The Journal of Physiological Sciences Advance Publication by J-STAGE doi:10.2170/physiolsci.RP001508 This version is to be replaced by the final version after page-setting and proofing.

Regular Paper Effects of Electrical Stimulation of the Superior Ovarian and Ovarian Plexus Nerve on the Ovarian Estradiol Secretion Rate in Rats

1,2 1 1 Fusako KAGITANI , Sae UCHIDA , and Harumi HOTTA

1Department of the , Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, 173-0015 Japan; and 2University of Human Arts and Sciences, Saitama, Saitama, 339-8539 Japan

Received on Jan 23, 2008; accepted on Mar 19, 2008; released online on Mar 22, 2008; doi:10.2170/physiolsci.RP001508 Correspondence should be addressed to: Fusako Kagitani, Department of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, 173-0015 Japan. Phone: +81-3-3964-3241 (Ext. 3086), Fax: +81-3-3579-4776, E-mail: [email protected]

Abstract: The present experiments examined the effects of electrical stimulation of the superior ovarian nerve (SON) and ovarian plexus nerve (OPN) on the ovarian estradiol secretion in rats. Rats were anesthetized on the day of estrous, and the ovarian venous blood was collected intermittently. The secretion rate of estradiol from the ovary was calculated from differences in the estradiol concentration between ovarian venous plasma and systemic arterial blood plasma, and from the flow rate of ovarian venous plasma. Either an SON or OPN, ipsilateral to the ovary that ovarian venous blood was collected, was electrically stimulated at a supra-maximal intensity for C-fibers. The secretion rate of estradiol was significantly decreased by 47 ± 6% during SON stimulation, but was not significantly changed during OPN stimulation. These results suggest that autonomic , which reach the ovary via the SON, have an inhibitory role in ovarian estradiol secretion. [The Journal of Physiological Sciences 58(2), 2008, in press]

Key words: superior ovarian nerve, ovarian plexus nerve, estradiol, secretion, rat.

Many studies have examined the relationship between hypothalamic-pituitary-ovarian hormones and ovarian functions. However, while several histological studies have described the innervation of vagal parasympathetic and sympathetic nerves including both afferents and efferents to the ovary, the roles of these autonomic nerves in ovarian function have not been clarified enough [1–6]. Histological studies by Burden et al. [1, 7] demonstrated that autonomic nerves enter the ovary via hilar perivascular plexuses, and tiny branches from these plexuses extend into the contiguous steroidogenic interstitial gland cells. Autonomic nerves to the rat ovary reach the organ by two routes: via the ovarian plexus nerve (OPN) along the ovarian artery and via the superior ovarian nerve (SON) in the suspensory ligament [8, 9]. Recently, we demonstrated that electrical stimulation of the splanchnic nerve or OPN produces vasoconstriction of ovarian arterioles and a reduction of ovarian blood flow in rats [10, 11]. This result suggests a vasoconstrictor role of ovarian autonomic nerves in regulating ovarian blood flow. On the other hand, earlier studies demonstrated that denervation of the OPN [12] or the SON [13] alters estradiol secretion from the ovary. These results suggest that ovarian autonomic nerves regulate ovarian estradiol secretion. However, no reports have directly examined the effect of stimulation of the autonomic nerves innervating the ovary on ovarian estradiol secretion. In the present study, we aimed to clarify the effects of electrical stimulation of the SON and OPN on the secretion rate of estradiol from the ovary in rats.

Previously, our laboratory developed methods for measuring the secretion rates of catecholamines from the adrenal medulla [14, 15] and thyroid hormones from the thyroid gland [16] in rats. In the present study, we applied these methods to measurement of the secretion rate of estradiol from the ovary. Although some previous reports suggested asymmetry in the autonomic regulation of ovarian functions [17], we studied on the right ovary in this study because of technical convenience. We used the rats on the day of estrous when concentration and secretion rate of estradiol in rat ovarian venous plasma are reportedly more stable than other stage [18].

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MATERIALS AND METHODS

Animals. Fifteen virgin female Wistar rats, 3–6 months old (body weight, 150–210 g), were used for the present experiments. The rats were kept in a room with a 12 h:12 h light-dark schedule, and rat chow and water were provided ad libitum. Daily vaginal smears were taken and those rats showing regular estrous cycles of 5 days were used in the experiments. This study was approved by the Animal Committee of our institution.

