We have investigated the seasonal regulation of the Ba current (IBa) through the L-type Ca2+ channels in Pleurodeles oocytes and its relation with the intracellular cAMP level. IBa-density remained relatively constant from January to February, increased from March to April (with a maximum increase in March) and decreased from May to July. While the permeable cAMP analogue 8Br-cAMP reduced IBa during the breeding season, it was without effect during the resting season. However, IBa recorded during the two seasons was increased by a dihydropyridine agonist Bay K 8644 and blocked by cadmium. The main conclusion is that the seasonal reduction in the L-type Ca2+ current amplitude may be correlated with a high intracellular cAMP level and may account for the inability of oocytes to mature during the resting season.

When capacitated human sperm were treated with hyaluronic acid (HA) for 30 min prior to the addition of progesterone or solubilised human zonae pellucidae, there was a significant increase in the percentage of acrosome reactions. Progesterone treatment alone increased acrosome reactions from 10.5% to 21.8% and pretreatment with 100 µg/ml HA resulted in 33.0% acrosome reactions. With zonae pellucidae treatment alone the increase was from 9.0% to 23.5% and with HA pretreatment it was 48.8%. HA treatment alone had no direct effect on acrosome reactions, and the enhancing effect of HA was not removed when sperm were washed prior to the addition of either acrosome reaction agonist. Experiments with sperm 5 min after HA treatment demonstrated that enhancement of acrosome reactions was apparent as early as 1 min after addition of zonae and within 5 min after addition of progesterone. When sperm were pretreated with Fab fragments of anti-PH-20 IgG, then with HA and then with progesterone or zonae pellucidae, there was no enhancement of the acrosome reaction. Fab treatment did not induce acrosome reactions and did not interfere with the action of either agonist in the absence of HA. Sperm that were treated with HA had significantly higher intracellular calcium levels, and pretreatment with Fab reduced this increase to 42.7%. Addition of progesterone to HA-treated sperm was followed by another large increase in intracellular calcium, which was lower when sperm were pretreated with Fab. These results suggest that HA interacts with the PH-20 protein to increase basal levels of intracellular calcium and thereby potentiates the acrosome reaction. The data support the hypothesis that HA in the cumulus matrix may act to prime the fertilising sperm for induction of the acrosome reaction by constituents of the cumulus and/or zona pellucida.

Ion-sensitive fluorophores are commonly used for quantitative measurements of intracellular ion concentrations. However, both the method of intracellular loading — which for many fluorophores involves endogenous esterase-mediated removal of hydrophobic groups such as acetoxymethyl esters (AM) — and fluorescence excitation of fluorophores in the cell, can produce toxic metabolites and reactive species. Techniques used to measure intracellular ion concentrations in mammalian eggs and embryos are being increasingly employed, yet little information is available about any detrimental effects of the use of fluorophores. We have therefore used in vitro fertilisation (IVF) to assess potential fluorophore toxicity in mouse eggs, and whole cell patch-clamp recordings to detect fluorophore-associated membrane damage in zygotes. Four fluorophores were examined: SNARF-1 and BCECF (pH indicators), Fura-2 (Ca2+) and MQAE (Cl−). Cleavage of AM groups alone had no effect either on the success of IVF or on membrane electrical properties of mouse zygotes. Intracellularly loaded BCECF, SNARF-1 and Fura-2 followed by fluorescence excitation were not cell-toxic under the conditions examined. In contrast, MQAE demonstrated significant toxicity both alone and in combination with fluorescence excitation.

The dorsoventral polarity of unequally cleaving spiralian embryos becomes established at an early stage. The factors determining the position of the dorsoventral axis are still unknown. We present data showing that the sperm entry point (SEP) in both normal development and under experimental conditions determines the position of the first cleavage furrow in Dreissena embryos. The position of the spindles at second cleavage is directed by the site of fertilisation also, and the large, dorsal D quadrant of the 4-cell stage always forms opposite the SEP. The spiral chirality at third cleavage seems to be independent of both the fertilisation point and the arrangement of the quadrants. Dextral and sinistral third cleavages are found in a single egg batch, but sinistral cleavages prevail. We postulate that two factors coordinate the proper positioning of the dorsoventral axis. The sperm entry point as an epigenetic factor determines the dorsal side of the embryo. But since the dorsoventral axis forms oblique to the first cleavage furrow, this first decision is still ambiguous, and a second decision is required that, due to the alternative chirality of spiral cleavage, finally sets up the dorsoventral axis.

