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Reviews in Developmental Biology have covered the pathways that generate the all-important intracellular calcium (Ca(2+)) signal at fertilization [Miyazaki, S., Shirakawa, H., Nakada, K., Honda, Y., 1993a. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel in Ca(2+) waves and Ca(2+) oscillations at fertilization of mammalian eggs. Dev. Biol. 158, 62-78; Runft, L., Jaffe, L., Mehlmann, L., 2002. Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237-254] and the different temporal responses of Ca(2+) in many organisms [Stricker, S., 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157-176]. Those reviews raise the importance of identifying how Ca(2+) causes the events of egg activation (EEA) and to what extent these temporal Ca(2+) responses encode developmental information. This review covers recent studies that have analyzed how these Ca(2+) signals are interpreted by specific proteins, and how these proteins regulate various EEA responsible for the onset of development. Many of these proteins are protein kinases (CaMKII, PKC, MPF, MAPK, MLCK) whose activity is directly or indirectly regulated by Ca(2+), and whose amount increases during late oocyte maturation. We cover biochemical progress in defining the signaling pathways between Ca(2+) and the EEA, as well as discuss how oscillatory or multiple Ca(2+) signals are likely to have specific advantages biochemically and/or developmentally. These emerging concepts are put into historical context, emphasizing that key contributions have come from many organisms. The intricate interdependence of Ca(2+), Ca(2+)-dependent proteins, and the EEA raise many new questions for future investigations that will provide insight into the extent to which fertilization-associated signaling has long-range implications for development. In addition, answers to these questions should be beneficial to establishing parameters of egg quality for human and animal IVF, as well as improving egg activation protocols for somatic cell nuclear transfer to generate stem cells and save endangered species.

Although recent studies have demonstrated the importance of calcium/calmodulin (Ca 2þ /CAM) signaling in mammalian fertilization, many targets of Ca 2þ /CAM have not been investigated and represent potentially important regulatory pathways to transduce the Ca 2þ signal that is responsible for most events of egg activation. A well-established Ca 2þ /CAM-dependent enzyme is myosin light chain kinase (MYLK2), the downstream target of which is myosin II, an isoform of myosin known to be important in cytokinesis. In fertilized mouse eggs, established inhibitors of MYLK2 and myosin II were investigated for their effects on events of egg activation. The MYLK2 antagonist, ML-7, did not decrease the activity of Ca 2 þ /CAM protein kinase II or the elevation of intracellular Ca 2 þ , and it did not delay the onset of Ca 2 þ oscillations. In contrast, ML-7 inhibited second polar body (PB) formation in a dose-dependent manner and reduced cortical granule (CG) exocytosis by a mean of approximately 50%. The myosin II isoform-specific inhibitor, blebbistatin, had similar inhibitory effects. Although both antagonists had no effect on anaphase onset, they inhibited second PB formation by preventing spindle rotation before telophase II and normal contractile ring constriction. To our knowledge, this is the first report that MYLK2 and myosin II are involved in regulating the position of the meiotic spindle, formation of the second PB, and CG exocytosis. The present results suggest that MYLK2 is one of a family of CAM-dependent proteins that act as multifunctional regulators and transduce the Ca 2þ signal at fertilization.

Elucidation of the biochemical mechanisms by which specific proteins transduce the all important intracellular calcium (Ca2+) signal at fertilization into events of egg activation will increase our understanding of the regulation of the onset of development and the extent to which these signals can be experimentally modified. Previously, we reported data supporting the hypothesis that mouse eggs have the capability to generate oscillations of the activity of Ca2+ and calmodulin-dependent kinase II (CaMKII), regulating the cell cycle and secretion. This study directly demonstrates transient increases of enzyme activity in relatively close synchrony with Ca2+ oscillations for the first hour of fertilization in single mouse eggs monitored for both Ca2+ and CaMKII activity. The extent of the enzyme activity increase was correlated with the level of intracellular Ca2+. After a rise in activity, the decrease in activity did not appear to be due to negative feedback from elevated Ca2+ or CaMKII activity over time, since enzyme activity persisted after 8 min of elevated Ca2+ from 7% ethanol activation. The contribution of CaMKII from a single sperm to the rise in CaMKII activity at fertilization appeared to be negligible. Also, long-term cell cycle inhibition was observed in fertilized eggs with the CaMKII antagonist myrAIP (50 μM), which did not inhibit the first large Ca2+ transient or subsequent early oscillations but did reduce the percentage of eggs fertilized. Thus, mammalian eggs appear to drive many activation events over time to completion with repeated short bursts of Ca2+ oscillation-dependent CaMKII activity, rather than by a steady-state, continuously elevated level of CaMKII activity that is maintained by periodic Ca2+ oscillations.

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.

Preovulatory, germinal vesicle (GV)-stage mouse oocytes are unable to undergo normal cortical granule (CG) secretion. Full secretory competence is observed by metaphase II (MII) of meiosis and involves the development of calcium response mechanisms. To identify the deficient or inhibited step in CG secretion, preovulatory GV-stage oocytes were stimulated and tested for their ability to undergo translocation, docking, and/or fusion. The mean CG distance to the plasma membrane was not reduced in fertilized or sperm fraction-injected, GV-stage oocytes relative to that in control GV-stage oocytes. In addition, analysis of individual CG distances to the plasma membrane indicated no subpopulation of CGs competent to translocate. Further analysis demonstrated that secretory incompetence likely is not due to a lack of proximity of CGs to the egg's primary calcium store, the endoplasmic reticulum. Calcium/calmodulin-dependent protein kinase II (CaMKII), which is reportedly involved in secretory granule translocation and secretion in many cells, including eggs, was investigated. A 60-kDa CaMKII isoform detected by Western blot analysis increased 150% during oocyte maturation. The CaMKII activity assays indicated that MII-stage eggs correspondingly have 110% more maximal activity than GV-stage oocytes. These data demonstrate that the primary secretory deficiency is due to a failure of CG translocation, and that a maturation-associated increase in CaMKII correlates with the acquisition of secretory competence and the ability of the egg to undergo normal activation.

At fertilization, the release of intracellular calcium is necessary and sufficient for most, if not virtually all, of the major events of egg activation that are responsible for the onset of embryonic development. In mammalian eggs, repetitive calcium oscillations stimulate egg activation events through calcium-dependent effectors, such as calmodulin, protein kinases, and specific proteins involved in exocytosis. One of the earliest calcium-dependent events is the exocytosis of cortical granules (CGs), a secretory event resulting in the block to polyspermy and the prevention of triploidy. Emerging studies suggest that CG release in mature eggs is dependent upon calcium-dependent proteins similar to those in somatic cells employed to undergo calcium-regulated exocytosis. In contrast, pre-ovulatory oocytes are incompetent to undergo CG exocytosis due to deficiencies in the ability to release and respond to increases in intracellular calcium. The development of competence to release and respond to calcium is relevant to both animal and human in vitro fertilization programs that largely utilize ovarian oocytes not all of which are fully activation competent.

The fertilization-induced exocytosis of egg cortical granules (CGs) is responsible for a block to polyspermy, crucial to the viability of many species. The contents of mammalian CGs have been an elusive target for analysis because of picogram quantities of CG proteins. By using media enriched in secreted CG contents from calcium ionophore-induced eggs as an immunogen, a monoclonal antibody was raised that immunolocalized to structures in the mouse egg cortex with all the hallmarks of CGs. These structures were the correct size, absent from the region over the metaphase II spindle, and greatly reduced after fertilization. Double-labeling experiments confirmed that the antibody recognized the same population of CGs as those recognized by Lens culinaris agglutinin. On Western blots, the antibody primarily recognized a 32-kDa protein (and secondarily one at ∼25 kDa) in mouse eggs. Analysis of biotin-labeled secreted proteins from activated eggs confirmed that CGs release only a small number of major proteins (45, 34, 32, 28, and ∼20 kDa by SDS-PAGE). We therefore propose that the 32-kDa protein identified by this antibody is likely to correspond to the 32-kDa protein released from activated eggs and that it may be involved in the block to polyspermy. These methods should make it possible to generate additional antibodies to study the structure of CG components as well as their roles in the polyspermy block and CG biogenesis.

Mammalian fertilization is characterized by the presence of long-lasting intracellular calcium ([Ca2+]i) oscillations that are required to induce oocyte activation. One of the Ca2+ channels that may mediate this Ca2+ release is the inositol 1,4, 5-trisphosphate receptor (IP(3)R). Three isoforms of the receptor have been described, but their expression in oocytes and possible roles in mammalian fertilization are not well known. Using isoform-specific antibodies against IP(3)R types 1, 2, and 3 and Western analysis, we determined the isoforms that are expressed in bovine metaphase II oocytes and ovaries. In oocytes, all isoforms are expressed, but type 1 is present in overwhelmingly larger amounts and is likely responsible for the majority of Ca2+ release at fertilization. In ovarian microsomes, all three isoforms appear well expressed, suggesting the participation of all IP(3)R isoforms in ovarian Ca2+ signaling. We then investigated whether the reported cessation/reduction in amplitude of fertilization-associated [Ca2+]i oscillations, which is observed as pronuclear formation approaches, corresponded with down-regulation of the IP(3)R-1 isoform. Fertilization resulted in approximately 40% reduction in the amount of receptor by 16 h postinsemination. In addition, injection of adenophostin A, a potent IP(3)R agonist that elicits high-frequency [Ca2+]i oscillations in mammalian oocytes, induced similar reduction in receptor numbers. Together, these data show that 1) the three IP(3)R isoforms are expressed in bovine oocytes; 2) IP(3)R-1 is likely to mediate most of the Ca2+ release during fertilization; 3) its down-regulation may explain the decline in amplitude of sperm-induced [Ca2+]i rises as fertilization progresses toward pronuclear formation; and 4) agonists of the IP(3)R induce down-regulation of the type-1 receptor in oocytes similar to that evoked by fertilization.

