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

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REGULAR PAPER J. Physiol. Sci. Vol. 58, No. 2; Apr. 2008; pp. 133–138 Online Mar. 22, 2008; doi:10.2170/physiolsci.RP001508

Fusako KAGITANI1,2, Sae UCHIDA1, and Harumi HOTTA1 1Department of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo ,173-0015 Japan; 2University of Human Arts and Sciences, Saitama, Saitama, 339-8539 Japan

Abstract: The present experiments examined the effects of rate of ovarian venous plasma. Either an SON or OPN, ipsilater- electrical stimulation of the superior ovarian nerve (SON) and al to the ovary from which ovarian venous blood was collected, the ovarian plexus nerve (OPN) on the ovarian estradiol secre- was electrically stimulated at a supramaximal intensity for C-fi- tion in rats. The rats were anesthetized on the day of estrus, and bers. The secretion rate of estradiol was significantly decreased the ovarian venous blood was collected intermittently. The se- by 47 ± 6% during SON stimulation, but it was not significantly cretion rate of estradiol from the ovary was calculated from dif- changed during OPN stimulation. These results suggest that au- ferences in the estradiol concentration between ovarian venous tonomic nerves, which reach the ovary via the SON, have an in- plasma and systemic arterial blood plasma, and from the flow hibitory role in ovarian estradiol secretion.

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

Many studies have examined the relationship between ulation of the autonomic nerves innervating the ovary on hypothalamic-pituitary-ovarian hormones and ovarian ovarian estradiol secretion. In the present study, we aimed functions. However, although several histological studies to clarify the effects of electrical stimulation of the SON have described the innervation of vagal parasympathetic and the OPN on the secretion rate of estradiol from the and sympathetic nerves including both afferents and effer- ovary in rats. ents to the ovary, the roles of these autonomic nerves in Previously, our laboratory developed methods for mea- ovarian function have not been sufficiently clarified [1– suring the secretion rates of catecholamines from the adre- 6]. Histological studies by Burden et al. [1, 7] demonstrat- nal medulla [14, 15] and thyroid hormones from the thy- ed that autonomic nerves enter the ovary via hilar perivas- roid gland [16] in rats. In the present study, we applied cular plexuses, and tiny branches from these plexuses ex- these methods to the measurement of the secretion rate of tend into the contiguous steroidogenic interstitial gland estradiol from the ovary. Although some previous reports cells. Autonomic nerves to the rat ovary reach the organ suggested asymmetry in the autonomic regulation of ova- by two routes: the ovarian plexus nerve (OPN) along the rian functions [17], we studied the right ovary in this study ovarian artery and the superior ovarian nerve (SON) in the because of technical convenience. We used the rats on the suspensory ligament [8, 9]. We recently demonstrated that day of estrus when the concentration and secretion rate of electrical stimulation of the splanchnic nerve or the OPN estradiol in rat ovarian venous plasma are reportedly more produces a vasoconstriction of ovarian arterioles and a re- stable than in other stages [18]. duction of ovarian blood flow in rats [10, 11]. This result suggests a vasoconstrictor role of ovarian autonomic MATERIALS AND METHODS nerves in regulating ovarian blood flow. On the other hand, earlier studies demonstrated that a denervation of Animals. Fifteen virgin female Wistar rats, 3–6 months the OPN [12] or the SON [13] alters estradiol secretion old (body weight, 150–210 g), were used for the present from the ovary. These results suggest that ovarian auto- experiments. The rats were kept in a room with a 12 h:12 nomic nerves regulate ovarian estradiol secretion. How- h light-dark schedule, and rat chow and water were pro- ever, no reports have directly examined the effect of stim- vided ad libitum. Daily vaginal smears were taken, and

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]

