Decreased AdipoR1 signaling and its implications for obesity-induced male infertility.

Obesity is among the risk factors for male infertility. Although several mechanisms underlying obesity-induced male subfertility have been reported, the entire mechanism of obesity-induced male infertility still remains unclear. Here, we show that sperm count, sperm motility and sperm fertilizing ability were decreased in male mice fed a high-fat diet and that the expression of the AdipoR1 gene and protein was decreased, and the expression of pro-apoptotic genes and protein increased, in the testis from mice fed a high-fat diet. Moreover, we demonstrate that testes weight, sperm count, sperm motility and sperm fertilizing ability were significantly decreased in AdipoR1 knockout mice compared to those in wild-type mice; furthermore, the phosphorylation of AMPK was decreased, and the expression of pro-apoptotic genes and proteins, caspase-6 activity and pathologically apoptotic seminiferous tubules were increased, in the testis from AdipoR1 knockout mice. Furthermore, study findings show that orally administrated AdipoRon decreased caspase-6 activity and apoptotic seminiferous tubules in the testis, thus ameliorating sperm motility in male mice fed a high-fat diet. This was the first study to demonstrate that decreased AdipoR1/AMPK signaling led to increased caspase-6 activity/increased apoptosis in the testis thus likely accounting for male infertility.

Impact of a Novel Digital Therapeutics System on Nonalcoholic Steatohepatitis: The NASH App Clinical Trial.

INTRODUCTION: Management of nonalcoholic steatohepatitis (NASH) is a currently unmet clinical need. Digital therapeutics (DTx) is an emerging class of medicine that delivers evidence-based therapeutic interventions. This study was aimed at investigating the efficacy of DTx in patients with NASH. METHODS: We conducted a multicenter, single-arm, 48-week trial in 19 patients with biopsy-confirmed NASH. All patients received a DTx intervention with a newly developed smartphone application. The primary endpoint was change in the nonalcoholic fatty liver disease activity score (NAS) without worsening of liver fibrosis. The secondary endpoints included improvement of the NAS by ≥2 points without worsening of liver fibrosis, change in the body weight, and regression of fibrosis. RESULTS: After the 48-week DTx intervention, improvement of the NAS was observed in 68.4% (13/19) of patients. The mean change in the NAS from baseline to the end of the intervention was -2.05 ± 1.96 ( P < 0.001 when compared with the threshold of -0.7). A decrease in the NAS by ≥ 2 points was achieved in 11 (57.9%). The average weight loss at the end of the intervention was 8.3% ( P < 0.001). Reduction of the fibrosis stage was observed in 58.3% when the analysis was limited to patients with stage F2/3 fibrosis. There were no serious adverse events that could be considered as being related to the DTx intervention. DISCUSSION: DTx for NASH was found to be highly efficacious and well-tolerated. Further evaluation of the DTx intervention for NASH in a phase 3 trial is warranted.

Orexin A Enhances Pro-Opiomelanocortin Transcription Regulated by BMP-4 in Mouse Corticotrope AtT20 Cells.

Orexin is expressed mainly in the hypothalamus and is known to activate the hypothalamic-pituitary-adrenal (HPA) axis that is involved in various stress responses and its resilience. However, the effects of orexin on the endocrine function of pituitary corticotrope cells remain unclear. In this study, we investigated the roles of orexin A in pro-opiomelanocortin (POMC) transcription using mouse corticotrope AtT20 cells, focusing on the bone morphogenetic protein (BMP) system expressed in the pituitary. Regarding the receptors for orexin, type 2 (OXR2) rather than type 1 (OX1R) receptor mRNA was predominantly expressed in AtT20 cells. It was found that orexin A treatment enhanced POMC expression, induced by corticotropin-releasing hormone (CRH) stimulation through upregulation of CRH receptor type-1 (CRHR1). Orexin A had no direct effect on the POMC transcription suppressed by BMP-4 treatment, whereas it suppressed Smad1/5/9 phosphorylation and Id-1 mRNA expression induced by BMP-4. It was further revealed that orexin A had no significant effect on the expression levels of type I and II BMP receptors but upregulated inhibitory Smad6/7 mRNA and protein levels in AtT20 cells. The results demonstrated that orexin A upregulated CRHR signaling and downregulated BMP-Smad signaling, leading to an enhancement of POMC transcription by corticotrope cells.

