The extracellular matrix and cell surface, mediators of cell interactions in chicken gastrulation.

This article reviews the factors that are involved in cell-cell and cell-matrix interactions during chicken gastrulation. The chemical nature of the extracellular matrix, the structure, composition, cellular origin and remodeling of the basement membrane, and the nature of the cell surfaces are successively analyzed in relation to a variety of cell biological processes, such as cell-to-cell and cell-to-substrate adhesion, specific binding to biological macromolecules and to receptors of the cell surface, promotion of cell migration and positioning, transmembrane triggering of intracellular events, and modulation of cell shape. These processes are the cellular basis of morphogenesis in the chicken blastoderm.

Cellular origin of the basement membrane in embryonic chicken/quail chimeras.

The cellular origin of the components of the basal lamina of an epithelium, the chicken epiblast, has been investigated in a model system resulting from the transplantation of metabolically labeled quail hypoblast (an epithelium without basal lamina), associated or not with individual mesoblast cells, into an unlabeled chicken blastoderm deprived of its own hypoblast. The ability to discriminate chicken from quail cells after nuclear staining, combined with autoradiographic labeling of the basal lamina components, made it possible to determine the origin of the cells and labeled compounds in tissue sections of the chimeras. The transplantation of 3H-glucosamine or 3H-fucose-labeled quail hypoblast into a chicken host embryo, and subsequent culture of the chimeric embryo for 5 h, led to the transfer of labeled macromolecules from the quail graft to the chicken basal lamina. Pre-treatment of sections with several glycosaminoglycan-degrading enzymes with different substrate specificities suggested that the 3H-glucosamine-labeled compounds that are deposited in the basal lamina are glycoproteins and/or heparan sulfate proteoglycan. However, in view of our current knowledge of the cellular origin of the latter compound, this molecule probably originates from the epithelium itself. Transfer of 3H-proline and 3H-hydroxyproline-containing molecules (mainly collagens) from the graft to the host basal lamina was not observed. Chasing the labeled compounds with unlabeled precursor during culture of the chimeras did not influence the final autoradiographic pattern. It is concluded that the basal lamina of the epiblast has a dual epithelial origin, resulting from the interaction of epiblast-derived materials and non-collagenous glycoproteins synthesized by the hypoblast. Evidence supporting the case of a non-epithelial, mesoblastic origin of non-collagenous glycoproteins was not found. An extensive review of literature on the epithelial vs non-epithelial origin of basement membrane components, mainly in mammalian species, is also provided.

The arrest of cell migration in the chicken blastoderm: experimental evidence for the involvement of a band of extracellular fibrils associated with the basal lamina.

This article overviews our current knowledge of the occurrence and distribution of oriented extracellular fibrils associated with the basal lamina, and their presumptive role in contact guidance of cells in early embryos. To investigate the role of the band of extracellular fibrils situated at the basal side of the epiblast at the cranial edge of the area pellucida of the chicken blastoderm, we determined the precise location and morphology of the fibrils using TEM and SEM, described the relationship between migrating mesoblast cells and the fibrillar band, and, finally, tested experimentally the behavior of homologous and heterologous tissues in the vicinity of the fibrillar band. The descriptive analysis demonstrated that a horseshoe-shaped, 170 microns-wide band of fibrils occurs at the cranial and lateral edges of the area pellucida and area opaca, the highest density being found in the area pellucida. Migrating mesoblast cells presented a surface morphology that was different from the morphology of cells reaching the fibrils at the lateral edge of the area pellucida. Mesoblast cells never crossed the fibrils, an observation that may explain why during gastrulation, mesoblast cells invade the area opaca only in the caudal region, where no fibrillar band is present. The experimental analysis, which involved transplantation and healing experiments, demonstrated that the arrest of cell migration, that occurred in all cases in the vicinity of the fibrillar band, was correlated with changes in surface morphology suggesting a decreased cell adhesion to the fibrils. From these observations emerged the view that the horseshoe-shaped fibrillar band functions as a barrier inhibiting migration of individual mesoblast cells and expansion of tissue sheets, rather than as an extracellular substrate mediating the oriented guidance of cells. In addition to its inhibitory role in cell migration, the extracellular band may also be regarded as a factor that stabilizes the polarity of the early embryo by determining the cranial and lateral limits between embryonic and extraembryonic tissues.

On the presence of proteolytic activity in glycosaminoglycan-degrading enzyme preparations.

