Rescue of Vitrified-Warmed Bovine Oocytes with Rho-Associated Coiled-Coil Kinase Inhibitor

Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.ArticleBIOLOGY OF REPRODUCTION. 89(2):26 (2013)journal articl

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (Tg′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the Tg′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer Tg′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of Tg′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both Tg′ of freeze-drying buffer and temperature during the drying phase.ArticleCRYOBIOLOGY. 68(3):354-360 (2014)journal articl

Quantitative Evaluation of Liver Function with Use of Gadoxetate Disodium-enhanced MR Imaging

Purpose: To determine whether liver function correlating with indocyanine green (ICG) clearance could be estimated quantitatively from gadoxetate disodium-enhanced magnetic resonance (MR) images. Materials and Methods: This retrospective study was approved by the institutional review board, and the requirement for informed consent was waived. Twenty-three consecutive patients who underwent an ICG clearance test and gadoxetate disodium-enhanced MR imaging with the same parameters as were used for a preoperative examination were chosen. The hepatocellular uptake index (HUI) from liver volume (V(L)) and mean signal intensity of the liver on contrastenhanced T1-weighted images with fat suppression (L(20)) and mean signal intensity of the spleen on contrastenhanced T1-weighted images with fat suppression (S(20)) on 3D gradient-echo T1-weighted images with fat suppression obtained at 20 minutes after gadoxetate disodium (0.025 mmol per kilogram of body weight) administration was determined with the following equation: V(L)[(L(20)/S(20)) 2 1]. The correlation of the plasma disappearance rate of ICG (ICG-PDR) and various factors derived from MR imaging, including HUI, iron and fat deposition in the liver and spleen, and spleen volume (V(S)), were evaluated with stepwise multiple regression analysis. The difference between the ratio of the remnant HUI to the HUI of the total liver (rHUI/HUI) and ratio of the liver remnant V(L) to the total V(L)(rV(L)/V(L)) was evaluated in four patients who had segmental heterogeneity of liver function. Results: HUI and V(S) were the factors significantly correlated with ICG-PDR (R = 0.87). The mean value and its 95% confidence interval were 0.18 and 0.01 to 0.34, respectively, for the following calculation: (rHUI/HUI) 2 (rV(L)/V(L)). Conclusion: The liver function correlating with ICG-PDR can be estimated quantitatively from the signal intensities and the volumes of the liver and spleen on gadoxetate disodium-enhanced MR images, which may improve the estimation of segmental liver function. (C) RSNA, 2011ArticleRADIOLOGY. 260(3):727-733 (2011)journal articl

LXR agonist increases apoE secretion from HepG2 spheroid, together with an increased production of VLDL and apoE-rich large HDL

<p>Abstract</p> <p>Background</p> <p>The physiological regulation of hepatic apoE gene has not been clarified, although the expression of apoE in adipocytes and macrophages has been known to be regulated by LXR.</p> <p>Methods and Results</p> <p>We investigated the effect of TO901317, a LXR agonist, on hepatic apoE production utilizing HepG2 cells cultured in spheroid form, known to be more differentiated than HepG2 cells in monolayer culture. Spheroid HepG2 cells were prepared in alginate-beads. The secretions of albumin, apoE and apoA-I from spheroid HepG2 cells were significantly increased compared to those from monolayer HepG2 cells, and these increases were accompanied by increased mRNA levels of apoE and apoA-I. Several nuclear receptors including LXRα also became abundant in nuclear fractions in spheroid HepG2 cells. Treatment with TO901317 significantly increased apoE protein secretion from spheroid HepG2 cells, which was also associated with the increased expression of apoE mRNA. Separation of the media with FPLC revealed that the production of apoE-rich large HDL particles were enhanced even at low concentration of TO901317, and at higher concentration of TO901317, production of VLDL particles increased as well.</p> <p>Conclusions</p> <p>LXR activation enhanced the expression of hepatic apoE, together with the alteration of lipoprotein particles produced from the differentiated hepatocyte-derived cells. HepG2 spheroids might serve as a good model of well-differentiated human hepatocytes for future investigations of hepatic lipid metabolism.</p

High revivability of vitrified-warmed bovine mature oocytes after recovery culture with alpha-tocopherol

online publication: 27 January 2015The objective of this study was to investigate whether developmental competence of vitrified–warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1–36.3% vs 19.2–25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified–warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.ArticleREPRODUCTION. 149(4):347-355 (2015)journal articl

