AJP - Endo Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 292: E1456-E1464, 2007; doi:10.1152/ajpendo.00226.2006
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Irusta, G.
Right arrow Articles by Tesone, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Irusta, G.
Right arrow Articles by Tesone, M.

Inhibition of cytochrome P-450 C17 enzyme by a GnRH agonist in ovarian follicles from gonadotropin-stimulated rats

Griselda Irusta ,1,* Fernanda Parborell,1,* and Marta Tesone1,2

1Instituto de Biología y Medicina Experimental-Consejo Nacional de Investigaciones Científicas y Técnicas; and 2Departamento de Química Biológica, Facultad de Ciencias Exactas, Universidad de Buenos Aires, Buenos Aires, Argentina

Submitted 11 May 2006 ; accepted in final form 26 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our objective was to study the direct action of a GnRH-I agonist, leuprolide acetate (LA), on ovarian steroidogenesis in preovulatory follicles obtained from equine chorionic gonadotropin (eCG)-treated rats. Previously, we have demonstrated an inhibitory effect of LA on steroidogenesis and follicular development. In this study, we tested the hypothesis that gonadotropin-releasing hormone (GnRH) exerts its negative effect on follicular development by inhibiting thecal cytochrome P-450 C17 (P450C17) {alpha}-hydroxylase expression and, consequently, androgen synthesis. Studies were carried out in prepubertal female rats injected with either eCG (control) or eCG plus LA (LA) and killed at different time points. Immunohistochemical studies indicated that LA induced steroidogenic acute regulatory protein (StAR) expression mainly in theca cells of preantral and antral follicles. In addition, serum progesterone levels increased significantly (P < 0.05), whereas those of androsterone decreased (P < 0.05) after 8 h of LA treatment. This inhibition caused by LA seemed to be a consequence of the decreased expression of follicular P450C17 {alpha}-hydroxylase, as demonstrated by Western blot and RT-PCR techniques. In vitro studies using follicles isolated from 48-h-eCG-treated rats and cultured with LA showed a significant (P < 0.05) inhibition of FSH-induced androsterone follicular content as well as P450C17 {alpha}-hydroxylase protein levels, as determined by Western analysis. However, LA increased StAR protein expression in these follicles without significant changes in P450scc enzyme levels. Taking all these findings into account, we suggest that GnRH-I exerts a direct inhibitory action on gonadotropin-induced follicular development by decreasing the temporal expression of the P450C17 enzyme and, consequently, androgen production, thus reducing the supply of estrogens available to developing follicles.

ovary; follicle; gonadotropin-releasing hormone agonist; steroidogenesis

THE SUPPRESSION OF GONADOTROPINS exerted by the administration of gonadotropin-releasing hormone (GnRH) analogs on the pituitary-gonadal axis is well known. The agonists initially stimulate the secretion of large amounts of gonadotropins, followed by a desensitization of the pituitary and the subsequent inhibition of gonadal steroid production (33, 62). However, in the last several years there has been increasing evidence that GnRH is an intraovarian regulatory factor. A GnRH-like peptide, GnRH receptors, and transcription products from their genes have been found in ovarian tissue (3, 7, 31, 65), and a direct inhibitory effect of this decapeptide has been observed on steroidogenesis in both granulosa cells (Gc) (31, 32, 35, 38, 40, 43, 58) and in corpus luteum (1, 62, 64). In addition, we (57) have demonstrated that in vitro treatment with a GnRH-I agonist (GnRH-I-a) produces an increase in apoptosis of preovulatory follicles by interfering with FSH, cAMP, and/or growth factors.

Our recent studies in prepubertal gonadotropin-stimulated rats have shown changes in the follicular expression of steroidogenic acute regulatory protein (StAR), a protein involved in the transfer of cholesterol from the outer to the inner mitochondrial membrane. In particular, 48 h of chronic in vivo GnRH-I-a treatment increases follicular concentrations of progesterone due to the enhancement of StAR expression and causes a decrease in follicular and serum concentrations of androgens and estradiol (34). Because estradiol is essential for normal follicular growth and atresia rescue (29), the previously observed GnRH-induced inhibition of folliculogenesis and increase in apoptosis appear to take place through alterations in the ovarian steroid pathway (1, 49, 56, 57).

Cytochrome P-450 C17 (P450C17) is a key branch point enzyme in the pathways of steroid hormone biosynthesis. It is associated with the smooth endoplasmic reticulum (SER) and catalyzes two sequential reactions: the hydroxylation of the C21 steroids progesterone or pregnenolone (17{alpha}-hydroxylase activity) and the cleavage of the two-carbon side chain (C17,20 lyase activity) to yield the C19 steroids androstenedione or dehydroepiandrosterone, respectively (19). Thus, this enzyme, confined to theca cells, is a crucial factor for the proper supply of androgens that are used as substrates for estrogen synthesis. Moreover, the P450C17 enzyme is considered a theca cell marker (23). The enzymes 5{alpha}-reductase and 3beta-hydroxysteroid dehydrogenase, also localized in SER, are responsible for the reduction and conversion of androstenedione and testosterone to androsterone and 5{alpha}-androstane-3{alpha},5beta-diol, respectively.

Available data indicate that thecal P450C17 expression is cAMP dependent in humans, cattle (14, 68), rats (21, 27), and pigs (9). LH appears to be essential for the maintenance of thecal cell P450C17 expression in all mammals studied to date (13). Many additional factors such as activin, inhibin, and insulin-like growth factor I also seem to regulate this enzyme (72).

There are several lines of evidence related to the direct effect of GnRH on gonadal androgen biosynthesis. Inhibition of testicular testosterone secretion in rats and toads has been reported (6, 69). Magoffin, Erickson, and colleagues (18, 48) have reported that GnRH treatment reduces basal and hCG-stimulated 17{alpha}-hydroxylase and C17,20 lyase in cultures of ovarian interstitial cells obtained from hypophysectomized immature rats. However, despite the increasing evidence of a GnRH effect on P450C17 activity in ovarian theca cells, little is known about the regulation of this enzyme expression at the protein level. Until now, no studies have been performed to establish whether GnRH agonists directly interfere with gonadotropin actions on P450C17 mRNA and protein expression in the ovary.

