{"paper_id":"1c817525-c29f-4e82-b1a3-ae140787a27b","body_text":"BioMed Central\nPage 1 of 6\n(page number not for citation purposes)\nReproductive Biology and \nEndocrinology\nOpen AccessResearch\nEvaluation of the true precocious puberty rats induced by neonatal \nadministration of Danazol: Therapeutic effects of nourishing \"Yin\"- \nremoving \"Fire\" Chinese herb mixture\nZhanzhuang Tian†1, Hong Zhao†1, Yan Sun1, Depei Cai2 and Boying Chen*1\nAddress: 1Department of Neurobiology and Integrative Medicine, Shanghai Medical College of Fudan University (Formerly Shanghai Medical \nUniversity), P.O. Box 291, 138 Yi-Xue-Yuan Road, 200032 Shanghai, P. R. China and 2Children's Hospital of Fudan University, 183 Feng-Lin Road, \n200032 Shanghai, P. R. China\nEmail: Zhanzhuang Tian - tianzhanzhuang@hotmail.com; Hong Zhao - zh2000h@hotmail.com; Yan Sun - sunyan7819@163.com; \nDepei Cai - tianz@umkc.edu; Boying Chen* - chen_bo_ying@hotmail.com\n* Corresponding author    †Equal contributors\nAbstract\nBackground: Nourishing \"Yin\"-Removing \"Fire\" Chines e Herb Mixture, a traditional herb-based\nformulation, has been succe ssfully used for the management of id iopathic true precocious puberty (IPP)\nfor more than thirty years. Precocious puberty rat model by neonatal administration of Danazol was used\nto investigate the effects of the he rb mixture on the advanced sexual  development of the rats, and the\nexpression of hypothalam ic gonadotropin-releasing hormone (GnR H), which is the important regulator\nfor the hypothalamus-pituitary-gonadal axis, particularly at puberty.\nMethods: Female Sprague-Dawley rats were divided into five groups: intact normal (N), IPP model (M),\nvehicle with no IPP (V), IPP model exposed to herb  mixture (HM) and IPP model exposed to saline (S).\nRats at 5 days of age were given a single subcutaneous injection of 300 microgram of Danazol dissolved in\n25 microliter vehicle of propylene glycol-ethanol (1 :1, v/v), to establish th e precocious puberty model.\nFrom the day 15, rats in HM and S groups were continuously fed with either Nourishing \"Yin\"-Removing\n\"Fire\" Chinese Herb Mixture 2 ml  or saline 2 ml, unt il 3 consecutive regular estrous cycles were\nestablished. The day of vaginal opening and the da y of setup regular estrous cycle of the rats were\nobserved. Blood concentration of estrogen was determined by radioimmunoassay. Immunohistochemistry\nand RT-PCR analysis were used to explore the expression of GnRH.\nResults: The day of vaginal opening and first estrous showed significant advancement in M compared with\nN and V (p < 0.05, respectively). The blood estrogen level increased significantly in M compared with those\nin other groups (about 28 days of age, at the time of vaginal opening in M rats ) (p < 0.05, respectively).\nGnRH cells in rostral medial septum (MS), Broca di agonal band nucleus (DBB) and the medial preoptic\narea (MPOA), were calculated. The number in M was less than those in N and V (p < 0.05, respectively).\nThe number was significantly higher in HM than that in M (p < 0.05). The GnRH mRNA expression\nincreased significantly in M compared with that in N and V (p < 0.05).\nConclusion: The true precocious puberty model by neonatal  administration of Danazol in female rats\nshowed augmented expression of hypothalamic GnRH ; the Nourishing \"Yin\"-Removing \"Fire\" Chinese\nHerb Mixture down-regulated the increased GnRH expression, and significantly delayed the sexual\ndevelopment of the precocious puberty rat.