The onset of activation responsiveness during maturation coincides with the formation of the cortical endoplasmic reticulum in oocytes of Xenopus laevis

Immature, Stage VI oocytes of Xenopus laevis fail to activate (i.e., to propagate a cortical reaction and elevate a fertilization envelope) when pricked or exposed to A23187. We determined the times during maturation when immature oocytes treated with progesterone in vitro developed the capacity to respond to pricking and to ionophore. Responsiveness to ionophore first appears at about 3.5-4.5 hr after progesterone treatment; all oocytes are activated by 8-9 hr after progesterone. The capacity to respond to pricking appears about 1.0-1.5 hr after first signs of ionophore responsiveness. We examined the cortical endoplasmic reticulum (CER) by TEM to determine whether the morphology of this component could be correlated with the development of responsiveness during maturation. Fully mature oocytes exhibit an extensive CER that (1) forms a "shell" around most cortical granules, (2) appears to interconnect cortical granules, and (3) forms junctions with the plasma membrane. The CER-plasma membrane junctions are especially obvious in preparations of isolated cortex. The elaborate CER is not present in immature oocytes. It first appears during maturation of progesterone-treated oocytes at 4.5-5.0 hr, coincident with the time when maturing oocytes develop their responsiveness to ionophore and to pricking. This temporal correlation is consistent with the hypothesis that the CER is one of the components required for regulation of intracellular free calcium in oocytes.     

Ultrastructure of the preparative phase of cell death in the larval fat body of Drosophila melanogaster

Progressive changes in the ultrastructure of the larval fat body of Drosophila melanogaster were studied during the third instar. In addition to electron microscopy, light microscopy and morphometric stereology were employed to evaluate the tissue at five 12-hr intervals: 48, 60, 72, 84, and 96 hr after hatching from the egg. Lipid and glycogen were found stored throughout the instar, whereas protein is stored in the form of cytoplasmic granules mainly during the final 24 hr. The cells increased in cross-sectional area, and there was a concomitant increase in the relative amounts of these substances. Based on morphological characteristics there were three types of protein granules which we called dense granules (D), heterogeneous granules (H), and autophagic vacuoles. The morphology, size range, time of appearance, and changes in frequency of these granules suggested that the H type arose from D granules, and that the autophagic vacuoles were derived from D and H types. Morphological evidence indicated D granules have the unusual characteristic of forming in the intercellular space before entering the cytoplasm.     

Vitrification of bovine oocytes with the open pulled straw method: ultrastructural consequences

The objective of the present study was to investigate the ultrastructural consequences of vitrification of bovine oocytes at the metaphase II (MII) stage by the so-called "Open Pulled Straw" method. Oocytes were matured in vitro for 22 hr and cryopreserved by vitrification. After warming and additional 2 hr of culture, the oocytes were inseminated in vitro. Oocytes were fixed for transmission electron microscopy immediately after warming, at 4 hr after warming (i.e., 2 hr post insemination [hpi]), at 26 hr after warming (i.e., 24 hpi), and at 74 hr after warming (i.e., 72 hpi). Control oocytes (i.e., nonvitrified oocytes) were processed at 22 hr after in vitro maturation and at 2, 22, and 72 hpi. Compared to the controls, the vitrified oocytes fixed immediately after warming presented an additional category of small membrane-bound vesicles and lacked the typical compartment of solitary cortical granules aligned along the oolemma. Instead, they presented clusters of cortical granules that displayed varying degrees of degeneration. In vitrified oocytes fixed at 2 hpi, the small vesicles were less abundant, and more advanced degeneration of the cortical granule clusters was noted. In vitrified oocytes fixed at 24 hpi, the small vesicles were practically absent, and polyspermic penetration was observed as were vacuoles containing degraded cortical granule content. In vitrified oocytes fixed at 72 hpi, lack of cleavage as well as vacuolization and degeneration of blastomeres were noted. Moreover, the nucleolar ultrastructure signaled aberrant activation of the ribosomal RNA genes. In conclusion, vitrification of bovine oocytes at the MII stage resulted in cell biological alterations in the oocyte after warming that apparently were reflected in the subsequent fertilization and embryonic development.     

