BACKGROUND
the major function of an ovary is to produce oocytes intended for fertilization as eggs and to create a favorable environment for the beginning of gestation.
in adult mammals, the ovary is a heterogeneous organ containing follicles at various stages of development and corpora lutea, either active or at various stages of involution.
the formation of primordial follicles  occurs during fetal development in many species including human, sheep   <cit> , cattle, and goat, or after birth like in rodents.
the primordial follicles represent a reserve of germ cells for the entire reproductive life of the female.
they remain dormant until their recruitment and irreversible growth towards the primary, secondary and tertiary stages .
the gradual exit of primordial follicles begins shortly after the formation of the primordial follicle pool and continues throughout the reproductive years  <cit> .
consequently, this early development is important as it regulates the size of the resting primordial follicle pool and the fate of the follicles, which, in turn, affects fertility and the reproductive life span.
early follicular development requires the appropriate expression of many genes at different developmental stages.
recent studies on natural mutations in sheep  <cit>  and on mutant mouse models demonstrated that the expression of different oocyte-specific genes is essential during early folliculogenesis in a stage specific expression pattern  <cit> .
figla, nobox, pten, foxo3a and nerve growth factor genes are involved in the formation of primordial follicles, whereas genes such as βfgf, gdf  <dig> and bmp  <dig> are involved in the transition from primordial to primary follicles.
other genes such as bmp <dig> are not expressed in the oocyte until the primary follicle stage and are involved in the transition from primary to secondary follicles, as shown in sheep .
this process also requires orchestrated communication between oocytes and granulosa cells , which are the structural components of the early follicle.
oocytes and gcs regulate follicle growth in an autocrine and paracrine manner via secreted factors and direct gap junctional communications.
on one hand, it is assumed that oocytes control the proliferation of gcs and later, their differentiation into steroid secreting cells and their metabolism  <cit> .
on the other hand, gcs are indispensable to oocyte growth, meiosis, cytoplasmic maturation and control of transcriptional activity within the oocyte  <cit> .
this dialog also takes place through gc cytoplasmic extensions that penetrate the zona pellucida  and form gap junctions with oocyte cell membrane  <cit> .
although some large scale expression studies have been conducted on rodent and human oocytes  <cit> , expression profiling of follicular compartments is difficult to accomplish due to the very small size of the follicles and the mixture of preantral stages in the ovary.
two studies in particular identified the specific transcriptome of human oocytes in the quiescent state  <cit> .
these studies highlighted enriched functions  and molecular mechanisms  in human oocytes, which were also identified in the only preliminary analysis conducted in sheep species to date  <cit> .
but exactly which granulosa cell factors play a specialized role in early follicular development is not known.
our knowledge of the key mechanisms and the bidirectional communication underlying early folliculogenesis is consequently still very limited and is mainly derived from rodents .
spatial and temporal information on gene expression patterns would facilitate the identification of the regulatory gene networks that underlie cell growth, differentiation, and the functional specification of each follicular compartment.
the purpose of this study was thus to 1- describe global gene expression during early ovarian folliculogenesis in each follicular compartment  in mono-ovulating species using sheep as the experimental model ; 2- identify differential and specific gene expression between these two follicular compartments; 3- investigate specific functions and pathways, and 4- explore bi-directional communication between oocytes and gcs.
this was achieved using laser capture microdissection  to isolate oocytes and gcs during the earliest stages of folliculogenesis.
gene expression profiles were monitored at each follicle stage according to cell type using rna-seq technology.
we report the successful isolation and generation of global gene expression data sets of pure oocytes and gc populations at key stages of early follicular development.
we describe the expression profile of  <dig>  <dig> genes with  <dig> % of genes displaying differential expression between the two compartments.
in addition, we identified oocyte and gc-specific gene expression and the pathways involved in oocyte-gc communication.
RESULTS
we combined laser capture microdissection   <cit>  and rna-sequencing technologies to detect separate gcs and oocyte global transcriptome for each stage of follicle development: primordial , primary , secondary  follicles and the small antral stage  as control.
three/four biological replicates were obtained per condition .
for the experimental protocol, see additional file 1: figure s <dig>
the rna-seq experiment generated around  <dig>  billion 100 bp reads with an average of  <dig>  million per lcm-derived amplified rna sample .
the result of the bioinformatics and annotation processing is summarized in figure  <dig> and detailed in additional file  <dig>  briefly, the genome assembly approach  generated a collection of  <dig>  <dig> fragments that aggregated  <dig> % of lcm-arna reads.
after 3′utr selection, the final dataset included  <dig>  <dig> genes.
<dig>  <dig> genomic fragments remained unannotated.
exploring global gene expression
the  <dig>  <dig> genes correspond to 62% of the bovine annotated genes in ensembl umd <dig>  release  <dig>  and to  <dig> % of the ovine annotated genes in csiro oarv <dig>  .
as expected, the number of expressed genes was uniformly distributed between chromosomes according to the number of expected genes except for chromosome  <dig> on which significantly fewer genes were expressed .
the percentage of genes transcribed for this chromosome was 57%, compared to an average of 80% for the other chromosomes .
the chromosome coverage of the expressed genes overlays oarv <dig>  annotated genes  except for chromosome  <dig> on which we identified two regions with fewer expressed genes.
the density of expressed genes in these two regions  was 75%-90% lower.
for each sample, the expression levels were measured in read counts per gene  and a normalized expression ) was calculated to describe the transcriptome of the tissue.
first, we visualized a dynamic range of gene expression between  <dig>  and  <dig>  <dig> rpm .
we estimated that 95% of the genes aggregated 56% of the reads.
we then examined the highest expressed genes and detected a clear distinction between cell types .
the highest oocyte-expressed genes contributed less to the total number of reads than gcs and multi-tissue samples .
for example, the ten highest oocyte-expressed genes contributed  <dig> % to  <dig> % of the total reads, while the highest expressed gc and mt genes contributed respectively 5% to  <dig> % and  <dig> % of the total reads.
in oocytes, the most expressed genes included zp <dig>  zp <dig> and gdf <dig>  genes known to be oocyte specific, but also actg, paip <dig>  pdk <dig>  in gcs, the most expressed genes included mainly ribosomal genes, such as rpl <dig>  rpl <dig>  rps <dig>  genes involved in cellular assembly and organization, such as actg, collagens, vim and cell growth regulators, such as mgp.
among these  <dig>  <dig> expressed genes, we observed a significant decrease in oocyte-expressed genes during early follicular development, i.e., from  <dig>  <dig> genes in primordial oocytes to  <dig>  <dig> genes in small antrum oocytes .
conjointly  <dig>  <dig> genes were found to be expressed in all the samples  and were considered as ubiquitously expressed genes.
however, their relative expression levels varied depending on the type of cell, for example ribosomal genes , vim, sparc, and low expressed genes such as bscl <dig>
in addition to this qualitative assessment, the transcriptome data set was analyzed using the r software package, deseq  <cit> .
principal component analysis of the deseq normalized data confirmed the relevance of the data .
the first axis explained 52% of expression variability and separated the two follicular compartments .
the second axis explained 15% of expression variability and separated the samples according to their follicular stages.
global differential gene expression
to identify differentially expressed genes, a generalized linear model was applied to the data after deseq normalization .
this model allowed statistical evaluation of the factor effects  and their interactions.
