Molecular Biology Reports

, Volume 39, Issue 9, pp 9167–9177 | Cite as

Analysis on DNA sequence of goat RFRP gene and its possible association with average daily sunshine duration

  • D. W. Huang
  • R. Di
  • J. X. Wang
  • M. X. ChuEmail author
  • J. N. He
  • G. L. Cao
  • L. Fang
  • T. Feng
  • N. Li


Goat RFRP gene was cloned and its mutations were detected in thirteen goat breeds whose reproductive seasonality and litter size were different. Then sequence characteristics were analyzed and association analyses were performed to reveal the relationships between mutations of RFRP gene and average daily sunshine duration, reproductive seasonality as well as litter size in goats. A 4,862 bp DNA fragment of goat RFRP gene was obtained and the complete CDS of 591 bp encodes 196 amino acids, having high homology with that of other mammals. The protein was predicted to be a secreted protein with a signal peptide of 21 amino acids. Moreover, two mutations (A712G, T1493C) in 5′ regulatory region and one mutation (A3438T) in exon 2 were detected. The test of genotype distribution in six selective goat breeds showed that there was no uniform significant association between the three polymorphisms and seasonal reproduction. The association just existed in some goat breeds for each locus. Interestingly, however, there was a strong positive correlation (r = 0.830, P = 0.003) between the G allele frequency of the A712G locus and average daily sunshine duration in ten local goat breeds, suggesting that RFRP gene has undergone a selective pressure in sunshine duration and may have indirect relationship with reproductive seasonality in goats. Additionally, no significant difference was found in litter size between genotypes in prolific Jining Grey goats.


RFRP Goat Polymorphism Photoperiod Seasonal reproduction Litter size 



This work was supported by the earmarked fund for China Agriculture Research System (CARS-39), National Key Technology Research and Development (R&D) Program of China (2008BADB2B01), National Natural Science Foundation of China (30871773), National High Technology R&D Program of China (2006AA10Z139), National Key Basic R&D Program of China (2006CB102105), Special Fund for Basic Scientific Research of Institute of Animal Science, Chinese Academy of Agricultural Sciences (2010jc-9).

Supplementary material

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Supplementary material 1 (DOC 258 kb)


