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Endocrinology Vol. 140, No. 2 632-640
Copyright © 1999 by The Endocrine Society

ARTICLES

Unique Regulation of CYP17 Expression in the Trophectoderm of the Preattachment Porcine Blastocyst1

Xin Chu2, C. Jo Corbin, Michael A. Kaminski3 and Alan J. Conley

Department of Animal and Range Sciences (X.C., M.A.K.), North Dakota State University, Fargo, North Dakota 58105; and Department of Population Health and Reproduction (C.J.C., A.J.C.), University of California, Davis, California 95616

Address all correspondence and requests for reprints to: Dr. A. J. Conley, VM-PHR, School of Veterinary Medicine, University of California at Davis. Davis, California 95616-8743. E-mail: ajconley{at}ucdavis.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of the gene encoding cytochrome P450 17{alpha}-hydroxylase, CYP17, is necessary for adrenal and gonadal steroidogenesis in most species. However, some animals, such as the pig, express CYP17 in the trophectoderm of the preattachment blastocyst, an event associated with estrogen synthesis and the establishment of pregnancy. How trophoblastic expression of CYP17 is regulated in the porcine blastocyst remains unknown and forms the basis of the following studies. The porcine CYP17 gene, including the complete coding and several kilobases of 5'-flanking regions, was cloned and sequenced. Blastocysts were examined by Northern analysis to verify the level of CYP17 transcript, and tissue-specific expression in the trophectoderm was confirmed by in situ hybridization. Primer extension, S1 nuclease protection, and 5'-rapid amplification of cDNA ends confirmed a common proximal transcription start site in adrenals and gonads (-48 bp) but identified a unique distal start site used in porcine trophectoderm (-182 bp). Additionally, reporter analysis of the CYP17 regulatory region demonstrated that constructs (-27 to -718 bp) were unresponsive to forskolin when expressed in porcine trophoblast cells, suggesting that trophoblast may not be able to respond to cAMP induction of this gene. The identification of this distal, previously undescribed, transcriptional start site suggests that unique mechanisms control the expression of CYP17 in porcine trophectoderm and possibly other genes important in implantation and early placental development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGEN, estrogen, and cortisol synthesis from pregnenolone is dependent on 17{alpha}-hydroxylase/C17,20 lyase cytochrome P450 (P450c17), a pivotal enzyme directing gonadal, adrenal, and in some species, placental steroidogenesis (1). Consequently, the regulation of P450c17 expression, particularly the induction by cAMP, has drawn considerable interest (2) based on the results of experiments showing the importance of transcriptional control in bovine adrenocortical cells (3). Many recent studies have identified the cAMP-response elements that promote the gonadal and adrenocortical expression of the gene (CYP17) encoding this enzyme in several species. Specifically, cAMP-response sequences have been reported for the human, bovine, rat, mouse, and porcine CYP17 genes (4, 5, 6, 7, 8, 9). Adrenal and gonadal CYP17 expression may be modulated by additional elements that bind trans-acting factors such as the orphan nuclear receptor SF-1 (10, 11, 12) or otherwise respond to hormones including angiotensin II (13, 14) and androgens (15).

Cytochrome P450c17 is also expressed in the placenta (16, 17, 18, 19, 20) and is particularly important in facilitating estrogen synthesis and the initiation of parturition in certain mammals (21). In addition, estrogen synthesis in porcine (22), equine (23, 24), and rabbit (25) embryos is seen as early as the blastocyst stage even before placental formation and is associated with the initial establishment of pregnancy (26). Results from previous studies in this laboratory and others suggest that CYP17 expression may be important in regulating estrogen synthesis by the porcine preattachment blastocyst. Specifically, CYP17 expression is highly correlated with estrogen content (27), transiently increasing in bilaminar blastocysts and decreasing as they transform morphologically from tubular to filamentous form (27, 28, 29). Immunocytochemical analyses indicate that expression is limited, almost exclusively, to a single germ cell layer, the outer trophectoderm, but not in the endoderm or embryonic disc (30). Therefore, CYP17 expression in the early porcine blastocyst appears to be tightly regulated both temporally and spatially. However, in contrast to CYP17 expression in the gonads and adrenal glands, little is known about the molecular control of CYP17 expression in embryonic tissues or placenta (26).

Based on these observations, and the general lack of knowledge about the regulation of gene expression in mammalian extraembryonic tissues, the following studies were conducted to 1) confirm the tissue-specific expression of CYP17 in the trophectoderm of the preattachment porcine blastocyst by in situ hybridization, 2) determine the transcriptional start site used in trophectoderm, and 3) examine the effect of cAMP stimulation on the CYP17 promoter activity in the porcine trophoblast cells. Analyses included gonadal and adrenal tissues for direct comparison. The results demonstrate the existence and utilization of a unique transcriptional start site used for CYP17 expression in the trophectoderm of the porcine blastocyst.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA isolation and Northern analysis
Total RNA was extracted in guanadinium thiocyanate by cesium chloride gradient centrifugation from porcine tissues that included day 11 blastocysts, adrenal cortex, testes, and theca interna from preovulatory follicles. Poly (A)+ was purified using spin columns (5Prime,3Prime, Boulder, CO), and both total and poly (A)+ RNA was quantified by A260. Northern analysis was performed as previously described (17, 27), and uniformity of loading across lanes was verified by ethidium bromide staining. Membranes were probed with a [35S] random-primed cDNA encoding porcine P450c17 (31).