Electrical Properties of Toad Oocytes During Maturation and Activation

The full-grown oocytes of the toad Bufo bufo japonicus , whether in follicular layer or not, had a membrane potential of about -50 mV in De Boer's solution (DB), but underwent a deep hyper-polarization of up to -90 mV when pretreated with Ca, Mg-free EDTA-solution. Regardless of the magnitude of their resting potentials, the defolliculated oocytes exposed to progesterone underwent a gradual depolarization before the germinal vesicle breakdown and retained membrane potential at a level of -10 mV until 18 hr post hormone treatment (PHT), the stage of the second meiotic metaphase. A positive-going activation potential of a magnitude of 70 mV was recorded in the oocytes when pricked at 18 hr PHT as well as in uterine eggs 3–5 min after insemination. A low magnitude of activation potential in response to pricking was recorded in 63% of the oocytes at 13 hr PHT, and premature oocytes exhibiting the activation potential always underwent cortical granule breakdown (CGBD) and perivitelline space formatión. Oocytes where the germinal vesicle had been removed before the hormone treatment exhibited an activation potential and underwent CGBD in response to pricking at 18 hr PHT, whereas those pulse-treated with cycloheximide (10 μg/ml) during the 8–11 hr PHT exhibited neither of these cortical responses. These results indicate that the syntheses of proteins independent of germinal vesicle taking place at 9–11 hr PHT enable the oocytes to undergo cortical responses.     

Differential Emergence of Cortical Granule Breakdown and Electrophysiological Responses during Meiotic Maturation of Toad Oocytes

Oocytes of the toad, Bufo bufo japonicus , at various stages of progesterone-induced maturation were stimulated by pricking or treatment with Ca-ionophore A23187. Upon pricking, oocytes 14 hr after hormone treatment (PHT) underwent sequential activation responses, such as development of an activation potential, cortical granule breakdown (CGBD), and formation of a perivitelline space (PVS), like those of mature oocytes (18 hr PHT). When oocytes were pricked at 14 hr PHT, it took about 10 min for the wave of CGBD to spread over all the oocyte surface, in contrast to the case with mature oocytes in which it took about 150 sec. The rate of spread of CGBD was significantly less in the vegetal hemisphere than in the animal hemisphere in both mature and immature oocytes. Treatment with A23187 (1 μM) for 5 min induced an activation potential, and PVS formation by the oocytes from 10 hr PHT, which was 3–4 hr earlier than the time when these responses could be induced by pricking. Oocytes at 8–9 hr PHT also showed CGBD in response to A23187, but without formation of an activation potential. Several patches of local PVS caused by the non-propagating CGBD were observed in oocytes treated with the ionophore 5–7 hr PHT. When a high concentration (10 μM) of A23187 was employed, CGBD without PVS formation was induced even in oocytes at 0 hr PHT. These results indicate that the responsiveness to a Ca²⁺ surge that is a prerequisite for both CGBD and genesis of an activation potential is acquired for the repective responses at different stages of oocyte maturation.     

Ultrastructural and histochemical study of the salivary glands of Aplysia depilans (Mollusca, Opisthobranchia)

The digestive system of the sea hare, Aplysia depilans , includes a pair of ribbon-shaped salivary glands. A central duct and a large blood vessel run close to each other along the length of these glands and both are surrounded by a layer of muscle cells. Three cell types form the glandular epithelium: granular cells, vacuolated cells and mucocytes. The granular cells possess cilia and spherical secretion granules, located primarily in the apical region. The granules of immature cells have a low electron density and are mainly formed by neutral polysaccharides with small amounts of proteins. The granules of mature cells are larger, have a high electron density and are mainly formed by proteins with lower amounts of neutral polysaccharides. Transition stages between immature and mature granular cells are observed. The vacuolated cells are large and frequently pyramidal in shape, but after the application of histochemical techniques almost all vacuoles remain uncoloured. The numerous vacuoles contain flocculent material in a clear background and the mitochondria possess large crystalline structures in the matrix. A pyramidal shape is also typical of the mucocytes, which are filled with vesicles containing granular masses surrounded by a network of secretion material. These large cells are strongly stained by Alcian blue, revealing the presence of acidic mucopolysaccharides. This is the first ultrastructural study of the salivary glands in opisthobranch gastropods.     

APPEARANCE AND DISTRIBUTION OF FERRITIN IN MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF HETEROLOGOUS ERYTHROCYTES

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.     

