Viable sperm cells of Nicotiana tabacum were isolated by the semi-vivo technique. After pollination, excised styles were floated, cut end immersed, in a solution of 15% sucrose with 0.01% boric and 0.03% Ca(NO3)2 at 27°C in a growth chamber until pollen tubes emerged. After sperm cells were formed (at least 8 h after pollination) tubes were immersed in a 9% mannitol solution. In this solution, sperm cells are nearly ellipsoidal and retain viability for over 6 h.

Over the past few years, we have witnessed a burgeoning series of papers addressing the role of calcium signalling in cell cycle control. In this review I will attempt to bring together all the diverse threads and discuss new concepts that have arisen from the most recent data. Because the major part of the data concerns mitosis/meiosis entry and exit, I have focused on these areas. I will jointly refer to meiotic and mitotic phases of the cell cycle as M-phase because these phases are highly comparable. Studies of the cell cycle involve a huge range of species, from plants to humans. I will, however, restrict this review to the work performed in early embryos. I apologise in advance to contributors to this field whose names I do not mention because they do not work on embryos.

Pretreatment of zona-free Chinese hamster (CH) oocytes with three kinds of lectin – concanavalin A (Con-A), phytohaemagglutinin-P (PHA) and wheat germ agglutinin (WGA) – was attempted in order to improve penetration by golden hamster (GH) spermatozoa in vitro. Con-A had no significant effect on penetration at 2 μg/ml, adequately facilitated oocyte-sperm fusion at 4 μg/ml, and caused excessive sperm binding and resultant severe polyspermy at 10 μg/ml. Neither PHA nor WGA had positive effects on sperm penetration at any concentrations (2–10 μg/ml) examined. Using the Con-A (4 μg/ml)pretreatment, high rates of interspecific fertilisation and subsequent chromosome analysis of hybrid 1-cell zygotes were achieved. Among 258 CH oocytes used, 212 (82.2%) were fertilised and 153 (72.2% of fertilised ova) developed to the first cleavage metaphase. Eventually, 132 CH-derived chromosome complements and 153 GH-derived ones were successfully karyoanalysed. Incidences of aneuploidy and structural anomaly were 3.1% and 2.3% in CH complements, and 1.4% and 6.5% in GH complements, respectively. These incidences were not significantly different from those obtained by intraspecific in vivo fertilisation, suggesting that our interspecific in vitro fertilisation system does not cause chromosome aberrations.

Mouse, sheep and bovine metaphase II oocytes were briefly preincubated in medium with 6-dimethyl-aminopurine (6-DMAP) and thereafter activated with ethanol before further culture in 6-DMAP supplemented medium. The presence of 6-DMAP enhances the efficiency of activation and the speed of pronuclear formation but has no effect on DNA synthesis. These results are discussed in the context of recently published data showing that the preincubation of condensed chromosomes with 6-DMAP blocks DNA synthesis when nuclei become reformed.

An electron microscopic study of the rabbit zygote has shown the presence of numerous paracrystalline structures (PSs) around the pronuclei. The majority of these structures are situated in the narrow space between pronuclei. The PSs during interphase are associated with small dense knobs, and filamentous material;some of them, namely those situated in the internuclear space, are also associated with striated rootlets. The PS and its appendages form a complex which nucleates microtubules during interphase and phase M. The structure of these complexes changes with the cell cycle. Striated rootlets disappear at G2/M. Dense Knobs and filamentous material separate from the PS, become loose and associate with numerous microtubules at the poles of the first mitotic spindle. PSs and their associated structures are considered to be a newly discovered morphological form of the centrosome.

We have investigated the quality of bovine IVM/IVF embryos co-cultured on Vero cells. Blastocyst cell numbers are very similar to those obtained in vivo, and higher than those obtained by co-culture with oviduct cells. The metabolism and conversion of fructose and glucose are not equivalent even though carbon dioxide production is similar and increasing from morula to blastocyst. Formation of free amino acids and incorporation into proteins are higher and faster for glucose than for fructose, but this conversion is rather stable with embryonic growth. Moreover, the by-products formed are not the same. Glucose at physiological concentrations (i.e.2 mM)seems to be a more appropriate fuel for the burst of embryonic development at the blastocyst stage in preparation for hatching.

