NEUROBIOLOGY & PHYSIOLOGY Animal Cells and Systems, 2014 Vol. 18, No. 2, 109–118, http://dx.doi.org/10.1080/19768354.2014.902863 Effects of various photoperiods on Kisspeptin and reproductive hormones in the goldfish, Carassius auratus a† a† a a a a b Hyun Suk Shin , Jin Ah Song , Ji Yong Choi ,NaNaKim , Young Jae Choi , Si Nae Sung , Mi Seon Park , b a* Byung Hwa Min and Cheol Young Choi a b Division of Marine Environment & BioScience, Korea Maritime and Ocean University, Busan 606-791, Korea; Aquaculture Management Division, National Fisheries Research & Development Institute, Busan 619-705, Korea (Received 22 September 2013; received in revised form 4 March 2014; accepted 5 March 2014) The study aimed to test differences in the hormones of the hypothalamus–pituitary–gonad axis, Kisspeptin (Kiss) and sex steroids, and the sexual maturation of goldfish (Carassius auratus) according to various photoperiods: 10 h light (L):14 h dark (D) (short day), 12L:12D (control), and 14L:10D (long day). To investigate the sexual maturation under various photoperiods, quantitative real-time PCR (QPCR) assays for salmon gonadotropin-releasing hormone, chicken gonadotropin- releasing hormone, gonadotropin hormones (GTHs), Kisspeptin 1 (Kiss1), and its receptor, G protein-coupled receptor 54 (GPR54), were developed. mRNA expression was monitored in cultured pituitary cells (in vitro) of various photoperiod groups. Furthermore, we injected or treated the animals with Kiss and found that Kiss treatment increases the mRNA expression levels of GTHs. We measured the plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), and 17α-hydroxypregnenolone levels. The gonadotropin-releasing hormones (GnRHs), GTHs, Kiss1/GPR54 mRNA expression, plasma FSH, LH, and 17α-hydroxypregnenolone levels in the 14L:10D group were significantly higher than in the other groups. These results suggest that light length regulates sex maturation by GnRH and Kiss1 in the brain of the goldfish. Keywords: gonadotropin-releasing hormone; kisspeptin; photoperiod; sex maturation; sex steroid hormone Introduction Sexual development and maturation in teleosts are regulated The role of GnRH, the hormone that regulates by various sex hormones in the hypothalamus–pituitary– sex maturation in the hypothalamus, is well characterized. gonad (HPG) axis, including gonadotropin-releasing hor- In contrast, Kisspeptin (Kiss) is a neuropeptide that has mone (GnRH), gonadotropin hormone (GTH), and steroid recently been detected in the hypothalamus (Lee et al. hormones, and elsewhere by neuroendocrine materials and 1996) and regulates sexual differentiation and spawn time gonadal hormones (Baroiller et al. 1999). in fish through the activation of the HPG axis (Funes et al. To date, the role of GnRH in the control of pituitary 2003; Seminara et al. 2003). Additionally, Kiss acts as a hormone secretion, in the direct GnRH actions on oocyte sex maturation-regulating factor by activating certain meiosis, and in the steroidogenesis (Habibi et al. 1988; nerves in the brain through the regulation of GnRH Pati & Habibi 2002) has been well documented. Fifteen expression (Chang et al. 2012). GnRH isoforms have been isolated from vertebrates There are two types of Kiss isoforms in teleosts and and protochordates (Fernald & White 1999; Adams et al. amphibians (Lee et al. 2009; Um et al. 2010). Kisspeptin 2002). Using the goldfish (Carassius auratus) as experi- 1 (Kiss1) is one of the important neuroendocrine factors mental fish, the presence of two distinct forms of GnRH, that regulate the sexual maturation of teleosts (Roa et al. namely, salmon GnRH (sGnRH) and chicken GnRH-II 2011), and it plays a role in the initiation of sexual (cGnRH-II), in the brain of a single fish has been character- maturation in the GnRH neurons of the hypothalamus ized (Yu et al. 1988). Generally, in certain teleosts, such as (Colledge 2009). Additionally, histological observations striped bass (Morone saxatilis), gilthead seabream (Sparus have shown that Kiss1 and its receptor, G protein-coupled aurata), and Nile tilapia (Oreochromis niloticus), sGnRH is receptor 54 (GPR54), are located with the GnRH neurons produced as a third form of GnRH in neuronal groups in the hypothalamus. Interactions between GnRH have localized in the ventral forebrain along the terminal nerve also been reported (Irwig et al. 2004; Messager et al. (Senthilkumaran et al. 1999). cGnRH-II neurons are localized 2005). According to these various research results, the in the midbrain tegmentum, project their axons widely Kiss1-GPR54 signaling system is one of