J . Zool., Lond. (1980) 192, 59-69

Yacuolated prolactin cells in the pituitary gland of several marine teleosts

M I C H A E L BENJAMIN Department of Anatomy, University College, CardqJ Wales

(Accepted 13 November 1979)

(With 4 plates and 4 figures in the text)

A morphometric study of prolactin cell ultrastructure in the pituitary gland of the Corkwing wrasse, Crenilabrus melops L., showed that cytoplasmic vacuoles, which accounted for 25 % of the cell volume, were associated with signs of decreased secretory activity. The Golgi apparatus, mitochondria and rough endoplasmic reticulum, all contributed relatively little to the total cell volume, and there was no sign of secretory-granule release by exocytosis. All vacuoles were intracellular and membrane-bound, and probably derived from rough endoplasmic reticulum, Golgi apparatus and the nuclear envelope. I t is thought that smaller vacuoles coalesce to form larger ones. The secretory granules were small and sparse, and this could account for the chromophobia of prolactin cells in lighl-microscopy prepara- tions. Similar vacuoles were reported in the prolactin cells of the gobiid fish, Chaparrudo Jlaveseens, Pomatoschistus pictus, Pomatoschistus minutus and Pomaroschistus mierops. The vacuoles in Chupurrudo were of similar ultrastructure to those in Crenilabrus.

Contents

Introduction . . . . . . . . . . Materials and methods . . . . . . . . Results . . . . . . . . . . . .

Corkwing wrasse . . . . . . . . Gobies . . . . . . . . . . . .

Discussion . . . . . . . . . . . . References . . . . . . . . . . . .

Page . . . . . . . . . . 59 . . . . . . . . . . 59 . . . . . . . . . . 61 . . . . . . . . . . 61 . . . . . . . . . . 61 . . . . . . . . . . 67 . . .. . . . . . . 68

Introduction In a light-microscope study of the pituitary gland of the Corkwing wrasse (Crenilabrus

melops), the cytology of the adenohypophyseal cells was described (Benjamin, 1979~). The widespread vacuoles recorded in the prolactin cell zone, are an unusual feature for a teleost pituitary gland, and their fine structure and significance are unknown. Indeed it is uncertain whether these vacuoles are intra- or extra-cellular. The present investigation clarifies these points by an ultrastructural study of the prolactin cells in Crrwilabrus, and also reports similar vacuoles in several species of gobies.

Materials and methods Corkwing wrasse (Crenilabrus melops), Two-spot gobies (Chaparrudo 3avescen.s) and Painted

gobies (Pomatoschistuspictus) were collected by hand-netting from a seawater lagoon at Southsea, Portsmouth, in January and April, 1977 and 1979. Sand gobies (Pomatoschistus minutus) were caught by hand-netting in the sea at Hayling Island, near Portsmouth, in December 1976, and Common gobies (Pomatoschistus microps) were collected from brackish water ( $ seawater) at Hiilsea, Portsmouth, in April 1977. Adult fish of both sexes were used throughout.

59

0022-5460/80/0Y0059 + 11 $02.00/0 GI 1980 The Zoological Society of London

60 M . B E N J A M l N

All fish were dccapitated immediately after collection and their pituitaries fixed for light or electron microscopy. All light microscopy techniques have been described previously (Benjamin, 1979~). Pituitaries for electron microscopy were fixed for 2 hours in 4 % glutaraldehyde in 0.1 M cacodylate buffer. After an overnight wash in buffer, they were postfixed in 1.33% osmium tetroxide in 0.1 M cacodylate buffer for 3 hours, bulk stained in 3”/, aqueous uranyl acetate, dehydrated in graded alcohols, treated with propylene oxide, and embedded in TAAB resin. Thin (silver-gold interference colour) sections were cut on an LKB Ultratome I 1 I , stained in Reynold’s (1963) lead citrate and examined on an AEI EM 801 electron microscope.

Four pituitaries of Cvenilubrus were randomly selected for a quantitative analysis of their prolactin cells. The cell components selected for analysis are listed in Table I . The precise interpre- tation of these terms has been given previously (Benjamin, 1976). The percentages of the total cell volume occupied by the various components shown in Table I1 were also estimated in order to provide additional information for the “reconstruction drawings” (Benjamin, 1974). In making the reconstruction drawings, no account was taken of the volume/surface ratio of the nucleus. Five electron micrographs were randomly taken from each pituitary and printed at a final magnification of x 11,368. The test system used for the point-counting volumetry consisted of a quadratic lattice of lines, whose cross-points served as markers for point-counting. On the final prints, the distance between the lines was 0.87 pm. All further details of the morphometric procedure have been described previously (Benjamin, 1976).

