26-hydroxycholesterol-stimulated DNA synthesis in smooth muscle cells and induction of endothelial injury using a coculture technique

We investigated the effects of 0.5, 2.5, and 10 micrograms/ml of cholesterol or 26-hydroxycholesterol on bovine aortic ECs and SMCs. Suppression of viable cell density and cytotoxic changes in both cells were induced by 2.5 and 10 micrograms/ml 26-hydroxycholesterol. ECs were more severely damaged than SMCs in the presence of 26-hydroxycholesterol. Levels of up to 10 micrograms/ml cholesterol had no effect on ECs or SMCs growth or cytotoxicity. Confluent ECs exposed to 2.5 or 10 micrograms/ml of 26-hydroxycholesterol secreted significant amounts of 6-ketoprostaglandin F1 alpha after a 24-hr incubation. An equivalent concentration of cholesterol had no such effect. SMCs cocultured with ECs exposed to 10 micrograms/ml 26-hydroxycholesterol, when compared with an equivalent level of cholesterol, synthesized DNA in significantly greater amounts during a 24-hr incubation. The cocultured ECs incubated in the presence of 2.5 and 10 micrograms/ml 26-hydroxycholesterol were partially detached due to cell death. However, no difference was observed in the DNA content of SMCs cultured without ECs in the presence of cholesterol or 26-hydroxycholesterol. The results suggest that 26-hydroxycholesterol produced not only cytotoxicity to ECs and SMCs but also stimulated DNA synthesis in SMCs through endothelial injury.     

Reaction of the cell center to exposure to the calcium ionophore A-23187

Calcium ionophore A23187, taken at a concentration of 0.1 microgram/ml, quickly uncouples mitochondria in PE culture cells in medium 199. The cell ultrastructure undergoes reversible changes (especially that of mitochondria): maximum changes occur 2 hours after the start of the treatment; in 8 hours they become less pronounced. The adaptation of cells does not involve the ionophore inactivation in the medium. 10 micrograms/ml of A23187 induces gradual but irreversible alterations. Microtubules in PE cells are not destroyed when incubated in medium 199 containing 10 micrograms/ml of A23187 and 11 mM Ca2+. The addition of 10 micrograms/ml ionophore to the normal 199 medium (1.26 mM Ca2+) results in the formation of electron dense bodies in the cell center 30 minutes after the start of incubation. These bodies disappear in the course of a subsequent incubation. The number of cells with primary cilia decreases. The percentage of centrioles located perpendicularly to the substrate increases 30 minutes following treatment with 0.1 microgram/ml A23187 in medium 199. 2 hours after the start of treatment with 0.1 microgram/ml ionophore no such changes are detected; an electron dense halo appears around the centriolar cylinders. 8 hours after the start of treatment the structure of the cell center does not differ from the normal one.     

Early activation and cell cycle entry of resting B cells after Fab-anti-Ig treatment: role of receptor crosslinking

