Exosomal and non-exosomal circulatory miRNAs in bovine follicular fluid : potential role of exosomal miRNAs in oocyte development

Growth and development of bovine follicle and oocyte is the result of series of complex and coordinated processes that involves extensive cell-to-cell communication in the follicle. This phenomenon involves enormous complex and heterogeneous biochemical substances existing in the oocytes and its surrounding cells and the follicular fluid. Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. The molecular mechanism of oocytes development and interaction between oocyte and follicular cells in the follicular micro-environment remains vague. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue Pro staining of the corresponding oocytes. Both ExoquickTM precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal portion of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNATM Universal RT miRNA PCR array system was used to profile miRNA expression. Western blot analysis against specific protein and electron microscopy imaging confirms the efficient separation of exosomal and non-exosomal fraction of follicular fluid. The real time qPCR array analysis revealed that a handful number of miRNAs are present in both exosomal and non-exosomal portion of bovine follicular fluid. Results revealed 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal portion of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus cells that may indicate the origin of extra-cellular miRNAs in follicular fluid. To further explore the potential roles of these extra-cellular miRNAs in follicular fluid, the potential targets were predicted using in silico based analysis, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. The present study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.Exosomale und non-exosomale zirkulierende miRNAs in der Follikelflüssigkeit beim Rind: Die Rolle von exosomalen miRNAs in der Oozytenentwicklung Wachstum und Entwicklung der Rinder Follikel und Eizellen ist das Ergebnis einer Reihe von komplexen und koordinierten Prozessen, die umfangreiche Zell-Zell-Kommunikation innerhalb der Follikel beinhaltet. Diese Interaktion beruht auf komplexen und heterogenen biochemischen Stoffen sowohl in den Eizellen, den benachbarten Zellen als auch in der Follikelflüssigkeit. Die Zell-Zell-Kommunikation innerhalb der Follikel ist geprägt durch viele Signalmoleküle. Dieser Prozess kann durch Sekretion und Aufnahme von Exosomen die mehrere bioaktive Moleküle einschließlich extrazellulärer miRNAs enthalten, vermittelt werden. Bisher jedoch, sind die molekularen Mechanismen der Eizellenentwicklung und der Interaktion zwischen Eizellen und Follikelzellen in der follikulären Umgebung noch nicht voll aufgeklärt. Follikelflüssigkeit und Zellen aus einzelnen Rinder Follikeln wurden auf der Grundlage einer Vitalfärbung der entsprechenden Eizellen mittels Brillant Cresyl blau Pro gruppiert. Sowohl eine Exoquick-Präzipitation als auch differentielle Ultrazentrifugation wurde verwendet, um die exosomalen und non-exosomalen Bestandteile der Follikelflüssigkeit zu trennen. Nach miRNA Isolation von beiden Fraktionen wurde mittels des humanen miRCURY LNATMUniversal RT miRNA PCR-Array-System, ein miRNA Expressionsprofil erstellt. Durch Western-Blot-Analysen und Elektronen-Mikroskopie konnte die effiziente Trennung von exosomalen und non-exosomalen Bestandteilen in der Follikelflüssigkeit bestätigt werden. Mittels Echtzeit-qPCR-Array-Analysen konnte einige miRNAs im exosomalen und non-exosomalen Teil der Rinder Follikelflüssigkeit nachgewiesen werden. Von diesen waren 25 miRNAs im exosomalen Teil differentiell exprimiert (16 rauf- und 9 runter reguliert) und 30 miRNAs waren im non-exosomalen Teil der Follikelflüssigkeit differentiell exprimiert (21 rauf- und 9 runter reguliert) im Vergleich zu den BCB- versus BCB+ Oozyten Gruppen. Im Anschluss daran, wurde die Expression ausgewählter miRNAs in Thecazellen, Granulosa- und Kumuluszellen ermittelt. Das Ergebnis könnte ein Hinweis auf die Herkunft der extrazellulären miRNAs in der Follikelflüssigkeit sein. Für eine genauere Erklärung der möglichen Funktion dieser extrazellulären miRNAs in der Follikelflüssigkeit wurden potenziellen Zielgene mit in silico Analyse, funktioneller Annotation und Pathway-Analysen untersucht. Dies ergab, dass die miRNAs überwiegend an Signalwegen der Regulation der follikulären Entwicklung und des Eizellen Wachstums beteiligt sind. Um die Ergebnisse in Bezug auf die exosomal vermittelte Zell-Zell-Kommunikation in der follikulären Mikroumgebung zu überprüfen, konnten wir nachweisen, dass aus der Aufnahme des Exosoms in die Zelle eine Erhöhung des miRNA-Levels mit nachträglicher Veränderung des mRNA-Niveaus von Targetgenen in Follikel-Zellen in vitro resultiert. Die vorliegende Studie zeigt zum ersten Mal, den von exosomalen oder non-exosomalen Bestandteilen vermittelten Transfer von miRNA in die bovine Follikelflüssigkeit. Weiterhin ist das Wachstum der Eizellen von den Variationen der extrazellulären miRNA Signaturen in der follikulären Umgebung abhängig

