Binding characteristics, hormonal regulation and identity of the sperm membrane progestin receptor in Atlantic croaker

Recently a novel cDNA was discovered in spotted seatrout ovaries encoding a protein with seven transmembrane domains that has the characteristics of the membrane progestin receptor (mPR) mediating maturation-inducing steroid (MIS) induction of oocyte maturation in this species. Preliminary results suggested the MIS also activates an mPR on the spermatozoa of spotted seatrout and a closely related species, Atlantic croaker, to stimulate sperm motility. We show here that plasma membranes of croaker sperm demonstrate high affinity (Kd approximately 20 nM), limited capacity (Bmax 0.08 nM), specific and displaceable binding for progestins that is characteristic of mPRs. The MIS (17,20beta,21-trihydroxy-4-pregnen-3-one, 20beta-S) displayed the greatest binding affinity for the sperm mPR among the steroids tested. Treatment of croaker testicular tissue in vitro with gonadotropin caused a several-fold increase in sperm mPR receptor concentrations that was partially blocked in the presence of cyanoketone, which suggests this action of gonadotropin is partially mediated by stimulation of steroidogenesis. Protein bands of the predicted sizes for the mPR and its dimer (40 and 80 kDa) were detected by Western blotting of croaker sperm membranes using a specific antibody to the novel seatrout mPR (mPRalpha). Immunocytochemistry of whole croaker spermatozoa with the mPRalpha antibody showed that staining was primarily localized on the midpiece, consistent with a role of the mPRalpha in mediating MIS stimulation of sperm motility. The results suggest that the mechanism of progestin action on fish sperm involving mPRs is basically similar to that in mammals and has been evolutionarily conserved amongst vertebrates.     

The effect of spawning salinity on eggs of spotted seatrout (Cynoscion nebulosus, Cuvier) from two bays with historically different salinity regimes

Spotted seatrout are capable of spawning in a wide range of salinities. Along the Texas Gulf Coast, bay salinities increase from an average of 14 ppt in Galveston Bay to an average of 40 ppt in Lower Laguna Madre due to the negative gradient of freshwater inflow from north to south. Tagging studies have shown that the majority of spotted seatrout do not migrate between adjacent bay systems. Spawning salinity has been shown to affect many properties of eggs including the diameter and salinity of neutral buoyancy. Spotted seatrout from two historically different salinity regimes (Matagorda Bay (MB) and Upper Laguna Madre (ULM)) were kept in the laboratory and induced to spawn in three salinities: 20, 30, and 40 ppt. The purpose of this study was to evaluate eggs at each of the three salinities and between the two bay systems. Two-way ANOVA showed a significant effect on the egg diameter of bay and spawning salinity, and a significant interaction between bays and spawning salinity. No significant difference in size at hatch was found between spawning salinities or between bays. Hatch rates in spawning salinity were >90% in all cases. Regression of wet weight on spawning salinity was highly significant for both bays. Eggs spawned in 20 ppt have the largest wet weight and eggs spawned in 40 ppt have the smallest wet weight, irrespective of parental bay origin. Percentage of water varied between 92% for 20 ppt spawned eggs and 86% in 40 ppt spawned eggs. Neutral Buoyancy Salinity (NBS) of eggs increased with increasing spawning salinity. Eggs spawned by the Upper Laguna Madre fish held at 20 ppt were not positively buoyant at 20 ppt. The results of this study suggest that spotted seatrout are locally adapted to the prevailing salinity regime within an estuary.     

Food, density, and microhabitat: factors affecting growth and recruitment potential of juvenile saltmarsh fishes

The relationships among microhabitat use, food habits, conspecific density and recent growth rate for estuarine-dependent juvenile spotted seatrout, Cynoscion nebulosus, and red drum, Sciaenops ocellatus, were studied to determine how nursery habitat influences early growth. Juvenile spotted seatrout and red drum were quantified along the marsh-edge ecotone from multiple drop samples, and their immediate environments characterized by a suite of physical and chemical variables along with substrate type and Spartina stem density. Recent daily growth of individual fish was modeled in a series of multiple regression analyses that considered the relative contributions of food, microhabitat, and conspecific density. The spotted seatrout model (p < 0.0001) included four independent variables, otolith radius, prey diversity, salinity, and a salinity-DO interaction term, and explained 67.9% of the variation in daily growth. All variables were significant (p < 0.05), and regression slopes were positive for all variables except salinity. The red drum model (p > 0.0001) included five independent variables, otolith radius, temperature, salinity, depth and substrate, and explained 63.3% of the variation in daily growth. All variables were significant (p > 0.05), and all regression slopes were positive. Fish size (as estimated by otolith radius) accounted for most of the variance in the spotted seatrout (60.2%) and red drum (44.8%) models, while the remaining environmental variables were significant and responsible for 7.7 and 18.5%, respectively. Density variables were not selected for either model, suggesting that density-dependence was not an important influence on recent daily growth. Generally, physico-chemical variables such as temperature, salinity, and dissolved oxygen contributed more to growth than diet or extrinsic factors such as grass stem density.     

Functional characteristics of membrane progestin receptor alpha (mPRalpha) subtypes: a review with new data showing mPRalpha expression in seatrout sperm and its association with sperm motility

A novel cDNA with characteristics of the membrane progestin receptor regulating induction of oocyte maturation by a non-genomic mechanism, named st-mPRalpha, was recently discovered in seatrout. Subsequently, both recombinant and native mPRalphas have been localized to the plasma membrane in several vertebrate models where they have been shown to bind progestins specifically, resulting in activation of G proteins. Non-genomic actions of progestins to stimulate hypermotility and the acrosome reaction in sperm have been identified, but the receptors which mediate these processes are unknown. Here, we demonstrate progestin stimulation of sperm motility in seatrout and expression of st-mPRalpha mRNA and protein in sperm with the receptor localized on the plasma membrane. Immunocytochemical staining of non-permeabilized sperm shows st-mPRalpha is localized to the midpiece with an extracellular N-terminal region, indicating its likely role in progestin regulation of sperm motility. Moreover, the abundance of the st-mPRalpha protein on sperm membranes from seatrout donors with low motility was significantly reduced compared to that of normal motile sperm. Finally, progestin treatment of sperm membranes caused activation of G proteins. These results suggest that st-mPRalpha is an intermediary in progestin stimulation of sperm motility in seatrout by an unknown mechanism involving G protein activation.     

Susceptibility and tolerance of spotted seatrout, Cynoscion nebulosus , and red snapper, Lutjanus campechanus , to experimental infections with Amyloodinium ocellatum

Amyloodinium ocellatum is a parasitic dinoflagellate that infects warm-water marine and estuarine fishes and causes mortalities in aquaculture. Its life cycle consists of 3 stages: a feeding trophont that parasitizes the gills and skin where it interferes with gas exchange, osmoregulation, and tissue integrity; a detached reproductive tomont; and a free-swimming infective dinospore. We compared the susceptibility and tolerance of juvenile spotted seatrout, Cynoscion nebulosus, and red snapper, Lutjanus campechanus, to this parasite by individually exposing fish in 3-L aquaria (at 25 C and 33 practical salinity units) to several dinospore doses over different time periods and quantified the size and number of resulting trophonts. We estimated the trophont detachment rate and trophont size at detachment, the 24-hr dinospore infection rate, the dinospore 48-hr median lethal dose (LD(50)), and the trophont lethal load at the 48-hr LD(50). There were no significant differences in dinospore infection rates or dinospore lethal doses between spotted seatrout and red snapper; however, trophonts remained attached longer and attained a larger size in red snapper than in spotted seatrout. The trophont lethal load was significantly higher in spotted seatrout than in red snapper. A proposed model simulating the trophont dynamics reflected our experimental findings and showed that A. ocellatum reproductive success is linked both to the number of dinospores and the size of the trophont, factors that, in turn, are linked to the time the trophont spends on the host and the number of trophonts the host can tolerate.     

