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Centre de Recherche en Reproduction Animale and Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6
Address all correspondence and requests for reprints to: Dr. Jean Sirois, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6. E-mail: siroisje{at}medvet.umontreal.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase/C17–20 lyase (P45017)
messenger RNAs (mRNAs) in vivo in equine preovulatory
follicles isolated during hCG-induced ovulation. Two distinct P450AROM
complementary DNAs (cDNAs) were isolated from an equine
preovulatory follicle cDNA library. One clone was 2682 bp in length and
included 115 bp of 5'-untranslated region (UTR), 1509 bp of open
reading frame encoding a well conserved 503-amino acid protein, and
1058 bp of 3'-UTR. Its 5'-most region represented the equine homolog of
exon 1f, previously designated brain specific. The other cDNA clone
encoded a truncated protein and contained a distinct 5'-UTR
characteristic of transcripts derived from promoter II, previously
identified as the predominant ovarian mRNA. Northern blot analyses were
performed using preovulatory follicles obtained during estrus between
0–39 h after the administration of hCG and with corpora lutea isolated
on day 8 of the estrous cycle (day 0 = day of ovulation). The
results showed a biphasic regulation of P450AROM mRNA expression:
levels were highest in follicles at 0 h post-hCG, decreased
significantly during the ovulatory process at 12 and 24 h
(P < 0.05), and increased again between 30–39 h
post-hCG and in corpora lutea. When oligonucleotides specific for
P450AROM mRNA variants were used as probes, a novel switching
phenomenon was observed. Promoter II-derived transcripts accounted for
the message present in follicles at 0 h post-hCG and in corpora
lutea, whereas promoter 1f-derived mRNA was expressed exclusively
during the ovulatory process (30–39 h post-hCG). Levels of P45017
mRNA were high in follicles at 0 h, but significantly decreased
after hCG treatment (P < 0.05), with lowest levels
in follicles at 36 and 39 h post-hCG and in corpora lutea.
Northern blots performed on isolated cellular preparations revealed
that P450AROM and P45017 transcripts were localized exclusively in
granulosa cells and theca interna, respectively. Equine aromatase
promoters II and 1f were cloned from a genomic library, and putative
transcription start sites were characterized by primer extension
assays. Sequence analyses identified distinct potential regulatory
elements in each promoter. Thus, this study identifies a novel
aromatase promoter-switching phenomenon in equine granulosa cells
during follicular luteinization and provides a new model in which
aromatase promoter switching is induced in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tissue-specific distribution of several aromatase transcripts has been linked in part to the use of different promoters (7, 9, 10, 12, 13, 18, 19, 20, 21). In humans, these promoters direct the synthesis of distinct aromatase messenger RNA (mRNA) variants that differ only by their 5'-noncoding termini. At least nine 5'-untranslated first exons, and thus mRNAs variants, have been identified, including exons I.1, I.2, I.3, I.4, I.5, I.6, PII, 2a, and 1f (22). They are alternatively spliced into a common 5'-splice acceptor site found 38 bp upstream of the translation start site in exon 2 and generate transcripts that have distinct, but overlapping, tissue distribution (7).
Increasing evidence suggests that switching of aromatase expression from one mRNA variant to another may be a key regulatory mechanism in several physiological and pathological processes. Distinct aromatase transcripts are expressed in early vs. midpregnancy in porcine placenta (12, 13, 23) as well as in fetal vs. adult human liver (24). Several studies have shown that a similar switch occurs in healthy vs. cancerous human breast adipose tissue (24, 25, 26, 27, 28, 29). It has been speculated that a switching mechanism may be involved in the ovulation/luteinization process (24). However, results from the study of Jenkins et al. (30) did not support this concept, as only promoter II-derived aromatase transcripts were detected in human follicles and corpora lutea.