Comparison of the effect of goat anti-rabbit Ig (GARIg) and its monovalent fragment (Fab-GARIg) demonstrates that surface Ig (sIg) crosslinking is not necessary to effect G0 to G1 transition in rabbit peripheral blood B cells but is required for induction of DNA synthesis. Five micrograms per milliliter or more of GARIg is sufficient to induce DNA synthesis but up to 50 micrograms/ml of Fab-GARIg is not. However, the monovalent reagent induces microscopically observable cytoplasmic and nuclear changes (blast transformation) in a dose-dependent manner. These differ qualitatively and quantitatively from the morphological changes seen with comparable doses of GARIg; Fab anti-Ig produces "small blasts" whereas complete GARIg induces large blasts. The monovalent reagent, in a wide range of concentrations, is as effective as the complete antibody in modulating sIg from rabbit B cells. Fab-GARIg treatment modulates sIg in a biphasic manner. It clears the high-density sIg within 5 min, whereas the remaining low-density receptors disappear after 4 hr. Cytosolic protein kinase C levels decline equally after treatment with either Fab-GARIg or whole anti-Ig. RNA synthesis, as measured by [3H]uridine incorporation, increases for the first 12 hr in cells activated with either reagent. It declines to basal levels in Fab-GARIg stimulated cells, but a continuous increase occurs in cells stimulated with 5 and 50 micrograms/ml of complete antibody. Simultaneous addition of 50 micrograms/ml Fab-GARIg with 5 microgram/ml of GARIg causes greater RNA synthesis for 12 hr after stimulation than is caused by GARIg alone. After 12 hr the monovalent reagent has an inhibitory effect on RNA synthesis. Fluorescence-activated cell sorter analysis of acridine orange-stained cells shows that Fab anti-Ig-stimulated cells have higher RNA content than resting cells, but lower than GARIg-activated cells. These findings suggest that rabbit B cells can be activated from the G0 stage of cell cycle to G1 by monovalent anti-Ig reagents but further cell cycle progression requires maintenance signals provided by receptor crosslinking. The implications of these results for B cell activation signalling are discussed in the context of the floating receptor model.     

The fibrinogenolytic activity of purified tryptase from human lung mast cells

The capacity of purified tryptase from human lung mast cells to metabolize human fibrinogen, fibrin, and plasminogen was evaluated. Tryptase (5 micrograms/ml) inactivated the thrombin-induced clotting activity of fibrinogen (100 micrograms/ml) with essentially similar t 1/2 values of 4.6 min in the absence of heparin and 5.8 min in the presence of heparin (20 micrograms/ml) that were not appreciably different than with lysine-Sepharose-purified plasmin (5 micrograms/ml). Fibrinogen treated with tryptase together with heparin lost all detectable clotting activity by 4 hr at 37 degrees C, whereas fibrinogen treated with tryptase alone resulted in destruction of only 80% of fibrinogen clotting equivalents after 16 hr. Tryptase alone was observed to cleave only the alpha-chains of fibrinogen by electrophoresis of tryptase-treated, denatured, and reduced fibrinogen in polyacrylamide gradient gels. Tryptase together with heparin cleaved first the alpha-chain and then the beta-chain, the latter cleavage corresponding to complete loss of fibrinogen clotting activity by 4 hr. No fibrinogen fragments with anticoagulant activity were generated by tryptase. In contrast, plasmin left no residual clotting activity after 4 hr of incubation and generated fibrinogen fragments with anticoagulant activity. Plasmin sequentially cleaved the alpha, beta, and gamma subunits of fibrinogen. Tryptase alone (6 micrograms/ml) or together with heparin (20 micrograms/ml) failed to activate plasminogen (0.6 mg/ml) after a 60-min incubation at 37 degrees C. Addition of urokinase to tryptase-treated or untreated plasminogen resulted in essentially identical plasmin activities (0.32 and 0.34 U/ml, respectively), indicating that tryptase neither activates nor destroys plasminogen. Tryptase (700 ng) also failed to substantially solubilize cross-linked fibrin (2.6 micrograms) or the corresponding amount of fibrinogen bound to plastic microtiter plates with or without heparin. The failure to solubilize fibrinogen and, possibly, fibrin is consistent with the observation that the apparent m.w. by SDS polyacrylamide gel electrophoresis of unreduced fibrinogen is not appreciably altered by prior treatment with tryptase, even though cleavage of alpha-and beta-chains is revealed after reduction. Fibrinogenolysis by tryptase complements other mast cell mediators with anticoagulant properties such as heparin and suggests a significant prevention of coagulation by activated mast cells.     

