 2018 ABSTRACT Purpose: Impotence is one of the major complications occurring in prostate cancer patients after radical prostectomy (RP). Self-repair of the injured nerve has been observed in animal models and in patients after RP. However, the downstream signalling is not well documented. Here, we found that the DAPK/CIP2A complex is involved in GAS6/AXL-related Schwann cell proliferation. Materials and Methods: The 3 groups were a sham group, a 14-day post-bilateral cavernous nerve injury (BCNI) group and a 28-day post-BCNI group. Erectile function was assessed and immunohistochemistry was performed. The rat Schwann cell RSC96 line was chosen for gene knockdown, cell viability, western blot, immunofluorescence and co-immunoprecipitation assays. Results: The intracavernosal pressure was low on the 14 th day after BCNI and partially increased by the 28 th day. GAS6 and p-AXL expression gradually increased in the cavernous nerve after BCNI. RSC96 cells incubated with a GAS6 ligand showed increased levels of p-ERK1/2 and p-AKT. Moreover, DAPK and CIP2A.p-AXL and p-DAPK and CIP2A complexes were identified by both immunoblotting and co-immunoprecipitation. Conclusion: The DAPK/CIP2A complex is involved in GAS6/AXL-related Schwann cell proliferation. CIP2A inhibits PP2A activity, which results in p-DAPK(S308) maintenance and promotes Schwann cell proliferation. CIP2A is a potential target for the treatment of nerve injury after RP.

Background: Measuring the fraction of inspired oxygen (FiO2) is challenging in spontaneously breathing patients with impaired respiratory mechanics during low-flow nasal cannula. Our study investigates the FiO2 with varied tidal volume (VT) and respiratory rate (RR) among different lung mechanics and provides equations to estimate the FiO2. Methods: Two training and test lungs were used in this study, and the three lung mechanics (normal (R5/C60), restrictive (R20/C80), obstructive (R5/C40)) were designed. Spontaneous breathing with VT (300, 500, and 700 mL) and RR (10, 20, and 30 breaths/min) was simulated. The flow rate of the nasal cannula was set to 1, 3, and 5 L per minute (LPM), and the FiO2 was measured at the carina. Results: The lowest and highest FiO2 were evident during high (700 mL) and low VT (300 mL), respectively, among normal, restrictive, and obstructive lung models. As RR increases, this decreases the FiO2. However, we found that VT and oxygen flow rate are the principal factors influencing measured FiO2 by multiple linear regression analysis. Conclusions: Our data suggest that the actual FiO2 is never as high in spontaneously breathing patients as that estimated. VT and oxygen flow rate had a substantial impact on the FiO2.

// Bou-Yue Peng 1 , Chi-Sheng Chiou 2, 3 , Navneet Kumar Dubey 4, 5 , Sung-Hsun Yu 4, 6 , Yue-Hua Deng 4, 7 , Feng-Chou Tsai 8 , Han-Sun Chiang 7 , Ying-Hua Shieh 10, 11 , Wei-Hong Chen 4 and Win-Ping Deng 2, 4, 9 1 Oral and Maxillofacial Surgery Section, Department of Dentistry, Taipei Medical University Hospital, Taipei, Taiwan 2 School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 3 Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan 4 Stem Cell Research Center, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 5 Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan 6 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan 7 Department of Life Science, Fu Jen Catholic University, Taipei, Taiwan 8 Department of Stem Cell Research, Cosmetic Clinic Group, Taipei, Taiwan 9 Graduate Institute of Basic Medicine, Fu Jen Catholic University, Taipei, Taiwan 10 Department of Family Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 11 Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan Correspondence to: Win-Ping Deng, email: wpdeng@tmu.edu.tw Keywords: osteoarthritis (OA), immortalized bone marrow stem cells (iBMSCs), cartilage regeneration, human papilloma virus (HPV)-16 E6/E7, molecular imaging Received: July 14, 2017 Accepted: August 27, 2017 Published: September 27, 2017 ABSTRACT Pathophysiology of osteoarthritis (OA) is characterized by progressive loss of articular cartilage in the knee-joints. To impart regenerative ability in lowly metabolizing chondrocytes, the bone marrow stem cells (BMSCs) has recently been recognized as a superior alternative treatment for OA. However, study of primary BMSCs-mediated chondrogenesis is difficult due to progressive cellular aging and replicative senescence. To obtain a therapeutic cell population for OA, BMSCs were immortalized by human papilloma virus (HPV)-16 E6/E7 along with mCherry luciferase (mCL), a gene marker for non-invasive imaging, and designated as iBMSCs-mCL. Next, their cell morphology, population doubling time (PDT) and colony forming ability (CFU) were evaluated. Furthermore, pluripotency and immunophenotypic markers were investigated. To deduce therapeutic ability, iBMSCs-mCL were intra-articularly injected into right knee of anterior cruciate ligament transaction (ACLT)-OA mice model and tracked through non-invasive bioluminescence imaging. Cell morphology of iBMSCs-mCL was similar to parental BMSCs. PDT and CFU ability of iBMSCs-mCLs were significantly increased. Pluripotency and immunophenotypic markers were highly expressed in iBMSC-mCL. Long-term survival and tri-lineage differentiation particularly chondrogenic potential of iBMSCs-mCL were also demonstrated in vitro and then in vivo which was monitored through non-invasive imaging. Intensive bioluminescent signals in iBMSCs-mCL administered knee-joint indicated a marked in vivo survival and proliferation of iBMSCs-mCL. Immunohistochemical staining for type II collagen (IHC of Col II) and alcian blue & safranin o staining of proteoglycans also corroborated cartilage regeneration by iBMSCs-mCL. Conclusively, iBMSCs-mCL maintains stemness and in vivo cartilage regeneration potential suggesting a promising avenue for development of OA therapeutics.

Male infertility is observed in approximately 50% of all couples with infertility. Intracytoplasmic sperm injection (ICSI), a conventional artificial reproductive technique for treating male infertility, may fail because of a severe low sperm count, immotile sperm, immature sperm, and sperm with structural defects and DNA damage. Our previous studies have revealed that mutations in the septin (SEPT)-coding gene SEPT12 cause teratozoospermia and severe oligozoospermia. These spermatozoa exhibit morphological defects in the head and tail, premature chromosomal condensation, and nuclear damage. Sperm from Sept12 knockout mice also cause the developmental arrest of preimplantation embryos generated through in vitro fertilization and ICSI. Furthermore, we found that SEPT12 interacts with SPAG4, a spermatid nuclear membrane protein that is also named SUN4. Loss of the Spag4 allele in mice also disrupts the integration nuclear envelope and reveals sperm head defects. However, whether SEPT12 affects SPAG4 during mammalian spermiogenesis remains unclear. We thus conducted this study to explore this question. First, we found that SPAG4 and SEPT12 exhibited similar localizations in the postacrosomal region of elongating spermatids and at the neck of mature sperm through isolated murine male germ cells. Second, SEPT12 expression altered the nuclear membrane localization of SPAG4, as observed through confocal microscopy, in a human testicular cancer cell line. Third, SEPT12 expression also altered the localizations of nuclear membrane proteins: LAMINA/C in the cells. This effect was specifically due to the expression of SEPT12 and not that of SEPT1, SEPT6, SEPT7, or SEPT11. Based on these results, we suggest that SEPT12 is among the moderators of SPAG4/LAMIN complexes and is involved in the morphological formation of sperm during mammalian spermiogenesis.

// Hung-Sheng Shang 1, 2, * , Yi-No Wu 3, * , Chun-Hou Liao 4, 5 , Tzong-Shi Chiueh 2 , Yuh-Feng Lin 1, 7, 8 and Han-Sun Chiang 3, 4, 6 1 Graduate Institute of Clinical of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 2 Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan 3 Graduate Institute of Basic Medicine, Fu Jen Catholic University, New Taipei City, Taiwan 4 Division of Urology, Department of Surgery, Cardinal Tien Hospital, Taipei City, Taiwan 5 College of Medicine, Fu Jen Catholic University, Taipei City, Taiwan 6 Department of Urology, Taipei Medical University Hospital, Taipei, Taiwan 7 Division of Nephrology, Department of Medicine, Shuang Ho Hospital, School of Medicine, College of Medicine, Taipei Medical University, New Taipei City, Taiwan 8 Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan * These authors have contributed equally to this work Correspondence to: Han-Sun Chiang, email: 053824@mail.fju.edu.tw Yuh-Feng Lin, email: linyf@s.tmu.edu.tw Keywords: apoptosis, corporal smooth muscle, erectile dysfunction, ketamine, nitric oxide synthase Received: November 09, 2015 Accepted: June 01, 2016 Published: July 20, 2016 ABSTRACT We investigated and evaluated the mechanisms of erectile dysfunction (ED) in a rat model of long-term ketamine administration. Adult male Sprague-Dawley rats (n = 32) were divided into four groups: namely the control group receiving intraperitoneal injection of saline, 1-month, 2-month and 3-month groups receiving daily intraperitoneal injection of ketamine (100 mg/kg/day) for 1, 2, and 3 month respectively. After treatment, animals underwent an erectile response protocol to assess intracavernosal pressure (ICP). Smooth muscle content was evaluated. Neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression were assessed using immunostaining assay. Ketamine-induced apoptosis was analyzed using TUNEL assay. Long-term ketamine administration caused significantly decreased erectile responses as measured by ICP. Smooth muscle content was significantly decreased in the ketamine-treated rats for 3 months. In the erectile tissue, ketamine administration significantly reduced nNOS expression and increased iNOS content compared with controls, whereas eNOS expression was not altered. Ketamine induced apoptosis in corpus cavernosum. The present study demonstrates that long-term ketamine administration led to erectile dysfunction in rat. The molecular mechanisms of ketamine-induced ED involved the increased apoptosis and up-regulated iNOS expression incorporating with loss of corporal smooth muscle content and reduced nNOS expression in cavernous nerve.