\nPublished: 22 August 2005\nReproductive Biology and Endocrinology 2005, 3:38 doi:10.1186/1477-7827-3-38\nReceived: 01 July 2005\nAccepted: 22 August 2005\nThis article is available from: http://www.rbej.com/content/3/1/38\n© 2005 Tian et al; licensee BioMed Central Ltd. \nThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), \nwhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\n\nReproductive Biology and Endocrinology 2005, 3:38 http://www.rbej.com/content/3/1/38\nPage 2 of 6\n(page number not for citation purposes)\nBackground\nSexual precocity is one of the most popular endocrine dis-\norders in children, incidence of which is about 0.6%\nthroughout the world [1]. It is 10 times more common in\ngirls than in boys of the disease. Precocious puberty is the\nappearance of the secondary sexual characteristics before\nage 8 years and can be mainly classified into true preco-\ncious puberty (GnRH-dependent sexual precocity) and\nincomplete isosexual precocity (GnRH-independent sex-\nual precocity). The term of true precocious puberty prop-\nerly applies only to sexual precocity mediated by\npremature activation of the hypothalamic-pituitary-ovar-\nian axis usually before 8 year-old. Though central nervous\nsystem tumors such as hamartomas and astrocytomas\nmay cause true precocious puberty, most cases have no\norganic disease, which is defined idiopathic true preco-\ncious puberty (IPP) [2].\nThough experimental models of precocious puberty have\nbeen induced in female rats by neonatal injection of tes-\ntosterone, estradiol or melatonin, these rats developed\npersistent vaginal estrous or disturbance of cyclicity (pre-\ndominance of estrous smear) shortly after the day of first\nestrous. It has been reported that the neonatal administra-\ntion of Danazol may affect the hypothalamic pituitary axis\nwith the rapid rate of maturation, which may serve as a\nmodel for analyzing true precocious puberty [3].\nSeveral medications have been reported to be effective\nagainst true precocious puberty, which include the GnRH\nanalogues [4], progesterone prescriptions and Chinese\nherbal medicine (CHM). Nourishing \"Yin\"-Removing\n\"Fire\" Chinese Herb Mixture, a traditional herb-based for-\nmulation, has been successfully used for the management\nof IPP by us for more than thirty years. It has been clini-\ncally verified to successfully modulate the course of puber-\ntal development and optimize skeletal development in\nchildren with precocious puberty, but without notable\nside effects [5]. The present study was to observe the\neffects of the herb mixture on the true precocious rat\nmodel.\nMethods\nAnimals\nFemale Sprague-Dawley rats at 3 days of age in company\nwith the maters were purchased from Medical Experimen-\ntal Animals Center of Fudan University (Shanghai,\nChina). Animals were housed under laminar flow in an\nisolated room with controlled temperature and at a 12 /12\n(light /dark) schedule. The model [3] litters at day 5 (the\nday of birth was termed day 1) were given a single subcu-\ntaneous injection of 300 µg of Danazol (Hualian Pharm\nLtd, Shanghai, China) dissolved in 25 µl vehicle of propyl-\nene glycol-ethanol (1:1, v/v), and allowed to grow with-\nout further treatment. The animals were weaned on day\n23, and were examined daily for vaginal opening after-\nwards, and then the daily vaginal smears were examined.\nAll experimental procedures involving the use of animals\nwere conducted in accordance with NIH Guidelines and\nwere reviewed and approved by the Animal Use and Care\nCommittee for the Fudan University.\nIn the Experiment 1, rats were used to observe the day of\nvaginal opening and the day of setup regular oestrous\ncycle, which were divided into five groups: intact normal\n(N), IPP model (M), vehicle with no IPP (V), IPP model\nexposed to herb mixture (HM) and IPP model exposed to\nsaline (S). Daily vaginal smears were checked until 3 con-\nsecutive regular 4 or 5 days estrous cycles were estab-\nlished. The day of diestrous I of the first cycle in the 3\nconsecutive regular estrous cycles was determined as the\nday of setup regular estrous cycle. From the day 15, rats in\nHM and S groups were continuously fed with either Nour-\nishing \"Yin\"-Removing \"Fire\" Chinese Herb Mixture 2 ml\nor saline 2 ml, until 3 consecutive regular estrous cycles\nwere established.