ABSTRACT
To assess the value of deep learning in selecting the optimal embryo for in vitro fertilization, a multicenter, randomized, double-blind, noninferiority parallel-group trial was conducted across 14 in vitro fertilization clinics in Australia and Europe. Women under 42 years of age with at least two early-stage blastocysts on day 5 were randomized to either the control arm, using standard morphological assessment, or the study arm, employing a deep learning algorithm, intelligent Data Analysis Score (iDAScore), for embryo selection. The primary endpoint was a clinical pregnancy rate with a noninferiority margin of 5%. The trial included 1,066 patients (533 in the iDAScore group and 533 in the morphology group). The iDAScore group exhibited a clinical pregnancy rate of 46.5% (248 of 533 patients), compared to 48.2% (257 of 533 patients) in the morphology arm (risk difference -1.7%; 95% confidence interval -7.7, 4.3; P = 0.62). This study was not able to demonstrate noninferiority of deep learning for clinical pregnancy rate when compared to standard morphology and a predefined prioritization scheme. Australian New Zealand Clinical Trials Registry (ANZCTR) registration: 379161 .
ABSTRACT
Preimplantation genetic testing for aneuploidies (PGT-A) is arguably the most effective embryo selection strategy. Nevertheless, it requires greater workload, costs, and expertise. Therefore, a quest towards user-friendly, non-invasive strategies is ongoing. Although insufficient to replace PGT-A, embryo morphological evaluation is significantly associated with embryonic competence, but scarcely reproducible. Recently, artificial intelligence-powered analyses have been proposed to objectify and automate image evaluations. iDAScore v1.0 is a deep-learning model based on a 3D convolutional neural network trained on time-lapse videos from implanted and non-implanted blastocysts. It is a decision support system for ranking blastocysts without manual input. This retrospective, pre-clinical, external validation included 3604 blastocysts and 808 euploid transfers from 1232 cycles. All blastocysts were retrospectively assessed through the iDAScore v1.0; therefore, it did not influence embryologists' decision-making process. iDAScore v1.0 was significantly associated with embryo morphology and competence, although AUCs for euploidy and live-birth prediction were 0.60 and 0.66, respectively, which is rather comparable to embryologists' performance. Nevertheless, iDAScore v1.0 is objective and reproducible, while embryologists' evaluations are not. In a retrospective simulation, iDAScore v1.0 would have ranked euploid blastocysts as top quality in 63% of cases with one or more euploid and aneuploid blastocysts, and it would have questioned embryologists' ranking in 48% of cases with two or more euploid blastocysts and one or more live birth. Therefore, iDAScore v1.0 may objectify embryologists' evaluations, but randomized controlled trials are required to assess its clinical value.
ABSTRACT
Cryopreservation of gametes and embryos is a growing technique in numerous reproductive fields including human-assisted reproduction. With improved understanding of embryo physiology and optimized culture conditions, there are now more embryos than ever to vitrify for potential use in subsequent cycles. Many gametes and embryos have been cryopreserved in open systems, but there are concerns with regard to contamination from the liquid nitrogen and also cross-contamination between patients' germplasm. The development of the Rapid-i™, a closed vitrification device that does not use direct contact with liquid nitrogen during vitrification or subsequent storage, will be discussed as well as clinical protocols for human oocytes and embryos.
Subject(s)
Cryopreservation/methods , Embryo, Mammalian , Vitrification , Blastocyst , Cryopreservation/instrumentation , Cryoprotective Agents , Female , Fertilization in Vitro , HumansABSTRACT
Cryopreservation facilitates long-term storage of gametes and embryos for numerous purposes. For example, cryobanking of unique mouse strains, particularly transgenic mice, offers important protection of valuable genetics. It also provides a practical solution for facilities trying to house large numbers of research animals or those looking to relocate without the risk of introducing an animal-derived pathogen. Furthermore, cryopreservation is currently being used for fertility preservation both in humans and as a safeguard for endangered animals. Ultrarapid vitrification offers an elegant, quick, and very reliable method for cryopreservation of mouse oocytes and embryos. Furthermore, research into the effects on mouse oocyte and embryo physiology has indicated that ultrarapid vitrification is superior to conventional slow freezing. High survival rates, embryo development, and viability are routinely achieved with the ultrarapid vitrification method described in this chapter.
