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Articles |
Induces a Rapid Decline in Progesterone Production and Steroidogenic Acute Regulatory Protein Expression in Isolated Rat Corpus Luteum Without Altering Messenger Ribonucleic Acid Expression1
a Reproductive Endocrinology, Infertility and Genetics Section, Department of Obstetrics and Gynecology, The Medical College of Georgia, Augusta, Georgia 30912
b Department of Physiology and Biophysics, The University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PGF2, n = 6) or saline (n = 6). Serum progesterone levels were determined by RIA, StAR and ß-actin mRNA expression by Northern analysis, and StAR and ß-actin protein expression by Western analysis. Adrenal, brain, and spleen from control animals were used as positive and negative controls for StAR expression. Scanning optical densitometry measurements were standardized by dividing the signal strength from each StAR autoradiogram lane by that from the corresponding ß-actin autoradiogram lane. ANOVA was used for significance testing, with set at 0.05. The 37-, 32-, and 30-kDa forms of StAR protein were expressed in all adrenal samples, whereas only the 37- and 30-kDa forms were found in CL. Serum progesterone levels and expression of the 30-kDa and 37-kDa forms of the StAR protein in CL were all found to be significantly lower in the PGF2-treated than the saline-treated group. StAR mRNA expression was not significantly different in the saline- and PGF2-treated rats. The rapid decline in StAR protein expression that accompanies PGF2 induced luteolysis, therefore, does not result from significant decline in mRNA expression.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One would expect a biochemical bottleneck to steroid production if, and when, the enzyme were present in limited quantities relative to available substrate. Long-term (4 h or more) application of steroidogenic stimuli has consistently been found to alter expression of protein and message for P450scc and other enzymes involved in steroidogenesis, in a transcription-dependent manner [4–17]. In contrast, acute stimulation of steroidogenesis has been determined to be independent of transcription, despite a requirement for de novo protein synthesis [18–27]. Moreover, it has now been established that the rate-limiting step in acute steroidogenesis is the delivery of cholesterol to the inner mitochondrial membrane where it can be acted upon by P450scc [28, 29].
On this basis, recent interest has focused on the study of factors that precede P450scc in the steroid hormone biosynthetic pathway as potential candidates for modulating the acute response to steroidogenic stimuli. The requirements for an ideal candidate include an extremely rapid response to stimulation by cAMP and to inhibition by cycloheximide and other protein synthesis inhibitors, increased expression independent of transcription, and expression specific to acutely regulated steroidogenic tissues. Among the various candidates, one protein that fulfills all of these criteria is steroidogenic acute regulatory protein (StAR) (for review see [30, 31]). StAR is a protein located in the inner mitochondrial membrane in its mature 30-kDa form. The precursor form of StAR is a 37-kDa protein found in the cytoplasm. As it associates with, and is transported into, the mitochondria, it is processed first to a 32-kDa form and then to the mature 30-kDa form of the protein, found within the mitochondrial matrix. The 37- and 32-kDa forms are transiently expressed relative to the more stable 30-kDa form of the protein.
Synthesis of StAR protein has been found to be disrupted in individuals affected with lipoid congenital adrenal hyperplasia, on the basis of a premature translation termination codon [32]. StAR is considered responsible for delivering cholesterol to P450scc within the mitochondria. The mechanism of action for this event has not been completely worked out; however, the 37-kDa precursor form of StAR is thought to facilitate cholesterol transfer through the mitochondrial membrane, making it available to P450scc [33,34]. The 37-kDa precursor form of StAR gives rise to the 30-kDa mature isoform, which remains associated with the inner mitochondrial membrane for a relatively long time and is believed to be no longer involved in cholesterol transfer [33, 34]. Thus, it is postulated that cholesterol transport across the mitochondrial membrane requires de novo synthesis of the 37-kDa form of StAR from a preexisting pool of mRNA, and that after transport the transporter molecule is cleaved to a smaller "mature" form that remains as an integral component of the inner mitochondrial membrane.
