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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, Centre de Recherche en Reproduction Animale, 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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17-kilobase clone obtained from the genomic library. The equine
PGHS-2 gene structure (10 exons and 9 introns; total length of 6991 bp)
is similar to its human homolog except for lacking sequence elements in
introns 4, 8, and 9 and in the 3'-UTR region of exon 10. To
characterize the regulation of PGHS-2 mRNA in equine follicles before
ovulation, preovulatory follicles were isolated during estrus, 0, 12,
24, 30, 33, 36, and 39 h (n = 4–5 follicles/time point)
after an ovulatory dose of hCG. Results from Northern blots showed
significant changes in steady state levels of PGHS-2 mRNA in
preovulatory follicles after hCG treatment (P <
0.05). The transcript remained undetectable between 0–24 h post-hCG,
first appeared (4 kilobases) only at 30 h, and reached maximal
levels 33 h post-hCG. PGHS-2 mRNA was selectively induced in
granulosa cells and not in theca interna. Thus, this study provides for
the first time the primary structure of the equine PGHS-2 gene,
transcript, and protein. It also demonstrates that the induction of
PGHS-2 gene expression in equine granulosa cells is a long molecular
process (30 h post-hCG), thereby providing a model to study the
molecular basis for the late transcriptional activation of PGHS-2 in
species with a long ovulatory process. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In recent years, several studies have implicated PGHS enzymes in
various reproductive functions, including luteolysis (17), embryonic
development and implantation (18, 19, 20, 21, 22, 23), and parturition (24, 25, 26, 27, 28, 29, 30, 31).
Ovulation is another physiological process during which PG synthesis is
required. In rat preovulatory follicles, there is a selective induction
of PGHS-2 messenger RNA (mRNA) and protein in granulosa cells before
ovulation (32, 33, 34, 35, 36). The induction is rapid (2–4 h post-hCG) and
transient, and precedes follicular rupture by approximately 10 h
(36). This molecular process is also present in species with a long
ovulatory process, such as cows (37, 38). However, PGHS-2 induction in
bovine granulosa cells is relatively delayed compared with that in
rats, being expressed only 18 h after hCG treatment (37).
Interestingly, as the interval from PGHS-2 induction to follicular
rupture is remarkably conserved in both species (10 h), we proposed
that PGHS-2 could be one of the determinants involved in dictating the
species-specific length of the ovulatory process (37).
Marked differences in the time course of PGHS-2 induction among species
have underscored the need to characterize the distinct molecular
mechanisms involved in PGHS-2 gene expression in large monoovulatory
species with a long ovulatory process. The mare is a valuable model to
study the hormonal control of follicular PG synthesis before ovulation.
The preovulatory follicle reaches a relatively large size (40–45 mm)
and can be identified in vivo by ultrasonography up to 7
days before ovulation (39, 40, 41). Ovulation can be induced by
administration of hCG, and the interval from gonadotropin injection to
follicular rupture is approximately 36–48 h (42, 43). It was recently
shown that the induction of PG synthetic activities in equine follicles
before ovulation is associated with the selective induction of PGHS-2
protein in granulosa cells (44). Its time course of induction (30 h
post-hCG) is further delayed compared with that in cows, but the
interval between PGHS-2 induction and ovulation remains similar (10
h), supporting the hypothesis that PGHS-2 induction could serve as an
important signal to control the mammalian ovulatory clock (44, 45).
To further increase our understanding of the molecular control of PG synthesis in equine follicles, the general objective of this study was to characterize the regulation of follicular PGHS-2 mRNA before ovulation. The specific objectives were to clone and determine the primary structure of equine PGHS-2, and to characterize the expression of PGHS-2 mRNA in a developmental series of equine preovulatory follicles isolated between 0–39 h after the administration of an ovulatory dose of hCG.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]deoxy (d)-CTP,
[-32P]dATP, [
-32P]ATP, and
[35S]dATP were obtained from Mandel Scientific-New
England Nuclear Life Science Products (Mississauga, Canada); QuikHyb
hybridization solution, Poly(A) Quick mRNA purification kit, and
ZAP-cDNA/Gigapack cloning kit 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,
and culture media were obtained from Life Technologies (Gaithersburg,
MD); RNAsin, Prime-a-Gene labeling system, DNA 5'-End Labeling System,
and AMV reverse transcriptase were purchased from Promega (Madison,
WI); Kodak X-Omat AR film was obtained from Eastman Kodak (Rochester,
NY); electrophoretic reagents were purchased from Bio-Rad Laboratories
(Richmond, CA); T4 polynucleotide kinase and all sequencing reagents
were obtained from Pharmacia Biotech (Baie D’Urfé, Canada).
Cloning and sequencing of the equine PGHS-2 complementary DNA
(cDNA) and gene
To clone the equine PGHS-2 cDNA, an equine expression library
was made with RNA extracted (TRIzol, Life Technologies) from a
preovulatory follicle isolated during estrus, 36 h after hCG
treatment (44). Polyadenylated [poly(A)+] RNA was
purified with the Poly(A) Quick mRNA purification kit (Stratagene), and
the library was constructed using the ZAP-cDNA/Gigapack cloning kit
(Stratagene) following the manufacturer’s protocol. One round of
300,000 plaques was screened with a 1.2-kb 5'-fragment of the mouse
PGHS-2 cDNA generated by EcoRI digestion (46). The probe was
labeled with [-32P]dCTP using the Prime-a-Gene
labeling system (Promega) to a final specific activity greater than
1 x 108 cpm/µg DNA, and hybridization was performed
at 55 C with QuikHyb hybridization solution (Stratagene). 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).
To clone the equine PGHS-2 gene, a genomic library (Stratagene) was screened according to the manufacturer’s protocol with the mouse PGHS-2 cDNA fragment described above. Seven positive clones were identified from an initial round of 400,000 phage plaques screened. They were purified and initially analyzed by restriction endonuclease mapping with SacI and XbaI and by Southern blot analyses with the mouse PGHS-2 cDNA probe. Fragments yielding a positive signal were subcloned into the vector pGEM 3ZF(-), partially sequenced and compared with the human homolog (47). One of the initial clones, clone 3-1b, was shown to contain the complete equine PGHS-2 gene as well as upstream and downstream DNA sequences. Additional restriction fragments from clone 3-1b were subcloned in pGEM 3ZF(-), and the entire gene (exons and introns) was sequenced. The exon/intron borders were determined by comparison between the genomic and cDNA sequences.
DNA sequencing was performed by the Sanger dideoxy nucleotide chain termination method (48) using the T7 Sequencing Kit (Pharmacia); vector-based primers (T3, T7, or SP6) and specific oligonucleotide primers synthesized as internal PGHS-2 sequences were obtained. Nucleotide and amino acid analyses were performed using the FASTA program of WI Package version 9.0 (Genetics Computer Group, Madison, WI) and the MacDNASIS software version 2.0 (Hitachi, Hialeah, FL).
Primer extension analysis
Primer extension analysis was performed in aqueous buffer as
described previously (49, 50). Briefly, total RNA was extracted with
TRIzol (Life Technologies) from preovulatory follicles isolated 0
h (negative control) or 36 h after administration of hCG. A 24-mer
antisense oligonucleotide (5'-GGCTGGGAGGCAGTGCTGGAGGAG-3') designed
from the equine PGHS-2 cDNA and located between +50 and +73 bp from the
beginning of the longest cDNA clone was end labeled and hybridized
(50,000 cpm/reaction) to 50 µg total RNA at 30 C overnight in 30 µl
buffer (1 M NaCl; 167 mM HEPES, pH 7.5; and
0.33 mM EDTA, pH 8.0). After precipitation, primer
extension was performed by adding 3.5 µl 4 mM dNTPs, 2.5
µl 10 x RT buffer (0.5 M Tris-Cl, pH 8.2; 50
mM MgCl2; 50 µM dithiothreitol;
0.5 M KCl; and 0.5 mg/ml BSA), 1.25 µl RNAsin, 18 µl
H2O, and 40 U AMV reverse transcriptase and incubating at
42 C for 90 min. After extraction and precipitation, the extension
product was analyzed by electrophoresis on a 6% polyacrylamide-7
M urea gel, and its size was determined by comparison with
two sequencing reactions run in adjacent lanes. One reaction involved
the same oligonucleotide used for primer extension and an equine PGHS-2
genomic clone spanning this region, whereas the other sequencing
reaction used an unrelated primer and a template of known sequence.
