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Centre de Recherche en Reproduction Animale, 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.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-[/4
isomerase (3ß-HSD) (9), steroidogenic acute
regulatory protein (StAR) (3), ACTH receptor (10, 11), müllerian inhibitory substance (12, 13),
LH ß-subunit (14, 15), GnRH receptor (16, 17), oxytocin (18, 19), Dax-1 (20, 21), and several others (22, 23, 24, 25, 26, 27, 28, 29, 30). Considering the
roles of these genes in various metabolic and developmental processes,
it is evident that the functions of SF-1 extend well beyond those that
originally prompted its discovery. Several studies on the regulation of
SF-1 activity have focused on posttranslational mechanisms, including
phosphorylation (31), potential ligands
(32, 33, 34), and associated proteins (12, 35, 36, 37, 38, 39, 40, 41). However, few studies have identified physiological
processes that modulate SF-1 activity at the transcriptional level.
GnRH has been reported to up-regulate SF-1 messenger RNA (mRNA) levels
in the pituitary (42), whereas a transient down-regulation
of SF-1 mRNA has been observed in ovarian cells after the LH surge
(43, 44, 45). Another member of the NR5A orphan nuclear receptor subfamily is NR5A2 (1), that has been previously termed hB1F (46), liver receptor homologous protein-1 (GenBank accession number M81385), PHR-1 (47), xFF1rA (48), fetoprotein transcription factor (49), and CYP7A promoter binding factor (50). NR5A2 and SF-1 have been found to share a high degree of structural similarity, notably within regions referred to as the hybrid P box, the A box, and the T box. Because these structures are directly or indirectly implicated in determining and interacting with the binding site, it was proposed that both receptors share identical DNA binding mechanisms and specificities (51). This has subsequently been demonstrated by several groups (27, 46, 48, 49, 50), and both nuclear receptors were found to transactivate at least one common promoter (27). Interestingly, NR5A2 has also been shown to transactivate a hepatic steroid hydroxylase gene (50). Although these data suggest a functional redundancy between the receptors, no overlap in their tissue distributions has been reported, and no role for NR5A2 in processes such as gonadal steroidogenesis has been proposed.
A recent series of studies examined the regulation and cellular
distribution of transcripts encoding various steroidogenic proteins and
enzymes throughout the equine ovulatory process (52, 53, 54).
It was shown that human CG (hCG) triggers a marked down-regulation of
StAR (54), P450scc (53), and P45017
(52) mRNA in theca interna before ovulation. Different
mRNA regulatory processes were observed within the granulosa cell
layer, in which P450arom expression was abrogated by administration of
hCG (52), whereas StAR (54) and P450scc
(53) were induced, and 3ß-HSD expression did not vary
(53). Considering the divergent regulation of
steroidogenic transcripts during the equine ovulatory process, it is
likely that factors other than SF-1 are involved, or that coactivators
and corepressors enter into play to activate and silence transcription
in a time-, tissue-, and gene-specific manner. The objectives of this
study were to clone equine SF-1 and to characterize the regulation of
its transcript in theca interna and granulosa cells after hCG
administration, to identify possible links between the regulation of
SF-1 mRNA and those of the various steroidogenic genes. As a
serendipitous finding, this paper also reports the cloning of equine
NR5A2 and its novel expression in granulosa and luteal cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]deoxycycidine triphosphate and
[-35S]deoxy-ATP were obtained from Mandel
Scientific-NEN Life Science Products (Mississauga,
Canada); QuikHyb hybridization solution and ExAssist/SOLR system were
purchased from Stratagene Cloning Systems (La Jolla, CA).
TRIzol total RNA isolation reagent, RNA ladder [0.24–9.5 kilobases
(kb)], 1-kb ladder, synthetic oligonucleotides, 5'-RACE System [for
rapid amplification of complementary DNA (cDNA) ends], SuperScript II,
and culture media were obtained from Life Technologies, Inc. (Gaithersburg, MD). Prime-a-Gene labeling system, Access
RT-PCR kit, pGEM-T easy Vector System I, and avian myeloblastosis virus
reverse transcriptase were purchased from Promega Corp.
(Madison, WI). X-OMAT AR film was obtained from Eastman Kodak Co. (Rochester, NY); electrophoretic reagents were
purchased from Bio-Rad Laboratories, Inc. (Richmond, CA).
Taq DNA polymerase, RNAguard, and all sequencing reagents
were obtained from Amersham Pharmacia Biotech (Baie
D’Urfé, Canada).
Isolation of equine tissues and RNA extraction
Equine preovulatory follicles and corpora lutea (CL) were
isolated at specific stages of the estrous cycle, from Standardbred and
Thoroughbred mares, as previously described (55). Briefly,
follicular development was monitored by real-time ultrasonography
during estrus. When preovulatory follicles reached 35 mm in diameter,
the ovulatory process was induced by injection of hCG (2500 IU, iv),
and unilateral ovariectomies were performed via colpotomy using a chain
ecraseur at 0, 12, 24, 30, 33, 36, or 39 h post hCG (n =
4/time point), as described (55). CL were isolated by the
same method on day 8 of the estrous cycle (n = 3; day 0 = day
of ovulation). Follicles were dissected into preparations of follicle
wall (theca interna with attached granulosa cells) or further dissected
into separate isolates of granulosa cells and theca interna. Male
gonadal tissues were obtained from the large animal hospital of the
Faculté de Médecine Vétérinaire
(Université de Montréal) following a routine castration
procedure, and other tissues were obtained at a local slaughterhouse.
