We analyzed the transcriptome profiles of bovine oocytes and their companion CCs and investigated transcriptome profile changes when either cell type matures with or without the other using Affymetrix GeneChip Bovine Genome Array. Here, we identified for the first time, transcripts that are exclusively expressed in bovine oocytes or CCs at GV and MII stages. Identifying specific transcription programs either in oocyte or CCs has a paramount importance in RNAi based gene function study. Knowledge of genes exclusively expressed either in oocyte or CCs would enable researchers to select the appropriate design for functional analysis of genes and route of introducing RNAi agents into COC. For instance, if a given gene is expressed only in CCs, transfection is the best method of introducing anti-sense oligomers into the complex. Identification of such genes would help also in understanding functional biological processes and pathways specific to either oocyte or CCs.
Additionally, we assessed the effect of removing the bi-directional communication axis on the gene expression profile of either cell during in vitro maturation and transcriptome profile changes associated with the transition of CCs from GV to MII stage at global scale. Identification of genes that are significantly affected when either the oocyte or CCs mature with or without one or the other would be vital to understand the most important cellular and molecular functions that are associated with the acquisition of developmental competence.
Specific expression program is exhibited by bovine oocyte and CCs
In addition to the previously identified ones [42, 59], this study identified several oocyte or CCs specific transcripts that may play important biological roles in the bi-directional communication of the two cell types during in vitro maturation and for the acquisition of developmental competence at latter stages. Hierarchical clustering of differentially expressed genes demonstrated that the expression profile of oocyte is markedly different from that of its companion CCs with the latter having more number of transcripts than the former.
Transcripts that are over expressed in oocytes are involved in processes leading to meiotic maturation. We found considerably higher (more than 1024 fold change) and exclusive expression of GDF9, BMP15, MOS, Zona Ppellucida Proteins (ZP2, 3, 4), NLRP5, RBM35A, TACSTD1, GAS7 and others in oocyte compared with CCs. Since the roles of GDF9 and BMP15 in oocyte growth and maturation have been widely addressed [2, 60, 61] we are not going to discuss them here. The c-mos proto-oncogene product MOS is believed to be an active component of the cytostatic factor that stabilizes and sustains the activity of maturation-promoting factor (MPF). Notable interspecies differences exist among different vertebrates regarding the physiological effects of MOS on oocyte maturation. Its higher expression in oocytes both at GV and MII stages in the current study supports previous claims that MOS is required both for the activation of MPF during meiosis I and II and for the meiotic arrest at meiotic MII [62, 63].
Trans-membrane proteins are involved in oocyte-granulosa cell regulatory loop and Rho proteins play a role in GTP-bound active state and can interact with a number of effectors to transduce signals leading to diverse biological responses including actin cytoskeletal rearrangements, regulation of gene transcriptions, cell cycle regulation, control of apoptosis and membrane trafficking [64, 65]. Phosphorylation and dephosphorylation of proteins are also crucial and control nearly every cellular activities, including metabolism, transcription and translation, cell-cycle progression, cytoskeletal rearrangement, protein-protein interactions, protein stability, cell movement, and apoptosis. These processes in turn depend on the highly regulated and opposing actions of protein kinases (PKs) and phosphatases (PPs) where the balance between the two plays an important role in the control of oocyte meiotic resumption . Consistent with this notion, we found oocyte specific expression of many of the members of trans-membrane proteins (TMEM30B, TMEM163, TMEM32, TMEM120B and TMEM52) and Rho GTPase activating proteins (ARHGAP10, 17, 18, 22, 24, 26, 27, 28), various members of the mitogene activated protein kinases (MAP4K2, MAPK10, MAPK8IP2), and phosphatases (PPP1R1B, PPP2R2B, PPP3R1, PPP1R3D) that may evidence the roles of these genes in meiotic maturation.
Like wise, some of the transcripts that were highly expressed in CCs relative to oocyte include IFIT5, BMP2, FSHr, GSTA1, FST, PTGR1, hormonal receptors and hormones such as INHA, INHBA, PGR and PGRMC2. The oocyte and CC genes expression study has revealed that the receptor of BMP2, also a receptor for GDF9, is expressed only in CCs . Similar studies have shown the expression of BMP2 receptor in bovine antral follicles and its potential role in the development and functioning of ovarian follicles . In support of these claims, we also detected this gene only in CCs suggesting its higher activity in CCs than in oocyte. GSTA1 is highly expressed in steroidogenically active cells of bovine ovarian follicle and suggested to intervene in folliculogenesis and oocyte maturation  and steroid receptor cells are found only in CCs evidencing the involvement of CCs derived GSTA1 in oocyte maturation. On the other hand, higher expression of FST and INHBA has been reported in cumulus oophorus that were obtained from in vivo produced COCs compared to these produced in vitro .
