Differential activation of IGF-II promoters P3 and P4 in Caco-2 cells during growth and differentiation.

BACKGROUND & AIMS: Insulin-like growth factor (IGF)-II gene is overexpressed in colon cancers. Transcriptional up-regulation may be the major mechanism contributing to its overexpression. IGF-II messenger RNA (mRNA) levels are up-regulated during proliferation followed by a significant decline during differentiation of Caco-2 cells. Mechanisms underlying transcriptional regulation of the IGF-II gene promoters (P1-P4) have yet to be examined in colon cancers, which was the basis for this study. METHODS: Ribonuclease protection assay was used to measure IGF-II mRNA derived from P1-P4. To determine if changes in the IGF-II transcripts reflected differences in promoter activity, transient transfection assays with the full-length P1-P4-luciferase expression vectors were performed. RESULTS: Both P3- and P4-derived transcripts were significantly up-regulated during the proliferative phase of the cells (days 3-6 in culture) and declined rapidly in cells undergoing differentiation (days 7-10); conversely, P1- and P2-derived transcripts were not detected. Similarly, transcriptional activity of P3 and P4 promoters reached peak levels by days 4-6 and declined rapidly thereafter. P1 and P2 were relatively inactive on all days. CONCLUSIONS: The activity of the P3 and P4 promoters may play a selective role in regulating IGF-II mRNA levels during growth and differentiation of colon cancer cells.

Loss of laminin and type IV collagen in uterine luminal epithelial basement membranes during blastocyst implantation in the mouse.

BACKGROUND: Removal of the uterine luminal epithelium and its basement membrane is necessary for successful implantation of invasive blastocysts. Few reports, however, have specifically addressed the penetration and loss of the uterine luminal epithelial basement membrane (UEBM). We investigated the loss of UEBM by examining the distribution of laminin and type IV collagen. METHODS: Blastocyst implantation sites were collected from mice on days 5, 6, and 7 of pregnancy. Paraffin sections were prepared from these tissues and processed with standard immunoperoxidase techniques to reveal the distribution of laminin and type IV collagen. RESULTS: On day 5 of pregnancy blastocysts were adherent to the uterine epithelium. The epithelium and UEBM were complete and uninterrupted. On day 6 the juxtaembryonic uterine epithelium was lost and focal discontinuities were seen along the UEBM. By 1200 hr the UEBM had receded to the region near the ectoplacental cone, but staining was reduced for both antigens over the entire region surrounded by decidual cells. This decreased staining of the UEBM occurred in areas not yet occupied by trophoblast cells. On day 7 the UEBM was lost over the entire embryonic half of the uterine lining, corresponding to the distribution of decidual cells. CONCLUSIONS: Progressive loss of the UEBM occurred in a consistent spatiotemporal pattern following the onset of blastocyst implantation. Diminished immunoreactivity of laminin and type IV collagen in the UEBM was closely correlated with the area occupied by decidualized endometrial stroma and occurred in areas not yet in contact with trophoblast cells. We conclude that decidual cells are instrumental in the removal of UEBM during early pregnancy.

Penetration of the uterine epithelial basement membrane during blastocyst implantation in the mouse.

For many species, blastocyst implantation is associated with a reduction in the number of cellular and extracellular matrix layers which separate the trophoblast from maternal vasculature. Following loss of uterine epithelial cells along the distal mural trophoblast, the mouse blastocyst encounters the residual epithelial basement membrane. This sheet of extracellular matrix must be breached and later removed prior to trophoblast invasion of the uterine stroma and formation of the placenta. The interactions between the trophoblast, luminal epithelial basement membrane, and decidual cells during the time when embryonic and uterine stromal cells first achieve contact were examined in this study. Distal mural trophoblast of activated delay blastocysts was in contact with the residual luminal epithelial basement membrane 36 hr after estrogen administration. This portion of the basement membrane contained areas in which the usual linear appearance was changed to an irregular, tortuous profile. The lamina densa frequently appeared flocculent and diffuse. Cytoplasmic processes from trophoblast and decidual cells simultaneously perforated the basement membrane at multiple discrete loci. With further development the basement membrane was lost, leaving trophoblast and decidual cells in close contact over large areas. In normally implanting blastocysts a similar stage of embryonic development, as described above, was attained by 0400 hr on day 6 of pregnancy. Regions of convoluted epithelial basement membrane were also seen in these implantation sites. However, only decidual cell processes were seen penetrating the residual basement membrane. These processes extended to the fetal side of the basement membrane and separated that matrix from overlying trophoblast. They contained organelles and formed rudimentary intercellular junctions with the trophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of laminin in hamster tracheal epithelial cell cultures.

