The 12S adenoviral E1A protein immortalizes avian cells and interacts with the avian RB product.

Quail cells were immortalized for the first time by using retroviruses expressing the 12S adenoviral E1A gene. In these cells, interaction between the 12S E1A product and the quail RB protein was shown, suggesting that the 12S adenoviral E1A product works in avian cells through similar biochemical pathways as in mammalian cells by interacting and inactivating host cellular proteins, including the RB product. These results confirm that the RB product exhibits a universal function among higher vertebrates in controlling cellular growth and tumor progression.

Cell mixing during the early development of mouse aggregation chimera.

Two different inbred strain combinations of mouse aggregation chimeras - C3H/HeN (H-2k) x C57BL/6N (H-2b) and C3H/HeN x BALB/cA (H-2d) were used for cell mixing analysis at two points in time - 24 h after aggregation (just prior to transplantation into foster mothers) and 7.5 days post coitum (p.c.). The cell proportion of two H-2 haplotypes at the blastocyst stage was studied using a fluorescence-labeled monoclonal antibody recognizing a C3H-specific alloantigen - CSA (C3H strain-specific antigen) and laser scanning confocal microscopy. The 7.5-day-old chimeras were sectioned and subsequently processed by sensitive biotinylated antibody - avidin peroxidase immunohistochemical technique. Our results showed that 24 h after aggregation (blastocyst stage), there was equal cell mixing and no mouse strain used in the present study was dominant at this time. In 7.5-day-old C3H/HeN x BALB/cA chimeras, cells of both genotypes were intermingled, but the C3H/HeN strain was dominant in all cases. In contrast, the combination C3H/HeN x C57BL/6N clearly showed reduced numbers of C3H/HeN cells (CSA-positive) in 83% of the chimeras evaluated. Generally, CSA positive cells were found only in randomly distributed small distinct areas representing less than 20% of embryonal cells. Surprisingly, the extraembryonal ectoplacental cone was uniformly CSA positive in some C3H/HeN x C57BL/6N chimeras. Furthermore, in 36% of normally implanted chimeras of both strain combinations progressive degeneration was observed. We suggest that the cell mixing pattern as well as the absolute number of cells derived from each strain in the aggregation chimera can be affected by specific immune interactions involving H-2 haplotype combinations of the allogeneic fetus and the fully immunocompetent host organism, at later points in development.

Isolation and characterization of a feeder-dependent, porcine trophectoderm cell line obtained from a 9-day blastocyst.

We have established in culture a feeder-dependent cell line, termed TE1, from a 9 day, pre-implantation, porcine embryo. TE1 cells were observed by light and electron microscopy, and characterized by immunocytochemistry: the morphology, cytology and ultrastructure of this cell line are described. The cells display epithelial characteristics, as revealed using immunofluorescence microscopy with antibody against cytokeratins of simple epithelia, but not with antibody against vimentin. The cells demonstrate many morphological and cytochemical features in common with trophectoderm of the intact porcine blastocyst. For example, TE1 cells are polarized and possess tight junctions at their borders, similar to those found in trophectoderm of the pre-implantation embryo. Moreover, TE1 cells label positively for the porcine trophectoderm-specific monoclonal antibody, SN1/38. Thus, by several important criteria TE1 is deduced to be a porcine trophectoderm cell line.

Conceptus attachment in the ewe: an ultrastructural study.

Scanning and transmission electron microscopic observations were made on trophoblast, caruncles and intercaruncular areas during the attachment of the conceptus. Three stages were determined: 1. From day 14 on, precontact was established and the conceptus appeared to be immobilized in the uterine lumen. On the centres of the caruncles which were depressed and folded the epithelial cells developed bulbous cytoplasmic protrusions. Throughout the free life of the conceptus, the trophoblast cells showed an abundant covering of microvilli. 2. On day 15, apposition occurred: most microvilli on the surface of the trophoblast disappeared. 3. Between days 16 and 18, adhesion began as a result of the interpenetration of the uterine microvilli and cytoplasmic projections of the trophoblast cells. During that stage trophoblast giant cells appeared and the uterine epithelium was turned into syncytial masses; however, it was apparently not destroyed later on. Between the caruncles, the trophoblast developed finger-like villi which invaded the lumen of the uterine glands from days 15 to 18. During their short life-time (they vanish at day 20), these trophoblastic differentiations may anchor the pre-attachment conceptus and absorb the histotrophic secretions of the glands.

Gap junction formation and connexin distribution in pig trophoblast before implantation.

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.