\nRIA of blood estrogen concentration\nAt the time of vaginal opening in model rats, the blood\nsamples of all the rats were collected from tail veins\nrespectively. The plasma was separated by centrifugation\nand stored at -80°C until assayed. Concentration of E 2\nwas determined by a double-antibody RIA using the kit\npurchased from the Shanghai Institute of Biological Prod-\nucts (Shanghai, China.). The sensitivity of the kit was 1.4\npg/ml, and the intra- and inter-assay coefficients of varia-\ntion were 3.7–8.0% and 4.74–7.7%.\nImmunohistochemistry analysis\nIn the Experiment 2, thirty rats divided into the same five\ngroups were used to investigate the expression of GnRH in\nthe rostral medial septum (MS), Broca diagonal band\nnucleus (DBB) and the medial preoptic area (MPOA) of\nthe rats by immunohistochemistry according to the Atlas\n[6]. At the days of vaginal opening in the model group,\nfollowing anesthesia, all of the animals were exsanguin-\nated with normal saline. Perfusion done, the brains were\nremoved and split equally along the third ventricle. One\nhalf of the brain was postfixed for >48 h in 4% parafor-\nmaldehyde in 0.1 M PB (PH 7.4) with 30% sucrose, and\ntheir sections were sliced at 35 µm thickness on a\nvibratome microslicer and stored at 4°C in tissue culture\nwells containing 0.1 M PBS (PH 7.4) plus 0.02% sodium\nazide until further processed. The other half of the brain\nwas snap-frozen in liquid nitrogen, and then stored at -\n80°C.\nWashed in PB for 30 min at room temperature (RT), the\nfloating sections were incubated in PB containing 10%\nbovine serum albumin (BSA) for 2 h at RT, then with both\n\nReproductive Biology and Endocrinology 2005, 3:38 http://www.rbej.com/content/3/1/38\nPage 3 of 6\n(page number not for citation purposes)\nrabbit anti-GnRH (1:2000, Chemicon International Inc.)\ndiluted in PB containing 1% BSA, 0.02% sodium azide,\nand 0.4% Triton-X 100 at 37°C for 2 h, and then at 4°C\nfor 70 h. When they had been rinsed in PB (× 3, 5 min\neach), the sections were incubated in secondary antibody\nsolutions (goat anti-rabbit IgG conjugated to peroxidase\n1:200, Sino-American Technology Company, China) with\nthe same blocking sera for 2 h at RT. After that, they were\nwashed (× 3, 30 min), and diaminobenzidine (DAB) was\nused as chromogen (Vectastain Elite kits, Vector Labs).\nTissue collection andtotal RNA preparation\nThe other half of the brain in Experiment 2 was used to\ninvestigate the expression of GnRH mRNA by RT-PCR.\nThe target regions, including mediobasal hypothalamus\nand the suprachiasmatic-preoptic area were dissected\n(limited anteriorly by the optic chiasma, laterally by the\nhypothalamic fissures, posteriorly by the mammilary\nbodies and in depth by the subthalamic sulcus). Total\nhypothalamic RNA was extracted using 'TRIzol Regent'\n(Biobasic Inc., Canada) according to the manufacturer's\ninstructions. The purity and integrity of the RNA were\nchecked spectroscopically and by gel electrophoresis\nbefore carrying out the analytical procedures.\nRNA analysis\nTissue RNA (2 µg) was reverse transcribed, in a final vol-\nume of 20 µl, using 200 IU M-MLV reverse transcriptase in\nthe presence of 25 pmol GnRH specific downstream\nprimer (Sangon Inc), 0.5 mM deoxy-NTP and 20IU Rna-\nsin (from Promega) for 60 min at 42°C, then heat dena-\ntured for 5 min at 95°C. 5 µl cDNAs were further\namplified by PCR using 25 pmol of upstream primer\n(Sangon Inc) for GnRH: 5'-ATT CTA CTG ACT TGG TGC\nGTG-3'; downstream, 5'-GGA ATA TGT GCA ACT TGG\nTGT-3' [7]. PCR was performed for 30 cycles (1 min at\n94°C, 45 sec at 62°C, 1 min at 72°C) in the presence of\nTaq DNA polymerase (3U per tube) and 2.2 mM magne-\nsium chloride (from Promega) in a final volume of 50 µl.