The importance of perivitelline fluid convection to oxygen uptake of Pseudophryne bibronii eggs

The ciliated epithelium of amphibian embryos produces a current within the perivitelline fluid of the egg that is important in the convective transfer of oxygen to the embryo's surface. The effects of convection on oxygen uptake and the immediate oxygen environment of the embryo were investigated in Pseudophryne bibronii. Gelatin was injected into the eggs, setting the perivitelline fluid and preventing convective flow. Oxygen consumption rate (M(.)o?) and the oxygen partial pressure (Po?) of the perivitelline fluid were measured in eggs with and without this treatment. M(.)o? decreased in eggs without convection at Gosner stages 17-19 under normoxia. The lack of convection also shifted embryos from regulators to conformers as environmental Po? decreased. A strong Po? gradient formed within the eggs when convection was absent, demonstrating that the loss of convection is equivalent to decreasing the inner radius of the capsule, an important factor in gas exchange, by 25%. M(.)o? also declined in stage 26-27 embryos without cilia-driven convection, although not to the extent of younger stages, because of muscular movements and a greater skin surface area in direct contact with the inner capsule wall. This study demonstrates the importance of convective flow within the perivitelline fluid to gas exchange. Convection is especially important in the middle of embryonic development, when the perivitelline space has formed, creating a barrier to gas exchange, but the embryos have yet to develop muscular movements or have a large surface area exposed directly to the jelly capsule.     

The influence of water pH on the perivitelline fluid of perch (Perca fluviatilis) eggs

The diameter, trans-chorion potential difference and perivitelline-fluid pH of the eggs of perch (Perca fluviatilis) have been measured in a range of water pH (8-4.5). The water used was relatively nutrient rich. Eggs transferred from pH 8 to pH 4.5 shrink; probably as a result of loss of water from perivitelline fluid. Trans-chorion potential differences are close to zero over the range of pH. Mean perivitelline-fluid pH is higher than external water pH at pH 6.7 and above. Below pH 6.7, mean perivitelline-fluid pH is approximately the same as external water pH. The results suggest a non-Donnan distribution of hydrogen ions. The results are compared with data on salmonid eggs in nutrient-poor waters.     

Adaptive value of the volume of perivitelline fluid in the eggs of euphausiids (Crustacea: Euphausiacea)

The perivitelline space of the euphausiid egg serves to maintain eggs buoyancy. It is believed that in oceanic species the volume of the perivitelline space is smaller than in neritic [32, 37, 40]. Published and original data suggest a more complicated tendency for perivitelline space volume and, correspondingly, egg buoyancy in euphausiids, which decrease in the direction: continental slope → shelf → ocean. This tendency is well explicable by adaptations of euphausiid species to the specific conditions of these biotopes. The possible mechanisms of these adaptations are discussed.     

Slow calcium waves accompany cytokinesis in medaka fish eggs

Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.     

Osmotic properties of the perivitelline fluid and some properties of the chorion of Atlantic salmon eggs (Salmo salar)

The chorion and perivitelline fluid of Atlantic salmon eggs were investigated by chemical and physical methods. The turgor pressure of the chorion in water hardened eggs was about 60 mm Hg. With newly stripped eggs in fresh water a similar pressure was achieved after about one day but the process of water uptake could be osmotically inhibited by adding high molecular weight substances to the external medium. Perivitelline fluid contained about 58 % water the remainder being a high molecular weight substance consisting mainly of protein but also containing significant quantities of carbohydrate and lipid. The functions of the chorion are discussed with reference to salmon eggs and also to marine pelagic fish eggs such as those of the plaice.     

Elemental composition of the perivitelline fluid in early Drosophila embryos

Samples of the perivitelline fluid in the polar pockets of preblastoderm Drosophila embryos were analyzed with an electron microprobe, and the results compared with analyses of adult hemolymph. The concentrations of sodium, magnesium, calcium, chlorine, and phosphorus are about the same in these two fluids; but potassium and sulfur are three to four times higher in perivitelline fluid. Moreover, the concentrations of these elements in the anterior and posterior pockets of the same embryos were compared. The former five elements seem to be about 10% more concentrated in the anterior pocket; but the latter two show no significant difference between pockets.     

Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes

Unfertilized eggs of the medaka fish (Oryzias latipes) were injected with pH-buffered calcium buffers. Medaka egg activation is accompanied by a transient increase in cytoplasmic free calcium (Gilkey, J. C., L. F. Jaffe, E. B. Ridgway, and G. T. Reynolds, 1978, J. Cell Biol., 76:448-466). The calcium buffer injections demonstrated that (a) the threshold free calcium required to elicit the calcium transient and activate the egg is between 1.7 and 5.1 microM at pH 7.0, well below the 30 microM reached during the transient, and (b) buffers which hold free calcium below threshold prevent activation of the buffered region in subsequently fertilized eggs. Therefore an increase in free calcium is necessary and sufficient to elicit the calcium transient, and the calcium transient is necessary to activate the egg. Further, these results are additional proof that the calcium transient is initiated and propagated through the cytoplasm by a mechanism of calcium- stimulated calcium release. Finally, a normal calcium transient must propagate through the entire cytoplasm to ensure normal development. Unfertilized eggs were injected with pH buffers to produce short-term, localized changes in cytoplasmic pH. The eggs were then fertilized at various times after injection. In other experiments, unfertilized and fertilized eggs were exposed to media containing either NH4Cl or CO2 to produce longer term, global changes in cytoplasmic pH. These treatments neither activated the eggs nor interfered with the normal development of fertilized eggs, suggesting that even if a natural change in cytoplasmic pH is induced by activation, it has no role in medaka egg development. The injected pH buffers altered the rate of propagation of the calcium transient through the cytoplasm, suggesting that the threshold free calcium required to trigger calcium-stimulated calcium release might be pH dependent. The results of injection of pH-buffered calcium buffers support this conjecture: for a tenfold increase in hydrogen ion concentration, free calcium must also be raised tenfold to elicit the calcium transient.     

Localization of a chymotrypsin-like protease to the perivitelline space of Xenopus laevis eggs.

A chymotrypsin-like protease is released from Xenopus laevis eggs at activation and is involved in conversion of the vitelline envelope to the fertilization envelope. To localize this enzyme in unactivated and activated eggs, we used the synthetic peptide substrate succinylalanylalanylprolylphenylalanyl-4-methoxy-2-naphthylamide whose product can be visualized using transmission electron microscopy. Protease product was localized within the perivitelline space of unactivated eggs, appearing as strings of beads. No protease activity was detected in activated eggs, which is consistent with the observation that the protease is released from the egg at activation.     

Vitrification of the inner perivitelline layer of chicken eggs for use in the sperm-egg interaction assay

The sperm-egg interaction assay is a good predictor of the fertilizing potential of rooster semen; the ability of chicken sperm to interact with the egg can be assessed by counting the number of holes in the inner perivitelline layer (IPVL) of a freshly laid egg. Atlhough isolated IPVL can be stored for up to 24 h, preservation of IPVL for prolonged intervals in liquid nitrogen would facilitate the sperm-egg interaction assay. The objective of this study was to adapt the technique of vitrifying swine oocytes for use with the IPVL. Our hypothesis was that vitrification would not alter the ability of the membrane to bind sperm; therefore, there would be no difference between vitrified and fresh IVPL in the number of hydrolysis holes made by sperm. Our hypothesis was supported; there were no differences in the mean ± SEM number of holes made by the same sample of sperm in vitrified and in fresh membranes (146.0 ± 17.7 holes/mm² IPVL and 159.5 ± 17.7 holes/mm² IPVL, respectively, P > 0.05; n = 123 IVPLs tested). Furthermore, 80% of frozen-thawed membranes were recovered intact. Because vitrification did not significantly change the ability of membranes to bind sperm, vitrified membranes can be safely used for the sperm-egg interaction assay. Vitrified IVPL would ensure availability for sperm evaluation and facilitate wide distribution of IPVL, enabling assays to be conducted even in the absence of facilities or expertise to prepare membranes.     

The surface structure of Antarctic fish eggs and its use in identifying fish eggs from the Southern Ocean

Summary The surface structure of eggs of Notothenia neglecta, Nototheniops larseni, Nototheniops nudifrons (Nototheniidae), Champsocephalus gunnari and Chaenocephalus aceratus (Channichthyidae), collected near Elephant Island (Atlantic sector of the Southern Ocean) and of Notothenia rossii rossii (Nototheniidae) from Kerguelen waters (Indian sector of the Southern Ocean) was studied by scanning electron microscopy (SEM). Eggs of N. larseni differed from the other species by containing a micropyle with two micropyle pits. External morphological characteristics of the egg envelope such as the ultrastructure of the zona radiata, its interpore distances and the diameter of the micropyle were species-specific and could aid in the identification of fish eggs collected from the Southern Ocean.     

