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Biology of Reproduction 64, 206-215 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Equine P450 Cholesterol Side-Chain Cleavage and 3ß-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase: Molecular Cloning and Regulation of Their Messenger Ribonucleic Acids in Equine Follicles During the Ovulatory Process1

Derek Boerboom a, and Jean Sirois2,a

a Centre de Recherche en Reproduction Animale and Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6

ABSTRACT

The preovulatory LH rise is the physiological trigger of follicular luteinization, a process during which the synthesis of progesterone is markedly increased. To study the control of follicular progesterone biosynthesis in mares, the objectives of this study were to clone and characterize the equine cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase (3ß-HSD), and describe the regulation and cellular localization of their transcripts in equine follicles during hCG-induced ovulation. Complementary DNA cloning and primer extension analyses revealed that the equine P450scc transcript is composed of a 5'-untranslated region (UTR) of 52 nucleotides, an open reading frame (ORF) of 1560 nucleotides, and a 3'-UTR of 225 nucleotides, whereas the equine 3ß-HSD mRNA consists of a 5'-UTR of 61 nucleotides, an ORF of 1119 nucleotides, and a 3'-UTR of 432 nucleotides. The equine P450scc and 3ß-HSD ORF encode 520 and 373 amino acid proteins, respectively, that are highly conserved (68–79% identity) when compared to homologs of other mammalian species. Northern blot analyses were performed with preovulatory follicles isolated 0, 12, 24, 30, 33, 36, and 39 h post-hCG, and corpora lutea obtained on day 8 of the cycle. Results showed that levels of P450scc mRNA in follicular wall (theca interna with attached granulosa cells) decreased after hCG treatment (30–39 h versus 0 h post-hCG, P < 0.05), and increased again after ovulation to reach their highest levels in corpora lutea (P < 0.05). Northern blots on isolated cellular preparations revealed that theca interna was the predominant site of P450scc expression in follicles prior to hCG (P < 0.05). However, transcript levels decreased in theca interna between 30–39 h (P < 0.05) and increased in granulosa cells at 39 h (P < 0.05), making the granulosa cell layer the predominant site of P450scc expression at the end of the ovulatory process. A different pattern of regulation was observed for 3ß-HSD, as transcript levels remained constant throughout the luteinization process (P > 0.05). Also, in contrast to other species, expression of 3ß-HSD mRNA in equine preovulatory follicles was localized only in granulosa cells and not in theca interna. Thus, this study characterizes for the first time the complete structure of equine P450scc and 3ß-HSD mRNA and identifies novel patterns of expression and regulation of these transcripts in equine follicles prior to ovulation.

follicle, follicular development, granulosa cells, ovary, theca cells

INTRODUCTION

The cholesterol side-chain cleavage cytochrome P450 (P450scc) and its associated electron-transport chain is the first rate-limiting and hormonally regulated step in the biosynthesis of steroids from cholesterol [1, 2]. The enzyme is located on the matrix side of inner mitochondrial membranes and catalyzes the conversion of substrate cholesterol to pregnenolone, a common precursor to all steroid hormones. The primary structure of P450scc has been deduced from its cloning in various species [3–7]. In humans, the P450scc gene spans more than 20 kilobases (kb), is split into nine exons, and encodes a transcript of about 2.0 kb and a protein of 521 amino acids [4, 8].

Once produced from cholesterol, pregnenolone proceeds either via the {Delta}5 steroidogenic pathway and undergoes 17{alpha}-hydroxylation to become 17-hydroxypregnenolone or enters the {Delta}4 pathway and is converted to progesterone [1, 2]. The enzyme 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4-isomerase (3ß-HSD) catalyzes the synthesis of progesterone from pregnenolone, as well as the conversion of other {Delta}5-3ß-hydroxysteroids into the corresponding {Delta}4-3-ketosteroids [9–12]. Thus, 3ß-HSD is essential for the biosynthesis of all classes of steroid hormones, including progesterone, androgens, estrogens, glucocorticoids, and mineralocorticoids [9–12]. The enzyme, located in the endoplasmic reticulum and in mitochondrial membranes [13–15], is expressed to high levels in classic steroidogenic tissues (i.e., gonads, adrenal cortex, and placenta), as well as in various peripheral tissues where it could play an important role in intracrine steroid synthesis [9–12, 16]. Multiple genes encoding distinct 3ß-HSD isoforms have been characterized in humans, rats, mice, and hamsters [9–12]. They are expressed in a tissue-specific manner and are under distinct mechanisms of regulation [9–12]. The adrenal/gonadal 3ß-HSD isoform in humans is referred to as type II, whereas the same isoform is designated type I in other species, in reference to the chronology of their cloning [10].