In the present report, we studied the role of GnRH in gonadotropin-stimulated prepubertal rats injected with either equine chorionic gonadotropin (eCG; control) or eCG plus GnRH-I-a [leuprolide acetate (LA)] and killed at different time points. Specifically, we 1) expanded our earlier study of StAR protein regulation to include its immunohistochemical localization in ovarian cells and 2) examined changes of follicular P450C17 expression and serum steroid levels. Additionally, we determined the in vitro effect of GnRH-I-a on androsterone content, StAR, P450C17, and P450scc protein expression in FSH-stimulated follicles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. LA, a GnRH-I-a, was obtained from Abbott Laboratories (Buenos Aires, Argentina). The original ampoule (2.8 mg/5 ml) was dissolved in saline to obtain the appropriate concentration. Syntex (Buenos Aires, Argentina) provided the eCG (Novormon). HEPES, SDS, and anti-rabbit secondary antibody conjugated with horseradish peroxidase were obtained from Sigma Chemical (St. Louis, MO). [1,2-3H(N)]progesterone (P4-3H) and [9,11-3H(N)]androsterone (A-3H) were obtained from New England Nuclear (Boston, MA). Dulbecco's modified Eagle medium (DMEM; 4.5 g glucose/l), Ham's F-12 nutrient mixture (F-12), fungizone (250 µg/ml), and gentamicin (10 mg/ml) were purchased from Invitrogen (Carlsbad, CA). All other chemicals were of reagent grade from standard commercial sources. StAR polyclonal rabbit antiserum was raised against a peptide fragment (amino acids 88–98) of the murine StAR sequence and was kindly donated by Dr. D. M. Stocco (Texas Tech University Health Sciences Center) (10). Rabbit antibody against bovine P450C17 was a kind gift from Dr. Anita Payne (Stanford University Medical Center, Stanford, CA). Rabbit-anti-porcine P450C17 antiserum was a generous gift from Dr. D. B. Hales (University of Illinois).

GnRH agonist treatment. Immature female 23- to 25-day-old Sprague-Dawley rats were allowed food and water ad libitum and kept at room temperature at a range of 21–23°C with a 12:12-h light-dark photo period in an air-conditioned environment. On the day of the experiment, all animals (n = 5/group for each time point) were injected sc at time 0, with 0.1 ml of eCG (25 IU/rat, control group) to induce multiple follicular growth. Animals belonging to the LA group were simultaneously injected with LA (2 µg/rat, LA group) and eCG, whereas control animals were injected with vehicle only (saline solution). The animals were killed by CO2 asphyxiation or decapitated to obtain blood samples for steroid extraction at 0 (untreated animals), 2, 8, 24, and 48 h after eCG or eCG plus LA injection. For the in vitro studies, animals were killed 48 h after the eCG injection (25 UI/rat), and the ovaries were removed and cleaned of adhering tissue for the subsequent assays. All animals were treated and cared for in accordance with standard international animal care protocols (Canadian Council of Animal Care, Guide to the Care and Use of Experimental Animals). The experimental protocols were approved by the Animal Experimentation Committee of the Instituto de Biología y Medicina Experimental.

Isolation of ovaries and follicles. One ovary from each rat (n = 5) was used in immunohistochemical studies. From the contralateral ovary, individual ovarian follicles (60–80 follicles/ovary) were dissected under a stereoscopic microscope, as previously described (1, 49, 56, 57). Accordingly, healthy antral follicles (AF; >200 µm in diameter) from five ovaries were isolated and divided into three pools, one for each of the different assays (RT-PCR, Western blot, or steroid extraction). Each experiment was repeated three times (n = 3).

Immunohistochemistry of StAR protein. Ovaries from the control and the LA-treated groups were immediately fixed in 4% neutral buffered formalin for 12 h and then embedded in paraffin. Five-micron step sections were mounted at 50-µm intervals onto microscope slides to prevent counting the same follicle twice according to Woodruff et al. (71). Tissue sections were deparaffinized in xylene and rehydrated by graded ethanol washes. Endogenous peroxidase activity was blocked with hydrogen peroxide in PBS, and nonspecific binding was blocked with 2% bovine serum albumin overnight at 4°C. Sections were incubated with rabbit polyclonal anti-StAR (1:100) overnight at 4°C. After washing, the slides were incubated with biotinylated anti-rabbit IgG and then with avidin-biotinylated horseradish peroxidase Complex (Vectastain ABC system; Vector Laboratories, Burlingame, CA) for 30 min. Immunoreactivity was visualized with diaminobenzidine staining. The reaction was stopped with distilled water, stained with hematoxylin, and dehydrated before being mounted with mounting medium (PMYR, Buenos Aires, Argentina). For the negative control, the primary antibody was omitted.

RT-PCR analysis. Total RNA was extracted from the control and LA groups (100 follicles/group) at different times using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For amplification of the rat P450C17 cDNA, the designed primers used were the sense primer 5'-AACTGGATCCTGGCTTTCCT-3' and the antisense primer 5'-CCTCTAAGATTGGGCTGTGG-3'. beta-Actin cDNA was amplified as an internal control using a commercial set of primers (the sense primer 5'-CGGAACCGCTCATTGCC-3' and the antisense primer 5'-ACCCACACTGTGCCCATCTA-3'; Biodynamics, Buenos Aires, Argentina). In rat follicular cells, beta-actin mRNA has been found to be expressed at a constant level independent of follicle status and size. In addition, its expression is not affected by growth factors or gonadotropins (42, 70). The molecular product sizes of the target genes (P450C17 and beta-actin) were ~647 bp and 289 bp, respectively. PCRs for P450C17 and beta-actin were run separately, and conditions for each set of primers were optimized. Validation of the RT-PCR assay was performed, testing different numbers of cycles to ensure semiquantification of the product in the linear phase of the amplification. Briefly, 1 µg of total RNA obtained from the different groups was treated with 1 µg of DNAase (Invitrogen). RT reaction was carried out at 37°C for 50 min in a 20-µl reaction volume using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and oligo primers (Biodynamics). PCR consisted of denaturation at 94°C for 1.5 min, annealing at 60°C for 30 s, and extension at 72°C for 2 min for 25–30 cycles total. A final cycle of extension at 72°C for 5 min was included. The optimized number of PCR cycles (linear phase) used were 30 (P450C17) and 25 (beta-actin), respectively. P450C17 and beta-actin PCR products were separated on a 2% agarose gel that was stained with ethidium bromide for 15 min to enhance sensitivity. Gels were visualized on a UV transilluminator and photographed, and the bands for P450C17 and beta-actin were quantified using the appropriate software (Scion Image). The PCR products were purified and sequenced at the ONPRC Molecular and Cell Biology Core facility using an ABI 3100 automated sequencer to confirm identity.

