1. Overview of gonadal development and maturation by principal component analysis: convergence of the maturing ovary to a testis-like profile
To focus on ovary maturation and aging in relation to gonadal development, we compared comprehensive gene expression profiles among a set of 141 microarray gonadal samples, of which 43 come from our study and 98 were reanalyzed from public databases deposited by 4 other laboratories in addition to ours (see Methods and below, section 2). The samples spanned the life history of the mouse ovary and testis from the bipotential gonad to adulthood, including several mouse models of premature ovarian failure. In order to identify major expression profiles associated with distinct genotypes and developmental stages, we used principal component analysis (PCA), an unsupervised clustering method that does not rely on a priori class assignment. Essentially, the method first identifies the group of genes with correlated expression profiles that account for the greatest amount of variance in the dataset (the first principal component, PC1), and then sequentially looks for additional co-expression profiles that are not correlated with the first group and account for decreasing amounts of variance (see Methods).
In Figure , two distinct groups of correlated profiles, represented by the first two principal components (PC1 and PC2), robustly discriminated gonadal samples into appropriate clusters of biological replicates, placing all testis (diamond) and ovary (circle) samples in trajectories according to developmental stages (details about the samples are given in additional files 1A–B
). In Figure , colored symbols are added to show the relative positions of clusters at specific times when particular genes of importance were ablated singly or in combination. The 300 most variable genes were sufficient for good discrimination, but better resolution was obtained with longer lists, i.e., about 6,000 top-ranking markers (Figure and see additional file 2A
Figure 1 First two principal components of PCA trained on the expression profiles of 6,455 developmental marker genes. Several published gonadal microarray datasets ("test dataset") have then been mapped onto the PCA space (our large panel of wild-type and mutant (more ...)
PC1, the first principal component, i.e., the horizontal axis of Figure , represented ~40% of the total variance of the developmental gonadal marker genes. In the bipotential gonad, at E10–E12, as expected, sets of genes were close by in ovary and testis. Then, along PC1, developing ovaries first diverged sharply from embryonic testes and subsequently moved back to values that were increasingly similar to newborn testes (E18 to adult ovary, as labeled at the top of Figure ). The trend is thus in keeping with morphological indications that some features of follicle maturation involve testis differentiation-like processes (see [16
] for a recent review).
The genes that showed sharp timing differences in their levels of expression along PC1 (see Methods) included those involved in meiotic activity as well as other early ovary somatic cell markers (e.g., Irx3
, and Dmc1h
) that had maximum values at the left of the PC1 axis. In addition, many top-scoring genes were involved in steroidogenesis and other early features of testis somatic cell differentiation (e.g., Dhh, Cyp17a1
), with maximum values at the right of the PC1 axis (Figure and see additional file 2B
). Furthermore, their PC1 positions in Figure illustrate that Wnt4-/-
ovaries and, to a lesser extent, Foxl2-/-
ovaries, mapped consistently closer to testis than did age-matched wild-type control ovaries. Also, double mutants showed additive or synergistic effects on "movement toward testis". However, the relative contribution of reduced meiotic activity vs
increased activity of steroidogenic genes and/or testis-like programs appears to differ in each type of mutant. Indeed, newborn ovaries lacking Kit, Lhx8
lie relatively close together along PC1 in spite of their very different composition in germ cell numbers and their highly divergent degrees of sex reversal (see below). Consistent with distinct roles of meiotic and testis genes along the PC1 axis, an adult, germ cell-depleted, Kitl
-deficient testis sample mapped at the far right of the graph (Figure ), i.e., nearer newborn testes than adult wild-type testes (the latter are located in the middle of the graph). Thus, multiple pathways, ranging from control of meiosis to somatic cell sex reversal, are involved in PC1.
Figure 2 Real-time PCR data for fetal and newborn gonads of all relevant genotypes. Top: Ovarian genes that require Foxl2; Middle: Testis genes that are derepressed in the absence of Foxl2; Bottom: Ovarian genes that are independent of Foxl2. Note that several (more ...)
