Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization.

The ER of eggs of the sea urchin Lytechinus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiIC18(3) (DiI) in soybean oil; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles. Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex. Since diffusion of DiI is confined to continuous bilayers, the spread of the dye supports the concept that the ER is a cell-wide, interconnected compartment. In time lapse observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was stationary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave apparently coincident with the calcium wave and becoming most marked by 2-3 min. By 5-8 min the ER returned to an organization similar to that of the unfertilized egg. The cortical network also changed at fertilization; it became disrupted and eventually recovered. DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis. DiI-stained membranes accumulated in the region of the microtubule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the egg; this accumulation persisted near the centrosomes and zygote nucleus throughout pronuclear fusion and the first two mitotic cycles. We have used a new method to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization.

Actin, microvilli, and the fertilization cone of sea urchin eggs.

Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges.

Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5-trisphosphate and GTP-binding protein.

To investigate the roles of inositol 1,4,5-trisphosphate (InsP3) and guanyl nucleotide binding proteins (G-proteins) in the transduction mechanism coupling fertilization and exocytosis of cortical vesicles in sea urchin eggs, we microinjected InsP3 and guanyl nucleotide analogs into eggs of Lytechinus variegatus. Injection of 28 nM InsP3 caused exocytosis. However, if the egg was first injected with EGTA ([Cai] less than or equal to 0.1 microM; EGTA = 1.6 mM), InsP3 injection did not cause exocytosis, supporting the hypothesis that InsP3 acts by causing a rise in intracellular free calcium. Injection of 28 microM guanosine-5'-0-(3-thiotriphosphate) (GTP-gamma-S), a hydrolysis-resistant analog of GTP, caused exocytosis, but exocytosis did not occur if the egg was pre-injected with EGTA. Injection of 3 mM guanosine-5'-0-(2-thiodiphosphate) (GDP-beta-S), a metabolically stable analog of GDP, prevented sperm from stimulating exocytosis. However, injection of GDP-beta-S did not prevent the stimulation of exocytosis by InsP3. These results suggested the following sequence of events. The sperm activates a G-protein, which stimulates production of InsP3. InsP3 elevates intracellular free calcium, which causes exocytosis.

Electrically mediated fast polyspermy block in eggs of the marine worm, Urechis caupo.

Previous work has established that the polyspermy block in Urechis acts at the level of sperm-egg membrane fusion. (J. Exp. Zool. 196:105). Present results indicate that during the first 5--10 min after insemination the block is mediated by a positive shift in membrane potential (the fertilization potential) elicited by the penetrating sperm, since holding the membrane potential of the unfertilized egg positive by passing current reduces the probability of sperm entry, while progressively reducing the amplitude of the fertilization potential by decreasing external Na+ progressively enhances multiple sperm penetrations. Also, a normal fertilization potential is correlated with a polyspermy block even under conditions (pH 7) in which eggs do not develop. We have investigated the mechanism of the electrical polyspermy block by quantifying the relationship between sperm incorporation, membrane potential and ion fluxes. Results indicate that the polyspermy block is mediated by the electrial change per se and not by the associated fluxes of Na+, Ca++, and H+.

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization.

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.

Calcium accumulations within the growing tips of pollen tubes.

Pollen of L. longiflorum was grown in 45Ca-labeled medium and washed with nonradioactive medium. Whole, labeled pollen was then frozen and autoradiographed at -78 degrees C. The autoradiographs show striking accumulations of 45Ca in the growing tips of the pollen tubes. This result is obtained when the pollen is labeled for times as short as 1 min, or as long as 5 h. In most cases, the tip concentration is about two to four times greater than that in the bulk of the pollen tube, and extends for a length of about 20 mum. In autoradiographs of tubes longer than 1 mm, a small fraction of cells show a distinctly larger 45Ca accumulation, the tip containing more than 100 times that in the rest of the cell. The 1- to 5-h labeling experiments show that calcium is relatively concentrated within the cytoplasm of the growing tip. The 1- to 3-min labeling experiments suggest that calcium may enter the tip faster than it enters other regions. These patterns of calcium accumulation and flux may be related to the localized secretion of vesicles at the grow;ng tip.

Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes.