In mammalian fertilization, inositol 1,4,5-trisphosphate receptor (IP 3 R)-dependent Ca 2+ release is a crucial signaling event that originates from the vicinity of sperm-egg interaction and spreads as a wave throughout the egg cytoplasm. While it is known that Ca 2+ is released by the type 1 IP 3 R in the egg cortex, the potential involvement of other isoform types responsible for the Ca 2+ rise in the mouse egg (interior) and their spatial distribution are not known. In addition, the biochemical basis has not been definitively established for the development of increased sensitivity to inositol 1,4,5-trisphosphate (IP 3 ) during meiotic maturation. Using specific antibodies to the type 1, 2, and 3 IP 3 R, we tested the hypotheses that different IP 3 R isoforms are responsible for the internal Ca 2+ elevation and that they contribute to the maturation-associated acquisition of IP 3 sensitivity. In both preovulatory oocytes and ovulated eggs of CF-1 mice, immunofluorescence revealed that types 1 and 2 isoforms were present in the cell cortex and interior. Type 1 was observed throughout the cytoplasm, and Western analysis indicated a 1.9-fold maturation-associated increase. In contrast, the signals detected for the type 2 (high-affinity) isoform and type 3 were present to a lesser extent, with type 2 restricted to isolated islands (similar to aggregates of vesicles detected by electron microscopy), which, in the cortex, may amplify early sperm-egg signaling events. The cortical-to-perinuclear localization of the receptor and cortical vesicle aggregates imply an efficient mechanism for propagating Ca 2+ release from the cortex into the interior of the egg to activate development, and the isoform localization analysis indicates a clear spatial and biochemical heterogeneity. Types 1 and 2 isoforms were also present in granulosa cells.

We have previously demonstrated that metaphase II-arrested eggs recovered from oviducts at increasing times after hCG administration display a time-dependent spontaneous entry into anaphase, as well as release of cortical granules (CGs) and the associated modifications of the zona pellucida (ZP), a decrease in histone H1 and mitogen-activated protein kinase activities, and the recruitment of maternal mRNAs [Xu et al., Biol Reprod 1997; 57:743-750). These changes are correlated with the time-dependent increase in susceptibility of these eggs to undergo parthenogenetic activation. We report here the effect of culture of ovulated eggs, retrieved 13 or 16 h post-hCG administration and cultured in vitro for various periods of time, on the aforementioned parameters of egg activation and cell cycle resumption. In contrast to extended residence of the eggs in the oviduct, culture in vitro retarded cell cycle events associated with completion of the second meiotic reduction and inhibited CG release and the associated modifications of the ZP, as well as the recruitment of maternal mRNAs. The retardation or inhibition of these changes during in vitro culture resulted in eggs that were less susceptible to parthenogenetic activation than eggs that resided in the oviduct for comparable time periods. Results of these experiments indicate that egg culture in vitro (which likely occurs under suboptimal conditions) inhibits, rather than accelerates, the progression into the interphase-like state as compared to that seen in eggs residing in the oviduct for increasing periods of time. These results also suggest that, for studies focused on in vitro fertilization or egg activation, the ovulated eggs should be placed under appropriate in vitro conditions as soon as possible.

The fertilization rates with ICSI range from 30% to 70% and suggest that, despite injecting sperm into mature oocytes, significant fertilization failure still occurs in humans. The objective of this study was to determine technical and physiological factors which may contribute to lower fertilization following ICSI. Eggs that failed to show two pronuclei (PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). The eggs were analyzed by staining with DNA fluorochromes, Hoescht 33258 and DAPI. The extent of sperm head as well as maternal chromatin decondensation in unfertilized ICSI eggs was determined by high resolution fluorescence microscopy. The average fertilization rate (FR) from all ICSI cycles in these two groups was 45%. The FR in Groups A and B were 35% and 59%, respectively (P < 0.05). In Group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In Group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin while 45% were partially decondensed. Sperm chromatin was not detected in 24% of all unfertilized eggs studied. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause for failure of fertilization rates in ICSI cycles. The increased percentage of eggs undergoing sperm head decondensation may be related to subtle changes in technique as experience is gained over time. The failure of sperm head decondensation in some of the ICSI eggs may be associated with cytoplasmic immaturity but not nuclear maturity.

