Serotonin receptors expression in caprine and ovine mammary gland by Real Time PCR-RT
The role of serotonin (5-HT) as a feedback inhibitor of lactation in humans, mice and cows has been previously reported. This action is mediated by 5-HT receptors, which are expressed in the mammary tissue. The 5-HTR subtype 7 is expressed in the three quoted species, but in bovine, subtypes 1B, 2A, 2B and 4 have been also found. The aim of this study was to identify the 5-HTR (subtypes 1A, 1B, 1D, 1E, 1F, 2A, 2B, 2C, 3A, 4, 5a, 6 and 7) expression in the goat and sheep mammary gland using Real Time PCR-RT. Lactating Holstein cows, Majorera dairy goats and Canarian dairy ewes were used to obtain hypothalamus and mammary gland samples. The sampling was carried out immediately after slaughter. Samples were kept in RNAlater, and subsequent RNA extraction was performed using a combination between TriPure reagent (Roche, Barcelona, Spain) and E.N.Z.A. ® total RNA kit (Omega, Nordic Naturals, inc) protocols. cDNA synthesis was conducted using iScript cDNA synthesis kit (BioRad, Madrid, Spain). The primers used in this study were obtained from published data (1B, 1F, 2A, 2B, 2C, 4, 5a and 7) or were designed using bovine CD sequences from GeneBank (1A, 1D, 1E, 3A and 6). Hypoxanthine phosphosibosyltransferase I (HPTR1), Ribosomal Protein (S18), β-Actin and Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) genes were utilized as internal control. Hypothalamus samples were used as positive control to evaluate the primers in the three studied species. Real Time PCR was performed at 57.5ºC of annealing temperature for all primers. The receptors found in bovine mammary gland were in agreement with previous studies. Receptors 1B, 1E, 2A, 2B, 4 and 7 were found in the three species. Additionally, receptors 1D and 5a were observed in goats and sheep. Moreover, 1A and 1F subtypes were only detected in ovine mammary gland. Nevertheless, subtypes 2C, 3A and 6 were found neither bovine, nor caprine or ovine mammary gland. In conclusion, the same 5-HTR subtypes previously described in cows are expressed in caprine and ovine mammary gland. In addition, in caprine mammary gland were identified two more (5-HTR1D and 5-HTR5a), and the most number of 5-HTR were found in ovine mammary gland. Further studies will be necessary to study more in deep the role of serotonin as inhibitor of lactation in small ruminants.
Keywords: Serotonin Receptors, Goat, Sheep