All animal procedures were approved by the institutional animal use and
care committee. Total RNA was isolated from tissues, using TRIzol
reagent (Life Technologies, Inc.), according to
manufacturer’s instructions, using a Kinematica PT 1200C Polytron
Homogenizer (Fisher Scientific, Montréal,
Canada).
Cloning of the equine SF-1 transcript
The equine SF-1 transcript was isolated in fragments using a
5-step cloning strategy (Fig. 1
). First,
a RT-PCR technique was performed using 5 µg of total RNA isolated
from adrenal gland, corpus luteum, and follicle wall (Fig. 1Ba). RT
reactions were done with poly-dT oligonucleotides and either
SuperScript II (Life Technologies, Inc.) or StrataScript
RNase H- reverse transcriptase
(Stratagene), essentially under the manufacturer’s
recommended conditions. These reactions were pooled and used as a
template in a PCR reaction that included primers designed by sequence
alignments of known SF-1 species homologues (Fig. 1C). Amplification
was performed as previously described (56) using
Taq polymerase (Amersham Pharmacia Biotech) and
an Omnigene TR3 SM5 thermal cycler (Hybaid Limited, Franklin,
MA) for 40 cycles of 94 C for 45 sec, 58 C for 1 min, and 72 C for 90
sec. After electrophoresis on a 1.2% TAE-agarose gel, the DNA fragment
was excised and ligated into the pGEM-T easy vector (Promega Corp.) according to the manufacturer’s instructions. DNA
sequencing was performed using the T7 Sequencing kit (Amersham Pharmacia Biotech) with vector-based (Sp6 and T7) and custom
oligonucleotide primers (Life Technologies, Inc.). A
545-bp equine SF-1 sequence was generated and submitted to GenBank
(accession number AF168796).
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b). RT was performed, as directed, using 5
µg of total RNA from granulosa cells. After TdT tailing, nested PCR
reactions were performed with appropriate oligonucleotides (Fig. 1C)
and Taq DNA polymerase (Amersham Pharmacia Biotech), using 35 (first reaction) or 30 (second reaction)
cycles of 94 C for 30 sec, 55 C for 1 min, and 72 C for 2 min, with a
final 7-min extension step at 72 C. The largest 5'-RACE products were
isolated and sequenced. A 3'-RACE technique was used to obtain
sequences downstream of the SF-1 PCR product (Fig. 1Bc). RT was
performed using 5 µg RNA from corpus luteum, to which 500 ng poly-dT
oligonucleotide (Fig. 1C) was added; and the mixture was heated to 70 C
for 5 min. RNAguard (39 U, Amersham Pharmacia Biotech),
AMV-RT (10 U, Promega Corp.), and deoxynucleotide
triphosphates (200 µM each final) were added;
and the reaction was incubated at 42 C for 1 h. Nested PCR
reactions were performed using appropriate oligonucleotides (Fig. 1C)
and 40 cycles of 94 C for 1 min, 60 C for 1 min, and 72 C for 4 min.
Because the 3'-end of the 3'-RACE product contained nonhomologous
sequences, RT-PCR was used to clone the balance of the coding region
(Fig. 1Bd). Oligonucleotide primers were designed from the 3'-RACE
product and 3'-UTR (untranslated regions) sequence alignments of all
known SF-1 species homologues (Fig. 1C). The RT-PCR reaction was
performed using the Access RT-PCR kit (Promega Corp.) as
described below, except 40 amplification cycles were used, and 100 ng
of granulosa cell total RNA served as the template. A final 3'-RACE was
performed to isolate the remainder of the 3'-UTR (Fig. 1Be). The
procedure was done as described above, except RT was performed with 4
µg of preovulatory granulosa cell RNA, appropriate primers (Fig. 1C),
and the cycling conditions were 94 C for 30 sec, 55 C for 1 min, and 72
C for 4 min.
