Morphometric and ultrastructural analysis of stage-specific effects of Sertoli and spermatogenic cells seen after short-term testosterone treatment in young adult rat testes.

The effects of testosterone treatment on spermatogenesis in the rat have been investigated by morphometric and structural analysis at the ultrastructural level in stages VII-IX. The aim has been to characterize the changes in Sertoli and spermatogenic cells to elucidate the mechanism of testosterone effects on spermatogenesis and to test the possibilities of developing male contraceptives. In stage VII, the morphometric parameters of volume and surface area in Sertoli cells (see abbreviations below): and the morphometric parameter of volume in the spermatogenic cells such as V(VPG,T), V(VPC,T), V(VrPT,T) and V(VelPT,T) decreased. In stage VIII, the respective values of Sertoli cells, VSN, and VSN/VSC decreased while SSJ increased, and the respective morphometric parameters in the spermatogenic cells, V(VPG,T), V(VPC,T), and V(VrPT,T) increased. In stage IX, in Sertoli cells VSC, VSN, VSN/VSC, and SSJ remained unchanged. In spermatogenic cells V(VPG,T), V(VPC,T), and V(VrPT,T) increased. Further, in all stages, a close apposition of mitochondria and rough endoplasmic reticulum in basolateral cytoplasm of Sertoli cells suggested active protein synthesis. In elongated spermatids in stage IX the microtubular manchette became disorganized. This disorganization and the unexpected shift after testosterone treatment from decrease in several morphometric parameters in stage VIII to increases in stage IX cannot be explained by alterations in testosterone (T), LH, FSH, and their respective receptors. Therefore, still unknown regulatory factors in spermatogenesis are apparently involved in the developmental interactions between Sertoli and spermatogenic cells.

Ultrastructural, morphometric, and hormonal analysis of the effects of testosterone treatment on Leydig cells and other interstitial cells in young adult rats.

Early effects of testosterone (T) treatment on the ultrastructure of testicular interstitium were analyzed by morphometry. In T-treated young adult rats the T-LH feed-back loop functioned as expected and the marked increase in peripheral T caused almost complete depletion of peripheral LH. Even though the peripheral LH concentration was almost undetectably low, the Leydig cells maintained regulatory interactions with macrophages, peritubular myoid cells and with the seminiferous epithelium lining the tubular lumina as indicated by the high correlations of morphometric parameters between the Leydig and other cell types. The morphometric alterations in the ultrastructure of Leydig cells suggest that the seminiferous tubules may signal by releasing inhibitory paracrine factors affecting the morphology and function of Leydig cells in T-treated young adult rats. The morphometry of Leydig cells in T-treated young adult rats showed a significant quantifiable reduction in nuclei and organelles involved in steroid synthesis and this analysis also offers a good basis for elucidation of the early effects of testosterone in terms of its contraceptive function as well as of different toxic compounds on reproductive functions.

A case of splenic abscess due to Chlamydia pneumoniae.

In this report, a case of chlamydial disease with splenic abscess associated with Chlamydia pneumoniae antigen and antibody was described. On spleen biopsy of the patient, an antigen specific to C.pneumoniae was detected by immunofluorescence staining with a monoclonal antibody. Serologic studies revealed a high antibody titer to C.pneumoniae in sera collected from the patient and her husband. Treatment with the antibiotic minocycline improved her condition.

Morphological changes in mouse accessory sex glands following neonatal estrogen treatment.

A single injection of beta-estradiol 17-cypionate into the mice within 5 hr after birth induced inflammation in all prostate lobes and the seminal vesicles. Neutrophils emigrated into the lumen through the basal lamina and epithelium of the seminal vesicle and the anterior prostate. Local infiltration of lymphocytes was observed in the stroma and epithelium of ventral prostates. Lymphocytes penetrated through smooth muscle cells into epithelium. This could support the hypothesis that smooth muscle cells are the target of the estrogen action of prostates in estrogenized animals.

Two types of three-dimensional structures formed by epithelial cell aggregates from the rat ventral prostate in collagen gel culture. The amount of activated charcoal for absorbing serum.

Cells taken from the rat ventral prostate and cultured formed a tubular structure inside the collagen gel in a medium containing activated charcoal-absorbed serum after a 14-day incubation. This might suggest that the active substances of serum could induce isolated epithelial cells to form such a spherical or tubular structure, depending on the amount of activated charcoal used for the absorption of serum.

