SITE OF 1-METHYLADENINE RECEPTORS IN THE MATURATION OF STARFISH OOCYTES

Maturation of vitelline coat-free (VCF) oocytes of the starfish, Asterina pectinifera , was studied. When the oocytes, the vitelline coats of which were elevated by adding the ionophorc A-23187, were forced through two sheets of copper mesh, the vitelline coats were completely removed from the oocytes. Although some of the VCF oocytes underwent germinal vesicle breakdown following this mechanical treatment, most of them retained the normal germinal vesicles. These VCF immature oocytes underwent breakdown of germinal vesicles after addition of 1-methyladenine (1-MA). Dose-response curves of VCF oocytes to 1-MA were similar to those of normal oocytes. These results indicate that 1-MA reacts with the plasma membrane and that the presence of the vitelline coat is not prerequisite for inducing oocyte maturation.     

不同发育时期哲罗鱼卵黄的超微结构

应用透射电镜观察了不同发育时期哲罗鱼(Hucho taimen)卵黄的超微结构.根据哲罗鱼卵黄物质在卵母细胞中的加工合成、积累以及卵母细胞中参与卵黄颗粒形成的细胞器的变化,可将该鱼卵黄发生分为4个特征时期,即卵黄发生前期、卵黄泡期、卵黄积累期和卵黄积累完成期.卵黄发生前期是指卵母细胞发育过程中的卵黄物质开始积累前的时期,此时期核仁不断分裂,出现线粒体云和早期的滤泡细胞层、基层和鞘细胞层;卵黄泡期特点主要是细胞器不断变化产生卵黄泡和皮层泡;卵黄积累期的滤泡膜由内向外依次为放射带、颗粒细胞层、基层和鞘细胞层,此时外源性卵黄前体物质不断经过血液汇集于鞘细胞层,后经微胞饮作用穿过胶原纤维组成的基层,经过多泡体作用转运至颗粒细胞内,在细胞内经过加工和修饰形成小的卵黄蛋白颗粒,卵黄蛋白颗粒经微胞饮穿过放射带进入卵母细胞边缘形成的空泡中,不断积累形成卵黄球;进入卵黄积累完成期,卵黄球体积变大,向细胞中心聚集,填满大部分卵母细胞,卵黄积累完毕.     

L'action exercée par l'appareil endocrine sur les phases tardives de l'ovogenèse chez Locusta migratoria

Most studies dealing with the endocrine control of oogenesis are focused on vitellogenesis, but the later events of oogenesis: vitelline membrane formation and choriogenesis have received less atention. In Locusta migratoria, the corpora allata evidently control vitellogenesis, but seem to have no bearing upon the formation of the vitelline membrane or the chorion. If the insects are deprived of their CA at the time when the oocyte length is around 3.5 to 4 mm, (normal length at egg laying: 6.5 mm) vitelline membrane formation and the chorion production are unaffected. In the same species, the electrocoagulation of the pars intercerebralis or excision of corpora cardiaca disturb oogenesis; vitelline membrane material is synthesized and secreted, but the primary granules fail to join each other and to transform into vitelline membrane and the production of chorionic material is disturbed.The rER of the follicle cell seems quite active but the substance elaborated accumulates in the Golgi saccules, and therefore the cytoplasm contains only very few secretory granules. The implantation of supernumerary pars intercerebralis and corpora cardiaca into immature females induces precocious deposition of the vitelline membrane which is complete in 5.0 mm oocytes (instead of 5.6 to 6.0 mm) and chorion formation is completed in 5.5 mm oocytes (instead of 6.0 to 6.3 mm). Ecdysone is secreted in the ovaries at the end of oogenesis. We tested the result of injecting ecdysone to immature females. It results in hastening oocyte growth during 24 hr, but this accelerated growth is followed by degeneration.     

Induction of germinal vesicle breakdown in Xenopus laevis oocytes: response of denuded oocytes to progesterone and insulin

Denuded oocytes freed of their vitelline envelope have been prepared by two methods, enzymatically with pronase and manually by microdissection. The response of denuded oocytes to progesterone, in terms of germinal vesicle breakdown (GVBD), was similar to that obtained with defolliculated oocytes (separated with collagenase from follicle cells, but still keeping their vitelline membrane). The same conclusion was drawn with respect to morphological features of the oocyte surface observed by transmission and scanning electron microscopy, before and after progesterone-induced GVBD. The synergistic effect of insulin and progesterone in denuded oocytes was comparable to that observed in defolliculated oocytes. Multiplication stimulating activity (MSA) had the same effect as insulin. These observations indicate that hormones act directly upon oocytes, without interference of the surrounding vitelline envelope and follicle cells.     

