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Centre de Recherche en Reproduction Animale (K.A.B., D.B., N.B., J.G.L. and J.S.) and Département de Pathologie et Microbiologie (M.D.), 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, 3200 Sicotte, Saint-Hyacinthe, Québec, Canada J2S 7C6. E-mail: jean.sirois{at}umontreal.ca.
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Introduction |
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17ßHSD4 is recognized as an unusual 17ßHSD because of its multifunctional domain structure and its unique localization to peroxisomes (13, 14, 15). It is a 80-kDa protein with three distinct functional domains: an N-terminal dehydrogenase domain sharing homology with other members of the SDR superfamily, a central hydratase domain similar to that of peroxisomal enzymes involved in ß-oxidation of fatty acids, and a C-terminal sterol carrier protein 2-like domain that potentially facilitates lipid and sterol transfer between membranes (16, 17, 18, 19). The N-terminal dehydrogenase domain is responsible not only for the oxidation of 17ß-estradiol, but also for the dehydrogenation of fatty acyl-coenzyme A. The full-length protein can be cleaved posttranslationally at its N terminus, resulting in a 32-kDa fragment containing the dehydrogenase domain, but the extent of the processing was shown to vary greatly among tissues. The enzyme is expressed ubiquitously, but little is known about the physiological regulators of 17ßHSD4 expression (20, 21, 22, 23). Mutated forms of 17ßHSD4 have been associated with a fatal peroxisomal genetic disorder in humans (24, 25), and the intact protein has recently been identified as a possible antigen in a neurological autoimmune disease (26).
In mammals, the preovulatory period is accompanied by dramatic changes in ovarian follicular steroidogenesis as the LH surge triggers follicular luteinization, a process during which the predominant steroid produced switches from 17ß-estradiol to progesterone (27, 28, 29). The loss in 17ß-estradiol biosynthetic capacity after the LH surge has been explained by a marked decrease in the expression of key steroidogenic enzymes involved in the follicular production of active estrogens (28, 30). However, there has been no attempt to determine whether an enzyme with 17ß-estradiol-inactivating capacity, such as 17ßHSD4, is also regulated during the luteinization process. In the present study the equine preovulatory follicle is used as model to investigate the regulation of 17ßHSD4 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The specific objectives were to clone equine 17ßHSD4 and determine the expression of its mRNA and protein in preovulatory follicles after hCG treatment.
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Materials and Methods |
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-32P]Deoxy-CTP was obtained from Mandel Scientific-NEN Life Science Products (Mississauga, Canada). TRIzol total RNA isolation reagent, 1-kb DNA ladder, and synthetic oligonucleotides were purchased from Invitrogen Life Technologies (Burlington, Canada). Biotrans nylon membranes (0.2 µm) were obtained from ICN Pharmaceuticals, Inc. (Montréal, Canada). Hybond-P polyvinylidene difluoride membranes, Rainbow molecular weight markers, ECL Plus, horseradish peroxidase-linked donkey antirabbit secondary antibody, pGEX-2T vector, BL-21 protease-deficient Escherichia coli strain, and glutathione-Sepharose beads were purchased from Amersham Pharmacia Biotech (Baie D’Urfé, Canada). The Vectastain ABC kit was obtained from Vector Laboratories, Inc. (Burlingame, CA). The Bio-Rad protein assay and all electrophoretic reagents were purchased from Bio-Rad Laboratories (Richmond, CA). hCG was obtained from The Buttler Co. (Columbus, OH). Diaminobenzidine tetrahydrochloride was purchased from Sigma-Aldrich Corp. (St. Louis, MO).
