ULTRASTRUCTURAL EFFECT OF GOSSYPOL ON THE ADRENAL CORTEX OF THE RATS / 解剖学报

This paper presents ultrastructural observations on the adrenal cortex of rats following gossypol administration of a daily dose of 10 and 25 mg/kg respectively for various time periods. Stereological analysis was olso carried out on the cellular components of zona glomerulosa.No detectable ultrastructural changes were observed in the glomerulosa cell organelles in the treated rats of 10 mg/kg dosage groups. However, changes, such as increase of the number of mitochondria, derangement and decrease in the number of their cristae, distension of endoplasmic reticulum and Golgi apparatus, and increase in lipid droplets were evident in the zona glomerulosa cells of experimental animals treated daily with 25 mg/kg of gossypol. Sstereological analysis of the zona glomerulosa cells also indicated that mitochondria and Golgi apparatus had a tendency to increase in number and size, although There was no significant difference statistically from that Seen in control animals. "Anomalous cells" that had never been seen in the control animals were observed in the zona fasciculata of almost all experimental animals treated daily with 25 mg/kg of gossypol. These cells were characterized by thier condensed cytoplasm and smaller size, shrinkage of nuclear membrane, decrease in amount of smooth endoplasmic reticulum as well as denature or decrease in mitochondrial matrix. Other compensatory changes such as the appearance of whorled membranous bodies and cholesterol crystals in the zona fasciculata cells were also discernible.No obvious changes in zona reticularis of rat adrenal cortex following gossypol treatment could be observed. The nature and the significance of the ultrastructural changes in the adrenal cortex following gossypol treatment are a nalysed and discussed.

ULTRA-CYTOCHEMICAL OBSERVATIONS ON THE EFFECT OF GOSSYPOL ON Na-K-ATPase OF THE RENAL CELL MEMBRANE OF RATS AND GUINEA PIGS / 解剖学报

Gossypol was found to have an inhibiting effect on spermatogenesis in animals and has been used as an antifertility agent in clinical trial. However, a very small proportion of subjects on gossypol were found to be hypokaleimia. The direct cause of it was due to renal potassium loss. The present study was designed to make an ultrastructural observation on the enzymatic activity of the membrane protein Na-K-ATPase, the so called sodium pump of renal tubular cells of rats and guinea pigs following gossypol treatment. Gossypol was administered to male animals at a dosage of 30 mg/kg body weight daily. Kidney tissues were removed at various time intervals after gossypol treatment and processed for ultra-cytochemical study and quantitative measurement of Na-K-ATPase. The results obtained were as follows.1. The enzymatic activity of Na-K-ATPase of kidney tissues was shown to decrease slightly following gossypol treatment in both rats and giunea pigs, but there was no significant difference between the gos ol treated and the control groups.2. Ultra-cytochemical observations on the localization of Na-K-ATPase demonstrated that the enzyme reactive products in renal distal convoluting tubules, ascending limbs of Henle's loop and collecting ducts desending through the cortex to the outer medulla were mainly localized to the basal plasma membranesand their retromembranous folds of the epithelial cells. A small amount of the reactive products associated with the luminal surface of the distal convoluting and collective tubules were also observed. The amount of enzyme reactive granules of the treated animals did not differ significantly from that of controls.3. The results of perfusion experiments with ouabain indicated that ouabain inhibited Na-K-ATPase specifically in renal distal convolutions, ascending limbs and collecting tubules in both the treated and control animals.4. The cytochemical reactive granules in the erythrocyte and cardiac cell membrenes decreased in number following gossypol treatment.

HISTOCHEMICAL OBSERVATIONS ON THE PATHOLOGICAL CHANGES OF THE VISCERAL ORGANS FOLLOWING ROUS SARCOMA VIRUS INFECTION IN THE CHICK EMBRYO / 解剖学报

