The zona pellucida surrounding the mammalian oocyte contains a major glycoprotein species, ZP3, that serves as a cell- and species-specific receptor for spermatozoa. In this study we have determined the primary amino acid structure of marmoset ZP3 (marZP3) and examined the expression of marZP3 mRNA within the ovary. The marZP3 gene possesses an open reading frame of 1272 nucleotides which is expressed specifically by the oocyte and encodes a polypeptide chain of 424 amino acids that exhibits 91% homology with the human ZP3 sequence. The disparity between these molecules was confined to a short domain spanning residues 322–352; otherwise the molecules were very similar, showing conservation of many structural features including the N–linked glycosylation sites, location and number of cysteine and proline residues and hydrophobicity profile. The results of this study have important implications for the use of the marmoset monkey as an animal model for the development of contraceptive vaccines targeting ZP3.

Round spermatids of the hamster and mouse were electrofused with homologous mature oocytes to examine the behaviour of their nuclei within the ooplasm. A single spermatid was inserted in the perivitelline space of a mature oocyte and an electric fusion pulse given. In the hamster, the best spermatid-oocyte fusion took place when the oocytes were pretreated with neuraminidase, subjected to 30 s AC (2 MHz, 20 V/cm) followed by a single fusion DC pulse (3000 V-cm, 10 μs) and another 30 s AC current. Inclusion of micromolar Ca2+ and Mg2+ in the fusion medium was essential for oocyte activation. Under these conditions all oocytes were activated and 20–40% fused with spermatids. Of these fused oocytes only 5–10% had fully developed spermatid (male) and oocyte (female) pronuclei. In the rest the spermatid-derived pronuclei remained small throughout the pronuclear stage. However, nucleolus-like structures appeared de novo and DNA synthesis occurred in these small pronuclei. Regardless of the size of male pronuclei, chromosomes from the spermatid and oocyte appeared to mingle and participate in the first cleavage. About 70% of the fused oocytes developed into the 2-cell stage. Electrofusion of mouse oocytes with spermatids was less efficient. Even under the best conditions tested, less than 10% of the oocytes fused with spermatids. Here again, most spermatid nuclei remained small throughout the pronuclear stage.

An erythrochyte anti-ankryin antibody was used to investigate the presence and distribution of ankryin in eggs and embryos of ascidian species with different modes of development. In eggs of the indirect developer Ascidia ceratodes anti-ankryin reacted with a 210 kDa polypeptide which has an electrophoretic mobility similar to the vertebrate ankryins. Immunofluorescence microscopy showed that the ankryin-like protein is co-distributed with the myoplasm throughout development. It is restricted to a thin layer under the plasma membrane in unfertilised eggs, undergoes ooplasmic segregation to the posterior pole of the zygote after fertilisation, and is distributed to the tail muscle cells during cleavage and embryogenesis. After gastrulation and neurulation, lower levels of the ankryin-like protein, presumably of zygotic origin, were observed in brain cells and in the apical margin of epidermal cells. The ankryin-like protein was also localised in the myoplasm in eggs and embryos of another indirect developing species, Halocynthia roretzi. The ankryin-like protein may link the cytoskeleton with the plasma membrane in ascidian eggs, as it does in vertebrate erythrocytes. In contrast to A. ceratodes and H. rorefzi, which are members of the families Ascidiidae and Pyuridae respectively, the pattern of ankryin-like protein expression was changed in five species in the family Molgulidae. These molgulid ascidians exhibit either indirect or direct development, and eggs of the direct developing species have lost or modified the myoplasm. The ankryir like protein was present in young oocytes but failed to persist during oogenesis and disappeared in mature eggs and embryos of these molgulid species. The change in ankryin-like protein expression may be a preadaptation for loss of the myoplasm and the evolution of direct development.

Staurosporine, an inhibitor of protein kinase activity, causes premature intercellular flattening of blastomeres but does not induce their premature polarisation. The flattening induced is calcium dependent, is reversed transiently at mitosis and requires the continuing presence of the drug. Staurosporine also blocks the decompacting effect of phorbol ester on 8-cell embryos.

