BACKGROUND
the activation of molecular pathways underlying oocyte to embryo transition  depends exclusively on maternal rnas and proteins accumulated during growth of the oocyte  <cit> .
during oet and preimplantation development in mice, the embryo becomes almost autonomous, and may gradually eliminate maternal components.
indeed, by the two cell stage, the major pathways regulated by maternal mrna are targeted protein degradation, translational control and chromatin remodelling  <cit> .
the recruitment of maternal mrna for translation has long been recognized as a widespread mechanism to generate newly synthesized proteins in maturing oocytes and fertilized eggs  <cit> .
conversely, rna that is no longer needed is actively degraded in the early embryo  <cit> .
moreover, careful regulation of proteolysis during the same period is likely to be important in oocytes, which are predominantly transcriptionally inactive and must often wait for long periods before fertilization in different species such as drosophila, xenopus, caenorhabditis and zebrafish  <cit> .
maternal transcripts that are present in the early pre-implantation embryo can be subdivided into two classes according to whether they are re-synthesized soon after embryonic genome activation or not.
the first is common to the oocyte and early embryo and is replenished after activation of the zygotic genome.
the second consists of oocyte-specific mrna that is not subsequently transcribed from zygotic genes in the embryo.
this class of mrna may be detrimental to early post-fertilization development  <cit> .
maternal effect genes have been found in several species ranging from invertebrates to mammals.
wide screening of mutants has been performed in invertebrates as drosophila melanogaster  <cit>  and caenorhabditis elegans  <cit>  where several mutations lead to arrest of early embryo development.
although females bearing this type of mutation are viable and appear to be normal, the development and survival of their embryos are compromised  <cit> .
maternal effect mutations have also been described in other vertebrates such as danio rerio for the nebel gene  <cit> , and xenopus laevis for the af gene  <cit> .
despite the fact that maternal effect mutations are well known in lower organisms, only a few examples have been reported in mammals.
all of them are based on knock-out experiments and concern three murine genes, i.e.
dnmt <dig>  hsf <dig> and mater  <cit> .
mater  is a single-copy gene that is transcribed in growing oocytes.
although its transcripts are degraded during meiotic maturation, mater protein persists into the blastocyst.
female mice lacking this  <dig> kda cytoplasmic protein produce no offspring because of an embryonic block at the early cleavage stage.
thus, mater is one of few documented genes for maternal effect in mammalian development  <cit> .
mater has been found in bovine models but there is no report in the literature on maternal effect genes conserved between species.
no information has been available to date on maternal effect genes in birds.
however, birds represent a good model to observe progressive accumulation of mrna in the oocyte before ovulation.
the embryonic genome of a model bird, i.e.
the chicken, is activated when the embryo contains  <dig> – <dig>  cells  <cit>   <dig> h after fertilization.
proteins and mrna, accumulated as the chicken oocyte matures, are essential not only for fertilization and first cleavage but also for supporting a high number of embryonic cell divisions before genome activation.
by comparison, the embryonic genome is activated at the 8-cell stage in bovines  <cit>  and at the 2-cell stage in the mouse  <cit> .
the avian oocyte consists of a large amount of yolk and a structure called the germinal disc   <cit> .
the gd is a white plaque of about 3– <dig> mm diameter on the top of the oocyte.
it contains the nucleus and 99% of oocyte organelles although it occupies less than 1% of the cell volume  <cit> .
structurally, and therefore functionally, the gd is mostly equivalent to the mammalian oocyte.
the ovary of the reproductively active hen consists of small pre-hierarchical follicles and maturing preovulatory follicles showing a hierarchy according to size   <cit> .
only a few studies have reported on gene expression in the oocyte and during early embryo development in the chicken.
the dynamics of the overall rna profile of the chicken oocyte through different maturation stages has been described by olzanska et al.
<cit> .
chicken vasa homolog protein  was hypothesized to be maternally inherited in the chicken embryo, since it has been localized in chicken oocytes and during first cleavage  <cit> .
another protein, epidermal growth factor, was found in f <dig> gd and its potential role in follicular development has also been investigated  <cit> .
since oocyte-specific genes expressed during follicular maturation and after ovulation are potentially involved in the fertilization process and in early embryo development, and almost no information is available on these genes in birds, the aim of this study was to identify avian oocyte-specific genes and then to investigate the temporal and spatial dynamics of their expression during follicular maturation and early embryogenesis.
we chose initially to focus on oocyte-specific genes because the accumulation of their transcripts in the oocyte should have greater consequences on fertilization and oet.
two different strategies were used to identify avian genes potentially involved in oocyte developmental competence.
the first was based on a candidate gene approach and consisted of a search for avian homologs of murine oocyte genes, previously identified by digital differential display  <cit> .
the second strategy involved a global transcriptomic approach based on chicken affymetrix microarray.
we report here several novel chicken genes with potential maternal effect identified using these two strategies.
we also describe the spatial and temporal dynamics of the expression of some of these genes as well as some potential mechanisms in which they could be involved.
we also compare chicken and murine orthologs in terms of their tissue specificity and their potential involvement in oocyte developmental competence and/or early embryogenesis.
RESULTS
in silico search for chicken homologs of murine oocyte genes
differential digital display analysis performed on murine tissues provided a list of  <dig> potentially oocyte-specific murine genes  <cit> .
bioinformatic analyses were performed on this list of genes in order to find potentially oocyte-specific chicken orthologs.
genes with a blast score higher than  <dig> were localized using mapview  <cit>  and blatsearch  <cit>  tools.
the syntenic regions were checked: chromosome localization of murine genes and chicken homologs were compared, in order to obtain the correct chicken ortholog of mice genes.
only genes with sufficient homology or whose localization was in accordance with syntenic regions were selected.
forty-one chicken genes were eliminated because of their poor homology with murine genes and  <dig> other genes were eliminated because they were localized outside the syntenic regions.
among the remaining  <dig> chicken genes the transcript of only  <dig> genes could be correctly amplified using real time or classic rt-pcr, of which the detailed study of two genes bmp <dig> and gdf <dig> has previously been reported  <cit> .
thus  <dig> avian genes were finally retained .
the homology with murine genes was strong for  <dig> of these genes  and was weak for  <dig> .
the last five chicken genes were nevertheless considered as potential orthologs of murine genes and kept as candidate genes because of their correct localizations with respect to the syntenic region.
eleven of the selected genes were localized in the expected syntenic region .
three other genes were localized in the vicinity of the expected syntenic region .
two genes were localized in the unexpected syntenic region, but they were identified with the same name as murine genes .
