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Haematoxylin and its oxidation products were studied by t.l.c., and i.r., n.m.r., u.v. and visible spectroscopy. Oxidation (‘ripening’) of Haematoxylin with oxygen, from the atmosphere or a cylinder, gave Haematein as the only major product; whereas with non-gaseous oxidants such as hydrogen peroxide, potassium permanganate and sodium iodate, further oxidation readily occurred. Such ‘over-ripening’ always produced the same major product: a yellowish quinone-carboxylic acid, which we term ‘Oxyhaematein’. Oxyhaematein did not form a complex with aluminium ions under staining conditions but did act as an acid dye. Thus the deleterious effects of the overripening of Haematoxylin and the reported yellow-orange acidophil background staining sometimes seen with metal-Haematoxylin stains may both be explained. However, an additional, or alternative, explanation is available for this orange staining: acidic solutions of Haematein were found to react with acidophils of tissue sections to give an orange-brown product, not extracted by the processing fluids.

Specimens of the annual salmonoid fish, Ayu, were collected from different places along the course of the Tassha-gawa River in Sado Island of the Japan Sea. The adenohypophysis was studied histologically to determine the cell types. For the purpose of examining the gland of marine life, some fish just entering the river were transferred to sea water.The rostral pars distalis consists mainly of two glandular cells: the dorsally shifted chromophobe cells facing the pars nervosa may correspond to the corticotrophs, and anteroventrally shifted acidophil cells are equivalent to the prolactin producing ones. Three types of cells are found in the proximal pars distalis: they are small, aldehyde fuchsin positive thyrotrophs bordering the pars nervosa; large, aldehyde fuchsin positive gonadotrophs which occur in the ventral region of the hypophysis and occupy an extensive portion of the pars distalis in parallel with sexual maturation; and the oval shaped orangenophil cells considered to be somatotrophs, lying between these two cell types mentioned above. The pars intermedia includes two cell types, larger and smaller ones, which could not be differentiated by their tinctorial responses.The acidophil cells in the rostral pars distalis undergo a marked hypertrophy and degranulation during the upstream migration of immature fish, whereas the cells in the fish kept in sea water showed only few of these changes.

Fifteen days after thyroidectomy, the pituitary acidophils of rats were definitely deprived of their large granules, about 350mμ in diameter each, and in due succession, they displayed the changes characterized by the features of “thyroidectomy cells” (TXcells). When these rats were given a single or three injections of thyroxine, 50mg/rat, most of TX-cells might revert to their original acidophils in a short time of 6 hr. The cytomorphological course of possible reversion of the TX-cells into acidophils was clearly demonstrated on our electron micrographs. It was learned that thyroxine exhibited a strong ability to produce large granules, 350mμ in diameter each, in TX-cells which might return to their original acidophils through the reduction of cell size probably due to contraction of ER and loss of intracisternal granules and through the re-production of the large granules in the ground matrix. If the rats, 15 days after thyroidectomy, were given dexamethasone (DM), 0.2mg/100g/day, for 14 days, most of TX-cells tended to slightly recover from their modifications due to thyroidectomy. DM had ability to produce both the small (130-150mμ in diameter) and the large (350mμ) granules in some TX-cells, but the rest of them remained unchanged and preserved a number of dilated cisternae and intracisternal granules. DM was greatly inferior to thyroxine in the ability to produce the acidophils of the large granule type (somatotrophs), while the former was superior to the latter in the ability to produce the acidophils of the small granule type. Our previous views that some TX-cells might originate from the degranulated acidophils was confirmed validly by the present observation. In conclusion, all TX-cells have not always been considered as hyperactive thyrotrophs. Mechanism of ER morphodynamics associated with vesiculation in TX-cells has been discussed with special reference to the development and reversion of TX-cells.

The accurate picture of the acidophil granules of the anterior pituitary which is provided by iron hematoxylin can be combined with the differential staining of the basophils by either the periodic acid-Schiff (PAS) or combined aldehyde-fuchsin-PAS procedures. To accomplish this the two stages of the iron hematoxylin technique are separated so that mordanting in iron alum precedes and application of hematoxylin follows the basophil procedures.

In general structure, the distal lobe closely resembles that of an amphibian, whereas the elaborate intermingling of nervous and intermediate lobes recalls the typical fish condition. In cell types, the majority of the intermediate-lobe cells have the same reactions as in amphibians but there is in addition another cell type which appears to be unique. In the distal lobe there are three types of basophils and two of acidophils, in many respects resembling the same five cell types found in amphibians. Developmental stages help to explain the structure of the neural-intermediate complex.

Several variants of surgical and radiotherapeutic technique have been employed in the treatment of pituitary adenoma, and this is reflected by the number of reports that have appeared. The earliest surgical approach was by the intracranial route (Horsley, 1906a, 1906b), which remains in use today, though the endonasal route was popular for a time (Cushing, 1912) and is still employed by some surgeons for ablation of the normal pituitary gland. Treatment of a series of patients by radiotherapy was first reported by Heinismann and Czerny (1926) ; sub sequently, primary treatment by radiotherapy became customary only for tumours that did not interfere with vision, while post operative irradiation was employed to reduce the incidence of recurrent tumour (Henderson, 1939). Despite the difference between patients treated surgically and those receiving radio therapy, particularly in the size of tumour and in the criteria adopted for diagnosis, most studies have reported both groups of patients together. For example, Henderson's (1939) report of 338 patients treated by Cushing includes patients operated on through both endonasal and intracranial routes, not all of whom received irradiation ; while another large study, that of Bakay (1950), comprises 292 patients similarly operated on by different methods and also given radiotherapy either before or after operation. Variations in treatment also occur in the studies of Horrax et al. (1952, 1955), N?rnberger and Korey (1953), Mogensen (1957), Ray and Patterson (1962), and Poppen (1963). By contrast, the 260 patients in the present series were treated by the same surgical technique, and, with few exceptions, by subsequent radiotherapy.

