Expression of Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid Is Limited to Theca of Healthy Bovine Follicles Collected during Recruitment, Selection, and Dominance of Follicles of the First Follicular Wave¹

^a Departments of Animal Sciences ^b and Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri 65211

	ABSTRACT

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Expression of mRNA encoding steroidogenic acute regulatory protein (StAR) in bovine follicles during recruitment and selection was examined. Dairy heifers (4–5/time period) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave (Time 0) after estrus. Follicles were collected and stored at -80°C until sectioning. Expression of StAR mRNA was localized by in situ hybridization and quantified by image analysis. Expression of StAR mRNA was first detected in theca interna of antral follicles as small as 0.5 mm in diameter and increased with increasing follicular size ( 4 mm; r = 0.75; p < 0.001). StAR mRNA was undetectable in granulosa of healthy follicles at any size or stage of follicular wave examined. However, granulosa or luteinized granulosa of some advanced or late atretic follicles expressed StAR mRNA. During recruitment, StAR mRNA expression in theca cells was similar among recruited follicles (4–8 mm). During selection of dominant follicles (36–48 h), StAR mRNA was expressed in theca of more than one follicle (7–9 mm); therefore, expression of StAR mRNA may not be associated with dominant follicle selection. StAR mRNA in theca was higher (p < 0.05) at 48 h after initiation of the first follicular wave than at 12, 24, and 36 h, and it remained elevated thereafter through 96 h. Dominant follicles expressed more (p < 0.01) StAR mRNA in theca than did subordinate healthy follicles. Healthy follicles expressed higher (p < 0.05) StAR mRNA in theca than atretic follicles. In summary, levels of StAR mRNA increased in theca with stage of follicular wave and size of follicles. Follicular atresia was associated with reduced expression of StAR mRNA in theca cells. The results indicate that expression of StAR mRNA in theca may not be the primary limiting factor for follicular recruitment and selection.

	INTRODUCTION

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Cytochrome P450 side chain cleavage enzyme (P450_scc), which catalyzes formation of pregnenolone from cholesterol within mitochondria, has long been considered the rate-limiting step in steroid hormone synthesis [1]. The preceding reaction occurs in the inner mitochondrial membrane and is stimulated by LH and FSH. Involvement of a steroid regulatory protein in translocation of cholesterol from outer to inner mitochondrial membranes has been known for over two decades [2, 3]. In recent years, steroidogenic acute regulatory protein (StAR) has been characterized and is believed to play an important role in steroidogenesis by transporting cholesterol from the outer to inner mitochondrial membrane [4–6].

StAR mRNA and protein have been detected in bovine luteal tissue, and the relative levels of mRNA and protein increase with luteal development and decrease during luteal regression; however, expression of StAR mRNA in bovine theca and granulosa cells was equivocal [7–9]. Little is known about the changes in expression of mRNA for StAR and regulation of StAR mRNA expression during follicular development in cattle, nor about the potential role of StAR during recruitment and selection of bovine ovarian follicles. Estradiol-17ß production is dependent upon sufficient amounts of androgen production by theca cells [10]. Androgen synthesis in theca cells is regulated, at least in part, by the sufficient amounts of cholesterol supplied to mitochondria and the activities of the steroidogenic enzymes, and possibly supplemented from pregnenolone and/or progesterone synthesized from granulosa [11].

In cattle, waves of follicular growth (recruitment, selection, and dominance) have been clearly characterized [12–14]. Each wave begins with growth of a cohort of follicles (recruitment). From this cohort, one is selected for further growth and becomes dominant over the others (selection).

Recently, we have systematically evaluated the expression of mRNAs for LH receptor (LHR), FSH receptor (FSHR), P450_scc, cytochrome P450 17-hydroxylase (P450_c17), cytochrome P450 aromatase (P450_arom), and 3-beta-hydroxysteroid dehydrogenase (3ß-HSD) enzymes in theca and granulosa cells of follicles during recruitment, selection, dominance, and atresia of bovine ovarian follicles harvested during the first follicular wave of the estrous cycle [15–18]. Expression of mRNAs for LHR, P450_scc, P450_c17, and 3ß-HSD in theca, and of FSHR, LHR, P450_scc, P450_arom, and 3ß-HSD in granulosa was differentially regulated. Two key observations were that mRNA expression of P450_scc and P450_arom was associated with recruitment of a cohort of follicles, and mRNA expression for LHR and 3ß-HSD was associated with selection of the dominant follicle. On the basis of the preceding observations and known functions of StAR, it was hypothesized that expression of StAR mRNA would be similar to the pattern of expression of P450_scc mRNA in theca and granulosa cells during recruitment and selection. Therefore, the present study was conducted to characterize, by in situ hybridization, changes in expression of StAR mRNA in bovine follicles collected at different times during recruitment and selection of the first follicular wave.

	MATERIALS AND METHODS

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Animals, Tissue Collection, and Tissue Preparation

Mature Holstein heifers received injections of 25 mg prostaglandin F₂ (PGF₂, Lutalyse; Upjohn, Kalamazoo, MI) to induce luteal regression. Starting from the second day after PGF₂ injection, heifers were checked 3 times daily for estrus. Initiation of the first follicular wave after estrus was identified by growth of a cohort of follicles 4 mm in diameter, using twice-daily real-time ultrasonography [19]. Groups of heifers (4–5/group) were ovariectomized through a flank incision at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave. The time of ovariectomy was chosen to obtain follicles at specific stages of follicular recruitment and selection. Immediately after removal, ovaries were placed on ice in saline and transported to the laboratory. Individual follicles 8 mm or blocks of ovarian tissue containing follicles 4 mm in diameter and smaller follicles between 2–3 mm in diameter were excised, frozen over liquid nitrogen, and stored at -80°C until sectioned. All follicles were frozen within 30 min of ovariectomy. The size of each follicle was measured with a caliper before freezing and confirmed, whenever possible, after sectioning.

Template Generation and cRNA Probe Synthesis

Generation of ovine StAR cDNA has been described previously [20]. Both antisense and sense [³⁵S]UTP-labeled cRNA probes were transcribed from linearized cDNA templates using a transcription kit (Stratagene, LaJolla, CA) according to the manufacturer's recommendations. The cRNA probes were purified by centrifugation on a Sephadex (Pharmacia and Upjohn, Kalamazoo, MI) G-50 column and used for hybridization within 2–3 days. Labeled probes were diluted in hybridization buffer (50% formamide, 0.3 M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, single-strength Denhardt's solution (single-strength = 0.02% [w:v] Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% [w:v] BSA), 10 mM dithiothreitol, 500 µg/ml yeast tRNA, and 10% dextran sulfate) to about 2 x 10⁷ cpm/ml.

Northern Analysis

RNA was isolated using Tri-reagent (Sigma, St. Louis, MO). Fifteen micrograms of follicular or luteal RNA was combined with RNA loading dye, denatured, and loaded onto a 1.5% agarose, 8.9-ml (37% w:w) formaldehyde, and 92-ml single-strength 3-(N-morpholino) propanesulfonic acid (MOPS) gel containing ethidium bromide. The gel was exposed for 3 h at 70 V in single-strength MOPS. The gel was then rinsed in 5-strength SSC (single-strength SSC = 0.3 M NaCl and 0.03 M sodium citrate, pH 7.0) in diethyl pyrocarbonate (DEPC)-treated water and blotted overnight onto a nylon membrane in 5-strength SSC. Positions of intact 18S and 28S RNA bands and DNA markers were marked onto the membrane. The membrane was then baked at 80°C for 2 h and prehybridized in buffer containing boiled herring sperm for at least 4 h at 55°C.

The StAR cRNA probe was synthesized using a Stratagene transcription kit with [³²P]CTP (DuPont NEN, Boston, MA). The probe was purified by centrifugation at 900 x g, diluted in NETS buffer (150 mm NaCl, 10 mm EDTA, 50 mm Tris, and 0.1% SDS), and boiled for 2 min; and approximately 50 million cpm was added to the prehybridization solution and hybridized overnight at 55°C. The blot was washed twice in double-strength SSC with 0.1% SDS for 20 min at 55°C and in 0.1-strength SSC with 0.1% SDS at 65°C for 20 min. The membrane was then exposed to x-ray film for 0.5–3 days at -70°C.