comparing oocyte and granulosa cells transcription profiles at final maturation steps using chicken affymetrix microarrays
the samples studied were: f <dig> gcs, corresponding to granulosa cells  of the largest follicles before ovulation ; f <dig> gdr, corresponding to the germinal disc region  of f <dig> follicles, and ov gdr, corresponding to the germinal disc region of ovulated oocytes  .
apart from the stage of maturation the main difference between these samples involved the presence of granulosa cells.
they were not present in gdr from ovulated oocytes, they were slightly present in f <dig> gdr, and they constituted a major component of f <dig> gcs .
statistical analysis of data obtained after affymetrix microarray hybridization provided lists of genes differentially expressed in three comparisons: f <dig> gdr and ov gdr, f <dig> gdr and f <dig> gcs, and ov gdr and f <dig> gcs .
there were only a few differentially expressed genes in each comparison  .
indeed, the first comparison, between the f <dig> gdr and ov gdr, showed  <dig> genes over-expressed at the f <dig> stage, including one of our in silico identified genes .
in the second comparison, between f <dig> gdr and f <dig> gcs,  <dig> differentially expressed genes were identified.
these genes involved  <dig> genes over-expressed in f <dig> gcs and  <dig> genes over-expressed in f <dig> gdr.
five of our in silico identified genes btg <dig>  chkmos, dazl, zpa and ktfn were found among the latter genes over-expressed in f <dig> gdr .
the third analysis compared the expression of genes between f <dig> gcs and ov gdr.
we obtained a set of  <dig> genes that were differentially expressed between f <dig> gcs and ov gdr, of which  <dig> genes were over-expressed in f <dig> gcs and  <dig> genes were over-expressed in ov gdr.
we found  <dig> of our in silico identified genes among these genes,  .
the venn diagram  shows overlapping differentially expressed genes between different dataset comparisons.
eighty-five differentially expressed genes were common for two comparisons .
only one gene was common for the comparisons between f <dig> gdr and f <dig> gcs, and f <dig> gdr and ov gdr and  <dig> gene was common for all three comparisons.
f <dig> gdr samples contained a quantity of granulosa cells in contrast to ov gdr samples which were free of granulosa cells.
analysis of the redundancy of over-expressed genes between different comparisons was therefore performed in order to distinguish between genes found over-expressed in f <dig> gdr samples due to granulosa cell contamination and those really over-expressed in the oocyte .
this analysis revealed that  <dig> of  <dig> genes over-expressed in f <dig> gdr were also over-expressed in f <dig> gcs, both compared to ov gdr, indicating that these genes characterized granulosa cell expression rather than variation in the expression between f <dig> gd and ov gd.
thus only  <dig> genes should be considered as overexpressed in f <dig> gd compared to ov gd.
moreover, of the  <dig> genes overexpressed in f <dig> gcs compared to f <dig> gdr,  <dig> were also overexpressed in f <dig> gcs compared to ov gdr.
consequently, taking into account the latter redundancy, a total of  <dig> genes was overexpressed in granulosa cells compared to oocytes.
on the other hand  <dig> genes were redundant among genes overexpressed in the ov gdr and f <dig> gcs, and f <dig> gdr and f <dig> gcs comparisons.
based on go annotation the genes upregulated in gcs were mostly related to metabolic processes, transport, proteolysis, regulation of transcription, immune response and cell adhesion, whereas genes preferentially expressed in gdr were preferentially involved in cell cycle, chromosome organization, phosphorylation of proteins, regulation of transcription, multicellular organism development and dna metabolic processes .
tissular pattern of gene expression
on the basis of the in silico and microarray approaches,  <dig> genes were retained for further study.
among these,  <dig> genes were found using the in silico approach as described above , of which  <dig> genes  were also found using the microarray approache  and  <dig> gene  was found only in the microarray approach.
the latter gene was chosen because of its strong involvement in the fertilization process  <cit> .
in contrast to zpa and zpc, the zpd gene was not found in the mouse  <cit> , explaining why it was absent from the list of murine potentially oocyte-specific genes.
four other genes already known for their involvement in final follicular maturation or early embryo development in the chicken or in other vertebrates were added  .
finally  <dig> chicken genes were further studied.
real time rt-pcr performed on  <dig> adult tissue samples  revealed differences in the specificity of their tissular expression patterns.
tissular expression profiles of some of these genes are presented on fig.
<dig>  seven of these genes  were specifically expressed in the ovary .
the specificity of zpc and chkmos has previously been described and our results concerning these genes  were in accordance with the literature .
eight genes were preferentially and strongly expressed in the ovary .
three genes from last group were slightly expressed in another tissue: fbox and zpd in the pituitary gland and ktfn in the muscle.
we also confirmed preferential ovarian expression of cvh  <cit>  and foxl <dig>  <cit> , as well as a low expression of the latter in the pituitary gland .
three other genes from this group  were slightly expressed in other tissues, in addition to the ovary.
the last  <dig> genes  were expressed as highly in the ovary as in other tissues .
rna state during follicular maturation and early embryo development
analysis of total rna, assessed with agilent rna nano chips , showed an atypical state of rna from gdr between ovulation and oviposition.
in our conditions, rrna seemed degraded from ovulation until oviposition.
with three different rna extraction methods , rneasy kit  and masterpure™ rna purification kit ), the rna profile of gdr from the ovulation stage and from the following embryonic stages remained degraded .
moreover, we assessed rrna 18s and 28s by real time pcr in order to confirm this atypical rna state .
these results confirmed our previous observations; 18s and 28s rrna expression showed a huge decrease from ovulation untill the oviposition.
we then performed labelled reverse transcription to investigate whether mrna was also degraded.
we compared three samples, i.e.
the rna from gdr of f <dig> stage, from a whole adult ovary that had a normal rrna profile and gdr of ovulation stage with had degraded rrna.
electrophoresis profiles of labelled cdna in denaturing agarose gel were almost identical for the three samples investigated and the smear corresponding to the reverse transcribed mrna was still present in all these samples .
gene expression during follicular maturation and early embryo development
a detailed study of the expression profiles during follicular maturation and early embryo development using real time rt-pcr was performed for  <dig> genes, for which tissue specificity of the expression was characterized.
first unsupervised hierarchical clustering of our data was performed in order to confirm the biological appropriateness of the selected genes and samples .
two other previously studied genes  were added to this analysis in order to facilitate the clustering process because of their already known oocyte-preferential localization  <cit> .
this analysis discriminated two major groups of samples.
the first included all granulosa samples from f <dig> to f <dig> .
the second group corresponded to gdr from f <dig> to the ovulation stage and to all embryo stages .
several subgroups could be distinguished within each group .
each subgroup corresponded to a different physiological state, indicated on fig 6a, suggesting that the genes selected were pertinent.
moreover, unsupervised clustering arranged samples in the perfect chronological order.
in order to group genes with similar expression patterns supervised clustering was performed with samples arranged first according to the nature of the sample  and then according to chronological order .
five clusters  were identified .
a representative example of the expression profile characterizing each cluster is shown on fig.