In situ hybridization and immunocytochemical staining
In situ hybridization was conducted essentially as described by Keeney et al. (32). PCR was used to amplify a 640-bp segment of DNA encoding the 5'-end of the porcine P450c17 testes cDNA, which was subsequently subcloned into pGEM-T (Promega Corp., Madison, WI). After linearization with SacII, a radiolabeled antisense cRNA probe was transcribed in vitro with Sp6 RNA polymerase (Stratagene, La Jolla, CA) and [35S]uridine triphosphate (Amersham Pharmacia Biotech, Arlington Heights, IL). The control sense cRNA probe was generated with T7 RNA polymerase after template linearization with SalI. Porcine day 11 blastocysts were fixed in 4% paraformaldehyde (in ribonuclease-free PBS, pH 7) and embedded in paraffin. A pair of serial sections, 5 µm thick, were dried on ProbeOn Plus slides (Fisher Scientific International, Inc., Santa Clara, CA). Prehybridized sections were deparaffinized and rehydrated followed by treatment with 4% paraformaldehyde, pronase E (protease type XIV; 110 µg/ml), and triethanolamine (0.1 M, pH 8.0) containing acetic anhydride. Separate hybridization mixtures of antisense and sense [35S]uridine triphosphate-labeled cRNA probes were applied to sections on one or the other half of each slide. After overnight hybridization at 50 C, sections were subjected to high-stringency washes and digestion with ribonuclease A (20 µg/ml). Sections were finally dehydrated, air dried, and dipped in Kodak NTB-2 nuclear track emulsion (Eastman Kodak Co., Rochester, NY). Slides were exposed for 3 days, developed photographically, and stained with hematoxylin and eosin. Immunocytochemical staining was also performed on adjacent sections as described previously (30, 33).

Cloning and sequencing of the porcine CYP17
A porcine genomic DNA library was constructed in EMBL3 {lambda}-phage vector according to the manufacturer (Stratagene) and screened by plaque hybridization using full-length pig P450c17 cDNA (31) as a probe. Two positive clones were isolated, subcloned into pUC18 at SalI/EcoRI and BamHI sites, and characterized by restriction mapping and Southern blot analysis. DNA sequence gene was obtained by double-strand dideoxy sequence analysis.

Primer extension
Primer-extended products were synthesized from 5 µg poly (A)+ RNA (from tubular and elongating day 11 blastocysts, as well as preovulatory theca interna) using 200 fmol of a 32P-end-labeled 21-mer oligonucleotide (oligo) primer complementary to the region around +68 bp of porcine CYP17 as a primer (oligo 1, Table 1Go). Templates were heated at 65 C for 15 min, chilled on ice for 2 min, and then incubated in a 50:l reaction mix containing primer, 10 mM dithiothreitol, 0.5 mM KCl, 0.5ml RNasin, 1 mM deoxynucleoside triphosphates, and 200 U MMLV reverse transcriptase (Gibco BRL, Gaithersburg, MD) at 37 C for 1 h. The products were separated on a 6.5% denaturing polyacrylamide gel alongside genomic sequence generated with the same primer.


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  		Table 1. Oligonucleotides (oligos) used for primer extension, S1 nuclease protection assay, 5'-RACE, and construction of reporter plasmids

 
S1 Nuclease protection assay
A 32P-end-labeled single-stranded cDNA fragment was generated by asymmetric PCR (34) using oligo 1 and oligo 2 (Table 1Go). Poly (A)+ RNA (5 µg) from porcine day 11 blastocysts, adrenal cortex, testes, and theca interna were coprecipitated with 0.01 pmol 32P-end-labeled single-stranded DNA, resuspended in 20 µl hybridization buffer (40 mM piperazine-N,N'-bis[2-ethanesulfonic acid], 400 mM NaCl, 1 mM EDTA, 80% formamide), heated at 85 C for 10 min, and rapidly transferred to 37 C water bath. After overnight hybridization, samples were digested with 37.5 U of S1 nuclease (Sigma Chemical Co., St. Louis, MO) in 150 µl digestion buffer (280 mM NaCl, 30 mM NaoAc, 2 mM ZnSO4, 50 µg/ml ssDNA) at 37 C for 1 h. Digestions were stopped by addition of 20 µl termination buffer (1.5 M NaoAc/pH 7, 25 mM EDTA, 15 mM MgCl2), and the products were separated on a 6.5% denaturing polyacrylamide gel together with a genomic sequence ladder starting at the 3'-end of the protected fragment.

5'-Rapid amplification of cDNA ends (RACE)
5'-RACE of porcine blastocyst messenger RNA (mRNA) (2 µg) was performed according to the manufacturer’s recommendation (Gibco BRL). The temperature for cDNA synthesis in these experiments was 42 C. Two nested primers (oligo 1 and oligo 3, Table 1Go) were used as gene-specific primers for cDNA synthesis and subsequent rounds of PCR amplification.

Construction of reporter plasmids
A series of deletion fragments of porcine CYP17 5'-region was generated by PCR using oligos listed in Table 1Go. Each amplified fragment was subcloned into a promoterless luciferase reporter plasmid pGL2-Basic (Promega Corp.). The integrity of inserts in each construct was confirmed by dideoxy sequencing.

Cell culture
The porcine Jag-1 trophoblast cell line (35) was grown in RPMI-1640 with 10% FBS with 1% penicillin/streptomycin, plated in 6-well dishes at a density of 1 x 105 cells per well. MA-10 mouse Leydig cells were grown in Waymouth’s MB 752/1 with 2.24 g NaHCO3/liter, 20 mM HEPES (pH 7.2) with 15% heat-inactivated horse serum, and 50 µg/ml gentamycin (36) in 24-well dishes at a density of 0.5 x 105 cells per well. All the culture media and serum were purchased from Gibco BRL, and all cells were incubated in humidified atmosphere of 5% CO2 at 37 C.

