Zinc-binding property of the major yolk protein in the sea urchin - implications of its role as a zinc transporter for gametogenesis

Major yolk protein (MYP), a transferrin superfamily protein that forms yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP in the coelomic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc-binding capacity that is diminished concomitantly with its incorporation from the coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus. Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EGMYP were present in nutritive phagocytes, where CFMYP bound more zinc than EGMYP. Saturation binding assays revealed that CFMYP has more zinc-binding sites than EGMYP. Labeled CFMYP injected into the coelom was incorporated into ovarian and testicular nutritive phagocytes and vitellogenic oocytes, and the molecular mass of part of the incorporated CFMYP shifted to 170 kDa. Considering the fact that the digestive tract is a major production site of MYP, we propose that CFMYP transports zinc, essential for gametogenesis, from the digestive tract to the ovary and testis through the coelomic fluid, after which part of the CFMYP is processed to EGMYP with loss of zinc-binding site(s).     

Egg transport in the coelom of the newt cynops pyrrhogaster. I. The ovulated egg is transported to the ostium by the ciliary movement of coelomic epithelial cells

We investigated the mechanism of egg transport in the newt not only by inserting various conditioned eggs into the recipient's body but also by placing them on the coelomic epithelia of the opened body cavity in the adult female newt. Most of the inserted coelomic eggs were oviposited, while 4 of 14 inserted de-jellied uterine eggs and 3 of 10 inserted de-jellied fertilized eggs were oviposited. The coelomic eggs placed on the coelomic epithelia were transported toward the ostium and entered the ostium. The de-jellied uterine eggs and the de-jellied fertilized eggs were transported to the ostium as well. Of all the eggs examined, the coelomic egg was transported the fastest. The transport speeds of coelomic eggs treated with periodic acid and the speed of boiled coelomic eggs were less than those of untreated coelomic eggs. In contrast, the transport speeds of coelomic eggs treated with trypsin and the speed of coelomic eggs removed from their vitelline envelopes (naked eggs) were faster than those of untreated coelomic eggs. Other experiments were carried out in order to ascertain the dependence of sexual activity on egg transport. The speed of coelomic egg transport in artificially sexually activated females was faster than in sexually inactive females, although the ciliary movement could always be observed in both sexually active females and sexually inactive females. This suggests that the speed of egg transport on the coelomic epithelia is controlled by the sexual activity of the female.     

A putative precursor to the major yolk protein of the sea urchin

We detected by electrophoresis, several glycoproteins in the eggs of three species of sea urchin. The major glycoprotein band disappears as development of the embryo proceeds. This protein is enriched in the yolk fraction obtained by zone sedimentation in 2.5–30% sucrose gradients. A fractionally larger glycoprotein has been found to be the major protein in the coelomic fluid of male and female gravid sea urchins. Partial proteolysis peptide mapping shows that the major coelomic fluid protein and the major yolk protein are related, presumably in a precursor-product relationship.     

保存介质和温度对西伯利亚鲟卵子短期保存的影响

研究了不同保存介质(体腔液CF、Hepes液、RMS液)、温度(4℃、16℃)和保存时间(4 h、8 h、16 h、24 h)对西伯利亚鲟卵子短期保存的影响.结果显示:保存介质、温度和时间对卵子受精率、孵化率和初孵仔鱼畸形率有显著性影响(P<0.05),受精率、孵化率均随保存时间的延长而下降,畸形率上升.16℃条件下保存卵子受精率、孵化率和畸形率均高于4℃,但4℃下卵子的保存时间较16℃下长.研究表明,采用根据西伯利亚鲟体腔液生化成分配制的Hepes液作为保存介质,于16℃下保存4 h为西伯利亚鲟卵子的最佳保存条件,此时受精率、孵化率和畸形率分别为86.36%、94.74%和0.     