Bone formation by cells from femurs cultured among three-dimensionally arranged hydroxyapatite granules.

In vitro bone formation by cells derived from adult rabbit femurs was investigated on or in several substrates with small porous hydroxyapatite granules (HAGs). When the bone fragments were cultured in HAG-packed glass tubes, which were inclined (5 degrees -30 degrees ) and rotated 90 degrees per day after one week of culture, thin lamellar tissues were newly formed in narrow spaces among the HAGs. By 11 days of culture, these tissues had been mineralized except for their periphery and had well developed collagen bundles and several monolayer cells. Some cells resided in bone lacuna-like spaces. By contrast, mineralization was negligible in 6-week cultures on two-dimensional glass and polystyrene plates with or without two-dimensionally arranged HAGs on their surfaces and in three-dimensional collagen gels with or without HAGs in spite of active cell proliferation. These results suggest that osteogenesis is accelerated in a specific three-dimensional constitution of extracellular matrix and/or under the effects of mechanical forces for the new tissue and that bioactive HAGs offer favorable three-dimensional spaces for osteogenic tissue formation.

Nuclear concentration of gold labeled-testosterone-bovine serum albumin conjugate injected intravenously in the hormone-target cells of rat.

We examined whether testosterone-bovine serum albumin conjugate (testosterone-BSA) showed similar distribution to radiolabeled testosterone in vivo, by injecting 2-nm colloidal gold labeled-testosterone-BSA (testosterone-BSA-gold) from rat tail vein. The testosterone-BSA-gold with the silver enhancement became visible as silver deposits under electron microscope in nuclei of Leydig cells, Sertoli cells, spermatogonia, spermatocytes and spermatids in the testis, those of the epithelial cells in the seminal vesicle and of the cardiac muscle cells in the heart of rat killed 2 h after the injection. Few deposits were present on the non-target cell nuclei in thymus and spleen. In the liver cells, the deposits were observed in the cytoplasm, but few in the nucleus. At high-power magnification without silver enhancement, the gold particles were found in the target cell nuclei in the testis. In control rat injected with BSA labeled with 2-nm colloidal gold, the percentages of nuclei showing the deposits were fewer than those in the rat injected testosterone-BSA-gold in the target cells. The deposits were also few in the nuclei of non-target cells in control rat. These results suggest that testosterone-BSA-gold is useful for morphological study of testosterone target cells, and imply that BSA conjugated with testosterone can enter the target cell nuclei of the rat.

The Golgi apparatus and acid phosphatase-negative cisternal portions of the trans-Golgi network: ultrastructural and cytochemical studies of secretory epithelial cells in the rat lateral prostate.

Ultrastructural and enzyme-cytochemical studies were performed on the Golgi apparatus in secretory cells of the lateral prostate of normal adult rats using serial ultra-thin sections. In the trans-Golgi area, a unique membrane complex composed of tubular portions and cisternal portions showing a rigid appearance is found. This corresponds to the GERL (Novikoff 1964) or the trans-Golgi network (Griffiths and Simons 1986). From their structural similarities, the cisternal portion found in this study is considered to be the same structure as the plate-like cisterna reported by Inoue and Kurosumi (1989). As we reported previously (Kimura and Ichihara 1985), there are at least two types of acid phosphatase (AcPase) in secretory cells of the rat lateral prostate: one is located only in the structural components involved in their secretory functions and reacts readily with naphthol AS-BI phosphate; the other is a lysosomal type, which reacts well with beta-glycerophosphate. Lysosomal AcPase activity demonstrated by Gomori's method (1952) was found in a few middle- to trans-Golgi cisternae and in lysosomes. The AcPase detectable with Gomori's method and thiamine pyrophosphatase seemed to exist in part in the same cisterna on the trans side. With Robinson and Karnovsky's method (1983) for lysosomal AcPase, however, the reaction products were found only in lysosomes that were occasionally tubular in shape. On the other hand, any activity of AcPases tested could not be detected in the cisternal portion with the rigid appearance. Thus, in secretory cells of the adult rat lateral prostate in normal condition, it is considered that the cisternal portion of GERL, or the trans-Golgi network, has no relation to the processing and/or transport of AcPases.