Transfection and luciferase reporter assays
Cells were grown for 24 h (50–70% confluent) and transfected with Lipofectamine (Gibco BRL) following the recommended procedure. Transfections were performed in either 6-well dishes with 0.5 µg plasmid DNA/well (porcine Jag-1 cells) or 24-well dishes with 0.25 µg plasmid DNA/well (MA-10 cells) at a ratio of 1:6 (micrograms of DNA to microliters of lipid) in 0.5 ml (24-well dishes) or 1 ml (6-well dishes) volume. ß-Galactosidase reporter plasmid pSV-ß-Galactosidase (Promega Corp.) was cotransfected with luciferase reporter constructs at a ratio of 1:1, and the resulting expression was used to normalize data. Medium was replaced after 6 h and incubation was continued for an additional 10 h. Fresh growth medium was then added with or without 30 µM forskolin and incubated for an additional 24 h. Luciferase assays were performed on lysed cells exactly as suggested by the manufacturer (Analytical Luminescence Laboratory, Ann Arbor, MI). Cotransfected ß-galactosidase activity was assayed using a chemiluminescent assay kit (CLONTECH, Palo Alto, CA). Light production was measured on a Monolight 2010C luminometer (Analytical Luminescence Laboratory). In addition, negative (pGL2-basic; promoterless) and positive (pGL2-control; SV-40 promoter) control plasmids were included in all transfection experiments with test constructs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genomic sequence analysis
Since the previously published porcine CYP17 genomic sequence (37) contained mismatches and a significant 27-bp deletion in exon II when compared with the published P450c17 cDNA (31), the porcine CYP17 gene was sequenced through the entire coding region. Two positive clones, isolated from a porcine EMBL3 genomic library made in our laboratory, were subcloned into pUC18. Restriction mapping and Southern blot analysis confirmed that these two clones contained all translated exons of CYP17 and several kilobases of 5'- and 3'-flanking sequence. Sequence analysis (GenBank accession numbers U41519, U41520, U41521, U41522, U41523, U41524, U41525) confirmed the genomic organization comprising 8 exons, similar to that of human (38), bovine (39), mouse (8), and rat (7) CYP17. Nucleotide alignment demonstrated that the coding sequence was identical to the published cDNA (31) including all of exon II. In fact, amplification across exon II using genomic DNA from several porcine breeds (data not shown) failed to find evidence of the deletion previously reported by Zhang et al. (37), which therefore most likely represents a library artifact. Overall, the comparison of porcine CYP17 coding sequence plus 500 bp of 5'-flanking regions with the corresponding sequence of the human, bovine, and rat CYP17 showed identities of 75%, 80%, and 69%, respectively.

Transcript analysis
Northern analysis and in situ hybridization. Northern analysis of total RNA, isolated from day 11 porcine blastocysts and theca interna of preovulatory ovarian follicles, demonstrated a strong hybridizing band of 1.7–1.8 kb that was present at comparable levels in both tissues (Fig. 1Go), as confirmed in subsequent transcript analyses. In addition, transcripts expressed in blastocysts appeared to migrate more slowly relative to those from the theca interna, suggesting a possible difference in size and/or sequence, reminiscent of the observation recently reported by Vianello et al. (40) for P450c17 mRNA expression in the rat liver. In situ hybridization (Fig. 2aGo) confirmed the tissue specificity of CYP17 expression as indicated by immunocytochemical analysis (Fig. 2bGo), demonstrating that CYP17 transcripts were expressed principally in the trophectoderm germ cell layer of the developing porcine blastocyst. Transcript abundance was notably lower in the trophectoderm adjacent to the embryonic disc and highest in regions of the trophoblast furthest from it.



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  		Figure 1. Northern analysis of total RNA (20 µg/lane) extracted from day 11 porcine blastocysts and theca interna dissected from preovulatory porcine follicles. Lanes 1 and 2 contain RNA from two independent pools of blastocysts in the elongation stage of development, and RNA from independent pools of preovulatory theca are represented in lanes 3 and 4. Details of the probe are included in the text. Equal loading was verified by ethidium bromide staining.

 


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  		Figure 2. Localization of CYP17 expression in day 11 porcine blastocysts by in situ hybridization (a) and immunocytochemistry (b). Tissue sections were hybridized with 35S-labeled antisense complementary RNA (cRNA) to porcine P450c17 or immunostained using polyclonal antisera raised against porcine P450c17. Note that CYP17 transcripts and protein are localized primarily in the trophectoderm (arrows), rather than the embryonic disc (D) or the endoderm (arrowheads). No hybridization was observed with sense RNA probe or in the absence of primary antibody (data not shown).

 
Transcription start sites in blastocysts, adrenal cortex, and gonads. Based on the results of Northern analysis, and a single polyadenylation site recognized on genomic analysis (data not shown), the possibility that transcripts in the blastocyst and theca interna differed in their 5'- ends was investigated further by primer extension analysis. This analysis (using a primer extending from +58 to +79 bp relative to the ATG; Table 1Go), yielded several larger products when reactions were performed with blastocyst mRNA as template (Fig. 3AGo) but only a single major band with theca interna mRNA (Fig. 3BGo). Of the multiple bands obtained using blastocyst mRNA, the largest suggested that the transcription start site used in blastocyst expression was located 134 bp further upstream than that used for expression in the theca. Since computer analysis indicated the potential for the formation of a high degree of secondary structure within this additional 134- bp region, it was considered likely that the smaller fragments represented premature termination of extension products. Regardless, these data were consistent with the slower migration of blastocyst transcripts on Northern analysis (Fig. 1Go) and the possibility that the transcriptional start site used for expression in porcine blastocysts was different from that used in theca.



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  		Figure 3. Primer extension of porcine blastocyst and theca mRNA. Reactions were performed with 5 µg mRNA from day 11 blastocysts (A) and preovulatory theca interna (B) using an end-labeled primer (oligo 1, Table 1Go), and the products were resolved by electrophoresis as described in the text. Genomic sequence, generated using the same primer, was loaded in adjacent lanes. Arrows indicate the primary product or largest fragment synthesized, and the position of the putative TATA box is also shown.

