Biomedical subjects

Rafael A Fissore

Publications and source records attributed to Rafael A Fissore.

At least 19 recordsLinked to original sources

PubMed2007

Activation of fertilized and nuclear transfer eggs.

In all animal species, initiation of embryonic development occurs shortly after the joining together of the gametes from each of the sexes. The first of these steps, referred to as "egg activation", is a series of molecular events that results in the syngamy of the two haploid genomes and the beginning of cellular divisions for the new diploid embryo. For many years it has been known that the incoming sperm drives this process, as an unfertilized egg will remain dormant until it can no longer sustain normal metabolic processes. Until recently, it was also believed that the sperm was the only cell capable of creating a viable embryo and offspring. Recent advances in cell biology have allowed researchers to not only understand the molecular mechanisms of egg activation, but to exploit the use of pharmacological agents to bypass sperm-induced egg activation for the creation of animals by somatic cell nuclear transfer. This chapter will focus on the molecular events of egg activation in mammals as they take place during fertilization, and will discuss how these mechanisms are successfully bypassed in processes such as somatic cell nuclear transfer.

Animals↗

PubMed2006

Regulation of fertilization-initiated [Ca2+]i oscillations in mammalian eggs: a multi-pronged approach.

The calcium ([Ca(2+)](i)) oscillations associated with mammalian fertilization and required to induce egg activation occur during M-phase stages of the cell cycle. The molecular mechanisms underlying this regulation remain unproven and may be multi-layered. Type 1 inositol 1,4,5-trisphosphate receptors (IP(3)R-1), which mediate [Ca(2+)](i) release during fertilization, have emerged as key regulatory units because they contain multiple phosphorylation consensus sites and undergo changes in cellular location and mass prior to and following fertilization. Hence, control of IP(3)R-1 function together with regulation of PLCzeta activity, the putative sperm factor, may combine to impart cell cycle and species-specific [Ca(2+)](i) oscillations characteristic of mammalian fertilization.

Animals↗

PubMed2006

Calcium oscillations and mammalian egg activation.

Fertilization in all species studied to date induces an increase in the intracellular concentration of free calcium ions ([Ca2+]i) within the egg. In mammals, this [Ca2+]i signal is delivered in the form of long-lasting [Ca2+]i oscillations that begin shortly after fusion of the gametes and persist beyond the time of completion of meiosis. While not fully elucidated, recent evidence supports the notion that the sperm delivers into the ooplasm a trigger of oscillations, the so-called sperm factor (SF). The recent discovery that mammalian sperm harbor a specific phospholipase C (PLC), PLCzeta has consolidated this view. The fertilizing sperm, and presumably PLCzeta promote Ca2+ release in eggs via the production of inositol 1,4,5-trisphosphate (IP3), which binds and gates its receptor, the type-1 IP3 receptor, located on the endoplasmic reticulum, the Ca2+ store of the cell. Repetitive Ca2+ release in this manner results in a positive cumulative effect on downstream signaling molecules that are responsible for the completion of all the events comprising egg activation. This review will discuss recent advances in our understanding of how [Ca2+]i oscillations are initiated and regulated in mammals, highlight areas of discrepancies, and emphasize the need to better characterize the downstream molecular cascades that are dependent on [Ca2+]i oscillations and that may impact embryo development.

Animals↗

PubMed2006

Intracytoplasmic sperm injection in the bovine induces abnormal [Ca2+]i responses and oocyte activation.

Fertilisation by intracytoplasmic sperm injection (ICSI), a technique that bypasses the membrane fusion of the gametes, has been widely used to produce offspring in humans and mice. Success with this technique has lent support to the hypothesis that in mammalian fertilisation, a factor from the sperm, the so-called sperm factor, is responsible for oocyte activation and that the fusion process is not involved in the generation of the hallmark [Ca2+]i signalling seen following fertilisation. However, the success of ICSI has largely eluded large domestic species, such as the bovine, porcine and equine, casting doubt on the current model of oocyte activation at fertilisation in these species. Using Ca2+ imagery and a series of treatments to manipulate the chemical structure of the sperm, we have investigated the early events of oocyte activation in response to ICSI in the bovine. Our results demonstrate, for the first time, that following ICSI, the majority of bovine oocytes are unable to mount [Ca2+]i oscillations, although, in few cases, the initiation of [Ca2+]i oscillations can occur in a manner indistinguishable from in vitro fertilisation. We also show that bull sperm possess a full complement of sperm factor. However, either the release and/or activation of the sperm factor are compromised after ICSI, leading to the delivery of a defective Ca2+ stimulus, which results in premature termination of embryo development.

Acrosome Reaction↗

PubMed2006

Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses at fertilization: a role for the MAP kinase pathway.

