The extraembryonic endoderm of the elongating ovine conceptus was analyzed by scanning and transmission electron microscopy and by whole mount actin staining and immunofluorescence. Morphological and functional differences between the visceral endoderm (VE), the founding cell layer, and the parietal endoderm (PE) are presented. During the elongation process, the PE differentiated to fusiform multinucleated cells aligned parallel to the elongation axis of the conceptus, whereas the VE cells retained the aspect of typical epithelial cells. The multinucleated PE cells however, expressed cellular and nuclear markers typical of endodermal and polarized epithelial cells. The proteins of the extracellular matrix, laminin, and fibronectin, were specifically expressed in the PE. The presence of pairs of nuclei linked by mid-bodies positively stained with tubulin antibodies, indicated that the syncytial differentiation of the PE was due to karyokinesis which was not followed by cytokinesis rather than by cell fusion.

The epithelial versus mesenchymal phenotypes of embryonic ectoderm and mesoderm cells of the prestreak stage pig embryos were examined by electron microscopy and molecular marker analysis. During this period the embryonic disc remained flat or slightly convex while becoming oval or pyriform in shape. Mesenchyme cells expressing vimentin were present between the embryonic disc and the underlying visceral endoderm before a primitive streak (or groove) was apparent. The migration of mesenchyme appeared to occur in lateral and posterior directions from a mass of quiescent cells located in the pointed end of the pyriform embryonic disc that expressed Brachyury; these cells are proposed to be the precursors of the primitive streak and/or form the equivalent of the mouse early gastrula organizer (EGO). Cells with the TEC‐1 (or SSEA‐1) epitope, the marker most frequently used to characterize pluripotent cells, were initially distributed randomly in the embryonic ectoderm and then were found to localize in an anterior crescent which may contain the precursor cells of ectoderm and neurectoderm. As mitotic figures were found only in the anterior crescent, it is proposed that at least some of these proliferating cells migrate toward the EGO. While cytokeratins were barely detectable in the embryonic ectoderm cells, vimentin expression was supposed to be associated with the migratory capacity of these cells. These findings indicate that the early step of gastrulation, migration of extraembryonic mesoderm, occurs at a prestreak stage during which the embryonic disc becomes polarized. genesis 38:13–25, 2004. © 2003 Wiley‐Liss, Inc.

This study describes the gap junctions in extraembryonic cell layers of the preimplantation pig embryo (trophectoderm and endoderm constituting the trophoblast). Using specific antibodies against connexins 31, 32 and 43, we found these connexins in embryos by immunodetection using Western blot and immunofluorescence analysis. By immunofluorescence, the first foci of connexin 31 were detected in the four-cell stage blastomeres, and the first diffuse gap junctions appeared at the eight-cell stage. Intercellular communication was observed with Lucifer yellow transfer to start also at the eight-cell stage around the onset of compaction. Typical gap junctions developed in the trophectoderm of blastocysts, as observed by transmission electron microscopy of thin sections and freeze-fracture replicas. Connexin proteins were differently expressed in time and space: connexin 31 was continuously present in trophectoderm, connexin 32 was essentially found in endoderm during elongation; connexin 43 was distributed in both trophectoderm and endoderm during blastulation and expansion. Connexin 43 was also found in two isoforms, phosphorylated or not, at day 14. Such developmentally regulated connexin expression may be essentially useful to control the exponential growth of trophoblast in preimplantation pig blastocysts.

