[Ciliogenesis in the mucous cells of the quail oviduct. I. Ultrastructural study in the laying quail]. / Ciliogenèse dans les cellules à mucus de l'oviducte de caille. I. Etude ultrastructurale chez la Caille en ponte

The luminal epithelium of the oviduct (magnum) of laying quails is composed of ciliated cells and mucous cells. Ciliogenesis was observed in some of the mucous cells. Both centrioles of the diplosome migrate to the top of the cell, and one of them induces the formation of a rudimentary cilium. In some of the other cells, that are filled with mucous granules, the formation of basal bodies by an acentriolar pathway was observed. In these cells, numerous, dense fibrous masses are associated with the forming face of the Golgi apparatus. In the Golgi zone, generative complexes composed of a deuterosome and some forming procentrioles were found. Cilia develop from completed basal bodies. During ciliogenesis, the Golgi apparatus is disorganized, and generally the production of mucous granules is arrested. The nucleus is also modified: it becomes larger and the chromatin is dispersed. It is assumed that mucous cells are able to be transformed into ciliated cells in the oviduct of laying quails.

[Ciliogenesis in the mucous cells of the quail oviduct. II. Hormonal control]. / Ciliogenèse dans les cellules à mucus de l'oviducte de caille. II. Contrôle hormonal

The hormonal control of ciliogenesis and transformation of mucous cells was studied in the oviduct (magnum) of ovariectomized quails. Estradiol benzoate induces ciliogenesis with doses varying from 10 mug/day to 100 mug/day after 6 days of treatment. With 100 mug/day, differentiation of some mucous cells is also induced as well as the formation of transitory "mixed cells" which are in the process of ciliogenesis and contain mucous granules. Associated with progesterone (1 mg/day), estradiol benzoate (10 mug/day) induces the differentiation of mucous cells and ciliated cells. The luminal epithelium of quails injected with this mixture is similar to the luminal epithelium observed in the oviduct of laying quails. With the same dose of progesterone (1 mg/day) and 20 mug/day of estradiol benzoate for 6 days, ciliogenesis is completely inhibited. All epithelial cells are secretory cells. Transformation of 50% of the mucous cells into ciliated cells is obtained by following the previous estradiol-progesterone treatment with the injection of estradiol benzoate (20 mug/day) for 3 days. Divisions of mucous cells were also observed. It is also possible to induce ciliogenesis in some mucous cells by withdrawing both hormones for 3 days. In this case, no cell divisions were observed.

Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody.

A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti-myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed.

Immunocytochemical localization of myosin during ciliogenesis of quail oviduct.

Myosin has been localized during ciliogenesis of quail oviduct by immunocytochemistry (immunofluorescence, immunoperoxidase, immunogold labeling) using a previously characterized monoclonal antibody. In ovariectomized quail oviduct many undifferentiated epithelial cells present a primary cilium arising from one of the diplosome centrioles. Myosin is associated with material located between the two centrioles. In contrast, in estrogen-stimulated quail oviduct, the material preceding the procentioles is never labeled. Basal bodies become labeled just before their migration toward the apical plasma membrane. During the anchoring phase, the labeling is mainly associated with the basal feet. In mature ciliated cells, myosin appears associated with an apical network embedding the basal bodies. This network is connected to a myosin-rich belt associated with the apical junctional complex which differentiates at the beginning of centriologenesis. The association of myosin with migrating basal bodies suggests that myosin could be involved in basal body movements.

Cytokeratin filament organization in the ciliated cells of the quail oviduct.

With the exception of keratinocytes and some types of cultured cells, ciliated cells appear to be the major cell type which contains the most developed cytokeratin meshwork. We report, here, on the intermediate filament (IF) organization in ciliated cells of the quail oviduct using ultrastructural and immunocytochemical techniques. Special attention was focused on the relationships between IF and other cell organelles. The meshwork of IFs appears as a subapical disk constituted of separate bundles mainly composed of interwoven 10-nm filaments. From this subapical region, a descending bundle connects the array of IFs occupying the basal part of the cell. The nucleus is maintained in a loose network of IFs. In ciliated cells there are no free centrioles, but IFs are related to centriolar appendages (striated rootlets).

