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Oryctolagus cuniculus

ABSTRACT: Rabbit limbal epithelial side population (SP) cells and non-SP cells

PROVIDER: PRJNA316450 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR3295310.fastq.gz	Fastqsanger.gz
	SRR3295320.fastq.gz	Fastqsanger.gz
	SRR3295330.fastq.gz	Fastqsanger.gz
	SRR3295340.fastq.gz	Fastqsanger.gz
	SRR3295311.fastq.gz	Fastqsanger.gz

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Project description:Side population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival side population cells by global differential gene expression to identify markers and signaling pathways associated with this cell phenotype. Four overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 3342 were cytometrically sorted into SP and non-SP cohorts. RNA was isolated and processed for microarray analysis using a commercial oligonucleotide array representing more than 55,000 transcripts derived from about 30,000 different genes. Selected microarray results were validated at the gene and protein levels by quantitative PCR, and immunological methods. Data mining methods were used to identify cellular processes relevant for stem cell function. Comparative analysis of transcripts expression based on expression levels and present/absent software calls across 4 replicate experiments identified 16993 expressed conjunctival epithelial transcripts including 10,266 unique known genes. Of those genes, 1254 and 363 were over expressed (> 2-fold ) or under expressed (< 0.5-fold), respectively, in the SP. The overexpressed set included genes coding for non-epithelial genes (e.g., CD62E/E-selectin and CD93), genes that have been associated with stem cell function in other cellular systems, including several homeodomain genes and genes whose over- or under-expression may underpin the stem cell slow cycling phenotype ( e.g., dual specificity phosphatases and cyclin kinases). Experiment Overall Design: Whole human conjunctivae from unidentifiable cadaver donors, aged 55 to 65, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) within 48 hours of collection. No donor details apart from age, sex, and cause of death were released. The use of human tissue in the study was in accordance with the provisions of the Declaration of Helsinki and sanctioned by the Institutional Review Board. Fresh rabbit tissue was obtained from local abattoirs within one hour of sacrifice. Unless stated otherwise all reagents were procured from Sigma (St. Louis, Mo). Experiment Overall Design: Eight microarray experiments using side population (Hoechst 33342-transporting cells; SP) or non side population (Hoechst 33342-stained cells; nSP) cells from four cadavers.

Project description:A greater understanding of the proteins involved in reproduction can benefit animal production. New advances in proteomics are having a major impact on our understanding of how spermatozoa acquire their capacity for fertilization [1]. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function [2]. The sperm cell is one of the most highly differentiated cells and is composed of a head with a highly compacted chromatin structure and a large flagellum with midpiece that contains the required machinery for movement and therefore to deliver the paternal genetic and epigenetic content to the oocyte [3]. By being so highly differentiated, spermatozoa are advantageous cells to study proteomics of specific compartments such as the membrane, which basically is the area of major importance for its role in interacting with the surroundings and the oocyte [4]. The fusion of a sperm and an oocyte is a sophisticated process that must be preceded by suitable changes in the sperm's membrane composition [5]. Recent studies of spermatozoa from the proteomic point of view have allowed the identification of different proteins in spermatozoa that are responsible for the regulation of normal/defective sperm functions [6]. While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics [7]. Using this method, detailed proteomic data are now available for human [8], macaque [9,10], mouse [11], rat [12], bull [13-15], stallion [16], fruit fly [17], Caenorhabditis elegans [18], carp [19], rainbow trout [20], mussel [21], ram [22], honeybee [23] and rooster [24] sperm membrane proteins. Rabbit (Oryctolagus cuniculus) is an important mammalian species worldwide, being at the same time of commercial interest and a research model animal. European rabbit meat production is approximately 500 thousand tons, corresponding to a 30% share of world production [25]. Besides, rabbits account for the seventh highest number of animals slaughtered per year in the European Union-27, with 347,603 × 1000 head in 2014 [26]. In a previous work, we identified and quantified rabbit seminal plasma proteins between two different genotypes [27], concluding the clear effect of genotype in the abundance of certain seminal plasma proteins. However, it is unknown at present whether these differences also exist at sperm proteome level. Therefore, the aim of the present study was to characterise rabbit sperm membrane proteins through NanoLC-MS/MS analysis focusing on the influence of the genetic origin.

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Oryctolagus cuniculus

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