Identification of differentially expressed orthologous genes in both species
Among the twenty-six genes studied in the QPCR-based comparative analysis, eight genes had similar expression profiles throughout late oogenesis in both rainbow trout and Xenopus. These results indicate that our strategy which consisted in reanalyzing a transcriptomic study in one species followed by a subsequent QPCR study in both species is relevant for the identification of orthologous genes that possibly participate in conserved molecular mechanisms among the two species. Among the eight genes exhibiting similar profiles in both species, five genes could be linked directly or indirectly to steroidogenesis, thus pointing out steroidogenic-related mechanisms as being possibly well conserved among fish and amphibians. Although gene expression of star, cyp19a1a and cyp17a1 have already been described in trout during late oogenesis [14, 26, 27], none of these genes have been described at the mRNA level during this period in Xenopus. Moreover no expression data regarding Hsd11b3 and apoC1 are available in any of the studied species. Due to the predominant role of sex steroids in the control of the reproductive process in teleosts and amphibians [2, 10], these 5 genes were further studied in the present study.
While our approach has been successful, it should however be pointed out that a significant number of genes over-expressed during maturation in trout did not exhibit a similar pattern in Xenopus. These differences could be due to species-specific differences or even correspond to non-conserved mechanisms. Another possible explanation for the discrepancy between trout and Xenopus is that in the present study, trout oocyte maturation occurred naturally in vivo. In contrast, Xenopus oocyte maturation was triggered in vitro by a 15-h incubation of isolated fully-grown follicles in medium supplemented with human chorionic gonadotropin (hCG). It is thus possible that the in vitro conditions did not trigger all the mechanisms that are naturally occurring in the preovulatory follicle, especially those involved in ovulation. Indeed, even if Xenopus injection of heterologous gonadotropin can lead to in vivo Xenopus oocyte maturation and ovulation , these processes can sometimes be uncoupled in vitro . This could explain, in part, our results.
Finally, in order to identify Xenopus genes related to the trout candidates we used, as a high throughput approach, a best blast hit strategy. The orthology relationships between trout and Xenopus cognate proteins were then validated only for the five candidates that we subsequently analyzed. Thus, we cannot totally rule out that a part of the discrepancy between trout and Xenopus profiles could be due to a misidentification of true orthologs in Xenopus. This is especially true for adam8a, adam22, foxo5 and etv5 genes that will require detailed phylogenic analyses.
Candidate gene analysis
Star. STeroidogenic Acute Regulatory protein
(Star) is involved in cholesterol shuttling across the mitochondrial membrane and its synthesis appears to be a limiting step in steroidogenesis [30–34]. In the present study, star was shown to be predominantly expressed in rainbow trout and Xenopus gonads. These results are in agreement with existing data in various vertebrates including teleosts [19, 20, 33]. Low expression levels were also detected in trout intestine and in Xenopus stomach. Although star expression has already been reported in rainbow trout intestine , no expression has been evidenced in digestive tract in any other species. While the over-expression during follicular maturation was previously documented in rainbow trout [19, 26], we showed for the first time that star is also over-expressed during hCG-induced oocyte maturation in Xenopus. Interestingly, star expression is induced in response to gonadotropin stimulation in mammals and birds [30, 35–38]. As oocyte maturation is induced in response to LH (Luteinizing Hormone) stimulation in fish and amphibians , it is likely that the strong star mRNA over-expression reported here in trout and Xenopus is also triggered by LH-mediated signaling pathway(s). In rainbow trout, it is also noteworthy that the circulating levels of the maturation-inducing steroid (MIS) detected during the preovulatory period  increase concomitantly with star mRNA levels. Likewise, in Xenopus laevis, progesterone and testosterone secretions are more important in stage VI follicles compared to stage IV follicles in agreement with star increased expression observed in this study . Together, these results point out the over expression of star by gonadotropin during oocyte maturation as a possible conserved mechanism among non-mammalian vertebrates and possibly all vertebrates. The nature of the corresponding protein emphasizes the importance of steroidogenesis in the control of late oogenesis in non-mammalian vertebrates.
Aromatase is an enzyme (CYP19; EC 126.96.36.199) that converts androgens to estrogens. In most species including humans, chicken, Xenopus and a cartilaginous fish, a single gene has been isolated [41–44]. In contrast, in most teleosts, two genes, cyp19a1a (also referred as cyp19a or cyp19a1) and cyp19a1b (also referred as cyp19b or cyp19a2), encode distinct proteins predominantly expressed in the ovary and the brain, respectively [17, 45–47]. In the present study, we show that cyp19a1a is only expressed in trout ovary, while p450arom-A is expressed mainly in Xenopus gonads and brain. The lack of cyp19a1a expression in rainbow trout brain is somewhat surprising as cyp19a1a has previously been detected in brain of various fish species [46–49]. The lack of expression of cyp19a1a in trout testis is, in contrast, in agreement with a previous study carried out in zebrafish . In our study, a weak expression of aromatase was detected in Xenopus testis. This observation is consistent with a previous study reporting a weak expression but no aromatase activity in Xenopus laevis adult testis . In Xenopus, we evidenced a very low expression in intestine and stomach, thus corroborating studies in human fetus which showed an aromatase expression in intestine [51, 52].
