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a Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In summary, this study demonstrates a distinct, cell-type specific regulation of StAR and P450scc mRNA in the two bovine luteal cell types.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ovulatory surge of LH triggers the differentiation of preovulatory follicular theca and granulosa cells into the highly steroidogenic cells of the corpus luteum (CL)—the small and the large luteal cells, respectively [10]. Gonadotropins are thought to be the primary regulators of luteinization in ruminants and in other species. However, in addition to this role of gonadotropins, important regulatory roles have also been assigned to an array of locally produced ligands such as insulin-like growth factor I (IGF-I) [11] and prostaglandin I2 [12].
Although both steroidogenic luteal cell types, i.e., the large and small luteal cells, are engaged in progesterone production, they exhibit diverse functional properties. One of the remarkable differences between these cell types is their differing basal and hormone-stimulated progesterone production. The molecular mechanisms underlying these luteal cell characteristics remain mostly obscure. Our studies have shown that granulosa and theca interna cells derived from preovulatory follicles can be induced to luteinize and acquire the characteristics of bovine small and large luteal cells, respectively [13–16]. This experimental system can be therefore used as a model for luteal cell function. The present study examined P450scc, StAR, and SF-1 mRNA expression in cells induced to luteinize under different hormonal (i.e., cAMP, insulin, and IGF-I) environments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco's Modified Eagle's medium:Ham's F-12 1:1 (v:v) nutrient mixture and Moloney murine leukemia virus (MMLV) SuperScript II H- reverse transcriptase were from Gibco BRL Life Technologies (Gaithersburg, MD); penicillin, streptomycin, neomycin, and fetal calf serum (FCS) were from Biological Industries (Beit HaEmek, Israel); forskolin and insulin were from Sigma (St. Louis, MO); deoxynucleotide triphosphates (dNTPs), random hexamers oligodeoxynucleotide, and Taq DNA polymerase were from Farmentas (Vilnius, Lithuania); and oligonucleotide primers were synthesized by Biotechnology General (Kiryat Weizmann, Rehovot, Israel). The enhanced chemiluminescence (ECL) Western blotting detection system was from Amersham (Buckinghamshire, UK); IGF-I was kindly provided by Prof. Arie Gertler, The Hebrew University of Jerusalem, Rehovot, Israel. Anti-bovine SF-1 antiserum was a generous gift from K. Morohashi of Kyushu University, Japan.
Cell Cultures
Granulosa and theca cells were isolated from healthy bovine preovulatory follicles as previously described [13, 14]. The experimental design involved long-term cultures in which granulosa and theca cells were cultured for 8 days with the treatments specified below. In the short-term cultures, cells were incubated with additional treatments for 24 more hours.
Long-term cultures Granulosa and theca cells were cultured for 8 days in 24-well plates, in media containing 1% FCS (basal medium) with or without insulin (2 µg/ml) and with varying concentrations of the adenylyl cyclase activator forskolin (0.1, 1, or 10 µM). In a separate set of experiments, cells were also cultured with physiological concentrations of insulin (20 ng/ml) and IGF-I (50 ng/ml). On Day 8, media were collected for progesterone determination, and DNA was extracted [17]. Cells were washed twice with ice-cold PBS, and total RNA was extracted.
Short-term cultures These studies were carried out only with cells cultured in basal media containing insulin (2 µg/ml) and forskolin (10 µM)—conditions previously shown to induce cell luteinization [13, 14]. On Day 8 of culture, cells were washed twice and incubated for 24 h in media containing the different treatments specified below. After the 24-h incubation period, cells were washed twice with ice-cold PBS, and total RNA was extracted.
RNA Extraction and Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)
Total RNA was extracted by the guanidinium thiocyanate method [18]. Total RNA (1 µg) was preheated for 5 min at 70°C, immediately cooled on ice, and reverse-transcribed for 50 min at 42°C in 20 µl of a reaction mixture containing single-strength RT buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol), 0.5 mM of each dNTP, 100 pmol random hexamers oligodeoxynucleotide, and 50 U of reverse transcriptase. The RT reaction was terminated by heating for 15 min at 70°C. The resulting cDNA templates were subjected to PCR amplification. Each cycle consisted of 30 sec at 95°C for denaturation; 30 sec at 58°C for annealing; and 1 min at 72°C in 25 µl of PCR reaction mixture for extension. The reaction mixture contained cDNA derived from the RT tube, 10 pmol of each oligonucleotide primer, 10 mM Tris-HCl (pH 9), 50 mM KCl, 2.5 mM MgCl2, 0.1% Triton X-100, 0.1 mM of each dNTP, and 2 U of Taq DNA polymerase. MgCl2 concentration, and amounts of dNTPs, oligonucleotide, and enzyme were precalibrated to ensure maximal reaction efficiency. PCR reaction products (20 µl of the 25-µl total reaction volume) were electrophoresed on 2% agarose gels, stained with ethidium bromide, and photographed by means of Fuji's BioSystem (Fuji Film Co., Tokyo, Japan). Band intensities were analyzed by computerized densitometry using NIH Image version 1.61 equipped with macros developed by Thomas Seebacher from the University of Konstanz, Germany.
Oligonucleotide Primers
PCR oligonucleotide primer pairs (19–22 nucleotides, 45–60% GC content) were designed on the basis of the cDNA sequence of the various target genes. All primer pairs were designed to span at least one intron, to avoid amplification of DNA contaminants. Several StAR mRNA transcripts have been described in cows and other species; these transcripts arise from alternative polyadenylation. Therefore, StAR primers were designed to amplify a cDNA fragment located 5' to the first polyadenylation sequence. A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) oligonucleotide primer pair (sense 5'-TGTTCCAGTATGATTCCACCC-3'; antisense 5'-TCCACCACCCTGTTGCTGTA-3') was synthesized as described by Tsai et al. [19]. Bovine SF-1 primers (sense5'-TGCATGGTCTTCAAGGAGC-3'; antisense 5'-TAGTGGCACAGGGTGTAGTC-3') were synthesized according to the cDNA sequence cloned by Honda et al. [7]. Bovine P450scc primers (sense 5'-AACGTCCCTCCAGAACTGTACC-3'; antisense 5'-CTTGCTTATGTCTCCCTCTGCC-3') were synthesized according to the cDNA sequence cloned by Morohashi et al. [20]. Bovine StAR primers (sense 5'-CATGGTGCTCCGCCCCTTGGCT-3'; antisense 5'-CATTGCCCACAGACCTCTTGA-3') were synthesized according the cDNA sequence cloned by Hartung et al. [21]. The expected PCR product lengths were 850 base pairs (bp) for G3PDH, 590 bp for StAR, 382 bp for SF-1, and 362 bp for P450scc. Because of the similar product lengths of SF-1 and P450scc, amplification was carried out using either G3PDH, SF-1, and StAR primers or G3PDH, StAR, and P450scc primers. Computer searches and sequence alignments were performed with software from Genetics Computer Group, Inc. (Madison, WI).
Calibration of Semiquantitative Multiple RT-PCR
Semiquantitative RT-PCR was carried out as described previously using the housekeeping gene G3PDH as an internal standard [15]. G3PDH is constitutively expressed in both granulosa-derived luteal cells and theca-derived luteal cells and has been used effectively in studies on the regulation of gene expression in ovarian cells [15, 19, 22]. The RT-PCR amplification of luteinized theca and luteinized granulosa cells (LTC and LGC, respectively) was calibrated using G3PDH and SF-1 or G3PDH, StAR, and P450scc primer pairs. The number of cycles was varied to determine the optimal number that would allow detection of the appropriate messages, while still keeping amplification for these genes in the log phase (primer dropping method [23]). The following amplification cycles were used to compare levels of expression between luteal cells (Fig. 1): G3PDH, 20 cycles; P450scc, 20 cycles; StAR, 24 cycles; and SF-1, 27 cycles. Because higher levels of P450scc and StAR were expressed in LGC than in LTC (Fig. 1), different cycles were used for the PCR reactions of LGC and LTC in all other experiments. In LTC, 21 cycles were used for P450scc and G3PDH and 25 cycles for StAR; in LGC, 21 cycles were used for G3PDH and StAR and 19 cycles for P450scc. SF-1 was amplified for 27 cycles in both cell types.
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Western Blot Analysis
Total cellular proteins were extracted in extraction buffer (50 mM Tris-HCl pH 7.5, 2% SDS) and briefly sonicated. Protein homogenates (20 µg) were electrophoresed by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The blots were incubated with anti-SF-1 antiserum and detected by the ECL method as previously described [24].
RIA for Progesterone
The assay for progesterone was carried out as described previously [13]. The intra- and interassay coefficients of variation were 5.2% and 12%, respectively, and the sensitivity limit was 1.9 pg/tube.