Cloning of the equine NR5A2 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) transcripts
The 5' SF-1 RT-PCR cloning product (Fig. 1Ba) was used to screen
an equine cDNA library prepared from a 36-h post-hCG preovulatory
follicle (55), with the intent to isolate a full-length
SF-1 cDNA clone. The probe was labeled to a specific activity of
greater than 108 cpm/µg of input DNA by means
of the Prime-a-Gene labeling kit (Promega Corp.),
following manufacturer’s instructions. Approximately 100,000 phage
plaques were screened, and hybridization was performed using QuikHyb
solution (Stratagene). Primary screening yielded seven
weak positive clones that required a 1-week exposure, at -70 C, to
X-OMAT AR film (Eastman Kodak Co.), to be clearly
identified, and only one clone was successfully purified through
secondary and tertiary rounds of screening. The ExAssist/SOLR system
(Stratagene) was used for in vivo excision,
producing the cDNA clone inserted in the pBluescript vector, and
sequencing was performed as described above. Comparison with available
GenBank sequence data revealed that the clone was the equine homologue
of NR5A2 (1). Because the NR5A2 cDNA clone was incomplete
at both the 5'- and 3'-ends (Fig. 2Ba),
the RACE procedures described for SF-1 were repeated. The conditions
used for 5'- and 3'-RACE were the same as for SF-1, except for the use
of gene-specific oligonucleotides (Fig. 2C) and a temperature of 58 C
for the annealing step in the 3'-RACE PCR reactions. Whereas the
5'-RACE was successful (Fig. 2Bb), an improperly spliced 3'-RACE
product was obtained for NR5A2 (Fig. 2Bc), requiring that an additional
RT-PCR cloning procedure be performed to obtain the downstream coding
regions (Fig. 2Bd). The reaction was performed as for SF-1, except that
100 ng of total RNA from a corpus luteum was used as a template. A
final 3'-RACE procedure, under the same conditions as for SF-1,
permitted the isolation of the remaining portion of the 3'-UTR (Fig. 2Be).
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Northern analysis
RNA samples (10 µg) were processed, electrophoresed on 1.2%
formaldehyde-agarose gels, and transferred to nylon membranes as
previously described (55). 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 to determine
the migration of standards. Hybridization was performed using QuikHyb
solution (Stratagene) and several equine cDNA probes,
including SF-1, NR5A2, P450arom (52), P450scc
(53), P45017 (52), 3ß-HSD
(53), StAR (54), and GAPDH. Each cDNA was
labeled using the Prime-a-Gene labeling system, as described above, and
stripping of hybridization signal between successive rounds of probing
was achieved by soaking filters in 0.1% SSC-0.1% SDS for 15 min at
90-95 C. After autoradiography at -70 C, a computer-assisted image
analysis system was used to capture and digitize the images (Collage
Macintosh program, Fotodyne, Inc., New Berlin, WI).
Semiquantitative RT-PCR and Southern analysis
The Access RT-PCR System (Promega Corp.) was used
for semiquantitative analysis of SF-1, NR5A2, and GAPDH levels in theca
interna and granulosa cells isolated between 0–39 h after hCG
treatment. Reactions were performed as directed by the manufacturer,
using the oligonucleotide pairs 5'-CCCGA GCTCA TCCTG CAGCT G-3' and
5'-CTGGC GGTCC AGCTG CAGCG-3' for SF-1, 5'-AGAAA GCGTT GTCCC TACTG
TCG-3' and 5'-TCTGG CTCAC ACTTC AAAAG TTCC-3' for NR5A2 and 5'-ATCAC
CATCT TCCAG GAGCG AGA-3' and 5'-GTCTT CTGGG TGGCA GTGAT GG-3' for
GAPDH. These reactions resulted in the generation of 429-, 539-, and
341-bp products, respectively. Each reaction was performed using 100 ng
of total RNA, and cycling conditions were 1 cycle of 48 C for 45 min,
and 94 C for 2 min, followed by a variable number of cycles of 94 C for
30 sec, 55 C for 1 min, and 68 C for 2 min. The number of cycles used
was optimized for each gene in preliminary experiments, to fall within
the linear range of PCR amplification, and included 16, 10, and 10
cycles for SF-1, NR5A2, and GAPDH, respectively. After PCR
amplification, samples were electrophoresed on 2% TAE-agarose gels and
transferred to nylon membranes, as previously described
(56). The membranes were probed with the corresponding
radiolabeled cDNA fragment, as described in Northern analysis. After
autoradiography, films were scanned using an IBM Flatbed Scanner and
Photo-Paint version 6.00 software (Corel Corp., Ottawa, Canada).
Signal strength was quantified by density analysis of the digital
images using NIH image software, version 1.61 (NIH, Bethesda, MA).
Statistical analysis
One-way ANOVA was used to test the effect of time, after hCG, on
levels of SF-1, NR5A2, and GAPDH transcript levels in theca interna and
granulosa cells. When ANOVAs indicated significant differences
(P < 0.05), Dunnett’s test was used for multiple
comparisons with the control (0 h post hCG). SF-1 and NR5A2 levels were
normalized with GAPDH, and results are expressed as means ±
SEM [n = 4 follicles (i.e.
mares)/time point]. No difference was observed in GAPDH levels at any
time point between 0 and 39 h post hCG (P <
0.05). 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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a) was then employed as a probe to
isolate a clone from an equine follicular expression library
(55). Because no SF-1 clones were obtained, 5'- and
3'-RACE were used to isolate the balance of the SF-1 transcript.
