WCRB2014 POSTER PRESENTATIONS (1) (335 abstracts)
Real-time observation of hormone-secretion using CFP, YFP-fused gonadotropin expression vector
Haruka Takanashi 1 , Naoto Nishimura 2 , Saishu Yoshida 2 , Masashi Higuchi 2 , Kohei Kawai 3 , Hiroki Ueharu 2 , Takako Kato 2 & Yukio Kato 2
1Division of Life Science, Graduate School of Agriculture, Meiji University, Kanagawa, Japan; 2Meiji University, Kanagawa, Japan; 3Department of Life Science, School of Agriculture, Meiji University, Kanagawa, Japan.
Introduction: FSH and LH are glycoprotein hormones and are synthesized in the anterior lobe of the pituitary gland and plays critical roles for maintenance of vital functions. FSH and LH are secreted to pituitary capillary vessel and exert their effects on reproductive organs of both sexes to stimulate steroidogenesis and gametogenesis. Secretion of FSH and LH is controlled mainly by GnRH released from hypothalamus. Many investigations on secretion of FSH and LH were performed by measurement of the hormone level in blood or in cultured medium of the pituitary primary cells or cell lines requiring much time. Herein, we developed a monitoring system by using the expression of fluorescence protein-fused FSH and LH.
Materials and methods: Expression vectors were constructed by fusing rat cDNA of FSHβ- and LHβ-subunit to the 5′ of CFP or YFP of the vector and transfected into gonadotrope-lineage cell line, LβT2, with FuGENE6. After time-laps observation of fluorescence microscopy, immunocytochemistry was performed with antibodies against α-, FSHβ-, and LHβ-subunit. Secretion was achieved with forskolin or phorbol ester followed by determination of the molecular sizes.
Results and discussion: By transfection of expression vectors, fluorescent signals were observed in the cytoplasmic granules and the presence of hormones were verified by immunocytochemistry. Addition of western blotting analysis of transfected cell lysates using anti-GFP antibody showed success of the synthesis of fluorescence-fused hormones. Thus, the expression vectors for CFP, YFP-fused FSH and LH are useful tool for real-time observation of the secretion of hormone.
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ISSN 2052-1472 (online)
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