ultrastructural studies on prolactin and growth hormone cells in anguilla pituitaries in vitro

Cell Tiss. Res. 174, 547-564 (1976) Cell and Tissue Research �9 by Springer-Verlag 1976

Ultrastructural Studies on Prolaetin and Growth Hormone Cells in Anguilla Pituitaries in vitro

Michael Benjamin Department of Cellular Biology and Histology, St. Mary's Hospital Medical School, Paddington, London, England

Bridget I. Baker

School of Biological Sciences, Bath University, Claverton Down, Bath, England

Summary. Eel hemi-pituitaries were cultured in vitro on high or low sodium media which are known to affect differentially prolactin and growth hormone release. Ultrastructural examination of the prolactin cells after 24 h culture showed the Golgi bodies were markedly more abundant and widely distributed in hemi-pituitaries from the low sodium medium. Secretory granule release profiles and dense bodies were also more frequent, but the percentage of the cytoplasmic volume occupied by secretory granules was lower than on the high sodium medium. RER was only slightly modified. Significant differences were noted in the shape and processes of the non-granulated (stellate) cells of the RPD, but there were only slight differences in the ultrastructure of the somatotropes.

Key words: Prolactin cell - A n g u i l l a - In vitro - EM morphometry.

Introduction

It has been shown in previous work that when eel pituitaries are maintained in vitro on culture medium with either a high or low sodium content and thus of a high or low osmotic pressure, the release of both prolactin and growth hormone is enhanced on the low osmotic pressure medium (Ingleton et al., 1973; Baker and Ingleton, 1975). However, it was not possible to demonstrate a difference in the rate of hormone synthesis between cultures from the two media, judging from radioactive leucine incorporation. The present work was therefore under- taken in the hope of obtaining further information about those aspects of cellular activity that were immediately affected by changes in osmotic pressure.

Send offprint requests to: Dr. M. Benjamin, present address: Department of Anatomy, University College, Cathays Park, Cardiff, Wales

548 M. Benjamin and B.I. Baker

Materials and Methods

Yellow eels (Anguilla anguilla) were obtained from commercial suppliers and kept in large stock tanks of fresh-water until required. The sea-water adapted eels had been maintained in sea-water for over 1 year.

Cultures. The culture medium was Kreb's bicarbonate Ringer with sodium concentrations of 110 mE/1 (= 110 Med) or 170 mE/1 (= 170 Med). The Ringer solution was gassed with 5 ~ CO 2 in oxygen and to it were added Eagle's MEM concentrated amino acids (Flow Laboratories) together with 2 mg/ml glucose and 100 U/ml penicilline and streptomycin. The final medium was sterilised by passage through a millipore filter.

Fish were killed by decapitation. Pituitaries were excised immediately and cut into sagittal halves. These were placed on small squares of millipore filter (8 ~tm pore size) supported by rafts of stainless steel mesh. Each raft was placed on a solid watchglass with a few drops of culture medium; the watchglass was covered with a glass lid, sealed with wax and incubated at 18 ~ C. Sterile technique was used throughout.

Electron microscopy. After 24 h culture, the pituitary halves were fixed for 2.5 h in Karnovsky's glutaraldehyde-paraformaldehyde mixture (Karnovsky, 1965) and post-fixed in 1 ~ osmium tetroxide in 1 ~ cacodylate buffer. After washing in 70~ alcohol, the glands were block-stained in saturated uranyl acetate, dehydrated and embedded in Spurr's resin (Spurr, 1969).

Blocks were sectioned on a Cambridge Huxley microtome using glass knives. For identifying cell types, adjacent thick (1.0 ~tm) and thin (silver interference colour) sections were cut. The sections were stained with a hot, alkaline solution of 0 .5~ azur II in 1 ~ borax and compared with the thin sections used for EM which were stained in lead citrate (Reynolds, 1963) and uranyl acetate. The thin sections were mounted on 100-mesh, collodion-coated copper grids, and examined on a Miles MR 60 C microscope.