Whereas the 5'-RACE reaction produced a cDNA fragment encompassing all
upstream coding regions and a considerable amount of 5'-UTR (Fig. 1Bb),
the 3'-RACE experiment generated a truncated product (Fig. 1Bc). This
latter product consisted of a few hundred bases of coding sequences
followed by a consensus splice junction and nonhomologous sequences,
and most likely resulted from inappropriate priming of intronic
sequences. An RT-PCR cloning strategy was therefore employed to obtain
all remaining downstream coding sequences (Fig. 1Bd). After this, the
3'-RACE protocol was successfully applied, generating a fragment
representing all the remaining 3'-UTR (Fig. 1Be). The deduced equine
SF-1 transcript includes a 5'-UTR of 161 bp, an open reading frame
(ORF) of 1386 bp that encodes a highly-conserved 461-amino acid
protein, and a 3'-UTR of 518 bp (Figs. 1A and 3A).
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a). A combination of 5'- and
3'-RACE was performed to further characterize the NR5A2 transcript.
Whereas all relevant 5' sequence information was obtained (Fig. 2Bb),
the 3'-end remained incomplete (Fig. 2Bc). To conclude the isolation of
the NR5A2 primary transcript, an RT-PCR product encompassing sequences
downstream of the first 3'-RACE product was generated (Fig. 2Bd), and
it was followed by a second 3'-RACE (Fig. 2Be). The complete deduced
NR5A2 transcript includes a 5'-UTR of 243 bp, an ORF of 1488 bp that
encodes a highly conserved 495-amino acid protein, and a 3'-UTR of 1358
bp (Figs. 2A and 3B).
When translated, the deduced primary transcripts were found to encode
orphan nuclear receptors that are more closely related to each other
than to any other known members of the nuclear receptor family. When
their sequences are aligned, an overall homology of approximately 60%
is observed, most of which is clustered within the putative DNA- and
ligand-binding domains (Fig. 4A). Direct
comparison of the DNA-binding domains of SF-1 and NR5A2 to each other
and to their human homologs reveals a 90% or higher degree of
identity, and a 99% or higher degree of similarity (Fig. 4B). This
includes the near-perfect duplication of the hybrid P box, A box, and T
box regions, which are critical determinants of DNA-binding specificity
(51). In addition, a serine residue located in the AF-1
domain of SF-1 whose phosphorylation has recently been implicated in
mediating cofactor recruitment (31) is also present in
NR5A2 (Fig. 4A).
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). Overexposures
showed low mRNA levels in liver, uterus, thymus, and the head of the
epididymis (data not shown), but no transcript was detected in the
adrenal gland. Conversely, SF-1 mRNA was detected in equine adrenals,
as well as in other major steroidogenic organs, albeit to considerably
lower levels than those of NR5A2 in preovulatory follicles and CL (Fig. 5). Low levels of SF-1 were detected in the spleen when the blot was
overexposed (data not shown). The same membrane was reprobed with
equine cDNAs encoding several steroidogenic enzymes. Results show a
relationship between the expression of SF-1 and that of the
steroidogenic enzymes, with a few notable exceptions such as the lack
of aromatase signal in the adrenal gland (Fig. 5). Also, an apparent
discrepancy occurred during the transition from the preovulatory
follicle to the corpus luteum, during which a drop in SF-1 mRNA levels
was associated with a marked increase in StAR and P450scc transcripts.
In contrast, NR5A2 expression remained at relatively high levels in the
preovulatory follicle and the corpus luteum.
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). Levels of
SF-1 mRNA in theca interna were high before hCG treatment (0 h), and
they subsequently decreased in a biphasic manner; as an initial 2-fold
drop induced within 12 h (P < 0.05) was followed
by another significant decrease between 33 and 36 h post hCG
(P < 0.05) (Fig. 6B). In granulosa cells, a
significant decrease in SF-1 mRNA levels was also observed after hCG
treatment (Fig. 6A). Levels observed at 24 h were approximately
2.5-fold lower than at 0 h (P < 0.05) and did not
vary significantly thereafter (Fig. 6A).
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).
Importantly, when considering the number of amplification cycles used
for SF-1 (16 cycles, Fig. 6) and NR5A2 (10 cycles, Fig. 7), it becomes
apparent that NR5A2 is much more highly expressed in granulosa cells
than SF-1. The administration of hCG caused a significant decrease in
NR5A2 transcript levels at 30, 36, and 39 h (P <
0.05, Fig. 7A). The absence of an effect at 33 h post hCG was
attributed to the heterogenous results obtained at this time point,
which resulted from two mares that apparently did not respond to hCG
(note high SEM at 33 h post hCG, Fig. 7A).
Contrary to granulosa cells, extremely low levels of NR5A2 were
observed in theca interna before hCG (Fig. 7B). Although a modest
increase in NR5A2 transcripts was observed at 36 and 39 h post
hCG, the physiological nature of this finding remains to be
verified.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(52), and P450arom (52). In response to this
stimulus, each of these transcripts was found to undergo a different
regulatory process, and their cellular localization within the follicle
wall varied in a gene-specific and time-dependent fashion
(52, 53, 54). Using other model systems, the orphan nuclear
receptor SF-1 has been proposed as a common transcriptional regulator
of all these key ovarian steroidogenic genes (3). However,
the precise mechanisms by which SF-1 could generate diverse mRNA
regulatory patterns in ovarian cells in response to hCG remain to be
elucidated. To determine whether the regulation of SF-1 mRNA represents
an important level of control in the equine ovary, this study reports
the cloning of equine SF-1 and the regulation of its transcript in the
follicle wall compartments during hCG-induced ovulation and
luteinization. Unexpectedly, our cloning efforts also led to the
serendipitous isolation of a cDNA encoding the orphan nuclear receptor
NR5A2, a member of the NR5A subfamily that also includes SF-1
(1).
