Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.     

Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea urchin fertilization

Anti-tubulin immunofluorescence microscopy is used here to demonstrate the configurations of the microtubule-containing structures which participate in the pronuclear movements of sea urchin fertilization. This technique shows that the egg is devoid of microtubules until after the fertilizing sperm is fully incorporated. All the microtubules which appear during the course of fertilization are organized around the base of the sperm head and the sperm aster thus formed behaves in a way that could account for the characteristic motions of the male and female pronuclei as documented by time-lapse video microscopy. Extension of astral microtubules appears to be responsible for the slow (ca. 2.5 μm min^?1) movement of the sperm aster into the cytoplasm of the egg; the rapid (ca. 15 μm min^?1) migration of the female pronucleus to the sperm aster seems to depend on connection of the female pronucleus to microtubules of the sperm aster. Continued extension of astral microtubules after the pronuclei are brought into conjunction can account for the centripetal motion of the paired (or fused) pronuclei and for the positioning of the zygote nucleus in the center of the egg. The behavior of astral microtubules during these motions suggests that they are capable of transmitting both pushing and pulling forces. All the pronuclear movements, and the assembly of detectable microtubules, are sensitive to the microtubule inhibitors griseofulvin and colchicine. Because of this sensitivity, and since all the observable microtubules within the egg during fertilization arise at the sperm aster, it is concluded that the pronuclear movements of fertilization result from the actions of the sperm aster. The pronuclear movements of sea urchin fertilization represent a simple but striking example of microtubule-mediated motility.     

Human sperm centrosomal function during fertilization, a novel assessment for male sterility

In human fertilization, the sperm introduces the centrosome-the microtubule organizing center-and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, the sperm aster, the functioning of which is essential for pronuclear movement for the union of the male and female genomes. We established functional assay for human sperm centrosomal function, by using heterologus ICSI system with bovine and rabbit eggs. After human sperm incorporation into mammalian egg, we observed that the sperm aster was organized from sperm centrosome, and the sperm aster enlarged as the sperm nuclei underwent pronuclear formation. The normal human sperm aster formation rate at 6 h post-ICSI were 60.0% in bovine egg and 36.1% in rabbit egg, respectively. However, sperm aster formation rate following heterologus ICSI into bovine eggs with teratozoospermia (globozoospermia, dysplasia of fibrous sheath) were low. These data indicate that human sperm centrosomal function is low in abnormal shaped sperm. Wherus, elucidation of human sperm centrosomal function can lead us to find a new type of failure in "post ICSI events in fertilization".     

Human sperm aster formation and pronuclear decondensation in bovine eggs following intracytoplasmic sperm injection using a Piezo-driven pipette: a novel assay for human sperm centrosomal function.

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8-12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.     

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.     

Role of the cytoskeleton in sperm entry during fertilization in the freshwater bivalve Dreissena polymorpha

The present study examined the role of the cytoskeleton in sperm entry and migration through the egg cytoplasm during fertilization in the zebra mussel, Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae). Fertilization in this freshwater bivalve occurs outside the mantle cavity, permitting detailed observations of fertilization. After its initial binding to the egg surface, the sperm is incorporated in two stages: (1) a gradual incorporation of the sperm nucleus into the egg cortex, followed by (2) a more rapid incorporation of the sperm axoneme, and translocation of the sperm head through the egg cytoplasm. Initial incorporation into the egg cortex was shown to be microfilament dependent. Microfilaments were found in the sperm's preformed acrosomal filament, the microvilli on the egg surface, and in an actin-filled insemination cone surrounding the incorporating sperm. Treatment of eggs with cytochalasin B inhibited sperm entry in a dose- and time-dependent manner. Microtubule polymerization was not necessary for initial sperm entry. Following incorporation of the sperm head, the flagellar axoneme entered the egg cytoplasm and remained active for several minutes. Associated with the incorporated axoneme was a flow of cytoplasmic particles originating near the proximal end of the flagella. Inhibition of microtubule polymerization prevented entry of the sperm axoneme, and the subsequent cytoplasmic current was not observed. After sperm incorporation into the egg cortex, no appreciable microfilaments were associated with the sperm nucleus. A diminutive sperm aster was associated with the sperm nucleus during its decondensation, but no obvious extension toward the female pronucleus was observed. The sperm aster was significantly smaller than the spindle associated with the female pronucleus, suggesting a reduced role for the sperm aster in amphimixis.     

Motility and centrosomal organization during sea urchin and mouse fertilization

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.     

