Mammalian Eggs Research Articles

Structural characterization of fish egg vitelline envelope proteins by mass spectrometry.

The extracellular coat, or vitelline envelope (VE), of rainbow trout (Oncorhynchus mykiss) eggs consists of three proteins, called VEalpha (M(r) approximately 52 kDa), VEbeta (M(r) approximately 48 kDa), and VEgamma (M(r) approximately 44 kDa). Each of these proteins is related to mammalian egg zona pellucida (ZP) glycoproteins ZP1-3 and possesses an N-terminal signal sequence, a ZP domain, and a protease cleavage site near the C-terminus. VEalpha and VEbeta also have a trefoil domain. All three proteins possess a relatively large number of cysteine residues (VEalpha, 18; VEbeta, 18; VEgamma, 12), of which 8 are present in the ZP domain and 6 are present in the trefoil domain of VEalpha and VEbeta. Here, several types of mass spectrometry were employed, together with gel electrophoresis of chemical and enzymatic digests, to identify intramolecular disulfide linkages, as well as the N- and C-terminal amino acids of VEalpha, VEbeta, and VEgamma. Additionally, these methods were used to characterize two high molecular weight proteins (HMWPs; M(r) > 110 kDa) of rainbow trout VEs that are heterodimers of individual VE proteins. These analyses have permitted assignment of disulfide linkages and identification of N- and C-terminal amino acids for the VE proteins and determination of the protein composition of two forms of HMWPs. These experiments provide important structural information about fish egg VE proteins and filaments and about structural relationships between extracellular coat proteins of mammalian and nonmammalian eggs.

Texture of the zona pellucida of the mature pig oocyte. The mammalian egg envelope revisited.

The zona pellucida (ZP) of mature pig oocytes is believed to consist of a dense filamentous meshwork, less compact on the inner and outer faces. The uneven surface of the ZP is made of unordered and stretched fibrils surrounding deep funnels which are the openings of the radial canaliculi. The topography of the ZP surface may contribute to the initial interplay between male and female gametes. Using cytochemical techniques for transmission electron microscopy (TEM), such as tannic acid and ruthenium red treatments, we found that the ZP of pig oocytes was essentially made of bundles of fibrils distributed in concentric layers (except in the innermost and outer parts). A correlation appears between the dense structure of the core layer of the ZP and its texture: it is constituted of superposed layers of fibril bundles, whereas only a random meshwork is found in a very thin innermost and in the outer layer. The fascicular configuration may control the permeability of the ZP, giving its semi-rigidity and elasticity, and may facilitate sperm penetration. The liquid crystal-like design of the core layer of the ZP is similar to textures found in the the vitelline envelope (zona radiata) of other vertebrates and possibly of all the deuterostomes. Such texture is probably related to the unique ZP protein composition and to a coordinated synthesis.

Are Src family kinases involved in cell cycle resumption in rat eggs?

The earliest visible indications for the transition to embryos in mammalian eggs, known as egg activation, are cortical granules exocytosis (CGE) and resumption of meiosis (RM); these events are triggered by the fertilizing spermatozoon through a series of Ca2+ transients. The pathways, within the egg, leading to the intracellular Ca2+ release and to the downstream cellular events, are currently under intensive investigation. The involvement of Src family kinases (SFKs) in Ca2+ release at fertilization is well supported in marine invertebrate eggs but not in mammalian eggs. In a previous study we have shown the expression and localization of Fyn, the first SFK member demonstrated in the mammalian egg. The purpose of the current study was to identify other common SFKs and resolve their function during activation of mammalian eggs. All three kinases examined: Fyn, c-Src and c-Yes are distributed throughout the egg cytoplasm. However, Fyn and c-Yes tend to concentrate at the egg cortex, though only Fyn is localized to the spindle as well. The different localizations of the various SFKs imply the possibility of their different functions within the egg. To examine whether SFKs participate in the signal transduction pathways during egg activation, we employed selective inhibitors of the SFKs activity (PP2 and SU6656). The results demonstrate that RM, which is triggered by Ca2+ elevation, is an SFK-dependent process, while CGE, triggered by either Ca2+ elevation or protein kinase C (PKC), is not. The possible involvement of SFKs in the signal transduction pathways that lead from the sperm-egg fusion site downstream of the Ca2+ release remains unclear.

Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs.

In mammalian eggs, sperm-induced Ca2+ oscillations at fertilization are the primary trigger for egg activation and initiation of embryonic development. Identifying the downstream effectors that decode this unique Ca2+ signal is essential to understand how the transition from egg to embryo is coordinated. Here, we investigated whether conventional PKCs (cPKCs) can decode Ca2+ oscillations at fertilization. By monitoring the dynamics of GFP-labeled PKCα and PKCγ in living mouse eggs, we demonstrate that cPKCs translocate to the egg membrane at fertilization following a pattern that is shaped by the amplitude, duration, and frequency of the Ca2+ transients. In addition, we show that cPKC translocation is driven by the C2 domain when Ca2+ concentration reaches 1–3 μM. Finally, we present evidence that one physiological function of activated cPKCs in fertilized eggs is to sustain long-lasting Ca2+ oscillations, presumably via the regulation of store-operated Ca2+ entry.

