the acth-interrenal axis in the freshwater stickleback, gasterosteus aculeatus form leiurus

Cell Tiss. Res., 155, 105--115 (1974) �9 by Springer-Verlag 1974

The ACTH-Interrenal Axis in the Freshwater Stickleback, Gasterosteus aculeatus

form leiurus

Michael Ben jamin and Michael P. I r e l a nd

Department of Zoology, University College of Wales, Aberystwyth, Wales

Received October 1, 1974

Summary. The ACTH-interrenal axis of the freshwater stickleback has been examined with the fish in a variety of physiological conditions. A morphometric analysis of ACTH cell ultrastructure in spring animals revealed that the only change from the winter condition was a significant decrease in the amount of perinuclear RER. The interrenal gland responded to metopirone treatment by an increase in both nuclear and cell size, although only a high dose of metopirone could degranulate the ACTH cells. Morphometry of the ACTH cells from metopirone-treated animals showed a significant increase in the amount of RER and a significant decrease in the number of free ribosomes and secretory granules, compared with control animals maintained in freshwater. Such ultrastructural changes may be expected of a cell that is stimulated to increase its secretion of polypeptide hormone. The ACTH-interrenal axis also responded to 70% seawater, as this treatment increased the interrenal cell and nuclear sizes.

Key words: ACTH-interrenal axis - - Freshwater stickleback - - Seasonal changes, Meto- pirone, Seawater adaptation - - Morphometry.

Introduction

Benjamin (1974 a) has r ecen t ly publ i shed a morphomet r i c s t u d y of the p i t u i t a r y cell t ypes in the f reshwater s t ickleback, Gasterosteus aculeatus form leiurus, in an a t t e m p t to replace the p seudoquan t i t a t i ve descr ip t ions stil l cus tomary among m a n y p i t u i t a r y morphologis ts wi th morphomet r i c da t a t ha t can be s ta t i s t i ca l ly tes ted . I n this publ ica t ion Ben jamin (1974a) p laced considerable emphasis on defining the cell t y p e s by the i r physiological condit ion.

The p resen t inves t iga t ion is pa r t of an a t t e m p t to ex tend the physiological condi t ions of the f reshwater s t ick leback in which the p i t u i t a ry g land and asso- c ia ted endocr ine organs have been s tudied. The ACTH cells from adu l t s t icklebacks collected in the spring have been sub jec ted to a morphomet r i c analysis, and var ious exper imen ta l procedures have been designed to a l te r the physiological s ta te of the ACTH- in te r rena l cell axis. Lea the r l and and L a m (1971) have examined the effect of ACTH and cortisol in jec t ions on var ious adenohypophysea l cell t ypes in the mig ra to ry s t ickleback, Gasterosteus aculeatus form trachurus.

Materials and Methods Collection o/Fish. Sticklebacks were collected from the river Rheidol, Cardiganshire as

described previously (Benjamin, 1974a).

Send o//print requests to: Dr. Michael Benjamin, Department of Cellular Biology and Histology, St. Mary's Hospital Medical School, Paddington, London, W. 2., England.

106 M. Benjamin and M. P. Ireland

Light Microscopy. The brains with pituitaries attached were dissected out and fixed in Bouin's fluid. 5 Izm paraffin wax sections were stained with lead haematoxylin (McConaill, 1947). For examination of the interrenal tissue of sticklebacks, the head kidneys were fixed in situ with Bouin's fluid, decalcified with 5% nitric acid in 80% alcohol and embedded in 52~ paraffin wax. Sections were cut at 8 ~zm and stained with haematoxylin and eosin.

Electron Microscopy. Only the ACTH cells were examined by electron microscopy. The methods have been described previously (Benjamin, 1974a).

Experimental Procedures. Adult sticklebacks of both sexes were collected in mid-Octeber and kept in well-aerated aquaria which contained 8 litres of tapwater, with 4 animals in each tank. Fish were acclimatized in the laboratory for 1 week before hormone administration. The drug metopirone (2-methyl-I, 2 di-3' pyridyl-propane-l-one, SU 4885: Ciba) was used in powder form as metopirone base. In the first experiment 5 mg/litre of metopirone base were added to the aquarium water. The pH was adjusted to 7.0 by adding drops of 1N sodium hydroxide. The aquarium water was changed every other day, and the fish were killed 2, 4 and 6 days after the beginning of each experiment. In the second experiment, 20 mg/litre of metopirone base were added to the aquarium water, and the animals were killed 2, 4 and 6 days after the beginning of the experiment. Most fish treated with this dose of metopirone showed signs of narcotization. In addition to the above LM investigations of mctopirone treatment, 5 animals were kept in tanks which contained 10 litres of tapwater to which were added 20 mg/litre of metopirone base. The ACTH cells of these animals and of 5 tapwater controls were examined by ultrastructural morphometry. LM measurements of possible cell size differences were not taken into account.

