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Articles |
a Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
b Department of Biochemistry and Cell Biology, Texas Tech Health Sciences Center, Lubbock, Texas 79430
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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hydroxylase [3] and up-regulation of the progesterone synthetic enzymes, cytochrome P450 side-chain cleavage enzyme (P450scc, [3, 4]) and 3ß-hydroxysteroid dehydrogenase [5]. The loss of estrogen synthesis by granulosa cells coincides with loss of expression of cytochrome P450 aromatase (P450arom [6]). The mobilization and delivery of cholesterol to the mitochondria is ligand-dependent in ovarian steroidogenic cells. A key step in the process is the transport of cholesterol from intracellular stores to the inner mitochondrial membrane, where it transformed to pregnenolone by P450scc. A protein that effects this transfer, steroidogenic acute regulatory protein (StAR), was identified in 1994 [7]. Its significance to steroidogenesis can be seen in the profound disruptions of gonadal and adrenal differentiation and of adrenocortical function that ensue when the StAR gene is mutated in humans [8] or in mice [9]. A role for StAR in luteinization can be inferred from its appearance in porcine follicles after the administration of ovulatory doses of gonadotropic hormone [4] and from the pattern of its expression in the corpus luteum (CL) during the bovine luteal phase [10]. In luteinized porcine granulosa cells, StAR expression is stimulated by FSH and cAMP, depends on ongoing protein synthesis, and is inhibited by phorbol 12 myristate 13-acetate (PMA [11]).
Growth factors have marked effects on the function of mammalian granulosa cells [12]. Epidermal growth factor (EGF) is a potent mitogen in pig granulosa cells in vitro [13, 14] and appears to be locally synthesized in the pig ovarian follicle [15]. Insulin-like growth factor I (IGF-I) expression can likewise be localized to the theca, stroma, and CL of the rat ovary [16] and has pleiotropic effects on ovarian cells. It interacts with gonadotropins in support of developing follicles in the bovine and ovine ovaries (reviewed in [17]). In the porcine ovary, IGF-I stimulates both proliferation and differentiation, usually in combination with FSH (for review, see [18]). In porcine granulosa cells in culture, IGF-I stimulates the activity of P450scc and P450arom [19] and acts in synergy with FSH in the induction of StAR gene expression [20].
As noted above, luteinization is a large-scale remodeling process that occurs in response to gonadotropin stimulation. Paracrine growth factors modulate both differentiation and proliferation, processes that appear to be mutually exclusive in luteinizing granulosa cells. The present experiments were conceived to test the hypothesis that EGF and IGF-I modulate luteinization in porcine granulosa cells, as indicated by the acquisition of the capacity to employ cholesterol for progesterone synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Porcine ovaries from adult and prepubertal gilts were collected from an abattoir. For determination of the variation in StAR expression associated with luteinization in vivo, CL were collected from one ovary each of cycling adult pigs at various stages of the estrous cycle. CL were placed in four categories corresponding to the stages of the luteal phase according to morphological criteria [21]: I, early; II, early midcycle; III, late midcycle; and R, regressed. Classification was confirmed by histological comparison of representative CL from the abattoir with CL taken from pigs slaughtered at known intervals after observed estrus. Three to five CL from each stage were collected for Northern and Western analysis. In the remainder of the experiments, granulosa cells, obtained by aspiration from medium-sized (3–5 mm) follicles from immature pigs, were employed. An aliquot of 3 x 106 viable cells was plated in 6-well plastic culture dishes in minimal essential medium (MEM; Gibco BRL, Burlington ON, Canada) containing 4.8 g/L Hepes (Gibco BRL), 2.2 g/L sodium bicarbonate (Sigma Chemical Co., St. Louis MO), 1 mg/L insulin (Sigma), 0.1 mM nonessential amino acids (Gibco BRL), 5 x 104 IU/L penicillin (Gibco BRL), 0.5 mg/L fungizone (Gibco BRL), and 10% fetal bovine serum (FBS; Gibco BRL). Incubations were carried out at 37°C in 95% humidified air with 5% CO2.