the circuits that throughout the central nervous system, and modulate sexual regulate GnRH secretion in the hypothalamus. and feeding behaviors (White et al. 1998). *Corresponding author. Email: email@example.com These authors contributed equally to this work. © 2014 Korean Society for Integrative Biology 110 H.S. Shin et al. Western blot (brain) (a) LRH13 52-kDa β-tubulin 55-kDa (b) sGnRH mRNA expression in hypothalamus 0.08 10:14 d3 12:12 0.06 14:10 c2 0.04 c2 0.02 b2 c1 b1 b1 a1 a1 a1 a1 a1 0 246 (c) cGnRH-II mRNA expression in hypothalamus 0.0025 10:14 d3 12:12 0.002 14:10 c3 0.0015 d1 0.001 b2 b2 b2 c1 b1 0.0005 a1 a1 a1 024 6 Time (months) Figure 1. Changes in the expression levels of the LRH13 protein (identiﬁed using a monoclonal mouse antiserum that recognizes most vertebrate GnRH forms; 52-kDa; Park & Wakabayashi 1986) in the brain (a) and sGnRH (b) and cGnRH-II (c) mRNA levels in the hypothalamus of goldﬁsh maintained under different photoperiods – 10L:14D (short day), 12L:12D (control), and 14L:10D (long day) – using a white ﬂuorescent bulb. Total hypothalamus RNA (2.0 µg) was reverse-transcribed and ampliﬁed. The results are expressed as normalized fold expression levels with respect to the β-actin levels in the same sample. Values marked with different characters are signiﬁcantly different at different times (months) in ﬁsh exposed to the same photoperiod (P < 0.05). The numbers indicate signiﬁcant differences between different photoperiods within the same month (P < 0.05). All values are means ± SD (n = 10). The synthesis and secretion of follicle-stimulating Various factors influence sex maturation, and external hormone (FSH) and luteinizing hormone (LH) in the factors such as photoperiod, temperature, pH, or feeding pituitary are stimulated by GnRH and Kiss1 protein behavior affect the endocrinological side (Baroiller et al. synthesis and secretion in the hypothalamus, and these 1999). Among these factors, photoperiod influences hormones play an important role in regulating reproduc- the regulation of diurnal endocrine rhythm (Duston & tion in teleosts (Andrews et al. 1988). There are two types Saunders 1990) and growth in fish (Gross et al. 1995) and of GTH in fish, FSH and LH (Van Der Kraak et al. 1998). sex maturation (Biswas & Takeuchi 2002). In particular, FSH and LH induce gonad development and gonad steroid photoperiod regulates sex maturation (Rodrlguez et al. hormone synthesis (Andrews et al. 1988). During the 2000) by strongly affecting neuroendocrine control and induction process, the precursor of gonad steroid hormone, the HPG axis (Koumoundouros et al. 2002). 17α-hydroxypregnenolone, is secreted, and cholesterol is Therefore, we investigated the changes in GnRH, GTHs, changed to 17α-hydroxypregnenolone by LH, which and Kiss1/GPR54 mRNA expression under different photo- affects the theca follicular cells and granulosa cells during periods using in vivo and in vitro (pituitary cell culture) the final stage of maturation. 17α-hydroxypregnenolone methods to examine the influence of light exposure time on also plays an important role in the synthesis of sex goldfish sex maturation. Additionally, we observed the hormones (estradiol and testosterone [T]), which interact changes in GTHs mRNA expression in pituitary cultures with FSH (Dickey and Swanson 2000; Yamato et al. 2010). treated with Kiss to investigate the influence of Kiss on the Normalized fold expression Normalized fold expression (relative of β-action) (relative of β-action) Animal Cells and Systems 111 Table 1. Primers used for QPCR amplification. Genes (accession no.) Primer DNA sequences cGnRH-II (U40567) Forward 5′-TTC AGA GGT TTC AGA AGA AAT CAA-3′ Reverse 5′-GCG TCC AGC AGT ATT GTC-3′ sGnRH (AB017272) Forward 5′-CCA ACA GAC GAG GAA GAG-3′ Reverse 5′-CGA TTC AGG ACG CAA ACT-3′ Kisspeptin (FJ236327) Forward 5′-TGA ACC TAC TTA CCA TAA TTT TGA TG-3′ Reverse 5′-CCTGAG ACC CTG GAG TGA -3′ GPR54 (FJ465139) Forward 5′-AGT GGT CAT TGT TGT TCT CTT-3′ Reverse 5′-AGG AGT TGG CAT AGG ACA T-3′ GTHα (D86552) Forward 5′- TAT CGG TGG TGC TGG TTA -3′ Reverse 5′- GCT GTC CTC AAA GTC GTT A -3′ FSHβ (D88023) Forward 5′-CCT GGA AAG TGA GGA ATG-3′ Reverse 5′GTT CTG GTA AGA CAG CAT CA-3′ LHβ (D88024) Forward 5′-TGT CCT ATT CTC TGT AAT TGT CC-3′ Reverse 5′-GTC TCA TTA ACT GGC TCA CA -3′ β-actin (AB039726) Forward 5′- TTC CAG CCA TCC TTC CTA T-3′ Reverse 5′- TAC CTC CAG ACA GCA CAG -3′ (a) Kiss1 mRNA expression in hypothalamus 0.01 10:14 12:12 d2 0.008 14:10 0.006 c1 c1 0.004 c2 b1 b1 b2 0.002 a1 a1 a1 a1 a1 0 2 46 (b) GPR54 mRNA expression in hypothalamus 0.25 d3 10:14 