The pathological changes of the visceral organs i.e. the liver, spleen, lung, meso-nephros and gonad of the Rous virus infected chick embryo have been investigated. Theorgan tissues were removed at various intervals from 6 hours to 10 days after virusinoculation. They were fixed in Carnoy's solution and the sections were stained withmethyl green-pyronin. Some of them were stained with Alcian blue-hematoxylin for acidmuco-polysaccharide and toluidine blue for metachromatic reaction. Sequential histologi-cal examination on the process of pathological changes and their histochemical reactionsof the visceral organs revealed the following facts: Hemorrhagic lesions appeared in 60.3% of inoculated host livers from the 5-7 daysonward. Distinctive focal necrobiotic change of the liver cells, destruction of the endo-thelial cells, distention, hyperaemia and extravasation of the blood vessels and sinusoidswhich characterized the hemorrhagic lesions had been observed. Other pathologicalchanges such as the distortion of the hepatic cords, nuclear pyknosis and loss of stainingreaction of the liver cells in the hemorrhagic lesions were also found. Hemorrhagiclesions also appeared in the mesonephros, lung, intestine and heart of some of the em-bryos having received virus inoculation. In some of the embryos, focal sarcoma-like proliferation or tumor-like nodules werefound in the liver, lung and mesonephros after 7-9 days of virus inoculation. Alcianblue staining of the nodules or foci demonstrated the presence of acid muco-polysaccharidereaction in the ground substance, indicating an early phase of growth of Rous sarcomain the infected mesodermal tissues in these organs. It was observed that a certain number of pyroninophilic round cells occurred in allof the visceral organs of the host. They formed aggregates around the blood vessels orscattered in the mesenchymal tissues. Sometimes these cellular aggregates were quite ex-tensive, forming large patches. The incidence of these cellular aggregates or patches inthe liver, spleen, lung, meseonephros and gonad were 52.57%, 39.37%, 31%, 41%, and37.77% respectively. The cells in these patches had an Alcian-blue positive reaction ofacid muco-polysaccharide and metachromatic toluidine-blue staining. Both of the stainscould be removed by 3--5 hours treatment with testicular hyaluronidase. These cellswere identical with the cells which we had found previously in the CAM-tumor inducedby the Rous virus, and were considered also to be belonging to the basophilic myelocyteseries. Their possible role in the pathological changes in the visceral organs was dis-cussed.

STUDIES ON THE REGIONAL RESPONSES TO INDUCTOR OF THE CHICK BLASTODERMS OF DIFFERENT AGES / 解剖学报

As an extension of previous investigation, the competence of the epiblast was sudiedin further detail, with special reference to the regional response of the chick blastodermto inductor. Transplantation of Hensen's node grafts were performed according to themethods described previously. The host blastoderms were removed and fixed at variousintervals from 19--40 hours after operation. They were then stained with diluted Dela-field hematoxylin, cleared and examined as whole mounts. Most of the specimens weresectioned in series at 8? for further observations. Of a total of 134 operated specimens, 46 showed an induction of secondary struc-tures from the host blastoderms, in various degrees of regional differentiation 37, pro-duced placodal thickenings and the remaining 51 had no response in the blastoderms. It was observed that secondary structures with regional differentiation were inducedexclusively in the epiblast of host blastoderms of stages 5 and 6. The responsiveness ofthe epiblast decreased rapidly in older blastoderms. The latter either produced onlyplacodal thickenings (stage 7--8) or no response (stage 8--9) to inductive stimu-lus. Our experiments demonstrated that the pattern of the induced structures was deter-mined by a regionally different responsiveness of the epiblast of the host blastoderms,accompanied by the process of aging, rather than the specific actions of the inductors.The evidence was that when a head organizer (node graft of stage 4) was transplantedto the host blastoderm, the secondary head structure was induced exclusively in the headregion of the host, and of the secondary trunk-tail structure appeared in the trunk region.In other words, the prospective head epiblast of host blastoderms responsed to the in-ductor by forming a brain structure, whereas the prospective trunk epiblast, responsedto the same inductor (head organizer) by forming a spinal cord. Further evidence wasfound in some instances that the induced trunk-tail structure was often provided withpart of hind brain when the inductors were transplanted to the prospective hind brainregion. In this case, the epiblast of that region responsed to the inductor by forming ahind brain structure. The possible role of patterning of the induced structures upon the responsiveness ofthe epiblast that associated with regional host influence was discussed.