Western Blot for StAR, P450scc, and cytochrome P450C17. Sixty healthy AF obtained from the control and LA-treated groups were homogenized at 4°C in 500 µl of lysis buffer (20 mM Tris·HCl, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, and 10% glycerol) with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 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-phenylethylchloromethyl ketone). The homogenate was centrifuged at 4°C for 10 min at 10,000 g, and protein concentration in the supernatant was determined by Bradford assay (5). Equal amounts of proteins (40 µg) were boiled for 5 min and resolved by 10% SDS-polyacrylamide gel at 25 mA for 2 h. The proteins were transferred for 2 h onto nitrocellulose membranes (Amersham Biosciences) in transfer buffer containing 20% methanol (vol/vol), 0.19 M glycine, and 0.025 M Tris base (pH 8.3) at 4°C. Blots were then blocked at 4°C in TBS (4 mM Tris·HCl, pH 7.5, 100 mM NaCl) containing low-fat powdered milk (5%) and Tween-20 (0.2%). Rabbit polyclonal anti-StAR (1:2,000), anti-P450scc (1:2,000), and anti-P450C17 (1:100) were used as primary antibodies. Protein bands were detected by incubation with anti-rabbit IgG (1:1,000 dilution, 1 h) conjugated to horseradish peroxidase (Sigma). beta-Actin was detected with anti-mouse (1:1,000 dilution, 1 h) secondary antibody (R&D Systems). Reactive protein bands were visualized by chemiluminescence, using the manufacturer's protocol (Amersham Pharmacia). Negative controls were obtained in the absence of the primary antibody. The levels of protein were compared in extracts from the control and LA groups and analyzed by densitometric studies. Optical density data are expressed as arbitrary units ± SE (n = 3). In the in vivo experiments, the proper loading was evaluated by staining the membranes with Ponceau S. As an internal control, the density of each protein was normalized to the density of a band that was observed on the protein transference pattern in all Ponceau S-stained membranes. This band was selected since it remained unchanged under different treatments. On the other hand, for the in vitro experiments, the density in each band was normalized using beta-actin (mouse monoclonal, Abcam) as an internal control. The Ponceau S method was validated in selected samples using beta-actin as control loading, since it was observed that the ratio of the desired protein band against beta-actin band was similar to the ratio obtained when the normalization was performed against the Ponceau S band (data not shown).

Culture of isolated ovarian follicles. To study the in vitro effects of LA on ovarian steroidogenic proteins, AF were dissected from ovaries collected following eCG treatment and cleansed of adhering tissue in culture medium. The culture was initiated within 1 h of ovary removal, and the follicular pools (containing 60 follicles/sample) were incubated in triplicate under serum-free conditions at 37°C in 1 ml DMEM-F-12 (1:1 vol/vol), containing 10 mM HEPES, supplemented with fungizone (250 µg/ml) and gentamicin (10 mg/ml). Follicles were incubated for 2, 8, 12, 24, and 48 h with FSH (20 ng/ml) in either the presence or the absence of LA (100 ng/ml) at 37°C under 95% O2 and 5% CO2, as previously described (50, 55). At the end of the cultures, media were discarded and follicular steroids extracted to determine endogenous androsterone content in each follicular pool.

Progesterone and androsterone assay. Serum steroid levels were measured by RIA following ether extraction of steroids at different times of death (n = 5 rats/group for each time point). Both progesterone and androsterone follicular contents were examined after acetone extraction, as previously described (34), at the indicated times of incubation. Briefly, each follicular pool was homogenized in acetone. Labeled steroids were added as internal standards, and the percentage of recovery was between 60 and 80%. Following centrifugation, the samples were evaporated to dryness. Distilled water was added, and samples were extracted twice with diethyl ether. The phase was transferred and evaporated to dryness and the remaining residue dissolved in methanol. After adding distilled water, the samples were submitted to a solvent partition. The upper layer was discarded and the lower phase evaporated. Finally, samples were stored in distilled water for later analysis by RIA. Progesterone was measured using a specific antibody supplied by Dr. G. D. Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO), and androsterone was determined by RIA using an antibody supplied by Dr. G. Barbe (Department of Physiology, University of Western Ontario, London, ON, Canada). Under these conditions, the intra-assay and interassay variations were 8.0 and 14.2% for progesterone and 8.1 and 14.5% for androsterone, respectively.

Data analysis. All experiments were repeated three times with five animals per group, and incubations were performed in triplicate using follicle pools. Data regarding densitometric analysis and in vitro experiments were expressed as means ± SE of pooled results obtained from three independent experiments (n = 3). Statistical analyses were performed using two-way ANOVA for experiments including two variables, such as time and treatments. When values were significant, ANOVA analysis was followed by either Tukey's or Bonferroni's multiple comparison test. Values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
GnRH-I-a effects on StAR expression. To determine the cell type-specific expression of StAR protein in prepubertal rat ovaries, immunohistochemistry was performed on ovarian sections derived from eCG- (control group) and eCG plus LA (LA group)-treated animals. When these animals were killed 2 or 8 h after the eCG injection (Fig. 1, A and B, respectively), immunostaining for StAR was observed in theca cells (Tc) and stroma (st) of preantral follicles (PF) and AF after only 8 h of eCG administration. In contrast, Gc showed no expression of this protein. After 8 h of eCG plus LA administration, the expression of StAR was increased in Tc of either PF or AF (Fig. 1D) compared with either the 2-h eCG plus LA treatment (Fig. 1C) or the 8-h control group (Fig. 1B). In addition, Gc of AF exhibited a weak immunostaining for StAR protein after 8 h of eCG plus LA treatment (Fig. 1D), whereas no positive staining was detected in the same type of cells in PF.