12:12 0.2 14:10 c2 0.15 c3 c1 b3 0.1 b2 b2 b1 b1 0.05 a1 a1 a1 024 6 Time (months) Figure 2. Changes in the mRNA expression levels of Kiss1 (a) and its receptor, GPR54 (b) in the hypothalamus in goldﬁsh maintained under different photoperiod conditions – 10L:14D (short day), 12L:12D (control), and 14L:10D (long day) – using a white ﬂuorescent bulb, as measured by QPCR. Total hypothalamus RNA (2.0 µg) was reverse-transcribed and ampliﬁed. The results are expressed as normalized fold expression levels with respect to the β-actin levels in the same sample. Values marked with different characters are signiﬁcantly different at different times (months) in ﬁsh exposed to the same photoperiod (P < 0.05). The numbers indicate signiﬁcant differences between different photoperiods within the same month (P < 0.05). All values are means ± SD (n = 10). Normalized fold expression Normalized fold expression (relative of β-action) (relative of β-action) 112 H.S. Shin et al. Western blot (pituitary) (a) GTHα 35-kDa β-tubulin 55-kDa (b) GTHα mRNA expression in pituitary 10:14 12:12 14:10 c3 b3 b3 b2 b2 b2 c1 bc1 b1 a1 a1 a1 (c) FSHβ mRNA expression in pituitary 1.2 10:14 12:12 14:10 d2 0.8 c3 b1 b3 b1 a2 a2 a1 a1 a1 a1 a1 0.4 (d) LHβ mRNA expression in pituitary 10:14 c3 12:12 c3 14:10 d2 c1 c2 b2 ab1 b1 b1 a1 a1 a1 024 6 Time (months) Figure 3. Changes in the expression levels of the GTHα protein (detected using anti-goldﬁsh GTHα; a polyclonal rabbit antibody; 35-kDa; Kobayashi et al. 2006) in the pituitary (a) and GTHα (b), FSHβ (c), and LHβ (d) mRNA in the pituitary of goldﬁsh maintained under different photoperiod conditions – 10L:14D (short day), 12L:12D (control), and 14L:10D (long day) – using a white ﬂuorescent bulb, as measured by QPCR. Total pituitary RNA (2.0 µg) was reverse-transcribed and ampliﬁed. The results are expressed as normalized fold expression levels with respect to the β-actin levels in the same sample. Values marked with different characters are signiﬁcantly different at different times (months) in ﬁsh exposed to the same photoperiod (P < 0.05). The numbers indicate signiﬁcant differences between different photoperiods within the same month (P < 0.05). All values are means ± SD (n = 10). expression of gonadal hormones secreted by the HPG axis. Korea). The goldfish were transferred into 300-L circula- Furthermore, we examined the difference of goldfish sex tion filter tanks in the laboratory and reared for 6 months. maturation by photoperiod regulation by measuring plasma The fish were randomly divided into three tanks, and each FSH, LH, and the 17α-hydroxypregnenolone levels. tank was exposed to one of three photoperiods (10 h light (L):14 h dark (D), 12L:12D, 14L:10D). The start of the light period for the 12L:12D and 14L:10D groups was Experimental fish and conditions 07:00 and for the 10L:14D group was 09:00. The light source was a white fluorescent bulb, and the lights were The immature goldfish (length 5.2 ± 1.2 cm, weight 8.1 ± 3.2 g) were purchased from Choryang aquarium (Busan, set 50 cm above the water surface. The water temperature Normalized fold expression Normalized fold expression Normalized fold expression (relative of β-action) (relative of β-action) (relative of β-action) Animal Cells and Systems 113 (a) Plasma FSH levels 10:14 c2 12:12 c2 c1 14:10 b2 c12 c1 bc1 b1 b1 a1 a1 a1 02 46 (b) Plasma LH levels 10:14 12:12 d3 14:10 c3 d2 b2 c2 c1 200 b1 b1 b1 a1 a1 a1 024 6 Time (months) Figure 4. Changes in the levels of plasma FSH (a) and LH (b) in goldﬁsh under different photoperiod conditions – 10L:14D (short day), 12L:12D (control), and 14L:10D (long day) – using a white ﬂuorescent bulb, as measured by a plate reader. Values with different characters are signiﬁcantly difference at different times (months) in ﬁsh exposed to the same photoperiod (P < 0.05). The numbers indicate signiﬁcant differences between different photoperiods within the same months (P < 0.05). All values are means ± SD (n = 10). was maintained at 22 ± 1°C. The goldfish were provided goldfish hypothalamus was significantly higher in 14L:10D with commercial feed twice daily (09:00 h and 17:00 h) at group compared with the other photoperiods by quantitative a4–5% wet body weight per day. real-time PCR (QPCR) (Figure 1b, c). The experiments were performed under these conditions for 6 months at intervals of two months. After 2, 4, and 6 Changes of Kiss1 and GPR54 mRNA expression levels months, female goldfish were analyzed in this study. Prior to To compare the expression levels of Kiss1 and GPR54 each experiment, the fish were anesthetized with 