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a Department of Zoology,
b Faculty of Science; Department of Physiology, Faculty of Medicine and Dentistry; University of Western Ontario, London, Ontario, Canada N6A 5C1
ABSTRACT
The stanniocalcin (STC) gene was recently found to be widely expressed in fish. In this study, we have characterized ovarian STC in the rainbow trout (Oncorhynchus mykiss) and cloned the ovarian cDNA. The STC gene expression was highest in early stage oocytes and diminished progressively as oocytes developed. At the cellular level, ovarian STC gene expression was most abundant in the ooplasm of early stage oocytes, but it was also weakly evident in the theca layer, interstitial cells, and vitellogenic oocytes. The STC protein was distributed in a pattern similar to that of gene expression but was also apparent in glycoprotein vesicles, nuclei, multivesicular bodies, and follicles undergoing atresia. Cloned cDNAs obtained from the corpuscles of Stannius (CS) and ovarian transcripts were nearly identical. However, Western blotting of the partially purified proteins revealed that ovarian STC was larger than CS STC. Further analysis revealed that ovarian STC had a much larger N-linked carbohydrate moiety (
12 kDa) compared to CS STC (7 kDa), indicating that the two hormones were differentially posttranslationally modified. To our knowledge, this is the first characterization of STC gene expression, cDNA, and protein distribution in the piscine ovary and the first evidence for any difference between alternative sources of the hormone in any species.
calcium, oocyte development, ovary
INTRODUCTION
Stanniocalcin (STC) is a homodimeric glycoprotein hormone that regulates calcium and phosphate homeostasis in fish [1–5]. It was originally identified in endocrine glands known as the corpuscles of Stannius (CS), where it is synthesized and secreted into the bloodstream in response to elevations in serum Ca2+. In the gill and gut, STC reduces Ca2+ uptake, whereas in the kidney, it stimulates phosphate reabsorption [6–8]. It is by these three mechanisms that STC prevents hypercalcemia in fish.
Originally, the STC gene was believed to be uniquely expressed in the CS glands of fish, because all attempts to detect STC elsewhere were unsuccessful. However, the recent discovery that the mammalian STC gene is widely expressed prompted the reexamination of this issue in fish. With the use of more sensitive probing techniques, we previously found that the fish gene was also widely expressed. In particular, the kidneys, testes, and ovaries all produce readily detectable STC transcripts [9].
In mammals, STC is produced abundantly in the ovary, where gene expression is discretely localized to the theca-interstitial cell layers. Once synthesized, mammalian STC appears to be secreted and targeted to developing oocytes and corpus lutea [10]. During pregnancy and lactation, gene expression is upregulated as much as 15-fold [11], and only during these physiological conditions can STC be detected in the serum. Whereas mammalian STC seems to have a conserved role in regulating calcium and phosphate homeostasis in the kidney and intestine [12–14], its precise roles in ovarian physiology and reproduction in general remain unknown.
Because of the high expression of STC in the mammalian ovary and its recent discovery in the fish reproductive system, this study focused on characterizing ovarian STC in fish. We have compared the proteins and cDNAs encoding the CS and ovarian forms of the hormone, considering the possibility that ovarian STC might not only have a different structure but also a different role. Furthermore, by identifying the sites of STC mRNA and protein localization in the fish ovary, we have set the stage for ultimately determining its role in fish reproduction.
MATERIALS AND METHODS
Animals and Tissue Preparation
For RNA extraction, in situ hybridization (ISH), immunocytochemistry (ICC), and histology, rainbow trout were obtained from Rainbow Springs hatchery in Thorndale, Ontario, Canada, between January and April 1999. Tissues were either immediately frozen on dry ice for RNA extraction or fixed in 4% (w/v) paraformaldehyde containing 0.15 M NaCl and 0.01 M NaPO4 (pH 7.4) for subsequent histological studies. For large-scale extraction of ovarian STC protein, ovaries were collected from 18- to 24-mo-old rainbow trout, kindly supplied by the Cole-Monroe fish processing plant in St. Thomas, Ontario, Canada, approximately 8 h postmortem, frozen on dry ice, and stored at -70°C until use.
