Phenotypic plasticity of reproductive effort in a colonial ascidian, Botryllus schlosseri.

Phenotypic plasticity is the capability of a genotype to produce different phenotypes in different environments. Previous studies have indicated phenotypic variability in asexual, male, and female reproduction in Botryllus schlosseri, a hermaphroditic, colonial ascidian, but not explicitly tested for genotype by environment interactions that indicate genetic variation in plastic responses. Consequently, clones derived from an estuarine population were deployed at their native site and a warmer, higher productivity site 10 km up-river. Male reproduction was assayed by testis size, female reproduction by the number of eggs produced, and asexual reproduction by colony growth rate. To test for ontogenetic effects, data were collected from two different generations of zooids born in the field. Analyses of variance indicated plasticity in asexual and female reproduction during the first zooid generation and plasticity in all three traits during the third zooid generation. Reaction norms varied significantly among genotypes in direction and magnitude for asexual reproduction at both times, implying that selection on asexual reproduction is weak. Sperm production during the third zooid generation was significantly lower at the nonnative site, but there was no genotype by environment interaction. The reaction norms for female reproduction varied significantly among genotypes in direction and magnitude during the first zooid generation, but only varied in magnitude during the third generation, with egg production being higher in all genotypes at the nonnative site. Comparisons of weighted frequency distributions between sites demonstrated that differences in egg production in the third generation were due to increases in the proportion of reproductive zooids within a colony. The greater emphasis on female reproduction at a site associated with higher food availability and temperature, and the greater emphasis on male reproduction at a colder, food-limited site, supports predictions from sex allocation theory.

Delayed insemination results in embryo mortality in a brooding ascidian.

We explored the effects of temporal variation in sperm availability on fertilization and subsequent larval development in the colonial ascidian Botryllus schlosseri, a brooding hermaphrodite that has a sexual cycle linked to an asexual zooid replacement cycle. We developed a method to quantify the timing of events early in this cycle, and then isolated colonies before the start of the cycle and inseminated them at various times. Colony-wide fertilization levels (assayed by early cleavage) increased from zero to 100% during the period when the siphons of a new generation of zooids were first opening, and remained high for 24 h before slowly declining over the next 48 h. Because embryos are brooded until just before the zooids degenerate at the end of a cycle, delayed fertilization might also affect whether embryos can complete development within the cycle. Consequently, we also determined the effect of delayed insemination on successful embryo development through larval release and metamorphosis. When fertilization was delayed beyond the completion of siphon opening, there was an exponential decline in the percentage of eggs that ultimately produced a metamorphosed larva at the end of the cycle. Thus, even though the majority of oocytes can be fertilized when insemination is delayed for up to 48 h, the resulting embryos cannot complete development before the brooding zooids degenerate.

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.

Development basis of phenotypic variation in egg production in a colonial ascidian: primary oocyte production versus oocyte development.

Colonies of the ascidian Botryllus schlosseri (a cyclical hermaphrodite) exhibit extreme variability in egg production, and there is a large genetic component to this phenotypic variation. Therefore, the developmental bases of variation among different genotypes was investigated. Colonies differing in egg production (assayed as number of eggs per asexual bud) were cultured in a common garden experiment, and buds were collected and fixed early in the reproductive cycle. The buds were serially sectioned, and the number and size of the oocytes in the developing ovaries were determined for the different genotypes. Because the buds were collected prior to the onset of vitellogenesis, they contained oocytes at the three previtellogenic stages. In reproductive colonies (>0.7 eggs per bud), there were negative relationships between the final number of eggs per bud and (1) the total number of oocytes present, (2) the number of stage 1 oocytes present, and (3) the number of stage 2 oocytes present. There was no relationship between these parameters in nonreproductive colonies (<0.3 eggs per bud). In contrast, the number of stage 3 oocytes per bud was positively correlated with the final number of eggs per bud in both reproductive and nonreproductive colonies. In reproductive animals there was a negative relationship between the total number of oocytes per bud and the percentage of oocytes at stage 3 in oogenesis. A principal component analysis revealed that a single vector equally weighted for the number of eggs per bud, the total number of oocytes per bud, and the percentage of oocytes at stage 3 accounted for 84% of the observed variation in reproductive colonies. These data indicate that the phenotypic variation in egg production among the B. schlosseri colonies in the Damariscotta River, Maine, is controlled by genetic variation in both the number of oocytes that populate developing ovaries, and the percentage of oocytes that reach stage 3 in oogenesis.