Microsatellite typing of sperm trapped in the perivitelline layers of avian eggs: a cautionary note

Recently Carter et al. (2000) have shown that it is possible to amplify microsatellite markers from the DNA of spermatozoa trapped in the perivitelline layers of avian eggs. This makes it possible to establish whether sperm reaches the ovum and to identify the males that contribute sperm competing to fertilise an egg. In this article we quantify the genotyping error rate associated with the biased amplification of differently sized alleles. We conclude that multiple marker loci should be used and that those markers where the competing males' alleles differ by more than ten base pairs should be avoided.     

The calcium transient characteristics induced by fluid shear stress affect the osteoblast proliferation

Ca²⁺ signaling is essential for bone metabolism. Fluid shear stress (FSS), which can induce a rapid release of calcium from endoplasmic reticulum (ER) to produce calcium transients, plays a significant role in osteoblast proliferation and differentiation. However, it is still unclear of how calcium transients induced by FSS activating a number of downstream signals which subsequently regulate cell functions. In this study, we performed a group of Ca²⁺ transients models, which were induced by FSS to investigate the effects of different magnitudes of Ca²⁺ transients in osteoblast proliferation. Further, we performed a global proteomic profile of MC3T3-E1 cells in different Ca²⁺ transients models stimulated by FSS. GO enrichment and KEGG pathway analysis revealed that the TCA cycle was activated in the proliferating process. The activation of TCA needed mitochondrial Ca²⁺ uptake which were influenced by the amplitude of Ca²⁺ transients induced by FSS. Our work elucidate that osteoblast proliferation induced by FSS was related to the magnitude of calcium transients, which further activated energetic metabolism signaling pathway. This work revealed further understanding the mechanism of osteoblast proliferation induced by mechanic loading and help us to design new methods for osteoporosis therapy.     

The spermatozoa and eggs of the cardinal fish

The gametes of the cardinal fish Apogon imberbis had the following characteristic: the semen contained both biflagellate (  c . 20% in the investigated samples) and monoflagellate (  c . 80%) spermatozoa. The spermatozoa were acrosomless, had an ovoid head, and a cylindrical midpiece. The midpiece contained a high number of mitochondria (seven–10). The distal centriole was fastened to the nucleus via electron-dense material. The proximal centriole was completely reduced. The eggs were spherical, had a smooth surface and one micropyle consisting of the micropylar channel, the vestibulum and a ring-like convulsion bordering the vestibulum. The zona radiata was formed out of four layers which could be distinguished by their fine structural and histochemical features. It was only c . 1·5 µm thick and therefore much thinner than in other marine teleosts. The internal organization of the eggs (homogenous protein yolk containing cortical granules and lipid droplets) as well as the qualitative and quantitative biochemical composition of proteins, lipids and carbohydrates was similar to that of the pelagic eggs of other marine species.     

Yolk utilization in stick insects entails the release of vitellin polypeptides into the perivitelline fluid

This study investigates the developmental fate of vitellin (Vt) polypeptides generated by limited proteolysis in an insect embryo. To this end, a number of polyclonal (pAb) and monoclonal antibodies (mAb) were raised against the yolk sac and the perivitelline fluid of late embryos of the stick insect Carausius morosus. Two dimensional immuno gel electrophoresis and Western blotting demonstrate that polypeptides resulting from Vt processing are present both in the yolk sac and the perivitelline fluid. At the confocal microscope, different labelling patterns were detected in the ooplasm depending on the stage of development attained by the embryo. At early developmental stages, label is associated with large unsegmented portions of the fluid ooplasm. During embryonic development, the fluid ooplasm is gradually transformed into yolk granules by intervention of vitellophages. Prior to dorsal closure, the yolk sac is separated from the perivitelline fluid by interposition of serosa cells (the so called serosa membrane). Several mAbs raised against the perivitelline fluid react specifically with this membrane suggesting that the release of Vt polypeptides from the yolk sac occurs by intracellular transit through the serosa cells. By immunocytochemistry, gold label appears associated with the cell surface and a number of vacuoles of the serosa membrane. These data are interpreted as suggesting that Vt polypeptides resulting from limited proteolysis in stick insect embryos are not exhaustively degraded within the yolk sac, but are instead transferred transcytotically to the perivitelline fluid through the serosa membrane.     

