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1.
Zoolog Sci ; 38(5): 451-458, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34664920

RESUMO

Viviparous fish, including white-edged rockfish (Sebastes taczanowskii), accumulate substantial yolk mass in the oocytes; however, the details of the molecular mechanisms underlying yolk formation are not yet fully understood, especially concerning multiplicity in the yolk precursor vitellogenin (Vtg). The present study aimed to reveal the hepatic transcriptional profiles of multiple vtg gene transcripts (vtgAa, vtgAb, vtgC) during the reproductive cycle in captive female white-edged rockfish reared in an aquarium under natural photo-thermal conditions. The serum estradiol-17ß concentration and the hepatic transcript levels of all vtg subtypes increased with the progress of vitellogenesis; both levels decreased at the beginning of oocyte maturation and remained low during the gestation period. Considering the similarity in the transcriptional profiles of vtg subtypes between Sebastes and Oncorhynchus, along with the differences between Sebastes and Morone, it is suggested that the transcription patterns of multiple vtg genes relate to neither their reproductive modes (viviparity versus oviparity) nor to teleost phylogeny.


Assuntos
Fígado/metabolismo , Perciformes/fisiologia , Vitelogeninas/metabolismo , Animais , Estradiol/sangue , Feminino , Ovário/fisiologia , Transcriptoma , Vitelogênese , Vitelogeninas/genética
2.
Fish Shellfish Immunol ; 82: 286-295, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30125707

RESUMO

The complement system plays an important role in immune regulation and acts as the first line of defense against any pathogenic attack. To comprehend the red sea bream (Pagrus major) immune response, three complement genes, namely, pmC1r, pmMASP and pmC3, belonging to the classical, lectin and alternative complement cascade, respectively, were identified and characterized. pmC1r, pmMASP, and pmC3 were comprised of 2535, 3352, and 5735 base mRNA which encodes 732, 1029 and 1677 aa putative proteins, respectively. Phylogenetically, all the three studied genes clustered with their corresponding homologous clade. Tissue distribution and cellular localization data demonstrated a very high prevalence of all the three genes in the liver. Both bacterial and viral infection resulted in significant transcriptional alterations in all three genes in the liver with respect to their vehicle control counterparts. Specifically, bacterial challenge affected the pmMASP and pmC3 expression, while the viral infection resulted in pmC1r and pmC3 mRNA activation. Altogether, our data demonstrate the ability of pmC1r, pmMASP and pmC3 in bringing about an immune response against any pathogenic encroachment, and thus activating, not only one, but all the three complement pathways, in red sea bream.


Assuntos
Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Dourada/genética , Dourada/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Iridoviridae/fisiologia , Filogenia
3.
Biol Reprod ; 96(4): 780-799, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28371886

RESUMO

Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical.


Assuntos
Peixes/fisiologia , Óvulo/fisiologia , Espermatozoides/fisiologia , Animais , Fertilização/fisiologia , Masculino , Inibidores de Serina Proteinase/farmacologia , Especificidade da Espécie , Capacitação Espermática/efeitos dos fármacos , Interações Espermatozoide-Óvulo
4.
Gen Comp Endocrinol ; 221: 9-15, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25660470

RESUMO

Fish egg yolk is largely derived from vitellogenins, which are synthesized in the liver, taken up from the maternal circulation by growing oocytes via receptor-mediated endocytosis and enzymatically processed into yolk proteins that are stored in the ooplasm. Lipid droplets are another major component of fish egg yolk, and these are mainly composed of neutral lipids that may originate from maternal plasma lipoproteins. This review aims to briefly summarize our current understanding of the molecular mechanisms underlying yolk formation in fishes. A hypothetical model of oocyte growth is proposed based on recent advances in our knowledge of fish yolk formation.


Assuntos
Proteínas do Ovo/metabolismo , Gema de Ovo/metabolismo , Peixes/metabolismo , Gotículas Lipídicas/metabolismo , Ovário/metabolismo , Vitelogeninas/metabolismo , Animais , Feminino
5.
Artigo em Inglês | MEDLINE | ID: mdl-26188170

RESUMO

Dietary compromises, especially food restrictions, possess species-specific effects on the health status and infection control in several organisms, including fish. To understand the starvation-mediated physiological responses in Edwardsiella tarda infected red sea bream, especially in the liver, we performed a 20-day starvation experiment using 4 treatment (2 fed and 2 starved) groups, namely, fed-placebo, starved-placebo, fed-infected, and starved-infected, wherein bacterial exposure was done on the 11th day. In the present study, the starved groups showed reduced hepatosomatic index and drastic depletion in glycogen storage and vacuole formation. The fed-infected fish showed significant (P<0.05) increase in catalase and superoxide dismutase activity in relation to its starved equivalent. Significant (P<0.05) alteration in glucose and energy metabolism, as evident from hexokinase and glucose-6-phosphate dehydrogenase activity, was recorded in the starved groups. Interestingly, coinciding with the liver histology, PPAR (peroxisome proliferator activated receptors) α transcription followed a time-dependent activation in starved groups while PPARγ exhibited an opposite pattern. The transcription of hepcidin 1 and transferrin, initially increased in 0dai (days after infection) starved fish but reduced significantly (P<0.05) at later stages. Two-color immunohistochemistry and subsequent cell counting showed significant increase in P63-positive cells at 0dai and 5dai but later reduced slightly at 10dai. Similar results were also obtained in the lysosomal (cathepsin D) and non-lysosomal (ubiquitin) gene transcription level. All together, our data suggest that starvation exerts multidirectional responses, which allows for better physiological adaptations during any infectious period, in red sea bream.


Assuntos
Edwardsiella tarda/crescimento & desenvolvimento , Infecções por Enterobacteriaceae/fisiopatologia , Doenças dos Peixes/fisiopatologia , Fígado/fisiopatologia , Dourada/fisiologia , Inanição , Animais , Catalase/metabolismo , Edwardsiella tarda/fisiologia , Metabolismo Energético , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Alimentos , Expressão Gênica , Glucose/metabolismo , Glicogênio/metabolismo , Interações Hospedeiro-Patógeno , Fígado/metabolismo , Fígado/microbiologia , PPAR alfa/genética , PPAR gama/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dourada/metabolismo , Dourada/microbiologia , Superóxido Dismutase/metabolismo , Vacúolos/metabolismo
6.
Biol Reprod ; 88(2): 47, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23303675

RESUMO

In some animals, such as fish, insects, and cephalopods, the thick egg coat has a narrow canal-a micropyle-through which spermatozoa enter the eggs. In fish, there is no indication that spermatozoa are attracted by eggs from a distance, but once spermatozoa come near the outer opening of the micropyle, they exhibit directed movement toward it, suggesting that a substance exists in this defined region to attract spermatozoa. Since Coomassie Blue (CB) binds preferentially to the micropyle region in flounder, herring, steelhead, and other fish, it probably stains this sperm guidance substance. This substance-a glycoprotein based on lectin staining-is bound tightly to the surface of the chorion, but can be removed readily by protease treatment. Although fertilization in fish (flounder) is possible after removal of this substance, its absence makes fertilization inefficient, as reflected by a drastic reduction in fertilization rate. The sperm "attraction" to the micropyle opening is species specific and is dependent on extracellular Ca(2+). Eggs of some insects, including Drosophila, have distinct micropyle caps with CB affinity, which also may prove to assist sperm entry. Our attempts to fertilize fly eggs in vitro were not successful.


Assuntos
Peixes/fisiologia , Glicoproteínas/fisiologia , Insetos/fisiologia , Oócitos/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologia , Animais , Bombyx , Borboletas , Cálcio/fisiologia , Córion/fisiologia , Drosophila , Feminino , Fertilização in vitro , Linguado , Técnicas In Vitro , Ionomicina/farmacologia , Masculino , Muscidae , Odonatos , Oncorhynchus mykiss , Oócitos/citologia , Oryzias , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos
7.
Fish Physiol Biochem ; 39(2): 373-90, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22918791

RESUMO

Elasmobranchs (sharks and rays) exhibit unique reproductive characteristics and, in contrast to the situation in teleosts, very little is known about the identity, structure and physical characteristics of their egg yolk proteins. The aims of this study were to (1) detect and purify the vitellogenin (Vtg; egg yolk precursor) and yolk proteins (YPs) of the cloudy catshark (Scyliorhinus torazame), (2) examine the relationships between Vtg and YPs and (3) characterize and classify the deduced primary structure of the Vtg transcript (vtg). The apparent molecular weights of purified Vtg and putative Vtg-related YPs (lipovitellin: Lv, phosvitin: Pv) were determined by gel filtration and were ~560, >669 and ~58 kDa, respectively. Following SDS-PAGE, these purified products (i.e., Vtg, Lv and Pv) appeared as bands of ~210, ~110 and ~22 kDa, respectively. On Western blots, antisera against purified Vtg, Lv and Pv recognized the ~210 kDa Vtg band. Catshark Pv, in contrast to teleost Pvs, had a very low serine content. The catshark Vtg cDNA sequence (vtg) appeared to contain an open-reading frame consisting of domains encoding Lv, Pv and ß'-component (ß'-c). A phylogenetic analysis, with a consideration of genome duplication events, placed catshark vtg into the 'vtgAB type.' It is concluded that at least a single major type of Vtg protein, which is transcribed and translated from catshark vtgAB gene, is the precursor of three egg yolk proteins (Lv, Pv and ß'-c) in catshark.


Assuntos
Proteínas do Ovo/genética , Fosvitina/genética , Tubarões/metabolismo , Vitelogeninas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting/veterinária , Cromatografia/veterinária , Cromatografia em Gel/veterinária , Análise por Conglomerados , Biologia Computacional , Primers do DNA/genética , DNA Complementar/genética , Proteínas do Ovo/química , Eletroforese em Gel de Poliacrilamida/veterinária , Dados de Sequência Molecular , Peso Molecular , Fosvitina/química , Filogenia , Análise de Sequência de DNA/veterinária , Vitelogeninas/análise , Vitelogeninas/química
8.
Reprod Biol Endocrinol ; 9: 71, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21615964

RESUMO

To elucidate the molecular mechanisms underling hydration during oocyte maturation, we characterized the structure of Japanese eel (Anguilla japonica) novel-water selective aquaporin 1 (AQP1b) that thought to be involved in oocyte hydration. The aqp1b cDNA encodes a 263 amino acid protein that includes the six potential transmembrane domains and two Asn-Pro-Ala motifs. Reverse transcription-polymerase chain reaction showed transcription of Japanese eel aqp1b in ovary and testis but not in the other tissues. In situ hybridization studies with the eel aqp1b cRNA probe revealed intense eel aqp1b signal in the oocytes at the perinucleolus stage and the signals became faint during the process of oocyte development. Light microscopic immunocytochemical analysis of ovary revealed that the Japanese eel AQP1b was expressed in the cytoplasm around the yolk globules which were located in the peripheral region of oocytes during the primary yolk globule stage; thereafter, the immunoreactivity was observed throughout the cytoplasm of oocyte as vitellogenesis progressed. The immunoreactivity became localized around the large membrane-limited yolk masses which were formed by the fusion of yolk globules during the oocyte maturation phase. These results together indicate that AQP1b, which is synthesized in the oocyte during the process of oocyte growth, is essential for mediating water uptake into eel oocytes.


Assuntos
Aquaporina 1/genética , Aquaporina 1/metabolismo , Enguias , Oócitos/metabolismo , Sequência de Aminoácidos , Animais , Aquicultura , Aquaporina 1/isolamento & purificação , Clonagem Molecular , Enguias/genética , Enguias/metabolismo , Enguias/fisiologia , Feminino , Expressão Gênica , Dados de Sequência Molecular , Oócitos/fisiologia , Oogênese/genética , Oogênese/fisiologia , Filogenia , Homologia de Sequência de Aminoácidos , Distribuição Tecidual
10.
Mar Biotechnol (NY) ; 11(2): 169-87, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18766402

RESUMO

Three complete cDNAs encoding different forms of vitellogenin (Vtg) were isolated from a white perch (Morone americana) liver cDNA library and characterized with respect to immunobiochemical and functional features of the three Vtgs and their product yolk proteins (YPs) in this species and in the congeneric striped bass (Morone saxatilis). The two longest cDNAs encoded Vtgs with a complete suite of yolk protein domains that, based on comparisons with vtg sequences from other species, were categorized as VtgAa and VtgAb using the current nomenclature for multiple teleost Vtgs. The shorter cDNA encoded a Vtg that lacked a phosvitin domain, had a shortened C-terminus, and was categorized as VtgC. Mapping of peptide sequences from the purified Vtgs and their derived YPs to Vtg sequences deduced from the cDNAs definitively identified the white perch VtgAa, VtgAb, and VtgC proteins. Detailed comparisons of the primary structures of each Vtg with partial or complete sequences of Morone yolk proteins or of Vtgs from other fishes revealed conserved and variant structural elements of teleost Vtgs with functional significance, including, as examples, signal peptide cleavage sites, dimerization sites, cathepsin D protease recognition sites, and receptor-binding domains. These comparisons also yielded an interim revision of the classification scheme for multiple teleost Vtgs.


Assuntos
Bass/genética , Peixes/genética , Vitelogeninas/genética , Sequência de Aminoácidos , Animais , Bass/classificação , Sítios de Ligação , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Sinais de Poliadenilação na Ponta 3' do RNA/genética , Alinhamento de Sequência , Homologia de Sequência , Vitelogeninas/química , Fator de von Willebrand/genética
11.
Int J Dev Biol ; 52(5-6): 753-7, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18649287

RESUMO

The fish egg is surrounded by a thick envelope called the chorion. The fertilizing spermatozoon enters the egg through a canal-like structure in the chorion, the micropyle. Examination of micropyle at fertilization is difficult if eggs are large and have no distinct landmarks surrounding the micropyle, or if they are positively buoyant in water. Eggs of many commercially important fishes (e.g., flounder, sea bream and eel) are buoyant in water or only slightly adhere to solid objects (e.g., sands, rock and water plants), which makes observation of spermatozoa at fertilization difficult. Here, we report that such eggs can be firmly attached to plastic and glass dishes that have been previously coated with poly-L-lysine. These adhering eggs can be fertilized and develop normally on the dishes. Observations of micropyles of three fish species, before and after sperm entry are presented.


Assuntos
Fertilização , Polilisina/farmacologia , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/fisiologia , Membrana Vitelina/metabolismo , Animais , Biologia do Desenvolvimento/instrumentação , Biologia do Desenvolvimento/métodos , Feminino , Peixes , Masculino , Modelos Biológicos , Peso Molecular , Oócitos/metabolismo , Capacitação Espermática , Motilidade dos Espermatozoides
12.
Int J Dev Biol ; 52(5-6): 743-52, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18649286

RESUMO

Sperm of the Pacific herring are immotile at spawning. Two egg-derived molecules are capable of initiating sperm motility. One is herring sperm activating protein(s) (HSAPs) and the other is sperm motility initiation factor (SMIF). These two motility initiators differ in their location and association with the chorion, and in their isoelectric points and molecular weights. In this study we have investigated the roles of these two inducers with respect to motility and fertilization. Using computer analysis of sperm motility, we found that HSAPs, as well as the C-terminal HSAPs peptide, elicit a linear motility pattern, while SMIF induced a highly circular and asymmetric pattern. HSAPs induced a two-fold increase in intracellular calcium, whereas SMIF induced a four-fold increase of motility initiation. SMIF-exposed sperm, preloaded with BAPTA-AM, showed a more linear motility and this motility trajectory decreased with their fertilizing capability. The difference in intracellular calcium levels between HSAPs and SMIF is consistent with the observed linear and circular motility. In the absence of SMIF, HSAPs do not support fertilization. Fertilization is rescued in these experiments if SMIF is reintroduced. We propose that diffusible HSAPs are not essential for fertilization, but enhance sperm-egg collisions via linear motility. SMIF, which is bound to the micropylar region of the chorion, is required for fertilization and induces circular motility that is a prerequisite for sperm to enter the micropylar canal and fertilize the egg.


Assuntos
Cálcio/metabolismo , Córion/metabolismo , Fertilização , Motilidade dos Espermatozoides , Animais , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Feminino , Peixes , Masculino , Modelos Biológicos , Óvulo/metabolismo , Capacitação Espermática , Transporte Espermático , Interações Espermatozoide-Óvulo
13.
Methods Mol Biol ; 1874: 475-487, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30353531

RESUMO

Microinjection is a powerful tool for studying embryonic development and analyzing gene functions in fish. This technique was first applied to model species of fish such as zebrafish and medaka whose egg chorions could be removed or softened before microinjection. Recent progress in genome editing using TALEN and CRISPR has opened the opportunity to analyze gene functions in a much wider range of fish including those important to marine aquaculture. Therefore, application of the microinjection technique is also required in these species. However, the characteristics of fish eggs vary widely between species and several technical difficulties need to be overcome in order to use microinjection in a wider range of species. To obtain consistent results with microinjection, an optimal method has to be developed for each target species. In this chapter, we describe the physical characteristics of the eggs of fish species that have been used in microinjection experiments in our laboratory and detail the microinjection system we developed for fish eggs with a hard chorion, such as those of marine species.


Assuntos
Peixes/embriologia , Microinjeções/métodos , Óvulo/metabolismo , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/crescimento & desenvolvimento , Desenvolvimento Embrionário , Feminino , Edição de Genes
14.
Stem Cell Reports ; 13(2): 419-433, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31412286

RESUMO

In vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor ß2 (ERß2) during embryogenesis of medaka, and here we investigated the functional importance of ERß2 in female gonad development and maintenance using a transgenerational ERß2-knockdown (ERß2-KD) line and ERß2-null mutants. We found that ERß2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERß2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERß2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.


Assuntos
Receptor beta de Estrogênio/metabolismo , Proteínas de Peixes/metabolismo , Gônadas/crescimento & desenvolvimento , Diferenciação Sexual , Animais , Cálcio/metabolismo , Proliferação de Células , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Embrião não Mamífero/metabolismo , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Feminino , Proteínas de Peixes/antagonistas & inibidores , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Células Germinativas/metabolismo , Gônadas/metabolismo , Masculino , Oryzias/crescimento & desenvolvimento , Oryzias/metabolismo , RNA Antissenso/genética , RNA Antissenso/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo
15.
Sci Rep ; 9(1): 13871, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554877

RESUMO

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Atum/fisiologia , Animais , Aquicultura/métodos , Feminino , Regulação da Expressão Gênica , Larva , Masculino , Mutagênese Sítio-Dirigida , Platina , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Natação/fisiologia , Atum/genética
16.
Mol Reprod Dev ; 75(8): 1307-17, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18324616

RESUMO

Disparate proteolytic patterns of yolk proteins, derived from three types of vitellogenin (VgA, VgB, and VgC), were observed in gray mullet. Immuno-biochemical analyses of extracts obtained from vitellogenic ovaries (VO) and ovulated eggs (OE) confirmed that a large proportion of VgA-derived lipovitellin (LvA) was degraded into free amino acids (FAAs) during ovarian follicle maturation. The maturation-associated alteration of VgB-derived Lv (LvB) involved only limited proteolysis; the heavy and light LvB chains were dissociated into at least three and one polypeptide fragments, respectively. The native mass of VgC-derived Lv (LvC) exhibited little difference between VO and OE, although it was apparent that the LvC was 'nicked' during maturation, resulting in the appearance of several bands in OE. Similar analyses confirmed that VgA-derived beta'-component (beta'-cA) and VgB-derived beta'-c (beta'-cB) decreased during maturation in both quantity and native mass, while phosvitin derived from either VgA (PvA) or VgB (PvB) appeared to be degraded into FAAs. The pattern of maturation-associated proteolysis of mullet yolk proteins is similar to that reported for other marine teleosts spawning pelagic eggs. However, the depository ratio of the three distinct types of Lv in the mullet VO appeared to be different from that estimated for another marine pelagophil, the barfin flounder. These results support a recent paradigm regarding the significance of Vg multiplicity upon successive physiological events in this group of fishes including the hydration of maturing oocytes, the acquisition of proper egg buoyancy, and the generation of requisite nutrient stocks for each stage of embryogenesis and larval development.


Assuntos
Proteínas do Ovo/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Peptídeo Hidrolases/metabolismo , Maturidade Sexual/fisiologia , Smegmamorpha/metabolismo , Vitelogeninas/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Proteínas do Ovo/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Imunoeletroforese , Dados de Sequência Molecular , Folículo Ovariano/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Smegmamorpha/fisiologia
17.
Environ Sci ; 14(5): 255-61, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17975537

RESUMO

The male three-spined stickleback (Gasterosteus aculeatus) produces a glue protein named "spiggin" that is used as a cementing substance for building its nest. The synthesis of spiggin is under the control of androgenic stimulation. Therefore, spiggin is an effective biomarker of any androgenic activity displayed by environmental chemicals, similarly to the use of vitellogenin as an estrogenic biomarker. The aim of this study was to establish a quantification system for spiggin mRNA to develop a highly sensitive system for evaluating environmental androgens. In this process, two different types of cDNA encoding spiggin (SPG-1 and SPG-2) were isolated. They closely resemble each other in primary structure and features. In addition, the transcriptions of both spiggin gene were induced by only androgenic stimulation in a receptor-mediated manner. These findings suggest the multiplicity albeit specificity of spiggin in the stickleback. The quantification system for spiggin mRNA was established using a real-time RT-PCR technique. This system enables accurate quantification within a wide range of spiggin mRNA from 10(1) to 10(6) copies.