In Situ Hybridization

Procedures for in situ hybridization have been described previously [17, 18]. Fourteen-micrometer sections of follicular tissue were cut at -20°C using a cryostat (JUNG Frigocut 2800N; Leica Inc., St. Louis, MO) and were mounted onto prechilled microscope slides (Superfrost/Plus; Fisher Scientific, Pittsburgh, PA). Slides were then air-dried and stored at -80°C in desiccated, airtight boxes until fixation and hybridization (within 1 mo). Before hybridization, sections were fixed in 4% formaldehyde in 0.01 M PBS for 5 min, washed in double-strength SSC for 2 min, acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min, rinsed in double-strength SSC, and dehydrated in increasing concentrations of ethanol. Slides were incubated in chloroform for 5 min, 100% ethanol for 2 min, and 95% ethanol for 2 min, and air-dried.

Hybridization was performed using 100 µl diluted probe in a humidified oven at 55°C for 20 h. After hybridization, slides were washed twice by shaking in double-strength SSC (45–52°C) for 15 min at room temperature, and treated with ribonuclease (RNase-A; 50 mg/ml in double-strength SSC) for 1 h at 37°C. Slides were then washed at 55°C in double-strength SSC containing 0.1% ß-mercaptoethanol (BME) for 15 min, single-strength SSC/0.1% BME for 15 min, single-strength SSC/50% formamide/0.1% BME for 30 min, and twice in 0.1-strength SSC/BME for 15 min each. The slides were dehydrated, air-dried, dipped in Kodak NTB-2 emulsion, and exposed for 14 days at 4°C. Slides were developed, lightly counterstained with hematoxylin and eosin, and mounted for microscopic examination. For each follicle, two sections were hybridized with the antisense probe, and one section was hybridized to the sense probe. Sections from animals ovariectomized at different times were balanced in each hybridization run to minimize biases due to variation among runs.

Classification of Follicles

Follicles were morphologically classified as healthy, or as early, advanced, or late atretic [15–18]. Briefly, early atretic follicles had a few minor degenerative changes, such as presence of a few pycnotic nuclei and/or local destruction of the basement membrane. Compared to early atretic follicles, advanced atretic follicles were characterized by a greater destruction of follicular structure, more degenerative granulosa cells, and/or a noticeable decrease in the number of granulosa cells. Follicles with more severe degenerative changes than advanced atretic follicles were classified as late atretic follicles.

Image Analysis

Hybridization intensity was quantified using the BIOQUANT image analysis system (R&M Biometrics Inc., Nashville, TN) as described previously by this laboratory [15–18]. For each follicle, four fields at roughly 90° angles were measured for both sections hybridized to the antisense probe and for the section hybridized to the sense probe. Specific hybridization intensity was defined as the average hybridization intensity for the two sections hybridized to the antisense probe minus the average hybridization intensity for the section hybridized to the sense probe. Intensity of hybridization was expressed as the percentage of pixels within a given marked area that was above a set grey threshold level.

Statistical Analysis

ANOVA was used to test the effects of stage of follicular development and type of follicle (health status) on the various parameters measured. Duncan's multiple-range procedure was used to compare means. When effects of follicle size on mRNA expression were tested, data from all follicles 4 mm in diameter and from some smaller follicles, irrespective of the stage of follicular wave, were examined. Pearson correlation was used to establish relationships between follicle size and level of mRNA expression. To test the effect of stage of the first follicular wave on expression of StAR mRNA, healthy follicles ( 4 mm in diameter) that expressed StAR mRNA within theca cells were used. All analyses were performed using SAS [21]. All data are reported as mean ± SEM.

	RESULTS

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Follicular Characteristics and Determination ofDominant Follicles

Follicular characteristics and determination of dominant follicles have been described recently [17, 18]. Briefly, initiation of the first follicular wave was detected from 24 to 72 h (mean ± SEM; 42.0 ± 2.6 h) after onset of estrus. At 12, 24, and 36 h after detection of a follicular wave (Time 0 = time of initiation of wave), three to seven healthy follicles were present on the ovaries of each animal (except for 1 cow with one healthy follicle). It was not possible to identify the dominant follicle on the basis of size at 12 or 24 h. At 36 h, dominant follicles were detected by size in some cows, but not in others. By 48 h after wave initiation, dominant follicles could be identified by size in all animals. Therefore, the 36- and 48-h time period after initiation of a follicular wave appeared to be a transition period for selection of the dominant follicle. Follicles ranging in size from 4 to 17 mm (dissected diameter) were classified into healthy, early atretic, advanced atretic, or late atretic follicles (Table 1). Average size of healthy follicles was greater (p < 0.01) than that of atretic follicles (Table 1). The size of healthy follicles was greater (p < 0.05) at 24 and 36 h compared to 12 h and continued to increase (p < 0.05) through 96 h after the initiation of the wave (Table 2).

