The phenomenon of developmental arrest at the 2-cell stage of zygotes obtained from certain mouse strains during in vitro culture is known as the 2-cell block. The effect of conditioned medium (CM) with rat hepatoma BRL cells on the 2-cell block of CD-1 mouse zygotes was investigated in comparison with that of CM with rat hepatoma Reuber H-35 cells. In control medium with EDTA, 75.4% of 2-cell embryos developed to the 4- to 8-cell stages. In the same conditions, the BRL Mr <10000 fraction inhibited the development of 2-cell embryos to the 4- to 8-cell stages (57.7%), although the inhibition by this fraction was weaker than by the Reuber Mr <10000 fraction (19.8%). As a result of reversed-phase column chromatography, a 2-cell stage specific inhibitor of the cleavage of mouse embryos (Fr.B-25), which separated into the Mr <10000 fraction of the Reuber CM, was detected at a low level in the BRL Mr <10000 fraction. On the other hand, the Mr >10000 fraction of BRL CM accelerated the development of the embryos (90.3%). This beneficial effect was also evident even in the absence of EDTA. RT-PCR analysis revealed that mRNAs encoding the β-A or β-B subunit of activins (Mr ~29000), which are well characterized cytokines that act as releasers of the 2-cell block, were expressed in BRL cells. These results indicate that BRL cells synthesize Fr.B-25 at low levels, and that activins contained in the BRL CM probably contributed to overcoming the 2-cell block of CD-1 zygotes cultured in vitro.

In Strongylocentrotus purpuratus the hyalins are a set of three to four rather large glycoproteins (hereafter referred to as ‘hyalin’), which are the major constituents of the hyaline layer, the developing sea urchin embryo's extracellular matrix. Recent research from our laboratories has shown that hyalin is a cell adhesion molecule involved in sea urchin embryo-specific cellular interactions. Other laboratories have shown it to consist of 2–3% carbohydrate and a cloned, sequenced fragment demonstrated repeat domains (HYR) and non-repeat regions. Interest in this molecule has increased because HYR has been identified in organisms as diverse as bacteria, flies, worms, mice and humans, as well as sea urchins. Our laboratories have shown that hyalin appears to mediate a specific cellular interaction that has interested investigators for over a century, archenteron elongation/attachment to the blastocoel roof. We have shown this finding by localizing hyalin on the two components of the cellular interaction and by showing that hyalin and anti-hyalin antibody block the cellular interaction using a quantitative microplate assay. The microplate assay, however, has limitations because it does not directly assess hyalin's effects on the adhesion of the two components of the interaction. Here we have used an elegant direct assay that avoids the limitations, in which we microdissected the two components of the adhesive interaction and tested their re-adhesion to each other, thereby avoiding possible factors in the whole embryos that could confound or confuse results. Using both assays, we found that mild periodate treatment (6 h to 24 h in sodium acetate buffer with 0.2 M sodium periodate at 4 °C in the dark) of hyalin eliminates its ability to block the cellular interaction, suggesting that the carbohydrate component(s) may be involved in hyalin's specific adhesive function. This first step is important in identifying the molecular mechanisms of a well known cellular interaction in the NIH-designated sea urchin embryo model, a system that has led to the discovery of scores of physiological mechanisms, including those involved in human health and disease.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

While vitrification has become the method of choice for preservation of human oocytes and embryos, cryopreservation of complex tissues and of large yolk-containing cells, remains largely unsuccessful. One critical step in such instances is appropriate permeation while avoiding potentially toxic concentrations of cryoprotectants. Permeation of water and small non-charged solutes, such as those used as cryoprotectants, occurs largely through membrane channel proteins termed aquaporins (AQPs). Substitution of a Thr by an Ala residue in the pore-forming motif of the zebrafish (Dario rerio) Aqp3b paralog resulted in a mutant (DrAqp3b-T85A) that when expressed in Xenopus or porcine oocytes increased their permeability to ethylene glycol at pH 7.5 and 8.5. The main objective of this study was to test whether ectopic expression of DrAqp3b-T85A also conferred higher resistance to cryoinjury. For this, DrAqp3b-T85A + eGFP (reporter) cRNA, or eGFP cRNA alone, was microinjected into in vivo fertilized 1-cell mouse zygotes. Following culture to the 2-cell stage, appropriate membrane expression of DrAqp3b-T85A was confirmed by immunofluorescence microscopy using a primary specific antibody directed against the C-terminus of DrAqp3b. Microinjected 2-cell embryos were then cryopreserved using a fast-freezing rate and low concentration (1.5 M) of ethylene glycol in order to highlight any benefits from DrAqp3b-T85A expression. Notably, post-thaw survival rates were higher (P<0.05) for T85A–eGFP-injected than for -uninjected or eGFP-injected embryos (73±7.3 vs. 28±7.3 or 14±6.7, respectively). We propose that ectopic expression of mutant AQPs may provide an avenue to improve cryopreservation results of large cells and tissues in which current vitrification protocols yield low survival.