In the case of the second axis (PC2) as well, multiple distinct pathways may be involved, but because they appear dissociated by PCA, they are likely different and relatively autonomous from those accounting for PC1. Of interest, PC2 pointed toward many genes involved in follicle formation. For example, newborn ovaries, which are enriched in primordial follicles (e.g., [1
]), were remarkably clustered at the top of PC2, and several genes involved in early follicle development, i.e., Lhx8, Figla
, and Sohlh1
]) strongly correlated with this axis (see additional file 2C
). However, follicle formation is only part of the underpinning of PC2, because some newborn ovary samples (e.g., Kit
mutants) were also high-scoring even though they are known to be largely devoid of follicles.
Overall, a representative "gonadal developmental transcriptome", trained on pre- and neonatal gonadal samples by PCA, appears to discriminate the range of wild-type gonadal variation throughout life. In addition, PCA suggests that dysgenetic ovaries activate programs that may represent the anticipation of a normal maturation/aging process and which may be a causative factor for associated pathology. However, PCA does not discriminate between possible different contributions to this aging-like process, in particular the effects of meiotic germ cell depletion vs
. the activation of bona fide
testis-like pathways in somatic cells. In addition, the expression profiles of some genes with important gonadal functions, including Foxl2
(see below), correlated only weakly with PCA coordinates. In particular, they both ranked > 100th
among genes that were ordered according to their degree of correlation with PC1 using the Focus classifier (see additional file 2C
; consistent results were obtained with "predictive analysis of microarray", PAM [29
], data not shown). We infer that functional studies of ovary development and maturation may be more incisive if one includes systematic comparisons to testis
samples in appropriately chosen developmental models. We thus present the molecular characterization of Foxl2
-null ovaries in the context of other models of ovarian dysgenesis, with special emphasis on the comparison of wild-type ovary and testis differentiation.
2.1. Foxl2 knockout ovaries
knockout ovaries have a histomorphologically normal appearance before birth. To detect molecular changes that may nonetheless predate postnatal anomalies, we sampled ovaries from prenatal and neonatal stages (13.5 and 16.5 days postcoitum, dpc, and birth). These sampling times covered the period of greatest variation in gonadal developmental genes (Figure , above) and could minimize minor stage-specific changes or small shifts in developmental rate that might bias single time points with age-matched samples. To this end, linear contrasts implemented in the Focus software work well [30
]: the 20 top-ranking differentially expressed genes (up or down) are listed in the left column of Table , and the full lists are given in additional files 3A–B
. We also obtained a combined gene list from standard pair-wise analyses, which was highly consistent but much less sensitive (see additional file 3C
and Methods). In addition, we sampled for comparison Wnt4
-null ovaries at 15.5 dpc, i.e., the stage at which partial sex reversal is well started but oocytes are still present and well differentiated.
Foxl2-null ("KO") ovaries relative to wild-type and in various biological contexts.
We first adopted a strategy to identify highly specific (though possibly indirect) targets of Foxl2, as follows.
2.2. Candidate genes for a primary role downstream of Foxl2: profiling of compound knockout models involving Foxl2
We reasoned that primary targets of Foxl2
would tend to be differentially expressed in all ovaries lacking Foxl2
either alone (from the above analysis) or in combination with the ablation of Wnt4
. At birth, these various mouse models show very different morphology, cell composition, and endocrine function, and may thus provide a stringent framework for the identification of Foxl2
-dependent genes [17
]. We compiled lists for genes that were differentially expressed in Wnt4-/-Foxl2-/-
double knockout ovaries relative to ovaries lacking Wnt4
function but harboring a functional Foxl2
allele (see additional file 4A–B
, the 20 top genes up- and down-regulated are given in Table ). We then intersected these lists with lists of genes associated with loss of Foxl2
alone (see above). That list comprises 190 and 78 genes that were systematically down or up-regulated in the absence of Foxl2
(see additional file 4C
; the 20 top genes up and down are given in Table , second column from right). These lists thus include highly specific genes that may mediate Foxl2
action, as follows.
