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

The transcriptional response of Caenorhabditis elegans to ivermectin exposure identifies novel genes involved in the response to reduced food intake

We have examined the transcriptional response of Caenorhabditis elegans following exposure to the anthelmintic drug ivermectin (IVM) using whole genome microarrays and real-time QPCR. Our original aim was to identify candidate molecules involved in IVM metabolism and/or excretion. For this reason the IVM tolerant strain, DA1316, was used to minimise transcriptomic changes related to the phenotype of drug exposure. However, unlike equivalent work with benzimidazole drugs, very few of the induced genes were members of xenobiotic metabolising enzyme families. Instead, the transcriptional response was dominated by genes associated with fat mobilization and fatty acid metabolism including catalase, esterase, and fatty acid CoA synthetase genes. This is consistent with the reduction in pharyngeal pumping, and consequential reduction in food intake, upon exposure of DA1316 worms to IVM. Genes with the highest fold change in response to IVM exposure, cyp-37B1, mtl-1 and scl-2, were comparably up-regulated in response to short–term food withdrawal (4 hr) independent of IVM exposure, and GFP reporter constructs confirm their expression in tissues associated with fat storage (intestine and hypodermis). These experiments have serendipitously identified novel genes involved in an early response of C. elegans to reduced food intake and may provide insight into similar processes in higher organisms

Vitrification of human immature oocytes before and after in vitro maturation: a review

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations

Source Imaging of a Moving Type-IV Solar Radio Burst and its Role in Tracking Coronal Mass Ejection From the Inner to the Outer Corona

Source imaging of solar radio bursts can be used to track energetic electrons and associated magnetic structures. Here we present a combined analysis of data at different wavelengths for an eruption associated with a moving type-IV (t-IVm) radio burst. In the inner corona, the sources are correlated with a hot and twisted eruptive EUV structure, while in the outer corona the sources are associated with the top front of the bright core of a white light coronal mass ejection (CME). This reveals the potential of using t-IVm imaging data to continuously track the CME by lighting up the specific component containing radio-emitting electrons. It is found that the t-IVm burst presents a clear spatial dispersion with observing frequencies. The burst manifests broken power-law like spectra in brightness temperature, which is as high as $10^7$-$10^9$ K while the polarization level is in-general weak. In addition, the t-IVm burst starts during the declining phase of the flare with a duration as long as 2.5 hours. From the differential emission measure analysis of AIA data, the density of the T-IVm source is likely at the level of 10$^8$ cm$^{-3}$ at the start of the burst, and the temperature may reach up to several MK. These observations do not favor gyro-synchrotron to be the radiation mechanism, yet in line with a coherent plasma emission excited by energetic electrons trapped within the source. Further studies are demanded to elucidate the emission mechanism and explore the full diagnostic potential of t-IVm bursts.Comment: 22 pages, 8 figures, Accepted for publication in AP

In vitro maturation of germinal vesicle oocytes in stimulated intracytoplasmic sperm injection cycles

Objective: This study evaluated in vitro maturation (IVM) of oocytes in the germinal vesicle (GV) stage in stimulated intracytoplasmic sperm injection (ICSI) cycles. Materials and Methods: A total of 26 women, aged 18-37 years, who were candidates for ICSI at the Fatemeh Zahra Infertility and Health Reproductive Research Center in 2007 were recruited for this study. We used the standard long protocol for ovarian stimulation. Follicles >11 mm were punctured 36-38 hours after administration of 10000 IU human chorionic gonadotrophin (hCG). Immature oocytes were cultured for 24-30 hours. Oocytes that liberated polar bodies were injected by sperm prepared within the previous day. IVM fertilized oocytes were cultured an additional 24-30 hours for cleavage. The rates of maturation, fertilization and cleavage in IVM oocytes were recorded and statistically compared to in vivo matured sibling oocytes. Results: There were 279 collected oocytes (mean±SD: 10.73 ±6.2), of which 4.08±2.79 were subjected to IVM. An average of 2.73 ±2.15 GV oocytes (70%) developed to metaphase II (MII). Although the maturation rate significantly differed between the IVM and in vivo Mil sibling oocyte groups (p=0.027), the numbers of fertilized oocytes (p=0.795) and cleaved embryos (p=0.529) were not significantly high in the in vivo group. Transfer of IVM embryos occurred in only three cases with one pregnancy that resulted in the delivery of a healthy baby. Conclusion: This study shows that culturing GV oocytes can produce acceptable numbers of four-cell embryos on the transfer day. The developmental competence of oocytes is not significantly different between early stage IVM and in vivo sibling embryos