\nTo check the presence of DNA contamination RT-PCR was\nperformed on 2 µg of total RNA without M-MLV reverse\ntranscriptase (negative control). An internal control ( β-\nactin, upstream, 5'-AAG CAG GAG TAT GAC GAG TCC G-\n3'; downstream, 5'-GCC TTC ATA CAT CTC AAG TTG G-\n3') for each RT-PCR was performed to account for proce-\ndural variations. For each sample 5 µl of PCR amplifica-\ntion products were analyzed on 2% agarose gels and\nstained with ethidium bromide. Standard DNA (100 bp\nDNA ladder Promega) was run to provide the appropriate\nsize marker. The RT-PCR products were extracted and\npurified from agarose gel by Golden Beads Gel Extraction\nkit (Sangon Inc., China) and sequenced using radioactive\ndideoxychain terminating method (Sangon Inc., China).\nThe intensities of the bands were evaluated by Image Mas-\nter Software (SYDR-1990, SYNGENE, U.S.A.).\nStatistical analysis\nAll data are presented as means ± S.E.M. Statistical analy-\nsis was performed on raw data using one-way analysis of\nvariance (ANOVA), with the significance concentrations\nof p < 0.05 in two-tailed testing chosen. Comparisons\namong groups were made using the Student's t-test.\nEffects of herb mixture on GnRH expression by immunohistochemistryFigure 1\nEffects of herb mixture on GnRH expression by \nimmunohistochemistry. N: intact normal, M: IPP model, \nV: vehicle with no IPP, HM: IPP model exposed to herb mix-\nture, S: IPP model exposed to saline. A: The statistical analy-\nsis of (spiny GnRH cells/total cells) % (n = 6). * p < 0.05 vs M; \n^ P < 0.05 vs HM. B: Total GnRH cells of the rats (n = 6). * p \n< 0.05 vs N and V, respectively; ^ P < 0.05 vs HM. C: Light \nmicrography illustrating the different types of GnRH cells at \nthe region of MS at low power (n = 6). Sm: smooth type cell; \nSp: spiny type cell.\n\n\nReproductive Biology and Endocrinology 2005, 3:38 http://www.rbej.com/content/3/1/38\nPage 4 of 6\n(page number not for citation purposes)\nResults\nThe day of vaginal opening and regular estrous cycle of the \nrats\nThe day of vaginal opening and regular estrous cycle were\nsignificantly (p < 0.01, respectively) advanced in M than\nthat in N and V, and there was no difference between N\nand V groups (Table 1). The day of vaginal opening and\nregular estrous cycle in HM were significantly (p < 0.01)\ndelayed than those in M and S (Table 1).\nBlood concentration of estrogen\nThe blood E 2 concentration in M is twofold higher than\nthat in N (p < 0.05). While the E 2 level was lower in HM\nthan in S (p < 0.05). There were no differences between N\nand V, M and S (Table 1).\nEffects of herb mixture on GnRH expression by \nimmunohistochemistry\nGnRH cells bodies, which were abundant in MS, DBB and\nMPOA, were calculated. The GnRH cells number in M was\nless than that in V (p < 0.05), and there was no difference\nbetween N and V (Fig 1). The number was significantly\nhigher in HM than that in S (p < 0.05), and no statistical\ndifference was observed between HM and N (Fig 1). There\nwas no significant difference between M and S (Fig 1). The\ntissue sections processed for immunohistochemistry\nusing antiserum after preabsorption with excessive anti-\ngens and omission of primary antibody showed the stain-\ning as expected.\nThe subtypes of all the observed GnRH neurons were dis-\ncerned under high microscope (× 400). The classification\nof spiny type and smooth type GnRH neurons was accord-\ning to the report by Witkin [8] (Fig 1). The percent of\nspiny type GnRH neurons was significantly higher in M\nthan those in N and HM (p < 0.05, respectively) (Fig 1).\nThere were no differences between N and V, M and S.\nEffects of herb mixture on GnRH mRNA expression by RT-\nPCR\nComparison of the amplified PCR fragment with rat\nGnRH sequence revealed 100% homology (data not\nshown). Densitometric analysis of the mRNA concentra-\ntion using GnRH/β-actin expressed as the mean with SEM.\nThe ratio of GnRH to β-actin in the M increased signifi-\ncantly compared with that in the N and V (p < 0.05), and\nthe ratio in the HM decreased, with no difference showed\nbetween HM and N. (Fig 2). There was no significant dif-\nference between M and S.\nDiscussion\nMammalian sexual maturation and adult reproductive\nfunction are centrally controlled by a subset of neuroen-\ndocrine neurons that produce GnRH. Recent ten years\nstudy indicated, GnRH neurons are the final common\npathway for the neuronal control of gonadotropin secre-\ntion, which will in turn stimulate gametogenesis and\ngonadal hormone secretion [9]. In humans and rats, the\nTable 1: The day of vaginal opening and regular estrous cycle of the rats\nN (n = 18) V (n = 18) M (n = 18) HM (n = 18) S (n = 18)\nDay of vaginal opening 34.00 ± 0.98 34.33 ± 1.09 25.97 ± 2.24* 32.56 ± 1.04 # 24.78 ± 1.01*\nDay of regular estrous cycle 41.26 ± 2.35 45.00 ± 1.19 32.43 ± 0.75* 40.58 ± 0.84 # 34.56 ± 0.89*\nBlood estrogen (pg/ml) 22.64 ± 3.69 24.58 ± 3.87 50.36 ± 5.54* 30.98 ± 7.02 # 51.14 ± 4.98*\n*p < 0.05 vs N and V, respectively; # p < 0.05 vs M and S, respectively\nN: intact normal, M: IPP model, V: vehicle with no IPP, HM: IPP model exposed to herb mixture, S: IPP model exposed to saline\nEffects of herb mixture on GnRH mRNA expression by RT-PCRFigure 2\nEffects of herb mixture on GnRH mRNA expression \nby RT-PCR. The upper picture shows the gel electrophore-\nsis of the RT-PCR products for the GnRH. Densitometric \nanalysis of the mRNA concentration using GnRH/β-actin \nexpressed as the mean with SEM bar (n = 6) in each column \nindicated in the lower panel. N: intact normal, M: IPP model, \nV: vehicle with no IPP, HM: IPP model exposed to herb mix-\nture, S: IPP model exposed to saline. *p < 0.05 vs N and V, \nrespectively; ^ p < 0.05 vs M and S, respectively.\n\n\nReproductive Biology and Endocrinology 2005, 3:38 http://www.rbej.com/content/3/1/38\nPage 5 of 6\n(page number not for citation purposes)\nonset of puberty is highlighted by the augmentation of\npulsatile GnRH secretion [10,11]. GnRH release is in a\nstate of low level before puberty, an increased pulsatile\nrelease of GnRH is essential for the onset of puberty. True\nprecocious puberty is commonly idiopathicly mediated\nby premature increasing secretion of GnRH, and is readily\ntreated with potent GnRH agonists [4].\nThough GnRH neurons in CNS are paucity, about 600–\n2,000 cells in mammals, the highest density of the cells is\nlocated in MS, DBB and MPOA, approximately 50 to 70%\nof which project to the median eminence (ME), thus con-\ntributing to the control of LH secretion from the adenohy-\npophysis [12]. From this point of view, we selected these\nregions to observe the change of GnRH expression. Moris-\nhito et al has reported the animal model that neonatal\ntreatment with Danazol may induce the true precocious\npuberty in female rats [3], but the hypothalamic GnRH\nexpression was not examined. In the present study, the\nhigh level of GnRH mRNA expression was observed in the\nM, but the number of the GnRH neuron decreased. The\npossible mechanism of the inconsistency change might\ndue to the super-release of hypothalamic GnRH in the\nmodel rats. Nevertheless, our results, the GnRH mRNA\nexpression showed a higher level in the precocious\npuberty model rats than in the same age normal rats, the\nblood estrogen concentration in the model group is two-\nfold higher than that in normal one, along with the day of\nvaginal opening and the onset of regular estrous cycle\nadvanced significantly in the model rats, give more evi-\ndence to sustain the hypothesis that the model induced by\nneonatal treatment with Danazol is a true precocious\npuberty model, and might be served to study the onset\nmechanism of puberty. Besides, though experimental\nmodels of precocious puberty have been induced in\nfemale rats from 15 to 30 days of age [11,13] or during\nneonate, by testosterone, estradiol, melatonin and so on,\nthis model appears the subsequent normal ovulatory\ncycles during adulthood [3].