Heterozygous knockout of Bile salt export pump ameliorates liver steatosis in mice fed a high-fat diet.

The incidence of nonalcoholic steatohepatitis (NASH) is increasing worldwide, including in Asian countries. We reported that the hepatic expression of bile salt export pump (BSEP) was downregulated in patients with NASH, suggesting that BSEP is involved in the pathogenesis of NASH. To identify the underlying mechanism, we analyzed Bsep heterozygous knock-out (Bsep+/- mice) and wild-type (WT) C57BL/6J mice fed a high-fat diet (HFD) (32.0% animal fat) or normal diet. We examined histological changes, levels of hepatic lipids and hepatic bile acids, and expression of genes related to bile acid and cholesterol metabolism. HFD-fed Bsep+/- mice exhibited milder hepatic steatosis and less weight gain, compared to HFD-fed WT mice. The concentrations of total bile acid, triglycerides, and cholesterols were reduced in the liver of HFD-fed Bsep+/- mice. Regarding hepatic bile acid metabolism, the expression levels of Farnesoid X receptor (Fxr) and Multidrug resistance-associated protein 2 were significantly upregulated in HFD-fed Bsep+/- mice, compared to HFD-fed WT mice. Furthermore, several alterations were observed in upstream cholesterol metabolism in the liver. The expression levels of bile acid metabolism-related genes were also altered in the intestine of HFD-fed Bsep+/- mice. In conclusion, HFD-fed Bsep+/- mice exhibited significant alterations of the expression levels of genes related to bile acid and lipid metabolism in both the liver and ileum, resulting in alleviated steatosis and less weight gain. These results suggest the importance of BSEP for maintenance of bile acid and cholesterol metabolism. Further investigations of the involvement of BSEP in the pathogenesis of NASH will provide greater insight and facilitate the development of novel therapeutic modalities.

Tumor Budding in Intrahepatic Cholangiocarcinoma: A Predictor of Postsurgery Outcomes.

Intrahepatic cholangiocarcinoma (ICC) is an extremely aggressive carcinoma. Useful predictors for the patients' prognosis after surgery have not been fully established. From the University of Tokyo Hospital pathology archives, we reviewed 107 cases of ICC, 54 cases of perihilar cholangiocarcinoma, and 40 cases of extrahepatic cholangiocarcinoma (ECC); we also investigated the significance of tumor budding in ICC, in comparison with perihilar cholangiocarcinoma and ECC. The tumor-budding frequencies were different by tumor location: 40.2% (43/107) in ICC, 70.4% (38/54) in perihilar cholangiocarcinoma, and 60.0% (24/40) in ECC. Tumor budding in ICC was associated with many pathologic indicators associated with invasion, such as major vascular invasion (P=0.012) and Union for International Cancer Control stage (P=0.007). Univariate and multivariate Cox regression analyses revealed tumor budding as a powerful prognostic factor for both recurrence-free survival (RFS) and overall survival (OS) in ICC by univariate (RFS: hazard ratio [HR]: 2.666; 95% confidence interval [CI]: 1.517-4.683, OS: HR: 4.206; 95% CI: 2.447-7.230) and by multivariate analyses (RFS: HR: 3.038; 95% CI: 1.591-5.973, OS: HR: 4.547, 95% CI: 2.348-8.805). Tumor budding was also a significant prognostic factor of perihilar cholangiocarcinoma, but not of ECC. When ICC was divided into 2 subtypes, type 1 (hilar) and type 2 (peripheral), tumor budding was the strong prognostic factor in type 2 ICC, but not in type 1 ICC, suggesting that some differences in biological behavior exist between type 1 ICC and perihilar cholangiocarcinoma. Tumor budding is prognostically important in ICC, and its pathogenetic role in biliary tract carcinomas might be different by anatomic location.

A Novel Non-invasive Method for Predicting Liver Fibrosis by Quantifying the Hepatic Vein Waveform.

The hepatic vein (HV) waveform by Doppler ultrasound reflects the severity of liver fibrosis. We conducted a proof-of-concept study of a new method for quantifying the HV waveform. We calculated the coefficient of variation (CV) of the HV flow velocity and created a new index "q-HV" (quantified HV) and analyzed its performance for predicting histologic liver fibrosis in 114 patients with chronic liver disease. The CV of the HV flow velocity was well associated with flattening of the waveform and the q-HV significantly increased with the progression of liver fibrosis. The areas under the curve for the prediction of fibrosis stage were 0.732 for F2, 0.772 for F3 and 0.805 for F4. Combined q-HV and FIB-4 index (widely used liver fibrosis score) increased the diagnostic accuracy for liver fibrosis. The q-HV showed good accuracy for predicting liver fibrosis; thus, q-HV is feasible and acceptable as a non-invasive tool for predicting liver fibrosis.

Orexin A modulates prolactin production by regulating BMP-4 activity in rat pituitary lactotorope cells.

The impact of orexins on anterior pituitary function has yet to be clarified. We studied the effects of orexin A and its interaction with the bone morphogenetic protein (BMP) system on the regulatory role of prolactin synthesis using rat lactotrope GH3 cells expressing BMP-4. Orexin type 1 receptor (OX1R), but not type 2 receptor (OX2R), was predominantly expressed in GH3 cells. Orexin A suppressed forskolin-induced, but not basal, prolactin mRNA expression without reducing cAMP levels. Of note, orexin A suppressed BMP-4-induced prolactin mRNA and cAMP synthesis. Impairment of the effects of orexin by chemical inhibitors suggested involvement of the P38 pathway in the OX1R activity that suppresses BMP-4-induced PRL expression. Given that inhibition of BMP-receptor signaling reduced prolactin mRNA levels, endogenous BMP action is likely to be linked to the activation of prolactin synthesis by GH3 cells. Orexin A was revealed to suppress Smad1/5/9 phosphorylation and Id-1 transcription induced by BMP-4, which was restored in the presence of orexin-receptor antagonists, suggesting that the inhibitory effect of orexin A occurred via OX1R. Orexin A also reduced ALK-3 expression but increased inhibitory Smad6/7 expression, while BMP-4 treatment downregulated OX1R expression. These results indicated that orexin A plays an inhibitory role in prolactin production through suppression of endogenous BMP activity in GH3 cells, suggesting that a new functional role of the interaction between orexin and BMP-4 is modulation of prolactin levels in lactotrope cells.

Regulatory role of melatonin and BMP-4 in prolactin production by rat pituitary lactotrope GH3 cells.

The effects of melatonin on prolactin production and its regulatory mechanism remain uncertain. We investigated the regulatory role of melatonin in prolactin production using rat pituitary lactotrope GH3 cells by focusing on the bone morphogenetic protein (BMP) system. Melatonin receptor activation, induced by melatonin and its receptor agonist ramelteon, significantly suppressed basal and forskolin-induced prolactin secretion and prolactin mRNA expression in GH3 cells. The melatonin MT2 receptor was predominantly expressed in GH3 cells, and the inhibitory effects of melatonin on prolactin production were reversed by treatment with the receptor antagonist luzindole, suggesting functional involvement of MT2 action in the suppression of prolactin release. Melatonin receptor activation also suppressed BMP-4-induced prolactin expression by inhibiting phosphorylation of Smad and transcription of the BMP-target gene Id-1, while BMP-4 treatment upregulated MT2 expression. Melatonin receptor activation suppressed basal, BMP-4-induced and forskolin-induced cAMP synthesis; however, BtcAMP-induced prolactin mRNA expression was not affected by melatonin or ramelteon, suggesting that MT2 activation leads to inhibition of prolactin production through the suppression of Smad signaling and cAMP synthesis. Experiments using intracellular signal inhibitors revealed that the ERK pathway is, at least in part, involved in prolactin induction by GH3 cells. Thus, a new regulatory role of melatonin involving BMP-4 in prolactin secretion was uncovered in lactotrope GH3 cells.

Melatonin regulates catecholamine biosynthesis by modulating bone morphogenetic protein and glucocorticoid actions.

