Publisher Summary This chapter discusses the electron microscopic morphology of oogenesis. The nucleus plays a very important role in regulation of cell life processes. In accordance with the theory that the nucleolus controls the synthesis of cytoplasmic RNA, it is noted in studies of oocyte nuclei that the nucleolus often shows signs of emitting granules into the nucleoplasm. In vitro nucleolar emission is observed in oocytes of the roach and this finding was correlated with a reticulate rim of the nucleolus as seen in the electron microscope. Two processes visibly affect the nuclear envelope during oogenesis. One is concerned with the partial or complete perforation of the envelope, possibly facilitating the passage of nuclear material to the cytoplasm; the other is the enlargement of the nuclear membrane area as well as its area relative to nuclear volume. 2. The literature on oogenesis makes little mention of smooth endoplasmic reticulum. It is generally accepted that this type of cytomembrane plays only a limited role in oocytes. The controversy in interpretation of Golgi structure and function may be equally applied to oogenetical studies. Electron microscopy has so far been the most reliable means for studying Golgi material.

Electron microscopic morphology of oogenesis.

The subcommissural organ (SCO) is a phylogenetically ancient and conserved structure. During ontogeny, it is one of the first brain structures to differentiate. In many species, including the human, it reaches its full development during embryonic life. The SCO is a glandular structure formed by ependymal and hypendymal cells highly specialized in the secretion of proteins. It is located at the entrance of the aqueduct of Sylvius. The ependymal cells secrete into the ventricle core-glycosylated proteins of high molecular mass. The bulk of this secretion is formed by glycoproteins that would derive from two different precursors of 540 and 320 kDa and that, upon release into the ventricle aggregate, form a threadlike structure known as Reissner's fiber (RF). By addition of newly released glycoproteins to its proximal end, RF grows caudally and extends along the aqueduct, fourth ventricle, and the whole length of the central canal of the spinal cord. RF material continuously arrives at the dilated caudal end of the central canal, known as the terminal ventricle or ampulla. When reaching the ampulla, the RF material undergoes chemical modifications, disaggregates, and then escapes through openings in the dorsal wall of the ampulla to finally reach local blood vessels. The SCO also appears to secrete a cerebrospinal fluid (CSF)-soluble material that is different from the RF material that circulates in the ventricular and subarachnoidal CSF. Cell processes of the ependymal and hypendymal cells, containing a secretory material, terminate at the subarachnoidal space and on the very special blood capillaries supplying the SCO. The SCO is sequestered within a double-barrier system, a blood-brain barrier, and a CSF-SCO barrier. The function of the SCO is unknown. Some evidence suggests that the SCO may participate in different processes such as the clearance of certain compounds from the CSF, the circulation of CSF, and morphogenetic mechanisms.

The subcommissural organ.

The subcommissural organs (SCO) of 76 specimens belonging to 25 vertebrate species (amphibians, reptiles, birds, mammals) were studied by use of the immunoperoxidase procedure. The primary antiserum was obtained by immunizing rabbits with bovine Reissner's fiber (RF) extracted in a medium containing EDTA, DTT and urea. Antiserum against an aqueous extract of RF was also produced. The presence of immunoreactive material in cell processes and endings was regarded as an indication of a possible route of passage. Special attention was paid to the relative development of the ventricular, leptomeningeal and vascular pathways established by immunoreactive structures.

Comparative immunocytochemical study of the subcommissural organ.

Publisher Summary This chapter highlights the cell biology of the subcommissural organ (SCO) and discusses its structure–function relationships to provide a basis for further experimental work. SCO is a complex of nonneuronal secretory cells covering and penetrating the posterior commissure. This complex protrudes toward the third ventricle, occupies the posterior portion of the diencephalic roof caudal to the pineal organ, and marks the entrance to the Sylvian aqueduct. SCO is formed by two populations of secretory cells, which, in many species, are arranged into two distinct layers: the ependyma and the hypendyma. The bulk of the secretory products of the SCO, apparently a complex containing different glycoproteins, are released into the ventricular cerebrospinal fluid (CSF). In the ventricle, the secretion is condensed into a thread-like structure, Reissner's fiber (RF) that terminates in the ampulla caudalis of the central canal. The blood vessels of the SCO represent a highly specialized vascular structure within the CNS. The SCO is sequestered within a double-barrier system of unknown functional significance, thereby indicating the unique character of this circumventricular organ.

