BACKGROUND
the early stages of mammalian development are sustained by the presence of transcripts and proteins that have been produced and stored in the oocyte during folliculogenesis.
this supply is used by the morula stage in sheep and rabbit preimplantation embryos; the 4- to 8-cell stage in humans and the 2-cell stage in the mouse, at which time zygotic gene activation  occurs and novel transcripts and proteins are expressed by the embryonic genome .
what is the exact maternal contribution during this period of transition from a maternal to an embryonic control of development, and how the maternal legacy of transcripts and proteins is orchestrated through the first mitotic divisions prior to zga, remains unclear.
an altered maternal contribution to a correct expression of zygotic genes leads to a developmental block at the time of zga  <cit> .
this developmental block is a feature of a group of fully grown antral oocytes present in the mammalian ovary.
based on their chromatin organisation, antral oocytes can be classified in sn  oocytes, with a ring of chromatin surrounding the nucleolus and in nsn  oocytes, with a more dispersed chromatin not surrounding the nucleolus .
these two types of gametes are present in the ovaries of all the mammalian species studied including rat  <cit> , monkey  <cit> , pig  <cit> , mouse  <cit>  and human  <cit> .
they have been extensively studied in the mouse and a number of morphological and functional differences have been described.
for example, in sn oocytes most of the centromeres localise around the compact nucleolus, whereas in nsn oocytes only the centromeres of chromosomes carrying the nucleolus organising regions are associated with the nucleolus  <cit> .
microtubule organising centres form around the nucleus of sn oocytes, but not around that of nsn oocytes  <cit> .
these morphological differences have a biological relevance as they have been correlated with changes in transcription  <cit> .
nsn antral oocytes are transcriptionally active and synthesise all classes of rna, whereas sn antral oocytes are transcriptionally inactive  <cit> .
the most striking difference between the two types of oocytes is their developmental competence.
when isolated from the mouse ovary, matured to metaphase ii  and fertilised in vitro, both types of oocytes  are capable of developing to the 2-cell stage, but only miisn oocytes are developmentally competent beyond the 2-cell stage  <cit>  and capable of reaching full term development  <cit> .
in this study we have compared the gene expression profile of developmentally incompetent miinsn oocytes with that of developmentally competent miisn oocytes, with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence.
to this end, fully grown nsn and sn antral oocytes were isolated from the ovaries, cultured in vitro to the mii stage and their profiles of gene expression compared.
we first focused our investigation on a group of maternal-effect genes whose altered expression leads to preimplantation developmental arrest.
then, using microarrays analysis and bioinformatic resources, we identified major groups of genes, gene expression networks and biochemical pathways characteristic of the developmentally incompetent miinsn oocytes.
RESULTS
stella, a maternal-effect protein required for the oocyte-to-embryo transition, is not detected in developmentally incompetent miinsn oocytes
our study was first focused on a specific group of gene transcripts and proteins accumulated during the oocyte growth to become necessary, after fertilisation, for successful embryogenesis.
these gene products and their effects are well documented in organisms such as drosophila melanogaster or xenopus laevis  <cit> , but in recent years, increasing evidence has been produced that maternal-effect genes are crucial in pre-  <cit>  and post-implantation  <cit>  development.
we analysed the profile of expression of the following five genes, known for playing a role during the oocyte-to-embryo transition: zar <dig>  npm <dig>  stella , smarca <dig>  and prei <dig>  <cit> .
the relative number of transcripts of the genes analysed resulted equally represented in the two types of mii oocytes, with the exception of stella that was  <dig> -fold down-regulated  in miinsn oocytes .
consistently with the down-regulation of gene expression, immunostaining with an antibody against stella did not detect the presence of the protein either in nsn antral or miinsn oocytes , whilst in sn antral oocytes stella co-localised around the nucleolus  and in miisn oocytes was dispersed within the whole ooplasm , as already shown in ovulated mii oocytes  <cit> .
stella down-regulation is a good candidate to explain the developmental block at the 2-cell stage that miinsn oocytes encounter following fertilisation  <cit> .
for this reason,, we wondered what induces the down-regulation of stella in miinsn oocytes and whether other genes could be involved in its regulation.
stella is positively regulated by oct- <dig> and nanog based on mouse oct- <dig> chip-pet data  <cit> , and it is also down-regulated upon rnai-mediated oct- <dig> knockdown in human embryonic stem  and embryo carcinoma  cells  <cit> .
a recent study has demonstrated a strong link between stella and oct- <dig>  showing that the expression of stella in mouse es cells is regulated by oct- <dig> which is necessary for maintaining a specific chromatin structure within the locus containing stella  <cit> .
the authors observed collapse of higher order chromatin structure throughout the locus and down-regulation of stella expression following depletion of the oct- <dig> gene.
additional studies using microarray analysis of gene transcription in es cells, have described oct- <dig> involvement in the regulation of chromatin modelling and chromatin-mediated transcription regulation  <cit> .
next in our investigation, we addressed the question whether the pattern of oct- <dig> gene and protein expression in developmentally incompetent miinsn and competent miisn oocytes, in concert with the knowledge already acquired on this gene, could establish oct- <dig> as a key regulator of stella expression during the late phases of oocyte maturation.
oct- <dig>  a chromatin-mediated regulator of stella transcription, is down-regulated in developmentally incompetent miinsn oocytes
oct- <dig> , a nuclear transcription factor that belongs to the pou family, is involved in the regulation of the expression of a number of developmental genes and is required for the maintenance of cell pluripotency .
oct- <dig> is expressed throughout folliculogenesis  <cit> ; following fertilisation, it remains of maternal origin until the 2-cell embryo, then at the 4- to 8-cell stage is first expressed by the embryonic genome  <cit> .
in an earlier study, we identified a 2-fold down-regulated expression of oct- <dig> in nsn antral oocytes compared to sn antral oocytes  <cit> .
in the present work, we found that down-regulation of oct- <dig> expression in miinsn oocytes correlated with a down-regulated expression of its encoded protein as revealed by immunocytochemistry.
figure 2m–2r illustrates the immunostaining pattern of the oct- <dig> protein in fully grown antral sn and nsn oocytes.