Use of Mammalian eggs for assessment of human sperm function: molecular and cellular analyses of fertilization by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI ) has been described as the 'cure' for male sterility because a single sperm can now be directly introduced into an egg with some chance of pregnancy. While ICSI has revolutionized the practice of assisted reproductive techniques (ART), there are few molecular and cellular studies about its safety and efficacy. Even by using ICSI, fertilization in humans succeeds only if the sperm effectively accomplishes a number of tasks including 'post-ICSI events' in fertilization. To assess the function of human sperm after ICSI, we used heterologous ICSI with human sperm into animal eggs. Egg activation, sperm decondensation and sperm centorosomal function were examined in sperm from fertile men and infertile patients. Sperm from fertile men and infertile patients were injected into hamster, rabbit and bovine eggs by Piezo micromanipulator, and studied in decondensation of sperm nuclei, egg activation and microtubule organization. Decondensation human sperm head following ICSI into hamster eggs occurred initially form basal lesion, and apical portion of sperm nuclei which is surrounded by acrosome and perinuclear theca, still condensed in early pronuclear stage. Radial array of microtubules from sperm centrosome 'sperm aster' which is essential for pronuclear movement was observed in 30% rabbit eggs following ICSI with human sperm. By heterologous ICSI system with fertile human sperm and bovine eggs, 83.3% of eggs was activated and 60% eggs had sperm aster, indicating that bovine Piezo ICSI system is appropriate for assessing human sperm oocyte activation ability and human sperm centrosomal function. Oocyte activation and sperm centrosomal function were significantly low in sperm from men with globozoospermia and men with dysplasia of fibrous sheath. These assays indicate differences of the process of fertilization between in vitro fertilization and ICSI, and reflect the human sperm function especially for the 'post-ICSI events' in fertilization. More molecular and cellular analyses in fertilization by ICSI are needed for improvement of ART.

Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ

In mammalian eggs, the fertilizing sperm evokes intracellular Ca 2+ ([Ca 2+] i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca 2+] 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 [Ca 2+] i oscillations. Based on findings showing that production of inositol 1,4,5-trisphosphate (IP 3) underlies the generation of [Ca 2+] i oscillations, it has been suggested that SF functions either as a phospholipase C (PLC), an enzyme that catalyzes the generation of IP 3, 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 IP 3 and the initiator of [Ca 2+] i oscillations in mammalian fertilization, with particular emphasis on the possible involvement of egg- and sperm-derived PLCs, including PLCζ, a novel sperm specific PLC.

The Art of ICSI: Animal Eggs

In this paper, we show the technical art of ICSI at our laboratory. We show the basic principal of ICSI on mammalian eggs, especially mouse and bovine ICSI. The use of Piezo-micromanipulator is useful for mouse and bovine ICSI, and how to use of piezo-electric actuator is shown carefully in this paper. The important point of mouse ICSI is the survival of eggs after sperm injection. On the other hand, in bovine ICSI, the accurate sperm injection and the less injection volume by using the piezo-electric actuator are important for successful bovine ICSI.

208.Zona pellucida vaccines for fertility control of brushtail possums in New Zealand

Introduced marsupial brushtail possums (Trichosurus vulpecula) are a major pest in New Zealand because of their impacts on conservation values and agricultural production. Immunologically-based fertility control (immunocontraception) offers an effective and humane alternative approach to possum management. The zona pellucida (ZP) is an extracellular coat around all mammalian eggs and an attractive target for the development of immunocontraceptive vaccines. Antibodies against ZP are ovary-specific and act by preventing sperm from binding and penetrating the ova and/or by disrupting the development of follicles or early embryo. The aim of these studies was to test the efficacy of possum-derived ZP antigens for their ability to elicit sustained immune responses and cause infertility, and to assess a range of options for development of a bait-delivered contraceptive vaccine. Alloimmunisation with possum ZP2 and ZP3 proteins showed that self-ZP antigens elicited strong humoral immune responses and reduced the fertility of female possums by 72-80%. Several potentially possum-specific immunocontraceptive peptides have been identified by linear epitope mapping and amino acid alignment and are being tested for their ability to reduce fertility. Recent trials have demonstrated that possums mount immune responses against ZP antigens delivered in transgenic plants and bacterial ghosts. Research on antigen and specific peptide identification, non-target effects and delivery systems is ongoing. Research supported by NZ Foundation for�Research Science & Technology, Marsupial CRC and NZ Animal Health Board.