In the third experiment fish were kept, after acclimatization, in 70% seawater for 2 and 6 days. In all three experiments, the diameters of the interrenal nuclei were measured on photographic prints enlarged so that 1 mm ~ 1.0 ~m. Since cell outlines were difficult to determine, changes in the size of interrenal cells were estimated by comparing the number of nuclei in 11 rectangular areas per animal (total a r e a : 4 1 0 0 ~tm2). The results were not statistically analyzed since the number of animals used was small (see Table 1) and the differences between the cell or nuclear sizes were large. Mean values were quoted however with their standard errors.

Reconstruction Drawings. In the morphometric study of the ACTH cells from animals collected in the spring, and the ACTH cells from animals treated for 2 days with 20 big/1 metopirone (and the control animals to this experiment) an attempt was made to visualise the quantitative data by a series of reconstruction drawings. The details of this technique have been described previously (Benjamin, 1974a).

Results

The A C T H Celh' in Spring. The results of the morphometr ic analysis of the

A C T H cells from freshwater st icklebacks collected in the spring are summarised

in Table 2. Fig. 1 is a semi-diagrammat ic reconst ruct ion of the cell type based

on the above information, the f requency distr ibut ions of the secretory granule

sizes (Fig. 2) and the surface-area: volume ratio of the nucleus (0.51 •

Essent ia l ly the only conspicuous change in the A C T H cell of the spring animal compared with the winter animal (Benjamin, 1974a) was a significant decrease

in the amount of perinuclear R E R . Experimental Studies. The sizes of the interrenal cells and their nuclei changed

according to the physiological s tate of the tissue. The results are summarised in

Table 1. Each dose of metopirone adminis tered for 2, 4 and 6 days caused the nuclear d iameter to increase. The size of the interrenal cells also increased in each case, as judged by the average number of nuclei per unit area. There was an obvious dose-dependent relationship, since the increase in nuclear and cell size

of the interrenal cells caused by t rea t ing the animals with 20 mg/l i t re of metopirone was considerably greater than tha t caused by t rea t ing them with 5 mg/l i t re

ACTH-Interrenal Axis of the Freshwater Stickleback 107

Fig. 1. Reconstruction drawing of an ACTH cell from an adult stickleback collected in the spring

40

2C

50 150 250 350 Fig. 2. Percentage frequency distribution of the profiles of secretory granule diameters in

ACTH cells from spring animals. Ordinate, percentage frequency; abscissa, diameter (nm)


Table 1. The response of the interrenal tissue to various experimental conditions

Treatment No. of Cell density Nuclear animals (No. of nuclei diameter

per uni t area) (t~m)

Control, FW, 2 days Control, FW, 4 days Control, FW, 6 days Metopirone, 5 mg/1, 2 days Metopirone, 5 mg/l, 4 days Metopirone, 5 mg/I, 6 days Metopirone, 20 mg/l, 2 days Metopirone, 20 mg/l, 4 days Metopirone, 20 rag/l, 6 days 70% SW, 2 days 70% SW, 6 days Widewater sticklebacks

4 86.0 ~ 5.6 3.71 • 0.05 4 70.3 :~ 4.0 3.87 • 0.06 3 98.0 • 14.6 3.63 • 0.21 4 63 .8 • 4.11 • 3 64.0 • 2.5 4.03 • 0.05 3 67.0 ~= 5.0 4.03 • 0.05 5 44.8 i - 6.5 4.74 • 0.07 3 42.7 • 3.8 4.55 • 0. I0 3 43.0 • 1.2 4.58 • 0.20 3 51.3 • 1.8 4.26 i 0.00 3 60.0 ~: 2.3 4.11 • 0.000 4 46.8 -~ 2.9 4.58 • 0.05

Table 2. The percentage volume of ACTH cells from animals collected in the spring, from animals t reated with 20 mg/litre of metopirone for 2 days and from tapwater controls,

occupied by various organelles (Means i s.e.)