One key function of P450AROM is to produce large amounts of estradiol
in mammalian preovulatory follicles (31), and the obligatory role of
estrogen synthesis in female reproduction was recently highlighted in
mice by targeted disruption of the CYP19 gene (32). In contrast to that
in other species, the molecular control of follicular steroidogenesis
in mares has remained largely uncharacterized. Yet, the equine
preovulatory follicle offers a good model for the study of ovarian gene
expression because it has a relatively large size (40–50 mm in
diameter), and its development can be precisely monitored in
vivo by ultrasound imaging (33, 34). Therefore, the objectives of
this study were to clone and determine the primary structure of equine
P450AROM, characterize the regulation of P450AROM and P450
17-hydroxylase/C17–20 lyase (P45017) mRNAs in a series of equine
preovulatory follicles isolated during hCG-induced ovulation, and
determine the cellular localization of each transcript.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]deoxy (d)-ATP, [-32P]dCTP,
[
-32P]ATP, and [35S]dATP were obtained
from Mandel Scientific NEN Life Science Products (Mississauga, Canada);
TRIzol total RNA isolation reagent, RNA ladder (0.24–9.5 kb),
synthetic oligonucleotides, and culture media were purchased from
Life Technologies (Gaithersburg, MD); T4 polynucleotide
kinase and all sequencing reagents were purchased from Pharmacia Biotech (Baie D’Urfé, Canada); Kodak film X-Omat AR was
obtained from Eastman Kodak Co. (Rochester, NY);
electrophoretic reagents were purchased from Bio-Rad Laboratories, Inc. (Richmond, CA); Lutalyse was obtained from
UpJohn (Kalamazoo, MI); hCG was purchase from The Buttler Co.
(Columbus, OH).
Cloning of equine cytochrome P450AROM and P45017 complementary
DNAs (cDNAs)
The equine P450AROM and P45017 cDNAs were cloned from an
expression library prepared from an equine preovulatory follicle
isolated 36 h after the administration of an ovulatory dose of hCG
(2500 IU), as previously described (35). Approximately 100,000 phage
plaques were screened, and hybridization was performed at 55 C with
QuikHyb hybridization solution (Stratagene). The probes,
including a 5', 1.0-kb EcoRI restriction fragment of the rat
P450AROM cDNA (36) and a 2.0-kb XhoI restriction fragment of
the bovine P45017 cDNA (37), were labeled with
[-32P]dCTP using the Prime-a-Gene labeling system
(Promega Corp.) to a final specific activity greater than
1 x 108 cpm/µg DNA. Positive clones were plaque
purified through secondary and tertiary screening, and pBluescript
phagemids containing the cloned DNA insert were excised in
vivo with the Ex-Assist/SOLR system (Stratagene). DNA
sequencing was performed by the Sanger dideoxy nucleotide chain
termination method (38) using the T7 Sequencing Kit (Pharmacia Biotech), vector-based primers (T3 and T7), and custom
oligonucleotide primers (Life Technologies, Inc.).
Nucleotide and amino acid analyses were performed using MacDNASIS
software (version 2.0, Hitachi, Hialeah, FL) and the FASTA program
of Wisconsin Package (version 9.0, Genetics Computer Group, Madison, WI).
Isolation of equine preovulatory follicles and Northern blot
analysis
Ovarian follicular development in Standardbred and Thoroughbred
mares was monitored daily by ultrasonography (33). When the
preovulatory follicle reached 35 mm in diameter during estrus,
ovulation was induced with hCG (2500 IU, iv). The ovary bearing the
presumptive preovulatory follicle was removed via colpotomy 0, 12, 24,
30, 33, 36, and 39 h post-hCG with a chain ecraseur (n = 4–5
follicles/time point) (35). Also, three corpora lutea were isolated on
day 8 of the estrous cycle using the same approach. The recovered ovary
was kept in ice-cold Eagles’s MEM supplemented with penicillin (50
U/ml)-streptomycin (50 µg/ml; Life Technologies, Inc.),
L-glutamine (2.0 mM; Life Technologies, Inc.), and nonessential amino acids (0.1 mM;
Life Technologies, Inc.). Preovulatory follicles were
dissected into preparations of follicle wall (theca interna with
attached granulosa cells) and isolated theca interna and granulosa
cells, as described (35). All samples were stored at -70 C until RNA
extraction. Animal procedures were approved by the institutional animal
use and care committee.
