Molecular pathways during marine fish egg hydration: the role of aquaporins

The pre‐ovulatory hydration of the oocyte of marine teleosts, a unique process among vertebrates that occurs concomitantly with meiosis resumption (oocyte maturation), is a critical process for the correct development and survival of the embryo. Increasing information is available on the molecular mechanisms that control oocyte maturation in fish, but the identification of the cellular processes involved in oocyte hydration has remained long ignored. During the past few years, a number of studies have identified the major inorganic and organic osmolytes that create a transient intra‐oocytic osmotic potential for hydrating the oocytes, whereas water influx was believed to occur passively. Recent work, however, has uncovered the role of a novel molecular water channel (aquaporin), designated aquaporin‐1b (Aqp1b), which facilitates water permeation and resultant swelling of the oocyte. The Aqp1b belongs to a teleost‐specific subfamily of water‐selective aquaporins, similar to mammalian aquaporin‐1 (AQP1) that has possibly evolved by duplication of a common ancestor and further neofunctionalization in oocytes of marine teleosts for water uptake. Strikingly, Aqp1b shows specific regulatory domains at the cytoplasmic tail, which are key to the vesicular trafficking and temporal insertion of Aqp1b in the oocyte plasma membrane during the phase of maximal hydration. These findings are revealing that the mechanism of oocyte hydration in marine teleosts is a highly regulated process based on the interplay between the generation of inorganic and organic osmolytes and the controlled insertion of Aqp1b in the oocyte surface. The discovery of Aqp1b in teleosts provides an important insight into the molecular basis of the production of viable eggs in marine fish.     

Molecular Characterization of Branchial aquaporin 1aa and Effects of Seawater Acclimation,Emersion or Ammonia Exposure on Its mRNA Expression in the Gills,Gut, Kidney and Skin of the Freshwater Climbing Perch,Anabas testudineus

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH₃ transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.     

Vertebrate vitellogenin gene duplication in relation to the "3R hypothesis": correlation to the pelagic egg and the oceanic radiation of teleosts

The spiny ray-finned teleost fishes (Acanthomorpha) are the most successful group of vertebrates in terms of species diversity. Their meteoric radiation and speciation in the oceans during the late Cretaceous and Eocene epoch is unprecedented in vertebrate history, occurring in one third of the time for similar diversity to appear in the birds and mammals. The success of marine teleosts is even more remarkable considering their long freshwater ancestry, since it implies solving major physiological challenges when freely broadcasting their eggs in the hyper-osmotic conditions of seawater. Most extant marine teleosts spawn highly hydrated pelagic eggs, due to differential proteolysis of vitellogenin (Vtg)-derived yolk proteins. The maturational degradation of Vtg involves depolymerization of mainly the lipovitellin heavy chain (LvH) of one form of Vtg to generate a large pool of free amino acids (FAA 150-200 mM). This organic osmolyte pool drives hydration of the ooctye while still protected within the maternal ovary. In the present contribution, we have used Bayesian analysis to examine the evolution of vertebrate Vtg genes in relation to the "3R hypothesis" of whole genome duplication (WGD) and the functional end points of LvH degradation during oocyte maturation. We find that teleost Vtgs have experienced a post-R3 lineage-specific gene duplication to form paralogous clusters that correlate to the pelagic and benthic character of the eggs. Neo-functionalization allowed one paralogue to be proteolyzed to FAA driving hydration of the maturing oocytes, which pre-adapts them to the marine environment and causes them to float. The timing of these events matches the appearance of the Acanthomorpha in the fossil record. We discuss the significance of these adaptations in relation to ancestral physiological features, and propose that the neo-functionalization of duplicated Vtg genes was a key event in the evolution and success of the teleosts in the oceanic environment.     

