Molecular genetic analysis of bovine oocytes with different developmental potentials

Poor oocyte developmental potential contributes to reduced fertility in livestock species and hampers the application of biotechnology techniques in cattle industry that could be used for spreading certain genotypes with production advantages. However, the molecular mechanisms regulating oocyte developmental potential are generally poorly understood. Therefore, the objective of this study was to identify differentially regulated genes in oocytes with different developmental potential that could be associated with their competence. For this, oocytes were selected based on phase of follicular turnover (growth vs. dominance) and BCB staining (BCB+ vs. BCB-) as two independent models for screening of oocyte competence. For each model, six pools of oocytes were used for mRNA isolation and subsequent RNA amplification. A custom-made cDNA array with ~ 2000 clones was used to compare the gene expression profiles of competent versus incompetent oocytes of each model. The Significance Analysis of Microarray (SAM) has been used for data generation. A total of 51 and 185 genes to be differentially expressed have been identified between the oocytes derived from growth compared to dominance follicular phase and BCB+ compared to BCB- ones, respectively. Based on biological process annotation, genes involved in protein biosynthesis as structural constituent of ribosome (RPL24, ARL6IP, RPS14 and RPS15), translation elongation factor activity (EEF1A1), chromosome organization and biogenesis (H2AFZ) and signal transduction (GNB2L1) were commonly up-regulated in competent compared to incompetent oocytes of both models. On the other hand, incompetent oocytes from both models were enriched with transcripts regulating transcription (PTTG1) and growth factor activity (BMP 15). Quantitative real-time PCR has confirmed the relative abundance of 8 out of 10 and 9 out of 10 genes to be in accordance with microarray analysis for follicular phase and BCB staining models, respectively. Overall, this study provides a genome-wide expression profiling of genes that could be associated with developmental competence of bovine oocytes. However, further functional investigations based on this data could help to define the exact key regulatory genes controlling oocyte quality that could be considered as good biomarkers.Molekulargenetische Analyse von Rinderoocyten mit unterschiedlichen Entwicklungspotentialen Ein geringes Entwicklungspotential von Oozyten führt zu einer verringerten Fruchtbarkeit in der Rinderhaltung. Gleichzeitig erschwert es moderne biotechnologie Zuchtmethoden, wie z.B. Embryotransfer, die für die schnelle Verbreitung von positiven Genotypen genutzt werden können. Allerdings sind die molekularen Mechanismen, die das Oozytenentwicklungspotential regulieren, bisher nur wenig bekannt. Ziel dieser Arbeit war es demzufolge, unterschiedlich regulierte Gene in Oozyten mit unterschiedlichem Entwicklungspotential zu identifizieren, die mit der Entwicklungskompetenz der Oozyten assoziiert sein könnten. Dafür wurden Oozyten auf Grund ihres follikularen Umsatzes (Wachstum gegenüber Dominanzverhalten) (Modell 1) bzw. auf Grund von BCB Färbung (BCB+ gegen BCB-) (Modell 2), ausgewählt. Für jedes Modell wurden sechs Pools von Oocyten für die mRNA-Isolierung und RNA-Amplifikation verwendet. Ein speziell angefertigter cDNA Array mit ~ 2000 Klonen wurde genutzt, um Genexpressionsprofile von kompetenten und inkompetenten Oocyten zu vergleichen. Das Programm Significance Analysis of Microarray (SAM) wurde für die Datenauswertung herangezogen. Insgesamt konnten 51 bzw. 185 unterschiedlich exprimierte Gene für die Oozyten mit unterschiedlichem follikularem Wachstum bzw. mit unterschiedlicher BCB Färbung identifiziert werden. Für die weiteren Untersuchungen wurden Gene, die in der Proteinbiosynthese als struktureller Bestandteil des Ribosoms vorkommen (RPL24, ARL6IP, RPS14 und RPS15), ein Translations-Elongations-Faktor (EEF1A1), ein Gen für Chromosomenorganisation und Biogenese (H2AFZ), sowie ein Gen für die Signal Transduktion (GNB2L1) ausgewählt. Diese Gene waren in kompetenten Oozyten höher exprimiert als in inkompetenten Oozyten. Des weiteren wurden ein Gen für die Transkriptionsregulierung (PTTG1) sowie ein Gen für die Wachstumsfaktor Aktivität (BMP15) ausgesucht, welche wiederum in inkompetenten Oozyten hochreguliert waren. Mittels quantitativer Real-time PCR wurde die relative Abundanz aus der Microarray Analyse bei acht von zehn Genen (Modell 1) bzw. neun von zehn Genen (Modell 2) bestätigt. Insgesamt beschreibt diese Arbeit ein genomweites Expressionsprofil von Genen, die mit der Entwicklungskompetenz von Rinderoozyten assoziiert werden können. Darüber hinaus könnten weitere funktionelle Analysen, basierend auf unseren Ergebnissen, helfen, die exakten regulierenden Schlüsselgene zu finden, welche die Oozytenqualität steuern, und die zukünftig als Biomarker verwendet werden könnten

