A CYTOLOGICAL STUDY OF THE RELATION OF THE CORTICAL REACTION TO SUBSEQUENT EVENTS OF FERTILIZATION IN URETHANE-TREATED EGGS OF THE SEA URCHIN, ARBACIA PUNCTULATA

Eggs of the sea urchin, Arbacia punctulata, treated with 3% urethane for 30 sec followed by 0.3% urethane and inseminated are polyspermic and fail to undergo a typical cortical reaction. Upon insemination the vitelline layer of urethane-treated eggs either does not separate or is raised only a short distance from the oolemma. 1–6 min after insemination, almost all of the cortical granules remain intact and are dislodged from the plasmalemma. Later (6 min to the two-cell stage) some cortical granules are released randomly along the surface of the zygote. Not all zygotes show the same degree of cortical granule dehiscence; most of them experience little if any granule release whereas others demonstrate considerably more. The thickness of the hyaline layer appears to be directly related to the number of cortical granules released. Subsequent to pronuclear migration, several male pronuclei become associated with the female pronucleus. Later the male and female pronuclear envelopes contact and the outer and the inner laminae fuse, thereby forming the zygote nucleus. The male pronuclei remaining in the cytoplasm increase in size and progressively migrate to, and fuse with, the zygote nucleus. By 60 min some zygotes appear to contain only one large zygote nucleus which subsequently enters mitosis. Other zygotes possess a number of male pronuclei which remain unfused, and later these pronuclei along with the zygote nucleus undergo mitosis. There does not appear to be a direct relation between the number of cortical granules a zygote possesses and the above mentioned dichotomy.     

Ultrastructural study on pronuclear development in fertilized eggs from goldfish, Carassius auratus

The formation of male and female pronuclei in physiologically monospermic fertilized eggs of the goldfish, Carassius auratus , has been investigated with transmission electron microscopy. Ultrastructural observations show that at 26°C the transformation of the sperm nucleus takes place very quickly. The sperm nuclear envelope degenerates and is replaced by a large number of smooth surface vesicles 1 min post-insemination. Concomitantly, most of the condensed sperm chromatin is dispersed and is surrounded by vesicles. Dispersion of the chromatin is followed by the fusion of vesicles and the formation of a new bilaminar pronuclear envelope. Within 5–10 min post-insemination, a spheroid male pronucleus with intranuclear annulate lamellae is produced. The formation of a female pronucleus is slightly different to that of the male pronucleus. The dispersing chromatin of the egg is divided into many groups, most of which are surrounded by multilaminar envelopes 5 min post-insemination. An ellipsoid female pronucleus with a continuous bilaminar pronuclear envelope and intranuclear annulate lamellae is formed 15 min post-insemination. Subsequently, the two pronuclei migrate towards one another. When the fully developed male and female pronuclei are located in the center of the blastodisc, each changes itself into a saccular complex 25 min post-insemination.     

The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro

The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies. Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona. Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova. Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.     

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.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.     

DNA synthesis in the fertilizing hamster sperm nucleus: sperm template availability and egg cytoplasmic control

To assess the role of the availability of sperm nuclear templates in the regulation of DNA synthesis, we correlated the morphological status of the fertilizing hamster sperm nucleus with its ability to synthesize DNA after in vivo and in vitro fertilization. Fertilized hamster eggs were incubated in 3H-thymidine for varying periods before autoradiography. None of the decondensed sperm nuclei nor early (Stage I) male pronuclei present after in vivo or in vitro fertilization showed incorporation of label, even in polyspermic eggs in which more advanced pronuclei were labeled. In contrast, medium-to-large pronuclei (mature Stage II pronuclei) consistently incorporated 3H-thymidine. To investigate the contribution of egg cytoplasmic factors to the regulation of DNA synthesis, we examined the timing of DNA synthesis by microinjected sperm nuclei in eggs in which sperm nuclear decondensation and male pronucleus formation were accelerated experimentally by manipulation of sperm nuclear disulfide bond content. Although sperm nuclei with few or no disulfide bonds decondense and form male pronuclei faster than nuclei rich in disulfide bonds, the onset of DNA synthesis was not advanced. We conclude the the fertilizing sperm nucleus does not become available to serve as a template for DNA synthesis until it has developed into a mature Stage II pronucleus, and that, as with decondensation and pronucleus formation, DNA synthesis also depends upon egg cytoplasmic factors.     

