Induction of spawning in Pacific white shrimp Litopenaeus vannamei (Boone, 1931) by injection of its molt inhibiting hormone isoform II produced in E. coli

The optimization of reproductive parameters in shrimp farming continues to be a challenge for most producing countries. Although the crustacean neuropeptides have been studied extensively in the last two decades, the functions of most of these neuropeptides remained putative. Among them, molt‐inhibiting hormone isoform II (MIH II) has shown an important role in vitellogenesis. In this study, the cDNA encoding mature MIH II peptide was isolated by RT‐PCR from the L. vannamei eyestalk. The cDNA was cloned into pET28a bacterial expression vector. Recombinant MIH II was obtained in the form of insoluble inclusion bodies and purified to ~88% purity. Two doses of rMIH II and a negative control group were assayed in vivo. The stages of ovarian maturation and spawning were recorded during 72 hr post‐injection. The results showed that ovarian maturation occurred approximately in 9% and 33% of females injected with rMIH II at the doses of 300 and 600 ng/gbw respectively. Neither maturation nor spawning was detected in the negative control group. Females injected with 600 ng/gbw, which showed vitellogenic stages III and IV, spawned. These preliminary results argue that the hormone rMIH II could be a promising candidate to induce spawning in L. vannamei shrimp.     

Reproduction phase-related variations in the GnRH immunoreactive fibers in the pineal of the Indian major carp Cirrhinus mrigala (Ham.)

We studied GnRH immunoreactivity in the pineal gland of the Indian major carp Cirrhinus mrigala during different phases of reproductive cycle. In the resting phase (December–January), GnRH immunoreactive (-ir) fibers were organized as paired fascicles above the posterior commissure that ascend in the stalk and distribute widely in the pineal gland. The GnRH-ir fiber density significantly declined (P<0.001) during the preparatory phase (February–April) and the fibers disappeared thereafter. While no GnRH fibers were seen during the prespawning (May–June) and spawning (July–August), isolated GnRH-ir fibers reappeared in the postspawning phase. Since no GnRH cell bodies were detected in the pineal, these GnRH-ir fibers seem to be of central origin. The results reveal a distinct reciprocal relationship between the GnRH immunoreactivity in the pineal and the status of the ovarian maturity; the fibers appeared in the pineal only during the period of ovarian quiescence. While the functional significance of these cyclic changes in GnRH is yet to be determined, we suggest that the decapeptide may serve as a transmitter of central origin that modulates the activity of the pineal gland.     

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.     

Distribution of three GnRHs in the brain and pituitary of the wild Japanese flounder Paralichthys olivaceus

ABSTRACT:   Wild adult maturing and immature female Japanese flounder Paralichthys olivaceus were collected in June 2004 and January 2005, respectively, to clarify a possible role of gonadotropin-releasing hormones (GnRHs) in reproduction. Levels of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and sea bream GnRH (sbGnRH) in the brain and pituitary were examined by time-resolved fluoroimmunoassay. Three forms of GnRHs were detected in the discrete brain at various levels. In the pituitary of both maturing and immature fish, sbGnRH was abundant together with a pronounced amount of sGnRH, whereas cGnRH-II was almost below the detectable limit. In maturing fish, levels of sbGnRH were high in the telencephalon, hypothalamus and pituitary, while levels of sbGnRH of immature fish were very low in these regions. These results indicate that sbGnRH is mainly responsible for gonadotropin secretion, and that sbGnRH in the anterior part of the brain is associated with gonadal maturation in the Japanese flounder.     

Control of Reproduction of the Shrimp Penaeus kerathurus Held in Captivity

The control of the reproductive cycle of the shrimp Penueus kerathurus , held in captivity from early juvenile stages without eyestalk ablation, was attempted in two sets of experimental conditions during two successive years. In the first set of experiments, light regime, sex ratio and presence of sand substratum were assessed in relation to ovarian maturation and successful mating. Examination of thelyca obtained from cast exoskeletons showed that 40–82% successful mating had taken place. A sex ratio of 2:l female/male gave a higher percentage of fertilized females than a ratio of 1:l. The induction to full ovarian development was observed for every treatment except the tank without sand substrate, where no mated females were observed.
In the second set of experiments, the role of diet, in particular the role played by a polychaete Nereis diversicolor , was assessed as a nutritive stimulator to induce maturation and spawning. With the same purpose, a parallel experiment in smaller aquaria was also conducted to assess the relative importance of mussel, squid, ragworm and crab and four composite diets consisting of two or three of the said food organisms. Nereis worm seemed to have a determinant role on the induction of shrimp ovarian maturation and spawning. The possible role of its fatty acids on shrimp reproduction is discussed. The experimental results suggest the possibility for extended control of the reproductive cycle of P. kerathurus in a controlled environment provided that proper food is available.     

