Japanese eel follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): production of biologically active recombinant Fsh and Lh by Drosophila S2 cells and their differential actions on the reproductive biology

Two gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), control gonadal steroidogenesis and gametogenesis in vertebrates, including teleost fish. Here, we report on the production of biologically active recombinant Fsh (rec-Fsh) and Lh (rec-Lh) in Japanese eel using Drosophila S2 cells. The three subunits composing Gths, i.e., glycoprotein hormone, alpha polypeptide (Cga), follicle-stimulating hormone, beta polypeptide (Fshb), and luteinizing hormone, beta polypeptide (Lhb), were at first independently produced and were proven to be glycosylated and secreted as the mature peptides. Each beta subunit, along with its Cga, was simultaneously coexpressed to produce heterodimeric rec-Fsh and rec-Lh that were subsequently highly purified. The biological activity of rec-Gths was demonstrated in various in vitro assays. The rec-Gths differentially activated their receptors, which resulted in an increase in 11-ketotestosterone (11KT) secretion, a differential alteration of gene expression of steroidogenic enzymes in immature testis, and the induction of the complete process of spermatogenesis in vitro. The data strongly suggest that Fsh and Lh differentially play important roles in the reproductive physiology of the Japanese eel. By contrast, these rec-Gths exhibited little activity in the gonad when administered in vivo. This difference between in vitro and in vivo bioactivity is probably due to the qualitative nature of glycosylation in S2 cells, which resulted in degradation of the recombinant protein in vivo. These differences in the carbohydrate moieties need to be elucidated and ameliorated.     

Effect of GnRHa on plasma levels of Fsh and Lh in the female greater amberjack Seriola dumerili

The effects of gonadotropin-releasing hormone agonist (GnRHa) on plasma levels of follicle-stimulating hormone (Fsh) and luteinising hormone (Lh) are reported for female greater amberjack Seriola dumerili with post-vitellogenic ovarian oocytes. Five females were implanted with pellets containing GnRHa (600 μg kg⁻¹ body weight), while five other females were injected with saline. All females implanted with GnRHa-containing pellets ovulated 36–42 h post-implantation. The GnRHa implants elevated Lh, but not Fsh plasma levels within 42 h of GnRHa administration.     

Ontogenic expression profiles of gonadotropins (fshb and lhb) and growth hormone (gh) during sexual differentiation and puberty onset in female zebrafish

In the zebrafish model, the ontogenic expression profiles of all pituitary hormones have been reported except gonadotropins, partly because they are not supposed to be expressed in the embryonic stage. The spatiotemporal expression patterns of gonadotropins, namely follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), in this species therefore remain largely unknown. As the master hormones controlling reproduction, the information on this issue would be valuable for understanding the roles of gonadotropins in early sexual development. Using double-colored fluorescent in situ hybridization (FISH) and real-time quantitative PCR (qPCR), this study was undertaken to analyze the ontogenic expression patterns of FSHbeta (fshb) and LHbeta (lhb) subunits in the zebrafish pituitary, with particular emphasis on the stage of sexual differentiation (～25-30 dpf [days postfertilization]) and puberty onset (～45 dpf). As a control, growth hormone (gh) was also examined throughout the study. The zebrafish were collected at different time points of early development, including 4, 5, 6, 8, 10, 13, 16, 19, 22, 25, 28, 38, 48, and 53 dpf. The head of each fish, including the brain and pituitary, was sampled for double-colored FISH analysis, whereas the body was fixed for histological examination of sex and gonadal developmental stage. Our results showed that the expression of fshb started much earlier than that of lhb, with its mRNA signal detectable (～2-3 cells per pituitary) shortly after hatching (4 dpf). In contrast, lhb expression became detectable much later, at the time of sex differentiation (～25 dpf). In female zebrafish, the first morphological sign for puberty is the first wave of follicle transition from the primary growth to previtellogenic stage, which occurs around 45 dpf and is marked by the appearance of cortical alveoli in the oocytes. Interestingly, the number of lhb-expressing cells was very low (～5-6 cells per pituitary) before this transition but increased dramatically during and after the transition. In contrast, the expression of fshb was abundant before puberty, with only a slight increase in cell number during puberty onset. The increased expression of fshb and lhb at puberty was also supported by real-time qPCR analysis at the single pituitary level. Interestingly, the fshb-expressing cells changed their spatial distribution significantly during puberty, from a predominantly peripheral to a central location. As the control, the expression of gh was abundant throughout prepubertal and pubertal periods. Our results strongly suggest an important role for Lh at the puberty onset of female zebrafish, similar to the situation in mammals, and its expression could be a sign for puberty at the pituitary level. However, the significance of the location change of Fsh cells during this period will be interesting to investigate.     

