Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production
Articolo
Data di Pubblicazione:
2016
Citazione:
Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production / C. Dieci, V. Lodde, R. Labreque, I. Dufort, I. Tessaro, M. Sirard, A.M. Luciano. - In: MOLECULAR HUMAN REPRODUCTION. - ISSN 1360-9947. - 22:12(2016 Dec), pp. 882-897.
Abstract:
STUDY QUESTION: Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte’s quality and response in culture during in-vitro embryo production (IVP).
SUMMARY ANSWER: The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP.
WHAT IS KNOWN ALREADY: Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte’s developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence.
STUDY DESIGN, SIZE, DURATION: Bovine cumulus–oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte’s chroma- tin compaction.
PARTICIPANTS/MATERIALS, SETTING, METHOD: RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, repre- senting an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted func- tions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes.
MAIN RESULTS AND THE ROLE OF CHANCE: Overall, quantitative RT-PCR results of a subset of five selected genes were consist- ent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as ‘cell cycle’ and ‘cell death and survival’, whereas genes included in cluster #5 were mostly related to ‘gene expression’. Interestingly, ‘lipid metabolism’ was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was ‘cell death and survival’. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apop- tosis increased significantly in CC from oocytes with condense
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
oocyte; chromatin; follicle; granulosa cells; cumulus cells; apoptosis; pre-maturation; embryo development
Elenco autori:
C. Dieci, V. Lodde, R. Labreque, I. Dufort, I. Tessaro, M. Sirard, A.M. Luciano
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