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Ca2+ influx during oocyte maturation and after sperm entry is necessary to fill the internal Ca2+ stores and for complete egg activation. We knocked out the transient receptor potential vanilloid member 3 (TRPV3) and the T-type channel, CaV3.2, to determine their necessity for maintaining these functions in mammalian oocytes/eggs. Double-knockout (dKO) females were subfertile, their oocytes and eggs showed reduced internal Ca2+ stores, and, following sperm entry or Plcz (also known as Plcz1) cRNA injection, fewer dKO eggs displayed Ca2+ responses compared to wild-type eggs, which were also of lower frequency. These parameters were rescued and/or enhanced by removing extracellular Mg2+, suggesting that the residual Ca2+ influx could be mediated by the TRPM7 channel, consistent with the termination of divalent-cation oscillations in dKO eggs by a TRPM7 inhibitor. In total, we demonstrated that TRPV3 and CaV3.2 mediate the complete filling of the Ca2+ stores in mouse oocytes and eggs. We also showed that they are required for initiating and maintaining regularly spaced-out oscillations, suggesting that Ca2+ influx through PM ion channels dictates the periodicity and persistence of Ca2+ oscillations during mammalian fertilization.

Compensatory endocytosis (CE) is one of the primary mechanisms through whichcells maintain their surface area after exocytosis. Considering that in eggs massiveexocytosis of cortical granules (CG) takes place after fertilization, the aim of this was to evaluate the occurrence of CE following cortical exocytosis in mouseeggs. For this purpose, we developed a pulse‐chase assay to detect CG membraneinternalization. Results showed internalized labeling in SrCl2‐activated and fertilized eggs when chasing at 37°C, but not at a nonpermissive temperature (4°C). The use of kinase and calcineurin inhibitors led us to conclude that this internal labeling corresponded to CE. Further experiments showed that CE in mouse eggs isdependent on actin dynamics and dynamin activity, and could be associated with atransient exposure of phosphatidylserine. Finally, CE was impaired in A23187ionophore‐activated eggs, highlighting once again the mechanistic differencesbetween the activation methods. Altogether, these results demonstrate for the firsttime that egg activation triggers CE in mouse eggs after exocytosis of CG, probably as a plasma membrane homeostasis mechanism. Fil: Gómez Elías, Matías Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Fissore, Rafael A.. University of Massachussets; Estados Unidos Fil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Ca(2+) oscillations and consequent Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation are required for embryogenesis, as well as neuronal, immunological, and cardiac signaling. Fertilization directly results in Ca(2+) oscillations, but the resultant pattern of CaMKII activity remains largely unclear. To address this gap, we first employed the one existing biosensor for CaMKII activation. This sensor, Camui, comprises CaMKIIα and therefore solely reports on the activation of this CaMKII variant. Additionally, to detect the activity of all endogenous CaMKII variants simultaneously, we constructed a substrate-based sensor for CaMKII activity, FRESCA (FRET-based sensor for CaMKII activity). To examine the differential responses of the Camui and FRESCA sensors, we used several approaches to stimulate Ca(2+) release in mouse eggs, including addition of phospholipase Cζ cRNA, which mimics natural fertilization. We found that the Camui response is delayed or terminates earlier than the FRESCA response. FRESCA enables assessment of endogenous CaMKII activity in real-time by both fertilization and artificial reagents, such as Sr(2+), which also leads to CaMKII activation. FRESCA's broad utility will be important for optimizing artificial CaMKII activation for clinical use to manage infertility. Moreover, FRESCA provides a new view on CaMKII activity, and its application in additional biological systems may reveal new signaling paradigms in eggs, as well as in neurons, cardiomyocytes, immune cells, and other CaMKII-expressing cells.

In mammals, fertilization initiates Ca(2+) oscillations in metaphase II oocytes, which are required for the activation of embryo development. Germinal vesicle (GV) oocytes also display Ca(2+) oscillations, although these unfold spontaneously in the absence of any known agonist(s) and their function remains unclear. We found that the main intracellular store of Ca(2+) in GV oocytes, the endoplasmic reticulum ([Ca(2+)](ER)), constitutively ‘leaks’ Ca(2+) through the type 1 inositol 1,4,5-trisphosphate receptor. The [Ca(2+)](ER) leak ceases around the resumption of meiosis, the GV breakdown (GVBD) stage, which coincides with the first noticeable accumulation of Ca(2+) in the stores. It also concurs with downregulation of the Ca(2+) influx and termination of the oscillations, which seemed underpinned by the inactivation of the putative plasma membrane Ca(2+) channels. Lastly, we demonstrate that mitochondria take up Ca(2+) during the Ca(2+) oscillations, mounting their own oscillations that stimulate the mitochondrial redox state and increase the ATP levels of GV oocytes. These distinct features of Ca(2+) homeostasis in GV oocytes are likely to underpin the acquisition of both maturation and developmental competence, as well as fulfill stage-specific cellular functions during oocyte maturation.

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca(2+), referred to as Ca(2+) oscillations. Maintenance of these oscillations requires Ca(2+) influx across the plasma membrane, which is mediated in part by T-type, Ca(V)3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca(2+) influx. Eggs lacking both TRPM7 and Ca(V)3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca(2+) influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca(2+) oscillations, which appears to require sperm–egg fusion. TRPM7 and Ca(V)3.2 channels almost completely account for Ca(2+) influx observed following store depletion, a process previously attributed to canonical store-operated Ca(2+) entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg(2+) and Ca(2+) concentrations and mediates the effects of these ions on Ca(2+) oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and Ca(V)3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca(2+) oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and Ca(V)3.2 as key mediators of Ca(2+) influx following fertilization provides a mechanistic basis for the rational design of culture media that optimize developmental potential in research animals, domestic animals, and humans.