The correlation between human zygote morphology and chromosomal anomalies after cleavage has not been well characterised. Commonly observed morphological qualities at the zygote stage have provided little insight into further development, and therefore selection for cryopreservation or transfer appears to be less specific than that at later stages of preimplantation development. The purpose of this work was to evaluate the relationship between aberrant pronuclear morphology and chromosomal anomalies after cleavage. Monospermic zygotes exhibiting two pronuclei where diameters differed by at least 4 μm were found to arrest at a significantly higher rate than zygotes with pronuclear diameters differing by less than 4 µm. In addition, a higher incidence of day 2 multinucleation was observed. Embryos deriving from zygotes with dysmorphic pronuclei that were not replaced or cryopreserved by day 3 of development were separated and fixed for fluorescence in situ hybridisation analysis of chromosomes X, Y, 13, 18 and 21. A significantly higher incidence of mosaicism was found in this group compared with others that had developed from zygotes with normal pronuclear morphology. Although the mechanism leading to this form of divergent pronuclear morphology is unclear, results suggest a correlation with oocyte cyto-plasmic immaturity.

We applied intracytoplasmic sperm injection (ICSI) to the rat comparing three different sperm injection techniques: conventional setup with a sharp needle bearing a spike (method 1), combination of partial zona dissection (PZD) needle and blunt pipette (method 2) and piezo-injection using a blunt pipette (method 3). We also investigated the timing of sperm pronuclear formation after injection. Survival rates after injection were 8%, 24% and 71% for the methods 1, 2 and 3, respectively. All surviving oocytes formed pronuclei by about 6 h after injection. Although the survival and activation rates following sperm injection using piezo-injection were high, the incidence of normal fertilisation, as evidenced by second polar body extrusion and formation of two pronuclei, was only 10%. The vast majority of the zygotes were multinucleated, although most of them subsequently underwent cleavage. Fixation and staining of injected oocytes at different times after injection revealed that replacement of sperm nuclear protamines by histones takes place by 15 min after injection, sperm head swelling occurs within 0.5–1 h after injection and pronuclei become fully developed by 7 h after injection. Although the rate of normal fertilisation in the rat following ICSI was low under the present experimental conditions, the results indicated that direct ICSI using a piezo-driven pipette would be a potentially valuable method of producing rat offspring.

We have injected soluble extracts of sperm from the ascidian Ciona intestinalis into oocytes of the same species to test whether these extracts can mimic the events of fertilisation. Injection of ascidian sperm extracts leads, after a delay of approximately 60 s, to a large calcium transient and repetitive pattern of calcium oscillations, mimicking the normal fertilisation response. The response was concentration-independent, suggesting a stimulatory mechanism in triggering the fertilisation response. We tested the pathway of calcium release in ascidian oocytes after injection of sperm extracts by preinjection of calcium release inhibitors. The data demonstrate that dual pathways to calcium release act at fertilisation in ascidians, as in other species. C. intestinalis oocytes are characterised by a nion channel in the plasma membrane that is gated uniquely by ADP ribose. We show that this channel is not gated by the injection of ascidian sperm extracts. Our data suggest that one metabolic pathway triggered by sperm, the release of nitric oxide, is not stimulated by sperm extracts and that several metabolic pathways are stimulated at fertilisation by more than one factor within sperm.

In vitro fertilisation (IVF) was used to assess the ability of solubilised sperm plasma membrane (PM) proteins to inhibit the interaction of intact gametes. This is a first step before evaluating the ability of individual isolated proteins to competitively inhibit sperm-oocyte interaction as part of the process of studying the molecular events of fertilisation. Porcine oocytes were aspirated from ovaries, matured for 48 h in Medium 199, and the zona pellucida (ZP) was removed by exposure to acid Tyrode's solution. ZP-free matured oocytes were exposed to 200–800 μg/ml sperm PM protein for 1 h prior to insemination and during gamete co-incubation. Twenty-four hours after insemination with 5 × 105 capacitated sperm/ml, the oocytes were fixed, stained and examined. Sperm PM protein clearly inhibited IVF in a concentration-dependent manner (r = −0.87). The inhibition index (I50%), representing the sperm PM protein concentration necessary to inhibit IVF to 50% of the control value, was 310 µg/ml. These results demonstrate that solubilised sperm PM protein inhibits the interaction of intact gametes as one might expect for receptor-ligand interactions. Furthermore, the complement of sperm PM proteins appeared maximally effective at a calculated concentration of 690 µm/ml, providing a foundation for further studies with individual proteins.