Cortical granule exocytosis in hamster eggs requires microfilaments.

Earlier work has demonstrated that hamster eggs that do not release a second polar body after fertilization in vitro lack a block to polyspermy (Stewart-Savage and Bavister, 1987: Gamete Res 18:333-338). Since polar body release requires microfilaments, the involvement of microfilaments in cortical granule exocytosis was examined. When hamster eggs were treated with cytochalsin B (CB) for 1 hr and then coincubated with sperm for 90 min, there was a dose-dependent increase in both the percentage of eggs with more than one sperm penetrating the zona pellucida and the mean number of sperm that penetrated the zona, with a maximum effect at 20 micrograms CB/ml (100% polypenetration, 3.0 +/- 0.3 sperm/egg). Cytochalasin-treated eggs retained 85% of their cortical granules 55 min after insemination, as compared to unfertilized eggs. Longer time periods did not result in any further reduction. As seen with the scanning confocal microscope, an extensive microfilament network was present in the cortex of untreated eggs, with the cortical granules located within the cortical network. The cortical microfilament network was highly reduced in CB-treated eggs. When viewed with the electron microscope, the same number of cortical granules were located next to the plasma membrane in both cytochalasin-treated and untreated, unfertilized eggs. These data indicate that intact microfilaments are required for normal cortical granule exocytosis in the hamster egg, but the role of the microfilaments in exocytosis is unresolved.

Life-History Variation in a Colonial Ascidian: Broad-Sence Heritabilities and Tradeoffs in Allocation to Asexual Growth and Male and Female Reproduction.

Intraspecific variation in life-history strategy provides a valuable opportunity for examining how natural selection acts on life-history variants to mold reproductive strategies. Evaluating the consequences of selection requires knowledge of the range of phenotypic variation in life histories, the extent to which variation is genetically based, and possible correlations among different traits that might constrain or promote the effect of selection on individual traits. We explored life-history variation in the colonial ascidian Botryllus schlosseri (a cyclical hermaphrodite) by growing clonal replicates of 18 genotypes in a common-garden experiment. Colonies of this species have previously been shown to vary in egg production and growth rate. We demonstrate that genotypes also vary in sperm production, which is manifested as variation in testis size. We then calculate broad-sense heritabilities for a suite of life-history traits and demonstrate correlations among traits that suggest a three-way tradeoff in resource allocation to asexual growth and sexual reproduction via male and female function. This correlation structure suggests that selection cannot act independently on individual life-history traits.

The timing of cortical granule fusion, content dispersal, and endocytosis during fertilization of the hamster egg: an electrophysiological and histochemical study.

To determine the temporal relationship between cortical granule exocytosis and the repetitive calcium transients, which are characteristic of mammalian fertilization, we monitored membrane addition from exocytosis during fertilization of hamster eggs. Continuous measurement of membrane capacitance by applying a 3.1-nA alternating current at 375 Hz showed addition of cortical granule membrane. Simultaneous measurement of membrane potential revealed each calcium transient by the appearance of transient hyperpolarizing responses due to calcium-activated potassium channels in the egg. The initial membrane capacitance of the eggs averaged 736 +/- 44 pF (mean +/- SD; n = 7) and an increase in capacitance of 61 +/- 19 pF occurred within 4 sec of the start of the first hyperpolarizing response (HR) after fertilization. Immediately after the first increase in capacitance there was a gradual decline in membrane capacitance in all eggs and in five/seven eggs the capacitance returned to the unfertilized level in 7.8 +/- 4.4 min. The gradual decline in capacitance after the first increase indicated endocytosis, which was confirmed by the internalization of fluorescently labeled dextran. Superimposed on the gradual decline in membrane capacitance were smaller increases in capacitance that occurred with the second and later HRs. The total increase in capacitance from the first three events averaged 72 +/- 19 pF, representing an average increase in capacitance of about 10% of the capacitance of the unfertilized egg. By labeling eggs before and after permeabilization with two different fluorochromes attached to Lens culinaris agglutinin, we demonstrate that the dispersal of the cortical granules contents does not occur immediately after exocytosis. Our results demonstrate that cortical granule exocytosis in hamster eggs is closely coupled to the periodic increases in calcium, that the contents of the cortical granules are slow to disperse, and that after exocytosis, the surface area of the egg returns to the unfertilized level because of a period of endocytosis.