Earlier studies (1) showed the fluorescein-conjugated antisera to human growth hormone (HGH) localized in acidophils of human, rat, and bovine pituitary glands, and that the fluorescence, which was inhibited when the specific antigen or the unconjugated antisera was applied before staining, was specific.During inhibition studies, one batch of anti-HGH (titer, 1/12,000: batch HGH-5) inhibited fluorescence caused by another batch of fluorescein-conjugated anti-HGH (titer 1/25,000; Hg-1) in the human but not the rat pituitary gland. Inhibition was repeated with anti-HGH batch HGH-5 and followed by 1:2 and 1:3 dilution of fluorescein-conjugated anti-HGH Hg-1 (which still produced fluorescence); again, fluorescence was not inhibited in the rat pituitary. However, when conjugated and unconjugated anti-HGH Hg-1 from the same batch were used, staining was completely inhibited in both rat and human pituitaries. The present studies were carried out to determine the cause of this discrepancy.Materials and Methods. T...

In addition to the cytoplasmic secretory activity in the cells of the preoptic nucleus of the eel, Anguilla japonica, which is responsible for the production of the so-called Gomori-positive neurosecretory material, a characteristic secretory activity is disclosed with the nuclei of the cells in question. Various aspects of nuclear transformation are observed, which are regarded to be concerned with the elaboration of a kind of acidophil, Gomori-negative, secretory material. The secretory material is conceded to be produced as the result of a kind of pyknotic change of the cell nucleus, and partially extruded into the blood capillaries without being subjected to further transformation within the cytoplasm. The modified ncleus presumably restores its integrity through a process of differentiation of basophil elements from the remainder of the acidophil material. Such nuclear change is not associated with any visible sign of cellular degeneration, and are considered to be of characteristic features in physiologically normal animal.

Adrenalectomy cells, previously demonstrated by autoradiography in the light microscope as the site of ACTH production in the adrenalectomized rat, were identified in the electron microscope by their peculiar morphology. The adrenalectomy cells were found to have certain characteristic fine structural features which aid in identifying and differentiating them from the other cell types. Their secretory granules, with a maximal diameter of about 200 mμ, are larger than thyrotroph granules, smaller than acidophil granules, and correspond in size roughly to the gonadotroph granules. Gonadotrophs are readily differentiated from adrenalectomy cells, however, by their typically rounded shape. (Endocrinology 76: 70, 1965)

Changes in the cytology of the pituitary gland of the palm squirrel, Funambulus pennanti (Wroughton), following gonadectomy, androgen administration and propylthiouracil treatment are described. Increase in gonadotroph cell number and granulation reaches its maximum by one week after gonad ablation and is maintained at this level during the next three weeks, following which there is a gradual decline. Female squirrels show hypertrophied cells with prominent Golgi apparatus at 16 weeks after gonadectomy whereas males show similar changes four weeks after castration, changes which are still clearly noticeable at the end of 48 weeks. Castration cells or “signet-ring cells,” so characteristically seen in rats, do not occur in gonadectomized squirrels. Thyrotrophs are not affected by gonadectomy. Acidophils quickly decrease in number following gonadectomy, and are maintained at this level throughout the post-castration period. Administration of testosterone propionate to one week castrates causes degranulation in some gonadotrophs while others are coarsely granulated and deeply stained. The degranulated cells are interspersed with the other deeply stained gonadotrophs in the lateral halves of the pars anterior. Testosterone propionate administration to 12-week castrates produces no marked changes in the granule content of the gonadotrophs. Propylthiouracil treatment causes degranulation of cells located in the anteromedian zone of the pars anterior. Degranulation is partial in animals treated for 15 days but is compete after 30 days of treatment. Acidophils are partially degranulated after 30 days of propylthiouracil treatment.

Cushing's syndrome in infants is a rare disorder, usually caused by adrenocortical carcinoma. Clinical and pathological features of a fatal case occurring in a female infant 8 weeks of age are reported. Initial abnormalities were glycosuria, granulocytosis, and failure to thrive. The marked retardation of skeletal maturation, with bone age interpreted as less than term, suggests a prenatal onset of the endocrine disorder. The infant died after adrenal exploration. Both adrenal glands were hyperplastic and nodular while generalized wasting, sparing heart and kidneys, characterized the anatomic changes generally. An unexpected preponderance of acidophils was discovered in cell counts of the anterior pituitary.

IN a previous paper (Zeckwer, 1953), it was shown that the accelerating effect of adrenalectomy on regrowth of hair in the rat does not depend on the presence in the anterior pituitary of granulated acidophils. Thus in thyroidectomized rats with pituitaries devoid of acidophil granules there was accelerated regrowth of hair following adrenalectomy, although such regrowth had a longer lag period than in adrenalectomized rats with thyroids intact. Evidence was presented that the regrowth of hair after adrenalectomy might be due to an increased secretion by the pituitary of somatotrophic hormone (STH). Whatever the growth promoting substance may be, and, in particular, whether or not it is STH, remains for future work to reveal. In the meantime it is of interest to find out whether the substance promoting hair growth is sufficiently increased in amount after adrenalectomy to pass across to a parabiotic partner.