Melatonin is functionally involved in the control of circadian rhythm and hormonal secretion. In the present study, we investigated the roles of melatonin in the interaction of catecholamine synthesis with adrenocortical steroids by focusing on bone morphogenetic protein (BMP)-4 expressed in the adrenal medulla using rat pheochromocytoma PC12 cells. Melatonin treatment significantly reduced the mRNA expression of catecholamine synthases, including the rate-limiting enzyme tyrosine hydroxylase (Th), 3,4-dihydroxyphenylalanine decarboxylase and dopamine-ß-hydroxylase expressed in PC12 cells. In accordance with changes in the expression levels of enzymes, dopamine production and cAMP synthesis determined in the culture medium and cell lysate were also suppressed by melatonin. The MT1 receptor, but not the MT2 receptor, was expressed in PC12 cells, and luzindole treatment reversed the inhibitory effect of melatonin on Th expression, suggesting that MT1 is a functional receptor for the control of catecholamine synthesis. Interestingly, melatonin enhanced the inhibitory effect of BMP-4 on Th mRNA expression in PC12 cells. Melatonin treatment accelerated BMP-4-induced phosphorylation of SMAD1/5/8 and transcription of the BMP target gene Id1. Of note, melatonin significantly upregulated Alk2 and Bmpr2 mRNA levels but suppressed inhibitory Smad6/7 expression, leading to the enhancement of SMAD1/5/8 signaling in PC12 cells, while BMP-4 did not affect Mt1 expression. Regarding the interaction with adrenocortical steroids, melatonin preferentially enhanced glucocorticoid-induced Th mRNA through upregulation of the glucocorticoid receptor and downregulation of Bmp4 expression, whereas melatonin repressed Th mRNA expression induced by aldosterone or androgen without affecting expression levels of the receptors for mineralocorticoid and androgen. Collectively, the results indicate that melatonin plays a modulatory role in catecholamine synthesis by cooperating with BMP-4 and glucocorticoid in the adrenal medulla.

[Effect of Ultra-fast-track Extubation in Cardiovascular Surgery].

Fast-track surgery has been widely implemented and allows such immediate extubation as in the operating room(OR). We retrospectively investigated the effect of routine ultra-fast-track(UFT) surgery extubating in the OR in patients undergoing cardiovascular operations. Among 333 consecutive patients, 224 (67.3%)were extubated in the OR. Five patients were re-intubated, but none were because of heart or respiratory failure. Independent predictors for failure of OR extubation were preoperative renal failure, pre-existent cerebrovascular disease, emergency surgery, and prolonged operation and/or cardiopulmonary bypass times. In patients extubated in the OR, postoperative pneumonia and delirium were less frequent, oral intake was facilitated, and lengths of stay in the intensive care unit as well as hospital were shortened. UFT in cardiovascular surgery can be safely and effectively performed in a majority of patients.

Negative pressure wound therapy induces early wound healing by increased and accelerated expression of vascular endothelial growth factor receptors.

BACKGROUND: Negative pressure wound therapy (NPWT) is commonly used to accelerate wound healing, especially following thoracic surgery; however, the mechanism remains elusive. Given the important role of vasculogenesis in wound healing, we evaluated whether NPWT might accelerate vasculogenesis in the wound area. Toward this end, we investigated the temporal expression of vascular endothelial growth factor receptors (VEGFRs) in an NPWT-wound healing rabbit model. METHODS: Rabbits were divided into an NPWT group and a non-NPWT control group, and tissue samples were collected around wounds made in the skin of each rabbit at five time points: 0, 7, 14, 21, and 28 days after wound creation. Cryopreserved samples were then immunostained and subject to image analysis to evaluate the temporal changes in VEGFR1, VEGFR2, and VEGFR3 expression in the wound-healing process. RESULTS: Results of histological analysis of the temporal changes in VEGFR expression throughout the healing process showed that compared to the control group, VEGFR2 and VEGFR3 were abundantly and rapidly expressed in the NPWT group, and were expressed earlier than VEGFR1. CONCLUSIONS: NPWT promotes the expression of VEGFR2 and VEGFR3, which provides insight into the mechanism by which NPWT accelerates wound healing. Level of Evidence: Not ratable.

BMP-6 modulates somatostatin effects on luteinizing hormone production by gonadrotrope cells.