Normal development depends on both the timing of fertilization and gamete quality, especially in assisted reproductive procedures. Recent studies of the proteins involved in the polyspermy block and cell cycle progression provide a cellular and biochemical basis for the short fertilizable lifespan of the mammalian egg in several species. Specifically, the status of cortical granules, zona proteins, cell cycle kinases, and intracellular calcium stores form a powerful panel of assays to monitor egg activation competence in eggs undergoing maturation and spontaneous activation events in mature eggs. An understanding of how these indicators are influenced by in vitro conditions and exogenous follicular stimulation should provide useful information for optimizing assisted reproductive procedures.

With increasing time after ovulation, mammalian eggs become more sensitive to agonists of activation in vitro or may undergo spontaneous activation in vivo. We have tested the hypothesis that postovulatory eggs undergo time-dependent cell cycle and cytoplasmic changes that result in a partially activated state, accounting for their time-dependent susceptibility to activate. In vivo changes in key activation markers in mouse eggs were quantified at 13, 16, and 22 h post-hCG (1, 4, and 10 h postovulation). Spontaneous activation was first detected at 16 h, with a 20-25% decrease in the activities of histone H1 and mitogen-activated protein (MAP) kinases and with 3% of eggs undergoing both anaphase onset and a partial loss of cortical granules. By 22 h, more than 60% of eggs were in anaphase, H1 and MAP kinase activities had decreased 40-50%, the extent of zona pellucida modification had increased, and proteins normally synthesized after fertilization had appeared. Pronuclear formation in response to inositol 1,4,5-trisphosphate injection increased dramatically from 10% at 13 h to about 40% and 90% at 16 h and 22 h, respectively. The partial decreases (less than those after fertilization) in H1 and MAP kinase activities provide a likely biochemical basis for the increased sensitivity of eggs to agonists, seen over time, that results in pronuclear formation. Also, all of these time-dependent changes caution against the use of mouse eggs > 16 h after hCG administration in studying the mechanism of normal fertilization and have implications for animal and human in vitro fertilization.

The purpose of this study was to determine at what stage of meiotic maturation mouse oocytes develop the ability to undergo sperm-induced activation, assayed by cortical granule (CG) release. Germinal vesicle breakdown (GVBD), prometaphase I (proMI), metaphase I (MI), and metaphase II (MII, the normal stage at fertilization) stages were evaluated by quantitative image analysis. At 2 hr, fertilized MII eggs underwent a mean CG loss of 69%; loss was global--in the entire cortex occupied by CGs. In contrast, fertilized GVBD and proMI oocytes had no significant CG loss in the cortex after 2 hr. After 4 hr, 63, 67, and 88% of fertilized GVBD, proMI, and MI oocytes, respectively, had localized CG release only in the vicinity of the fertilizing sperm. At 4 hr, GVBD oocytes had small sperm-associated, CG-depleted domains, approximately 100 microns 2 (< 1% of the oocyte cortex), whereas those for proMI oocytes were approximately 2500 microns 2. The CG density was reduced by 90% in these domains, whereas the remaining cortex showed no evidence of CG loss. Both the extent and time course of CG loss were altered in oocytes fertilized before MII and were not significantly affected with 25 microM thimerosal, which sensitizes egg calcium stores. Unlike the ability to undergo CG release, the ability to initiate sperm chromatin decondensation was not stage specific and the extent of decondensation was cell cycle related. Competence to undergo CG loss develops in two phases: the ability to undergo localized CG release increases between the GV and MI stages, whereas the mechanism of propagating a normal wave of global CG loss from the site of sperm entry develops between MI and MII.

Although the important subplasmalemmal localization of cortical granules (CG) has been documented, little is known about the timing and mechanism of granule translocation to the oocyte cortex. A fluorescent CG probe, confocal microscopy, and image analysis were used to obtain kinetic, spatial, and temporal information about CG migration during mouse oogenesis. The mean CG density in the cortex increased exponentially, from 3 to 14 CG/100 microns 2, during oocyte growth from 40-50 microns to 70-80 microns in diameter, respectively. Full-grown, > or = 80-microns, germinal vesicle-intact oocytes had a density of 30-35 CG/100 microns 2. During growth from 40-50 microns to > or = 80 microns, the mean total number of CG in the cortex per oocyte increased from 6000. When analyzed in terms of the stage of germinal vesicle chromatin organization, the mean CG density increased from 3 to 21 CG/100 microns 2 from stage I to early stage IV, respectively. In 50-60-microns oocytes, there was a crescent-shaped area of perinuclear staining containing both granules and Golgi apparatus-like structures, which were also more sparsely distributed in the subcortical cytoplasm. It is likely that these were sites of CG production from which translocation takes place; they were not observed in ovulated metaphase II eggs. This study demonstrates that CG number in the cortex increases continually during mouse oocyte growth (in contrast to that in several other species), that there is a greater than twofold increase in CG number during the final phase of growth, and that subcortical production ceases after ovulation.

