Effects of testosterone replacement therapy on metabolic syndrome among Japanese hypogonadal men: A subanalysis of a prospective randomised controlled trial (EARTH study).

We investigated the effects of testosterone replacement therapy (TRT) on metabolic factors among hypogonadal men with a metabolic syndrome. From the study population of the EARTH study, which was a randomised controlled study in Japan, 65 hypogonadal patients with a metabolic syndrome, comprising the TRT group (n = 32) and controls (n = 33), were included in this study analysis. The TRT group was administered 250 mg of testosterone enanthate as an intramuscular injection every 4 weeks for 12 months. Waist circumference, body mass index, body fat volume and blood pressure were measured in all patients at baseline and at 12 months. In addition, blood biochemical data, including total cholesterol, triglyceride (TG), HDL cholesterol, fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c) levels, were also evaluated. Changes in these categories from baseline to 12 months were compared between the TRT and control groups, with significant differences observed in waist circumference, body fat percentage, FPG, TG and HbA1c levels. No significant differences were observed in other parameters. TRT for 1 year was associated with improvements in some metabolic factors among Japanese men with hypogonadism and metabolic syndrome.

SIN3A mutations are rare in men with azoospermia.

A loss of function of the murine Sin3A gene resulted in male infertility with Sertoli cell-only syndrome (SCOS) phenotype in mice. Here, we investigated the relevance of this gene to human male infertility with azoospermia caused by SCOS. Mutation analysis of SIN3A in the coding region was performed on 80 Japanese patients. However, no variants could be detected. This study suggests a lack of association of SIN3A gene sequence variants with azoospermia caused by SCOS in humans.

A novel approach inducing transplant tolerance by activated invariant natural killer T cells with costimulatory blockade.

Invariant natural killer T (iNKT) cells are one of the innate lymphocytes that regulate immunity, although it is still elusive how iNKT cells should be manipulated for transplant tolerance. Here, we describe the potential of a novel approach using a ligand for iNKT cells and suboptimal dosage of antibody for CD40-CD40 ligand (L) blockade as a powerful method for mixed chimerism establishment after allogenic bone marrow transplantation in sublethally irradiated fully allo recipients. Mixed-chimera mice accepted subsequent cardiac allografts in a donor-specific manner. High amounts of type 2 helper T cytokines were detected right after iNKT cell activation, while subsequent interferon-gamma production by NK cells was effectively inhibited by CD40/CD40L blockade. Tolerogenic components, such as CD11c(low) mPDCA1(+) plasmacytoid dendritic cells and activated regulatory T cells (Tregs) expressing CD103, KLRG-1 and PD-1, were subsequently augmented. Those activating Tregs seem to be required for the establishment of chimerism because depletion of the Tregs 1 day before allogenic cell transfer resulted in a chimerism brake. These results collectively suggest that our new protocol makes it possible to induce donor-specific tolerance by enhancement of the innate ability for immune tolerance in place of the conventional immunosuppression.

Single-nucleotide polymorphisms in the LRWD1 gene may be a genetic risk factor for Japanese patients with Sertoli cell-only syndrome.

Genetic mechanisms have been implicated as a cause of some cases of male infertility. Recently, ten novel genes involved in human spermatogenesis, including human LRWD1, have been identified by expression microarray analysis of human testictissue. The human LRWD1 protein mediates the origin recognition complex in chromatin, which is critical for the initiation of pre-replication complex assembly in G1 and chromatin organization in post-G1 cells. The Lrwd1 gene expression is specific to the testis in mice. Therefore, we hypothesized that mutation or polymorphisms of LRWD1 participate in male infertility, especially azoospermia. To investigate whether LRWD1 gene defects are associated with azoospermia caused by SCOS and meiotic arrest (MA), mutational analysis was performed in 100 and 30 Japanese patients by direct sequencing of the coding regions, respectively. Statistical analysis was performed for patients with SCOS and MA and in 100 healthy control men. No mutations were found in LRWD1; however, three coding single-nucleotide polymorphisms (SNP1-SNP3) could be detected in the patients. The genotype and allele frequencies in SNP1 and SNP2 were notably higher in the SCOS group than in the control group (P < 0.05). These results suggest the critical role of LRWD1 in human spermatogenesis.

Effects of bethanechol chloride and distigmine bromide on postvoiding residual volume in patients with underactive bladder.

AIM: The efficacy of cholinergic drugs for reduction of post-voiding residual volume (PVR) in patients with underactive bladder is still controversial. This study was performed to examine whether cholinergic drugs have such an effect on PVR. METHODS: Patients with underactive bladder treated for more than two months with cholinergic drugs, which were later discontinued, were extracted retrospectively based on their charts. The changes in PVR, cholinesterase activity (ChE), renal function, and voiding function before and after discontinuation of cholinergic drugs were reviewed and analyzed. RESULTS: Twenty-nine patients were included in this study. In multiple linear regression analysis, the discontinuation of distigmine bromide (DB) was indicated as a significant covariate for PVR increase and ChE increase, while bethanechol chloride (BC) was not a significant covariate. The increase in ChE was significantly correlated with both PVR and voided volume after discontinuation of cholinergic drugs. CONCLUSION: DB could reduce PVR via a decrease in ChE. However, BC at doses up to 60 mg did not reduce PVR. DB may be recommended for the reduction of PVR in patients with underactive bladder.

Reduced-intensity unrelated cord blood transplantation for treatment of metastatic renal cell carcinoma: first evidence of cord-blood-versus-solid-tumor effect.

We report a 69-year-old man with cytokine-resistant metastatic renal cell carcinoma treated with reduced-intensity unrelated cord blood transplantation. The patient achieved durable donor engraftment with minimal graft-versus-host disease. The patient showed regression of metastatic disease, providing the first evidence of a graft-versus-tumor effect on a solid tumor resulting from cord blood graft.

Effects of transforming growth factor-beta released from gastric carcinoma cells on the contraction of collagen-matrix gels containing fibroblasts.

To investigate the mechanisms underlying contraction of the stomach wall in cases of gastric scirrhous carcinoma, we have developed an in vitro model for gastric cancer, in which both fibroblasts and gastric carcinoma cells are embedded within a collagen matrix. Gastric carcinoma cells of the scirrhous type (KATO-III) but not the nonscirrhous type (MKN-28) markedly enhanced the ability of human intestine, human lip, and mouse kidney fibroblasts to contract collagen gels. KATO-III cells released transforming growth factor-beta (TGF-beta) into culture media in an activated form, whereas the MKN-28 cells produced a latent form. The role of TGF-beta produced by gastric cancer cells from the scirrhous type was clarified by adding TGF-beta (receptor grade) into collagen gels embedded with fibroblasts, contraction being enhanced. Other growth factors tested, including transforming growth factor-alpha and epidermal growth factor, did not enhance the contraction of collagen gels containing embedded human and rodent fibroblasts. These results suggest that the activated form of TGF-beta released from gastric scirrhous carcinoma cells stimulates fibroblasts to contract the collagenous stroma of the stomach wall, which leads to the so-called "linitis plastica" stomach condition.

Transformation of epithelial Madin-Darby canine kidney cells with p60(v-src) induces expression of membrane-type 1 matrix metalloproteinase and invasiveness.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed both in carcinoma cells and in surrounding stromal fibroblasts. MT1-MMP localizes to the surface of tumor cells and is thought to play an important role in tumor invasion. To analyze the mechanism of MT1-MMP gene expression in epithelial tumor cells, the dog kidney epithelial cell line Madin-Darby canine kidney (MDCK) was transformed by oncogenes, including v-src, and expression of MT1-MMP was examined. Transformation of MDCK cells with v-src resulted in loss of cell-to-cell contacts and morphological change. Expression of MT1-MMP in v-src-transformed cells was identified by Northern and Western blotting. Gelatin zymography analysis showed that progelatinase A in the culture medium was processed from latent to activated form by MDCK cells transformed with v-src. The MDCK cells transformed by v-src were tumorigenic in the subcutis (ectopic) and kidney (orthotopic) of nude mice and spontaneously metastasized to the lung after orthotopic implantation. These results suggest that MT1-MMP induced by v-src transformation may promote invasiveness of transformed cells.