<dig>  the graphical representation of expression profiles of all other genes with the corresponding statistical analysis of variation of their expression is supplied in additional files .
the chkmos, btg <dig>  wee, zpa, dazl, hsf <dig>  fbox and bmp <dig> genes, forming cluster c <dig>  were preferentially expressed in gdr.
these genes showed a significant increase in mrna expression in gdr during follicular maturation and a steady decrease after ovulation, becoming nearly undetectable at  <dig> h after fertilization.
the cvh, ktfn and zar <dig> genes included in cluster c <dig> were also preferentially expressed in gdr but, in contrast to genes from cluster c <dig>  they were constantly expressed in the early embryo.
moreover, ktfn and zar <dig> genes displayed a significant increase in expression from  <dig> to  <dig> hours after fertilization.
the genes from cluster c <dig> were expressed in gcs, in gdrs and in the embryo.
moreover, mark <dig>  igf <dig>  gdf <dig> and transfact <dig> genes showed a significant decrease in expression during the last stages of follicular maturation in both gdr and gcs.
cluster c <dig>  including discs <dig>  znfingerriz, foxl <dig> and mtprd genes, was characterized by expression that was fairly similar to that of cluster c <dig>  the difference consisted of less pronounced variations in gene expression in gcs and more pronounced variations in gene expression in gdr for cluster c <dig> compared to genes in cluster c <dig> through the stages investigated.
genes belonging to cluster c <dig> were expressed in gcs and gdr but, in contrast to the clusters c <dig> and c <dig>  their expression dropped dramatically at ovulation, especially for zpc and zpd whose transcripts showed increasing expression in gcs during the last stages of follicular maturation and a less marked increase in the expression in gdr.
in contrast, the levels of mcmip decreased progressively during the same period.
the last gene  was not clustered.
its expression profile was fairly similar to that of genes belonging to cluster c <dig>  but its expression increased significantly  <dig> h after fertilization.
localization of gene expression in the ovary by in situ hybridization
we performed in situ hybridization on mature and immature ovarian sections.
all the probes were assessed on the two stages, but only the most significant results are shown on fig.
<dig>  for all the genes studied we detected homogeneous signals of mrna expression in oocytes similar to those we have previously described for bmp <dig> and gdf <dig>  <cit> .
the mrna of four of eight genes localized by in situ hybridization  were detected with a high intensity signal in the oocytes from follicles of  <dig> μm –  <dig> μm from both immature  and mature  ovaries.
no significant expression was detected in somatic cells.
for four other genes  a signal was found in oocytes from the largest follicles of  <dig> μm- <dig> mm of mature ovaries .
the signal was particularly high for chkmos and zpa , whereas zpc and especially zpd had weaker signals in both oocyte and somatic cells .
no significant signal was detected with the corresponding sense probes.
discussion
in the present study we identified and characterized for the first time several genes expressed in the chicken oocyte during follicular maturation and/or in early embryo.
moreover, we showed that our candidate gene approach and microarray approach were complementary in finding new avian genes potentially involved in follicular maturation and/or early embryo development.
five genes preferentially and highly expressed in the oocyte  were identified using both microarray analysis and digital differential display on murine genes.
moreover,  <dig> genes , identified as overexpressed in gcs by microarray analysis, were also confirmed by real time pcr analysis to be highly preferentially expressed in chicken gcs compared to gdr.
microarray analysis identified a total of  <dig> genes upregulated in the hen f <dig> and ovulated oocytes both compared to f <dig> gcs.
among these,  <dig> overexpressed genes were common to ov gdr and and f <dig> gdr, both compared to f <dig> gcs, and therefore represent particular interesting candidate oocyte genes for further exploration of their potential role in oocyte maturation, fertilization and oet.
the fact that we found almost five times fewer genes overexpressed in the oocyte at the ovulation stage  than in the oocyte at f <dig> stage , compared to granulosa cells from f <dig> follicles, means that for some genes mrna expression in the oocyte decreased between f <dig> and ovulation stages.
this change in the mrna expression levels between f <dig> and ovulated oocytes is probably insufficient to be detected in the comparison between ov gdr and f <dig> gdr by microarray hybridization, since only  <dig> differentially expressed genes were identified in this comparison.
on the other hand microarray analysis enabled us to detect the presence of gcs in f <dig> gdr samples, revealed by the redundant overexpressed genes in f <dig> gdr and in f <dig> gcs compared to ov gdr.
the functions of overexpressed genes in the various comparisons according to go categories revealed clear differences between gcs and mature oocytes.
for gcs overexpressed genes these functions were mostly related to metabolic processes, transport, proteolysis, regulation of transcription, immune response and cell adhesion, whilst for the mature oocyte they were mostly related to cell cycle, chromosome organization, phosphorylation and dephosphorylation of proteins, multicellular organism development and dna metabolic process.
these presumed functions of genes overexpressed in the oocyte are consistent with the physiological processes that it must undergo: i.e.
fertilization, cleavage, chromatin remodeling, and supporting early embryo development.
the use of both bioinformatics and microarray approaches provided information on the molecular mechanisms through which oet is driven in the hen.
the expression of several oocyte-specific genes increased during final follicular maturation, suggesting that transcription was still effective.
after ovulation, despite the fact that 18s and 28s ribosomal rna subunits were degraded, we showed by both labelled reverse transcription and microarray analysis that the integrity of mrna was almost unaffected.
in fact, mrna levels were nearly the same for many genes because only  <dig> of  <dig> genes were differentially expressed between gdrs before and after ovulation, of which only  <dig> really corresponded to the oocyte genes.
the high number of replicates performed and the different extraction methods used strongly indicate that the difference in quality between oocyte rrna and mrna after ovulation is not the artifact of the experiment but reflects a real physiological feature of chicken oet, consisting probably of the arrest and degradation of the oocyte translational machinery.
it could thus be hypothesized that the maternal translational system has to be replaced by the embryonic translational system.
indeed, the maternal ribosome in the embryo must be degraded before activation of the genome, in other words, before the beginning of transcription, and translation, when new embryonic ribosomes are required.