  1. 1.
    Clarke IJ, Sari IP, Qi Y, Smith JT, Parkington HC, Ubuka T, Iqbal J, Li Q, Tilbrook A, Morgan K, Pawson AJ, Tsutsui K, Millar RP, Bentley GE (2008) Potent action of RFamide-related peptide-3 on pituitary gonadotropes indicative of a hypophysiotropic role in the negative regulation of gonadotropin secretion. Endocrinology 149(11):5811–5821CrossRefPubMedGoogle Scholar
  2. 2.
    Smith JT, Shahab M, Pereira A, Pau KY, Clarke IJ (2010) Hypothalamic expression of KISS1 and gonadotropin inhibitory hormone genes during the menstrual cycle of a non-human primate. Biol Reproduc 83(4):568–577CrossRefGoogle Scholar
  3. 3.
    Ubuka T, Morgan K, Pawson AJ, Osugi T, Chowdhury VS, Minakata H, Tsutsui K, Millar RP, Bentley GE (2009) Identification of human GnIH homologs, RFRP-1 and RFRP-3, and the cognate receptor, GPR147 in the human hypothalamic pituitary axis. PLoS One 4(12):e8400CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Fukusumi S, Habata Y, Yoshida H, Iijima N, Kawamata Y, Hosoya M, Fujii R, Hinuma S, Kitada C, Shintani Y, Suenaga M, Onda H, Nishimura O, Tanaka M, Ibata Y, Fujino M (2001) Characteristics and distribution of endogenous RFamide-related peptide-1. Biochim Biophys Acta 1540(3):221–232CrossRefPubMedGoogle Scholar
  5. 5.
    Hinuma S, Shintani Y, Fukusumi S, Iijima N, Matsumoto Y, Hosoya M, Fujii R, Watanabe T, Kikuchi K, Terao Y, Yano T, Yamamoto T, Kawamata Y, Habata Y, Asada M, Kitada C, Kurokawa T, Onda H, Nishimura O, Tanaka M, Ibata Y, Fujino M (2000) New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals. Nat Cell Biol 2(10):703–708CrossRefPubMedGoogle Scholar
  6. 6.
    Clarke IJ, Qi Y, Puspita Sari I, Smith JT (2009) Evidence that RF-amide related peptides are inhibitors of reproduction in mammals. Front Neuroendocr 30(3):371–378CrossRefGoogle Scholar
  7. 7.
    Sari IP, Rao A, Smith JT, Tilbrook AJ, Clarke IJ (2009) Effect of RF-amide-related peptide-3 on luteinizing hormone and follicle-stimulating hormone synthesis and secretion in ovine pituitary gonadotropes. Endocrinology 50(12):5549–5556CrossRefGoogle Scholar
  8. 8.
    Dardente H, Birnie M, Lincoln GA, Hazlerigg DG (2008) RFamide-related peptide and its cognate receptor in the sheep: cDNA cloning, mRNA distribution in the hypothalamus and the effect of photoperiod. J Neuroendocr 20(11):1252–1259CrossRefGoogle Scholar
  9. 9.
    Ubuka T, Inoue K, Fukuda Y, Mizuno T, Ukena K, Kriegsfeld LJ, Tsutsui K (2011) Identification, expression, and physiological functions of Siberian hamster gonadotropin-inhibitory hormone. Endocrinology. doi: 10.1210/en.2011-1110
  10. 10.
    Kriegsfeld LJ, Mei DF, Bentley GE, Ubuka T, Mason AO, Inoue K, Ukena K, Tsutsui K, Silver R (2006) Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals. Proc Natl Acad Sci USA 103(7):2410–2415CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Johnson MA, Tsutsui K, Fraley GS (2007) Rat RFamide-related peptide-3 stimulates GH secretion, inhibits LH secretion, and has variable effects on sex behavior in the adult male rat. Horm Behav 51(1):171–180CrossRefPubMedGoogle Scholar
  12. 12.
    Smith JT, Coolen LM, Kriegsfeld LJ, Sari IP, Jaafarzadehshirazi MR, Maltby M, Bateman K, Goodman RL, Tilbrook AJ, Ubuka T, Bentley GE, Clarke IJ, Lehman MN (2008) Variation in kisspeptin and RFamide-related peptide (RFRP) expression and terminal connections to gonadotropin-releasing hormone neurons in the brain: a novel medium for seasonal breeding in the sheep. Endocrinology 149(11):5770–5782CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Qi Y, Oldfield BJ, Clarke IJ (2009) Projections of RFamide-related peptide-3 neurones in the ovine hypothalamus, with special reference to regions regulating energy balance and reproduction. J Neuroendocr 21(8):690–697CrossRefGoogle Scholar
  14. 14.
    Ubuka T, McGuire NL, Calisi RM, Perfito N, Bentley GE (2008) The control of reproductive physiology and behavior by gonadotropin-inhibitory hormone. Integr Comp Biol 48(5):560–569CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Pineda R, Garcia-Galiano D, Sanchez-Garrido MA, Romero M, Ruiz-Pino F, Aguilar E, Dijcks FA, Blomenröhr M, Pinilla L, van Noort P, Tena-Sempere M (2010) Characterization of inhibitory roles of RFRP3, the mammalian ortholog of GnIH, in the control of gonadotropin secretion in the rat: in vivo and in vitro studies. Am J Physiol Endocrinol Metab 299(1):E39–E46CrossRefPubMedGoogle Scholar
  16. 16.