Presence of prolactin receptors in eel liver and carp kidney and growth hormone receptor in eel liver.

1. 125I-labelled ovine prolactin and bovine growth hormone were used to test for the presence of prolactin and growth hormone receptors in membrane prepared from tissues of the white eel Anguilla japonica, the carp Ctenopharynogodon idellus and the ricefield eel Monopterus albus. 2. High levels of specific 125I-labelled ovine prolactin binding were found in white eel liver membranes and carp kidney membranes. 3. High levels of specific 125I-labelled bovine growth hormone binding were detected in white eel liver membranes. 4. Tissues of the ricefield eel did not bind 125I-labelled ovine prolactin or bovine growth hormone. 5. The results suggest the presence of prolactin receptors in white eel liver and carp kidney membranes and growth hormone receptors in white eel liver membranes.     

Immunocytochemical identification and ontogeny of adenohypophyseal cells in a cave fish,Phreatichthys andruzzii (Cypriniformes: Cyprinidae)

The morphogenesis of the pituitary gland and the chronological appearance of adenohypophyseal cells were investigated for the first time in the Somalian cave fish Phreatichthys andruzzii by immunocytochemistry. The adult adenohypophysis contained: a rostral pars distalis, with prolactin (PRL) cells arranged in follicles and adrenocorticotropic (ACTH) cells, a proximal pars distalis with somatotropic (GH), β‐thyrotropic (TSH), β‐gonadotropic type I (FSH) and type II (LH) cells and a pars intermedia with α‐somatolactin (SL), α‐melanotropic (MSH) and β‐endorphin (END) cells. All regions were deeply penetrated by neurohypophyseal branches. At hatching (24 h post‐fertilization) the pituitary was an oval cell mass, close to the ventral margin of diencephalon. The first immunoreactive cells appeared as follows: PRL at 0·5 days after hatching (dah), GH and SL at 1·5 dah, END at 2 dah, TSH, ACTH and MSH at 2·5 dah, FSH at 28 dah and LH at 90 dah. The neurohypophysis appeared at 5 dah and branched extensively inside the adenohypophysis at 130 dah, but there was no boundary between rostral pars distalis and proximal pars distalis at this stage. The potential indices of prolactin and growth hormone production increased until 28 and 60 dah, respectively. The potential index of growth hormone production correlated positively with total length. Activity of PRL and GH cells, measured as ratio of cell area to nucleus area, was significantly higher in juveniles than in larvae.     

不同生态因子对黄鳝受精卵孵化率的影响

This paper studied the effects of some ecological factors (temperature, pH, hatching method and ammonia) on the embryonic development of ricefield eel. The results indicated that the optimum hatching temperature of ricefield eel was 25～28℃, and no significant difference was found about the effects of pH within the range of pH 5.5～9.5 on the hatching rate of oosperm. Dripping water incubation had a higher hatching rate of oosperm than staticing water incubation, and the hatching rate of oosperm decreased with increasing ammonia content. The present study provided an available suggestion for the all-artificial and half-artificial breeding of ricefield eel.     

Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development

Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.     