The present study is the first to document the concomitant regulation
of SF-1 in both granulosa cells and theca interna throughout the
ovulation/luteinization process in vivo. Although our data
indicate that SF-1 transcripts were present in both follicular cell
types, the equine theca interna seemed as the predominant site of SF-1
mRNA expression, as previously observed by in situ analysis
in murine follicles (57). The expression and regulation of
thecal SF-1 mRNA, before and after hCG administration, closely
paralleled those of the equine thecal steroidogenic genes StAR,
P450scc, and P45017 (52, 53, 54). Most notably, a marked
decrease in the mRNA levels of all these genes was observed a few hours
before ovulation (i.e. at 36 h and 39 h post hCG).
As previously pointed out for thecal steroidogenic genes
(52, 53, 54), the down-regulation of SF-1, with the approach
of ovulation, coincides with the proposed degeneration of the thecal
layer, a process unique to the equine follicle, that leads to the
formation of a corpus luteum derived solely from granulosa cells
(58, 59). This finding could provide a putative mechanism
for the transcriptional down-regulation of steroidogenic genes in
thecal cells, in which the level of SF-1 expression represents a key
rate-limiting factor. It is also tempting to propose that the decrease
in SF-1 mRNA could represent a consequence of degenerative (and likely
apoptotic) signaling processes that presumably occur in the equine
theca interna before ovulation. In granulosa cells, the down-
regulation of equine SF-1 mRNA by hCG is in agreement with similar
reports in rats that demonstrated losses of SF-1 transcripts, protein,
and DNA-binding activity in response to gonadotropin
(43, 44, 45). This down-regulation of SF-1 in rat granulosa
cells has been correlated with the LH/hCG- induced abrogation of
P450arom expression (43, 44, 45). The same relationship is not
as clear in equine follicles, because the near complete loss of
P450arom mRNA (12 h post hCG; 52) occurred before the
first significant drop in SF-1 transcript (24 h post hCG; this study).
Also, transcripts for other steroidogenic genes, such as StAR and
P450scc, were shown to be induced in granulosa cells after hCG
treatment in vivo (53, 54), whereas 3ß-HSD
levels were not found to vary in equine granulosa cells after hCG
(53). Thus, mechanisms other than regulation of SF-1 mRNA
must come into play to ensure gene-specific control of steroidogenesis
in equine granulosa cells and are likely to include posttranscriptional
mechanisms such as the regulation of SF-1 translation, phosphorylation,
and association with cofactors.
This report is also the first to demonstrate the expression of NR5A2 in gonadal tissues, as well as hormonal regulation of its mRNA in ovarian cells. NR5A2 expression has previously been localized in liver and pancreas but not in reproductive organs (46, 49, 50). Interestingly, results from the present study indicate that follicular NR5A2 expression is primarily, if not solely, localized to the granulosa cell layer. Even more unforeseen, levels of NR5A2 mRNA far surpassed those of SF-1, thus making it the predominant NR5A subfamily receptor present in granulosa cells. NR5A2 was also the predominant NR5A nuclear receptor mRNA present in the corpus luteum. Considering that the equine corpus luteum is thought to be derived solely from granulosa cells (58, 59), our results suggest that NR5A2 could function as an important transcriptional regulator in these cells at various stages of differentiation (i.e. in both unluteinized and luteinized cells). For example, the elevated expression of NR5A2 transcripts in equine granulosa cells and the corpus luteum closely parallels that of promoter II-derived P450arom mRNA (52). The very high levels of expression of other steroidogenic genes, such as StAR and P450scc, in the equine corpus luteum (53, 54) also coincide well with those of NR5A2. Although these evidences remain circumstantial, the potential role of another nuclear receptor, closely related to SF-1, involved in the transcription of steroidogenic genes should be considered. In contrast to granulosa and luteal cells, the expression of NR5A2 transcripts in theca interna was extremely low. The modest increase in thecal mRNA levels observed in the hours just before ovulation [i.e. at 36 and 39 h post hCG; ovulation occurs between 39 and 42 h post hCG in this model (60, 61)] should be interpreted with caution. In fact, we believe that this finding could be artifactual, because the complete separation of granulosa cells from the theca interna becomes increasingly difficult between 30 and 39 h post hCG in mares, as a result of the copious synthesis of mucosubstances by granulosa cells (58). The presence of only a few contaminating granulosa cells in the theca interna preparation would likely be sufficient to generate the low levels of NR5A2 transcript observed by RT-PCR. In situ hybridization or immunohistochemistry analyses will be required to resolve this issue.