the following criteria including fdr <  <dig> % to 1%  and a fold change > <dig> were chosen to determine significant differentially expressed genes.
using these criteria,  <dig> % of genes  were significantly differentially expressed between the two compartments;  <dig> % of genes were significantly differentially expressed during early development .
we observed differences in expression profiles during early folliculogenesis between the two compartments for  <dig> % of the genes .
for example, the expression of the kcnn <dig> gene decreased in gcs during early development whereas its expression increased in oocytes.
differential expression is detailed in table  <dig>  which, interestingly, shows that genes were under expressed in oocytes compared to in gcs .
focus on differential expression between compartments
we focused our analysis on the  <dig>  <dig> significant differentially expressed genes  between compartments .
the aim was to provide an overview of specific gene expression, mechanisms and functions in these cell types, which may be involved in the dialog between the oocyte and gcs, and regulate follicle development.
first, we observed numerous genes on the deg list already known to have cell-type specific expression.
the expression of  <dig> genes known to be compartment specific was investigated and the expression of  <dig> of them was confirmed by the rna-seq statistical analysis .
last, the expression of 33% of the genes  varied during early follicular development.
supervised hierarchical clustering was performed to identify groups of genes with similar expression profiles.
this led to a clear separation between the two compartments and identified clusters of genes according to the stage of follicular development, suggesting a difference in the dynamics of transcription between oocytes and gcs .
compartment specific expression
previous studies that were mainly devoted to rodent species identified oocyte-specific genes with important functions related to oocyte development and folliculogenesis.
however, few data are available for gcs.
to gain insight into the specific expression of each compartment, we performed a pair-wise comparison of the transcriptome of each compartment and both mt and the other compartment transcriptomes.
in this way, we identified  <dig> highest differentially expressed genes in gcs  and  <dig> highest differentially expressed genes in oocytes  and the gc-restricted expression of four transcription regulators .
for a list of the highest differentially expressed genes, see additional file  <dig>
validation of differential expression by quantitative real time rt-pcr
to validate the rna-seq analysis, the expression profiles of a subset of genes of interest were monitored using qrt-pcr.
these genes were selected as follows:  genes showing significant up or down expression in oocytes ,  genes involved in particular canonical pathways  or  compartment-specifically expressed genes .
statistical analysis confirmed the differential expression between compartments observed in rna-seq data for all the genes tested  and the compartment-specific expression of  <dig> out of  <dig> genes .
the accuracy of our gene expression measurement was also confirmed by the similarity between rna-seq and qrt-pcr fold change .
figure 2d illustrates the new compartment-specific expressions checked by qrt-pcr in oocytes  and in gcs .
*: pval <  <dig> ; **: pval < <dig> ; ***: pval <  <dig> .
*: pval <  <dig> ; ***: pval <  <dig> .
1: expression only detected in oocytes.
2: expression only detected in gcs.
biological trends
biological functions
to investigate the biological functions associated with the two compartments, the deg list was subjected to ingenuity pathway analysis  and tested for function enrichment .
in the oocyte compartment , we found a high statistical enrichment of genes involved in the cell cycle , gene regulation, cell morphology and organization, and the reproductive system.
the cell cycle function  was also enriched in gcs.
last, we observed different involvement of oocytes and gcs in the reproductive system.
on one hand, oocytes over-expressed  <dig> genes that have been shown to play important roles in fertility.
the genes over-expressed in gcs  were found to be mainly related to cellular proliferation and atresia , cell morphology  and movement, lipid metabolism and the reproductive system such as bmp <dig>  gdf <dig>  boll, figla, mos, piwil <dig>  insl <dig>  on the other hand, gcs over-expressed  <dig> genes involved in ovarian tumors of which eight were related to gc cancer .
figure 3a is a graphic representation of the deg biological functions in which the  <dig> main categories  are shown.
canonical pathways
based on the results of the ipa canonical pathway analysis, we identified  <dig> significant pathways that encompassed metabolism, regulatory and cell signaling.
these pathways are shown in figures 3b-d. this analysis underlined two different cell cycle checkpoint regulations according to the compartment: enrichment of genes involved in the g1/s checkpoint for gcs and in the g2/m dna damage checkpoint for oocytes .
we observed significant cellular proliferation and apoptosis mechanisms in gcs, and gene enrichment in nuclear signaling receptors such as pparg, lrx/rxr and prx/rxr involved in lipid metabolism.
we also identified gc-specific enrichment pathways such as camp-mediated, p <dig> mapk, wnt/β-catenin and pdgf signaling pathways.
figures 3c-d provide further evidence for the over-expression of growth hormone-encoding genes in gcs compared to oocytes during early ovarian folliculogenesis, and for the involvement of pten, calcium, enos and shh signaling pathways in gcs.
last, we observed enrichment of genes involved in ampk signaling related to gc energy regulation.
cell-cell interactions
in mammals, bidirectional communications between oocyte-gc and gc-gc are required for meiotic maturation, acquisition of oocyte competence, and follicle development.
analysis of the canonical pathways identified molecular events involved in these communications including igf <dig>  vegf, fgf, notch and tight junction signaling pathways.
figure  <dig> illustrates oocyte-gc cross-talk with the over expression of igf1r gene in oocytes  and the over expression of igf <dig> gene in gcs .
regulation of the igf <dig> system was then achieved through the over expression of igfbp <dig>  igfbp <dig>  igfbp <dig>  igfbp <dig> genes in gcs.
in the same way, vegfa was highly expressed in gcs  while its receptor flt <dig> was over expressed in oocytes  .
the involvement of fgf <dig> in oocyte-gc dialog was also evidenced by the over expression of fgf <dig> in oocytes and of fgfr <dig> and fgfr <dig> in gcs.
we also revealed for the first time over expression of fgf family members such as fgf <dig> , fgf <dig>  in oocytes and fgf <dig> , fgf <dig> , fgf <dig>  in gcs.
notch signaling was assumed to be involved in two kinds of interactions.
firstly, the abundance of notch <dig> transcripts in gcs and of its ligand jag <dig> in oocyte confirmed compartmental interactions .
secondly, this pathway could be involved in gc interactions via its functional ligand cntn <dig> .
finally, we detected the differential expression of genes involved in cellular connections and adhesions.
gja <dig> , which is over expressed in gcs, and gja <dig> , which is over expressed in oocytes , are known to participate to gap junctions and connect oocytes with gcs.
we also observed over expression of genes involved in the adherent junctions  and in the tight junctions  .
transcription regulators
oocyte and gc transcription factors  play an important role in folliculogenesis  <cit> .
they control both oocyte development and gc function.
to identify new transcription regulators, we performed ipa upstream regulator analysis on the deg data set.
for each compartment, we predicted the transcriptional regulators that were over expressed and involved in the regulation of this deg:  <dig> gc regulators and  <dig> oocyte regulators .
among them, the oocyte transcription regulator nobox and  <dig> gc transcription regulators  were predicted to have their protein activated.
discussion
to our knowledge, this is the first comprehensive exploration of transcriptome from oocyte and granulosa cells separately and at several key stages corresponding to in vivo major transitions during early folliculogenesis in sheep.
the study resulted in reliable transcriptional profiling in time and space.
we explored changes in gene expression between the two follicular compartments and identified specific transcriptional programs in either oocyte or granulosa cells.
in addition, we identified pathways involved in bidirectional communication.
thanks to mouse genetic models, a significant number of targeted molecular data  has been accumulated during the past years.
recently, these data were complemented by mouse and human oocyte transcriptome data that give us more information about the molecular mechanisms involved in early folliculogenesis.
our study expanded transcriptome data to gc and enabled us to determine the precise expression of genes involved in these mechanisms in a mono ovulating species.
oocyte and gc transcriptional profiling
this study documented the expression of  <dig>  <dig> genes  in ovarian follicles during early follicular development.
thanks to the sensitivity of rnaseq, we estimated a larger number of genes expressed in oocytes  than other microarray studies .
in our study,  <dig>  <dig> genes were also estimated to be expressed in gcs.
these high numbers of expressed genes were close to the higher number of genes expressed in testis  compared to the other human and mouse tissues   <cit> .
the high number of expressed genes in both oocytes and gc evidenced strong transcriptional activity.
interestingly, this analysis revealed a slight decrease in the number of oocyte expressed genes during follicular development from sc follicles that coincides with the gain in methylation from pm to sc follicle transition   <cit> .
the detection of a large number of ubiquitous genes  was previously reported by ramköld et al.
