| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Centre for Biomedical Research,3 University of Victoria, Victoria, British Columbia, Canada V8W 3N5
Great Lakes Water Institute,4 University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53204
Simon Fraser University,5 Burnaby, British Columbia, Canada V5A 1S6
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, LHß, FSHß, thyroid-stimulating hormone ß, and retinol-binding protein in both the ovary and testis of trout.
cDNA, microarray, ovary, rainbow trout, testis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bidirectional communication occurs between both the oocytes and the somatic follicular cells. Oocyte-secreted factors regulate granulosa cell differentiation, proliferation, and function, whereas granulosa cell paracrine activities ensure the growth and development of the oocyte [5, 6]. Changes in expression of the components that comprise the connective tissue matrix also participate in follicular maturation and function [3, 7]. Some evidence also indicates that immune cells interact with and coordinate the function of the somatic cells associated with germ cells [8, 9]. These complex processes must provide the precise regulatory and physiological milieu for production of functional gametes.
One interesting phenomenon in a small percentage of juvenile salmon is that they are ready to undergo spawning at least a year ahead of their siblings. These precocious males and females undergo dramatic increases in growth and development of their testes and ovaries compared to their normal ("less mature") cohorts. This provides an opportunity to compare and characterize the genes expressed in immature, normal, and precocious reproductive tissues of the same age.
To understand what genes are involved in these dynamic developmental processes, we undertook the following study. First, to identify some of the genes expressed differentially in normal and precocious ovary, we constructed subtracted cDNA libraries using immature tissue as the reference cDNA population. Second, we used 3557-gene salmonid cDNA microarrays to profile gene expression at three stages of precocious ovary development (June, August, and October) relative to reference (normal; June) ovary. We also followed the expression of several genes heretofore considered to be absent from or only weakly expressed during ovarian development.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Each gonadal tissue used in the present study was obtained from 1.5-to 1.8-yr-old male or female rainbow trout (Oncorhynchus mykiss) raised in an open lake fed by a natural stream in Sooke (BC, Canada; Mountain Trout Sales). Treatment of the fish used in the present study was in compliance with the regulations of the University of Victoria Animal Care Committee. Most trout ovulate and spawn for the first time at 3 yr of age and then continue to spawn annually. However, 10–20% of trout mature precociously, beginning at approximately 1.5 yr of age, and may spawn at 2 yr of age, a year ahead of their normal ("less mature") cohorts. Precocious maturation is a normal reproductive state in which offspring can be produced. Fish were judged to be precociously mature based on the weight of the gonads and on other defining characteristics, such as visible eggs, orange coloration, and larger size of ovaries in comparison to their normal cohorts. Gonadal tissue was considered to be immature if it was sexually indeterminate by visual inspection.
Tissue and RNA Extraction
Fish were exsanguinated for several minutes. The tissues were removed, flash-frozen in liquid nitrogen, and stored at –80°C until RNA extraction. Flash-frozen tissues were ground using baked (220°C, 5 h) mortars and pestles under liquid nitrogen. Then, total RNA was extracted in TRIzol reagent (Invitrogen, Carlsbad, CA), and poly(A)+ RNA was purified using MicroPoly(A)Pure kits (Ambion, Austin, TX).
Subtractive Hybridization
Total RNAs extracted from April precocious ovaries and testes, normal ovaries and testes, and immature tissues were obtained from several animals (except for precocious tissues) because of quantity differences based on the different maturation states of each tissue. Poly(A)+ RNAs were converted into cDNAs, and reference (driver) and experimental (tester) cDNAs were subjected to suppression subtractive hybridization (SSH) using the PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. An SSH library is enriched for cDNAs that are more abundant in the tester than in the driver. In subtractive hybridizations, precocious ovary and testis as well as normal ovarian and testicular cDNAs were used individually as tester against driver naïve cDNA. In addition, reciprocal SSH libraries were generated from normal ovarian and normal testicular cDNAs.
