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BOR - Papers in Press, published online ahead of print February 25, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025205
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BIOLOGY OF REPRODUCTION 71, 73–82 (2004)
DOI: 10.1095/biolreprod.103.025205
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Targeted Gene Expression Profiling in the Rainbow Trout (Oncorhynchus mykiss) Ovary During Maturational Competence Acquisition and Oocyte Maturation

Julien Bobe1, Thaovi Nguyen, and Bernard Jalabert

Institut National de la Recherche Agronomique, SCRIBE, Campus de Beaulieu, 35042 Rennes Cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A real-time polymerase chain reaction-based gene expression survey was performed using 37 target genes and 22 female rainbow trout sampled during follicular maturational competence (FMC) acquisition or during oocyte maturation. In females sampled before meiosis resumption, FMC was estimated using an in vitro assay. Several growth factors, bone morphogenetic proteins, steroidogenic enzymes, cathepsins, genes known to play a role in the fish preovulatory ovary, as well as previously unstudied genes, were analyzed in this survey. Gene expression profiling was performed using a supervised clustering analysis in order to identify groups of genes exhibiting similar expression profiles in the ovary during FMC acquisition and follicular maturation. From the clustering analysis, three clusters exhibiting a specific expression during FMC acquisition or at the time of oocyte maturation were identified. Cluster 1 was characterized by a progressive increase in gene expression during FMC acquisition, whereas cluster 2 exhibited an increased expression at the time of oocyte maturation. In contrast, cluster 3 was characterized by a decreased mRNA expression at the time of oocyte maturation. Among the 37 target genes used in this survey, 18 were significantly regulated during maturational competence acquisition or at the time of oocyte maturation. Among these 18 genes, 16 belonged to one of the three clusters identified. Although the results allowed a global description of gene expression profiles, they also suggest an important role for several factors, including some previously unstudied bone morphogenetic proteins, in the paracrine control of FMC acquisition and meiosis resumption.

follicle, follicular development, gene regulation, growth factors, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In fish, as in many other vertebrates, oocyte (or meiotic) maturation and ovulation processes are closely linked. They are triggered primarily by an endocrine signal that is characterized by a gonadotropic surge [1]. However, in order to produce a developmentally competent oocyte that can be fertilized and undergo subsequent normal development, this signal must be received by a fully differentiated postvitellogenic ovarian follicle [2, 3]. As a matter of fact, the differentiation stage reached by the whole follicle (oocyte, granulosa, theca) is determinant and includes the acquisition of specific characteristics such as enzymatic equipment, endocrine and paracrine signaling capacities, and mechanical properties of the follicle [1]. The enzymatic equipment of the postvitellogenic follicle includes enzymes involved in the synthesis of the maturation-inducing steroid (MIS) and prostaglandins, as well as the proteolytic enzymes required for ovulation [4–6]. Endocrine and paracrine signals are necessary for the coordination of the different follicular compartments and depend on the receptivity to MIS and gonadotropins, and for the modulation of intercellular communication such as that of gap junctions [1, 7]. As long as this differentiation stage, or follicular maturational competence (FMC) [8] is not reached, a precocious stimulation of the oocyte by the MIS or of the follicle by the gonadotropins can result in no oocyte maturation, oocyte maturation not followed by ovulation [2, 3, 9], or ovulation of poor-quality oocytes exhibiting a poor developmental competence [9]. Thus, maturational competence implies that each cellular component of the follicle is able to receive and correctly translate an external signal (receptivity) within a normal physiological range (sensitivity) and is subsequently capable of giving the appropriate response (responsiveness) [1].

