Cell Tiss. Res. 191, 161-170 (1978) Cell and TissueResearch© by Springer-Verlag 1978

Ultrastructural Studies on Prolactinand Growth Hormone Cells in Anguilla Pituitariesin Long Term Cultures

Michael Benjamin*Department of Anatomy, University College, Cathays Park, Cardiff, Wales

Bridget I. BakerSchool ofBiological Sciences, Bath University, Claverton Down, Bath, England

Summary. Eel hemi-pituitaries were cultured in vitro on high or low sodiummedia, previously shown to affect differentially prolactin and growth hormonerelease. After 6 days culture, there were marked differences in the ultrastructureo f both prolactin and growth hormone cells from the two groups. Morphome-tric data on the prolactin cells from SW-adapted eels showed a greaterabundance of RER and paucity o f secretory granules in cells from the lowsodium medium. The size o f the Golgi apparatus and the number of exocytosedsecretory granules did not differ markedly between experimental groups, incontrast to previous findings on short-term cultures. Differences in the profilediameters o f secretory granules are recorded between the experimental groupsand the pattern differs markedly from that previously recorded for short-termcultures. The growth hormonecells from low sodium media were characterisedby abundant, vesiculated RER, a prominent Golgi apparatus (in SW-adaptedanimals) and relatively few secretory granules. The activity o f these growthhormone cells is in marked contrast to previous findings relating to short-termcultures. The shape and size of the non-granulated (stellate) cells of the RPD wasagain affected by the osmotic pressure of the medium.

Key words: Prolactin cells - Growth hormone cells - Pituitary (Angui l la) - Invitro - EM morphometry.

Introduction

When eel pituitaries are cultured in vitro, the rate of prolactin and growth hormonerelease is related to the osmotic pressure of the medium, and is clearly more intenseon medium with a low osmotic pressure and sodium concentration (Ingleton et al.1973; Baker and Ingleton, 1975). However it has not been possible to detect any

Send offprint requests to: Dr. M. Benjamin, Department ofAnatomy, University College, Cathays Park,CardiffCF1 1XL, Wales* I should like to thank Mr. P.F. Hire for his photographic assistance

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162 M. Benjaminand B.I. Baker

obvious c h a n g e in the rate of radioactive leucine incorporation into the hormonesu n d e r these two culture conditions. Ultrastructural studies of prolactin cells frompituitaries cultured for only 24 h (Benjamin and Baker, 1976), showed that in thisshort period there was a rapid enlargement of the G o l g iapparatus on a low sodiumm e d i u m together with abundant profiles of granule-exocytosis. This suggestedintense hormone packaging and release - though at this time there was only limitedevidence of an increase in RER. In short term cultures there were no obviousdifferences in the ultrastructure of the growth hormone cells from different media.The present work continues the earlier s tudy (Benjamin and Baker, 1976) andexamines the ultrastructure of eel hemi-pituitaries cultured on high or low sodiumm e d i a for a longer time - six days.

M a t e r i a l s and M e t h o d s

Yellow eels (Anguilla anguilla) were caught in the Severn estuary. Some were kept in the laboratoryaquarium intap water, while others were transferred to sea water. Both groups were allowed toadaptforseveral weeks before use.

Cultures. Eels werekilled by decapitation, the pituitaries removed aseptically and cut sagitallyin half.Thehalveswerecultured independently as described previously (Benjamin and Baker,1976) onmediumcontaining either ll0mE Na÷ (110 Med; approximately 240mOsm) or 170mE Na ÷ (170Med;approximately 340m Osm). Medium was renewed every secondday and the tissue fixed for electronmicroscopy onthe sixth day. EM processing techniques and morphometry werethesame as described ina previous paper (Benjamin and Baker, 1976).

R e s u l t s

Although the pituitaries in this s tudy were taken from either freshwater (FW)- orseawater (SW)-adapted eels, the differences arising as a result of 6 days culture on110 Med or 170 Med were similar, regardless of the previoushistory of the eel. Thusthe two groups will be considered together.

