BACKGROUND
many studies have been carried out to identify the mechanisms controlling early folliculogenesis .
these early stages are important in regulating the size of the resting primordial follicle pool and the fate of the follicles, which in turn affects reproductive life span and fertility.
even if the events of folliculogenesis are well-conserved among mammals, differences exist between species in the timing of specific developmental changes and more specifically, the role of several genes was shown to be different between mono-ovulating and poly-ovulating species.
the formation of primordial follicles occurs within a few days after birth in rodents and during fetal development in primates and ruminants such as sheep   <cit> .
the primordial follicles are composed of diplotene oocytes surrounded by flattened pregranulosa cells.
transcriptomic studies in human and in rodents, showed that a number of genes, such as transcription factors   <cit> , zona proteins, meiosis-specific enzymes and nerve growth factors  <cit>  have already been identified as being involved in primordial follicle assembly.
once formed, primordial follicles remain in a dormant phase until they are recruited to initiate growth towards the primary stage.
orchestrated communication between oocytes and somatic cells  is required during this transition and during the subsequent growth of follicles.
different components of the extra-cellular matrix, such as growth factors and cytokines, acting in an autocrine and paracrine manner, are involved in this cross talk.
for example, cytokines such as the kit-ligand, expressed in granulosa cells, and its receptor c-kit, expressed in oocyte and theca cells  <cit> , are involved in the formation of primary follicles.
then, oocyte-secreted growth factors such as gdf <dig> and bmp <dig> are involved in primary to secondary follicle transition  <cit> .
they co-operate to regulate proliferation of granulosa cells  <cit> .
in addition, other factors such as foxl <dig>  amh or ngf also play a role in these different steps of early folliculogenesis  <cit> .
until now, efforts to discover genes have mainly focused on rodents and only a small number of genes are currently known, meaning that our basic understanding of the gene expression patterns driving early folliculogenesis is still very poor.
more specifically, the expression of various oocyte-specific genes was demonstrated to be essential at a specific time during early folliculogenesis but little is known exactly about which granulosa cell factors play a specialized role in follicular development.
moreover, all findings in rodent species are not necessarily applicable to other mammals, especially mono-ovulating species.
indeed, whereas mutations in the bmp <dig> gene cause infertility in ewes due to defects in folliculogenesis  <cit>  and have been associated with premature ovarian failure  in women  <cit> , most defects in female mice lacking the bone morphogenetic protein bmp <dig> are confined to the ovulation process  <cit> .
finally, most transcriptomic studies of early folliculogenesis are based on homogenized tissues   <cit> , and do not take the multiplicity of cell types used in the analyses into consideration.
consequently, potentially valuable spatial information is lost and the minor cell components, expressed only in few cell types may be diluted below the level of detection.
laser capture microdissection  allows precise microscopic isolation of pure cell populations from heterogeneous tissues for subsequent extraction and analysis of nucleic acids  <cit> .
conducting lcm experiments for gene expression profiling requires an acceptable tissue morphology to allow for histological selection of the desired cell type and to guarantee rna integrity.
particularly, single cell lcm requires a lot of time to search for and identify cells of interest in the tissue section.
preserving rna quality during this long process is challenging.
indeed, most existing protocols  <cit>  do not guarantee the integrity of rna during the long time required for microdissection.
in the present study, lcm was used to isolate oocytes and granulosa cells  from new born sheep ovaries and their respective transcriptomes were compared using a  <dig> k bovine microarray and quantitative real-time pcr .
we developed an lcm method allowing for a longer microdissection time without rna degradation, to gain specific access to the follicular compartments, i.e.
gc and oocytes, at each follicular developmental stage  <cit> .
starting from amplified rna, gene expression profiles were monitored for each follicle population, according to cell type on an affymetrix genechip bovine array.
our results showed that microdissected samples were representative of the different compartments and stages and demonstrated the feasibility of using lcm and array hybridization to identify cell type and/or stage gene expression signatures in developing ovaries.
RESULTS
lcm and rna amplification
the purpose of any tissue preparation protocol for lcm is to obtain tissue sections allowing for unambiguous identification and successful capture of the cells of interest, while maintaining rna integrity.
four staining protocols: toluidin blue, hematoxylin-eosin , histogen® and cresyl violet® were evaluated in terms of tissue morphology, maintenance of molecular integrity and capture success.
figure  <dig> shows that the best tissue morphology was provided by cresyl violet® and h&e staining.
the rna integrity number  analysis revealed significant rna degradation with toluidin blue and histogen® staining .
finally, although the cresyl violet® protocol was the best both for identifying structures and for maintaining the best rna quality, the ovary sections were fixed too tightly to the glass and capture did not exceed 50%.
consequently, we developed a new cold fixation protocol , allowing efficient single-cell capture .
in these conditions, rna quality did not decrease more than 10% during the staining and microdissection period .
the rin values typically obtained with rna extracted from stained sections ranged between  <dig> and  <dig>  and were similar to those obtained with standard fixation protocols .
