Glutathionyl-biliverdin IXα, a new heme catabolite in a marine annelid: Sex and cell specific accumulation

We have isolated and characterized the green pigment accumulating in coelomic cells (eleocytes) of the common clam worm Nereis virens by means of RP-HPLC and ESI-tandem mass spectrometry. This pigment is a novel biliverdin-glutathione conjugate in which the glutathione is linked to the biliverdin-backbone via a thioether bond. The yolk precursor vitellogenin, a female-specific high-density lipoprotein (ρ = 1179 kg/m³) with a native molecular mass of ∼500 kDa and a subunit mass of ∼150 kDa, is capable of transporting this pigment as well as heme. The sex-independent large discoidal lipoprotein present in the coelomic fluid, which is sequestered by the eleocytes, could also be shown to transport heme. This renders both lipoproteins as heme-lipoproteins. As the vitellogenin is secreted by the eleocytes, this suggests the eleocytes as a metabolic hub linking the uptake of the potent pro-oxidant heme thought to arise from aged hemoglobin via the large discoidal lipoprotein and its conversion to a bile pigment-conjugate. The conjugate is exported from the eleocytes to the oocytes via vitellogenin leading to the accumulation of green yolk protein. In contrast, no such export route exists in male eleocytes resulting in an accumulation of the biliverdin-conjugate in these cells.     

Regulation of vitellogenesis in Nereis virens (Annelida: Polychaeta): effect of estradiol-17beta on eleocytes

In the marine polychaete Nereis virens, the yolk protein precursor vitellogenin (Vg) is synthesized in specialized coelomic cells (eleocytes) during oogenesis. This process was visualized by immunohistochemistry using antibodies raised against the yolk protein. Transversal sections from male and female worms confirmed that eleocytes from females but not from males produce Vg. In order to investigate the hormonal regulation of Vg synthesis, eleocytes were incubated in vitro with estradiol-17beta (E(2)) at a concentration of 1 microg/l for up to three days. A strong increase in Vg secretion was detected by ELISA in culture media of treated eleocytes from vitellogenic females. In contrast, no response to the hormonal treatment was detectable in immature worms. Our results showed that Vg synthesis is under a complex regulation, which involves endocrine factors like estrogens. The role of E(2) in vitellogenesis of N. virens rather resembles the situation found in vertebrate than the one in insects.     

Nucleotide pool changes in coelomic cells (eleocytes) of the polychaete Nereis virens during sexual maturation

Eleocytes (a type of coelomic cell) of the polychaete Nereis virens can store large amounts of adenine nucleotides at certain times. Since eleocytes have specific functions related to gametogenesis, we tested whether the presence of these large nucleotide stores in eleocytes is specific to gender or related to specific events during gametogenesis. Nucleotide pools in eleocytes isolated at different stages of sexual maturation from N. virens were analysed using high-performance liquid chromatography. Eleocytes from immature and male animals had extremely high concentrations of both AMP and ADP (each > 10 μmol/ml of packed cell volume). In eleocytes from male animals, the high nucleotide stores were maintained throughout the maturation phase and decreased at a late stage, while in female animals the nucleotides were degraded at an early stage of maturation. In male eleocytes, the decrease in the adenine nucleotide pool may be the result of its conversion to inosine which is then released by the eleocytes and reutilized by male germ cells for nucleic acid biosynthesis, as has been suggested previously. Our study shows that the time of degradation of the adenine nucleotide pool coincides with the period of spermatogonia proliferation which involves intense nucleic acid synthesis. ATP levels (0.4–1.5 μmol/ml packed cell volume) and the guanine nucleotide pool (GTP+GDP+GMP; 0.08–0.18 μmol/ml packed cell volume) were similar in both sexes, did not change during germ cell development and were decreased only in eleocytes from prespawning females. The GTP/GDP ratios were initially higher (up to 14) in eleocytes from females compared to ratios in eleocytes from immature (4–9) and male animals (up to 8), and decreased during the maturation phase of the animals. GTP levels were correlated with those of ATP; this correlation was much closer in eleocytes from females than from males. The results further support the hypothesis that the adenine nucleotide stores in the eleocytes are maintained as a supply of purine precursors for the growing germ cells.     