Spermatozoa from the mollusc Ostrea edulis are capable of fusing to and entering de-chorionated ascidian eggs. During interaction they generate activation currents, comparable to the fertilisation currents induced by homologous spermatozoa. Activation currents are inward at − 80 mV, with a mean initial slope of 111 ± 124 pA/s for Ciona intestinalis eggs and 47 ± 25 pA/s for Phallusia mammillata eggs, while the mean peak currents are 2782 ± 1132 pA and 1523 ± 1668 pA, respectively. The fertilisation and activation currents reverse at a holding potential of 0 mV to + 20 mV, suggesting that oyster sperm and ascidian sperm gate the same channel precursor, a non-specific, large conductance channel described previously (Dale & DeFelice, 1984). In contrast to homologous fertilisation, the activation current is not followed by a polarised contraction of the egg surface, nor other signs of egg activation. Staining eggs with Hoechst 33342 after insemination shows the female nucleus and a single oyster sperm nucleus at the antipode. This suggests a specialised predetermined site at the vegetal pole for sperm entry. Homologous and heterologous spermatozoa delivered, in a large pipette, to localised areas of the egg surface generate fast inward currents of 200–2000 pA, but do not induce contraction of the egg surface. This shows that although channel precursors are located globally over the egg surface, channel activation does not necessarily trigger the contraction wave. Subsequent induction of both a fertilisation current and a contraction by homologous sperm added to the bath, implies a regionalised activation site with an accumulation of channel precursors and a ‘pacemaker’ for the initiation of the contraction wave.

Oviductal oocytes (n = 795) were obtained from ovulation-induced prepubertal gilts. In the first experiment, different parameters related to in vitro fertilisation (IVF) were compared in the presence and absence of cumulus matrix (which is shed with the oocytes at ovulation.) The results show that the presence of this matrix is beneficial because the rates of fertilisation (69%) (number of monospermic oocytes/total number of healthy mature oocytes; 42%), and the median number of spermatozoa per oocyte (1.52±0.06), were improved with respect to those obtained in its absence (54%, 22% and 2.33 ± 0.08, respectively; p<0.01). In the second experiment the effect of two different volumes of co-incubation medium (2 ml and 0.4 ml) on the same parameters of porcine IVF were compared. No significant differences between volumes were observed, except in the mean number of spermtozoa per oocyte and the percentage of dispermic oocytes.

In the marine brown alga, Fucus, two poles are differentiated before cell division determining the future rhizoid or thallus. We have used a combination of the Ca2+ -sensitive dye Calcium Green and the pH-sensitive dye SNARF monitored at pH-insensitive wavelengths to obtain confocal ratio images of free cytoplasmic calcium distribution at different stages in polarising Fucus zygotes. These dyes have the advantage that they can be used in most confocal microscopes and their longer excitation wavelengths greatly reduce autofluorescence problems. Dyes of varying molecular weights (free acid form, 10 000 mol.wt or 70 000 mol.wt dextran-conjugated) were pressure microinjected into early zygotes which were allowed to polarise in unidirectional light. Dextran-conjugated dyes remained non-compartmentalised and fluorescence could be monitored for up to 3 days following microinjection. Currently we have been able to detect Ca2+ gradients at the tip of the rhizoid, confirming earlier results. Localised Ca2+ elevations have also been observed at the rhizoid pole of the polarising zygote before the onset of rhizoid germination. Limitations of this technique and the significance of these Ca2+ gradients are discussed.

M5 ganglioside (NeuGcα2–6Glcβl-' Cer) is the predominant glycosphingolipid in sea urchin eggs. Distribution of M5 ganglioside was studied in unfertilised and fertilised eggs of the sea urchin Hemicentrotus pulcherrimus by indirect immunofluorescence microscopy. In the cortices of unfertilised eggs, anti-M5 antibody strongly stained the submembranous, polygonal and tubular network of endoplasmic reticulum that was revealed by a membrane-staining dye, DiIC18(3). In addition to the cortical network of endoplasmic reticulum, at least two morphologically distinct vesicles were positive to the antibody. In the cortices isolated from fertilised eggs 30 min after insemination, the antibody stained only a similar network of endoplasmic reticulum, presumably the one reconstructed 5–10 min after fertilisation. During mitosis the endoplasmic reticulum is known to aggregate within the asters of the mitotic apparatus. Indeed, the antibody stained the asters and (more strongly) the vesicular components attaching to the periphery of the mitotic apparatus.