During spermatogenesis the activity of intracellular Ca2+-release channels is likely to play an important role in different specific cellular functions. Accordingly, messenger RNAs for the three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes were found to be present throughout spermatogenesis. Immunocytochemical analysis revealed distinct distribution patterns of the mature IP3Rs during sperm differentiation. At early stages, IP3Rs are distributed throughout the cytoplasm, and as differentiation proceeds they become selectively localised to the Golgi complex. Consistently, spermatogonia underwent large intracellular Ca2+ release in response to thapsigargin (TG), while smaller responses were detected in late spermatocytes and spermatids. The distribution of IP3Rs and the larger Ca2+-release responses found in spermatogonia, suggest that IP3Rs may be involved in cell proliferation at this stage. This notion is supported by our observations in a spermatogenic cell line that depletion of intracellular Ca2+ pools using TG inhibits cell division, and that incubation with an IP3R-I antisense oligonucleotide completely inhibited proliferation. Furthermore, the three genes encoding ryanodine receptor proteins (RyRs) are expressed at all stages of spermatogenesis. However, immunocytochemical studies with specific antibodies against each of the RyR subtypes detected types 1 and 3 in spermatogenic cells and only type 3 in mature sperm. In contrast to IP3Rs, RyRs remain scattered in the cytoplasm throughout differentiation. Functional responses to caffeine and ryanodine were absent in spermatogenic cells and in mature sperm. These findings suggest that IP3Rs have significantly more important roles in spermatogenesis than RyRs, and that one of these roles is crucial for cell proliferation.

Xenopus laevis eggs are surrounded by an extracellular matrix consisting of a vitelline envelope, and three jelly layers, J1, J2, and J3 (from egg surface outward). The jelly layers vary in thickness (about 150, 15 and 200 μm for J1, J2 and J3 respectively) but all are translucent allowing observation of sperm penetration. Video microscopy demonstrated that sperm are able to penetrate and traverse J3 at velocities approaching 30 μm/s. Sperm swim through jelly in a corkscrew-like manner with their rotational and forward velocities being tightly coupled at about 30°/μm forward travel. They are propelled by whip-like power strokes involving hairpin bends in the flagellum that are generated every 180° of rotation and which are propagated from base to tip. The overall trajectories of individual sperm are quite variable. Many sperm head directly for J2 but some do not, these swimming circumferentially, or even away from the egg surface. Most sperm (over 97%) that enter the jelly do not get to the egg surface but are stopped at a variety of positions within J3 or at the outer surface of J2. Efficient sperm penetration and passage through the jelly layers requires a low electrolyte concentration in the surrounding medium, and is inhibited by the lectin wheat germ agglutin (WGA) in a dose-dependent manner. WGA does not block sperm penetration of J3 but does block further progression towards the egg surface. This observation suggests that sperm motility within the jelly is dependent on the carbohydrate moieties of the large glycoconjugates present, and that their alteration by WGA binding accounts for the inability of sperm to reach the egg surface and fertilise the egg.

In mammals, the restoration of rRNA transcription after fertilisation is accompanied by a gradual differentiation of the nucleolar structure by a process called embryonic nucleogenesis. During cleavage, the nucleolar components appear sterically related to a class of nuclear bodies already detectable in pronuclei. These structures, due to their apparent function as centres of nucleolus formation, have been designated nucleolus precursor bodies (NPBs). It was found recently not only that the size and morphology of the NPBs differ among mammalian species, but that the pattern of embryonic nucleologenesis and even the molecular composition of different NPB compartments vary from one species to another. Accordingly we assumed that at least two definitely different types of NPBs exist, namely the mouse-type NPB and cow-type NPB. In the mouse-type NPB, the original compact material of the NPB remains detectable in the early functional nucleolus. This NPB core does not contain DNA or typical Ag-NOR nucleolar proteins. At the onset of rRNA transcription, the nucleolonema is formed at the periphery of the NPB. The cow-type NPB shows a homogeneous distribution of typical nucleolar proteins throughout its body from the pronucleolar to the early 8-cell stage. At the beginning of rRNA transcription, the cow-type NPB is penetrated by perinucleolar DNA and rRNA synthesis is detectable deep inside the nucleolus. In this case, the entire NPB is readily transformed into a typical nucleolus. These processes are recognisable using fine-structure analysis of preimplantation mammalian embryos. For this reason this approach is often used as a method of evaluating the state of experimental embryos; in such studies, the species differences must be taken into account.