J. Cell Sci. 3> 357-364 (1968) 357

Printed in Great Britain

THE FINE STRUCTURAL LOCALIZATION OF

ACID PHOSPHATASE IN THE PROLACTIN

CELL OF THE EEL PITUITARY

C. R. HOPKINSDepartment of Histology, Liverpool University, Brownlotv Hill, Liverpool 3

AND BRIDGET I. BAKERDepartment of Zoology, Liverpool University, Brownlow Hill, Liverpool 3

SUMMARY

In the prolactin cell of the eel adenohypophysis acid phosphatase activity occurs within themajority of the Golgi cisternae and developing secretory granules. Acid phosphatase is alsopresent within larger membrane-bound bodies, most of which are similar to the lyric densebodies described in other cell types. In discussing the functional significance of this enzymedistribution particular attention is paid to its association with the secretory mechanisms of theprolactin cell.

INTRODUCTION

In recent years a number of reports (Novikoff, 1963; Novikoff & Essner, 1962;Novikoff, Essner, Goldfischer & Heus, 1962; Novikoff, Essner & Quantina, 1964;Poux, 1963; Smith, 1963; Osinchak, 1964; Brandes, 1965; Smith & Farquhar, 1966)have suggested that, in certain secretory cells, acid phosphatase activity is associatedwith the formation of the secretory product.

Cells in which such a relationship is believed to exist include those of exocrine andendocrine tissues. In the endocrine tissues studied, an acid phosphatase which can berelated to secretory activity is most consistently found in the cells of the adenohypo-physis (Sobel, 1961a, b, 1962, 1964; Schrieber, 1962; Smith, 1963, Hinoshige,Nakatzugawa, Matsuoka & Itoh, 1966; Smith & Farquhar, 1966). Most of the studieson the adenohypophysis have employed methods of biochemical assay (Sobel, 1961 a;Schrieber, 1962; Kobayashi, 1966; Hinoshige et al. 1966) and light-microscopehistochemical staining procedures (Sobel, 1961a, b, 1962), but the most definitiveresults have been provided by ultrastructural cytochemistry (Smith, 1963; Smith &Farquhar, 1966). Using these techniques, Smith & Farquhar (1966) have been able toinvestigate the fine-structural localization of acid phosphatase within a single specificcell type of the rat adenohypophysis under a variety of experimental conditions. Theirresults have shown that lytic activity, as indicated by the distribution of acid phos-phatase, is a major feature of the cell's secretory mechanism, and seems to be con-cerned not only with the formation of secretory product, but also with an hithertounsuspected intracellular digestive pathway for the modulation of secretory activity.

23 Cell Sci. 3

358 C.R. Hopkins and B. I. Baker

Whether or not such lytic activity is to be found in the other cell types of the adeno-hypophysis remains to be shown.

Few previous enzyme histochemical studies (Legait, Legait & Mercier, 1964) havebeen made on the fish adenohypophysis; the aim of the present study is to examine thedistribution of acid phosphatase in a single adenohypophysial cell type of a specieswithin this vertebrate class and to attempt to correlate its localization with thesecretory mechanism of these cells.

The tissue comprising the anterior rostral part of the eel adenohypophysis waschosen because the majority of its cells are of one type only—prolactin (Olivereau &Ball, 1964), and because a basis for an electron microscope cytochemical study ofthese cells has been provided in the recent fine-structural investigation of the eeladenohypophysis by Knowles & Volrath (1966).

MATERIALS AND METHODS

Young, immature, (25-28 cm) eels (Anguilla anguilla) were collected from Angleseyby electric fisher and allowed to acclimatize for one month in laboratory aquaria.Following the rapid removal of the pituitary, the rostral part was excised and placedm 3 % glutaraldehyde in 0-067 M cacodylate buffer at 5 °C. In the glutaraldehyde thetissue was cut into longitudinal strips and allowed to fix for 1 h. Following fixation thetissue was given three 20 min rinses in 0-067 M cacodylate-buffered 5 % sucrose beforebeing prepared either for routine postfixation in osmium tetroxide (2 %) and embed-ment in Maraglas (Spurlock, Kattine & Freeman, 1963) or for histochemistry. Tissueemployed in histochemical procedures was either sectioned at 8 /i on a cryostat forlight-microscope examination or sectioned at 40 /i on a modified Mclllvain chopper(Smith & Farquhar, 1965). Cryostat sections were incubated for varying periods oftime in the standard Gomori acid phosphatase substrate medium (Gomori, 1952),reacted with ammonium sulphide and, after rinsing, mounted in glycerine. Controlsections were incubated in substrate medium lacking either sodium /?-glycerophos-phate, or lead ions, or to which o-oi M sodium fluoride had been added. The 40 jisections were incubated in the Gomori substrate medium for times determined by theresults of the cryostat section procedure (30-60 min) but were not reacted withammonium sulphide. After rinsing in 0-067 M cacodylate buffer these sections werepost-osmicated for 1 h at 5 °C before being dehydrated in cold ethanol and embeddedin Maraglas.

All Maraglas-embedded tissues were sectioned with a diamond knife on a Huxleyultramicrotome, mounted on unsupported grids and stained with lead citrate alone(Reynolds, 1963), or with ethanolic uranyl acetate followed by lead citrate. The sec-tions were examined in a Siemens Elmiskop I electron microscope and photographedat magnifications of between x 2300 and x 10000.

Acidphosphatase in the eel adenohypophysis 359

OBSERVATIONS

The prolactin cells of the eel adenohypophysis are grouped into discrete follicles.The cells are elongate pyramids with basal nuclei and contain an extensive distribu-tion of rough endoplasmic reticulum within their peripheral cytoplasm (Fig. 1; seeKnowles & Volrath, 1966). Typically (Knowles & Volrath, 1966), these cells containabundant membrane-bound secretory granules (diam. 280 m/i), the electron densityof which varies with the preparative procedure employed (Figs. 1, 2, 5, 7, 8).

Of particular interest to the present study is the well-developed Golgi apparatuswhich lies close to the nucleus in the apical cytoplasm (Fig. 1). Appropriate sectionsshow that this structure is cup- or bowl-shaped, its outer convex cisternae often lyingclosely parallel to the adjacent cisternae of rough endoplasmic reticulum (Fig. 8). Theinner concave Golgi cisternae surround an area of cytoplasm within which a numberof free and forming membrane-bound bodies occur (Figs. 2-4).

Usually there are between six and eight parallel cisternae, most of them terminatingas bulbous expansions (Figs. 3, 4, 8). These expansions may be smooth surfaced orbear a coat of short spines; often they contain electron-opaque secretory product(Fig. 3). The largest accumulations of secretory material occur within the innermostcisternae and there are frequent profiles to suggest that it is from these cisternae thatthe free secretory granules arise. The contents of the smaller, presumably newlyformed secretory granules are not homogenously electron-opaque since they have athin electron-lucent rim beneath their limiting membrane (Figs. 2, 3). In the fullydeveloped secretory granule the limiting membrane is closely applied to an homo-genously electron-opaque core (Fig. 8).

Small vesicles (diam. 400-800 A) with electron-lucent contents also arise from theexpanded terminations of the innermost Golgi cisternae. These vesicles, which maybe smooth surfaced or bear a spinous coat, are similar to those termed Golgi vesiclesby Novikoff et al. (1964).

Profiles suggesting that Golgi vesicles may fuse with maturing secretory granulesare frequently observed (Figs. 1, inset, 4, 8). There is, however, no evidence to suggestthat developing secretory granules coalesce with one another.

Other, larger membrane-bound bodies are also especially numerous in the vicinityof the Golgi apparatus. Most of these bodies have evenly granular contents (Figs. 4, 9)and are morphologically identical to the dense bodies described from other cell types(Novikoff et al. 1964). The remainder of the larger membrane-bound bodies are moreirregular in outline and heterogenous in content (Figs. 5-7). Frequently these bodiescontain membranous configurations, and, in addition, there are occasionally densespherical areas, similar in size and density to a mature secretory granule (Fig. 6).

Acid phosphatase distribution

With the light microscope acid phosphatase is seen to be localized in small (1 fi)particles within the apical cytoplasm. The fine structural localization of reactionproduct confirms this observation and in addition shows acid phosphatase activity tobe present within the Golgi apparatus. No acid phosphatase reaction product is ob-

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360 C. R. Hopkins and B. I. Baker

served within the endoplasmic reticulum or in association with the cell plasmamembrane.