Following the demonstration of high levels of interferon-gamma (IFN-gamma) synthesis by the pig trophectoderm around implantation, an adequate experimental system was established so as to study the regulation of endogenous IFN-gamma gene. Several stable cell lines have been isolated from Day 14 and 15 pig trophoblast, that could withstand indefinite growth when cultured on collagen-coated supports. Since no feeder cells were used for culture, and cell lines could be successfully cloned, these lines represent the first pure porcine trophoblastic (TB) cell lines isolated so far. These cells were shown to exhibit most differentiation markers of epithelial cells and in addition to express the porcine trophectoderm-specific antigen SN1-38. The TB cell line A (TBA) was characterized in more details upon culture on microporous filters, where a high apico-basal polarity could be obtained. In those conditions, a transient and acute interferon-gamma secretion was detected only in the apical medium. Moreover, the two trophoblast-specific mRNA of 1.3 and 1.4 kb that have been described in the blastocyst collected in vivo were shown to be synchronously transcribed. This polarized synthesis and secretion of IFN-gamma was correlated with the acquisition of maximal levels of electric resistance of TBA monolayers on filters, and was not observed on the same cells cultured on plastic. This differentiation was maintained over 30 passages, demonstrating that the induction of IFN-gamma secretion by pig conceptus is not maternally controlled. This cellular model will be of prime importance for studies on the developmental regulation of the IFN-gamma gene, and more generally for studies on relationship between secretion and polarity in transporting epithelia.

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.

Cloned blastocysts developed in vitro for 7 d had a mean number of cells (82.86 +/- 5.35) as evaluated by nuclei counting in serial optical sections using confocal microscopy, after staining with propidium iodide. This number was not significantly different from that of control IVF embryos cultured under the same conditions during the same period (mean = 88.89 +/- 7.53). Semi-thin sections revealed that most of the blastocysts had an inner cell mass (10/12) and a blastocoele. Under transmission electron microscopy, the trophectoderm appeared well differentiated as a polarized epithelium with apical microvilli and lateral junctions including desmosomes with bound intermediate filaments. The cytoplasm sometimes contained immature mitochondria or a large number of residual bodies. About half of the blastocysts examined had a large amount of cellular debris in the perivitelline space or inside the blastocoele cavity. The cloned blastocysts were also able to hatch in vitro by day 8 and SEM indicated a normal morphology of the trophectoderm cells with numerous apical microvilli. The high number of excluded or degenerating cells found in some embryos may partially explain early embryonic mortality that follows transfer. However, these observations do not give a clear explanation for the high incidence of fetal losses.

Biology of the CellVolume 84, Issue 3 p. 222-222 Application of different techniques of microscopy for the study of early differentiation in mammalian embryos: cells junctions Jacques Flechon, Jacques Flechon Biologie Cellulaire et Moléculaire, INRA, 78350 JOUY-EN-JOSAS, FRANCESearch for more papers by this authorBernadette Flechon, Bernadette Flechon Biologie Cellulaire et Moléculaire, INRA, 78350 JOUY-EN-JOSAS, FRANCESearch for more papers by this authorJéril Degrolard, Jéril Degrolard Biologie Cellulaire et Moléculaire, INRA, 78350 JOUY-EN-JOSAS, FRANCESearch for more papers by this authorSolange Delasalle, Solange Delasalle Biologie Cellulaire et Moléculaire, INRA, 78350 JOUY-EN-JOSAS, FRANCESearch for more papers by this author Jacques Flechon, Jacques Flechon Biologie Cellulaire et Moléculaire, INRA, 78350 JOUY-EN-JOSAS, FRANCESearch for more papers by this authorBernadette Flechon, Bernadette Flechon Biologie Cellulaire et Moléculaire, INRA, 78350 JOUY-EN-JOSAS, FRANCESearch for more papers by this authorJéril Degrolard, Jéril Degrolard Biologie Cellulaire et Moléculaire, INRA, 78350 JOUY-EN-JOSAS, FRANCESearch for more papers by this authorSolange Delasalle, Solange Delasalle Biologie Cellulaire et Moléculaire, INRA, 78350 JOUY-EN-JOSAS, FRANCESearch for more papers by this author First published: 1995 https://doi.org/10.1016/0248-4900(96)89437-8AboutPDF 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. Volume84, Issue31995Pages 222-222 RelatedInformation