The cytochrome P450 aromatase expression decreases dramatically during late oogenesis in both Xenopus and trout resulting in a barely detectable aromatase expression during maturation. Previous studies already indicated a decrease of aromatase expression [14, 53–57] and activity  as well as a reduction of circulating E2 levels [26, 27, 39, 59] throughout late oogenesis in several fish species. However, we report here for the first time that aromatase transcript expression decreases dramatically in Xenopus post-vitellogenic, immature, follicles throughout late oogenesis. This observation is consistent with the decrease of aromatase and E2 production by the ovarian follicle during the post-vitellogenic period [40, 60]. Together, these observations suggest that the drop of aromatase mRNA expression in the late oogenetic follicle is possibly a conserved molecular mechanism among non-mammalian vertebrates. In addition, existing data on the inhibition of oocyte maturation by E2 obtained in fish  as well as in another amphibian, Rana pipiens [62, 63] suggest that this mechanism of inhibition of precocious meiosis resumption could contribute to oocyte maturational competence acquisition in non-mammalian vertebrates.
The cytochrome P450 17A1, for which the official symbol is cyp17a1 is a member of the large superfamily of cytochrome P450. This enzyme (CYP17A1; EC = 188.8.131.52) acts as a 17α-hydroxylase and a 17-20-lyase. The 17α-hydroxylase activity converts progesterone and pregnenolone to 17α-hydroxyprogesterone and 17α-hydroxypregnenolone, respectively while the 17,20-lyase activity converts 17α-hydroxypregnenolone to dehydroepiandrosterone (DHEA) and 17α-hydroxyprogesterone to androstenedione. DHEA and androstenedione are precursors of testosterone and estrogen synthesis while 17α-hydroxyprogesterone is a precursor of different progestins and cortisol. In teleosts, two genes have been identified : Cyp17a1 (previously referred as P450-I) and cyp17a2 (previously referred as P450-II). Cyp17a1 encodes a protein exhibiting both activities, as in other vertebrates, whereas cyp17a2 encodes a protein lacking 17,20 lyase activity.
Cyp17a1 is expressed in the gonads of both species, consistently with previous data in mammals , birds , and fish [67, 68], including rainbow trout [26, 27, 69, 70] but reported here for the first time in an amphibian species. In a previous study, a strong mRNA expression was also reported in rainbow trout kidney . These authors also detected a low expression in various tissues using semi-quantitative RT-PCR that we were not able to confirm in the present work using QPCR. In both species, ovarian mRNA levels decrease dramatically throughout late oogenesis. In rainbow trout, the cyp17a1 profile was previously been documented [26, 27] and found to be consistent with previous Northern blot data indicating that cyp17a1 was abundant in the post-vitellogenic ovary as a result of an increase of expression that occurred during vitellogenesis . Moreover, it has been shown in tilapia that cyp17a1 expression in granulosa cells decreases during late oogenesis, while cyp17a2 expression increases . It should be however noticed that cyp17a1 expression during late oogenesis can be variable depending on the species [67, 71]. Nevertheless, a low cyp17a1 mRNA expression was also reported in female fat head minnow close to full sexual maturity  and in two catfish populations, cyp17 mRNA level was shown to decrease throughout the period of spawning . In rat, both cyp17a1 mRNA and protein abundance increase throughout folliculogenesis and subsequently decrease after hCG stimulation of preovulatory follicles . Similarly, cyp17a1 mRNA expression decreased after LH surge in bovine preovulatory follicles . Consistent with these observations was the report of a decrease of cyp17a1 mRNA expression in follicular layers of the preovulatory chicken follicles [56, 74]. Together, existing and present observations suggest that even though species-specificity may exist, the decrease of cyp17a1 mRNA expression towards the end of oogenesis may be a well conserved molecular mechanism, possibly related to maturational competence acquisition. Indeed, Cyp17a1 could participate in the shift from E2 to maturation-inducing steroid production observed in fish and amphibians during late oogenesis [40, 75]. In addition, Cyp17a1 could also participate in the production of androgens that have been evidenced to play important roles during late oogenesis in both amphibians and fish [76–78].
