Maturation and fertilization in Lottia gigantea oocytes: intracellular pH, Ca(2+), and electrophysiology.

Intracellular pH and Ca(2+) were measured with BCECF- and Calcium Green-dextran during maturation and fertilization of oocytes of the limpet Lottia gigantea. Maturation of oocytes from prophase to metaphase I of meiosis was induced in seawater adjusted to pH 9 with NH(4)OH. Intracellular pH rose during maturation induction, and maturation was also induced by microinjecting pH 8, but not pH 7, HEPES buffer. Intracellular Ca(2+) rose during NH(4)OH-induced maturation, but maturation was not inhibited when the increase was blocked by microinjection of BAPTA. When the metaphase I oocytes were fertilized(), there was an abrupt increase in intracellular Ca(2+), and activation (polar body formation) failed to occur in BAPTA-injected oocytes. Intracellular pH did not rise during fertilization. These observations show that maturation from prophase to metaphase I of meiosis is pH-dependent and activation of the metaphase I oocytes is Ca(2+)-dependent. A Ca(2+) action potential was present in both immature and mature oocytes but was more prominent in mature oocytes whose input resistance was higher. Fertilization produced a long-lasting (17-20 min) Na(+)-dependent fertilization potential with superimposed oscillations resembling Ca(2+) action potentials.

Inactivation of Ca(2+) action potential channels by the MEK inhibitor PD98059.

In a previous study involving the inhibition of mitogen-activated protein kinase (MAPK) activation during fertilization of the marine worm Urechis caupo, we found that PD98059, but not U0126, caused multiple sperm penetrations in oocytes (Gould and Stephano, 1999, Dev. Biol. 216, 348-358). Since these oocytes are protected against polyspermy by a positive shift in membrane potential at fertilization (Gould-Somero et al., 1979, J. Cell Biol. 82, 426-440), we investigated the effects of PD98059 on the electrical properties of the oocyte membrane. PD98059, but not U0126, selectively blocked the voltage-dependent Ca(2+) channels that participate in the electrical polyspermy block. We also noted previously that PD98059 had more serious effects than U0126 on chromosome behavior during meiosis. This, too, could be explained by the effect on Ca(2+) channels, since when U0126-treated eggs were fertilized in low Ca(2+) seawater to reduce Ca(2+) uptake, similar effects were produced. These results show that PD98059 has side effects unrelated to the inhibition of MAPK activation and underscores the need for caution in interpreting the results of experiments with this widely used MEK inhibitor.

MAP kinase, a universal suppressor of sperm centrosomes during meiosis?

We reported previously that inhibition of MAP kinase during meiosis in Urechis caupo eggs caused premature sperm aster formation and we reviewed indirect evidence that the suppression of sperm asters by MAPK during meiosis might be a universal mechanism (M. C. Gould and J. L. Stephano, 1999, Dev. Biol. 216, 348-358). We tested this proposition with oyster (Crassostrea gigas) and starfish (Asterina miniata) eggs, utilizing the MEK inhibitors U0126 and PD98059. Centrosomes, asters, and meiotic spindles were visualized by normal epifluorescence and confocal microscopy following indirect immunocytochemical staining for anti-beta-tubulin. When MAPK activation was inhibited, sperm asters in both species developed prematurely and tended to move toward the egg centrosomes, sometimes even fusing with the egg spindle or centrosomes. Meiotic spindles and polar body formation were also abnormal when MAPK was inhibited.

MAP kinase, meiosis, and sperm centrosome suppression in Urechis caupo.