Cell biology of the subcommissural organ.

1.

Die Zona radiata der Eizelle der Frosche und Schwanzlurche ist gleichartig aufgebaut. Eine extrazellulare Grundsubstanz enthalt zwei zellulare Anteile: Mikrovilli der Eizelle und Cytoplasmafortsatze der Follikelzellen. Die Zona radiata ist in zwei Schichten geteilt, die sich histochemisch und bei den Urodelen auch elektronenmikroskopisch unterscheiden lassen. Bei jungen Oocyten ist die Oberflache an bestimmten Stellen mit dem Follikelepithel durch Desmosomen verbunden. Zwischen diesen flachenhaften Anheftungen entsteht die erste Grundsubstanz. Sie wird als ein Produkt des Eies, aber auch der Follikelzelle aufgefast.




2.

Die Feinstruktur des Melanin-Pigmentes ist je nach Tierart verschieden. Jedes Pigmentgranulum ist von einer Membran umgeben. Das Internum kann aus Einzelgranula bestehen (Triton), einen homogenen Aufbau zeigen oder Innenstrukturen aufweisen (Xenopus). Die Pigmentgenese wird als Funktion des „vakuolaren Apparates“ der Zelle erklart. In Blaschen dieses Apparates werden Stoffe abgelagert, die zusammen mit der Melanin-Komponente das Pigmentkorn bilden. Das Melanin-Pigment ist eine hochorganisierte Struktur, die mit einem Komplex-Cytosom vergleichbar ist. Die ubrigen Pigmententstehungstheorien lassen sich mit der von uns entwickelten vereinbaren und erklaren.




3.

Das Dotterplattchen setzt sich aus mehreren Teilen zusammen, wobei das Zentrum ein kristalloides Gefuge von hohem Ordnungsgrad ist. Dieser Biokristall besteht aus parallel orientierten Micellen. Je nach Schnittrichtung durch dieses Gefuge resultieren unterschiedliche Strukturbilder. Dem kristallinen Internum folgt eine ebenfalls dichte, aber granulierte Schale, die nach Behandlung mit Phosphorwolframsaure an Kontrast zunimmt und sich basophil gegenuber dem acidophilen Internum verhalt. Zwischen dem dichten Anteil und der umgebenden Membran liegt eine lockere Zone mit Granula und feinen Blaschen.




4.

Die Dotterbildung wurde als ein Prozes erkannt, an dem fast samtliche Teile der Zelle aktiv eingesetzt sind. Das Material, das den Dottervorstufen zugefuhrt wird, ist zuerst in Cytoplasma-Vesikeln elektronenmikroskopisch sichtbar. Ein Teil der Blaschen entsteht durch Pinocytose an der Basis der Mikrovilli, ein anderer Teil vermutlich im Bereich der Golgi-Lamellen. In der Dottervorstufe fliest das gesamte Material zusammen: Sie ist ein groses Cytosom (Komplex-Cytosom). Innerhalb des Komplex-Cytosoms kommt es zur Organisation des Materials.




5.

Der Dotterkern besteht aus dicht gelagerten Mitochondrien. In den Mitochondrien entstehen Stoffe. Wir nehmen an, das Teile als Cytosomen abgeschnurt und den Dottervorstufen zugefuhrt werden.




6.

Der Begriff „vakuolarer Apparat“, der sich in seiner Konzeption als sehr nutzlich fur die Erklarung der Pigment- und Dotterbildung erwiesen hat, wird in Anlehnung an die Vorstellungen und Untersuchungen von Schmidt (1961) besprochen. Er beinhaltet die Summe aller Blaschen, Cytosomen und Komplex Cytosomen des Cytoplasmas. Diesen Gebilden soll eine Aufgabe bei der Aufnahme, Anreicherung, Verarbeitung und Abgabe von Stoffen zukommen.