Change in acid phosphatase and N-acetyl-beta-glucosaminidase activity in mouse lymphocytes induced by concanavalin A

The treatment with concanavalin A (5 micrograms/ml) of mouse lymphocytes containing 70-72% of T cells entails an increase in the activity of acid phosphatase and a decrease in the activity of N-acetyl-beta-glucosaminidase. These changes were detectable 15 h after lymphocyte incubation with Con A. After 24 h of incubation acid phosphatase activity rose 2-fold whereas that of N-acetyl-beta-glucosaminidase dropped 45-50%. Possible mechanisms of these changes are discussed.     

Rhodamine 123 alters the mitochondrial ultrastructure of cultured L1210 cells

Exposure of exponentially growing L1210 cells in vitro to 5-10 micrograms/ml of rhodamine 123 (R123) for 16-48 hr inhibits cell proliferation and induces cell arrest in the G1A phase of the cell cycle. The cells remain viable during the arrest and resume growth after removal of R123; extended exposure to R123 is cytotoxic. Exposure to R123 results in morphological alterations in mitochondria of all cells observed; specifically, mitochondria of R123-treated cells are characterized by a distention of the intracristal spaces and a significant increase in the number of matrix granules. Gross morphological changes of mitochondria include formation of extended organelles and the appearance of doughnut-shaped structures.     

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.     

Features of the ultrastructure of loach embryos under the influence of norfloxacin

Results from a study of the ultrastructure of embryos of loach, Misgurnus fossilis L., on the stages of the first and tenth blastomere division under the control and in the presence of the fluoroquinolone series antibiotic norfloxacin (5 and 25 μg/ml) in the incubation medium are presented. The action of norfloxacin leads to ultrastructural changes in the cell organelles, such as hypertrophy of the rough and smooth endoplasmic reticulum and disorganization of the mitochondria and the plasma membranes of the embryos. It is established that fluoroquinolone inhibits biosynthesis processes that directly influence the biosynthesis structure of the blastomeres. Destructive changes in the organelles are a consequence of disturbances in assimilation processes that ultimately lead to death of the embryos. Thus, the results that have been obtained indicate that high embryotoxicity is characteristic of norfloxacin.     

Characterization of ATP-dependent Ca2+ uptake by canine brain microsomes with saponin

ATP-dependent Ca2+ uptake by brain microsomes was classified into two fractions according to the sensitivity to saponin. Properties of each fraction of Ca2+ uptake were examined and compared with those of inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum. The concentration of saponin for 50% inhibition (IC50) of major saponin-sensitive Ca2+ uptake was 11 micrograms/ml, and this uptake was enhanced by calmodulin. The minor saponin-insensitive Ca2+ uptake fraction (IC50; 90 micrograms/ml) was not affected by calmodulin but was enhanced by oxalate or 0.1 M KCl. The IC 50 of saponin for inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum was 11.3 and 114.8 micrograms/ml, respectively. A characteristic ring-like saponin-cholesterol micellar structure was observed electron microscopically in most membrane vesicles of brain microsomes and erythrocyte membrane vesicles but not in the cardiac sarcoplasmic reticulum. These observations indicate that saponin-sensitive and insensitive Ca2+ uptake was derived from plasma membranes and endoplasmic reticulum, respectively. Saponin proved useful for distinguishing the Ca2+ transport activity of plasma membrane from the Ca2+ uptake of other cellular organelles in the membrane preparations.     

Direct effect of gossypol on TR-ST cells: perturbation of rhodamine 123 accumulation in mitochondria

