| Objective: In this paper, We discussed the distribution of Ghrelin and its receptor GHS-R1a in the hypothalamus - pituitary– gonad axis, and analyzed the expression of Ghrelin and its receptor GHS-R1a mRNA in the reproductive axis during estrous cycle, and investigated the effection of exogenous Ghrelin on gonadotropin, gonadal hormone secretion and gene expression. The aim to reveal the mechanism and route of Ghrelin inflencing reproductive axis at the hormone, histological and molecular level.Methods: (1) Hypothalamus, pituitary, ovary and testis were separated from male and female adult SD rats, then fixed in 4% paraformaldehyde, made into paraffin sections for immunohistochemistry to observe the distribution of the Ghrelin and its receptor GHS-R1a. (2)The distribution of Ghrelin mRNA in the hypothalamus, pituitary, ovary and testis were observed by situ hybridization using digoxigenin labeled oligonucleotide Ghrelin probes. (3) The expression of Ghrelin mRNA and GHS-R1a mRNA were analyzed in the hypothalamus, pituitary, ovary during estrous cycle by real-time fluorescence quantitative PCR. (4) The rats were treated with intraperitoneal injection using 1% sodium pentobarbital ( 1mL), then were fixed in a stereotaxic apparatus. male rats and female rats during estrous cycle were intracerebroventricularly injected with different doses of Ghrelin (0.3nmol, 1nmol, 3nmol and saline control group). The rats were decapitated 15min later. The serum were collected for determinating the hormone levels. The luteinizing hormone, follicle-stimulating hormone levels were measured by enzyme-linked immunosorbent assay(ELISA), and serum testosterone, estrogen and progesterone concentrations were measured by radioimmunoassay(RIA). (5) The hypothalamus, pituitary, ovary and testis were separated for estimating the expression of FSH, LH, GnRH, AR, ERβand PRA+B mRNA using real-time quantitative PCR (6) Adult male rats and 25d old female rats were killed, testicular interstitial cells, luteal cells and granulosa cells were collected and cultured in CO2 incubator. Cell culture media were collected 6h later, The concentration of LH, FSH, testosterone in the culture media were determinated by ELISA and RIA, respectively. The real-time quantitative PCR was used to analyze AR, ERβand PRA+B mRNA expression. Results: (1) Ghrelin and its receptor GHS-R1a were mainly distributed in the arcuate nuclei, ventromedial nucleus, median eminence, adenohypophysis, leydig cells, spermatocyte, sertoli cells, oocytes and among others. (2) In the hypothalamus, pituitary, testis, Ghrelin mRNA displayed a consistent positioning with Ghrelin. In the ovary, the results showed the distribution of Ghrelin mRNA and Ghrelin was not consistent. (3) Ghrelin and GHS-R1a mRNA were expressed in hypothalamus and pituitary of rats during different estrous cycles, the abundance of expression changed with estrous cycles.The expression of GHS-R1a mRNA were not detected throughout the estrous cycle. (4) Intracerebroventricular injection of Ghrelin significantly inhibited gonadotropin, gonadal hormone secretion, which was regulated by the the phase of estrous cycle, dose and gender. (5) Intracerebroventricular injection of Ghrelin significantly inhibited the expression of GnRH mRNA,FSH mRNA,LH mRNA,ERβmRNA,PRA+B mRNA,AR mRNA, which was regulated by the the phase of estrous cycle and dose. (6) In vitro, Ghrelin significantly inhibited progesterone and estrogen secretion and the expression of ERβ, PRA+B mRNA. But Ghrelin had no effect on testosterone secrete and the expression of AR mRNA.Conclusion: Ghrelin, GHS-R1a and Ghrelin mRNA were all distributed in rat hypothalamus, pituitary and gonads, Ghrelin inhibits the secretion of gonadotropin and gonadal hormone and gene expression of gonadotropin-releasing hormone, gonadotropin and gonadal hormone receptor through its receptor at the central level and peripheral level. The inhibitions were regulated by the estrous cycle, dose and gender.
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