Prolactin Cells and Associated Non-Granulated Cells. Previous studies (Benjaminand Baker, 1976) showed that after only one day of culture, prolactin cells from alow sodium m e d i u m had multiple, well-developed G o l g i bodies in contrast to thesingle, inconspicuous G o l g i body of prolactin cells from the high sodium medium.This difference was not maintained after six days of culture (Table 1) by which timethe G o l g i apparatus appeared as a single, fairly prominent structure in cells fromboth media. Thus, although in the particular pair of hemi-pituitaries illustrated inFigs. 1 and 2, the G o l g i apparatus is clearly more conspicuous in 110 Med, this wasnot the case in all pituitaries. Signs of immature, secretory granule formation wereevident in the G o l g i regions from both groups.

Another difference between long-term and short-term cultures was the numberof figures of granule exocytosis. It was noted previously (Benjamin and Baker,1976) that after 1 day exposure to l l 0 M e d , figures of granule exocytosis weremuch more c o m m o n than on 170Med, although differences in cytoplasmicgranulation were not apparent at this stage. After 6 days culture however, the

Prolactin and Growth Hormone Cells in Anguilla Pituitary 163

Figs.1 and 2. Prolactincells from opposite halves of a SW-adapted eel pituitary culturedon 110 Med(Fig. 1) and 170 Med (Fig. 2). Note theprominent nucleolus (N) and RER and the sparsity of secretorygranules in 110 Med. In this pituitary, the Golgi apparatus (G) is dearly larger in cells from 110 Med.Some irregularly shaped secretory granules can be seen in Fig. 2 (arrows). DB dense bodies, x 7200

cytoplasm was very markedly degranulated in four out of five cultures, but thefrequency ofexocytotic granules was now rather similar in the two groups (Table 1;Figs. 1, 2). These ultrastructural features are in keeping with assays of rates ofprolactin release into the culture m e d i u m(Baker and Ingleton, 1975). The secretorygranules were often slightly irregular in shape, particularly in pituitaries taken fromSW-adapted eels (Fig. 2). Measurements of granule diameters indicated a tendencytowards larger granules in cells from 110 Med after 6 days culture (Figs. 3, 4). Thusin FW-adapted eels, over 70 ~ of granules had diameters greater ~than 200 nm,compared with only a b o u t 30 ~ of the granules in cells from 170 Med.

164 M. Benjamin and B,I. Baker

4O

35 [ ~ 11(1 Med

30 [ ~ 171] Med

~ 25

~ 2O

10

5

0 50 100 150 200 250 300 350 400 450

Diameter (nm)

Fig. 3. Histogram showingthe size distribution of theprolactin secretory granules fromcellson 110 Medand 170 Med. SW-adapted eel

41]

35

30

25

20

15

10

5

\ \~ . . ~ 111] Med

~ - \ \ " IN'N] 170 Med

N \ \ , ,\ \ \ - .

50 100 150 2110 250 300 350 400 450

Diameter (nm)

Fig. 4. Histogram showingthe size distribution ofthe prolaetin secretory granules fromcellson 110 Medand 170 Med. FW-adapted eel

Prolactin and Growth Hormone Cells in Anguilla Pituitary 165

In keeping with light microscope observations (Baker and Ingleton, 1975), theRER was much better developed in prolactin cells from a low sodium medium(Table 1; Figs. 1, 2). It comprised both parallel cisternae near the nucleus and a fewsmall tubules scattered through the cytoplasm. The nucleolus was also moreprominent in such cells (Fig. 1).

In contrast to the earlier study relating to 1 day cultures (Benjamin and Baker,1976), there were no marked differences between the number ofdense (lysosomal?)bodies and lipid droplets in 110 Med and 170 Med (Table 1).

