Heikki J. Helminen

Studies on mammary gland involution: I. On the ultrastructure of the lactating mammary gland

Four types of cells constitute the alveoli of rat lactating mammary glands: secretory epithelial cells, myoepithelial cells, “pale cells” (possibly lymphocytes), and macrophages. The milk protein in the secretory cells appears to correspond to 80–90 A thick branching fibrils in the lumens of rough-surfaced ER and Golgi vacuoles. In the latter, the fibrils are condensed to form secretion droplets with characteristic ultrastructure; the droplets move toward the luminal plasma membrane, appear to fuse with the membrane, and thereby release their contents. Some droplets may form directly from apically located ER. Mature secretion droplets are bordered by a triple-layered, ∼ 100 thick membrane resembling the plasma membrane. Cytosomes—believed to correspond to lysosomes—are also bordered by ∼ 100 A thick triple-layered membranes. They are sparse and small in all cell types except the macrophages. Cytosomes are distinguished from secretion droplets by the regular mosaic pattern of milk protein in the droplets. The extent of apparent autophagocytosis is low in the secretory epithelial cells. Fat droplets are transferred to the alveolar lumen through apocrine secretion. The findings suggest that the membrane bordering the milk proteins undergoes a change in thickness of the order of 30–40 A during the transport of proteins from ER to alveolar lumen; this change appears to occur at the level of the Golgi apparatus.

Ultrastructural studies on prostatic involution in the rat. Mechanism of autophagy in epithelial cells, with special reference to the rough-surfaced endoplasmic reticulum

The fine structural mechanisms involved in autophagy during cellular remodeling were studied in rat prostatic epithelial cells following castration. Alterations in groups of rough-surfaced endoplasmic reticulum cisternae were shown to precede their sequestration in autophagic vacuoles. Rough-surfaced endoplasmic reticulum was the most common and major component of the autophagic vacuoles. Early autophagic vacuoles were often bordered by two parallel smooth-surfaced membranes and did not appear to contain acid phosphatase. Degradation of the cytoplasmic components sequestrated in the autophagic vacuoles—notably the rough-surfaced endoplasmic reticulum cisternae—resulted in the formation of “residual bodies” showing presence of acid phosphatase and morphologic appearance reminiscent of lipofuscin granules. The findings suggest (a) that the process of autophagy is discriminative with respect to the rough surfaced endoplasmic reticulum and triggered by functional and/or structural modifications of this organelle; (b) that hormonal factors, indirectly, exert control over the autophagic process; (c) that the major mechanism for the reduction in size of the rough-surfaced endoplasmic reticulum is cellular autophagy; (d) that lipofuscin-like bodies can result from lytic breakdown and structural reorganization of organelles enclosed in autophagic vacuoles; and (e) that autolysosomes were created through fusion between an autophagosome and a lysosome. Lysosomes accidentally trapped in whorls of endoplasmic reticulum cisternae might contribute enzyme to forming autophagic vacuoles.

Studies on mammary gland involution: II. Ultrastructural evidence for auto- and heterophagocytosis

During enforced mammary gland involution in the rat, greatly increased numbers of cytosegresomes appear in the secretory epithelial cells 1–3 days after weaning. These cytosegresomes are interpreted as indicative of autophagocytosis. At 3 days and later, the cytosegresomes are few and appear to be replaced by large cytosomes, which are more numerous than in the lactating parenchyma. These findings support the theory that cytosegresomes are transformed to cytosomes. Increased numbers of macrophages were present between the epithelial cells in the alveoli during involution. Apparent heterophagocytic uptake of portions of epithelial cell cytoplasm and milk protein droplets in these macrophages appeared to be most pronounced at late intervals (3–10 days) after weaning.

On the mechanism of lysosomal enzyme secretion. Electron microscopic and histochemical studies on the epithelial cells of the rat's ventral prostate lobe

Mature secretion granules produced by the epithelial cells in the rats ventral prostate lobe contain acid phosphatase and seem to represent primary lysosomes. These granules take their origin from the Golgi apparatus where acid phosphatase-positive condensing vacuoles appear to pinch off from dilated portions of likewise enzyme-containing Golgi cisternae. The observations suggest that these apparent specialized forms of primary lysosomes move toward the apex of the cells and release their contents into the alveolar lumen by way of membrane fusion, without participating in intracellular auto- or heterophagic digestive events. Some of the granules may fuse with other primary lysosomes—usually represented by acid phosphatase-positive “Golgi vesicles” — prior to their fusion with the apical plasma membrane.

Studies on mammary gland involution: IV. Histochemical and biochemical observations on alterations in lysosomes and lysosomal enzymes

The activities of four lysosomal enzymes—acid phosphatase, aryl sulfatase, cathepsin D, and acid deoxyribonuclease—were measured during involution of the rat mammary gland. Acid phosphatase and aryl sulfatase were studied histochemically in perfusion-fixed mammary glands. The results indicated that there was increased activity of lysosomal enzymes both in secretory epithelial cells and macrophage-like cells during involution. This increase appears to be due to induced synthesis of the enzymes. It is suggested that such an induction may be triggered by the occurrence of “autophagosomes” and “heterphagosomes” in the epithelial cells and the macrophage-like cells, respectively. Acid phosphatase and aryl sulfatase seemed to be present together in the same “lysosome”.