Purpose: The current study aims at evaluating penile erectile function after nerve-sparing radical prostatectomy (RP) based on evidence from a reliable animal model. The previous model in which the cavernous nerve (CN) is temporarily crushed is reviewed and compared to the present method of prostate lobes removal near the CN to determine which is more relevant to the clinical situation. Materials and Methods: Twenty-four rats were divided into three groups. One group was subjected to temporary CN crushing, one group was subject to prostate lobes removal near the CN, and the last group was preserved (sham group). All rats were re-examined at 4 weeks after the first surgical procedure. The pathological changes of the CNs were evaluated by their gross appearance and immunohistochemistry. Intracavernosal pressure (ICP) was measured as a parameter of male erectile function. Results: The results of the current study demonstrate that the removal of the prostate lobes near the CN led to the degeneration of CN, even with careful sparing techniques. Four weeks after the first surgical procedure, the rats' abdomens were reopened, and CNs were identified in only 60% of the rats with prostate lobes removal near the CN. Furthermore, the remaining CNs in this group were found to be histologically degenerated, with poorer erectile function presented by ICP. In contrast, the CNs after temporary crush were only mildly injured and demyelinated, with evidence of regeneration. The changes in the rats with prostate lobes removal near the CN are much more similar to those in rats with clinical RP with CN sparing. Conclusion: The current study concluded that, in the rat model, CNs will be injured, degenerated, and eventually disappear after prostate lobes removal near the CN; this is very similar to what is observed in clinical RP. Protection of the CNs for erectile function preservation should be investigated further using this animal model.

Background.The pathophysiology of Taiwanese congenital bilateral absence of the vas deferens (CBAVD) is different from that in Caucasians. In particular, major cystic fibrosis transmembrane conductance regulator (CFTR) mutations and cystic fibrosis are absent in the former. Instead, deficiency in solute carrier family 9 sodium/hydrogen exchanger isoform 3 (SLC9A3) may play a role by generating obstructive azoospermia and degraded epithelial structure in the reproductive tract.Objectives.The objective of the study was to test whether SLC9A3 variants cause Taiwanese CBAVD.Materials and Methods.Six-month-oldSlc9a3−/−male mice were used to evaluate the effect of long-term SLC9A3 loss on the reproductive system. A case-control cohort of 29 men with CBAVD and 32 fertile men were genotyped for SLC9A3 variants.Results.SLC9A3 was expressed and localized in the apical border of the epithelium of human vas deferens and glandular epithelium of the seminal vesicle. SLC9A3 deficiency specifically induces atrophy of vas deferens and unfolding of seminal vesicle mucosa in mice. Loss of SLC9A3 increased the incidence of CBAVD in humans from 3.1% to 37.9% (p < 0.001). Up to 75.9% of CBAVD patients carry at least one variant in either SLC9A3 or CFTR.Discussion.Our findings build upon previous data associated with CBAVD pathogenesis. Here, we now report for the first time an association between CBAVD and loss ofSLC9A3and propose that specific defects in the reproductive duct due toSLC9A3variants drive CBAVD development.Conclusion.The data implicate loss of SLC9A3 as a basis of Taiwanese CBAVD and highlight SLC9A3 function in reproduction.

Radical prostatectomy causes erectile dysfunction (ED) and irreversible morphologic changes, including induction of endothelial and smooth muscle cell (SMC) apoptosis in the corpus cavernosum (CC). The injection of smooth muscle progenitor cells (SPCs) thickens the vascular intima and has demonstrated therapeutic benefit in cardiovascular disease animal. Herein, we investigated the effect of SPCs on the recovery of erectile function (EF) in rat models with bilateral cavernous nerve (CN) injury. Twenty-four male Sprague-Dawley rats were randomized into sham, vehicle only, or SPC treatment groups. Rats in the SPC treatment and vehicle groups were subjected to bilateral CN injury before intracavernosal injection. Intracavernosal injections of SPCs increased all EF parameters at day 28 after injury and simultaneously reduced apoptosis of the SMCs. Ultrastructural analysis revealed that SPCs maintained the integrity of the CC by preserving the structure of the adherens junctions. Tracking transplanted SPCs labeled with EdU showed that transplanted SPCs remained in the CC 28 days after treatment. Intracavernosal SPC injection restored EF after bilateral CN injury by reducing SMC apoptosis, which favored the maintenance of the structure of adherens junctions and regulated the stability of corporal vessels. These findings demonstrate the therapeutic potential of SPCs for treating ED in humans.

Few papers have focused on small guanosine triphosphate (GTP)-binding proteins and their regulation during spermatogenesis. TBC1D21 genes (also known as male germ cell RAB GTPase-activating protein MGCRABGAP) are related to sterility, as determined through cDNA microarray testing of human testicular tissues exhibiting spermatogenic defects. TBC1D21 is a protein specifically expressed in the testes that exhibits specific localizations of elongating and elongated spermatids during mammalian spermiogenesis. Furthermore, through co-immunoprecipitation (co-IP) and nano liquid chromatography&ndash, tandem mass spectrometry (nano LC&ndash, MS/MS), Rap1 has been recognized as a potential TBC1D21 interactor. This study determined the possible roles of Rap1 and TBC1D21 during mammalian spermiogenesis. First, the binding ability between Rap1 and TBC1D21 was verified using co-IP. Second, the stronger signals of Rap1 expressed in elongating and elongated murine spermatids extracted from testicular sections, namely spermatogonia, spermatocytes, and round spermatids, were compared. Third, Rap1 and TBC1D21 exhibited similar localizations at postacrosomal regions of spermatids and at the midpieces of mature sperms, through isolated male germ cells. Fourth, the results of an activating Rap1 pull-down assay indicated that TBC1D21 overexpression inactivates Rap1 activity in cell models. In conclusion, TBC1D21 may interact with and potentially regulate Rap1 during murine spermatogenesis.

Congenital bilateral absence of vas deferens (CBAVD) is a special entity in obstructive azoospermia. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are involved in Taiwanese CBAVD but most heterozygous 5T variant. The solute carrier family 9 isoform 3 (SLC9A3) is the Na+/H+ exchanger, which interacts with CFTR and regulates the Ca2+ homeostasis. Loss of SLC9A3 decreases CFTR protein and causes obstructive azoospermia in mice. It also causes mal-reabsorption by the efferent tubules, which leads to the obstructive phenomenon and eventually results in testicular atrophy. In 6-month old SLC9A3 deficiency mice, the atrophy of their vas deferens and seminal vesicles become more prominent. Decreases of CFTR expression in the reproductive organ in the SLC9A3 deficient (−/−) mice prove the interaction between CFTR and SLC9A3 in the reproductive tract. Most of Taiwanese CBAVD have at least one variant of SLC9A3 deletion and CFTR IVS8-5T, which co-contribute to Taiwanese CBAVD. The report indicates SLC9A3 deficiency can reverse the pathological changes in the gastrointestinal tract of CF mice. Further research can explore the definite mechanism of SLC9A3 and its role interacting with CFTR in different organ systems, which can contribute to novel treatment for the patients with cystic fibrosis and CBAVD. Keywords: Congenital bilateral absence of vas deferens, Cystic fibrosis transmembrane conductance regulator, Solute carrier family 9 isoform 3

Solute carrier family 9 isoform 3 (SLC9A3), a Na⁺/H⁺ exchanger, regulates the transepithelial absorption of Na⁺ and water and is primarily expressed on the apical membranes of the intestinal epithelium, renal proximal tubule, epididymis, and vas deferens. Loss of the Slc9a3 allele in mice enhances intestinal fluid and causes diarrhoea as a consequence of diminished Na⁺ and HCO₃- absorption. Hence, the loss also causes male infertility and reveals the abnormal dilated lumen of the rete testis and calcification in efferent ductules. However, whether loss of Slc9a3 alleles also disrupts mammalian spermatogenesis remains unknown. First, through immunoblotting, we determined that SLC9A3 is highly expressed in the murine testis compared with the small intestine, epididymis, and vas deferens. During murine spermatogenesis, SLC9A3 is specifically expressed in the acrosome region of round, elongating, and elongated spermatids through immunostaining. Furthermore, SLC9A3 signals are enriched in the acrosome of mature sperm isolated from the vas deferens. In Slc9a3 knockout (KO) mice, compared with the same-aged controls, the number of spermatids on the testicular section of the mice progressively worsened in mice aged 20, 35, and 60 days. Sperm isolated from the epididymis of Slc9a3 KO mice revealed severe acrosomal defects. Our data indicated that SLC9A3 has a vital role in acrosomal formation during spermiogenesis.