T A B L E I Morphornetric model of the prolactin cells in Crenilahrus

Prolactin cell

I I 1-

I Diameters Volunres Numbers

--Nuclei -Mitochondria --Secretory granules m mitochondria -Multivesicular bodies -Rough endoplasmic reliculum (RER) --Vacuoles -Dense bodies (presumptive lysosomes) --Multivesicular bodies mi free ribosomes

-Exocytosed secretory granules

T A B L t 1 1

Cell components selected for point-counting volumetry in order to provide udititionul duta for the “reconstruction drawings”

(mean values not quoted)

I . Nucleolus 2. Parallel arrays of RER around the nucleus 3. Small, isolated pieces of RER, without dilated cavities 4. Parallel arrays of RER in the cell, next to the cell membrane 5. Small, Golgi-vesicles 6. Large, dilated, Golgi-saccules 7. Flattened, Golgi-cisternae

TELEOST PROLACTIN CELLS 61

Results Corkwing wrasse

Figure 1 and Table IIT summarize the morphometric data on the prolactin cells, and Fig. 3 is a reconstruction drawing of this cell, based partly on the above information (see also “Materials and methods”) and on the secretory-granule, profile-diameters (Fig. 2).

‘The conspicuous vacuoles in the prolactin-cell region were entirely intracellular (Plate I). They were irregular in size and shape (0.2-6.0 pm greatest diameter), and there could be many per cell (Plate I). They accounted for one quarter of the total cell volume and were restricted to the cytoplasm--though some were continuous with the nucllear envelope (Plate II(a)). All were devoid of electron-dense contents. Because vacuoles were frequently

TABLE I I I

Numerical density of various organelle profiles per unit area of cytoplasm (means k s.e.) in the prolactin cells of Crenilubrus

Organelle Numerical density ~ _ ~ ~ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ Mitochondria 0.1 8 & 0.04/ wm2 Dense bodies (presumptive lysosomes) 3.87 f 1.07,’lOO pm2 Multivesicular bodies 0.47 f0.24,1100 pm2 Exocytosed secretory granules 0.00 0,001 pm2

PLATE I. Conspicuous intracellular vacuoles (V) in the prolactin cells of Crenikabrcrs. Note the enormous vacuole indenting the nucleus (N) in one cell. Secretory granules (SG) are sparse and unevenly distributed. Some mitochondria (M) are very large. BV, blood vessel; DB, dense body: NUC, nucleolus ( ~ 2 5 0 0 ) .

67 M . B E N J A M I N

PLATI: 1 1 . (a) Juxtaposed vacuoles (V) in the prolactin cells of Crenilubrus, separated only by their limiting membranes. Note one large vacuole that is continuous with the nuclear membrane at N, and another that is closely associated with a mitochondrion (M). ( x 5800.) (b) The Golgi region of a prolactin cell in Crenilnhrus. C, centriole; DR, dense body: MV, multivesicular body; SG, small Golgi vesicles; V, vacuole ( I1,500).

TELEOST P R O L A C T I N CELLS 63

juxtaposed and separated only by their limiting membranes (Plate II(a)), it seems likely that smaller vacuoles coalesce to form larger ones. All were membrane-bound, and the few ribosomes attached to their outer surface suggested that some arose from RER. However, their ad.ditiona1 presence in the Golgi region (Plate Il(b)), could mean that they also arose from this organelle. Larger vacuoles (5.0-6.0 pm greatest diameter) were usually restricted to profiles including a nucleus, and they often indented this organelle (Plate I).

Another characteristic feature of the prolactin cells in Crenilabrus, were tlhe enormous (approximately 2.0-3.0 pm diameter) mitochondria (Plate I). Indeed these organelles were sometimes closely associated with vacuoles (Plate II(a)). They were packed with tubular or vesicular cristae. Other, smaller mitochondria were present. Although con- spicuous, the mitochondria accounted for only a small fraction of the total cell volume (Fig. 1).