this suggests that, because there are no maternal ribosomes at the stage between ovulation and oviposition, there is probably no translation or only translation of a few specific genes.
if this is the case, maternal proteins should be the major essential components that support early embryo development after fertilization.
this is supported by the fact that, based on go annotation, a considerable number of the genes overexpressed in the mature oocyte are related to protein phosphorylation.
further investigation is required onto whether accumulated proteins have such an important role during these early stages of development in birds or if de novo protein synthesis still occurs and is dependent on the oocyte pool of ribosomes as in mammals  <cit> .
on the basis of in silico and microarray approaches and analysis of the literature,  <dig> chicken genes were chosen in this study for further investigation of their expression using real time pcr and in situ hybridization.
all these genes showed state-specific and/or cell type-specific expression patterns throughout the period, beginning from the first stages of final follicular maturation until embryonic genome activation.
this suggests that these genes have different functions and have a role at the different physiological stages investigated.
the observed decrease in the mrna expression of almost all genes studied between ovulation and oviposition, which corresponds to late genome activation in chicken embryo  <cit> , is consistent with a potential arrest of transcription and progressive maternal mrna degradation occurring during meiotic maturation  <cit> .
however, the rate of maternal mrna degradation in the chicken seems to be considerably lower than that of rrna, as demonstrated by the present study.
five genes  belonging to cluster c <dig>  were no longer expressed after activation of the embryonic genome, and thus transcripts of these genes are only maternally inherited.
moreover, chkmos, btg <dig> and wee genes are known to be involved in the cell cycle in other species.
in our study these maternally inherited genes were increasingly expressed during follicular maturation and thus are probably used during the last stages of final follicular maturation and/or early embryo development.
chkmos is the chicken homolog of mos  <cit> , protein kinase required for meiotic maturation in vertebrates  <cit>  and for mitosis in xenopus laevis  <cit> .
meiotic maturation is brought about by steroids using redundant pathways involving synthesis of mos, which regulates the activity of mpf .
the mos-mapk pathway has long been implicated in the arrest of mitosis in vertebrate eggs  <cit> .
the b cell translocation gene  <dig>  belongs to a family of cell-cycle inhibitors.
in the mouse and bovine, btg <dig> is preferentially expressed in the oocyte  <cit>  where it exerts a marked antiproliferative activity,  <cit> .
wee is a conserved gene from invertebrates to mammals and regulates meiotic maturation during oocyte development  <cit> .
the transcripts of dazl are also maternally inherited in the medaka embryo  <cit> , and in adult medaka fish the expression of dazl was detected exclusively in the ovary and in the testis  <cit> .
zpa, zpc and zpd, that belong to the zp  gene family, are known to be involved in oogenesis, fertilization and preimplantation development  <cit> .
in our study the expression pattern of zpa was different from that of zpd and zpc using both real time pcr analysis, where they were distributed in different clusters , and in situ hybridization analysis.
in contrast to chicken zpd and zpc, which were expressed in oocytes and somatic cells, chicken zpa was found to be specific to the oocyte, as is the case in the mouse  <cit> , and expressed earlier than zpd and zpc.
in the mouse the expression of zpa also precedes that of zpc  <cit> .
indeed in our in situ hybridization experiments zpa expression detected in small follicles of the mature ovary was oocyte-specific, whereas that of zpc and zpd was weaker and found in oocytes and in the somatic cells of the same follicles.
real time rt-pcr, showed increasing expression of zpc and zpd in both gcs and gdr from f <dig> to f <dig>  our finding on the dynamics of zpc in gcs are in accordance with a previously reported study  <cit> .
moreover at the f <dig> stage both zpc and zpd were significantly more highly expressed in gcs than in gd and this was consistent with our results for microarray hybridization, but expression decreased dramatically after ovulation.
in contrast to the cellular expression pattern found for chicken zpc, in several mammals zpc  is specifically expressed in the oocyte.
however, in the equine species, zpc protein synthesis is completely taken over by cumulus cells  <cit> .
these findings indicate species specificity of zpc distribution inside the follicle.
zpc protein plays a crucial role in the fertilization process in mammals and birds,  <cit> .
as zpa other genes  were preferentially expressed in the oocyte and might play role in fertility  or in germ cell specification .
both zar <dig> and ktfn were expressed at higher levels after activation of the embryonic genome.
we could therefore hypothesize that these genes might be involved not only in oocyte maturation, but also in early embryo development, just after maternally inherited genes.
of these  <dig> genes, only zar <dig> has been studied in reproduction.
it is one of the few known oocyte-specific maternal-effect genes essential for oet in mice.
in mammals and humans it is hypothesized to be involved in the initiation of embryo development and fertility control  <cit> .
cvh protein has been previously proposed to be a part of the mechanism for germ cell specification in birds  <cit> .
our results concerning the spatio-temporal expression of cvh mrna during follicular maturation and early embryo development are consistent with previously reported studies on the cvh protein.
the genes belonging to clusters c <dig> and c <dig> were all preferentially expressed in the ovary in both gcs and gdr and had quite similar expression patterns.
except for foxl <dig>  their expression declined during follicular maturation in gcs and less in gdr and persisted at low levels in the early embryo.
this suggests that they are especially involved in the first stages of final follicular maturation as well as in oocyte maturation.
the chicken homolog of the mouse par-1a-like gene, i.e.
mark <dig>  is required for oocyte differentiation and microtubule organization in the drosophila  <cit> , and its role in cell polarity and wnt signaling is conserved from invertebrates to mammals  <cit> .
the expression of another gene , which did not belong to any cluster, also decreased during follicular maturation in gcs and in gd but, as for zar <dig> and ktfn, it showed a significant increase after embryonic genome activation.
msh <dig> is known to be involved in mediating recombination of homologous chromosomes and dna mismatch repair in the mouse  <cit> .
these events occur during the meiotic prophase, the stage where oocytes are blocked for a long time before meiotic maturation.
CONCLUSIONS
in conclusion, the findings of the present study on spatio-temporal expression of  <dig> chicken oocyte specific genes  were consistent with our hypothesis that oocyte-specific genes in the chicken should play a major role in oocyte maturation, fertilization and early embryo development as in the mouse  <cit> .
other genes, whose mrna expression were found in our study to be more specific for gcs or detected in both gcs and gdr depending on the stage, seem to be involved in follicular maturation  and fertilization  rather than in early embryo development.
moreover, the microarray approach provided allowed the discovery of a set of new potential chicken mature oocyte and chicken granulosa cell markers for future studies.
interestingly, 40% of these genes had no homologs in the gene databases and some of them probably correspond to specific chicken mechanisms such as hierarchical follicular maturation or rapid yolk accumulation.