Figure 1
View larger version (158K):
[in this window]
[in a new window]

  		Fig. 1. Immunohistochemical localization of steroidogenic acute regulatory protein (StAR) in follicles of different stages of development in equine chorionic gonadotropin (eCG)- and/or leuprolide acetate (LA)-treated rat ovaries. Ovaries from eCG [control (C)]- or eCG + LA (LA)-treated rats were fixed at indicated times after the beginning of the treatments. A–D: ovarian sections obtained from C and LA rats, respectively, that were killed at 2 (A and C) or 8 h (B and D) after treatments. PF, preantral follicles; AF, antral follicles; Tc, theca cells; Gc, granulosa cells; st, stroma. Arrowheads indicate StAR immunoreactive cells. Scale bar, 0.1 mm (A–D). D, inset: negative control in which the primary antibody was omitted.

 
Surprisingly, after 8 h of LA treatment, the theca layer in PF was thicker than that observed in ovarian sections from eCG-treated rats (Fig. 1, B vs. D).

In agreement with previous studies performed by Ronen-Fuhrmann et al. (61), the expression of StAR was not detected at time 0 (without treatment; data not shown).

Effects of GnRH-I-a on serum progesterone and androsterone levels. To determine whether the increase in ovarian StAR expression after the agonist treatment was correlated with steroid levels, we performed RIA in serum collected from both eCG- and eCG plus LA-treated animals. As shown in Fig. 2A, serum levels of progesterone increased significantly (P < 0.05) after 8 h of eCG plus LA administration but were not different from the corresponding control at other time points (2, 24, and 48 h). In addition, we analyzed serum levels of androsterone, the principal androgen produced by prepubertal rat ovaries (18). Figure 2B illustrates serum androsterone levels at different periods of treatment. The injection of eCG (control group) produced a time-dependent stimulation in androsterone production. Cotreatment with LA significantly inhibited eCG-stimulated androsterone production by 8 h (P < 0.05) of treatment.


Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. In vivo effect of gonadotropin-releasing hormone-I agonist (GnRH-I-a) treatment on serum steroid levels. As described in MATERIALS AND METHODS, eCG- or eCG + LA-treated rats were killed at different time points after the beginning of the treatment. Serum was collected, and progesterone (A) and androsterone (B) levels were determined by RIAs. The group of untreated immature rats is represented by 0 h. Values are means ± SE. *P < 0.05 vs. corresponding control group; n = 5 rats/group for each time point.

 
In vivo effect of LA on ovarian cytochrome P450C17 gene and protein expression. To determine whether the decrease in serum androsterone by LA treatment was due to a decrease in P450C17 enzyme levels, we performed Western blot analyses of follicles obtained from the eCG- and eCG plus LA-treated animals (Fig. 3A). As shown, the GnRH-I-a treatment significantly decreased the relative intensity of P450C17 in the LA group compared with the control group at 8 h. To establish whether P450C17 regulation by the agonist also occurred at the mRNA level, we carried out RT-PCR analyses at all the time points mentioned. Figure 3B shows that mRNA levels were decreased after 8 h of eCG plus LA treatment, similar to the results observed with protein expression.


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 3. In vivo effect of GnRH-I-a on cytochrome P-450 C17 (P450C17) expression in antral follicles. A, top: representative immunoblot of P450C17 protein content in preovulatory follicles from control rats and those treated with LA. After homogenization, proteins were extracted and subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes. P450C17 was visualized using an anti-P450C17 antibody. Bottom: densitometric quantification of P450C17 follicular content. Data represent means ± SE of 3 experiments. P < 0.05 vs. corresponding control group. B: RT-PCR analysis of P450C17. Top: representative example of an individual run illustrates the P450C17 (647 bp) and beta-actin (289 bp) mRNA levels. Bottom: summary of densitometric values normalized to beta-actin. Data are presented as means ± SE of 3 experiments. *P < 0.05 vs. corresponding control.

 
Effect of LA on follicular androsterone in ovarian follicle cultures. Ovarian follicles isolated from 48-h-eCG-treated rats were cultured with FSH or FSH plus LA for 8, 12, 24, and 48 h. This model has the advantage of keeping the integrity of the follicle and is currently used to investigate the pathways that control steroidogenesis, apoptosis, and follicular atresia (57, 66). By means of this model, we expected to analyze steroidogenesis regulation by the GnRH-I-a and to confirm whether the agonist affects this process directly. As shown in Fig. 4, follicular androsterone content was significantly decreased (P < 0.05) when follicles were coincubated for 24 h with FSH plus LA (Fig. 4).


Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 4. In vitro effect of GnRH-I-a on follicular content of androsterone. AF from rats treated with eCG for 48 h were isolated by microdissection and cultured with either FSH (20 ng/ml) or FSH (20 ng/ml) + LA (100 ng/ml) for different periods of time. Follicular steroids were extracted as described in MATERIALS AND METHODS. Androsterone content was determined by RIA. Values are means ± SE. *P < 0.05 vs. corresponding control group.

 
In vitro effect of LA on ovarian StAR, P450scc, and P450C17 protein expression. To study the direct effects of LA on the expression of ovarian steroidogenic proteins, AF obtained from ovaries of eCG-stimulated rats were cultured for different periods of time (2, 8, 12, 24, and 48 h) in serum-free medium with FSH (20 ng/ml) in either the presence or absence of LA (100 ng/ml) (Fig. 5). Follicular content of StAR, P450scc, and P450C17 were determined by Western blotting. The levels of StAR detected in protein extracts obtained from LA-incubated follicles increased significantly at 12 h (FSH: 0.23 ± 0.09, FSH + LA: 1.67 ± 0.15, P < 0.05; Fig. 5A). This protein was not detected after 24 and 48 h of incubation in FSH-incubated follicles in either the presence or absence of LA. No changes were observed in P450scc levels at the different periods of time studied (data not shown). Interestingly, follicles incubated with FSH plus LA showed a significant decrease in P450C17 protein expression at 12 h when compared with follicles incubated with FSH alone (FSH: 0.51 ± 0.04, FSH + LA: 0.16 ± 0.03, P < 0.05; Fig. 5B).


