Fertilisation in the marine alga Fucus serratus is accompanied by increased influx of Ca2+ from the external medium. The onset of this increase, monitored with the Mn2+ fluorescence quench technique, corresponded with the depolarisation phase of the fertilisation potential. External Ca2+ was necessary for the onset of the fertilisation potential and the early activation events, including cell wall exocytosis. Removal of Ca2+ from, or addition of Sr2+ to, the external medium during the fertilisation potential reduced the magnitude of the depolarisation and prolonged its duration. While fertilisation potentials could not be elicited in the presence of 0.1 mM Ca2+, addition of Ba2+ in the presence of 0.1 mM Ca2+ allowed normal fertilisation potential and egg activation. Microinjection of ryanodine or cyclic guanosine 5'-monophosphate (cGMP) did not induce cytoplasmic Ca2+ elevation or egg activation. Inositol 1,4,5-triphosphate [Ins(l,4,5)P3] produced a transient elevation of cytoplasmic Ca2+, monitored using ratio photometry, but did not cause cell wall exocytosis except at the site of microinjection. The results demonstrate an essential role for Ca2+ influx during Fucus egg activation. The relative importance of influx and intracellular Ca2+ release in Fucus egg activation is discussed.

Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertion of three ion channel types. They could be discerned on the basis of their selectivity, conductance, gating and voltage-dependent properties. The presence of a previously reported large, Ca2+-selective channel was confirmed. Here, it is reported that the Ca21-selective channel from mouse sperm plasmamembrane displayed a pNa+/Pk+ = 1.6 ± 0.2(n=4) and was blocked by micromolar concentrations of ruthenium red. Fusion yielded also a cation-selective channel (PNa+/Pk+ = 2.5±0.3, n=3) with a main open conductance substate of 103 pS and a smaller open substate of 51 PS(600mM K+cis/100 mM Na+trans). The channel inserted into bilayers in two stable fashions: a high-activity mode (open probability = 0.57 ± 0.02, n=3), and a low activity mode (open probability <1%, n=4). In high mode, the channel displayed bursting kinetics and burst length was voltage independent. In addition, a perfectly anion-selective channel, with a slope conductance of 83 PS (600KCI cis/100KCI trans), was identified. It displayed a high, nearly constant open probability (∼0.90)in the 0 to –80 mV range.

The distribution of different classes of acetylcholine (ACh) receptor-like molecules in sperms of different invertebrate and vertebrate species is described. ACh receptor molecules belong to one of two classes: muscarinic receptors (mAChRs), associated with signal transduction mechanisms in the inner domain of the cell, and nicotinic receptors (nAChRs), capable of opening Na+ channels when activated by the ligand. Molecules immunologically related to mAChRs and to ACh can be identified by specific antibodies, and revealed by immunofluorescent or immunogold staining; the nicotinic receptor-like molecules are localised as curare-sensitive affinity sites for α-bungarotoxin. In all species studied, both classes of receptors were found, with a similar distribution. Muscarinic-like molecules were found mainly in the sperm head regions of most species; such a localisation may be correlated to a function in sperm–egg interaction, for instance in the regulation of the block to polyspermy. Nicotinic-like molecules are present mainly in the tail and in the post-acrosomal region of most animals, thus confirming their function in the regulation of sperm propulsion, but are also present at the acrosomal region of most species. The distribution patterns of the different classes of molecules indicate that both may be in sperm–egg interactions, in addition to their known function in the regulation of sperm propulsion.

We have used confocal microscopy and a fluorescent calmodulin probe to examine the mechanism of localisation of calmodulin during the first cell cycle of the sea urchin zygote. Using fluoresceincalmodulin, calmodulin can be observed within the nucleus and interphase astral microtubule arrays as cells approach mitosis. During mitosis, calmodulin redistributes to the mitotic apparatus and to condensed chromosomes. Quantitative analysis with reference to a control dye (fluorescein-dextran) shows that the distribution of calmodulin is specific. We used a competitive inhibitor of calcium-dependent calmodulin binding (Trp-peptide; Török & Trentham (1994) Biochemistry 33, 12807–20) to test whether the cell cycle localisation of calmodulin was due to its binding to targets on activation. The Trp-peptide eliminates localisation of calmodulin within the nucleus. However, microtubule localisation persists in the presence of the Trp-peptide. These data show that calmodulin can localise by calcium (and hence activation)-dependent as well as calcium-independent mechanisms. This suggests that distinct mechanisms of localisation may be involved in the regulation of the differential functions of calmodulin, at least during the cell cycle.

