Changes in brain and pituitary GnRH levels during ovarian maturation in wild female Japanese flounder

To elucidate the role of gonadotropin-releasing hormone (GnRH) in gonadal maturation in wild female Japanese flounder Paralichthys olivaceus, we monitored changes in the levels of seabream GnRH (sbGnRH) in the olfactory bulb, telencephalon, hypothalamus, and pituitary during ovarian development together with changes in plasma levels of testosterone (T), estradiol-17β (E₂), and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP). Fish were caught offshore of the northern mainland of Japan in the Pacific Ocean at 3- to 4-week intervals between April and September by gill net. The netted fish were categorized into six groups based on ovarian stages: previtellogenic (April–early May), early yolk (April–late May), late yolk (late May–June), early spawning (June–August), late spawning (September), and termination (September) stages. The gonadosomatic index significantly increased from the previtellogenic to early spawning stages and decreased thereafter. In the olfactory bulb, no significant differences were observed in sbGnRH levels among the developmental stages. In contrast, sbGnRH levels in the telencephalon and hypothalamus were very high in the previtellogenic stage, lower in the early spawning stage, and relatively high in latter stages. sbGnRH levels in the pituitary were high in the previtellogenic stage and low in the early spawning stage. In addition, the relatively high levels of pituitary sbGnRH were found together with high plasma T, E₂, and DHP levels in fish in the late yolk stage. These results indicate that sbGnRH in the telencephalon, hypothalamus, and pituitary is involved in ovarian maturation and that sbGnRH may play an important role in the initiation of ovarian recrudescence in wild Japanese flounder.     

Thyroid hormone deficiency in abnormal larvae of the Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   In large-scale rearing of juveniles of the Japanese flounder Paralichthys olivaceus, a certain morphological abnormality occurred spontaneously in 4% of the fish. These fish showed a slight but clearly different appearance from any developmental stage of this species, and did not settle when all the other juveniles in the same tank completed metamorphosis and had settled. From comparisons of external and internal structures between the normal and the abnormal fish, the abnormality was attributed to unbalanced progress of metamorphosis, mainly due to metamorphic stasis. The thyroid of the abnormal fish was apparently activated morphologically. In addition, serum thyroxine (T4) concentrations in the abnormal fish were reduced to less than 1/10 of that of normal fish. After 14 days of T4 treatment (0.1 p.p.m) of the abnormal fish, all the abnormal characteristics disappeared, and the fish recovered to normal, suggesting normal responsiveness to thyroid hormones in peripheral tissues, whereas thiourea treatment (30 p.p.m., 14 days) further delayed metamorphosis. These results suggest that these abnormal fish were suffering from thyroid hormone deficiency, and were unable to secrete a sufficient amount of thyroid hormone to complete metamorphosis.     

石鲽、牙鲆精子冷冻保存研究及其在人工杂交中的应用

对石鲽(Kareius bicoloratus )和牙鲆(Paralichthys olivaceus)精子冷冻保存技术进行了研究,筛选到一个优良的稀释液MPRS,用MPRS冷冻保存石鲽、牙鲆精子,冻后成活率在70%以上。在石鲽冻精和牙鲆卵的杂交实验中,杂交组受精率是 28 4%±4 55(n=3),孵化率是 42 7%±7 35(n=3),胚胎发育正常;5 000尾杂交鱼苗有102尾成功度过变态期。在牙鲆冻精和大菱鲆卵的杂交实验中,杂交组的受精率只有2 7%,部分受精卵虽能孵化出膜,但仔鱼全为畸形。这两个杂交实验,特别是石鲽冻精和牙鲆卵的杂交实验,证实了冷冻保存的精子完全可以应用在杂交育种工作中。     

Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder

ABSTRACT:   To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus , localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic–hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.     