Nuclear export of recombinant baculovirus nucleocapsids through small pore or nuclear-pore-like structure in Sf9 cells.

Translocation of baculovirus nucleocapsids (45 nm in diameter, approximate length of 280-300 nm) from nucleoplasm to cytoplasm was studied morphologically using cryofixation and gold labeled wheat germ agglutinin (WGA-gold) during recombinant Autographa californica nuclear polyhedrosis virus infection in Sf9 cells. Nucleocapsids formed in the nucleoplasm migrated into protrusions of the nuclear envelope, but not into nuclear pore complexes. We found cross-like membranous structures. Small pores seemed to be in the protruding nuclear double membranes. The middle piece of a nucleocapsid was located within the small pore whereas the upper part was in the cytoplasm. Other nucleocapsids were situated within pores without colocalization of WGA-gold in the nuclear envelope. These results suggest that baculovirus nucleocapsids use small pores in the nuclear-derived membranes or incomplete nuclear pores in the nuclear envelope to migrate from the nucleoplasm to the cytoplasm, but not complete nuclear pore complexes proper.

Reactivity of lysosomes to inside-out cell membrane vesicles in a cell-free system.

To study ultrastructurally the mechanisms of lysosome reactions to cell membrane-derived intracellular membranes we developed a cell free system using small inside-out and rightside-out cell membrane vesicles (IOVs and ROVs) as a target of the reactions. The IOVs were generated from rat erythrocyte ghosts in a low ionic strength alkaline solution in the absence of divalent cations after erythrocytes were reacted with wheat germ agglutinin-coated colloidal gold [WGA (CG)], while ROVs were from ghosts homogenized in a buffer with MgSO4 and bovine serum albumin-coated CG [BSA (CG)]. WGA (CG)s bound to the cell surface were rearranged on the membrane and distributed irregularly on the inner surface of generated small IOVs. A coat structure derived from the ghost's submembranous coat was almost depleted from their outer surface. By contrast, BSA (CG)-binding to the membranes was negligible in the process of ROV formation. When isolated rat liver lysosomes were incubated with these WGA (CG)-binding small IOVs at 37 degrees C, CG particles were found in several lysosomes under electron microscopy. Some lysosomes adhered to the IOVs, and their limiting membranes were found to collapse and disappear partially at the adhering region, suggesting their fusion. This reaction seems to occur even in cytosol-free solution. By contrast, the lysosomes indicated very low reaction to BSA (CG)-containing ROV, and to WGA (CG) or BSA (CG) alone. Therefore, it is suggested that isolated liver lysosomes react, at least to fuse, in a cytosol-independent fashion, with surface coat-depleted IOVs derived from WGA (CG)-bound and then -rearranged erythrocyte membranes.

The effect of androgen and estrogen on secretory epithelial cells and basal cells of the rat ventral prostate after long-term castration.

After long-term castration, rats were injected with cotton seed oil, testosterone- and estradiol-17 beta-cypionate (CS, TC and EC). The height of the epithelial cells of the ventral prostates from the castrated rats increased after TC and EC-injection. The secretory and basal cells formed two layers of epithelium, an inner layer near the lumen with pale nuclei and another layer with dark nuclei. These two layers could result from a reduction of secretory epithelial cells. Castration decreased the ratio of secretory cells to basal cells (S/B). TC-injection increased the ratio of S/B because of the secretory epithelial cell growth. Longer dark cells may be transient cells, appearing during the differentiation of basal cells into secretory epithelial cells. A sheet branching off from the basal lamina was observed. Androgen may stimulate the synthesis of the lamina, but whether it induces the synthesis or turnover of the basal lamina has not been established. EC increased the ventral prostatic weight and secretory epithelial cell height and induced the appearance of crystalline granules. Increase in S/B ratio may result from an increase in the secretory epithelial cells, but not from basal cell multiplication due to squamous metaplasia. The ratio is significantly correlated to the weight of the ventral prostate, but not to the secretory epithelial cell height. Its value could indicate the multiplication of secretory epithelial cells, differentiation of basal cells into epithelial cells, or both. It is probable that basal cells do not change in number, but control the size of the rat ventral prostate in response to the hormone level.

Ultrastructure and morphometry of testicular Leydig cells and the interstitial components correlated with testosterone in aging rats.