The predominant expression of NR5A2 over SF-1 [NR5A1a
(1)] in granulosa cells and the corpus luteum raises the
obvious question of its role in the ovary. Equine SF-1 and NR5A2 share
important structural features, such as 99%-similar DNA binding domains
featuring nearly identical hybrid P box, A box, and T box elements. The
A box is of particular interest, because it seems to dictate the
overall DNA-binding specificity of the receptor by contacting DNA
regions 5' of the hexamer half-site (51). It therefore
seems likely that both equine SF-1 and NR5A2 will be found to share the
same DNA-binding properties, as demonstrated for their human homologs
(46, 48, 49, 50). Another key structural element that is very
similar in equine SF-1 and NR5A2 is the putative ligand-binding domain,
which for SF-1 has been suggested to bind a small molecule ligand
(33) or to mediate protein-protein interactions that
modulate transcriptional activity (12, 35, 36, 38). The
conserved phosphorylatable serine residue in the AF-1 domain of NR5A2
could also be involved in recruiting transcriptional cofactors such as
GRIP1 and SMRT, as reported for SF-1 (31). Interestingly,
human SF-1 and NR5A2 [known as FTF (49)] have recently
been shown to transactivate at least one promoter in common
(27). Thus, based on the marked expression of NR5A2 in
granulosa/luteal cells, on the mounting evidence of a potential
functional redundancy between SF-1 and NR5A2 and on the
demonstrated ability of the human homolog of equine NR5A2 to
transactivate a hepatic steroid hydroxylase gene (cholesterol
7-hydroxylase; 50), we believe that NR5A2 may play
a key role in the regulation of gonadal steroidogenesis.
In summary, this study reports the cloning and characterization of two members of the equine NR5A nuclear receptor subfamily and the regulation and cellular localization of their transcripts in equine follicles during hCG-induced ovulation in vivo. The most significant finding of this study resides in the novel localization and elevated expression of NR5A2 transcript in gonadal cells and in its potential implication in the control of ovarian steroidogenesis. Future studies will be required to demonstrate that NR5A2 can transactivate classic steroidogenic target genes in granulosa cells and that gonadal expression of this nuclear receptor is conserved in other species.
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Acknowledgments |
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Footnotes |
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2 Supported by a Canadian Institutes of Health Research Doctoral
Research Award.
3 Supported by a Fonds pour la Formation de Chercheurs et l’Aide
à la Recherche Doctoral Research Scholarship.
4 Supported by a bursary from the Ministry of Culture and Higher
Education of the Government of Iran.
5 Supported by a Canadian Institutes of Health Research Investigator
Award.
Received May 31, 2000.
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  C. Andreu-Vieyra, R. Chen, and M. M. Matzuk Conditional Deletion of the Retinoblastoma (Rb) Gene in Ovarian Granulosa Cells Leads to Premature Ovarian Failure Mol. Endocrinol., September 1, 2008; 22(9): 2141 - 2161. [Abstract] [Full Text] [PDF]
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 C. Mataki, B. C. Magnier, S. M. Houten, J.-S. Annicotte, C. Argmann, C. Thomas, H. Overmars, W. Kulik, D. Metzger, J. Auwerx, et al. Compromised Intestinal Lipid Absorption in Mice with a Liver-Specific Deficiency of Liver Receptor Homolog 1 Mol. Cell. Biol., December 1, 2007; 27(23): 8330 - 8339. [Abstract] [Full Text] [PDF]
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 C. Labelle-Dumais, J.-F. Pare, L. Belanger, R. Farookhi, and D. Dufort Impaired Progesterone Production in Nr5a2+/ Mice Leads to a Reduction in Female Reproductive Function Biol Reprod, August 1, 2007; 77(2): 217 - 225. [Abstract] [Full Text] [PDF]
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 J. Guo, S.-X. Tao, M. Chen, Y.-Q. Shi, Z.-Q. Zhang, Y.-C. Li, X.-S. Zhang, Z.-Y. Hu, and Y.-X. Liu Heat Treatment Induces Liver Receptor Homolog-1 Expression in Monkey and Rat Sertoli Cells Endocrinology, March 1, 2007; 148(3): 1255 - 1265. [Abstract] [Full Text] [PDF]
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 K. Sayasith, N. Bouchard, M. Dore, and J. Sirois Cloning of equine prostaglandin dehydrogenase and its gonadotropin-dependent regulation in theca and mural granulosa cells of equine preovulatory follicles during the ovulatory process Reproduction, February 1, 2007; 133(2): 455 - 466. [Abstract] [Full Text] [PDF]
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 K. A Brown, K. Sayasith, N. Bouchard, J. G Lussier, and J. Sirois Molecular cloning of equine 17{beta}-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo J. Mol. Endocrinol., January 1, 2007; 38(1): 67 - 78. [Abstract] [Full Text] [PDF]