SINCE the thioflavine T (T.T.) fluorochrome method1 was first recommended for the demonstration of amyloid its use has been extended widely. Uptake of the dye has been shown to occur with myeloma casts2, muciphages in the intestinal mucosa3,4, keratin4, mast cells5,6, myelin figures in neurones of infantile amaurotic idiocy and some of the component cells in the granular cell myoblastoma7, acidophils in the pituitary8, juxtaglomerular apparatus cell granules in the kidney6,9 and zymogen granules in the pancreas of mice6.

Koneff's ('38) modification of Mallory-azan reveals the presence of azocarmine, orange G, aniline blue, and chromophobic cells in the pars distalis of the frog. A strong periodic acid Schiff (PAS) reaction, apparently of carbohydrate-containing material, uniformly distributed throughout the cytoplasm, is found in the azocarmine cells and in the aniline blue cells. Certain of the orange G cells contained PAS-positive material confined to cytoplasmic vesicles. The PAS-positive material is probably not glycogen. It was not possible to distinguish between the various PAS-reactive sites with the cytochemical methods employed in this study.

Male rats were thyroidectomized at 26 days of age by a combination of surgery and I131. Seventy days later, when growth stasis was complete, they were injected with 5 mg of iodide (as KI) daily for 28 days. At weekly intervals after the onset of injections, 5 rats were killed for study of their pituitary acidophils. A limited regranulation of acidophils was observed at the end of the 1st week of treatment. Thereafter, the response increased to maximal at 14 days, and then began to decline, being definitely reduced after 4 weeks. Weight gains and skeletal growth closely paralleled the changes in the acidophil population of the pituitary.

The cytological changes in the pars distalis of pituitary of male and female green frog, Rana esculenta during the reproductive cycle have been studied by examining the form, cytoplasmic granulation and numerical proportions of the different cell types. Some sex differences were recorded, particularly in relative total number of cells in a median sagittal section of the pars distalis. No extensive cytological changes occur during the hibernation period, but there are striking alterations at the time of spring awakening, during spawning months and during replenishment of the spent gonads i.e. during late summer and autumn months. Important changes are to be observed in the acidophils type one (A1) and basophils type two (B2) and three (B3). Changes in the A1 cells during breeding season have been speculated as being in support of the spawning activity of the animal. The B2 cells exhibit most profound changes which correlate precisely with the morphological changes in gonads, and these cells are considered as the FSH-gonadotrops. The functional significance of the B3 cells is discussed and it is suggested that they might be the source of an ICSH- or LH-like hormone.

Pituitaries of Syrian hamsters with and without chronic hyperglycaemia were studied with morphometric and karyometric methods. The hyperglycaemic state was induced by low doses of alloxan, and maintained for 25 to 37 days. It had been potentiated in one group by a resection of at least 50% of pancreatic tissue prior to alloxan administration. A marked rise in the volume of the acidophils was found (p< 0.005). On the other hand, the karyometric studies revealed a definite diminution of the mean nuclear area of the acidophils in the diabetic animals, indicating decreased activity. The combination of an increase in mass of acidophils and a decrease in their nuclear activity supports the theory of an increased storage rather than hypersecretion of a hormone. The hormone which was stored in the acidophils is probably identical with growth hormone. If we consider the role of this hormone in carbohydrate and lipid metabolism, such a storage in chronic hyperglycaemia seems to be of importance.

This report deals with a new technique developed by the author to isolate various types of anterior pituitary cells. Some of the principal procedures involved in this technique are: 1) initial digestion of pituitary tissues by trypsin in a calcium-and magnesium-free buffer solution of Rinaldini, 2) filtration of the dissociated cells through the stainless steel mesh, 3) treatment of the filtered cells with elastase and collagenase to prevent their aggregation and aggultination, 4) and final separation of the cells according to their types by centrifuging dextran A on the discontinuous gradient to which the filtered cells were added. Light and electron microscopic examinations of all the fractions after centrifugation confirmed a perfectseparation of chromophobes, undifferentiated cells of ambiguous nature, and intermediate cells between chromophobes and chromophils. However, the acidophils and basophils isolated by our technique were found intermingled with a small number of the intermediate cells.

The cytology of the pars distalis of pituitary green frog, Rana esculenta, was studied using various staining techniques. In general, besides the irregular occurrence of chromophobes, five chromophilic cell types were readily distinguishable (acidophils types 1 and 2, and basophils types 1, 2, and 3). The distribution of various cell types in the pars distalis has been described. The cytological modifications in the pars distalis of the male and female green frogs, gonadectomized during different periods of the sexual cycle, were studied at short and long intervals after gonadectomy. Administration of different doses of testosterone propionate and estradiol benzoate in the long-term castrates has been found effective in restoring the postgonadectomy changes. Similar treatment in the recently gonadectomized frogs prevented the occurrence of postgonadectomy changes. In general the basophils type 2 (B 2) exhibited the most profound modifications following gonadectomy and steroid hormone administration in both the male and female green frogs. The functional significance of the B 2 cells is discussed. It is suggested that these cells are the source of gonadotropic hormone(s) in this species.

Anterior pituitary glands from broody turkey hens were shown to have 4 times as much prolactin per milligram of fresh tissue as glands from laying and nonlaying turkeys. The prolactin activity was determined by intradermal injections over the crop glands of White King pigeons and estimated from a dose-response curve. Electron micrographs of sections of the pituitary glands from broody turkeys showed a highly developed endoplasmic reticulum in acidophils as compared with little development in these cells in nonlaying turkeys. This suggests a high rate of protein synthesis in the glands of broody turkeys that may be correlated with the increase in the prolactin content of these glands. The granules appear to be smaller in the acidophilic cells of the glands from broody birds as compared with the granules from the pituitary of nonlaying turkeys.