using this protocol, the follicular compartments, i.e.
gc and oocytes, were captured for each follicle stage: primordial , primary , secondary  follicles and the first antral stage as control .
after rna extraction and amplification, the lcm-derived anti-sense-rna  samples appeared as a highly reproducible smear, ranging from  <dig> to  <dig> bases in length across all samples .
table  <dig> summarizes the amount of estimated microdissected sections input and the arna yield for each lcm-sample.
estimated number of microdissection sections input and corresponding yield of arna for each sample.
arna: amplified rna
validation of sample specificity
the purity of the lcm-derived arna samples and the microarray data quality were examined by investigating specific gene expression of nine genes using quantitative real-time pcr .
among them, six were oocyte-specific genes  and three known to be expressed in gc .
table  <dig> summarizes the results of the statistical analyses  for each gene.
we identified significant interaction effects between compartments  and between stages  and the first antral stage ) for six genes.
significant differential expression between the compartments was confirmed for all the genes tested.
moreover, four genes showed a significant increase in expression associated with early follicular development .
for example, bmp <dig>  amh and mael expression increased between the primary and secondary developmental stage respectively.
by contrast, vasa, kl and sohlh <dig> displayed no significant differences between stages .
these results demonstrated that the lcm-derived arna samples were representative of the cell type and/or developmental stage concerned.
sohlh2
vasa
<dig>
***
mael
mater
gdf9
bmp15
<dig>
<dig>
<dig>
<dig>
<dig>
*
***
***
<dig>
<dig>
<dig>
***
*
*
***
**
*
kl
gata 4
las1l
- <dig>
gc only
- <dig>
- <dig>
*
*
amh
o/gc fold change corresponds to the highest oocyte expression of the  <dig> follicular stages versus the lowest ones.
for genes overexpressed in gc, a minus sign was added to the gc/o fold change.
follicular stage fold change corresponds to the highest expression stage versus the lowest within a compartment.
for each gene, a one-way anova model was fitted with the  <dig> factors: "stage"  and "compartment"  with interactions using r statistical software.
the expression profile column visualizes the expression level of each compartment: bold line is used for oocyte expression and double line for gc expression.
the slope symbolizes a significant increase of the expression during the follicular development.
p-value class: *: p <  <dig> ; **: p <  <dig> ; ***:p <  <dig> ; ns: non specific
abbreviations: o: oocyte , gc: granulosa cells
global gene expression analysis
to explore global gene expression in the cell types and/or early developmental stages, the ovine lcm-derived arna samples were hybridized to the affymetrix bovine genome array, as generic microarray is not available in sheep, and the bovine and ovine genomes are phylogenetically close.
first, to assess the reliability of the heterologous hybridization on the bovine microarray, we compared hybridizations with rna from bovine and ovine fetal gonads.
hybridization with the ovine fetal gonad rna sample revealed an average of  <dig> % of the probe sets called as "present"  compared to an average of  <dig> % for the bovine counterpart sample.
around 90% of these probe sets were shared .
the percentage of ovine transcripts detected in this control experiment is similar to a previous report using ovine mrna on the affymetrix genechip bovine array  <cit> .
this result demonstrated the possibility of using heterologous hybridization in our conditions.
next, in order to keep lcm-derived arna samples for further expression experiments, we assessed the quality of arcturus turbo™ labeling  compared to that of the affymetrix labeling kit.
ovine fetal gonad rna was subjected to three different labeling protocols  and hybridized to the affymetrix bovine genome array.
the sensitivity and reliability of these protocols were evaluated using three measures of genechip performance  provided by the affymetrix algorithm .
the double round amplification data revealed a slight reduction in the number of "present" calls.
the scale factor was similar in all hybridizations.
finally in labeling protocols from amplified rna , the 3'/5' signal ratio was higher than the 3'/m ratio signal for two housekeeping genes , demonstrating that the mean size of the arna obtained was shorter than affymetrix standard labeling kit.
similar observations have been reported by others  <cit> .
since each round of amplification shortens the length of the rna transcript, eventually resulting in the loss of 5' regions, the signal ratio between 3' and 5' probe sets consequently increases.
in addition, the intensity signal of arcturus turbo™ labeling arna showed a high level of concordance with the affymetrix ones .
the analysis of signal intensity values for the four bacillus subtilis control transcripts that were added to all lcm-derived rna samples before amplification  showed comparable amplification in all the lcm-derived rna samples and the ovine fetal ovary .
cell profiling
different follicle population hybridizations enabled us to detect  <dig>  probe sets corresponding to  <dig> annotated transcripts, split into  <dig> to  <dig> probe sets according to the samples .
distribution of the detected probe sets and unique annotated genes among the lcm-derived rna samples.