Gene Expression Analysis by Northern Blotting
Total RNA was isolated from rainbow trout ovaries using Trizol (Life Technologies, Burlington, ON, Canada) according to the manufacturer's instructions and stored at -70°C until use. Northern blotting was performed as previously described [10] with minor changes. In brief, 30 µg of RNA per lane were loaded for all stages except the perinucleolar and late vitellogenic stages, for which, due to low RNA levels, RNA pooled from four animals was used, loading 7 µg/lane. Blots were prehybridized in Ambion ULTRAhyb (Ambion, Austin, TX) at 42°C for 2 h before the addition of a random prime 32P-labeled salmon STC cDNA probe encoding base pairs (bp) -44 to 1015 of the published sequence [15] encompassing the entire protein-coding region. The blot was probed overnight in ULTRAhyb at 42°C and washed under high stringency as described [10]. The same hybridization protocol was followed to determine 18S RNA levels using an 18S RNA probe. The STC:18S RNA ratios were determined using Image Master VDS quantitation software version 2.0 (Amersham Pharmacia Biotech, Baie d'Urfé, PQ, Canada).
Tissue Embedding and In Situ Hybridization
In preparation for in situ hybridization (ISH), ovarian tissue was fixed, embedded, and sectioned according to standard methods [16]. Sense and antisense digoxigenin-uridine triphosphate (Roche Scientific, Laval, PQ, Canada)-labeled riboprobes were synthesized by in vitro transcription of the coding region of the Coho salmon STC cDNA according to the manufacturer's directions. In preparation for probing, ovarian tissue sections were dewaxed and rehydrated in a descending ethanol gradient. To expose RNA for hybridization, slides were subjected to two treatments of 2 min each with 2x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate), followed by one 10-min digestion with 20 µg/ml of proteinase K. Slides were then washed in 2x SSC and refixed in 4% (w/v) paraformaldehyde. Nonspecific binding of the probe to positive amino acid groups was reduced with a 0.25% acetic anhydride treatment, followed by a wash in 2x SSC. Tissues were then dehydrated using an increasing gradient of ethanol and air-dried. RNase guard (1 µl [37.7 U] per 300 ml) was added to all solutions except those containing ethanol and xylene.
Hybridization was performed overnight at 50°C in 0.5 M NaCl, 0.03 M Tris-HCl, 0.75 mM EDTA, 1.5x Denhardt solution, 15% (w/v) dextran sulfate, and 15 mM dithiothreitol (DTT), all dissolved in 50% (v/v) deionized formamide in sealed boxes containing the hybridization buffer solution without probe to prevent desiccation of probe to sections. Riboprobes were applied at a concentration of 1–2 ng/µl in hybridization buffer. The next day, slides were washed for 15 min in 2x SSC at 50°C, followed by two washes of 1x SSC at 50°C. Tissues were then treated with RNase to degrade any unbound probe, first by rinsing them twice in RNase buffer (final concentration: 0.01 M PIPES, 0.05 M NaCl, and 0.1% (v/v) Tween 20 [pH 7.2]) for 5 min each time at 37°C, followed by digestion in 20 µg/µl of RNase (in the same buffer) at 37°C twice for 15 min each time. Following RNase treatment, the sections were rinsed twice in 1x Tris-buffered saline (TBS) for 5 min each time. For immunological detection, slides were blocked with 3% (v/v) normal sheep serum (NSS), and digoxigenin (DIG) was probed using anti-DIG IgG at a dilution of 1:250 with 3% NSS at 4°C overnight. The DIG color detection was finally performed on the third day using NBT/BCIP (Roche) with levamisole following a wash in 1x TBS (pH 9.5) as directed for 1–5 h at room temperature as necessary. Finally, sections were dehydrated, mounted, and photographed. For ISH, Northern blotting, and ICC, oocytes were classified into six stages based on the scheme described by Bromage and Cumaranatunga [17].