More than 15 years have elapsed since the identification of phospholipase C ζ1 (PLCζ) from a genomic search for mouse testis/sperm-specific PLCs. This molecule was proposed to represent the sperm factor responsible for the initiation of calcium (Ca2+ ) oscillations required for egg activation and embryo development in mammals. Supporting evidence for this role emerged from studies documenting its expression in all mammals and other vertebrate species, the physiological Ca2+ rises induced by injection of its messenger RNA into mammalian and nonmammalian eggs, and the lack of expression in infertile males that fail intracytoplasmic sperm injection. In the last year, genetic animal models have added support to its role as the long sought-after sperm factor. In this review, we highlight the findings that demonstrated the role of Ca2+ as the universal signal of egg activation and the experimental buildup that culminated with the identification of PLCζ as the soluble sperm factor. We also discuss the structural-functional properties that make PLCζ especially suited to evoke oscillations in eggs. Lastly, we examine unresolved aspects of the function and regulation of PLCζ and whether or not it is the only sperm factor in mammalian sperm.

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whetherin vitromaturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI,in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, althoughin vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation byin vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu ofin vitro-matured oocytes and to replicate the molecular changes associated within vivocapacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospho- lipase C, zeta (PLCz), the sperm factor thought to induce the Ca 2+ oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCz in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCz and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCz is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca 2+ oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCz in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equa- torial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCz in globozoospermic sperm is thus con- sistent and reinforces the role of PLCz as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2-globozoospermic sperm and the compromised developmental potential of embryos obtained using sperm from patients with a deletion of the DPY19L2 gene.

This study was conducted to determine if expression of the testis-specific phospholipase C Zeta1 (PLCZ1) correlated with low success or fertilization failure after ICSI in patients with normal parameters after standard semen analysis (SA).Couples43 years with one or two failed or low fertilization ICSI cycles. Standard Semen Analysis (SA) was performed to determine sperm parameters in male partners, whereas females were evaluated for antral follicle counts (AFC), day 3 FSH levels and peak Estradiol (E2) levels. The presence of PLCZ1 in sperm was ascertained using Western blotting and Immunofluorescence (IF) analysis. The ability of sperm to initiate changes in the intracellular concentrations of free calcium ([Ca(2+)]i), which is characteristic of mammalian sperm, was performed after injection of human sperm into mouse eggs loaded with the Ca(2+) sensitive dye fura-2 AM.Male partners of couples with failed or low success ICSI fertilization but with normal SA parameters showed low expression levels of PLCZ1 as determined by western blotting and reduced fluorescent signal during IF studies. In addition, fewer of these males' sperm showed PLCZ1 expression and were able to initiate robust [Ca(2+)]i oscillations upon injection into eggs.Our data suggest that in patients with normal SA parameters but with repeated low fertilization or outright failed fertilization results after ICSI, abnormal PLCZ1 function should be considered as the underlying mechanism responsible for the failure of fertilization.

SummaryIn mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a transient receptor potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, because TrpV3−/− eggs failed to conduct Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation.

The activation of the developmental program in mammalian eggs relies on the initiation at the time of fertilization of repeated rises in the intracellular concentration of free calcium ([Ca(2+)](i)), also known as [Ca(2+)](i) oscillations. The ability to mount the full complement of oscillations is only achieved at the end of oocyte maturation, at the metaphase stage of meiosis II (MII). Over the last decades research has focused on addressing the mechanisms by which the sperm initiates the oscillations and identification of the channels that mediate intracellular Ca(2+) release. This review will describe the up-to-date knowledge of other aspects of Ca(2+) homeostasis in mouse oocytes, such as the mechanisms that transport Ca(2+) out of the cytosol into the endoplasmic reticulum (ER), the Ca(2+) store of the oocyte/egg, into other organelles and also those that extrude Ca(2+). Evidence pointing to channels in the plasma membrane that mediate Ca(2+) entry from the extracellular milieu, which is required for the persistence of the oscillations, is also discussed, along with the modifications that these mechanisms undergo during maturation. Lastly, we highlight areas where additional research is needed to obtain a better understating of the molecules and mechanisms that regulate Ca(2+) homeostasis in this unique Ca(2+) signaling system.

Changes in Ca2+ homeostasis render oocytes competent to undergo [Ca2+]i oscillations and activation. During mouse oocyte maturation Ca2+ influx and SOCE are down-regulated, whereas [Ca2+]ER content increases. Bypassing the down-regulation of Ca2+ influx disturbs oocyte maturation., In preparation for fertilization, mammalian oocytes undergo optimization of the mechanisms that regulate calcium homeostasis. Among these changes is the increase in the content of the Ca2+ stores ([Ca2+]ER), a process that requires Ca2+ influx. Nevertheless, the mechanism(s) that mediates this influx remains obscure, although is known that [Ca2+]ER can regulate Ca2+ influx via store-operated Ca2+ entry (SOCE). We find that during maturation, as [Ca2+]ER increases, Ca2+ influx decreases. We demonstrate that mouse oocytes/eggs express the two molecular components of SOCE—stromal interaction molecule 1 (Stim1) and Orai1—and expression of human (h) Stim1 increases Ca2+ influx in a manner that recapitulates endogenous SOCE. We observe that the cellular distribution of hStim1 and hOrai1 during maturation undergoes sweeping changes that curtail their colocalization during the later stages of maturation. Coexpression of hStim1 and hOrai1 enhances influx throughout maturation but increases basal Ca2+ levels only in GV oocytes. Further, expression of a constitutive active form of hStim1 plus Orai1, which increases basal Ca2+ throughout maturation, disturbs resumption of meiosis. Taken together, our results demonstrate that Ca2+ influx and SOCE are regulated during maturation and that alteration of Ca2+ homeostasis undermines maturation in mouse oocytes.

BACKGROUND: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS: Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS: Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

International audience; In mammals, sperm-oocyte fusion initiates Ca(2+) oscillations leading to a series of events called oocyte activation, which is the first stage of embryo development. Ca(2+) signaling is elicited by the delivery of an oocyte-activating factor by the sperm. A sperm-specific phospholipase C (PLCZ1) has emerged as the likely candidate to induce oocyte activation. Recently, PAWP, a sperm-born tryptophan domain-binding protein coded by WBP2NL, was proposed to serve the same purpose. Here, we studied two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intracytoplasmic sperm injection. Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, c.1465A>T; p.Ile489Phe, converting Ile 489 into Phe. We showed the mutation is deleterious, leading to the absence of the protein in sperm, mislocalization of the protein when injected in mouse GV and MII oocytes, highly abnormal Ca(2+) transients and early embryonic arrest. Altogether these alterations are consistent with our patients' sperm inability to induce oocyte activation and initiate embryo development. In contrast, no deleterious variants were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans, the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes. This opens the door to structure-function studies to identify the conserved amino acids of the C2 domain that regulate the targeting of PLCZ1 and its selectivity for its lipid substrate(s).