'total detected' refers to the whole detected probe sets within the lcm-derived rna oocyte samples.
lcm-derived rna oocyte samples were compared to pan et al.
data .
we also identified  <dig> transcripts specifically expressed in gc and  <dig> transcripts expressed only in oocytes during early follicular development.
five genes  known as having compartment-specific expression were confirmed by microarray data and qpcr analyses .
five additional genes  showing a new spatiotemporal expression were checked by qpcr and  <dig> of them were validated.
moreover, we observed a significant increase in sirt <dig>  mael and fst expression during early follicular development .
the biological relevance of the two specifically expressed gene lists  was evaluated in silico using ingenuity pathway analysis .
a total of  <dig> genes were identified as eligible by ipa and used to generate networks.
seven significant biological networks for oocytes and ten significant biological networks for gc were selected .
these networks identified common and specific top functions  with their associated pathways .
the "diseases and disorders" catalog was specific to gc and included the most significant genetic disorder class, which contained 72% of the eligible genes.
genes from gc were also mainly involved in "cell growth and proliferation", "cell death" and "lipid metabolism" classes.
we also identified gc functions involved in "cell movement and cell interactions".
the oocyte-specific gene list highlighted two specific functions: "reproductive system and function" that clearly targets folliculogenesis  and the "hematopoiesis function" that mainly includes differentiation processes.
discussion
gene expression profiling through the use of oligoarrays is a powerful approach for characterizing the whole population of transcripts in a tissue, and amplification methods now allow these techniques to be used for very rare material.
nevertheless, it is still difficult to obtain cell type specific expression profiles from heterogeneous tissues.
for these reasons, in the context of early folliculogenesis, our current knowledge has been largely derived from rodent whole ovaries  <cit>  and the few isolation protocols available referred to oocytes  <cit>  or whole follicles  <cit>  and did not differentiate gc.
although the expression profile of some compartment-specific genes could be inferred, the presence together of different cell types confused the analysis.
consequently, little information about the role of gc during early folliculogenesis is available.
the strength of the present study is to develop a method for obtaining reliable specific expression profiles and to characterize gc and oocytes separately at several key stages corresponding to major transitions during the development of the follicles.
quality of the lcm-derived arna
to obtain reliable microarray results, lcm must produce a sufficient amount of high quality rna.
in the case of single cell lcm, we have to face the long microdissection period required and the extraction of only a limited amount of rna.
in this study, our modified protocol allowed us to capture the cells and to preserve the rna integrity of the two follicular compartments  throughout microdissection .
first, we showed that, as previously described in other tissues  <cit> , histological and rna quality of the staining are tissue specific  and probably depend on cell component reactions and intracellular rnase content.
the success of capture depends on a number of tissue-slide adhesion factors such as the type of slides used or the method and temperature of fixation  <cit> .
sluka et al.
<cit>  found that maintaining testis tissue at a cold temperature during fixation and subsequent processing  was important to maximize preservation of tissue morphology.
as recently described in mammary gland  <cit> , we showed in this study, that cold temperature was also important for the capture process.
finally, our data showed that 500- <dig> single gcs were sufficient to produce enough arna to perform both microarray hybridization and qpcr experiments .
the decrease in the required number of oocytes captured according to the stage of follicular development is also in agreement with an increase in the diameter of the oocyte   <cit>  and in their rna content  <cit> .
gene expression patterns
in this study, we documented nine ovine gene expression patterns in specific follicular compartments within in vivo microenvironment .
we confirmed cell type-specific expression in all the genes tested.
as described by luzzy et al.
<cit> , we identified variability between the replicates  that could be associated with technical processing and/or with biological microheterogeneity and individual variability.
the expression profiles of ovine bmp <dig>  gdf <dig> mater, vasa and sohlh <dig> oocyte-specific genes and ovine kl, gata <dig>  amh gc-specific genes are consistent with previous reports on sheep fetal ovaries  <cit> , germinal vesicle  oocytes  <cit>  or other species  <cit> .
in addition, we report here an increase in the expression of four cell type-specific genes during the early ovine folliculogenesis.
our data provide additional clues about species specificities.
they underline differences in sohlh <dig> and mael gene expression patterns from those identified in rodent species .
the sohlh <dig> gene is a spermatogenesis- and oogenesis-specific transcription factor that was recently discovered in mouse species with a restricted pattern of expression in oocytes of primordial and primary ovarian follicles in immature ovaries  <cit> .
by contrast with mouse species, we observed constant expression of sohlh <dig> until the sa stage.
likewise, mael is a drosophila spindle-class gene required for drosophila oogenesis that localizes to nuage  and is implicated in mirna/rnai pathways  <cit> .
mael is expressed in the early drosophila germ line  <cit> .
in mouse, gallardo et al.
showed that expression of mael is high in primordial oocytes but rapidly decreases during follicle growth to disappear in the sa follicles  <cit> .
we revealed that the expression of mael is maintained until small antral follicles in sheep.
such sohlh <dig> and mael expression patterns suggest the existence of different mechanisms as a function of species that will need further investigation.
this underlines the importance of acquiring expression data from different species and highlights certain species specificities.
accuracy of array data
the originality of this preliminary study was to identify global gene expression in oocyte and gc separately for key stages of early folliculogenesis.
several observations support the accuracy of the expression data.
first, the level of "present" call probes from the oocyte samples  and the size of the annotated transcripts detected  were in accordance with mouse oocyte data   <cit>  and bovine oocyte data   <cit> .
in addition, we also identified the great majority of genes previously found to be expressed in the four following studies: dadé et al.