<cit>  suggesting that functional specialization is not associated with a large population of genes but results in more subtle and complex control.
rna-seq has made it possible to identify novel transcripts.
in this study, we uncovered  <dig>  <dig> genomic fragments that now need to be annotated.
some of them present a high expression level and compartment-specific expression  suggesting they play an important role in early folliculogenesis.
further exploration using bioinformatics and gene expression validation will be necessary to refine these results.
differential expression between compartments reflects the fate of the cell types
the development of the follicle and the maturation of oocyte require orchestrated communication between oocytes and gcs at all stages of early folliculogenesis.
they also require the expression of compartment-specific genes.
to investigate these molecular processes, we focused our analysis on differences in gene expression between oocytes and gcs and identified a large number  of differentially expressed genes.
we identified functional differences between oocytes and gcs and described the characteristics of each compartment that lead to two specific cell fates.
as described in our previous study  <cit> , the top functions of differentially expressed genes illustrated the three main cellular events known to be involved in early follicular development: the change in the shape of the gc cell from flattened to cuboidal , the marked increase in the number of gcs  and oocyte enlargement  <cit>  .
deg comparison of genes identified in five previous studies validated this list .
in addition to the information provided by our previous study  <cit> , ipa rna-seq analysis increased available information thanks to the identification of additional deg.
meiotic regulation
in most mammals, meiosis is initiated during fetal life and stops prenatally in the diplotene stage of prophase i at the g2/m transition until development into a graafian follicle .
this study highlights oocyte over expression of genes involved in the regulation of meiotic progression, acquisition of meiotic maturation, and maintenance of meiotic arrest.
for instance, spo <dig> is involved in initiating meiotic recombination by catalyzing dna double strand breaks  <cit> .
btrc loss of function in mice results in slow progression through meiosis and abnormal divisions in spermatocytes  <cit> .
we also observed a significant increase in sheep ovol <dig> mrna  and asz <dig> mrna  during early folliculogenesis.
ovol <dig> is known to be involved in regulating the pachytene progression of male germ cells  <cit>  and shown to be expressed during sheep early folliculogenesis  <cit> .
in mice, the asz <dig> protein is a germ cell-specific protein located in the cytoplasm of oocytes at all stages of development that may act as an important signaling protein and/or transcriptional regulator during germ cell maturation  <cit> .
the function of these genes is poorly understood in mammalian ovary development but their expression in early follicular development suggests they play a role in the regulation of female meiosis.
finally, we confirmed the expression of genes already known to be involved in the regulation of the oocyte and/or spermatocyte meiotic arrest .
the establishment of gc steroid synthesis
this study clearly shows that gcs over-expressed genes involved in steroid synthesis  and in steroid regulation ).
nrs include orphan receptors , steroid hormone receptors , fxr ), and retinoid acid receptors , all of which are over expressed in gcs during preantral follicle stages.
nr5a <dig> , which has already been described in sheep gcs  <cit> , is a master gene that regulates the expression of numerous genes including reproductive and steroidogenic enzyme-encoding genes  <cit> .
gc specific sf- <dig> ko mice are infertile whose ovaries contain a reduced number of follicles and reduced expression of amh, which partially correlates with the reserve of ovarian follicles  <cit> .
e <dig> and androgens hormones exert their influence on early folliculogenesis via their respective receptors, esr and ar.
like in mice, e <dig> probably plays a role in controlling the primordial follicle pool  <cit>  and androgen signaling is crucial for normal folliculogenesis  <cit> .
we observed strong expression of ar in gcs, like in humans  <cit> .
finally, for the first time, we identified over expression of lxrb, fxra and rxra in gc preantral stages.
rxr is a retinoid x receptor that binds as heterodimers  and becomes transcriptionally active only in the presence of a ligand .
lxrs are activated by oxysterols ) and are key regulators of ovarian steroidogenesis at antral stages  <cit> .
fxrs are activated by bile acid, sterol and different lipids and are possibly important regulators of androgen homoeostasis in male gonads  <cit> .
complex regulation of follicular atresia
atresia is an important process in folliculogenesis and concerns the majority of the follicles.
apoptosis is found in the oocytes of primordial follicles and progressively extends to gcs of growing follicles.
we characterized the expression of different genes of the bcl <dig> family either over expressed in oocytes , bcl2l <dig> ) or in gcs  and that mainly prevent oocyte loss.
for example, the bcl2l <dig> gene was shown to play a crucial role in the survival of germ cells  <cit> .
bcl2l <dig> may antagonize bax activities and may also play other roles related to cell cycle control and oocyte maturation .
in sheep, the high expression of bcl2l <dig> in oocytes and its up-regulation during preantral stages reinforces its already important role in oocyte development.
by decreasing gc atresia, the bcl <dig> gene family expressed in gcs, such as bcl <dig>  was also believed to increase the number of germ cells and enhance their development.
lastly, bok mrna, was found to be restricted to reproductive tissue and was detected in gcs from pm to antral follicles in rat species  <cit> .
gc signaling pathways
the molecular pathways were mainly linked to gc proliferation, steroid production, and morphological change.
sheep gcs over expressed members of the sonic hedgehog  signaling pathway , which is consistent with a potential role for the hh pathway in increased gc proliferation during preantral stages  <cit> .
last, genes of the wnt family and their receptors  and rho-gtpase activating proteins  were shown to be over expressed in gcs.
in the ovary, the β-catenin dependant canonical pathway of wnt <dig> leads to ovarian determining pathways.
wnt proteins can also signal via rho-gtpases, using non-canonical pathways that involve changes in polarized cell shape and cell migrations  <cit> .
this pathway may promote changes in cell shape that affect gcs during early folliculogenesis.
cell-cell communications
follicular growth depends on close interactions between oocytes and gcs but also between gcs themselves.
two kinds of cell interactions are described in this study demonstrating the existence of complex dialogs between compartments: a molecular dialog  and physical communications .
signaling pathways: gc regulation of oocyte development
gcs are known to play a role in supporting the growth of oocytes, regulating the progression of meiosis, and modulating global oocyte transcription activity.
nevertheless, so far, few factors have been identified that support these assumptions.
to date, the most widely studied ligand-receptor systems  are the kit/kitl and bmp pathways.
below we describe the involvement of other signaling pathways.
