Journal of Histochemistry and Cytochemistry, Vol. 47, 769-776, June 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

Immunolocalization and Expression of the Steroidogenic Acute Regulatory Protein During the Transitional Stages of Rat Follicular Differentiation

Winston E. Thompsona, Jacqueline Powella, Kewlyn H. Thomasb, and Joseph A. Whittakerb
a Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, Georgia
b Neuroscience Institute and Department of Anatomy, Morehouse School of Medicine, Atlanta, Georgia

Correspondence to: Winston E. Thompson, Dept. of Obstetrics and Gynecology, Morehouse School of Medicine, 720 Westview Dr. SW, Atlanta, GA 30310.


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

This study was designed to determine the pattern of expression and cellular distribution of the steroidogenic acute regulatory protein (StAR) during the transitional stages of follicular differentiation in rat ovary. Using specific antisera against the StAR, immunohistochemistry, Western blotting, and immunoprecipitation analyses provide evidence confirming the localization and expression of StAR in granulosa cells (GCs) of juvenile rat ovaries before and after PMSG treatment. The results also show that StAR expression occurs in theca intersitial cells surrounding preantral, antral, and larger antral follicles in adult diestrous ovaries. Furthermore, we have demonstrated heterogenous StAR immunoreactivity in the granulosa cell layers and cells of the corpora lutea. A novel finding presented here is that, during ongoing growth and differentiation of the follicle, the immunoreactivity of StAR tends to shift from the GC of early antral follicles to the theca cell layers in the adult. The spatiotemporal changes or shifts in StAR expression and cellular localization also coincide with the appearance of more acidic isoforms of the 30-kD protein, as determined by two-dimensional gel electrophoresis. Although the functional implications of these observations remain unclear, the acute temporal changes in StAR expression and localization may not only reflect the dynamic steroidogenic capacity of follicular cells but may also support a possible role for FSH in the induction of follicular maturation. (J Histochem Cytochem 47:769–776, 1999)

Key Words: rats, StAR, immature, adult diestrus, immunohistochemistry, immunoprecipitation, Western blotting, ovaries


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
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Ovarian follicular development and the production of a viable oocyte depend on the granulosa and theca cell layers and on regulatory hormonal and nonhormonal signals that influence the growth and differentiation of the follicle (Richards 1980 ; Hsueh et al. 1989 ). During folliculogenesis, follicles previously identified as steroidogenically quiescent are known to proliferate and then differentiate, leading to the maturation and release of the ovum. Because these processes are highly dependent on steroid hormone production, it is essential to elucidate the cellular and molecular mechanisms underlying steroidogenesis in these tissues.

The differentiation of rat granulosa cells isolated from preantral and early antral follicles has been reported to be associated with the expression of a 25-kD intracellular protein (Thompson et al. 1997 ). This protein was subsequently identified as the mitochondria-associated steroidogenic acute regulatory protein (StAR). The StAR protein was first cloned from MA-10 mouse Leydig tumor cells by Clark and colleagues in 1994. Functionally, this protein is considered a regulator of the rate-limiting transfer of cholesterol from the outer to the inner mitochondrial membrane, which is a prerequisite for the production of steroids (Clark et al. 1994 ). Earlier reports by Epstein and Orme-Johnson 1991 and by Stocco and Sodeman 1991 indicated that StAR is synthesized as a 37-kD cytosolic precursor protein that is imported into the mitochondria to produce four 30-kD mature isoforms (Epstein and Orme-Johnson 1991 ; Stocco and Sodeman 1991 ; Clark et al. 1994 ). More recent evidence suggests that the active form of StAR is the 37-kD precursor protein (King et al. 1995 ). Interestingly, information relevant to the functional significance of StAR during steroidogenesis has come from Lin et al. 1995 , who demonstrated that a mutation in the StAR gene resulted in the acquisition of lipoid congenital adrenal hyperplasia. This disease, which is fatal if not diagnosed and treated early, is characterized by inability of the adrenal glands and the gonads to synthesize steroids and is due mainly to inefficient transport of cholesterol into mitochondria. However, the mechanism by which StAR facilitates mitochondrial cholesterol transport is still unclear and is currently the subject of intensive investigation.