牙鲆减数分裂与有丝分裂雌核发育的遗传差异

利用同一尾牙鲆亲鱼的同一批卵子诱导减数分裂雌核发育二倍体和有丝分裂雌核发育二倍体,同时用牙鲆精子做人工授精制备普通二倍体,作为对照组。利用10对微卫星引物对普通二倍体和两种雌核发育二倍体进行遗传分析。结果表明,母本基因型在8个位点为杂合,2个位点为纯合。普通二倍体有6种基因型,等位基因均来自父母本的随机结合,类型丰富;母本与子代、子代个体之间的遗传相似系数分别为0.452 8和0.560 3,接近随机交配群体的遗传相似度。减数分裂雌核发育二倍体有3种基因型,除在1个位点出现异于母本的纯合基因型外,其他所有位点的基因型与母本完全一致;母本与子代、子代个体之间遗传相似系数分别为0.976 6和0.959 5,接近近交系的遗传相似度。有丝分裂雌核发育二倍体有2种基因型,且全部为纯合型;母本与子代、子代个体之间遗传相似系数分别为0.806 2和0.742 5,有丝分裂雌核发育二倍体全部为纯合个体。减数分裂雌核发育二倍体具有高度的遗传相似性,适于固定母本性状;有丝分裂雌核发育二倍体纯合度高,适于作为制备克隆的亲本;两者具有明显不同的遗传特性,均可作为特征不同的育种材料。     

乳酸菌对牙鲆稚鱼养殖水体和肠道菌群的影响

在牙鲆稚鱼中投喂添加由单一鼠李糖乳杆菌P15制备的微生态制剂和黄霉素,在60d的投喂期内,采用平板计数法检测好氧性异养菌的总数、总弧菌数和乳酸菌的数量。结果表明,在投喂菌液和冻干菌粉后,养殖水体和牙鲆肠道的乳酸菌数均呈上升趋势,在30d后乳酸菌数量达到稳定并在肠道内定植。同时,由于乳酸菌的抑制作用,弧菌的数量下降,以肠道中的弧菌最明显。乳酸菌对养殖水体和牙鲆肠道的好氧性异养菌没任何影响。乳酸菌对养殖水体和肠道菌群的影响与抗生素具有相近的效果,表明乳酸菌作为饲料添加剂可以取代抗生素应用在牙鲆的养殖中。     

Distribution of immunoglobulin-positive cells in the spleen and kidney of Japanese flounder Paralichthys olivaceus

SUMMARY: The distribution of surface immunoglobulin-positive (sIg⁺) cells in the spleen, the head-kidney, and the trunk-kidney of adult Japanese flounder Paralichthys olivaceus was investigated by a flow cytometric analysis using a monoclonal antibody FB17 against flounder immunoglobulin M. Although the percentage of sIg⁺ cells varied among organs, a good correlation was revealed between the percentage in the head-kidney and that in the trunk-kidney ( r = 0.996, P < 0.001). In contrast, no correlation was obtained between the percentage of sIg⁺ cells in the spleen and the head-kidney or trunk-kidney. The head-kidney and the trunk-kidney contained cells bearing fewer sIg that were stained dimly by FB17 in addition to brightly stained sIg⁺ cells regarded as mature B cells, while the spleen contained only sIg⁺ cells stained brightly. Moreover, the immunocytochemical analyses showed that the head-kidney contains plasma cells with strongly stained cytoplasm in addition to B cells with stained cell surface, which suggests that the cells with fewer sIg detected by flow cytometric analysis are presumed to be at a stage during the processes of differentiation to plasma cells. These results suggest that there are some differences between the spleen and kidney in the role of the immune system and it is considered that differentiation of B cells to plasma cells occurs in the kidney.     