Integrated microsystem for non-invasive electrophysiological measurements on Xenopus oocytes

We propose a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. Xenopus oocyte is a well-known expression system for various kinds of ion channels, that are potential tools in drug screening. In the traditional “Two Electrode Voltage Clamp” (TEVC) method, delicate micromanipulation is required to impale an oocyte with two microelectrodes. In our system, a non-invasive electrical access to the cytoplasm is provided by permeabilizing the cell membrane with an ionophore (e.g. nystatin). Unlike the classical patch-clamp or “macropatch” techniques, this method does not require removal of the vitelline membrane. Cell handling is significantly simplified, resulting in more robust recordings with increased throughput. Moreover, because only part of the oocyte surface is exposed to reagents, the required volume of reagent solutions could be reduced by an order of magnitude compared to the TEVC method. The fabrication process for this disposable microchip, based on poly-dimethylsiloxane (PDMS) molding and glass/PDMS bonding, is cost-efficient and simple. We tested this new microdevice by recording currents in oocytes expressing the human Epithelial Sodium Channel (hENaC) for membrane potentials between −100 and +50 mV. We recorded benzamil-sensitive currents with a large signal-to-noise ratio and we also obtained a benzamil concentration–inhibition curve displaying an inhibition constant IC₅₀ of about 50 nM, comparable to previously published values obtained with the TEVC technique.     

The N-terminal prodomain of sV23 is essential for the assembly of a functional vitelline membrane network in Drosophila

The vitelline membrane is a specialized extracellular matrix that surrounds and protects the oocyte. Recent studies indicate that it also serves as a storage site for embryonic pattern determinants. sV23, a major vitelline membrane protein, is essential for the morphogenesis of the vitelline membrane as sV23 protein null mutants lay flaccid, infertile eggs. By analyzing a series of sV23 mutant transgenes in the sV23 protein null genetic background, we have shown that sV23 is secreted as a proprotein in functional excess and that C- and N-terminal prodomains are removed successively, following its deposition in the extracellular space. Although a target site for subtilisin-like convertases is essential for N-terminal processing, N-terminal processing is not necessary for the assembly of a functional vitelline membrane layer. While C-terminal truncations were tolerated, the removal of N-terminal sequences lead to the production of flaccid, infertile eggs with a soluble, rather than insoluble, vitelline membrane network. We propose that the hydrophobic N-terminal prodomain plays an early and essential role in aligning molecules within the vitelline membrane network, much like hydrophobic domains within elastin drive the assembly and alignment of molecules within elastin-based extracellular matrices.     

Surface ultrastructural alterations of bovine oocytes after parthenogenetic activation

Oocyte activation is a critical component of the current animal cloning scheme. This study was designed to examine surface characteristics of bovine oocytes by scanning electron microscopy (SEM) after activation by calcium ionophore A23187 (A23187) and electric pulse combined with cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) treatments. In vitro matured (IVM) oocytes were activated then harvested at 0 to 19 hours after the onset of treatments for SEM processing. The zona pellucida (ZP) of untreated IVM oocytes exhibited an open mesh structure. The ZP surface showed little changes after A23187 alone, but dramatically changed to a less porous surface 3 hours after combined treatments with CHX or 6-DMAP. The vitelline membrane of IVM oocytes was covered with well-developed microvilli (MV). The MV became shorter (0.83 vs. 1.35 microm, p < 0.01) 8 hours after A23187 treatment alone. The vitelline membrane was altered in all oocytes examined 3 hours after incubation with A23187 plus CHX or 6-DMAP. A 1.5-fold increase in the diameter of MV in the CHX group and a higher incidence of large cytoplasmic protrusions (more than 1 microm width) in the 6-DMAP group were observed. After removal of inhibitors, the surface morphologies of the ZP and vitelline membrane were returned nearly to those of untreated IVM oocytes in both groups. The present study clearly showed that surface characteristics of the bovine oocyte were more profoundly changed by a combination of agents for parthenogenetic stimulation, and that the ultrastructural effects were reversible.     

