Fertilization of the eggs ofCynops pyrrhogaster (japanese newt) after immersion in water

Summary Cynops pyrrhogaster oviducal eggs with and without jelly envelopes (jelly egg and dejellied egg respectively) were immersed in water, and then inseminated artificially. After 1 h of immersion in water, more than half the dejellied eggs were fertilized and developed, but no jelly eggs developed. The rapid decrease in the ability of jelly eggs to be fertilized after immersion in water is not due to a deficiency in the egg. Our results make it clear that hydrated jelly envelopes prevent the eggs from fertilizing. The ability of the egg to be fertilized decreases for a long time in water and this decrease proceeds more slowly in De Boer's solution or Holtfreter's balanced salt solution than in water.     

Microscopic organization of the sperm storage tubules in the oviducal gland of the female gummy shark (Mustelus antarcticus), with observations on sperm distribution and storage

Oviducal gland morphology, the microscopic organization of the terminal zone, and sperm storage were described in the female gummy shark (Mustelus antarcticus). Mustelus antarcticus is a nonplacental viviparous hound shark, which displays minimal histotrophy during embryonic development. The animals examined represented all stages of maturity and gestation. The oviducal gland was found to have the same fundamental zonation as in most chondrichthyans. Using recent terminology, the oviducal gland of chondrichthyans has an anterior club zone, followed by a papillary zone, both of which produce jelly that surrounds the egg, a baffle zone that elaborates the tertiary egg envelope and a terminal zone, where sperm storage occurs. Each zone is composed of simple tubular glands that connect to transverse grooves, which extend the full width of the gland. The exception is the terminal zone, which does not have transverse grooves but consists of individual tubules. The microscopic organization and histochemical nature of the zones display similar patterns to those of other chondrichthyan genera. Tubules of the terminal zone contain four types of cell: ciliated cells, alcian blue‐positive secretory cells, periodic acid‐Schiff and alcian blue‐negative secretory cells, and secretory columnar cells. These tubules end in recesses, the sperm storage tubules, which extend beyond the periphery of the baffle zone. Sperm were stored in the sperm storage tubules of all maturing and mature animals examined. Of note is the observation of stored sperm in an animal 1 year prior to first ovulation. Sperm were also observed throughout the uterine sphincter, body of the uterus, isthmus, and oviduct of maturing and mature animals, and in the uterine sphincter of an immature animal. These sperm represent immediately postcopulation aggregations of sperm and sperm in the process of migrating to the site of storage or to the site of fertilization. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Factors influencing the embryonic development and hatchling size of the oceanic squid Illex coindetii following in vitro fertilization

Egg masses of oceanic squid accidently collected in the wild have been observed only from a few spawning events in aquaria, and as a consequence, the study of their embryos and hatchlings is very limited. Here, we used in vitro fertilization techniques to understand the abiotic factors that influenced egg development and hatchling performance of the ommastrephid squid Illex coindetii from the Mediterranean Sea. Egg and hatchling sizes of these ommastrephids are close to the minimum for cephalopod species, and therefore, short embryonic developmental periods were expected under a Mediterranean temperature regime. Low incubation densities and the use of antibiotics in the incubation medium resulted in relatively high survival rates to hatchling. Hatching time was inversely proportional to incubation temperature and ranged from 5 to 11 days at 21 °C and 13 °C, respectively. The addition of oviducal jelly to the eggs immediately after fertilization, followed by another addition at the beginning of organogenesis, resulted in better chorionic expansion, with an increase in egg diameter, delayed hatching and higher hatchling weight, which suggests a better use of yolk reserves. Larger hatchlings in length and weight also tended to be produced at low temperature, probably also due to better yolk absorption. As this species spawns all year round, size differences between summer and winter hatchlings may be suspected. In vitro fertilization techniques used in the laboratory proved to be useful in shedding some light on the early life of this oceanic squid. Further research is needed to elaborate on tests to predict gamete quality and to develop methods to avoid premature hatching.     