At the time of fertilization, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP(3)R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca(2+) release. The sensitivity of IP(3)R1, that is, its Ca(2+) releasing capability, is increased during oocyte maturation so that the optimum [Ca(2+)](i) response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP(3)R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca(2+) concentration ([Ca(2+)](ER)). Here, we evaluated using mouse oocytes how each of these factors affected IP(3)R1 sensitivity. The capacity for IP(3)-induced Ca(2+) release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP(3)R1 sensitivity was underpinned by an increase in [Ca(2+)](ER) and receptor phosphorylation(s) but not by changes in IP(3)R1 cellular distribution, as inhibition of the former factors reduced Ca(2+) release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca(2+)](ER) and IP(3)R1 phosphorylation during maturation enhance IP(3)R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP(3)R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca(2+) homeostasis also shape the pattern of oscillations in mammalian eggs.

Cryopreservation of mature eggs is a useful technique that can be applied in assisted reproductive technology. However, the method has some limitations, such as cryodamage induced by biophysical modifications during the cryopreservation process. To assess these biophysical damage, we analyzed the relationship between intracellular calcium ([Ca2+]i) oscillatory activity via type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) distribution after vitrification and efficiency of cryopreservation according to cryoprotectant (CPA) composition. In immunostaining, results of IP(3)R1 with eggs after the vitrification performed using ethylene glycol (EG) alone or EG + dimethylsulfoxide (DMSO) as CPAs, CPA-treated, and fresh eggs displayed a homogeneous IP(3)R1 distribution which is spread uniformly throughout cytoplasm with clusters on the cortex. However, after vitrification and warming process, more than 60% of eggs displayed a heterogeneous distribution which is non-uniform distribution with patches and disconnection of IP(3)R1. In 90-min incubation for recovery from cryodamage, eggs from the EG + DMSO group recovered from with a heterogeneous IP(3)R1 distribution to the homogeneous distribution, but not in EG alone group. In ICSI experiments, vitrified eggs in the EG-alone group presented significantly low blastocyst formation compared to those of the fresh and EG + DMSO groups. These results suggest that the vitrification process influences IP(3)R1 distribution, and subsequently, [Ca2+]i oscillatory activity and embryonic development. Accordingly, we propose that IP(3)R1 distribution and [Ca2+]i oscillatory activity are correlated with egg quality and developmental potential after vitrification, and may thus be applied as an effective indicator to evaluate the efficiency of oocyte cryopreservation methods.

One of the most important processes in fertilization is the fusion of egg and sperm; however, the molecular mechanisms involved in this process are not well understood. So far, using genetic approaches, only two proteins have been demonstrated to be necessary for this process: Izumo in sperm and CD9 in the egg. Here we demonstrate that sperm produced by Tssk6 (Sstk)-null mice present defects that prevent the successful fertilization of eggs in vitro and the fusion to zona-pellucida-free eggs. Tssk6 is a member of the testis-specific serine kinase family of proteins and is expressed postmeiotically in male germ cells. In order for fusion to occur, during the process known as acrosome reaction Izumo needs to relocate from the anterior head to other regions, including the postacrosomal compartment. Tssk6-null sperm fails to relocate Izumo during the acrosome reaction. Agents that interfere with actin dynamics blocked the acrosome-reaction-associated translocation of Izumo that is required for fusion in wild-type sperm. Additionally, actin polymerization was compromised in Tssk6-null sperm. Taken together, our results indicate that Tssk6 is involved in sperm-egg fusion through the regulation of actin polymerization and changes in Izumo localization.

We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase blastocyst development (0 and 4.5%) over a control treatment (injection of sperm extract after ionomycin exposure; 5.6%). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in five pregnancies, all lost before 70 days of gestation. In Experiment 3, cells from a second biopsy sample from the same horse produced significantly more blastocysts than did the original sample (4/44 vs. 0/58; p0.05). Transfer of these four blastocysts produced two viable foals. In Experiment 4, blastocyst development rates did not differ between oocytes in metaphase I or II at the time of nuclear transfer (16.7 and 3.0%, respectively). A healthy foal was produced from a blastocyst originating from a metaphase I oocyte.

Xenopus oocyte death is partly controlled by the apoptotic initiator, caspase-2. We reported previously that oocyte nutrient depletion activates caspase-2 upstream of mitochondrial cytochrome c release. Conversely, nutrient-replete oocytes inhibit caspase-2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. We now show that caspase-2 phosphorylated at S135 binds 14-3-3ζ, thus preventing caspase-2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1, which directly binds caspase-2. Although caspase-2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3ζ from caspase-2 is controlled by metabolism and allows for caspase-2 dephosphorylation. Accordingly, a caspase-2 mutant unable to bind 14-3-3ζ is highly susceptible to dephosphorylation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine caspase-2 by phosphorylation and 14-3-3 binding in mouse eggs. These findings provide an unexpected evolutionary link between 14-3-3 and metabolism in oocyte death.

During fertilization of mammalian eggs a factor from the sperm, the sperm factor (SF), is released into the ooplasm and induces persistent [Ca2+]ioscillations that are required for egg activation and embryo development. A sperm-specific phospholipase C (PLC), PLCz, is thought to be the SF. Here, we investigated whether the SF activity and PLCζare simultaneously and completely released into the ooplasm soon after sperm entry. To accomplish this, we enucleated sperm heads within 90 min of intracytoplasmic sperm injection (ICSI) and monitored the persistence of the [Ca2+]ioscillations in eggs in which the sperm had been withdrawn. We also stained the enucleatedsperm heads to ascertain the presence/absence of PLCζ. Our results show that by 90 min all the SF activity had been released from the sperm, as fertilized enucleated eggs oscillated as fertilized controls, even in cases in which oscillations were prolonged by arresting eggs at metaphase. In addition, we found that the released SF activity became associated with the pronucleus (PN), as induction of PN envelope breakdown evoked comparable [Ca2+]iresponses in enucleated and non-manipulated zygotes. Lastly, we found that PLCzlocalized to the equatorial area of bull sperm and to the post-acrosomal region of mouse sperm and that by 90 min after ICSI all the sperm’s PLCζimmunoreactivity was lost in both species. Altogether, our findings show that during fertilization the SF activity and PLCζimmunoreactivity are simultaneously released from the sperm, suggesting that PLCζmay be the only [Ca2+]ioscillation-inducing factor of mammalian sperm.