Mouse oocyte cortical granule (CG) activation and distribution were investigated during in vivo meiotic maturation to determine the onset of competence to undergo the cortical reaction, which is considered responsible for the block to polyspermy. In the present study, the resumption of oocyte maturation was stimulated by hCG administration. Competence to undergo the cortical reaction (assessed with calcium ionophore A23187) was undetectable (0% loss) in germinal vesicle-stage oocytes 0.5 h after hCG administration. When germinal vesicle breakdown and metaphase I had taken place (3 and 7 h post hCG, respectively), approximately 30% CG loss was observed. Maximal (A23187-inducible) levels of CG loss, 67% and 72%, were present at 10 and 13 h, respectively, during metaphase II. Cortical granule distribution changed dramatically during metaphase I, polar body formation, metaphase II, and post-ovulatory aging in vivo. A stable metaphase II distribution was present from 13 to 18 h. After 24 and 32 h, 28% and 83% of the eggs, respectively, exhibited major alterations in the cortical distribution of CGs, some of which did not appear to be susceptible to release by A23187. These data support the hypothesis that just before ovulation the egg cortex completes the development of its normal structure and physiological competence, which are maintained for only a brief period of time afterward. The implications are discussed for normal fertilization and polyspermy in mammals, including humans.

Fertilization results in cortical granule exocytosis, which is thought to be involved in modifications of the zona pellucida that constitute the zona pellucida block to polyspermy. A previous report demonstrated that a decrease in the number of Lens culinaris agglutinin-staining granules, which are likely to be cortical granules, occurred during in vivo mouse oocyte maturation with arrest at metaphase II, as well as the formation of a cortical granule-free domain in the area of the metaphase II spindle (T. Ducibella, E. Anderson, D.F. Albertini, J. Aalberg, and S. Rangarajan, 1988, Dev. Biol. 130, 184-197). We extend these observations by reporting here that germinal vesicle-intact oocytes matured in vitro to metaphase II in either the absence or the presence of serum develop a cortical granule-free domain and have reduced numbers of cortical granules when compared to germinal vesicle-intact oocytes; these changes are similar to those of oocytes matured in vivo. The reduction in the number of cortical granules requires germinal vesicle breakdown, since it is prevented by dibutyryl cAMP, which inhibits germinal vesicle breakdown in vitro. The ability of oocytes to respond to the calcium ionophore A23187 with a reduction in the number of cortical granules is also associated with meiotic maturation and develops between 7 and 12 hr after initiation of maturation. The maturation-associated reduction in the number of cortical granules is likely to represent cortical granule exocytosis, since this reduction is accompanied by the formation of a cortical granule-free domain and a conversion of ZP2 to ZP2f when the oocytes are matured in vitro in serum-free medium; this zona pellucida modification occurs following fertilization and is thought to be due to cortical granule exocytosis. In contrast, the loss of cortical granules and development of the cortical granule-free domain of oocytes matured in vitro in the presence of serum is not accompanied by the modification of ZP2. The inhibitory effect of serum on the ZP2 modification may afford in vivo a physiological mechanism to prevent a precocious modification of the zona pellucida that could result in a premature block to polyspermy and hence inhibit fertilization.

Novel methods of egg activation in human assisted reproductive technologies and animal somatic cell nuclear transfer are likely to alter the signalling process that occurs during normal fertilization. Intracytoplasmic sperm injection (ICSI) bypasses the normal processes of the acrosome reaction, sperm-egg fusion, and processing of the sperm plasma membrane, as well as alters some parameters of intracellular calcium ([Ca 2+ ] i ) dynamics (reported previously by Kurokawa and Fissore (2003)). Herein, we extend these studies to determine if ICSI alters the activity of the Ca 2+ -dependent protein, Ca 2+ /calmodulin-dependent kinase II (CaMKII), which is responsible for the completion of meiosis in vertebrate eggs. After ICSI or in vitro fertilization (IVF), individual mouse eggs were monitored for their relative changes in both [Ca 2+ ] i and CaMKII activity during the first [Ca 2+ ] i rise and a subsequent rise associated with second polar body extrusion. The duration of the first [Ca 2+ ] i rise was greater in ICSI than in IVF, but the amplitude of the rise was transiently higher for IVF than ICSI. However, a similar mean CaMKII activity was observed in both procedures. During polar body extrusion, the amplitude and duration of the Ca 2+ rises were increased by a small amount in ICSI compared with IVF, whereas the CaMKII activities were similar. Thus, compared with IVF, ICSI is not associated with decreased or delayed CaMKII activity in response to these Ca 2+ signals in the mouse.

The mammalian egg appears to transduce the duration, amplitude, and temporal presentation of the increase in the intracellular calcium concentration ([Ca(2+)](i)) upon fertilization. These Ca(2+) parameters have important short-term effects on the initiation and completion of early events of egg activation, as well as much later consequences for the extent of peri-implantation development. Recent studies have begun to shed light on how the egg quantitatively interprets the Ca(2+) signal (e.g., by summation of individual Ca(2+) rises) and the mechanisms by which down-stream Ca(2+) effectors, such as Ca(2+)/Calmodulin (CaM)-dependent protein kinase II (CaMKII), utilize this ionic signal to promote biological events that initiate development.

