In the present study we were able to identify orthologous genes exhibiting similar expression profiles in the somatic cells surrounding the oocyte during developmental competence acquisition in vertebrates. Our results rely on genome-wide transcriptome analyses carried out, in parallel, in four different vertebrate species using (i) a similar technology and (ii) similar key steps of developmental competence acquisition. The transcriptome analysis was followed by an OrthoMCL analysis to specifically identify orthologous genes exhibiting similar expression profiles. To our knowledge such an approach has never been used to investigate the oogenetic process in evolutionary distant vertebrate species.
A surprisingly high number of orthologous genes exhibiting evolutionary-conserved expression profiles
We identified a surprisingly high number of orthologous genes exhibiting similar expression profiles at key steps of oocyte-developmental competence in different vertebrate species. This is especially true in mice and cow in which ~1500 and ~1200 orthologous genes were identified, respectively. This is also the case in tetrapod species in which ~400 orthologous genes were found to be differentially expressed in the 3 species among a total of 4000–8000 differentially expressed genes in each species. It has to be stressed that the highest number of differentially expressed genes (~8000) was found in the mouse in which the number of spotted probes was twice as high as in the 2 other species. Together, this suggests that approximately 10% of the differentially expressed genes have orthologs in the 2 other tetrapod species that behave similarly during oocyte developmental competence acquisition. It should be noted that the percentage of orthologous genes with similar profiles is remarkably high when considering the major differences in follicle ultrastructure and physiological regulations that exist between amphibians and mammals. This suggests a strong conservation -and possibly a crucial role - of the corresponding molecular mechanisms during vertebrate evolution despite the presence of an antrum in the mammalian ovarian follicle and the absence of antrum in non-mammalian vertebrates. In agreement with this hypothesis is the significant enrichment in genes involved in cellular energy metabolism, cell-to-cell communications, and meiosis observed in the three tetrapod species and the two mammalian species. Among the four vertebrate species studied herein, a much more limited set of common differentially expressed genes could be identified, in comparison to the large number of common genes shared by the three tetrapod species or by the two mammalian species. This can largely be explained by the evolutionary distance between the species, especially between rainbow trout and mammals. Several methodological reasons also exist that have possibly reduced the number of common genes identified. These reasons are mostly due to the lack of genome sequence availability in rainbow trout and its consequences on the design of the oligonucleotides and on subsequent in silico analysis. Despite these difficulties, we were able to highlight genes and pathways involved in oocyte developmental competence acquisition in the four vertebrate species (Figures
7). In consistency with data obtained in tetrapods and mammals, our results stress the importance of the nutritive role of the somatic cells surrounding the oocyte and of the mechanisms involved in extracellular matrix formation. In addition, more than 200 common genes were identified in rainbow trout and Xenopus as highlighted in the result section (Figure
In the present study, we have deliberately chosen to highlight some of the genes exhibiting similar profiles in the somatic compartment during oocyte developmental competence acquisition in several vertebrate species. Many groups of orthologous genes (i.e. OrthoMCL groups) were however identified that could not be discussed here due to space limitations. All corresponding expression profiles and OrthoMCL information are however available (see additional files) and can be searched for specific genes or mechanisms. We believe that this unique dataset will be very useful to investigators in the fields of reproductive biology and evolutionary biology. Similarly, the large species-specific datasets generated in the present study can be further analyzed by other investigators to address species-specific questions.
As indicated above, our approach has led to the identification of more than 1200 orthologous genes that deserve further analysis. In the present study, we have deliberately chosen to focus our analysis on common genes shared by tetrapods or vertebrates in agreement with the main objectives of our study. We will however briefly highlight in this section some of the identified genes differentially expressed in both mouse and cow in order to stress the biological relevance of our approach and to clearly demonstrate the methodological validity of our results.