Spawning site selection by spotted seatrout,Cynoscion nebulosus,and black drum,Pogonias cromis,in Louisiana

Synopsis Spawning site selection by spotted seatrout and black drum was studied by locating drumming aggregations through the use of a hydrophone. From March 1987 to October 1990, 315 sound observations were made to identify and characterize spawning seasons and environmental requirements of both species in the Barataria, Caminada, and eastern Timbalier Bay systems of Louisiana. The sounds produced by the spawning aggregations were identified and verified against known recordings. Spawning was verified on several occasions by capturing and rearing zygotes (eggs) into identifiable larvae. Spotted seatrout formed drumming aggregations from late May to early October at salinities ranging from 7.0 to 25.8 ppt and temperatures from 24.5 to 33.5° C. Black drum formed drumming aggregations between January and April in salinities from 10.0 to 27.0 ppt and temperatures from 15.0 to 24.0°C. Large drumming aggregations of spotted seatrout were located from 1800 to 2400h and from 1800 to 2200h for black drum. Spotted seatrout aggregation size was highly correlated with water temperature and for black drum with dissolved oxygen concentrations. Spawning sites for both species were frequently located in deep moving water between barrier islands as well as in channels in open water where water depth ranged from 3 to 50 m. Spawning site selection depended on a particular range of environmental conditions and spawning locations varied seasonally and yearly depending upon hydrological variation.Correspondence to D.M. Baltz     

Habitat-related predation on juvenile wild-caught and hatchery-reared red drum Sciaenops ocellatus (Linnaeus)

We examined the patterns of habitat-specific mortality for newly settled red drum (Sciaenops ocellatus) using an experimental mesocosm approach. Experiments were designed to analyze prey vulnerability and fish rearing-type (wild-caught or hatchery-reared) in estuarine habitats of varying structural complexity including marsh (Spartina alterniflora Loisel), oyster reef (Crassostrea virginica Gmelin), seagrass (Halodule wrightii Aschers), and nonvegetated sand bottom. We used two different predators, pinfish (Lagodon rhomboides Linnaeus) and spotted seatrout (Cynoscion nebulosus Cuvier). For both predators, vulnerability of wild-caught red drum was significantly lower in structurally complex habitats such as seagrass and oyster reef; the highest vulnerability was associated with the nonvegetated bottom. This habitat effect was not apparent for hatchery-reared prey. In trials using a combination of both rearing-types, there was no significant habitat effect on prey selection, but hatchery-reared red drum suffered higher overall mortality than wild-caught fish from pinfish predators. In these trials, spotted seatrout did not select for either prey type. Differences we observed in prey vulnerability were likely caused by behavioral differences between wild-caught and hatchery-reared red drum. Our results reinforce the conclusion that structural complexity in estuarine habitats increases survival of newly settled fishes. Our data also suggest that hatchery-reared red drum may be more vulnerable to predation than natural fishes, and that survival of stocked fish may be enhanced through habitat-related behavior modification.     

Multiple rapid progestin actions and progestin membrane receptor subtypes in fish

Progestin hormones exert rapid, nongenomic actions on a variety of target tissues in fish. The induction of oocyte maturation and the progestin membrane receptor (mPR) that mediates this action of progestins have been well characterized in fishes. Progestins also act on Atlantic croaker spermatozoa via an mPR to rapidly increase sperm motility. Preliminary results indicate that progestins can also exert rapid actions in the preoptic anterior hypothalamus (POAH) in this species to down-regulate gonadotropin-releasing hormone (GnRH) secretion. Recently, we reported the cloning, sequencing and characterization of a novel cDNA in a closely related species, spotted seatrout, that has the characteristics of the mPR involved in the progestin induction of oocyte maturation. Three distinct mPR subtypes, named alpha, beta, and gamma, have been identified in both fishes and mammals. The tissue distribution of the mPRalpha protein in seatrout suggests the alpha-subtype mediates progestin actions on GnRH secretion, sperm motility and oocyte maturation. However, mPRbeta antisense experiments in zebrafish oocytes suggest the beta-subtype also participates in the control of oocyte maturation in zebrafish.     