Morphometry. Fourteen micrographs of prolactin and associated non-granulated cells ( = stellate, follicular or neck cells) were taken at random from each hemi-pituitary from 5 animals. Calibration of the microscope showed that the final magnification of the printed micrographs was x 5730. The relative volumes of the cells (or their cytoplasmic portions) occupied by the various cell components was determined by the point-counting method of Weibel (1969) using a coherent double lattice test system (4 : 1). The distance between the thick lines on the printed micrograph was 1.75 ~tm and each point on the test system represented an area equivalent to 3.05 ~tm 2. The volume/surface (v/s) estimates on the non-granulated cells were determined by combined point and intersection counts (Chalkley et al., 1949) using a multipurpose test system (Weibel et al., 1966) of line length equivalent to 1.75 ~tm on the printed micrograph.

Results

General Morphology of the Prolactin Cells, RPD Non-granulated Cells and Somatotropes

Prolactin Cells and Associated Non-granulated Cells. T h e p r o l a c t i n cel ls a r e

h i g h l y p o l a r i s e d a n d a r e a r r a n g e d in fo l l ic les a r o u n d a c e n t r a l l u m e n . T h e n u c l e u s

is s i t u a t e d b a s a l l y n e a r t h e b a s e m e n t m e m b r a n e a n d b l o o d vessels , a n d t h e cel ls

t a p e r t o w a r d s t h e i r a p e x a t t h e fo l l ic le l u m e n . T h e R E R is o r g a n i z e d in p e r i n u c l e a r

a r r a y s a n d is a l so s c a t t e r e d t h r o u g h o u t t h e c y t o p l a s m b o t h a b o v e a n d b e l o w t h e

n u c l e u s (F ig . 1). T h e G o l g i a p p a r a t u s is i n v a r i a b l y s u p r a n u c l e a r a n d c o n s i s t s o f

f l a t t e n e d c i s t e r n a e , s m a l l ves ic les a n d a f ew l a rge v a c u o l e s . I n t h e a p i c a l r e g i o n

o f t h e p r o l a c t i n cel l a re n u m e r o u s s m a l l ves ic les a n d a l so a c e n t r i o l e a s s o c i a t e d w i t h a t y p i c a l (9 + 2) c i l i u m a n d c i l i a ry r o o t l e t s (F ig . 2).

Prolactin and Growth Hormone Cells in Anguilla Pituitary 549

Fig. 1. General morphology of prolactin cells. FW-adapted eel. 110 Med. Elongated cells with many basal secretory granules (SG 1) and few supranuclear (apical) granules (SG 2). Prominent rough endoplasmic reticulum (RER); non-granulated cells (NGC) at apical and basal poles interdigitate between the prolactin cells, x 6,000

The non-granulated cells of the R P D are small cells found in all follicles. They intercalate between the prolactin cells with their nuclei situated either at the base or at the apex of the follicle. Their cytoplasm contains relatively few organelles, with no secretory granules, little organised R E R and surprisingly few microfilaments or microtubules. However, there are often conspicuous Golgi bodies, many free ribosomes and fat droplets and some very conspicuous mitochondria (Fig. 3). These cells also contain phagocytic vacuoles enclosing partially digested clusters of endocrine secretory granules, presumably prolactin granules


Fig. 2. General morphology of an RPD follicle from a FW-adapted eel (110 Med). The follicle is sectioned through its apical (luminal) pole, showing prolactin (PRL) and non-granulated cells (NGC). Note microviUi (MV) and cilia (C) projecting into the lumen (L). Note also centrioles (Ct) and ciliary rootlets (R) and junctional complexes between all cells (arrows). x 20,000

Fig. 3. General morphology of an RPD non-granulated cell. FW-adapted eel (110 Med). Note the prominent Golgi apparatus comprising flattened cisternae (FC) and small vesicles (V), many free ribosomes (R) and conspicuous mitochondria (M). x 45,000