Comparative study of the distribution of fuzzy coat, lectin receptors, and intramembrane particles of the ciliary membrane.

Cilia from seminal vesicles of Discoglossus (Anura, Amphibia) are partially covered with a fuzzy coat easily stained by the PATAg technique. The freeze-fractures of the ciliary membrane show large intramembrane particles (IMPs) not randomly distributed. At the base of the cilium, 3 or 4 rows of large IMPs form the ciliary necklace. Above the necklace, a 0.1 micrometer high region is devoid of particles after which the shaft membrane exhibits large IMPs coarsely lined up in 9 longitudinal rows 0.6 micrometer in height. Smaller IMPs appear randomly dispersed. The fuzzy coat is well developed in regions possessing large IMPs: the necklace region and the basal portion of the shaft. Between the necklace and the shaft, the region lacking in IMPs is also devoid of fuzzy coat. The distribution of Con A receptors is similar to that of large IMPs and to that of filaments in the fuzzy coat. On the contrary, wheat germ agglutinin (WGA) receptors are randomly distributed all along the ciliary membrane. Finally, the relationships between fuzzy coat, lectin receptors, IMPs and fibrillar links between plasma membrane and microtubules of the axoneme are illustrated.

The effect of hypophysectomy on the quail's oviduct response to low and high doses of estradiol benzoate.

The response of the quail's (Coturnix coturnix Japonica) oviduct to different doses of estradiol benzoate (EB) was explored in ovariectomized and ovariectomized-hypophysectomized animals. Doses of EB ranged from 10 microgram to 1 mg/animal daily for 6 consecutive days; animals were sacrificed 24 h after the last injection. Controls were injected with olive oil. Parameters thought to reflect stimulation of the oviduct were 1) wet and dry weight, 2) DNA, 3) RNA, 4) soluble proteins, 5) ovalbumin, and 6) morphological changes at the level of magnum's mucosa. A statistical significant difference in several of the parameters listed was found between the effect of different doses of EB and whether the injection was done in ovariectomized or in ovariectomized-hypophysectomized animals. The dose response with EB for cell proliferation and induced proteins accumulation (ovalbumin) show that it takes considerably more EB to maximally stimulate ovalbumin accumulation when compared with cell proliferation. Hypophysectomy decreases the oviduct responses to high EB doses. Ultrastructural studies of the magnum mucosa confirm these biochemical results. The evidence presented suggesting an EB dose-dependent difference induction of specific cellular responses in the presence and absence of the pituitary in quails are the basis to postulate the contributing role of the pituitary in the parameters measured.

Effect of estrogen on adenosine 3'5',cyclic monophosphate in quail oviduct: possible involvement in estradiol-activated growth.

Previous studies have shown that estradiol indirectly stimulated the proliferation of oviduct epithelial cells in the quail. The present study was undertaken to investigate the effects of estradiol and other steroid hormones on the cAMP concentration. The ability of forskolin, a specific activator of the catalytic subunit of adenylate cyclase, to induce oviduct cell proliferation and specific protein synthesis (progesterone receptor) in the absence of estrogen was also tested. Administration of estradiol benzoate (EB) to immature female quails produced a transient surge in oviduct cAMP concentration. After EB injection, cAMP concentration increased by 36.7% after 6 h and returned to control values after 12 h. This rise in oviduct cAMP concentration preceded the beginning of DNA synthesis. The same effect was observed even in the absence of increased plasma estradiol, when the hormone was perfused through the hepatic portal vein. Estriol, estrone, and testosterone failed to elevate cAMP concentrations. After repeated EB injections, the oviduct cAMP concentration declined below the control value (-66% after 72 h). A similar drop in the cAMP concentration was observed in developing quails during the proliferative phase of the luminal epithelial and glandular cells. Administration of forskolin to immature female quail pretreated with EB rapidly increased the oviduct cAMP concentration, induced a burst of DNA synthesis, and shortened the prereplicative period. In addition, forskolin administration did not increase the progesterone receptor concentration. These results demonstrate that cAMP is involved in the mechanism by which estradiol indirectly stimulates oviduct epithelial cell proliferation in the quail. The events that may take place during the prereplicative period and the antiproliferative effect of progesterone through a sustained increase in the cAMP concentration in the oviduct are discussed.

