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Departments of Oncology (R.V., G.E.D.), Biochemistry (R.V., G.E.D.), and Physiology (C.K.C.W., H.D., G.F.W.), University of Western Ontario, and the London Regional Cancer Center (R.V., G.E.D.), London, Ontario, Canada N6A 4L6
Address all correspondence and requests for reprints to: Gabriel E. DiMattia, Ph.D., London Regional Cancer Center, 790 Commissioners Road, London, Ontario, Canada N6A 4L6. E-mail: dimattia{at}julian.uwo.ca
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Introduction |
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STC was regarded as unique to fishes until immunological and biological assays revealed its existence in mammals (8). STC-immunoreactive cells and proteins were first identified in human and rat kidney using antibodies to salmon STC (8). The complementary DNAs (cDNAs) encoding human and mouse STC (mSTC) were isolated shortly thereafter and shown to encode a 247-amino acid protein with a high degree (73%) of sequence similarity to fish STC (9, 10, 11). Like the fish hormone, mammalian STC regulates the renal transport of phosphate through its stimulator effects on NaPi-2 cotransporter activity (7, 9, 12, 13). As in fishes, STC also regulates Ca2+ and phosphate fluxes across the mammalian gut (1, 14). Human STC (hSTC) is also bioactive in fish, suggesting that the receptor-binding domain is conserved (9, 15). It appears that STC has similar functions in both mammals and fish; however, the expression pattern of mSTC implies that a new function(s) has been acquired in mammals (10).
Although STC cDNAs have been isolated from human (9, 10) and mouse (11) tissues, there is no information on gene structure or regulation in mammals or fishes. Therefore, we have characterized and compared the mouse and human STC genes and mapped the overall expression pattern of STC in the mouse. Our findings suggest that the human and mouse genes are highly homologous, but are distinct in important functional respects. Our observations also suggest that STC plays a previously unknown role in mammalian reproduction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FixII
(Stratagene, La Jolla, CA). A total of about 5 x
105 plaque-forming units were plated, transferred to
nitrocellulose membranes (Schleicher & Schuell, Inc.,
Keene, NH), and hybridized with a random primer (Life
Technologies, Grand Island, NY) [
-32P]deoxy-CTP
(3000 Ci/mmol)-labeled hSTC cDNA in 5 x SSC (0.75 M
NaCl and 0.075 M sodium citrate), 40% formamide, 1 x
Denhardt’s solution (0.02% each of Ficoll, polyvinylpyrrolidone, and
BSA), 0.02 M Tris-HCl (pH 7.6), and 0.1% SDS at 42 C, with
final filter washes in 0.5 x SSC and 0.1% SDS at 55 C. Positive
clones were purified to homogeneity using standard methods (16),
subcloned into pBKSII (Stratagene), and analyzed by
restriction enzyme digestion and Southern blot hybridization using
subfragments of the hSTC cDNA to identify exon-containing gene
fragments. These fragments were then subcloned and sequenced using the
T7 Sequenase version 2.0 kit (Amersham, Arlington Heights,
OH) or by an PE Applied Biosystems automated
sequenator. hSTC genomic clones were isolated from a human genomic
library in FIXII (Stratagene) and characterized in the
same manner. Rat STC genomic clones were obtained from a DASHII
library (Stratagene) using a 222-bp mSTC exon 1 fragment
as a probe and were subsequently analyzed as described above to locate
exon 1.
mSTC cDNAs were isolated from a mouse kidney gt11 cDNA library
(Clontech, Palo Alto, CA) as described above. Additional
mSTC cDNA clones were isolated from a mouse ovary cDNA library (Dr.
Jurrien Dean, NIDDK, NIH, Bethesda, MD) constructed in the ZAP
vector (Stratagene). ZAP cDNA clones were excision-rescued
using Exassist helper phage following the manufacturer’s instructions
(Stratagene). cDNA clones encompassing the mSTC-coding
region were generated using RT-PCR from ovary total RNA. RT using
Superscript II (Life Technologies) was performed using an
oligo(deoxythymidine) primer. The mSTC-coding region was amplified
using primers that spanned the region from nucleotide 154
(5'-ATGCTCCAAAACTCAGCAGTGATTC-3') to 973 (5'-ACACTCAAAGTTGGTGTG-3').
