Nitric oxide regulates ovarian blood flow in the rat during the periovulatory period

Kenrokuro Mitsube1,2,3, Ulf Zackrisson1 and Mats Brännström1

1 Department of Obstetrics and Gynecology, Göteborg University, Göteborg, Sweden and 2 Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, Sapporo, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The aim of this study was to characterize the roles of nitric oxide (NO) on the rat ovarian blood flow (OBF) during the preovulatory period. METHODS AND RESULTS: Immature Sprague-Dawley rats were primed with pregnant mares’ serum gonadotrophin (PMSG, 15 IU) and given hCG (15 IU) 48 h later. The ovary was exposed 48–56 h after PMSG, a laser Doppler probe was attached to the ovarian surface and OBF was measured at two time periods: preovulatory (PO) 48 h after PMSG and ovulatory (OV) 6–8 h after hCG. A non-selective NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), was injected i.v. (4 and 10 mg/kg) or intrabursally (1 mg/kg). Intravenous administration of L-NAME to OV rats rapidly increased blood pressure and reduced OBF by 30%, which returned to the pretreatment level within 30 min. L-NAME given into the ovarian bursa of both PO and OV rats did not affect blood pressure and reduced OBF by nearly 40%, which remained low throughout the experiment. Intravenous injection of hCG to PO rats increased OBF to 116.1% at 5 min and 133.5% at 30 min in relation to the pretreatment level. When L-NAME was given intrabursally, subsequent hCG injection was without effect. CONCLUSIONS: These results indicate that locally produced NO is important for the maintenance and increase of rat OBF during the preovulatory period.

Key words: blood flow/laser Doppler flowmetry/nitric oxide/NOS inhibitor/ovary


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ovary is a highly vascularized organ and there are distinct cycle-dependent changes in the localization and extent of vascularization (Abisogun et al., 1988Go). During the growth of the ovarian follicles, the capillaries in the theca interna, which surround the avascular antrum/granulosa cell compartment, proliferate and anastomose to form a basket-like structure (Murakami et al., 1988Go). Shortly after the preovulatory surge of LH, ovarian blood flow (OBF) increases (Janson, 1975Go; Brännström et al., 1998Go) and there is a marked dilatation of the vessels around the ovulating follicles (Kranzfelder et al., 1992Go). These changes in the OBF are accompanied by signs of increased permeability in the follicular microvasculature (Okuda et al., 1983Go), extravasation of blood components into the pericapillary space (Brännström et al., 1993Go) and formation of oedema in the ovarian stroma (Bjersing and Cajander, 1974Go).

Our previous studies with intravital microscopy in vivo and in vitro indicated that a sustained high blood flow to the ovulating follicle is of importance for the final rupture of the follicular wall (Löfman et al., 1989Go; Zackrisson et al., 2000aGo). In experiments where the OBF of the rat was reduced by ligation of any of the two major arteries feeding the ovary, the number of ovulations was decreased (Zackrisson et al., 2000bGo). The dependence on blood flow was also demonstrated in the in-vitro perfused rat ovary, where intentionally lowered perfusion pressure reduced ovarian perfusion flow and subsequently decreased ovulation number (Bonello et al., 1996Go). Luteal steroidogenesis is also affected by OBF (Janson et al., 1981Go; Sogn et al., 1984Go).