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K. Sayasith, M. Dore, and J. Sirois Molecular characterization of tumor necrosis {alpha}-induced protein 6 and its human chorionic gonadotropin-dependent induction in theca and mural granulosa cells of equine preovulatory follicles Reproduction, January 1, 2007; 133(1): 135 - 145. [Abstract] [Full Text] [PDF]
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 G. Benoit, A. Cooney, V. Giguere, H. Ingraham, M. Lazar, G. Muscat, T. Perlmann, J.-P. Renaud, J. Schwabe, F. Sladek, et al. International Union of Pharmacology. LXVI. Orphan Nuclear Receptors Pharmacol. Rev., December 1, 2006; 58(4): 798 - 836. [Abstract] [Full Text] [PDF]
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K. A. Brown, M. Dore, J. G. Lussier, and J. Sirois Human Chorionic Gonadotropin-Dependent Up-Regulation of Genes Responsible for Estrogen Sulfoconjugation and Export in Granulosa Cells of Luteinizing Preovulatory Follicles Endocrinology, September 1, 2006; 147(9): 4222 - 4233. [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 induction of an equine aldo-keto reductase (AKR1C23) with 20{alpha}-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo. J. Mol. Endocrinol., June 1, 2006; 36(3): 449 - 461. [Abstract] [Full Text] [PDF]
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J. Weck and K. E. Mayo Switching of NR5A Proteins Associated with the Inhibin {alpha}-Subunit Gene Promoter after Activation of the Gene in Granulosa Cells Mol. Endocrinol., May 1, 2006; 20(5): 1090 - 1103. [Abstract] [Full Text] [PDF]
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 Y. Li, M. Choi, K. Suino, A. Kovach, J. Daugherty, S. A. Kliewer, and H. E. Xu Structural and biochemical basis for selective repression of the orphan nuclear receptor liver receptor homolog 1 by small heterodimer partner PNAS, July 5, 2005; 102(27): 9505 - 9510. [Abstract] [Full Text] [PDF]
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W. Wang, C. Zhang, A. Marimuthu, H. I. Krupka, M. Tabrizizad, R. Shelloe, U. Mehra, K. Eng, H. Nguyen, C. Settachatgul, et al. The crystal structures of human steroidogenic factor-1 and liver receptor homologue-1 PNAS, May 24, 2005; 102(21): 7505 - 7510. [Abstract] [Full Text] [PDF]
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 J. W. Kim, J. C. Havelock, B. R. Carr, and G. R. Attia The Orphan Nuclear Receptor, Liver Receptor Homolog-1, Regulates Cholesterol Side-Chain Cleavage Cytochrome P450 Enzyme in Human Granulosa Cells J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1678 - 1685. [Abstract] [Full Text] [PDF]
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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. Schoonjans, L. Dubuquoy, J. Mebis, E. Fayard, O. Wendling, C. Haby, K. Geboes, and J. Auwerx Liver receptor homolog 1 contributes to intestinal tumor formation through effects on cell cycle and inflammation PNAS, February 8, 2005; 102(6): 2058 - 2062. [Abstract] [Full Text] [PDF]
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L. J. Martin and J. J. Tremblay The Human 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase Type 2 Promoter Is a Novel Target for the Immediate Early Orphan Nuclear Receptor Nur77 in Steroidogenic Cells Endocrinology, February 1, 2005; 146(2): 861 - 869. [Abstract] [Full Text] [PDF]
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K. L. Britt, P. G. Stanton, M. Misso, E. R. Simpson, and J. K. Findlay The Effects of Estrogen on the Expression of Genes Underlying the Differentiation of Somatic Cells in the Murine Gonad Endocrinology, August 1, 2004; 145(8): 3950 - 3960. [Abstract] [Full Text] [PDF]
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P. Jeyasuria, Y. Ikeda, S. P. Jamin, L. Zhao, D. G. de Rooij, A. P. N. Themmen, R. R. Behringer, and K. L. Parker Cell-Specific Knockout of Steroidogenic Factor 1 Reveals Its Essential Roles in Gonadal Function Mol. Endocrinol., July 1, 2004; 18(7): 1610 - 1619. [Abstract] [Full Text] [PDF]
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V. Pezzi, R. Sirianni, A. Chimento, M. Maggiolini, S. Bourguiba, C. Delalande, S. Carreau, S. Ando, E. R. Simpson, and C. D. Clyne Differential Expression of Steroidogenic Factor-1/Adrenal 4 Binding Protein and Liver Receptor Homolog-1 (LRH-1)/Fetoprotein Transcription Factor in the Rat Testis: LRH-1 as a Potential Regulator of Testicular Aromatase Expression Endocrinology, May 1, 2004; 145(5): 2186 - 2196. [Abstract] [Full Text] [PDF]
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H. Hiroi, L. K. Christenson, L. Chang, M. D. Sammel, S. L. Berger, and J. F. Strauss III Temporal and Spatial Changes in Transcription Factor Binding and Histone Modifications at the Steroidogenic Acute Regulatory Protein (StAR) Locus Associated with StAR Transcription Mol. Endocrinol., April 1, 2004; 18(4): 791 - 806. [Abstract] [Full Text] [PDF]