Figure 5
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 5. In vitro effect of GnRH-I-a on StAR and P450C17 protein content of antral follicles. Representative immunoblots of StAR (A) and P450C17 (B) protein content in AF incubated with FSH (20 ng/ml) in either the presence or the absence of LA (100 ng/ml) for 2, 8, 12, 24, and 48 h. Similar results were obtained in 3 independent experiments. Densitometric quantification of the immunoblots is shown in A and B, bottom. Data are expressed as means ± SE of 3 experiments. *P < 0.05 vs. respective controls.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present study demonstrate, for the first time, that GnRH-I-a administered in vivo to gonadotropin-treated prepubertal rats induces an increase in StAR protein and a decrease in P450C17 expression and, consequently, in androgen production. The demonstration of this paradoxical effect in steroidogenic proteins and enzymes is novel. These findings were supported by in vitro studies confirming a direct action of GnRH on follicular steroidogenesis.

StAR is especially involved in the acute regulation of steroid synthesis in response to the gonadotropin surge. Ronen-Fuhrmann et al. (61) have demonstrated a biphasic increase in the levels of this protein after eCG administration in ovarian homogenates from immature rats. In this regard, we (34) have previously shown an increase in StAR protein content and messenger levels in healthy antral or preovulatory follicles from superovulated LA-treated rats after either acute or chronic treatment. These observations led us to study the time-dependent effect of a GnRH-I-a on steroidogenic proteins and steroid production.

In the present study, we demonstrated by immunohistochemical studies that the administration of a GnRH-I-a (LA) to gonadotropin-treated prepubertal rats produces an increase in ovarian StAR expression localized in theca cells of preantral and antral follicles. The results obtained in eCG-treated rats were in agreement with previous reports (61) in which a bright pattern of StAR staining in interstitial and theca cells was observed after gonadotropin treatment without expression of this protein in granulosa cells. However, in our study, after cotreatment with LA, a weak StAR staining in granulosa cells was observed. In addition, when we quantified serum levels of progesterone, the increase of this progestagen in eCG plus LA-treated rats was parallel to StAR expression. In contrast, Yang et al. (73) have recently reported an inhibition in serum progesterone production in pregnant rats treated with a GnRH agonist for 8 h. In that study, the agonist was continuously administered and a higher dose was used. In addition, GnRH agonists stimulate progesterone accumulation in granulosa cells from Graffian follicles and plasma progesterone in acutely hypophysectomized rats (17, 28).

These reports indicate the existence of different responses to GnRH depending on the ovarian cell types and dose and route of GnRH administration. In antral follicles, a failure to express aromatase in granulosa cells is characterized by a high androgen-to-estrogen ratio in follicular fluid, an event that causes follicular atresia. Therefore, the accurate supply of androgens is critical for normal folliculogenesis beyond the antral stage (20). Furthermore, estrogen is necessary for both normal folliculogenesis and maintenance of the female phenotype of the somatic cells within the ovaries (20). Given the importance of androgens as follicular estrogen substrates (4, 12, 26, 44), we studied plasma androsterone levels in animals treated with the eCG plus GnRH-I-a, since this androgen has been reported to be one of the main 5{alpha}-metabolites in the immature rat ovary (47). In our experimental model, GnRH-I produced a marked decrease in serum androsterone in animals killed 8 h after the administration of the agonist. Our observations of the steroidogenic profile of the GnRH-I-treated animals support that the inhibition of the P450C17 enzyme accounts for the decreased androgen synthesis despite the increase in serum progesterone levels produced as a consequence of the increase of StAR expression. In this sense, it has been suggested that GnRH switches the interstitial cells from androgen- to progesterone-secreting cells (18, 46).

We also demonstrated in this work a negative effect on P450C17 follicular expression at messenger and protein levels caused by in vivo treatment with a GnRH agonist in gonadotropin-treated rats. Although 17{alpha}-hydroxylase/C17,20 lyase activity has been determined under different conditions, both in vivo and in vitro (15, 16, 39, 45, 51, 53), until now, few studies have been carried out on the expression of this enzyme at the protein level. In this regard, the relevance of our work resides in the correlation of P450C17 mRNA and protein expression levels and its regulation by GnRH-I. This is the first in vivo study in which different regulation points of P450C17 have been investigated in prepubertal gonadotropin-stimulated rats treated with a GnRH-I-a.

In addition, to elucidate whether the LA effect was due to a direct effect on ovarian cells and to discard a possible action of the agonist at the pituitary level, in vitro experiments were performed. These studies showed a significant inhibition of the FSH-induced follicular androsterone content in follicles incubated for 24 h with FSH plus GnRH-I-a. This effect may be a consequence of the direct effect of the agonist on P450C17 enzyme expression. In agreement with this hypothesis, blotting analyses showed a decrease in the P450C17 follicular expression after 12 h of culture in the presence of LA. Similarly to the in vivo effect of LA on StAR expression, the agonist also induced an increase in this protein after 12 h of incubation. Furthermore, in agreement with our previous in vivo results (34), no changes in P450scc protein expression were detected after in vitro LA follicle treatment.

There are several lines of evidence about GnRH-like peptides playing autocrine or paracrine regulatory roles in the ovary. It has been reported that an intrinsic GnRH system, with endogenous GnRH, GnRH receptor, and biological response, exists in rodent (2, 24, 52) and human (8, 11, 22, 30, 36, 37, 59) ovaries. It is well documented that GnRH-I possesses antigonadotropic effect in the rat ovary, downregulating the expression of FSH and LH receptors (67, 60), inhibiting gonadotropin-stimulated cAMP production (54, 41), and suppressing steroidogenic enzymes (32, 63). In addition, we demonstrated that GnRH-I-treatment interferes with follicular recruitment, growth, and luteinization induced by gonadotropins (1, 25, 30). Moreover, we demonstrated that the GnRH antagonist treatment of gonadotropin-treated rats improves follicular development, suggesting a binding impairment of the GnRH-like peptide synthesized in the ovary to its receptors (1, 49, 56, 57).

Considering all these findings, we propose a possible mechanism by which GnRH-I exerts a direct inhibitory action on follicular development through the alteration of key steroidogenic proteins and their products. These lines of evidence, together with our previous reports demonstrating that antral follicles from gonadotropin-treated rats are more sensitive toward undergoing an apoptotic process when they have been exposed to a GnRH-I-a treatment (55, 56), suggest that GnRH could act as an intraovarian factor by interfering with gonadotropin induction of follicular development.