A solid-phase protease assay has been used to screen different commercial preparations of glycosaminoglycan-degrading enzymes for the presence of proteolytic activity. Proteases cannot be detected in preparations of testicular hyaluronidase and of chondroitinase at the concentration used for histochemical purposes. Commercial Streptomyces hyaluronidase contains proteolytic contaminants detectable at the concentration used for histochemistry. At higher concentrations, all preparations appear to be contaminated with proteases. The results obtained using this assay suggest that addition of a mixture of proteinase inhibitors containing N-ethylmaleimide, EDTA, pepstatin, and phenylmethanesulfonylfluoride or soybean trypsin inhibitor has little effect on the proteolytic activity of the glycosaminoglycan-degrading enzyme preparations, irrespective of the pH used. Moreover, the use of EDTA in this mixture is questionable. This study also describes two testicular hyaluronidase preparations that may be particularly useful in functional studies of the living organism, as they are only slightly contaminated.

Localization of apoptotic cells by direct immunogold detection of digoxigenin-labeled genomic DNA in semithin sections.

In this study, which correlates apoptosis with avian ovarian physiology, we modified an in situ DNA nick end-labeling method using immunogold reagents to detect apoptotic cells in semithin sections of quail ovaries embedded in glycol methacrylate resin. Special attention was paid to the prevention of background staining. The results are comparable with those of the ApopTag peroxidase kit in paraffin-embedded ovaries.

Thromboembolic events after aortic valve replacement in elderly patients with a Carpentier-Edwards Perimount pericardial bioprosthesis.

OBJECTIVES: Thromboembolic events after aortic valve replacement with a bioprosthesis were the most frequently occurring complications in elderly patients. Whether this was valve related or dependent on other factors needed further exploration. METHODS: Five hundred patients with a median age of 73 years were followed retrospectively after aortic valve replacement with a pericardial prosthesis for occurrence of thromboembolism. Of these, 348 also underwent coronary artery bypass grafting. Twenty-five factors were investigated for their potential effect by using univariate and multivariate analysis. RESULTS: Univariate analysis revealed 6 significant factors: preoperative endocarditis (P =.0001), preoperative cerebrovascular accident (P =.002), use of postoperative warfarin sodium (Coumadin, DuPont Merck; P =.006), arterial hypertension (P =.023), size of valve prosthesis of 27 mm or larger (P =.023), and hospital thromboembolism (P =.040). There was a trend toward increased fatal thromboembolism in patients without medication. With a multivariate analysis, 4 factors remained significant: preoperative cerebrovascular accident (risk ratio, 4.8; P =.0016), warfarin sodium (risk ratio, 3.0; P =.0028), preoperative endocarditis (risk ratio, 5.6; P =.006), and hospital thromboembolism (risk ratio, 6.1; P =.016). Hypertension had a borderline effect. Age, sex, diabetes, 4 coronary artery factors, 3 other valvular factors, atrial fibrillation, and carotid artery disease had no significant effect. CONCLUSIONS: Some emboli seemed triggered by the valve prosthesis. A proper anticoagulant protocol but also a treatment of hypertension is important in the prevention of thromboembolism after aortic valve replacement with a bioprosthesis. We did not find a significant role of atrial fibrillation and carotid artery disease.

Gastric emptying rate measurement after vertical banded gastroplasty.

BACKGROUND: In vertical banded gastroplasty (VBG), a small proximal gastric pouch is created, which is believed to fill rapidly and to empty slowly. METHODS: In 13 patients who underwent VBG, gastric emptying rate was measured. A radiolabelled solid test meal was used before and 2 weeks after operation. From a region of interest above the whole stomach, the proximal pouch and the distal stomach, half emptying time as well as retention percentage were derived. RESULTS: All patients experienced early satiety and gastric fullness after ingestion of a small test meal. The proximal pouch emptied rapidly. The evacuation of the test meal from the whole gastric region as well as the distal stomach were not altered significantly by the operation. CONCLUSION: VBG is a safe operation which reduced weight significantly. Early satiety, however, induced by this technique, cannot be explained alone by slow emptying of the proximal pouch. The nature of the outlet of the pouch as well as the behavior of its wall must be considered.

Primary cilia associated with striated rootlets in granulated and folliculo-stellate cells of the avian adenohypophysis.

During observation of the ultrastructure of adenohypophyses of normal and experimentally-manipulated quails, primary cilia were found in secretory cells as well as in non-granulated, folliculo-stellate cells of both cephalic and caudal lobes of the gland. These solitary cilia shared morphological characteristics with those observed in other cell types and species, i.e. they arose from a basal body, which basically had a centriolar structure and 9 doublets of microtubules and no central tubules in their axoneme. A 8 + 1 arrangement of microtubules was exceptionally observed. A 9 + 2 pattern, which is commonly described in motile cilia, was never found. The cilia extended in extracellular spaces between secretory cells, but not in the follicular cavities nor in the blood vessels. In addition to the basal body, a single centriole was frequently present in its vicinity. The basal body was often associated with a basal foot or satellite from which microtubules radiated, and with ladderlike structures corresponding to the classical description of striated rootlets. The presumptive roles of primary cilia in general, and of their morphological features in particular, are discussed in view of our results and compared to the several observations reported in mammalian adenohypophyses. As the evidence gained in favour of a given function of primary cilia has, so far, always been circumstantial, extreme caution in interpretation must be exercise.