Fig. 4. General morphology of an RPD non-granulated cell. FW-adapted eel (110 Med). The cell contains a lipid droplet (L) and several phagocytic vacuoles enclosing partially digested clusters of prolactin secretory granules (P). x 15,000


Fig. 5. General morphology of somatotropes (STH). Note the rounded nuclei of the STH cells and their large, electron-dense secretory granules. GtH gonadotropes, x 8,000

(Fig. 4). Cell processes of varying width penetrate between the prolactin cells and associate with the perivascular space at the periphery of the follicle. At the luminal border of the follicle the non-granulated cells often have a zone of interlocking cell membranes with other non-granulated cells or with prolactin cells. Also at this border, junctional complexes are found between prolactin cells and between prolactin and non-granulated cells (Fig. 2).


Somatotropes. These are slightly elongated cells with a very conspicuous, rounded nucleus located centrally within the cell. Their cytoplasm is densely packed with mainly round, very electron-dense secretory granules which are larger than the granules of the neighbouring gonadotropes (Fig. 5). The Golgi apparatus is juxtanuclear and the small amount of laminar RER is also arranged around the nucleus.

The Effect of Culture on Medium with High or Low Sodium Content

Because of the great variation in the secretory state of cells in different fish, only opposite halves of the same pituitary were compared. As a check on the subjective assessment of changes in the secretory state, the EM data for the prolactin and non-granulated cells from FW-adapted eels were quantified using morphometric procedures (cf. Materials and Methods).

A. Prolactin and Non-granulated Cells from FW-adapted Eels. The morphometric data suggest that prolactin cells from all hemi-pituitaries cultured on 110 Med are more active than prolactin cells from their opposite halves cultured on 170 Med. The most pronounced differences concerned the development of the Golgi apparatus with its associated vesicles and the number of profiles of exocytosed secretory granules.

The percentage of the cytoplasmic volume occupied by Golgi bodies was significantly greater (P < 0.05) in prolactin cells from 110 Med than 170 Med, and many more groups of Golgi cisternae and vesicles were apparent (compare Figs. 6 and 7); they were dispersed throughout the apical region of the cells and were frequently located very close to the lateral plasmalemma (Fig. 8). Secretory granule formation, i.e., the presence of immature secretory granules within the cavities of the Golgi apparatus, was more evident on the low sodium medium. Occasionally the membrane of a secretory granule was extended into a tubule that approached the lateral plasmalemma. No unequivocal continuity between these tubules and the plasmalemma could be demonstrated, but some very close associations were noted (Fig. 9). Similar observations have been made in the MSH cells of the eel pars intermedia (Thornton and Howe, 1974).

Secretory granule release profiles were much more frequent in 4 of the 5 eels examined from 110 Med, but in one exceptional pair of pituitary halves the reverse was the case (Table 1). In hemi-pituitaries from both media the released secretory granules found in the intercellular spaces were often small and of low electron density. There were occasional signs of 'multiple release figures' from the prolactin cells (Fig. 12) and such groups were sometimes engulfed by the non- granulated cells.

In spite of the greater release of prolactin granules on 110 Med, differences in cytoplasmic granulation between the two groups of pituitaries were not striking. Morphometric measurements of the cytoplasmic volume occupied by granules showed that this is greater on 170 Med, but while this could be due to a larger number of granules it might also be attributed to the differences in granule size;

Figs. 6 and 7. Prolactin cells from opposite halves of a FW-adapted eel pituitary cultured on 110 Med (Fig. 6) and 170 Med (Fig. 7). Note the prominent and widely dispersed Golgi bodies (arrows) on 110 Med, SG-exocytosed secretory granules; NGC, non granulated cell. x 15,000

Fig. 8. Detail ofprolactin cell from 110 Med, showing a Golgi apparatus (arrow) close to cell membrane (CM), FW-adapted eel. x 30,000