The Golgi apparatus in the cells observed is consistently acid phosphatase-positive.In any one Golgi area, however, although most of the cisternae are positive, singlecisternae lacking reaction product also frequently occur (Figs. 3, 4). The expandedterminations of the cisternae, whether or not these terminations contain secretoryproduct, together with the free elements to which they give rise, may also be acidphosphatase-positive (Figs. 3, 4, 8). In those cisternae and free bodies which containsecretory product, the acid phosphatase reaction product is usually peripheral, lyingimmediately below the limiting membrane (Figs. 2-4). Those Golgi vesicles which areacid phosphatase-positive are filled with reaction product (Figs. 2-4). It is interestingto note, however, that, as in the Golgi cisternae, only a proportion of the developingsecretory granules and Golgi vesicles contain reaction product.

All of the various forms of larger membrane-bound body present in the cytoplasmof the prolactin cell may contain acid phosphatase (Figs. 1, 2, 4, 5, 7). These bodies,however, are not always acid phosphatase positive and, although the heavy depositionof lead phosphate often makes it difficult to discern the morphological details of thesestructures, it can be said that the bodies which least often contain reaction product arethose with membranous or fibrous contents. The dense body is the most frequentlyencountered form of reactive body.

The acid phosphatase activity concerned in the interaction between the smallGolgi vesicles and the maturing secretory granules varies. Thus profiles have beenseen which suggest that acid phosphatase-positive vesicles fuse with acid phosphatase-positive and acid phosphatase-negative secretory granules (Figs. 4, 8). It is unusualfor demonstrable acid phosphatase activity to occur within those secretory granuleswhich, because of their size and content of secretory material, can be regarded asfully developed (Figs. 1-8).

DISCUSSION

Recent work on the fish and amphibian pituitary by Knowles and others (Knowles,1965; Knowles & Bern, 1966; Knowles, Volrath & Nishoika, 1967; Iturriza, 1964;Cohen, 1967) has suggested that the secretory activity within the adenohypophysis isunder dual neurosecretory control and may depend upon the interplay betweeninhibitory and stimulatory impulses. Having provided a morphological basis for thiscontrol by describing two structurally different kinds of synaptic ending upon thesecretory cells (Knowles, 1965; Knowles & Volrath, 1966), it has been further sug-gested that the control may be concerned with two distinct phases of secretoryactivity, namely the processes of synthesis and release of the secretory product(Knowles & Bern, 1966; Cohen, 1967).

In interpreting the fine structure of the prolactin cell observed in the present studyin the light of these suggestions, features such as the cell volume, size of the nucleus,abundance of endoplasmic reticulum and the development of the Golgi apparatus, allsuggest a relatively high level of synthesis. At the same time, however, the abundance

Acid phosphatase in the eel adenohypophysis 361

of mature secretory granules, together with the paucity of release figures, indicatesthat the release of the secretory product is only occurring at a low level.

In the present study the main focus of interest has been on the Golgi area of theprolactin cell since, with the techniques employed, the most informative changes con-cerned with the synthesis of the secretory product occur within this organelle.

The cup shape of the Golgi apparatus observed in the prolactin cell, and its associa-tion with the surrounding endoplasmic reticulum and forming secretory granules,suggests that it may be functionally polarized. Such an interpretation was originallyput forward by Mollenhauer & Whaley (1963) for a similar Golgi configurationwithin the plant cell, and it has since been applied by a number of workers on animaltissues (Novikoff & Shin, 1964; Bainton & Farquhar, 1966; Jamieson & Palade, 1966;Smith & Farquhar, 1966). According to this interpretation, the outer convex surfaceof the Golgi is closely associated with the endoplasmic reticulum from which it re-ceives newly synthesized, membrane-bound materials such as secretory product pre-cursors. At the inner, concave surface it is believed that the concentrated productsarise from the cisternae as free membrane-bound elements; these include the secretorygranules and Golgi vesicles. A similar intracellular pathway through the Golgi hasbeen outlined by the extensive studies of Palade and co-workers (Palade, Siekvitz &Caro, 1962; Caro & Palade, 1964; Jamieson & Palade, 1967a, b) in their combinedbiochemical and autoradiographical studies of the synthesis of 'protein for export' inthe pancreatic zymogen cell.