The RT-PCR product was subsequently cloned into pBKSII
(Stratagene). The mSTC cDNA consensus sequence was
determined from nine independent cDNA clones, genomic clone exons, and
ovarian STC RT-PCR products. DNA sequence was obtained from both
strands of each clone. Nucleotide sequence comparison of the human and
mSTC was performed using the LALIGN program
(http://genome.eerie.fr/home.html.). The complete mSTC cDNA has been
submitted to GenBank.
Southern and Northern blot analyses
To confirm mSTC gene structure and copy number, mouse DNA (20
µg) was digested with 30 U of each restriction endonuclease, and
Southern blots were prepared as described previously (17).
Hybridization was performed at high stringency using a 815-bp fragment
of mSTC cDNA encompassing the entire protein coding sequence as a
probe. Filters were washed twice for 20 min each time in 2 x
SSC-0.1% SDS at room temperature, then twice for 20 min each time in
0.1 x SSC-0.1% SDS at 65 C. Filters were then exposed to Kodak
XAR-5 film (Eastman Kodak Co., Rochester, NY) with an
intensifying screen at -80 C for 24–72 h.
Northern blots were generated using 50 µg total RNA isolated either from embryos (6–18 days postconception) or 5- to 8-week-old CD-1 mouse tissues. Animals were killed by cervical dislocation, and the following dissected tissues were frozen on dry-ice and stored at -80 C: brain, thymus, heart, lung, kidney, spleen, adrenal, pituitary, bone marrow, liver, stomach, small intestine, large intestine, bladder, prostate, seminal vesicle, ovary, uterus, pituitary, adipose, and skeletal muscle. All tissues were collected separately from each sex. Embryos were dissected from the placenta and stored as described above. Total RNA was purified following the Chromczynski and Sacchi method (18). Embryo RNA was prepared from multiple conceptus, with the exception of that on days 16 and 18, which were prepared from single embryos. Northern blots were hybridized with a random primed, 32P-labeled, 815-bp mSTC cDNA fragment under high stringency conditions, then subjected to autoradiography as described previously (19). The integrity and uniform transfer of RNA were verified by hybridization of each Northern blot with a random primed, 32P-labeled, 400-bp rat cyclophilin cDNA fragment (20). The relative level of STC messenger RNA (mRNA) in ovary and kidney was determined from six different blots using a PhosphorImager SI (Molecular Dynamics, Inc., Sunnyvale, CA). Densitometric analysis of relative STC mRNA levels from autoradiograms was determined using the gel analysis software, Matrix version 2.1 (QuantaVision Canada, Montréal, Canada). The ovary STC mRNA signal was set at 100% because it was the highest expresser, and the levels in all other tissues were determined relative to that in ovary.
Primer extension analysis
A 24-base oligonucleotide complementary to the N-terminal amino
acids, 1–8 of mouse preSTC, (5'-AATCACTGCTGAGTTTTGGAGCAT-3', mSTC-N)
was end labeled with [
-32P]ATP (6000 Ci/mmol) using T4
polynucleotide kinase (New England Biolabs, Inc., Beverly,
MA) according to the manufacturer’s instructions and was separated
from unincorporated isotope with ProbeQuant G-50 microspin columns
(Pharmacia Biotech, Baie d’Urfe, Canada). Labeled
oligonucleotide (1 x 106 dpm) was annealed to 25 µg
total RNA for 90 min at 65 C in hybridization buffer [0.15
M KCl, 0.01 M Tris-HCl (pH 8.3), and 0.001
M EDTA). Yeast transfer RNA (25 µg) was used as a
negative control, and a -32P-labeled 18S primer
(5'-ACCAAAGGAACCATAACTG-3'), when used in place of the mSTC-N primer as
a positive control, produced a 124-bp cDNA as expected. Primer
extension was performed with 20 U Superscript reverse transcriptase
(Life Technologies) for 90 min at 42 C in extension buffer
(0.023 M Tris-HCl (pH 8.3), 10 mM
MgCl2, 5 mM dithiothreitol, 2.25 mg actinomycin
D, 150 mM dNTPs, 0.5 mM KCl, and 0.33
mM EDTA). The reaction mixture was subsequently treated
with ribonuclease A (RNase A; 0.2 U), extracted in
phenol-chloroform-isoamyl alcohol, and ethanol precipitated.