200 mg/L mRNA in the three photoperiod groups, we performed tricaine methanesulfonate (MS-222, Sigma, USA), and QPCR (Table 1) using cDNA extracted from goldfish their body masses and total lengths were recorded. hypothalamus in each experimental group. As a result, Kiss1 and GPR54 mRNA expression levels in 14L:10D were significantly higher compared with the other groups Results (Figure 2). Additionally, we observed increases in the Changes in sGnRH, cGnRH-II mRNA, and GnRH Kiss1 and GPR54 mRNA expression levels in the three protein expression levels photoperiod groups as rearing time increased. GnRH protein levels using protein extracted from goldfish brain were increased in all three photoperiod groups as Changes of GTHs mRNA and GTHα protein expression rearing time increased. In particular, the levels in levels 14L:10D were high compared with the other photoperiods (Figure 1a). Additionally, the expression of sGnRH and The GTHα protein levels measured in protein extracted cGnRH-II mRNA levels using cDNA extracted from from the goldfish pituitary increased with rearing time in Plasma LH (mIU/mL) Plasma FSH (mIU/mL) 114 H.S. Shin et al. Plasma 17α-hydroxypregnenolone levels c3 10:14 c3 12:12 14:10 d2 c2 d1 b3 c1 b2 b1 a1 a1 a1 024 6 Time (months) Figure 5. Changes in the levels of plasma 17α-hydroxypregnenolone in goldﬁsh under different photoperiod conditions – 10L:14D (short day), 12L:12D (control), and 14L:10D (long day) – using a white ﬂuorescent bulb, as measured by a plate reader. Values marked with different characters are signiﬁcantly different at different times (months) in ﬁsh exposed to the same photoperiod (P < 0.05). The numbers indicate signiﬁcant differences between different photoperiods within the same month (P < 0.05). All values are means ± SD (n = 10). all three photoperiod groups. In particular, the levels in nM Kiss. However, the GTH mRNA expression levels 14L:10D were high compared with those in the other were significantly decreased in the group treated with photoperiods (Figure 3a). Additionally, we performed 1000 nM Kiss. In particular, the GTH mRNA expression QPCR using cDNA extracted from goldfish pituitary in levels in 14L:10D were significantly lower than those of each experimental group to compare the expression levels the other groups (Figure 6). of GTHs (GTHα,LHβ, and FSHβ) mRNA. The levels of GTHα,LHβ, and FSHβ mRNA expressions were signifi- Body weight cantly higher in the 14L:10D group, which was illuminated We have compared the body weight to investigate the sex longer than the other photoperiod groups, compared with maturation levels of goldfish according to rearing period the other photoperiods (Figure 3b, c,and d). in each photoperiod experimental group. The body weight at the initial stage was about 9.0 ± 0.5 g, but rearing time Plasma FSH, LH, and 17α-hydroxypregnenolone levels increased, the weights were significantly increased in all experimental groups. The highest value of weight of 25.07 Plasma FSH, LH, and 17α-hydroxypregnenolone levels ± 1.30 g was observed in 14L:10D compared with the were increased in all three photoperiod groups as rearing other groups (Figure 7). time increased. In particular, the levels in 14L:10D were high compared with those in the other photoperiod groups. The FSH levels increased from 1.55 ± 0.08 mIU/mL to Discussion 3.13 ± 0.10 mIU/mL, and the LH levels increased from 103.03 ± 6.18 mIU/mL to 350.14 ± 13.11 mIU/mL To investigate the effects of photoperiod on the sexual (Figure 4). Plasma 17α-hydroxypregnenolone levels were maturation of goldfish, we reared the goldfish under significantly increased from 2.05 ± 0.10 ng/mL to 10.10 ± three photoperiod conditions (10L:14D, 12L:12D, and 14L:10D) for 6 months and examined various parameters 0.13 ng/mL after 6 months (Figure 5). every two months. We examined the mRNA levels of GnRH, GTHs, the neuropeptide Kiss1 and its receptor Changes of GTHs mRNA expression levels by treated GPR54, and the changes in GTH mRNA levels in an in Kiss (in vitro) vitro pituitary culture treated with Kiss. Furthermore, we investigated the changes in plasma FSH, LH, and 17α- We investigated the changes in the GTHs (GTHα,LHβ, hydroxypregnenolone levels. and FSHβ) mRNA expression levels by pituitary culturing treated with Kiss (0, 100, and 1000 nM) for 5 days under First, we observed that the GnRH protein and the the three photoperiod conditions: 10L:14D, 12L:12D, and mRNA expression levels of two types of GnRH, sGnRH, 14L:10D. As a result, GTH mRNA expression levels were and cGnRH-II were significantly higher in the 14L:10D group (Figure 