1 Department of Obstetrics and Gynecology and 2 Department of Surgery, Göteborg University, Sweden
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Abstract |
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Key words: blood flow/laser Doppler/ovary/ovulation/rat
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Introduction |
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In parallel with well described structural changes during the ovulatory process, involving the action of several proteases (Brännström and Janson, 1991), dramatic blood flow alterations occur in the ovary. Increased blood flow has been suggested to be of importance for follicular development, rupture, and corpus luteum formation (Janson, 1975
). The pre-ovulatory luteinizing (LH) surge is known to induce ovarian production or release of several mediators, such as eicosanoids (LeMaire, 1980), histamine (Schmidt, 1988), neuropeptides (Kannisto, 1992) and nitric oxide (Zackrisson et al., 1996
), all of which may have effects on the vascular system of the pre-ovulatory follicle. It is unknown to what extent the ovulation-promoting actions of these mediators act directly on the extracellular matrix of the follicle wall or via indirect vascular mechanisms.
A number of different techniques, such as venous drainage measurements (Piacsek and Huth, 1971; Janson and Albrecht, 1975
), corrosion cast combined with electron microscopy (Kitai et al., 1985
), indirect measurements using radioactive isotopes, such as ovarian content of radio-iodinated serum albumin (Ellis, 1961
), 133Xe clearance (Janson and Jansson, 1997
), indicator fractionation using 86Ru (Janson and Albrecht, 1975
) or radioactive microspheres (Janson, 1975
), thermocouple methods (Makinoda et al., 1988
), and analysis of haemoglobin content in the ovary (Tanaka et al., 1989
) have been utilized in attempts to study cyclic changes of ovarian blood flow. These methods have shown various limitations since they are indirect and give only a single recording of blood flow. Thus, these methods may not be optimal for measuring the short and long term variations in blood flow, which undoubtedly occur during the ovulatory process. Recently, it has been shown that laser Doppler flowmetry can be used for recordings of ovarian blood flow in the rat (Brännström et al., 1997
). With this method and intravital microscopy the existence of regular short term variations of ovarian blood flow, termed vasomotion, were demonstrated. The phenomenon of vasomotion has previously been described in the testis (Damber et al., 1982
), where laser Doppler flowmetry was used to study blood flow in a longitudinal manner.
Intravital microscopy studies of ovulation in vitro (Löfman et al., 1989) and in vivo (Brännström et al., 1997
) indicate that a sustained high blood flow to the follicle base in combination with increased vascular permeability is of importance during the stages of the ovulatory process immediately preceding the follicular rupture. These vascular changes are postulated to prevent the follicle from collapsing and to ensure the extrusion of the oocytecumulus complex from the interior of the follicle. Further support for the importance of high ovarian blood flow is provided by a study utilizing the in-vitro perfused rat ovary model, where a nitric oxide inhibitor decreased the perfusate flow and subsequently suppressed the ovulation rate (Bonello et al., 1996
). This effect was replicated in perfusions with an intentional reduction of the perfusion pressure, resulting in a decreased number of ovulations. Therefore, a continuous high perfusion pressure appears to be critical to successful ovulation.
It appears that only one study has attempted to investigate a possible causal relationship of blood flow changes and the rate of ovulation in vivo. A reduced ovulation rate in rats was seen when examined more than 10 days after ligation of the uterine artery at different levels (Peppler, 1972). The author suggested that blood supply to the ovary via the uterine artery is important for a complete ovulatory response. The results may not necessarily be due to a change in the blood flow, since a possible reduction of the ovarian blood flow can be reversed within this time frame by the remaining ovarian artery (Blasco et al., 1973
). Furthermore, since counter-current mechanisms between the uterine vein and the ovarian artery have been shown to exist in the human (Bendz, 1977
) and to affect ovarian function in the sheep (McCracken et al., 1971
), it is possible that vascular ligations may affect ovulation by similar mechanisms. The aims of the present study were to quantify acute blood flow changes, at the pre-ovulatory stage, after ligation of one or both of the two major arteries supplying the ovary and to determine the effect of these ligations on ovulation.