One important result of egg activation is the establishment of blocks to prevent polyspermic fertilization; these blocks are established on the zona pellucida and the egg plasma membrane. This study examines what the sperm brings to the egg to induce the establishment of the membrane block to polyspermy, building on past evidence that membrane block establishment does not occur in response to parthenogenetic stimuli that induce a single transient increase in cytosolic Ca2+ or intracytoplasmic sperm injection (ICSI). We test the hypotheses that (i) sperm-associated Ca2+ release activity triggers membrane block establishment; (ii) introduction of sperm contents via variations on ICSI protocols (resulting in improved Ca2+ transients, egg activation and embryo development over traditional ICSI protocols) triggers membrane block establishment and (iii) sperm adhesion [binding of an extracellular sperm ligand(s) to an egg receptor(s)] combined with sperm-associated Ca2+ release activity triggers membrane block establishment. Interestingly, none of these stimuli induced establishment of the membrane block to polyspermy in mouse eggs. However, the sperm-associated remodeling of the egg cortical cytoskeleton differs between conventionally fertilized and ICSI-fertilized eggs; taken with our previous data implicating actin microfilaments in membrane block establishment, this raises the possibility that cortical reorganization may be a contributing factor. In sum, fertilization-like Ca2+ transients, either alone or combined with sperm-egg binding, are not sufficient for membrane block establishment, but that an event(s) associated with gamete interaction plays a role in this membrane function change.

The oocytes of B6.YTIR sex-reversed female mice can be fertilized but the resultant embryos die at early cleavage stages. In the present study, we examined chromosome segregation at meiotic divisions in the oocytes of XY female mice, compared to those of XX littermates. The timing and frequency of oocyte maturation in culture were comparable between the oocytes from both types of females. At the first meiotic division, the X- and Y-chromosomes segregated independently and were retained in oocytes at equal frequencies. However, more oocytes retained the correct number of chromosomes than anticipated from random segregation. The oocytes that had reached MII-stage were activated by fertilization or incubation with SrCl2. As expected, the majority of oocytes from XX females completed the second meiotic division and reached the 2-cell stage in 24 h. By contrast, more than half of oocytes from XY females initially remained at the MII-stage while the rest precociously entered interphase after SrCl2 activation; very few oocytes were seen at the second anaphase or telophase and they often showed impairment of sister–chromatid separation. Eventually the majority of oocytes entered interphase and formed pronuclei, but very few reached the 2-cell stage. Similar results were obtained after fertilization. We conclude that the XY chromosomal composition in oocyte leads to impairment in the progression of the second meiotic division.

Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs. Fil: Bello, Oscar Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Cappa, Andrea Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: de Paola, Maria Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Zanetti, Maria Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Fukuda, Mitsunori. Tohoku University; Japón Fil: Fissore, Rafael A.. Dep. Veterinary And Animal Science-univ.massachusetts A; Estados Unidos Fil: Mayorga, Luis Segundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Michaut, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentina

Fertilization in mammals is initiated when a sperm fuses with a mature MII oocyte, also known as egg, and triggers a plethora of finely controlled processes identified as egg activation. The completion of all events of egg activation is driven by and depends on a series of repetitive calcium (Ca(2+)) increases (Ca(2+) oscillations), which rely on Ca(2+) influx from the extracellular media. Ca(2+) channels on the egg plasma membrane (PM) are thought to mediate this influx. The TRP Ca(2+) channel TRPV3 is differentially expressed during oocyte maturation, being most active at the MII stage. Specific stimulation of TRPV3 channels promotes Ca(2+) influx sufficient to induce egg activation and parthenogenesis. Here, we explore the function and distribution dynamics of the TRPV3 channel protein during maturation. Using dsRNA, TrpV3 overexpression, and inhibitors of protein synthesis, we modified the expression levels of the channel and showed that the TRPV3 protein is synthesized and translocated to the PM during maturation. We demonstrated that 2-APB at the concentrations used here to promote Ca(2+) influx in eggs, specifically and reversibly targets TRPV3 channels without blocking IP3R1. Finally, we found that the activity of TRPV3 channels is dependent upon an intact actin cytoskeleton, suggesting an actin-based regulation of its expression and/or function on the PM. Collectively, our results show TRPV3 is a target of 2-APB in eggs, a condition that can be used to induce parthenogenesis. The need of an intact actin cytoskeleton for the function of TRPV3 channels in oocytes is a novel finding and suggests the rearrangements of actin that occur during maturation could regulate both the presence on the PM and/or the function of TRPV3 and of other Ca(2+) channels involved in oocyte maturation and fertilization.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) mediates increases in the intracellular concentration of Ca(2+) ([Ca(2+)]i) during fertilization in mammalian eggs. The activity of IP3R1 is enhanced during oocyte maturation, and phosphorylations by M-phase kinases are thought to positively regulate the activity of IP3R1. Accordingly, we and others have found that IP3R1 is phosphorylated at S(421), T(799) (by Cdk1) and at S(436) (by ERK). Nevertheless, the effects of these phosphorylations on the function of the receptor and their impact on [Ca(2+)]i oscillations in eggs have not been clearly examined. To address this, we expressed in mouse oocytes an IP3R1 variant with the three indicated phosphorylation sites replaced by acidic residues, IIIE-IP3R1, such that it would act like a constitutively phosphorylated IP3R1, and examined [Ca(2+)]i parameters in response to stimuli. We found that overexpression of wild type (wt-IP3R1) or IIIE-IP3R1 in oocytes containing endogenous receptors caused dominant negative-like effects on Ca(2+) release and oscillations. Therefore, we first selectively removed the endogenous IP3R1, and subsequently expressed the exogenous receptors. We found that in response to injection of PLCζ cRNA, eggs without endogenous IP3R1 failed to mount persistent Ca(2+) oscillations, although expression of wt-IP3R1 restored their [Ca(2+)]i oscillatory activity. We also observed that the Ca(2+) oscillatory ability and the sensitivity to IP3 in eggs expressing IIIE-IP3R1 were greater than in those expressing wt-IP3R1. Lastly, we found that exogenous IP3R1s are resistant to downregulation and support longer oscillations and of higher amplitude. Altogether, our results show that phosphorylations by Cdk1 and MAPK enhance the activity of IP3R1, which is consistent with its maximal activity observed at the time of fertilization and the role of Ca(2+) release in egg activation.

