THE EXPRESSION OF TSH BETA mRNA AND PROTEIN BY RAT PITUITARY THYROTROPHS IS CRITICAL ON TRIDIMENSIONAL ORGANIZATION OF ANTERIOR PITUITARY GLAND

Program: Late-Breaking Abstracts
Session: SAT-LB-Late-Breaking Poster Session 1
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-LB-08
Paula Bargi-Souza*1, Marek Kucka2, Ivana Bjelobaba2, Melanija Tomic2, Maria Tereza Nunes1 and Stanko S Stojilkovic2
1University of São Paulo, Brazil, 2NICHD, NIH, Bethesda, MD
Here we analyzed basal and TRH-stimulated expression of TSH beta mRNA (Tshb) and protein (TSHB) in rat pituitary thyrotrophs in vivo and in vitro. To do this, qRT-PCR, western blot, RIA, ELISA, and single cell calcium measurements were used. The expression of Tshb in intact pituitaries increased during neonatal and infantile age in both sexes with comparable rates, reaching the peak response at 28 days in females and 35 days in males. In peripubertal and adult animals, male pituitaries always exhibited significantly higher Tshb expression than female pituitaries. We further found that primary pituitary cell cultures from 6 and 8 week-old animals have significantly lower Tshb expression compared to intact tissue. Cell dispersion already accounted for about 50% of decrease, while culturing cells for 68 h was accompanied with further decrease in the expression of Tshb, dropping to only 2% of that observed in intact pituitaries. Substitution of horse serum with BSA only slightly slowed down this decay. The TSHB content also decreased during cell preparation to about 50%, indicating a massive TSH release initiated by chemical and mechanical dispersion. TSHB content further decreased in cultured cells 24 h after dispersion to about 5% of that observed in intact male and female pituitaries. This low residual intracellular hormone content was still sufficient for immunocytochemical detection, revealing 11% TSHB positive cells in 24 and 48 h old cultures. In perifused pituitary cells from 24 h old female and male cultures, ELISA analysis showed biphasic TRH (100 nM)-induced TSH secretory response. However, the peak amplitude of TSH response by thyrotrophs was low, about 3 ng/ml for female and 15 ng/ml for male perifused cells, in contrast to about 50 ng/ml LH release by GnRH-stimulated gonadotrophs (100 nM), also representing about 11% of cultured cells. TRH (100 nM) failed to stimulate Tshb mRNA expression immediately after dispersion and in 24 and 48 h old cultures independently of the pattern of delivery, in contrast to GnRH triggering upregulation of Fshb and Lhb. Single cell calcium analysis revealed that 42% of immunocytochemically-identified thyrotrophs did not respond to TRH, and qRT-PCR analysis showed over 60% drop in Trhr expression 16 h after cell preparation. In contrast, TRH-induced calcium signaling and secretion was not affected in lactotrophs. These results indicate that the sex specific pattern of Tshb expression is established during juvenile period. Furthermore, thyrotrophs are more sensitive than gonadotrophs and lactotrophs to cell dispersion and culturing conditions, causing a massive loss of TSH content and Tshb and Trhr expression. This indicates that tridimensional organization of pituitary is critical for thyrotroph function, which limits the usage of rat primary cultures to investigate the signaling-secretion coupling in these cells.

Nothing to Disclose: PB, MK, IB, MT, MTN, SSS

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) #2012/06643-8; Intramural Research Program of the National Institute of Child Health and Human Development