Precipitin reactions in vitro have shown that antibody to ovine prolactin is capable of binding ovine prolactin and a prolactin-like fraction prepared from the pituitary glands of the carp (Cyprinus carpio) and the pollack (Pollachius virens). It seems reasonable to postulate that it would also bind prolactin of other teleosts. Hence the globulin fraction of antiovine prolactin serum was bound to fluorescein isothiocyanate, purified, and the antibody solution used to localize prolactin within sections of frozen pituitary of Fundulus heteroclitus. Sections so prepared show the presence of green fluorescent antigen-antibody complex only in the acidophils of the rostral pars distalis. Control staining with fluorescent normal globulin failed to give this result. Serial sections adjacent to the fluorescent antibody-treated preparaions were stained histologically and the erythrosinophilic eta cells identified as those which contain prolactin. Since no other cells of the pituitary gave these reactions it can be concluded that the eta cells are the prolactin cells. Similar preparations from the pituitaries of fresh water adapted fundulus show that in the enlarged rostral zone the erythrosinophilic eta cells contain prolactin.

Recent findings have shown that thymus atrophy can be induced in young mice by a single intraperitoneal injection of rabbit antimouse pituitary serum. These mice also show inhibition of body growth and a cachectic syndrome with features similar to those in mice wasting after neonatal thymectomy [1]. It was observed that the degree of thymus involution with concomitant loss of small lymphocytes in the cortex parallels the inhibition of body growth. In animals which overcome the syndrome and gain weight, a repopulation of the thymus cortex and normalization of the thymus structure and weight is observed [2]. It has been shown that this antipituitary serum contains antibodies directed against the acidophilic, growth hormone-producing cells of the mouse anterior pituitary [3]. The observation of a degranulation of acidophils in the pituitary gland of neonatally thymectomized mice [4] and the findings on the reconstitution produced by gonadectomy in lymphoid tissue of neonatally thymectomized mice [5] have suggested that perinatal thymus is under direct or indirect influence of one or several hypophyseal hormones. These hormones are probably responsible for the turnover of the cells of the thymus cortex and for the high mitotic rate of this organ in the period of body growth until and after sexual maturation [2, 4]. The concept of perinatal thymus as target gland of the hypophysis has been strengthened by the observation that hereditary pituitary dwarf mice have a hypotrophic thymus and peripheral lymphoid tissue [6, 7].

The ultrastructure of the secretory cells of the adenohypophysis of juvenile sockeye salmon was investigated. Pituitary glands were collected from immature fish transferred experimentally to sea water and subsequently returned to fresh water. The rostral pars distalis contained three cell types: ACTH cells, prolactin cells, and non-secretory cells. The prolactin and non-secretory cells were joined together in the form of follicles by desmosomes and they both had cilia and microvilli projecting into the follicle lumen. Various follicular structures such as lumen, multivesicular structures, and peripheral basement membrane are discussed as possible sites of prolactin cell granule release. The columnar ACTH cells were found at the junction of the rostral pars distalis and the neurohypophysis. The cytoplasmic granules in these cells were characteristically separated from their limiting membrane by a clear space. Multivesicular structures were also found in association with this cell type. The caudal pars distalis also contained three cell types: one acidophil (putative somatotrop) and two basophils (putative thyrotrops and gonadotrops), all of which were similar to those described in adult fish. The pars intermedia contained only one cell type. They appeared to be active cells and were characterized by containing membrane-bounded granules similar to those found in the ACTH cells. Changes in ambient salinity had no apparent effect on any cell type described.

SummaryEffects of severe stress, immersion in water at 70°C for 5 seconds, were studied on the male albino rat in relation to response of the anterior pituitary. Using 5 different staining methods, no significant changes in percentage of acidophils, basophils or chromophobes were found 1, 3 or 12 hours after stress, although there were marked increases in number of cells per field in the 12 hour post-stress rats. Fresh weight of the pituitary showed no significant change in 1 and 3 hour post-stress animals, but the 12 hour group displayed a marked reduction, which was evident on both an absolute and a relative to body weight basis. Comparison of relative dry weight and amount of fluid lost in drying in unstressed and 12 hour stressed animals revealed that the weight decrease consisted of both fluid loss and reduction in solids.

The cytological changes in the pars distalis of female and male green frogs, Rana esculenta, were studied following varying periods of confinement in open air vivaria and inside the laboratory with the temperature regulated at 18–20°. In general, after long confinement up to a period of nearly 1 year, the basophils type two (B2) showed partial degranulation, total loss of their orangeophilic granules, reduction in size, and an insignificant decrease in their percentage. The acidophils and chromophobes also showed no significant changes in their percentage. The effect of confinement upon pituitary cytology was thus more morphological than “numerical.” The results have been discussed in the light of our earlier observations on this gland under different experimental conditions.

1. 1. Cytological examination was made of pituitary glands and ultimobranchial bodies from specimens of Diemyctylus (Triturus) viridescens killed at various times after surgical thyroidectomy. Pituitary and thyroid glands were studied after various periods of treatment with radioiodine, thiourea, and potassium perchlorate. 2. 2. Approximately two weeks after surgical removal of the thyroid, large chromophobic thyroidectomy cells began to appear in the pars distalis of the pituitary. They were abundant after the first month and persisted to at least the eighth month in some animals. In other animals sacrificed six or more months after thyroidectomy, none of these cells was present. The disappearance of the thyroidectomy cells is discussed. 3. 3. The formation of thyroidectomy cells was inhibited by keeping the thyroidectomized animals immersed in solutions of thyroxine. 4. 4. The source of the thyroidectomy cells seen is uncertain, but it is suggested that they arise from acidophils whereas it is generally agreed that in the mammal, thyroidectomy cells come from basophilic thyrotrophs. 5. 5. Following radiological damage to the thyroid, thyroidectomy cells developed apparently at the same rate as after surgical thyroidectomy. 6. 6. Continuous immersion in solutions of either thiourea (0.25%) or potassium perchlorate (0.005%) brought about the involution of the thyroid gland and, consequently, the development of thyroidectomy cells within 5 months (thiourea) or 6 months (potassium perchlorate). 7. 7. Molting was inhibited by surgical or radiological thyroidectomy and by immersion in a solution of potassium perchlorate but not by immersion in a solution of thiourea. Thiourea apparently has a direct stimulatory effect on the skin since inhibition of molting was not seen in animals injected with thiourea until thyroidal involution occurred.

