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Puerarin acted as a weak estrogen-active compound in the GnRH pulse generator.In the medial preoptic area, puerarin and E2B downregulated ERα mRNA expression.E2B decreased pituitary GnRHR mRNA and serum LH levels, while puerarin did not.Puerarin and E2B stimulated the size of the very estrogen-sensitive organ, uterus.Puerarin exerts E2B-like anoretic action mediated partly via ERα in preoptic area.Effects of puerarin on the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator function is investigated, for the first time, in ovariectomized rats at the level of mRNA expression of estrogen-responsive genes, e.g., estrogen receptor (ER), GnRH and its receptor (GnRHR). Rats were treated orally for 90days either with a soy-free diet containing two different doses of puerarin (low dose of 600mg/kg and high dose of 3000mg/kg) or estradiol benzoate (E2B) at either low dose (4.3mg/kg) or high dose (17.3mg/kg). Levels of mRNA expression in the medial preoptic area/anterior hypothalamus (MPOA/AH), mediobasal hypothalamus/median eminence (MBH/ME) and adenohypophysis were measured by quantitative TaqMan® real-time RT-PCR. Plasma levels of luteinizing hormone (LH) and prolactin (PRL) were measured by radioimmunoassay. In the MPOA/AH, both puerarin and E2B decreased ERα mRNA levels without any significant changes in ERβ and GnRH mRNA levels. Both puerarin and E2B did not significantly alter the expression levels of ERα, ERβ and GnRHR in the MBH/ME. E2B exerted significant effects on the down-regulation of adenohypophyseal GnRHR mRNA transcripts and serum LH levels. Puerarin did not cause significant changes in pituitary GnRHR mRNA transcripts and serum LH and PRL levels. This is the first study to demonstrate that in ovariectomized rat models of ovarian hormone deprivation, puerarin acted as a weak estrogen-active compound in the hypothalamic GnRH pulse generator through the downregulation of MPOA/AH ERα mRNA expression.