Although progesterone is the maturation inducer in amphibians, it has been demonstrated that in Bufo arenarum oocytes resumed meiosis with no need of exogenous hormonal stimulus if derived of their enveloping, follicle cells. This phenomenon, called spontaneous maturation, is quite rare in amphibians. In B. arenarum, spontaneous maturation took place only in oocytes obtained during the reproductive period (spring-summer). During this period the oocytes also demonstrated a respiratory activity characteristic of mature oocytes. Interestingly, full-grown B. arenarum oocytes always responded to progesterone regardless of the season in which they were obtained and of their respiratory activity. The disposition of oocytes competent or not competent to mature spontaneously provides a useful system for the study of molecular mechanisms involved in the maturation process. The data presented here indicate that the activation of protein kinase C (PKC) induces germinal vesicle breakdown (GVBD) in denuded oocytes unable to mature spontaneously (winter oocytes) and is involved in the in vitro spontaneous maturation of B. arenarum full-grown oocytes. The inhibition of PKC by 1-(5-isoquinolynyl-sulphonyl-2-methyl-piperazine (H-7) impeded spontaneous maturation in a dose-dependent manner, thus supporting the participation of the PKC pathway during this process. Interestingly phorbol 12-myristate-13-acetate (PMA)-induced GVBD is inhibited by the incubation of the oocytes in dibutyryl cAMP (dbcAMP), indicating that both pathways, PKC and protein kinase A (PKA), are related at a certain point. However, spontaneous GVBD is less sensitive than PMA-induced GVBD to dbcAMP. This fact would support the suggestion that in spontaneous GVBD mechanisms different from activation of PKC are at work.

The acrosome reaction in Bufo arenarum spermatozoa has been visualised only by electron microscopy. Different staining procedures for spermatozoa are described in the present study. By light microscopy the acorsomal status cannot be determined, In some reacted sperm, stained with Coomassie blue, a filament that could be the ‘perforatorium’ was observed, as seen by electron microscopy. Several fluorescent lectins were used, but only FITC-PNA binds to acrosomal proteins. However, the fluorescence observed was weak. Indirect immunofluorescence, with antibodies raised against acrosomal matrix, showed different staining patterns between acrosome-reacted and intact spermatozoa. The technique is specific for evaluating acrosomal status in a large population of spermatozoa. Moreover, immunostaining, in contrast with lectin staining, can be carried out in the presence of glycoconjugates such as oocyte extracellular matrix without interference.

Fresh cytosols extracted from unfertilised eggs of Xenopus laevis contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+ and is designated primary CSF (1°CSF). During storage of Ca2+-containing cytosols at 2°C, a stable CSF activity appears which is designated secondary CSF (2°CSF). In the present study, we report that 2°CSF activity can be induced in cytosols extracted from stage VI oocytes, unfertilised eggs, electrically activated eggs or blastulae, in the presence of Ca2+. Both the intensity and the rate of 2°CSF development are dependent on the concentration of Ca2+ ions added to the cytosol. At Ca2+ concentrations of 5–10 mM, 2°CSF activity reaches a maximum in about 7 days. Secondary CSF is relatively resistant to heat but loses all activity after 5 min at 70°C. When stored at −80°C, 2°CSF activity remains detectable for about 6 weeks. Cytological observations show that blastomeres arrested by microinjection of 2°CSF developed in cytosols of unfertilised eggs, activated eggs or blastulae contain metaphase chromosomes embedded in a bipolar spindle that has no asters developed at its poles. In contrast, blastomeres arrested by 2°CSF in cytosols of stage VI oocytes contain condensed chromosomes but no spindle is formed. The mechanisms of the development of 2°CSF in Ca2+-containing cytosols and its mode of action are discussed.

To enhance potential use of the Chinese hamster, Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques for in vitro fertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37°C under 5% CO2 in air. They were then treated with ionophore A23187 (20¼M) for 10min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37°C under 5% CO2 in air. In this study, 245 oocytes ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1mM hypotaurine under the same gas phase. Within 30h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage(‘2-cell block’) was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.