The effect of parental acclimation to spawning salinity on the survival of larval Cynoscion nebulosus

The yolk and oil depletion of eggs and larvae of spotted seatrout Cynoscion nebulosus , produced by fish collected from two bays with historically different salinity regimes (Matagorda Bay (MB; 18-24%) and Upper Laguna Madre (ULM; 40–50%), Texas, U.S.A. and spawned in salinities of 20, 30 and 40%, differed in their response to both salinity and history. Time to 90% yolk depletion was significantly longer for low salinity bay fish (MB) kept at 20%, but not for high salinity bay fish (ULM) at 20%. The neutral buoyancy salinity of 1 and 2 day old MB 20% larvae was significantly lower than that of MB larvae spawned in 30 or 40%. Overall, eggs and larvae spawned by MB fish were able to hatch out and survive to 3 days post-hatch in lower salinities than those from ULM. Furthermore, the tolerance of eggs and larvae to very low salinities increased with decreasing spawning salinity. The ability of 1–9 day old ULM, but not MB, larvae to survive 18 h exposure to salinities above or below that of spawning exhibited an age-dependent pattern with day 3 being the most sensitive. This study shows that the response of spotted seatrout eggs and larvae to changes in salinity is dependent upon the spawning salinity of the adults and the prevailing salinity regime within the bay.     

Plasma vitellogenin in landlocked Atlantic salmon (Salmo salar Ouananiche): isolation, homologous radioimmunoassay and immunological cross-reactivity with vitellogenin from other teleosts

Vitellogenin was isolated by affinity chromatography and gel filtration from landlocked Atlantic salmon plasma. Vitellogenin was labelled with iodine-131 using iodogen and an homologous radioimmunoassay was developed. There was poor immunological cross-reactivity with vitellogenin or plasma from other teleosts. Parallelism of the vitellogenin standard to the displacement by plasma of vitellogenic salmon allowed the assay to be used to evaluate the seasonal concentration profile of vitellogenin in female adult salmon. Extracts of liver or ovary from female Atlantic salmon also yielded displacements parallel to the vitellogenin standard in the assay.     

Progestin membrane receptors involved in the meiotic maturation of teleost oocytes: a review with some new findings

The endocrine control of oocyte maturation in fish has proven to be a valuable model for investigating rapid, nongenomic steroid actions at the cell surface. Considerable progress has been made over the last decade in identifying and characterizing progestin membrane receptors mediating these actions in fish, in understanding the hormonal regulation and physiological roles of these receptors in oocyte maturation, in elucidating the signal transduction pathways they activate, and in determining their nature. Recent advances on these topics are briefly reviewed. New data demonstrating the involvement of pertussis toxin-sensitive inhibitory G-proteins in induction of oocyte maturation by the maturation-inducing steroid (MIS) in teleosts is also presented. In addition, the cloning strategy to isolate the MIS receptor gene from spotted seatrout ovaries and the characteristics of a novel gene and protein discovered by this approach are discussed. Current evidence suggests this G-protein-coupled receptor-like protein is the long sought after MIS receptor mediating meiotic maturation of teleost oocytes.     

Development and Application of an Effective Detection Method for Fish Plasma Vitellogenin Induced by Environmental Estrogens

Vitellogenin is a protein induced by estrogens, including environmental chemicals with estrogenic activity. To measure the effects of environmental estogens, we developed an effective and rapid one-step method of detecting and purifying fish plasma vitellogenin using a high-performance anion-exchange chromatography column, POROS-HQ. Vitellogenin in a plasma of estradiol-treated male fish (mummichog and red sea bream) was eluted as a single peak with a retention time of 10 minutes from the column, which gives an almost pure preparation as assessed by SDS-PAGE. The lowest detectable amount of vitellogenin was 2 μg per assay. The method was used to analyze the plasma vitellogenin level of aquacultured red sea breams caught in August, when the spawning season is over, and usually no vitellogenin is detected in either females or males, physiologically. However, the data showed that in addition to a few females, some male fish synthesized vitellogenin, suggesting that some chemicals or unknown factors with estrogenic activity have induced fish in the ocean to produce vitellogenin.     