 the activation of molecular pathways underlying oocyte to embryo transition depends exclusively on maternal rnas and proteins accumulated during growth of the oocyte. during oet and preimplantation development in mice, the embryo becomes almost autonomous, and may gradually eliminate maternal components. indeed, by the two cell stage, the major pathways regulated by maternal mrna are targeted protein degradation, translational control and chromatin remodelling. the recruitment of maternal mrna for translation has long been recognized as a widespread mechanism to generate newly synthesized proteins in maturing oocytes and fertilized eggs. conversely, rna that is no longer needed is actively degraded in the early embryo. moreover, careful regulation of proteolysis during the same period is likely to be important in oocytes, which are predominantly transcriptionally inactive and must often wait for long periods before fertilization in different species such as drosophila, xenopus, caenorhabditis and zebrafish. maternal transcripts that are present in the early pre-implantation embryo can be subdivided into two classes according to whether they are re-synthesized soon after embryonic genome activation or not. the first is common to the oocyte and early embryo and is replenished after activation of the zygotic genome. the second consists of oocyte-specific mrna that is not subsequently transcribed from zygotic genes in the embryo. this class of mrna may be detrimental to early post-fertilization development. maternal effect genes have been found in several species ranging from invertebrates to mammals. wide screening of mutants has been performed in invertebrates as drosophila melanogaster and caenorhabditis elegans where several mutations lead to arrest of early embryo development. although females bearing this type of mutation are viable and appear to be normal, the development and survival of their embryos are compromised. maternal effect mutations have also been described in other vertebrates such as danio rerio for the nebel gene, and xenopus laevis for the af gene. despite the fact that maternal effect mutations are well known in lower organisms, only a few examples have been reported in mammals. all of them are based on knock-out experiments and concern three murine genes, i.e. dnmt hsf and mater. mater is a single-copy gene that is transcribed in growing oocytes. although its transcripts are degraded during meiotic maturation, mater protein persists into the blastocyst. female mice lacking this kda cytoplasmic protein produce no offspring because of an embryonic block at the early cleavage stage. thus, mater is one of few documented genes for maternal effect in mammalian development. mater has been found in bovine models but there is no report in the literature on maternal effect genes conserved between species. no information has been available to date on maternal effect genes in birds. however, birds represent a good model to observe progressive accumulation of mrna in the oocyte before ovulation. the embryonic genome of a model bird, i.e. the chicken, is activated when the embryo contains – cells h after fertilization. proteins and mrna, accumulated as the chicken oocyte matures, are essential not only for fertilization and first cleavage but also for supporting a high number of embryonic cell divisions before genome activation. by comparison, the embryonic genome is activated at the 8-cell stage in bovines and at the 2-cell stage in the mouse. the avian oocyte consists of a large amount of yolk and a structure called the germinal disc. the gd is a white plaque of about 3– mm diameter on the top of the oocyte. it contains the nucleus and 99% of oocyte organelles although it occupies less than 1% of the cell volume. structurally, and therefore functionally, the gd is mostly equivalent to the mammalian oocyte. the ovary of the reproductively active hen consists of small pre-hierarchical follicles and maturing preovulatory follicles showing a hierarchy according to size. only a few studies have reported on gene expression in the oocyte and during early embryo development in the chicken. the dynamics of the overall rna profile of the chicken oocyte through different maturation stages has been described by olzanska et al.. chicken vasa homolog protein was hypothesized to be maternally inherited in the chicken embryo, since it has been localized in chicken oocytes and during first cleavage. another protein, epidermal growth factor, was found in f gd and its potential role in follicular development has also been investigated. since oocyte-specific genes expressed during follicular maturation and after ovulation are potentially involved in the fertilization process and in early embryo development, and almost no information is available on these genes in birds, the aim of this study was to identify avian oocyte-specific genes and then to investigate the temporal and spatial dynamics of their expression during follicular maturation and early embryogenesis. we chose initially to focus on oocyte-specific genes because the accumulation of their transcripts in the oocyte should have greater consequences on fertilization and oet. two different strategies were used to identify avian genes potentially involved in oocyte developmental competence. the first was based on a candidate gene approach and consisted of a search for avian homologs of murine oocyte genes, previously identified by digital differential display. the second strategy involved a global transcriptomic approach based on chicken affymetrix microarray. we report here several novel chicken genes with potential maternal effect identified using these two strategies. we also describe the spatial and temporal dynamics of the expression of some of these genes as well as some potential mechanisms in which they could be involved. we also compare chicken and murine orthologs in terms of their tissue specificity and their potential involvement in oocyte developmental competence and/or early embryogenesis.  in silico search for chicken homologs of murine oocyte genes differential digital display analysis performed on murine tissues provided a list of potentially oocyte-specific murine genes. bioinformatic analyses were performed on this list of genes in order to find potentially oocyte-specific chicken orthologs. genes with a blast score higher than were localized using mapview and blatsearch tools. the syntenic regions were checked: chromosome localization of murine genes and chicken homologs were compared, in order to obtain the correct chicken ortholog of mice genes. only genes with sufficient homology or whose localization was in accordance with syntenic regions were selected. forty-one chicken genes were eliminated because of their poor homology with murine genes and other genes were eliminated because they were localized outside the syntenic regions. among the remaining chicken genes the transcript of only genes could be correctly amplified using real time or classic rt-pcr, of which the detailed study of two genes bmp and gdf has previously been reported. thus avian genes were finally retained. the homology with murine genes was strong for of these genes and was weak for. the last five chicken genes were nevertheless considered as potential orthologs of murine genes and kept as candidate genes because of their correct localizations with respect to the syntenic region. eleven of the selected genes were localized in the expected syntenic region. three other genes were localized in the vicinity of the expected syntenic region. two genes were localized in the unexpected syntenic region, but they were identified with the same name as murine genes. comparing oocyte and granulosa cells transcription profiles at final maturation steps using chicken affymetrix microarrays the samples studied were: f gcs, corresponding to granulosa cells of the largest follicles before ovulation; f gdr, corresponding to the germinal disc region of f follicles, and ov gdr, corresponding to the germinal disc region of ovulated oocytes. apart from the stage of maturation the main difference between these samples involved the presence of granulosa cells. they were not present in gdr from ovulated oocytes, they were slightly present in f gdr, and they constituted a major component of f gcs. statistical analysis of data obtained after affymetrix microarray hybridization provided lists of genes differentially expressed in three comparisons: f