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Endocrinology Vol. 140, No. 2 667-674
Copyright © 1999 by The Endocrine Society

ARTICLES

Human Chorionic Gonadotropin Induces an Inverse Regulation of Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid in Theca Interna and Granulosa Cells of Equine Preovulatory Follicles1

Abdurzag Kerban2, Derek Boerboom3 and Jean Sirois

Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6

Address all correspondence and requests for reprints to: Dr. Jean Sirois, Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, Québec, Canada J2S 7C6. E-mail: siroisje{at}medvet.umontreal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The time- and gonadotropin-dependent regulation of steroidogenic acute regulatory protein (StAR) has not been characterized in vivo in preovulatory follicles of large monoovulatory species or sexually mature animals. The objectives of this study were to clone equine StAR and describe the regulation of its messenger RNA (mRNA) in equine follicles after the administration of an ovulatory dose of hCG. The screening of an equine follicle complementary DNA (cDNA) library with a mouse StAR cDNA probe revealed two forms of equine StAR that differ only in the length of their 3'-untranslated region (3'-UTR); a long form of 2918 bp and a short form of 1599 bp. The StAR long form cDNA contains a 5'-UTR of 117 bp, an open reading frame (ORF) of 855 bp, and a 3'-UTR of 1946 bp. Primer extension analysis showed that the cDNA clone lacked the first 10 bp of the primary transcript, giving a total of 127 bp for the complete StAR 5'-UTR. The ORF encodes a 285-amino acid protein that is 86–90% identical to StAR of other species characterized to date. The regulation of StAR mRNA in vivo was studied in equine preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h (n = 4–5 follicles/time point) after an ovulatory dose of hCG. Results from Northern blots showed no significant changes in StAR mRNA levels after hCG treatment when analyses were performed on intact follicle wall (theca interna with attached granulosa cells). However, Northern blots performed on isolated follicle cells revealed an unexpected regulation of StAR mRNA. In granulosa cells, StAR transcripts were undetectable at 0 h but were significantly increased at 30 h post-hCG, and this induction was associated with a rise in follicular fluid concentrations of progesterone (P < 0.05). In contrast, StAR mRNA levels were high in theca interna at 0 h, remained unchanged until 33 h post-hCG, and dropped dramatically thereafter (P < 0.05). Thus, this study describes the primary structure of equine StAR, documents the regulation of StAR mRNA in vivo in preovulatory follicles of a large monoovulatory species, and identifies a novel inverse regulation of StAR transcripts in theca interna and granulosa cells of equine follicles before ovulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BIOSYNTHESIS of all steroid hormones begins in mitochondria with the conversion of cholesterol to pregnenolone by the cytochrome P450 cholesterol side-chain cleavage enzyme complex (P450scc) (1, 2). Adequate delivery of hydrophobic cholesterol to the inner mitochondrial membrane, where resides P450scc, is a key rate-limiting step in the acute regulation of steroidogenesis (3, 4, 5). Although the mechanism of intracellular transport of cholesterol to the mitochondrion remains unresolved, its translocation from the outer to the inner mitochondrial membrane appears to involve a protein originally described by Orme-Johnson and collaborators (6, 7) and recently purified, cloned, and named steroidogenic acute regulatory protein (StAR) by Clark et al. (8). StAR is a phosphoprotein synthesized in the cytosol as a short-lived 37-kDa precursor that is processed into more stable 30-kDa proteins after mitochondrial import (9, 10, 11). Interestingly, the 37-kDa precursor protein is believed to represent the active form of StAR involved in moving cholesterol across mitochondrial membranes, whereas the role, if any, of the 30-kDa proteins remains unknown (12, 13). The deduced amino acid sequence of the StAR protein has been characterized in mouse (8), human (14), cow (15), rat (16, 17, 18, 19), sheep (20), pig (21), and hamster (22).

The critical role of StAR in steroid hormone synthesis has been clearly demonstrated using various models, including a biochemically defined in vitro system (11), cultures of intact cells (14, 23), and a targeted gene disruption approach to generate StAR knockout mice (24). Moreover, the finding that mutations within the StAR gene are responsible in humans for congenital lipoid adrenal hyperplasia, an autosomal recessive disease in which the synthesis of all adrenal and gonadal steroid is severely impaired, further underscores the importance of the protein (25, 26).