Determination of Fsh quantity and bioactivity during sex differentiation and oogenesis in European sea bass

Follicle-stimulating hormone (FSH) is a glycoprotein hormone that plays a key role in the regulation of gonadal functions in vertebrates. The present study reports the monitoring of pituitary and plasma Fsh levels during sex differentiation and oogenesis in European sea bass (Dicentrarchus labrax) using a homologous immunoassay and an in vitro bioassay. Both assays were used complementarily for the first time in a fish species. High levels of Fsh bioactivity in plasma were found during the initial phases of sexual differentiation. Plasma and pituitary Fsh (quantity and bioactivity) levels and biological to immunological (B:I) ratios were higher in females than in males, suggesting sexual dimorphism in the synthesis and potency of Fsh. In females, the B:I ratios in adult were lower than during sex differentiation indicating that Fsh would be less biopotent in the adult stage. Plasma Fsh bioactivity levels increased during vitellogenesis, suggesting that Fsh would be involved in the regulation of the midphases of oogenesis, whereas luteinizing hormone would be responsible for the final events.     

Follicle-stimulating hormone induces spermatogenesis mediated by androgen production in Japanese eel, Anguilla japonica

Follicle-stimulating hormone (FSH) plays important roles in spermatogenesis. However, the biologic activity of FSH can vary in different vertebrate classes, and the definitive function of FSH has not been established. In this study, we investigated the functions of FSH on spermatogenesis using an in vitro culture system for Japanese eel testis. The eel Fsh receptor was expressed in testis tissue during the whole process of spermatogenesis, mainly by Leydig cells that produce steroid hormones and by Sertoli cells surrounding type A spermatogonia and early type B spermatogonia. In an in vitro organ culture, recombinant eel Fsh (r-eFsh) induced complete spermatogenesis from the proliferation of spermatogonia to spermiogenesis during 36 days of culture; also, spermatozoa were observed in the testicular fragments. Spermatogenesis induced by r-eFsh was inhibited by trilostane, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. However, trilostane did not inhibit spermatogenesis induced by 11-ketotestosterone. These results clearly show that the main function of FSH in eel is to induce spermatogenesis via stimulating androgen production.     

黄鳝的繁殖生态学研究

以洞庭湖稻区黄鳝为调查研究对象 ,通过野外调查和室内实验系统研究了黄鳝的繁殖生态。对黄鳝的性腺发育周年变化、繁殖行为、自然产卵过程、产卵条件及繁殖洞穴的构造进行了观察和记录 ,产卵室是黄鳝繁殖洞穴特有的构造 ,繁殖洞穴泥土p H平均值为 6 .73± 1.0 12。亲鳝有护卵习性 ,通过对 30尾守洞亲鳝的性腺观察和切片验证 ,守洞鳝多为雄鳝 (占 6 1.3% ) ,少数为兼性偏雄性 (占 38.7% ) ,守洞鳝 1:0 0～ 2 :0 0全在外面活动 ,守洞鳝体长 L (cm)与体重 W (g)回归方程为 :W=1.2 5×10 - 2 L1 .4 2 (r=0 .76 )。人工模拟条件下 ,泡沫组受精卵的平均孵化率 (85 .2 % )极显著地高于对照组 (2 5 .4 % ) (t=8.18,t0 .0 5=2 .4 5 ) ,仔鱼平均成活率 (6 4 .0 % )显著高于对照组 (14 .0 % ) (t=3.73) ,同时对繁殖季节黄鳝为孵卵而所吐的泡沫的作用进行了分析。另外 ,证实了黄鳝产卵的最适放养密度为 2～ 3尾 / m2。为黄鳝的全人工繁殖和半人工繁殖提供了合理化的建议     

大型多钩槽绦虫与隐藏新棘虫种间关系研究

大型多钩槽绦虫和隐藏新棘虫单独在黄鲜肠道内分布时,其分布益十分相似,相似性系数ｒ＝０．９２２６（Ｐ〈０．０１）,当两种寄生蠕虫在黄鳝中共同感染时,随着大型多钩槽绦虫的生长,其分布位置明显前移而隐藏新棘虫的分布位置则显著后退,生态２位重叠明显下降。两种寄生蠕虫种间正关联显著,它们在宿主人共存并不改变它们的感染速度。     