Recently, we studied the mRNA expression of six candidate genes during FMC acquisition using deyolked ovaries composed of follicular layers and extrafollicular tissue [8]. We observed an increased expression of both FSH receptor (FSHr) and insulin-like growth factor II (IGF-II) in females exhibiting high maturational competence. It seems difficult, however, to fully comprehend the complex mechanisms associated with FMC acquisition by monitoring only the expression of a limited number of genes. We therefore decided to extend this analysis to a larger number of target genes (~40) and to a larger number of females sampled not only during FMC acquisition, but also at the time of oocyte maturation. However, extracting information from a large amount of data can be difficult. In addition, the progressive differentiation of the preovulatory follicle is putatively associated with progressive or step-wise changes in the expression of specific genes [8]. Therefore, in order to identify the groups of genes exhibiting similar expression profiles, including progressive changes during FMC acquisition, and to better visualize their expression profiles, we performed a supervised clustering analysis that allows a color representation of gene expression profiles across the sample set [10].

This gene expression survey was performed using not only genes known to be expressed in the ovarian follicle at the time of meiosis resumption or suspected to be associated with events occurring in the ovary at that time, but also other transcripts the expression of which had not been studied in the ovary during FMC acquisition or oocyte maturation. To do this, target genes were chosen not only among previously characterized rainbow trout mRNA sequences, but also among recently released rainbow trout expressed sequence tags (ESTs).

The IGF system appears to play a paracrine role during maturational competence acquisition and oocyte maturation in several fish species. For example, intrafollicular germinal vesicle breakdown (GVBD) can be induced by IGF-I or IGF-II in several species [11, 12]. In addition, it was recently shown that IGF-II mRNA levels were increased during maturational competence acquisition in rainbow trout [8]. Therefore, several members of the IGF system, such as IGF-I, IGF-II, IGF receptor 1a (IGFR1a), and the previously unstudied IGF binding protein 2 (IGFBP-2), were included in the present study.

Members of the transforming growth factor ß (TGFß) superfamily, such as TGFß, activin, inhibin, growth differentiation factor 9 (GDF9), and several bone morphogenetic proteins (BMPs), have been shown to have a role in controlling ovarian follicle development in mammals [13]. In contrast, little is known about their influence on ovarian functions in fish. In zebrafish (Danio rerio), the activin system is suspected of promoting oocyte maturation and maturational competence in a paracrine manner [14]. However, no information is currently available regarding the expression of BMPs in the fish ovarian follicle. Therefore, the expression profiles of several TGFß family members, including TGFß, activinßA, inhibin, GDF9, BMP4, and BMP7 were investigated in the present study.

In order to account for steroidogenic events occurring in the follicular layers, the expression profiles of the steroidogenic enzymes 20ß hydroxysteroid dehydrogenase (20ßHSD), 3ßHSD, aromatase, cholesterol side-chain cleavage cytochrome P450 (P450scc), 11ß hydroxylase (11ßH), 17 hydroxylase (17H), estrogen receptor {alpha} (ER{alpha}) , androgen receptors {alpha} and ß (AR{alpha} and ARß), as well as steroidogenic acute regulatory (StAR) protein were analyzed in the present survey.

In addition, LH receptor (LHr), FSHr, a connexin (connexin 43), as well as the two enzymes designated prostaglandin endoperoxide synthase-1 (PGS-1) and -2 (PGS-2) that catalyze the initial conversion of arachidonic acid in the biosynthetic pathway for prostaglandin synthesis, were also analyzed in the present survey because of their suspected role in the preovulatory ovary [4, 15].

Finally, other genes such as p53; p62; the cathepsins D, S, L, B, Z, and K; caspase 3ß; glyceraldehyde-3-phosphate dehydrogenase (GAPDH); and {alpha} actinin were also studied regardless of any suspected role they had in the control of ovarian function during this period.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal and Tissue Collection

Investigations and animal care were conducted in compliance with the laws of France on the care and use of laboratory animals. Twenty-two postvitellogenic rainbow trout (Oncorhynchus mykiss) were obtained during their first reproductive season from the INRA/SEDI fish farm (Sizun, France) and held in a natural photoperiod in a recirculated water system at 12°C in INRA experimental facilities (Rennes, France). Trout were deeply anesthetized in 2-phenoxyethanol, killed by a blow on the head, and bled by gill arch section. Ovaries were then dissected out of the body cavity under sterile conditions. A part of the ovary was dissected and deyolked as previously described [16]. Ovarian aliquots were frozen in liquid nitrogen and stored at –80°C until RNA extraction. Ovaries were used immediately for in vitro oocyte maturation analysis.

