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Ovary |
Instituto de Biología y Medicina Experimental (IBYME)-CONICET,3 Facultad de Ciencias Exactas, Universidad de Buenos Aires y Nacional de la Plata, 1428 Buenos Aires, Argentina
Department of Cell Biology and Biochemistry,4 Texas Tech University Health Sciences Center, Lubbock, Texas 79430
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, follicular development, gonadotropin-releasing hormone, ovary, steroid hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The synthesis of sex steroids is one of the most important functions of ovarian follicular cells. Trophic hormones stimulate the synthesis of steroid hormones through increases in cAMP levels [13, 14]. Also, the steroidogenic acute regulatory protein (StAR), a mitochondrial phosphoprotein [15–18], is acutely induced by trophic hormones. StAR mediates the rate-limiting step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane, where it is cleaved to pregnenolone by the inner membrane-bound P450scc enzyme [15]. The essential role of StAR in steroidogenesis was demonstrated by the fact that mutations in its gene cause the lipoid form of congenital adrenal hyperplasia in humans, a potentially lethal disease resulting from an inability to synthesize steroids [19, 20]. StAR has been described in the ovaries of rats, humans, rabbits, and mice [21–23]. Sridaran et al. demonstrated that administration of GnRH-a suppressed luteal steroidogenesis in pregnant rats through inhibition of StAR, P450scc, and the peripheral-type benzodiazepine receptor proteins [24]. However, to our knowledge, no studies have been performed on the effects of GnRH-a on StAR and P450scc follicular expression and steroid production in gonadotropin-stimulated immature rats.
The hypothesis addressed in this study is whether GnRH has a role in regulating ovarian function by interfering with steroidogenic pathways stimulated by gonadotropins. Consequently, the aim of the present study was to examine the acute and chronic in vivo effects of a GnRH-a (leuprolide acetate, LA) on the expression of StAR, P450scc, and steroid content in antral and preovulatory ovarian follicles obtained from prepubertal equine choriogonadotropin (eCG)-treated rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LA was obtained from Abbott Laboratories (Buenos Aires, Argentina). The original ampule (2.8 mg/5 ml) was dissolved in saline to obtain the appropriate concentration. SYNTEX SA (Buenos Aires, Argentina) provided the equine chorionic gonadotropin (Novormon). Hepes, SDS, extravidin-peroxidase, anti-rabbit IgG biotin conjugate, and diaminobenzidine were purchased from Sigma Chemical Co. (St. Louis, MO). Progesterone [1,2-3H(N)] (P4-3H), testosterone [1,2-3H(N)] (T-3H), androsterone [9,11-3H (N)] (A-3H), estradiol [2,4,6,7-3H(N)] (E2-3H), and hydroxypregn-4ene-3-ona,20
[1,2-3H (N)] (20-OH-P-3H) were obtained from New England Nuclear (Boston, MA). Dulbecco modified eagle medium (4.5 g glucose/L), Ham F-12 nutrient mixture, fungizone (250 µg/ml), and gentamicin (10 mg/ml) were purchased from Gibco BRL (Grand Island, NY). All other chemicals were of reagent grade from standard commercial sources. Rabbit antibody against bovine cytochrome P450scc was a generous gift from Dr. Anita H. Payne (Stanford University Medical Center, Stanford, CA). StAR polyclonal rabbit antiserum was raised against a peptide fragment (amino acids 88–98) of the mouse StAR protein [25].
In Vivo eCG/LA Treatment and Superovulation
Female Sprague-Dawley rats, 23–25 days old, were allowed food and water ad libitum and kept at room temperature in a range of 21–23°C. To study the chronic effects of LA, animals were injected s.c. with eCG (25 IU/rat, referred to as the control group) to induce multiple follicular growth. At Time 0, LA was injected at 0.5 µg/rat, and then at 12 h intervals, only LA was given at the same dose. The last LA injection was performed 3 h before sacrifice. Following 48 h of eCG injection, the animals were killed by decapitation and blood samples were collected. To study the acute effects of LA, rats were injected subcutaneously with either a single dose of eCG (25 IU/rat, control group) or eCG (25 IU/rat) plus LA (2 µg/rat; referred to as the LA group), and animals were killed at different intervals (0, 2, 4, and 8 h). The ovaries were removed and cleaned in culture medium prior to subsequent assays. Healthy antral (200–400 µm, acute effect) or preovulatory follicles (>500 µm, chronic effect) were dissected microscopically using fine needles, then were collected and pooled (40–60 follicles for steroid determinations or 100 follicles for Western blot analysis). All experimental protocols were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by a local committee.