Tectonic Evolution of the Sambagawa Schists and its Implications in Convergent Margin Processes

The Sambagawa schists as high P /T metamorphic rocks are a member of Mesozoic accretionary complexes developed in the southern front of the Kurosegawa-Koryoke continent of Southwest Japan. The Mesozoic accretionary complexes are divided into four megaunits developed as nappes, Chichibu megaunit II, Sambagawa megaunit, Chichibu megaunit I and Shimanto megaunit in descending order of structural level. The Chichibu megaunit II consists of three accretionary units developed as nappes, late early Jurassic unit, late middle Jurassic unit and latest Jurassic unit (Mikabu unit) in descending order of structural level. The Chichibu megaunit I consists of five accretionary units developed as nappes, late middle Jurassic unit (Niyodo unit), late Jurassic unit, Valanginian unit, Barremian unit and Albian unit in descending order of structural level. The Shimanto megaunit, which just underlies the Chichibu megaunit I, is Cenomanian-Turonian accretionary unit and Coniacian-Campanian accretionary unit. The schists, which underlie the Chichibu megaunit II, all have been so far called the Sambagawa schists. These are divided into six units, Saruta unit, Fuyunose unit, Sogauchi unit, Sakamoto unit, Oboke unit and Tatsuyama unit in descending order of structural level, which show different tectono-metamorphic history and different radiometric ages from each other. The Sakamoto unit, Oboke unit and Tatsuyama unit have been assumed with reference to their radiometric ages and structural relations to belong to the late middle Jurassic accretionary unit of the Chichibu megaunit I (high pressure equivalent of the Niyodo unit), the Cenomanian-Turonian accretionary unit of the Shimanto megaunit and the Coniacian-Campanian accretionary unit of the Shimanto megaunit respectively in this paper. The upper member of the Sambagawa schists, Saruta unit, Fuyunose unit and Sogauchi unit, is therefore called the Sambagawa megaunit in this paper. The northern half and the southern half of the Sambagawa megaunit are intercalated as nappes between the Chichibu megaunit II and the Oboke unit and between the Chichibu megaunit II and the Sakamoto unit respectively. The constituent units of the Chichibu megaunit II, Sambagawa megaunit and Shimanto megaunit clearly show a downward younging age polarity, as compared with each other with reference to the oldest one of radiometric ages ( = metamorphic ages) of each unit. The Chichibu megaunit II and the Chichibu megaunit I show the same radiometric ages as compared between them with the same fossil age. The Saruta unit, Fuyunose unit and Sogauchi unit have therefore been assumed to be high pressure equivalent of Valanginian unit, that of Barremian unit and that of Albian unit of the Chichibu megaunit I respectively. These high pressure units were exhumed, separating the Chichibu supermegaunit into the Chichibu megaunit II and the Chichibu megaunit I and thrusting up onto the Chichibu megaunit I. On the basis of the growth history of amphibole in hematite-bearing basic schists of the Sambagawa megaunit, it has been assumed that the highest temperature metamorphism of the Fuyunose unit occurred, when it had been coupled with the Saruta unit which was exhuming, and that of the Sogauchi unit did through its coupling with the Fuyunose and Saruta units which were exhuming. In the subduction zone which was responsible for the formation of the Sambagawa megaunit, namely, the peak metamorphism of a newly subducted unit appears to have occurred when it had been coupled with previously subcreted units which were exhuming. It has been also clarified that the subduction of a new unit occurred mixing the lower pressure part of the pre-existing subcretion unit as tectonic blocks. There is a distinct difference in the oldest one of radiometric ages between constituent units of the Sambagawa schists, showing a downward younging age polarity. The oldest one of radiometric ages of each unit appears to approximate to the age of the ending of peak metamorphism and to the age (Eh age) of the beginning of its exhumation. Such the tectonics of the Sambagawa megaunit would be explained in term of two-way street model. Because the age (Sub age) of the beginning of the subduction of each unit can be assumed from its fossil age, the average velocity of the subduction and that of the exhumation of the Sambagawa megaunit in Shikoku have roughly been estimated to be ca. 0.9 mm/year and ca. 2.0 mm/year respectively. Deformation of quartz, whose style depends strongly upon strain rate, resulted in type I crossed girdle without conentration in Y even in the depth part of more than 10kb of the subduction zone, which was placed under temperature condition of much higher than 500°C, unlike the cases of magma-arcs where quartz c-axis fabrics with maximum concentration in Y are found in gneisses produced under temperature condition of lower than 500°C. Quartz deformation in the depth part of 15-17kb of the subduction zone appears to have occurred as dominant prism slip. The hanging wall of the Kurosegawa-Koryoke continent, which was placed at the depth of ca. 15-17 kb, thrust onto the Saruta unit at the depth of ca. 10-11 kb, accompanying intermingling of constituent rocks of the former and the latter and also mixing of various depth parts of the latter. The highest temperature metamorphism of the Saruta unit, which appears to have occurred under metamorphic condition of lower P /T than under that related to the formation of the general type of high P /T type metamorphic rocks, is ascribed to a contact metamorphism related to the overthrusting of the Kurosegawa-Koryoke continent. The thrusting of the Kurosegawa-Koryoke continent is ascribed to its collision with the Hida continent. The coupling of the previously subcreted Saruta unit with the newly subcreted Fuyunose unit occurred accompanying nearly isobaric cooling of the former. The great exhumation of the Saruta nappe (I + II) and Fuyunose nappe schists with great volume began together with the subcretion of the Sogauchi unit. The beginning age of the exhumation of the Sambagawa schists with great volume appears to coincide with that of the subduction of the Kula-Pacific ridge in Kyushu-Shikoku, which has been assumed by Kiminami et al. (1990). Namely, their great exhumation occurred with the progress of the subduction of the Kula-Pacific ridge with an eastward younging age polarity. The exhumation units, which were developed after the Mikabu unit, clearly show an eastward younging age polarity. Namely, these comparable with the Saruta unit, Fuyunose unit and Sogauchi unit are not found in central Japan and the Kanto Mountains. Rock deformation in the deformation related to the exhumation of the Sambagawa schists and their underlying schists appears to have commonly been of flattened type in mean strain. During the Ozu phase when the Kula-Pacific ridge subducted to the greater depth, the collapse of the Kurosegawa-Koryoke continent took again place, accompanying that of the pile nappe structures of the Sambagawa megaunit, Chichibu megaunit I and Oboke unit, and the thermal gradient along the plate boundary greatly changed, giving rise to medium P/T type metamorphism in the subduction zone (formation of the Tatsuyama nappe schists). The geological structures of the Sambagawa megaunit consist thus of two types of pile nappe structures, pre-Ozu phase pile nappe structures and Ozu phase pile nappe structures. The former is structures related to the coupling of the exhuming units ( = previously subcreted units) with the newly subcreted unit. The latter is structures showing the collapse of the former. The Ozu phase pile nappe structures are further divided into the pile nappe structures formed during the earlier stage (Tsuji stage) of the Ozu phase and these formed during the later stage (Futami stage). The former is disharmonic with reference to movement picture with the latter: The deformation related to the formation of the former, accompanying exhumation of the Oboke nappes, appears to contain a component of northward displacement, while that for the latter does a component of southward displacement. After the Ozu phase deformation the Sambagawa megaunit suffered the Hijikawa-Oboke phase folding, forming a series of sinistral en echelon upright folds. The relationship between the above-mentioned tectonic events of the Sambagawa megaunit and its surroundings and their radiometric ages is summarized as follows: [Original table is skipped. For more details, please refer to the full text.