The effects of somatostatin analogs and roles of BMP-6 in the regulation of luteinizing hormone (LH) secretion were investigated using mouse gonadotrope LßT2 cells. LH mRNA expression and LH secretion induced by GnRH were suppressed by treatments with somatostatin analogs, including octreotide and pasireotide, in LßT2 cells. Of note, the inhibitory effects of somatostatin analogs on LH secretion were enhanced by the action of BMP-6. BMP-6 increased the expression levels of somatostatin receptor (SSTR)5, suggesting that BMP-6 upregulates SSTR activity that leads to reduction of GnRH-induced LH secretion. In addition, GnRH-induced phosphorylation of MAPKs including ERK, but not P38 or SAPK, was suppressed by pasireotide in the presence of BMP-6. Given that each inhibitor of ERK, JNK or P38 signaling suppressed GnRH-induced LH transcription, MAPKs are individually involved in the induction of LH production by LßT2 cells. Somatostatin analogs also impaired BMP-6-induced Smad1/5/8 phosphorylation by suppressing BMPRs and augmenting Smad6/7 expression. Collectively, the results indicate that somatostatin analogs have dual effects on the modulation of GnRH-induced MAPK signaling and BMP activity. The pituitary BMP system may play a regulatory role in GnRH-induced LH secretion by tuning the responsiveness to somatostatin analogs in gonadotrope cells.

Mutual effects of melatonin and activin on induction of aldosterone production by human adrenocortical cells.

Melatonin has been reported to suppress adrenocorticotropin (ACTH) secretion in the anterior pituitary and cortisol production in the adrenal by different mechanisms. However, the effect of melatonin on aldosterone production has remained unknown. In this study, we investigated the role of melatonin in the regulation of aldosterone production using human adrenocortical H295R cells by focusing on the activin system expressed in the adrenal. Melatonin receptor MT1 mRNA and protein were expressed in H295R cells and the expression levels of MT1 were increased by activin treatment. Activin increased ACTH-induced, but not angiotensin II (Ang II)-induced, aldosterone production. Melatonin alone did not affect basal synthesis of either aldosterone or cortisol. However, melatonin effectively enhanced aldosterone production induced by co-treatment with ACTH and activin, although melatonin had no effect on aldosterone production induced by Ang II in combination with activin. These changes in steroidogenesis became apparent when the steroid production was evaluated by the ratio of aldosterone/cortisol. Melatonin also enhanced dibutyryl-AMP-induced aldosterone/cortisol levels in the presence of activin, suggesting a functional link to the cAMP-PKA pathway for induction of aldosterone production by melatonin and activin. In accordance with the data for steroids, ACTH-induced, but not Ang II-induced, cAMP synthesis was also amplified by co-treatment with melatonin and activin. Furthermore, the ratio of ACTH-induced mRNA level of CYP11B2 compared with that of CYP17 was amplified in the condition of treatment with both melatonin and activin. In addition, melatonin increased expression of the activin type-I receptor ALK-4 but suppressed expression of inhibitory Smads6/7, leading to the enhancement of Smad2 phosphorylation. Collectively, the results showed that melatonin facilitated aldosterone production induced by ACTH and activin via the cAMP-PKA pathway. The results also suggested that mutual enhancement of melatonin and activin receptor signaling is involved in the induction of aldosterone output by adrenocortical cells.

Interaction of pituitary hormones and expression of clock genes modulated by bone morphogenetic protein-4 and melatonin.

Functional interaction of clock genes and pituitary hormones was investigated by focusing on bone morphogenetic protein (BMP)-4 and melatonin actions in anterior pituitary cells. A significant correlation between the mRNA expression of proopiomelanocortin (POMC) and Per2 was revealed in serial cultures of corticotrope AtT20 cells. Knockdown of Per2 expression by siRNA in AtT20 cells resulted in a significant reduction of POMC mRNA level with or without corticotropin-releasing hormone (CRH) stimulation. Treatments with BMP-4 and melatonin, both of which suppress POMC expression, reduced Per2 mRNA as well as protein levels in AtT20 cells. On the other hand, in lactosomatotrope GH3 cells, an expressional correlation was found between prolactin (PRL) and Clock mRNA levels, which was attenuated in the presence of forskolin treatment. The siRNA-mediated knockdown of Clock expression, but not that of Bmal1, significantly reduced PRL mRNA levels in GH3 cells. Interestingly, Clock mRNA and protein levels did not fluctuate with melatonin, BMP-4 or forskolin treatment, although Bmal1 expression was significantly increased by forskolin treatment. Collectively, a significant correlation between the expression of POMC and Per2 and that between PRL and Clock were uncovered in corticotrope and lactosomatotrope cells, respectively. Per2 expression was inhibited by POMC modulators including melatonin and BMP-4, while Clock expression was steadily maintained. Thus, the effects of melatonin and BMP-4 on clock gene expression may imply differential stability of circadian rhythms of adrenocorticotropin (ACTH) and PRL secreted from the anterior pituitary.

