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To evaluate potential involvement of clathrin in endocytosis of vitellogenin (Vtg) by teleost oocytes, cDNAs encoding clathrin heavy chain (cltc) were cloned from ovaries of cutthroat trout. Quantitative PCR revealed three types of cltc (cltc-a1, cltc-a2, cltc-b) to be expressed in 10 different tissues including the ovary. The cltc-a1 alone exhibited a significant decrease in ovarian expression during vitellogenesis; this was correlated with a corresponding decrease in transcripts encoding the major Vtg receptor (Vtgr). No development-related changes in ovarian cltc-a2 or cltc-b transcript levels were observed. In situ hybridization revealed a strong ctlc signal in pre-vitellogenic oocytes, but not in vitellogenic oocytes. Western blotting using a rabbit antiserum (a-Cltc) raised against a recombinant Cltc preparation detected a polypeptide band with an apparent mass of ~170kDa in vitellogenic ovary extracts. Immunohistochemistry using a-Cltc revealed Cltc to be uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, translocated to the periphery of lipid droplet stage oocytes, and localized to the oolemma during vitellogenesis. These patterns of cltc/Cltc distribution and abundance during oogenesis, which are identical to those previously reported for vtgr/Vtgr in this species, constitute the first empirical evidence that cltc-a1/Cltc-a1 is involved in Vtg endocytosis via the Vtgr in teleost fish., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The multiple vitellogenin (Vtg) system of striped bass, a perciform species spawning nearly neutrally buoyant eggs in freshwater, was investigated. Vitellogenin cDNA cloning, Western blotting of yolk proteins (YPs) using Vtg and YP type-specific antisera, and tandem mass spectrometry (MS/MS) of the YPs revealed the complex mechanisms of yolk formation and maturation in this species. It was discovered that striped bass possesses a tripartite Vtg system (VtgAa, VtgAb, and VtgC) in which all three forms of Vtg make a substantial contribution to the yolk. The production of Vtg-derived YPs is generally similar to that described for other perciforms. However, novel amino-terminal labeling of oocyte YPs prior to MS/MS identified multiple alternative sites for cleavage of these proteins from their parent Vtg, revealing a YP mixture far more complex than reported previously. This approach also revealed that the major YP product of each form of striped bass Vtg, lipovitellin heavy chain (LvH), undergoes limited degradation to smaller polypeptides during oocyte maturation, unlike the case in marine fishes spawning buoyant eggs in which LvHAa undergoes extensive proteolysis to osmotically active free amino acids. These differences likely reflect the lesser need for hydration of pelagic eggs spawned in freshwater. The detailed characterization of Vtgs and their proteolytic fate(s) during oocyte growth and maturation establishes striped bass as a freshwater model for investigating teleost multiple Vtg systems.

A cDNA encoding a vitellogenin receptor with 8 ligand binding repeats (vtgr) was cloned from ovaries of the cutthroat trout, Oncorhynchus clarki. In situ hybridization and quantitative PCR analyses revealed that the main site of vtgr mRNA expression was the oocytes. Expression was strongly detected in perinucleous stage oocytes, gradually decreased as oocytes grew, and became hardly detectable in vitellogenic oocytes. A rabbit antibody (a-Vtgr) was raised against a recombinant Vtgr protein in order to immunologically detect and localize Vtgr within the ovarian follicles. Western blotting using a-Vtgr detected a bold band with an apparent mass of ~95-105kDa in an ovarian preparation that also bound Sakhalin taimen, Hucho perryi, vitellogenin in ligand blots. Immunohistochemistry using a-Vtgr revealed that the Vtgr was uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, subsequently translocated to the periphery of lipid droplet stage oocytes, and became localized to the oolemma during vitellogenesis. We provide the first characterization of Vtgr at both the transcriptional and the translational levels in the cutthroat trout, and our results suggest that this receptor is involved in uptake of Vtg by oocytes of this species., (© 2013 Elsevier Inc. All rights reserved.)

