EFFECT OF DNP ON ACCELERATION OF THE CLEAVAGE FOLLOWING INSUFFICIENT PARTHENOGENETIC ACTIVATION IN THE SEA URCHIN EGG*

Unfertilized eggs of sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus, were treated with 4–5% butyric acid-sea water for 40–60 sec so that they were activated partheno-genetically without visible cortical changes. When these insufficiently activated eggs were inseminated 90–120 min after butyric acid-treatment, they divided much earlier than the control eggs in the first cleavage cycle. In the present paper, it becomes clear that if eggs are put into m /2,000-m /16,000 DNP-sea water at 60 min after insufficient activation and 30 min later, returned to normal sea water and then inseminated, they still show acceleration of the first cleavage in the same degree as the eggs which are not treated with DNP, while if eggs are exposed to DNP for 30 min prior to the insufficient activation or within 60 min after the activation, they do not show any acceleration of the cleavage. From these results, it may be concluded that some preparations for cleavage acceleration which are arrested by DNP become ready in the eggs at an early period in the first cleavage cycle and these preparations cannot be cancelled by DNP-treatment once they have been completed.     

Separation of microvilli from Tubifex eggs upon activation: Its inhibition by concanavalin A hinders ooplasmic segregation and cleavage

The surface of mature eggs of the freshwater oligochaete Tubifex exhibits numerous microvilli. Upon activation, microvilli become narrower at their base and separated from the ooplasmic surface. Here it is shown that concanavalin A (Con A) reversibly inhibits the separation of microvilli from activated Tubifex eggs. The Con A-treated eggs undergo meioses and mitoses at a normal rate. Microvilli on these eggs change their length in a meiotic cycle-dependent manner; their core bundles of microfilaments elongate significantly during the second meiosis. The Con A-treated eggs fail to complete polar body formation, ooplasmic segregation and cleavages. Treatment with Con A of eggs that have accomplished microvillar separation does not exert any inhibitory effect on their development. Succinyl-Con A, a dimeric derivative of Con A, does not prevent microvillar separation, suggesting that the tetravalent form of Con A is essential for Con A to exert its inhibitory effect on microvillar separation.     

Ultrastructural studies of oocytes and embryos derived from females flies carrying the grandchildless mutation in Drosophila subobscura

The maternal effect mutant grandchildless in Drosophila subobscura has been analyzed with the electron microscope. The original mutation was linked to a visible genetic marker and established in a balanced stock. Oocytes and early embryos were examined by both transmission and scanning electron microscopy. The earliest defect is seen in mutant eggs and occurs at the end of oogenesis. In the cortex, at both the anterior and the posterior tips, regions appear which are free of ribosomes, mitochondria, and other cytoplasmic organelles. Most of the polar granules are included in these regions at the posterior tip. Following oviposition, this cytoplasmic segregation is no longer observed and most polar granules have disappeared. The few remaining granules are presumed to derive from the peripheral polar plasm which does not become segregated. During embryogenesis there is a retarded movement of nuclei to the anterior and posterior cortices. At the posterior tip nuclei are delayed in reaching the lateral sides and never move directly into the posterior polar plasm. Pole cells never form. After the last syncytial division the lateral nuclei move under the posterior polar plasm to complete the blastoderm. The posterior polar plasm itself protrudes during blastoderm formation as long cytoplasmic extensions which separate from the blastoderm as cytoplasmic blebs. Neither polar granules nor mitochondria are found in these blebs. The grandchildless phenotype is due to the failure of nuclei to migrate directly into the posterior polar plasm. The defect in the polar plasm presumably is related to the process in mature eggs whereby portions of the cortex become segregated at both anterior and posterior tips. This process may change the properties of the posterior polar plasm so that nuclei do not penetrate into it.     

Second meiotic division and polar body formation in mouse eggs fertilized in vitro

The second meiotic division and polar body formation in mouse eggs fertilized in vitro were observed by phase-contrast and polarizing microscopy, and recorded by time-lapse cinematography. Eggs were collected from oviducts of mice that had been superovulated by injections of PMS and HCG. Some eggs, inseminated with spermatozoa that had been collected from caudae epididymides of mature male mice and cultured for two to three hours before insemination, were observed continuously on a glass slide under a phase microscope. Other eggs were inseminated in Petri dishes in a 5% CO₂ incubator and examined every 20 minutes for 180 minutes. Compatible results in both sets of eggs showed that formation of the second polar body began 25–40 minutes after fusion of spermatozoon with the vitellus; it was completed 40–60 minutes later; anaphase II lasted approximately five minutes before the appearance of the furrow abstricting the second polar body. It is suggested that the furrowing associated with second polar body formation is guided by the same kind of forces that divide a cell mitotically.     