Sperm penetration through the zona pellucida of salt-stored hamster eggs is delayed.

One of the problems in assaying sperm capacitation is that live eggs mount a block to polyspermy at the zona pellucida. To circumvent this problem, researchers have started to use salt-stored eggs to assay sperm capacitation (Boatman et al., Gamete Res 19:19-29, 1988; Yoshimatsu et al., Gamete Res 21:115-126, 1988). These researchers have demonstrated that the zonae of salt-stored eggs were penetrable by capacitated sperm, but they did not examine whether the timing of penetration of fully capacitated sperm was altered. This report shows that zona penetration by fully capacitated sperm is delayed in both short-term salt-treated eggs (2 hr) and in eggs stored in salt for > 2 days. These results indicate that the zona is modified when eggs are treated with a high-salt solution.

Effect of bovine serum albumin concentration and source on sperm capacitation in the golden hamster.

Since virtually all experiments on sperm capacitation routinely include serum albumin in the culture media, the relative effect of BSA concentration on sperm capacitation was investigated. The fertile life of hamster sperm, as measured by the ability to penetrate the zona pellucida, decreased from 5 to 3 h as the concentration of BSA was increased from 3 to 18 mg/ml. To determine the extent of sperm capacitation in sperm populations that resulted in 100% penetration, the rate and timing of sperm penetration through the zona pellucida was determined. For each insemination, the rate of sperm penetration (%/min), the time when 50% of the eggs were zona-penetrated (T50), and the sperm capacitation index (T50/rate) were calculated. The extent of sperm capacitation as measured by all three parameters increased as the duration of preincubation or the BSA concentration was increased. The data on the duration of preincubation and the BSA concentration matrix indicated two properties of the effect of BSA on sperm capacitation: 1) the extent of sperm capacitation after the first hour of preincubation was independent of BSA concentration; and 2) when sperm were preincubated for two or more hours, the capacitating effectiveness of BSA was saturable; the saturating concentration of BSA decreased as the duration of preincubation increased. To determine whether the first hour of preincubation is independent of BSA, sperm were preincubated for 1 h without BSA and then 2 h with BSA. The extent of sperm capacitation of these sperm was equal to that of the 3-h control sperm; thus the first hour of capacitation is independent of BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cumulus maturity on sperm penetration in the golden hamster.

The influence of the cumulus on gamete interaction was investigated in naturally and precociously ovulated eggs from superstimulated (eCG) hamsters. Inseminations were performed in which the absolute gamete ratio (sperm/egg) was held constant, but the relative gamete ratio (sperm x egg-1 x ml-1) was varied 10-fold. Sperm penetration to the perivitelline space was significantly higher at the higher relative gamete ratio, indicating that there is no chemoattractant in the cumulus and that gamete interaction in cumulus-intact eggs is a function of collision frequency, which varies according to the relative gamete ratio. The level of successful gamete interaction as a function of the relative gamete ratio was determined for naturally and precociously ovulated eggs. The level of sperm penetration reached saturation at a relative gamete ratio of 10(3.3) for naturally ovulated eggs and 10(4.0) sperm x egg-1 x ml-1 for precociously ovulated eggs. At suboptimal relative gamete ratios, there was no difference in the level of penetration between partially and fully capacitated sperm. These results indicate that the larger, more mature cumulus of naturally ovulated eggs is successfully penetrated at lower collision frequencies than the denser, less mature cumulus of precociously ovulated eggs. To determine whether the maturity of the cumulus affects the timing of sperm penetration, cumulus-free and cumulus-intact eggs were inseminated at their optimal relative gamete ratios. The rate of sperm penetration of partially capacitated sperm was not different between cumulus-free and cumulus-intact eggs, regardless of ovulation method. However, the period of time between insemination and when half the eggs were penetrated (T50) was longer in cumulus-intact eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course and pattern of cortical granule breakdown in hamster eggs after sperm fusion.