Ultrastructural study of the adenohypophysis of the male Chinese quail.

The ultrastructural analysis of the adenohypophysis in the male Chinese quail reveals seven different types of granular cells, and agranular folliculo-stellate cells. The cell types are assumed to be endocrine cells and are classified as: Type I cells (presumptive LH-gonadotrophs), with dilated endoplasmic reticulum, perinuclear spaces, and granules of 150--260 nm; Type II cells (presumptive FSH-gonadotrophs), with regularly-shaped cytoplasmic cisterns and small granules (80--150 nm); Type III cells (presumptive thyrotrophs), very close in appearance to the type II cells of normal birds; Type IV cells (presumptive prolactin cells), with very large secretory granules (up to 400 nm), Type V cells (presumptive corticotrophs), with abundant and electron-dense granules (160--300 nm); Type VI cells, with irregularly-shaped granules; Type VII cells (presumptive somatotrophs), with abundant granules (130--220 nm) and less cytoplasmic structures. Cytological characteristics of the nucleus, and more particularly the presence of a Feulgen-postive nucleolus with a very particular ultrastructure are here reported. It is proposed that heterospecific associations of Chinese quail cells with chick cells can be used in embryological work for the study of cellular interactions.

Peripheral avian yolk assemblage and its persistence in the blastoderm, studied by trypan blue-induced fluorescence.

Shortly after subcutaneous or intraperitoneal injection of nontoxic quantities of trypan blue into laying Japanese quails, red fluorescent yolk granules appear in the peripheral ooplasm of their oocytes at the end of the lampbrush stage or subsequently. Later a red fluorescence can be observed in the apical cytoplasm of the granulosa cells. The results obtained by this method confirm our previous results (Callebaut 1979) obtained by autoradiography after 3H-leucine administration and furnish interesting additional data. The trypan blue-induced fluorescence method gives a good indication of the permeability of the oocytal cortex and its derivative the germinal disc. The avian yolk which is, or has been peripherally assembled (primordial, true white and yellow yolk) can be characteristically labelled by the administration of trypan blue. The injection of higher, still nontoxic quantities of trypan blue has a prolonged "retarding" effect and permits the marking of a broader part of the germinal disc or eventually of the blastoderm which develops from it.

Determinants of survival after aortic valve replacement as treatment for symptomatic aortic valve disease in the elderly.

BACKGROUND AND AIM OF THE STUDY: The effect of concurrent disease and cardiac comorbidity on survival after bioprosthetic valve replacement in elderly patients was assessed retrospectively. Risk factors were categorized as general, non-cardiac (age, diabetes, previously treated carcinoma) and cardiac (LVEF, three-vessel disease, previous CABG or valve replacement, and endocarditis). METHODS: A total of 400 elderly patients (median age 73 years; range: 71-76 years) was studied. Medical history included diabetes, previous CABG or aortic valve replacement (AVR), endocarditis and treatment of previous carcinoma. A left ventricular ejection fraction (LVEF) of <0.66 and presence of three-vessel disease were also investigated. Hospital deaths (and cause) were recorded; survival or date and cause of death after discharge were obtained by questionnaire. Kaplan-Meier univariate and Cox proportional hazards multivariate regression analyses were carried out. RESULTS: Mortality during follow up was 28.3%; hospital mortality was 3.8%. Univariate analysis showed five factors significantly to affect survival: LVEF, history of endocarditis, carcinoma, age and three-vessel disease. Fifteen of 38 patients with history of carcinoma died, 10 due to a malignancy. Of 76 patients with three-vessel disease, 26 died. A history of diabetes and previous CABG did not influence survival significantly. Four of eight patients with preoperative endocarditis died, all in hospital. Six of 11 patients died after redo-AVR, none before 36 months follow up. By Cox regression analysis, LVEF and histories of carcinoma and endocarditis remained significant. CONCLUSION: AVR should be performed before ventricular deterioration occurs. Previous CABG is not a contraindication for AVR. Endocarditis impaired survival. Long-term mortality after redo-AVR in this population was relatively high, but acceptable. AVR should also be performed in elderly patients with aortic valve disease. Since prognosis of symptomatic aortic valve disease is poor in the short term, AVR is indicated in patients treated for carcinoma.