半滑舌鳎(Cynoglossus semilaevis) gnrh2基因克隆、组织分布及卵巢成熟过程中表达分析

为了研究下丘脑神经肽促性腺激素释放激素(Gonadotropin-releasing hormone 2,GnRH2)在半滑舌鳎(Cynoglossus semilaevis)卵巢成熟过程中的生理作用,本研究通过RT-PCR及RACE方法获得了半滑舌鳎GnRH2全长cDNA序列;通过实时荧光定量PCR(qPCR)对gnrh2 mRNA的组织分布以及卵巢成熟过程中的时空表达特性进行了分析.结果显示,半滑舌鳎GnRH2全长cDNA序列为538 bp(不包括polyA尾),其中,5'非编码区(Untranslated region,UTR)为154 bp,3'UTR为126 bp,开放阅读框(Open reading frame,ORF)为258 bp,编码85个氨基酸的前体多肽,其分子量及等电点分别为9.69 kDa和8.55.GnRH2前体多肽由信号肽、GnRH2十肽、酶切位点(GKR)以及GnRH相关肽共4部分组成.序列比对分析发现,GnRH2在鱼类中同源性极高,尤其是十肽(QHWSHGWYPG)在所有硬骨鱼类中完全相同.半滑舌鳎GnRH2与鲈形目同源性最高(89.41％-90.5 9％),其次为鲽形目、鲑形目和鲍形目(78.82％-85.88％),与鲤形目同源性最低(61.18％-71.76％).gnrh2 mRNA主要在脑中表达,在垂体及其他外周组织中表达量极低.此外,组织学分析显示,半滑舌鳎卵巢发育共分为5个时期(Ⅱ、Ⅲ、Ⅳ、Ⅴ和Ⅵ期).在卵巢成熟过程中,脑gnrh2 mRNA表达量在卵黄生成期(Ⅲ期)显著性增加,达到峰值;随后表达量急剧下降,在成熟期(Ⅴ期)达到最小值;在排卵后期(Ⅵ期)又显著性增加.然而,在卵巢成熟过程中,垂体gnrh2 mRNA表达量在卵黄生成后期(Ⅳ期)显著性降低,随后在成熟期(Ⅴ期)有所增加,但在排卵后期(Ⅵ期)又急剧下降.上述研究结果表明,脑GnRH2可能参与了半滑舌鳎卵巢发育过程.     

Gonadotropin-releasing hormones in the African catfish: molecular forms, localization, potency and receptors

Two gonadotropin releasing hormones (GnRHs) were identified in the African catfish: chicken GnRH-II (cGnRH-II) and catfish GnRH (cfGnRH). Immunological screening of HPLC fractions from pituitary extracts indicated a third GnRH which co-eluted with lamprey GnRH-III. However, mass determination and amino acid sequencing identified this material as isotocin. This underlines the risk of identifying multiple forms of GnRH in tissue extracts on the basis of immunoreactivity in HPLC fractions. In vivo and in vitro studies demonstrated that cGnRH-II is an over 100-fold more potent gonadotropin (GTH) secretagogue than cfGnRH. This correlates with the respective receptor affinities. The presence of both GnRHs in the pituitary gland suggests that they may modulate each other's GTH release activity. Sub-threshold or low doses of cGnRH-II partly inhibited cfGnRH-induced GTH II secretion. Conversely, combinations of sub-threshold or low doses of cfGnRH with effective doses of cGnRH-II led to increases in GTH II levels similar to those induced by cGnRH-II alone. Combinations of submaximally effective dose of the 2 peptides resulted in additive effects. Hence, both GnRHs participate in the regulation of GTH II release, and their relative concentrations may determine the overall effect. Immunocytochemistry, using anti-bodies against the respective recombinant GnRH associated peptides (GAPs), as well as in situ hybridization showed that cfGnRH neurones are scattered in the ventral forebrain and project into the pituitary gland, while cGnRH-II neurones are confined to the midbrain tegmentum and without projections to the pituitary gland. Transfection experiments with GnRH receptor cDNA shows ligand activation characteristics similar to those of the native GnRH-R. Autoradiographic studies and hormone release studies indicate that GnRH-Rs in the African catfish pituitary gland are restricted to the gonadotrophs.     

Characterization of three GnRH cDNA sequences in the pejerrey fish Odontesthes bonariensis

In this work we have characterized cDNAs fragments of GnRH preprohormones by 3′RACE. This characterization confirms the presence and expression of three GnRH forms in brain tissue and constitutes an initial step in the study of the physiological role of GnRHs during the sexual differentiation, maturation and reproduction of pejerrey.     