Inspection of the top genes in the lists (Table ) indicates that a large fraction of Foxl2
-dependent genes have no previously assigned role in gonadal development but have been associated with either neuronal or vascular development (as indicated by asterisk or underline, respectvely, in Table , column 3 from the left). Some show female-specific upregulation in supporting or steroidogenic cells of embryonic gonads, including a transcription factor, ODZ4, and several plasma transmembrane proteins, including plexin (PLXNC1), the cadherin-domain containing Calsyntenin 2 (CLSTN2), and a leucine-rich repeat protein (LRRC4) (bold font, Table ). Because some of these genes are involved in brain cortical patterning [31
], they might conceivably mediate the formation of the ovarian cortico-medullary axis, which is essential for follicle dynamics and reproductive longevity in mammals, and has no histoanatomical counterpart in the testis [33
Top-ranking down-regulated genes also included Grip1
(glutamate receptor interacting protein 1), a nuclear repressor required for estrogen receptor alpha activity, and the gene encoding the AKR1C14 aldo-keto reductase, which can metabolize the most potent natural androgen, dihydrotestosterone [34
-dependence of these genes during embryonic development (Figure ) might reflect an early role for Foxl2
in regulating sex-specific steroidogenesis, which may be particularly important in higher mammals. Furthermore, starting around 16.5 dpc, upregulation of aromatase/Cyp19a1
and the nuclear receptor Nr5a2/LRH-1
, related to the master regulator of steroidogenesis, Nr5a1/Steroidogenic factor-1
, was strongly reduced in Foxl2
-null ovaries relative to controls.
Additional hits further down the lists included sphingomyelin synthase Sgms2
(see additional file 4C
). It may be a positive regulator of primordial follicle formation and/or maintenance, given that loss of a sphingomyelinase, which performs the reverse metabolic reaction, was reported to enhance primordial follicle maintenance in mice [36
]. Another hit, encoding the cytoskeletal protein BICD1 that is related to a maternal fertility factor in Drosophila
, was enriched in primordial follicles of both oocytes and somatic cells (data from [37
]). It may complement the action of a paralogue, BICD2, that we have previously shown to be expressed in growing oocytes [39
Expression levels were sometimes altered starting from 13.5 dpc, as validated by real-time PCR (Figure ). There the levels in E13 testis, first three bars at the left of each row, are compared to levels in the ovary collected at various developmental stages in wild-type and mutant mice; the corresponding microarray data (see additional file 5
) are concordant in every case. Their relevance as candidate Foxl2
targets was further substantiated by experiments in which an added Foxl2
transgene was provided to wild-type mice (see below).
The comparison of ovaries lacking Foxl2 alone or in combination with either of two other genes therefore prioritizes candidate target genes that show sensitivity to Foxl2 loss in the independent mouse models. Candidate Foxl2 targets may also be inferred by their response to multiple ovarian regulatory pathways, as follows.