Fig. 9. Prolactin cell (FW-adapted eel) with secretory granule (SG) whose membrane extends into a neighbouring tubule (arrows) near the cell membrane (CM). x 30,000

Figs. 10 and 11. Prolactin cells from opposite halves o f a FW-adapted eel pituitary cultured on 110 Med (Fig. 10) or 170 Med (Fig. 11). Note the variation in number of dense (lysosomal?) bodies (DB) between the two media. • 6,000


Table 1. Morphometric data on prolactin cells from hemi-pituitaries of FW-adapted eels after 24 h culture. Pairs of results from opposite halves of the same pituitary

Morphometric parameter Na + 110 mEq/1 Na + 170 mEq/1 (235 mOsm) (337 mOsm)

Volume density of the nucleus (~o)

13.7 12.8 17.1 17.1 15.7 20.5 14.5 17.2 14.0 17.5

Percentage of the total cytoplasmic volume occupied by secretory granules

19.5 24.9 21.0 23.0 17.4 18.4 26.2 27.7 18.0 20.4

Percentage of the total cytoplasmic volume occupied by the Golgi apparatus and its associated vesicles

2.9 2.2 2.6 0.9 4.7 1.5 4.2 0.5 4.7 1.9

Percentage of the total cytoplasmic volume occupied by mitochondria

6.1 7.9 4.2 5.0 5.4 6.2 8.8 5.3 6.7 5.4

Percentage of the total cytoplasmic volume occupied by dense (lysosomal?) bodies and lipid droplets

1.2 0.5 1.2 0.8 1.8 0.4 0.8 0.4 0.9 0.6

5.4 0.6 Number of exocytosed secretory 0.8 0.0 granule profiles/100 gm 2 of prolactin 0.7 1.2 cell 3.6 0.0

7.4 0.0

Statistical analysis: p values based on comparing pairs of results from opposite halves of the same pituitary. Significant differences (p<0.05) are marked with an asterisk. Data assessed by paired test (Burn et al., 1950)

measurements of granule size (200 granules/culture) showed that there were consistently more small granules on 110 Med (Fig. 13).

Prolactin cells from the two culture conditions also differed in the number of dense (lysosomal?) bodies and lipid droplets they contained (Figs. 10, 11 ; Table 1). There were no consistent differences in the cytoplasmic volume occupied by mitochondria, the amount of RER or the nuclear size, although both latter features were modified after 8 days culture (Ingleton et al., 1973).

The only change shown by the non-granulated cells of the RPD in response to the culture medium was a change in the width of the cell processes projecting


Fig. 12. Prolactin cell from a FW-adapted eel, showing multiple granule release, A released granules; B secretory granules engulfed by a non-granulated cell; G Golgi apparatus; NGC non-granulated ceil; PRL prolactin cell. x 22,500


13 s5

60

55

5O

45

40

> , 35 U e-

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u. 25

2o

15

10

5

~IIII / / / / t . . . .

I I I I i f / / / t

r

0 50

[ ] 110 Med

[ ] 170 Med

100 150 200 250 300 350 lO0 450

Diameter (nm)

Fig. 13. Histogram showing the size distribution of the prolactin secretory granule profiles from cells on 110 Med and 170 Med. FW-adapted eels

between the prolactin cells. Scrutiny of many photographs suggests that these processes were broader in cells from 170 Med and this is reflected by the increased volume/surface ratio (Table 2). Cells from 110 Med had processes that tended to be markedly narrower (Figs. 14, 15).

B. Prolactin and Non-granulated Cells from SW-adapted Eels. Prolactin cells from SW-adapted fish tended to have fewer secretory granules than prolactin cells from FW-adapted fish; the granules also had a lesser and more variable electron density and were more elongated and irregular in shape.