 
Additional 5'-RACE and nuclease protection analyses were performed to verify the existence and utilization of this putative, unique transcription start site(s) in the blastocyst, to determine whether transcript splicing was involved, and to extend the comparison with other tissues expressing CYP17. Sequence analysis of several cloned inserts resulting from 5'-RACE of blastocyst mRNA (Fig. 4Go) exactly matched the contiguous upstream region of the gene (data not shown), suggesting that splicing was not involved in the synthesis of these transcripts. In addition, the sequence at the 5'-ends of all clones was consistent with the results of primer extension data, suggesting a distal start site of transcription located 182 bp from the initiation codon. This distal transcriptional start site, used for CYP17 expression in trophectoderm, was verified by nuclease protection, comparing transcripts isolated from the blastocyst with those expressed in theca interna, testis, and adrenal glands. Using an asymmetric PCR- generated, single stranded cDNA probe (-194 to +79 bp), a common transcription start site was confirmed at the thymidine position -48 (relative to the ATG) in porcine adrenal, testis, and theca interna (Fig. 5Go). However, S1 nuclease protection assays of porcine blastocyst mRNA using the same probe generated a much larger protected fragment (Fig. 5Go) consistent with transcription initiated from position -182, the most 5'-site suggested by the results of the primer extension and 5'-RACE analyzes. These results demonstrate conclusively that CYP17 expression in the preattachment blastocyst utilizes a unique, alternative transcription initiation site upstream of that used for expression in the porcine adrenal cortex and gonads (Fig. 6Go).



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  		Figure 4. 5'-RACE of mRNA from porcine blastocyst. Shown is an ethidium bromide-stained, 2% agarose gel demonstrating a clear, single amplified fragment that was subsequently cloned and sequenced. The sequence of all clones analyzed exactly matched the genomic sequence indicating that the CYP17 transcript was generated in the blastocyst without alternative splicing.

 


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  		Figure 5. S1 nuclease protection assays of mRNA (5 µg/reaction) from different porcine tissues: testes (lane 1), theca interna (lane 2), blastocyst (lane 3), and adrenal gland (lane 4) generated protected fragments (arrowheads) using a single-stranded cDNA probe generated by asymmetric PCR (oligos 1 and 2, Table 1Go). The adjacent sequence ladder was generated from a cloned fragment of the porcine CYP17 gene using the same downstream primer (oligo 1). These data confirm the alternative start site of transcription used for CYP17 expression in the trophectoderm of the porcine preimplantation blastocyst. In addition, the results further verify that CYP17 expression levels in porcine blastocysts are similar to those seen in gonadal and adrenal tissues.

 


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  		Figure 6. Sequence of porcine CYP17 gene extending upstream from the coding region (+1). The transcription start site used for expression in the adrenal glands and gonads (arrow at -48 bp) is shown in relation to the start site used in the trophectoderm (arrow at -182 bp). The putative TATA element is in boldface type, the conserved SF-1 cis-element is double underlined, and an AP-2 and inverted Sp-1 site are also indicated further upstream. Note that there is no obvious TATA-like element 5' of the distal trophectoderm start site.

 
Gene reporter analysis-cAMP response
Reporter constructs were developed to examine the cAMP responsiveness of putative upstream promoter(s) driving porcine CYP17 expression. Although it is not known whether or not cAMP up-regulates CYP17 expression in trophoblast, it clearly does so in the adrenal cortex and gonads. Therefore, transfection experiments with reporter constructs were conducted in the presence or absence of forskolin (30 µM) not only in porcine Jag-1 trophoblast cells, but also in the mouse MA-10 Leydig tumor cell. Four CYP17/reporter constructs were used (p-27/-152, p-27/-189, p-27/-260, and p-27/-718; Fig. 7AGo) so as to include the proximal putative TATA box, trophoblastic, and adrenal/gonadal transcription start sites and the region believed to mediate cAMP response in the human, bovine, and mouse CYP17 genes. However, despite an increase in basal promoter activity with additional CYP17 gene sequence, none of the constructs tested in Jag-1 cells responded to the addition of forskolin (Fig. 7BGo). This was not due to any inherent or apparent defect in the cell line or the transfection process. Transfection of Jag-1 cells with constructs that included the SV-40 enhancer linked to the promoter region increased reporter activity a further 20- to 30-fold, almost twice the response obtained with these same constructs in MA-10 cells (data not shown). In contrast to lack of cAMP response observed in Jag-1 cells, forskolin increased relative luciferase activity after transfection of MA-10 mouse Leydig cells with the p-27/-189, p-27/-260, and p-27/-718 plasmids by 1.5- to more than 3.5-fold of vehicle controls (Fig. 7CGo). Therefore, although reporter gene responsiveness to forskolin was evident in MA-10 cells, none was evident in the porcine trophoblast cells. As expected, little luciferase activity was detected in either cell line transfected with the promoterless (pGL2-basic) construct with or without forskolin. The pGL2-control exhibited the anticipated high luciferase activity that was generally 10- to 20-fold higher than the most active CYP17/reporter constructs.



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  		Figure 7. Functional luciferase reporter gene analysis of the promoter region of porcine CYP17. In addition to negative, promoterless (pGL2-basic), and positive (SV-40 promoter; not shown) controls, the four porcine CYP17 constructs that were tested included deletion fragments from -27 bp up to -718 bp (A) designated relative to the translation initiation site (+1), and all contained the native TATA-like element. The transcription start sites used for CYP17 expression in porcine adrenal glands (thin arrow, -48 bp) and trophectoderm (thick arrow, -182 bp) are shown. These constructs were transiently expressed in both porcine Jag-1 trophoblast cells (B) and mouse MA-10 Leydig tumor cells (C), treated or not with forskolin (30 µM). Luciferase activity was normalized to the activity of a cotransfected plasmid containing the ß-galactosidase gene. Shown are the means ± SEM of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of in situ hybridization confirmed that the majority of transcripts associated with the first appearance of CYP17 expression in the pig are localized in the trophectoderm at the blastocyst stage of embryonic development, before formation of the embryonic mesoderm (27, 30). Trophectodermal expression in the porcine preattachment blastocyst utilizes a unique, alternative transcriptional start site (at position -182 bp relative to the ATG), which is distinct from that used for expression in the adrenal cortex or gonads (at position -48 bp relative to the ATG). Mapping failed to find evidence for splicing of untranslated exons, a mechanism known to control the tissue-specific expression of other steroid hydroxylases, notably aromatase cytochrome P450 (41). Reporter gene analysis demonstrated that porcine CYP17 constructs (-27 to -718 bp) with promoter activity did not respond to forskolin when transfected into a porcine trophoblast cell line (Jag-1), even though the same constructs were responsive in mouse MA-10 cells. Importantly, a similar lack of cAMP response was reported by Yamamoto et al. (20) in a rat trophoblast cell line. It is important to note also that no TATA-like element was found upstream of the distal transcription start site used for CYP17 expression in porcine trophectoderm. Collectively, these data are consistent with the need and possible existence of a unique mechanism controlling CYP17 expression in the embryonic trophoblast. In fact, to our knowledge, this is the first report to document an alternative transcriptional start site for CYP17 expressed in any tissues of any species examined to date.