    Pineda R, Garcia-Galiano D, Sanchez-Garrido MA, Romero M, Ruiz-Pino F, Aguilar E, Dijcks FA, Blomenröhr M, Pinilla L, van Noort PI, Tena-Sempere M (2010) Characterization of the potent gonadotropin-releasing activity of RF9, a selective antagonist of RF-amide-related peptides and neuropeptide FF receptors: physiological and pharmacological implications. Endocrinology 151(4):1902–1913CrossRefPubMedGoogle Scholar
  17. 17.
    Murakami M, Matsuzaki T, Iwasa T, Yasui T, Irahara M, Osugi T, Tsutsui K (2008) Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats. J Endocrinol 199(1):105–112CrossRefPubMedGoogle Scholar
  18. 18.
    Johnson MA, Fraley GS (2008) Rat RFRP-3 alters hypothalamic GHRH expression and growth hormone secretion but does not affect KiSS-1 gene expression or the onset of puberty in male rats. Neuroendocrinology 88(4):305–315CrossRefPubMedGoogle Scholar
  19. 19.
    Ducret E, Anderson GM, Herbison AE (2009) RFamide-related peptide-3, a mammalian gonadotropin-inhibitory hormone ortholog, regulates gonadotropin-releasing hormone neuron firing in the mouse. Endocrinology 150(6):2799–2804CrossRefPubMedGoogle Scholar
  20. 20.
    Kadokawa H, Shibata M, Tanaka Y, Kojima T, Matsumoto K, Oshima K, Yamamoto N (2009) Bovine C-terminal octapeptide of RFamide-related peptide-3 suppresses luteinizing hormone (LH) secretion from the pituitary as well as pulsatile LH secretion in bovines. Domest Anim Endocrinol 36(4):219–224CrossRefPubMedGoogle Scholar
  21. 21.
    Sethi S, Tsutsui K, Chaturvedi CM (2010) Age-dependent variation in the RFRP-3 neurons is inversely correlated with gonadal activity of mice. Gen Comp Endocrinol 168(3):326–332CrossRefPubMedGoogle Scholar
  22. 22.
    Sethi S, Tsutsui K, Chaturvedi CM (2010) Temporal phase relation of circadian neural oscillations alters RFamide-related peptide-3 and testicular function in the mouse. Neuroendocrinology 91(2):189–199CrossRefPubMedGoogle Scholar
  23. 23.
    Paul MJ, Pyter LM, Freeman DA, Galang J, Prendergast BJ (2009) Photic and nonphotic seasonal cues differentially engage hypothalamic kisspeptin and RFamide-related peptide mRNA expression in siberian hamsters. J Neuroendocrinol 21(12):1007–1014CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Mason AO, Duffy S, Zhao S, Ubuka T, Bentley GE, Tsutsui K, Silver R, Kriegsfeld LJ (2010) Photoperiod and reproductive condition are associated with changes in RFamide-related peptide (RFRP) expression in syrian hamsters (Mesocricetus auratus). J Biol Rhythms 25(3):176–185CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Revel FG, Saboureau M, Pévet P, Simonneaux V, Mikkelsen JD (2008) RFamide-related peptide gene is a melatonin-driven photoperiodic gene. Endocrinology 149(3):902–912CrossRefPubMedGoogle Scholar
  26. 26.
    Gingerich S, Wang X, Lee PK, Dhillon SS, Chalmers JA, Koletar MM, Belsham DD (2009) The generation of an array of clonal, immortalized cell models from the rat hypothalamus: analysis of melatonin effects on kisspeptin and gonadotropin-inhibitory hormone neurons. Neuroscience 162(4):1134–1140CrossRefPubMedGoogle Scholar
  27. 27.
    Huang DW, Chu MX (2011) Research progress in molecular mechanism of animal seasonal reproduction. Hereditas (Beijing) 33(7):695–706CrossRefGoogle Scholar
  28. 28.
    Maeda KI, Adachi S, Inoue K, Ohkura S, Tsukamura H (2007) Metastin/kisspeptin and control of estrous cycle in rats. Rev Endocr Metab Disord 8(1):21–29CrossRefPubMedGoogle Scholar
  29. 29.
    Caraty A, Smith JT, Lomet D, Ben Saïd S, Morrissey A, Cognie J, Doughton B, Baril G, Briant C, Clarke IJ (2007) Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes. Endocrinology 148(11):5258–5267CrossRefPubMedGoogle Scholar
  30. 30.
    Clarke IJ, Smith JT, Caraty A, Goodman RL, Lehman MN (2009) Kisspeptin and seasonality in sheep. Peptides 30(1):154–163CrossRefPubMedGoogle Scholar
  31. 31.
    Smith JT (2011) The role of kisspeptin and gonadotropin inhibitory hormone in the seasonal regulation of reproduction in sheep. Domest Anim Endocrinol. doi: 10.1016/j.domaniend.2011.11.003
  32. 32.
    Khan AR, Kauffman AS (2011) The role of kisspeptin and RFRP-3 neurons in the circadian-timed preovulatory luteinizing hormone surge. J Neuroendocrinol. doi:  10.1111/j.1365-2826.2011.02162.x
  33. 33.
    Molnár CS, Kalló I, Liposits Z, Hrabovszky E (2011) Estradiol down-regulates RF-amide-related peptide (RFRP) expression in the mouse hypothalamus. Endocrinology 152(4):1684–1690CrossRefPubMedGoogle Scholar
  34. 34.
    Quennell JH, Rizwan MZ, Relf HL, Anderson GM (2010) Developmental and steroidogenic effects on the gene expression of RFamide related peptides and their receptor in the ratbrain and pituitary gland. J Neuroendocrinol 22(4):309–316CrossRefPubMedGoogle Scholar
