Structural aspects of bovine oocyte maturation in vitro.

Bovine cumulus-oocyte complexes (COCs) were collected from 4-8 mm follicles and graded into four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of oocytes that had matured in vivo and were collected 20-23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphological characteristics. After in vitro maturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: 1) vacuolization, 2) flattening of cumulus cells, and 3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: 1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, 2) retardation of some aspects of the cytoplasmic maturation, and 3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.

Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes.

The integrity of the cumulus cell processes were studied in four categories of bovine cumulus oocyte complexes (COCs) selected on their morphological characteristics. Three different types of cumulus cell process endings (CCPEs) were identified, one penetrating the cortex, another not penetrating the cortex, and a third form was intermediate and more rare in appearance. The process endings that penetrated the cortex frequently made gap junctions with the oolemma. The division of the three types of CCPEs over the four different COC categories was specific for three of the four categories. The first-category COC predominantly possessed the penetrating CCPE, the fourth-category COC possessed predominantly the nonpenetrating CCPE, and the second and third categories had both types of CCPEs. The metabolic coupling of the cumulus-oocyte contacts was assessed by means of incorporation of 3H-choline into the oocyte. The majority of category 4 COCs transferred low levels of choline into the oocyte while the majority of the oocytes of the other three categories transferred high levels of choline into the oocyte. Category 4 includes a smaller proportion of oocytes capable of cleaving after fertilization than the other three categories. This reduced developmental capacity is probably due to the loss of metabolic coupling before the onset of culture.

Morphology of immature bovine oocytes.

Bovine cumulus oocyte complexes (COCs) as used for in vitro maturation and fertilization can be classified into different categories by light microscopical inspection. We have distinguished four categories based on compactness and transparency of the cumulus investment and homogeneity and transparency of the ooplasm. The four categories were studied for their morphological characteristics at the ultrastructural level and for their developing capacity in an in vitro maturation system. In categories 1 and 2 oocytes, organelles were evenly distributed. In categories 3 and 4, oocytes organelles were clustered and the distribution of the organelles mimicked the characteristics of oocytes during final maturation. Cumulus cell process endings penetrated the cortex of the oocyte or were located superficial to the cortex of the oocyte. In category 1 oocytes, most of the process endings penetrated the cortex. In category 4 oocytes, most of the process endings did not penetrate. In categories 2 and 3 oocytes, both forms of process endings did occur. After in vitro maturation, only category 4 oocytes showed a decreased developing capacity. Categories 1-3 oocytes showed equal developing capacity in an in vitro maturation system.

Isolation of autonomously growing dog mammary tumor cell lines cultured in medium supplemented with serum treated to inactivate growth factors.

Conventional methods for isolation of cell lines from carcinomas suffer inherently from a lack of advantage for proliferation of transformed cells as opposed to contaminating fibroblasts and normal epithelial cells. To isolate cell lines from metastases of estrogen receptor-negative mammary carcinomas in dogs, we applied a novel method using medium supplemented with serum treated to inactivate growth factors. Under these conditions, autonomously growing tumor cells are selectively allowed to proliferate. In this way, four autonomously growing tumor cell lines were obtained from metastases of two dogs. Tumors formed from cells implanted in C3H nude mice closely resembled the original dog tumors, indicating that the main result of this selective procedure was suppression of normal cell proliferation. Serum treated to inactivate growth factors seems to be an important medium supplement for isolation of autonomously growing tumor cell lines, which may be valuable tools for future studies on regulation of cell proliferation in advanced hormone-independent mammary tumors.

Ultrastructural localization of platelet-derived growth factor and related factors in normal and transformed cells.

Visualization of platelet-derived growth factor (PDGF) and PDGF-like growth factors in cultured cells has been achieved by cryo-ultramicrotomy in combination with immunogold labeling. Immunogold staining of cryosections requires a mild chemical fixation in order to ensure preservation of antigenicity and ultrastructural details. Therefore the effect of several chemical fixatives on the antigenic properties of PDGF and PDGF-like growth factors was studied by indirect immunofluorescence using a polyclonal anti-PDGF antiserum. These studies demonstrated that formaldehyde has no effect on antigenicity, in contrast to glutaraldehyde or acrolein. For this reason formaldehyde was used as the only fixative for the visualization of PDGF in cryosections. PDGF was visualized in cryosections of normal human fibroblasts, preincubated with PDGF under various conditions. Preincubation at 4 degrees C with PDGF resulted in partial internalization of the growth factor. During subsequent warming of the cells to 37 degrees C PDGF was translocated to the nucleus. PDGF was also detected in the cytoplasm of tumor cells producing endogenous PDGF-like growth factors (neuroblastoma and simian sarcoma virus-transformed cells) but in these cases no significant amounts of these growth factors were present in the nucleus or at the extracellular surface of these cells. These results will be discussed in view of the intracellular routing of PDGF in normal responsive cells and of PDGF-like growth factors in factor-producing cells.

Immunocytochemical demonstrations of cytoplasmic and cell-surface EGF receptors in A431 cells using cryo-ultramicrotomy, surface replication, freeze-etching and label fracture.

In this paper we describe the use of a number of complimentary methods to visualize cytoplasmic and cell-surface located epidermal growth factor (EGF) receptors in cultured A431 cells. Cryo-ultramicrotomy in combination with immuno-gold labelling will be shown to provide an excellent method in visualizing cytoplasmic located EGF receptors in addition to cell-surface located EGF receptors. An important aspect in this method involves the possible effects of the fixatives on antigenicity. Using radioactive labelled anti EGF receptor antibodies, it was shown that formaldehyde as a fixative had no significant effect on label-efficiency. The density and lateral distribution of EGF receptors at the cell surface has been studied by three methods, i.e. surface replication, freeze etching and label fracture, all methods in conjunction with immuno-gold labelling. These methods allow in principle a quantitation of the surface distribution of the EGF receptors. The surface-replication method involves, however, dehydration and critical-point drying steps, and using radioactive labelled anti EGF receptor antibodies it was shown that in particular OsO4 fixation and dehydration caused a significant loss of cell-associated antibodies. This disadvantage is overcome by freeze etching and the label-fracture method, and as such these techniques provide the best methods for quantitative analysis of the planar distribution of cell-surface located EGF-receptors.

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling.

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.

Ultrastructural aspects of rapid plasma membrane growth in mitotic neuroblastoma cells.

In murine C1300 neuroblastoma cells, clone Neuro 2A, the major fraction of the necessary increase in cell surface area during the cell cycle occurs within a short period around mitosis. During this period cell cycle-related modulations in a number of structural, dynamic and transport properties are most prominent. In this study we have examined the mechanism of rapid plasma membrane growth during mitosis, and the resulting changes in the ultrastructural features of the plasma membrane, by scanning and freeze-fracture electron microscopy as well as by electron microscopy of ultrathin sections. Our observations show that plasma membrane growth occurs by the fusion with and the incorporation into the plasma membrane of cytoplasmic multilamellar, lipidic membrane vesicles. Such vesicles are not observed at other times in the cell cycle. As a consequence, IMP-free domains appear transiently in the mitotic and early post-mitotic plasma membrane. Comparison of replicas prepared from glutaraldehyde-fixed cells and unfixed, ultrarapidly frozen cells showed that aldehyde fixation artefactually induces a bleb-like appearance of these domains. The IMP-free domains disappear in the G1-phase as a result of the mobilization and lateral redistribution of membrane components. It is argued that mitotic membrane growth by preferential incorporation of membrane lipids not only serves to accomodate for the necessary increase in cell surface area, but also provides a mechanism for plasma membrane-mediated regulation of the cell cycle.