Inhibition of the onset of vitellogenesis by abnormal retention of mature eggs in the stick insect,Carausius morosus

Mature eggs stored in the lateral oviducts are unusual in Carausius morosus. When egg laying is experimentally prevented, ovarian production is inhibited by the accumulation of mature eggs within the lateral oviducts. Such storage affects only the very small terminal oocytes and not the vitellogenic follicles, which attain to term and are ovulated. The fact that unilateral retention of eggs affects only the corresponding ovary strongly suggests that the mature eggs themselves inhibit ovarian production in the stick insect without the involvement of an endocrine organ. It is likely that this mode of action, different from that suggested for the housefly or Rhodnius prolixus, is correlated with the independence of ovarian development from the corpus allatum hormone in the stick insect.     

Hydrozoan eggs can only be fertilized at the site of polar body formation

Hydrozoan eggs are normally fertilized at the site of polar body formation. The female pronucleus is just under the cell membrane at this site. Sperm are attracted to the eggs and aggregate at this site. This paper demonstrates that this site is the only region on the egg surface where the sperm can fuse with the egg. This has been done by cutting unfertilized eggs into fragments containing the site of polar body formation and fragments without this region. Sperm were added to the fragments and their ability to be fertilized was assayed by noting whether or not they cleaved. Only fragments containing the site of polar body formation cleaved. The absence of cleavage in fragments lacking the site of polar body formation cannot be attributed to the inability of these fragments to attract sperm. Such fragments attract sperm for several hours while fragments which contain the site of polar body formation stop attracting sperm a few minutes after fertilization. Cytological studies of egg fragments which do not contain the site of polar body formation show that they do not contain sperm nuclei. The lack of cleavage in these fragments cannot be attributed to the lack of a female pronucleus. By using centrifugation it is possible to move the female pronucleus away from the site of polar body formation. By cutting these centrifuged eggs in an appropriate way it is possible to create egg fragments with the site of polar body formation that lack the female pronucleus and egg fragments that lack the site of polar body formation but contain a female pronucleus. Only fragments which contain the site of polar body formation can be fertilized.     

In vivo fertilization and development of medaka eggs initiated by artificial insemination

Fertilization and development in the ovarian cavity of oviparous fish, Oryzias latipes, were examined using the S-rR strain in which the sex genotype can be easily distinguished by the body color of the fish. Mature eggs were fertilized within the ovarian cavity after a sperm suspension was artificially introduced with a small bore-pipette through the urinogenital opening. Three batches of eggs ovulated within 48 hrs were fertilized and began to develop in the ovarian cavity, while eggs ovulated 72 hrs post-insemination (PI) were no longer fertilized. These observations indicate that ovulation occurs irrespective of the existence of developing embryos within the ovarian cavity. All embryos developing in the ovarian cavity were, however, retarded and ceased development before the stage of initiation of blood circulation at room temperature. These embryos developed normally and hatched after they were transferred from the ovarian cavity into regular saline 48 or 72 hrs PI. When these individuals matured sexually, their sex differentiation was found to be normal, and sex reversal was not observed.     

Brefeldin A disrupts asymmetric spindle positioning in mouse oocytes

Polar body formation in oocytes is an extreme form of asymmetric cell division, but what regulates the asymmetric spindle positioning and cytokinesis is poorly understood. During mouse oocyte maturation, the metaphase I spindle forms at the center but then moves to the cortex prior to anaphase I and first polar body emission. We show here that treating denuded mouse oocytes with brefeldin A, an inhibitor of Golgi-based membrane fusion, abolished the asymmetric positioning of the metaphase I spindle and resulted in the formation of two half-size metaphase II eggs, instead of a full-sized egg and a polar body. The normal metaphase II spindle is similarly asymmetrically positioned in the mature egg, where the spindle lies with its axis parallel to the cortex but becomes perpendicular before anaphase II and emission of the second polar body. When ovulated eggs were activated with strontium in the presence of brefeldin A, the metaphase II spindle failed to assume perpendicular position, and the chromosomes separated without the extrusion of the second polar body. Remarkably, symmetric cytokinesis began following a 3 h delay, forming two half-size eggs each containing a pronucleus. BFA-sensitive intracellular vesicular transport is therefore required for spindle positioning in both MI and MII.     