EM examination of 28 day cultures of enzymatically dissociated hamster tracheal epithelial (HTE) cells grown on collagen coated millipore filters reveals that fragments of basal lamina may be present at the basal plasmalemma. Since the basal lamina consists of several major components including type IV collagen, heparan sulfate proteoglycans, entactin/nidogen, and laminin, questions naturally arise concerning the presence of such a structure in this cell culture system. When immunocytochemical procedures utilizing anti-laminin antibody and PAP techniques are carried out with paraffin sections of HTE culture at 1,2,3, and 4 weeks in vitro, LM analysis reveals that a thin, dense line of reaction product is present between the basal surface of the HTE cells and the underlying collagen substrate. Immunoblotting evaluation carried out with supernatants of 7d HTE cell homogenates and HTE cell conditioned media also indicate that laminin is being produced by the tracheal cells. Thus, the presence of basal lamina-like fragments, the immunocytochemical localization of laminin, and immunoblot identification of laminin in hamster tracheal epithelial cell cultures, suggest that, although basal lamina components may be produced by HTE cells, at the time points tested, they are not yet being organized into a complete basal lamina.

Blastocyst implantation in the Chinese hamster (Cricetulus griseus).

Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete area. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well-developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert's membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert's membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents.

Endometrial response to deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone: an ultrastructural study.

The present work continues our aim of establishing an experimental model to study the decidual cell reaction to an artificial deciduogenic stimulus in the long-term ovariectomized rhesus monkey treated with oestrogen followed by progesterone. The fine structural details of decidual, granular and plaque cells, which constituted the endometrial cellular response to the deciduogenic stimulation in the present study, revealed striking similarities with those reportedly present in an endometrial response to blastocyst implantation in the rhesus monkey. Plaque epithelia showed a significant degree of hypertrophy, hyperplasia and differentiation followed by a steady degeneration by day 32 (equivalent to day 16 after trauma) of treatment. The plaque cells were shown to contain numerous regular-shaped mitochondria, polyribosomes and large amounts of rough endoplasmic reticulum (RER) in their cytoplasm and were characteristically arranged in clusters or acini formation surrounded by discrete basal laminae. As early as day 28 of treatment, the initiation of stromal decidual cell transformation was noted and, by day 48, a sizeable pool of decidual cells was found. The decidual cells had rounded nuclei and elaborate arrangements of interconnected cisternae of RER which were often moderately dilated and filled with amorphous, electron-dense material. Granular cells were characterized by eccentrically located nuclei and numerous membrane-bound, electron-dense granules in their cytoplasm and were found in increasing numbers in the stroma around decidual cells, blood vessels and glandular epithelia.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA synthesis in the mouse blastocyst during the beginning of delayed implantation.

The spatiotemporal pattern of DNA synthesis in the mouse embryo at the beginning of metabolic dormancy was examined. Embryos were recovered from females at intervals following ovariectomy at 1100 hours on day 4 of pregnancy, incubated in vitro for 1 h in the presence of [3H]thymidine, and prepared for light microscopic autoradiography. The proportion of labeled cells in the embryo remained high (40-60%) for 18 h after ovariectomy and then declined gradually to 12% by 96 h. However, analysis of individual cell subpopulations showed that the decline was not uniform in all regions of the blastocyst. Labeling was high over the inner cell mass (ICM) during all time intervals in the study, while labeling over the mural trophoblast cells declined sharply by 24 h after ovariectomy. Labeling over the polar trophoblast also declined but had values that were intermediate between the ICM and mural trophoblast regions of the blastocyst. These regional differences in DNA synthesis during the arrest of development suggest that intermediate steps are involved in control of DNA synthesis in the embryo and that the ICM may play a role in the different responses of the trophoblast cell populations.

Resumption of DNA synthesis in delayed implanting mouse blastocysts during activation in vitro.

The resumption of DNA synthesis in delayed implanting mouse embryos undergoing metabolic activation in vitro was examined. Blastocysts were recovered from ovariectomized mice, incubated for various intervals in basal Eagle's medium, exposed to 3H-thymidine, and prepared for light microscopic autoradiography. Following incubation the proportion of labeled cells increased from 4% at 1 hr to 30% by 24 hr. This increase in labeling was not uniform in all regions of the blastocyst, i.e., labeling was initially highest over the inner cell mass (ICM) but remained low over the polar and proximal mural trophoblast for 6 and 12 hr, respectively, and then began to increase. This pattern in the resumption of DNA synthesis during activation in vitro is similar to that reported in vivo (Given and Weitlauf, '81) and suggests that the mechanism responsible in intrinsic to the blastocyst rather than being a differential response to the intrauterine milieu. Furthermore, it appears that the ICM may play an essential role in the resumption of synthesis in the surrounding trophoblast.