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D. Boerboom, K. A. Brown, D. Vaillancourt, P. Poitras, A. K. Goff, K. Watanabe, M. Dore, and J. Sirois Expression of Key Prostaglandin Synthases in Equine Endometrium During Late Diestrus and Early Pregnancy Biol Reprod, February 1, 2004; 70(2): 391 - 399. [Abstract] [Full Text] [PDF]
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T. Fayad, V. Levesque, J. Sirois, D. W. Silversides, and J. G. Lussier Gene Expression Profiling of Differentially Expressed Genes in Granulosa Cells of Bovine Dominant Follicles Using Suppression Subtractive Hybridization Biol Reprod, February 1, 2004; 70(2): 523 - 533. [Abstract] [Full Text] [PDF]
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N. Peng, J. W. Kim, W. E. Rainey, B. R. Carr, and G. R. Attia The Role of the Orphan Nuclear Receptor, Liver Receptor Homologue-1, in the Regulation of Human Corpus Luteum 3{beta}-Hydroxysteroid Dehydrogenase Type II J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6020 - 6028. [Abstract] [Full Text] [PDF]
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R. C. Fowkes, M. Desclozeaux, M. V. Patel, S. J. B. Aylwin, P. King, H. A. Ingraham, and J. M. Burrin Steroidogenic Factor-1 and The Gonadotrope-Specific Element Enhance Basal and Pituitary Adenylate Cyclase-Activating Polypeptide-Stimulated Transcription of the Human Glycoprotein Hormone {alpha}-Subunit Gene in Gonadotropes Mol. Endocrinol., November 1, 2003; 17(11): 2177 - 2188. [Abstract] [Full Text] [PDF]
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L. J. Whale, D. C. Eckery, and J. L. Juengel Determination of Steroidogenic Potential of Ovarian Cells of the Brushtail Possum (Trichosurus vulpecula) Biol Reprod, September 1, 2003; 69(3): 947 - 958. [Abstract] [Full Text] [PDF]
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R. Marchal, M. Caillaud, A. Martoriati, N. Gerard, P. Mermillod, and G. Goudet Effect of Growth Hormone (GH) on In Vitro Nuclear and Cytoplasmic Oocyte Maturation, Cumulus Expansion, Hyaluronan Synthases, and Connexins 32 and 43 Expression, and GH Receptor Messenger RNA Expression in Equine and Porcine Species Biol Reprod, September 1, 2003; 69(3): 1013 - 1022. [Abstract] [Full Text] [PDF]
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A. E. Falender, R. Lanz, D. Malenfant, L. Belanger, and J. S. Richards Differential Expression of Steroidogenic Factor-1 and FTF/LRH-1 in the Rodent Ovary Endocrinology, August 1, 2003; 144(8): 3598 - 3610. [Abstract] [Full Text] [PDF]
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D. L. Liu, W. Z. Liu, Q. L. Li, H. M. Wang, D. Qian, E. Treuter, and C. Zhu Expression and Functional Analysis of Liver Receptor Homologue 1 as a Potential Steroidogenic Factor in Rat Ovary Biol Reprod, August 1, 2003; 69(2): 508 - 517. [Abstract] [Full Text] [PDF]
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T. Suzuki, M. Kasahara, H. Yoshioka, K.-i. Morohashi, and K. Umesono LXXLL-Related Motifs in Dax-1 Have Target Specificity for the Orphan Nuclear Receptors Ad4BP/SF-1 and LRH-1 Mol. Cell. Biol., January 1, 2003; 23(1): 238 - 249. [Abstract] [Full Text]
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A. E. Stock, N. Bouchard, K. Brown, A. P. Spicer, C. B. Underhill, M. Dore, and J. Sirois Induction of Hyaluronan Synthase 2 by Human Chorionic Gonadotropin in Mural Granulosa Cells of Equine Preovulatory Follicles Endocrinology, November 1, 2002; 143(11): 4375 - 4384. [Abstract] [Full Text] [PDF]
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N. R. Stallings, N. A. Hanley, G. Majdic, L. Zhao, M. Bakke, and K. L. Parker Development of a Transgenic Green Fluorescent Protein Lineage Marker for Steroidogenic Factor 1 Mol. Endocrinol., October 1, 2002; 16(10): 2360 - 2370. [Abstract] [Full Text] [PDF]
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 K. L. Parker, D. A. Rice, D. S. Lala, Y. Ikeda, X. Luo, M. Wong, M. Bakke, L. Zhao, C. Frigeri, N. A. Hanley, et al. Steroidogenic Factor 1: an Essential Mediator of Endocrine Development Recent Prog. Horm. Res., January 1, 2002; 57(1): 19 - 36. [Abstract] [Full Text] [PDF]
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J. S. Richards, D. L. Russell, S. Ochsner, M. Hsieh, K. H. Doyle, A. E. Falender, Y. K. Lo, and S. C. Sharma Novel Signaling Pathways That Control Ovarian Follicular Development, Ovulation, and Luteinization Recent Prog. Horm. Res., January 1, 2002; 57(1): 195 - 220. [Abstract] [Full Text] [PDF]
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J. S. Richards Perspective: The Ovarian Follicle--A Perspective in 2001 Endocrinology, June 1, 2001; 142(6): 2184 - 2193. [Full Text] [PDF]
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