The 11beta-hydroxysteroid dehydrogenase isoenzymes HSD11B mainly catalyze the interconversion of active glucocorticoid (cortisol and corticosterone) and inactive 11-keto forms (cortisone and 11-dehydrocorticosterone) . Three proteins have been described in mammals: HSD11B1, HSD11B2 and HSD11B3 (previously known as HSD11B1like or HSD11B1L). Previous analyses have indicated that hsd11b1 was not present in teleost species  and could only be found in amphibians, birds and mammals. In fish, Hsd11b2 has been characterized and shown to be expressed in various tissues such as gill, heart, intestine, ovary, testis and skin  and studied in more details in the ovary during late oogenesis [79, 81]. Interestingly, hsd11b3 was phylogenetically characterized  but no expression data was available to date in fish and amphibians. In the present study, we show that hsd11b3 mRNA is expressed in a large number of tissues but not in Xenopus skin while it is expressed mainly in brain, skin and intestine in trout. Thus, rainbow trout hsd11b3 mRNA appears to have a more restricted tissue distribution than hsd11b2 and tissue expression differs greatly between trout and Xenopus. Within the ovary, we evidence a decrease of hsd11b3 transcript level throughout late oogenesis in both rainbow trout and Xenopus whereas hsd11b2 mRNA has been reported to accumulate at the same time in trout female gonad [79, 81].
At the functional level, HSD11B2 acts in mammals as a reductase and converts inactive cortisone to active cortisol. In fish, this isoform is also able to convert 11β hydroxy-testosterone into 11keto-testosterone (11-KT), which has been shown to be a major androgen steroid in fish . In contrast, mammalian HSD11B1 predominantly acts in an opposite way, as likely does HSD11B3 according to the evolutionary history of the protein , since no functional study has been reported on this isoform. Likewise, fish Hsd11b3 is likely to act as the mammalian HSD11B1 and/or HSD11B3 (Additional file 4). Together, the expression profiles of hsd11b2 [79, 81] and hsd11b3 (present results) in the rainbow trout preovulatory ovary suggest a combined role of these enzymes in gonad protection against any deleterious effect of stress-induced cortisol. Despite the lack of data on glucocorticoid levels and effects during oogenesis in amphibians, we may also hypothesize a similar role of Hsd11b3 during Xenopus late oogenesis. We cannot totally rule out a role of Hsd11b3 in regulating 11 ketotestosterone levels during follicular development and oocyte growth. Indeed, 11KT, besides its well-known action in fish spermatogenesis, has been demonstrated as controlling oocyte growth, likely through lipid accumulation in eel  and in atlantic cod . However, this cannot be extended to all teleosts as 11KT circulating levels are low or even undetectable in salmonid females [85–87]. Further investigations including protein expression and enzyme activity measurements are thus required to conclude on the exact physiological role(s) of these 11beta-hydroxysteroid dehydrogenase isoenzymes in ovarian functions during late oogenesis.
The apolipoprotein C1 belongs to the family of soluble apolipoproteins involved in cholesterol transport and uptake in vertebrates . Interestingly, in vitro studies have also suggested that apoC1 could stimulate lecithin-cholesterol acyltransferase (LCAT) activity , thereby increasing the formation of esterified cholesterol as well as estradiol ester. In the present study, the tissue expression analysis reveals that apoC1 is primarily expressed in trout and Xenopus liver, but also in stomach, intestine and gonad. This result is consistent with previous studies performed in mammals [90–92] and in another teleost, Hemibarbus mylodon . Only in the orange-spotted grouper, apoC1 could not be detected in the liver but it was nonetheless expressed in gonad and in brain .
Within the ovary, we show that apoC1 mRNA levels dramatically increase during late oogenesis in trout and Xenopus. This result confirms previous findings in trout  and in orange-spotted grouper ; only Tingaud-sequeira et al. recently published in a marine flatfish ovary a different pattern of apoC1 expression, where its transcript levels significantly increase solely during follicle atresia following ovulation but not during follicle growth or maturation . This study is also the first report on apoC1 expression in an amphibian. In mammals, ApoC1 expression in ovary during follicle development has not been studied to our knowledge but the increase of LCAT activity, correlated with a decrease of estradiol/progesterone ratio in antrum of human growing ovarian follicles  may indirectly reflect an increase of apoC1 expression. The function of ApoC1, accumulating during late oogenesis, remains to be elucidated. First, this apolipoprotein may have a role in remaining yolk degradation by the oocyte companion cells during follicle atresia  or yolk degradation as a nutrient source for early embryo development . ApoC1 could also be involved in the regulation of steroidogenesis at least in two ways: (i) as an inhibitor of lipoprotein uptake via inhibition of lipoprotein binding to their receptors ; it may then modulate the ovarian cholesterol uptake during oogenesis; (ii) cholestryl and estradiol esters, formed upon LCAT activation by apoC1 , remain a source of bioactive metabolites in the steroid synthesis pathways. Interestingly, estradiol esters may also be involved in antioxydation processes . Further studies will be required to fully understand the role(s) of ApoC1 during ovarian follicle development in vertebrates.