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Regular article |
a Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
b Investigative Treatment Division, National Cancer Center Research Institute East, Kashiwa, Japan
c Department of Histology and Cell Biology, Nagasaki University School of Medicine, Nagasaki, Japan
ABSTRACT
Our own recent studies have demonstrated that inducible nitric oxide synthase (iNOS) is predominantly localized in granulosa cells of healthy immature follicles in the rat ovary, whereas granulosa cells of either healthy mature follicles or follicles destined to be atretic are devoid of iNOS. These findings suggest that iNOS is pivotal for immature follicles to remain dormant. To test this hypothesis, we examined the effects of a GnRH agonist (buserelin), a proapoptotic substance, and epidermal growth factor (EGF), a mitogenic and, consequently, antiapoptotic factor, on the amount of iNOS mRNA in rat granulosa cells. Administration of buserelin in immature female rats transiently diminished iNOS mRNA levels in the ovaries as determined by Northern blot analysis. In cultured rat granulosa cells, buserelin and EGF increased the incidence of apoptosis and DNA synthesis, respectively, whereas both reduced iNOS mRNA levels as determined by reverse transcription-coupled polymerase chain reaction. The concomitant addition of S-nitroso-N-acetyl-DL-penicillamine, an NO donor, together with buserelin or EGF eliminated the observed effects of these substances (i.e., induction of apoptosis and stimulation of DNA synthesis, respectively). These results suggest that the changes in developmental status of immature follicles either into development or atresia are associated with reduced iNOS levels in granulosa cells, thus reinforcing the notion of NO as a cytostatic factor in ovarian follicles.
apoptosis, follicle, follicular development, GnRH, granulosa cells, growth factors, hypothalamic hormones, neuropeptides/neurotransmitters, nitric oxide
INTRODUCTION
In the ovary, gonadotropin stimulates development of a set of follicles, most of which undergo atretic degeneration through apoptosis of granulosa cells and oocytes, whereas the remaining follicles, which evade atresia, are destined to ovulate [1]. Follicular development is considered to be regulated by various factors such as cytokines, growth factors, and locally produced hormones. Recently, nitric oxide has also emerged as a potential regulator of follicular development because of its involvement in the regulation of several physiological functions of the ovary, including ovulation and steroidogenesis [2–19].
In our own recent studies, inducible nitric oxide synthase (iNOS) has been predominantly localized in the granulosa cells of most immature follicles, whereas granulosa cells from either mature, healthy follicles or follicles destined to be atretic are devoid of iNOS [15, 16]. In addition, the iNOS mRNA level in immature rat ovaries transiently decreased after the administration of gonadotropin [15]. These findings lead us to postulate that the presence of iNOS is a prerequisite for immature follicles to stay quiescent, whereas the absence of iNOS might set them in motion toward either developed (i.e., growing) follicles or apoptotic (i.e., atretic) follicles.
In this study, we attempted to reinforce our hypothesis regarding iNOS as a cytostatic factor for immature follicles by looking at the effects of agents that drive immature follicles into different developmental stages in relation to changes in the iNOS mRNA level. More specifically, we employed two agents. The first, epidermal growth factor (EGF), is a mitogenic and antiapoptotic factor for granulosa cells and is supposed to be involved in follicular development, presumably, in part, by suppressing granulosa cell apoptosis [1, 20, 21]. The second, buserelin, is a GnRH agonist and is known to induce apoptosis and inhibit cell proliferation in granulosa cells [1, 21]. Here, we demonstrate that EGF and buserelin, which produce cell proliferation and apoptotic changes in granulosa cells, respectively, concomitantly reduce the level of iNOS mRNA, thus providing further support for the notion that iNOS in immature follicles may keep them in a quiescent state.
MATERIALS AND METHODS
Chemicals
The GnRH agonist [D-Ser(t-Bu)6, des-Gly10-ethylamide] GnRH (buserelin) was synthesized and supplied by Hoechst (Frankfurt, Germany). DMEM-F12 medium and fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY). All other chemicals, unless otherwise mentioned, were obtained from Sigma Chemical Co. (St. Louis, MO).