TaMe 1. Morphometric data on prolactin cells from hemi-pituitaries of SW-adapted eels after 6 daysculture. Pairs of results from opposite halves of the same pituitary

Morphometric parameter Na+ ll0mEq/1 Na+ 170mEq/1(235 mOsm) (337 mOsm)

Percentage of the total cytoplasmic volumeoccupied by the Golgi apparatus and itsassociated vesicles

2.3 2.53.4 2.13.1 N.S. 2.72.5 2.73.5 4.4

Percentage of the total cytoplasmic volumeoccupied by secretory granules

1.9 5.912.5 11.62.2 N.S. 8.8.0.7 12.73.6 10.0

Percentage o f the total cytoplasmic volumeoccupied by RER

19.9 3.221.5 Significant 6.014.6 p<0.001 9.618.8 6.218.1 6.4

Percentage of the total cytoplasmic volumeoccupied by mitochondria

9.9 5.58.2 8.66.3 N.S. 9.9

13.4 9.611.2 6.2

Percentage of the total cytoplasmic volumeoccupied by dense (lysosomal?) bodies andlipid droplets

0.6 0.60.5 0.91.2 N.S. 0.61.8 0.91.3 2.4

Number of exocytosed secretory granule profiles/100 ~tm2 of prolactin cell

1.2 0.41A 1.15.4 N.S. 2.01.6 1.61.6 0.2

Statistical analysis: p values based on comparing pairs of results from opposite halves of the samepituitary. Data assessed as previously (Benjamin and Baker, 1976)

166 M. Benjaminand B.I. Baker

The non-granulated (stellate) cells were consistently larger in hemi-pituitariescultured on 170 Med and their processespenetrating between cells were narrower incells from 110 Med. This was confirmed by quantitative measurements of volumesand volume/surface ratios of non-granulated cells (Table 2). Lipid droplets anddense bodies, which were a conspicuous feature of non-granulated cells in short-term cultures, were less conspicuous in these 6 day cultures (Table 2).

Growth Hormone Cells. In bo th FW and SW-adapted eels, the ultrastructuraldifferences between growth hormone cells were very marked compared withprevious findings relating to one day cultures (Benjamin and Baker, 1976). In 110Med the cells were markedly degranulated and some of the remaining granules wereof low electron densi ty(Fig. 5). Somatotropes on 110 Med also contained far moreRER than those on 170 Med (Figs. 5, 6). This RER was arranged bo th as parallelar rays ofcisternae and as small tubulesscattered throughout the degranulated par t sof the cell(Figs. 5, 7). In pituitaries taken from eels previously adapted to seawater,the G o l g i apparatus was better developed and newly-formed granules were morec o m m o n on 110 Med (Fig. 7), but this difference was less evident in pituitaries takenfrom freshwater-adapted eels. The number of dense (lysosomal?) bodies wasvariable, but the trend was for a greater number in 110 Med. Exocytosed secretorygranules were never seen.

Table2. Morphometric data on non-granulatedcells ofthe RPD, from hemi-pituitaries of SW-adaptedeels after 6 days culture. Pairs of results from opposite halves of the same pituitary

Morphometric parameter Na÷ 100mEq/1 Na+ 170mEq/l(235 mOsm) (337 mOsm)

Ratio of prolactincell volume tonon-granulated cell volume

5.7 4.410.2 5.46.9 Significant 3.6

10.9 p<0.001 2.73.5 2.0

Relative non-granulated cell volumes 1 . 0 : 1 . 3

1 . 0 : 1 . 51.0 : 1 . 5

1.0 : 4.41.0 : 1.4

Volume surface (llma/lam 2) ratio ofnon-granulated cells (i.e., volume ofnon-granulated cells/surface in contact withprolactincells)

0.62 0.940.48 0.820.71 Significant 0.940.68 p<O.O01 0.870.41 0.65

Percentage of the total cytoplasmic volumeoccupied by dense (lysosomal?) bodies andlipid droplets