Effects of epidermal growth factor and follicle-stimulating hormone during in vitro maturation on cytoplasmic maturation of porcine oocytes

The present study was undertaken to investigate the influence of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) during in vitro maturation on cytoplasmic maturation of porcine oocytes as revealed by the success of fertilization and by the changes in the pattern of protein synthesis in oocytes and cumulus cells. For fertilization studies, oocyte-cumulus cell complexes (OCC) were cultured in media containing human recombinant EGF (1 ng/ml) or FSH (1.5 μg/ml) or both for 44 hr prior to fertilization with fresh sperm for 6–8 hr. The oocytes were then fixed, stained, and examined as whole mounts following an additional 14 hr of culture. Addition of EGF, FSH, and EGF + FSH significantly increased the proportion of oocytes reaching MII stage. The addition of EGF alone significantly decreased the percentage of polyspermic oocytes and increased the proportion of monospermic oocytes forming 2 normal pronuclei. FSH abolished these effects of EGF and significantly increased the percentage of polyspermic oocytes forming more than 2 pronuclei when added alone or with EGF. For protein analysis, OCC were cultured in media containing the above hormones for 6, 24, and 44 hr and exposed to 0.5 mCi/ml L-[³⁵S]methionine during the last 3 hr of cultures. The oocytes and cumulus cells were separated prior to lysis in SDS sample buffer, and denatured polypeptides were separated by 1-dimensional SDS-PAGE. In the oocyte, addition of EGF and FSH alone stimulated the synthesis of 34, 45, and 97 kDa proteins after 6 hr of culture; however, the addition of EGF and FSH together was without any effect. After 24 hr, EGF alone inhibited the synthesis of these peptides, whereas FSH alone and with EGF maintained the stimulation of synthesis of 34 and 45 kDa proteins. Two additional peptides corresponding to 66 and 200 kDa appeared at this time as a result of exposure to FSH alone or with EGF. After 44 hr of culture, these 2 new peptides were observed in all groups and the stimulatory effect of FSH and FSH + EGF was still evident. An additional peptide of 26 kDa appeared at this time as a result of FSH and EGF + FSH treatments. In the cumulus cells, EGF and FSH each alone induced the synthesis of a new peptide of 26 kDa after 6 hr of culture. FSH when added alone or with EGF induced the synthesis of an additional peptide of 29 kDa, the synthesis of which remained unchanged at 24 and 44 hr. After 24 hr, FSH alone and in combination with EGF induced the synthesis of an additional 38 kDa peptide and its synthesis was still maintained at 44 hr. EGF alone had no effect on protein synthesis in cumulus cells at 24 and 44 hr. These studies indicate that EGF may have a physiological role in the regulation of cytoplasmic maturation of porcine oocytes. Mol. Reprod. Dev. 46:401–407, 1997. © 1997 Wiley-Liss, Inc.     

Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-³H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.     

Effects of superovulatory doses of pregnant mare serum gonadotropin on oocyte quality and ovulatory and steroid hormone responses in rats

Immature rats (aged 28 days) were injected with 4, 20, or 40 IU pregnant mare serum gonadotropin (PMSG) and sacrificed every 6 or 12 hr. Control rats (4 IU) ovulated between 60 and 72 hr, whereas rats given superovulatory doses of PMSG (20 and 40 IU) ovulated between 24 and 72 hr. The oocyte count from the superovulated rats increased slightly between 24 and 36 hr and markedly between 48 and 72 hr. Degenerated oocytes were recovered 48 and 36 hr after administration of 20 and 40 IU PMSG, respectively. Thereafter, the proportion of degenerated oocytes was dose dependent and reached a maximum at 72 (30.9%, 20 IU) and 60 hr (61.0%, 40 IU). 17β-estradiol content of the superovulated ovaries increased significantly (P < 0.01) from 36 hr and was maximal at 60 (20 IU) or 54 hr (40 IU), when compared to the control regimen. Administration of 40 IU PMSG resulted in a biphasic increase of progesterone content with the peaks at 36 and 60 hr. Androgen content of the superovulated ovaries was lower than control levels during the first 36 hr but was significantly (P < 0.01) higher thereafter. The results suggest that these alterations in the steroid response (particularly androgens) from 36 hr onward following superovulation may be responsible for the coincidental occurrence of abnormal oocytes, possibly by disturbing the specific intrafollicular steroid environment essential for complete maturation. In addition, oocyte aging that is due to earlier activation by the exogenous luteinizing hormone activity may be a contributing factor.     