Quantitative Evaluation of Liver Function with Use of Gadoxetate Disodium–enhanced MR Imaging

Purpose: To determine whether liver function correlating with indocyanine green (ICG) clearance could be estimated quantitatively from gadoxetate disodium-enhanced magnetic resonance (MR) images. Materials and Methods: This retrospective study was approved by the institutional review board, and the requirement for informed consent was waived. Twenty-three consecutive patients who underwent an ICG clearance test and gadoxetate disodium-enhanced MR imaging with the same parameters as were used for a preoperative examination were chosen. The hepatocellular uptake index (HUI) from liver volume (V(L)) and mean signal intensity of the liver on contrastenhanced T1-weighted images with fat suppression (L(20)) and mean signal intensity of the spleen on contrastenhanced T1-weighted images with fat suppression (S(20)) on 3D gradient-echo T1-weighted images with fat suppression obtained at 20 minutes after gadoxetate disodium (0.025 mmol per kilogram of body weight) administration was determined with the following equation: V(L)[(L(20)/S(20)) 2 1]. The correlation of the plasma disappearance rate of ICG (ICG-PDR) and various factors derived from MR imaging, including HUI, iron and fat deposition in the liver and spleen, and spleen volume (V(S)), were evaluated with stepwise multiple regression analysis. The difference between the ratio of the remnant HUI to the HUI of the total liver (rHUI/HUI) and ratio of the liver remnant V(L) to the total V(L)(rV(L)/V(L)) was evaluated in four patients who had segmental heterogeneity of liver function. Results: HUI and V(S) were the factors significantly correlated with ICG-PDR (R = 0.87). The mean value and its 95% confidence interval were 0.18 and 0.01 to 0.34, respectively, for the following calculation: (rHUI/HUI) 2 (rV(L)/V(L)). Conclusion: The liver function correlating with ICG-PDR can be estimated quantitatively from the signal intensities and the volumes of the liver and spleen on gadoxetate disodium-enhanced MR images, which may improve the estimation of segmental liver function. (C) RSNA, 2011ArticleRADIOLOGY. 260(3):727-733 (2011)journal articl