Studies on mammary gland involution: III. Alterations outside auto- and heterophagocytic pathways for cytoplasmic degradation

Early alterations in secretory epithelial cells during mammary gland involution (detectable within 24 hours after weaning) include occurrence of large vacuoles containing numerous protein secretion droplets, and reorganization and reduction in size of the rough endoplasmic reticulum with concomitant focal loss of ribosomes and dilatation of some of the cisternae. These alterations are more extensive at 48 hours when a general pronounced reduction in the number of cytoplasmic organelles outside the autophagic-lysosomal system is noted; nuclei appear unaltered, however. It is suggested that these alterations are unassociated with previously described lysosomal pathways for intracellular digestion. Apparent sloughing of some of the altered epithelial cells into alveolar lumens occurs after 48–72 hours. The results of this and previous study (9) suggest that the degenerative alternations in the glandular epithelial cells during involution are dependent upon one lysosomal and one apparently non lysosomal component working simultaneoously during the early phase. Following sloughing of some whole cells or portions of cells, heterophagocytosis in macrophages appears to play a dominating role in involution during the late, DNA-reducing phase.

Ultrastructural studies on prostatic involution in the rat. Evidence for focal irreversible damage to epithelium, and heterophagic digestion in macrophages

A small number of lymphocyte-like cells and macrophages are interspersed between the epithelial cells in the acinar lining of the rats ventral prostate. During castration-induced involution, cellular autophagy in the epithelium is the most conspicuous alteration in the prostate. However, occasional epithelial cells undergo more severe, irreversible degenerative changes presumed to lead to necrosis. Some of these degenerating cells are engulfed and digested within heterolysosomes in macrophages, others are expelled into the acinar lumen. The macrophages occur in increased numbers during involution, while the frequency of lymphocyte-like cells appears to be unaltered.

Ultrastructural studies on prostatic involution in the rat changes in the secretory pathways

The fine-structural changes in the secretory pathways were studied in the rat prostatic epithelial cells after castration. Alterations were first noticed in the rough-surfaced endoplasmic reticulum (on day 2). A gradual reduction in the number of Golgi-associated, smooth-surfaced vesicles, vacuoles with granular content (“condensing vacuoles”), and secretory granules occurred from day 3 onward. The vacuoles with granular content appeared to be replaced in part by “empty” vacuoles. The Golgi apparatus proper showed decreased size on days 3–5, and later seemed to lose its structural polarity in the cells. Apparent acid phosphatase activity was present in the Golgi apparatus (cisternae and some vesicles), vacuoles (“empty” and with granular content) associated with the Golgi apparatus, secretory granules, apical “empty” vacuoles, lysosomes, and the alveolar lumen at intervals up to day 20 after castration. The observations suggest (a) that the occurrence of small smooth-surfaced vesicles at the periphery of the Golgi complex, condensing vacuoles, and secretory granules is dependent on the continuous production of the normal secretory product by the rough-surfaced endoplasmic reticulum; (b) that cessation of major androgenic hormone action results in quantitative and morphologic changes in the secretory product while the pathways for the intracellular transport are essentially unaltered; (c) that the reduction in size of the Golgi apparatus is relatively slow and gradual and is unrelated to autophagy; and (d) that the loss of polarity of the Golgi apparatus is due to the combined effects of diminished function in secretion and retained function in, for example, random lysosome production.

Observations on the localization of arylsulphatase activity in renal cortical tubules

SummaryThe fine structural localization of arylsulphatase in rat kidney cortex was investigated using p-nitrocatechol sulphate as substrate and lead as capturing ion. The studies included observations of the effects of different modes of fixative application in order to define optimal conditions for the histochemical procedure. Reaction product (lead sulphate) was constantly precipitated in the cytosomes in cells of proximal and distal convoluted tubules and collecting ducts. Previous studies have demonstrated that these same organelles contain acid phosphatase and appear to correspond to lysosomes in biochemically isolated fractions from renal cortex. The observations are compatible with the notion that most of the cytosomes in renal cortical tubules and collecting ducts contain both acid phosphatase and arylsulphatase. The constant absence of precipitate in microbodies of proximal tubule cells supported the assumption that these organelles are functionally different from cytosomes, and may correspond to “peroxisomes”.

Histochemical and biochemical studies on the localization and activity of lysosomal enzymes in fracture callus

SummaryQuantitative biochemical studies on the activities of four lysosomal hydrolases during different stages of fracture healing in the rat were performed, and the results obtained were integrated with those of histochemical observations relating to changes in the localization of acid phosphatase in the same tissue.The findings showed presence of all the four lysosomal enzymes assayed in the callus; during early callus formation the enzyme activities calculated on a DNA basis increased up to about 12 days after the fracture. The enzyme activities appeared to be roughly reflected histochemically by the acid phosphatase staining. The increasing activity during early callus formation seemed to depend on the presence of numerous macrophage-like cells in the tissue containing many large lysosomes. A decrease in enzyme activity was found after day 12. Comparison with the histochemical and ultrastructural findings suggested that this decrease was due to a reduction in the number of macrophage-like cells and a concomitant increase in osteogenic cells with a lower enzyme content.