In vitro study of a putative role of gonad‐inhibiting hormone in oocyte growth stimulation in Penaeus monodon

Ovarian development in crustacean is controlled by several factors, among which a neuropeptide gonad‐inhibiting hormone (GIH) is known to inhibit vitellogenin (Vg) synthesis in the ovary. It has been postulated that GIH may control Vg synthesis by inhibiting the release of gonad‐stimulating factor (GSF) from brain and thoracic ganglia. To prove this hypothesis, this study was primarily aimed to investigate the influence of GIH on the release of GSF from thoracic ganglia of Penaeus monodon. Our result showed that GIH did not suppress the release of putative GSF from thoracic ganglia by calcium ionophore A23187 as the induction of oocyte growth in the ovary explants that were cocultured with thoracic ganglia in the presence of A23187 was not affected by the addition of recombinant GIH protein. In addition and interestingly, when the ovary explants were incubated with the recombinant GIH alone, the oocyte growth was increased at the rate comparable to that induced by A23187 in the presence of thoracic ganglia. Hence, our in vitro study demonstrated that the stimulation of GSF released from thoracic ganglia is independent of GIH, and that the GIH has a dual function in oocyte growth stimulation and inhibition of Vg synthesis in the early stage of ovarian development. This expands our knowledge on the regulation of ovarian development in shrimp by GIH. Further in vivo studies in this novel aspect of GIH function will be useful for the improvement of shrimp ovarian maturation in the future.     

Intracellular mechanisms mediating gonadotropin and growth hormone release in the goldfish,Carassius auratus

Evidence for the involvement of Ca²⁺, protein kinase C, cAMP, and arachidonic acid metabolism in mediating gonadotropin (GTH) and growth hormone (GH) release in the goldfish is reviewed. Models for the signal transduction pathways mediating GTH-releasing hormone (GnRH) and dopamine actions on GTH and GH secretion are postulated. A novel hypothesis that two GnRHs which bind to the same receptor type activate different transduction cascade in two different cell types (GTH vs. GH) as well as within the same cell type (GTH) is presented.

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Résumé Cette revue présente les données expérimentales démontrant l'implication de Ca⁺⁺, de la protéine kinase C et du métabolismes de l'acide arachidonique dans les mécanismes régulant la sécrétion des hormones gonadotrope (GTH) et de croissance (GH). Des modèles de signaux de transduction de l'action de la gonadolibérine (GnRH) et de la dopamine sur la sécrétion de GTH et de GH sont proposés. Les deux GnRHs existant chez le poisson rouge pourraient se lier au même type de récepteur et activer différentes voies de transduction dans deux différents types cellulaires (GTH vs. GH) ou dans un seul type (GTH).

     

Positive feedback control by the gonads on gonadotropin (GTH) and gonadoliberin (GnRH) levels in experimentally matured female silver eels,Anguilla anguilla

Treatment of sham-operated female silver eels with carp pituitary extract stimulated ovarian development and induced increases in pituitary gonadotropin (GTH) and gonadoliberin (GnRH) contents. Both effects of carp pituitary extract were abolished in ovariectomized eels, indicating the involvement of the gonads. Endogenous sexual steroids, the secretion of which was increased during sexual maturation, should be responsible for the stimulation of GTH and GnRH levels. Ovariectomy itself had no significant effect on pituitary GTH and GnRH contents, reflecting the fact that, at the silver stage, sexual steroid levels are too low to exert any significant effect on pituitary GTH and GnRH. The positive feedback control exerted by the gonads on GTH and GnRH levels during sexual maturation, in the eel as well as in some other teleosts, would produce an amplification of the pubertal stimulation of the hypothalamo-pituitary-gonadal axis.     

GnRH isoforms expression in relation to the gonadal cycle and to dominance rank in the Gilthead seabream, Sparus aurata

The manner in which behavior influences the gonadotropin-releasing hormone (GnRH) axis in hermaphroditic fishes is not understood. The Gilthead seabream, Sparus aurata, is a protandrous hermaphrodite with a complex gonadal cycle consisting of a quiescent, pre-spawning, spawning, and post-spawning stage. On two separate experiments, I used real-time quantitative PCR to measure the mRNA expression of three GnRH isoforms in homogenized seabream whole-brain extracts. In the first experiment, I measured the levels of GnRH-1, GnRH-2, and GnRH-3 mRNA throughout the gonad cycle. All three GnRH mRNAs increase around the peak of the spawning season (December). GnRH-3 mRNA expression is also elevated in August, which coincides with the beginning of gonad differentiation. All three GnRH mRNAs have the lowest expression levels in the month of September. There was no difference between males and females in the expression levels of any of the three GnRH mRNA. In the second experiment, I measured individual dominance ranks in six groups of fish, three during quiescence and three during spawning. GnRH-1 mRNA expression was positively correlated with dominance rank only during the quiescent period. The more dominant fish tended to have higher GnRH-1 mRNA expression. The existence of a quiescent-only correlation between GnRH-1 mRNA and dominance rank suggests a mechanism by which activation of gonad maturation could occur first in the most dominant ambisexual fish.     