A sperm-induced intracellular Ca2+ signal ([Ca2+]i) underlies the initiation of embryo development in most species studied to date. The inositol 1,4,5 trisphosphate receptor type 1 (IP3R1) in mammals, or its homologue in other species, is thought to mediate the majority of this Ca2+ release. IP3R1-mediated Ca2+ release is regulated during oocyte maturation such that it reaches maximal effectiveness at the time of fertilization, which, in mammalian eggs, occurs at the metaphase stage of the second meiosis (MII). Consistent with this, the [Ca2+]i oscillations associated with fertilization in these species occur most prominently during the MII stage. In this study, we have examined the molecular underpinnings of IP3R1 function in eggs. Using mouse and Xenopus eggs, we show that IP3R1 is phosphorylated during both maturation and the first cell cycle at a MPM2-detectable epitope(s), which is known to be a target of kinases controlling the cell cycle. In vitro phosphorylation studies reveal that MAPK/ERK2, one of the M-phase kinases, phosphorylates IP3R1 at at least one highly conserved site, and that its mutation abrogates IP3R1 phosphorylation in this domain. Our studies also found that activation of the MAPK/ERK pathway is required for the IP3R1 MPM2 reactivity observed in mouse eggs, and that eggs deprived of the MAPK/ERK pathway during maturation fail to mount normal [Ca2+]i oscillations in response to agonists and show compromised IP3R1 function. These findings identify IP3R1 phosphorylation by M-phase kinases as a regulatory mechanism of IP3R1 function in eggs that serves to optimize [Ca2+]i release at fertilization.

Amino Acid Sequence↗

PubMed2005

Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm.

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.

Animals↗

PubMed2005

Fertilization and inositol 1,4,5-trisphosphate (IP3)-induced calcium release in type-1 inositol 1,4,5-trisphosphate receptor down-regulated bovine eggs.

It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP(3)) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca(2+)](i)) seen during mammalian fertilization. IP(3) promotes Ca(2+) release in eggs by binding to its receptor, the type-1 IP(3) receptor (IP(3)R-1, also known as ITPR1), a ligand-gated Ca(2+) channel located in the membrane of the endoplasmic reticulum, the main Ca(2+) store of the cell. While IP(3)R-1 has been shown to mediate all Ca(2+) release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca(2+) release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a approximately 70%-80% reduction in the number of intact IP(3)R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP(3) analogue, adenophostin A. Functional Ca(2+) release analysis revealed that IP(3)R-1 is the predominant Ca(2+) release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca(2+)](i) oscillations in response to fertilization, expression of PLCzeta (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP(3) and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca(2+)](i) oscillations in this reduced IP(3)R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP(3)R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP(3)R-1-mediated Ca(2+) release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca(2+)](i) oscillations at the level of IP(3)R-1.

Adenosine↗

PubMed2005

In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system.

BACKGROUND: Deficiencies remain in the ability of in vitro-matured human oocytes to acquire full developmental competence and give rise to a healthy pregnancy. A clear deficiency of current systems utilizing human oocytes has been the absence of cumulus cells. In the present study, a three-dimensional (3D) co-culture system exploiting an extracellular matrix was developed and compared to conventional methods for its ability to support maturation of human oocytes. METHODS AND RESULTS: Cumulus cells were embedded into a 3D collagen gel matrix with individual oocytes added to each gel. Oocytes from the same patient cultured in the gel matrix matured to metaphase II at rates similar to those of cumulus-free oocytes cultured in individual microdrops. Following maturation of oocytes and fixation of intact gels, chromatin and cytoskeletal elements were assessed in oocytes and cumulus cells. The activities of the key cell cycle kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were compared in oocytes matured under the two culture conditions. Compared with denuded oocytes, co-cultured oocytes exhibited increased MAPK activity, but no difference in MPF levels. CONCLUSIONS: This work characterizes a novel and efficacious culture system that takes advantage of the unique properties of the extracellular matrix, a 3D microenvironment, and the presence of cumulus cells for maturing human oocytes in vitro.

Adult↗

PubMed2004

Transgenic RNA interference reveals role for mouse sperm phospholipase Czeta in triggering Ca2+ oscillations during fertilization.

A sperm-specific phospholipase (PL) C, termed PLCzeta, is proposed to be the soluble sperm factor that induces Ca(2+) oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCzeta mRNA have reduced amounts of PLCzeta protein. Sperm derived from these transgenic mice trigger patterns of Ca(2+) oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca(2+) oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCzeta is the physiological trigger of Ca(2+) oscillations required for activation of development.

Animals↗

PubMed2004

Cell cycle-coupled [Ca(2+)](i) oscillations in mouse zygotes and function of the inositol 1,4,5-trisphosphate receptor-1.

