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.

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.

Light and electron microscope histochemistry of alkaline phosphatase in the ventral prostate of rats.

Alkaline phosphatase was studied in the ventral prostate of rats by light and electron microscopy. In light microscopy, the enzyme activity was found in the luminal secretion and also on the luminal edge of the acinar walls as well as in the basal area of the acinar walls. It was also found in the walls of periacinar blood capillaries. The enzyme activity was abolished with addition of either L-phenyl-alanine or L-homoarginine and also of L-tartaric acid into the incubating medium. In electron microscopy, the enzyme activity was observed in the luminal secretory material and further it was observed strongly in the sharply localized pittings of apical surface of acinar secretory epithelial cells. The lower lateral and basal surface of the cells showed presence of rather weak activity of the enzyme. The basal cells showed strong activity of the enzyme in pinocytotic vesicles. Further, intense activity of the enzyme was observed on the cell surface of the fibrocytes immediately surrounding acinar basal lamina and also in pinocytotic vesicles of periacinar blood capillaries.

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.

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.

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.

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.

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.

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.

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.

Some ultrastructural effects of testosterone and insulin on the ventral prostate of rats in organ culture.

The fine structure of the secretory epithelial cells of rat's ventral prostate has been studied following organ culture. Culturing with either testosterone or insulin alone, and with the two hormones combined, were carried out to investigate how insulin modifies the action of testosterone on the maintenance of cellular integrity. After 4 days in hormone-free culture, the secretory epithelial cells showed signs of cellular atrophy and regression, involving loss of the apical microvilli, absence of the apical secretory vacuoles, atrophy of the Golgi apparatus, decrease in rough endoplasmic reticulum and the appearance of autophagic vacuoles. The presence in the medium of either testosterone or insulin alone, or combined, prevented cellular atrophy and regression. The best maintenance of cellular integrity was obtained in a culture containing both hormones. The effects of insulin was approximately equivalent to those of testosterone in the maintenance of cellular integrity.

The fine structure of the epithelium of prostate glands in adult female mastomys erythroleucus Temm.

The prostate glands in adult female Mastomys were studied with the light and the electron microscope. In the light microscopy, each prostatic acinus is lined by a single layer of tall cuboidal secretory epithelial cells which are surrounded by a capsule composed of a few layers of elongated and flattened cells. The acinar secretory epithelial cells show no presence of the supranuclear light areas in their cytoplasm and tend to be more readily stained with toluidine blue in the perinuclear and the upper basal cytoplasm than in the rest cytoplasm. In the electron microscopy, sharply localized pittings are occasionally found on the luminal surface and further the secretory granules which are closely situated to the apical cell surface are recognized. In the supranuclear cytoplasm, several well-developed Golgi apparatuses appear individually and early developing secretory granules are also observed. In the perinuclear and the upper basal cytoplasm the rough endoplasmic reticulum is well developed while in the lower basal cytoplasm the smooth endoplasmic reticulum is conspicuously developed and predominates.

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.

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.

Cytochemical studies of acid phosphatases in the rat lateral prostate with special reference to secretory apparatus and lysosome system.

A cytochemical study of acid phosphatase (AcP-ase) in the lateral prostate of the rat was performed to investigate whether AcP-ase in the secretory apparatus can be distinguished from AcP-ase in lysosomes and their related structures. Two types of AcP-ase were observed in the rat lateral prostate. One was found in the secretory apparatus (Golgi saccules and some Golgi vesicles, condensing and secretory vacuoles), and reacted well with naphthol AS-BI phosphate (N AS-BI P) as substrate; the other was found in the lysosomes and Golgi-associated endoplasmic-reticulum-lysosome system (GERL)-like structure, and reacted well with beta-glycerophosphate (beta GP) as substrate. Although the AcP-ase which reacted well with N AS-BI P was also observed in certain portions of pleomorphic lysosomes, it was concluded that it was the same as the AcP-ase found in the condensing and secretory vacuoles, since a lysosome engulfing a condensing vacuole was often observed. Therefore, it is concluded that the AcP-ase in the secretory apparatus in the rat lateral prostate is different from the AcP-ase in lysosomes. Condensing vacuoles appear to originate from particular portions of Golgi saccules, but not from the GERL or GERL-like structure, at least in the rat lateral prostate.

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.

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 fine structure of ventral prostatic secretory epithelial cells in older rats.

The ventral prostatic secretory epithelial cells in older rats were studied by light and electron microscopy. The cells vary in height in different parts of the same organ, and ultrastructurally they show the presence of a developed secretory apparatus such as well-developed Golgi body and abundant rough endoplasmic reticulum. They also show signs of a depressed secretory activity, involving occasional emiocytosis of apical secretory vacuoles and a paucity of condensing vacuoles in the Golgi region and above it. Further, they are characterized by the frequent occurrence of supra and paranuclear pleomorphic lysosomes.