Cytoplasmic activation of starfish oocytes by sperm and divalent ionophore A-23187

The relationship between onset of the early cytoplasmic stages of oocyte activation (vitelline membrane separation and elevation) and nuclear meiotic maturation was investigated in starfish oocytes after their exposure to divalent ionophore (A-23187) or sperm. Meiotically mature oocytes, isolated in calcium-free seawater, underwent activation in response to sperm or ionophore as previously reported. Large, immature starfish oocytes, arrested in prophase I of meiosis (germinal vesicle stage), underwent vitelline membrane elevation when treated with divalent ionophore A-23187 or starfish sperm. Histological studies demonstrated that cortical granule breakdown in the oocyte cortex was associated with vitelline membrane elevation after these treatments. Activation of oocytes by sperm occurred only in response to starfish sperm. Sea urchin, sand dollar, surf clam, or marine worm sperm did not induce vitelline membrane elevation of either immature or mature starfish oocytes. Sperm- or ionophore-activated immature oocytes underwent nuclear maturation after addition of the meiosis-inducing hormone, l-methyladenine; however, parthenogenetic development did not occur and embryonic development was markedly inhibited. In contrast to previous studies, the present results indicate that cytoplasmic activation can be initiated before and without hormone induction of the nuclear maturation process. Differentiation of the oocyte cell surface or cortex reactivity therefore appears to occur during oogenesis rather than as a consequence of maturation. The data further support the view that divalent ions mediate certain of the early activation responses initiated by sperm at the time of fertilization and that synchronization of fertilization to the meiotic process in the oocyte is important for the occurrence of normal development.     

Integrated microsystem for non-invasive electrophysiological measurements on Xenopus oocytes

We propose a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. Xenopus oocyte is a well-known expression system for various kinds of ion channels, that are potential tools in drug screening. In the traditional “Two Electrode Voltage Clamp” (TEVC) method, delicate micromanipulation is required to impale an oocyte with two microelectrodes. In our system, a non-invasive electrical access to the cytoplasm is provided by permeabilizing the cell membrane with an ionophore (e.g. nystatin). Unlike the classical patch-clamp or “macropatch” techniques, this method does not require removal of the vitelline membrane. Cell handling is significantly simplified, resulting in more robust recordings with increased throughput. Moreover, because only part of the oocyte surface is exposed to reagents, the required volume of reagent solutions could be reduced by an order of magnitude compared to the TEVC method. The fabrication process for this disposable microchip, based on poly-dimethylsiloxane (PDMS) molding and glass/PDMS bonding, is cost-efficient and simple. We tested this new microdevice by recording currents in oocytes expressing the human Epithelial Sodium Channel (hENaC) for membrane potentials between −100 and +50 mV. We recorded benzamil-sensitive currents with a large signal-to-noise ratio and we also obtained a benzamil concentration–inhibition curve displaying an inhibition constant IC₅₀ of about 50 nM, comparable to previously published values obtained with the TEVC technique.     

Changes in the protein components of vitelline envelope during oogenesis of a tubiculous polychaete,Schizobranchia insignis

In sabellid polychaetes the vitelline envelopes, in which microvilli with glycocalyx structures at the tips are invested, change in structure during oogenesis. Vitelline envelopes isolated from Schizobranchia oocytes 25–100 μ m and 160–185 μ m in diameter, were analyzed in protein components by iodination, electrophoresis, Western blotting and radioactive labeling technique. The observations demonstrate that the membrane proteins of the vitelline envelopes are not consistent but variable in components during oogenesis. Most of these proteins, particularly the high molecular weight proteins, are PAS-positive glycoproteins, which may have specific carbohydrate residues binding to wheat germ agglutinin. The proteins could be labeled with [³H]valine within 36 h by incubating the whole oocytes in sea water to a high level, indicating that the proteins are actively synthesized by the growing oocytes. Synthetic rates of the proteins differ from each other at one stage and are higher in the small than in the large oocytes in general, suggesting that the membrane proteins are involved in the function of the vitelline envelopes during oogenesis.     

Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. Here we show that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. Our results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell.     