The LH preovulatory rise is the physiological trigger of follicular luteinization, a process during which progesterone biosynthesis is markedly increased [17]. The molecular basis of this event has been studied in various species, with results often revealing differences in the regulation and cellular distribution of key steroidogenic enzymes across species [18–24]. The transcriptional regulation of three steroidogenic genes, including the steroidogenic acute regulatory protein (StAR), P450scc, and 3ß-HSD genes, are thought to play a major role in the control of progesterone biosynthesis [9, 25, 26]. A recent study in the horse documented a unique inverse regulation of StAR mRNA in theca interna and granulosa cells of equine follicles prior to ovulation [27]. However, in contrast to other species, the gonadotropin-dependent control of P450scc and 3ß-HSD expression in equine follicular cells has remained largely uncharacterized. Therefore, the general objective of this study was to describe the regulation of P450scc and 3ß-HSD in equine preovulatory follicles. The specific objectives were to clone and characterize the primary structure of equine P450scc and 3ß-HSD, and describe the regulation and cellular localization of their transcripts in equine follicles during hCG-induced ovulation.

MATERIALS AND METHODS

Materials

Rompun was obtained from Haver (Bayvet Division, Shawnee, KS); dormosedan was purchased from SmithKline Beecham, Animal Health (West Chester, PA); hCG was obtained from The Buttler Company (Columbus, OH); torbugesic was purchased from Fort Dodge Laboratories Inc. (Fort Dodge, IA); lutalyse was purchased from UpJohn (Kalamazoo, MI); [{alpha}-32P]dCTP, [{gamma}-32P]ATP, and [35S]dATP were obtained from Mandel Scientific NEN Life Science Products (Mississauga, Ontario); TRIzol total RNA isolation reagent, RNA ladder (0.24–9.5 kb), synthetic oligonucleotides, and culture media were purchased from Life Technologies Inc. (Gaithersburg, MD); avian myeloblastosis virus (AMV) reverse transcriptase, RNAsin, DNA 5'-End Labeling System, Prime-a-Gene labeling system, and AMV reverse transcriptase were obtained from Promega (Madison, WI); Biotrans nylon membranes (0.2 µm) were purchased from ICN Pharmaceuticals (Montreal, Quebec, Canada); QuikHyb hybridization solution was obtained from Stratagene Cloning Systems (La Jolla, CA); T4 polynucleotide kinase and all sequencing reagents were purchased from Pharmacia Biotech Inc. (Baie D'Urfé, Québec, Canada); electrophoretic reagents were purchased from Bio-Rad Laboratories (Richmond, CA); and Kodak film X-OMAT AR was obtained from Eastman Kodak Company (Rochester, NY).

Cloning of Equine Cytochrome P450scc and 3ß-HSD cDNAs

The equine P450scc and 3ß-HSD cDNAs were cloned using an expression library prepared with mRNA extracted from an equine preovulatory follicle isolated during estrus and with the ZAP-cDNA/Gigapack cloning kit (Stratagene), as previously described [28]. Approximately 100 000 phage plaques were screened with a 1.2-kb EcoRI restriction fragment of the rat P450scc cDNA [5] and a 1.5-kb EcoRI restriction fragment of the bovine 3ß-HSD cDNA [29]. Probes were labeled with [{alpha}-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 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 [30] was performed using the T7 Sequencing Kit (Pharmacia), vector-based primers (T3 and T7), and custom oligonucleotide primers (Gibco BRL). Nucleotide and amino acid analyses were performed with the FASTA program of Wisconsin Package Version 9.0 (Genetics Computer Group, Madison, WI) and the MacDNASIS software version 2.0 (Hitachi, Hialeah, FL).