Background: The striped bass and its relatives (genus Morone) are important fisheries and aquaculture species native to estuaries and rivers of the Atlantic coast and Gulf of Mexico in North America. To open avenues of gene expression research on reproduction and breeding of striped bass, we generated a collection of expressed sequence tags (ESTs) from a complementary DNA (cDNA) library representative of their ovarian transcriptome., Results: Sequences of a total of 230,151 ESTs (51,259,448 bp) were acquired by Roche 454 pyrosequencing of cDNA pooled from ovarian tissues obtained at all stages of oocyte growth, at ovulation (eggs), and during preovulatory atresia. Quality filtering of ESTs allowed assembly of 11,208 high-quality contigs ≥ 100 bp, including 2,984 contigs 500 bp or longer (average length 895 bp). Blastx comparisons revealed 5,482 gene orthologues (E-value < 10-3), of which 4,120 (36.7% of total contigs) were annotated with Gene Ontology terms (E-value < 10-6). There were 5,726 remaining unknown unique sequences (51.1% of total contigs). All of the high-quality EST sequences are available in the National Center for Biotechnology Information (NCBI) Short Read Archive (GenBank: SRX007394). Informative contigs were considered to be abundant if they were assembled from groups of ESTs comprising ≥ 0.15% of the total short read sequences (≥ 345 reads/contig). Approximately 52.5% of these abundant contigs were predicted to have predominant ovary expression through digital differential display in silico comparisons to zebrafish (Danio rerio) UniGene orthologues. Over 1,300 Gene Ontology terms from Biological Process classes of Reproduction, Reproductive process, and Developmental process were assigned to this collection of annotated contigs., Conclusions: This first large reference sequence database available for the ecologically and economically important temperate basses (genus Morone) provides a foundation for gene expression studies in these species. The predicted predominance of ovary gene expression and assignment of directly relevant Gene Ontology classes suggests a powerful utility of this dataset for analysis of ovarian gene expression related to fundamental questions of oogenesis. Additionally, a high definition Agilent 60-mer oligo ovary 'UniClone' microarray with 8 × 15,000 probe format has been designed based on this striped bass transcriptome (eArray Group: Striper Group, Design ID: 029004).

Full-length cDNAs encoding three forms of vitellogenin (Vg) were obtained from a liver cDNA library of estrogen-treated red seabream, Pagrus major. Two of the three Vg sequences had high homology with type-A and -B Vgs (VgA and VgB) of other teleosts. The third red seabream Vg was classified as a type-C or phosvitinless (Pvl) Vg due to its lack of a phosvitin (Pv) domain. Two Vg preparations (610 and 340 kDa) from blood serum of estradiol-treated fish were biochemically characterized. Analyses of precursor-product relationships by examination of N-terminal amino acid sequences verified cleavage of the 610 kDa Vg into a 540 kDa lipovitellin (Lv) and a 32 kDa beta'-component. Each of these yolk preparations comprising both VgA- and VgB-derived polypeptides. The 340 kDa Vg, which was immunologically verified to be a PvlVg, was accumulated by vitellogenic oocytes with no alterations to its native molecular mass. During oocyte maturation, the VgA- and VgB-derived yolk proteins were differentially processed, presumably to generate a pool of free amino acids for oocyte hydration or for allocation of specific types of nutrients, amino acids, and proteins, to the developing embryo. Conversely, the 340 kDa Vg-derived yolk protein is unlikely to contribute to oocyte hydration or diffusible nutrients since the molecule underwent only minor proteolytic nicking during oogenesis. The present study elucidates for the first time specific functions of three different forms of Vg and their product yolk proteins in a higher taxonomic group of marine teleosts that spawn pelagic eggs., (Copyright 2006 Wiley-Liss, Inc.)

A combination of recombinant human (rh) insulin-like growth factor-I (IGF-I) (25 nM) and the maturation-inducing hormone (MIH), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S; 72.5 nM), induced germinal vesicle breakdown (GVBD) in ovarian follicles of white bass incubated in vitro, whereas a four times greater concentration of each hormone was ineffective alone. These results indicate that IGF-I induces oocyte maturational competence (OMC) but not meiotic resumption in white bass. Culture medium concentrations of 20beta-S remained below detection limits for ovarian fragments incubated with rhIGF-I. Actinomycin D blocked GVBD in response to hCG but not to rhIGF-I plus 20beta-S, suggesting that IGF-I requires de novo translation but not transcription to induce OMC. Gap junction uncouplers, 1-octanol and 1-heptanol, and the phosphatidylinositiol 3-kinase (PI 3-K) inhibitors, wortmannin and LY 294002, attenuated hCG-, 20beta-S-, and rhIGF-I plus 20beta-S-induced GVBD. Although these inhibitors reduced hCG-induced progestin release, PI 3-K inhibitors did not alter MIH synthesis in some incubations and addition of 20beta-S to the incubations did not fully overcome the effects of either class of inhibitors, suggesting that decreasing MIH production is not their only inhibitory effect on gonadotropin (GtH) action. Our data suggest that gap junctions and PI 3-K activity are necessary for GtH and IGF-I to induce and maintain OMC in white bass. The induction of OMC but not meiotic resumption by IGF-I in white bass, compared with the induction of meiotic resumption but not OMC by IGF-I discovered in the congeneric striped bass suggests rapid evolution of the reproductive actions of IGF-I among temperate basses (genus Morone).