Cloning of the equine 17ßHSD4 cDNA
Isolation of equine 17ßHSD4 cDNA was performed by a combination of RT-PCR and cDNA library screening. A cDNA fragment was first isolated using 500 ng pooled equine ovarian RNA, oligonucleotide primers (sense primer, 5'-AAAGCAGTGGCCAACTATGA TTCAG-3'; antisense primer, 5'-TTGCTGGCATTGTCAAAGTCACAGA-3') designed by sequence alignments of known 17ßHSD4 species homologs, and the Access RT-PCR kit (Promega), as directed by the manufacturer. Ovarian RNA samples were prepared from preovulatory follicles isolated before or after hCG treatment and a corpus luteum obtained on d 8 of the cycle (d 0 = day of ovulation), as previously described (31). The cDNA fragment was subcloned into the pGEM-T Easy plasmid vector (Promega Corp.) and sequenced by Service de Séquençage de l’Université Laval (Québec, Canada). As a second approach, an equine cDNA library prepared with mRNA from a preovulatory follicle obtained 36 h after hCG treatment was screened with the equine RT-PCR 17ßHSD4 cDNA fragment as previously described (32). The probe was labeled with [-32P]deoxy-CTP using the Prime-a-Gene labeling system (Promega Corp.) to a final specific activity greater than 1 x 108 cpm/µg DNA, and hybridization was performed at 68 C with QuickHyb hybridization solution (Stratagene). Positive clones were plaque-purified through secondary and tertiary screenings, pBluescript phagemids containing the cloned DNA inserts were excised in vivo with the Ex-Assist/SOLR system (Stratagene), and DNA sequencing was performed commercially as described above.
Equine tissues and RNA extraction
Equine preovulatory follicles and corpora lutea were isolated at specific stages of the estrous cycle from standardbred and thoroughbred mares, 3–10 yr old and weighing approximately 375–450 kg, as previously described (32). Briefly, when preovulatory follicles reached 35 mm in diameter during estrus, the ovulatory process was induced by injection of hCG (2500 IU, iv), and ovariectomies were performed via colpotomy using an ovariotome at 0, 12, 24, 30, 33, 36, or 39 h post-hCG (n = 4–6 mares/time point). 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, as previously described (33). Ovariectomies were also performed on d 8 of the estrous cycle (d 0 = day of ovulation) to obtain corpora lutea (n = 3 mares). Testicular tissues were obtained from the Large Animal Hospital of Faculté de Médecine Vétérinaire (Université de Montréal) after routine castration, whereas other nonovarian tissues were collected at a local slaughterhouse. All animal procedures were approved by the institutional animal use and care committee. Total RNA was isolated from tissues with TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions using a Kinematica PT 1200C Polytron homogenizer (Fisher Scientific, Montréal, Canada).
Semiquantitative RT-PCR and Southern analysis
The Access RT-PCR System (Promega) was used for semiquantitative analysis of 17ßHSD4 and rpL7a mRNA levels (control gene) in equine tissues. Reactions were performed as directed by the manufacturer, using sense (5'-TAAGCCACTTCCCAGAACGGG-3') and antisense (5'-AGCTC CCCACCCTTAGAGGG-3') primers specific for equine 17ßHSD4. Sense (5'-ACAGGACATCCAGCCCAAACG-3') and antisense (5'-GCTCCTTTGTCTTCCGAGTTG-3') primers specific for equine rpL7a were designed from a published sequence deposited in GenBank (accession no. AF508309). These reactions resulted in the production of 17ßHSD4 and rpL7a DNA fragments of 624 and 516 bp, respectively. Each reaction was performed using 100 ng total RNA, and cycling conditions were one 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, 58 C for 1 min, and 68 C for 2 min. The number of cycles used was optimized for each gene to fall within the linear range of PCR amplification and were 15 and 18 cycles for 17ßHSD4 and rpL7a, respectively. After PCR amplification, samples were electrophoresed on 2% Tris-acetic acid-EDTA-agarose gels, transferred to nylon membranes, and hybridized with corresponding radiolabeled 17ßHSD4 and rpL7A cDNA fragments using QuikHyb hybridization solution (Stratagene). Membranes were exposed to a phosphor screen, and signals were quantified on a Storm imaging system using the ImageQuant software version 1.1 (Molecular Dynamics, Sunnyvale, CA).
