Role of Korean red ginseng total saponins in rat infertility induced by polycystic ovaries

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1 Role of Korean red ginseng total saponins in rat infertility induced by polycystic ovaries Sok Cheon Pak, Ph.D., a Sung Chul Lim, M.D., Ph.D., b Seung-Yeol Nah, D.V.M., Ph.D., c Jiae Lee, M.S., d Joseph A. Hill, M.D., Ph.D., e and Chun Sik Bae, D.V.M., Ph.D. f a New Zealand College of Oriental Medicine, Hamilton, New Zealand; b Department of Pathology and Research Center for Resistant Cells, Chosun University Medical School, Gwangju, South Korea; c Research Laboratory for the Study of Ginseng Signal Transduction and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul, South Korea; d Creation and Love Women s Hospital, Gwangju, South Korea; e Fertility Centers of New England, Reading, Massachusetts; and f College of Veterinary Medicine, Biotechnology Research Institute, Chonnam National University, Gwangju, South Korea Objective: To investigate the effect of Korean red ginseng total saponins (GTS) on ovarian morphology and nerve growth factor (NGF) expression in the ovaries, pituitary, and hippocampus. Design: Polycystic ovary (PCO) rat model induced by estradiol valerate (EV). Setting: University research laboratory. Patient(s): Thirty sexually mature female Sprague-Dawley rats weighing g. Intervention(s): Female Sprague-Dawley rats ( g) were separated into three groups: EV control (n 10), EV plus GTS (n 10), and oil control (n 10). Main Outcome Measure(s): Ovarian morphology and NGF protein expression. Result(s): Polycystic ovary was fully developed in rats with a single intramuscular injection of EV. Increased expression of NGF was noted in the ovaries and the brain of rats with PCO. GTS administration attenuated NGF expression in the ovaries but not in the brain. Conclusion(s): Our findings suggest a role for GTS in the regulation of NGF expression in female rats with PCO. (Fertil Steril 2005;84(Suppl 2): by American Society for Reproductive Medicine.) Key Words: Ginseng total saponins, polycystic ovary syndrome, nerve growth factor, estradiol valerate, immunohistochemistry Polycystic ovary syndrome (PCOS) is one of the most common types of endocrinopathy among reproductive age women (1). This syndrome has been defined in a number of different ways since its initial description in 1935 (2, 3). The presence of a polycystic ovarian morphology has been recently included in the list of criteria for defining PCOS (4). The major clinical features of PCOS are hyperandrogenism, anovulation and metabolic disturbances. Women with PCOS tend to have a higher luteinizing hormone/ follicle-stimulating hormone (LH/FSH) ratio than normal women, which causes a disruption in the ovarian follicular development and anovulation (5). Furthermore, the disrupted estrogen levels along with the severe oligomenorrhea and amenorrhea can also result in endometrial hyperplasia and cervical cancer (6). The creation of an all-purpose animal model is complicated by the multifactorial etiology of PCOS (7 10). A rat model for PCOS based on the establishment of polycystic ovaries after an injection of estradiol (E 2 ) valerate (EV) has been described (7). Received November 16, 2004; revised and accepted April 9, This study was financially supported by research fund of Chonnam National University in The first two authors contributed equally to this work. Reprint requests: Chun Sik Bae, D.V.M., Ph.D., College of Veterinary Medicine, Biotechnology Research Institute, Chonnam National University, Yongbongdong, Bukgu, Gwangju , South Korea (FAX: ; csbae210@chonnam.ac.kr). The neurotrophin family including the nerve growth factor (NGF) reportedly involved the NGF receptor (11) and NGF mrna (12) in ovulation and in the pathophysiology of PCOS. A polycystic ovary is reported to exhibit a high density of intraovarian nerve fibers that are associated with sympathetic hyperresponsiveness (12). In the rat ovary, NGF is mainly synthesized in the follicular wall cells (13). The activation of NGF might be involved in enhancing the norepinephrine outflow in an E 2 -induced polycystic ovary (11). Ginseng (the roots of Panax ginseng C.A. Meyer, Araliaceae) is a traditional Eastern Asian herbal medicine taken orally for a tonic and slowing down of the aging process. Ginseng is a popular dietary supplement throughout the world. The major active ingredients of ginseng are the ginseng saponins, which are composed of various ginsenosides (14, 15). To date, more than 30 ginsenosides have been identified (16). This study produced a murine PCO model using E 2 valerate to investigate the effect of Korean red ginseng total saponins (GTS) on the ovarian morphology and NGF protein expression in a rat PCO model. MATERIALS AND METHODS Animals Sprague-Dawley female rats (n 30) weighing g and with regular 4-day estrous cycles were purchased from the Daehan Bio Link Co. (Taejon, South Korea). The rats /05/$30.00 Fertility and Sterility Vol. 84, Suppl 2, October 2005 doi: /j.fertnstert Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. 1139