Subject(s)
Embryonic Development , Oocytes/growth & development , Vitrification , Animals , Cryopreservation , Embryo, Mammalian/cytology , Mice , Molecular Biology/methodsABSTRACT
The cryopreservation of gametes and embryos is vital to numerous fields of reproductive biology, including assisted human reproduction. With improved culture conditions, there are an increasing number of embryos to cryopreserve for potential use in subsequent cycles. Many of the gametes and embryos in human IVF are cryopreserved in open systems. Because liquid nitrogen is not sterile, concerns have been raised with regard to contamination from the liquid nitrogen and also cross-contamination between patients' germplasm. Human gamete and embryo cryopreservation are discussed, with recommendations on how to minimize and eliminate contamination, emphasizing the benefits of closed vitrification devices.
ABSTRACT
OBJECTIVE: To develop a closed vitrification device (i.e., one that requires no direct contact with liquid nitrogen) for successful cryostorage of embryos. DESIGN: Prospective laboratory research study. SETTING: University-based research laboratory. ANIMAL(S): F1 mice and mouse embryos. INTERVENTION(S): Mouse embryos were vitrified using two methods and compared with nonvitrified controls. Embryos were vitrified on a device by either [1] presealing it within a straw before plunging into liquid nitrogen or [2] placing the straw into liquid nitrogen so that the air inside the straw is super-cooled before inserting the device holding the embryos. MAIN OUTCOME MEASURE(S): Survival, subsequent embryo development, and cell number were determined. Embryos were also cryopreserved for 12 months to assess long-term storage. Synchronized ETs were performed to compare viability with nonvitrified control embryos. RESULT(S): All embryos survived with both techniques. Day-4 and -5 embryo development was comparable between the two vitrification methods. Use of the presealing method resulted in a significantly lower mean cell number than the postsealing method and control. Long-term storage did not affect subsequent embryo development or cell number. The implantation and fetal development rates of embryos vitrified with super-cooled air were comparable to those for nonvitrified control embryos. CONCLUSION(S): These data demonstrate that a closed vitrification device (Rapid-i), which does not require direct liquid nitrogen contact for vitrification, is appropriate for vitrification and long-term storage of mouse embryos.
Subject(s)
Air , Cold Temperature , Embryonic Development/physiology , Mice/embryology , Vitrification , Animals , Embryo Transfer/methods , Female , Humans , Male , Pregnancy , Prospective StudiesABSTRACT
Male and female preimplantation mammalian embryos differ not only in their chromosomal complement, but in their proteome and subsequent metabolome. This phenomenon is due to a finite period during preimplantation development when both X chromosomes are active, between embryonic genome activation and X chromosome inactivation, around the blastocyst stage. Consequently, prior to implantation male and female embryos exhibit differences in their cellular phenotype. Manifestations of such differences include altered total activity of specific X-linked enzymes and the metabolic pathways they regulate. Subsequently, one would expect to be able to determine differences in the rate of consumption and utilization of specific nutrients between male and female embryos. Data to date on animal models support this, with sex-specific differences in glucose and amino acid utilization being reported for the mouse and cow blastocysts. Such differences in metabolic phenotype may logically be involved in the reported differences in growth rates between preimplantation embryos of different sex. As the fields of proteomics and metabolomics are being increasingly applied to human assisted conception it is prudent to consider how such technologies may be applied to identify sex differences in the human embryo. Such data would have implications far beyond current invasive technologies used to identify the sex of an embryo conceived in vitro for the diagnosis of X-linked diseases.
