Molecular cloning, characterization of porcine IZUMO1, an IgSF family member.

IZUMO1, belonging to the family of mammalian immunoglobulin proteins, has been well characterized in the mouse. Here, we describe the molecular cloning and expression analysis of porcine IZUMO1 (pIZUMO1). Partial sequence information published in the National Center for Biotechnology Information (NCBI) database was used to generate the full-length sequence for IZUMO1 using rapid amplification of cDNA ends (RACE). A search of the porcine genomic sequence in the NCBI database identified a bacterial artificial chromosome (BAC) encoding the pIZUMO1 gene. This BAC is derived from porcine chromosome 6 and is syntenic with the corresponding regions of mouse, bovine, and human genomes encoding the IZUMO gene family. This BAC was found to encode an IZUMO1 protein with a predicted amino acid sequence having high similarity with mouse and human IZUMO1. Western blot analysis of proteins from porcine tissues indicated that pIZUMO1 was specifically expressed in the sperm. Furthermore, to confirm whether pIZUMO1 forms complexes, we overexpressed pIZUMO1 in HEK293 cells. The recombinant pIZUMO1 from cell extracts was found to form complexes. Our finding suggests that pIZUMO1 forms homodimeric complex on the sperm membrane. Furthermore, an IVF inhibition assay with an antibody for the porcine IZUMO1 Ig-like domain showed that Ig-like domain effectively prevented pig sperm-egg interactions.

Development of a surface conductivity measurement system for ultrahigh vacuum transmission electron microscope.

The surface conductivity measurement system using a micro-four-point probe (M4PP) had been developed for the ultrahigh vacuum transmission electron microscope (UHV-TEM). Since the current distribution in the sample crystals during the current voltage measurement by the M4PP is localized within the depth of several micrometers from the surface, the system is sensitive to the surface conductivity, which is related with the surface superstructure. It was installed in the main chamber of the TEM and the surface conductivity can be measured in situ. The surface structures were observed by reflection electron microscopy and diffraction (REM-RHEED). REM-RHEED enables us to observe the surface superstructures and their structure defects such as surface atomic steps and domain boundaries of the surface superstructure. Thus the effects of the defects on the surface conductivity can be investigated. In the present paper we present the surface conductivity measurement system and its application to the Si(111)-square root(3) x square root(3)-Ag surface prepared on the Si(111) vicinal surfaces. The result clearly showed that the surface conductivity was affected by step configuration.

Human round spermatids from azoospermic men exhibit oocyte-activation and Ca2+ oscillation-inducing activities.

During mammalian fertilization, intracellular Ca2+ oscillations are important for both oocyte activation and embryonic development. As the ability of round spermatids (ROS) to induce Ca2+ oscillations and oocyte activation is different between species, we examined Ca2+ oscillation- and oocyte activation-inducing abilities of human ROS originating from patients with non-obstructive azoospermia. Human ROS from 11 non-obstructive azoospermic patients were collected during their TESE-ICSI cycles. Following injection into mature unfertilized mouse oocytes, we examined the oocyte-activating and Ca2+ oscillation-inducing activities of ROS by using Ca2+ imaging and confocal laser scanning microscopy (mouse test). In these 11 cases, clinical TESE-ICSI using mature testicular spermatozoa was successful, with the exception of one case in which only one sperm-injected oocyte was not fertilized. The mean fertilization rate was 70.1% (40-100%); the mean cleavage rate was 97.9% (46/47). Two pregnancies were established from 10 transfer cycles (PR; 20%). When the ROS from these patients were injected into mouse oocytes, the ROS from all patients induced at least some intracellular Ca2+ oscillations (25-100%). In all patients, 40 out of 82 oocytes injected with ROS exhibited normal oscillation patterns of [Ca2+]i. Human spermatogenetic cells acquired oocyte-activating and Ca2+ oscillation-inducing abilities at the round spermatid stage, an earlier stage than found for rodent cells. These data indicate that human ROS might be useful for clinical treatments of non-obstructive azoospermic patients exhibiting mature spermatozoa in biopsied specimens.

Controlling the dissociative ionization of ethanol with 800 and 400 nm two-color femtosecond laser pulses.