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Materials and methods |
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Animals
The experiments were approved by the animal ethics committee of Göteborg University and were carried out according to the principles and procedures outlined in the NIH Guiding Principles for the Care and Use of Research Animals. Female immature SpragueDawley rats were obtained from B-K (Stockholm, Sweden). The animals were kept under controlled conditions with lights on from 05.00 to 19.00 and they had free access to water and pelleted food. At 09.00 on day 28 of age, the rats were given 15 IU eCG s.c. to induce maturation of a first generation of pre-ovulatory follicles. Forty-eight hours later the rats were injected with 15 IU HCG s.c. to induce ovulatory changes with follicular rupture, which was estimated to occur 1015 h later (Tanaka et al., 1989).
Histology and ovarian weights
Initial experiments were performed with bilateral sham operation of ovarian artery/vein (OS) or bilateral ligation and severance of the ovarian artery/vein (OL) at 3 h after HCG and the ovaries were examined about 17 h later with ovulation rate assessed by counting the number of rupture sites as previously described (Bonello et al., 1996) and then also by histology. The number of rupture sites, defined as reddish, non-transparent bulging structures, was counted prior to paraffin embedding. Ovaries used for histology were first placed for 24 h in paraformaldehyde (4%), and then embedded in paraffin followed by staining with haematoxylin and eosin. Under a dissecting microscope, the ovaries were examined morphologically by two different persons blind to experimental procedures. Some ovaries were excised 3 h after OL and OS and were weighed after blotting (immediately after excision) and drying (room temperature in a dust-free chamber for 4 weeks).
Experimental design to study ovulation rate
The rats were divided into treatment groups: bilateral sham operation of ovarian artery/vein (OS), bilateral ligation and severance of ovarian artery/vein (OL), bilateral sham operation of ovarian branch of uterine artery/vein (US), bilateral ligation and severance of the ovarian branch of uterine artery (UL), bilateral sham of both the ovarian artery and the ovarian branch of the uterine artery (OS+US), and bilateral ligation and severance of both the ovarian artery/vein and the ovarian branch of the uterine artery (OL+UL). Bilateral operations were performed because preliminary experiments with these animals showed a large variation in ovulation number beween the right and left ovary, with a fairly constant number of ovulations per rat. Each experimental day the same number of rats from age, weight and litter matched rats were allocated to respective sham and ligated groups to minimize variations. Operations were performed immediately prior to (0 h), 3 h after, 6 h after, or 9 h after HCG injection.
The rats were anaesthetized with i.p. injection of a combination of ketamine and xylazine (40 and 7 mg/kg body weight). Prior to laparotomy the fur was carefully washed with 70% ethanol. Paramedial incisions (~20 mm in length) were then performed bilaterally over the ovaries. The ovaries were identified and care was taken not to directly manipulate the ovary, since this procedure has previously been shown to increase ovarian blood flow in the rabbit (Janson 1988). The peritoneum was gently opened and the adipose tissues surrounding either the ovarian artery/vein (OS, OL) or ovarian branch of the uterine artery/vein (US, UL) were removed close to their entry into the ovary by gentle traction with dissection forceps. Due to the small size of the arteries supplying the ovary, it was not possible to ligate these vessels separately from the paired veins. Two Autosuture titanium premium surgiclips s-9.0 (Autosuture, Sollentuna, Sweden) were placed to ligate the vessels, after which the vessels were severed with dissection scissors (Figure 1
). An identical procedure, except for placement of clips and severance, was performed with the sham operated animals. The peritoneum and the skin were then separately sutured with Vicryl 40 sutures. After surgery, the rats were kept in separate cages. Twenty hours after HCG injection the animals were anaesthetized a second time, as previously described, and the chest was opened to sample blood by cardiac puncture. Serum was stored at 20°C for later analysis of progesterone concentrations. Ovaries and oviducts were then removed and placed in phosphate-buffered saline (PBS). The oviducts were separated from the ovaries and the ampullary region was identified. The oocyte/cumulus cell masses were identified within the oviduct which was then incised to allow extrusion of the oocytecumulus complexes. The adhesive cell mass, containing oocytes and cumulus cells, was placed for 3 min in a drop of PBS containing hyaluronidase (60 IU/ml) to separate the cumulus cells from the oocytes. The oocytes were then counted under a microscope. The observers counting the oocytes were blind to the specific experimental procedures.