The ultrastructure of testicular interstitium in young and aged adult rats was analysed using morphometric methods, and the plasma testosterone concentration was measured. With increasing age there was an augmentation in the volume of collagen fibrils in the intercellular matrix and in blood vessels. During the aging process (approximately two years) the average volume of the Leydig cell decreased from 1364 microns 3 to 637 microns 3, but the number of Leydig cells in paired testes increased from 53 x 10(6) to 113 x 10(6). The absolute volume of smooth surfaced endoplasmic reticulum (SER) per Leydig cell amounted in aged rats to 78% of that in young adult rats. The total amount of SER in paired testes increased by 62% with aging. The present analysis suggests that the ability of SER to maintain peripheral testosterone concentration decreases with age. In young adult rats the absolute volume of peroxisomes per Leydig cell correlated significantly with the concentration of testosterone in blood and also with the absolute volume of SER per Leydig cell. These results combined with ultrastructural observations of close apposition of peroxisomes and SER suggest that peroxisomes have a role in testosterone secretion by Leydig cells.

Interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into CV-1 cell nucleus.

The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4 degrees C, and then chased for 2 h at 37 degrees C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membrane of some vacuoles seemed to be in contact with the outer nuclear membrane. Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40 near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membranes were often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4 degrees C, chased for 2 h at 37 degrees C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membrane as well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane, and further indicate that some endocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.

Migration of simian virus 40 from the vascular system into the glomerular mesangial region.

The behavior of simian virus 40 (SV40) injected into the vascular system was investigated in the rat glomerulus. The kidney was perfused via the abdominal aorta with a serum-free culture medium for 5 min, with PBS solution containing SV40 and then with the same medium for 15 min at 37 degrees C. In the glomeruli, SV40 particles were detected in the lumen of the capillaries, fenestrations of endothelial cells and lamina rara interna of the glomerular basement membrane. They were also found in the mesangial matrix and mesangial cells. Invaginations of their membrane were observed on several surface areas where SV40 particles were localized close to the surface. Similarly, when the particles were injected into the tail vein, they were detected in the lamina rara interna, the mesangial matrix, and in vacuoles of mesangial cells at 2 h after the injection. These results indicate that SV40 particles migrate from the vascular system into the mesangial matrix, and are then endocytosed in vivo by mesangial cells.

Immunological analysis and characterization of lymphocyte subsets in specimens of human hepatocellular carcinomas and metastatic liver cancers.

The distribution and number of CD2 (Coulter T11)+ cells, CD16 (Leu 11b)+ cells, Leu 7+ cells, CD8 (OKT 8)+ cells, CD11 (Leu 15)+ cells, CD4 (Leu 3a + 3b)+ cells and Leu 10+ or Leu 14+ cells in the liver of patients with hepatocellular carcinoma (HCC) and metastatic liver cancer (MLC) were investigated using monoclonal antibodies and immunohistological methods. In the majority of those with HCC and MLC, CD8 (OKT 8)+, Leu 7+ and CD16 (Leu 11b)+ cells were present both in the tumor and non-tumor tissues. The CD8 (OKT 8)+ cells were more numerous than Leu 7+ and CD16 (Leu 11b)+ cells. No significant difference was observed in the distribution and number of Leu 7+ and CD16 (Leu 11b)+ cells, in any area, in both groups. The number of CD8 (OKT 8)+ cells predominated in the non-tumor area, in both groups. CD11 (Leu 15)+ cells and CD8 (OKT 8)+ cells were present in the ratio of 1:3 or 1:4. The number of CD4 (Leu 3a + 3b)+ cells was less than that of CD8 (OKT 8)+ cells in both groups, especially in the tumor area. A few Leu 10+ or Leu 14+ cells were present in all areas, in both groups. In most cases of MLC, the CD8 (OKT 8)+ cells were absent in the tumor area. There was no correlation between the distribution and number of these cells and anti-tumor chemotherapy or non-specific immunotherapy.

Correlation of anionic residue mobility at the filopodial plasma membranes with multilayer formation in transformed 3T3 cells.