Establishment of self-sterility of eggs in the ovary of the solitary ascidian,Halocynthia roretzi

When unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, are released naturally they are strictly self-sterile, whereas almost all ovarian eggs isolated after spawning are self-fertile. Self-sterile eggs are prepared within a relatively short period of several hours before the spawning. The morphological changes in ovarian eggs during late oogenesis were studied with special reference to the establishment of self-sterility. Four types of eggs at serial developmental stages were classified according to the morphology of their external envelopes. Self-sterility was established in the last stage, from the ovarian egg type 3 (OVE3) to UFE stages. Ovarian eggs which had become committed to UFE were denoted as full-grown ovarian eggs (FOE). FOE were able to differentiate into self-sterile UFE in vitro, whereas OVE3 could not. Several morphological differences between OVE3 and UFE were found. OVE3 had a germinal vesicle (GV), a type of vitelline coat (VC-OVE3) and no expanded perivitelline space, whereas UFE had completed germinal vesicle break down (GVBD), had another type of coat (VC-UFE) and showed an expanded perivitelline space. There were also some differences in the mode of fertilization between OVE3 and UFE. In UFE, sperm became bound firmly to the vitelline coat and passed through the coat with the help of follicle cells, whereas in OVE3, sperm did not bind so strongly and entered the perivitelline space without the aid of follicle cells. The relationships between the establishment of self-sterility and these morphological and functional changes in ovarian eggs are discussed.     

Description of the fully mature oocyte of Drosophila melanogaster.

We detected fully mature undescribed oocytes in Drosophila melanogaster ovaries. The fully mature oocytes were rehydrate in ovaries. In the oocyte of stage 14, chromosomes make globular mass, while the chromosome shows meiotic metaphase I in fully mature oocytes. Both mature oocytes and stage 14 oocytes were activated by hypotonic treatment. When the mature oocytes or the stage 14 oocytes were activated, telophase II figures were observed in former oocytes but meiosis in latter oocyte stopped at late metaphase I, suggesting the stage 14 activated oocyte cannot pass the second checkpoint of meiosis.     

Development of pronuclei from human spermatozoa injected microsurgically into frog (Xenopus) eggs

Human spermatozoa were demembranated with Triton X-100 (TX) and injected into the mature eggs of Xenopus laevis. The nuclei of these spermatozoa decondensed and developed into pronuclei. Chromosomes did not appear in the eggs until the end of a 5-hr incubation period. When the demembranated human spermatozoa were further treated with dithiothreitol (DTT) before they were injected into the eggs, the sperm nuclear decondensation and pronuclear development took place considerably faster than in spermatozoa treated with the detergent alone. By the end of the 5-hr incubation period, decondensed chromatin threads or chromosome-like structures appeared, but none of the eggs cleaved. When human spermatozoa were injected into full-grown ovarian oocytes with intact germinal vesicle (GV) or oocytes which had matured without GV, the nuclei of a proportion of TX-treated and all TX-DTT-treated sperm decondensed but showed no sign of developing into pronuclei. Sperm nuclei injected into maturing oocytes formed condensed chromatin fragments as long as the oocytes were not activated, but they transformed into pronuclei when the oocytes were stimulated with electric shock. These results indicate that the cytoplasmic factors responsible for the decondensation of human sperm nuclei are present in egg cytoplasm independent of GV-materials. We also suggest that the factors controlling development of decondensed sperm nuclei into pronuclei are dependent on GV materials.     