Experiments
The first experiment was conducted to determine the characteristics of in vitro luteinization of granulosa cells. Granulosa cells were treated with either dibutyryl cAMP (cAMP, 300 µM; Sigma) or medium alone, and cultures were terminated at 1, 6, 12, and 24 h. The cAMP dose was based on optimal stimulation of steroidogenic proteins in previous studies [11]. In the second trial, the effects of human IGF-I were assessed by incubating freshly isolated cells with 0, 1, 10, or 100 ng/ml human IGF-I (Gibco BRL), with cultures terminated at 24 or 48 h after initiation of treatment. In subsequent trials, the effects of cAMP (300 µM) and IGF-I (10 ng/ml) on the expression of factors associated with luteinization were assessed over 96 h from the time of isolation of cells. Cultures were terminated at 12, 24, 48, 72, or 96 h. As in previous studies [11], cells were incubated with FBS for the first 48 h and were serum-free for the final 48 h of culture. Subsequent trials were conducted in the luteinized cells that had been cultured for 96 h. These cells were treated with medium alone, cAMP (300 µM), or EGF (10 nM; Sigma), and incubated for a further 0, 12, 18, or 30 h.
RNA Analysis
CL and cultured cells were homogenized in 4 M guanidine isothiocyanate (Gibco BRL), 2.6 mM sodium acetate (Sigma), and 0.12 M fl-mercaptoethanol (Sigma), and stored at -70°C until analysis. Total RNA was purified by CsCl gradient ultracentrifugation, and aliquots of 15 µg were subjected to Northern analysis as previously described [11]. Probes were labeled with 32P to specific activity of 1.5–3.0 x 109 dpm/µg with a random primer synthesis kit (Boehringer-Mannheim, Laval, PQ, Canada). These and all remaining blots were hybridized in sequence with three probes, porcine StAR cDNA [11], P450scc cDNA [22], and 28s rRNA. Duplicate blots from all trials were hybridized with a P450arom probe corresponding to nucleotides 850–1497 of the bovine sequence [23]. To further characterize the response to EGF, representative blots were hybridized with a porcine LH receptor probe [24]. Quantitative estimates of mRNA abundance for the genes of interest were made as we have previously reported [10, 11]. For StAR, the two major positive bands on the consequent autoradiograms were scanned individually using a computer imaging system. The areas under the resultant curves were summed and integrated by means of the Macintosh program Collage (Fotodyne Inc., New Berlin, WI). The single P450scc, P450arom, and 28s bands were likewise scanned, and the latter served as control for loading and transfer of RNA.
Western Blots
CL from each of the four stages of the luteal phase described above were homogenized in 0.25 M sucrose, 10 mM Tris, and 0.1 mM EDTA. The mitochondrial fraction was isolated by centrifugation [10], and the protein content was assayed by the Lowry method [25]. Western immunoblotting was performed as previously described [10]. Membranes bearing electrophoretically separated proteins were incubated with primary antibodies specific to StAR [7]. The second antibody, donkey anti-rabbit IgG conjugated to horseradish peroxidase, was added in the presence of the blocking agent. The signal was detected by chemiluminescence as previously described [7], and the optical density of the immunospecific bands was quantitated by means of a BioImage (Ann Arbor, MI) Visage 2000 computer-assisted analysis system.
Morphological Observations
Cells were examined under phase contrast microscopy at 24-h intervals from the time of their isolation through 96 h. Cells were also observed at 6, 12, and 24 h after treatment with EGF. Photomicrographs of representative fields were taken.
Progesterone Assay
Culture medium samples taken at 48 h in the luteinization experiment and at all times following EGF treatment were subjected to dilution and RIA as previously described [26]. The intraassay coefficient of variation, calculated between duplicate samples, was 9% or less, and the interassay value, calculated from four samples present in all assays, ranged from 5% to 11%.
Statistical Analysis
Northern blot data, comprising the ratio of optical density values for the mRNA in question to that of 28s, and progesterone concentrations in culture medium were evaluated in three or four independent replicates of each experiment, each initiated on different days, in different pools of cells. For statistical analysis, Shapiro's test was employed to determine normality of distribution of optical density data, and when normality was absent, data were logarithmically transformed before analysis. Homogeneity of variance was confirmed for each experiment by Bartlett's test, and statistical significance was evaluated by ANOVA. In the presence of a significant overall F value, preplanned comparisons between treatments and controls were made by the least-significant-difference method. The relation of StAR mRNA to its protein abundance was determined by correlation analysis. The level of probability accepted as statistically significant was p < 0.05.
