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.
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 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.
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.
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