An Overview of One Health Concept Focusing on Toxoplasmosis

The "One Health" concept is a universal approach to sustainably balancing and optimizing the health of humans, animals, and ecosystems. This approach is based on the health of humans, domestic and wild animals, and plants in a wider environment in which self-renewable ecosystems exist, with essential characteristics of integration, unifying and holistic perspective. Toxoplasmosis, one of the most common zoonotic infections in both terrestrial and oceanic ecosystems in the world, is an ideal model disease for the “One Health” approach. Toxoplasmosis is a zoonotic disease caused by the obligate intracellular pathogen protozoan Toxoplasma gondii. In the life cycle of T. gondii, the definitive host is domestic cats and felines, and the intermediate hosts are all mammals (including humans), birds and reptiles. The infected cats have primary importance and play a crucial role in the contamination of habitats in the ecosystems with T. gondii oocysts. Thus, ecosystems with domestic cats and stray cats are contaminated with cat feces infected with T. gondii oocytes. T. gondii positivity has been scientifically demonstrated in all warm-blooded animals in terrestrial and aquatic habitats. The disease causes deaths and abortions in farm animals, resulting in great economic losses. However, the disease causes great problems in humans, especially pregnant women. During pregnancy, it may have effects such as congenital infections, lesions in the eye and brain of the fetus, premature birth, intrauterine growth retardation, fever, pneumonia, thrombocytopenia, ocular lesions, encephalitis, and abortion. The mechanism of death and abortion of the fetus in a pregnant woman infected with T. gondii occurs as a result of complete disruption of the maternal immune mechanism. The struggle against toxoplasmosis requires the universal collaboration and coordination of the World Organization for Animal Health, the World Health Organization and the World Food Organization in the "One Health" concept and integrative approaches of all responsible disciplines. Establishing universal environmental safety with the prevention and control of toxoplasmosis requires the annihilation of the feces of the infected cats using suitable techniques firstly. Then routinely, the monitoring and treatment of T. gondii positivity in cats, avoiding contact with contaminated foods and materials, and development of modern treatment and vaccine options. Particularly, mandatory monitoring or screening of T. gondii positivity during the pregnancy period in humans should be done. It would be beneficial to replace the French model, especially in the monitoring of disease in humans. In this article, the ecology of toxoplasmosis was reviewed at the base of the "One Health" concept