<cit> , arraztoa et al.
<cit> , pan et al.
<cit>  and gallardo et al.
<cit>  .
the results of these studies mainly derived from oocytes .
figure 6a shows that 41% of the oocyte genes reported in these studies were expressed in our sheep oocyte samples.
our data on gc during early folliculogenesis are quite unique, as even if some information about gc expression was previously available  <cit> , it only concerned very few genes .
figure 6b shows that 33% of these genes were also detected in our gc data sets.
these percentages take into account the percentage of undetected genes  which could be partly due to the relative specificity of heterologous hybridizations and the high percentage of missing genes .
this underlines the fact that genomics of livestock animals have to face with incomplete arrays and/or incomplete genome annotations  <cit> .
biological significance
to confirm that these data are biologically meaningful and a rich source for functional analysis, two lists of compartment-specific genes were generated and subjected to ingenuity pathway analysis .
this functional analysis identified the three main cellular events known to be involved in early follicle development   <cit> .
first, we observed three cellular functions in gc  probably associated with the first cellular event, the switch of cell shape from flattened to cuboidal  <cit> .
second, the identification of genes involved in "cellular growth and proliferation" and "cell death" is correlated with the second cellular event: the marked increase in the number of gc   <cit> , a key process in the initiation and the development of primordial follicles.
finally, in oocytes, the "axonal guidance signaling" pathway  and the "cellular development" function are consistent with the third cellular event: oocyte enlargement.
axonal guidance signaling refers to the different receptors and signal transduction cascades that drive axons towards their synaptic targets and finally lead to the reorganization of the cytoskeleton and confer the adhesive properties the cell requires for its spatial organization.
this pathway was previously mentioned in the analysis of regulatory gene networks involved in primordial follicle development  <cit> .
in addition, the importance of the crosstalk between the oocyte and gc during follicular development  <cit>  may be illustrated by the expression of genes of the bmp signaling pathway in gc in response to oocyte bmp <dig> production .
this crosstalk was also highlighted by the "cell to cell signaling and interaction" function in both gc and oocytes  and "inflammatory and immune response" mechanisms found in gc.
these mechanisms featured the "cytokine signaling" pathway, "cellular immune response" pathways and the "genetic disorder" class .
they include members of the tgfβ gene family such as amh  <cit> , and cytokines encoding genes such as kl  <cit>  both expressed in gc and known to interact with oocytes and to play an essential role in early folliculogenesis.
we also identified the specific gc expression of different interleukins such as the proinflammatory cytokine il1-β previously reported in antral follicles for their involvement in ovarian steroidogenesis, in the ovulation process and in oocyte maturation  <cit>  and which are up-regulated during primordial-primary follicle transition   <cit> .
interestingly, the il <dig> receptor gene  appears to be only expressed in oocytes, which supports the hypothesis of cytokine oocyte-gc crosstalk.
however, the exact function of these genes in this context remains to be elucidated.
thanks to these specific gene lists, we were able to show for the first time that expression of the sirt <dig> gene is confined to the oocyte in sheep follicles .
this gene was found to be expressed in most mouse and human tissues including -at a lower level of expression- the ovary  <cit> .
the detailed expression pattern of the current study completes a previous rat expression pattern comparing ovaries enriched in different follicle populations  <cit> .
the sirt <dig> gene belongs to the sirtuin family and is involved in the regulation of a wide range of biological processes such as gene silencing, aging, cellular differentiation, and metabolism  <cit> .
in human, the colocalization of sirt <dig> with polymerase i and the nucleolar transcription activator ubf suggest that sirt <dig> is a positive regulator of pol i transcription  <cit> .
its oocyte expression is consistent with the high demand for protein synthesis for oocyte growth.
like the basonuclin gene  <cit> , this expression suggests a role for sirt <dig> in the transcriptional regulation of sheep oocyte rdna that will need further experiments to verify nuclear localization.
finally, this study underlines the precise expression pattern in gc of the fst gene that plays an important role in female fertility by regulating activin and members of the bmp subfamily  <cit> .
different studies have shown induction of the fst gene expression by recombinant human bmp <dig>  <cit>  and suggested an inhibitory action of fst on bmp <dig> function  <cit> .
its strongly increased expression at the secondary stage in sheep  combined with the bmp <dig> oocyte-expression pattern corroborate the results of these studies and are consistent with the bmp <dig> function to enable the primary-secondary transition.