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.

Nhlh2 is a member of the basic helix-loop-helix (bHLH) transcription factor family and is expressed in developing and adult neuroendocrine tissues such as the pituitary and hypothalamus. Targeted deletion of Nhlh2 (N2KO) in mice results in hypogonadism and obesity. While gonadally intact male N2KO mice are infertile and lack male sexual behavior, female N2KO mice can become pregnant and carry litters to full term. Unlike normal females in which fertility averages 8–12 months with approximately one pregnancy per month, N2KO females have a shorter reproductive span with most females supporting only three to four pregnancies in a 9-month period. In addition, N2KO females exhibit abnormal estrous cycles characterized by a truncated estrus and a prolonged proestrus. We have found that while young female N2KO mice ovulate the same number of oocytes as normal females in response to exogenous hormones, the number of oocytes released by aged N2KO females is reduced over 50%. Interestingly, oocytes from N2KO females are equally competent for in vitro fertilization assays when compared to oocytes from similarly aged normal and heterozygous mice. We have further demonstrated that both young and old N2KO females show at least a 50% reduction in hormone-stimulated sexual behavior as measured by their lordosis quotient. This suggests that N2KO females show a lifelong behavioral hyporesponsiveness to exogenous steroid hormones accompanied by a reduction in reproductive longevity via reduced ovulation with aging. Potential gene regulatory mechanisms that involve the action of the Nhlh2 transcription factor on female fertility and sexual behavior are discussed.

The various inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that of IP(3)R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca(2+) inhibited its phosphorylation (IC(50)

Recent evidence in marine invertebrate, frog, and zebrafish eggs suggests the involvement of a Src family kinase (SFK) in fertilization-induced Ca2+release. In the present study, we have investigated whether activation of an SFK is required for initiation of intracellular Ca2+([Ca2+]i) oscillations in mouse fertilization. We detected a Hck-like protein and tyrosine-phosphorylated proteins in soluble and insoluble sperm fractions, respectively. However, the presence of these proteins did not correspond to the active fractions of porcine sperm extracts (pSE). Moreover, [Ca2+]ioscillations induced by pSE in mouse eggs were unaltered by pre-incubation of pSE with specific SFK inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]-pyrimidine (PP2) or lavendustin A, despite the fact that the inhibitors were shown to be active bothin vivoandin vitro. Another SFK inhibitor, peptide A, blocked oscillations when incubated with pSE prior to injection into eggs, but this inhibition required more than ten times the concentration reportedly required to inhibit SFK activity. In addition, pre-injection or pre-incubation of eggs with these inhibitors did not affect the ability of pSE to trigger [Ca2+]ioscillations in mouse eggs. Microinjection of a recombinant c-Src protein or mRNAs encoding constitutively active Src proteins did not induce [Ca2+]irelease. Finally, when sperm and eggs, both of which were pre-treated with PP2, were fertilized, [Ca2+]ioscillations occurred normally. We can therefore conclude that activation of an SFK is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]ioscillations.

In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.

ICSI bypasses not only fusion of the gametes but also a series of signalling events that occur in the sperm prior to and during interaction with the oocyte's vestments. The effect of this altered encounter of the gametes on the fertilization-associated intracellular calcium ([Ca2+]i) oscillations has not been thoroughly investigated. Here, ICSI and IVF were performed using gametes from two mouse strains, B6D2F1 and CD1, and in-vitro development, pattern of [Ca2+]i oscillations and down-regulation of inositol 1,4,5-trisphosphate receptor-1 (IP3R-1) in the produced embryos were compared. ICSI and IVF resulted in comparable rates of activation and pre-implantation development. However, ICSI-generated zygotes cleaved at a slower rate, had lower cell numbers and lower hatching rates. The deleterious effects of ICSI could not be exclusively attributed to the injury by the injection since sham-injected IVF zygotes only exhibited delayed progression to the blastocyst stage. ICSI and IVF induced similar initial [Ca2+]i responses, although ICSI zygotes exhibited shorter durations of [Ca2+]i oscillations and showed diminished degradation of IP3R-1. Importantly, sperm manipulation affected the pattern of oscillations, which further decreased pre-implantation developmental rates. Our results demonstrate that ICSI-induced [Ca2+]i responses are not equivalent to those initiated by IVF and that this may have developmental consequences.

International audience; We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia. Non-genetically characterized cases of globozoospermia were associated with DNA alterations, suggesting that DPY19L2-dependent globozoospermia maybe associated with poor DNA quality. However the origins of such defects have not yet been characterized and the consequences on the quality of embryos generated with globozoospermic sperm remain to be determined. Using the mouse model lacking Dpy19l2, we compared several key steps of nuclear compaction. We show that the kinetics of appearance and disappearance of the histone H4 acetylation waves and of transition proteins are defective. More importantly, the nuclear invasion by protamines does not occur. As a consequence, we showed that globozoospermic sperm presented with poor sperm chromatin compaction and sperm DNA integrity breakdown. We next assessed the developmental consequences of using such faulty sperm by performing ICSI. We showed in the companion article that oocyte activation (OA) with globozoospermic sperm is very poor and due to the absence of phospholipase C zeta; therefore artificial OA (AOA) was used to bypass defective OA. Herein, we evaluated the developmental potential of embryos generated by ICSI + AOA in mice. We demonstrate that although OA was fully rescued, preimplantation development was impaired when using globozoospermic sperm. In human, a small number of embryos could be generated with sperm from DPY19L2-deleted patients in the absence of AOA and these embryos also showed a poor developmental potential. In conclusion, we show that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Most of the DNA breaks were already present when the sperm reached the epididymis, indicating that they occurred inside the testis. This result thus suggests that testicular sperm extraction in Dpy19l2-dependent globozoospermia is not recommended. These defects may largely explain the poor embryonic development of most mouse and human embryos obtained with globozoospermic sperm.