Immunolocalization of laminin during estrogen-induced differentiation of quail oviduct epithelial cells.

During estrogen-induced development of the quail oviduct, tubular glands are formed by evagination of epithelial cells into the stroma. The distribution of laminin was studied during the early stages by means of immunofluorescence and immunoperoxidase techniques. Ultrastructural changes in the basal lamina were studied by electron microscopy. Basement membranes at all stages of development were delineated with 3 polyclonal antilaminin antisera. However, in ovariectomized birds, laminin could not be detected by one of the polyclonal antilaminin antisera. Subsequently, this antibody detected laminin as epithelial cell evaginations were induced by estradiol benzoate. The heavy and light chains of Engelbreth Holm sarcoma (EHS) laminin were revealed in immunoblotting by all antibodies. By electron microscopy after the immunoperoxidase technique with antilaminin antisera laminin appears to be accumulated mainly in the lamina densa. Furthermore, the thickness of the basal lamina increases during oviduct development. These data indicate that basal lamina organization is modified during oviduct cell differentiation and that immunoreactivity of epithelial basement membrane laminin changes during development.

[The shell of the quail's egg: ultrastructural and crystallographic study (author's transl)]. / Coquille de l'oeuf de caille: étude ultrastructurale et cristallographique.

The egg-shell of Japanese quail was studied by several techniques. Semithin sections (1 micron thick) of non-decalcified shell were observed by normal and polarized light microscopy. Thin sections of non-decalcified shell, examined by transmission electron microscopy, permitted us to observe the forms and dimensions of crystals of calcite within different layers of the shell: mammilary layer, layer of cones, palissade layer and surface crystal layer. There appears to be two distinct zones in the layer of cones as well as in the superficial crystal layer. Electron microdiffraction revealed the orientation of calcite crystals in the columns. Some crystal defects (twins?) were described and the possibility of their artefactual formation during ultramicrotomy is discussed. Localization of Ca, Mg, P and S were made by X-ray microanalysis of semithin sections. This technique shows that shell membranes, and chiefly the true cuticle, are also mineralized but, in these layers, minerals are not crystallized. Otherwise the distribution of Mg is not uniform throughout the shell thickness; it is less concentrated in the external zone of the layer of cones. These results together with observation of developing shells by scanning electron microscopy allowed us to propose a scheme for shell organization of the quail egg. This organization was related with decalcification which occurs during hatching.

Evolutionary conservation of an epitope associated with striated rootlets in different epithelial ciliated cells.

In ciliated cells of metazoa, striated rootlets associated with basal bodies anchor the ciliary apparatus to the cytoskeleton. We have used here a monoclonal antibody against a 175 kDa protein associated with the striated rootlets of quail ciliated cells, to study ciliated cells of different species. In mussel gill epithelium the antibody recognized a protein of 92 kDa which shows a periodic distribution along the striated rootlets. In frog ciliated palate epithelium, two different rootlets are associated with basal bodies, both are decorated and only one protein of 48 kDa is recognized on immunoblot. The antigen is arranged in a helix around the striated rootlets. In rabbit ciliated oviduct epithelium, we detected the presence of very small and thin rootlets which are weakly labeled. We have shown that an epitope associated with the striated rootlets is preserved through evolution although the molecular weight of the peptide varies. We have also observed the appearance of this epitope on protein associated with junctional complexes in rabbit and cytoskeleton component in quail oviduct.

