RESUMEN
In vertebrates, gonadotropin-releasing hormone (GnRH) regulates gonadal maturation by stimulating the synthesis and release of pituitary gonadotropins. GnRH has also been identified in invertebrates. Crustacea consists of several classes including Cephalocarida, Remipedia, Branchiopoda (e.g., tadpole shrimp), Hexanauplia (e.g., barnacle) and Malacostraca (e.g., shrimp, crab). In the malacostracan crustaceans, the presence of GnRH has been detected in several species, mainly by immunohistochemistry. In the present study, we examined whether a GnRH-like peptide exists in the brain and/or nerve ganglion of three classes of crustaceans, the tadpole shrimp Triops longicaudatus (Branchiopoda), the barnacle Balanus crenatus (Hexanauplia), and the hermit crab Pagurus filholi (Malacostraca), by immunohistochemistry using a rabbit polyclonal antibody raised against chicken GnRH-II (GnRH2). This antibody was found to recognize the giant freshwater prawn Macrobrachium rosenbergii GnRH (MroGnRH). In the tadpole shrimp, GnRH-like-immunoreactive (ir) cell bodies were located in the circumesophageal connective of the deuterocerebrum, and GnRH-like-ir fibers were detected also in the ventral nerve cord. In the barnacle, GnRH-like-ir cell bodies and fibers were located in the supraesophageal ganglion (brain), the subesophageal ganglion, and the circumesophageal connective. In the hermit crab, GnRH-like-ir cell bodies were detected in the anterior-most part of the supraesophageal ganglion and the subesophageal ganglion. GnRH-like-ir fibers were observed also in the thoracic ganglion and the eyestalk. These results suggest that a GnRH-like peptide exists widely in crustacean species.
Asunto(s)
Crustáceos/anatomía & histología , Crustáceos/metabolismo , Ganglios/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Animales , Inmunohistoquímica , Péptidos/análisisRESUMEN
Euphausia pacifica is a good candidate for a resource of marine n-3 PUFA. However, few reports exist of the lipid and fatty acid composition of E. pacifica. To examine the potential of E. pacifica as a resource of marine n-3 PUFA, we analyzed E. pacifica oil. We extracted lipids from E. pacifica harvested from the Pacific Ocean near Sanriku, Japan. Lipid classes of E. pacifica oil were analyzed by TLC-FID and the fatty acid composition of the oil was analyzed by GC/MS. Free fatty acids and hydroxy-fatty acids were analyzed by LC/QTOFMS. The lipid content of E. pacifica ranged from 1.30% to 3.57%. The ratios of triacylglycerols, phosphatidylcholine, phosphatidylethanolamine and free fatty acids in E. pacifica lipids were 5.3-23.0%, 32.6-53.4%, 8.5-25.4% and 2.5-7.0%, respectively. The content of n-3 PUFA in E. pacifica lipids was 38.6-46.5%. We also showed that E. pacifica contains unusual fatty acids and derivatives: C16-PUFAs (9,12-hexadecadienoic acid, 6,9,12-hexadecatrienoic acid and 6,9,12,15-hexadecatetraenoic acid) and hydroxy-PUFAs (8-HETE and 10-HDoHE). E. pacifica is a good resource of marine n-3 PUFA. Moreover, E. pacifica can provide C16-PUFA and hydroxy-PUFAs.
Asunto(s)
Euphausiacea/química , Lípidos/análisis , Animales , Cromatografía Liquida , Cromatografía en Capa Delgada , Ionización de Llama , Japón , Lípidos/aislamiento & purificación , Espectrometría de Masas , Océano PacíficoRESUMEN
We examined whether a gonadotropin-releasing hormone (GnRH)-like peptide exists in the central nervous system (CNS) of the kuruma prawn, Marsupenaeus japonicus, by reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoassay (TR-FIA) analysis and by immunohistochemistry. The displacement curve obtained for serially diluted extracts of the kuruma prawn brain paralleled the chicken GnRH-II (cGnRH-II) standard curve obtained by cGnRH-II TR-FIA using the anti-cGnRH-II antibody, which cross-reacts not only with cGnRH-II but also with lamprey GnRH-II (lGnRH-II) and octopus GnRH (octGnRH). Extracts of kuruma prawn brains and eyestalks showed a similar retention time to synthetic lGnRH-II and octGnRH in rpHPLC combined with TR-FIA analysis. Using this antibody, we detected GnRH-like-immunoreactive (ir) cell bodies in the anterior-most part of the supraesophageal ganglion (brain), the protocerebrum. Furthermore, GnRH-like-ir fibers were observed in the protocerebrum and deutocerebrum. In the eyestalk, GnRH-like-ir cell bodies were detected in the medulla interna, and GnRH-like-ir fibers were distributed in the medulla interna, medulla externa, and lamina ganglionalis. In the thoracic ganglion, GnRH-like-ir fibers, but not GnRH-like-ir cell bodies, were detected. No GnRH-like-ir cell bodies or fibers were detected in the abdominal ganglion or ovary. Thus, we have shown the existence and distribution of a GnRH-like peptide in the CNS of the kuruma prawn.