The purpose of this study was to determine the pattern of expression and cellular distribution of StAR protein with respect to the transitional stages of follicular differentiation in rat ovary. We examined StAR protein localization and expression in preantral follicles, antral follicles, and the terminally differentiated state of the corpus luteum in infant, juvenile, and adult rat ovaries, respectively. The potential functional significance of the differential expression of StAR protein during follicular development and steroidogenesis is discussed.


  Materials and Methods
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Summary
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Materials and Methods
Results
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Literature Cited

Immunofluorescence Confocal Microscopy
The handling of animals in this study was approved by the Institutional Animal Care and Use Committee in accordance with the guidelines of the National Institutes of Health and the US Department of Agriculture.

Female Sprague–Dawley rats (Taconic Farms; Germantown, NY) ages 15 days (n = 4), 25 days (n = 4), and 10 weeks diestrus (n = 2), were sacrificed by an overdose of sodium pentobarbital (5 mg/100 g body weight). A fourth group of 23-day-old rats (n = 5) received single SC injections of pregnant mare serum gonadotropin (PMSG, 50 IU) and were sacrificed 48 hr after injection. The adult estrous cycle stage was determined by vaginal smears that showed a predominance of small leukocytes interspersed by few nucleated epithelial cells. The transitional stages of follicular differentiation were defined as described by Ojeda et al. 1986 and by Ojeda and Urbanski 1994 for 15-day-old (infant), 25-day-old (juvenile), and adult rats. Ovaries from both the PMSG-treated and nontreated groups were removed, immediately embedded in Tissue-Tek OCT compound (Miles Labs; Elkhart, IN), and cut into 15-µm cryostat sections. Sections were then fixed with 2% paraformaldehyde in PBS for 30 min, washed in PBS, and incubated in ice-cold methanol for 5 min at -20C. These were subsequently treated with 50 mM NH4Cl in PBS for 10 min, washed in PBS, and permeabilized with 0.2% Triton X-100 for 5 min. After blocking nonspecific (antibody) binding with 10% calf serum in PBS, sections were incubated for 1 hr with the anti-StAR peptide antibody at 1:100 dilution (generously provided by Dr. Douglas Stocco; Texas Tech University) in PBS. The cellular localization of StAR was visualized with a fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (Pierce; Rockford, IL). Sections were then mounted in glycerol containing 50 µg/ml N-propyl gallate and examined using a laser scanning confocal microscopic imaging system (Carl Zeiss; Thornwood, NY). Classification of follicular and corpus luteum morphology was based on criteria previously described by Peters and McNatty 1980 .

Western Blot Analysis
Fifty µg of ovarian protein extracts from 15-day-old (n = 20), 25-day-old (n = 10), PMSG-treated (n = 5), and adult rats (n = 4) was subjected to one- and two-dimensional gel electrophoresis. Proteins separated by 12% SDS-PAGE were subsequently transferred to 0.2-µm nitrocellulose membranes (Sigma; St Louis, MO) using the Royal Genie electrophoretic blotter (Idea Scientific; Minneapolis, MN) at 350 mA for 5 hr. Blots were preincubated in Tris-buffered saline (TBS) with 0.05% Tween-20 and 5% nonfat dried milk and then incubated overnight at 4C with rabbit anti-peptide antibody to StAR (1:1000) and rabbit anti-peptide antibody to StAR signal sequence (amino acids 1–26; 1:1000). Incubation with the secondary antibody was for 1 hr at room temperature (RT) and antibody binding was subsequently revealed with chemiluminescence (Amersham; Arlington Heights, IL). Total proteins were determined by a dye binding assay (Bio-Rad; Richmond, CA). All samples were normalized to total protein.