Although recent studies have demonstrated the importance of calcium/calmodulin (Ca(2+)/CAM) signaling in mammalian fertilization, many targets of Ca(2+)/CAM have not been investigated and represent potentially important regulatory pathways to transduce the Ca2+ signal that is responsible for most events of egg activation. A well-established Ca(2+)/CAM-dependent enzyme is myosin light chain kinase (MYLK2), the downstream target of which is myosin II, an isoform of myosin known to be important in cytokinesis. In fertilized mouse eggs, established inhibitors of MYLK2 and myosin II were investigated for their effects on events of egg activation. The MYLK2 antagonist, ML-7, did not decrease the activity of Ca(2+)/CAM protein kinase II or the elevation of intracellular Ca2+, and it did not delay the onset of Ca2+ oscillations. In contrast, ML-7 inhibited second polar body (PB) formation in a dose-dependent manner and reduced cortical granule (CG) exocytosis by a mean of approximately 50%. The myosin II isoform-specific inhibitor, blebbistatin, had similar inhibitory effects. Although both antagonists had no effect on anaphase onset, they inhibited second PB formation by preventing spindle rotation before telophase II and normal contractile ring constriction. To our knowledge, this is the first report that MYLK2 and myosin II are involved in regulating the position of the meiotic spindle, formation of the second PB, and CG exocytosis. The present results suggest that MYLK2 is one of a family of CAM-dependent proteins that act as multifunctional regulators and transduce the Ca2+ signal at fertilization.

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.

Fertilization-induced intracellular calcium (Ca(2+)) oscillations stimulate the onset of mammalian development, and little is known about the biochemical mechanism by which these Ca(2+) signals are transduced into the events of egg activation. This study addresses the hypothesis that transient increases in Ca(2+) similar to those at fertilization stimulate oscillatory Ca(2+)/calmodulin-dependent kinase II (CaMKII) enzyme activity, incrementally driving the events of egg activation. Since groups of fertilized eggs normally oscillate asynchronously, synchronous oscillatory Ca(2+) signaling with a frequency similar to fertilization was experimentally induced in unfertilized mouse eggs by using ionomycin and manipulating extracellular calcium. Coanalysis of intracellular Ca(2+) levels and CaMKII activity in the same population of eggs demonstrated a rapid and transient enzyme response to each increase in Ca(2+). Enzyme activity increased 370% during the first Ca(2+) rise, representing about 60% of maximal activity, and had decreased to basal levels within 5 min from the time Ca(2+) reached its peak value. Single fertilized eggs monitored for Ca(2+) had a mean increase in CaMKII activity of 185%. One and two ionomycin-induced Ca(2+) transients resulted in 39 and 49% mean cortical granule (CG) loss, respectively, while CG exocytosis and resumption of meiosis were inhibited by a CaMKII antagonist. These studies demonstrate that changes in the level of Ca(2+) and in CaMKII activity can be studied in the same cell and that CaMKII activity is exquisitely sensitive to experimentally induced oscillations of Ca(2+) in vivo. The data support the hypothesis that CaMKII activity oscillates for a period of time after normal fertilization and temporally regulates many events of egg activation.

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281-309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(3) binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP(3)R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o(-) cells that predominantly express type 3 IP(3)R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP(3)R-1 directly and may therefore be a useful tool in the study of IP(3)R functional regulation.

Objective : To determine the extent of paternal and maternal chromatin decondensation in unfertilized eggs after intracytoplasmic sperm injection (ICSI). Design : Eggs that failed to show two pronuclei (2-PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). Patient(s) : Forty-nine patients undergoing IVF cycles. Main Outcome Measure(s) : The unfertilized eggs were studied by chromatin staining. Result(s) : The average fertilization rate from all ICSI cycles in these two groups was 45%. The fertilization rates in groups A and B were 35% and 59%, respectively. In group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin, whereas 45% were partially decondensed. In these two groups, no sperm chromatin was detected in 24% of the unfertilized eggs. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. Conclusion(s) : These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause of failure of fertilization rates in ICSI cycles. Rather, it is likely to be the failure to complete both the maternal and paternal chromatin transitions that occur with normal fertilization.