Products from secondary PCRs amplified using the Advantage cDNA PCR kit (Clontech) were size-fractionated on a 1.0% agarose gel. Insert sizes of cDNA libraries were determined by visual comparison of clone restriction fragments with the DNA size markers HindIII and a 1-kilobase ladder. High-molecular-weight (500–1500 base pair [bp]) and low-molecular-weight (200–500 bp) cDNAs were subcloned into pCR4-TOPO vector and transformed using Top10 electrocompetent cells (Invitrogen). Next, 7936 randomly selected clones from the 12 sublibraries (high- and low-molecular-weight libraries) were extracted and sequenced by BigDye Terminator (ABI, Foster City, CA) cycle sequencing on an ABI 3700 sequencer using conventional procedures and M13 forward and M13 reverse primers. Base-calling from chromatogram traces was performed using PHRED [10, 11]. Vector, poly-A tails, and low-quality regions were trimmed from each sequence; sequences that had less than 100 good-quality bases after trimming were discarded.
Microarray Fabrication and Quality Control
Library construction, gene selection, microarray fabrication, and quality control of the array used in the present study have been described in detail previously [12]. Briefly, 3557 cDNA clones from 18 high-complexity salmonid cDNA libraries/library groups were selected and printed as double, side-by-side spots on ArrayIt Superamine slides (Telechem International, Sunnyvale, CA) with the Biorobotics Microgrid II microarray printer (Apogent Discoveries, Hudson, NH). Microspot 10K quill pins (Biorobotics, Cambridge, U.K.) in a 48-pin tool were used to deposit approximately 0.5 nl (0.2 ng of cDNA) per spot onto the slide. The resulting microarrays have a 4 x 12 subgrid layout, with 132 spots per subgrid and each spot having an approximate diameter and pitch of 100 and 250 µm, respectively. The slides were cross-linked in a UV Stratalinker 2400 (Stratagene, La Jolla, CA) at 120 mJ. Spot morphology was assessed by visual inspection or by staining with SYBR Green 1 (Molecular Probes, Eugene, OR).
Microarray Hybridization and Analysis
This microarray experiment was designed to comply with MIAME guidelines [13]. To minimize technical variability, all targets were synthesized in one round. Total RNA was extracted (TRIzol) from flash-frozen precocious (June, August, and October) and normal (June) ovarian tissues collected from rainbow trout in the second year. Extracted total RNAs were cleaned using MEGAclear (Ambion) and then quantified and quality-checked by spectrophotometer and agarose gel, respectively. The microarray experiment used June normal ovary as reference and included three replicates (two identical and one dye-flip) for comparison of each precocious ovary stage with the reference sample. Nine microarrays were used in total: three June precocious versus June normal ovaries, three August precocious versus June normal ovaries, and three October precocious versus June normal ovaries.
Hybridizations were performed using the Genisphere Array50 version 2 kit and instructions (Genisphere, Hatfield, PA). Briefly, 11 µg of total RNA were reverse transcribed using oligo-d(T) primers with unique 5'-sequence overhangs for the cyanine fluor Cy5- or Cy3-labeling reactions. Microarrays were prepared for hybridization by washing twice for 5 min each time in 0.1% SDS, washing five times for 1 min each time in Milli-Q H2O, (Millipore, Billerica, MA) immersing for 3 min in 95°C Milli-Q H2O, and drying by centrifugation (5 min at 2000 rpm in a 50-ml conical tube). The cDNA was hybridized to the salmon cDNA microarray in a formamide-based buffer (25% formamide, 4x SSC [1x SSC: 0.15 sodium chloride and 0.015 sodium citrate], 0.5% SDS, 2x Denhardt solution) for 16 h at 48°C. The arrays were washed once for 10 min in 48°C (2x SSC, 0.1% SDS), twice for 5 min each time in (2x SSC, 0.1% SDS) at room temperature (RT), twice for 5 min each time in 1x SSC at RT, twice for 5 min each time in 0.1x SSC at RT, and dried by centrifugation. The Cy5 and Cy3 three-dimensional fluorescent molecules (3DNA capture reagent; Genisphere) were hybridized to the bound cDNA on the microarray with 3DNA capture reagents bound to their complementary cDNA capture sequences on the oligo-d(T) primers. The second hybridization was done for 3 h at 48°C and then washed and dried as described above.