Regulatory role of BMP-9 in steroidogenesis by rat ovarian granulosa cells.

BMPs expressed in the ovary differentially regulate steroidogenesis by granulosa cells. BMP-9, a circulating BMP, is associated with cell proliferation, apoptosis and differentiation in various tissues. However, the effects of BMP-9 on ovarian function have yet to be elucidated. Here we investigated BMP-9 actions on steroidogenesis using rat primary granulosa cells. BMP-9 potently suppressed FSH-induced progesterone production, whereas it did not affect FSH-induced estradiol production by granulosa cells. The effects of BMP-9 on FSH-induced steroidogenesis were not influenced by the presence of oocytes. FSH-induced cAMP synthesis and FSH-induced mRNA expression of steroidogenic factors, including StAR, P450scc, 3ßHSD2 and FSHR, were suppressed by treatment with BMP-9. BMP-9 mRNA expression was detected in granulosa cells but not in oocytes. BMP-9 readily activated Smad1/5/8 phosphorylation and Id-1 transcription in granulosa cells. Analysis using ALK inhibitors indicated that BMP-9 actions were mediated via type-I receptors other than ALK-2, -3 and -6. Furthermore, experiments using extracellular domains (ECDs) for BMP type-I and -II receptor constructs revealed that the effects of BMP-9 were reversed by ECDs for ALK-1 and BMPRII. Thus, the functional receptors for BMP-9 in granulosa cells were most likely to be the complex of ALK-1 and BMPRII. Collectively, the results of the present study showed that BMP-9 can affect luteinization and that there are two possible sources of BMP-9, serum and granulosa cells in the ovary.

[Stuck valve detected during aortic valve replacement; report of a case].

Aortic valve replacement, ascending aorta replacement and coronary artery bypass grafting were performed in a 61-year-old woman with aortic stenosis, ascending aortic aneurysm, and angina pectoris. However, immediately after surgery, transient hypotension and pulmonary hypertension repeated every few beats.Transesophageal echocardiography revealed a stuck valve, and reimplantation was carried out. The patient's postoperative course was uneventful. We present a case of successful treatment of valve dysfunction immediately after valve replacement.

MicroRNA-200a locally attenuates progesterone signaling in the cervix, preventing embryo implantation.

Although cervical pregnancy and placenta previa, in which the embryo and placenta embed in or adjacent to the cervix, are life-threatening complications that result in massive bleeding and poor pregnancy outcomes in women, the incidence of these aberrant conditions is uncommon. We hypothesized that a local molecular mechanism is normally in place to prevent embryo implantation in the cervix. The ovarian hormones progesterone (P(4)) and estrogen differentially direct differentiation and proliferation of endometrial cells, which confers the receptive state for implantation: P(4) dominance causes differentiation of the luminal epithelium but increases stromal cell proliferation in preparation of the uterus for implantation. In search for the cause of cervical nonresponsiveness to implantation, we found that the statuses of cell proliferation and differentiation between the uterus and cervix during early pregnancy are remarkably disparate under identical endocrine milieu in both mice and humans. We also found that cervical levels of progesterone receptor (PR) protein are low compared with uterine levels during this period, and the low PR protein levels are attributed to elevated levels of microRNA(miR)-200a in the cervix. These changes were associated with up-regulation of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (200α-HSD) and down-regulation of its transcriptional repressor signal transducer and activator of transcription 5 in the cervix. The results provide evidence that elevated levels of miR-200a lead to down-regulation of P(4)-PR signaling and up-regulation of (200α-HSD) in the cervix, rendering it nonresponsive to implantation. These findings may point toward not only the physiological but also the pathological basis of the cervical milieu in embryo implantation.

Melatonin counteracts BMP-6 regulation of steroidogenesis by rat granulosa cells.