Isolation and dissection of equine preovulatory follicles
Standardbred and Thoroughbred mares were teased daily with a
pony stallion for detection of estrus, and ovarian follicular
development was monitored daily by transrectal real-time
ultrasonography, as previously described (44). During estrus and when
the preovulatory follicle reached 35 mm in diameter, hCG (2500 IU, iv)
was administered, and ovariectomy was performed via colpotomy 0, 12,
24, 30, 33, 36, and 39 h post-hCG treatment with a chain ecraseur
(n = 4–5 follicles/time point) (44). During the procedure,
neuroleptanalgesia was induced with a combination of xylazine (Rompun;
0.65 mg/kg, iv), butorphanol (Torbugesic; 0.005 mg/kg, iv), and
detomidine (Dormosedan; 0.02 mg/kg, iv), as previously described (44).
The recovered ovary was immersed in ice-cold Eagles’s MEM supplemented
with penicillin (50 U/ml)-streptomycin (50 µg/ml; Life Technologies),
L-glutamine (2.0 mM; Life Technologies), and
nonessential amino acids (0.1 mM; Life Technologies). All
animal procedures were approved by the comité de
déontologie animale of the University of Montreal.
The preovulatory follicle was dissected into three cellular preparations using a methodology previously described (44). They included pieces of follicle wall (theca interna with attached granulosa cells) and isolated preparations of theca interna and granulosa cells. All samples were stored at -70 C until RNA extraction.
RNA extraction and Northern blot analysis
Total cellular RNA was extracted from equine tissues using
TRIzol (Life Technologies) 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 50%
deionized formamide-6% formaldehyde, electrophoresed in a 1%
formaldehyde-agarose gel, and transferred onto a nylon membrane as
previously described (36, 37). 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. The membrane was first hybridized to the
32P-labeled equine PGHS-2 cDNA probe using QuikHyb solution
(Stratagene) as described above. After stripping the radioactivity with
0.1% SSC (standard saline citrate)-0.1% SDS for 30 min at 100 C, the
same blot was subsequently hybridized with a rat elongation factor Tu
(EFTu) cDNA as a control gene for RNA loading and transfer (51).
Statistical analysis
Changes in relative levels of mRNA during the ovulatory process
were quantified by determining on autoradiograms the optical density of
the PGHS-2 band with a computer-assisted image analysis system (Collage
Macintosh program, Fotodyne, New Berlin, WI). The EFTu signal was also
scanned and used to normalize results. For each cellular preparation,
data were expressed as ratios of PGHS-2 mRNA to EFTu and are presented
as the mean ± SEM (n = 4 follicles/time point).
One-way ANOVA was used to test the effect of time after hCG treatment
on relative PGHS-2 mRNA levels. When ANOVAs indicated significant
differences (P < 0.05), Dunnett’s test was used for
multiple comparisons with the control (0 h post-hCG). Data were
transformed to logarithms before analysis when heterogeneity of
variance was observed with the Hartley test. Statistical analyses were
performed using the JMP Sortware (SAS Institute, Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
large 3'-UTR was found to contain numerous (n = 11) repeats of the
Shaw-Kamen’s sequence (ATTTA) (52), a motif typically associated with
short half-life of mRNAs (Fig. 1B).
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). Results showed that the open reading
frame encodes an equine protein that is identical in length (604 amino
acids) to PGHS-2 of other mammalian species and is 1 amino acid longer
than chicken PGHS-2. The equine enzyme appears to share all the
important structural and functional domains implicated in PGHS function
(Fig. 2). Comparisons between equine PGHS-2 and other mammalian
homologs revealed more than 80% identity at the amino acid and nucleic
acid levels.
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) using a
24-mer antisense oligonucleotide (5'-GGCTCCGAGGCAGTGCTGGAGGAG-3') and
RNA extracted from a preovulatory follicle isolated 36 h post-hCG
treatment. The extension products were resolved on a 6% denaturing
polyacrylamide gel, and their sizes were characterized by comparison
with an adjacent sequencing reaction. Results showed the presence of
two extension products that differed by only 1 base, with their sizes
corresponding to 81 and 82 nucleotides (Fig. 3). The 81-nucleotide
extension product was considered as the principal transcription
initiation site because the band had a greater intensity (Fig. 3B).