Asunto(s)
Sistema Nervioso Central/metabolismo , Crustáceos/anatomía & histología , Crustáceos/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Inmunohistoquímica , AnimalesRESUMEN
The growth rate of fish shows extensive plasticity in response to various environments. Metabolic responses of fish to excessive nutritional shortages such as starvation have been reported, but the effects of moderate nutrient shortage remain unclear. We examined expression levels of some genes related to ATP metabolism and to myogenesis, the RNA/DNA ratio, and the protein/DNA ratio of fish under different feeding conditions: a diet of 212-432% (frequent feeding, FR) or 32-82% (restricted feeding, RE) of initial body weight per week was supplied. The expression levels of nucleoside diphosphate kinase (NDK)-Z2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and myogenin genes of RE fish were higher than those of FR fish, although the RNA/DNA ratio and the protein/DNA ratio were unaffected by the feeding amount. Moreover, expression levels of NDK-Z2 and GAPDH were upregulated to a greater extent than those for myogenin and myostatin 1 under restricted feeding. Together, our results show that gene expression is more sensitive to nutrient conditions of fish than traditional indicators such as the RNA/DNA ratio. The ATP metabolic system is more sensitive to moderate nutrient shortages than the myogenic system.
Asunto(s)
Adenosina Trifosfato/metabolismo , Privación de Alimentos/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Miogenina/genética , Miostatina/genética , Nucleósido-Difosfato Quinasa/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Peso Corporal , ADN/análisis , Ingestión de Energía , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , ARN/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Pez Cebra/crecimiento & desarrolloRESUMEN
In penaeid shrimp species, vitellogenin (VTG) synthesis in the ovary and hepatopancreas is under the inhibitory regulation of a neuroendocrine system, the X-organ/sinus gland complex in the paired eyestalks, and eyestalk ablation (removal of the X-organ/sinus gland complex) is widely used for inducing ovarian development. However, the difference in effects of bilateral and unilateral ablation on VTG gene expression has not been clarified so far. In the present study, VTG synthesis was monitored over a 16-day period after ablation and compared between replicates of immature female kuruma prawns, Marsupenaeus (Penaeus) japonicus, that had been bilaterally or unilaterally ablated and control specimens. After bilateral ablation, ovarian development was induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary increased significantly. Significant VTG mRNA increase was detected 12 h after bilateral ablation. In contrast, after unilateral ablation, ovarian development was not induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary did not change significantly from the control. These results indicate that in immature female prawns, unilateral ablation does not induce VTG gene expression, whereas bilateral ablation induces rapid VTG gene expression (<12 h). The ineffectiveness of unilateral ablation suggests that the remaining X-organ/sinus gland complex in the unilaterally ablated female prawns may secrete sufficient VIH to suppress VTG synthesis.
Asunto(s)
Regulación de la Expresión Génica , Ovario/metabolismo , Penaeidae/metabolismo , ARN Mensajero/metabolismo , Vitelogeninas/biosíntesis , Animales , Glucemia , Proteínas Portadoras/metabolismo , Cartilla de ADN/genética , Glándulas Endocrinas/metabolismo , Femenino , Fluoroinmunoensayo , Hormonas de Invertebrados/metabolismo , Ovario/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vitelogeninas/sangreRESUMEN
In penaeid shrimps, vitellogenin (VTG), the precursor of vitellin, is synthesized in the ovary and hepatopancreas and accumulated in oocytes during ovarian development. In the present study, VTG gene expression levels and hemolymph VTG levels were determined throughout ovarian development in female kuruma prawn, Marsupenaeus japonicus. Hemolymph VTG levels and VTG mRNA levels in the ovary and hepatopancreas were high during vitellogenesis, remained high until final maturation, and then decreased after oviposition. This profile suggests that VTG synthesis activity increases during vitellogenesis and decreases after oviposition. Absence of a significant increase in ovary size in final maturation suggests cessation of yolk accumulation and low activity of VTG synthesis in spite of high VTG mRNA levels. VTG mRNA levels in ovary and hepatopancreas were both highly correlated during vitellogenesis. Thus, their contribution to yolk accumulation seems to be similar. In contrast, VTG mRNA levels in the hepatopancreas increased more slowly at the start of vitellogenesis and declined more sharply after oviposition than in the ovary. This suggests a difference in the regulation of VTG synthesis between the ovary and the hepatopancreas.