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (Tg′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the Tg′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer Tg′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of Tg′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both Tg′ of freeze-drying buffer and temperature during the drying phase.ArticleCRYOBIOLOGY. 68(3):354-360 (2014)journal articl

Fructo-oligosaccharides ameliorate steatohepatitis, visceral adiposity, and associated chronic inflammation via increased production of short-chain fatty acids in a mouse model of non-alcoholic steatohepatitis

Background: Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome. Within the spectrum of NAFLD, non-alcoholic steatohepatitis (NASH) in combination with hepatic inflammation and fibrosis can lead to liver cirrhosis and hepatocellular carcinoma. Dysbiosis was reported to contribute to NASH pathogenesis. This study aimed to determine the effects of fructo-oligosaccharides (FOS) on steatohepatitis and visceral adiposity in an obese mouse model of NASH. Methods: Twelve newborn C57BL/6 J male mice were subcutaneously injected with monosodium glutamate (MSG) to induce obesity on a conventional diet. Six mice were also administered 5% FOS via drinking water from 10 weeks of age. At 18 weeks, histological characteristics of the liver and epididymal fat were compared between the groups. Hepatic mRNA expression of lipid metabolism enzymes and SCFA in feces and sera were measured. Results: Hepatic steatosis, inflammatory cell infiltration, and hepatocyte ballooning in the liver and increased hepatic mRNA expression of fatty acid synthase and glycerol-3-phosphate acyltransferase were observed in the MSG-treated mice. FOS treatment improved the liver pathology and blunted the increases in the mRNA expression levels of lipid metabolism enzymes. In addition, FOS inhibited adipocyte enlargement and formation of crown-like structures and reduced the M1 macrophage frequency in the epididymal fat of the MSG mice (39.4% ± 3.0% vs. 22.8% ± 0.7%; P = 0.001). FOS increased not only the fecal concentrations of n-butyric acid (0.04 ± 0.01 vs. 0.38 ± 0.14 mg/g, P = 0.02), propionic acid (0.09 ± 0.03 vs. 0.42 ± 0.16 mg/g, P = 0.02), and acetic acid (0.65 ± 0.16 vs. 1.48 ± 0.29 mg/g, P = 0.03) but also the serum concentration of propionic acid (3.9 ± 0.5 vs. 8.2 ± 0.5 μmol/L, P = 0.001). Conclusions: FOS ameliorates steatohepatitis, visceral adiposity, and chronic inflammation by increasing SCFA production

Malaria Parasites Hijack Host Receptors From Exosomes to Capture Lipoproteins

Malaria parasites cannot multiply in host erythrocytes without cholesterol because they lack complete sterol biosynthesis systems. This suggests parasitized red blood cells (pRBCs) need to capture host sterols, but its mechanism remains unknown. Here we identified a novel high-density lipoprotein (HDL)-delivery pathway operating in blood-stage Plasmodium. In parasitized mouse plasma, exosomes positive for scavenger receptor CD36 and platelet-specific CD41 increased. These CDs were detected in pRBCs and internal parasites. A low molecular antagonist for scavenger receptors, BLT-1, blocked HDL uptake to pRBCs and suppressed Plasmodium growth in vitro. Furthermore, platelet-derived exosomes were internalized in pRBCs. Thus, we presume CD36 is delivered to malaria parasites from platelets by exosomes, which enables parasites to steal HDL for cholesterol supply. Cholesterol needs to cross three membranes (RBC, parasitophorous vacuole and parasite’s plasma membranes) to reach parasite, but our findings can explain the first step of sterol uptake by intracellular parasites