Sperm incorporation,the pronuclear migrations,and their relation to the establishment of the first embryonic axis: Time-lapse video microscopy of the movements during fertilization of the sea urchin Lytechinus variegatus

The movements during fertilization have been investigated with differential interference optics and recorded by time-lapse video microscopy of the clear egg of the sea urchin Lytechinus variegatus. Sperm-egg binding occurs rapidly, and following a time when the sperm gyrates on the egg surface, gamete fusion occurs. A rapid cortical contraction radiates from the fusion site and is succeeded by the elevation of the fertilization coat. Sperm incorporation occurs in two stages: the fertilization cone enlarges around and above the erect and immotile sperm and then the sperm head, midpiece, and tail are displaced along the subsurface region of the egg at an average rate of 3.5 μm/min. The formation of the sperm aster moves the male pronucleus from the subsurface region of the egg toward the egg center at a rate of 4.9 μm/min. When the rays of the radial sperm aster appear to contact the female pronucleus, the female pronucleus migrates at a rate of 14.6 μm/min to the center of the sperm aster. The now adjacent pronuclei are moved to the egg center by the continuing enlargement of the sperm aster at a rate of 2.6 μm/min. Syngamy is usually preceded by the disassembly of the sperm aster. The centripetal migration of the pronuclei appears involved in the establishment of the first embryonic axis; cleavage occurs within 8° of the direction of this centering motion.     

Centrosomal function assessment in human sperm using heterologous ICSI with rabbit eggs: a new male factor infertility assay

Sperm centrosomal function was assessed by immunocytochemical analysis after the injection of human sperm into mature rabbit eggs. Three hours after intracytoplasmic sperm injection (ICSI), an astral microtubule array from the base of the human sperm was observed in the rabbit eggs. This sperm aster expanded in the egg cytoplasm, concomitant with pronuclear formation, and a dense microtubule array was organized at the time of pronuclear centration. Using fertile donor sperm, the sperm aster formation rate at 3 hr after ICSI was 35.0 +/- 1.5%. Using sperm from infertile patients, the average aster formation rate was lower (25.4 +/- 14.8%, P<0.05). Among infertile cases, there was no correlation between sperm aster formation rates and conventional parameters of semen analysis. However, the sperm aster formation rate correlated with the embryonic cleavage rate following human in vitro fertilization (IVF). These data suggest that this assay reflects sperm function during embryonic development after sperm entry and that reproductive success during the first cell cycle requires a functional sperm centrosome. Furthermore, sperm centrosomal function cannot be predicted from conventional parameters of semen analysis. We propose that insufficient centrosomal function could be the cause of certain cases of idiopathic infertility. These assays may lead to the discovery of new types of infertility, which have previously been treated as "unexplained infertility," and may also lead to the treatment of infertility incurable even by ICSI. Consequently, an accurate and relevant assay to help assure couples of the success of fertilization is warranted, perhaps prior to ICSI therapy.     

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.     

Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization

Unfertilized eggs of the rose bitterling (Rhodeus ocellatus ocellatus) were squeezed out of females that had an elongated ovipositor and were dechorionated mechanically with fine forceps in physiological saline. The dechorionated eggs were transferred into fresh water then inseminated at once by spermatozoa of the same species. A large number of spermatozoa was found on the surface of eggs that had not yet had cortical reaction following insemination. The surface of the naked eggs responded by formation of many small cytoplasmic protrusions (viz., fertilization cones) at sperm attachment sites. The formed fertilization cones were rosettelike structures formed by the aggregation of some bleblike swellings devoid of microvilli and microplicae. About 10 min after insemination, the fertilization cones retracted, but marks of their presence characterized by less microvilli and microplicae remained in the eggs 15 min after insemination. Many spermatozoa penetrated into the cytoplasm of each naked egg. The sperm nuclear envelope disappeared by means of vesiculation resulting from fusion of the inner and outer membranes. The sperm nucleus decondensed and developed into a larger male pronucleus. Smooth-surfaced vesicles surrounded the decondensing sperm nucleus and formed the new male pronuclear envelope. Sperm mitochondria and flagella were found in the egg 15 min after insemination. The response of the egg surface to sperm entry and pronucleus formation are discussed.     

Microtubules are required for centrosome expansion and positioning while microfilaments are required for centrosome separation in sea urchin eggs during fertilization and mitosis