Quercetin is one of the natural components from natural plant and it induces cell apoptosis in many human cancer cell lines. However, no available reports show that quercetin induces apoptosis and altered associated gene expressions in human gastric cancer cells, thus, we investigated the effect of quercetin on the apoptotic cell death and associated gene expression in human gastric cancer AGS cells. Results indicated that quercetin induced cell morphological changes and reduced total viability via apoptotic cell death in AGS cells. Furthermore, results from flow cytometric assay indicated that quercetin increased reactive oxygen species (ROS) production, decreased the levels of mitochondrial membrane potential (ΔΨm ), and increased the apoptotic cell number in AGS cells. Results from western blotting showed that quercetin decreased anti-apoptotic protein of Mcl-1, Bcl-2, and Bcl-x but increased pro-apoptotic protein of Bad, Bax, and Bid. Furthermore, quercetin increased the gene expressions of TNFRSF10D (Tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain), TP53INP1 (tumor protein p53 inducible nuclear protein 1), and JUNB (jun B proto-oncogene) but decreased the gene expression of VEGFB (vascular endothelial growth factor B), CDK10 (cyclin-dependent kinase 10), and KDELC2 (KDEL [Lys-Asp-Glu-Leu] containing 2) that are associated with apoptosis pathways. Thus, those findings may offer more information regarding the molecular, gene expression, and signaling pathway for quercetin induced apoptotic cell death in human gastric cancer cells.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF) and are associated with congenital bilateral absence of the vas deferens (CBAVD), which is the major cause of infertility in male patients with CF. However, most Taiwanese patients with CBAVD do not carry major CFTR mutations. Some patients have a single copy deletion of the solute carrier family 9 isoform 3 (SLC9A3) gene. SLC9A3 is a Na+/H+ exchanger, and depleted Slc9a3 in male mice causes infertility due to the abnormal dilated lumen of the rete testis and efferent ductules. Furthermore, SLC9A3 interacts with CFTR in the pancreatic duct and functions as a genetic modifier of CF. However, SLC9A3 function and its relation to CFTR expression in the male reproductive tract in vivo remain elusive. In the present study, we found that CFTR expression was dramatically decreased in the epididymis and vas deferens of Slc9a3 knockout mice. Adult Slc9a3-/- mice showed not only significantly decreased epididymis and vas deferens weight but also increased testis weight. Furthermore, Slc9a3-/- mice developed obstructive azoospermia because of abnormal abundant secretions and calcification in the lumen of the reproductive tract. Ultrastructural analysis of the epithelium in Slc9a3–/–epididymis and vas deferens displayed disorganized and reduced number of stereocilia and numerous secretory apparatuses. Our data revealed that interdependence between SLC9A3 and CFTR is critical for maintaining a precise microenvironment in the epithelial cytoarchitecture of the male reproductive tract. The Slc9a3-deficient mice with impaired male excurrent ducts in this study provide proof for our clinical findings that some Taiwanese of CBAVD carry SLC9A3 deletion but without major CFTR mutations., Author summary Cystic fibrosis (CF) is the most common inherited life-threatening disease in Caucasians. The most well-known cause of CF is a genetic defect in CFTR, an apical membrane chloride and bicarbonate channel. The symptoms of CF include defects in the respiratory, digestive, and male reproductive systems. Most male patients with CF are infertile due to congenital bilateral absence of the vas deferens (CBAVD), which leads to obstructive azoospermia. Nevertheless, Taiwanese patients with CBAVD do not carry the common mutations of CFTR found in Caucasians. We have identified a potential candidate, SLC9A3, of which a single copy is lost in Taiwanese patients with CBAVD. In addition to the previously reported role of SLC9A3 in the digestive system and efferent ductules, we now report that the SLC9A3 deficiency causes obstructive azoospermia and impairs the epithelial structure of the reproductive tract. Loss of SLC9A3 also leads to dramatic reduced expression of CFTR in the reproductive tract. We suggest that the interplay between SLC9A3 and CFTR is responsible for CF-related infertility. Thus, we have characterized a potential critical player in the pathogenesis of CBAVD and provide a new diagnostic candidate for Asian patients with CBAVD.

The septin gene belongs to a highly conserved family of polymerizing GTP-binding cytoskeletal proteins. SEPTs perform cytoskeletal remodeling, cell polarity, mitosis, and vesicle trafficking by interacting with various cytoskeletons. Our previous studies have indicated that SEPTIN12+/+/+/− chimeras with a SEPTIN12 mutant allele were infertile. Spermatozoa from the vas deferens of chimeric mice indicated an abnormal sperm morphology, decreased sperm count, and immotile sperm. Mutations and genetic variants of SEPTIN12 in infertility cases also caused oligozoospermia and teratozoospermia. We suggest that a loss of SEPT12 affects the biological function of microtublin functions and causes spermiogenesis defects. In the cell model, SEPT12 interacts with α- and β-tubulins by co-immunoprecipitation (co-IP). To determine the precise localization and interactions between SEPT12 and α- and β-tubulins in vivo, we created SEPTIN12-transgene mice. We demonstrate how SEPT12 interacts and co-localizes with α- and β-tubulins during spermiogenesis in these mice. By using shRNA, the loss of SEPT12 transcripts disrupts α- and β-tubulin organization. In addition, losing or decreasing SEPT12 disturbs the morphogenesis of sperm heads and the elongation of sperm tails, the steps of which are coordinated and constructed by α- and β-tubulins, in SEPTIN12+/+/+/− chimeras. In this study, we discovered that the SEPTIN12-microtubule complexes are critical for sperm formation during spermiogenesis.

Objective To show the flexibility in magnetic resonance imaging (MRI) of seminal vesicle (SV) and intra-abdominal segment of vas deferens for the patients with congenital absence of the vas deferens (CAVD), including congenital bilateral absence of the vas deferens (CBAVD) and congenital unilateral absence of vas deferens (CUAVD). Methods Fourteen consecutive patients with CAVD had transrectal ultrasonography (TRUS) and further MRI evaluations. TRUS was performed using a 7.5-MHz transducer, and images of the SVs were obtained, calculated in the transaxial plane. MRI studies were performed with a 1.5-7 superconducting system, T1- and T2-weighted axial, coronal, and sagittal imaging of the pelvis was obtained. If the SVs were present, then their size was measured for the morphologic classification and diagnosis. All of the patients also received cystic fibrosis transmembrane conductance regulator ( CFTR ) gene mutation testing. Results In a series of 12 men with CBAVD, only 4 were found to have bilateral SV agenesis using MRI. The remaining 8 men with unilateral hypoplasia still had SV remnants. MRI also detected the intra-abdominal segment of the vas deferens. Through our study of MRI, SV agenesis is not well associated with the presence of CFTR mutation in patients with CAVD. Conclusion MRI provides a precise imaginal diagnosis of SV defect, which is superior to the TRUS examination for the patients with CAVD. Compared with the previous inaccurate examination method of TRUS, this study demonstrates that MRI can provide better images for the patients with CAVD for the clinical diagnosis of existing defects of internal seminal tract and internal organs.