Functionalized polymers of intrinsic microporosity for highly energy- intensive gas separations

Membrane-based gas separation is a rapidly emerging technology that has been established in the purification of air and hydrogen streams and is showing an increasingly larger role in natural gas sweetening and vapor/gas separations. One strategy actively pursued to generate polymers with combinations of high permeability and high selectivity is the introduction of microporosity (pores \u3c 20 Å) in the polymer matrix. It has been shown that rigid ladder-type chains comprising fused rings joined by sites of contortion pack inefficiently in the solid state to produce polymers of intrinsic microporosity (PIMs). Recently, a successful integration of monomers contorted by spirobisindane, ethanoanthracene, Tröger’s base and triptycene moieties into polyimide structures has also generated highly permeable intrinsically microporous polyimides (PIM-PIs). Some of these PIM-PIs have shown significantly enhanced performance for O2/N2, H2/N2 and H2/CH4 separations with properties defining the most recent 2015 permeability/selectivity upper bounds. Here, we will discuss several series of novel PIM-PIs and ladder PIMs based on rigid and bicyclic moieties, which are solution processable to form mechanically robust films with high internal surface areas (up to 1100 m2/g). Gas permeation and physisorption data indicate the development of an ultramicroporous structure that is tunable for different gas separation applications. Specific emphasis will be placed on the potential use of hydroxyl- and carboxyl-functionalized PIMs for highly-energy demanding applications for natural gas treatment and olefin/paraffin separation

BLOOD LIPID DISORDER IN MEN WITH INCREASED WAIST CIRCUMFERENCE COMPARED TO MEN HAVING NORMAL WAIST CIRCUMFERENCE WITHIN THE SAME CATEGORY OF BMI

Background: No local studies have been performed yet to investigate the influence of central or abdominal obesity on serum lipids in men having increased Waist Circumference (WC) compared to men with normal Waist Circumference values within the same BMI (Body Mass Index) category. Objective:To examine whether the prevalence of dyslipidemia, (defined as Hypercholesterolemia (Total Cholesterol level ≥240 mg/dl), high LDL-C level (≥160 mg/dl), low HDL-C level (<35 mg/dl), or Hypertriglyceridemia (TG level ≥200 mg/dl)), is higher in men having high Waist Circumference compared to others with normal WC values within the same BMI category. Methods: The study was conducted between September 2013 and July 2014. Eighty-eight overweight men (BMI = 25-29.9) were grouped by WC as follows: 28 with high values (>102 cm) and 60 with normal values (≤ 102cm). Blood samples were drawn and assayed for total cholesterol, triglyceride, HDL-C, and LDL-C,at the department of Laboratory in the Faculty of Public Health, Lebanese University. All assays were performed by enzymatic colorimetric methods using Hitachi-704. Results: Overweight men with high WC values (according to cutoff points internationally adopted) were the most likely to have dyslipidemia with its subsequent increased health risk compared with those having normal WC values. Conclusion: we showed in this study that the prevalence of dyslipidemia in men with high WC values is greater compared to those with normal WC values within the same BMI category. This finding leads us to the importance of the incorporated evaluation of WC in addition to the BMI in clinical practice

BLOOD LIPID DISORDER IN MEN WITH INCREASED WAIST CIRCUMFERENCE COMPARED TO MEN HAVING NORMAL WAIST CIRCUMFERENCE WITHIN THE SAME CATEGORY OF BMI

Background: No local studies have been performed yet to investigate the influence of central or abdominal obesity on serum lipids in men having increased Waist Circumference (WC) compared to men with normal Waist Circumference values within the same BMI (Body Mass Index) category. Objective:To examine whether the prevalence of dyslipidemia, (defined as Hypercholesterolemia (Total Cholesterol level ≥240 mg/dl), high LDL-C level (≥160 mg/dl), low HDL-C level (<35 mg/dl), or Hypertriglyceridemia (TG level ≥200 mg/dl)), is higher in men having high Waist Circumference compared to others with normal WC values within the same BMI category. Methods: The study was conducted between September 2013 and July 2014. Eighty-eight overweight men (BMI = 25-29.9) were grouped by WC as follows: 28 with high values (>102 cm) and 60 with normal values (≤ 102cm). Blood samples were drawn and assayed for total cholesterol, triglyceride, HDL-C, and LDL-C,at the department of Laboratory in the Faculty of Public Health, Lebanese University. All assays were performed by enzymatic colorimetric methods using Hitachi-704. Results: Overweight men with high WC values (according to cutoff points internationally adopted) were the most likely to have dyslipidemia with its subsequent increased health risk compared with those having normal WC values. Conclusion: we showed in this study that the prevalence of dyslipidemia in men with high WC values is greater compared to those with normal WC values within the same BMI category. This finding leads us to the importance of the incorporated evaluation of WC in addition to the BMI in clinical practice

