Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ∼40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (∼5–10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ∼3 mM Ca2+ (SW is ∼10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.

Oogenesis and ovarian development cycle of the spiny lobster Panulirus japonicus (Decapoda:Palinuridae)

Oogenesis and ovarian development in female Panulirus japonicus were examined by light and electron microscopy. Eight substages (oogonium, bouquet, chromatin nucleolus, oil globule, pre-yolk platelet, yolk platelet, pre-maturation and maturation) were distinguished in the typical process of oogenesis (multiplication, pre-vitellogenesis, vitellogenesis and maturation stages). Yolk accumulation started at the late pre-yolk platelet substage, when electron-dense granules appeared. Yolk granules seemed to be accumulated in two ways, being produced endogenously by the abundant rough endoplasmic reticulum during vitellogenesis and exogenously by micropinocytosis from the yolk platelet to pre-maturation substages. Ovulation occurred after oocytes became mature (i.e. after the metaphase of the primary maturation division was reached). The diameter of mature oocytes was 465–477 µm. The seasonal ovarian development cycle was divided into seven stages: inactive, developing, ripe, re- developing, re-ripe, spawned and recovery. The morphological characteristics relating to the gonadosomatic index (GSI) of each stage are described (GSI was calculated by the formula I = W × 105/L3, where I represents GSI, W is the gonad weight in grams, and L is the carapace length in millimetres). Estimated GSI values ranged from 11.3 to 12.4 in individuals with mature oocytes, and the 99% confidence intervals for GSI values of adjacent oogenesis substages did not overlap. GSI values at the developing and ripe stages were significantly larger than those at the re-developing and re-ripe stages, respectively.

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.

Changes in yolk platelet pH during Xenopus laevis development correlate with yolk utilization. A quantitative confocal microscopy study

The variations of the pH in Xenopus yolk platelets have been estimated by fluorescence confocal microscopy and computer image processing. For pH measurements in vitellogenic oocytes, the pH-sensitive fluorescent dye, DM-NERF, was coupled to vitellogenin, and the DM-NERF-vitellogenin was taken up by oocytes via receptor-mediated endocytosis. Dual emission ratio measurements of internalized DM-NERF-vitellogenin indicated that the mature yolk platelets are mildly acidic (pH 5.6). Their precursors, the primordial yolk platelets, have a similar pH. This pH is probably sufficiently low for the partial cleavage of vitellogenin to yolk proteins, but not for yolk degradation. The yolk platelet pH at various developmental stages was estimated by measuring the accumulation of Acridine Orange, both in isolated yolk platelets and in disaggregated embryonic cells. During oogenesis, the yolk platelets accumulated a constant amount of Acridine Orange, corresponding to a pH of around 5.7. During embryogenesis, however, yolk platelets became progressively much more acidic (pH < 5). Acidification correlated with yolk degradation in the various tissues examined, and yolk utilization was blocked when acidification was inhibited with bafilomycin, an inhibitor of vacuolar H+-ATPase. Bafilomycin also inhibited differentiation of cells isolated from stage 13–15 embryos. These data show that the yolk platelet pH is developmentally regulated and is involved in triggering yolk degradation. Also, yolk acidification and degradation appeared to be associated with cell differentiation and with the formation of the endosomal/lysosomal compartment, typical of adult cells, but absent in early embryos.

Deep cytoplasmic rearrangements during early development in Xenopus laevis

The egg of the frog Xenopus is cylindrically symmetrical about its animal-vegetal axis before fertilization. Midway through the first cell cycle, the yolky subcortical cytoplasm rotates 30 degrees relative to the cortex and plasma membrane, usually toward the side of the sperm entry point. Dorsal embryonic structures always develop on the side away from which the cytoplasm moves. Details of the deep cytoplasmic movements associated with the cortical rotation were studied in eggs vitally stained during oogenesis with a yolk platelet-specific fluorescent dye. During the first cell cycle, eggs labelled in this way develop a complicated swirl of cytoplasm in the animal hemisphere. This pattern is most prominent on the side away from which the vegetal yolk moves, and thus correlates in position with the prospective dorsal side of the embryo. Although the pattern is initially most evident near the egg's equator or marginal zone, extensive rearrangements associated with cleavage furrowing (cytoplasmic ingression) relocate portions of the swirl to vegetal blastomeres on the prospective dorsal side.