While investigations into StAR expression under circumstances of acute stimulation of steroidogenesis have been intense, the effects of steroid synthesis inhibition (e.g., during prostaglandin-induced luteolysis) upon StAR have been less well studied. The effect of prostaglandin F2 (PGF2) on StAR mRNA expression has been studied in the ovine corpus luteum by Juengel et al. [35], in bovine corpus luteum by Pescador et al. [36], in whole postovulatory rat ovary by Sandhoff and McLean [37], and in human corpus luteum by Chung et al. [38]. All these groups report a significant time-dependent decrement in expression of the message for this protein that appears to parallel the decrease in serum progesterone levels. Expression of the StAR protein itself has yet to be evaluated during prostaglandin-induced luteolysis.
In consideration of the possibility that the 37-kDa StAR precursor protein may be the actual cholesterol transmitochondrial membrane transporter protein, we believe that it is important to elucidate changes in the various forms of StAR protein expression under luteolytic conditions. We therefore have undertaken to study serum progesterone levels and the concomitant expression of mRNA and protein for StAR in corpora lutea isolated 1 h after luteolytic injection of rats with PGF2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Use of rats for this study was approved by the Medical College of Georgia Committee on Animal Use for Research and Education, and the animals were handled in accordance with the guidelines established at the Medical College of Georgia.
Female Sprague-Dawley rats (Harlan-Sprague-Dawley, Indianapolis, IN) were housed in environmentally controlled rooms at 22°C with a 14L:10D cycle, fed Purina rodent laboratory chow (Ralston-Purina Co., St. Louis, MO), and allowed ad libitum access to tap water.
A preliminary study, documenting expression of progesterone throughout the luteal phase after synchronization of ovulation in our rat model, was carried out as follows. To synchronize ovulation, 78 animals were injected s.c. with 10 IU of eCG between 0800 and 1000 h on Day 28 of life [39]. On each of postovulatory Days 1–13, between 1100 and 1300 h, six rats were anesthetized with ether, and 1 ml of blood was obtained by venous puncture and held on ice for serum progesterone determination. This was followed by rapid surgical removal of ovaries. While still anesthetized, the animals were killed by cervical dislocation. Corpora lutea (CL) were then rapidly isolated from ovaries under a dissecting microscope in small amounts of ice-cold Dulbecco's PBS, using an ice-cold dissecting stand, to determine the number of CL produced.
Animal handling and tissue procurement proceeded as follows for the remainder of the experiments concerning effects of PGF2 on StAR expression. To synchronize ovulation, 12 animals were injected s.c. with 10 IU of eCG between 0800 and 1000 h on Day 28 of life [39]. On postovulatory Day 7 between 1000 and 1200 h, rats were injected i.p. with either 250 µg of PGF2 (n = 6) or an equal volume of PBS (control group, n = 6). One hour after injection, the animals were anesthetized with ether, and 1 ml of blood was obtained by venous puncture and held on ice, followed by rapid surgical removal of ovaries. While still anesthetized, the animals were killed by cervical dislocation. Adrenal, spleen, and cortical brain tissue were harvested from control animals immediately after cervical dislocation. These tissues were used as positive (adrenal) and negative (spleen and cortical brain) controls for StAR expression in Northern and Western analyses.
CL were rapidly isolated from ovaries under a dissecting microscope in small amounts of ice-cold PBS, using an ice-cold dissecting stand. CL and other tissues were immediately snap frozen in liquid nitrogen and stored at -70°C until the time of RNA and protein extraction.
Progesterone RIA
The serum fraction was obtained from blood samples by centrifugation at 2000 x g for 20 min at 4°C after CL isolation. Serum samples were stored at -70°C until the time of RIA. To allow for assessment of intraassay variability, all samples were run in triplicate.
