ARTICLE |
Correspondence to: Winston E. Thompson, Dept. of Obstetrics and Gynecology, Morehouse School of Medicine, 720 Westview Dr. SW, Atlanta, GA 30310.
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Summary |
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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 47:769776, 1999)
Key Words: rats, StAR, immature, adult diestrus, immunohistochemistry, immunoprecipitation, Western blotting, ovaries
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Introduction |
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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 (
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 (
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.
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Materials and Methods |
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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 SpragueDawley 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
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 126; 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
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 ASepharose (
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, MannWhitney 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.
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Results |
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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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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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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 24). 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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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 126) 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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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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Discussion |
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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 (
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
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
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 (
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 (
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 (
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.
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Acknowledgments |
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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.
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