Once the secretory granules become free from the Golgi cisternae they increase insize. Unlike the situation described in the rat prolactin cell (Smith & Farquhar, 1966),in the material examined here there is no evidence to suggest that the larger secretorygranules arise as a result of the smaller, immature granules coalescing with one another.In the eel prolactin cell the most likely source of additional material for the enlargingsecretory granule appears to be derived from the Golgi vesicles.

When compared with the localization of acid phosphatase activity within the Golgiapparatus described in earlier reports (Smith, 1963; Novikoff, 1963; Miller & Palade,1964; Osinchak, 1964; Novikoff et al. 1964; Gordon, Miller & Bensch, 1965; Smith &Farquhar, 1966; Bertolini & Hassan, 1967), that observed in the prolactin cell of theeel adenohypophysis is of particular interest. In these earlier reports acid phosphataseactivity is restricted to the innermost one or two Golgi cisternae, whereas in the eelprolactin cell reaction product is consistently observed in the majority of theseelements. Only in the recent publication of Wetzel, Spicer & Horn (1967), in whichthe Golgi-associated acid phosphatase of the developing heterophil leucocyte in therabbit was studied, has a similar distribution been described.

The presence of peripheral acid phosphatase activity within the developing secre-tory granules has been described in a number of tissues (Novikoff & Essner, 1962;Novikoff et al. 1962; Novikoff, 1963; Osinchak, 1964; Smith & Farquhar, 1966), butin all of these tissues, as in the prolactin cell, the fully formed secretory granules areusually acid phosphatase-negative (Novikoff et al. 1964).

In attempting to account for acid phosphatase activity within Golgi cisternae anddeveloping secretory granules it has been suggested that the real significance of such

362 C. R. Hopkins and B. I. Baker

activity may be concerned with the production of primary lysosomes, and that itspresence within the secretory granule is fortuitous (Osinchak, 1964; Smith & Farquhar,1966). While it is difficult to discount this possibility, the recent observations ofWetzel et al. (1967) and to a lesser extent those of Smith & Farquhar (1966) suggestthe contrary, since in these investigations it has been shown that the distribution ofacid phosphatase activity within the Golgi alters with changes in the secretory activityof the cell.

Also related to the possible functional significance of the Golgi- and secretorygranule-associated acid phosphatase is the finding that only in a proportion of theseelements can acid phosphatase activity be demonstrated. This arrangement implies thatthere may be two kinds of secretory granule, dissimilar in content and possibly in fate.It would be of interest to know if the proportion of acid phosphatase-containingcisternae and secretory granules varies directly with changes in secretory activity.

The knowledge that only a proportion of the developing secretory granules containacid phosphatase has further implications because it also allows for the possibility thatthe enzyme is, as in other situations (de Duve & Wattiaux, 1966), actively lytic. Thissuggestion, which was not included in the review of possible functions outlined bySmith & Farquhar (1966), implies that only those developing secretory granules whichlack acid phosphatase will become fully developed. It is interesting to view thissuggestion in the context of what is known about the secretory state of the prolactincells studied. Thus, as discussed above, although the morphological features of theprolactin cells suggest that secretory granules are continuing to be produced, theaccumulation of these granules indicates that they are only being slowly released.Under these conditions it seems reasonable to suggest that the majority of granulesarising from the Golgi should be destined for breakdown before they become mature.

A number of publications, reviewed by de Duve & Wattiaux (1966), have suggestedthat the acid phosphatase content of the Golgi cisternae is also concerned with theproduction of acid phosphatase-positive Golgi vesicles. In the present study the pro-files observed support this view. In accordance with the functional classification oflysosomes made by de Duve & Wattiaux (1966) these vesicles may be termed primarylysosomes, since they have not yet been associated with any digestive activity.

The observation that coated and uncoated Golgi vesicles may contain acid phos-phatase is in agreement with the recent observations of Holtzman, Novikoff & Villi-verde (1967) made on nerve cells. The significance of the spinous coat, however,remains obscure.

The association of acid phosphatase-positive Golgi vesicles with developing secre-tory granules is of interest, but the implications of this association will remain unclearuntil the significance of acid phosphatase activity within the developing secretorygranule can be more fully understood.

