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The morphogenesis of the fibrous sheath in the rat spermatid has been studied by electron microscopy and by EM radioautogaphy following injection of 3H-proline. The appearance of the developing fibrous sheath from the distal to proximal end of the principal piece has been examined in spermatids in each of the 19 steps of spermiogenesis. The precise timing of the various steps of its formation has been established and related to radioautographic observations on the synthesis and incorporation of proteins into the developing fibrous sheath. The longitudinal columns form slowly over a period of 15 days, appearing first at the distal end of the principal piece in step 2 and gradually extending in a proximal direction, ending at the level of the annulus in step 17. Throughout this 15-day period, proline-containing proteins are synthesized and incorporated into the growing columns. All of the ribs, on the other hand, arise from anlagen which are assembled along the length of the principal piece during a much shorter (4.5-day) period between steps 11 and 15 of spermiogenesis. New rib anlagen seemingly originate from bundles of proteinaceous filamentous material which are synthesized in the cytoplasm of step 11-15 spermatids and become aligned along the plasma membrane of the principal piece, starting at the distal end. These observations suggest that the two components of the fibrous sheath are assembled by means of two independent mechanisms that proceed asynchronously except during an overlap period of 2.5 days between steps 12 and 14.

The morphogenesis of the outer dense fibers (ODF) in rat spermatids has been studied by electron microscopy, and the synthesis and incorporation of proteins into the ODF during this process have been followed by radioautography using 3H-proline and 3H-cystine as precursors for ODF proteins. In the first phase (step 8-14), nine very fine fibers termed anlagen of the ODF develop in association with the microtubule doublets. These first appear along the most proximal portion of the axoneme in step 8 of spermiogenesis; during steps 9-14 they gradually increased in length in a proximal-to-distal direction, being first observable along the forming midpiece and later along the principal piece as well. In the second phase (steps 15-16), the rudimentary fibers suddenly increase in diameter, with the most dramatic growth occurring in step 16, and assume a close resemblance to the mature ODF. This striking transformation, which appears to result from simultaneous deposition of electron-dense material along the length of anlagen of the ODF, coincides with a period of rapid incorporation of 3H-proline-and 3H-cystine-containing proteins, which become permanent structural components of the ODF. These proteins, which comprise the bulk of the ODF, are synthesized in the cytoplasm of spermatids during the acrosome and early maturation phases. In the final phase (steps 17-19) the fibers continue to enlarge very slowly, assuming their definitive form in step 19 of spermiogenesis. Thus formation of the ODF in the rat is a lengthy multistep procedure, requiring from step 8-19 of spermiogenesis and utilizing proteins synthesized throughout most of this period.

In acute myocardial infarction, myocardial salvage is dependent on rapid restoration of blood flow. Pharmacologic (streptokinase, recombinant tissue-type plasminogen activator), mechanical (percutaneous transluminal coronary angioplasty, guide wire perforation) or combined forms of reperfusion therapy can accomplish this goal, but their effects on infarcted myocardium and vessel occlusion site have not been compared at necropsy. The heart of 19 necropsy patients who had received various forms of acute reperfusion therapy was studied: 14 had pharmacologic or combined forms of reperfusion therapy (13 streptokinase and 1 tissue-type plasminogen activator, including 4 with combined balloon angioplasty) and 5 had had purely mechanical (balloon angioplasty) reperfusion therapy. Reperfusion was initially clinically successful in all 19 patients with the average time from onset of symptoms to reperfusion being 3.7 hours. Necropsy observations separated the 19 patients into distinct subgroups based on changes in the myocardium and infarct-related coronary arteries. Of the 19 patients, 14 (74%) had hemorrhagic myocardial infarction and they all received pharmacologic or combined forms of reperfusion therapy. The remaining five patients (26%) had nonhemorrhagic (anemic) infarction and were treated with balloon angioplasty therapy alone. Increased luminal cross-sectional area was present in 8 of 9 patients with acute balloon angioplasty but severe coronary atherosclerotic plaque remained in 9 of 10 patients without acute balloon angioplasty. Severe hemorrhage surrounded angioplasty sites in all four patients who also received streptokinase or tissue-type plasminogen activator. Severe bleeding at the angioplasty site compromised the dilated coronary lumen in one patient. No patient with angioplasty alone had intraplaque bleeding. Thus, acute coronary balloon angioplasty reperfusion therapy alone appears to avoid the potentially adverse effects of myocardial and intraplaque hemorrhage while simultaneously increasing luminal cross-sectional area at the site of acute occlusion.