Granulosa Cell Collection and Culture
Granulosa cells (GCs) were isolated from rat ovaries using a method previously described by Sanbuissho et al. 1993 . Rats of ages 15 days (n = 20), 25 days (n = 10), and PMSG-treated rats (n = 5) were sacrificed by an overdose of sodium pentobarbital (5 mg/100 g body weight). Ovaries from both the treated and nontreated groups were then removed, placed in ice-cold McCoy's 5A medium (Gibco BRL; Grand Island, NY), punctured with fine needles, and incubated in 2 ml of Cell Dissociation Solution (a nonenzymatic solution) (Sigma) at 37C for 10 min. This method allowed GC separation but prevented the dissociation of thecal and stromal cells. In PMSG-treated rats, only large antral follicles were punctured. Ovaries were then transferred to hypertonic McCoy's 5A medium containing 0.5 M sucrose for 5 min at RT (Campbell 1979 ). GCs were harvested by gently pressing the ovaries against the wall of a polypropylene test tube with a Teflon pestle in ice-cold McCoy's medium containing 1% bovine serum albumin (BSA Fraction V) and filtered through 50-mm nylon mesh. The cell suspension was pelleted by centrifugation at 200 x g for 10 min at 4C. The pellet was then resuspended in serum-free medium and immediately cultured for 5.5 hr with 250 µCi [35S]-methionine (Du Pont NEN; Boston, MA) (Anderson and Lee 1993 ). Cell viability was determined by the erythrosin B exclusion method (Phillips 1973 ).

Immunoprecipitation
Cells were subsequently lysed in Radio-Immuno-Precipitation Assay (RIPA) buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.15 M NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, and 1 mM PMSF). Approximately 1 x 106 cpm of material was precipitated with trichloroacetic acid and incubated overnight at 4C with 2.5 µl of anti-StAR peptide antibody coupled to protein A–Sepharose (Ausubel et al. 1994 ). Then the immunoprecipitated complexes were washed four times in 500 µl RIPA buffer. The immunoprecipitate was then dissociated by boiling in SDS-PAGE sample buffer. The protein solution was next analyzed by SDS-PAGE, the gel fixed, and signals detected by a Molecular Dynamic PhosphorImager (Molecular Dynamics; Sunnyvale, CA).

Data Analysis
Experiments were repeated at least three times and representative autoradiographs are presented. The optical density of each band was quantitated by means of a BioImage Whole Band Analyzer (BioImage; Ann Arbor, MI) computer-assisted analysis system. Estimations were carried out with a minimum of three replicate blots. Wilcoxon, Mann–Whitney equal variance and unequal variance t-tests were used to analyze changes in the levels of StAR expression. Significance was considered when p<0.05.


  Results
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Materials and Methods
Results
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Literature Cited

Confocal Microscopic Localization of StAR in Rat Ovary
The cellular localization of StAR in rat ovary during the transitional stages of follicular differentiation was examined using anti-peptide StAR polyclonal rabbit antiserum and immunohistochemical techniques. In ovaries containing preantral follicles obtained from infant rats, we found no detectable StAR localization pattern within the tripartite structure of the ovarian follicle or in the interstitial cells surrounding preantral follicles (Figure 1A). As shown in Figure 1B, ovaries from juvenile animals contained a mixed population of preantral and early antral follicles. In these follicles, a heterogenous staining pattern for StAR was observed. In addition, some antral follicles expressed the StAR protein. Figure 1B clearly demonstrates that some mural GCs (Figure 1B, arrow) exhibited punctate staining for the StAR protein, whereas others obviously did not (Figure 1B, arrowhead). StAR immunoreactivity was not observed in the theca interstitial cells (Figure 1B).



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Figure 1. Confocal microscopic examination of StAR localization during follicular differentiation. Frozen ovarian sections (15 µm) from 15-, 25-day-old, and PMSG-treated rats were reacted with an antipeptide StAR antibody. (A) A 15-day-old rat ovary. (B) Punctate (arrow) forms of StAR immunoreactivity in 25-day-old rat ovary. (C) StAR protein localization in PMSG-treated rat ovary. (D) Negative control of ovarian section obtained from a 25-day-old rat antral follicle. Arrow, punctate StAR localization; arrowhead, no StAR localization; a, antrum; O, oocyte; GC, granulosa cell layers; T, thecal interstitial cells. Bars = 50 µm.