Subject(s)
Blastocyst/metabolism , Animals , Epigenesis, Genetic , Female , Humans , Male , Metabolome/physiology , Mice , Phenotype , Sex FactorsABSTRACT
OBJECTIVE: To examine the effect of exposure to 1,2-propanediol (PrOH) on the mammalian oocyte proteome. DESIGN: Experimental study. SETTING: Research laboratory. ANIMAL(S): Four- to five-week-old F1 (C57BL/6 x CBA/Ca) female mice. INTERVENTION(S): Analysis by time-of-flight mass spectrometry. MAIN OUTCOME MEASURE(S): Protein profiles of denuded mouse metaphase II (MII) oocytes. RESULT(S): Metaphase II oocytes exposed to 1.5 M PrOH for 20 minutes with or without seeding (i.e., equilibration, dehydration with or without seeding) exhibited significantly altered protein profiles in comparison to controls (i.e., 37 degrees C for 20 minutes and room temperature for 20 min). In total, 11 protein-biomarkers displayed profiles of statistically significantly lower abundance, whereas 8 protein-biomarkers were observed to be statistically significantly up-regulated. Protein profiles of MII oocytes maintained at room temperature for 20 minutes were more similar to those that were incubated for 20 minutes at 37 degrees C. CONCLUSION(S): Analysis of the mouse MII oocyte at different stages of the slow-freezing protocol revealed a negative impact on oocyte physiology after PrOH exposure. In contrast, temperature changes per se (during cooling to room temperature) have limited effect on the oocyte proteome.
Subject(s)
Cryoprotective Agents/adverse effects , Oocytes/drug effects , Oocytes/metabolism , Propylene Glycol/adverse effects , Proteome/drug effects , Animals , Cryopreservation , Female , Freezing , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Oocytes/physiology , Time FactorsABSTRACT
In contrast to the preimplantation mammalian embryo, it has been notoriously difficult to cryopreserve the metaphase II oocyte. The ability to store oocytes successfully at -196 degrees C has numerous practical and financial advantages, together with ethical considerations, and will positively impact animal breeding programs and assisted conception in the human. Differences in membrane permeability and in physiology are two main reasons why successful oocyte cryopreservation has remained elusive. It is proposed, therefore, that rather than relying on technologies already established for the preimplantation embryo, the development of cryopreservation techniques suitable for the mammalian oocyte needs to take into account the idiosyncratic physiology of this cell. Analysis of intracellular calcium, for example, has revealed that exposure to conventional permeating cryoprotectants, such as propanediol, ethylene glycol and DMSO, all independently result in an increase in calcium, which in turn has the potential to initiate oocyte activation, culminating in zona hardening. Quantification of the metabolome and proteome of the oocyte has revealed that whereas slow freezing has a dramatic effect on cell physiology, vitrification appears to have limited effect. This is plausibly achieved by the limited exposure to cryoprotectants. Analysis of meiotic spindle dynamics and embryo development following IVF, also indicate that vitrification is less traumatic than slow freezing, and therefore has the greatest potential for successful oocyte cryopreservation.
Subject(s)
Breeding , Cryopreservation/standards , Meiosis/physiology , Oocytes/physiology , Animals , Calcium/metabolism , Calcium Signaling , Cryopreservation/methods , Cryopreservation/veterinary , Cryoprotective Agents/adverse effects , Cryoprotective Agents/pharmacology , Energy Metabolism/physiology , Humans , Oocytes/cytology , Oocytes/metabolism , Time FactorsABSTRACT
The ability to routinely cryopreserve human oocytes and embryos represents a significant advancement in the field of assisted reproductive technology. Although the method of slow freezing is commonly employed, research on the alternative technique of vitrification is promising. Vitrification involves incubation of the cell in a cryoprotectant rich solution, which permits a glass-like state to occur almost instantaneously in liquid nitrogen. A number of different techniques have been invented for holding oocytes and embryos in the cryoprotectant solution during rapid vitrification and subsequent storage. Most of these involve direct contact with liquid nitrogen. Recently, concerns have been raised regarding the sterility of such a method and the potential of viral contamination from the liquid nitrogen. The present study shows that the previously reported Cryoloop method can be used to vitrify and store embryos without direct liquid nitrogen contact (during vitrification and storage). When such vitrified embryos are warmed, they are capable of subsequent development comparable with non-vitrified embryos.