2 were housed under controlled conditions of temperature (25 C 1 C), with access to food and water ad libitum, and were cared for in accordance with the Animal Care Board at the Chonnam National University. Institutional Review Board approval was obtained before in vivo experiments. PCO Induction Twenty rats received a single 4-mg IM injection of E 2 valerate (Sigma, St. Louis, MO) in 0.2 ml of sesame oil (Sigma) to induce a polycystic ovary, and ten received oil only for the control, as previously described (7). To obtain a fully developed polycystic ovary, a duration of 60 days after the injection was chosen. Ginseng Total Saponin The GTS contained at least 11 glycosides, namely, Rb1 (18.26%), Rb2 (9.07%), Rc (9.65%), Rd (8.24%), Re (9.28%), Rf (3.48%), Rg1 (6.42%), Rg2 (3.62%), Rg3 (4.7%), Ro (3.82%), and Ra (2.91%), as well as other minor ginsenosides (20.55%). The GTS was a kind gift from the Korea Ginseng and Tobacco Research Institute, Taejon, Korea. The GTS was dissolved in saline and administered intraperitoneally. Study Protocol The animals were randomized into the two treatment groups in a blind manner: ten rats in the EV control group and ten in the GTS-treated group (and ten in the oil control group). The GTS-treated EV group rats were administered 100 mg/kg GTS intraperitoneally every other day for 60 days, beginning 1 day after the EV injection. Nerve Growth Factor Measurement by Immunohistochemistry The dissected ovary, pituitary, and hippocampus were stored in fixative at room temperature for 2 days. The fixed tissue samples were embedded in paraffin for microtome slicing into 4- m thick slices, and the sliced tissue sections were mounted on X-tra slides (Surgipath, Richmond, VA). To observe the immunohistochemical reaction to NGF, the slides were deparaffinated and hydrated by immersing sequentially in xylene and decreasing concentrations of ethanol (100%, 95%, 90%, and 80%) for 5 minutes each followed by distilled water for 10 minutes. The level of antigen retrieval was enhanced by pretreating the sections in a microwave (Pelco laboratory microwave oven; Ted Pella, Redding, CA) in 0.01 mol/l citrate buffer (ph 6.0) 3 times for 5 minutes at 360 W. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 15 minutes. After each step, the sections were washed with 50 mmol/l Tris buffered saline (TBS, ph 7.5) 3 times for 10 minutes each. The tissue sections were then covered with a blocking antibody for 10 minutes and incubated overnight at 4 C with a rabbit antimouse NGF antibody (clone 2.5s; Serotec, Kidlington, Oxford, UK) at a dilution of 1:50. For each case, a corresponding section was incubated in TBS without the primary antibody as a control for nonspecific staining. The biotinylated rabbit antimouse secondary antibody was added for 10 minutes, which was followed by adding the avidin-biotinylated peroxidase complex for an additional 10 minutes. After washing, the sections were stained with 3-amino-9-ethyl carbazole (AEC; Vector Laboratories, Burlingame, CA), counterstained with Mayor s hematoxylin and mounted. The positive control for NGF was the cerebral cortical neurons of the rat. Instead of the primary antibody, TBS was used as the negative control. Ovarian Morphology All 30 rats were killed by transcardial perfusion with chloral hydrate (500 mg/kg) anesthesia containing a 4% paraformaldehyde solution in 0.1 mol/l of sodium cacodylate buffer and 4% sucrose added at ph 7.4. The dissected ovaries were bisected midsagittally and placed in the same fixative overnight at room temperature. The ovaries were then totally embedded in paraffin. The samples were sectioned at 4 m and stained with hematoxylin and eosin. The number of follicles containing an oocyte with a nucleus were counted and analyzed by the same individual blinded to the treatment group. If ovum degeneration or at least one pyknotic granulosa cell was observed, the follicle populations were classified as being atretic. The morphologic characteristics of follicular atresia included scattered pyknotic nuclei in the granulosa cell layer, detachment of the granulosa cell layer from the basement membrane, fragmentation of the basal lamina, and the presence of cell debris in the antrum of the follicle. Analysis and Interpretation of Immunohistochemical Staining Two independent observers without any knowledge of the experiments assessed the stained sections. Consensus scores were assigned for each case by reviewing the slides with scoring discrepancies. The NGF was decoded according to the staining intensity and the number of positive stained cells as follows: 0, negative staining; 1, a few positive staining; 2, diffuse weakly positive staining; 3, moderately positive staining; 4, strongly positive staining. Each score was added together and mean SD was calculated in each group. Statistics All the values for the weights of body and ovaries, and the scores for the NGF immunostaining in the ovary, pituitary, and hippocampus were compared using analysis of variance. The significance level was Pak et al. PCO treatment with genseng saponins Vol. 84, Suppl 2, October 2005