Synthesized cDNAs were resuspended in 40% formamide loading buffer and
resolved on a denaturing 6% polyacrylamide gel. Sequencing reactions
with the same 24-bp primer (mSTC-N) performed on mSTC exon 1 and
5'-flanking DNA were loaded in adjacent lanes for estimation of product
size. The gel was dried, and autoradiography carried out at -80 C with
an intensifying screen and Kodak XAR-5 film for 24–48 h.
Characterization of additional STC 5'-untranslated region (5'UTR)
sequence
As none of the mSTC cDNA clones isolated from cDNA libraries
contained a complete 5'UTR, rapid amplification of 5'-cDNA ends
(5'RACE) and PCR from genomic DNA were performed to amplify that
region. Mouse ovarian polyadenylated mRNA was purified from total RNA
using the PolyATract mRNA isolation system (Promega Corp.,
Madison, WI) following the manufacturer’s instructions. Amplification
of the mSTC 5'UTR was performed using the Marathon 5'/3' RACE system
(Clontech) using an adaptor-specific primer in conjunction
with the gene-specific mSTC-N primer. Three different first strand
synthesis reactions were performed using random hexamers, an
STC-specific primer, or a modified lock-docking oligo(deoxythymidine)
primer (Clontech). PCR from the adaptor-linkered cDNAs
produced for use in 5'RACE was used to amplify the 5'UTR and a portion
of the coding region using two primers spanning the 5'UTR
(5'-CACACACACACACACACACAAATT-3') to nucleotide 510
(5'-CCTCTGGAAAGTCGAACACCTCCG-3') of our mSTC cDNA sequence. PCR was
performed with the Advantage PCR cDNA cloning system
(Clontech) and Taq DNA polymerase (Life
Technologies) under the following conditions: 2.0 µM of each
primer, 200 µM deoxy-NTPs, 1.5 mM Mg2+, and 1
U Taq polymerase in a 50-µl total volume. Each PCR
reaction consisted of an initial denaturation (2 min, 94 C) followed by
30 cycles of denaturation (30 sec, 94 C), annealing (30 sec, 56 C),
elongation (1 min, 72 C), and a final elongation (10 min, 72 C).
Amplified products of the predicted size (510 bp) were subcloned using
the TA cloning kit (Clontech) into pCR2.1 and sequenced as
described above.
PCR amplification of the 5'UTR from genomic DNA was carried out using 0.5 U Taq DNA polymerase (Life Technologies) and mSTC-V primer (11) (5'-CAGCAGCCGCCTGCCAGCCAGCCAGC-3') with mSTC-N according to the manufacturer’s instructions. Genomic DNA for use as PCR templates was isolated from the BALB/C, CD-1, and C57BL/6J x DBA/2J strains of mice as previously described (21). After the PCR reactions (30 cycles; annealing temperature, 56 C), products were size fractionated on 1% agarose gels, and primary products were isolated using the QiaexII DNA isolation kit (Qiagen, Mississauga, Canada), subcloned using a TA cloning kit (Invitrogen, San Diego, CA), and sequenced.
PCR from genomic and cDNA templates was performed using a variety of DNA polymerase enzymes containing 3',5'-exonuclease activity, including Expand DNA polymerase (Boehringer Mannheim, Québec, Canada), ID-Proof (ID-Labs, London, Canada), and KlenTaq-1 (Clontech), using annealing temperatures of 56–58 C. The primary PCR product was subcloned and sequenced as described above.
In situ hybridization
An 815-bp cDNA fragment containing 247 amino acids of mSTC was
used to generate sense and antisense riboprobes as previously described
(22). The plasmid was linearized with restriction enzymes for the
synthesis of sense and antisense RNAs to a specific activity of
2–4 x 108 dpm/mg RNA with [35S]UTP
according to the manufacturer’s instructions (Pharmacia
Biotech, Piscataway, NJ). Probe size was estimated to be full
length by electrophoresis in 6% polyacrylamide gels. In
situ hybridization was then performed as described for the
localization of STC mRNA in rat kidney (22). Briefly, after proteinase
K treatment, tissue sections were prehybridized for 2 h at 50 C in
50% formamide containing 0.2 M sodium chloride, 20
mM Tris-HCl (pH 8.0), 1 mM EDTA, 10 x
Denhardt’s solution, and 50 µg/ml denatured salmon sperm DNA.
