Characterization of gonadotropin-releasing hormone and gonadotropin in jack mackerel (Trachurus japonicus): Comparative gene expression analysis with respect to reproductive dysfunction in captive and wild fish
Introduction
As in other vertebrates, the brain–pituitary–gonad (BPG) axis regulates gametogenesis in teleosts. Gonadotropin (GtH) is synthesized in and secreted from the pituitary gland, acting on the gonads to stimulate the production of sex steroids (Swanson et al., 2003). The two forms of GtHs, the follicle-stimulating hormone (FSH) and the luteinizing hormone (LH), share a common α-subunit but possess unique β-subunits (Pierce and Parsons, 1981, Yaron et al., 2003). During oogenesis, primarily in salmonids, FSH is believed to act on vitellogenesis via estradiol-17β (E2) production, while in several teleost species, LH induces final oocyte maturation (FOM) and ovulation via production of the maturation-inducing steroid (Nagahama and Yamashita, 2008, Prat et al., 1996, Swanson et al., 1991, Tyler et al., 1991). The gonadotropin-releasing hormone (GnRH) decapeptide in the brain is the upstream regulator of GtH (Lethimonier et al., 2004, Zohar et al., 2010). Phylogenetic analysis shows that three molecular forms of GnRH (GnRH1, GnRH2, and GnRH3) exist among teleost species, with two or three forms coexisting within the brain of individual species. Multiple GnRH1 homologs (e.g., catfish GnRH [cfGnRH], herring GnRH [hgGnRH], mammalian GnRH [mGnRH], medaka GnRH [mdGnRH] or pejerrey GnRH [pjGnRH], and seabream GnRH [sbGnRH]) have been identified in teleosts, while GnRH2 (chicken GnRH II [cGnRH-II]) and GnRH3 (salmon GnRH [sGnRH]) show common amino acid sequences among species (Somoza et al., 2002). GnRH1-expressing neurons are distributed in the preoptic area and the hypothalamus, innervating into the pituitary, and GnRH1 is suggested to play a major role in the stimulation of GtH (Zohar et al., 2010).
Gamete production is critical to successful aquaculture; however, most cultured female fish exhibit some degree of reproductive dysfunction. Inhibition of FOM, which occurs prior to ovulation and spawning, is the most common form of dysfunction (Zohar and Mylonas, 2001). FOM/ovulation failure may be caused by a lack of LH stimulus from the pituitary due to weakened hypothalamic GnRH-stimulated LH secretion (Mylonas et al., 2010). Hence, in many cultured fish species, FOM/ovulation is induced by a variety of hormonal treatments, mainly those using human chorionic gonadotropin (hCG) and GnRH analog (GnRHa) (Mylonas et al., 2010). hCG shows high structural similarity with LH and acts directly on the ovary, while GnRHa elevates serum LH levels by stimulating pituitary LH secretion promoting the initiation of FOM and ovulation (Forniés et al., 2003, Ludwig et al., 2002, Mikolajczyk et al., 2003, Mylonas et al., 1998). Conversely, vitellogenesis impairment is considered to be a more serious type of reproductive dysfunction, and it occurs in certain fish species, such as freshwater eel (genus Anguilla) and Mediterranean greater amberjack (Seriola dumerili) (Micale et al., 1999, Mylonas et al., 2004, Ohta et al., 1997). It has been suggested that stress induced by captive-rearing (e.g., confinement) negatively affects GnRH synthesis (Zohar and Mylonas, 2001). However, the mechanisms underlying captivity stress-induced vitellogenesis dysfunction are not well understood.