After stimulation of follicular differentiation with PMSG for 48 hr, it became evident that many follicles were recruited to the preovulatory stage (Figure 1C). In the ovaries of these hormonally treated animals, all preovulatory follicles expressed StAR immunoreactivity. Similar to the early antral follicles of untreated juvenile rat, some mural GCs extending from the basement membrane to the antrum of preovulatory follicles showed a punctate StAR immunostaining pattern (Figure 1C, arrow). The theca interstitial cells (Figure 1C) and at least some GCs (Figure 1C, arrowhead), at this stage of follicular differentiation did not stain positively for StAR. A representative control section from an antral follicle is shown in Figure 1D.

Further examination of StAR immunolocalization in adult rat ovaries indicated a reduction in protein expression in GCs during the luteal phase in early antral follicles (Figure 2). This is in contrast to the elevated immunoreactivity levels observed in early antral GCs of the juvenile ovary (Figure 1B). Punctate StAR immunostaining could be observed within some theca cells surrounding these early antral follicles (Figure 2A, arrow). Interestingly, in the mature corpus luteum there were some cells that exhibited strong immunoreactivity to StAR, whereas other cells had comparatively lower levels of immunostaining (Figure 2A and Figure 2B). In the large antral follicles of cycling ovaries, we found scattered punctate staining patterns of StAR in the mural granulosa cell layers (Figure 2B). This is in contrast to the patterns observed in the theca cell layers that surround early antral follicles (Figure 2A). The oocyte was not positive for StAR (Figure 1A and Figure 2A). Figure 2C shows an example of a negative control micrograph of a corpus luteum.



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Figure 2. StAR localization in an adult diestrous rat ovary as imaged by confocal microscopy. Frozen ovarian sections (15 µm) from cycling rat ovaries were reacted with an anti-peptide StAR antibody. (A) Early antral follicle and corpus luteum. Note punctate staining (arrow) patterns localized to some theca and corpus luteum cells, but undetectable in GC layer and some corpus luteum cells (arrowhead). Bar = 40 µm. (B) Large antral follicle and corpus luteum. At this stage, sparsely distributed punctate expression of StAR immunoreactivity was evident in GCs. (D) Negative control of a corpus luteum. O, oocyte; GC, granulosa cells; T, thecal interstitial cells; arrow, StAR punctate localization; arrowhead, no StAR localization. Bars = 20 µm.

Western Blot Analysis of StAR in Rat Ovaries
A 37-kD protein was observed in all protein extracts derived from infant to adult ovaries, although there was a decrease in the amount of the 37-kD protein in PMSG-treated juvenile and adult rat ovaries (Figure 3A). A 30-kD protein was also detected in the juvenile, juvenile treated with PMSG, and adult ovaries (Figure 3A, Lanes 2–4). This 30-kD protein was undetectable in infant rat ovaries (Figure 3A, Lane 1) but was significantly increased in juvenile and adult ovaries (Figure 3B). Changes in protein levels during the various stages of follicular differentiation were determined and quantified using a BioImage Whole Band Densitometer. Figure 3B shows relative densitometric values for the 30-kD protein expressed among all groups tested in this study. Relative to untreated juvenile and infantile ovaries, there was a 10- to 14-fold increase in the 30-kD protein in PMSG-treated juveniles and untreated adult ovaries (Figure 3B). The appearance of the 30-kD protein was observed to coincide with the localization and punctate staining pattern for StAR in GCs (see Figure 1 and Figure 2).



