Recruitment Of Maternal mRNAs Research Articles

Biochemical alterations in the oocyte in support of early embryonic development.

Notwithstanding the enormous reproductive potential encapsulated within a mature mammalian oocyte, these cells present only a limited window for fertilization before defaulting to an apoptotic cascade known as post-ovulatory oocyte aging. The only cell with the capacity to rescue this potential is the fertilizing spermatozoon. Indeed, the union of these cells sets in train a remarkable series of events that endows the oocyte with the capacity to divide and differentiate into the trillions of cells that comprise a new individual. Traditional paradigms hold that, beyond the initial stimulation of fluctuating calcium (Ca2+) required for oocyte activation, the fertilizing spermatozoon plays limited additional roles in the early embryo. While this model has now been drawn into question in view of the recent discovery that spermatozoa deliver developmentally important classes of small noncoding RNAs and other epigenetic modulators to oocytes during fertilization, it is nevertheless apparent that the primary responsibility for oocyte activation rests with a modest store of maternally derived proteins and mRNA accumulated during oogenesis. It is, therefore, not surprising that widespread post-translational modifications, in particular phosphorylation, hold a central role in endowing these proteins with sufficient functional diversity to initiate embryonic development. Indeed, proteins targeted for such modifications have been linked to oocyte activation, recruitment of maternal mRNAs, DNA repair and resumption of the cell cycle. This review, therefore, seeks to explore the intimate relationship between Ca2+ release and the suite of molecular modifications that sweep through the oocyte to ensure the successful union of the parental germlines and ensure embryogenic fidelity.

P-Body Loss Is Concomitant with Formation of a Messenger RNA Storage Domain in Mouse Oocytes1

In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.

The γ isoform of CaM kinase II controls mouse egg activation by regulating cell cycle resumption

Fertilization triggers a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the egg that initiates a series of events known as egg activation. These events include cortical granule exocytosis that establishes a block to polyspermy, resumption of meiosis, and recruitment of maternal mRNAs into polysomes for translation. Several calcium-dependent proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII), have been implicated in egg activation. However, the precise role of CaMKII in mediating specific events of egg activation and the identity of the isoform(s) present in mouse eggs have not been unequivocally established. Through targeted deletion of the gamma isoform of CaMKII, we find that CaMKIIgamma is the predominant CaMKII isoform in mouse eggs and that it is essential for egg activation. Although CaMKIIgamma(-/-) eggs exhibit a normal pattern of Ca(2+) oscillations after insemination and undergo cortical granule exocytosis, they fail to resume meiosis or to recruit maternal mRNAs. Surprisingly, we find that the recruitment of maternal mRNAs does not directly depend on CaMKII, but requires elevated [Ca(2+)](i) and metaphase II exit. We conclude that CaMKIIgamma specifically controls mouse egg activation by regulating cell cycle resumption.

CaMKII can participate in but is not sufficient for the establishment of the membrane block to polyspermy in mouse eggs

Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).

Calmodulin-dependent protein kinase II triggers mouse egg activation and embryo development in the absence of Ca 2+ oscillations

Fertilization in mammalian eggs is accompanied by oscillatory changes in intracellular Ca 2+ concentration, which are critical for initiating and completing egg activation events and the developmental program. Ca 2+/Camodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is postulated to be the downstream transducer of the Ca 2+ signal in many cell types. We tested the hypothesis that CaMKII is the major integrator of Ca 2+-induced egg activation events and embryo development by microinjecting a cRNA that encodes a constitutively active (Ca 2+-independent) mutant form of CaMKII (CA-CaMKII) into mouse eggs. Expression of this cRNA, which does not increase intracellular Ca 2+, induced a sustained rise in CaMKII activity and triggered egg activation events, including cell cycle resumption, and degradation and recruitment of maternal mRNAs; cortical granule exocytosis, however, did not occur normally. Furthermore, when mouse eggs were injected with sperm devoid of Ca 2+-releasing activity and activated with either CA-CaMKII cRNA or by SrCl 2, similar rates and incidence of development to the blastocyst stage were observed. These results strongly suggest that CaMKII is a major integrator of the Ca 2+ changes that occur following fertilization.

