In mammalian oocytes, calcium plays an important role in the regulation of meiotic maturation. In our study, we used the mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, to mobilise intracellular calcium deposits during in vitro maturation of pig oocytes. The CPA treatment of maturing oocytes significantly accelerated the progress of their maturation. Oocytes entered the CPA-sensitive period after 21 h of in vitro culture. A very short (5 min) exposure to CPA (100 mM) is sufficient to accelerate maturation and it seems that accelerated maturation can be triggered by a transient elevation of intracellular calcium levels. The effect of CPA is not mediated through the cumulus cells, because maturation is accelerated by CPA treatment even in oocytes devoid of cumulus cells. Culture of oocytes with the calcium channel blocker verapamil (concentrations ranging from 0.01 to 0.04 mM) blocked the progress of oocyte maturation beyond the stage of metaphase I. This block can be overcome by the mobilisation of intracellular calcium deposits after CPA treatment (100 nM). The microinjection of heparin (20 pl, 50.1 mg/;ml), the inhibitor of inositol triphosphate receptors, before CPA treatment prevented the acceleration of oocyte maturation. This indicates that CPA mobilises the release of calcium deposits through inositol trisphosphate receptors. On the other hand, the microinjection of procaine (20 pl, 200 nM) or the microinjection of ruthenium red (20 pl, 50 mM), both inhibitors of ryanodine receptors, did not prevent accelerated maturation in CPA-treated oocytes. If present in pig oocytes, ryanodine receptors evidently play no part in the liberation of calcium from intracellular stores after CPA treatment.

We have used monoclonal antibodies specific for different sets of human cytokeratins and the anti-IFA (Intermediate Filament Antigen) antibody to investigate the expression of intermediate filament proteins in the mature oocyte of the teleostCyprinus carpio. Several polypeptides have been identified, showing molecular weights ranging from 43 to 65kDa. Two-dimensional analysis of the immunoreactive species revealed the presence of at least six major protein spots and a series of minor components, grouped in quite a narrow pI range from 5.52 to 6.28. The general complexity of the carp oocyte cytokeratin-related cytoskeleton appears to be higher than those described for oocytes of other vertebrate species.

Immature mouse oocytes (germinal vesicle stage, GV), oocytes at different stages during maturation (prometaphase to anaphase I) and matured oocytes (metaphase II arrested) were cultured in 6-dimethylaminopurine (6-DMAP)-supplemented medium also containing bromodeoxyuridine for the assessment of DNA replication in these cells. Immature oocytes remained arrested at the GV stage and DNA replication was never detected in them. On the other hand, oocytes at the prometaphase to anaphase-telophase I stages responded to 6-DMAP treatment by forming nuclei which synthesised DNA. Mature (metaphase II) oocytes did not respond to 6-DMAP and their chromatin remained condensed. DNA synthesis could even be induced in GV-staged oocytes, but only when they were fused to freshly activated oocytes and incubated in 6-DMAP-supplemented medium.

This study evaluates the ability of the cytoplasm to determine the nature of the division cycle (meiotic or mitotic) in nuclei obtained from mitotically dividing cells. Using mouse oocytes in different stages of development two types of cytoplasm were prepared: firstly, early meiotic ooplasts were obtained by enucleation of non-matured, prophase-stage oocytes; secondly, mitotic cytoplasts were prepared by enucleation and activation of metaphase II (Mll)-stage oocytes. These two types of cytoplasts were then used in fusion experiments, in which mouse primitive type A spermatogonia (prospermatogonia) or mouse fibroblasts were used as a source of donor nuclei. While the fusion of prospermatogonia with mitotic cytoplasts resulted, as expected, in normal premature chromosome condensation (PCC) and subsequent pronuclear formation (58.1%), the majority of hybrids obtained by fusion of prospermatogonia with early meiotic ooplasts (40.3%) displayed unique morphology consisting of two sets of chromosomes organised in two spindle centres connected by microtubules. Each set of chromosomes contained the haploid (1n) number of chromosomes as revealed by chromosome analyses. The same morphology was observed also in 44.2% of hybrids in which the differentiated nuclei of fibroblasts were used as a source of donor mitotic nuclei. In both cases the hybrids were blocked at this stage with high activity of maturation promoting factor (MPF), resistant to any kind of activation and not able to undergo further development. These results suggest that the early meiotic ooplasm was able to induce the initiation of a meiosis-like reducing division in mitotic nuclei originating both from the germline cells and from more differentiated somatic cells.

Sea urchin spermatozoa were injected into mature mouse oocytes to determine whether they can activate mouse oocytes and, if so, how they behave within the oocyte cytoplasm of such a distant species. While injection of a single spermatozoon into each oocyte did not activate any of the oocytes, injection of 10 spermatozoa activated about 20%. Within the cytoplasm of unactivated oocytes, sperm heads commonly transformed into chromosome-like structures. When a single spermatozoon was injected, and oocytes were then activated by Sr2+, about 30% of the activated oocytes had both female (mouse) and male (sea urchin) pronuclei when examined 8 h after sperm injection. These results indicated that sperm-borne oocyte activating factor(s) and the cytoplasmic factors controlling the development of the sperm pronucleus are not strictly species-specific.

