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a Institut National de la Recherche Agronomique, S.C.R.I.B.E., Campus de Beaulieu, 35042 Rennes Cedex, France
b University of Notre Dame, Department of Biological Sciences, Notre Dame, Indiana 46556
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Mature brook trout (300–400 g) were purchased during the reproductive season from a commercial hatchery in Grand Haven, MI, and held under natural photoperiods in 1100-liter tanks supplied with flow-through well water at 12°C. Prior to collecting tissue, the reproductive stage of individual trout was determined by sampling follicles in vivo as previously described [11]. For in vitro incubations with PMA, ovarian tissue was collected from females after vitellogenesis but prior to the resumption of meiosis. Therefore, the ovaries contained fully mature follicles in which the germinal vesicle (nucleus) was approximately four fifths the distance from the center to the periphery of the oocyte. Follicles at this particular stage were chosen for differential-display PCR (DDPCR) analysis since it had previously been shown that they could be stimulated with PMA and A23187 to produce prostaglandins [9]. For Northern analysis of ovarian RNA taken at different reproductive stages, ovarian tissue was collected from females during ovulation (20–100% of the ovary ovulated at the time of sampling) and 48 h, 7 days, and 4–5 wk postovulation. To collect ovarian tissue, trout were overanesthetized in 2-phenoxyethanol and decapitated. Ovaries were removed and either placed in ice-cold Cortland medium [12] for in vitro incubation (PMA/A23187) or frozen and stored in liquid nitrogen before RNA extraction or in situ analysis.
In Vitro Incubation for Phorbol Ester Stimulation
The follicles from both ovaries of each female were dissected in large groups (20–40 follicles) in which the extrafollicular tissue was opened to ensure that all follicles were exposed to the medium. The dissected follicles from both ovaries of a given female were mixed together and then split into two equal portions based on weight. Each portion (approximately 400 follicles) was then incubated in individual 1-liter flasks containing 200 ml of Cortland medium. One of the two flasks received a combined treatment of 0.05 µg/ml PMA (Sigma Chemical Co., St. Louis, MO) and 0.05 µg/ml A23187 (Calbiochem, La Jolla, CA). These agents were initially dissolved at high concentration in dimethylsulfoxide (DMSO), and an aliquot of the DMSO stock was added to the medium to obtain the desired final concentration of the agonist. The DMSO concentration was held constant at 0.5 µl/ml medium. The other flask served as a control and received an equivalent amount of DMSO alone. The flasks were incubated at 12°C for 24 h under intermittent agitation. After incubation, the ovarian tissue was processed for RNA as described below.
RNA Extraction and Poly(A)+ mRNA Isolation
After incubation, the ovarian tissue was de-yolked prior to RNA extraction by pressing the entire tissue between two stainless steel screens while continuously applying ice-cold Cortland medium with a squirt bottle. The de-yolked tissue was immediately homogenized (Polytron; Brinkmann, Westbury, NY) in Tri Reagent (Molecular Research Center, Cincinnati, OH) at a ratio of 50 mg tissue/1.0 ml of reagent. This reagent was used to extract total RNA as previously described [13,14]. The RNA obtained was dissolved in RNase-free water and held at -70°C prior to mRNA isolation. Poly(A)+ RNA were isolated using the PolyAtract mRNA Isolation System (Promega, Madison, WI).