In Vitro Oocyte Maturation

In vitro oocyte maturation was performed as previously described [8, 17]. Briefly, groups of two to five follicles were dissected out of the ovary and incubated in 6-well culture plates (Corning, Corning, NY) at a ratio of 25 follicles per 3 ml of incubation medium (IM8/300) (133 mM NaCl, 3.09 mM KCl, 0.28 mM MgSO4, 0.98 mM MgCl2, 3.40 mM CaCl2, 5.55 mM glucose, 20 mM HEPES pH 8.0, 300 mOsm). After a 2-h preincubation step, partially purified gonadotropins (PPGs) were added to a final concentration of 0, 6, 12, 23, 47, 94, 188, 375, 750, 1500, or 3000 ng of protein per milliliter of incubation medium. In vitro maturation was performed in duplicate for each dilution. After a 60-h incubation period, the percentage of GVBD was measured in each well by direct observation as previously described [18, 19]. For each female, the PPG efficient concentration for 50% GVBD (EC50) was calculated as previously described [19].

RNA Extraction and Reverse Transcription

Ovarian tissue was homogenized in Trizol reagent (Invitrogen, Cergy Pontoise, France) at 100 mg/ml of reagent. Total RNA was extracted as previously described [20, 21]. For each individual female, 3 µg of total RNA were reverse transcribed using 200 units of Moloney murine Leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI) and 0.5 µg random hexamers (Promega) in a reverse transcription master mix containing 2 mM dNTPs, 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, pH 8.3. Twenty-five units of RNase inhibitor (RNasin, Promega) were added to the reaction. Briefly, RNA and dNTPs were denatured for 6 min at 70°C, then chilled on ice for 5 min before the reverse transcription master mix was added. Reverse transcription was performed at 37°C for 1 h and 15 min followed by a 15-min incubation step at 70°C. Control reactions were run without MMLV reverse transcriptase and used as negative controls in the real-time polymerase chain reaction (PCR) study.

Real-Time PCR

Real-time PCR was performed using an I-Cycler IQ (Bio-Rad Laboratories, Hercules, CA) as previously described [8]. Reverse transcription products were diluted to 1/50, and 5 µl were used for each real-time PCR reaction. Triplicates were run for each female. Real-time PCR was performed using a real-time PCR kit provided with a SYBR Green fluorophore (Eurogentec, Seraing, Belgium) according to the manufacturer's instructions. Primer concentration ranged from 300 to 600 nM depending on the target gene. After a 2-min incubation step at 50°C and a 10-min incubation step at 95°C, the amplification was performed using the following cycle: 95°C for 20 sec; 60°C for 1 min; 40 times. A pool of reverse transcribed ovarian RNA originating from postvitellogenic females was serially diluted and used to check for PCR efficiency. For all studied genes, elongation factor 1{alpha} (EF1{alpha}) was used as an internal standard and the relative abundance of target cDNA was calculated using the I-Cycler IQ software. After amplification, a fusion curve was obtained using the following protocol: 10 sec holding followed by a 0.5°C increase, repeated 80 times, and starting at 55°C. Primer sequences, GenBank accession number of the target gene and PCR product sizes are presented in Table 1. When possible, one of the primers was designed on an exon boundary, or primers were located across an intron. Reverse transcription reactions were performed without adding reverse transcriptase and run as negative controls for all target genes.


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  		TABLE 1. Nucleotide sequence of real-time PCR primers, GenBank accession number, and PCR product size for target genes

Clustering Analysis

For supervised clustering analysis, data were log transformed, median centered, and an average linkage clustering was performed using CLUSTER software (http://rana.lbl.gov/EisenSoftware.htm). Clusters were visualized using TREEVIEW software [10].

