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.

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.

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.

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.

The fertilisation calcium wave in sea urchin eggs triggers the onset of development. The wave is an explosive increase in intracellular free calcium concentration that begins at the point of sperm entry and crosses the egg in about 20 s. Thimerosal is a sulphydryl reagent that sensitises calcium release from intracellular stores in a variety of cell types. Treatment of unfertilised eggs with thimerosal causes a slow increase that results eventually in a large, spontaneous calcium transient and egg activation. At shorter times after thimerosal treatment, egg activation and the calcium transient can be triggered by calcium influx through voltage-gated calcium channels, a form of calcium-induced/calcium release (CICR). Thimerosal treatment also reduces the latency of the fertilisation calcium response and increases the velocity of the fertilisation wave. These results indicate that thimerosal can unmask CICR in sea urchin eggs and suggest that the ryanodine receptor channel based CICR may contribute to explosive calcium release during the fertilisation wave.

The objective of the current experiments was to investigate whether all or only some blastomeres from precompacted mouse embryos were affected by zona photoablation. The microbeam of xenon chloride excimer laser (308 nm) was guided through an inverted microscope (non-contact system). Topical effects of lasing were determined by microinjection of a vital fluorescent dye of high molecular weight (fluorescein isothiocyanate [FITC] dextran) into the cell immediately adjacent to the site of zona photoablation. This dye is only passed onto daughter blastomeres and therefore allows study of specific cell lines. Embryonic growth was assessed following cell separation at the morula and blastocyst stage. Four-cell embryos treated with the laser had significantly fewer cells 12 h after zona photoablation than control embryos. A similar effect was noted after 24 h between dye injected embryos and those injected and lased simultaneously, indicating potential toxic effects of the laser treatment on the embryo. Effects on the blastomere closest to the site of ablation were evaluated by calculating the ratio of dyed cells to the total number of cells at specific time intervals. The ratios were similar in the dye and laser + dye groups of treated 4-cell embryos 36 h after treatment (0.22 and 0.23, respectively), indicating that the dye was still present in approximately 25% of the cells and that the negative effect of photoablation was evenly distributed among the blastomeres. It is concluded that zona photoablation may have long-term detrimental effects of a non-topical nature on precompacted mouse embryos in spite of the apparent precision of the laser spot size.

We have used the whole-cell recording technique to compare three stages of primary and secondary oocytes from F1 hybrid mice (C57BL/6J x SJL/J):neonatal germinal vesicle (NGV) stage primary oocytes from 10- to 20-day-old, prepubescent mice; mature germinal vesicle (MGV) stage primary oocytes from 12-week-old, post-pubescent, superovulated mice; first polar body (FPB) stage secondary oocytes from 12-week-old, post-pubescent mice during the normal oestrus cycle or following superovulation. NGV, MGV and FPB oocytes all exhibit two voltage-dependent currents: an inward, rapidly activating/inactivating current, and an outward, slowly activating/non-inactivating current. In 1.5 mmol/1 external Ca the average peak inward current is − 2.9, − 12.4 and − 13.8 μA/cm2 in NGV, MGV and FPB oocytes, respectively. In 20 mmol/1 Ca these currents increase and the reversal potential shifts to the right. The outward current decreases slightly with growth and development: at 40 mV test potentials, NGV oocytes have average outward currents of 8.9 μA/cm2, and MGV and FPB oocytes have currents of 5.0 and 5.5 μA/cm2, respectively. Thus, MGV oocytes express FPB current patterns. The reversal potentials, kinetics and pharmacology of the currents indicate that Ca channels carry the inward current and K channels carry the outward current. During growth in vivo a gradual depolarisation accompanies maturation. Resting potentialsranged from − 45 to − 30 mV in NGV oocytes to − 35 to − 17 mV in MGV oocytes to − 20 mV to − 3 mV in FPB oocytes. These data suggest that a selective increase occurs in the number of Ca channels during oocyte growth. This increase precedes nuclear maturation and coincides with the acquisition of meiotic competence.