Cytoplasmic transfer in Mammalian eggs and embryos.

Cytoplasmic transfer in Mammalian eggs and embryos.

011.Waking up the egg. How the sperm regulates exit out of the meiotic cell cycle

A series of calcium spikes are induced in the mammalian egg cytoplasm at fertilisation. These calcium spikes, which last for several hours, are the necessary and sufficient signal that stimulates the egg to escape from arrest at metaphase of the second meiotic division. Metaphase arrest is achieved by preventing the destruction of cyclin B1, the regulatory component of Maturation (M-Phase) Promoting Factor, and securin, which prevents segregation of sister chromatids. Both these proteins are destroyed by tagging with ubiquitin, using an E3 ligase the Anaphase-Promoting Complex (APC). Ubiquitination tags them for proteolysis by the 26S proteasome. Work from my lab has demonstrated that the sperm calcium signal works through activating the APC, not the 26S proteasome. Although we do not know which APC component is affected by calcium, this activation appears specific to a metaphase-arrested cell cycle state. More recently we have found that the APC is differently regulated at specific points during exit from meiosis II. Before extrusion of the second polar body it is the APC activator cdc20 that regulates APC activity. However, following extrusion of the second polar body cdh1 appears the major regulator. It is probable, therefore, that the calcium spiking affects the activity of both APCcdc20 and APCcdh1. This swap in APC activator at the time of second polar body extrusion has not been reported in eggs of other species, in fact non-mammalian eggs all lack cdh1. Since APCcdc20 and APCcdh1 have different substrate specificities, the function of APCcdh1 in mammalian eggs warrants further investigation.

Epigenetic Mechanisms in Early Mammalian Development

begins well before fertilization because the molecules and controlling mechanisms that direct early development are put in place during oogenesis. The informational content of a mammalian egg at fertilization is not only restricted to its DNA sequence, but also to various DNA and chromatin modifications, specific macromolecules (RNAs and proteins), and possibly the characteristic architecture of the cytoplasm and plasma membrane. These sources of stored, necessary information can be viewed as epigenetic controlling mechanisms (Fig. 1). In the ensuing text we will briefly touch on these subjects, delineating what is presently known about them and, more important, what still remains to be elucidated. The literature on these subjects is vast and space restrictions prevent us from citing each relevant paper, for which we apologize.

Recombinant phospholipase Czeta has high Ca2+ sensitivity and induces Ca2+ oscillations in mouse eggs.

Sperm-specific phospholipase Czeta (PLCzeta) is known to induce intracellular Ca(2+) oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCzeta (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCzeta protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCzeta protein into mouse eggs induced serial Ca(2+) spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca(2+) release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCdelta1 also induced Ca(2+) oscillations, but a 20-fold higher concentration was required compared with PLCzeta. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca(2+)]), PLCzeta exhibited a significant activity at [Ca(2+)] as low as 10 nm and had 70% maximal activity at 100 nm [Ca(2+)] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCdelta1 increased at a [Ca(2+)] between 1 and 30 microm. EC(50) was 52 nm for PLCzeta and 5.7 microm for PLCdelta1. Thus, PLCzeta has an approximately 100-fold higher Ca(2+) sensitivity than PLCdelta1. The ability of purified PLCzeta protein to induce Ca(2+) oscillations qualifies PLCzeta as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca(2+) sensitivity of PLC activity as PLCzeta that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca(2+) release first, and maintains Ca(2+) oscillations.

Reassessing the molecular biology of sperm-egg recognition with mouse genetics.

The zona pellucida is an extracellular coat that surrounds mammalian eggs and early embryos. This insoluble matrix separates germ from somatic cells during folliculogenesis and plays critical roles during fertilization and early development. The mouse and human zona pellucida contain three glycoproteins (ZP1 or ZPB, ZP2, ZP3), the primary structures of which have been deduced by molecular cloning. Targeted mutagenesis of endogenous mouse genes and transgenesis with human homologues provide models to investigate the roles of individual zona components. Collectively, the genetic data indicate that no single mouse zona pellucida protein is obligatory for taxon-specific sperm binding and that two human proteins are not sufficient to support human sperm binding. An observed post-fertilization persistence of mouse sperm binding to "humanized" zona pellucida correlates with uncleaved ZP2. These observations are consistent with a model for sperm binding in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.