Spatial and temporal changes in the nekton of the Terminos Lagoon, Campeche, Mexico

This study describes the current state of the nektonic communities in Terminos Lagoon in relation to the main environmental factors, and documents variations in number of fish species, dominance and abundance, compared to studies performed in 1980. Information on several environmental factors (temperature, salinity, oxygen, pH and transparency) in the Terminos Lagoon between October 1997 and March 1999 allowed the classification of the lagoon into five zones or subsystems. The hydrological characteristics of these subsystems showed spatial differences compared with studies carried out during the 1980s. During the study period, the number of fish species was 105, compared with 154 species reported in the 1980s. Five fish assemblages were identified and appeared to be associated with the five zones or subsystems. Five fish species considered important based on their abundance in the lagoon during the 1980s declined in numbers: pig fish Orthopristis chrysoptera , Atlantic croaker Micropogonias undulates , striped burrfish Chilomycterus schoepfi , spotted seatrout Cynoscion nebulosus and Gulf toadfish Opsanus beta . Six fish species, previously of minor importance, however, were very abundant: star drum Stellifer lanceolatus , Atlantic anchoveta Cetengraulis edentulus , gafftopsail catfish Bagre marinus , sand seatrout Cynoscion arenarius , striped mojarra Eugerres plumieri and ground croaker Bairdiella ronchus . Among the dominant species, the silver croaker Bairdiella chrysoura and the sea bream Archosargus rhomboidalis experienced significant changes in average abundance and distribution patterns. These changes were probably related to the impact of human activities in the Terminos Lagoon and natural variability.     

Implication of growth hormone in experimental induction of vitellogenesis by estradiol-17 beta in female silver eel (Anguilla anguilla L.)]

The effect of different preparations of growth hormone (GH) was assayed with 17 beta-estradiol on vitellogenesis in hypophysectomized or normal female silver eels. Vitellogenin (Vg) plasma levels were taken as the index of hepatic vitellogenesis. The E2 doses were chosen to give the same pattern for the plasma Vg level as in the controls. They decreased or remained undetectable in hypophysectomized or normal animals. GH also failed to induce alone a significant modification. When E2 was injected together with a GH, plasma Vg levels were 5.13 +/- 1.30 times higher with salmon GH in hypophysectomized eels and 2.01 +/- 0.25 times higher with bovine GH in normal eels. GH is shown to enhance the effects of E2 on hepatic vitellogenesis induction in a teleost.     

Purification of insect vitellogenin and vitellin by gel-immobilized ferric chelate.

Vitellogenin and vitellin of Manduca sexta and some other insect species were purified by immobilized metal ion affinity chromatography. Ferric ion was chosen as the immobilized metal ion. Agarose-bound carboxymethylpicolylamine was used as the chelating adsorbent for the ferric ion. Vitellogenin and vitellin, both phosphorylated lipoproteins, were shown to bind specifically to the iron. The general applicability of immobilized ferric ion affinity chromatography for the purification of insect vitellogenin and vitellin is suggested.     

Influence of egg production on zinc, copper and iron metabolism in the turkey hen (Meleagris gallopavo)

1. Serum concentrations of zinc, copper, iron, calcium and protein increased significantly with the onset of egg production in turkey hens. 2. Hepatic concentrations of zinc, copper and iron declined significantly with the onset of egg production, whereas the total hepatic content of zinc and copper increased significantly. 3. When plasma from laying and nonlaying hens was subjected to gel-permeation column chromatography, a high molecular weight protein fraction was detected in laying but not in nonlaying plasma which bound significant amounts of zinc, copper and iron and which coeluted with purified vitellogenin. 4. Vitellogenin purified from the plasma of laying hens is a metalloprotein which upon analysis was found to contain 0.54, 0.09 and 0.72 micrograms of zinc, copper and iron, respectively, per mg of protein.     

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.     

In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA.

Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.