CONCLUSIONS
in the present study, lcm and microarray analysis were used as tools to identify distinct gene expression patterns in ovary cell populations and led to the identification of genes of potential significance in female fertility.
the data obtained here are in agreement with the literature regarding oocyte and gc-specific transcript lists.
we identified specific expression patterns in sheep .
our findings show that the mrna repertoire in the different cell types is regulated dynamically during the transition from primordial to intermediate follicles.
this study is a starting point for further investigation of the gc compartment in particular.
specific gene expression patterns in the oocyte and gc could be of great interest for deciphering the critical molecular processes and the complexity of the communication between these two compartments, who enable the folliculogenesis process to occur as it is expected.

 many studies have been carried out to identify the mechanisms controlling early folliculogenesis. these early stages are important in regulating the size of the resting primordial follicle pool and the fate of the follicles, which in turn affects reproductive life span and fertility. even if the events of folliculogenesis are well-conserved among mammals, differences exist between species in the timing of specific developmental changes and more specifically, the role of several genes was shown to be different between mono-ovulating and poly-ovulating species. the formation of primordial follicles occurs within a few days after birth in rodents and during fetal development in primates and ruminants such as sheep. the primordial follicles are composed of diplotene oocytes surrounded by flattened pregranulosa cells. transcriptomic studies in human and in rodents, showed that a number of genes, such as transcription factors, zona proteins, meiosis-specific enzymes and nerve growth factors have already been identified as being involved in primordial follicle assembly. once formed, primordial follicles remain in a dormant phase until they are recruited to initiate growth towards the primary stage. orchestrated communication between oocytes and somatic cells is required during this transition and during the subsequent growth of follicles. different components of the extra-cellular matrix, such as growth factors and cytokines, acting in an autocrine and paracrine manner, are involved in this cross talk. for example, cytokines such as the kit-ligand, expressed in granulosa cells, and its receptor c-kit, expressed in oocyte and theca cells, are involved in the formation of primary follicles. then, oocyte-secreted growth factors such as gdf and bmp are involved in primary to secondary follicle transition. they co-operate to regulate proliferation of granulosa cells. in addition, other factors such as foxl amh or ngf also play a role in these different steps of early folliculogenesis. until now, efforts to discover genes have mainly focused on rodents and only a small number of genes are currently known, meaning that our basic understanding of the gene expression patterns driving early folliculogenesis is still very poor. more specifically, the expression of various oocyte-specific genes was demonstrated to be essential at a specific time during early folliculogenesis but little is known exactly about which granulosa cell factors play a specialized role in follicular development. moreover, all findings in rodent species are not necessarily applicable to other mammals, especially mono-ovulating species. indeed, whereas mutations in the bmp gene cause infertility in ewes due to defects in folliculogenesis and have been associated with premature ovarian failure in women, most defects in female mice lacking the bone morphogenetic protein bmp are confined to the ovulation process. finally, most transcriptomic studies of early folliculogenesis are based on homogenized tissues, and do not take the multiplicity of cell types used in the analyses into consideration. consequently, potentially valuable spatial information is lost and the minor cell components, expressed only in few cell types may be diluted below the level of detection. laser capture microdissection allows precise microscopic isolation of pure cell populations from heterogeneous tissues for subsequent extraction and analysis of nucleic acids. conducting lcm experiments for gene expression profiling requires an acceptable tissue morphology to allow for histological selection of the desired cell type and to guarantee rna integrity. particularly, single cell lcm requires a lot of time to search for and identify cells of interest in the tissue section. preserving rna quality during this long process is challenging. indeed, most existing protocols do not guarantee the integrity of rna during the long time required for microdissection. in the present study, lcm was used to isolate oocytes and granulosa cells from new born sheep ovaries and their respective transcriptomes were compared using a k bovine microarray and quantitative real-time pcr. we developed an lcm method allowing for a longer microdissection time without rna degradation, to gain specific access to the follicular compartments, i.e. gc and oocytes, at each follicular developmental stage. starting from amplified rna, gene expression profiles were monitored for each follicle population, according to cell type on an affymetrix genechip bovine array. our results showed that microdissected samples were representative of the different compartments and stages and demonstrated the feasibility of using lcm and array hybridization to identify cell type and/or stage gene expression signatures in developing ovaries.  lcm and rna amplification the purpose of any tissue preparation protocol for lcm is to obtain tissue sections allowing for unambiguous identification and successful capture of the cells of interest, while maintaining rna integrity. four staining protocols: toluidin blue, hematoxylin-eosin, histogen® and cresyl violet® were evaluated in terms of tissue morphology, maintenance of molecular integrity and capture success. figure shows that the best tissue morphology was provided by cresyl violet® and h&e staining. the rna integrity number analysis revealed significant rna degradation with toluidin blue and histogen® staining. finally, although the cresyl violet® protocol was the best both for identifying structures and for maintaining the best rna quality, the ovary sections were fixed too tightly to the glass and capture did not exceed 50%. consequently, we developed a new cold fixation protocol, allowing efficient single-cell capture. in these conditions, rna quality did not decrease more than 10% during the staining and microdissection period. the rin values typically obtained