Ethanol molecules were irradiated with a pair of temporally overlapping ultrashort intense laser pulses (10(13)-10(14) Wcm(2)) with different colors of 400 and 800 nm, and the dissociative ionization processes have been investigated. The yield ratio of the C-O bond breaking with respect to the C-C bond breaking was varied in the range of 0.17-0.53 sensitively depending on the delay time between the two laser pulses, and the absolute value of the yield of the C-O bond breaking was found to be increased largely when the Fourier-transform limited 800 nm laser pulse overlaps the stretched 400 nm laser pulse, demonstrating an advantage of the two-color intense laser fields in controlling chemical bond breaking processes.

Dissociative ionization of ethanol by 400 nm femtosecond laser pulses.

The dissociative ionization of ethanol in short-pulsed laser fields at approximately 400 nm is investigated. The yield ratio of the C-O bond breaking with respect to the C-C bond breaking increases sharply as the temporal width increases from 60 to 400 fs, and the yield ratio is two to three times as large as that at 800 nm in the entire pulse-width range of 60-580 fs. The enhancement of the C-O bond breaking of singly charged ethanol at 400 nm and the bond elongation prior to the Coulomb explosion of doubly charged ethanol occurring in the relatively weak light field intensity of 10(12)-10(13) W cm(2) is interpreted by the efficient light-induced coupling among the electronic states at the shorter wavelength of 400 nm. From the double pulse experiment, in which ethanol is irradiated with a pair of short pulses (<80 fs), the most efficient coupling occurs at Deltat=160 fs that is much earlier than Deltat=250 at 800 nm, where Deltat denotes the temporal separation of the two pulses, indicating that the nonadiabatic field-induced potential crossings of singly charged ethanol occurs much earlier at 400 nm than at 800 nm.

Hydrodynamics-based gene delivery of naked DNA encoding fetal liver kinase-1 gene effectively suppresses the growth of pre-existing tumors.

Antiangiogenic gene therapy is a promising strategy for cancer treatment, which generally requires highly efficient delivery systems. To date, success of this strategy has depended almost exclusively on the delivery of high titers of viral vectors, which can result in effective transgene expression. However, their cytotoxicity and immunogenicity are a major concern for clinical applications. Recent advances in delivery efficiency of naked DNA could potentially meet the requirement for both high transgene expression and minimal side effects. To investigate whether naked DNA can be used for antiangiogenic cancer therapy, an expression plasmid was generated that encodes a soluble form of fetal liver kinase-1 (Flk-1) gene, a receptor for vascular endothelial growth factor (VEGF). Hydrodynamic injection of this plasmid resulted in close to 0.1 mg/ml of soluble Flk-1 protein in mouse serum and blocked VEGF-driven angiogenesis in matrigel in vivo. The same delivery significantly suppressed the growth of two different pre-existing subcutaneous tumors, Renca renal cell carcinoma and 3LL lung carcinoma. CD31 immunohistochemistry revealed that the tumor-associated angiogenesis was also highly attenuated in soluble Flk-1-treated mice. Thus, expression of genes by hydrodynamics-based gene delivery of naked DNA appears to be a promising approach for antiangiogenic cancer gene therapy.

Results and complications of laparoscopic Palomo varicocelecctomy.

The laparoscopic Palomo varicocelectomy were performed in 38 males with left-sided varicocele. The mean operation time was 37 (25-56) min. There were no intra-abdominal visceral or vascular complications during operation. Neither testicular atrophy nor recurrence was observed postoperatively. However, hydrocele formation was found in two (5.3%) patients. These findings suggest that laparoscopic Palomo varicocelectony is a safe and effective procedure for patients with varicocele.

Absence of microdeletions in the Y chromosome in patients with Prader-Willi syndrome with cryptorchidism.

Unilateral or bilateral cryptorchidism is found in 80-100% of male patients with Prader-Willi syndrome (PWS). Recently, the relationship between Yq deletions and cryptorchidism has been assessed. However, the relationship between Yq deletions and PWS patients with cryptorchidism remains unclear. Polymerase chain reaction (PCR) amplification of 51 DNA loci encompassing all of the regions for azoospermia factor (AZF) of the Y chromosome, including the deleted in azoospermia (DAZ) and ribonucleic acid-binding motif (RBM) genes, were examined for microdeletions in 10 PWS males with cryptorchidism and 20 healthy control male subjects. No microdeletions of 51 loci were found in any of the PWS males. The present study therefore suggests that microdeletions in the AZF regions of the Y chromosome do not relate to the occurrence of cryptorchidism in PWS patients.

Beta amyloid peptide (Abeta42) is internalized via the G-protein-coupled receptor FPRL1 and forms fibrillar aggregates in macrophages.

