Immunocytochemical studies of chicken somatotrophs and somatotroph granules before and after hatching

Immunocytochemical methods were used to gain information about the embryonic development of chicken somatotrophs before and after hatching. To localize growth hormone, anterior pituitary sections were incubated with growth-hormone antibody, and then an indirect peroxidase method was used for light microscopy and an immunogold method for electron microscopy. The earliest evidence of embryonic somatotrophs was seen at 12 days. At this stage somatotrophs were sparse (0.2% of parenchymal cells) and their granules were pleomorphic with elongated ovoid and lozenge shapes predominating. Few of the immunogold-labeled somatotroph granules of the embryo were spherical until 15 days after fertilization. At 18 days, most of the granules were spherical (their shape in the adult chicken). During the six days between the 15-day-old embryo and the 1-day-old chick, the number of gold particles per granule section approximately doubled suggesting an increase in growth hormone content of the granules. This rise was the result of increases in the size of the granule sections and in the concentration of gold particles in the sections. During the embryonic period of 12–20 days, somatotrophs were not more than 3.6% of the anterior pituitary cell population. During the following two days, between the 20-day-old embryo and the 1-day-old chick, the percentage of somatotrophs in the pituitary parenchymal cell population rose rapidly from 3.6% to 20.7% and then increased slowly to 24.6% during the period of 1–5 days after hatching. Both the sharp percentage rise in somatotrophs (20-day-old embryo to 1-day-old chick) and the rise in growth hormone content of the granules (15-day-old embryo to 1-day-old chick) suggested by gold-particle counts occur close to the time of hatching. These morphological changes may reflect an increased synthesis of growth hormone that is responsible for the rise in plasma growth-hormone concentration that begins about the same time and is especially abrupt two days later (1–3 days after hatching).     

Immunogold identification of the somatotrophs of domestic fowl of different ages

Summary The somatotrophs of the pituitary gland of the male domestic fowl were identified by means of an immuno-electron-microscopic method based on gold as the electron-opaque label and an antibody to growth hormone. Gold particles indicating sites of growth hormone were restricted to cells in which virtually all of the granules were labelled. Little, if any, gold label was found outside the granules in these cells designated as somatotrophs, or at sites outside these cells. The size of these gold-labelled secretory granules presumed to contain growth hormone decreased with age, from a mean sectional diameter of 256±6.2 nm (SEM) at 4–6 weeks to 221±5.7 nm at 11–18 weeks and 205±8.6 nm at 24–30 weeks of age. On the basis of these values for mean sectional diameters the change between the first two periods represents a decrease in granule volume of about 36%. However, during the same period the growth hormone concentration of the granules increased. Accordingly, growth hormone content per granule changed little if at all. In contrast, from 11–18 weeks to 24–30 weeks of age there was a decrease of 31% in growth hormone content per granule. These data indicate that growth hormone packaging in the chicken somatotroph changes with age. The first change results in the production of smaller granules of higher growth hormone concentration. During this period growth hormone content per granule remains relatively constant. The later change results in the production of granules of lower growth hormone content than that of younger animals.This is a paper of the Journal Sciences, New Jersey Agriculture Experimental Station supported in part by the State and Hatch Act Funds and a grant from the National Science Foundation (PMC-8022727)     

Age-related changes of the somatotrophs of the domestic fowl Gallus gallus

Summary The ultrastructure of the somatotrophs of the caudal pituitary of the domestic fowl was studied quantitatively. Two age groups of male chickens were compared: 4–6 weeks and 24–30 weeks post-hatching. With age, somatotrophs decreased from about 40% to about 30% of the pituitary cell population. Their volume density decreased similarly. Mean volume of a somatotroph was the same in young and adult animals. Because the granule volume density of the somatotrophs was unchanged, but the somatotroph volume density of the gland declined, the granule volume density of the caudal pituitary gland dropped in parallel with that of the somatotrophs. Thus the volume of the gland comprised of somatotroph granules fell about 32%: from 6.57% to 4.45%. This lowered pool of stored hormone may be linked to the lowered circulating levels of growth hormone found in older animals by other investigators.The granule volume density of the somatotrophs was unchanged but the numerical density approximately doubled; thus the mean granule size decreased by 47% with age. The relationship of the size reduction of the granules to the lowered plasma growth hormone levels is not understood at present.Supported in part by Hatch and State funds from the New Jersey State Agricultural Experimental Station and NSF grants PCM 8,0227,27 and PCM 8,302197.     

