In-situ characterization of porcine fibroblasts in response to silver ions by Raman spectroscopy and liquid scanning transmission electron microscopy.

Since silver ion is known for its antimicrobial function, most of the research has focused mainly on toxicity effects rather than the role of silver ion in general biology and the behind mechanism of actions of silver ion in mammalian cells. Moreover, a conventional in vitro approach to estimate the effects of silver ion on cells does not provide information about the biochemical changes and might accompany artifacts due to invasive and destructive sample preparation processes. In the present study, in-situ real time approaches were applied to evaluate the impact of silver ion (0.

Effects of electrical biostimulation and silver ions on porcine fibroblast cells.

The medical applications of electrical biostimulation and silver ions have been evaluated in laboratory experiments and clinical studies for more than two decades. Their effects on preventing infection and promoting wound healing have been described. However, little is known about the role of electrical biostimulation and/or silver ion on changes in cellular transcriptome dynamics.

Serial Culture Is Critical for In Vitro Development of Parthenogenetic Embryos in the Golden Syrian Hamster.

Unlike oocytes of many other mammalian species, parthenogenetically activated hamster oocytes have not been reported to develop beyond the two-cell stage. This study investigated the in vitro development into blastocysts of parthenogenetic embryos of Golden Syrian hamsters. We observed that hamster oocytes could easily be artificially activated (AA) by treatment with ionomycin plus 6-dimethylaminopurine + cycloheximide + cytochalasin B as assessed by embryo cleavage in HECM-9 (63.

Cytokine gene expression at the maternal-fetal interface after somatic cell nuclear transfer pregnancies in small ruminants.

The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure.

Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.

The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters.

SGO1 maintains bovine meiotic and mitotic centromeric cohesions of sister chromatids and directly affects embryo development.

Shugoshin (SGO) is a critical factor that enforces cohesion from segregation of paired sister chromatids during mitosis and meiosis. It has been studied mainly in invertebrates. Knowledge of SGO(s) in a mammalian system has only been reported in the mouse and Hela cells.

Decreased expression of CD9 in bovine oocytes after cryopreservation and the relationship to fertilization capacity.

This study was conducted to investigate the effect of vitrification of bovine metaphase-II (MII) oocytes on CD9 expression and fertilization capacity. Surviving vitrified/warmed oocytes were used to detect CD9 distribution (fluorescence microscopy), CD9 mRNA (qRT-PCR), and CD9 protein expression (Western blot), and to analyze in vitro fertilization rates (number of sperm bound to or that penetrated the oocytes) after removing the zona pellucida. Fresh oocytes acted as control.

Enucleation of demecolcine-treated bovine oocytes in cytochalasin-free medium: mechanism investigation and practical improvement.

Demecolcine-assisted/induced enucleation has been used in nuclear transfer cloning procedures for many species, yet its mechanism of action remains unclear. Primarily because oocytoplasm protrusion induced by demecolcine is inhibited by the presence of cytochalasin, its use has had limited application. In this experiment, we investigated the microtubule and microfilament alterations in bovine oocytes after demecolcine and/or cytochalasin B (CB) treatments by immunocytochemical staining.

Effect of cryopreservation on acetylation patterns of lysine 12 of histone H4 (acH4K12) in mouse oocytes and zygotes.

Purpose: to determine the effect of cryopreservation on acH4K12 in oocytes and their respective zygotes.

Methods: AcH4K12 in fresh or vitrified-warmed oocytes and their respective zygotes at 70 min-12 h post-fertilization were assessed using fluorescent staining.

Results: 1.

In vitro development and chromosomal configuration of bovine somatic cloned embryos with nonenucleated metaphase II oocytes.

This study was designed to examine the effects of the presence of oocyte nuclei on the donor cell nuclear remodeling, including premature chromosome condensation (PCC) and DNA configuration, and subsequent embryo development. The results showed that: (1) the presence of oocyte MII spindles was more likely to induce donor cell PCC. (2) The positional relationship between the fused donor cell and the oocyte metaphase spindle had an effect on oocyte PB2 extrusion.

Changes in acetylation on lysine 12 of histone H4 (acH4K12) of murine oocytes during maternal aging may affect fertilization and subsequent embryo development.

Objective: To compare acH4K12 levels in oocytes during mouse aging and then assess how such changes might affect the developmental potential of oocytes.

Design: Experimental animal study.

Setting: State key laboratory and university research laboratory.

Colcemid-treatment of heifer oocytes enhances nuclear transfer embryonic development, establishment of pregnancy and development to term.

Four experiments were designed to examine the effects of colcemid, a microtubule assembly inhibitor, on the development of bovine nuclear transfer (NT) embryos in vitro and in vivo. Recipient oocytes matured at different times were exposed to colcemid. Approximately 80-93% of the exposed oocytes, with or without the first polar body (PB1), developed obvious membrane projections.

Nicotine combined with okadaic acid or taxol adversely affects bovine oocyte maturation and subsequent embryo development.