Extra-cellular matrix remodeling linked to ovulation
Cathepsin S (Ctss) and Cathepsin B (Ctsb) are lysosomal cysteine proteinases involved in degradation of extracellular matrix. Their transcripts (OG_10260 and OG_10174, respectively) were over-expressed in cumulus complexes (CC) of mature (C2) oocytes in mice and cow in comparison to CCs surrounding developmentally incompetent oocytes (NC1). In fact, Ctss and Ctsb genes were reported as markers of oocyte developmental competence in cow and their expression in CCs of metaphase-II oocytes was shown to be high in comparison to immature oocytes
. Both cathepsins were also detected and localized in mice ovary
. Secreted acidic cysteine rich glycoprotein (Sparc) is a matricellular protein expressed during tissue morphogenesis and reorganization, and we observed an increase of transcript level (OG_10175) between NC1 and C2 stages in both mice and cow. In agreement with our observations, high expression levels of Sparc were previously reported in mice cumulus oophorus
. In bovine CCs, the upregulation of Sparc was related to cumulus expansion in response to growth factors complementation during IVM
Ligands and receptors of the developing follicle
Follistatin (Fst) is involved in inhibition of FSH release, and was also found to be expressed in mice and bovine OCs
[26, 27]. Our transcriptomic analysis revealed the high expression of Fst gene (OG_11886) in immature CCs in both species and a significant decrease after maturation. Similarly, the transcript levels of Anti-Mullerian hormone (Amh, OG_11935) and one of its receptors AMH type II receptor (Amhr2; OG_11318) dramatically decreased in CCs upon cow and mouse oocyte maturation (C2 stage compared to NC1 and C1 stages). Such an expression profile of the hormone has already been recently described in cow ovary
 as well as in many other mammalian species (sheep, rat, primates, and humans - reviewed by
). To our knowledge, Amhr2 expression pattern has not been studied in cow ovaries but in rat as well as in mouse, this receptor was shown to be mainly expressed in granulosa cells of preantral and early antral follicles
[30, 31]. Similar observations can be made for the FSH receptor (OG_10792) for which a down-regulation was observed herein at C2 stage in both species as previously observed at least in cow ovaries
Intra-cellular cell signaling
Phosphodiesterases (PDEs) regulate intracellular cyclic nucleotide concentration by converting cyclic nucleotides into 5' nucleotides. Their activity was found in mice and bovine granulosa/cumulus cells
[33, 34] and inhibition of PDE3/PDE4 activity blocks meiosis in bovine oocytes
. In our study, several genes encoding PDEs were found to be similarly expressed in mice and bovine. Thus, mRNA level of Pdea4 and Pde6d (OG_10100 and OG_11911) increased during maturation, and that of Pde5a and Pde7a (PL_10380530) decreased. Similarly, ERβ transcript (OG_11926) is down regulated between C1 and C2 stage in bovine and mouse cumulus in the present study in agreement with previous reports demonstrating a predominant expression of this ER isoform in small rodent preovulatory follicles
[36, 37] and a down-regulation by gonadotropins
. Such a regulated pattern of expression was also reported in bovine follicles
Among the genes exhibiting a conserved profile during oocyte developmental competence acquisition, some were previously known to play an important role as illustrated hereafter. This further validates the approach used in the present study. For instance, growth hormone receptor (Ghr) has been shown to be expressed in ovarian follicles in many vertebrate species and involved in folliculogenesis, particularly by promoting secondary follicle growth and antrum formation in mammals as shown in cases of Ghr deficiency in cow
 and in mice
 for review). In the present study, the corresponding transcript (OG_10283) was highly expressed in NC1 and or C1 stage granulosa cells and was down-regulated in C2 stage in the 3 tetrapod species. These observations are in full agreement with previous reports in cow, mice and even fish. Indeed, a study conducted at the protein level in rainbow trout showed that ovarian growth hormone receptor concentration was high during the early phases of follicular development and subsequently decreased during oocyte and follicular growth to reach a minimal value during oocyte maturation
In addition to genes known to participate in ovarian functions, our study also shed light on a large number of molecular actors for which a role in ovarian physiology was previously unsuspected or poorly investigated. This is for instance the case of a decoy TNF receptor gene up-regulated at C2 stage (Tnfrsf23; OG_10201, Figure
4). The importance of the TNF system in folliculogenesis is well known and many TNF ligands and “classical” receptors are differentially regulated during oogenesis in a wide variety of vertebrate species
[42–44]. Although the precise role of Tnfrsf23 in the mammalian ovary remains uncharacterized, it is thought to be a decoy TNF receptor
. Decoy TNF receptors have the ability to bind TNF ligands without inducing cell death. Interestingly, alternatively spliced variants of Tnfrsf23 are expressed in the chicken ovary
 and a decoy TNF receptor was found to be differentially expressed in the fish ovary during final oocyte maturation
. An increased expression of a set of genes, including sphingomyelin synthase 1 (Sgms1), syntaxin 1A (Stx1a), and Pacsin3, which are implicated to a different extent in exo/endocytosis and protein trafficking, was also observed in our study, particularly at C2 stage (Figure
4). To the best of our knowledge, none of these genes has ever been reported to play a role in ovarian follicle/oocyte development. Sgms1, by regulating cellular levels of ceramide and diacylglycerol, can affect the proliferation/apoptosis balance and it has only been shown to participate in TNF-mediated apoptosis in Chinese Hamster Ovary cells
. Stx1a is rather expressed in neurons while the expression of other syntaxins, such as Stx4a or Stx7/avalanche, has been reported in human granulosa cells or in drosophila follicle cells, respectively
[49, 50]. Finally, Pacsin3 besides its role in vesicular transport, membrane dynamics, and cytoskeleton organization also participates in addressing some ion channels and glucose transporters to the plasma membrane
. In the present study, down-regulation of a cornichon-related gene (Cnih2 also known as Cni1) expression was evidenced in the somatic compartment during oocyte competence acquisition in the 3 tetrapod species (Figure
4). Cornichon (Cni) proteins have been studied in Drosophila and Yeast, Cni being involved in Drosophila embryo polarization
. Four genes exist in vertebrates (named Cnih, Cnih2, Cnih3, and Cnih4 in the mouse). Recent evidence suggested that metazoans cornichon proteins have a similar subcellular localization and regulate transport of TGFα family ligands
. The transcripts of Cnih are highly abundant in full grown oocyte and unfertilized egg in mice. A role in ovarian follicular regulation has, however, never been reported in any vertebrate species to date. Interestingly, Cni plays an important role in Drosophila oogenesis and more specifically in a signal transduction pathway that transmits information between the germline cells and the somatic follicle cells of the ovary
. Together, these observations suggest an important, yet unsuspected, role of the Cnih family in the somatic compartment of the follicle during oocyte competence acquisition in tetrapods.
