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Mechanisms of Hormone Action |
Department of Physiology3 and Neuroscience Institute
Department of Anatomy and Neurobiology,4 Morehouse School of Medicine, Atlanta, Georgia 30310-1495
School of Anatomy and Human Biology and the Western Australian Institute of Medical Research,5 University of Western Australia, Nedlands, Perth, Western Australia 6907, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, corpus luteum, gonadotropin-releasing hormone, nitric oxide, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nitric oxide (NO), a highly reactive free radical molecule generated in a biological system, is synthesized from L-arginine by nitric oxide synthase (NOS) isozymes [11]. To date, three different isoforms of NOS have been characterized, two of which were first identified in the endothelium (eNOS) and brain (nNOS) as constitutively expressed isoforms [12]. An inducible isoform (iNOS) has been found in many cells and is correlated with cytostatic and cytotoxic events [13]. Nitric oxide is now recognized as an important intracellular and intercellular messenger molecule known to have diverse physiological and pathological roles in multiple systems [14]. Recently, NOS isozymes have been found in follicles and CL of human [15], mouse [16], and rabbit [17]. Nitric oxide has been known to be associated with a variety of female reproductive functions that include follicular development [18, 19], ovulation [20, 21], and oocyte meiotic maturation [22]. Specifically, NO has emerged as a potential regulator of ovarian function suppressing the production of estradiol and progesterone in the follicle and corpora lutea [15, 23].
The inhibitory effects of GnRH and NO on steroidogenesis in the ovary lead us to postulate that NO might act as a messenger molecule for GnRH to suppress progesterone production in the CL of the pregnant rat. Therefore, the objectives of this study were 1) to investigate the correlative changes occurring between the luteal concentrations of NO and NO synthases and the serum progesterone and pregnenolone concentrations after in vivo GnRH-Ag treatment and 2) to confirm that the suppressive effects of GnRH-Ag on luteal NO synthesis and steroidogenesis are directly on the CL utilizing an in vitro model system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Timed-pregnant Holtzman Sprague-Dawley rats were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). They were housed at the animal facilities of Morehouse School of Medicine in a temperature of 23–25°C and exposed to a daily photoperiod of 14L:10D (lights on at 0500 h and off at 1900 h) as previously described [8]. The day of insemination, identified by a sperm plug, was designated as Day 1 of pregnancy. Animal protocols were approved by our Institutional Animal Care and Use Committee, and studies were conducted in accordance with the principles and procedures of the NIH guide for the care and use of laboratory animals.
GnRH Agonist Treatment
GnRH-Ag ([pyro]-Glu-His-Trp-Ser-Tyr-D-Trp-NmeLeu-Arg-Pro-ethylamide-luteinizing hormone releasing hormone; Wyeth-40972) was a gift from Wyeth-Ayerst Laboratories (Philadelphia, PA). GnRH-Ag was administered continuously at a delivery rate of 5 µg/24 h using osmotic minipumps (Model 2001; Alza, Palo Alto, CA) starting on the morning of Day 8 of pregnancy, and sham-operated control rats received no treatment as described by us previously [8, 9]. Briefly, each rat was implanted s.c. on the dorsal surface of the neck with an osmotic minipump under Metofane anesthesia. Rats were killed at 0, 4, 8, and 24 h after the commencement of treatment. Prior to killing, under anesthesia, blood from the jugular vein was obtained for the measurement of progesterone and pregnenolone. Serum was separated from the blood and stored at -20°C. At autopsy, ovaries were removed and fixed in 4% paraformaldehyde for immunohistochemisty. CL were separated from the ovaries and snap frozen in liquid nitrogen and stored at -70°C for nitric oxide assay and Western blot.
Luteal Cell Separation and Culture
To determine if the suppressive effects of GnRH-Ag are directly on the CL, ovaries were removed on Day 8 of pregnancy while the rats were under Metofane anesthesia. CL were separated, cleaned of any adhering follicle, and placed in Medium 199. Luteal cells were obtained by dissociating the pooled CL as previously described [24, 25]. Luteal cells (0.5 million/well) were plated on a 24-well culture plate (Becton Dickinson Labware, Lincoln Park, NJ). The luteal cells were precultured to adhere on the plate for 90 min, and medium was not replaced. Media and cells were collected at 4, 8, 12, and 24 h after the treatment of GnRH-Ag (10-6 M). It should be noted that an additional time point at 12 h after the GnRH-Ag treatment was added in the in vitro study to determine the effects of GnRH-Ag treatment between 8 and 24 h. Media were stored at -70°C for nitric oxide and progesterone assays, and cells were stored at -70°C for Western blotting and fixed in 4% paraformaldehyde solution for immunohistochemisty. The experiment was repeated at least twice with two to three replicates per point in each experiment.
Progesterone and Pregnenolone Assay
Progesterone concentrations in the serum and media were measured using a radioimmunoassay kit obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX). The sensitivity of the kit was 0.12 ng/ml. The coefficients of variation between and within assays were 7.2% and 5.2%, respectively. Pregnenolone concentrations in the serum and media were measured using a specific antibody prepared against pregnenolone-3-monohemisuccinate: HAS (catalog no. 07-172016, ICN Biomedicals, Inc., Costa Mesa, CA). The antisera were previously titered with a chromatographically pure isotope (pregnenolone 3H, ICN Biochemicals, Inc.) to bind between 40% and 50%. This has shown <0.03% cross-reactivity with other steroids. The sensitivity of the standard curve was 10–25 pg/ml. The serum samples were purified prior to the assay by extraction with hexane. The coefficients of variation between and within assays were 8.4% and 5.7%, respectively. Concentrations of progesterone and pregnenolone secreted by the cultured luteal cells were normalized for the amount of total protein, estimated by Lowry's method, present in the cells and expressed as concentrations per milligram of protein.
Nitric Oxide Assay
The final products of NO in vivo are nitrite and nitrate, and the best index of the total NO is the sum of both nitrite and nitrate. Nitric oxide concentrations in the CL and cultured media were assessed by measuring the concentration of nitrate and nitrite using a nitrate/nitrite colorimetric assay kit (Alexis Biochemicals, San Diego, CA). CL were homogenized in PBS and centrifuged. Supernatant and media collected from cultures were ultrafiltered using a 10-kDa molecular mass cutoff filter (Amicon, Millipore Co., Bedford, MA). The filtrate was incubated with nitrate reductase mixture and enzyme cofactor mixture. After adding sulfanilamide and N-[1-naphthyl] ethylenediamine, the absorbance was measured at 540 nm using the microplate reader (Spectra Max 250, Molecular Devices, Sunnyvale, CA). Concentrations of NO in the CL and cultured luteal cells were normalized for the amount of total protein present in the CL and cultured cells and expressed as concentrations per milligram of protein.
Immunohistochemisty for eNOS, iNOS, and nNOS
Ovaries fixed in 4% paraformaldehyde were dehydrated and embedded in paraffin wax. Sections were cut to 5 µm and deparaffinized by xylene, and then sections were rehydrated through series of alcohol solutions. For eNOS and iNOS staining, sections were first boiled in citric acid solution (10 mM citrate, pH 6.0) for 1 min using microwave and immersed in 3% hydrogen peroxide solution for 5 min. Sections were preincubated with goat normal serum for 30 min at room temperature and then reacted with 1:200 rabbit polyclonal anti-rat eNOS, iNOS, or nNOS (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. After washing in PBS, sections were incubated with biotinylated anti-rabbit IgG (ABC kit; Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature and washed. Then the sections were incubated with avidin biotin-peroxidase complex (ABC kit; Vector Laboratories) for 30 min. The sites of peroxidase complex were visualized by DAB solution (DAB kit; Vector Laboratories). The nuclei were counterstained with hematoxylin. For negative controls, primary antibodies were replaced with the PBS during the reaction. For immunofluorescence staining of eNOS and iNOS in cultured luteal cells, the luteal cells fixed with 1% paraformaldehyde were incubated with rabbit polyclonal anti-rat eNOS or iNOS for 1 h at room temperature. After washing in PBS, the luteal cells were reacted with FITC conjugated anti-rabbit IgG (Santa Cruz Biotechnology). We observed the cells stained with FITC under confocal microscope (Olympus, Japan).
