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Mechanisms of Hormone Action; |
Facultad de Ciencias,3 Oceanología, Montevideo 11400, Uruguay
INRA, SCRIBE,4 IFR 140, Cédex Rennes 65342, France
ABSTRACT
The present study was designed to obtain new insights into fish gonadal sex differentiation by comparing the effects of two different masculinizing treatments on some candidate gene expression profiles. Masculinization was induced in rainbow trout, Oncorhynchus mykiss, genetic all-female populations using either an active fish androgen (11betaAnd, 11beta-hydroxyandrostenedione) or an aromatase inhibitor (ATD, 1,4,6-androstatriene-3,17-dione). The expression profiles of 100 candidate genes were obtained by real-time RT-PCR, and 46 profiles displayed a significant differential expression between control populations (males and females) and ATD/11betaAnd-treated populations. These expression profiles were grouped in four temporally correlated expression clusters. Among the common responses shared by the two masculinizing treatments, the inhibition of some early female differentiating genes (cyp19a1, foxl2a, fst, and fshb) appears to be crucial for effective masculinization, suggesting that these genes act together via a short regulation loop to maintain high sex-specific ovarian expression of cyp19a1. This simultaneous down-regulation of female-specific genes could be triggered by some testicular genes, such as dmrt1, nr0b1 (also known as dax1), and pdgfra, which are quickly up-regulated by the two masculinizing treatments. In contrast to 11betaAnd, ATD quickly restored the expression levels of steroidogenesis related genes (cyp11b2.1, cyp11b2.2, hsd3b1, cyp17a, star, and nr5a1) and some Sertoli cell markers (sox9a2 and amh) to the expression levels observed during control testicular differentiation. This demonstrates that these genes are probably not needed for active masculinization and that the inhibition of endogenous estrogen synthesis produces a much more complete and specific testicular pattern of gene expression than that observed following androgen-induced masculinization.
early development, gene regulation, ovary, steroid hormones, testis
The important role of sex steroid hormones during gonadal differentiation in nonmammalian vertebrates is now clearly established [1–6]. This key role of steroids was initially supported by the fact that steroid treatments are able to provoke complete or partial sex inversion of the gonadal phenotype of many vertebrates. Some gonadal steroid-induced inversions, albeit only partial, have even been observed in mammals [7, 8]. Aside from the effects of these steroid treatments, much other evidence has strengthened this steroid hypothesis, including the early steroid synthesis potentiality and receptivity of the differentiating gonads. Among the sex steroids, the implication of estrogens in ovarian differentiation is now especially well described in many vertebrates, including fish. This key role of estrogen in ovarian differentiation has been supported by the fact that the inhibition of estrogen synthesis in the differentiating ovaries can trigger partial or complete masculinization in fish [9, 10], amphibians [11, 12], reptiles [13, 14], and birds [15, 16]. However, androgens have also been shown to be very potent masculinizing steroids in fish [17–20] and also in some other nonmammalian vertebrates [21, 22]. The molecular mechanisms by which these two treatments trigger their masculinizing effects are, however, still not well understood, and the question remains whether the lack of estrogens is a necessary and sufficient condition for efficient masculinization or whether androgens are needed to functionally complete this process. Concerning masculinization with an aromatase inhibitor in fish, there is currently only one report showing that cyp19a1 (the fish ovarian aromatase) was down-regulated after the treatment [23]. For androgen-induced masculinization, two reports clearly demonstrated that androgens completely down-regulate most genes encoding steroid enzymes, including cyp19a1 [19, 24], leading to the hypothesis that at least one part of this androgen-induced masculinization is mediated through a cyp19a1 inhibition. However, evidence in support of this is scarce and does not provide conclusive evidence as to whether the absence of estrogen synthesis capacity by gonads or the presence of androgens is responsible for testicular differentiation.