while in sn oocytes the protein was intensely present and mostly localised around the nucleolus , in nsn antral oocytes it was undetectable .
following in vitro maturation to the mii phase, oct- <dig> continued to be absent in miinsn oocytes , whereas miisn oocytes maintained the presence of the transcription factor localised around the mii plate and partially within the cytoplasm .
in summary, since a sound experimental evidence has demonstrated that oct- <dig> regulates the expression of stella, oct- <dig> and stella down-regulation in miinsn oocytes may contribute to the inability of these gametes to develop beyond the 2-cell stage.
our results suggest a novel role of maternal oct- <dig> in the regulation of the developmental competence of the female gamete.
oct-4-dependent transcriptional networks have been described regulating self-renewal and pluripotency in human  <cit>  and mouse  <cit>  es and ec cells and in human mesenchymal cells  <cit> .
these studies demonstrate that oct- <dig> may interact with other genes to regulate specific biological process.
we next asked the question, is oct- <dig> a key regulator of genes associated with or implicated in establishing developmental competence on oocytes?
to address this, we first analysed by microarrays, global differences in gene expression between the two types of oocytes, and then searched for genes known to be regulated by oct- <dig>  in the array-derived list of regulated genes.
microarray-based analysis of the transcription profiles of developmentally competent and incompetent mii oocytes
the microarray platform that we used allowed the analysis of a total of  <dig>  genes and gene sequences .
of these,  <dig>  genes were expressed only in miinsn oocytes;  <dig>  genes were expressed specifically in miisn oocytes and  <dig>  genes were expressed in both types of oocytes.
a fold-change cut-off of at least  <dig>  and a p value <  <dig>  was set to filter the genes whose expression was differentially regulated.
in the comparison between miinsn and miisn oocytes, we identified a total of  <dig> regulated genes, of these  <dig> were up-regulated in miinsn oocytes, and  <dig> down-regulated .
using the gene ontology  annotations implemented in the illumina platform  and also by david   <cit> , we identified  <dig> major biological themes that characterised the group of regulated and annotated genes : transcription regulation , protein biosynthesis , cellular transport , metabolism , development , signal transduction , cell cycle , carbohydrate metabolism , dna metabolism , cytoskeleton organisation , apoptosis , chromatin/nuclear organisation  and various go ; also, some genes fell into a group with undetermined go .
an inherent weakness of the bioinformatic programs available to analyse the patterns of gene expression obtained following microarrays analysis is that they are limited to those genes with assigned annotations or published relationships, and depend on the accuracy of these annotations.
the number of down-regulated and up-regulated genes in each of the  <dig> categories and the list of these genes are shown in figure 3b and additional file  <dig>  respectively.
to determine whether and how individual genes are inter-related or interact with each other and to search for biological pathways and the inter-relationships between network genes, we used the ingenuity pathways analysis  version  <dig>  analysis tool .
the genes of each gene group  were uploaded as tab-delimited text files and ipa queried the ingenuity pathways knowledge base for interactions between focus genes and all other objects stored in the knowledge base and generated a set of networks.
those networks with a score ≥  <dig> were considered for further analyses .
genes up-regulated in miinsn oocytes
the great majority  of the regulated genes was up-regulated.
when ipa was performed on the up-regulated focus genes, it generated  <dig> networks  with a score of  <dig> or higher, of these,  <dig> had  <dig> or more focus genes .
because a score of  <dig> or higher has a confidence of at least 99% of not being generated by chance, the high confidence level obtained, particularly in the top nine networks, reveals the strong interrelation and interaction among the genes that are up-regulated in miinsn oocytes.
figure in additional file  <dig> shows the top  <dig> gene expression networks obtained.
the specificity of these networks to the biology of the ovary was confirmed using ipa to enquire public ovarian gene expression data sets.
network  <dig> has  <dig> focus genes and of these  <dig>  are known to be expressed in ovarian tissue.
in networks 2– <dig>  the percentage of ovarian genes decreased from  <dig> %  to 50% .
comparison with data sets of somatic tissues, namely liver, kidney, heart, lung and spleen gave a lower matching .
network
*: in bold are oct-4-regulated genes .
the gene expression networks described were associated to top functions such as gene expression, protein synthesis, cancer, immunological disease, genetic disorder, dermatological disease and reproductive system disease, highlighting the complexity of the different transcriptional profile between developmentally incompetent miinsn and competent miisn oocytes.
ipa was then interrogated to obtain a list of biochemical pathways that are representative of the functions in which the genes are involved.
the genes up-regulated in miinsn oocytes were assigned to more than  <dig> pathways;  <dig> of these were considered statistically significant  and the top six, together with the gene expression networks they belong to, are shown in table  <dig>  these six pathways included a total of  <dig> different genes, the majority of which belonged to network  <dig> , network  <dig>  and network  <dig> , and were associated with oxidative phosphorylation, mitochondrial dysfunction, galactose metabolism, purine metabolism, protein ubiquitination and gaba receptor signalling.
some of these genes are expressed within different pathways.
pathway
genes down-regulated in miinsn oocytes
ipa analysis of the focus genes down-regulated in miinsn oocytes generated  <dig> networks  with a score ≥  <dig> .
the top functions associated with these gene expression networks include cell cycle, cell death, embryonic development and cancer.
when ipa was interrogated to generate the biochemical pathways that were most representative of the functions attributed to the genes involved in the  <dig> networks, it created ten major pathways represented by  <dig> genes,  <dig> of which are associated with network  <dig>  and network  <dig>  .
of these  <dig> genes, atm and mdm <dig>  are involved in the g2/m dna damage checkpoint regulation.
the protein encoded by the prdx <dig> gene is a member of the thiol-specific antioxidant protein family, involved in the regulation of phospholipids turnover as well as in the protection against oxidative injury.
to identify the inter-relationship among up- and down-regulated genes, we next carried out an ipa analysis of all the regulated genes.
genes regulated in miinsn oocytes
the ipa of all the regulated genes, documented  <dig> gene expression networks with a score ≥  <dig>  of these  <dig> showed a score ≥  <dig> .
since most of the regulated genes are up-regulated , as expected, the top functions represented by these networks are those already identified with the separated analysis of the up- and down-regulated genes , but it also revealed new categories .