Dosing pole recommendations for lymphatic filariasis elimination: A height-weight quantile regression modeling approach

BACKGROUND: The World Health Organization (WHO) currently recommends height or age-based dosing as alternatives to weight-based dosing for mass drug administration lymphatic filariasis (LF) elimination programs. The goals of our study were to compare these alternative dosing strategies to weight-based dosing and to develop and evaluate new height-based dosing pole scenarios. METHODOLOGY/PRINCIPAL FINDINGS: Age, height and weight data were collected from \u3e26,000 individuals in five countries during a cluster randomized LF clinical trial. Weight-based dosing for diethylcarbamazine (DEC; 6 mg/kg) and ivermectin (IVM; 200 ug/kg) with tablet numbers derived from a table of weight intervals was treated as the gold standard for this study. Following WHO recommended age-based dosing of DEC and height-based dosing of IVM would have resulted in 32% and 27% of individuals receiving treatment doses below those recommended by weight-based dosing for DEC and IVM, respectively. Underdosing would have been especially common in adult males, who tend to have the highest LF prevalence in many endemic areas. We used a 3-step modeling approach to develop and evaluate new dosing pole cutoffs. First, we analyzed the clinical trial data using quantile regression to predict weight from height. We then used weight predictions to develop new dosing pole cutoff values. Finally, we compared different dosing pole cutoffs and age and height-based WHO dosing recommendations to weight-based dosing. We considered hundreds of scenarios including country- and sex-specific dosing poles. A simple dosing pole with a 6-tablet maximum for both DEC and IVM reduced the underdosing rate by 30% and 21%, respectively, and was nearly as effective as more complex pole combinations for reducing underdosing. CONCLUSIONS/SIGNIFICANCE: Using a novel modeling approach, we developed a simple dosing pole that would markedly reduce underdosing for DEC and IVM in MDA programs compared to current WHO recommended height or age-based dosing

Increased expression of a microRNA correlates with anthelmintic resistance in parasitic nematodes

Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3′ UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3′ UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species

Pharmacokinetics, safety, and efficacy of a single co-administered dose of diethylcarbamazine, albendazole and ivermectin in adults with and without Wuchereria bancrofti infection in Cote d\u27Ivoire

BackgroundA single co-administered dose of ivermectin (IVM) plus diethylcarbamazine (DEC) plus albendazole (ALB), or triple-drug therapy, was recently found to be more effective for clearing microfilariae (Mf) than standard DEC plus ALB currently used for mass drug administration programs for lymphatic filariasis (LF) outside of sub-Saharan Africa. Triple-drug therapy has not been previously tested in LF-uninfected individuals from Africa. This study evaluated the pharmacokinetics (PK), safety, and efficacy of triple-drug therapy in people with and without Wuchereria bancrofti infection in West Africa.MethodsIn this open-label cohort study, treatment-naïve microfilaremic (>50 mf/mL, n = 32) and uninfected (circulating filarial antigen negative, n = 24) adults residing in Agboville district, Côte d’Ivoire, were treated with a single dose of IVM plus DEC plus ALB, and evaluated for adverse events (AEs) until 7 days post treatment. Drug levels were assessed by liquid chromatography and mass spectrometry. Persons responsible for assessing AEs were blinded to participants’ infection status.FindingsThere was no difference in AUC0-inf or Cmax between LF-infected and uninfected participants (P>0.05 for all comparisons). All subjects experienced mild AEs; 28% and 25% of infected and uninfected participants experienced grade 2 AEs, respectively. There were no severe or serious adverse events. Only fever (16 of 32 versus 4 of 24, PConclusionsModerate to heavy W. bancrofti infection did not affect PK parameters for IVM, DEC or ALB following a single co-administered dose of these drugs compared to uninfected individuals. The drugs were well tolerated. This study confirmed the efficacy of the triple-drug therapy for clearing W. bancrofti Mf and has added important information to support the use of this regimen in LF elimination programs in areas of Africa without co-endemic onchocerciasis or loiasis.Trial registrationClinicalTrials.gov NCT02845713.</div