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.

We used patch clamp electrophysiology and concurrent imaging with the Ca(2+)-sensitive dye, fura-2, to study the temporal relationship between membrane capacitance and conductance and intracellular free Ca(2+) concentration ([Ca(2+)](i)) during mouse egg fertilization. We found an approximately 2 pF step increase in egg membrane capacitance and a minor increase in conductance with no change in [Ca(2+)](i) at sperm fusion. This was followed approximately 1 min later by a rise in [Ca(2+)](i) that led to larger changes in capacitance and conductance. The most common pattern for these later capacitance changes was an initial capacitance decrease, followed by a larger increase and eventual return to the approximate starting value. There was some variation in this pattern, and sub-microM peak [Ca(2+)](i) favored capacitance decrease, while higher [Ca(2+)](i) favored capacitance increase. The magnitude of accompanying conductance increases was variable and did not correlate well with peak [Ca(2+)](i). The intracellular introduction of porcine sperm factor reproduced the postfusion capacitance and conductance changes with a similar [Ca(2+)](i) dependence. Raising [Ca(2+)](i) by the intracellular introduction of IP(3) initiated fertilization-like capacitance changes, but the conductance changes were slower to activate. Capacitance decrease could be induced when [Ca(2+)](i) was increased modestly by activation of an endogenous Ca(2+) current, with little effect on resting conductance. These results suggest that net turnover of the mouse egg surface membrane is sensitive to [Ca(2+)](i) and that sperm and the active component of sperm factor may be doing more than initiating the IP(3)-mediated release of intracellular Ca(2+).

Changes in the intracellular concentration of free calcium ([Ca2+]i) regulate diverse cellular processes including fertilization. In mammalian eggs, the [Ca2+]i changes induced by the sperm unfold in a pattern of periodical rises, also known as [Ca2+]i oscillations. The source of Ca2+ during oscillations is the endoplasmic reticulum ([Ca2+]ER), but it is presently unknown how [Ca2+]ER is regulated. Here, we show using mouse eggs that [Ca2+]i oscillations induced by a variety of agonists, including PLCζ, SrCl2 and thimerosal, provoke simultaneous but opposite changes in [Ca2+]ER and cause differential effects on the refilling and overall load of [Ca2+]ER. We also found that Ca2+ influx is required to refill [Ca2+]ER, because the loss of [Ca2+]ER was accelerated in medium devoid of Ca2+. Pharmacological inactivation of the function of the mitochondria and of the Ca2+-ATPase pumps PMCA and SERCA altered the pattern of oscillations and abruptly reduced [Ca2+]ER, especially after inactivation of mitochondria and SERCA functions. We also examined the expression of SERCA2b protein and found that it was expressed throughout oocyte maturation and attained a conspicuous cortical cluster organization in mature eggs. We show that its overexpression reduces the duration of inositol-1,4,5-trisphosphate-induced [Ca2+]i rises, promotes initiation of oscillations and enhances refilling of [Ca2+]ER. Collectively, our results provide novel insights on the regulation of [Ca2+]ER oscillations, which underlie the unique Ca2+-signalling system that activates the developmental program in mammalian eggs. ispartof: Journal of Cell Science vol:126 issue:24 pages:5714-5724 ispartof: location:England status: published

Summary Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca 2+ to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.

Apoptosis in most cell types is accompanied by altered Ca(2+) homeostasis. During apoptosis, caspase-3 mediated cleavage of the type 1 inositol 1,4,5-trisphosphate receptor (IP3 R1) generates a 95-kDa C-terminal fragment (C-IP3 R1), which represents the channel domain of the receptor. Aged mouse eggs display abnormal Ca(2+) homeostasis and express C-IP3 R1, although whether or not C-IP3 R1 expression contributes to Ca(2+) misregulation or a decrease in developmental competency is unknown. We sought to answer these questions by injecting in mouse oocytes and eggs cRNAs encoding C-IP3 R1. We found that: (1) expression of C-IP3 R1 in eggs lowered the Ca(2+) content of the endoplasmic reticulum (ER), although, as C-IP3 R1 is quickly degraded at this stage, its expression did not impair pre-implantation embryo development; (2) expression of C-IP3 R1 in eggs enhanced fragmentation associated with aging; (3) endogenous IP3 R1 is required for aging associated apoptosis, as its down-regulation prevented fragmentation, and expression of C-IP3 R1 in eggs with downregulated IP3 R1 partly restored fragmentation; (4) C-IP3 R1 expression in GV oocytes resulted in persistent levels of protein, which abolished the increase in the ER releasable Ca(2+) pool that occurs during maturation, undermined the Ca(2+) oscillatory ability of matured eggs and their activation potential. Collectively, this study supports a role for IP3 R1 and C-IP3 R1 in regulating Ca(2+) homeostasis and the ER Ca(2+) content during oocyte maturation. Nevertheless, the role of C-IP3 R1 on Ca(2+) homeostasis in aged eggs seems minor, as in MII eggs the majority of endogenous IP3 R1 remains intact and C-IP3 R1 undergoes rapid turnover.

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.

