SPERM FACTORS AND EGG ACTIVATION: ICSI and the discovery of the sperm factor and PLCZ1.

The discovery of PLCZ1 nearly 20 years ago as the primary Ca2+ oscillation-inducing factor in the sperm of mammals represented a significant breakthrough in our quest to elucidate the molecules and pathways that promote egg activation during fertilization. The advent of the intracytoplasmic sperm injection (ICSI) technique, which made fertilization possible without sperm capacitation, acrosome reaction, and gamete fusion, strengthened the research that led to the discovery of PLCZ1 and became an essential clinical tool for humans. The use of ICSI combined with the detection of PLCZ1 expression and mutations in infertile patients established the fundamental role of PLCZ1 in human fertility while leading to the discovery of novel components of the perinuclear theca, the site of the residence of PLCZ1 in sperm before fertilization. Remarkably, the more extensive use of ICSI in species other than humans and mice revealed poor success and exposed gaps in our understanding of PLCZ1 release and/or activation. Similarly, fertilization using sperm from mouse models lacking Plcz1 has produced striking results whose true implications are yet to be determined. Nevertheless, answers to these unresolved questions will produce a complete picture of the adaptations and molecular players that mammalian species employ to ensure the success of the triggering event of embryo development that has linked generations since the beginning of times.

Constitutive IP3R1-mediated Ca2+ release reduces Ca2+ store content and stimulates mitochondrial metabolism in mouse GV oocytes.

In mammals, fertilization initiates Ca2+ oscillations in metaphase II oocytes, which are required for the activation of embryo development. Germinal vesicle (GV) oocytes also display Ca2+ 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 Ca2+ in GV oocytes, the endoplasmic reticulum ([Ca2+]ER), constitutively 'leaks' Ca2+ through the type 1 inositol 1,4,5-trisphosphate receptor. The [Ca2+]ER leak ceases around the resumption of meiosis, the GV breakdown (GVBD) stage, which coincides with the first noticeable accumulation of Ca2+ in the stores. It also concurs with downregulation of the Ca2+ influx and termination of the oscillations, which seemed underpinned by the inactivation of the putative plasma membrane Ca2+ channels. Lastly, we demonstrate that mitochondria take up Ca2+ during the Ca2+ oscillations, mounting their own oscillations that stimulate the mitochondrial redox state and increase the ATP levels of GV oocytes. These distinct features of Ca2+ 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.

Metabolic activation of CaMKII by coenzyme A.

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.

TRPM7 and CaV3.2 channels mediate Ca2+ influx required for egg activation at fertilization.

The success of mammalian development following fertilization depends on a series of transient increases in egg cytoplasmic Ca2+, referred to as Ca2+ oscillations. Maintenance of these oscillations requires Ca2+ influx across the plasma membrane, which is mediated in part by T-type, CaV3.2 channels. Here we show using genetic mouse models that TRPM7 channels are required to support this Ca2+ influx. Eggs lacking both TRPM7 and CaV3.2 stop oscillating prematurely, indicating that together they are responsible for the majority of Ca2+ influx immediately following fertilization. Fertilized eggs lacking both channels also frequently display delayed resumption of Ca2+ oscillations, which appears to require sperm-egg fusion. TRPM7 and CaV3.2 channels almost completely account for Ca2+ influx observed following store depletion, a process previously attributed to canonical store-operated Ca2+ entry mediated by STIM/ORAI interactions. TRPM7 serves as a membrane sensor of extracellular Mg2+ and Ca2+ concentrations and mediates the effects of these ions on Ca2+ oscillation frequency. When bred to wild-type males, female mice carrying eggs lacking TRPM7 and CaV3.2 are subfertile, and their offspring have increased variance in postnatal weight. These in vivo findings confirm previous observations linking in vitro experimental alterations in Ca2+ oscillatory patterns with developmental potential and offspring growth. The identification of TRPM7 and CaV3.2 as key mediators of Ca2+ 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.

FRET-based sensor for CaMKII activity (FRESCA): A useful tool for assessing CaMKII activity in response to Ca2+ oscillations in live cells.

Ca2+ oscillations and consequent Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation are required for embryogenesis, as well as neuronal, immunological, and cardiac signaling. Fertilization directly results in Ca2+ 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 Ca2+ 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 Sr2+, 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.

Compensatory endocytosis occurs after cortical granule exocytosis in mouse eggs.

