BACKGROUND
an ovarian primordial follicle is composed of an inactive oocyte surrounded by granulosa cells all enclosed by a basal lamina.
the granulosa cells of the ovarian follicle support and nurture the oocyte, and secrete oestrogens which are necessary for normal reproductive function.
in mammals, the latter stage of follicle development can involve an approximate hundred fold increase in diameter,  <dig> doublings of granulosa cell numbers  <cit>  and formation of a fluid-filled antrum  <cit> .
in cattle, the growth of follicles is tightly regulated, since two or three groups or waves of follicles emerge from a pool of follicles larger than 5 mm in diameter during each oestrous cycle  <cit> .
in these waves, follicles continue to enlarge over several days until one follicle grows faster and larger than the others and hence gains ‘dominance’  <cit> .
this deviation in size occurs when the follicles are around 7–8 mm in diameter  <cit> .
these processes of follicular growth occur largely due to the stimulatory influence of fsh, through its receptor localised exclusively to the granulosa cells, though other factors produced locally, for example growth differentiation factor - <dig>  <cit>  and bone morphogenetic protein - <dig> from the oocyte  <cit> , are also involved.
instead of one primordial follicle growing to ovulatory size and then ovulating, many follicles commence growing during the course of the cycle.
most of these growing follicles become atretic, resulting in cows and humans, in only one or occasionally two follicles ovulating each cycle.
the highest rates of atresia in follicular development occur around the time of antrum formation.
it has been shown that the atretic process begins with cell death in the membrana granulosa initially by an apoptotic process  <cit> .
generally, apoptosis may be instigated intracellularly by cytotoxic stress, possibly due to free radicals or calcium influx  <cit>  which cause mitochondrial changes that eventually also lead to caspase activation.
apoptosis can be initiated externally to the cell by the binding of ‘death’ ligands such as fas ligand, tumour necrosis factor α  or trail to specific receptors  <cit> .
in follicular atresia it is unlikely that cell death occurs on a cell-by-cell basis because numerous pyknotic nuclei are observed during atresia  <cit> .
therefore it is probable that atresia is initiated by either the presence or absence of a particular external signal.
tnfα can initiate apoptosis in granulosa cells  <cit> .
the expression of tnfα receptors on granulosa and theca cells has been shown to be increased in atretic follicles when compared with healthy small or preovulatory follicles  <cit> .
studies on atretic follicles so far have shown that many of the genes/pathways involved are common to those stimulated by tnfα, as recently reviewed by matsuda et al.
<cit> .
investigation of the effects of various agents on granulosa in vitro is dependent on the follicle stage at which the cells were isolated and the composition of the culture medium.
it has been demonstrated that granulosa from small antral follicles are more responsive to fsh in serum-free culture and are capable of increasing oestradiol production over a six day period  <cit> .
this is an important consideration for studying granulosa cells as they have a propensity to differentiate into granulosa-lutein cells in a process called luteinisation, if cultured in serum-supplemented medium  <cit> .
such cells are completely unresponsive to fsh.
a previous study showed that tnfα was able to block the effects of fsh in serum-free culture of rat granulosa cells  <cit> .
we were interested in the effect of fsh and tnfα on steroid production and global gene expression in bovine granulosa cells to help elucidate the mechanisms of action of these compounds at the transcriptional level.
interest in the action of pro-inflammatory signals like tnfα has been augmented by recent reports that ovarian granulosa cells of cattle and other species fulfil an innate immunity role, being capable of detecting and responding to bacterial pathogens  <cit> .
we therefore cultured granulosa cells from small bovine follicles with or without fsh and tnfα, alone and in combination, assayed steroid hormone production by immunoassay and conducted microarray analyses using genechip bovine genome arrays.
RESULTS
cell culture and hormone assays
a dose response culture experiment was performed to determine the optimal concentrations of fsh and tnfα  to be used for the microarray experiments.
on the basis of this experiment it was shown that oestradiol production was highest with a fsh dose of  <dig>  ng/ml, being significantly different from values observed at all other fsh concentrations in the absence of tnfα.
ten ng/ml of tnfα was sufficient to reduce this effect to the control level.
progesterone levels and viable cell number at the end of the culture period were not significantly  affected by either fsh or tnfα treatment.
on the basis of these results,  <dig>  ng/ml fsh and 10 ng/ml tnfα were selected for the culture of cells to be used for microarray analyses.
the results of the hormone secretion in the cultures of granulosa cells which were used for the microarray analyses are shown in figure  <dig>  fsh stimulation of the cultures produced a greater than 20-fold induction  of oestradiol secretion by the granulosa cells compared with the control cells .