gdr and ov gdr, f gdr and f gcs, and ov gdr and f gcs. there were only a few differentially expressed genes in each comparison. indeed, the first comparison, between the f gdr and ov gdr, showed genes over-expressed at the f stage, including one of our in silico identified genes. in the second comparison, between f gdr and f gcs, differentially expressed genes were identified. these genes involved genes over-expressed in f gcs and genes over-expressed in f gdr. five of our in silico identified genes btg chkmos, dazl, zpa and ktfn were found among the latter genes over-expressed in f gdr. the third analysis compared the expression of genes between f gcs and ov gdr. we obtained a set of genes that were differentially expressed between f gcs and ov gdr, of which genes were over-expressed in f gcs and genes were over-expressed in ov gdr. we found of our in silico identified genes among these genes,. the venn diagram shows overlapping differentially expressed genes between different dataset comparisons. eighty-five differentially expressed genes were common for two comparisons. only one gene was common for the comparisons between f gdr and f gcs, and f gdr and ov gdr and gene was common for all three comparisons. f gdr samples contained a quantity of granulosa cells in contrast to ov gdr samples which were free of granulosa cells. analysis of the redundancy of over-expressed genes between different comparisons was therefore performed in order to distinguish between genes found over-expressed in f gdr samples due to granulosa cell contamination and those really over-expressed in the oocyte. this analysis revealed that of genes over-expressed in f gdr were also over-expressed in f gcs, both compared to ov gdr, indicating that these genes characterized granulosa cell expression rather than variation in the expression between f gd and ov gd. thus only genes should be considered as overexpressed in f gd compared to ov gd. moreover, of the genes overexpressed in f gcs compared to f gdr, were also overexpressed in f gcs compared to ov gdr. consequently, taking into account the latter redundancy, a total of genes was overexpressed in granulosa cells compared to oocytes. on the other hand genes were redundant among genes overexpressed in the ov gdr and f gcs, and f gdr and f gcs comparisons. based on go annotation the genes upregulated in gcs were mostly related to metabolic processes, transport, proteolysis, regulation of transcription, immune response and cell adhesion, whereas genes preferentially expressed in gdr were preferentially involved in cell cycle, chromosome organization, phosphorylation of proteins, regulation of transcription, multicellular organism development and dna metabolic processes. tissular pattern of gene expression on the basis of the in silico and microarray approaches, genes were retained for further study. among these, genes were found using the in silico approach as described above, of which genes were also found using the microarray approache and gene was found only in the microarray approach. the latter gene was chosen because of its strong involvement in the fertilization process. in contrast to zpa and zpc, the zpd gene was not found in the mouse, explaining why it was absent from the list of murine potentially oocyte-specific genes. four other genes already known for their involvement in final follicular maturation or early embryo development in the chicken or in other vertebrates were added. finally chicken genes were further studied. real time rt-pcr performed on adult tissue samples revealed differences in the specificity of their tissular expression patterns. tissular expression profiles of some of these genes are presented on fig. seven of these genes were specifically expressed in the ovary. the specificity of zpc and chkmos has previously been described and our results concerning these genes were in accordance with the literature. eight genes were preferentially and strongly expressed in the ovary. three genes from last group were slightly expressed in another tissue: fbox and zpd in the pituitary gland and ktfn in the muscle. we also confirmed preferential ovarian expression of cvh and foxl, as well as a low expression of the latter in the pituitary gland. three other genes from this group were slightly expressed in other tissues, in addition to the ovary. the last genes were expressed as highly in the ovary as in other tissues. rna state during follicular maturation and early embryo development analysis of total rna, assessed with agilent rna nano chips, showed an atypical state of rna from gdr between ovulation and oviposition. in our conditions, rrna seemed degraded from ovulation until oviposition. with three different rna extraction methods, rneasy kit and masterpure™ rna purification kit ), the rna profile of gdr from the ovulation stage and from the following embryonic stages remained degraded. moreover, we assessed rrna 18s and 28s by real time pcr in order to confirm this atypical rna state. these results confirmed our previous observations; 18s and 28s rrna expression showed a huge decrease from ovulation untill the oviposition. we then performed labelled reverse transcription to investigate whether mrna was also degraded. we compared three samples, i.e. the rna from gdr of f stage, from a whole adult ovary that had a normal rrna profile and gdr of ovulation stage with had degraded rrna. electrophoresis profiles of labelled cdna in denaturing agarose gel were almost identical for the three samples investigated and the smear corresponding to the reverse transcribed mrna was still present in all these samples. gene expression during follicular maturation and early embryo development a detailed study of the expression profiles during follicular maturation and early embryo development using real time rt-pcr was performed for genes, for which tissue specificity of the expression was characterized. first unsupervised hierarchical clustering of our data was performed in order to confirm the biological appropriateness of the selected genes and samples. two other previously studied genes were added to this analysis in order to facilitate the clustering process because of their already known oocyte-preferential localization. this analysis discriminated two major groups of samples. the first included all granulosa samples from f to f. the second group corresponded to gdr from f to the ovulation stage and to all embryo stages. several subgroups could be distinguished within each group. each subgroup corresponded to a different physiological state, indicated on fig 6a, suggesting that the genes selected were pertinent. moreover, unsupervised clustering arranged samples in the perfect chronological order. in order to group genes with similar expression patterns supervised clustering was performed with samples arranged first according to the nature of the sample and then according to chronological order. five clusters were identified. a representative example of the expression profile characterizing each cluster is shown on fig. the graphical representation of expression profiles of all other genes with the corresponding statistical analysis of variation of their expression is supplied in additional files. the chkmos, btg wee, zpa, dazl, hsf fbox and bmp genes, forming cluster c were preferentially expressed in gdr. these genes showed a significant increase in mrna expression in gdr during follicular maturation and a steady decrease after ovulation, becoming nearly undetectable at h after fertilization. the cvh, ktfn and zar genes included in cluster c were also preferentially expressed in gdr but, in contrast to genes from cluster c they were constantly expressed in the early embryo. moreover, ktfn and zar genes displayed a significant increase in expression from to hours after fertilization. the genes from cluster c were expressed in gcs, in gdrs and in the embryo. moreover, mark igf gdf and transfact genes showed a significant decrease in expression during the last stages of follicular maturation in both gdr and gcs. cluster c including discs znfingerriz, foxl and mtprd genes, was characterized by expression that was fairly similar to that of cluster c the difference consisted of less pronounced variations in gene expression in gcs and more pronounced variations in gene expression in gdr for cluster c compared to genes in cluster c through the stages investigated. genes belonging to cluster c were expressed in gcs and gdr but, in contrast