Gonadotropin-Activated Androgen-Dependent and Independent Pathways Regulate Aquaporin Expression during Teleost (Sparus aurata) Spermatogenesis

The mediation of fluid homeostasis by multiple classes of aquaporins has been suggested to be essential during spermatogenesis and spermiation. In the marine teleost gilthead seabream (Sparus aurata), seven distinct aquaporins, Aqp0a, -1aa, -1ab, -7, -8b, -9b and -10b, are differentially expressed in the somatic and germ cell lineages of the spermiating testis, but the endocrine regulation of these channels during germ cell development is unknown. In this study, we investigated the in vivo developmental expression of aquaporins in the seabream testis together with plasma androgen concentrations. We then examined the in vitro regulatory effects of recombinant piscine gonadotropins, follicle-stimulating (rFsh) and luteinizing (rLh) hormones, and sex steroids on aquaporin mRNA levels during the spermatogenic cycle. During the resting phase, when plasma levels of androgens were low, the testis exclusively contained proliferating spermatogonia expressing Aqp1ab, whereas Aqp10b and -9b were localized in Sertoli and Leydig cells, respectively. At the onset of spermatogenesis and during spermiation, the increase of androgen plasma levels correlated with the additional appearance of Aqp0a and -7 in Sertoli cells, Aqp0a in spermatogonia and spermatocytes, Aqp1ab, -7 and -10b from spermatogonia to spermatozoa, and Aqp1aa and -8b in spermatids and spermatozoa. Short-term in vitro incubation of testis explants indicated that most aquaporins in Sertoli cells and early germ cells were upregulated by rFsh and/or rLh through androgen-dependent pathways, although Aqp1ab in proliferating spermatogonia was also activated by estrogens. However, expression of Aqp9b in Leydig cells, and of Aqp1aa and -7 in spermatocytes and spermatids, was also directly stimulated by rLh. These results reveal a complex gonadotropic control of aquaporin expression during seabream germ cell development, apparently involving both androgen-dependent and independent pathways, which may assure the fine tuning of aquaporin-mediated fluid secretion and absorption mechanisms in the seabream testis.     

Teleost fish with specific genome duplication as unique models of vertebrate evolution

Whole-genome duplication (WGD) is believed to be one of the major evolutionary events that shaped the genome organization of vertebrates. Here, we review recent research on vertebrate genome evolution, specifically on WGD and its consequences for gene and genome evolution in teleost fishes. Recent genome analyses confirmed that all vertebrates experienced two rounds of WGD early in their evolution, and that teleosts experienced a subsequent additional third-round (3R)-WGD. The 3R-WGD was estimated to have occurred 320–400 million years ago in a teleost ancestor, but after its divergence from a common ancestor with living non-teleost actinopterygians (Bichir, Sturgeon, Bowfin, and Gar) based on the analyses of teleost-specific duplicate genes. This 3R-WGD was confirmed by synteny analysis and ancestral karyotype inference using the genome sequences of Tetraodon and medaka. Most of the tetrapods, on the other hand, have not experienced an additional WGD; however, they have experienced repeated chromosomal rearrangements throughout the whole genome. Therefore, different types of chromosomal events have characterized the genomes of teleosts and tetrapods, respectively. The 3R-WGD is useful to investigate the consequences of WGD because it is an evolutionarily recent WGD and thus teleost genomes retain many more WGD-derived duplicates and “traces” of their evolution. In addition, the remarkable morphological, physiological, and ecological diversity of teleosts may facilitate understanding of macrophenotypic evolution on the basis of genetic/genomic information. We highlight the teleosts with 3R-WGD as unique models for future studies on ecology and evolution taking advantage of emerging genomics technologies and systems biology environments.     

The maturational disassembly and differential proteolysis of paralogous vitellogenins in a marine pelagophil teleost: a conserved mechanism of oocyte hydration