with rna extracted from stained sections ranged between and and were similar to those obtained with standard fixation protocols. using this protocol, the follicular compartments, i.e. gc and oocytes, were captured for each follicle stage: primordial, primary, secondary follicles and the first antral stage as control. after rna extraction and amplification, the lcm-derived anti-sense-rna samples appeared as a highly reproducible smear, ranging from to bases in length across all samples. table summarizes the amount of estimated microdissected sections input and the arna yield for each lcm-sample. estimated number of microdissection sections input and corresponding yield of arna for each sample. arna: amplified rna validation of sample specificity the purity of the lcm-derived arna samples and the microarray data quality were examined by investigating specific gene expression of nine genes using quantitative real-time pcr. among them, six were oocyte-specific genes and three known to be expressed in gc. table summarizes the results of the statistical analyses for each gene. we identified significant interaction effects between compartments and between stages and the first antral stage ) for six genes. significant differential expression between the compartments was confirmed for all the genes tested. moreover, four genes showed a significant increase in expression associated with early follicular development. for example, bmp amh and mael expression increased between the primary and secondary developmental stage respectively. by contrast, vasa, kl and sohlh displayed no significant differences between stages. these results demonstrated that the lcm-derived arna samples were representative of the cell type and/or developmental stage concerned. sohlh2 vasa *** mael mater gdf9 bmp15 * *** *** *** * * *** ** * kl gata 4 las1l - gc only - - * * amh o/gc fold change corresponds to the highest oocyte expression of the follicular stages versus the lowest ones. for genes overexpressed in gc, a minus sign was added to the gc/o fold change. follicular stage fold change corresponds to the highest expression stage versus the lowest within a compartment. for each gene, a one-way anova model was fitted with the factors: "stage" and "compartment" with interactions using r statistical software. the expression profile column visualizes the expression level of each compartment: bold line is used for oocyte expression and double line for gc expression. the slope symbolizes a significant increase of the expression during the follicular development. p-value class: *: p <; **: p <; ***:p <; ns: non specific abbreviations: o: oocyte, gc: granulosa cells global gene expression analysis to explore global gene expression in the cell types and/or early developmental stages, the ovine lcm-derived arna samples were hybridized to the affymetrix bovine genome array, as generic microarray is not available in sheep, and the bovine and ovine genomes are phylogenetically close. first, to assess the reliability of the heterologous hybridization on the bovine microarray, we compared hybridizations with rna from bovine and ovine fetal gonads. hybridization with the ovine fetal gonad rna sample revealed an average of % of the probe sets called as "present" compared to an average of % for the bovine counterpart sample. around 90% of these probe sets were shared. the percentage of ovine transcripts detected in this control experiment is similar to a previous report using ovine mrna on the affymetrix genechip bovine array. this result demonstrated the possibility of using heterologous hybridization in our conditions. next, in order to keep lcm-derived arna samples for further expression experiments, we assessed the quality of arcturus turbo™ labeling compared to that of the affymetrix labeling kit. ovine fetal gonad rna was subjected to three different labeling protocols and hybridized to the affymetrix bovine genome array. the sensitivity and reliability of these protocols were evaluated using three measures of genechip performance provided by the affymetrix algorithm. the double round amplification data revealed a slight reduction in the number of "present" calls. the scale factor was similar in all hybridizations. finally in labeling protocols from amplified rna, the 3'/5' signal ratio was higher than the 3'/m ratio signal for two housekeeping genes, demonstrating that the mean size of the arna obtained was shorter than affymetrix standard labeling kit. similar observations have been reported by others. since each round of amplification shortens the length of the rna transcript, eventually resulting in the loss of 5' regions, the signal ratio between 3' and 5' probe sets consequently increases. in addition, the intensity signal of arcturus turbo™ labeling arna showed a high level of concordance with the affymetrix ones. the analysis of signal intensity values for the four bacillus subtilis control transcripts that were added to all lcm-derived rna samples before amplification showed comparable amplification in all the lcm-derived rna samples and the ovine fetal ovary. cell profiling different follicle population hybridizations enabled us to detect probe sets corresponding to annotated transcripts, split into to probe sets according to the samples. distribution of the detected probe sets and unique annotated genes among the lcm-derived rna samples.'total detected' refers to the whole detected probe sets within the lcm-derived rna oocyte samples. lcm-derived rna oocyte samples were compared to pan et al. data. we also identified transcripts specifically expressed in gc and transcripts expressed only in oocytes during early follicular development. five genes known as having compartment-specific expression were confirmed by microarray data and qpcr analyses. five additional genes showing a new spatiotemporal expression were checked by qpcr and of them were validated. moreover, we observed a significant increase in sirt mael and fst expression during early follicular development. the biological relevance of the two specifically expressed gene lists was evaluated in silico using ingenuity pathway analysis. a total of genes were identified as eligible by ipa and used to generate networks. seven significant biological networks for oocytes and ten significant biological networks for gc were selected. these networks identified common and specific top functions with their associated pathways. the "diseases and disorders" catalog was specific to gc and included the most significant genetic disorder class, which contained 72% of the eligible genes. genes from gc were also mainly involved in "cell growth and proliferation", "cell death" and "lipid metabolism" classes. we also identified gc functions involved in "cell movement and cell interactions". the