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.     

Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration

A male pronucleus migrates toward the center of an egg to reach the female pronucleus for zygote formation. This migration depends on microtubules growing from two centrosomes associated with the male pronucleus. Two mechanisms were previously proposed for this migration: a "pushing mechanism," which uses the pushing force resulting from microtubule polymerization, and a "pulling mechanism," which uses the length-dependent pulling force generated by minus-end-directed motors anchored throughout the cytoplasm. We combined two computer-assisted analyses to examine the relative contribution of these mechanisms to male pronuclear migration. Computer simulation revealed an intrinsic difference in migration behavior of the male pronucleus between the pushing and pulling mechanisms. In vivo measurements using image processing showed that the actual migration behavior in Caenorhabditis elegans confirms the pulling mechanism. A male pronucleus having a single centrosome migrated toward the single aster. We propose that the pulling mechanism is the primary mechanism for male pronuclear migration.     

Analysis of the Role of Astral Rays in Pronuclear Migration in Sand Dollar Eggs by the Colcemid-UV Method

The formation and migration of the sperm aster, and the migration of male and female pronuclei during fertilization were investigated in the eggs of the sand dollar, Clypeaster japonicus using the Colcemid-UV method. When an egg in Colcemid sea water was irradiated locally with UV light (about 365 nm wavelength) at a limited region containing sperm head, a sperm aster formed in this region, and migrated to the center of the UV-irradiated region during its formation. When the UV-irradiated region was displaced or its shape was changed after the formation of the sperm aster, the aster migrated to the center of the new UV-irradiated region. The direction of the migration of the sperm aster coincided with the direction of the longest astral rays. Direct contact between astral rays and the egg surface was not essential for sperm aster migration. When a region containing both the sperm centrosome and the female pronucleus was irradiated with UV light, the female pronucleus migrated toward the center of the sperm aster after they were connected by astral rays. The migration was suppressed when UV light was shaded over the region between the aster and the female pronucleus. These results suggest that the female pronucleus migrates to the sperm aster by attractive force between them.     

Fertilization and early development in Beroe ovata

Fertilization in the clear egg (1 mm in diameter) of the ctenophore Beroe ovata and, in particular, the positioning and movements of pronuclei, and their relationship to the larval oral-aboral axis have been observed. Fertilization can take place anywhere on the egg surface. The sperm pronucleus remains at its entry site and becomes surrounded by a specialized zone (30–50 μm in diameter) beneath the surface referred to as the sperm pronuclear zone or SPZ and devoid of large cortical granules. Polyspermy has been observed to be frequent; each pronucleus is surrounded by its own SPZ. Only the egg pronucleus migrates with a continuous velocity (averaging 18 μm/min) and moves beneath the surface directly toward the immobile sperm pronucleus. In polyspermic eggs, the egg pronucleus can probe several SPZ, each containing a single sperm nucleus, before it finally enters one SPZ and fuses with the chosen sperm pronucleus. These migrations of the egg pronucleus occur over several millimeters and take hours, but the mechanism underlying the motion or how the egg pronucleus decides which SPZ to enter is not yet known. Under our experimental conditions the mitotic apparatus and the first cleavage plane which defines the oral-aboral axis of the larva (see Reverberi (1971). “Experimental Embryology of Marine and Fresh-Water Invertebrates.” North-Holland, Amsterdam. for review) pass through the point of sperm entry. During fertilization and cleavage, movements of a cortical autofluorescent material are clearly seen. This material is segregated into micromeres as cleavage progresses.     

The movements and fusion of the pronuclei at fertilization of the sea urchin Lytechinus variegatus: Time-lapse video microscopy

The penetration of the sperm into the egg, and the movements of the male and female pronuclei were followed from sperm attachment through pronuclear fusion, using time-lapse video microscopy of gametes and zygotes of the sea urchin Lytechinus variegatus (23° C). The pronuclei move in four stages: I. Sperm Entry Phase, following sperm-egg fusion and a rapid radiating surface contraction (5.9 ± 1.3 μm/second) when egg microvilli engulf the sperm head, midpiece, and tail to form the fertilization cone and the sperm tail beats in the egg cytoplasm; II. Formation of the Sperm Aster, which pushes the male pronucleus centripetally at a rate of 4.9 ± 1.7 μm/minute starting 4.4 ± 0.5 minutes after sperm-egg fusion, as the male pronucleus undergoes chromatin decondensation; III. Movement of the Female Pronucleus, the greatest and fastest of the pronuclear motions at a rate of 14.6 ± 3.5 μm/minute at 6.8 ± 1.2 minute after sperm-egg fusion, which establishes the contact between the pronuclei; and IV. Centration of the Pronuclei to the egg center at a rate of 2.6 ± 0.9 μm/minute by 14.1 ± 2.6 minutes after sperm-egg fusion. Pronuclear fusion typically occurs after stage IV and proceeds rapidly starting 14.7 ± 3.6 minutes after sperm-egg fusion with the male pronucleus coalescing into the female pronucleus at a rate of 14.2 ± 2.6 μm/minute.     