Identification and characterization of miRNAs expressed in the bovine ovary

<p>Abstract</p> <p>Background</p> <p>MicroRNAs are the major class of gene-regulating molecules playing diverse roles through sequence complementarity to target mRNAs at post-transcriptional level. Tightly regulated expression and interaction of a multitude of genes for ovarian folliculogenesis could be regulated by these miRNAs. Identification of them is the first step towards understanding miRNA-guided gene regulation in different biological functions. Despite increasing efforts in miRNAs identification across various species and diverse tissue types, little is known about bovine ovarian miRNAs. Here, we report the identification and characterization of miRNAs expressed in the bovine ovary through cloning, expression analysis and target prediction.</p> <p>Results</p> <p>The miRNA library (5'-independent ligation cloning method), which was constructed from bovine ovary in this study, revealed cloning of 50 known and 24 novel miRNAs. Among all identified miRNAs, 38 were found to be new for bovine and were derived from 43 distinct loci showing characteristic secondary structure. While 22 miRNAs precursor loci were found to be well conserved in more than one species, 16 were found to be bovine specific. Most of the miRNAs were cloned multiple times, in which let-7a, let-7b, let-7c, miR-21, miR-23b, miR-24, miR-27a, miR-126 and miR-143 were cloned 10, 28, 13, 4, 11, 7, 6, 4 and 11 times, respectively. Expression analysis of all new and some annotated miRNAs in different intra-ovarian structures and in other multiple tissues showed that some were present ubiquitously while others were differentially expressed among different tissue types. Bta-miR-29a was localized in the follicular cells at different developmental stages in the cyclic ovary. Bio-informatics prediction, screening and Gene Ontology analysis of miRNAs targets identified several biological processes and pathways underlying the ovarian function.</p> <p>Conclusion</p> <p>Results of this study suggest the presence of miRNAs in the bovine ovary, thereby elucidate their potential role in regulating diverse molecular and physiological pathways underlying the ovarian functionality. This information will give insights into bovine ovarian miRNAs, which can be further characterized for their role in follicular development and female fertility as well.</p

Alterations in transcript abundance of bovine oocytes recovered at growth and dominance phases of the first follicular wave

<p>Abstract</p> <p>Background</p> <p>Oocyte developmental competence is highly affected by the phase of ovarian follicular wave. Previous studies have shown that oocytes from subordinate follicles recovered at growth phase (day 3 after estrus) are developmentally more competent than those recovered at dominance phase (day 7 after estrus). However, the molecular mechanisms associated with these differences are not well elucidated. Therefore, the objective of this study was to investigate transcript abundance of bovine oocytes retrieved from small follicles at growth and dominance phases of the first follicular wave and to identify candidate genes related to oocyte developmental competence using cDNA microarray.</p> <p>Results</p> <p>Comparative gene expression analysis of oocytes from growth and dominance phases and subsequent data analysis using Significant Analysis of Microarray (SAM) revealed a total of 51 differentially regulated genes, including 36 with known function, 6 with unknown function and 9 novel transcripts. Real-time PCR has validated 10 transcripts revealed by microarray analysis and quantified 5 genes in cumulus cells derived from oocytes of both phases. The expression profile of 8 (80%) transcripts (ANAXA2, FL396, S100A10, RPL24, PP, PTTG1, MSX1 and BMP15) was in agreement with microarray data. Transcript abundance of five candidate genes in relation to oocyte developmental competence was validated using Brilliant Cresyl Blue (BCB) staining as an independent model. Furthermore, localization of mRNA and protein product of the candidate gene MSX1 in sections of ovarian follicles at days 0, 1, 3 and 7 of estrous cycle showed a clear fluorescent signal in both oocytes and cumulus cells with higher intensity in the former. Moreover, the protein product was detected in bovine oocytes and early cleavage embryos after fertilization with higher intensity around the nucleus.</p> <p>Conclusion</p> <p>This study has identified distinct sets of differentially regulated transcripts between bovine oocytes recovered from small follicles at growth and dominance phases of the first follicular wave. The validation with independent model supports our notion that many of the transcripts identified here may represent candidate genes associated with oocyte developmental competence. Further specific functional analysis will provide insights into the exact role of these transcripts in oocyte competence and early embryonic development.</p

Ovarian mRNA and miRNA Transcriptome Profiling of Domestic Sheep Breeds

201

Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries)

Background: The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. Results: Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. Conclusions: The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation.Peer reviewe

Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries)

Abstract Background The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. Results Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. Conclusions The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation