When analyzing reprogramming after nuclear transfer, it is interesting to focus on the nucleolar compartment, which is the most morphologically well-defined compartment in the nucleus. As with many messenger RNA-encoding genes, the ribosomal RNA genes are expressed in the nuclei of cells used for nuclear transfer. We suppose that a successful passage from the expression of genes specific to somatic cells to those characteristic of an early embryo implies the transient arrest of any expression under the effect of the oocyte cytoplasm. After nuclear transfer, it is possible to observe using electron microscopy the changes in nucleoli that reflect their activity. In successful cases, the nucleoli are deactivated effectively before the end of the one-cell stage. Sometimes however, incomplete changes or delays in the process may be observed that eventually are associated with abnormal development. It is possible to confirm the diagnosis using other techniques, three examples of which are given: the loss of reticulated structure of nucleoli may be quickly detected by immunofluorescence; proteins that are specific for a nucleolar component can be tracked during nucleolar changes by immunochemistry on thin sections; and the presence of deoxyribonucleic acid inside a nucleolus (indispensable for its activity) can be verified by ultrastructural cytochemistry.

The extracellular matrix (ECM) of porcine mature oocytes was revealed by transmission electron microscopy (TEM) after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS) and on the surface of the zona pellucida (ZP), it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells) or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA) bound to proteoglycans – for various times (with or without chase) and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose) and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI) and metaphase II (MII) and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a favourable factor for sperm penetration.

As it was shown earlier, resumption of rRNA transcription in early mouse embryo is localized in the peripheral region of nucleolus precursor body/NPB/during the two‐cell stage. Recently, nucleolar phosphoprotein Nopp140 was presented to shuttle between the nucleolus and cytoplasm as chaperone of snoRNPs. Nopp140 interacts with RNA polymerase I in nucleolus and also accumulates in CBs, suggesting a pathway between the two organelles. The aim of the study was to describe the changing location of Nopp140 during the first cleavage stages of mouse embryos and its re‐location after inhibition of rRNA synthesis with actinomycin D. Light microscope immunocytochemical staining showed Nopp140 in the periphery of NPBs before activation of rDNA transcription and in addition confirmed its localization in CBs. Immunolabelling with antibodies against RNA Pol I and UBF gave co‐localization of these proteins, implicating that Nopp140 may actively participate to rDNA transcription. We suggest that fundamental differences in molecular organization of rDNA synthesis and postranscriptional processes between cycling somatic and pre‐implantation embryonic cells may be in selective transport of transcription and/or processing‐complexes of proteins to the nucleolar organizer regions (NOR). Mol. Reprod. Dev. 59:277–284, 2001. © 2001 Wiley‐Liss, Inc.

SummaryGerm cells were isolated from rabbit fetal gonads between 18 and 22 dayspost coitumand 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 cellsin 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.

Changes in cell-to-cell contact and distribution of cytoskeletal components were investigated during in vitro culture of cattle oocyte cumulus complexes (OCC). Freeze-fracture analysis (FF), microinjections of the fluorescent dye Lucifer Yellow (LY), immunofluorescence, and ultrastructural immunocytochemistry were used. The cumulus cells (CC) remained in close contact via gap junctions (GJ) constituted of connexin43 (Cx43) during the entire culture time. Whereas the GJ decreased in diameter after 24 h of culture, their number was still substantially great at that time. The Cx43-positive GJ, localized between corona radiata cell projections and oolemma, disappeared after 6 h of culture. Concomitantly, the OCC lost the ability to transfer LY from cumulus to oocyte, and connexin32 (Cx32) became detectable in the oocytes. Both the changes in corona-oocyte coupling and cumulus expansion were preceded by the redistribution of F-actin in cytoplasm of CC. These data indicate that functional GJ linked the CC until the second meiotic arrest. However, the removal of Cx43-positive GJ interconnecting cytoplasmic projections of corona radiata cells with the oocyte was temporally correlated with germinal vesicle breakdown. The present results suggest the pivotal role of the cytoskeleton (F-actin) in cumulus expansion.

The organization of the cytoskeleton during early pig embryogenesis was investigated by using fluorescence and electron microscopy. The early morphogenesis of the pig embryo differed from that of the mouse, the standard model of the early mammalian development. In the pig, both compaction and polarization were gradual, and definitive polarization of cell surface microville occurred first shortly before blastocyst formation; the compaction and polarization of the mouse embryo are completed as early as at the 8 cell stage. Furthermore, the pig morula undergoes cycles of compaction and decompaction throughout its development. Distinct changes in the distribution of actin and the actin-associated proteins alpha-fodrin, vinculin and E-cadherin coincided with these events. In the pig, all these molecules were evenly distributed at all aspects of the blastomeres during early cleavage and then gradually accumulated in regions of intercellular contacts toward the blastocyst stage; microfilaments in trophectoderm cells formed a cortical meshwork associated with apical microvilli and adherent junctions (zonula adherens). In the mouse, the corresponding changes occur earlier, at the 8 cell stage. Microtubules formed a network-like cortical layer beneath the microvilli at the free outer surfaces of pig blastomeres. Cytokeratin bundles were not observed until the early blastocyst, where they characteristically associated with newly formed desmosomes. In both species a close correlation between morphologically defined developmental stages and the organization of the cytoskeleton: actin and actin-associated proteins are involved in polarization and compaction, whereas the appearance of intermediate filament bundles coincides with the building of the first epithelium, the trophectoderm; it is in the timing of events that a contrast between species is observed.