2.3. Candidate Foxl2 target genes that respond to Wnt4 or other ovarian pathways
We cross-compared all mouse models of ovarian dysgenesis that have been studied by microarray expression profiling to date. They include knockouts of the somatic genes Emx2
, and the oocyte genes Lhx8, Nobox, Foxo3
]. We reanalyzed the respective datasets compared to their respective controls (see Methods and additional files 6A–J
). As expected for genes that are all required for fetal and newborn ovary development, the resulting lists of differentially expressed genes indicated similar alterations, reflecting synergistic effects. In particular, several well-known ovarian markers were comparably changed. Inferred putative interactions are represented in Figure , along with others that involve important novel candidate targets of Foxl2
, and Sgms2
, see above). Arrows
indicate gene dependencies in terms of ranking by statistical significance in the microarray analyses; thus, the arrowhead points to gene(s) that score among the top hits (i.e., for thick
arrows, ranks 1 through 50) in the list of genes affected by the gene from which the arrow originates. [Some gene dependencies that have been shown to be strong in Rspo1
-null ovaries, for which microarray data are not available, are indicated with blue arrows.] For example, Wnt4
scored among the top hits in the respective lists of Emx2
- and Wnt4
-dependent genes, respectively, consistent with previous reports [40
]. Similarly, zona pellucida genes Zp2
were identified among the genes most strongly down-regulated in the absence of Lhx8, Nobox
. In addition, thin
arrows indicate progressively lower degrees of dependence (i.e., rankings arbitrarily set at 50–150 and > 150, resp.); for instance, intermediate (thin arrow) dependencies include that of Dax1/Nr0b1
, a dependence that is known to be only partial [47
Figure 3 Putative interactions among developmental genes inferred from the comparison of several knockout models of ovarian dysgenesis. Arrows indicate positive interactions. Most cases of competitive or antagonistic interactions are not represented, because all (more ...)
Overall, Figure shows that in spite of a widespread network of positive correlations among well-known ovarian genes, they are affected to a different degree in distinct pathological conditions, with a quite clear-cut separation of the main candidate targets of somatic genes Emx2/Rspo1/Wnt4/Foxl2 from those of oocyte genes Lhx8/Nobox/Figla/Foxo3 (green/pink vs red boxes, resp., in Figure ; different colors are used for Foxl2 and Wnt4-related genes because of their partial independence, see below). This is consistent with the proposal that somatic cells and oocytes may act on largely distinct though complementary pathways to promote ovary development. The relation of these genes to testis-like markers (blue boxes in Figure ) was more complex (see below).
We then extended the analysis from known markers to the complete gene lists obtained by microarray profiling, and thus determined their degree of overlap by chi-square (Table and see Methods). As expected, most lists were positively correlated with one another (i.e., genes up- or down-regulated in the absence of one ovarian gene tended to change in the same direction in the absence of the others; see additional files 6A–H
). Nevertheless, one exceptional trend was notable: the lists of genes that depend on Foxl2
and those dependent on Wnt4
were negatively correlated. In other words, genes that were up-regulated in the absence of Wnt4
were preferentially down-regulated in the absence of Foxl2
, and vice versa
(p < 0.001, chi-square; see additional file 7
). In particular, important developmental genes that were up in Foxl2
-null ovaries and down in Wnt4
-null ovaries included known or likely Wnt4
-dependent genes in the ovary [Nr0b1/Dax1, Amhr2
, and Podxl;
the last of which is also a candidate Wnt4
target in the kidney (see additional file 7D
and data not shown)].
Degree of concordance between lists of differentially expressed genes in several models of ovarian dysgenesis.
These differences were reflected in the relative distribution of several pathways listed in the Gene Ontology and KEGG databases (at FDR 10%; Table and see additional file 8
). In particular, glucose metabolism and protein synthesis were strongly enriched in Foxl2
-null ovaries but depleted in Wnt4
-null ovaries, respectively, whereas cell-cell interactions and neuronal-like pathways were depleted in Foxl2
-null ovaries but enriched in the absence of Wnt4
. Consistent with reduced cell-cell signaling, loss of Foxl2
apparently led to reduced cell migratory activities, as inferred from the down-regulation of MAPK and several cancer-related pathways that are presumably involved in ovarian tissue remodeling, as well as apoptotic factors that may have homeostatic actions in these processes. This is consistent with the notion that Foxl2
is a critical determinant of ovary histogenesis, notably follicle formation.
KEGG analysis on gene lists for Foxl2-null or Wnt4-null fetal ovaries.
Of interest, Foxl2 loss also affected Wnt-signaling. Global alterations associated with oocyte genes were much less pronounced (none scored at the 10% FDR threshold). Consistent with a complementary action of oocytes and somatic cells, loss of oocyte genes led to a mixture of Foxl2- and Wnt4-associated features (not shown).