Immunocytochemical Localization of STC Protein
A polyclonal antiserum against salmon STC [18] was used for immunocytochemical localization of STC protein on adjacent tissue sections using the avidin-biotin peroxidase method (Vectastain; Vector Laboratories, Inc., Burlingame, CA) as previously described [11]. Controls included the application of nonimmune rabbit serum and primary antiserum preabsorbed with STC (30 µg/ml) in lieu of primary antiserum alone.
Extraction and Partial Purification of Ovarian STC
Ovarian STC protein was purified on concanavalin A (ConA) sepharose as previously described for CS STC with minor modifications [2, 4]. Ovarian tissue was homogenized in 10 volumes of ice-cold, 50 mM acetic acid using a blender and then a Polytron homogenizer (Brinkman Instruments, Rexdale, ON, Canada) to obtain a uniform liquid homogenate. The homogenate was centrifuged at 14 000 x g for 30 min at 4°C, and the supernatant was filtered through Whatman #1 filter paper (Whatman International Ltd., Maidstone, England). The pellet was re-extracted twice as described above, and the supernatants were pooled and lyophilized. The lyophilized crude extract was dissolved in ice-cold ConA-binding buffer (50 mM Tris-HCl, 0.5 M NaCl, 1 mM CaCl2, and 1 mM MgCl2) at a concentration of 1 mg/ml, filtered through Whatman #1 filter paper, and passed three times through a 2.5- x 25-cm column of ConA sepharose maintained at 4°C. The column was washed extensively with ConA buffer before elution of bound proteins with 0.5 M methyl 
-D-mannopyranoside. Protein-containing fractions (as determined by A280) were pooled, dialyzed against 0.01 M ammonium bicarbonate, filtered through Whatman #1 filter paper, and lyophilized as described above. The ovarian ConA-bound fraction was dissolved in 50 mM Tris-HCl/50% glycerol and stored at -20°C. Yields were approximately 20 mg of protein per gram of tissue (wet weight). The CS STC was prepared in the same manner. Void fractions of the ConA column did not contain any immunoreactive STC.
Western Blotting
Protein samples were subjected to SDS-PAGE using 12% discontinuous polyacrylamide gels [18]. Proteins were electroblotted onto a polyvinylidene fluoride membrane (Life Technologies) for immunodetection using enhanced chemiluminescence (ECL; Roche) according to the manufacturers' directions. The primary salmon STC antibody [19] was applied overnight at a dilution of 1:20 000 at room temperature. A donkey anti-rabbit horseradish peroxidase-linked secondary antibody at a dilution of 1:20 000 (Amersham Pharmacia Biotech) was applied the following day for 30 min at room temperature. Blots were extensively washed with 1x Tris-buffered saline containing Tween (TTBS) between antibody treatments and before detection by ECL.
Reverse Transcription-Polymerase Chain Reaction, Cloning, and Sequence Analysis of STC Transcripts
Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA isolated from CS and ovarian tissues using Trizol as described above. To prepare the template for the RT reaction, 1 µg of oligo-dT as added to 10 µg of RNA and incubated for 10 min at 70°C. The reaction was placed on ice for 5 min to anneal the primer to the template. First-strand synthesis was initiated by adding the primer/template preparation to reverse transcriptase buffer at a final concentration of 50 mM Tris-HCl (pH 8.3 at room temperature), 75 mM KCl, 3 mM MgCl2, 0.01 M DTT, 0.5 M deoxyribonucleotide triphosphates, and 10 U/µl of Superscript II reverse transcriptase (Life Technologies) in a total volume of 40 µl. The solution was mixed, centrifuged briefly, overlaid with paraffin oil, and incubated for 90 min at 43°C. Following first-strand synthesis RT, the product was incubated at 95°C for 5 min to destroy the Superscript II enzyme.
