The effect of leukaemia inhibitory factor (LIF) on bovine embryo development in vitro : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Animal Science at Massey University

The aim of the study was to investigate the effect of Leukaemia Inhibitory Factor (LIF) either during in vitro maturation (IVM) or in vitro culture (IVC) on bovine embryo development. Three main experiments were conducted using oocytes aspirated from 2-8 mm diameter follicles collected from cows slaughtered at local abattoirs, Hamilton. The oocytes were matured in a modified TCM-199 containing 10 µ/ml of FSH and LH, and 1 µg/ml E2, fertilised in TALP and cultured in SOF/AA/BSA. Experiment 1 examined the effect of LIF (0, 500, 1000 or 2000 U/ml) and various time periods of IVM (18, 22 or 28 h), in a 4 × 3 factorial design on oocyte maturation. Following maturation, oocytes were stripped out of cumulus cells, then denuded oocytes were stained in 1% lacmoid for determination of maturation stage while the cumulus cells were examined for the incidence of apoptosis by observation of DNA fragmentation using gel electrophoresis procedures. Experiment 2 comprised two parts, (a) the effect of LIF (0, 500, 1000 or 2000 U/ml) at 24 h IVM in a randomised block design on in vitro development of embryos, (b) comparison of 20 vs 24 h IVM in the presence of LIF (0, 500, 1000 or 2000 U/ml) in a 2 × 4 factorial experiment on embryo development. In the two studies, the proportion of bovine oocytes that cleaved and developed to blastocyst stage was recorded. In addition, cell numbers of blastocysts after Giemsa staining were counted. Experiment 3 examined the effect of LIF during IVM (0 vs 1000 U/ml) or IVC (0, 500, 1000 or 2000 U/ml) in a 2 × 4 factorial design on development of embryos. The incidence of cleavage and blastocyst development and cell numbers of blastocysts were recorded. In addition, blastocysts were further categorised into early, expanded and hatched blastocyst stages and cell numbers of blastocyst inner cell mass (ICM) and trophectoderm (TE) after differential staining with Hoechst 33342 and propidium iodide were determined. In Experiment One, an interaction of LIF concentration and duration of IVM was not observed for the proportion of immature oocytes reaching metaphase II (P>0.05). The presence of LIF (500, 1000 or 2000 U/ml) increased the proportion of oocytes at metaphase II at 18 h (50%, 52% or 58%, respectively, compared to without LIF= 27%), indication that LIF may accelerate the maturation process in vitro. Supplementation of LIF during IVM did not affect the incidence of apoptosis of the cumulus cells. In Experiment Two, compared to 24 h IVM in the presence of LIF, 20 h IVM significantly increased blastocyst rates (Σ blastocysts : Σ cleaved, P0.05), however the data show that treatment groups of 20 h IVM in LIF resulted in higher cell numbers of blastocysts than achieved by 24 h IVM. In Experiment Three, there was a correlation between LIF during IVM and LIF during IVC in the proportion of blastocysts (P0.05). However, blastocysts derived from oocytes matured without LIF had significantly increased cell numbers (121 cells) compared to those matured in 1000 U/ml LIF (109 cells, P0.05). However, a concentration of 2000 U/ml LIF during IVC accelerated blastocyst development with more blastocysts hatching (60%, P0.05). A concentration of 1000 U/ml LEF during IVC resulted in higher cell numbers of ICM (P<0.05). This study suggests that LIF of 500, 1000 or 2000 U/ml increased the proportion of metaphase II bovine oocytes and even reduced the time course of IVM. Supplementation of LIF during IVM may suppress the incidence of apoptosis of the cumulus cells. IVM for 20 h in the presence of LIF resulted in a higher number of blastocysts and 1000 U/ml LIF during IVM and culture in LIF increased the proportion of blastocysts. A higher concentration of LIF is required for reaching the hatched blastocyst stage. A level of 1000 U/ml LIF during IVC promoted higher cell numbers of ICM

Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF

Injury to peripheral nerves results in the infiltration of immune cells, which remove axonal- and myelin-derived material. Schwann cells could play a key role in this process by regulating macrophage infiltration. We show here that medium conditioned by primary denervated Schwann cells or the Schwannoma cell line RN22 produces chemotactic activity for macrophages. The presence of blocking antibodies to macrophage chemoattractant protein-1 (MCP-1) or leukemia inhibitory factor (LIF) reduced this activity to similar to35 and 65% of control levels, respectively, and only 15% remained in the presence of both antibodies. The presence of chemotactic LIF in Schwann cell-conditioned medium was confirmed by using cells from lif-/- mice. Although interleukin-6 (IL-6) is not itself a chemotactic factor, we found that medium from il-6-/- nerves showed only 40% of the activity secreted by wild-type nerves. Furthermore, IL-6 rapidly induced LIF mRNA in primary Schwann cells, and LIF rapidly induced MCP-1 mRNA expression. Treatment of RN22 Schwannoma cells with IL-6 or LIF enhanced the secretion of the chemotactic activity of these cells.These observations show that Schwann cells attract macrophages by secreting MCP-1 and LIF. They also provide evidence for an autocrine-signaling cascade involving IL-6, LIF, and MCP-1, which amplifies the Schwann cell-derived chemotactic signals gradually, in agreement with the delayed entry of macrophages to injured nerves

Determining the role of tumor-derived leukemia inhibitory factor in cancer cachexia using a genetic approach

Cachexia is a multifactorial metabolic wasting syndrome that affects a large percentage of cancer patients and results in the involuntary loss of skeletal muscle and adipose tissue. The consequences of this condition include metabolic imbalances and fatigue, which are strongly associated with poor prognosis. While the specific mechanism for skeletal muscle wasting is still undefined, LIF secreted by C26 colon carcinoma cells has recently be found to induce atrophy in treated myotubes. The purpose of this study is to determine the necessity of LIF for inducing atrophy in mouse myotubes by producing a knockout of Lif in C26 cells using CRIPSR-Cas9. Media was collected from these cells and used to treat myotubes. Measurements of myotube diameters were made and atrophy was compared between myotubes that received medium from C26 and C26Lif-/- cells. A dosage of recombinant mouse LIF was also added to LIF-deficient medium in order to determine if LIF alone was sufficient to induce atrophy. At study endpoint, myotubes that were treated with media taken from C26 cells showed significant signs of atrophy compared to myotubes that were treated C26Lif-/- media. LIF was also shown to be sufficient to induce myotube atrophy on its own, with atrophy being rescued in myotubes that received a dosage of LIF added to C26Lif-/- media. These results demonstrate that LIF is required for atrophy to be induced in mouse myotubes treated with media taken from cancer cells, and can do so independent of other secreted factors

Interleukin 1 Signaling Is Regulated by Leukemia Inhibitory Factor (LIF) and Is Aberrant in Lif−/− Mouse Uterus

This study addresses the regulation of the interleukin 1 (IL1) system in the murine uterine luminal epithelium (LE) and stroma by leukemia inhibitory factor (LIF). Using RT-PCR we compared expression of Il1a, Il1b, Il1rn, Il1r1, and Il1r2 during the pre- and peri-implantation periods of pregnancy in wild-type (WT) and LIF-null LE and stroma. In WT LE, Il1a transcripts were down-regulated on Day 4 of pregnancy (D4), with renewed expression by the evening of D4 (D4 pm). In Lif−/− LE there was a gradual decrease in expression on D2, and expression became undetectable by D6. Il1b and Il1r1 expression were similar in WT and null mice, but Il1rn expression was almost completely lost during the peri-implantation period in Lif−/− LE. In the stroma, Il1a was sharply down-regulated on D4 and reappeared on D4 pm but was only expressed from D3 to D5 in the null mice. Stromal Il1r1 and Il1r2 were also misregulated. Il1rn showed constitutive expression in null stroma in contrast to the loss of expression on D4 in the WT mouse. In Lif-deficient mice, immunostaining indicated a reduction of endometrial IL1A at the time of implantation and of IL1B in stroma. LE-stromal coculture revealed that LIF stimulated the apical secretion of both IL1A and PTGES2 by LE cells without affecting basal secretion of IL1A and with only a small effect on basal PTGES2 secretion. We conclude that Il1a and Il1rn in LE and Il1a, Il1rn, and Il1r1 in stroma are regulated by LIF, which stimulates apical secretion of IL1A by LE

Aircraft based four-channel thermal dissociation laser induced fluorescence instrument for simultaneous measurements of NO2, total peroxy nitrate, total alkyl nitrate, and HNO3

