- Research article
- Open Access
Search for the genes involved in oocyte maturation and early embryo development in the hen
© Elis et al; licensee BioMed Central Ltd. 2008
Received: 12 September 2007
Accepted: 29 February 2008
Published: 29 February 2008
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 18 chicken homologs of mouse potential oocyte genes found by digital differential display. Using the chicken Affymetrix microarray, we identified 461 genes overexpressed in granulosa cells (GCs) and 250 genes overexpressed in the germinal disc (GD) 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 21 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, 8 genes (btg4, chkmos, wee, zpA, dazL, cvh, zar1 and ktfn) were preferentially expressed in the maturing occyte and cvh, zar1 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.
The activation of molecular pathways underlying oocyte to embryo transition (OET) 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. Dnmt1, Hsf1 and Mater . Mater (Maternal antigen that embryos require) 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 125 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 30,000–50,000 cells  24 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 (GD) . The GD is a white plaque of about 3–4 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 (F6 to F1) .
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. [13, 19–22]. Chicken vasa homolog protein (CVH) 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 F2 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
Accession numbers of murine sequences used and of homolog chicken sequences found. Bold text represents chicken genes whose syntenic regions are conserved with the appropriate murine homologs.
similar to zinc finger protein RIZ. partial
similar to mtprd protein – mouse
similar to hypothetical protein
similar to MutS homolog 4
similar to Discs. large homolog 5
similar to Kruppel-like transcription factor neptune
similar to mast cell maturation inducible protein 1
similar to p30 B9.10
similar to MAP/microtubule affinity-regulating kinase 3 long isoform
Gallus gallus similar to zygote arrest 1
similar to transcription factor 20 isoform 1
deleted in azoospermia-like
Chicken c-mos proto-oncogene
similar to zona pellucida A
similar to Wee1A kinase
zona pellucida C protein
Comparing oocyte and granulosa cells transcription profiles at final maturation steps using chicken Affymetrix microarrays
Genes differentially expressed in Affymetrix experiment. Bold text represents genes that were further studied using real time RT-PCR
Gene name found by Blast search
Abbreviation of genes studied
Number of overexpressed genes
Gallus gallus wingless-type MMTV integration site family, member 4
F1 GCs and Ov GDR
56 genes overexpressed in Ov GDR
Gallus gallus mRNA for hypothetical protein, clone 32l2
Finished cDNA, clone ChEST746h6
similar to Interferon regulatory factor 6
aldo-keto reductase family 1, member D1 (delta 4-3-ketosteroid-5-beta-reductase)
Gallus gallus tumor-associated calcium signal transducer 1
Gallus gallus claudin 1
Finished cDNA, clone ChEST914o3
Amyotrophic lateral sclerosis 2 chromosomal region candidate gene protein 7
B-cell translocation gene 4
zona pellucida protein D
F1 GCs and Ov GDR
392 genes overexpressed in F1 GCs
Gallus gallus similar to adrenodoxin homolog
Gallus gallus finished cDNA, clone ChEST974b18
Gallus gallus similar to chromosome 9 open reading frame 61
Gallus gallus 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase
Gallus gallus finished cDNA, clone ChEST591g11
similar to hypothetical protein FLJ22662
Gallus gallus finished cDNA, clone ChEST537h21
Gallus gallus similar to LRTS841
Gallus gallus similar to CG8947-PA
Gallus gallus wingless-type MMTV integration site family, member 4
F1 GDR and F1 GCs
238 genes overexpressed in F1 GDR
similar to Interferon regulatory factor 6
Gallus gallus similar to CG31613-PA
Gallus gallus similar to carbonic anhydrase 9
Gallus gallus tumor-associated calcium signal transducer 1
Gallus gallus deleted in azoospermia-like
B-cell translocation gene 4
Gallus gallus similar to Kruppel-like transcription factor neptune
Gallus gallus similar to zona pellucida A
oocyte maturation factor Mos
F1 GDR And F1 GCs
104 genes overexpressed in F1 GCs
Gallus gallus similar to CG8947-PA
similar to relaxin 3 preproprotein
Gallus gallus finished cDNA, clone ChEST699k2
Gallus gallus similar to CG8947-PA
Gallus gallus nuclear receptor subfamily 5, group A, member 2
Gallus gallus similar to chromosome 9 open reading frame 61
Gallus gallus finished cDNA, clone ChEST159o8
Gallus gallus similar to Ephx1 protein
Gallus gallus reversion-induced LIM protein
Gallus gallus zona pellucida protein D
F1 GDR And Ov GDR
92 genes overexpressed in F1 GDR
similar to adrenodoxin homolog – chicken
Gallus gallus 3beta-hydroxysteroid dehydrogenase delta5-delta4 isomerase
Finished cDNA, clone ChEST591g11
similar to LRTS841
weak similarity to HUMAN Putative protein X123
Finished cDNA, clone ChEST974b18
Finished cDNA, clone ChEST738j4
similar to hypothetical protein FLJ22662
Gallus gallus zona pellucida glycoprotein 3
Tissular pattern of gene expression
Accession number of chicken genes selected on the basis of the literature
Govoroun et al. 2004