The mechanism by which all-trans retinoic acid (RA) stimulates gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line. IAR203, was investigated. When RA, at 0.1 microM for 24-48 h, enhanced the dye transfer in IAR203 cells (x 1.4), it increased the amount of connexin43 (Cx43) in the cell-cell contact regions of the plasma membrane, as evidenced by analysis by Western blot and by immunofluorescence. It had no effect on the level of Cx43 mRNA. Freeze-fracture analysis of the size of gap junctions revealed an increase of the proportion of small gap junctions in RA-treated cells. We conclude that, in IAR203 cells, RA stimulates GJIC by acting at the post-transcriptional level of Cx43 regulation. The possibility that RA acts indirectly on the regulation of Cx43 expression, and increases the half-life of Cx43 by inducing adhesion molecules is discussed.

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.

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

Biology of the CellVolume 73, Issue 2-3 p. 10a-10a Parietal endoderm cells of elongating ovine blastocysts are plurinucleate, observations on the cytoskeleton JacquesE. Flechon, JacquesE. Flechon Laboratoire de Biologie Cellulaire et MoléculaireSearch for more papers by this authorBernadette Flechon, Bernadette Flechon Laboratoire de Biologie Cellulaire et MoléculaireSearch for more papers by this authorJeril Degrolard, Jeril Degrolard Laboratoire de Biologie Cellulaire et MoléculaireSearch for more papers by this authorSolange Delasalle, Solange Delasalle Laboratoire de Biologie Cellulaire et MoléculaireSearch for more papers by this authorMichel Guillomot, Michel Guillomot Laboratoire d'Endocrinologie de l'Embryon, INRA, 78352 Jouy-en-Josas, Cedex, France.Search for more papers by this author JacquesE. Flechon, JacquesE. Flechon Laboratoire de Biologie Cellulaire et MoléculaireSearch for more papers by this authorBernadette Flechon, Bernadette Flechon Laboratoire de Biologie Cellulaire et MoléculaireSearch for more papers by this authorJeril Degrolard, Jeril Degrolard Laboratoire de Biologie Cellulaire et MoléculaireSearch for more papers by this authorSolange Delasalle, Solange Delasalle Laboratoire de Biologie Cellulaire et MoléculaireSearch for more papers by this authorMichel Guillomot, Michel Guillomot Laboratoire d'Endocrinologie de l'Embryon, INRA, 78352 Jouy-en-Josas, Cedex, France.Search for more papers by this author First published: 1991 https://doi.org/10.1016/0248-4900(91)90125-7AboutPDF 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 onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume73, Issue2-31991Pages 10a-10a RelatedInformation

In cows undergoing spontaneous oestrous cycles and mated during the first 6 hours of oestrus, the distribution of spermatozoa in the oviduct isthmus and changes in their surface membranes and neighbouring epithelium have been examined shortly before and after ovulation. In agreement with previous histological studies, relatively few spermatozoa were detected in the oviduct lumen: most were located in the caudal isthmus before ovulation, frequently among folds and in the presence of a viscous secretion. A majority of spermatozoa in this region showed strands and droplets of secretory material distributed over the anterior portion of an intact head before ovulation. whereas distribution of material over the post-nuclear cap of spermatozoa close to vesiculation or already acrosome-reacted was characteristic of the post-ovulatory situation. These changes in sperm head membranes were viewed as an expression of the completion of capacitation, and seemingly permit microvillous engagement with the rostral tip of the head. In conjunction with a narrow lumen and viscous secretions in the caudal isthmus, microvilli may thus serve to regulate periovulatory sperm progression towards the site of fertilisation, and be the basis of intermittent phases of adhesion to the oviduct epithelium as seen by phase-contrast microscopy. Although cilia do not similarly engage the heads of bull spermatozoa (cf. boar spermatozoa), they may act to regulate progression of capacitated spermatozoa by contacting the principal piece of the flagellum. In the light of these observations, changes in the molecular composition of sperm surface domains during the process of capacitation in vivo now require specific definition.