  35. 35.
    Tu YR (1989) The Sheep and Goat Breeds in China. Shanghai Science and Technology Press, Shanghai, pp 88–90, 98–101, 112-114Google Scholar
  36. 36.
    Malan SW (2000) The improved Boer goat. Small Rumin Res 36(2):165–170CrossRefPubMedGoogle Scholar
  37. 37.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599CrossRefPubMedGoogle Scholar
  38. 38.
    Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL, Kolchanov NA (1998) Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26(1):362–367CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Schulz HL, Stoehr H, White K, van Driel MA, Hoyng CB, Cremers F, Weber BH (2002) Genomic structure and assessment of the retinally expressed RFamide-related peptide gene in dominant cystoid macular dystrophy. Mol Vis 19(8):67–71Google Scholar
  40. 40.
    Zhao S, Zhu E, Yang C, Bentley GE, Tsutsui K, Kriegsfeld LJ (2010) RFamide–related peptide and messenger ribonucleic acid expression in mammalian testis: association with the spermatogenic cycle. Endocrinology 151(2):617–627CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Hromas R, Collins SJ, Hickstein D, Raskind W, Deaven LL, O’Hara P, Hagen FS, Kaushansky K (1991) A retinoic acid-responsive human zinc finger gene, MZF-1, preferentially expressed in myeloid cells. J Biol Chem 266(22):14183–14187PubMedGoogle Scholar
  42. 42.
    de Jong R, van der Heijden J, Meijlink F (1993) DNA-binding specificity of the S8 homeodomain. Nucleic Acids Res 21(20):4711–4720CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Hyatt GA, Dowling JE (1997) Retinoic acid. A key molecule for eye and photoreceptor development. Invest Ophthalmol Vis Sci 38(8):1471–1475PubMedGoogle Scholar
  44. 44.
    Khanna H, Akimoto M, Siffroi-Fernandez S, Friedman JS, Hicks D, Swaroop A (2006) Retinoic acid regulates the expression of photoreceptor transcription factor NRL. J Biol Chem 281(37):27327–27334CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Shearer KD, Goodman TH, Ross AW, Reilly L, Morgan PJ, McCaffery PJ (2010) Photoperiodic regulation of retinoic acid signaling in the hypothalamus. J Neurochem 112(1):246–257CrossRefPubMedGoogle Scholar
  46. 46.
    Ross AW, Helfer G, Russell L, Darras VM, Morgan PJ (2011) Thyroid hormone signaling genes are regulated by photoperiod in the hypothalamus of F344 rats. PLoS One 6(6):e21351CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Ji LD, Xu J, Wu DD, Xie SD, Tang NL, Zhang YP (2010) Association of disease-predisposition polymorphisms of the melatonin receptors and sunshine duration in the global human populations. J Pineal Res 48(2):133–141CrossRefPubMedGoogle Scholar
  48. 48.
    Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery GW, Beattie AE, Davis GH, Ritvos O (2000) Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner. Nat Genet 25(3):279–283CrossRefPubMedGoogle Scholar
  49. 49.
    Chu MX, Liu ZH, Jiao CL, He YQ, Fang L, Ye SC, Chen GH, Wang JY (2007) Mutations in BMPR-IB and BMP-15 genes are associated with litter size in small tailed han sheep (Ovis aries). J Anim Sci 85(3):598–603CrossRefPubMedGoogle Scholar
  50. 50.
    Chu MX, Jiao CL, He YQ, Wang JY, Liu ZH, Chen GH (2007) Association between PCR-SSCP of bone morphogenetic protein 15 gene and prolificacy in Jining Grey goats. Anim Biotechnol 18(4):263–274CrossRefPubMedGoogle Scholar
  51. 51.
    Hanrahan PJ, Gregan SM, Mulsant P, Mullen M, Davis GH, Powell R, Galloway SM (2004) Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and belclare sheep (Ovis aries). Biol Reprod 70(4):900–909CrossRefPubMedGoogle Scholar
  52. 52.
    Davis GH, Montgomery GW, Allison AJ, Kelly RW, Bray AR (1982) Segregation of a major gene influencing fecundity in progeny of booroola sheep. NZJ Agric Res 25(4):525–529CrossRefGoogle Scholar
  53. 53.
    Singh P, Krishna A, Sridaran R, Tsutsui K (2011) Immunohistochemical localization of GnRH and RFamide-related peptide-3 in the ovaries of mice during the estrous cycle. J Mol Histol 42(5):371–381CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • D. W. Huang
    • 1
  • R. Di
    • 1
  • J. X. Wang
    • 1
  • M. X. Chu
    • 1
    Email author
  • J. N. He
    • 1
  • G. L. Cao
    • 1
  • L. Fang
    • 1
  • T. Feng
    • 1
  • N. Li
    • 2
  1. 1.Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation of Ministry of Agriculture, Institute of Animal ScienceChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  2. 2.State Key Laboratory of Agricultural BiotechnologyChina Agricultural UniversityBeijingPeople’s Republic of China

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