Primer Extension Analysis

Primer extension assays were performed in aqueous buffer, as described [28, 31]. The primers included a 30-mer antisense oligonucleotide 5'-CTTTGACCAAGACTGAGCGCAGAGGAAGCC-3' corresponding to the region located between 28 and 57 base pairs (bp) from the beginning of the longest P450scc cDNA clone, and a 30-mer antisense oligonucleotide 5'-CACCCAGCCATGGGTAAACCTGTTAGAGTG-3' corresponding to the region located between 21 and 50 bp from the beginning of the longest 3ß-HSD cDNA clone. The primers were end-labeled (DNA 5'-End Labeling System; Promega) and hybridized (50 000 cpm/reaction) to 50 µg of total RNA extracted from a corpus luteum (10 µg; Day 8 of cycle), and RNA extracted from spleen (negative control) at 30°C overnight in 30 µl of 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 of 4 mM dNTPs, 2.5 µl of 10x RT buffer (0.5 M Tris-Cl, pH 8.2, 50 mM MgCl2, 50 µM dithiothreitol, 0.5 M KCl, 0.5 mg/ml BSA), 1.25 µl RNAsin, 18 µl H2O, 40 U AMV reverse transcriptase, and incubating at 42°C for 90 min. After extraction and precipitation, extension products were analyzed by electrophoresis on a 6% polyacrylamide/7 M urea gel, and their size was determined by comparison with the products of either an unrelated equine sequencing reaction that served as a nucleotide ladder (3ß-HSD), or a sequencing reaction that used a corresponding equine P450scc genomic clone (obtained by genomic library screening; Boerboom and Sirois, unpublished data).

Isolation of Equine Preovulatory Follicles and Corpora Lutea

Equine preovulatory follicles and corpora lutea were isolated from Standardbred and Thoroughbred mares at precise stages of equine estrous cycle, as previously described [28, 32]. Ovulation was induced with hCG (2500 IU, i.v.) during estrus when the preovulatory follicle reached 35 mm in diameter. 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 per time point) [28]. In this model, ovulation occurs between 39 and 42 h after hCG treatment [28, 32]. Additional hemiovariectomies were performed during the luteal phase (Day 8 of cycle) to isolate three corpora lutea. The recovered ovary was kept in ice-cold Eagles's minimal essential medium (MEM) supplemented with penicillin (50 U/ml)-streptomycin (50 µg/ml), L-glutamine (2.0 mM), and nonessential amino acids (0.1 mM). Preovulatory follicles and corpora lutea were dissected from the surrounding ovarian tissues with a scalpel. Follicles were dissected into three cellular preparations using a methodology previously described [28, 33]. Briefly, the follicle was cut into several pieces, and under a dissecting microscope, the theca externa and other surrounding tissues were dissected away from the theca interna using fine forceps. The resulting theca interna with attached granulosa cells was subsequently referred to as a follicular wall preparation. Some pieces of follicular wall were further dissected into isolated preparations of granulosa cells and theca interna by gently scraping the theca interna with a bent glass Pasteur pipette. Granulosa cells were recovered by centrifugation. With this approach, the relative purity of each cellular preparation is thought to exceed 95% based on the selective expression of P450 17{alpha}-hydroxylase-C17–20 lyase and P450 aromatase mRNAs by theca interna and granulosa cells, respectively [34]. All samples were stored at -70°C until RNA extraction. Animal procedures were approved by the institutional animal use and care committee.

Ribonucleic Acid Extraction and Northern Blot Analysis

Ribonucleic acid was extracted from equine tissues using TRIzol (Life Technologies) and a Kinematica PT 1200C Polytron Homogenizer (Fisher Scientific). Northern blot analyses were performed as described [28, 35]. The RNA samples (10 µg) were denatured at 55°C for 15 min in denaturing buffer, electrophoresed on a 1% formaldehyde-agarose gel, and transferred by capillarity to a nylon membrane [28, 35]. A ladder of RNA standards was run with each gel, and ethidium bromide (10 µg) was added to each sample prior to electrophoresis to compare RNA loading and determine migration of standards. Hybridization was performed using QuikHyb solution (Stratagene) and the following cDNA probes: a 0.7-kb HindIII/PstI fragment of the equine P450scc cDNA, a 0.9-kb HindIII/SacI fragment of the equine 3ß-HSD cDNA, and the rat elongation factor Tu cDNA (EFTu) as a control gene for RNA loading [36]. Each cDNA was labeled by random oligonucleotide-primed synthesis to a final specific activity greater than 1 x 108 cpm/µg DNA using [{alpha}-32P]dCTP and the Prime-a-Gene labeling system (Promega), and following the manufacturer's protocol. Stripping of hybridization signal between successive rounds of probing was achieved by soaking filters in 0.1% SSC (SSC is 0.15 M NaCl and 0.015 M sodium citrate) and 0.1% SDS for 20 min at 100°C.

