Introduction. Calcium signals and developmental patterning

Calcium ions generate ubiquitous cellular signals. Calcium signals play an important role in development. The most obvious example is fertilization, where calcium signals and calcium waves are triggered by the sperm and are responsible for activating the egg from dormancy and cell cycle arrest. Calcium signals also appear to contribute to cell cycle progression during the rapid cell cycles of early embryos. There is increasing evidence that calcium signals are an essential component of the signalling systems that specify developmental patterning and cell fate. This issue arises from a Discussion Meeting that brought together developmental biologists studying calcium signals with those looking at other patterning signals and events. This short introduction provides some background to the papers in this issue, setting out the emerging view that calcium signals are central to dorsoventral axis formation, gastrulation movements, neural specification and neuronal cell fate.     

Could modifications of signalling pathways activated after ICSI induce a potential risk of epigenetic defects?

A calcium signal during oocyte or egg activation is a conserved event in virtually all species analyzed so far. This signal, that is in the form of calcium oscillations in mammals, is spatially and temporally controlled and is mainly supported by calcium release from internal calcium stores, but how it is triggered after fertilization is far from understood. The sperm factor hypothesis of egg activation postulates that sperm delivers a calcium-releasing factor into the egg following sperm-egg fusion. Among the many potential sperm factors, PLCzeta is the strongest bona fide sperm factor candidate. However, how sperm-oocyte fusion occurs prior to PLCzeta delivery and oocyte activation is not entirely known. We propose in the first part of this review the possibility that other pathways such as those involving G-proteins, tyrosine kinases or integrins could be activated besides sperm factor injection and could be upstream mechanisms involved in later embryonic development. Among different assisted reproductive technologies (ARTs), intracytoplasmic sperm injection (ICSI) is considered as the best and easiest therapeutic technique to circumvent severe male infertility. Although most reports are reassuring, some recent data suggest a greater incidence of abnormalities in children conceived by ART compared with those conceived normally. Spatio-temporal signals may be missing or abnormal during ICSI, perhaps because membrane fusion and signalling events are bypassed. We discuss in the second part of this review the hypothesis that potential perturbations during the ICSI procedure may have repercussions on epigenetic processes, inducing not only alterations of embryonic development, but also diseases in young children and, perhaps, in adults.     

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.     

Egg activation: upstream of the fertilization calcium signal

Interaction of sperm and egg at fertilization induces well-coordinated molecular events including specific recognition between species, adhesion and fusion, that lead to the formation of a zygote, a totipotent cell that develops into a new individual. A calcium signal, common to a great number of species, from marine invertebrates to mammals, is essential to activate the metabolism of the unfertilized oocyte. However, how fertilization triggers this calcium signal and initiates development of the early embryo is far from understood. The signaling pathways activated in eggs may be similar to those described in somatic cells, since changes in intracellular free calcium and in mitosis activating protein (MAP) kinase activity occur in both systems after activation. Several hypotheses are currently proposed, implying a spermatic ligand binding to a specific receptor expressed at the egg surface, or where the fused sperm either allows the transit of external calcium into the egg or injects one (or several) activating factor(s). It is still not known which of these ideas is true. We concentrate in this review on the possible signaling pathways involving IP3 (inositol trisphosphate), since its production is involved in most species to generate the fertilization calcium wave.     

Calcium waves and oscillations in eggs

Eggs from several protostomes (molluscs, annelids, nemerteans, etc.) and two deuterostomes (mammals and ascidians) display repetitive calcium signals. Oscillations in the level of intracellular calcium concentration are occasionally triggered by maturing hormones (as in some molluscs) and mostly observed after fertilization which occurs at different stages of the meiotic cell cycle (oocytes are arrested in prophase, metaphase I or metaphase II). In most eggs examined so far, calcium oscillations last until the end of meiosis just before male and female pronuclei form. This ability depends on the sensitivity of InsP3 channels and on the permeability of the plasma membrane to extracellular calcium. In eggs that undergo cytoplasmic reorganization at fertilization (annelids, nemerteans, ascidians, etc.) the repetitive calcium signals are waves that originate from localized cortical sites that become calcium waves pacemakers. In ascidians we have identified the site of initiation of repetitive calcium waves as an accumulation of endoplasmic reticulum sandwiched between the plasma membrane and an accumulation of mitochondria. We compare and discuss the generation of calcium signals in the different eggs, their relationship with the cell cycle and the possible roles they play during development.     

On the transition from the meiotic to mitotic cell cycle during early mouse development

Here, we outline the mechanisms involved in the regulation of cell divisions during oocyte maturation and early cleavages of the mouse embryo. Our interest is focused on the regulation of meiotic M-phases and the first embryonic mitoses that are differently tuned and are characterized by specifically modified mechanisms, some of which have been recently identified. The transitions between the M-phases during this period of development, as well as associated changes in their regulation, are of key importance for both the meiotic maturation of oocytes and the further development of the mammalian embryo. The mouse is an excellent model for studies of the cell cycle during oogenesis and early development. Nevertheless, a number of molecular mechanisms described here were discovered or confirmed during the study of other species and apply also to other mammals including humans.     