Ontogeny of the feeding apparatus of hatchery‐reared white margined sole,Dagetichthys marginatus (Soleidae): implications for cultivation

The aim of this study was to describe the ontogeny of the feeding apparatus of hatchery‐reared Dagetichthys marginatus and to discuss development in relation to generalized early development in (flat)fish and the potential influence of reproduction and/or rearing practices used. Fertilized eggs were obtained from hormone‐induced wild females of the white margined sole, D. marginatus. Subsequent larvae were reared until shortly after metamorphosis between 35 and 45 dah (days‐after‐hatching) when mortalities occurred. At 4 dah a small maxilla and two paired branchial arches appeared. Metamorphosis was completed at 22 dah while the left/migrating eye still remained in its initial position at 31 dah. Based on the likely disadvantages for survival it is assumed that the observed development indicates underdevelopment rather than an evolutionary adaptation. The influence of reproduction and rearing practices used on development and mortalities are discussed.     

Differentiation of steroid-producing cells and folliculogenesis in the developing ovary of the Nile tilapia Oreochromis niloticus

Differentiation and development of steroid-producing cells (SPCs) and folliculogenesis during ovarian differentiation in the Nile tilapia Oreochromis niloticus were immunohistochemically and ultrastructurally examined. Clusters of immunopositive cells (IPCs) against antibodies (ABs) of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), and cytochrome P450aromatase (P450arom) only appeared in the area near blood vessels in the fish ovaries at 50-60 days after hatching (dah). Ultrastructural results showed that differentiation and development of SPCs from undifferentiated to maturation occurred in the area near blood vessels, indicating that it would be the original site of SPCs. At 70-80 dah, IPC clusters invaded the interstices among oocytes at the perinucleolar stage from the area near the blood vessels. IPCs increased in number in the interstices among the previtellogenic oocytes, and some clusters began to enclose the outer thecal layer of the previtellogenic oocytes at 90 dah. The process of folliculogenesis was ultrastructurally observed. SPCs enclosed by fibroblastic cells invaded the interstitial areas among oocytes and some reached the surfaces of oocytes. The upper portions of these elongations opened and began to enclose the outer surfaces of developed oocytes to become thecal layer. Later, newly migrated SPCs reach the thecal layer to become thecal cells. These results indicate that steroid-producing thecal cells originate from the SPCs in the area near blood vessels. After thecal layer formation, an immunopositive reaction against P450arom AB, but not against P450scc or 3β-HSD ABs, appeared first in the granulosa cells enclosing the vitellogenic oocytes at 100 dah. At this time, estrogen production in serum levels rapidly increased. Thus, folliculogenesis could be essential for active production of estrogen in the ovary.     

黄鳝饲料蛋白质需求量的研究

为确定黄鳝(Monopterus albus)对饲料蛋白质的需求量, 试验分7组, 每组4个重复。每个重复放养初始体重为(17.1±0.2) g的幼鳝25尾, 分别以鱼粉和酪蛋白为主要蛋白源, 配制蛋白质含量为32.74%(T1)、38.50%(T2)、42.26%(T3)、48.35%(T4)、52.65%(T5)、57.87%(T6)和62.31%(T7)的7种等能不等氮饲料, 养殖期为60d。以生产性能、蛋白质保留率和生理生化指标等综合评价确定蛋白质的需求量。结果: (1)随饲料蛋白质含量升高, 饲料系数呈先降低后升高; 增重率(WGR)、特定生长率(SGR)、蛋白保留率(PRR)与蛋白质效率(PER)先升高后降低; 饲料蛋白质含量57.87%组获得最好的生长性能, 52.65%组蛋白质保留率最高。(2)随饲料蛋白质含量升高, 黄鳝血清中尿素氮(BUN)含量升高, 谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性先降低后保持稳定, 总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)及血糖(GLU)含量降低; 丙二醛(MDA)含量呈现先降低后升高的趋势, 在52.65%时最低。(3)随饲料蛋白质含量升高, 黄鳝肝胰脏、胃和肠蛋白酶活性先显著上升后呈缓慢上升趋势; 肝胰脏脂肪酶活性先显著下降后呈缓慢下降趋势, 胃和肠脂肪酶活性各试验组之间无显著性差异; 胃淀粉酶活性无显著差异, 肝胰脏和肠呈先显著下降后缓慢降低的趋势。综上: 当饲料蛋白质含量低时, 黄鳝会加大摄食量满足蛋白质需求, 在饲料蛋白质含量过高时, 氧化应激增加, 黄鳝通过降低摄食量减少蛋白质摄入。以WGR、PER、SGR及饲料系数(FCR)为评价指标确定幼鳝对饲料蛋白质的适宜需求量为48.51%—52.60%。     

