We have measured the amounts of Na+, K+ and C− in preimplantation mouse embryos (1-cell, 2-cell and morula) using electron probe X-ray microanalysis. The levels of these ions do not vary much over this period, and are approximately the same as those found in other mammalian cells, contrary to previous reports. We have confirmed that preimplantation embryos exhibit Na+/K+-ATPase activity at all stages examined, and have shown that the ATPase maintains high K+/Na+ ratios (12–16) in all these embryonic stages, comparable to those seen in other healthy cells; this is in contrast to the low ratios reported in earlier work. Inhibition of the Na+/K+-ATPase results in the slow exchange of intracellular K+ for extracellular Na+ (half-time approximately 5 h), indicating that Na+/K+-ATPase activity maintains steep Na+ and K+ gradients in preimplantation mouse embryos as it does in most other cells.

We have recently shown that the incubation of Xenopus laevis oocytes in procaine-containing solutions induced germinal vesicle breakdown without white spot formation and, in some cases, with the appearance of spindle and chromosomes in the cytoplasm. The present study was performed to determine whether M-phase promoting factor was involved in this unusual maturation. Procaine failed to induce maturation in the presence of 6-dimethylamino purine or roscovitine, which are both known to inhibit p34cdc2kinase. Histone H1 kinase activity was detected in procaine-treated oocytes but it was always lower than in progesterone-treated controls. A shift in p34cdc2 was observed in oocytes that had been exposed to procaine for 16h, but it was not detected in those exposed for 24h. Finally, cytoplasm transfer experiments demonstrated that the maturation promoting activity that occurred in oocytes incubated in procaine for 16h could induce maturation of recipient stage VI oocytes. This transferable activity was weaker than that from progesterone-treated controls since only 30% of the recipients underwent germinal vesicle breakdown and only a few spindles were observed, which were not always correctly located. Taken together these results demonstrate that M-phase promoting factor is involved in the procaine maturing effect despite some differences compared with progesterone-treated oocytes which might explain the particular type of maturation induced by this substance. The discovery of the mechanisms by which procaine is able to activate M-phase promoting factor might now help in the understanding of some steps in progesterone-induced maturation that have still to be elucidated.

Larval testes of four Lepidoptera species were examined using electron microscopy. The testes of one species, the Mediterranean mealmoth Ephestia kuehniella (Pyralidae), were devoid of intracellular pathogens and serve as a control. In this species, metaphase spindles of primary spermatocytes showed a thick layer of perispindle membranes. The membranes were structurally very similar to the agranular endoplasmic reticulum. Membranes of this type occurred also at high frequency throughout the spindle matrix. The analysis of larval testes of Pieris brassicae (Pieridae) revealed virus-like particles within spermatocytes. In another species, Philudoria potatoria (Lasiocampidae), the spermatocytes possessed intracellular bacteria. Whereas the pathogens were found within the germ cells in these species, a fourth species, Plutella xylosteUa (Plutellide), showed microsporidia within somatic cells of the testis sheath. In all the infected animals, the mass of perispindle membranes was reduced in comparison with spermatocytes of E. kuehniella. However, spindle structure appeared regular in the infected animals. This indicates that a thick layer of perispindle membranes is not decisive for spindle assembly and function in male meiosis of Lepidopera

The isolation of viable egg cells of rape (Brassica napus L.) has been achieved from microdissected ovules. The non-gametic cells of the embryo sac, synergids and central cells have also been isolated. Their morphology corresponded to that of these cells in situ, making a discrimination from isolated sporophytic cells possible. Two hours after isolation the egg cells were still viable. Viable egg cells have been reproducibly isolated with afrequency of 25% per dissected ovule.

During fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entery into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been apoint of dispute. To resolve this issue, the fate of the germinal vesicle stage(GV oocytes) or fertilised eggs at the pronuclear stage(PN eggs). The SINEs disappeared quikly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelops around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle. i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase Promoting factor (MPF) is absent from the ooplasm.

Germ cells were isolated from rabbit fetal gonads between 18 and 22 days post coitum and examined morphologically, ultrastructurally and for immunocytochemical and cytochemical characteristics. Observations were compared with the information available from the corresponding cells of other mammalian species. The general morphology and ultrastructure of healthy isolated rabbit fetal germ cells were found to be very similar to those of the rabbit and mouse diploid germ cells in situ. Moreover, rabbit fetal germ cells shared common immunocytochemical characteristics with mouse undifferentiated embryonic stem cells or embryonic carcinoma cells, such as the presence of TEC-1 (SSEA-1) antigens, a peripheral network of F-actin, the absence of cytokeratins 8/18 and lamins A/C and an alkaline phosphatase activity. No difference between the sexes was observed. Morphological and physiological similarities with the migrating and cultured primordial germ cells of the mouse also suggest that diploid rabbit germ cells would be good candidates for deriving pluripotential embryonic germ cells (EG cells) if favourable culture conditions could be found. In conclusion, the rabbit may be suitable model for investigations on EG cells in domestic mammals with delayed meiosis.

The objective of this study was to determine the effects of oviductal fluid and heparin on sperm penetration and the characteristics of spermatozoa. The addition of oviductal fluid and heparin to the fertilisation medium decreased sperm penetration and the mean number of spermatozoa in penetrated eggs. The number of spermatozoa firmly bound to zona pellucida was also decreased in the presence of oviductal fluid and heparin. Chlortetracycline (CTC) fluorescence patterns were used to determine the incidence of capacitation and the acrosome reaction. The proportion of capacitated and acrosome-free spermatozoa increased when spermatozoa were exposed for 1.5 and 3 h to oviductal fluid and heparin. In contrast heparin alone did not increase the number of capacitated spermatozoa at these time points. These results suggest that factor(s) in oviductal secretions reduce polyspermic fertilisation and the number of spermatozoa that will penetrate porcine oocytes. The reduction of polyspermic penetration by oviductal secretions may be due to a reduced number of spermatozoa in the fertilisation medium with an intact acrosome.

Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM). When examined after 24, 56, 64 and 80h of culture, embryos developed to the 2-cell (100%), 4-cell (93%), 4-cell to 8-cell (97%) and ≥8-cell (95%) stages, respectively. When 0.4 M phosphate (NaH2PO4) was added to the medium after 0, 24, 56 and 64 h of culture, percentages (0–67%) of embryos that developed to the blastocyst stage after 115h of culture were lower than that (86%) in the medium without phosphate. However, addition of phosphate after 80h of culture accelerated blastocyst formation; a significantly higher percentage (94%) of blastocysts was obtained after 110 h of culture in this medium compared with when phosphate was not added (67%). When phosphate was added to the medium after 64h, almost all (97%) 8-cell embryos developed to the blastocyst stage by 100h of culture but development of 4-cell to 7-cell embryos was inhibited (22–63%). Acceleration of blastocyst formation was caused by addition of phosphate rather than by the exchange of the medium. The stimulatory effect of phosphate on embryo development was observed at concentrations of 0.1–1.2mM. The mean numbers of cells (54.5–60.9 cells) in blastocysts examined after 115 h of culture were increased by the addition of 0.4–1.6mM phosphate at 80 h as compared with blastocysts from cultures without phosphate (46.6 cells).

The dependence of pig oocyte activation (both nuclear activation and cortical granule exocytosis) induced by staurosporine on intracellular Ca2+ rise and spindle assembly was studied. Nuclear activation was evaluated by pronuclear (PN) formation, cleavage and their developmental ability, and cortical granule (CG) exocytosis was assessed by electron microscopy and laser confocal microscopy of oocytes labelled with fluorescein isothiocyanate-peanut agglutinin. Exposure of pig oocytes of 0.3 and 3μM protein kinase inhibitor staurosporine for 30 min resulted in the nuclear activation in 71.8% and 85.7% of the oocytes, respectively. The pronuclei in activated oocytes contained several compact nucleoli. When the cleaved 2-cell oocytes were further cultured in vitro, 93.5% developed beyond the 4-cell stage, and 12.9% developed to the morula stage after 4 days of culture. Of the oocytes treated with 3μM staurosporine, 62.5% and 9.4% released their CGs partially and completely, respectively. The nuclear activation induced by staurosporine was overcome by the prior treatment of oocytes with okadaic acid, resulting in only 33.3% of the oocytes undergoing nuclear activation. However, when oocytes were exposed first to 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethanal ester), a cell permeate calcium chelator, or Colcemid, a meiotic spindle disrupter, and then to staurosporine, nuclear activation was observed in 74.2% and 82.3% of the oocytes, espectively. These data were the same as those in oocytes treated only with staurosporine (85.7%). The present study indicates that pig oocytes can be activated by the inhibition of staurosporine-sensitive protein kinase(s), and that this activation is dependent upon mitogen-activated protein kinase but independent of the intracellular Ca2+ rise and spindle integrity.

Microinjection of anti-α-tubulin (Dm1A) inhibited progesterone-induced meiotic maturation in large follicle-enclosed oocytes of the frog, Rana pipiens. DM1A (46nl; 10mg/ml) injection significantly increased the ED50 value for progesterone as determined by germinal vesicle dissolution (GVD) bioassay. By contrast, low doses of microinjected DM1A (46nl; 2.5mg/ml), anti-actin (clone KJ43A), anti-cytokeratin (C-11), anti-intermediate filament antibody (IFA), generic IgG (46 nl; 20 mg/ml) or sodium azide (46 nl; 1 mg/ml), an antibody preservative, were without inhibitory effect in this bioassay. Microinjected, affinity-purified DM1A (46n1; 7.5mg/ml) was also inhibitory, but preabsorption with pure tubulin prior to injection significantly reduced the inhibitory effect. DM1A injection had no effect on centrifugation-induced germinal vesicle migration (GVM). Previous work indicated that drugs (e.g. demecolcine and nocodazole), which destabilise microtubules, enhance both centrifugation-induced GVM and progesterone-induced GVD in Rana oocytes. Taking these results together, it is suggested that DM1A injection may have differential effects on microtubules in this cell. Thus, while the majority of microtubules were apparentl depolymerised by DM1A (46nl; 10mg/ml) injection, a small subpopulation appeared to be stabilised as bundles. Confocal immunofluorescence microscopy of follicle-enclosed oocytes after DM1A injection revealed a major loss of microtubules throughout the cell however, apparent sparse bundles of nucrotubules arranged in an approximately 600 μm shell were associated with the injectate region 24h post-injection. By contrast, control follicle-enclosed oocytes topically labelled with DM1A post-fixation had extensive microtubule arrays similar to those previously reported in Xenopus oocytes. Intracellular recording after DM1A injection and progesterone treatment yielded an intermediate membrane potential (Vm= −31.8mV) compared with control (immature) DMIA-injected cells (Vm= −44.7 mV) or potassium balanced salt solution (KBS)-injected cells matured with progesterone (Vm= −13.9mV). These results suggest that DM1A injection does not completely inhibit electrophysiological changes initiated by progesterone. Working hypotheses are proposed that suggest a role for microtubules in the action of progesterone which normally lifts the prophase I block in the Rana follicle-enclosed oocyte.