Objective: To investigate the effects of nicotine in combination with okadaic acid (OA) or taxol on bovine oocyte maturation and subsequent embryonic development.

Design: Prospective randomized study.

Setting: University research laboratory.

A novel approach to identify bovine sperm membrane proteins that interact with receptors on the vitelline membrane of bovine oocytes.

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions.

Nicotine alters bovine oocyte meiosis and affects subsequent embryonic development.

The effects of nicotine on nuclear maturation and meiotic spindle dynamics of bovine oocytes and subsequent embryonic development were investigated. Maturation rates (85%-94%) derived from nicotine treatments at 0.01 to 1.

Effect of nicotine on in vitro maturation of bovine oocytes.

The putative effect of nicotine on maturation and the chromosomal complement of bovine oocytes were investigated in the present study. Cumulus-enclosed oocytes were incubated in maturation medium with 0, 0.5, 1.

Specific integrin subunits in bovine oocytes, including novel sequences for alpha 6 and beta 3 subunits.

Integrins facilitate attachment of cells to the extra-cellular matrix, often binding the arginine-glycine-aspartic acid tri-peptide motif, thus facilitating cell migration, mediating cell-cell adhesion, linking the extracellular matrix (ECM) with cytoskeletal elements, and acting as signaling molecules. Adhesion activates signaling mechanisms that regulate integrin function, cytoskeletal assembly, cell behavior, and protein synthesis. Immunofluorescence was used to determine the presence of integrin alpha and beta subunits on the surface of bovine oocytes using a panel of monoclonal antibodies (mAbs) specific for alphaL, alphaM, alphaX, alphaV, alpha2, alpha4, alpha6, beta1, beta2, and beta3 antigens, with multiple antibodies for each subunit.

Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development.

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1).

The developmental competence of bovine nuclear transfer embryos derived from cow versus heifer cytoplasts.

Due to its economic importance, the production of cattle by nuclear transfer has been a primary research focus for many researchers during the past few years. While many groups have successfully produced cattle by nuclear transfer, and progress in this area continues, nuclear transfer remains a very inefficient technology. This study evaluates the effect of the oocyte source (cow and heifer) on the developmental competence of nuclear transfer embryos.

Molecular phylogenetics and biogeography of Lepus in Eastern Asia based on mitochondrial DNA sequences.

In spite of several classification attempts among taxa of the genus Lepus, phylogenetic relationships still remain poorly understood. Here, we present molecular genetic evidence that may resolve some of the current incongruities in the phylogeny of the leporids. The complete mitochondrial cytb, 12S genes, and parts of ND4 and control region fragments were sequenced to examine phylogenetic relationships among Chinese hare taxa and other leporids throughout the World using maximum parsimony, maximum likelihood, and Bayesian phylogenetic reconstruction approaches.

Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air-dry karyotyping method.

Of the few published studies on the cytogenetic analyses of bovine nuclear transferred (NT) embryos, results differ between air-dry and fluorescent in situ hybridization (FISH) procedures. A modified air-dry procedure is reported in this study that provides more metaphase plates for analysis. Day 5 and Day 7 bovine NT embryos were cultured in colcemid-containing CR1aa for 10-12 or 16-18 h, then treated in hypotonic sodium citrate for 3-5 min.

Asymmetric division of spindle microtubules and microfilaments during bovine meiosis from metaphase I to metaphase III.

The kinetics of spindle and chromosomes during bovine oocyte meiosis from meiosis I to meiosis III is described. The results of this study showed that (1) oocytes began to extrude the first polar body (Pb1) at the early anaphase I stage and the Pb1 totally separated from the mother cell only when oocytes reach the MII stage; (2) the morphology of the spindle changed from barrel-shaped at the metaphase stage to cylinder-shaped at early anaphase, and then to a thin, long triangle-shaped cone at late anaphase and telophase stages; (3) chromosome morphology went from an individual visible stage at metaphase to a less defined chromatin state during anaphase and telophase stages, and then back to visible individual chromosomes at the next metaphase; (4) chromatin that connected with the floor of the cone became the polar bodies and expelled, and almost all of the microtubules (MTs) and microfilaments (MFs) composing the spindles moved towards and contributed to the polar bodies; and (5) the size of the metaphase I (MI) spindle was larger than the metaphase II (MII) and metaphase III (MIII) spindles. The MII spindle, however, is more barrel-shaped than the MI spindle.

Cloned horse pregnancies produced using adult cumulus cells.

The objectives of the present study were to: (1) clone horses using adult cumulus cells; and (2) determine whether the cumulus cell donor affected the outcome. In vivo-matured cumulus-oocyte complexes were obtained using transvaginal ultrasound-guided follicle aspiration; oocytes were used as cytoplasts, whereas cumulus cells (from one of three different mares) were used as donor cells. Immediately following nuclear transfer and activation procedures, cloned embryos were transferred surgically to the oviduct of recipient mares (n = 2-5 embryos per recipient) that had ovulated within 24 h prior to the transfer.