Ultrastructure of pituitary somatotrophs of male rats during liver carcinogenesis by 3'-methyl-4-dimethylaminoazobenzene

Somatotrophs of male Wistar rats fed a carcinogenic dose of the potent hepatocarcinogen 3'methyl-4-dimethylaminoazobenzene-(3'MeDAB) in a complete diet for up to 21 weeks, showed ultrastructural changes within one week of the start of the experiment. Morphometric analysis showed that the area of the somatotrophs occupied by hormone storage granules was reduced significantly at all stages up to 21 weeks, when the experiment was terminated. The cell area occupied by rough endoplasmic reticulum (RER) was significantly increased after three weeks and RER surface density increased after eight weeks, whilst granule size was reduced after four weeks. Pituitary content of radioimmunoassayable growth hormone (RIA-GH) was also lower in treated rats than in controls, from one week after the start of the experiment, but only significantly so at eight and twelve weeks, owing to large standard errors. Serum levels of RIA-GH were similarly lower in 3'MeDAB treated rats than in controls. Withdrawal of 3'MeDAB diet after twelve weeks treatment permitted partial recovery of some cell organelles, in that the content of hormone storage granules increased and the area of the RER decreased compared to rats fed 3'MeDAB continuously. There was also a partial, but non-significant, increase in pituitary RIA-GH. The pituitaries of rats pair-fed 3'MeDAB diet for 15 weeks contained significantly less RIA-GH than matched controls. These results suggest that 3'MeDAB, or its metabolites, may react with pituitary somatotrophs and impair their ability to synthesise and secrete hormone.     

In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell

The ultrastructural localization of growth hormone and prolactin in cow anterior pituitary was studied by double immunocytochemical labeling using specific antibodies and protein A-gold particles of different sizes. The two hormones were found in specific somatotrophs and mammotrophs as well as in somatomammotropic cells which were multinucleated and predominantly arranged in clusters in the central area of the lobules. In these mixed cells the two hormones were packaged (a) in different granules of the same cell, (b) in the same granules where they were segregated in different portions of the granule content, or (c) in the same granules but evenly intermixed. The relative proportion of these three types of granules varied in somatomammotrophs of different animals. A single large Golgi complex was generally present in somatomammotrophs. Small, immature granules containing either growth hormone or prolactin or both hormones were found randomly distributed along Golgi stacks. This suggests that in these cells the two hormones are processed in the same Golgi cisternae and that mechanism(s) exist(s) to sort out the two hormones from each other.     

Pituitary growth hormone and somatotrophs in rats bearing chemically induced hepatomas.

Measurement of pituitary growth hormone (GH) content by polyacrylamide gel electrophoresis (PAGE) showed that rats bearing diethylnitrosamine (DENA) induced hepatomas contained significantly less GH than age-related controls. Light microscopy of these pituitaries revealed many apparently dying somatotrophs in tumour bearing rats. By electron microscopy the somatotrophs of DENA hepatoma rats were relatively inactive and some clearly dying, which could account for the reduced hormone content. However, electron microscopy of somatotrophs of rats with 2-acetyl-aminofluorene (2-AAF) hepatic tumour nodules revealed many very active cells with greatly increased content of free ribosomes and many granules poised for imminent release. Other somatotrophs of these rats showed lysosomal activity indicating reduced secretion after an active phase.     

Possible sequential transformation of a series of acidophils in the pituitary autografts in the renal capsule of male rats in association with ACTH cell.