On the day of assay, 20 µl of serum was taken from each thawed sample and diluted 1:20 with phosphate buffer with gelatin, pH 7.0; 100 µl of the resulting solution was added to each tube. To each tube was added 100 µl (10 000 cpm) of [1,2,6,7-3H]progesterone (generously provided to us by Dr. Thomas Mills [The Medical College of Georgia, Augusta, GA], after purification on a Celite column; original specific activity 109 Ci/mmol; Dupont NEN Research Products, Boston, MA) and antibody, highly specific to 11ß-hydroxyprogesterone [40] (GDN-337; kindly provided to us by Dr. Gordon Niswender, Colorado State University, Fort Collins, CO), in a 1:5000 final dilution in the assay.
All tubes were vortexed briefly and incubated overnight at 4°C. Free and bound progesterone were separated by addition of 500 µl of dextran-coated activated charcoal. The samples were then incubated on ice for 30 min followed by centrifugation at 2000 x g for 20 min at 4°C to pellet the charcoal. Supernatant (700 µl) was transferred into scintillation vials, mixed well with 4 ml Scintiverse BD (Fisher Scientific, Pittsburgh, PA), and counted in a Beckman LS 7500 scintillation counter (Beckman Instruments, Fullerton, CA). The standard curve and sample serum concentrations of progesterone were calculated using Beckman ImmunoFit software.
RNA Isolation and Quantitation
Total RNA was isolated from the CL using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the supplier's instructions. The resulting pellet was washed in ice-cold 75% ethanol by vortexing and was stored as an ethanol precipitate at -70°C. On the day of gel loading, samples were centrifuged at 12 000 x g for 5 min at 4°C, after which the resulting pellets were solubilized in diethyl pyrocarbonate-treated H2O for determination of optical density (OD) and subsequent Northern analysis.
Because of the small amounts of tissue available for RNA extraction from CL, Microcarrier Gel-TR (Molecular Research Center) was added prior to homogenization of CL samples for isolation of total RNA according to the instructions provided by the supplier.
The RNA was quantitated and monitored for purity by evaluating the 260:280-nm absorption with Beckman DU-65 Spectrophotometer. To quantitate RNA from CL samples that contained Microcarrier Gel-TR, the OD readings from these samples were compared to those from blanks containing equal amounts of Microcarrier Gel-TR that had been taken through the entire extraction process in parallel to the CL samples.
Northern Analysis
Total RNA (30 µg) from each sample was fractionated by electrophoresis on a denaturing 0.8% agarose/2.2 M formaldehyde gel at 100 volts for 3 h, and transferred overnight to a Zeta Probe nylon membrane (Bio-Rad Laboratories, Richmond, CA) followed by UV cross-linking of RNA (UV Stratalinker 1800; Stratagene Cloning Systems, La Jolla, CA). The MOPS running buffer consisted of 20 mM 3-[N-morpholino]propanesulfonic acid, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0; the 20-strength SSC transfer buffer consisted of 3 M NaCl and 300 mM sodium citrate, pH 7.0. The nylon membranes were hybridized with a StAR cDNA probe (5 x 106 cpm probe/ml hybridization buffer) at 42°C for 18–20 h after a 3- to 4-h prehybridization incubation. The prehybridization and hybridization buffer consisted of 200 mM phosphate buffer, pH 7.2, 50% deionized formamide, 1% BSA (fraction V), 7% SDS, and 1 mM EDTA, pH 8.0.
After hybridization, the membranes were washed under high-stringency conditions: two 30-min washes in double-strength SSC with 0.1% SDS, the first at 22°C and the second at 60°C, were followed by two 20-min washes in 0.2-strength SSC with 0.1% SDS at 60°C. The membranes were then exposed to Fuji RX x-ray film for 19 h (Fuji Photo Film, Tokyo, Japan) at -70°C.