Fluorescent images of hybridized arrays were acquired immediately at 10-mm resolution using ScanArray Express (PerkinElmer, Wellesley, MA). The Cy3 and Cy5 cyanine fluors were excited at 543 and 633 nm, respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in the present study (Cy3, PMT 73; Cy5, PMT 67). Fluorescent intensity data were extracted from TIFF images using ImaGene 5.5 software (Biodiscovery, El Segundo, CA). Quality statistics were compiled in Excel (Microsoft, Redmond, WA) from raw ImaGene fluorescence intensity report files. Elements were sorted (7356 salmonid spots representing 3557 different cDNAs, 20 Arabidopsis spots representing five different cDNAs, and 1356 other control spots), and median signal values and mean numbers of salmonid elements passing threshold were determined for Cy3 and Cy5 data separately. Data analyses (background correction, Lowess normalization, and fold-change gene list formation) were performed in GeneSpring 6.1 (Silicon Genetics, Redwood City, CA). For a microarray feature to be included in an informative transcript list, its background-corrected, Lowess-normalized (BCLN) Cy5:Cy3 ratio had to be either greater than 2.0 (Table 1) or less than 0.5 (Table 2) in all three pertinent slides. For Tables 1 and 2, fold-change values (ratios) were calculated with the dominant channel (the higher expression sample; i.e., precocious for Table 1 and normal for Table 2) in the numerator. For Tables 3–6, all fold-change values were calculated with BCLN precocious sample values in the numerator. For each transcript of interest, fold-change values were entered into an Excel spreadsheet. Mean, SD, and SEM were made across replicate microarrays in Excel. All scanned microarray TIFF images, extracted ImaGene grid files, the gene identification file, ImaGene quantified data files, and quality statistics are available on-line as supplemental data (http://web.uvic.ca/cbr/grasp).
|
|
|
|
|
|
Polymerase Chain Reaction
The primers used to amplify common glycoprotein (Cg) , LHß, FSHß, thyroid-stimulating hormone (TSH) ß, retinol-binding protein (RBP), and ubiquitin (control) were designed specifically against the sequences provided for each rainbow trout gene obtained from http://www.ncbi.nlm.nih.gov with the following accession numbers: AB050834 for Cg, AB050836 for LHß, AB050835 for FSHß, D14692 for TSHß, AF257326 for RBP, and AB036060 for ubiquitin. For each gene, sequences of the forward and reverse primers used in each respective polymerase chain reaction (PCR) were as follows: Cg, 5'-CAACATCATGCAGTGTACAGG-3' and 5'-ATCAGTATTCAATTCATACAG-3', respectively; LHß, 5'-GATGTTAGGTCTTCATGTAGG-3' and 5'-CAAGTACATTCACATACAACC-3', respectively; FSHß, 5'-TGCCGACTAAACAACATGACC-3' and 5'-TGCAATAGCACATCAACAATG-3', respectively; TSHß, 5'-CTGCTCTTCAGCCAAGCTGTG-3' and 5'-AACACACGAGTACGACAATGC-3', respectively; RBP, 5'-CAATGTCGTCGCTCAGTTCT-3' and 5'-TCAACTGCTTTCACAGAAAC-3', respectively; and ubiquitin, 5'-ATGTCAAGGCCAAGATCCAG-3' and 5'-TAATGCCTCCACGAAGACG-3', respectively.
The cDNAs were synthesized in 25-µl reactions that contained 200 ng of poly(A)+ RNA or 1.0 µg of total RNA using Omniscript RT (Qiagen, Mississauga, ON) according to the manufacturer's instructions. The reactions were incubated at 37°C for 90 min, and the transcriptase was heat-inactivated at 70°C for 30 min. Approximately 200 ng of cDNA were used in each 25-µl PCR reaction containing 1.25 U of Taq polymerase, 1x Taq buffer, 1.25 mM MgCl2, 10 mM dNTPs (Invitrogen), and 15 pmol of each gene-specific 5' and 3' primer. Each PCR was carried out under the following cycling parameters: 94°C for 2 min, followed by 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min using a PerkinElmer 9600. The PCR products were separated by electrophoresis on 1.0% agarose gels, and photos were stored using an Eagle Eye II still video system (Stratagene). Representative products were isolated and cloned into pCR4-TOPO vector and sequenced to confirm gene identities.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To identify potential gonad-specific and sex-specific genes, we used SSH as a technique to create 12 sublibraries. From these libraries, a total of 7936 clones were M13 forward sequenced and quality checked. Of these clones, 5778 cDNAs passed quality-filtering processing. (Access to data related to each of these gene fragments can be found at http://web.uvic.ca/cbr/grasp.) The two ovarian tissue classes examined (precocious and normal) had 1722 different cDNAs (see libraries rtah, rtal, rtch, and rtcl); the testicular counterparts had 2318 different genes reported (see libraries rtbh, rtbl, rtdh, and rtdl). We also reported 639 and 1099 genes that are differentially expressed between normal ovary and testis (see libraries rteh and rtel) and between normal testis and ovary (see libraries rtfh and rtfl), respectively.