Results from recent studies have documented the pattern of expression and regulation of StAR in ovarian cells during various physiological processes. High levels of StAR messenger RNA (mRNA) and protein were observed in corpora luteum of sheep (20), cows (15, 21, 27), rats (18, 28), humans (29, 30), and pigs (31). Luteal StAR transcripts were increased by LH and GH in hypophysectomized sheep (20) and by 17ß-estradiol in rabbits (32). In contrast, regression of the corpus luteum is accompanied by a marked decrease in StAR expression (20, 27, 28, 30). StAR is also regulated in a gonadotropin-dependent and stage-specific manner during follicular development (17, 29, 31, 33). Experiments in vitro showed that gonadotropins and activators of the protein kinase A pathway up-regulate StAR expression in granulosa cells (16, 27, 29, 33, 34, 35, 36), whereas PGF2{alpha} and phorbol 12-myristate 13-acetate appeared to be negative regulators of StAR expression in vivo and in vitro (20, 28, 29, 33). The equine CG/hCG-treated immature rat model was used to study the control of StAR expression in vivo in preovulatory follicles (17, 33). However, the regulation of follicular StAR in a more physiological system using sexually mature animals has not been characterized, and in-depth studies in large monoovulatory species are lacking. Therefore, the general objective of this study was to use the equine preovulatory follicle as a model to study the cell-specific and time-dependent regulation of StAR by gonadotropins in vivo. The specific objectives were to clone and characterize equine StAR, describe the regulation of its mRNA in preovulatory follicles after the administration of an ovulatory dose of gonadotropins, and determine the contribution of theca interna and granulosa cells to follicular StAR expression.


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

Cloning and sequencing of equine StAR
To clone the equine StAR complementary DNA (cDNA), an expression library prepared with equine follicle mRNA (37) was screened with a mouse StAR cDNA (8). The probe was labeled with [{alpha}-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. Approximately 100,000 phage plaques were screened, and hybridization was performed at 55 C with QuikHyb hybridization solution (Stratagene). Positive clones were plaque purified through secondary and tertiary screening, and pBluescript phagemids containing the cloned DNA insert were excised in vivo with the Ex-Assist/SOLR system (Stratagene). DNA sequencing was performed by the Sanger dideoxynucleotide chain termination method (38) using the T7 sequencing kit (Pharmacia Biotech) and vector-based primers (T3 and T7), and specific primers synthesized as internal StAR sequences were obtained. Nucleotide and amino acid analyses were performed using the FASTA program of Wisconsin Package version 9.0 (Genetics Computer Group, Madison, WI) and Mac-DNASIS software version 2.0 (Hitachi, Hialeah, FL).

Primer extension analysis
Primer extension analysis was performed in aqueous buffer as previously described (37, 39). The reaction used total RNA extracted with TRIzol (Life Technologies) from a corpus luteum isolated on day 8 of the estrous cycle and from spleen (negative control), and a 30-mer antisense oligonucleotide 5'-GGCTCCGAGGCAGTGCTGGAGGAG-3' corresponding to 46–75 bp of the longest StAR cDNA clone (Fig. 1Go). The extension product was analyzed by electrophoresis on a 6% polyacrylamide-7 M urea gel, and its size was determined by comparison with the products of an unrelated sequencing reaction run in adjacent lanes.



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  		Figure 1. Primary structure of equine StAR cDNA. A, Schematic representation of two forms of equine StAR; the short and the long form differ in the lengths of their 3'-UTR. B, Complete nucleotide sequence of the equine StAR long form obtained from clone 1-2 as described in Materials and Methods. The ORF is indicated by uppercase letters, the translation initiation (ATG) and stop (TAA) codons are highlighted in bold, the 5'-UTR and 3'-UTR are shown in lowercase letters, and numbers on the left refer to the first nucleotide on that line. The first (c) and the last (c) nucleotide of the equine StAR short form cDNA are underlined and in boldface. Nucleotide sequences were submitted to GenBank (accession no. AF031696 and AF031697).

