Hiromichi Ishikawa

Cadmium nephrotoxicity and evacuation from the body in a rat modeled subchronic intoxication

Background: Cadmium (Cd) is an important industrial pollutant, although its mechanism of toxicity has not been completely clarified. We studied Cd‐induced subchronic nephrotoxicity and the cadmium evacuation system in rats and cultured human renal tubular cells.

A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility

Significance Polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5) belongs to the pp-GalNAc-T family, but its in vivo activity has not yet been identified. To investigate the functions of GALNTL5, we attempted to establish Galntl5-deficient mice and found that the heterozygous mutation of Galntl5 causes infertility in male mice because of immotile sperm. In these mice, glycolytic enzymes required for sperm motility were decreased, their protein loading into acrosomes was disrupted, and aberrant localization of the ubiquitin–proteasome system was observed. We found a patient diagnosed with asthenozoospermia, poor sperm motility, who had a mutation of the GALNTL5 gene in sperm and blood cells. Our data suggest that GALNTL5 is an essential functional molecule for sperm development, and the GALNTL5 mutation may cause human asthenozoospermia. For normal fertilization in mammals, it is important that functionally mature sperm are motile and have a fully formed acrosome. The glycosyltransferase-like gene, human polypeptide N-acetylgalactosaminyltransferase-like protein 5 (GALNTL5), belongs to the polypeptide N-acetylgalactosamine-transferase (pp-GalNAc-T) gene family because of its conserved glycosyltransferase domains, but it uniquely truncates the C-terminal domain and is expressed exclusively in human testis. However, glycosyltransferase activity of the human GALNTL5 protein has not been identified by in vitro assay thus far. Using mouse Galntl5 ortholog, we have examined whether GALNTL5 is a functional molecule in spermatogenesis. It was observed that mouse GALNTL5 localizes in the cytoplasm of round spermatids in the region around the acrosome of elongating spermatids, and finally in the neck region of spermatozoa. We attempted to establish Galntl5-deficient mutant mice to investigate the role of Galntl5 in spermiogenesis and found that the heterozygous mutation affected male fertility due to immotile sperm, which is diagnosed as asthenozoospermia, an infertility syndrome in humans. Furthermore, the heterozygous mutation of Galntl5 attenuated glycolytic enzymes required for motility, disrupted protein loading into acrosomes, and caused aberrant localization of the ubiquitin–proteasome system. By comparing the protein compositions of sperm from infertile males, we found a deletion mutation of the exon of human GALNTL5 gene in a patient with asthenozoospermia. This strongly suggests that the genetic mutation of human GALNTL5 results in male infertility with the reduction of sperm motility and that GALNTL5 is a functional molecule essential for mammalian sperm formation.

Germ Cell Apoptosis in Undescended Testis: The Origin of its Impaired Spermatogenesis in the TS Inbred Rat

PURPOSE Undescended testis is one of the most important congenital anomalies in male urogenital organs that may cause male infertility. We examined the process of spermatogenesis of TS inbred rats, of which approximately 70% of male newborns have congenital undescended testes. MATERIALS AND METHODS Unilaterally affected animals at ages 4, 6 and 8 weeks were analyzed in this study. Histopathological evaluation of spermatogenesis was performed by periodic acid-Schiff-hematoxylin staining. To elucidate the pathophysiology of seminiferous tubule damage germ cell apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and electron microscopy. Four animals per group were used for staining and nick end-labeling. RESULTS Testicular weight significantly decreased on the affected side at ages 6 and 8 weeks. Impaired spermatogenesis was observed as early as age 4 weeks. Germ cell apoptosis was significantly more frequent on the affected side in all age groups with the most prominent incidence at age 6 weeks. Most apoptotic germ cells were considered spermatocytes. Electron microscopy revealed apoptosis of spermatocytes with condensation of chromatins and agglutination of cytoplasmic contents. CONCLUSIONS This study suggests the efficacy of early intervention in patients with undescended testes.