Resumption of DNA synthesis during activation of delayed implanting mouse blastocysts.

Cell division ceases in mouse blastocysts during the extended dormant period associated with delayed implantation but resumes following activation of the embryos by administration of 17 beta-estradiol to the mother. To determine the temporal and spatial aspects of the resumption of DNA synthesis during activation, blastocysts were recovered from delayed implanting females at various intervals after an injection of 17 beta-estradiol, incubated with 3H-thymidine in vitro, and prepared for light microscopic autoradiography. Although less than 4% of the cells were labeled in delayed implanting embryos, the proportion of labeled cells increased soon after the administration of 17 beta-estradiol and reached a maximum of over 50% by 24 hours. This increase in labeling was not uniform in all regions of the embryo, i.e., the labeling index of the inner cell mass began a steady increase immediately after the injection of 17 beta-estradiol while labeling of the polar and proximal mural trophoblast remained depressed for 6 and 12 hours, respectively, and only then began to increase. No labeling was present over the distal mural trophoblast in delayed implanting or activated blastocysts although cytological changes characteristic of primary giant cell transformation were present in activated embryos. These results indicate that the resumption of DNA synthesis is part of the overall increase in metabolic activity associated with activation. Furthermore, the sequential pattern of resumption of synthesis suggests that the ICM may influence the initiation of DNA synthesis in the surrounding trophoblast.

Mouse uterine glands during the peri-implantation period. II. Autoradiographic studies.

Protein synthesis and secretion in mouse uterine glands during the peri-implantation period were studied, by both light and electron microscopic autoradiography, after the in vivo administration of tritiated leucine (3H-leucine) and proline (3H-proline). Light microscopic autoradiography revealed that the time course of synthesis and secretion of labeled proteins was constant during days four, five, and six of pregnancy. Labeled material could be detected in the glandular lumen by 45 minutes after administration and in higher concentrations by 90 minutes after administration. Analysis of electron microscopic autoradiographs from days five and six of pregnancy showed that high levels of activity were initially present over the rough endoplasmic reticulum and Golgi complexes and subsequently declined at the longer time intervals (45 and 90 minutes), while activity over the glandular lumen increased with time. The pathway of intracellular transport to the glandular lumen appeared to be via small cytoplasmic vesicles on both days five and six of pregnancy. Additional pathways for transport of the labeled protein to the glandular lumen appeared to be present in the form of the large vesicles on day five and granules on day six of pregnancy. Throughout the peri-implantation period, mouse uterine glands were active secretory structures in which the mode of secretion was similar to other exocrine cells. Thus, the uterine glands of the mouse must be considered a source of uterine fluid proteins at the time of implantation that may contribute to quantitative changes in these proteins.

Cytology of the endometrium of delayed and early implantation with special reference to mice and mustelids.

Although it is apparent that the uterus is involved in the control of embryonic diapause, in no instance do we know the precise nature of that control. The most direct control would be through synthesis of proteins or other macromolecules by the uterus itself. Whereas luminal contents have been shown to vary significantly near the time of implantation, neither the role of the luminal fluid constituents, nor the cellular origins of these molecules are fully known, nor have the mechanisms of storage and secretion of proteins in the endometrium been fully established. Cellular structures that may be indicative of functional activity include secretory granules or vesicles, increase or dilation of granular endoplasmic reticulum and increase in size, and associated vesicles of the Golgi complex. Storage of glycogen or lipid appears more indicative of lowered activity. However, despite striking changes in these cytological characteristics in different animals during delay and early implantation, there is no single pattern during the delay period, and no predictable alteration in the peri-implantation period. In general, the structure of the uterus of an animal during delay resembles a modification of that of a closely related species that does not delay more than it resembles that of other species that do show delay.

Differentiation and migration of endoderm in the rat and mouse at implantation.

The initial differentiation of endoderm at the time of onset of implantation, and the subsequent rapid differentiation of visceral and parietal endoderm were studied in the rat and mouse. Transmission electron microscopy illustrates the reorientation and loosening of embryonic cell mass cells during implantation, as well as cytological evidence that endoderm cells have differentiated. Using scanning electron microscopy, parietal endoderm consists of individual stellate cells with numerous peripheral branching filopodia. As these cells migrate abembryonically, the rest of the embryonic cell mass becomes recompacted. The visceral endoderm proliferates and forms a columnar epithelium which has the cytological characteristic of an absorptive epithelium and is able to ingest exogenous proteins. Thus, by 24 hours after implantation, the two endodermal derivatives have assumed widely diverse shapes and different types of associations and rates of replication, and are probably performing different functions.