1). The present results are in agreement significantly higher in the 14L:10D group treated with 100 Plasma 17α-hydroxypregnenolone (ng/mL) Animal Cells and Systems 115 (a) GTHα mRNA expression in cultured pituitary 0.4 10:14 c3 c3 c2 12:12 c3 0.3 14:10 b3 b2 b2 b3 b1 b2 b3 b1 c1 0.2 b3 b1 a2 b1 b1 a3 a3 0.1 a1 a1 a1 a2 a2 a1 a1 a1a1 a1 a1 a1a1 a1 a1 a1 0 100 1000 0 100 1000 0 100 1000 0 100 1000 0 1 3 5 (b) FSHβ mRNA expression in cultured pituitary 0.008 c2 10:14 c3 b3 b1 c3 c3 b3 12:12 c1 0.006 14:10 b3 c2 a2 b2 b2 a2 a1 a1 b12 b1 c1 0.004 b1 a3 a2 b1 a1 0.002 a2 a1 a1a1a1 a1 a1 a1 a1a1a1 0 100 1000 0 100 1000 0 100 1000 0 100 1000 0 1 3 5 (c) LHβ mRNA expression in cultured pituitary 0.008 10:14 b3 b3 12:12 c3 0.006 14:10 b1 b3 c1 b3 c1 b3 c2 b3 c2 b3 0.004 a3 b2 a2 a2 b1 b2 a1 b1 a1 b1 a1 a2 a1 0.002 a1 a1 a1a1 a1 a1 a1 a1a1 a1 0 100 1000 0 100 1000 0 100 1000 0 100 1000 0 1 3 5 Time (days) and treatment (nM) Figure 6. Changes in the expression levels of GTHα (a), FSHβ (b), and LHβ (c) mRNA in the cultured pituitary (kiss treatment) of goldﬁsh under different photoperiod conditions – 10L:14D (short day), 12L:12D (control), and 14L:10D (long day) – using a white ﬂuorescent bulb, as measured by quantitative real-time PCR. Total cultured pituitary RNA (2.0 μg) was reverse-transcribed and ampliﬁed. The results are expressed as normalized fold expression levels with respect to the β-actin levels in the same sample. Values with different characters are signiﬁcantly different at different Kisspeptin treatment concentrations (nM) in ﬁsh exposed to the same days (P < 0.05). The numbers indicate signiﬁcant differences between different photoperiods within the same days (P < 0.05). All values are means ± SD (n = 5). with a previous report by Carrillo et al. (2010), which illuminated by long light exposure (Cowan et al. 2012). demonstrated growth and sexual maturation of European Additionally, the sexual maturation and Kiss1 expression sea bass (Dicentrarchus labrax) illuminated with light for levels in a mammal, the Syrian hamster, were significantly long period (18L:6D) were significantly higher than increased by a long-day photoperiod (Revel et al. 2006). natural photoperiod. According to a report from Irwig et al. (2004), Furthermore, the changes in the neuropeptide Kiss1, histological observations show that Kiss1 is localized which affects the mRNA expression levels of GnRH and within the GnRH neurons in the hypothalamus. Colledge its receptor GPR54 in the hypothalamus, were signifi- (2009) also reported that the interaction between GnRH and cantly higher in 14L:10D, and the results were similar to Kiss1 is a function of the GnRH regulator, which regulates the changes in the GnRH mRNA expression (Figure 2). the gonad steroid hormones; these hormones interact with These results demonstrate that a longer exposure to light Kiss1, and their signals are conveyed to the brain through a positively affects sexual maturation, and the results are in feedback mechanism. According to these results, GTHα agreement with those of a study that showed high levels of protein and GTHα, FSHβ,LHβ mRNA expression levels Kiss1 mRNA expression in Atlantic cod (Gadus morhua) were significantly higher in 14L:10D, the group that Normalized fold expression Normalized fold expression Normalized fold expression (relative of β-action) (relative of β-action) (relative of β-action) 116 H.S. Shin et al. c2 10:14 12:12 c1 d1 14:10 b2 b3 b1 c1 b2 b1 a1 a1 a1 0 24 6 Time (months) Figure 7. Changes in the body weight in goldﬁsh maintained under different photoperiod conditions – 10L:14D (short day), 12L:12D (control), and 14L:10D (long day) – using a white ﬂuorescent bulb. Values marked with different characters are signiﬁcantly different at different times (months) in ﬁsh exposed to the same photoperiod (P < 0.05). The numbers indicate signiﬁcant differences between different photoperiods within the same month (P < 0.05). All values are means ± SD (n = 10). showed the highest expression levels of Kiss1 and GPR54 increased concentration of Kiss in the HPG axis. In (Figure 3). Additionally, plasma FSH and LH levels were particular, we hypothesized that the observed decrease in significantly increased in the 14L:10D group (Figure 4). GTHs in only 14L:10D means that 14L:10D is sufficient to Meanwhile, Kiss1 is reported to be expressed in stimulate the secretion of the reproductive hormones the hypothalamus of various fish (Kanda et al. 2008; required for sexual maturation without external Kiss Kitahashi et al. 2009). Kiss functions as a GnRH regulator treatment based on high