Noriko Usui

Ultrastructural Basis of the Tension Increase in Sea‐Urchin Eggs Prior to Cytokinesis

Cortices of sea‐urchin eggs were studied by electron microscopy to identify the structure responsible for the rise in tension at the egg surface prior to cleavage. During anaphase the tension increased and fine filaments of 70–90 Å in diameter appeared in the cell cortex forming a thin mesh‐work beneath the cell membrane. The meshwork spread all around the egg cortex without reference to the mitotic axis and the number of filaments seemed to increase up to telophase. Immediately before appearance of the cleavage furrow, the meshwork in the anticipated furrow region became dense. As the furrow appeared the tension began to decrease and the meshwork disappeared. In the progressing furrow region fine filaments of the same size as that of the meshwork‐filament were oriented in a bundle to form a contractile ring. Treatment with cytochalasin B suppressed both the tension increase and the formation of the filamentous meshwork. These results suggest that the component filament of the meshwork is an actin microfilament, and that the tension increase at anaphase is due to formation of a meshwork of actin microfilaments from which a contractile ring is subsequently derived at late telophase.

Breakdown of starfish ovarian follicle induced by maturation-promoting factor.

Immature starfish oocytes are surrounded by envelopes consisting of follicular cells. These cells adhere to each other and to the oocyte, immobilizing the latter within the ovary. When isolated oocytes in their follicles are treated with 1-methyladenine (1-MeAde), germinal vesicle breakdown (GVBD) and follicular envelope breakdown (FEBD) occur simultaneously. The 1-MeAde acts on the oocyte surface to produce a maturation-promoting factor (MPF) in the cytoplasm, which brings about GVBD. In the present study, MPF was found to induce FEBD as well as GVBD when injected into immature oocytes with their follicles in Asterina pectinifera. Although GVBD was induced by MPF in the presence of cytochalasin D, this drug prevented MPF-induced FEBD, and each follicular cell remained in situ on the surface of the oocyte. However, desmosomes connecting the processes of the follicle cell with the oocyte surface were disrupted following MPF injection even in the presence of cytochalasin D, and the processes became detached from the oocyte. FEBD occurred in these oocytes when cytochalasin D was removed, resulting in the formation of a small follicular clump by microfilament-mediated contraction of the follicle cells. These results show that FEBD is not brought about by the direct action of 1-MeAde but by the action of MPF. Therefore, in starfish, spawning as well as oocyte maturation is directly triggered by MPF produced under the influence of 1-MeAde.

Specific induction of self‐discrimination by follicle cells in Ciona intestinalis oocytes

Self‐incompatibility, a mechanism that prevents self‐fertilization in ascidians, is based on the ability of the oocyte vitelline coat to distinguish and accept only heterologous spermatozoa. In Ciona intestinalis self‐discrimination is established during late oogenesis and is contributed or controlled by products of the overlying follicle cells. In this study we have further investigated the role of the follicle cells in the onset of self‐discrimination by using in vitro maturation of ovarian oocytes deprived of the follicle cells and incubated with either autologous or heterologous follicle cells. Fertilization assays demonstrate that the action of the follicle cells is exerted even when they are detached from the vitelline coat and that only autologous follicle cells can promote the induction of self‐sterility on the egg coat. Electron microscopy of the oocytes during maturation reveals that the switch from self‐fertility to self‐sterility is accompanied by the appearance of a thin electron‐dense layer on the outer surface of the vitelline coat. We suggest that the formation of this layer is the result of the interaction between products of the follicle cells and the autologous vitelline coat.

Purification and immunocytochemical localization of the vitelline coat lysin of abalone spermatozoa

Abstract Spermatozoa of the abalone Haliotis discus were treated with high-calcium seawater to induce the acrosome reaction. The soluble components released from the sperm acrosomal vesicles showed potent lytic activity on the egg vitelline coat. A vitelline coat lysin was purified by salting-in, preparative polyacrylamide gel electrophoresis, and high-performance liquid chromatography. Its molecular weight was 15,500 and its isoelectric point 9.6. These properties were similar to those of other molluskan vitelline coat lysins. The lysin was immunocytochemically localized using a protein A-gold technique, in the posterior half of the acrosomal vesicle.

Sperm nuclear envelope: breakdown of intrinsic envelope and de novo formation in hamster oocytes or eggs.

During fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entry into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been a point of dispute. To resolve this issue, the fate of the SINEs of hamster sperm nuclei was traced after incorporation into immature oocytes at the germinal vesicle stage (GV oocytes) or fertilised eggs at the pronuclear stage (PN eggs). The SINEs disappeared quickly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelopes around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3 h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle, i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase promoting factor (MPF) is absent from the ooplasm.