Fertilization of newt coelomic eggs in the absence of jelly envelope material

Summary Fertilization ofCynops pyrrhogaster (Japanese newt) coelomic eggs was studied in the absence of jelly envelope material or synthetic high polymer. An undiluted sperm fluid from the vas deferens fertilized coelomic eggs in the absence of the jelly envelope material or synthetic high polymer. The fertilized eggs developed beyond gastrulae and formed tail bud embryos. These results indicate that the fertilization process does not depend upon the presence of jelly envelope material or synthetic high polymer and that the sperms within the vas deferens are already capable of fertilizing the eggs inC. pyrrhogaster. The sperm suspension in Holtfreter's balanced salt solution (H-sperm) fertilized the coelomic eggs without the jelly envelope material or synthetic high polymer. These eggs had been suspended in Holtfreter's balanced salt solution (H) or in 1/20 strength H (1/20 H) prior to insemination (H-eggs or 1/20 H-eggs). In contrast, the sperm suspension in 1/20 H (1/20 H-sperm) did not fertilize 1/20 H-eggs, but dit H-eggs. In the latter case, H surrounding the eggs may affect sperms, allowing them to be fertilized. The 1/20 H-sperms regained their ability to fertilize 1/20 H-eggs on re-exposure to H. The 1/20 H-sperm also fertilized jelly eggs. The results of the dejellied egg experiment showed the same pattern. These results indicate that the sperms within the vas deferens lose their capacity to fertilize 1/20 H-eggs on exposure to low ionic strength solution (1/20 H); this capacity is restored by exposure to high ionic strength solution (H) or to jelly envelope.     

Influence of short-term fasting during the luteal phase of the estrous cycle on ovarian follicular development during the ensuing proestrus of ewes

In order to study the effects of storage media and time of storage on the viability of unfertilized eggs of endangered Caspian brown trout (Salmo trutta caspius), the ova of this fish was stored in coelomic fluid and Cortland artificial media at 2–3 °C for 120 h. In this research, Cortland artificial medium was buffered with 20 mM of three different buffers: Hepes (C₈H₁₈N₂O₄S), Tris–HCl (C₄H₁₁NO₃–HCl) and sodium salt Hepes (C₈H₁₇N₂O₄SNa). The pH of these media were adjusted according to natural pH of coelomic fluid. The eggs that stored in these media fertilized at times 0 h (eggs fertilized prior to storage), 48, 72 and 120 h of post-stripping, using fresh and pooled sperm obtained from four to six males. According to the results of present study, time of storage showed a significant (p < 0.05) main effect on eyeing, hatching and eyed eggs mortality rates. Eyeing and hatching rates significantly (p < 0.05) decreased from 97.4 ± 2.1% and 95.1 ± 4.4% at time 0 (eggs fertilized prior to storage) to 77.9 ± 3% and 65.5 ± 5% after 120 h of storage. Within a similar period of time, eyed eggs mortality significantly (p < 0.05) increased from 2.4 ± 2.4% to 17.2 ± 3.9%. No significant (p > 0.05) main effect was found among media buffered with Tris–HCl (82.8 ± 3.2%, 73.4 ± 5.4%, 12.1 ± 4.5%), Hepes (88.2 ± 3.4%, 80.7 ± 5.5%, 9.3 ± 3.4%), sodium salt Hepes (77.8 ± 3.8%, 69.3 ± 5.7%, 12.2 ± 3.9%) and coelomic fluid (84.8 ± 3.8%, 77.7 ± 5.1%, 8.9 ± 2.7%) for eyeing, hatching and eyed eggs mortality rates. There was a negative correlation (r = −0.895, p < 0.001) between eyed eggs mortality and hatching rates. In conclusion, unfertilized eggs of endangered Caspian brown trout can be successfully stored for 48 h without significant loss of fertility. But, storage for 120 h results in the falling of hatching rate. In addition, no significant difference was found between viability rates of ova stored in coelomic fluid and artificial media, 120 h post-storage. It reveals that artificial media could be substituted for coelomic fluid as storage medium at least for 120 h in Caspian brown trout.     

Analyses of Oviductal Pars Recta-Induced Fertilizability of Coelomic Eggs in Xenopus laevis