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Figure 3. Western blot of StAR protein in rat ovaries during follicular differentiation (A,C). Ovarian homogenates (50 µg) were subjected to 12% SDS-PAGE followed by transfer of protein to 0.2-µm nitrocellulose membranes. Lanes 1, 15-day-old ovary; Lanes 2, 25-day-old ovary; Lanes 3, PMSG-treated ovary; Lanes 4, adult diestrous ovary. The blots were probed with an anti-peptide StAR antibody (A) and an anti-peptide StAR antibody to the signal sequence (amino acids 1–26), followed by a secondary antibody conjugated to horseradish peroxidase. Specific signal detection was via chemiluminescence and was quantitated by computer-assisted densitometry (B). Data points represent the mean ± SEM of three or four independent gel runs. Different superscripts represent significant differences (p<0.05).

To determine the specificity of the 37-kD protein observed in total ovarian extracts (Figure 3A), we used an anti-peptide antibody to the signal sequence (peptides 1–26) of StAR. Western blot analysis revealed a single band of the 37-kD protein in extracts from all four groups of ovaries evaluated (Figure 3C). This band corresponds to the characteristic protein band observed in Figure 3A. This 37-kD protein showed a marked reduction in expression during ovarian differentiation (Figure 3A and Figure 3C). This is in contrast to that of the 30-kD protein, whose levels of expression increased as the follicles differentiated (Figure 3A and Figure 3B).

Samples of the respective protein extracts were also utilized in two-dimensional Western blot analyses to determine whether changes in StAR isoforms occur during the transitional stages of follicular differentiation. Two-dimensional gel analyses delineated four polypeptide species of the 30-kD protein (Figure 4). For convenience of description, these isoforms were arbitrarily assigned the numbers 1, 2, 3, and 4. These had isoelectric points (pI) of 6.2, 6.0, 5.8, and 5.6, respectively. Although not detectable in protein samples obtain from the infantile ovaries, the more basic of these isoforms [numbers 1 (pI 6.2) and 2 (pI 6.0)] were more highly expressed in juvenile ovaries compared to those of adults. However, in adult ovaries, as with PMSG-treated juveniles, a shift to the more acidic isoforms of the 30-kD protein appears to be favored (Figure 4).



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Figure 4. Two-dimensional gel electrophoresis patterns for StAR expression. Sample of rat ovaries (15-day, 25-day, PMSG-treated, and adult diestrus) (100 µg) were focused for 16,000 v.h with a mixture of pH 3–10 and pH 5–7 ampholyte and following the second dimension spots were detected by Western blotting procedure. The left side of the membrane was basic and the right side acidic.

Expression of StAR in Isolated Granulosa Cells
The cell type-specific expression of StAR in GCs cells was examined during the transitional stages of follicular differentiation, using polyclonal anti-StAR peptide antisera. Granulosa cells were obtained from infantile and juvenile ovaries and from large preovulatory follicles of PMSG-treated juvenile ovaries (Figure 5A). Analysis of the protein immunoprecipitates extracted from these cells, as shown in Figure 5B, revealed the presence of the 30-kD protein but not the 37-kD form. There was an apparent fourfold increase in the expression of the 30-kD protein in hormonally primed GCs from juveniles compared to nonprimed follicles (Figure 5C). In infant GCs, the 30-kD protein was undetectable.



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Figure 5. Immunoprecipation analysis of rat ovarian granulosa cells isolated and immediately cultured with [35S]-methionine for 5.5 hr. Shown are cells viewed with Nomarski optics (A). Cells were harvested after lysis in RIPA buffer with 0.2% SDS, and 1 x 106 cpm of material was precipitated with trichloroacetic acid (TCA) and then incubated with a rabbit polyclonal antibody to StAR. Precipitated proteins were resolved on 12% PAGE under reducing conditions (B). Lanes 1, 15-day-old; Lanes 2, 25-day-old; Lanes 3, PMSG-treated. Signals were detected with a Molecular Dynamic PhosphoImager and quantitated by computer-assisted densitometry (C). Data points represent the mean ± SEM of three independent gel runs. Different superscripts represent significant differences at p<0.05.