Statistical Analysis

Statistical analysis was performed using an ANOVA followed by a Tukey honest significant difference test between the following groups: females exhibiting no or little maturational competence (n = 8), females exhibiting high maturational competence (n = 8), and females undergoing oocyte maturation (n = 6).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Oocyte Maturation

Among the 22 females used in the present study, five were sampled at the time of oocyte maturation (GVBD completed) and were therefore not assayed for FMC. One female spontaneously matured in vitro even without any gonadotropin addition and was considered to have already begun oocyte maturation. Among the 16 other females assayed in vitro for FMC, two never matured even at the highest gonadotropin concentration and were therefore considered to be meiotically incompetent. The EC50 of PPG of the 14 other females ranged from 19 to 450 ng of PPG per milliliter of incubation medium.

For the supervised clustering analysis, the females were ordered as follows: 2 incompetent females, 14 females of increasing competence (decreasing EC50), 1 spontaneously maturing female, and 5 females undergoing oocyte maturation.

For statistical analysis, the spontaneously maturing female was grouped with the 5 females undergoing maturation while the 16 remaining females were arbitrarily divided into 2 groups. The no-or-poor-competence group was composed of the two incompetent females and the six females (EC50 140–450) exhibiting the lowest competence, while the high-competence group was composed of the eight females exhibiting the highest competence (EC50 19– 115).

Supervised Clustering Analysis

From the supervised clustering analysis, three clusters could be identified (Fig. 1). Cluster 1 consisted of nine genes: activinßA, cathepsin K, IGF-I, IGF-II, IGFBP-2, 11ßH, 3ßHSD, P450scc, and connexin 43. The cluster was characterized by decreased expression in incompetent or poorly competent (EC50 >250) females, and a progressive rise in gene expression during maturational competence acquisition or at the time of oocyte maturation. In contrast, cluster 2 was characterized by a strong increase in mRNA levels at the time of oocyte maturation or in the very last steps of maturational competence acquisition (EC50 <65). Cluster 2 consisted of inhibin, FSHr, BMP7, fibroblast growth factor 2 (FGF2), cathepsin D, cathepsin L, and StAR. Finally, cluster 3 was characterized by a decreased expression at the time of oocyte maturation but no significant variation in expression levels during FMC acquisition. Cluster 3 consisted of 17H, LHr, ER{alpha}, AR{alpha} and ARß.



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  		FIG. 1. Supervised clustering analysis of mRNA expression of 37 genes in ovarian tissue sampled from 2 incompetent females (Inc), 14 females of increasing competence, and 6 females undergoing oocyte maturation (Maturation). For each gene, the expression levels across the samples are indicated using a color scale

Statistical Analysis

Among the 37 genes assayed in the present expression survey, 18 exhibited a significant difference in their mRNA levels between at least 2 of the groups defined above: no or poor competence, high competence, and maturation. These results are summarized in Table 2.


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  		TABLE 2. Significant differences (P values) between females exhibiting no or poor competence (n = 8), high compentence (n = 8), and females undergoing oocyte maturation (n = 6)

More specifically, the mRNA expression of three genes was significantly higher in high competence females than in no or poor competence females. Thus, IGF-II and cathepsin K exhibited a 50% increase, and activinßA exhibited a 70% increase during FMC acquisition (Fig. 2).



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  		FIG. 2. Messenger RNA expression levels (mean ± SEM) of IGF-I, IGF-II, activinßA, connexin 43, 11ßH, and cathepsin K in ovarian tissue sampled from eight incompetent or poorly competent females (Low), eight highly competent females (High), and six females undergoing oocyte maturation (Mat). Bars sharing the same letter or letters are not significantly different at P < 0.05. P values are shown in Table 2. The target gene is indicated for each graph

In addition, five genes exhibited a higher expression in maturing females than in females exhibiting a high competence, whereas three genes exhibited an opposite pattern. Thus, we observed a 170% increase in 11ßH expression, a 180% increase in BMP4 expression, a 250% increase in FSHr expression, a 100% increase in FGF2 expression, and a 400% increase in inhibin levels at the time of oocyte maturation. In contrast, we observed a 100% decrease in LHr expression, a 120% decrease in 17H, and a 140% decrease in ER{alpha} at the time of oocyte maturation (Figs. 2– 4).