In summary, we suggest that GnRH-I exerts a direct inhibitory action on gonadotropin-induced follicular development by causing a decrease in the temporal expression of the P450C17 enzyme and consequently in androgen production, thus reducing the supply of estrogens available to the developing follicles.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Agencia Nacional de Promocion Cientifica y Tecnologica (BID 1728/OC-AR PICT 2004 5-26047), CONICET (PIP 5471), University of Buenos Aires (EX237), and Roemmers Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. C. P. Lantos (University of Buenos Aires) and Dr. J. T. Jensen (Oregon National Research Primate Center-Oregon Health and Science University) for carefully reading this manuscript. We thank Dr. Douglas Stocco (Texas Tech University Health Sciences Center) for the kind gift of anti-StAR antibody for immunohistochemical studies and Dr. Anita Payne for the P450scc antibody (Stanford University Medical Center, Stanford, CA). We also thank Dr. D. H. Hales (University of Illinois) for donation of the anti-CYP17 antisera and Dr. G. Barbe (University of Western Ontario) for the androsterone antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Tesone, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina (e-mail: mtesone{at}dna.uba.ar)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* G. Irusta and F. Parborell contributed equally to this article. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Andreu C, Parborell F, Vanzulli S, Chemes H, Tesone M. Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: relationship between steroidogenesis and apoptosis. Mol Reprod Dev 51: 287–294, 1998.[CrossRef][ISI][Medline]
  2. Aten RF, Williams AT, Behrman HR. Ovarian gonadotropin-releasing hormone-like protein(s): demonstration and characterization. Endocrinology 118: 961–967, 1986.[Abstract]
  3. Behrman HR, Preston SL, Hall AK. Cellular mechanism of the antigonadotropic action of luteinizing hormone-releasing hormone in the corpus luteum. Endocrinology 107: 656–664, 1980.[ISI][Medline]
  4. Billig H, Furuta I, Hsueh AJ. Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133: 2204–2212, 1993.[Abstract]
  5. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
  6. Canosa LF, Pozzi AG, Somoza GM, Ceballos NR. Effects of mGnRH on testicular steroidogenesis in the toad Bufo arenarum. Gen Comp Endocrinol 127: 174–180, 2002.[CrossRef][ISI][Medline]
  7. Chamson-Reig A, Pignataro OP, Libertun C, Lux-Lantos VA. Alterations in intracellular messengers mobilized by gonadotropin-releasing hormone in an experimental ovarian tumor. Endocrinology 140: 3573–3580, 1999.[Abstract/Free Full Text]
  8. Cheng CK, Leung PC. Molecular biology of gonadotropin-releasing hormone (GnRH)-I, GnRH-II, and their receptors in humans. Endocr Rev 26: 283–306, 2005.[Abstract/Free Full Text]
  9. Chu X, Kaminski MA, Corbin CJ, Coney AJ. Effects of LH and forskolin on proliferation and cytochrome P450 alpha-hydrolase (P450C17) expression of primary porcine theca cell culture (Absract). Proc Int Congr Endocrinol 10th, 1996.
  10. Clark BJ, Wells J, King SR, Stocco DM. The purification, cloning, and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 269: 28314–28322, 1994.[Abstract/Free Full Text]
  11. Clayton RN, Eccleston L, Gossard F, Thalbard JC, Morel G. Rat granulosa cells express the gonadotrophin-releasing hormone gene: evidence from in-situ hybridization histochemistry. J Mol Endocrinol 9: 189–195, 1992.[Abstract/Free Full Text]
  12. Clayton RN, Harwood JP, Catt KJ. Gonadotropin-releasing hormone analogue binds to luteal cells and inhibits progesterone production. Nature 282: 90–92, 1979.[CrossRef][Medline]
  13. Conley AJ, Bird IM. The role of cytochrome P450 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the delta 5 and delta 4 pathways of steroidogenesis in mammals. Biol Reprod 56: 789–799, 1997.[CrossRef][ISI][Medline]
  14. Demeter-Arlotto M, Rainey WE, Simpson ER. Maintenance and regulation of 17 alpha-hydroxylase expression by bovine thecal cells in primary culture. Endocrinology 132: 1353–1358, 1993.[Abstract]
  15. Eckstein B, Greenbaum O, Cohen S. Kinetic studies on ovarian C-17,20-lyase activity: effect of luteinizing hormone surge. Endocrinology 117: 2376–2382, 1985.[Abstract]
  16. Eckstein B, Tsafriri A. The steroid C-17,20-lyase complex in isolated Graafian follicles: effects of human chorionic gonadotropin. Endocrinology 118: 1266–1270, 1986.[Abstract]
  17. Ekholm C, Hillensjo T, Isaksson O. Gonadotropin releasing hormone agonists stimulate oocyte meiosis and ovulation in hypophysectomized rats. Endocrinology 108: 2022–2024, 1981.[Abstract]
  18. Erickson GF, Magoffin DA, Dyer CA, Hofeditz C. The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev 6: 371–399, 1985.[CrossRef][ISI][Medline]
  19. Fevold HR, Lorence MC, McCarthy JL, Trant JM, Kagimoto M, Waterman MR, Mason JI. Rat P450(17 alpha) from testis: characterization of a full-length cDNA encoding a unique steroid hydroxylase capable of catalyzing both delta 4- and delta 5-steroid-17,20-lyase reactions. Mol Endocrinol 3: 968–975, 1989.[CrossRef][ISI][Medline]
  20. Findlay JK, Drummond AE, Dyson ML, Baillie AJ, Robertson DM, Ethier JF. Recruitment and development of the follicle; the roles of the transforming growth factor-beta superfamily. Mol Cell Endocrinol 191: 35–43, 2002.[CrossRef][ISI][Medline]