Biology of the CellVolume 76, Issue 2 p. 226-226 Changes in intercellular contracts, distribution of cytoskeleton and extracellular matrix in cattle oocyte-cumulus-complex during in vitrp maturation Sutovsky Peter, Sutovsky Peter Laboratoire de Biologie Cellulaire et Moleculaire, I.N.R.A., 78352 Jouy-en-Josas Cedex — FranceSearch for more papers by this authorFlechon Jacques-E, Flechon Jacques-E Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this authorFlechon Bernadette, Flechon Bernadette Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this authorPeynot Nathalie, Peynot Nathalie Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this authorMotlik Jan, Motlik Jan Laboratoire de Biologie Cellulaire et Moleculaire, I.N.R.A., 78352 Jouy-en-Josas Cedex — FranceSearch for more papers by this authorHeyman Yvan, Heyman Yvan Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this authorChesne Patrick, Chesne Patrick Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this author Sutovsky Peter, Sutovsky Peter Laboratoire de Biologie Cellulaire et Moleculaire, I.N.R.A., 78352 Jouy-en-Josas Cedex — FranceSearch for more papers by this authorFlechon Jacques-E, Flechon Jacques-E Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this authorFlechon Bernadette, Flechon Bernadette Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this authorPeynot Nathalie, Peynot Nathalie Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this authorMotlik Jan, Motlik Jan Laboratoire de Biologie Cellulaire et Moleculaire, I.N.R.A., 78352 Jouy-en-Josas Cedex — FranceSearch for more papers by this authorHeyman Yvan, Heyman Yvan Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this authorChesne Patrick, Chesne Patrick Institute of Animal Physiology and Genetics, 27721 Libechov, CzechoslovakiaSearch for more papers by this author First published: 1992 https://doi.org/10.1016/0248-4900(92)90278-9AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume76, Issue21992Pages 226-226 RelatedInformation

Development, Growth & DifferentiationVolume 28, Issue s1 p. 21-22 Free Access Acrosome Reaction and Changes in Membrane Properties of the Mammalian Sperm Head in Relation to Gamete Fusion J.-E. FLÉCHON, J.-E. FLÉCHON Department of animal Physiology, I. N. R. A., 78350. Jouy-en-Josas, FranceSearch for more papers by this author J.-E. FLÉCHON, J.-E. FLÉCHON Department of animal Physiology, I. N. R. A., 78350. Jouy-en-Josas, FranceSearch for more papers by this author First published: August 1986 https://doi.org/10.1111/j.1440-169X.1986.00021.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Yanagimachi , R. , 1981. In “Fertilization and Embryonic Development In Vitro” (eds. L. Mastoianni and J. D. Biggers ), pp. 82– 84. Plenum, New York . 2 Yanagimachi , R. , and Y. D. Noda , 1970. J. Ultrastruct. Res., 31, 486– 493. 3 Phillips , D. M. and R. Yanagimachi , 1982. Dev. Growth. Differ., 24, 543– 551. 4 Piko , L. and A. Tyler , 1964. In: “Proc. 5th Int. Congr. Anim. Reprod. A. I. Trento”, 2, pp. 372– 377. 5 Barros , C. J. M. Bedford , L. F. Franklin and C. R. Austin , 1967. J. Cell Biol., 34, C1– C5. 6 Shams-Bohran , G. and R. A. P. Harrison , 1981. Gamete Res., 4, 407– 432. 7 Fléchon , J. E. , 1985. Am. J. Anat., 174, 239– 248. 8 Fléchon , J. E. , R. A. P. Harrison , B. Fléchon and J. Escaig , 1986. J. Cell Sci., 81, 43– 63. 9 Friend , D. S. , L. Orci , A. Perrelet and R. Yanagimachi , 1977. J. Cell. Biol., 74, 561– 577. 10 Yanagimachi , R. and F. Suzuki , 1985. Gamete Res., 11, 29– 40. 11 Bearer , E. and D. S. Friend , 1982. J. Cell Biol., 92, 604– 615. 12 Myles , D. G. and P. Primakoff , 1984. J. Cell. Biol., 99, 1634– 1641. 13 Yanagimachi , R. and D. M. Phillips , 1984. Gamete Res., 9, 1– 19. 14 Pavlok , A. and J. E. Fléchon , 1985. J. Repord. Fert., 74, 597– 604. 15 Koehler , J. K. , D. Smith and L. K. Karp , 1982. Gamete Res., 9, 197– 205. 16 Bedford , J. M. , 1983. Biol. Reprod., 28, 108– 120. 17 Huang , T. T. F. Jr. , and R. Yanagimachi , 1985. Am. J. Anat., 741, 249– 268. Volume28, Issues1August 1986Pages 21-22 ReferencesRelatedInformation

The expression of beta-2 microglobulin (beta 2m), a protein associated with the histocompatibility antigens of the pig complex (SLA), was studied in preimplantation embryos between the segmentation stage and the beginning of elongation (day 12). The antigen was visualized at the ultrastructural level by immunocytochemical techniques using peroxidase or colloidal gold particles as labels. beta 2m expression appeared to parallel trophoblast differentiation. No positive reaction was obtained before the early blastocyst stage. From this stage on, labelling was observed on the apical surface of the trophectoderm cells.