RNA was extracted from equine tissues using TRIzol (Life Technologies, Inc.) and a Kinematica PT 1200C Polytron
Homogenizer (Fisher Scientific, Fairlawn, NJ). For
Northern analysis, RNA samples (10 µg) were denatured at 55 C for 15
min in denaturing buffer, electrophoresed on a 1.2% agarose, and
transferred by capillarity to a nylon membrane, as previously described
(35). A ladder of RNA standards was run with each gel, and ethidium
bromide (10 µg) was added to each sample before electrophoresis to
compare RNA loading and determine the migration of standards.
Hybridization was performed using the QuikHyb solution
(Stratagene) and the following cDNA probes: a 0.5-kb
EcoRI fragment of the equine P450AROM cDNA (clone A17), a
2.0-kb EcoRI/HindIII fragment of the equine
P45017 cDNA, and the rat elongation factor Tu cDNA (EFTu) as a
control gene for RNA loading and transfer (39). Each cDNA was labeled
using the Prime-a-Gene labeling system as described above, and
stripping of hybridization signal was achieved by soaking filters in
0.1% SSC (standard saline citrate)-0.1% SDS for 30 min at 100 C.
To study the specific regulation of promoter II- and promoter
1f-derived aromatase transcripts in granulosa cells, two
oligonucleotides complementary to unique sequences located at the
5'-end of each transcript were labeled using the DNA 5'-End Labeling
System (Promega Corp.), and used as probes in Northern
blot analysis. They included a 24-mer antisense oligonucleotide
5'-GTCTGCTGGTCAC TTCTAGTTTCC-3' complementary to nucleotides 50 and 73
in promoter 1f-derived transcript (clone A17; Fig. 1
), and a 23-mer oligonucleotide
5'-CCAAAAGGTACAT CTAGGACTCC-3' complementary to nucleotides 5 and 27 in
promoter II-derived P450AROM transcript (clone A1; Fig. 1).
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). To clone the aromatase promoter 1f, the same genomic phage plaques
were screened with a radioactive DNA fragment generated by PCR and
corresponding to the first 77 bases of the equine aromatase promoter
1f-derived cDNA (clone A17; Fig. 1). The latter probe was generated by
inclusion of 0.5 mCi [-32P]dATP (6000 Ci/mmol) in a
standard PCR reaction mixture (40) and by using clone A17 as template
DNA, sense primer 5'-TTCCTAACAGCCGTGCATCATTAG-3', and antisense primer
5'-CTGAGTCTGCTGGTCACTTCTAGT-3'. The DNA fragment was purified by
electrophoresis on a 2% agarose gel. Six exon II- and three
exon 1f-containing clones were isolated from the initial round and were
purified through secondary and tertiary screening. After digestion with
SacI, Southern blot analyses were performed using
oligonucleotide probes corresponding to the 5'-end of each equine
aromatase transcript (same oligonucleotides described for Northern
blots). Fragments yielding a positive signal were subcloned into the
pBluescript plasmid vector (Stratagene) and sequenced
using a T7 Sequencing Kit (Pharmacia Biotech) and custom
oligonucleotide primers (Life Technologies, Inc.).
Primer extension analysis
Primer extension analyses were performed in aqueous
buffer, as previously described (35). To determine the putative
transcription start site of the promoter II-derived transcript, the
primer extension assay used RNA extracted from a corpus luteum (50
µg; day 8 of cycle), a tissue known to contain high levels of
transcripts, and a 30-mer antisense oligonucleotide
5'-GGCGAAGCAATGTAAAGGCCTGTGGAA ATC-3' corresponding to the region
located between +51 and +80 bp from the beginning of clone A1 (Fig. 1b). RNA isolated from spleen served as a negative control. The
putative transcription start site of the promoter 1f-derived aromatase
transcript was determined using RNA extracted from granulosa cells of a
preovulatory follicles isolated 30 h post-hCG (30 µg) and a
30-mer antisense oligonucleotide (5'-GGCCTGAGTCTGCTGGTCACTTCTAGTTTC-3')
corresponding to the region located between +51 and +80 bp from the
beginning of clone A17 (Fig. 1b). RNA isolated from spleen and corpus
luteum served as the negative control. The extension products were
analyzed by electrophoresis on a 6% polyacrylamide-7 M
urea gel, and the putative sites of transcription initiation were
determined by comparisons with adjacent sequencing reactions that used
the same oligonucleotides as primers and two corresponding aromatase
genomic clones that contained these regions as templates.