Microsomal fractions of Xenopus oocytes release preloaded 45Ca2+ when treated with inositol triphosphate (InsP3). The effective concentration of InsP3 required for half-maximal release (EC50) is 59 nM and maximal release occurs at ∼ 2 μM InsP3. Uptake and release of 45Ca2+ are not altered by the catalytic subunit of protein kinase A, dibutyrl cyclic adenosine monophosphate, protein kinase A peptide inhibitor or nocodazole. In contrast, taxol decreases the sensitivity of the microsomal fraction to InsP3, shifting the EC50 for InsP3-induced Ca2+ release from 59 to 259 nM. In lysates of oocytes, InsP3-induced Ca2+ release causes the tyrosine phorphorylation of a 42000 (Mr 42k) protein identified as 42k mitogen-activated protein (MAP) kinase. InsP3-induced tyrosine phosphorylation of MAP kinase is prevented by BAPTA and taxol, but not by nocodazole. Thus, microtubule polymerisation modifies InsP3-induced Ca2+ release, thereby inhibiting phosphorylation of MAP kinase.

Previously we have shown that mitogen-activated protein (MAP) kinase activity abruptly increases at the first metaphase (M1) and remains significantly higher than that at the germinal vesicle (GV) stage until the second metaphase (M2) in porcine oocytes cultured in vitro. The present paper describes how the mechanism of the blockage of meiotic maturation by protein sythesis inhibition involves MAP kinase regulation. Cycloheximide arrested both germinal vesicle breakdown (GVBD) and the normal transition from M1 to M2. MAP kinase activation was also reduced in these maturation-inhibited oocytes. By using immunofluorescence microscopy with the monoclonal antibody raised against rat α-tubulin, we showed that cycloheximide caused morphological abnormality in a spindle at M1, but not at M2. All these results indicate that in porcine oocytes: (1) GV blockage by protein synthesis inhibition involves the suppression of both histone H1 kinase and MAP kinase activation, (2) during the transition from M1 to M2, maintenance of a normal metaphasic spindle and high MAP kinase activity require protein synthesis, and (3) once the M2 cytoskeletal structures have been completed, and/or after the ‘critical period’, cytostatic factor activity is independent of protein synthesis.

The influence of unprotected freezing of mammalian spermatozoa on their oocyte activating capacity and chromosome integrity is unknown. However, this type of sperm treatment has been used in assisted reproduction by intracytoplasmic sperm injection in cattle and humans. The mouse oocyte injection test was used to analyse the influence of unprotected freezing of human spermatozoa on their reproductive characteristics. Mouse oocytes were microinjected with intact human spermatozoa or spermatozoa treated with two cycles of unprotected freeze-thawing. Oocytes surviving the injection were either cultured without further treatment or exposed to ethanol solution to induce parthenogenetic activation. Both injected and activated oocytes were used for sperm chromosome analysis. The results revealed a significant reduction in oocyte activating capacity and a tenfold increase in the incidence of structural chromosomal abnormalities in human spermatozoa treated by unprotected freezing. We conclude that unprotected freezing of human spermatozoa has a detrimental effect on their reproductive characteristics. Our data also provide a new perspective on the stability of mammalian spermatozoa to physical factors and demonstrate the importance of detailed analysis of the stability of sperm structures for successful development of new approaches in assisted reproduction.

The chorion is the acellular envelope surrounding mature eggs of teleostean fish. The macromolecular composition of the zebrafish (Danio rerio) egg chorion, organised as a three-layered structure, has been analysed. SDS-PAGE analysis, under reducing conditions, of isolated and purified chorions revealed a reproducible pattern of four major polypeptides (116, 97, 50 and 43kDa) and several minor bands. Lectin binding assays showed that both the 116 kDa and 50kDa proteins were recognised by concanavalin agglutinin (Con A), Galanthus nivalis agglutinin (GNA), Sambucus nigra bark agglutinin (SNA) and Ricinus communis agglutinin (RCA 120), suggesting that these polypeptides are N-linked glycoproteins. By contrast, neither the 97 kDa nor the 43 kDa polypeptides were stained by these lectins, indicating that these polypeptides are not glycosylated. Amino acid analysis also showed significant differences in the average content of some amino acids, for example serine and proline, when compared with previous reports.