 
Isolation and dissection of equine preovulatory follicles
Standardbred and Thoroughbred mares were teased daily with a pony stallion for detection of estrus, and ovarian follicular development was monitored daily by transrectal real-time ultrasonography (40). Ovulation was induced during estrus with hCG (2500 IU, iv) when the preovulatory follicle reached 35 mm in diameter. Ovariectomy was performed via colpotomy 0, 12, 24, 30, 33, 36, and 39 h post-hCG with a chain ecraseur (n = 4–6 follicles/time point) (41). During the procedure, neuroleptanalgesia was induced with a combination of xylazine (Rompun; 0.65 mg/kg, iv), butorphanol (Torbugesic; 0.005 mg/kg, iv), and detomidine (Dormosedan; 0.02 mg/kg, iv), as described previously (42). The recovered ovary was kept in ice-cold Eagles’s MEM supplemented with penicillin (50 U/ml)-streptomycin (50 µg/ml; Life Technologies), L-glutamine (2.0 mM; Life Technologies), and nonessential amino acids (0.1 mM; Life Technologies). Each preovulatory follicle was dissected into three cellular preparations, as previously described (42, 43). They included pieces of follicle wall (theca interna with attached granulosa cells) and isolated preparations of theca interna and granulosa cells. All samples were stored at -70 C until RNA extraction. Animal procedures were approved by the animal research committee of the University of Montreal.

RNA extraction and Northern blot analysis
Total RNA was extracted with TRIzol (Life Technologies) using a Kinematica PT 1200C Polytron homogenizer (Fisher Scientific International, Inc., Pittsburgh, PA) from equine tissues. For Northern analysis, RNA samples (10 µg) were denatured at 55 C for 15 min in 50% deionized formamide-6% formaldehyde, electrophoresed in a 1% formaldehyde-agarose gel, and transferred onto a nylon membrane as previously described (37). A ladder of RNA standards was run with each gel, and ethidium bromide (10 µg) was added to each sample before electrophoresis to compare RNA loading and determine the migration of standards. The membrane was first hybridized to the 32P-labeled equine StAR cDNA probe using QuikHyb solution (Stratagene, La Jolla, CA). After stripping the radioactivity with 0.1% SSC-0.1% SDS for 30 min at 100 C, the same blot was subsequently hybridized with a rat elongation factor Tu (EFTu) cDNA as a control gene for RNA loading and transfer (44).

Progesterone RIA
Nonextracted aliquots of follicular fluid were assayed for progesterone by a specific RIA (45). The sensitivity of the assay was 7.29 pg/assay tube, and the intra- and interassay coefficients of variations were 11.4% and 18.6%, respectively.

Statistical analysis
One-way ANOVA was used to test the effect of time after hCG administration on relative StAR mRNA levels and concentrations of progesterone in follicular fluids. When ANOVAs indicated significant differences (P < 0.05), Dunnett’s test was used for multiple comparisons with the control (0 h post-hCG). Data were transformed to logarithms before analysis when heterogeneity of variance was observed with the Hartley test. Statistical analyses were performed using software from SAS Institute, Inc. (Cary, NC). Relative levels of StAR mRNA were quantified by determining the optical density of the StAR band on autoradiograms with a computer-assisted image analysis system (Collage Macintosh program, Fotodyne, Inc., New Berlin, WI). The EFTu signal was also quantified and used to normalize results. For each cellular preparation, data were expressed as ratios of StAR mRNA to EFTu and are presented as the mean ± SEM (n = 4 follicles/time point).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the equine StAR cDNA
Twelve positive clones were isolated from an equine follicle cDNA library after an initial screening of approximately 100,000 phage plaques. DNA sequencing analyses revealed that the clones represent two forms of equine StAR, a short form composed of 1599 bp (clone 10-1) and a long form of 2918 bp (clone 1-2). The short and long clones had 5'-untranslated regions (5'-UTR) of 114 and 117 bp, respectively, and a common open reading frame (ORF) of 855 bp (Fig. 1Go). However, they differed in the lengths of their 3'-UTR, corresponding to 630 and 1946 bp in the short and long forms, respectively (Fig. 1Go).