Inhibition of the activation reaction of Xenopus laevis eggs by the lectins WGA and SBA

Ionophore A23187 and electrical activation of dejellied mature eggs of Xenopus laevis are both prevented by the lectins wheat germ agglutinin (WGA) and soya bean agglutinin (SBA). However, this inhibition is not total since one of the events associated with egg activation, the activation potential, still occurs under lectin treatment. After 10 min of incubation in 50 μg/ml WGA or 100 μg/ml SBA, the cortical reaction, cortical contraction, and second polar body emission are totally impaired, whereas the activation potential, although different from the normal one, still proceeds. At the ultrastructural level, the lectin binding sites are localized on the vitelline envelope and on the plasma membrane. The inhibitory effects of these lectins are not detected in jellied eggs. Also, spermatozoa are strongly agglutinated by WGA at concentrations as low as 2.5 μg/ml, but not by SBA. This suggests that inhibition of fertilization in WGA-treated eggs is due to an effect of the lectin on the sperm.     

Surface alterations of fertilized surf clam (Spisula solidissima) eggs induced by concanavalin A

Fertilized Spisula eggs, incubated in ConA, were examined at periodic intervals to determine the effects of lectin binding on events of fertilization and cleavage. ConA was localized to specific regions of the vitelline layer and plasma membrane by reacting lectin-treated eggs with horseradish peroxidase and diaminobenzidine. In contrast to eggs, little reaction product was associated with the plasma membrane of spermatozoa. Sperm that fused with ConA-treated eggs failed to move into the cortex of the ovum and were observed as bulbous appendages at the surface of the zygote. Reorganization of sperm nuclei was inhibited, and male pronuclei failed to develop. ConA also inhibited polar body formation and cleavage. The maternally derived chromatin underwent meiosis, and the chromosomes normally taken into the first and second polar bodies were retained within the zygote. All of the maternally derived chromatin was organized within four or more female pronuclei which subsequently entered mitosis. The effects of ConA binding on events at the surface of fertilized Spisula eggs were abrogated by α-methyl-d-mannoside; succinyl-ConA only partially inhibited fertilization-related processes. The effects of ConA are discussed in terms of possible cross-linking of surface components of fertilized Spisula eggs which may inhibit deformation of the zygote cortex.     

Fertilization Reaction without Changes in Intracellular Ca2+ in Medaka Eggs—An Experiment with Acetone-Treated Eggs

To investigate whether or not causal relationship exists between the increase in intracellular Ca²⁺ and other cortical reactions at fertilization in the medaka, Oryzias latipes , intracellular Ca²⁺ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca²⁺. Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone-treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca²⁺ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca²⁺ and exocytosis.     

Localization of morphogenetic determinants in a special cytoplasm present in the polar lobe ofBithynia tentaculata (Gastropoda)

Summary In the first polar lobe ofBithynia eggs a special plasm, the vegetal body, is present. It consists of a cupshaped aggregate of small vesicles. Centrifugation of eggs prior to first cleavage may result in displacement of the vegetal body. In about 50% of thecentriguged eggs the vegetal body is found outside the polar lobe, in one of the blastomeres. Removal of the polar lobe from non-centrifuged eggs always leads to severe defects in development. When the lobe is removed from centrifuged eggs, however, about 50% of the eggs develop into normal embryos. It is concluded that the presence of the vegetal body in a blastomere suffices to ensure normal development and, hence, that the polar lobe-specific morphogenetic determinants are contained within the vegetal body.     

Microtubule cycles in oocytes of the surf clam, Spisula solidissima: an immunofluorescence study

Oocytes of the surf clam, Spisula solidissima, underwent germinal vesicle breakdown and two meiotic divisions to give off polar bodies when they were fertilized or parthenogenetically activated with KCl. Fertilized eggs further proceeded to mitosis and cleaved, while parthenogenetically activated eggs remained uncleaved. We examined changes in microtubule-containing structures during meiotic divisions and subsequent mitotic processes by immunofluorescence. A monoclonal anti-tubulin antibody was applied to alcohol-fixed eggs from which the vitelline membrane had been removed by protease digestion. Up to the stage of second polar body formation, the pattern of microtubule organization in the first and second meiotic spindles was identical in both fertilized and parthenogenetically activated eggs. However, while fertilized eggs formed a sperm aster and mitotic spindles later, activated eggs formed only monaster- or ring-shaped microtubule-containing structures which underwent cycles of alternating formation and breakdown. Lactoorecin staining of parthenogenetically activated eggs revealed that the chromosome cycle could occur in these eggs, in phase with this microtubule cycle.     

