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.

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.

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.

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.

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.

Mouse oocytes penetrated by spermatozoa during germinal vesicle (GV) breakdown undergo maturation and are arrested at metaphase of the second meiotic division despite the presence of sperm nuclei within the ooplasm. When these oocytes were re-inseminated, none was penetrated by newly added spermatozoa. When GV oocytes were inseminated and cultured in the presence of dibutyryl cAMP, the oocytes remained at GV stage, yet they did not permit entry of additional spermatozoa. These observations suggest that the plasma membrane of maturing oocytes is modified by precociously penetrating spermatozoa independently from cortical granule exocytosis. Sperm components incorporated into the oocytes seem to be responsible for the modification of the oocyte's plasma membrane.

Hypoxanthine can block preimplantation mouse embryo development in vitro at the 2- to 4-cell stages, and this has recently been shown to be reversed by cAMP-elevating agents. However, the extent of this hypoxanthine-induced arrest is determined by the culture conditions and strain of mouse. Whitten's and KSOM/AA are two embryo culture media that support preimplantation development to the blastocyst stage. This study was undertaken to examine the influence of several components in these media on hypoxanthine-arrested preimplantation mouse embryos and to test the hypothesis that reversal of the hypoxanthine block by cAMP-elevating agents requires cooperative interaction with the chelator, EDTA. Initial experiments demonstrated that embryo development was blocked in the presence of hypoxanthine in Whitten's medium but not in KSOM/AA; furthermore, removal of EDTA from KSOM/AA rendered this medium incapable of supporting high levels of development to blastocyst (9%), whereas high numbers of blastocysts (80%) formed in Whitten's medium, which does not contain the chelator. Consequently, Whitten's medium was used to test our hypothesis. It has previously been demonstrated that the phosphodiesterase inhibitor, IBMX, can reverse the developmental arrest imposed by hypoxanthine in EDTA-supplemented Earle's basic salt solution, but in the present study the addition of IBMX to Whitten's medium resulted in a block to development and failed to reverse the hypoxanthine arrest. These disparate effects can be explained by the presence or absence of EDTA. Supplementing Whitten's medium with EDTA reverses the IBMX effect, but not the hypoxanthine-induced block. While IBMX alone is unable to reverse the hypoxanthine block in Whitten's medium, development is greatly enhanced by the simultaneous addition of EDTA and IBMX. Similar results were obtained with the cAMP analogue, 8-AHA-cAMP. The data therefore support our hypothesis that the reversal of the hypoxanthine-induced arrest by cAMP-elevating agents is critically dependent on the presence of EDTA. We contrast this with the situation in mouse oocytes, where the hypoxanthine-induced meiotic arrest is not reversed by the addition of EDTA and/or cAMP-elevating agents.

Parthenogenetically activated mammalian oocytes have been used in the past decade as cytoplasts, in an attempt to support the development of nuclear transplant embryos. The present experiments were undertaken to study the DNA synthesis and the organisation of microtubules, nuclear envelope and chromatin during the first cell cycle of electrically activated porcine oocytes (parthenotes) matured in vitro by using immunocytochemistry and laser scanning confocal microscopy. The results showed that pronuclear-like (PN) formation began 4–5 h post-activation (hpa), whilst DNA synthesis as revealed by bromodeoxyuridine incorporation was initiated 5–6 hpa, with a maximum number of labelled oocytes (73%) around 11 hpa, and persisted in some parthenotes until 15–16 hpa. In the metaphase II (MII) oocytes, microtubules were detected only in the metaphase II spindle; no lamin A/C antigen was observed. Electrical DC pulses resulted in 91% of MII oocytes being activated and confocal microscopy Indicated that microtubules were assembled in the spindle first for the extrusion of a second polar body, and for the second time for division from one to two cells. Nuclear envelope, indicated by anti-lamin A/C stain, was formed around the time of PN formation and surrounded the nuclear chromatin of 1- and 2-cell parthenogenotea. These results demonstrate that the apparent normality in both DNA synthesis and dynamics of microtubules and nuclear envelope is involved with chromosomal organisation in the parthenotes. In addition, the use of electrically activated IVM oocytes for both nuclear transfer and parthenogenetic studies in pigs is discussed.

In this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation and in vitro fertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.

Prophase-arrested oocytes of Ruditapes philippinarum cannot be fertilised or stimulated by excess KCl, in contrast to the situation found in other bivalve species such as Barnea and Spisula. However, these oocytes can be triggered to reinitiate meiosis following treatment by serotonin or several pharmacological agents (calcium ionophores, thapsigargin, weak bases) which promote an intracellular calcium surge. Ruditapes oocytes further arrest in metaphase I, at which stage they can be activated either by sperm or by excess KCl. This suggests that functional voltage-operated calcium channels are expressed in this species during the course of maturation. Using pharmacological tools and direct binding of specific dihydropyridines, we demonstrate that these channels are L-type calcium channels which become functional after serotonin stimulation, their number increasing before germinal vesicle breakdown. Moreover we establish that: (1) the addition of 20 μM S(—)Bayκ8644, an agonist of L-type calcium channels, to metaphase-arrested oocytes releases them from metaphase block; (2) incubating these oocytes with PN200-110, a potent blocker of L-type calcium channels, inhibits their activation by both excess KCl and fertilisation. Taken together these data suggest that the absence of L-type calcium channels in the membrane of prophase-arrested oocytes of Ruditapes may account for their inability to be fertilised.

Adducin is a cytoskeletal protein that can function in vitro to bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. The Drosophila Adducin-like (Add) locus (also referred to as hu-li tai shao (hts)) encodes a family of proteins of which several are homologous to mammalian adducin (Ding et al., Proc. Natl. Acad. Sci. USA90, 2512–16, 1993; Yue & Spradling, Genes Dev.6, 2443–54, 1992). We report the identification of two novel adducin isoforms: a 95 × 103Mr form (ADD-95) and an 87 × 103Mr form (ADD-87). We present a detailed analysis of the distribution patterns of ADD-95 and ADD-87 during oogenesis and embryogenesis. The isoforms are co-expressed in several cell- and tissuetypes; however, only ADD-87 is present in mid- to late-stage oocytes. ADD-87 is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is detectable only at the anterior pole from stage 11 onward, correlated with localisation of Add-hts mRNA first to the cortex and then to the anterior pole of the oocyte. ADD-87 co-localises with F-actin and spectrin in the cortex of the oocyte through stage 10 of oogenesis, consistent with a possible role in cytoskeletal assembly or function predicted by mammalian studies.