conversely, tnfα treatment caused a > 65% reduction in the amount of oestradiol secretion under basal conditions  and completely abolished the fsh-induced rise in oestradiol secretion .
progesterone levels averaged approximately 200 ng/ml across all cultures, trending higher for the fsh-treated culture, but no significant differences were observed between the treatments .
this confirms that the cultured granulosa cells maintained a hormone production profile characteristic of non-luteinised cells, and responded to fsh and tnfα as anticipated.
hierarchical clustering and principal component analyses
these analyses were performed on the sixteen microarrays, four from each treatment group, as presented in figure  <dig>  and additional file 1: figure s <dig> .
these figures show that culture sample g <dig> from the ‘control’ group was quite different in signal intensity across most probe sets on the chip from the other controls, and in fact all other arrays.
this result prompted us to consider this array as a statistical outlier, and it was excluded from further analyses.
the other arrays formed two clusters on the basis of distribution of signal intensity, the ‘tnfα and tnfα + fsh’ groups , and the ‘control and fsh’ groups .
pca was also conducted for the tnfα and the tnf + fsh-treated groups , and then for the control and the fsh-treated groups .
in both cases, for each comparison no differences were observed between the fsh treated and the non fsh-treated cells .
anova demonstrated that no genes were different by more than 2-fold with an fdr of p <  <dig> .
we can therefore assume that under our experimental culture conditions, fsh alone did not have a substantial effect on granulosa cell total gene expression, whereas tnfα had a major effect and actually overrode any effects of fsh when treated in combination.
quantitation of gene expression by rt-pcr
figure  <dig> shows the quantitative rt-pcr results for some of the genes which were significantly affected by fsh and/or tnfα treatment of granulosa cells in culture: fshr , the steroidogenic genes cyp19a <dig> , cyp11a <dig> , hsd3b <dig> , hsd17b <dig>  and star , and other genes inha , inhba  and fst .
these genes are known to be active during antral follicular expansion when the granulosa cells are responsive to higher levels of fsh  <cit> .
all genes except star, cyp11a <dig> and hsd3b <dig> were up regulated by fsh treatment alone.
tnfα, whether alone or in combination with fsh, decreased expression of the following genes below the level of the control: fshr, star, inha, inhb and fst and the steroidogenic genes hsd17b <dig> and cyp19a <dig> to the level of the control.
the decrease in cyp19a <dig>  the gene encoding aromatase, the key enzyme of oestradiol synthesis, explains the observed decline in oestradiol production in the cultures under the influence of tnfα .
differential gene expression analyses
comparison of all treatments against the control group by anova with a three-fold change and a statistical cut-off of p <  <dig>  produced a differentially-regulated list of  <dig> genes for the tnfα-treated arrays compared with the controls, and  <dig> genes were common to both tnfα datasets and regulated in the same direction and to approximately the same degree .
these numbers reflect the results of the unsupervised statistical analyses.
although fsh was shown by qrt-pcr and microarray analyses to statistically alter a number of specific genes , comparison of all treatments by anova determined that no genes were differentially regulated by fsh treatment more than  <dig> fold with an fdr of p <  <dig> .
additionally the hierarchical clustering and pca analyses  did not indicate any major effects of fsh.
we therefore compared tnfα ± fsh treated cultures  with the control ± fsh treated cultures  and generated lists of differentially expressed genes as shown in table  <dig>  lists of genes which were three-fold differentially expressed between these conditions are presented in tables  <dig> and  <dig> and the full annotations of these genes are presented in additional file 5: table s <dig>  this list was imported into ingenuity pathway analysis  and gene ontology enrichment analysis software tool kit  for functional pathway and network analyses.
numbers were determined by a one-way anova with a fdr p <  <dig>  for multiple comparisons using partek.
genes are categorised by function and listed in descending order of fold change in each category.
the association of genes with functional terms in this table was determined manually for each gene based on information obtained through the entrez gene database of ncbi.
the level of fold change and statistical significance  were determined using the step up benjamini-hochberg post-hoc test for multiple corrections following one way anova.
efna5
the level of fold change and statistical significance  was determined by one-way anova with post-hoc benjamini-hochberg testing for multiple corrections.
the association of genes with functional terms in this table was determined manually for each gene based on information obtained through the entrez gene database of ncbi.
genes are listed in descending order of fold change in each category.
ipa and go enrichment analysis of tnfα regulated dataset
genes from the dataset that were differentially regulated >  <dig> fold and p <  <dig>  between tnfα ± fsh and control ± fsh were mapped to canonical pathways in ipa and shown in figure 5a.