A structural analysis of the differential proteolysis of vitellogenin (Vtg)-derived yolk proteins in the maturing oocytes of a marine teleost that spawns very large pelagic eggs is presented. Two full-length hepatic cDNAs (hhvtgAa and hhvtgAb) encoding paralogous vitellogenins (HhvtgAa and HhvtgAb) were cloned from nonestrogenized Atlantic halibut, and the N-termini of their subdomain structures were mapped to the oocyte and egg yolk proteins (Yps). The maturational oocyte Yp degradation products were further mapped to the free amino acid (FAA) pool in the ovulated egg. The deduced amino acid sequences conformed to the linear NH(2)-(LvH-Pv-LvL-beta'-CT)-COO(-) structure of complete teleost Vtgs. However, the Yps did not match the expected cleavage products of complete Vtgs. Specifically, the phosvitin subdomain of the HhvtgAa paralogue remains covalently attached to the lipovitellin light chain, while the phosvitin subdomain of the HhvtgAb paralogue remains covalently attached to a C-terminal fragment of the lipovitellin heavy chain (LvH). During oocyte hydration, the LvH of the HhvtgAa paralogue is disassembled and extensively degraded to FAA. In the HhvtgAb paralogue, the LvH is nicked in the C-sheet in a manner similar to that seen in lamprey and other teleosts. A small part of the C-terminal end of the LvH-Ab undergoes proteolysis to FAA, together with the phosvitin, beta' component, and much ( approximately 65%) of the lipovitellin light chain (LvL-Ab). The independently measured FAA pool in the ovulated egg corroborates that calculated from differential proteolysis of the Yps. Based on the 3:1 (HhvtgAb:HhvtgAa) Yp expression ratio, each paralogue contributes approximately equal amounts of FAA to the organic osmolyte pool of the hydrating oocyte during maturation.     

Rearrangement rate following the whole-genome duplication in teleosts

It is now clear that a whole-genome duplication (WGD) occurred at the base of the teleost fish lineage. Like the other anciently polyploid genomes investigated so far, teleost genomes now behave like diploids with chromosomes forming pairs at meiosis. The diploidization process is currently poorly understood. It is associated with many gene deletions, such that one of the duplicates is lost at most loci and has also been proposed to coincide with an increase in genomic instability. Here we ask whether WGD is a determinant of the genomic rearrangement rate in teleosts. We study variability of the rates of rearrangement along a vertebrate phylogenetic tree, composed of 3 tetrapods (human, chicken, and mouse) and 3 teleost fishes (zebrafish, Tetraodon, and Takifugu), whose complete genome sequences are available. We devise a simple parsimony method for counting rearrangements, which takes into account various methodological complications caused by the WGD and the subsequent gene losses. We show that there does appear to be an increase in rearrangement rate after WGD, but that there is also a great deal of additional variability in rearrangement rates across species.     

Zebrafish enhancer trap line recapitulates embryonic aquaporin 1a expression pattern in vascular endothelial cells

Aquaporin 1 (Aqp1) is a water channel protein, expressed widely in microvascular endothelial cells and implicated in mammalian tumor angiogenesis. However, its developmental expression has not yet been characterized in great detail. An enhancer trap screen was performed using a Tol2-derived GFP reporter in zebrafish embryos. An insertional Et(GBT-B1)tpl1 line was identified that has reporter insertion in the vicinity of the aqp1a gene. We further characterized the embryonic expression pattern of this GFP reporter line, as well as that of endogenous aqp1a. Both endogenous aqp1a and reporter GFP expression were restricted to the vascular endothelial cells within the dorsal aorta, cranial, intersegmental and other secondary vessels, but were absent in the axial venous vasculature. In addition, endogenous aqp1a expression was observed in both primitive and definitive hematopoietic erythroid progenitors, as well as in the otic vesicle, swim bladder, pneumatic duct, intestine and a subset of neurons within the retina and the midbrain-hindbrain region. We further show that gata1 and etsrp/etv2 function is required for hematopoietic and endothelial aqp1a expression, respectively. Aqp1a expression is restricted to endothelial and erythroid cells during early embryogenesis. The transgenic Et(GBT-B1)tpl1 line recapitulates endogenous endothelial aqp1a expression. Because currently very few reporter lines can differentiate between arterial and venous endothelial cells, the Et(GBT-B1)tpl1 transgenic line and characterization of the aqp1a expression pattern will be useful for future studies of endothelial and arterial-venous differentiation.     