Statistical Analysis
All experiments were repeated at least three times; data are presented as means ± SEM. One-way ANOVAs were used to determine statistical difference between effects of treatments (basal medium, three forskolin concentrations, insulin, and their combinations) as indicated in the text. Interactions between effects of insulin and forskolin on progesterone production and P450scc and StAR mRNA levels were analyzed by a two-way ANOVA. The relationship between levels of mRNA and progesterone levels was determined by correlation analyses. All statistical analyses were performed using JMP software [25]. A value of p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mRNA levels of P450scc, StAR, and SF-1 mRNA in the two luteal cell types was determined. The mean values (in arbitrary units; n = 6) of StAR and P450scc mRNA levels were 17.26 ± 4.6 and 15.18 ± 3.0, respectively, for LGC, and 3.71 ± 0.95 and 3.04 ± 0.51, respectively, for LTC (Fig. 1). Indeed, the data show that both P450scc and StAR were expressed at higher levels in LGC (4.6-fold and 5-fold, respectively, p < 0.001) than in LTC. In contrast, SF-1 was expressed in equal amounts in both cell types: the mean values (n = 6) of SF-1 expression for LGC and LTC were 7.5 ± 0.5 and 8.9 ± 1.1, respectively (Fig. 1).
Hormonal Control of Steroidogenesis in Cells Undergoing Luteinization
Next, we studied the regulatory roles of forskolin and insulin on the steroidogenic capacity of cells undergoing luteinization (Fig. 2). On Day 8, progesterone production during the last 24 h of culture was measured, and P450scc, StAR, and SF-1 mRNA levels were determined by RT-PCR (Figs. 2 and 3).
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Culture of theca cells for 8 days with insulin alone did not significantly influence StAR or P450scc mRNA levels (Fig. 2, a and b); a high concentration of forskolin (10 µM) increased both progesterone production and StAR mRNA (3.8 ± 1.8 and 0.02 ± 0.01 vs. 39.8 ± 8.2 and 0.09 ± 0.04, respectively; p < 0.05). However, the combined treatment of forskolin and insulin induced a synergistic stimulation of progesterone production, P450scc mRNA, and StAR mRNA; the effect was significantly different from that of forskolin alone (p < 0.01; Fig. 2, a–c). In the presence of insulin, forskolin stimulated StAR mRNA, P450scc mRNA, and progesterone production in a dose-dependent manner, with maximal increases being 130-, 3.6-, and 33-fold, respectively, over basal levels. Progesterone biosynthesis by LTC, which were induced to luteinize in the various hormonal milieus, was highly and positively correlated (r = +0.91) with the steady-state mRNA levels of StAR and to a lesser degree with those of P450scc (r = +0.47).
In contrast to the synergistic interaction of forskolin plus insulin in LTC, each of these regulators independently affected steroidogenic functions in LGC (Fig. 2, d–f). Insulin significantly (p < 0.01) up-regulated progesterone production and StAR mRNA levels (Fig. 2, d and f), which were 2–3 times higher in LGC incubated with insulin (159.9 ± 21.7 and 1.12 ± 0.2, respectively) than in basal media only (61.1 ± 16.0 and 0.47 ± 0.14, respectively). Forskolin, on the other hand, did not significantly affect StAR mRNA levels, but it stimulated a dose-dependent increase in P450scc mRNA expression (Fig. 2e, p < 0.01). There was no additive or synergistic effect of forskolin and insulin stimulation on P450scc or StAR mRNA in LGC. However, the independent actions of forskolin (up-regulating P450scc) and insulin (up-regulating StAR) were combined in their effect on progesterone production by LGC (Fig. 2f). In this cell type, insulin and forskolin each significantly (p < 0.01) elevated progesterone synthesis. However, while 10 µM of forskolin alone was necessary to exert a significant effect, progesterone could be stimulated to its maximal levels by 1/100 of this concentration in the presence of insulin.
The expression of SF-1 in luteal cells is demonstrated in Figure 3. SF-1 protein content did not vary with luteal cell type or in the presence of either forskolin or insulin (Fig. 3a). Similarly, SF-1 mRNA levels were not altered by the hormonal treatment in either LTC or LGC (Fig. 3b). The induction of StAR mRNA was clearly visible in LTC incubated with forskolin plus insulin.