Oocyte germinal vesicle breakdown (GVBD) was induced in striped bass ovarian fragments when tissues were incubated with 100-nM recombinant human insulin-like growth factor-I (rhIGF-I), 25-IU human chorionic gonadotropin (hCG) ml(-1), or 290 nM of the maturation-inducing steroid (MIS), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S). Inhibitors of phosphatidylinositol 3-kinase (PI 3-K), wortmannin (100 nM) and LY 294002 (50 microM), inhibited GVBD induced by these hormones. Furthermore, the inhibitors attenuated hCG-induced steroid hormone synthesis. Previous studies report that gap junction uncouplers inhibit GVBD induced by hCG, but not by rhIGF-I, in striped bass. We show that 20beta-S-induced GVBD is also attenuated by 1 mM 1-heptanol or 1-octanol without being affected by incubation with 3 mM ethanol. Thus, the effects of inhibiting PI 3-K activity on GtH and MIS actions are similar to effects of uncoupling gap junctions. These data suggest that PI 3-K activity is required for GtH- MIS- and IGF-I induction of GVBD in striped bass. Our data are also consistent with the notion that a ligand that regulates PI 3-K activity, possibly an IGF, participates in maintenance of gap junctional communication required for maximal GtH and MIS action.

Recombinant human (rh) insulin-like growth factor-I (IGF-I) was more potent than rhIGF-II at inducing in vitro germinal vesicle breakdown (GVBD), a marker for resumption of meiosis, in oocytes of striped bass. Treatment of ovarian fragments containing oocytes in intact follicles with rhIGF-I increased concentrations of estradiol-17beta and maturation-inducing steroid (MIS) 17,20beta, 21-trihydoxy-4-pregnen-3-one (20beta-S) in the culture medium and decreased testosterone levels. The follicles were too immature for oocytes to complete GVBD in response to 20beta-S (MIS incompetent) or hCG. Addition of 20beta-S to cultures did not increase the percentage of oocytes completing GVBD in response to rhIGF-I or rhIGF-II. Bovine insulin was without effect on GVBD or steroid production. Incubation of MIS-competent follicles with actinomycin D, cyanoketone, trilostane, 1-heptanol, or 1-octanol had no effect on rhIGF-I-induced GVBD, but attenuated hCG-induced GVBD and 20beta-S production. Cycloheximide inhibited rhIGF-I-induced GVBD. Collectively, these observations indicate that IGF-I can induce GVBD via MIS- and transcription-independent pathways without coupled gap junctions between oocytes and granulosa cells or among granulosa cells, but requires protein synthesis to do so. An rhIGF-I analogue that does not bind IGF-binding proteins, des(1,3)IGF-I, was more potent than rhIGF-I in inducing GVBD, suggesting ovarian IGF-binding proteins may inhibit IGF-I action.

Two forms of vitellogenin (Vg), Vg A and Vg B, were identified in serum from estrogen-treated barfin flounder (Verasper moseri). Structural changes of lipovitellins (Lvs) derived from the two Vgs were examined during vitellogenesis and oocyte maturation. Two Lvs, vLv A and vLv B, were identified electrophoretically and immunologically in postvitellogenic oocytes. Each appeared to be composed of distinct heavy chains (vLvH A, M(r) 107,000, and vLvH B, M(r) 94,000) and light chains (vLvL A, M(r) 30,000, and vLvL B, M(r) 28,000) when analyzed by SDS-PAGE. Results from N-terminal amino acid sequencing and Western blotting using antisera to vLvH A and vLvH B verified that there are two Vg polypeptides in serum from estrogen-treated fish, Vg A (M(r) 168,000) and Vg B (M(r) 175,000), which give rise to vLvH A-vLvL A and vLvH B-vLvL B, respectively. N-terminal sequencing revealed two sequences for both phosvitin and beta'-component, supporting the concept of duality for all three classes of Vg-derived yolk proteins. During oocyte maturation, native dimeric vLv B was dissociated into a native M(r) 170,000 monomer (oLv B). Meanwhile, vLv A was extensively cleaved including complete degradation of vLvH A into free amino acids. We propose that the quantitative ratio of vLv A to vLv B in postvitellogenic oocytes regulates the buoyancy of the spawned pelagic eggs by controlling availability of free amino acids which function as osmotic effectors during oocyte hydration. The vLv A/vLv B ratio likely also controls the proportional availability of different types of nutrients, free amino acids versus Lv, for use during embryonic development., (Copyright 1999 Academic Press.)

