Marc Callebaut

Immunohistochemical localization of S-100 protein, glial fibrillary acidic protein, and neuron-specific enolase in the pars distalis of quail, rat, and human hypophyses

SummaryIn the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.

Plain water as a rinsing agent preferable to sulfurous acid after the Feulgen nucleal reaction.

After staining for the Feulgen nucleal reaction with Schiffs reagent, slides were immediately submerged in running distilled or tap water and washed for 30 sec or longer. Rapid and complete removal of residual Schiffs reagent from the stained tissue will give preparations which show all details characterizing the nucleal reaction, and which are more durable in storage than those processed with the customary washing in a solution of SO2. Care must be taken to insure that all parts of the slides are thoroughly washed and that, on the surface of the sections, no spilled adhesive or other interfering coating retards the washing. Standardization of the procedure for quantitative DNA determination may be facilitated by this modification.

The avian ovary is an open organ

SummaryAfter an intraperitoneal injection of diluted yolk in female quails, young chicks or chick embryos (killed with minimal loss of blood), the absorption of a considerable quantity of yolk granules by the ovarian medullary lacunae can be demonstrated. This can be confirmed by the i.p. injection of killed Hela cells. From our investigation it may be concluded that the avian ovary, by the progressive expansion of its medullary lacunae, develops into a contractile sac-like structure with small openings (communicating with the peritoneal cavity) on its dorsal side. The lacunar system seems to function as an expansion room for the enlarging follicles (also the large pediculated follicles) and intervenes in follicle stalk formation. The relatively rapid uptake by means of an ovarian pumping mechanism of particles and fluid from the peritoneal cavity into the lacunae, suggests also that the latter play a role as diffusion chambers in the supply of nutritious substances to and elimination of wastes from the follicles.

Origin, Fate, and Function of the Components of the Avian Germ Disc Region and Early Blastoderm: Role of Ooplasmic Determinants

In the avian oocytal germ disc region, at the end of oogenesis, we discerned four ooplasms (α, β, γ, δ) presenting an onion‐peel distribution (from peripheral and superficial to central and deep. Their fate was followed during early embryonic development. The most superficial and peripheral α ooplasm plays a fundamental role during cleavage. The β ooplasm, originally localized in the peripheral region of the blastodisc, becomes mainly concentrated in the primitive streak. At the moment of bilateral symmetrization, a spatially oblique, sickle‐shaped uptake of γ and δ ooplasms occurs so that γ and δ ooplasms become incorporated into the deeper part of the avian blastoderm. These ooplasms seem to contain ooplasmic determinants that initiate either early neurulation or gastrulation events. The early neural plate‐inducing structure that forms a deep part of the blastoderm is the δ ooplasm‐containing endophyll (primary hypoblast). Together with the primordial germ cells, it is derived from the superficial centrocaudal part of the nucleus of Pander, which also contains δ ooplasm. The other structure (γ ooplasm) that is incorporated into the caudolateral deep part of the blastoderm forms Raubers sickle. It induces gastrulation in the concavity of Raubers sickle and blood island formation exterior to Raubers sickle. Raubers sickle develops by ingrowth of blastodermal cells into the γ ooplasm, which surrounds the nucleus of Pander. Raubers sickle constitutes the primary major organizer of the avian blastoderm and generates only extraembryonic tissues (junctional and sickle endoblast). By imparting positional information, it organizes and dominates the whole blastoderm (controlling gastrulation, neurulation, and coelom and cardiovascular system formation). Fragments of the horns of Raubers sickle extend far cranially into the lateral quadrants of the unincubated blastoderm, so that often Raubers sickle material forms three quarters of a circle. This finding explains the regulative capacities of isolated blastoderm parts, with the exception of the anti‐sickle region and central blastoderm region, where no Raubers sickle material is present. In avian blastoderms, there exists a competitive inhibition by Raubers sickle on the primitive streak and neural plate‐inducing effects of sickle endoblast. Avian primordial germ cells contain δ ooplasm derived from the superficial part of the nucleus of Pander. Their original deep and central ooplasmic localization has been confirmed by the use of a chicken vasa homologue. We conclude that the unincubated blastoderm consists of three elementary tissues: upper layer mainly containing β ooplasm, endophyll containing δ ooplasm, and Raubers sickle containing γ ooplasm). These elementary tissues form before the three classic germ layers have developed. Developmental Dynamics 233:1194–1216, 2005.

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail

SummaryIt was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induced by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhains fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5×10−3 mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured.It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.

Immunohistochemical localization of desmin in the quail ovary

SummaryWe have localized desmin in the quail ovary, by the unlabelled antibody peroxidase-antiperoxidase technique, using two monoclonal and one polyclonal antisera. Special attention has been paid to the influence of fixation and of proteolytic pretreatment of sections. It appeared that the immunostaining of desmin largely depends on the nature of the fixative. Carnoy fluid, Bouins fixative, and a paraformaldehyde-acetic acid fixative preserved the histological structure very efficiently. However, trypsin pretreatment proved to be necessary to unmask the antigenic sites in the ovaries fixed in Bouins fixative and the paraformaldehyde-acetic acid fixative. Desmin immunoreactivity was detected in the tunica albuginea and the chordae, a number of which surrounding the blood vessels, from the hilus to the thecal surface of the follicles. Small branches of chordae connected them with the tunica albuginea, forming a suspensory apparatus. Desmin was also localized in the smooth-muscle cells of the blood vessels. In the theca, immunoreactivity was detected in the wall of arterioles, of venules, and of capillaries. Further experimental and immunohistochemical research have to be performed to establish if the suspensory apparatus is a myoid tissue.