The present investigation was designed to better understand this steroid-induced masculinization mechanism by searching for a common molecular pathway between these two functional masculinization treatments. For this purpose, we compared gonadal gene expression profiles following masculinization with a natural nonaromatizable [17] fish androgen (11β-hydroxyandrostenedione) and with an aromatase inhibitor (1,4,6-androstatriene-3,17-dione), using genetic all-male and all-female rainbow trout populations. These temporal expression profiles were analyzed by real-time RT-PCR [25] on 100 genes that were previously investigated during sex differentiation in rainbow trout [25, 26] and that are known to play a potentially important role in the vertebrate sex differentiation cascade.
Research involving animal experimentation conformed to the principles for the use and care of laboratory animals, in compliance with French and European regulations on animal welfare. Genetically all-male and all-female rainbow trout larvae were obtained from the INRA experimental fish farm (Drennec, France), as previously described [10]. Fish were maintained at 10°C from fertilization until 24 days postfertilization (dpf) and transferred to the experimental installations, that is, 0.3-m3 tanks with a recirculating water system, at 10 ± 0.1°C, under a constant photoperiod (12L:12D). After complete yolk resorption by 53 dpf, four batches of 800 fish were transferred to 12°C tanks and fed daily ad libitum with a commercial diet (dry pellet food; Biomar) with or without treatments. Treatments were applied during 3 mo, from the first feeding (53 dpf) to 144 dpf. The groups were as follows: female—all female fish were fed with a diet treated with ethanol (control group); male—all male fish were fed with a diet treated with ethanol (control group); F-ATD—all female fish were fed with a diet supplemented by 1,4,6-androstatriene-3,17-dione (ATD) in ethanol at 50 mg/kg of food; and F-11βAnd—all female fish were fed with a diet supplemented by 11β-hydroxyandrostenedione (11βAnd; Sigma) at 10 mg/kg of food. The masculinizing efficiency of these two treatments has been already investigated [10, 19], and the resulting males were found to be fertile. Samples were taken for real-time PCR at the following stages of development: 53, 60, 69, 83, 116, 144, and 164 dpf. For each sampling date, gene expression profiles were obtained for two independent pools (biological replicates) of gonads, each containing 20–100 gonads that were immediately frozen in liquid nitrogen and stored at –80°C until RNA extraction. Additional gonads were fixed in glutaraldehyde, embedded in Epon, and cut at 2 µm [26] for histological analysis.
Total RNA was extracted using TRIzol reagent (Invitrogen) as previously described [19]. The cDNA synthesis was carried out on 1 µg of total RNA. Total RNA was denaturated in the presence of random hexamers (0.5 µg) for 5 min at 70°C and then chilled on ice. RT was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega) as described by the manufacturer.
Real-time RT-PCR was carried out on an iCycler iQTM (BioRad). Reactions were performed in 20-µl samples with 300 nM of each primer. The gene list, the primer selection, and the complete procedure were as previously described [25].
In situ hybridization was carried out as previously described [26] with digoxigenin-labeled antisense RNA probes (Promega T3/T7 RNA polymerase Riboprobe Combination System). ISH was performed using the In situ Pro, Intavis AG robotic station. The digoxigenin signal was revealed with an antidigoxigenin antibody, conjugated with alkaline phosphatase (Roche Diagnostics) and a NBT/BCIP revelation system (Roche Diagnostics). Slides were mounted with mowiol 4–88 (Calbiochem).
The 100 gene expression profiles obtained by real-time RT-PCR were first log transformed, median centered (relative to the median expression across all samples), and normalized in order to characterize their relative variation rather than absolute difference. This data set was then analyzed with the Significance Analysis of Microarrays (SAM) software [27] implemented in the TIGR MultiExperiment Viewer [28]. Multiclass SAM analysis was run to discriminate significant differential expression profiles across the four classes of samples (females, males, F-11βAnd, and F-ATD). The lowest delta value, producing 0% median number of falsely significant genes, was selected for further analysis. Using the significant gene expression profiles, characterized by SAM analysis, we then classified the relationship between the different biological samples by unsupervised hierarchical clustering using a centroid linkage clustering with the Pearson uncentered correlation as a similarity metric. Gene clusters were distinguished using the nonhierarchical unsupervised learning k-means algorithm as implemented in the Cluster 3 program [29], with a maximum cycle parameter of 100. The optimal minimal k number of clusters, corresponding to the stability of the k-means clustering, was empirically set at four, based on previous analysis of unsupervised clustering. Results were displayed using the Java TreeView program [30].