Production of an antiequine 17ßHSD4 antibody
A pair of sense (5'-GATGGATCCAGAGTTCATTTGCGGGGCTCC-3') and antisense (5'-CAGAATTCCTGATGTGGCCGTTGATGCCGC-3') primers that incorporated a BamHI and an EcoRI restriction site, respectively, was designed from the equine 17ßHSD4 open reading frame to generate a fragment spanning the region from Arg121 to Ser322 The fragment was amplified by PCR using the Expand High Fidelity polymerase (Roche, Indianapolis, IN), and following the manufacturer’s protocol. The fragment was isolated after electrophoresis, digested with BamHI and EcoRI, subcloned into pGEX-2T in-frame with the glutathione-S-transferase (GST) coding region (Amersham Pharmacia Biotech, Arlington Heights, IL), and sequenced to confirm its identity. Protease-deficient E. coli BL-21 (Amersham Pharmacia Biotech) were transformed with the 17ßHSD4/pGEX-2T construct, expression of recombinant 17ßHSD4/GST fusion protein was induced with isopropyl-ß-D-thiogalactoside, and bacterial protein extracts were obtained after sonication and centrifugation, as previously described (34, 35). The 17ßHSD4/GST fusion protein was purified by affinity on glutathione-Sepharose beads (Amersham Pharmacia Biotech), digested with thrombin to release the 17ßHSD4 fragment, resolved by one-dimensional SDS-PAGE, transferred on nitrocellulose, and stained with Ponceau S Red (33, 34). The 17ßHSD4 band (molecular weight, 21,800) was cut and used to immunize rabbits.
Cell extracts and immunoblot analysis
Cell extracts were prepared as previously described (35). Briefly, tissues were homogenized and sonicated on ice in TED buffer [20 mM Tris (pH 8.0), 50 mM EDTA, and 0.1 mM diethyldithiocarbamic acid) containing 1.0% Tween. The sonicates were centrifuged at 16,000 x g for 15 min at 4 C. The recovered supernatant (whole cell extract) was stored at -80 C until electrophoretic analyses were performed. Protein concentration was determined by the method of Bradford (36) (Bio-Rad protein assay). Samples (60 µg proteins) were resolved by one-dimensional SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (35). Membranes were incubated with the polyclonal antiequine 17ßHSD4 antibody (1:1,000), and immunoreactive proteins were visualized on Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) after incubation with the horseradish peroxidase-linked donkey antirabbit secondary antibody (1:10,000 dilution) and the enhanced chemiluminescence system (ECL Plus), following the manufacturer’s protocol (Amersham Pharmacia Biotech). Autoradiograph images were digitized using a ScanMaker 4 flatbed scanner (Microtek Lab, Inc., Redondo Beach, CA), and signal intensities were quantified using ImageQuant software version 1.1 (Molecular Dynamics).
Immunohistochemical localization of 17ßHSD4
Immunohistochemical staining was performed using the Vectastain ABC kit (Vector Laboratories, Inc.), as previously described (37). Briefly, formalin-fixed tissues were paraffin-embedded, and 3 µm-thick sections were prepared and deparaffined through a graded alcohol series. Endogenous peroxidase was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min. After rinsing in PBS for 15 min, sections were incubated with diluted normal goat serum for 20 min at room temperature. The anti-17ßHSD4 antibody was diluted in PBS (1:1000 dilution) and applied, and sections were incubated overnight at 4 C. Control sections were incubated with PBS. After rinsing in PBS for 10 min, a biotinylated goat antirabbit antibody (1:222 dilution; Vector Laboratories, Inc.) was applied, and sections were incubated for 45 min at room temperature. Sections were washed in PBS for 10 min and incubated with avidin DH-biotinylated horseradish peroxidase H reagents for 45 min at room temperature. After washing with PBS for 10 min, the reaction was revealed using diaminobenzidine tetrahydrochloride as the chromogen. Sections were counterstained with Gill’s hematoxylin stain and mounted.