expression levels of Kiss1, GnRH, in the hypothalamus (Colledge 2009), and it plays a and GTH mRNA. Therefore, negative feedback action positive and negative feedback role by causing sex occurred in 14L:10D due to the treatment with Kiss. hormones to be secreted (Colledge 2008). Therefore, we Additionally, the levels of the precursor of gonad hypothesized that the significant high expression levels of steroid hormone secreted in the final stage of maturation, Kiss1 and GPR54 mRNA in the hypothalamus and GTH 17α-hydroxypregnenolone, were significantly higher in mRNA in the pituitary suggested that the increased Kiss1 14L:10D (Figure 5). Plasma steroids are converted to 17α- in 14L:10D enhanced GTH secretion in the pituitary, and hydroxypregnenolone during steroidogenesis by stimula- then the secretion of reproductive hormones are signifi- tion of LH and FSH (Hu et al. 2001); therefore, we cantly increased in 14L:10D compared with the other hypothesized that the levels of this hormone have a close photoperiod conditions. To examine this hypothesis, we relationship with sexual maturation because its expression examined the expression of GTH mRNA in the pituitary level patterns are similar to those of FSH and LH. culture (in vitro) treated by Kiss and observed signifi- These results are in agreement with previous studies cantly high levels (Figure 6). This result is consistent with showing that sexual maturation under continuous light the hypothesis that Kiss enhances GTH secretion in the conditions for a few months is enhanced through a significant pituitary and previous reports that show increasing LH increase in sex hormones and the number of oocytes and mRNA expression levels from HPG axis stimulation of developing oocytes in Atlantic salmon (Salmo salar)(Tar- Kiss when the pituitaries of mature Wistar rats (Gutiérrez- anger et al. 1999), Atlantic cod (Hansen et al. 1995), and Pascual et al. 2007) and goldfish (Bo et al. 2010) are rainbow trout (Duston & Bromage 1986). Also, several treated with Kiss. Additionally, previous pharmacological previous studies have shown that long photoperiods stimulate research has demonstrated that Kiss is able to stimulate reproduction in the goldfish and golden shiner (e.g. De FSH secretion in the rat (Messager et al. 2005). Vlaming 1975;Stacey et al. 1979; Delahunty et al. 1980). In However, we observed that the GTH mRNA expression addition, Bon et al. (1999) reported that a photoperiod levels were weakly decreased in the 14L:10D group treated involving a longer day length simulates initial reproduction with a high concentration (1000 nM) of Kiss. This result is development. Thus, this study results supported the theory in agreement with Rance (2009), who reported a decrease in that there is a relationship between light and sexual matura- GTH expression through a decrease in GnRH expression tion (Stacey et al. 1979; Duston & Bromage 1986; Hansen according to the action of negative feedback from an et al. 1995; Revel et al. 2006). Body weight (g) Animal Cells and Systems 117 spermiation and enhances growth in male European sea In conclusion, we hypothesized that Kiss1 is an bass (Dicentrarchus labrax). Aquaculture. 299:157–164. important factor in the mechanism regulating FSH, LH, Chang JP, Mar A, Wlasichuk M, Wong AOL. 2012. Kisspeptin-1 and GnRH. Furthermore, based on these results, GnRHs, directly stimulates LH and GH secretion from goldfish 2+ GTHs, Kiss1/GPR54 mRNA expression, plasma FSH, LH, pituitary cells in a Ca -dependent manner. Gen Comp Endocrinol. 179:38–46. and 17α-hydroxypregnenolone levels were significantly Colledge WH. 2008. GPR54 and kisspeptins. Results Probl Cell higher in 14L:10D, the longer day length photoperiod. As Differ. 46:117–143. the results, we have examined the possibility of female sex Colledge WH. 2009. Kisspeptins and GnRH neuronal signaling. maturation by various photoperiods. The maturation stage Trends Endocrinol Metab. 