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Mechanisms of Hormone Action; |
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel
ABSTRACT
Established gap junctional communication (GJC) in the ovarian follicle is essential for maintaining the oocytes in meiotic arrest. Alternatively, LH-induced reinitiation of meiosis is subsequent to breakdown of GJC. It was recently reported that nitric oxide (NO) inhibits maturation in rat follicle-enclosed oocytes and elevates GJC in cultured mesangial cells. Taking these observations into account, we hypothesized that NO prevents reinitiation of meiosis by antagonizing the effect of LH on GJC in the ovarian follicle. Indeed, we found that NO interferes with LH-induced disruption of GJC as well as with the decrease of the expression of the gap junction protein GJA1 (previously known as CONNEXIN43). We also demonstrated that NO prevents activation of LH-induced mitogen-activated protein kinases (MAPKs) 1 and 2 and inhibits cumulus expansion. Along this line, incubation of ovarian follicles with an inhibitor of soluble guanylate cyclase, which is a downstream NO effector, induced on its own oocyte maturation as well as cumulus expansion. Unlike previous studies, we show here that elevation of NO resulted in inhibition of ovulation. We conclude that the mechanism by which NO inhibits LH-induced oocyte maturation possibly involves a negative effect on MAPK activation and, in turn, interference with interruption of GJC. This action of NO in the ovarian follicle is apparently mediated by cGMP. In addition, the negative effect of NO on ovulation may be subsequent to its inhibitory effect on cumulus expansion. Together, this study suggests that the preovulatory decrease in NO concentrations is a prerequisite for the ovarian response to LH.
follicle, gap junction, luteinizing hormone, nitric oxide, oocyte, ovarian follicle, ovulation
Nitric oxide (NO) has been identified as a major intracellular as well as intercellular signaling molecule involved in diverse physiological processes. It is produced from L-arginine by three different NO synthase (NOS) enzymes: neuronal nitric oxide synthase 1 (NOS1), endothelial cell nitric oxide synthase 3 (NOS3), and inducible nitric oxide synthase 2 (NOS2). Both NOS3 and NOS2, but not NOS1, are expressed in the ovary (reviewed in Rosselli et al. [1]). It was recently reported that following injection of hCG, the concentrations of NO within the ovarian follicle decrease [2]. Additionally, it was demonstrated that although Nos3 mRNA was mainly detected in the theca cells and increased in response to hCG, Nos2 mRNA was predominately expressed in the granulosa and significantly decreased in response to this hormone [2].
The full-grown oocytes in the mammalian ovary are arrested at prophase of the first meiotic division, awaiting the surge of LH that induces reinitiation of meiosis followed by ovulation [3]. However, resumption of meiosis can also occur spontaneously in oocytes liberated from their follicular environment. Both LH-induced and spontaneous oocyte maturation are associated with a decrease in intraoocyte cAMP concentrations (reviewed in Dekel [4]). It has been shown that intraoocyte concentrations of cGMP also decrease during spontaneous maturation and that microinjection of this nucleotide into rat oocytes inhibits resumption of meiosis [5]. This last observation apparently represents the inhibitory effect of cGMP on the activity of the oocyte-specific phosphodiesterase 3A (PDE3A [6]). The production of cGMP is upregulated by NO via activation of a soluble guanylate cyclase1 (GUCY1, reviewed in Blaise et al. [7]).
An extensive gap junctional communication (GJC) network is present among the cumulus/granulosa cells as well as between the oocyte and the somatic cells of the ovarian follicle [8–12]. Gap junctions are composed of proteins encoded by the connexin gene family, the most abundant of which in the ovary is GJA1 (previously known as CONNEXIN43 [13]). GJA1 is mostly expressed by the somatic ovarian cells (reviewed by Kidder and Mhawi [14]), whereas the expression of GJA4 (previously known as CONNEXIN37) is restricted to the oocyte [15, 16]. One role of GJC within the ovarian follicle is to allow the supply of nutrients from the somatic compartment supporting oocyte growth [17, 18]. In addition, gap junctions mediate the transfer of cAMP from the granulosa/cumulus cells to the oocyte [19, 20] which, in turn, maintains the oocyte in meiotic arrest. Alternatively, reinitiation of oocyte maturation in response to LH is preceded by interruption of GJC in the ovarian follicle.