Hybridization reactions were carried out in the same buffer solution
containing 10% dextran sulfate, 500 µg/ml yeast transfer RNA, 20
mM dithiothreitol, and 0.1 µg labeled complementary RNA
probe/ml solution. Single stranded, nonspecifically bound riboprobe was
digested in 20 µg/ml RNase A and 10 U/ml RNase T1 for 30 min at 37 C,
followed by washing in 2 x SSC four times for 15 min each time at
22 C and in 0.1 x SSC at 65 C for 30 min. The slides were
dehydrated in graded ethanols containing 0.3 M ammonium
acetate and dipped in Amersham LM-1 emulsion (diluted 1:1
with 0.3 M ammonium acetate). Emulsion-coated slides were
stored at 4 C in light-tight boxes containing silica gel as desiccant.
Slides were developed (Kodak D-19) and counterstained with
hematoxylin.
Immunocytochemistry (ICC)
The antiserum employed for ICC was generated in rabbits against
recombinant hSTC and has been characterized for specificity by ICC and
Western blot analysis (9, 12, 22, 23). The staining procedure involved
pretreatment of tissue sections with 10% normal goat serum in diluent
buffer (10 mM Tris, pH 7.5, containing 150 mM
NaCl) to reduce nonspecific staining, followed by an overnight
incubation at 4 C with antiserum (1:1000 dilution). The sites of
antibody binding were then visualized with the ABC glucose oxidase
detection system according to the manufacturer’s instructions
(Vector Laboratories, Inc., Burlingame, CA), which
produces a blue precipitate. Slides were washed three times for 15 min
each time in the diluent buffer after each reagent application. The
immunostained slides were counterstained with methyl green, dehydrated,
and mounted under coverslips. Control procedures included the
application of preimmune rabbit serum or antiserum preabsorbed with
hSTC in lieu of antiserum alone.
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Results |
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along with the genomic
clones used to reconstruct each of the genes and their intron-exon
junction sequences. A comparison of their DNA and protein sequences is
shown in Fig. 2. The close structural
similarities between the human and mSTC genes are immediately apparent
from Fig. 1. Both are composed of four exons that are nearly identical
except for the UTRs: exon 1 encodes the 5'UTR and the first 39 residues
(118 bp) of preSTC, exon 2 contains 48 residues (144 bp), exon 3
encodes 71 residues (213 bp), and exon 4 codes for the last 89 amino
acids as well as the 3'UTR in both genes. These exons were separated by
introns of 1.9 kb (intron A), 0.68 kb (intron B), and 6.1 kb (intron C)
in the human gene. By comparison, the introns in the mouse gene were
1.83, 0.6, and 6.35 kb, respectively. These findings are also
summarized in Fig. 1B, where it can be seen that the intron/exon
junctions are identical, and the splice donor-acceptor sites are very
similar in both species. These junction sequences are essential for PCR
amplification of specific exons for the identification of sequence
changes that may correlate with naturally occurring mouse mutant
phenotypes that map to the chromosomal region of the STC gene. The
intronic sequences in the 2 species diverged markedly in the regions
between the splice donor-acceptor sites (data not shown). The composite
mSTC cDNA sequence obtained from overlapping cDNAs, isolated from
different cDNA libraries and by 5'RACE, was in agreement with the mouse
genomic sequence, confirming that the mouse gene is composed of 4 exons
that are transcribed to produce a single transcript.
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. There was 91% identity over the
protein-coding region at the DNA level and 96% identity at the protein
level. In total, nine amino acid substitutions were found, most of
which were conservative, except for Glu
His at position 244 in the
mouse. All the cysteine residues that participate in the dimerization
of the STC monomer (15) were conserved in both species, as was the
N-linked glycosylation consensus sequence at residues 62–64
(Asn-Ser-Thr).
The long 3'UTR of hSTC mRNA (8) is surprisingly well conserved between
human and mouse, showing 83.5% homology. The 3'UTR of mSTC was deduced
from six overlapping cDNAs isolated from ovary and kidney libraries. It
proved to be slightly longer than that of the human gene (2919
vs. 2858 bp), such that exon 4 was 3190 bp in the mouse and
3125 bp in the human. Thereafter, the 3'-flanking sequences of human
and mouse STC diverge completely about 35 bp downstream of exon 4 (Fig. 2).