Although MAP kinase is an important regulatory enzyme in many somatic cells, almost nothing is known about its functions during meiosis, except in frog and mouse oocytes. We investigated MAPK activation and function in oocytes of the marine worm Urechis caupo that are fertilized at meiotic prophase. Activity was first detected at 4-6 min after fertilization in immunoblots with anti-active MAPK, prior to germinal vesicle breakdown (GVBD). MAPK activation did not require new protein synthesis and was dependent on the increases in both intracellular pH and intracellular Ca(2+) that normally occur during activation. When MAPK activation was inhibited with PD98059 or U0126, GVBD still occurred, but meiosis was abnormal and there was a dramatic premature enlargement of sperm asters, which normally do not appear until second polar body formation. Failure of polar body formation and premature sperm aster enlargement also occurred when MAPK activation was inhibited by an entirely different treatment which involved lowering the pH of external seawater to interrupt the normal cytoplasmic pH increase. Thus, in Urechis, active MAPK appears to be required for (1) normal meiotic divisions and (2) suppressing the paternal centrosome until after the egg completes meiosis, a general phenomenon whose mechanism has been unknown.

The intracellular calcium increase at fertilization in Urechis caupo oocytes: activation without waves.

The intracellular Ca2+ (Cai) increase at fertilization of the marine worm Urechis caupo (Echiura) was studied with conventional and confocal epifluorescence microscopy in oocytes microinjected with calcium green dextran or dually labeled with the calcium-insensitive dye tetramethylrhodamine dextran. Calcium green fluorescence was also measured with a photomultiplier system while the oocyte membrane potential was recorded and manipulated. The results show that Cai rises simultaneously around the oocyte cortex and peaks slightly later in the nucleoplasm. The Cai rise coincides with the initiation of the fertilization potential and we conclude that it is due primarily to external Ca2+ entering through the voltage-gated Ca2+ action potential channels that open during the fertilization potential because: (1) current clamping the oocyte membrane potential to positive values in the absence of sperm produces a similar Cai increase, (2) external Ca2+ is required, (3) and the confocal images are consistent with this mechanism. External application of sperm acrosomal peptide (P23) also caused a Cai increase that was inhibited in the presence of CoCl2. Cai and pHi (measured with BCECF dextran) were manipulated in experiments employing microinjection of BAPTA (to chelate Cai), external application of NH4Cl (to increase pHi) and CoCl2 (to block Ca2+ channels), and fertilization of eggs in pH 7 seawater (Cai increase without pHi increase). The results showed that increases in both Cai and pHi are required for GVBD; neither alone is sufficient. However, although nuclear and cytoplasmic Ca2+ levels tended to parallel each other in oocytes fertilized at pH 7, and during the initial Cai response in oocytes fertilized at pH 8, there was a disproportionate fluorescence increase in the nucleoplasm of the latter prior to GVBD which could not be explained by any artifact we tested, suggesting there may be a selective increase in nuclear Ca2+ associated with GVBD. Finally, electrophysiological experiments with BAPTA-injected oocytes showed that the opening of the fertilization potential Na+ channels was Ca2+-independent, (although they did not close at the normal time). These and earlier results suggest that Urechis sperm may activate oocytes by interacting directly with the Na+ channels or associated receptors.

Parthenogenesis in Urechis caupo (Echiura). II. Role of intracellular pH in parthenogenesis induction.

A peptide (P23) isolated from sperm acrosomal protein initiates development in eggs of the marine worm Urechis caupo. We have shown previously that eggs exposed to P23 for > or = 3 min complete meiosis but fail to cleave. However, a brief (1.5-2 min) exposure to P23 at pH 8, followed by either acidification of the seawater of pH 7 or dilution of P23 at pH 8 causes germinal vesicle breakdown (GVBD), but eggs fail to complete meiosis and many then later advance to mitosis. In the present study we investigated the hypothesis that partial activation leading to parthenogenesis occurs when there is a partial intracellular alkalinization. Measurements with the fluorescent pH indicator bis(carboxyethyl)-carboxyfluorescein (BCECF) showed that P23 induces a pHi increase similar to that occurring during fertilization and the parthenogenesis-inducing treatments interrupt this rise in pHi. In eggs exposed to P23 for > 3 min the pHi increase was 0.31-0.49 units, slightly higher than in fertilized eggs. In partially activated eggs exposed to P23 for 1.5-2 min at pH 8, pHi began to rise but then returned to control values or remained only partially elevated (< 0.2 pH units average increase). Electrophysiological measurements revealed that removal of P23 during the first few minutes of exposure caused the activation potential to terminate and experiments with [14C]-P23 confirmed that dilution results in a rapid unbinding of P23 from eggs. If proton export is driven by membrane potential as well as the pH gradient, these results explain why dilution of P23 at pH 8 also interrupts the pHi increase.

