Daily examination of the vaginal smears of mature C3H mice was carried out more than 1 year. The control 21 mice showed almost regular estrous cycles in the duration of the age of 2 to 12.5 months. Twenty-eight mice, injected by 2.5 mg of DMBA at the age of 52 or 59 days, could not reveal a regular appearance of estrous cycle in the whole life after the single injection. The DMBA-injected mice were begun to be examined the vaginal smears at the age of 45 days, and devided to 4 experimental groups, depending on the stage of estrous cycle in which the single injection was performed. These groups were neither connected to the type of following irregular estrous cycles, to the incidence of the ovarian tumors, nor to the histological pattern of the established ovarian tumors.
DMBA promoted to reveal an estrus-stage immediately after the single injection, and produced a diestrus-dominant stage 1-4 weeks after the injection. This biological phenomen suggested DMBA could injure the estrogen-producing cells directly.
Four types of irregularity of the following estrous cycles after the injection of DMBA were classified. The 1st type was almost regular as same as the controls and could not be observed on the experimental groups. The 2nd type was very irregular and showed a continuous diestrus in the latter half period of the experiment. The 3rd type was extremely irregular and showed a continuous estrus in the latter half period. Number of the mice in the 2nd or 3rd type was almost the same each other, and less half of the treated animals.The 4th type, about 10% of the injected mice, showed no estrus in the life after the injection. The last type was subdivided a- and b-subtype. IV-a type revealed never estrus in all life span. IV-b type showed few estrus in the course after the injection, even estrus was almost always detected immediately after the single injection. The mice showing II type had atrophic ovaries, small luteoma, cystoma and blood yst without thecal cell proliferation, or follicular type granulosa cell tumors. Incidence of the macroscopic tum or in II type was 33%. The mice showing III type beared diffuse. type of undifferentiated granulosa cell tumor, or the abnormal ovaries with thecal cell proliferation, in which lutein cells hardly be detected. The induction rate of ovarian tumors was macroscopically 69%in III type. The IV-typed mice had hypoplastic ovaries and no ovarian tumors macroscopically.
Estrogen-producing cells in the DMBA-induced ovarian tumors of C3H mice would be the proliferated thecal cells and interstitial gland cells in the tumor, or undifferentiated tumor cells close-related by sex-cord mesenchymal cells.

View full abstract

[in Japanese]

View full abstract

[in Japanese]

View full abstract

[in Japanese]

View full abstract

Vol. 21 (1969) No. 4 p. 171-172

View full abstract

The ovaries of each 2 C3H mice of the age of 2 months or 12 months were observed by an electron microscope. In comparison of the findings on these controls, the effects of DMBA upon the ovarian tissues were investigated electron-microscopically. The 4 C3H mice, injected by 5 mg of DMBA-emulsion at the age of 45 days, were studied 7 days after the single injection. Some degenerative alterations in the mitochondrion, the polysomes and the smooth-surfaced endoplasmic reticula were detected on the ovum and the granulosa cells. Many dilatated ergastoplasms, coarse lipid granules and giant dense bodies could be found in the theca' cells and the interstitial gland cells. But, any changes were not demonstrated in the lutein cells. The ovaries of each 2 C3H mice, injected with 2.5 mg of DMBA-emulsion at the age of 2 months, were studied 2 or 5months after the single injection. Neither any ovum nor mature granulosa cells were detected in the ovaries. Some luteinizing sex-cord mesenchymal cells having both the tubular-structured mitochondrion and the small vesicular, smooth-surfaced endoplasmic reticula were seen in the abundunt collagenous stroma.
Pathological ovarian tissues of 13 old C3H mi ce, injected by 2.5 mg of DMBA-emulsion at the age of 52 or 59 days, were observed electron-microscopically 7-11.5 months after the single intravenous injection. In 3 cases of follicular type of granulosa cell tumor, more loose attachment of cytoplasmic membranes of tumor cells, nuclear and nucleolar predominances, abnormally dilatated rough-surfaced endoplasmic reticula, less polysomes, and decrease of the mitochondrion were indicated on the tumor cells. In 6 cases of diffuse type of undifferentiated granulosa cell tumor, the tumor cells indicated nuclear predominance, smaller vesicular smooth-surfaced ergastoplasms and spindle-shaped mitochondrion having some papillary cristae in their narrow cytoplasms. The polysome in them was less than the follicular granulosa tunir cells, but the rough-surfaced endoplasmi 'reticula were more detected in the tumor cells than inthe follicular granulosa tumor. In the focus of reactive cell proliferation accompanied by 1 ease of ovarian blood cyst, many electronmicrographs similar to the thecal cells were seen. Three cases of the atrophic ovaries revealed many luteinizing mesenchymal cells in the collagenous tissues. Electron-micrographs of the DMBA-induced ovarian tumors in C3H mice would indicate that the tumor cell of diffuse type of undifferentiated granulosa cell tumor is resemble to the undifferentiated sex-cord mesenchymal cells of the ovary, and that the tumor cell of follicular type of granulosa cell tumor is similar to the granulosa cells of growing follicles of the ovary.