The transcription start site of the hSTC gene was ascertained by primer
extension analysis using RNA from the human fibrosarcoma cell line,
HT1080, and the human endometrial stromal cell line, St-2 (24), both of
which contain the highest levels of steady state STC mRNA of 30
different cell lines tested (data not shown). Using a primer extending
from the initiator methionine, 2 major cDNA products were obtained from
HT1080 RNA, yielding 5'UTRs of 153 and 271 nucleotides (Fig. 3A), whereas St-2 RNA produced only the
153-nucleotide product (data not shown). Primer extension experiments
using different mouse tissue RNAs reproducibly yielded a cDNA product
(153 bp) denoting a 5'UTR identical in length to the smaller of the 2
seen for the human gene (Fig. 3B). Sequence analysis of about 1 kb of
hSTC 5'-flanking DNA showed that the 5'UTR is contiguous with the exon
1 coding sequence (Fig. 4). In addition,
the proximal 5'-region of the hSTC gene proved to be rich in the
trinucleotide repeat, CAG, with 19 such repeats clustered within 102
nucleotides of the proximal transcription start site. This same region
represents 5'UTR sequence relative to the distal transcription start
site 271 nucleotides 5' of the initiator methionine (Fig. 4).
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); however, this region was not repeated in our genomic clone 6B
or the rSTC genomic clones. In fact, the rSTC 5' UTR was 95% identical
to the mSTC sequence in that region (data not shown). Furthermore, our
analysis of 5'RACE and genomic PCR products encompassing this region of
the mSTC gene clearly showed that the 36-nucleotide repeat was not
present in the mSTC 5'UTR (data not shown).
A comparison of the rSTC 5'UTR and flanking DNA to that of human (867
bp) showed 70% nucleotide identity (Fig. 4). The CAG trinucleotide
repeats in this region (described above) were not precisely conserved,
because in the rat the repeats were found in two blocks of 6 and 18
nucleotides separated by 3 CAA repeats. The human gene contained 3
shorter, interrupted blocks of CAG repeats. Interestingly, the rSTC
5'-flanking DNA contains a 62-bp CA dinucleotide repeat not found in
the human sequence that began 311 bp upstream of the putative start
site (Fig. 4). These repeats are found in the promoters of many genes
and constitute regions that can affect DNA structure and modulate gene
expression (24, 25).
mSTC gene expression pattern
The expression pattern of the STC gene in adult mice is shown in
Figs. 3B and 5. Northern analysis of
total tissue RNA showed measurable levels of expression in ovary,
prostate, bladder, kidney, adrenal, lung, heart, uterus, and pituitary
(Fig. 5, A and B). Two transcripts of about 4 and 2 kb were seen in
ovary, but only the larger transcript was detectable in most other
tissues. The sensitivity of primer extension was such that gene
activity was seen in all tissues, except for spleen and seminal vesicle
(Fig. 3B); however, these data were not quantitative and therefore were
not directly comparable to the Northern blot data presented in Fig. 5A.
The expression pattern between male and female nonreproductive tissues
did not differ significantly (data not shown). However, there was a
distinct difference in the level of expression in reproductive tissues.
The highest levels of steady state mRNA by Northern blot were found in
the ovary, where the level of expression was 5- to 10-fold higher than
that in the kidney (the next highest expressing tissue; Fig. 5A). In
contrast, STC expression in male reproductive tissue (prostate and
testes) was markedly lower. Thus, the STC gene was expressed in a
near-ubiquitous manner in adult mouse, with the highest levels of
expression occurring in ovary (Fig. 5B). STC mRNA was also readily
detectable in whole embryo RNA from days 6–18 postconception,
indicating that the gene is highly expressed throughout embryogenesis
(Fig. 5C).