Polarized porcine trophoblastic cell lines spontaneously secrete interferon-gamma.

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.

Expression of beta 2-microglobulin on preimplantation pig embryos.

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.

Spermiation and epididymal maturation of spermatozoa in the bonnet macaque (Macaca radiata) as viewed by scanning electron microscopy.

Spermiation and epididymal maturation of spermatozoa were studied in the bonnet monkey (Macaca radiata) by light and scanning electron microscopy. The morphology, location, and release of residual body were observed during spermiation. Along the epididymal duct, the shape of spermatozoa changed, the constriction at the anulus disappeared, the marginal thickening diminished in length, and the cytoplasmic droplet regressed and moved toward the posterior end of the middle piece. Mature spermatozoa were very similar to those of other Cercopithecidae, and they showed a forward motility and a drop in eosin stainability.

Surface ultrastructure of preimplantation baboon embryos.

Scanning and transmission electron microscopy were used to study baboon preimplantation embryos 3 to 5 days E.F.A. (estimated fertilization age), ranging from about 16 to more than 60 cells. The peripheral blastomers were covered with microvilli scattered on the convex outer surface and along the borders of the intercellular furrows. In younger morulae, some longer microvilli may bridge over the furrow separating contiguous blastomeres. A few blastomeres showed poorly developed microvilli. Blastomeres of smaller diameter than the others may arise from more recent cleavage. Cell junctions as well as small intercellular spaces were noted at the apposition of the blastomere plasma membranes whereas surface intercellular ridges were not observed.

Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions.

We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes. The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analyses. The cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemistry. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in serum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d. Confluent oviduct cells also maintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography. Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins.

Distribution of spermatozoa in the utero-tubal junction and isthmus of pigs, and their relationship with the luminal epithelium after mating: a scanning electron microscope study.

The epithelial cell morphology and distribution in the utero-tubal junction and isthmus of pigs was documented by scanning electron microscopy around ovulation. In animals mated at different times before slaughter, our observations confirmed that the utero-tubal junction and posterior part of the isthmus regulate the transport of spermatozoa. The utero-tubal junction appears clearly as a form of mechanical valve strongly limiting the number of sperm cells penetrating the oviduct. The isthmus, and especially its posterior part poor in ciliated cells, is a storage place for spermatozoa which appear as though trapped in the epithelial folds. It remains to be demonstrated if they stay in such reservoirs due to the constriction of the lumen by the thick muscular wall of the duct, or to some chemotactic attraction by tubal secretions, or simply due to adhesion on the epithelium. Our study supports the hypothesis that transport of spermatozoa in the isthmus towards the site of fertilization depends in part on ciliary motion. The instant direction of propagation appears random for spermatozoa escaping from the reservoirs. Other factors such as tubal contractions probably ensure that the resultant movement is a progressive ascent.

Distribution, morphology and epithelial interactions of bovine spermatozoa in the oviduct before and after ovulation: a scanning electron microscope study.

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.

Pre- and peri-ovulatory distribution of viable spermatozoa in the pig oviduct: a scanning electron microscope study.

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 utero-tubal 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-tubal 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.

Sperm surface changes during the acrosome reaction as observed by freeze-fracture.

The mammalian acrosome reaction is an exocytotic process that can be analyzed by the technique of freeze-fracture; only sperm cells capacitated in vitro or treated to elicit the acrosome reaction in vitro have been studied, and all pictures published are from material fixed before freezing. All the authors point out the appearance of particle-free areas in the plasma membrane of the acrosomal region during capacitation and before any fusion. This is interpreted as an increase in membrane fluidity as suggested by studies on membrane lipid composition in guinea-pig sperm. We have recently described the induced acrosome reaction in ram spermatozoa. Fusion starts at the limit of the anterior and equatorial segments and progresses forward in the anterior segment along ramified paths, resulting in a fenestration gradient of the acrosomal cap. Fusion propagation may be controlled by fluidity increase in the plasma membrane of the anterior segment, and it is probably inhibited in the equatorial segment by the ordered structure of the acrosomal membrane.

A scanning electron microscope study of the hatching of bovine blastocysts in vitro.

The blastocysts were recovered from cows 7--10 days after oestrus and cultured. The zona pellucida has a spongy fibrous structure. Hatching begins, about 24 h after culture, through a relatively small hole out of which the blastocyst appears to escape by its own activity. Later the zona becomes split and the two edges of the slit surround part of the blastocyst. The emerging cells were large or small blastomeres which were generally covered by a dense mass of microvilli.