AN INVESTIGATION INTO THE PRoBLEM OF"CELL FORMATION FROM YOLK SPHERE"DURING THE DEVELOPMENT OF THE CHICK EMBRYO II.ON THE FORMATION OF ENTODERM AND OF THE "COLUMNAR EPITHELIUM"OF GERM WALL AND YoLK SAC WALL / 解剖学报

As an extension of previous investigations, the possibility of the yolk spheres of chick embryo developing into cells under normal and experimental conditions was studied in further detail. It was observed that the deeper cells of the early blastoderm made their morphological differentiation and gra- dually became separated by delamination from the superficial cells to form the primary entoderm; the yolk spheres that entered the subgerminal cavity, although located adjacent to or even intermingled with the deeper cells of the blastoderm, became disintegrated in later stages and did not take any part in the process of entoderm formation. The so-called "columnar epithelial cells" of the germ wall and of the yolk sac lining were not derived from the cells or the yolk spheres of that region, but were transformed from a structure of multinucleated syncytium, with the yolk sphere or granules in its meshes, which is histologically characteristic of the germ wall and the area vitellina interna of the yolk sac. They had an uncertain number of nuclei (from 1 to 3, or none at all) and an extraordinary large body size incomparable to any kind of embryonic cells (average 40-50? in length and 10-15? in diameter; the larger ones even reach 75-80? in length and 18? in diameter). The formation of these "cells" was found to be parallel with the expansion of the vitelline blood vessels along the yolk sac wall. Local cauterization indicated that outside the burned region in which the blood vessels were absent, the "columnar cells" failed to differentiate subsequently. The arrival of the blood vessels seems to induce the surrounding syncytial structure and yolk spheres to arrange themselves from an irregular state into long columns, and in such a way that the syncytial protoplasm and nuclei were moulded by the yolk spheres into a columnar structure, the so-called "columnar epithelial cells", but actually only a structure of syncytium. During this process, the yolk spheres underwent disintegration and liquefaction; some of them broke down into granules, droplets, and fragments of unequal sizes, with positive Feulgen's nuclear reaction. Some even showed an appearance of a pyknotic nucleus; but judging from their morphological detail and disintegration in the sucessive stages, they did not really show the characteristics of a nucleus. These results furnished further evidence that the yolk spheres of chick embryo could not develop into any kind of embryonic cell.

ULTRASTRUCTURAL OBSERVATIONS ON THE GONADOTROPHIC CELLS OF ADENOHYPOPHYSIS AND THE INTERSTITIAL CELLS OF TESTIS IN RATS FOLLOWING GOSSYPOL TREATMENT / 解剖学报

Observations of the ultrastructural changes on the gonadotrophic cells of adenohypophysis and the interstitial cells of testis were carried out in rats following gossypol treatment.It was found that four types of gonadotrophs in the rat adenohypophysis could be classified under the electron microscopic observation. The contral groups mainly consisted of the types Ⅰ and Ⅱ gonadotrophic cells and the latter appear to be the most numerous cell type, which constitute about 65% of the total gonadotrophs. Following gossypal administration, the number of type Ⅱ cells decreased obviously while that of the type Ⅲ cells increased steady and became the principal type of gonadotrophic cells in gossypol-treated animals. The proportion of the type Ⅲ cells were calculated as 56%, 44% and 58% of the total gonadotrophs after Ⅰ, 2 and 12 months of gossypol treatment, respectively. The type Ⅳ cells were characterized by the appearance of dilated vesicles derived from rough endoplasmic reticulum which occupied almost the entire cytoplasm. Morphologically, these cells were similar to "castration cells" which appeared in the rat adenohypophysis after gonadectomy. They were observed in the experimental animal groups following two months of gossypol treatment, and their proportion was about 13% of the gonadotrophs.No significant ultrastructural changes in the interstitial cells of the treated rat testis could be observed.The significance of the above mentioned changes and the possible mechanism of gossypol effect on the gonadotrophs were discussed.

ULTRASTRUCTURAL OBSERVATIONS OF THE GOSSYPOL EFFECT ON THE SERTOLI CELLS IN RAT / 解剖学报

The present study was designed to investigate the ultrastructural changes of Sertoli cells of rat testis following gossypol administration at a dosage of 30 mg/kg/day for 4～6 weeks. Results indicate that 4 weeks after gossypol treatment, a series of ultrastructural changes concerning with phagocytic activity such as increased in amount of lysosomes, lipid droplets, ring or cup shaped mitochondria as well as the multiform changes of mitochondria and lysosomes were evident in the Sertoli cell. However, by 6 weeks after treatment, the Sertoli cells became "inactive" and began to show degenerative changes: distension and vesiculization of endoplasmic reticulum, accumulation of lipid droplets, cellular debris and lysosomes in varying sizes and phases, and the occurrence of atrophic changes of mitochondria. The nature of these changes in Sertoli cells following gossypol treatment and its significance was discussed.