DDPCR
DDPCR was performed on the RNA from 4 incubations in the presence or absence of PMA/A23187 using a kit (RNAmap; GenHunter, Nashville, TN). Prior to DDPCR, total RNA was first treated with DNase (MessageClean; GenHunter) to remove contaminating DNA. The purified RNA was then reverse transcribed using Moloney murine leukemia virus reverse transcriptase and an anchored oligo(dT) primer. The resulting cDNA was then subjected to PCR using the anchor primer and a number of random 10-base pair (bp) primers (AP-1-AP-8; GenHunter) in the presence of [33P]dATP (2000 Ci/mmol; DuPont NEN, Boston, MA). The labeled products were separated on 6% denaturing polyacrylamide gels at 2500 V for 5 h. Gels were blotted without fixing and exposed to x-ray film for 2 days. The film and original gel were aligned, and bands of interest were then excised with a razor blade. Pieces of gel (attached to filter paper) were placed in 2.0-ml microcentrifuge tubes (screw cap) and soaked in 100 µl of distilled water for 10 min. They were boiled for at least 20 min and spun in a microcentrifuge, and the DNA was precipitated in the presence of glycogen using 3 M sodium acetate and absolute ethanol. The samples were centrifuged to pellet DNA and washed, and an aliquot of the DNA was reamplified with the same primer pair that had been used to generate the original DDPCR band. The reamplified PCR product was then separated on an agarose gel, and the band was cut and gel purified (Qiaex II; Qiagen, Chatsworth, CA). The gel-purified DNA was ligated to pCRII (Invitrogen, San Diego, CA), and competent Escherichia coli INV
F' cells (Invitrogen) were transformed with the ligated vector. Positive colonies were screened by PCR using colonies as a template and vector and/or DDPCR primers. Plasmid preparations were then prepared for several clones and sequenced using the dideoxy chain termination method [15] with Cy5-labeled primers (Pharmacia Biotech, Piscataway, NJ) and T7 DNA polymerase. The sequence reactions were separated and analyzed using an ALFexpress Sequencer (Pharmacia Biotech). After confirming that the sequences of several positive clones obtained from the same DDPCR band were identical, two of the clones were thoroughly sequenced on both strands. One of these clones was used as a probe for Northern analysis to substantiate that it truly represented an up-regulated cDNA as observed during the original DDPCR.
Cloning of Full-Length cDNAs
Using ZAP Express (Stratagene, La Jolla, CA), we constructed a library of ovarian cDNAs generated by stimulating trout ovaries with PMA/A23187 for 12 hours, which was then screened using the cDNA from the original DDPCR band. A number of positive plaques were obtained that were then rescreened once to homogeneity. Positives plaques were converted into pBK-CMV (Stratagene) phagemids by in vivo excision, and the phagemids were used to transfect XLOLR cells. The cells were grown on plates containing kanamycin, and resistant colonies were selected and grown for plasmid DNA preparation. From the rescreening, 12 clones were obtained, and 6 were sequenced on both strands as described above. For two of the clones, the central part of the cDNA was amplified by PCR (Advantage cDNA Polymerase Mix; Clontech, Palo Alto, CA) with two different primer sets. Each PCR product was cloned in pCR 2.1 Topo (Invitrogen) and used to transform competent E. coli TOP10 cells (Invitrogen). From this, several clones were fully sequenced as previously described.
Northern Blot Analysis
For Northern analysis, mRNA samples were separated on formaldehyde-agarose gels (1.6% agarose, 2.2 M formaldehyde, and single-strength 3-[N-morpholino]propanesulfonic acid; MOPS). Messenger RNA samples in formazol were mixed 1:1 with a reaction/loading buffer containing 4.2 M formaldehyde, bromophenol blue (0.05 mg/ml), 10% glycerol, and double-strength MOPS and heated at 55°C for 15 min prior to loading. Gels were run at 55 V until the dye front had migrated 8–9 cm. After washing, mRNA was transferred to nylon membranes (MagnaCharge-MSI, Westborough, MA) by downward capillary elution using 20-strength SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.2). After washing, membranes were cross-linked (Stratalinker; Stratagene) and either stored under vacuum or immediately hybridized. Nylon membranes were prehybridized for at least 2 h in roller tubes at 42°C in a buffer containing 5-strength SSPE (0.75 M NaCl, 0.05 M sodium phosphate monobasic, 5 mM EDTA, pH 7.40), 0.1% SDS, 5-strength Denhardt's solution, 50% formamide, and 150 µg/ml sonicated calf thymus DNA. Northern blots were probed with a radiolabeled, double-strand insert obtained by EcoRI excision from pCRII. The insert was labeled with [32P]dATP (> 3000 Ci/mmol; ICN, Costa Mesa, CA) using Klenow fragment and random primer (Prime-It II; Stratagene). The labeled probe was purified using a gel filtration spin column (Centri-Sep; Princeton Separations, Adelphia, NJ) and heat denatured in boiling water for 5 min. After heat denaturation, the labeled probe was added to the prehybridization buffer, and the Northern was incubated at 42°C overnight. After hybridization, Northern blots were washed for 15 min twice at medium stringency (single-strength SSPE, 0.1% SDS, 45°C), followed by two 15-min washes at high stringency (0.1-strength SSPE, 0.5% SDS, 65°C). Northern blots were exposed to phosphorimaging screens (Eastman Kodak, Rochester, NY) and visualized using a Storm 840 phosphorimager (Molecular Dynamics, Sunnyvale, CA). When necessary, radioactive mRNA bands were quantified using ImageQuant (Molecular Dynamics), and the results were analyzed statistically using a Student's test for PMA/A23187 stimulation, or ANOVA followed by Tukey's test to evaluate significant differences in mRNA levels between reproductive stages.