The results thus support our previous findings that Foxl2
act independently during ovary differentiation, and further substantiate the notion that they may partly antagonize each other and/or regulate mutually competitive pathways. Nevertheless, some known gonadal genes were top-ranking candidate targets of both Foxl2
, including Kitl
(pink-green color in Figure and see additional file 1D
). Novel genes of potential interest that were strongly down-regulated in ovaries lacking Foxl2
, and thus responsive to the sum of their actions, included Gpc4
(see additional file 7A
and see Discussion). Additional hits included several transcription factors, e.g., Msx1, Grip1, Tcf4
, and Foxp1
(see additional file 7A
). Although they scored relatively low, these genes were up-regulated in Foxl2
-transgenic mouse embryos (see below). Thus all of these genes, many of them expressed in somatic cells, now become candidates for a Foxl2
-dependent role in coordinating ovarian histogenesis and/or steroidogenesis.
2.4. Anti-testis roles of Foxl2 and Wnt4, and of other possible regulatory genes
We next focused on genes that were significantly up-regulated in ovaries lacking Foxl2
alone or in combination with Kit
. They included both ovarian and testis genes. The ovarian genes included Nr0b1/Dax1, Wnt4
as well as novel candidates, such as Zbtb7c;
the testis genes included Sox9, Dhh, Dmrt1
, and Cyp26b1
(Table , Figure , and see additional file 3
). It is notable that these and other top-scoring ovarian and testis genes have important roles in sex determination and/or early sexual dimorphism (reviewed by, e.g., [18
]). Thus, in the absence of Foxl2
, the exacerbation of Foxl2
-independent ovarian pathways(s) coincided with the partial de-repression of testis-like genes. This started during embryonic development, even in single knockout ovaries that are morphologically normal until birth (see Discussion). Similarly, and consistent with much earlier onset of morphological anomalies, Wnt4
-null single knockout ovaries showed de-repression of some testis genes, e.g., Dhh
; and although Foxl2
was not up-regulated in these dysgenetic ovaries by whole-organ microarray (see additional file 5
), its levels remained high compared to other ovarian genes and were increased by real-time PCR (Figure , and see below). In addition, FOXL2 protein expression was strongly detected in the persisting cord-like and follicle-like structures (data not shown).
The expression levels of some early testis-like genes showed opposite directions of change in the absence of distinct ovarian genes. In particular, Inhbb
was up in fetal ovaries lacking either Wnt4
, but down in ovaries lacking the oocyte gene Lhx8
and in the most masculinized phenotype that we have studied, i.e., Wnt4-/-Foxl2-/-
double knockout newborn ovaries (Figures and ). Similarly, Robo1
, which is up-regulated in Wnt4
-null ovaries ([43
] and see additional file 6B
), showed reduced levels in ovaries lacking Foxl2
(Figure and see additional file 3A
). Therefore, regulation of these testis-like genes, and their impact on the ovarian phenotype, may depend on the conditional interactions of multiple ovarian factors.
Similar to loss of Foxl2
, loss of Lhx8
led to up-regulation of a few genes that are normally expressed in testes (e.g., Stra6
see additional file 6F
). However, this effect did not extend to genes known to be involved in testis sex determination, suggesting that Foxl2
- and Wnt4
-null ovaries are better suited to identify novel anti-testis genes.