7.

Als Ursache fur die Cytoplasma-Basophilie wurden im elektronenmikroskopischen Bereich freie Ribosomen und 100 mμ grose Blaschen mit granularem Inhalt erkannt. Diese Blaschen durften mit den Chromidien der alteren Autoren identisch sein. Sie stammen vom Kern und uben eine in den Einzelheiten nicht zu bestimmende Funktion bei der Vitellogenese aus.




8.

Die Kern-Plasma-Grenze besteht aus zwei Membranen, von denen die innere haufig dicker als die ausere ist und in denen sich in regelmasiger und dichter Anordnung Poren von etwa 500 A Lichtungsdurchmesser befinden. Im Bereich der Poren last sich bei OsO4-Fixation (nicht nach KMnO4-Fixation) durchtretendes Kernmaterial nachweisen.




9.

Durch histochemische Methoden lassen sich wahrend der Dotterbildung zwei Nucleolen-Typen ermitteln. Eine kleine Form, die ahnlich dem Nucleolus vor Beginn der Dotterbildung keine eindeutig positive Reaktion zeigt und ein groser, meist peripher im Kern liegender Typ, der sich stark mit Pyronin und Gallocyanin-Chromalaun anfarbt und auf NH2- und SH-Gruppen-Nachweisen positiv reagiert. Elektronenmikroskopisch ist mit Beginn der Dotterbildung ein deutlicher Wechsel in der Struktur des Nucleolus sichtbar. Dieser Wechsel lauft parallel mit der Zunahme der nachweisbaren RNS und locker gebauter Bezirke. Weitere dichte Bezirke enthalten vorwiegend fibrillares Material. Sie bilden vor der Dotterbildung den Hauptteil des Nucleolus. Der Begriff Nucleolonema wird verworfen.

Electron microscopic and histochemical studies on the oogenesis of amphibia egg cells

The secretion of the subcommissural organ (SCO) has been studied immunocytochemically by use of the following antisera: (1) antiserum against an aqueous extract of bovine Reissner's fiber (RF), (anti-RF-DC antiserum); (2) antiserum against the protein fraction F1 obtained by gel electrophoresis of the aqueous RF-extract (RF-DF1-antiserum); (3) antiserum against the protein fraction F2 prepared in the same manner (RF-DF2-antiserum). As shown by immunological and/or immunocytochemical experiments in bovines and rats, the three antisera are of high specificity, i.e., react exclusively with the secretion of the SCO, which appears to be a unique product of the vertebrate organism. Concerning the distribution of the reaction products within the SCO-cells, no differences were found light microscopically after use of the RF-DC-antiserum, the RF-DF1-antiserum, or the RF-DF2-antiserum. Comparative studies were carried out with the RF-DC-antiserum only. A positive immunocytochemical reaction of the SCO-secretion was obtained in many vertebrate species (mammals, birds, reptiles, amphibians, bony fishes, sharks, and cyclostomes). RF gives a positive reaction in mammals only; to date RF of non-mammalian vertebrates did not react immunocytochemically with the present antiserum. Comparative immunocytological studies have shown that (1) the SCO-cells of the ependymal layer as well as the SCO-cells of the hypendymal layer contain immunoreactive material, (2) in the majority of vertebrates hypendymal structures are more common than has been previously supposed, and (3) RF or constituents of this structure are produced by the SCO. The immunocytochemical studies have led to the impression that the SCO-secretion is not only discharged into the cerebrospinal fluid, but also in hypendymal vessels and/or leptomeningeal spaces, as has been postulated previously by Oksche and others (for review, see Oksche 1969).

The secretion of the subcommissural organ. A comparative immunocytochemical investigation.

The results of a preliminary immunocytochemical investigation on the subcommissural organ (SCO) in rats show that (1) Reissner's fiber (RF) or essential compounds of the RF are produced by the SCO, (2) the immunoreactive material is produced in the epithelial cells of the SCO as well as in the hypendymal cells, and (3) the immunoreactive material of the SCO belongs to a category of endogenous peptides to date not demonstrable immunocytochemically in other brain structures.