While the mechanisms underlying the increase and the maintenance of ovarian perfusion during the periovulatory phase have not been fully elucidated, the involvement of several LH-induced intra-ovarian mediators has been suggested (Abisogun et al., 1988Go; Kranzfelder et al., 1992Go). Nitric oxide (NO) is one of these mediators that may contribute to the regulation of OBF. In various extra-ovarian tissues, NO has been established as a molecule that controls basal vascular tonus and microcirculation (Moncada et al., 1991Go). Depletion of NO synthesis by chemical inhibitors of NO synthase (NOS) or by gene targeting results in a severe constriction of blood vessels in most organs and an increased systemic blood pressure (Wang et al., 1992Go; Huang et al., 1995Go). Vasomotion, a short time variation of blood flow observed in vascular beds of actively functioning organs, including the rat ovary, may also be modulated by NO (Griffith 1994Go; Zackrisson et al., 2000bGo). Concerning the ovary, two isoforms of NOS—endothelial and inducible NOS—are expressed mainly in the highly vascularized theca cell layer of the larger follicles during the periovulatory period (Zackrisson et al., 1996Go; Jablonka-Shariff et al., 1997Go). The ovarian endogenous production of NO markedly increases after LH/hCG stimulation with an up-regulation of NOS protein (Bonello et al., 1996Go; Jablonka-Shariff et al., 1997Go). Pharmacological inhibition of NOS caused a reduction in ovulation number in vivo (Shukovski and Tsafriri, 1994Go) and in perfused ovaries in vitro (Bonello et al., 1996Go; Yamauchi et al., 1997Go). The importance of NOS-generated NO in ovulation was later confirmed in NOS knockout mice (Jablonka-Shariff et al., 1998Go).

Previously, we have demonstrated that an NO donor increases the velocity of human perifollicular and intrauterine blood flow, suggesting a vasodilatating effect of NO in the human reproductive organs (Zackrisson et al., 1998Go). Combined with the results of other studies in the ovary and extra-ovarian tissues, modulating blood perfusion in the ovary may be one of the significant mechanisms by which NO can affect various aspects of ovarian function (Bonello et al., 1996Go; Rosselli et al., 1998Go). The aim of the present study was to investigate the roles of NO in the regulation of blood flow in the rat ovary by using laser Doppler flowmetry, which allows direct and continuous measurement of tissue perfusion (Zackrisson et al., 2000bGo).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hormones and chemicals
hCG was purchased from Serono (Rome, Italy). Pregnant mares’ serum gonadotrophin (PMSG) and the non-selective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma Chemical Company (St Louis, MO, USA). Stock solutions of hCG and PMSG were prepared in 0.9% NaCl and stored at –70°C until used. L-NAME was dissolved in 0.9% NaCl just prior to the injection.

Experimental groups and OBF measurement
Immature female Sprague-Dawley rats (B&K Universal, Sollentuna, Sweden) were kept under controlled light (14 h light, 10 h dark) and had free access to pelleted food and water. All experiments were carried out according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Ethics Committee of Göteborg University. At 26 days of age, all rats were treated with 15 IU of PMSG s.c. to promote growth and maturation of a first generation of preovulatory follicles. Some animals were given hCG (15 IU, i.p.) 48 h later to induce the ovulatory cascade with predicted ovulation 12–15 h later.

Longitudinal measurement of OBF was performed by laser Doppler flowmetry (Zackrisson et al., 2000). OBF was measured 46–48 h after PMSG administration, when the ovary had reached a preovulatory stage (PO group), or 6–8 h after hCG, which is a time approximately half-way through the ovulatory stage (OV group). This was estimated to correspond to a time when OBF had reached maximal levels after hCG stimulation (Abisogun et al., 1988Go; Makinoda et al., 1988Go).

The rats were anaesthetized with s.c. injection of ketamine and xylazine (50 and 10 mg/kg respectively) and placed on a heating pad to maintain body temperature at 37°C. Tracheal intubation was performed to maintain patent airways, and the iliac artery and femoral vein on the left side were cannulated with PE-20 polyethylene catheters. Arterial blood pressure (BP) was measured directly through the iliac artery cannula using a Grass polygraph (Grass instruments, Quincy, MA, USA). The animals were continuously infused through the femoral vein with ketamine (0.5 mg/kg/min) and xylazine (0.1 mg/kg/min) in 0.9% NaCl.

One ovary was exposed by a flank incision and the ovary was stabilized by a ligature tied to the periovarian adipose tissue. A laser Doppler probe (Probe 407; fibre separation = 0.25 mm; Perimed AB, Stockholm, Sweden) with an adhesive miniholder was placed on the ovarian surface at a site with no larger blood vessels for measurement of relative changes in OBF. The probe and the incision wound were covered with an aluminium foil shield to reduce possible effects of the external light. The signal was analysed by a laser Doppler flowmeter (PeriFlux System 5000 with PF5010 laser Doppler perfusion monitor units; Perimed AB) and was continuously recorded by PeriSoft software for Windows (Perimed AB). The OBF was quantified by laser Doppler flowmetry as an arbitrary perfusion unit, which is proportional to the number and velocity of moving blood cells in an tissue volume of ~1 mm3 (Lissbrant et al., 1997Go). As the OBF measured in different preparations could not be directly compared, the average flow between –5 to 0 min in relation to the injection time was used as a basal level and the relative OBF values at 5 and 30 min time-points were used for analysis. A stable blood flow signal was recorded at least for 10 min prior to the administration of any drugs/hormones and all procedures were completed within 90 min after the laparotomy.

Experimental protocols (Figure 1Go)
Protocol 1
A non-selective NOS inhibitor, L-NAME, was given i.v. to OV-stage rats through the femoral vein (4 mg/kg, n = 7 or 10 mg/kg, n = 4 in 100 µl saline versus saline control; n = 4) to produce a systemic inhibition of NO synthesis (Powers et al., 1995Go; Lissbrant et al., 1997Go).



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Figure 1. Schematic drawings of the three different experimental protocols used in the present study.

 
Protocol 2
To minimize the systemic effect, L-NAME was administrated locally into the ovarian bursa (intrabursally; i.b.). The experiment was conducted in rats of both PO group (L-NAME, n = 5 and control, n = 5) and OV group (L-NAME, n = 9 and control, n = 5). A polytetrofluoroethylene (PTFE) tube (diameter 0.4 mm; Cole-Parmer International, Vernon Hills, IL, USA), with the tip cut sharp, was attached to a microsyringe filled with either of L-NAME (1 mg/kg) or 0.9% NaCl in a total volume of 5 µl. The PTFE tube was threaded through the periovarian adipose tissue into the ovarian bursa and the laser Doppler probe was applied on to the ovarian surface. After 10 min of stable recordings, L-NAME or saline (5 µl) was injected into the bursal cavity and the changes in OBF were analysed in the same way as in protocol 1. In order to confirm that the tube had been actually inserted into the bursal cavity, at the end of each experiment 30–50µl of solution and/or air was injected through the tube to ensure that the expected bulging of the capsule wall took place.

Protocol 3
The effect of L-NAME on the immediate changes in OBF after hCG injection was studied. Initially, L-NAME (1mg/kg in 5µl of saline) or the same volume of saline was administrated i.b. to the PO-stage rats (first injection; L-NAME group and saline group respectively). After stable levels of OBF were observed for at least 5 min, 15 IU of hCG (in 100 µl of saline) or saline for control was injected into either of the groups through the femoral vein (second injection). These treatments resulted in four groups (first injection–second injection); saline–saline (n = 5), saline–hCG (n = 5), L-NAME–saline (n = 6) and L-NAME–hCG (n = 5). The changes in OBF in relation to the basal level prior to any of the treatments (PT) were measured at 5 min time-points after the first injection, and at 5 and 30 min time-points after the second injection. The effect of hCG on OBF in the presence or absence of L-NAME was assessed by comparing OBF levels between hCG treated and untreated rats, in either of the L-NAME group and the saline group separately.

Statistical analysis
Values are presented as mean ± SEM. The results were analysed using repeated measures analysis of variance (ANOVA) followed by Scheffé’s test for comparison within a group at different time-points. Differences among multiple groups in response to the treatment were evaluated by one-factor ANOVA followed by Scheffé’s test. P < 0.05 was considered to be statistically significant.


    Results
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 Materials and methods
 Results
 Discussion
 References
 
Intravenous injection of L-NAME (protocol 1)
Intravenous administration of L-NAME in the rats of OV stage resulted in a rapid and significant (P < 0.01) increase of the mean arterial BP that lasted throughout the 30 min examination period (Figure 2Go). The increase of BP was not statistically different in rats given the higher dose (10 mg/kg) of L-NAME as compared with those with the lower dose (4 mg/kg). A marked and rapid decrease in OBF was observed with a nadir that reached 70% of the initial level around the 5 min time-point at both doses (Figure 3Go). The OBF was rapidly reversed and returned to the pretreatment level within 30 min (Figures 3 and 4GoGo). Vasomotion activity was observed (Figure 4Go) but no significant changes in its amplitude or frequency could be detected (data not shown).



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Figure 2. Changes in mean arterial pressure in response to i.v. administration of a non-specific NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME: 4 mg/kg, n = 7 or 10 mg/kg, n = 4) as compared with saline-treated controls (n = 4). Rats were pretreated sequentially with PMSG and hCG (54–56 and 6–8 h prior to the experiments respectively). PT = pretreatment value; mean OBF between –5 and 0 min in relation to the injection was used as a basal level, to which the changes in OBF was standardized. ++Significantly (P < 0.01) higher than corresponding basal level (PT) of the same group. **Significantly (P < 0.01) higher than control at corresponding time-points.

 


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Figure 3. Changes in ovarian blood flow (OBF) in response to i.v. administration of a non-specific NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME: 4 mg/kg, n = 7 or 10 mg/kg, n = 4) as compared with saline-treated controls (n = 4). Rats were pretreated sequentially with PMSG and hCG (54–56 and 6–8 h prior to the experiments respectively). Relative values to the pretreatment basal levels (–5 to 0 min; PT) are presented as means ± SEM. ++Significantly (P < 0.01) lower than corresponding basal level (PT) of the same group. **Significantly (P < 0.01) lower than control at corresponding time-points.

 


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Figure 4. A representative result of ovarian blood flow (OBF) measurement by laser Doppler flowmetry. The mark indicates i.v. injection of L-NAME (10 mg/kg). The OBF values are presented in an arbitrary perfusion unit.

 
Intrabursal injection of L-NAME (protocol 2)
In the initial experiments with i.v. administration of L-NAME (protocol 1), it was not clear if the recovery of OBF after the initial decrease was caused by local ovarian mechanisms or by the systemic reaction to the NOS inhibitor. Thus in the experiments of protocol 2, L-NAME was administrated locally into the ovarian bursa in order to minimize the systemic effects. The experiments were carried out in rats of both PO and OV stages (Figure 1Go). There was no detectable change in systemic BP (Figure 5Go). In both treatment groups, i.b. administration of L-NAME rapidly reduced OBF (at 5 min time-point, 63.6 ± 13.0% in PO group, Figure 6Go; 60.0 ± 5.6% in OV group, Figure 7Go). In contrast to the results of previous experiments with i.v. injection (protocol 1), the OBF remained low during the 30 min of observation (at 30 min time-point; 68.6 ± 4.3% in PO group, Figure 6Go; 72.8 ± 4.8% in OV group, Figure 7Go). These i.b. treatments did not significantly change the vasomotion activity (Figure 8Go).



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Figure 5. Mean arterial pressure after i.b. administration ofL-NAME (1 mg/kg, n = 9) as compared with saline-treated controls (n = 5). As the results in preovulatory and ovulatory stage animals were identical, only those in the latter group were presented. Rats were pretreated sequentially with PMSG and hCG (54–56 and 6–8 h prior to the experiments respectively). No significant changes in blood pressure were observed in either of the groups. PT = pretreatment value; between –5 and 0 min in relation to the injection.

 


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Figure 6. Ovarian blood flow (OBF) in response to i.b. administration of L-NAME (1 mg/kg, n = 5) as compared with saline-treated controls (n = 5). Rats were pretreated with PMSG 46–48 h prior to the experiment. Relative values to the pretreatment basal levels (–5 to 0 min; PT) are presented as means ± SEM. ++Significantly (P < 0.01) lower than corresponding basal level (PT) of the same group. **Significantly (P < 0.01) lower than control at corresponding time-points.

 


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Figure 7. Changes in ovarian blood flow (OBF) in response to i.b. administration of L-NAME (1 mg/kg, n = 9) as compared with saline-treated controls (n = 5). Rats were pretreated sequentially with PMSG and hCG (54–56 and 6–8 h prior to the experiments respectively). Relative values to the pretreatment basal levels(–5 to 0 min; PT) are presented as means ± SEM. +, ++Significantly (+P < 0.05, ++P < 0.01) different from corresponding basal level (PT) of the same group. **Significantly (P < 0.01) lower than control at each time-point.

 


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Figure 8. A representative result of ovarian blood flow (OBF) measurement by laser Doppler flowmetry. The mark indicates i.b. injection of L-NAME (1mg/kg). The OBF values are presented in an arbitrary perfusion unit.

 
Effects of L-NAME on hCG-induced increase of OBF (protocol 3)
Changes in OBF in the experiment according to protocol 3 are presented in Figure 9Go. No significant changes in BP were observed in any of the treatment groups (data not shown). The initial i.b. treatment with L-NAME (first injection) reduced OBF to the similar levels as observed in the previous experiments of protocol 2. In the rats administrated i.b. with saline, a subsequent i.v. injection of hCG (second injection) increased OBF significantly to 116.1 ± 2.3% at 5 min and 133.5 ± 6.6% at 30 min in relation to the basal pretreatment level. However, in the presence of L-NAME, the injection of hCG did not alter OBF from those injected with saline.



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Figure 9. Ovarian blood flow (OBF) responses after i.b. administration of L-NAME (1 mg/kg) or saline (first injection) followed by hCG (15 IU) or saline (second injection) in rats pretreated with PMSG 46–48 h prior to the experiments. Relative values to the pretreatment basal levels (PT; average OBF between–5 to 0 min in relation to the first injection) are presented as means ± SEM. +, ++Significantly (+P < 0.05, ++P < 0.01) higher than corresponding OBF levels of the same group at the time-point5 min after the first injection of L-NAME or saline.*, **Significantly (*P < 0.05, **P < 0.01) different from the saline-saline treated group at each time-point.

 

    Discussion
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 Materials and methods
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 References
 
Recent studies in both animal and human indicate that various reproductive events in the ovary are dependent on NO-associated pathways (Rosselli et al., 1998Go). Involvement of locally produced NO in the regulation of follicular development (Anteby et al., 1996Go; Jablonka-Shariff et al., 1999Go), ovulation (Shukovski and Tsafriri, 1994Go; Bonello et al., 1996Go), oocyte meiotic maturation (Jablonka-Shariff et al., 1998Go) and ovarian steroidogenesis (van Voorhis et al., 1994Go; Jablonka-Shariff et al., 1999Go) have been demonstrated. Since NO is a potent vasodilator, it has been implied that NO exerts part of its physiological actions in the ovary through modulation of OBF, possibly by facilitating an increase or maintenance of local blood perfusion in the ovary (Zackrisson et al., 1998Go). These inferences were mostly based on rather indirect evidences obtained from in-vitro perfusion studies of the rat ovary (Bonello et al., 1996Go) and on studies conducted on non-ovarian tissues (Wang et al., 1992Go). Since several observations indicate that the response to NOS inhibitors may be heterogeneous among different vascular beds (Lissbrant et al., 1997Go; Hernández et al., 1999Go), the present study was conducted to elucidate the specific features of the vascular bed of the rat ovary.

In the present study, we measured immediate changes in OBF as well as vasomotion activity in the rat ovary in the presence of the non-selective NOS inhibitor, L-NAME. The laser Doppler flowmetry technique was used since this method enables real-time and longitudinal measurements of OBF with minor experimental manipulations of the ovary (Zackrisson et al., 2000bGo). However, it should be noted that in most of the experiments, a tendency of increased OBF was observed in the control groups at the 30 min time-point, when the ovary had been exposed for 40–60 min. This OBF increase may be due to some effects of the experimental procedures, such as secretion of vasoactive substances due to manipulation and changes in temperature or humidity, although we attempted to minimize these factors by careful preparation and by the shield to enclose the peritoneal cavity. Taking this observation into consideration, the experiments were only conducted up to the 30 min time-point and the results were compared with controls with the same preparation.

Intravenous injection of L-NAME to rats of OV stage resulted in a rapid decline in OBF and an increase in the systemic BP. This response is in line with previous results obtained in studies of other organs (Wang et al., 1992Go) and with the accepted notion that NO is an important mediator for the maintenance of local blood perfusion as well as the regulation of BP (Wang et al., 1992Go; Huang et al., 1995Go). However, in our longitudinal observation, reduction of OBF caused by systemic administration of L-NAME proved to be a reversible phenomenon, since OBF returned to the pretreatment level within 30 min after the injection. Since the half-life of L-NAME is ~250 min (Whiting et al., 1994Go) and an observed increase in BP by this NOS inhibitor remained high during the entire experiment, it is unlikely that the recovery of OBF was due to the loss of L-NAME activity in the ovary. Therefore, the results of the experiments with systemic administration of the NOS inhibitor suggest that the role of NO in the regulation of ovarian microcirculation is very limited and transient in itself, or that mechanisms exist that counteract the vasoconstrictive property of L-NAME in the ovary.

In order to elucidate whether this restoration of OBF occurred because of any local ovarian events or as a systemic reaction to the NOS inhibition, L-NAME was administrated locally into the ovarian bursa in the second set of experiments. With this method, the effect of L-NAME on the systemic circulation was minimized, as indicated by the unchanged BP after treatment with the NOS inhibitor. After i.b. administration of L-NAME, OBF declined immediately to the same extent (~40% reduction in both the PO and the OV groups) as observed in the rats treated i.v. with L-NAME. In contrast to i.v. injection, local administration of L-NAME caused a reduction in OBF that persisted throughout the 30 min observation period. These results indicate that the restoration of OBF observed in the first set of experiments was caused by the systemic and extra-ovarian effects of L-NAME. Therefore we could conclude that endogenously produced NO in the ovary contributes to the maintenance of a high ovarian blood perfusion during the preovulatory period. The mechanisms that caused the rapid normalization of OBF when L-NAME was given i.v. are unclear. It is conceivable that redistribution of blood flow between different vascular beds occurred during this period. This may be a reaction to the sustained high systemic BP or through a modulation of autonomic functions by reduced NO production in the nervous system (Zanzinger, 1999Go).

The doses of L-NAME used i.v. in the present study were in the same range as those which reduced ovulation number in previous in-vivo studies (Shukovski and Tsafriri, 1994Go; Powers et al., 1995Go). The results of previous in-vivo experiments and in-vitro ovarian perfusion (Bonello et al., 1996Go) suggested that the reduction in OBF might be one of the mechanisms responsible for the lower ovulation number caused by the NOS inhibitors. While the methods of the present study did not allow longitudinal observations for >30 min, it could be inferred that the OBF during the preovulatory phase was not so severely reduced by the systemic treatment with L-NAME as expected before. Therefore, the effects of NOS inhibitors on ovulation in the previous in-vivo studies may not be through the reduction in OBF. This hypothesis is supported by a study with ovarian perfusion in vitro, where reduced ovarian perfusion flow resulted in lower ovulation number, while the level of inhibition was not to the same degree as that observed in the L-NAME-treated ovary (Bonello et al., 1996Go). The difference in the level of inhibition suggests the importance of non-vasodilatating functions of NO in the ovulatory process, such as modulation of ovarian vascular permeability (Powers et al., 1995Go), mobilization of local mediators (Faletti et al., 1999Go) and the altered apoptosis in the ovary (Matsumi et al., 2000Go). Recently, we have demonstrated that ovarian intra-follicular pressure (IFP) of the rat increases prior to ovulation (Matousek et al., 2001Go). Local application of L-NAME to the ovary completely negated this preovulatory elevation of IFP, suggesting that NO may promote the ovulatory process partly through the modulation of IFP (Matousek et al., 2001Go).

It has been established that OBF increases within minutes after the preovulatory LH surge (Janson, 1975Go). Several other studies using a variety of different techniques consistently indicate a steep increase in ovarian perfusion and capillary dilatation after LH/hCG stimulation, that reaches maximal levels ~3–6 h after the gonadotrophin injection (Abisogun et al., 1988Go; Makinoda et al., 1988Go; Tanaka et al., 1989Go). Our experiment with i.b. administration of L-NAME (according to protocol 2) was conducted in two groups of rats (PO and OV) to compare the involvement of NO in the regulation of OBF in the presence or absence of hCG stimulation. We observed that OBF decreased to the same level in both groups (60% of the pretreatment value) when L-NAME was injected i.b., indicating that the NO-regulated portion of the total OBF was in the same range regardless of hCG stimulation. Considering the LH/hCG-induced increase in the absolute OBF values reported in previous studies (Janson, 1975Go; Makinoda et al., 1988Go; 275% in rabbits and 155% in rats respectively), the present results could only be explained when both NO-dependent and NO-independent fractions of OBF increase proportionally at this period (4–6 h) after hCG. While the actual mechanisms behind the NO-independent increase of OBF need to be elucidated, the contribution of several other ovarian local mediators, such as eicosanoids (Tsafriri and Reich, 1999Go) and vascular epithelial growth factor (Agrawal et al., 1999Go) have been suggested.

In the experiments according to protocol 3, we examined the immediate increase of OBF following hCG stimulation. The changes in OBF at this earlier period might be highly dependent on NO up-regulation, since a delay has been observed for most of the other ovarian local mediators that are activated by gonadotrophin stimulation (Richards et al., 1998Go). In rats treated i.b. with saline, the subsequent i.v. injection of hCG caused a rapid and significant increase in OBF, which was agreement with previous studies (Janson, 1975Go; Abisogun et al., 1988Go; Makinoda et al., 1988Go; Tanaka et al., 1989Go). Pretreatment with L-NAME immediately reduced OBF by 40% and the subsequent hCG injection did not cause any changes in OBF. This observation indicated that an NO-associated pathway in the ovary was prerequisite for the rapid elevation of OBF after hCG, and furthermore suggested that the up-regulation of NO production was responsible for the initial change in the ovarian perfusion.

In conclusion, the present study indicates that locally produced NO is of importance for the maintenance of rat OBF during the preovulatory period. While the extent of the contribution of NO to the increase in OBF after the LH surge is not clear, the constitutive or up-regulated secretion of NO in the ovary is necessary for the vascular dilatation observed during this period. Systemic inhibition of NOS may mobilize compensatory pathways that counteract the ovarian local vasoconstriction. The roles of NO in the ovulatory process do not seem to be restricted to modulation of the OBF, suggesting that other NO-regulated pathways are also critical for the full completion of a normal ovulatory process.


    Notes
 
3 To whom the correspondence should be addressed at: Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, N-15, W-7, Kita-ku, Sapporo, 060-8638, Japan. E-mail: ken_mitsube{at}hotmail.com Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 8, 2002; accepted on June 8, 2002.





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