many of the pathways were associated with immune cell function and inflammatory response pathways.
several of these pathways contain genes such as the nuclear factor-kappa- b complex  genes, nfkb1a and nfkb <dig>  interleukin receptor  <dig> and the cell survival gene pik3r <dig>  the go enrichment analysis for this dataset , also showed over-representation of processes such as antigen presentation, inflammation and stress response.
we also used ipa upstream regulator analysis to identify upstream transcriptional regulators.
upstream regulators were predicted using a fisher’s exact t-test to determine the probability that genes from the dataset correspond with targets which are known to be activated or inhibited by those molecules based on current knowledge in the ingenuity database.
table  <dig> reveals a number of upstream regulators which were predicted to be influenced by tnfα treatment.
it contained the inflammatory response mediators il1b and if1b, and the matrix encoding gene fn <dig>  interestingly, the interleukin receptor antagonist il1rn was predicted to be down regulated, but it was shown to be up regulated according to the array intensity data.
p
†genes >3-fold differentially regulated between tnfα  and control  were used for this analysis.
the p value of overlap is the calculated statistical significance of overlap between genes from the dataset and genes that are known to be regulated by the upstream regulator using fisher’s exact test.
the bias-corrected z-score is used to infer the activation states of transcriptional regulators.
it is calculated from the proportions of genes which are differentially regulated in an expected direction based on the known interactions between the regulator and the genes present in the ingenuity database.
those genes with a z-score greater or less than two are considered to be either activated or inhibited respectively.
the two highest scoring gene networks generated in ipa from our dataset for the effect of tnfα on the cultured granulosa are displayed in figure  <dig>  network a shows an emphasis on innate immune response genes including several which are induced by interferon such as ifit <dig>  ifi <dig>  ifih <dig> and ifi <dig>  which are all up regulated.
other genes in this network have some involvement with apoptotic signalling, namely ripk <dig>  foxs <dig> and bex <dig>  the molecule forming most interactions in this network is the nf-κb complex, located within the nucleus.
the other network  shows a focus around tgfb <dig> with interactions between other genes such as pdgf, col16a <dig> and adamtsl <dig>  there is also connectivity with the down-regulated genes inha, inhba and fst, which are all known to play a role in folliculogenesis as previously mentioned.
other genes which were highly activated due to tnfα treatment , included gpr <dig> and slp <dig> , prkcb , col6a <dig> , krt <dig>  and hsd11b <dig> .
amongst the down-regulated group of genes, chst <dig>   may have a novel intra-follicular role.
discussion
this study examined the effects of the treatment with fsh and tnfα, separately or combined, on granulosa cells cultured under serum-free conditions and maintained in a non-luteinised state.
the effect of fsh alone at  <dig>  ng/ml paralleled the result of a previous study by glister et al.
<cit> , with similar increases in oestradiol production and expression of follistatin and inhibin a confirming the robustness of this physiologically relevant in vitro model used in the current experiments.
moreover, our qrt-pcr findings confirmed the ability of fsh to up regulate expression of its cognate receptor  as reported previously  <cit>  although a statistically significant difference was not detected by microarray analysis.
with the exception of fshr, there was excellent agreement between microarray and qrt-pcr data with respect to treatment effects on the other eight transcripts used for validation purposes.
the ability of tnfα to suppress the production of oestradiol in our experiment was also expected from results of previous studies in serum-free  <cit>  and serum-supplemented  <cit>  culture systems.
the unsupervised array analyses and the numbers of genes differentially regulated, show surprisingly perhaps, that in our experiment fsh alone had a minor effect on total gene expression, compared with tnfα, where many genes were differentially regulated.
the effects of fsh were limited to stimulation of energy metabolism and steroidogenesis in overall terms, in comparison with tnfα which mainly influenced inflammatory pathways and molecules.
clearly the main specific effect of fsh treatment was to stimulate oestradiol production by up regulation of aromatase expression .
the production of oestradiol and the concomitant activation of the folliculogenesis regulating genes for inhibin a and follistatin occur through recognised fsh cascade signalling involving cyclic amp and protein kinase a  <cit> .
the relatively low dose of fsh  used to treat the cells in our microarray experiment was selected as being optimal for promoting oestradiol secretion and was insufficient to induce an increase in progesterone synthesis or proliferation of the granulosa cells in culture.
however, there was transcriptional activation of the cyclin b <dig> and b <dig> genes, which indicates an increase in mitotic activity, although a net increase in cell number was not observed under the culture conditions used.
the endocrine functions of granulosa cells appeared to be down regulated upon tnfα treatments with reductions in fst , inha , inba , amh .
