Incread expression of kisspeptin and GnRH forms in the brain of scombroid
fish during final ovarian maturation and ovulation
Reproductive Biology and Endocrinology 2012,10:64doi:10.1186/1477-7827-10-64
Sethu Selvaraj Dr (lvaraj@agr.kyushu-u.ac.jp)
Hajime Kitano Dr (h-kitano@agr.kyushu-u.ac.jp)
Masafumi Amano Dr (amanoma@kitasato-u.ac.jp)
海洋能Hirofumi Ohga Mr (hohga@agr.kyushu-u.ac.jp)
Michio Yoneda Dr ()
Akihiko Yamaguchi Dr (akihikoy@agr.kyushu-u.ac.jp)
Akio Shimizu Dr (jp)
老师用英语怎么说Michiya Matsuyama Dr ()
ISSN
1477-7827Article type
Rearch Submission date
31May 2012Acceptance date春夏秋冬教案
22August 2012Publication date
27August 2012Article URL /content/10/1/64
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Incread expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation
Sethu Selvaraj1
Email: lvaraj@agr.kyushu-u.ac.jp
Hajime Kitano1
Email: h-kitano@agr.kyushu-u.ac.jp
Masafumi Amano2
Email: amanoma@kitasato-u.ac.jp
Hirofumi Ohga1
Email: hohga@agr.kyushu-u.ac.jp
Michio Yoneda3
Email:
Akihiko Yamaguchi1
Email: akihikoy@agr.kyushu-u.ac.jp
Akio Shimizu3
Email: jp
幼儿园图片大全Michiya Matsuyama1*
* Corresponding author
Email: rinya_m@agr.kyushu-u.ac.jp
一尾鱼
1 Laboratory of Marine Biology, Kyushu University, Fukuoka 812-8581, Japan
沃尔玛商城2 School of Marine Biosciences, Kitasato University, Sagamihara, Kanagawa
252-0373, Japan
3 Fisheries Rearch Agency, Kanazawa, Yokohama 236-8648, Japan
Keywords
Kisspeptins, Kiss, GnRH, Mackerel, Brain, Pituitary, Spawning cycle
Background
In vertebrates, including teleosts, reproductive process are regulated by the preci coordination of neuroendocrine hormones acting through the brain-pituitary-gonad (BPG) axis. A neurohormone, gonadotropin-releasing hormone (GnRH), plays a central role by stimulating the synthesis and relea of the pituitary gonadotropins (GtHs). The pituitary GtHs, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), act on the gonads to stimulate steroidogenesis, which is responsible for progression of ovarian growth and maturation [1,2]. However, in recent years, kisspeptins, a member of the RF-amide family, have been shown to act as an upstream endogenous regulator of GnRH neurons in mammals [3,4]. Recent studies indicate that their role in teleostean fish is also conrved [5,6]. Kisspeptins primarily act at the level of GnRH neurons, which express kisspeptin receptor (GPR54 or Kiss1r) [7,8].
Studies in teleosts have revealed the prence of multiple kisspeptin forms (Kiss1, Kiss2) in the brain [6]. Moreover, teleosts brain express multiple GnRH forms (GnRH1, GnRH2, and GnRH3) wi
陕西臊子面th one or two forms regulating pituitary function [9,10]. The multiplicities have complicated our understanding of their physiological roles in the gonadal growth and maturation in teleosts, especially in females as they exhibit different forms of reproductive dysfunctions when reared in captivity [11].
The experimental scombroid fish model, chub mackerel (Scomber japonicus), is a multiple batch-spawning pelagic fish. It is one of the most commercially important marine fish species in Japan. This species has been targeted for aquaculture in recent years owing to a sharp decline in the wild population, high consumer demand, u in the tuna fishing industry as bait, and high early growth potential [12]. In southwestern Japan, wild-caught fish are being ud for aquaculture production in a pens [13]. However, vitellogenic females fail to undergo final ovarian maturation (FOM) and ovulation in aquaculture conditions [13,14]. Therefore, characterization and understanding of neuroendocrine pathways acting via BPG axis is critical to clarify the reproductive dysfunction in female chub mackerel [15]. Our group already standardized a protocol bad on sustained GnRH analogue delivery system to induce FOM and ovulation in outdoor tanks during natural spawning ason (April-June) [16]. This system allows us to sample fish at different stages of FOM and ovulation.
The chub mackerel brain express kiss1 and kiss2. During the asonal ovarian cycle, kiss2 mRNA levels decrea during vitellogenic and ovarian regression stages [17]. Also, the prence of three GnRH forms, namely GnRH1, GnRH2, and GnRH3 (previously abream GnRH, chicken GnRH-II, and salmon GnRH forms, respectively [18]) in the brain were demonstrated previously [19,20]. An increa in the pituitary peptide levels of GnRH1 was obrved during ovarian growth and regression stages [20], in agreement with our immunocytochemical obrvation of den GnRH1-immunoreactive (ir) fibers localized clo to FSH- and LH-producing cells in the pituitary [19]. In female gilthead abream (Sparus aurata), which also express three GnRH forms as that of chub mackerel, an increa in the levels of all three forms of GnRH-encoding mRNAs in the brain was reported during FOM [21]. In the prent study, to clarify the possible involvement of kisspeptin and GnRH system in the regulation of FOM and ovulation, we analyzed the expression profiles of kiss and gnrh mRNAs in the brain as well as corresponding GnRH peptides in the pituitary of chub mackerel after initial administration of GnRH analogue.