In Situ Hybridization
In situ hybridization was performed on the ovaries of female brook trout taken just after the completion of ovulation. Frozen tissues were stored in liquid nitrogen before they were embedded in histo-prep solution (Fisher Scientific, Pittsburgh, PA). Tissue sections were cut at 30 µm and immediately fixed for 5 min in 4% paraformaldehyde in PBS (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 8.1 mM; KH2PO4, 1.5 mM). Hybridizations were performed using the in situ hybridization and detection system (Gibco, Gaithersburg, MD) according to the instructions of the manufacturer. Briefly, sections were hybridized overnight at 47°C in the presence of vanadyl ribonucleoside complex (Gibco), 20% dextran sulfate, hybridization buffer (Gibco), and biotinylated cDNA probe (9 µg/ml, Bioprime; Gibco). A 374-bp fragment (base pairs 78 to 451) was produced by PCR of the full-length PMA/A23187 up-regulated clone and was used to make the biotinylated probe. A negative control probe was made using pBR322 DNA/RsaI fragments (Bioprime; Gibco). After hybridization, sections were rinsed briefly in 3 changes of 0.2-strength SSC (30 mM sodium chloride, 3 mM sodium citrate, pH 7.0) and for 15 min twice in 0.2-strength SSC. For detection of the probe, tissue sections were incubated for 15 min in 200 µl of blocking solution to ensure specific binding of the streptavidin-alkaline phosphatase conjugate to the hybridized biotinylated probe, and for 15 min in 100 µl of the streptavidin-alkaline phosphatase conjugate solution. Slides were then rinsed twice in TBS (Tris base, 100 mM; sodium chloride, 150 mM; pH 7.5) for 15 min at room temperature and once in alkaline substrate buffer (Tris base, 100 mM; sodium chloride, 150 mM; MgCl2, 50 mM; pH 9.5) for 5 min. Sections were then incubated in alkaline substrate buffer at 37°C with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3 indolylphosphate p-toluidine salt. Slides were checked regularly until the appearance of the hybridization signal in the tissue. The color development was stopped after the same time interval for experimental samples and negative controls, by rinsing the sections in several changes of deionized water.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The full-length TDcR cDNA obtained from DDPCR was 2267 bp in length and contained several minor and one major open reading frame (Fig. 1). The first ATG codon existed at position 7 of the cDNA, and a stop codon was present at position 913. The cDNA contained a very short 5' untranslated region and a 1357-bp 3' untranslated region. The open reading frame presumably encodes a protein of 302 amino acids with a calculated molecular mass of 34034 daltons. On the basis of the amino acid sequence, TDcR was generally similar to members of the tumor necrosis factor (TNF) receptor superfamily and specifically with decoy receptor 3 (DcR3, 35% identity), osteoprotegerin (OPG, 45% identity), osteoclastogenesis inhibitory factor (OCIF, 45% identity), and CD40 (35% identity). The amino terminal region of TDcR contained several cysteine-rich domains (CRDs) (Fig. 2). Within these CRDs, all the cysteines were aligned with those present in the human DcR3 and OPG (Fig. 2).