Surgical procedures. Rats on the day of estrus (Fig. 1A) were anesthetized with urethane (1.1 g/kg, i.p.). The trachea was cannulated and respiration was artificially maintained using a respirator (model 683, Harvard, Holliston, Massachusetts, USA). The end-tidal CO2 concentration, which was monitored using a gas monitor (Microcap, Oridion Medical, Jerusalem, Israel), was kept at 3–4% by controlling respiratory volume and frequency. The systemic blood pressure was continuously recorded through a cannula in a common carotid artery with a strain gauge (TP-400T, Nihon Kohden, Tokyo, Japan). The jugular vein was cannulated for infusion of necessary solutions. The animal was immobilized by administration of gallamine triethiodide (20 mg/kg, i.v., as required, Sigma, Missouri, USA). The core body temperature, which was monitored in the rectum, was maintained at around 37.5°C using a body temperature control system containing a thermostatically-regulated DC current heating pad and an infrared lamp (ATB-1100, Nihon Kohden, Tokyo, Japan). During the experiments, urethane (10% of the dose used for initial anesthesia) was administered i.v. every 1–2 h.

Collecting of ovarian venous blood samples. Following a midline laparotomy, a polyethylene catheter (external diameter 0.5 mm, internal diameter 0.2 mm, Natsume Co. Ltd., Tokyo, Japan) was inserted into the right ovarian vein (Fig. 1B). The uterine vein at the anastomosis with ovarian vein and other veins connected to the right ovarian vein were occluded either by thin threads or an electric coagulator (model 440E, Radionics, Massachusetts, USA). The animals were infused continuously with a heparin sodium solution (200 IU/kg/h, Shimizu Pharm. Co. Ltd., Shizuoka, Japan) to insure the free flow of blood through the tubing, and ovarian venous blood samples were collected into hematocrit tubes by capillary action and venous pressure. As blood was taken from the ovarian vein, a solution of 4% Ficoll 70 was administered at a rate of 2 ml/h to the jugular vein by an infusion pump (STC-521, Terumo, Tokyo, Japan). When samples were not being collected, the ovarian venous blood was shunted into the right femoral vein through a catheter. Since the dead space volume was approximately 30 µl with this method, two drops (about 40 µl) of blood was removed before collecting each sample. A 60–70 µl blood sample was collected into heparinized hematocrit capillary tubes (Clay Adams, New Jersey, USA). After centrifuging the blood samples for 5 min at 11,000 rpm, plasma samples were collected and ethylene diamine tetraacetate disodium was added (l–2 mg/ml plasma). The samples were frozen and stored at –80°C until estradiol measurement was carried out. The secretion rate of estradiol was calculated from the absolute estradiol concentration of the ovarian venous plasma (taking into consideration the concentration in the arterial blood entering the ovary) and the ovarian venous plasma flow rate. In rats that received either the SON or OPN stimulation, five samples were taken, with a sample taken 5 min before stimulation. The other four samples were taken at 1, 10, 20 and 30 min after onset of the 5 min stimulation period. Each rat received either an SON or OPN stimulation. In control rats that received no SON or OPN stimulation, blood samples were taken during rest on the same schedule as the rats that received SON or OPN stimulation.

Collecting of systemic arterial and venous blood. After collecting all samples from ovarian venous blood, samples (about 140 µl) of systemic arterial and venous blood were collected into hematocrit capillary tubes through a polyethylene tube, which was inserted into the right femoral artery and femoral vein, respectively. In preliminary experiments, we confirmed a stability of estradiol concentration in systemic arterial plasma between samples collected both at the beginning and at the end of experiment.

Measurement of blood estradiol. After the plasma samples were diluted 2–11 times with EIA buffer, plasma estradiol levels were measured by enzyme immunoassay utilizing an estradiol (17β-estradiol) EIA kit (Cayman Chemical Co., Michigan, USA).

Stimulation of the superior ovarian nerve (SON) and ovarian plexus nerve (OPN). In 5 rats, the right SON running along the suspensory ligament together with the suspensory ligament was cut at approximately 5 mm from the ovary (Fig. 2A). In the other 5 rats, the right OPN running along the ovarian artery was cut at

2 The Journal of Physiological Sciences Advance Publication by J-STAGE doi:10.2170/physiolsci.RP001508 This version is to be replaced by the final version after page-setting and proofing. approximately 20 mm from the ovary. The peripheral end was placed on a bipolar platinum-iridium wire stimulating electrode, and the nerve was covered with warm liquid paraffin. Electrical square pulse stimulation of 0.5 ms width, 20 Hz, 20 V (for SON) or 5 V (for OPN) was applied to these nerves for 5 min. In this study all stimulations were of 5 min duration to allow time for sampling of ovarian venous blood during the stimulus period. In rats used for SON stimulation, OPN was kept intact. In rats used for OPN stimulation, SON was kept intact. In 4 control rats, both SON and OPN were kept intact.

In one rat, evoked volleys were recorded from the SON and OPN. The SON was cut as described above, and the proximal cut end was placed on stimulating electrodes. Furthermore, the SON was cut at about 27 mm from the ovary and the peripheral cut end was placed on the recording electrodes. The OPN was cut as described above, and the peripheral cut end was placed on stimulating electrodes. Furthermore, the OPN was cut at about 8 mm from the ovary, and the proximal cut end was placed on the recording electrodes. Evoked volleys were recorded from the SON and OPN by applying electrical square pulse stimuli of 0.5 ms width, varying intensity (0.1–20 V). The distance between the stimulating electrodes and recording electrodes of the SON was 22 mm and that of the OPN was 12 mm. Action potentials were amplified (time constant 0.3 s) and displayed on a storage oscilloscope. The maximum conduction velocities of unmyelinated fibers in the SON and the OPN were calculated by dividing the distance by the latency of respective action potentials evoked.

Data analysis. Values were expressed as means ± SEM. Statistical analysis was performed by two-way repeated ANOVA followed by Bonferroni post-test, or Student’s t-test. Statistical significance was set at p < 0.05.

RESULTS

Secretion rate of estradiol from the ovary under resting conditions The mean concentration of estradiol in the ovarian venous plasma in 4 control rats was 134.0 ± 13.3 pg/ml (Fig. 1C). Estradiol concentrations in systemic arterial plasma and systemic venous plasma were approximately half of that observed in ovarian venous plasma (69.8 ± 6.9 and 69.9 ± 5.3 pg/ml, respectively) (Fig. 1C). The secretion rate of estradiol was calculated from the absolute estradiol concentration of the ovarian venous plasma (systemic arterial estradiol concentration was subtracted from ovarian venous estradiol concentration) and the ovarian plasma flow rate. Each value of the ovarian plasma flow rate, the estradiol concentration of ovarian venous plasma, and the secretion rate of estradiol in 5 successive samples taken during 35 min under resting conditions in each rat was stable. In 4 control rats, the ovarian plasma flow rate, estradiol concentration of ovarian venous plasma, and the secretion rate of estradiol ranged from 26.0 to 28.7 µl/min, 115.8 to 173.0 pg/ml, and 1.5 to 2.4 pg/min, respectively. The mean basal level of the mean arterial pressure (MAP) was 109.1 ± 14.9 mmHg (n = 4), and the MAP level was stable during collection of ovarian venous blood samples for 35 min.

Evoked potentials recorded from SON and OPN Figure 2, B–E, shows typical recordings of evoked potentials of unmyelinated C fibers in the SON and OPN. The evoked potential of the C fibers in the SON appeared with a stimulus strength above 3 V and reached a maximum at 15 V. The maximum conduction velocity of the C fibers in SON was 1.2 m/s. The evoked potential of the unmyelinated C fibers in the OPN appeared with a stimulus strength above 0.8 V and reached a maximum at 2 V. The maximum conduction velocity of the C fibers in OPN was 1.1 m/s.

In the following experiments, the stimulus intensity was set at a supramaximal intensity for C-fibers (20 V for SON and 5 V for OPN). Stimulus frequency was set at 20 Hz, because effector responses usually reached nearly maximum when autonomic efferent C-fibers were stimulated at 20Hz [19, 20].