Asunto(s)
Hemolinfa/química , Oviposición/fisiología , Penaeidae/genética , Penaeidae/fisiología , Vitelogénesis/genética , Vitelogeninas/genética , Animales , Femenino , Regulación de la Expresión Génica , Ovario/crecimiento & desarrollo , Ovario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vitelogeninas/biosíntesis , Vitelogeninas/metabolismoRESUMEN
For shrimp immune defences, prophenoloxidase (proPO) activating system and antimicrobial peptides in circulating haemocytes play important roles. In the present study, the effects of lipopolysaccharide (LPS) injection on gene expression of penaeidins, crustin, serine proteinase and proPO in haemocytes were determined using real-time reverse transcription-polymerase chain reaction (PCR) in the Pacific white shrimp Litopenaeus vannamei. After injection of LPS, mRNA levels of antimicrobial peptides, penaeidin 2 (PEN2), penaeidin 3 (PEN3), penaeidin 4 (PEN4) and crustin decreased in a dose-dependent manner, while mRNA levels of serine proteinase and proPO did not change significantly. In a time-course experiment, injection of LPS caused significant depression in mRNA levels of PEN2, PEN3, PEN4 and crustin at 4h post-injection, and the depressed mRNA levels returned to initial levels by 72h post-injection. On the other hand, mRNA levels of serine proteinase and proPO did not show a significant change after injection. These results suggest that the antimicrobial peptide system responds to LPS injection at a gene expression level while the proPO system does not respond at a gene expression level.
Asunto(s)
Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Penaeidae/efectos de los fármacos , Penaeidae/fisiología , Animales , Catecol Oxidasa/efectos de los fármacos , Catecol Oxidasa/genética , Cartilla de ADN/química , Relación Dosis-Respuesta a Droga , Precursores Enzimáticos/efectos de los fármacos , Precursores Enzimáticos/genética , Expresión Génica/inmunología , Hemocitos/efectos de los fármacos , Hemocitos/inmunología , Lipopolisacáridos/administración & dosificación , Péptidos/efectos de los fármacos , Péptidos/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/efectos de los fármacos , Serina Endopeptidasas/genética , Factores de TiempoRESUMEN
To investigate the nutritional regulation of lipid metabolism in fish, molecular characterization of lipases was conducted in red sea bream Pagrus major, and the effects of fasting and refeeding on their gene expression was examined. Together with data from a previous study, a total of four lipase genes were identified and characterized as lipoprotein lipase (LPL), hepatic lipase (HL) and pancreatic lipase (PL). These four lipase genes, termed LPL1, LPL2, HL and PL, share a high degree of similarity. LPL1 and LPL2 genes were expressed in various tissues including adipose tissue, gill, heart and hepatopancreas. HL gene was exclusively expressed in hepatopancreas. PL gene expression was detected in hepatopancreas and adipose tissue. Red sea bream LPL1 and LPL2 gene expression levels in hepatopancreas were increased during 48 h of fasting and decreased after refeeding, whereas no significant change in the expression levels of LPL1 and LPL2 was observed in adipose tissue, indicating that LPL1 and LPL2 gene expression is regulated in a tissue-specific manner in response to the nutritional state of fish. HL and PL gene expression was not affected by fasting and refeeding. The results of this study suggested that LPL, HL and PL gene expression is under different regulatory mechanisms in red sea bream with respect to the tissue-specificities and their nutritional regulation.
Asunto(s)
Lipasa/genética , Lipoproteína Lipasa/genética , Hígado/enzimología , Páncreas/enzimología , Dorada/genética , Dorada/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Ayuno , Expresión Génica , Lipasa/química , Lipoproteína Lipasa/química , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.