In order to validate the microarray data, specifically for those transcripts exclusively expressed in oocyte or CCs, we analyzed the expression of some selected genes using semi-quantitative PCR. A 2% agarose gel pictures showing transcripts that are specific to oocyte or CCs are shown in Figure 20. Previous studies have suggested that higher expression of HAS2, PTX3, TNFAIP6, PTGS2, CD44, INHBA and BTC in CCs can be used as molecular bio-markers to select quality embryo in women  and cow . Higher expression of PTX3, PTGS2, ADAMTS1, INHA and INHBA was also reported in human CCs . But, data that clearly demonstrate whether these transcripts are oocyte or CC specific or expressed in both is not available. Here, we show that none of HAS2, PTX3, INHA, INHBA and CD44 are CC specific as they are expressed in both samples (supplemental tables S3 and S6, Figure 20).
Additionally, we report for the first time that POU class 5 homeobox 1 (POU5F1), interferon regulatory factor 6 (IRF6), sex determining region Y box2 (SOX2) and insulin like growth factor 2 binding protein 3 (IGF2BP3) are expressed only in oocytes while secreted protein, acidic, cysteine-rich (SPARC), glutamate pyruvate transaminase (GPT), ADAM metallopeptidase with thrombospondin type 1 (ADAMTS1) and heparanase (HPSE) are expressed only in CCs. Significantly higher expression of POU5F1 has been reported in developmentally competent bovine oocytes and its loss of function has resulted in preimplantation lethality in mouse embryos as it is a central regulator of pluripotency . Similarly, SOX2, which is expressed only in oocyte, is a developmental pluripotency marker and has been hypothesized as a regulator of POU5F1 controlled genes  suggesting a possible synergetic effect of the two genes on oocyte maturation.
IRF6 has been suggested as a key mediator of cellular proliferation and differentiation in mammary epithelial cells by facilitating entry into the G0 phase of the cell cycle . It regulates cell proliferation and differentiation in different cell types and its higher expression in oocyte sample in the present study at both mRNA and protein levels (Figure 21) may show IRF6 as a maternal candidate transcript that play a role in acquiring developmental competence. ADAMTS1 is also expressed only in CCs but more abundantly at MII compared to at GV stage (Figure 20). Previously, increased expression of ADAMTS1 protein has been reported in mouse GCs in response to preovulatory LH surge  where it targets Versican (VCAN), one of the proteins that cross link hyaluronic acid (HA) rich CCs matrix and contributes to oocyte maturation, ovulation and/or fertilization .
Transcripts specifically expressed in CCs also include SPARC, a multifunctional calcium-binding glycoprotein that modulates extracellular matrix interactions and influences cell-cell adhesion, migration and invasion in vitro and in vivo . Although the role of SPARC in the biology of oocytes is not documented, the fact that it is expressed only CCs supports previous reports where it has been expressed only in the somatic cells of germarium and follicles during oogenesis .
The absence of CCs during in vitro maturation alters the gene expression profile of MII oocyte
Despite the fact that we did not observe significant morphological differences between oocyte matured with or without the surrounding cumulus cells including the polar body extrusion, previous study in bovine  has shown that oocytes matured without the surrounding cumulus cells resulted in significantly reduced blastocysts rates compared to those matured in the presence of the surrounding cumulus cells. Therefore, we hypothesized that the absence of cumulus cells during maturation can affect the nuclear and molecular maturation of the resulting oocytes. Maternal gene expression is an important biological process in oocyte maturation. If the oocyte is to complete normal maturation processes, the underlying transcriptional mechanism must be robust. Interestingly, some of the genes that are under expressed due to removal of CCs before in vitro maturation have vital roles in gene expression. The most important one is RNA polymerase II, an enzyme that plays a significant role in gene transcription. Reduced expression of this gene due to removal of CCs before in vitro maturation means the expression of other genes is greatly affected and hence the developmental competence of such oocytes is compromised.
In vitro studies have shown that follicle stimulating hormone (FSH) dependent cyclic adenosine monophosphate (cAMP), the activator of MAPK signaling, is produced by CCs and diffuses to the oocyte via the gap junction [75, 76]. The activated MAPK in turn activates the components of MPF to initiate meiotic resumption  and simulate mos mRNA cytoplasmic polyadenylation during Xenopus [78, 79] and mouse  oocyte maturation. Low expression of molecules that play roles in biochemistry of oocyte maturation (MAP3K2, MAP3K3 and MAP4K14) in OO-CCs samples may imply defects in the maturation process due to removal of CCs before maturation.
