Abstract
Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here, we aim to identify molecular and functional markers associated with oocyte developmental potential when selected based on G6PDH activity. Immature compact cumulus–oocyte complexes were stained with brilliant cresyl blue (BCB) for 90 min. Based on their colouration, oocytes were divided into BCB− (colourless cytoplasm, high G6PDH activity) and BCB+ (coloured cytoplasm, low G6PDH activity). The chromatin configuration of the nucleus and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement. The abundance and phosphorylation pattern of protein kinases Akt and MAP were estimated by Western blot analysis. A bovine cDNA microarray was used to analyse the gene expression profiles of BCB+ and BCB− oocytes. Consequently, marked differences were found in blastocyst rate at day 8 between BCB+ (33.1±3.1%) and BCB− (12.1±1.5%) oocytes. Moreover, BCB+ oocytes were found to show higher phosphorylation levels of Akt and MAP kinases and are enriched with genes regulating transcription (SMARCA5), cell cycle (nuclear autoantigenic sperm protein, NASP) and protein biosynthesis (RPS274A and mRNA for elongation factor 1α, EF1A). BCB− oocytes, which revealed higher mitochondrial activity and still nucleoli in their germinal vesicles, were enriched with genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10) and growth factor activity (bone morphogenetic protein 15, BMP15). This study has evidenced molecular and subcellular organisational differences of oocytes with different G6PDH activity.
Introduction
In modern animal agriculture, with increasing milk production there is a continuous decline in the fertility of dairy cows leading to higher economic loss (Macmillan et al. 1996). This decline in fertility can be explained by management changes within the dairy industry and also negative genetic correlations between milk production and reproduction. One of the primary mechanisms that depresses fertility in lactating cows is abnormal pre-implantation embryo development, which that may be a result of poor oocyte quality (Snijders et al. 2000, Lucy 2007). Oocyte developmental competence is defined as the ability of an oocyte to resume meiosis, to cleave following fertilisation, to develop to the blastocyst stage, to induce a pregnancy and bring offspring to term in a good health (Krisher 2004, Sirard et al. 2006). This competency is acquired gradually during the course of folliculogenesis as the oocyte grows and its companion somatic cells differentiate (Eppig et al. 1994).
Many factors have been shown to affect the oocyte's developmental potential, including follicle size (Lonergan et al. 1994), health of the follicle (Blondin & Sirard 1995, Vassena et al. 2003), phase of follicular wave (Hagemann 1999, Machatková et al. 2004), hormonal stimulation (Blondin et al. 2002; for review Sirard et al. 2006), maturation environment (Warzych et al. 2007; for review Sutton et al. 2003), season (Al-Katanani et al. 2002, Sartori et al. 2002), nutrition (Fouladi-Nashta et al. 2007) and age (Rizos et al. 2005). Although previous studies support the notion that oocyte competence depends on multiple factors, it remains difficult to draw clear and reliable criteria for oocyte selection.
Morphological assessment of oocytes based on thickness, compactness of the cumulus investment and the homogeneity of the ooplasm (Gordon 2003) is a relatively popular and convenient way of evaluating oocyte quality in practice. However, results derived from this non-invasive approach are often conflicting, largely due to subjectivity and inaccuracy. Morphological evaluation alone is insufficient to distinguish competent oocytes that have the ability to bring about full-term pregnancy (Lonergan et al. 2003, Coticchio et al. 2004, Krisher 2004). With the urgent need for establishing non-invasive and non-perturbing means for oocyte selection, the brilliant cresyl blue (BCB) staining test has been successfully used to differentiate oocytes with different developmental capacity in various species, including pig (Ericsson et al. 1993, Roca et al.1998, Wongsrikeao et al. 2006), goat (Rodríguez-González et al. 2002) and cattle (Alm et al. 2005, Bhojwani et al. 2007).
During the course of their growth, immature oocytes are known to synthesise a variety of proteins, including glucose-6-phosphate dehydrogenase (G6PDH; Mangia & Epstein 1975, Wassarman 1988). The activity of this protein is decreased once this phase has been completed and oocytes are then likely to have achieved developmental competence (Wassarman 1988, Tian et al. 1998). BCB is a dye that can be degraded by G6PDH (Ericsson et al. 1993, Tian et al. 1998); thus, oocytes that have finished their growth phase show decreased G6PDH activity and exhibit cytoplasm with a blue colouration (BCB+), while growing oocytes are expected to have a high level of active G6PDH, which results in colourless cytoplasm (BCB−).
In our previous studies, it has been shown that oocytes screened based on BCB staining differ in their developmental potential to reach blastocyst stage (Alm et al. 2005) and efficiency in utilisation for somatic cell nuclear transfer (Bhojwani et al. 2007). Moreover, oocytes screened with BCB staining were reported to differ in various oocyte quality markers like cytoplasmic volume and mitochondria DNA copy number (El-Shourbagy et al. 2006). However, little is known about the molecular and the subcellular characteristics of these oocytes. Therefore, the aim of this study was to characterise these oocytes at the subcellular level (dissolution of nucleoli and mitochondrial activity), molecular level (gene expression profile) and functionally (activity of protein kinase). The results of the present study evidence the prevailing differences of these oocyte groups in relative abundance transcripts and mitochondrial and MAPK activities contributing to their differences in developmental potential.
Results
Chromatin configuration and mitochondrial activity in BCB+ and BCB− oocytes
Because of their importance as parameters for oocyte quality, we investigated the status of nuclei and mitochondria in BCB+ and BCB− oocytes before in vitro maturation (IVM). A larger proportion of oocytes with high G6PDH activity (BCB−) were found to be in early stage of diplotene with clear visible nucleoli (Dipl+Nuc) in their germinal vesicle than the BCB+ oocytes (Table 1; P<0.005). However, a significantly lower number of a BCB− oocytes was found to be in more progressed diakinesis stage after germinal vesicle breakdown (GVBD) compared with their BCB+ counterparts.
Chromatin configuration and mitochondrial activity (fluorescence intensity/oocyte based on vital labelling of metabolic active mitochondria) in brilliant cresyl blue (BCB+) and BCB− oocytes (n=337).
| Chromatin configuration in %±s.e.m. | Mitochondrial activity | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Oocyte group | Number of oocytes | Dipl+Nuc | Dipl | CC | Dia | MI | MII | Pyc | Fluorescence intensity/oocyte in μA±s.e.m. |
| BCB+ | 169 | 1.8±1.0* | 17.2±2.9 | 49.1±3.8 | 20.1±3.1‡ | 7.1±2.0 | 0.6±0.6 | 4.1±1.4 | 358.4±18.9∥ |
| BCB− | 168 | 21.4±3.1† | 16.7±2.9 | 42.9±3.8 | 8.9±2.2§ | 4.2±1.5 | 1.2±0.8 | 4.7±1.6 | 539.1±19.0¶ |
*:†, ‡:§P<0.005; ∥:¶P<0.001. Dipl+Nuc, diplotene with nucleolus; Dipl, diplotene; CC, condensed chromatin in GV; Dia, diakinesis; MI, metaphase I; MII, metaphase II; Pyc, pycnotic chromatin.
To confirm that the fluorescence intensity of the emission light from the fixed MitoTracker-labelled oocytes was stable during the time of storage, a preliminary study was conducted to measure the fluorescence intensity of 40 oocytes in intervals of 7 days during 6 weeks. The measured fluorescence intensity was not influenced by the storage.
The data in Table 1 demonstrate that the fluorescence intensity in the oocytes pre-labelled by the vital mitochondrial-specific probe chloromethyl tetramethylrosamine (CMTM Ros) and measured by fluorescence intensity for 570 nm emission/oocyte is associated with their G6PDH activity (P<0.001). The highest fluorescence intensity/oocyte was found in BCB− oocytes compared with the BCB+ ones.
Detection of abundance and phosphorylation of protein kinases Akt and MAP
In order to elucidate the activities of protein kinases that contribute in the regulation of gene expression, we have analysed the abundance and phosphorylation state of the MAPKs ERK1, ERK2 and Akt. As indicated in Fig. 1, the abundance of MAPK and Akt1 was not different between BCB+ and BCB− oocytes. In contrast to these observations, BCB+ oocytes show a higher phosphorylation of ERK1, ERK2 and Akt at all phosphorylation sites compared with their BCB− counterparts (Fig. 1).