We have determined the temporal relationship between sperm fusion and cortical granule breakdown in the hamster egg. Sperm fusion was determined by the Hoechst-transfer method (Stewart-Savage and Bavister: Dev Biol 128:150-157, 1988), and cortical granules were visualized with fluorescein isothiocynate-conjugated Lens culinaris agglutinin (Cherr et al. J Exp Zool 246:81-93, 1988). By 55 min after insemination, there was an 85% reduction in the density of cortical granules (fewer than four granules/100 microns2). Taking this value as the completion of the cortical reaction, analysis of the data indicate that the cortical reaction was completed 9 min after sperm fusion and 3 min after the formation of the zona and cell surface blocks to polyspermy. There was no obvious spatial pattern of granule loss in eggs that had a Hoechst-positive sperm but had not completed the cortical reaction.

Polarity of the surface and cortex of the amphibian egg from fertilization to first cleavage.

The anuran egg is polarized along its animal-vegetal axis and becomes bilaterally symmetrical before first cleavage. Functional sperm entry is regionally restricted to the animal hemisphere of the egg, and functional sperm entry does not occur after egg activation. This regional and functional restriction in sperm entry correlates with the presence of long, slender microvilli and with the presence of the filamentous component of the glycocalyx. After sperm fusion, the egg undergoes activation, including a depolarization of the membrane potential and exocytosis of granules in the cortex. Both of these activation responses are the result of a propagated increase in intracellular calcium. The egg's ability to undergo a propagated activation response develops after germinal vesicle breakdown and depends on the development of the cortical endoplasmic reticulum. Once activated, the radial symmetric egg acquires bilateral symmetry due to a rotation of the egg cortex relative to the inner cytoplasm. A transient array of parallel microtubules forms near the vegetal cortex and may be part of the motor driving the cortical rotation.

Success of fertilization in golden hamsters is a function of the relative gamete ratio.

Gamete concentrations can be expressed as either absolute gamete ratios (sperm/egg), the concentration of the motile sperm (sperm/ml), or as a relative gamete ratio (sperm/egg x ml). We demonstrate that the success of hamster fertilization in vitro is a function of the relative gamete ratio and that any effect of the insemination medium geometry is minimal. Consistent fertilization occurs when the relative gamete ratio is above 10(3.5) sperm/egg x ml, but becomes variable above 10(5.0) sperm/egg x ml. At suboptimal relative gamete ratios, there is a strong sperm concentration effect on both the consistency and level of fertilization, whereas the absolute gamete ratio only affects the overall level of fertilization. These effects are seen when the sperm concentration is below 10(3.5) sperm/ml and when the absolute gamete ratio is below 10(2.3) sperm/egg. These influences are probably due to reduced sperm survival at low sperm concentrations and due to a sampling error that occurs when small numbers of sperm are transferred. When the absolute gamete ratios found in vivo in the hamster [Cummins and Yanagimachi, 1982, Gamete Res 5, 239-256] are converted to relative gamete ratios, they are similar to our in vitro results. Thus relative gamete ratios allow, for the first time, comparison between in vitro and in vivo data in the hamster, and between other rodents.

A cell surface block to polyspermy occurs in golden hamster eggs.