chst <dig> was also down regulated in the tnfα-responsive datasets.
the encoded enzyme is a sulphur transferase that sulphates n-acetylgalactosamine β <dig>  linked with n-acetylglucosamine  moieties on certain glycoproteins prior to secretion  <cit> .
sulphation of these structures can modulate the activity of these molecules by affecting the kinetics of binding  <cit>  and increases their rate of clearance from the body  <cit> .
glycodelin, an immune mediator, is produced by the granulosa cells at antral stages and possesses these lacdinac moieties.
glycodelin is taken up by the cumulus cells, where it is deglycosylated, loses immunosuppressive function and acquires properties beneficial to the fertilisation process  <cit> .
it may be possible that sulphation may also play a role in determining the activity of glycodelin, but confirmation of this would require further investigation.
it is known that tnfα exerts its apoptotic effects through the type i receptor , whereas other pro-inflammatory actions on growth and differentiation are mediated via the type ii receptor  as previously reviewed by matsuda et al.
<cit> .
since we did not observe any effect on viable cell number after  <dig> days exposure to tnfα, this might suggest that tnfα did not induce apoptosis in the granulosa cells and may act predominantly via tnfrii in our culture system to activate a pro-inflammatory cascade that modifies other aspects of cell function including matrix remodelling and up regulation of antigen presentation molecules.
many of the genes whose expression in granulosa cells was up regulated by tnfα are often associated with innate immune responses.
this reinforces recent evidence that granulosa cells can act as immune sensors and play an active role in initiating protective inflammatory responses to bacterial pathogens, recognised via interaction of pathogen-associated molecular patterns  such as lipopolysaccharide, with toll-like receptor  <dig>  on the cell surface  <cit> .
indeed, bovine granulosa cells were recently shown to express a full complement of tlrs.
moreover, functional inflammatory responses to pamps interacting with tlr <dig> and tlr <dig> were demonstrated  <cit> .
the genes influenced by tnfα treatment were generally associated with endocrine function, apoptosis, inflammation, and degradation as were expected from previous studies.
in our culture system, tnfα alone did not cause any net loss of cells.
some pro-apoptotic , but more anti-apoptotic genes , in fact, appeared to be stimulated by tnfα.
it is likely that the genes which act to block the apoptotic process are responding as a cellular survival mechanism, although some may be directly activated via the tnfα signalling pathway.
the blocking effect of tnfα on fsh-induced oestradiol production has been shown previously to be suppressed by treatment with peroxisome proliferator-activated receptor  γ ligands  <cit> , and in this study pparg expression was inhibited by tnfα, indicating that this lipid metabolism pathway was also involved.
hsd11b <dig> encodes a key enzyme in glucocorticoid metabolism and has been previously shown to be activated by tnfα via the regulatory gene cebpb in cultured cells  <cit> .
the genes identified as being most highly up regulated by tnfα include tgm <dig> , gpr <dig> , slpi  and tnc , none of which have previously been noted in granulosa cells.
tgm <dig> catalyses the cross-linking of proteins and the conjugation of polyamines to proteins.
it is also implicated as a positive regulator of the inflammatory response, nf-κb signalling and cell adhesion  <cit> .
gpr <dig>  is one of several receptors for the c5a molecule, a major chemotactic and pro-inflammatory product of the complement cascade activated during the innate immune response  <cit> .
slpi  was first characterised as a protease inhibitor but is now recognised as having additional properties including antimicrobial and immunomodulatory activities associated with the innate immune response.
slpi is up regulated by pro-inflammatory mediators and appears to have a tissue protective role  <cit> .
tnc  is an extracellular matrix molecule that is highly expressed during embryonic development but is normally present in low amounts in adult tissues.
however, tnc expression is up regulated in pathological situations involving tissue injury, wound healing, inflammation and cancer.
tnc influences cell migration, proliferation and cell signalling pathways through a variety of mechanisms including induction of pro-inflammatory cytokines  <cit> .
CONCLUSIONS
in vitro, the transcriptome of granulosa cells responded minimally to fsh compared with the response to tnfα.
the response to tnfα indicated a reduction in endocrine function and an active process akin to tissue repair and remodelling as would occur upon atresia.
additionally there was an inflammatory response to tnfα that displays many features normally associated with immune cells.