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  		FIG. 4. Messenger RNA expression levels (mean ± SEM) of 17{alpha}H, ER{alpha}, and LH receptor in ovarian tissue sampled from eight incompetent or poorly competent females (Low), eight highly competent females (High), and six maturing females (Mat). Bars sharing the same letter or letters are not significantly different at P < 0.05. P values are shown in Table 2. The target gene is indicated for each graph

Finally, the mRNA expression of 13 genes was significantly higher in maturing females than in females exhibiting no or little maturational competence. Thus, IGF-I exhibited an 80% increase, IGF-II a 70% increase, 11ßH a 300% increase, activinßA a 100% increase, connexin 43 a 400% increase, BMP4 a 110% increase, cathepsin Z a 170% increase, BMP7 a 170% increase, StAR an 1100% increase, cathepsin D a 130% increase, cathepsin L a 170% increase, FSHr a 350% increase, and FGF2 a 250% increase (Figs. 2 and 3).



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  		FIG. 3. Messenger RNA expression levels (mean ± SEM) of Star; FSH receptor; inhibin; FGF2; BMP7; BMP4; and cathepsins D, L, and Z in ovarian tissue sampled from eight incompetent or poorly competent females (Low), eight highly competent females (High), and six maturing females (Mat). Bars sharing the same letter or letters are not significantly different at P < 0.05. P values are shown in Table 2. The target gene is indicated for each graph

It is interesting that among the 18 genes exhibiting significant changes in their expression during FMC acquisition or at the time of oocyte maturation, 16 belonged to one of the 3 clusters identified above. Thus, in cluster 1, six genes among nine exhibited a significant increase in mRNA expression during FMC acquisition or at the time of oocyte maturation. In cluster 2, all the genes exhibited a significant increase in mRNA expression during FMC acquisition or at the time of oocyte maturation. Finally, in cluster 3, three genes among five exhibited a lower expression in females undergoing oocyte maturation than in females exhibiting high FMC.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The purpose of the present study was to identify genes in the ovary exhibiting an expression profile that could be associated with maturational competence acquisition and oocyte maturation. The biological material used for RNA extraction consisted of deyolked ovary that includes not only the granulosa and theca layers of full-grown follicles, but also extrafollicular ovarian tissue, including previtellogenic follicles. Therefore, the gene expression monitored here are not necessarily located in the full-grown follicles, but can also occur elsewhere in the ovary.

The clustering analysis clearly shows that several genes are characterized by a sharp increase at the time of oocyte maturation or in the very last steps of FMC acquisition (cluster 2). In contrast, other genes exhibit a progressive rise in their expression levels during FMC acquisition (cluster 1). Taken together, these observations are in complete agreement with the hypothesis of a progressive differentiation of the ovarian follicle all along the process of FMC acquisition. While the genes turned on or off at the time of oocyte maturation are probably important for the completion of the process as well as for the later steps of oogenesis (i.e., ovulation), genes belonging to cluster 1 are likely to be involved in preparing the ovary for the gonadotropic-dependent events that lead to the resumption of meiosis. These two types of expression profiles further point out that it is difficult to obtain homogeneous groups of females. Thus, the use of clustering analysis, which takes into account the expression levels in all females and not only the average value for a class of competence, is quite valuable. Indeed, although the use of groups (i.e., high competence, maturation, etc.) is necessary to allow a statistical analysis, it is somewhat limiting in describing a progressive and intricate process such as FMC acquisition.

In the present study, EF1{alpha} was used as an internal standard. In addition, GAPDH, another commonly used internal standard, was included in the expression study in order to strengthen our observations. Indeed, GAPDH mRNA levels were not significantly different between groups, and GAPDH did not belong to any of the gene clusters associated with a varying expression during FMC acquisition or oocyte maturation. Finally, this expression study is exclusively focused on mRNA levels of target genes, and neither the corresponding protein levels nor their functionality were assessed. For that reason, the mRNA expression profiles reported here suggest only the involvement of corresponding gene products in physiological mechanisms occurring within the ovary simultaneously with FMC acquisition or oocyte maturation.