Organelle Spring animals Metopirone Control

Nucleus 20.8 • 25.5 ~ 2 . 7 4 20.0 • 1.87

Nucleolus 0.34 i 0.22 0.42 • 0.14 0.22 • 0.09

Golgi apparatus (a) Small vesicles 2.5 • 3.4 • 1.2 •

(b) large, dilated vacuoles 0.69 • 0.50 0.81 • 0.23 1.7 • 0.18

(c) f lat tened cistcrnae 0.00 • 0.00 0.07 • 0.04 0.07 • 0.03 (d) total Golgi app. 3.2 • 1.01 4.27 • 0.72 3.0 • 0.28

Immature secretory granules 0.00 • 0.00 0.02 i 0.02 0.02 ~- 0.01

Acanthosomes 0.00 i 0.00 0.00 • 0.00 0.00 • 0.00

Muir• bodies 0.21 • 0.10 0.16 i 0.06 0.52 • 0.08

Dense bodies 0.91 • 0.06 0.49 • 0.22 0.40 • 0.06

R E R membrane profiles (a) isolated small pieces without 4.3 • 0.69 4.9 • 0.77 4.9 -+- 0.68

dilated cavities (b) isolated small pieces with 5.0 i 1.53 5.6 • 1.04 4.2 • 0.95

dilated cavities (c) per• arrays 0.76 • 0.08* 5.3 • 1.02 3.8 • 0.59 (d) curvilinear whorls 6.7 • 1.12 5.7 • 1.36 5.4 • 1.28 (e) total R E R 16.8 • 2.57 21.5 • 1.74 18.3 • 1.84

Mature secretory granules 11.6 • 4.0 i 0 . 6 1 10.8 • 1.02

Mitochondria 5.7 • 3.7 • 4.8 •

Free ribosomes 15.5 • 1.90 11.1 • 1.08 16.6 • 1.46

Cytoplasmic ground substance 23.4 • 2.05 26.2 i 1.83 28.4 • 2.08

Significant differences (p > 0.05) between control animals and metopirone-treated animals are underlined. The only significant difference between the values for ACTH cells in spring and winter animals (Benjamin, 1974a) is marked with an asterisk.


Fig. 3. The RPD of a stickleback kept in tapwater for 2 days (control experiment). Note the moderately granulated ACTH cells (arrows). Lead haematoxylin

Fig. 4. The RPD of a stickleback kept in 20 mg/litre of metopirone for 2 days. Note the degranulatcd ACTH cells (arrows). Lead haematoxylin

(for 2, 4 and 6 days). With each dose level of metopirone, treatment for different lengths of time did not cause large differences in cell or nuclear sizes. The cell and nuclear sizes of animals kept in 70% seawater for 2 and 6 days increased, while animals collected from the land-locked saline lagoon at Widewater, also had enlarged interrenal cells and nuclei. Although all the experimental conditions produced an increase in interrenal cell and nuclear size, only the greater dose of metopirone (for 2, 4 and 6 days) caused a slight degranulation of the ACTH cells (Figs. 3-6).


Fig. 5. The head kidney of a stickleback kept in tapwater for 2 days (control experiment). h, haemopoietic tissue; ic, interrenal cells. Haematoxylin and Eosin

Fig. 6. The head kidney of a stickleback kept in 20 mg/litre of metopirone for 2 days showing cellular and nuclear hypertrophy of the interrenal cells. Note the mitotic figure (arrow) in

one interrenal cell. h, haemopoietic tissue; ic interrenat cells. Haematoxylin and Eosin

A quantitative ultrastructural examination of ACTH cells from animals treated with 20 mg/litre of metopirone for 2 days showed the changes in volume density of the orgenelles indicated in Table 2 (compared with control animals kept in freshwater for 2 days). Fig. 7 shows the frequency distribution of the granule sizes in the metopirone-treated animals and Fig. 8 is the reconstruction drawing based on the information given in Table 2, Fig. 7 and on the surface-area: volume ratio of the nucleus (0.58 Jr 0.06). The differences between the control animals kept in freshwater for 2 days and winter animals killed immediately they arrived in the laboratory (Benjamin, 1974a) were slight. Consequently it was