We have examined the frequency and fate of supernumerary sperm in the perivitelline space (PVS) of in vitro fertilized hamster eggs to determine if there is a cell surface block to polyspermy. The zona pellucida block to polyspermy is very effective since only one sperm penetrated the zona pellucida in 72.8% of the 876 fertilized eggs examined. Of the polypenetrated eggs, 41.6% had a supernumerary sperm within the PVS. The proportion of polypenetrated eggs with PVS sperm did not change when the duration of coincubation was increased from 3 to 6 hr. PVS sperm were found in 67% of the inseminations. From these data we conclude that there is a cell surface block to polyspermy in the hamster. To investigate the mechanism of the cell surface block, we used the Hoechst-transfer technique (R. Hinkley, B. Wright, and J. Lynn, 1986, Dev. Biol. 118, 148-154) to monitor sperm-egg fusion. We first demonstrated that dye transfer from zona pellucida-free eggs to sperm only occurred when fusion was possible, i.e., in the presence of calcium, and that dye was transferred to all fused sperm. When cumulus-free, zona-intact eggs were preloaded with Hoechst dye and viewed 3 hr postinsemination, three classes of eggs with supernumerary sperm in the PVS were observed: eggs with only Hoechst-positive sperm (62%), eggs with only Hoechst-negative sperm (27%), and eggs with both a Hoechst-positive and a Hoechst-negative sperm (11%). Because of the limited time resolution of the Hoechst-transfer technique, the cell surface block could operate by preventing sperm fusion (Hoechst-negative), by the failure of the eggs to incorporate fused sperm (Hoechst-positive), and/or by the "unfusing" of fused sperm (Hoechst-positive and Hoechst-negative). We are unable at this time to differentiate between these mechanisms.

Deterioration of stored culture media as monitored by a sperm motility bioassay.

A hamster sperm motility bioassay was used to monitor medium quality during storage. The media studied were (1) TL-PVA, a modified Tyrode's solution; (2) TLP-PVA, a defined medium that supports hamster sperm motility but does not support capacitation; and (3) TALP-PVA (TLP-PVA + serum albumin), which supports hamster sperm survival and capacitation. Each medium was stored at 5 and -20 degrees C and tested every two weeks. All of the media deteriorated with increased storage time, but at different rates (TLP-PVA greater than TL-PVA greater than TALP-PVA). The deterioration of the media correlated with an increase in pH during storage, probably due to a loss of CO2 and bicarbonate. Albumin, added after storage, was able to rescue frozen TL-PVA.

Failure of hamster eggs fertilized in vitro to extrude the second polar body correlates with high levels of polyspermy.

The occurrence of second polar body (PB2) retention in in-vitro fertilized hamster eggs is reported. Of 2,872 eggs examined, 6.9% (199 eggs) failed to extrude PB2. Eggs that failed to extrude PB2 were more likely to be polyspermic than eggs with PB2 (63% and 14.6%, respectively), and the number of fusing sperm per egg was higher in the abnormal eggs. These data indicate that eggs which fail to extrude PB2 have an impaired block to polyspermy. The level of PB2 retention varied between females and ranged from all eggs extruding PB2 to 20% of the eggs failing to extrude PB2 (41% and 4% of the females, respectively). There was no correlation within a female between the percentage of eggs that failed to extrude PB2 and the level of polyspermy in the sister eggs with PB2. Therefore, regardless of the condition of their sister eggs, eggs that fail to extrude PB2 have an impaired block to polyspermy and eggs that extrude PB2 have a normal block.

Loss of functional sperm entry into Xenopus eggs after activation correlates with a reduction in surface adhesivity.