Macronutrient modulation of mRNA and microRNA function in animals: A review

Dietary macronutrients have been regarded as a basic source of energy and amino acids that are necessary for the maintenance of cellular homeostasis, metabolic programming as well as protein synthesis. Due to the emergence of "nutrigenomics", a unique discipline that combines nutritional and omics technologies to study the impacts of nutrition on genomics, it is increasingly evident that macronutrients also have a significant role in the gene expression regulation. Gene expression is a complex phenomenon controlled by several signaling pathways and could be influenced by a wide variety of environmental and physiological factors. Dietary macronutrients are the most important environmental factor influencing the expression of both genes and microRNAs (miRNA). miRNA are tiny molecules of 18 to 22 nucleotides long that regulate the expression of genes. Therefore, dietary macronutrients can influence the expression of genes in both direct and indirect manners. Recent advancements in the state-of-the-art technologies regarding molecular genetics, such as next-generation sequencing, quantitative PCR array, and microarray, allowed us to investigate the occurrence of genome-wide changes in the expression of genes in relation to augmented or reduced dietary macronutrient intake. The purpose of this review is to accumulate the current knowledge focusing on macronutrient mediated changes in the gene function. This review will discuss the impact of altered dietary carbohydrate, protein, and fat intake on the expression of coding genes and their functions. In addition, it will also summarize the regulation of miRNA, both cellular and extracellular miRNA, expression modulated by dietary macronutrients. (C) 2020, Chinese Association of Animal Science and Veterinary Medicine. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd

Relative abundance of extra-cellular miRNAs in bovine follicular fluid: Implication for cell–cell communication during oocyte growth

Here we aimed to investigate the expression patternof the circulating extra-cellular miRNAs in exosome andnon-exosomal fraction of follicular fluid consisted of fullygrown or growing oocytes and to validate exosome medi-ated Here we aimed to investigate the expression patternof the circulating extra-cellular miRNAs in exosome andnon-exosomal fraction of follicular fluid consisted of fullygrHere we aimed to investigate the expression patternof the circulating extra-cellular miRNAs in exosome andnon-exosomal fraction of follicular fluid consisted of fullygrown or growing oocytes and to validate exosome medi-ated cell–cell communication between follicular cells. Forthis, follicles of 5–8mm diameter (n=120) were isolatedand individual COCs were subjected to brilliant cresyl blue(BCB) staining and classified as BCB+ (fully grown,n=60)and BCB−(growing,n=60) groups. The correspondingfollicular fluid, granulosa cells and theca cells were usedfor further molecular analysis. miRNAs isolated fromexosomal and nonexosomal portion of follicular fluidfrom the two categories was used for cDNA synthesis andsubsequent analysis using a human miRNA PCR array (with745 miRNA).own or growing oocytes and to validate exosome medi-ated cell–cell communication between follicular cells. Forthis, follicles of 5–8mm diameter (n=120) were isolatedand individual COCs were subjected to brilliant cresyl blue(BCB) staining and classified as BCB+ (fully grown,n=60)and BCB−(growing,n=60) groups. The correspondingfollicular fluid, granulosa cells and theca cells were usedfor further molecular analysis. miRNAs isolated fromexosomal and nonexosomal portion of follicular fluidfrom the two categories was used for cDNA synthesis andsubsequent analysis using a human miRNA PCR array (with745 miRNA).cell–cell communication between follicular cells. Forthis, follicles of 5–8mm diameter (n=120) were isolatedand individual COCs were subjected to brilliant cresyl blue(BCB) staining and classified as BCB+ (fully grown,n=60)and BCB−(growing,n=60) groups. The correspondingfollicular fluid, granulosa cells and theca cells were usedfor further molecular analysis. miRNAs isolated fromexosomal and nonexosomal portion of follicular fluidfrom the two categories was used for cDNA synthesis andsubsequent analysis using a human miRNA PCR array (with745 miRNA).</p

Intracellular drug delivery by exosomes: Present condition and future perspectives