0.7 0.00.4 0.30.0 N.S. 0.02.8 0.80.1 0.8

Statistical analysis: p values based on comparingpairs of results from opposite halves of the samepituitary. Data assessed as previously (Benjamin and Baker, 1976)

Prolactin and Growth Hormone Cells in Anguilla Pituitary 167

Figs.5 and6. Growth hormone cellsfrom opposite halves o fa SW-adapted eelpituitary cultured on 110Med (Fig. 5) and 170 Med (Fig. 6).Note the prominent, yet fragmented, RER in Fig. 5 and the relativescarcity of secretory granules. In Fig. 5 occasional secretory granules are of low electron density(arrows). x 8000

168 M. Benjamin and B.I. Baker

Fig.7. The prominent Golgi apparatus in a growth hormone cell from 110 Med(SW-adapted eel)showing many signs ofsecretory granule formation (arrows). Note the tubularand vesicular RER nearthe plasma membrane (P). x 25,600

Discussion

These observations confirm that when eel pituitaries are maintained in vitro, bothprolactin and growth hormone cellsshow a greater degranulation and developmento f RER on a medium with low sodium and osmotic pressure. In the prolactin cells,granule exocytosis is an immediate response to lowered osmotic pressure and ismarked after 24h culture (Benjamin and Baker, 1976); but development of newRER takes longer. Such a time lag has been noted inseveral pituitary cell types afterthe application of a stimulus (Ball and Ingleton, 1973; Weatherhead and Whur,1972). However a more immediateresponse in RER development has been recordedin vitro(Zambrano et al., 1974). The presence of numerous, small and peripherallyplaced Golgi bodies, which was so striking a feature after one day in vitro on 110Med, was no longer seen after 6 days. It is possible that Golgi duplication is astructural response to the need for rapid granule formation at a time when the RERis poorly developed. Thus, after 6 days culture on 110 Med when RER is welldeveloped, multipleGolgi bodies are no longer apparent. Although reports o fGolgiapparatus duplication in animal cells do not appear to be common, such a responsehas been noted in mammalian GH3 cells following stimulation by TRH (Tixier-Vidal, 1975).

Many authors have noted that the prolactin secretory granules tend to be largerin fish adapted to freshwater than to seawater, both in eels (Knowles and Vollrath,

Prolactin and Growth Hormone Cells in AnguillaPituitary 169

1966) and in other species (Nagahama et al., 1973; Leatherland et al., 1974;Abraham et al., 1977). This change was noted also after 6 days in vitro, in spite ofthe tendency towards smaller granules on 110 Med after only 1 day culture. Thereason for this divergence in granule size in association with different secretoryactivities is obscure.

The marked effect o f different salinities on the ultrastructure of the growthhormonecells also reflects what has been observed in vitro and suggests a possibleosmo - or ionoregulatory role of the hormone. Environmental salinity affects thegrowth hormone cells in Anguilla (Olivereau and Ball, 1970), in Tilapia spp.(Leatherland et al., 1974), in Oncorhynchus kisutch (Nagahama et al., 1977) and inPungitiuspungitius(Benjamin, 1978). As a result o f in vitro observations on Tilapiamossambiea pituitaries, Zambrano et al. (1974) were doubtful if the growthhormone cells also reflects what has been observed in vivo and suggests a possiblethey could observe no changes in these cells after 6 h culture on a hypotonicmedium. However, earlier and present observations on Anguilla pituitaries in vitro,suggest that the growth hormone cells respond to osmotic change much moreslowly than do the prolactin cells; even in the eel differences on 110 Med and 170Med were scarcely evident after 24h culture (Benjamin and Baker, 1976).