the notch signaling pathway plays many roles in cell communication by influencing cell proliferation, differentiation, and apoptosis  <cit> .
in sheep, we found over expression of notch1-2- <dig> in gcs and over expression of the notch ligands jag <dig> and dll <dig> in oocytes that perfectly illustrate oocyte-gc crosstalk  and that may influence oocyte apoptosis, like in postnatal mice  <cit> .
in addition, we suggest that notch is involved in gc proliferation  <cit>  through notch1/cntn <dig> binding .
vascular endothelial growth factor  was already known to promote early folliculogenesis  <cit> .
in this study, we identified over expression of members of vegf pathway including vegfa and nrp <dig>  in gcs, and over expression of flt <dig>  in oocytes.
these gene expressions suggest a role for the vegf pathway in gc-oocyte and gc-gc crosstalks .
as already described, the vegf pathway may protect gc against atresia  <cit> .
the crucial role of the igf system is largely described during terminal follicular growth  <cit>  but is poorly documented in preantral stages.
ovarian igf <dig> production was shown to vary with the species.
igf <dig> is mainly expressed by gcs in rat and pig species  <cit>  but its protein was also detected in the oocyte of rat preantral follicles  <cit> .
moreover, the igf <dig> transcript was not detected in adult sheep  <cit>  or cattle ovaries  <cit>  but its receptor igf1r was expressed from primary follicles and its expression increased with follicular growth  <cit> .
our study provides a precise description of members of the igf system in sheep species at preantral stages .
the igf <dig> pathway  suggests a role for igf <dig> in oocytes and gcs in agreement with studies on mice.
indeed, although insulin/igf signaling is not essential in oocyte conditional knockout mice  <cit> , igf- <dig> significantly increases oocyte maturation  <cit> .
in gcs, the expression of igfr <dig> and the over expression of the downstream signaling molecules irs <dig> and akt <dig> are also in agreement with the involvement of igf <dig> in the regulation of gc survival  <cit> .
additional immunohistochemistry to detect igf <dig> and igf1r proteins and in vitro early follicle cultures could be used to evaluate the effect of igf <dig> on sheep preantral growth.
in vitro early follicle cultures are described in mochida et al  <cit> .
signaling pathways: oocyte regulation of gc function
numerous studies have shown that oocytes are potent stimulators of gc proliferation.
a compilation of oocyte-secreted factors that regulate gc function was published in  <dig>  <cit> .
among these factors, we describe the over expression of fgf <dig> in sheep oocytes and fgfr1-2-l <dig> receptors in gcs that may influence primordial follicle development in accordance with in vitro goat and rat studies  <cit> .
we also report the over expression of fgf16- <dig> in oocytes for the first time in mammals.
a recent study on nile tilapia species suggests that fgf16- <dig> are involved in early oocyte development in the female  <cit> .
in other mammal tissues, fgf <dig> and fgf <dig> promote cell proliferation and differentiation.
physical interactions
transzonal projections  are gc specialized extensions corresponding to long cell projections and microvilli that are in close contact with the surface of the oolemma or invaginate the cortical area of the oocyte.
these projections establish communicative  and adhesive junctions  both with the oocyte and neighboring transzonal projections.
in this study, we observed an enrichment of genes involved in the formation of cellular protrusions in both gcs and oocytes .
we confirmed over expression of two gap junction genes that are indispensable for the regulation of folliculogenesis and oogenesis: gja <dig>  in gcs and gja <dig>  in oocytes.
moreover in our study, zp2- <dig> were seen to significantly increase during preantral stages as shown in human fetal ovaries before the formation of zona pellicida <cit> .
regulators
in the present experiment, we identified a set of upstream transcriptional regulators able to regulate follicle gene expression and ovarian development.
in sheep oocytes, nobox, which is known to promote follicular growth in mice, was detected in this study at medium expression level in preantral oocytes and was predicted to increase gene expression of bmp <dig>  dnmt <dig>  gdf <dig>  h1foo, mos, zar <dig> genes  <cit> .
analysis of ipa upstream regulators also predicted activation of ahr in oocytes .
ahr protein may act on oocyte gene expression through the receptor nuclear translocator arntl <dig> over expressed in oocytes.
these findings are consistent with ahr regulation of follicular growth by affecting germ cell death during female gametogenesis  <cit> .
in gcs, transcriptional regulators referred to sterol regulation  hormone response , cellular growth , cellular differentiation , and increased transcription .
here we describe for the first time, over expression of nfia and hif1a in gcs of preantral follicles.
nfia is a transcription factor involved in the control of cell growth in both humans and model systems  <cit> .
hif1a is an arnt interacting protein that activates the transcription of target genes involved in energy metabolism, angiogenesis, and apoptosis  <cit> .
we were able to predict the regulation of  <dig> genes by hif1a and we observed an increased in its expression during early folliculogenesis, assuming it plays a role in preantral development.
genes preferentially expressed according to cell type
oocyte-specific genes and their role in folliculogenesis have been extensively studied in murine knockout models  <cit>  but there is a relative paucity of information concerning gc-specific genes.
in this study, we completed existing data by highlighting the expression of  <dig> preferentially expressed genes , including genes whose function in ovarian folliculogenesis is unknown.
nevertheless, gcs exhibited fewer preferentially expressed genes than oocytes.
in gcs, the defb <dig> gene is described for the first time as preferentially expressed.
this gene is poorly documented but is reported to have a regionalized expression in the epididymis in rat and in human and to be related to sperm maturation  <cit> .
as already described in sheep, the preferential expression of bmp <dig>  which promotes bmp signaling via chordinase activities  <cit> , was confirmed.
finally, the molecular chaperone tcp <dig>  which assists the folding of proteins upon atp hydrolysis , was specifically detected at a slight but constant level of expression during the preantral stage.
tcp <dig> appears to play a key role in the cytodifferentiation of spermatids that relies on the high plasticity of microtubules  <cit> .
our result may be associated to the remodeling of the microtubule cytoskeleton during the change in cell shape.
in oocytes, we identified the preferential expression of genes such as btg <dig>  although much about the function of btg <dig> remains unknown, it is known to be involved in cell cycle arrest and to be endowed with antiproliferative properties.
in mice, its expression is restricted to the olfactory epithelium, testis, and oocyte  <cit> .
the role of btg <dig> during preantral stages in sheep requires further investigation.
another gene of interest is patl <dig>  a rna-binding protein  that was originally characterized as being oocyte specific in xenopus laevis, where it acts as a translational repressor in a cpeb-containing complex  <cit> .
in xenopus laevis, patl <dig> is maternally expressed in immature oocytes, but disappears upon meiotic maturation.
some authors suggest that patl <dig> plays a role in regulating the translation of specific maternal mrnas required for the progression of xenopus laevis oocyte maturation  <cit> .
the expression of patl <dig> has not yet been documented in mammals; but here we describe for the first time increased specific expression of this gene in oocytes during preantral stages in sheep.
in comparison with the role of patl <dig> in xenopus laevis, our finding suggests it plays an important role in the regulation of the translation in preantral oocytes in sheep.
finally, we identified over expression of musk in oocytes at preantral stages.
musk is a receptor tyrosine kinase  which is induced during skeletal muscle differentiation, and is dramatically down-regulated in mature muscle  <cit> .
this study highlighted involvement of new gene expression in early folliculogenesis.
further investigations are underway to estimate the implication of these genes in mammalian folliculogenesis.