To identify novel candidate anti-testis genes with a "master" regulatory role comparable to Foxl2
, we used a simple statistical approach (Methods) that detects genes that are both testis-depleted and up- or partially down-regulated in the dysgenetic ovary relative to the wild-type ovary. The gene candidates for "independent anti-testis" gene action in the Foxl2
-null or Wnt4
-null ovaries are listed in additional files 9A–C
, with the top-ranking hits given in Table . As expected as an internal control, consistent with their relative independence [17
was top-ranking in Foxl2
-null ovaries, and Foxl2
-null ovaries. In addition, the overlap between the two lists detected Rspo1
] and numerous oocyte genes as female-enriched and independent of both Wnt4
[the latter may again reflect the overall independence of oocytes from loss of either Wnt4
(before birth) or Foxl2
]. In addition, several other genes were expressed in somatic cells, i.e., Runx1, Irx3
, a Foxl2
-antisense transcript, Wnt9a
, and Zbtb7c
. The relative levels of all of them were validated by multiple microarray probe sets and/or by real-time PCR (as well as in studies of Foxl2
transgenic mice, below). These genes may thus have a role in autonomous pathways capable of antagonizing testis differentiation independently of Foxl2
The thirty top-ranking genes for an action independent of Foxl2 and Wnt4.
3. Foxl2 transgenic mice
We used Foxl2
-transgenics to see if candidate Foxl2
targets inferred from the microarray analyses presented above were indeed responsive to Foxl2 (Figure and ; there the levels of individual genes are compared in gonads from wild-type males, wild-type females, transgenic males, and transgenic females). As expected, Foxl2
expression was absent or at background levels in gonads from XY wildtype embryos. But in XY transgenic embryos, gonadal levels of Foxl2
were similar to wildtype ovaries, and XX transgenic ovaries showed even stronger expression (nearly 2-fold, see additional file 10
). This correlated with sex-specific effects, as follows.
Figure 4 Expression levels of several gonadal genes in 13.5 dpc gonads from Foxl2 transgenic embryos compared to littermates. A: genes known for a role in early testis differentiation, some of which are also expressed in the adult ovary; B: novel or known ovarian (more ...)
All female markers normally up-regulated during early ovarian differentiation, whether sexually dimorphic or not, were expressed at consistently higher levels in 13.5 dpc Foxl2
-transgenic ovaries relative to wild-type littermates (21 genes tested, Figures and and see additional file 11
). That included Rspo1
. In XX embryos, up-regulation overtly extended to the meiotic markers Sycp3
. The status of Foxl2
candidate targets inferred from the microarray analysis of Foxl2
knockout mice is thereby validated, and there are hints of feed-forward interactions with Foxl2
-independent genes, such as Wnt4
Furthermore, several ovarian genes were up-regulated to various extents in 13.5 dpc transgenic XY gonads, which contained severely disorganized seminiferous tubules [17
]. These changes are consistent with the induction of a limited degree of male-to-female sex reversal in Foxl2
transgenics. Among the statistically significant genes were some expressed in meiotic germ cells (Sycp3
) and others in somatic cells, i.e., Zbtb7c, Clstn2, Grip1, Lrrc4
and aromatase (Cyp19a1
). In particular, induction of aromatase and Clstn2
transgenic XY gonads was remarkably strong relative to testes (Figure ). This is in agreement with previous in vitro
studies indicating that Foxl2
can transactivate the aromatase promoter (e.g., [48
]). Conversely, 13.5 dpc Foxl2
-transgenic XY gonads showed reduced expression of the male sex determining genes Sox9
. In addition, strong repression was seen for downstream embryonic testis-specific genes of endocrine relevance (e.g., the antimullerian hormone, Amh
, and Cyp11a1/P450ssc
, which were reduced 3- and 18-fold, respectively). Repression was also strikingly pronounced for the two male germ cell markers tested, Xmr
, which were reduced 26- and 4-fold, respectively (Figure ).
A few results were unanticipated; e.g., the Foxl2 transgene down-regulated the expression of Wnt4 and Dax1/Nr0b1 while increasing expression of Inhbb. These unanticipated effects may result from an exacerbation of the inferred partial antagonism between Foxl2 and Wnt4, and possibly relate to the complex responses shown by Inhbb attendant on the loss of various ovary genes alone or in combination (see above). In any case, the preponderance of the data provide independent support for an early anti-testis action of Foxl2 – whether by delaying development of XY gonads or by promoting male-to-female sex reversal. Most important, the Foxl2-mediated disruptive effects, including the suppression of Wnt4, were confined to XY embryos. The notion of a specific and early sexually dimorphic role for Foxl2 is thereby supported.