in network  <dig>  nf-kb is one of the principal nodes, together with insulin, calmodulin and protein phosphatase 2a .
correct expression of nf-kb is required for the development of the mouse preimplantation embryo, as inhibition of its expression blocks development at the 2-cell stage  <cit> .
ppa <dig> is one of the four major ser/thr phosphatases implicated in the negative control of cell growth and division.
further analysis of the most representative biochemical pathways identified nine top pathways,  <dig> of which are shown in additional file  <dig>  the most representative pathways were those assigned to protein ubiquitination, oxidative phosphorylation, ubiquinone biosynthesis, g2/m dna damage checkpoint regulation, fructose and mannose metabolism, valine, leucine and isoleucine degradation, attributed mainly to network  <dig> , network  <dig>  and network  <dig> .
genes expressed solely in miinsn or miisn oocytes
the comparison of the expression profiles of miinsn with that of miisn oocytes, resulted in the identification of a small number of focus genes transcribed exclusively by miinsn oocytes   or miisn oocytes  .
three of the miinsn oocyte-specific genes have an assigned go function as negative regulators of cell proliferation  or apoptosis , whereas two annotated miisn oocyte-specific genes are involved in processes of positive regulation of the cell cycle .
out of  <dig> focus genes expressed only in miinsn oocytes,  <dig> were assigned to a single gene expression network , with top functions that included hair and skin development and function, organ morphology and cellular development.
the number of miisn-specific genes was so low that it was not considered for further analysis.
rt-pcr analysis of the relative number of transcripts of two miinsn oocyte-specific genes  and two miisn oocyte-specific genes  confirmed the microarrays data and the presence of a different number of transcripts in the two types of oocytes; however, whilst jam <dig> and ogfr were shown to be expressed only in miinsn oocytes ; crebbp and fnip <dig> were not exclusive to miisn oocytes, but instead showed respectively a  <dig> -fold and  <dig> -fold, higher number of transcripts in these gametes .
the presence of transcripts of crebbp and fnip <dig> genes in miinsn oocytes is due to the overt single-cell sensitive of nested-pcr that amplified the low number of transcripts that were not detected by the less sensitive microarray analysis.
when the number of the second pcr cycles was reduced to  <dig>  and  <dig> , expression was not detected in the miinsn oocyte samples.
in summary, the results of the microarray analysis as a whole, indicate that developmentally incompetent miinsn oocytes have activated, among others, gene expression networks implicated in the regulation of biochemical pathways representative of the adverse biological status of these oocytes.
these pathways include, oxidative phosphorylation, mitochondrial dysfunction and apoptosis.
to identify oct- <dig> target genes among the list of regulated genes, we compared our list with the published mouse and human chip datasets of oct-4-regulated genes  <cit> .
eighteen oct-4-regulated genes are associated with adverse gene expression networks in developmentally incompetent miinsn oocytes
this analysis identified a total of  <dig> target genes.
when comparing miinsn with miisn oocytes, all these genes, with the exception of odz <dig> and zfp36l <dig> , were up-regulated in miinsn oocytes with fold changes ranging from  <dig>   to  <dig>   .
eighteen of these genes were part of gene expression networks  <dig> to  <dig> generated from the ipa of up-regulated genes in miinsn oocytes , and thus involved in the activation of the same biochemical pathways described above.
when ipa was specifically interrogated with the list of these  <dig> genes, including oct- <dig>  it generated  <dig> networks  with a score ≥  <dig>  of these, the first two contained  <dig> and  <dig>  focus genes respectively, and had top functions centred around biological themes such as gene expression, cell death, cancer and reproductive system disease.
oct- <dig> was part of network  <dig> , which included the tnf  pathway, a multifunctional proinflammatory cytokine that acts on several different signalling pathways to regulate apoptosis , nf-kb activation of inflammation, and activation of stress-activated protein kinases .
network
*: in bold are the oct-4-regulated focus genes.
underlined is the oct- <dig>  gene.
interestingly, zfp36l <dig> , one of the two oct-4-regulated genes that was down-regulated in miinsn oocytes, is a member of the tis <dig> family of early response genes, of which zfp36l <dig>  another maternal-effect gene crucial for early embryonic development, is also a member  <cit> .
perhaps, zfp36l <dig> is a novel maternal-effect gene whose expression is regulated by oct- <dig> and whose down-regulation may affect the developmental competence of miinsn oocytes.
the microarray analysis revealed a  <dig> -fold down-regulation of oct- <dig> expression in miinsn oocytes.
however, this data was not included in the list of regulated genes  because its p value was equal to  <dig> , thus just slightly above the set cut-off limit of p <  <dig> .
to understand how the expression of the oct- <dig> gene was related with the adverse gene expression networks that we found activated in miinsn oocytes, we next included the expression data for oct- <dig> obtained by microarray analysis  into the list of up-regulated or down-regulated genes and performed an ipa analysis.
with the group of down-regulated genes, oct- <dig> was included in network  <dig> ; with the up-regulated genes, oct- <dig> was integrated into network  <dig>  which has top functions as post-translational modification, cancer, genetic disorder and activates adverse pathways such as oxidative phosphorylation and mitochondrial dysfunction, see table  <dig> and table 2).
in both cases the gene fell into the two top networks highlighting the central role it plays.
in summary, down-regulation of oct- <dig> expression in miinsn oocytes induces the up-regulation of a group of  <dig> oct-4-regulated genes that are part of the top gene expression networks involved in the activation of adverse pathways, i.e., oxidative phosphorylation, mitochondrial dysfunction and apoptosis.
discussion
the maternal contribution of transcripts and proteins supplied to the zygote is crucial for the successful progression of preimplantation development.
an aberrant oocyte inheritance leads to developmental arrest, mostly at the time of zga.
to gain a better understanding of the genes, gene expression networks and biochemical pathways that may be activated/inactivated to compromise the developmental competence of mii oocytes, we compared two types of mouse mii oocytes, one which is developmentally competent and reaches full term development , the other that ceases development at the 2-cell stage .
our study began with the analysis of the profile of expression of five maternal-effect genes.