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). Figure 6 shows low power
darkfield micrographs of the mouse ovary after hybridization to sense
(Fig. 6a) and antisense probes (Fig. 6b). The dense signal over the
islands of interstitial cells (double arrow) and theca
interna cells (single arrow) bordering the follicles is
readily apparent in Fig. 6b. Whether all cells of theca interna express
STC is presently unknown, because it consists of theca cells, smooth
muscle cells, as well as connective and fibroblastic elements that are
not readily distinguishable with conventional in situ
hybridization techniques. Correlative in situ hybridization
and immunohistochemistry with other thecal cell-specific markers will
help clarify this question. In contrast, the corpora lutea and
developing follicles did not contain significant STC mRNA. Figure 6
shows one group of follicles under brightfield (Fig. 6c) and darkfield
(Fig. 6d) illuminations; it can be seen that STC mRNA is absent in
oocytes and follicular granulosa cells.
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, e and f). Interstitial cells and theca interna
cells both contained high levels of immunoreactive STC (Fig. 6f), as
expected in view of their high STC mRNA levels. However, STC protein
was also observed in several cell types exhibiting little or no STC
gene expression. For example, corpus luteal cells in some cases
contained extremely high levels of immunoreactive STC, as did the
oocytes of secondary and tertiary follicles (Fig. 6f). On the other
hand, the primary follicles and granulosa cells were devoid of STC
staining (Fig. 6f). The use of nonimmune serum or antiserum preabsorbed
with hSTC abolished staining in all cell types (Fig. 6e). |
Discussion |
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The CAG triplet repeats in the hSTC gene are of interest because of the growing number of genetic diseases associated with unstable G+C-rich repeats, such as Huntington’s disease (32), spinobulbar muscular atrophy (33), and myotonic dystrophy (34, 35). It is noteworthy that CAG trinucleotide repeat expansion can have deleterious effects in both coding (36) and noncoding (35) regions of a gene. Consequently, it is tempting to speculate that the CAG trinucleotide repeats in the STC gene may be susceptible to expansion, which, in turn, may alter its expression pattern and lead to a disease state. In the case of hSTC, the triplet repeats are relatively small, consisting of 3–6 repeats separated by 6–15 nucleotides; however, the sequence that interrupts the CAG triplets is GC rich, consisting of 6 CGG triplets, which translates to an overall GC content of 72%. The rSTC gene also contains the CAG repeat region in a different configuration: (CAG)6-(CAA)3-(CAG)18. The fact that the CAG repeats are conserved implies that this region of the mammalian STC gene may represent a transcriptional control domain, as seen for the CGG triplet repeat in the 5'UTR of the FMR1 gene (fragile X syndrome), which when hypermethylated leads to a loss of gene expression (38). The CAG repeat-rich region immediately upstream of the hSTC proximal transcription start site and the putative rSTC initiation site may represent a transcriptional control region functioning in the absence of an obvious TATA box that is usually located about 30 nucleotides 5' of the mRNA start site. The closest TATA box is 39 nucleotides upstream of the hSTC distal transcription start site and could therefore function in facilitating transcription from that site. In contrast, transcription initiation from the downstream site in the hSTC gene may be considered TATA-less, as would the rat gene start site, because both are about 155 bp downstream of the nearest consensus TATA box. Interestingly, the human endometrial stromal cell line, St-2, which contains STC mRNA levels equivalent to those in the HT1080 line, generated a single primer-extended product indicative of proximal transcription start site usage only. The functional significance of these start sites with regard to cell-specific gene regulation remains to be determined. Additionally, these findings provide the unique opportunity to assess what effect the CA repeat of the rodent STC gene might have on the basal and cell-specific expression of hSTC through promoter studies in vitro and in vivo where the rodent CA repeat is placed in the hSTC promoter. The sequence 5' and 3' of the rSTC CA repeat is highly conserved with the human gene and should facilitate the interpretation of such studies with regard to the functional contribution of the CA repeat. These analyses of the mammalian STC gene provide the framework for future studies, using it as a model gene to examine ovarian interstitial-specific transcriptional regulation. The delineation of critical cis-active elements governing gene expression in the ovarian stroma will facilitate transgenic ectopic expression experiments designed to provide new information regarding ovarian development and interstitial cell function.