The 42 amino acid form of beta amyloid (Abeta42) plays a pivotal role in neurotoxicity and the activation of mononuclear phagocytes in Alzheimer's disease (AD). Our recent study revealed that FPRL1, a G-protein-coupled receptor, mediates the chemotactic and activating effect of Abeta42 on mononuclear phagocytes (monocytes and microglia), suggesting that FPRL1 may be involved in the proinflammatory responses in AD. We investigated the role of FPRL1 in cellular uptake and the subsequent fibrillar formation of Abeta42 by using fluorescence confocal microscopy. We found that upon incubation with macrophages or HEK293 cells genetically engineered to express FPRL1, Abeta42 associated with FPRL1 and the Abeta42/FPRL1 complexes were rapidly internalized into the cytoplasmic compartment. The maximal internalization of Abeta42/FPRL1 complexes occurred by 30 min after incubation. Removal of free Abeta42 from culture supernatants at 30 min resulted in a progressive recycling of FPRL1 to the cell surface and degradation of the internalized Abeta42. However, persistent exposure of the cells to Abeta42 over 24 h resulted in retention of Abeta42/FPRL1 complexes in the cytoplasmic compartment and the formation of Congo red positive fibrils in macrophages but not in HEK 293 cell transfected with FPRL1. These results suggest that besides mediating the proinflammatory activity of Abeta42, FPRL1 is also involved in the internalization of Abeta42, which culminates in the formation of fibrils only in macrophages.

Effect of testosterone replacement therapy on serum PSA in patients with Klinefelter syndrome.

The effect of testosterone replacement therapy on serum prostate-specific antigen (PSA) was investigated in 11 patients with Klinefelter syndrome. Significant increases in serum testosterone level and prostate volume were observed after testosterone replacement therapy. However, serum PSA level did not change after testosterone replacement therapy. It would appear that serum PSA is not influenced by exogenous testosterone in patients with Klinefelter syndrome.

Stress and testicular germ cell apoptosis.

Chronic immobilization stress provokes an increase in serum corticosterone, which causes the decline in testosterone concentration. The stress and glucocorticoid administration induce germ cell apoptosis in rat testes. The cell type that has been shown to undergo apoptosis is spermatogonia. Since a potent glucocorticoid receptor agonist completely suppressed glucocorticoid induced germ cell apoptosis, the regulation of transcription of gene mediated by the glucocorticoid receptor may enhance apoptosis of testicular germ cells. The apoptosis of testicular germ cells would involve certain specific gene activities and proteins, including Bcl-2 family, p53, and Fas. Molecular investigations may help to determine whether the different pathways possess mechanisms of germ cell apoptosis induced by somatic stress and glucocorticoids.

Successful treatment of a patient with lymphomatoid papulosis by methotrexate.

We report a case of lymphomatoid papulosis (LyP) that occurred in a 44-year-old Japanese male patient. Reddish papules with a small number of pustules and nodules were observed on the extremities, chest and upper back. Most lesions were also associated with central necrosis, ulceration and crusting, and regressed spontaneously within 4 to 6 weeks. Histopathological examination revealed wedge-shaped dense cellular infiltrate in the dermis, which was mixed with large atypical lymphoid cells, small lymphocytes, eosinophils and neutrophils. These large atypical cells expressed CD30 on their cell membrane and cytoplasm. Rearrangement of the T-cell receptor (TcR) beta-chain gene was detected in the skin lesion. Lymphadenopathy with histopathologic change similar to the skin lesions, but without TcR gene rearrangement, was found at the left inguinal area. Systemic administration of methotrexate (7.5-15.0 mg/week) was found to be dramatically effective in resolution of skin lesions and prevention of their recurrence.

Reduction of serum interleukin-5 levels reflect clinical improvement in patients with atopic dermatitis.