Analysis of the abundance and phosphorylation state of the protein kinases Akt (A) and MAPK (B) in BCB differentiated oocytes. Fifty oocytes each, BCB+ and BCB−, were analysed for the abundance Akt (total Akt) and MAPK (total MAPK) and the phosphorylation state as indicated by Western blotting. As a control, in A and B right panels, the phosphorylation state of MI-stage oocytes (where Akt is the highest phosporylated) and MII-stage oocytes (where MAPK is the highest phosphorylated) is depicted.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Analysis of the abundance and phosphorylation state of the protein kinases Akt (A) and MAPK (B) in BCB differentiated oocytes. Fifty oocytes each, BCB+ and BCB−, were analysed for the abundance Akt (total Akt) and MAPK (total MAPK) and the phosphorylation state as indicated by Western blotting. As a control, in A and B right panels, the phosphorylation state of MI-stage oocytes (where Akt is the highest phosporylated) and MII-stage oocytes (where MAPK is the highest phosphorylated) is depicted.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Analysis of the abundance and phosphorylation state of the protein kinases Akt (A) and MAPK (B) in BCB differentiated oocytes. Fifty oocytes each, BCB+ and BCB−, were analysed for the abundance Akt (total Akt) and MAPK (total MAPK) and the phosphorylation state as indicated by Western blotting. As a control, in A and B right panels, the phosphorylation state of MI-stage oocytes (where Akt is the highest phosporylated) and MII-stage oocytes (where MAPK is the highest phosphorylated) is depicted.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Developmental competence of bovine oocytes depending on their G6PDH status
In order to evaluate the developmental competence of BCB+ and BCB− oocytes, developmental phenotypes were assessed until day 8 following IVM, in vitro fertilisation (IVF) and in vitro culture (IVC). There were no significant differences among the groups in cleavage rate 2 days after IVF. Significant differences (P<0.05) among the groups were observed in blastocyst rate at day 8, where the BCB+ oocytes resulted in significantly higher (35.7%) blastocyst rate compared with the BCB− groups (13.2%). In addition, the number of nuclei in the resulting blastocysts was higher for BCB+ oocytes compared with the BCB− ones (Table 2).
Developmental competence of bovine oocytes depending on their glucose-6-phosphate dehydrogenase status (brilliant cresyl blue, BCB+, BCB−) before in vitro maturation (n=259).
| Oocyte group | Number of oocytes | Cleavage rate day 2 p.IVF in % ±s.e.m. | Blastocyst rate day 8 p.IVF in % ±s.e.m. |
|---|---|---|---|
| BCB+ | 172 | 78.4±8.3 | 33.1±3.1* |
| BCB− | 87 | 75.0±4.7 | 12.1±1.5† |
*:†P<0.05.
Genes differentially expressed between BCB+ and BCB− oocytes
To identify candidate genes related to oocyte developmental competence, oocytes screened based on G6PDH activity (BCB+ and BCB−) were analysed using bovine cDNA microarray platform. After LOWESS normalisation of the data, log value of Cy5 total intensities was compared with the log value of Cy3 total intensities for both the target and the respective dye-swap hybridisations. The coefficient of determination was high and consistent between target (R2=0.98) and dye-swap (R2=0.99) hybridisations.
To obtain a highly confident set of differentially expressed genes, we used a rigorous combination of P values (P≤0.05) and false discovery rate (FDR≤5%). The SAM analysis revealed that a total of 185 genes to be differentially expressed between the BCB+ and the BCB− oocytes (with ≥1.9-fold change). Of these, 85 genes were up-regulated (Tables 3 and 4) and 100 were down-regulated (Tables 5 and 6) in BCB+ compared with BCB− oocytes. Comparative analysis of the magnitude of differential gene expression between the two oocyte groups showed that, while the up-regulated genes were in the range of 1.9- to 7.8-fold change, down-regulated genes were in the range of 2.0- to 11.5-fold change in BCB+ compared with BCB− oocytes.
Genes up-regulated in brilliant cresyl blue (BCB+) compared with BCB− oocytes.
| Gene name | Accession no. in GenBank | Fold change | Gene function (biological process) |
|---|---|---|---|
| Homo sapiens zinc finger protein 91 homologue (mouse; ZFP91), transcript variant 1, mRNA | NM_053023 | 7.8 | DNA binding (transcription) |
| Homo sapiens zinc finger protein 519, mRNA, complete cds (ZNF519) | BC024227 | 4.4 | DNA binding (transcription) |
| Homo sapiens high-mobility group nucleosomal binding domain 2, mRNA (HMGN2) | BC071707 | 6.3 | DNA binding (transcription) |
| Homo sapiens DEAD (Asp-Glu-Ala-Asp) box polypeptide 10, mRNA (DDX10) | NM_004398 | 5.0 | RNA binding |
| Homo sapiens tudor and KH domain containing, mRNA, with apparent retained intron (TDRKH) | BC022467 | 4.4 | RNA binding |
| TPA_exp: Mus musculus regulator of sex-limitation candidate 2, mRNA, complete cds (Rslcan2) | BK001637 | 4.4 | Nucleic acid binding (transcription) |
| Bovine mRNA for histone H2A.Z (H2AFZ) | X52318 | 4.4 | DNA binding (chromosome organisation and biogenesis) |
| Homo sapiens proliferation-associated 2G4, 38 kDa, mRNA, complete cds (PA2G4) | BC007561 | 4.2 | Transcription factor activity |
| Homo sapiens zinc finger protein 85 (HPF4, HTF1), mRNA (ZNF85) | BC047646 | 4.0 | Transcription factor activity |
| Bos taurus partial stat3 gene for signal transducer and activator of transcription 3 (STAT3) | AJ620667 | 4.0 | Transcription factor activity |
| Bos taurus DNA (cytosine 5) methyltransferase 1, mRNA (DNMT1) | NM_182651 | 3.9 | Transcription factor binding |
| Homo sapiens fibronectin type 3 and ankyrin repeat domains 1, mRNA (FANK1) | BC024189 | 3.9 | Transcription factor binding |
| Homo sapiens SWI/SNF-related, matrix-associated, actin- dependent (SMARCA5) | NM_003601 | 6.7 | RNA polymerase II transcription factor activity |
| Homo sapiens ring finger protein 10, mRNA, complete cds (RNF10) | BC016622 | 3.2 | Protein binding |
| Homo sapiens v-ral simian leukaemia viral oncogene homologue A (ras-related; RALA) | BC039858 | 3.6 | Protein binding (signal transduction) |
| Homo sapiens related RAS viral (r-ras) oncogene homologue 2, mRNA (RRAS2) | BC013106 | 3.9 | Protein binding (signal transduction) |
| Homo sapiens cell adhesion molecule with homology to L1CAM (close homologue of L1; CHL1) | NM_006614 | 3.6 | Protein binding (signal transduction) |
| S. scrofa mRNA encoding G-beta like protein (GNB2L1) | Z33879 | 2.9 | Protein binding (signal transduction) |
| Canine rab11 mRNA for ras-related GTP-binding protein (RAB11A) | X56388 | 2.8 | Protein binding (plasma membrane to the endosome) |
| Homo sapiens occluding mRNA (OCLN) | NM_002538 | 2.6 | Protein binding (protein complex assembly) |
| Canis familiaris occluding 1B mRNA, complete cds (OCLN) | AF246976 | 2.4 | Protein binding |
| Homo sapiens chaperonin-containing TCP1, subunit 8 (theta), mRNA (CCT8) | BC012584 | 2.8 | Unfolded protein binding (protein folding) |
| Homo sapiens ADP-ribosylation factor-like 6 interacting protein (ARL6IP1) | BC010281 | 2.7 | Protein binding (protein targeting membrane) |
| Homo sapiens protein regulator of cytokinesis 1, mRNA (PRC1) | BC003138 | 2.7 | Protein binding (cell cycle) |
| Homo sapiens nuclear autoantigenic sperm protein (histone-binding) mRNA (NASP) | BT006757 | 2.6 | Hsp90 protein binding (cell cycle, blastocyst development) |
| Homo sapiens mutL homologue 1, colon cancer, non-polyposis type 2 (E. coli), mRNA (MLH1) | NM_000249 | 2.4 | Protein binding (cell cycle) |
| Homo sapiens ubiquitin-like, containing PHD and RING finger domains, 2 (UHRF2), mRNA | NM_152896 | 2.7 | Ubiquitin-protein ligase activity (cell cycle) |
| Homo sapiens ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homologue), (UBE2D3) | BC003395 | 2.6 | Ubiquitin-protein ligase activity (cell cycle) |
| Mus musculus ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homologue, yeast), mRNA (Ube2d3) | NM_025356 | 2.6 | Ubiquitin-protein ligase activity (cell cycle) |
| Homo sapiens aurora kinase A, transcript variant 4, mRNA (AURKA) | NM_198435 | 2.2 | Ubiquitin-protein ligase activity (cell cycle) |
| Bos taurus mRNA sequence (CCNB1) | L26548 | 2.4 | (Regulation of progression through cell cycle) |
| Homo sapiens M-phase phosphoprotein 9, mRNA (MPHOSPH9) | NM_022782 | 2.1 | (Regulation of progression through cell cycle) |
| Homo sapiens centrin, EF-hand protein, 3 (CDC31 homologue, yeast), (CETN3) | BC005383 | 4.1 | Calcium ion binding (cell cycle) |
| Bos taurus isolate Cow1 ASPM mRNA, partial cds (ASPM) | BC010658 | 2.2 | Phosphoprotein phosphatase activity (cell cycle) |
| Homo sapiens nucleolar and spindle-associated protein 1, mRNA (NUSAP1) | BC011008 | 2.5 | (Establishment of mitotic spindle localisation) |
| Homo sapiens discs, large homologue 7 (Drosophila), mRNA (DLG7) | AY485424 | 2.1 | Calmodulin binding (cell cycle) |
| Homo sapiens IQ motif containing GTPase-activating protein 1 (IQGAP1) | NM_003870 | 2.1 | Calmodulin binding (signal transduction) |
| Homo sapiens regulator of G-protein signalling 16, mRNA (RGS16) | NM_002928 | 2.6 | Calmodulin binding (signal transduction) |
Differentially expressed genes were identified by SAM at a false discovery rate (FDR) of ≤5% and P≤0.05.