Changes in teleost yolk proteins during oocyte maturation: correlation of yolk proteolysis with oocyte hydration

Yolk proteins of prematuration occytes and postmaturation eggs were compared by SDS gel electrophoresis in several teleosts, including freshwater species that produce demersal eggs, estuarine and marine species with demersal eggs, and marine species with pelagic eggs. In certain teleosts distinct changes in yolk protein banding patterns during oocyte maturation are suggestive of extensive secondary proteolysis of yolk proteins at this time; proteolysis is most pronounced in marine fishes with pelagic eggs. In many teleosts the oocyte swells by hydration during maturation; this hydration is also most pronounced in marine fishes with pelagic eggs. The extent of yolk proteolysis is well correlated with the extent of oocyte hydration during maturation.     

Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by an H+-ATPase inhibitor: effects on the hydration mechanism

In teleost oocytes, yolk proteins (YPs) derived from the yolk precursors vitellogenins are partially cleaved into free amino acids and small peptides during meiotic maturation before ovulation. This process increases the osmotic pressure of the oocyte that drives its hydration, which is essential for the production of buoyant eggs by marine teleosts (pelagophil species). However, this mechanism also occurs in marine species that produce benthic eggs (benthophil), such as the killifish (Fundulus heteroclitus), in which oocyte hydration is driven by K+. Both in pelagophil and benthophil teleosts, the enzymatic machinery underlying the maturation-associated proteolysis of YPs is poorly understood. In this study, lysosomal cysteine proteinases potentially involved in YP processing, cathepsins L, B, and F (CatL, CatB, and CatF, respectively), were immunolocalized in acidic yolk globules of vitellogenic oocytes from the killifish. During oocyte maturation in vitro induced with the maturation-inducing steroid (MIS), CatF disappeared from yolk organelles and CatL became inactivated, whereas CatB proenzyme was processed into active enzyme. Consequently, CatB enzyme activity and hydrolysis of major YPs were enhanced. Follicle-enclosed oocytes incubated with the MIS in the presence of bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, underwent maturation in vitro, but acidification of yolk globules, activation of CatB, and proteolysis of YPs were prevented. In addition, MIS plus bafilomycin A1-treated oocytes accumulated less K+ than those stimulated with MIS alone; hence, oocyte hydration was reduced. These results suggest that CatB is the major protease involved in yolk processing during the maturation of killifish oocytes, whose activation requires acidic conditions maintained by a vacuolar-type H+-ATPase. Also, the data indicate a link between ion translocation and YP proteolysis, suggesting that both events may be equally important physiological mechanisms for oocyte hydration in benthophil teleosts.     

Evidence for a role of Na+,K(+)-ATPase in the hydration of Atlantic croaker and spotted seatrout oocytes during final maturation.

During final maturation the oocytes of many marine teleosts swell four to five times their original size due to uptake of water. The involvement of active inorganic ion transport and Na+,K(+)-ATPase in oocyte hydration in Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus), marine teleosts which spawn pelagic eggs, was investigated by examining changes in the inorganic ion content of ovarian follicles containing mainly oocytes, by performing in vitro incubations of the follicles with ion channel blockers, and by assaying membrane preparations of ovaries containing hydrating and non-hydrating oocytes for Na+,K(+)-ATPase activity and content. There were marked increases in the contents of K+, Mg++, and Ca++, but not Na+, in oocytes of M. undulatus and C. nebulosus during hydration. Incubation of follicle-enclosed oocytes in K(+)-free medium or with ouabain or amiloride, inhibitors of Na+,K(+)-ATPase and Na+ channels, respectively, blocked gonadotropin-induced oocyte hydration in M. undulatus. In addition, Na+,K(+)-ATPase activity increased threefold and the concentration of the enzyme increased 50% in ovarian tissue during oocyte hydration. These results strongly suggest a major role for active ion regulation by a ouabain-sensitive Na+,K(+)-ATPase system in oocyte hydration in two species of sciaenid fishes.     