Time-lapse cinephotomicrography, videography, and videomicrography of the avian blastoderm.

Publisher Summary This chapter discusses the time-lapse cinephotomicrography, videography, and videomicrography of the avian blastoderm. Morphogenetic movements can be revealed by time-lapse cinephotomicrography or videography. Time-lapse cinephotomicrography or videography is now in widespread use in research and is an essential tool in the modern teaching of developmental biology. The goal of the research is to understand the mechanisms and dynamics of morphogenetic movements during gastrulation and neurulation. For the study described in the chapter, the setups for time-lapse videography and videomicrography were developed. Videography has superseded cinephotomicrography because sequences are copied faster, tape does not break as easy as film and does not scratch, video sequences can be observed forward and backward and as still images, and editing video sequences is less time-consuming. Video registrations have led to the new concept of mitotic pressure as a major mechanism of morphogenetic movements during gastrulation and neurulation. Video registration is a basic technique in the study of developmental biology as only video registration allows morphogenetic movements to be observed directly. It allows access to the fourth dimension: the evolution of shape in time, as morphogenesis may be called.

Localization and transport of lipids in the avian ovarian follicular layers and the structural relationship of theca and granulosa to the basement-membrane

We describe the localization of lipids in the wall and superficial ooplasm of the largest avian ovarian follicles by the use of different fixatives and light and electron microscopy. We demonstrate that each yolk globule is always accompanied by one or more highly osmiophilic and sudanophilic alcohol insoluble yolk masses, which we have called satellite yolk. Together with the protein containing yolk globule it forms an integral morphological part of a compartmentalized, bipartite yolk system. Cytochemical, histoautoradiographic, biochemical, and light and electron microscopical aspects of satellite yolk were studied. At the start of satellite yolk formation in the 3–4 mm diameter follicle (when the oocyte begins to yellow) the distribution of the microcirculation of the follicle wall becomes printed on the underlying superficial ooplasm of the oocyte. The oocyte then presents so‐called yolk mountains (containing satellite yolk), only localized below the thecal capillary sinus and not below the efferent and radially perforating thecal veins (black hole regions). We also describe the structural continuity between the thecal intercellular spaces and the microvilli‐associated extracellular spaces of the granulosa cells via the basement membrane. The thecal cells present centripetal extensions into the basement membrane and the basement membrane material extends centripetally into the granulosa microvillar channels. Therefore, at least two cellular barriers are crossed when fat or fat precursors are transported from the thecal capillary sinus to the ooplasm.

Immunohistochemical localization of transforming growth factor-β1 and β2 during folliculogenesis in the quail ovary

SummaryImmunohistochemical methods were used to show the presence and distribution of transforming growth factor-β1 and β2 during folliculogenesis in quail ovarian tissues. The results indicated that both transforming growth factor-β subtypes are present. Immunolabelling for transforming growth factor-β1 demonstrated that prelampbrush oocytes are immunoreactive in the Balbiani complex, and developing and pre-ovulatory oocytes in the zona radiata. Immunolabelling was also associated with granulosa cells. The number of stained granulosa cells decreased during folliculogenesis. In the pre-ovulatory follicles, immunolabelling was found predominantly in the theca interna. Immunolabelling for transforming growth factor-β2 was associated with the zona radiata of developing and preovulatory follicles, and with stromal interstitial cells. Moderate immunoreactivity was found in the Balbiani complex of prelampbrush oocytes. Weak immunolabelling was localized in the granulosa cells of prelampbrush follicles, and in a few cells of the theca interna of pre-ovulatory follicles. The immunolocalization of transforming growth factor-β1 and-β2 in the quail ovary supports their autocrine and/or paracrine role in avian ovarian processes.

IMMUNOHISTOCHEMICAL LOCALIZATION OF EPIDERMAL GROWTH FACTOR IN THE OVARY OF THE ADULT JAPANESE QUAIL

SummaryThe present study focuses on the immunohistochemical localization of epidermal growth factor in the ovary of the adult Japanese quail. Immunoreactivity was predominantly found in the smooth muscle cells of blood vessels and of chordae, in granulosa cells of pre-lampbrush follicles, in interstitial cells, in the Balbiani complex of pre-lampbrush oocytes, and in ganglia. In developing follicles, immunoreactivity was also detected in some granulosa and thecal cells, in the zona radiata, and especially in cell clusters localized in the thecal periphery. The number of immunostained cells in the granulosa decreased during folliculogenesis, and increased after ovulation. In the ooplasm of oocytes, immunoreactivity was shifted from the Balbiani complex to the zona radiata during development. These observations support the hypothesis that epidermal growth factor acts primarily on less differentiated follicles. It is also suggested that epidermal growth factor can modulate ovarian contractility. Finally, in one ovary, we detected immunostained bodies in the ooplasm of small developing oocytes.