The Isolation and Fractionation of Chicken Egg White Ovomucin

Three fractions of chicken egg white ovomucin were obtained from ovomucin precipitated by the classical method of dilution of egg white with water. The first fraction was obtained by dilute salt extraction of the precipitated ovomucin and consisted of smaller components of ovomucin. The second fraction was obtained by extraction with 1 M KCl of the precipitate remaining after dilute salt extraction and consisted of a heterogeneous mixture of components of larger mass. The third fraction, the ovomucin remaining after concentrated salt extraction, was insoluble.

The two soluble fractions were found to be unstable with time (pH 8, μ 0.2). The rate of breakdown was slower in concentrated salt solution. The final breakdown product(s) for both fractions appeared to be a 6 S component (s) of molecular mass 1.5 × 10⁵ daltons.     

The sexual mechanism of the reef bass Pseudogramma bermudensis and its implications in the classification of the Pseudogrammidae (Pisces: Perciformes)

The reef bass, Pseudogramma bermudensis, is an hermaphroditic percoid in which the testicular tissue is confined to a median lobe in the mid-dorsal wall of the oviduct. Development of an invagination of the ventral oviducal wall is correlated with the presence of maturing ova. These unique features indicate that Pseudogramma has had a separate evolutionary history and is not closely related to either the hermaphroditic Serranidae or the Grammistidae.     

INDUCTION OF THE ACROSOME REACTION ON THE EGG SURFACE OF DE-JELLIED SEA URCHIN EGGS BEFORE AND AFTER FERTILIZATION*

The capacity of the surface of sea urchin eggs to induce the acrosome reaction was assayed by estimating the rate of acrosome reaction of supernumerary spermatozoa in the presence of variously treated eggs before and after fertilization. DTT-disruption of the vitelline coat did not eliminate the acrosome reaction-inducing capacity. This capacity was retained after fertilization in eggs of both H. pulcherrimus and A. crassispina. The acrosome reaction-inducing capacity of the eggs was markedly decreased by treatment with trypsin. The low capacity of the trypsin-treated eggs was maintained after fertilization in H. pulcherrimus, but in A. crassispina the capacity returned to the pre-trypsin treatment level after fertilization. Fertilized eggs from which the fertilization membrane was mechanically removed retained the inducing capacity to a considerable extent, independent of the presence or absence of the hyaline layer, but the capacity diminished rapidly as cleavage proceeded. It was concluded from these data that the acrosome reaction of spermatozoa actually occurred at the surface of de-jellied eggs and that the inducing substance resides in the plasma membrane in addition to the fertilization membrane. A chemical difference between the inducing substance of egg surface and jelly substance is discussed.     

NMDA‐type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm

The N‐methyl d ‐aspartate type glutamate receptor (NMDAR) is a ligand‐gated cation channel that causes Ca²⁺ influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA‐seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster. Glutamate of at least 0.7 ± 0.5 mM was detected in the egg‐jelly substances along with acrosome reaction‐inducing substance (ARIS) and sperm motility‐initiating substance (SMIS). In the egg‐jelly extract (JE) that included the ARIS and SMIS, the acrosome reaction was inhibited by a NMDAR antagonists, memantine and MK801. MK801 also inhibited the spontaneous acrosome reaction in Steinberg's salt solution (ST). Furthermore, memantine and MK801 suppressed the progressive motility of the sperm in JE and spontaneous waving of the undulating membrane, which is the tail structure giving thrust for forward motility, in ST. The spontaneous waving of the undulating membrane was promoted when Mg²⁺, which blocks Ca²⁺ influx through gated NMDARs, was removed from the ST. In addition, the ARIS‐induced acrosome reaction was inhibited by a selective antagonist of the transient receptor potential vanilloid 4, whose activation might result in the membrane depolarization to release Mg²⁺ from the NMDAR. These results suggest that NMDAR acts together with other cation channels in the induction of the acrosome reaction and motility of the sperm during the fertilization process of C. pyrrhogaster.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U-¹⁴C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U-¹⁴C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions.     