cDNA cloning and characterization of two gelatinases from Japanese flounder

SUMMARY: Toughness is one of the most important elements that define the commercial value of the raw meat of fish. Degradation of the extracellular matrix is thought to be a cause of postmortem tenderization of fish meat. A previous study has suggested that this tenderization is caused mainly by metalloproteinases. The present study seeks to identify the proteinase(s) involved in tenderization; hence, cloned cDNA of two gelatinases from Japanese flounder, which showed high homology with mammalian matrix metalloproteinase (MMP)-2 and MMP-9, were designated as jfMMP-2 and jfMMP-9 , respectively. Northern blot analysis revealed that jfMMP-2 mRNA was expressed almost ubiquitously in adult tissues including the brain, muscle, gill, heart, gut, kidney, spleen, testis, and ovary. In contrast, the expression of jfMMP-9 mRNA was observed in those tissues which were abundant in blood cells, such as kidney, spleen, heart, and gill. Both recombinant proteins (jfMMP-2 and jfMMP-9) produced with the COS-7 cell system exhibited gelatin-degrading activity that was sensitive to 1,10-phenanthroline, a typical metalloproteinase inhibitor.     

Effects of photoperiod on gonadotropin-releasing hormone levels in the brain and pituitary of underyearling male barfin flounder

ABSTRACT:   A pleuronectiform fish, the barfin flounder Verasper moseri , expresses three gonadotropin-releasing hormone (GnRH) forms in the brain: salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To clarify the effects of photoperiod on GnRH systems, changes in brain and pituitary GnRH peptide levels were examined using time-resolved fluoroimmunoassays. In experiment 1, 5-month-old male barfin flounder (mean total length 9.0 cm, body weight 11.0 g) were divided into short (8:16 h light : dark [L:D] cycle; lights on 08.00–16.00 hours) and long photoperiod (16:8 h L:D cycle; lights on 04.00–20.00 hours) groups in mid September and maintained until November under natural water temperature (19.3–15.2°C). Brain sGnRH concentrations were significantly higher in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in total length, body weight, plasma testosterone concentration, brain cGnRH-II concentration and pituitary sbGnRH content. In experiment 2, 7-month-old male barfin flounder (mean total length 16.5 cm, body weight 76.8 g) were divided into short and long photoperiod groups in mid December and maintained until February under natural water temperature (12.5–6.6°C). Total length, body weight and condition factor were significantly greater in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in plasma testosterone concentration and GnRH levels in the brain and pituitary. These results indicate that levels of sGnRH in barfin flounder are influenced by photoperiodic treatment dependent on water temperature and/or body size.     

Effects of dietary arginine and lysine levels on growth performance and biochemical parameters of juvenile Japanese flounder Paralichthys olivaceus

ABSTRACT: To investigate the effects of disproportionate levels of dietary arginine and lysine on juvenile Japanese flounder Paralichthys olivaceus , growth performance and biochemical parameters were evaluated by feeding five test diets, comprising different levels of arginine and lysine, to triplicate groups of juveniles (initial bodyweight 1.85 g) for 40 days. Crystalline amino acids were supplemented to test diets to correspond to the amino acid pattern found in the whole body protein of the Japanese flounder, except for arginine and lysine. After the feeding trials, plasma arginine, lysine and urea levels, excreted ammonia-N, and liver arginase activity were analysed. Survival, specific growth rate, feed conversion efficiency, and apparent protein retention were adversely affected ( P  <   0.05) by the deficiency in dietary arginine or lysine concentrations. An excess of either lysine or arginine in the diet did not depress growth when the diets contained adequate levels of either arginine or lysine, proving that there is no evidence for an arginine–lysine antagonism. Results for plasma arginine, lysine and urea levels, excreted ammonia, and liver arginase activity also demonstrated that Japanese flounder juveniles are not sensitive to excess dietary levels of lysine and arginine.     

Solubilization of type I and V collagens in Japanese flounder muscle during chilled storage

ABSTRACT:   The post-mortem changes of type I and V collagens in Japanese flounder muscle during chilled storage were examined. The muscle softened significantly within 12 h under chilled storage. Both type I and V collagens isolated by ion-exchange chromatographies showed no remarkable changes in band patterns even after 24 h of storage, suggesting that degraded collagen molecules may be removed from the collagen fraction by a conventional preparation method. In contrast, type I and V collagen molecules were detected by Western blotting with each specific antibody in a saline extract and gradually increased during chilled storage. These results suggest that both type I and V collagens may participate in the post-mortem softening of fish muscle.