The nuclear DNA content of intact, live or fixed, human and mouse oocytes and blastomeres has been measured rapidly and reliably. Chromosomal DNA has been stained with DAPI, the fluorescent emission from which has been measured photocytometrically. In vitro fertilised mouse oocytes and embryos at various stages of development were assessed for their DNA content. The mean values of 1C, 2C and 4C DNA content were clearly different, and it was possible to assign correctly individual values for DNA content to each class with 92%, 61% and 81% confidence respectively. Maintaining the cells as whole mounts allowed other morphological and structural features to be examined. When formation of multiple micronuclei was induced in mouse oocytes by their insemination in the presence of nocodazole, the additive signal from all the micronuclei in one zygote was equivalent to the expected DNA content. Application to early human blastomeres of this photocytometric technique for measurement of the total cellular DNA content revealed that multinucleated blastomeres contained 2C to 4C DNA levels, consistent with a diploid DNA content.

In the starfish, spermatozoa undergo the acrosome reaction upon encountering the jelly coat of eggs. A highly sulphated glycoprotein in the jelly coat is called acrosome-reaction-inducing substance (ARIS) because it is the key signal molecule to trigger the acrosome reaction. The activity of ARIS is mainly attributed to its sulphate and saccharide residues. The extremely large molecular size and speciesspecific action of ARIS suggest the presence of a specific ARIS receptor on the sperm surface, but no experimental evidence for the receptor has been presented. We therefore measured specific binding of ARIS and its pronase digest (P-ARIS), which retains the full activity of ARIS, to homologous spermatozoa by using fluorescien-isothiocyanate-labelled ARIS and125 I-labelled P-ARIS, respectively. The spermatozoa had the ability to bind ARIS, as well as P-ARIS, specifically. The binding was species-specific, and mostly localised to the head region of spermatozoa. Scatchard plot analysis indicated the presence of one class of ARIS receptor on the surface of acrosome-intact speramatozoa. Furthermore, the specific binding of P-ARIS to the anterior region of sperm heads was microscopically confirmed by using P-ARIS conjugated to polystyrene latex beads with intense fluorescence. It is concluded that starfish spermatozoa have a specific receptor for ARIS on the surface of the anterior region of heads.

It is now accepted that sperm trigger deuterostome egg activation by causing an increase in egg cytoplasmic Ca2+ levels (Jaffe, 1983; Whitaker & Swann, 1993). This increase generally takes the form of a single wave of Ca2+ release that crosses the egg from the point of sperm-egg interaction (Jaffe, 1983)eggs Ca2+ waves occur repetitively and soon turn into synchronous pulses, or homogeneous oscillations, that last for hours after sperm-egg fusion (Miyazaki et al., 1993b; Homa et al., 1993). Despite their extensive characterisation it is still not established how sperm trigger these Ca2+ changes in eggs. The signal transduction mechanism is missing. There is a proliferation of overlapping and complex schemes for how the sperm may initiate Ca2+ release (Miyazaki et al., 1993b;Whitaker & Swann, 1993). Here, my aim is to present one simple scheme in its generic form. The brevity of this essay restricts citations and necessitates using reviews to reference original work.