Egg jelly influences sperm motility in the externally fertilizing frog,Crinia georgiana

Recent in vitro fertilization studies have revealed female and male × female interaction effects on the probability of fertilization. These findings suggest a mechanism of cryptic female choice via sperm–egg interactions. The egg jelly of anuran amphibians contains proteins that facilitate the chemoattraction and binding of sperm for fertilization. Here we show that egg jelly also influences the onset of motility and swimming velocity of motile sperm in the frog Crinia georgiana. Moreover, we found significant among female variation in the effects of egg jelly on sperm motility. We discuss this finding with respect to male and female effects on nonrandom fertilization observed in this species.     

Distribution of Lectin Binding Sites in Xenopus laevis Egg Jelly

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.     

Using citizen science to obtain data on large,floating gelatinous spheres from NE Atlantic,attributed to egg mass of ommastrephid squid (Oegopsida,Cephalopoda, Mollusca)

A total of 27 large, gelatinous spherical masses observed in coastal Norwegian waters from Nordland to Aust-Agder Counties in Norway, and off Lysekil in Sweden, Muljica Island in Croatia, Gulf of Naples in Italy, Reqqa Point in Malta, and Saint Mandrier in France, during the months of April to September 2001 to 2017, are reported. Individual spheres measured 0.3–2 m in diameter, averaging one metre (n?=?24, ±0.53 m), with all but four sighted in suspension in the water column between 0.5 and 52 m depth, in water temperatures ranging between 10–21°C. About half of all spheres contained a yellow-red streak through their gelatinous core. Tissue samples were not obtained. We attribute these gelatinous spheres to the egg masses of squid (Cephalopoda, Oegopsida), and most likely to the ommastrephid Todarodes sagittatus, given similarities with egg masses of T. pacificus.     

On the contents of the cortical granules from Xenopus laevis eggs

The extruded contents of the cortical granules in eggs of Xenopus laevis were solubilized by exposure to divalent metal ion chelators. Chelator extraction of cortical granule (CG) material from intact fertilized or artificially activated eggs was quantitated by fluorescence spectroscopy. The isolated fertilization envelope, formed upon interaction between CG material and the preexisting vitelline envelope, was also subject to extraction. An ultrastructural analysis revealed that chelator exposure resulted in the disruption of the structural integrity of the CG-derived F-component of the fertilization envelope. CG material was isolated from Xenopus ova by three procedures: (1) extrusion from artificially activated, dejellied eggs; (2) extraction of intact, fertilized eggs; and (3) extraction of isolated fertilization envelopes. Only 4–5% of the CG protein recovered by extrusion or by extraction of the intact fertilized egg could be associated with the isolated fertilization envelopes. One predominant polypeptide fraction with an identical relative mobility was demonstrated in all CG preparations upon polyacrylamide gel electrophoresis in SDS. Polymeric forms of CG protein were detected in chelator extracted preparations. The presence of an intact jelly coat during CG breakdown was a prerequisite to the transformation of the vitelline envelope to a fertilization envelope with altered physicochemical characteristics. Further, the CG-derived F-component of the fertilization envelope did not appear to play a critical role in determining the physicochemical properties of the fertilization envelope.     

The regulation of growth in the mesonephric kidney of adult Xenopus laevis by an endogenous inhibitor of proliferation.

The effect of dorsal lymph sac implanted tissue fragments, of a 100,000g kidney supernatant, and of various kidney-derived ultrafiltrate fractions on the percentage of DNA synthesizing cells in the mesonephric kidney of Xenopus laevis following partial unilateral nephrectomy was investigated autoradiographically. Using Amicon filters with cut-off values of MW 50,000 and 10,000, three ultrafiltrate fractions were obtained: a fraction containing molecules of MW 50,000 and less, a fraction containing molecules of MW 10,000 and less, and one containing molecules in the range of MW 10,000 to 50,000. The ultrafiltrates containing molecules of less than 10,000 MW were found to depress DNA synthetic activity on the sixth postoperative day by 30 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 showed no significant effect. It has been concluded that an endogenous inhibitor of proliferation, with the attributes of a chalone, is present in the fraction of less than 10,000 MW. The loss of inhibitor action following Pronase treatment of the ultrafiltrate suggests that the inhibitor substance may be a protein or polypeptide, or that such constituent may be the carrier for the active agent. Since a depression in DNA synthetic activity of 60% was obtained in normal adult mesonephric kidneys following the injection of the ultrafiltrate, it is concluded that both compensatory growth and reparative growth in the kidney of Xenopus laevis are regulated by a G₁ kidney chalone of less than 10,000 MW.     