Oocytes of Xenopus laevis undergo maturation when injected with an affinity-purified antibody against the COOH-terminal decapeptide of the alpha subunit of the G-protein Gs, an antibody that inhibits Gs activity. Germinal vesicle breakdown, chromosome condensation, and polar body formation occur, with a time course similar to that for oocytes treated with progesterone. The alpha S antibody-injected oocytes also acquire the ability to be activated by sperm. Coinjection of the catalytic subunit of cAMP-dependent protein kinase, or incubation with cycloheximide, inhibits maturation in response to injection of the alpha S antibody; these experiments show that the alpha S antibody acts at an early point in the pathway leading to oocyte maturation, before formation of maturation promoting factor, and like progesterone, its action requires protein synthesis. Immunogold electron microscopy shows that alpha S is present in the yolk platelet membranes as well as the plasma membrane. These results support the hypothesis that progesterone acts by inhibiting alpha S, and suggest that the target of progesterone could include yolk platelet membranes as well as the plasma membrane.

Oocyte maturation in starfish is mediated by the beta gamma-subunit complex of a G-protein.

The stimulation of meiotic maturation of starfish oocytes by the hormone 1-methyladenine is mimicked by injection of beta gamma subunits of G-proteins from either retina or brain. Conversely, the hormone response is inhibited by injection of the GDP-bound forms of alpha i1 or alpha t subunits, or by injection of phosducin; all of these proteins should bind free beta gamma. alpha-subunit forms with reduced affinity for beta gamma (alpha i1 or alpha t bound to hydrolysis-resistant GTP analogs, or alpha i1-GMPPCP treated with trypsin to remove the amino terminus of the protein) are less effective inhibitors of 1-methyladenine action. These results indicate that the beta gamma subunit of a G-protein mediates 1-methyladenine stimulation of oocyte maturation.

Calcium release at fertilization in starfish eggs is mediated by phospholipase Cgamma.

Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP3. To distinguish between a role for PLCgamma, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCbeta, which is activated by a G protein, we injected starfish eggs with a PLCgamma SH2 domain fusion protein that inhibits activation of PLCgamma. In these eggs, Ca2+ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCgammaSH2 protein is a specific inhibitor of PLCgamma in the egg, since it did not inhibit PLCbeta activation of Ca2+ release initiated by the serotonin 2c receptor, or activation of Ca2+ release by IP3 injection. Furthermore, injection of a PLCgamma SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cgamma by an SH2 domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.

Fertilization events induced by neurotransmitters after injection of mRNA in Xenopus eggs.

Fertilization initiates in the egg a dramatic increase in intracellular calcium that opens ion channels and causes exocytosis. To explore the possibility that these events might involve a receptor-mediated pathway, receptors for serotonin or acetylcholine (M1 muscarinic) were expressed in the Xenopus egg; serotonin or acetylcholine then could initiate a series of responses similar to those normally initiated by sperm. Thus, there may be an endogenous receptor in the egg membrane that is activated by sperm, and the serotonin or M1 muscarinic receptor may replace the sperm receptor in this pathway.

Ionic mechanism of the fertilization potential of the marine worm, Urechis caupo (Echiura).

Microelectrode and tracer flux studies of the Urechis egg during fertilization have shown: (a) insemination causes a fertilization potential; the membrane potential rises from an initial level of -33 +/- 6 mV to a peak at +51 +/- 6 mV (n = 16), falls to a plateau of about +30 mV, then returns to the original resting potential 9 +/- 1 min (n - 10) later; (b) the fertilization potential results from an increase in Na+ permeability, which is amplified during the first 15 s by a Ca++ action potential; (c) the maximum amplitude of the fertilization potential, excluding the first 15 s, changes by 51 mV for a 10-fold change in external [Na+]; (d) in the 10 min period after insemination, both Na+ and Ca++ influxes increase relative to unfertilized egg values by factors of 17 +/- 7 (n = 6) and 34 +/- 14 (n = 4), respectively; the absolute magnitude of the Na+ influx is 16 +/- 6 times larger than that of Ca++; (e) in the absence of sperm these same electrical and ionic events are elicited by trypsin; thus, the ion channels responsible must preexist in the unfertilized egg membrane; (f) increased Na+ influx under conditions of experimentally induced polyspermy indicates that during normal monospermic fertilization, only a fraction of available Na+ channels are opened; we conclude that these channels are sperm-gated; (g) Ca++ influx at fertilization is primarily via the membrane potential-gated channel, because kinetics are appropriate, and influx depends on potential in solutions of varying [Na+], but is independent of number of sperm incorporations in normal sea water.