Changes in levels of GnRH in the brain and pituitary and GTH in the pituitary in male masu salmon,Oncorhynchus masou,from hatching to maturation

Levels of two types of gonadotropin-releasing hormone (salmon GnRH and chicken GnRH-II) in the brain and pituitary, and content of gonadotropin (GTHIβ and IIβ) in the pituitary were measured in male masu salmon from hatching to gonadal maturation for three years in order to clarify the involvement of GnRHs in precocious maturation. Underyearling precocious males were distinguishable in summer of year 1 and were marked by an increased GSI. Spermiation was observed among these individuals thereafter every autumn. Pituitary GTHIβ content in both precocious and immature males, and GTHIIβ content in precocious males showed seasonal fluctuations — high in autumn and low in winter. Pituitary GTHIIβ content was low in immature males. Pituitary sGnRH content in precocious males increased from spring to autumn during the three-year period. sGnRH concentrations in discrete brain areas showed seasonal changes — high during autumn to winter and low in summer. Concentrations in the olfactory bulbs and hypothalamus increased significantly in association with testicular maturation during year 3. sGnRH concentrations in the hypothalamus were significantly higher in precocious males than in immature males; this was possibly due to positive feedback of steroid hormones. cGnRH-II was undetectable in the pituitary and no distinct changes were observed in its concentration in the brain in relation to maturation. The phenomenon of underyearling precocious maturation is considered to be triggered before the onset of early summer. It is suggested that males which mature precociously are larger in size and contain much sGnRH in the pituitary before the outward signs of precocity appear; sGnRH may stimulate GTH II synthesis and induce precocious maturation.     

Breeding and life cycle of the ornamental freshwater shrimp Neocaridina davidi in a biofilm‐based culture system

Neocaridina davidi is a popular shrimp in the aquarium industry; however, information regarding its husbandry is scarce. In this study, we investigated the contribution of biofilm to its life cycle, comprising three successive phases: (1) the evaluation of biofilm growth on plastic nets (PN), plastic sheets (PS) and agrovelo (AV); (2) the reproduction of adult shrimp to get juveniles (JI); and (3) the effects of biofilm on the survival and growth performance of JI. Trials were performed in aquaria with zero water exchange and natural environmental conditions. Biofilm was composed mainly of microalgae, diatoms, cyanobacteria and ciliates and used as the sole diet. Survival, biomass and biochemical reserves of JI reared in this culture system were significantly higher in the presence of PN and AV substrates. The occurrence of ovarian maturation and egg incubation of female shrimp in these treatments indicate that biofilm supplied the energy required for somatic growth and fecundity. Harvested females also displayed the size and the red pigmentation associated with premium pricing. Based on these results, it is concluded that N. davidi can complete the life cycle and display characteristic life history traits in a low‐cost biofilm technology system without losing economic value as ornamental species.     

Organization and promoter analysis of a tiger shrimp Penaeus monodon single WAP domain-containing protein gene

The main function of the single whey acidic protein domain (SWD)-containing protein in shrimp is unknown. To elucidate the function of the SWD-containing protein in vivo, the SWD-containing protein gene was isolated and characterized. A 9.3-kb shrimp SWD-containing protein gene, and a 3.9-kb 3′-flanking region. The shrimp SWD-containing protein gene contained three exons and two introns. Different fragments of the shrimp SWD-containing protein 5′-flanking region were transfected into HeLa cells. The promoter activities were assayed by basal human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone (LRH), and gonadotropin-releasing hormone (GnRH) treatments. The in vitro actions of the SWD-containing protein promoter expression pattern were studied by transfection of an SWD-containing protein promoter (1 kb)-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene into the HeLa cell line, which was then microinjected into zebrafish Danio rerio embryos. These results indicate that the shrimp SWD-containing protein promoter might play an important role in gene regulation of sex hormones in mammalian cell lines and in gene regulation of developmental stages in zebrafish.     