Nanotechnology plays a crucial part in the detection, diagnosis, andtreatment of a wide variety of human diseases including cancer. The recentdecades have witnessed a rapid development of nanocarriers as drug deliverysystem. Using nanocarriers have several pharmacokinetics and safety profiles inaddition to the increased bioavailability of the entrapped molecules. However,there are several obstacles in attaining the goal of successful clinicaltranslation of nanocarrier-based drug delivery system because of the safety,stability and delivery efficiency of nanocarriers. In this scenario, exosomecould be an excellent alternative to conventional drug delivery agents. Exosomesare bi-layered nanovesicles of 40-100 nm diameter and contain differentbioactive molecules including microRNAs, mRNAs, DNA fragments, and proteins.Exosomes are actively released by healthy cells in the extracellular matrix andcan be taken up by the surrounding or distant cells. Due to their inherentability to carry bioactive molecules, exosomes emerged as a versatile drugvehicle and have attracted the attention of many researchers. The most impressivecharacteristics of exosomes are nanosized, non-immunogenic due to similarcomposition as body’s own cells, high biocompatibility, readily bioavailable,ability to penetrate biological barriers, and encapsulation of various cargowhich significantly distinguish them from other conventional nanocarriers. Itis important to note that recently exosome-based nanocarriers containing smalldrug molecules and bioactive macromolecules have been developed for thetreatment of several prevalent diseases such as cancer, central nervous systemdisorders, and other degenerative diseases. Nanocarriers based on exosomes haveenormous prospects in overcoming several difficulties faced in gene and drugdelivery. This article will briefly highlight the advancement as well as thechallenges of exosome-based nanocarriers as drug delivery system. In addition,it will also discuss the future perspectives of exosome-based drug deliverysystem. Special attention will be placed on the exosomal biogenesis, releasemechanisms, and advantages of using exosomes containing special cargo, as adrug delivery system, in treating obstinate diseases. The aim is to offer newinsights for exploring exosomes in the emerging field of drug delivery.&nbsp;</p

The Expression of IL-1β gene In Response to Mannheimia haemolytica Bacteria in Sheep Alveolar Macrophages in vitro

Small ruminants, particularly sheep, are suffering from many respiratory diseases.Among the causative agents of respiratory diseases, the most important microorganismsare Mannheimia haemolytica. In the case of a most severe form of infection which iscommonly known as the severe fibrinous pleuropneumonia characterized by fibrindeposition and merging, intra-alveolar hemorrhage, forceful leukocyte infiltration inalveoli of the lungs. Being a Gram-negative bacterium, Mannheimia haemolytica iscausing respiratory diseases in animals and pneumonia which is the must dangersdiseases that make a great economic loss. The sheep immune system is responsible forthe detection, capturing and elimination of foreign bodies including bacteria. However,there are no substantial studies explaining the molecular mechanism of respiratorydiseases and immune system response in sheep lung. Therefore, this study wasconducted to investigate the expression pattern of IL-1β, one of the important genes inimmune system, to understand the molecular mechanism underlying in M. haemolyticainduced infection in sheep lung.&nbsp;</p

Extracellular/Circulating MicroRNAs: Release Mechanisms,Functions and Challenges

AbstractMicroRNAs (miRNAs) are endogenously initiated, small non-coding RNAs and typically regulate the expression of mRNAs in post transcriptional level either via translational repression or mRNA degradation. Aberrant expression of miRNAs is observed in diverse disease and altered physiological states. Recently, it has been revealed that miRNAs are not only present in cells but also in extracellular milieu especially in different bio-fluids including blood plasma, follicular fluid and even in cell culture media. Such extracellular miRNAs (ECmiRNAs) are remarkably stable in the extracellular harsh environment with the presence of high RNAse activity. Although the precise mechanisms of release of cellular miRNAs to extracellular environment remain largely unknown, recent studies suggest that the expression of these ECmiRNAs can be associated with patho-physiological condition of an organism. Moreover, these ECmiRNAs may deliver to the recipient cells via certain pathways where they can regulate translational activity of target genes. This review will discuss the nature and stability of ECmiRNAs along with their release mechanisms. Furthermore, based on recent evidences, it also summarizes the possible function of these ECmiRNAs in distant cell-to-cell communication and the difficulties we may face during ECmiRNA research