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The experiments were carried out for OL and OS (0 h) as described above and the animals were allowed to recover from this surgery for 3 h after which animals were once more anaesthetized by i.p. injection of a combination of ketamine and xylazine (40 and 7 mg/kg body weight respectively). The right common carotid artery was exposed and cannulated with a PE 25 polyethylene cannula followed by injection of 1 ml of the microsphere suspension over 1 min. To obtain a homogeneous mixture of microspheres, the suspension was mechanically stirred during the injection. Before the intracardiac injection, the microspheres were sonicated for 5 min and then diluted with saline to a concentration of 40 000 to 60 000 spheres per ml to obtain an adequate number of spheres in the tissue, to allow reliable calculation. This was based on previous investigations demonstrating that >384 microspheres must be present in a tissue to permit blood flow to be measured at a 95% confidence level with a precision of 10% (Buckberg et al., 1971).
Initial experiments (n = 8) revealed that the right ventricle of the heart was frequently perforated by the carotid artery cannulae. In subsequent experiments, the tip of the cannula was withdrawn at a standardized length from the mandibula. The animals were killed through an air embolus 2 min after the microsphere injection and both ovaries and kidneys were dissected out, weighed, and then counted in a gamma counter. A reference sample was made each experimental day by adhering microspheres to a greased millimetre-squared paper after which the number of adhered spheres was counted under a microscope. The radioactivity of the reference sample was measured, allowing calculation of the exact number of entrapped spheres in the analysed tissue. The weight of the animals varied from 71 to 98 g. Due to the small sizes of the animals and their vessels it was not possible to cannulate the femoral artery to obtain a blood-withdrawal reference sample. Instead, ovarian blood flow was calculated in relation to the kidney blood flow, since it has been shown in rabbits that blood flow to the kidneys is unaffected by LH injection (Janson 1975). Ovarian blood flow index was calculated according to the formula:
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Although both ovaries were pooled for each animal, a high number of the ovarian pairs contained <384 entrapped spheres. Additionally, the distribution of microspheres between the right and left ovary frequently showed uneven distribution, indicating an incomplete mixture of microspheres with the blood. These methodological difficulties may be caused by variations in the exact positioning of the injection cannula, leading to uneven distribution of spheres in peripheral blood and thus also reflected in the tissue.
Progesterone assay
Twenty hours after HCG injection, serum was sampled after heart puncture during anaesthesia and stored at 20°C until further analysis. Concentrations of progesterone in serum were measured with a Delfia kit (Wallac, Turku, Finland) with an intra-assay variation of <6%. The Delfia kit had been validated and found suitable for the measurements of progesterone in rat serum by serial dilution experiments and in measurements of progesterone concentrations in serum of ovariectomized rats which had been spiked with known concentrations of progesterone. No modifications had to be made.
Statistics
Results are presented as mean % ± SEM and mean ± SEM. For laser Doppler flowmetry experiments, blood flow is presented as the percentage of the mean flow before ligation. Estimations of ovarian blood flow in microsphere experiments are related to kidney blood flow, and are presented as the percentage in relation to kidney flow of the same animals. Statistical differences for laser Doppler flowmetry experiments were calculated by the Friedman test followed by Wilcoxon signed-rank test. The KruskalWallis test followed by the MannWhitney U-test were used for comparison of sham groups and ligated groups. These distribution-free tests were used despite the knowledge that blood flow data and the data for number of ovulations were normally distributed. P < 0.05 was considered significant.
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Results |
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The blood pressure remained stable throughout the experiments (mean arterial pressure 9095 mmHg). The blood flow demonstrated rhythmical short-term variations (vasomotion) in all preparations, with a frequency between 7 and 12 per min and an amplitude of 27.0 ± 10.8% of the mean blood flow value (Figure 3 Top). The vasomotion activity remained with unchanged frequency and amplitude after OL or UL. Ligation of the ovarian branch of the uterine artery (UL) performed during the interval 03 h after HCG caused a decrease of blood flow with mean values of 83.9 ± 5.4% of initial values which occurred within 1520 s after ligation (Figure 3
Middle, Table I
). A similar decrease was seen after OL (Figure 3
Lower, Table I
). Ovarian blood flow continued to decrease until stable values at lower levels were reached 1520 min after treatment (Table I
). When UL or OL were performed at later stages of the ovulatory phase (69 h after HCG), the reduction was somewhat more pronounced (Table I
). In preparations with both UL and OL (03 h), blood flow was immediately reduced to a stable value of ~1/5 of the value prior to ligation (Table I
). The same pattern was observed after UL+OL 69 h after HCG (Table I
). In all but one of UL+OL vasomotion was absent.