Animal Treatment
Guidelines for the care and use of animals as approved by the local institution were followed. Twenty-five-day-old, immature, female Wistar rats weighing 50–60 g were purchased from Charles River Japan, Inc. (Yokohama, Japan) and housed in a temperature-controlled room with a 12L:12D schedule. Pelleted food and water were provided ad libitum. On the 26th day of age, five rats were killed as controls (time, 0 h). To induce apoptosis of granulosa cells in the follicles, rats were injected intraperitoneally with 100 µg of buserelin, which was dissolved in 0.2 ml of saline containing 0.1% dimethyl sulfoxide (DMSO), and were killed 6, 12, 24, and 48 h later by cervical dislocation. Similarly, rats that were treated with 0.2-ml injection vehicle alone instead of buserelin were killed 6, 12, 24, and 48 h later. At each time point, six rats were killed. Removed ovaries were immediately cleaned of surrounding connective tissues as described by Hakuno et al. [22]. Ovaries were snap-frozen in liquid nitrogen and stored at -80°C until used for Northern blot analysis.
RNA Isolation and Northern Blot Analysis
Northern blot analysis of iNOS mRNA expression was performed using 15 µg of total RNA and essentially based on the method described by Chomczynsky and Sacchi [23–25]. The total RNA was extracted by the guanidine thiocyanate method [23–25]. Fifteen µg of total RNA were separated by electrophoresis on a 1% agarose gel containing 6% formaldehyde and transferred to a Hybond-N membrane (Amersham, Little Chalfont, UK). Prehybridization and hybridization were performed as described by Ogura et al. [25]. A 700-base pair (bp) fragment of the 5' portion of cloned rat liver iNOS cDNA was labeled with [
-32P] dCTP (3000 Ci mmol-1) using a random prime labeling kit (Amersham). The membranes were washed with 2x standard saline citrate (SSC; 1x SSC = 0.15 M NaCl, 0.015 M sodium citrate) and 0.1% SDS containing 0.2% sodium pyrophosphate at 58°C four times for 30 min and then exposed to an imaging plate (Fuji Photo Film Co., Kanagawa, Japan). The relative intensities of Northern blot hybridization signals were determined using a Fujix BAS 2000 Bio-Image Analyzer (Fuji Photo Film Co.). To normalize iNOS mRNA levels based on ß-actin mRNA level, the filter was rehybridized with a 32P-labeled cDNA probe of cloned rat ß-actin [24, 25]. The iNOS mRNA levels were then compared among the treatment groups after normalization by ß-actin mRNA content.
Preparation and Culture of Granulosa Cells
On the 26th day of age, rats were injected intraperitoneally with 10 IU of pregnant mare serum gonadotropin (PMSG; Teikokuzoki, Tokyo, Japan) in 0.2 ml of saline. Forty-eight hours after PMSG administration, granulosa cells were collected from the ovaries as described by Hakuno et al. [22]. Granulosa cells were suspended in DMEM-F12 medium supplemented with 10% FBS, unless otherwise stated, and cultured in a humidified atmosphere of 5% CO2 in 95% air at 37°C in all granulosa cell-culture experiments.