Statistical analysis
Relative levels of P450AROM and P45017 mRNAs were quantified
by densitometric analysis of autoradiogram bands using a
computer-assisted image analysis system (Collage Macintosh program,
Fotodyne, Inc., New Berlin, WI). The EFTu signal was also
quantified and used to normalize results. Data were expressed as ratios
of P450AROM to EFTu and P45017 to EFTu (n = 4 follicles/time
point). One-way ANOVA was used to test the effect of time after hCG on
relative levels of P450AROM and P45017 mRNAs. When ANOVAs indicated
significant differences (P < 0.05), Dunnett’s test
was used for multiple comparisons with the control (0 h post-hCG).
Statistical analyses were performed using JMP software (SAS Institute, Inc., Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNAs). Clone A17 was 2682
bp in length and included a 5'-untranslated region (UTR) of 115 bp, an
open reading frame of 1509 bp, and a 3'-UTR of 1058 bp. The open
reading frame encoded a 503-amino acid protein that included all
conserved features characteristic of cytochrome P450AROM, such as a
membrane-spanning region, an I helix, a heme-binding region, as well as
a domain encompassing a putative cAMP-dependent protein kinase
phosphorylation site (Fig. 1). The 5'-end of clone A17 (bases 1–77;
Fig. 1B) was found to be the equine homolog of aromatase exon 1f, often
designated as brain specific (21, 41, 42, 43). Clone A17 was designated as
a promoter 1f-derived aromatase transcript. In contrast, the 5'-end of
clone A1 (bases 1–61; Fig. 1B) represented the region immediately
upstream of exon II. This latter region has previously been cloned by
RT-PCR from equine tissues (9), and is known to be the primary mRNA
species present in granulosa cells of several species (9, 18, 30, 36).
Clone A1 was designated a promoter II-derived transcript. However,
clone A1 was short and encoded a truncated 347-amino acid protein
lacking important 3'-structural elements such as the heme-binding
domain (data not shown). Database homology searches showed that
homologous sequences ceased after the splice junction between exons 8
and 9, suggesting that the clone may be a splice variant or an artifact
of the cloning process.
Twelve P45017 clones were isolated from the equine cDNA library
using a bovine homologous probe. Partial DNA sequencing of one selected
clone showed that it was identical to an equine testicular P45017
cDNA previously characterized (data not shown) (44).
Regulation of equine P450AROM and P45017 mRNAs in preovulatory
follicles
Changes in levels of aromatase mRNA during the
ovulation-luteinization process were studied by Northern blots using a
cDNA probe common to both transcripts. Results showed a biphasic
pattern of aromatase expression after gonadotropin treatment (Fig. 2). High levels of aromatase mRNA were
detected in walls of follicles isolated before hCG (0 h; Fig. 2A), but
a marked drop in transcripts was observed 12 and 24 h post-hCG
(P < 0.05). Then, aromatase levels increased again
between 30 and 39 h post-hCG and were elevated in mature equine
corpora lutea (Fig. 2A). To assess the cellular localization of the
aromatase message within the follicle wall, Northern blots were
performed on isolated preparations of granulosa cells and theca
interna. Results showed that expression of aromatase transcripts was
restricted to the granulosa cell layer and followed a pattern similar
to that observed in follicle wall (Fig. 3). The size of the P450AROM mRNA was
approximately 3.0 kb in most samples, with the exception of those
isolated before hCG treatment (0 h post-hCG), where transcripts of
about 4.0 and 1.2 kb were also observed. No aromatase transcripts were
detected in theca interna (Fig. 3).
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mRNA in equine preovulatory
follicles and corpora lutea was studied by Northern blots using the
same membranes. Levels of P45017 mRNA were high in follicles at
0 h, but significantly decreased after hCG treatment
(P < 0.05), with lowest levels in follicles at 36 and
39 h post-hCG and in corpora lutea (Fig. 2B). When Northern blots
were performed with isolated cellular preparations, no P45017 mRNAs
were detected in granulosa cells, except for a relatively weak signal
in a few samples, probably resulting from contamination by theca
interna cells (Fig. 3). In contrast, P45017 expression was high in
theca interna of follicles isolated between 0–33 h post-hCG, but
dropped markedly thereafter (P < 0.05; Fig. 3).
