Aim of this work was to evaluate changes in the transcriptome (mRNA levels) during in vitro maturation of oocytes deriving from adult and prepubertal ovine donors using a customized ovine cDNA microarray. In collaboration with Porto Conte Ricerche S.r.l. and Illumina, Inc., we generated a medium density microarray based on Illumina DASL technology; it comprises 1536 independent assays addressing 1039 transcripts relevant to several cell functions such as transcription, cell cycle regulation, signal transduction and immune response. Sequences were selected in the OAGI (Ovis Aries Gene Index) database of the Dana-Farber Cancer Institute Gene Index Gene Ontology (GO) section (http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=sheep). Immature (GV) and in vitro matured (IVM) oocytes deriving from adult and prepubertal ovine donors (3 groups of 20 oocytes for each class) were subjected to mRNA isolation with Dynal microbeads kit (Oslo, Norway) and subsequently hybridized to the customized microarray. Comparison of mRNA content between adult and prepubertal GV oocytes revealed 14 differentially expressed genes, mainly upregulated in prepubertal gametes, while 20 genes were differentially expressed at the MII stage (13 upregulated in adults and 7 in prepubertal oocytes), (P<0.01). After in vitro maturation, 44 genes showed different abundance between the GV and the MII stage of adult-derived oocytes (41 transcripts more abundant in GV and 9 in MII stage), while 31 mRNA showed different levels in prepubertal gametes (19 transcripts more abundant in GV and 12 in MII), (P<0.01). Notably, only one transcript was seen to vary during meiotic progression of both adult and prepubertal gametes. Quantitative reverse transcription Real Time PCR validated the expression profile of six selected transcripts. This study confirms differences in the transcriptome of GV and IVM oocytes deriving from adult or prepubertal sheep. The differences observed during meiotic progression suggest that prepubertal and adult oocytes may have different needs to undergo meiosis.
Custom microarray analysis of gene expression in oocytes derived from adult and prepubertal sheep during in vitro maturation / Bebbere, D; Bogliolo, Luisa; Ariu, F; Pescatori, M; Uzzau, Sergio; Ledda, Sergio. - (2011), pp. 40-40. (Intervento presentato al convegno Proceeding of GEMINI Group III Workshop tenutosi a Jerusalem, Israel nel 31st May - 3rd June, 2011).
Custom microarray analysis of gene expression in oocytes derived from adult and prepubertal sheep during in vitro maturation
Bebbere D;BOGLIOLO, Luisa;UZZAU, Sergio;LEDDA, Sergio
2011-01-01
Abstract
Aim of this work was to evaluate changes in the transcriptome (mRNA levels) during in vitro maturation of oocytes deriving from adult and prepubertal ovine donors using a customized ovine cDNA microarray. In collaboration with Porto Conte Ricerche S.r.l. and Illumina, Inc., we generated a medium density microarray based on Illumina DASL technology; it comprises 1536 independent assays addressing 1039 transcripts relevant to several cell functions such as transcription, cell cycle regulation, signal transduction and immune response. Sequences were selected in the OAGI (Ovis Aries Gene Index) database of the Dana-Farber Cancer Institute Gene Index Gene Ontology (GO) section (http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=sheep). Immature (GV) and in vitro matured (IVM) oocytes deriving from adult and prepubertal ovine donors (3 groups of 20 oocytes for each class) were subjected to mRNA isolation with Dynal microbeads kit (Oslo, Norway) and subsequently hybridized to the customized microarray. Comparison of mRNA content between adult and prepubertal GV oocytes revealed 14 differentially expressed genes, mainly upregulated in prepubertal gametes, while 20 genes were differentially expressed at the MII stage (13 upregulated in adults and 7 in prepubertal oocytes), (P<0.01). After in vitro maturation, 44 genes showed different abundance between the GV and the MII stage of adult-derived oocytes (41 transcripts more abundant in GV and 9 in MII stage), while 31 mRNA showed different levels in prepubertal gametes (19 transcripts more abundant in GV and 12 in MII), (P<0.01). Notably, only one transcript was seen to vary during meiotic progression of both adult and prepubertal gametes. Quantitative reverse transcription Real Time PCR validated the expression profile of six selected transcripts. This study confirms differences in the transcriptome of GV and IVM oocytes deriving from adult or prepubertal sheep. The differences observed during meiotic progression suggest that prepubertal and adult oocytes may have different needs to undergo meiosis.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