Background Oocyte activation is a crucial step that comprises the release of the oocyte from meiotic arrest, pronuclear formation and subsequent embryo development. Oocytes are activated by repetitive increases in the intracellular concentration of free Ca(2+), [Ca(2+)](i) oscillations, which are triggered during fertilization by the introduction of the sperm-specific phospholipase C zeta 1 (PLCZ1). Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce [Ca(2+)](i) oscillations or oocyte activation. We first purified recombinant human PLCZ1 (hPLCZ1) protein and evaluated its [Ca(2+)](i) oscillation activity in mouse and human oocytes with the view to investigate its application in the clinic for assisted oocytes activation in lieu of chemical agents. Methods Recombinant hPLCZ1 was synthesized using the Escherichia coli system, and subjected to immunoblot analysis with anti-PLCZ1 and anti-His tag antibodies. [Ca(2+)](i) oscillations by microinjection of recombinant hPLCZ1 into mouse or human oocytes were examined by [Ca(2+)](i) monitoring with Fluo 4. Ploidy of the oocytes with recombinant hPLCZ1 injection was confirmed with fluorescence in situ hybridization. Results A band of 68 kDa on recombinant protein was detected with both antibodies. Injection of recombinant hPLCZ1 induced [Ca(2+)](i) oscillations in a dose-dependent manner in both mouse and human oocytes. These oscillations, which closely resembled those initiated by the sperm upon fertilization, triggered activation and cleavage in oocytes of both species, although further development of the mice embryos was low. U73122, a PLC inhibitor, blocked the ability of hPLCZ1 to initiate oscillations. Microinjection of recombinant hPLCZ1 into ICSI-failed human oocytes rescued fertilization failure in five of eight attempts. Conclusions Repeated [Ca(2+)](i) oscillations and oocyte activation were induced in mouse and human oocytes by microinjection of recombinant hPLCZ1 synthesized in E. Coli. Injection of recombinant protein could thus provide a biological solution for inducing artificial activation of oocytes.

BACKGROUND: Male factor and idiopathic infertility contribute significantly to global infertility, with abnormal testicular gene expression considered to be a major cause. Certain types of male infertility are caused by failure of the sperm to activate the oocyte, a process normally regulated by calcium oscillations, thought to be induced by a sperm-specific phospholipase C, PLCzeta (PLCζ). Previously, we identified a point mutation in an infertile male resulting in the substitution of histidine for proline at position 398 of the protein sequence (PLCζ(H398P)), leading to abnormal PLCζ function and infertility. METHODS AND RESULTS: Here, using a combination of direct-sequencing and mini-sequencing of the PLCζ gene from the patient and his family, we report the identification of a second PLCζ mutation in the same patient resulting in a histidine to leucine substitution at position 233 (PLCζ(H233L)), which is predicted to disrupt local protein interactions in a manner similar to PLCζ(H398P) and was shown to exhibit abnormal calcium oscillatory ability following predictive 3D modelling and cRNA injection in mouse oocytes respectively. We show that PLCζ(H233L) and PLCζ(H398P) exist on distinct parental chromosomes, the former inherited from the patient's mother and the latter from his father. Neither mutation was detected utilizing custom-made single-nucleotide polymorphism assays in 100 fertile males and females, or 8 infertile males with characterized oocyte activation deficiency. CONCLUSIONS: Collectively, our findings provide further evidence regarding the importance of PLCζ at oocyte activation and forms of male infertility where this is deficient. Additionally, we show that the inheritance patterns underlying male infertility are more complex than previously thought and may involve maternal mechanisms.

Background: Mammalian oocyte activation occurs via a series of intracellular calcium (Ca 2) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLCζ gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLCζ residue 398 (PLCζ H398P), leading to abnormal Ca 2 release profiles and reduced oocyte activation efficiency.Methods: AND Results: In the present study, we used HEK293T cells to produce recombinant human wild-type PLCζ (PLCζ WT) protein which, upon microinjection into mouse oocytes, induced Ca 2 oscillations characteristic of oocyte activation. Injection of recombinant PLCζ H398P was unable to elicit Ca 2 oscillations in mouse oocytes. Loss of activity mutations, such as PLCζ H398P and an artificially induced frameshift mutation (PLCζ ΔYC2) did not affect Ca 2 release when over-expressed in HEK293T cells, whereas PLCζ WT inhibited adenosine triphosphate-activated Ca 2 release. Confocal imaging of fluorescently tagged PLCζ isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLCζ WT > PLCζ H398P > PLCζ ΔYC2, indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLCζ immunofluorescence from the patient exhibiting PLCζ H398P compared with fertile controls. Conclusions: We demonstrate, for the first time, the production of active recombinant human PLCζ protein which retained the ability to elicit characteristic Ca 2 oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLCζ, and provide a critical step forward in obtaining purified PLCζ protein as a potential therapeutic agent for oocyte activation deficiency. © 2011 The Author. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

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.

Injection of mammalian sperm extracts or cRNA of the sperm-specific phospholipase C zeta 1 (PLCZ1) has been shown to trigger repetitive oscillations in the concentration of free calcium ([Ca(2+)](i)), leading to oocyte activation and embryo development in all mammals studied to date. While PLCZ1 has cross-species activity, it has also been observed that species-specific differences may exist in the frequency and pattern of the resulting [Ca(2+)](i) oscillations following PLCZ1 cRNA injection into oocytes of different species. Accordingly, we used a crossover design strategy to directly investigate the activity of murine and bovine PLCZ1 in both murine and bovine oocytes. In murine oocytes, injection of murine Plcz1 cRNA induced [Ca(2+)](i) oscillations at 10-fold lower concentrations than bovine PLCZ1, although in bovine oocytes bovine PLCZ1 was more effective than murine Plcz1 at inducing [Ca(2+)](i) oscillations. Investigation of ITPR1 (IP(3)R1) down-regulation in bovine oocytes by PLCZ1 cRNA also showed that bovine PLCZ1 was more active in homologous oocytes. To determine whether these PLCZs exhibited similar cellular distribution, Venus-tagged PLCZ1 cRNA was injected into oocytes, and PLCZ1 was overexpressed. Bovine PLCZ1 failed to accumulate in the pronucleus (PN) of bovine or murine zygotes, despite possessing a putative nuclear localization signal. Conversely, murine PLCZ1 accumulated in the PN of both murine and bovine zygotes. These results demonstrate that murine PLCZ1 and bovine PLCZ1 possess species-specific differences in activity and suggest potential differences in the mode of action of the protein between the two species. Variation in sperm PLCZ1 protein content among species, along with oocyte-specific differences in the localization and availability of PLCZ1 substrates, may further contribute to optimize the activation stimulus to enhance embryo development.

