the cell types in the adenohypophysis of the marine teleost crenilabrus melops

Acta Zool. (Stockh.) 60: 105-113 (1979)

The Cell Types in the Adenohypophysis of the Marine Teleost Crenilabrus melops

Michael Ben jamin

Department of Anatomy, University College, Cardiff, Wales (Accepted November 27, 1978)

Abstract Benjamin, M . 1979. The cell types in the adenohypophysis of the marine teleost Crenilabrus melops. (Department of Anatomy, University College, Cardiff, Wales.) - Acta zool. (Stockh.) 6 0 ( 2 ) : 105-113.

Sagittal sections from pituitaries of winter and spring fish were stained with a number of methods including alcian blue-PAS-orange G, Herlant's tetrachrome, Lux01 fast blue-PAS-orange G and lead haematoxylin. By these methods seven different endocrine cell types were readily demonstrated in the adenohypophysis. In the rostra1 pars distalis (RPD), there were conspicuous vacuoles (intra- and possibly extracellular) in the prolactin cell zone of all animals. The ACTH cells in the RPD were very numerous. In the proximal pars distalis (PPD), the growth hormone cells were arranged in coiling bands that were predominantly dorsal and juxtaposed to neurohypophysis. Two types of basophils were readily distinguished: both were predominantly ventral in the proximal pars distalis, though the type I basophil also formed a constant group in the dorsal region of the pituitary, between the PPD and the RPD. The pars intermedia contained two cell types - a lead haematoxylin-positive cell and a PAS-positive cell. The pituitary morphology is compared with that of other teleosts.

Michael Benjamin, Department of Anatomy, University College, Cardiff CFl , l X L , Wales.

Introduction

Wrasses are common inshore fish that are widespread throughout temperate and tropical seas. A large number of species are known, the most abundant of which in British waters is the corkwing, Crenilabrus melops. This is a small fish, usually less than 15 cm in length, that is fre- quently found on rocky shores, in rock pools and below tide level (Wheeler 1969). I t favours shallower waters than other wrasses and is thus caught by hand-netting. I t is a fish of little economic importance and perhaps partly for this reason, there has been no description of its pituitary gland. The present paper provides an account of the normal cytology of its adenohypo- physeal cell types, with a view to establishing

whether the pituitary is worth further attention. The pituitary cytology of numerous teleosts has been described and the reader is referred to reviews by Ball and Baker 1969, Sage and Bern 1971, Schreibman et al. 1973 and Holmes and Ball 1974, for further details.

Materials and Methods

Corkwing wrasse (Crenilabrus melops L.) of both sexes were collected by hand-netting from a seawater lagoon at Southsea, Portsmouth, in January and April, 1977. All fish were 6-8 cm long. They were killed by decapitation, their lower jaws re- moved and a hole cut in the roof of the skull. The heads were fixed in Bouin-Hollande for 3 days and

106 Michael Benjamin

stored in 70 % alcohol. In some fish the brains with attached pituitaries were dissected out; in others the heads were decalcified in 5 "/o nitric acid in 80 % alcohol. The dehydration was com- pleted, the material cleared in terpineol and em- bedded in 58°C paraffin wax. Sagittal sections were cut at 6 p m and stained in lead haematoxylin (McConaill 1947) with or without PAS, Gomori's aldehyde fuchsin (after oxidation with acidified permanganate) , Herlant's ( 1960) tetrachrome, Brookes's (1968) trichrome and PAS-orange G combined with luxol fast blue (Shanklin et al. 1959) or alcian blue (Kerr 1965).

F i g . 1. Mid-sagittal section through the pituitary gland of Crenilabrus melops, showing the two strands of tissue (S) connecting the pituitary with the brain and the four areas into which the gland can be divided. N neurohypophysis, PI pars intermedia, PPD proximal pars distalis, RPD rostra1 pars distalis. Herlant's tetrachrome. X 120. Fig. 2. The distribution of the cell types in the adenohypophysis of Crenilabrus melops. 9 ACTH cells, Prolactin cells, 0 Growth hormone cells, @ PPD type 1 basophil, A PPD type 2 basophil, 0 PI 1 cell, a PI 2 cell.