we showed that of these, only the expression of stella was mis-regulated and its protein absent in miinsn oocytes.
the maternal inheritance of stella is needed for correct preimplantation development  <cit> .
when oocytes from stella-/- females are inseminated with wild-type sperm, while fertilisation proceeds normally, development is severely altered resulting in a progressive decline in the number of blastocysts, with the majority of embryos that do not develop beyond the 2-cell stage.
stella encodes for a basic protein with a sap-like domain and a splicing factor-like motif thought to have a role in chromosomal organisation or rna metabolism.
stella binds both rna and dna in vitro, supporting its involvement in linking chromatin with rna-related processes, as it is the case for other sap-domain proteins  <cit> .
a recent study has shown that stella protects against inappropriate dna demethylation in the maternal genome and at certain imprinted loci during epigenetic reprogramming after fertilisation  <cit> .
down-regulation of stella, is likely to be partly causal of the developmental arrest at the 2-cell stage of miinsn oocytes.
lack of stella protein in miinsn oocytes is a consequence of the down-regulation of oct- <dig>  which is known to regulate stella expression in es and ec cells.
this implies a pivotal role for maternal oct- <dig> in the establishment of the developmental legacy of female gametes.
the down-regulation of oct-4/stella in nsn gv oocytes compromises their developmental competence.
in favour of the hypothesis- "factors present in miisn oocytes, but absent in miinsn oocytes, govern their developmental competence", inoue and collaborators  <cit>  have micromanipulated fully grown sn and nsn antral oocytes to demonstrate that the developmental competence of sn oocytes is dependent on unknown cytoplasmic factor that are released from the germinal vesicle at the time of mii formation and that these ooplasmic factors are not contained in the gv of nsn oocytes.
both stella and oct- <dig> proteins localise within the germinal vesicle of the sn antral oocytes; following meiosis resumption and germinal vesicle break-down, oct- <dig> is circumscribed mainly around the mii-plate area, while stella is released within the ooplasm.
the association of oct- <dig>  with the nucleolus in antral sn oocytes may have a functional significance.
it was previously shown that oct- <dig> is closely associated with rna polymerase ii in the nucleolus of oocytes   <cit> ; since nlbs are the only coilin-containing structures at the end of pre-ovulatory stages of oogenesis, they may represent sites for the storage of the transcriptional machinery needed after fertilisation and of which oct- <dig> may be a key component  <cit> .
this association of oct- <dig> with the nlbs may secure a transfer of the maternally inherited protein through the nlb to the zygote  <cit> , where oct- <dig> perhaps continues to be necessary as a regulator of the molecular events leading to zga.
our microarray analysis indicate that the role of oct- <dig> as a regulator of gene expression in mii oocytes extends beyond the regulation of stella expression and has an inhibitory effect on the expression of oct-4-regulated genes in developmentally competent miisn oocytes.
down-regulation of oct- <dig> in miinsn oocytes correlates with the up-regulation of  <dig> oct-4-regulated genes involved in the activation of gene expression networks and biochemical pathways that most represent the negative legacy of miinsn oocytes.
these pathways include oxidative phosphorylation, mitochondrial dysfunction and cell death.
following fertilisation, these negative features might be passed on to the zygote and constitute a detrimental maternal legacy that affects further development.
oxidative phosphorylation and mitochondrial dysfunction are biochemical pathways principal to the first two top gene expression networks described for the group of up-regulated genes , which emphasises the central role that these genes and mitochondria play in the biochemistry of the developmentally incompetent miinsn oocytes.
in addition to producing most of the cell's atp, recent studies have demonstrated that mitochondria have a central role in cellular processes such as the regulation of intracellular redox potential and control of apoptosis in oocytes and preimplantation embryos .
accumulation of reactive oxygen species  induces apoptosis in mouse zygotes, and mitochondria are involved in the early phase of oxidative stress-induced apoptosis  <cit> , that leads to developmental arrest and alterations of the zga  <cit> .
also, changes in the levels of ros regulate a number of transcription factors critical in early development, these include pebp <dig>  ap- <dig>  p <dig> and nf-kb  <cit> .
the two-fold role for mitochondria, to maintain life or to commit to cell death, may represent a quality control system in the female gamete and in the early embryo that will determine whether the embryo proceeds further into development or is quickly eliminated  <cit> .
if down-regulation of oct- <dig> activates these negative pathways, what does upstream of oct- <dig> regulate its expression?
we have previously shown an effect of pmsg  on oct- <dig> gene expression during folliculogenesis, demonstrating a hormonal regulation of oocyte-specific gene expression  <cit> .
pmsg, which was used in this study to prime females before oocytes isolation, may enter the gamete with the involvement of a fsh-r signaling pathway that, through the modulation of c-kit on the surface of oocytes  <cit> , conveys the gonadotropin signal into the nucleus and then act upon the transcription of several genes.
one candidate could be gcnf , a member of the nuclear receptor family, critical in controlling basal activities or hormonal responsiveness of numerous target genes and known to repress oct- <dig> expression  <cit> .
gcnf may be a target of pmsg action that leads to the down-regulation of oct- <dig> expression in miinsn oocytes.
this hypothesis needs to be tested, however, it is interesting that in our microarray analysis, gcnf was  <dig> -fold up-regulated in miinsn, although, because of its p value >  <dig> , it was excluded from the list of regulated genes.
CONCLUSIONS
in summary, our study of developmentally competent and incompetent mouse mii oocytes has identified a small number of genes, gene expression networks and biochemical pathways whose activation is critical for the correct progression of the early stages of preimplantation development and regulate the 2-cell block in miinsn oocytes.
our data constitute a framework for further exploration of the genes and related transcriptional networks dedicated to establishing and preservating the developmental competence of mammalian oocytes.
based on our results, we have drawn a working hypothesis that assigns a role to some of the molecular players that have been described in this and other studies.
the use of a model study in which the mii oocyte ceases development consistently at the 2-cell stage has allowed to illustrate a role for the maternal oct- <dig> that has never been described before.
oct- <dig> emerges as a key regulator of the molecular processes that govern the establishment of the maternal legacy of mii oocytes required for the transition from a gametic to an embryonic control of development.
a sound knowledge of the orchestrating molecules and the biochemical pathways entailed during the very early stages of development will allow a better understanding of the molecular mechanism that determine the block of development at the time of zga, which in humans is the cause of a loss of a high percentage of embryos obtained by in vitro fertilisation.
a thorough functional analysis of these genes in preimplantation embryos derived from miinsn and miisn oocytes is our next challenge.