During fertilization in mammalian species, a sperm-induced intracellular Ca(2+) signal ([Ca(2+)](i)) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP(3)R1), the channel responsible for Ca(2+) release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP(3)R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP(3)R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP(3)R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34(cdc2) kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP(3)R1 phosphorylation, although inactivation of p34(cdc2) kinase by roscovitine dramatically reduced IP(3)R1 phosphorylation. Neither inhibitor affected total expression of IP(3)R1. Altogether, our results show that IP(3)R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization. ispartof: Animal Science Journal vol:81 issue:1 pages:34-41 ispartof: location:Australia status: published

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca(2+)](i)), which is almost entirely mediated by inositol 1,4,5-trisphosphate receptor 1 (IP(3)R1). In mammalian eggs, fertilization-induced [Ca(2+)](i) responses exhibit a periodic pattern that are called [Ca(2+)](i) oscillations. These [Ca(2+)](i) oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP(3)R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP(3)R1 degradation and examined the impact of the IP(3)R1 levels on the pattern of [Ca(2+)](i) oscillations. Using microinjection of IP(3) and of its analogs and conditions that prevent the development of [Ca(2+)](i) oscillations, we show that IP(3)R1 degradation requires uniform and persistently elevated levels of IP(3). We also established that progressive degradation of the IP(3)R1 results in [Ca(2+)](i) oscillations with diminished periodicity while a near complete depletion of IP(3)R1s precludes the initiation of [Ca(2+)](i) oscillations. These results provide insights into the mechanism involved in the generation of [Ca(2+)](i) oscillations in mouse eggs. ispartof: Journal of Cellular Physiology vol:222 issue:1 pages:238-247 ispartof: location:United States status: published

Egg activation and further embryo development require a sperm-induced intracellular Ca(2+) signal at the time of fertilization. Prior to fertilization, the egg's Ca(2+) machinery is therefore optimized. To this end, during oocyte maturation, the sensitivity, i.e. the Ca(2+) releasing ability, of the inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1), which is responsible for most of this Ca(2+) release, markedly increases. In this study, the recently discovered specific Polo-like kinase (Plk) inhibitor BI2536 was used to investigate the role of Plk1 in this process. BI2536 inactivates Plk1 in oocytes at the early stages of maturation and significantly decreases IP(3)R1 phosphorylation at an MPM-2 epitope at this stage. Moreover, this decrease in Plk1-dependent MPM-2 phosphorylation significantly lowers IP(3)R1 sensitivity. Finally, using in vitro phosphorylation techniques we identified T(2656) as a major Plk1 site on IP(3)R1. We therefore propose that the initial increase in IP(3)R1 sensitivity during oocyte maturation is underpinned by IP(3)R1 phosphorylation at an MPM-2 epitope(s). ispartof: CELL CALCIUM vol:46 issue:1 pages:56-64 ispartof: location:Netherlands status: published

Methods presently used to activate mare oocytes for assisted reproduction technologies provide low rates of advanced embryonic development. Because phospholipase Czeta (PLCzeta) is the postulated sperm-borne factor responsible for oocyte activation at fertilisation, the aim of the present study was to investigate the pattern of [Ca(2+)](i) oscillations and developmental rates achieved by microinjection of three concentrations of mouse PLCzeta complementary (c) RNA (1, 0.5 or 0.25 microg microL(-1)) into mare oocytes. The frequency of [Ca(2+)](i) oscillations was no different (P0.05) after injection of 1, 0.5 or 0.25 microg microL(-1) PLCzeta cRNA (41.1 +/- 5.3, 47 +/- 4.0 and 55.4 +/- 9.0, respectively). However, [Ca(2+)](i) oscillations persisted longest (P0.05) for oocytes injected with 0.5 microg microL(-1) PLCzeta cRNA (570.7 +/- 64.2 min). There was no significant difference in cleavage rates after injection of the three concentrations of PLCzeta (P0.05; range 97-100%), but the proportion of oocytes reaching advanced stages of embryonic development (64 nuclei) was significantly lower for oocytes injected with 0.25 microg microL(-1) PLCzeta cRNA (3%) than for those injected with 1 microg microL(-1) PLCzeta cRNA (15%). Based on these results, microinjection of PLCzeta may prove an effective and consistent method for the parthenogenetic activation of mare oocytes for nuclear transfer and provides a physiologically relevant tool with which to study fertilisation-dependent [Ca(2+)](i) signalling in this species.

Background During natural fertilization, sperm fusion with the oocyte induces long lasting intracellular calcium oscillations which in turn are responsible for oocyte activation. PLCZ1 has been identified as the factor that the sperm delivers into the egg to induce such a response. We tested the hypothesis that PLCZ1 cRNA injection can be used to activate bovine oocytes. Results Mouse and bovine PLCZ1 cRNAs were injected into matured bovine oocytes at different concentrations. Within the concentrations tested, mouse PLCZ1 injection activated bovine oocytes at a maximum rate when the pipette concentration of cRNA ranged from 0.25 to 1 μg/μL, while bovine PLCZ1 was optimal at 0.1 μg/μL. At their most effective concentrations, PLCZ1 induced parthenogenetic development at rates similar to those observed using other activation stimuli such as Ionomycin/CHX and Ionomycin/DMAP. Injection of mouse and bovine PLCZ1 cRNA induced dose-dependent sperm-like calcium oscillations whose frequency increased over time. Injection of bovine and mouse PLCZ1 cRNA also induced IP3R-1 degradation, although bovine PLCZ1 cRNA evoked greater receptor degradation than its mouse counterpart. Conclusion Injection of PLCZ1 cRNA efficiently activated bovine oocytes by inducing a sperm-like calcium oscillatory pattern. Importantly, the high rate of aneuploidy encountered in parthenogenetic embryos activated by certain chemical means was not observed in PLCZ1 activated embryos.

Background information. The IP3R (inositol 1,4,5-trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca2+ release in virtually all cell types. We have previously demonstrated that caspase-3-mediated cleavage of IP3R1 during cell death generates a C-terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP3R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase-3-cleaved C-terminus of IP3R1 increased the rate of thapsigargin-mediated Ca2+ leak and decreased the rate of Ca2+ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca2+ stores. We detected the truncated IP3R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP3R1 blocked sperm factor-induced Ca2+ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase-3-mediated truncation of IP3R1 enhanced the Ca2+ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca2+ leak is largely compensated by enhanced Ca2+-uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca2+ leak acts as a feed-forward mechanism to divert the cell into apoptosis.