Male factor infertility accounts for approximately 50 percent of infertile couples. The male factor-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile sperm, immature sperm, abnormally structured sperm, and sperm with nuclear damage. Our knockout and knock-in mice models demonstrated that SEPTIN12 (SEPT12) is vital for the formation of sperm morphological characteristics during spermiogenesis. In the clinical aspect, mutated SEPT12 in men results in oligozoospermia or teratozoospermia or both. Sperm with mutated SEPT12 revealed abnormal head and tail structures, decreased chromosomal condensation, and nuclear damage. Furthermore, several nuclear or nuclear membrane-related proteins have been identified as SEPT12 interactors through the yeast 2-hybrid system, including NDC1 transmembrane nucleoporin (NDC1). NDC1 is a major nuclear pore protein, and is critical for nuclear pore complex assembly and nuclear morphology maintenance in mammalian cells. Mutated NDC1 cause gametogenesis defects and skeletal malformations in mice, which were detected spontaneously in the A/J strain. In this study, we characterized the functional effects of SEPT12–NDC1 complexes during mammalian spermiogenesis. In mature human spermatozoa, SEPT12 and NDC1 are majorly colocalized in the centrosome regions; however, NDC1 is only slightly co-expressed with SEPT12 at the annulus of the sperm tail. In addition, SEPT12 interacts with NDC1 in the male germ cell line through coimmunoprecipitation. During murine spermiogenesis, we observed that NDC1 was located at the nuclear membrane of spermatids and at the necks of mature spermatozoa. In male germ cell lines, NDC1 overexpression restricted the localization of SEPT12 to the nucleus and repressed the filament formation of SEPT12. In mice sperm with mutated SEPT12, NDC1 dispersed around the manchette region of the sperm head and annulus, compared with concentrating at the sperm neck of wild-type sperm. These results indicate that SEPT12–NDC1 complexes are involved in mammalian spermiogenesis.

According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

There is an unmet need for treatment of erectile dysfunction resulting from radical prostatectomy and cavernous nerve (CN) injury. Given the neuroprotective properties of docosahexaenoic acid (DHA), we investigated its effect on penile functional and structural recovery in a rat model of bilateral cavernous nerve injury. Rats were subject to CN injury and received intraperitoneal administration of either vehicle or a DHA nanoemulsion (nano-DHA) at 10, 50, or 250 μg/kg. Functional testing and histological analyses were performed at 28 days post-injury. The maximum intracavernosal pressure (ICP) and other measures of erectile function were significantly higher in the nano-DHA groups than in the vehicle group (p p

Endothelial cells contribute to the function and integrity of the vascular wall, and a functional aberration may lead to atherogenesis. There is increasing evidence on the atheroprotective role of androgens. Therefore, we studied the effect of the androgens-testosterone and dihydrotestosterone-and estradiol on human coronary artery endothelial cell (HCAEC) function. We found by MTT assay that testosterone is not cytotoxic and enhances HCAEC proliferation. The effect of testosterone (10-50 nM), dihydrotestosterone (5-50 nM), and estradiol (0.1-0.4 nM) on the adhesion of tumor necrosis factor-α (TNF-α)-stimulated HCAECs was determined at different time points (12-96 h) by assessing their binding with human monocytic THP-1 cells. In addition, the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), was determined by ELISA and Western blot analysis. Both testosterone and dihydrotestosterone attenuated cell adhesion and the expression of VCAM-1 and ICAM-1 in a dose- and time-dependent manner. Furthermore, androgen treatment for a longer duration inhibited cell migration, as demonstrated by wound-healing assay, and promoted tube formation on a Matrigel. Western blot analysis demonstrated that the expression of phosphorylated endothelial nitric oxide synthase (eNOS) increased, whereas that of inducible nitric oxide synthase (iNOS) decreased following the 96-h steroid treatment of TNF-α-stimulated HCAECs. Our findings suggest that androgens modulate endothelial cell functions by suppressing the inflammatory process and enhancing wound-healing and regenerative angiogenesis, possibly through an androgen receptor (AR)-dependent mechanism.

Limited donor-cell engraftment to the injured tissue restricts therapeutic efficacy of stem cell transplantation. Herein, we proposed an alternative strategy by using in utero transplantation (IUT) to create mixed-chimerism environment in recipients and to facilitate donor-cell engraftment followed by postnatal secondary boost with the same cells. Mouse bone marrow stromal cells (BMSCs) were used as the xenogenic donor cells and given into rat fetus as an early exposure of IUT treatment. The engraftment potential was analyzed for the presence of BMSCs by flow cytometry or PCR in recipient tissues. The function of a second boost of mouse BMSCs, in terms of cardioprotection, was tested by given 1×10⁶ cells to rat IUT hearts with ischemia/reperfusion (IR) injury that was induced by a 45 min of left coronary ligation and released for 72 h. Mouse BMSCs demonstrated an immunosuppressive effect when mixed with mouse or rat lymphocytes. IUT treatment only caused few BMSCs engrafted to fetal (embryonic day 20) and adult (4 weeks after birth) rat organs including heart, but engraftment was increased in hearts of the IUT rats after second boost. This was coincided with attenuation of cardiac injury caused by IR. Interestingly, an up-regulation of CXC chemokine receptor type 4 (CXCR4) was seen when BMSCs were exposed to hypoxia. This indicates that enhanced engraftment of mouse BMSCs to post-ischemic rat hearts possibly is dependent on CXCR4. Moreover, results of flow cytometry demonstrated that the presence of CD34⁺ cells in rat IUT hearts with IR injury was increased. These observations suggest that enhanced engraftment of donor BMSCs to rat IR hearts by CXCR4 may recruit endogenous CD34⁺ cells of recipients which in turn protects heart against IR. This also supports the notion of fetal preconditioning with BMSC enhances the efficiency of progenitor cell-mediated organ protection after a postnatal second boost in xeno-transplantation.

Non-hematopoietic CD45+ precursor cells are not known to differentiate into cardiomyocytes. We found that CD45+/CD34-/lin- stromal cells isolated from mouse bone marrow (BMSCs) potentially differentiated into cardiomyocyte-like cells in vitro. Therefore, we hypothesized that the CD45+/CD34-/ lin- BMSCs might protect rat hearts against ischemia/reperfusion (IR) injury following xeno-transplantation. In the present study, BMSCs were isolated by immunoselection and their cellular phenotype and biochemical properties were characterized. The immunological inertness of BMSCs was examined by the allogeneic and xenogeneic mixed lymphocyte reaction (MLR). The potential role of BMSCs for cardioprotection was evaluated by intravenous introduction of 1 x 10(6) cells into rat IR hearts, induced by left coronary ligation for 45 min and released for 72 h. Changes in cardiac contractility and the degree of myocardial injury were assessed. Our findings indicated that BMSCs expressed the muscle-cell marker alpha-actinin after 5-azacytidine treatment. CD45+/CD34-/lin- stromal cells were characterized as mesenchymal progenitor cells based on the expression of Sca-1 and Rex-1. The MLR assay revealed an immunosuppression of BMSCs on mouse and rat lymphocytes. After xeno-transplantation, the BMSCs engrafted into the infarct area and attenuated IR injury. However, increases in intracardial TGF-beta and IFN-gamma contents of IR hearts were not affected by BMSC treatment. Interestingly, ex vivo evidence indicated that CXCR4, SDF-1 and TGFbeta-1 receptors were up-regulated after the cells were exposed to tissue extracts prepared from rat post-IR hearts. In addition, IFN-gamma treatment also markedly increased Sca-1 expression in BMSCs. Mechanistically, these results indicated that CXCR4/SDF-1 and TGF-beta signals potentially enhanced the interaction of BMSCs with the damaged myocardium, and increased IFN-gamma in post-ischemic hearts might cause BMSC to behave more like stem cells in cardioprotection. These data show that CD45+/CD34-/lin- BMSCs possess cardioprotective capacity. Evidently, the accurate production of soluble factors TGF-beta and IFN-gamma in parallel with increased expression of both TGF-beta and Sca-1 receptors may favor BMSCs to achieve a more efficient protective capacity.

Purpose Oxidative stress and endoplasmic reticulum (ER) stress may induce renal apoptosis and contribute to the pathogenesis of the kidney with unilateral ureteral obstruction (UUO). Materials and Methods We induced UUO the female Wistar rats by ligation of the left ureter at the ureteropelvic junction. The UUO kidney was performed from 4 hr to 7 days course. At the indicated time, we measured the arterial blood pressure and renal blood flow in each rat, renal ROS measurement in vivo by a chemiluminescence analyzer. We performed immunohistochemistry of monocyte/macrophage (ED-1) stain for leukocyte infiltration, 4-hydroxynoneal (4-HNE) stain for ROS products, and apoptosis by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and Western blot to analyze ER stress-associated and apoptosis-related proteins expression in the UUO kidney. Results We found that UUO decreased renal blood flow and increased renal vascular resistance and renal ROS. UUO decreased renal manganese superoxide dismutase (MnSOD) and catalase protein expression in a time-dependent manner. Increased 4-HNE stain in the renal tubules and ED-1 stain in the renal tubulointerstitial compartment occurred after 4 hr of UUO in the kidney. UUO significantly enhanced ER stress markers like ER stress-response protein 25 and glucose-regulated protein 78 and ER-associated apoptosis proteins, c-JUN NH2-terminal kinase, and caspase 12, in the kidney. Subsequently, UUO enhanced renal pro-apoptotic Bax and caspase 3 expression and decreased anti-apoptotic Bcl-2 expression, leading to renal tubular apoptosis. Conclusions Our data suggest that renal tubular apoptosis induced by oxidative stress and ER stress occurred in the UUO kidney. Neurourol. Urodynam. 30:472–479, 2011. © 2011 Wiley-Liss, Inc.