The acquisition of fertilizability in coelomic eggs of Xenopus laevis has been shown to be correlated with the physical, biochemical, and ultrastructural alterations of the egg envelope [coelomic envelope (CE)] induced during the passage of eggs through the pars recta portion of the oviduct. However, no direct evidence that the pars recta renders eggs fertilizable has yet been presented. In this study, we show that coelomic eggs are highly fertilizable when they are incubated with continuous shaking for 4 h at 15 degrees C in pars recta extract (PRE) derived from females prestimulated by pregnant mare serum gonadotropin. The PRE from pituitary-stimulated Bufo japonicus was as potent as homologous PRE in rendering Xenopus eggs fertilizable. Incubation of coelomic eggs in PRE for 30 min induced a dramatic increase in the rates of sperm binding to the envelope to a level equivalent to that exhibited by the envelope from uterine eggs (VEs). The CE-to-VE ultrastructural conversion and a 43k-to-41k hydrolysis of the envelope glycoprotein component started 5 min after, and were completed by 15 min after, the start of incubation in PRE and were accompanied by an exposure of a new N-terminal sequence typical to gp41. Thus, the biochemical and ultrastructural conversions and the sperm-binding activity of the envelope induced by PREs, although being prerequisite, were not sufficient to render coelomic eggs fully accessible to fertilizing sperm.     

Concentrations of erythromycin and azithromycin in mature Chinook salmon Oncorhynchus tshawytscha after intraperitoneal injection, and in their progeny

A single dose (40 mg kg(-1)) of erythromycin or azithromycin dihydrate was injected intraperitoneally into maturing female fall Chinook salmon 12 to 32 d before spawning to observe the distribution, retention and clearance of the drugs in plasma, kidney, coelomic fluid and egg vitellin, and their persistence in alevins derived from these fish. Salmon administered prophylactic dosages of erythromycin as subadults were also included to investigate potential interactive effects of oral and injected treatments on reproductive performance and antibiotic clearance. Erythromycin was rapidly cleared from plasma and coelomic fluid, but was detected in the kidney (3.52 to 12.40 microg g(-1)) and egg vitellin (5.32 to 8.87 microg ml(-1)) of all fish at spawning. High, stable concentrations of azithromycin were detected in plasma (14.66 to 20.33 microg ml(-1)), kidney (43.16 to 59.96 microg g(-1)), coelomic fluid (2.52 to 5.50 microg ml(-1)) and egg vitellin (12.65 to 23.51 microg ml(-1)). Oral administration of erythromycin to subadult salmon did not significantly affect tissue concentrations of either erythromycin or azithromycin administered by prespawning injection. Reductions in the percentage of eggs that yielded live embryos at the eyed stage of development occurred among eggs derived from females that had received orally administered erythromycin as subadults. Erythromycin was not detected in unfed fry derived from adults injected with the drug prespawning, but azithromycin was present for more than 2 mo after the onset of exogenous feeding.     

Purification of the most abundant protein in the coelomic fluid of a sea urchin which immunologically cross reacts with 23S glycoprotein in the sea urchin eggs

The most abundant glycoprotein in the coelomic fluid of sea urchin Anthocidaris crassispina was purified and its subunit structure, molecular form in the native state, amino acid composition, and electron micrographic image were studied. The results showed that the protein in its native state was basically a tetramer with a total molecular weight of about 700,000, which was in equilibrium with a high molecular weight form corresponding to an octamer. The electron micrograph of the tetramer showed two ellipsoidal units aligned in parallel with a wide gap in between. The subunits all had the same molecular weight of 180,000 +/- 10,000 and were disulfide bonded in pairs. The carbohydrate content was about 16% with mannose and fucose as the two most abundant sugars. Although this protein accounted for 70% of the total protein in the coelomic fluid, it did not take part in the known activities of the fluid, namely hemagglutination and coagulation. Despite its structural similarity to the mammalian alpha-2-macroglobulin or reptilian and avian ovomacroglobulins it did not interact with bovine trypsin or chymotrypsin. This protein showed immunological cross reactivity with 23S glycoprotein purified from sea urchin eggs which, we believe, corresponds to the previously described 22-27S protein particles in eggs.     

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.     

Alteration of structure and penetrability of the vitelline envelope after passage of eggs from coelom to oviduct in Xenopus laevis.