Immunocytochemical investigation of the subcommissural organ in the rat

The secretion of the subcommissural organ (SCO) of the rat was studied by means of immunocytochemistry at the electron-microscopic level with the use of (1) the polar embedding medium Lowicryl K4M at -30 degrees C, (2) the protein A-gold technique, and (3) a rabbit antiserum against bovine Reissner's fiber (see Sterba et al. 1981). Two different substructures of the ependymal and the hypendymal SCO-cells display a positive immunocytochemical reaction: (1) sacs containing flocculent secretion, which originate from the granular endoplasmic reticulum, and (2) vacuoles filled with fine granular secretion, which are pinched off from the Golgi apparatus. The secretory material of the sacs and the vacuoles is discharged both (i) apically into the cerebrospinal fluid and (ii) basally into intercellular spaces of the SCO-hypendyma. The apically released secretion is condensed to a lamina-like formation, which more caudally assumes the form of Reissner's fiber. The route of the basally released secretion remains, however, vague. The "periodically striated bodies", which were thought to be morphological mediators of the discharge of the secretion into the capillaries, are never labeled by gold particles.

Preparation and discharge of secretion in the subcommissural organ of the rat. An electron-microscopic immunocytochemical study.

Low-temperature-embedded tissue of the subcommissural organ (SCO) of the rabbit was analyzed for the basal route of secretory product by means of indirect immuno-metal cytochemistry (protein A-gold technique) at the electron-microscopic level By use of (1) an antiserum against bovine Reissner's fibre (see Sterba et al 1981) and, thereafter, (2) particulate gold-marker solution, immunoreactive sites could be clearly visualized within the extracellular matrix of both (a) the basal part of the ependymal cell layer, and (b) the hypendyma proper Abundant secretory material was identified within (i) dilated intercellular spaces (a + b) as well as (ii) branching basal lamina labyrinths and distinct perivascular spaces (b) All these compartments are thought to belong to a system of extracellular channels, which may function in secretion directed toward hypendymal blood vessels

Immuno-electron-microscopic analysis of the basal route of secretion in the subcommissural organ of the rabbit.

1.

Die Oozyten von Melanotaenia maccullochi zeigen von Anfang an eine bipolare Differenzierung. Der um das Cytozentrum entstehende, zunachst sehr kompakte Dotterkern lockert sich zu einer schalenartigen retikularen Zone um den Kern auf, die spater das ganze Zellplasma bis auf einen schmalen Randsaum infiltriert.




2.

Als Differenzierung der primaren Oozytenmembran entstehen noch vor der Ausbildung der ersten Rindenvakuolen geiselartige Haftfaden, die zwischen Oozytenmembran und Follikel vordringend stark in die Lange wachsen und die Oozyten schlieslich knauelartig umgeben.




3.

Die Corticalschicht ist eine Bildung der Oozyte innerhalb der Oozytenmembran. Sie entsteht zunachst am animalen Pol unmittelbar nach den ersten Rindenvakuolen und erstreckt sich dann erst auf die ubrigen Eibezirke. Wahrend der weiteren Entwicklung nimmt die Dicke der Corticalschicht zu; die typische Radiarstreifung tritt bereits kurz nach der Anlage auf.




4.

Der Follikel liefert keine Eihulle. Das Ei zieht beim Verlassen des Follikels den Haftfadenschopf hinter sich her. Primare Oozytenmembran und Corticalschicht werden bei der Befruchtung von der Oozyte — die jetzt eine neue Dotterhaut bildet — durch den perivitellinen Raum getrennt.

https://link.springer.com/content/pdf/10.1007/BF00339374.pdf

Günther Sterba

Papers

The secretion of the subcommissural organ. A comparative immunocytochemical investigation.

Immunocytochemical investigation of the subcommissural organ in the rat

Preparation and discharge of secretion in the subcommissural organ of the rat. An electron-microscopic immunocytochemical study.

Immuno-electron-microscopic analysis of the basal route of secretion in the subcommissural organ of the rabbit.

[Differentiation of the egg-membrane in Melanotaenia maccullochi].