Effects of the Treatments on Gonadal Morphology
Comparison of morphological changes observed in gonads undergoing natural development in genetic all-female (F) and all-male (M) trout populations and those subjected to masculinizing treatments, that is, the aromatase inhibitor (F-ATD) and the natural androgen 11β-hydroxyandrostenedione (F-11βAnd) treatments, is shown in Figure 1. Seven days after the beginning of the masculinizing treatments (at 60 dpf), gonads of male and female control fish remained histologically undifferentiated, and no difference, as compared to the control groups, was detected in F-ATD and F-11βAnd at that stage. At 69 dpf, ovaries start to be clearly recognizable with a specific spatial distribution of the connective tissue around the future ovarian lamellae. No difference is observed between male and female germ cells at this stage, and the testis-type gonad is recognizable only by the absence of the ovarian lamellae formation (Fig. 1). After 14 days of treatments (69 dpf, the gonads of the treated fish are similar to the testis-type gonads of the control males, with a well-developed connective tissue containing some germ cells. The later histological changes are observed by 116 dpf, when the first previtellogenetic oocytes, characterized by their large size and a nucleus with multiple nucleoli, are detected within control ovaries. Concomitantly, testis cords containing groups of germ cells and delimited by an extracellular fibrous connective tissue are observed within the control testis. At 144 dpf and older (164 dpf), control gonads are characterized by well-structured ovarian lamellae containing previtellogenic oocytes in females and testicular cords, filled with mitotic spermatogonia surrounded by Sertoli cells in males. Gonads from 144- and 164-dpf ATD-treated fish (F-ATD) were histologically indistinguishable from the immature control testis (e.g., 144 dpf in Fig. 1), whereas gonads from 144- and 164-dpf 11βAnd-treated fish were characterized by precocious spermatogenesis with the presence of numerous spermatocytes and spermatids that were not observed in control testis at these stages.
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Comparison of the Biological Samples Based on Gene Expression Clustering
The full gene expression profile data set has been deposited online and is available in the Sigenae Web site (http://sigenae.org) as supplementary files via the link "citations." Following multiclass SAM analysis, 46 genes were retained with differentially significant expression between the four groups (control males, control females, F-11βAnd, and F-ATD). The unsupervised classification of the biological samples, using these 46 gene expression profiles, clusters these samples into three major branches (Fig. 2). Branch A clusters all the female samples (correlation coefficient R = 0.10); branch B, all the females treated with 11β-hydroxyandrostenedione (F-11βAnd, R = 0.14); and branch C, all the male samples (R = 0.16). The clustering of the females masculinized with an aromatase inhibitor (F-ATD) reveals that these F-ATD samples do not cluster as a homogeneous group but are instead clustered with either control females, F-11βAnd, or control males, depending on the sampling period. One week after the beginning of the masculinizing treatment (60 dpf), F-ATD samples were more similar to early control female samples (53 and 60 dpf). Later (i.e., 69 and 83 dpf), they were more similar to F-11βAnd and at further stages (i.e., 144 and 164 dpf) very similar to the control males. Surprisingly, none of the F-11βAnd samples cluster with control males, showing that this masculinizing treatment clearly induces a highly different temporal gene expression pattern with regard to the final testicular phenotype produced by androgens. Finally, it can be noticed that among the 25 biological duplicate samples, 17 are tightly clustered together, demonstrating a good correlation of most of the duplicate samples based on the expression levels of the 46 different genes.