The differences between pituitary halves from different culture media resembled those noted for FW-adapted eels, but were less intense. Thus, there was a slight increase in the development and dispersion of the Golgi apparatus on 110 Med but only a few signs of granule formation by the Golgi apparatus in either group. Exocytosis figures were very rare on either medium, although measurements of hormone in the medium shows that release is enhanced by 110 Med (Ingleton

Prolactin and Growth Hormone Ceils in Anguilla Pituitary 559

Table 2. Morphometric data on non-granulated cells of the RPD, from hemi- pituitaries of FW-adapted eels. Pairs of results from opposite halves of the same pituitary

Morphometric parameter Na + II0 mEq/1 Na + 170 mEq/l (235 mOsm) (337 mOsm)

Ratio of prolactin cell volume to stellate cell volume

8.0 5.5 4.7 3.3 4.7 5.3 4.5 4.7 6.9 4.0

Relative non-granulated cell volumes

1.0 1.0 1.0 1.0 1.0

1.2 1.5 0.9 1.0 1.0

Volume/surface (~m3/gm 2) ratio of non-granulated cells (i.e., volume of non-granulated cells/surface in contact with prolactin cells)

0.47 0.84 0.49 0.71 0.67 0.78 0.52 0.93 0.57 0.77

Percentage of total cell volume occupied by the nucleus (%)

18.2 18.2 15.3 14.7 21.3 18.7 31.7 31.4 22.3 22.1

Percentage of total cytoplasmic volume occupied by dense (lysosomal?) bodies and lipid droplets

6.7 6.0 3.3 4.0 5.4 6.0 5.2 6.4 6.3 10.0

Statistical analysis: p values based on comparing pmrs of results from opposite halves of the same pituitary. Significant differences (p < 0.05) are marked with an asterisk. Data assessed by paired test (Burn et al., 1950)

et al., 1973). As before, dense ( lysosomal?) bodies and lipid droplets were more a b u n d a n t on 110 Med.

C. Somatotropes from FW and SW-adapted Eels. In F W - a d a p t e d eels the ultra- s t ructural differences between the STH cells f rom the different media were very slight. The general conclus ion was that the STH cells were slightly more active on 110 Med. The Golgi appara tus was bet ter developed and there was sometimes more RER. Somatot ropes f rom both media showed signs of secretory granule fo rmat ion within the Golgi cisternae, bu t in nei ther group were there figures of exocytosis. Some STH cells f rom 110 Med were par t ia l ly degranula ted (compare Figs. 16 and ! 7) bu t this was no t a consis tent difference between cells f rom the two media. Dense ( lysosomal?) bodies and lipid droplets seemed slightly more n u m e r o u s on the low sod ium medium, bu t there were no conf i rmatory mor pho -

560 M, Benjamin and B.I. Baker

Figs. 14 and 15. Cells from 110 Med (Fig. 14) and 170 Med (Fig. 15) of a FW-adapted eel, showing differences in the size of non-granulated cell processes (NGC) between prolactin cells (PRL). x 7,000


Figs. 16 and 17. Somatotropes (STH) from opposite halves of a FW-adapted eel pituitary cultured on 110 Med (Fig. 16) and 170 Med (Fig. 17). Note only slight differences in granulation on the two media. G Golgi apparatus; GtHgonadotrope; RER rough endoplasmic reticulum, x 10,000


metric measurements. No differences were discernible between STH cells taken from SW-adapted fish whose hemi-pituitaries were cultured on 110 Med or 170 Med. On neither medium was there clear evidence of secretory granule formation or hormone release, although studies of 3H leucine incorporation show that these processes do occur. Changes in the ultrastructure of these cells are thus less sensitive indicators of secretory response than are measurements of the hormone in the medium.