Comparative analysis of calf and cow oocytes during in vitro maturation

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3–5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 ± 1.15 versus 122.83 ± 0.74 μm). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [³⁵S]methionine and [³⁵S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence. Mol. Reprod. Dev. 49:168–175, 1998. © 1998 Wiley-Liss, Inc.     

SEQUENTIAL DEGRANULATION OF THE TWO TYPES OF POLYMORPHONUCLEAR LEUKOCYTE GRANULES DURING PHAGOCYTOSIS OF MICROORGANISMS

The sequential discharge of neutrophilic polymorphonuclear leukocyte (PMN) granules—azurophils and specifics—was investigated by electron microscopy and cytochemistry. Thus the enzyme content of PMN phagocytic vacuoles was determined at brief intervals after phagocytosis of bacteria, utilizing peroxidase as a marker enzyme for azurophil granules, and alkaline phosphatase for specifics. At 30 s, approximately half the phagocytic vacuoles were reactive for alkaline phosphatase, whereas none contained peroxidase. Peroxidase-containing vacuoles were rarely seen at 1 min, but by 3 min, vacuoles containing both enzymes were consistently present. Alkaline phosphatase was found in both small and large vacuoles, whereas peroxidase was visible only in large ones. By 10 min, very big phagocytic vacuoles containing considerable amounts of reaction product for both enzymes were evident. These observations indicate that the two types of PMN granules discharge in a sequential manner, specific granules fusing with the vacuole before azurophils. In an earlier paper, we reported that the pH of phagocytic vacuoles drops to 6.5 within 3 min and to ~4 within 7–15 min. Substances known to be present in specific granules (alkaline phosphatase, lysozyme, and lactoferrin) function best at neutral or alkaline pH, whereas most of those contained in azurophil granules (i.e., peroxidase and the lysosomal enzymes) have pH optima in the acid range. Hence the sequence of granule discharge roughly parallels the change in pH, thereby providing optimal conditions for coordinated activity of granule contents.     

Ultrastructural modifications in bovine oocytes maintained in meiotic arrest in vitro using roscovitine or butyrolactone

Butyrolactone-I (BL-I) and roscovitine (ROSC) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes for 24 hr without having a negative affect on subsequent development to the blastocyst stage. The aim of the present study was to describe the morphological changes occurring in fully grown immature and in vitro matured bovine oocytes following exposure to either BL-I or ROSC for 24 hr at concentrations known to be consistent with normal development. Immature bovine cumulus oocyte complexes, recovered from the ovaries of slaughtered heifers, were incubated for 24 hr in the presence of one of the inhibitors. They were then either fixed immediately and processed for transmission electron microscopy (TEM), or cultured for a further 24 hr in the absence of the inhibitor, in conditions permissive to maturation, and subsequently processed for TEM. A control group of oocytes were processed for TEM immediately upon recovery (0 hr) or following in vitro maturation (IVM) for 24 hr. In general, incubation with either inhibitor disrupted the integrity of the surrounding cumulus cells and affected their subsequent expansion during IVM. Within the oocyte cytoplasm, swelling of the mitochondrial cristae was immediately noticeable following meiotic inhibition in the presence of ROSC, while an increased population of pleomorphic mitochondria and mitochondria with electron lucent matrices following BL-I treatment was not observed until after the subsequent IVM period. Both inhibitors caused degeneration of the cortical granules, effectively reducing the population, most noticeably following IVM. At the level of the nucleus, both inhibitory treatments caused convolution of the nuclear membrane, furthermore, aberrant structures were observed within the nucleoplasm of ROSC-treated cumulus oocyte complexes (COCs). In conclusion, while it has been shown that inhibition of meiotic resumption using specific cdk inhibitors is possible and that such oocytes are capable of undergoing maturation, fertilization, and early embryo development, there is as yet no definitive proof that oocytes treated in this way can ultimately give rise to normal offspring. We have shown here that some modifications are induced in the oocytes at the ultrastructural level. Whether or not these modifications are compatible with normal gestation and the birth of a live calf remain to be elucidated.     