The nuclei were large and round, contained mainly euchromatin, and had prominent nucleoli (Fig. 1 and Plate I). RER was poorly represented in the cell (Fig. 1) and consisted of small, isolated fragments, scattered throughout the cytoplasm. The Golgi apparatus was also insignificant (Fig. I ) and consisted of a few flattened cisternae and more numerous, small vesicles. However, there were signs of secretory-granule formation (Plate 111). There were few, mature secretory granules (Fig. 1) and these were unevenly distributed in the cytoplasm (Plate I). They were very small (Fig. 2) and uniformly electron-dense. There was no evidence of exocytosis (Table 111). Dense bodies (presumptive lysosomes)

FIG. 1 . The percentage of the total cell volume in the prolactin cells of CrenilubruJ occupied by various organelles (means+ s.e.). D&M, dense bodies and multivesicular bodies (0.29 kO.06 and 0 14 kO.08 respeclively); G, Golgi apparatus; FR, free ribosomes; N, nucleus; SG, secretory granules; RER, rough endoplasmic reticulum; V, vacuoles.

64 M . BENJAMIN

151

V E a, l o -

8 5- a"

r

rn C .I-

I J 50 100 150 200 250

Diameter Cnm)

Frc. 2. The percentage frequency of secretory-granule, profile-diameters in the prolactin cells of Cwnilnbnrs.

FIG. 3. A reconstruction drawing of the prolactin cell in Cvenilubrus, based on average, quantitative data. DB, dense body; G , Golgi apparatus; M, mitochondrion; MB, multivesicular body; NUC, nucleolus: R, free ribosomes; RER, rough endoplasmic reticulum; SG, secretory granule; V, vacuole.

TELEOST P R O L A C T I N CELLS 65

RPD PPD PI 0.1 mm N H \ \

0.1 mm N H FIG. 4. The distribution of the cell types in the adenohypophysis of Chaparrudo flavescens, Pomatoschistuspictus,

P . sninutus and P. microps. A mid-sagittal section. 0 ACTH cells; 0, prolactin cells; *, growth hormone cells; B1, PPD type 1 basophil; a, PPD type 2 basophil; H, hypothalamus; N, neuroliypophysis; PI, pars intermedia; PPL), proximal pars distalis; RPD, rostra1 pars distalis.

P,La.rE 111. Evidence of secretory-granule formation (arrows) in the Golgi region of a prolactin cell in Creuilabrirs. FG, flattened Golgi cisternae; SG, small Golgi vesicles; V, vacuole ( x 17,000).

66 M. B E N J A M I N

PLAT^ 1V. (a) Vacuolated prolactin cells (arrows) in the rostra1 pars distalis (RPD) of Chnpurrudo f l ~ ~ v e s c r ~ ~ s . H, hypothalamus; PPD, proximal pars distalis. Herlant's tetrachrome. Orange filter ( x 375). (b) Vacuoles of various sizes (arrows) in the prolactin cells of C. jhvesc'ens. Two adjacent vacuoles are separated only by their membranes (M). The cells contain only a few, small, secretory granules (SG) and these are irregularly distributed. There is little rough endoplasmic reticulum (RER) and the Golgi apparatus is small (G) ( x 5800).

TELEOST PROLACTIN CELLS 67

were fairly common (Plates I and Il(b); Table Ill), although multivesicular bodies were rare (Plate II(b) and Table 111). Centrioles were also present in the Golgi iregion (Plate IT@) ).

Gobies 1\11 four species of goby had similar pituitaries in which the adenohypophysial cell

types were distributed as shown in Fig. 4. The glands were embedded in the hypothalamus, shaped like egg-plants, and easily divisible into a rostral pars distalis, proximal pars distalis, pars intermedia, and neurohypophysis. The rostral pars distalis contained pro- lactin cells, ACTH cells, and stellate (non-granuIated) cells, although the prolactin cells were most abundant. They were rounded or oval cells that were weakly acidophilic and could be distinguished tinctorially from the growth hormone cells of the proximal pars distalis by careful application of Herlant’s tetrachrome. The growth hormone cells had a greater affinity for orange G, while the prolactin cells had a slight affinity for erythrosin.

In all fish a large number of prolactin cells were vacuolated (IJlate IV(a)). The vacuoles were devoid of stainable contents, rounded in light-microscopy sections and often juxta- posed to nuclei. In all respects they resembled those previously described in Crenilabrus melops (Benjamin, 1979a). The fine structure of the vacuolated prolactin cells was examined only in Chaparrudo flavescens (Plate IV(b)) and the details of vacuole morphology, including the secretory state of the parent cells were similar to those described above for Crenilabrus.