The regulation of CYP17 in the adrenal glands and gonads of many species shares some common features that are reflected at the molecular level. High-sequence homology is evident in the 5'-flanking regions (>67% identity in the first 500 bp) of human, mouse, bovine, and porcine CYP17. As shown in Fig. 9, transcripts in these species are all initiated within an 8-bp region (-48 bp for the porcine) downstream of a poorly conserved TATA-like promoter sequence believed to drive expression, at least in adrenal glands and gonads (Refs. 4, 5, 8, 9 and this study). Moreover, similarities extend to cis-regulatory elements. A consensus SF-1 binding sequence exists at almost the same position in all species (Fig. 8Go), consistent with the essential role of this transcription factor in the regulation of adrenal and gonadal steroidogenesis (42, 43). In addition, an inverted Sp-1 site, overlapping with an AP-2 site further upstream (-229 to -242 bp) of the porcine and bovine CYP17, has been reported to positively affect both basal and cAMP- regulated expression (9, 44) and is consistent with the results of the present study. However, species differences are also evident. For example, nonconsensus cAMP response elements have been identified in the human (4), bovine (5, 6), mouse (8), and porcine CYP17 (Ref. 9 and this study), but at different positions in 5'-flanking region. Negative regulatory elements inhibiting CYP17 expression are also activated by androgen in the mouse, and as a result of chicken ovalbumin upstream promoter transcription factor binding in the rat (45) and bovine (10) genes. The lack of androgen inhibition of CYP17 expression in porcine Leydig cells (46) suggests that negative regulation may be more species specific and less conserved than the molecular mechanisms involved in cAMP induction.



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  		Figure 8. Sequence alignment of the regulatory regions of the mouse (M), human (H), bovine (B), and porcine (P) of the CYP17 genes. The putative TATA box is shown in boldface type, and underlined is a putative SF-1 recognition site (thick underline) and an inverted SP1 (thin underline) and an AP-2 site (dotted underline) found upstream in the porcine gene. Also shown are the transcription start sites in boldface (T). Note that homology is high, particularly closer to the downstream transcription start site used in adrenal and gonadal CYP17 expression.

 
The identification of a trophoblast-specifc transcriptional start site strongly suggests the utilization of an alternative promoter driving expression of CYP17 in the porcine blastocyst. The use of alternative promoters for transcription has been reported for several eukaryotic genes and may not be uncommon among genes expressed during early development (47). For instance, expression of the gene encoding alcohol dehydrogenase during embryonic development of Drosophila melanogaster is regulated in a tissue-specific and ontogenic fashion using distal or proximal promoters in different tissues during embryonic stages. Only the distal promoter is used for alcohol dehydrogenase expression in adult tissues (48, 49). The expression of other genes with different start sites of transcription is more often regulated through differential splicing of alternatively used, untranslated exons. Expression of CYP19, the gene encoding aromatase cytochrome P450 (P450arom), is a case in point. Several studies have demonstrated that expression of human P450arom involves alternate transcriptional start sites, promoters, and splicing of untranslated, tissue-specific exons (41). CYP19 is also expressed in the porcine blastocyst (27, 50), and expression is correlated with that of CYP17 (27). However, unlike CYP17 and CYP19 in other mammals, multiple copies of porcine CYP19, clustered on chromosome 1 (51, 52, 53), encode tissue-specific isozymes (52). There appears to be few examples among eukaryotic genes wherein alternative start sites exist on the same exon within just a few hundred bases of one another. Therefore, although the CYP17 and CYP19 are expressed coordinately during porcine blastocyst expansion (27, 28, 29), the expression of CYP17 is still regulated in a fundamentally different fashion from that of CYP19.

Past studies on the regulation of expression of genes encoding the steroid hydroxylase enzymes suggest that the binding of specific transcription factors mediates gonadal and adrenal expression, which is further induced by cAMP. For instance, SF-1, an orphan nuclear receptor originally shown to be critical for fetal adrenal and gonadal development (54, 55), was thought to be essential for initiating steroidogenic enzyme expression in all tissues. However, our data indicate that CYP17 expression in the preattachment porcine blastocyst is unlikely to involve SF-1 or cAMP stimulation. There was no effect of forskolin on promoter activity in trophoblast cells (Fig. 7BGo), and it seems unlikely that the SF-1 binding site located downstream of the promoter driving trophoblastic expression could be involved in regulating transcription in the blastocyst. This is consistent with the observation that the levels of SF-1 are negligible in human trophoblast cells (26) and undetectable in porcine blastocysts by immunoblot analysis (our unpublished observations). What molecular signal initiates CYP17 expression at this early stage of development is unknown. However, an alternative mechanism of transcriptional regulation of CYP17 expression may be necessitated by a lack of SF-1 expression at this stage of development, or by an inability of the trophoblast to respond to cAMP stimulation (20) in general. Strauss et al.. (26) hypothesized the existence of "trophoblast-specific" transcription factors, and several candidate factors have been identified that appear to control trophoblastic differentiation. These include two members of the GATA family of proteins, GATA-2 and GATA-3 (56), and two basic helix-loop-helix factors, Mash-2 (57) and a protein encoded by Hxt (58). Whether or not unique transcription factors bind to a trophoblast-specific promoter region of porcine CYP17 remains to be determined.