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RESULTS |
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The abundance of mRNA coding for both StAR and P450scc, depicted in Figure 1, varied during the luteal phase in CL (p < 0.05). Both were detectable in the earliest CL recovered, and their levels were elevated during the later part of the midluteal phase (p < 0.05). No message for either gene was detectable in regressed CL. StAR protein was also detected in the earliest CL collected. Mean protein abundance was 3- to 4-fold greater during the midluteal phase (p < 0.05), and no protein was detectable in Western blots from regressed CL (Fig. 1, inset). StAR message and StAR protein were correlated (r = 0.79, p < 0.03). P450arom mRNA was not detectable in any CL samples.
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Luteinization In Vitro
At isolation from porcine follicles, granulosa cells contained P450arom mRNA, while both StAR and P450scc mRNA was undetectable. Stimulation of these cells for 1 h with 300 µM cAMP resulted in up to 5-fold increases in the message coding for P450arom (p < 0.05; Fig. 2). By 6 h after isolation, both steady-state and cAMP-stimulated P450arom message were barely present, while P450scc mRNA had begun to appear. At 12 and 24 h, the P450arom message had disappeared, and it could not be found at any time thereafter through 120 h after cell isolation. The first cAMP induction of P450scc, 1.6-fold, was present at 6 h after the beginning of culture. Transcript abundance had increased to 4.5-fold at 12 h (Fig. 2).
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Cells underwent progressive morphological changes consistent with luteinization (Fig. 3). At 24 h after plating, the cultures were not yet confluent, and cells were found in clumps, with most maintaining the rounded shape present at isolation, and a few displaying cytoplasmic projections (Fig. 3A). By 48 h, large scale remodeling had occurred, confluence had been achieved, round cells had become rare, and a flattened, epithelial form was the most common phenotype. By 72 h, virtually all of the cells had attained the epithelial form, and by 96 h, an epithelial, block-like form was the most common phenotype (Fig. 3B).
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StAR mRNA was not detected in control cultures until 48 h after plating (Fig. 4), whereas P450scc mRNA, first observed at 6 h, was found to be continuously present through 96 h of culture (Fig. 5). Incubation with 300 µM cAMP hastened the appearance of the message coding for StAR by at least 24 h. The same dose of cAMP significantly elevated both StAR and P450scc message abundance at every time evaluated, from 24 to 96 h of culture (Figs. 4 and 5, p < 0.01). IGF-I (10 ng/ml) also induced precocious occurrence of StAR message relative to control cultures, as evidenced by a detectable signal that was present at 24 h. The effects of IGF-I on StAR mRNA were not dose-dependent, as responses of the same magnitude were present at 1, 10, and 100 ng/ml (data not shown), nor were the effects of cAMP and IGF-I additive (Fig. 4). IGF-I also increased P450scc message abundance at 24 and 96 h (p < 0.05), with a tendency toward increase at 48 and 72 h (p = 0.08; Fig. 5). As above, the effects of IGF-I on the P450scc message were not dose-dependent within the range of 1–100 ng/ml (data not shown). At 48 h, progesterone accumulation in medium was greater in cultures treated with cAMP, IGF-I, and cAMP+IGF-I relative to controls (p < 0.01; Fig. 6). IGF-I had no apparent effect on the disappearance of P450arom that occurred with luteinization (data not shown).
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Effects of EGF on Luteinized Cells
EGF induced remarkable changes in the luteinized epithelioid phenotype (Fig. 7A). Within 6 h, the first signs of morphological change, the lengthening of the cytoplasmic processes, were evident. By 12 h, cells with the flattened epithelial phenotype had been largely replaced by star-shaped cells with multiple thin projections (Fig. 7B). By 18 h and 24 h, these cells were present in clumps, and the projections had further thinned.