After development of autoradiograms, the membranes were stored at -70°C for 18 wk to allow adequate radioactive decay of the signal prior to rehybridization of each membrane with a ß-actin cDNA probe. Adequate decay was confirmed by a 24-h exposure of each membrane to Fuji RX x-ray film at -70°C. This was followed by rehybridization of each membrane with the ß-actin cDNA probe (5 x 106 cpm probe/ml hybridization buffer). Rehybridization of membranes with the ß-actin cDNA probe was carried out according to the procedure described above for the StAR probe. Subsequent exposure to Fuji RX x-ray film at -70°C lasted 5 h.
Preparation of cDNA Probes
The 1.5-kilobase (kb) StAR cDNA insert [41] was cleaved from pSPORT plasmid (Gibco-BRL, Grand Island, NY) by overnight enzymatic digestion with SalI (Promega Corporation, Madison, WI) and NotI (Promega) at 37°C. The resulting fragments were separated electrophoretically on a 1% agarose gel, and the insert was recovered using a Geneclean Kit (Bio 101, La Jolla, CA). Adequate purification and molecular weight of the insert were confirmed on a minigel. The concentration of cDNA insert was determined spectrophotometrically at 260 nm.
The 1.1-kb ß-actin insert [42] was isolated from its pBR322 vector (Gibco-BRL) with PstI (Promega). The remainder of the purification and quantitation procedure for this insert proceeded according to the methods described above for StAR cDNA insert isolation.
Radiolabeled cDNA probes were each prepared for StAR and ß-actin by random primer method using a High Prime labeling kit (Boehringer Mannheim, Indianapolis, IN) as follows. Fifty nanograms of linear template DNA in sterilized H2O was incubated at 100°C for 10 min followed by rapid chilling in an ice/ethanol bath. An appropriate volume of High Prime solution and 50 µCi of [-32P]dCTP (3000 Ci/mmol; ICN Biomedicals, Irvine, CA) was added, and incubation was performed for 3 h at 37°C. The reaction was terminated by addition of EDTA to a concentration of 20 mM. Nonincorporated [-32P]dCTP was removed by passage through a NucTrap push column (Stratagene). Activity (cpm/µl) of the effluent was determined using a Beckman LS 7500 scintillation counter. Immediately prior to hybridization of membranes, each probe was incubated at 100°C for 10 min followed immediately by rapid chilling on ice for 5 min.
Protein Isolation and Quantitation
Tissue samples were homogenized on ice by hand using a glass homogenizer in ice-cold lysis buffer, pH 7.0, consisting of 10 mM Tris-HCl, 2% SDS, 40 µM PMSF, 0.3 µM aprotinin, and 1 µM leupeptin. This was followed by a 30-min incubation on ice and centrifugation at 2000 x g for 20 min at 4°C. The supernatant was transferred to new tubes, which were held on ice during protein quantitation. Protein was quantitated according to the methods described by Lowry et al. [43] using a Beckman DU-65 Spectrophotometer; then samples were appropriately aliquoted and stored at -70°C until the time of electrophoresis.
