GnRH mRNA levels in male three-spined sticklebacks, Gasterosteus aculeatus, under different reproductive conditions ☆ Yi Ta Shao a,d , Yung Che Tseng b, ⁎, Chia-Hao Chang c , Hong Young Yan d , Pung Pung Hwang e , Bertil Borg a, ⁎⁎ a Department of Zoology, Stockholm University, Stockholm S-10691, Sweden b Department of Life Science, National Taiwan Normal University, Taipei 11676, Taiwan c Department of Biological Science and Technology, National Chiao Tung University, Taiwan d Marine Research Station, Academia Sinica, I-lan 262, Taiwan e Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115 29, Taiwan abstract article info Article history: Received 29 May 2014 Received in revised form 9 October 2014 Accepted 16 October 2014 Available online 4 November 2014 Keywords: Gonadectomy Photoperiod GnRH2 GnRH3 Stickleback Feedback mRNA In vertebrates, reproduction is regulated by the brain–pituitary–gonad (BPG) axis, where the gonadotropin- releasing hormone (GnRH) is one of the key components. However, very little is known about the possible role of GnRH in the environmental and feedback control of fish reproduction. To investigate this, full-length gnrh2 (chicken GnRH II) and gnrh3 (salmon GnRH) sequences of male three-spined sticklebacks (Gasterosteus aculeatus), which are clustered with the taxa of the same GnRH type as other Euteleostei, were cloned and anno- tated. gnrh1 is absent in this species. The mRNA levels of gnrh2 and gnrh3 in the sticklebacks' brain were mea- sured under breeding and post-breeding conditions as well as in castrated and sham-operated breeding fish and castrated/sham-operated fish kept under long-day (LD 16:8) and short-day (LD 8:16) conditions. Fully breeding males had considerably higher mRNA levels of gnrh2 and gnrh3 in the thalamus (Th) and in the telen- cephalon and preoptic area (T + POA), respectively, than post-breeding males. Sham-operated breeding males have higher gnrh3 mRNA levels than the corresponding castrated males. Moreover, higher gnrh2 mRNA levels in the Th and higher gnrh3 mRNA levels in the T + POA and hypothalamus (HypTh) were also found in long- day sham-operated males than in sham-operated fish kept under an inhibitory short day photoperiod. Neverthe- less, gnrh2 and gnrh3 mRNA levels were not up-regulated in castrated males kept under long-day photoperiod, which suggests that positive feedbacks on the brain-pituitary-gonad axis are necessary for this response. © 2014 Elsevier Inc. All rights reserved. 1. Introduction In vertebrates, reproductive rhythms are regulated by the brain–pi- tuitary–gonad (BPG) axis. The secretion of gonadotropins (GtH) from the pituitary is stimulated by gonadotropin-releasing hormone (GnRH) from the brain (Goos, 1991; Peter et al., 1991), although other factors are also involved. To date, 14 forms of GnRH have been identified in vertebrates, belonging to three groups (GnRH 1, 2 and 3) (reviewed in Okubo and Nagahama, 2008). To date, all studied fish species have at least two GnRH forms (GnRH2 and GnRH3) in the central nervous system, and many bony fishes have all three forms (reviewed in Okubo and Nagahama, 2008; Somoza et al., 2002). Based on available genome database (the Ensembl Genome Browser system; Ensembl release 75-February 2014), two types of GnRH, GnRH2 (chicken GnRH II) and GnRH3 (salmon GnRH), have been assembled in the stickleback genome. However, GnRH1 is absent. Even though it's neighbor genes were found on chromosome group XII, no possible GnRH homolog was present in the appropriate locus as has also recently been found in the zebrafish (Danio rerio)(Tostivint, 2011). Numerous studies have shown that GnRH treatments can induce elevated plasma GtH levels and sexual maturation in many fishes, e.g., goldfish (Carassius auratus), African catfish (Clarias gariepinus), gilthead sea breams (Sparus aurata) and salmonids (reviewed in Peter et al., 1991). In several studies, brain GnRH peptide contents have been measured in relation to gonadal maturation in goldfish, eels (Anguilla anguilla), platyfish (Xiphophorus maculatus) and salmo- nids, but the results vary among species (reviewed in Amano et al., 1997). Nevertheless, recent studies showed that mRNA levels of hypophysiotropic gnrh increase at sexual maturation in red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegeli) (Senthilkumaran et al., 1999), salmonids (Onuma et al., 2010) and Atlantic cod (Gadus morhua)(Hildahl et al., 2011). Very little is known about the possible role of GnRH in the environmental and BPG-axis feedback control of seasonal reproduction. The three-spined stickleback, Gasterosteus aculeatus, is one of the most studied fishes with regard to the control of seasonal reproductive cycles (Borg, 2010). Its natural breeding season is late spring to early Comparative Biochemistry and Physiology, Part A 180 (2015) 6–17 ☆ Supported by the Swedish Research Council FORMAS. ⁎ Corresponding author. Tel.: +886 2 77346324. ⁎⁎ Corresponding author. Tel.: +46 8 164082. E-mail addresses: yct@ntnu.edu.tw (Y.C. Tseng), bertil.borg@zoologi.su.se (B. Borg). http://dx.doi.org/10.1016/j.cbpa.2014.10.008 1095-6433/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa