RESUMO
For externally fertilizing fishes, interactions between male and female gametes have been shown to have remarkable impacts on sperm performance. Ovarian fluid (OF) and its ability to alter the swimming behavior of fish sperm makes it a determining factor of fertility. With the expansion of channel catfish (Ictalurus punctatus) â × blue catfish (Ictalurus furcatus) â hybrid aquaculture, it is essential to understand the impacts during fertilization and the magnitude such gametic interactions have on sperm performance and subsequent male fertility potential. This study was conducted to address the following: 1) activate blue catfish sperm with/without channel catfish OF to determine impacts on sperm performance and 2) assess if sperm behave differently when activated in the OF from individual females. Sperm (n = 4 males) were activated without OF (control) and with diluted OF from unique females (n = 6), creating 24 experimental crosses. Sperm motility (%), velocity (VCL), and longevity were analyzed using computer assisted sperm analyses software. With OF incorporated in the activation media, sperm velocity was significantly higher than the control at 10, 20, and 30 s post-activation. OF did not have an impact on motility for any females at 10 s and 20 s post-activation but became significantly higher than the control at 30 s. In all cases, OF treatments greatly increased longevity. Male × female interactions were highly significant, such that motility, velocity, and longevity were dependent on specific male-female pairs. This information shows that OF should be incorporated in aquatic media to simulate natural spawning conditions and accurately assess the fluid mechanics of sperm propulsion for each male. Additionally, there are mechanisms that drive gamete interactions that need to be explored further, which may improve selection of male-female pairs for in-vitro fertilization. On a broad scale, our results also help to shed light on the complexities of fertilization and fish reproduction overall, which may have implications for recruitment variability and recovery strategies of threatened and/or endangered freshwater species.
Assuntos
Fertilidade/fisiologia , Ictaluridae/fisiologia , Ovário/fisiologia , Reprodução/fisiologia , Espermatozoides/fisiologia , Animais , Aquicultura/métodos , Sobrevivência Celular/fisiologia , Líquido Extracelular/fisiologia , Feminino , Masculino , Motilidade dos Espermatozoides/fisiologia , Interações Espermatozoide-Óvulo/fisiologiaRESUMO
The type-II cAMP-dependent protein kinase (A-Kinase) partitions primarily into the particulate fraction in gastric parietal cells. Localization of this kinase to particular subcellular domains is mediated through the binding of the regulatory subunit (RII) dimer to A-Kinase-anchoring proteins (AKAPs). Using a [32P]RII overlay assay, we have screened a rabbit gastric parietal cell cDNA library and have isolated a single RII-binding protein clone. Sequence analysis revealed an open reading frame coding for 1022 amino acids (AKAP120). Recombinant fragments of the full-length clone were prepared and the RII-binding region mapped to an area between amino acids 489 and 549. This area contained a putative alpha-helical RII-binding region between amino acids 503 and 516. Incubation of [32P]RII with a synthetic peptide of AKAP120-(489-522) completely inhibited the binding of [32P]RII to the recombinant AKAP120 fragments that demonstrated RII binding. In vitro RII-binding affinity studies indicated a high-affinity interaction between AKAP120 and RII with a Kapp between 50 and 120 nM for the three recombinant fragments that bound [32P]RII. RNase-protection analysis revealed that AKAP120 is a widely distributed protein, with the highest levels of mRNA observed in gastric fundus. The presence of this novel high-affinity AKAP in gastric parietal cells suggests that it may regulate RII subcellular sequestration in this cell type.
Assuntos
Proteínas de Transporte , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Parietais Gástricas/metabolismo , Proteínas/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteína Quinase Tipo II Dependente de AMP Cíclico , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Dados de Sequência Molecular , Proteínas/genética , Proteínas/metabolismo , Coelhos , Análise de SequênciaRESUMO
cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas do Citoesqueleto/química , Células Parietais Gástricas/química , Fosfoproteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Citoesqueleto/metabolismo , Dados de Sequência Molecular , Células Parietais Gástricas/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Recent investigations have suggested that subcellular compartmentalization of second messenger responsive enzyme systems may be responsible for specific patterns of cellular activation. The type II cAMP-dependent kinase (A-kinase) is localized to particular subcellular domains through the binding of the regulatory subunit (RII) dimer to A-kinase anchoring proteins (AKAPs). Using a [32P]RII overlay assay, we have investigated the presence of AKAPs throughout the gastrointestinal tract, with specific emphasis focused on the gastric parietal cell. All gastrointestinal tissues contained at least one detectable AKAP (60 kDa), with five AKAPs (50-140 kDa) in fundic and antral mucosa. Isolated gastric glands contained four AKAPs. Two AKAPs (50 and 78 kDa) were detected in purified parietal cells, with the 78 kDa AKAP (AKAP78) specific to parietal cell enriched populations. RII-binding to all of these AKAPs was abolished by preincubation of [32P]RII with a synthetic peptide representing the RII-binding region of the AKAP, HT-31. AKAP78 was distributed throughout all membrane fractions of subfractionated parietal cells, with the largest amount of RII-binding detected in the light membrane fraction. Identification of A-kinase regulatory subunits by photoaffinity labeling with 8-azido-[32P]cAMP demonstrated that RII segregated into the same parietal cell subfractions as AKAP78. A majority (approximately 60%) of AKAP78 was detected in the Triton X-100-insoluble fraction, suggesting that this protein resides in a cytoskeletal domain. AKAP78 may be involved in localizing the type II A-kinase to specific intracellular locations in the parietal cell.