 an ovarian primordial follicle is composed of an inactive oocyte surrounded by granulosa cells all enclosed by a basal lamina. the granulosa cells of the ovarian follicle support and nurture the oocyte, and secrete oestrogens which are necessary for normal reproductive function. in mammals, the latter stage of follicle development can involve an approximate hundred fold increase in diameter, doublings of granulosa cell numbers and formation of a fluid-filled antrum. in cattle, the growth of follicles is tightly regulated, since two or three groups or waves of follicles emerge from a pool of follicles larger than 5 mm in diameter during each oestrous cycle. in these waves, follicles continue to enlarge over several days until one follicle grows faster and larger than the others and hence gains ‘dominance’. this deviation in size occurs when the follicles are around 7–8 mm in diameter. these processes of follicular growth occur largely due to the stimulatory influence of fsh, through its receptor localised exclusively to the granulosa cells, though other factors produced locally, for example growth differentiation factor - and bone morphogenetic protein - from the oocyte, are also involved. instead of one primordial follicle growing to ovulatory size and then ovulating, many follicles commence growing during the course of the cycle. most of these growing follicles become atretic, resulting in cows and humans, in only one or occasionally two follicles ovulating each cycle. the highest rates of atresia in follicular development occur around the time of antrum formation. it has been shown that the atretic process begins with cell death in the membrana granulosa initially by an apoptotic process. generally, apoptosis may be instigated intracellularly by cytotoxic stress, possibly due to free radicals or calcium influx which cause mitochondrial changes that eventually also lead to caspase activation. apoptosis can be initiated externally to the cell by the binding of ‘death’ ligands such as fas ligand, tumour necrosis factor α or trail to specific receptors. in follicular atresia it is unlikely that cell death occurs on a cell-by-cell basis because numerous pyknotic nuclei are observed during atresia. therefore it is probable that atresia is initiated by either the presence or absence of a particular external signal. tnfα can initiate apoptosis in granulosa cells. the expression of tnfα receptors on granulosa and theca cells has been shown to be increased in atretic follicles when compared with healthy small or preovulatory follicles. studies on atretic follicles so far have shown that many of the genes/pathways involved are common to those stimulated by tnfα, as recently reviewed by matsuda et al.. investigation of the effects of various agents on granulosa in vitro is dependent on the follicle stage at which the cells were isolated and the composition of the culture medium. it has been demonstrated that granulosa from small antral follicles are more responsive to fsh in serum-free culture and are capable of increasing oestradiol production over a six day period. this is an important consideration for studying granulosa cells as they have a propensity to differentiate into granulosa-lutein cells in a process called luteinisation, if cultured in serum-supplemented medium. such cells are completely unresponsive to fsh. a previous study showed that tnfα was able to block the effects of fsh in serum-free culture of rat granulosa cells. we were interested in the effect of fsh and tnfα on steroid production and global gene expression in bovine granulosa cells to help elucidate the mechanisms of action of these compounds at the transcriptional level. interest in the action of pro-inflammatory signals like tnfα has been augmented by recent reports that ovarian granulosa cells of cattle and other species fulfil an innate immunity role, being capable of detecting and responding to bacterial pathogens. we therefore cultured granulosa cells from small bovine follicles with or without fsh and tnfα, alone and in combination, assayed steroid hormone production by immunoassay and conducted microarray analyses using genechip bovine genome arrays.  cell culture and hormone assays a dose response culture experiment was performed to determine the optimal concentrations of fsh and tnfα to be used for the microarray experiments. on the basis of this experiment it was shown that oestradiol production was highest with a fsh dose of  ng/ml, being significantly different from values observed at all other fsh concentrations in the absence of tnfα. ten ng/ml of tnfα was sufficient to reduce this effect to the control level. progesterone levels and viable cell number at the end of the culture period were not significantly affected by either fsh or tnfα treatment. on the basis of these results,  ng/ml fsh and 10 ng/ml tnfα were selected for the culture of cells to be used for microarray analyses. the results of the hormone secretion in the cultures of granulosa cells which were used for the microarray analyses are shown in figure  fsh stimulation of the cultures produced a greater than 20-fold induction of oestradiol secretion by the granulosa cells compared with the control cells. conversely, tnfα treatment caused a > 65% reduction in the amount of oestradiol secretion under basal conditions and completely abolished the fsh-induced rise in oestradiol secretion. progesterone levels averaged approximately 200 ng/ml across all cultures, trending higher for the fsh-treated culture, but no significant differences were observed between the treatments. this confirms that the cultured granulosa cells maintained a hormone production profile characteristic of non-luteinised cells, and responded to fsh and tnfα as anticipated. hierarchical clustering and principal component analyses these analyses were performed on the sixteen microarrays, four from each treatment group, as presented in figure  and additional