Discussion

Previous experiments involving hormone assay have shown that more prolactin is released on 110 Med than on 170 Med (Ingleton et al., 1973; Baker and Ingleton, 1975) and the present observations that the release ofprolactin granules is enhanced on 110 Med is in line with these assay results. The consistent shift towards smaller prolactin granules on 110 Med has also been noted in vivo in Poecilia (Hopkins, 1969) following transfer from sea-water to fresh-water. It is not known whether the smaller size results from increased formation of small granules on 110 Med or more rapid release of larger, stored granules. In support of both possibilities are the observations that on 110 Med there were more images of new granule- formation within the Golgi cisternae and also a more intense release of both granules and hormone. The converse finding, seen in long-term adapted fish, in which prolactin granules tend to be smaller in sea-water adapted fish than in freshwater adapted individuals (review by Schreibman et al., 1973), probably has a different physiological interpretation.

The enlargement and fragmentation of the Golgi apparatus was a more immediate and striking response to low sodium conditions than were changes in the RER which, although striking after 8 days culture (Ingleton et al., 1973) were not always apparent after only 24 h culture. This inconsistent difference in the RER doubtless helps explain the failure to demonstrate a difference in 3H leucine incorporation on the two media after 24 h culture (Ingleton et al., 1973). A frag- mented Golgi apparatus, i.e., multiple groups of Golgi cisternae and vesicles, has been noted in the prolactin cells of other teleosts e.g., Gillichthys (Nagahama et al., 1973), Salmo gairdneri (Nagahama, 1973) and Poecilia (Batten et al., 1975), as well as in certain mammal cells e.g., GH 3 tumour cells (Tixier-Vidal, 1975) and also in chondrocytes (Moskalewski et al., 1975). The finding that the frag- mented condition was enhanced during increased secretory activity on low sodium has not been noted in other fish, but was observed in mammalian GH 3 cells following stimulation by TRH (Tixier-Vidal, 1975).

The increased number of dense bodies, identified previously by Hopkins and Baker (1968) as lysosomes, that was observed in prolactin cells on 110 Med, appears contrary to what has been observed in vivo in Poecilia (Hopkins, 1969) and to what is now regarded as an important crinophagic role of lysosomes in conditions of reduced hormonal release (Smith and Farquhar, 1966; Farquhar, 1970). It is possible that the increase in the number of dense bodies with increased secretory activity is an abnormality associated with in vitro conditions. However, it is worth mentioning here that an increase in lysosomal activity is not invariably


associated with reduced hormone release even in vivo, since Hopkins (1970) has described an increase in lysosomes accompanying an increase in secretory activity in the pars intermedia of Xenopus. A similar increase in dense bodies associated with increased cellular activity has been observed in stimulated GH 3 cells in vitro (Tixier-Vidal, 1975).

Several functions have been suggested for the non-granulated cells in the pituitary of mammals and teleosts, the best documented role being a phagocytic one (see reviews by Farquhar et al., 1975; Leatherland and Percy, 1976). In the present work, the inclusion of large vacuoles within the non-granulated cells, with some of these vacuoles clearly containing secretory granules from prolactin cells, supports the suggestion of such a function. Inspection of photomicrographs together with morphometric measurements, suggests a difference in the shape of these cells between cultures from 110 Med and 170 Med, possibly related to the change in secretory activity of the prolactin cells. A mention of variations in the size, or prominence of these cells among the prolactin cells of fish from different salinities can be found in a number of papers (Leatherland et al., 1974; Batten et al., 1975). However, it is not possible to determine from the various descriptions, whether these changes are comparable in the different investigations, nor thus what is their physiological significance.

References

Baker, B.I., Ingleton, P.M.: Secretion of prolactin and growth hormone by teleost pituitaries in vitro II. Effect of salt concentration during long-term organ culture. J. comp. Physiol. 100, 269-282 (1975)

Batten, T., Benjamin, M., Ball, J.N.: Ultrastructure of the adenohypophysis in the teleost Poecilia latipinna. Cell Tiss. Res. 161, 239-261 (1975)

Burn, J.H., Finney, D.J., Goodwin, L.G.: Biological standardization, 2nd edn. Oxford: Oxford University Press 1950

Chalkley, H.W., Cornfield, J., Park, H.: A method for estimating volume-surface ratios. Science 110, 295-297 (1949)