Although pharmacological agonists of protein kinase C (PKC) stimulate some events of mammalian egg activation, including cortical granule (CG) exocytosis, it is not known if these events are dependent on PKC activation during the normal process of fertilization. In order to examine the potential role of PKC in CG exocytosis, this study investigated whether PKC agonists faithfully mimic CG release and whether PKC antagonists block fertilization-induced CG release in mature mouse eggs. Phorbol ester (TPA, 2.5 ng/ml) treatment resulted in an atypical pattern of CG release in which there was a greater net loss of CGs in the equatorial region of the egg than in the region opposite the spindle. This pattern also was in contrast to that during fertilization, in which CG release occurred randomly throughout the cortex. Fertilization experiments utilized two different PKC inhibitors, bisindolyl-maleimide (5 μM) and chelerytherine (0.8 μM), targeted to both the “conserved” substrate and ATP binding domains of PKC. Simultaneous use of both inhibitors at maximal concentrations (compatible with fertilization and above their IC50S) resulted in no detectable inhibition of CG release in treated fertilized eggs compared to controls. In addition, no inhibition of anaphase onset was observed in treated fertilized eggs. Activity of the inhibitors was verified by demonstrating that they blocked the induction of CG loss by TPA. Moreover, 1 μM staurosporine, a potent but less specific antagonist of PKC, also did not block CG loss, whereas the metaphase-anaphase transition was temporarily inhibited. The results indicate that TPA does not faithfully mimic CG release in fertilized eggs, that a role for PKC in CG release at fertilization remains to be established, and that other calcium-dependent effectors may be involved in CG exocytosis. Mol Reprod Dev 46:216–226, 1997. © 1997 Wiley-Liss, Inc.

Results of numerous experiments indicate that the transient rise in intracellular Ca2+ following sperm-egg fusion is essential for the subsequent events that constitute egg activation. Some events of egg activation, e.g., cortical granule exocytosis, however, appear more sensitive to intracellular Ca2+ than other events, e.g., cell cycle resumption. To examine if specific events of egg activation have different thresholds for Ca2+, we manipulated buffered intracellular Ca2+ concentrations by microinjecting Ca2+-BAPTA buffers and then examined the effect on the cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. We find that whereas cortical granule exocytosis occurs over a narrow threshold range of injected free Ca2+ concentrations between 0.5 and 1.0 microM, recruitment of maternal mRNAs is only partially stimulated at injected free Ca2+ concentrations of 2.5 microM, and no evidence for cell cycle resumption was observed (up to 2.5 microM Ca2+). Although the Ca2+- and phospholipid-dependent protein kinase, protein kinase C, is implicated in aspects of egg activation, calmodulin is also a potential target for the transient increase in Ca2+ that occurs following fertilization. Whereas incubation of eggs in the presence of the calmodulin antagonist W-7 followed by insemination does not block cortical granule exocytosis, cell cycle resumption, as assessed by the metaphase-to-anaphase transition, a decrease in histone H1 kinase activity and the time course for the emission of the second polar body are significantly delayed/inhibited.

Successful intracytoplasmic sperm injection (ICSI) is dependent upon the competence of the oocyte to respond to the injection of a spermatozoon in the presence of calcium. This study determined if oocytes that failed to become fertilized (absence of any pronuclei) failed to undergo cytoplasmic activation, as ascertained by an electrophoretic shift in the zona pellucida (zona) protein, huZP2. Of 48 zonae individually analysed, 58% did not have a detectable huZP2 shift. Three patterns were observed. In seven patients, none of the unfertilized oocyte huZP2 shifted (16 out of 48); in two, all zonae exhibited a huZP2 shift (five out of 48); in eight, there was a mixture of shifted and unshifted (27 out of 48) in each case. There was no clear relationship between the presence of the huZP2 shift and maternal age, pregnancy outcome, male-factor infertility, or fertilization rate. However, in six couples diagnosed as having no detectable male-factor infertility, 73% (11 out of 15) of the zonae had unshifted huZP2, suggesting that an oocyte defect is involved in some cases of failed fertilization after ICSI. One likely cause for the absence of the huZP2 shift is a failure of cytoplasmic activation. Since oocytes were injected at metaphase II, we hypothesize that some oocytes that failed to fertilize have completed nuclear (meiotic) without cytoplasmic maturation.

The basis for the incompetence of the cortical reaction in germinal vesicle stage (GV) mouse oocytes was studied by evaluating cortical granules (CGs) and vesicles in GV and mature oocyte cortices. Dark and light CGs had a similar mean distance of 0.4-0.6 micron from the plasma membrane for GV and mature cortices. The cortex of mature oocytes had a large population of membrane-bounded, 0.1-1.0 micron (diameter) vesicles. More than three times as many vesicles were observed in the CG domains of mature oocytes as were observed in GV oocytes. This lack of cortical vesicles (with their potential to store calcium) and not CG depth may account for cortical reaction incompetence in GV oocytes.