file 1: figure s. these figures show that culture sample g from the ‘control’ group was quite different in signal intensity across most probe sets on the chip from the other controls, and in fact all other arrays. this result prompted us to consider this array as a statistical outlier, and it was excluded from further analyses. the other arrays formed two clusters on the basis of distribution of signal intensity, the ‘tnfα and tnfα + fsh’ groups, and the ‘control and fsh’ groups. pca was also conducted for the tnfα and the tnf + fsh-treated groups, and then for the control and the fsh-treated groups. in both cases, for each comparison no differences were observed between the fsh treated and the non fsh-treated cells. anova demonstrated that no genes were different by more than 2-fold with an fdr of p < . we can therefore assume that under our experimental culture conditions, fsh alone did not have a substantial effect on granulosa cell total gene expression, whereas tnfα had a major effect and actually overrode any effects of fsh when treated in combination. quantitation of gene expression by rt-pcr figure  shows the quantitative rt-pcr results for some of the genes which were significantly affected by fsh and/or tnfα treatment of granulosa cells in culture: fshr, the steroidogenic genes cyp19a, cyp11a, hsd3b, hsd17b and star, and other genes inha, inhba and fst. these genes are known to be active during antral follicular expansion when the granulosa cells are responsive to higher levels of fsh. all genes except star, cyp11a and hsd3b were up regulated by fsh treatment alone. tnfα, whether alone or in combination with fsh, decreased expression of the following genes below the level of the control: fshr, star, inha, inhb and fst and the steroidogenic genes hsd17b and cyp19a to the level of the control. the decrease in cyp19a the gene encoding aromatase, the key enzyme of oestradiol synthesis, explains the observed decline in oestradiol production in the cultures under the influence of tnfα. differential gene expression analyses comparison of all treatments against the control group by anova with a three-fold change and a statistical cut-off of p <  produced a differentially-regulated list of genes for the tnfα-treated arrays compared with the controls, and genes were common to both tnfα datasets and regulated in the same direction and to approximately the same degree. these numbers reflect the results of the unsupervised statistical analyses. although fsh was shown by qrt-pcr and microarray analyses to statistically alter a number of specific genes, comparison of all treatments by anova determined that no genes were differentially regulated by fsh treatment more than fold with an fdr of p < . additionally the hierarchical clustering and pca analyses did not indicate any major effects of fsh. we therefore compared tnfα ± fsh treated cultures with the control ± fsh treated cultures and generated lists of differentially expressed genes as shown in table  lists of genes which were three-fold differentially expressed between these conditions are presented in tables  and and the full annotations of these genes are presented in additional file 5: table s this list was imported into ingenuity pathway analysis and gene ontology enrichment analysis software tool kit for functional pathway and network analyses. numbers were determined by a one-way anova with a fdr p <  for multiple comparisons using partek. genes are categorised by function and listed in descending order of fold change in each category. the association of genes with functional terms in this table was determined manually for each gene based on information obtained through the entrez gene database of ncbi. the level of fold change and statistical significance were determined using the step up benjamini-hochberg post-hoc test for multiple corrections following one way anova. efna5 the level of fold change and statistical significance was determined by one-way anova with post-hoc benjamini-hochberg testing for multiple corrections. the association of genes with functional terms in this table was determined manually for each gene based on information obtained through the entrez gene database of ncbi. genes are listed in descending order of fold change in each category. ipa and go enrichment analysis of tnfα regulated dataset genes from the dataset that were differentially regulated > fold and p <  between tnfα ± fsh and control ± fsh were mapped to canonical pathways in ipa and shown in figure 5a. many of the pathways were associated with immune cell function and inflammatory response pathways. several of these pathways contain genes such as the nuclear factor-kappa- b complex genes, nfkb1a and nfkb interleukin receptor and the cell survival gene pik3r the go enrichment analysis for this dataset, also showed over-representation of processes such as antigen presentation, inflammation and stress response. we also used ipa upstream regulator analysis to identify upstream transcriptional regulators. upstream regulators were predicted using a fisher’s exact t-test to determine the probability that genes from the dataset correspond with targets which are known to be activated or inhibited by those molecules based on current knowledge in the ingenuity database. table  reveals a number of upstream regulators which were predicted to be influenced by tnfα treatment. it contained the inflammatory response mediators il1b and if1b, and the matrix encoding gene fn interestingly, the interleukin receptor antagonist il1rn was predicted to be down regulated, but it was shown to be up regulated according to the array intensity data. p †genes >3-fold differentially regulated between tnfα and control were used for this analysis. the p value of overlap is the calculated statistical significance of overlap between genes from the dataset and genes that are known to be regulated by the upstream regulator using fisher’s exact test. the