  21. Fournet N, Weitsman SR, Zachow RJ, Magoffin DA. Transforming growth factor-beta inhibits ovarian 17 alpha-hydroxylase activity by a direct noncompetitive mechanism. Endocrinology 137: 166–174, 1996.[Abstract]
  22. Fraser HM, Bramley TA, Miller WR, Sharpe RM. Extra pituitary actions of LHRH analogues in tissues of the human female and investigation of the existence and function of LHRH-like peptides. Prog Clin Biol Res 225: 29–54, 1986.[Medline]
  23. Gelety TJ, Magoffin DA. Ontogeny of steroidogenic enzyme gene expression in ovarian theca-interstitial cells in the rat: regulation by a paracrine theca-differentiating factor prior to achieving luteinizing hormone responsiveness. Biol Reprod 56: 938–945, 1997.[Abstract]
  24. Goubau S, Bond CT, Adelman JP, Misra V, Hynes MF, Schultz GA, Murphy BD. Partial characterization of the gonadotropin-releasing hormone (GnRH) gene transcript in the rat ovary. Endocrinology 130: 3098–3100, 1992.[Abstract]
  25. Guerrero HE, Stein P, Asch RH, de Fried EP, Tesone M. Effect of a gonadotropin-releasing hormone agonist on luteinizing hormone receptors and steroidogenesis in ovarian cells. Fertil Steril 59: 803–808, 1993.[ISI][Medline]
  26. Harwood JP, Clayton RN, Chen TT, Knox G, Catt KJ. Ovarian gonadotropin-releasing hormone receptors. II. Regulation and effects on ovarian development. Endocrinology 107: 414–421, 1980.[ISI][Medline]
  27. Hedin L, Rodgers RJ, Simpson ER, Richards JS. Changes in content of cytochrome P450(17)alpha, cytochrome P450scc, and 3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian follicles and corpora lutea: correlation with theca cell steroidogenesis. Biol Reprod 37: 211–223, 1987.[Abstract]
  28. Hillensjo T, LeMaire WJ, Clark MR, Ahren K. Effect of gonadotrophin-releasing hormone (GnRH) and GnRH agonists upon accumulation of progesterone, cAMP and prostaglandin in isolated preovulatory rat follicles. Acta Endocrinol (Copenh) 101: 603–610, 1982.[Abstract/Free Full Text]
  29. Hsueh AJ, Adashi EY, Jones PB, Welsh TH Jr. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5: 76–127, 1984.[CrossRef][ISI][Medline]
  30. Hsueh AJ, Erickson GF. Extrapituitary action of gonadotropin-releasing hormone: direct inhibition ovarian steroidogenesis. Science 204: 854–855, 1979.[Abstract/Free Full Text]
  31. Hsueh AJ, Jones PB. Extrapituitary actions of gonadotropin-releasing hormone. Endocr Rev 2: 437–461, 1981.[CrossRef][ISI][Medline]
  32. Hsueh AJ, Schaeffer JM. Gonadotropin-releasing hormone as a paracrine hormone and neurotransmitter in extra-pituitary sites. J Steroid Biochem 23: 757–764, 1985.[ISI][Medline]
  33. Imai A, Iida K, Tamaya T. Tight coupling of gonadotropin-releasing hormone receptor to stimulated phosphoinositide turnover and antigonadotropic action in granulosa cells. Gynecol Obstet Invest 33: 36–41, 1992.[CrossRef][ISI][Medline]
  34. Irusta G, Parborell F, Peluffo M, Manna PR, Gonzalez-Calvar SI, Calandra R, Stocco DM, Tesone M. Steroidogenic acute regulatory protein in ovarian follicles of gonadotropin-stimulated rats is regulated by a gonadotropin-releasing hormone agonist. Biol Reprod 68: 1577–1583, 2003.[Abstract/Free Full Text]
  35. Jones PB, Hsueh AJ. Direct effects of gonadotropin releasing hormone and its antagonist upon ovarian functions stimulated by FSH, prolactin, and LH. Biol Reprod 24: 747–759, 1981.[Abstract]
  36. Kaiser UB, Zhao D, Cardona GR, Chin WW. Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor. Biochem Biophys Res Commun 189: 1645–1652, 1992.[CrossRef][ISI][Medline]
  37. Kang SK, Choi KC, Cheng KW, Nathwani PS, Auersperg N, Leung PC. Role of gonadotropin-releasing hormone as an autocrine growth factor in human ovarian surface epithelium. Endocrinology 141: 72–80, 2000.[Abstract/Free Full Text]
  38. Kang SK, Tai CJ, Nathwani PS, Leung PC. Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid in human granulosa-luteal cells. Endocrinology 142: 182–192, 2001.[Abstract/Free Full Text]
  39. Khan I, Sridaran R, Johnson DC, Gibori G. Selective stimulation of luteal androgen biosynthesis by luteinizing hormone: comparison of hormonal regulation of P45017 alpha activity in corpora lutea and follicles. Endocrinology 121: 1312–1319, 1987.[Abstract]
  40. Knetch M, Amsterdam A, Catt KJ. Inhibition of granulosa cell differentiation by gonadotropin-releasing hormone. Endocrinology 110: 865–872, 1982.[ISI][Medline]
  41. Knetck M, Ranta T, Feng P, Sinohara O, Catt KJ. Gonadotropin-releasing hormone as a modulator of ovarian functions. J Steroid Biochem Mol Biol 23: 771–778, 1985.[ISI]
  42. Lapolt PS, Piquette GN, Soto D, Sincich C, Hsueh AJ. Regulation of inhibin subunit messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells. Endocrinology 127: 823–831, 1990.[Abstract]
  43. Maeda K, Kitawaki J, Yokota K, Noguchi T, Urabe M, Yamamoto T, Honjo H. [Effects of gonadotropin-releasing hormone and its analogue (buserelin) on aromatase in cultured human granulosa cells]. Nippon Sanka Fujinka Gakkai Zasshi 48: 89–95, 1996.[Medline]
  44. Magoffin DA, Erickson GF. Dynamics of Ovarian Function, edited by Schwartz NB and Hunzicker-Dunn M. New York: Raven, 1981.
  45. Magoffin DA, Erickson GF. Mechanism by which 17 beta-estradiol inhibits ovarian androgen production in the rat. Endocrinology 108: 962–969, 1981.[Abstract]
  46. Magoffin DA, Erickson GF. Mechanism by which GnRH inhibits androgen synthesis directly in ovarian interstitial cells. Mol Cell Endocrinol 27: 191–198, 1982.[CrossRef][ISI][Medline]