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Histology and ovarian weights
In initial experiments a reduction of the number of visible corpora lutea per rat was found when OL (3 h; n = 7) and OS (3 h; n = 7) were compared 20 h after HCG injection (17.5 ± 3.6 and 37.0 ± 2.5, respectively). Microscopic examination indicated an increased number of entrapped oocytes in ovaries of the ligated groups (Figure 4). There were no other obvious morphological differencies between sham operated and ligated ovaries. The wet weights for OL (3 h; n = 7) and OS (3 h; n = 7) were similar (30.1 ± 0.5 and 30.7 ± 0.7 mg respectively). Dry weights were ~1/5 of the wet weight and did not show any difference between OL and OS (5.4 ± 0.9 and 5.4 ± 1.2 mg respectively).
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Discussion |
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Observations of blood flow with laser Doppler flowmetry showed a rapid and persistent reduction after both OL and UL independent of the stage of the ovulatory process. The blood flow stabilized within 20 min after the procedures at values ~5778% of the initial blood flow. The anaesthesia only allowed the recording for a time up to ~1 h; we cannot, from these experiments, determine whether these reductions in blood flow remained during the entire ovulatory process. However, the experiments utilizing radioactive microspheres pointed towards a decreased blood flow for at least 3 h after ligation.
The blood flow reduction after OL was slightly more pronounced than after UL and this difference was observed both in the early and late phase of the ovulatory process. Although the difference was not significant, the findings of this study contradict earlier studies that have suggested the uterine artery to be the major contributor of blood flow to the rat ovary (Del Campo and Ginther, 1972). In a recent study, using venous outflow measurements, the contribution of ovarian blood in the non-pregnant cycling rat was found to be equal between ovarian and uterine arteries with an increase in uterine contribution during pregnancy (Massa and Bruce, 1997
).
A low blood flow of ~1/5 of the normal value was observed despite the ligation of both major arterial supplies to the ovary. This demonstrates that there is a small, but significant, collateral circulation in the ovary, where a major part may come through the suspensory ligamental tissue.
In all experiments with laser Doppler flowmetry, regular short term variations of blood flow (vasomotion) were observed with a frequency of 712 per min. The functional role of these rhythmical blood flow changes is not clear but it has been speculated that they may facilitate transcapillary fluid exchange since increased flow would promote fluid efflux to the interstitial space, and decreased flow would promote fluid influx to the capillaries (Intaglietta and Gross, 1982). This mechanism may be of importance to enhance the transcapillary and interstitial exchange of various hormones and cytokines between the different cell types of the ovary. The fact that vasomotion activity was not recorded after UL+OL procedure, demonstrates that the registration of short term variations are due to an intra-ovarian regulation of blood flow rather than mechanical influence on the laser Doppler probe by direct transmission of uterine/oviductal contractions or intestinal motions.
The significant reduction in ovulation rate when UL or OL were performed during the early ovulatory phase indicates that this stage of the process may be most sensitive to haemodynamic disturbances. It is possible that the ligations at 0 h would reduce the delivery of HCG to the pre-ovulatory follicles and thereby reduce ovulation rate. During later stages of the ovulatory process, the reductions of the ovulation rate in OL were more pronounced compared to UL, indicating an increasing role with time for blood flow through the ovarian artery, in facilitating follicular rupture. The ovarian artery may have a greater capability to compensate for a relative reduction of blood flow, since there was no reduction of the ovulation rate in UL at 6 and 9 h. Another explanation for decreased ovulation rate after OL during late ovulatory phase is based upon the assumption that the hydrostatic pressure gradient over the blood/follicle barrier is required for and also generates the driving force for final follicular rupture and extrusion of the oocyte (Löfman et al., 1989). Ligation of the ovarian artery late in the ovulatory process may reduce blood flow and thereby intrafollicular pressure below a value where it is not able to break the partly degraded follicle apex.
Of interest are reports (Peppler 1972, 1975
) of a reduced ovulation rate in the rat, when hysterectomy or ligation of the uterine artery was performed. In these studies, the blood flow was not analysed and the ovulation rate was not measured until 1116 days after surgery, which theoretically could allow the remaining artery to compensate for a reduction of blood flow (Blasco et al., 1973
). In a recently published study (Massa and Bruce, 1997
), ovarian blood flow was examined by venous effluent technique in the rat during dioestrus and pregnancy. In the non-pregnant rat, there was no major difference between the two major ovarian arteries regarding vascular resistance and contribution of ovarian blood flow. However, these variables of circulation tended to be more pronounced for the ovarian artery than for the uterine artery. In the same study, ligation of the individual vessels also indicated that blood flow reduction was larger when ligation was performed on the ovarian artery compared to the uterine artery in line with findings of the present study. Moreover, the blood flow reductions after ligations were in the same range as those observed in our laser Doppler flowmetry experiments during the early ovulatory phase.