Massive macrophage lipid accumulation presenting as hepatosplenomegaly and lymphadenopathy associated with long-term total parenteral nutrition therapy for short bowel syndrome.

We present a unique case of massive splenomegaly, hepatomegaly, and lymphadenopathy caused by lipid-laden macrophages in a 50 year old white female with short-bowel syndrome treated with long-term total parenteral nutrition. Using transmission electron microscopy and special stains we were able to show that the total parenteral nutrition lipid component was composed of lipid droplets and micelles morphologically identical to those found in lipid-laden macrophages which had accumulated in the patient's reticuloendothelial system leading to massive splenomegaly, hepatomegaly (without evidence of steatosis) and lymphadenopathy. While this phenomenon has been reported in animal models, no human cases have been previously reported.

In situ hybridization for Aspergillus and Penicillium in allergic fungal sinusitis: a rapid means of speciating fungal pathogens in tissues.

Allergic fungal sinusitis (AFS) is a serious form of sinonasal fungal disease that is commonly associated with Aspergillus or Dematiaceous fungi. This study was performed to determine the incidence of Aspergillus or Penicillium in AFS by using in situ hybridization (ISH) for Aspergillus and Penicillium ribosomal RNA (rRNA). The Fontana-Masson melanin stain (FMMS) was also used to detect pigmented fungi (A. niger and Dematiaceous fungi). ISH was performed on 26 patients: 17 AFS cases with histologic evidence of fungi, 5 AFS cases without histologic evidence of fungi, 3 cases of invasive fungal sinusitis (IFS), and 1 case of fungus ball. Nine AFS specimens with histologic evidence of fungi were ISH positive. Positivity was also noted in two of three IFS cases, while no staining was seen in the fungus ball and in six AFS specimens without fungi demonstrable by silver stains. Six ISH-positive cases were FMMS positive, suggesting A. niger. Five ISH-negative AFS specimens were FMMS positive, suggesting Dematiaceous fungi. In summary, many AFS patients in our institution demonstrate Aspergillus/Penicillium organisms. Ancillary techniques may help identify fungi responsible for AFS if cultures are negative or not performed. ISH for rRNA is a useful means for rapidly speciating fungi in human tissues.

Cerebral spinal fluid involvement by Hodgkin's disease diagnosed by CSF cytology and immunocytochemistry.

A 39-yr-old man with stage IV Hodgkin's disease (HD) involving bone marrow was being evaluated for autologous bone marrow transplantation when he developed diplopia, prompting a lumbar puncture tap for cerebral spinal fluid (CSF) examination. Cytologic examination of the CSF revealed numerous Reed-Sternberg (RS) cells in a polymorphous inflammatory background of small lymphocytes, monocytes, rare plasma cells, and eosinophils. However, magnetic resonance imaging (MRI) studies of the brain and spinal cord failed to reveal evidence of leptomeningeal disease or intracranial masses. Repeat CSF examination again demonstrated cytologic evidence of HD. Immunocytochemical stains established that the RS cells and mononuclear Hodgkin's cells were positive for CD30 and CD20 but negative for CD15; this phenotype was identical to that of RS cells in the initial diagnostic bone marrow biopsy, confirming CSF involvement by HD. The patient was treated with intrathecal methotrexate, 15 mg, 6 days after his bone marrow transplant. After treatment, all subsequent CSF cytology specimens were negative for tumor. In this case of disseminated HD, cytologic examination allowed for early detection of CNS involvement by lymphoma prior to development of radiographically detectable lesions.

First messengers at fertilization.

To investigate the 'first messengers' that pass between the spermatozoon and egg to initiate development, the function of G-proteins and membrane potential at fertilization have been examined. G-proteins are present in eggs, and activating them with GTP-gamma-S, cholera toxin, or receptors for serotonin or acetylcholine (expressed following mRNA injection) causes activation responses in eggs similar to those occurring at fertilization. ADP-ribosylation of most of the pertussis-sensitive G-proteins in Xenopus eggs does not block the responses to spermatozoa or serotonin. These results suggest that activation of a pertussis-insensitive G-protein may initiate activation responses in the egg at fertilization. In many species, one of these responses is a change in the egg's membrane potential, which prevents entry of additional spermatozoa. Results of cross-species fertilizations between voltage-sensitive and voltage-insensitive species indicate that the voltage-dependence of fertilization is due to the presence of a voltage-sensitive component in the sperm membrane, suggesting that the 'first messenger' is a positively charged component of the sperm membrane that inserts into the egg membrane to initiate sperm-egg fusion and egg activation.