forkhead box L2
Aegerter et al. 2005, Heck et al. 2005
Insulin growth factor 2
Tsunekawa et al. 2000
Gallus gallus DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4)
Hsf1 Anckar et al. 2007
chicken heat shock factor protein 1
RNA state during follicular maturation and early embryo development
Gene expression during follicular maturation and early embryo development
Localization of gene expression in the ovary by in situ hybridization
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 (btg4, chkmos, dazL, zpA and ktfn) were identified using both microarray analysis and digital differential display on murine genes. Moreover, 2 genes (zpC and zpD), 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 245 genes upregulated in the hen F1 and ovulated oocytes both compared to F1 GCs. Among these, 49 overexpressed genes were common to Ov GDR and and F1 GDR, both compared to F1 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 (comparison between Ov GDR and F1 GCs) than in the oocyte at F1 stage (comparison between F1 GDR and F1 GCs), compared to granulosa cells from F1 follicles, means that for some genes mRNA expression in the oocyte decreased between F1 and ovulation stages. This change in the mRNA expression levels between F1 and ovulated oocytes is probably insufficient to be detected in the comparison between Ov GDR and F1 GDR by microarray hybridization, since only 7 differentially expressed genes were identified in this comparison. On the other hand microarray analysis enabled us to detect the presence of GCs in F1 GDR samples, revealed by the redundant overexpressed genes in F1 GDR and in F1 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 92 of 28000 genes were differentially expressed between GDRs before and after ovulation, of which only 7 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, 21 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 (chkmos, btg4, wee, dazL and zpA) belonging to cluster C1 (Fig. 6B) were no longer expressed after activation of the embryonic genome, and thus transcripts of these genes are only maternally inherited. Moreover, chkmos, btg4 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 [41, 42] 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 (M-phase promoting factor). The Mos-MAPK pathway has long been implicated in the arrest of mitosis in vertebrate eggs . The B cell translocation gene 4 (btg4) belongs to a family of cell-cycle inhibitors. In the mouse and bovine, btg4 is preferentially expressed in the oocyte [44, 45] where it exerts a marked antiproliferative activity, . Wee is a conserved gene from invertebrates to mammals and regulates meiotic maturation during oocyte development [38, 41–43]. 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 (zona pellucida) 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 (C1 for zpA and C5 for zpD and zpC respectively), 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 [50, 51], and expressed earlier than zpD and zpC. In the mouse the expression of zpA also precedes that of zpC [50, 51]. 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 F6 to F1. Our finding on the dynamics of zpC in GCs are in accordance with a previously reported study . Moreover at the F1 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 (murines, bovines and porcines)zpC (zp3) 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, [31, 37, 49].
As zpA other genes (zar1, ktfn and cvh) were preferentially expressed in the oocyte and might play role in fertility (zar1, ktfn) or in germ cell specification (cvh). Both zar1 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 2 genes, only zar1 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 [54, 55]. CVH protein has been previously proposed to be a part of the mechanism for germ cell specification in birds [16, 23]. 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 C3 and C4 were all preferentially expressed in the ovary in both GCs and GDR and had quite similar expression patterns. Except for foxL2, 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. mark3, 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 [57, 58]. The expression of another gene (msh4), which did not belong to any cluster, also decreased during follicular maturation in GCs and in GD but, as for zar1 and ktfn, it showed a significant increase after embryonic genome activation. Msh4 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 8 chicken oocyte specific genes (chkmos, btg4, wee, zpA, dazL, cvh, zar1 and ktfn) 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 (foxl2, transfac 20, mark3, ...) and fertilization (zpD and zpC) 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.
Laying breed hens aged 60–70 weeks (ISA Brown, egg layer type, Institut de Selection Animale, Saint Brieuc, France) were housed individually in laying batteries with free access to feed and water and were exposed to a 15L:9D photoperiod, with lights-on at 8.00 pm. Individual laying patterns were monitored daily. For in situ hybridization, these hens and younger ones of (10 weeks old) were used to provide mature and immature ovaries, in order to study follicles at each stage. Hens used to provide fertilized eggs were bred in the same conditions and inseminated once a week.