In the present work the degranulated growth hormone cells from 110 Med werepacked with rough endoplasmic reticulum (RER). It is noteworthy that much of thisRER was in the form of small tubules and vesicles. Similar findings have beenreported in actively secreting, rat mammotrophs (Chang and Nikitovitch-Winer,1976). These authors suggest that the structural collapse, yet proliferation, o f theRER may relate to a direct release of hormone on to the cell surface, without anintermediary passage through the secretory granule phase. The hypothesis iscertainly an attractive one for the growth hormonecells of Anguilla, in view of ourfailure to detect any evidence ofexocytosis - although granule formation within theGolgi apparatus was evident.

References

Abraham, M., Dinari-Lavie, V., Lotan, R.: The pituitary ofAphanius dispar (R~ippell) from hypersalinemarshes and freshwater. II. Ultrastructure of the rostral pars distalis. Cell Tiss. Res. 179, 317-330(1977)

Baker, B.I., Ingleton,P.M.: Secretion of prolactin and growth hormone by teleostpituitaries invitro II.Effect of salt concentration during long-term organ culture. J. comp. Physiol. 100, 26%282 (1975)

Ball, J.N., Ingleton,P.M.: Adaptivevariations inprolactin secretion in relation to external salinity in theteleost Poecilia latipinna (Teleostei). Gen. comp. Endocr. 20, 312-325 (1973)

Benjamin,M.: Cytologicalchanges inprolactin,ACTH and growthhormone cells ofthe pituitary glandofPungitiuspungitius L., in response to increased environmental salinities. Gen. comp. Endocr. Inpress (1978)

Benjamin, M., Baker, B.I.: Ultrastructural studies on prolactin and growth hormone cells in Anguillapituitaries in vitro. Cell Tiss. Res. 174, 547-564 (1976)

Chang, N.G., Nikitovitch-Winer, M.B.: Correlation between suckling-induced changes in theultrastructure of mammotrophs and prolactin release. Cell Tiss. Res. 166, 389-406 (1976)

Ingleton,P.M., Baker, B.I., Ball, J.N. :Secretion of prolactin and growthhormone by teleost pituitariesin vitro. I. Effect of sodium concentration and osmotic pressure during short-term incubations. J.comp. Physiol.87, 317-328 (1973)

Knowles, F., Vollrath, L.: Neurosecretory innervation of the pituitary of the eels Anguilla and Conger.II. The structure and innervation of the pars distalis at different stages of the life-cycle. Phil. Trans.250, 329-342 (1966)

170 M. Benjamin and B.I. Baker

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

Nagahama, Y., Clarke, W.C., Hoar, W.S.: Influence ofsalinityon ultrastructure ofthe secretory cells ofthe adenohypophysealpars distalis inyearlingcoho salmon (Oncorhynchus kisutch). Canad. J. Zool.55, 183-198 (1977)

Nagahama, Y., Nishioka, R.S., Bern, H.A.: Responses ofprolactincells of twoeuryhalinemarine fishes,Gillichthys rnirabilis and Platichthys stellatus, to environmental salinity. Z. Zellforsch. 136, 153-167(1973)

Olivereau, M., Ball, J.N.: Pituitary influences on osmoregulation in teleosts. In: Hormones and theEnvironment. Mem. Soc. Endocr. 18, 57-82 (1970)

Tixier-Vidal, A.: Ultrastructure o fanteriorpituitarycells in culture.In: The anteriorpituitary(A. Tixier-Vidal and M.G. Farquhar, eds.), pp. 181-229. New York: Academic Press 1975

Weatherhead, B., Whur,P.: Quantification ofthe ultrastructural changes in the'melanocyte-stimulatinghormone cell' of the pars intermedia of the pituitary of Xenopus laevis, produced by change ofbackground colour. J. Endocr. 53, 303-310 (1972)

Zambrano, D., Clarke, W.C., Hajek, A., Sage, M., Bern, H.A.: Influence of medium concentration onprolactin and growth hormone cells during short-term incubation of pituitary glands from Tilapiamossambica. Acta zool. (Stockh.) 55, 205-216 (1974)

Accepted March 23, 1978