Western Analysis
Western blot analysis began with SDS-PAGE on a 5% stacking and a 10% resolving gel in a pH 8.4 buffer consisting of 0.25 M Tris base, 1.92 M glycine, and 35 mM SDS at 60 volts (19 amps) for 18 h at 4°C. Each lane was loaded with 25 µg of protein. After electrophoresis, the gels were blotted to an Immobilon-P polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA) in single-strength Tris-glycine buffer, pH 8.2, with 20% methanol at 25 volts for 23 h at 4°C. Next, membranes were fixed in 99% methanol for 15 min followed by 15 min in distilled H20 at 22°C. They were then blocked for 1 h in 5% nonfat dry milk at 22°C, followed by two brief rinses and three 10-min washes in Tris-buffered saline with 0.1% Tween 20, pH 7.5 (TBS-T), all at 22°C. Next, membranes were incubated with the primary antibody for 1 h in TBS-T at 22°C, followed again by two brief rinses and three 10-min washes in TBS-T at 22°C. The rabbit anti-mouse StAR specific polyclonal antibody was used in a 1:2000 dilution. This antibody has been shown to recognize the 37-, 32-, and 30-kDa forms of StAR protein [41]. After washing, the membranes were incubated in TBS-T for 1 h at 22°C with peroxidase-labeled anti-rabbit antibody (StAR, 1:3000 dilution; Amersham Life Science, Little Chalfont, England). The blots were then rinsed briefly in TBS-T and incubated overnight at 4°C in TBS-T. A final wash was then undertaken with TBS (lacking Tween 20) for removal of excess Tween 20 from the membrane. Detection was carried out using an enhanced chemiluminescence (ECL; Amersham) Western blotting analysis system according to the manufacturer's instructions. Membranes were exposed to Fuji RX x-ray film at 22°C. Membranes blotted with anti-StAR antibody were exposed initially for 10 sec to optimize density of the 30-kDA StAR protein signal, followed by a 5-min exposure to optimize density of the 37-kDa StAR protein signal.
After completion of blotting and detection of StAR protein signals, the membranes were soaked in 99% methanol for 2 min, followed by 5 min in distilled water. They were then soaked for 30 min with gentle agitation in a stripping buffer containing 2-mercaptoethanol, 2% SDS, and Tris-HCl, pH 6.7. Membranes were then subjected to two 10-min washes in TBS-T, followed by blocking for 1 h in 5% nonfat dry milk, all at 22°C.
Each membrane was then blotted with anti-mouse actin (Ab-1)-specific monoclonal antibody (Oncogene Science, Manhassett, NY), used in a 1:400 dilution, according to the same protocol outlined above for blotting with anti-StAR antibody. The secondary antibody was anti-mouse antibody (1:3000 dilution; Amersham Life Science). Signal detection was carried out using an ECL Western blotting analysis system. Membranes blotted with anti-actin antibody were exposed for 2 h to Fuji RX x-ray film at 22°C.
Radiographic Signal Quantitation
Relative signal intensities from films generated by Northern and Western analysis were determined via a Shimadzu (Columbia, MD) CS-9301PC Dual Wavelength Flying Spot Scanning Densitometer at 560 nm, using a 1 x 10-mm beam for films from Northerns and a 1 x 5-mm beam for films from Westerns. Each scan was repeated three times, with removal from and replacement of the autoradiogram in the cassette prior to each scan. To account for 1) possible differences in the quantities of RNA and/or protein loaded in each well of the gels and 2) possible variability in efficiency of protein or RNA transfer from wells in the same gel to the membranes, peak signal strengths (area under the curve) from each lane of StAR autoradiograms were each divided by the signal strength from the corresponding lane in the ß-actin autoradiogram generated with the same membrane in each of the Northern and Western blots.
Thus, relative scanning OD measurements were standardized for between-group comparisons. These measurements are henceforth referred to as "standardized OD readings."
Statistical Analysis
A repeated-measures ANOVA was used to determine the presence of a significant difference in mean serum progesterone levels in PGF2- and saline-treated rats, and to test for significant differences among the triplicate measures of progesterone obtained from each rat serum sample subjected to RIA.
Northern and Western blot analyses were carried out according to a randomized complete block experimental design [44]. Each of the six autoradiograms generated for 1) StAR mRNA, 2) 37-kDa StAR protein, and 3) 30-kDa StAR protein constituted a block. ANOVA was used to determine the presence of significant differences in mean standardized OD readings in PGF2- and saline-treated rats, separately for each of the above three parameters. This analysis also allows testing for differences between repeated densitometer measurements within autoradiograms to demonstrate the presence of any significant difference between the three standardized OD readings taken from each lane on each of the autoradiograms.
The significance level () for all comparisons was set at 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mean serum progesterone levels on each of postovulatory Days 1 through 13, after synchronization of ovulation with eCG, are shown in Figure 1.
