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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: blood flow/laser Doppler flowmetry/nitric oxide/NOS inhibitor/ovary
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1989; Zackrisson et al., 2000a
). 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., 2000b
). 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., 1996
). Luteal steroidogenesis is also affected by OBF (Janson et al., 1981
; Sogn et al., 1984
).
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., 1988; Kranzfelder et al., 1992
). 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., 1991
). 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., 1992
; Huang et al., 1995
). 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 1994
; Zackrisson et al., 2000b
). Concerning the ovary, two isoforms of NOSendothelial and inducible NOSare expressed mainly in the highly vascularized theca cell layer of the larger follicles during the periovulatory period (Zackrisson et al., 1996
; Jablonka-Shariff et al., 1997
). The ovarian endogenous production of NO markedly increases after LH/hCG stimulation with an up-regulation of NOS protein (Bonello et al., 1996
; Jablonka-Shariff et al., 1997
). Pharmacological inhibition of NOS caused a reduction in ovulation number in vivo (Shukovski and Tsafriri, 1994
) and in perfused ovaries in vitro (Bonello et al., 1996
; Yamauchi et al., 1997
). The importance of NOS-generated NO in ovulation was later confirmed in NOS knockout mice (Jablonka-Shariff et al., 1998
).
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., 1998). 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., 1996
; Rosselli et al., 1998
). 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., 2000b
).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1215 h later.
Longitudinal measurement of OBF was performed by laser Doppler flowmetry (Zackrisson et al., 2000). OBF was measured 4648 h after PMSG administration, when the ovary had reached a preovulatory stage (PO group), or 68 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., 1988; Makinoda et al., 1988
).
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., 1997). 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 1)
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., 1995; Lissbrant et al., 1997
).
|
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 injectionsecond injection); salinesaline (n = 5), salinehCG (n = 5), L-NAMEsaline (n = 6) and L-NAMEhCG (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 Scheffés 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 Scheffés test. P < 0.05 was considered to be statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 2000b). 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 4060 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., 1992) 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., 1992
; Huang et al., 1995
). 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., 1994
) 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, 1999).
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, 1994; Powers et al., 1995
). The results of previous in-vivo experiments and in-vitro ovarian perfusion (Bonello et al., 1996
) 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., 1996
). 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., 1995
), mobilization of local mediators (Faletti et al., 1999
) and the altered apoptosis in the ovary (Matsumi et al., 2000
). Recently, we have demonstrated that ovarian intra-follicular pressure (IFP) of the rat increases prior to ovulation (Matousek et al., 2001
). 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., 2001
).
It has been established that OBF increases within minutes after the preovulatory LH surge (Janson, 1975). 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 ~36 h after the gonadotrophin injection (Abisogun et al., 1988
; Makinoda et al., 1988
; Tanaka et al., 1989
). 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, 1975
; Makinoda et al., 1988
; 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 (46 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, 1999
) and vascular epithelial growth factor (Agrawal et al., 1999
) 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., 1998). 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, 1975
; Abisogun et al., 1988
; Makinoda et al., 1988
; Tanaka et al., 1989