The results of functional in vitro reporter assays must also be interpreted with certain reservations. First, quantitative comparisons of reporter activity between cell lines are complicated by potential differences in transfection, transcription, or translation efficiencies. For instance, the positive control (pGL2-control) construct expressed consistently 10-fold higher luciferase activity in the Jag-1 than in the MA-10 cells, but the ß-galactosidase construct, using the same SV-40 promoter, exhibited 100-fold higher galactosidase activity in Jag-1 compared with MA-10 cells. Thus, although useful to correct for transfection efficiency between different constructs within cell lines, inclusion of ß-galactosidase in reported calculations of the relative luciferase activities decreased values for Jag-1 compared with those of the MA-10 cells. Second, it is important to ask whether Jag-1 cells adequately represent the porcine trophoblast cells under study. Although Jag-1 cells are cytokeratin positive and vimentin negative, consistent with an epidermal origin, they do not express appreciable levels of P450c17 (data not shown). This may be a consequence of their derivation from blastocysts recovered on day 14 postmating when CYP17 expression is low (27, 28, 29) or may reflect phenotypic alterations that arise during prolonged growth in vitro. Indeed, as noted earlier, CYP17 expression in vivo is only very transiently elevated in the trophectoderm during blastocyst elongation, a period of perhaps 12–24 h. This is in contrast to the extended period of Cyp17 expression in the trophoblast of the rat, which is seen throughout most of the second half of gestation (18, 19, 20). In addition, it is important to note the results of recent studies in our laboratory indicating that porcine conceptus CYP17 expression is more dependent on the day of gestation than on blastocyst diameter or stage of development (59). This suggests that CYP17 gene expression may be induced in the porcine preattachment conceptus by an as yet unknown, but precisely timed, uterine signal rather than representing a programmed differentiative event in the trophoblast. Further studies to define the promoter element(s) driving trophoblast CYP17 expression may help to clarify these issues.

In conclusion, these data suggest that the regulation of CYP17 expression in porcine preattachment blastocysts involves a novel, tissue-specific transcription start site not previously recognized or identified in the gene of any other species. This observation suggests that trophoblast most likely utilizes a different promoter and possibly unique trans-acting factors than those used for the regulation of this gene in the adrenal cortex or gonads, highlighting the novel or potentially complex control of gene expression in extraembryonic tissues. Studies are in progress to carefully define basal promoter activity and nuclear protein binding to regulatory elements that drive CYP17 expression in the preattachment blastocyst.


   Acknowledgments
 
We thank Dr. Mario Ascoli for providing MA-10 cells and Dr. Robert J. Christopherson for providing Jag-1 cells. We also thank Dr. Claire Mazow Gelfman for assistance with the transfection studies, Dr. Duane Davis for blastocyst tissue collection, and Dr. Diane Keeney for help in establishing in situ hybridization.


   Footnotes
 
1 This work was Supported by USDA Grant 94–37203-1300 (to A.J.C.). Back

2 Current address: Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0412. Back

3 Current address: Department of Cell Biology, Neurobiology, and Anatomy, College of Medicine, University of Cincinnati, Cincinnati Ohio 45267-0521. Back