20:115–121. for this experimental period was for the initial maturation Cowan M, Davie A, Migaud H. 2012. Photoperiod effects on the expression of Kisspeptin and gonadotropin genes in Atlantic stage, not the final sex maturation stage in this study. cod, Gadus morhua, during first maturation. Comp Biochem Furthermore, we have plans to investigate the further Physiol A. 163:82–94. research about final sex maturation by various photoper- Delahunty G, Schreck CB, De Vlaming VL. 1980. Effects iods. These results (1) suggest that a longer day length of photoperiod on plasma corticoid levels in the goldfish, Carassius auratus-role of the pineal. Comp Biochem photoperiod affects the stimulation of the sexual matura- Physiol A. 65:355–358. tion of goldfish and (2) provide the basic information De Vlaming VL. 1975. Effects of photoperiod and temperature needed for the investigation of the mechanism through on gonadal activity in the cyprinid teleost, Notemigonus which Kiss mediates sexual maturation. Moreover, as one Crysoleucas. Biol Bull. 148:402–415. of the genes that regulate maturation in an epistatic Dickey JT, Swanson P. 2000. Effects of salmon gonadotropin- releasing hormone on follicle-stimulating hormone secretion fashion, Kiss is one of the important factors in the and subunit gene expression in coho salmon (Oncorhynchus regulation of sexual maturation. kisutch). Gen Comp Endocrinol. 118:436–449. Duston J, Bromage N. 1986. Photoperiodic mechanisms and Acknowledgments rhythms of reproduction in the female rainbow trout. Fish Physiol Biochem. 2:35–51. This study was supported by a grant from the National Fisheries Duston J, Saunders RL. 1990. The entrainment role of photo- Research & Development Institute [NFRDI; RP-2013-AQ-222], period on hypoosmoregulatory and growth related aspects of and by the Information Technology Research Center (ITRC) smolting in Atlantic salmon (Salmo salar). Can J Zool. support program supervised by the National IT Industry Promo- 68:707–715. tion Agency (NIPA) [2013-H0301-13-2009]. Fernald RD, White RB. 1999. Gonadotropin-releasing hormone genes: phylogeny, structure, and functions. Front Neuroen- References docrinol. 20:224–240. Funes S, Hedrick JA, Vassileva G, Markowitz L, Abbondanzo S, Adams BA, Vickers ED, Warby C, Park M, Fischer WH, Grey- Golovko A, Yang S, Monsma FJ, Gustafson EL. 2003. The Craig A, Rivier JE, Sherwood NM. 2002. Three forms of Kiss-1 receptor GPR54 is essential for the development of gonadotropin-releasing hormone, including a novel form, in the murine reproductive system. Biochem Biophys Res a basal salmonid, Coregonus clupeaformis. Biol Reprod. Commun. 312:1357–1363. 67:232–239. Gross WL, Roelofs EW, Fromm PO. 1995. Influence of Andrews WV, Mauer R, Conn PM. 1988. Stimulation of rat photoperiod on growth of green sunfish, Lepomis cyanellus. luteinizing hormone β- messenger RNA levels by gonado- J Fish Res Board Can. 22:1379–1386. tropin-releasing hormone. J Biol Chem. 263:13755–13761. Gutiérrez-Pascual E, Martínez-Fuentes AJ, Pinilla L, Tena- Baroiller JF, Guiguen Y, Fostier A. 1999. Endocrine and Sempere M, Malagón MM, Castaño JP. 2007. Direct pituitary environmental aspects of sex differentiation in fish. Cell effects of kisspeptin: activation of gonadotrophs and somato- Mol Life Sci. 55:910–931. trophs and stimulation of luteinizing hormone and growth Biswas AK, Takeuchi T. 2002. Effect of different photoperiod cycles on metabolic rate and energy loss of both fed and hormone secretion. J Neuroendocrinol. 19:521–530. unfed adult tilapia (Oreochromis niloticus) Part II. Fish Sci. Habibi HR, Van der Kraak G, Peter RE. 1988. Effect of teleost 68:543–553. GnRH on reinitiation of oocyte meiosis in goldfish, in vitro. Bo Y, Quan J, Ting C, Wendy KW, Ko AOLW. 2010. Am J Physiol. 255:R268–R273. Goldfish kisspeptin: molecular cloning, tissue distribution Hansen T, Kjesbu OS, Holm JC, Karlsen Ø. 1995. Growth, of transcript expression, and stimulatory effects on prolactin, gonadal development and spawning time of Atlantic cod growth hormone and luteinizing hormone secretion and gene (Gadus morhua) reared under different photoperiods. In: expression via direct actions at the pituitary level. Gen Comp Goetz FW, Thomas P, editors. The 5th International Sympo- Endocrinol. 165:60–71. sium on Reproductive Physiology of Fish. Austin, TX: Bon E, Breton B, Govoroun MS, Le Menn F. 1999. Effects of University of Texas; p. 186. accelerated photoperiod regimes on the reproductive cycle of Hu M-C, Chiang EF-L, Tong S-K, Lai W, Hsu N-C, Wang LC- K, Chung B-C. 2001. Regulation of steroidogenesis in the female rainbow trout: II. Seasonal variations of plasma transgenic mice and zebrafish. Mol Cell Endocrinol. gonadotropins (GTH I and GTH II) levels correlated with 171:9–14. ovarian follicle growth and egg size. Fish Physiol Biochem. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, 20:143–154. Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA. Carrillo M, Begtashi I, Rodríguez L, Marin MC, Zanus S. 2010. 2004. Kisspeptin activation of gonadotropin releasing Long photoperiod on sea cages delays timing of first 118 H.S. Shin et al. hormone neurons and regulation of Kiss-1 mRNA in the photoperiodic control of reproduction in hamsters. Curr male rat. Neuroendocrinology. 