Genes up-regulated in brilliant cresyl blue (BCB+) compared with BCB− oocytes.
| Gene name | Accession no. in GenBank | Fold change | Gene function (biological process) |
|---|---|---|---|
| Homo sapiens regulator of G-protein signalling 2, 24 kDa (RGS2) | NM_002923 | 2.2 | Calmodulin binding (signal transducion) |
| Bos taurus EF1A mRNA for elongation factor 1α, complete cds (EEF1A1) | AB060107 | 2.5 | Translation elongation factor activity (translation) |
| Bos taurus mRNA for elongation factor 1α (EEF1A1) | AJ238405 | 2.0 | Translation elongation factor activity (translation) |
| Homo sapiens ribosomal protein S15 mRNA (RPS15) | NM_001018 | 2.0 | Structural constituent of ribosome (translation) |
| Bos taurus mRNA for similar to ribosomal protein S14, partial cds (RPS14) | AB099089 | 2.1 | Structural constituent of ribosome (translation) |
| Bos taurus mRNA for similar to ubiquitin-S27a fusion protein (RPS27A) | AB098891 | 1.9 | Structural constituent of ribosome |
| Bos taurus ribosomal protein S29, mRNA (RPS29) | NM_174804 | 2.4 | Structural constituent of ribosome |
| Bos taurus ribosomal protein S29 mRNA, complete cds (RPS29) | U66372 | 1.9 | Structural constituent of ribosome |
| Bos taurus mRNA for similar to ribosomal protein L18a, partial cds, (RPL18A) | AB098916 | 2.4 | Structural constituent of ribosome |
| Bos taurus mRNA for similar to ribosomal protein L9, partial cds, (RP L9) | AB099048 | 1.9 | Structural constituent of ribosome |
| Bos taurus ribosomal protein L24 mRNA (RPL24) | NM_174455 | 2.0 | Structural constituent of ribosome (translation) |
| Bos taurus type 4 mucus-type core 2 (GCNT3) | AY283766 | 2.3 | Glucosyltransferase activity |
| Homo sapiens asparagine-linked glycosylation 6 homologue (ALG6) | BC001253 | 2.4 | Glucosyltransferase activity (N-linked glycosylation) |
| Homo sapiens RIO kinase 3 (yeast) transcript variant 1, mRNA (RIOK3) | NM_003831 | 2.1 | Transferase activity (phosphorylation) |
| Bos taurus S-adenosylmethionine decarboxylase 1 mRNA (AMD1) | NM_173990 | 2.1 | Lyase activity (spermine biosynthetic process) |
| Homo sapiens tectorin-β mRNA, complete cds (TECTB) | AF312827 | 1.9 | Glycosylphosphatidylinositol anchor binding |
| Homo sapiens galactokinase 2, mRNA (GALK2) | NM_002044 | 1.9 | Galactokinase activity (galactose metabolic process) |
| Bos taurus ornithine decarboxylase (ODC1), mRNA | NM_174130 | 2.1 | Ornithine decarboxylase activity (polyamine biosynthetic) |
| Bos taurus seryl-tRNA synthetase mRNA, complete cds (SARS) | AF297553 | 2.0 | Ligase activity (seryl-tRNA aminoacylation) |
| Bos taurus mitochondrion, complete genome | AY526085 | 2.0 | Oxidoreductase activity (electron transport) |
| Homo sapiens degenerative spermatocyte homologue, lipid desaturase (Drosophila), mRNA (DEGS1) | BC000961 | 2.2 | Electron carrier activity (lipid metabolic process) |
| Homo sapiens tropomyosin 3, mRNA (TPM3) | NM_153649 | 2.3 | Actin binding (cell motility) |
| Homo sapiens cytoplasmic dynein intermediate chain mRNA, complete cds (DYNC1I2) | AY037160 | 2.0 | Microtubule motor activity (microtubule movement) |
| Homo sapiens kinesin family member 20A, mRNA (KIF20A) | BC012999 | 2.9 | Microtubule motor activity (microtubule movement) |
| Bos taurus zona pellucida glycoprotein 4, mRNA (ZP4) | NM_173975 | 2.0 | Receptor activity (fertilisation) |
| Homo sapiens tripartite motif-containing 51, mRNA (SPRYD5) | BC005014 | 2.0 | Unknown |
| Bos taurus p97 protein mRNA (CFDP2) | NM_174800 | 2.0 | Unknown |
| Homo sapiens haematological and neurological expressed 1, mRNA (HN1) | BC039343 | 2.0 | Unknown |
| Arabidopsis thaliana T-DNA flanking sequence, left border, clone | AJ521477 | 2.0 | Unknown |
| Homo sapiens transforming, acidic coiled-coil containing protein 3 (TACC3) | NM_006342 | 2.3 | Unknown |
| Homo sapiens WW domain containing adaptor with coiled-coil, mRNA (WAC) | BC004258 | 2.3 | Unknown |
Differentially expressed genes were identified by SAM at a false discovery rate (FDR) of ≤5% and P≤0.05.
Genes down-regulated in brilliant cresyl blue (BCB+) compared with BCB− oocytes.
| Gene name | Accession no. in GenBank | Fold change | Gene function (biological process) |
|---|---|---|---|
| Homo sapiens neuronal pentraxin II mRNA (NPTX2) | NM_002523 | 11.5 | Calcium ion binding (synaptic transmission) |
| Bos taurus S100 calcium-binding protein A10 mRNA (S100A10) | NM_174650 | 11.2 | Calcium ion binding |
| Homo sapiens S100 calcium-binding protein A14 mRNA (S100A14) | NM_020672 | 10.6 | Calcium ion binding |
| Homo sapiens S100 calcium-binding protein A16, mRNA (S100A16) | BC019099 | 10.5 | Calcium ion binding |
| Homo sapiens chloride intracellular channel mRNA 1 (CLIC1) | NM_001288 | 10.4 | Chloride ion binding (chloride transport) |
| Human cysteine-rich intestinal protein mRNA, complete cds (CRIP1) | U58630 | 9.7 | Metal ion binding |
| Homo sapiens hypothetical protein DKFZp564K0822, mRNA (ECOP) | BC016650 | 9.5 | Signal transducer activity |
| Homo sapiens basigin long isoform mRNA, complete cds (BSG) | AF548371 | 9.8 | Signal transducer (cell surface receptor linked signal transduction) |
| Bos taurus mRNA for similar to galactose-binding lectin, partial (LGALS1) | AB099039 | 6.3 | Signal transducer (regulation of apoptosis) |
| Bos taurus T cell receptor alpha gene, J segments and C region (TCRA) | AY227782 | 9.1 | Transferase activity (apoptosis) |
| Bos taurus arachidonate 15-lipoxygenase (ALOX15), mRNA | NM_174501 | 6.3 | Lipoxygenase activity (anti-apoptosis) |
| Homo sapiens, clone IMAGE:4428430, mRNA (PARP12) | BC044660 | 6.0 | Transferase activity (protein amino acid ADP-ribosylation) |
| Bos taurus conserved helix–loop–helix ubiquitous kinase mRNA (CHUK) | NM_174021 | 7.4 | Transferase activity (immune response) |
| Homo sapiens fumarate hydratase, mRNA (cDNA clone MGC:15363 (FH) | BC017444 | 8.6 | Fumarate hydratase activity (cell cycle) |
| Homo sapiens nucleophosmin (nucleolar phosphoprotein B23 numatrin), mRNA (NPM1) | BC016768 | 7.4 | RNA binding (anti-apoptosis) |
| Homo sapiens poly(A) polymerase gamma (PAPOLG), mRNA | NM_022894 | 3.7 | RNA binding (RNA polyadenylation) |
| Homo sapiens KIAA0020 mRNA, complete cds (KIAA0020) | D13645 | 5.9 | RNA binding |
| Homo sapiens maelstrom homologue (Drosophila) mRNA (MAEL) | BC028595 | 7.3 | DNA binding |
| Homo sapiens zinc finger, BED domain containing 4, mRNA (ZBED4) | NM_014838 | 7.2 | DNA binding |
| Homo sapiens pituitary tumour-transforming 1, mRNA (PTTG1) | NM_004219 | 3.7 | Transcription factor binding |
| Homo sapiens centromere protein F, 350/400 kDa (mitosin; CENPF) | NM_016343 | 7.1 | Chromatin binding (G2 phase of mitotic cell cycle) |
| Mus musculus ADP-ribosylation factor 4 mRNA (Arf4) | NM_007479 | 6.7 | Nucleotide binding |
| Homo sapiens RAN, member RAS oncogene family, mRNA (RAN) | BC014901 | 6.7 | GTP binding (DNA metabolic process) |
| Homo sapiens F-box only protein 5 mRNA (FBXO5) | NM_012177 | 5.4 | Protein binding (cell cycle) |
| Bos taurus BTAB2MDS3 β-2-microglobulin gene, 3′ UTR (B2M) | AY325771 | 4.9 | Protein binding (immune response) |
| Homo sapiens NACHT, leucine-rich repeat and PYD containing 2, mRNA (NLRP2) | BC001039 | 4.4 | Protein binding (apoptosis) |
| Homo sapiens chromosome 15 open reading frame 23 (C15orf23), mRNA | NM_033286 | 6.7 | Protein binding |
| Homo sapiens GrpE-like 1, mitochondrial (E. coli), mRNA (GRPEL1) | BC024242 | 4.6 | Unfolded protein binding |
| Homo sapiens ralA-binding protein 1, mRNA (RALBP1) | BC013126 | 7.1 | Protein binding (signal transduction) |
| Homo sapiens F-box only protein 34, mRNA (FBXO34) | NM_017943 | 3.3 | Protein transport |
| Bos taurus non-selenium glutathione phospholipid hydroperoxide (AOP2) | AF090194 | 2.3 | Oxidoreductase activity (response to reactive oxygen species) |
| Bos taurus prostaglandin G/H synthase-2 mRNA, complete cds (PGHS-2) | AF031698 | 3.5 | Oxidoreductase activity (prostaglandin biosynthetic process) |
| Homo sapiens retinol dehydrogenase 11 (all-trans and 9-cis), mRNA (RDH11) | BC026274 | 6.1 | Oxidoreductase activity (metabolic process) |
| Bos taurus NADH dehydrogenase (ubiquinone) 1 α-subcomplex, 7 (NDUFA7) | NM_176658 | 2.5 | Oxidoreductase activity (mitochondrial electron transport) |
| Bos taurus cytochrome c oxidase subunit VIIa polypeptide 2 (liver; COX7A2) | NM_175807 | 5.3 | Cytochrome c oxidase activity (electron transport) |
| Bos taurus isolate FL405 mitochondrion, partial genome (FL405) | AY308069 | 3.1 | Oxidoreductase activity (mitochondrial electron transport) |
| Bos taurus ATP synthase, H+ transporting, mitochondrial F0 complex (ATP5G2) | NM_176613 | 2.4 | Hydrogen ion transporting ATPase activity (ATP synthesis) |
| Bos taurus ATP synthase, H+ transporting, mitochondrial F1 complex (ATP5A1) | NM_174684 | 3.8 | Hydrogen ion transporting ATPase activity (ATP synthesis) |
Differentially expressed genes were identified by SAM at a false discovery rate (FDR) of ≤5% and P≤0.05.
Genes down-regulated in brilliant cresyl blue (BCB+) compared with BCB− oocytes.
| Gene name | Accession no. in GenBank | Fold change | Gene function (biological process) |
|---|---|---|---|
| Homo sapiens lectin, galactoside-binding, soluble, 3 (galectin 3), (LGALS3) | BC001120 | 2.4 | Immunoglobulin binding of the IgE isotype |
| Bos taurus mRNA for StAR protein | Y17259 | 2.4 | Cholesterol binding (regulation of steroid biosynthetic process) |
| Homo sapiens bone morphogenetic protein 15 precursor gene (BMP15) | AF082350 | 5.1 | Growth factor activity (female gamete generation) |
| Bos taurus bone morphogenetic protein 15 mRNA, partial cds (BMP15) | AY304484 | 3.9 | Growth factor activity |
| Bos taurus partial mRNA for bone morphogenetic protein 15 (BMP15) | AJ534391 | 2.7 | Growth factor activity |
| Bovine mRNA fragment for cytokeratin A (no. 8; KRT8) | X12877 | 3.1 | Structural molecule activity |
| Homo sapiens calmodulin 2 (phosphorylase kinase, delta) (CALM2) | NM_001743 | 2.0 | Unknown |
| Human DNA sequence from clone RP11-146N23 on chromosome 9, complete (DENND4C) | AL161909 | 2.3 | Unknown |
| Bos taurus BAC CH240-454H24 complete sequence | AC150492 | 2.4 | Unknown |
| Bovine thymus satellite I (1.715 g/ml) DNA | J00037 | 2.5 | Unknown |
| Bovine satellite DNA fragment | V00121 | 2.2 | Unknown |
| Homo sapiens chromosome 8 clone CTC-369M3 map 8q24.3, complete sequence | AF186190 | 2.2 | Unknown |
| Homo sapiens chromosome 16 clone RP11-19H6, complete sequence | AC012175 | 2.4 | Unknown |
| Dictyostelium discoideum extrachromosomal palindromic rRNA | AY171067 | 2.0 | Unknown |
| Bos taurus clone rp42-194o5, complete sequence | AC098687 | 5.1 | Unknown |
| Bos taurus clone RP42-351K5, complete sequence | AC092727 | 5.6 | Unknown |
| Bos taurus butyrophilin gene, complete cds (BTN1A1) | AF005497 | 2.2 | Unknown |
| O. aries mRNA for thyroid hormone receptor β1 (ERBA β1) | Z68307 | 5.0 | Unknown |
| Bos taurus DNA for SINE sequence Bov-tA | X64124 | 2.9 | Unknown |
| Bos taurus X-inactivation centre region, Jpx and Xist genes (XIST) | AJ421481 | 2.1 | Unknown |
| B. taurus DNA for SINE sequence Bov-2 | X64125 | 2.5 | Unknown |
| Bos taurus clone RP42-400M23, complete sequence | AC090976 | 2.2 | Unknown |
| Bos taurus clone RP42-221D7, complete sequence | AC136966 | 2.0 | Unknown |
| Bos taurus clone rp42-513g13, complete sequence | AC107065 | 2.0 | Unknown |
| Homo sapiens placenta-specific 8, mRNA (PLAC8) | NM_016619 | 2.7 | Unknown |
| B. taurus cosmid-derived repetitive DNA (clone IDVGA-50; subclone3Rev) | X89421 | 2.3 | Unknown |
| Homo sapiens chromosome 5 clone CTC-448D22, complete sequence | AC093206 | 2.2 | Unknown |
| Mouse DNA sequence from clone RP23-44F9 on chromosome 11, complete | AL935275 | 2.5 | Unknown |
| Mus musculus 11 days embryo gonad cDNA, RIKEN full-length (7030402D04Rik) | AK078561 | 2.1 | Unknown |
| B. primigenius mRNA for α-cop coat protein | X96768 | 2.0 | Unknown |
| Homo sapiens G antigen, family C 1, mRNA (PAGE4) | NM_007003 | 2.0 | Unknown |
| Canis β-galactosides-binding lectin (LGALS3) mRNA, 3′ end | L23429 | 2.4 | Unknown |
| Gallus gallus finished cDNA, clone ChEST201k3 | BX950233 | 2.0 | Unknown |
Differentially expressed genes were identified by SAM at a false discovery rate (FDR) of ≤5% and P≤0.05.
A combination of hierarchical clustering and heatmap of differentially regulated genes (Fig. 2) was used to show the overall expression pattern of the target genes in replicate hybridisation. The average linkage clustering analysis revealed the presence of many subgroups within the up- and down-regulated genes (or clusters) sharing similar expression pattern.