Received May 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Conley AJ, Bird IM 1997 The role of cytochrome P45017{alpha}-hydroxylase and 3ß-hydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the {Delta}5 and {Delta}4 pathways of steroidogenesis in mammals. Biol Reprod 56:79–799
  2. Waterman MR 1994 Biochemical diversity of cAMP-dependent transcription of steroid hydroxylase genes in the adrenal cortex. J Biol Chem 269:27783–27786[Free Full Text]
  3. John ME, John MC, Boggaram V, Simpson ER, Waterman MR 1986 Transcriptional regulation of steroid hydroxylase genes by ACTH. Proc Natl Acad Sci USA 83:4715–4719[Abstract/Free Full Text]
  4. Brentano ST, Picado-Leonard J, Mellon SH, Moore CCD, Miller WL 1990 Tissue-specific, cyclic adenosine 3',5'-monophosphate-induced, and phorbol ester-repressed transcription from the human P450c17 promoter in mouse cells. Mol Endocrinol 4:1972–1979[Abstract/Free Full Text]
  5. Lund J, Ahlgren R, Wu D, Kagimoto M, Simpson ER, Waterman MR 1990 transcriptional regulation of the bovine CYP17 (P-45017{alpha}) gene. J Biol Chem 225:3304–3312
  6. Demeter-Arlotto M, Michael MD, Kilgore MW, Simpson ER 1996 17{alpha}-hydroxylase gene expression in the bovine ovary: mechanisms regulating expression differ from those in adrenal cells. J Steroid Biochem Mol Biol 59:21–29[CrossRef][Medline]
  7. Givens CR, Zhang P, Bair SR, Mellon SH 1994 Transcriptional regulation of rat cytochrome P450c17 expression in mouse Leydig MA-10 and adrenal Y-1 cells: identification of a single protein that mediates both basal and cAMP-induced activities. DNA Cell Biol 13:1087–1098[Medline]
  8. Youngblood GL, Payne AH 1992 Isolation and characterization of the mouse P450 17{alpha}-hydroxylase/C17–21 lyase gene (Cyp17): transcriptional regulation of the gene by cyclic adenosine 3',5'-monophosphate in MA-10 Leydig cells. Mol Endocrinol 6:927–934[Abstract/Free Full Text]
  9. Zhang P, Han X, Mellon SH, Hall PF 1996 Expression of the gene for cytochrome P-450 17{alpha}-hydroxylase/C17–20 lyase (CYP17) in porcine Leydig cells: identification of a DNA sequence that mediates cAMP response. Biochim Biophys Acta 1307:73–82[Medline]
  10. Bakke M, Lund J 1995 Mutually exclusive interactions of two nuclear orphan receptors determine activity of a cyclic adenosine 3',5'-monophosphate-responsive sequence in the bovine CYP17 gene. Mol Endocrinol 9:327–339[Abstract/Free Full Text]
  11. Zhang P, Mellon SH 1996 The orphan nuclear receptor steroidogenic facter-1 regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17{alpha}-hydroxylase/C17–20 lyase). Mol Endocrinol 10:147–158[Abstract/Free Full Text]
  12. Lund J, Bakke M, Mellgren G, Morohashi K, Drskeland S 1997 Transcriptional regulation of the bovine CYP17 gene by cAMP. Steroids 62:43–45[CrossRef][Medline]
  13. Bird IM, Mason JI, Oka K, Rainey WE 1993 Angiotensin-II stimulates an increase in cAMP and expression of 17{alpha}-hydroxylase cytochrome P450 in fetal bovine adrenocortical cells. Endocrinology 132:932–934[Abstract/Free Full Text]
  14. Mason JI, Bird IM, Rainey WE 1995 Adrenal androgen biosynthesis with special attention to P450c17. Proc Natl Acad Sci USA 774:47–58
  15. Burgos-Trinidad M, Youngblood GL, Maroto MR, Scheller A, Robins DM, Payne AH 1997 Repression of cAMP-induced expression of the mouse P450 17{alpha}-hydroxylase/C17–20 lyase gene (Cyp17) by androgens. Mol Endocrinol 11:87–96[Abstract/Free Full Text]
  16. Mason JI, France JT, Magness RR, Murry BA, Rosenfeld CR 1989 Ovine placental steroid 17{alpha}-hydroxylase/C-17,20-lyase, aromatase and sulphatase in dexamethasone-induced and natural parturition. J Endocrinol 122:351–359[Abstract/Free Full Text]
  17. Conley AJ, Head JR, Stirling D, Simpson ER, Mason JI 1992 Expression of steroidogenic enzymes in bovine placenta and fetal adrenals throughout gestation. Endocrinology 130:2641–2650[Abstract/Free Full Text]
  18. Durkee TJ, McLean MP, Hales DB, Payne AH, Waterman MR, Khan I, Gibori G 1992 P450(17{alpha}) and P450scc gene expression and regulation in the rat placenta. Endocrinology 130:1309–1317[Abstract/Free Full Text]
  19. Johnson DC 1992 Cellular localization and factors controlling rat placental cytochrome P45017{alpha} (CYP17):17{alpha}-hydroxylase/C17,20-lyase activity. Biol Reprod 46:30–38[Abstract]
  20. Yamamoto T, Chapman BM, Johnson DC, Givens CR, Mellon SH, Soares MJ 1996 Cytochrome P450 17{alpha}-hydroxylase gene expression in differentiating rat trophoblast cells. J Endocrinol 150:161–168[Abstract/Free Full Text]
  21. Conley AJ, Mason JI 1993 Steroidogenic enzymes. In: Rice GE, Brennecke SP (eds) Molecular Aspects of Placental and Fetal Membrane Autacoids. CRC Press, Boca Raton, FL, pp 1–25
  22. Perry JS, Heap RB, Amoroso EC 1973 Steroid hormone production by pig blastocyst. Nature 245:45–47[CrossRef][Medline]
  23. Zavy MT, Mayer R, Vernon MW, Bazer FW, Sharp DC 1979 An investigation of the uterine luminal environment of nonpregnant and pregnant pony mares. J Reprod Fertil [Suppl] 27:403–411
  24. Heap RB, Hamon MH, Allen WR 1991 Oestrogen production by the preimplantation donkey conceptus compared with that of the horse and the effect of between-species embryo transfer. J Reprod Fertil 93:141–147[Abstract/Free Full Text]
  25. George FW, Wilson JD 1978 Estrogen formation in the early rabbit embryo. Science 199:200–201[Abstract/Free Full Text]
  26. Strauss III JF, Matinez F, Kiriakidou M 1996 Placental steroid hormone synthesis: unique features and unanswered questions. Biol Reprod 54:303–311[Abstract]
  27. Conley AJ, Christenson RK, Ford SP, Geisert RD, Mason JI 1992 Steroidogenic enzyme expression in porcine conceptuses during and after elongation. Endocrinology 131:896–902[Abstract/Free Full Text]
  28. Green ML, Simmen RCM, Simmen FA 1995 Developmental regulation of steroidogenic enzyme gene expression in the peri-implantation porcine conceptus: a paracrine role for insulin-like growth factor-I. Endocrinology 136:3961–3970[Abstract]
  29. Yelich JV, Pomp D, Geisert RD 1997 Ontogeny of elongation and gene expression in the early developing porcine conceptus. Biol Reprod 57:1256–1265[Abstract]
  30. Conley AJ, Christenson LK, Ford SP, Christenson RK 1994 Immunocytochemical localization of cytochromes P450 17{alpha}-hydroxylase and aromatase in embryonic cell layers of elongating porcine blastocysts. Endocrinology 135:2248–2254[Abstract/Free Full Text]
