Mammalian Egg Activation Research Articles

Sperm-Induced Ca2+ Release in Mammalian Eggs: The Roles of PLCζ, InsP3, and ATP.

Mammalian egg activation at fertilization is triggered by a long-lasting series of increases in cytosolic Ca2+ concentration. These Ca2+ oscillations are due to the production of InsP3 within the egg and the subsequent release of Ca2+ from the endoplasmic reticulum into the cytosol. The generation of InsP3 is initiated by the diffusion of sperm-specific phospholipase Czeta1 (PLCζ) into the egg after gamete fusion. PLCζ enables a positive feedback loop of InsP3 production and Ca2+ release which then stimulates further InsP3 production. Most cytosolic Ca2+ increases in eggs at fertilization involve a fast Ca2+ wave; however, due to the limited diffusion of InsP3, this means that InsP3 must be generated from an intracellular source rather than at the plasma membrane. All mammalian eggs studied generated Ca2+ oscillations in response to PLCζ, but the sensitivity of eggs to PLCζ and to some other stimuli varies between species. This is illustrated by the finding that incubation in Sr2+ medium stimulates Ca2+ oscillations in mouse and rat eggs but not eggs from other mammalian species. This difference appears to be due to the sensitivity of the type 1 InsP3 receptor (IP3R1). I suggest that ATP production from mitochondria modulates the sensitivity of the IP3R1 in a manner that could account for the differential sensitivity of eggs to stimuli that generate Ca2+ oscillations.

PMCA1 depletion in mouse eggs amplifies calcium signaling and impacts offspring growth†.

Egg activation in mammals is triggered by oscillations in egg intracellular calcium (Ca2+) level. Ca2+ oscillation patterns can be modified in vitro by changing the ionic composition of culture media or in vivo by conditions affecting mitochondrial function, such as obesity and inflammation. In mice, disruption of Ca2+ oscillations in vitro impacts embryo development and offspring growth. Here we tested the hypothesis that, even without in vitro manipulation, abnormal Ca2+ signaling following fertilization impacts offspring growth. Plasma membrane Ca2+ ATPases (PMCA) extrude cytosolic Ca2+ to restore Ca2+ homeostasis. To disrupt Ca2+ signaling in vivo, we conditionally deleted PMCA1 (cKO) in oocytes. As anticipated, in vitro fertilized cKO eggs had increased Ca2+ exposure relative to controls. To assess the impact on offspring growth, cKO females were mated to wild type males to generate pups that had high Ca2+ exposure at fertilization. Because these offspring would be heterozygous, we also tested the impact of global PMCA1 heterozygosity on offspring growth. Control heterozygous pups that had normal Ca2+ at fertilization were generated by mating wild type females to heterozygous males; these control offspring weighed significantly less than their wild type siblings. However, heterozygous offspring from cKO eggs (and high Ca2+ exposure) were larger than heterozygous controls at 12week-of-age and males had altered body composition. Our results show that global PMCA1 haploinsufficiency impacts growth and support that abnormal Ca2+ signaling after fertilization in vivo has a long-term impact on offspring weight. These findings are relevant for environmental and medical conditions affecting Ca2+ handling and for design of culture conditions and procedures for domestic animal and human assisted reproduction.

Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca2+) oscillations that induce egg activation and early embryonic development during mammalian fertilization. In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca2+ release via the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca2+ oscillations within mammalian eggs. Moreover, studies describing PLCζ “knockout” mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca2 + oscillations and egg activation in mammals, while also discussing the nature of a potential “alternative” sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.

The sperm phospholipase C-ζ and Ca2+ signalling at fertilization in mammals.