oocyte-specific gene list highlighted two specific functions: "reproductive system and function" that clearly targets folliculogenesis and the "hematopoiesis function" that mainly includes differentiation processes. discussion gene expression profiling through the use of oligoarrays is a powerful approach for characterizing the whole population of transcripts in a tissue, and amplification methods now allow these techniques to be used for very rare material. nevertheless, it is still difficult to obtain cell type specific expression profiles from heterogeneous tissues. for these reasons, in the context of early folliculogenesis, our current knowledge has been largely derived from rodent whole ovaries and the few isolation protocols available referred to oocytes or whole follicles and did not differentiate gc. although the expression profile of some compartment-specific genes could be inferred, the presence together of different cell types confused the analysis. consequently, little information about the role of gc during early folliculogenesis is available. the strength of the present study is to develop a method for obtaining reliable specific expression profiles and to characterize gc and oocytes separately at several key stages corresponding to major transitions during the development of the follicles. quality of the lcm-derived arna to obtain reliable microarray results, lcm must produce a sufficient amount of high quality rna. in the case of single cell lcm, we have to face the long microdissection period required and the extraction of only a limited amount of rna. in this study, our modified protocol allowed us to capture the cells and to preserve the rna integrity of the two follicular compartments throughout microdissection. first, we showed that, as previously described in other tissues, histological and rna quality of the staining are tissue specific and probably depend on cell component reactions and intracellular rnase content. the success of capture depends on a number of tissue-slide adhesion factors such as the type of slides used or the method and temperature of fixation. sluka et al. found that maintaining testis tissue at a cold temperature during fixation and subsequent processing was important to maximize preservation of tissue morphology. as recently described in mammary gland, we showed in this study, that cold temperature was also important for the capture process. finally, our data showed that 500- single gcs were sufficient to produce enough arna to perform both microarray hybridization and qpcr experiments. the decrease in the required number of oocytes captured according to the stage of follicular development is also in agreement with an increase in the diameter of the oocyte and in their rna content. gene expression patterns in this study, we documented nine ovine gene expression patterns in specific follicular compartments within in vivo microenvironment. we confirmed cell type-specific expression in all the genes tested. as described by luzzy et al., we identified variability between the replicates that could be associated with technical processing and/or with biological microheterogeneity and individual variability. the expression profiles of ovine bmp gdf mater, vasa and sohlh oocyte-specific genes and ovine kl, gata amh gc-specific genes are consistent with previous reports on sheep fetal ovaries, germinal vesicle oocytes or other species. in addition, we report here an increase in the expression of four cell type-specific genes during the early ovine folliculogenesis. our data provide additional clues about species specificities. they underline differences in sohlh and mael gene expression patterns from those identified in rodent species. the sohlh gene is a spermatogenesis- and oogenesis-specific transcription factor that was recently discovered in mouse species with a restricted pattern of expression in oocytes of primordial and primary ovarian follicles in immature ovaries. by contrast with mouse species, we observed constant expression of sohlh until the sa stage. likewise, mael is a drosophila spindle-class gene required for drosophila oogenesis that localizes to nuage and is implicated in mirna/rnai pathways. mael is expressed in the early drosophila germ line. in mouse, gallardo et al. showed that expression of mael is high in primordial oocytes but rapidly decreases during follicle growth to disappear in the sa follicles. we revealed that the expression of mael is maintained until small antral follicles in sheep. such sohlh and mael expression patterns suggest the existence of different mechanisms as a function of species that will need further investigation. this underlines the importance of acquiring expression data from different species and highlights certain species specificities. accuracy of array data the originality of this preliminary study was to identify global gene expression in oocyte and gc separately for key stages of early folliculogenesis. several observations support the accuracy of the expression data. first, the level of "present" call probes from the oocyte samples and the size of the annotated transcripts detected were in accordance with mouse oocyte data and bovine oocyte data. in addition, we also identified the great majority of genes previously found to be expressed in the four following studies: dadé et al., arraztoa et al., pan et al. and gallardo et al.. the results of these studies mainly derived from oocytes. figure 6a shows that 41% of the oocyte genes reported in these studies were expressed in our sheep oocyte samples. our data on gc during early folliculogenesis are quite unique, as even if some information about gc expression was previously available, it only concerned very few genes. figure 6b shows that 33% of these genes were also detected in our gc data sets. these percentages take into account the percentage of undetected genes which could be partly due to the relative specificity of heterologous hybridizations and the high percentage of missing genes. this underlines the fact that genomics of livestock animals have to face with incomplete arrays and/or incomplete genome annotations. biological significance to confirm that these data are biologically meaningful and a rich source for functional analysis, two lists of compartment-specific genes were generated and subjected to ingenuity pathway analysis. this functional analysis identified the three main cellular events known to be involved in early follicle development. first, we observed three cellular functions in gc probably associated with the first cellular event, the switch of cell shape from flattened to cuboidal. second, the identification of genes involved in "cellular growth and proliferation" and "cell death" is correlated with the second cellular