Gonadotrophs and castration cells in the male rat pituitary showed nuclear concentration of radioactivity 1 hour after [ 1,2,6,7-3H ] testosterone injection. Thyrotrophs and acidophils did not retain radioactivity; also the cells of the intermediate and posterior lobes did not accumulate radioactivity. The autoradiographic results suggest a direct and selective action of androgen on gonadotrophs, which contrasts with the action of estradiol which was shown earlier to bind not only to basophils but to acidophils and chromophobes as well.

Six types of cells were identified in the pars distalis of the sheep adenohypophysis using various tinctorial methods and cytochemical reactions: They were two types of acidophils (alpha and epsilon), two types of amphophils (zeta and delta 1) and two types of basophils (beta and delta 2). Various experiments such as castration, thyroidectomy and adrenalectomy were carried out to examine the changes in each cell type.The alpha cells were stained orange with orange G in trichrome or tetrachrome methods and showed acid hematein positive but PAS negative reaction. They were located throughout the gland except the zona tuberalis.The epsilon cells were stained red with acid fuchsin, azocarmine or erythrosin and showed positive reaction for the acid hematein test and PAS method. They were larger than the alpha cells and were distributed throughout the pars distalis especially in its peripheral area.The zeta cells were amphophilic cells stained purple with aniline blue and acid fuchsin in trichrome method. Their granules were PAS positive, but AF and AT negative, and were R type after the PFA/AB/PAS/OG method. The zeta cells were large, irregular or polygonal cells distributed in the central portion of the pars distalis. They were considered to be the only source of ACTH, since they increased in number and size and showed a marked degranulation after adrenalectomy.The beta cells were large polygonal basophils which were confined to the zona tuberalis. The PAS positive granules of the beta cells were stained selectively by AF and alcian blue but not by AT or methyl blue. The beta cells were considered to be thyrotrophs, because, after thyroidectomy, they became enlarged and degranulated cells identical with thyroidectomy cells.The delta 1 cells were small, round or oval amphophilic cells stained reddish purple with both aniline blue and acid fuchsin in the trichrome method. They were distributed throughout the pars distalis. Their granules were coarse and stained blue-black or purple in AT, purple in PAS and methyl blue, but not stained by AF.The delta 2 cells were large, oval or rounded basophilic cells and were stained light blue with aniline blue in the trichrome method. The granules of delta 2 cells showed a weak positive reaction for PAS and AT but not for AF. After castration, the delta 1 and delta 2 cells increased in number and size, degranulated, and developed into castration cells, which contained larger or smaller vacuoles in their cytoplasm. These cells were considered to be gonadotrophs.

Summary and Conclusions 1. The biologic and pathologic effects of Piperonyl butoxide, a synthetic organic insecticidal material used synergistically with pyrethrins, were studied by an acute toxicity test on rats, by subacute toxicity tests on rats, by graded-dosage chronic toxicity tests on rats and dogs, by a low-dosage chronic test on a goat and by a short term experiment of low and moderate dosages on monkeys. 2. Piperonyl butoxide was found to be a material of a relatively low order of toxicity for the tested warm-blooded animals. There was relatively little difference between sexes and between species of animals (rats, dogs, monkeys, goat) in susceptibility to this material, and no indication of cumulative toxic effect upon the second and third generations of rats fed food containing up to 10,000 parts per million of Piperonyl butoxide. 3. The outstanding biologic findings of moderate toxicity caused by chronic ingestion of relatively large doses of Piperonyl butoxide (10,000 and 25,000 p.p.m.) were anorexia, wasting, reduced food consumption, reduced or lost ability to reproduce and accentuation of concomitant natural disease. 4. Biologic and pathologic findings in a two-year, graded dosage, paired-rat experiment, in which Piperonyl butoxide was added to a standard diet and fed ad libitum, indicated the following degrees of toxicity: a) 100 p.p.m. were nontoxic. b) 1000 p.p.m. were tolerated in good health. c) The addition of one-sixth as much pyrethrins to a ration containing 1000 p.p.m. Piperonyl butoxide produced no additional toxicity. d) 10,000 p.p.m. were tolerated with moderate toxicity. e) 25,000 p.p.m. were fatal to a group of 24 rats during a period of 4 to 68 weeks. 5. Biologic and pathologic findings in a one-year graded-dosage dog experiment, in which Piperonyl butoxide was fed six times a week by capsule, indicated the following converted degrees of toxicity: a) Approximately 80 p.p.m. were nontoxic. b) Approximately 700 p.p.m. were tolerated in good health. c) Approximately 3000 p.p.m. were tolerated with moderate toxic effect. d) Approximately 11,000 p.p.m. were fatal in 4 to 15 weeks. 6. Subbiologic toxic lesions observed in the livers of rat, dog, goat and monkey in good health were acidophil and hyalin-necrosis cells, hydropic swelling and slight dystrophy and dysplasia. These lesions were nonspecific and no significant change occurred in incidence and degree, as compared to lesions in the livers of the control rats and dogs. 7. In moderate biologic toxicity, the outstanding lesions of rat and dog were moderate numbers of acidophil and hyalin-necrosis cells, moderate dystrophy and dysplasia and advanced hydropic swelling; the lesions were accentuated in instances of concomitant natural disease. 8. Biochemical Balance studies on a dog indicated that when a large dose of Piperonyl butoxide is fed by stomach-tube it is quickly eliminated in the feces, and that only traces appear in the urine. 9. Piperonyl butoxide was not carcinogenic for the liver. It did not exert a malignant tumorogenic effect upon the general tissues, nor upon the endocrine glands and breasts, and did not have an indirect general tumorogenic effect through disturbance of the endocrine glands. 10. The safe human tolerance for chronic ingestion of Piperonyl butoxide was estimated, after allowing for a 100-fold safety factor, to be 42 parts per million in all the food eaten.