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Mean serum progesterone levels from PGF2-treated rats were significantly lower than those from the saline-treated rats (p = 0.0001) as shown in Figure 2, consistent with cessation of progesterone synthesis in response to PGF2. As described in Materials and Methods, each rat serum sample was subjected to progesterone RIA in triplicate. Progesterone levels were not found to be significantly different among these triplicate measures.
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Northern analysis for StAR mRNA yielded two autoradiographic bands per lane, and that for ß-actin yielded one autoradiographic band per lane (Fig. 3). Therefore standardized OD for StAR mRNA could be calculated using either the peak signal strength (area under the curve) from the 1.6-kb band, that from the 3.5-kb band, or the sum of the peak signal strengths of the 1.6-kb and 3.5-kb bands prior to dividing by the peak signal strength from the ß-actin band from the same lane in the corresponding autoradiogram. Mean standardized OD readings for StAR mRNA in CL were not found to be significantly different in PGF2- and saline-treated rats regardless of which of the three methods was used to determine the standardized OD. For the data shown in Figure 4, peak signal strengths from the 1.6-kb and 3.5-kb bands in each lane were summed prior to dividing by the peak signal strength from the ß-actin band from the same lane in the corresponding autoradiogram.
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The 37-, 32-, and 30-kDa forms of StAR protein were expressed in all adrenal samples from our rats. On the other hand, only the 37- and 30-kDa forms of the protein could be detected in CL from these rats regardless of treatment group (Fig. 5).
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Mean standardized OD readings for the 37-kDa StAR protein (Fig. 6, top) were significantly lower in PGF2-treated than in saline-treated rats (p = 0.0002). Mean standardized OD readings for the 30-kDa StAR protein (Fig. 6, bottom) were also significantly lower in PGF2-treated than in saline-treated rats (p = 0.0126).
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All triplicate standardized OD readings were in agreement; i.e., no significant differences were found among the three standardized OD readings taken from each lane of each autoradiogram for StAR mRNA, the 37-kDa StAR protein, and the 30-kDa StAR protein.
StAR mRNA (Fig. 3) and StAR protein (Fig. 5) were detectable in CL and adrenal but undetectable in spleen and cortical brain. This pattern of detection was consistent with the known steroidogenic capacity of these tissues and their patterns of StAR expression [36], and served to validate the function of this StAR cDNA probe and antibody.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, without a concomitant change in expression of StAR RNA. We chose to study serum progesterone levels and StAR protein and mRNA expression in isolated CL at 1 h postinjection in order to evaluate the acute effects of PGF2 upon steroidogenesis and StAR expression. Consistent with findings of others [33], the 37-, 32-, and 30-kDa forms of StAR protein were expressed in all adrenal samples from our rats, whereas only the 37- and 30-kDa forms of the protein could be detected in CL from these rats regardless of treatment group. Our failure to detect the 32-kDa intermediate form of the protein in the rat CL is, most likely, indicative of a much more rapid turnover of the 32-kDa intermediate form to the 30-kDa mature form in CL than in adrenal.
We anticipated that we would find a decline in expression of the 37-kDa StAR protein along with a possible decline in expression of the mature form(s) of the protein in response to PGF2. We indeed found a decline in both the 37- and 30-kDa forms of the protein. We also expected to find a decrement in the expression of StAR mRNA; however, our data do not demonstrate any significant change in StAR mRNA expression in rat CL within 1 h of PGF2 injection, despite a significant decline in serum progesterone levels and expression of the StAR proteins over the same time period.