Effect of electrical stimulation of the SON or OPN on the secretion rate of estradiol from the ovary In the 5 rats used for SON stimulation, the prestimulus (5 min before the stimulation) basal values of the ovarian plasma flow rate, the estradiol concentration of ovarian venous plasma and the secretion rate of

3 The Journal of Physiological Sciences Advance Publication by J-STAGE doi:10.2170/physiolsci.RP001508 This version is to be replaced by the final version after page-setting and proofing. estradiol were not significantly different from the values observed in control rats. As shown by the filled circles and solid line in Fig. 3A, repetitive electrical stimulation of the SON at the supramaximal intensity for C fibers (0.5 ms, 20 V, 20 Hz) produced a decrease in the ovarian plasma flow rate, reaching 76 ± 3% of the prestimulus values during stimulation. The decreased plasma flow rates returned to the prestimulus basal level 5 min after the end of stimulation. SON stimulation had a tendency to reduce the estradiol concentration in ovarian venous plasma, but the response was not statistically significant (Fig. 3B). During stimulation of the SON, the secretion rate of estradiol from the ovary was decreased in all 5 rats tested. The reduction of the secretion rate of estradiol reached 31–65% (53 ± 6%) of the prestimulus values during the stimulation (Fig. 3C). The decreased response of the secretion rate of estradiol returned to the prestimulus basal level 5 min after the end of stimulation. No significant response of MAP was noted following stimulation of the SON.

In the 5 rats used for OPN stimulation, the prestimulus (5 min before the stimulation) basal values of the ovarian plasma flow rate, the estradiol concentration of ovarian venous plasma and the secretion rate of estradiol were not significantly different from the values observed in control rats. As shown by the filled triangles and the broken line in Fig. 3A, repetitive electrical stimulation of the OPN at the supramaximal intensity for C fibers (0.5 ms, 5 V, 20 Hz) produced a decrease in the ovarian plasma flow rate, reaching 74 ± 9% of the prestimulus values during stimulation. The decreased plasma flow rates returned to the prestimulus basal level 5 min after the end of stimulation. OPN stimulation had no significant effect on the estradiol concentration in ovarian venous plasma or the secretion rate of estradiol (Fig. 3, B and C). No significant response of MAP was noted following stimulation of the OPN.

DISCUSSION

This study demonstrated that the secretion rate of estradiol from the ovary was decreased by electrical stimulation of the SON, but not of the OPN at the supramaximal intensity for C fibers (Fig. 4). Ovarian estradiol production is considered synonymous with release, because steroid hormones, once produced, can freely cross the cell membrane without having to be packed in granules and actively exocytosed [21]. Therefore, activation of the SON may decrease estradiol synthesis in the ovary. In the present results, the ovarian venous plasma flow rate was equally decreased by stimulation of either the SON or OPN (Fig. 4). We can assume that the reduction of the secretion rate of estradiol by SON stimulation was due to direct inhibitory effects of the SON on estradiol synthesis in the ovary rather than due to a reduction of ovarian blood flow. The ovary obtains efferent innervation both from the sympathetic system and from the parasympathetic system via the vagus nerve [5, 6, 22]. Concerning the neural regulation of ovarian blood flow, our previous study demonstrated that electrical stimulation of splanchnic (sympathetic) nerve but not vagal (parasympathetic) nerve decreases ovarian blood flow [10]. The decrease in ovarian blood flow by electrical stimulation of splanchnic nerve was completely abolished by intravenous injection of alpha adrenoceptor antagonist. We also demonstrated that electrical stimulation of the OPN decreases ovarian blood flow [11]. Our present study showed electrical stimulation of either the SON or OPN produces decrease in ovarian blood flow (ovarian venous plasma flow rate). Therefore, we can assume that sympathetic adrenergic vasoconstrictor fibers may travel through both the SON and OPN to the ovary. Using ex vivo celiac ganglion-SON-ovary system and superior mesenteric ganglion-OPN-ovary system, it was demonstrated that chemical stimulation of celiac ganglion or superior mesenteric ganglion increases the release of nitric oxide in the ovarian compartment [23, 24]. Since electrical stimulation of the SON and OPN produces decrease in ovarian blood flow in the present results, adrenergic vasoconstrictor effect seems more dominant than vasodilatory effect of nitric oxide, even if nitric oxide was released in the present experimental condition.

A histological studyreported that most adrenergic nerves derived from the OPN are perivascular, while those derived from the SON innervate both blood vessels and steroidogenic interstitial gland cells in the rat ovary [8]. Our present result showed that OPN stimulation decreases ovarian blood flow without affecting ovarian estradiol secretion, while SON stimulation decreases both ovarian blood flow and ovarian estradiol secretion. These present results support the histological study by Lawrence and Burden [8]. However, since vagal nerve may also travel through SON and/or OPN, we cannot exclude the possible contribution of vagus nerve in the regulation of ovarian estradiol secretion by the SON in the present study.