Western Blot Analyses for eNOS, iNOS, and nNOS
Frozen CL or the luteal cells were homogenized in ice-cold homogenization buffer containing 50 mM Tris-base (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.1% Tween-20, and protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). The homogenates were centrifuged at 12 000 x g for 30 min at 4°C. The protein concentration in the supernatant was determined by the DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of proteins (20 µg) were resolved by 7% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then incubated in a blocking solution of 5% nonfat dry milk in Tris-buffered saline (TTBS) containing 10 mM Tris (pH 7.6), 150 mM NaCl, and 0.1% Tween-20 for 1 h at room temperature. Then the membranes were incubated with rabbit polyclonal anti-rat eNOS, iNOS, or nNOS antibodies (Santa Cruz Biotechnology) diluted to 1:1000 in blocking solution overnight at 4°C. Following incubation, the membranes were washed in TTBS and incubated with anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology) diluted to 1:1000 in blocking solution for 1 h at room temperature. After washing the membranes in TTBS for 30 min, the membrane was treated with enhanced chemiluminescence solution (ECL kit; Amersham Life Science, Buckinghamshire, England) for 1 min and exposed to x-ray film (Hyperfilm, Amersham Life Science). To monitor the amount of protein loaded into each lane, the membranes were reprobed with an antibody against actin. After stripping the membranes with a stripping buffer (0.1 M glycine, pH 2.5), they were incubated with 1:5000 dilution of mouse monoclonal anti-actin antibody (Sigma, St. Louis, MO) at room temperature for 1 h. Subsequently, the membranes were incubated with secondary antibody and processed as described previously. Films were developed immediately, and the protein bands were analyzed densitometrically.
Statistical Analyses
The data on production of progesterone, pregnenolone, and NO were collected from three to four rats in the in vivo study and four to six replicates per point in the in vitro study. Values concerning Western blots of NOS were expressed as a percentage of 0-h controls (rats treated with no surgery). All data were presented as means ± SEM. The results on progesterone, pregnenolone, and NO were analyzed using ANOVA followed by Fisher protected least significant difference test. Wilcoxon Mann-Whitney equal variance and unequal variance t-tests were used to analyze changes in the amount of eNOS and iNOS proteins [26]. A P-value less than 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The administration of GnRH-Ag caused a decrease in serum concentrations of progesterone compared with those of the sham control. Although there was no change in the progesterone at 4 h, it decreased dramatically 8 h after the administration of GnRH-Ag showing a significant difference between the sham control and the GnRH-Ag treatment. This decrease in serum concentrations of progesterone was more pronounced at 24 h after the treatment (Fig. 1A) as has been demonstrated by us previously [7–10]. In addition, pregnenolone concentrations were decreased in the GnRH-Ag-treated group compared to the respective sham controls at 8 and 24 h after the treatment (Fig. 1B).
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Effects of GnRH-Ag Treatment on the Production of NO in the CL
Similar to progesterone and pregnenolone, luteal concentrations of NO were also significantly decreased relative to sham controls at 8 and 24 h after the administration of GnRH-Ag, respectively (Fig. 2).