In vitro induction of luteinizing hormone synthesis by estrogen in organ-cultured pituitary glands of the Japanese eel,Anguilla japonica

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) beta protein immunohistochemically. In a basal medium (Leibovitz L-15), LH beta-immunoreactive cells were very scarce, but after addition of estradiol-17beta (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH beta synthesis were dose (1-100 ng/ml)- and time (1.5-7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.     

Immunohistochemically detected ontogeny of prolactin and growth hormone cells in the African catfish Clarias gariepinus

The chronological appearance of selected endocrine cells in the pituitary of African catfish Clarias gariepinus (Burchell, 1822) was studied morphologically, histologically and immunohistochemically by using antisera raised against catfish growth hormone (cgGH) and recombinant tilapia prolactin I (tPRL). cgGH- and tPRL-like immunoreactive cells were visible from day 1 post fertilisation (hatching) throughout the juvenile and the adult stage. From 1 to 90 days after hatching, the larval pituitary is oval in shape with a distinctly shaped rostral pars distalis, proximal pars distalis and pars intermedia. From day 120 onwards allometric growth of the rostral and proximal pars distalis extended the prolactin and growth hormone cells anteriorily and posteriorily, respectively. Size and activity of the prolactin and growth hormone cells, measured by the ratio of cell surface to nuclear surface remained constant until day 40 and showed a growth spurt thereafter. Growth hormone content, measured with a catfish-specific radio-immunoassay from hatching until 60 h post hatching, increased exponentially between 30 and 60 h.     

The effect of different exogenous kisspeptins on sex hormones and reproductive indices of the goldfish (Carassius auratus) broodstock

Despite several studies on fish hormone therapy, finding new candidates may provide more reproductive efficiency in artificial propagation. Kisspeptins, being upstream of the hypothalamic–pituitary-gonadal axis, appear to play a key role in the reproduction process. In the present study, the effect of different variants of kisspeptide, including goldfish (Carassius auratus) kiss1 kisspeptin (Kiss1), human kisspeptin (Hkiss), and their combination (Kiss1 + H), on the reproductive indices of goldfish broodstock in comparison to Ovaprim (a typical synthetic Gnrh hormone) was investigated. Peptides (Kiss1 and Hkiss) were synthesized using a solid-phase synthesis approach. Kiss1 and Hkiss were injected at a dose of 100 μg kg⁻¹ body weight, blood samples were taken 6 h after injection and sex hormones (E2, Dhp, and 11-Kt), gonadotropins (Lh and Fsh), cortisol and reproductive indices (fecundity, fertilization and hatching percentage) were measured. The results showed a significant increase of plasma sex hormones and gonadotropins in fish treated with kisspeptins. In addition, the cortisol and lipoprotein lipase in Kiss1, Hkiss and Kiss1 + H were remarkably increased compared to Ovaprim. In conclusion, kisspeptins could be a more suitable candidate than Ovaprim for accelerating and synchronizing oocyte maturation in the fisheries industry.