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Effects of the Treatments on Global Gene Expression Profiling
In order to better individualize clusters of genes with similar expression profiles, the 46 significant expression profiles were grouped in four clusters of correlated expression, using a k-means clustering. The k number of clusters (k = 4) was chosen after visual analysis of supervised hierarchical clustering of these 46 expression profiles (available at http://sigenae.org). Among these four k-means clusters (Fig. 3), cluster 1 (C1) is characterized by a specific and sustained high expression level in control females when compared to control male expressions. This C1 cluster includes the following genes: bmp4, ovol1, cbln1, fshb, cyp19a1, fst, foxl2a, and foxl2b (Fig. 3). Following both ATD and 11βAnd treatments, most of these early female-specific gonadal genes are very quickly (i.e., before 83 dpf) down-regulated. This quick inhibition is detected for cyp19a1, fst, and foxl2a (Fig. 4), with, in particular, a remarkable complete down-regulation of cyp19a1 (aromatase) and foxl2a in F-11βAnd animals, inhibition that preceded the development of a testis-type gonad. In F-ATD animals this inhibition is slightly delayed and less pronounced. The C2 cluster contains 14 genes that are overexpressed in control males during testicular development, in comparison with ovarian development: inhbb, cebpa, bad, madh7 (smad7), tcf21 (pod1), cav1, pdgfrb, pdgfra, esr2 (erβ), nr0b1, esr1 (er
), igf2, cyp11a1, and dmrt1 (Fig. 3). Most of these are also highly up-regulated following masculinization with either ATD or 11βAnd. Interestingly, for some of these genes (e.g., nr0b1 or dax1, dmrt1, and pdgfra), this up-regulation is even observed before the development of a testis-type gonad and concomitantly with the down-regulation of key ovarian genes in cluster 1 (Fig. 4). The C3 cluster contains genes up-regulated during previtellogenesis (from 116 to 164 dpf), including the following genes: bzrp (pbr), tial1, alox5, birc5a, akr1b1, vldlr (vtgr), aldob, nup62 (p62), sox24, casp3b, figla, and vim. Some of these genes are not affected by the 11βAnd treatment (bzrp, tial1, alox5, birc5a, and akr1b1), whereas others are clearly down-regulated (vldlr, aldob, nup62, sox24, casp3b, figla, and vim). The effect of ATD treatment on this cluster of genes is a slight but not complete regulation (see figla, vim, and vldlr in Fig. 5). Cluster 4 contains genes overexpressed during natural testis development, as compared to ovarian development, and includes many genes encoding steroid enzymes (cyp17a, hsd3b1, cyp11b2.1, and cyp11b2.2), or known regulators of steroidogenesis (nr5a1 or sf1 and star) along with genes known for their implication in gonadal sex differentiation in vertebrates (lhx9, sox9a2, and amh). Several days after the development of a testis-type gonad, the ATD treatment restores a pattern of gene expression, as observed during natural testicular differentiation with the genes involved in androgen synthesis (cyp11b2, hsd3b1, cyp17a, star, and nr5a1) and some Sertoli cell markers (sox9a2 and amh) (Fig. 3). In contrast, with the ATD treatment, 11βAnd induced a profound alteration of these testicular-specific gene expression profiles, with all genes from cluster C4 remaining strongly down-regulated throughout all treatments see pax2a, amh, and cyp11b2.1 in Fig. 5).
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Effects of the Treatments on Some Key Gene Expressions
Two genes, cyp19a1 and amh, representatives of clusters 1 and 4, respectively, were further investigated by in situ hybridization to support their temporal gene expression profiles (Fig. 6). At 69 dpf, expression of cyp19a1 was restricted to a cluster of somatic cells located in the ventral side of female control gonads. In agreement with its ovarian-specific expression, no signal was detected in the control male gonads at that time. After 2 wk of treatment (69 dpf), cyp19a1 was not detected in either ATD- or 11βAnd-treated animals, confirming the quick down-regulation of this gene following both ATD and 11βAnd masculinizing treatments. At 83 dpf, a strong amh expression was found both in control males and in ATD-treated animals. In contrast, no expression was detected in 11βAnd animals. In control males this expression was found in many somatic cells surrounding some germ cells. In ATD-treated males, the location of amh is mainly peripheral and not organized in pretestis cords as in the male gonads. At this stage, amh expression in females was found to be low and restricted mainly to somatic cells located outside the future ovarian lamellae.