Subject(s)
Cryopreservation/methods , Cryoprotective Agents/chemistry , Culture Media/chemistry , Oocytes , Reproductive Techniques, Assisted , Analysis of Variance , Animals , Cryopreservation/instrumentation , Female , Mice , Nitrogen , Time FactorsABSTRACT
Despite the success of embryo cyropreservation, routine oocyte freezing has proved elusive with only around 200 children born since the first reported birth in 1986. The reason for the poor efficiency is unclear, but evidence of zona pellucida hardening following oocyte freezing indicates that current protocols affect oocyte physiology. Here we report that two cryoprotectants commonly used in vitrification procedures, dimethyl sulfoxide (DMSO) and ethylene glycol, cause a large transient increase in intracellular calcium concentration in mouse metaphase II (MII) oocytes comparable to the initial increase triggered at fertilization. Removal of extracellular calcium from the medium failed to affect the response exacted by DMSO challenge, but significantly reduced the ethylene glycol-induced calcium increase. These results suggest that the source of the DMSO-induced calcium increase is solely from the internal calcium pool, as opposed to ethylene glycol that causes an influx of calcium across the plasma membrane from the external medium. By carrying out vitrification in calcium-free media, it was found that zona hardening is significantly reduced and subsequent fertilization and development to the two-cell stage significantly increased. Furthermore, such calcium-free treatment appears not to affect the embryo adversely, as shown by development rates to the blastocyst stage and cell number/allocation. Since zona hardening is one of the early activation events normally triggered by the sperm-induced calcium increases observed at fertilization, it is possible that other processes are negatively affected by the calcium rise caused by cryoprotectants used during oocyte freezing, which might explain the current poor efficiency of this technique.
Subject(s)
Calcium/metabolism , Cryopreservation/methods , Cryoprotective Agents/pharmacology , Zona Pellucida/metabolism , Animals , Calcium/analysis , Chelating Agents/pharmacology , Cleavage Stage, Ovum , Dimethyl Sulfoxide/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Embryonic Development , Ethylene Glycol/pharmacology , Female , Fertilization in Vitro , Intracellular Fluid/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Sperm-Ovum Interactions , Zona Pellucida/drug effectsABSTRACT
An increase in intracellular Ca2+ concentration ([Ca2+]i) has been shown to drive sea-urchin embryos and some fibroblasts through nuclear-envelope breakdown (NEBD) and the metaphase-to-anaphase transition. Mitotic Ca2+ transients can be pan-cellular global events or localized to the perinuclear region. It is not known whether Ca2+ is a universal regulator of mitosis or whether its role is confined to specific cell types. To test the hypothesis that Ca2+ is a universal regulator of mitosis, we have investigated the role of Ca2+ in mitosis in one-cell mouse embryos. Fertilized embryos generate Ca2+ transients during the first mitotic division. Imposing a Ca2+ transient by photorelease of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] resulted in acceleration of mitosis entry, suggesting that a [Ca2+]i increase is capable of triggering mitosis. Mitotic Ca2+ transients were inhibited using three independent approaches: injection of intracellular Ca2+ buffers; downregulation of Ins(1,4,5)P3 receptors; and removal of extracellular Ca2+. None of the interventions had any effects on the timing of NEBD or cytokinesis. The possibility that NEBD is driven by localized perinuclear Ca2+ transients was examined using two-photon microscopy but no Ca2+-dependent increases in fluorescence were found to precede NEBD. Finally, the second mitotic division took place in the absence of any detectable [Ca2+]i increase. Thus, although an induced [Ca2+]i increase can accelerate mitosis entry, neither cytosolic nor perinuclear [Ca2+] increases appear to be necessary for progression through mitosis in mouse embryos.
Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/physiology , Mitosis/physiology , Animals , Calcium Channels/metabolism , Chelating Agents/chemistry , Chelating Agents/metabolism , Female , Fertilization/physiology , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Fura-2/chemistry , Fura-2/metabolism , Inositol 1,4,5-Trisphosphate/chemistry , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate Receptors , Mice , Microinjections , Microscopy, Fluorescence , Receptors, Cytoplasmic and Nuclear/metabolismABSTRACT
The sperm-specific phospholipase C-zeta (PLCzeta) elicits fertilization-like Ca2+ oscillations and activation of embryo development when microinjected into mammalian eggs (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development (Camb.) 129, 3533-3544; Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623). PLCzeta may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLCzeta is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLCzeta structure-function, we assessed the ability of PLCzeta and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca2+ changes in intact mouse eggs. PLCzeta and the closely related PLCdelta1 had similar K(m) values for phosphatidylinositol 4,5-bisphosphate, but PLCzeta was around 100 times more sensitive to Ca2+ than was PLCdelta1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLCzeta constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca2+ sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca2+ oscillations. These data suggest that the exquisite Ca2+-dependent features of PLCzeta regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca2+ oscillations in intact mouse eggs.
Subject(s)
Calcium/metabolism , Cytoplasm/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Protein Conformation , Type C Phospholipases/chemistry , Type C Phospholipases/metabolism , Animals , Cytoplasm/chemistry , Female , Humans , Hydrogen-Ion Concentration , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Microinjections , Molecular Sequence Data , Oocytes/cytology , Oocytes/physiology , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphoinositide Phospholipase C , Phospholipase C delta , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Type C Phospholipases/geneticsABSTRACT
A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently, MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos, both kinases show a similar activation profile, peaking at the time of mitosis entry. We tested whether the activity of both kinases is required for mitosis entry and whether either kinase controls mitotic calcium signals. We found that reducing the activity of either mitotic kinase prevents nuclear envelope breakdown, despite the presence of a calcium transient, when cdk1/cyclin B kinase activity is alone inhibited. When MAP kinase activity alone was inhibited, the calcium signal was absent, suggesting that MAP kinase activity is required to generate the calcium transient that triggers nuclear envelope breakdown. However, increasing intracellular free calcium by microinjection of calcium buffers or InsP(3) while MAP kinase was inhibited did not itself induce nuclear envelope breakdown, indicating that additional MAP kinase-regulated events are necessary. After MAP kinase inhibition early in the cell cycle, the early events of the cell cycle (pronuclear migration/fusion and DNA synthesis) were unaffected, but chromosome condensation and spindle assembly are prevented. These data indicate that in sea urchin embryos, MAP kinase activity is part of a signaling complex alongside two components previously shown to be essential for entry into mitosis: the calcium transient and the increase in cdk1/cyclinB kinase activity.
Subject(s)
Calcium/metabolism , MAP Kinase Signaling System , Mitosis , Animals , Blotting, Western , Butadienes/pharmacology , CDC2 Protein Kinase/metabolism , Calcium/chemistry , Cell Nucleus/metabolism , Chromatin/chemistry , Chromosomes/ultrastructure , Cyclin B/metabolism , Dextrans/pharmacology , Dose-Response Relationship, Drug , Egtazic Acid/chemistry , Enzyme Inhibitors/pharmacology , Fertilization , Immunoprecipitation , Lytechinus , Mitogen-Activated Protein Kinase Phosphatases , Nitriles/pharmacology , Protein Tyrosine Phosphatases/metabolism , Purines/pharmacology , Recombinant Fusion Proteins/chemistry , Roscovitine , Time Factors , Xenopus Proteins/metabolismABSTRACT
Fertilization of sea urchin eggs results in a large, transient increase in intracellular free Ca2+ concentration that is responsible for re-initiation of the cell division cycle. We show that activation of ERK1, a Ca2+-dependent MAP kinase response, is required for both DNA synthesis and cell cycle progression after fertilization. We combine experiments on populations of cells with analysis at the single cell level, and develop a proxy assay for DNA synthesis in single embryos, using GFP-PCNA. We compare the effects of low molecular weight inhibitors with a recombinant approach targeting the same signalling pathway. We find that inhibition of the ERK pathway at fertilization using either recombinant ERK phosphatase or U0126, a MEK inhibitor, prevents accumulation of GFP-PCNA in the zygote nucleus and that U0126 prevents incorporation of [3H]-thymidine into DNA. Abrogation of the ERK1 signalling pathway also prevents chromatin decondensation of the sperm chromatin after pronuclear fusion, nuclear envelope breakdown and formation of a bipolar spindle.