In Xenopus, the plasma membrane of the unactivated egg is receptive to sperm only in the animal hemisphere (R. Grey, M. Bastiani, D. Webb, and E. Schertel, 1982, Dev. Biol. 89, 475-487). The reinsemination experiments of investment-free eggs reported in this paper demonstrate that functional sperm entry is lost after activation. Supernumerary sperm were excluded even though the fertilization envelope was absent and the membrane potential had returned to the level found in the unfertilized egg. Even when the electrical block to polyspermy was suppressed by 40 mM NaI (which reduces the membrane potential), polyspermy could be induced only if denuded eggs were initially inseminated in this medium. We estimate that the loss of functional sperm entry, independent of the electrical block, occurs during the first 10 min following fertilization. Sperm readily adhere to the surface of the animal hemisphere of unactivated eggs divested of their extracellular coats, but they do not adhere to the surface of activated eggs. Denuded eggs also adhere to each other, with the surface of the animal hemisphere of unactivated eggs exhibiting the greatest degree of adhesivity. We used electric field-induced fusion (EFIF), without prior dielectrophoresis, to quantify the regional and temporal adhesiveness of eggs. At electric field strengths greater than 8 V/cm, the probability of fusion during EFIF is highest with the animal hemisphere of unactivated eggs, moderate with both the vegetal hemisphere of unactivated eggs and the animal hemisphere of activated eggs, and lowest with the vegetal hemisphere of activated eggs. When pairs of eggs are constructed with different hemispheres in contact, the fusion characteristics of the pair are similar to the more adhesive member of the pair. The regional and temporal differences in the adhesiveness of the Xenopus egg surface correlate with its receptivity to sperm and could possibly account for the plasma membrane's activation-induced loss of functional sperm entry.

The Cell Cycle Governs the Onset of Spherulation of Xenopus Eggs Fused by an Electric Field: (amphibian egg/cell cycle/cortex/electic field-induced fusion).

The mechanism of electric field-induced fusion has been studied in detail (Zimmermann, Biochim. Biophys. Acta. 694 (1982) 227), but little is known about the process by which the two fused cells become a single entity, a process we term spherulation. We observe a clear difference between activated and unactivated Xenopus eggs in the time after electic field (EF) application when spherulation starts, and in the time required for spherulation to be completed. In unactivated eggs, spherulation started 7 min after EF application and was completed within 5 1/2 min. In activated eggs, the lag between EF application and the start of spherulation increased with the cell cycle. At the end of the first cell cycle spherulation started 78 min after EF application and was competed 30 min later. The lag period is not due to delayed fusion, for electric coupling between activated eggs can be recorded before the start of spherulation. The morphology of the contact zone between paired eggs, as observed by light and electron microscopy, is also described. We suggest that the difference in the timing of spherulation reflects a difference in the lability of the cytoskeleton through the cell cycle.

Fertilization of investment-free Xenopus eggs.

The vitelline envelope of unfertilized Xenopus egg can be removed manually after treating the dejellied eggs for 10 min with 20% (w/v) sucrose in F-1 saline. Fertilization occurred in 52% of the eggs denuded in this way when UV-solubilized jelly was added to the sperm-egg mixture; without the jelly the level of fertilization was only 6%. Fertilization did not occur synchronously in the denuded eggs; the average delay between insemination and fertilization was 19 +/- 18 min.

The temporal and spatial relationships between cortical contraction, sperm trail formation, and pronuclear migration in fertilizedXenopus eggs.

The cortical contraction begins 4 min after insemination and one minute after prick activation. During the next 4 min, the pigment margin moves 15 degrees toward the animal pole. The cortex then relaxes to the prefertilization level over the next 10 min. Contrary to earlier estimations, the cortical contraction occurs during the same time span as the wave of cortical granule exocytosis. We suggest that the two events may result from a common stimulus. The sperm trail (ST) forms during the relaxation of the cortex. The ST first appears as a conically-shaped trail of pigment in the cytoplasm; it then elongates into a funnel-shaped trail as the male pronucleus migrates into the egg. The base of the cytoplasmic ST can be seen on the surface of the egg as a circular condensation of pigment. The male and female pronuclei migrate at a constant rate of 12 µm per minute. The male pronucleus migrates by the enlargement of its aster, whereas, it appears that the female pronucleus is dependent on the male aster for its motion.