A series of intracellular oscillations in the free cytosolic Ca(2+) concentration is responsible for activating mammalian eggs at fertilization, thus initiating embryo development. It has been proposed that the sperm causes these Ca(2+) oscillations after membrane fusion by delivering a soluble protein into the egg cytoplasm. We previously identified sperm-specific phospholipase C (PLC)-ζ as a protein that can trigger the same pattern of Ca(2+) oscillations in eggs seen at fertilization. PLCζ appears to be the elusive sperm factor mediating egg activation in mammals. It has potential therapeutic use in infertility treatments to improve the rate of egg activation and early embryo development after intra-cytoplasmic sperm injection. A stable form of recombinant human PLCζ could be a prototype for use in such invitro fertilization (IVF) treatments. We do not yet understand exactly how PLCζ causes inositol 1,4,5-trisphosphate (InsP3) production in eggs. Sperm PLCζ is distinct among mammalian PI-specific PLCs in that it is far more potent in triggering Ca(2+) oscillations in eggs than other PLCs, but it lacks a PH domain that would otherwise be considered essential for binding to the phosphatidylinositol 4,5-bisphosphate (PIP2) substrate. PLCζ is also unusual in that it does not appear to interact with or hydrolyse plasma membrane PIP2. We consider how other regions of PLCζ may mediate its binding to PIP2 in eggs and how interaction of PLCζ with egg-specific factors could enable the hydrolysis of internal sources of PIP2.

Molecular characteristics of horse phospholipase C zeta (PLCζ)

A sperm-specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca(2+) ]i ) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT-PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long-lasting [Ca(2+) ]i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca(2+) ]i oscillation-inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.

Calcium signaling in mammalian egg activation and embryo development: The influence of subcellular localization

Calcium (Ca(2+) ) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca(2+) signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca(2+) sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca(2+) in many cell types and of the impact of cellular localization on Ca(2+) signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca(2+) is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca(2+) release and effectors of Ca(2+) signals. We then summarize studies exploring how Ca(2+) directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca(2+) signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe for future research.

Identification and functional analysis of an ovarian form of the egg activation factor phospholipase C zeta (PLCζ) in pufferfish

Recent studies suggest that egg activation in mammals is triggered by a sperm-specific phospholipase C, PLCzeta. In other vertebrate species such as medaka fish, chickens, and quail, PLCzeta is also expressed as a testis-specific mRNA. Functional studies suggest that PLCzeta plays a similar role as a trigger of egg activation in these species. Here, we report the identification of PLCzeta orthologues in pufferfish species Takifugu rubripes (Fugu) and Tetraodon nigroviridis (Tetraodon). Unexpectedly in these species PLCzeta is expressed not in the testis, but in ovary and brain. Injection of pufferfish PLCzeta copy ribonucleic acid (cRNA) into mouse eggs failed to trigger calcium oscillations, unlike medaka PLCzeta cRNA. Our findings provide the first evidence that PLCzeta may be expressed in the egg, rather than the sperm, in some vertebrate species, and that its mechanism of action and physiologic role at fertilization may differ in different vertebrate species.

Molecular mechanisms of sperm-egg interactions and egg activation.

The binding of acrosome reacted mammalian sperm to the egg plasma membrane initiates a series of signaling events in the egg, termed "egg activation", which lead to the completion of meiosis II and the initiation of a mitotic cell cycle. Many of these signaling events have characteristics of classical signal transduction events in somatic cells. Currently, there are two hypotheses for how sperm-induced egg activation is initiated. In the "receptor" hypothesis, the fertilizing sperm interacts with a specific egg surface receptor, and this interaction leads to signal transduction and effector activation. In the "fusion" hypothesis it is postulated that following fusion of the sperm and egg plasma membranes a soluble sperm-derived factor enters the egg's cytoplasm and activates pathways leading to egg activation. This chapter will provide an overview of the processes of cell-cell interaction and signal transduction leading to mammalian egg activation. It will concentrate on specific molecules proposed to be involved in sperm-egg interaction, signal transduction and effector mechanisms involved in egg activation, and a discussion of sperm-associated factors that have been implicated in egg activation.

PLCζ(zeta): A sperm protein that triggers Ca2+ oscillations and egg activation in mammals

At fertilization in mammals, the sperm activates development by causing a prolonged series of intracellular Ca(2+) oscillations that are generated by increased production of inositol trisphosphate (InsP(3)). It appears that the sperm initiates InsP(3) generation via the introduction of a sperm factor into the egg after gamete membrane fusion. We recently identified a sperm-specific form of phospholipase C (PLC), referred to as PLCzeta(zeta). We review the evidence that PLCzeta represents the sperm factor that activates development of the egg and discuss the characteristics of PLCzeta that distinguish it from the somatic forms of PLC.

Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis

To use in vivo gene transfer into the testis by electroporation to express a fluorescent recombinant form of a testis-specific gene in the mature epididymal sperm of mice and thus study the pattern of gene localization. Controlled animal study. Research laboratory at the University of Oxford. Four- to 6-week-old male mice. Phospholipase C zeta (PLCzeta), the putative mammalian egg activation factor, was fused to enhanced yellow fluorescent protein (EYFP), and in vivo gene transfer by electroporation was used to introduce this transgene (PLCzeta-EYFP) into mouse testis. Transgene expression in testis and sperm were analyzed at 20 and 40 days after electroporation. Transgene expression in testis and epididymal sperm was analyzed by fluorescence microscopy and an excitation light source suitable for EYFP. Phospholipase C zeta-EYFP was successfully expressed in epididymal sperm when analyzed 40 days after gene transfer and was localized to the head and midpiece regions. Our results provide the first demonstration that in vivo gene transfer can be used to study the localization of proteins in mature sperm and that this represents a powerful new technique for studying male infertility and gene function in sperm.

Egg activation is the result of calcium signal summation in the mouse

Egg activation in mammals is caused by cytosolic Ca(2+) oscillations that are essential for development. However, despite increasing knowledge about signal transduction mechanisms, the functional linkage between frequency number, amplitude and duration of the Ca(2+) signal and the kinetics of pronucleus formation has not yet been defined. While a wide range of Ca(2+) signal parameters are efficient in causing egg activation, the basic rules governing how the egg integrates these signalling events are not yet clear. Thus, in the perspective of better understanding how the egg processes Ca(2+) signalling events, the objective of this study was to determine experimentally whether the efficiency of egg activation and the subsequent early developmental stages rely on Ca(2+) signalling summation. Non-fertilized, but freshly ovulated mouse eggs, were subjected to a series of repetitive Ca(2+) influxes of various patterns modulated by a non-invasive membrane electropermeabilization method. Using a combination of two suboptimal treatments we have shown that mouse eggs can sum up the effects caused by various patterns of intracellular Ca(2+) concentrations transient during the period of egg activation. In addition, overloading the intracellular milieu by repetitive Ca(2+) influxes did not seem to inhibit the process of activation. The kinetics of pronuclear formation among a population of eggs treated in the same conditions became accelerated when the total dose of Ca(2+) signal 'experienced' by the eggs was increased. The results suggested that summation of the biological effects of all Ca(2+) signals constitutes an important mode of Ca(2+) signal integration.

Mammalian egg activation: from Ca2+ spiking to cell cycle progression

Mammalian eggs arrest at metaphase of the second meiotic division (MetII). Sperm break this arrest by inducing a series of Ca(2+) spikes that last for several hours. During this time cell cycle resumption is induced, sister chromatids undergo anaphase and the second polar body is extruded. This is followed by decondensation of the chromatin and the formation of pronuclei. Ca(2+) spiking is both the necessary and solely sufficient sperm signal to induce full egg activation. How MetII arrest is established, how the Ca(2+) spiking is induced and how the signal is transduced into cell cycle resumption are the topics of this review. Although the roles of most components of the signal transduction pathway remain to be fully investigated, here I present a model in which a sperm-specific phospholipase C (PLCzeta) generates Ca(2+) spikes to activate calmodulin-dependent protein kinase II and so switch on the Anaphase-Promoting Complex/Cyclosome (APC/C). APC/C activation leads to securin and cyclin B1 degradation and in so doing allows sister chromatids to be segregated and to decondense.

Expression and possible involvement of calpain isoforms in mammalian egg activation

At fertilization in mammals, the spermatozoon triggers a unique signal transduction mechanism within the egg, leading to its activation. It is well accepted that the earliest event observed in all activated eggs is an abrupt rise in intracellular calcium concentrations. However, little is known regarding the downstream proteins that are activated by this rise in calcium. Calpains constitute a family of intracellular calcium-dependent cysteine proteases whose members are expressed widely in a variety of cells. We investigated the expression and possible role of the calpain isoforms mu and m throughout egg activation. Both calpains were expressed in the rat egg and localized at the egg cortex as well as in the meiotic spindle. m Calpain translocated to the membrane and to the spindle area during parthenogenetic egg activation and during in vivo fertilization, upon sperm binding to the egg. The cytoskeletal protein alpha-spectrin (fodrin) was proteolysed by calpain during the egg-activation process, as demonstrated by specific calpain-breakdown products. Following parthenogenetic activation by ionomycin or puromycin, the calpain-selective permeable inhibitor, calpeptin, inhibited the resumption of meiosis and cortical reaction in a dose-dependent manner. Calpeptin was also effective in inhibiting in vitro fertilization. These results may imply a correlation between calpain activation and mammalian egg activation at fertilization and a possible role for calpain in the cascade of cellular events leading to resumption of meiosis.

