The capacity of sister blastomeres of mouse embryos for induced fusion changed during the 2-cell stage. It was at low level (24%) at the early 2-cell stage, increased and reached 98.5% at the middle 2-cell stage and fell sharply to 31% at the late 2-cell stage. At the time corresponding to the G2/Mphase of the cell cycle the blastomeres fused in only 8% of cases. Vital staining of 2-cell embryos by rhodamine 123 showed that the mitochondria were dispersed throughout the cytoplasm with a ringlike (around the nucleus) or spot-like (over the metaphase plate) concentration in the centre of each blastomere. At the periphery of blastomeres the mitochondrial content was low. The behaviour of the mitochondria reflected the subsequent events of structural and functional integration of the sister blastomeres under induced fusion: a discernible boundary between partners during 30 min after electrofusion or 1 h after fusion with polyethylene glycol; movement of the two ‘rings’ to the centre of the blastomere fusion products (BFP) to form one large bright ‘spot’ over the common metaphase plate; mitochondria outlining the shape of the spindle and connection between sister blastomeres until completion of the first mitosis of BFP The data obtained suggest that fusion of the blastomeres does not lead to extensive changes in the hybrid cytoplasm and integration of nuclear material is taking place only at metaphase stage Cytogenetic examination of BFP at the 2-cell stage confirmed reconstruction of the tetraploid embryos and found that sister blastomeres of such embryos could asynchronously enter the next cleavage division similarly to normal diploid 2-cell embryos.

Oocytes from eight inbred strains of mice were screened for the timing of germinal vesicle breakdown (GVB) in vitro. This characteristic varied between strains, reaching most extreme values in oocytes from AKR and BALB/c mice (3.1 and 1.6h after release from dibutyryl cAMP block, respectively; p<0.0001). The difference between AKR and BALB/c mice was confirmed in experiments in which GVB was induced in vivo by stimulation with exogenous gonadotrophins. Analysis of the rate of GVB in hybrids obtained after fusion of nuclear and cytoplamic fragments of oocytes from both strains suggests that the factor responsible for the difference between AKR and BALB/c mice is located in the cytoplasm of the proghase oocytes. Finally, in oocytes from both strains stimulated to resume meiotic maturation with okadaic acid, an inhibitor of protein phosphatases types 1 and 2A the rate of GVB was the same (2.2h and 2.3h for AKR and BALB/c, respectively; p= 0.48). This suggests that the difference between strains is not related to the amount or quality of the pre-MPF (Maturation Promoting Factor) stored in the prophase oocyte, but to the factor(s) acting upstream of the dephosphorylation of p34cdc2. kinase in the pathway leading to pre-MPF activation.

Although the piezo-electrically driven pipette is much more effective than a conventional pipette for direct sperm injection into mouse oocytes, the piezo-pipette driving unit is expensive and may not be readily accessible to most basic laboratories. We reprot here that the inefficiency of the conventional pipette can be improved by performing the operation in a medium with high serum concentration.

Porcine oocyte-cumulus complexes were cultured in bovine serum albumin(BSA)-free North Carolina State University (NCSU)23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml)and hormonal supplements (eCG and hCG: 10IU/ml each) for 22h. They were then cultured in the same medium but without hormonal supplements for an additional 22h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM)199 containing caffeine (5mM), fetal calf serum (FCS; 10%) and varying concentrations (26–56 mM)of NaHCO3 for 9h(experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3 concentrations. Experiment 3 examined the effect of FCS (1% and 10%)and NaHCO3 (26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p<0.05)at 36–56mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p< 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56mM NaHCO3, respectively. The fertilisation medium containing 46mM NaHCO3 and 1% FCS showed a higher penetration rate (84%)with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3 stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.

During the early mitotic cell cycles of the sea urchin embryo, the cell oscillates between S-phase and M-phase. In the presence of aphidicolin, a DNA synthesis inhibitor, a checkpoint control blocks the activation of the p34cdc2 protein kinase, by keeping it in the inactive, tyrosine phosphorylated form, and the embryos do not enter mitosis. Caffeine has been shown to bypass the G2/M-phase checkpoint in mammalian cells and in cycling Xenopus extracts and to induce mitosis despite the presence of damaged or unreplicated DNA. In this study we show that caffeine also induces mitosis and cell division in sea urchin embryos, in the presence of unreplicated DNA, by stimulating the tyrosine dephosphorylation of p34cdc2 and switching on its protein kinase activity. We also show that the caffeine-induced activation of the p34cdc2 protein kinase is not mediated by either of the two second messengers, calcium and cAMP, or by inhibition of the p34cdc2 tyrosine kinase. Thus, none of the mechanisms proposed for caffeine's action can explain how it overrides the S-phase checkpoint in the early cell cycles of the sea urchin embryo.

Primordial germ cells (PGCs) are described from the gonad of c. 2 cm juvenile Branchiostoma virginiae; early oocytes (c. 10 μm) and enlarging, previtellogenic oocytes (c. 35 μm) are described from the ovary of c. 5 cm adults. The germinal epithelium of the juvenile gonad and adult ovary is composed of both germinal and somatic cells. In the juvenile, somatic cells retain contact with the basal lamina of the germinal epithelium though their perikarya may be displaced towards the lumen; the germinal epithelium is, therefore, a simple but pseudostratified epithelium. In the adult ovary, somatic cells may lose contact with the basal lamina and the epithelium appears to become stratified. PGCs and oocytes are identified as germ cells by the presence of nuage. PGCs and oocytes are polarised epithelial cells. They rest on a basal lamina, extend apically towards a lumen, form adhering junctions with neighbouring cells, and exhibit apical-basal polarity. PGCs and early oocytes have an apical flagellum with an associated basal body, accessory centriole, and one or more striated rootlet fibres. The flagellum is surrounded by a collar of microvilli. Once oocytes begin to enlarge and bulge basally into the connective tissue layer, the flagellum is lost, but the basal bodies and ciliary rootlets are present at the apex of 35 μm oocytes. Similarities of the oogenic pattern in cephalochordates and echinoderms indicate that the establishment of egg polarity in deuterostomes is influenced by the polarity of the germinal epithelium.