 the early stages of mammalian development are sustained by the presence of transcripts and proteins that have been produced and stored in the oocyte during folliculogenesis. this supply is used by the morula stage in sheep and rabbit preimplantation embryos; the 4- to 8-cell stage in humans and the 2-cell stage in the mouse, at which time zygotic gene activation occurs and novel transcripts and proteins are expressed by the embryonic genome. what is the exact maternal contribution during this period of transition from a maternal to an embryonic control of development, and how the maternal legacy of transcripts and proteins is orchestrated through the first mitotic divisions prior to zga, remains unclear. an altered maternal contribution to a correct expression of zygotic genes leads to a developmental block at the time of zga. this developmental block is a feature of a group of fully grown antral oocytes present in the mammalian ovary. based on their chromatin organisation, antral oocytes can be classified in sn oocytes, with a ring of chromatin surrounding the nucleolus and in nsn oocytes, with a more dispersed chromatin not surrounding the nucleolus. these two types of gametes are present in the ovaries of all the mammalian species studied including rat, monkey, pig, mouse and human. they have been extensively studied in the mouse and a number of morphological and functional differences have been described. for example, in sn oocytes most of the centromeres localise around the compact nucleolus, whereas in nsn oocytes only the centromeres of chromosomes carrying the nucleolus organising regions are associated with the nucleolus. microtubule organising centres form around the nucleus of sn oocytes, but not around that of nsn oocytes. these morphological differences have a biological relevance as they have been correlated with changes in transcription. nsn antral oocytes are transcriptionally active and synthesise all classes of rna, whereas sn antral oocytes are transcriptionally inactive. the most striking difference between the two types of oocytes is their developmental competence. when isolated from the mouse ovary, matured to metaphase ii and fertilised in vitro, both types of oocytes are capable of developing to the 2-cell stage, but only miisn oocytes are developmentally competent beyond the 2-cell stage and capable of reaching full term development. in this study we have compared the gene expression profile of developmentally incompetent miinsn oocytes with that of developmentally competent miisn oocytes, with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence. to this end, fully grown nsn and sn antral oocytes were isolated from the ovaries, cultured in vitro to the mii stage and their profiles of gene expression compared. we first focused our investigation on a group of maternal-effect genes whose altered expression leads to preimplantation developmental arrest. then, using microarrays analysis and bioinformatic resources, we identified major groups of genes, gene expression networks and biochemical pathways characteristic of the developmentally incompetent miinsn oocytes.  stella, a maternal-effect protein required for the oocyte-to-embryo transition, is not detected in developmentally incompetent miinsn oocytes our study was first focused on a specific group of gene transcripts and proteins accumulated during the oocyte growth to become necessary, after fertilisation, for successful embryogenesis. these gene products and their effects are well documented in organisms such as drosophila melanogaster or xenopus laevis, but in recent years, increasing evidence has been produced that maternal-effect genes are crucial in pre- and post-implantation development. we analysed the profile of expression of the following five genes, known for playing a role during the oocyte-to-embryo transition: zar npm stella, smarca and prei. the relative number of transcripts of the genes analysed resulted equally represented in the two types of mii oocytes, with the exception of stella that was -fold down-regulated in miinsn oocytes. consistently with the down-regulation of gene expression, immunostaining with an antibody against stella did not detect the presence of the protein either in nsn antral or miinsn oocytes, whilst in sn antral oocytes stella co-localised around the nucleolus and in miisn oocytes was dispersed within the whole ooplasm, as already shown in ovulated mii oocytes. stella down-regulation is a good candidate to explain the developmental block at the 2-cell stage that miinsn oocytes encounter following fertilisation. for this reason,, we wondered what induces the down-regulation of stella in miinsn oocytes and whether other genes could be involved in its regulation. stella is positively regulated by oct- and nanog based on mouse oct- chip-pet data, and it is also down-regulated upon rnai-mediated oct- knockdown in human embryonic stem and embryo carcinoma cells. a recent study has demonstrated a strong link between stella and oct- showing that the expression of stella in mouse es cells is regulated by oct- which is necessary for maintaining a specific chromatin structure within the locus containing stella. the authors observed collapse of higher order chromatin structure throughout the locus and down-regulation of stella expression following depletion of the oct- gene. additional studies using microarray analysis of gene transcription in es cells, have described oct- involvement in the regulation of chromatin modelling and chromatin-mediated transcription regulation. next in our investigation, we addressed the question whether the pattern of oct- gene and protein expression in developmentally incompetent miinsn and competent miisn oocytes, in concert with the knowledge already acquired on this gene, could establish oct- as a key regulator of stella expression during the late phases of oocyte maturation. oct- a chromatin-mediated regulator of stella transcription, is down-regulated in developmentally incompetent miinsn oocytes oct-, a nuclear transcription factor that belongs to the pou family, is involved in the regulation of the expression of a number of developmental genes and is required for the maintenance of cell pluripotency. oct- is expressed throughout folliculogenesis; following fertilisation, it remains of maternal origin until the 2-cell embryo, then at the 4- to 8-cell stage is first expressed by the embryonic genome. in an earlier study, we identified a 2-fold down-regulated expression of oct- in nsn antral oocytes compared to sn antral oocytes. in the present work, we found that down-regulation of oct- expression in miinsn oocytes correlated with a down-regulated expression of its encoded protein as revealed by immunocytochemistry. figure 2m–2r illustrates the immunostaining pattern of the oct- protein in fully grown antral sn and nsn oocytes. while in sn oocytes the protein was intensely present and mostly localised around the nucleolus, in nsn antral oocytes it was undetectable. following in vitro maturation to the mii phase, oct- continued to be absent in miinsn oocytes, whereas miisn oocytes maintained the presence of the transcription factor localised around the mii plate and partially within the cytoplasm. in summary, since a sound experimental evidence has demonstrated that oct- regulates the expression of stella, oct- and stella down-regulation in miinsn oocytes may contribute to the inability of these gametes to develop beyond the 2-cell stage. our results suggest a novel role of maternal oct- in the regulation of the developmental competence of the female gamete. oct-4-dependent transcriptional networks have been described regulating self-renewal and pluripotency