Determination of first cleavage plane: the relationships between the orientation of the mitotic apparatus for first cleavage and the position of meiotic division-related structures in starfish eggs

In order to understand when the orientation of the first cleavage plane is fixed along the animal-vegetal axis in starfish eggs, the behavior of the sperm aster was examined by indirect immunofluorescence staining. After duplication, the sperm aster organizes the mitotic apparatus for first cleavage perpendicular to the cleavage plane. The sperm aster located in the egg periphery just after fertilization and moved to the site close to the animal pole rather than the egg center by meiosis II. At early metaphase II, duplication of the sperm aster was detected but the axis of the resultant sperm diaster randomly pointed. Subsequently, its axis had already turned perpendicular to the animal-vegetal axis before pronucleus fusion. These results indicate that the orientation processes of the sperm diaster consist of positioning before its duplication and successive determining its azimuth. Furthermore, the azimuth and position of the mitotic apparatus for first cleavage did not change by shifting or eliminating the meiotic division-related structures such as the germinal vesicle, meiotic spindle, and female pronucleus by micromanipulation. These results show that none of them determines the first cleavage plane. Therefore, we discuss the pointing mechanism of the first cleavage plane without the influence of these meiotic division-related structures.     

"Spiral asters" and cytoplasmic rotation in sea urchin eggs: induction in Strongylocentrotus purpuratus eggs by elevated temperature

"Spiral asters" composed of swirls of subcortical microtubules were recently described in fertilized eggs of the sea urchin Strongylocentrotus purpuratus. In our study, these structures did not occur at culture temperatures below 16 degrees C. When the culture temperature was elevated, however, "spiral asters" routinely appeared during a susceptible period before mitotic prophase when the sperm aster-diaster normally exists. A massive and protracted rotation of the cytoplasm (excluding an immobile cortex and perinuclear region) began within 1 min of exposure to elevated temperature. Fibrils of the "spiral aster" could be seen within this rotating mass even by bright-field microscopy. The identity of microtubules in these structures was confirmed by indirect immunofluorescence microscopy. A mechanistic association between "spiral aster" formation and cytoplasmic rotation was indicated by the simultaneous inhibitory effects of microtubule and dynein poisons. Inhibitors of microfilaments, however, had no effect. We infer that elevated temperature induces unique changes in the microtubules of the pre-prophase sperm aster-diaster, resulting in cytoplasmic rotation and the spiral configuration of microtubules. Comparative cytological evidence supports the idea that "spiral asters" do not normally occur in fertilized sea urchin eggs. Biogeographic evidence for S. purpuratus indicates that fertilization and development naturally occur below 15 degrees C, hence "spiral asters" in eggs of this species should be regarded as abnormalities induced in the laboratory by unnaturally elevated temperatures.     

Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage

Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.     

Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs.

Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185-197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.     

Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs.

Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.     

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.     

Independence of two microtubule systems in fertilized frog eggs: the sperm aster and the vegetal parallel array

Two microtubule-containing structures are implicated in dorsoventral polarization of the frog egg, and we examined the relationship between them. The sperm aster provides a directional cue for a cortical rotation specifying polarity, and a vegetal cortical array of parallel microtubules is likely part of the rotational machinery. The growing aster has an accumulation of microtubules marking the path of the sperm pronucleus, and its microtubules extend into the egg cortex as well as the cytoplasm. To test whether the vegetal parallel array was an extension of astral cortical growth, fertilized or activated eggs were bisected into animal and vegetal fragments. The vegetal fragments formed parallel arrays, even when isolated within a few minutes of egg activation. Neither the sperm centrosome nor another microtubule organizing center in the animal half of the egg is required for formation of the parallel array, but some animal half activity is involved in its disappearance. Correspondence to: R.P. Elinson     

Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs

Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis . Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV-irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2–0.3 μm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 μm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule-dependent, perhaps sharing the same basic mechanisms.     

FERTILIZATION PROCESS IN THE HEART-URCHIN,CLYPEASTER JAPONICUS OBSERVED WITH A DIFFERENTIAL INTERFERENCE MICROSCOPE*

The observations of the fertilization process in the heart-urchin, Clypeaster japonicus with a differential interference microscope indicate that the sperm pronucleus is carried to the center of the egg by the growth of the sperm aster as stated by Chambers (5), and that the egg pronucleus is carried to the center of the aster by a filamentous structure formed between them. The curved path of egg pronucleus in the fertilized egg is interpreted as the combination of the movement of the center of the aster and the movement of the egg pronucleus toward the center of the aster. The movement and the rotation of the sperm head result from pushing by the tail being engulfed in the egg.     

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.     

Fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection

We demonstrated normal fertilization processes (as determined by pronuclear formation, pronuclear apposition and syngamy) in porcine oocytes either following intracytoplasmic spermatozoon (ICSI) or isolated sperm head injection. Microtubule organization and chromatin configuration were investigated in these oocytes during the first cell cycle. Following ICSI, the microtubular aster was organized from the neck of the spermatozoon and filled the whole cytoplasm. These male-derived microtubules appear to move both pronuclei to the center of oocytes. These cytoskeletal changes are analogous to those seen following conventional fertilization. In contrast, following isolated sperm head injection, the sperm aster was not seen. Instead, the microtubule matrix was organized from the cortex and then filled the whole cytoplasm in all cases in normally fertilized oocytes following injection (n = 35). This organization is similar to what has been shown in the parthenogenetically activated oocytes. Chromosome analysis revealed that the oocytes injected with isolated sperm heads were fertilized normally. At 7 days following injection, the incidence of blastocoele formation following ICSI (38%) and isolated sperm head injection (22%) was higher than that following sham injection (2%). These results suggested that successful fertilization and preimplantation development occurred in porcine oocytes following either ICSI or isolated sperm head injection. Our results also indicated that fertilization processes can occur by self-assembled microtubules within cytoplasm in the absence of a sperm centrosome. Mol. Reprod. Dev. 51:436–444, 1998. © 1998 Wiley-Liss, Inc.     

Chromatin and microtubule morphology during the first cell cycle in bovine zygotes

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage. © 1993 Wiley-Liss, Inc.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

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.     

Selective identification of the paternal mitochondrion in living sea urchin eggs and embryos by chlorotetracycline

When sea urchin eggs are pretreated with fluorescent chelate probe chlorotetracycline (CTC) and then fertilized with unlabeled sperm, a small, brightly fluorescent particle resembling the mitochondrion of free-swimming sperm both in size and fluorescent staining characteristics appears in the egg cytoplasm. This particle first appears near the base of the insemination cone and, like the paternal mitochondrion identified in previous ultrastructural studies, remains closely associated with the male pronucleus during its microtubule-dependent migration toward the egg center. These similarities strongly suggest that the fluorescent particle observed in the cytoplasm of living, CTC-pretreated sea urchin eggs is, in fact, the mitochondrion of the fertilizing sperm.     

The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation

We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 microns/s. A similar wave is seen in egg fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in sperm-activated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30-40 s after the calcium wave starts, a slower (1.4 microns/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 degrees away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.     

Intracellular pH shift leads to microtubule assembly and microtubule-mediated motility during sea urchin fertilization: correlations between elevated intracellular pH and microtubule activity and depressed intracellular pH and microtubule disassembly

The regulation of the microtubule-mediated motions within eggs during fertilization was investigated in relation to the shift in intracellular pH (pHi) that occurs during the ionic sequence of egg activation in the sea urchins Lytechinus variegatus and Arbacia punctulata. Microtubule assembly during formation of the sperm aster and mitotic apparatus was detected by anti-tubulin immunofluorescence microscopy, and the microtubule-mediated migrations of the sperm and egg nuclei were studied with time-lapse video differential interference contrast microscopy. Manipulations of intracellular pH were verified by fluorimetric analyses of cytoplasmic fluorescein incorporated as fluorescein diacetate. The ionic sequence of egg activation was manipulated i) to block the pHi shift at fertilization or reduce the pHi of fertilized eggs to unfertilized values, ii) to elevate artificially the pHi of unfertilized eggs to fertilized values, and iii) to elevate artificially or permit the normal pHi shift in fertilized eggs in which the pHi shift at fertilization was previously prevented. Fertilized eggs in which the pHi shift was suppressed did not assemble microtubules or undergo the normal microtubule-mediated motions. In fertilized eggs in which the pHi was reduced to unfertilized levels after the assembly of the sperm aster, no motions were detected. If the intracellular pH was later permitted to rise, normal motile events leading to division and development occurred, delayed by the time during which the pH elevation was blocked. Microtubule-mediated events occurred in eggs in which the intracellular pH was elevated, even in unfertilized eggs in which the pH was artificially increased. These results indicate that the formation and normal functioning of the egg microtubules is initiated, either directly or indirectly, by the shift in intracellular pH that occurs during fertilization.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.     

Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo

We show that certain events of the cell cycle can still occur in starfish oocytes or fertilized eggs from which the germinal vesicle (the prominent nucleus of prophase-arrested oocytes) has been removed before the induction of meiotic maturation. Two meiotic asters develop following hormonal induction of meiotic maturation in these enucleated oocytes. The asters then divide to form a transient tetrapolar figure. When enucleated oocytes are fertilized, the sperm centrosome duplicates at the times corresponding to each cleavage in control nucleated embryos. Periodic changes in the organization of the asters and in the morphology of the cell surface also occur in synchrony with controls. Decondensation of the sperm nucleus, spindle formation, and cleavage do not occur when enucleated oocytes are fertilized. Ultimately the number of asters increases to approximately 520 (about 2(9] before the pseudo-embryo arrests and cytolyzes. Fertilized eggs from which both pronuclei but not the sperm aster have been removed undergo nine cleavages and then cease cell division. The cessation of division may be related to the events that cause the midblastula transition after seven cleavages in normal nucleated embryos.