Ontogeny of StAR mRNA Expression duringFollicular Growth

StAR was localized to theca interna cells, but not to granulosa cells, of healthy antral follicles (Fig. 1; Table 2). Expression of StAR mRNA was first detected in the theca interna of small antral follicles after antrum formation (Fig. 1, A and B). Irrespective of stage of follicular wave, expression of StAR mRNA in theca cells of healthy follicles 4 mm increased with increasing size of follicles (r = 0.75; p < 0.001; Fig. 1, C–F). Granulosa cells of healthy follicles did not express StAR mRNA (Fig. 1, A–F). Compared to expression of StAR mRNA in theca interna cells of healthy follicles, StAR mRNA expression in atretic follicles was lower (p < 0.001; Table 1). Expression of mRNA for StAR in theca cells of early atretic follicles was not different from that of advanced or late atretic follicles (Table 1). Granulosa cells of early atretic follicles did not express StAR mRNA. However, granulosa cells or luteinized granulosa cells of some large advanced and late atretic follicles expressed StAR mRNA (Fig. 1, G–J). The morphology of granulosa cells expressing StAR mRNA appeared hypertrophied and/or luteinized (Fig. 1, G–J).

View larger version (133K): [in this window] [in a new window]   FIG. 1. In situ hybridization of StAR mRNA in bovine follicles collected at different stages of development (brightfield and darkfield views). A and B) A 1-mm nonrecruited healthy antral follicle, showing specific hybridization in theca interna layer but not in granulosa layer; C and D) a 6-mm recruited follicle showing hybridization in the theca interna; E and F) a 12-mm dominant follicle collected at 60 h, showing an increased intensity of hybridization in the theca interna; G–J) 8- and 10-mm atretic follicles, showing hybridization in the granulosa cells; K and L) a dominant follicle (14 mm) hybridized to the sense probe, showing no specific hybridization in either theca or granulosa cells. T, Theca interna layer; G, granulosa layer. Bar = 100 µm for all panels except G and H, in which bar = 50 µm.

Northern Analysis

Using Northern analysis, the antisense cRNA hybridized to a main 1.9-kilobase (kb) band (> 95%) in follicular and luteal tissues (data not shown). In some luteal and follicular tissues, there was evidence of hybridization to a minor 2.9-kb band (one sample showed evidence of a band at 1.3 kb).

Expression of StAR mRNA at Different Stages after Initiation of a Follicular Wave

In theca cells, expression of StAR mRNA changed (p < 0.001) with the stage of the first follicular wave (Table 2). There were no differences in expression of StAR mRNA in theca cells among follicles collected at 12 h through 36 h. However, expression of StAR mRNA was higher (p < 0.05) in theca interna cells of dominant follicles collected at 48 h through 96 h compared to follicles at 12 through 36 h. StAR mRNA was expressed in theca cells of each healthy follicle ( 4 mm) within the cohort, and level of expression was similar among follicles. Overall, theca cells of dominant follicles (42.9 ± 4.5, n = 26) expressed greater (p < 0.05) amounts of StAR mRNA than did recruited healthy follicles (16.4 ± 1.5, n = 36). In contrast to theca cells, expression of StAR mRNA in granulosa cells was undetectable in any healthy follicles including nonrecruited small follicles, recruited follicles, and dominant follicles harvested during the first follicular wave (Table 1; Fig. 1).

Correlation of StAR mRNA Expression to mRNAs for Gonadotropin Receptor and Steroidogenic Enzymes, and Size of Follicles

Expression of StAR mRNA in ovarian theca cells was correlated with expression of mRNAs for gonadotropin receptors and steroidogenic enzymes that was reported previously from this laboratory [17, 18]. Expression of StAR mRNA in theca cells was positively correlated with the size of follicles and expression of mRNAs for gonadotropin receptors and steroidogenic enzymes (Table 3). The order of correlation (the highest to the smallest) was follicular size > P450_arom in granulosa > LHR in granulosa > 3ß-HSD in granulosa > LHR in theca > P450_c17 in theca > FSHR in granulosa > P450_scc in granulosa > 3ß-HSD in theca > P450_scc in theca.