In rainbow trout, it is possible to use an in vitro assay to estimate the FMC of each female [22]. This allows discrimination of FMC postvitellogenic females that do not exhibit any morphological differences in terms of ovarian development, whereas gene expression profiling can be performed on ovarian tissue directly sampled from each female. Thus the measured mRNA expression reflects the in vivo gene expression in contrast to investigations performed in other fish species such as Atlantic croaker (Micropogonias undulatus) or red seabream (Pagrus major), in which maturational competence was acquired through in vitro gonadotropic stimulation [23–26]. Finally, the rainbow trout species has undergone major sequencing efforts in the last few years. Therefore, due to its biological specificities and the numerous ESTs available in public databases, rainbow trout as a model offers powerful opportunities for studying FMC acquisition.

Gonadotropin Receptors

In a previous study, we observed an increased expression of FSHr by the end of the FMC acquisition process [8]. We now show that this FSHr mRNA expression also remains important during oocyte maturation, thus suggesting a role for it in oocyte maturation and, possibly, in ovulation. This is consistent with the rise in FSH circulating levels observed in rainbow trout before oocyte maturation [27]. In contrast, we show a slight decrease in LHr mRNA expression during oocyte maturation compared to expression levels in females exhibiting the highest maturational competence. However, the mRNA levels of LHr are not significantly lower during oocyte maturation than in females exhibiting no or little maturational competence. This would also be consistent with a little peak in LH circulating levels observed in rainbow trout 4 days before meiosis resumption [27]. LH is the determining factor regulating the production of the maturation-inducing steroid and the induction of meiosis resumption in salmonids, whereas FSH has a significant but lower effect [28]. However, the physiological role of FSH in the preovulatory ovary remains unclear and requires further functional investigation. Indeed, FSHr mRNA expression could possibly occur in the previtellogenic follicles present in the ovary at that time.

Insulin-Like Growth Factor System

From clustering and statistical analyses alike, we observe a progressive increase in the mRNA expression of IGF-I and IGF-II during FMC acquisition and oocyte maturation. This observation suggests that both IGF-I and IGF-II are important not only for FMC acquisition, but also during oocyte maturation. This is in agreement with previous studies reporting GVBD after stimulation in vitro, by either recombinant human IGF-I or IGF-II, depending on the species [11, 12, 29]. It is interesting that IGFBP-2 is closely clustered with IGF-I and IGF-II. Taken together, these results are consistent with the hypothesis of a paracrine role for the IGF system during FMC acquisition and oocyte maturation. From the clustering analysis, we can speculate that this role could be important quite early in the process of FMC acquisition.

Activin/Inhibin System

The activin system has been suspected to play a role, in a paracrine manner, in the control of oocyte maturation in several fish species. In zebrafish (Danio rerio) full-grown follicles, both activinßA and activinßB mRNAs are expressed in the follicle layers, and the entire activin signaling system is expressed in the oocyte [14], whereas in rainbow trout, activinßA was detected by in situ hybridization in the theca cells of late-vitellogenic follicles [30]. Our results are consistent with these previous observations. In addition, we show that activinßA and inhibin have different expression profiles. We observed a sharp increase in inhibin mRNA expression at the time of oocyte maturation while activinßA expression progressively increased during FMC acquisition.

Connexins and Gap Junctions

Gap junctions are aggregates of intracellular channels composed of the protein connexin between adjacent cells. In Atlantic croaker and red seabream, an increase in homologous and heterologous gap junction contacts was observed after a gonadotropic stimulation of late-vitellogenic follicles [26, 31]. More precisely, the hCG-dependent formation of gap junctions observed in vitro was shown to be associated with increased levels of connexin 32.2 mRNA levels in Atlantic croaker [32]. In the present study, we show an increase in the mRNA levels of connexin 43 during FMC acquisition and oocyte maturation. While this observation is consistent with prior observations in other species, further studies are needed to understand the respective role of the numerous members of the connexin family in the occurrence of heterologous and homologous gap junctions in the preovulatory differentiation of fish follicles.