Effects of Insulin and IGF-I on StAR mRNA Levels
Using pharmacological concentrations, insulin can exert its effects via both insulin and type 1 IGF-I receptors. To better define the mechanism of insulin action in LGC, cells were cultured with physiological and pharmacological concentrations of insulin (20 and 2000 ng/ml, respectively); in addition, cells were cultured with IGF-I (Fig. 4). IGF-I, similar to insulin at 2 µg/ml, significantly increased StAR mRNA levels in LGC (p < 0.05). Also in LTC, the effects induced by IGF-I on StAR mRNA levels were similar to those exerted by 2 µg/ml of insulin (data not shown). Together, these data suggest that the effects of insulin are exerted via type 1 IGF I receptors.
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Maintenance of P450scc and StAR mRNA Levels in Luteal Cells
We next examined the roles of forskolin and insulin in the maintenance of P450scc and StAR mRNA expression in luteal cells that had already acquired high steroidogenic capacity (i.e., cells cultured in the presence of 10 µM forskolin and 2 µg/ml insulin). On Day 8 of culture, forskolin, insulin, or both were withdrawn from the media, and incubation was continued for an additional 24 h (Fig. 5). In LTC, the removal of forskolin caused a significant reduction in both StAR and P450scc mRNA levels (45% and 70%, respectively, p < 0.01), and a small, nonsignificant decrease in the expression of both genes was evident in cells incubated for 24 h without insulin (Fig. 5). In contrast, in LGC, insulin appeared to play a major role in maintaining StAR mRNA levels; its withdrawal reduced StAR mRNA levels by 50% (p < 0.01). As observed for LTC, P450scc mRNA levels were significantly reduced in LGC (by 30%, p <0.05) as a result of the removal of forskolin. In keeping with the results obtained in the long-term cultures, variations in the levels of P450scc mRNA were smaller than those observed for StAR mRNA. There was no additional reduction in either StAR or P450scc mRNA levels when both insulin and forskolin were withdrawn from the culture media.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since the discovery of StAR a few years ago [3], it has been argued that cAMP (being a mediator of tropic hormones) was sufficient for the induction of StAR mRNA [4, 26, 27]. For instance, incubation of transformed Leydig cells [27] or human granulosa and theca-lutein cells [26] with cAMP analogues dramatically elevated StAR mRNA. These results seem to conflict with the present findings for bovine luteal cells, demonstrating that forskolin alone (in both cell types) only marginally affected the induction of StAR. However, in these previous studies, cells were cultured in the presence of 15% serum, which may contain considerable amounts of several growth factors including IGF-I; therefore, the necessity for IGF-I in StAR gene expression cannot be ruled out. The role of IGF-I in StAR mRNA induction was also demonstrated in a study carried out by Balasubramanian et al. [28]. Using immature porcine granulosa cells under serum-free conditions, they reported that a substantial increase in cAMP-induced StAR mRNA levels could not be observed unless IGF-I was concomitantly added. A similar synergistic effect involving cAMP and insulin was demonstrated in the present study only for LTC. In LGC, on the other hand, it was insulin alone, rather than its combination with cAMP, that markedly elevated StAR. The difference between the present findings and those previously reported [28] may be attributed to the use of granulosa cells of medium versus large follicles. In the present study, the cells induced to luteinized were mature granulosa cells derived from preovulatory follicles. Such cells had already acquired the ability to produce low levels of progesterone and to express StAR gene [29], in contrast to the immature granulosa cells of medium follicles [28, 30]. Therefore, it may be suggested that the initial induction of StAR mRNA (in immature cells) is regulated by different mechanisms from those that control its continuous transcription (in mature cells).
StAR is believed to play a key role in the acute regulation of steroidogenesis; this acute stimulation might be regulated by changes in message stability or translation of mRNA [27] or by posttranslational modification of this protein (e.g., phosphorylation [31]). The observations presented here address the longer-term regulation of StAR, which may be particularly relevant to the process of luteinization. Indeed, during luteal development there is a gradual up-regulation in StAR mRNA as well as P450scc levels, which reach a plateau at mid-luteal phase (Days 8 and 12 for pigs [32] and cows [21], respectively).