The positive effect of strontium ions (Sr2+) on sperm motility, capacitation and acrosome reaction has been demonstrated in the mouse, human, guinea pig and hamster. In the present study, we have evaluated the effect of Sr2+ on the viability and acrosome morphology of boar spermatozoa, and on the fertilization and development after the microinjection of Sr2+-treated spermatozoa into porcine oocytes. Before incubation, 79% of spermatozoa were classified as propidium iodide (PI)-negative (live) using the LIVE/DEAD Sperm Viability Kit. After incubation with strontium chloride (SrCl2), 39% (0 mM; no divalent cations), 25% (1.9 mM) and 24% (7.5 mM) of them were classified as PI-negative. The proportion of spermatozoa that had initiated the acrosome reaction was higher in Sr2+-containing medium than in Sr2+-free medium, when assessed by electron microscopy. There was no significant difference in percentage of spermatozoa initiating the acrosome reaction between Sr2+-treated groups (1.9 mM: 22%, 7.5 mM: 33%, p > 0.05). After the microinjection of spermatozoa incubated with SrCl2, 67% (1.9 mM) and 61% (7.5 mM) of injected oocytes were successfully fertilized, and then 43% (1.9 mM) and 41% (7.5 mM) contained a fully decondensed sperm head. Sham-injected oocytes were significantly activated at a lower rate than Sr2+-treated groups (27%, p < 0.05). Next, after microinjection of spermatozoa incubated with 1.9 mM SrCl2 (n = 51), 45% of injected oocytes cleaved on day 2, and 18% developed to blastocysts on day 7 (sham-injection, n = 48: 15% to cleavage and 0% to blastocyst). These results demonstrate that Sr2+ is likely to positively affect the fertilizing capacity of spermatozoa in the pig.

The objective of this study was to elucidate the signalling pathways initiated by cAMP once inside the Xenopus laevis oocyte, where it triggers and maintains vitellogenin endocytic uptake. Our results showed the presence of Xepac transcripts at all stages of oogenesis and we demonstrated that a cAMP analogue that exclusively activates Xepac, 8-CPT, was able to rescue the endocytic activity in oocytes with uncoupled gap junctions. Inhibition experiments for the IP3/Ca2+ signalling pathway showed either a complete inhibition or a significant reduction of the vitellogenic process. These results were confirmed with the rescue capability of the A-23187 ionophore in those oocyte batches in which the IP3/Ca2+ pathway was inhibited. Taking our findings into account, we propose that the cAMP molecule binds Xepac protein enabling it to activate the IP3/Ca2+ pathway, which is necessary to start and maintain X. laevis vitellogenin uptake.

The objective of this study was to investigate the effects of different levels of seminal plasma (SP) on boar sperm quality, antioxidant capacity and bacterial concentrations during liquid storage at 17°C. Boar sperm was diluted with Beltsville Thawing Solution (BTS) consisting of 0, 25, 50 and 75% (v/v) of SP. Total motility, progressive motility and dynamic parameters were assessed by the computer assisted sperm analysis (CASA) system. Acrosome and plasma membrane integrity were measured by FITC-PNA/DAPI and SYBR-14/PI staining, respectively. In addition, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, and reactive oxygen species (ROS) levels were detected using commercial assay kits. Bacterial concentrations were assessed by turbidimetric assay. Our results showed that 25% SP markedly improved total motility, progressive motility, sperm dynamic parameters, acrosome integrity compared with 0, 50 and 75% SP (P < 0.05). In addition, 25% SP significantly increased T-AOC but decreased MDA content and ROS levels compared with 0, and 75% SP (P < 0.05). Moreover, 25% SP significantly decreased the bacterial concentrations in extended semen compared with 50% and 75% SP, however, which was higher than with 0% SP (P < 0.05). These results suggest that 25% SP can promote boar sperm quality through enhancing its antioxidant capacity during liquid storage.

The aim of this study was to characterize the protein profile of ovarian follicular fluid (FF) of brown brocket deer (Mazama gouazoubira). Five adult females received an ovarian stimulation treatment and the FF was collected by laparoscopy from small/medium (≤3.5 mm) and large (>3.5 mm) follicles. Concentrations of soluble proteins in FF samples were measured and proteins were analyzed by 1-D SDS-PAGE followed by tryptic digestion and tandem mass spectrometry. Data from protein list defined after a Mascot database search were analyzed using the STRAP software tool. For the protein concentration, no significant difference (P > 0.05) was observed between small/medium and large follicles: 49.2 ± 22.8 and 56.7 ± 27.4 μg/μl, respectively. Mass spectrometry analysis identified 13 major proteins, but with no significant difference (P > 0.05) between follicle size class. This study provides insight into elucidating folliculogenesis in brown brocket deer.