The vitelline envelope (VE) that surrounds an egg released from the ovary into the coelom of Xenopus laevis differs markedly, in structure and penetrability, from the VE surrounding an oviposited egg. In a coelomic egg, the filaments that form the VE are arranged in distinct fascicles or bundles. The exterior surface of the VE is irregular in contour and is permeated by channels. In an oviposited egg, the filaments are evenly dispersed and lack a fasciculated arrangement; the exterior surface is smooth and no channels are present. The fascicular arrangement of fibrils in the coelomic VE is maintained only at neutral pH, and is not visibly altered by the cortical reaction. VEs from coelomic eggs retain their fasciculated morphology after isolation from the egg. In an in vitro test system, sperm penetrated VEs isolated from oviposited eggs, but failed to penetrate VEs isolated from coelomic eggs. The structural transformation of the VE from the coelomic type to the oviposited type occurs in the first 1-cm segment of the oviduct, prior to addition of jelly to the egg. Neither intact jelly, solubilized jelly, nor jelly extracts were capable of altering the structural organization of coelomic VEs, suggesting that the structural transformation of the VE is effected by some oviducal factor other than jelly.     

The Properties of the Oviducal Pars Recta Protease which Mediates Gamete Interaction by Affecting the Vitelline Coat of a Toad Egg

SDS-PAGE analyses of the vitelline coats (VCs) of coelomic eggs (CEVC) and uterine eggs (UEVC) of Bufo japonicus revealed that the UEVC lacks the 40K–52K molecular weight components present in the CEVC; this is concomitant with the increased stainability of a 39K component and the appearance of a 36K component. These macromolecular alterations, accompanied by the acquisition of egg fertilizability, were induced when coelomic eggs were treated with the contents of secretory granules obtained from the oviducal pars recta (PRG). Gel-filtration of PRG in combination with hydrolytic assays employing either fluorescamine-labeled CEVC or a variety of synthetic substrates showed that the CEVC to UEVC alterations are ascribable to the action of a protease hydrolyzing specifically peptidyl-Arg-MCAs in a highly Ca²⁺-dependent way. This enzyme, which has an optimal pH of 8.0–8.2, is inhibited by soybean trypsin inhibitor and leupeptin, as well as by such serine protease inhibitors as DFP and p-APMSF. On the basis of a SDS-PAGE analysis, its molecular weight is estimated to be 66K. Treatment of coelomic eggs with the partially purified PR protease did not render the eggs fertilizable, although CEVC to UEVC macromolecular alterations were effected. We conclude that the action of this oviducal protease in partially hydrolyzing the VC is a prerequisite but insufficient in itself to render the coelomic eggs fully accessible to a fertilizing sperm.     

Immunoelectron Microscopic Demonstration of Cortical Granule Lectins in Coelomic, Unfertilized and Fertilized Eggs of Xenopus laevis

Immunoelectron microscopic studies demonstrated cortical granule lectins (CGLs) in coelomic, unfertilized and fertilized eggs of Xenopus laevis . An antiserum raised against purified cortical granule lectin 1 specifically reacted with the CGLs in immunoblotting and agar diffusion tests. When ultrathin sections were treated with the antiserum and protein A-gold solution, gold particles, indicating antigenic sites, were seen over cortical granules of coelomic and unfertilized eggs, and over the perivitelline space, the vitelline coat and the condensed region of the fertilization layer of fertilized eggs. The pre-fertilization layer immediately adjacent to the outer margin of the vitelline coat in unfertilized eggs was free from gold particles. These observations suggest that released CGLs permeate through the vitelline coat of fertilized eggs and interact with the pre-fertilization layer mainly at the outer margin of the vitelline coat, resulting in formation of the fertilization layer which acts as a block to polyspermy.     

Hemolytic function of opsonizing proteins of earthworm's coelomic fluid

Synthetic 2-hydroxyethylmethacrylate copolymer particles (HEMA) can be opsonized in the coelomic fluid of Eisenia foetida earthworms. The incomplete coelomic fluid (i.e. the coelomic fluid after incubation with HEMA particles) exerts a lower level of hemolytic activity compared to complete coelomic fluid. The decreased hemolysis can be compensated by the addition of isolated opsonins. On the other hand, isolated opsonins do not possess direct hemolytic capacity. It can be suggested that at least one of the isolated opsonins is involved in the hemolytic process. These results support the hypothesized cooperation of humoral and cellular mechanisms in earthworm defence.     