Microarray Analysis
Differential gene expression in the three developmental stages of precocious ovary (June, August, and October) relative to June normal ovary was determined using a microarray presenting 3557 different cDNAs selected from 18 high-complexity salmonid cDNA libraries [12]. Genes from libraries of ovarian or testicular origin have 281 representatives on this array. The majority of cDNAs selected for the chip came from a normalized mixed-tissue library (Salmo salar spleen, kidney, and brain).
Data analysis executed in GeneSpring 6.1 permitted the passage of 2852 genes. We found 263, 164, and 304 genes greater than twofold up-regulated and 220, 146, and 348 genes greater than twofold down-regulated in June, August, and October precocious ovary (relative to June normal ovary), respectively (Tables 1 and 2). Only those cDNAs that were above or below the twofold lines in two or more stages of analysis were included in these tables. In cases of multiple hits for the same gene name, only the best candidate was included in Table 1 or 2. The presence of multiple entries of some genes served to provide an internal validation of our microarray results. For example, we found five prostaglandin D synthase, two fatty acid-binding protein H-FABP, and two simple type II keratin K8a microarray elements in the original "genes up-regulated in precocious ovary relative to normal ovary" gene list contributing to Table 1. Also, those genes that possibly were of little interest to the focus of this experiment (e.g., ribosomal RNAs and general housekeeping genes) were not included in Tables 1 and 2.
The most highly up-regulated transcript in the present study was the complement receptor type 2 (CR2) (average, 27.53-fold; SEM, 5.79) (Table 1). Several other immunoregulatory genes (e.g., several histocompatibility antigens, complement components, and immunoglobulins) also were found to be up-regulated in precocious relative to normal ovary. We present data for 31 other potential immunoregulatory genes that were not differentially expressed between precocious and normal ovary (Table 3).
We also observed the steady-state expression of a number of ubiquitin-proteasome components, cell-division regulators, and apoptotic factors (Table 4). Expression of some of these genes could point to both proteolytic and nonproteolytic activities, some of which might be key to meiotic and/or mitotic control mechanisms. Coexpression of at least five of these genes (Table 4, bold) defines an important period in which follicular maturation undergoes a steroidogenic shift. Furthermore, the products of genes such as elastase IIIA, cathepsins, and nidogen (Table 1) as well as 2-macroglobulins, alveolin and TIMP2 (Table 2) have been implicated in cellular assembly and editing. Six more genes with similar functions that were expressed at steady-state levels are included in Table 5.
|
|
Only cDNAs having significant (E < 10–5) BLASTX hits against the current GenBank databases are described for genes in Tables 1 and 2 (greater than twofold up- or down-regulated in precocious ovary relative to normal ovary) or Tables 3–6 (similar expression levels in precocious and normal ovary). For each table, a GenBank accession number is provided for each expressed sequence tag (EST) corresponding to each microarray element. Not available (N/A) indicates when the EST has not yet been submitted. To identify potentially informative genes, the degree of similarity (length and percentage identity over aligned region) between salmonid microarray element EST translations and their most significant (most negative E-value) BLASTX hits are presented (Tables 1–6). If a salmonid EST has no significant BLASTX hit, then the most significant BLASTN hit (n) is shown.
Changes in transcription of informative genes are provided for each stage (June, August, and October) of precocious ovary development relative to normal June ovary and are shown as the mean fold-change (MFC) with SEM (Tables 1–6). The MFC values presented in each table are organized in descending order by June precocious ovary MFC.
