| Summary: | Thyroid stimulating hormone (TSH), also known as thyrotropin, is a pituitary hormone which stimulates the thyroid gland to synthesize and secrete the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). In tetrapod vertebrates, the regulation of pituitary hormone production and secretion is accomplished by a portal system which delivers thyrotropin-releasing hormone to positively stimulate the pituitary to release more TSH. However, in teleost fish such as the red drum (Sciaenops ocellatus), TSH production appears to be negatively controlled through direct neurosecretory innervations of the pituitary by the hypothalamus. Further research has yet to establish the importance of precise regulation of TSH expression in the red drum pituitary gland via hypothalamic TSH inhibitory factors (TIFs). Examining pituitary glands in in vitro incubations should provide a direct method for testing possible TIFs while controlling for other factors. Unfortunately, the pituitary gland of the red drum provides a very small amount of tissue, and it can be difficult to extract enough RNA from single pituitary glands to use in common mRNA expression analysis techniques such as Northern blotting. My objective is to determine if more sensitive techniques, dot blot and real-time PCR, are suitable for analyzing mRNA expression of red drum TSH. Dot blot was found to be successful in determining relative quantification of TSH mRNA in samples of at least two pooled pituitary glands following in vitro incubation. Real-time PCR with TaqMan probes was also successful at amplifying a TSH mRNA signal from 50ng of red drum pituitary mRNA. Thus, real-time PCR should provide a sensitive technique to measure mRNA expression of TSH in single pituitary glands and allow further investigation of the existence of hypothalamic TSH inhibitory control factors.
|
|---|