Subject(s)
Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/enzymology , Fertilization , Mitogen-Activated Protein Kinase 3/metabolism , S Phase , Sea Urchins/embryology , Sea Urchins/enzymology , Animals , Aphidicolin/pharmacology , Butadienes/pharmacology , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/metabolism , Calcimycin/pharmacology , Cell Nucleus/metabolism , Chromatin/drug effects , Chromatin/metabolism , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Enzyme Activation/drug effects , Female , Flavonoids/pharmacology , MAP Kinase Signaling System , Male , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase Phosphatases , Nitriles/pharmacology , Ovum/drug effects , Ovum/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/metabolism , Protein Tyrosine Phosphatases/pharmacology , Recombinant Proteins/metabolism , S Phase/drug effects , Sea Urchins/cytology , Spermatozoa/drug effects , Spermatozoa/metabolism , Xenopus Proteins/pharmacologyABSTRACT
During the first cell cycle Ca2+ oscillations are regulated in a cell cycle-dependent manner, such that the oscillations are unique to M phase. How the Ca2+ oscillations are regulated with such cell cycle stage-dependency is unknown, despite their importance for egg activation and embryo development. We recently identified a novel, sperm-specific phospholipase C (PLCzeta; PLCzeta) that triggers Ca2+ oscillations similar to those caused by sperm. We show that PLCzeta-induced Ca2+ oscillations also occur exclusively during M phase. The cell cycle-dependency can be explained by PLCzeta's localisation to the pronuclei, which depends specifically upon a nuclear localisation signal sequence. Preventing pronuclear localisation of PLCzeta by mutation of the nuclear localisation signal, or by inhibiting pronuclear formation/import, can prolong Ca2+ oscillations or allow them to occur during interphase. These data suggest a novel mechanism for regulating a PLC through nuclear sequestration and may explain the cell cycle-dependent regulation of Ca2+ oscillations following fertilisation.
Subject(s)
Calcium/metabolism , Cell Cycle/physiology , Cell Nucleus/metabolism , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Type C Phospholipases/metabolism , Amino Acid Substitution , Animals , Calcium/analysis , Cell Division , Cells, Cultured , Female , Fluorescein-5-isothiocyanate , Fluorescent Antibody Technique, Direct , Fluorescent Dyes , Glutamine/metabolism , Mice , Mice, Inbred Strains , Microinjections , Microscopy, Confocal , Models, Biological , Oocytes/metabolism , Parthenogenesis , Phosphoinositide Phospholipase C , Type C Phospholipases/geneticsABSTRACT
Established studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. In echinoderms, egg activation appears to involve an egg phospholipase C gamma (PLCgamma). However, numerous studies in mammalian species suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific PLC isoform with distinctive properties (PLCzeta). Homologues of PLCzeta have since been isolated from human and cynomolgus monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species such as chicken, Xenopus, and a flowering plant. Here we report evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia Oreochromis niloticus (L). Using an established bioassay for calcium release, the sea urchin egg homogenate, we demonstrate that protein extracts obtained from tilapia spermatozoa exhibit PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, tilapia sperm extracts induced calcium oscillations when injected into mouse oocytes.
Subject(s)
Calcium/metabolism , Oocytes/metabolism , Ovum/metabolism , Spermatozoa/enzymology , Tilapia/embryology , Type C Phospholipases/metabolism , Animals , Biological Assay , Calcium Signaling , Cell Extracts/administration & dosage , Cell Extracts/pharmacology , Cytosol/enzymology , Injections , Kinetics , Male , Mice , Ovum/drug effects , Proteins/administration & dosage , Proteins/analysis , Proteins/pharmacology , Sea Urchins/embryology , Spermatozoa/chemistry , Type C Phospholipases/analysisABSTRACT
Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.