Ultrastructure of post-fertilization development in the red alga Nienburgia andersoniana (Delesseriaceae, Ceramiales, Rhodophyta)

The ultrastructure of post-fertilization development in Nienburgia andersoniana (J. Ag.) Kyl. is described. Above the auxiliary cell there is a group of four sterile cells. The presence of abundant storage products (starch granules, lipid bodies and protein crystals) in these cells indicates that the sterile cells function as nutrient suppliers to the young auxiliary and gonimoblast cells of the carposporophyte during its early steps of development. Following fertilization and transfer of the diploid nucleus to the auxiliary cell, the trichogyne disappears and large multinucleate gonimoblast initials are produced. These subsequently produce generative gonimoblast cells which cleave successively to form young carpospores. Those of the gonimoblast cells which will not differentiate into carpospores are transformed into cells producing mucilage. Both kinds of gonimoblast cells contain plastids, starch granules, cytoplasmic concentric membrane bodies and small vesicles. Dark-staining spherical masses occurring in the cytoplasm of the auxiliary and gonimoblast cells may represent degenerating haploid nuclei. Septal plugs interconnecting the auxiliary cell and gonimoblast cells increase considerably in size during carposporophyte development. The fusion cell at the late stage of carposporophyte development appears degenerative. Young carpospores have plastids and mitochondria, and concentric membrane bodies that will form mucilage sacs. Medium-aged carpospores have fully developed plastids, starch granules and fibrous vacuoles. Mature carpospores possess, in addition, cored vesicles. The inner pericarp cells contribute large amounts of mucilage to the cytostocarpic cavity and eventually are consumed. © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 289–299.     

Oogenesis in pig ovaries during the prenatal period: ultrastructure and morphometry

Oogenesis in fetal pig ovaries comprises the successive changes from the primordial germ cells to the dictyotene oocytes in primordial ovarian follicles. In this study the observations were carried out with an electron microscope and stereological analysis was performed. At the ultrastructural level there are no differences between the primordial germ cells and oogonia, but oogonia are connected with the intercellular bridges. The onset of the dictyotene phase was accompanied by the changes in the cytoplasm of oocytes. Near the nucleus, the yolk nucleus is formed containing numerous Golgi bodies, endoplasmic reticulum (ER), mitochondria and granules. ER proliferates in contact with the external leaflet of the nuclear envelope forming the narrow ER cisterns. Between the nuclear envelope and ER cisterns, the vesicles with grey content are visible. The proliferating ER forms numerous concentric cisterns around the nucleus. Next, the most external cisterns fragment, detach, and then form the cup-like structures. These structures separate the distinct areas of cytoplasm-compartments, which contain mitochondria, ribosomes and lipid droplets. The cells of cortical sex cords of the ovary, which encloses the oocyte, form the follicles. The volume of oocytes in forming follicle increases due to the increase in the number of the cell inclusions: lipid droplets, vacuoles and yolk globules. In the oocytes of primordial ovarian follicles, the compartments are transformed into the yolk globules, which are encountered by a sheath of ER cisterns and the grey vesicles; they contain the mitochondria, lipid droplets and light vacuoles. The role of the compartments and yolk globules as metabolic units is discussed in comparison with similar structures of the mature eggs of pigs and other mammal species.     

Changes in membrane hydrogen and sodium conductances during progesterone-induced maturation of Ambystoma oocytes

A voltage-gated hydrogen ion-selective conductance has been previously described in the immature oocyte of the urodele amphibian Ambystoma. The present study was prompted by reports that changes in membrane voltage and internal pH, as well as in internal sodium ion concentration, occur during the hormone-induced maturation of oocytes from other amphibians. As activation of membrane currents might mediate changes in internal ion concentrations in addition to altering the membrane voltage, microelectrode recording techniques have been employed to examine changes in membrane conductances which occur during maturation of Ambystoma oocytes. It was observed that during the first 5 hr of maturation the magnitude of the hydrogen ion conductance gradually decreased, and that subsequently there was an increase in the amplitude of a voltage-dependent noninactivating sodium conductance. After 6 to 7 hr, after the loss of the hydrogen conductance and at about the time of germinal vesicle breakdown, the resting potential of the oocyte spontaneously shifted from approximately -10 mV to approximately +30 mV, where it remained until at least 24 hr after the initiation of maturation. This voltage transition was due to the appearance of mechanisms generating inward current in the oocyte membrane; part of this inward current was due to the tonic activation of the sodium conductance. Changes in internal pH and internal sodium ion concentration occurred during maturation, as judged from shifts in the reversal potentials of both hydrogen and sodium currents. A gradual decrease in internal hydrogen ion concentration was observed up until the time of disappearance of the hydrogen conductance (change in internal pH from about 7.15 in immature oocytes to about 7.40 by 3 hr after application of progesterone). This was followed, as sodium conductance increased, by an apparent rise in the internal sodium ion concentration (from about 6 mM to about 17 mM by 10 hr postprogesterone).     