Western Blotting
Pooled follicles obtained from control and LA groups were lysed for 20 min at 4°C in lysis buffer (20 mM Tris-Cl, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, and 10% glycerol) supplemented with protease inhibitors (0.5 mM PMSF, 0.025 mM N-CBZ-L-phenylalanine chloromethyl ketone, 0.025 mM N'-p-tosyl-lysine chloromethyl ketone, 0.025 mM L-1-tosylamide-2-phenyl-ethylchloromethyl ketone). The lysate was centrifuged at 4°C for 10 min at 10 000 x g and the pellet was discarded. Protein concentrations in the supernatant were measured by the Bradford assay (Bio-Rad, Hercules, CA). After boiling for 5 min, 100 µg of protein from each sample was applied to a 15% SDS-polyacrylamide gel, and electrophoresis was performed at 25 mÅ for 1.5 h. The separated proteins were transferred onto nitrocellulose membranes in transfer buffer (20% methanol, vol/vol; 0.19 M glycine; 0.025 M Tris-base; pH 8.3) for 2 h at 4°C. Blots were blocked for 1 h in TBS (4 mM TRIS-Cl, pH 7.5, 100 mM NaCl) containing low-fat powered milk (2%) and Tween 20 (0.2%) at room temperature. Rabbit polyclonal anti-StAR and anti-P450scc (1:2000 and 1:2500, respectively, overnight) were used as primary antibodies. Protein bands were visualized by incubating the blots with a biotin-conjugated secondary anti-rabbit IgG (1:500, 1 h), followed by extravidin-peroxidase complex and diaminobenzidine solution. At different times, the levels of specific proteins in extracts obtained from control and LA-treated groups were compared by densitometry. Consistency of protein loading was evaluated by staining the membranes with Ponceau-S.
Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA from control and LA groups (100 follicles per group) at different time periods was extracted using Trizol reagent (Gibco BRL) according to the manufacturer's instructions. For amplification of the rat StAR cDNA, the primers used were the sense primer, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and the antisense primer, 5'-TAGCTGAAGATGGACAGACTTGC-3' [25]. To evaluate the variation in reverse transcription-polymerase chain reaction (RT-PCR) efficiency, the primers for L19 ribosomal protein gene (an internal control) were coamplified in each sample, using the sense primer 5'-GAAATCGCCAATGCCAACTC-3' and the antisense primer 5'-TCTTAGACCTGCGAGCCTCA-3', as described previously [26]. The molecular sizes of the target genes (StAR and L19) were approximately 980 and 405 base pairs, respectively.
RT and PCR were run sequentially in the same assay tube for amplifying target genes under optimized conditions [27, 28]. Briefly, 2.5 µg of total RNA obtained from different groups were reverse transcribed and the cDNAs generated were further amplified by PCR using the above primer pairs in a 50-µl reaction mixture containing 1 nM of each oligo primer, 200 mM of deoxy-NTP mixtures including [32P]-dCTP (3000 Ci/mmol), 20 U RNasin, 4.5 U AMV-RT, and 2.5 U Taq-DNA polymerase in 1x PCR buffer (Promega, Madison, WI). The reaction was initiated at 50°C for 16 min (RT) followed by denaturation at 97°C for 5 min. The PCR was then performed with the steps of amplification defined by denaturation at 96°C for 1.6 min, annealing at 56°C for 1.5 min, and extension at 72°C for 3 min (PTC-100; Peltier Thermal Cycler, MJ Research, Waltham, MA). The number of PCR cycles used was 22, which was optimized to be in the exponential phase in the PCR. A final cycle of extension at 72°C for 18 min was included. StAR and L19 PCR products were separated on 1.2% agarose gels and the gels vacuum dried for 45–60 min. Dried gels were exposed to Hyperfilm (Amersham International PLC, Buckinghamshire, UK) at 4°C for 1–3 h, and the autoradiograms were analyzed for StAR and L19 mRNAs. The relative levels of the different signals were quantitated using a computer-assisted image-analysis system (Visage 2000; BioImage, Ann Arbor, MI).
Measurement of Ovarian and Serum Steroids
Ovarian steroids were extracted following the method described previously [29, 30]. Each preovulatory follicular pool from both groups (control and LA) was homogenized in acetone with an Ultra-Turrax (IKA Werk, Breisgau, Germany) homogenizer. Known quantities of labeled steroid hormones, i.e., 3H-progesterone, 3H-20-hydroxy-progesterone (20-OH-P-3H), 3H-testosterone, 3H-androsterone, and 3H-estradiol in acetone (approximately 3000 cpm), were added to each sample as internal standards. An aliquot was taken from each homogenate for protein measurement. After complete homogenization, the samples were centrifuged (1600 x g for 10 min) and the resultant supernatant was evaporated to dryness. Following the addition of distilled water and vortexing, the samples were twice extracted with diethyl ether and the upper ether phase was transferred to conical tubes and again evaporated to dryness. The remaining residue was dissolved in methanol and, after adding distilled water, the samples were submitted to a solvent partition with n-hexane and dichloromethane and the upper layer was discarded and the lower phase evaporated. Finally, samples were stored in distilled water for later analysis by radioimmunoassay. This method was validated using increasing numbers of follicles in the follicular pool and different extraction volumes; in these assays, linearity in steroid concentrations was observed (not illustrated).