We have investigated the fertilisation competence, protein synthesis, histone H1 kinase and myelin basic protein (MBP) kinase activities in three categories of bovine oocytes (derived from three size categories of follicles: M–medium, 2.5–5.0 mm; S–small, 1.5–2.5 mm; T – tiny, 1.0–1.5mm). In contrast to more or less normal meiotic maturation (85.6%) and fertilisation (70.8%) of M oocytes cultured for 24h, the fertilisation of M oocytes cultured for 40h was associated with increased rates of retarded male pronuclear development and retention of the second polar body. The S and T oocytes cultured for 24h or 40h were mostly arrested at defective late diakinesis - metaphase I (77.5–100%) stage. After fertilisation of S and T oocytes cultured for 24h no polar body was extruded and formation of one, three or four female pronuclei, together with mostly normal male pronuclei, was observed. The fertilisation of S and T oocytes after 40h culture resulted in a higher number of female and a decreased number of male pronuclei. A major change in the pattern of protein synthesis was associated with the resumption of meiosis. There were no significant differences in the profile of protein synthesis between oocyte categories in all groups either matured or fertilised. The H1 kinase activity reached comparable increased levels in oocytes of all categories matured for 24h and decreased during the 40h culture, most significantly in M oocytes. The MBP kinase activity was at approximately the same high level in all categories of oocytes after 24h of culture and remained stable until 40h. The fertilisation after 24h of culture resulted, in M oocytes, in low levels of both H1 and MBP kinase activities; in S oocytes, only H1 kinase was completely inactivated while MBP kinase activity decreased to some extent; in T oocytes, both H1 and MBP kinase activity decreased. Fertilisation of all oocyte categories after 40h culture resulted in complete inactivation of both these kinases to their basal levels.

The objective of this study was to investigate the influence of sperm mitochondrial destruction on sperm head decondensation, male pronuclear formation, cleavage and embryonic development. In the study two models were used:heterologous (hamster ICSI assay: human sperm injected into a hamster oocyte) for evaluation of sperm head decondensation and pronuclear formation, and homologous (mouse model) for the study of fertilisation and development. Destruction of mitochondria of the sperm was achieved by exposure to cyanide, a respiratory poison. Rhodamine 123 was used to evaluate the functional integrity of mitochondria. Sperm head decondensation was found to be not statistically significantly affected by mitochondrial damage (p = 0.8), with 62.8% and 67.9% condensa tion in the experimental and control groups respectively. Male pronucleus formation was seen in 40.2% and 44.4% of the injected oocytes in the experimental and control groups respectively. In the mouse experiments 45.5% and 49.7% of the injected oocytes were fertilised in the mitochondria-damaged and live-intact sperm groups respectively(p = 0.53)Development to blastocyst was achieved in 53.5% and 59.4% of the experimental and control groups respectively; the difference was not significant (p = 0.71). Inner cell mass (ICM) cell number were 15.7 ± 4.02 and 43.1 ± 11.3 respectively in the mitochondria damaged group; the equivalent numbers were 14.12±4.12 and 39.3 ± 12.6 in the control group. However, the differences in ICM and total cell counts between these two groups were not significant. Of the blastocysts transferred to pseudopregnant mice, 51.3% (20/36) implanted and 33.4% (12/36) developed to live fetuses in the mitochondria-damaged group. These rates were 60.5% (23/38) and 39.5% (15/38) in the control group. In conclusion, this study shows that functional integrity of thesperm mitochondria is not necessary in the process of fertilisation and development when the sperm is deposited into the ooplasm. Fertilisation and development can be achieved by injection of sperm at the very early stage of necrosis in which only the mitochondria have been destroyed and the rest of the cell including the plasma membrane is still intact.

Oocyte maturation in mammals follows a highly conserved pattern of release from arrest through to the extrusion of the first polar body and formation of the second metaphase spindle. Oscillations in cytoplasmic calcium concentration precede the events of maturation in many species. These calcium ions interact with and activate calcium-binding proteins, including calmodulin, within the cell. Thus, it was of interest to us to examine whether calcium acted through calmodulin in the initial stages of maturation in rabbit oocytes or whether calmodulin was required for continuation through metaphase I on to metaphase II. Using the calmodulin inhibitor W-7 we found a significant (p < 0.05) decrease in the percentage of oocytes that underwent germinal vesicle breakdown. Calmidazolium did not prevent germinal vesicle breakdown; however, it caused a significant (p < 0.05) decrease in the proportion of oocytes with fully elaborated spindles and taxol-induced cytoplasmic asters. Both inhibitors caused a significant (p < 0.05) reduction in the proportion of oocytes that extruded their first polar bodies. The kinase inhibitor 6-DMAP caused a significant reduction in the proportion of oocytes with spindles and condensed chromatin, indicating the necessity for phosphorylation events in the resumption of meiosis. In rabbit oocytes calmodulin may play a role in the release from prophase arrest, and it is necessary for spindle preservation and continuation through metaphase I to metaphase II. The varying effects of the two inhibitor stems from their different binding sites on the calmodulin molecule thus causing a differential effect on its downstream effectors.

Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2–6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM 199 supplemented with 10 μg each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5°C) for 4.5 h during transportation. Then, oocytes were transferred into 75 μl of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5°C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37°C water bath for 15s. Motile fractions were selected by swim-up, then incubated for 90 mm in TALP with 10 μg heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 μl) were added to 10 μl medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM 199 + 20% FBS at 37°C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 μl of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.

Full-grown ovarian oocytes of the amphibian Bufo arenarum were induced to mature in vitro by removing the follicular layers (spontaneous maturation) or by treatment with progesterone (hormone-induced maturation). These oocytes were then treated with trypsin and inseminated with homologous spermatozoa. Oocytes matured in vivo that had not undergone any influence of the oviducts (coelomic oocytes), inseminated under the same experimental conditions, were used as controls. The results show that oocytes induced to mature in vitro and exhibiting apparently normal signs of activation were polyspermic. In fact, 2 h after insemination numerous functioning pronuclei could be observed in the animal hemisphere. These results suggest that even though the oocytes which matured in vitro were able to undergo activation after insemination, they were unable to establish an effective block to polyspermy.

As glycine is one of the most concentrated amino acids in the female genital tract, we investigated its uptake by bovine in vitro matured/in vitro fertilised blastocysts in the presence of increasing concentrations of radiolabelled glycine. We also determined methionine uptake by in vitro and in vivo produced embryos. In our study, the hypothesis of more than one site of enzyme activity for glycine substrate was not validated. We determined a Vmax of 23.4fmol/min per embryo and a Km value of 13.3μM. No significant difference was observed either between in vivo and in vitro derived embryos or between grade 1 and grade 2 embryos for methionine uptake. The methionine and glycine uptake of a day 7 bovine was similar to that of a day 4 mouse blastocyst. This is rather low if we consider the relative cell numbers.

The addition of acetylcholine (ACh, 100 μl of 10 μM) to the bath in the vicinity of unfertilised sea urchin eggs (Lytechinus variegatus) suspended in sea water (SW) abruptly depolarises the membrane potential (Vm) of the eggs from the resting value of approximately −70 mV. This results in the firing of the egg's action potential, followed by partial repolarisation. Similar addition of ACh to eggs voltage clamped at −70 mV induces an inward current of abrupt onset with peak amplitude of −1.26 ± 0.20 nA (SE, n = 81). When the eggs are clamped at a Vm more positive than −70 mV, the peak amplitude of the ACh-induced inward current decreases, becoming 0 at a clamped Vm of approximately −20 mV. Further positive shift of the Vm fails to cause reversal of the current. Oocytes clamped at −70 mV exhibit similar inward current responses following application of ACh. Since ACh stimulates both nicotinic and muscarinic receptors (nACh-R and mACh-R, respectively), the effects of exposing eggs to the agonists and antagonists for each type of receptor were examined. For unfertilised eggs clamped at −70 mV the application of 100 μM (−)-nicotine hydrogen tartrate, an agonist of the nACh-R, induces an inward current response similar to that elicited by 10 μM ACh, but of smaller peak amplitude. In contrast, the application of (+)-muscarine chloride, an agonist of the mACh-R, fails to induce any response. Antagonists of the nACh-R inhibit either the neuronal type of nACh-R or the skeletal muscle type of nACh-R. The effect of the antagonists on the amplitudes of the ACh-induced inward current response was determined by supervising individual eggs clamped at −70 mV with the desired antagonist dissolved in SW, followed by the addition of 100 μl of 10 μM ACh in the vicinity of the egg. Mecamylamine chloride, an antagonist of the neuronal nACh-R at a concentration of 1 μM, markedly decreases the response to ACh, while at a concentration of 10 μM the response to ACh is abolished. Hexamethonium chloride, another inhibitor of the nACh-R of the neuronal type, also diminishes the ACh-induced response, but at a concentration of 10 μM the response is not completely abolished. Exposure of eggs to α-bungarotoxin, an antagonist of the skeletal muscle nACh-R at concentrations up to 250 nM for periods of 30 min, has no effect on the ACh-induced response. The effects of two antagonists of the mACh-R, atropine sulphate and QNB (R-(−)-3-quinuclidinyl benzilate) were also examined. Exposure of eggs to 1 μM atropine does not affect the ACh-induced response, but at concentrations of 10 μM atropine the amplitude of the ACh-induced inward current is significantly reduced. The exposure of eggs to QNB, a highly specific antagonist of the mACh-R, at concentrations up to 50 nM, has no effect on the ACh-induced response. Consequently, the likely explanation for the inhibitory effect of atropine is that at high concentrations atropine cross-reacts with the nACh-R. These findings reveal the presence in unfertilised sea urchin eggs of an ACh-R resembling the neuronal nACh-R. No evidence could be obtained that these receptors have a role in sperm entry, activation of the egg, or early development.