Methods
Fish and tissue sampling
Adult chub mackerel (2-year-old) were caught from the wild using pur ine during autumn 2008 and reared for six months in a pens at a fish farm in the Oita prefecture, Kyushu Island. During the following spawning ason (April-June), fish were transferred to Fishery Rearch Laboratory of Kyushu University and moved into 3-ton outdoor concrete tanks circulated with running awater. The fish were acclimated and reared under natural photoperiod and temperature. Our previous studies indicated that female chub mackerel fail to undergo FOM and ovulation spontaneously in this captive system [13,14]. An induced spawning protocol bad on sustained relea GnRH delivery system was adopted from previous study [16].
After 3 days of acclimation, fish were anaesthetized with 2-phenoxyethanol (100 mg/l) and females with late vitellogenic oocytes (600–650 μm in diameter) were lected by ovarian biopsy using a plastic catheter tube (2 mm internal diameter), as described previously [13,16]. Males oozing milt under gentle abdominal pressure were lected. After lection of required number of females and males, intramuscular injection with the GnRH agonist (D-Ala6, des-Gly10)-LHRH ethylamide (Sigma-Aldrich, St. Louis, USA) at 400 μg/kg body weight were performed on April 30th, May 13th, May 21st, and May 22nd, 2009 to obtain different ovarian stages, namely germinal vesicle migration (GVM), oocyte hydration (HY), ovulation (OV), and post-ovulation (POV), respectively. The sampling times w
ere 13.00, 16.00, 20.00, and 6.00 h of the day, respectively. In all cas, injections were performed at 11.00 h. Sampling times were fixed bad on our previous data on time cour of FOM and ovulation in chub mackerel induced by GnRHa [16]. Fish sampling for the analysis was performed on day 8, bad on previous reports showing the decline in the plasma concentration of GnRH agonist on day 5 after intramuscular injection with the GnRH agonist suspended in coconut oil in Plaice, Pleuronectes platessa[22,23]. The first spawning was obrved 34–36 h post-injection, and subquent daily spawning occurred between 22.00 and 24.00 h. In the following experimental system, daily spawning of chub mackerel is obrved for 20–30 days during the spawning ason, when the water temperature ranged between 18-23°C (Yoneda et al., unpublished obrvations). The late vitellogenic (LV) stage fish were sampled before the start of induced spawning experiment.
Fish ud in the experiment were sacrificed in accordance with the guidelines for animal experiments propod by the Faculty of Agriculture and Graduate Cour at Kyushu University and according to the laws (No. 105) and notifications (No. 6) of the Japane government. The fork length, body, and gonad weights of each individual were measured before tissue sampling. The brain and pituitary of each fish were removed following decapitation, snap-frozen in liquid nitrogen, and stored at −80°C until further analysis. For ovarian histological evaluation, ovary midctions from indi
vidual fish were fixed in Bouin’s solution. To analyze the changes in kiss and gnrh mRNA levels in the whole brain and GnRH peptides in the whole brain and pituitary, two experimental ts of fish samples were ud (Table 1). The brain tissue from the first t was ud for mRNA analysis, and the cond t was ud for GnRH peptide analysis. Male fish were excluded from the analysis.
Table 1Fork length, body weight, and gonadosomatic index of female chub mackerel analyzed in the study period Analys Parameters Ovarian stages
LV GVM HY OV POV
Kiss/GnRH mRNAs Fork length (cm) 33.6 ± 0.4 34.7±0.7 34.6±0.5 33.0±0.4 34.7±0.6
Body weight (g) 523.6 ±24.1 637.9±54.7 692.3±38.1 522.1±13.7 591.5±26.0
GSI (%) 7.3 ± 1.4 7.7±1.6 13.7±2.8 7.0±0.8 6.2±1.1
n 6 6 5 6 6
GnRH peptides Fork length (cm) 33.5 ± 0.5 34.6 ± 0.8 34.9 ± 0.5 33.2 ± 0.4 35.3 ± 1.3
Body weight (g) 521.9 ±24.1 578.6 ± 30.0 659.9 ± 44.0 513.5 ± 19.3 694.7 ± 94.4
GSI (%) 6.7 ± 0.8 4.5 ± 0.66 8.2 ± 1.8 6.9 ± 1.2 8.0 ± 2.7
n 6 5 4 6 4
Values are expresd as the mean ± SEM. LV, late vitellogenesis; GVM, germinal vesicle migration; HY, hydration; OV, ovulation; POV, post-ovulation; GSI, gonadosomatic index (GSI=gonad weight/body weight without gonads x 100).