RESULTS Ovarian Morphology Normal-appearing ovaries were observed in the oil control group, as identified by numerous primary, secondary, and growing follicles in the cellular stroma.

They consisted of multiple dilated follicular cysts, atretic follicles, abundant cortical stroma, and well-developed theca interna. Atretic follicles also showed well-developed theca interna.

These morphological observations were in accord with those reported by Brawer et al. (17).

3 RESULTS Ovarian Morphology Normal-appearing ovaries were observed in the oil control group, as identified by numerous primary, secondary, and growing follicles in the cellular stroma. Antral and atretic follicles were also observed infrequently. The ovaries in the EV control group exhibited multiple cystic follicles, which were consistent with fully developed PCO. They consisted of multiple dilated follicular cysts, atretic follicles, abundant cortical stroma, and well-developed theca interna. Atretic follicles also showed well-developed theca interna. There were almost no primary and secondary follicles observed. Focal areas of stromal hyperthecosis were also noted. These morphological observations were in accord with those reported by Brawer et al. (17). The numbers of corpora lutea and corpora albicantia in the EV plus GTS group were significantly higher than the EV control while the number of cystic follicles was lower. A larger number of growing secondary follicle numbers was also noted compared with the EV control (Fig. 1). Nerve Growth Factor The tissues from the ovary, pituitary, and hippocampus were used for the NGF measurements by immunohistochemistry. In the oil control group, a few positive scattered stromal cells for NGF in the ovaries were observed. The hippocampus had scattered positive cells that stained positive for NGF. However, NGF expression was absent in the pituitary. In the EV control group, there were many thecal and stromal cells that strongly stained NGF positive, some scattered positive follicular cells in the ovary (Fig. 2), and some scattered positive FIGURE 1 Estradiol valerate plus ginseng total saponins group. Microscopic finding shows many corpora lutea (stars), 40. FIGURE 2 Estradiol valerate control group. Strong positive immunoreactivity for nerve growth factor is noted in many follicular cells (arrows). ABC method, counterstained by hematoxylin, 40. neuronal cells in the hippocampus and pituitary gland. In the EV plus GTS group, the thecal and stromal NGF immunoreactivities were lower in the ovaries than in the ovaries of the EV controls (Fig. 3). Scattered positive immunoreactivity for NGF was observed in the neuronal cells of the hippocampus and pituitary. The intensity of NGF staining in the pituitary and hippocampus was similar to that observed in the EV control tissues. FIGURE 3 Estradiol valerate plus ginseng total saponins group. Weakly positive immunoreactivity for nerve growth factor is noted in follicular cells (arrows) of the ovary. ABC method, counterstained by hematoxylin, 40. Fertility and Sterility 1141