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Earlier studies demonstrated that denervation of the OPN [12] or the SON [13] alters estradiol secretion from the ovary. Kawakami et al. [12] reported that increase in estradiol secretion from the ovary elicited by electrical stimulation of medial basal prechiasmatic area is abolished by transection of the OPN in proestrous rats, but the authors did not test in estrous rats. Aguado and Ojeda [13] reported SON section decreases ovarian estradiol secretion in proestrous stage, but does not alter ovarian estradiol secretion in estrous stage. The left ovary was used in the studies of Kawakami et al. [12] and Aguado and Ojeda [13]. Although asymmetry in the autonomic regulation of ovarian functions has been suggested [17], our present results on the right ovary of estrous rats that SON section did not alter ovarian estradiol secretion are in agreement with the results on the left ovary of estrous rats [13]. There is a possibility that present results of the responses of ovarian estradiol secretion by transection or by stimulation of the SON or OPN in estrous rats, are different in proestrous rats.

In the present study, evoked potentials of unmyelinated C-fibers were recorded from the SON and OPN. These electrophysiological results are consistent with the studies using electron microscopy that demonstrated both the SON and OPN consist mainly of unmyelinated fibers with only a small percentage being myelinated [8, 25].

We are grateful to Prof. Yuko Sato of the University of Human Arts and Sciences for her encouragement in the performance of this study.

REFERENCES

1. Burden HW, Leonard M, Smith CP, Lawrence Jr IE. The sensory innervation of the ovary: A horseradish peroxidase study in the rat. Anat Rec. 1983;207:623-7. 2. Burden HW. The adrenergic innervation of mammalian ovaries. In: Ben-Jonathan N, Bahr JM, Weiner RI, editors. Serono Symposia Publications from Raven Press. Vol 18. Catecholamines as Hormone Regulators. New York: Raven Press; 1985. p. 261-78. 3. Traurig HH, Papka RE. Autonomic efferent and visceral sensory innervation of the female reproductive system: special reference to the functional roles of nerves in reproductive organs. In: Maggi CA, editor. Nervous Control of the Urogenital System. Switzerland: Harwood academic publishers; 1993. p. 103-41. 4. Owman C, Stjernquist M. Origin, distribution, and functional aspects of aminergic and peptidergic nerves in the male and female reproductive tracts. In: Björklund A, Hökfelt T, Owman C, editors. Handbook of Chemical Neuroanatomy. Vol 6. The peripheral nervous system. Amsterdam: Elsevier; 1988. p. 445-544. 5. Gerendai I, Tóth IE, Boldogköi Z, Medveczky I, Halász B. Neural labeling in the rat brain and spinal cord from the ovary using viral transneuronal tracing technique. Neuroendocrinology. 1998;68:244-56. 6. Gerendai I, Tóth IE, Boldogköi Z, Medveczky I, Halász B. CNS structures presumably involved in vagal control of ovarian function. J Auton Nerv Syst. 2000;80:40-5. 7. Burden HW. Adrenergic innervation in ovaries of the rat and guinea pig. Am J Anat. 1972;133:455-61. 8. Lawrence JrIE, Burden HW. The origin of the extrinsic adrenergic innervation to the rat ovary. Anat Rec. 1980;196:51-9. 9. Baljet B, Drukker J. The extrinsic innervation of the abdominal organs in the female rat. Acta Anat (Basel). 1979;104:243-67. 10. Uchida S, Hotta H, Kagitani F, Aikawa Y. Ovarian blood flow is reflexively regulated by mechanical afferent stimulation of a hindlimb in nonpregnant anesthetized rats. Auton Neurosci. 2003;106:91-7. 11. Uchida S, Hotta H, Hanada T, Okuno Y, Aikawa Y. Effects of thermal stimulation, applied to the hindlimb via a hot water bath, upon ovarian blood flow in anesthetized nonpregnant rats. J Physiol Sci. 2007;57: 227-33. 12. Kawakami M, Kubo K, Uemura T, Nagase M, Hayashi R. Involvement of ovarian innervation in steroid secretion. Endocrinol. 1981;109:136-45.