Results from the laboratory of Dr. G.D. Niswender have suggested that PGF2 exerts its antisteroidogenic and "luteolytic" effects in the CL via two distinct second messenger systems: the induction of apoptosis realized after treatment with PGF2 appears to occur via activation of phospholipase C, which in turn results in increased levels of free intracellular calcium [45]. Increasing levels of free intracellular calcium are believed to take part in activation of a Ca2+/Mg2+-dependent endonuclease responsible for cleavage of DNA into the characteristic 185-base pair oligonucleosomes [46]. On the other hand, reversible inhibition of steroidogenesis appears to be modulated by activation of the luteal protein kinase C (PKC) system [47–49]. The precise mechanism by which activation of the PKC pathway by PGF2 modulates steroidogenesis is yet to be determined, but it does not appear to involve a decrease in the expression of mRNA for P450scc. Proposed mechanisms include decreasing levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA, modulation of the activity of 3ß-HSD, and decreasing transport of cholesterol to the mitochondrial membrane [50]. Our findings suggest that one mechanism by which PGF2 may exert its antisteroidogenic effect in the CL is via inhibition of StAR protein expression. Moreover, it is conceivable that PKC mediates the PGF2-induced decline in StAR protein expression that we have observed.
Our findings are consistent with the findings of others who have studied StAR expression during PGF2-induced luteolysis in isolated CL. In bovine CL, Pescador et al. [36] studied StAR mRNA expression following injection of PGF2. The earliest postinjection time point that they examined was 12 h, at which point they observed a significant decline in StAR mRNA. They did not determine progesterone levels in their study. Juengel and coworkers [35] evaluated serum progesterone levels and StAR mRNA expression in ovine CL on postovulatory Day 10 or 11 (corresponding to the time of peak luteal progesterone production, or midluteal phase, in the ewe) at 4, 12, and 24 h after PGF2 injection. They reported that progesterone levels decreased significantly by 4 h postinjection and continued to decline, whereas a significant decrement in StAR mRNA levels was not yet apparent by 4 h postinjection but was present 8 h later.
Our findings with regard to StAR mRNA appear to be at odds with those of Sandhoff and McLean [37], who found that expression of mRNA declined significantly within 30 min of PGF2 injection and continued to decline thereafter. It must be recognized that their study was in whole rat ovaries, which contain developing and atretic follicles in addition to CL.
Another, perhaps more important, consideration is that our studies were carried out on postovulatory Day 7 (midluteal phase), whereas Sandhoff and McClean [37] investigated animals on postovulatory Day 10. By postovulatory Day 10, CL (closer to the time of spontaneous luteal regression) may become more sensitive than Day 7 CL to the phospholipase C-induced effects of PGF2, leading to a more global decrement in transcriptional activity or to a global increase in the rate of mRNA degradation. This same group of investigators (Chung et al. [38]) have recently studied the effects of incubating isolated human CL in varying concentrations of PGF2 on expression of StAR mRNA and progesterone levels in early and midluteal phase. Consistent with the hypothesis proposed above, they did not find decline in StAR mRNA expression in early luteal phase CL in response to incubation with PGF2. However, they demonstrated a significant decline in StAR mRNA expression after 4 h of incubation with PGF2 in midluteal phase CL.
Whereas it would seem that the finding of Chung et al. [38] of a significant decline in StAR mRNA expression in response to PGF2 in midluteal phase contradicts our results, one must consider the following issues, which would suggest that their data do not necessarily disagree with our data or with the above hypothesis. In their study, only 4 CL were assayed in the midluteal phase, which they defined as menstrual cycle Days 20 through 24 (corresponding with postovulatory Days 6 through 11). This includes both postovulatory Days 7 and 10. Their wide range of midluteal cycle days combined with the small number of midluteal CL assayed makes it difficult to compare their human midluteal phase data with our postovulatory Day 7 rat CL data or with postovulatory Day 10 data from their study in whole rat ovaries [37].
In their midluteal phase CL, after 4 h of incubation, a significant decline in StAR mRNA expression was seen only at the two highest concentrations of PGF2, while a significant decline in progesterone was observed in these CL at all four concentrations of PGF2 tested [38]. This too supports the possibility that there is a mechanism by which PGF2 effects a rapid decrease in progesterone production that does not involve a significant decline in StAR mRNA expression.