Production of androgens and estrogens by tubular adenomas which developed in ovaries of mutant mice of Sl/Slt genotype.

Bilateral tubular adenomas develop spontaneously in ovaries of WB X C57BL/6 F1-Sl/Slt mice after exponential loss of oocytes. We investigated the ability of this tumor to produce sex steroids. Incubation of tubular adenoma tissue with [3H] progesterone resulted in production of [3H]androstenedione and [3H]-testosterone. The amount of androgens produced by the tumor tissue was much greater than that produced by the normal ovarian tissue. The concentration of testosterone in the serum of the Sl/Slt mice bearing tubular adenomas, measured by radioimmunoassay, was about five times as high as the value in the serum of the congenic +/+ mice. Although incubation of the tumor tissue with [3H]androstenedione resulted in production of 3H-estrogens, the aromatase activity of the tumor tissue was about one-fifth of that of the normal ovarian tissue. Therefore, the major sex steroid secreted by tubular adenomas seemed to be testosterone. These endocrinological features of tubular adenomas were consistent with the following pathological features of the Sl/Slt mice with the tumors: (a) although the weight of uterus of the Sl/Slt mice was about one-half of the value observed in the +/+ mice, it decreased markedly after oophorectomy; and (b) submandibular glands of the Sl/Slt mice were significantly heavier than those of the +/+ mice.

A PLK4 mutation causing azoospermia in a man with Sertoli cell-only syndrome.

About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo-like kinase 4 (PLK-4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell-only syndrome (SCOS) identified one man with a heterozygous 13-bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild-type PLK4-transfected cells, but was hampered in PLK-4-mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia.

Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility.

Many differentiating spermatogenic cells die by apoptosis during the process of mammalian spermatogenesis. However, very few apoptotic spermatogenic cells are detected by histological examination of the testis, probably due to the rapid elimination of dying cells by phagocytosis. Previous in vitro studies showed that Sertoli cells selectively phagocytose dying spermatogenic cells by recognizing the membrane phospholipid phosphatidylserine (PS), which is exposed to the surface of spermatogenic cells during apoptosis. We examined here whether PS-mediated phagocytosis of apoptotic spermatogenic cells occurs in vivo. For this purpose, the PS-binding protein annexin V was microinjected into the seminiferous tubules of normal live mice, and their testes were examined. The injection of annexin V caused no histological changes in the testis, but significantly increased the number of apoptotic spermatogenic cells as assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. The number of Sertoli cells did not change in the annexin V-injected testes, and annexin V itself did not induce apoptosis in primary cultured spermatogenic cells. These results indicate that annexin V inhibited the phagocytic clearance of apoptotic spermatogenic cells and suggest that PS-mediated phagocytosis of those cells occurs in vivo. Furthermore, the injection of annexin V into the seminiferous tubules brought about a significant reduction in the number of spermatogenic cells and epididymal sperm in anticancer drug-treated mice. This suggests that the elimination of apoptotic spermatogenic cells is required for the production of sperm.

Detection of human telomerase reverse transcriptase messenger RNA in voided urine samples as a useful diagnostic tool for bladder cancer.

Activation of telomerase and stabilization of telomeres are thought to be required for cellular immortality and oncogenesis. Telomerase activity is detected in >90% of various cancers, including urothelial cancers. Of the three subunits comprising telomerase complex, human telomerase reverse transcriptase (hTERT) is a rate-limiting determinant of the enzymatic activity of telomerase. In the present study, spontaneously voided urine specimens from 33 patients with bladder cancer and 26 without bladder lesions were examined for the expression of hTERT mRNA, and the usefulness of detecting hTERT mRNA in urine samples for screening of bladder cancer was evaluated. RT-PCR analysis revealed that approximately 80% of urinary sediments from patients with bladder cancer expressed hTERT mRNA, regardless of clinical stage or pathological grade, whereas only 4% of sediments from patients without urothelial lesions did. Interestingly, hTERT mRNA expression was observed, even in some urine samples from bladder cancer patients with negative urinary cytology. These findings suggest that the expression of hTERT in urine sample may be a useful diagnostic marker for bladder cancer.

Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer.

The activation of telomerase and stabilization of telomeres are thought to be required for cellular immortality and oncogenesis. Three major components of human telomerase--human telomerase RNA (hTERC), telomerase-associated protein (TEP1), and human telomerase catalytic subunit (hTERT)-have recently been identified. However, the roles played by these subunits in the regulation of telomerase activity are still unclear. In the present study, a total of 37 urothelial cancers, including one metastatic lesion, and adjacent normal tissues as well as cell lines derived from bladder cancers were examined for the expression of each telomerase subunit. Reverse transcription-PCR analysis revealed that more than 90% of urothelial cancers expressed hTERT mRNA, whereas less than 20% of normal adjacent tissues did. In contrast, hTERC and TEP1 mRNA were commonly expressed in both cancers and normal tissues. All of the three cell lines derived from bladder cancer expressed each of the telomerase subunits, whereas the two normal primary fibroblast cell lines expressed hTERC and TEP1 mRNA but not hTERT mRNA. Telomerase activity was examined using telomeric repeat amplification protocol assay. All of the cancers examined exhibited telomerase activity, whereas only 2 of 12 normal tissues exhibited weak activity. There was a significant association of telomerase activity with hTERT mRNA expression but not with hTERC or TEP1 mRNA expression. These findings provide strong evidence that the expression of hTERT is a rate-limiting determinant of the enzymatic activity of human telomerase and that the up-regulation of hTERT expression may play a critical role in human carcinogenesis.

Demethylating reagent 5-azacytidine inhibits telomerase activity in human prostate cancer cells through transcriptional repression of hTERT.

Telomerase activation is thought to be a critical step in cellular immortality and oncogenesis. Several reagents including differentiation-inducing and antineoplastic agents are known to inhibit telomerase activity, although the molecular mechanisms through which they inhibit telomerase activity remain unclear. Demethylating reagents have recently been used as potential antineoplastic drugs for some types of cancers including those of the prostate. In the present study, we examined the effect of the demethylating reagent 5-azacytidine (5-aza-CR) on telomerase activity using cells of two prostate cancer cell lines, DU-145 and TSU-PR1. 5-aza-CR treatment significantly reduced telomerase activity in TSU-PR1 cells, but not in DU-145 cells, although growth inhibition was observed to a similar extent in both cell lines. Reverse transcription-PCR analyses revealed that inhibition of telomerase activity was accompanied by down-regulation of telomerase catalytic subunit (hTERT) mRNA expression. Transient expression assays showed that 5-aza-CR repressed the transcriptional activity of the hTERT promoter and that the E-box within the core promoter was responsible for this down-regulation. Western blot analyses revealed that 5-aza-CR reactivated p16 expression and repressed c-Myc expression in TSU-PR1 cells but not in DU-145 cells. Overexpression of p16 in TSU-PR1 cells led to significant repression of c-Myc transcription. These findings suggest that 5-aza-CR inhibits telomerase activity via transcriptional repression of hTERT, in which p16 and c-Myc may play a key role.

Mechanism of synergy between granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in colony formation from human marrow cells in vitro.