Table 4 summarizes the cell-specific and developmental stage-specific expression pattern of the mRNAs for gonadotropin receptors, steroidogenic enzymes, and StAR in healthy bovine follicles obtained during recruitment and selection. Messenger RNAs for FSHR and P450_arom were exclusively localized in granulosa cells, while mRNAs for P450_c17 and StAR were solely detected in theca cells. Messenger RNAs for LHR, P450_scc, and 3ß-HSD were expressed in both granulosa and theca cells [17, 18].

During follicular development, FSHR mRNA was the only mRNA detected in granulosa cells of nonrecruited follicles whereas mRNAs for LHR, P450_c17, P450_scc, 3ß-HSD, and StAR were expressed in theca cells of nonrecruited follicles. In a recruited cohort of follicles, all the mRNAs that were expressed in nonrecruited follicles were continuously expressed in their respective cell types, and expression of mRNAs for P450_scc and P450_arom was initiated in granulosa cells. During selection, mRNAs for LHR and 3ß-HSD were expressed in granulosa cells, and expression of mRNAs for all parameters tested was increased in selected dominant follicles (Table 4).

	DISCUSSION

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In the current study, expression of StAR mRNA in theca and granulosa cells was localized and quantified during development of the bovine first follicular wave. StAR mRNA was localized only in theca interna, not in granulosa, of healthy follicles. StAR mRNA was first expressed in theca cells of antral follicles of 0.5–1 mm in size, a size to which follicles grow without gonadotropin stimulation [22–24]. In a previous study using the same follicular tissues, mRNA expression for LHR, P450_scc, P450_c17, and 3ß-HSD was first detected when theca interna was first formed around the time of antrum formation. Therefore, it appears that expression of mRNAs for LHR and steroidogenic enzymes (P450_scc, P450_c17, and 3ß-HSD) in theca cells occurs slightly earlier than that of StAR [15–18]. These results suggest that cholesterol may be transported to mitochondria and metabolized to pregnenolone for subsequent conversion to androgen in follicles as small as 0.5–1 mm ([15–18], present study). StAR mRNA expression increased with increasing size of healthy follicles, indicating that cholesterol transport may be enhanced coincident with increased theca cell androgen production during follicular growth [14–18].

After initiation of the first follicular wave in the present study, expression of StAR mRNA and size of healthy follicles increased with stage of follicular wave. There were no differences in expression of StAR mRNA in theca cells among healthy follicles collected at 12 h through 36 h, suggesting that changes in expression of StAR mRNA in theca cells are not associated with follicular recruitment. However, expression of StAR mRNA was greater in theca interna cells of healthy follicles collected at 48 h through 96 h compared to 12, 24, and 36 h. Expression of LHR and 3ß-HSD mRNA in granulosa cells occurs around the time of follicular selection (36–48 h) and increases with continued growth of selected dominant follicles [17, 18]. Increased StAR mRNA expression in theca cells at the time of or after follicular selection indicates that increased estradiol-17ß production by granulosa cells may require increased cholesterol transport within theca cells to produce the estradiol-17ß substrate, androgen. Therefore, increases in expression of StAR mRNA in theca of dominant follicles may be related to increases in estradiol-17ß production by granulosa cells [11].

Follicular granulosa cells also produce progesterone, and its production is regulated by LH, FSH, P450_scc, and 3ß-HSD. Expression of mRNAs and proteins for LHR, P450_scc, and 3ß-HSD has been detected in granulosa cells of follicles in many mammalian species [15–18, 25–36]. However, expression of StAR mRNA was not localized in the granulosa cells of healthy follicles collected from the first follicular wave in the current study. This agrees with a recent study in which StAR mRNA was found only in theca interna, not in granulosa cells of bovine ovarian follicles collected from superovulated animals [9]. Another earlier study from the same laboratory found StAR mRNA in both theca and granulosa cells of bovine follicles collected at slaughter [8]. In contrast, Hartung et al. [7] reported that StAR mRNA was absent in both bovine theca and granulosa cells harvested from follicles collected at an abattoir. In a personal communication, Murphy [8] expressed the belief that the granulosa cells used to characterize StAR mRNA were luteinized or mixed with luteinized granulosa cells. From the study by Hartung et al. [7], why no expression of StAR mRNA was detected is unknown, but it may be due to sensitivity of the assay used. We hypothesized that expression of StAR mRNA in theca and granulosa cells would be similar to expression of P450_scc mRNA in the bovine theca and granulosa cells during the first follicular wave. Specifically, StAR mRNA in theca cells would first be detected around the same time as P450_scc mRNA expression in theca (around the time of antrum formation), and expression of StAR mRNA in theca would increase with increasing size of the follicles and advancing stages of the follicular wave. Similarly, expression of StAR mRNA in granulosa cells would be seen first in the recruited cohort of follicles after the initiation of the first follicular wave and continue to be expressed as the stage of the wave advances. Results from the present study supported the first part of our hypothesis&; the pattern of StAR mRNA expression in theca cells followed expression of P450_scc in the theca cells [17]. Since StAR mRNA was not detected in granulosa cells, the second part of the hypothesis&; the pattern of StAR mRNA expression in granulosa cells would follow expression of P450_scc in granulosa cells—was rejected [17]. Therefore, StAR mRNA expression in theca cells during the follicular wave may not be associated with the mechanisms of follicular recruitment and selection. However, the increased expression of StAR mRNA in theca cells observed from the time of selection onward may be necessary for increased steroid production and maintenance of the dominant follicle.