Identification and Confirmation of UniquelyExpressed Genes
Microarray analysis revealed the steady-state expression of various important growth factors, cytokines, and hormones (Table 6). One unexpected finding was the hybridizations to the array of transcripts that encode the pituitary glycoprotein hormone subunits shown in Table 6 (bold). To confirm these results and investigate how broadly some of these transcripts might be expressed, we used PCRs to amplify cDNA taken from tissues at various development states (Fig. 1). The expression of RBP also was followed, because the presence of this gene had not previously been unequivocally demonstrated in either ovary or testis of fish [14, 15]. The PCR products of the following sizes were generated using specific primer sets for each gene: Cg, 462 bp; LHß, 587 bp; FSHß, 414 bp; TSHß, 549 bp; RBP, 417 bp; and ubiquitin, 158 bp.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Regulation of Ovarian Cellular Organizationand Modeling
A fine balance of the spatiotemporal expression of some of these messages must occur during organization and modeling of the supporting tissues and during oocyte development. For example, differences in the timing and expression levels of cathepsins K, L, and S are shown in Table 1. These cysteine proteinases have been demonstrated to possess collagenolytic activities that degrade ECM and basement membranes [2]. Cathepsin L, together with cathepsin D (expressed between twofold lines), have activities that have been associated with yolk processing during vitellogenesis in rainbow trout as well [18].
A trout ovulatory protein (TOP)-2 with potential antielastase or anticathepsin activity [19] also is expressed at dramatically increasing levels during the period from June to October. Interestingly, the strong expression of the TOP-2 transcript coincides with increased expression of a pancreatic elastase transcript (Table 1). The marked increase in expression of a serine protease through the period of the present study also correlates with Northern blot and densitometric analyses of this transcript in preovulatory and ovulatory brook trout ovarian tissue [20].
The expression of transcripts that inhibit proteolysis, such as tissue inhibitor of metalloproteinase (TIMP) 2 and 2-macroglobulins 1 and 3, are down-regulated in these tissues (Table 2). Concurrent with the declining expression of TIMP2, we observe decreased expression of alveolin (a metalloproteinase) and steady-state expressions of various elastases (Tables 2 and 5).
Interestingly, a variety of matrix metalloproteinases, elastases, and inhibitors were isolated from normal ovary-specific subtracted libraries using normal testicular cDNAs as the reference population (see http://web.uvic.ca/cbr/grasp). These activities were not identified in the testis-specific subtracted libraries. Although less than 2000 genes in this category were sampled, this observation could point to differences in the timing of the transcription of these morphogenic factors between normal ovary and testis of the same age.
Presence of Immunoregulators in the Developing Ovary
Up-regulation of CR2 and various complement factors and immunoglobulins were detected in the present study (Table 1). Many immune factors potentially involved in the development of the ovary that were expressed at steady-state levels also are shown in Table 3. The complement system is activated primarily by two pathways: the classical pathway, and the alternative pathway. The classical pathway is triggered by antigen-antibody complexes, and the alternative pathway is initiated on cell surfaces in the absence of antibodies. The regulated and steady-state expressions of CR2, complement C1q, and various downstream complement components and immunoglobulins (Tables 1 and 3) indicate potential complement activation by both pathways. Both of these arms of the complement cascade could be initiated for tagging and removal of apoptotic cells and cellular debris from tissues undergoing considerable growth and remodeling. Some members of the complement cascade also may be involved in modulating changes in the ECM through proteolytic activities that modify the actions of various cytokines and growth factors in different cell types [21]. The terminal complement components (C5–C9) and formation of membrane attack complex also have been shown to be important for the release of proinflammatory mediators but also could point to nonlethal cell signaling and induction of cell proliferation [22, 23]. Active complement proteins have been associated with mammalian preovulatory follicular fluid [24] and uterus [25]. Both complement factor B and complement C3 mRNA have been detected in mouse uterus, but not ovary, and gene expression, particularly that of C3, is significantly increased by estrogen [26]. The complement C4 identified in Table 1 has 44% identity with carp complement C4A, but it also shares approximately 25% identity with trout complement C3A. Interestingly, at least three different C3 molecules exist in trout serum, each possessing distinct binding specificities [27]. The specific roles of these various immune effectors in the developing piscine ovary as well as in postovulatory stages, as evidenced by mammalian investigations, need to be elucidated.