Statistical analysis
One-way ANOVA was used to test the effect of time after hCG administration on levels of 17ßHSD4 mRNA in samples of follicle wall, corpora lutea, theca interna, and granulosa cells. 17ßHSD4 mRNA levels were normalized with the control gene rpL7a before analysis. One-way ANOVA was also used to test the effect of time after hCG treatment on levels of 17ßHSD4 protein in follicle wall preparations. When ANOVAs indicated significant differences (P < 0.05), Dunnett’s test was used for multiple comparisons of individual means. Statistical analyses were performed using JMP software (SAS Institute, Inc., Cary, NC).
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). When this fragment was used as a probe to screen an equine follicular cDNA library, five positive clones were obtained from a screen of approximately 125,000 phage plaques, of which two (clones 4 and 5) were selected for purification and DNA sequencing. Results revealed that the equine 17ßHSD4 cDNA was composed of a 5'-untranslated region of 103 bp, an open reading frame of 2208 bp (including the stop codon), and a 3'-untranslated region of 341 bp containing two overlapping 5'-AATAAA-3'polyadenylation signals (Fig. 1).
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). The equine 17ßHSD4 sequence was shown to be highly conserved when compared with other species homologues, with a greater than 81% identity observed at the amino acid level and greater than 82% identity at the nucleic acid level. The N-terminal dehydrogenase, central hydratase, and C-terminal sterol carrier protein 2 (SCP2)-like domains that characterize the multifunctional domain structure of 17ßHSD4 are conserved in the equine protein (Fig. 2). Moreover, the localization of equine 17ßHSD4 to peroxisomes is also predicted based on the Ala-Lys-Leu tripeptide peroxisomal targeting signal present at the C terminus (Fig. 2) (15, 41). The equine protein was shown to contain several consensus glycosylation sites (Fig. 2), as observed in other species (13, 38, 40), but their biological significance remains unclear, because other reports indicate that 17ßHSD4 is not glycosylated (42).
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). High levels of 17ßHSD4 mRNA were observed in testis and in a preovulatory follicle isolated 36 h after hCG; moderate levels were detected in brain, heart, liver, kidney, and skeletal muscle; and low to very low levels were present in other tissues studied (Fig. 3).
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). This increase appeared transient, as levels of 17ßHSD4 in a d 8 corpus luteum returned to those present before hCG treatment (Fig. 4A). When results from multiple follicles and corpora lutea were expressed as ratios of 17ßHSD4 to rpL7a, a significant increase in 17ßHSD4 transcript was detected in follicles 24 and 36 h after hCG treatment (P < 0.05), but not in follicles 12 h after hCG or in corpora lutea (Fig. 4C). No significant change was observed in levels of rpL7a transcript in follicle wall and corpora lutea preparations (Fig. 4B).
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). The results revealed that both cell types were involved in 17ßHSD4 mRNA expression within the follicle, with the granulosa cell layer apparently providing the predominant site of 17ßHSD4 induction. In granulosa cells, a significant increase in 17ßHSD4 mRNA was observed 30, 36, and 39 h after hCG (P < 0.05; Fig. 5A). In contrast, the increase in 17ßHSD4 transcript in theca interna was more modest and became significant only 24 h after hCG treatment (P < 0.05), and levels tended to decrease 36 and 39 h after hCG treatment.
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). Interestingly, Western blot analyses revealed that the 80,000 molecular weight signal increased significantly between 24–39 h after hCG treatment compared with that at 0 h (P < 0.05; Fig. 6). In contrast, the 45,000 molecular weight band remained constant between 0–30 h and decreased at 36 and 39 h after hCG administration, whereas no significant difference was observed across time for the 32,000 molecular weight band (Fig. 6).