Cytokines, in particular IL-4 and IL-5, regulate IgE synthesis and eosinophil activation in atopic dermatitis (AD). To elucidate whether the serum levels of IL-4 and IL-5 are related to the serum IgE level, eosinophilia, or clinical severity of the disease, 25 cases with AD were studied. Blood samples were isolated from two groups of donors: 1) patients with AD (n = 25); 2) non-allergic individuals (NA, n = 20) with serum IgE levels below 100 IU/ml and with blood eosinophil counts below 250/microliter. Each parameter was evaluated at least twice in AD patients at the beginning of the study and after 4, 8 or 12 weeks of treatment. IL-4 was hardly detected in AD and NA, but IL-5 was increased (> 10 pg/ml) in most cases (22/25) of AD group with 513.6 pg/ml as the mean. AD with normal serum IgE levels exhibited increased levels of IL-5, whereas AD with high serum IgE levels did not necessarily have elevated IL-5 levels. The IL-5 level tended to change in parallel with the clinical severity in each AD case, although the level itself was not correlated with the clinical severity per se. A significant decrease of IL-5 was observed in AD when the clinical severity decreased. Eosinophils also decreased along with the improvement of AD, whereas the serum level of IgE did not change during the observation period. Our results suggest that IL-5 is involved in the regulation of clinical courses of AD and that its kinetics at the serum level reflects the clinical activity of AD.

Oocyte activation and Ca(2+) oscillation-inducing abilities of mouse round/elongated spermatids and the developmental capacities of embryos from spermatid injection.

To investigate differences in fertilization mechanisms and the potential clinical use of round/elongated spermatid, we conducted detailed studies of oocyte activation and Ca(2+) oscillation-inducing abilities in these immature sperm cells and compared these abilities against those of mature spermatozoa. When round spermatids from B(6)D(2)F(1) mice were injected, none of the oocytes was activated and no intracellular Ca(2+) ([Ca(2+)](i)) increases were observed. Elongated spermatids could induce activation normally in 87% of injected oocytes, but Ca(2+) oscillation could not be induced at all and most of the oocytes (94%) exhibited only several transient [Ca(2+)](i) rises (transient patterns). Because normal offspring could be obtained when embryos through elongated spermatid injection were transferred to foster mothers, it seems that a normal oscillation pattern of [Ca(2+)](i) is not essential for normal fertilization and embryo development. [Ca(2+)](i) patterns of injected oocytes changed from transient patterns to oscillation patterns while the injected immature sperm cells were maturing to spermatozoa. Dissociations were seen between the timing of appearance of oocyte activation and that of Ca(2+) oscillation-inducing abilities in maturing sperm cells. These dissociations may be due to differences in the thresholds to oocyte activation and Ca(2+) oscillation-inducing factor for inducing oocyte activation and Ca(2+) oscillation.

Glucocorticoid hormone can suppress apoptosis of rat testicular germ cells induced by testicular ischemia.

OBJECTIVE: To evaluate the effect of dexamethasone, a potent synthetic glucocorticoid hormone, on apoptosis of testicular germ cells and vascular neutrophil adhesion after repair of testicular torsion in rats. DESIGN: Controlled laboratory study. SETTING: Department of Urology, Yamagata University School of Medicine, Yamagata, Japan. ANIMAL(S): Fifty-one male Sprague-Dawley rats. INTERVENTION(S): Dexamethasone, 10 mg/kg of body weight. MAIN OUTCOME MEASURE(S): Testicular germ-cell apoptosis (percentages of apoptotic tubules and apoptotic cells) and vascular neutrophil adhesion were assessed by using DNA nick-end labeling and the endothelial-neutrophil adhesion score, respectively. RESULT(S): Intravenous administration of dexamethasone at repair of 90-minute testicular torsion significantly inhibited testicular germ-cell apoptosis and vascular neutrophil adhesion. This inhibition was suppressed by intravenous injection of mifepristone, a glucocorticoid-receptor antagonist. CONCLUSION(S): Glucocorticoids can be administered for torsion in addition to conventional torsion repair.

Sutureless vasovasostomy using new vascular closure staples in rats.

To evaluate the efficacy of sutureless vasovasostomy using vascular closure staples, 20 male Sprague-Dawley rats were used for the study. In each rat, a sutureless vasovasostomy using vascular closure staples was performed on a randomly selected side, and a conventional end-to-end microsutured anastomosis was performed on the opposite side. Operation time was significantly shorter for sutureless vasovasostomy using vascular staples (p < .001). Four weeks after the surgery, however, patency rate and granuloma formation were not different for conventional end-to-end vasovasostomy vs. sutureless vasovasostomy. These findings suggest that sutureless vasovasostomy is simple to perform and requires fewer special technical skills. This procedure may provide an alternative to the conventional vasovasostomy.

Reevaluation of testicular biopsies of males with nonobstructive azoospermia in assisted reproductive technology.