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First, our results demonstrate that one of the shared responses found after both ATD and 11βAnd treatments is the strong and quick repression of some early expressed female-specific genes, especially cyp19a1, foxl2a, and fst. Among these genes, cyp19a1 is a key enzyme involved in the production of endogenous estrogen, and its implication in fish ovarian differentiation is now well documented [10, 23, 26, 31, 32]. Therefore, its down-regulation after 11βAnd treatment, leading in turn to an inhibition of endogenous estrogen production, is probably a key event for an active masculinization. The fact that the ATD treatment also suppressed cyp19a1 expression, albeit in a slightly delayed fashion, was quite intriguing. But such an inhibition has been also observed in gonads of Japanese flounders following masculinization with another aromatase inhibitor [23], strongly suggesting that cyp19a1 would be under estrogen regulation either directly or indirectly. Of interest in this context is the concomitant down-regulation of foxl2a that we observed either with 11βAnd or with ATD. Foxl2 is one of the few important ovarian determining genes and has been characterized in the differentiating ovary of mammals [33], birds [34], and fish [35, 36]. In trout we have previously shown that this gene was strongly estrogen dependent [35] and temporally coexpressed with cyp19a1 during the initial steps of ovarian differentiation [26]. The fact that foxl2 is able to up-regulate cyp19a1 both in mammals and in fish [37, 38] and that estrogens up-regulate foxl2 in fish [35] suggests a positive feedback loop regulating these two genes. This positive feedback loop has already been proposed to explain the down-regulation of Foxl2 following masculinization of chickens with an aromatase inhibitor [39]. This loop would also explain both the down-regulation of cyp19a1 by ATD and the huge gene expression sex difference observed for cyp19a1 and foxl2a during natural early gonadal differentiation. Among the other female-specific genes, follistatin (fst) was also repressed temporally in the same way as cyp19a1 and foxl2a. This reinforces the strong temporal correlation that we have previously reported between these three gene expression profiles during early ovarian differentiation [26]. In mouse, Fst has been proposed as an early ovarian differentiation gene [40, 41], acting downstream of Wnt4 [41] to antagonize the testis-specific formation of the coelomic vessels [42]. However, in human, FST has been also characterized as an important local regulator of ovarian steroidogenesis that is able to directly enhance estradiol production [43]. It is hypothesized that concerted action of the activin/inhibin pathway acts as a cofactor, along with Foxl2, to regulate estrogen production during goat gonadal differentiation [37]. This leads us to propose that fst might be involved in the positive regulation of estrogen production, along with foxl2a, during fish ovarian differentiation. The β subunit of follicle stimulating hormone (fshb) is another down-regulated gene in this female-specific cluster. Interestingly, the transcriptional regulation of cyp19a1 during gonadal sex differentiation in fish might also be under the regulation of both fsh and foxl2 signaling [44]. This would make fshb another important candidate gene for early regulation of ovarian estrogen production.