Coexpression of Genes Important in FollicularMaturation Events
One interesting feature of this microarray analysis was the capture of the expression of a number of transcripts whose roles are intimately connected. The present study revealed the transcription at steady-state levels of various cell division regulators (cdc2 and cyclin B) and ubiquitin-mediated proteolysis components (ubiquitin-conjugating enzyme E2–E23 and cyclin-selective ubiquitin carrier E2-C) that selectively mark and degrade these factors (Table 4). Furthermore, the expression of the enzyme carbonyl reductase/20ß-hydroxysteroid dehydrogenase (20ß-HSD) also was concurrently expressed at these levels. The expression of 20ß-HSD marks a steroidogenic shift in postvitellogenic follicles from the production of estradiol-17ß to synthesis of a progesterone derivative, 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-DP) [28]. In postvitellogenic follicles, these changes indicate the end of rapid oocyte growth associated with vitellogenesis in response to estradiol and the start of a period of oocyte maturation influenced by a maturation-promoting factor (MPF). The 17,20ß-DP exerts its action through oocyte membrane receptors to activate the formation of a complex of the two components of the MPF, cdc2 and cyclin B [28]. Postvitellogenic oocytes (arrested in prophase) require active MPF for resumption of meiotic maturation and, during meiotic arrest at the metaphase II stage, to become fertilizable [29].
It is possible that we have captured a small glimpse of these processes at the gene-expression level. Our work does not indicate whether each of these transcripts are translated in these tissues at this stage of development. It also could be that the concurrent expression of cdc2, cyclin B, and 20ß-HSD (and, presumably, 17,20ß-DP) is an indicator of somatic (follicle) cells undergoing mitotic divisions. Cell-cycle transitions may be controlled by regulation of the ubiquitin carrier and cyclin ligase destruction machinery. To date, and to our knowledge, no reports have precisely detailed cDNA expression of each ubiquitin-proteasome component in piscine follicular cells, but some of this proteolytic machinery has been isolated from goldfish oocytes [30, 31]. Cyclin B transcript also is present in goldfish and zebrafish immature oocytes, but it is not translated until later, when the oocyte meiotic maturation phase is initiated [28, 32]. It therefore is possible that similar posttranscriptional controls, as well as other regulatory constraints [33, 34], are placed on the transcripts that encode the proteolytic machinery that selectively degrades cyclins. The culmination of expression of this particular group of transcripts points to an interesting stage of salmon ovarian development that could, when coupled with immunodetection, lead to a greater understanding of the machinery involved in controlling mitosis and meiosis in immature and preovulatory follicles.
Expression of RBP in Salmon Ovary and Testis
To our knowledge, this is the first presentation of strong evidence for RBP gene expression in the piscine ovary (Table 1 and Fig. 1). Reports concerning other teleosts indicate only weak, if any, expression of RBP in ovary [14, 15]. In fact, we observed RBP cDNA expression in immature (data not shown), normal (Fig. 1), and precocious (Table 1) tissues. Locally expressed RBP may serve to deliver retinol to the developing oocyte. The metabolites of the retinol then could be used during embryogenesis. It also is possible that delivery of retinol to the ovary from the liver (the major vertebrate storage site of retinol) is by a more general carrier, such as with vitellogenin, albumin, or low-density lipoproteins. Once in the ovary, RBP may be required for the transport of retinol to specific cell-types to participate in ovarian maturation. In support of this argument, expression of RBP in granulosa cells [35] and Sertoli cells [36] of the rat has been demonstrated. To date, and to our knowledge, a complete understanding of how retinol and other nutritional and regulatory substances are deposited in the oocyte yolk has not been elucidated [14, 15].