Centrosomes undergo cell cycle-dependent changes in shape and separations, changes that govern the organization of the cytoskeleton. The cytoskeleton is largely organized by the centrosome; however, this investigation explores the importance of cytoskeletal elements in directing centrosome shape. Since the sea urchin egg during fertilization and mitosis displays dramatic and synchronous changes in centrosome shape, the effects of cytoskeletal inhibitors on centrosome compaction, expansion, and separation were explored by the use of anticentrosome immunofluorescence microscopy. Centrosome expansion and separation was studied during two phases: the transition after sperm incorporation, when the compact sperm centrosome enlarges and the sperm aster develops, and from prometaphase to telophase, when the compact spindle poles enlarge. Compaction was investigated when the dispersed centrosome at interphase condenses into the two spindle poles at prometaphase. Although centrosome expansion and separation typically occur concurrently, beta-mercaptoethanol results in centrosome separation independent of expansion. Microtubule inhibitors prevent centrosome expansion and separation, and expanded centrosomes collapse. Since pronuclear union is arrested by microtubule inhibitors, this treatment also affords the opportunity to explore the relative attractiveness of the male and female pronuclei for these centrosomal antigens. Both pronuclei acquire centrosomal material; though only the male centrosome is capable of organizing a functional bipolar mitotic apparatus at first division, the female centrosome nucleates a monaster. Microfilament inhibition (cytochalasin D) prevents centrosome separation but not expansion or compaction. These results demonstrate that as the centrosome shapes the cytoskeleton, the cytoskeleton alters centrosome shape.     

Effects of cytoskeletal inhibitors on ooplasmic segregation and microtubule organization during fertilization and early development in the ascidian Molgula occidentalis

The effects of microtubule and microfilament inhibitors on ooplasmic segregation and microtubule organization were examined during fertilization, parthenogenetic activation, and early development in the ascidian Molgula occidentalis. At fertilization the egg cortex contracts as the first phase movement and shortly after mitochondria migrate as the myoplasmic crescent develops in the second phase. The microtubule inhibitors colcemid and nocodazole inhibit the second phase, but not the first phase, of ooplasmic segregation. The microfilament inhibitor cytochalasin E has the reciprocal effect of inhibiting the first, but not the second, phase. It appears that sperm may initially bind at any site on the egg surface and that the contractile activities at the first phase and during polar body formation occur independent of the microtubule system. Since the second phase migration occurs as the sperm astral microtubules assemble and since microtubule, but not microfilament, inhibitors arrest this aspect of ooplasmic segregation, microtubules appear necessary for mitochondrial migration. These results demonstrate that the two phases of ascidian ooplasmic segregation are mediated by different systems, the first by microfilaments and the second by microtubules. The microtubule and microfilament systems appear to operate independent of one another and their combined actions result in the completion of ooplasmic segregation. A model is proposed in which the cortical contraction following fertilization is important not only as the motive force for the first phase movement but also as a method to unite the myoplasm with the entering sperm which can initially bind anywhere on the egg surface. The association between myoplasmic components and the growing sperm aster would ensure that the migration and the spatial distribution of myoplasm in the second phase results in the formation of the myoplasmic crescent.     

Effects of cytochalasins B and D on the fertilization of zebrafish (Brachydanio) eggs

The effects of selected concentrations of cytochalasins B (1-10 micrograms/ml; CB) and D (10, 50 micrograms/ml; CD) on the morphology and fertilization of zebra danio (Brachydanio) eggs were studied primarily with light and scanning electron microscopy. Eggs pretreated with either CB (10 micrograms/ml) or CD (10, 50 micrograms/ml) prepared in Fish Ringer's solution-0.5% DMSO showed a flattened shape, alterations in the form of surface microplicae and microvilli, and occasional spontaneous exocytosis of cortical granules. All eggs preincubated in either CB or CD were activated upon transfer to tap water, showing cortical granule exocytosis, elevation of the chorion, and formation of a fertilization cone. When eggs were pretreated for 5 minutes with 1-5 micrograms/ml CB or 10 micrograms/ml CD and inseminated, they incorporated the fertilizing sperm and typically developed to the two-cell stage. A single sperm cell attached to and fused with the sperm entry site microvilli but failed to enter the cytoplasm in eggs preincubated with 10 micrograms/ml CB. Eggs that were immersed continuously in either CB (10 micrograms/ml) or CD (50 micrograms/ml) 15 seconds after insemination also failed to incorporate the fertilizing sperm. Treatment of eggs after insemination with CD (10 micrograms/ml), however, did not prevent sperm cell incorporation or fertilization cone formation. Our drug data suggest the presence of actin-containing filaments in the danio egg before and following fertilization. These filaments appear to play a role in maintaining the shape of the egg cell and its surface specializations and in the incorporation of the fertilizing sperm. The fertilization cone appears to form independently of actin polymerization.     