Statistical Analysis

Relative levels of P450scc, 3ß-HSD, and EFTu mRNAs were quantified by densitometric analysis of autoradiogram bands using a computer-assisted image analysis system (Collage Macintosh program, Fotodyne Inc., New Berlin, WI). Data were expressed as ratios of P450scc to EFTu, and 3ß-HSD to EFTu prior to analyses (n = 4 follicles [or mares]/time point). Statistical analyses were performed using JMP Software (SAS Institute Inc., Cary, NC). One-way ANOVA was used to test the effect of time after hCG on relative levels of P450scc and 3ß-HSD mRNAs. When ANOVAs indicated significant differences (P < 0.05), the Tukey-Kramer test was used to compare individual means.

RESULTS

Characterization of Equine Cytochrome P450scc

Twelve positive clones isolated from the primary screening were selected for purification and in vivo excision, and extensive DNA sequencing was performed on the three longest cDNA clones. Results revealed that the longest equine P450scc cDNA consisted of a 5'-untranslated region (5'-UTR) of 14 bp, an open reading frame (ORF) of 1560 bp, and a 3'-UTR of 225 bp (Fig. 1). The coding region encodes a 520-amino acid protein, which is identical in length to that of goat [7], sheep [7], cow [3], and pig [6] P450scc, but 1 and 6 amino acids shorter than that of the human [4] and rat protein [5], respectively (Fig. 2).



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  		FIG. 1. Isolation and characterization of the nucleotide sequence of the equine P450scc cDNA. A) Schematic representation of the isolated equine P450scc cDNA clone. The cDNA is composed of a 5'-UTR of 14 bp, an ORF of 1560 bp, and a 3'-UTR of 225 bp. B) Complete nucleotide sequence of the P450scc cDNA clone. The ORF is indicated by uppercase letters, the translation initiation (ATG) and stop (TGA) codons are highlighted in bold, the 5'-UTR and 3'-UTR are shown in lowercase letters, the polyadenylation signal is underlined, and numbers on the right refer to the last nucleotide on that line. The nucleotide sequence was submitted to GenBank (accession number AF031664)



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  		FIG. 2. Predicted amino acid sequence of equine P450scc and comparison with known mammalian homologs. The deduced amino acid sequence of the equine (equ) P450scc is aligned with the caprine (cap), ovine (ovi), bovine (bov), porcine (por), human (hum), and rat homologs. Identical residues are indicated by a printed period. The putative mitochondrial leader sequence cleavage site is indicated by an inverted arrowhead. The first three boxed regions represent proposed substrate binding regions A, B, and C, whereas the fourth box represents the proposed heme-binding region. Note that the third box also encompasses the domain proposed to bind adreno-ferredoxin. Numbers on the right refer to the last amino acid residue on that line

Primer extension analysis was used to determine the size of the complete P450scc 5'-UTR. A major 95-nucleotide extension product was produced when the primer was hybridized to a sample known to contain P450scc mRNA (corpus luteum) (Fig. 3A). From this result, it is deduced that the longest isolated P450scc cDNA appeared to lack the first 38 nucleotides of the full-length transcript, giving a complete 5'-UTR of 52 bp.



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  		FIG. 3. Primer extension analyses of equine P450scc and 3ß-HSD mRNAs. Two antisense oligonucleotides corresponding to regions located within the first 60 nucleotides of the cloned P450scc and 3ß-HSD cDNAs were hybridized to RNA samples containing (corpus luteum [CL], Day 8 of cycle) and not containing (spleen) P450scc and 3ß-HSD, and reverse transcription was performed as described in Materials and Methods. Extension products were analyzed on a 6% polyacrylamide gel, and their size was determined by comparison with the products of adjacent sequencing reactions. Results reveal a 95-nucleotide (A) and a 72-nucleotide (B) extension product corresponding to major transcription start sites of P450scc and 3ß-HSD mRNA, respectively

Characterization of Equine 3ß-HSD

The three longest cDNAs obtained from an initial group of 12 positive clones were used for the characterization of equine 3ß-HSD. Sequencing results showed that the longest equine 3ß-HSD clone consisted of a 5'-UTR of 39 bp, an ORF of 1119 bp, and a 3'-UTR of 432 bp (Fig. 4). The 3ß-HSD coding region encodes a 373-amino acid protein that is highly homologous to the adrenal-gonadal 3ß-HSD isoform of other mammalian species [29, 37–41] (Fig. 5). Putative functional regions include two YXXXK motifs that are characteristic of short-chain alcohol dehydrogenase active sites, an amino-terminal GXXGXXG motif thought to form a hydrophobic pocket involved in binding NAD+, and two hydrophobic domains involved in anchoring 3ß-HSD to membranes [9, 11, 42, 43] (Fig. 5).



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  		FIG. 4. Isolation and characterization of the nucleotide sequence of the equine 3ß-HSD cDNA. A) Schematic representation of the isolated equine 3ß-HSD cDNA. The cDNA is composed of a 5'-UTR of 39 bp, an ORF of 1119 bp, and a 3'-UTR of 432 bp. B) Complete nucleotide sequence of the 3ß-HSD cDNA clone. The ORF is indicated by uppercase letters, the translation initiation (ATG) and stop (TGA) codons are highlighted in bold, the 5'-UTR and 3'-UTR are shown in lowercase letters, the polyadenylation signal is underlined, and numbers on the right refer to the last nucleotide on that line. The nucleotide sequence was submitted to GenBank (accession number AF031665)



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  		FIG. 5. Predicted amino acid sequence of equine 3ß-HSD and comparison with known mammalian homologs. The deduced amino acid sequence of the equine (equ) 3ß-HSD is aligned with the murine (mur), rat, hamster (ham), human (hum), macaque (mac), and bovine (bov) homologs. Only primary adrenal-gonadal isoforms are represented. Boxed regions represent YXXXK motifs characteristic of short-chain alcohol dehydrogenase active sites. Double-overlined sequences are hydrophobic and may be involved in anchoring to membranes. The glycines involved in the formation of the hydrophobic cofactor-binding pocket are underlined. Numbers on the right refer to the last amino acid residue on that line

The length of the complete 5'-UTR of the equine 3ß-HSD mRNA was determined by primer extension analysis. Results showed that a single 72-nucleotide extension product was produced when the primer was hybridized to RNA extracted from a corpus luteum (Fig. 3B). Therefore, our longest 3ß-HSD cDNA clone appeared to lack the first 22 nucleotides of the full-length transcript, giving a complete 5'-UTR of 61 bp.

Regulation of Equine P450scc and 3ß-HSD mRNAs in Preovulatory Follicles

To characterize the regulation of P450scc and 3ß-HSD mRNAs during the equine ovulatory process, Northern blot analyses were performed with preovulatory follicles isolated between 0 and 39 h after an ovulatory dose of hCG, and corpora lutea were obtained on Day 8 of the cycle (Fig. 6). The equine P450scc mRNA appeared primarily as a transcript of 2.0 kb in size, but a less abundant transcript of approximately 4.0 kb was detected in samples containing more P450scc (Fig. 6). Two transcripts of comparable intensities, 1.8 and 3.9 kb, were observed for 3ß-HSD in follicular extracts (Fig. 6). When data from all follicular wall samples (n = 4/time point) were quantified by densitometric analyses and corrected with the control gene EFTu, results showed significant changes in levels of P450scc but not in 3ß-HSD during the ovulatory process. Administration of hCG caused a decrease in follicular P450scc mRNA, with levels at 30, 33, 36, and 39 h being significantly lower than at 0 h (P < 0.05). Following ovulation, a significant increase in P450scc mRNA was observed in corpora lutea (P < 0.01), whereas levels of 3ß-HSD remained constant during this period (P > 0.05).