Discussion This report confirms the presence of vacuoles in the prolactin-cell zone of Crenilabrus

and clearly shows they are intracellular. The larger vacuoles reported in a previous, light-microscopy study (Benjamin, 1979a), probably arose by the confluence of vacuoles in neighbouring cells, and were unlikely to be extracellular. Thus the vacuoles in Crenilabrus anti in the gobies studied, are very different from the “extracellular cysts” thal characterize the rostral pars distalis in the N ine-spined stickleback, Pungitius pungitius (Benjamin & Williams, 1979). All the vacuoles examined in Crenilabrus and Chaparrudo with the electron microscope, were devoid of electron-dense contents, and no satisfact ory explana- tion can be offered for the occasional, alcian blue-positive droplets reported previously (Bt:nj amin, 1 9 7 9 ~ ) .

The morphometric data on Crenilabrus suggest the vacuoles are associated with inactive prolactin cells. When the present data are compared with those available for prolactin cells in other teleosts, e.g. Gasterosteus aculeutus (Benjamin, 1974); Poecilia latipinnu (Ben-jamin, 1976; Batten & Ball, 1977); Poecilia reticulata (Ethridge & Benjamin, 1977); Anguillu anguillu (Benjamin & Baker, 1976) and Pungitius pungitius (Benjamin, 1979b), the Golgi apparatus, mitochondria, and RER, evidently contribute relatively little to the total cell volume. One would expect these organelles to be prominent in actively-secreting cells (Palade, 1975). The paucity of mature, secretory granules in conjunction with the absence of exocytotic figures, could be evidence for a low rate of hormone synthesis. It also accounts for the chromophobia of the prolactin cells in the previous, light-microscopy study (Benjamin, 1979~) .

The presence of vacuoles in the prolactin cells of C. j7avescens, P. pictus, P. minutus and P. microps, is surprising in view of the failure of other authors to report them in related

6X M . B E N J A M I N

species (Moiseeva, 1971u, 6 ; Nagahama et ul., 1973; Tsuneki & Ichikawa, 1973; Oota & Shinkai, 1978; Yoshie & Honma, 1978). It is interesting that the prolactin cells were still vacuolated in P. microps collected from 5 seawater. Shih (1979) has reported similar vacuoles in P. niicrops collected from full strength seawater. Evidently the vacuoles do not disappear with lowered salinities.

A few other authors have commented on vacuolated prolactin cells in teleosts. Clarke & Nagahama (1977) described vacuoles in inactive prolactin cells in the pituitary glands of juvenile Coho salmon (Oncorhynchus kisutch), as these fish approached their spawning grounds. They were interpreted as evidence of cell degeneration. Aoki & Umeura (1970) noted large vacuoles that were present in a few prolactin cells of Oryzius lutipes. Evidently, vacuoles are also found in prolactin cells in vitro, for they appeared when pituitaries of Pseudocrenilubrus philander were cultured for 48-96 hours (Rawdon, 1978). They, t o q were associated with evidence of secretory inactivity.

One should not assume however, that vacuolated prolactin cells are always inactive. Batten & Ball (1977) reported vacuoles in the prolactin cells of Sailfin mollies (Poeciliu lutipinnaj transferred from dilute seawater to freshwater, where the cells showed signs of active secretion, e.g. enlarged RER and Golgi apparatus, many exocytoses and immature secretory-granules. This was also the case with occasional vacuoles noted in the prolactin cells of Mugil plutunus (Zambrano, 1975) and Tilapia mossumbica (Dharmamba & Nishioka, 1968).

Batten & Ball (1977) consider the vacuoles in the prolactin cells of Poecilia may be autophagic vacuoles, derived from the Golgi apparatus. Those in Crenilubrus and Chupar- rudo could arise from the Colgi apparatus, but additional origins from the nuclear envelope and RER are likely. Zambrano (1975) associates the vacuoles in Mugil with dilated RER.

It can only be assumed that the large nucleoli in the prolactin cells of Crenilabrus reflect synthetic processes other than those concerned with hormone secretion. The large mito- chondria are similar to those described by Nagahama et al. (1973) in Plutichthys and to the multilamellar organelles described by Leatherland (1970) in Gasterosteus. The small size of the secretory granules in the prolactin cells of Crenilubrus and Chupurrudo seems typical of marine teleosts (Holmes & Ball, 1974).

I wish to thank Mr P. F. Hire for his help with the photography.