Bovine embryonic viability after in vitro fertilisation (IVF) was affected by addition of platelet-derived growth factor (PDGF) or growth factor combinations to gonadotrophin-supplemented serum-free medium for in vitro maturation (IVM). Thus, 0.1, 1.0 or 10.0 ng PDGF/ml combined with 500 ng follicle-stimulating hormone (FSH)/ml enhanced oocyte maturation as reflected by greater proportions (p < 0.05) of matured and inseminated oocytes developing to blastocysts in vitro than were afforded by PDGF alone, i.e. 32.4%, 28.2% and 31.0% respectively vs. 11.4%, 13.5% and 8.6% respectively. By contrast, luteinising hormone (LH, 500 ng/ml) combined with the same concentrations of PDGF did not increase the proportions of oocytes developing to blastocysts (16.2%, 12.5% and 16.9%). Epidermal growth factor (EGF) + insulin-like growth factor-I (IGF-I) with or without PDGF when added to low concentrations of FSH or LH did not significantly improve cleavage or morula development. Significant improvement (p & 0.05) in blastocyst formation was seen when FSH was supplemented with either EGF (38.0%) or EGF + IGF-I + PDGF (40.7%) compared with FSH alone (27.4%) or FSH + LH (24.3%), but no improvement followed FSH + EGF + IGF-I (33.6%) or FSH + LH + EGF + IGF-I + PDGF (29.2%) treatments for IVM. Results revealed a positive influence on blastocyst development of FSH and PDGF or EGF ± PDGF during IVM and suggest the possibility of a concerted action of gonadotrophins with growth factors in physiological (functional) oocyte maturation.

Egg activation at fertilisation is composed of a complex, choreographed series of events, the initiation of which still is not understood. Two major hypotheses have emerged as explanations for the mechanism of egg activation (reviewed by Nuccitelli, 1991; Whitaker & Swann, 1993). The first holds that the sperm delivers an ‘activating factor’ (e.g. inositol trisphosphate, calcium, or a protein) that diffuses into the egg cytoplasm after gamete fusion. The second hypothesis holds that sperm bind to receptors spanning the egg plasma membrane which then transduce a signal to second messenger enzymes. We present here the evidence for receptor-mediated signal transduction in egg activation at fertilisation. By ‘receptor’ we mean only molecules that bind an extra-membrane ligand and which transduce a signal to molecules residing on the cytoplasmic side of the egg membrane. It is critical to be aware that several alternative activating mechanisms are not mutually exclusive and that species differences may exist. In fact, as more has been learnt about the molecules involved in gamete recognition and binding and of the signalling pathways in the egg, it seems likely that multiple pathways exist to trigger complete egg activation.

The effect of zinc and copper on the in vitro development of mouse preimplantation embryos and on metallothionein (MT) levels was studied by exposing the embryos to 100 μM concentrations of the metals for 24 h at the 1-cell,2-cell, 6-8-cell, morula and blastocyst stages. Zinc affected embryo development in the early but not in the late stages, whereas copper affected it more generally. The combined presence of both metals caused a stronger embryotoxicity. MT levels were measured by radioimmunoassay and were found to be similar at all developmental stages, though possibly higher at the blastocyst stage. The exposure of embryos to zinc and copper increased MT levels significantly only at the blastocyst stage, supporting previously published results on MT mRNA levels.

Intracellular membranes isolated from fully grown immature oocytes, mature oocytes (eggs) and activated eggs of Xenopus laevis were fractionated through a discontinuous sucrose density gradient into light, intermediate and heavy fractions. Electron microscopy showed that the light and intermediate fractions consisted mainly of smooth membranes, while the heavy fraction consisted mainly of rough membranes and mitochondria. Variations in the proteins associate with samples taken at different stages were observed by SDS-PAGE. The following differences were consistently observed: a 200 kDa protein was present only in the intermediate fraction of activated eggs, 29 and 44 kDa proteins were present only in the intermediate fractions of immature oocytes and activated eggs, and 120 and 145 kDa proteins were present only in the heavy fractions of mature oocytes and activated eggs. Examination of Western blots showed that cyclins A and B2 did not associate with membrane fractions at any stage of meiosis. Instead, cyclin A was present in the cytosols of mature oocytes and cyclin B2 was present in the cytosols of immature and mature oocytes. c-mos protein was detected in the cytosols and occasionally in the light fractions of mature oocytes and activated eggs. While α- and β-tubulins were detected in the light and intermediate fractions at all the stages of meiosis examined, only β-tubulin was present in the heavy fraction. βtubulin present in the heavy fraction was detected only at interphase, i.e. in immature oocutes and activated eggs, and not in mature oocytes. Using immunogold labelling we confirmed these results and found evidence to suggest that β-tubulin associates with the rough endoplasmic reticulum of interphase cells by a linking protein.