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  		FIG. 6. Regulation of equine P450scc and 3ß-HSD mRNAs in equine preovulatory follicles during hCG-induced ovulation. Preparations of follicular wall were obtained from preovulatory follicles isolated between 0 and 39 h after hCG, and corpora lutea (CL) were isolated on Day 8 of the estrous cycle. Samples of total RNA (10 µg/lane) were analyzed by Northern blotting using an equine P450scc cDNA probe (A), an equine 3ß-HSD cDNA probe (B), and the rat EFTu as a control gene for RNA loading (C). Markers on the right indicate the size of the transcripts. Filters in A, B, and C were exposed to film at -70°C for 15, 24, and 13 h, respectively

Cellular Localization of P450scc and 3ß-HSD Expression in Equine Follicles

Northern blot analyses were performed on isolated preparations of granulosa cells and theca interna to study the relative contribution of each steroidogenic cell type in follicular P450scc and 3ß-HSD mRNA expression. Prior to hCG treatment (0 h), theca interna was clearly the predominant site of P450scc expression in the follicle, as levels of transcripts were higher in theca than in granulosa cells (P < 0.01, Fig. 7). Levels of P450scc mRNA remained unchanged between 0 and 24 h in theca interna, but a significant decrease was observed at 30–39 h post-hCG (P < 0.05, Fig. 7). In granulosa cells, levels of P450scc transcripts were relatively low and remained unchanged between 0 and 36 post-hCG, but a significant increase was observed at 39 h post-hCG (P < 0.05, Fig. 7). Interestingly, the predominant site of P450scc mRNA expression in the preovulatory follicle switched from the theca interna layer at 0 h to the granulosa cell layer at 39 h post-hCG (Fig. 7).



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  		FIG. 7. Relative changes of P450scc and 3ß-HSD mRNA levels in equine follicle cells isolated between 0 and 39 h after hCG treatment. Samples (n = 10 µg) of total RNA extracted from granulosa cells and theca interna were analyzed by Northern blotting with the equine P450scc cDNA, the equine 3ß-HSD cDNA, and the rat EFTu cDNA as a control gene for RNA loading. After autoradiography (films not shown), the signal intensity was quantified by densitometric analysis, and data from steroidogenic transcripts were normalized with the control gene EFTu. Results are presented as a ratio of P450scc to EFTu ([P450scc/EFTu] x 100) and a ratio of 3ß-HSD to EFTu ([3ß-HSD/EFTu] x 100) (mean ± SEM; n = 4 follicles [i.e., mares]/time point). Columns marked with an asterisk are significantly different (P < 0.05) from 0 h post-hCG

The cellular localization and regulation of 3ß-HSD transcripts in equine ovarian cells differed from that observed for P450scc. Expression of 3ß-HSD mRNA was observed predominantly, if not exclusively, in granulosa cells (Fig. 7). The administration of hCG had no significant effect on 3ß-HSD transcripts, with levels remaining unchanged in granulosa cells throughout the ovulatory process (P > 0.05, Fig. 7). A very weak 3ß-HSD signal was detected in few theca interna samples that likely resulted from contaminating granulosa cells.

DISCUSSION

In contrast to other species, the molecular control of follicular steroidogenesis has remained largely uncharacterized in mares. Yet, this species provides an interesting model for the study of gonadotropin-dependent gene expression in the ovary, considering the large size of the equine preovulatory follicle (40–45 mm in diameter), and the ability to monitor precisely follicular development by ultrasound imaging [44, 45]. We have recently described the regulation of transcripts coding for key steroidogenic proteins and enzymes in equine follicles during the ovulatory process, including mRNAs for StAR, cytochrome P450 17{alpha}-hydroxylase-C17–20 lyase (P45017{alpha}), and cytochrome P450 aromatase (P450arom) [27, 34]. To provide a more complete understanding of the control of equine follicular steroidogenesis, the present study reports the molecular cloning and characterization of equine P450scc and 3ß-HSD and the regulation and cellular localization of corresponding transcripts in a series of preovulatory follicles isolated between 0 and 39 h after an ovulatory dose of hCG. The equine P450scc and 3ß-HSD mRNAs were found to encode 520- and 373-amino acid proteins, respectively, which is highly similar in length to corresponding enzymes in other mammalian species [3–7, 29, 37–41]. The amino acid sequence of the equine P450scc showed a high degree of conservation when compared to that of other mammalian homologs (71–79% identical to goat, sheep, cow, pig, human, and rat P450scc), particularly within regions proposed to be involved in binding the substrate and a prosthetic heme group [3–7]. The deduced amino acid sequence of equine 3ß-HSD was also highly similar to other species homologs, being 68–79% identical to rat [37], mouse [38], hamster [41], human [40], macaque [39], and cow [29] adrenal-gonadal-type 3ß-HSD. Additional studies will be needed to determine whether multiple isoforms of 3ß-HSD are present in the horse, as observed in numerous species [9–12].