Spawning, in vitro Maturation, and Changes in Oocyte Electrophysiology Induced by Serotonin in Tivela stultorum.

Spawning was induced in both male and female Pismo clams by injecting 0.4 ml of 5 mM serotonin into the gonad. Spawned oocytes had already matured to metaphase I of meiosis or were undergoing germinal vesicle breakdown at the time of release. Prophase-arrested oocytes scraped from the ovaries of uninjected clams were induced to undergo germinal vesicle breakdown in vitro by incubating them with 0.22--0 µM serotonin in seawater; the former concentration was optimal. In vitro matured oocytes were fertilizable and developed to larvae, whereas sperm penetrated prophase-arrested oocytes without activating them. Fertilization was more successful in slightly alkaline seawater (pH 8.5). The electrical response of oocytes to serotonin was studied by means of intracellular microelectrode recording. Resting potentials of prophase-arrested oocytes were between -60 and -80 mV and there was no immediate electrical response to perfusion with serotonin. However, about 10-15 min later (before germinal vesicle breakdown), membrane potentials usually began to drift slowly in the positive direction (net change by 40 min + 9 +/- 6.6 mV (SD; n = 8), whereas resting potentials of oocytes perfused with seawater alone usually drifted in the negative direction (-3 +/- 6.1 mV; n = 7). A dramatic increase in input resistance was consistently observed in oocytes induced to mature with serotonin, probably due to the inactivation of K+ channels, although this was not tested. Action potentials were always (7 out of 7 cases) present in maturing oocytes, but were detected only sometimes (7 of 14 cases) in prophase-arrested oocytes, presumably due to their lower input resistances.

Parthenogenesis in Urechis caupo (Echiura). I. Persistance of functional maternal asters following activation without meiosis.

A peptide (P23) isolated from sperm acrosomal protein initiates development in eggs of the marine worm Urechis caupo (Echiura). However, in these earlier experiments the eggs were exposed continuously to P23 and did not cleave unless they were provided with a sperm centrosome. The present study reports that parthenogenetic cleavage can be induced when the eggs are exposed to P23 for 1.5 to 2 min in seawater at pH 8 followed by acidifying the seawater to pH 7 or washing off the peptide at pH 7 or 8. The mechanism of parthenogenesis induction was studied by comparing the behavior of maternal and paternal centrosomes (microtubule organizing centers), chromosomes, nuclei and nucleoli in fertilized eggs, P23-activated eggs that failed to cleave, and P23-activated eggs that cleaved, using bright-field, phase-contrast, and fluorescence microscopy following anti-tubulin and bis-benzimide staining. Parthenogenetic cleavage to mostly diploid embryos occurred in eggs that underwent germinal vesicle breakdown without polar body formation. The centrosomes used for cleavage were the persistent maternal centrosomes that did not cycle through meiosis. When cytochalasin B was used to suppress polar body formation without inhibiting the meiotic cycles, eggs did not subsequently cleave even though all four maternal centrosomes were retained. When fertilized eggs were transferred to pH 7 seawater at 1.5 to 2 min after insemination, they underwent a partial activation like the P23-activated eggs and did not form polar bodies. Subsequent cleavage was very abnormal due to the presence of multiple asters formed by the persistant active maternal centrosomes. These results show that maternal centrosomes are normally inactivated by a maternal mechanism associated with meiotic cycling.

Nuclear and cytoplasmic pH increase at fertilization in Urechis caupo.