Finally, up-regulation of Tribbles homolog 1 (Trib1) was evidenced herein during oocyte maturation (i.e. between C1 and C2 stages) in surrounding somatic cells (Figure
4). Trib1 belongs to a family of genes (Tribbles 1, 2, 3, and 4) that has been implicated in the development of many diseases including cancers (reviewed by Kiss-toth
); for instance, Trib1 is involved in the onset of acute myeloid leukemia (AML) by regulating C/EBP transcription factor levels and activity through a MAPK (MEK1/ERK)-dependent pathway
, but is also associated with hyperlipidemia
. Interestingly, Yamamoto et al.
 have observed that Trib1-deficient drosophila and mouse females are infertile; in drosophila, such a phenotype is likely due to the fact that Trib1 has been shown to regulate migration of a cluster of ovarian follicle (epithelial) cells, called border cells, leading to micropyle formation required for oocyte fertilization. In this case, Trib1 function would also involve a post-translational regulation of the drosophila C/EBP homologue, slbo
. In mammals, the mechanisms underlying Trib1 participation in ovarian folliculogenesis and oocyte development remain however to be elucidated.
Together, our results demonstrate that tetrapods share a large number of common molecular actors from somatic origin that have similar expression profiles during developmental competence acquisition. This suggests that many conserved mechanisms exist in the ovary that are required or important for the acquisition of a full developmental competence. We also showed that many of the new players presently identified in the context of oocyte competence acquisition in our study were previously unstudied in the ovary and deserve further investigations. Finally, our results reveal that some of the important molecular actors from somatic origin found in tetrapods are also shared by some non-vertebrate metazoan species. This suggests that some of the key signals from somatic origin involved in oocyte developmental competence acquisition would be shared, not only by vertebrates, but also by metazoans.
Among the four vertebrate species studied, a total of 42 groups of orthologous genes could be identified that shared similar expression profiles. As for tetrapods, our study shed light on novel molecular players from somatic origin. This is for instance the case of the kelch family for which different members are differentially expressed, depending on the species. The Kelch protein was initially discovered in Drosophila in which it is known to play an important role during oogenesis and more specifically in the communications between somatic cells and oocyte. Such communications are required for the development of a normal oocyte and mutation of kelch leads to female infertility
. The kelch protein family is a large multigenic family and over 30 Kelch-like proteins (KLHL) exist in humans. A comprehensive picture of the entire family is however currently lacking in vertebrates and little is known about the role of Kelch proteins during oogenesis. Our results, evidencing a conserved regulation of Kelch gene expression in the somatic compartment are thus extremely original and suggest an evolutionary-conserved important role of kelch proteins in the development of a normal oocyte in insects and vertebrates. Our results are also consistent with the abundant expression of KLHL5 previously reported in human ovary
Similarly, the Krüppel-like factor (KLF) is also a large multigenic family named after the Drosophila Krüppel protein and 17 KLF genes can be found in the human genome
[62, 63]. The KLFs are transcription factors that regulate a wide variety of biological processes including proliferation, growth, development, survival, and responses to external stress (see
 for review). Their role in reproductive functions has, in contrast, received very little attention. In the present study, we report the up-regulation of Klf13 mRNA expression during oogenesis in the four species. This pattern is consistent with the previous report of Klf13 mRNA expression in swine granulosa cells
. In addition, several Klf genes were reported to be differentially expressed in the ovarian follicle under normal or pathological conditions
[65–67]. Together, this suggests an important role of the KLF family in vertebrate oogenesis and a conserved pattern of Klf13 expression in the somatic compartment during intrafollicular competence acquisition.
Finally, we have chosen to highlight the up-regulation of genes belonging to the Adamts1 (a disintegrin and metalloproteinase with thrombospondin like repeats 1) OrthoMCL group at C2 stage in the four vertebrate species studies. In mammals, Adamts1 plays an important role during ovulation in mammals to prepare the oocyte for fertilization
. Such role in the inflammatory-like ovulatory process is in agreement with the profiles reported here in mice and cow. In non-mammalian vertebrates, the mechanisms underlying ovulation are less documented. The inflammatory nature of the ovulatory process has, however, been established in fish
. The present work strongly suggests that the important role played by Adamts1 in mammals to prepare the oocyte for fertilization would also be found by non-mammalian vertebrates.
Together, the observations made in the 4 vertebrate species studied further support the results obtained in the 3 tetrapod species that suggest that some of the key signals from somatic origin involved in oocyte developmental competence acquisition would be shared, not only by vertebrates, but also by metazoans.