Simian virus 40-transformed 3T3 (SV40-3T3) cells formed multilayers on a Falcon dish and had numerous filopodial projections, some of which intertwined with those of adjacent cells, in contrast to the few projections of their nontransformed counterparts. When these cells were incubated with polycationic ferritin (0.5 mg/ml), ferritin particles, representative of anionic sites, were spread widely on their surfaces at 4 degrees C, while they formed clusters at 37 degrees C, especially on filopodial surface areas opposing adjacent projections in SV40-3T3 cells. These findings demonstrate an increase in the mobility of molecules with anionic residues on filopodial plasma membranes in SV40-3T3 cells, thus suggesting a role for these projections in the formation of multilayered cell aggregates.

Primary culture of epithelial cells derived from the rat ventral prostate: formation of three-dimensional acinus-like structure in collagen gel.

Rat ventral prostate epithelial cells were cultured in collagen gel after collagenase digestion. The primary cultures were mainly composed of single and spherical cells. After 10 days incubation in growth medium containing insulin, transferrin, and cholera toxin, there was a 3.8-fold increase in cell numbers, aggregates of which formed three-dimensional acinus-like structures. These structures consisted of one layer of cells surrounding the lumen. The cells were joined together with a junctional complex and had microvilli on the luminal surface and secretory vacuoles in the cytoplasm facing the lumen. The ultrastructural features of the cells were not altered by growth medium containing steroids. This culture system may prove to be very useful in elucidating proliferation, organization, and differentiation of prostatic epithelial cells in relation to the extracellular matrix and stromal cells.

Possible microapocrine-type secretion developing in prostatic secretory epithelial cells of older rats in the early stage of castration.

Secretory epithelial cells at 1 and 2 d after castration became higher than those of control as the result of convex protrusion of the apical cytoplasm into the acinar lumen, which was quickly followed by pinching off the small balloon-like cytoplasmic process developing on its surface. At the same time, the secretory vacuoles became more numerous, while the well-developed Golgi region, prominent rough endoplasmic reticulum and its dilated end facing the Golgi region remeined. On 7 and 14 d after castration, secretory epithelial cells became markedly low and they had very few secretory vacuoles, regressed Golgi region and a markedly reduced amount of rough endoplasmic reticulum. At 7 d after castration, marcrophage-like cells appeared among secretory epithelial cells and acinar basal lamina. These findings might suggest that the involutionary process of secretory epithelial cells was started by apical convex protrusion and accompanied by microapocrine secretion of its subsurface cytoplasm followed by phagocytosis of the remaining disintegrated cytoplasm by macrophage-like cells.

Fusion of SV40-induced endocytotic vacuoles with the nuclear membrane.

The interaction between simian virus 40(SV40)-induced endocytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4 degrees C. Following incubation of these modified cells at 37 degrees C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37 degrees C in the same medium, many various-sized vacuoles were present that contained membrane-bound CF, Con A and SV40. After 2 h of incubation at 37 degrees C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane.

Immunohistochemical characterization of inflammatory infiltrates at the site of bile duct injury in primary biliary cirrhosis.

Surgically obtained liver specimens from four patients with primary biliary cirrhosis (PBC), Stages I-II, were studied immunohistochemically using a broad panel of monoclonal antibodies. At the site of bile-duct injury (CNSDC), the inflammatory cells were recognized to be Coulter T-11+ (directed against all T-cells) and OKT-8+ (directed against cytotoxic/suppressor: C/S T-cells) cells. The MHC-class I antigen (i.e. HLA-A, B, C) was expressed weakly in the cytoplasm of the minority of damaged bile-duct epithelial cells, and the MHC-class II antigen (i.e. HLA-DR) was not expressed. Thus, OKT-8+ cells may play an important role in the immunologically mediated destruction of ductular epithelium in PBC. Strong MHC-antigen expression and OKT-8+ cell infiltration in destructive bile-duct lesions were not simultaneously observed. In the portal lymphocyte-rich areas, OKT-4+ (directed against helper/inducer: H/I T-cells) predominated over OKT-8+ cells. B-lymphocytes were present predominantly in the peripheral zones of the lymphoid aggregates. The distribution of helper/inducer T-cells and B-lymphocytes indicates that they may play a role in the induction of immunoglobulin in the lymphocyte-rich areas. The inflammatory mononuclear cells within the granuloma observed in one patient were Coulter T-11+, OKT-8+ and Leu-3a+3b+ (directed against helper/inducer T-cells) cells.