Periodic changes in the rigidity of activated anuran eggs depend on germinal vesicle materials

The eggs of Xenopus laevis and Bufo bufo japonicus matured in vitro by progesterone were denuded and activated by electrical shock or pricking, and their diameters were determined periodically until the postactivation time equivalent to the 8- to 16-cell stage. Normally matured eggs showed a quick rounding-up and flattening immediately after activation (activation response), followed by a periodic flattening and rounding-up (postactivation response) corresponding to the intervals of early cleavage cycles. When denuded eggs that were induced to mature after removal of the germinal vesicle (GV) were activated, they showed a normal activation response, but retained the rounded-up state without exhibiting the periodic postactivation changes. Injection of GV materials back into enucleated Bufo oocytes and determination of the postactivation flattening 120 min after activation revealed that the extent of flattening correlated well with the amount of GV materials injected, provided injection was made before the first polar body was emitted. Thus, postactivation flattening, but not activation flattening, is an active property of eggs established under the influence of the GV and is linked to the periodical changes of rigidity during early cleavage cycles.     

The influence of group size on ovarian development in adult and neotenic reproductives of the termite Zootermopsis angusticollis (Hagen) (Hodotermitidae)

Termite imagines of Zootermopsis angusticollis (Hagen), while still in the colony as winged individuals, do not show ovary development, although vitellogenin is present in the blood at low levels. When removed from the colony, artificially dealated, and paired, vitellogenin levels rise: subsequently ovaries develop resulting in oviposition approximately 15–16 days after removal from the colony. Imagines removed from the colony, dealated and held together in a heterosexual group, however, show little or no ovary development after 25 days, although vitellogenin is present in the haemolymph in low levels. Newly developed neotenic reproductives, on the other hand, removed from the colonly 1–2 days after the neotenic moult, and isolated in heterosexual pairs for 60 days, had ovaries which did not become larger than 0.02 mm³, and no eggs were laid. This is in contrast to the normal ovary development of neotenic reproductives produced in groups of larvae, as described earlier. Vitellogenin levels in isolated pairs of neotenics were low until 25 days after isolation, while vitellogenin levels increased 7–9 days after the moult in female neotenics kept in a colony with larvae. The results of this study on ovarian development and vitellogenesis in neotenic and adult reproductives reflect the normal biology of the two reproductive types where the latter leave the colony to found new colonies, while neotenics become sexually mature within the colony in which they developed.     

Oocyte Maturation and Furrow Formation in an Unfertilized Egg by Fusion with a Fertilized Egg or Blastomeres in the Ascidian, Ascidia sydneiensis divisa

A technique for fusing an ascidian egg with blastomeres using a chemical fusiogen was established and then used to identify cytoplasmic factors that regulate the process of oocyte maturation in ascidian eggs. Unfertilized eggs fused with fertilized eggs or blastomeres in hypotonic artificial sea water containing 20% polyvinyl alcohol within 10 min. After fusion polar bodies were extruded from the unfertilized portion of the fused eggs. Furrows were formed not only in the fertilized portion but also in the unfertilized portion in the fused eggs. No polar body extrusion and furrow formation occur in either portion of fused unfertilized eggs. These results suggest that fertilized eggs and blastomeres contain a factor that induces oocyte maturation. Polar body extrusion and furrow formation were not suppressed in the fertilized portion of fused eggs, suggesting that unfertilized eggs do not contain a factor that inhibits oocyte maturation.     

Factors inducing closure of the micropylar canal in the chum salmon egg

The micropylar canal of the chum salmon egg was almost completely closed following egg activation caused by incubation in a hypotonic salt solution (HSS) for I h. The closure occurred in both inseminated and parthenogenetically activated eggs. Incubation of isolated envelopes from non-activated eggs in HSS or perivitelline fluid (PVF) did not induce any modification in micropylar structure, indicating that normal organization of the egg is essential for inducing closure. To reduce the volume of the perivitelline fluid, the eggs were activated in PVF or HSS containing 8 mM Dextran, Although the envelope showed hardening, closure of the micropyle was not observed in these eggs. The wall of the micropylar canal, however, possessed a slightly rough surface. Following activation in a Ca-free hypotonic salt solution with 10 mM EDTA, hardening of the egg envelope was completely inhibited. Although such eggs possessed an apparent perivitelline space, neither closure of the micropylar canal nor roughening of the canal surface were detected. We conclude that the synergistic action of perivitelline turgor pressure and perivitelline material is responsible for the closure of the micropyle.