title: Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep.
abstract: BACKGROUND
  The gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA).
  
  
  METHODS
  Antibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference.
  
  
  RESULTS
  In adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01).
  
  
  CONCLUSIONS
  Evidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep.
authors.affiliation: School of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, WA, Australia. d.miller@murdoch.edu.au
authors.forename: David W
authors.initials: DW
authors.lastname: Miller
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Harrison
authors.forename: Yvonne A
authors.initials: YA
authors.lastname: Brown
authors.forename: Una
authors.initials: U
authors.lastname: Doyle
authors.forename: Alanna
authors.initials: A
authors.lastname: Lindsay
authors.forename: Clare L
authors.initials: CL
authors.lastname: Adam
authors.forename: Richard G
authors.initials: RG
authors.lastname: Lea
mesh_terms: D000018:Abomasum / Q000502:physiology; D000818:Animals; D004702:Endocrine Glands / Q000502:physiology*; D005260:Female; D005333:Fetus / Q000378:metabolism; D005835:Genitalia / Q000502:physiology*; D054439:Ghrelin; D007031:Hypothalamus / Q000502:physiology; D007150:Immunohistochemistry; D008297:Male; D010053:Ovary / Q000502:physiology; D019899:Paracrine Communication / Q000502:physiology*; D036361:Peptide Hormones / Q000276:immunology / Q000502:physiology*; D017440:Photoperiod; D010903:Pituitary Gland, Anterior / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000276:immunology; D054440:Receptors, Ghrelin; D012249:Rhombencephalon / Q000502:physiology; D012756:Sheep / Q000502:physiology*; D019089:Stem Cell Factor / Q000032:analysis / Q000276:immunology; D013737:Testis / Q000502:physiology
chemical_list: D054439:Ghrelin; D036361:Peptide Hormones; D018809:Proliferating Cell Nuclear Antigen; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D019089:Stem Cell Factor