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Northern Blots of Brook Trout Tissue Probed with TDcR
On all Northern blots of ovarian mRNA probed with TDcR, a single band of 2.2 kilobases (kb) was observed, and this corresponded to the size of the isolated cDNA (Figs. 3 and 4). TDcR mRNA was strongly up-regulated after 12 h of phorbol ester stimulation in the ovaries of all females investigated (Fig. 3). However, after 6 h of incubation with PMA/A23187, the TDcR transcript was up-regulated in only half of the females. Thus, the mean expression level of the 2.2-kb transcript increased significantly only after 12 h of PMA/A23187 stimulation (Fig. 3).
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On Northern blots of ovulatory and postovulatory ovarian mRNA, the expression of the 2.2-kb transcript increased significantly within 24 h of the completion of ovulation (Fig. 4). In contrast, the signal was very low during ovulation. After 48 h postovulation there was a general down-regulation of the transcript, though it did not appear to decrease to ovulatory levels until a number of weeks postovulation.
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On Northern blots of mRNA from other tissues, TDcR was detected only in the testis (Fig. 5). The level of the transcript observed in the testis was comparable to that detected in ovaries during ovulation.
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In Situ Hybridization of Ovarian Tissue Sections with TDcR
In sections of ovarian tissue taken within 24 h of the end of ovulation, TDcR mRNA was strongly detected in the granulosa cells of ovulated follicles (Fig. 6). No hybridization was observed using the control biotinylated probe.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In mammals, TNF receptor family members display no more than 25% homology overall at the protein level [17]. However, TNF receptor family members are grouped together by the presence of conserved cysteine residues in their extracellular domain [17]. In the present study, we isolated a cDNA (TDcR) from the trout ovary presumably encoding for a protein showing similarity with several members of the TNF receptor family. As with other members, these similarities are greatest with the alignment of cysteine residues localized in the ligand-binding domain. Within the TNF receptor family, TDcR is actually most similar to DcR3, OPG, and OCIF. Besides the similarities in the cysteine-rich repeats of the amino-terminal region, TDcR also shares sequence identity with DcR3 in the carboxy terminus and is nearly identical in size. In contrast, no similarities were found between TDcR and the carboxy-terminal portion of any other TNF receptor homologues.
TNF receptor members contain several CRDs defined by the presence of approximately 6 cysteine residues spread within a stretch of 30–40 amino acids in the ligand-binding domain [21]. OPG and DcR3 have 4 CRDs, each containing 4, 6, 4, and 5 cysteine residues [19]. In TDcR, all the cysteine residues could be aligned with those present in OPG and DcR3 (Fig. 2). OPG and DcR3 are soluble TNF receptors lacking an apparent transmembrane domain [18,19]. Similarly, TDcR did not have an apparent transmembrane domain, and thus it is possible that TDcR is also a secreted TNF receptor like OPG and DcR3. Several of the TNF receptor family proteins share homology in a conserved intracellular motif of approximately 80 amino acids that is called the death domain [17]. This domain is associated with the activation of apoptotic signaling pathways. TDcR did not have any regions similar to the death domains of any TNF receptor member. Thus it is likely that TDcR does not have a death domain and this is also the case for OPG and DcR3. Based on sequence similarities with the secreted decoy TNF receptors DcR3 and OPG, the lack of a clear transmembrane domain, and the absence of a death domain, the cDNA we isolated from the trout ovary was tentatively called a trout decoy receptor.
The proposed function of decoy TNF receptors is the inhibition of cell death by the binding of several cytotoxic ligands such as FasL (Fas ligand), LIGHT (herpes virus entry mediator ligand), and TRAIL (TNF-related apoptosis inducing ligand). Thus, certain tumors expressing DcR3 can escape FasL-dependent immune-cytotoxic attack by expressing this decoy receptor that blocks FasL action [19]. DcR3 is also able to suppress LIGHT-mediated apoptosis by is ability to bind LIGHT [20]. OPG also behaves as a decoy receptor and is able to inhibit TRAIL-induced apoptosis by binding the cytotoxic ligand TRAIL [18]. The absence of a death domain in the TDcR amino acid sequence suggests that this protein may also be involved in the inhibition of cell death. However, further investigations are needed to identify what cytotoxic ligand, if any, is able to bind TDcR.