AN AUTORADIOGRAPHIC STUDY ON THE UPTAKE OF RADIOSULPHATE (S~(35))——METHIONINE IN THE EMBRYONIC TISSUE VERSUS YOLK MATERIAL OF THE DEVELOPING CHICK EMBRYO / 解剖学报

Saline solution (0.9%),containing 5—10?c of radiosulphate(S~(35))——methio-nine(0.2—0.4ml)was injected into the yolk sacs of unincubated to 8—day chickembryos.Fixation was made after 6—24 hours of incubation in various fixa-tives (Bouin's,Carnoy's,neutral formalin,methyl or 95% ethyl alcohol).Mostof the embryos were stained in toto with Ehrlich's hematoxylin and eosin,butsome were stained with Wallback's Giemsa mixture of Mallory's modificationafter sectioning.Serial sections(5—7?) were used for contrast autoradiography.It was observed that a remarkable radiosulphate uptake in the embryonic tissuefollows the cellular pattern and was concentrated in proportion to the cellulardensity of tissue,the tracer is noticeable in the autographs by its ?-tracts andgrains over cytoplasm and nuclei of cells;while the yolk spheres entered thesubgerminal cavity and in the space between ectoderm and entoderm in thegerm wall were not radioactive.The blood vessels,erythroblasts and the mul-tinucleated syncytium,including the so-called “columnar epithelial cells” of theyolk sac lining and of the germ wall,also show high incorporation of radio-sulphate,but not at all in the yolk material.Furthermore,the yolk granules,droplets of unequal size with positive Feulgen reaction,and those even withan appearence of pyknotic nucleus in the “transitional zone” of yolk sac liningas described in the previous investigation,also show no tracer uptake.It isobvious,that radiosulphate is not incorporated into the yolk material.Fromthe above,it may be assumed that no protein synthesis or resynthesis(“??-????????”) occurs in the yolk sphere.These results are in good agreementwith our previous findings and afford further evidence to the conclusion that theyolk spheres are not a living substance incapable of developing into embryoniccell.

ULTRASTRUCTURAL OBSERVATION OF THE TOXIC EFFECT OF GOSSYPOL ON RATS / 解剖学报

The present study was designed to examine the toxic effect of gossypol on the heart, liver, kidney,adrenal and testis of the Wistar rats with the electron microscope. Gossypol was administered orally at a daily dose of 30mg/kg body weight for 1-3 weeks, the tissues were secured at various time intervals and prepared for observation. It was shown that no significant ultrastructural changes could be observed in the heart muscle, kidney, and adrenal of animals fed on gossypol at the dosage that had damaged to testis. The latter demonstrated obvious degenerative changes in spermatids and spermatocytes 3 weeks following treatment and appeared to be more sensitive to the effect of gossypol poisoning. Gossypol induced distension of the endoplasmic reticulum and caused an increases in amount of smooth endoplasmic reticulum as well as lysosomes of the hepatic cells. Other subtle ultrastructural changes in the hepatic cells including the decrease of glycogen granules, displacement of ribosomes from rough endoplasmic reticulum and the distension of canaliculi biliferi were also discernible. Based on the above results, the toxic efficacy of gossypol was analysed and discussed.