to the clusters c and c their expression dropped dramatically at ovulation, especially for zpc and zpd whose transcripts showed increasing expression in gcs during the last stages of follicular maturation and a less marked increase in the expression in gdr. in contrast, the levels of mcmip decreased progressively during the same period. the last gene was not clustered. its expression profile was fairly similar to that of genes belonging to cluster c but its expression increased significantly h after fertilization. localization of gene expression in the ovary by in situ hybridization we performed in situ hybridization on mature and immature ovarian sections. all the probes were assessed on the two stages, but only the most significant results are shown on fig. for all the genes studied we detected homogeneous signals of mrna expression in oocytes similar to those we have previously described for bmp and gdf. the mrna of four of eight genes localized by in situ hybridization were detected with a high intensity signal in the oocytes from follicles of μm – μm from both immature and mature ovaries. no significant expression was detected in somatic cells. for four other genes a signal was found in oocytes from the largest follicles of μm- mm of mature ovaries. the signal was particularly high for chkmos and zpa, whereas zpc and especially zpd had weaker signals in both oocyte and somatic cells. no significant signal was detected with the corresponding sense probes. discussion in the present study we identified and characterized for the first time several genes expressed in the chicken oocyte during follicular maturation and/or in early embryo. moreover, we showed that our candidate gene approach and microarray approach were complementary in finding new avian genes potentially involved in follicular maturation and/or early embryo development. five genes preferentially and highly expressed in the oocyte were identified using both microarray analysis and digital differential display on murine genes. moreover, genes, identified as overexpressed in gcs by microarray analysis, were also confirmed by real time pcr analysis to be highly preferentially expressed in chicken gcs compared to gdr. microarray analysis identified a total of genes upregulated in the hen f and ovulated oocytes both compared to f gcs. among these, overexpressed genes were common to ov gdr and and f gdr, both compared to f gcs, and therefore represent particular interesting candidate oocyte genes for further exploration of their potential role in oocyte maturation, fertilization and oet. the fact that we found almost five times fewer genes overexpressed in the oocyte at the ovulation stage than in the oocyte at f stage, compared to granulosa cells from f follicles, means that for some genes mrna expression in the oocyte decreased between f and ovulation stages. this change in the mrna expression levels between f and ovulated oocytes is probably insufficient to be detected in the comparison between ov gdr and f gdr by microarray hybridization, since only differentially expressed genes were identified in this comparison. on the other hand microarray analysis enabled us to detect the presence of gcs in f gdr samples, revealed by the redundant overexpressed genes in f gdr and in f gcs compared to ov gdr. the functions of overexpressed genes in the various comparisons according to go categories revealed clear differences between gcs and mature oocytes. for gcs overexpressed genes these functions were mostly related to metabolic processes, transport, proteolysis, regulation of transcription, immune response and cell adhesion, whilst for the mature oocyte they were mostly related to cell cycle, chromosome organization, phosphorylation and dephosphorylation of proteins, multicellular organism development and dna metabolic process. these presumed functions of genes overexpressed in the oocyte are consistent with the physiological processes that it must undergo: i.e. fertilization, cleavage, chromatin remodeling, and supporting early embryo development. the use of both bioinformatics and microarray approaches provided information on the molecular mechanisms through which oet is driven in the hen. the expression of several oocyte-specific genes increased during final follicular maturation, suggesting that transcription was still effective. after ovulation, despite the fact that 18s and 28s ribosomal rna subunits were degraded, we showed by both labelled reverse transcription and microarray analysis that the integrity of mrna was almost unaffected. in fact, mrna levels were nearly the same for many genes because only of genes were differentially expressed between gdrs before and after ovulation, of which only really corresponded to the oocyte genes. the high number of replicates performed and the different extraction methods used strongly indicate that the difference in quality between oocyte rrna and mrna after ovulation is not the artifact of the experiment but reflects a real physiological feature of chicken oet, consisting probably of the arrest and degradation of the oocyte translational machinery. it could thus be hypothesized that the maternal translational system has to be replaced by the embryonic translational system. indeed, the maternal ribosome in the embryo must be degraded before activation of the genome, in other words, before the beginning of transcription, and translation, when new embryonic ribosomes are required. this suggests that, because there are no maternal ribosomes at the stage between ovulation and oviposition, there is probably no translation or only translation of a few specific genes. if this is the case, maternal proteins should be the major essential components that support early embryo development after fertilization. this is supported by the fact that, based on go annotation, a considerable number of the genes overexpressed in the mature oocyte are related to protein phosphorylation. further investigation is required onto whether accumulated proteins have such an important role during these early stages of development in birds or if de novo protein synthesis still occurs and is dependent on the oocyte pool of ribosomes as in mammals. on the basis of in silico and microarray approaches and analysis of the literature, chicken genes were chosen in this study for further investigation of their expression using real time pcr and in situ hybridization. all these genes showed state-specific and/or cell type-specific expression patterns throughout the period, beginning from the first stages of final follicular maturation until embryonic genome activation. this suggests that these genes have different functions and have a role at the different physiological stages investigated. the observed decrease in the mrna expression of almost all genes studied between ovulation and oviposition, which corresponds to late genome activation in chicken embryo, is consistent with a potential arrest of transcription and progressive maternal mrna degradation occurring during meiotic maturation. however, the rate of maternal mrna degradation in the chicken seems to be considerably lower than that of rrna, as demonstrated by the present study. five genes belonging to cluster c were no longer expressed after activation of the embryonic genome, and thus transcripts of these genes are only maternally inherited. moreover, chkmos, btg and wee genes are known to be involved in the cell cycle in other species. in our study these maternally inherited genes were increasingly expressed during follicular maturation and thus are probably used during the last stages of final follicular maturation and/or early embryo development. chkmos is the chicken homolog of mos, protein kinase required for meiotic maturation in vertebrates and for mitosis in xenopus laevis. meiotic maturation is brought about by steroids using redundant pathways involving synthesis of mos, which regulates the activity of mpf. the mos-mapk pathway has long been implicated in the arrest of mitosis in vertebrate eggs. the b cell translocation gene belongs to a family of cell-cycle inhibitors. in the mouse and bovine, btg is preferentially expressed in the oocyte where it exerts a marked antiproliferative activity,. wee is a conserved gene from invertebrates to mammals and regulates meiotic maturation during oocyte development. the transcripts of dazl are also maternally inherited in the medaka embryo, and in adult medaka fish the expression of dazl