The structure of the chorion and associated surface filaments in Oryzias—evidence for the presence of extracellular tubules

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Abstract
The structure of the chorion with its associated surface filaments has been examined in Oryzias latipes using several techniques, including scanning and transmission electron microscopy, enzymatic digestion, and sodium dodecylsulfate‐polyacrylamide gel electrophoresis. The chorion of the recently fertilized egg was found to be organized into three zones: an outer, fuzzy electron‐lucent zone that was continuous over the surface of filaments, a middle, homogeneous electron‐dense zone, and an inner zone of ten to 12 horizontal, fibrous lamellae.Two topographically distinct types of filaments were found on the chorionic surface: nonattaching and attaching. Nonattaching filaments showed a regular spatial distribution over the chorion with an interfilament distance of about 60–70 μm. Attaching filaments originated from a localized portion of the chorion and united with those of neighboring eggs to anchor the egg cluster to the gonoduct of the female. Both nonattaching and attaching filaments were morphologically regionalized into basal and distal segments. Internally, nonattaching and attaching filaments were constructed of unbranched, packed tubules with an average outside diameter of approximately 19.5 and 18.8 nm, respectively. Using the attaching filament for further study, it was determined by rotational analysis (Markham et al., '63) that the wall of each tubule was a cylinder composed of 14 globular subunits. Two structural types of attaching filaments were identified. The type I attaching filament was similar in internal organization to the nonattaching filament and consisted of only tubules. The type II attaching filament, however, showed a highly osmiophilic, electrondense bar surrounded by packed tubules.Tubules of attaching filaments of the adult were resistant to the action of Triton X‐100 and colchicine, but sensitive to a 0.1% protease solution. However, colchicine‐treated ovary tissue showed an absence and pattern of disorganization of tubules at the periphery of developing filaments. Solubilized attaching filament samples electrophoresed on 7.5% polyacrylamide‐SDS gels were resolved into a pair of Coomassie‐blue‐positive bands that comigrated with purified porcine brain tubulin. The apparent molecular weight of the attaching filament polypeptide was determined to be approximately 55,000 daltons. These data suggest that the extracellular, tubular components of attaching filaments (as well as nonattaching filaments) are proteinaceous and show properties similar to those of cytoplasmic microtubules.Tubular precursor material was electron‐dense and appeared to originate in the cisternae of the rough endoplasmic reticulum of ovarian follicle cells. With increased synthesis, regions of the endoplasmic reticulum appeared to become dilated into membrane‐limited vesicles. The contents of these vesicles in single planes of section included electron‐dense aggregates of various shapes and crystalloid inclusions. The crystalloid inclusions contained parallel arrays of tubules and resembled the tubular structure described for filaments of the chorion. The crystalloid inclusions appeared to be continuous with the lighter‐staining portion of the electron‐dense bodies, suggesting that the latter may initiate the formation of the filament. Developing filaments appeared to be extruded into the extracellular space by exocytosis. Data from this study, when interpreted with existing literature, indicates that the chorion of Oryzias is produced by both the oocyte and the surrounding follicle cells.