The capacity of the oocyte to metabolize glucose is positively correlated with its developmental potential and this depends on the presence of companion somatic cells [81, 82]. Glucose is a pivotal metabolite for COCs and is metabolized via various pathways. During oocyte maturation, a large proportion of total glucose is metabolized in the CCs via the glycolytic pathway to provide substrates such as pyruvate for energy production . Consistent with this, some genes that are involved in carbohydrate metabolism are under expressed due to removal of CCs indicating defective energy metabolism in the groups cultured without CCs and hence poor developmental competence.
In general, removal of companion CCs at GV stage appeared to affect the gene expression of MII oocytes as a number of genes are over expressed in oocytes cultured with CCs relative to those cultured without. As explained above, some of these genes have been implicated to be involved in various biological processes that are pertinent to oocyte meiotic resumption and maturation supporting the notion that the presence of CCs during in vitro maturation is crucial for oocyte developmental competence. However, the majority of these over expressed genes are uncharacterized and/or their functions, particularly with regard to oocyte development and maturation, are poorly understood. Paradoxically, several previously identified and biologically important OSFs (GDF9, BMP6, 15, TGFBs), zona pellucida proteins (ZP2, 3, 4), the components of MPF (CDK1 and Cyclin B1) and others are missing from the list of genes over expressed in OO + CCs.
From these results, it can be argued that either these over expressed genes have functional redundancy with those missing genes or the expression of the latter is completed prior to CCs removal at GV stage and consequently they are detected as equally as those cultured without their CCs. One plausible explanation inline of the latter argument is the fact that bovine oocytes are transcriptionally active during folliculogenesis and transcriptional activity decreases at later stages of follicular development . Additionally, our present microarray data analysis between GV and MII oocytes (data not shown) reveals that only GDF9 and CDK1 are slightly over expressed (fold change = 2.46) at MII relative to GV stage. Interestingly, while BMP15 and TGFB2 are over expressed at GV stage, ZP2, 4 and Cyclin B1 are equally expressed between the two stages. Interestingly several genes have been found to be over expressed in MII stage relative to GV stage including MPV17, ATP6V1D, TMEM127, NUDT14, UQCR, EXOSC6, MAPK10, CSNK1D, DBNL, FILIP1L, YIF1A and ARHGAP27. Considering the transcriptional activity of bovine oocytes, the over-expression of transcripts at MII stage compared to the GV stage need further investigation in terms of transcriptional regulation of genes during oocyte maturation.
The absence of oocyte during in vitro maturation alters the gene expression profile of CCs
Notable interspecies differences exist whether OSFs are mandatory for FSH induced CCs expansion in vitro. In rat, the presence of OSFs are crucial for in vitro CCs expansion as oocytectomized complexes (CCs - OO) failed to expanded their cumulus oophorus . In cattle and pig, CCs expansion doesn't depend on the presence of these factors as CCs - OO expanded as equally as the intact ones [46, 47]. In the present study the results of IPA showed that some of the genes under expressed due to removal of oocyte before in vitro maturation are classified into cellular growth and proliferation (VEGFA, GADD45A, FOS, EGR1, HAS2), cell cycle (CCND2, CDCA8, CDK6), and gene expression (FOSB, TGFB2, ATF3) functional groups (Figure 9). These genes are also mapped into a complex gene network that includes genes involved in cellular development such as Cyclin D, HPSE, JUNB and others (Figure 9). Some of these genes are not well characterized and their direct role in the biology of CC is poorly understood. However, results of previous studies in different species have shown that some of these genes are involved in cumulus expansion and oocyte maturation. For instance, genes from FOS family have been implicated as regulators of cell proliferation, differentiation and transformation and IGFPB proteins stimulate the growth promoting effects of IGF1 which in turn is important for oocyte growth and maturation and granulosa cells proliferation .
Although a number of genes were differentially expressed between CCs that were cultured with or without their enclosed oocytes, it is not easy to conclude that removal of oocyte completely changes the expression of CCs genes at MII stage. For instance, except HAS2, the majority of CCs genes previously identified as molecular bio markers for developmental competence including INHβA, EGFR, BTC, CD44, TNFAIP6, PTX3 and PTGS2 [40, 48] were not differentially expressed between the two sample groups. From these results, we hypothesize that either these differentially expressed genes predict oocyte developmental competence better than the previously indentified ones or the transcription of these previously identified genes is completed earlier at GV stage before the ooplasm is removed and hence they are not differentially expression at MII stage. However, the assertion of both hypotheses requires further investigation.