Chromophobes isolated by the original method from the rat anterior pituitaries morphologically differentiated into acidophils when cultured in a chemically defined medium, NCTC-109, supplemented with various amounts of CRF (0.15 to 15μg/2ml) and colchicine. An electrophoresis and a paper chromatography revealed that ACTH was a main hormone produced by the cells maintained in the supplemented medium for as long as 12 days. The addition of larger amounts of CRF shortened a duration to reach to a certain maximal level of ACTH concentration in the cells, and this maximal level was equivalent in the cases of the variable amounts of CRF. A maximal release of the ACTH from the cells into the medium took place always 3 days after reaching to a maximal concentration of ACTH in the cells. The chromophobes produced α-MSH only in an earlier phase of differentiation without discharging it into the medium. The intracellular granules isolated from the cells kept in the control medium were equally 100 to 150 mp in diameter and regarded as the common storage carriers of six trophic hormones because they showed commonly all their low activities. Three different granules were successfully isolated from the cells differentiating in the CRF-medium from the chromophobes into the acidophils with negligible cross-contaminations. They were small (100 to 200mμ in diameter), large (300 to 450 mμ) and huge and irregularly shaped granules (450 to 1000 mμ) respectively. All the granules were confirmed by bio-and immuno-assays to contain ACTH activity alone regardless of their dimension.

It was preliminarily revealed by a histogram made from the electron micrographs of acidophils on the tissue sections of anterior pituitaries in normal rats that the two different sizes of storage granules were distributed in high population in many acidophils. These two granules, small and large in size, were isolated from the pellet of isolated acidophils and separated each other by filtrations through membrane filters with various pore sizes and by discontinuous sucrose density gradient centrifugations. The other histogram made from the electron micrographs of these separated granules coincided with the above histogram. ACTH, GH and prolactin activities were bioassayed on the free granules. GH activity was found only in the large granules with diameter between 300 and 450mμ. ACTH activity was detected only in the small granules with diameter between 100 and 200 mμ, though the content of ACTH contained in the isolated granules represented only a few per cent of the total ACTH content of the homogenate of anterior pituitary. No prolactin activity was found in the both kinds of granules. These findings strongly suggested that the two kinds of granules originated from the acidophils of the rat anterior pituitaries were involved in GH and ACTH storage and the acidophils were responsible for ACTH production. The findings were also discussed related to the mode of intracellular localization of the hormones.

DURING the course of investigations into the histochemistry of fish pituitaries, the distribution of periodic acid–Schiff positive material1 was noted in the parrot fish, Pseudoscarus guacamaia. Whereas in mammalian pituitaries periodic acid–Schiff positive material is associated only with basophils (aniline-blue positive after Heidenhain's ‘Azan’ procedure), namely, the gonadotrophs and thryotrophs2, in Pseudoscarus the mid-glandular region of the adenohypophysis has two types of acidophils, one of which is strongly and uniformly periodic acid–Schiff positive and the other negative. This large polygonaly shaped periodic acid–Schiff positive acidophil of Pseudoscarus stains a deep orange after the Gomori aldehyde–fuchsin orange G method for the demonstration of neurosecretory material and is carminophilic after treatment with Heidenhain's ‘Azan’ stain. In addition to this type of cell, two types of basophils are present in the mid-glandular region which are periodic acid–Schiff positive as in mammals and other teleost fish. Furthermore, although some of the periodic acid–Schiff positive basophils stained selectively after aldehyde–fuchsin as in other teleosts3, the periodic acid–Schiff positive acidophil gave a negative aldehyde–fuchsin reaction. It would appear, then, that in this fish, unlike mammals, a carbohydrate-containing hormone is possibly produced by cells which are ‘acidophilic’ after application of the conventional staining procedures.

In the adenohypophysis of Cichlasoma biocellatum, which may be divided into a pro-, meso-, and meta-adenohypophysis, seven cell types can be distinguished. Treatment with deionized- and salt water effects three of these cell types. The rostral acidophils, which were proved to produce prolactin by means of an immunochemical method, are distinctly stimulated in deionized water and markedly inactivated in 25% sea-water. The putative ACTH cells, bordering neurohypophysial branches in the pro-adenohypophysis, are moderately stimulated by both treatments. The presumptive TSH cells, which in control animals appear to be very active, do not show marked changes in deionized water; they are strikingly inactivated, however, in salt water specimens.

THIS report presents preliminary results obtained by comparing consecutive sections of human pituitary glands stained with a variety of histologic and histochemical stains. This investigation has a twofold objective: 1) to determine the staining properties of the histologically defined groups of cells, and 2) to determine the homogeneity or lack of homogeneity of these cell groups, on the basis of cell-by-cell comparison of consecutive sections stained by different means. Demonstration of specific cellular sites of formation of the pituitary hormones has not kept pace with the number of hormones isolated from this gland. This discrepancy may be considered as casting doubt on either the distinctness of the hormones isolated or the functional unity of the cell types as defined by staining properties. Doubt appears to be more justified in the latter instance, but the acidophils, basophils and chromophobes of the pituitary body have, in the main, resisted further breakdown.

