1 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK and 2 Current address: Bourn Hall, Bourn, Cambridge CB3 7TR, UK
3 To whom correspondence should be addresed. Email: geraldine.hartshorne{at}warwick.ac.uk
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Abstract |
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Key words: culture/follicle/inhibitors/nitric oxide/ovulation
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Introduction |
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The aim of this study was to analyse the effects of a variety of NOS inhibitors, NOS stimulators and NO donors upon mouse follicle development and ovulation in vitro. The effects upon ovarian function of pharmacological agents that modulate NO have been studied previously using various approaches. NOS inhibitors including aminoguanidine (AG) and N G-methyl-L-arginine (L-NMMA) suppress ovulation in rats in vivo (Shukovski and Tsafriri, 1994) and in vitro-perfused ovaries (Hesla et al., 1997
). However, ours is the first study to test effects upon cultured follicles. The absence of systemic influences upon cultured follicles enables local effects to be studied and enables the relative effects of intra- and extrafollicular regulation to be distinguished.
Most NOS inhibitors vary in their affinities for the isoforms of NOS (iNOS, nNOS and eNOS) (Rosselli et al., 1998). Analogues of L-arginine act as competitive inhibitors reversible by addition of excess L-arginine, although prolonged exposure may cause irreversible inhibition (Olken et al., 1994
). N G-Nitro-L-arginine (L-NNA) has greatest affinity for nNOS, less for eNOS, with little effect upon iNOS (Reif and McCreedy, 1995
). N G-Nitro-L-arginine methyl ester (L-NAME) is the methyl ester of L-NNA and inhibits eNOS (Rees et al., 1990
) and nNOS (Xia et al., 1996
) potently but iNOS weakly (McCall et al., 1991
). L-NMMA is approximately equipotent against all three isoforms in humans (Vallance, 1996
) and inhibits the enzymatic activity of iNOS induced by interleukin (IL)-1
pre-treatment in rats (Corbett and McDaniel, 1996
). S-Methylisothiourea sulphate (SMT) is a non-amino acid analogue of L-arginine, which acts as a competitive inhibitor of NOS, having some selectivity for iNOS over eNOS (Szabo et al., 1994
).
The normal physiological profile of NO production cannot easily be replicated. Chemical donors can raise levels of NO, but toxic effects occur at high concentrations because NO is a free radical. Interpretation of experiments using NO donors can be complex as NO exerts negative feedback on its own production, inhibiting NOS (Buga et al., 1993) by interacting with the haem group (Hurshman and Marletta, 1995
) and by interfering with nuclear factor (NF)-
B (Park et al., 1997
), a key transcription factor in the promoter region of the iNOS gene.
Other factors may stimulate iNOS production. For example, lipopolysaccharide (LPS) is a component of bacterial cell walls and an endotoxin, which induces the production of inflammatory mediators such as tumour necrosis factor (TNF
), interferon
and IL-1
(Griffith and Stuehr, 1995
). Both LPS and interferons can increase iNOS mRNA via actions on specific promoter regions of the iNOS gene (Laubach et al., 1995
). In rat and rabbit perfused ovaries, IL-1
can induce ovulation even in the absence of an LH stimulus (Brannstrom et al., 1993
) and further increasing the LH-stimulated ovulation rate (Bonello et al., 1996
). These actions appear to be mediated by NO because ovulation was sharply reduced when L-NAME was added together with LH and IL-1
.
Our previous studies detected iNOS and eNOS in the thecal cells and oocyte and also weakly in the granulosa cells of whole mouse ovaries (Mitchell et al., 2004). Cultured mouse follicles were also positive for iNOS, and the requirement for NOS action for normal follicle growth in vitro was demonstrated by manipulation of levels of NOS substrate (L-arginine), intermediate (NG-hydroxy-L-arginine) and end-product (L-citrulline) (Mitchell et al., 2004
). In the present study, selective inhibition of iNOS and eNOS has therefore been attempted by applying carefully chosen concentrations of inhibitors to cultured mouse follicles in order to distinguish their effects upon follicle growth and ovulation.
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Materials and methods |
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Inhibitors of NOS
NOS inhibitors and information on their relative inhibitory activities were obtained from Calbiochem (USA). Stock concentrations prepared in basal culture medium were used to supplement follicle growth media. Where necessary, stock aliquots were stored at 20°C. Inhibitors were present for the duration of the culture at the following final concentrations: L-NNA, 0, 1, 10, 100 µmol/l (6669 follicles per group, seven experiments); L-NAME. 0, 0.5, 5, 50 µmol/l (4956 follicles per group, four experiments); 100, 1000, 10 000 µmol/l (3234 follicles per group, three experiments); L-NMMA, 0, 0.39, 3.9, 39 µmol/l (102105 follicles per group, eight experiments) and 60, 600, 6000 µmol/l (3640 follicles per group, two experiments); SMT, 0, 6, 60, 600, 6000 µmol/l (66130 follicles per group, five to nine experiments).
L-NNA is an L-arginine analogue and is a potent, cell-permeable, reversible, competitive inhibitor of nNOS and eNOS at low concentrations (IC50=25 and 90 nmol/l respectively) and inhibits the activity of iNOS at higher concentrations (IC50=8.1 µmol/l).
L-NAME is the more soluble methyl ester of L-NNA and is a competitive inhibitor of eNOS at IC50=0.5 µmol/l and iNOS at >1000 µmol/l. L-NAME was used at low (0.550 µmol/l) and high (0.110 mmol/l) concentrations. At the low concentrations, only eNOS would be inhibited, whereas at the higher concentrations both iNOS and eNOS would be affected.
L-NMMA is a competitive inhibitor of all three NOS isoforms (IC50=650 nmol/l for nNOS, IC50=700 nmol/l for eNOS and IC50=3.9 mmol/l for iNOS). L-NMMA was used at low (0.3939 µmol/l) and high (0.066 mmol/l) concentrations. The low concentrations would be expected to inhibit all three NOS isoforms; however, Griffith and Kilbourn (1996) reported that when the culture medium contains L-arginine, the L-NMMA concentration should be 510-fold higher than that of L-arginine, i.e. up to 6 mmol/l, hence the higher concentration set was applied also.
SMT is a selective inhibitor of iNOS with EC50 of 26 µmol/l (Szabo et al., 1994).
NO donors
NO donors (Calbiochem) were prepared in basal culture medium immediately prior to use, because they are unstable in solution. The following donors were included in follicle growth media for the duration of culture at the concentrations listed: 3-morpholino-sydnonimine, HCl (SIN-1), 0, 10, 100, 1000 µmol/l (7983 follicles per group, seven experiments); S-nitroso-N-acetylpenicillamine (SNAP), 0, 13, 130, 1300 nmol/l (8995 follicles per group, seven experiments); 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazine (NOC-18), 0, 1, 10, 100 µmol/l and 10 mmol/l [3150 follicles per group, three experiments (two for 10 mmol/l)].
The concentrations of SIN-1 (101000 µmol/l) were selected based upon previous publications with rat neurons, liver microsomes and human hepatoma cell cultures (de Groot et al., 1993; Lipton et al., 1993
; Gergel et al., 1995
).
The concentrations of SNAP (0.013, 0.13 and 1.3 µmol/l) were selected based upon the work of Henry et al. (1989) showing an EC50 of 130 nmol/l in bovine arteries.
NOC-18 is a NO donor with a relatively long half-life for NO release (t=78 mins in PBS at 37°C; Hrabie et al., 1993
) and each molecule results in the release of two molecules of NO. NOC-18 was added at concentrations between 1 µmol/l and 10 mmol/l.
NOS stimulators
Escherichia coli lipopolysaccharide (LPS, Calbiochem) was added to cultured follicles at final concentrations of 0, 20, 200, 2000 pg/ml; 7577 follicles comprised each group (five experiments). The stock solution of LPS (2 mg/ml) in culture medium was stored at 4°C.
IL-1 (Calbiochem) was added to follicle cultures at final concentrations of 0, 1, 10 and 100 pg/ml, with 4347 follicles per group (four experiments). These doses were chosen as other researchers have used similar concentrations with human chondrocytes requiring 100500 pg/ml IL-1 for maximum NO production (Hauselmann et al., 1994
). Stock solution was prepared in serum-free culture medium and aliquots were stored for up to 3 months at 70°C.
Monitoring follicle growth
All follicles were observed daily and their growth recorded using extensively characterized morphological criteria of attachment, basement membrane integrity, granulosa cell outgrowth, antrum formation, oocyte appearance, ovulation and cumulus presence and appearance, as described in detail previously (Mitchell et al., 2002). At the end of culture, follicles were considered to have survived or not based upon the appearance of the oocyte and granulosa cells. Follicles in which the oocyte was spherical and centrally located, in granulosa which was not overtly degenerate, were considered to have survived. Timely ovulation was defined as the release of the oocyte within 16 h after the hCG stimulus, and delayed ovulation defined as the release of the oocyte between 16 and 48 h of the hCG stimulus. Such ovulation has previously been shown to occur in response to hCG rather than as a result of follicle deterioration. Occasional oocytes extruded from degenerating follicles could be distinguished by their lack of cellular investments and differences in the follicle appearance (Mitchell et al., 2002
).
Statistical analysis
Follicle survival and ovulation were analysed using contingency tables with 2, with P<0.05 considered significant.
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Results |
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N G-Monomethyl-L-arginine, monoacetate salt (L-NMMA)
Follicle survival rates were similar at all L-NMMA concentrations. In the first set of experiments, the highest timely ovulation rate was observed in the control group (71%) compared with 59% in 0.39 µmol/l L-NMMA , 67% in 3.9 µmol/l L-NMMA (NSD) and 53% with 39 µmol/l L-NMMA
(Figure 1c). Interestingly, at the end of culture, one follicle in this group had retained an intact basement membrane and follicular architecture similar to that reported previously for follicles cultured in the absence of L-arginine (Mitchell et al., 2004
). No significant effects of L-NMMA were observed at the higher concentrations (0.066 mmol/l).
S-Methylisothiourea sulphate (SMT)
Survival rates of 8997% occurred for follicles cultured in 60 µmol/l SMT, whereas survival was significantly
reduced with 600 and 6000 µmol/l SMT (Figure 1d). Timely ovulation rates were also drastically reduced with SMT
600 mmol/l. Follicles cultured with 600 µmol/l SMT appeared degenerative by day 7, whereas 6000 µmol/l SMT resulted in dark and granular follicles by day 2.
NO donors
3-Morpholino-sydnonimine, HCl (SIN-1)
The survival and ovulation rates were similar (8992%) at all concentrations of SIN-1 (Figure 2a).
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1-Hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazine (NOC-18)
NOC-18 had no effect on follicle survival (8594%) at up to 100 µmol/l; however, only 26% survived with 10 mmol/l NOC-18 (Figure 2c). The timely ovulation rates with 1, 10 and 100 µmol/l NOC-18 were significantly (P<0.02, P<0.05, P<0.05 respectively) reduced compared to control. At 10 and 100 µmol/l NOC-18, all follicles that had not degenerated by day 10 released a nude oocyte in response to hCG.
NOS stimulators
Lipopolysaccharide (LPS)
The apparent increases in follicle survival and timely ovulation rates were not significant. Follicles cultured with LPS were healthy in appearance and similar to control follicles throughout the culture period (Figure 3a).
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Significantly fewer follicles formed antra when cultured with IL-1 >1 pg/ml, compared to the control group. For follicles with 0, 1, 10 and 100 pg/ml IL-1
, antrum formation occurred in 67, 72, 44%
and 19%
respectively. Examples of antral follicles in each group are shown in Figure 4. Control follicles formed antra from day 5 onwards and developed large antra by day 9. Follicles cultured with 1 pg/ml IL-1
formed antra from day 4 onwards which had grown to a similar large size by day 9. Follicles cultured with 10 and 100 pg/ml IL-1
started to form antra from day 5 onwards, and these became larger by the end of culture, but did not expand to the same extent as controls. Generally fewer follicular cells were present than in the control.
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Discussion |
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In the present study, we investigated the role of the different NOS isoforms using pharmacological inhibitors having different relative affinities. All these experiments were performed in medium containing L-arginine, to ensure that the NOS pathway was intact. The low numbers of cells present precluded measuring nitrite using the Griess reaction or by real-time electrochemical measurement of NO (G.M.Hartshorne, unpublished data). Hence, the local effectiveness of the inhibitors and other agents could not be quantified by direct NO measurement.
NOS inhibitors competitively block NO production. Their functional groups can interact with iron groups, which may produce non-specific reactions (Peterson et al., 1992). We found no effect of L-NNA upon follicle survival or ovulation. Reif and McCreedy (1995)
demonstrated that L-NNA did not affect purified iNOS at the concentrations used here although eNOS was inhibited. In our experiments, L-NNA transport may have been inhibited by L-leucine and L-phenylalanine present in the culture media (Griffith and Kilbourn, 1996
). While higher concentrations might improve availability, specificity for eNOS would be lost. We therefore sought other inhibitors.
L-NAME is more soluble than L-NNA (Griffith and Kilbourn, 1996) and was used at 0.5, 5 and 50 µmol/l to inhibit eNOS. Under these conditions, total ovulation rates remained similar. While the percentage of follicles that ovulated on time appeared lower with L-NAME, this observation was not significant. A significant reduction in timely ovulation occurred with higher levels of L-NAME (0.110 mmol/l) affecting both eNOS and iNOS (Moncada et al., 1991
). Delayed ovulation seemed to be increased by all concentrations of L-NAME.
L-NMMA inhibits all isoforms of NOS. While two of the low doses significantly reduced timely ovulation, this was not dose-dependent. L-NMMA is required at a concentration 510-fold > L-arginine for >90% inhibition of NOS (Griffith and Kilbourn, 1996), however, L-NMMA is converted to L-citrulline and NO at 5% efficiency relative to L-arginine (Olken and Marletta, 1993
). We previously showed that L-citrulline can partially restore follicle survival and ovulation in vitro if L-arginine is absent (Mitchell et al., 2004
).
SMT is a 1030-fold more potent inhibitor of iNOS than L-NMMA (Szabo et al., 1994). SMT at
600 µmol/l sharply reduced follicle survival and ovulation compared to L-NMMA at the same concentration. No effect was evident at
60 µmol/l SMT. The most likely explanation is that SMT is toxic, as the highest dose caused degeneration within 2 days, i.e. shorter than the 5 days taken for L-arginine deprivation to affect follicle development in vitro (Mitchell et al., 2004
). However, SMT is not toxic up to 10 mmol/l in mouse macrophages and rat smooth muscle (Szabo et al., 1994
).
In summary, the results obtained with NOS inhibitors were not conclusive, but suggested that eNOS does not have a role in the in vitro follicle culture system, since its inhibition appeared not to affect ovulation. The role of other isoforms such as iNOS is less clear. Some evidence of reducing efficiency of ovulation occurred with iNOS inhibitors; however, a clear dose response was lacking and the magnitude of effects was small. Prolonged exposure to competitive inhibitors may have caused accommodation by the cells, and a brief exposure around the time of ovulation might give different results.
The physiological production of NO is impossible to reproduce experimentally because of NO's local production and short half-life. High NO concentrations (10100 µmol/l) are potentially toxic to cells (Griffith and Kilbourn, 1996) and may be obtained using NO donors or when stimulating iNOS with IL-1
or LPS. High NO levels can cause DNA strand breaks and fragmentation (Forstermann et al., 1995
). Chemical donors result in high levels which decline exponentially, effectively producing a pulse of NO at each medium change. de Groot et al. (1993)
reported that SIN-1 at 1, 10 and 100 µmol/l released NO to levels of 1±0.2, 6.5±1 and 26.4±2.4 µmol/l in 30 min respectively. The concentrations of NO produced by donors cannot be measured in our system, but it is clear that their dynamics would be different to that arising from enzymatic conversion of L-arginine.
NO donors did not significantly affect follicle survival at the concentrations tested and did not appear to be toxic. SIN-1 at 1 mmol/l (the highest dose used here) was slightly toxic to human hepatoma cells (Gergel et al., 1995). SIN-1 also produces superoxide which may have biological effects (Hogg et al., 1992
). NOC-18 was reported not to produce toxic metabolites (Hrabie et al., 1993
), but caused dose-dependent apoptosis in mouse macrophages (Shimoaka et al., 1995
).
NO donors resulted in few effects upon ovulation. There was a tendency towards a dose-dependent reduction in timely ovulation, which might have occurred due to damage of some cells by artificially elevated NO. However, ovulation was not promoted, as might potentially have been anticipated. In addition to the non-physiological mode of NO presentation, these results might also be explained by NO exerting negative feedback on NOS enzymes (Buga et al., 1993).
Factors stimulating local NO production would potentially produce a more physiologically relevant result. LPS is a component of the cell wall of gram negative bacteria and is known to induce iNOS mRNA in macrophages (Weisz et al., 1994) and hepatocytes (Geller et al., 1993
). With LPS from 20 to 2000 ng/ml, non-significant increases in timely ovulation (810% higher) and survival rates (36% higher) were observed. These results appeared to have a different profile to the other NO modulators employed but were non-significant. This might support the notion that local NO production might promote ovulation.
IL-1 is a cytokine that stimulates iNOS induction by binding to cell surface receptors on theca and granulosa cells (Adashi, 1997
). IL-1
, at 10100 ng/ml, is reported to be a potent survival factor for rat pre-ovulatory follicles, mediating its effects via NO (Chun et al., 1995
); however, here, IL-1
decreased timely ovulation, and survival rates were reduced with levels >10 pg/ml. Possible explanations for this interesting difference include the use by Chun et al. (1995)
of short term (1 day) cultures of individually dissected, gonadotrophin-stimulated pre-ovulatory follicles, which might be less sensitive to the potentially toxic effects of IL-1
, which may be stage-specific (Markstrom et al., 2002
). Differences between rat and mouse follicles, the duration of exposure and the possibility of other IL-1
effects exerted via different pathways may also be important. Our data suggest that high concentrations of IL-1
may be toxic to mouse follicles in vitro. Timely ovulation was inhibited, although total ovulation was not affected. The features of follicles cultured with IL-1
(reduced cell number and antrum expansion, but no major effect on survival or darkening of granulosa cells), were different to those when L-arginine was omitted (major reduction in survival, darkened granulosa; Mitchell et al., 2004
) which might suggest that IL-1
produced the observed results other than via its effects on NO.
The role of the different isoforms of NOS in follicle growth and ovulation has proved difficult to clarify using the present approach, for several likely reasons. NO is potentially toxic and so mechanisms to ensure its control, including homeostatic feedback from end-products (Buga et al., 1993) via NOS inhibition, and potentially via other intracellular regulators may occur. Other parallel pathways might also have to be inhibited before a significant effect on ovulation would be evident. Other authors, using mice genetically modified to be deficient in either iNOS or eNOS, recently concluded that eNOS was the major isoform affecting the number of oocytes released by ovulation induction (Hefler and Gregg, 2002
). However, <50% inhibition of ovulation occurred in all mice, indicating that eNOS was not the only factor involved. Since the vascular role of eNOS remained intact in iNOS-deficient mice, unlike in the follicle culture system, minor local effects of iNOS, such as were evident in this study, may not have been apparent in their study.
Overall, our study suggests that while NO clearly has a role in follicle growth and ovulation, the importance of the local intrafollicular mechanism, principally involving iNOS, is secondary to the role of effects mediated by extrafollicular eNOS. The profound inhibition of ovulation achieved with NOS inhibitors in vivo was not reproduced in vitro.
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Acknowledgements |
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Submitted on December 8, 2003; accepted on April 4, 2004.
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