CONCLUSIONS
this work generated a significant amount of expression data that accurately document early folliculogenesis in sheep.
the abundance of expressed genes and the differential expression profiles found in oocytes confirm the hypothesis that oocytes are dynamic cells.
the decrease in the number of expressed genes following the development of oocytes is in accordance with a general decrease in transcription.
this study identified specific functions and molecular mechanisms involved in each cell type during early folliculogenesis and in the dialog between the different cell types.
we confirm and incorporate a large number of individual studies in different species.
last, we highlight new gene expressions that support and extend current knowledge on early folliculogenesis.
this study is the first thorough, integrated overview of the molecular mechanisms involved in early folliculogenesis in a mono-ovulating species.
it provides a valuable data base for future functional studies in mono-ovulating species including humans.

 the major function of an ovary is to produce oocytes intended for fertilization as eggs and to create a favorable environment for the beginning of gestation. in adult mammals, the ovary is a heterogeneous organ containing follicles at various stages of development and corpora lutea, either active or at various stages of involution. the formation of primordial follicles occurs during fetal development in many species including human, sheep, cattle, and goat, or after birth like in rodents. the primordial follicles represent a reserve of germ cells for the entire reproductive life of the female. they remain dormant until their recruitment and irreversible growth towards the primary, secondary and tertiary stages. the gradual exit of primordial follicles begins shortly after the formation of the primordial follicle pool and continues throughout the reproductive years. consequently, this early development is important as it regulates the size of the resting primordial follicle pool and the fate of the follicles, which, in turn, affects fertility and the reproductive life span. early follicular development requires the appropriate expression of many genes at different developmental stages. recent studies on natural mutations in sheep and on mutant mouse models demonstrated that the expression of different oocyte-specific genes is essential during early folliculogenesis in a stage specific expression pattern. figla, nobox, pten, foxo3a and nerve growth factor genes are involved in the formation of primordial follicles, whereas genes such as βfgf, gdf and bmp are involved in the transition from primordial to primary follicles. other genes such as bmp are not expressed in the oocyte until the primary follicle stage and are involved in the transition from primary to secondary follicles, as shown in sheep. this process also requires orchestrated communication between oocytes and granulosa cells, which are the structural components of the early follicle. oocytes and gcs regulate follicle growth in an autocrine and paracrine manner via secreted factors and direct gap junctional communications. on one hand, it is assumed that oocytes control the proliferation of gcs and later, their differentiation into steroid secreting cells and their metabolism. on the other hand, gcs are indispensable to oocyte growth, meiosis, cytoplasmic maturation and control of transcriptional activity within the oocyte. this dialog also takes place through gc cytoplasmic extensions that penetrate the zona pellucida and form gap junctions with oocyte cell membrane. although some large scale expression studies have been conducted on rodent and human oocytes, expression profiling of follicular compartments is difficult to accomplish due to the very small size of the follicles and the mixture of preantral stages in the ovary. two studies in particular identified the specific transcriptome of human oocytes in the quiescent state. these studies highlighted enriched functions and molecular mechanisms in human oocytes, which were also identified in the only preliminary analysis conducted in sheep species to date. but exactly which granulosa cell factors play a specialized role in early follicular development is not known. our knowledge of the key mechanisms and the bidirectional communication underlying early folliculogenesis is consequently still very limited and is mainly derived from rodents. spatial and temporal information on gene expression patterns would facilitate the identification of the regulatory gene networks that underlie cell growth, differentiation, and the functional specification of each follicular compartment. the purpose of this study was thus to 1- describe global gene expression during early ovarian folliculogenesis in each follicular compartment in mono-ovulating species using sheep as the experimental model; 2- identify differential and specific gene expression between these two follicular compartments; 3- investigate specific functions and pathways, and 4- explore bi-directional communication between oocytes and gcs. this was achieved using laser capture microdissection to isolate oocytes and gcs during the earliest stages of folliculogenesis. gene expression profiles were monitored at each follicle stage according to cell type using rna-seq technology. we report the successful isolation and generation of global gene expression data sets of pure oocytes and gc populations at key stages of early follicular development. we describe the expression profile of genes with % of genes displaying differential expression between the two compartments. in addition, we identified oocyte and gc-specific gene expression and the pathways involved in oocyte-gc communication.  we combined laser capture microdissection and rna-sequencing technologies to detect separate gcs and oocyte global transcriptome for each stage of follicle development: primordial, primary, secondary follicles and the small antral stage as control. three/four biological replicates were obtained per condition. for the experimental protocol, see additional file 1: figure s the rna-seq experiment generated around billion 100 bp reads with an average of million per lcm-derived amplified rna sample. the result of the bioinformatics and annotation processing is summarized in figure  and detailed in additional file briefly, the genome assembly approach generated a collection of fragments that aggregated % of lcm-arna reads. after 3′utr selection, the final dataset included genes. genomic fragments remained unannotated. exploring global gene expression the genes correspond to 62% of the bovine annotated genes in ensembl umd release and to % of the ovine annotated genes in csiro oarv. as expected, the number of expressed genes was uniformly distributed between chromosomes according to the number of expected genes except for chromosome on which significantly fewer genes were expressed. the percentage of genes transcribed for this chromosome was 57%, compared to an average of 80% for the other chromosomes. the chromosome coverage of the expressed genes overlays oarv annotated genes except for chromosome on which we identified two regions with fewer expressed genes. the density of expressed genes in these two regions was 75%-90% lower. for each sample, the expression levels were measured in read counts per gene and a normalized expression ) was calculated to describe the transcriptome of the tissue. first, we visualized a dynamic range of gene expression between and rpm. we estimated that 95% of the genes aggregated 56% of the reads. we then examined the highest expressed genes and detected a clear distinction between cell types. the highest oocyte-expressed genes contributed less to the total number of reads than gcs and multi-tissue samples. for example, the ten highest oocyte-expressed genes contributed % to % of the total reads, while the highest expressed gc and mt genes contributed respectively 5% to % and % of the total reads. in oocytes, the most expressed genes included zp zp and gdf genes known to be oocyte specific, but also actg, paip pdk in gcs, the most expressed genes included mainly ribosomal genes, such as rpl rpl rps genes involved in cellular assembly and organization, such as actg, collagens, vim and cell growth regulators, such as mgp. among these expressed genes, we observed a significant decrease in oocyte-expressed genes during early follicular development, i.e., from genes in primordial oocytes to genes in small antrum oocytes. conjointly genes were found to be expressed in all the samples and were considered as ubiquitously expressed genes. however, their relative expression levels varied depending on the type of cell, for example ribosomal genes, vim, sparc, and low expressed genes such as bscl in addition to this qualitative assessment, the transcriptome data set was analyzed using the r software package, deseq. principal component analysis of the deseq normalized data confirmed the relevance of the data. the first axis explained 52% of expression variability and separated the two follicular compartments. the second axis explained 15% of expression variability and separated the samples according to their follicular stages. global differential gene expression to identify differentially expressed genes, a generalized linear model was applied to the data after deseq normalization. this model allowed statistical evaluation of the factor effects and their interactions. the following criteria including fdr <  % to 1% and a fold change > were chosen to determine significant