We evaluated the association between serum sex hormone levels and prostate volume in Taiwanese men.A cross-sectional study was conducted in 505 men (aged 40-79 years, mean age 58 years). Serum total testosterone (TT), free testosterone (FT), dihydrotestosterone (DHT) and estradiol (E2) levels were measured. Total prostate volume (TPV) and transition zone volume (TZV) were measured by transrectal ultrasonography. Body mass index (BMI), DHT/TT and E2/TT were calculated. Correlations were determined using univariate and multivariate regression analyses.Apart from DHT, an age-dependent change of sex hormone levels were observed. On univariate analyses, age, BMI, serum DHT level and DHT/TT ratio, as well as serum E2 level and E2/TT ratio, but not serum TT and FT levels showed a significant association with prostate volume. On multivariate analysis, however, only serum DHT level and DHT/TT ratio remained significant. Logistic regression analysis showed that the odds ratios (95% confidence interval) of the second, third, and fourth quartiles of serum DHT levels for benign prostatic hyperplasia (defined as TPV ≥ 20 ml) risk were 2.06 (1.21-3.51), 2.66(1.56-4.53) and 7.15(4.0-12.6), respectively (p0.001).Higher serum DHT level and DHT/TT ratio were associated with larger prostate volume and higher prevalence of BPH in Taiwanese men.

The purpose of this study is to characterize the smooth muscle differentiation of purified human muscle-derived cells (hMDCs). The isolation and purification of hMDCs were conducted by modified preplate technique and Dynal CD34 cell selection. Smooth muscle cell differentiation was induced by the use of smooth muscle induction medium (SMIM) and low-serum medium. The gene expressions at the mRNA and protein levels of undifferentiated and differentiated hMDCs were tested by RT-PCR, Western blot and immunofluorescence studies. Western blot and immunofluorescence studies demonstrated the purified hMDCs cultured in SMIM for 4 weeks and expressed significant amount of smooth muscle myosin heavy chain (MHC) and α-smooth muscle actin (ASMA). The cells cultured in low-serum medium for 4 weeks also expressed ASMA, while the control group did not. RT-PCR analysis showed increased gene expression of smooth muscle markers, such as ASMA, Calponin, SM22, Caldesmon, Smoothelin and MHC when purified hMDCs were exposed to SMIM for 2 and 4 weeks when compared to the controls. In conclusion, we confirmed the smooth muscle differentiation capability of purified hMDCs. The gene expression of smooth muscle differentiation of purified hMDCs was characterized. These cells may be potential biomaterials for human tissue regeneration.

Aims We hypothesize that increased H2O2 in the urinary bladder may affect ATP and acetylcholine release and activate urothelial purinergic and muscarinic signaling consequently leading to hyperactive bladder. Materials and Methods We infused 0.3% and 1.5% H2O2 to the urinary bladder to evaluate the voiding function and frequency, pelvic afferent (PANA) and pelvic efferent nerve activity (PENA) simultaneously in the urethane anesthetized rats. We measured ATP and acetylcholine content in the rat urinary bladder under saline or H2O2 stimulation. Hyaluronic acid, muscarinic, and purinergic receptor antagonists were used to evaluate their effects and mechanisms on H2O2-induced hyperactive bladder. Results Intravesical H2O2 administration increased the frequency of voiding and the maximal amplitude of intravesical pressure (IVP) in the urethane anesthetized rats. Intravesical H2O2 decreased the threshold level of PANA and PENA for triggering micturition and increased ATP and acetylcholine contents in the H2O2-treated bladder in the rat. Hyaluronic acid in vitro directly scavenged H2O2 in a dose-dependent manner. Intravesical hyaluronic acid for 30 min attenuated H2O2-elicited excitatory effects on the frequency of voiding, amplitude of IVP and the sensitization of PANA and PENA in the rats. Hyaluronic acid treatment reduced H2O2-induced ATP and acetylcholine release in the urinary bladder. Intravesical administration of muscarinic receptor antagonist atropine methyl nitrate (50 µM) or purinergic receptor antagonist PPADS (1 mM) ameliorated H2O2-induced hyperactive bladder. Conclusions These results indicate that hyaluronic acid treatment can ameliorate H2O2-induced bladder hyperactivity possibly via the antioxidant activity and the inhibition of activating purinergic and muscarinic signaling pathway. Neurourol. Urodynam. 29:765–770, 2010. © 2010 Wiley-Liss, Inc.

Propofol is now the most commonly used intravenous anesthetic-for general anesthesia and sedation because of its rapid onset and recovery. Besides the well-known adverse effects of cardiovascular and respiratory depression, recent studies indicate that propofol may cause excitatory phenomena such as myoclonus, opisthotonus, and even seizure. However, the mechanisms of these excitatory effects of propofol have not been elucidated. Considering glutamate as the principle excitatory neurotransmitter in the central nervous system and excessive glutamatergic synaptic transmission can cause seizure, we examined the effect of propofol on the release of glutamate from rat cerebral cortex nerve terminals (synaptosomes). Results showed that subanesthetic concentration propofol facilitated 4-aminopyridine (4-AP), but not KCl- or ionomycin-evoked glutamate release from nerve terminals. The facilitation of 4-AP-evoked glutamate release by propofol also occurred in the calcium chelation and significantly attenuated by glutamate transporter inhibitors, DL-threo-β-benzyloxyaspartic acid (DL-TBOA) and L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC). In addition, propofol increased 4-AP-evoked depolarization of the plasma membrane potential. Furthermore, protein kinase C (PKC) inhibition suppressed propofol-mediated facilitation of glutamate release. These results suggest that subanesthetic concentration propofol facilitates glutamate release from rat cerebrocortical glutamatergic terminals by increasing nerve terminal excitability, likely through the activation of PKC pathway. This finding may provide an explanation for propofol-induced excitatory phenomena. Synapse 63:773–781, 2009. © 2009 Wiley-Liss, Inc.

Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, is one of the most common autosomal recessive diseases in Caucasians. We screened for the CFTR gene mutation in a Caucasian father with congenital bilateral absence of the vas deferens (CBAVD), a Taiwanese mother, and twins resulting from an intracytoplasmic single sperm injection (ICSI) procedure. DNA fragments that showed abnormal banding patterns on temporal temperature gradient gel electrophoresis analysis followed by analysis of DNA sequence was used. The Caucasian father with CBAVD had ΔF508 and p.L375F mutations. The two children were heterozygous for the ΔF508 and p.L375F mutations, respectively. Mutation analysis of the CFTR gene should always be recommended for infertile couples seeking ICSI. The possibility of the children resulting from ICSI being a victim or carrier of CBAVD or CF, especially when the father is Caucasian with CBAVD, should be discussed during genetic counseling.

Androgen and the androgen receptor (AR) have been shown to play critical roles in male fertility. Our previous data demonstrated that mice lacking AR (AR(-/y)) revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in Leydig cells remain largely unknown. Using a Cre-LoxP conditional knockout strategy, we generated a tissue-specific knockout mouse (L-AR(-/y)) with the AR gene deleted by the anti-Müllerian hormone receptor-2 (Amhr2) promoter driven Cre expressed in Leydig cells. Phenotype analyses show that the outside appearance of L-AR(-/y) mice was indistinguishable from wild type mice (AR(+/y)), but with atrophied testes and epididymis. L-AR(-/y) mice were infertile, with spermatogenic arrest predominately at the round spermatid stage and no sperm could be detected in the epididymis. L-AR(-/y) mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone and follicle-stimulating hormone concentrations than AR(+/y) mice. Further mechanistic studies demonstrated that hypotestosteronemia in L-AR(-/y) mice is not caused by reducing numbers of Leydig cells, but instead by the alterations of several key steroidogenic enzymes, including 17beta-HSD3, 3beta-HSD6, and P450c17. Together, L-AR(-/y) mice provide in vivo evidence that functional AR in Leydig cells is essential to maintain normal spermatogenesis, testosterone production, and required for normal male fertility.