  Discussion
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Materials and Methods
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In this study, immunological techniques were used to characterize StAR expression patterns during the transitional stages of follicular differentiation. Our data provide evidence confirming the localization and expression of StAR in the granulosa cells of juvenile rat ovaries before and after PMSG treatment. We have also shown that StAR expression also occurs in theca intersitial cells surrounding preantral, antral, and larger antral follicles in adult diestrous ovaries. Furthermore, we have demonstrated heterogeneous StAR immunoreactive staining in the granulosa cell layers and in cells of the corpus luteum. A novel finding presented here is that, during ongoing growth and differentiation of the follicle, the immunoreactivity of StAR tends to shift from the GCs of early antral follicles to the theca cell layers in the adult. The present findings are consistent with previous reports demonstrating that, in the rat, the spatial and temporal patterns of ovarian StAR expression appear to coincide with the steroidogenic potential of the follicle during folliculogenesis (Ronen-Fuhrmann et al. 1998 ).

After PMSG administration, StAR immunoreactivity increased only in the granulosa cells of preovulatory follicles. No evidence for StAR immunostaining was obvious in GCs of preantral follicles of 15-day-old infants, 25-day-old juveniles, or juvenile ovaries treated with PMSG. In addition, StAR immunostaining could not be detected in theca interstitial cells of infant, juvenile, or juvenile rat ovaries treated with PMSG. However, StAR was localized to theca interstitial layers in adult animals, indicating a GC-to-theca maturational shift in follicular StAR expression. Immunoprecipitation of the StAR protein from isolated GCs and immunohistochemistry unequivocally demonstrate that StAR expression occurs in these cells. In addition, these data rule out the possibility of artifactual StAR localization in the granulosa cells in immunohistochemistry studies. Taken together, these results suggest that the appearance of StAR in granulosa cells could potentially signal early functional maturation of the rat ovarian antral follicles.

According to a report by Ronen-Fuhrmann et al. 1998 , StAR expression was not detectable within juvenile rat ovarian granulosa cells before or 48 hr after PMSG administration. The apparent discrepancy in the immunodetection of the StAR protein between the present study and that of Ronen-Fuhrmann et al. 1998 is probably due to differences in fixation and immunohistochemical treatment of the tissue. Preliminary studies in which cryostat sections were fixed with paraformaldehyde and permeabilized with Triton X-100 indicate that immunoreactivity to StAR was very weak (not shown). However, fixation of proteins with both paraformaldehyde and methanol enhanced detection of the punctate cytoplasmic accumulation of the StAR antigen. The use of laser scanning confocal microscopy for image analysis also facilitated improved visualization and resolution of StAR localization in contrast to the conventional immunofluorescence microscopic techniques that were used by Ronen-Fuhrmann et al. 1998 .

In contrast to its immunolocalization in juvenile rat ovaries, StAR expression was not found in granulosa cell layers of early antral follicles of the adult diestrous rat. However, preovulatory follicles exhibited sparsely distributed punctate StAR immunoreactivity in some GCs. This punctate pattern increased in the adult theca interstitial cell layers surrounding the preantral, antral, and preovulatory follicles. Some cells in the corpus luteum also exhibited high levels of this punctate staining, consistent with observations reported in the human ovary by Pollack et al. 1997 and by Kiriakidou et al. 1996 . To our knowledge, the results from the present study provide the first direct visualization of StAR in the adult diestrous rat ovary. The data strongly suggest differential regulation of StAR expression in the developing ovary.

Recent studies report the existence of two subpopulations of GCs in the juvenile rat ovary on the basis of size, which appear to respond differentially to hormonal and growth factor stimulation (Kasson et al. 1985 ; Rao et al. 1991 ; Sanbuissho et al. 1993 ). Microheterogeneity of StAR expression was revealed in granulosa cells of individual follicles and the corpus luteum. This apparent heterogeneous expression of StAR may be indicative of the steroidogenic potential of the immnolabeled cells. The presence of two different cell sizes in the corpus luteum has also been demonstrated in rat ovary (Wilkinson et al. 1976 ). These observations suggest that steroidogenesis in these cell types may be regulated by different mechanisms and, as a result, could be important in regulating the functional lifespan of the antral follicle and the corpus luteum. In part, differential StAR regulation could be a function of the local hormonal microenvironment and/or of follicular responsiveness to gonadotropins (Richards 1980 ; Ojeda et al. 1986 ), growth factors, and steroids (Leung and Steele 1992 ).