ACTH-Interrenal Axis of the Freshwater Stickleback

40

111

20

50 150 250 350 Fig. 7. Percentage frequency distribution of the profiles of secretory granule diameters in ACTH cells from sticklebacks treated with 20 mgflitre of metopirone for 2 days. Ordinate,

percentage frequency; abscissa, diameter (nm)

Fig. 8. Reconstruction drawing of an ACTH cell from an adult stickleback treated with 20 mg/litre of metopirone for 2 days


unnecessary to have a separate reconstruction drawing of the ACTH cells from control animals, or a separate frequency histogram of secretory granule sizes.

Discussion

According to Leatherland (1970) the ACTH cells of the migratory stickleback, Gasterosteus aculeatus form trachurus were most active in spring fish collected in freshwater, and were significantly larger than in juvenile and winter fish. The ACTH cells of these spring fish were more heavily granulated than the winter or juvenile fish. In the freshwater stickleback, G. aculeatus form leiurus, there was no such difference in the granulation of winter and spring fish, the only significant difference between winter and spring animals being a decrease in the amount of perinuclear RER in the latter. This, in keeping with the findings on the prolactin cell of the freshwater stickleback (Benjamin, 1974b), suggests that the seasonal changes in the RPD are not so pronounced as they are in the migratory stickleback.

Although the ACTH cells of the freshwater stickleback did not respond markedly to salinity changes, there was a definite increase in the cell and nuclear volumes of interrenals cells from animals transferred to seawater for either 2 or 6 days. In addition, these parameters were much greater in sticklebacks collected from the land-locked lagoon at Shoreham than in freshwater-maintained animals. There was a similar stimulation of the interrenal in Salmo gairdneri, when the animals were kept in seawater (Olivereau, 1962). In this case there was also an increase in the size and granulation of the ACTH cells. In contrast with these results, Fleming et al. (1971) found marked degenerative changes in the interrenal tissue of Fundulus kansae after exposing the fish to 0.4 M saline for fourteen days, suggesting that not much ACTH was secreted. There was an even more drastic lack of correspondence between the reactions of the ACTH and interrenal cells to altered environmental salinities in Anguilla anguilla (Hanke et al., 1967). A histological examination showed that there was a decrease in interrenal cell activity but an increase in ACTH cell activity. Furthermore, injections of ACTH or cortisol caused an increase in the activity of the ACTH cells. Lcatherland and Lam (1971), who investigated the effect of various hormones on the pituitary- interrenal axis of the migratory stickleback, found that injections of ACTH stimulated the interrenal tissue of winter fish. This was also accompanied however by signs of decreased ACTH cell activity. In summer fish, ACTH injections did not stimulate the interrenal cells, but still affected the ACTH cells.

This is the reverse of the situation in freshwater animals, where it was the ACTH cells that were unaffected by the various experiments. All these findings emphasize the difficulty of interpreting histological changes in terms of changes in secretory activity. I t may not be entirely appropriate to use interrenal nuclear diameters as an objective measurement of interrenal response, for mean nuclear diameter may reflect the rate of growth of a tissue as well as its secretory activity (Alfertetal., 1955). As pointed out by Rennelsetal. (1971), the quantity of hormone stored in the cytoplasm of any endocrine cell reflects the balance between hormone synthesis and hormone release. Consequently there will only be histological changes in the state of activity of an endocrine cell (e.g., increased


or decreased cell size and granulation) if the experimental procedures differentially affect synthesis and release. I t is therefore possible that the output of ACTH from the stickleback pituitary is increased in seawater-adapted animals, but that histological signs of this are absent. Although lead haematoxylin is considered to be the best available staining technique for ACTH cells, it is not specific for the ACTH molecule (Solcia etal . , 1969). Consequently it does not necessarily follow that staining intensities are directly related to hormone levels. The ACTH cells of the freshwater stickleback stain only moderately with lead haematoxylin, and it may well be that this teleost is not the most suitable for physiological examination. The mediocre staining properties of these cells with lead haematoxylin could be due to the fixation technique employed. Solcia et al. (1969) found that Bouin-fixed endocrine granules are readily stained by basic dyes, but react poorly with lead haematoxylin unless previously treated with hydrochloric acid.

