Document Type : Original Article


1 Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

2 Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran.

3 Department of Animal Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran


Several studies have shown that neuropeptide Y (NPY) is considered to be one of the key regulators of the hypothalamic-pituitary-gonadal axis in the mammals. Also, kisspeptin is a powerful upstream regulator of gonadotropin-releasing hormone neurons in the hypothalamus. The present study aims to investigate the effects of the intracerebroventricular (ICV) injection of NPY and BIBP3226 (NPY receptor antagonist) on the reproductive axis (either hormonal or behavioral) of the male rats. Furthermore, to see whether NPY signals can be relayed through the pathway of KiSS1/GPR54, the gene expression of these peptides in the arcuate nucleus was measured. The ICV injection of NPY decreased the latencies and increased the frequencies of sexual parameters of the male rats in a significant way. Results obtained from LH and testosterone measurement showed that NPY had a significant increase in comparison with the control group. In this line, BIBP3226 antagonized the stimulative effects of NPY. Furthermore, data from real-time quantitative PCR showed that injection of NPY significantly increased the gene expression of KiSS1 and GPR54, while treatment with BIBP3226 controlled the stimulative effects of NPY on gene expression of KiSS1 and GPR54. Summing up, NPY can exert its impacts on the reproductive axis, this occurs at least partly through affecting KiSS1/GPR54 system.