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). The administration of hCG caused a marked expansion of the granulosa cell layer and severe edema and vascular changes in thecal layers, as previously characterized (43). A marked increase in 17ßHSD4 immunoreactivity was observed in granulosa and thecal cells of follicles isolated 39 h after hCG treatment (Fig. 7, B–D). Also, the elevated levels of 17ßHSD4 mRNA detected by RT-PCR in the testis (Fig. 3
) appeared associated with intense 17ßHSD4 immunostaining in Leydig cells of equine testicular sections (Fig. 7E).
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and P450arom, which contribute to reducing estrogen biosynthesis, and an increase in steroidogenic acute regulatory protein and cytochrome P450 cholesterol side-chain cleavage, which promote enhanced progesterone synthesis (28, 30, 44, 45). Although differences in cellular localization and time-course regulation of these enzymes have been observed among species, they are clearly recognized as part of the common biochemical basis for the switch in predominantly 17ß-estradiol to progesterone production occurring after the LH surge, including in the equine preovulatory follicle (31, 46, 47). Results from the present study suggest that the induction of an 17ß-estradiol-inactivating enzyme such as 17ßHSD4 may represent a novel and complementary mechanism contributing to the reduction in levels of active estrogens during follicular luteinization. Previous investigations of the expression of 17ßHSD4 in the ovary have primarily been limited to tissue distribution analyses using commercial human RNA membranes (13, 48) or Northern blots and immnohistochemistry performed on various porcine tissues collected randomly at the slaughterhouse (42). Overall, these studies revealed that 17ßHSD4 was expressed at low to moderate levels in ovarian tissues, which agrees with levels observed in the wall of equine preovulatory follicles collected before hCG treatment. However, this is the first study to investigate the expression of the enzyme in a developmental series of preovulatory follicles collected during gonadotropin treatment and to establish the presence of high levels of 17ßHSD4 36 h after hCG treatment. High levels of 17ßHSD4 were also observed in the equine testis, with expression located primarily in Leydig cells, as observed in the pig (42). The ubiquitous pattern of expression of the enzyme in equine tissues as well as differences in the predominant sites of 17ßHSD4 expression among tissues are also in keeping with observations in other species (13, 21, 42, 48, 49). However, the presence of moderate levels of 17ßHSD4 in equine liver, heart, kidney, and muscle compares with reports in humans (13, 48).
The control of 17ßHSD4 expression in vivo remains largely uncharacterized. Progesterone has been identified as a positive regulator of the enzyme in the pig uterus (21, 50), but it is not clear whether this effect is conserved in other species (51). Several environmental toxicants acting as peroxisome proliferators have also been shown to affect steroid metabolism and up-regulate the expression of 17ßHSD4, a peroxisomal enzyme (23, 52). The net effect of these chemicals in female rats was to reduce serum levels of 17ß-estradiol, which was originally proposed to involve the increased expression of estrogen-inactivating 17ßHSD4 in the liver (23). Interestingly, peroxisome proliferators have also recently been shown to suppress P450arom and increase 17ßHSD4 in cultured rat granulosa cells, providing evidence for a concomitant effect in the ovary (51, 53). The present study identifies high/ovulatory levels of gonadotropins as a physiological regulator of 17ßHSD4 in the preovulatory follicle. Basal expression and hCG-dependent regulation of follicular 17ßHSD4 mRNA was observed in both granulosa and theca interna cells, but the up-regulation of the transcript clearly appeared more pronounced in the granulosa cell compartment. The development and use of an equine anti-17ßHSD4 antibody revealed the presence of three immunoreactive bands thought to correspond to full-length (80,000 molecular weight band) and proteolytic C-terminal (45,000 molecular weight band = hydratase + SCP2-like domain) and N- terminal (32,000 molecular weight band = hydrogenase domain) fragments, based on reports in other species (18, 54). The hCG-dependent increase in full-length 17ßHSD4 protein (80,000 molecular weight band) detected by immunoblots was in keeping with results identified for the transcript. However, the precise biochemical basis for the apparent reduction in the 45,000 molecular weight band and unchanged low levels of the 32,000 molecular weight band remains unclear and will require further investigations. Likewise, the relatively robust increase in 17ßHSD4 immunoreactivity observed in thecal cells 39 h after hCG treatment was somewhat surprising given the relatively low levels of transcripts at that time, and additional studies will be needed to resolve this issue.