(-)-Epicatechin-3-gallate (ECG) is a polyphenolic compound similar to (-)-epigallocatechin-3-gallate (EGCG) which is abundant in green tea. Numerous workers have proposed that EGCG protects epidermal cells against UVB-induced damage. However, little has been known about whether ECG protects keratinocytes against UVB-induced damage. We decided to investigate the protective effects and underlying mechanisms of ECG on UVB-induced damage. Cell viability was determined by the MTT assay. Activation of ERK1/2, p38 and JNK was analyzed by Western blotting. Intracellular H2O2 production and DNA content was analyzed by flow cytometry. Lipid peroxidation was assayed by colorimetry. In our study, we found that ECG dose-dependently attenuated UVB-induced keratinocyte death. Moreover, ECG markedly inhibited UVB-induced cell membrane lipid peroxidation and H2O2 generation in keratinocytes, suggesting that ECG can act as a free radical scavenger when keratinocytes were photodamaged. In parallel, H2O2-induced the activation of ERK1/2, p38 and JNK in keratinocytes could be inhibited by ECG. UVB-induced pre-G1 arrest leading to apoptotic changes of keratinocytes were blocked by ECG. Taken together, we provide here evidence that ECG protects keratinocytes from UVB-induced photodamage and H2O2-induced oxidative stress, possibly through inhibition of the activation of ERK1/2, p38 and JNK and/or scavenging of free radicals.

The aqueous extract of Psidium guajava L. (PE) inhibited the cancer cell DU-145 in a dose- and time-dependent manner. At 1.0 mg/mL, PE reduced the viability of PCa DU-145 (the androgen independent PCa cells) to 36.1 and 3.59%, respectively after 48 h and 72 h of incubations. The absolute cell viability suppressing capability (VSC)(AC) could reach 262.5 cells-mL-h/mg on exposure to PE for 72 h, corresponding to the safe ranges, i.e. the percent viability suppressing rates (PVSR) of 2.72 and 2.41 folds for DU-145 comparing to PZ-HPV-7 cells when treated with PE at 0.5 and 1.0 mg/mL respectively for 72 h. In addition, the colony forming capability of DU-145 cells was apparently lowered. The suppressing rates of which reached 8.09 and 5.96 colony/mg/day for D-145 and PZ-HPV-7 cells, respectively within the concentration range of PE at 0.1 asymptotically equal to 0.25 mg/mL. Cell cycle arrests at G0/G1 phase in both cells were observed by TUNEL assay and flow cytometric analysis, yet more prominently evident in DU-145. In addition, suppression of the matrix metalloproteinases MMP-2 and MMP-9, and the upregulation of active caspase-3 at 0.10 to 1.0 mg/mL in DU-145 were also effected in a dose-dependent manner by PE at 0.25 to 1.0 mg/mL, implicating a potent anti-metastasis power of PE. Conclusively, we ascribe the anticancer activity of PE to its extraordinarily high polyphenolic (165.61 +/- 10.39 mg/g) and flavonoid (82.85 +/- 0.22 mg/g) contents. Furthermore, PE might be useful for treatment of brain derived metastatic cancers such as DU-145, acting simultaneously as both a chemopreventive and a chemotherapeutic.

Background/purpose To estimate the age-specific prevalence rates of phimosis and circumcision in an urban sample of Taiwanese boys. Methods A convenience sample of 1145 boys aged from 7 to 13 years was enrolled and cross-sectionally evaluated for preputial retractability and status of circumcision. Another convenience sample of 59 newborn male infants was enrolled from the infant room of a city municipal hospital. These infants were examined for preputial development at birth. Results None of the newborn male infants had a completely retractable prepuce (i.e. type 3). The prevalence rate of type 3 prepuce increased with age from 71.7% (95% confidence interval [CI], 66.5-75.5%) for boys aged 7 years to 72.4% (95% CI, 67.3-77.0%) for boys aged 10 years and 84.1% (95% CI, 79.6-88.0%) for boys aged 13 years. In contrast, the prevalence rate of type 1 prepuce decreased with age from 83.1% (95% CI, 71.0-91.6%) for newborn infants to 0.3% (95% CI, 0.0001-1.8%) for boys aged 13 years. On the other hand, the prevalence of circumcision slightly increased with age from 7.2% (95% CI, 5.3-10.8%) for boys aged 7 years to 8.7% (95% CI, 6.5-13.3%) for boys aged 13 years. Conclusion Nonretractability of the prepuce was very common among the Taiwanese newborns. Among the school boys, the degree of preputial separation and exposure of glans increased with age and progressed even more rapidly in adolescence. Very few boys still suffered from unretractable prepuce by the age of 13. [ J Formos Med Assoc 2007;106(4):302-307]

It has been reported that ultraviolet B (UVB) irradiation causes the loss of E-cadherin of melanocytes, leading them to escape from neighboring keratinocytes during melanoma development. However, little has been paid on its effect on E-cadherin of keratinocytes. In the present study we therefore focus on whether UVB affects expression of E-cadherin-catenin complex in human HaCaT keratinocytes. We found that E-cadherin, beta-, and gamma-catenin but not alpha-catenin were proteolytically cleaved in UVB-irradiated HaCaT keratinocytes. The effect was only observed in keratinocyte undergoing apoptosis. Cleavage of beta- and gamma-catenin was fully abolished by caspase-3 and caspase-8 inhibitors, whereas cleavage of E-cadherin was inhibited by neither caspase nor metalloproteinase inhibitors. Functional analysis showed that the cleavage resulted in the disruption of the physical association between E-cadherin and catenins, indicating that E-cadherin signaling was compromised in UVB-irradiated HaCaT keratinocytes. Because E-cadherin in keratinocytes plays important roles in mediating cell-cell adhesion in epidermis of skin, the loss of E-cadherin and signaling components in keratinocytes may lead to the disruption of skin integrity after UVB exposure.

Introduction: Those patients featuring a large prostate and undergoing transurethral resection typically face a longer duration of surgery, they have a higher degree of hemorrhage and a higher irrigant volume required for the procedure, and they tend to reflect a higher incidence of morbidity than is the case for a small prostate. We report on the relative safety and efficacy of transurethral electrovapor resection of the prostate (TUVRP) as compared with standard transurethral resection of the prostate (TURP) for Taiwanese patients having a prostate size >50 ml. Patients and Methods: Seventy-six symptomatic benign prostatic hyperplasia patients featuring a prostate size >50 ml were randomized and underwent either TUVRP using wedge loop or standard TURP. Perioperative parameters, treatment outcome, and adverse events associated with the surgical procedure are assessed herein. Results: Mean operation time, changes in hemoglobin levels, resected prostate weight, perioperative irrigant volume needed, and incidences of recatheterizations and readmissions, all differed significantly when the two surgical procedures were compared. The average expense differences for the two procedures were impressive (p < 0.0001). No postoperative intergroup sexual dysfunction differences were noted. Clinical improvements following either TUVRP or TURP were significant and sustained for at least a period of 2 years. Intergroup comparison of International Prostate Symptom Score, quality of life, peak flow rate, and postvoid residual volume revealed no significant differences at 2 years (p = 0.45, 0.48, 0.12, and 0.29, respectively). The need for postoperative medical retreatment and/or reoperation did not differ significantly between the two groups (p = 0.62 and p = 0.56, respectively, at 6 months and p = 0.47 and p = 0.48, respectively, at 2 years). Conclusions: The TUVRP procedure offers advantages as regards a number of perioperative parameters when compared with the TURP procedure, and it seems that TUVRP might provide at least an equivalent treatment outcome as is the case for TURP.

It is increasingly evident that the stromal cells are involved in key metastatic processes of melanoma and some malignant solid tumors. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, has been shown to have anti-tumor activity, inhibiting adhesion, migration, and proliferation of tumor cells. However, little attention has been paid on its effects on stromal cells. In the present study, we determined the effects of EGCG on stromal fibroblasts. We showed that fibroblast adhesion to collagen, fibronectin, and fibrinogen were inhibited by EGCG. One of the possible mechanisms is binding of EGCG to fibronectin and fibrinogen but not to collagen. We then focused how EGCG affected fibroblast adhesion to collagen. EGCG treatment attenuated the antibody binding to fibroblast's integrin alpha2beta1, indicating EGCG may affect the expression and affinity of integrin alpha2beta1. Moreover, intracellular H2O2 level was decreased by EGCG treatment, suggesting that the tonic maintenance of intracellular H2O2 may be required for cell adhesion to collagen. In parallel, collagen-induced FAK phosphorylation, actin cytoskeleton reorganization in fibroblasts, migration and matrix metalloproteinase(s) (MMPs) activity were also affected by EGCG. Tubular networks formed by melanoma cells grown on three-dimensional Matrigel were also disrupted when fibroblasts were treated with EGCG in a non-contact coculture system. Taken together, we provided here the first evidence that EGCG is an effective inhibitor on behaviors of the stromal fibroblasts, affecting their adhesion and migration. The inhibitory activity of EGCG may contribute to its anti-tumor activity. The findings and concepts disclosed here may provide important basis for a further experiment towards understanding tumor-stroma interaction.