Body mass index for chronic hemodialysis patients: Stable hemodialysis and mortality

Abstract Body mass index (BMI) is used as a reference for weight control programs in the general population and in morbidity and mortality studies in diabetes patients. However, the implications of BMI in chronic hemodialysis patients is unclear. We studied the BMI of chronic hemodialysis patients, focusing on problems encountered during outpatient hemodialysis therapy and on 2‐year mortality. Outpatients with chronic hemodialysis (n = 258; 144 men, 114 women) were divided into four groups: (i) patients with stable hemodialysis; (ii) patients with marked hypotension requiring catecholamine infusion during hemodialysis; (iii) patients with excessive interdialysis weight gain requiring occasional additional hemodialysis; and (iv) patients with troublesome hemodialysis due to other causes. The statistical differences between the average BMI among these groups were evaluated, and were subdivided into sex, age and the duration of hemodialysis history. The 2‐year mortality rates of these patients were also studied according to their BMI. In patients under 60 years of age, those with excessive interdialysis weight gain had statistically larger BMI (23.2; n = 35) compared to patients with good hemodialysis control (20.1; n = 178), regardless of gender and hemodialysis history. The mortality rate was at a minimum at approximately 20 BMI in patients under 60 years of age. However, lower BMI was associated with a greater mortality rate in patients 60 years or over. For chronic hemodialysis patients, the BMI associated with stable hemodialysis and minimum mortality is approximately 20, in those under 60 years of age. The BMI of aged hemodialysis patients should be considered separately in morbidity and mortality studies.

Cadmium-induced testicular damage in a rat model of subchronic intoxication

BackgroundCadmium (Cd)-induced testicular damage in relation to spermatogenesis has not been well studied. We studied the mechanism of Cd-induced testicular damage in a rat model of subchronic intoxication.MethodsMale Sprague-Dawley rats were subcutaneously injected with 0.6 mg Cd/kg per day for 6 weeks. The concentration of Cd in urine, serum and testes was measured by using atomic absorption spectrophotometry. Testicular damage was evaluated by counting the spermatogonia (SG) and spermatocytes (SC) on one cut-surface of five seminiferous tubules in stages VII or VIII of spermatogenesis every week. The location of intratesticular cadmium was determined by using oxine-fluorescent cytochemistry.ResultsThere were no differences in the testes/bodyweight ratio between the study and control groups. The concentration of Cd in the testes increased more than 100-fold that in serum after week 2, suggesting active testicular Cd accumulation (1–3 mg/g tissue). Cadmium accumulation was detected in SG and SC. The number of SG and SC diminished significantly in the study group (week 2: SG 74%, SC 90%; week 4: SG 47%, SC 75%; week 6: SG 30%, SC 54% of the control, respectively).ConclusionsCadmium accumulated in SG and SC, consequently reduced the number of these cells, and disturbed the spermatogenesis in this rat model of subchronic Cd intoxication. Therefore, the number of SG decreased in this rat model of subchronic Cd intoxication.

Improvement of sensitivity and selectivity of high-performance liquid chromatography for anti-retroviral drugs (non-reverse transcriptase inhibitors) by diamond-electrode electrochemical and fluorescence detection

The new non-reversed transcriptase inhibitor (NRTI) drugs for treatment of acquired immunodeficiency syndrome (AIDS) are reported. An improvement in the sensitivity and selectivity of high-performance liquid chromatography was obtained by diamond electrode-electrochemical detector and fluorescence detector owing to different structural information. The four anti-retroviral NRTI drugs (abacavir, didanosine, lamivudine and zidovudine) were separated on a CapcellPak C18 UG120 column (250 mm x 4.6 mm I.D., 5 microm) with an acetonitrile-25 mM potassium dihydrogenphosphate buffer (pH 8.0; 1:9, v/v) as the mobile phase. We applied dual detection (electrochemical detection and florescence detection) for improving the peak identification and also for improved selectivity, which assisted monitoring by trace-volume samples (e.g., plasma). The electrochemical detector, equipped with a diamond electrode, was set at 2000 mV (applied voltage) and the fluorescence detector was set at excitation wavelength 275 nm and emission wavelength 315 nm. The detection limits of the four NRTIs in spiked plasma were 1-100 ng/ml by electrochemical detection and 5-10 pg/ml by fluorescence detection. The calibration graphs were linear up to 20 microg/ml by electrochemical detection and 10 microg/ml by fluorescence detection. This is the first report of LC analysis of NRTIs by electrochemical detection, also combined with fluorescence detection. The detection limits of didanosine, lamivudine and zidovudine were improved 20-fold by electrochemical detection and 500-fold by fluorescence detection compared to previous reports on UV detection. The selectivity was also improved by dual detection. The proposed method was applied to the preliminary monitoring of NRTIs in plasma.

Preliminary Monitoring of Bisphenol A and Nonylphenol in Human Semen by Sensitive High Performance Liquid Chromatography and Capillary Electrophoresis After Proteinase K Digestion

Abstract To investigate human exposure by bisphenol A (BPA) and nonylphenol (NP), BPA and NP in human semen were preliminary monitored by sensitive high performance liquid chromatography (HPLC) by fluorescence pre-labeling with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole (CDB) and capillary electrophoresis (CE) based on miceller electrokinetic chromatography after proteinase K digestion. The semen samples were collected from 57 patients who attended in vitro embryo fertilization program (IVF-ET). They were classified into nine groups according to their properties by semen character (e.g., normozoospermia) and statistical analysis (e.g., sperm count and sperm mobility). The proteinase K digestion was effective in obtaining high recovery value on the extraction process. In this time, BPA and NP were not detected in nine groups (57 samples, limits of detections = 1 and 7 pg/mL). It was suspected that BPA and NP levels in these semen were less than 1 pg/mL and 7 pg/mL, respectively. There were no significant exposures to BPA and NP on semen samples in this time experiments. Further large scale monitoring was needed for diagnosis effect of endocrine disruptor to human sperm production.

Single-Cell Pulsed-Field Gel Electrophoresis to Detect the Early Stage of DNA Fragmentation in Human Sperm Nuclei

Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.

Surgical treatment for an idiopathic renal arteriovenous fistula with a large aneurysm

Abstract Interventional embolization is currently the first line treatment for arteriovenous fistulas; however, the efficacy of repeated embolization remains controversial. A case is reported of an idiopathic renal arteriovenous fistula with large aneurysmal dilatation treated by surgery following the failure of interventional embolization. A 42‐year‐old woman was admitted to Ichikawa General Hospital, Japan, with gross hematuria containing many clots. Right renal arteriogram showed an aberrant vessel arising from the main trunk of the renal artery, which was followed by the formation of an aneurysm and rapidly ended in arteriovenous shunting. The aneurysm was approximately 35mm in size. As interventional embolization had failed, surgical treatment including extracorporeal renal surgery was performed. Postoperative renal angiography revealed excellent renal function and beautiful vascular architecture. Although the indication for surgical treatment of renal vascular disease has decreased owing to the advancement of interventional techniques, there are still some risky cases for which surgical repair should be recommended.

Copy-number variations in Y-chromosomal azoospermia factor regions identified by multiplex ligation-dependent probe amplification.

Although copy-number variations (CNVs) in Y-chromosomal azoospermia factor (AZF) regions have been associated with the risk of spermatogenic failure (SF), the precise frequency, genomic basis and clinical consequences of these CNVs remain unclear. Here we performed multiplex ligation-dependent probe amplification (MLPA) analysis of 56 Japanese SF patients and 65 control individuals. We compared the results of MLPA with those of conventional sequence-tagged site PCR analyses. Eleven simple and complex CNVs, including three hitherto unreported variations, were identified by MLPA. Seven of the 11 CNVs were undetectable by conventional analyses. CNVs were widely distributed in AZF regions and shared by ~60% of the patients and ~40% of the controls. Most breakpoints resided within locus-specific repeats. The majority of CNVs, including the most common gr/gr deletion, were identified in the patient and control groups at similar frequencies, whereas simple duplications were observed exclusively in the patient group. The results imply that AZF-linked CNVs are more frequent and heterogeneous than previously reported. Non-allelic homologous recombination likely underlies these CNVs. Our data confirm the functional neutrality of the gr/gr deletion in the Japanese population. We also found a possible association between AZF-linked simple duplications and SF, which needs to be evaluated in future studies.