Comparative transcriptome analysis of developmental stages of the Limonium bicolor leaf generates insights into salt gland differentiation

With the expansion of saline land worldwide, it is essential to establish a model halophyte to study the salt‐tolerance mechanism. The salt glands in the epidermis of Limonium bicolor (a recretohalophyte) play a pivotal role in salt tolerance by secreting excess salts from tissues. Despite the importance of salt secretion, nothing is known about the molecular mechanisms of salt gland development. In this study, we applied RNA sequencing to profile early leaf development using five distinct developmental stages, which were quantified by successive collections of the first true leaves of L. bicolor with precise spatial and temporal resolution. Specific gene expression patterns were identified for each developmental stage. In particular, we found that genes controlling salt gland differentiation in L. bicolor may evolve in a trichome formation, which was also confirmed by mutants with increased salt gland densities. Genes involved in the special ultrastructure of salt glands were also elucidated. Twenty‐six genes were proposed to participate in salt gland differentiation. Our dataset sheds light on the molecular processes underpinning salt gland development and thus represents a first step towards the bioengineering of active salt‐secretion capacity in crops.     

The occurrence of free and bound cytokinins in clubroots and Plasmodiophora brassicae infected turnip tissue cultures

This paper deals with the quantitative determination of free and bound cytokinins in clubroot tissue and in Plasmodiophora brassicae Woron, infected Brassica campestris L. callus tissue. The fractions were separated in a butanol soluble fraction containing the free cytokinins such as zeatin and zeatin riboside and a water soluble fraction containing the bound cytokinins. The butanol fraction was extensively purified and analysed by high pressure liquid chromatography (HPLC). The butanol fraction contained cytokinins which cochromatographed with zeatin and zeatin riboside and not with dihydrozeatin. Zeatin and zeatin riboside were quantitatively determined by HPLC. Recovery of the cytokinins varied between 30–50%. Clubs contained 50–160 ng zeatin and 210–300 ng zeatin riboside per g dry weight. Callus tissue contained 133 ng zeatin and 169 ng zeatin riboside per g dry weight. Clubs, callus as well as healthy tissue contain large amounts of bound cytokinins. Upon treatment of the water soluble fraction first with alkaline phosphatase and then with β-glucosidase biologically active fractions were found which coeluted with zeatin and zeatin riboside on Sephadex LH₂₀ in 20% ethanol. Evidence is presented for a novel cytokinin in the water soluble fraction which yields free zeatin and glucose-6-phosphate after treatment with β-glucosidase.     

Bracelet salt glands of the recretohalophyte Limonium bicolor: Distribution,morphology, and induction

Halophytes complete their life cycles in saline environments. The recretohalophyte Limonium bicolor has evolved a specialized salt secretory structure,the salt gland, which excretes Na⁺to avoid salt damage. Typical L. bicolor salt glands consist of 16 cells with four fluorescent foci and four secretory pores. Here, we describe a special type of salt gland at the base of the L. bicolor leaf petiole named bracelet salt glands due to their beaded-bracelet-like shape of blue auto-fluoresc...     

Xenopus laevis Egg Jelly Contains Small Proteins That Are Essential to Fertilization