Following oviposition and/or fertilization in fieshwater, the soft vitelline membrane surrounding the carp oocyte elevates and becomes tough (fertilization envelope). We have studied the biochemical (protease digestion) and morphological (analysis scanning electron microscopy) changes associated with this transformation. The vitelline envelope is easily digested by most proteolytic enzymes; following elevation, the chorion becomes very resistant to proteolyse. This chorion is much thinner and smoother than the vitelline membrane, but retains a characteristic porous appearance. These results are compared with previous observations obtained by transmission electron microscopy and are discussed in relation to the blockage to polyspermy.

Mouse oocytes at the germinal vesicle (GV) stage were fused with maturing oocytes in which GVs were no longer visible. The fused cells were fixed at different time-intervals after the initiation of fusion and prepared for scanning electron microscope (SEM) observation. Concomitantly, some fused cells were prepared for light microscope evaluation. Our SEM observations showed no significant differences in surface morphology between immature and maturing oocytes. However, immediately after fusion was initiated, dramatic changes occurred on the surface of the maturing oocytes. The microvilli were shortened or disappeared locally and the plasma membrane was deeply ruffled. One hour after fusion, when the giant cells were nearly spherical, the microvilli reappeared and the ruffling gradually disappeared. In some areas, the microvilli were extremely long. Three hours after fusion, the fused cells were perfectly round and their surfaces were generally covered with microvilli of equal length. No further ruffling was observed. It is suggested that cytoplasmic mechanisms regulate the surface morphology of the oocytes during fusion.

Using sexually mature animals, the distribution of spermatozoa has been examined at the utero-tubal junction and in the distal and proximal portions of the oviduct isthmus. Mating occurred during early oestrus and, with one exception, specimens were prepared shortly before or after ovulation. Distinct reservoirs of spermatozoa were identified in furrows between the terminal folds of the isthmus, and particularly within the troughs and transverse ridges of this region. The density of spermatozoa diminished steeply from the uterotubal junction towards the isthmus, especially in the pre-ovulatory specimens. The membranes of most spermatozoa in the isthmus were intact up to the time of ovulation, suggesting that the acrosome reaction is a peri- or post-ovulatory event. Whilst the flagella of spermatozoa in the reservoirs were usually straight or only slightly curved, those on the surface of the epithelial folds were undulating (S-shaped). Specific microenvironments may therefore exist in the distal portion of the isthmus to regulate sperm motility; droplets of secretion were a notable feature in this region. In specimens prepared 24 hr after ovulation, spermatozoa were almost absent from the utero-tutnrl junction and isthmus. However, denuded eggs were observed in the proximal portion of the isthmus in this animal, and they had spermatozoa associated with the zona pellucida. Arguments are presented for a peri-ovulatory endocrine regulation of sperm redistribution and capacitation.

The process of cumulus mucification in pig preovulatory follicles was examined by scanning electron microscopy. The localization of extracellular material and changes in the granulosa cell surface were observed at 0, 16, 20 and 40 h after hCG injection. At 0 h the pig oocyte cumulus complex was closely attached to the parietal layer of granulosa cells. Cumulus and parietal granulosa cells had microvilli and cytoplasmic projections connecting neighbouring cells. The network of extracellular amorphous material was observed for the first time at 16 h after hCG around the cells that formed a stalk between the parietal granulosa and the cumulus oophorus. At 20 h after hCG, the intercellular matrix was thicker and extended to almost all the cumulus oophorus surface; when visible, cells were often covered by blebs and ruffling membranes. All oocytes examined by light microscopy at 16 and 20 h after hCG were at the germinal vesicle stage with condensing bivalents. Therefore, it was concluded that oocyte nuclear maturation started at the same time that a mucified peduncle was forming between the cumulus oophorus and the parietal granulosa. Shortly before ovulation (40 h after hCG) the matrix material filled all intercellular spaces and mucification had extended to the corona cell layer and the zona pellucida surface.