Phospholipase Cζ, the trigger of egg activation in mammals, is present in a non-mammalian species

The activation of the egg to begin development into an embryo is triggered by a sperm-induced increase in intracellular egg Ca2+. There has been much controversy about how the sperm induces this fundamental developmental event, but recent studies suggest that, in mammals, egg activation is triggered by a testis-specific phospholipase C: PLCzeta. Since the discovery of PLCzeta, it has been unclear whether its role in triggering egg activation is common to all vertebrates, or is confined to mammals. Here, we demonstrate for the first time that PLCzeta is present in a non-mammalian vertebrate. Using genomic and cDNA databases, we have identified the cDNA encoding a PLCzeta orthologue in the domestic chicken that, like the mammalian isoforms, is a testis-specific gene. The chicken PLCzeta cDNA is 2152 bp in size and encodes an open reading frame of 639 amino acids. When injected into mouse oocytes, chicken PLCzeta cRNA triggers Ca2+ oscillations, indicating that it has functional properties similar to those of mammalian PLCzeta. Our findings suggest that PLCzeta may have a universal role in triggering egg activation in vertebrates.

The cytosolic sperm factor that triggers Ca2+ oscillations and egg activation in mammals is a novel phospholipase C: PLCζ

When sperm activate eggs at fertilization the signal for activation involves increases in the intracellular free Ca2+ concentration. In mammals the Ca2+ changes at fertilization consist of intracellular Ca2+ oscillations that are driven by the generation of inositol 1,4,5-trisphosphate (InsP3). It is not established how sperm trigger the increases in InsP3 and Ca2+ at fertilization. One theory suggests that sperm initiate signals to activate the egg by introducing a specific factor into the egg cytoplasm after membrane fusion. This theory has been mainly based upon the observation that injecting a cytosolic sperm protein factor into eggs can trigger the same pattern of Ca2+ oscillations induced by the sperm. We have recently shown that this soluble sperm factor protein is a novel form of phospholipase C (PLC), and it is referred to as PLCzeta(zeta). We describe the evidence that led to the identification of PLCzeta and discuss the issues relating to its potential role in fertilization.

299OOCYTE ACTIVATION IS THE RESULT OF CALCIUM SIGNAL SUMMATION

Egg activation in mammals is caused by a series of cytosolic Ca2+ oscillations that are essential for development. However, the specific functional significance of the number or the frequency of the Ca2+ signals is not clear. The objective of this study was to determine whether the efficiency of egg activation relies on the frequency or on the number of signals. Given the repetitive nature of the signal, it is nearly impossible to discriminate the specific effect of the frequency from the number because increasing the time interval between signals directly reduces their number in a given time unit and vice versa. To bypass such difficulty, we subjected non-fertilized mouse oocytes, which are not capable of releasing Ca2+ from intracellular stores, to a coherent series of four treatments. Repetitive intracellular Ca2+ signals were introduced into freshly ovulated mouse oocytes by electropermeabilisation of Ca2+ ions by means of a microfluidic processor . The first regime (T1) was a monotonic [Ca2+]i increase lasting about 10min and provoked by 4 consecutive electrical pulses (1.45kVcm−1) given at 2-min intervals. This large calcium signal gave a poor activation rate: 6% (4/67). The second regime (T2) was made up of six lower pulses (1.12kVcm−1) given at 30-min intervals. The rate of egg activation reached 38% (75/195). The third treatment (T3) consisted in subjecting oocytes consecutively to the T1 and the T2 treatments. In that case, the egg activation reached 88% (121/138). The remarkable increase in egg activation efficiency can be attributed to the increase in the total [Ca2+]i load, which is the only change in T3. Thus, it appears that the large monotonic increase (T1) potentiates the response as revealed by the periodic stimulation delivered by T2. It therefore seems that the process downstream of the signal is incremental in nature, and entrance in interphase of the cell cycle is reached when the summation of individual signals reaches a sufficient level. We next evaluated whether the putative summation effect makes it possible to regularly get 100% of egg activation by increasing the cytosolic Ca2+load. Oocytes were subjected to a last treatment (T4) that consisted of T1 and then 24 pulses (1.45kVcm−1) given every 8min. In that case 100% of the oocytes were activated (80/80). Thus, it appears that all oocytes are subject to egg activation if they receive a sufficient amount of calcium signal. Moreover, the frequency does not seem to play a crucial role for entrance into interphase because reducing the time interval from 30min (T3) to 8min (T4) resulted in high activation rates in both cases (88% and 100%, respectively). The question remains as to whether the distribution of the Ca2+ signal throughout the process of egg activation might affect some epigenetic modifications that impact developmental processes.