Differential expression of promoter II- and 1f-derived aromatase
transcripts
The potential regulation of two distinct aromatase mRNAs in
granulosa cells was studied using mRNA-specific, end-labeled
oligonucleotide probes representing either promoter II- or promoter
1f-derived transcripts. The results suggested the presence of
successive aromatase promoter-switching events during follicular
luteinization and corpus luteum formation. Levels of promoter
II-derived transcripts were high before hCG treatment (0 h), dropped
drastically after hCG treatment, and remained very low before ovulation
(Fig. 4A). Conversely, promoter
1f-derived mRNAs were undetectable at 0 h, but were induced
between 30–39 h post-hCG (Fig. 4B). Interestingly, the process
reversed itself after ovulation, as promoter 1f-derived mRNA
disappeared and promoter II-derived transcripts reappeared in the
corpus luteum (Fig. 4). The summation of promoter II- and promoter
1f-derived aromatase mRNAs seemed to represent the overall
aromatase expression detected using a nonspecific probe (Fig. 3).
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and 6). The equine aromatase promoter II
contains a highly conserved adrenal 4 binding protein/steroidogenic
factor-1 (SF-1) element involved in cAMP-dependent and -independent
regulation in granulosa cells of other species (45, 46, 47) (Figs. 5 and 7A). However, a cAMP response element
(CRE)-like region known to bind cAMP-binding protein (CREB) in
rat and human (45, 48), but not in bovine (49), ovarian tissues is not
conserved in the horse (Fig. 7B). Alignment of the first 250 bases of
the equine promoter 1f with the corresponding human and mouse regions
shows a remarkable level of homology among species (90% and 96%
identity between equine and murine, and equine and human sequences,
respectively). This region features several perfectly conserved
elements, notably overlapping c-myc and GRE consensus sites.
Although genomic clones ranged in size from about 12–25 kb, no single
clone was found to include both promoters, indicating that these equine
aromatase promoters are separated by at least several thousand bases as
reported in humans (7).
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and 8). This cytosine residue was positioned
16 bp upstream of the first base of the aromatase cDNA clone A1 (Fig. 1), indicating that the total length of the 5'-UTR of promoter
II-derived transcripts is 115 bp. Primer extension analysis of promoter
1f-derived transcripts identified a major putative transcription
initiation site at a cytosine residue located 31 bp downstream of a
consensus TATA box (Figs. 6 and 8). The criteria used to select this
band as a major site was the presence of a strong signal with the
granulosa cell RNA extract (which contains promoter 1f-derived mRNA)
and the absence of a signal with the corpus luteum extract (which does
not contain promoter 1f-derived mRNA; Fig. 4). The cytosine residue was
located 16 bp upstream of the first base of the aromatase cDNA clone
A17, giving an overall length of 93 bp for exon 1f and a total length
of 131 bp for the 5'-UTR of promoter 1f-derived transcripts. Longer,
but less abundant, extension products were observed with promoter
1f-derived transcripts (data not shown). These products were apparently
transcribed downstream from two additional TATAA elements found in the
promoter 1f at positions -87/-92 and -111/-116 (+1 = major
putative transcription initiation site). These TATA boxes are also
conserved in the mouse and human promoter 1f (21, 41), but their
relative functional significance has not been determined.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The elevated expression of promoter II-derived aromatase transcripts in equine follicles before hCG treatment is in keeping with previous reports showing that it is the predominant mRNA species in granulosa cells (18, 30, 36, 49). Also, the selective localization of aromatase mRNA in granulosa cells agrees with the immunohistochemical localization of the protein in equine follicles (55, 56), and the steroidogenic capacities of isolated equine follicular cells in vitro (57), thus resolving a previous controversy on the site of estrogen biosynthesis in the equine follicle (34). This study reports the cloning and characterization of the equine aromatase promoter II. Some of the molecular mechanisms involved in the regulation of this promoter have been studied in other species, and at least two cis-elements appear crucial for cAMP-dependent and -independent expression in human and rat granulosa cells (45, 46, 47, 48). A first element, a consensus SF-1-binding site conserved in all species, appears to mediate both constitutive and inducible aromatase transcription (45, 46, 47). A second element, a CRE-like element to which CREB can bind, appears required to achieve optimal transcriptional activity in rats and humans (44, 47). However, this latter element is poorly conserved in other species and contains a 1-bp deletion in the equine (this study), bovine (49), and porcine (16) promoters. In cattle, this deletion was initially thought to be responsible for the lack of aromatase expression in bovine luteal cells. However, a site-directed mutagenesis study designed to render the bovine CRE-like site identical to its human counterpart resulted only in partial restoration of cAMP-inducible promoter activity in luteal cells, suggesting that other elements are involved (49). The marked drop in promoter II-derived aromatase transcripts in equine follicles after hCG treatment agrees with a similar down-regulation in other species after the LH surge in vivo (36, 58, 59, 60, 61). In rat granulosa cells, the decrease in promoter II activity and aromatase mRNA was associated with a drop in SF-1 and in the A kinase regulatory subunit type II (RIIß), but not in CREB expression or binding activity (47, 60, 62).