BACKGROUND In Taiwan, an area with a very low incidence of cystic fibrosis (CF), we first screened for the most common mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and looked for clinical correlations in 27 patients with clinically diagnosed congenital bilateral absence of the vas deferens (CBAVD). METHODS AND RESULTS The clinical results showed that none of the 27 patients had CF symptoms. We did not detect any definite renal anomaly ultrasonographically. Mutation analysis was carried out on these 27 cases and 46 normal fertile males as controls. No mutations of Delta F508 or R117H were identified in any of the samples analysed. In the screening of IVS8-poly T, five of the 27 CBAVD patients showed the homozygous genotype for 5T/5T, 14 showed the heterozygous genotype for 5T/7T and eight showed the homozygous genotype for 7T/7T. The frequency of 5T alleles was 44.4%, which was significantly higher than in the 46 normal fertile males, for which there was a 5T frequency of 5.4%. CONCLUSIONS The absence of major mutations of CFTR genes could be related to the much lower CF incidence in Taiwan. Further investigations into differences in the mutation spectrum of other CFTR genes are needed for a better understanding of the development of Taiwanese-Oriental CBAVD.

To investigate whether the therapeutic effect of Ginkgo biloba extract (GBE) in a rat model can improve erectile dysfunction after bilateral cavernous nerve injury.Forty-three male Sprague-Dawley rats underwent cavernous nerve crush injury and were randomized into 4 groups, including: vehicle only, high-dose GBE, medium-dose GBE, and low-dose GBE. Eight animals underwent sham operation. Four weeks later, erectile function was assessed by cavernous nerve electrostimulation, and penile tissue was collected for histologic analysis.Significant recovery of erectile function was observed in the high-dose GBE group in a dose-dependent manner as compared with the vehicle-only group (P.001). The high-dose GBE group had a significant increase in neurofilament-1 expression (P.001), preservation of neural nitric oxide synthase nerve fibers of the dorsal penile nerve (P.05), and increased smooth muscle cell content (P.001) compared with the vehicle-only group. In addition, high-dose GBE markedly augments the smooth muscle-to-collagen ratio (P.05) and reduces the apoptotic index.Administration of GBE increases neuron survival and preserves the neural nitric oxide synthase nerve fiber and contents of the corpus cavernosum after bilateral cavernous nerve injury. These implications indicate the beneficial effects of GBE use in the repair of the cavernous nerve and recovery of erectile function after radical prostatectomy.

Purpose: We evaluated the long-term results of penile prosthesis implantation during the last 10 years. Special considerations were examined concerning Asian corporeal diameter, device survival and sociocultural factors influencing patient-partner satisfaction.Materials and Methods: We followed 331 patients who underwent penile prosthesis implantation from December 1985 to May 1996. Surgical data, postoperative complications and information from a survey on satisfaction are reported.Results: The most prevalent corporeal diameter in our patients was 11.0 to 11.5 cm. Postoperatively wound infection was the most serious surgical complication. The 5-year device survival was similar in the malleable and inflatable types except for a poorer outcome with the self-contained inflatable Hydroflex † †American Medical Systems, Minnetonka, Minnesota. penile prosthesis. Overall patient satisfaction with surgery was 86.6%.Conclusions: Penile prosthesis implantation still has its place as definitive therapy in some patie...

The percutaneous delivery of PGE1 and its alkyl esters in alcoholic saline solution through hairless mouse skin was compared. The quantification of alkyl esters was based on the same principle as that for PGE1, which was converted to PGB1 to enhance the sensitivity and minimize the interference. Results showed that it was PGE1 that appeared in the receiver compartment for all alkyl esters examined. The flux of all alkyl esters of PGE1 in the same concentration was higher than PGE1 itself at most of saline vehicle with various fractions of alcohol. The maximal flux for a fixed concentration of each alkyl ester appeared at different fractions of alcohol. When the fractions of alcohol was kept constant, the alkyl ester that showed the maximal flux at this concentration appeared to have a longer chain length with increasing the fraction of alcohol. But isopropyl ester deviated from this order. It was concluded that the alkyl ester derivatives promoted the penetration of PGE1 mainly as a result of enhancing the drug partitioning into the stratum corneum. The alcohol fraction that needed to achieve the maximal flux at the same concentration increased with the increase of alkyl chain length, which resulted in the decrease of solubility parameter. It is necessary to optimize the fraction of alcohol in the saline solution in order to achieve the maximal flux at a fixed concentration for these alkyl esters with different alkyl chain length.

Purpose: We review our experience with traumatic tunical rupture repair with and without simultaneous penile curvature correction.Materials and Methods: Since November 1987, 11 men 23 to 39 years old have presented to us with tunical rupture, of whom 10 underwent surgical repair. All except 1 of the 8 men injured during sexual activity reported a curved penile appearance during erection. After patient 3 in our series sustained repeat rupture 5 months postoperatively due to penile curvature the next 7 underwent simultaneous curvature correction, which since 1996 has been done using 6-zero nylon.Results: Recovery was uneventful in 2 of the 3 men who underwent simple tunical repair with 4-zero polyglactin or polyglycolic acid. All 7 of subsequent patients in whom curvature correction was performed simultaneously recovered satisfactorily with resumed erectile capability.Conclusions: Although coital position may be an important factor in tunical rupture during sexual activity, penile curvature may be contribut...

Thirty spinal cord-injured (SCI) males were studied for evaluation of their pituitary-testicular and pituitary-thyroid axes using combined luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) tests and electroejaculated semen analyses. Thirty age-matched normal male volunteers served as controls. There were four subjects with low serum triiodothyronine (T3) levels, one with elevated serum follicle-stimulating hormone (FSH) level, eight with elevated serum testosterone levels, and 11 with elevated serum prolactin levels. There were significantly elevated luteinizing hormone (LH) responses to LHRH in SCI subjects when compared with normal controls. There were 16 (53.3%) SCI subjects who had exaggerated and/or prolonged LH responses. Among them, six subjects also had elevated FSH responses. There were eight and four subjects whose thyrotropin (TSH) and prolactin responses to TRH were exaggerated, respectively. Marked impaired motility was observed in 56 electroejaculated semen samples from 16 SCI subjects. There was a significant correlation between LH and total sperm count. Our data suggest that there is a reduced central dopaminergic tone in SCI subjects.

A series of 21 patients who underwent pelvic exenteration for local advanced or recurrent gynecological malignancies and urinary diversions was retrospectively reviewed. The major disease process was carcinoma of the cervix. Early complications included sepsis with wound infection and dehiscence in 4 patients, intestinal insuffency in 2 patients, 4 urinary fistulas and 3 ureteral obstructions. Six patients had a late urinary complication of stenosis of the ureterointestinal anastomosis; stones in the urinary reservoir and pyelonephritis were also noted. Factors responsible for these complications are identified and discussed with a view to reducing the morbidity related to urinary diversion in such patients.

Male infertility affects approximately 50% of all infertile couples. The male-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile or immature sperm, and sperm with structural defects such as those caused by premature chromosomal condensation and DNA damage. Our previous studies based on a knockout mice model indicated that SEPT12 proteins are critical for the terminal morphological formation of sperm. SEPT12 mutations in men result in teratozospermia and oligozospermia. In addition, the spermatozoa exhibit morphological defects of the head and tail, premature chromosomal condensation, and nuclear damage. However, the molecular functions of SEPT12 during spermatogenesis remain unclear. To determine the molecular functions of SEPT12, we applied a yeast 2-hybrid system to identify SEPT12 interactors. Seven proteins that interact with SEPT12 were identified: SEPT family proteins (SEPT4 and SEPT6), nuclear or nuclear membrane proteins (protamine 2, sperm-associated antigen 4, and NDC1 transmembrane nucleoproine), and sperm-related structural proteins (pericentriolar material 1 and obscurin-like 1). Sperm-associated antigen 4 (SPAG4; also known as SUN4) belongs to the SUN family of proteins and acts as a linker protein between nucleoskeleton and cytoskeleton proteins and localizes in the nuclear membrane. We determined that SEPT12 interacts with SPAG4 in a male germ cell line through coimmunoprecipitation. During human spermiogenesis, SEPT12 is colocalized with SPAG4 near the nuclear periphery in round spermatids and in the centrosome region in elongating spermatids. Furthermore, we observed that SEPT12/SPAG4/LAMINB1 formed complexes and were coexpressed in the nuclear periphery of round spermatids. In addition, mutated SEPT12, which was screened from an infertile man, affected the integration of these nuclear envelope complexes through coimmunoprecipitation. This was the first study that suggested that SEPT proteins link to the SUN/LAMIN complexes during the formation of nuclear envelopes and are involved in the development of postmeiotic germ cells.