  31. Conley AJ, Graham-Lorence SE, Kagimoto M, Lorence MC, Murry BA, Oka K, Sanders D, Mason JI 1992 Nucleotide sequence of a cDNA encoding porcine testis 17{alpha}-hydroxylase cytochrome P-450. Biochim Biophys Acta 1130:75–77[Medline]
  32. Keeney DS, Jenkins CM, Waterman MR 1995 Developmentally regulated expression of adrenal 17{alpha}-hydroxylase cytochrome P450c17 in the mouse embryo. Endocrinology 136:4872–4879[Abstract]
  33. Conley AJ, Kaminski MA, Dubowsky SA, Jablonka-shariff A, Redmer DA, Reynolds LP 1995 Immunohistochemical localization of 3ß-hydroxysteroid dehydrogenase and P450 17{alpha}-hydroxylase during follicular and luteal development in pigs, sheep, and cows. Biol Reprod 52:1081–1094[Abstract]
  34. McCabe PC 1990 Production of single-stranded DNA by asymmetric PCR. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols, a Guide to Methods and Applications. Academic Press Inc., San Diego, FL, pp 76–83
  35. Ramsoondar J, Christopherson RJ, Guilbert LJ, Wegmann TG 1993 A porcine trophoblast cell line that secretes growth factors which stimulate porcine macrophages. Biol Reprod 49:681–694[Abstract]
  36. Wang H, Ascoli M 1990 Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA-10 cells with a mutant gene of the type I regulatory subunit of the cAMP-dependent protein kinase. Mol Endocrinol 4:80–90[Abstract/Free Full Text]
  37. Zhang P, Nason TF, Han X, Hall PF 1992 Gene for 17{alpha}-hydroxylase/C17–20 lyase P450:complete nucleotide sequence of the porcine gene and 5' upstream sequence of the rat gene. Biochim Biophys Acta 1131:345–348[Medline]
  38. Picado-Leonard J, Miller WL 1987 Cloning and sequence of the human gene for P450c17 (steroid 17{alpha}-hydroxylase/17–20 lyase): similarity with the gene for P450c21. DNA 6:439–448[Medline]
  39. Bhasker CR, Adler BS, Dee A, John ME, Kagimoto M, Zuber MX, Ahlgren R, Wang X, Simpson ER, Waterman MR 1989 Structural characterization of the bovine CYP17 (17{alpha}-hydroxylase) gene. Arch Biochem Biophys 271:479–487[CrossRef][Medline]
  40. Vianello S, Waterman MR, Valle LD, Colombo L 1997 Developmentally regulated expression and activity of 17{alpha}-hydroxylase/C-17,20-lyase cytochrome P450 in rat liver. Endocrinology 138:3166–3174[Abstract/Free Full Text]
  41. Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Hinshelwood MM, Granham-Lorence S, Amarneh B, Ito Y, Fisher CR, Michael MD, Mendelson CR, Bulun SE 1994 Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev 15:342–355[Abstract/Free Full Text]
  42. Rice DA, Mouw AR, Bogerd AM, Parker KL 1991 A shared promoter element regulates the expression of three steroidogenic enzymes. Mol Endocrinol 5:1552–1561[Abstract/Free Full Text]
  43. Morohashi K, Honda S, Inomata Y, Handa H, Omura T 1992 A common trans-acting factor, ad4-binding protein, to the promoters of steroidogenic P-450s. J Biol Chem 267:17913–17919[Abstract/Free Full Text]
  44. Borroni R, Liu Z, Simpson ER, Hinshelwood MM 1997 A putative binding site for Sp-1 is involved in transcriptional regulation of CYP17 gene expression in bovine ovary. Endocrinology 138:2011–2020[Abstract/Free Full Text]
  45. Zhang P, Mellon SH 1997 Multiple orphan nuclear receptors converge to regulate rat P450c17 gene transcription: novel mechanisms for orphan nuclear receptor action. Mol Endocrinol 11:891–904[Abstract/Free Full Text]
  46. Clark AM, Chuzel F, Sanchez P, Saez JM 1996 Regulation by gonadotropins of the messenger ribonucleic acid for P450 side-chain cleavage, P45017{alpha} -hydroxylase/C17,20-lyase, and 3ß-hydroxysteroid dehydrogenase in cultured pig leydig cells. Biol Reprod 55:347–354[Abstract]
  47. Schibler U, Sierra F 1987 Alternative promoters in developmental gene expression. Annu Rev Genet 21:237–257[CrossRef][Medline]
  48. Fisher JA, Maniatis T 1986 Regulatory elements involved in Drosophila muelleri alcohol dehydrogenase gene. Nucleic Acids Res 13:6899–6917[Abstract/Free Full Text]
  49. Savakis C, Ashburner M, Willis JH 1986 The expression of the gene coding for alcohol dehydrogenase during the development of Drosophila melanogaster. Dev Biol 114:194–207[CrossRef]
  50. Choi I, Simmen, RCM, Simmen FA 1996 Molecular cloning of cytochrome P450 aromatase complementary deoxyribonucleic acid from preimplantation porcine and equine blastocysts identifies multiple novel 5'-untranslated exons expressed in embryos, endometrium and placenta. Endocrinology 137:1457–1467[Abstract]
  51. Choi I, Troyer DL, Cornwell DL, Kirby-Dobbels KR, Collante WR, Simmen FA 1997 Closely related genes encode developmental and tissue isoforms of porcine cytochrome P450 aromatase. DNA Cell Biol 16:769–777[Medline]
  52. Conley AJ, Corbin CJ, Smith T, Hinshelwood MM, Liu Z, Simpson ER 1997 Porcine aromatases: studies on tissue-specific, functionally distinct isozymes from a single gene? J Steroid Biochem Mol Biol 61:407–413[CrossRef][Medline]
  53. Lahbib-Mansais Y, Barbosa A, Yerle M, Parma P, Milan D, Pailhoux E, Gellin J, Cotinot C 1997 Mapping in pig of genes involved in sexual differentiation: AMH, WT1, FTZF1, SOX2, SOX9, AHC, and placental and embryonic CYP19. Cytogenet Cell Genet 76:109–14[Medline]
  54. Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[CrossRef][Medline]
  55. Hatano S, Takayama K, Imai T, Waterman MR, Takakusu A, Omura T, Morohashi K 1994 Sex-dependent expression of a transcription factor, ad4BP, regulating steroidogenic P-450 genes in the gonads during prenatal and postnatal rat development. Development 120:2787–2797[Abstract]
  56. Ma GT, Roth ME, Groskop JC, Tsai F, Orkin SH, Grosveld F, Engel JD, Linzer DH 1997 GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo. Development 124:907–914[Abstract]
  57. Guillemot F, Nagy A, Auerbach A, Rossant J, Joyner AL 1994 Essential role of Mash-2 in extraembryonic development. Nature 371:333–336[CrossRef][Medline]
  58. Cross JC, Flannery ML, Blanar MA, Steingrimsson E, Jenkins NA, Copeland NG, Rutter WJ, Werb Z 1995 Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development. Development 121:2513–2523[Abstract]
  59. Kaminski MA, Ford SP, Conley AJ 1997 Differences in the expression of cytochromes P450 17{alpha}-hydroxylase and aromatase in day 10.5–14 Meishan and Yorkshire conceptuses. J Reprod Fertil 111:213–219[Abstract/Free Full Text]



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