Egg activation events are regulated by the duration of a sustained [Ca 2+] cyt signal in the mouse

Although the dynamics of oscillations of cytosolic Ca 2+ concentration ([Ca 2+] cyt) play important roles in early mammalian development, the impact of the duration when [Ca 2+] cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca 2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca 2+] cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca 2+] cyt change for 15, 25, or 50 min by means of repetitive Ca 2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca 2+] cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca 2+] 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. Ca 2+ influx into unfertilized eggs causes neither Ca 2+ release from intracellular stores nor rapid removal of cytosolic Ca 2+. Thus, the total Ca 2+ 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 [Ca 2+] cyt.

Egg-to-Embryo Transition Is Driven by Differential Responses to Ca2+ Oscillation Number

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.

Egg-to-Embryo Transition Is Driven by Differential Responses to Ca 2+ Oscillation Number

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.

In Vitro Culture Retards Spontaneous Activation of Cell Cycle Progression and Cortical Granule Exocytosis That Normally Occur in In Vivo UnfertilizedMouse Eggs1

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.

Evidence That GqFamily G Proteins Do Not Function in Mouse Egg Activation at Fertilization

Embryonic development is initiated after the fertilizing sperm contacts the egg and triggers a process termed “egg activation,” resulting in calcium release, cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. Heterotrimeric guanine nucleotide-binding proteins (G proteins) may be involved in mouse egg activation since inhibition of G protein βγ subunits partially inhibits sperm-induced cell cycle resumption. In addition, specific events of egg activation can be initiated in the absence of sperm by acetylcholine stimulation of mouse eggs overexpressing the human m1 muscarinic receptor, a G protein-coupled receptor. In somatic cells, G proteins in the Gqfamily couple ligand stimulation of the m1 muscarinic receptor to activation of phospholipase C, resulting in the production of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated release of intracellular calcium. Since IP3-mediated calcium release is involved in egg activation at fertilization, we have examined the role of Gqfamily G proteins in both sperm-independent (muscarinic receptor-mediated) and sperm-induced egg activation using a function-blocking antibody raised against the common C-terminal region of Gqand G11proteins. We show that this antibody effectively inhibits Gqfamily G proteins in mouse eggs by demonstrating that the antibody inhibits egg activation in response to stimulation of the m1 muscarinic receptor. This same antibody, however, does not inhibit sperm-induced egg activation events. These results indicate that although activation of Gqfamily G proteins can result in egg activation in the mouse, it is unlikely that these proteins are used by the sperm to initiate egg activation at fertilization.

Effects of Calcium-BAPTA Buffers and the Calmodulin Antagonist W-7 on Mouse Egg Activation

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 μM,recruitment of maternal mRNAs is only partially stimulated at injected free Ca2+concentrations of 2.5 μM,and no evidence for cell cycle resumption was observed (up to 2.5 μMCa2+). 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.

Complete Mouse Egg Activation in the Absence of Sperm by Stimulation of an Exogenous G Protein-Coupled Receptor

Sperm-induced egg activation has several features in common with signal transduction pathways mediated by ligand-receptor-effector systems. G proteins are postulated to act as signal transducers regulating the plethora of cellular responses constituting egg activation that occur in response to sperm. We report that acetylcholine treatment of mouse eggs expressing the human m1 muscarinic receptor, a G protein-coupled receptor, results in the full complement of events of egg activation, including ZP2 to ZP2f conversion, recruitment of maternal mRNAs, pronuclear formation with subsequent DNA synthesis, cleavage to the two-cell stage, and activation of the embryonic genome. The induction of a full complement of sperm-induced egg activation events by ACh in the absence of sperm strongly suggests that sperm-induced egg activation is mediated by egg G proteins.