We reported previously that in mouse testis calmodulin-dependent protein phosphatase (calcineurin) is localised in the nuclei of round and elongating spermatids (Cell Tissue Res. 1995; 281: 273-81). In this study, we studied the immunohistochemical localisation of calcium/calmodulin-dependent protein kinase (CaM kinase II) using antibodies against CaM kinase IIγ from chicken gizzard and specific antibodies raised against the amino acid sequence Ileu480–Ala493 of this enzyme, and compared it with the distribution of calmodulin. Indirect immunofluorescence was most concentrated in early spermatocytes and localised in the outermost layer of seminiferous tubules where the calmodulin level was relatively low. Measurements of immuno-gold particle densities on electron micrographs revealed that CaM kinase II is transiently increased in the nucleus of zygotene spermatocytes. These observations suggest the involvement of CaM kinase II in the meiotic chromosomal pairing process. An extremely high concentration of calmodulin in spermatogenic cells undergoing meiosis may not be directly related to activation of calmodulin-dependent kinases and phosphatases.

The present study evaluated the possible embryotrophic role of fructose supplementation in chemically defined protein-free KSOM on in vitro development of bovine transgenic cloned embryos. Bovine fetal fibroblasts transfected with expression plasmids for bovine prion protein (PrP) mutant gene with GFP marker gene were used as donor nuclei for reconstruction of slaughterhouse-derived in vitro matured oocytes. The reconstructed oocytes were cultured in KSOM supplemented with 0.01% PVA (KSOM–PVA) at 39 °C in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 for 192 h. In Experiment 1, when reconstructed oocytes were cultured in KSOM–PVA supplemented with glucose (0.2 mM), fructose (1.5 mM) or combined glucose and fructose (0.2 and 1.5 mM, respectively), significantly (p < 0.05) higher blastocyst (19.2%) and hatching/hatched blastocyst (13.1%) formation rates were obtained in combined fructose and glucose supplemented medium than glucose supplemented counterpart (10.0% and 5.7%, respectively). In Experiment 2, when reconstructed oocytes were cultured in KSOM–PVA supplemented with 0.0, 0.2, 1.5, 3.0 and 5.6 mM fructose in combination with 0.2 mM glucose, the blastocyst formation rate was significantly higher (17.6%) in 1.5 mM fructose supplemented group than that of no fructose supplemented counterpart (9.7%; p > 0.05). In conclusion, supplementation of combined fructose (1.5 mM) and glucose (0.2 mM) in chemically defined protein-free KSOM enhances the in vitro development of bovine transgenic cloned embryos.

An animal egg such as amphibian, mammalian or sea urchin egg receives only a single sperm at fertilisation. After binding of the first sperm, the egg is prevented from allowing the entry of additional sperm. In fact, polyspermy results in aborted development of the zygote. It has been generally accepted that a molecule(s) released from cortical granules participates in the block to polyspermy. As one such molecule, a cortical granule lectin has been isolated from unfertilised Xenopus eggs (Xenopus cortical granule lectin; XCGL). XCGL is released into the perivitelline space after fertilisation, and forms a complex with J1 jelly molecules to form an F layer, resulting in a block to additional sperm penetration.

A lectin molecule has also been purified from the eggs of several species of fish. The fish egg lectin is located in the cortical alveoli and is released from them after fertilisation. However, its biological function is unclear. We isolated cortical alveolar lectin from unfertilised eggs of Chinook salmon through affinity column chromatography (salmon egg lectin; SEL). The lectin activity was estimated by haemagglutination. The activity of the purified SEL was most strongly inhibited by L-rhamnose and D-galactose, but not by EDTA. Further analysis by C4 reverse-phase column chromatography using HPLC revealed that the lectin was composed of three subunit proteins: 24K, 26Ka and 26Kb proteins. In addition, we cloned cDNAs for them by RT-PCR. The deduced amino acid sequence of the 26Ka protein was homologous with that of the 26Kb protein (identity, 96.4%). Identities of the 24K with the 26Ka and the 26Kb proteins were 55.9% and 66.7%, respectively. A database search revealed that a lectin molecule similar to the SEL had been identified in Anthocidaris crassispina egg (sea urchin egg lectin; SUEL). The SUEL is composed of 105 amino acids, and is similar to both amino-terminal and carboxyl-terminal halves of the SELs. Thus, the SEL molecule is composed of two repeats of such SUEL-like domains, suggesting that the SEL gene was produced by gene duplication.