Characterization of lipovitellin in eggs of the polychaete Neanthes arenaceodentata

The ooplasm of mature oocytes of the polychaete Neanthes arenaceodentata is characteristically filled with yolk platelets. A major component of these structures is lipovitellin, which provides energy and materials required by newly hatched larvae. The lipovitellin isolated and purified from the fertilized eggs of this polychaete was a high-density lipoprotein composed of protein (57%), lipid (42%) and carbohydrate (1%). The lipid component included phospholipids (92% of lipid), triacylglycerol (3% of lipid) and cholesterol (3% of lipid), while sodium dodecyl sulfate-gel electrophoresis showed the major protein component was a 120-kDa peptide. Microscopically, mature oocytes were present in the coelom along with phagocytic eleocytes. The presence of muscle fragments and oil droplets in eleocytes suggests that eleocytes play an important role in providing the protein and lipid needed for the assembly of lipovitellin in the oocytes.     

Autofluorescence in eleocytes of some earthworm species

Immunocompetent cells of earthworms, coelomocytes, comprise adherent amoebocytes and granular eleocytes (chloragocytes). Both cell populations can be expelled via dorsal pores of adult earthworms by exposure to an electric current (4.5 V) for 1 min. Analysis by phase contrast/fluorescence microscopy and flow cytometry demonstrated that eleocyte population of several species exhibits a strong autofluorescence. A high percentage (11-35%) of autofluorescent eleocytes was recorded in Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion sp. (O. cyaneum, O. tyrtaeum tyrtaeum and O. tyrtaeum lacteum). In contrast, autofluorescent coelomocytes were exceptionally scarce (less than 1%) in representative Aporrectodea sp. (A. caliginosa and A. longa) and Lumbricus sp. (L. castaneus, L. festivus, L. rubellus, L. terrestris). Thus, this paper for the first time describes profound intrinsic fluorescence of eleocytes in some--but not all--earthworm species. The function (if any) and inter-species differences of the autofluorescent coelomocytes still remain elusive.     

Beta-Alanine and other free amino acids during salinity adaptation of the polychaete Nereis japonica

The free amino acid pool was measured in the body wall muscle and in coelomic cells (eleocytes) of the polychaete Nereis japonica following adaptation to salinities between 6 and 44 per thousand. Beta-Alanine and glycine were the major amino acids comprising 35-60% of the total free amino acid pool in the body wall. In eleocytes, glutamate and lysine in addition to beta-alanine and glycine were the dominant free amino acids. In the body wall, the concentrations of beta-alanine were closely correlated with the ambient salinity between 12 and 35 per thousand. The concentrations of glycine rose initially but remained unchanged at concentrations above 26 per thousand. In both body wall and eleocytes, the mean total primary amine concentrations were correlated with the ambient salinities between 12 and 35 per thousand. The sum of amino acids determined by HPLC showed the same correlation in both tissues, but accounted only for 60-85% of the concentrations of total primary amines. The total protein content of the body wall was slightly higher at 44 per thousand compared to the lower salinities indicating dehydration of the tissues. Eleocytes swell at 6 per thousand and showed irregular amino acid concentrations indicating a loss of metabolic integrity.     

Riboflavin content in autofluorescent earthworm coelomocytes is species-specific

We have recently shown that a large proproportion of earthworm coelomocytes exhibit strong autofluorescence in some species (Dendrobaena veneta, Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion spp.), while autofluorescent coelomocytes are very scarce in representatives of Lumbricus spp. and Aporrectodea spp. Riboflavin (vitamin B2) was identified as a major fluorophore in Eisenia jetida coelomocytes. The main aim of the present experiments was to quantify riboflavin content in autofluorescent coelomocytes (eleocytes) from several earthworm species through a combination of flow cytometric and spectrofluorometric measurements. Spectrofluorometry of coelomocyte lysates showed that riboflavin was non-detectable in the coelomocytes of Aporrectodea spp. and Lumbricus spp., but was a prominent constituent of lysates from species with autofluorescent eleocytes. In the latter case, riboflavin content was the highest in E. fetida, followed by Octolasion spp. > A. chlorotica > D. rubidus. The riboflavin content of coelomocytes correlates positively with eleocyte autofluorescence intensity measured by flow cytometry and visible with fluorescence microscopy.     