Reverse Transcription-coupled Polymerase Chain Reaction
Granulosa cells were seeded onto 10-cm plastic culture dishes (Beckton Dickinson Co., Lincoln Park, NJ) at a density of 106 cells/dish and cultured. After 24 h, the medium was replaced with fresh medium containing buserelin or human recombinant EGF (Wakunaga, Hiroshima, Japan). Buserelin was dissolved in DMSO and diluted with the medium. The final concentration of DMSO never exceeded 0.05%. After an additional 6, 12, and 24 h incubation, granulosa cells were harvested for RNA extraction. Reverse transcription-coupled polymerase chain reaction (RT-PCR) of RNA samples was performed essentially as described by Fujisawa et al. [24] and Ogura et al. [25]. First-strand cDNA was synthesized in a reaction volume of 15 µl containing 5 µg of total RNA and 0.2 µg of random hexamer primers using a commercial kit according to the manufacturer's instructions (Pharmacia Biotech, Uppsala, Sweden). For the detection of iNOS and G3PDH mRNAs, PCR amplification was performed with the following oligonucleotide primers: iNOS, 5'-TCCAACCTGCAGGTCTTCGATGC-3' (sense), and 5'-GGACCAGCCAAATCCAGTCTGC-3' (antisense); and G3PDH, 5'-ATCCGCAAAGACCTGTACGC-3' (sense), and 5'-TGTGTGGACTTGGGAGAGGA-3' (antisense). Denaturation, annealing, and elongation in the PCR reaction were performed at 94, 57, and 72° for 30 sec, 1 min, and 2 min, respectively, for 40 cycles in the case of iNOS and for 25 cycles in the case of G3PDH. The amplified DNA fragments were separated on a 3% agarose gel and then visualized by ethidium bromide staining. Densitometry was used to quantify the relative signal densities. The iNOS mRNA levels were compared among the treatment groups after normalization by G3PDH mRNA content.
[3H]Thymidine Incorporation Assay
Granulosa cells were seeded into Costar 24-well plates at a density of 4 x 104 cells/well and then cultured. After 24 h, the culture medium was replaced with fresh medium supplemented with 0.1% FBS. After an additional 24 h, the medium was further replaced with fresh medium containing various concentrations of EGF and S-nitroso-N-acetyl-DL-penicillamine (SNAP), an NO donor. At the same time, 1 µCi of [methyl-3H]thymidine (Amersham) was concomitantly added per well. The medium was supplemented with 0.1% FBS when EGF and SNAP were added and with 10% FBS when only SNAP was added. After incubation for 24 h, the cells were washed three times with PBS and four times with 5% trichloroacetic acid at 4°C. The precipitate was solubilized in 0.1 N NaOH with 0.1% SDS, and the radioactivity was measured in a liquid scintillation counter.
Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling
Granulosa cells were seeded into Costar 24-well plates at a density of 4 x 104 cells/well and then cultured. After 24 h, the medium was replaced with the medium containing 10-6 M buserelin, with or without 0.5 mM SNAP. After a further 24 h, the cells were collected by trypsin treatment, placed on silane-coated glass slides, and fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. To analyze DNA fragmentation histochemically, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was performed using an Apop Tag kit according to the manufacturer's instructions (Intergen Co., Purchase, NY). Briefly, after washing in PBS three times for 5 min each, the slides were immersed in 3% (v/v) H2O2 in PBS for 5 min at room temperature to inactivate the endogenous peroxidase. The slides were next rinsed twice with PBS and covered with equilibration buffer alone at room temperature for 30 sec. Then, the buffer containing terminal deoxynucleotidyl transferase, digoxigenin-dUTP, and dATP was added onto the slides to catalyze digoxigenin-dUTP and dATP to 3'-ends of fragmented DNA. The slides were incubated in a humidified atmosphere at 37°C for 1 h. To terminate the reaction, the slides were transferred to the stopping buffer for 10 min at room temperature and then rinsed with PBS. After incubation with antidigoxigenin peroxidase conjugate for 30 min at room temperature, the slides were washed in PBS, and the sites of peroxidase were visualized by the addition of 3,3'-diaminobenzidine substrate. The frequency of TUNEL-positive cells was calculated by counting the number of stained cells per more than 200 cells.
Statistical Analysis
Data are presented as the mean ± SEM from at least quadruplicate experiments. The statistical analysis was performed by Mann-Whitney rank sum test.