Intracellular pH (pHi) was measured in Urechis caupo (Echiura) eggs during fertilization using the pH-sensitive dye BCECF [bis(carboxyethyl)carboxyfluorescein] and fluorescence microscopy. When eggs were inseminated at pH 8, pHi began to rise 22-36 sec (n = 7) after sperm contact and reached a plateau by 3 min (2.8 +/- 1 SD; n = 14). The net increased was 0.25-0.3 pH units and the alkalinization persisted through 1 hr after insemination (after second polar body formation). Separate measurements of germinal vesicles and cytoplasm revealed that pH rose dramatically within the nuclei well before germinal vesicle breakdown (GVBD), as well as in cytoplasm. When eggs were fertilized at pH 6.9 (previously shown to inhibit proton release and egg activation despite sperm entry; see Paul, 1970, 1975; Holland et al., 1984), there was no net pH increase in either cytoplasm or germinal vesicles. When fertilization was at pH 7.45, the average pHi increase in whole eggs was 58% of that at pH 8, and 61% of the eggs activated partially or completely. These results show that the pHi rise is correlated with GVBD and egg activation.

Peptides from sperm acrosomal protein that initiate egg development.

How sperm initiate egg development is being investigated with gametes of the marine worm Urechis. Sperm acrosomal protein, previously shown to activate eggs (Gould et al., 1986, Dev. Biol. 117, 306-318; Gould and Stephano, 1987, Science 235, 1654-1656), was enzymatically cleaved into soluble peptide fragments. When this mixture was added to eggs they activated, and parthenogenetic cleavage often occurred. An active peptide (P23) was purified from the mixture and its sequence was determined to be Val-Ala-Lys-Lys-Pro-Lys. Synthetic peptide had the same biological activity. P23 induced eggs to undergo the complete sequence of changes that normally follows fertilization, including the fertilization potential, completion of meiosis, and DNA replication. When a sperm centrosome was introduced into eggs by prefertilization without activation, and the eggs were subsequently activated by P23, they developed normally to trochophore larvae (the contribution of another sperm component is not ruled out by this experiment). P23 covalently coupled to bovine serum albumin also activated eggs, showing that it acted on the external surface of the egg. The peptide did not activate sea urchin eggs, but did cause oyster eggs to undergo germinal vesicle breakdown.

Quantitation of endotoxin in products using the LAL kinetic turbidimetric assay.

The data presented here show the kinetic turbidimetric LAL assay to be a highly quantitative and effective method for determining endotoxin concentrations in products. The assay allows for the accurate assessment of inhibiting or enhancing effects in products when related to a LRW standard curve. However, designating some products as inhibitors or enhancers can be both misleading and erroneous unless qualified as to the dilution and/or endotoxin concentration. Our results demonstrate that some products can yield both inhibiting and enhancing results when related to water. Due to the enhanced resolution of the kinetic turbidimetric assay, these complicating inhibition/enhancing effects can usually be avoided by diluting to the WED. Alternatively, products could be related to a PSC in which the endotoxin response is defined and quantified within the product itself. The practicality of a PSC, however, depends upon the pass/fail limit established, the "cleanliness" of the product used as a standard and the degree of product "lot to lot" variability. Current FDA Guidelines consider a PSC valid providing the value of the "unspiked" product extrapolated from the regression line of the PSC is less than 10% of lambda, the lowest endotoxin concentration used to construct the standard. All products in which LAL kinetics have not been previously analyzed will require a characterization similar to that used with the four products described. From these data, the optimal methodology for kinetically testing the product (dilution to a WED or generation of a PSC) can be determined. Although analysis of endotoxin in a product will always require a characterization of the kinetics of the LAL-endotoxin-product reaction, subsequent testing should be rapid and straightforward. More importantly, the kinetic turbidimetric assay allows the user to quantitatively assess product endotoxin levels with a degree of precision greater than that of any other methodology currently in use.

Biochemical, hematologic, and histopathologic studies in rabbits intrarectally inseminated.

Rabbits subjected three times a week for 7 months to administrations of fresh homologous semen, colonic enemas, or semen (previously frozen at -70 degrees) preceded by enema were monitored by a battery of biochemical and hematologic tests and histopathologic examination. No biochemical, hematologic or histopathologic changes were observed. Negative blood cultures and serum endotoxin determinations excluded any possibility of systemic bacterial infection caused or induced by the treatments. Wide temporary variations in the hematologic parameters, including concentrations of T and B cells, were observed in all treated groups. The relevance of these findings to immune status is not yet certain.