Roles of cAMP in regulation of both MAP kinase and p34(cdc2) kinase activity during meiotic progression, especially beyond the MI stage

Time-dependent changes in the level of adenosine cyclic AMP (cAMP) in porcine oocytes during meiotic progression from the germinal vesicle stage (GV stage) to the metaphase II stage (MII stage) were examined using reversed-phase HPLC with UV detection. The concentration of cAMP in oocytes reached a peak at 8 hr of cultivation of cumulus-oocyte complexes (COCs), but it was dramatically decreased after 12-hr cultivation. After a 28-hr cultivation period, the level of cAMP in the oocytes had significantly reduced further, and the basal level of cAMP was observed in oocytes cultured at 32 hr and for up to 48 hr. When phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase C (PKC) in cumulus cells [which were required for meiotic progression to the MII stage in porcine oocytes (Shimada and Terada, 2001: Biol Reprod 64:1106-1114)] was suppressed by each specific inhibitor following initial 24-hr cultivation of COCs, cAMP level in the oocytes was significantly increased. After 24-hr cultivation in the maturation medium, COCs, which were cultured for an additional 24 hr in the presence of either forskolin or 3-isobutyl-1-methylxanthine (IBMX), exhibited a significant increase in the oocyte cAMP level to the similar level of that in oocytes cultured with PI 3-kinase inhibitor or PKC inhibitor, and the addition of each agent significantly suppressed meiotic progression from the MI to the MII stage and the activity of mitogen-activated protein kinase (MAPK) and p34(cdc2) kinase. These results demonstrated that when transported into oocytes from the cumulus cells via gap junctions, cAMP plays an important role not only in meiotic resumption, but also in the regulation of meiotic progression beyond the MI stage in porcine oocytes.     

On the ultrastructure of the sinus venosus in Chimara monstrosa L. (Elasmobranchii: Holocephali)

Summary The wall of the sinus venosus in an elasmobranchian species, Chimaera monstrosa L. is described.Endocardial cells contain numerous large vacuoles, as well as a number of membrane-bounded, moderately electron dense bodies (MDB). Myocardial cells lie closely packed into bundles surrounded by a basal lamina of about 20 nm thickness, and by large amounts of collagen fibres. These cells are connected by desmosomes of 1–2 µm length and with an intermembranous gap of 10–20 nm. Myocardial cells poor in myofibrils are intermingled with cells containing a well developed contractile material. Atrial specific granules are scarce. Vesiculated nerve processes occur at a distance of about 20 nm from the myocardial sarcolemma. Myocardial cells of the sino-atrial junction appear ultrastructurally similar to those located elsewhere in the sinus venosus. Epicardial cells contain large vacuoles, and have fibrecoated protrusions extending into the pericardial space.The possibility of pacemaker activity in the elasmobranchian sinus venosus is discussed.     

Localization of selenium deposits in meristematic cells of Allium sativum L. roots treated with selenium salts

Ultrastructural analysis of garlic roots treated for 24 h with sodium selenate or sodium selenite at the concentrations 80, 160, 320 microM revealed the presence of selenium deposits in meristematic cells. They appeared as small and large granules or aggregates of electron-dense material. Many small granules were localised in plastids but some in mitochondria, endoplasmic reticulum as well as in Golgi apparatus, nucleus and cytoplasm. Sometimes the large granules were seen in cytoplasm but aggregates of electron-dense material only in vacuoles. It seems possible that these deposits represent a non-dissolved form of selenium, i.e. elemental selenium or its complexes with other ions.