Nascent protein requirement for completion of meiotic maturation and pronuclear development: examination of fertilized and A-23187-activated surf clam (Spisula solidissima) eggs

The involvement of newly synthesized proteins and calcium in meiotic processes, sperm nuclear transformations, and pronuclear development was examined in emetine-treated, fertilized, and A-23187-activated Spisula eggs by observing changes in the morphogenesis of the maternal and paternal chromatin. Emetine treatment (50 micrograms/ml) initiated 30 min before fertilization or A-23187 activation inhibited incorporation of [3H]leucine into TCA-precipitable material and blocked second polar body formation. Sperm incorporation and the initial enlargement of the sperm nucleus were unaffected; however, the dramatic enlargement and transformation of the sperm nucleus into a male pronucleus, which normally follow polar body formation, were delayed 10 to 20 min. Unlike the situation in untreated, control eggs, male pronuclear development took place while the maternally derived chromosomes remained condensed. It was not until approximately 20 min after the normal period of pronuclear development that the maternal chromosomes dispersed and formed a female pronucleus in emetine-treated, fertilized eggs. Formation of pronuclei, however, was unaffected in both emetine-treated, A-23187-activated eggs and fertilized eggs incubated with A-23187. These observations indicate that germinal vesicle breakdown, first polar body formation, and initial transformations of the sperm nucleus are independent of newly synthesized proteins. Inhibition of second polar body formation and the delay in pronuclear development brought about by emetine, as well as the appearance of silver grains over pronuclei in autoradiographs of control eggs incubated with [3H]leucine demonstrate that nascent proteins are involved with the completion of meiotic maturation and the development of male and female pronuclei. The ability of A-23187 to override the inhibitory effects of emetine on pronuclear development suggests that both nascent protein and calcium signals are involved in regulating the status of the maternal and paternal chromatin during pronuclear development.     

THE FINE STRUCTURE OF PRONUCLEAR DEVELOPMENT AND FUSION IN THE SEA URCHIN, ARBACIA PUNCTULATA

Fertilization events following coalescence of the gamete plasma membranes and culminating in the formation of the zygote nucleus were investigated by light and electron microscopy in the sea urchin, Arbacia punctulata. Shortly after the spermatozoon passes through the fertilization cone, it rotates approximately 180° and comes to rest lateral to its point of entrance. Concomitantly, the nonperforated nuclear envelope of the sperm nucleus undergoes degeneration followed by dispersal of the sperm chromatin and development of the pronuclear envelope. During this reorganization of the sperm nucleus, the sperm aster is formed. The latter is composed of ooplasmic lamellar structures and fasciles of microtubules. The male pronucleus, sperm mitochondrion, and flagellum accompany the sperm aster during its migration. As the pronuclei encounter one another, the surface of the female pronucleus proximal to the advancing male pronucleus becomes highly convoluted. Subsequently, the formation of the zygote nucleus commences with the fusion of the outer and the inner membranes of the pronuclear envelopes, thereby producing a small internuclear bridge and one continuous, perforated zygote nuclear envelope.     

Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes

We tested the ability of chromosomes in a mitotic cytoplasm to organize a bipolar spindle in the absence of centrosomes. Sea urchin eggs were treated with 5 X 10(-6) colcemid for 7-9 min before fertilization to block future microtubule assembly. Fertilization events were normal except that a sperm aster was not formed and the pronuclei remained up to 70 microns apart. After nuclear envelope breakdown, individual eggs were irradiated with 366-nm light to inactivate photochemically the colcemid. A functional haploid bipolar spindle was immediately assembled in association with the male chromosomes. In contrast to the male pronucleus, the female pronucleus in most of these eggs remained as a small nonbirefringent hyaline area throughout mitosis. High-voltage electron microscopy of serial semithick sections from individual eggs, previously followed in vivo, revealed that the female chromosomes were randomly distributed within the remnants of the nuclear envelope. No microtubules were found in these pronuclear areas even though the chromosomes were well-condensed and had prominent kinetochores with well-developed coronas. In the remaining eggs, a weakly birefringent monaster was assembled in the female pronuclear area. These observations demonstrate that chromosomes in a mitotic cytoplasm cannot organize a bipolar spindle in the absence of a spindle pole or even in the presence of a monaster. In fact, chromosomes do not even assemble kinetochore microtubules in the absence of a spindle pole, and kinetochore microtubules form only on kinetochores facing the pole when a monaster is present. This study also provides direct experimental proof for the longstanding paradigm that the sperm provides the centrosomes used in the development of the sea urchin zygote.     

Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes

Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.     

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

The application of hybrid vigor and crossbreeding is conventional and proved effective. Nevertheless, the phenotype of the progeny of hybrids, which carry hybrid vigor and produce their offspring through bisexual reproduction, will segregate inevitably and their hybrid vigor will de-crease in subsequent generations. The more serious consequence might result in destroying com-pletely those endemic populations when hybrids are released into open water bodies, because hy-brids will cross with the…     

Two unisexual artificial polyploid clones constructed by genome addition of common carp (<Emphasis Type="Italic">Cyprinus carp</Emphasis>) and crucian carp (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.     

Derivation of the membrane comprising the male pronuclear envelope in inseminated sea urchin eggs

Investigations were conducted in an effort to determine the origin of the membrane comprising the male pronuclear envelope of inseminated sea urchin eggs. The events of fertilization in zygotes treated with 200 μg/ml of puromycin are not impaired even though incorporation of [³H]leucine is inhibited up to 80% when compared to control specimens. Developing male pronuclei in zygotes treated with puromycin form nuclear envelopes structurally similar to and within the same period as controls. In puromycin-treated and untreated zygotes morphologically recognizable portions of the sperm nuclear envelope are incorporated into the structure of the male pronuclear envelope. Pronuclear development was also examined in inseminated ova where most of the endoplasmic reticulum (ER) was confined to a specific area of the zygote. Eggs were centrifuged in order to stratify their organelles into specific layers (stratified eggs); with further centrifugation stratified eggs are bisected to form nucleate (rich in ER) and nonnucleate halves (containing little ER). Observations of inseminated stratified eggs and nucleate and nonnucleate halves demonstrate an inverse relation between the amount of ER present in the vicinity of a reorganizing sperm nucleus and the time it takes to form the male pronuclear envelope. Computation of the maximum quantity of membrane in the male pronucleus that may be derived from the sperm nuclear envelope is approximately 15%. These investigations suggest that a major portion of the male pronuclear envelope is derived from endoplasmic reticulum within the egg and only a small portion (up to 15%) originates from the sperm nuclear envelope.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

Analysis of fertilization and polyspermy in serotonin-spawned eggs of the zebra mussel,Dreissena polymorpha