Patterns of haemolymph vitellogenin and ovarian vitellin in the German cockroach, and the role of Juvenile Hormone

Abstract. The concentrations of fat body and haemolymph vitellogenin and ovarian vitellin during the first gonadotropic cycle of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) have been studied. For these purposes, a polyclonal antibody against B. germanica vitellogenin and vitellin has been obtained, and an ELISA to quantify these proteins has been developed. Ovarian vitellin levels follow a pattern which parallels those of basal oocyte growth and Juvenile Hormone production by the corpora allata. This suggests that Juvenile Hormone regulates vitellogenin uptake into oocytes. Fat body and haemolymph vitellogenin levels give cyclic and parallel patterns. However, the cycle of Juvenile Hormone appears delayed with respect to that of vitellogenin. We suggest that the production of Juvenile Hormone, although cyclic in profile, does not modulate alone the cycle of vitellogenin. At least a supplementary mechanism, apparently independent of Juvenile Hormone, may be involved in the decline of vitellogenin production at the end of the vitellogenic cycle.     

Isolation and sequence of a partial vitellogenin cDNA from the cockroach,Blattella germanica (L.) (Dictyoptera,Blattellidae), and characterization of the vitellogenin gene expression

A partial cDNA clone of the vitellogenin gene from the cockroach Blattella germanica has been isolated from a cDNA expression library using an anti-vitellin–vitellogenin antiserum probe. The analysis of cDNA inserts gave a sequence of 2,645 nucleotides corresponding to the 3′ region. The deduced amino acid sequence is 825 residues long and is similar to the homologous portion of the vitellogenin of other insect species, especially that of the mosquito Aedes aegypti. RNA hybridization studies indicated that the vitellogenin gene expression is limited to the fat body of adult females. The pattern of expression during the first vitellogenic cycle was approximately parallel to that of vitellogenin production by the fat body previously described. The availability of a cDNA probe for the B. germanica vitellogenin gene represents a useful tool to study the molecular action of hormones affecting vitellogenin synthesis in this species. Arch. Insect Biochem. Physiol. 38:137–146, 1998. © 1998 Wiley-Liss, Inc.     

Vitellogenin regulates hormonal dynamics in the worker caste of a eusocial insect

Functionally sterile honey bee workers synthesize the yolk protein vitellogenin while performing nest tasks. The subsequent shift to foraging is linked to a reduced vitellogenin and an increased juvenile hormone (JH) titer. JH is a principal controller of vitellogenin expression and behavioral development. Yet, we show here that silencing of vitellogenin expression causes a significant increase in JH titer and its putative receptor. Mathematically, the increase corresponds to a dynamic dose-response. This role of vitellogenin in the tuning of the endocrine system is uncommon and may elucidate how an ancestral pathway of fertility regulation has been remodeled into a novel circuit controlling social behavior.     

Quantitation of vitellogenin messenger RNA in the liver of male xenopus toads during primary and secondary stimulation by estrogen

From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males.Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen.During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of ³²PO₄ into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA.In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be <0.03% of the cytoplasmic poly(A)-containing RNA. Secondary stimulation by estrogen of these animals results in an at least 30 fold faster accumulation of vitellogenin mRNA in the cytoplasm within the initial 12 hr of hormone treatment. This may explain the faster appearance of vitellogenin in the blood plasma.     

Estrogen induces tissue specific changes in the chromatin conformation of the vitellogenin genes in Xenopus.

Nuclei from male Xenopus liver were digested extensively with DNase I and the residual amount of the four vitellogenin genes measured by hybridization with a moderate excess of vitellogenin cDNA. The saturation value was about twofold lower in chromatin isolated from liver cells of estrogen treated than from untreated males or from erythrocytes. Analyzing the disappearance of several defined restriction fragments specific for the A1 and A2 vitellogenin genes, after limited digestion with DNase I, suggested that the entire A1 and A2 vitellogenin genes are about twofold more sensitive to DNase I in chromatin of hepatocytes isolated from estrogen treated than from untreated males. Using the same assay no change in the DNase I sensitivity of the two vitellogenin genes in erythrocyte chromatin was observed. Analysis of the beta 1-globin and an albumin gene demonstrated that the DNase I sensitivity of these genes in both cell types is not altered by estrogen. All these data indicate that estrogen stimulation results in an increased DNase I sensitivity specific for the vitellogenin genes in hepatocytes.     

Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout,Oncorhynchus mykiss

The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (¹²⁵iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (K _d=8.2·10^-9 mol·1^-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin.Abbreviations B _max maximum number of binding sites available - BSA bovine serum albumin - bt-VTG brown trout vitellogenin - c-VTG earp vitellogenin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - K _d dissociatian constant - NCM nitrocellulose membranes - PMSF phenylmethylsulphonylfluoride - rt-VTG rainbow trout vitellogenin - VTG vitellogenin     

Identification of a large precursor of vitellogenin mRNA in the liver of estradiol-treated chicks.

Studies were performed to determine whether vitellogenin mRNA from avian liver has a precursor molecule or not. Total cellular RNA was prepared from estradiol-treated chicken liver in the presence of 8 M guanidine HCl, 2-mercaptoethanol and aurintricarboxylic acid. After denaturation, RNA was fractionated on sodium dodecylsulfate-sucrose gradients and large size RNA was analyzed under stringent conditions on 85% formamide-sucrose gradients at 25 degrees C. RNA fractions collected from the gradients were hybridized with vitellogenin (3H)-cDNA. Besides mature vitellogenin mRNA (32S, 7,000 nucleotides) vitellogenin sequences were also found in RNA fractions ranging from 38-50S with a peak at 45-50S (12-15,000 nucleotides). Only 5-10% of the putative 38-50S pmRNA is polyadenylated. We calculated that the half-life of vitellogenin pmRNA is about 3-4 minutes. We conclude that vitellogenin mRNA has a precursor which is twice the size of the mature mRNA.     

Vitellogenin synthesis in blood-fed Aedes aegypti in the absence of the head,thorax and ovaries

A blood meal initiates oöcyte maturation in Aedes aegypti, and we have used rocket immunoelectrophoresis to investigate the function of midgut, ovaries, and head in the onset of vitellogenin synthesis. Non-blood-fed females and those fed blood (by enema) containing soybean trypsin inhibitor never contained vitellogenin. This demonstrates that the pressure of an undigested blood meal on stretch receptors of the midgut plays no role in the induction of vitellogenin synthesis, rather the stimulus is a digestion product of blood.When females were ovariectomized or decapitated and then fed blood, the haemolymph contained newly synthesized vitellogenin 24 h later. This was also demonstrated in isolated ovariectomized abdomens. Apparently, induction of vitellogenin synthesis does not require factors from either the head, thorax, or ovaries. When ovariectomy or decapitation was postponed after a blood meal, the level of vitellogenin in the haemolymph rose. Therefore, interaction of factors from the head and ovaries maintain the synthesis needed for oöcyte maturation.     

Molecular Characterization of a cDNA Encoding Vitellogenin and Its Expression in the Hepatopancreas and Ovary during Vitellogenesis in the Kuruma Prawn, Penaeus japonicus

In Crustacea, reproductive function and mechanisms regulating vitellogenesis have not been fully elucidated. This is due in great part to a lack of information concerning the biochemical nature of the vitellogenin molecule, the hemolymph precursor of yolk protein, vitellin, as well as the functional expression of the vitellogenin-encoding gene. We have therefore cloned a cDNA encoding vitellogenin in the kuruma prawn, Penaeus japonicus based on the N-terminal amino acid sequence of the 91 kDa subunit of vitellin. The open reading frame of this cDNA encoded 2,587 amino acid residues. This is the first investigation reporting a full-length cDNA and its corresponding amino acid sequence for vitellogenin in any crustacean species.Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in both the follicle cells in the ovary and the parenchymal cells in the hepatopancreas. In nonvitellogenic females, vitellogenin mRNA levels were negligible in both the ovary and hepatopancreas, but in vitellogenic females, levels were dramatically increased in both tissues. In the ovary, highest levels were observed during the early exogenous vitellogenic stage, and thereafter rapidly decreased, whereas in the hepatopancreas, high levels were maintained until the onset of the late vitellogenic stage. Differing profiles of vitellogenin mRNA levels in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenin synthesis harbor separate and complementary roles during vitellogenesis.