The coding region of equine StAR cDNA encodes a 285-amino acid protein, which is identical in length to human (14), pig (21), and bovine StAR (15, 21), but is one amino acid longer than those of the mouse (8), rat (17, 18, 19), and hamster (22) proteins (Fig. 2Go). Comparison across species indicates that the amino acid sequence of equine StAR is highly similar to that of other mammalian homologs, being 90%, 89%, 88%, 87%, 87%, 86%, and 88% identical to the human, porcine, bovine, murine, rat, hamster, and ovine StAR. Computer analysis of the StAR protein sequence using Prosite PC/Gene (Oxford Molecular Group, Inc., Oxford, UK) identified several potential phosphorylation sites, including two cAMP- and cGMP-dependent protein kinase (Ser56 and Ser195), three protein kinase C (Thr5, Ser13, Ser186), and four casein kinase II (Ser61, Ser69, Thr204, Thr263) phosphorylation sites. Also, a putative mitochondrial transit peptide was predicted in positions 1–55.



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  		Figure 2. Deduced amino acid sequence of equine (equ) StAR and comparison with human (hum), pig, bovine (bov), mouse (mou), rat, hamster (ham), and ovine (ovi) homologs. Ovine StAR has not been fully characterized, and only a partial sequence is shown. Identical residues are indicated by a printed period. Potential phosphorylation sites for cAMP- and cGMP-dependent protein kinase (A), protein kinase C (B), and casein kinase II (C) are highlighted in bold uppercase letters.

 
Length of the StAR 5'-UTR
Primer extension analysis was used to determine the full length of the StAR 5'-UTR. One major extension product was produced with RNA extracted from a corpus luteum, whereas none was generated when negative control spleen RNA was used (Fig. 3Go). The size of the extension product, as determined by comparisons with an unrelated sequencing reaction run in adjacent lanes, was 85 nucleotides. Therefore, our longest StAR cDNA clone (clone 1-2) appears to lack 10 nucleotides of the primary transcript, suggesting a full-length 5'-UTR of 127 nucleotides.



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  		Figure 3. Primer extension analysis of equine StAR mRNA. A labeled 30-mer antisense oligonucleotide complementary to the region from 46–75 bp of the StAR long form cDNA (Fig. 1Go) was hybridized to RNA samples containing (corpus luteum) and not containing (spleen) StAR, and primer extension was performed as described in Materials and Methods. Reactions were analyzed on a 6% polyacrylamide gel, and the size of the extended product was determined by comparison with the products of an unrelated sequencing reaction shown on the left. The results show a 85-nucleotide extension product corresponding to a major transcription initiation site. No extension product was detected with RNA isolated from spleen (negative control).

 
Inverse regulation of StAR mRNA in granulosa cells and theca interna
To study potential changes in StAR mRNA levels during equine follicular luteinization, preovulatory follicles were isolated between 0–39 h post-hCG and Northern blot analyses were performed on RNA extracted from intact follicle wall (theca interna with attached granulosa cells). StAR transcripts of approximately 3.0 kb were detected in walls of follicles isolated at 0 h, and administration of hCG had no significant effect on relative transcript levels (Fig. 4AGo). However, higher levels of StAR mRNA were observed in two corpora lutea obtained on day 8 of the estrous cycle (Fig. 4AGo).



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  		Figure 4. Regulation of StAR mRNA by hCG in equine preovulatory follicles. Preparations of follicle wall (theca interna with attached granulosa cells) were obtained from preovulatory follicles isolated 0, 12, 24, 30, 33, 36, and 39 h after hCG treatment, as described in Materials and Methods. In addition, two corpora lutea (CL) were isolated on day 8 of the estrous cycle. Samples of total RNA (10 µg/lane; two follicles per time point) were analyzed by Northern blotting using a 32P-labeled equine StAR cDNA probe (A). The same blot was stripped of radioactivity and hybridized with a cDNA encoding the rat EFTu as a control gene for RNA loading (B). Brackets on the left show migration of 28S and 18S ribosomal bands, and markers on the right indicate the migration of RNA standards. Filters in A and B were exposed to film at -70 C for 4 and 2 h, respectively.