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Effects of GnRH-Ag Treatment on the Production of eNOS and iNOS Proteins in the CL
To determine whether the decrease in luteal production of NO by GnRH-Ag treatment was due to an inhibition of NOS proteins, we performed immunohistochemical staining and Western blot analyses of eNOS and iNOS in the CL. As shown in Figure 3, immunohistochemical staining indicated that eNOS and iNOS were present in the CL of pregnant rats on Day 8. Staining of eNOS and iNOS was more intense in the CL sham controls (Fig. 3, c and g) than in those of the GnRH-Ag-treated groups (Fig. 3, d and h) 8 h after the administration of GnRH-Ag. Negative control sections showed no immunostaining (Fig. 3, a and e). However, this was only a qualitative analysis. Using the Western blot analyses, we examined quantitative changes that occurred in eNOS and iNOS protein content of the CL. Figure 4 (top) and Figure 5 (top) illustrate the Western blots of the CL at different periods after GnRH-Ag treatment. Western blots of eNOS and iNOS were repeated three times, respectively, and subsequently analyzed using densitometry. As shown in Figures 4 (bottom) and 5 (bottom), relative intensity of eNOS and iNOS decreased significantly by about 20% in the GnRH-Ag-treated group compared to its sham control group at 4 and 8 h.
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Effects of GnRH-Ag Treatment on the Production of nNOS Protein in the CL
Figure 6 shows the results of immunohistochemical staining and Western blot of nNOS in the CL. Staining intensity of nNOS in the CL was more than those of eNOS and iNOS. In addition, we observed that nNOS immunostaining was localized in cytoplasm (Fig. 6, A and b), whereas negative control sections show no specific staining (Fig. 6, A and a). As shown in the figure, expression of nNOS in the CL is similar in the sham control and the GnRH-Ag treatment groups throughout the experiment. Using the Western blot analyses (Fig. 6B), we examined quantitatively changes that occurred in nNOS protein content of the CL. The content of nNOS protein in the CL at different periods after GnRH-Ag treatment was not found to be significantly different from the respective sham control groups (data not shown).
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Effects of GnRH-Ag on Progesterone and NO Production in the Cultured Luteal Cells
GnRH-Ag decreased the production of progesterone and pregnenolone by cultured luteal cells compared with those of the saline control. Although their concentrations did not change 4 h after the GnRH-Ag treatment similar to in vivo study, it decreased significantly at 8 and 12 h after the treatment. However, their concentrations recovered to those of the saline control 24 h after the treatment (Fig. 7). Corresponding to progesterone and pregnenolone concentrations, NO produced by the cultured luteal cells also decreased significantly 8 and 12 h after the treatment compared with the saline control and recovered at 24 h (Fig. 8).
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Effects of GnRH-Ag on the Expression of eNOS and iNOS in the Cultured Luteal Cells
To confirm whether eNOS and iNOS proteins were expressed in the cultured luteal cells as well as to determine if their expression is regulated by GnRH-Ag treatment in vitro as well as in vivo, we performed immunofluorescence staining using confocal microscopy and Western blot of eNOS and iNOS in the cultured luteal cells. Confocal images of eNOS and iNOS after immunofluorescence staining (Figs. 9A and 10A, respectively) show the presence of eNOS and iNOS in the cultured luteal cells. In Western blot analyses, eNOS and iNOS proteins decreased significantly in the GnRH-Ag-treated group compared with its saline control group at 4 and 8 h and then recovered at 12 and 24 h after the GnRH-Ag treatment (Figs. 9C and 10C, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dong et al. [29] previously reported that diethylenetriamine (EDTA)/NO, a NO donor, caused a dose-dependent increase in progesterone synthesis in the rat ovary. This observation supports that a decrease in serum concentrations of progesterone and pregnenolone could be due to a reduction in the luteal production of NO triggered by the administration of GnRH-Ag as has been demonstrated in this study.
We have reported that suppression of progesterone production in cultured luteal cells by GnRH-Ag could be mediated via a decrease in luteal PBR and StAR resulting from reduction of NO synthesis [30]. In addition, we have also demonstrated the presence of PBR and StAR in the pregnant CL and that the coordinated suppression of these proteins by GnRH-Ag lead to reduced ovarian steroidogenesis [9]. PBR protein is known to exist in mitochondria and is implicated in regulating the movement of cholesterol across the mitochondrial membrane [31]. StAR also is known to be necessary for mobilization of cholesterol into the outer/inner contact sites of mitochondrial membranes resulting in increased steroid formation [32, 33]. Based on these results, a decrease in progesterone and pregnenolone production by GnRH-Ag may result from diminished bioactivity or production of PBR and StAR involved in the pathway mediating the conversion of cholesterol to progesterone. This reduction of PBR and StAR production could be due to NO, which might modulate the production of PBR and StAR as a messenger molecule for GnRH action in the CL. However, additional studies are necessary to substantiate the regulatory role that NO might play in the production of PBR and/or StAR.