Hierarchical clustering and heatmap of differentially expressed genes. The red blocks represent up-regulated genes, while the green blocks represent down-regulated genes in BCB+ compared with BCB− oocytes. Columns represent individual hybridisations, rows represent individual genes.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Hierarchical clustering and heatmap of differentially expressed genes. The red blocks represent up-regulated genes, while the green blocks represent down-regulated genes in BCB+ compared with BCB− oocytes. Columns represent individual hybridisations, rows represent individual genes.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Hierarchical clustering and heatmap of differentially expressed genes. The red blocks represent up-regulated genes, while the green blocks represent down-regulated genes in BCB+ compared with BCB− oocytes. Columns represent individual hybridisations, rows represent individual genes.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Functional classification of target genes
The ontological classification of differentially regulated genes in BCB+ versus BCB− oocytes was performed based on the criteria of Gene Ontology Consortium classifications (http://www.geneontology.org), which annotates transcripts with regard to their molecular functions. The resulting data were supplemented with additional information from Centre and CowBase at the AgBase database (www.agbase.msstate.edu). The differentially regulated genes between the two groups of oocytes were found to represent genes with known function (57.3%; 106/185), with unknown function (18.4%; 34/185) and novel transcripts (24.3%; 45/185).
We observed that certain functional annotations were more represented in either BCB+ (Fig. 3) or BCB− oocytes (Fig. 4). The BCB+ oocytes were found to be enriched with genes related to protein binding (RALA), enzymatic activity (RIOK3), structural constituent of ribosome (RPS14), nucleic acid binding (H2AFZ), transcription (SMARCA5), ubiquitin–protein ligase activity (UHRF2), calmodulin binding (RGS16), translation elongation factor activity (EEF1A1) and microtubule motor activity (DYNC1I2) in BCB+ oocytes (Fig. 3). On the other hand, transcripts involved in protein binding (NLRP2), ion binding (NPTX2), nucleic acid binding (PAPOLG), oxidoreductase activity (PGHS2), enzymatic activity (ALOX15), signal transduction (LGALS1), growth factor activity (BMP15) and hydrogen ion transporting ATPase activity (ATP5A1) were found to be highly abundant in BCB− oocytes compared with BCB+ ones (Fig. 4).
Differentially expressed genes in BCB+ as classified based on the Gene Ontology Consortium classifications (http://www.geneontology.org).
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Differentially expressed genes in BCB+ as classified based on the Gene Ontology Consortium classifications (http://www.geneontology.org).
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Differentially expressed genes in BCB+ as classified based on the Gene Ontology Consortium classifications (http://www.geneontology.org).
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Differentially expressed genes in BCB− as classified based on the Gene Ontology Consortium classifications (http://www.geneontology.org).
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Differentially expressed genes in BCB− as classified based on the Gene Ontology Consortium classifications (http://www.geneontology.org).
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Differentially expressed genes in BCB− as classified based on the Gene Ontology Consortium classifications (http://www.geneontology.org).
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Real-time PCR validation
Real-time PCR analysis using a set of samples distinct from those used in microarray experiment validated the mRNA transcript abundance of ten genes (Fig. 5). The relative abundance of the GAPDH gene was tested and showed no variability between the samples under investigation. Accordingly, five up-regulated genes namely EEF1A1, ODC1, RPS27A, NASP and SMARCA5 showed higher transcript abundance (P≤0.05) in BCB+ than BCB− oocytes as observed in array analysis. Similarly, the relative abundance for ATP5A1, FL405, S100A10 and PTTG1 were greater (P≤0.05) in BCB− than BCB+ oocytes. The transcript abundance for BMP15 was also confirmed but the differences between the two oocyte groups were not statistically significant.
Quantitative real-time PCR validation of ten differentially expressed genes in BCB+ and BCB− oocytes as identified by microarray analysis (A and B). The relative abundance of mRNA levels represents the amount of mRNA compared with the calibrator (with the lowest normalised value). Bars with different superscripts (a and b) are significantly different at P<0.05.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Quantitative real-time PCR validation of ten differentially expressed genes in BCB+ and BCB− oocytes as identified by microarray analysis (A and B). The relative abundance of mRNA levels represents the amount of mRNA compared with the calibrator (with the lowest normalised value). Bars with different superscripts (a and b) are significantly different at P<0.05.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Quantitative real-time PCR validation of ten differentially expressed genes in BCB+ and BCB− oocytes as identified by microarray analysis (A and B). The relative abundance of mRNA levels represents the amount of mRNA compared with the calibrator (with the lowest normalised value). Bars with different superscripts (a and b) are significantly different at P<0.05.
Citation: REPRODUCTION 135, 2; 10.1530/REP-07-0348
Discussion
The success of in vitro production of bovine transferable blastocysts using oocytes aspirated from slaughterhouse ovaries does not exceed 40–50%. Various studies have shown the quality of the oocyte to be the main determinant of blastocyst rate, while the culture environment affects their quality (Rizos et al. 2002, Lonergan et al. 2003). Therefore, selection and further use of good quality or developmentally competent oocytes is vital for the success of various embryo technologies. The use of BCB staining based on the presence of active G6PDH in immature oocytes has proven to be efficient tool to screen developmentally competent or incompetent oocytes for various species including cattle (Alm et al. 2005, Bhojwani et al. 2007). The present study further evidenced differences in subcellular organisations and transcript abundance between the two oocyte groups.
In terms of biological processes, the expression profiles of BCB+ oocytes were markedly different from those of BCB− ones. The majority of expressed genes in BCB+ oocytes are associated with regulation of the cell cycle (NASP, MLH1, PRC1, UHRF2, UBE2D3, CCNB1, MPHOSPH9, CETN3, ASPM, NUSAP1 and AURKA), transcription (SMARCA5, ZFP91, ZNF519, ZNF85, HMGN2, PA2G4, STAT3, DNMT1 and FANK1) and translation (EEF1A1, RPS27A, RPS14, RPS15, RPS29, RPL18A, RPL9 and RPL24); while BCB− oocytes encoded genes controlling ATP synthesis (ATP5A1), mitochondrial electron transport (FL405) and calcium ion binding (S100A10).
Numerous factors involved in cell cycle regulation have been more recognised in BCB+ than BCB− oocytes. Among these cell cycle regulators, a NASP was first identified as a nuclear-associated protein in rabbit testis (Welch & O'Rand 1990, Welch et al. 1990). This gene has high homology with Xenopus histone-binding protein, N1/N2, which is expressed in oocytes (Kleinschmidt et al. 1986, Kleinschmidt & Seiter 1988). NASP is an H1 histone-binding protein that is cell cycle regulated and occurs in two major forms: tNASP, found in gametes, embryonic cells and transformed cells; and sNASP, found in all rapidly dividing somatic cells (Richardson et al. 2000). Moreover, it was strongly expressed in mouse embryos developed under non-blocking culture conditions in which embryos do not exhibit developmental arrest at the two-cell stage; however, the function of this transcript in early embryonic development remains unknown (Minami et al. 2001). NASP was one of the genes with increased expression in very fast moving bovine oocytes, which showed higher blastocyst rate compared with the slow groups after dielectrophoretic separation (Dessie et al. 2007).
It is not surprising that cell cycle regulator genes category is the one of the largest highly expressed transcripts in BCB+ oocytes. The embryo has to divide thrice to reach maternal zygotic transition (MZT) in conditions of very low transcription (Barnes & First 1991). Therefore, the competent oocyte must store enough mRNA coding for cell cycle proteins like CCNB1 (Tremblay et al. 2005) to ensure that these proteins will not be limiting the embryo progression.
Both the assembly of transcriptional machinery and organisation of appropriate chromatin structure are critical for establishing the programme of early mouse development shortly after fertilisation (Sun et al. 2007). Changes in chromatin structure are thought to play an important role in reprogramming gene expression during zygotic genome activation (ZGA) (Schultz & Worrad 1995, Kanka 2003). For example, an apparent increase in histone acetylation accompanies the one- to two-cell transition in the mouse (Sarmento et al. 2004). Chromatin remodelling enzymes belong to the SNF2 family of DNA-dependent ATPases, all of which have a helicase-like ATPase domain (Henikoff 1993). The SWI/SNF ATP-dependent chromatin remodelling complexes are example of these families and SMARCA5 represents one of its members. Mammalian SWI/SNF-related chromatin remodelling complexes regulate transcription and are good candidates for being involved in ZGA in mice (Bultman et al. 2006). The expression of SMARCAL1 as another member of this family was increased in eight-cell embryos compared with MII oocytes, which suggest a potential role in regulation of embryonic genome activation (Misirlioglu et al. 2006). In addition, the balance of chromatin remodelling factors present in the early cleavage stages can dramatically affect embryo development (Magnani & Cabot 2007). Homozygous SMARCA4 knockout mouse embryos arrest during pre-implantation development (Bultman et al. 2000). Several other subunits of SWI/SNF-related complexes, often referred to as BRG1-associated factors, have also been knocked out and confer periimplantation lethality as well (Klochendler-Yeivin et al. 2000, Guidi et al. 2001). Consistent with this, greater mRNA abundance (6.7-fold change) of the SMARCA5 transcript was detected in BCB+(with higher developmental competence) when compared with BCB−oocytes. Alterations in the expression of some of genes encoded chromatin regulatory factors in rhesus monkey oocytes of different developmental potentials suggest that the expression of such transcripts could provide useful markers of oocyte quality (Zheng et al. 2004).