Effect of Schistosoma mansoni infection on fecundity and perivitelline fluid composition in Biomphalaria glabrata

Egg production in the snail, Biomphalaria glabrata, infected with Schistosoma mansoni declined on day 23 postinfection, and was significantly lower than uninfected control snails by day 28 and thereafter. Protein and galactogen content of eggs produced by infected snails did not change during the period of reduced fecundity. This suggests that decreased hemolymph nutrient levels (rather than depleted albumen gland reserves) are responsible for inhibition of snail egg production. Growth rates of infected and uninfected snails were indistinguishable from days 14 through 35 postinfection. The hatching success of eggs produced by infected snails decreased slightly beginning at day 21 postinfection.     

Selective sequestration of an oviductal fluid glycoprotein in the perivitelline space of mouse oocytes and embryos

Previously, we identified a 215 kd glycoprotein, GP215, which is associated with postovulatory oocytes and embryos, but not with preovulatory oocytes (Kapur and Johnson, '85). In this paper a polyclonal antibody that specifically recognizes GP215 has been used to study the distribution of the molecule in association with ova and preimplantation embryos and in the female reproductive tract. GP215 is present in epithelial cells lining the cranial portions of the oviduct and in oviductal fluid, ovarian bursal fluid, and medium conditioned by oviductal tissue in vitro. Immunofluorescence assays of the ovum and early embryo show that GP215 is sequestered in the perivitelline space. Since preovulatory oocytes exposed to bursal fluid in vitro acquire GP215, we hypothesize that GP215 is synthesized and secreted by the oviductal epithelium and secondarily associates with the ovulated oocyte. Sequestration of GP215 within the perivitelline space is relatively specific since mouse serum albumin, a major constituent of oviductal fluid, and other high molecular weight proteins are not similarly retained. These observations indicate that the composition of the perivitelline space may be significantly different from the greater environment external to the zona pellucida such that fertilization and early development of mammalian ova potentially take place in a distinct perivitelline microenvironment.     

Quantification of spermatozoa in the sperm-storage tubules of turkey hens and the relation to sperm numbers in the perivitelline layer of eggs

This study was conducted to determine the number of spermatozoa residing in the oviduct sperm-storage tubules (SST) and the relationship between these numbers and the number of spermatozoa embedded in the perivitelline layer of oviductal eggs after a single insemination of 200 x 10(6) spermatozoa. The SST of hens inseminated within one week before the expected onset of egg production were filled faster (4 h vs. 2 days) and possessed more spermatozoa (4.1 vs. 2.0 x 10(6)) than the SST of hens inseminated after the onset of egg production. Furthermore, for hens in egg production, significantly fewer spermatozoa were recovered from the SST if the hen was inseminated within 2 h before or after oviposition than if inseminated more than 2 h before or after the oviposition. There was a strong positive correlation between the number of spermatozoa in the SST and the number of spermatozoa embedded in the perivitelline layer of the oviductal eggs (r = 0.85, p less than 0.01). These data show that the population of spermatozoa actually accepted by the SST is quite small relative to the number of spermatozoa inseminated and that maximum sperm-storage is achieved when the hen is inseminated just prior to the onset of egg production. It is suggested that the sperm-storage capacity of the oviduct and the quality of the semen sample can be estimated on the basis of numbers of spermatozoa embedded in the egg perivitelline layer.     

First proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition

Pomacea canaliculata is a freshwater snail that deposits eggs on solid substrates above the water surface. Previous studies have emphasized the nutritional and protective functions of the three most abundant perivitelline fluid (PVF) protein complexes (ovorubin, PV2, and PV3) during its embryonic development, but little is known about the structure and function of other less abundant proteins. Using 2-DE, SDS-PAGE, MALDI TOF/TOF, and LC-MS/MS, we identified 59 proteins from the PVF of P. canaliculata, among which 19 are novel. KEGG analysis showed that the functions of the majority of these proteins are "unknown" (n = 34), "environmental information processing" (10), 9 of which are related to innate immunity, and "metabolism" (7). Suppressive subtractive hybridization revealed 21 PVF genes to be specific to the albumen gland, indicating this organ is the origin of many of the PVF proteins. Further, the 3 ovorubin subunits were identified with 30.2-35.0% identity among them, indicating their common origin but ancient duplications. Characterization of the PVF proteome has opened the gate for further studies aiming to understand the evolution of the novel proteins and their contribution to the switch to aerial oviposition.