The morphology of normal and neoplastic acidophil cells of the rat pituitary has been studied by electron microscopy with special reference to the size and shape of the secretory granules. In the female rats, pregnant or non-pregnant, growth hormone (GH) cells and prolactin cells are easily separated, but in the male rats this separation is very uncertain. Acidophil tumours with granules similar to the GH type or to the prolactin type can be induced with stilboestrol treatment. These results indicate a close relationship between the two types of acidophil cells. They may be derived from a common progenitor which can be differentiated into either GH or prolactin cell or they may represent one cell type capable of producing both hormones.

Summary. Percentages of cell types in the adenohypophysis were compared with the content of sth and prolactin at different reproductive stages (e.g. immature, oestrous cycle, pregnancy, lactation and after hysterectomy) in the pig. Acidophils represent sth and prolactin activities. Chromophobes are undifferentiated, inactive or depleted cells. Acidophils represented half (53\m=.\5%)the cell population. Percentages of sth cells were higher in 17-day-old pigs than in mature animals. This is a period of rapid growth in the young pig. During the cycle, during pregnancy and after hysterectomy, the percentages of prolactin cells were higher than those found in immature or lactating pigs. The chromophobes represented 13\m=.\1% of the cell population. Chromophobes were the predominant cell type during lactation, but these seemingly inactive cells may have been active acidophils that were synthesizing and secreting prolactin. By rapid turnover of hormone, the cytoplasmic granules in prolactin cells lack differentiation. The sth activity was similar in pigs during pregnancy and lactation and after hysterectomy. There were no significant correlations in the percentages of sth cells and the concentration or content of sth during pregnancy and lactation. There was a trend of increased percentages of prolactin cells in later stages of pregnancy and after hysterectomy. Exogenous oestrogen caused a marked increase in the pituitary content ofsth and prolactin in hysterectomized pigs as compared with uninjected hysterectomized animals. Prolactin levels remained relatively constant during pregnancy and after hysterectomy, but declined during lactation. Exogenous oestrogen caused a consistently higher trend in prolactin activity in hysterectomized animals.

The cytology and cytochemistry of the pitiutary gland of the palm squirrel Funambulus pennanti during different phases of reproductive activity are described. Two types each of basophils and acidophils are recognized. The basophils include gonadotrophs and thyrotrophs and acidophils include orangeopils and carminophils. The gonadotrophs which are ovoid, coarsely granulated and mainly located on the sinusoidal borders of the lateral halves of the pars anterior are more numerous in males than in females. They are PAS-positive and AF-negative. They cannot be distinguished tinctorially into FSH and LH secreting cells as in the pituitary of the rat. The thyrotrophs which are large, rounded or polyhedral, finely granulated cells are confined to the antero-median zone of the pars anterior. They are PAS and AF-positive. The basophils can be tinctorially differentiated by the performic acid-alcian blue-PAS-orange G staining in which the gonadotrophs are stained purple and the thyrotrophs are red. The acidophils are differentiated tinctorially into two types. The orangeophils or STH secreting cells are small, rounded, and coarsely granulated. Carminophils or LTH secreting cells which appear in the pituitaries of females in early pregnancy and increase in number during later pregnancy and lactation, are large and coarsely granulated. The acidophils are specifically stained with mercury-bromphenol blue.

The pure chromophobes isolated from adenohypophyseal cells of rats were successfully cultured by the roller tube method in a serum-free NCTC-109 medium supplemented with nucleic acids and l-thyroxine. Upon light and electron microscopy, some chromophobes cultured in the control media proliferated by mitosis and others differentiated into immature ambiguous cells. The assay of ACTH, GH, and prolactin showed that these control explants produced a small amount of ACTH and GH, but a large amount of prolactin. When cultured in a CRF (corticotrophin releasing factor) medium, most chromophobes differentiated continuously into acidophils, but not into basophils. Most cells cultured in a CRF medium accumulated granules of various sizes. Since the incorporation of 3H-thymidine into DNA of chromophobes and ambiguous cells reached a maximum at an early stage of their culture, these two cells were assumed to be the maternal cells with the proliferating activity. When the chromophobes were kept in the media containing CRF and colchicine, their cell division was arrested at metaphase. With evident decrease in number of the chromophobes and ambiguous cells, many cells of the explants turned into acidophilic cells providing protein synthesizing ability. Thus the virtual transformation of the chromophobes into acidophils was induced. The explants in a CRF-colchicine medium produced a large amount of ACTH exclusively, and produced neither GH nor prolactin irrespective of the different sizes of granules. During the time course of CRF culture the 3H-thymidine incorporation into nucelar DNA was low when the ACTH content of explants was high, and vice versa. As CRF content of the media was increased, the peak of volumetric percentage of acidophils in the explants appeared earlier. The chromophobes may be dynamic rather than static in a mode of transformation into acidophils following the addition of CRF in to the medium. The cells which were differentiating on the acidophil axis could be, therefore, regarded as ACTH producers in reference to bioassay data.

SummaryA single electro-convulsive shock administered to 5 pregnant rats 12 hours after mating had no effect on the course of pregnancy. Of 5 rats shocked once daily for 6 days beginning 12 hours after copulation, on the eleventh day 2 showed some degranulation of pituitary acidophils, slight reduction in size of basophils, small or degenerating corpora lutea and degenerating retarded embryos in the uteri. All animals similarly shocked for 11 days, showed the same pituitary and ovarian changes, and, in addition, complete destruction of the fetuses with necrosis and sloughing of decidua into the uterine lumen.