Compared to expression of StAR mRNA in theca interna cells of healthy follicles, StAR mRNA expression in atretic follicles was lower, suggesting that follicular atresia is associated with reduction of StAR mRNA and a decrease in cholesterol transport from outer to inner mitochondrial membranes. Expression of mRNA for P450_scc and steroid production are also greatly decreased in atretic follicles [15, 17]. Granulosa cells of early atretic follicles did not express StAR mRNA in the current study. However, granulosa cells of some large advanced and late atretic follicles did express detectable StAR mRNA. Granulosa cells expressing StAR mRNA appeared to be hypertrophied and/or luteinized. It is possible that spontaneous luteinization of these remaining granulosa cells after degeneration of the oocyte might result in StAR mRNA expression in the same way that luteinization is caused by the preovulatory LH surge [37]. This is comparable to expression of P450_scc and 3ß-HSD mRNA in granulosa cells of atretic follicles [15, 18].

The present study suggests that StAR-stimulated cholesterol transport from the outer to inner mitochondrial membrane occurs in theca cells but not in granulosa cells. Previously, we have shown that P450_scc mRNA is expressed in granulosa cells of recruited (< 9 mm in size) and selected dominant follicles ( 9 mm in size) and that 3ß-HSD mRNA is expressed in granulosa cells of selected dominant follicles ( 9 mm in size) [17, 18]. These results indicate that granulosa cells of a recruited cohort of follicles (< 9 mm) are able to convert cholesterol to pregnenolone, but not pregnenolone to progesterone until a follicle is selected to become dominant. The main function of StAR is involved in acute production of steroid hormones [6]. StAR mRNA is not expressed in granulosa cells, indicating that pregnenolone and progesterone production by granulosa cells of recruited and selected dominant follicles, respectively, may not be an efficient system or may not be acutely regulated. On the other hand, detection of StAR mRNA in theca cells in the present study suggests that high levels of cholesterol are required within mitochondria of theca cells to synthesize androgen, which then is metabolized into estradiol-17ß in the granulosa cells. Therefore, theca androgen production may be acutely regulated at the level of StAR-stimulated cholesterol transport to provide substrate for high levels of estradiol-17ß biosynthesis by granulosa cells of dominant follicles ([6, 11], present study).

In summary, StAR mRNA is localized in theca cells, but not in granulosa cells, of healthy follicles harvested during the first bovine follicular wave. Levels of StAR mRNA in theca cells increased with increasing size of follicles and advancing stages of follicular wave. Follicular atresia was accompanied by reduced expression of StAR mRNA in theca cells.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge Dr. G.D. Niswender at the University of Colorado, Fort Collins, for providing StAR cDNA probes for this study. We also wish to thank Jennifer Cresswell and Moretta Leivan for technical assistance.

	FOOTNOTES

 
¹ This work was supported in part by USDA Grant USDA CSRS 93–37203–9331. This is a contribution from the Missouri Agricultural Experiment Station; Journal Series Number 12,655.

² Correspondence: H. Allen Garverick, Department of Animal Sciences, 163 Anim. Sci. Res. Center, University of Missouri, Columbia, MO 65211. FAX: 573 882 6827; allen_garverick{at}muccmail.missouri.edu

³ Current address: Center for Biomedical Research, The Population Council, 1230 York Avenue, New York, NY 10021.

⁴ Current address: The Procter&Gamble Co., 11810 E. River Rd., Ross, OH 45061.

Accepted: June 4, 1998.

Received: June 19, 1997.

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