Distribution of apoptosis-related proteins in the quail ovary during folliculogenesis: BCL-2, BAX and CPP32.

It is suggested that follicular apoptosis is driven by the status of the BCL-2: BAX rheostat, and that CPP32 is a key effector of granulosa cell death. In the present study, we have immunohistochemically localized two BCL-2 family members, BCL-2 and BAX, and one caspase, CPP32, in the quail ovary during folliculogenesis. BCL-2 was predominantly found in the granulosa cells of developing follicles. BAX was detected in some follicular cells of atretic follicles, and in the nucleus of some prelampbrush oocytes. Expression of CPP32 was detected in leukocytes and in follicular cells of atretic follicles. Immunostaining was also found in interstitial cells, in surface epithelial and vascular endothelial cells, and in some thecal cells of post-ovulatory follicles. In the granulosa cells of non-growing and small prehierarchal follicles, a weak immunostaining was observed. We can conclude that in the avian ovary, BAX and CPP32 are involved in atresia. The present results support the BCL-2: BAX rheostat hypothesis.

Role of age, gender and association of CABG on long-term results after aortic valve replacement with a Carpentier-Edwards bioprosthesis in the elderly.

AIM: The problem of postoperative sudden death and the effect of age, gender and association of coronary artery bypass grafting were studied after implantation of a bioprosthetic valve in aortic position. DESIGN: retrospective investigation during 13 year. SETTING: general hospital. PATIENTS: 500 mostly symptomatic patients who received this bioprosthesis had a follow-up of 2,022 patient-year for 499 patients. INTERVENTION: aortic valve replacement with a Carpentier-Edwards pericardial prosthesis. MEASURES: hospital complications and mortality, long-term mortality with focus on sudden death and its risk factors, valve related complications and other cardiac events. RESULTS: Within the hospital: atrial fibrillation was the most frequent complication, for which only gender had an effect (p=0.014). The most occurring valve related complications were thrombo-embolic events. Mortality was adversely affected by male gender (p=0.040). Long-term results: thrombo-embolic events were the most important valve related complications. These events and haemorrhage, endocarditis and reoperation rate were not affected by the association of a CABG. Univariate analysis in patients over 73 showed that the need for CABG significantly increased global mortality (p=0.0001), occurrence of cardiac fatality (p=0.0003) and congestive heart failure (p=0.0036). Non-valve related factors seemed most responsible for postoperative sudden death. CONCLUSION: Age, male gender and the association of a CABG remain important determinants for postoperative survival, but not for the occurrence of valve related complications. Sudden death seems not always related to the valve prosthesis, but is as such classified by convention.

Gastric emptying rate for solid and for liquid test meals in patients with dyspeptic symptoms after partial gastrectomy and after vagotomy followed by partial gastrectomy.

BACKGROUND/AIMS: Gastric emptying rate for solid and for liquid test meals was investigated retrospectively in patients with longstanding epigastric distress after partial gastrectomy, either as primary treatment or after failure of vagotomy for peptic ulcer in order to find an explanation for the postoperative symptoms. METHODOLOGY: Radionuclide-labeled liquid and solid test meals were used to evaluate gastric emptying rate, at least one year after surgery. RESULTS: The lag phase for liquid test meals disappeared in all operated patients. Partial gastrectomy usually lead to fast emptying but this resective procedure, if performed after vagotomy, lead to stasis in a significant number of patients. Gastric emptying rate for solids increased in only a few of these symptomatic patients. In most of them however, there was a normal to decreased emptying rate. If a vagotomy had preceded the resective procedure, gastric emptying rate decreased significantly. CONCLUSIONS: In all these symptomatic patients, gastric emptying had been disturbed for at least one type of test meal. This makes investigation for both meals necessary, especially since there is a lack of correlation. Furthermore, if vagotomy fails to prevent ulcer recurrence, one should carefully consider all options before performing partial gastrectomy since gastric emptying rate after these consecutive procedures worsens considerably.

The effect of stress on gastric emptying rate measured with a radionuclide tracer.