Determination of ciliary polarity precedes differentiation in the epithelial cells of quail oviduct.

In quail oviduct epithelium, as in all metazoan and protozoan ciliated cells, cilia beat in a coordinated cycle. They are arranged in a polarized pattern oriented according to the anteroposterior axis of the oviduct and are most likely responsible for transport of the ovum and egg white proteins from the infundibulum toward the uterus. Orientation of ciliary beating is related to that of the basal bodies, indicated by the location of the lateral basal foot, which points in the direction of the active stroke of ciliary beating. This arrangement of the ciliary cortex occurs as the ultimate step in ciliogenesis and following the oviduct development. Cilia first develop in a random orientation and reorient later, simultaneously with the development of the cortical cytoskeleton. In order to know when the final orientation of basal bodies and cilia is determined in the course of oviduct development, microsurgical reversal of a segment of the immature oviduct was performed. Then, after hormone-induced development and ciliogenesis, ciliary orientation was examined in the inverted segment and in normal parts of the ciliated epithelium. In the inverted segment, orientation was reversed, as shown by a video recording of the direction of effective flow produced by beating cilia, by the three-dimensional bending forms of cilia immobilized during the beating cycle and screened by scanning electron microscopy, and by the position of basal body appendages as seen in thin sections by transmission electron microscopy. These results demonstrate that basal body and ciliary orientation are irreversibly determined prior to development by an endogenous signal present early in the cells of the immature oviduct, transmitted to daughter cells during the proliferative phase and expressed at the end of ciliogenesis.

Relationships between cytokeratin filaments and centriolar derivatives during ciliogenesis in the quail oviduct.

In the quail oviduct, the mature ciliated cells contain a well developed and polarized cytokeratin network which is bound to desmosomes and in close contact with the striated rootlets associated with basal bodies. In ovariectomized quail, the immature epithelial cells of oviduct present a rudimentary cytokeratin network associated with the centrioles of the diplosome (one of them forming a primary cilium) and with the short striated rootlets. The development of the cytokeratin network which occurs simultaneously with the ciliogenesis was observed by electron microscopy and immunocytochemistry (immunofluorescence and immunogold staining) using a prekeratin antiserum. During estrogen-induced ciliogenesis, cytokeratin intermediate filaments are always found associated with the different ciliogenic structures i.e. [dense granules, deuterosomes, procentrioles and centrioles]. In ciliogenic cells, the procentrioles and centrioles seem to be associated with the intermediate filaments by their pericentriolar material. These direct contacts decrease once the centrioles/basal bodies are anchored to the plasma membrane. Simultaneously the striated rootlets develop and associate with cytokeratin. The ciliogenic cells appear as a suitable system for studying in vivo, the possible association between centrioles and intermediate filaments and its functional meaning.

In vitro effects of benzodiazepines on ciliogenesis in the quail oviduct.

Immature oviduct implants from quails stimulated by estrogen to induce ciliogenesis were submitted to the in vitro action of benzodiazepines in organotypic culture. Diazepam and medazepam were added to the culture medium for 24 or 48 hours and tissues were examined by transmission and scanning electron microscopy for alterations in ciliary differentiation. Ciliogenesis was inhibited by both diazepam and medazepam, which affected mainly the migration of the basal bodies. Assembly of basal bodies was achieved normally in the cytoplasm, but their separation from generative complexes and migration toward the apical membrane were prevented. They remained in clusters around a deuterosome or eventually anchored to the close lateral plasma membrane. Furthermore, the drugs affected mature beating cilia, which then appeared lying tangentially to the cell surface. Relation between basal bodies and cortical cytoskeleton seemed to be altered by the drugs, which implies that the bearing of cilia and probably the ciliary beating movement were modified. Microvillus development was also altered by the action of these drugs.

Immunological detection of actin in isolated cilia from quail oviduct.