    1. Kageyama H, Takenoya F, Hirako S, et al. Neuronal circuits involving neuropeptide Y in hypothalamic arcuate nucleus-mediated feeding regulation. Neuro-peptides 2012;46(6):285-289.
    2. Raposinho PD, Broqua P, Pierroz DD, et al. Evidence that the inhibition of luteinizing hormone secretion exerted by central administration of neuropeptide Y (NPY) in the rat is predominantly mediated by the NPY-Y5 receptor subtype. Endocrinology 1999; 140(9):4046-4055.
    3. Leupen SM, Besecke LM, Levine JE. Neuropeptide Y Y1-receptor stimulation is required for physiological amplification of preovulatory luteinizing hormone surges. Endocrinology 1997;138(7):2735-2739.
    4. Jain MR, Pu S, Kalra PS, et al. Evidence that stimulation of two modalities of pituitary luteinizing hormone release in ovarian steroid-primed ovariectomized rats may involve neuropeptide Y Y1 and Y4 receptors. Endocrinology 1999;140(11):5171-5177.
    5. Crowley WR, Hassid A, Kalra SP. Neuropeptide Y enhances the release of luteinizing hormone (LH) induced by LH-releasing hormone. Endocrinology 1987;120:941-945.
    6. Dhillon SS, Gingerich S, Belsham DD. Neuropeptide Y induces gonadotropin-releasing hormone gene expression directly and through conditioned medium from mHypoE-38 NPY neurons. Regul Pept 2009; 156(1-3):96-103.
    7. Urban JH, Bauer-Dantoin AC, Levine JE. Neuropeptide Y gene expression in the arcuate nucleus: Sexual dimorphism and modulation by testosterone. Endocrinology 1993;132(1):139-145.
    8. Sahu A, Crowley WR, Tatemoto K, et al. Effects of neuro-peptide Y, NPY analog (norleucine4-NPY), galanin and neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen, progesterone-treated rats. Peptides 1987;8:921-926.
    9. Bauer-Dantoin AC, McDonald JK, Levine JE. Neuro-peptide Y potentiates luteinizing hormone (LH)-releasing hormone-induced LH secretion only under conditions leading to preovulatory LH surges. Endocrinology 1992;131:2946-2952.
    10. Kalra PS, Bonavera JJ, Kalra SP. Central administration of antisense oligodeoxynucleotides to neuropeptide Y (NPY) mRNA reveals the critical role of newly synthesized NPY in regulation of LHRH release. Regul Pept 1995;59:215-220.
    11. Navarro VM, Castellano JM, Fernández-Fernández R, et al. Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. Endocrinology 2004;145(10):4565-4574.
    12. Han S-K, Gottsch ML, Lee KJ, et al. Activation of gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty. J Neurosci 2005;25(49):11349-11356.
    13. d'Anglemont de Tassigny X, Fagg LA, Dixon JPC, et al. Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci USA 2007; 104(25):10714-10719.
    14. Navarro VM, Castellano JM, Fernández-Fernández R, et al. Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 2005;146(1):156-163.
    15. de Roux N, Genin E, Carel J-C, et al. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100(19):10972-10976.
    16. Pinilla L, Aguilar E, Dieguez C, et al. Kisspeptins and and reproduction: physiological roles and regulatory mechanisms. Physiol Rev 2012;92(3):1235-1316.
    17. Lehman MN, Hileman SM, Goodman RL. Neuroanatomy of the kisspeptin signaling system in mammals: Comparative and developmental aspects. Adv Exp Med Biol 2013;784:27-62.
    18. Mikkelsen JD, Simonneaux V. The neuroanatomy of the kisspeptin system in the mammalian brain. Peptides 2009;30(1):26-33.
    19. Lehman MN, Coolen LM, Goodman RL. Minireview: kisspeptin/neurokinin B/dynorphin (KNDy) cells of the arcuate nucleus: A central node in the control of gonadotropin-releasing hormone secretion. Endocrinology 2010;151(8):3479-3489.
    20. Wahab F, Atika B, Shahab M. Kisspeptin as a link between metabolism and reproduction: Evidences from rodent and primate studies. Metabolism 2013; 62(7):898-910.
    21. Rissman EF. Behavioral regulation of gonadotropin-releasing hormone. Biol Reprod 1996;54(2):413-419.
    22. Saito TR. Effects of LHRH on copulatory behavior and locomotor activity in sexually inexperienced male rats. Jikken Dobutsu 1988;37(4):489-492.
    23. Awoniyi CA, Reece MS, Hurst BS, et al. Maintenance of sexual function with testosterone in the gonadotropin-releasing hormone-immunized hypogonadotropic infertile male rat. Biol Reprod 1993;49(6):1170-1176.
    24. Paxinos G, Watson C. The rat brain in stereotaxic co-ordinates. San Diego, USA: Academic Press 2006; 90-97.
    25. Agmo A, Paredes R, Fernández H. Differential effects of GABA transaminase inhibitors on sexual behavior, locomotor activity, and motor execution in the male rat. Pharmacol Biochem Behav 1987;28(1):47-52.
    26. Salehi MS, Namavar MR, JafarzadehShirazi MR, et al. A simple method for isolation of the anteroventral peri-ventricular and arcuate nuclei of the rat hypothalamus. Anatomy 2012-2013;(6-7):48-51.
    27. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25(4):402-408.
    28. van Furth WR, van Emst MG, van Ree JM. Opioids and sexual behavior of male rats: Involvement of the medial preoptic area. Behav Neurosci 1995;109(1):123-134.
    29. Babaei-Balderlou F, Khazali H. Effects of ghrelin on sexual behavior and luteinizing hormone beta-subunit gene expression in male rats. J Reprod Infertil 2016; 17(2):88-96.
    30. Agmo A. Sexual motivation--an inquiry into events determining the occurrence of sexual behavior. Behav Brain Res 1999;105(1):129-150.
    31. Davidson JM. Activation of the male rat's sexual behavior by intracerebral implantation of androgen.
      Endocrinology 1966;79:783-794.
    32. Garelick T, Swann J. Testosterone regulates the density of dendritic spines in the male preoptic area. Horm Behav 2014;65(3):249-253.
    33. Poggioli R, Vergoni AV, Marrama D, et al. NPY-induced inhibition of male copulatory activity is a direct beha-vioural effect. Neuropeptides 1990; 16(3):169-172.
    34. Dupont J, Maillard V, Coyral-Castel S, et al. Ghrelin in female and male reproduction. Int J Pept 2010;158102. doi: 10.1155/2010/158102.
    35. Stoyanovitch AG, Johnson MA, Clifton DK, et al. Galanin-like peptide rescues reproductive function in the diabetic rat. Diabetes 2005;54:2471-2476.
    36. Horvath TL, Naftolin F, Kalra SP, et al. Neuropeptide-Y innervation of beta-endorphin-containing cells in the rat mediobasal hypothalamus: A light and electron microscopic double immunostaining analysis. Endocrinology 1992;131(5):2461-2467.
    37. Kauffman AS, Smith JT. Kisspeptin signaling in reproductive biology. New York, USA: Springer 2013; 113-131.
    38. Irwig MS, Fraley GS, Smith JT, et al. Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 2004;80(4):264-272.
    39. Zeydabadi Nejad S, Ramezani Tehrani F, Zadeh-Vakili A. The role of kisspeptin in female reproduction. Int J Endocrinol Metab 2017;15(3):e44337. doi: 10.5812/ ijem.44337.
    40. Tonsfeldt KJ, Goodall CP, Latham KL, et al. Oestrogen induces rhythmic expression of the kisspeptin-1 receptor GPR54 in hypothalamic gonadotrophin-releasing hormone-secreting GT1-7 cells. J Neuro-endocrinol 2011;23(9):823-830.
    41. Kotani M, Detheux M, Vandenbogaerde A, et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 2001;276(37):34631-34636.
    42. Glanowska KM, Burger LL, Moenter SM. Development of gonadotropin-releasing hormone secretion and pituitary response. J Neurosci 2014;34(45):15060-15069.
    43. Rudolf K, Eberlein W, Engel W, et al. The first highly potent and selective non-peptide neuropeptide Y Y1 receptor antagonist: BIBP3226. Eur J Pharmacol 1994;271(2-3):R11-13.
    44. Raposinho PD, Broqua P, Hayward A, et al. Stimulation of the gonadotropic axis by the neuropeptide Y receptor Y1 antagonist/Y4 agonist 1229U91 in the male rat. Neuroendocrinology 2000;71:2-7.
    45. Pielecka-Fortuna J, Chu Z, Moenter SM. Kisspeptin acts directly and indirectly to increase gonadotropin-releasing hormone neuron activity and its effects are modulated by estradiol. Endocrinology 2008;149(4): 1979-1986.
    46. Luque RM, Kineman RD, Tena-Sempere M. Regulation of hypothalamic expression of KiSS-1 and GPR54 genes by metabolic factors: Analyses using mouse models and a cell line. Endocrinology 2007;148(10):4601-4611.
    47. Li C, Chen P, Smith MS. Morphological evidence for direct interaction between arcuate nucleus neuro-peptide Y (NPY) neurons and gonadotropin-releasing hormone neurons and the possible involvement of NPY Y1 receptors. Endocrinology 1999;140(11):5382-5390.
    48. Rodriguez-Sierra JF, Jacobowitz DM, Blake CA. Effects of neuropeptide Y on LH, FSH and TSH release in male rats. Peptides 1987;8:539-542.
    49. Kinoshita M, Tsukamura H, Adachi S, et al. Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology 2005;146(10):4431-4436.
    50. Kim GL, Dhillon SS, Belsham DD. Kisspeptin directly regulates neuropeptide Y synthesis and secretion via the ERK1/2 and p38 mitogen-activated protein kinase signaling pathways in NPY-secreting hypothalamic neurons. Endocrinology 2010;15(10)1:5038-5047.