The second shared response, between ATD and 11βAnd masculinizing treatments, concerns a gene cluster displaying a strong and rapid up-regulation following the two treatments. This cluster contains some markers of trout testicular differentiation like dmrt1, nr0b1 (dax1), and pdgfra [25]. This early up-regulation might be interpreted as an essential step required for active masculinization of the fish gonad. In this context, nr0b1 has recently been shown to down-regulate cyp19a1 expression in the fish ovary [45], and Nr0b1 disruption in Dax1-deficient mice triggers the up-regulation of Cyp19 [46]. Its rapid and strong up-regulation, induced by the two treatments, in trout suggests that nr0b1 could be a key factor in antagonizing the ovarian pathway by down-regulating cyp19a1 expression. These two masculinizing treatments also stimulated the up-regulation of some other testicular genes, such as inhibin-βB (inhbb) and igf2, which have been shown to be involved in the modulation of germ cell proliferation in fish [47]; madh7, a signaling molecule of the transforming growth factor-beta (TGF-beta) pathway [48]; tcf21 (pod1), a helix-loop-helix transcription factor that is able to repress steroidogenic factor 1 (Sf1/Nr5a1) [49]; and the side chain cleavage steroid enzyme, cyp11a1. All these genes were up-regulated during the process of masculinization of the female gonads regardless of the treatment, and they may be involved in the process of ovary-to-testis transdifferentiation.
Apart from these common responses shared by the two treatments, one gene cluster was characterized by highly differential gene expression profiles between the ATD and 11βAnd masculinizing treatments. This cluster groups genes that have been previously identified as fish testis markers [25] and have been localized in differentiating gonads, either in pre-Leydig cells (cyp11b2.1, hsd3b1, cyp17a, star, and nr5a1) or in pre-Sertoli cells (sox9a2 and amh) [26]. Their expression profile in females treated with ATD is quickly up-regulated to levels similar to those recorded in control male gonads. In contrast, 11βAnd strongly and quickly inhibits this set of genes, consistent with some previous results focusing on genes encoding steroid enzymes [19]. These results indicate that a disturbed gene expression pattern involves genes that are probably not necessary for complete and functional masculinization. In fact, the deregulation of steroidogenesis may be compensated for by the exogenous androgen supply provided by the 11βAnd treatment. But the down-regulation of some Sertoli cell markers, like sox9a2 and amh, is more surprising. In mammals, Amh expression is triggered by Sox9 in Sertoli cells at the onset of testicular differentiation [50], and Amh acts as an inhibitor of certain steroidogenesis steps [51] and may also be repressed by androgens [52]. The known function of amh in fish currently highlights the regulation of germ cell development [53, 54]. In our experiments 11βAnd specifically induced a repression of amh among other genes as well as a precocious stimulation of spermatogenesis [55]. The causal relationship between amh repression and the stimulation of spermatogenesis following androgen treatment is well supported by studies already carried out on the Japanese eel (Anguilla japonica), which demonstrated that amh is a "preventing spermatogenetic substance" that can be down-regulated by androgens [54].
In conclusion, these results provide new insights into gonadal sex differentiation and steroid-induced masculinization in rainbow trout. The inhibition of estrogen production is probably the key step required for this effective masculinization. Based on our data, we propose that the down-regulation of cyp19a1 probably involves the simultaneous down-regulation of some other key ovarian differentiation genes, such as foxl2a, fst, and fshb. These genes may act together within a short regulation loop to maintain high sex-specific ovarian expression of cyp19a1. This down-regulation would be triggered by an overexpression of some testicular genes, such as nr0b1 (dax1). We also clearly demonstrated that in rainbow trout, masculinization induced by inhibition of endogenous estrogen synthesis, using ATD, produces a much more complete and specific testicular pattern of gene expression than that observed following androgen-induced masculinization. However, it should be mentioned that these experiments do not rule out a possible implication of the special sex chromosome makeup (XX) of these masculinized populations and that some of the differences observed between gene expressions in masculinized XX females and normal XY males could be the result of the XX genotype of these animals.
FOOTNOTES
1Supported by the Institut National de la Recherche Agronomique (INRA) and a CIPA-OFIMER-INRA (AGENOP) grant. D.V. received financial support from CSIC (Universidad de la República of Uruguay) and a fellowship from the PHASE Animal Physiology and Breeding System INRA department. 
Correspondence: 2FAX: 33 2 234 85020; e-mail: yann.guiguen{at}rennes.inra.fr
Received: 9 October 2007.
First decision: 7 November 2007.
Accepted: 4 January 2008.
REFERENCES
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