BACKGROUND/AIMS: Stress can delay gastric emptying rate. This phenomenon has not yet been investigated with a physiological solid test meal or a regional analysis. METHODOLOGY: We investigated the gastric emptying rate in beagle dogs using a radio-labeled solid test meal and a gamma camera. The transport to an unknown environment served as a stress stimulus. This offers the advantage to mimic fairly well the clinical situation just before a diagnostic or therapeutic procedure. Control values were obtained after the third investigation, in which all dogs were accustomed to the environment. RESULTS: Regional analysis of the emptying curves from the whole gastric region revealed that the length of the lag phase increased with stress, but the post-initial emptying rate remained unchanged. Emptying of the fundus changed hardly, but the antropyloral motor activity decreased during stress. CONCLUSIONS: Gastric emptying rate is impeded during stress. It is necessary to limit the unnatural stress-stimuli, in order to mimic the clinical situation. Measurement techniques that influence directly gastric emptying or act as a stress-stimulus by themselves are undesirable. A radionuclide solid test meal is preferable and a regional analysis reveals the impairment of the antral motility as the mechanism of the delay of gastric emptying.

Comparison of anterior and geometric mean liquid and solid gastric emptying rates in dogs and in patients after partial gastrectomy.

OBJECTIVE: The purpose of this study was to determine if geometrical correction is necessary in the study of gastric emptying rate (GER) for liquids, using a low-energy radionuclide, such as 99mTc. Solid test meals were considered the reference. METHODS: Both solid and liquid GERs were investigated using regional analysis. Anterior data were compared with geometrically corrected values in 15 dogs and 9 partial gastrectomy patients. RESULTS: Anterior and geometrically corrected measurements differed significantly for solid food in the whole gastric region and in the antrum. Geometrically corrected values differed slightly from anterior data after partial gastrectomy. No difference was found for liquid food. Liquids redistributed much faster than solids within the stomach. CONCLUSION: Measurement of GER using a single-phase liquid meal does not require geometrical correction. This is due to the rapid intragastric redistribution of the liquid. Geometrical correction for solid food can be omitted only after partial gastrectomy.

Effect of gravity on the interaction between the avian germ and neighbouring ooplasm in inverted egg yolk balls.

The developmental capacities of an avian germ (from before symmetrization to the moment of laying) are strongly diminished after inversion of its egg yolk ball followed by culture in egg white. Our present experiments show that even when the avian germ is completely horizontally inverted (without an upper or lower border) below its egg yolk ball before symmetrization, symmetrization and gastrulation phenomena take place. The germ grows slower and becomes smaller than after normal incubation. After culture of inverted unincubated germs, localized on freshly laid eggs, the closure of the neural tube is impaired and it remains open over a long distance. Although a primitive streak (PS) develops, mesoderm migration (mainly from the lateral part of the area pellucida) is also impaired. On sections through the germinal disc one can see the abnormal upward migration into the depth of the ooplasm and yolk of cells from the germ wall and the development of large cellular extensions encircling the yolk globules. Most prominent is the loss of contact between the superficial cell layers and the deep layer elements (junctional endoblast and yolk endoblast in the area opaca). Large areas without deep layer elements (even visible on surface micrographs) develop in the area vasculosa and area vitellina interna. The margin of overgrowth grows and extends normally over the egg yolk ball. An autoradiographic study after labelling of the yolk layers in inverted egg yolks reveals that mainly compression of the peripheral subgerminal and perigerminal ooplasm takes place. This suggests that the compression by the neighbouring yolk and upwards growth of cells are at the origin of the impaired development. After return to the normal upward orientation of the germ on the topmost part of the egg yolk ball, a more or less pronounced restoration to normal development takes place (depending on the duration of the inversion period and the age of the germ).

Localisation of epidermal growth factor receptor in the quail ovary.

The present study focuses on the distribution of EGFR in the follicular and stromal compartments of the Japanese quail (Coturnix coturnix japonica) ovary, using immunohistochemical methods and a histological ligand binding assay. EGFR was predominantly present in granulosa cells during each stage of the folliculogenesis. Furthermore, EGFR was also detected in thecal cells and in oocytes. In some cells of the vascular endothelium, of the ovarian smooth muscle, and of the surface epithelium, the presence of EGFR was detected as well. The presence of EGFR allows to formulate the hypothesis that EGFR-ligands are involved in the autocrine and/or paracrine regulation of oocyte maturation and of folliculogenesis in the quail, and possibly in all birds.

Presence and function of growth factors in the avian ovary.

It is now evident that a number of growth factors and cytokines are intimately involved in ovarian processes. This review focuses on these autocrine/paracrine modulators in terms of describing their presence and function in avian ovaries.

Smooth muscle cells in the peritubular tissue of the quail testis.

The aim of the present study was to reveal the presence of smooth muscle cells in the quail testis. We used an immunocytochemical and ultrastructural approach. The features of smooth muscle cells were present in the cells of the peritubular tissue and of the tunica albuginea. The peritubular cells formed a pseudo-stratified tissue. We suggest that the smooth muscle cells of the quail testis may be important in testicular contractile processes.