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In Situ Hybridization Detection of TSHβ Subunit Gene Expression in the Serum‐Free Primary Culture of the Adult Rat Pituitary
Author(s) -
Koibuchi Noriyuki,
Ohtake Hideki,
Sakai Mikako,
Kato Yukio,
Yamaoka Sadao
Publication year - 1994
Publication title -
journal of neuroendocrinology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.062
H-Index - 116
eISSN - 1365-2826
pISSN - 0953-8194
DOI - 10.1111/j.1365-2826.1994.tb00620.x
Subject(s) - medicine , endocrinology , paraformaldehyde , triiodothyronine , messenger rna , gene expression , in situ hybridization , trh stimulation test , prohormone , chemistry , hormone , biology , thyrotropin releasing hormone , gene , biochemistry , organic chemistry
This study was designed to construct the primary culture system to detect the change in TSHβ subunit (TSHβ) gene expression in individual cells. Adult, male Wistar rats were sacrificed by transcardial perfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight). Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3 or 6 days with or without 1 nM triiodothyronine (T 3 ) under the serum‐free condition. In some cultures, TRH was added to a final concentration of 1 μM on 6, 12 or 24 h before fixation. Then the culture media were removed to measure TSH concentration. Cells were fixed with paraformaldehyde and hybridized with 35 S‐labeled RNA probe complementary to TSHβ mRNA. Emulsion autoradiography was subsequently performed. T 3 treatment markedly suppressed relative cellular levels of TSH4bT mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6. TRH treatment increased TSHβ mRNA on 12 and 24 h after the treatment on day 2 and 3 but did not increase TSHβ mRNA on day 6. TSH concentration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, and 24 h on day 6. On day 2 and 3, although T 3 treatment suppressed basal level of TSHβ mRNA, TRH‐induced increase in TSHβ mRNA was not suppressed by T 3 treatment. These results show that the thyroid hormone and TRH regulate TSHβ gene expression independently. Our culture system may provide a useful model to examine the action of individual substances on a specific subpopulation of the anterior pituitary cells.