It has recently been proposed that some of the processes induced by fertilisation in mammals may be mediated by integrins. By peforming immunofluorescence labelling and Western blots with antibodies directed against some of the α and β subunits of integrins, we show here the presence of some of these proteins in human and hamster oocytes. Among them, α2 and α5 were also present on in vitro preparations of sea urchin egg cortices. In addition, antibodies raised against these two proteins were the most effective at inhibiting attachment and fusion of human spermatozoa with hamster oocytes. We suggest that α2 and α5 integrin chains may be common mediators in adhesion-fusion mechanisms triggered by fertilisation. Using similar techniques, we show that eggs are rich three cytoskeletal proteins known to be linked to the β chain of integrins: talin, vinculin and α-actinin. Moreover, we found that talin and α-actinin were associated with proteins phosphorylated on tyrosine after fertilisation in sea urchin eggs. We suggest that integrins might be involved during fertilisation and trigger egg activation through cytoskeletal structures.

In the mouse, mature oocytes injected with prespermatozoal cell nuclei remain unactivated. Additional stimulation is needed to trigger oocyte activation leading to embryo development. We compared various electrical stimulations, treatment with cycloheximide alone or in combination with electrical stimulation, and injection of sperm-borne oocyte-activating factor (oscillogen) in terms of their oocyte activation and embryo development rates. Of all the treatments tested, a single electrical pulse (1.0 kV / cm, 128 μs) was the simplest, yet very effective, in allowing the development of the oocytes injected with spermatid nuclei.

This study aimed to achieve high frequencies of nuclear maturation and penetrability through the zona pellucida of hamster oocytes cultured under protein-free conditions. Completion of nuclear maturation by cumulus-intact, immature oocytes (79% metaphase II stage) was depressed (37% p < 0·05) by adding four amino acids (glutamine, isoleucine, methionine and phenylalanine) reported necessary for nuclear maturation of cumulus-free oocytes. Following in vitro maturation, cumulus cells were removed and oocytes were inseminated with capacitated sperm, but after 6 h sperm:egg co-incubation, only 24% of in vitro matured oocytes were penetrated compared with 60% of in vivo matured oocytes (p < 0·05). Time required for zona lysis by α-chymotrypsin was not significantly different among in vitro and in vivo matured oocytes and 1-cell embryos. Addition to the maturation medium of soybean trypsin inhibitor or fetuin, both known to inhibit the zona reaction in vitro, did not improve penetrability of in vitro matured oocytes, implying that in hamsters, unlike other rodent species, a premature zona reaction is unlikely to be responsible for inhibiting sperm penetration. When oocytes were incubated with 20% periovulatory oviductal fluid (OF) for another 3 h after maturation, penetration was significantly improved (60% vs 37% with and without OF, respectively; p < 0·05), but was not equivalent to penetration of in vivo matured follicular oocytes similarly treated with OF (84%, p < 0·05)However, zona penetration was further improved by increasing sperm concentration from 1·0 × 104 (66%) to 5·0 or 10·0 × 104 sperm/ml (89%, p < 0·05). This study shows that nuclear maturation of hamster oocytes can occur in chemically defined medium, and indicates that a deficiency in the zona of in vitro matured oocytes can be overcome by preincubation with OF and insemination at high sperm conccentration.

Expression of various developmentally regulated markers was screened throughout the preimplantation stages of in vitro-derived bovine embryos. This was done by investigating the distribution of several nuclear, cytoplasmic and extracellular proteins by means of immunofluorescence microscopy. While lamin B appeared as a constitutive component of nuclei of all preimplantation stages, lamins A/C had a stage-related distribution. The early cleavage stage nuclei contained lamins A/C which generally disappeared in the following stages, with the possible exception of a few positive nuclei in the morula and early blastocyst stage. In the expanded blastocyst stage the nuclei of trophectoderm cells became positive while no positivity was observed in the inner cell mass cells. Starting from day 6, the appearance and/or polarised distribution of various cytoskeletal and cytoskeleton-related components such as Factin, α-catenin and E-cadherin gave an insight into the timing of events related to compaction of bovine e bryos. Compaction was correlated with the first differentiation event, i.e. the formation of trophectoderm; this is the first embryonic epithelium, characterised by cytokeratins and desmoplakin. Extracellular fibronectin was first detected in the early blastocyst stage shortly before the morphological differentiation of primitive endoderm, and in the later stages it was localised at the interface between trophectoderm and extraembryonic endoderm. Laminin and collagen IV were expressed by the endoderm cells and contributed to the extracellular matrix underlying the trophectoderm. This study is a first attempt to characterise the cells of in vitro-derived bovine embryos valid for cell line derivation.