Gossypol has deleterious effects directly on TR-ST cells originating from a rat testicular tumor. Exposure of TR-ST cells to gossypol (5 micrograms/ml) decreases their rate of protein synthesis approximately 30% within 1 h and 65% by greater than 10 h, causes intracellular vacuolation, changes cell shape from cobblestone to a rounded conformation and inhibits cell proliferation. Yet, these gossypol-treated cells remain viable, as assessed by their ability to hydrolyze fluorescein diacetate. Gossypol also perturbs mitochondrial transmembrane potential in TR-ST cells, as demonstrated by marked changes in rhodamine 123 staining. Mitochondria of control TR-ST cells avidly accumulate rhodamine 123, but those in cells exposed to gossypol (greater than or equal to 5 micrograms/ml) for greater than 1 h fail to sequester the fluorochrome. Instead, the cell cytoplasm shows a light and diffuse staining with rhodamine 123. Rat spermatozoa show a similar response. Conversely, at concentrations of 20 micrograms/ml, gossypol has minimal effects on rhodamine 123 accumulation by primary cultures of hepatocytes and by rat spermatogenic cells, including primary spermatocytes and spermatids (Steps 1-12). Moreover, TR-ST cells exhibit reduced mitochondrial staining with gossypol at an ED50 of 7.6 micrograms/ml, while those for the nontesticular Rat-1, AnAn, 3T3 and PtK2 cell lines are 13.1, 21.5, 28.5 and 26.4 micrograms/ml, respectively.     

In vitro studies of the testis: the effect of LH on cyclic nucleotides.

This study evaluated the relationship between LH, cyclic AMP, cyclic GMP, and testosterone using in vitro incubation of decapsulated rat testes and sampling incubation medium. With added LH (1.0, 5.0, 100, and 500 mIU/ml) there were statistically significant increases in cyclic AMP at 5 mIU/ml or more LH, and progressively greater titers of this nucleotide were produced as LH was increased. For cyclic GMP all levels of added LH caused significant increments in titers of nucleotide; however, peak cyclic GMP concentrations occurred with 5 mIU/ml of LH. The addition of 10(-3) and 10-(4)M 8-bromo-cyclic AMP caused significant increases in testosterone production, while no changes in production of this androgen were found with 10(-3), 10(-4), or 10(-5)M 8-bromo-cyclic GMP. Neither cyclic AMP nor cyclic GMP titers were altered by the addition of 1 to 50 micrograms/ml of testosterone to medium bathing the rat testes. The dose response curves of cyclic AMP and cyclic GMP to LH are different. Progressive increments in added LH cause parallel increases of cyclic AMP and a biphasic change of cyclic GMP, 8-bromo-cyclic GMP does not cause testosterone generation, suggesting that cyclic GMP does not result in androgen synthesis. However, cyclic GMP may be involved in other Leydig cell functions.     

Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.     

Filamentous actin in paramecium cells: functional and structural changes correlated with phalloidin affinity labeling in vivo

Rhodaminylated (R)-phalloidin microinjected into Paramecium tetraurelia cells at a final concentration of greater than or equal to 20 micrograms/ml produces considerable functional and structural changes. F-actin bundles (with 20 micrograms/ml phalloidin within 15 min) are formed, which subsequently (greater than 30 min) are sequestered into autophagic vacuoles; simultaneously, the originally intense fluorescence of a narrow cortical layer becomes more and more diminished. When such microinjected cells are processed for electron microscopy, they display concomitant ultrastructural alterations, namely, the formation of transcellular bundles of 5-7 nm-thick filaments, which subsequently appear in autophagosomes, as well as a considerable reduction of filamentous materials in the cortex. This, in turn, entails a considerable restructuring of the cortex, enabling free access of various structural components to the cortex. Higher doses of R-phalloidin abolish cytoplasmic streaming (e.g., 50 micrograms/ml after 20-30 min); although the cells may survive, new secretory organelles (trichocysts) are no longer docked to the cell membrane. In contrast, exocytosis of docked trichocysts (as well as subsequent membrane resealing and retrieval) is not impaired under any conditions. Cortical F-actin may account for the cytoplasmic streaming that may normally guarantee the delivery of new trichocysts to free docking sites at the cell membrane. When docking is inhibited by high R-phalloidin doses, excess free trichocysts are sequestered into autophagosomes (crinophagy). One of the most sensitive cell functions is food vacuole formation (assayed by prelabeling with India ink), which correlates with the presence of R-phalloidin labeling in the cytostomal region and around food vacuoles. The main conclusions from this work are that filamentous actin may be involved in structuring of the cortex and in cytoplasmic streaming, and may therefore influence the formation, and possibly the transcellular transport (cyclosis), of food vacuoles, as well as the docking of trichocysts, whereas it does not play a role in exocytosis per se or in the steps immediately following.     