The multifunctional nature of the 17ßHSD4 protein led investigators to question the predominant role of the enzyme under physiological conditions. Because the rate of fatty acyl-coenzyme A oxidation is several-fold higher than that of 17ß-estradiol, some proposed that the enzyme may be primarily involved in fatty acid metabolism, and that estrogen inactivation may represent only a secondary activity in vivo (4, 18, 19). However, the enzymatic parameters (maximum velocity and Km) of 17ßHSD4 for 17ß-estradiol oxidation are very similar to those of 17ßHSD2, another 17ßHSD isoform expressed in numerous tissues, essentially involved in inactivating circulating androgens and estrogens, and generally thought to protect peripheral tissues from excessive steroid hormone actions (9, 10, 18). Based on previous investigations and results from the present study, it is tempting to propose that the predominant physiological role of 17ßHSD4 may be tissue- and stage-dependent. For example, its expression in liver may serve primarily functions linked to lipid metabolism, whereas the gonadotropin-dependent induction of 17ßHSD4 in preovulatory follicles may relate predominantly to estrogen metabolism. There is a tremendous amount of 17ß-estradiol in the follicular fluid of equine preovulatory follicles, with estimates ranging from 60–135 µg (2–3 µg 17ß-estradiol/ml; total volume of fluid, 30–45 ml) (55, 56, 57). The concentration of the hormone in equine follicles (7.3–11.0 µM) is therefore several-fold higher than the reported Km for this substrate in other species (0.2 µM) (13, 18). Thus, the dramatic reduction in levels of 17ß-estradiol during luteinization may result not only from a decrease in the expression of biosynthetic enzymes, but also from the induction of an intraovarian 17ßHSD4-dependent estrogen-inactivating system. However, one should not exclude that the other domains of the multifunctional protein are also involved in ovarian functions. The localization of 17ßHSD4 in peroxisomes, which are known to play a major role in cholesterol biosynthesis (58, 59), and the presence of an SCP2-like transfer domain in the protein remain intriguing and point to a potential role in cholesterol transport.
In summary, this study is the first to characterize the primary structure of equine 17ßHSD4, to demonstrate the regulation of the enzyme during follicular luteinization, and to identify ovulatory levels of gonadotropins as a positive regulator of 17ßHSD4 expression. The precise physiological significance and molecular control of 17ßHSD4 induction in preovulatory follicles remain to be elucidated. Interestingly, the luteinization/ovulatory process is also accompanied by an induction of follicular progesterone synthesis, progesterone receptors, and peroxisome proliferator-activated receptors (31, 59, 60, 61, 62). Given the putative role of progesterone and peroxisome proliferator-activated receptors in 17ßHSD4 expression (21, 23, 50, 52, 53), it will be important to determine whether they are intermediates in the gonadotropin-dependent induction of the enzyme in follicular cells. With its relatively large size (40–45 mm in diameter) and long ovulatory process (39–42 h after hCG), the equine preovulatory follicle will provide an interesting model to investigate some of these issues.
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Footnotes |
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Abbreviations: GST, Glutathione-S-transferase; hCG, human chorionic gonadotropin; 17ßHSD4, 17ß-hydroxysteroid dehydrogenase type 4; SCP2, sterol carrier protein 2; SDR, short-chain dehydrogenase/reductase.
Received December 17, 2003.
Accepted for publication January 7, 2004.
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-hydroxysteroid dehydrogenase activity. J Biol Chem 268:12964–12969
5-4 isomerase: molecular cloning and regulation of their messenger ribonucleic acids in equine follicles during the ovulatory process. Biol Reprod 64:206–215 and PPAR
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