The recent work of Smith & Farquhar (1966) on the prolactin' cell of the ratadenohypophysis has suggested that when release of the secretory product is abruptlyinhibited by removal of the suckling young the secretory granules become over-abundant. At this time, the surplus granules are rapidly degraded along an intra-cellular digestive pathway with which large lytic bodies are concerned, but in which

Acid phosphatase in the eel adenohypophysis 363

the Golgi-associated acid phosphatase has not been implicated. As the mature granulesare degraded they are contained within the membrane-bound lytic bodies, and Smith &Farquhar (1966) have described the morphological changes which occur within thesebodies during the successive phases of degeneration. In view of these findings it is ofinterest to note that in the prolactin cells of the eel adenohypophysis large acid phos-phatase-containing bodies are also present. Indeed, the morphology of these structuresso closely resembles the forms of lytic body described by Smith & Farquhar (1966)in the rat prolactin cell that it is not unlikely that a similar digestive pathway alsooccurs within these cells. However, since it is not possible to subject the eel to experi-mental procedures similar to those employed on the rat it is not possible to demonstrateunequivocally the presence of such a pathway.

We would like to thank Dr. J. W. Jones for assistance in procuring the eels, Miss MargaretPenny for technical assistance and Professor N. M. Hancox for helpful criticism of the manu-script.

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{Received 28 September 1967)

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For legends see next page.C R. HOPKINS AND B. I. BAKER (Facing p. 364)

Fig. i. Apical portions of three prolactin cells showing a nucleus (n) surrounded byendoplasmic reticulum cisternae (er), and abundant secretory granules (sg). Acidphosphatase-positive Golgi cisternae (g) and lytic bodies (Ib) are present within theapical cytoplasm, cm, cell membrane. Inset ( x 60000 approx.) shows developingsecretory granule (sg) fusing with a ' coated' Golgi vesicle (arrow). 40 /* non-frozensection incubated in acid phosphatase medium 30 min. Glutaraldehyde/osmiumfixation, x 14000 approx.

Fig. 2. Golgi area from a similar preparation showing at higher magnification reactionproduct within the Golgi cisternae (g), developing secretory granules (dg) and largerlysosomal bodies (Ib). Note the absence of acid phosphatase activity in one developingsecretory granule (arrow), cm, cell membrane. Preparation as for Fig. 1. x 36000approx.

Fig. 3. Golgi area showing acid phosphatase reaction product within some of theGolgi cisternae, their bulbous terminations and, at the periphery, in some of thedeveloping secretory granules (dg). Structures perhaps representing Golgi vesicles(gv) also contain reaction product. 40 /(• non-frozen section incubated in acid phos-phatase medium 1 h. Glutaraldehyde/osmium fixation, x 60000.

Fig. 4. Golgi region showing a similar distribution of reaction product to that shownin Fig. 3. Note acid phosphatase within the majority of the Golgi cisternae, Golgivesicles (gv) and developing secretory granules (dg). Arrows indicate points of fusionof possibly acid phosphatase-positive Golgi vesicles with a secretory granule. Ib, largelytic-dense body. Preparation as for Fig. 3. x 52000 approx.

Fig. 5. Lytic body with fine fibrous content containing reaction product. Preparationas for Fig. 3. x 14000 approx.

Fig. 6. Lytic body containing electron-dense inclusion (arrow) similar in size andform to the surrounding secretory granules. Preparation as for Fig. 3. x 18000 approx.

Fig. 7. Larger lytic body with a dense fibrous content containing reaction product.Preparation as for Fig. 3. x 22000 approx.

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Fig. 8. Portion of the prolactin cell apical cytoplasm showing the close relationshipbetween the rough endoplasmic reticulum (rer), smooth endoplasmic reticulum (ser),Golgi cisternae (g) and Golgi vesicles. Note that a number of the Golgi cisternae con-tain acid phosphatase reaction product, especially within their expanded terminations.Arrows indicate the points of fusion between two Golgi vesicles and a developing secre-tory granule. 40 fi non-frozen section incubated for 1 h in acid phophatase medium.Glutaraldehyde/osmium fixation, x 54000 approx.

Fig. 9. Lytic bodies (Ib) in non-incubated tissue; three of them (W>'J~I have evenlygranular contents and are regarded as dense bodies, dg, developing secretory granule;rer, rough endoplasmic reticulum;#, Golgi area; in, mitochondrion. Glutaraldehyde/osmium fixation, x 30000 approx.

C. R. HOPKINS AND B. I. BAKER