Library

Notes: The perforatorium is the subacrosomal portion of the perinuclear theca that encapsulates the nucleus of spermatozoa. In the rat, the perforatorium is a curved pointed structure, which in cross section is triangular in outline over the apical half and beyond the tip of the nucleus. The perforatorium, composed of several proteins, appears as a distinct structural entity only at the very end of spermiogenesis. In this study, polyclonal antibodies prepared against the entire isolated perforatorial fraction and against the major 16 and 34 kDa perforatorial polypeptides were used to determine the distribution of perforatorial proteins in germinal cells at various steps of differentiation. Immunoperoxidase staining at the LM level and quantitative immunogold labeling at the EM level were used. The labeling patterns with all three antibody preparations were identical. The immunolabeling first appeared in early pachytene spermatocytes and increased progressively, with a statistically significant upward trend, in both the nuclei and cytoplasm of spermatocytes and spermatids until step 9 of spermiogenesis. Up to this step the labeling concentration was significantly higher over the nucleus than over the cytoplasm. During nuclear condensation in steps 9 and 12 spermatids, there was a progressive loss of all the labeling over the nucleus and a corresponding increase of labeling over the cytoplasm. During steps 16-18, the early signs of condensation of perforatorial proteins occurred next to the inner acrosomal membrane. Then during step 19 there was a sudden condensation of perforatorial proteins into a definitive perforatorium. Thus proteins destined to form this cytoskeletal structure reside in both the nucleus and cytoplasm of spermatocytes and spermatids until nuclear condensation of the latter. Thereafter, they are restricted to the spermatid's cytoplasm and finally condense around the elongated nucleus at the end of spermiogenesis.

Notes: The initial segment of the epididymis of rats, fixed with glutaral-dehyde, was postfixed with reduced osmium, a technique that clearly delineates the membranes of cisternae of the endoplasmic reticulum (ER) and the various elements of the Golgi apparatus, or with tannic acid to enhance the coats of vesicles and ribosomes on ER cisternae. The material was also treated to demonstrate various phosphatase activities (NADPase, TPPase, CMPase, G-6-Pase) or impregnated with osmium tetroxide.In osmium-impregnated material, the Golgi apparatus of the epithelial principal cells of the initial segment appeared in the light microscope as a branching, anastomosing ribbon forming a large network in the supranuclear region. In the electron microscope, ER were of two types: the heavily granulated, flattened, rough ER seen in the infranuclear and juxtanuclear regions and the distended, tubular, sparsely granulated ER, showing only few ribosomes, seen interlaced with the Golgi ribbon in the supranuclear region and at the apical pole of the cell.Of particular interest in this cell was the fact that the sparsely granulated ER approximated the Golgi stack on both its cis- and trans-faces. On the cis-face of the Golgi stack, the sparsely granulated ER cisternae showed the usual finger- or bud-like protrusions directed toward the cis element of the Golgi stack and around which numerous small 80 nm vesicles or membranous tubules were clustered. The Golgi stack consisted of the following elements in a cis-trans axis: the cis osmio-philic element, a first saccule slightly dilated, saccules two to four (S2-S4), which were NADPase-positive, and saccules five to seven and the eight Golgi element, which were TPPase-positive. On the trans-aspect of the Golgi stacks, several (up to four) CMPase-positive trans-Golgi networks were observed often in close apposition to the sparsely granulated ER cisternae. One of the trans-Golgi networks showed a “peeling-off” configuration, i.e. part of it was closely apposed to the overlying Golgi element of the stack, whereas the remaining part was separated from the stack by a space occupied by a cisterna of sparsely granulated ER. The other trans-Golgi networks were completely separated from the stack and were often seen sandwiched between sparsely granulated ER cisternae. Thus, ER cisternae showed extensive areas of close apposition but no continuity with the trans-Golgi networks. Although the saccules of the Golgi stacks showed NADPase and/or TPPase activity, the trans-Golgi networks displayed CMPase activity, thus facilitating their identification from the closely associated unreactive sparsely granulated ER cisternae.The trans-Golgi networks were variable in appearance but often showed sporadic electron-lucent dilations along their length that were comparable to uncoated vesicles of similar size (150-300 nm) and appearance found in the trans-region of the Golgi apparatus and at the apical pole of the cells. Such vesicles appeared to arise from the trans-Golgi networks that eventually undergo fragmentation. Their identification as possible secretory vesicles will be discussed.

Notes: In order to analyse, at the electron microscope level, the three-dimensional configuration of the trans compartment of the Golgi apparatus rat dorsal root ganglia were treated to demonstrate cytidine monophosphatase (CMPase) activity. The localization of enzymatic activity in the Golgi apparatus varied according to cell types. In type A and C cell, CMPase was exclusively located in the transmost sacculotubular element, whereas in type B cells all the saccules of the stacks forming the Golgi ribbon and the trans-Golgi networks were impregnated. Numerous dense bodies seen at proximity were also CMPase positive. In 3 μm thick sections of type A cells examined at low magnification, the impregnated element was scattered throughout the cytoplasm and never formed a continuous structure. In type B cells, the strongly reactive trans-Golgi networks did not follow the entire length of the impregnated Golgi ribbon but were preferentially located in the concavity of its arched portions. At higher magnification and in all cell types some tubular portions of the trans-Golgi networks took the apperance of spheroidal cage-like structures, the CMPase positive anastomotic tubules forming the bars of the cage. Anastomotic tubules separated from the trans-Golgi networks formed fenestrated spheres, while nearby CMPase-reactive dense bodies exhibited a paler hilus. These observations were taken to indicate that in ganglion cells, some CMPase positive dense bodies, presumably lysosomes, formed by fragmentation of the trans-Golgi networks.