An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male)

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.     

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.     

Effects of motility inhibitors during sea urchin fertilization : Microfilament inhibitors prevent sperm incorporation and restructuring of fertilized egg cortex, whereas microtubule inhibitors prevent pronuclear migrations

The sensitivity of specific stages of fertilization to microfilament inhibitors (cytochalasins B (CB), D (CD), and E (CE) and phalloidin) and to inhibitors of microtubule assembly (colcemid (CMD), colchicine (CLC), griseofulvin (GSF), maytansine (MAY), nocodazole (NCD), podophyllotoxin (PDP), and vinblastine (VB)) was investigated using differential interference contrast, time-lapse video microscopy of the sea urchin Lytechinus variegatus. Cytochalasins (CDCE>CB) will prevent sperm incorporation if added prior to or simultaneous with insemination. Sperm-egg fusion and the cortical reaction appear normal, but then the subsequent elevation of the fertilization coat lifts and eventually detaches the ‘fertilizing’ sperm from the egg plasma membrane. When the cytochalasins are added after fusion, the forming fertilization cone is rapidly resorbed, and the lateral displacement of the sperm along the egg cortex is terminated; the pronuclear migrations and mitoses occur normally though cytokinesis is never observed. Cytochalasin treatment before or within 2 min of insemination results in the development of aberrant egg cortices, whereas cytochalasin treatments after 2 min post-fusion have little effect. Phalloidin results in large and long-lasting fertilization cones and a retardation of the rate of sperm incorporation. Eggs exposed to any of the microtubule inhibitors 15 min prior to insemination will incorporate the spermatozoon, though the formation of the sperm aster and the accompanying pronuclear migrations are prevented. Interestingly, the final stage of sperm incorporation involving a lateral displacement of the sperm along the egg cortex is greater (27.1 vs 12.4 μm in controls) and faster (5.4 vs 3.5 μm/min in controls) in microtubule-inhibited eggs. GSF and VB, which readily permeate fertilized eggs, will prevent the formation of the sperm aster if added 3 min after sperm-egg fusion, they will prevent the migration of the female pronucleus if added 5 or 7 min after sperm-egg fusion, pronuclear centration if added 10 min post-fusion, and syngamy if added 12 min post-fusion. CLC- or CMD- treated eggs will develop normally if these drugs are photochemically inactivated with 366 nm light within 4 min post-fusion, arguing that sperm incorporation is completely independent of assembling microtubules. These results indicate that microfilament inhibitors will prevent sperm incorporation and the restructuring of the fertilized egg cortex, and that microtubule inhibitors will prevent the formation and functioning of the sperm aster during the pronuclear migrations; an interplay between cortical microfilaments and cytoplasmic microtubules appears required for the successful completion of fertilization.     

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.     

Studies on fertilization in the teleost. I. Dynamic responses of fertilized medaka eggs

In order to understand the mechanisms of fertilization in the teleost, the movements of the egg cortex, cytoplasmic inclusions and pronuclei were observed in detail in fertilized medaka Oryzias latipes eggs. The first cortical contraction occurred toward the animal pole region following the onset of exocytosis of cortical alveoli. The cortical contraction caused movement of oil droplets toward the animal pole where the germinal vesicle had broken down during oocyte maturation. The movement of oil droplets toward the animal pole region was frequently twisted in the right or left direction. The direction of the twisting movement has been correlated with the unilateral bending of non-attaching filaments on the chorion. The female pronucleus, which approached the male pronucleus from the vicinity of the second polar body, took a course to the right, left or straight along the s-p axis connecting the male pronucleus and the second polar body. The course of approach by the female pronucleus correlated with the bending direction of the non-attaching filaments that had been determined by rotation of the oocyte around the animal–vegetal axis during oogenesis. The first cleavage furrow also very frequently coincided with the axis. These observations suggest that dynamic responses of medaka eggs from fertilization to the first cleavage reflect the architecture dynamically constructed during oogenesis.     

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.     

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     

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.     

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.     

Cargo Transport by Cytoplasmic Dynein Can Center Embryonic Centrosomes

To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell’s center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.     

Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tubifex (Annelida,Oligochaeta): I. Meiotic apparatus formation

The fine structure of the animal pole cortex is examined in the fertilized Tubifex egg undergoing the formation of the second meiotic apparatus (MA). The fully formed MA which orients its axis at right angles to the surface is found at the animal pole about 40 min after formation of the first polar body. It is composed of a spindle and asters at its poles; a centriole is found in the inner aster, but not in the peripheral aster adjacent to the surface. During the formation of the MA, the animal pole surface is lined with a 0.15-μm-thick, electron-dense cortical layer, which is rich in microfilaments. The arrangement of the filaments in the layer changes from a parallel array to a meshwork with progressive formation of the MA. Microtubules of the peripheral aster terminate in the cortical layer. When a jet stream of glycerol/dimethyl sulfoxide solution is applied to an egg fragment glued on a polylysine-coated coverslip, an egg cortex-MA complex is isolated on the coverslip; the MA appears to be tethered to the egg surface by the structural connection between the filamentous cortical layer and microtubules of the peripheral aster. Cytochalasin B (50 μg/ml), when administrated at early phase of the MA formation, does not show any effect on the structure of the cortical layer and the MA; however, if eggs shortly before the termination of the first polar body formation are immersed in the same test solution, the cortical layer of the animal pole becomes thinner, and the filamentous material is not observed in it. Furthermore, in these eggs, the peripheral aster and the spindle are not structurally discernible because of the suppression of microtubule assembly, whereas microtubules on kinetochores and in the inner aster are normally developed. These results are discussed in relation to the role of the animal pole cortex in fixing of the MA to the egg surface and in forming of the MA.     

Migration and division of cleavage nuclei in the gall midge,Wachtliella persicariae

Summary In the eggs ofWachtliella persicariae the cleavage nuclei move relative to the surrounding ooplasm. This active migration is caused by an organelle whose ultrastructure was studied throughout the mitotic cycle. It consists of a greatly enlarged polar cytaster derived from the mitotic apparatus, linked to the nucleus by 100 Å filaments. The microtubules of the cytaster were found only during periods of active nuclear migration, i.e., from the onset of anaphase to the early prophase of the next mitotic cycle. They are always solitary and follow the course of the astral rays, which are known to temporarily adhere to peripheral structures of the egg cell and to exert tractive forces. In contrast to the cytaster microtubules, the microtubules in the spindle are bundled and persist from early metaphase through late telophase.During ontogenesis the first migration cytaster is built up between 3 and 12 min after oviposition near the anterior egg pole, in the vicinity of the sperm nucleus. In non-inseminated eggs time lapse films show a migration cytaster to develop autonomously in a region free from nuclei, but it does not follow the normal path of the male pronucleus. In several cases the female pronucleus, which remains without a cytaster of its own, was observed to move to the cytaster generated in the absence of the male pronucleus. Whether or not it is adhering to a nucleus, the cytaster divides into two at the correct time, i.e, corresponding to the first cleavage division in fertilized eggs. In some non-inseminated eggs this type of pseudocleavage has been observed to occur repeatedly, giving rise to an increasing number of anucleate cytasters.     

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.     

In vitro fertilization in ctenophores: sperm entry, mitosis, and the establishment of bilateral symmetry in Beroe ovata.

We have found ways to control in vitro fertilization in a ctenophore (Beroe ovata) for the first time. This is based on the existence of a partial block to self-fertilization at the time of gamete release which can be overcome by removal of the egg envelope. It has allowed us to exploit the excellent optical properties of Beroe eggs to make detailed observations on all events from sperm penetration or penetrations in these physiologically polyspermic eggs to first cleavage, and to extend our initial observations (Carré and Sardet, 1984). Sperm entry is characterized by local modifications of the egg cortex in a 70-microns zone around the penetration site or sites. Upon sperm entry, the egg surface contracts and relaxes locally, then a fertilization cone forms and disappears. These events are accompanied by localized exocytosis, growth of a ring of microvilli, thickening of the egg cortex, and gathering of mitochondria around the sperm pronuclei. The female pronucleus then migrates beneath the egg surface toward one or successive sperm pronuclei. The fusion of pronuclei, sperm and egg chromatin intermixing, and mitosis were also observed with exceptional clarity. Furthermore, we have noticed that the direction of the last trajectory of the female pronucleus tends to define the orientation of the mitotic spindle, and as a consequence the position of first unipolar cleavage furrow. This in turn determines the future sagittal plane of the embryo and of the adult B. ovata.