Cilia from quail oviduct were isolated with their membrane. The ultrastructural study revealed a good preservation of cilia in the purified fraction. Electrophoresis on SDS-PAGE showed a reproducible pattern of ciliary proteins, the major bands being those of tubulins 57 kDa and dyneins above 250 kDa. Among the minor bands, an immunological study was focused on a 43 kDa molecular mass protein, using monospecific antibodies against actin. Presence of actin was then detected by immunoblotting of isolated cilia fractions as well as of demembranated cilia, suggesting that actin is associated with the axoneme. The presence of actin in the cilia was confirmed by immunofluorescence. The cilia were found stained only on the proximal part, suggesting an heterogeneous distribution of actin within the axonemal length.

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium.

The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.

The orientation of ciliary basal bodies in quail oviduct is related to the ciliary beating cycle commencement.

In mature ciliated cells, the basal feet associated to the basal bodies point out in the direction of the effective stroke of the ciliary beating. In contrast, during ciliogenesis, the basal feet of the newly anchored basal bodies are randomly oriented. The reorientation of basal bodies occurs during the beginning of the coordinated beating cycle of the cilia.

In vitro effects of taxol on ciliogenesis in quail oviduct.

When induced by in vivo oestrogen stimulation, ciliogenesis continues in culture in vitro of quail oviduct implants. Ultrastructure of ciliogenic cells was compared after culture for 24 or 48 h in the presence or absence of 10(-5) M-taxol. Taxol, which promotes polymerization and stabilization of microtubules, disturbed ciliogenesis, but formation of basal bodies was unaffected by the drug. Conversely, their migration towards the apical surface seemed to be slowed down or blocked and axonemal doublets polymerized onto the distal end of cytoplasmic basal bodies. They elongated and often constituted a more or less complete axoneme, extending between organelles in various orientations. These axonemes, often abnormal, were not surrounded by a membrane, with the exception of the transitional or neck region between the basal body and axoneme. The formation of membrane in this area resulted from the binding of some vesicles to the anchoring fibres of the basal body. They fused in various numbers, occasionally forming a ring, at the site of the transitional region, and exhibited the characteristics of the ciliary necklace. The association of basal bodies with vesicles or with the plasma membrane appeared to be a necessary signal for in situ polymerization of axonemal doublets. In addition, taxol induced polymerization of numerous microtubules in the cytoplasm, especially in the apical part of the cell and in the Golgi area. This network of microtubules may prevent basal body migration.

In vitro effects of colchicine and nocodazole on ciliogenesis in quail oviduct.

Oviduct implants from quails which were primarily stimulated in vivo by estrogen so as to induce ciliogenesis in some epithelial cells were cultured in vitro in the presence or absence of colchicine or nocodazole. After 24 or 48 hr of culture, implants were examined by transmission and scanning electron microscopy to determine drug-induced alterations in ciliogenesis. After 24 hr of 10(-5) M colchicine treatment, the formation of basal bodies was totally inhibited, though the precursor material of generative complexes was unchanged. The inhibitory effect was not reversed when colchicine was removed in a 24 hr recovery culture. Treatment with 10(-6) M nocodazole for 24 hr, partially inhibited the assembly of basal bodies, which exhibited altered morphology. The assembly of basal bodies was restored during the 24 hr recovery period, after removal of nocodazole. Colchicine and nocodazole did not prevent polarized migration towards the apical surface of basal bodies formed prior to drug treatment. They anchored to the plasma membrane, but the formation of cilia was strongly disturbed in the presence of the drug. Numerous cells possessed anchored basal bodies which failed to induce the formation of cilia. The elongation of cilia was inhibited, as seen by their abnormal capping structure. In the enlarged tip, microtubules diverged. In contrast, these very short cilia possessed a mature ciliary necklace which was constructed during drug treatment. Differentiation of this membrane ciliary structure appeared to be unrelated to axoneme growth.