We address the developmental activation, in the zebrafish embryo, of intrinsic cell-cycle checkpoints which monitor the DNA replication process and progression through the cell cycle. Eukaryotic DNA replication is probably carried out by a multiprotein complex containing numerous enzymes and accessory factors that act in concert to effect processive DNA synthesis (Applegren, N. et al. (1995) J. Cell. Biochem. 59, 91–107). We have exposed early zebrafish embryos to three chemical agents which are predicted to specifically inhibit the DNA polymerase α, topoisomerase I and topoisomerase II components of the DNA replication complex. We present four findings: (1) Before mid-blastula transition (MBT) an inhibition of DNA synthesis does not block cells from attempting to proceed through mitosis, implying the lack of functional checkpoints. (2) After MBT, the embryo displays two distinct modes of intrinsic checkpoint operation. One mode is a rapid and complete stop of cell division, and the other is an ‘adaptive’ response in which the cell cycle continues to operate, perhaps in a ‘repair’ mode, to generate daughter nuclei with few visible defects. (3) The embryo does not display a maximal capability for the ‘adaptive’ response until several hours after MBT, which is consistent with a slow rranscriptional control mechanism for checkpoint activation. (4) The slow activation of checkpoints at MBT provides a window of time during which inhibitors of DNA synthesis will induce cytogenetic lesions without killing the embryo. This could be useful in the design of a deletion-mutagenesis strategy.

The primary spermatocytes used were male germ cells at prophase I. The present study was undertaken to see whether bivalent chromosomes of mouse primary spermatocytes can undergo meiotic divisions within maturing oocytes and participate in subsequent embryonic development. Primary spermatocytes (pachytene to diplotene) freshly collected from the testes of mature males were electrofused with immature oocytes shortly before or after germinal vesicle breakdown. After culture in MEM-α medium for 15 h, most (> 90%) of the oocytes containing spermatocyte chromosomes underwent maturation and arrested at metaphase II (Mil). Among 23 Mil oocytes examined, 17 (74%) had one group of chromosomes and one polar body, indicating that male chromosomes had intermingled with those of the females and completed the first meiotic division. Chromosome analyses of these Mil oocytes demonstrated their diploidy. The metaphase chromosomes were transferred to enucleated Mil oocytes freshly recovered from superovulated mice. After artificial activation, the reconstructed Mil oocytes resumed meiosis and developed to the morula/blastocyst stage. However, no pups were born following embryo transfer into recipient females. These findings indicate that the chromosomes of primary spermatocytes undergo meiotic divisions in maturing oocytes and participate in the formation of diploid embryos.

Cumulus-free pig oocytes at the germinal vesicle (GV) stage were incubated in modified Brackett & Oliphant's medium with 5% fetal calf serum and 5mM caffeine with or without cryopreserved, ejaculated spermatozoa. When oocytes were transferred into modified tissue culture medium (TCM- 199B at pH 7.4) supplemented with 1OIU/ml eCG, 1OIU/ml hCG and 1 μg/ml oestradiol-17p after 8h of incubation with spermatozoa and cultured for 0–48 h, 86–99% of oocytes were penetrated. Most (95–100%) oocytes penetrated 0–16 h after transfer had decondensed sperm chromatin. However, 24 h after transfer 47% and 33% of penetrated oocytes contained recondensed sperm chromatin and sperm metaphase chromosomes, respectively. The proportion of penetrated oocytes containing sperm metaphase chromosomes increased after 36–48 h of transfer (51–65%). The transformation of sperm nuclei to metaphase chromosomes was obtained in 75% and 79% of anaphase I (AI) to telophase I (TI) and metaphase II (Mil) oocytes, respectively, but only in 38% of metaphase I (MI) oocytes. Moreover, such transformation was observed only in 1 of 30 oocytes at the stages of GV breakdown to prometaphase I and none of 69 oocytes at the GV stage. The transformation of sperm nuclei into metaphase chromosomes was completely inhibited in oocytes penetrated by eight or more spermatozoa. Well-developed male and female pronuclei were observed in only 3 (4%) of 77 oocytes penetrated 48 h after transfer. The proportion of oocytes reaching Mil was greatly inhibited by sperm penetration; only 18% of penetrated oocytes, but 87% of non-inseminated oocytes, reached Mil by 48 h after transfer. None of the oocytes penetrated by seven or more spermatozoa reached MIL Most (75%) oocytes were inhibited from the transition from MI to Mil even though they were cultured for 48 h. The present results indicate that: (1) the cytoplasm of maturing oocytes possesses an activity for transforming sperm nuclei into metaphase chromosomes, (2) immature pig oocytes penetrated by spermatozoa can undergo meiotic maturation to MI, and (3) the transition of such oocytes from MI to Mil is inhibited, suggesting that an activity of mitogen-activated protein kinase may be retarded.