The bovine oocyte, zygote and embryo have a profound need for protein synthesis. However, the mRNA transcripts for these proteins are not synthesised throughout development, but rather during specific phases (Hyttel et al. 2001). In mammals, synthesis of RNA, up to 60–65% of which is ribosomal (rRNA), increases during oocyte growth and reaches a peak at the beginning of follicular antrum formation (Wassarman & Kinloch 1992). This is in accordance with our investigation concerning meiotic configuration in BCB− oocytes (Table 1). These oocytes with insufficient cytoplasmic maturation, under the control of high G6PDH activity, and in the end of oocyte growth showed a proportion of 21.4% with morphological features for rRNA synthesis – nucleoli. In contrast, in BCB+ oocytes only a small proportion (1.8%) showed germinal vesicles with nucleoli. This process of nucleolus remodelling in GV-containing oocytes is a marker for the finished r-RNA synthesis for the establishment of sufficient ribosomes for the following protein synthesis during the final oocyte maturation after GVBD. In our previous studies, we found an increased level in protein synthesis during final oocyte maturation after GVBD, not before (Tomek et al. 2002a, 2002b).
Elongation factor 1α is a component of the eukaryotic translational apparatus and it is also a GTP-binding protein that catalyses the binding of aminoacyl tRNAs to the ribosome (Tatsuka et al. 1992). The tRNA carries the amino acid to the ribosome, which is then used in protein synthesis, thereby inferring a crucial role for this factor in the translation process in protein biosynthesis. Acquisition of high developmental capacity in mammalian oocytes is dependent on high rates of RNA and protein synthesis, imprinting processes and biogenesis of organelles such as mitochondria (Eichenlaub-Ritter & Peschke 2002). Consistent with this, oocytes with greater developmental potential (BCB+) showed higher mRNA transcript abundance for RPS27A and EEF1A1 that represent members of ribosomal and translation related genes respectively. Collectively, it is possible to conclude that BCB+ oocytes have greater stores of cell cycle, transcription and protein biosynthesis transcripts that could be used for resuming meiosis (Tatemoto & Horiuchi 1995) and supporting maternal to zygotic transition (Hyttel et al. 2001). This is in accordance with the results obtained with respect to the developmental competence of BCB+ and BCB− oocytes (Table 2).
Concerning the activity of cell cycle proteins in oocytes, it has been shown previously that maturing bovine oocytes posses the highest phosphorylation of MAPKs in MII and of Akt in MI stage (Tomek & Smiljakovic 2005, Bhojwani et al. 2006). Furthermore, it has been shown that these phosphorylations are tightly correlated with the activities of the kinases. Therefore, from our observations (Fig. 1), it can be concluded that BCB+ GV stage oocytes have a higher basal activity regarding MAPK and Akt, which probably positively influences their developmental competence and which is well reflected by corresponding gene expression.
The reduced developmental capacity of early embryonic development has been associated with mitochondrial dysfunction and low ATP in mammalian oocytes and embryos (Keefe et al. 1995, Barnett et al. 1997, Van Blerkom et al. 1998, Van Blerkom 2004). Recently, the amount of mitochondrial DNA and transcripts has been quantified in bovine oocytes and embryos (May-Panloup et al. 2005) showing that bovine oocytes that failed to cleave contained significantly lower transcripts implicated in mitochondrial biogenesis. A global down-regulation of mitochondrial transcripts has been reported in human compromised oocytes and embryos (Hsieh et al. 2004). In the pig, competent BCB+ oocytes contain more copies of mtDNA and are more likely to be fertilised than incompetent BCB− oocytes (El-Shourbagy et al. 2006). However, supplementation of BCB− oocytes with mitochondria from BCB+ oocytes, and subsequent improved fertilisation outcome, again demonstrates the association between mitochondrial number and fertilisation outcome. Mouse BCB+ oocytes gained better cytoplasmic maturity than BCB− oocytes as determined by a higher intracellular glutathione (peroxidase 1) level, fully polarised mitochondrial distribution (most of mitochondria aggregated in the oocyte hemisphere around the MII spindle). In this study, it is remarkable that oocytes with high G6PDH activity (BCB−) had an increased level of mitochondrial fluorescence intensity and up-regulation of mitochondrial transcripts (ATP5A1 and FL405) compared with BCB+ oocytes. One can speculate that the reason for the higher fluorescence intensity of labelled mitochondria in BCB− oocytes is likely the increased respiratory activity to provide ATP for still unfinished processes in cytoplasmic maturation. In a recent study, incompetent (BCB−) oocytes exhibited a delay in mtDNA replication due to the delayed onset of expression of their nuclear-encoded replication factors and the oocyte attempts to rescue this during the final stages of maturation. Consequently, oocyte competence in terms of mtDNA replication and composition is not fully synchronised and will result in either failed fertilisation or developmental arrest (Spikings et al. 2007). In addition, it could be possible that the higher level of mitochondrial fluorescence intensity in BCB− oocytes may be due to increased oxidative stress in these oocytes. ATP5A1 is a nuclear-encoded gene whose protein contributes to the overall function of the ATP synthase and it is the universal enzyme for cellular ATP synthesis (Pedersen 1994). It has been reported that null mutations in ε-subunit of mitochondrial ATP synthase gene in Drosophila lead to embryonic death (Kidd et al. 2005). ATP6V1E1 transcript was up-regulated at two-cell block mouse embryos (Jeong et al. 2006). From the above-mentioned facts, it is clear that alterations in mitochondrial distribution, DNA replication, copy number and transcripts may lead to overall dysfunction for the mitochondria and influence the ability of embryos to scavenge free radicals and also induce an oxidative stress response, which contributes to impaired development. It seems also that the competency of oocytes is highly dependent on distinct set of genes mainly regulating transcription, translation, cell cycle, chromatin remodelling and mitochondrial machineries which may interact to fulfil this task.
Overall, this study provides a genome-wide expression profiling of genes that could be associated with functional relevance for the establishment of developmental competence in oocytes. However, further functional investigations based on these data could help to define the exact key regulatory genes controlling oocyte quality, which could be considered as good biomarkers for oocytes with high or low developmental competence.
Materials and Methods
Oocyte recovery and BCB staining
Oocytes aspirated from slaughterhouse ovaries were used for BCB staining. The procedure of BCB staining was done as described in our previous studies (Alm et al. 2005, Bhojwani et al. 2007). Briefly, a total of 2128 morphologically good quality compact cumulus–oocyte complexes (COCs) were subjected to 26 μM BCB (B-5388, Sigma–Alderich) diluted in mDPBS for 90 min at 38.5 °C in humidified air atmosphere. After washing, the stained COCs were examined under stereomicroscope and categorised into two groups according to their cytoplasm colouration: oocytes with any degree of blue colouration in the cytoplasm (BCB+; n=1167) and oocytes without visual blue colouration (BCB−; n=961). From each group, oocytes were used for: analysis of chromatin configuration and mitochondrial activity (n=337); detection of abundance and phosphorylation of protein kinases Akt and MAP (n=500); investigation of gene expression (n=1032) and assessment of in vitro development during IVM, IVF and IVC (n=259).
In vitro maturation, fertilisation and culture (n=259 COCs)
After classification in BCB− and BCB+, the COCs were washed twice in maturation medium (TCM 199 supplemented with 20% (v/v) heat-treated fetal calf serum and 10 μg/ml follicle-stimulating hormone (Ovagen; Auckland, ICP, New Zealand) and then incubated in maturation medium for 24 h at 38.5 °C in 5% CO2 in air.
After IVM, oocytes were fertilised in vitro using frozen-thawed bovine semen. A motile sample of sperm was obtained by swim-up separation based on the method of Lonergan et al. (1994). Approximately 0.25 ml cryopreserved semen was layered under 1 ml capacitation base medium (modified Ca2+-free Tyrode's medium). Following incubation for 1 h, the uppermost 0.5–0.8 ml of medium containing motile spermatozoa was removed and washed twice with 2–3 ml capacitation base medium followed by centrifugation at 500 g for 7 min. The resulting pellet was measured using an adjustable micropipette. A 50–60 μl aliquot of the swim-up separated spermatozoa were then diluted with an equal volume of capacitation medium containing 200 μg/ml heparin (H 3393). After incubation for 15 min, the suspension was further diluted with capacitation base medium to reduce the concentration of capacitation inductors and to obtain the desired final concentration of spermatozoa for IVF.
