Synergistic effects of salmon gonadotropin-releasing hormone and estradiol-17b on gonadotropin subunit gene expression and release in masu salmon pituitary cells in vitro Hironori Ando, a, * Penny Swanson, b Tomoko Kitani, c Nobuhisa Koide, d Houji Okada, e Hiroshi Ueda, c and Akihisa Urano a,c a Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan b Northwest Fisheries Science Center, National Marine Fisheries Service, 2725 Montlake Boulevard East, Seattle, Washington 98112, USA c Field Science Center for Northern Biosphere, Hokkaido University, Sapporo, Hokkaido 060-0809, Japan d Hokkaido Fish Hatchery, Eniwa, Hokkaido 061-1433, Japan e Hokkaido Wakkanai Fisheries Experimental Station, Wakkanai, Hokkaido 097-0001, Japan Received 6 November 2003; revised 4 February 2004; accepted 25 February 2004 Abstract Effects of salmon gonadotropin-releasing hormone (sGnRH) and estradiol-17b (E2) on gene expression and release of go- nadotropins (GTHs) were examined in masu salmon (Oncorhynchus masou) using primary pituitary cell cultures at three repro- ductive stages, initiation of sexual maturation in May, pre-spawning in July, and spawning in September. Amounts of GTH subunit mRNAs were determined by real-time polymerase chain reaction, and levels of GTH released in the medium were determined by RIA. In control cells, the amounts of three GTH subunit mRNAs (a2, FSHb, and LHb) peaked in July prior to spawning. FSH release spontaneously increased with gonadal maturation and peaked in September, whereas LH release remained low until July and extensively increased in September. Addition of E2 to the culture extensively increased the amounts of LHb mRNA in May and July in both sexes. It also increased the a2 mRNA in July in the females. In contrast, sGnRH alone did not have any significant effects on the amounts of three GTH subunit mRNAs at all stages, except for the elevation of a2 and FSHb mRNAs in July in the females. Nevertheless, synergistic effects by sGnRH and E2 were evident for all three GTH subunit mRNAs. In May, sGnRH in combination with E2 synergistically increased the amounts of LHb mRNA in the males and a2 mRNA in the females. However, in July the combination suppressed the amounts of a2 and FSHb mRNAs in the females. sGnRH alone stimulated LH release at all stages in both sexes, and the release was synergistically enhanced by E2. Synergistic stimulation of FSH release was also observed in May and July in both sexes. These results indicate that a functional interaction of sGnRH with E2 is differently involved in synthesis and release of GTH. The synergistic interaction modulates GTH synthesis differentially, depending on subunit, stage, and gender, whereas it potentiates the activity of GnRH to release GTH in any situation. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Gonadotropin; Gonadotropin-releasing hormone; Estrogen; Pituitary; Gene expression; Release; Salmon 1. Introduction The teleost pituitary secretes two gonadotropins (GTHs), follicle-stimulating hormone (FSH, formerly designated as GTH I in fish) and luteinizing hormone (LH, formerly GTH II). GTHs are heterodimeric gly- coproteins composed of a common a subunit and a unique b subunit. In salmonids, they are produced in different cells in the pituitary (Naito et al., 1991; Nozaki et al., 1990a), and are secreted in a different manner during reproductive cycle. A number of studies on pi- tuitary contents (Naito et al., 1991; Nozaki et al., 1990a,b; Sumpter and Scott, 1989; Suzuki et al., 1988) and plasma levels (Gomez et al., 1999; Oppen-Berntsen et al., 1994; Prat et al., 1996; Saligaut et al., 1998; Slater et al., 1994; Sumpter and Scott, 1989; Suzuki et al., 1988; Swanson, 1991; Swanson et al., 1989) of GTHs have * Corresponding author. Fax: +81-11-706-4448. E-mail address: hando@sci.hokudai.ac.jp (H. Ando). 0016-6480/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.02.012 www.elsevier.com/locate/ygcen General and Comparative Endocrinology 137 (2004) 109–121 GENERAL AND COMPARATIVE ENDOCRINOLOGY