Evidence for a divergence in function between two glucocorticoid receptors from a basal teleost

ABSTRACT: BACKGROUND: Duplicated glucocorticoid receptors (GR) are present in most teleost fish. The evolutionary advantage of retaining two GRs is unclear, as no subtype specific functional traits or physiological roles have been defined. To identify factors driving the retention of duplicate GRs in teleosts, the current study examined GRs in representatives of two basal ray-finned fish taxa that emerged either side of the teleost lineage whole genome duplication event (WGD) event, the acipenseriform, Acipenser ruthenus, (pre-WGD) and the osteoglossimorph, Pantodon buchholzi, (post-WGD). RESULTS: The study identified a single GR in A. ruthenus (ArGR) and two GRs in P. buchholzi (PbGR1 and PbGR2). Phylogenetic analyses showed that ArGR formed a distinct branch separate from the teleosts GRs. The teleost GR lineage was subdivded into two sublineages, each of which contained one of the two P. buchholzi GRs. ArGR, PbGR1 and PbGR2 all possess the unique 9 amino acid insert between the zinc-fingers of the DNA-binding domain that is present in one of the teleost GR lineages (GR1), but not the other (GR2). A splice variant of PbGR2 produces an isoform that lacked these 9 amino acids (PbGR2b). Cortisol stimulated transactivation activity of ArGR, PbGR2b and PbGR1 in vitro; with PbGR2b and PbGR1, the glucocorticoid 11-deoxycortisol was a more potent agonist than cortisol. The hormone sensitivity of PbGR2b and PbGR1 differed in the transactivation assay, with PbGR2b having lower EC50 values and greater fold induction. CONCLUSIONS: The difference in transactivation activity sensitivity between duplicated GRs of P. buchholzi suggests potential functional differences between the paralogs emerged early in the teleost lineage. Given the pleiotropic nature of GR function in vertebrates, this finding is in accordance with the hypothesis that duplicated GRs were potentially retained through subfunctionalisation followed by gene sharing. A 9 amino acid insert in the DNA-binding domain emerged in basal ray-finned fish GRs. However, the presence of a PbGR2 splice variant that lacks this insert, as well as the loss of the exon encoding these amino acids in the genes encoding for other teleost GR2 suggests the selection of two receptors with different DNA-binding domain structures in teleosts.     

Structural and functional divergence of two fish aquaporin-1 water channels following teleost-specific gene duplication

Background  

Teleost radiation in the oceans required specific physiological adaptations in eggs and early embryos to survive in the hyper-osmotic seawater. Investigating the evolution of aquaporins (AQPs) in these vertebrates should help to elucidate how mechanisms for water homeostasis evolved. The marine teleost gilthead sea bream (Sparus aurata) has a mammalian aquaporin-1 (AQP1)-related channel, termed AQP1o, with a specialized physiological role in mediating egg hydration. However, teleosts have an additional AQP isoform structurally more similar to AQP1, though its relationship with AQP1o is unclear.     

Derivation of major yolk proteins from parental vitellogenins and alternative processing during oocyte maturation in Fundulus heteroclitus

Various Coomassie blue-staining yolk proteins (YPs) present in oocytes and eggs of Fundulus heteroclitus, a teleost that produces low hydrated, demersal eggs (benthophil species), were subjected to N-terminal microsequencing. Four YPs were N-terminally blocked, while five yielded sequence information. Of the latter, four corresponded to internal sequences of vitellogenin 1 (Vg1), whereas a fifth band corresponded to the N-terminal sequence of Vg2. Phosphorylated YPs (phosvitins and phosvettes) derived from the polyserine domain of Vg were not successfully sequenced. The major N-terminally blocked 122-and 103-kDa YPs both represented the lipovitellin heavy chain of Vg1 (LvH1), and thus most of the oocyte YPs were derived from Vg1. During oocyte maturation in vivo and in vitro, the LvH1 122 is degraded, concomitant with an increased enzymatic activity of cathepsin B, while the 45-kDa YP is converted to a 42-kDa YP. The LvH1 122 was found to contain a consensus site for proteolytic degradation (PEST) near its C-terminus, which is missing from its stable, but truncated twin sequence, LvH1 103. We suggest that this site becomes exposed to cathepsin B during the hydration process that accompanies oocyte maturation and renders the LvH1 122 susceptible to proteolysis. PEST sites are found in Vg sequences from other benthophil fish, whereas, interestingly, they are missing in marine teleosts that spawn highly hydrated, pelagic eggs (pelagophil species), displaying a different pattern of Vg incorporation into YPs and LvH1 and LvH2 processing to that found in F. heteroclitus. Thus, different models of Vg/YP precursor/product relationship and further processing during oocyte maturation and hydration are proposed for pelagophil and benthophil teleosts.     