Our analysis of STC gene expression in the mouse revealed that the highest levels of mRNA were found in ovary, followed by nearly equivalent levels in kidney, bladder, prostate, adrenal, heart, and lung. However, in contrast with an earlier study in which a high level of expression was reported in spleen, we were unable to detect a transcript in spleen even by primer extension (11). The two transcripts we observed in ovary are probably due to alternative usage of polyadenylation signals in the long 3'UTR, because we did not detect alternative splicing of coding exons among the various STC cDNAs isolated and sequenced. In addition, RT-PCR amplification of the coding region of mSTC using primers bracketing the mSTC-coding region (154–968 nucleotides) produced a single cDNA product of the expected size, 815 bp. This also suggests that the mSTC gene is not alternatively spliced within the coding region of the gene. The pattern of STC gene expression in humans, as deduced by Chang et al. (10), showed that STC mRNA was widespread, with the highest levels appearing in ovary, prostate, and thyroid. However, Olsen et al. (9) found the highest levels in kidney and barely detectable levels in other tissues, but did not examine ovary. The discrepancies are not surprising given the fact that the tissue RNAs came from a variety of sources and from donors who varied in age and health, and were most likely extracted from different regions of the organs in question. These factors may have also contributed to differences in STC expression pattern in the human and the mouse.
Steady state STC mRNA levels during development were examined in mouse whole embryo RNA from different days of gestation and were higher than those in all individual adult tissues except ovary. Whether STC gene expression is ubiquitous or confined to a few embryonic tissues is presently under investigation. However, the fact that STC mRNA was so readily detected in whole embryos implies that the protein serves a unique role in development.
The high level of STC gene expression in the ovary suggests that STC has an important role in mammalian reproduction. To identify cellular sources of STC production, we carried out immunocytochemistry and in situ hybridization analyses on normal cycling ovaries, and the findings were particularly intriguing. STC protein and gene expressions were observed in the secondary interstitial and theca interna cells, whereas only protein was seen in oocytes and corpus luteal cells. There are two possible explanations for this difference. The first is that our in situ procedure was insufficiently sensitive to detect extremely low levels of message in oocytes and luteal cells. The second possibility is that oocytes and luteal cells do not express the gene and are instead targets of STC that have sequestered the hormone. Indeed, there are several precedents for this phenomenon. The intracellular sequestering of secreted proteins has been reported in the case of corticosteroid-binding globulin and basic fibroblast growth factor (37, 38) and more recently for STC. In the kidneys, STC is synthesized and secreted by the collecting ducts and accumulates in proximal tubules, a site where we have been unable to find any evidence of gene expression and yet which is known to be a target of the hormone (9, 12, 22). These findings in kidney have led us to conclude that STC can be sequestered by its target cells, and the present findings concur by showing that developing oocytes and corpus luteum are targets of thecal and/or interstitial cell-derived STC in ovary. Currently, functional data defining a role for STC in the ovary are lacking, yet it is interesting to note that all sites of gene and protein expression are LH regulated.
The thecal cells that express the STC gene are often described as having been recruited from the ovarian stroma closely apposed to the developing follicle (39) that differentiate into thecal cells by an unknown mechanism. Both the thecal and secondary interstitial cells are distinguished by their ability to produce androgen in response to stimulation by LH. In this respect, it is noteworthy that the pattern of STC mRNA distribution was identical to that described in rat ovary for cytochrome P450c17a(C17–20-lyase) (40), an enzyme essential for androgen production. The enzyme is part of the requisite pathway for supplying testosterone to the granulosa cells in the developing oocyte. The androgens are then converted into estrogen by the granulosa cell aromatase complex to support follicular growth. The apparent production of STC in the same cells indicates that STC may play a role in androgen synthesis or secretion and/or maintenance of the follicle in the context of the stroma, forming the structural framework of the ovary. There is evidence to support a mineral regulatory function for STC in mammals analogous to fish in both kidney (9, 12, 22, 23) and intestinal epithelium (7, 14). In ovary, calcium has been shown to be important in several processes, including the response of granulosa cells to androsteinedione (41), the dissociation of the surface epithelium of the mature follicle during ovulation (42), the resumption of meiosis in quiescent oocytes (43), and potentially the regulation of blood flow to the ovary (44). The possibility that STC plays a role in regulating calcium availability to support any of the mechanisms described above is under active investigation.