Observations

The pituitary of Crenilabrus was cone-shaped and had a prominent infundibular recess (Fig. 1). It was suspended from the brain by two strands of tissue; a thinner, anterior connection composed of fibrous connective tissue anteriorly and ependyma posteriorly, and a thicker, posterior connection consisting of fibrous connective tissue posteriorly and ependyma anteriorly. Occasionally small portions of the saccus vasculosus adhered to the caudal aspect of the posterior connection, though

Adenohypophysis of Crenilabrus melops 107

the main bulk of this organ was more distant. Between the two pituitary connections the gland was covered with cuboidal ependymal cells. The neurohypophysis was usually separated from the adenohypophysis by a thin membrane staining positively with PAS, alcian blue, aniline blue and aldehyde fuchsin. The adenohypophysis consisted of a rostra1 and proximal pars distalis ( R P D and PPD respectively) and a pars intermedia (Figs. 1 and 2) . The neurohypophysis penetrated the pars intermedia (PI) extensively, the PPD slightly and the RPD minimally. Gomori-positive neuro- gecretory material was restricted mainly to the neurohypophysis adjoining the PI and was particularly prominent in spring animals.

Rostra1 Pars Distalis

The RPD contained two types of endocrine cells (Figs. 2 and 3) . The prolactin cells were virtually chromophobic, with only a slight affinity for acid dyes. They occupied the ventral portions of the RPD and were small cells, with round/oval nuclei and inconspicuous nucleoli. They were closely packed and not arranged in follicles. Conspicuous vacuoles were found throughout the prolactin cell region in all animals (Fig. 3 ) . As no flattened (endothelial cell) nuclei surrounded these pro- files, they could not be mistaken for capillaries. However it was difficult to be certain whether particular vacuoles were intra- or extra-cellular. AS some vacuoles were next to nuclei, it was

Fig. 3. Detail of the RPD showing the two cell types in this region. The prolactin cells ( P ) are ventrally situated and characterised by numerous intra- (and possibly extra-) cellular vacuoles. The more dorsal ACTH cells are not vacuolated and have a slight affinity for acid alizarine blue. Her- lant’s tetrachrome. X 750. Inset - intracellular vacuoles in prolactin cells. Luxol fast blue-PAS- orange G. Red filter. x 1250.

assumed these were intra-cellular. However in other cases the size of the vacuoles suggested either a confluence of intra-cellular vacuoles from several cells or extracellular vacuoles between stellate prolactin cells. These vacuoles occasionally contained PAS and alcian blue-positive droplets, especially1 in the spring. ACTH cells were particularly numerous and arranged in a thick layer dorsal to the prolactin cells and adjacent to the neurohypophysis (Figs. 2 and 3 ) . They were usually smaller than prolactin cells, but had larger and rounder nuclei (Fig. 3 ) . ACTH cells had a slightly greater affinity for acid dyes than did prolactin cells, but were best characterised by staining with lead haematoxylin or Herlant’s tetrachrome. Unlike the prolactin cells, ACTH cells were not vacuolated.

Proximal Pars Distalis

The PPD was situated largely between the RPD and the PI, though in several animals, in both January and April, it also extended ventral to the P I (Figs. 2 and 4). Three cell types could be


Fig. 4. The ventral extension of the PPD beneath the PI that was characteristic of several animals in both January and April. Both types of basophils (presumptive thyrotrophs and gonadotrophs) are found in this area. Alcian blue-PAS-orange G. Red filter. x 300. Fig. 5. The growth hormone cells ( G H ) of the PPD arranged in a coiling band, adjacent to a portion of neurohypophysis (N) . Alcian blue-PAS- orange G. Red filter. x 1200.

distinguished by their shape, distribution and staining reactions (Fig. 2). T h e acidophilic, growth hormone cells were mainly found in the dorsal regions and were often arranged in coiling

bands next to strands of neurohypophysis (Fig. 5). When sectioned in a suitable plane, they were columnar in shape, with a central/basal ovoid nucleus. The long axis of the cells lay at right angles to the adjoining nervous tissue.