Amplification of the cDNA was performed by adding 1 µl of the RT reaction to the PCR buffer at a final concentration of 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.8% Nonidet P40, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 0.2 mM 5' primer, and 0.2 mM 3' primer in a total volume of 100 µl. All primer pairs spanned five introns, ensuring that any contaminating DNA, if amplified, would be a different size than the expected product. A hot-start strategy was employed by adding 2.5 U of normal recombinant Taq polymerase (MBI Fermentas, Burlington, ON, Canada) to the above-described PCR mix during the first cycle at 70°C. The cycle profile consisted of 5 min for initial denaturing, 40 cycles of 95°C for 30 sec, 60°C annealing for 30 sec, 72°C extension for 1 min, and a 10-min final extension. One forward primer, (fSTC44–65F) 5'-CCT GTC CAA CCT ATC CCA TCG-3', and three reverse primers, (fSTC1015–996R) 5'-TGT GTG TCA GTG TGC GTG TGA-3', (fSTC1766–1746R) 5'-CCT GAC TAG CAC ACA TTC C-3', and (fSTC1875–1856R) 5'-CGT TTA ATC ACC AGT GAG TCT CGA C-3', were used to amplify the ovarian and CS cDNAs.
The RT-PCR products were subjected to agarose gel electrophoresis, excised from the gels, and isolated using a Sephaglas BandPrep kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Purified RT-PCR products were ligated into pGEM-T plasmid vectors (Amersham Pharmacia Biotech) and cloned according to the manufacturer's directions in DH5 subcloning efficiency competent cells (Life Technologies). Plasmid amplified cDNAs were purified using miniprep kits (Qiagen, Mississauga, ON, Canada) and sequenced on an Applied Biosystems automated sequencer at the Robart's Research Institute sequencing facility in London, Ontario, Canada. In addition to the SP6 and T7 promoters in the pGEM-T vector, two sequencing primers were used, (fSTC488F) 5'-CAG TAG ACT GGA CAT CT and (fSTC525R) TGA AAGAAC AGT TGA CAG TAT CGT GC, where F and R indicate forward and reverse, respectively. Sequences were analyzed using the Baylor College of Medicine ClustalW 1.8 multiple sequence alignment program (http://dot.imgen.bcm.tmc.edu:9331/multi-align/multialign.html).
RESULTS
Comparative Analysis of STC Gene Expression During Oocyte Development
The STC gene expression was examined in the rainbow trout ovary during the course of oocyte maturation by Northern blotting. Levels of STC mRNA were highest during early oocyte development and then diminished to undetectable levels by late vitellogenesis (Fig. 1). The STC mRNA levels were eightfold higher during the perinucleolar stage (stage 2) than the vesicular stage (stage 3), dropping to less-than-detectable levels in all subsequent stages of development. Stages were assigned based on histological examination of ovary sections.
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Characterization of CS and Ovarian STC Proteins and Carbohydrate Moieties
Western blotting of ovarian and CS STC revealed marked differences between the relative sizes of the two. Ovarian STC was evident as a single, approximately 44-kDa product, which was some 5 kDa larger than the main CS STC product, and as a doublet approximately 39 kDa in size. A smaller and less abundant product of approximately 23 kDa was also visible in the CS STC lane.
Because CS STC has a carbohydrate moiety attached at a single consensus N-linked glycosylation site (at residues Asn29-Ser30-Thr31 of the mature protein core), the glycosylation patterns of CS and ovarian STC were compared. To this end, protein extracts from each tissue were subjected to peptide-N-glycosidase F (PNGase F) digestion to remove N-linked carbohydrate moieties before Western blotting (Fig. 2). Ovarian STC proved to be much more highly glycosylated than CS STC, because it underwent a molecular weight reduction of approximately 12 kDa following deglycosylation as compared to a reduction of only approximately 7 kDa for the CS hormone. The deglycosylated protein cores were approximately the same size (32 kDa). As a control, the same incubation procedure was applied to the extracts in the absence of enzyme and demonstrated no discernable differences (data not shown).