\nDanazol, an isoxazol derivative of 17 α -ethinylestoster-\none, is known to have various effects on the reproductive\nsystem. Danazol binds to androgen and progesterone\nreceptors, and possesses weak androgenic activity in rats.\nBut the mechanism and the action site of Danazol, admin-\nistered during the neonatal period, induces a true preco-\ncious puberty is not clear. It is generally accepted that the\nrat hypothalamus is immature at birth and that manipu-\nlations of maturation process are possible between days 1\nand 10 of life. From these viewpoints, it is possible that\nthe neonatal administration of Danazol may affect the\nhypothalamic pituitary axis with the rapid rate of matura-\ntion, producing a true precocious puberty.\nGnRH neurons display a range of shapes from smooth-\ncontoured to extremely irregular or spiny, which reflects\nor can be modified by the gonadal steroid milieu [8]. The\nspiny on the GnRH neuron has been identified the syn-\napse by its ultrastructure, suggesting that more synapse\nconnections exist among GnRH neurons and other com-\nponents in the GnRH network. In the present results, the\npercent of spiny type GnRH neuron was significantly\nhigher in M than those in N and HM, which might\nindicate that, on one hand, the gonadal steroid concentra-\ntion in the local brain region was high; on the other hand,\nmore factors were involved in the process. The local brain\naromatization has been observed to be increased in the M\n[14], which indirectly reflects the increased estrogen con-\ncentration. The factors controlling the increased and\nabnormal secretion of GnRH at the onset of precocious\npuberty are now being investigated in the same model by\nus, in which TGF might be an important factor. But further\nstudy is carried out. The animals in M and HM groups dis-\nplayed neural changes to the different degree, suggesting\nthat the herb mixture played certain regulatory effects on\nthe precocious puberty rats.\nWe have, successfully used the Nourishing \"Yin\"-Remov-\ning \"Fire\" Chinese Herb Mixture, for the management of\nIPP for more than thirty years. In 1990', one hundred and\nsix clinical cases have been reported [5]. Before and after\ntreatment, GnRH stimulating test, size of uterus and\novary, X-ray bone age measurement were adopted to esti-\nmate the effect of the herb mixture on HPOA and devel-\nopment of internal genitalia and skeleton. Clinical studies\nindicate that the herb mixture could markedly reduce the\nblood levels of FSH, LH, E 2 of the patients, and the vol-\nume of uterus and ovary as well. The herb mixture could\nalso inhibit the excessive functional activities of osteob-\nlasts and the decelerated linear growth of skeleton.\nBesides, the cheap-effective therapeutic approach is more\nsuitable to our national condition than the high-price\nGnRH agonist.\nThough the physiology of human and rodent is not same,\nthe therapeutic effects of the herb mixture on not only true\nprecocious puberty patient but also rat have been\nobserved. The mechanism of the effective drug has been\nfurther studied focusing on the neuroendocrine gene\nexpression with modern medical techniques. Recently,\nseven differential displayed genes between M and HM rats\nin the same target brain region, have been screened out,\nby the means of RNA arbitrarily primed PCR (RAP-PCR)\n(unpublished data). Hopefully, with the all-right thera-\npeutic effect, the herb mixture compound might bring\nmore profits for patients throughout the world.