80:264–272. Biol. 16:1730–1735. Kanda S, Akazone Y, Matsunaga T, Yamamoto N, Yamada S, Roa J, Navarro TM, Tena-Sempere M. 2011. Kisspeptins in Tsukamura H, Maeda K, Oka Y. 2008. Identification of reproductive biology: consensus knowledge and recent KiSS-1 product kisspeptin and steroid-sensitive sexually developments. Biol Reprod. 85:650–660. dimorphic kisspeptin neurons in medaka (Oryzias latipes). RodrÍguez L, Begtashi I, Zanuy S, Carrillo M. 2000. Develop- Endocrinology. 149:2467–2476. ment and validation of an enzyme immunoassay for testo- Kitahashi T, Ogawa S, Parhar IS. 2009. Cloning and expression sterone: effects of photoperiod on plasma testosterone levels of kiss2 in the zebrafish and medaka. Endocrinology. and gonadal development in male sea bass (Dicentrarchus 150:821–831. labrax L.) at puberty. Fish Physiol Biochem. 23:141–150. Kobayashi M, Morita T, Ikeguchi K, Yoshizaki G, Suzuki T, Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Watabe S. 2006. In vivo biological activity of recombinant JS Jr, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof goldfish gonadotropins produced by baculovirus in silkworm KM, Hendrick AG, et al. 2003. The GPR54 gene as a larvae. Aquaculture. 256:433–442. regulator of puberty. N Engl J Med. 349:1614–1627. Koumoundouros G, Pavlidis M, Anezaki L, Kokkari C, Sterioti Senthilkumaran B, Okuzawa K, Gen K, Ookura T, Kagawa H. A, Divanach P, Kentouri M. 2002. Temperature sex deter- 1999. Distribution and seasonal variations in levels of three mination in the European sea bass, Dicentrarchus labrax native GnRHs in the brain and pituitary of perciform fish. J (L., 1758) (Teleostei, Perciform, Moronidae): critical sensit- Neuroendocrinol. 11:181–186. ive ontogenic phase. J Exp Zool. 292:573–579. Stacey NE, Cook AF, Peter RE. 1979. Ovulatory surge of Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman gonadotropin in the goldfish, Carassius auratus. Gen Comp BE, Welch DR. 1996. KiSS-1, a novel human malignant Endocrinol. 37:246–249. melanoma metastasis-suppressor gene. J Natl Cancer Inst. Taranger GL, Haux C, Hansen T, Stefansson SO, Björnsson BT, 88:1731–1737. Walther BT, Kryvl H. 1999. Mechanisms underlying photo- Lee YR, Tsunekawa K, Moon MJ, Um HN, Huang JI, Osugi T, periodic effects on age at sexual maturity in Atlantic salmon, Otaki N, Sunakawa Y, Kim K, Vaudry H, et al. 2009. Salmo salar. Aquaculture. 177:47–60. Molecular evolution of multiple forms of Kisspeptins and Um HN, Han JM, Hwang JI, Hong SI, Vaudry H, Seong JY. GPR54 receptors in vertebrates. Endocrinology. 150:2837–2846. 2010. Molecular coevolution of Kisspeptins and their Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zhen D, receptors from fish to mammals. Ann NY Acad Sci. Dixon J, Thresher RR, Malinge I. 2005. Kisspeptin directly 1200:67–74. stimulates gonadotropin-releasing hormone via G protein Van Der Kraak G, Chang JP, Janz DM. 1998. Reproduction. In: coupled receptor 54. Proc Natl Acad Sci USA. 102: Evans DH, editor. The physiology of fishes. New York: CRC 1761–1766. Press; p. 465–488. Park MK, Wakabayashi K. 1986. Preparation of a monoclonal White RB, Eisen JA, Kasten TL, Fernald RD. 1998. Second gene antibody to common amino acid sequence of LHRH and its for gonadotropin-releasing hormone in humans. Proc Natl application. Endocrinol Jpn. 33:257–272. Acad Sci USA. 95:305–309. Pati D, Habibi HR. 2002. The involvement of protein kinase c Yamato S, Nakagawa S, Yamazaki N, Aketo T, Tachikawa E. and arachidonic acid pathways in the gonadotropin-releasing 2010. Simultaneous determination of pregnenolone and 17α- hormone regulation of oocyte meiosis and follicular steroi- hydroxypregnenolone by semi-micro high-performance dogenesis in the goldfish ovary. Biol Reprod. 66:813–822. liquid chromatography with an immobilized cholesterol Rance EN. 2009. Menopause and the human hypothalamus: oxidase as a pre-column reactor: application to bovine evidence for the role of kisspeptin/neurokinin B neurons in adrenal fasciculata cells. J Chromatogr B. 878:3358–3362. the regulation of estrogen negative feedback. Peptides. Yu KL, Sherwood NM, Peter RE. 1988. Differential distribution 30:111–122 of two molecular forms of gonadotropin-releasing hormone Revel FG, Saboureau M, Masson-Pevet M, Pevet P, Mikkelsen in discrete brain areas of goldfish (Carassius auratus). JD, Simonneaux V. 2006. Kisspeptin mediates the Peptides. 9:625–630.
Animal Cells and Systems
– Taylor & Francis
Published: Mar 4, 2014
Keywords: gonadotropin-releasing hormone; kisspeptin; photoperiod; sex maturation; sex steroid hormone