The synergy of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human granulocyte colony-stimulating factor (G-CSF) in the colony formation derived from human marrow cells was studied. The colony formation stimulated by GM-CSF plus G-CSF was dependent on the dose of each CSF, with the plateau for the number of GM colonies being higher than the sum of the individual plateaus by GM-CSF or G-CSF. Analysis of the colonies formed by GM-CSF plus G-CSF revealed efficient formation of neutrophil and monocyte colonies. To study the effect of GM-CSF and G-CSF on the maintenance of the progenitors that respond to the synergy of the CSFs, addition of each CSF to the medium of clonal cell culture was delayed. The progenitors that formed colonies on day 7 due to synergy of the CSFs were perfectly maintained by GM-CSF for at least 72 h and the progenitors that formed colonies on day 14 due to synergy of the CSFs were partly maintained by G-CSF or GM-CSF. The DNA synthetic rate of the progenitor cells that respond to GM-CSF plus G-CSF was significantly lower than those that respond to GM-CSF or G-CSF. According to light scatter analysis of phagocyte-depleted marrow mononuclear cells (PD-MMCs) using a flow cytometer, the peak population of progenitors that respond to GM-CSF plus G-CSF was in the smaller part of the PD-MMCs than those to GM-CSF or G-CSF. These results indicated that the progenitors to the synergy of GM-CSF and G-CSF are in a different proliferative state than those to each CSF. The synergy of GM-CSF and G-CSF depends on each CSF maintaining the viability of a different population of GM progenitors that can form GM colonies by both CSFs together.

Y chromosome haplogroup D2a1 is significantly associated with high levels of luteinizing hormone in Japanese men.

The association between the Y chromosome haplogroup D2 and risk of azoospermia and low sperm motility has been previously studied, and it was indicated that haplogroups DE (YAP lineage) are associated with prostate cancer risk in Japanese males. Our assumption had been that Y chromosome haplogroups may be associated with sex hormone levels, because sex hormones have been deemed responsible for spermatogenesis and carcinogenesis. In this study, we assessed the association between Y chromosome haplogroups and sex hormone levels, including those of testosterone, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin-B, and calculated free testosterone (cFT), in 901 young men from the general Japanese population (cohort 1) and 786 Japanese men of proven fertility (cohort 2). We found that the haplogroup D2a1 was significantly associated with high LH levels in a combined analysis involving two cohorts (ß = 0.068, SE = 0.025, p = 0.0075), following correction for multiple testing. To date, this result is the first evidence that implicates Y chromosome haplogroups in an association with sex hormone levels.

Stimulatory effect of prolactin on luteinizing hormone-induced testicular 5 alpha-reductase activity in hypophysectomized adult rats.

Two pituitaries from 7-week-old female rats were grafted under the capsule of the left kidney of 50-day-old male rat to induce hyperprolactinemia. All of the pituitary-grafted and sham-operated rats were hypophysectomized at 56 days of age. The hypophysectomized rats in groups of 4 were given daily sc injections of saline or 9 micrograms NIADDK-ovine-(o)LH-23 for 4 and 5 days starting from days 58 and 70, respectively (short and long term hypophysectomized groups). The metabolism of [3H]progesterone or [14C]androstenedione by testicular homogenates, concentrations of testosterone and 5 alpha-androgens (androsterone plus 5 alpha-androstane-3 alpha, 17 beta-diol) in the serum and testes, and testicular LH receptors were estimated. Hypophysectomy caused significant decreases in testicular enzyme activities per gram of tissue, androgen production, and testicular LH receptors. In the testes of hypophysectomized rats, LH treatment significantly stimulated 5 alpha-reductase and 17-hydroxylase activities. Although pituitary grafts alone showed little or no effect on these testicular enzyme activities, hyperprolactinemia induced by the grafts markedly enhanced the LH-stimulated 5 alpha-reductase activity in both groups, especially in the long term hypophysectomized group. Therefore, androsterone and 5 alpha-androstane-3 alpha,17 beta-diol were shown to be the major C19-steroid products (immature type of testicular androgen production) in the LH- and PRL-stimulated testes of long term hypophysectomized adult rats. On the other hand, hyperprolactinemia was associated with a significant inhibition and a slight increase of the LH-stimulated 17-hydroxylase activities in the short and long term hypophysectomized groups, respectively. This difference can be attributed to both a PRL-induced increase in testicular LH receptors and a PRL-induced inhibition of 17-hydroxylase via a postreceptor mechanism(s). The present findings demonstrate for the first time that PRL directly stimulates LH-induced 5 alpha-reductase activity in the testes. It appears that PRL may play a role in the increased production of 5 alpha-C19-steroids and the parallel decrease of testosterone production in immature rat testes.

Forebrain muscarinic control of micturition reflex in rats.