View full abstract

The control 21 C3H female mice, aged 1 -2 years, had neither nodular hyperplasia nor adenoma in the adrenal cortex. Intravenous injection of 5 mg of DMBA-emulsion failed to cause any immediate acute toxic effects on the adrenals of 8 C3H female mice aged 45days. In the 35 C3H female mice, injected singly by 2,5 mg of DMBA-emulsion at the age of 45,52 or 59 days,2 cases of type A cell adenoma of the adrenal cortex and 1of the same cell hyperplasia were detected at the age of 11-15 months. The electronmicrographs of the tumor cells looked to be the undifferentiated subcapsular mesenchymal cells of the adrenal gland. Any type B cell adenoma or hyperplasia was not recognized, in the same experimental group, but 1 adrenocortical calcification, following by an acute necrosis, was found.
Ovariectomy of all 3 C3H mice at the age of 43 days led to the bilateral type B cell hyperplasia at the age of 10 months, but no adrenal tumors appeared in the same mice. Four female C3H mice out of 7 survivors, received with ovariectomy at the age of 43days and with single intravenous injection of 2.5 mg of DMBA-emulsion 2 days later, had unilateral type B cell adenoma at the age of 10 months.
In berief, DMBA would hardly have acute necrotizing effect on the adrenocortical cells of C3H female mice, but it could cause a neoplastic transformation in them. Ovariectomy or presenile changes evoked by DMBA would accerelate the development of the adrenocortical tumor.

View full abstract

The serum enzymatic activity of phosphohexose isomerase (PHI), lactic dehydrogenase (LDH), isocitric dehydrogenase (ICD), GOT, GPT, leucine aminopeptidase (LAP), alkaline phosphatase (ALP) and β-glucuronidase (βG) was studied in 53 cases of acute hepatitis,42 cases of chronic hepatitis (active type 5, inactive type 37),27 cases of liver cirrhosis,18 cases of liver cancer,4 cases of bile duct cancer,23 cases of bile duct and gall bladder disease,40 cases of chronic gastritis,54 cases of gastric and duodenal ulcer and 39 cases of gastric cancer.
Experimen tal studies were made of serum enzymatic activity in rats with Yoshida ascites sarcoma biliary duct ligation and CC14 hepatic disturbances.
The results obtained were as follows:
1) Cases of acute hepatitis showed a marked increase in enzymatic activity, especially in ICD, GPT, βG, GOT and LAP.
2) Cases of chronic hepatitis showed a slight increase in the serum activity of ICD, LAP and βG.
3) Cases of liver cirrhosis showed an enzymatic pattern similar to that in cases of chronic hepatatis.
4) C ases of liver cancer showed a marked increase in the serum enzymatic activity of PHI, LDH, ICD, LAP and βG.
5) Cases of bile duct cance r showed an enzymatic pattern similar to that in cases of liver cancer, but the former showed a slight increased level of LAP, ALP and βG over the latter.
6) Cases of bile duct and gall bladder disease showed a normal enzymatic activity, except for a moderate increase in ALP and LAP.
7) Cases of gastric and duodenal ulcer sh owed a normal enzymatic activity.
8) Cases of gastric cancer showed an increase in the activity of PH I, LDH and ALP.
9) An increase was observed in the ICD and ALP activify in rats with Cat hepatic disturbances in the ALP and βG in rats with biliary duct ligation and in the LDH and βG in rats with Yoshida ascites sarcoma.
This approach seems to be useful as a supplementary means of the differencial diagnosis of digestive diseases.

View full abstract

Fructose 1,6-diphosphate (FDP) or fructose 1-phosphate (FIP) is used as substrate in determining aldolase (ALD) activity. An FDP/FIP activity ratio, toward the substrates of FDP and FIP is about 1 for ALD B, and about 50 for ALD A. The malignant tissue shows an increase in ALD A resulting in an increase of FDP/FIP activity, ratio in the serum.
Presently determination of FDP/FIP was made in digestive diseases cases' serum, tissue and the serum of rats with experimental hepatic disturbances. The ALD zymogram was made of the organ tissues of human subjects and animals.
1) Serum FDP/FIP activity ratio was 2.7 in healthy subjects,9.3 in gastric cancer cases and 10.4 in hepatic cancer cases. It was low, being almost equal to normal, in cases of benign digestive diseases.
2) Tissue FDP/FIP acti vity ratio proved to be 45.5 for the skeletal muscle,10.4 for the brain and 1.27 for the liver. It was 13.7 in the cancerous region,2.0 in the grossly normal region of liver cancer and 1.7 in the liver tissue of cancer patients without liver metastasis. It was markedly high in the region with a hepatic cancer infiltration.
3) FDP/FIP activity ratio in serum of rats with experimental hepatic disturbanc es. In cases of CC14 hepatic disturbances or common bile duct ligation. FDP/FIP was decreased, but in the regenerated liver the ratio which had decreased post-operatively showed a gradual, daily increase regaining the previous ratio in five days.
4) The ALD zymogram. Liver: In human subjects there were the A band and B band; a hybrid was observed near to the B band. In rats, only the B band was observed. Skeletal muscle: Human subjects and rats showed the A band near to the origin in the anode side. Human hepatic cancer: In the cancerous region only the A band was observed. DAB hepatoma: The B band became light and the A band appeared. Serum: On electrophoresis of acute hepatitis cases' sera a band was found at the B region. Determination of an FDP/FIP activity of serum and tissue A LD seems valuable in differentially diagnosing between the malignant and benign nature of digestive disease.