Asunto(s)
Adipocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Insulina/farmacología , Lipoproteína Lipasa/metabolismo , Dorada/metabolismo , Células del Estroma/efectos de los fármacos , Triyodotironina/farmacología , Vitaminas/farmacología , Animales , Células Cultivadas , Expresión Génica , Grasa Intraabdominal/citologíaRESUMEN
In crustaceans, vitellogenin (VTG, the precursor of major yolk protein) is synthesized in the ovary and/or hepatopancreas, and its synthesis is considered to be under the negative control of the vitellogenesis-inhibiting hormone (VIH), a neuropeptide secreted from the X-organ/sinus gland complex in the eyestalks. In the present study, the effects of pharmacological agents on VTG mRNA levels in incubated ovarian fragments of the kuruma prawn Marsupenaeus japonicus were examined to determine the intracellular signalling pathways for VTG synthesis. After 24 h incubation, A23187 (calcium ionophore), dibutyl-cAMP (cAMP analogue), dibutyl-cGMP (cGMP analogue), forskolin (adenylate cyclase activator), 3-isobutyl-1-methylxanthine (IBMX, phosphodiesterase inhibitor), and phorbol 12-myristate 13-acetate (PMA, protein kinase C activator) decreased VTG mRNA levels in the ovarian fragments. This result suggests that cyclic nucleotides, Ca2+, and protein kinase C are involved in the signalling pathways for the regulation of VTG mRNA levels in the ovaries. Furthermore, the inhibitory effects of sinus gland extract and the pharmacological agents on VTG mRNA were larger in previtellogenic ovaries than in vitellogenic ovaries. This result suggests that the degree of responsiveness to VIH changes during ovarian development and that the changes in responsiveness to VIH involve maturity-related changes in cellular signalling mechanisms in the ovaries.
Asunto(s)
Calcio/farmacología , Nucleótidos Cíclicos/farmacología , Ovario/efectos de los fármacos , Penaeidae/metabolismo , Ésteres del Forbol/farmacología , Vitelogeninas/genética , Vitelogeninas/metabolismo , Animales , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Ionóforos/farmacología , Neuropéptidos/farmacología , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
In penaeid shrimp species, ovarian development is characterized by the accumulation of a major yolk protein (vitellin) and the formation of cortical rods in the oocytes. The process is considered to be under the control of a neuroendocrine organ in the eyestalk (the X-organ sinus gland complex). In the present study, the synthesis of vitellogenin (VTG, precursor of vitellin) and two kinds of cortical rod proteins (cortical rod protein, CRP; thrombospondin, MjTSP) was induced by bilateral eyestalk ablation (removal of the X-organ sinus gland complex) in immature female kuruma prawn, Marsupenaeus japonicus, and the synthesis process was monitored over a 7-day period after the ablation. The ovarian weight and hemolymph VTG levels increased in the ablated females. The VTG mRNA levels in the ovary increased concomitantly with vitellin accumulation in the ovary after eyestalk ablation. On the other hand, the CRP and MjTSP protein levels in the ovary increased after eyestalk ablation, whereas the CRP and MjTSP mRNA levels in the ovary did not change concomitantly. The results suggest that the regulatory mechanism of gene expression by eyestalk hormones is different between VTG (transcriptional control) and CRP-MjTSP (translational control).
Asunto(s)
Vitelogeninas/biosíntesis , Animales , Western Blotting , Peso Corporal , Proteína C-Reactiva/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Inmunoensayo , Oocitos/metabolismo , Ovario/metabolismo , Ovario/patología , Penaeidae , Células Fotorreceptoras de Invertebrados/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trombospondinas/metabolismo , Factores de Tiempo , Transcripción Genética , Vitelinas/metabolismo , VitelogénesisRESUMEN
Recombinant peptides related to vitellogenesis-inhibiting hormone (VIH) of the American lobster Homarus americanus were expressed in bacterial cells, and then purified after being allowed to refold. Biological activities of the recombinant VIHs having an amidated C-terminus (rHoa-VIH-amide) and a free carboxyl-terminus (rHoa-VIH-OH) were examined using an ovarian fragment incubation system derived from the kuruma prawn, Marsupenaeus japonicus. The rHoa-VIH-amide significantly reduced vitellogenin mRNA levels in the ovary, while rHoa-VIH-OH had no effect. This is the first report that describes the production of a crustacean VIH having biological activity and the importance of the C-terminal amidation for its vitellogenesis-inhibiting activity.