Immunocytochemical detection of acrosomal damage following cold shock: loss of acrosin from the acrosomal region of ram, bull and boar spermatozoa.

Samples of ram, bull and boar semen were subjected to cold shock and then stained using an indirect immunocytochemical method for detecting acrosin. It was found that the shock treatment abolished staining of the acrosomal region in all but a very few cells. This finding was interpreted as a great loss of proacrosin or acrosin from damaged acrosomes. Using a fluorescent label in the system, sperm cells in both control and treated samples could be scored easily and reproducibly as "stained" or "unstained". Results using a peroxidase label were less satisfactory because staining was not as consistent. Biochemical assessment of sperm acrosin content before and after cold shock confirmed that such treatment caused acrosin loss from the cells into the seminal plasma, but as a quantitative measure of cellular integrity the biochemical method exhibited a number of deficiencies compared with the immunofluorescent cytochemical method. Preliminary data are presented for semen evaluation comparing this latter method with a "live-dead" stain.

Distribution of sterols and anionic lipids in human sperm plasma membrane: effects of in vitro capacitation.

Filipin, a membrane beta-hydroxysterol probe, and polymyxin B (PXB), a probe for anionic lipids, were used to study human sperm plasma membrane (PM) with particular reference to changes induced by capacitation in vitro. In washed but noncapacitated spermatozoa the density of filipin/sterol complexes (FSC) was uniformly high in the PM overlying the acrosome, without any differences between its anterior and equatorial regions. The postacrosomal region was poor in FSC. The exclusion of FSC from small areas of the PM covering the two acrosomal regions and reduction of their number in the postacrosomal region were the main changes induced by sperm capacitation. Unlike filipin, PXB produced a capacitation-independent reaction pattern characterized by a high reactivity of the PM covering the anterior portion of the acrosome, probably conferring to this region its potential fusigenicity temporarily antagonized by the elevated sterol content. Only an exceptional and slight PXB binding was seen in the nonfusigenic equatorial and postacrosomal regions.

Ultrastructural study of the interactions and fusion of ram spermatozoa with zona-free hamster oocytes.

The interaction of preincubated ram sperm with zona-free hamster oocytes was studied by scanning and transmission electron microscopy. The behaviour of the sperm cells was quite different, depending on whether the acrosomal reaction had taken place or not. The apical ridge of intact spermatozoa contacted the oocyte surface, and egg microvilli spread onto the anterior segment but no fusion ensued. When the acrosomal cap was fenestrated, microvilli were also found on its surface but were then spread over the surface of the postacrosomal region and the equatorial segment of the sperm head lying flat on the egg surface; fusion with the oocyte occurred in the equatorial segment and extended to the postacrosomal region. Contacts between the microvilli and the inner acrosomal membrane were infrequent and no fusion occurred in the anterior segment. These observations confirm that local changes in the adhesiveness and fusibility of the sperm plasma surface occurred during the acrosome reaction.

Immunocytochemical characterisation of rabbit and mouse embryonic fibroblasts.

By using indirect immunofluorescence we demonstrated the localisation of extracellular matrix (ECM) proteins (laminin--LAM, collagen IV--COL IV, fibronectin--FN) and the basic fibroblast growth factor (bFGF) in rabbit and mouse primary embryonic fibroblasts (PEF). Proliferating mitotically arrested PEF (by mitomycin C) were compared in both species. The stability of protein expression was ascertained during the first five successive passages. In addition, STO cells (i.e. permanent line of irradiated mouse fibroblasts) were similarly analysed. Rabbit PEF showed very high extracellular staining for FN and a negligible cytoplasmic positivity for LAM and COL IV. A totally reversed staining pattern for ECM proteins was found in mouse PEF. A dense cytoplasmic granulation (concentrated around the nucleus) was revealed for LAM and COL IV and almost no reaction for FN. The staining patterns were very stable at the culture conditions we applied. They were maintained during the first five successive passages in proliferating as well as non-proliferating mouse and rabbit PEF and were independent of cell concentration (individually dispersed cells versus cells in a confluent layer). STO cells showed the same staining for ECM proteins as the mouse PEF, thus confirming their origin from the same animal species. Light granular staining for bFGF was found in the cytoplasm of proliferating and mitotically arrested rabbit and mouse PEF and STO cells. The differences in expression of ECM proteins between the rabbit and mouse PEF, as well as the synthesis of bFGF, should be taken into consideration when these cells are used in vitro as a feeder layer for various cells (e.g. embryonic stem cells).