RESULTS
Effect of GnRH Agonist Administration on iNOS Expression in the Rat Ovary
Using Northern blot analysis, we examined the changes of iNOS mRNA levels in the whole ovaries after buserelin administration. Figure 1 illustrates the representative result of Northern blot analysis, demonstrating that iNOS mRNA was constitutively expressed in the ovary from immature rats. Six hours after buserelin administration, iNOS mRNA levels decreased to undetectable levels and, thereafter, increased gradually to 40% of the untreated levels by 48 h after buserelin administration. In the untreated control group, iNOS mRNA levels remained unchanged throughout the experimental period (data not shown).
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Effects of EGF and GnRH Agonist Treatment on iNOS Expression in Cultured Rat Granulosa Cells
The effects of EGF and buserelin on in vitro iNOS mRNA levels were investigated by RT-PCR in cultured granulosa cells. As shown in Figure 2, RT-PCR demonstrated that a 700-bp DNA fragment from untreated granulosa cells was amplified. The PCR product was sequenced and confirmed to be identical to the sequence of iNOS obtained from rat liver [26]. Interestingly, both 100 ng/ml EGF and 10-5 M buserelin decreased the iNOS mRNA level in granulosa cells with time in culture of up to 24 h, whereas iNOS mRNA levels marginally decreased in the control culture. At 24 h of treatment, the amount of iNOS mRNA was dose-dependently decreased by EGF at 1–100 ng/ml and by buserelin at 10-7–10-5 M.
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Effect of SNAP on Incorporation of [3H]Thymidine by Cultured Rat Granulosa Cells
The effect of SNAP, an NO donor, on DNA synthesis was examined in cultured granulosa cells. As shown in Figure 3, SNAP at 0.05, 0.2, and 0.5 mM produced a dose-dependent inhibition of [3H]thymidine incorporation into DNA. At 0.5 mM, SNAP caused a 90% inhibition in DNA synthesis. As shown in Figure 4, EGF at 10 ng/ml induced a 144.0 ± 3.7% (P < 0.001 vs. control) increase in [3H]thymidine incorporation into DNA. Addition of SNAP (0.5 mM) with EGF (10 ng/ml) resulted in a precipitous decline in DNA synthesis that was, by far, smaller relative to the control.
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Effect of SNAP on Apoptosis of Cultured Rat Granulosa Cells
The effect of SNAP on the incidence of apoptotic cells as determined by TUNEL-positive nuclei in cultured granulosa cells was also examined (Fig. 5). The in vitro induction of granulosa cell apoptosis by 10-6 M buserelin (Fig. 5, B and D) was markedly inhibited by the addition of 0.5 mM SNAP (Fig. 5C). As shown in Figure 6, the percentage of apoptotic cells was significantly increased by the addition of 10-6 M buserelin (71.5 ± 1.7 %; P < 0.001 vs. control) compared with that in the control (3.5 ± 0.6 %), and this increment was suppressed by concurrent supplement of 0.5 mM SNAP (16.0 ± 4.2 %; P < 0.001 vs. buserelin alone).
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DISCUSSION
In the present study, we demonstrated that iNOS mRNA levels in rat ovaries significantly decreased with administration of the GnRH agonist buserelin, an atretogenic agent for ovarian follicles [1, 21]. In addition, both EGF, a mitogen and prodevelopmental substance for granulosa cells [1, 20, 21], and buserelin decreased iNOS mRNA levels in cultured rat granulosa cells. Along with our recent findings that iNOS mRNA and its protein are predominantly localized in the granulosa cells in immature follicles, but not in mature follicles with an antrum or atretic follicles [15, 16], the present data further strengthen our hypothesis that NO produced by iNOS in immature follicles acts as a cytostatic factor [16].
The present study also revealed that EGF and buserelin lowered iNOS mRNA levels in cultured granulosa cells in the absence of gonadotropin. In our own recent study, however, administration of gonadotropin in immature rats was associated with a marked reduction in iNOS mRNA levels in ovaries [15], which is consistent with the finding by Van Voorhis et al. [6] that iNOS mRNA levels were maximum in unstimulated, immature rat ovaries and reduced 48 h after gonadotropin administration. At present, we cannot exclude the likelihood that gonadotropin may stimulate production of endogenous EGF or GnRH-like substance locally, both or either of which, in turn, could reduce iNOS expression.