bias-corrected z-score is used to infer the activation states of transcriptional regulators. it is calculated from the proportions of genes which are differentially regulated in an expected direction based on the known interactions between the regulator and the genes present in the ingenuity database. those genes with a z-score greater or less than two are considered to be either activated or inhibited respectively. the two highest scoring gene networks generated in ipa from our dataset for the effect of tnfα on the cultured granulosa are displayed in figure  network a shows an emphasis on innate immune response genes including several which are induced by interferon such as ifit ifi ifih and ifi which are all up regulated. other genes in this network have some involvement with apoptotic signalling, namely ripk foxs and bex the molecule forming most interactions in this network is the nf-κb complex, located within the nucleus. the other network shows a focus around tgfb with interactions between other genes such as pdgf, col16a and adamtsl there is also connectivity with the down-regulated genes inha, inhba and fst, which are all known to play a role in folliculogenesis as previously mentioned. other genes which were highly activated due to tnfα treatment, included gpr and slp, prkcb, col6a, krt and hsd11b. amongst the down-regulated group of genes, chst may have a novel intra-follicular role. discussion this study examined the effects of the treatment with fsh and tnfα, separately or combined, on granulosa cells cultured under serum-free conditions and maintained in a non-luteinised state. the effect of fsh alone at  ng/ml paralleled the result of a previous study by glister et al., with similar increases in oestradiol production and expression of follistatin and inhibin a confirming the robustness of this physiologically relevant in vitro model used in the current experiments. moreover, our qrt-pcr findings confirmed the ability of fsh to up regulate expression of its cognate receptor as reported previously although a statistically significant difference was not detected by microarray analysis. with the exception of fshr, there was excellent agreement between microarray and qrt-pcr data with respect to treatment effects on the other eight transcripts used for validation purposes. the ability of tnfα to suppress the production of oestradiol in our experiment was also expected from results of previous studies in serum-free and serum-supplemented culture systems. the unsupervised array analyses and the numbers of genes differentially regulated, show surprisingly perhaps, that in our experiment fsh alone had a minor effect on total gene expression, compared with tnfα, where many genes were differentially regulated. the effects of fsh were limited to stimulation of energy metabolism and steroidogenesis in overall terms, in comparison with tnfα which mainly influenced inflammatory pathways and molecules. clearly the main specific effect of fsh treatment was to stimulate oestradiol production by up regulation of aromatase expression. the production of oestradiol and the concomitant activation of the folliculogenesis regulating genes for inhibin a and follistatin occur through recognised fsh cascade signalling involving cyclic amp and protein kinase a. the relatively low dose of fsh used to treat the cells in our microarray experiment was selected as being optimal for promoting oestradiol secretion and was insufficient to induce an increase in progesterone synthesis or proliferation of the granulosa cells in culture. however, there was transcriptional activation of the cyclin b and b genes, which indicates an increase in mitotic activity, although a net increase in cell number was not observed under the culture conditions used. the endocrine functions of granulosa cells appeared to be down regulated upon tnfα treatments with reductions in fst, inha, inba, amh. chst was also down regulated in the tnfα-responsive datasets. the encoded enzyme is a sulphur transferase that sulphates n-acetylgalactosamine β linked with n-acetylglucosamine moieties on certain glycoproteins prior to secretion. sulphation of these structures can modulate the activity of these molecules by affecting the kinetics of binding and increases their rate of clearance from the body. glycodelin, an immune mediator, is produced by the granulosa cells at antral stages and possesses these lacdinac moieties. glycodelin is taken up by the cumulus cells, where it is deglycosylated, loses immunosuppressive function and acquires properties beneficial to the fertilisation process. it may be possible that sulphation may also play a role in determining the activity of glycodelin, but confirmation of this would require further investigation. it is known that tnfα exerts its apoptotic effects through the type i receptor, whereas other pro-inflammatory actions on growth and differentiation are mediated via the type ii receptor as previously reviewed by matsuda et al.. since we did not observe any effect on viable cell number after days exposure to tnfα, this might suggest that tnfα did not induce apoptosis in the granulosa cells and may act predominantly via tnfrii in our culture system to activate a pro-inflammatory cascade that modifies other aspects of cell function including matrix remodelling and up regulation of antigen presentation molecules. many of the genes whose expression in granulosa cells was up regulated by tnfα are often associated with innate immune responses. this reinforces recent evidence that granulosa cells can act as immune sensors and play an active role in initiating protective inflammatory responses to bacterial pathogens, recognised via interaction of pathogen-associated molecular patterns such as lipopolysaccharide, with toll-like receptor on the cell surface. indeed, bovine granulosa cells were recently shown to express a full complement of tlrs. moreover, functional inflammatory