Integration and modularity of teleostean pectoral fin shape and its role in the diversification of acanthomorph fishes

Phenotypic integration and modularity describe the strength and pattern of interdependencies between traits. Integration and modularity have been proposed to influence the trajectory of evolution, either acting as constraints or facilitators. Here, we examine trends in the integration and modularity of pectoral fin morphology in teleost fishes using geometric morphometrics. We compare the fin shapes of the highly diverse radiation of acanthomorph fishes to lower teleosts. Integration and modularity are measured using two‐block partial least squares analysis and the covariance ratio coefficient between the radial bones and lepidotrichia of the pectoral fins. We show that the fins of acanthomorph fishes are more tightly integrated but also more morphologically diverse and faster evolving compared to nonacanthomorph fishes. The main pattern of shape covariation in nonacanthomorphs is concordant with the main trajectory of evolution between nonacanthomorphs and acanthomorphs. Our findings support a facilitating role for integration during the acanthomorph diversification. Potential functional consequences and developmental mechanisms of fin integration are discussed.     

Goldsinny wrasse (Ctenolabrus rupestris) is an extreme vtgAa-type pelagophil teleost

During oocyte maturation in the goldsinny wrasse (Ctenolabrus rupestris) extensive proteolysis of yolk proteins generates a large pool of free amino acids that drive hydration of the pelagic egg. By cloning hepatic vitellogenins (vtg) and using mass spectrometry, N-terminal microsequencing, and Western-immunoblotting to identify the yolk proteins (Yp), we show that multiple forms of vitellogenin mRNAs (vtgAa, vtgAb, and vtgC) are expressed in the liver, but only a single major class of the Yps derived from vtgAa predominates in the oocytes. Some Yps derived from vtgAb and vtgC appear also to be incorporated in the oocytes and eggs, but only at background levels. During oocyte hydration the vtgAa-derived lipovitellin heavy chain (LvH-Aa) and its cleavage variants are completely degraded leaving only a processed lipovitellin light chain (LvL-Aa) fragment as the major yolk protein for embryonic development. The maturational cleavage site of the LvL-Aa is identified as two amino acids downstream from the conserved Tyr(1168) of VtgAa in Atlantic halibut. In addition, although a beta'-component (approximately 18 kDa) is present in the oocytes, it is not fully degraded during the hydration process.     

Comparative genomics of duplicate γ-glutamyl transferase genes in teleosts: medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), green spotted pufferfish (Tetraodon nigroviridis), fugu (Takifugu rubripes), and zebrafish (Danio rerio)

The availability of multiple teleost (bony fish) genomes is providing unprecedented opportunities to understand the diversity and function of gene duplication events using comparative genomics. Here we examine multiple paralogous genes of γ-glutamyl transferase (GGT) in several distantly related teleost species including medaka, stickleback, green spotted pufferfish, fugu, and zebrafish. Through mining genome databases, we have identified multiple GGT orthologs. Duplicate (paralogous) GGT sequences for GGT1 (GGT1 a and b), GGTL1 (GGTL1 a and b), and GGTL3 (GGTL3 a and b) were identified for each species. Phylogenetic analysis suggests that GGTs are ancient proteins conserved across most metazoan phyla and those paralogous GGTs in teleosts likely arose from the serial 3R genome duplication events. A third GGTL1 gene (GGTL1c) was found in green spotted pufferfish; however, this gene is not present in medaka, stickleback, or fugu. Similarly, one or both paralogs of GGTL3 appear to have been lost in green spotted pufferfish, fugu, and zebrafish. Syntenic relationships were highly maintained between duplicated teleost chromosomes, among teleosts and across ray-finned (Actinopterygii) and lobe-finned (Sarcopterygii) species. To assess subfunction partitioning, six medaka GGT genes were cloned and assessed for developmental and tissue-specific expression. On the basis of these data, we propose a modification of the "duplication-degeneration-complementation" model of subfunction partitioning where quantitative differences rather than absolute differences in gene expression are observed between gene paralogs. Our results demonstrate that multiple GGT genes have been retained within teleost genomes. Questions remain, however, regarding the functional roles of multiple GGTs in these species.     