The role of microtubules immediately preceding compaction in early 8-cell stage blastomeres (8-CSB) was investigated in the mouse embryo. The results of a previous study [12] were extended by initiating colchicine treatment at the 5–7-cell stage when the first 8-CSB appear. In 2.5 × 10 −5 or 2.5 × 10 −6 M drug, 8-CSB became a completely compacted group, whereas the 4-CSB remained separate and spherical. EM analysis demonstrated the absence of cortical microtubules in colchicine-treated embryos, the presence of normal close cell-cell apposition by 8-CSB, the development of apical localization of microvilli by 8-CSB, the failure of 8-CSB to spread over 4-CSB, and the metaphase arrest of 4-CSB. In addition, some blastomeres developed shallow, abnormally-located cleavage furrows suggesting that microfilaments were still able to organize. In summary, (1) microtubules are not required for compaction and the development of cell polarity, supporting other recent studies in which the role of microtubules in a cell shape change is not clear;(2) the surface of arrested 4-CSB is not a suitable substrate over which 8-CSB can spread which may be due to insufficient adhesiveness at the 4-CS or alterations during mitosis.

We have evaluated the distribution of mitochondria and acidic organelles using, respectively, the specific vital fluorescent dyes rhodamine 123 and acridine orange during preimplantation embryonic development in the mouse. Under conditions used to visualize organelles in living embryos, staining with either dye was found to have no effect on either the rate or extent of in vitro development of five- to eight-cell embryos up to the blastocyst stage. Mitochondria were randomly distributed throughout the cytoplasm and located around nuclei in blastomeres of uncompacted embryos. During compaction, mitochondria initially reorganized to the blastomere cortex; however, these organelles were later confined to the perinuclear region in the trophectoderm (TE) of expanded blastocysts. Acidic organelles were randomly distributed in the cytoplasm of uncompacted embryos, but following compaction, they were concentrated in cortical and perinuclear locations. Moreover, in TE cells of expanded blastocysts, acidic organelles were found exclusively in a tight perinuclear pattern. Microtubules and microfilaments in TE cells were localized in fixed embryos stained with antitubulin antibodies and rhodamine phalloidin, respectively; these structures were found primarily in the cortical cytoplasm at areas of cell-cell contact and secondarily in a perinuclear location. Thus mitochondria and acidic organelles undergo stage-specific redistributions from a diffuse or cortical pattern at the eight-cell stage to a tight perinuclear localization in the TE. We conclude that the polarized distributions of some organelles and cytoskeletal proteins during compaction may not be reliable permanent markers of the mature TE.

The mechanism of fluid transport in the developing preimplantation mouse embryo has been studiedin vitro by inhibiting zonular tight junction formation. Compaction, the morphogenetic process permitting zonular blastomere adhesions at the 8-cell stage, was suppressed by lowering extracellular calcium (Ca). The Ca threshold required for compaction is 0.04–0.06 mM, and in concentrations above the threshold, the rate of compaction is concentration dependent, whereas the rate of blastocyst formation is not and proceeds normally. At 0.02 mM Ca, both compaction and blastocyst development are completely prevented. Although focal tight and gap junctions are present, zonular tight junctions do not develop. We conclude that Ca is required for the maximization of cell-cell contact, but not for focal tight junction and gap junction formation. When early morulae are cultured in 0.02 mM Ca, small trophoblastic vesicles develop frequently with intracellular fluid vacuoles. If early 8-cell embryos are similarly cultured, cell division continues and many blastomeres acquire small intracellular membrane-bounded vaculoes. These coalesce, the cell volume increases, and the nucleus becomes eccentrically positioned, resulting in a giant vacuolated blastomere reminiscent of a miniaturized blastocyst. We propose that (1) vacuole formation may be an exaggeration of an intermediate intracellular step in fluid transport and (2) normal cell polarity established by zonular tight junctions is required for transcellular fluid transport.

Objective To determine the extent of biochemical changes in the zona pellucida (ZP) and cortical granule release in eggs failing to fertilize in IVF. Design After insemination, unactivated eggs without two pronuclei (PN) were studied by high resolution microscopy and fluorescent probes to determine cortical granule density and meiotic stage. After ZP isolation, proteins from individual ZPs were biotinylated, electrophoresed, and visualized by western blots with avidin-chemiluminescence. Controls included mouse unfertilized and fertilized eggs and human germinal vesicle stage oocytes and ≥ 3PN eggs. Setting University medical center and hospital tertiary care IVF-ET program. Results Many of the unfertilized eggs were in metaphase and had relatively low cortical granule densities indicative of cortical granule loss. Approximately one half of the ZPs showed evidence of biochemical hardening with a modification of a 90 to 100 × 103 molecular (weight) ratio (Mr) ZP protein. The 3PN eggs had few cortical granules and their ZPs had a pronounced 90 to 100 × 103 Mr modification that was not detected in germinal vesicle stage ZP controls. Conclusion The observed changes in ZP biochemistry and cortical granule quantitation demonstrate that failed fertilization is frequently associated with spontaneous cytoplasmic, but not nuclear, activation. In affected eggs, these changes could prevent fertilization in routine IVF and in cases of reinsemination. The relationship of changes in the ZP and cortical granules to infertility and in vitro culture requires further investigation. The 90 to 100 × 103 Mr ZP protein modification can be detected biochemically with 1 10 of a human ZP.