The observed induction of promoter 1f-derived aromatase transcripts in equine granulosa cells is novel. Exon 1f-containing mRNA was originally cloned from brain tissues and has been described as brain specific (21, 41, 42, 43). However, several aromatase expression studies have shown a vast tissue distribution for many aromatase mRNA species (7, 10, 24), suggesting that the tissue specificity model for aromatase promoter usage may be oversimplified. This view is also supported by recent reports, including this one, that show the use of alternative aromatase promoters in a given tissue (10, 12, 20, 23, 24, 25, 26, 27). The molecular mechanisms involved in promoter 1f-derived aromatase expression have not been characterized, which contrasts with studies on promoter II. Although promoter 1f has previously been cloned in humans (41) and mice (21), no regulatory elements have been functionally identified. Potential cis-acting elements include the highly conserved, overlapping c-myc and glucocorticoid response element identified in horses (this study), humans (41), and mice (21). Recent studies have shown an increase in c-myc expression in rat granulosa cells during hCG-induced luteinization (62, 63). Also, a glucocorticoid response cis-element present in the human aromatase promoter I.4 is required (in conjunction with glucocorticoids) for cytokine-induced transcription in adipocytes via the Janus kinase/STAT (signal transducer and activator of transcription) pathway (64). Whether these pathways are involved in promoter 1f-derived aromatase expression in equine granulosa cells remains to be determined.
This study provides a first characterization of the regulation and
cellular localization of cytochrome P45017 transcripts in equine
preovulatory follicles. Selective expression of P45017 mRNA in the
theca interna layer is similar to reports in other species (58, 59, 61, 65) and supports studies in vitro showing that secretion of
androgens was observed in cultures of equine theca interna cells, but
not in those of granulosa cells (57). No major changes were observed in
levels of P45017 mRNA in theca interna between 0 and 33 h
post-hCG, but a marked drop occurred thereafter. Comparable loss of
P45017 mRNA has been observed in bovine (58), porcine (61), and rat
(66) theca interna after the LH surge. Interestingly, the time course
of P45017 mRNA disappearance (between 33–36 h post-hCG) was
distinct from that of promoter II-derived aromatase transcripts in
granulosa cells (between 0–12 h post-hCG), suggesting the presence of
separate down-regulatory mechanisms in each follicular cell type.
However, the decrease in P45017 transcript in theca interna
coincides precisely with the loss of steroidogenic acute regulatory
protein mRNA in theca interna (67). This apparent reduction in thecal
steroidogenic capacity could relate to the reported demise of the layer
at the time of ovulation in mares (34, 68). Despite high levels of
aromatase transcripts in the equine corpus luteum, estrogen
biosynthesis is very limited during the luteal phase (34). Insufficient
luteal P45017 expression, and thus aromatizable androgen substrates,
has been proposed to be rate-limiting in luteal estrogen synthesis
(69).