Indirect immunofluorescence and antibodies to tubulin were used to visualize the distribution of microtubules (MT) in short-term primary cultures of retinal pigment epithelial (RPE) cells isolated from the eyes of control RCS rats and those with inherited retinal degeneration. At all stages of cell spreading in vitro, the pigment granules (PG) in these cells remained tightly clustered in the perinuclear region, surrounded by numerous MT. Initially, in relatively round cells, the perinuclear region was encompassed by a closely woven ring of circularly arranged MT. At later stages of spreading, the ring had disappeared and MT running in various directions could be observed. Cells that were plated singly assumed at first a discoid, then stellate, shape during spreading, whereas, those plated in small groups formed colonies of wedge-shaped cells. In well-spread single cells or colonies, a brightly fluorescent zone that appeared to contain the highest density of MT within the cells was located between the PG-containing perinuclear region and the spreading edges of the cells. A single prominent star-shaped structure from which a number of MT radiated was situated among the PG in the perinuclear region of each cell; we believe this structure corresponds to the centrioles. It was generally well-separated from the regions of highest MT density. No differences in cell shape or in the distribution of MT, centriole-containing regions or PG in spreading or fully spread cells were detected when RPE cells from normal and dystrophic RCS rats were compared. Thus, the distribution of MT and their assembly during cell spreading appear normal in RPE cells of the dystrophic rat.

A cytochemical method was developed for localization in isolated rod outer segments of manganese-dependent pyrimidine 5'-nucleotidase (MDPNase), an enzyme activity with possible relevance to shedding that we recently reported in photoreceptors and retinal pigment epithelial (RPE) cells in the intact rat retina. The purpose of this study was to eliminate the possibility that the previously observed cytochemical staining of the rods was due to diffusion of reaction product from the RPE cell lysosomes, which were also heavily stained. Rod outer segments (ROS) were isolated on continuous sucrose gradients from retinal homogenates prepared from rats raised in cyclic light (12 hr light:12 hr dark) and killed during the first 2 hr after light onset. ROS-containing bands were removed from the gradients and the isolated rods were fixed in 0.25% glutaraldehyde and pelleted. Chopped sections of the pellets were incubated in cytochemical medium for MDPNase activity and processed for light- and electron-microscopic localization of the enzyme activity. Two patterns of cytochemical staining were seen in ROS isolated from retinas obtained at this time of day. A few of the pellets contained clusters of ROS that were heavily coated along their surfaces and seemingly interconnected by thick strands of highly reactive extracellular material that displayed a punctate pattern of cytochemical staining. This material may have originated from the apical processes of the RPE cells, which were heavily stained in tissue fixed in situ around the time of light onset. The second staining pattern, visible only by electron microscopy, was more commonly observed. In the majority of the isolated ROS profiles, discrete streaks of cytochemical reaction product were seen in association with the internal aspects of the discs, at sites that seemed to correspond to the rims, and to narrow zones within the disc interiors. This distribution of reactive sites closely resembled that observed over most of the length of the ROS in the intact retina fixed at the same time of day. Occasionally, ROS profiles were encountered in which additional reactive sites were localized to the interdisc spaces between the plasma membrane and the rims of the discs. The latter pattern resembled the distribution of reaction product seen during this period over the tips of the ROS fixed in situ. As in the intact retinas, the cytochemical staining of the isolated ROS was inhibited by fluoride ions and strongly stimulated by manganese ions.(ABSTRACT TRUNCATED AT 400 WORDS)