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Localization of Ovarian STC Gene Expression and Protein Distribution by ISH and ICC
To characterize STC gene expression and protein distribution at the histological level, sections of trout ovary were subjected to ISH and ICC (Fig. 3). Both STC and STC mRNA were abundant in the ooplasm of small perinucleolar oocytes during early development (Fig. 3, A and B). In perinucleolar oocytes, STC immunoreactivity and mRNA appeared to be uniformly distributed in the ooplasm (Fig. 3, C and D). The STC gene expression was considerably lower in larger mature oocytes, namely the vesicular and vitellogenic stages (Fig. 3E). In contrast to the weak STC gene expression in large oocytes, theca, and interstitial cell layers (Fig. 3E), STC immunoreactivity remained relatively intense (Fig. 3F). Remarkably, STC protein was widely distributed in structures such as nuclei, multivesicular bodies, and glycoprotein vesicles, none of which was correlated with the pattern of gene expression (Fig. 3H). Controls consisted of sense riboprobes and preimmune serum in lieu of primary antibody and yielded no specific hybridization or staining, respectively (Fig. 3, A and B). An additional control for ICC included a preincubation of the primary antibody with excess STC, which also produced no staining (data not shown). A summary of the relative mRNA and protein levels during oocyte development are provided in Table 1.
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Comparison of CS and Ovarian cDNA Sequences
Having observed obvious differences in the carbohydrate moieties of the CS and ovarian forms of STC, we proceeded to clone and sequence the cDNAs to determine if differences in primary structure also existed. The strategy used to amplify and clone the ovarian and CS cDNAs is provided in Figure 4. Ovarian (GenBank accession no. AF326317) and CS STC (GenBank accession no. AF326318) transcripts exhibited only a few minor differences at the nucleotide level and were identical at the deduced amino acid level (Fig. 5). The most notable differences between ovarian and CS sequences were in the 3' untranslated region (UTR), where the ovarian transcript contained a 6-bp insert between bp 887–888 of the CS sequence. In comparison to the published Coho salmon CS cDNA sequence, the trout sequence contained a 50-bp insert in the 3' UTR between bp 1646–1647 of the published salmon cDNA. All three sequences were identical at the deduced amino acid level.
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DISCUSSION
This study was prompted by the recent discovery that the STC gene is, in fact, widely expressed in fish and not confined to the CS glands as was previously accepted. In this paper, we provide the first characterization, to our knowledge, of STC in one of these novel sites, the ovary. Our interest in piscine STC in the ovary stems not only from its recent discovery but also from the mammalian STC gene being highly expressed in this tissue [10]. A comparative study of the hormone in the two systems should enhance our understanding of its function in both.
The findings presented here suggest that the patterns of gene expression and protein distribution change dramatically in the fish ovary in accordance with the developmental stage. This study also establishes that CS and ovarian STC are differentially posttranslationally processed, with ovarian STC being substantially larger than the CS-derived hormone. This is due to differential N-linked glycosylation, as revealed by PNGase F digestion. Ovarian STC proved to be more highly glycosylated, shifting by approximately 12 kDa following carbohydrate digestion as compared to only approximately 7 kDa for the CS form of the hormone. This alternative glycosylation pattern may be the basis for differences in the biology of CS and ovarian STC with respect to both function and mechanism of action. Indeed, in numerous cases, the carbohydrate moiety of a ligand has been shown to be of biological significance. These include prolactin, hCG, and FSH, which have altered clearance kinetics, potency, signal transduction, secretory characteristics, and target specificity solely on the basis of differing degrees of glycosylation [20–22]. Consequently, the degree of glycosylation could also have great importance to the physiology of STC.