The exact significance of the stimulation of the interrenal tissue of freshwater sticklebacks by seawater is difficult to assess, as Olivereau (1967) and Olivereau and Olivereau (1968) found that conditions of stress stimulated the ACTH-interrenal axis. LeLoup-Hs (1964), in comparing the adrenal reaction of several species of teleosts subjected to various altered environmental salinities, found that the response was independent of the euryhalinity of the species studied or the salinity of the altered environment. He concluded that adrenocortical activation is a stress response and may reflect other metabolic changes. The large nuclear diameters (4.58 :~ 0.05 ~m 2) of interrenal cells from freshwater sticklebacks naturally living in a highly saline environment (Widewater) perhaps indicate that the ACTH- interrenal axis is osmoregulatory in this species.

Only treating the fish with metopirone caused a change in the ACTH cells as well as the interrenal cells. After two, four or six days of treating the fish with the larger dosage of metopirone, the ACTH cells degranulated and the interrenal cells hypertrophied. The smaller dosage of metopirone did not change the ACTH cells, although the interrenal cells were again st imulated--but to a lesser extent. Numerous investigators have used metopirone to show that a pituitary-interrenal axis exists in various teleosts (e.g., Olivereau and Ball, 1963; Ball and Olivereau, 1966; Fagerlund et al., 1968; Mattheij, 1968) and in all these reports metopirone increased the activity of the ACTH-interrenal axis. The stimulation of this axis in the freshwater stickleback makes it likely that the cells lining the dorsal region of the RPD in this animal also produce ACTH. In contrast to the numerous publications on the LM effect of metopirone on the ACTH cells, no work has been published to the author's knowledge on the ultrastructural effect of metopirone on the ACTtt cells in teleosts. The decrease in the number of secretory granules in the ACTH cells of freshwater sticklebacks treated with metopirone could be caused by a faster rate of release than synthesis under the action of the drug. Under these conditions of increased release of the polypeptide hormone, ACTH, by metopirone treatment, there was a significant decrease ( p ) 0 . 0 5 ) in the number of free ribosomes and a significant increase (p~0.55) in the relative volume of the cell occupied by RER. According to Birbcck and Mercer (1961) ribosomes attached to the membranes of the ER (i.e., RER) synthesize protein for export from the cell, whereas those lying free in the cytoplasm synthesize protein for intracellular use. Consequently the increased demand for ACTH

8 ( :e l l T i s s . R e s . 155


caused by metopi rone t r e a t m e n t could be ref lected by the change in the re la t ive amounts of free r ibosomes and R E R . I t mus t be r emembered though t h a t no account has been t a k e n here of possible changes in the volume of the whole cell af ter metopi rone t l e a t m e n t . According to Pr ies t ley and Malt (1969) there is no change in the predominance of free r ibosomes in the k idney dur ing compensa to ry renal growth. Al though there was a slight increase in the volume of the Golgi appa ra tu s in me top i rone - t r ea t ed s t icklebacks, this was not significant (p < 0.05). Poss ib ly the exis t ing amoun t of Golgi appa ra tu s in the ACTH cell was sufficient to cope with the increased demands on i ts ac t iv i ty brought abou t by metopi rone .

The lysosomal sys tem has recen t ly been impl ica ted in the remova l of unwan ted secre tory granules from p i t u i t a r y cells (Smith and Fa rquhar , 1966; Hopk ins and Baker , 1968; Hopkins , 1969). I t is widely held t h a t mul t ives icu la r bodies are pa r t of the lysosomal system, and thus the i r s ignif icant decrease (p~0.O5) in the me top i rone - t r ea t ed animals would be expec ted if there were no unwan ted secre tory granules in the cytoplasm. However , there was no signif icant change (p < 0.05) in the number of dense bodies, which are also lysosomal.

