Development of Recombinant PLC-Zeta Protein as a Therapeutic Intervention for the Clinical Treatment of Oocyte Activation Failure.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca ionophores.

Sperm-Induced Ca Release in Mammalian Eggs: The Roles of PLCζ, InsP, and ATP.

Mammalian egg activation at fertilization is triggered by a long-lasting series of increases in cytosolic Ca concentration. These Ca oscillations are due to the production of InsP within the egg and the subsequent release of Ca from the endoplasmic reticulum into the cytosol. The generation of InsP is initiated by the diffusion of sperm-specific phospholipase Czeta1 (PLCζ) into the egg after gamete fusion.

Sperm induce a secondary increase in ATP levels in mouse eggs that is independent of Ca2+ oscillations.

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred ∼1 h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations.

SPERM FACTORS AND EGG ACTIVATION: The structure and function relationship of sperm PLCZ1.

In 2002, sperm-specific phospholipase C zeta1 (PLCZ1) was discovered and through these 20 years, it has been established as the predominant sperm oocyte-activating factor. PLCZ1 cRNA expression or direct protein microinjection into mammalian oocytes triggers calcium (Ca2+) oscillations indistinguishable from those observed at fertilization. The imperative role of PLCZ1 in oocyte activation is revealed by the vast number of human mutations throughout the PLCZ1 gene that have been identified and directly linked with certain forms of male infertility due to oocyte activation deficiency.

Dynamic shapes of the zygote and two-cell mouse and human.

Mouse zygote morphokinetics were measured during interphase, the mitotic period, cytokinesis, and two-cell stage. Sequences of rounder-distorted-rounder shapes were revealed, as were changing patterns of cross section area. A calcium chelator and an actin-disrupting agent inhibited the area changes that occurred between pronuclear envelope breakdown and cytokinesis.

A primary effect of palmitic acid on mouse oocytes is the disruption of the structure of the endoplasmic reticulum.

Exposure of mouse oocytes to saturated fatty acids (FAs) such as palmitic acid (PA) has been shown to increase lipid content and cause an endoplasmic reticulum (ER) stress response and changes in the mitochondrial redox state. PA can also disrupt Ca2+ stores in other cell types. The links between these intracellular changes, or whether they are prevented by mono-unsaturated FAs such as oleic acid (OA), is unclear.

Vitrifying multiple embryos in different arrangements does not alter the cooling rate.

Vitrification is the most common method of cryopreservation of gametes in fertility clinics due to its improved survival rates compared to slow freezing techniques. For the Open Cryotop® vitrification device, the number of oocytes, or embryos, mounted onto a single device can vary. In this work, a mathematical model is developed for the cooling of oocytes and embryos (samples).

The role of ATP in the differential ability of Sr2+ to trigger Ca2+ oscillations in mouse and human eggs.

At fertilization in mice and humans, the activation of the egg is caused by a series of repetitive Ca2+ oscillations which are initiated by phospholipase-C(zeta)ζ that generates inositol-1,4,5-trisphophate (InsP3). Ca2+ oscillations and egg activation can be triggered in mature mouse eggs by incubation in Sr2+ containing medium, but this does not appear to be effective in human eggs. Here, we have investigated the reason for this apparent difference using mouse eggs, and human eggs that failed to fertilize after IVF or ICSI.

The soluble sperm factor that activates the egg: PLCzeta and beyond.

PLCzeta(ζ) initiates Ca2+ oscillations and egg activation at fertilization in mammals, but studies in mouse eggs fertilized by PLCζ knockout (KO) sperm imply that there is another slow acting factor causing Ca2+ release. Here, I propose a hypothesis for how this second sperm factor might cause Ca2+ oscillations in mouse eggs.

Dynamic label-free imaging of lipid droplets and their link to fatty acid and pyruvate oxidation in mouse eggs.

Mammalian eggs generate most of their ATP by mitochondrial oxidation of pyruvate from the surrounding medium or from fatty acids that are stored as triacylglycerols within lipid droplets. The balance between pyruvate and fatty acid oxidation in generating ATP is not established. We have combined coherent anti-Stokes Raman scattering (CARS) imaging with deuterium labelling of oleic acid to monitor turnover of fatty acids within lipid droplets of living mouse eggs.

Mitochondria and lipid metabolism in mammalian oocytes and early embryos.

Mammalian oocytes and early cleavage-stage embryos are critically dependent on their ˜100,000 mitochondria to develop from ovulation to compacted morula stage. They rely almost solely on oxidative phosphorylation of multiple intracellular substrates- namely pyruvate, fatty acids and glutamine- for production of ATP. Increasing evidence exists for the requirement of both fatty acids and pyruvate for mammalian developmental potential.

Electrical-assisted microinjection for analysis of fertilization and cell division in mammalian oocytes and early embryos.