In summary, we have found that PGF2 induces an acute decline in StAR protein expression in the rat CL. This is accompanied by a rapid decline in progesterone production and may contribute significantly to luteolysis. Our findings and those of others suggest that acute inhibition of steroidogenesis by PGF2 may be the result of a decrement in the expression of StAR protein that is partially mediated through some mechanism not involving an immediate change in mRNA expression. While our study does not rule out other mechanisms for acute inhibition of steroidogenesis, it clearly points out that the rapid decline in StAR protein expression accompanying PGF2-induced luteolysis does not necessarily involve significant suppression of transcription. Future studies should be directed toward elucidating this important mechanism.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence and current address: Iqbal Khan, Department of Obstetrics and Gynecology, The Medical College of Georgia, Augusta, GA 30912. FAX: 706 721 6830; ikhan{at}mail.mcg.edu
Accepted: April 15, 1999.
Received: July 24, 1998.
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V. K. Yadav, R. R. Sudhagar, and R. Medhamurthy Apoptosis During Spontaneous and Prostaglandin F2{alpha}-Induced Luteal Regression in the Buffalo Cow (Bubalus bubalis): Involvementof Mitogen-Activated Protein Kinases Biol Reprod, September 1, 2002; 67(3): 752 - 759. [Abstract] [Full Text] [PDF]
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A. C. Nackley, W. Shea-Eaton, D. Lopez, and M. P. McLean Repression of the Steroidogenic Acute Regulatory Gene by the Multifunctional Transcription Factor Yin Yang 1 Endocrinology, March 1, 2002; 143(3): 1085 - 1096. [Abstract] [Full Text] [PDF]
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A. Zetser, T. Kisliouk, E. Ivakin, and M. Lahav Dependence on Prolactin of the Luteolytic Effect of Prostaglandin F2{alpha} in Rat Luteal Cell Cultures Biol Reprod, October 1, 2001; 65(4): 1082 - 1091. [Abstract] [Full Text] [PDF]
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C. O. Stocco, J. Chedrese, and R. P. Deis Luteal Expression of Cytochrome P450 Side-Chain Cleavage, Steroidogenic Acute Regulatory Protein, 3{beta}-Hydroxysteroid Dehydrogenase, and 20{alpha}-Hydroxysteroid Dehydrogenase Genes in Late Pregnant Rats: Effect of Luteinizing Hormone and RU486 Biol Reprod, October 1, 2001; 65(4): 1114 - 1119. [Abstract] [Full Text] [PDF]
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K. K. Olson, L. E. Anderson, M. C. Wiltbank, and D. H. Townson Actions of Prostaglandin F2{{alpha}} and Prolactin on Intercellular Adhesion Molecule-1 Expression and Monocyte/Macrophage Accumulation in the Rat Corpus Luteum Biol Reprod, March 1, 2001; 64(3): 890 - 897. [Abstract] [Full Text]
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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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L. P. Walsh and D. M. Stocco Effects of Lindane on Steroidogenesis and Steroidogenic Acute Regulatory Protein Expression Biol Reprod, April 1, 2000; 63(4): 1024 - 1033. [Abstract] [Full Text]
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 X. Wang, L. P. Walsh, A. J. Reinhart, and D. M. Stocco The Role of Arachidonic Acid in Steroidogenesis and Steroidogenic Acute Regulatory (StAR) Gene and Protein Expression J. Biol. Chem., June 23, 2000; 275(26): 20204 - 20209. [Abstract] [Full Text] [PDF]
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C. O. Stocco, L. Zhong, Y. Sugimoto, A. Ichikawa, L. F. Lau, and G. Gibori Prostaglandin F2alpha -induced Expression of 20alpha -Hydroxysteroid Dehydrogenase Involves the Transcription Factor NUR77 J. Biol. Chem., November 17, 2000; 275(47): 37202 - 37211. [Abstract] [Full Text] [PDF]
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