The Steroidogenic Control of FMC Acquisition and Oocyte Maturation

The endocrine regulation of oocyte maturation and ovulation has received long-term interest [1, 7, 33, 34]. Both in vitro studies and in vivo monitoring of circulating levels have shown the eminent role of steroid hormones [34–36]. In salmonids, the preovulatory follicle is able to produce progestins, androgens, estrogens, and corticosteroids [35]. In rainbow trout, the maturation-inducing steroid 17,20ß-dihydroxy-4-pregnen-3-one, shows a sharp rise at the precise time of oocyte maturation [37]. In contrast, estradiol-17ß (E2) levels decrease drastically before maturation [38, 39], whereas testosterone levels, though decreasing, still appear high or even show a slight peak before ovulation [39].

In the present study, we observed that P450scc and 3ßHSD belong to cluster 1, thus suggesting a progressive increase in the mRNA expression of both genes during FMC acquisition and oocyte maturation. In contrast, 20ßHSD expression is extremely variable depending on the female. Taken together, these results are consistent with previous observations of strong daily fluctuations in MIS circulating levels in female rainbow trout undergoing oocyte maturation [40]. In addition, it is noteworthy that two closely related rainbow trout 20ßHSD cDNAs, both expressed in vitellogenic ovarian follicles, were previously identified [41]. Enzymatic characterization using recombinant proteins produced in Escherichia coli showed that only the product of one of the two cDNAs had 20ßHSD activity [41]. However, because these two cDNAs are highly similar (99% identity at the nucleotide level), they were both amplified by the primers used in the present study.

In contrast, 17H and ER{alpha} mRNA levels were significantly lower in females undergoing oocyte maturation than in females exhibiting high FMC. In addition, the highest aromatase mRNA levels were observed in meiotically incompetent or poorly competent females, whereas the lowest mRNA levels were observed in maturing or highly competent females (Fig. 1). These observations are consistent with the drop in E2 circulating levels observed during FMC acquisition in rainbow trout [38, 39].

Both androgen receptors {alpha} and ß genes belonged to cluster 3, thus suggesting a decreased expression of these genes during oocyte maturation, which is consistent with the drop in testosterone circulating levels observed in rainbow trout prior to ovulation [42].

The steroidogenic acute regulatory protein (StAR) is responsible for the delivery of cholesterol to P450scc. In brook trout (Salvelinus fontinalis), it was recently shown that StAR mRNA was barely detectable in ovarian follicles before the resumption of meiosis, and that its expression increased after GVBD [43]. The present study is in complete agreement with this previous work, and a dramatic increase in StAR mRNA levels was observed in the last steps of FMC acquisition.

We also observed, interestingly, a progressive rise in 11ß-hydroxylase (11ßH) mRNA expression during FMC acquisition. In vertebrates, this steroidogenic enzyme acts at the final steps of biosynthesis of glucocorticoids and mineralocorticoids. In salmonids, the preovulatory follicle is able to produce corticosteroids [35]. However, a rise in cortisol circulating levels is not observed at the time of GVBD, but it is observed after ovulation [44]. In rainbow trout, deoxycorticosterone was only able to induce incomplete maturation without GVBD [45, 46]. While further investigations are needed to elucidate the putative roles of corticosteroids in preovulatory follicle, the observed increase in 11ßH expression during FMC acquisition could be related to the hydration of the oocyte observed at that time in some fish species [47]. The rise in 11ßH mRNA expression observed at the time of oocyte maturation could also be related to 11-ketotestosterone (11-KT) production. However, a recent study showed that 11-KT levels were extremely low in female rainbow trout sampled during spawning season [48].