Farquhar, M.G.: Processing of secretory products by cells of the anterior pituitary gland. In: Sub- cellular organization and function in endocrine tissues. Mem. Soc. Endocr. 19, 79-122 (1971)

Farquhar, M.G., Skutelsky, E.H., Hopkins, C.R.: Structure and function of the anterior pituitary and dispersed pituitary cells. In vitro studies. In: The anterior pituitary (Tixier-Vidal, A., Farquhar, M.G., eds.), pp. 83-135. New York: Academic Press 1975

Hopkins, C.R. : The fine structural localization of acid phosphatase in the prolactin cell of the teleost pituitary following the stimulation and inhibition of secretory activity. Tissue & Cell 1, 653-671 (1969)

Hopkins, C.R.: Studies on secretory activity in the pars intermedia of Xenopus laevis 2. A biochemical and electron cytochemical investigation of acid hydrolase activity following the stimulation of secretory activity in vivo. Tissue & Cell 2, 71-81 (1970)

Hopkins, C.R., Baker, B.I.: The fine structural localization of acid phosphatase in the prolactin cells of the eel pituitary. J. Cell Sci. 3, 357-364 (1968)

Ingleton, P.M., Baker, B.I., Ball, J.N. : Secretion ofprolactin and growth hormone by teleost pituitaries in vitro. I. Effect of sodium concentration and osmotic pressure during short-term incubations. J. comp. Physiol. 87, 31%328 (1973)

Karnovsky, M.J.: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A (1965)

Leatherland, J.F., Ball, J.N., Hyder, M.: Structure and fine structure of the hypophyseal pars distalis in indigenous African species of the genus Tilapia. Cell Tiss. Res. 149, 245-266 (1974)

Leatherland, J.F., Percy, R.: Structure of the nongranulated cells in the hypophyseal rostral pars distalis of cyclostomes and actinopterygians. Cell Tiss. Res. 166, 185-200 (1976)


Moskalewski, S., Thyberg, J., Lohmander, S., Friberg, U.: Influence of colchicine and vinblastine on the Golgi complex and matrix deposition in chondrocyte aggregates. Exp. Cell Res. 95, 440454 (1975)

Nagahama, Y. : Histo-physiological studies on the pituitary gland of some teleost fishes, with special reference to the classification of hormone-producing cells in the adenohypophysis. Mem. Fac. Fish. Hokkaido Univ. 21, 1-63 (1973)

Nagahama, Y., Nishioka, R.S., Bern, H.A.: Responses of prolactin cells of two euryhaline marine fishes, Gillichthys mirabilis and Platichthys stellatus, to environmental salinity. Z. Zellforsch. 136, 153-167 (1973)

Reynolds, E.S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963)

Schreibman, M.P., Leatherland, J.F., McKeown, B.A.: Functional morphology of the teleost pituitary gland. Amer. Zool. 13, 71%742 (1973)

Smith, R.E., Farquhar, M.G.: Lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland. J. Cell Biol. 31, 319 347 (1966)

Spurr, A.R.: A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3143 (1969)

Thornton, V.F., Howe, C.: The effect of change of background colour on the ultrastructure of the pars intermedia of the pituitary of the eel (Anguilla anguilla). Cell Tiss. Res. 151, 103-115 (1974)

Tixier-Vidal, A.: Ultrastructure of anterior pituitary cells in culture. In: The anterior pituitary (Tixier- Vidal, A., Farquhar, M.G., eds.), pp. 181-229. New York: Academic Press 1975

Weibel, E.R.: Stereological principles for morphometry in electron microscopic cytology. Int. Rev. Cytol. 26, 235-302 (1969)

Weibel, E.R., Kistler, G.S., Scherle, W.F.: Practical stereological methods for morphometric cytology. J. Cell Biol. 30, 23-38 (1966)

Accepted June 8, 1976

ultrastructural studies on prolactin and growth hormone cells in anguilla pituitaries in vitro

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