Previous studies have shown that blood plasma levels of 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S) increase in striped bass (Morone saxatilis) undergoing final oocyte maturation (FOM). Both hormones are produced by ovarian fragments undergoing hCG-induced germinal vesicle breakdown (GVBD) in vitro. In the present study, we investigated binding of DHP and 20beta-S to ovarian membranes from striped bass undergoing FOM. Saturable binding sites for DHP were not detected. Saturation of 20beta-S binding sites with 5 nM [3H]20beta-S occurred within 40 min at 0 degrees C (at 3 min, half of the maximum specific binding of steroid was calculated to have occurred), and the binding was pH-dependent. Scatchard analyses revealed the presence of a single class of high-affinity (dissociation constant [Kd] = 1.4 +/- 0.2 nM), limited-capacity (estimated concentration [Bmax] = 2.7 +/- 0.3 pmol/g ovary) 20beta-S binding sites on membranes from striped bass ovaries undergoing FOM. In contrast, only low levels of specific binding (Bmax < 0.04 pmol/g tissue) were detected on membranes from testes, liver, brain, and muscle. Ovarian membranes prepared from vitellogenic females also had low levels (Bmax < 0.1 pmol/g ovary) of specific 20beta-S binding, less than 5% of that found during FOM. Results of competition assays showed that DHP was approximately 250 times less effective than 20beta-S for displacing 20beta-S from ovarian membranes. In contrast, 20beta, 21-dihydroxy-4-pregnen-3-one was a very effective competitor, although it is only a weak inducer of oocyte GVBD in vitro. Of several other steroids tested, only progesterone showed affinity for the 20beta-S binding site within a physiological range of concentrations. Taken together with previous studies of striped bass FOM, these findings indicate that 20beta-S is the oocyte maturation-inducing steroid hormone in striped bass.

An in vitro culture system was developed to investigate hormonal control of final oocyte maturation (FOM) in striped bass (Morone saxatilis). Isolated ovarian fragments exposed to human chorionic gonadotropin (hCG), dibutyryl cAMP, or forskolin produced significant amounts of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP) and the oocytes underwent germinal vesicle breakdown (GVBD). Slight increases in 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) production were also observed. Production of testosterone and estradiol-17 beta was relatively high at the beginning of in vitro treatment with hCG but decreased as production of DHP increased and GVBD was initiated. Inhibitors of protein transcription (actinomycin-D), translation (cycloheximide), and steroidogenesis (trilostane) completely blocked hCG-induced DHP and 20 beta-S production and the associated GVBD. FOM, assessed from the progress of GVBD, proceeded in trilostane-treated but not in cycloheximide-treated follicle-enclosed oocytes when DHP or 20 beta-S was added to the cultures. Structure-activity experiments revealed that DHP and 20 beta-S were more potent at inducing GVBD than 14 other structurally similar C21 steroids that were tested. These results demonstrate that FOM in striped bass is induced by gonadotropin-mediated production of a delta 4 steroid through an adenylate-cyclase pathway which requires protein synthesis. DHP and 20 beta-S are implicated as final oocyte maturation-inducing steroid hormones in striped bass.

Levels of estradiol-17 beta (E2), testosterone (T), 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), and 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) were measured by radioimmunoassay (RIA) in blood plasma of striped bass undergoing final oocyte maturation (FOM). Females were captured just prior to, or in the early stages of, FOM and induced to complete maturation and ovulation with injected human chorionic gonadotropin, synthetic salmon gonadotropin-releasing hormone analogue (sGnRHa; [D-Arg6-Pro9 NEt]-sGnRH), sGnRHa plus the dopamine receptor antagonist, domperidone (DOM), or OVAPRIM, a commercial preparation of sGnRHa + DOM. Their plasma levels of immunoreactive DHP and 20 beta-S were significantly greater at ovulation relative to the time of hormone injection, whereas the plasma levels of E2 and T were greatest at injection and decreased by ovulation and 24 hr thereafter. Plasma levels of 20 beta-S, but not DHP, were sustained at high levels after ovulation. Fish injected only with DOM did not undergo FOM, its associated changes in plasma steroid levels, or ovulation. In females captured at various natural stages of FOM, plasma levels of 20 beta-S and DHP were low during germinal vesicle migration (GVM), peaked coincident with germinal vesicle breakdown, and then decreased near the time of ovulation. Plasma levels of E2 and T were greatest during GVM and decreased as DHP and 20 beta-S levels increased. Analyses of conjugated versus free plasma steroids showed 64-79% of the various hormones to be in the free fraction. RIA of plasma fractionated by reversed-phase HPLC showed that half of the 20 beta-S immunoreactivity coeluted with 5 beta-pregnan-3 alpha,17,20 beta,21-tetrol, a putative 20 beta-S metabolite with 99.7% cross-reactivity in the 20 beta-S RIA. These results indicate that striped bass follow the typical profile of changing plasma steroid levels seen in other teleosts during FOM, with a clear shift from C18 and C19 steroids to C21 steroids. They suggest that both DHP and 20 beta-S, both potent inducers of striped bass FOM in vitro, may play a role in regulating FOM in this species.