Hemagglutinin activity in Nereis coelomic fluid

1. Nereis coelomic fluid agglutinates rat, mouse, chicken, guinea pig and rhesus monkey erythrocytes (RBC). 2. Lipid fractions of the particulate matter from coelomic fluid are hemagglutinins exhibiting different activity inhibition profiles with complex polysaccharides. 3. The high mol. wt hemagglutinin from coelomic fluid supernatant is not a protein and is inhibited by bovine submaxillary mucin (BSM), thyroglobulin, transferrin and their asialo derivatives. 4. Coelomic fluid supernatant has a population of low mol. wt protein hemagglutinins inhibited by BSM, fetuin, antiserum to coelomic fluid and some mannan preparations. 5. Hemagglutination by lipids characterized by RBC specificity and specificity for inhibition by carbohydrate is noteworthy and may be significant in studies of cellular interactions and immunity in invertebrates.     

Oviducal pars recta-induced degradation of vitelline coat proteins in relation to acquisition of fertilizability of toad eggs

In the toad Bufo bufo japonicus the vitelline coat (VC) of the uterine egg (UEVC) is more readily lysed by the sperm lysin than the VC of coelomic egg (CEVC). Fluorometric determinations of released proteins after incubation of the VC with the sperm lysin in vitro revealed that the CEVC is not completely refractory to the lysin but increases in susceptibility after treatment with a pars recta extract (PRE). Experiments employing isolated pars recta granules showed that both this increase of VC susceptibility and the acquisition of egg fertilizability are ascribable to the contents of the granules. SDS-PAGE analyses of VC proteins revealed that in comparison with CEVC, UEVC lacks 40–52K proteins concomitant with the increased stainability of 39K protein and the appearance of 36K protein. These changes in SDS-PAGE profiles were observed either in oviducal eggs after passage through the pars recta or in coelomic eggs treated with PRE but were inhibited when coelomic eggs were treated with PRE containing soybean trypsin inhibitor (SBTI) and leupeptin. Likewise, the acquisition of fertilizability by treatment of coelomic egg with PRE was inhibited by SBTI. Dejellied uterine eggs were successfully fertilized when pretreated with trypsin inhibitors before insemination but were not fertilized when pre-treated with concanavalin A. We propose that the hydrolytic degradation of certain VC proteins due to the tryptic activity of pars recta granules renders the VC susceptible to the sperm lysin, so that the eggs are made receptive to a fertilizing sperm.     

Identité immunologique et métabolique entre le liquide coelomique,les coelomocytes et les ovocytes de Perinereis cultrifera (Annélide Polychète)

Immunological study of Perinereis cultrifera reveals the existence of an identical antigen in the coelomic fluid of females (oocyte diameter > 140 (μm), in oocytes, and in coelomocytes. This factor is not found in the body fluid of males or young females.

The elution patterns obtained after Sephadex chromatography shows a similar glycoprotein fraction (fraction I) in the coelomic fluid, in coelomocytes, and in soluble oocyte extracts. This fraction includes the main part of the antigenic components of the coelomic fluid.

Gas chromatography reveals that identical monosaccharides are present, albeit in varying proportions, in samples of fraction I obtained from the different sources.

The metabolic interrelationships of coelomocytes, coelomic fluid and oocytes is discussed. Glycoprotein synthesized by coelomocytes may be discharged into the coelomic fluid and contribute to the development of the cortical alveoli of the oocytes. No evidence of an involvement of this material in yolk synthesis has been obtained.     

Viviparity of larvae,a new type of development in phoronids (Lophophorata: Phoronida)

A new type of phoronid development, viviparity of larvae, has been discovered in a new phoronid species that lives as a commensal of digging sand shrimps in Vostok Bay, the Sea of Japan. The embryos develop in the mother’s trunk coelom up to the young larva stage. During development, embryos increase in size twice and probably obtain nutriment from the mother’s coelomic fluid. Spawning occurs by young larvae, which are released through nephridiopores. The new type of development is described in a phoronid that has a small body size but a high fertility, producing large amounts of extremely small eggs. The combination of viviparity and large number of eggs increases the number of competent larvae that can undergo metamorphosis in the burrows of shrimps.     

Measurement of oxygen consumption and sampling of body fluids of echinoderms in situ

A number of echinoderm species common to the coast of Malta have been subjected to respirometry measurement and coelomic fluid sampling. Experiments were carried out in situ by SCUBA divers and the results compare favourably with parallel laboratory investigations. One species of sea urchin had low coelomic fluid pO₂ when consuming more oxygen at night. The pH of the coelomic fluid of all the species was acid to the sea water. The value of making in situ measurements is discussed.