The jack mackerel (Trachurus japonicus), which belongs to the order Perciformes and the family Carangidae, is widely distributed in east Asian seas and provides an important fish source in Japan, China, Korea, and Taiwan (Sassa et al., 2008, Zhang and Lee, 2001). These fish are mainly caught by purse seining in the East China Sea, along the Japan Sea coast, and off the Pacific coast of southern Japan; the Japanese stock has been managed since 1997 using a total allowable catch (TAC) system (Nishida, 2004). Due to increased demand for this species, juvenile and adult fish are captured in the wild and reared for an appropriate duration in sea cages in southwestern regions of Japan, after which the fish are marketed (Tamotsu et al., 2012). However, stable seed production has not yet been established. Like other cultured fish species, jack mackerel do not undergo spontaneous spawning because FOM does not occur after the completion of vitellogenesis (Oka and Mori, 2006). Furthermore, the impairment of vitellogenesis in jack mackerel has been observed in our previous rearing experiments. When adult fish were reared in outdoor concrete tanks for more than a year, no female fish completed vitellogenesis, with the majority displaying pre-vitellogenic ovaries (Nyuji et al., 2008). Of the adult female fish caught in the wild and maintained in sea cages for approximately 1 month before the spawning season, 75% failed to complete vitellogenesis (Nyuji et al., 2012b). Thus, understanding the negative impacts of captivity on reproductive dysfunction, especially vitellogenesis, is important for jack mackerel aquaculture; however, physiological studies have not been conducted previously in this species.
For the physiological assessment of jack mackerel reproduction, endocrinological factors involved in the BPG-axis were analyzed with respect to reproductive dysfunction. First, we identified the different GnRH peptides expressed in brain tissues of jack mackerel using high performance liquid chromatography (HPLC) combined with time-resolved fluoroimmunoassay (TR-FIA). We then characterized and cloned cDNAs encoding jack mackerel GnRHs and GtH subunits. To elucidate the endocrinological causes of vitellogenesis and FOM/ovulation impairment in the BPG-axis, we measured gene expression levels of GnRHs and GtH subunits, serum E2 concentrations in captive and wild female jack mackerel sampled during the spawning season.
Section snippets
TR-FIA
For immunological identification of jack mackerel GnRHs, wild fish (344–420 mm fork length [FL], 462–703 g body weight [BW]) were purchased from a fish market at Fukuoka, Japan. The method for separating GnRH peptides from brain extract and TR-FIA detection of GnRHs were followed by Selvaraj et al. (2009). Briefly, GnRH peptides were extracted from the brains according to the procedure described by Okuzawa et al. (1990), filtered, and subjected to HPLC, using an Asahipak Gel ODP-50 column (Asahi
Immunological identification of GnRHs in jack mackerel
TR-FIA was used to identify jack mackerel GnRH peptides. The jack mackerel brain tissues expressed three GnRH peptides: sbGnRH, cGnRH-II, and sGnRH (Fig. 1). The immunoreactivity peaks seen in Fig. 1 correspond primarily to the synthetic sbGnRH, cGnRH-II, and sGnRH, respectively.
Molecular characterization of GnRHs and GtH subunits in jack mackerel
The full jack mackerel gnrh1, gnrh2, and gnrh3 cDNAs (GenBank Accession No. KC818623, KC818624, KC818625) were 404 bp, 578 bp, and 441 bp (without the poly(A) tail), respectively. These cDNAs contained open reading frames
Discussion
Teleost fish possess two or three forms of GnRH peptides (Zohar et al., 2010). HPLC and TR-FIA experiments revealed that the sbGnRH, cGnRH-II, and sGnRH peptides are expressed in jack mackerel brain. Similarly, this HPLC/immunoassay procedure demonstrated the presence of the same three GnRH forms in other perciform species, such as red seabream (Pagrus major) and Nile tilapia (Oreochromis niloticus) (Senthilkumaran et al., 1999). We also determined the cDNA nucleotide sequences encoding jack
Acknowledgments
We thank the students in the Laboratory of Marine Biology, Kyushu University, for their experimental support. This study was performed as part of the Establishment of Rearing Systems in Jack Mackerel Program, which is supported by the Fisheries Agency of Japan. These studies were also supported in part by a Grant-in-Aid for Scientific Research (23658163) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. M. N. is supported by JSPS Research Fellowship for Young
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- 1
These authors contributed equally to this work.
- 2
Present address: National Research Institute of Aquaculture, Fisheries Research Agency, Minamiise, Watarai, Mie 516-0193, Japan.