differentially expressed genes. using these criteria, % of genes were significantly differentially expressed between the two compartments; % of genes were significantly differentially expressed during early development. we observed differences in expression profiles during early folliculogenesis between the two compartments for % of the genes. for example, the expression of the kcnn gene decreased in gcs during early development whereas its expression increased in oocytes. differential expression is detailed in table  which, interestingly, shows that genes were under expressed in oocytes compared to in gcs. focus on differential expression between compartments we focused our analysis on the significant differentially expressed genes between compartments. the aim was to provide an overview of specific gene expression, mechanisms and functions in these cell types, which may be involved in the dialog between the oocyte and gcs, and regulate follicle development. first, we observed numerous genes on the deg list already known to have cell-type specific expression. the expression of genes known to be compartment specific was investigated and the expression of of them was confirmed by the rna-seq statistical analysis. last, the expression of 33% of the genes varied during early follicular development. supervised hierarchical clustering was performed to identify groups of genes with similar expression profiles. this led to a clear separation between the two compartments and identified clusters of genes according to the stage of follicular development, suggesting a difference in the dynamics of transcription between oocytes and gcs. compartment specific expression previous studies that were mainly devoted to rodent species identified oocyte-specific genes with important functions related to oocyte development and folliculogenesis. however, few data are available for gcs. to gain insight into the specific expression of each compartment, we performed a pair-wise comparison of the transcriptome of each compartment and both mt and the other compartment transcriptomes. in this way, we identified highest differentially expressed genes in gcs and highest differentially expressed genes in oocytes and the gc-restricted expression of four transcription regulators. for a list of the highest differentially expressed genes, see additional file validation of differential expression by quantitative real time rt-pcr to validate the rna-seq analysis, the expression profiles of a subset of genes of interest were monitored using qrt-pcr. these genes were selected as follows: genes showing significant up or down expression in oocytes, genes involved in particular canonical pathways or compartment-specifically expressed genes. statistical analysis confirmed the differential expression between compartments observed in rna-seq data for all the genes tested and the compartment-specific expression of out of genes. the accuracy of our gene expression measurement was also confirmed by the similarity between rna-seq and qrt-pcr fold change. figure 2d illustrates the new compartment-specific expressions checked by qrt-pcr in oocytes and in gcs. *: pval < ; **: pval <; ***: pval < . *: pval < ; ***: pval < . 1: expression only detected in oocytes. 2: expression only detected in gcs. biological trends biological functions to investigate the biological functions associated with the two compartments, the deg list was subjected to ingenuity pathway analysis and tested for function enrichment. in the oocyte compartment, we found a high statistical enrichment of genes involved in the cell cycle, gene regulation, cell morphology and organization, and the reproductive system. the cell cycle function was also enriched in gcs. last, we observed different involvement of oocytes and gcs in the reproductive system. on one hand, oocytes over-expressed genes that have been shown to play important roles in fertility. the genes over-expressed in gcs were found to be mainly related to cellular proliferation and atresia, cell morphology and movement, lipid metabolism and the reproductive system such as bmp gdf boll, figla, mos, piwil insl on the other hand, gcs over-expressed genes involved in ovarian tumors of which eight were related to gc cancer. figure 3a is a graphic representation of the deg biological functions in which the main categories are shown. canonical pathways based on the results of the ipa canonical pathway analysis, we identified significant pathways that encompassed metabolism, regulatory and cell signaling. these pathways are shown in figures 3b-d. this analysis underlined two different cell cycle checkpoint regulations according to the compartment: enrichment of genes involved in the g1/s checkpoint for gcs and in the g2/m dna damage checkpoint for oocytes. we observed significant cellular proliferation and apoptosis mechanisms in gcs, and gene enrichment in nuclear signaling receptors such as pparg, lrx/rxr and prx/rxr involved in lipid metabolism. we also identified gc-specific enrichment pathways such as camp-mediated, p mapk, wnt/β-catenin and pdgf signaling pathways. figures 3c-d provide further evidence for the over-expression of growth hormone-encoding genes in gcs compared to oocytes during early ovarian folliculogenesis, and for the involvement of pten, calcium, enos and shh signaling pathways in gcs. last, we observed enrichment of genes involved in ampk signaling related to gc energy regulation. cell-cell interactions in mammals, bidirectional communications between oocyte-gc and gc-gc are required for meiotic maturation, acquisition of oocyte competence, and follicle development. analysis of the canonical pathways identified molecular events involved in these communications including igf vegf, fgf, notch and tight junction signaling pathways. figure  illustrates oocyte-gc cross-talk with the over expression of igf1r gene in oocytes and the over expression of igf gene in gcs. regulation of the igf system was then achieved through the over expression of igfbp igfbp igfbp igfbp genes in gcs. in the same way, vegfa was highly expressed in gcs while its receptor flt was over expressed in oocytes. the involvement of fgf in oocyte-gc dialog was also evidenced by the over expression of fgf in oocytes and of fgfr and fgfr in gcs. we also revealed for the first time over expression of fgf family members such as fgf, fgf in oocytes and fgf, fgf, fgf in gcs. notch signaling was assumed to be involved in two kinds of interactions. firstly, the abundance of notch transcripts in gcs and of its ligand jag in oocyte confirmed compartmental interactions. secondly, this pathway could be involved in gc interactions via its functional ligand cntn. finally, we detected the differential expression of genes involved in cellular connections and adhesions. gja, which is over expressed in gcs, and gja, which is over expressed in oocytes, are known to participate to gap junctions and connect oocytes with gcs. we also observed over expression of genes involved in the adherent junctions and in the tight junctions. transcription regulators oocyte and gc transcription factors play an important role in folliculogenesis. they control both oocyte development and gc function. to identify new transcription regulators, we performed ipa upstream regulator analysis on the deg data set. for each compartment, we predicted the transcriptional regulators that were over expressed and involved in the regulation of this deg: gc regulators and oocyte regulators. among them, the oocyte transcription regulator nobox and gc transcription regulators were predicted to have their protein activated. discussion to our knowledge, this is the first comprehensive exploration of transcriptome from oocyte and granulosa cells separately and at several key stages corresponding to in vivo major transitions during early folliculogenesis in sheep. the study resulted in reliable transcriptional profiling in time and space. we explored changes in gene expression between the two follicular compartments and identified specific transcriptional programs in either oocyte or granulosa cells. in addition, we identified pathways involved in bidirectional communication. thanks to mouse genetic models, a significant number of targeted molecular data has been accumulated during the past years. recently, these data were complemented by mouse and human oocyte transcriptome data that give us more information about the molecular mechanisms involved in early folliculogenesis. our study expanded transcriptome data to gc and enabled us to determine the precise expression of genes involved in these mechanisms in a mono ovulating species. oocyte and gc transcriptional profiling this study documented the expression of genes in ovarian follicles during early follicular development. thanks to the sensitivity of rnaseq, we estimated a larger number of genes expressed in oocytes than other microarray studies. in our study, genes were also estimated to be expressed in gcs. these high numbers of expressed genes were close to the higher number of genes expressed in testis compared to the other human and mouse tissues. the high number of expressed genes in both oocytes and gc evidenced strong transcriptional activity. interestingly, this analysis revealed a slight decrease in the number of oocyte expressed genes during follicular development from sc follicles that coincides with the gain in methylation from pm to sc follicle transition. the detection of a large number of ubiquitous genes was previously reported by ramköld et al. suggesting that functional specialization