Having established posttranslational differences between the two proteins, the cDNAs were compared to determine if any differences existed in the open reading frame of the primary transcript. This proved not to be the case, because the CS and ovarian STC cDNAs amplified by RT-PCR displayed more than 99% nucleotide sequence identity, and the deduced amino acid sequences were identical. Both trout STC cDNAs were also identical to Coho salmon STC [15] in the protein coding region and in the 26 bp of 5' UTR sequenced. Of the few notable differences observed, most occurred exclusively in the 3' UTR. In particular, a number of insertions were found in the trout sequences compared with the Coho sequence. It would thus appear that the STC 3' UTR is less conserved than the protein coding region and 5' UTR among salmonids. Other differences included a single nucleotide substitution in the coding region of one of three CS clones, resulting in a single amino acid change, and an AC repeat in the 3' UTR of the ovarian cDNA sequence. Both these differences may be due to alternatively expressed alleles or, more likely, amplification errors, because they were only found in one of three clones. Thus, the minor sequence differences observed may not be of biological significance.
The Northern blotting data indicated that STC gene expression was highest during early oocyte development, an observation fully supported by the ISH results. Transcripts were most abundant in perinucleolar (stage 2) oocytes, decreased sharply by the vesicular stage (stage 4), and were less than the limits of detection by late vitellogenesis (stage 6). This expression pattern for STC correlates with the normal expression pattern of several other mRNAs within developing trout oocytes. These include the vitellogenin receptor, ß-actin [23], and cathepsin D [24]. In general, RNA levels in lower vertebrate oocytes reach a maximum early during the first meiotic arrest [25, 26], which corresponds to the high STC levels found at the perinucleolar stage in rainbow trout.
The STC transcripts localized by DIG ISH appeared to be largely confined to the ooplasm of early stage oocytes. Hybridization was also evident to a lesser extent in middle-stage vesicular oocytes, but by late vitellogenesis, little or no evidence of STC mRNA was found. The low RNA levels observed in these larger oocytes were likely due to decreased transcription, because overall gene expression was quite low, as indicated by 18S hybridization signal on Northern blots. In the theca and interstitial layers surrounding oocytes, there also appeared to be very low levels of gene expression. This is in marked contrast to the mammalian ovary, where STC is expressed almost exclusively in these cell types [10]. Thus, the pattern of STC gene expression was consistent with other mRNAs in the fish oocyte, but the source of the expression differed drastically from what occurs in the mammalian ovary.
The STC protein distribution was examined by ICC and was evident during all stages of oocyte development examined (stages 1–6; chromatin nucleolar to late vitellogenic inclusive). For the most part, STC was uniformly distributed throughout the ooplasm of small developing oocytes. However, in some instances, the hormone localized to the peripheral ooplasm, multivesicular bodies (or Balbiani bodies), and cell nuclei. Protein localization to such diverse structures suggests that STC may be involved in a number of processes. Balbiani bodies, for instance, are believed to be associated with formation of organelles such as mitochondria, the Golgi apparatus, and the endoplasmic reticulum [27, 28]. In larger vesicular and early vitellogenic oocytes, STC localized to the ooplasm between the lipid droplets that are characteristic of these stages. Because this was long after gene expression had diminished, it appears that STC is somehow stabilized in the ooplasm or is still produced de novo from low levels of transcript. In amphibians, it has been observed that low levels of transcription occur in late vitellogenic oocytes [29]; thus, the same may be true of STC in fish.
The mammalian ovary also contains high levels of STC in oocytes [10, 11]. Thus, although the site of synthesis differs, it seems that in both vertebrate classes, STC protein is accumulated in abundance in the oocyte. In mammals, STC protein levels appear to be relatively constant through oocyte development, but in fish, STC is clearly much more abundant in small oocytes. Because of the importance of calcium and phosphate in reproduction and the defined role of STC in mineral homeostasis, it seems reasonable to propose that STC may also somehow regulate the levels of these minerals in the piscine ovary. Moreover, because of the variety of structures to which STC localizes, it may also play a part in some additional processes.