Maturation of the starfish oocyte cortex to produce an effective cortical granule reaction and fertilisation envelope is believed to develop in three phases: (1) pre-methyladenine (1-MA) stimulation; (2) post-1-MA stimulation, pregerminal vesicle breakdown; and (3) post-germinal vesicle breakdown. The present study was initiated to identify what each of these phases may encompass, specifically with respect to structures associated with the oocyte cortex, including cortical granules, microvilli and vitelline layer. 1-MA treatment brought about an orientation of cortical granules such that they became positioned perpendicular to the oocyte surface, and an ∼ 4-fold decrease in microvillar length. A-23187 activation of immature oocytes treated with (10 min; pregerminal vesicle breakdown) or without 1-MA resulted in a reduction in cortical granule number of 21% and 41%, respectively (mature oocytes underwent a 96% reduction in cortical granules). Elevation of the fertilisation envelope in both cases was significantly retarded compared with activated mature oocytes. In activated mature oocytes, the vitelline layer elevated 20.0 ± 5.4 μm from the egg's surface, whereas in immature oocytes treated with just A-23187 or with 1-MA (10 min) and A-23187, it lifted 0.35 ± 0.1 and 0.17 ± 0.04 μm, respectively. The fertilisation envelopes of activated (or fertilised) immature oocytes also differed morphologically from those of mature oocytes. In activated, immature oocytes, the fertilisation envelope was not uniform in its thickness and possessed thick and thin regions as well as fenestrations. Additionally, it lacked a complete electron-dense stratum that characterised the fertilisation envelopes of mature oocytes. The nascent perivitelline space of immature oocytes was also distinguished by the presence of numerous vesicles which appeared to be derived from microvilli. Differences in the morphology of cortices from activated (fertilised) and non-activated, immature and mature oocytes substantiate previous investigations demonstrating three phases of cortical maturation, and are consistent with physiological changes that occur during oocyte maturation, involving ionic conductance of the plasma membrane, establishment of slow and fast blocks to polyspermy and elevation of a fertilisation envelope.

We describe a rapid and reproducible method for cloning cDNA amplified from 10 mouse oocytes. The procedure consists in priming cDNA synthesis from a crude cellular extract using an oligo d(T) containing primer and submitting the size-limited cDNA first strand to poly(dG) tailing. The whole cDNA population is then polymerase chain reaction (PCR) amplified using two primers complementary to oligo d(A) and oligo d(G) ends of the cDNA. In this procedure no purification steps are required. We obtained about 5 ×106 clones from 10 oocytes. Screening of the library showed that the relative abundance of the transcripts was preserved during amplification and cloning and that the procedure allows cloning of low-abundance sequences at least as rare as 0.008% of the mRNA. The repeatable generation of representative cDNA libraries from reduced numbers of oocytes or embryos should open new opportunities for obtaining genetic information from mammalian preimplantation embryos.

Fertilisation of ascidian eggs induces the rapid release of a cell surface N-acetylglycosaminidase that blocks sperm binding to vitelline coat sperm receptors resulting in a block to polyspermy. Fertilisation also triggers a large contraction of the egg (thus stimulating ooplasmic segregation) that is completed within 5 min of insemination. In eggs of the ascidian Phallusia mammillata, glycosidase release and cortical contractions are blocked by BAPTA-AM [bis-(o-aminophenoxy-ethane-N,N,N',N' -tetraacetic acid, tetra(acetoxymethyl)-ester], a cell-permeant calcium chelator, indicating that both processes are probaly dependent on a rise in intracellular calcium levels. Both glycosidase release and the cortical contraction are induced by treatment of the egg with the protein synthesis inhibitor emetine, while only the glycosidase release is induced by isoproterenol, carbachol or acetylcholine. Previous work with ryanodine demonstrated that ryanodine also caused glycosidase release but not the cortical contraction Inversely, activation by ionomycin in calcium-free sea water causes cortical contractions but not glycosidase release. Thus the two processes can be activated independently. Dextran-coupled (10kDa) calcium green-1 injected eggs show an increase in intracellular calcium 30–40s before the cortical contraction is triggered by fertilisation or ionomycin- induced activation. This confirms previous findings that the cortical contraction is a consequence of the activation calcium the triggered by te sperm. The extracellular calcium requirement for the glycosidase release suggests that calcium influx may be more important for this phase of egg activation. Thus activation eggs appears to involve two independent pathways involving calcium.

Mast cells of connective tissue type are scattered in the interstitial compartment of the lizard Podarcis s. sicula. Their number varies during the year, showing peaks in spring and in winter, respectively.Thermal manipulation affects mast cell number (MCN): high temperature decreased MCN in both January and May, while low temperature increased MCN only in January. Ethane dimethane sulphonate, a toxin which specifically destroys Leydig cells, induced an increase in MCN on days 3 and 7 of treatment. Oestradiol treatment provoked a strong increase in MCN that was blocked by tamoxifen. Blocking androgen receptors with cyproterone acetate resulted in an increase in MCN, while testosterone injection provoked a strong decrease. These results suggest a relationship between the presence of mature Leydig cells and mast cell proliferation and/or differentiation.

To investigate the involvement of mitogen-activated protein kinase(MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using basic protein(MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0–20 h of culture. An abrupt increase was observed at metaphase I(30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.

Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.