Functional contribution of the cholinergic pathway between the frontal cortex and basal nucleus of Meynert to micturition reflex was investigated. Male Wistar rats were subjected to bilateral lesion of the basal forebrain by ibotenic acid (IA) injection (7.5 microg/rat on each side) (BF rats). Phosphate buffered saline (PBS) was injected into control rats (sham operated rats; SO rats). Cystometrograms were obtained from conscious BF and SO rats 7-10 days after IA/PBS injection. Bladder capacity (BC) of BF rats was significantly smaller than that of SO rats (approximately 43.7%) and was accompanied by decrease in choline-acetyltransferase activity in the frontal cortices. Oxotremorine M, a muscarinic receptor agonist, increased BC in BF rats, while pirenzepine, an M1 muscarinic receptor antagonist, counteracted the effect of the oxotremorine M-induced increase in BC. Injection of oxotremorine M into the dorsal pontine tegmentum (DPT) reduced BC in BF and SO rats, while injection of pirenzepine had no effect on cystometrograms. These findings indicate that the M1 muscarinic receptor plays a part in the forebrain inhibitory mechanisms involved in the micturition reflex and that muscarinic receptor in the DPT contributes to excitatory control of micturition reflex.

Optical monitoring of synaptic summation along the dendrites of CA1 pyramidal neurons.

The primary function of neurons is to integrate synaptic inputs and to transmit the results to other cells. Recent studies with somatic whole-cell recordings have shown that separate excitatory inputs to hippocampal or cortical pyramidal neurons are summated non-linearly. In the present study, we examined how postsynaptic potentials (PSPs) are summated along the dendrites employing fast optical voltage imaging techniques. Rat hippocampal slices were stained with a fluorescent voltage-sensitive dye (JPW1114) and optical signals were monitored with a 16 x 16 photodiode array system. Two independent input pathways were stimulated individually or in pairs through glass electrodes such that different locations of the dendrites received separate synaptic inputs. We found that (1) the summation of PSPs was sub-linear along the entirety of dendrites, (2) the blockade of GABA(A) receptors suppressed sub-linearity and (3) further blockade of GABA(B) receptors suppressed sub-linearity of the summation of separate inputs on apical dendrites. Our study demonstrates that pyramidal neurons integrate PSPs linearly along the entirety of dendrites; moreover, GABAergic inputs are responsible for maintaining sub-linear summation in CA1 pyramidal neurons.

Role of protein kinase C in central muscarinic inhibitory mechanisms regulating voiding in rats.

To evaluate the role of protein kinase C in central muscarinic mechanisms regulating voiding, cystometry was performed in conscious rats. Oxotremorine methiodide, a muscarinic agonist was injected i.c.v. in a dose (0.1 microg/rat) shown previously to alter voiding function. Oxotremorine methiodide was also tested after i.c.v. injection of chelerythrine chloride (a protein kinase C inhibitor, 2 microg/rat) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, a protein kinase inhibitor, 5 nmol/rat). In untreated rats, oxotremorine methiodide elicited a bimodal response consisting of an initial increase in bladder capacity, maximal voiding pressure, pressure threshold and post voiding intravesical pressure, but reduced voiding efficiency and bladder compliance. The second response consisted of a decrease in bladder capacity and bladder compliance, increases in maximal voiding pressure and post voiding intravesical pressure, but no change in pressure threshold or voiding efficiency. However, approximately 20 min after pre-treatment with chelerythrine chloride or H-7 in doses that did not alter voiding function, oxotremorine methiodide decreased bladder capacity, increased maximal voiding pressure, but did not change pressure threshold or voiding efficiency. These results indicate that inhibitory and facilitatory muscarinic mechanisms in the brain that control voiding function involve different second messenger systems. Inhibitory mechanisms which are blocked by chelerythrine chloride or H-7 must involve protein kinase C and normally be inactive because the protein kinase inhibitors alone did not alter voiding. On the other hand, facilitatory muscarinic mechanisms which previous studies showed were tonically active are not mediated by chelerythrine chloride or H-7 sensitive signaling pathways.