Pleiotropic effect of okadaic acid on maturing mouse oocytes.

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

Involvement of microtubules and microfilaments in the control of the nuclear movement during maturation of mouse oocyte.

We confirm that the centrifugal migration of the chromosomes in maturing mouse oocytes depends on a microfilament-mediated process. We investigated the role of the cytoskeleton in the germinal vesicle (GV) behavior of oocytes prevented from resuming meiosis by either activators of protein kinase A or activators of protein kinase C. A time-lapse microcinematography study demonstrates that GV immobilization by isobutylmethylxanthine (IBMX) is overcome by colcemid (COL), nocodazole (NOC), and taxol and that cytochalasin D (CCD) reversibly immobilizes the GV of oocytes treated with either IBMX + COL (or NOC) or 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, known to allow a programmed GV cortical translocation. An immunofluorescence analysis shows that the disorganization of a perinuclear microtubule network is the very first cytological clue of maturation. IBMX promotes its persistence while NOC, COL, and taxol induce its immediate disappearance. It is concluded that elements of the cytoplasmic microtubular complex (CMTC) are passively involved in the control of the setting up of a "centrifugal displacement property" (CDP) by counteracting a motive force provided by the microfilament cytoskeleton. Finally, TPA induces a clearcut reorganization instead of a total disorganization of the CMTC. This reorganization is, however, sufficient to allow the microfilaments to drive the GV displacement.

Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C.

A passive erratic movement of the germinal vesicle (GV), already visible in small incompetent oocytes, is followed by an active scalloping of the nuclear membrane soon before GV breakdown (GVBD) in cultured competent oocytes. Maturation can be inhibited by activators of protein kinase A (PK-A) and protein kinase C (PK-C). Our time-lapse cinematography analysis allowed us to describe an unexpected behaviour of the GV when PK-C, but not PK-A, is activated: GV undergoes a displacement toward the cortex according to the same biological clock which triggers the programmed translocation of the spindle in control oocytes. It is concluded that, when oocytes become committed to undergo maturation, the cytoplasm acquires a PK-A-controlled "centrifugal displacement property" which is not restricted to the spindle.

Histogenesis in 11-day mouse embryo limb buds explanted in organ culture.

Fore- and hindlimb buds from 11-day mouse embryos with 40 to 52 somites (including the four occipital pairs) were explanted in organ culture and submitted to systematic histological analysis. Chondrogenesis occurs normally in culture in all preskeletal rudiments which were already represented by condensed blastemas before explantation. In the proximal territories, the progress of cartilage differentiation occurs according to the normal pattern and can be revealed histologically much earlier than in bulk preparations. In all explanted hindlimbs as well as in forelimbs from embryos with less than 50 somites, a primary coalescence occurs between the IId and IIId digital rays, leading to various fusions from soft tissue syndactyly to oligosyndactyly. This is the result of two combined unfavorable effects of the culture conditions: the lack of simultaneous volumetric growth of the foot- or handplate, which normally would provide the necessary space for the laying down of a pentadactylous pattern, and a loss of cells resulting from abnormal cell death affecting selective mesodermal sites in the zeugopod and in the marginal subridge area, the latter being more severely affected in hindlimb buds. Several observations suggest that the preferential sensitivity of the marginal mesoderm might be related to early changes in the apical ectoderm, which itself becomes excessively necrotic and rapidly looses its pseudostratified configuration. The forelimb buds from embryos with 50 somites and more usually develop a pentadactylous pattern with a better individuation of digital structures. In all explants, the prospective mesoderm of digit I exhibits stronger regulatory tendencies.

The behaviour of cow blastocyst in vitro: cinematographic and morphometric analysis.

The behaviour of the cow blastocyst in vitro was studied by time-lapse cinematography and analysed by morphometry. Three types of behaviour were observed: continuous expansion followed by hatching; discontinuous expansion interrupted by few contractions and followed by hatching; discontinuous expansion interrupted by several rapid contractions without hatching. This demonstrated that the pulsatile activity of the blastocyst is not a necessary condition of hatching but also that only a moderate pulsatile activity is compatible with normal hatching. The time of hatching in vitro corresponded approximately with the time of zona loss in vivo (9-10 days). Rupture of the zona occurred at any point of the trophoblast layer. Hatching by herniation through a reduced opening of the zona was occasionally observed. The behavior of the embryos from a particular animal was very similar but differences were noted between embryos from different animals.

Time-lapse cinematographic analysis of hatching of normal and frozen-thawed cow blastocysts.

This paper describes time-lapse cinematography on cattle blastocysts hatching from the zona in vitro. Four normal morulae were taken on Day 6 and cultured for 5 days. During this period 3 formed normal blastocysts of which 2 hatched from the zona without undergoing any contractions. Early blastocysts were taken on Day 7, frozen and stored at low temperature. After thawing they were cultured; only 1 of the 11 behaved like the normal embryos, and the others underwent contractions and failed to hatch. These contractions could be interpreted as a sign of an alteration of the trophoblastic structure and properties.

[Cinematographic and morphometric analysis of in vitro behavior of blastocysts of the mouse]. / Analyse cinématographique et morphométrique du comportement in vitro du blastocyste de la souris.

The behaviour of the mouse blastocyst in vitro was studied by time-lapse cinematography and analysed by morphometry. Three types of behaviour were observed: discontinuous expansion interrupted by rapid contractions and followed or not by hatching; practically continuous expansion followed by hatching. This demonstrates that the pulsatile activity of the blastocyst is not a necessary condition of hatching. Analysis of the rate and delay of hatching in vitro as compared to the conditions in utero is in favour of the existence of a uterine factor insuring partial lysis of the zona pellucida and helping the mechanical action of the blastocyst. Hatching by herniation through a reduced opening of the zona was occasionally observed. The process of in vitro implantation could be followed by cinematography.

Cell division and cell allocation in early mouse development.

Cell division was observed in intact and dissociated mouse embryos between the 2-cell stage and the blastocyst in embryos developing in culture. Division to the 4-cell stage was usually asynchronous. The first cell to divide to the 4-cell stage produced descendants which tended to divide ahead of those cells produced by its slow partner at all subsequent stages of development up to the blastocyte stage. The descendants of the first cell to divide to the 4-cell stage did not subsequently have short cell cycles. The first cell or last cell to divide from the 4-cell stage was labelled with tritiated thymidine. The embryo was reassembled, and it was found that the first pair of cells to reach the 8-cell stage contributed disproportionately more descendants to the ICM when compared with the last cell to divide to the 8-cell stage.

Ultrastructural localization of non-specific alkaline phosphatase during cleavage and blastocyst formation in the mouse.

The localization of non-specific alkaline phosphatase activity during cleavage and blastocyst formation has been investigated in the mouse by electron microscopy. The activity is detectable for the first time at the two-cell stage and is localized on the surface of the interblastomeric plasma membranes and on small cytoplasmic inclusions. It increases in the following stages, predominantly on the interblastomeric membranes, the outside membranes remaining devoid of reaction. From the four-cell stage on, small reactive grains are also observed in the crystalloid plates of the cytoplasm. At the morula stage, the plasma membranes of the inner mass cells are entirely marked by the reaction whereas the trophoblastic cells are polarized, with their inner surfaces positive and outside surfaces negative. At the blastocyst stage the enzyme is gradually eliminated from the membranes bordering the blastocoel and from the interblastomeric furrows of the trophoblast and primary endoderm. The significance of the differential localization of the enzyme is discussed, especially in relation to the differentiation mechanisms of the trophoblast and inner cell mass.