was detected exclusively in the ovary and in the testis. zpa, zpc and zpd, that belong to the zp gene family, are known to be involved in oogenesis, fertilization and preimplantation development. in our study the expression pattern of zpa was different from that of zpd and zpc using both real time pcr analysis, where they were distributed in different clusters, and in situ hybridization analysis. in contrast to chicken zpd and zpc, which were expressed in oocytes and somatic cells, chicken zpa was found to be specific to the oocyte, as is the case in the mouse, and expressed earlier than zpd and zpc. in the mouse the expression of zpa also precedes that of zpc. indeed in our in situ hybridization experiments zpa expression detected in small follicles of the mature ovary was oocyte-specific, whereas that of zpc and zpd was weaker and found in oocytes and in the somatic cells of the same follicles. real time rt-pcr, showed increasing expression of zpc and zpd in both gcs and gdr from f to f our finding on the dynamics of zpc in gcs are in accordance with a previously reported study. moreover at the f stage both zpc and zpd were significantly more highly expressed in gcs than in gd and this was consistent with our results for microarray hybridization, but expression decreased dramatically after ovulation. in contrast to the cellular expression pattern found for chicken zpc, in several mammals zpc is specifically expressed in the oocyte. however, in the equine species, zpc protein synthesis is completely taken over by cumulus cells. these findings indicate species specificity of zpc distribution inside the follicle. zpc protein plays a crucial role in the fertilization process in mammals and birds,. as zpa other genes were preferentially expressed in the oocyte and might play role in fertility or in germ cell specification. both zar and ktfn were expressed at higher levels after activation of the embryonic genome. we could therefore hypothesize that these genes might be involved not only in oocyte maturation, but also in early embryo development, just after maternally inherited genes. of these genes, only zar has been studied in reproduction. it is one of the few known oocyte-specific maternal-effect genes essential for oet in mice. in mammals and humans it is hypothesized to be involved in the initiation of embryo development and fertility control. cvh protein has been previously proposed to be a part of the mechanism for germ cell specification in birds. our results concerning the spatio-temporal expression of cvh mrna during follicular maturation and early embryo development are consistent with previously reported studies on the cvh protein. the genes belonging to clusters c and c were all preferentially expressed in the ovary in both gcs and gdr and had quite similar expression patterns. except for foxl their expression declined during follicular maturation in gcs and less in gdr and persisted at low levels in the early embryo. this suggests that they are especially involved in the first stages of final follicular maturation as well as in oocyte maturation. the chicken homolog of the mouse par-1a-like gene, i.e. mark is required for oocyte differentiation and microtubule organization in the drosophila, and its role in cell polarity and wnt signaling is conserved from invertebrates to mammals. the expression of another gene, which did not belong to any cluster, also decreased during follicular maturation in gcs and in gd but, as for zar and ktfn, it showed a significant increase after embryonic genome activation. msh is known to be involved in mediating recombination of homologous chromosomes and dna mismatch repair in the mouse. these events occur during the meiotic prophase, the stage where oocytes are blocked for a long time before meiotic maturation.  in conclusion, the findings of the present study on spatio-temporal expression of chicken oocyte specific genes were consistent with our hypothesis that oocyte-specific genes in the chicken should play a major role in oocyte maturation, fertilization and early embryo development as in the mouse. other genes, whose mrna expression were found in our study to be more specific for gcs or detected in both gcs and gdr depending on the stage, seem to be involved in follicular maturation and fertilization rather than in early embryo development. moreover, the microarray approach provided allowed the discovery of a set of new potential chicken mature oocyte and chicken granulosa cell markers for future studies. interestingly, 40% of these genes had no homologs in the gene databases and some of them probably correspond to specific chicken mechanisms such as hierarchical follicular maturation or rapid yolk accumulation. 
the initial stages of development depend on mrna and proteins accumulated in the oocyte, and during these stages, certain genes are essential for fertilization, first cleavage and embryonic genome activation. using the chicken affymetrix microarray, we identified genes overexpressed in granulosa cells and genes overexpressed in the germinal disc of the hen oocyte. moreover, detailed study of the expression of some of these genes revealed promising candidates for maternal effect genes in the chicken. the aim of this study was first to search for avian oocyte-specific genes using an in silico and a microarray approaches, then to investigate the temporal and spatial dynamics of the expression of some of these genes during follicular maturation and early embryogenesis. according to their expression patterns all these genes are involved in different stages of final follicular maturation and/or early embryogenesis in the chicken. among them, genes were preferentially expressed in the maturing occyte and cvh, zar and ktfn were also highly expressed in the early embryo. we showed that in silico and affymetrix microarray approaches were relevant and complementary in order to find new avian genes potentially involved in oocyte maturation and/or early embryo development, and allowed the discovery of new potential chicken mature oocyte and chicken granulosa cell markers for future studies. the in silico approach allowed us to identify chicken homologs of mouse potential oocyte genes found by digital differential display. finally we studied the spatial and temporal dynamics of the expression of chicken genes. six genes were identified using both in silico and microarray approaches.
 the initial stages of development depend on mrna and proteins accumulated in the oocyte, and during these stages, certain genes are essential for fertilization, first cleavage and embryonic genome activation. the aim of this study was first to search for avian oocyte-specific genes using an in silico and a microarray approaches, then to investigate the temporal and spatial dynamics of the expression of some of these genes during follicular maturation and early embryogenesis.  the in silico approach allowed us to identify chicken homologs of mouse potential oocyte genes found by digital differential display. using the chicken affymetrix microarray, we identified genes overexpressed in granulosa cells and genes overexpressed in the germinal disc of the hen oocyte. six genes were identified using both in silico and microarray approaches. based on go annotations, gc and gd genes were differentially involved in biological processes, reflecting different physiological destinations of these two cell layers. finally we studied the spatial and temporal dynamics of the expression of chicken genes. according to their expression patterns all these genes are involved in different stages of final follicular maturation and/or early embryogenesis in the chicken. among them, genes were preferentially expressed in the maturing occyte and cvh, zar and ktfn were also highly expressed in the early embryo.  we showed that in silico and affymetrix microarray approaches were relevant and complementary in order to find new avian genes potentially involved in oocyte maturation and/or early embryo development, and allowed the discovery of new potential chicken mature oocyte and chicken granulosa cell markers for future studies. moreover, detailed study of the expression of some of these genes revealed promising candidates for maternal effect genes in the chicken. finally, the finding concerning the different state of rrna compared to that of mrna during the postovulatory period shed light on some mechanisms through which oocyte to embryo transition occurs in the hen. 