The dynamics of CCs transcriptome during the transition of COCs from GV to MII stages is associated with functional changes
Massive transcript degradation during in vitro maturation of bovine , human [42, 59] and mouse  oocytes has been reported. Similarly, considerable shift of transcripts and associated functional changes were observed in CCs during the transition of COCs from GV to MII stage. For instance, while transcripts that are involved in cell cycle, DNA replication, metabolic process, steroid and cholesterol biosynthesis, signal transduction and regulation of catalytic activity are over expressed in CCs at GV stage, these involved in cell adhesion, protein metabolic process, regulation of cellular component organization and biogenesis and actin filament polymerization are over expressed in CCs at MII stage (Figure 13). The most interesting finding of this experiment is not only the change in the number of transcripts but also the identity of transcripts that are involved in a given GO term at the two developmental stages.
Cell cycle and DNA replication are the two successive events which play significant roles in meiotic process resulting in the formation of four haploid cells. The functioning of a cell depends upon its ability to extract and use chemical energy stored in organic molecules via metabolic pathways. On the other hand, MAPK activation in CCs rather than in oocytes exerts essential functions during mammalian oocyte meiotic resumption [66, 87] and steroids such as progesterone have been suggested to induce cAMP dependent MAPK signaling cascade leading to meiotic resumption . Hence, over expression of transcripts that are involved in cell cycle, metabolic and steroid biosynthetic pathways in CCs at GV stage suggest that these pathways are more active in GV than in MII stage.
As the oocyte lacks gonadotropin receptors, it has been hypothesized that FSH exerts its effect via a positive meiosis factor (EGF) synthesized by CCs indirectly via signal transduction pathway that involves cAMP dependent MAPK to induce meiotic resumption . From these findings, we propose that over expression of transcripts that are involved in signal transduction pathway at GV stage CCs relative to MII is an indication that this pathway is more active at the former than the latter.
Focal adhesions are large macromolecular assemblies through which both mechanical force and regulatory signals are transmitted . They serve not only to anchor the cell, but also to carry signals, which inform the cell about the condition of the ECM and thus affect their behaviour . In view of their increased expression at MII relative to their expression at GV stage, we propose that these genes are involved in one of the regulatory network that connects the oocyte to its companion CCs during in vitro maturation.
Actin filaments localize to specific regions within mammalian oocytes and their modelling including polymerization are important for oocyte maturation, fertilization and embryo development [76, 92, 93]. Interestingly, we found higher expression of transcripts that are involved in actin filament polymerization at MII than in GV CCs. Consistent with the notion that in vitro meiotic resumption in bovine oocytes is triggered by FSH [76, 93]; we observed higher expression of FSHR mRNA in GV CCs than in MII stage. Prior to in vitro meiotic resumption, FSH is received by CGCs via FSHR and this activates the release of cAMP and MAPK signaling pathways to initiate meiotic resumption .
CCs expansion is a key biological event for successful oocyte maturation, ovulation and fertility [40, 48, 94, 95]. HAS2 is an important enzyme for the biosynthesis of HA to form stable matrix during CCs expansion . The binding proteins of HA (TNFAIP6 and PTX3) and its receptor protein (CD44) plays significant roles in attaining full CC expansion. CD44 is a widely expressed cell adhesion molecule that binds the extracellular matrix component, HA in a tightly regulated manner . The interaction between HA and CD44 is the key molecular mechanism for the activation of signalling cascades that contribute to cell adhesion, proliferation, migration and differentiation [98, 99]. This interaction is also important for MAPK signalling pathway in oocyte and may promote meiotic resumption . Oocytes can't attain cytoplasmic maturation when cultured in the absence of their companion CCs as they can't store sufficient mRNAs, proteins and transcription factors that are important for maturation process due to removal of the b-idirectional communication axis. The interaction between HA-CD44 is critical for modification of this communication axis during CCs expansion  and relatively higher expression of these molecules in cultured CCs is consistent with the notion that HA-CD44 interactive effect is vital for oocyte maturation .
The majority of genes differentially expressed between different oocyte and CCs samples are poorly characterized and their roles in the biology of bovine oocyte are not known. Moreover, due to the dynamic nature of gene expression in different species, tissues and follicular stages, what have been reported so far in other organisms may not necessarily hold true for bovine oocytes and CCs. Therefore, we recommend detailed gene by gene study to unveil specific roles of these genes in the biology of bovine COCs.