Sperm entry in mammalian eggs initiates oscillations in the concentration of free calcium ([Ca(2+)](i)). In mouse eggs, oscillations start at metaphase II (MII) and conclude as the zygotes progress into interphase and commence pronuclear (PN) formation. The inositol 1,4,5-trisphosphate receptor (IP(3)R-1), which underlies the oscillations, undergoes degradation during this transition, suggesting that one or more of the eggs' Ca(2+)-releasing machinery components may be regulated in a cell cycle-dependent manner, thereby coordinating [Ca(2+)](i) responses with the cell cycle. To ascertain the site(s) of interaction, we initiated oscillations at different stages of the cell cycle in zygotes with different IP(3)R-1 mass. In addition to sperm, we used two other agonists: porcine sperm factor (pSF), which stimulates production of IP(3), and adenophostin A, a non-hydrolyzable analogue of IP(3). None of the agonists tested induced oscillations at interphase, suggesting that neither decreased IP(3)R-1 mass nor lack of production or excessive IP(3) degradation can account for the insensitivity to IP(3) at this stage. Moreover, the releasable Ca(2+) content of the stores did not change by interphase, but it did decrease by first mitosis. More importantly, experiments revealed that IP(3)R-1 sensitivity and possibly IP(3) binding were altered at interphase, and our data demonstrate stage-specific IP(3)R-1 phosphorylation by M-phase kinases. Accordingly, increasing the activity of M-phase kinases restored the oscillatory-permissive state in zygotes. We therefore propose that the restriction of oscillations in mouse zygotes to the metaphase stage may be coordinated at the level of IP(3)R-1 and that this involves cell cycle stage-specific receptor phosphorylation.

Adenosine↗

PubMed2004

Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C.

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)<or=2microM) and this inhibition was further potentiated by calmodulin (CaM), while the Ca(2+)-independent CaM mutant CaM(1234) was ineffective. Ca(2+) and CaM, however, did not inhibit IP(3)R3 phosphorylation by PKC. Taken together, these findings show that Ca(2+) and CaM differentially regulate the PKC-mediated phosphorylation of IP(3)R1 and IP(3)R3 and are indicative for a role for the inhibition of IP(3)R1 phosphorylation by Ca(2+) and CaM in the negative slope of the bell-shaped effect of Ca(2+) on IP(3)R function.

Animals↗

PubMed2004

Mammalian fertilization: from sperm factor to phospholipase Czeta.

In mammalian eggs, the fertilizing sperm evokes intracellular Ca2+ ([Ca2+]i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca2+]i oscillations still remains unclear, accumulating studies suggest that a presently unknown substance, termed sperm factor (SF), is delivered from the fertilizing sperm into the ooplasm and triggers [Ca2+]i oscillations. Based on findings showing that production of inositol 1,4,5-trisphosphate (IP3) underlies the generation of [Ca2+]i oscillations, it has been suggested that SF functions either as a phospholipase C (PLC), an enzyme that catalyzes the generation of IP3, or as an activator of a PLC(s) pre-existing in the egg. This review discusses the role of SF as the molecule responsible for the production of IP3 and the initiator of [Ca2+]i oscillations in mammalian fertilization, with particular emphasis on the possible involvement of egg- and sperm-derived PLCs, including PLCzeta, a novel sperm specific PLC.

Animals↗

PubMed2004

The N-terminal Ca2+-independent calmodulin-binding site on the inositol 1,4,5-trisphosphate receptor is responsible for calmodulin inhibition, even though this inhibition requires Ca2+.

Calmodulin (CaM) is a ubiquitous Ca(2+)-sensor protein that plays an important role in regulating a large number of Ca(2+) channels, including the inositol 1,4,5-trisphosphate receptor (IP(3)R). CaM binds to the IP(3)R at Ca(2+)-dependent as well as at Ca(2+)-independent interaction sites. In this study, we have investigated the Ca(2+)-independent CaM-binding site for its role in the regulation of the Ca(2+)-dependent bell-shaped activation curve of the IP(3)R. Suramin, a polysulfonated napthylurea, displaced CaM in both the presence and the absence of Ca(2+). Suramin competed with CaM for binding to different peptides representing the previously identified CaM-binding sites on IP(3)R1. By interacting with the N-terminal Ca(2+)-independent CaM-binding site, suramin mimicked the functional effect of CaM and induced an allosteric but competitive inhibition of IP(3) binding. Therefore, suramin also potently inhibited IP(3)-induced Ca(2+) release (IICR) from permeabilized cells predominantly expressing IP(3)R1 (L15 fibroblasts) or IP(3)R3 (Lvec fibroblasts), even though the IP(3)R3 does not contain Ca(2+)-dependent CaM-binding sites. Furthermore, we have found that CaM(1234), a CaM mutated in its four EF hands, inhibited IICR in a Ca(2+)-dependent way with the same potency as CaM. We conclude that CaM inhibits IICR via the N-terminal binding site. The inhibition requires Ca(2+) but CaM itself is not the Ca(2+) sensor for the inhibition of the IP(3)R.

Animals↗

PubMed2004

Evidence that activation of Src family kinase is not required for fertilization-associated [Ca2+]i oscillations in mouse eggs.

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+]i oscillations 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 both in vivo and in 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+]i oscillations in mouse eggs. Microinjection of a recombinant c-Src protein or mRNAs encoding constitutively active Src proteins did not induce [Ca2+]i release. Finally, when sperm and eggs, both of which were pre-treated with PP2, were fertilized, [Ca2+]i oscillations occurred normally. We can therefore conclude that activation of an SFK is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]i oscillations.

Animals↗

PubMed2003

Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse.

In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.

Animals↗

PubMed2003

Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization.

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.

Animals↗

PubMed2003

Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs.

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.

Animals↗

PubMed2003

Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm.

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

Acrosome↗