In our electron microscopy, acidiphils in the pituitary autografts placed in the renal capsules of immature male rats underwent a sequential transformation with the lapse of time: Within 3 and 6 days, all the somaotrophs packed with the large granules of about 350 mmu diameter dispersed. The size and number of the granules in somatotrophs were quickly and markedly reduced with severe modification of cell shape. There was evidence during this time course that Siperstein's or Moriarty's corticotrophs might be synonymous with the stellate shape of acidophils with the arrangement of small granules 150-200 mmu in diameter along the cell acidophils. The "acidophils of the small granule type" possibly related to ACTH production according to Yoshimura et al. (1974) were frequently detected in the grafts as elongated or irregularly shaped cells. Their minute granules 100-150 mmu in diameter were also distributed in row in the cytoplasmic peripheral area. Gradual loss of the minute granules below 100 mmu in diameter eventually made the acidophils to transform into agranular cells. Our own idea that ACTH secretion might correlate with a series of cells transforming along the acidophil-axis was indirectly supported by the present observation on pituitary grafts. On the other hand, basophils rapidly degenerated and died away. Ten and 20 days after autografting, the graft cells which might be principally composed of the cells of acidophil origin enormously proliferated through mitotic division, showing the homologous fine structure, without the normal cell individuality. They always contained three different size and shape of granules simulataneously. Significance of such a rapid and strong response of acidophils to the ectopic replacement in the immature male rats was discussed from the view-point of hypothalamic regulation to simple protein hormones.     

Effects of growth hormone-releasing hormone on somatotrophs in anterior pituitary gland allografts in hypophysectomized,orchidectomized hamsters

Summary We investigated the influences of growth hormone-releasing hormone (GHRH) on the percentage, size, and shape of somatotrophs in ectopic anterior pituitary tissue. Entire pituitary glands removed from 7-week-old male hamsters were placed beneath the renal capsules of 12-week-old hamsters that had been hypophysectomized and castrated 3 weeks previously. Beginning 6 days after each host had received a single allograft, each was injected subcutaneously twice daily with 4 g GHRH in 100 l of vehicle or 100 l of vehicle for 16 days. Six hosts in each group were killed by decapitation on day 17, 16 h after the last injection. Nine normal male hamsters were also decapitated and their pituitary glands were removed. Sections of anterior pituitary tissue were stained for GH and with hematoxylin. The percentage of anterior pituitary cells that stained for growth hormone was similar in the 3 groups. In contrast, somatotrophs in grafts had a smaller mean cross-sectional area than those observed in glands in situ. This effect was reversed by GHRH. Analysis of the shape of somatotrophs in both groups of grafts disclosed that they were less circular in cross-section than those in glands in situ. The results suggest that GHRH may not play a role in maintaining the percentage of somatotrophs among anterior pituitary cells, but that it does play a role in maintaining their size.     

Dynamics of secretory granules in somatotrophs of rats after stimulation with growth hormone-releasing factor: a stereological analysis

The anterior pituitary tissue of male rats injected with growth hormone-releasing factor (GRF) was either processed for stereology at the light-and electron-microscopic levels, or homogenized for growth hormone (GH) assay 2–60 min after GRF injection. Secretory granules of somatotrophs became smaller but increased in numerical density 2 min after GRF injection. Their volume density began to increase at 5 min. The frequency of exocytosis of the granules was most prominent as early as 2 min after GRF injection and reduced thereafter. GH levels in the tissue were lowest at 2–5 min, and returned to the control value by 60 min. Serum GH levels were highest at 15 min; even at 60 min, this value was higher than in the controls. These findings suggest that secretory granules in somatotrophs are stimulated to divide by GRF, resulting in a decrease in size and an increase in number. The discrepancy between the earlier formation of new secretory granules and the later restoration of intracellular GH levels implies that GRF first stimulates the synthesis of constituents of granules other than GH, and only later the synthesis of GH, and that newly formed small secretory granules contain less GH. From the clearance rate of serum GH and the frequency of granule exocytosis, it can be estimated that about a half million granules are released to maintain 1 ng/ml of serum GH in rats.     

Fine structure of somatotrophs and mammotrophs in the pituitary pars distalis of the little (lit) mutant mouse

The cellular pathology and postnatal differentiation of somatotrophs and mammotrophs in the pars distalis of little (lit/lit) and normal (+/+; lit/+) mice were studied by means of electron microscopy. The results indicate that in little the pituitary at birth contains recognizable somatotrophs and mammotrophs; however, between 14 and 24 days of age a contrast between little and normal becomes conspicuous with respect to the somatotrophs. Little somatotrophs are less heavily populated with granules, have granules of smaller size, and show less developed organelles than do normal somatotrophs at comparable stages. Beyond 24 days the little somatotrophs become more difficult to locate, and those that do occur show a minimal increase in complexity from that present at 14 days. In contrast, the mammotrophs in little are similar in appearance to normal mammotrophs and show increasing complexity as development proceeds, often sending forth cellular processes between neighboring cells, as do normal mammotrophs.     