Assuntos
Androgênios/metabolismo , Disruptores Endócrinos/análise , Disruptores Endócrinos/toxicidade , Smegmamorpha/metabolismo , Sequência de Aminoácidos , Androgênios/agonistas , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Feminino , Proteínas de Peixes/química , Proteínas de Peixes/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Rim , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/genética , Padrões de Referência , Homologia de Sequência de Aminoácidos , Caracteres Sexuais , Smegmamorpha/genética
18.
Artigo em Inglês | MEDLINE | ID: mdl-28163184

RESUMO

Dietary regime modifications have been an integral part of health and healing practices throughout the animal kingdom. Thus, to assess the effects of periodic starvation and refeeding schedule on the physiological and immunological perturbations in Edwardsiella tarda infected red sea bream, we conducted a 20day experiment using 4 treatment groups, namely, pre-fed placebo (PFP); pre-starved placebo (PSP); pre-fed infected (PFI); and pre-starved infected (PSI), wherein a 5h E. tarda infection was done on the 11th day. In the present investigation, the pre-starved groups showed significant (P<0.05) alterations in the liver Hexokinase and Glucose-6-phosphatase activity. The pre-starved fish also exhibited significant (P<0.05) increment in the hepatosomatic index, along with increased hepatic glycogen content, in a time dependent fashion. The PPAR (peroxisome proliferator activated receptors)α transcription in the pre-starved group decreased significantly (P<0.05) by 10dai, while the PPARγ showcased a reverse pattern. The transcription of Hepcidin1 and Transferrin (iron homeostasis related genes), and Cathepsin D and Ubiquitin (programmed cell death related genes) portrayed a time responsive decrease and increase in PSI and PFI groups, respectively. Additionally, in comparison to the PFI group, the PSI fish demonstrated substantially reduced oxidative stress level. Fluorescent Immunohistochemistry showed significant (P<0.05) increase in p63 positive cells in the 10dai PFI fish in relation to the PSI group. Therefore, these findings provide new insight into the beneficial role of alternating starvation and refeeding schedule, preferably short-term starvation prior to an infection, in order to obtain better capability to battle against E. tarda infection in red sea bream.


Assuntos
Edwardsiella tarda , Infecções por Enterobacteriaceae/veterinária , Métodos de Alimentação/veterinária , Doenças dos Peixes/prevenção & controle , Perciformes , Inanição/veterinária , Animais , Infecções por Enterobacteriaceae/prevenção & controle , Imuno-Histoquímica , Estresse Oxidativo/fisiologia
19.
Sci Rep ; 7(1): 3183, 2017 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-28600501

RESUMO

Environmental stressors, gonadal degenerative diseases and tumour development can significantly alter the oocyte physiology, and species fertility and fitness. To expand the molecular understanding about oocyte degradation, we isolated several spliced variants of Japanese anchovy hatching enzymes (AcHEs; ovastacin homologue) 1 and 2, and analysed their potential in oocyte sustenance. Particularly, AcHE1b, an ovary-specific, steroid-regulated, methylation-dependent, stress-responsive isoform, was neofunctionalized to regulate autophagic oocyte degeneration. AcHE1a and 2 triggered apoptotic degeneration in vitellogenic and mature oocytes, respectively. Progesterone, starvation, and high temperature elevated the total degenerating oocyte population and AcHE1b transcription by hyper-demethylation. Overexpression, knockdown and intracellular zinc ion chelation study confirmed the functional significance of AcHE1b in autophagy induction, possibly to mitigate the stress effects in fish, via ion-homeostasis. Our finding chronicles the importance of AcHEs in stress-influenced apoptosis/autophagy cell fate decision and may prove significant in reproductive failure assessments, gonadal health maintenance and ovarian degenerative disease therapy.


Assuntos
Gônadas/química , Metaloendopeptidases/química , Oócitos/química , Isoformas de Proteínas/genética , Animais , Apoptose , Autofagia , Linhagem da Célula/genética , Fragmentação do DNA , Fertilidade/genética , Gônadas/crescimento & desenvolvimento , Metaloendopeptidases/genética , Oócitos/crescimento & desenvolvimento , Progesterona/genética , Isoformas de Proteínas/química , Proteólise , Vertebrados/crescimento & desenvolvimento
20.
Zoolog Sci ; 23(11): 1021-9, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17189915

RESUMO

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.


Assuntos
Linguado/metabolismo , Oócitos/metabolismo , Fosvitina/química , Fosvitina/metabolismo , Vitelogeninas/metabolismo , Animais , Cálcio/metabolismo , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Isoformas de Proteínas
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