After maturation, oocytes were transferred to modified TALP medium and cumulus cells were removed mechanically by gentle pipetting. Five oocytes were placed in a 45 μl droplet of fertilisation medium (TALP; Lonergan et al. 1994) and 5–8 μl of the final sperm suspension were added to each droplet to have a final concentration of ∼1.0×106 motile sperm/ml in the fertilisation droplet. Fertilisation was carried out for 24 h at 38.5 °C under 5% CO2 in 100% humidified air.
After 20 h coincubation with spermatozoa, presumptive zygotes were denuded and transferred to TCM 199 containing 5% oestrous cow serum. Another 24 h later, the embryos were cultured in synthetic oviductal fluid medium (Minitüb, Tiefenbach, Germany) supplemented with 10% oestrous cow serum and covered with mineral oil. Embryo culture was performed at 38.5 °C in 5% CO2, 5% O2 and 90% N2 and development was evaluated at 48 h (cleavage rate) and at 192 h (day 8; blastocyst rate).
The cleavage rate (number of eggs that had cleaved to the two-cell stage or beyond at 48 h after IVF) and the proportion of blastocysts developing at the end of the 8-day culture period were compared among groups. The number of blastomeres (nuclei) in embryos was determined using the Hoechst staining technique (Alm & Hinrichs 1996).
Parallel fluorescence labelling of oocytes for the analysis of chromatin configuration and mitochondrial activity (n=337 COCs)
Oocyte processing
Oocytes were processed for fluorescence labelling of mitochondria according to the procedure described previously for porcine and horse oocytes (Torner et al. 2004, 2007). Briefly, COCs were incubated for 30 min in PBS containing 3% (w/v) BSA and 200 nM MitoTracker Orange-fluorescent tetramethylrosamine (M-7510; Molecular Probes, Eugene, OR, USA) under culture conditions. The mitochondrial-specific fluorescent and cell-permeant probe MitoTracker Orange (M-7510) is readily sequestered only by actively respiring organelles, depending upon their oxidative activity. Following exposure of COCs to the probe, cumulus cells were mechanically removed from the oocytes by repeated pipetting and subsequent treatment with 3% sodium citrate. The denuded oocytes were washed thrice in pre-warmed PBS without BSA. The oocytes were then fixed for 15 min at 37 °C using freshly prepared 2% (v/v) paraformaldehyde in Hank's balanced salt solution. The thiol-reactive chloromethyl moiety of the probe can react with accessible thiol groups on peptides and proteins of active mitochondria to form an aldehyde-fixable fluorescent conjugate, which is retained after cell fixation over a period of 8 weeks. Immediately after fixation, the same oocytes were prepared for the further staining of chromatin configuration. They were washed thrice in PBS and then mounted between slide and cover slip in a mixture of Moviol V4-88 (133 mg/ml, Hoechst, Frankfurt, Germany) and n-propyl gallate (5 mg/ml, Sigma) containing 2.5 μg/ml bis-benzimide (Hoechst 33342, Sigma) to detect chromatin configuration. The slides were kept for 2–3 weeks at 4 °C in darkness until oocyte analysis.
Oocyte analysis
An epifluorescence microscope (Jenalumar, Carl Zeiss, Jena, Germany) was used for all experiments. First, the chromatin configuration in each oocyte was evaluated under u.v. fluorescence at 410 nm. The chromatin configuration was classified according to the onset of meiotic stages into diplotene with nucleolus (Dipl+Nuc), diplotene (Dipl), condensed chromatin in germinal vesicle (CC), diakinesis (Dia), metaphase I (MI), metaphase II (MII) and degenerated pycnotic chromatin configuration (Pyc).
For subsequent evaluation of mitochondrial activity at ×500 magnification, the emission wavelengths were separated by a 540 nm dichroic mirror followed by further filtering through a 570 nm long pass filter (red emission). Only the labelled mitochondria that were actively respiring were recorded. The fluorescence intensity per oocyte (μA) was measured by the Nikon Photometry System P 100 (Nikon, Düsseldorf, Germany) as described in pig, horse and bovine oocytes (Torner et al. 2004, 2007, Kuzmina et al. 2007). For measurement of intensity, we placed the whole oocyte (thickness around 20 μm) with the eyepiece of the Photometer head P 100 in a defined area of measurement (same size for all observation). The measured intensity was not influenced by the focus of objectives, e.g. different levels of observation (0–20 nm in the oocyte) led to the same quantitative measurement of emitted fluorescence light, because the Photometer measured all emission light from the whole oocyte in the area of frame. To exclude unspecific or artificial effects of the fluorescence probe, we stored different categories of COCs immediately after recovery in the refrigerator (4 °C) for 5 days. Following staining and fixing with the same protocol as described, we determined only the fluorescence intensity of the oocytes in the same level like background fluorescence. We used the level of amplification for photomultiplier, which allow estimation of the highest and the lowest intensity of light emission in a measurement area of linearly progression. Microscope adjustments and photomultiplier settings were kept constant for all experiments.
Detection of abundance and phosphorylation of protein kinases Akt and MAP (n=500 COCs)
BCB+ and BCB− oocytes were analysed for the abundance and phosphorylation state of the MAPKs, ERK1, ERK2 and the protein kinase Akt by Western blotting. The MII stage oocytes (for MAPK) where MAPK shows the highest phosphorylation (Tomek et al. 2002a) and MI stage oocytes (for Akt) where Akt shows the highest phosphorylation (Tomek & Smiljakovic 2005) were used as positive controls. Denuded oocytes (50 each) were separated on 10% SDS-gels and transferred to PVDF membranes as described (Vanselow et al. 2006). After blocking the membrane with 5% fat-free dry milk in TTBS, the blot were incubated overnight at 4 °C with antibodies against MAPKs (diluted 1:2500, sc-94; Santa Cruz Biotechnology, Heidelberg, Germany), phospho-MAPK Thr282, Tyr294, Akt1 (diluted 1:1000, CST 9272; Cell Signalling Technologies, Frankfurt a. Main, Germany) phospho-Akt Ser473 (diluted 1:800, CST 9271) and phosphor-Akt Thr308 (diluted 1:800; CST 9275). The blots were washed and incubated with a second HRP-conjugated anti-rabbit IgG (diluted 1:4000) as described previously (Tomek et al. 2002a, Bhojwani et al. 2006). The bands were visualised with ECL according to the manufacturer's instruction (GE Healthcare, Freiburg, Germany). The experiments were repeated once and representative blots are shown in Fig. 1.
Investigation of gene expression (n=580 COCs)
From each BCB group, three pools of oocytes (each with 110 oocytes and a total of 330) were used for mRNA isolation and subsequent array hybridisation after removal of cumulus cells. The remaining oocytes from each group were used as independent samples for array results validation using real-time PCR. In this study, a bovine cDNA array (BlueChip v.2 with ∼2000 clones or genes; Sirard et al. 2005) was used to investigate the gene expression profiles.
Oocyte denudation and storage
The surrounding cumulus cells were removed from the oocytes of each group by treatment with hyaluronidase 1 mg/ml (Sigma) and gentle pipetting in maturation medium. Separation of cumulus cells was carefully checked under a stereomicroscope. Cumulus-free oocytes and the corresponding cumulus cells of each group were washed twice in PBS (Sigma) and snap frozen separately in cryotubes containing 20 μl lysis buffer (0.8% IGEPAL (Sigma), 40 U/μl RNasin (Promega), 5 mM dithiothreitol (Promega)). Finally, samples were stored at −80 °C until RNA extraction.
RNA isolation
mRNA isolation of oocytes and cumulus cells was performed at two different points during the whole experiment. (1) A total of six pools, each containing 110 oocytes, was used for array analysis after amplification. (2) A total of 10 pools, each containing 25 oocytes, was used for real-time validation of array results. The mRNA isolation was performed using Dynabead oligo (dT)25 (Dynal Biotech, Oslo, Norway) according to manufacturer's instructions. Briefly, oocytes in lysis buffer were mixed with 40 μl binding buffer (20 mM Tris–HCl with pH 7.5, 1 M LiCl, 2 mM EDTA with pH 8.0) and incubated at 70 °C for 5 min to obtain complete lysis of the oocytes and to release RNA. Ten microlitres of oligo (dT)25 attached magnetic bead suspension was added to the samples, and incubated at room temperature for 30 min. The hybridised mRNA and magnetic beads were washed thrice using washing buffer (10 mM Tris–HCl with pH 7.5, 0.15 mM LiCl, 1 mM EDTA with pH 8.0). For each sample, cDNA synthesis has been performed using oligo (dT)23 primer and superscript reverse transcriptase II (Invitrogen) except for samples used in array analysis where the RT was performed using T7 promotor attached oligo d(T)21 primer.