In amphibian eggs the formation of a capsular chamber is one of the most striking events occurring either upon oviposition or after fertilisation. In the egg of the anuran Discoglossus pictus a capsular chamber forms following fertilisation or activation; the egg with its vitelline envelope rotates in this chamber according to gravity. Previous work showed that the chamber is the product of plug dissolution. The plug is a lens-shaped jelly coat, typical of Discoglossus, covering only part of the animal hemisphere. Its dissolution is caused by material released from the egg about 15 min after fertilisation through exocytosis of at least two types of vacuoles. Liquefaction of the plug correlates with the reduction of disulphide bonds present in the jelly matrix. In this study we investigated the nature of the substances released from the egg and some changes occurring in the plug during liquefaction. SDS-PAGE showed that the proteic profile of the plug changes dramatically after fertilisation, confirming proteic cleavage in the plug matrix during its dissolution. Through in vitro tests and electrophoretic analysis of the Ringer solution in which the egg exudate was collected, an increase in the activity of the solution was determined in the presence of hydrogen peroxide, and peroxidase activity was depicted in the egg exudate. The presence of free thiol groups and cysteic acid residues (or cysteine sulphinic acid) in the plugs of activated eggs was established, suggesting that during plug dissolution some disulphide bonds are oxidatively opened. This suggests that enzyme(s) with peroxidase activity are released following fertilisation. We surmise that such enzymes are contained in the intraovular vacuoles the exocytosis of which triggers the onset of plug liquefaction. The possible release of hydrogen peroxide from the egg is discussed.

Male adult rabbits and rats treated with Spartium junceum showed a significant decrease in fertility, demonstrated by a lower number of pregnancies. The target of the drug seems to be the acrosomal protease system, the activity of which appears greatly reduced, while the morphology of testicular cells and epididymal spermatozoa is only partially affected. The antifertility effect is completely reversible.

The sperm plasma membrane over the equatorial segment of the acrosome gains the ability to fuse with the oolemma some time during, or after, the acrosome reaction. Since acrosin is a major component of the acrosome matrix that dissolves during the acrosome reaction, we sought to determine the effect of acrosin inhibitors on the sperm's ability to fuse with the oolemma. Five acrosin inhibitors (soybean trypsin inhibitor (SBTI), leupeptin, benzamidine, N-p-tosyl-1-lysin-chloromethyl ketone (TLCK) and phenylmethylsulphonyl fluoride (PMSF) and one non-acrosin inhibitor (N-p-tosyl-1-phenylalanine chloromethyl ketone (TPCK) were tested at non-toxic levels (below motility-disturbing concentrations). These inhibitors were added at three different times: (1) during the acrosome reaction of spermatozoa, (2) during sperm-oocyte contact and fusion, and (3) soon after sperm-oocyte fusion was completed. TLCK prevented sperm-oocyte fusion by inhibiting the acrosome reaction.PMSF inhibited gamete fusion, without inhibiting the acrosome reaction. SBTI, leupeptin and benzamidine also inhibited gamete fusion, but they had no effect if spermatozoa were allowed to acrosome-react in inhibitor-free medium. TPCK was without any inhibitory effects, suggesting that chymotrypsin-like enzymes are not involved in gamete fusion. Although acrosin inhibitors prevented acrosome-reacted spermatozoa from becoming fusion-competent, acrosin (and trypsin) alone could not make the plasma membrane of acrosome-intact spermatozoa fusion-competent. The data suggest that (1) the plasma membrane of the acrosomal region first undergoes dramatic changes immediately before or during the acrosome reaction and (2) acrosin released from the acrosome during the acrosome reaction further alters biophysical and biochemical characteristics of the plasma membrane over the equatorial segment. Such dual changes make the plasma membrane of this specialised region of the spermatozoon competent to fuse with the oolemma. Acrosin may not be the only acrosomal enzyme to participate in these changes.