). 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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agrawal, R., Conway, G.S., Sladkevicius, P., Payne, N.N., Bekir, J., Campbell, S., Tan, S.L. and Jacobs H.S. (1999) Serum vascular endothelial growth factor (VEGF) in the normal menstrual cycle: association with changes in ovarian and uterine Doppler blood flow. Clin. Endocrinol. (Oxf.), 50, 101106.[ISI][Medline]
Anteby, E.Y., Hurwitz, A., Korach, O., Revel, A., Simon, A., Finci-Yeheskel, Z., Mayer, M. and Laufer, N. (1996) Human follicular nitric oxide pathway: relationship to follicular size, oestradiol concentrations and ovarian blood flow. Hum. Reprod., 11, 19471951.[Abstract]
Bjersing, L. and Cajander, S. (1974) Ovulation and the mechanism of follicle rupture. VI. Ultrastructure of theca interna and the inner vascular network surrounding rabbit graafian follicles prior to induced ovulation. Cell. Tissue. Res., 153, 3144.[ISI][Medline]
Bonello, N., McKie, K., Jasper, M., Andrew, L., Ross, N., Braybon, E., Brännström, M. and Norman, R.J. (1996) Inhibition of nitric oxide: effects on interleukin-1 beta-enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat. Biol. Reprod., 54, 436445.[Abstract]
Brännström, M., Mayrhofer, G. and Robertson, S.A. (1993) Localization of leukocyte subsets in the rat ovary during the periovulatory period. Biol. Reprod., 48, 277286.[Abstract]
Brännström, M., Zackrisson, U., Hagstrom, H.G., Josefsson, B., Hellberg, P., Granberg, S., Collins, W.P. and Bourne, T. (1998) Preovulatory changes of blood flow in different regions of the human follicle. Fertil. Steril., 69, 435442.[ISI][Medline]
Faletti, A., Perez Martinez, S., Perotti, C. and de Gimeno, M.A. (1999) Activity of ovarian nitric oxide synthase (NOs) during ovulatory process in the rat: relationship with prostaglandins (PGs) production. Nitric Oxide, 3, 340347.[ISI][Medline]
Griffith, T.M. (1994) Modulation of blood flow and tissue perfusion by endothelium-derived relaxing factor. Exp. Physiol., 79, 873913.[ISI][Medline]
Hernández, I., Carbonell, L.F., Quesada, T. and Fenoy, F.J. (1999) Role of angiotensin II in modulating the hemodynamic effects of nitric oxide synthesis inhibition. Am. J. Physiol., 277, R104111.
Huang, P.L., Huang, Z., Mashimo, H., Bloch, K.D., Moskowitz, M.A., Bevan, J.A. and Fishman, M.C. (1995) Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature, 377, 239242.[ISI][Medline]
Jablonka-Shariff, A. and Olson, L.M. (1997) Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary. Endocrinology, 138, 460468.
Jablonka-Shariff, A. and Olson, L.M. (1998) The role of nitric oxide in oocyte meiotic maturation and ovulation: meiotic abnormalities of endothelial nitric oxide synthase knock-out mouse oocytes. Endocrinology, 139, 29442954.
Jablonka-Shariff, A., Ravi, S., Beltsos, A.N., Murphy, L.L. and Olson, L.M. (1999) Abnormal estrous cyclicity after disruption of endothelial and inducible nitric oxide synthase in mice. Biol. Reprod., 61, 171177.
Janson, P.O. (1975) Effects of the luteinizing hormone on blood flow in the follicular rabbit ovary, as measured by radioactive microspheres. Acta Endocrinol. (Copenh.), 79, 122133.[ISI][Medline]
Janson, P.O., Damber, J.E. and Axen, C. (1981) Luteal blood flow and progesterone secretion in pseudopregnant rabbits. J. Reprod. Fertil., 63, 491497.[Abstract]
Kranzfelder, D., Reich, R., Abisogun, O. and Tsafriri, A. (1992) Preovulatory changes in the perifollicular capillary network in the rat; Role of eicosanoids. Biol. Reprod., 46, 379385.
Lissbrant, E., Löfmark, U., Collin, O. and Bergh, A. (1997) Is nitric oxide involved in the regulation of the rat testicular vasculature? Biol. Reprod., 56, 12211227.[Abstract]
Löfman, C.O., Brännström, M., Holmes, P.V. and Janson, P.O. (1989) Ovulation in the isolated perfused rat ovary as documented by intravital microscopy. Steroids, 54, 481490.[Medline]
Makinoda, S., Tabata, M., Yamaguchi, T., Nakajin, K., Koyama, T. and Ichinoe, K. (1988) Ovarian blood flow and oxygen transport to the follicle during the preovulatory period. Adv. Exp. Med. Biol., 222, 689697.[Medline]
Matousek, M., Carati, C., Gannon, B. and Brännström, M. (2001) Novel method for intrafollicular pressure measurements in the rat ovary: increased intrafollicular pressure after hCG stimulation. Reproduction, 121, 307314.
Matsumi, H., Yano, T., Osuga, Y., Kugu, K., Tang, X., Xu, J.P., Yano, N., Kurashima, Y., Ogura, T., Tsutsumi, O. et al. (2000) Regulation of nitric oxide synthase to promote cytostasis in ovarian follicular development. Biol. Reprod., 63, 141146.