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those rats showing regular estrous cycles of 5 days were Ltd., Tokyo, Japan) was inserted into the right ovarian used in the experiments. This study was approved by the vein (Fig. 1B). The uterine vein at the anastomosis with Animal Committee of our institution. ovarian vein and other veins connected to the right ovari- Surgical procedures. Rats on the day of estrus (Fig. 1A) an vein were occluded either by thin threads or an electric were anesthetized with urethane (1.1 g/kg, i.p.). The trachea coagulator (model 440E, Radionics, Massachusetts, was cannulated and respiration was artificially maintained USA). The animals were infused continuously with a hep- using a respirator (model 683, Harvard, Holliston, Massa- arin sodium solution (200 IU/kg/h, Shimizu Pharmaceuti-

chusetts, USA). The end-tidal CO2 concentration, which cal Co. Ltd., Shizuoka, Japan) to ensure the free flow of was monitored using a gas monitor (Microcap, Oridion blood through the tubing, and ovarian venous blood sam- Medical, Jerusalem, Israel), was kept at 3–4% by control- ples were collected in hematocrit tubes by capillary action ling respiratory volume and frequency. The systemic blood and venous pressure. As blood was taken from the ovarian pressure was continuously recorded through a cannula in a vein, a solution of 4% Ficoll 70 was administered at a rate common carotid artery with a strain gauge (TP-400T, Ni- of 2 ml/h to the jugular vein by an infusion pump (STC- hon Kohden, Tokyo, Japan). The jugular vein was cannulat- 521, Terumo, Tokyo, Japan). When samples were not be- ed for an infusion of necessary solutions. The animal was ing collected, the ovarian venous blood was shunted into immobilized by an administration of gallamine triethiodide the right femoral vein through a catheter. Since the dead (20 mg/kg, i.v., as required, Sigma, Missouri, USA). The space volume was approximately 30 µl with this method, core body temperature, which was monitored in the rectum, two drops (about 40 µl) of blood were removed before was maintained at around 37.5°C using a body temperature each sample was collected. A 60–70 µl blood sample was control system containing a thermostatically regulated DC collected in heparinized hematocrit capillary tubes (Clay current heating pad and an infrared lamp (ATB-1100, Ni- Adams, New Jersey, USA). After the blood samples were hon Kohden, Tokyo, Japan). During the experiments, ure- centrifuged for 5 min at 11,000 rpm, plasma samples were thane (10% of the dose used for initial anesthesia) was ad- collected and ethylene diamine tetra-acetate disodium ministered i.v. every 1–2 h. was added (l–2 mg/ml plasma). The samples were frozen Collecting of ovarian venous blood samples. Following and stored at –80°C until estradiol measurement was car- a midline laparotomy, a polyethylene catheter (external ried out. The secretion rate of estradiol was calculated diameter 0.5 mm, internal diameter 0.2 mm, Natsume Co. from the absolute estradiol concentration of the ovarian

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

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Ovarian Nerves Regulate Estradiol Secretion

venous plasma (taking into consideration the concentra- Stimulation of the superior ovarian and ovarian plexus tion in the arterial blood entering the ovary) and the ovari- nerves. In 5 rats, the right SON running along the suspen- an venous plasma flow rate. In rats that received either the sory ligament together with the ligament was cut at ap- SON or the OPN stimulation, five samples were taken, proximately 5 mm from the ovary (Fig. 2A). In 5 other one 5 min before stimulation. The other four samples were rats, the right OPN running along the ovarian artery was taken at 1, 10, 20, and 30 min after the onset of the 5-min cut at approximately 20 mm from the ovary. The peripher- stimulation period. Each rat received either an SON or al end was placed on a bipolar platinum-iridium wire- OPN stimulation. In control rats that received neither stimulating electrode, and the nerve was covered with stimulation, blood samples were taken during rest on the warm liquid paraffin. Electrical square pulse stimulation same schedule as the rats that received SON or an OPN 0.5 ms wide, 20 Hz, 20 V (for SON) or 5 V (for OPN) was stimulation. applied to these nerves for 5 min. In this study, all stimula- Collecting systemic arterial and venous blood. After all tions were of 5 min duration to allow time for the sam- samples from ovarian venous blood were collected, sam- pling of ovarian venous blood during the stimulus period. ples (about 140 µl) of systemic arterial and venous blood In rats used for SON stimulation, the OPN was kept intact. were collected in hematocrit capillary tubes through a In rats used for OPN stimulation, the SON was kept intact. polyethylene tube, which was inserted into the right femo- In 4 control rats, both the SON and the OPN were kept in- ral artery and femoral vein. In preliminary experiments, tact. we confirmed a stability of estradiol concentration in sys- In one rat, evoked volleys were recorded from the SON temic arterial plasma between samples collected both at and the OPN. The SON was cut as described above, and the beginning and at the end of the experiment. the proximal cut end was placed on stimulating elec- Measurement of blood estradiol. After the plasma sam- trodes. Furthermore, the SON was cut at about 27 mm ples were diluted 2–11 times with EIA buffer, plasma es- from the ovary, and the peripheral cut end was placed on tradiol levels were measured by enzyme immunoassay, the recording electrodes. The OPN was cut as described utilizing an estradiol (17β-estradiol) EIA kit (Cayman above, and the peripheral cut end was placed on stimulat- Chemical Co., Michigan, USA). ing electrodes. Furthermore, the OPN was cut at about 8