Bafilomycin A1 inhibits proteolytic cleavage and hydration but not yolk crystal disassembly or meiosis during maturation of sea bass oocytes

Oocytes of the black sea bass, Centropristes striata, were enlarged in volume more than three-fold over a 24-hr period during oocyte maturation, both in vivo and in vitro. At the same time, the opaque oocytes clarified while the crystalline yolk inclusions lost their ordered structure, fused with one another, and formed a continuous electron-lucent mass. The oocyte size increase was due almost entirely to water uptake, which was accompanied by the accumulation of Na+, K+, and free amino acids (FAAs). The absolute amounts of each of these small molecular weight osmotic effectors increased 2x, 4x, and over 10x, respectively, indicating that the generation of FAAs is the major cause of water uptake during maturation. Amino acid analyses indicated that the amounts of all amino acids except taurine increased, so that selective amino acids were not produced during maturation. The increase in FAAs was accompanied by the loss of certain high-molecular-weight yolk proteins and the generation of many smaller peptides. Oocytes stimulated to undergo maturation in the presence of bafilomycin A1, a specific inhibitor of the vacuolar ATPase-dependent proton pump, clarified and underwent maturation but did not increase significantly in size. Cytological examination revealed that yolk crystals fused and became homogeneous but maintained their electron density. No evidence of proteolysis was found in bafilomycin A1-treated oocytes and the generation of FAAs together with hydration was inhibited in a dose-dependent manner (I50 = 3 nM bafilomycin A1). Taken together, we postulate that the pronounced oocyte hydration in marine teleosts that spawn pelagic (floating) eggs is accomplished by a two-step process whereby (i) K+ influx promotes yolk crystal disassembly and yolk sphere fusion and (ii) acidification of the yolk spheres activates yolk proteolysis and concomitant hydration. Bafilomycin A1 inhibits only the second step so that many of the events of oocyte maturation, including germinal vesicle breakdown, occur in its presence but oocyte hydration is suppressed.     

Lipovitellins derived from two forms of vitellogenin are differentially processed during oocyte maturation in haddock (Melanogrammus aeglefinus)

In the process of cloning vitellogenin (Vtg) cDNAs from haddock (Melanogrammus aeglefinus), two related, but distinct, mRNAs were identified. Full-length cDNA sequences were determined for both Vtg types (Had1 and Had2), and the deduced amino acid sequences were found to be 54% identical to each other and 48-58% identical to other teleost Vtgs. To investigate the expression of the two Vtg mRNAs, proteins from prehydrated oocytes and fertilized eggs were separated on SDS-polyacrylamide gels. Only a single lipovitellin I band was detected in each sample, and the egg lipovitellin I was smaller (97 vs. 110 kDa) than the oocyte protein, indicative of proteolytic processing during oocyte hydration. Mass spectrometric (MALDI-TOFMS and tandem mass spectrometry) analyses of tryptic fragments from the haddock oocyte and egg lipovitellin I revealed that the lipovitellin I from prehydrated oocytes contained tryptic fragments that matched the sequences of both types of Vtg, suggesting that there were two proteins in this band, while the egg lipovitellin I contained tryptic fragments that only matched the Had1 cDNA sequence, indicating that the Had2 lipovitellin had been degraded during hydration. Physiological data from haddock oocytes and eggs demonstrate that, as in other marine fish that spawn pelagic eggs, the free amino acid content increases during oocyte hydration and apparently contributes to hydration by driving the osmotic uptake of water. The correlation of the disappearance of one lipovitellin I with the increase of free amino acids in the oocyte suggests that this protein is a major source of the free amino acids for oocyte hydration.