in human and mouse es and ec cells and in human mesenchymal cells. these studies demonstrate that oct- may interact with other genes to regulate specific biological process. we next asked the question, is oct- a key regulator of genes associated with or implicated in establishing developmental competence on oocytes? to address this, we first analysed by microarrays, global differences in gene expression between the two types of oocytes, and then searched for genes known to be regulated by oct- in the array-derived list of regulated genes. microarray-based analysis of the transcription profiles of developmentally competent and incompetent mii oocytes the microarray platform that we used allowed the analysis of a total of genes and gene sequences. of these, genes were expressed only in miinsn oocytes; genes were expressed specifically in miisn oocytes and genes were expressed in both types of oocytes. a fold-change cut-off of at least and a p value < was set to filter the genes whose expression was differentially regulated. in the comparison between miinsn and miisn oocytes, we identified a total of regulated genes, of these were up-regulated in miinsn oocytes, and down-regulated. using the gene ontology annotations implemented in the illumina platform and also by david, we identified major biological themes that characterised the group of regulated and annotated genes: transcription regulation, protein biosynthesis, cellular transport, metabolism, development, signal transduction, cell cycle, carbohydrate metabolism, dna metabolism, cytoskeleton organisation, apoptosis, chromatin/nuclear organisation and various go; also, some genes fell into a group with undetermined go. an inherent weakness of the bioinformatic programs available to analyse the patterns of gene expression obtained following microarrays analysis is that they are limited to those genes with assigned annotations or published relationships, and depend on the accuracy of these annotations. the number of down-regulated and up-regulated genes in each of the categories and the list of these genes are shown in figure 3b and additional file respectively. to determine whether and how individual genes are inter-related or interact with each other and to search for biological pathways and the inter-relationships between network genes, we used the ingenuity pathways analysis version analysis tool. the genes of each gene group were uploaded as tab-delimited text files and ipa queried the ingenuity pathways knowledge base for interactions between focus genes and all other objects stored in the knowledge base and generated a set of networks. those networks with a score ≥ were considered for further analyses. genes up-regulated in miinsn oocytes the great majority of the regulated genes was up-regulated. when ipa was performed on the up-regulated focus genes, it generated networks with a score of or higher, of these, had or more focus genes. because a score of or higher has a confidence of at least 99% of not being generated by chance, the high confidence level obtained, particularly in the top nine networks, reveals the strong interrelation and interaction among the genes that are up-regulated in miinsn oocytes. figure in additional file shows the top gene expression networks obtained. the specificity of these networks to the biology of the ovary was confirmed using ipa to enquire public ovarian gene expression data sets. network has focus genes and of these are known to be expressed in ovarian tissue. in networks 2– the percentage of ovarian genes decreased from % to 50%. comparison with data sets of somatic tissues, namely liver, kidney, heart, lung and spleen gave a lower matching. network *: in bold are oct-4-regulated genes. the gene expression networks described were associated to top functions such as gene expression, protein synthesis, cancer, immunological disease, genetic disorder, dermatological disease and reproductive system disease, highlighting the complexity of the different transcriptional profile between developmentally incompetent miinsn and competent miisn oocytes. ipa was then interrogated to obtain a list of biochemical pathways that are representative of the functions in which the genes are involved. the genes up-regulated in miinsn oocytes were assigned to more than pathways; of these were considered statistically significant and the top six, together with the gene expression networks they belong to, are shown in table these six pathways included a total of different genes, the majority of which belonged to network, network and network, and were associated with oxidative phosphorylation, mitochondrial dysfunction, galactose metabolism, purine metabolism, protein ubiquitination and gaba receptor signalling. some of these genes are expressed within different pathways. pathway genes down-regulated in miinsn oocytes ipa analysis of the focus genes down-regulated in miinsn oocytes generated networks with a score ≥. the top functions associated with these gene expression networks include cell cycle, cell death, embryonic development and cancer. when ipa was interrogated to generate the biochemical pathways that were most representative of the functions attributed to the genes involved in the networks, it created ten major pathways represented by genes, of which are associated with network and network. of these genes, atm and mdm are involved in the g2/m dna damage checkpoint regulation. the protein encoded by the prdx gene is a member of the thiol-specific antioxidant protein family, involved in the regulation of phospholipids turnover as well as in the protection against oxidative injury. to identify the inter-relationship among up- and down-regulated genes, we next carried out an ipa analysis of all the regulated genes. genes regulated in miinsn oocytes the ipa of all the regulated genes, documented gene expression networks with a score ≥ of these showed a score ≥. since most of the regulated genes are up-regulated, as expected, the top functions represented by these networks are those already identified with the separated analysis of the up- and down-regulated genes, but it also revealed new categories. in network nf-kb is one of the principal nodes, together with insulin, calmodulin and protein phosphatase 2a. correct expression of nf-kb is required for the development of the mouse preimplantation embryo, as inhibition of its expression blocks development at the 2-cell stage. ppa is one of the four major ser/thr phosphatases implicated in the negative control of cell growth and division. further analysis of the most representative biochemical pathways identified nine top pathways, of which are shown in additional file the most representative pathways were those assigned to protein ubiquitination, oxidative phosphorylation, ubiquinone biosynthesis, g2/m dna damage checkpoint regulation, fructose and mannose metabolism, valine, leucine and isoleucine degradation, attributed mainly to network, network and network. genes expressed solely in miinsn or miisn oocytes the comparison of the expression profiles of miinsn with that of miisn oocytes, resulted in the identification of a small number of focus genes transcribed exclusively by miinsn oocytes or miisn oocytes. three of the miinsn oocyte-specific genes have an assigned go function as negative regulators of cell proliferation or apoptosis, whereas two annotated miisn oocyte-specific genes are involved in processes of positive regulation of the cell cycle. out of focus genes expressed only in miinsn oocytes, were assigned to a single gene expression network, with top functions that included hair and skin development and function, organ morphology and cellular development. the