Interestingly, our data also demonstrated, for the first time, the presence of nNOS protein in the CL of pregnant rats. Among NOS isoforms, the expression of eNOS and iNOS in the rat ovary has been known, but there has been no previous report indicating the nNOS expression in the CL of the pregnant rat [34, 35]. Recently, Srivastava et al. [36] reported the presence of nNOS in the prepubertal rat ovary and measured the amount of nNOS protein in the ovary by Western blotting. This observation supports our present data demonstrating the expression of nNOS in the CL of pregnant rats, thus suggesting that nNOS may be an important factor in the CL during pregnancy in the rat. However, the data from this study failed to demonstrate that nNOS synthesis was affected by GnRH-Ag treatment.
Nitric oxide is known to play a role not only as a modulator of steroidogenesis but also as a survival (antiapoptotic) factor in ovary [37]. However, the role of NO in apoptosis in the ovary has been controversial until now [38]. High concentrations of NO inhibit DNA fragmentation in the cells, whereas low concentrations stimulate cellular apoptosis only in granulosa cells from bovine follicles [39]. These studies suggest that the concentration of intracellular NO could be a critical factor in cell survival and function. In cultured granulosa cells, the addition of S-nitroso-N-acetyl-DL-penicillamine, a NO generator, directly inhibits spontaneously occurring apoptosis [40]. Chun et al. [41] also demonstrated that in antral follicles treated with the NO generator, sodium nitroprusside, apoptosis was suppressed as effectively as that induced by FSH. These results suggest that NO plays an important role as a survival factor preventing apoptosis in the ovary. Based on the present results showing that GnRH-Ag caused a decrease in NO concentrations in the luteal cells and the results from our previous study displaying that administration of GnRH-Ag increased the rate of DNA degradation in the CL [8], we propose that an increase in apoptosis in the CL by GnRH-Ag administration may be due to a decrease in intraluteal concentrations of NO.
In conclusion, we have demonstrated that GnRH-Ag directly suppresses NO synthesis by luteal cells leading to a decrease in the production of progesterone and pregnenolone by the CL in the pregnant rat. These results suggest that GnRH-Ag could directly suppress steroidogenesis and induce apoptosis in the CL via NO and that GnRH may be involved in luteolysis of the CL in pregnant rats as an important regulator.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Rajagopala Sridaran, Department of Physiology, Morehouse School of Medicine, 720 Westview Dr., S.W., Atlanta, GA 30310-1495. FAX: 404 752 1045; sridaran{at}msm.edu
Received: 21 September 2002.
First decision: 10 October 2002.
Accepted: 21 January 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In: Program of the 11th International Congress of Endocrinology; 2000; Sydney, Australia. Abstract P1218 This article has been cited by other articles:
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  A. Sengupta, T. Baker, N. Chakrabarti, J. A. Whittaker, and R. Sridaran Localization of Immunoreactive Gonadotropin-releasing Hormone and Relative Expression of Its mRNA in the Oviduct During Pregnancy in Rats J. Histochem. Cytochem., May 1, 2007; 55(5): 525 - 534. [Abstract] [Full Text] [PDF]
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 G. Irusta, F. Parborell, and M. Tesone Inhibition of cytochrome P-450 C17 enzyme by a GnRH agonist in ovarian follicles from gonadotropin-stimulated rats Am J Physiol Endocrinol Metab, May 1, 2007; 292(5): E1456 - E1464. [Abstract] [Full Text] [PDF]
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 M Casais, S M Delgado, Z Sosa, and A M Rastrilla Pregnancy in rats is modulated by ganglionic cholinergic action. Reproduction, June 1, 2006; 131(6): 1151 - 1158. [Abstract] [Full Text] [PDF]
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