Ovarian re‐maturation following the first spawning in the Chinese mitten crab,Eriocheir sinensis (H. Milne‐Edwards)

The present study investigated the external and histological changes in the ovary and measured the gonadosomatic index (GSI) and hepatosomatic index (HSI) during the re‐maturation of the Chinese mitten crab Eriocheir sinensis. The results show that the ovarian re‐maturation cycle of this crab species can be divided into four stages, i.e. Stage I: the ovary had an X shape and appeared milky white or with a mottled buff colour, and the dominant gametocyte at the stage were oogonia (OG, 28.3%) and previtellogenic oocytes (PR, 31.6%); Stage II: the ovary appeared light yellow or tan in colour, dominated by endogenous vitellogeic oocytes (EN, 69.3%). The yolk globules of unreleased mature oocytes (MO) from the previous spawn were absorbed, leaving many vacuoles in the retrogressing MO; Stage III: the ovary appeared crimson red or deep purple, and although the main type of gametocyte was exogenous vitellogenic oocytes (EX), near‐mature oocytes (NO) could also be found in the late phase of the ovarian development stage; Stage IV: the ovary was ripe and filled with MO that contained large yolk globules. During the period of the second ovarian maturation cycle, the GSI increased significantly from the time just after the first spawning (P<0.01). However, although HSI appeared to decrease within days after spawning (DAS), no significant correlation was found between GSI and HSI or between HSI and DAS (P>0.05). Furthermore, the final GSI and the volume of MO of E. sinensis during the second maturation cycle were drastically lower than those of the first maturation cycle.     

Ovarian histology of stunted pond-reared Macrobrachium rosenbergii females

The present study describes the ovarian histology of stunted freshwater prawn Macrobrachium rosenbergii. The ovarian maturation of stunted animals was examined and compared with similar‐aged normal females. Ten animals of the stunted group and each maturation stage of the normal group were sampled from the same pond and had their ovaries removed for histological analysis. Body weight, body length, ovarian weight and gonadosomatic index (GSI) were recorded for each female. The diameters of the different oocyte types were compared among groups through histological assessments. The ovarian histology of stunted M. rosenbergii females indicated that although the somatic growth is severely affected (7.6 g), some energy has been placed on the vitellogensis. Stunted females showed the simultaneous occurrence of previtellogenic, vitellogenic and mature oocytes in their ovarian tissue, but overall oocyte diameter and GSI (1.02%) were significantly affected when compared with normal females.     

Induced Ovarian Maturation of Penaeus vannamei by Implantation of Lobster Ganglion

Induction of ovarian maturation in Penaeus vannamei , by implantation of ganglion prepared from female lobster, Homarus americanus , with developing ovaries was investigated under tank culture conditions. Four of six females with thoracic ganglion implants were maturing while only two of thirteen females of the control groups with abdominal ganglion or no implant matured. Two ripe stage V were found 18 days after implantation of lobster's thoracic ganglion. This indicates that ovarian maturation of P. vannumei in tanks can be induced and accelerated by implantation of thoracic ganglion prepared from maturing females of another species. Ovarian maturation may be induced by a gonad-stimulating hormone, secreted by the thoracic ganglion of maturing females. This gonnd-stimdating hormone is not species specific in activity in the shrimp and lobster.     

Molecular cloning of cDNA of mammalian and chicken II gonadotropin-releasing hormones (mGnRHs and cGnRH-II) in the beluga (<Emphasis Type="Italic">Huso huso</Emphasis>) and the disruptive effect of methylmercury on gene expression

Two gonadotropin-releasing hormone (GnRH) isoforms were identified in the beluga (Huso huso) brain by cDNA sequencing: prepro-mammalian GnRH (mGnRH) and prepro-chicken GnRH-II (cGnRH-II). The nucleotide sequences of the beluga mGnRH and cGnRH-II precursors are 273 and 258 base pairs (bp) long, encoding peptides of 91 and 86 amino acids, respectively. To investigate the effect of methylmercury (MeHg) on GnRH gene expression, animals were fed with four diets containing increasing levels of MeHg (0 mg kg⁻¹ [control]; 0.76 mg kg⁻¹ [low]; 7.8 mg kg⁻¹ [medium]; 16.22 mg kg⁻¹ [high]) for 32 days. The effects of MeHg on brain GnRH mRNA levels were evaluated by real-time PCR. A significant decrease in brain mGnRH and cGnRH-II mRNA levels were detected in fish receiving high dietary MeHg dose compared to controls on day 11 (P < 0.05). On day 18 and 32, all treatment groups had significantly lower brain mGnRH and cGnRH-II mRNA levels compared to the control group (P < 0.05). These findings demonstrate a disruptive role of MeHg on the level of brain mGnRH and cGnRH-II mRNAs in immature beluga.