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Equally profound changes in gene expression followed EGF treatment of luteinized granulosa cells (Fig. 8). StAR mRNA, present in low abundance at the initiation of treatment, was reduced to the limit of detection at 6 h. Steady-state P450scc message was elevated from 6 to 18 h relative to pretreatment controls (p < 0.05). In EGF-treated cells, mean P450scc mRNA was also higher from 6 to 18 h relative to pretreatment controls (p < 0.05), but was not significantly elevated relative to contemporary control values. StAR and P450scc messages were increased by incubation with cAMP, with changes from 12- to 35-fold for StAR and from 7- to 20-fold for P450scc (p < 0.001). EGF eliminated the StAR response to cAMP at 6 and 12 h and appeared to attenuate it at 18 and 30 h. Progesterone accumulation in culture medium was elevated from 12 to 30 h by cAMP (p < 0.02) but remained unchanged relative to control in EGF- and EGF+cAMP-treated cultures (Fig. 9). LH receptor mRNA was probed for comparison, and it was progressively reduced by EGF treatment, with significant reduction at 6 h and virtual elimination of the message by 24 h (Fig. 10). No P450arom expression could be documented, either before or after EGF treatment of the cultures (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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StAR transcripts and protein varied in the CL throughout the estrous cycle in mature pigs, with maximum expression during the mid to late luteal phase and no detectable signal in regressed CL. The pattern of StAR mRNA concurs with that observed by LaVoie et al. [4] in the CL of gilts induced to ovulate with eCG and hCG, and with our previous results in the bovine CL [10]. StAR protein is correlated with transcript abundance in the pig, indicating coupling of transcription and translation, as in the bovine CL [10]. Correspondence between StAR expression and progesterone concentrations in follicular and luteal tissue [4] is consistent with its important role in the acquisition of steroidogenic capability during luteinization.
LaVoie et al. [4] demonstrated basal levels of StAR transcripts in whole follicles in unstimulated pig ovaries. We previously reported a strong presence of StAR mRNA in thecal cells from porcine follicles, but none in porcine granulosa cells [11]. It is therefore expected that the StAR is restricted to the thecal compartment in follicles.
In Vitro Luteinization
One purpose of this study was to determine how faithful the model of granulosa cell luteinization in vitro is to the development of the CL in vivo. Estrogen production and P450arom activity in granulosa cells declines rapidly after the LH surge in both in the pig [27, 28] and the cow [29, 30], and P450 aromatase gene expression is decreased by the LH surge in the rat [6]. In the present study, no P450arom mRNA could be detected in the CL from the porcine estrous cycle, and there was loss of both the steady-state and cAMP-inducible P450arom mRNA during the early stages of in vitro luteinization, within 12 h after plating of granulosa cells. Together these findings indicate that there is functional concordance between the in vitro and in vivo models.
StAR mRNA was undetectable in untreated cultures before 48 h, consistent with reports of unstimulated bovine granulosa cells [31]. This contrasts with the report of Balasubramanian et al. [20], who reported detection of steady-state StAR mRNA in porcine granulosa cells at 30 h after plating in 3% FBS. The difference may reflect their use of the ribonuclease (RNase) protection assay, expected to be more sensitive than the Northern analysis we employed. Induction of StAR by cAMP was first detectable at 24 h in the present study, similar to FSH stimulation at 30 h reported by Balasubramanian et al. [20]. In contrast to StAR, P450scc was present and cAMP-inducible as early as 6 h after plating in the present study. Progesterone accumulation in medium of porcine granulosa cells has been demonstrated as early as 24 h in this model [32]. It has been suggested that steroidogenesis can proceed in the absence of previous or concurrent StAR expression in both adrenal [33] and Leydig [34] cells. It is possible that this occurs early in the luteinization process in granulosa cells. However, it is more likely that StAR is expressed in low but sufficient abundance to allow for basal progesterone synthesis during early luteinization.
The present study clearly demonstrates that IGF-I brings about earlier expression of StAR mRNA and increased progesterone accumulation at 48 h, and thus, accelerated luteinization. The effects appear to be binomial rather than dose-dependent, as the mRNA responses were the same through a 100-fold range of IGF-I doses. The combination of IGF-I and cAMP was neither consistently additive nor synergistic. These results differ from those of Balasubramanian et al. [20], who found little direct effect of IGF-I on StAR expression, and who reported a marked synergism between IGF-I and either FSH, a ligand that employs cAMP as a second messenger, or 8-bromo-cAMP. Part of this effect may be attributed to enhanced cAMP levels in FSH-treated granulosa cells [35]. Nonetheless, there appear to be effects of IGF-I on StAR promoter activity that are downstream from cAMP stimulation of protein kinase A [35]. IGF-I mRNA is implicated in granulosa cell proliferation in the rat, while its absence is related to luteinization [36], providing a contrast to present findings in the pig. This difference may be explained by the fact that IGF-I mRNA and IGF-I protein do not colocalize in the rodent ovary [16].