View full abstract

Terminal ductules of the resting mammary glands of young adult female rats were studied by an electron microscope to determine the limits of normal ultrastructural variability.
Fine pieces of the abdominal mammary-fatty tissues of Sprague-Dawley fem a le rats, aged 50-150 days, were packed into a small wire-meshed cago, and then fixed satisfactorily in Karnovsky's fixatives and postfixed in osmium tetroxide buffered with veronal-acetate. Sufficient dehydration in graded alcohols and propylene oxide was followed by embedding in Epon. After the careful trimmings, sections were cut on an ultrathin microtome and stained with uranyl acetate and lead citrate.

View full abstract

Distribution of the histochemical activity of phosphatases in the resting mammary glands of young adult female rat of Sprague-Dawley's strain was observed under an electronmicroscope, by means of Mayahara et al's method for nonspecific alkaline phosphatase (alk-P), Ogawa et al's method for adenosinetriphosphatase in high alkaline medium (ATPase) and Gomori's method for acid phosphatase (acid-P).
Abundant deposition of the reaction product for alk-P is seen along the folded plasma membrane of the epithelial cell. Intensity of the reaction is strongest at the infolded basal plasma membrane, but along the lateral plasma membrane interdigitation are sparsely present rather small precipitates. Deposition on the lateral plasma membrane becomes less and less as it approaches the luminal edge. No precipitates are found along the luminal surface, nor on the microvilli. Intracytoplasmic organelles of the epithelial cell are free from the deposits in exception of few lysosomal dense bodies located in basal portions of the epithelial cell. In the myoepithelial cell, myofilaments do not react after the method for alk-P and only parts of the surface plasma membrane, especially the pinocytotic vesicles, bear the deposits of reaction product. Intracytoplasmic organelles of the myoepithelial cell are also free from the deposits.
Histochemical activity of ATPase is recognized only along the lateral plasma membrane of the epithelial cell after the method for ATPase in high alkaline medium. The reaction product hardry be detected at the infolded basal plasma membrane of the epithelial cell, in the pinocytotic vesicles of the myoepithelial cell or on the myofilaments. Intracytoplasmic organells of both cells are free from the precipitates.
Reaction product for acid-P is seen in some lysoso mal dense bodies of both cells of the ductule, which are found more in the epithelial cell. It is detected on few basal plasma membrane of the epithelial cell. The other parts of both cells are free from the products.

View full abstract

Electron-microscopic observations were carried out on the ultrastructure and the localization of phosphatases in ductules of the mammary gland of Sprague-Dawley female rats, intubated singly by 20 mg of 7,12-DMBA at the age of 50-60 days, in the course of 3months after the administration. Induced mammary carcinomas with the treatment were also observed by the same methods.
Alterations of the ultrastru cture in the epithelial cells of ductules of the mammary gland could not be seen 18-24 hours after the intubation. Irregular dilatation of the perinuclear spaces in the epithelial cells was seen 2-7 days after the single treatment. In the same period, dilatation of the ergastoplasms and the Golgi's complex and increase of the secretion granules were also found.
General electron-microsc opic findings upon the ductules of the mammary gland at the stage of 3 weeks,1 or 2 months after the single administration indicated to be atrophic; bizar-figured, pycnotic nuclei, reduced Golgi bodies, and decrease of rough-surfaced endoplasmic reticulums and free ribosomes were observed. Abundant reaction products for alk-P could be seen still at the basal plasma membrane of the epithelial cells. The distinguished changes could not be detected in the myoepithelial cells.
Ultrastructure of the induced mammary cancer with 7,12 -DMBA suggested to be anaplastic epithelial cells from the ductule of the mammary gland. Intracytoplasmic organellaes of the tumor cells, e. g. the nucleus, the rough-surfaced endoplasmic reticulums or the ribosomes were immature. Especially the Golgi complex was small, multiple, and it revealed no similarity in distribution. Definite attachments of the connecting epithelial cells, mentioned above on the resting mammary gland, lost in the most of the tumor tissues. Histochemical activity of alk-P could not be proved at the plasma membrane of many tumor cells, even at the basal plasma membrane. Alterations on the Golgi complex and the basal plasma membrane indicated that the tumor cells did lose their polarities.

View full abstract

[in Japanese]

View full abstract