During follicle development, interactions between somatic and germ cells are critical for the acquisition of oocyte competence. Moreover, granulosa cells serve a critical role in the modulation of chromatin configuration changes that occurs before meiotic resumption. Importantly, chromatin configurations within the oocyte germinal vesicle (GV) are indicative of oocytes metabolic state as well as developmental potential. However, the mechanisms acting in the somatic compartment that regulate oocyte competence are far from being understood. This study was aimed to assess gene expression profile by microarray platform in cumulus cells (CC) isolated from ovarian follicles, which are linked to the oocyte developmental competence. To this aim we determined the expression profile of CC of oocytes at different stage of differentiation and selected according to their chromatin configuration as GV0, GV1, GV2 and GV3. CC belonging to each group were collected, RNA was extracted, amplified and hybridized on a bovine embryo-specific 44K Agilent slide (EmbryoGene). The GV1, GV2 and GV3 classes were each hybridized against the GV0, which represents a stage of early oocyte differentiation with poor development competence. Data were normalized with Loess and Fold changes (FC) of differentially expressed genes were determined with Limma procedure. Finally, the regulation pattern genes and function predicted were studied with Ingenuity Pathway Analysis software. Transcriptome analysis (FC >1.5; p<0.05) reveals 1441 genes differentially expressed in GV1 vs GV0 (684 down and 757 upregulated), 1474 in GV2 vs GV0 (699 down and 775 upregulated) and 2047 in GV3 vs GV0 (981 down and 1066 upregulated). Data analysis indicated a deregulation of several transcripts associated to extracellular matrix formation and stabilization such as: Hyaluronan synthase 2 (HAS2), Serpine 2 (SERPINE2) and Progesterone receptor (PGR). Moreover, along with the increase in chromatin compaction, we noticed an upregulation of the gene solute carrier family 39 member 8 (SLC39A8), which is a zinc transporter, an upregulation of inhibin alpha subunit (INHA), which is implicated in hormonal regulation as well as a down-regulation of G-protein signalling 2 (RGS2), which is implicated in the G protein coupled receptor pathway. Importantly, another group of differentially regulated genes was represented by apoptosis-related transcripts such as apoptosis-related cysteine peptidase (CASP3), Thrombospondin-1 (THBS1) and proliferating cell nuclear antigen (PCNA). Microarray data were validated by quantitative RT-PCR. Relative expression levels of target genes such as THBS1, SERPINE2, regulator of RGS2, INHA and SLC39A8 were calculated with the delta-delta Ct method using B ACTIN, GAPDH and HPRT1 as reference genes for normalization. Statistical analysis of the data were conducted by ANOVA followed by Newman-Keuls post hoc test. To further confirm that CC belonging to oocytes with increasing degree of chromatin compaction are more prone to apoptotic events, CC belonging to each GV group were cultured for 3hrs in defined conditions and apoptotic cells were assessed by CaspaTag Pan-Caspase in situ Assay kit. Our results indicated that a low percentage of CC from GV0 oocytes are undergoing apoptosis, while this percentage significantly increases in oocytes with more condensed chromatin, reaching the highest level in CC of GV3 oocytes (one way ANOVA p<0.05). This study further confirms how the process of oocyte competence acquisition is profoundly influenced by the somatic compartment and provides multiple non-invasive biomarkers that can predict oocyte developmental potential. This has important implications in treating human infertility as well as developing breeding schemes in domestic mammals. Work supported by the NSERC Strategic Network EmbryoGENE, Canada.

Gene expression profiles and functionality of bovine cumulus cells derived from oocytes with different chromatin configuration / A.M. Luciano, C. Dieci, R. Labrecque, I. Dufort, I. Tessaro, M.A. Sirard, V. Lodde. ((Intervento presentato al 48. convegno Annual Meeting of the Society for the Study of Reproduction tenutosi a San Joan nel 2015.