is not associated with a large population of genes but results in more subtle and complex control. rna-seq has made it possible to identify novel transcripts. in this study, we uncovered genomic fragments that now need to be annotated. some of them present a high expression level and compartment-specific expression suggesting they play an important role in early folliculogenesis. further exploration using bioinformatics and gene expression validation will be necessary to refine these results. differential expression between compartments reflects the fate of the cell types the development of the follicle and the maturation of oocyte require orchestrated communication between oocytes and gcs at all stages of early folliculogenesis. they also require the expression of compartment-specific genes. to investigate these molecular processes, we focused our analysis on differences in gene expression between oocytes and gcs and identified a large number of differentially expressed genes. we identified functional differences between oocytes and gcs and described the characteristics of each compartment that lead to two specific cell fates. as described in our previous study, the top functions of differentially expressed genes illustrated the three main cellular events known to be involved in early follicular development: the change in the shape of the gc cell from flattened to cuboidal, the marked increase in the number of gcs and oocyte enlargement. deg comparison of genes identified in five previous studies validated this list. in addition to the information provided by our previous study, ipa rna-seq analysis increased available information thanks to the identification of additional deg. meiotic regulation in most mammals, meiosis is initiated during fetal life and stops prenatally in the diplotene stage of prophase i at the g2/m transition until development into a graafian follicle. this study highlights oocyte over expression of genes involved in the regulation of meiotic progression, acquisition of meiotic maturation, and maintenance of meiotic arrest. for instance, spo is involved in initiating meiotic recombination by catalyzing dna double strand breaks. btrc loss of function in mice results in slow progression through meiosis and abnormal divisions in spermatocytes. we also observed a significant increase in sheep ovol mrna and asz mrna during early folliculogenesis. ovol is known to be involved in regulating the pachytene progression of male germ cells and shown to be expressed during sheep early folliculogenesis. in mice, the asz protein is a germ cell-specific protein located in the cytoplasm of oocytes at all stages of development that may act as an important signaling protein and/or transcriptional regulator during germ cell maturation. the function of these genes is poorly understood in mammalian ovary development but their expression in early follicular development suggests they play a role in the regulation of female meiosis. finally, we confirmed the expression of genes already known to be involved in the regulation of the oocyte and/or spermatocyte meiotic arrest. the establishment of gc steroid synthesis this study clearly shows that gcs over-expressed genes involved in steroid synthesis and in steroid regulation ). nrs include orphan receptors, steroid hormone receptors, fxr ), and retinoid acid receptors, all of which are over expressed in gcs during preantral follicle stages. nr5a, which has already been described in sheep gcs, is a master gene that regulates the expression of numerous genes including reproductive and steroidogenic enzyme-encoding genes. gc specific sf- ko mice are infertile whose ovaries contain a reduced number of follicles and reduced expression of amh, which partially correlates with the reserve of ovarian follicles. e and androgens hormones exert their influence on early folliculogenesis via their respective receptors, esr and ar. like in mice, e probably plays a role in controlling the primordial follicle pool and androgen signaling is crucial for normal folliculogenesis. we observed strong expression of ar in gcs, like in humans. finally, for the first time, we identified over expression of lxrb, fxra and rxra in gc preantral stages. rxr is a retinoid x receptor that binds as heterodimers and becomes transcriptionally active only in the presence of a ligand. lxrs are activated by oxysterols ) and are key regulators of ovarian steroidogenesis at antral stages. fxrs are activated by bile acid, sterol and different lipids and are possibly important regulators of androgen homoeostasis in male gonads. complex regulation of follicular atresia atresia is an important process in folliculogenesis and concerns the majority of the follicles. apoptosis is found in the oocytes of primordial follicles and progressively extends to gcs of growing follicles. we characterized the expression of different genes of the bcl family either over expressed in oocytes, bcl2l ) or in gcs and that mainly prevent oocyte loss. for example, the bcl2l gene was shown to play a crucial role in the survival of germ cells. bcl2l may antagonize bax activities and may also play other roles related to cell cycle control and oocyte maturation. in sheep, the high expression of bcl2l in oocytes and its up-regulation during preantral stages reinforces its already important role in oocyte development. by decreasing gc atresia, the bcl gene family expressed in gcs, such as bcl was also believed to increase the number of germ cells and enhance their development. lastly, bok mrna, was found to be restricted to reproductive tissue and was detected in gcs from pm to antral follicles in rat species. gc signaling pathways the molecular pathways were mainly linked to gc proliferation, steroid production, and morphological change. sheep gcs over expressed members of the sonic hedgehog signaling pathway, which is consistent with a potential role for the hh pathway in increased gc proliferation during preantral stages. last, genes of the wnt family and their receptors and rho-gtpase activating proteins were shown to be over expressed in gcs. in the ovary, the β-catenin dependant canonical pathway of wnt leads to ovarian determining pathways. wnt proteins can also signal via rho-gtpases, using non-canonical pathways that involve changes in polarized cell shape and cell migrations. this pathway may promote changes in cell shape that affect gcs during early folliculogenesis. cell-cell communications follicular growth depends on close interactions between oocytes and gcs but also between gcs themselves. two kinds of cell interactions are described in this study demonstrating the existence of complex dialogs between compartments: a molecular dialog and physical communications. signaling pathways: gc regulation of oocyte development gcs are known to play a role in supporting the growth of oocytes, regulating the progression of meiosis, and modulating global oocyte transcription activity. nevertheless, so far, few factors have been identified that support these assumptions. to date, the most widely studied ligand-receptor systems are the kit/kitl and bmp pathways. below we describe the involvement of other signaling pathways. the notch signaling pathway plays many roles in cell communication by influencing cell proliferation, differentiation, and apoptosis. in sheep, we found over expression of notch1-2- in gcs and over expression of the notch ligands jag and dll in oocytes that perfectly illustrate oocyte-gc crosstalk and that may influence oocyte apoptosis, like in postnatal mice. in addition, we suggest that notch is involved in gc proliferation through notch1/cntn binding. vascular endothelial growth factor was already known to promote early folliculogenesis. in this study, we identified over expression of members of vegf pathway including vegfa and nrp in gcs, and over expression of flt in oocytes. these gene expressions suggest a role for the vegf pathway in gc-oocyte and gc-gc crosstalks. as already described, the vegf pathway may protect gc against atresia. the crucial role of the igf system is largely described during terminal follicular growth but is poorly documented in preantral stages. ovarian igf production was shown to vary with the species. igf is mainly expressed by gcs in rat and pig species but its protein was also detected in the oocyte of rat preantral follicles. moreover, the igf transcript was not detected in adult sheep or cattle ovaries but its receptor igf1r was expressed from primary follicles and its expression increased with follicular growth. our study provides a precise description of members of the igf system in sheep species at preantral stages. the igf pathway suggests a role for igf in oocytes and gcs in agreement with studies on mice. indeed, although insulin/igf signaling is not essential in oocyte conditional knockout mice, igf- significantly increases oocyte maturation. in gcs, the expression of igfr and the over expression of the downstream signaling molecules irs and akt are also in agreement with the involvement of igf in the regulation of gc survival. additional immunohistochemistry to detect igf and igf1r proteins and in vitro early follicle cultures could be used to evaluate the effect of igf on sheep preantral growth. in vitro early follicle cultures are described in mochida et al. signaling pathways: oocyte regulation of gc function numerous studies have shown that oocytes are potent stimulators of gc proliferation. a compilation of oocyte-secreted factors that regulate gc function was published in. among these factors, we