Possible effects of the ligations on ovarian nerve supply must also be taken into consideration. The ovarian nerves reach the ovary via two routes. The major portion of the nerve supply forms a plexus parallel to and around the ovarian vascular pedicle and a smaller portion of the ovarian nerve supply reaches the ovary via the ovarian suspensory ligament (Burden and Lawrence, 1978; Burden et al., 1983
). The nerves enter the ovary at the hilus and some are terminated in the theka layer of the follicle (Owman and Sjöberg, 1966
). The ovarian nerves have been suggested to be important for follicular contractility and vascular regulation (Owman et al., 1979
) and several transmitters may be functional in the ovulatory process and regulation of ovarian blood flow (Kannisto et al., 1992
). During OL procedure, it is likely that the ovarian nerves are damaged and this may directly influence ovulation. It was recently demonstrated that intrabursal administration of a noradrenergic neurotoxin reduces the ovulation rate (Goldman et al., 1996
). It has also been shown that an
-adrenoreceptor blocker prevents ovulation in the rabbit when injected into the aorta at a site just above the branching of the ovarian arteries (Virutamasen, 1971). On the other hand, transection of the suspenory ligament (Selstam et al., 1985
) or complete sympathetic denervation in rats due to freezing of the ovarian vascular pedicle and suspensory ligament (Wylie et al., 1985
) do not affect ovulation rates, indicating that the final stages of follicular growth as well as the mechanism of follicular rupture are unaffected by these treatments.
In the present study, the time-dependent gradual decrease of the ovulation rate after OL suggests mechanisms other than denervation. In these cases of acute denervation, equal inhibition of ovulation would be expected without any relation to the time span from HCG to the denervation. If the damaged nerves were important during the entire ovulatory process, the reduced ovulation rate would be expected to be more pronounced when OL was performed during early stages of the ovulatory process. In the experiments in this study, the suspensory ligament was kept intact and the the ovary would not be totally devoid of nerves. Other factors arguing against denervation as an important factor contributing to the decreased ovulation rate are that ovulation is also achieved in the rat ovarian in-vitro perfusion model (Bonello et al., 1996). Furthermore, in the present study a reduction of ovulation rate was also observed after severance of the ovarian branches of the uterine arteries. This treatment should not interfere with the ovarian nerve supply since no ovarian nerves are found in this portion of the vessel (Lawrence and Burden, 1980
).
The close anatomical position of the artery and vein did not allow separate ligations, prompting the placement of clips over both the corresponding ovarian vein and artery as well as the corresponding vein and artery of the ovarian branch of the uterine vessels. Obstruction of postcapillary vessels with one remaining artery intact could cause obstruction of flow through the remaining vein. This may lead to a pathological interstitial oedema. However, no apparent differences in oedema formation or blood congestion were observed in histological sections or in measurements of ovarian weights comparing OL and OS 3 h, when the ovaries were excised 20 h after HCG injection.
Luteal steroidogenesis is positively related to ovarian blood flow in vivo in rabbits (Janson et al., 1981) and linearly related to flow of perfusate in vitro in rat ovaries during vascular perfusion (Sogn et al., 1984
) indicating that an adequate rate of blood flow is important for an optimal ovarian function. This is also indicated by the present study where reduced serum progesterone was seen after OL. However, after UL no significant changes of serum progesterone concentrations were observed, although ovulation rate was inhibited in UL at 0 and 3 h. This indicates that the ovarian artery has a major role in the regulation of steroid output and the luteinization process. Furthermore, ovarian progesterone production may not only be the result of the numbers of formed corpora lutea but also by the absolute blood flow to the ovary, where a larger blood flow reduction after OL would cause decreased progesterone secretion, which would be unaffected by the smaller blood flow reduction after UL.
In conclusion, the present study demonstrates the existence of vasomotion in the ovarian microvasculature and that the ovulation process is sensitive to disturbances in the blood flow to the ovary.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Göteborg University, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden
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References |
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Submitted on June 1, 1999; accepted on October 15, 1999.