Therefore, we conclude that the longest cDNA clone (clone 6) begins 9
bp downstream of the start site, and that the full-length 5'-UTR of the
equine PGHS-2 mRNA has 128 bp. No extension product was detected when
follicular RNA isolated 0 h post-hCG was used (Fig. 4B).
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). The sizes of
all internal exons (exons 2–9) and the coding region of exons 1–10
are identical between the 2 species. However, the length of the 5'-UTR
of exon 1 and that of the 3'-UTR of exon 10 differed (Fig. 4). The
coding nucleotide sequence of the genomic clone was identical to that
of the cDNA clones. Figure 5 shows that
intron size varies from 119 bp (intron 2) to 802 bp (intron 1), and
that each splice site agrees with the consensus donor/acceptor (GT/AG)
sequence.
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). No PGHS-2 mRNA was
detected between 0–24 h post-hCG. Transcripts (4 kb) first appeared
30 h post-hCG treatment, reached maximal levels at 33 h, and
progressively decreased thereafter. Hybridization of the same membrane
with a cDNA encoding rat EFTu confirmed uniform RNA loading (Fig. 6B).
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). No
transcripts were detected in theca interna, except for a relatively
weak signal in one sample (36 h post-hCG; Fig. 7) that probably
resulted from contamination by granulosa cells.
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). In addition to
PGHS-2, the EFtu band was also scanned to normalize for RNA loading and
transfer. The results showed a significant increase (P
< 0.05) in PGHS-2 mRNA levels between 30–36 h post-hCG in follicle
wall and granulosa cells compared with levels present at 0 h (Fig. 8, A and B). In contrast, PGHS-2 signals remained very low or
undetectable in all theca interna samples, and no significant
differences were observed at any time point (Fig. 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One interesting structural feature of the equine PGHS-2 transcript is the presence of numerous Shaw-Kamen’s sequences (5'-ATTTA-3') in the 3'-untranslated region. This motif has previously been shown to be present in several immediate early genes and to confer instability to mRNAs (52, 55). The number of repeats in equine PGHS-2 (n = 11) is comparable to the numbers observed in PGHS-2 of other species (n = 8–16) (8, 9, 11, 13, 14). Interestingly, although the position of several motifs varies among species, a group of five Shaw-Kamen’s repeats is consistently found within the first 80 nucleotides downstream of the translation termination codon of all PGHS-2 transcripts (9, 11, 13, 14), suggesting their greater relative importance in mediating mRNA degradation. Rapid turnover of PGHS-2 mRNA has previously been shown in different cell types and probably relates to the need for a tight regulation of gene expression considering the potent biological effects of prostanoids (3, 4, 5).
To date, the primary structure of the PGHS-2 gene had been characterized only in human (56) and mouse (57). This study documents the exon/intron organization of the equine gene. Compared with those of mouse and human PGHS-2, the genomic structure of equine PGHS-2 is highly conserved, with 10 exons and nine introns (56, 57). Internal exons 2–9 and the coding regions of exons 1 and 10 of the equine gene are identical in size to their human and mouse counterparts. However, differences are observed in the size of the untranslated region of exon 1: 129, 134, and 122 nucleotides in length for the equine, human, and mouse genes, respectively (11, 56). Transcription of the equine PGHS-2 gene starts at an adenosine residue, which is identical to the that in the rat (50) and mouse (11) but distinct from the human cap site identified as a cytidine (56). Also, important variations are observed among species in the length of the 3'-UTR in exon 10, which correlates with overall differences observed in the sizes of cloned cDNAs. Although rat and mouse PGHS-2 cDNAs are approximately 4.0 kb (9, 11), equine and human cDNAs are relatively shorter, only 3.4 kb (13). Results from genomic sequencing in the horse show that the stretch of adenosines found at the end of our cDNA clones may, in fact, correspond to a 21-base adenosine repeat present in the corresponding region of the PGHS-2 gene. This finding suggests that our cDNA clones may have been reverse transcribed from an internal poly(A)+ sequence in the 3'-UTR instead of the poly(A)+ tail, and therefore, the full-length cDNA could be longer than reported herein. Similar conclusions can be drawn for the apparent small size of the human PGHS-2 cDNA (13, 56).