The ovarian bone morphogenetic protein (BMP) system is a physiological inhibitor of luteinization in growing ovarian follicles. BMP-6, which is expressed in oocytes and granulosa cells of healthy follicles, specifically inhibits FSH actions by suppressing adenylate cyclase activity. In the present study, we studied the role of melatonin in ovarian steroidogenesis using rat primary granulosa cells of immature female rat ovaries by focusing on the interaction with BMP-6 activity. Treatment with melatonin had no direct effect on FSH-induced progesterone or estradiol production by granulosa cells, and the results were not affected by the presence of co-cultured oocytes. In addition, synthesis of cAMP by granulosa cells was not significantly altered by melatonin treatment. To elucidate the interaction between activities of melatonin and BMPs, the effect of melatonin treatment on suppression of progesterone synthesis by BMP-6 was investigated. Interestingly, the inhibitory effect of BMP-6 on FSH-induced progesterone production was impaired by co-treatment with melatonin. Granulosa cells express higher levels of MT1 than MT2, and BMP-6 had no significant effect on MT1 expression in granulosa cells. However, BMP-6-induced Smad1/5/8 phosphorylation and Id-1 transcription were suppressed by melatonin, suggesting that melatonin has an inhibitory effect on BMP receptor signaling in granulosa cells. Although the expression levels of ALK-2, -6, ActRII and BMPRII were not affected by melatonin, inhibitory Smad6, but not Smad7, expression was upregulated by melatonin. Thus, melatonin plays a role in the regulation of BMP-6 signal intensity for controlling progesterone production in the ovary. These findings suggest that the effect of melatonin on maintenance of ovarian function is, at least in part, due to the regulation of endogenous BMP activity in granulosa cells.

Concurrent activation of acetylation and tri-methylation of H3K27 in a subset of hepatocellular carcinoma with aggressive behavior.

Analysis of acetylation and tri-methylation of the same residue of histone molecules might identify a subset of hepatocellular carcinoma (HCC) with aggressive behavior. In the present study, we examined acetylation and tri-methylation of lysine 27 on histone H3 (H3K27ac and H3K27me3, respectively) because these two modifications are known to exhibit opposite effects (enhancing and silencing) on gene expression. Neoplastic and non-neoplastic tissues from 198 HCC cases were immunostained with specific monoclonal antibodies against H3K27ac and H3K27me3. The stained tissues were evaluated by an image analyzing program to generate histological scores (H-scores, range 0-300), which were determined by multiplying the percentage of positive-stained cells with the classified immunohistochemical marker intensity (0-3). HCC tissues showed significantly higher H3K27ac (156.7±86.8) and H3K27me3 H-scores (151.8±78.1) compared with the background liver (40.3±33.0 and 64.7±45.6, respectively) (both P<0.001). The cases with H-scores of high-H3K27ac/high-H3K27me3 (n = 54) showed significant correlation with poor differentiation of morphology (P<0.01) and p53-positive staining (P<0.05), and poor prognosis (P<0.01). Confocal microscopy revealed segregated intranuclear localization of both modifications in the individual cancer cells: H3K27ac localization in central euchromatin regions and H3K27me3 in peripheral heterochromatin regions. Concurrent acetylation and methylation at H3K27 occurs in HCC cells in association with p53 abnormalities. These findings demonstrate that image analyzer-assisted H-scores of H3K27ac and H3K27me3 identified an aggressive subgroup of HCC, and could serve as a prognostic marker for HCC.

The TRK-fused gene is mutated in hereditary motor and sensory neuropathy with proximal dominant involvement.

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal-dominant neurodegenerative disorder characterized by widespread fasciculations, proximal-predominant muscle weakness, and atrophy followed by distal sensory involvement. To date, large families affected by HMSN-P have been reported from two different regions in Japan. Linkage and haplotype analyses of two previously reported families and two new families with the use of high-density SNP arrays further defined the minimum candidate region of 3.3 Mb in chromosomal region 3q12. Exome sequencing showed an identical c.854C>T (p.Pro285Leu) mutation in the TRK-fused gene (TFG) in the four families. Detailed haplotype analysis suggested two independent origins of the mutation. Pathological studies of an autopsied patient revealed TFG- and ubiquitin-immunopositive cytoplasmic inclusions in the spinal and cortical motor neurons. Fragmentation of the Golgi apparatus, a frequent finding in amyotrophic lateral sclerosis, was also observed in the motor neurons with inclusion bodies. Moreover, TAR DNA-binding protein 43 kDa (TDP-43)-positive cytoplasmic inclusions were also demonstrated. In cultured cells expressing mutant TFG, cytoplasmic aggregation of TDP-43 was demonstrated. These findings indicate that formation of TFG-containing cytoplasmic inclusions and concomitant mislocalization of TDP-43 underlie motor neuron degeneration in HMSN-P. Pathological overlap of proteinopathies involving TFG and TDP-43 highlights a new pathway leading to motor neuron degeneration.