responses to pamps interacting with tlr and tlr were demonstrated. the genes influenced by tnfα treatment were generally associated with endocrine function, apoptosis, inflammation, and degradation as were expected from previous studies. in our culture system, tnfα alone did not cause any net loss of cells. some pro-apoptotic, but more anti-apoptotic genes, in fact, appeared to be stimulated by tnfα. it is likely that the genes which act to block the apoptotic process are responding as a cellular survival mechanism, although some may be directly activated via the tnfα signalling pathway. the blocking effect of tnfα on fsh-induced oestradiol production has been shown previously to be suppressed by treatment with peroxisome proliferator-activated receptor γ ligands, and in this study pparg expression was inhibited by tnfα, indicating that this lipid metabolism pathway was also involved. hsd11b encodes a key enzyme in glucocorticoid metabolism and has been previously shown to be activated by tnfα via the regulatory gene cebpb in cultured cells. the genes identified as being most highly up regulated by tnfα include tgm, gpr, slpi and tnc, none of which have previously been noted in granulosa cells. tgm catalyses the cross-linking of proteins and the conjugation of polyamines to proteins. it is also implicated as a positive regulator of the inflammatory response, nf-κb signalling and cell adhesion. gpr is one of several receptors for the c5a molecule, a major chemotactic and pro-inflammatory product of the complement cascade activated during the innate immune response. slpi was first characterised as a protease inhibitor but is now recognised as having additional properties including antimicrobial and immunomodulatory activities associated with the innate immune response. slpi is up regulated by pro-inflammatory mediators and appears to have a tissue protective role. tnc is an extracellular matrix molecule that is highly expressed during embryonic development but is normally present in low amounts in adult tissues. however, tnc expression is up regulated in pathological situations involving tissue injury, wound healing, inflammation and cancer. tnc influences cell migration, proliferation and cell signalling pathways through a variety of mechanisms including induction of pro-inflammatory cytokines.  in vitro, the transcriptome of granulosa cells responded minimally to fsh compared with the response to tnfα. the response to tnfα indicated a reduction in endocrine function and an active process akin to tissue repair and remodelling as would occur upon atresia. additionally there was an inflammatory response to tnfα that displays many features normally associated with immune cells. 
to investigate the effects of stimulatory and inhibitory factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3– initially dose–response experiments were performed to determine the optimal concentrations of fsh and tnfα to be used for the microarray experiments. oocytes mature in ovarian follicles surrounded by granulosa cells. during follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. tnfα treatment reduced the endocrine activity of granulosa cells with reductions in expression of fst, inha, inba and amh. in vitro, the transcriptome of granulosa cells responded minimally to fsh compared with the response to tnfα. the top-ranked canonical pathways and go biological terms for the tnfα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. additionally there was reduction in endocrine function and induction of an inflammatory response to tnfα that displays features similar to immune cells. the effect of tnfα on gene expression dominated that of fsh, with substantially more genes differentially regulated, and the pathways and genes regulated by tnfα being similar to those of fsh plus tnfα treatment. for array experiments cells were cultured under control conditions, with fsh, with tnfα, or with fsh plus tnfα and rna was harvested for microarray analyses. the two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. the response to tnfα indicated an active process akin to tissue remodelling as would occur upon atresia.
 oocytes mature in ovarian follicles surrounded by granulosa cells. during follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. however, most follicles cease growing and undergo atresia or regression instead of ovulating. to investigate the effects of stimulatory and inhibitory factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3– initially dose–response experiments were performed to determine the optimal concentrations of fsh and tnfα to be used for the microarray experiments. for array experiments cells were cultured under control conditions, with fsh, with tnfα, or with fsh plus tnfα and rna was harvested for microarray analyses.  statistical analysis showed primary clustering of the arrays into two groups, control/fsh and tnfα/tnfα plus fsh. the effect of tnfα on gene expression dominated that of fsh, with substantially more genes differentially regulated, and the pathways and genes regulated by tnfα being similar to those of fsh plus tnfα treatment. tnfα treatment reduced the endocrine activity of granulosa cells with reductions in expression of fst, inha, inba and amh. the top-ranked canonical pathways and go biological terms for the tnfα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. the two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. upstream regulator analyses also predicted tnf, interferons γ and β and interleukin 1β.  in vitro, the transcriptome of granulosa cells responded minimally to fsh compared with the response to tnfα. the response to tnfα indicated an active process akin to tissue remodelling as would occur upon atresia. additionally there was reduction in endocrine function and induction of an inflammatory response to tnfα that displays features similar to immune cells. ovarymicroarray analysisbovine granulosa cellsfollicles 