It has been shown recently that the addition of a NO donor to cultured mesangial cells upregulates Gja1 expression [21]. Moreover, this study also demonstrated that this NO donor upregulated GJC between these cells. In addition, it has also been reported that NO inhibits the LH-induced maturation in rat follicle-enclosed oocytes, whereas the addition of aminoguanidine (AG), an NOS2 inhibitor, mimics the effect of LH in this system [22]. Combining the results of the above-mentioned reports, we hypothesized that NO inhibits LH-induced oocyte maturation by interfering with the disruption of GJC within the ovarian follicle. Our present study was designed to test this hypothesis. We also took into consideration that NO may affect the oocyte directly by upregulation of cGMP concentrations that inhibit the PDE3A activity and prevent, in turn, cAMP degradation. In addition, we examined the effect of elevated concentrations of NO on other well-known ovarian responses to LH, such as cumulus expansion and ovulation.
Leibovitz L-15 tissue culture medium and fetal bovine serum (FBS) were purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Antibiotics were purchased from Bio-Lab Ltd. (Jerusalem, Israel), eCG from Chrono-gest Intervet (Boxmeer, The Netherlands), and ovine LH (o-LH-26) from NHPP (Harbor-UCLA Medical Center, Torrance, CA). Phenylmethyl-sulfonylfluoride (PMSF), leupeptin, aprotinin, and dithiothreitol were purchased from Sigma. Monoclonal mouse anti-pMAPK antibodies were kindly provided by Professor Rony Seger (Weizmann Institute of Science, Rehovot, Israel). Polyclonal rabbit anti-MAPK antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal anti-GJA1 antibodies were purchased from Transduction Laboratories (Lexington, KY). Monoclonal mouse anti-
and β-tubulin antibodies were purchased from Sigma (Rehovot, Israel). Goat anti-mouse peroxidase-conjugated antibodies were purchased from Jackson Immunoresearch Laboratories Inc. (West Grove, PA), S-nitroso-L-acetyl penicillamine (SNAP) from Sigma, 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one (ODQ) from Alexis (San Diego, CA), aminoguanidine (AG) from Sigma, RAF1 kinase inhibitor II (BAY 43-9006) from Calbiochem, hCG (Pregnyl NV) from Organon (Oss, Holland), penicillin and streptomycin from Bio-Lab (Jerusalem, Israel), and 8-bromoguanosine-3',5'-cyclomonophosphate sodium salt (8-Bromo-cGMP) from Sigma.
Follicle-enclosed oocytes were recovered from sexually immature, eCG-primed, 25-day-old female Wistar rats. The rats were killed by cervical dislocation 48 h after eCG administration, their ovaries were removed, and the large antral follicles were separated. The isolated intact follicles were incubated in suspension in L-15 tissue culture medium containing 5% FBS, penicillin (100 IU/ml), and streptomycin (50 µg/ml) in 25-ml flasks, supplied with 50% O2 and 50% N2. Incubations were carried out at 37°C in an oscillating water bath in the presence or the absence of either 1.5 µg/ml of ovine LH with or without 500 µM of SNAP that was added 15 min prior to the addition of the hormone. At the end of the incubation period, the follicles were incised and the cumulus-oocyte complexes (COCs) were recovered. The oocytes were monitored by differential interference contrast microscopy (standard WL research microscope; Carl Ziess). The breakdown of the germinal vesicle (GVB) served as a morphological marker for reinitiation of meiosis.
All experiments were conducted in accordance with the Guides for the Care and Use of Laboratory Animals (National Research Council, National Academy of Sciences, Washington, DC).
Protein Extraction and Western Blot Analysis
To determine the extent of MAPK activation, the ovarian follicles were lysed in RIPA buffer containing 50 mM β-glycerophosphate, 20 mM Tris, pH 7.4, 137 mM NaCl, 10% glycerol, 0.1% SDS, 1% Triton X-100, 1.5 mM EGTA, 1 mM EDTA, 1 mM Na-orthovanadate, 1 mM benzamidine, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 2 µg/ml pepstatin. The lysates were then centrifuged for 10 min, after which the supernatants were collected and their protein concentrations determined. Equal amounts of protein (30 µg) were dissolved in protein sample buffer (2% β-mercaptoethanol, 2% SDS, 50 mM Tris, HCL, pH 6.8, 10% glycerol, and 0.01% bromophenol blue), boiled, and loaded on 10% SDS-PAGE. For better resolution of the different phosphorylation forms of GJA1, the bisacrylamide in the monomer mixture was reduced from 8% to 0.12%. After electrophoretic separation, the proteins were transferred to a nitrocellulose membrane, washed for 1 h with a blocking solution (5% milk, 0.05% Tween in PBS), and then incubated for 1 h with either anti-GJA1 monoclonal antibodies (1:250) or with two anti-MAPK antibodies. One MAPK antibody immunoreacts with the phosphorylated (active) MAPK (pMAPK), whereas the second immunoreacts with both the active and the inactive MAPK (total MAPK). The relative amount of phosphorylated MAPK in each sample represents the extent of MAPK activation. The membrane was then washed several times and incubated with anti-mouse horseradish peroxidase-conjugated antibodies (1:1000). Chemiluminescent signals were generated by incubation with the ECL reagent (Amersham).
Granulosa cells were recovered from the ovaries of the aforementioned female rats. The cells were plated onto serum-coated 24-multiwell dishes (Nunc, Copenhagen, Denmark) containing 0.5 ml of L-15 medium with 5% FBS, penicillin (100 IU/ml), and streptomycin (50 µg/ml). Cell cultures were kept at 37°C in a humidified incubator for 48 h to obtain a confluent cell layer.
Scrape Loading Dye Transfer Assay
The cultured granulosa cells were incubated with or without LH (10 µg/ml) in the presence or absence of SNAP (500 µM) for 30 min. After incubation, the plates were washed, and PBS containing a mixture of 0.7 mg/ml lucifer yellow (LY; Sigma) and 5 mg/ml rhodamin dextran (Rh; Molecular Probes, Eugene, OR) was added as previously described [23]. The plates were mechanically scratched with a sharp scalpel. After an additional 4 min of incubation, the cells were washed several times with PBS and fixed with 3% paraformaldehyde. The cells were then viewed by a laser scanning confocal imaging system (radiance 2000/AGR-3; Bio-Rad) connected to a Nikon T200 microscope (Melville, NY).
To study the effect of NO on ovulation, intrabursal injection was performed using sexually immature, 25-day-old, eCG-primed Wistar female rats [24]. Rats were lightly anesthetized, and one of their ovaries was exteriorized through a small lumbosacral incision. Phosphate-buffered saline (100 µl) with or without SNAP (500 µM) was injected through a 30-gauge needle threaded into the ovarian bursa via the adjoining fat pad. After injection, the ovary was returned to the abdominal cavity, and the skin was clipped. This procedure was followed by an hCG (10 IU) intraperitoneal injection. Twenty-two hours later, the rats were killed by cervical dislocation, the ampullae of the oviducts were excised, and the oocytes were released and counted under the microscope.
The number of repetitions of each individual experiment is indicated in the figure legends. Statistical significance determined by ANOVA was used to assess differences between multiple experimental groups.
LH-Induced, Follicle-Enclosed Oocyte Maturation and MAPK Activation Is Inhibited by NO
As previously mentioned, it was reported that the addition of an NO donor to follicle-enclosed oocytes inhibits hCG-induced maturation [22]. Our initial experiment was designed in order to confirm these findings. As expected, LH induced GVB in 85% of the oocytes, an effect that was markedly inhibited upon the addition of the NO donor, SNAP (Fig. 1A). All of the follicle-enclosed oocytes incubated in the presence or the absence of the NO donor remained meiotically arrested, as indicated by the presence of the GV.
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MAPK is involved in the resumption of meiosis of both mouse and rat oocytes [25, 26]. The upstream regulator of MAPK is MAPK2K1 (MEK1). Phosphorylation and activation of MAP2K1 are catalyzed by RAF1 kinase (reviewed by Seger and Krebs [27]). However, in gametes, MAP2K1 is uniquely subjected to regulation by MOS kinase [28]. Under these conditions, a specific inhibitor of RAF1 will prevent MAPK activation exclusively in the granulosa cells, with no such effect on the oocyte. This experiment will confirm that it is the granulose, not the oocyte MAPK that should be activated for the induction of oocyte maturation. As seen in Figure 1B, the addition of the specific RAF1 inhibitor BAY 43-9006 [29] significantly interfered with LH-induced, follicle-enclosed oocyte maturation.
It has been reported that NO inhibits MAPK activation [30]. Since MAPK activation mediates reinitiation of meiosis, we examined the activity of MAPK in the ovarian follicle in the presence of LH under conditions of elevated NO. As seen in Figure 1C, NO inhibited the LH-induced MAPK activation.
LH-Induced Disruption of GJC Is Inhibited by NO
We have shown in our previous study that MAPK mediates the LH-induced disruption of GJC within the ovarian follicle [25]. As mentioned previously, NO increases GJC in mesangial cells [21]. Since disruption of GJC within the ovarian follicle is essential for resumption of meiosis [25, 31], we examined the possible effect of NO on GJC. For this purpose, we employed the scrape-loading dye transfer assay using cultured granulosa cells. This assay takes into consideration the fact that LY and Rh do not penetrate intact cells but can be incorporated into injured cells. However, since LY has a molecular weight of less than 1 kDa, it flows transcellularly through gap junctions, whereas Rh, which is a larger molecule (10 kDa), cannot leave the injured cells. As seen in Figure 2, Rh staining was restricted to the injured cells, as expected. The spread of LY to neighboring cells can be observed clearly under control conditions and, as shown previously [25], is substantially reduced in the presence of LH. The addition of the NO donor restored the dye spread to control levels. Incubation with AG, an NOS2 inhibitor, mimicked the effect of LH and reduced the dye spread.
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LH-Induced GJA1 Downregulation Is Partially Inhibited by NO
It was previously shown that LH stimulates GJA1 downregulation [32] via inhibition of its translation and that this effect of LH is mediated by MAPK [33]. Since NO inhibits LH-induced MAPK activation, the abundance of GJA1 following incubation of the ovarian follicles with LH in combination with the NO donor was examined. As seen in Figure 3, in the presence of LH, GJA1 was almost completely eliminated. Addition of the NO donor partially reduced this effect.
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Human CG-Induced Ovulation Is Inhibited by NO
Several studies reported that inhibition of NO production blocked hCG-induced ovulation [34–36]. Unexpectedly, we found that intrabursal injection of NO donors totally inhibited hCG-induced ovulation (Fig. 4A). Histological examination of ovarian sections of the injected animals revealed some oocytes entrapped within forming corpora lutea, with no other morphological abnormalities (Fig. 4B).
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LH-Induced Cumulus Expansion Is Inhibited by NO
It is commonly accepted that the expansion of the cumulus is absolutely essential for normal ovulation (reviewed in Richards [37]). It was also demonstrated that MAPK activation is a prerequisite for this process [26]. As intrabursal injection of an NO donor blocked ovulation, and since we detected a reduction in MAPK activation in the presence of this reagent, we examined the possibility that NO affects cumulus expansion. For this purpose, we incubated intact ovarian follicles in the presence of LH with or without an NO donor. At the end of incubation, the COCs were analyzed for cumulus expansion. As seen in Figure 5, NO donor inhibited LH-induced cumulus expansion.
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Cyclic GMP Inhibits MAPK Activation, Oocyte Maturation, and Cumulus Expansion
It is well established that NO induces the production of cGMP via activation of GUCY1 (reviewed in Rosselli et al. [1]). As mentioned, microinjection of cGMP into oocytes prevents their maturation [5]. In addition, cGMP inhibits the activity of PDE3A [6]. Taking all this information into account, we examined the effect of the GUCY1 inhibitor, ODQ, on ovarian follicles. As seen in Figure 6A, ODQ mimicked LH and induced MAPK activation. Moreover, this inhibitor of GUCY1 also stimulated follicle-enclosed oocyte maturation as well as cumulus expansion (Fig. 6, A and B). Furthermore, the stimulatory effect of ODQ on oocyte maturation was totally prevented by 8-Bromo-cGMP (Fig. 6C).
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Our present study demonstrates that NO inhibits LH-induced MAPK activation in the ovarian granulosa cells. We also show that NO prevents the LH-induced downregulation of the gap junction protein GJA1 as well as the breakdown of the ovarian follicle GJC in response to this gonadotropin. This mechanism could possibly account for the previously reported and herein confirmed NO inhibition of LH-induced oocyte maturation. In addition, we found that the inhibitory action of NO on oocyte maturation in the ovarian follicle is mediated by an active GUCY1 and its product, cGMP. We report here for the first time that NO inhibits LH-stimulated cumulus expansion and that this effect apparently leads to impaired ovulation.
Previous studies from our [25, 31] as well as other laboratories [11, 38] demonstrated that LH/hCG interrupts cell-to-cell communication between the oocyte and the somatic follicular cells. In a more recent study, we showed that closure of GJC within the ovarian follicle is associated with a decrease in the intraoocyte concentrations of cAMP and the subsequent oocyte maturation [39]. Taken together, these findings suggested that breakdown of communication may serve as the mechanism for LH-induced oocyte maturation. It has been previously reported [22] and herein confirmed that NO prevents hCG-induced maturation of follicle-enclosed oocytes. In this study, we explored the mechanism that underlies this effect. We specifically examined the hypothesis that NO inhibits LH-induced breakdown of GJC in the ovarian granulosa cells. The scrape load dye transfer assay indeed confirmed our assumption. These findings agree with a previous report that NO elevates Gja1 expression and increases GJC in mesangial cells [21].
As mentioned previously, GJA4 is also expressed in the ovarian follicle, but its expression is restricted to the oocyte [16]. We have recently demonstrated that oocytes that lack GJA1 stay meiotically arrested and communicate with the cumulus cells [15]. Therefore, it is apparently the oocyte GJA4 and the cumulus GJA1 that compose the heterotypic gap junction channels at the interface between the germ cell and the somatic compartment of the ovarian follicle. However, cumulus-enclosed oocytes removed from the ovarian follicle resume meiosis spontaneously, whereas the follicle-enclosed oocytes remain meiotically arrested. Taken together, these observations suggest that it is not the cumulus but rather the granulosa that provides the inhibitory amounts of cAMP. The granulosa cell cAMP uses the abundantly expressed GJA1 gap junctions to reach the cumulus mass and, in turn, affects the oocyte. Therefore, breakdown of GJA1-medited GJC is sufficient to significantly reduce the supply of cAMP to the oocyte and allows the resumption of meiosis independently of the functional status of GJA4.
Luteinizing hormone-induced breakdown of GJC is mediated by MAPK [25]. We demonstrate herein that LH-induced MAPK activation in the ovarian follicles is inhibited by NO. It has been shown elsewhere that MAPK activation in the ovarian follicle is dependent on the presence of an active PKA [40]. Downregulation of MAPK activity by NO may, therefore, represent its previously reported negative effect on adenylyl cyclase [41]. Alternatively, it has been reported that NO inhibits MAPK activation via generation of cGMP [30]. Since we provide evidence that in the ovarian follicle, NO acts via activation of GUCY1 and, in turn, cGMP production, it is possible that a drop in cGMP concentrations upon LH administration enables the activation of MAPK in granulosa cells. Our findings indeed suggest that cGMP is involved in the action of NO on the granulosa cells but cannot rule out a cGMP-mediated effect that is directly elicited on the oocyte. However, since incubation of denuded oocytes with an NO donor [22] or microinjection of oocytes with cGMP [5] did not prevent but only postponed spontaneous maturation, it seems that a cGMP-mediated effect is not directly elicited at the oocyte level.