Effect of dimethyl sulphoxide and some antibiotics on cultured human T-lymphoma cells as measured by microcalorimetry

The influence of dimethyl sulphoxide (I), penicillin/streptomycin (II), gentamicin (III), and amphotericin B (IV) on growing human T-lymphoma cells was measured by microcalorimetry. There was a dose-dependent decrease in the heat production rate of the cells after 24 h of incubation with I in concentrations ranging from 0-2% (v/v). At 3.6%, about half of the cells died. II and III had no effect on the cells after incubation for 6 days, at concentrations from 1 to 10 times that of the normal (50-500 IU/ml; 50-500 micrograms/ml). IV was used in combination with II (50 IU/ml; 50 micrograms/ml) and III (50 micrograms/ml), respectively, at concentrations between 0.25 and 7.5 micrograms/ml. After 6 days of incubation, the results were similar to those obtained with II and III separately.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

SCE induction and cell-cycle delay by toxaphene

Toxaphene is genotoxic in mammalian cell systems and also inhibits cell replication. It was therefore used to investigate possible masking of SCE induction due to cell-cycle delay. In this study, toxaphene-treated Chinese hamster lung (Don) cells exhibited a dose-dependent decrease in cell-cycle progression compared with untreated cells. At high, nontoxic toxaphene levels (15 micrograms/ml), cell cycling also slowed as the toxaphene treatment time was increased. Toxaphene induced significantly higher numbers of SCEs in treated cells, demonstrating a dose- and treatment time-relationship. Slopes of dose-response curves were 0.29, 0.43 and 0.77 SCE/micrograms toxaphene for 20.5 h, 24.5 h and 28.5 h incubation, respectively. There were no changes in SCE values in control cultures even when slower dividing cells were sampled e.g. at longer incubation times. Thus, higher SCE values in Chinese hamster cells were not associated per se with slower or more delayed cells. The results demonstrate that longer toxaphene treatment times were not necessary for obtaining sufficient harlequin-stained cells for SCE analysis, but that higher numbers of SCEs occurred in slower dividing cells, following prolonged incubation of cultures treated with toxaphene.     

Induction of chromosome aberrations and sister-chromatid exchanges in CHO-K1 cells by o-phenylphenol

o-Phenylphenol (OPP), is used in Japan as a fungicide in food additives for citrus fruits. The induction of chromosome aberrations and sister-chromatid exchanges (SCEs) by OPP in cultured Chinese hamster ovary (CHO-K1) cells was studied. Cells were exposed to various concentrations of OPP ranging from 50 to 175 micrograms/ml for 3 h, and further incubated for 27 and 42 h. These incubation periods are almost equal to 2 and 3 cell cycles. SCEs and chromosome aberrations were induced by OPP at concentrations of 100, 125 and 150 micrograms/ml after the incubation for 27 h. For chromosome aberrations, chromatid breaks and exchanges there was a dose-dependent increase. Diplochromosomes due to endoreduplication were also caused by the same concentrations of OPP in a dose-dependent manner. After incubation for 42 h, chromosome aberrations were also increased by OPP at concentrations of 100 and 125 micrograms/ml, but the frequencies of SCEs were not significantly different from those of the control. These results suggest that OPP has a cytogenetic toxicity, and that the DNA damage resulting in SCEs induced by OPP is relatively short-lived and can be repaired during the longer incubation time.     

Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode

In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.     

Developmental and ultrastructual characteristics of mouse oocytes grown <Emphasis Type="Italic">in Vitro</Emphasis> from primordial germ cells

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.