Moncada, S., Palmer, R.M. and Higgs, E.A. (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev., 43, 109142.[ISI][Medline]
Murakami, T., Ikebuchi, Y., Ohtsuka, A., Kikuta, A., Taguchi, T. and Ohtani, O. (1988) The blood vascular wreath of rat ovarian follicle, with special reference to its changes in ovulation and luteinization: a scanning electron microscopic study of corrosion casts. Arch. Histol. Cytol., 51, 299313.[ISI][Medline]
Okuda, Y., Okamura, H., Kanzaki, H. and Takenaka, A. (1983) Capillary permeability of rabbit ovarian follicles prior to ovulation. J. Anat., 137, 263269.[ISI][Medline]
Powers, R.W., Chen, L., Russell, P.T. and Larsen, W.J. (1995) Gonadotropin-stimulated regulation of blood-follicle barrier is mediated by nitric oxide. Am. J. Physiol., 269, E290E298.
Richards, J.S., Russell, D.L., Robker, R.L., Dajee, M. and Alliston, T.N. (1998) Molecular mechanisms of ovulation and luteinization. Mol. Cell. Endocrinol., 145, 4754.[ISI][Medline]
Rosselli, M., Keller, P.J. and Dubey, R.K. (1998) Role of nitric oxide in the biology, physiology and pathophysiology of reproduction. Hum. Reprod. Update, 4, 324.
Shukovski, L. and Tsafriri, A. (1994) The involvement of nitric oxide in the ovulatory process in the rat. Endocrinology, 135, 22872290.[Abstract]
Sogn, J., Abrahamsson, G. and Janson, P.O. (1984) Release of cyclic AMP and progesterone from the isolated perfused luteal ovary of the PMSG treated rat. Acta Endocrinol. (Copenh.), 106, 265270.[ISI][Medline]
Tanaka, N., Espey, L.L. and Okamura, H. (1989) Increase in ovarian blood volume during ovulation in the gonadotropin-primed immature rat. Biol. Reprod., 40, 762768.[Abstract]
Tsafriri, A. and Reich, R. (1999) Molecular aspects of mammalian ovulation. Exp. Clin. Endocrinol. Diabetes, 107, 111.[ISI][Medline]
Van Voorhis, B.J., Dunn, M.S., Snyder, G.D. and Weiner, C.P. (1994) Nitric oxide: an autocrine regulator of human granulosa-luteal cell steroidogenesis. Endocrinology, 135, 17991806.[Abstract]
Wang, Y.X., Gavras, I., Wierzba, T., Lammek, B. and Gavras, H. (1992) Inhibition of nitric oxide, bradykinin, and prostaglandins in normal rats. Hypertension, 19 (Suppl. 2), 255261.[Abstract]
Whiting, M.J., Rutten, A.J., Williams, P. and Bersten, A.D. (1994) Determination of NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester in plasma by high-performance liquid chromatography. J. Chromatogr. B. Biomed. Appl., 660, 170175.[Medline]
Yamauchi, J., Miyazaki, T., Iwasaki, S., Kishi, I., Kuroshima, M., Tei, C. and Yoshimura, Y. (1997) Effects of nitric oxide on ovulation and ovarian steroidogenesis and prostaglandin production in the rabbit. Endocrinology, 138, 36303637.
Zackrisson, U., Mikuni, M., Wallin, A., Delbro, D., Hedin, L. and Brännström, M. (1996) Cell-specific localization of nitric oxide synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation. Hum. Reprod., 11, 26672673.[Abstract]
Zackrisson, U., Brännström, M., Granberg, S., Janson, P.O., Collins, W.P. and Bourne, T.H. (1998) Acute effects of a transdermal nitric oxide donor on perifollicular and intrauterine blood flow. Ultrasound Obstet. Gynecol., 12, 5055.[ISI][Medline]
Zackrisson, U.J., Mikuni, M. and Brännström, M. (eds) (2000a) Ovulation. Evolving Scientific and Clinical Concepts. Springer-Verlay, New York, USA, pp. 222242.
Zackrisson, U., Mikuni, M., Peterson, M.C., Nilsson, B., Janson P.O. and Brännström, M. (2000b) Evidence for the involvement of blood flow-related mechanisms in the ovulatory process of the rat. Hum. Reprod., 15, 264272.
Zanzinger, J. (1999) Role of nitric oxide in the neural control of cardiovascular function. Cardiovasc. Res., 43, 63949.[ISI][Medline]
Submitted on April 8, 2002; accepted on June 8, 2002.