The apparent sequestration of STC in luteal cells and oocytes suggests that it may be a new signaling molecule in the ovary. Our studies have provided the foundation to more accurately define a role for STC in mammalian reproduction through its potential effects on luteal cell function and oocyte maturation. Equally intriguing will be the investigation of how and when STC is taken up by corpora lutea and oocytes. Clinically, the study of STC levels in women with polycystic ovary disease is of considerable interest, because hypertrophy of ovarian stroma is one of its pathological hallmarks, and the expression of STC by stromal cells may be altered by this disease process.
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Acknowledgments |
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Footnotes |
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Received March 30, 1998.
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References |
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phage to generate
plaques. In: Ausubel FA, Brent R, Kingston RE, Moore DD, Seidman
JG, Smith JA, Struhl K (eds) Current Protocols in Molecular Biology.
Wiley and Sons, New York, pp 1.11.1–1.11.3
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  A. C.-M. Chang, J. Hook, F. A. Lemckert, M. M. McDonald, M.-A. T. Nguyen, E. C. Hardeman, D. G. Little, P. W. Gunning, and R. R. Reddel The Murine Stanniocalcin 2 Gene Is a Negative Regulator of Postnatal Growth Endocrinology, May 1, 2008; 149(5): 2403 - 2410. [Abstract] [Full Text] [PDF]
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 G. Dole, E. E Nilsson, and M. K Skinner Glial-derived neurotrophic factor promotes ovarian primordial follicle development and cell-cell interactions during folliculogenesis Reproduction, May 1, 2008; 135(5): 671 - 682. [Abstract] [Full Text] [PDF]
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 C. Chen, M. S. Jamaluddin, S. Yan, D. Sheikh-Hamad, and Q. Yao Human Stanniocalcin-1 Blocks TNF-{alpha}-Induced Monolayer Permeability in Human Coronary Artery Endothelial Cells Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 906 - 912. [Abstract] [Full Text] [PDF]
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 A. Chakraborty, H. Brooks, P. Zhang, W. Smith, M. R. McReynolds, J. B. Hoying, R. Bick, L. Truong, B. Poindexter, H. Lan, et al. Stanniocalcin-1 regulates endothelial gene expression and modulates transendothelial migration of leukocytes Am J Physiol Renal Physiol, February 1, 2007; 292(2): F895 - F904. [Abstract] [Full Text] [PDF]
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L.-J. Xiao, J.-X. Yuan, X.-X. Song, Y.-C. Li, Z.-Y. Hu, and Y.-X. Liu Expression and regulation of stanniocalcin 1 and 2 in rat uterus during embryo implantation and decidualization. Reproduction, June 1, 2006; 131(6): 1137 - 1149. [Abstract] [Full Text] [PDF]
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 G. Song, F. W. Bazer, G. F. Wagner, and T. E. Spencer Stanniocalcin (STC) in the Endometrial Glands of the Ovine Uterus: Regulation by Progesterone and Placental Hormones Biol Reprod, May 1, 2006; 74(5): 913 - 922. [Abstract] [Full Text] [PDF]
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 S. Wu, Y. Yoshiko, and F. De Luca Stanniocalcin 1 Acts as a Paracrine Regulator of Growth Plate Chondrogenesis J. Biol. Chem., February 24, 2006; 281(8): 5120 - 5127. [Abstract] [Full Text] [PDF]
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 A. C.-M. Chang, J. Cha, F. Koentgen, and R. R. Reddel The Murine Stanniocalcin 1 Gene Is Not Essential for Growth and Development Mol. Cell. Biol., December 1, 2005; 25(23): 10604 - 10610. [Abstract] [Full Text] [PDF]
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H. Y. Yeung, K. P. Lai, H. Y. Chan, N. K. Mak, G. F. Wagner, and C. K. C. Wong Hypoxia-Inducible Factor-1-Mediated Activation of Stanniocalcin-1 in Human Cancer Cells Endocrinology, November 1, 2005; 146(11): 4951 - 4960. [Abstract] [Full Text] [PDF]
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 C. P. Hasilo, C. R. McCudden, J. R. J. Gillespie, K. A. James, E. R. Hirvi, D. Zaidi, and G. F. Wagner Nuclear targeting of stanniocalcin to mammary gland alveolar cells during pregnancy and lactation Am J Physiol Endocrinol Metab, October 1, 2005; 289(4): E634 - E642. [Abstract] [Full Text] [PDF]
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