Spermatogenesis was investigated in seminiferous tubules of 100 males with nonobstructive azoospermia. Forty-four (44%) cases had Sertoli-cell-only syndrome, 23 (23%) had spermatogonium in the tubules, 17 (17%) had primary spermatocyte in the tubules, and 16 (16%) had round or late spermatid in the tubules. No cases showed secondary spermatocyte present in the tubules. The mean serum follicle-stimulating hormone (FSH) in males with nonobstructive azoospermia was significantly higher than that in males with obstructive azoospermia (p < .001). The mean concentrations of serum FSH in cases with Sertoli-cell-only syndrome and spermatogonium in the tubules were significantly higher than those in cases with primary spermatocyte and spermatid in the tubules (p < .05-.001). The results indicate that the evaluation of testicular histology using the type of germ cells present in seminiferous tubules is available for assisted reproductive technology.

The neurotoxic prion peptide fragment PrP(106-126) is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1.

Prion diseases are transmissible and fatal neurodegenerative disorders which involve infiltration and activation of mononuclear phagocytes at the brain lesions. A 20-aa acid fragment of the human cellular prion protein, PrP(106-126), was reported to mimic the biological activity of the pathologic isoform of prion and activates mononuclear phagocytes. The cell surface receptor(s) mediating the activity of PrP(106-126) is unknown. In this study, we show that PrP(106-126) is chemotactic for human monocytes through the use of a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1), which has been reported to interact with a diverse array of exogenous or endogenous ligands. Upon stimulation by PrP(106-126), FPRL1 underwent a rapid internalization and, furthermore, PrP(106-126) enhanced monocyte production of proinflammatory cytokines, which was inhibited by pertussis toxin. Thus, FPRL1 may act as a "pattern recognition" receptor that interacts with multiple pathologic agents and may be involved in the proinflammatory process of prion diseases.

Influence of sperm immobilization on onset of Ca(2+) oscillations after ICSI.

Sperm immobilization prior to intracytoplasmic sperm injection (ICSI) is thought to be necessary for efficient fertilization. A variety of methods of sperm immobilization (pipetting, squeezing and piezo application) are currently employed in ICSI. The effect of differences in immobilization method on the timing of initial Ca(2+) oscillations of oocytes in ICSI was investigated. Motile spermatozoa were immobilized in eosin Y solution using pipetting, squeezing and piezo application. Complete staining of the sperm head was achieved after 220.7, 42.2 and 5.0 s respectively. Oscillations after ICSI were measured fluorometrically for each method. The onset of Ca(2+) oscillations was observed at 4.8 to 80.4 min after ICSI. Ca(2+) oscillations developed earlier with the piezo method (14.4 +/- 6.4 min) than other methods (pipetting, 43.1 +/- 20.2 min, P < 0.01; squeezing, 18.4 +/- 3.8 min, P = NS). The piezo method produced the earliest staining of the sperm head and may have caused the most damage to the sperm membrane. A more rapid onset of Ca(2+) oscillations was also observed with the piezo method. The method of sperm immobilization may be important for the rapid release of sperm factors that initiate oocyte activation. This study also showed that Ca(2+) oscillations develop earlier in human oocytes treated by ICSI than indicated in previous reports.

Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay.

Oocyte activation and Ca2+ oscillation-inducing abilities of round spermatid (ROS) and elongated spermatid (ELS) of some rodents and human were assessed by their injection into mouse (B6D2F1) oocytes (mouse test). With mice (B6D2F1, ICR) and rat, ROS displayed no oocyte activation or Ca2+ oscillation-inducing abilities. Although ELS could induce activation at 87, 86 and 31% of injected oocytes respectively, most of the intracellular calcium concentration ([Ca2+]i) responses of ELS-injected oocytes did not show oscillation patterns; only several transient [Ca2+]i rises (transient pattern) were seen. Similarly, with hamster, rabbit and human, while ROS could induce oocyte activation efficiently (70, 71 and 52% respectively), most of the [Ca2+]i patterns of injected oocytes were transient patterns, and not oscillation patterns. When ROS nuclei only from these latter species were injected into mouse oocytes, most of the oocytes could not be activated. [Ca2+]i patterns of oocytes injected with immature sperm cells changed from transient pattern to oscillation pattern while the cells were maturing into spermatozoa. With hamster ROS, oocyte-activating factor was found to be distributed mainly in the cytoplasm. It was interesting that there is a dissociation between the timings of appearance of oocyte activation and that of Ca2+ oscillation of oocytes injected with developing immature sperm cells.