Ultrastructure of Pharyngostomoides procyonis Harkema 1942 (Diplostomatidae). II. The female reproductive system

Several components of the female reproductive system of Pharyngostomoides procyonis, including the vitellaria and vitelline duct, ovary and oviduct, Laurer's canal, and Mehlis' gland and associated ducts, were observed with the electron microscope. Vitelline follicles contain cells in various stages of development. Mature vitelline cells contain membrane-delimited clusters of vitelline globules near the plasma membrane. Cilia are present in the vitelline duct. The ovary contains germ cells in various stages of maturation. Oogonia are found in the peripheral region. Mature oocytes contain numerous dense bodies near the plasmalemma. Also included in the cytoplasm of mature oocytes are "nucleolus-like bodies," myelin-like bodies, and mitochondria containing dense granules and few cristae. The epithelium of the oviduct is ciliated. Sperm are present in the oviduct and in Laurer's canal. Two types of secretory cells found in Mehlis' gland are described.     

Spermatolysins in Bufo arenarum: their activity on oocyte surface

The activity of spermatolysins from Bufo arenarum spermatozoa on spawned dejellied oocytes is studied at structural and ultrastructural levels. After adding spermatolysins to spawned dejellied oocytes, a wrinkling of the animal hemisphere is first observed under a stereomicroscope. Two or three minutes later, the vitelline envelope in the animal hemisphere is completely digested, which produces oocyte flattening. The vitelline envelope covering the vegetal hemisphere is not modified with the treatment. Ultrastructural observations indicate that while the vitelline envelope of the vegetal hemisphere remains unaltered, the animal counterpart gradually loses its components and finally all structures disappear. Scanning microscope observations reveal that the microvilii of the plasma membrane in the animal hemisphere decreases in number and length, while the vegetal region is not altered by the enzyme.     

Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na⁺-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification. expression studies; aquaporin 2 mutations     

东方杯叶吸虫卵黄腺和卵巢的超微结构研究

本文应用透射电镜观察了东方杯叶吸虫卵黄腺和卵巢的超微结构,并与体外培养成虫进行比较。根据形态特征和内含物的存在情况,将卵黄细胞和卵母细胞的发育均分为不同时期,详细描述了各期的形态特征,探讨了卵黄球和皮质颗粒等内含物的生理功能。体外培养成虫成熟卵黄细胞中有散在的卵黄物质,成熟卵母细胞中线粒体囊泡化,这些可作为体外培养的评价指标。     

Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants

Investigation into fish oocyte membrane permeability is essential for developing successful protocols for their cryopreservation. The aim of the present work was to study the permeability of the zebrafish (Danio rerio) oocyte membrane to water and cryoprotectants before cryopreservation protocol design. The study was conducted on stage III and stage V zebrafish oocytes. Volumetric changes of stage III oocytes in different concentrations of sucrose were measured after 20 min exposure at 22 degrees C and the osmotically inactive volume of the oocytes (Vb) was determined using the Boyle-van't Hoff relationship. Volumetric changes of oocytes during exposure to different cryoprotectant solutions were also measured. Oocytes were exposed to 2 M dimethyl sulphoxide (DMSO), propylene glycol (PG), and methanol for 40 min at 22 degrees C. Stage III oocytes were also exposed to 2 M DMSO at 0 degrees C. Oocyte images were captured on an Olympus BX51 cryomicroscope using Linkham software for image recording. Scion Image was used for image analysis and diameter measurement. The experimental data were fitted to a two-parameter model using Berkeley Madonna 8.0.1 software. Hydraulic conductivity (L(p)) and solute (cryoprotectant) permeability (Ps) were estimated using the model. The osmotically inactive volume of stage III zebrafish oocytes was found to be 69.5%. The mean values+/-SE of Lp were found to be 0.169+/-0.02 and 0.196+/-0.01 microm/min/atm in the presence of DMSO and PG, respectively, at 22 degrees C, assuming an internal isosmotic value for the oocyte of 272 mOsm. The Ps values were 0.000948+/-0.00015 and 0.000933+/-0.00005 cm/min for DMSO and PG, respectively. It was also shown that the membrane permeability of stage III oocytes decreased significantly with temperature. No significant changes in cell volume during methanol treatment were observed. Fish oocyte membrane permeability parameters are reported here for the first time. The Lp and Ps values obtained for stage III zebrafish oocytes are generally lower than those obtained from successfully cryopreserved mammalian oocytes and higher than those obtained with fish embryos and sea urchin eggs. It was not possible to estimate membrane permeability parameters for stage V oocytes using the methods employed in this study because stage V oocytes experienced the separation of outer oolemma membrane from inner vitelline during exposure to cryoprotectants.     