BMPs and Other TGFß Superfamily Members

The BMPs form a group of growth and differentiation factors belonging to the TGFß superfamily. BMPs and their receptors are known to have important roles in mammalian ovarian folliculogenesis [13, 49]. One of the most novel findings of the present study is, for the first time in fish, the report of an mRNA expression of BMP4 and BMP7 in ovarian tissue during FMC acquisition or oocyte maturation. BMP4 and BMP7 are expressed by theca cells in rats [50] and cows [13]. In chickens, both BMP4 and BMP7 were recently detected in both granulosa and theca cells [51]. In the present study, BMP7 shows an increased expression in highly competent and maturing females, whereas BMP4 expression is increased later, at the time of oocyte maturation. During chicken follicular development, both BMP4 and BMP7 expression increase in granulosa cells, whereas BMP7 expression is increased in the theca [51]. It is interesting that BMP4 and BMP7 both lead to IGF-I-stimulated, and gonadotropin-stimulated progesterone production by cultured chicken granulosa cells [51]. However, the specific roles of BMP4 and BMP7 in rainbow trout follicular differentiation need to be further investigated.

In addition, we also report in the present study an expression of growth differentiation factor 9 (GDF9) and TGFß in the preovulatory ovary. However, neither of these genes exhibited any specific expression profile associated with FMC acquisition or oocyte maturation. In fact, GDF9 is an oocyte-derived growth factor involved in mammalian follicle development [13]. The GDF9 mRNA expression detected here could therefore be located in the previtellogenic follicles present in the trout periovulatory ovary.

Cathepsins

In the present study, we observed an ovarian expression of cathepsins B, D, K, L, S, and Z. In addition, cathepsins D, L, and Z exhibited increased mRNA expression at the time of oocyte maturation. Indeed, cathepsins D and L belong to cluster 2 and exhibited an increased expression in highly competent females. These observations are in agreement with the report of an ovarian expression of cathepsins D, L, and B in rainbow trout females undergoing oocyte maturation [52]. However, the role of these enzymes in follicular layers or extrafollicular ovarian tissue remains unknown. Cathepsin L is a member of the papain family, and its expression is, interestingly, selectively induced in granulosa cells of mouse preovulatory follicles by the LH surge [53].

Prostaglandin Endoperoxide Synthases 1 and 2

PGS1 and PGS2, the two enzymes that catalyze the initial conversion of arachidonic acid in the biosynthetic pathway for prostaglandin synthesis were both expressed, although not differentially, during FMC acquisition and oocyte maturation. Using Northern blot analysis, it was previously reported that only PGS1 but not PGS2 was expressed in the brook trout preovulatory ovary [6]. Using real-time PCR, a highly sensitive technique, we observed that PGS1 expression is much higher than PGS2 expression. Our results are therefore in agreement with this earlier study.

Other Genes

From the other genes analyzed in the present profiling study, fibroblast growth factor 2 (FGF2) was the only one exhibiting a significant increase in mRNA expression during the process of FMC acquisition and oocyte maturation. FGF2 is, interestingly, expressed in bovine follicles during their final growth to the preovulatory follicle stage [54], where it could be involved in the proliferation of capillaries.

Conclusions

In conclusion, we performed a profiling analysis that allows a better description of the ovarian gene expression sequence associated with the differentiation of the ovarian follicle during FMC acquisition and meiosis resumption. Our results strongly suggest that many paracrine factors such as IGFs, FGF2, BMPs, and other members of the TGFß family are involved in the preovulatory differentiation of the follicle or the control of intrafollicular oocyte maturation. In the future, such an approach should be extended to a systematic analysis of the transcriptome using microarray technology. In addition, complementary investigations should be performed to identify the cellular compartments that express each target gene and possibly identify dialogs between the different follicular or ovarian cellular compartments.


   ACKNOWLEDGMENTS
 
We thank Y. Guiguen and D. Baron for their helpful input in the clustering analysis and for designing several primer pairs, and S. Aegerter for designing several primer pairs.


   FOOTNOTES
 
1 Correspondence: Julien Bobe, Institut National de la Recherche Agronomique, SCRIBE, Campus de Beaulieu, 35042 Rennes Cedex, France. FAX: 33 2 23 48 50 20; bobe{at}beaulieu.rennes.inra.fr Back

Received: 5 November 2003.

First decision: 4 December 2003.

Accepted: 13 February 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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