number of miisn-specific genes was so low that it was not considered for further analysis. rt-pcr analysis of the relative number of transcripts of two miinsn oocyte-specific genes and two miisn oocyte-specific genes confirmed the microarrays data and the presence of a different number of transcripts in the two types of oocytes; however, whilst jam and ogfr were shown to be expressed only in miinsn oocytes; crebbp and fnip were not exclusive to miisn oocytes, but instead showed respectively a -fold and -fold, higher number of transcripts in these gametes. the presence of transcripts of crebbp and fnip genes in miinsn oocytes is due to the overt single-cell sensitive of nested-pcr that amplified the low number of transcripts that were not detected by the less sensitive microarray analysis. when the number of the second pcr cycles was reduced to and, expression was not detected in the miinsn oocyte samples. in summary, the results of the microarray analysis as a whole, indicate that developmentally incompetent miinsn oocytes have activated, among others, gene expression networks implicated in the regulation of biochemical pathways representative of the adverse biological status of these oocytes. these pathways include, oxidative phosphorylation, mitochondrial dysfunction and apoptosis. to identify oct- target genes among the list of regulated genes, we compared our list with the published mouse and human chip datasets of oct-4-regulated genes. eighteen oct-4-regulated genes are associated with adverse gene expression networks in developmentally incompetent miinsn oocytes this analysis identified a total of target genes. when comparing miinsn with miisn oocytes, all these genes, with the exception of odz and zfp36l, were up-regulated in miinsn oocytes with fold changes ranging from to. eighteen of these genes were part of gene expression networks to generated from the ipa of up-regulated genes in miinsn oocytes, and thus involved in the activation of the same biochemical pathways described above. when ipa was specifically interrogated with the list of these genes, including oct- it generated networks with a score ≥ of these, the first two contained and focus genes respectively, and had top functions centred around biological themes such as gene expression, cell death, cancer and reproductive system disease. oct- was part of network, which included the tnf pathway, a multifunctional proinflammatory cytokine that acts on several different signalling pathways to regulate apoptosis, nf-kb activation of inflammation, and activation of stress-activated protein kinases. network *: in bold are the oct-4-regulated focus genes. underlined is the oct- gene. interestingly, zfp36l, one of the two oct-4-regulated genes that was down-regulated in miinsn oocytes, is a member of the tis family of early response genes, of which zfp36l another maternal-effect gene crucial for early embryonic development, is also a member. perhaps, zfp36l is a novel maternal-effect gene whose expression is regulated by oct- and whose down-regulation may affect the developmental competence of miinsn oocytes. the microarray analysis revealed a -fold down-regulation of oct- expression in miinsn oocytes. however, this data was not included in the list of regulated genes because its p value was equal to, thus just slightly above the set cut-off limit of p <. to understand how the expression of the oct- gene was related with the adverse gene expression networks that we found activated in miinsn oocytes, we next included the expression data for oct- obtained by microarray analysis into the list of up-regulated or down-regulated genes and performed an ipa analysis. with the group of down-regulated genes, oct- was included in network; with the up-regulated genes, oct- was integrated into network which has top functions as post-translational modification, cancer, genetic disorder and activates adverse pathways such as oxidative phosphorylation and mitochondrial dysfunction, see table and table 2). in both cases the gene fell into the two top networks highlighting the central role it plays. in summary, down-regulation of oct- expression in miinsn oocytes induces the up-regulation of a group of oct-4-regulated genes that are part of the top gene expression networks involved in the activation of adverse pathways, i.e., oxidative phosphorylation, mitochondrial dysfunction and apoptosis. discussion the maternal contribution of transcripts and proteins supplied to the zygote is crucial for the successful progression of preimplantation development. an aberrant oocyte inheritance leads to developmental arrest, mostly at the time of zga. to gain a better understanding of the genes, gene expression networks and biochemical pathways that may be activated/inactivated to compromise the developmental competence of mii oocytes, we compared two types of mouse mii oocytes, one which is developmentally competent and reaches full term development, the other that ceases development at the 2-cell stage. our study began with the analysis of the profile of expression of five maternal-effect genes. we showed that of these, only the expression of stella was mis-regulated and its protein absent in miinsn oocytes. the maternal inheritance of stella is needed for correct preimplantation development. when oocytes from stella-/- females are inseminated with wild-type sperm, while fertilisation proceeds normally, development is severely altered resulting in a progressive decline in the number of blastocysts, with the majority of embryos that do not develop beyond the 2-cell stage. stella encodes for a basic protein with a sap-like domain and a splicing factor-like motif thought to have a role in chromosomal organisation or rna metabolism. stella binds both rna and dna in vitro, supporting its involvement in linking chromatin with rna-related processes, as it is the case for other sap-domain proteins. a recent study has shown that stella protects against inappropriate dna demethylation in the maternal genome and at certain imprinted loci during epigenetic reprogramming after fertilisation. down-regulation of stella, is likely to be partly causal of the developmental arrest at the 2-cell stage of miinsn oocytes. lack of stella protein in miinsn oocytes is a consequence of the down-regulation of oct- which is known to regulate stella expression in es and ec cells. this implies a pivotal role for maternal oct- in the establishment of the developmental legacy of female gametes. the down-regulation of oct-4/stella in nsn gv oocytes compromises their developmental competence. in favour of the hypothesis- "factors present in miisn oocytes, but absent in miinsn oocytes, govern their developmental competence", inoue and collaborators have micromanipulated fully grown sn and nsn antral oocytes to demonstrate that the developmental competence of sn oocytes is dependent on unknown cytoplasmic factor that are released from the germinal vesicle at the time of mii formation and that these ooplasmic factors are not contained in the gv of nsn oocytes. both stella and oct- proteins localise within the germinal vesicle of the sn antral oocytes; following meiosis resumption and germinal vesicle break-down, oct- is circumscribed mainly around the mii-plate area, while stella is released within the ooplasm. the association of oct- with the nucleolus in antral sn oocytes may have a functional significance. it was previously shown that oct- is closely associated with rna polymerase ii in the nucleolus of oocytes; since nlbs are the only coilin-containing structures at the end of pre-ovulatory stages of oogenesis, they may represent sites for the storage of the transcriptional machinery needed after fertilisation