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [≈1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum + 0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.

The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen–thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE–PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE–PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE–PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE–PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.

Primary mesenchyme cells (PMCs) are skeletogenenic cells that produce a calcareous endoskeleton in developing sea urchin larvae. The PMCs fuse to form a cavity in which spicule matrix proteins and calcium are secreted forming the mineralized spicule. In this study, living sea urchin embryos were stained with fluorescently conjugated wheat germ agglutinin, a lectin that preferentially binds to PMCs, and the redistribution of this fluorescent tag was examined during sea urchin development. Initially, fluorescence was associated primarily with the surface of PMCs. Subsequently, the fluorescent label redistributed to intracellular vesicles in the PMCs. As the larval skeleton developed, intracellular granular staining diminished and fluorescence appeared in the spicules. Spicules that were cleaned to remove membranous material associated with the surface exhibited bright fluorescence, which indicated that fluorescently labelled lectin had been incorporated into the spicule matrix. The results provide evidence for a cellular pathway in which material is taken up at the cell surface, sequestered in intracellular vesicles and then incorporated into the developing spicule.

During sperm/egg interaction, the sperm of Sicyonia ingentis undergoes a unique biphasic acrosome reaction. After acrosomal vesicle exocytosis, the sperm generates an acrosomal filament, over a period of 4-6 min, that is approximately 10 μm in length. Neither actin filaments nor normal microtubules have been demonstrated at the ultrastructural level of this unusual filament. Using a battery of cytoskeleton-directed antibodies the biochemical nature of this filament has been investigated. Antibodies to actin and tubulin do not label the subacrosome or acrosomal filament, but do recognise actin and tubulin in other shrimp tissues. Antibodies to tau, MAP2, and neurofilament medium and heavy subunits were all localised to the subacrosomal region of the sperm. It is interesting, however, that only the two clones of neurofilament monoclonal antibodies recognised the acrosomal filament. Electrophoretic and Western blot analysis in conjunction with amino acid sequencing revealed that the proteins localised to the acrosomal filament are of a unique sequence and may represent a new type of protein of centrosomal origin.

The aim of this study was to establish a functional freezing–thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen–thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against α-tubulin 4–6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.

The enzymatic activities of commercially prepared glycosidases were verified by direct chemical assays using defined substrates and fixed and live sea urchin (Lytechinus pictus) embryos to determine if a model cellular interaction of interest to developmental biologists for over a century (interaction of archenteron tip and roof of the blastocoel) was mediated by glycans. Glycosidases (active and denatured) were incubated with microdissected archenterons and blastocoel roofs in a direct assay to learn if their enzymatic activities could prevent the normal adhesive interaction. Of the five glycosidases tested only β-amylase (an exoglycosidase) immediately inhibited the interaction at relatively low unit activity. α-Amylase (an endoglycosidase) had no measurable effect, while other glycosidases (α-glucosidase, β-glucosidase, β-galactosidase) only substantially inhibited adhesion after a 12-h incubation. We demonstrated that the five glycosidases were active (not inhibited) in the presence of embryo materials, and that cleaved sugars could be detected directly after incubation of some enzymes with the embryos. The biochemical purity of the enzymes was examined using gel electrophoresis under denaturing conditions, and the absence of contaminating proteases was confirmed using Azocoll™ substrate. As we cannot entirely rule out the presence of minor contaminating enzymatic activities, only inhibitions of adhesion after very short incubations with enzyme were considered significant and biologically relevant. Although glycans in indirect experiments have been implicated in mediating the interaction of the tip of the archenteron and roof of the blastocoel, to our knowledge, this is the first study that directly implicates polyglucans with terminal 1,4-linked glucose residues in this adhesive event.

Production of embryos that are free of tough outer coats facilitates studies that are not possible with embryos surrounded by impenetrable envelopes. This report describes a new procedure for preventing formation of fertilisation membranes in the sea urchin (Lytechinus pictus) model. This procedure involves treating unfertilised eggs with the enzyme alpha-amylase, which cleaves alpha-1,4 glucosidic bonds in the vitelline layer. A major advantage of this method is that it is very well defined and completely controllable with alpha-amylase inhibitor. The results suggest that intact alpha-1,4 glucosidic bonds are essential for vitelline layer integrity required for formation of the fertilisation membrane. Eggs treated with alpha-amylase possessed the same surface lectin receptors as untreated eggs and, as shown by light and transmission electron microscopy, produced healthy, cleaving embryos that were free of fertilisation envelopes.