Radioimmunoassay
Following ether extraction, serum steroid levels were measured by RIA after 48 h eCG+LA treatment (chronic effect). After tissue or serum steroid extractions, progesterone, 20-OH-P, testosterone, androsterone, and estradiol levels were determined by RIA as described previously [31]. Progesterone, 20-OH-P, testosterone, and estradiol were measured using specific antibodies supplied by Dr. G.D. Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO). Androsterone was determined by RIA using an antibody supplied by Dr. G. Barbe (Department of Physiology, University of Western Ontario, London, Canada).
Under these conditions, the intraassay and interassay variations were 8.0% and 14.2% for P; 7.5% and 13.2% for 20-OH-P; 7.2% and 12.5% for estradiol; 7.3% and 13.2% for testosterone, and 8.1% and 14.5% for androsterone, respectively.
Statistical Analysis
All experiments were repeated three times with at least six animals per group. Incubations were performed in triplicate. One-way ANOVA was used to compare the mean values among the treatments. Two-way ANOVA was employed for statistical analysis in specific experiments that included two variables such as time and different treatments. When the values were significant, ANOVA analysis was followed by Scheffe multiple range test to compare the means from different treatments. Values of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Follicular contents of StAR and P450scc proteins following in vivo eCG (control group) and eCG+LA (LA group) treatment of prepubertal rats were measured by Western blotting. Treatment with eCG had no effect at 2 h but induced significant increases (P < 0.05) in StAR protein content by 4 and 8 h. Coadministration of eCG with a single LA injection produced a significant increase in StAR protein levels at 2, 4, and 8 h when compared with the control group (Fig. 1, panel A). Conversely, no changes in P450scc protein levels in fresh follicles were observed in either the control or LA groups at these times (Fig. 1, panel B). The results obtained were qualitatively similar using longer periods of eCG plus LA treatment (chronic effect). Figure 1 also shows the levels of follicular StAR (panel C) and P450scc (panel D) protein content 48 h after LA injection. It can be observed that LA induced an increase in StAR protein content while no change was seen in the P450scc protein levels.
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Effect of LA on StAR Gene Expression
To determine if the increase in StAR protein content induced by LA treatment is a reflection of StAR gene expression, the StAR mRNA levels in follicular extracts were measured by RT-PCR analysis in eCG- and eCG+LA-treated prepubertal rats at different times. Figure 2 shows that StAR mRNA levels were detectable in follicles prior to eCG administration with the RT-PCR assay conditions used here. When compared with noninduced controls, StAR mRNA levels showed significant increases at 2 and 4 h following eCG treatment, then appeared to level off at 8 h poststimulation. Administration of eCG+LA resulted in an even more rapid increase in the rate of StAR mRNA expression in the first 2 h when compared with eCG alone. StAR expression in eCG+LA-treated animals then leveled off at 4 and 8 h but remained significantly elevated over samples taken at similar times following eCG treatment only (Fig. 2).
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Ovarian and Serum Progesterone, Androgens, and Estradiol Levels
The chronic effect of LA treatment (48 h) in eCG-treated rats demonstrated a significant increase (P < 0.05) in follicular progesterone levels (363%), while significantly decreased testosterone (78%), androsterone (63%), and estradiol (72%) levels were observed (Fig. 3). These results show that LA treatment produces a dramatic rise in StAR expression and, as a consequence, an increase in ovarian progesterone production. However, the decrease in follicular levels of both androgens and estradiol indicates an alteration of the steroid pathway somehow producing these apparently controversial results. We measured the progesterone inactive metabolite, 20-OH-P, and it also exhibited a pattern similar to progesterone, being significantly increased (P < 0.05) in the LA group (362.9 ± 43.5 pg/follicle) when compared with the eCG-treated control (89.0 ± 24.9). Similar results were obtained when steroid levels were measured in whole ovaries or when the results were expressed per milligram of follicular or ovarian protein (data not shown).