Collection of tissues, oocytes and embryos
Hens aged 60 weeks were used. Tissue samples were collected from the ovary, spleen, intestine, gizzard, liver, heart, skin, brain, pectoralis muscle, lung and pituitary gland. Germinal disc regions (GDR) and granulosa cells (GCs) surrounding the GDR (Fig. 1) were collected from different preovulatory follicles (F1 to F6), just ovulated oocytes and early embryos at 6.5 h, 12 h, 24 h, 36 h and 48 h post ovulation. The GDR and GCs surrounding the GDR were carefully dissected in the same way under a binocular microscope using fine forceps and scissors (World Precision Instruments) as previously described for quail oocytes . After washing in phosphate buffer saline (PBS, Gibco, Cergy Pontoise, France) GDR and GCs were frozen in liquid nitrogen and then were stored at -80°C until use. For the last two stages, eggs were incubated at 37.8°C for 12 and 24 h, respectively. The 6.5 h, 12 h, and 24 h stages of embryo development correspond to stages I, V and X of the Eyal-Giladi and Kochav classification, respectively . The 36 h and 48 h stages correspond to stages 3 and 6 of the Hamburger-Hamilton classification, respectively . During follicular maturation the germinal disc is closely associated with its overlying granulosa cells (GCs) and forms a structure called the germinal disc region (GDR) (Figure 1). The GDR from F6-F1 follicles used for these studies consisted not only of the germinal disc but also of the overlying layer of GCs, because GD and overlying GCs cannot be completely separated [60, 63, 64] and the number of GCs in GDR preparations could not be counted.
A differential digital display analysis has already been performed with mouse ESTs [25–27], providing a list of murine oocyte-specific genes. Using this murine gene list, we systematically searched for chicken orthologs of these genes in international public databases pubmed . Blast bit scores higher than 100 were retained. Moreover, the physical localization of genes identified on chicken chromosomes was retrieved from both mapview  and from blat search . We also verified that chicken homologs were localized in the syntenic genomic regions conserved with that of mouse species to have a better chance that true orthologs were studied with ensembl .
RNA isolation and Microarray Analysis
Total RNA was extracted from GDR of F1 and ovulated oocytes, and from GCs of F1 follicles as described above. We thus had 3 samples with a biological replicate of each sample. The RNeasy Mini Kit (QIAGEN, Hilden, Germany) was used according to the manufacturer's instructions. The tissues (GDR or GC) from 25 hens were pooled for each stage investigated to obtain enough RNA for probe synthesis. Two such a pools were constituted for each sample in order to achieve two biological replicates of microarray hybridization. All RNA samples were checked for their integrity on the Agilent 2100 bioanalyzer according to Agilent Technologies guidelines (Waldbroon, Germany). Two micrograms of total RNA were reverse transcribed with the One-cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA), according to the manufacturer's procedure. Clean up of the double-stranded cDNA was performed with Sample Cleanup Module (Affymetrix, Santa Clara, CA) followed by in vitro transcription (IVT) in the presence of biotin-labelled UTP using GeneChip® IVT labelling Kit (Affymetrix). The quantity of the cRNA labelled with RiboGreen® RNA Quantification Reagent (Turner Biosystems, Sunnyvale, CA) was determined after cleanup by the Sample Cleanup Module (Affymetrix). Fragmentation of 15 μg of labelled-cRNA was carried out for 35 minutes at 94°C, followed by hybridization for 16 hours at 45°C to Affymetrix GeneChip® Chicken Genome Array, representing approximately 32,773 transcripts, corresponding to over different 28,000 Gallus gallus genes. After hybridization, the arrays were washed with 2 different buffers (stringent: 6X SSPE, 0.01% Tween-20 and non-stringent: 100 mM MES, 0.1 M [Na+], 0.01% Tween-20) and stained with a complex solution including Streptavidin R-Phycoerythrin conjugate (Invitrogen/molecular probes, Carlsbad, CA) and anti Streptavidin biotinylated antibody (Vectors laboratories, Burlingame, CA). The washing and staining steps were performed in a GeneChip® Fluidics Station 450 (Affymetrix). The Affymetrix GeneChip® Chicken Genome Arrays were finally scanned with the GeneChip® Scanner 3000 7G piloted by the GeneChip® Operating Software (GCOS).
All these steps were performed on Affymetrix equipement at INRA-URGV, Evry, France.
