Limits...
Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells.

Regassa A, Rings F, Hoelker M, Cinar U, Tholen E, Looft C, Schellander K, Tesfaye D - BMC Genomics (2011)

Bottom Line: Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes.Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2).In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Germany.

ABSTRACT

Background: The bi-directional communication between the oocyte and its companion cumulus cells (CCs) is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa.

Results: We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV) oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII) oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO+CCs) and without (OO-CCs) CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs+OO and CCs-OO, respectively.While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18), translation (EIF2AK1, EIF4ENIF1) and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI), protein metabolic processes (IHH, APOA1, PLOD1), steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7). Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2).

Conclusion: In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes. Moreover, the presence or absence of oocyte and CC factors during bovine oocyte maturation can have a profound effect on transcript abundance of each cell types, thereby showing the prevailing molecular cross-talk between oocytes and their corresponding CCs.

Show MeSH

Related in: MedlinePlus

Representative images showing CCs from which the enclosed oocyte was completely removed at GV stage using oocytectomy (A) and the oocyte from which the companion CCs were completely removed mechanically at GV stage using pipettes (B) and intact COC (C). Images were taken using Leica DM-IRB microscope with a magnification of 100×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045333&req=5

Figure 2: Representative images showing CCs from which the enclosed oocyte was completely removed at GV stage using oocytectomy (A) and the oocyte from which the companion CCs were completely removed mechanically at GV stage using pipettes (B) and intact COC (C). Images were taken using Leica DM-IRB microscope with a magnification of 100×.

Mentions: In order to increase the homogeneity of the experimental samples, COCs were further screened for developmental competence using brilliant cresyl blue (BCB) staining as described in [50-52]. BCB positive (BCB+) COCs were assigned randomly into the following four experiments. Each experiment had three pools of biological replicates; each replicate containing 50 oocytes or CCs samples (Figure 1). In experiment 1a, CCs were mechanically removed from oocytes at GV stage by repeated in and out pipetting and the resulting denuded oocytes (DOs), designated by number 1 and their companion CCs by number 2, were frozen. In experiment 1b, intact COCs were cultured; their companion CCs were mechanically removed and the resulting denuded MII oocytes, designated by number 4 and their companion CCs by number 3, were frozen. In experiment 2, CCs were mechanically removed from their enclosed oocytes at GV stage and the resulting (OO - CCs), designated by number 5 and other intact COCs (OO + CCs) were cultured separately. After 22 hr culture period, CCs were mechanically removed from the intact oocytes and the resulting denuded MII oocytes (number 6) were frozen. In experiment 3, oocytes were micro surgically removed from their companion CCs at GV stage as described previously [17,46,47]. Briefly, using micromanipulators with a holding pipette (20 pm diameter hole) and a fine glass needle, the complex is held under negative pressure and the oocyte pierced through both sides of the zona pellucida. The contents of the oocyte are then aspirated through the holding pipette while removing the glass needle, resulting in temporary deformation of the zona pellucida. Almost all of the oocyte contents are removed in this way, leaving an intact complex without the oocyte, the oocytectomised complex (CCs - OO). The resulting complexes (CCs - OO), designated by number 7 and other intact complexes (CCs + OO) were cultured. After 22 hr culture, oocytes were micro surgically removed from their intact cumulus oocyte complexes (CCs + OO), as described above and the resulting MII CCs without their enclosed oocytes, designated by number 8 were frozen. In experiment 4, CCs were mechanically removed from their enclosed oocytes both at GV (number 9) and MII stages (number 10) and frozen for subsequent total RNA isolation. In both cases, cells were cultured for 22 hrs and each experiment was repeated three times. Complete removal of either cell from one or the other was confirmed by microscopic examination of the corresponding samples (Figures 2A and 2B) as described in [53]. Cells were cultured in groups of 50 in 400 μl MPM medium supplemented with 12% estrus cow serum and 10 μg mL-1 FSH for 22 hr at 39°C in an incubator with humidified atmosphere containing 5% CO2. Samples were stored at -80°C until subsequent RNA isolation.


Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells.

Regassa A, Rings F, Hoelker M, Cinar U, Tholen E, Looft C, Schellander K, Tesfaye D - BMC Genomics (2011)

Representative images showing CCs from which the enclosed oocyte was completely removed at GV stage using oocytectomy (A) and the oocyte from which the companion CCs were completely removed mechanically at GV stage using pipettes (B) and intact COC (C). Images were taken using Leica DM-IRB microscope with a magnification of 100×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045333&req=5

Figure 2: Representative images showing CCs from which the enclosed oocyte was completely removed at GV stage using oocytectomy (A) and the oocyte from which the companion CCs were completely removed mechanically at GV stage using pipettes (B) and intact COC (C). Images were taken using Leica DM-IRB microscope with a magnification of 100×.
Mentions: In order to increase the homogeneity of the experimental samples, COCs were further screened for developmental competence using brilliant cresyl blue (BCB) staining as described in [50-52]. BCB positive (BCB+) COCs were assigned randomly into the following four experiments. Each experiment had three pools of biological replicates; each replicate containing 50 oocytes or CCs samples (Figure 1). In experiment 1a, CCs were mechanically removed from oocytes at GV stage by repeated in and out pipetting and the resulting denuded oocytes (DOs), designated by number 1 and their companion CCs by number 2, were frozen. In experiment 1b, intact COCs were cultured; their companion CCs were mechanically removed and the resulting denuded MII oocytes, designated by number 4 and their companion CCs by number 3, were frozen. In experiment 2, CCs were mechanically removed from their enclosed oocytes at GV stage and the resulting (OO - CCs), designated by number 5 and other intact COCs (OO + CCs) were cultured separately. After 22 hr culture period, CCs were mechanically removed from the intact oocytes and the resulting denuded MII oocytes (number 6) were frozen. In experiment 3, oocytes were micro surgically removed from their companion CCs at GV stage as described previously [17,46,47]. Briefly, using micromanipulators with a holding pipette (20 pm diameter hole) and a fine glass needle, the complex is held under negative pressure and the oocyte pierced through both sides of the zona pellucida. The contents of the oocyte are then aspirated through the holding pipette while removing the glass needle, resulting in temporary deformation of the zona pellucida. Almost all of the oocyte contents are removed in this way, leaving an intact complex without the oocyte, the oocytectomised complex (CCs - OO). The resulting complexes (CCs - OO), designated by number 7 and other intact complexes (CCs + OO) were cultured. After 22 hr culture, oocytes were micro surgically removed from their intact cumulus oocyte complexes (CCs + OO), as described above and the resulting MII CCs without their enclosed oocytes, designated by number 8 were frozen. In experiment 4, CCs were mechanically removed from their enclosed oocytes both at GV (number 9) and MII stages (number 10) and frozen for subsequent total RNA isolation. In both cases, cells were cultured for 22 hrs and each experiment was repeated three times. Complete removal of either cell from one or the other was confirmed by microscopic examination of the corresponding samples (Figures 2A and 2B) as described in [53]. Cells were cultured in groups of 50 in 400 μl MPM medium supplemented with 12% estrus cow serum and 10 μg mL-1 FSH for 22 hr at 39°C in an incubator with humidified atmosphere containing 5% CO2. Samples were stored at -80°C until subsequent RNA isolation.

Bottom Line: Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes.Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2).In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Germany.

ABSTRACT

Background: The bi-directional communication between the oocyte and its companion cumulus cells (CCs) is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa.

Results: We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV) oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII) oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO+CCs) and without (OO-CCs) CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs+OO and CCs-OO, respectively.While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18), translation (EIF2AK1, EIF4ENIF1) and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI), protein metabolic processes (IHH, APOA1, PLOD1), steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7). Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2).

Conclusion: In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes. Moreover, the presence or absence of oocyte and CC factors during bovine oocyte maturation can have a profound effect on transcript abundance of each cell types, thereby showing the prevailing molecular cross-talk between oocytes and their corresponding CCs.

Show MeSH
Related in: MedlinePlus