Compensatory endocytosis (CE) is one of the primary mechanisms through which cells maintain their surface area after exocytosis. Considering that in eggs massive exocytosis of cortical granules (CG) takes place after fertilization, the aim of this study was to evaluate the occurrence of CE following cortical exocytosis in mouse eggs. For this purpose, we developed a pulse-chase assay to detect CG membrane internalization. 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 is dependent on actin dynamics and dynamin activity, and could be associated with a transient exposure of phosphatidylserine. Finally, CE was impaired in A23187 ionophore-activated eggs, highlighting once again the mechanistic differences between the activation methods. Altogether, these results demonstrate for the first time that egg activation triggers CE in mouse eggs after exocytosis of CG, probably as a plasma membrane homeostasis mechanism.

The eggstraordinary story of how life begins.

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.

Molecular Characterization and Comparison of Phospholipase C zeta (PLCZ1) Gene Between Swamp (Bubalus carabanensis) and Riverine (Bubalus bubalis) Buffaloes: Its Implications and Future Perspectives.

Phospholipase C zeta, a novel sperm-specific protein which is widely known to induce oocyte activation following fertilization, had already been characterized in various mammalian species, but not in water buffaloes thus far. The present study was conducted to initially characterize and compare the sequences of PLCZ1 gene of swamp and riverine buffaloes. Semen samples were collected; total RNA was extracted and reverse-transcribed. PLCZ1 cDNA was then amplified, and submitted for sequencing. Buffalo PLCZ1 gene yielded a sequence of 1905 base pair nucleotides translated into 634 bp amino acids. In general, the buffalo PLCZ1 gene was found to have high sequence identity with cattle and other domestic species. Similarly, significant residues and motifs in PLCZ1 gene sequence are found conserved in water buffaloes. However, there are variations in sequences identified between types of water buffaloes that may play a role in species-specific differences in terms of gene and protein expression, physiological mechanisms, and biological functions. The molecular information on buffalo PLCZ1 gene is highly valuable in subsequent works such as correlation studies on the identified gene variations with semen quality and fertility, and the development of biomarkers for bull fertility.

Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs.

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.

Conotoxins as Tools to Understand the Physiological Function of Voltage-Gated Calcium (CaV) Channels.

Voltage-gated calcium (CaV) channels are widely expressed and are essential for the completion of multiple physiological processes. Close regulation of their activity by specific inhibitors and agonists become fundamental to understand their role in cellular homeostasis as well as in human tissues and organs. CaV channels are divided into two groups depending on the membrane potential required to activate them: High-voltage activated (HVA, CaV1.1-1.4; CaV2.1-2.3) and Low-voltage activated (LVA, CaV3.1-3.3). HVA channels are highly expressed in brain (neurons), heart, and adrenal medulla (chromaffin cells), among others, and are also classified into subtypes which can be distinguished using pharmacological approaches. Cone snails are marine gastropods that capture their prey by injecting venom, "conopeptides", which cause paralysis in a few seconds. A subset of conopeptides called conotoxins are relatively small polypeptides, rich in disulfide bonds, that target ion channels, transporters and receptors localized at the neuromuscular system of the animal target. In this review, we describe the structure and properties of conotoxins that selectively block HVA calcium channels. We compare their potency on several HVA channel subtypes, emphasizing neuronal calcium channels. Lastly, we analyze recent advances in the therapeutic use of conotoxins for medical treatments.

TMTC1 and TMTC2 are novel endoplasmic reticulum tetratricopeptide repeat-containing adapter proteins involved in calcium homeostasis.

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.

Regulation of endoplasmic reticulum Ca(2+) oscillations in mammalian eggs.

Changes in the intracellular concentration of free calcium ([Ca(2+)]i) regulate diverse cellular processes including fertilization. In mammalian eggs, the [Ca(2+)]i changes induced by the sperm unfold in a pattern of periodical rises, also known as [Ca(2+)]i oscillations. The source of Ca(2+) during oscillations is the endoplasmic reticulum ([Ca(2+)]ER), but it is presently unknown how [Ca(2+)]ER is regulated. Here, we show using mouse eggs that [Ca(2+)]i oscillations induced by a variety of agonists, including PLCζ, SrCl2 and thimerosal, provoke simultaneous but opposite changes in [Ca(2+)]ER and cause differential effects on the refilling and overall load of [Ca(2+)]ER. We also found that Ca(2+) influx is required to refill [Ca(2+)]ER, because the loss of [Ca(2+)]ER was accelerated in medium devoid of Ca(2+). Pharmacological inactivation of the function of the mitochondria and of the Ca(2+)-ATPase pumps PMCA and SERCA altered the pattern of oscillations and abruptly reduced [Ca(2+)]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 [Ca(2+)]i rises, promotes initiation of oscillations and enhances refilling of [Ca(2+)]ER. Collectively, our results provide novel insights on the regulation of [Ca(2+)]ER oscillations, which underlie the unique Ca(2+)-signalling system that activates the developmental program in mammalian eggs.