TNF receptor I and II expression is modulated by various activators of PKC such as PMA. In the pulmonary epithelial cell line A549, the mRNA level of TNF receptor I is up-regulated by phorbol ester [22]. In the human histiocytic cell line U-937, PMA up-regulates the 80-kDa transcript of TNF receptor [23]. In contrast, studies on activated T lymphocytes showed a rapid down-regulation of TNF membrane receptors induced by several activators of PKC, including PMA and calcium ionophore [24]. Recently, the mRNA expression of the decoy receptor DcR3 was reported to be up-regulated by PMA/ionomycin in Jurkat T leukemia cells [20]. In the present study we showed that TDcR was also strongly up-regulated by PMA/A23187 in the trout ovary. The exogenous activation of PKC by phorbol esters was shown to affect several ovarian processes in fish, including ovulation [1,2], prostaglandin synthesis [3], and steroidogenesis [4]. However, since TDcR is up-regulated at the completion of ovulation, it is unlikely that it is involved in the process of ovulation.
In mammals, TNF- is involved in several ovarian processes including follicular development and atresia, ovulation, and corpus luteum function [25]. TNF- is one of the main cytokines involved in the process of follicle rupture [26]. In addition, Fas and TNF- receptor are involved in apoptosis of mammalian granulosa cells [27]. In the present study, we clearly demonstrate that TDcR is expressed in granulosa cells of the ovulated trout ovary. This suggests the presence of a TNF/TNF receptor-like interaction in the granulosa cells of postovulatory ovarian tissue. Since TDcR does not have a death domain, it is unlikely that TDcR is an effector of apoptosis in the granulosa cells. However, the presence of a decoy TNF receptor in the ovary just after the completion of ovulation strongly suggests a down-regulation of the action of a TNF-like ligand following ovulation. The temporal expression pattern of TDcR in the ovaries of naturally ovulating brook trout supports this hypothesis. For example, the TDcR level was low during ovulation but increased significantly by 24 h postovulation.
In conclusion, an mRNA was isolated from the trout ovary using DDPCR. This transcript was gonad specific and was up-regulated by phorbol ester stimulation. It was elevated after the completion of ovulation and remained high for 24 h postovulation. The mRNA presumably encodes for a 302-amino acid protein that is homologous with the TNF receptor superfamily, and more specifically with DcR3 and OPG, two decoy TNF receptors. To date, 4 decoy TNF receptors have been identified, all in mammals. Thus, to our knowledge, this is the first description of a member of the TNF receptor family from a lower vertebrate and the first report of a decoy TNF receptor in the vertebrate ovary.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by USDA grant #95-37203-1962. 
2 Correspondence: Frederick William Goetz, Department of Biological Sciences, University of Notre Dame, P.O. Box 369, Notre Dame, IN 46556-0369. FAX: 219 631 7413; goetz.1{at}nd.edu
Accepted: September 17, 1999.
Received: June 17, 1999.
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REFERENCES |
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, 20ß beta-dihydroxy-4-pregnen-3-one and phorbol ester. Gen Comp Endocrinol 1989; 75:454–465.[CrossRef][Medline]This article has been cited by other articles:
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  H.-F. Chen, J.-S. Chen, C.-T. Shun, Y.-F. Tsai, and H.-N. Ho Decoy receptor 3 expression during the menstrual cycle and pregnancy, and regulation by sex steroids in endometrial cells in vitro Hum. Reprod., June 1, 2009; 24(6): 1350 - 1358. [Abstract] [Full Text] [PDF]
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 F. W. Goetz The "ups" and "downs" in Using Subtractive Cloning Techniques to Isolate Regulated Genes in Fish Integr. Comp. Biol., December 1, 2003; 43(6): 786 - 793. [Abstract] [Full Text] [PDF]
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 M. Kusakabe, T. Todo, H. J. McQuillan, F. W. Goetz, and G. Young Characterization and Expression of Steroidogenic Acute Regulatory Protein and MLN64 cDNAs in Trout Endocrinology, June 1, 2002; 143(6): 2062 - 2070. [Abstract] [Full Text] [PDF]
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 J. Bobe and F. William Goetz An Ovarian Progastricsin Is Present in the Trout Coelomic Fluid after Ovulation Biol Reprod, April 1, 2001; 64(4): 1048 - 1055. [Abstract] [Full Text]
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