Pituitaries of female mice in anestrus resulting from colony housing were characterized by a 58.0-percent (acidophil content. Subsequent exposure to restrained male mice for one and two nights failed to evoke significant acidophilic degranulation and resulted in pituitary acidophil values of 57.4 and 55.1 percent respectively. Exposure to released males on the third night produced marked acidophilic degranulation resulting in a significant decline in pituitary acidophils to 38.0 percent. These findings support the view that female pheromone suppresses and male pheromone favors the secretion of follicle-stimulating hormone and indicate that luteotrophic hormone is secreted at its assigned time in the sequence of cyclic ovarian events initiated by the secretion of follicle-stimulating hormone.

The high energy of growth of the larvae of Pelobates juscus and the intensive differentiation and early metamorphosis of the larvae of Bufo viridis are directly dependent upon specific differences in the functional maturity of the neurosecretory cells of the preoptic nucleus. Differentiation of the basophils of the adenohypophysis and the general mosaic of its cytological zonation depend upon local features of its vascularization. Metamorphosis, which is associated with dehydration, appears to be more intensive in the toad larvae; this is obviously related to neurosecretory activity. Extirpation of the preoptic nucleus results in a sharp increase in the hydrophilia of the tissues and the absence of metamorphosis. Predominance of acidophils over basophils is characteristic of the pituitary gland of the larvae of P. fuscus, while in the toad larvae there is a predominance of basophils. At a later period of development these correlations are reversed, this reversal being reflected in the rate of growth and in the final sizes of individuals of the two species.

Antisera to six mammalian pituitary hormones and a synthetic corticotrophin (Synacthen) were prepared and tested for specificity. The gamma-globulin fractions of the antisera were conjugated with fluorescein isothiocyanate and the labelled antibodies were isolated by column chromatography. Fresh-frozen sections of pituitary glands of adult migratory sockeye salmon (Oncorhynchus nerka) were incubated with the antibody solutions and examined with a fluorescence microscope. Anti-ACTH and anti-Synacthen appeared to be bound by theepsilon cells, whereas anti-prolactin reacted with theeta cells. Anti-GH was bound to the acidophil cells of the proximal pars distalis. Anti-LH reacted with some of the basophil cells of the proximal pars distalis. Antibodies to FSH or TSH failed to react.

The six kinds of cells, viz. chromophobes, ambiguous cells, acidophilic and basophilic intermediate cells, typical acidophils and basophils, were isolated from the rat fresh anterior pituitaries by the procedure of Ishikawa (1969) with some modifications. Bioassays of ACTH, prolactin, and a-MSH, and radioimmunoassay of GH were carried out for a mass of each isolated cell type. The chromophobes showed no hormonal activity. The ambiguous cells contained only α-MSH. Both ACTH and GH were localized predominantly in the acidophilic intermediate cells and typical acidophils, but prolactin was found only in the latter. Only a trace amount of ACTH, GH, and prolactin was found in the basophilic intermediate cells and typical basophils. It was elucidated that the cells on the acidophilic axis were involved in the producing cells of ACTH.

Publisher Summary This chapter provides an overview of two types of polymorphonuclear phagocytes—neutrophils and eosinophils. Neutrophils of a living human, rabbit, and chicken are viewed by phase contrast microscopy. The cells range from 10 to 15 microns in diameter. The shape of the moving cell changes constantly; the leading edge may take the form of a broad ruffled border or a pseudopod, and commonly at the trailing portion there is a constriction into a tail-like structure, often with thin strands extending from it. The unusual nuclear shape of neutrophils is one of the most striking features of these cells. The molecular mechanism for the development and maintenance of a nucleus divided into oval or sausage-shaped lobes connected to each other by thin strands is completely unknown. Around 100 years ago eosinophils were recognized as a distinct type of polymorphonuclear leukocyte in blood and tissues. This recognition was based on certain morphologic features of the cell and especially on the affinity of its cytoplasmic granules for acid aniline dyes. This latter characteristic led to the name acidophil or eosinophil.

Anterior pituitary glands of adult male rats weighing 175–500 g were cultured for periods up to 13 h in Medium 199 containing 2 μc/ml of 3H-thymidine. Extraction of the nucleic acids from pituitary homogenates by conventional biochemical procedures indicated that the 3H-thymidine was incorporated into DNA. In addition, radioautographs showed that the newly synthesized DNA was nuclear and in pituitary parenchymal cells. Labeled mitotic figures were found, and labeled acidophils and basophils were also observed. These results indicate that pituitary cells committed to the synthesis of specific hormones can also synthesize DNA.

IN previous investigations on the anterior pituitary of rats intoxicated with benzene we found histochemical changes suggesting an increased hormone secretion from both the acidophil cells and the beta cells1. Since the beta cells are believed to be concerned in the rat with the secretion of thyrotrophic hormone and are therefore also called thyrotrophs2,3, it seemed of interest to determine whether the hyperfunction of the pituitary beta cells is paralleled by cytological signs of augmented activity in their target gland, the thyroid.

The demonstration of intracellular hemoglobin in permanent preparations has long been a problem. The affinity of hemoglobin for iron hematoxylin is well known but this stain also colors yolk, chromatin, and other structures and is therefore not a reliable criterion. The presence of hemoglobin has been associated with an acidophil cytoplasm which stains a characteristic color, but a careful inspection of living cells in early hematopoetic or embryological stages demonstrates that hemoglobin is present in the erythrocytes which are quite basophilic. In the course of some research on the blood of embryonic frogs it became desirable to demonstrate the presence of hemoglobin in cells by means of a specific staining reaction.