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

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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 0.5 ms wide, with varying intensity (0.1–20 V). The distance between the stimulating and recording electrodes of the SON was 22 mm; of the OPN it 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. The values were expressed as means ± SEM. A statistical analysis was performed by two-way re- peated ANOVA followed by a Bonferroni posttest or a 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 the concentration 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 Fig. 3. Effects of electrical stimulation of the SON and OPN plasma (systemic arterial estradiol concentration was sub- on ovarian venous plasma flow rate (A), ovarian venous es- tracted from ovarian venous estradiol concentration) and tradiol concentration (B), and estradiol secretion rate from the the ovarian plasma flow rate. Each value of the ovarian ovary (C). Stimulation of 5-min duration is indicated by the up- plasma flow rate, the estradiol concentration of ovarian per horizontal bar. Magnitudes of responses during and after venous plasma, and the secretion rate of estradiol in 5 suc- stimulation are expressed as percentages of the prestimulus values (5 min before the stimulation). Each point and vertical cessive samples taken during 35 min under resting condi- bar represents the mean ± SEM. *P < 0.05; **P < 0.01; signifi- tions in each rat was stable. In 4 control rats, the ovarian cantly different from the response of control using two-way re- plasma flow rate, estradiol concentration of ovarian peated ANOVA followed by Bonferroni posttests. 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 and 5 V for OPN). Stimulus frequency was set at 20 Hz, mean arterial pressure (MAP) was 109.1 ± 14.9 mmHg (n because effector responses usually reached nearly maxi- = 4), and the MAP level was stable for 35 min during the mum when autonomic efferent C fibers were stimulated at collection of ovarian venous blood samples. 20 Hz [19, 20].

Evoked potentials recorded from SON and OPN Effect of electrical stimulation of the SON or OPN Figure 2, B–E, shows typical recordings of evoked po- on the secretion rate of estradiol from the ovary tentials of unmyelinated C fibers in the SON and the OPN. In the 5 rats used for SON stimulation, the prestimulus The evoked potential of the unmyelinated C fibers in the (5 min before the stimulation) basal values of the ovarian SON appeared with a stimulus strength of more than 3 V plasma flow rate, the estradiol concentration of ovarian and reached a maximum at 15 V. In the OPN it appeared venous plasma, and the secretion rate of estradiol were not with a strength of more than 0.8 V and reached a maxi- significantly different from the values observed in control mum at 2 V. The maximum conduction velocity of the C rats. As shown by the filled circles and solid line in Fig. fibers in the SON was 1.2 m/s. In the OPN it was 1.1 m/s. 3A, repetitive electrical stimulation of the SON at the su- In the following experiments, the stimulus intensity was pramaximal intensity for C fibers (0.5 ms, 20 V, 20 Hz) set at a supramaximal intensity for C fibers (20 V for SON produced a decrease in the ovarian plasma flow rate,