MAGNESIUM ION-REQUIRING STEP IN FERTILIZATION OF SEA URCHINS*

The magnesium ion‐requiring step in fertilization of sea urchins was investigated. When eggs were inseminated in Mg‐free sea water, several spermatozoa were found to bind to each egg surface with their reacted acrosomes without elevation of fertilization membrane. The number of binding jelly‐treated spermatozoa to an egg did not differ regardless of the presence or virtual absence of magnesium ions. Although fertilization did not occur in Ca, Mg‐deficient sea water (CM‐deficient SW) even when jelly‐treated spermatozoa were employed, some eggs could be fertilized by the addition of magnesium to the CM‐deficient SW 60 sec after insemination, when jelly‐treated spermatozoa had completely lost their fertilizing capacity in the CM‐deficient SW. The acrosomal process of jelly‐treated spermatozoa appeared to penetrate the vitelline layer in the CM‐deficient SW. DTT‐ or pancreatin‐treated eggs could not be fertilized in the virtual absence of magnesium. Re‐fertilization using the fertilized eggs deprived of fertilization membrane did not occur under conditions of magnesium deficiency. These results suggest that external magnesium ions are indispensable at least for the fertilization process following penetration of the vitelline layer by the spermatozoa, such as fusion of the plasma membrane between an egg and a reacted spermatozoon, or the subsequent step(s) such as sperm penetration into egg interior and egg activation which precedes the cortical reaction.

THE SURFACE EVENTS AT FERTILIZATION OF THE SEA URCHIN EGG I. EVENTS ON THE SURFACE OF THE VITELLINE COAT1

Sperm‐egg interaction during normal fertilization in the sea urchins, Strongylocentrotus intermedius and Hemicentrotus pulcherrimus, was studied by scanning and transmission electron microscopy. Several seconds after insemination, acrosome‐reacted spermatozoa were found attached to the surface of the vitelline coat on each egg. Soon, several bulges of the vitelline coat appeared surrounding the fertilizing spermatozoon. These bulges then spread over the surface increasing in number, while they became fewer and disappeared around the sperm head. Thin sections of the bulging areas revealed discharging cortical granules. As the bulging vitelline coat was elevated, the sperm head was incorporated into the perivitelline space, passing through a small hole in the coat that resulted from penetration of the sperm acrosomal process immediately before fusion of the gametes. When the spermatozoon disappeared beneath the fertilization membrane, a hole was left in the membrane and the cortical reaction had finished on the other hemispheric surface. Mechanical removal of the membrane at that time exposed a spermatozoon protruding perpendicularly from the egg plasma membrane surface. The anterior tip of the sperm head was smoothly connected with the egg surface, and neither microvillous projections nor cytoplasmic covering of the egg cytoplasm could be found around the spermatozoon.

Quantitative analysis of modulations in intramembrane particles during maturation of starfish oocytes

The cell membrane of oocytes of the starfish Asterina pectinifera was investigated during maturation by freeze-fracture electron microscopy. On the micrographs of freeze-fractured oolemmas at various stages of maturation induced by 1-methyladenine, the intramembrane particles (IMPs) were counted and the size of each IMP was measured using an image analyzer. A 5-min treatment with the hormone lead to a decrease in IMP density on the E face and an increase on the P face. The most remarkable change in the density, an increase of 87% on the E face and a decrease of 42% on the P face, occurred within the following 5 min, the late “hormone-dependent period.” The mean value of IMP size first decreased on both faces, then gradually increased until values similar to those of the immature oocyte were reached. The results revealed that the hormonal signal to the surface receptors induces reorganization of the membrane components at the time of its action on the ooplasm, and that the oolemma also changes as a result of maturation of the ooplasm.

Ultrastructural changes in Hamster spermatogenic cell nuclei after incorporation into homologous oocytes by electrofusion

Isolated hamster spermatogenic cells at various stages of spermatogenesis were examined by thin‐sectioning techniques after electrofusion with activated homologous oocytes. The nuclei and attached organelles of the cells remained almost unchanged within the ooplasm three to five hours after the fusion pulse, by which time the oocytes had developed to the pronuclear stage. Only the spherical nuclei of primary spermatocytes and early spermatids in the Golgi and cap phases underwent modifications in their fine structures. They gained the morphological characteristics of well‐developed mammalian pronuclei; e.g., electron‐dense round nucleolus‐like bodies and blebbing of the nuclear envelope. In contrast, the elongated nuclei of later spermatids in the acrosome and maturation phases retained their original features, except that their acrosomes were deformed. Thus, ooplasm‐mediated transformation within activated oocytes at the pronuclear stage occurred only in nuclei containing dispersed chromatin and having nuclear pores in the envelope. Mol. Reprod. Dev. 52:66–73, 1999.

Membrane differentiations in echinoderm spermatozoa before and during the acrosome reaction as revealed by freeze fracture

Abstract Spermatozoa of sea urchin and starfish were examined before and during the acrosome reaction by means of thin sectioning and freeze fracturing with particular emphasis on the regions where membrane events occur upon the reaction. The two specialized regions demonstrated in earlier thin-section studies showed regional specialization in freeze-fracture study also. (1) In the trigger region, the anterior apex where the acrosomal granule (AG) and plasma membranes were closely apposed, an area poor in intramembrane particles (IMPs) was observed on the P face ofthe plasma membrane. Fusion occurred in this area, but the IMP-rich region around it remained unchanged in acrosome-reacting spermatozoa, suggesting that the IMP distribution in the plasma membrane is not modified except in the trigger region. (2) In the acrosomal process (AP) membrane precursor region, the posterior half of the AG where a layer(s) differing in electron density was demonstrated, compact IMP clusters were found uniformly covering the E face of the AG membrane. These IMP clusters disappeared from the AP membrane of reacting spermatozoa, indicating that the IMPS had been redistributed throughout the AP membrane. It is suggested that the intrinsic proteins in the AP membrane are derived from the IMP clusters of the AG membrane E face.