Structural and biochemical analysis of the Leptinotarsa decemlineata (Coleoptera; Chrysomeloidea) crystalline chorionic layer

The developmental aspects of the Leptinotarsa decemlineata crystalline chorionic layer (CCL) morphogenesis, its composition and its supramolecular structure were studied. The mature Leptinotarsa decemlineata eggshell consists of the vitelline membrane and the CCL, while the follicle cell remnants following their degeneration after oogenesis completion constitute the outer chorionic layer. The vitelline membrane and the CCL layers are formed through continuous material deposition from the follicular epithelium, whereas the main morphogenic factor during most insect eggshell formation, namely the follicle cell and oocyte microvilli, are seemingly involved only in vitelline membrane formation. Analysis of the CCL morphogenesis showed that this layer is assembled from a fiber-like pre-crystalline material, which accumulates at the vitelline membrane-follicle cell interface. The mature CCL is about 1 microm thick and exhibits a periodicity of approximately 10 nm, while computer image analysis studies of thin-sectioned CCL revealed the existence of crystalline layers parallel to the CCL surface. Finally, SDS-PAGE-electrophoresis of purified CCLs showed that this crystalline layer is of a proteinaceous nature and is most likely composed of 3-5 polypeptides with a molecular weight ranging in between 28-60 kDa. Overall, these data exemplify for the first time the nature and supramolecular arrangement of a crystalline layer and its constituent molecules in Coleoptera.     

Ultrastructure of the eggshell and its formation in Planipapillus mundus (Onychophora: Peripatopsidae)

Although the majority of onychophorans are viviparous or ovoviviparous, oviparity has been described in a number of species found exclusively in Australia and New Zealand. Light microscopy and scanning and transmission electron microscopy were used to examine developing eggs and the reproductive tract of the oviparous Planipapillus mundus. Deposited eggs and fully developed eggs dissected from the terminal end of the uteri have an outer thick, slightly opaque chorion, and an inner thin, transparent vitelline membrane. The chorion comprises an outermost extrachorion, sculptured with domes equally spaced over the surface; a middle exochorion, with pores occurring in a pattern of distribution equivalent to that of the domes of the extrachorion above; and an innermost, thick endochorion consisting of a spongelike reticulum of cavities comparable to the respiratory network found in insect eggs. The vitelline membrane lies beneath the chorion, from which it is separated by a fluid‐filled space. The vitelline membrane tightly invests the developing egg. Examination of oocytes in the ovary and developing eggs at various stages of passage through the uterus indicate that the majority of chorion deposition occurs in the midregion of the uterus, where vast networks of endoplasmic reticulum are present in the columnar epithelium. The vitelline membrane, however, is believed to begin its development as a primary egg membrane, surrounding the developing oocytes in the ovary. The vitelline membrane is transformed after fertilization, presumably by secretions from the anterior region of the uterus; hence, it should be more accurately referred to as a fertilization membrane. Aspects of the reproductive biology of P. mundus are also included. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

An integrated field-effect microdevice for monitoring membrane transport in Xenopus laevis oocytes via lateral proton diffusion

An integrated microdevice for measuring proton-dependent membrane activity at the surface of Xenopus laevis oocytes is presented. By establishing a stable contact between the oocyte vitelline membrane and an ion-sensitive field-effect (ISFET) sensor inside a microperfusion channel, changes in surface pH that are hypothesized to result from facilitated proton lateral diffusion along the membrane were detected. The solute diffusion barrier created between the sensor and the active membrane area allowed detection of surface proton concentration free from interference of solutes in bulk solution. The proposed sensor mechanism was verified by heterologously expressing membrane transport proteins and recording changes in surface pH during application of the specific substrates. Experiments conducted on two families of phosphate-sodium cotransporters (SLC20 & SLC34) demonstrated that it is possible to detect phosphate transport for both electrogenic and electroneutral isoforms and distinguish between transport of different phosphate species. Furthermore, the transport activity of the proton/amino acid cotransporter PAT1 assayed using conventional whole cell electrophysiology correlated well with changes in surface pH, confirming the ability of the system to detect activity proportional to expression level.