To our knowledge, the overall regulation of P450scc mRNA in equine follicles prior to ovulation is unique and thus adds to the diversity of paradigms observed for the control of P450scc expression in preovulatory follicles of other species [5, 19–21, 46–51]. Prior to hCG treatment, P450scc mRNA was relatively high in theca interna and low in granulosa cells of equine follicles, which compares with observation in pigs [21, 50] but differs from reports in humans [19] and cows [20, 48] who express high levels of transcripts in granulosa cells prior to LH-hCG surge. This finding suggests that the equine theca interna plays a major role in follicular steroidogenesis prior to the gonadotropin rise, as implied previously from the theca cell-selective expression of StAR and P45017{alpha} transcripts in equine follicles at this stage [27, 34]. Induction of the ovulatory-luteinization process with hCG leads to a downregulation of follicular P450scc mRNA and a unique cellular redistribution of the transcript. Similar studies in other species revealed that a downregulation of P450scc also occurs in cows [20, 48] after the LH surge, whereas transcript levels remain unchanged in porcine [21] and ovine [46] follicles and increase in rat follicles after the surge [47, 51]. However, the cellular redistribution of P450scc mRNA, defined as the disappearance of the transcript in theca interna and the concurrent increased expression in granulosa cells, is unprecedented in other species. This phenomenon could be related to the putative degeneration of the theca interna at the time of ovulation in mares, which is unique to this species and leads to the formation of a corpus luteum solely derived from granulosa cells [52]. The timing of the loss of P450scc mRNA in theca interna coincides with the disappearance of StAR and P45017{alpha} transcripts in this cell type [27, 34], providing further biochemical evidence for a putative demise of the equine theca interna prior to ovulation. The marked increase in P450scc expression observed in the equine corpus luteum is in keeping with observations in other species [20, 46, 49, 51, 53–55].

The present study documents a novel pattern of 3ß-HSD mRNA expression in equine follicular cells during the ovulatory process. The presence of 3ß-HSD transcript in equine granulosa cells and its absence or very low expression in theca interna contrast with findings in other species [19–21, 23, 24, 46, 56, 57]. However, this pattern agrees with a previous report showing that 3ß-HSD activity was present in granulosa cells of large follicles isolated during estrus but absent in theca interna of all equine follicles tested [58]. Thus, the equine theca interna presumably does not produce much progesterone in vivo, although elevated expression of StAR [27] and P450scc mRNAs in this cell type suggests that it synthesizes large amounts of pregnenolone precursors. This model is further supported by studies in vitro showing that cultures of equine theca interna secrete negligible amounts of progesterone, and that gonadotropins have no effect on its secretion [33]. The apparent lack of modulation of 3ß-HSD mRNA expression in equine granulosa cells during the ovulatory process contrasts with the downregulation observed in cows [20]. In other species such as the pig and the sheep, 3ß-HSD is not detectable in granulosa cells prior to ovulation but is induced in the corpus luteum [21, 23, 46]. The constant levels of 3ß-HSD mRNA in equine granulosa cells suggest a nonlimiting role for this enzyme during equine terminal follicular steroidogenesis. However, the present study does not exclude the likelihood that the 3ß-HSD expression undergoes regulatory processes at other developmental stages, as reported in other species [24, 46, 57, 59–61]. The detection of two 3ß-HSD transcripts of comparable intensities was unexpected. Whereas the smaller transcript is in keeping with the size of the cloned cDNA, the precise nature of the larger transcript remains unknown but could represent a product derived from an alternative polyadenylation site, as observed for chicken 3ß-HSD [62], or a hybridization artifact. Likewise, the precise nature of a larger, albeit less abundant, P450scc mRNA in some follicular samples remains unknown.