We compared the 2-year safety and efficacy of two transurethral resection techniques-transurethral vapor resection of the prostate (TUVRP) and conventional loop transurethral resection of the prostate (TURP)-in the surgical management of benign prostatic hyperplasia.Between August 1997 and September 2002, 441 patients underwent transurethral prostatectomy, either TUVRP (n = 221) or TURP (n = 220). TUVRP was performed using a "RollerLoop" resection loop. All patients were assessed preoperatively by means of International Prostate Symptom Score (IPSS), quality of life (QOL) score, prostate volume, peak urinary flow (Qmax) and post-void residual volume (PVR) measurements and a sexual function questionnaire. Patients were followed up for 3, 6, 12 and 24 months after surgery, and this was followed by a comparison of the incidences of sexual dysfunction, complications and re-treatment.After 2 years of follow-up, no differences were noted between the TUVRP and TURP groups with respect to average IPSS (p = 0.9), QOL scores (p = 0.56), Qmax (p = 0.89) or PVR (p = 0.55), as well as the incidences of bladder neck contracture or urethral stricture (p = 0.34), re-treatment (p = 0.49) or sexual dysfunction (p = 0.57). However, significant reductions in operative time (p = 0.005), decrease in serum hemoglobin levels (p0.001), catheterization time (p0.001), postoperative hospital stay (p0.001) and hospitalization costs (p0.001) were observed in the TUVRP group compared to the TURP group.Our results suggest that TUVRP provides equivalent safety and efficacy to TURP during a 2-year follow-up period, in which short-term advantages in perioperative morbidity and cost savings were also demonstrated.

High levels of (+)-catechin are found in the skin and seed of many fruits such as apples and grapes. Dietary supplementation with (+)-catechin has been demonstrated to protect epidermal cells against damage induced by ultraviolet B (UVB) radiation. However, the underlying mechanisms are not well understood yet. To determine whether (+)-catechin protects keratinocytes from UVB-induced damage, the viability of UVB- and H2O2-treated cells was determined by cell viability assay. Intracellular H2O2 level was measured by flow cytometry. UVB- or H2O2-induced signaling pathways were detected by Western blotting. The results indicated that (+)-catechin inhibited UVB- and H2O2-induced keratinocyte death. In parallel, intracellular H2O2 generation in keratinocytes irradiated by UVB was inhibited by (+)-catechin in a concentration-dependent manner. (+)-Catechin also inhibited UVB- and H2O2-induced JNK activation in keratinocytes. However, it had little inhibitory effect on UVB- and H2O2-induced ERK and p38 activation even at a higher concentration, suggesting indirectly that JNK activation is required for the induction of apoptosis in keratinocytes exposed to UVB. Finally, we compared the cytotoxicity of (+)-catechin and (−)-epigallocatechin-3-gallate (EGCG) on keratinocytes. Cell viability assay showed that (+)-catechin was relatively nontoxic at higher doses. Taken together, our results demonstrate that (+)-catechin inhibits UVB- and oxidative stress-induced H2O2 production and JNK activation and enhances human keratinocyte survival. However, although it seems that (+)-catechin and EGCG are equally effective in preventing keratinocyte death, (+)-catechin is relatively nontoxic and thus is suitable for developing as an anti-ageing agent for skin care.

(−)-Epigallocatechin-3-gallate (EGCG), a constituent of green tea, has been extensively studied and shown to be a powerful antioxidant protecting skin cells against photodamage. In this study, however, we demonstrated that another gallated catechin, (−)-epicatechin-3-gallate (ECG), was also able to protect human keratinocytes against damage induced by ultraviolet A (UVA) light. We found that ECG dose-dependently inhibited UVA-induced keratinocyte death as determined by cell viability assay. Moreover, ECG had similar potency to EGCG in inhibiting UVA-induced cell death. Therefore, the mechanism of action of ECG was further investigated. As assayed by flow cytometry, UVA-induced hydrogen peroxide (H2O2) production in keratinocytes was inhibited by ECG in a concentration-dependent manner, suggesting that ECG can act as a free radical scavenger while keratinocytes were photodamaged. The scavenging effect of ECG was confirmed by the fact that ECG treatment attenuated cell damage induced by H2O2 and hypoxanthine-xanthine oxidase. In a parallel experiment, UVA-induced activation of extracellular signal-regulated kinase in keratinocytes was blocked by ECG. We provided here the first evidence that ECG is a potent protectant that protects keratinocytes from photodamage. Because ECG is abundant in green tea, we believe that this compound is beneficial for skin care.

We describe the 2 unique Asian cases of pure urinary bladder lymphoepithelioma-like carcinoma (LELCA). One patient had metastatic pelvic lymphoadenopathy and the other with superficial tumor. The EBER in situ hybridization both showed negative results despite racial or geographical factors might influence the peculiar susceptibility of individuals of Asian ancestry to Epstein-Barr virus-associated lymphoepithelioma-like carcinoma. The 2 patients were treated pertinently with satisfactory outcomes and intravesical BCG instillation is performed to prevent superficial LELCA recurrence rationally.

Objectives We applied interphase fluorescence in situ hybridization (FISH) to testis sections to examine the evidence of spermatogenesis in patients with nonobstructive azoospermia. This technique was evaluated and compared with conventional testicular histopathologic findings for the possibility of additional clinical applications. Methods Thirty-seven consecutive patients with nonobstructive azoospermia were carefully evaluated clinically. Testes were biopsied for both sperm extraction and histopathologic examination. FISH staining was performed with a CEP 18 SpectrumAqua/CEP X SpectrumGreen/CEP Y SpectrumOrange probe. Results Eight of 11 cases (sensitivity 73%) that were found to have spermatids on the histopathologic slides also were proven to produce haploid cells by FISH staining. On the other hand, 21 of the 26 cases (specificity 81%) for which no spermatids could be found on the histopathologic slides also had only diploid cells by FISH staining. On the basis of the good correlation between the FISH staining and conventional histopathologic findings, we could confirm the diagnosis of spermatogenesis using both methods. Conclusions FISH staining of testicular sections allows more reliable prediction of spermatogenesis and provides benefits for a patient’s decision regarding fertility counseling.

Growth arrest-specific (Gas) genes are expressed during serum starvation or contact inhibition of cells grown in culture. Here we report the isolation and characterization of Gas8, a novel gene identified on the basis of its growth arrest-specific expression in murine fibroblasts. We show that production of Gas8 mRNA and protein occurs in adult mice predominantly in the testes, where expression is regulated during postmeiotic development of male gametocytes. Whereas a low level of Gas8 mRNA was detected by Northern blotting in testes of murine male neonates and young adolescents, Gas8 mRNA increased rapidly postmeiotically. In adult males, both Gas8 mRNA and protein reached steady state levels in testes that were 10-fold higher than in other tissues. Immunohistochemical analyses showed that Gas8 protein accumulates in gametocytes as they approach the lumen of seminiferous tubules and is localized to the cytoplasm of round spermatids, the tails of elongating spermatids, and mature spermatid tail bundles protruding into the lumen; in epididymal spermatozoa Gas8 protein was present in the flagella. However, premeiotic murine gametocytes lacked detectable Gas8 protein, as did seminiferous tubules in biopsy specimens from seven human males having cytological evidence of non-obstructive azoospermia secondary to Sertoli cell-only syndrome. Our findings, which associate Gas8 production developmentally with the later stages of spermatogenesis and spatially with the sperm motility apparatus, collectively suggest that this growth arrest-specific gene product may have a role in sperm motility. This postulated role for Gas8 is supported by our observation that highly localized production of Gas8 protein occurs also in the cilia of epithelial cells lining pulmonary bronchi and fallopian tubes and by the flagellar association of a Trypanosoma brucei ortholog of Gas8.

The optimization of percutaneous delivery of PGE1 and its ethyl ester in alcoholic buffer solution through hairless mouse skin was investigated. A reversed-phase HPLC system with a photodiode array detector was used to differentiate the UV spectra of the penetration products. By comparison of the UV spectrum for each chromatographic peak, the conversion of PGE1 ethyl ester to PGE1 by enzymatic hydrolysis was found to be the predominant degradation pathway during the in vitro penetration. The quantification of ethyl ester was developed based on the same principle as that for PGE1. It was then applied to monitor the penetration of prostaglandins through hairless mouse skin from the vehicles containing various fractions of alcohol. Results demonstrated that the alkyl group promoted the penetration mainly as a result of enhancing the drug partitioning into the stratum corneum at its maximal thermodynamic activity. The alcohol fraction around 20% seemed to be optimal for the percutaneous delivery of the ethyl ester. The use of collagen gel to carry PGE1 ethyl ester for percutaneous application was included for comparison. The effect of adding alcohol in the collagen gel on the penetration of PGE1 ethyl ester was found to be slightly lower than that from the same vehicle without collagen.