Structural changes of the endoplasmic reticulum of sea urchin eggs during fertilization.

The endoplasmic reticulum (ER) of the sea urchin egg includes a system of cisternae, a cortical tubular network, and the nuclear envelope. In previous work (Terasaki and Jaffe, 1991, J. Cell. Biol. 114, 929-940) we showed that the long chain fluorescent lipophilic dye DiI spread from an intracellularly injected oil drop into all of these parts of the ER. In this previous study, we observed that in the first minute after fertilization, the ER cisternae became more finely partitioned; the large cisternae then reformed by 5-8 min after fertilization. To investigate whether these changes indicated a breaking up of the ER into discontinuous pieces, we have now examined the ability of DiI to spread in the ER at various times after fertilization. In eggs injected after fixation with aldehydes, we find that DiI spreads through the ER in unfertilized eggs and in eggs at 10 min postinsemination. However, DiI does not spread out of the oil drop in eggs fixed at 1 min postinsemination. We also find that in live eggs, the spreading of DiI is slower in eggs at 1-3 min postinsemination than in unfertilized eggs or in eggs later after fertilization. We interpret these observations to indicate that in the first few minutes after fertilization, the egg's ER is fragmented. This may be functionally significant for the early events of egg activation.

Proteases stimulate fertilization-like responses in starfish eggs.

Application of proteases to eggs of the starfish, Asterina miniata, caused several responses like those seen at fertilization. Cortical granule exocytosis and fertilization envelope elevation occurred within about 1 min after exposure to trypsin, chymotrypsin, or pronase; protease inhibitors prevented these responses. Kallikrein caused cortical granule exocytosis and fertilization envelope elevation, but this response required more time (congruent to 30 min). Exocytosis was also seen in response to a recombinant trypsin, but not to a point-mutated trypsin without proteolytic activity. The extent of exocytosis was similar to that seen at fertilization, as measured by the fluorescent dye FM 1-43. In addition to causing exocytosis, application of trypsin, chymotrypsin, or pronase caused an increase in intracellular free calcium, detected by calcium green dextran, and stimulation of DNA synthesis, detected by incorporation of bromodeoxyuridine. Exocytosis also occurred when trypsin or chymotrypsin was applied in artificial sea water in which the free calcium was reduced to a low level (40-70 nM) such that Ca influx would be reduce by > 10,000-fold; this indicated that the proteases did not act by damaging the eggs and causing external calcium to leak into the cytoplasm. These findings show that there is an extracellularly exposed protein that when proteolyzed can induce fertilization-like responses; this protein may be a receptor that transduces a signal from the sperm to initiate egg activation at fertilization.

Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization.

The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization.

Evidence that the voltage-dependent component in the fertilization process is contributed by the sperm.

To investigate the mechanisms that account for the voltage dependence of fertilization and provide an electrical block to polyspermy, we studied cross-fertilizations between three species of amphibians having different degrees of voltage dependence. Anurans, such as the toad Bufo japonicus, as well as the primitive urodele Hynobius nebulosus, have voltage-dependent fertilization; other urodeles, such as Cynops pyrrhogaster, have voltage-independent fertilization (Y. Iwao, 1989, Dev. Biol. 134, 438-445). Entry of Hynobius sperm into Cynops eggs was blocked by clamping the egg's membrane potential at +40 mV, as is the case for fertilization of Hynobius eggs with Hynobius sperm, but not for fertilization of Cynops eggs with Cynops sperm. Therefore, fertilization was voltage dependent in an experimental condition where only the sperm could be contributing this characteristic. The voltage-dependent properties of fertilization between Bufo eggs and Hynobius sperm were also characteristic of the sperm species; fertilization was blocked at +50 mV as in Hynobius fertilization, but not at +20 mV as in Bufo fertilization. These results support the conclusion that the voltage dependence of fertilization results from a component contributed by the sperm.