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

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

Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats.

Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future

14-3-3 proteins stabilize LGI1-ADAM22 levels to regulate seizure thresholds in mice

新たなてんかん治療戦略を提案 --脳の過剰興奮を阻止するタンパク質ADAM22の量が鍵--. 京都大学プレスリリース. 2021-12-15.What percentage of the protein function is required to prevent disease symptoms is a fundamental question in genetic disorders. Decreased transsynaptic LGI1-ADAM22 protein complexes, because of their mutations or autoantibodies, cause epilepsy and amnesia. However, it remains unclear how LGI1-ADAM22 levels are regulated and how much LGI1-ADAM22 function is required. Here, by genetic and structural analysis, we demonstrate that quantitative dual phosphorylation of ADAM22 by protein kinase A (PKA) mediates high-affinity binding of ADAM22 to dimerized 14-3-3. This interaction protects LGI1-ADAM22 from endocytosis-dependent degradation. Accordingly, forskolin-induced PKA activation increases ADAM22 levels. Leveraging a series of ADAM22 and LGI1 hypomorphic mice, we find that ∼50% of LGI1 and ∼10% of ADAM22 levels are sufficient to prevent lethal epilepsy. Furthermore, ADAM22 function is required in excitatory and inhibitory neurons. These results suggest strategies to increase LGI1-ADAM22 complexes over the required levels by targeting PKA or 14-3-3 for epilepsy treatment

Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

Neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism, involve altered synaptic transmission and plasticity. Functional characterization of their associated genes is vital for understanding physio-pathological brain functions. LGI3 is a recently recognized ID-associated gene encoding a secretory protein related to an epilepsy-gene product, LGI1. Here, we find that LGI3 is uniquely secreted from oligodendrocytes in the brain and enriched at juxtaparanodes of myelinated axons, forming nanoscale subclusters. Proteomic analysis using epitope-tagged Lgi3 knockin mice shows that LGI3 uses ADAM23 as a receptor and selectively co-assembles with Kv1 channels. A lack of Lgi3 in mice disrupts juxtaparanodal clustering of ADAM23 and Kv1 channels and suppresses Kv1-channel-mediated short-term synaptic plasticity. Collectively, this study identifies an extracellular organizer of juxtaparanodal Kv1 channel clustering for finely tuned synaptic transmission. Given the defective secretion of the LGI3 missense variant, we propose a molecular pathway, the juxtaparanodal LGI3-ADAM23-Kv1 channel, for understanding neurodevelopmental disorders.<br/

A POS-based preordering approach for English-to-Arabic statistical machine translation

In this work, we present a POS-based preordering approach that tackles both long- and short-distance reordering phenomena. Syntactic unlexicalized reordering rules are automatically extracted from a parallel corpus using only word alignment and a source-side language tagging. The reordering rules are used in a deterministic manner; this prevents the decoding speed from being bottlenecked in the reordering procedure. A new approach for both rule filtering and rule application is used to ensure a fast and efficient reordering. The tests performed on the IWSLT2016 English-to-Arabic evaluation benchmark show a noticeable increase in the overall Blue Score for our system over the baseline PSMT system

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