 
To determine the relative contribution of each steroidogenic cell type in follicular StAR mRNA expression, Northern blots were prepared from isolated preparations of granulosa cells and theca interna. Results revealed an unexpected reciprocal regulation of StAR transcripts by hCG in each cell type. In granulosa cells, StAR was undetectable or present at very low levels between 0–12 h post-hCG, but the transcript increased between 24–39 h (Fig. 5AGo). In contrast, levels of StAR mRNA in theca interna were high before hCG injection (0 h), remained relatively constant until 33 h post-hCG, and then dropped dramatically at 36 and 39 h post-hCG (Fig. 6AGo). Uniform RNA loading in all Northern blots was confirmed by hybridization with a cDNA encoding the rat elongation factor Tu (Figs. 4BGo, 5BGo, and 6BGo).



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  		Figure 5. Regulation of StAR mRNA by hCG in granulosa cells of equine preovulatory follicles. Isolated preparations of granulosa cells were obtained from equine preovulatory follicles isolated between 0–39 h after hCG treatment, as described in Materials and Methods. In addition, preparations of theca interna (TI; 0 h) and corpus luteum (CL; day 8 of cycle) were isolated. Samples of total RNA (10 µg/lane; n = 2 follicles/time) were analyzed by Northern blotting using a 32P-labeled equine StAR cDNA probe (A). The same blots were stripped of radioactivity and hybridized with a cDNA encoding the rat EFTu as a control gene for RNA loading (B). Brackets on the left show the migration of 28S and 18S ribosomal bands, and markers on the right indicate the migration of RNA standards. Filters in A and B were exposed to film at -70 C for 6 and 2 h, respectively.

 


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  		Figure 6. Regulation of StAR mRNA by hCG in theca interna of equine preovulatory follicles. Isolated preparations of theca interna were obtained from equine preovulatory follicles isolated between 0–39 h after hCG treatment, as described in Materials and Methods. In addition, samples of granulosa cells (GC; 39 h) and corpus luteum (CL; day 8 of cycle) were isolated. Samples of total RNA (10 µg/lane; n = 2 follicles/time) were analyzed by Northern blotting using a 32P-labeled equine StAR cDNA probe (A). The same blots were stripped of radioactivity and hybridized with a cDNA encoding the rat EFTu as a control gene for RNA loading (B). Brackets on the left show migration of 28S and 18S ribosomal bands, and markers on the right indicate migration of RNA standards. Filters in A and B were exposed to film at -70 C for 4 and 2 h, respectively.

 
To provide a quantitative estimate of the relative changes in StAR mRNA during the gonadotropin-induced luteinization process, Northern blots were prepared from follicle wall, theca interna, and granulosa cells of four separate preovulatory follicles isolated at each time point between 0–39 h post-hCG, and results were quantified by densitometric analyses. No significant changes were observed in the relative levels of StAR transcripts in follicle wall (Fig. 7AGo). However, this apparent constant expression of follicular StAR concealed a significant increase in message levels in the granulosa cell layer, first detected at 30 h post-hCG (Fig. 7BGo), and a significant drop in StAR transcripts in the theca interna layer at 36 and 39 h post-hCG (Fig. 7CGo).



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  		Figure 7. Relative changes in StAR mRNA levels in equine follicle cells isolated between 0–39 h after hCG treatment. Samples (n = 10 µg) of total RNA extracted from follicle wall (A), granulosa cells (B), and theca interna (C) were analyzed by Northern blotting with the equine StAR cDNA and subsequently with the rat EFTu cDNA as a control gene for RNA loading. After autoradiography (films not shown), the StAR signal intensity was quantified by densitometric analysis and normalized with the control gene EFTu. Results are presented as StAR mRNA levels relative to EFTu (mean ± SEM; n = 4 follicles/time point). Columns marked with an asterisk are significantly different (P < 0.05) from 0 h post-hCG.