The .raw CEL files were imported in R software for data analysis. All raw and normalized data are available from the Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information (NCBI) , accession number GSE7805. Gene Ontology annotations were performed with NetAffx.
RNA extraction and reverse transcription
Total RNA was extracted from whole adult tissues (ovary, spleen, intestine, gizzard, liver, heart, skin, brain, pectoralis muscle, lung and pituitary gland) using Tri-reagent (Euromedex, Mundolsheim, France) according to the manufacturer's procedure. RNA quality and quantity were then assessed by using RNA nano chips and Agilent RNA 6000 nano reagents (Agilent Technologies, Waldbronn, Germany) according to the manufacturer's instructions. Samples were stored at -80°C until use. Reverse transcription (RT) was performed to test the expression of candidate genes in different tissues and at different stages of follicular maturation and embryo development using polymerase chain reactions (PCR). One microgram of total RNA extracted from tissues or GDR was digested by RQ1 DNase (Promega, Madison, WI, USA) and reverse transcribed to first-strand cDNA using Moloney Murine Leukemia Virus reverse transcriptase I with an oligo dT-random primer mix (Promega, Madison, WI, USA) according manufacturer's instructions.
Labelled RT was performed in order to assess mRNA quality in GDR of F1 stage and just ovulated oocytes. Ten μCi αP32 dCTP was added to the reverse transcription mix in order to label cDNA. Labelled cDNA was then separated on 1.2% denaturing agarose gel (50 mM NAOH, 1 mM EDTA) by electrophoresis. A storage phosphor screen (Amersham Biosciences, Bucks, UK) was placed on the gel in an exposure cassette (Amersham Biosciences, Bucks, UK). The signal was detected one hour later with a STORM 840 (Molecular Dynamics), a phosphor screen imaging system.
Real time RT-PCR
Oligonucleotide primer sequences
Real time PCR
zn finger RIZ
trans fact 20
Govoroun et al. 2004
Heck et al. 2003
AGC AGA CTT TGT GAC CTT GCC
TGA CAT GAG ACA GAC GGT TGC
in situ hybridization
The hierarchical classification of data obtained using real time RT-PCR was performed with the Cluster 3.0 program using unsupervised single linkage or supervised complete linkage clustering in order to classify biological samples or to group together genes with a similar expression pattern, respectively .
In situ hybridization
Female chickens were sacrificed at different stages of sexual development. Two types of tissue were used, i.e. mature ovaries, containing follicles of different sizes (50 μm-7 mm) from 60-week-old hens (most follicles being larger than 300 μm) and immature ovaries, containing a majority of small follicles (25–500 μm) from 10-week-old hens (most follicles being smaller than 100 μm). Mature and immature ovaries were then collected and included in Tissue-Tek (Sakura Finetek Europe BV, Zoeterwoude, The Netherlands). Frozen ovaries were serially sectioned with a cryostat (thickness 10 μm) to perform in situ hybridization experiments using 35S-labeled chicken gene cRNA. The gene antisense and sense constructs used for in situ hybridization were generated by inserting 700 – 800 bp fragments of chicken gene cDNA into the pGEM-T vector (Promega, Madison, WI, USA), and selecting a clone with the appropriate antisense or sense orientation. The gene cDNA fragments were generated by RT-PCR from chicken ovary mRNA using forward and reverse primers (Table 4). The in situ hybridization was performed as previously described . Hybridization specificity was assessed by comparing signals obtained with the cRNA antisense probe and the corresponding cRNA sense probe.
Data obtained after Affymetrix microarray hybridization analysis were normalized with the gcrma algorithm , available in the Bioconductor package . Differential analysis was performed with the varmixt package of R . A double-sided, unpaired t-test was computed for each gene between the two conditions. Variance of the difference in gene expression was split between subgroups of genes with homogeneous variance . The raw P values were adjusted by the Bonferroni method, which controls the Family Wise Error Rate (FWER) . A gene is declared differentially expressed if the Bonferroni-corrected P-Value is less than 0.05.
All other experimental data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used to test differences. If ANOVA revealed significant effects, the means were compared by Fisher's test, with P < 0.05 considered significant. Different letters indicate significant differences.
We thank Svétlana Uzbekova and Rozenn Dalbies-Tran for helpful discussion, Sonia Métayer for igf2 primers and Frederic Mercerand and Jean-Didier Terlot-Bryssine for expert animal care. This study was supported by the "Institut National de la Recherche Agronomique". S Elis was supported by a fellowship from the Institut National de la Recherche Agronomique and "Région Centre".
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