Signalling in mammalian egg activation: role of protein kinases

Embryonic development is initiated after the fertilizing spermatozoon enters the egg and triggers a process known as ‘egg activation’. Activation results in an increase in intracellular calcium concentration, cortical granule exocytosis (CGE), cell cycle resumption and recruitment of maternal mRNA. Various treatments can induce parthenogenetic activation characterized by the same manifestations. Signal transduction pathways similar to those known for somatic cells mediate the mammalian egg activation. This review focuses on the signal transduction pathways that occur during mammalian fertilization and during parthenogenetic egg activation. We discuss the possibility that members of the protein tyrosine kinase (PTKs) families, the Src family PTKs in particular, operate during egg activation and that protein kinase C can induce CGE.

Biochemical and biological effects of KN-93, an inhibitor of calmodulin-dependent protein kinase II, on the initial events of mouse egg activation induced by ethanol.

Calmodulin-dependent protein kinase II (CaMKII) is transiently activated in mouse eggs by the increase in calcium that occurs upon activation with ethanol. This study investigated the biological and biochemical effects of KN-93, a reported selective inhibitor of CaMKII, to explore the potential role of this kinase in the initial events of egg activation. Mouse eggs were incubated for 30 min in the presence of different concentrations of KN-93 and induced to activate by 7% ethanol. KN-93 elicited a dose-dependent inhibition of polar body emission that resulted from the failure of the eggs to undergo meiosis resumption and inactivation of maturation-promoting factor (MPF). Furthermore, 15 mumol KN-93 l-1 produced a marked reduction in ethanol-induced loss of cortical granules. In vivo biochemical analysis revealed that 15 mumol KN-93 l-1 was responsible for significant inhibition of ethanol-stimulated CaMKII. The activity of the enzyme remained at a resting value, in spite of the presence of a calcium signal similar to that measured in control activated eggs. The inhibitory effects of KN-93 on the parameters tested in this study could not be mimicked by the inactive analogue KN-92. These results show that in mouse eggs, when ethanol-induced CaMKII activation was prevented, cortical granule exocytosis and meiosis resumption were inhibited. This suggests that CaMKII acts as a switch in the transduction of the calcium signal triggering mammalian egg activation.

Early events in mammalian egg activation.

The fertilizing spermatozoon initiates a series of events in the mammalian egg, referred to as 'egg activation'. These biochemical and morphological events include a transient rise in intracellular Ca2+ concentration ([Ca2+]i) that leads to the cortical reaction (CR) and the establishment of the block to polyspermy on the one hand, and to the resumption of meiosis and later embryonic mitotic divisions on the other. The initial increase in [Ca2+]i appears to be critical for the initiation of egg activation. However, another second messenger, protein kinase C (PKC), was also suggested as a possible inducer of some aspects of egg activation. The review focuses on early events during mammalian sperm-egg interaction and discusses possible roles of Ca2+ and PKC in egg activation.

Profile of protein kinase C isozymes and their possible role in mammalian egg activation

Western blot analysis was used to investigate protein kinase C (PKC) profile of rat eggs. The presence of eight PKC isozymes was demonstrated: conventional PKC α, β and γ; novel PKC δ, ϵ and μ; atypical PKC ζ and λ. PKC α was detected by RT-PCR as well. PKC translocation from the cytosol to the plasma membrane served as a marker for enzyme activation. Immunofluorescence confocal microscopy demonstrated a relatively uniform distribution of PKC α, βI, and βII throughout the cytosol of metaphase II arrested eggs. PKC accumulation at the plasma membrane was detected 5 min after exposure to 12- O-tetradecanoyl phorbol-13-acetate and increased with time, thus demonstrating activation of these PKCs.