Microtubule and centrosome distribution during sheep fertilization

The distribution of microtubules and centrosomes was studied during sheep fertilization by electron and immunofluorescence microscopy. Tubulin and centrosomal material was identified with monoclonal anti-alpha-tubulin and MPM-2 antibodies, respectively. In ovulated eggs, microtubules were exclusively found in the meiotic spindle and centrosomal material at each of its poles. At fertilization, sperm centrosomes were incorporated into the egg and organized the sperm astral microtubules. During pronuclear development and migration, the sperm aster increased in size; microtubules of the sperm aster extended from the male pronucleus to the egg center and towards the female pronucleus. The position of the sperm aster during pronuclear migration suggests that it plays a role in this process. When the pronuclei were in apposition in the egg center, a dense array of microtubules and the centrosomal material were present between the two pronuclei. The proximal centriole of the sperm was identified by electron microscopy, between the apposed pronuclei. The centrosomal material extending around the centriole and the sperm neck and proximal mid-piece, apparently contained several foci from which microtubules radiated. These data suggest that in sheep unlike in mice, centrosomal material originating from the sperm is involved in the fertilization events.     

Microtubules in ascidian eggs during meiosis, fertilization, and mitosis

The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic crescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.     

Microtubule assembly is required for the formation of the pronuclei,nuclear lamin acquisition,and DNA synthesis during mouse,but not sea urchin,fertillization

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.     

Physiological responses of sea urchin eggs to stimulation by calcium ionophore A23187 analysed with protease inhibitors

Protease inhibitors were used to study certain physiological responses (secretion of the cortical granule protease, altered resceptively to sperm penetration, initiation of cell division and embryogenesis) of sea urchin eggs to stimulation by calcium ionophore A23187. Protease activity in the secretory product released from the eggs 5 min after insemination or parthenogenetic activation with ionophore was completely inhibited by soybean trypsin inhibitor (SBTI), antipain (Ap), and leupeptin (Lp). A barrier was established to prevent subsequently added sperm from penetrating (fertilizing) ionophore-activated eggs, co-incident with the elevation of the fertilization membrane. These processes were retarded by inhibitors of the cortical granule protease in ionophore-activated eggs, just as they are when eggs are initially stimulated by sperm at fertilization. A23187-activated eggs did not divide unless they had been secondarily fertilized by sperm, even if the ionophore was subsequently removed by extensive washing. However, ionophore-activated eggs that were penetrated by a single spermatozoan in SBTI developed into normal larvae under similar conditions. These results suggest that A23187 may be an incomplete parthenogenetic agent because it cannot stimulate eggs to assemble centrioles required to organize the mitotic apparatus. The centrioles are normally provided by the sperm during fertilization. A23187 may also be toxic to the eggs. Furthermore, since cortical granules are secretory organelles, the data suggest a possible functional relationship between calcium ions and protease activation in stimulus-secretion coupling in sea urchin eggs at fertilization.     

Axis establishment and microtubule-mediated waves prior to first cleavage in Beroe ovata

The single axis (oral-aboral) and two planes of symmetry of the ctenophore Beroe ovata become established with respect to the position of zygote nucleus formation and the orientation of first cleavage. Bisection of Beroe eggs at different times revealed that differences in egg organisation are established in relation to the presumptive oral-aboral axis before first cleavage. Lateral fragments produced after but not before the time of first mitosis developed into larvae lacking comb-plates on one side. Time-lapse video demonstrated that waves of cytoplasmic reorganisation spread through the layer of peripheral cytoplasm (ectoplasm) of the egg during the 80 minute period between pronuclear fusion and first cleavage, along the future oral-aboral axis. These waves are manifest as the progressive displacement and dispersal of plaques of accumulated organelles around supernumerary sperm nuclei, and a series of surface movements. Their timing and direction of propagation suggest they may be involved in establishing cytoplasmic differences with respect to the embryonic axis.Inhibitor experiments suggested that the observed cytoplasmic reorganisation involves microtubules. Nocodazole and taxol, which prevent microtubule turnover,blocked plaque dispersal and reduced surface movements.The microfilament-disrupting drug cytochalasin B did not prevent plaque dispersal but induced abnormal surface contractions. We examined changes in microtubule organisation using immunofluorescence on eggs fixed at different times and in live eggs following injection of rhodamine-tubulin. Giant microtubule asters become associated with each male pronucleus after the end of meiosis. Following pronuclear fusion they disappear successively, those nearest the zygote nucleus shrinking first, to establish gradients of aster size within single eggs. Regional differences in microtubule behaviour around the time of mitosis were revealed by brief taxol treatment, which induced the formation of small microtubule asters in the region of the nucleus or spindle during both first and second cell cycles. The observed wave of change may thus reflect the local appearance and spreading of mitotic activity as the zygote nucleus approaches mitosis.     

Biparental Inheritance of γ-Tubulin during Human Fertilization: Molecular Reconstitution of Functional Zygotic Centrosomes in Inseminated Human Oocytes and in Cell-free Extracts Nucleated by Human Sperm

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents γ-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. γ-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal γ-tubulin. Recruitment of maternal γ-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm “priming” induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.