Both the remaining cell types in the PPD were basophilic, though readily distinguishable from one another in sections carefully stained with alcian blue-PAS-orange G (Fig. 6, Table 1 ) . Type 1 basophil was mainly found in the ventral regions of the PPD and in its narrow extension beneath the PI. I t was a rounded cell with a central nucleus and stained a characteristic purple

Fig. 6 . Type 1 (presumptive gonadotrophs) and t ype 2 (presumptive thyrotrophs) basophils in the PPD near a blood vessel (BV) . Alcian blue-PAS- orange G. Red filter. x 1200. Fig. 7. The PPD flanked by the RPD and the PI. Note the predominance of both types of basophils in the ventral region of the PPD and the conspicuous group of strongly alcian blue-positive, type 2 basophils in the dorsal region of the pituitary between the PPD and RPD (arrow). Alcian blue- PAS-orange G. Red filter. x 300.

colour with alcian blue-PAS-orange G. All such basophils were far more prominent and heavily stained in animals collected in spring. For this reason, it was provisionally regarded as a gonado-

8 - Act3 Zool. 1979: 2


troph. Type 2 basophil was strongly alcian blue -- positive, even after greatly reduced oxidation times. I t therefore stained dark blue with alcian blue-PAS-orange G (Fig. 6, Table 1) . I t was a markedly angular cell, with a coarsely granulated cytoplasm, and was often located next to strands of neurohypophysis. In such cells, the nuclei were eccentric and directed away from the nervous tissue. The type 2 basophils were found both ventrally and dorsally within the PPD. There was a particularly constant group of cells in the dorsal region of the pituitary, between the RPD and PPD (Fig. 7). Their appearance did not differ in spring and winter animqls.


Fig. 8. Polygonal, lead haematoxylin-positive cells (PI 2 cells) and columnar (obliquely sectioned) PAS-positive cells ( P I 1 cells) in the pars intermedia. PAS - lead haematoxylin. Red filter. x 1200. Fig. 9. Detail of the pars intermedia showing PAS- positive type 1 cells (arrows) bordering the neurohypophysis ( N ) . Alcian blue-PAS-orange G. Green filter. x 375.

also stained strongly with orange G and was thus readily characterised by a number of staining methods (Table 1). The P I 1 cell usually bor- dered the neurohypophysis (Fig. 9), though in several glands the cells were more randomly dis-

Pars Intermedia

The PI contained two cell types; one was PAS- positive and the other lead haematoxylin-positive (Figs. 2 and 8) . The PAS-positive cell ( P I 1 cell)

Adenohypophysis of Crenilabrus melops 1 1 I

Table 1. The staining reactions of the adenohypophysial cell types in Crenilabrus melops.

Cell Type Herlant’s Brookes’s Alcian-blue- Luxol-PAS- Gomori’s PAS-Lead tetrachrome trichrome PAS-orange G orange G aldehyde haematoxylin

fuchsin

Prolactin ACTH Growth

Hormone Type 1 PPD

basophil Type 2 PPD

basophil PI 1 cell PI 2 cell

chromophobic blue-purple dark yellow

dull blue

bright blue

dull red blue-grey

dull red light green bright red

light green

light green

dull red light green

chromophobic light green chromophobic chromophobic very pale yellow light green chromophobic dark grey bright orange bright green light purple grey

purple dull brown dark purple red

dark blue red dark purple red

magenta dark green light purple red light brown light green chromophobic dark grey

tributed. I t was a large, columnar cell with a basal nucleus and a supra-nuclear mass of stain- able secretory granules. The lead haematoxylin- positive cell (PI 2 cell) was more numerous than the PI 1 cell (Fig. 8 ) . It was a polygonal cell with processes extending between adjacent PI 1 cells.