R E F E R E N C E S

Aoki, K . & Umeura, H. (1970). Cell types in the pituitary of the medaka, Oryzius lutipes. Endocu. jupon. 17: 45-55. Batten, T. F. C. & Ball, J. N . (1977). Quantitative ultrastructural evidence of alterations in prolactin secretion

Benjamin, M. (1974). A morphometric study of the pituitary cell types in the freshwarer stickleback, Gusterosteus

Benjamin, M. (1976). Ultrastructure of the endocrine cell types in the adenohypophysis of the teleost, Poeciliu

Benjamin, M. (19790). The cell types in the adenohypophysis of the marine teleost Crenilabvus melops. Actu zool.,

Benjamin, M. (1 9796). Comparative ultrastructural srudies on the prolactin cells in the pituitary of nine-spined

related to external salinity in a teleost fish (Poeciliu latipinnu). Cell Tiss. Res. 185: 129-145.

uculeutus, form Ieiurus. Cell Tiss. Res. 152 : 69-92.

latipinnu-a morphometric model. Cell Tiss. Res. 167: 125-146.

Srockh. 60: 105-113.

sticklebacks, Pungitius pungitius L., with and without adenohypophyseal cysts. Zoornorphologie 593: 125-1 35.

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

TELEOST PROLACTIN CELLS 69

Benjamin, M. & Williams, J. G . (1979). Pituitary cysts in the nine-spined stickleback, Pungitius pitngitius L. 11. Light and electron microscopy. .4cta zool., Stockh. 60: 241-250.

Clarke, W. C . & Nagahama, Y . (1977). Effect of premature transfer to sea water- on growth and morphology of the pituitary, thyroid, pancreas, and interrenal in juvenile coho salmon (Oncorhynchus kisutch). Can. J .

Dharmamba, M. & Nishioka, R. S. (1968). Response of "prolactin-secreting" cells of Tilupia mossambica to

Ethridge, L. & Benjamin, M. (1977). The time-sequence of response of the prolactin cells of the freshwater teleost,

Holmes, R. L. & Ball, J. N. (1974). The pituitary gland: a comparative account. Cambridge: University Press. Leatherland, J. F. (1970). Seasonal variation in the structure and ultrastructure: of the pituitary of the marine

form (Trachurus) of the threespine stickleback, Gasterosteus aculeatus L. Rostra1 pars distalis. 2. Zellforsch. mikrosk. Anat. 104: 301-317.

Moiseeva, E. B. (1971~). Differential identification and some morphological features of cells of the proadenohypo- physis of gobies. Dokl. Akad. Nauk SSSR 197: 501-503.

Moiseeva, E. B. (19716). Morphological comparison of the pituitary glands of Gobius melanostomus Fall. and Gobius batrachocephalus Pall., spawning intermittently and simultaneously. Uokl. Akad. Nauk SSSR 198: 467-470.

Nagahama, Y., Nishioka, R. S. &Bern, H. A. (1973). Responses of prolactin cells of two euryhaline marine fishes, Gillichthys mirabilis and Platichthys stellatus, to environmental salinity. 2. Zellforsch. mikrosk. Anat. 136:

Oota, Y . & Shinkai, T. (1978). The cell types in the adenohypophysis of the goby, Chaenogobius urotaenia. Rep.

Palade, G. (1975). Intracellular aspects of the process of protein synthesis. Scienc;e, Wash. 189: 347-358. Rawdon, B. B. (1978). Ultrastructure of the non-granulated hypophysial cells in the teleost Pseudocrenilabrus

philander (Hemihaplochromis philander), with particular reference to cyrological changes in culture. Acta zool., Stockh. 59: 25-33.

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

Shih, S. H. (1979). Pituitary structure and cycles of the Common goby, Pomatoachistus microps (Kr~yer). Ph.D. Thesis, University of Bristol.

Tsuneki, K. & Ichikawa, T. (1973). The cell types in the adenohypophysis of the teleost, Chasmichthys dolicho- gnathus. Annotnes zool. jap. 46: 173-182.

Yoshie, S. & Honma, Y. (1978). Experimental demonstration of the cell types in the adenohypophysis of the gobiid fish, Rhinogobius brunneus. Arch. hist. jap. 41: 129-140.

Zambrano, D. (1975). The ultrastructure, catecholamine, and prolactin contents of the rostra1 pars distalis of the fish Mugil platanus after reserpine or 6-hydroxydopamine administration. Cell Tiss. Res. 162: 551-563.

2001. 55: 1620-1630.

environmental salinity. Gen. comp. Endocr. 10: 409420.

Poecilia reticulata, to an altered environmental salinity. Cell Tisay. Res. 182:: 371-382.

153-167.

Fac. Sci. Shizuoka Univ. 12: 87-97.