It has been shown that knock out of the Nos3 gene severely impairs both oocyte maturation and ovulation [42, 43]. This study seems to suggest that NO is essential for both of these responses of the ovarian follicle to LH, opposing the conclusion of our study. To overcome this apparent contradiction, it should be taken into consideration that in the Nos3 knockout animals, the ovaries are deprived of NO from the very early stages of their development. Indeed, this study reports that the ovaries are small in size, contain a lower number of antral follicles, and produce a small amount of steroid hormones. Impaired oocyte maturation and ovulation in that case is apparently only secondary to the defective ovarian phenotype. This is not the case in our study, in which properly developed antral ovarian follicles are examined. At this advanced stage of folliculogenesis, NO seems to be detrimental, negatively regulating the response to LH.
Our studies were extended to include exploration of the possible effect of NO on ovulation. We show that a direct application of an NO donor to the ovary resulted in total inhibition of hCG-induced follicle rupture. The normal ovarian histology, demonstrated in our study, rules out a possibly toxic effect of this agent. In disagreement with our findings, other studies that used NOS inhibitors for intrabursal injection prevented follicular rupture, thus claiming that NO is required for normal ovulation [34–36]. Several attempts made throughout our study to administer the NOS2 inhibitor AG to the ovarian follicles (data not shown) severely damaged the follicular cells as well as the oocytes. In the absence of histological examination of the ovaries in the studies mentioned above, a toxic effect of the NOS inhibitor remains possible. It is also possible that the opposite influence of NO on ovulation described by us and reported previously represents the well-known biphasic dose response to this agent demonstrated in different biological systems [44], including mouse oocytes [45, 46].
It is well established that cumulus expansion is a prerequisite for normal ovulation [37]. Therefore, the inhibitory effect of NO on ovulation demonstrated by us is further supported by the substantial reduction in cumulus expansion in intact ovarian follicles incubated with LH in the presence of a NO donor. Moreover, previous findings that MAPK activation is required for cumulus expansion [26] also agree with our observation.
Taking our results into account, we suggest an optional model for the involvement of NO in LH-induced oocyte maturation (Fig. 7): Upon LH administration, the concentrations of NO in the ovarian follicle drop, GUCY1 activity is downregulated, and the levels of cGMP decline. This, in turn, allows the sequential activation of RAF1, MAP2K1, and MAPK and the subsequent disruption of GJC in the somatic follicular compartment. Breakdown of GJC stops the transfer of the inhibitory cAMP from the granulosa cells to the oocyte, enabling the resumption of meiosis. In addition, the reduction in cGMP concentrations may also lead to the removal of its inhibitory effect on PDE3A, increasing the efficiency of degradation of the intraoocyte cAMP.
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To summarize, we explored in this study the involvement of NO in the key processes of female reproduction. Based on our results, we suggest that the previously reported drop of the ovarian NO concentration after hCG administration [2] seems to be essential for allowing the stimulation of both oocyte maturation as well as ovulation by this gonadotropin.
ACKNOWLEDGMENTS
We thank Ms. Merav Persky for her assistance in the experiments with BAY 43-9006.
FOOTNOTES
1Supported by the Dwek Fund for Biomedical Research. N.D. is the incumbent of the Phillip M. Klutznick Professorial Chair of Developmental Biology. 
Correspondence: 2Nava Dekel, Department of Biological Regulation, 15 Weizmann Institute of Science, Rehovot 76100, Israel. FAX: 972 8 934 4116; e-mail: nava.dekel{at}weizmann.ac.il
Received: 10 September 2007.
First decision: 2 October 2007.
Accepted: 31 January 2008.
REFERENCES
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