Re~erences Alfert, M., Bern, H. A., Kahn, R. It . : Hormonal influence on nuclear synthesis IV. Karyo-

metric and microspectro-photometric studies of rat thyroid nuclei in different functional states. Acta anat. (Basel) 23, 185-205 (1955)

Ball, J. 1~., Olivereau, M.: Identification of ACTH cells in the pituitary of two teleosts, Poecilia latipinna and Anguilla anguilla: correlated changes in the interrenal and in the pars distalis resulting from administration of metopirone (SU 4885). Gen. comp. Endocr. 6, 5-18 (1966)

Benjamin, M. : A morphometric study of the pituitary cell types in the freshwater stickleback, Gasterosteus aculeatus, form leiurus. Cell Tiss. Res., 152, 69-92 (1974a)

Benjamin, M. : Seasonal changes in the prolactin cell of the pituitary gland of the freshwater stickleback, Gasterosteus aculeatus form leiurus. Cell Tiss. Res., 152, 93-102 (1974b)

Birbeck, M. S. C., Mercer, E. H. : Cytology of cells which synthesize protein. Nature (Lond.) 189, 558-560 (1961)

Fagerlund, U. It. M., McBride, J. R., Donaldson, E. M. : Effect of metopirone on pituitary- interrenal function in two teleosts, sockeye salmon (Oncorhynchus nerka) and rainbow trout (Salmo gairdneri). J. Fish. Rs. Bd. Canada 25, 1465-1474 (1968)

Fleming, W. R., Ball, J. N., Conaway, C. H.: The effects of a saline environment and ACTH on the interrenal of Fundulus kansae. Z. vergl. Physiol. 74, 121-126 (1971)

Hanke, W., Bergerhoff, K., Chan, D. K. 0.: Histological observations on pituitary ACTtt- cells, adrenal cortex, and the corpuscles of Stannius of the european eel (Anguilla anguilla L.) Gen comp. Endocr. 9, 64-75 (1967)

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

Leatherland, J. F. : Seasonal variation in the structure and ultrastructure of the pituitary of the marine form (Trachurus) of the threespine stickleback, Gasterosteus aculeatus L. 1. Rostral pars distalis. Z. Zellforsch. 104, 301-317 (1970)

Leatherland, J. F., Lam, T. J.: Effects of prolactin, corticotrophin and cortisol on the adenohypophysis and interrenal gland of anadromous threespine sticklebacks, Gasterosteus aculeatus L. form trachuru8, in winter and summer. J. Endocr. 51, 425-436 (1971)


LeLoup-Hgtey, J. : Etude du determinisme de l 'activation de l 'interrenal ant6rieur observ6e chez quelques t616ost6ens soumis s un accroissement de la salinit6 du milieu ext6rieur. Arch. Sci. physiol. 18, 293-324 (1964)

Mattheij, J . A . M . : The ACTH cells in the adenohypophysis of the mexican cave fish, Anoptichthys jordani, as identified by metopirone (SU 4885) treatment. Z. Zellforsch. 92, 588-595 (1968)

McConaill, M. A. : Staining of the central nervous system with lead haematoxylin. J. Anat. (Lond.) 81,371-372 {1947)

Olivereau, M.: Cytologic de l'hypophyse du cyprin (Carassius auratus L.). C.R. Acad. Sci. (Paris) 255, 2007-2009 (1962)

Olivereau, M. : R6actions observ6es ehez l'Anguille maintenue dans un milieu priv6 d'61eetro- lytes, en particulier au niveau du systbme hypothalamo-hypophysaire. Z. Zellforsch. 80, 264-285 (1967)

Olivereau, M., Ball, J. N. : Fonction corticotrope et cytologic hypophysaire chez deux t616- ost6ens MoUienisia latipinna Le Sueur et Anguilla anguilla L. C.R. Acad. Sci. (Paris) 266, 3766-3769 (1963)

Olivereau, M., Olivereau, J.: Effects de l'interr6nalactomie sur la structure histologique de l'hypophyse et de quelques tissues de l'anguille. Z. Zellforsch. 84, 44-58 (1968)

Priestley, G. C., Malt, R. A. : Membrane-bound ribosomes in kidney. Methods of estimation and effect of compensatory renal growth. J. Cell Biol. 41,886-893 (1969)

Rennels, E. G., Bogdanove, E.M., Arimura, A., Saito, M., Schally, A. V. : Ultrastructural observations of rat pituitary gonadotrophs following injection of purified porcine LH-RH. Endocrinology 88, 1318-1326 (1971)

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)

Solcia, E., Capella, C., Vassallo, G. : Lead-haematoxylin as a stain for endocrine cells. Signifi- cance of staining and comparison with other selective methods. Histochemie 20, 116-126 (1969)

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the acth-interrenal axis in the freshwater stickleback, gasterosteus aculeatus form leiurus

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