Developmental regulation of granule size and numbers in larval salivary glands of drosophila by steroid hormone ecdysone

The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well-known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, approximately 3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5x10(-6) m ecdysone.     

Morphological effects of somatostatin on rat somatotrophs previously activated by growth hormone-releasing factor

Summary Correlative morphological and physiological analysis was carried out in order to clarify the role of somatostatin in the inhibition of the secretion of growth hormone (GH) from somatotrophs of the rat anterior pituitary gland in vivo. Transmission electron microscopy combined with immunogold labelling showed an increased number of exocytotic GH-containing secretory granules in somatotrophs fixed between 2 and 10 min after injection of GH-releasing factor (GRF). Injection of GRF also induced the appearance of immunopositive material in cisternae of the Golgi apparatus, many coated vesicles and multivesicular bodies. Microtubules were observed more frequently throughout the cytoplasm, particularly in and near the Golgi region. At 2 and 10 min after injection of somatostatin (SRIF), both the number of exocytotic figures in the somatotrophs previously stimulated by GRF and the amount of radioimmunoassayable GH in the plasma were clearly decreased. Undulation of the plasma membrane (PM) induced by GRF rapidly disappeared, and the number of granules just beneath the plasma membrane was significantly reduced. After injection of SRIF, parallel bundles of microfilaments were often observed in the space between the granules and the plasma membrane. SRIF did not cause a noticeable decrease in the amount of immunopositive material, coated vesicles and multivesicular bodies in the Golgi areas or any significant changes in the distribution of microtubules. SRIF therefore appears to inhibit hormone release mainly at the level of the plasma membrane, probably through changes in the distribution of microfilaments.     

Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in long term cultures

Summary Eel hemi-pituitaries were cultured in vitro on high or low sodium media, previously shown to affect differentially prolactin and growth hormone release. After 6 days culture, there were marked differences in the ultrastructure of both prolactin and growth hormone cells from the two groups. Morphometric data on the prolactin cells from SW-adapted eels showed a greater abundance of RER and paucity of secretory granules in cells from the low sodium medium. The size of the Golgi apparatus and the number of exocytosed secretory granules did not differ markedly between experimental groups, in contrast to previous findings on short-term cultures. Differences in the profile diameters of secretory granules are recorded between the experimental groups and the pattern differs markedly from that previously recorded for short-term cultures. The growth hormone cells from low sodium media were characterised by abundant, vesiculated RER, a prominent Golgi apparatus (in SW-adapted animals) and relatively few secretory granules. The activity of these growth hormone cells is in marked contrast to previous findings relating to short-term cultures. The shape and size of the non-granulated (stellate) cells of the RPD was again affected by the osmotic pressure of the medium.I should like to thank Mr. P.F. Hire for his photographic assistance     

Somatotrophs and lactotrophs: an immunohistochemical study of Gallus domesticus pituitary gland at different stages of induced moult

The objective of this study was to determine the distribution of somatotrophs and lactotrophs and conduct a morphometrical analysis of immunoreactive somatotrophs and lactotrophs in the pituitary glands of White Leghorn Hens (Gallus domesticus) during the period of induced moult. We divided the periods of induced moulting into three phases viz. 7, 14 and 21 days. The labeled alkalinephsphatase method with anti-GH (growth hormone) and anti-PRL (prolactin) as a primary antibody was used to detect somatotrophs and lactotrophs, in the midsagital sections of chicken adenohypophysis. Immunohistochemistry showed that somatotrophs are not only confined to the cephalo-caudal axis but can also be found in the caudal lobe; while lactotrophs were distributed in both lobes of the anterior pituitary gland at all stages of moulting (7, 14 and 21 days). Lactotrophs were of different shapes but somatotrophs were oval to round in morphology. At the given stages of induced moulting, some hypertrophied lactotrophs were also present after 7 days of induced moult in the anterior pituitary gland. However, there were moulting-related changes: from 7 to 21 days of induced moulting the immunoreactive-PRL cell population decreased, while the mean lactotroph size was more than that of somatotrophs. Basic quantitative and morphological information relating to somatotrophs and lactotrophs during the period of induced moult in laying hens is reported here and the changes brought about by induced moulting are restricted to PRL positive cells rather than GH positive cells.Key words: Moult, pituitary gland, somatotrophs, lactotrophs, chicken.     

Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary

Summary Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.     

Thyroid hormone differentially regulates cellular development in neonatal rat heart and kidney.

The role of thyroid hormone in the control of cardiac and renal cell development was examined in neonatal rats made hyperthyroid by administration of triiodothyronine (T3, 0.1 mg/kg s.c. on postnatal days 1-5) or hypothyroid by administration of propylthiouracil (PTU, 20 mg/kg s.c. given to dams on gestational day 17 through postnatal day 5 and to pups on postnatal days 1-5). Indices of total cell number (total DNA per tissue), cell packing density (DNA per g tissue), and relative cell size (protein/DNA ratio) were evaluated from birth through young adulthood. PTU administration led to primary shortfalls in cell number that were of similar magnitude in both tissues, but persisted somewhat longer in the kidney than in the heart. Deficits in cell packing density and cell size in the hypothyroid animals were secondary to the effect on cell number, displaying smaller magnitudes of effect and a lag in appearance and disappearance of the deficits compared to that for total DNA; indeed, the phase in which tissues were restoring their cell numbers was accompanied by increased cell packing density, reflecting a more rapid restitution of cell numbers than tissue weight or cell size. In contrast to the relatively similar effects of PTU on developing cardiac and renal cells, the effects of T3 were selective for the heart. Although T3 caused general growth impairment, it evoked marked cardiac overgrowth that was accompanied by a striking increase in cell number and a small increase in cell size. The cardiac hyperplasia is unique to the developing animal, as post-replicative heart cells in adult animals show only hypertrophy in response to thyroid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postnatal development of somatotrophs and mammotrophs in the pars distalis of the C57BL mouse

Summary The pars distalis of the pituitary gland of the C57BL mouse was studied by means of electron microscopy during postnatal stages of males ranging in age from newborn through 24 days, with particular emphasis on the somatotrophs and mammotrophs. During this period, growth curves were plotted in order to correlate postnatal growth patterns with the state of differentiation of the somatotrophs in the pars distalis. In the newborn, the somatotrophs show well developed organelles, including rough endoplasmic reticulum and Golgi complexes. These cells are not as densely packed with granules as the adult somatotrophs; however, from days 5 through 24, they show a progressive accumulation of granules. Although mammotrophs are scarce in the newborn, they are readily distinguishable in the pars distalis at 5 days. Male mice in small litters show a progressive increase in body weight between birth and 11–12 days, at which time the rate slackens until 18–19 days when the rate again increases. Growth curves for mice from large litters are similar to those from smaller litters, except that the transitory decrease in rate is more prominent.Supported by NIH grant no. HD12308 from the National Institute of Child Health and Human Development, USPHSThe authors wish to thank Pratima Ganguly for technical assistance     

Reversibility of omeprazole-evoked changes in the ultrastructure of ECL cells in the rat stomach

The ECL cells are histamine- and peptide hormone-producing endocrine cells in the rat oxyntic mucosa. They are rich in secretory vesicles and also contain microvesicles and electron-dense granules. They operate under the control of circulating gastrin. In the present study, we examined the ECL-cell ultrastructure after long term treatment with omeprazole, which is known to induce hypergastrinemia, and after withdrawal of the drug. Rats received omeprazole (400 µmol/kg per day, orally) for 16 days and were killed 1, 5, 20, or 40 days after the last dose of the drug. Oxyntic mucosal specimens were processed for electron microscopy. Electron micrographs of ECL-cell profiles were analyzed planimetrically. The ECL-cell profile area increased promptly in response to omeprazole, the secretory vesicles and granules were reduced in number and volume density, the microvesicles were unchanged in number but reduced in volume density, and vacuoles appeared. Within a week after stopping the omeprazole treatment, the numbers and volume densities of secretory vesicles and microvesicles returned to pre-stimulation values. Also, the vacuoles disappeared promptly. The ECL-cell profile area decreased below the pre-stimulation level within five days after stopping treatment, while, in contrast, the granules increased in number and volume density. Somewhat surprisingly, the cell size and the granule compartment did not return to normal until 40 days after stopping treatment.     

Immunocytochemical localisation of prolactin and growth hormone in the perinatal sheep pituitary

Summary The peroxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestational age of the fetus may influence the size range of prolactin secretory granules.