 
Follicular fluid concentrations of progesterone
Concentrations of progesterone were measured in follicular fluid of preovulatory follicles isolated between 0–39 h post-hCG to assess whether the regulation of StAR mRNA in granulosa and theca interna related to changes in progesterone biosynthesis. Progesterone levels were relatively low in follicles isolated before gonadotropin treatment (0 h post-hCG; 53.6 ± 21.1 ng/ml) and remained unchanged at 12 and 24 h (Fig. 8Go). However, follicular fluid concentrations of progesterone were significantly increased at 30 h and reached maximal levels at 39 h post-hCG (783.0 ± 246.1 ng/ml).



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  		Figure 8. Follicular fluid concentrations of progesterone in equine preovulatory follicles. Preovulatory follicles were isolated between 0–39 h after hCG treatment, and follicular fluid concentrations of progesterone were determined by specific RIAs. Results are shown as the mean ± SEM (n = 5 follicles/time point, 0–30 and 36 h post-hCG; n = 6 follicles, 33 h post-hCG; n = 3 follicles, 39 h post-hCG). Columns marked with an asterisk are significantly different (P < 0.05) from 0 h post-hCG.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous reports have used the equine CG/hCG-primed immature rat model to study the regulation of StAR in follicles in vivo (17, 33) and cultures of granulosa cells to investigate in vitro some of the molecular mechanisms of StAR expression (16, 27, 29, 33, 34, 35, 36, 46). This study is the first to document the precise time-course and cell-specific regulation of StAR mRNA in vivo in preovulatory follicles of a large monoovulatory species and sexually mature animals. The mare was selected as an animal model for several reasons: the preovulatory follicle reaches a relatively large size (40–45 mm in diameter), its development can be precisely monitored in vivo by ultrasound imaging, and the process of equine follicular recruitment, selection, and dominance is similar to that of other species (40, 47). Results provide evidence for a novel inverse regulation of StAR transcripts in theca interna and granulosa cells of equine preovulatory follicles in response to an ovulatory dose of hCG. Also, the study reports for the first time the cloning of equine StAR from which the complete primary structure of the StAR protein is deduced.

Comparative analyses underscore the highly conserved nature of StAR across species, with the amino acid sequence of the equine protein being more than 86% identical to that of other species (12, 13). However, although the equine protein appears to contain a putative mitochondrial transit peptide within the first 55 amino acids, it does not have the consensus motif for mitochondrial two-step cleavage identified in mouse StAR (8). This divergence from the murine sequence is also observed in human (14), bovine (15), and porcine StAR (21) and argues against a critical role for this region in StAR action. Indeed, mounting evidence in the mouse clearly shows that the steroidogenic action of StAR instead involves C-terminal domains (48, 49). The activity of the protein appears to lie outside of the mitochondria, and mitochondrial import is not required for StAR action (48, 49). Computer analysis of the equine StAR amino acid sequence also revealed several potential phosphorylation sites that could modulate the activity of the protein. Although the functional role of each site remains to be established, results indicate that the potential phosphorylation site located at serine 195, shown in human and mouse to regulate StAR activity (50), is conserved in the equine protein.

Northern blot analyses revealed the presence of one major StAR transcript of about 3.0 kb in follicular extracts and a minor band of about 1.8 kb in a few samples. The finding of multiple equine StAR transcripts is in agreement with the results in other species. Three mRNAs have been observed in mice and rats, including two major bands of 3.4 and 1.6 kb (17, 51). One major transcript of 1.6 kb and two minor mRNAs of 4.4 and 7.5 kb have been reported in human tissues (14, 52), two transcripts of 3.0 ad 1.8 kb were detected in bovine tissues (15, 21, 34, 52), and up to three transcripts have been reported in the pig (21, 34). Only one StAR mRNA of 2.8 kb has been observed in sheep (20). Our cloning results suggest that differences in the lengths of transcripts are attributable to variations in the 3'-UTR, as the short form equine StAR measuring 1.6 kb appeared derived from an internal polyadenylation signal (5'-AATAAA-3') located 22 bp from the end of the clone. However, the functional significance, if any, of multiple StAR transcripts remains unknown.