In summary, this study reports the cloning and characterization of two
equine aromatase transcripts, as well as approximately 1 kb of genomic
sequences putatively involved in their transcription. This study
provides a first characterization of the regulation and cellular
localization of the P450AROM and P45017 mRNAs during the ovulatory
process, with results indicating that the classic two-cell (theca
interna/granulosa cells), two-gonadotropin (LH/FSH) model for estradiol
production is operative in the mare. Most importantly, we report the
presence of a novel aromatase promoter-switching phenomenon in equine
granulosa cells during follicular luteinization, characterized by a
down-regulation of promoter II- and an up-regulation of promoter
1f-derived transcripts after hCG treatment. This phenomenon apparently
reverses itself after ovulation, as promoter II-derived mRNAs become
elevated, whereas those derived from promoter If disappear in the
corpus luteum. Considering the potential role of aromatase promoter
switching in various physiological and pathological processes and the
uncharacterized nature of its molecular control, we propose that the
equine preovulatory follicle provides a valuable model system to study
this phenomenon.
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Acknowledgments |
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cDNA,
and Dr. R. Levine (Cornell University, Ithaca, NY) for the rat EF-Tu
cDNA. |
Footnotes |
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2 Supported by a Medical Research of Canada Doctoral Research Award.
3 Supported by a fellowship from Al-Fateh University.
Received December 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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of gene expression by ACTH and elucidation of primary sequence. J
Biol Chem 261:2475–2482-hydroxylase/C17,20 lyase messenger
ribonucleic acid. Biol Reprod [Monogr 1] 52:615–622
-hydroxylase and P450
aromatase in preovulatory bovine follicles decrease after the
luteinizing hormone surge. Endocrinology 132:2239–2245, cytochrome P450scc, and
3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian
follicles and corpora lutea:correlation with theca cell
steroidogenesis. Biol Reprod 37:211–223[Abstract]
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 K. Sayasith, N. Bouchard, D. Boerboom, K. A. Brown, M. Dore, and J. Sirois Molecular Characterization of Equine P-Selectin (CD62P) and Its Regulation in Ovarian Follicles During the Ovulatory Process Biol Reprod, March 1, 2005; 72(3): 736 - 744. [Abstract] [Full Text] [PDF]
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K. A. Brown, D. Boerboom, N. Bouchard, M. Dore, J. G. Lussier, and J. Sirois Human Chorionic Gonadotropin-Dependent Regulation of 17{beta}-Hydroxysteroid Dehydrogenase Type 4 in Preovulatory Follicles and Its Potential Role in Follicular Luteinization Endocrinology, April 1, 2004; 145(4): 1906 - 1915. [Abstract] [Full Text] [PDF]
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K. Golovine, M. Schwerin, and J. Vanselow Three Different Promoters Control Expression of the Aromatase Cytochrome P450 Gene (Cyp19) in Mouse Gonads and Brain Biol Reprod, March 1, 2003; 68(3): 978 - 984. [Abstract] [Full Text] [PDF]
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M. Kishida and G. V. Callard Distinct Cytochrome P450 Aromatase Isoforms in Zebrafish (Danio rerio) Brain and Ovary Are Differentially Programmed and Estrogen Regulated during Early Development Endocrinology, February 1, 2001; 142(2): 740 - 750. [Abstract] [Full Text] [PDF]
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D. Boerboom and J. Sirois Equine P450 Cholesterol Side-Chain Cleavage and 3{beta}-Hydroxysteroid Dehydrogenase/{{Delta}}5-{{Delta}}4 Isomerase: Molecular Cloning and Regulation of Their Messenger Ribonucleic Acids in Equine Follicles During the Ovulatory Process Biol Reprod, January 1, 2001; 64(1): 206 - 215. [Abstract] [Full Text]
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D. Boerboom, N. Pilon, R. Behdjani, D. W. Silversides, and J. Sirois Expression and Regulation of Transcripts Encoding Two Members of the NR5A Nuclear Receptor Subfamily of Orphan Nuclear Receptors, Steroidogenic Factor-1 and NR5A2, in Equine Ovarian Cells during the Ovulatory Process Endocrinology, December 1, 2000; 141(12): 4647 - 4656. [Abstract] [Full Text] [PDF]
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K. W. Walters, C. J. Corbin, G. B. Anderson, J. F. Roser, and A. J. Conley Tissue-Specific Localization of Cytochrome P450 Aromatase in the Equine Embryo by In Situ Hybridization and Immunocytochemistry Biol Reprod, May 1, 2000; 62(5): 1141 - 1145. [Abstract] [Full Text]
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