The eggs of Xenopus laevis are surrounded by investment layers of egg jelly that interact with the sperm immediately prior to fertilization. Components of these egg jelly layers are necessary for the fertilization of the egg by incoming sperm. Eggs which are stripped of their jelly layers are refractile to fertilization by sperm, but the addition of solubilized jelly promotes fertilization. We have shown previously that the egg jelly layers are composed of a fibrous network of glycoconjugates which loosely hold smaller diffusible components. Extracts of these diffusible components were prepared by incubation of freshly ovulated eggs in high-salt buffers for 12 h at 4°C. This diffusible component extract, when incubated with sperm, promoted the sperm's ability to fertilize dejellied eggs in a dose-dependent manner. In contrast, the high-molecular-weight “structural” glycoconjugates of jelly that remain after extraction of the diffusible components did not increase fertilization efficiency of dejellied eggs nor did nonspecific proteins, carbohydrate polymers, or organic polymers. The diffusible components, analyzed by SDS–PAGE, consisted of a mixture of proteins from 4 to 180 kDa. The protein responsible for fertilization rescue appeared to be <50 kDa and appeared to self-aggregate or to bind to larger proteins. This protein component was required during sperm binding to the egg, its action required an intact egg vitelline envelope, and its action was independent of large soluble polymers such as Ficoll.     

The development of the oviducal gland in the Rajid thornback ray, <Emphasis Type="Italic">Raja clavata</Emphasis>

The reproductive processes of chondrichthyans are complex. Knowledge of the development and maturation of the oviducal gland is vital for understanding the reproductive biology of a species. This study represents the first contribution of this subject for skates. In the oviparous thornback ray, Raja clavata, oviducal gland development begins early in the developing stage with the formation of gland tubules and the distinct lamellae of each zone: club, papillary, baffle and terminal. Oviducal development is complete by the end of the developing stage when the storage and secretion of products is evident within the gland tubules of each zone. Periodic acid-Schiff and alcian blue histological staining showed that the secretory mucous cells of the club and papillary zones produce neutral and sulfated acid mucins. The last row of gland tubules of the papillary zone stains intensely for sulfated acid mucins. The baffle zone, which is responsible for the production of the egg capsule, represented 60–80% of the glandular zone of the oviducal gland. Sperm bundles were observed in the deeper recesses of the baffle zone during the maturation process, and during capsule extrusion, sperm were detected near the lumen. The terminal zone was composed of two types of gland tubules: serous (producing protein fibres) and mucous glands (producing sulfated acid mucins).     

Properties of the Cortical Granule Lectin Isolated from Xenopus Eggs

The cortical granule lectin that participates in forming the fertilization layer in Xenopus laevis was isolated and partially characterized. About 400 μg of lectin was purified from 5 mg of crude exudate by chromatography on Sepharose 6B and Concanavalin A-conjugated Sepharose 4B columns and electrophoretic separation on polyacrylamide gel. The lectin has a molecular weight of 550 Kd and is composed of two species of polypeptides (46 Kd and 42 Kd). The lectin gave a single precipitin line against material in the prefertilization layer in an agglutination reaction on an agarose plate. The agglutination reaction involved D-galactoside residues and metal ions. The lectin formed an electron-dense layer on the outer surface of the vitelline coat of oviducal eggs covered with the prefertilization layer, but on the outer surface of jelly layer, not on that of the vitelline coat of jellied eggs. Although the jelly could be agglutinated by the lectin, the possibility that the jelly layer is the site of fertilization layer formation was excluded by the fact that the prefertilization layer is the first to meet the cortical granule lectin during normal fertilization.     

Non-Specific Induction of Pisatin and Local Resistance in Pea Leaves by Elicitors from Mycosphaerella pinodes, M. melonis and M. ligulicola and the Effect of Suppressor from M. pinodes

The accumulation of pisatin was induced non-specifically by elicitors prepared from the high molecular weight fraction (molecular weight: more than 10,000 daltons) of the spore germination fluid of three species of Mycosphaerella-plant pathogens in pea leaves with epidermis removed, regardless of the pathogenicity of the fungi to pea. Before the elicitation of pisatin synthesis, local resistance to infection by Mycosphaerella pinodes was induced by elicitors again non-specifically inpea leaves in which wax had been removed from the leaf surface. The substance responsible for local resistance could be extracted with ethylacetate from the elicitor-containing drop diffusate which was placed on pea leaves. The substance prevented the penetration of M. pinodes through heat-killed pea epidermis, but did not affect spore germination. The suppressor prepared from the low molecular weight fraction (molecular weight: less than 10,000 daltons) of the spore germination fluid of M.pinodes counteracted the ability of elicitors to induce both phases of resistance mechanisms.