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13. Aguado LI, Ojeda SR. Ovarian adrenergic nerves play a role in maintaining preovulatory steroid secretion. Endocrinol. 1984;114:1944-6. 14. Araki T, Ito K, Kurosawa M, Sato A. Responses of adrenal sympathetic nerve activity and catecholamine secretion to cutaneous stimulation in anesthetized rats. Neurosci. 1984;12:289-99. 15. Sato A, Sato Y, Suzuki A, Uchida S. Reflex modulation of catecholamine secretion and adrenal sympathetic nerve activity by acupuncture-like stimulation in anesthetized rat. Jpn J Physiol. 1996;46:411-21. 16. Hotta H, Ooka H, Sato A. Changes in basal secretion rates of thyroxine and 3,3',5-triiodothyronine from the thyroid gland during aging of the rat. Jpn J Physiol. 1991;41:75-84. 17. Morales L, Ricardo B, Bolaños A, Chavira R, Domínguez R. Ipsilateral vagotomy to unilaterally ovariectomized pre-pubertal rats modifies compensatory ovarian responses. Reprod Biol Endocrinol. 2007;5:24. 18. Shaikh AA. Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. Biol Reprod. 1971;5:297-307. 19. Hotta H, Nishijo K, Sato A, Sato Y, Tanzawa S. Stimulation of lumbar produces vasodonstriction of the vasa nervorum in the sciatic nerve via α-adrenergic receptors in rats. Neurosci Lett. 1991;133:249-52. 20. Sato Y, Hotta H, Nakayama H, Suzuki H. Sympathetic and parasympathetic regulation of the uterine blood flow and contraction in the rat. J Auton Nerv Syst. 1996;59:151-8. 21. Ojeda SR, Griffin JE. Organization of the endocrine system. In: Griffin JE, Ojeda SR, editors. Textbook of Endocrine Physiology. 5th ed. Oxford: Oxford University Press; 2004. p.1-16. 22. Klein CM, Burden HW. Anatomical localization of afferent and postganglionic sympathetic neurons innervating the rat ovary. Neurosci Lett. 1988;85:217-22. 23. Delgado SM, Casais M, Sosa Z, Rastrilla AM. Ganglionic adrenergic action modulates ovarian steroids and nitric oxide in prepubertal rat. Endocr J. 2006;53:547-54. 24. Orozco AV, Sosa Z, Fillipa V, Mohamed F, Rastrilla AM. The cholinergic influence on the mesenteric ganglion affects the liberation of ovarian steroids and nitric oxide on oestrus day rats: characterization of an ex vivo system. J Endocrinol. 2006;191:587-98. 25. Payer AF. Ultrastructural study of the accompanying the ovarian artery and vein in the rat. Anat Rec. 1978;190:47-63.

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Fig. 1. A: Typical changes of estrous cycle by observation of a vaginal smear for 8 successive days. E: estrus, P: proestrus, D: diestrus. The experiment was performed in the rat on the day of estrus. B: Illustration showing the experimental procedures for collecting ovarian venous blood. C: Estradiol concentrations in ovarian venous plasma, systemic arterial plasma and systemic venous plasma in 4 rats (mean ± SEM).

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Fig. 2. Evoked compound action potentials following electrical stimulation of the SON and OPN. (A) Anatomical illustration showing ovarian innervation by the SON and OPN. (B, D) Specimen records of evoked potentials by C fibers in the SON (B) and in the OPN (D). Arrows at the bottom indicate the time that a single shock was delivered. (C, E) Strength-response curves of evoked potentials of C-fibers in the SON (C) and in the OPN (E). Abscissa: stimulus intensity in V. Ordinate: Amplitudes of evoked potentials expressed in % of the maximum evoked potentials obtained for C-fibers, evoked in the SON and OPN by a single shock to the respective nerves.

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Fig. 3. Effects of electrical stimulation of the SON and OPN on ovarian venous plasma flow rate (A), ovarian venous estradiol concentration (B) and estradiol secretion rate from the ovary (C). Stimulation of 5 min duration is indicated by the upper horizontal bar. Magnitudes of responses during and after stimulation are expressed as percentages of the prestimulus values (5 min before the stimulation). Each point and vertical bar represents the mean ± SEM. *P < 0.05; **P < 0.01; significantly different from the response of control using two-way repeated ANOVA followed by Bonferroni posttests.

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Fig. 4. Schematic diagram of a possible mechanism for the regulation of estradiol secretion and blood flow of the ovary by stimulation of the SON and OPN.

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