The pattern of STC gene expression and protein localization appears to be uncorrelated with the levels of serum gonadotropins in fish. In the trout, oocyte development occurs independently of these hormones until the first meiotic arrest or, roughly, the perinucleolar stage (stage 2). Whereas GTHI, the piscine homologue of FSH, is present throughout the reproductive cycle, it peaks at the time of vitellogenic yolk accumulation [30]. This is much later than the time of maximal STC gene expression and protein accumulation. Moreover, GTHII does not become important for oocytes until final maturation, much in the same fashion as the LH surge before ovulation in mammals. At this time (late vitellogenesis; stage 6), STC gene expression was undetectable.
Our findings do little to elucidate the relationship, if any, between the ovarian and CS forms of the hormone. For example, one issue that needs to be addressed is whether ovarian STC is released from oocyte stores locally or systemically. Until quite recently, CS was accepted as the sole source of STC in fish. However, previous studies have shown that adult trout contain multiple molecular weight forms of STC, and it is yet to be proven that all of these derive from CS [31]. Furthermore, it is also noteworthy that serum STC immunoreactivity is not completely abolished following surgical removal of the CS glands [32]. It is thus feasible that tissues such as the ovary provide alternative sources of STC that may be released into the blood. This may either counteract the loss of CS STC or serve a function distinct from that of the CS-derived hormone. In the case of ovarian STC, it may signal other tissues involved in regulating reproductive development. Finally, whereas the source of bloodborne forms of STC remains debatable, the origin of ovarian STC seems to be more defined. Despite high circulating levels of CS STC (1–2 nM) [31], the high level of gene expression and unique size of ovarian STC suggest that most, if not all, of the protein found in the ovary is locally produced.
In this report, we have presented evidence for differential posttranslational processing of ovarian STC and provided, to our knowledge, the first characterization of STC protein distribution and gene expression in the fish ovary. Collectively, the evidence suggests that the biology of ovarian STC is considerably different than that which has been established in the CS glands, and that ovarian STC may be a heretofore unknown hormone involved in the reproductive biology of fish.
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ACKNOWLEDGMENTS
We would like to thank Faridah Sadat for assistance with ISH and ICC as well as Mark Paciga and Kathi James for critically reviewing the manuscript. We also thank Cole-Monroe for kindly supplying the rainbow trout tissue used for protein purification.
FOOTNOTES
First decision: 13 March 2001.
1 Supported by the Canadian Institute of Health Research and the Natural Sciences and Engineering Council of Canada grants to G.F.W. 
2 Correspondence. FAX: 519 661 3827; graham.wagner{at}med.uwo.ca
Accepted: April 12, 2001.
Received: February 27, 2001.
REFERENCES
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  M. Paciga, E. R. Hirvi, K. James, and G. F. Wagner Characterization of big stanniocalcin variants in mammalian adipocytes and adrenocortical cells Am J Physiol Endocrinol Metab, August 1, 2005; 289(2): E197 - E205. [Abstract] [Full Text] [PDF]
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 C.-W. Luo, K. Kawamura, C. Klein, and A. J. W. Hsueh Paracrine Regulation of Ovarian Granulosa Cell Differentiation by Stanniocalcin (STC) 1: Mediation through Specific STC1 Receptors Mol. Endocrinol., August 1, 2004; 18(8): 2085 - 2096. [Abstract] [Full Text] [PDF]
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 C. R. McCudden, K. A. James, C. Hasilo, and G. F. Wagner Characterization of Mammalian Stanniocalcin Receptors. MITOCHONDRIAL TARGETING OF LIGAND AND RECEPTOR FOR REGULATION OF CELLULAR METABOLISM J. Biol. Chem., November 15, 2002; 277(47): 45249 - 45258. [Abstract] [Full Text] [PDF]
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 M. Paciga, A. J. Watson, G. E. DiMattia, and G. F. Wagner Ovarian Stanniocalcin Is Structurally Unique in Mammals and Its Production and Release Are Regulated through the Luteinizing Hormone Receptor Endocrinology, October 1, 2002; 143(10): 3925 - 3934. [Abstract] [Full Text] [PDF]
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