describe the over expression of fgf in sheep oocytes and fgfr1-2-l receptors in gcs that may influence primordial follicle development in accordance with in vitro goat and rat studies. we also report the over expression of fgf16- in oocytes for the first time in mammals. a recent study on nile tilapia species suggests that fgf16- are involved in early oocyte development in the female. in other mammal tissues, fgf and fgf promote cell proliferation and differentiation. physical interactions transzonal projections are gc specialized extensions corresponding to long cell projections and microvilli that are in close contact with the surface of the oolemma or invaginate the cortical area of the oocyte. these projections establish communicative and adhesive junctions both with the oocyte and neighboring transzonal projections. in this study, we observed an enrichment of genes involved in the formation of cellular protrusions in both gcs and oocytes. we confirmed over expression of two gap junction genes that are indispensable for the regulation of folliculogenesis and oogenesis: gja in gcs and gja in oocytes. moreover in our study, zp2- were seen to significantly increase during preantral stages as shown in human fetal ovaries before the formation of zona pellicida. regulators in the present experiment, we identified a set of upstream transcriptional regulators able to regulate follicle gene expression and ovarian development. in sheep oocytes, nobox, which is known to promote follicular growth in mice, was detected in this study at medium expression level in preantral oocytes and was predicted to increase gene expression of bmp dnmt gdf h1foo, mos, zar genes. analysis of ipa upstream regulators also predicted activation of ahr in oocytes. ahr protein may act on oocyte gene expression through the receptor nuclear translocator arntl over expressed in oocytes. these findings are consistent with ahr regulation of follicular growth by affecting germ cell death during female gametogenesis. in gcs, transcriptional regulators referred to sterol regulation hormone response, cellular growth, cellular differentiation, and increased transcription. here we describe for the first time, over expression of nfia and hif1a in gcs of preantral follicles. nfia is a transcription factor involved in the control of cell growth in both humans and model systems. hif1a is an arnt interacting protein that activates the transcription of target genes involved in energy metabolism, angiogenesis, and apoptosis. we were able to predict the regulation of genes by hif1a and we observed an increased in its expression during early folliculogenesis, assuming it plays a role in preantral development. genes preferentially expressed according to cell type oocyte-specific genes and their role in folliculogenesis have been extensively studied in murine knockout models but there is a relative paucity of information concerning gc-specific genes. in this study, we completed existing data by highlighting the expression of preferentially expressed genes, including genes whose function in ovarian folliculogenesis is unknown. nevertheless, gcs exhibited fewer preferentially expressed genes than oocytes. in gcs, the defb gene is described for the first time as preferentially expressed. this gene is poorly documented but is reported to have a regionalized expression in the epididymis in rat and in human and to be related to sperm maturation. as already described in sheep, the preferential expression of bmp which promotes bmp signaling via chordinase activities, was confirmed. finally, the molecular chaperone tcp which assists the folding of proteins upon atp hydrolysis, was specifically detected at a slight but constant level of expression during the preantral stage. tcp appears to play a key role in the cytodifferentiation of spermatids that relies on the high plasticity of microtubules. our result may be associated to the remodeling of the microtubule cytoskeleton during the change in cell shape. in oocytes, we identified the preferential expression of genes such as btg although much about the function of btg remains unknown, it is known to be involved in cell cycle arrest and to be endowed with antiproliferative properties. in mice, its expression is restricted to the olfactory epithelium, testis, and oocyte. the role of btg during preantral stages in sheep requires further investigation. another gene of interest is patl a rna-binding protein that was originally characterized as being oocyte specific in xenopus laevis, where it acts as a translational repressor in a cpeb-containing complex. in xenopus laevis, patl is maternally expressed in immature oocytes, but disappears upon meiotic maturation. some authors suggest that patl plays a role in regulating the translation of specific maternal mrnas required for the progression of xenopus laevis oocyte maturation. the expression of patl has not yet been documented in mammals; but here we describe for the first time increased specific expression of this gene in oocytes during preantral stages in sheep. in comparison with the role of patl in xenopus laevis, our finding suggests it plays an important role in the regulation of the translation in preantral oocytes in sheep. finally, we identified over expression of musk in oocytes at preantral stages. musk is a receptor tyrosine kinase which is induced during skeletal muscle differentiation, and is dramatically down-regulated in mature muscle. this study highlighted involvement of new gene expression in early folliculogenesis. further investigations are underway to estimate the implication of these genes in mammalian folliculogenesis.  this work generated a significant amount of expression data that accurately document early folliculogenesis in sheep. the abundance of expressed genes and the differential expression profiles found in oocytes confirm the hypothesis that oocytes are dynamic cells. the decrease in the number of expressed genes following the development of oocytes is in accordance with a general decrease in transcription. this study identified specific functions and molecular mechanisms involved in each cell type during early folliculogenesis and in the dialog between the different cell types. we confirm and incorporate a large number of individual studies in different species. last, we highlight new gene expressions that support and extend current knowledge on early folliculogenesis. this study is the first thorough, integrated overview of the molecular mechanisms involved in early folliculogenesis in a mono-ovulating species. it provides a valuable data base for future functional studies in mono-ovulating species including humans. 
finally, we highlighted several transcription regulators and specifically expressed genes that likely play an important role in early folliculogenesis. successful early folliculogenesis is crucial for female reproductive function. collectively, our data advance our understanding of early folliculogenesis in mono-ovulating species and will be a valuable resource for unraveling human ovarian dysfunction such as premature ovarian failure. it requires appropriate gene specific expression of the different types of ovarian cells at different developmental stages. we used a laser capture microdissection method combined with rna-seq technology to explore the transcriptome in oocytes and granulosa cells during development of the sheep ovarian follicle. to our knowledge, this is the first comprehensive exploration of transcriptomes derived from in vivo oocytes and gcs at key stages in early follicular development in sheep. in mono-ovulating species, few studies have addressed gene expression profiles and mainly concerned human oocytes. we first documented the expression profile of genes, then focused on the genes showing differential expression between oocytes and gcs.
 successful early folliculogenesis is crucial for female reproductive function. it requires appropriate gene specific expression of the different types of ovarian cells at different developmental stages. to date, most gene expression studies on the ovary were conducted in rodents and did not distinguish the type of cell. in mono-ovulating species, few studies have addressed gene expression profiles and mainly concerned human oocytes.  we used a laser capture microdissection method combined with rna-seq technology to explore the transcriptome in oocytes and granulosa cells during development of the sheep ovarian follicle. we first documented the expression profile of genes, then focused on the genes showing differential expression between oocytes and gcs. enriched functional categories such as oocyte meiotic arrest and gc steroid synthesis reflect two distinct cell fates. we identified the implication of gc signal transduction pathways such as shh, wnt and rho gtpase. in addition, signaling pathways and gc transzonal projections suggest the existence of complex cell-cell interactions. finally, we highlighted several transcription regulators and specifically expressed genes that likely play an important role in early folliculogenesis.  to our knowledge, this is the first comprehensive exploration of transcriptomes derived from in vivo oocytes and gcs at key stages in early follicular development in sheep. collectively, our data advance our understanding of early folliculogenesis in mono-ovulating species and will be a valuable resource for unraveling human ovarian dysfunction such as premature ovarian failure. rna-seqearly folliculogenesismolecular dialogtranscriptomesheepoocytegranulosa cellsmicrodissection 