RNA amplification
First- and second-strand cDNA synthesis was carried out as described in our previous study (El-Sayed et al. 2006). The resulting double-stranded cDNA was purified and used for in vitro transcription using AmpliScribe T7 transcription kit (Epicentre technologies, Oldendorf, Germany) according to manufacturer's instructions. Then, the amplified RNA (aRNA) was purified using RNeasy Mini kit (Qiagen) according to the manufacturer's recommendations. Finally, the aRNA was eluted in 30 μl RNase-free water from which 8 μl was taken to estimate the yield, purity of aRNA by gel electrophoresis and u.v. absorbance reading at A260/280 using Ultrospec 2100 pro u.v./Visible Spectrophotometer (Amersham Bioscience).
Labelling and array hybridisation
Minimum information about microarray experiments guidelines were adhered to in the experimental design. Two independent labelling reactions were carried out per aRNA sample pertinent to each biological replicate for dye-swap hybridisations. Accordingly, 3 μg aRNA from each oocyte pool representing each oocyte group (BCB+ or BCB−) was used as a template in RT reactions incorporating amino-modified dUTPs into the cDNA using the CyScribe Post-Labelling Kit (Amersham Biosciences) as described previously (El-Sayed et al. 2006). The aminoallyl-labelled cDNA samples from BCB+ and BCB− oocytes were differentially labelled indirectly using N-hydroxysuccinate-derived Cy3 and Cy5 dyes and incubated for 1.5 h at room temperature in darkness. At the end of incubation, non-reacting dyes were quenched by adding 15 μl of 4 M hydroxylamine solution (Sigma) and incubated for 15 min at room temperature in darkness. To avoid variation due to dye coupling, aRNA samples from the same follicular phase were labelled reversibly either with Cy3 or Cy5 for dye-swap hybridisations. The reaction was then purified with CyScribe GFX purification kit (Amersham Biosciences). Samples were finally eluted in 60 μl elution buffer.
Pre-hybridisation of the slides was performed by placing the array slides into a corning GAPS II slide container as described in El-Sayed et al. (2006). Hybridisation and post-hybridisation washes were carried out as previously described elsewhere (Hedge et al. 2000) with slight modifications as described in Ghanem et al. (2007). Samples that were going to be hybridised on specific array were mixed and dried in speedvac centrifuge (Savant Instruments Inc., Holbrook, NY, USA) and then the pellet was re-suspended in pre-warmed (42 °C) formamide-based hybridisation buffer (15 μl hybridisation buffer (Amersham Bioscience), 30 μl 100% formamide and 15 μl DEPC water). Yeast tRNA (4 mg/ml) and 2.5 μl Cot-human DNA (1 mg/ml; Invitrogen) were used in the reaction in a volume of 2.5 μl each to avoid non-specific hybridisation.
Array scanning and data analysis
The slides were scanned using Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA, USA). The GenePix Pro 4.0 software (Axon Instruments) was used to process the images, to find spots, to integrate robot-spotting files and finally to create reports of spot intensity data. The LOWESS normalisation of microarray data was performed using GProcessor 2.0a software (http://bioinformatics.med.yale.edu/group). The normalised data were used to calculate intensity ratios of all replicates and to obtain one value per clone. Ratios were finally log2 transformed and submitted to SAM analysis. Microarray data analysis was performed using SAM (Significance Analysis for Microarray), a free software developed at Stanford University (http://www-stat.stanford.edu/∼tibs/SAM/). Hierarchical clustering and heatmap of log2-transformed data for up- and down-regulated genes was generated using PermutMatrix (version 1.8.2) available at (http://www.lirmm.fr/%7Ecaraux/PermutMatrix/). Genes expressed equally in both samples were not included in the hierarchical clustering.
Quantitative real-time PCR analysis
To validate microarray results, ten candidate genes were selected for further analysis by real-time PCR (Table 7). Quantitative analysis of cDNA samples was performed using ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). The cDNA synthesised from BCB+ and BCB− samples were subjected to real-time PCR using GAPDH primer to test for any variation in the expression of this internal control gene. The real-time PCR was performed as described in El-Sayed et al. (2006). Final quantitative analysis was done using the relative standard curve method and results were reported as the relative expression or n-fold difference to the calibrator after normalisation of the transcript amount relative to the endogenous control (Tesfaye et al. 2004).
Details of primers used for real-time PCR quantitative analysis.
| Gene name | GenBank accession number | Primer sequences | Annealing temperature (°C) | Product size (bp) |
|---|---|---|---|---|
| BMP15 | AY304484 | F: 5′-CTGACGCAAGTGGACACCCTA-3′ | 60 | 396 |
| R: 5′-GACACACGAAGCGGAGTCGTA-3′ | ||||
| PTTG1 | NM_004219 | F: 5′-GAAGAGCACCAGATTGCGC-3′ | 55 | 194 |
| R: 5′-GTCACAGCAAACAGGTGGCA-3′ | ||||
| S100A10 | NM_174650 | F: 5′-GGATTTCTGAGCATATGGGACC-3′ | 55 | 131 |
| R: 5′-GAGCAAGAGGATGCAAGCAATA-3′ | ||||
| NASP | BT006757 | F: 5′-CCTAGAGCTTGCCTGGGATATG-3′ | 55 | 198 |
| R: 5′-TCGTGGGCTTCCAGGTACTG-3′ | ||||
| SMARCA5 | NM_003601 | F: 5′-AGTGAACTTTCGCCCATCTTG-3′ | 55 | 194 |
| R: 5′-AGGCTTGTGGATCAGAATCTG-3′ | ||||
| EEF1A1 | AB060107 | F: 5′-CCATGGCATATTAGCACTTGGTT-3′ | 55 | 214 |
| R: 5′-GCTTACACCCTGGGTGTGA-3′ | ||||
| ODC1 | NM_174130 | F: 5′-CAAAGGCCAAGTTGGTTTTAC-3′ | 55 | 201 |
| R: 5′-CAGAGATGGCCTGCACAAAG-3′ | ||||
| ATP5A1 | NM_174684 | F: 5′-CTCTTGAGTCGTGGTGTGCG-3′ | 55 | 184 |
| R: 5′-CCTGATGTTGGCTGATAACGTG-3′ | ||||
| RPS27A | AB098891 | F: 5′-TGCAGATTTTCGTGAAGACCCT-3′ | 54 | 203 |
| R: 5′-TTCTTTATCCTGGATCTTGGCC-3′ | ||||
| GAPDH | BC102589 | F: 5′-ACCCAGAAGACTGTGGATGG-3′ | 60 | 247 |
| R: 5′-ACGCCTGCTTCACCACCTTC-3′ |
Statistical analysis
In all cases, the data of the three independent experiments were statistically analysed; differences of P≤0.05 were considered to be significant. The data of the Western blots, morphological analysis of oocytes before IVM and results after IVM/IVF/IVC were expressed as means±s.e.m. Statistical analysis was done using the SAS system for Windows (release 8.02).
The relative mRNA expression data were analysed using General Linear Model (GLM) of the Statistical Analysis System (SAS) software package version 8.0 (SAS Institute Inc., Cary, NC, USA). Differences in mean values were tested using ANOVA followed by a multiple pairwise comparison using t-test.
Declaration of interest
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
Funding
This work was supported by the Deutsche Forschungsgemeinschaft (DFG; To 138/5-1). This article is based on research presented at the 2nd International Meeting on Mammalian Embryogenomics, which was sponsored by the Organisation for Economic Co-operation and Development (OECD), Le conseil Régional Ile-de-France, the Institut National de la Recherche Agronomique (INRA), Cogenics-Genome Express, Eurogentac, Proteigene, Sigma-Aldrich France and Diagenoda sa. All authors declare that they have no relationship with any of the meeting sponsors.
Acknowledgements
The authors would like to thank Dr Andreas Waha (Institute of Neuropathology, University of Bonn) for facilitating the use of GenePix scanner and programme during microarray analysis.
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This article was presented at the 2nd International Meeting on Mammalian Embryogenomics, 17–20 October 2007. The Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems of The Organisation for Economic Co-operation and Development (OECD) has supported the publication of this article. The meeting was also sponsored by Le conseil Régional Ile-de-France, the Institut National de la Recherche Agronomique (INRA), Cogenics-Genome Express, Eurogentec, Proteigene, Sigma-Aldrich France and Diagenode sa.