4. Gene dosage effects in Foxl2+/- heterozygous ovaries
Loss of a single copy of Foxl2 might represent a model for the heterozygous FOXL2 mutations that affect patients. We looked at the extent to which heterozygotes expressed molecular anomalies like those seen in Foxl2-null ovaries. We focused on two stages, 16.5 dpc and 7 dpn (days post-natum), because the corresponding Foxl2-null ovaries showed well-differentiated morphological states that were either normal or well engaged in abnormal development.
In order to identify molecular anomalies associated with heterozygous loss of function, we generated oocyte- vs
somatic cell gene lists enriched in fetal vs postnatal ovaries of heterozygous mice. The lists were based on published microarray data, supplemented by a list of the probes for known testis determining genes (Methods and see additional file 12
). We then evaluated differential enrichment in these lists by a sensitive algorithm that detects non-random distributions in pair-wise comparisons (Gene Set Enrichment Analysis, or GSEA, [50
]; all these results are presented in additional file 13
; see Methods).
We first tested testis-determining genes (including Dmrt1, Sox9, Dhh, Fgf9, and Sf1/Nr5a1), which, as a group, were significantly up-regulated in 16.5 dpc Foxl2+/- heterozygous ovaries relative to wild-type (p < 0.03, GSEA), with levels that were comparable to those in Foxl2-null ovaries (p = 0.20, GSEA). These findings raise the possibility that loss of a single allele of Foxl2 may be sufficient for a significant though weak derepression of testis genes in the fetal mouse ovary. Nevertheless, this effect appeared to be transient. Indeed, in 7 dpn Foxl2+/- heterozygous ovaries, the microarrays evinced no significant trend toward the expression of testis markers (p > 0.2, GSEA). This contrasted with Foxl2-null ovaries, which, consistent with our previous studies showing delayed postnatal oocyte growth and partial sex reversal (see above), showed a significant enrichment of testis determining genes at 7 dpn (p = 0.02, GSEA).
Consistent with ongoing sex reversal, 7 dpn Foxl2
-null ovaries also showed a sharp extensive repression of postnatal oocyte and somatic follicle cell markers compared to both Foxl2+/-
heterozygous and wild-type ovaries (p < 0.001, GSEA). Compared to Foxl2
-null ovaries, Foxl2+/-
heterozygous ovaries exhibited normal expression of oocyte growth genes but showed clear dosage effects for somatic follicle markers. The latter were inhibited almost as much in heterozygotic ovaries as in Foxl2
-null ovaries compared to wild-type (p < 0.001, GSEA). Furthermore, as expected for a dosage-related response, the inhibition in heterozygous ovaries was less strong than in Foxl2
-null ovaries (p < 0.001). Top-scoring genes were often known or likely to be involved in early steps of follicle growth, i.e., Inhba, Inhbb, Cyp11a1/P450ssc, Cyp17a1, Nr5a2
(see additional file 14
), as well as novel genes with a likely role in somatic cells, e.g., Odz4
(see above). Several of these genes are known to be involved in steroidogenesis. Thus, in the absence of one Foxl2
allele, strong dosage effects in somatic follicle growth genes contrasted sharply with the normal expression of oocyte growth genes.
In addition, in 16.5 dpc and 7 dpn Foxl2+/- heterozygous ovaries, we found significant up-regulation of markers for fetal ovary development, independent of whether they were germline- or somatic cell-enriched (e.g. Figla, Sohlh1, Sycp3, vs Rspo1, Irx3, Gng13, p < 0.001 and p < 0.01, resp., GSEA). This suggests that Foxl2+/- heterozygous ovaries undergo a developmental delay starting in fetal life.