Eggs isolated from animals spawned with 10⁻³ M serotonin were inseminated with sperm concentrations ranging from 10³–10⁶ sperm/ml. Multiple sperm attached to the surface of the egg and sperm incorporation occurred within 3 min postinsemination (PI). Sperm mitochondria, centrioles, and flagellum were also incorporated. Incorporation was essentially complete by 6 min PI. In the egg cortex, the sperm head rotated 180°, and a rapid translocation of the sperm through the cytoplasm towards the egg interior began by 5–6 min PI. In heavily polyspermic inseminations, translocations of the sperm were either minimal or nonexistent. In monospermic eggs, nuclear decondensation occurred after translocation was complete, beginning by 9–10 min PI. A male pronucleus began to develop in the cytoplasm by 21 min PI and enlarged to 20 μm before fusing with the female pronucleus. Oscillation of the egg cytoplasm and mitotic spindle apparatus was observed immediately prior to cleavage. Cleavage occurred at 60 min PI. Sperm incorporation and pronuclear formation were confirmed with fluorescent and confocal microscopy using the DNA-specific dyes Hoescht 33342 and 7-aminoactinomycin D. In sperm concentrations >10⁴ sperm/ml, 26–76% of the eggs exhibited polyspermy. The high incidence of polyspermy suggests that rapid, effective blocks to polyspermy were not present or were ineffective in a significant proportion of serotonin-spawned eggs. © 1996 Wiley-Liss, Inc.     

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.     

Cytological events leading to the cleavage of golden hamster zygotes.

Fertilized golden hamster eggs were examined between 6 and 20 hours post-ovulation to determine the events leading to the two-cell stage. Following their migration the pronuclei remain in the central region of the zygote for approximately ten hours. The morphologically, indistinguishable male and female pronuclei remain relatively unchanged during this period, i.e., they do not interdigitate or fuse with one another as described for the zygotes of other organisms. Following this period and at the time of pronuclear breakdown elongate vesicles appear along the nucleoplasmic surface of the pronuclear envelopes. Later the pronuclear envelopes fragment into elongate cisternae; these and the vesicles formed along the inner lamina of the pronuclear envelopes remain closely associated and constitute quadrilaminar structures. The chromosomes which condense prior to and during pronuclear envelope breakdown, migrate to the equatorial plate of the forming cleavage spindle. After cytokinesis the chromosomes in the blastomere nuclei disperse. Increase in the nuclear envelope to accommodate this dispersion may involve the addition of membrane from the quandrilaminar structures.     

Sperm aster in rabbit zygotes: its structure and function

Microscope observations of rabbit zygotes demonstrate that a sperm aster forms in association with the male pronucleus approximately 1 h postinsemination and consists of two regions. One, the centrosphere, contains a dense aggregation of cisternae of smooth endoplasmic reticulum and microtubules. The second consists of fascicles of microtubules which emanate from the centrosphere. Fertilized rabbit eggs were cultured in medium containing colcemid in order to determine its effects on various events of fertilization, such as movements of the male and female pronuclei and DNA synthesis. No evidence was obtained to indicate that a sperm aster is formed in colcemid-treated zygotes. In addition, migration and close apposition of the pronuclei do not take place. Breakdown of the pronuclear envelopes and condensation of the maternally and paternally derived chromosomes occur even though the pronuclei fail to migrate centrad. Autoradiographic analysis of the synthesis of DNA by both pronuclei demonstrates that their migration into close apposition to one another is not required for the incorporation of tritiated thymidine.     

天然雌核发育银鲫卵子控制异源精核发育的受精学机制

作者对两性融合生殖鱼和雌核发育银鲫脱膜卵受精的精核发育进行了观察,并采用鱼类卵子无细胞系对以上两类卵质提取物体外诱导经Ｔｒｉｔｏｎ—Ｘ１００处理的精子及其发育进行了初步研究,结果表明在两性融合生殖型脱膜鱼卵中精核通过解凝最终形成原核,而在雌核发育的银鲫脱膜卵子中部分精核体积虽有一定程度的增加,但始终没有观察到原核的发育;在体外诱导实验中,经Ｔｒｉｔｏｎ—Ｘ１００处理的精子在两类卵质提取物中充分发育,都出现了类似体内原核的状态。该现象提示在银鲫卵质中存在有促使精核形成原核的因子,但在正常受精状态下,由于银鲫卵质促使精核核膜解体的功能的异常,使覆盖精子头部的核膜不能象在两性融合生殖受精卵子中进行崩解,精核进一步的原核发育受到抑制。另外,建立体外诱导系统的重要意义,在于它为研究雌核发育调控的分子学机制提供了一条有效途径。