Microinjection is an essential approach in the study of mammalian oocytes and early embryos, and is useful for the introduction of many molecules and reagents. Whereas microinjection into germinal vesicle stage oocytes is relatively simple using various microinjection setups, metaphase-II mouse eggs are notoriously fragile, and nondamaging microinjection can be difficult to achieve. Here we describe a microinjection method that is based on electrophysiology, which vastly reduces microinjection damage, especially in metaphase-II eggs.

The role of Ca in oocyte activation during In Vitro fertilization: Insights into potential therapies for rescuing failed fertilization.

At fertilization the mature mammalian oocyte is activated to begin development by a sperm-induced series of increases in the cytosolic free Ca concentration. These so called Ca oscillations, or repetitive Ca spikes, are also seen after intracytoplasmic sperm injection (ICSI) and are primarily triggered by a sperm protein called phospholipase Czeta (PLCζ). Whilst ICSI is generally an effective way to fertilizing human oocytes, there are cases where oocyte activation fails to occur after sperm injection.

PLCζ Induced Ca Oscillations in Mouse Eggs Involve a Positive Feedback Cycle of Ca Induced InsP Formation From Cytoplasmic PIP.

Egg activation at fertilization in mammalian eggs is caused by a series of transient increases in the cytosolic free Ca concentration, referred to as Ca oscillations. It is widely accepted that these Ca oscillations are initiated by a sperm derived phospholipase C isoform, PLCζ that hydrolyses its substrate PIP to produce the Ca releasing messenger InsP. However, it is not clear whether PLCζ induced InsP formation is periodic or monotonic, and whether the PIP source for generating InsP from PLCζ is in the plasma membrane or the cytoplasm.

Male infertility-linked point mutation reveals a vital binding role for the C2 domain of sperm PLCζ.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the Ca oscillation-inducing activity and the biochemical properties of human PLCζ.

Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm.

Study Question: Is it possible to improve clinical visualization of phospholipase C zeta (PLCζ) as a diagnostic marker of sperm oocyte activation capacity and male fertility?

Summary Answer: Poor PLCζ visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLCζ, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols.

What Is Known Already: Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca) oscillations induced by sperm-specific PLCζ. PLCζ represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility.

Molecular triggers of egg activation at fertilization in mammals.

In mammals, the sperm activates the development of the egg by triggering a series of oscillations in the cytosolic-free Ca(2+) concentration (Ca(2+) i). The sperm triggers these cytosolic Ca(2+i) oscillations after sperm-egg membrane fusion, as well as after intracytoplasmic sperm injection (ICSI). These Ca(2+) i oscillations are triggered by a protein located inside the sperm.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos.

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

A series of intracellular oscillations in the free cytosolic Ca(2+) concentration is responsible for activating mammalian eggs at fertilization, thus initiating embryo development. It has been proposed that the sperm causes these Ca(2+) oscillations after membrane fusion by delivering a soluble protein into the egg cytoplasm. We previously identified sperm-specific phospholipase C (PLC)-ζ as a protein that can trigger the same pattern of Ca(2+) oscillations in eggs seen at fertilization.

Ca(2+) dynamics in oocytes from naturally-aged mice.

The ability of human metaphase-II arrested eggs to activate following fertilisation declines with advancing maternal age. Egg activation is triggered by repetitive increases in intracellular Ca(2+) concentration ([Ca(2+)]i) in the ooplasm as a result of sperm-egg fusion. We therefore hypothesised that eggs from older females feature a reduced ability to mount appropriate Ca(2+) responses at fertilisation.

Egg Activation at Fertilization by a Soluble Sperm Protein.

The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca(2+) concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca(2+) observed within the egg cytoplasm.

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2).

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca(2+) oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP2) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes.

Rescue of failed oocyte activation after ICSI in a mouse model of male factor infertility by recombinant phospholipase Cζ.

Artificial oocyte activation to overcome failed fertilization after intracytoplasmic sperm injection (ICSI) in human oocytes typically employs Ca(2+) ionophores to produce a single cytosolic Ca(2+) increase. In contrast, recombinant phospholipase Czeta (PLCζ) causes Ca(2+) oscillations indistinguishable from those occurring during fertilization, but remains untested for its efficacy in a scenario of ICSI fertilization failure. Here, we compare PLCζ with other activation stimuli in a mouse model of failed oocyte activation after ICSI, in which heat-treated sperm are injected into mouse oocytes.

Functional disparity between human PAWP and PLCζ in the generation of Ca2+ oscillations for oocyte activation.

Mammalian oocyte activation is mediated by cytosolic calcium (Ca(2+)) oscillations initiated upon delivery of a putative 'sperm factor' by the fertilizing sperm. Previous studies suggest the identity of this sperm factor as the testis-specific phospholipase C-zeta (PLCζ). Recently, a post-acrosomal sheath WW domain-binding protein (PAWP) has been proposed as an alternative sperm factor candidate, following a report that human PAWP protein and cRNA elicited Ca(2+) oscillations in mouse and human oocytes.