event: the marked increase in the number of gc, a key process in the initiation and the development of primordial follicles. finally, in oocytes, the "axonal guidance signaling" pathway and the "cellular development" function are consistent with the third cellular event: oocyte enlargement. axonal guidance signaling refers to the different receptors and signal transduction cascades that drive axons towards their synaptic targets and finally lead to the reorganization of the cytoskeleton and confer the adhesive properties the cell requires for its spatial organization. this pathway was previously mentioned in the analysis of regulatory gene networks involved in primordial follicle development. in addition, the importance of the crosstalk between the oocyte and gc during follicular development may be illustrated by the expression of genes of the bmp signaling pathway in gc in response to oocyte bmp production. this crosstalk was also highlighted by the "cell to cell signaling and interaction" function in both gc and oocytes and "inflammatory and immune response" mechanisms found in gc. these mechanisms featured the "cytokine signaling" pathway, "cellular immune response" pathways and the "genetic disorder" class. they include members of the tgfβ gene family such as amh, and cytokines encoding genes such as kl both expressed in gc and known to interact with oocytes and to play an essential role in early folliculogenesis. we also identified the specific gc expression of different interleukins such as the proinflammatory cytokine il1-β previously reported in antral follicles for their involvement in ovarian steroidogenesis, in the ovulation process and in oocyte maturation and which are up-regulated during primordial-primary follicle transition. interestingly, the il receptor gene appears to be only expressed in oocytes, which supports the hypothesis of cytokine oocyte-gc crosstalk. however, the exact function of these genes in this context remains to be elucidated. thanks to these specific gene lists, we were able to show for the first time that expression of the sirt gene is confined to the oocyte in sheep follicles. this gene was found to be expressed in most mouse and human tissues including -at a lower level of expression- the ovary. the detailed expression pattern of the current study completes a previous rat expression pattern comparing ovaries enriched in different follicle populations. the sirt gene belongs to the sirtuin family and is involved in the regulation of a wide range of biological processes such as gene silencing, aging, cellular differentiation, and metabolism. in human, the colocalization of sirt with polymerase i and the nucleolar transcription activator ubf suggest that sirt is a positive regulator of pol i transcription. its oocyte expression is consistent with the high demand for protein synthesis for oocyte growth. like the basonuclin gene, this expression suggests a role for sirt in the transcriptional regulation of sheep oocyte rdna that will need further experiments to verify nuclear localization. finally, this study underlines the precise expression pattern in gc of the fst gene that plays an important role in female fertility by regulating activin and members of the bmp subfamily. different studies have shown induction of the fst gene expression by recombinant human bmp and suggested an inhibitory action of fst on bmp function. its strongly increased expression at the secondary stage in sheep combined with the bmp oocyte-expression pattern corroborate the results of these studies and are consistent with the bmp function to enable the primary-secondary transition.  in the present study, lcm and microarray analysis were used as tools to identify distinct gene expression patterns in ovary cell populations and led to the identification of genes of potential significance in female fertility. the data obtained here are in agreement with the literature regarding oocyte and gc-specific transcript lists. we identified specific expression patterns in sheep. our findings show that the mrna repertoire in the different cell types is regulated dynamically during the transition from primordial to intermediate follicles. this study is a starting point for further investigation of the gc compartment in particular. specific gene expression patterns in the oocyte and gc could be of great interest for deciphering the critical molecular processes and the complexity of the communication between these two compartments, who enable the folliculogenesis process to occur as it is expected. 
the ovary is a complex mixture of different cell types. enrichment in specific cell types was controlled by qrt-pcr analysis of known genes: six oocyte-specific genes and three granulosa cell-specific genes. successful achievement of early folliculogenesis is crucial for female reproductive function. despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of lcm-derived rna. the process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. the recently developed laser capture microdissection technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. however, one main challenge was to preserve the rna quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of rna. lcm and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations. we developed a new tissue fixation protocol ensuring efficient single cell capture and rna integrity during the microdissection procedure.
 successful achievement of early folliculogenesis is crucial for female reproductive function. the process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. the very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult. the recently developed laser capture microdissection technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. however, one main challenge was to preserve the rna quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of rna. using a new lcm method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.  we developed a new tissue fixation protocol ensuring efficient single cell capture and rna integrity during the microdissection procedure. enrichment in specific cell types was controlled by qrt-pcr analysis of known genes: six oocyte-specific genes and three granulosa cell-specific genes. a global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine affymetrix chip. most notably, the granulosa cell dataset is unique to date. the comparison of oocyte vs. follicular cell transcriptomes revealed transcripts specific to the granulosa cell and specific to the oocyte. functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of lcm-derived rna.  the ovary is a complex mixture of different cell types. distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. lcm and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations. 