The molecular control of P450scc and 3ß-HSD gene expression in equine ovarian cells remains to be characterized. Several reports in other species have implicated the orphan nuclear receptor steroidogenic factor-1/adrenal 4-binding protein (SF-1/Ad4BP) in the transcriptional regulation of genes encoding steroidogenic enzymes, including P450scc [63–66] and 3ß-HSD [10, 67]. Recent studies have demonstrated that the LH-hCG surge results in a pronounced downregulation of SF-1 mRNA in rat granulosa cells [68, 69]. Thus, the downregulation of P450scc mRNA in equine follicles after the hCG treatment could potentially result, at least in part, from a decrease in transcriptional activity due to a decline in SF-1. However, the fact that 3ß-HSD mRNA levels were unaffected by hCG in equine follicles suggests that, although SF-1 likely plays a role in the control of equine steroidogenesis, additional cell type- and promoter-specific regulatory factors must be involved in the complex differential regulation of steroidogenic genes in theca and granulosa cells.

A working model for the control of equine follicular steroidogenesis is proposed based on results presented in this report and in previous studies [27, 32–34, 52, 58, 70] (Fig. 8). During the early follicular phase, the theca interna layer appears to be the site of very active steroidogenesis; the layer consists of plump polyhedral cells [32, 52] in which high levels of StAR [27], P450scc (this study) and P45017{alpha} mRNAs [34] are either predominantly or exclusively expressed. Because the theca interna expresses very low or undetectable levels of 3ß-HSD mRNA (this study) or activity [58] and produces negligible amounts of progesterone in vitro [33], steroidogenesis likely proceeds primarily via the {Delta}5 pathway to generate the androgen dehydroepiandrosterone (DHEA) (Fig. 8). Estrogens would then be synthesized from DHEA by granulosa cells that are the predominant, if not the only, follicular cells that express 3ß-HSD mRNA (this study) and activity [58], P450arom mRNA [55] and protein [70], and estradiol synthetic capacity [33] (Fig. 8). At the end of the ovulatory process, morphological studies revealed that the equine theca interna undergoes a putative degenerative process [32, 52]. Biochemically, this process is accompanied by an apparent loss of StAR [32], P450scc (this study) and P45017{alpha} [34] in the theca interna layer, and an upregulation of StAR [32] and P450scc mRNA (this study) in granulosa cells, thus putting all enzymes required for progesterone synthesis (i.e., StAR, P450scc, and 3ß-HSD) in the same cell type (Fig. 8). These gonadotropin-dependent changes in enzyme expression would redefine the principal steroidogenic pathway from {Delta}5 to {Delta}4, with progesterone becoming the obligatory end product. An accessory role of the {Delta}4 pathway during the early follicular phase is not excluded; low-level expression of P450scc in granulosa cells could lead to appreciable amounts of progesterone production in that tissue. Then, progesterone would be converted into androstenedione after diffusion to the theca interna layer (Fig. 8). Although this model attempts to integrate current knowledge on the regulation of equine follicular steroidogenesis, its should not be viewed as the definitive paradigm but rather as a working model from which hypotheses can be generated and tested. Importantly, additional studies will be needed to establish a complete relationship between changes in transcripts, proteins, and enzymatic activities, and to unravel the molecular basis for steroidogenic gene expression in equine ovarian cells.



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  		FIG. 8. Proposed model for the regulation of equine follicular steroidogenesis during the early (0 h post-hCG, A) and late follicular phase (39 h post-hCG, B). The model is based on the regulation and cellular localization of transcripts involved in equine follicular steroidogenesis as reported herein and in previous reports [27, 34], as well as on the cellular localization of steroidogenic enzymes [70] or activities [58], the steroidogenic capacity of equine follicular cells in vitro [33], and the histology of equine follicular cells during the ovulatory process [32, 52]. For more details see the text

ACKNOWLEDGMENTS

We thank Dr. J.S. Richards (Baylor College of Medicine, Houston, TX) for the rat P450scc cDNA, Drs. F. Labrie and V. Luu-The (Université Laval, Québec, Canada) for the bovine 3ß-HSD cDNA, and Dr. R. Levine (Cornell University, Ithaca, NY) for the rat EF-Tu cDNA.

FOOTNOTES

First decision: 25 July 2000.

1 This study was supported by Natural Sciences and Engineering Research Council of Canada grant OPG0171135. D.B. is supported by a Medical Research Council (MRC) of Canada Doctoral Research Award. J.S. is supported by an MRC of Canada Scientist Award. Back

2 Correspondence. FAX: 450 778 8103; siroisje{at}medvet.umontreal.ca Back

Accepted: August 22, 2000.

Received: June 13, 2000.

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