and of which oct- may be a key component. this association of oct- with the nlbs may secure a transfer of the maternally inherited protein through the nlb to the zygote, where oct- perhaps continues to be necessary as a regulator of the molecular events leading to zga. our microarray analysis indicate that the role of oct- as a regulator of gene expression in mii oocytes extends beyond the regulation of stella expression and has an inhibitory effect on the expression of oct-4-regulated genes in developmentally competent miisn oocytes. down-regulation of oct- in miinsn oocytes correlates with the up-regulation of oct-4-regulated genes involved in the activation of gene expression networks and biochemical pathways that most represent the negative legacy of miinsn oocytes. these pathways include oxidative phosphorylation, mitochondrial dysfunction and cell death. following fertilisation, these negative features might be passed on to the zygote and constitute a detrimental maternal legacy that affects further development. oxidative phosphorylation and mitochondrial dysfunction are biochemical pathways principal to the first two top gene expression networks described for the group of up-regulated genes, which emphasises the central role that these genes and mitochondria play in the biochemistry of the developmentally incompetent miinsn oocytes. in addition to producing most of the cell's atp, recent studies have demonstrated that mitochondria have a central role in cellular processes such as the regulation of intracellular redox potential and control of apoptosis in oocytes and preimplantation embryos. accumulation of reactive oxygen species induces apoptosis in mouse zygotes, and mitochondria are involved in the early phase of oxidative stress-induced apoptosis, that leads to developmental arrest and alterations of the zga. also, changes in the levels of ros regulate a number of transcription factors critical in early development, these include pebp ap- p and nf-kb. the two-fold role for mitochondria, to maintain life or to commit to cell death, may represent a quality control system in the female gamete and in the early embryo that will determine whether the embryo proceeds further into development or is quickly eliminated. if down-regulation of oct- activates these negative pathways, what does upstream of oct- regulate its expression? we have previously shown an effect of pmsg on oct- gene expression during folliculogenesis, demonstrating a hormonal regulation of oocyte-specific gene expression. pmsg, which was used in this study to prime females before oocytes isolation, may enter the gamete with the involvement of a fsh-r signaling pathway that, through the modulation of c-kit on the surface of oocytes, conveys the gonadotropin signal into the nucleus and then act upon the transcription of several genes. one candidate could be gcnf, a member of the nuclear receptor family, critical in controlling basal activities or hormonal responsiveness of numerous target genes and known to repress oct- expression. gcnf may be a target of pmsg action that leads to the down-regulation of oct- expression in miinsn oocytes. this hypothesis needs to be tested, however, it is interesting that in our microarray analysis, gcnf was -fold up-regulated in miinsn, although, because of its p value >, it was excluded from the list of regulated genes.  in summary, our study of developmentally competent and incompetent mouse mii oocytes has identified a small number of genes, gene expression networks and biochemical pathways whose activation is critical for the correct progression of the early stages of preimplantation development and regulate the 2-cell block in miinsn oocytes. our data constitute a framework for further exploration of the genes and related transcriptional networks dedicated to establishing and preservating the developmental competence of mammalian oocytes. based on our results, we have drawn a working hypothesis that assigns a role to some of the molecular players that have been described in this and other studies. the use of a model study in which the mii oocyte ceases development consistently at the 2-cell stage has allowed to illustrate a role for the maternal oct- that has never been described before. oct- emerges as a key regulator of the molecular processes that govern the establishment of the maternal legacy of mii oocytes required for the transition from a gametic to an embryonic control of development. a sound knowledge of the orchestrating molecules and the biochemical pathways entailed during the very early stages of development will allow a better understanding of the molecular mechanism that determine the block of development at the time of zga, which in humans is the cause of a loss of a high percentage of embryos obtained by in vitro fertilisation. a thorough functional analysis of these genes in preimplantation embryos derived from miinsn and miisn oocytes is our next challenge. 
the maternal contribution of transcripts and proteins supplied to the zygote is crucial for the progression from a gametic to an embryonic control of preimplantation development. oct- emerges as a key regulator of the molecular events that govern the establishment of the developmental competence of mouse oocytes. we report that: 1) the transcription factor oct- is absent in miinsn oocytes, accounting for 2) the down-regulation of stella, a maternal-effect factor required for the oocyte-to-embryo transition and of which oct- is a positive regulator; 3) eighteen oct-4-regulated genes are up-regulated in miinsn oocytes and are part of gene expression networks implicated in the activation of adverse biochemical pathways such as oxidative phosphorylation, mitochondrial dysfunction and apoptosis. here we compared the transcriptional profiles of two types of mouse mii oocytes, one which is developmentally competent, the other that ceases development at the 2-cell stage, with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence. the use of a model study in which the mii oocyte ceases development consistently at the 2-cell stage has allowed to attribute a role to the maternal oct- that has never been described before. the down-regulation of oct- plays a crucial function in a sequence of molecular processes that leads to the developmental arrest of miinsn oocytes.
 the maternal contribution of transcripts and proteins supplied to the zygote is crucial for the progression from a gametic to an embryonic control of preimplantation development. here we compared the transcriptional profiles of two types of mouse mii oocytes, one which is developmentally competent, the other that ceases development at the 2-cell stage, with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence.  we report that: 1) the transcription factor oct- is absent in miinsn oocytes, accounting for 2) the down-regulation of stella, a maternal-effect factor required for the oocyte-to-embryo transition and of which oct- is a positive regulator; 3) eighteen oct-4-regulated genes are up-regulated in miinsn oocytes and are part of gene expression networks implicated in the activation of adverse biochemical pathways such as oxidative phosphorylation, mitochondrial dysfunction and apoptosis.  the down-regulation of oct- plays a crucial function in a sequence of molecular processes that leads to the developmental arrest of miinsn oocytes. the use of a model study in which the mii oocyte ceases development consistently at the 2-cell stage has allowed to attribute a role to the maternal oct- that has never been described before. oct- emerges as a key regulator of the molecular events that govern the establishment of the developmental competence of mouse oocytes. 