It could be questioned whether EGF and buserelin exerted their biological effects (i.e., stimulation of cell proliferation and induction of apoptosis, respectively) via a reduction of iNOS mRNA (i.e., a loss of NO) or another, as-yet-unidentified mechanisms unrelated to NO. Here, we demonstrated that SNAP, an NO donor, attenuated both EGF-stimulated DNA synthesis and buserelin-induced apoptosis in cultured granulosa cells. These findings suggest a direct implication of NO in regulating the developmental status of immature follicles.
In our own recent study [16], SNAP directly inhibited spontaneously occurring apoptosis in cultured granulosa cells, which is in keeping with the data reported by Chun et al. [7]. So far, several studies have dealt with the link between NO and apoptosis in a diverse range of tissues and cells [15, 16], documenting both pro- and antiapoptotic effects of NO. Higher concentrations of NO, which generally are observed in pathological settings, may induce apoptosis [27]. In light of our recent observation of an inverse relationship between the incidence of apoptosis and iNOS expression in rat ovarian follicles [16], NO may play a role as an antiapoptotic factor among immature follicles in physiological paradigm.
There is an extensive literature on the possible roles of NO in regulation of cell proliferation and the cell cycle [28–32]. For instance, NO arrests the neuronal cell division required for terminal differentiation [28], and it acts as an antiproliferative agent during Drosophila development [29]. Furthermore, NO has been shown to block the cell cycle at the G2+M phase in mouse macrophage-like cells [30]. In this connection, recent studies have shown NO to inhibit the activity of cyclin-dependent kinase 2, a key regulator of the G1 and S phases of the cell cycle, in vascular smooth muscle cells [31, 32]. These findings on the actions of NO to suppress cell growth and the cell cycle are well in line with the idea that NO may prevent immature follicles from undergoing differentiation.
Among NOS isoforms, the presence of endothelial NOS (eNOS) as well as iNOS in the ovary is already known [6, 11–13, 17–19]. The regulatory mechanisms for the expression of iNOS and eNOS, however, seem to be different. The eNOS mRNA level is highest in rat ovaries containing ovulatory follicles, whereas the expression of iNOS is hardly detectable [6]. Given the localization of eNOS in the theca cell layer, stroma, and blood vessels in the ovary, along with its mRNA being induced by estrogen, eNOS appears to play a role in the event of ovulation [6, 11, 13, 17–19]. By use of eNOS-knockout female mice, which exhibit fewer large antral follicles compared with wild-type mice during proestrus, eNOS-derived NO is assumed to be a modulator of oocyte meiotic maturation, steroidogenesis, and ovulation [17, 18]. In contrast, iNOS-knockout mice have as many large antral follicles as wild-type mice [18]. Hence, eNOS and iNOS seem to be differently involved in the regulation of follicle development.
In conclusion, the present study suggests that locally produced NO, via iNOS, might act as a cytostatic factor in immature follicles, with a loss of NO being associated with the conversion into a different developmental status, in whichever direction (i.e., progression into differentiation, or demise via apoptotic process). The precise molecular mechanisms whereby iNOS expression is regulated and NO keeps immature follicles arrested await further study.
ACKNOWLEDGMENTS
We thank Shinobu Ikeda and Noriko Ide for their technical assistance.
FOOTNOTES
First decision: 19 January 2000.
1 Supported by the Yamada Scholar Award from the Yamada Scholarship Foundation (H.M.) and in part by grants-in-aid from the Ministry of Education, Science and Culture, from the Science and Technology Agency, and from the Ministry of Health and Welfare for the second-term Comprehensive 10-Year Strategy for Cancer Control, Japan. 
2 Correspondence: Tetsu Yano, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. FAX: 81 3 3816 2017; tetu-tky{at}umin.ac.jp
Accepted: February 18, 2000.
Received: December 15, 1999.
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