A four-channel thermal dissociation laser induced fluorescence (TD-LIF) instrument has been developed for simultaneous measurements of nitrogen dioxide (NO2), total peroxy nitrate (∑PNs), total alkyl nitrate (∑ANs) and nitric acid (HNO3). NO2 is measured directly by LIF at 532 nm, whereas organic nitrates and nitric acid are thermally dissociated at distinct temperatures in the inlet to form NO2, which is then measured by LIF. The concentrations of each dissociated species are derived by the differences in measured NO2 relative to the reference colder inlet channel. The TD-LIF was adapted to fly on board the UK Facility for Airborne Atmospheric Measurements (FAAM) BAe 146-301 atmospheric research aircraft in summer 2010, and to date has successfully flown in five field campaigns. This paper reports novel improvements in the TD-LIF instrumentations, including (1) the use of a single wavelength laser, which makes the system compact and relatively cheap; (2) the use of a single beam laser that allows easy alignment and optical stability against the vibrational aircraft environment; and (3) the optical assembly of four detection cells that allow simultaneous and fast (time resolution up to 0.1 s) measurements of NO2, ∑PNs, ∑ANs and HNO3. Laboratory-generated mixtures of PNs, ANs and HNO3 in zero air are converted into NO2 and used to fix the dissociation temperatures of each heated inlet to test the selectivity of the instrument and potential interferences due to recombination reactions of the dissociated products. The effectiveness of the TD-LIF was demonstrated during the RONOCO aircraft campaign (summer 2010). A chemiluminescence system that was measuring NO2 and a broadband cavity enhanced absorption spectrometer (BBCEAS) that was measuring one of the PNs (N2O5) were installed on the same aircraft during the campaign. The in-flight intercomparison of the new TD-LIF with the chemiluminescence system for NO2 measurements and the intercomparison between ∑PNs measured by the TD-LIF and N2O5 by the BBCEAS are used to assess the performance of the TD-LIF

Growth of Self Organized Eutectic Fibers from LiF-Rare Earth Fluoride Systems

Eutectic fibers consisting of an ordered arrangement of LiF fibrils inside a LiREF4 matrix (RE = Y, Gd) can be grown with the micro-pulling-down method at sufficiently large pulling rate exceeding 120 mm/h. The distance between individual fibrils could be scaled down to 1 micrometer at 300 mm/h pulling. LiF-LiYF4 has stronger tendency to form facetted eutectic colonies than LiF-LiGdF4, explained by the larger entropy of melting of the former.Comment: 6 pages, 5 figures, Talk on MRS Fall 2012 Bosto

Fluoroacidity evaluation in molten salts

The fluoroacidity of several alkaline fluoride media was studied by monitoring the concentration of electroactive species which is decreasing versus time due to a gas species release, such as silicon fluorides, as indicated by the reaction: SiF(4+x)x- = SiF4(g) + x F- This article relates the Si(IV) reaction study to define a relative fluoroacidity scale by studying the silicon ions stability in different melts. Electrochemical techniques allow the measurement of SiF4+xx- concentration evolution and thus the reaction rate constant to be calculated at different temperatures and for several fluoride media. The article shows that the free F- content depends on the fluoride mixture and that the rate values are correlated with the fluoroacidity allowing a qualitative estimation. Then a fluoride solvents fluoroacidity scale was proposed, scaling the different eutectic melts from basic melt to acidic one: NaF-KF < LiF-KF < NaF-MgF2 < NaF-CaF2 < LiF-NaF < LiF < LiF-CaF2

Effect of 1 wt% LiF additive on the densification of nanocrystalline Y2O3 ceramics by spark plasma sintering

Densification of nanocrystalline cubic yttria (nc-Y2O3) powder, with 18 nm crystal size and 1 wt% LiF as a sintering additive was investigated. Specimens were fabricated by spark plasma sintering at 100 MPa, within the temperature range of 700–1500 °C. Sintering at 700 °C for 5 and 20 min resulted in 95% and 99.7% dense specimens, with an average grain size of 84 and 130 nm, respectively. nc-Y2O3 without additive was only 65% dense at 700 °C for 5 min. The presence of LiF at low sintering temperatures facilitated rapid densification by particle sliding and jamming release. Sintering at high temperatures resulted in segregation of LiF to the grain boundaries and its entrapment as globular phase within the fast growing Y2O3 grains. The sintering enhancement advantage of LiF was lost at high SPS temperatures