Redistribution of Mn++-dependent pyrimidine 5'-nucleotidase (MDPNase) activity during shedding and phagocytosis.

Ultrastructural cytochemistry was used to localize a previously undescribed retinal enzyme activity, ie, "manganese-dependent pyrimidine 5'-nucleotidase" (MDPNase) activity in retinas from rats raised in cyclic light and killed at various times in the lighting cycle. In retinas fixed during the last 3 hr of darkness, at the beginning of the shedding period, heavy cytochemical staining was observed over the extracellular surfaces of the apical processes of the retinal pigment epithelium (RPE) cells. The adjacent distal ends of the rod outer segments (ROS), ensheathed by the apical processes, were also stained along their surfaces. During the first 3 hr after light onset, at the time of maximal shedding and phagocytic activity, the tips of the ROS and ROS phagosomes were heavily labeled with reaction product distributed throughout the interdisc spaces. The RPE apical processes, previously heavily stained, were at this time weakly reactive relative to the ROS tips and phagosomes. After the shedding peak (4-8 hr after light onset), the tips of the ROS were no longer labeled with reaction product, and the apical processes were unreactive. The proximal portions of the ROS were weakly stained throughout the lighting cycle. The observed patterns of redistribution of MDPNase activity before, during, and after the shedding peak suggest that the presence of the enzyme in the ROS tips may be correlated with shedding. Changes in the staining of the RPE apical processes in relation to cyclic light further suggest that this enzyme may be transferred from the apical processes to the ROS tips prior to shedding.

Distribution of microtubules in cultured RPE cells from normal and dystrophic RCS rats.

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.

Cytochemical localization of Mn2+-dependent pyrimidine 5'-nucleotidase activity in isolated rod outer segments.

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)

Synthesis and assembly of connecting-piece proteins as revealed by radioautography.

The synthesis and assembly of connecting-piece proteins have been studied during spermiogenesis in the rat by electron microscopy and radioautography following intratesticular injection of radiolabeled amino acids [3H]proline and [3H]cystine. Early in spermiogenesis (steps 1-7) the two centrioles that give rise to the connecting piece are essentially unmodified. During the 6.5-day period between steps 8 and 15, the major elements of the connecting piece (striated columns and capitulum) gradually become assembled from an electron-dense material that is deposited around the walls of the centrioles; throughout this period, protein molecules containing proline and cystine are synthesized by the step 8-15 spermatids and incorporated into the developing neck region. These proteins subsequently become permanent structural components of the connecting piece. Following completion of the major elements in step 15, few additional proteins are added to the connecting piece during the final steps 16-19 of spermiogenesis.

Formation of the outer dense fibers during spermiogenesis in the rat.

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.

Kinetics of fibrous sheath formation in the rat spermatid.

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.

Biochemical evidence for Mn2+-dependent 5'-nucleotidase activity in isolated rod outer segments.

Recently cytochemical evidence has been presented for a novel enzyme activity, i.e. 'manganese-dependent pyrimidine 5'-nucleotidase (MDPNase)' activity in the rod outer segments (ROS) of rat retinas in situ and in isolated rat ROS. The present biochemical study was undertaken to seek further evidence for this enzyme activity using an independent method. A series of enzyme assays was carried out to test for MDPNase activity in Triton extracts of rat ROS isolated by sucrose density gradient centrifugation. Hydrolysis of the substrate, cytidine-5'-monophosphate, was measured spectrophotometrically and expressed as microgram of released inorganic phosphorus hr-1 mg-1 protein in the sample. The results showed that the ROS extracts contained enzyme activity (18.1 +/- 3.8) that was increased 5-6-fold (102.3 +/- 10.6) in the presence of 7.4 mM MnCl2. The enzyme activity was not enhanced by Mg2+ ions (19.0 +/- 7.7) and was strongly inhibited by 10-20 mM NaF (11.8 +/- 2.9). Assays for substrate specificity revealed that the Mn2+-stimulated phosphatase activity was specific for 5'-nucleotides. Pyrimidine nucleotides (5'-CMP and 5'-UMP) were the preferred substrates. Comparison of enzymatic hydrolysis of 5'-CMP and 5'-AMP over a pH range from 4.5 to 8.0 revealed that at acid pH, the majority of the observed 5'-nucleotidase activity (82% at pH 5.0, 58% at pH 5.5) was manganese dependent, whereas at neutral pH and above, most of the enzyme activity was unaffected by the presence of Mn2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)