The effects of EGF on luteinized granulosa cells were acute and profound. They included reduction in the abundance of the LH receptor message within 6 h and reduction to undetectable levels by 24 h. EGF has a similar inhibitory effect on hCG binding and LH receptor mRNA in pig Leydig cells [37] and in MA-10 Leydig tumor cells [38], in which the effect is reduction in transcription of the LH receptor gene [39]. StAR mRNA declined to barely detectable levels within 12 h of EGF treatment, and the ability of cAMP to stimulate StAR and progesterone accumulation was drastically compromised by EGF. The cAMP stimulation of steroidogenic enzymes is likewise neutralized by EGF in pig Leydig cells [40].
The cAMP induction of P450scc was only marginally affected by EGF, consistent with earlier reports that EGF up-regulates P450scc mRNA in porcine granulosa cells [41]. These findings indicate that EGF has differential effects on steroidogenic proteins, with stimulation or no effect on P450scc and profound down-regulation of StAR expression. Elevated cellular temperature and consequent heat shock protein synthesis have similar differential effects: StAR protein abundance is severely reduced, with little change in P450scc protein [42].
The protein kinase-C (PKC) pathway plays a role in EGF-mediated changes in porcine granulosa cells [43]. In addition, phorbal ester activation of PKC interferes with ligand-induced StAR expression [11], and PKC activation interferes with differentiation [44]. These effects may occur at the level of StAR transcription, as another growth factor, transforming growth factor-ß, down-regulates StAR gene expression in bovine adrenal cortex cells, via Smad protein regulation of its gene [45]. There are potential inhibitory elements on the StAR gene; for example, the nuclear receptor DAX-1 binds DNA and acutely down-regulates StAR transcription in MA-10 cells [46]. The mechanisms of EGF interference with StAR expression await further investigation.
Structural Changes in Granulosa Cells during Luteinization and after EGF Treatment
At isolation, granulosa cells are round, and at attachment, they take a fibroblastic form. After 72 h in culture, they have a blocky appearance, often described as epithelioid. These changes are accompanied by, and may be due to, rearrangement of junctional elements and concurrent reorganization of the cytoskeleton [47, 48]. Phenotypic remodeling is an essential element of luteinization, believed to be necessary for maximal progesterone synthesis [47]. The morphological changes induced by EGF, especially the establishment of long cytoplasmic processes, are suggestive of a return to the fibroblastic form and the involvement of the cytoskeleton. Similar morphology was documented by Hatey et al. [44] in phorbol ester-treated pig granulosa cells, implicating the PKC pathway and EGF in structural modification of granulosa cells. A mechanism for EGF-induced cytoskeletal remodeling can be found in the presence of an actin binding domain on the EGF receptor [49].
In summary, the present investigation demonstrates that in pig ovarian tissues, the onset of StAR and the loss of P450arom gene expression accompany luteinization in vivo and in vitro. The acquisition of StAR can be precociously induced by addition of IGF-I to pig granulosa cell cultures. EGF causes structural changes in luteinized granulosa cells and eliminates the steady-state levels of StAR and LH receptor mRNA, with no effect on P450scc. Together these results suggest the IGF-I enhances, and EGF reverses, the process of luteinization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 450 778 8103; murphyb{at}medvet.umontreal.ca
Accepted: January 20, 1999.
Received: July 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5,4 isomerase in ovine corpora lutea during the estrous cycle. Biol Reprod 1994; 51:380–384.[Abstract]-hydroxylase and aromatase messenger ribonucleic acids in cattle undergoing superovulation. Biol Reprod 1996; 55:1419–1426.[Abstract]This article has been cited by other articles:
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  M. M Seneda, M. Godmann, B. D Murphy, S. Kimmins, and V. Bordignon Developmental regulation of histone H3 methylation at lysine 4 in the porcine ovary Reproduction, June 1, 2008; 135(6): 829 - 838. [Abstract] [Full Text] [PDF]
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 N. Gevry, K. Schoonjans, F. Guay, and B. D. Murphy Cholesterol supply and SREBPs modulate transcription of the Niemann-Pick C-1 gene in steroidogenic tissues J. Lipid Res., May 1, 2008; 49(5): 1024 - 1033. [Abstract] [Full Text] [PDF]
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 L. Miranda-Jimenez and B. D. Murphy Lipoprotein receptor expression during luteinization of the ovarian follicle Am J Physiol Endocrinol Metab, October 1, 2007; 293(4): E1053 - E1061. [Abstract] [Full Text] [PDF]
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 D. C. Woods, M. J. Haugen, and A.L. Johnson Actions of Epidermal Growth Factor Receptor/Mitogen-Activated Protein Kinase and Protein Kinase C Signaling in Granulosa Cells from Gallus gallus Are Dependent upon Stage of Differentiation Biol Reprod, July 1, 2007; 77(1): 61 - 70. [Abstract] [Full Text] [PDF]
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E. Needle, K. Piparo, D. Cole, C. Worrall, I. Whitehead, G. Mahon, and L. T. Goldsmith Protein Kinase A-Independent cAMP Stimulation of Progesterone in a Luteal Cell Model Is Tyrosine Kinase Dependent but Phosphatidylinositol-3-Kinase and Mitogen-Activated Protein Kinase Independent Biol Reprod, July 1, 2007; 77(1): 147 - 155. [Abstract] [Full Text] [PDF]
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J M Silva, M Hamel, M Sahmi, and C A Price Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro. Reproduction, December 1, 2006; 132(6): 909 - 917. [Abstract] [Full Text] [PDF]
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 M. A. Lazzaro, D. Pepin, N. Pescador, B. D. Murphy, B. C. Vanderhyden, and D. J. Picketts The Imitation Switch Protein SNF2L Regulates Steroidogenic Acute Regulatory Protein Expression during Terminal Differentiation of Ovarian Granulosa Cells Mol. Endocrinol., October 1, 2006; 20(10): 2406 - 2417. [Abstract] [Full Text] [PDF]
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R. Rusovici, Y. Y. Hui, and H. A. LaVoie Epidermal Growth Factor-Mediated Inhibition of Follicle-Stimulating Hormone-Stimulated StAR Gene Expression in Porcine Granulosa Cells Is Associated with Reduced Histone H3 Acetylation Biol Reprod, April 1, 2005; 72(4): 862 - 871. [Abstract] [Full Text] [PDF]
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Z. T. Ruiz-Cortes, Y. Martel-Kennes, N. Y. Gevry, B. R. Downey, M.-F. Palin, and B. D. Murphy Biphasic Effects of Leptin in Porcine Granulosa Cells Biol Reprod, March 1, 2003; 68(3): 789 - 796. [Abstract] [Full Text] [PDF]
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A.L. Johnson, E.V. Solovieva, and J.T. Bridgham Relationship Between Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Hen Granulosa Cells During Follicle Development Biol Reprod, October 1, 2002; 67(4): 1313 - 1320. [Abstract] [Full Text] [PDF]
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 N. Gevry, D. Lacroix, J.-H. Song, N. Pescador, M. Dobias, and B. D. Murphy Porcine Niemann Pick-C1 Protein Is Expressed in Steroidogenic Tissues and Modulated by cAMP Endocrinology, February 1, 2002; 143(2): 708 - 716. [Abstract] [Full Text] [PDF]
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A. L. Johnson and J. T. Bridgham Regulation of Steroidogenic Acute Regulatory Protein and Luteinizing Hormone Receptor Messenger Ribonucleic Acid in Hen Granulosa Cells Endocrinology, July 1, 2001; 142(7): 3116 - 3124. [Abstract] [Full Text] [PDF]
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N. Sekar, H. A. LaVoie, and J. D. Veldhuis Concerted Regulation of Steroidogenic Acute Regulatory Gene Expression by Luteinizing Hormone and Insulin (or Insulin-Like Growth Factor I) in Primary Cultures of Porcine Granulosa-Luteal Cells Endocrinology, November 1, 2000; 141(11): 3983 - 3992. [Abstract] [Full Text] [PDF]
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G. Zhang, J. C. Garmey, and J. D. Veldhuis Interactive Stimulation by Luteinizing Hormone and Insulin of the Steroidogenic Acute Regulatory (StAR) Protein and 17{alpha}-Hydroxylase/17, 20-Lyase (CYP17) Genes in Porcine Theca Cells Endocrinology, August 1, 2000; 141(8): 2735 - 2742. [Abstract] [Full Text] [PDF]
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B. D. Murphy Models of Luteinization Biol Reprod, July 1, 2000; 63(1): 2 - 11. [Abstract] [Full Text]
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