The present investigation has identified both a 37- and a 30-kD protein in rat ovarian cells that correspond to the known molecular weight of StAR. These results are consistent with those previously reported for bovine corpora lutea (Pescador et al. 1996 ) and rat granulosa cells cultured on an extracellular matrix (Aharoni et al. 1997 ). Two-dimensional gel electrophoretic analyses have further delineated four isoforms of the 30-kD protein. The more acidic of these isoforms were found to have increased during ovarian differentiation and have been previously identified as phosphorylated products (Pon and Orme-Johnson 1988 ; Stocco 1997 ). Phosphorylation of the StAR protein is associated with an increase in its biological activity (Arakane et al. 1997 ). Unlike the 30-kD protein, however, we were unable to immunoprecipitate the 37-kD protein from isolated GCs. Although inefficient immunoprecipitation of the 37-kD protein might have occurred under the present experimental conditions, this observation could also indicate that the 37-kD protein is immediately processed, after de novo synthesis, to form the 30-kD protein in 25-day-old juvenile GCs. Another likely scenario is that we and others (Pescador et al. 1996 ; Aharoni et al. 1997 ) have identified a novel 37-kD protein expressed in rat granulosa and bovine corpora lutea cells that has sequence homology to StAR. Clarification of this discrepancy will require the isolation and sequence analysis of this 37-kD protein. These studies are now in progress in our laboratory.

Functionally, StAR has been considered a regulator of the rate-limiting transfer of cholesterol from the outer to the inner mitochondrial membrane in steroid hormone biosynthesis. This biosynthetic mechanism is responsive to tropic hormones via a cAMP-dependent pathway (Clark et al. 1994 ). Our results indicate a possible intraovarian regulatory role for StAR in follicular differentiation (Leung and Steele 1992 ). The demonstrated shift in StAR expression suggests a possible maturational shift in gonadotropic hormone responsiveness. The data imply that primary StAR-dependent steroidogenic activity is confined largely to the GC compartment during the early stages of follicular differentiation. Consistent with this is the notion that the steroidogenic capacity of granulosa cells may confer some level of synergism with FSH to bring about induction of functional maturation or recruitment for ovulation (Ronen-Fuhrmann et al. 1998 ).

In summary, we have provided evidence that StAR is first expressed and localized in the juvenile antral follicles of the rat (without exogeneous hormonal stimulation). StAR is also expressed and localized to some cells of antral follicles of unstimulated and stimulated juvenile rat ovaries, and to the corpora lutea of the adult diestrous rat. Spatiotemporal changes or shifts in StAR expression and cellular localization coincide with the appearance of more acidic isoforms of the 30-kD protein. Although the functional implications of these observations are still speculative, the acute temporal changes in StAR expression and localization may reflect not only the dynamic steroidogenic capacity of follicular cells but also possible FSH induction of follicular maturation. A number of questions remain to be answered concerning the subpopulation (i.e., small and large cells) of granulosa and luteal cells in which StAR first appears. It is also unclear what other regulatory factors, in addition to gonadotropins, modulate (i.e., intragonadal signals such as growth factors) StAR expression in early antral follicles and corpora lutea during the transitional stages of follicular differentiation.


  Acknowledgments

Supported in part by Baxter International Corporation (WET) and by an NIH grant U54-NS34194 (JAW), and by an RCMI institutional grant to the Molecular Genetic Core Facility (RRAI03034).

Special thanks are extended to Dr Douglas Stocco for his generous gifts of StAR antibodies. We also thank Drs Craig Bond, Marlene MacLeish, and Judith Gwathmey (Boston University) for their editorial comments.

Received for publication September 21, 1998; accepted January 19, 1999.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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