Presence of Uniquely Expressed Genes in Salmon Ovary and Testis
We also report the expression of cDNAs that encode Cg as well as LHß, FSHß, and TSHß in the salmonid ovary. The LH, FSH, and TSH each share the Cg subunit and acquire their unique attributes by heterodimeric binding through the hormone-specific ß subunits. These glycoprotein hormones are more commonly associated with expression and synthesis in the pituitary; therefore, detection of their hybridizations to the array throughout ovarian development was unexpected (Table 6). Expression of these cDNAs were further demonstrated in ovarian and testicular cDNAs at different developmental stages by PCR (Fig. 1). These findings also are supported by mammalian investigations that demonstrated FSH expression in both ovary [37] and testis [38]. Although evidence exists for the expression of both Cg and LHß in the rat testis [39], no corresponding reports, to our knowledge, have appeared for LHß expression in the mammalian ovary. Therefore, the present report appears to be the first to indicate the potential for synthesis of both Cg and LHß in the ovary for any species. The lack of any discernible mRNA for any of these transcripts in the unfertilized egg as well as expression in the testes (except for TSH) implicate that these molecules serve specific functions in the gonads rather than being produced as agents for subsequent embryogenesis.
Hypothalamic GnRH controls and modulates the release of LH and FSH in the pituitary, and GnRH synthesis occurs in both the ovary and testis [40]. Unfortunately, the microarray employed in the present study did not contain any prepro-GnRH cDNA elements. However, expression of a prepro-thyrotropin-releasing hormone (the hypothalamic activator of TSH) was observed throughout the present study (Table 2). Investigations to determine the physiological roles of each of the glycoprotein hormones as well as their activators and receptors within the gonad clearly are required in piscine and mammalian models.
In conclusion, we have shown the utility of using microarrays to identify genes important in the development and maturation of the trout ovary. Our salmonid gene-specific microarray analysis revealed changes that occur in the expression of genes involved in cellular organization and modeling, immunoregulation, cell cycling, and other areas of interest. The present study enabled the tracking of specific cDNA expressions that potentially mark a crucial phase in follicular maturation. Microarrays also can serve as useful tools to detect unexpected tissue-specific expression of genes.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Ben F. Koop, Centre for Biomedical Research, University of Victoria, P.O. Box 3020 STN CSC, Victoria, British Columbia V8W 3N5, Canada. FAX: 250 472 4075; bkoop{at}uvic.ca
Received: 3 August 2004.
First decision: 15 September 2004.
Accepted: 29 September 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
,20ß-Dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: mechanisms of synthesis and action. Steroids 1997 62:190-196[CrossRef][Medline]-subunits are expressed in mouse testis, as determined in wild-type mice and those transgenic for the FSH ß-subunit/herpes simplex virus thymidine kinase fusion gene. Endocrinology 1995 136:4769-4775[Abstract]This article has been cited by other articles:
|
|
 |
|
  L. Levi, B. Levavi-Sivan, and E. Lubzens Expression of Genes Associated with Retinoid Metabolism in the Trout Ovarian Follicle Biol Reprod, September 1, 2008; 79(3): 570 - 577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Derome, B. Bougas, S. M. Rogers, A. R. Whiteley, A. Labbe, J. Laroche, and L. Bernatchez Pervasive Sex-Linked Effects on Transcription Regulation As Revealed by Expression Quantitative Trait Loci Mapping in Lake Whitefish Species Pairs (Coregonus sp., Salmonidae) Genetics, August 1, 2008; 179(4): 1903 - 1917. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Hook, A. D. Skillman, B. Gopalan, J. A. Small, and I. R. Schultz Gene Expression Profiles in Rainbow Trout, Onchorynchus mykiss, Exposed to a Simple Chemical Mixture Toxicol. Sci., March 1, 2008; 102(1): 42 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L Rise, S. E Douglas, D. Sakhrani, J. Williams, K V. Ewart, M. Rise, W. S Davidson, B. F Koop, and R. H Devlin Multiple microarray platforms utilized for hepatic gene expression profiling of GH transgenic coho salmon with and without ration restriction. J. Mol. Endocrinol., October 1, 2006; 37(2): 259 - 282. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. N. Morrison, G. A. Cooper, B. F. Koop, M. L. Rise, A. R. Bridle, M. B. Adams, and B. F. Nowak Transcriptome profiling the gills of amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.): a role for tumor suppressor p53 in AGD pathogenesis? Physiol Genomics, September 14, 2006; 26(1): 15 - 34. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-J. Yu, P. Hogan, and V. Sundaresan Analysis of the Female Gametophyte Transcriptome of Arabidopsis by Comparative Expression Profiling Plant Physiology, December 1, 2005; 139(4): 1853 - 1869. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