4 TABLE 1 ANOVA table for the differences among the mean scores of each group. Source Type III SS df MS F P value Model Group Block Error Total Note: SS sum of squares; df degrees of freedom; MS mean squares; F F statistic. The differences among the mean scores of the oil control group, the EV control group, and the EV plus GTS group were tested by ANOVA. Table 1 shows the ANOVA result generated by SPSS (SPSS, Chicago, IL). The F value is Because the P value is.000, the mean scores of the oil control group, EV control group, and EV plus GTS group are not all the same under the significance level.05. We also concluded that the groups are different from each other by the Duncan s multiple comparison test under the significance level.05. Weights of Body and Ovaries Table 2 shows the ovarian weights in all groups. The ovarian weight in the EV plus GTS group was lower than that in the oil control and EV control groups, and there was a statistically significant difference between the EV plus GTS group and the oil control group (P.01). No fluctuations in the rat body weight were observed over an 8-week period (data not shown). DISCUSSION This study shows that GTS administered intraperitoneally attenuated the NGF expression level in the rat ovaries with PCO. However, the brain areas of the pituitary and hippocampus were unaffected by GTS. This suggests that the action site of the ginseng saponins in the regulation of NGF expression may be in the periphery rather than centrally TABLE 2 Weights of ovary measured at day 60. Weight (g, mean SD) Oil control (n 10) EV control (n 10) EV GTS treated (n 10) a a P.01, EV plus GTS vs. oil control. within the brain. The peripheral noradrenergic and peptidergic responses have been implicated in the highly systematic ovarian function (18). The findings of high levels of NGF expression in the ovaries and brain tissue support recent reports showing that the ovarian NGF concentrations in the rats with experimentally induced PCO were higher (11, 19). The administration of E 2 valerate is known to overproduce NGF, which is related to the hyperactivation of the ovarian sympathetic input. This enhanced activity of the neurotrophic neurogenic control system contributes to the process by which EV induces ovarian cysts and disrupts ovulation in the rats, resulting in infertility. Because the sympathetic nerves arrive at the ovary via the superior ovarian nerves, the delivery of GTS appeared to disrupt the ovary-related nervous system at the early stages of the ovarian changes because increased ovarian NGF concentrations have been shown to precede the morphologic changes in the rats with PCO (11). Furthermore, the cyclicity and ovulatory capacity in the EV-treated rats were restored when the superior ovarian nerves were transected (20). This was the reason why the GTS was administered from 1 day after the EV injection. The changes in the immunohistochemical reaction as a result of GTS demonstrated that NGF protein expression in rats could be regulated by these ginseng saponins. The results showed that a single IM injection of EV could induce the appearance of PCO in rats. The messenger for the induction of PCO was hyperactivity in the sympathetic nervous system, as suggested by the expression of NGF in the ovaries and the central nervous system. Using an experimental PCO model, GTS was found to partially reverse the abundance of NGF in the EV-treated rats. REFERENCES 1. Carmina E, Lobo RA. Polycystic ovary syndrome (PCOS): arguably the most common endocrinopathy is associated with significant morbidity in women. J Clin Endocrinol Metab 1999;84: Stein IF, Levental ML. Amenorrhea associated with bilateral polycystic ovaries. Am J Obstet Gynecol 1935;29: Pak et al. PCO treatment with genseng saponins Vol. 84, Suppl 2, October 2005

5 3. Zawadzki JK, Dunaif A. Diagnostic criteria for polycystic ovary syndrome: towards a rational approach. In: Dunaif A, Givens JR, Haseltine FP, Merriam GR, eds. Polycystic ovary syndrome: current issues in endocrinology and metabolism. Vol. 4. Boston: Blackwell Scientific; p Chang R. Polycystic ovary syndrome: diagnostic criteria. In: Chang R, Heinder J, Duanif A, eds. Polycystic ovary syndrome. New York: Marcel Dekker; p Taylor AE, McCourt B, Martin KA, Anderson EJ, Adams JM, Schoenfeld D, et al. Determinants of abnormal gonadotropin secretion in clinically defined women with polycystic ovary syndrome. J Clin Endocrinol Metab 1997;82: Norman RJ, McVeigh E. Polycystic ovary syndrome and implications for the menopause. Climacteric 1999;2: Bai YH, Lim SC, Song CH, Bae CS, Jin CS, Choi BC, et al. Electroacupuncture reverses nerve growth factor abundance in experimental polycystic ovaries in the rat. Gynecol Obstet Invest 2004;57: Barraclough CA, Gorski RA. Evidence that the hypothalamus is responsible for androgen-induced sterility in the female rat. Endocrinology 1961;68: Bagavandoss P, England B, Asirvatham A, Bruot BC. Transient induction of polycystic ovary-like syndrome in immature hypothyroid rats. Proc Soc Exp Biol Med 1998;219: Campbell CS, Schwartz NB. The impact of constant light on the estrous cycle of the rat. Endocrinology 1980;106: Lara HE, Dissen GA, Leyton V, Paredes A, Fuenzalida H, Fiedler JL, et al. An increased intraovarian synthesis of nerve growth factor and its low affinity receptor is a principal component of steroid-induced polycystic ovary in the rat. Endocrinology 2000;141: Stener-Victorin E, Lundeberg T, Cajander S, Aloe L, Manni L, Waldenstrom U, et al. Steroid-induced polycystic ovaries in rats: effect of electro-acupuncture on concentrations of endothelin-1 and nerve growth factor (NGF), and expression of NGF mrna in the ovaries, the adrenal glands, and the central nervous system. Reprod Biol Endocrinol 2003;1: Dissen GA, Hill DF, Costa ME, Dees WL, Lara HE, Ojeda SR. A role for trka nerve growth factor receptors in mammalian ovulation. Endocrinology 1996;137: Kim DH, Moon YS, Lee TH, Jung JS, Suh HW, Song DK. The inhibitory effect of ginseng saponins on the stress-induced plasma interleukin-6 level in mice. Neurosci Lett 2003;353: Tachikawa E, Kudo K, Hasegawa H, Kashimoto T, Sasaki K, Miyazaki M, et al. In vitro inhibition of adrenal catecholamine secretion by steroidal metabolites of ginseng saponins. Biochem Pharmacol 2003; 66: Bae EA, Han MJ, Kim EJ, Kim DH. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch Pharm Res 2004;27: Brawer JR, Munoz M, Farookhi R. Development of the polycystic ovarian condition (PCO) in the estradiol valerate treated rat. Biol Reprod 1986;35: Lara HE, McDonald JK, Ojeda SR. Involvement of nerve growth factor in female sexual development. Endocrinology 1990;126: Stener-Victorin E, Lundeberg T, Waldenstrom U, Manni L, Aloe L, Gunnarsson S, et al. Effects of electro-acupuncture on nerve growth factor and ovarian morphology in rats with experimentally induced polycystic ovaries. Biol Reprod 2000;63: Barria A, Leyton V, Ojeda SR, Lara HE. Ovarian steroidal response to gonadotropins and beta-adrenergic stimulation is enhanced in polycystic ovarian syndrome: role of the sympathetic innervation. Endocrinology 1993;133: Fertility and Sterility 1143

The Effect of Herbal Medicine on Nerve Growth Factor in Estradiol Valerate-induced Polycystic Ovaries in Rats

The American Journal of Chinese Medicine, Vol. 31, No. 6, 885 895 2003 World Scientific Publishing Company & Institute for Advanced Research in Asian Science and Medicine The Effect of Herbal Medicine