Henri Alexandre

Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos

The development of the nucleoli and the sites of rDNA transcription have been studied by high-resolution autoradiography during the cleavage stages of mouse embryos. The appearance of fibrillar centres at the periphery of the fibrillar primary nucleoli has been observed at the 4-cell stage. Several fibrillar centres, interconnected by electron-dense fibrillar strands, form a reticulated region around the fibrillar mass at the 6-to 8-cell stage. After a 10 min pulse with [3H]uridine, only this peripheral network is labelled. At the late morula and at the blastocyst stage, the fibrillar component (nucleolonema) of the reticulated nucleoli is labelled after 10 min [3H]uridine incorporation. When the embryos are reincubated for 2 h in cold medium, the label is localized mainly in the granular component. Fibrillar centres are not labelled. Autoradiograms of in vitro developed embryos pulsed for 2 h with [3H]uridine confirm that the central fibrillar core of the nucleoli of 6-to 8-cell embryos is never labelled. Thus, the fibrillar constituent of this core is not homologous to the fibrillar component of the nucleoli of later stage embryos, which is the site of active rDNA transcription. An interpretation of nucleologenesis during early mouse embryogenesis is proposed.

Mammalian Heat Shock Factor 1 Is Essential for Oocyte Meiosis and Directly Regulates Hsp90α Expression

Heat shock transcription factor 1 (HSF1) is the main regulator of the stress response that triggers the transcription of several genes encoding heat shock proteins (Hsps). Hsps act as molecular chaperones involved in protein folding, stability, and trafficking. HSF1 is highly expressed in oocytes and Hsf1 knock-out in mice revealed that in the absence of stress this factor plays an important role in female reproduction. We previously reported that Hsf1-/- females produce oocytes but no viable embryos. Consequently, we asked whether oocytes require HSF1 to regulate a particular set of Hsps necessary for them to develop. We find that Hsp90α (Hspaa1) is the major HSF1-dependent chaperone inasmuch as Hsf1 knock-out resulted in Hsp90-depleted oocytes. These oocytes exhibited delayed germinal vesicle breakdown (or G2/M transition), partial meiosis I block, and defective asymmetrical division. To probe the role of Hsp90α in this meiotic syndrome, we analyzed meiotic maturation in wild-type oocytes treated with a specific inhibitor of Hsp90, 17-allylamino-17-demethoxy-geldanamycin, and observed similar defects. At the molecular level we showed that, together with these developmental anomalies, CDK1 and MAPK, key meiotic kinases, were significantly disturbed. Thus, our data demonstrate that HSF1 is a maternal transcription factor essential for normal progression of meiosis.

Effect of genistein on the temporal coordination of cleavage and compaction in mouse preimplantation embryos.

Initially, we investigated the effect of genistein, an inhibitor of protein tyrosine kinases, on compaction of the mouse embryo since tyrosine phosphorylation of the cadherin-catenins complex was suggested to down-regulate its adhesive function. Genistein prevented cleavage from the 2- to the 4-cell stage in a concentration-dependent manner. The next cleavage is inhibited at all concentrations used. Time course of intercellular flattening is however identical for both control 8-cell embryos and 4-cell arrested embryos. This confirms that compaction takes place according to a biological clock that does not depend on completion of the third cell cycle. Our results also suggest that, since, in contrast to genistein, protein kinases C modulators are known to cause a premature compaction, diacylglycerol-dependent kinases but not protein tyrosine kinases might be upregulators of compaction.

Respective roles of protein tyrosine kinases and protein kinases C in the upregulation of β-catenin distribution, and compaction in mouse preimplantation embryos: a pharmacological approach

The cellular distribution of β‐catenin was determined by western blotting and laser confocal scanning microscopy in both control and pharmacologically‐manipulated mouse preimplantation embryos. Most of the stored maternal β‐catenin is Triton X—100‐extractable and distributed throughout the cytoplasm. In 2‐cell stage embryos, the remaining molecules are concentrated in regions of cell contact and, to a lesser extent, at non apposed surfaces. Association of β‐catenin with the cortex of non apposed membranes decreases as cleavage proceeds, and is lost at compaction. In contrast to the rapid cross‐linking of cell surfaces induced by wheat germ agglutinin, the diacylglyceride‐induced compaction‐like adhesion of 2‐ and 4‐cell embryos correlates with complete restriction of β‐catenin to the apposing membranes. On the contrary, tyrphostin B46, a specific protein tyrosine kinase inhibitor, fails to induce both premature β‐catenin relocalisation and compaction. In addition, we show that orthovanadate induces a dramatic increase in the level of phosphotyrosine labelling of cell‐cell junctions in compacted 8‐cell stage embryos without inducing their decompaction. However, most of these orthovanadate tyrosine‐phosphorylated proteins are detergent‐soluble, while β‐catenin restricted to the apposing membranes is not.

The origin of the nascent blastocoele in preimplantation mouse embryos ultrastructural cytochemistry and effect of chloroquine

SummaryMouse morulae are known to undergo cavitation as soon as some external cells have entered the sixth cell cycle (Garbutt et al. 1987). Since the early cytological features of cavitation are still unclear, we undertook a careful ultrastructural analysis of late morulae-nascent blastocysts. In addition, since maturation of lysosomes might be involved in the first step of cavity formation, we focused our attention on these organelles by means of the cytochemical localization of trimetaphosphatase activity and by the study of the effects of chloroquine on precavitation embryos. Our results suggest that cavitation starts in a few external cells (presumably competent cells entering the sixth cell cycle), by the chloroquine-sensitive formation of degradative autophagic vacuoles engulfing lipid droplets and vacuoles containing osmiophilic material. These complex structures enlarge (as a result of lipid metabolism?) and so transform into intrablastomeric cavities which, by means of a membrane fusion process, very rapidly become extracellular cavities that coalesce. The abembryonic pole of the blastocyst is determined in this way. Moreover, we suggest that the juxtacoelic cytoplasmic processes covering the inner cell mass (ICM) cells, which are known to restrict the expression of their totipotency during early cavitation (Fleming et al. 1984), are the latest remnants of the walls of the growing intrablastomeric cavities.

Effect of taxol and okadaic acid on microtubule dynamics in thimerosal-arrested primary mouse oocytes: a confocal study.

A pulse of thimerosal (TMS), a sulfhydryl reagent, induces an instantaneous, complete and long‐lasting microtubule interphasic network disassembly in mouse primary oocytes, correlated with the irreversible inhibition of meiosis reinitiation This inhibition is bypassed by dithiothreitol (DTT) while thiosalicylic acid, an analog of TMS, does induce neither microtubules depolymerisation nor inhibition of reinitiation and resumption of meiosis. This strongly suggests that the dramatic and pleiotropic inhibitory effect of TMS is specifically related to its sulfhydryl group oxidising activity of critical molecules among which tubulin. In contrast to DTT, okadaic acid (OA), known to bypass the inhibitory effect of drugs interfering with protein kinase activities, induces a late chromatin condensation and GVBD in TMS‐pulsed oocytes as compared to the control situation, with no significant concomitant microtubule assembly. These cytological features are suggested to be indirectly induced by a late MAPK activation and confirm that a very early thiol oxidation induced by TMS exerts a much more dramatic effect on resumption of meiosis than any pharmacological manipulation of protein kinase activities leading to activation of MPF. Finally, taxol was shown to promote tubulin polymerisation even when microtubules were irreversibly disassembled by thiol oxidation but fails to restore the ability to undergo maturation.

Étude au microscope électronique de l'effet des rayons x sur l'évolution des structures nucléaires et cytoplasmiques au cours de la segmentation des oeufs de Pleurodeles waltlii

Summary X irradiation of Pleurodeles embryos, at the beginning of cleavage, inhibits the lobulisation of the nuclei and the formation of small cytoplasmic invaginations which appear in the normal advanced blastulae. This inhibition seems to be closely linked to the inhibition of nuclear metabolism. An inhibition of the appearance of the outer granular constituent in the nucleoli would correspond to a decrease in rRNA synthesis. This inhibition is proportional to the X-ray dose. The lysis of the embryos is characterized by the existence of annulated lamellae either in or outside the nuclei and especially by the appearance of cytolysomes containing degenerated mitochondriae, vesicles of glycogen, lamellar structures. Finally, we have observed the presence of glycogen granules associated into chains in the intercellular spaces of both control and irradiated embryos. However, the irradiated embryos seem to be characterized by a higher content of glycogen; the inhibition of the utilisation of glycogen might explain this fact.

Nucleic acid synthesis in preimplantation mouse embryos. Autoradiographic evidence of uridine and deoxyuridine utilization in DNA synthesis

SummaryMouse embryos were collected at the 2-cell stage, cultured in vitro in the presence of3H deoxyuridine or uridine for 6 or 4 h and autoradiographed.Deoxyuridine is actively incorporated into the DNA of cleaving mouse embryos indicating the existence of thymidylate synthetase activity at least at the 4-cell stage and presumably already before this.RNAase treatment of embryos squashed on slides shows a weak but obvious incorporation of uridine into DNA of cleaving mouse embryos, from the 4-cell stage onwards; this incorporation is totally inhibited by hydroxyurea. The reduction of ribonucleotides to deoxyribonucleotides is a metabolic pathway already required for cleavage, as shown by hydroxyurea experiments.The second polar pody, known to incorporate thymidine, is unable to incorporate either deoxyuridine or uridine.

Gametogenesis and Maturation: The Formation of Eggs and Spermatozoa

The formation of the egg, in the ovary, is an extremely important period in ontogeny. It is much more than an accumulation of food material, which will be progressively utilized until the embryo becomes capable of feeding itself. It is also a period of intensive genetic activity, which leads to the synthesis of many important macromolecules (RNA, proteins) which will be used at later stages of development only.

Fertilization: How the Sleeping Egg Awakes

Fertilization of the egg is an all-important event for the life of all organisms that reproduce sexually. Not only does it start the machinery for embryonic development, but it also has fundamental genetic consequences: the diploid (2n) state is restored and the paternal hereditary characters are introduced into the fertilized egg (also called the zygote). In most species, the sex of the future adult is established at fertilization.