IP3 Responsiveness Is Regulated in a Meiotic Cell Cycle Dependent Manner: Implications for Fertilization Induced Calcium Signalling

Fertilization of mammalian eggs is known to trigger a series of transient rises in cytosolic calcium, known as calcium oscillations, the initiation and duration of which are crucial for meiotic exit and subsequent entry into embryogenesis. It is not known how these calcium oscillations are terminated when the zygote exits meiosis; it is thought that responsiveness to inositol 1,4,5-trisphosphate (IP3) is involved, since the oscillations are known to be mediated by an IP3 dependent mechanism. Here we report that IP3 responsiveness is maintained throughout meiotic maturation and falls very rapidly after meiotic exit. We also show that inhibition of the major cell cycle kinase, CDK1, has no effect on responsiveness, but that prolonging CDK1 activity prevents the decline in responsiveness normally seen at meiotic exit. We conclude that CDK1 plays a role, but is not the only factor involved in controlling IP3 responsiveness during the meiotic cell cycle.

Golgi dynamics during meiosis are distinct from mitosis and are coupled to endoplasmic reticulum dynamics until fertilization

One current theory of the Golgi apparatus views its organization as containing both a matrix fraction of structural proteins and a reservoir of cycling enzymes. During mitosis, the putative matrix protein GM130 is phosphorylated and relocalized to spindle poles. When the secretory pathway is inhibited during interphase, GM130 redistributes to regions adjacent to vesicle export sites on the endoplasmic reticulum (ER). Strikingly, meiotic maturation and fertilization in nonrodent mammalian eggs presents a unique experimental environment for the Golgi apparatus, because secretion is inhibited until after fertilization, and because the centrosome is absent until introduced by the sperm. Here, we test the hypothesis that phosphorylated GM130 associates not with meiotic spindle poles, but with ER clusters in the mature bovine oocyte. At the germinal vesicle stage, phosphorylated GM130 is observed as fragments dispersed throughout the cytoplasm. During meiotic maturation, GM130 reorganizes into punctate foci that associate near the ER-resident protein calreticulin and is notably absent from the meiotic spindle. GM130 colocalizes with Sec23, a marker for ER vesicle export sites, but not with Lens culinaris agglutinin, a marker for cortical granules. Because disruption of vesicle transport has been shown to block meiotic maturation and embryonic cleavage in some species, we also test the hypothesis that fertilization and cytokinesis are inhibited with membrane trafficking disruptor brefeldin A (BFA). Despite Golgi fragmentation after BFA treatment, pronuclei form and unite, and embryos cleave and develop through the eight-cell stage. We conclude that, while the meiotic phosphorylation cycle of GM130 mirrors that of mitosis, absence of a maternal centrosome precludes Golgi association with the meiotic spindle. Fertilization introduces the sperm centrosome that can reorganize Golgi proteins, but neither fertilization nor cytokinesis prior to compaction requires a functional Golgi apparatus.

Structural Characterization of Native Mouse Zona Pellucida Proteins Using Mass Spectrometry

The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.

Phospholipase C-dependent Ca 2+ release by worm and mammal sperm factors

Egg activation in all animals evidently requires the synthesis of inositol 1,4,5-trisphosphate (InsP 3) from phosphatidylinositol 4,5-bisphosphate (PIP 2) by phospholipase C (PLC). Depending on the organism, InsP 3 elicits either calcium oscillations or a single wave, which in turn initiates development. A soluble component in boar sperm that activates mammalian eggs has been suggested to be a PLC isoform. We tested this hypothesis in vitro using egg microsomes of Chaetopterus. Boar sperm factor elicited Ca 2+ release from the microsomes by an InsP 3-dependent mechanism. The PLC inhibitor U-73122, but not its inactive analog U-73343, blocked the response to sperm factor but not to InsP 3. U-73122 also inhibited the activation of fertilized and parthenogenetic eggs. Chaetopterus sperm also contained a similar activity. These results strongly support the hypothesis that sperm PLCs are ubiquitous mediators of egg activation at fertilization.

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.

Cellular and molecular mechanisms leading to cortical reaction and polyspermy block in mammalian eggs.

Following fusion of sperm and egg, the contents of cortical granules (CG), a kind of special organelle in the egg, release into the perivitelline space (cortical reaction), causing the zona pellucida to become refractory to sperm binding and penetration (zona reaction). Accumulating evidence demonstrates that mammalian cortical reaction is probably mediated by activation of the inositol phosphate (PIP(2)) cascade. The sperm-egg fusion, mediated by GTP-binding protein (G-protein), may elicit the generation of two second messengers, inositol 1,4,5 triphosphate (IP(3)) and diacylglycerol (DAG). The former induces Ca(2+) release from intracellular stores and the latter activates protein kinase C (PKC), leading to CG exocytosis. Calmodulin-dependent kinase II (CaMKII) may act as a switch in the transduction of the calcium signal. The CG exudates cause zona sperm receptor modification and zona hardening, and thus block polyspermic penetration. Oolemma modification after sperm-egg fusion and formation of CG envelope following cortical reaction also contribute to polyspermy block.

Avian chromosomes can be examined by injection of erythrocyte nuclei into mouse oocytes.

Avian chromosomes can be examined by injection of erythrocyte nuclei into mouse oocytes.