The numerous effects of insulin and IGF-I are mediated via the tyrosine kinase activities of their receptors; these two peptides also increased phosphatidylinositol-3-kinase activity and DNA synthesis in bovine luteal cells [33]. The activation by gonadotropins/cAMP, on the other hand, involves the protein kinase A pathway. The point at which these signaling pathways interact to ultimately increase StAR mRNA in LTC is as yet unknown. However, in LTC this synergism was also apparent in P450scc gene expression and in progesterone production; therefore, it is reasonable to assume that it occurs at an early step(s) of their signaling pathways. Potentiation of forskolin-activated adenylyl cyclase by insulin/IGF-I may account for such a phenomenon [34]. No such synergism could be detected in LGC, in which the stimulation of P450scc was cAMP-dependent and that of StAR was insulin-dependent. Different mechanisms may, therefore, operate to control the expression of these genes in the two respective luteal cell types. In this regard, it is worth noting that the expression of the LH receptor also differed between the two luteal cell types [15]. The existence of cell-type specific, cis-acting factors might explain such observations.
IGF-I is produced locally within the bovine CL [11]; in addition, IGF-I produced by the liver also reaches the ovary, where it can up-regulate StAR expression in steroidogenic luteal cells. Support for this contention comes from the studies of Juengel et al. [35]. They demonstrated that growth hormone (GH) injection (which is likely to increase IGF-I release from the liver [36] and also from the ovary [37]) significantly elevated luteal StAR mRNA levels (and plasma progesterone concentrations) in hypophysectomized ewes. Moreover, the combined administration of LH and GH restored StAR mRNA levels to those measured in the intact animal [35]. The hormonal regulation of StAR mRNA in the two luteal cell types, defined in the present study, may explain the molecular/cellular mechanisms underlying the in vivo effects of LH and GH mentioned above.
Numerous reports have shown that granulosa-derived large luteal cells exhibit unique functional characteristics as compared with other steroidogenic cell types or even with theca-derived small luteal cells [16, 38]. In vivo, large luteal cells, which constitute only a minority of luteal cells, contribute the major portion of total progesterone output from the CL [38, 39]. Previously, we [16] and others [40] have shown that this is partially due to high P450scc mRNA levels and a stable protein content, which is maintained in the absence of cAMP-inducing agents. Data presented in this study further expand our understanding of the steroidogenesis in large luteal cells. In addition to P450scc, the large luteal cell also contained high levels of StAR mRNA, induced and maintained by insulin/IGF-I. IGF-I is produced by the granulosa-derived luteal cells [11], and since these cells also express IGF-I receptors [41, 42], it can act in an autocrine way. Together, these facts may explain the "autonomous" production of progesterone by bovine large luteal cells, i.e., secretion of elevated levels of progesterone independently of external stimuli. The physiological importance of IGF-I in stimulating and maintaining steroidogenesis in the large luteal cell is reinforced by the fact that these cells express very low levels of LH receptor and have a low LH-induced cAMP response [43], rendering these cells highly dependent on insulin and/or IGF-I.
Our findings indicate that the inductive nature of steroidogenesis in LTC is in fact a reflection of its StAR, rather than P450scc, gene expression. Nevertheless, even after stimulation, theca-derived luteal cells contained lower StAR mRNA levels (and progesterone production) than granulosa-derived luteal cells.
The orphan nuclear receptor SF-1 was initially shown to be an essential regulator of the cytochrome P450 steroid hydroxylases [7] and was subsequently linked to various genes expressed throughout the hypothalamic-pituitary-steroidogenic organ axis [9]. Analyses of the human [44] and mouse [45] StAR gene promotors revealed sequence elements that bind SF-1, thus implicating SF-1 in the expression of StAR gene as well. Whether SF-1 expression is hormonally regulated is still an open question. Using bovine luteal cells, Michael et al. [46] found that forskolin elevated the protein and mRNA expression of SF-1; in contrast, SF-1 was constitutively expressed and did not vary during the various stages of follicular and luteal development in the rat [6]. Our present findings also support the notion that SF-1 is constitutively expressed in steroidogenic cells. Hormonal treatments that caused vast changes in P450scc and StAR mRNA levels (and in progesterone production) were not accompanied by alterations in SF-1 mRNA levels. In addition, we could not observe differences in SF-1 mRNA levels between the two luteal cell types.
In summary, the present study provides an insight into the elaborate control of steroid hormone synthesis in each of the two luteal cell types. An understanding of such regulatory mechanisms may eventually lead to better control of normal or abnormal (insufficient) CL function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: October 13, 1998.
Received: July 24, 1998.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R. Mamluk, N. Levy, B. Rueda, J. S. Davis, and R. Meidan Characterization and Regulation of Type A Endothelin Receptor Gene Expression in Bovine Luteal Cell Types Endocrinology, May 1, 1999; 140(5): 2110 - 2116. [Abstract] [Full Text]
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