\n\nPublish with BioMed Central   and  every \nscientist can read your work free of charge\n\"BioMed Central will be the most significant development for \ndisseminating the results of biomedical research in our lifetime.\"\nSir Paul Nurse, Cancer Research UK\nYour research papers will be:\navailable free of charge to the entire biomedical community\npeer reviewed and published immediately upon acceptance\ncited in PubMed and archived on PubMed Central \nyours — you keep the copyright\nSubmit your manuscript here:\nhttp://www.biomedcentral.com/info/publishing_adv.asp\nBioMedcentral\nReproductive Biology and Endocrinology 2005, 3:38 http://www.rbej.com/content/3/1/38\nPage 6 of 6\n(page number not for citation purposes)\nConclusion\nThe true precocious puberty model by neonatal adminis-\ntration of Danazol in female rats showed augmented\nexpression of hypothalamic GnRH; the Nourishing \"Yin\"-\nRemoving \"Fire\" Chinese Herb Mixture down-regulated\nthe increased GnRH expression, and significantly delayed\nthe sexual development of the precocious puberty rat.\nAuthors' contributions\nZhanzhuang Tian and Hong Zhao designed the study, per-\nformed the animal and molecular genetic studies, and\ndrafted the manuscript. Yan Sun performed the statistical\nanalysis. Depei Cai participated in the coordination. Boy-\ning Chen conceived of the study, and participated in its\ndesign and coordination. All authors read and approved\nthe final manuscript.\nAcknowledgements\nThis project was financed by the Younger Scientific Fund of Fudan Univer-\nsity (2004) and Shanghai Medical College (2004).\nReferences\n1. Ge QS: Precocious puberty.  J Reprod Med 2000, 9:377-380.\n2. Samuel Yen SC: Reproductive Endocr inology: physiology,\npathophysiology and c linical management.  4th edition. W.B.\nSaunders Company; 2001:388-412. \n3. Morishito H, Takemoto M, Kondo H, Higuchi K, Aono T: Induction\nof true precocious puberty by neonatal treatment with dan-\nazol in female rats.  Neurosci Lett 1993, 157:33-36.\n4. Heger S, Sippell WG, Partsch CJ: Gonadotropin-releasing hor-\nmone analogue treatment for precocious puberty. Twenty\nyears of experience.  Endocr Dev 2005, 8:94-125.\n5. Cai D, Zhang W: Regulative actions of the Chinese drugs for\ntonifying the kidney on gene expression of the hypothalamic\nGnRH, pituitary FSH, LH and osteoblastic BGP.   J Tradit Chin\nMed 2005, 25:58-61.\n6. Bao XM, Shu SY: Stereostic Brain Atlas.  Beijing People's Health\nPress; 1991:22-30. \n7. Tian ZZ, Zhao H, Chen BY: Expression of GnRH and GnRH\nreceptor mRNA in female rats with danazol-induced preco-\ncious puberty.  Chin J Endocrinol Metab 2003, 19:399-401.\n8. Witkin J: Morphology of luteinizin g hormone-releasing hor-\nmone neurons as a function of age and hormonal condition\nin the male rat.  Neuroendocrinology 1989, 49:344-348.\n9. Terasawa EI, Fernandez DL: Neurobiological mechanism of the\nonset of puberty in primates.  Endocrin Rev 2001, 22:111-150.\n10. Clive MD, Jonathan R, Timothy WJ, Robert PM: Prepubertal\nincrease in gonad otropin-releasing hormone mRNA, gona-\ndotropin-releasing hormone precursor, and subsequent\nmaturation of precursor processing in rats.   J Clin Invest 1992,\n90:2496-2501.\n11. Pinyerd B, Zipf WB: Puberty-timing is everything!  J Pediatr Nurs\n2005, 20:75-82.\n1 2 . Z h a o  H ,  T i a n  Z Z ,  C h e n  B Y :  Progress on GnRH cells model\n(review).  Prog Physiol 2004, 35:185-187. (in Chinese)\n13. Pinilla L, Lopez F, Gonzalez D, Fe rnandez C, Criado JES, Aguilar E:\nLuteinizing-hormone-mediated precocious puberty induced\nin female rats by a prepubertal pituitary graft.  Neuroendocrinol-\nogy 1989, 50:495-499.\n14. Tian ZZ, Zhao H, Chen BY: Decreased hypothalamic aromatase\nin female rats of precocious puberty.   Neurosci Lett  2004,\n366:92-96.","source_license":"CC0","license_restricted":false}