Asunto(s)
Proteínas Portadoras/química , Hormonas de Invertebrados/química , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/metabolismo , Femenino , Hormonas de Invertebrados/metabolismo , Masculino , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Nephropidae , Ovario/metabolismo , Plásmidos/metabolismo , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
The mature penaeid oocytes possess cortical rods that contain two related cortical rod proteins (CRP, 28.6 kDa and 30.5 kDa). In the present study, localization of CRP mRNA and gene expression profiles of CRP and vitellogenin (Vg) during ovarian development were examined in kuruma prawn, Marsupenaeus japonicus, an economically important species for shrimp and prawn farming. Northern blot analysis revealed that CRP mRNA was expressed in the ovary. In situ hybridization showed strong signals for CRP transcripts in the oocytes at early developmental stages in both immature and mature ovaries. Quantitative analysis by real-time polymerase chain reaction revealed that CRP mRNA levels were higher in the previtellogenic and endogenous (primary) vitellogenic stages than in more advanced stages. Unlike CRP mRNA, Vg mRNA levels were low in the ovary and hepatopancreas in previtellogenic females. When the ovary developed into the endogenous vitellogenic stage, ovarian Vg mRNA levels increased significantly, followed by rapid decrease in more advanced stages. The Vg mRNA levels in the hepatopancreas, on the other hand, tended to be high in the exogenous (secondary) vitellogenic and maturation stages, in which ovarian Vg mRNA levels were decreased. Our findings indicate that CRP mRNA is highly expressed before the onset of vitellogenesis, suggesting that the transcription, translation, and cortical-rod formation of CRP occur at different phases of oocyte development. The endogenous vitellogenic stage is a crucial stage for the initiation of CRP and Vg syntheses. The coincidence of these protein syntheses suggests that CRP and Vg syntheses are regulated by closely-related mechanisms.
Asunto(s)
Proteínas del Huevo/genética , Penaeidae/metabolismo , ARN Mensajero/metabolismo , Animales , Proteínas del Huevo/biosíntesis , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Oocitos/metabolismo , Ovario/crecimiento & desarrollo , Penaeidae/crecimiento & desarrollo , Vitelogeninas/biosíntesisRESUMEN
In order to determine the function of molt-inhibiting hormone (MIH) in vivo, we examined the effects of injecting of a recombinant MIH on the molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus. The injection of recombinant MIH significantly prolonged the molt interval (9.0 +/-0.4 days in the control group, 9.5+/-0.5 days in the 2500 ng/g-body weight/injection-group, mean+/-SD), and significantly decreased the hemolymph ecdysteroid level (ratio of levels between after and before injection: 1.94+/-1.09 in the control and 1.28+/-0.39 in the 3000 ng/g-body weight/injection-group, mean+/-SD). These results conclusively show the inhibitory effects of MIH on molting in vivo.
Asunto(s)
Ecdisteroides/sangre , Hemolinfa/metabolismo , Hormonas de Invertebrados/farmacología , Muda/efectos de los fármacos , Penaeidae/metabolismo , Proteínas Recombinantes/farmacología , Animales , Hormonas de Invertebrados/metabolismo , Japón , Muda/fisiología , Proteínas Recombinantes/metabolismo , Factores de TiempoRESUMEN
Most pandalid shrimps exhibit protandric hermaphroditism, and detailed information on ovarian development of pandalid species is important for a better understanding of vitellogenesis in crustacean species. In the present study, we characterized ovarian development under light and electron microscopy and examined the hemolymph vitellogenin levels in the coonstriped shrimp, Pandalus hypsinotus under laboratory conditions. To measure vitellogenin levels, a time-resolved fluoroimmunoassay (TR-FIA) was developed after purification of vitellin and production of the anti-vitellin antiserum. The TR-FIA showed wide assay range (0.98-2000 ng/ml), high sensitivity (0.5 ng/ml), and low assay variability (0.9-6.4% of intraassay coefficients, 1.4-5.1% for interassay coefficients). Female P. hypsinotus had non-vitellogenic ovaries in March after the eggs attached to the abdomen hatched, and started yolk accumulation in the ovaries during April-October. During yolk accumulation, yolk globules appeared and increased in the ooplasm. After yolk accumulation, gonadosomatic index (GSI) reached 8.3-8.5 just before oviposition. Females spawned and were ovigerous during June-July of the next year. Hemolymph vitellogenin levels were low (0.006+/-0.008 mg/ml, mean+/-SD) before the yolk accumulation, and became significantly higher (2.66 +/-0.93 mg/ml) during yolk accumulation (GSI, 2-8). Just before oviposition, levels declined to low levels (0.040+/-0.012 mg/ml). Vitellogenin levels were significantly correlated to GSI during the yolk accumulation. The obtained results show that the process of vitellogenesis during the female phase of P. hypsinotus is similar to other crustacean species that do not change sex.