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In addition, serum progesterone levels measured at 48 h following eCG/LA treatment were found to be unaltered, while significant decreases (P < 0.05) in circulating estradiol, testosterone, and androsterone levels were observed when compared with the eCG treated control group (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The enzymatic conversion of cholesterol to pregnenolone, catalyzed by the cytochrome P450scc enzyme, is considered as an important step in steroid biosynthesis in steroidogenic tissues [32, 33]. However, it has long been recognized that, prior to the catalytic activity of P450scc, the process of steroidogenesis requires the active delivery of the substrate cholesterol to its enzymatic site of action in the inner mitochondrial membrane [34]. This function is performed by the StAR protein, which has been implicated in the regulation of cholesterol uptake by the mitochondria [16, 17, 35]. Utilizing immunoblotting, an increase in the StAR protein content was observed in healthy antral or preovulatory follicles from LA-treated rats. Interestingly, this effect was observed after either acute or chronic LA treatment. In addition, these results demonstrate the increased expression of StAR mRNA levels as measured by RT-PCR. These data are the first demonstration that, in antral and preovulatory follicles, StAR expression is up-regulated by a GnRH agonist. As such, this observation represents an important contribution to our understanding of the effects of GnRH on ovarian follicular growth. In contrast with the effect on StAR, we observed that the follicular P450scc protein levels did not change after LA treatment. These results are in contrast with previous studies performed in rat corpora lutea that demonstrated that GnRH-a treatment decreased progesterone synthesis and interrupted pregnancy in vivo [8, 36]. Moreover, the latter study demonstrated a decrease in StAR, P450scc, and 3ß-hydroxysteroid dehydrogenase levels and, as a consequence, a reduction in luteal steroidogenesis during pregnancy. In addition, we earlier described a decrease in the mitochondrial P450scc content in corpora lutea from gonadotropin pseudopregnant rats after GnRH treatment [1]. Taken together, these data suggest that GnRH-a can produce different responses, which are dependent on the stage of ovarian cell differentiation (follicular vs. luteal cells).
Previous reports indicate that the negative effects of GnRH on LH-stimulated androgen biosynthesis in ovarian interstitial cells are mediated through a selective inhibition of the 17-hydroxylase and C17,20 lyase enzymes [37, 38]. In this study, we show that in vivo GnRH treatment decreases the follicular content of androgens as well as estrogen. Moreover, similar results are obtained when serum androgens and estradiol are measured, an observation in general agreement with previous findings [37, 38]. In addition, a direct effect of LHRH analog has been demonstrated to repress rat ovarian steroidogenesis by inhibiting the microsomal enzyme activities of 3ß-hydroxysteroid dehydrogenase, 17 hydroxylase, 17,20 desmolase, 17 keto-steroid reductase, and aromatase [39]. In contrast, follicular progesterone levels were increased in comparison with the other steroids, perhaps as a result of the increased StAR content and the inhibition of androgen biosynthesis. This increase in follicular progesterone content following GnRH-a treatment appears to be due to two mechanisms: 1) enhanced StAR expression and 2) inhibition of androgen biosynthesis. In addition, GnRH-a inhibits follicular estradiol content, likely as a consequence of the decrease in androgen production. It is known that estradiol is essential for normal follicular growth and is a crucial factor involved in ovarian follicular apoptosis rescue [10]. In this regard, the inhibition of folliculogenesis and the increase in ovarian follicular apoptosis by GnRH treatment [1, 12, 40] may be explained through alterations in these steroid pathways. In comparison with the serum steroid data observed, similar androgen and estradiol changes were obtained in follicular tissue. However, the increase in progesterone levels seen in the follicles from the LA group was not reflected in serum, where progesterone concentrations similar to the control group were detected. This apparent paradox could be explained by alterations in ovarian capillary permeability or in the metabolic clearance of progesterone in response to LA treatment.
Maintenance of various ovarian functions has been demonstrated to be influenced by many other hormones and locally produced factors, including GnRH or GnRH-like substances [41–44]. Studies have demonstrated the presence of specific high-affinity GnRH receptors in the rat ovary [45, 46], and direct effects of GnRH on rat ovarian steroidogenesis have also been reported [39, 47]. In our experimental model, the effects of LA on StAR expression and steroid biosynthesis were compared following eCG treatment, and it is postulated that the agonist acts directly at the ovarian level through GnRH receptors. A central unanswered question as to whether the effects of LA influence other hormones and/or factors involved in modulating ovarian function needs further investigation. However, an indirect action of this agonist via changes in pituitary gonadotropin release or through modification of the effects of local growth factors in the ovary cannot be excluded. Based on these considerations, studies on the mechanism of LA effects on ovarian function are currently underway and are an area of obvious interest in our future investigations.
In summary, we suggest that the inhibitory effect of LA on ovarian function is related to changes in the follicular levels of the StAR protein and subsequent follicular steroid production.
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FOOTNOTES |
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2 Correspondence: Marta Tesone, Instituto de Biología y Medicina Experimental, Obligado 2490, 1428 Buenos Aires, Argentina. FAX: 54 011 4786 2564; mtesone{at}dna.uba.ar
Received: 1 August 2002.
First decision: 21 August 2002.
Accepted: 18 November 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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