06-JPS58-2-RP001508.fm 133 ページ ２００８年４月１４日 月曜日 午後３時２２分 REGULAR PAPER J. Physiol. Sci. Vol. 58, No. 2; Apr. 2008; pp. 133–138 Online Mar. 22, 2008; doi:10.2170/physiolsci.RP001508 Effects of Electrical Stimulation of the Superior Ovarian Nerve and the Ovarian Plexus Nerve on the Ovarian Estradiol Secretion Rate in Rats 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] The Journal of Physiological Sciences Vol. 58, No. 2, 2008 133 06-JPS58-2-RP001508.fm 134 ページ ２００８年４月１４日 月曜日 午後３時２２分 F. KAGITANI et al. 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 ex- periment was performed on the rat on the day of estrus. B: Illus- tration showing the experimen- tal procedures for collecting ovarian venous blood. C: Estra- diol concentrations in ovarian venous plasma, systemic arteri- al plasma, and systemic venous plasma in 4 rats (mean ± SEM). 134 The Journal of Physiological Sciences Vol. 58, No. 2, 2008 06-JPS58-2-RP001508.fm 135 ページ ２００８年４月１４日 月曜日 午後３時２２分 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.