  47. Malozowski S, Cassorla F, Gelato M, Nicoletti M, D'Agata R, Mericq V, Loriaux D. Direct effect of the luteinizing hormone releasing hormone analog D-Trp6-Pro9-Net-LHRH on rat ovarian steroidogenesis. Horm Metab Res 17: 321–322, 1985.[ISI][Medline]
  48. McGee E, Spears N, Minami S, Hsu SY, Chun SY, Billig H, Hsueh AJ. Preantral ovarian follicles in serum-free culture: suppression of apoptosis after activation of the cyclic guanosine 3',5'-monophosphate pathway and stimulation of growth and differentiation by follicle-stimulating hormone. Endocrinology 138: 2417–2424, 1997.[Abstract/Free Full Text]
  49. Munir I, Yen HW, Geller DH, Torbati D, Bierden RM, Weitsman SR, Agarwal SK, Magoffin DA. Insulin augmentation of 17alpha-hydroxylase activity is mediated by phosphatidyl inositol 3-kinase but not extracellular signal-regulated kinase-1/2 in human ovarian theca cells. Endocrinology 145: 175–183, 2004.[Abstract/Free Full Text]
  50. Oikawa M, Dargan C, Ny T, Hsueh AJ. Expression of gonadotropin-releasing hormone and prothymosin-alpha messenger ribonucleic acid in the ovary. Endocrinology 127: 2350–2356, 1990.[Abstract]
  51. Okuda K, Okazaki T, Saeki M, Mori H. The activity of 17alpha-hydroxylase/C17–C20 lyase in the ovaries of immature hypophysectomized rats treated with recombinant FSH combined with various doses of human chorionic gonadotropin. Eur J Endocrinol 137: 530–536, 1997.[Abstract]
  52. Orly J, Rei Z, Greenberg NM, Richards JS. Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids. Endocrinology 134: 2336–2346, 1994.[Abstract]
  53. Parborell F, Dain L, Tesone M. Gonadotropin-releasing hormone agonist affects rat ovarian follicle development by interfering with FSH and growth factors on the prevention of apoptosis. Mol Reprod Dev 60: 241–247, 2001.[CrossRef][ISI][Medline]
  54. Parborell F, Irusta G, Vitale AM, Gonzalez O, Pecci A, Tesone M. GnRH antagonist Antide inhibits apoptosis of preovulatory follicle cells in rat ovary. Biol Reprod 72: 659–666, 2005.[Abstract/Free Full Text]
  55. Parborell F, Pecci A, Gonzalez O, Vitale A, Tesone M. Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by EGF and the expression of Bcl-2-related genes. Biol Reprod 67: 481–486, 2002.[Abstract/Free Full Text]
  56. Parinaud J, Beaur A, Bourreau E, Vieitez G, Pontonnier G. Effect of a luteinizing hormone-releasing hormone agonist (Buserelin) on steroidogenesis of cultured human preovulatory granulosa cells. Fertil Steril 50: 597–602, 1988.[ISI][Medline]
  57. Peng C, Fan N, Ligier M, Vaananen J, Leung PC. Expression and regulation of gonadotropin-releasing hormone (GnRH) receptor messenger ribonucleic acids in human granulosa-luteal cells. Endocrinology 135: 1740–1746, 1994.[Abstract]
  58. Piquette GN, Lapolt PS, Oikawa M, Hsueh AJ. Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells. Endocrinology 128: 2449–2456, 1991.[Abstract]
  59. Ronen-Fuhrmann T, Timberg R, King SR, Hales KH, Hales DB, Stocco DM, Orly J. Spatio-temporal expression patterns of steroidogenic acute regulatory protein (StAR) during follicular development in the rat ovary. Endocrinology 139: 303–315, 1998.[Abstract/Free Full Text]
  60. Sridaran R, Lee MA, Haynes L, Srivastava RK, Ghose M, Sridaran G, Smith CJ. GnRH action on luteal steroidogenesis during pregnancy. Steroids 64: 618–623, 1999.[CrossRef][ISI][Medline]
  61. Sridaran R, Philip GH, Li H, Culty M, Liu Z, Stocco DM, Papadopoulos V. GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy. J Mol Endocrinol 22: 45–54, 1999.[Abstract]
  62. Srivastava RK, Sridaran R. Inhibition of luteal steroidogenesis by two LHRH antagonists (Nal-Glu and Nal-Arg antagonists) in the pregnant rat. Endocr Res 20: 365–376, 1994.[ISI][Medline]
  63. Stojilkovic SS, Reinhart J, Catt KJ. Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15: 462–499, 1994.[CrossRef][ISI][Medline]
  64. Tilly JL, Billig H, Kowalski KI, Hsueh AJ. Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 6: 1942–1950, 1992.[Abstract]
  65. Tilly JL, Kowalski KI, Schomberg DW, Hsueh AJ. Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase. Endocrinology 131: 1670–1676, 1992.[Abstract]
  66. Voss AK, Fortune JE. Levels of messenger ribonucleic acid for cytochrome P450 17 alpha-hydroxylase and P450 aromatase in preovulatory bovine follicles decrease after the luteinizing hormone surge. Endocrinology 132: 2239–2245, 1993.[Abstract]
  67. Wanderley MI, Negro-Vilar A. Pretreatment with phorbol ester and an LHRH agonist reduces testosterone production and protein kinase C activity in rat Leydig cells challenged with PDBu and LHRH. Braz J Med Biol Res 29: 1557–1565, 1996.[ISI][Medline]
  68. Weiner KX, Dias JA. Regulation of ovarian ornithine decarboxylase activity and its mRNA by gonadotropins in the immature rat. Mol Cell Endocrinol 92: 195–199, 1993.[CrossRef][ISI][Medline]
  69. Woodruff TK, Lyon RJ, Hansen SE, Rice GC, Mather JP. Inhibin and activin locally regulate rat ovarian folliculogenesis. Endocrinology 127: 3196–3205, 1990.[Abstract]
  70. Woodruff TK, Mather JP. Inhibin, activin and the female reproductive axis. Annu Rev Physiol 57: 219–244, 1995.[CrossRef][ISI][Medline]
  71. Yang H, Bhat GK, Wadley R, Wright KL, Chung BM, Whittaker JA, Dharmarajan AM, Sridaran R. Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat. Biol Reprod 68: 2222–2231, 2003.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Irusta, G.
Right arrow Articles by Tesone, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Irusta, G.
Right arrow Articles by Tesone, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.