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reaching 76 ± 3% of the prestimulus values during stimu- (sympathetic) nerve but not the vagal (parasympathetic) lation. The decreased plasma flow rates returned to the nerve decreases ovarian blood flow [10]. The decrease in prestimulus basal level 5 min after the end of stimulation. ovarian blood flow by the electrical stimulation of the SON stimulation had a tendency to reduce the estradiol splanchnic nerve was completely abolished by the intra- concentration in ovarian venous plasma, but the response venous injection of alpha adrenoceptor antagonist. We was not statistically significant (Fig. 3B). During stimula- also demonstrated that the electrical stimulation of the tion of the SON, the secretion rate of estradiol from the OPN decreases ovarian blood flow [11]. Our present study ovary was decreased in all 5 rats tested. The reduction of showed that an electrical stimulation of either the SON or the secretion rate of estradiol reached 31–65% (53 ± 6%) the OPN produces a decrease in ovarian blood flow (ova- of the prestimulus values during stimulation (Fig. 3C). rian venous plasma flow rate). Therefore we can assume The decreased response of the secretion rate of estradiol that sympathetic adrenergic vasoconstrictor fibers may returned to the prestimulus basal level 5 min after the end travel through both the SON and the OPN to the ovary. of stimulation. No significant MAP response was noted Using the ex vivo celiac ganglion-SON-ovary system and following stimulation of the SON. the superior mesenteric ganglion-OPN-ovary system, it In the 5 rats used for OPN stimulation, the prestimulus was demonstrated that a chemical stimulation of the celiac (5 min before stimulation) basal values of the ovarian ganglion or superior mesenteric ganglion increases the re- plasma flow rate, the estradiol concentration of ovarian lease of nitric oxide in the ovarian compartment [23, 24]. venous plasma, and the secretion rate of estradiol were not Since an electrical stimulation of the SON and the OPN significantly different from the values observed in control produces a decrease in ovarian blood flow in the present rats. As shown by the filled triangles and the broken line results, the adrenergic vasoconstrictor effect seems more in Fig. 3A, repetitive electrical stimulation of the OPN at dominant than the vasodilatory effect of nitric oxide, even the supramaximal intensity for C fibers (0.5 ms, 5 V, 20 if nitric oxide was released in the present experimental Hz) produced a decrease in the ovarian plasma flow rate, condition. reaching 74 ± 9% of the prestimulus values during stimu- A histological study reported that most adrenergic lation. The decreased plasma flow rates returned to the nerves derived from the OPN are perivascular, and those prestimulus basal level 5 min after the end of stimulation. derived from the SON innervate both blood vessels and OPN stimulation had no significant effect on the estradiol steroidogenic interstitial gland cells in the rat ovary [8]. concentration in ovarian venous plasma or on the secre- Our present results showed that OPN stimulation decreas- tion rate of estradiol (Fig. 3, B and C). No significant es ovarian blood flow without affecting ovarian estradiol MAP response was noted following stimulation of the secretion, but that SON stimulation decreases both ovari- 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 mem- brane without having to be packed in granules and active- ly exocytosed [21]. Therefore the 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 the OPN (Fig. 4). We can assume that the reduction of the estradiol secretion rate by SON stimulation was due to the direct inhibitory effects of the SON on estradiol synthesis in the ovary rather than 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 regu- Fig. 4. Schematic diagram of a possible mechanism for the lation of ovarian blood flow, our previous study demon- regulation of estradiol secretion and blood flow of the ovary strated that the electrical stimulation of the splanchnic by stimulation of the SON and OPN.

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an blood flow and ovarian estradiol secretion. These re- 4. Owman C, Stjernquist M. Origin, distribution, and functional aspects of aminergic sults support the histological study by Lawrence and Bur- and peptidergic nerves in the male and female reproductive tracts. In: Björklund A, Hökfelt T, Owman C, editors. Handbook of Chemical Neuroanatomy. Vol 6. den [8]. However, since the vagal nerve may also travel The peripheral nervous system. Amsterdam: Elsevier; 1988. p. 445-544. through the SON and/or the OPN, we cannot exclude the 5. Gerendai I, Tóth IE, Boldogköi Z, Medveczky I, Halász B. Neuronal labeling in possible contribution of the vagus nerve in the regulation the rat brain and spinal cord from the ovary using viral transneuronal tracing technique. Neuroendocrinology. 1998;68:244-56. of ovarian estradiol secretion by the SON in the present 6. Gerendai I, Tóth IE, Boldogköi Z, Medveczky I, Halász B. CNS structures study. presumably involved in vagal control of ovarian function. 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