Gene expression profiles and functionality of bovine cumulus cells derived from oocytes with different chromatin configuration

A.M. Luciano
Primo
;
C. Dieci
Secondo
;
I. Tessaro;V. Lodde
Ultimo
2015

Abstract

During follicle development, interactions between somatic and germ cells are critical for the acquisition of oocyte competence. Moreover, granulosa cells serve a critical role in the modulation of chromatin configuration changes that occurs before meiotic resumption. Importantly, chromatin configurations within the oocyte germinal vesicle (GV) are indicative of oocytes metabolic state as well as developmental potential. However, the mechanisms acting in the somatic compartment that regulate oocyte competence are far from being understood. This study was aimed to assess gene expression profile by microarray platform in cumulus cells (CC) isolated from ovarian follicles, which are linked to the oocyte developmental competence. To this aim we determined the expression profile of CC of oocytes at different stage of differentiation and selected according to their chromatin configuration as GV0, GV1, GV2 and GV3. CC belonging to each group were collected, RNA was extracted, amplified and hybridized on a bovine embryo-specific 44K Agilent slide (EmbryoGene). The GV1, GV2 and GV3 classes were each hybridized against the GV0, which represents a stage of early oocyte differentiation with poor development competence. Data were normalized with Loess and Fold changes (FC) of differentially expressed genes were determined with Limma procedure. Finally, the regulation pattern genes and function predicted were studied with Ingenuity Pathway Analysis software. Transcriptome analysis (FC >1.5; p<0.05) reveals 1441 genes differentially expressed in GV1 vs GV0 (684 down and 757 upregulated), 1474 in GV2 vs GV0 (699 down and 775 upregulated) and 2047 in GV3 vs GV0 (981 down and 1066 upregulated). Data analysis indicated a deregulation of several transcripts associated to extracellular matrix formation and stabilization such as: Hyaluronan synthase 2 (HAS2), Serpine 2 (SERPINE2) and Progesterone receptor (PGR). Moreover, along with the increase in chromatin compaction, we noticed an upregulation of the gene solute carrier family 39 member 8 (SLC39A8), which is a zinc transporter, an upregulation of inhibin alpha subunit (INHA), which is implicated in hormonal regulation as well as a down-regulation of G-protein signalling 2 (RGS2), which is implicated in the G protein coupled receptor pathway. Importantly, another group of differentially regulated genes was represented by apoptosis-related transcripts such as apoptosis-related cysteine peptidase (CASP3), Thrombospondin-1 (THBS1) and proliferating cell nuclear antigen (PCNA). Microarray data were validated by quantitative RT-PCR. Relative expression levels of target genes such as THBS1, SERPINE2, regulator of RGS2, INHA and SLC39A8 were calculated with the delta-delta Ct method using B ACTIN, GAPDH and HPRT1 as reference genes for normalization. Statistical analysis of the data were conducted by ANOVA followed by Newman-Keuls post hoc test. To further confirm that CC belonging to oocytes with increasing degree of chromatin compaction are more prone to apoptotic events, CC belonging to each GV group were cultured for 3hrs in defined conditions and apoptotic cells were assessed by CaspaTag Pan-Caspase in situ Assay kit. Our results indicated that a low percentage of CC from GV0 oocytes are undergoing apoptosis, while this percentage significantly increases in oocytes with more condensed chromatin, reaching the highest level in CC of GV3 oocytes (one way ANOVA p<0.05). This study further confirms how the process of oocyte competence acquisition is profoundly influenced by the somatic compartment and provides multiple non-invasive biomarkers that can predict oocyte developmental potential. This has important implications in treating human infertility as well as developing breeding schemes in domestic mammals. Work supported by the NSERC Strategic Network EmbryoGENE, Canada.
giu-2015
Settore VET/01 - Anatomia degli Animali Domestici
Gene expression profiles and functionality of bovine cumulus cells derived from oocytes with different chromatin configuration / A.M. Luciano, C. Dieci, R. Labrecque, I. Dufort, I. Tessaro, M.A. Sirard, V. Lodde. ((Intervento presentato al 48. convegno Annual Meeting of the Society for the Study of Reproduction tenutosi a San Joan nel 2015.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/293843
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