早期胚胎发育合子基因组激活调控

动物早期胚胎发育始于分化成熟的雌雄配子经受精后重编程为全能性合子。在胚胎发育的初期,合子基因组的转录水平处于静默状态,母源物质调控占据主导地位。随着胚胎发育的进行,母源物质会经历分阶段的降解,合子基因组开始逐渐激活转录,标志着早期胚胎发育从母源性调控向合子基因组调控的转变,也称为母源-合子转换（maternal-zygotic transition,MZT）。其中一个关键的转折性事件就是合子基因组激活（zygotic genome activation,ZGA）,ZGA的正确发生对于早期胚胎发育和细胞命运决定至关重要。然而,目前对于ZGA的调控因子和具体的分子机制仍知之甚少。研究表明,ZGA在不同物种中存在较大差异,可能受到DNA甲基化、组蛋白修饰、非编码RNA、染色质重塑以及ZGA相关因子等多种调控因素的影响。本文探讨了上述几种调控因素影响合子基因组激活的研究进展,对进一步研究早期胚胎ZGA的相关机制具有借鉴意义。     

From oogenesis through gastrulation: developmental regulation of apoptosis

Apoptosis is a mechanism employed by multicellular organisms throughout development as a means of eliminating damaged or otherwise unwanted cells. From oogenesis through fertilization and gastrulation, organisms use an array of cell- and tissue-specific mechanisms to regulate the apoptotic program in response to stress or developmental cues. Since cell death regulation is tightly interwoven with cell cycle and checkpoint controls, and embryos of the fly, fish and frog exhibit unique embryonic cell cycle regulation, it is of great interest to understand how early embryos coordinate these cellular functions.     

TBX5 is required for embryonic cardiac cell cycle progression

Despite the critical importance of TBX5 in normal development and disease, relatively little is known about the mechanisms by which TBX5 functions in the embryonic heart. Our present studies demonstrate that TBX5 is necessary to control the length of the embryonic cardiac cell cycle, with depletion of TBX5 leading to cardiac cell cycle arrest in late G(1)- or early S-phase. Blocking cell cycle progression by TBX5 depletion leads to a decrease in cardiac cell number, an alteration in the timing of the cardiac differentiation program, defects in cardiac sarcomere formation, and ultimately, to cardiac programmed cell death. In these studies we have also established that terminally differentiated cardiomyocytes retain the capacity to undergo cell division. We further show that TBX5 is sufficient to determine the length of the embryonic cardiac cell cycle and the timing of the cardiac differentiation program. Thus, these studies establish a role for TBX5 in regulating the progression of the cardiac cell cycle.     

The biology and dynamics of mammalian cortical granules

Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals.     

Fertilization stimulates long-lasting oscillations of CaMKII activity in mouse eggs

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 microM), 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.     

A C-type lectin associated and translocated with cortical granules during oocyte maturation and egg fertilization in fish

Oocyte maturation and egg fertilization in both vertebrates and invertebrates are marked by orchestrated cytoplasmic translocation of secretory vesicles known as cortical granules. It is thought that such redistribution of cellular content is critical for asymmetrical cell division during early development, but the mechanism and regulation of the process is poorly understood. Here we report the identification, purification and cDNA cloning of a C-type lectin from oocytes of a freshwater fish species gibel carp (Carassius auratus gibelio). The purified protein has been demonstrated to have lectin activity and to be a Ca(2+)-dependent C-type lectin by hemagglutination activity assay. Immunocytochemistry revealed that the lectin is associated with cortical granules, gradually translocated to the cell surface during oocyte maturation, and discharged to the egg envelope upon fertilization. Interestingly, the lectin becomes phosphorylated on threonine residues upon induction of exocytosis by fertilization and returns to its original state after morula stage of embryonic development, suggesting that this posttranslational modification may represent a critical molecular switch for early embryonic development.     

A ménage à trois made in heaven: G-protein-coupled receptors, lipids and TRP channels

Recent technical and conceptual advances in lipid analysis have given us a glimpse into the true versatility of the lipidome and the complexity of lipid signaling species. Progress alike in protein chemistry and genetics has presented us with new signal pathways and molecular mechanisms for the lipid actions. G-protein-coupled receptors (GPCR) appear to play a central role in the regulation of many lipid signals and are also themselves targets for some of these. TRP channels have recently been acknowledged as one of the most important GPCR effectors; in many cases the signals from GPCRs to TRPs are mediated via lipid signals. This review aims at presenting a view into the complex lipid signaling networks, their possible regulation by GPCRs and the signals transmitted to the TRP channels. Critical views and possible shortcomings in the composition of the studies are also presented.     

Roles of reactive oxygen species and antioxidants in ovarian toxicity

Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.     

Comparative biology of calcium signaling during fertilization and egg activation in animals.

During animal fertilizations, each oocyte or egg must produce a proper intracellular calcium signal for development to proceed normally. As a supplement to recent synopses of fertilization-induced calcium responses in mammals, this paper reviews the spatiotemporal properties of calcium signaling during fertilization and egg activation in marine invertebrates and compares these patterns with what has been reported for other animals. Based on the current database, fertilization causes most oocytes or eggs to generate multiple wavelike calcium oscillations that arise at least in part from the release of internal calcium stores sensitive to inositol 1,4,5-trisphosphate (IP3). Such calcium waves are modulated by upstream pathways involving oolemmal receptors and/or soluble sperm factors and in turn regulate calcium-sensitive targets required for subsequent development. Both "protostome" animals (e.g., mollusks, annelids, and arthropods) and "deuterostomes" (e.g., echinoderms and chordates) display fertilization-induced calcium waves, IP3-mediated calcium signaling, and the ability to use a combination of external calcium influx and internal calcium release. Such findings fail to support the dichotomy in calcium signaling modes that had previously been proposed for protostomes vs deuterostomes and instead suggest that various features of fertilization-induced calcium signals are widely shared throughout the animal kingdom.