Role of caspase-3 cleaved IP3 R1 on Ca(2+) homeostasis and developmental competence of mouse oocytes and eggs.

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.

Protein phospholipase C Zeta1 expression in patients with failed ICSI but with normal sperm parameters.

PURPOSE: 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). METHODS: Couples <43 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. RESULTS: 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. CONCLUSION: 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.

Zn2+ is Essential for Ca2+ Oscillations in Mouse Eggs.

Changes in the intracellular concentration of free calcium (Ca2+) underpin egg activation and initiation of development in animals and plants. In mammals, the Ca2+ release is periodical, known as Ca2+ oscillations, and mediated by the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). Another divalent cation, zinc (Zn2+), increases exponentially during oocyte maturation and is vital for meiotic transitions, arrests, and polyspermy prevention. It is unknown if these pivotal cations interplay during fertilization. Here, using mouse eggs, we showed that basal concentrations of labile Zn2+ are indispensable for sperm-initiated Ca2+ oscillations because Zn2+-deficient conditions induced by cell-permeable chelators abrogated Ca2+ responses evoked by fertilization and other physiological and pharmacological agonists. We also found that chemically- or genetically generated eggs with lower levels of labile Zn2+ displayed reduced IP3R1 sensitivity and diminished ER Ca2+ leak despite the stable content of the stores and IP3R1 mass. Resupplying Zn2+ restarted Ca2+ oscillations, but excessive Zn2+ prevented and terminated them, hindering IP3R1 responsiveness. The findings suggest that a window of Zn2+ concentrations is required for Ca2+ responses and IP3R1 function in eggs, ensuring optimal response to fertilization and egg activation.

Zn2+ is essential for Ca2+ oscillations in mouse eggs.

Changes in the intracellular concentration of free calcium (Ca2+) underpin egg activation and initiation of development in animals and plants. In mammals, the Ca2+ release is periodical, known as Ca2+ oscillations, and mediated by the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). Another divalent cation, zinc (Zn2+), increases exponentially during oocyte maturation and is vital for meiotic transitions, arrests, and polyspermy prevention. It is unknown if these pivotal cations interplay during fertilization. Here, using mouse eggs, we showed that basal concentrations of labile Zn2+ are indispensable for sperm-initiated Ca2+ oscillations because Zn2+-deficient conditions induced by cell-permeable chelators abrogated Ca2+ responses evoked by fertilization and other physiological and pharmacological agonists. We also found that chemically or genetically generated eggs with lower levels of labile Zn2+ displayed reduced IP3R1 sensitivity and diminished ER Ca2+ leak despite the stable content of the stores and IP3R1 mass. Resupplying Zn2+ restarted Ca2+ oscillations, but excessive Zn2+ prevented and terminated them, hindering IP3R1 responsiveness. The findings suggest that a window of Zn2+ concentrations is required for Ca2+ responses and IP3R1 function in eggs, ensuring optimal response to fertilization and egg activation.

Essential role of Mg2+ in mouse preimplantation embryo development revealed by TRPM7 chanzyme-deficient gametes.

TRPM7 (transient receptor potential cation channel subfamily M member 7) is a chanzyme with channel and kinase domains essential for embryo development. Using gamete-specific Trpm7-null lines, we report that TRPM7-mediated Mg2+ influx is indispensable for reaching the blastocyst stage. TRPM7 is expressed dynamically from gametes to blastocysts; displays stage-specific localization on the plasma membrane, cytoplasm, and nucleus; and undergoes cleavage that produces C-terminal kinase fragments. TRPM7 underpins Mg2+ homeostasis, and excess Mg2+ but not Zn2+ or Ca2+ overcomes the arrest of Trpm7-null embryos; expressing Trpm7 mRNA restores development, but mutant versions fail or are partially rescued. Transcriptomic analyses of Trpm7-null embryos reveal an abundance of oxidative stress-pathway genes, confirmed by mitochondrial dysfunction, and a reduction in transcription factor networks essential for proliferation; Mg2+ supplementation corrects these defects. Hence, TRPM7 underpins Mg2+ homeostasis in preimplantation embryos, prevents oxidative stress, and promotes gene expression patterns necessary for developmental progression and cell-lineage specification.

Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation.

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.

A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility.

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.

Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes.

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.