EXPERIMENTAL ANALYSIS ON THE REACTIVE CAPACITY OF THE EPIBLAST OF THE CHICK BLASTODERM / 解剖学报

It is well known that the competence of the prospective ectoderm of early amphi-bian gastrulae for neuralization changes with time and region of the embryo. In chickembryo, Woodside (1937) demonstrated also that the neural competence of the epiblastdecreases with increasing age. However, no experimental data bearing on the regionaldifference in competence have been offered. This report represents an attempt to givean analysis of the reactive capacity of the ectoblast of the chick blastoderm with specialreference to time and space. The grafts of Hensen's node at the primitive streak stage, ranging in size from0.3--0.4 mm square were transplanted to the host blastoderms of different ages, varyingfrom the early head process stage up to 4 somites stage (i. e. corresponding to the deve-lopmental stages of 5, 6, 7 and 8 of Hamburger and Hamilton). The grafts were placed underncath the ectoderm at various levels (i.c. antero-latcral to the node or lateral to thenode and streak) of the host. They were then removed at various intervals from 6--30hours after operation, preserved in Bouin's fixatives and stained with dilute Delafieldhematoxylin. It was observed that neural formation, e.g. neural epithelium, neural plate or neuralfold could be induced in the whole epiblast of host blastoderms of stages 5 and 6. Thereactivity changed quickly during the neural fold formation stage (stage 7), the epiblast atthis stage produced only placodal thickenings, except in a few cases the neural formationwas induced in the anterior epiblast of the blastoderm. The placodal thickening couldalso be called forth in the anterior epiblast of host blastoderm of stage 8 (2--4 somitesstage), but the posterior epiblast was incapable of any response to inductors. Ifl otherwords, the competence for neuralization vanished shortly after the late head fold stage(stage 6--7), and the capacity for placodal thickening response vanished in the 4-somitesstage (stage 8). These results clearly demonstrated that the reactive capacity of the epi-blast to inductors decreased with increasing age of the host at the time of transplanta-tion. The results presented here together with those obtained in our previous findingsshowed that there was no regional difference in neural competence in the epiblast of theprimitive streak stage, but such difference did develop gradually with increasing age ofthe blastoderms. Our data indicated that the frequencies of neural response to inductorsof the epiblast were higher at regions anterior and lateral to the Hensen's node andlower at the posterior region in the blastoderm of stages 5 and 6. The frequenciestended to decrease along a gradient axis in a cephalocaudal direction, and appeared tobe no difference in capacity in the epiblast between the left and right side of the blasto-derm. Finally, we also observed in some instances that when a node graft was transplantedto the posterior border of the pellucida area of the blastoderm, a gut-like epitheliumcould be induced from host germ wall endoderm which had been in contact with thechordal mesoderm of the graft. The possible role of endoderm differentiation uponchordal mesoderm and the significance of their relationship are discussed.

HISTOCHEMICAL OBSERVATIONS OF THE NUCLEIC ACID AND ACID POLYSACCHARIDE ON THE PATHOGENESIS OF THE ROUS SARCOMA INDUCED BY ROUS VIRUS ON THE CHORIO-ALLANTOIS MEMBRANE OF THE CHICK EMBRYO / 解剖学报

Preparations of cell-free supernatant from Rous sarcoma "?????" were inocu-lated onto the chorio-allantoic membranes (CAM) of the chick embryos of 7--9 days in-cubation. The inoculated portions of the CAM were removed at various intervals from6 hours to 10 days afterwards. The tissues were fixed in Carnoy's solution and werestained with methyl green-pyronin for nucleic acid, Alcian blue-hematoxylin or Alcianblue-alum cochineal for acid polysaccharide and toluidine blue for metachromatic reac-tion. The pathogenesis of the Rous sarcoma of the inoculated CAM and the histo-chemical reaction of the nucleic acid and acid polysaccharide have been followed by his-tological examinations. Recognizable macroscopic lesions consisting of membrane opacity, focal thickening,oedema and discrete nodules in the inoculated CAM appeared from the first day on-ward. Distinctive hemorrhagic lesions and pocks were observed on the 3rd to the 5thday. Tumor masses of different sizes appeared from the 7th to the 9th day. In casethe CAM were inoculated with horse serum-saline, no or only slight reaction was ob-served, and the normal thickness and structures of the CAM were generally maintained. Three successive stages of the Rous sarcoma on the inoculated CAM weredistinguished: (1) Focal ecto-mesodermal proliferation with formation of earlytumor-like nodules; (2) Extreme ectodermal hyperplasia and early mesodermalsarcoma-like proliferation; (3) Ectodermal necrosis and extensive malignant growthof the mesodermal neoplastic cells. The fibroblasts, pyroninophilic round cells andthe ectodermal cells were the main types of cells concerned in the developmentof tumors following infection of the CAM Rous virus. The fibroblasts transformedinto tumor cells and became the most conspicuous elements of the Rous sarcoma. Thepyroninophilic round cells were considered to be belonging to the basophilic myelocyteseries, according to their morphological and staining characteristics. Their possible rolein the tumor infiltration was discussed. The intensity of the nucleic acid in the CAM cells increased along with the increaseof histopathological changes at the inoculated area of the membrane. The possible sig-nificance of their parallel relationship was discussed. Alcian blue staining of the growing tumor tissue demonstrated the presence of acidpolysaccharide reaction in the ground substance. The intensity increased with the increaseof the malignancy of the neoplastic cells. This material is non-metachromatic whenstained with toluidine blue and could be removed by treatment with testicular hyaluroni-dase for 3--5 hours. The mucin present is considered to be of a hyaluronic acid nature.