A unique time course of induction of PGHS-2 mRNA was observed in a series of equine preovulatory follicles isolated between 0–39 h after hCG treatment. Induction of PGHS-2 transcript in granulosa cells was first detected only 30 h post-hCG. This impressive delay in agonist induction of PGHS-2 gene expression is unprecedented. In other cell types, the regulation of PGHS-2 is more rapid, being induced within 1 h by 12-O-tetradecanoylphorbol-13-acetate or lipopolysaccharide in fibroblasts, macrophages, endothelial cells, and mesangial cells (11, 13, 58, 59). Although PGHS-2 is considered an early response gene in fibroblasts, its very delayed induction in equine granulosa cells suggests that it does not serve this role in ovarian cells. Wong et al. (60) have shown that induction of PGHS-2 transcript by gonadotropins in rat granulosa cells is dependent on protein synthesis, which shows that it is not an early response gene in follicular cells. The cellular localization of PGHS-2 mRNA in equine follicles complements a similar result recently reported for the PGHS-2 protein (44). Also, the delayed induction of the transcript coincides with the late detection of the protein and of follicular PG synthetic activities (44, 61). Collectively, these results clearly suggest that the transcriptional regulation of the PGHS-2 gene in equine granulosa cells is a relatively long molecular process (30 h post-hCG) compared with its regulation in rat (2–4 h post-hCG) (36) and bovine (18 h post-hCG) (37) preovulatory follicles. The apparent relationship between the progressively delayed induction of PGHS-2 transcripts in species with long ovulatory processes further supports a putative role of PGHS-2 as a determinant of the mammalian ovulatory clock (45).
In summary, this study documents for the first time the primary structure of the equine PGHS-2 gene, transcript, and protein, and comparative analyses further underscore the highly conserved structure of the enzyme across species. Studies of the regulation of PGHS-2 mRNA in equine follicles during the ovulatory process reveal a time-dependent (30 h post-hCG) and granulosa cell-specific induction of the transcript. The regulation of PGHS-2 gene expression in equine granulosa cells is a uniquely delayed molecular event compared with its regulation in follicles of other species with shorter ovulatory processes and its rapid agonist-dependent induction in other cell types. The characterization of the equine PGHS-2 promoter and the development of homologous ovarian cell culture system are currently underway to provide a model to study the molecular basis for the delayed transcriptional activation of PGHS-2 in species with a long ovulatory process.
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Acknowledgments |
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Footnotes |
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Received October 6, 1997.
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References |
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secretion in cultured endometrial cells.
Endocrinology 138:237–247 receptor in bovine preovulatory follicles.
Endocrinology 137:3348–3355[Abstract]
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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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J. Liu, M. Antaya, D. Boerboom, J. G. Lussier, D. W. Silversides, and J. Sirois The Delayed Activation of the Prostaglandin G/H Synthase-2 Promoter in Bovine Granulosa Cells Is Associated with Down-regulation of Truncated Upstream Stimulatory Factor-2 J. Biol. Chem., December 3, 1999; 274(49): 35037 - 35045. [Abstract] [Full Text] [PDF]
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D. Boerboom, A. Kerban, and J. Sirois Dual Regulation of Promoter II- and Promoter 1f-Derived Cytochrome P450 Aromatase Transcripts in Equine Granulosa Cells during Human Chorionic Gonadotropin-Induced Ovulation: A Novel Model for the Study of Aromatase Promoter Switching Endocrinology, September 1, 1999; 140(9): 4133 - 4141. [Abstract] [Full Text]
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A. Kerban, D. Boerboom, and J. Sirois Human Chorionic Gonadotropin Induces an Inverse Regulation of Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid in Theca Interna and Granulosa Cells of Equine Preovulatory Follicles Endocrinology, February 1, 1999; 140(2): 667 - 674. [Abstract] [Full Text]
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F. Filion, N. Bouchard, A. K. Goff, J. G. Lussier, and J. Sirois Molecular Cloning and Induction of Bovine Prostaglandin E Synthase by Gonadotropins in Ovarian Follicles Prior to Ovulation in Vivo J. Biol. Chem., August 31, 2001; 276(36): 34323 - 34330. [Abstract] [Full Text] [PDF]
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