One key finding of the present study is the reciprocal regulation by gonadotropins of StAR transcripts in theca interna and granulosa cells of equine preovulatory follicles. This result clearly highlights the importance of defining the contribution of each steroidogenic cell type, as Northern blots from whole follicular wall extracts could have erroneously lead to the conclusion that hCG had no effect on StAR expression in equine follicles. The pattern of induction of StAR mRNA in equine granulosa cells compares with that observed in the immature rat model after hCG administration in vivo (17, 33) and with the ability of agonists of the protein kinase A pathway to up-regulate StAR expression in granulosa cells in vitro (16, 27, 29, 33, 34, 35, 36). Also, absence of the transcript in equine granulosa cells isolated before gonadotropin treatment is consistent with results obtained in cattle (53). Interestingly, the increase in steady state levels of StAR mRNA caused by hCG paralleled a significant rise in follicular fluid concentrations of progesterone, suggesting a link between StAR expression in granulosa cells and the onset of follicular luteinization in vivo. A more precise understanding of its relative role in the equine follicle should result from further studies on the characterization and gonadotropin regulation of key enzymes involved in equine follicular steroidogenesis. However, a relationship between StAR and steroid hormone production has been clearly established in various systems (11, 14, 23).

In contrast to granulosa cells, high levels of StAR mRNA were observed in theca interna of equine preovulatory follicles isolated before hCG treatment. This observation is not surprising considering the hypertrophied and highly steroidogenic appearance of the theca interna layer in equine preovulatory follicles isolated during early estrus, as characterized under light microscopy (54). Also, elevated levels of StAR transcripts have been reported in theca interna of large follicles in rats (17, 33), cows (53), and humans (29, 30). However, whereas StAR mRNA remained relatively constant until 33 h post-hCG, a dramatic loss in the transcript occurred thereafter in theca interna. This loss is cell type specific, as a concomitant increase in StAR was observed in the neighboring granulosa cell layer. To our knowledge, this is the first time that such a reciprocal regulation of StAR mRNA was simultaneously observed in distinct cellular compartments of ovarian follicles or any other steroidogenic tissue. The loss of StAR transcript in equine theca interna occurred 6–9 h before the expected time of ovulation (37). Although the biological significance of the loss of StAR transcript remains to be precisely established, we believe that it could represent the first biochemical consequence of a putative degenerative process in theca interna of equine follicles just before ovulation (54, 55). Van Niekerk et al. (54) reported that, in contrast to other species, the theca interna degenerates at the time of ovulation in mares, and therefore does not contribute to the formation of the corpus luteum. Interestingly, the abrupt disappearance of StAR between 33–36 h post-hCG could suggest a timing for the onset of the degenerative process, thereby providing a paradigm to study its molecular regulation.

In summary, this study describes the primary structure of equine StAR and reports the cloning of two transcripts that differ primarily in the length of the 3'-UTR. The equine protein is composed of 285 amino acids, and its sequence is highly homologous to that of other species. The gonadotropin-dependent and cell-specific regulation of StAR mRNA in vivo was studied in a series of preovulatory follicles isolated before and after hCG treatment. The results revealed a unique inverse regulation of StAR mRNA in equine follicular cells, with hCG causing an induction of StAR transcripts in granulosa cells and the disappearance of the message in theca interna. Although these changes are believed to relate to the luteinization of granulosa cells and a putative degeneration of theca interna before ovulation in mares, future studies are needed to better understand the precise role of StAR during equine follicular steroidogenesis.


   Acknowledgments
 
We thank Dr. Douglas M. Stocco (Texas Tech University, Lubbock, TX) for the mouse StAR cDNA, Dr. R. Levine (Cornell University, Ithaca, NY) for the rat EF-Tu cDNA, and Dr. Alan K. Goff (Université de Montréal, Montréal, Canada) for the progesterone antibody.


   Footnotes
 
1 This work was supported by Natural Sciences and Engineering Research Council of Canada Grant OGP0171135 (to J.S.). The nucleotide sequences reported in this paper have been submitted to GenBank with accession numbers AF031696 and AF031697. Back

2 Supported by a fellowship from Al-Fateh University. Back

3 Supported by a Medical Research Council of Canada Doctoral Research Award. Back

Received July 31, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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