Asunto(s)
Fluoroinmunoensayo/métodos , Ovario/crecimiento & desarrollo , Pandalidae/crecimiento & desarrollo , Vitelogénesis/fisiología , Vitelogeninas/sangre , Animales , Electroforesis en Gel de Poliacrilamida , Femenino , Técnicas Histológicas , Immunoblotting , Inmunohistoquímica , Masculino , Microscopía Electrónica , Ovario/ultraestructuraRESUMEN
In order to determine the primary structure of vitellogenin in a protandric species, the coonstriped shrimp Pandalus hypsinotus, we previously purified four vitellin components (designated as VnA, VnB, VnC, and VnD, respectively), and chemically analyzed their partial amino acid sequences. In this study, we subsequently cloned a cDNA encoding vitellogenin in this species based on the N-terminal and internal amino acid sequences of VnA, as well as the N-terminal sequence of VnC. The open reading frame of this cDNA encoded a pro-vitellogenin in which vitellins were arranged as follows: NH2-VnA-VnB-VnC/D-COOH. The deduced amino acid sequence possessed a single consensus cleavage sequence, R-X-K/R-R, along the lines of vitellogenins reported in other crustaceans and insects, and the N-terminal sequence of VnB was immediately preceded by this sequence. The comparison of primary structures revealed the existence of a basic and characteristic structure for the vitellogenin molecule in decapod crustacean species, and phylogenetic analysis reflected the current taxonomic classifications of Crustacea. An approximately 8 kb-long transcript of the vitellogenin gene was detected in the hepatopancreas of female shrimps having a gonadosomatic index higher than 1.0 by Northern blot analysis, but was not observed in the hepatopancreas and gonads of male shrimps and the hepatopancreas of female shrimps having a gonadosomatic index lower than 1.0. These results indicate that the hepatopancreas is responsible for vitellogenin synthesis.
Asunto(s)
Pandalidae/genética , Filogenia , ARN Mensajero/metabolismo , Vitelogeninas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Análisis por Conglomerados , Cartilla de ADN , ADN Complementario/genética , Femenino , Componentes del Gen , Hepatopáncreas/metabolismo , Japón , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia , Vitelogeninas/metabolismoRESUMEN
The freshwater prawn Macrobrachium rosenbergii shows three male morphotypes: blue-claw males (final stage having high mating activity), orange-claw males (transitional stage showing rapid somatic growth), and small males (primary stage showing sneak copulation). This morphotypic differentiation is considered to be controlled by androgenic gland hormone, which is probably a peptide hormone. However, its physiological roles are not fully understood. In the present study, we examined the correlation of androgenic gland cell structure to spermatogenic activity and morphotypic differentiation histologically in M. rosenbergii. spermatogenic activity showed close correlation to the molt cycle in orange-claw males and small males. spermatogonia increased in number in the late premolt stage, becoming spermatocytes in the postmolt stage, and spermatocytes differentiated into spermatozoa in the intermolt and early premolt stages. Ultrastructure of the androgenic gland was additionally compared among the molt stages, but, distinct histological changes were not observed in relation to spermatogenesis during the molt cycle. On the other hand, among the three morphotypes, the androgenic gland was largest in the blue-claw males, containing developed rough endoplasmic reticulum in the cytoplasm. These results suggest that, during spermatogenesis which is related to the molt cycle, the androgenic gland hormone is at rather constant levels and plays a role in maintaining spermatogenesis rather than directly regulating the onset of a specific spermatogenesis stage and that, during the morphotypic differentiation, the androgenic gland is most active in the blue-claw males and plays a role in regulating the observed high mating activity in M. rosenbergii.