Discussion

The pituitary gland of Crenilabrus shares several features with the pituitaries of other percomorph fish. It seems a common characteristic of this order that the gland is conical in shape and that the growth hormone cells lie in the dorsal regions of the PPD along neurohypophysial boundaries (Kerr 1942, Olivereau 1968). Further- more, in several percomorphs there is a zone of basophils between the RPD and PPD (Olivereau 1968, Nagahama et al. 1973, Yoshie and Honma 1978). Olivereau ( 1968) suggested that such cells in Mugil are thyrotrophs, because of their response to thyroxine and thiouracil. Basophils in a similar position have also been recorded in several Pleuronectiformes (Kerr 1942, Benjamin 1975).

The prolactin cells of marine teleosts are gener- ally chromophobic (Ball and Baker 1969). In accordance with the widely accepted role of teleost prolactin in osmoregulation (see reviews by Ball 1969, Ensor and Ball 1972, Johnson 1973), this chromophobia could mean that the cells neither form nor release many secretory granules. It was therefore no surprise to find that the prolactin cells in Crenilabrus were chromophobic.

Nevertheless the widespread vacuolation in the prolactin cell region of every pituitary was less expected. While the author is confident that some of these vacuoles were intracellular, a complete understanding of vacuole morphology ultimately depends on resolving the cell membrane. Thus it awaits an ultrastructural study. Vacuoles are an unusual feature of teleost prolactin cells, though they are common in gonadotrophs (Olivereau 1963, Overbeeke and McBride 1976, Mattheij 1968, Benjamin 1975), where they may signal impending cellular exhaustion (Olivereau and Fontaine 1966). Vacuolated prolactin cells have been found in certain gobies-also living in seawater (Benjamin in preparation). There are extra-cellular vacuoles or ‘cysts’ of varying sizes among the prolactin cells in Pungitius pungitius (Benjamin 1978). Such cysts may or may not be lined by a PAS-positive membrane, and in winter animals they form in response to increased environmental salinities. Thus they may represent extreme signs of prolactin cell inactivity.

In many teleosts, including Poecilia Zatipinna and AnguiZla angzrilla (Ball and Olivereau 1966), Salmo gairdneri and Onchorhynchus nerka (Fagerlund et al. 1968) and Anoptichthys jordani (Mattheij 1968), there is a palisade layer of lead haematoxylin-positive cells in the dorsal region of the RPD immediately above the prolactin cells. Most authors now regard these distinctive cells as corticotrophs (ACTH cells), though Boddingius (1975) is critical of this view. The ACTH cells are usually smaller and considerably less numerous than the prolactin cells. This is not so in Crenilabrus, for the ACTH cells are nearly as


common as the prolactin cells and their nuclei a t least, are larger.

The alcian blue-PAS-orange G technique has proved most useful for distinguishing two types of basophils (presumably gonadotrophs and thyrotrophs) in the pituitaries of many teleosts besides Crenilabrus. I t has been usefully applied to the pituitaries of Rutilus rutilus, Scardinius eryth- ropthalamus, Abramis brama and Gobio gobio (Arme 1968), Anoptichthys jordani and Cichlu- soma biocellatum (Mattheij 1968, Mattheij et al. 1971a, 1971b) and Phoxinus phoxinus and Pleu- ronectes flesus (Benjamin 1975). Despite these works a large number of authors continue to ignore the technique.

Most researchers on the fish pituitary gland have concentrated largely on freshwater or migra- tory fish. This is understandably linked with the inland location of many research institutions. Yet the pituitaries of marine fish deserve more attention-a fact recognised by Nagahama et al. (1973). In particular, we do not understand the role of prolactin in seawater fish. Crenilabrus melops is a most suitable marine teleost that is worthy of further study. I t is large enough to allow blood samples to be easily taken, yet small enough to be kept in considerable numbers in limited space. It has a pituitary gland that can be satisfactorily stained with a variety of tech- niques and one that remains attached to the brain when the latter is dissected from the skull.

Acknowledgements

I wish to thank Professor J. D. Lever for his critical appreciation of the manuscript and Mr. P. F. Hire for his photographic assistance.

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the cell types in the adenohypophysis of the marine teleost crenilabrus melops

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