Cell Tiss. Res. 162, 551--563 (1975) �9 by Springer-Verlag 1975

The Uhrastructure, Catecholamine, and Prolactin Contents of the Rostral Pars distalis of the Fish Mugil platanus After Reserpine or 6-Hydroxydopamine Administration

D a v i d Z a m b r a n o *

Departamento de Ciencias Biol6gicas, Facultad de Farmacia y Bioqulmica, Universidad de Buenos Aires, Argentina

Received June 6, 1975

Summary. The rostral pars distalis (RPD) of the teleost Mugil platauus from animals pretreated with reserpine or 6-hydroxydopamine (6-HODA) were assayed for dopamine (DA) or noradrenaline (NA) or for prolactin hormone. Such determinations were coupled with electron microscopy. I t was found that reserpine and 6-HODA produced a significant decrease in the content of DA, NA, and prolactin. Electron microscope studies revealed that prolactin cells became activated as judged by ultrastructural criteria. After 6-HODA treatment type " B " neurosecretory fibers entering the RPD became selectively destroyed. These observa- tions lead us to suggest that prolactin secretion is under inhibitory control by type " B " neurosecretory fibers of adrenergic nature.

Key words: Hypophysis - - Rostral pars distalis - - Mugil platanus - - Animals - - Prolactin hormone secretion.

In t roduct ion

Dur ing the last few years an increasing body of l i t e ra ture has evidenced t h a t the h y p o t h a l a m u s exer ts control of adenohypophys i a l funct ion in te leost fish (Peter, 1970; Bal l et al., 1972). As opposed to lower ac t inop te ryg ians and te t ra - pods in which there is an indirect , neurovascula r p a t t e r n of communica t ion be tween the h y p o t h a l a m u s and the p i t u i t a r y (Lagios, 1970; Zambrano , 1971), the te leos t h y p o t h a l a m u s appears to exercise its control b y way of a more d i rec t t y p e of inne rva t ion of the p i t u i t a r y g landula r cells (Foll6nius, 1965; Zambrano , 1972).

Several e lec t ron microscope s tudies have shown t h a t one t y p e of neuro- secre tory axons ( type " B " according to Knowles , 1965) conta in ing dense-cored vesicles (DGV), 900-1000 A in d iamete r , is the most common k ind of axon con- t ac t ing all cell t y p e s of te leos t p i t u i t a ry . His tochemica l (Zambrano, 1970; Zam- brano et al., 1972) as well as r ad ioau tog raph ic s tudies (Foll6nius, 1968, 1971) have ind ica ted t h a t these axons m a y be of adrenergic na ture , a l though t h e y m a y conta in substances o ther t han biogenic amines (Zambrano, 1970). Fu r the rmore , there are indica t ions t h a t the h y p o t h a l a m u s might control an te r io r p i t u i t a r y

Send o//print requests to: Prof. Dr. David Zambrano. Orientaci6n Histologia Humana, Departa- mento de Ciencias Biol6gicas, Facultad de Farmacia y Bioquimica, Junfn 956, 5 ~ piso, Buenos Aires, Argentina.

* Member of the Scientific Career, Consejo Nacional de Investigaciones Cientificas y T6cnicas, Argentina.

552 D. Zambrano

secret ion th rough this t y p e of neurosecre to ry fibers (Zambrano, 1972; Zambrano et al., 1971, 1974).

I n the presen t paper we have inves t iga ted whether t he hypo tha l a mus controls pro lac t in secret ion in the teleost fish Mugil platanus. I t ~vas decided to use th is species because i t has been prev ious ly shown tha t in M. platanus, t y p e " B " fibers come into d i rec t contac t wi th pro lac t in cells (Zambrano, 1971), and because i t has been shown in Mugil idae t h a t pro lac t in hormone, as in o ther euryha l ine fishes, p lays an i m p o r t a n t osmoregu la to ry role (Abraham, 1971). Such exper i - ments were per formed because conflict ing d a t a exis t as to whether pro lac t in secret ion is under hypo tha l amic control or not . Whereas some au thors have shown the presence of an inh ib i to ry control in some species (Sundarara j and N a y y a r , 1969; Sage, 1970; Z a m b r a n o et al., 1972, 1974), o thers have fai led to de tec t any control mechanism (Olivereau, 1971).

The presen t s t u d y descr ibes the effect of two drugs which are known to affect ca techolamine content in the central nervous sys tem. One, reserpine, produces deple t ion of monoamine-con ta in ing terminals , b y interfer ing with the up take - s torage mechanism of the amine granules (Sulser and Sunders-Bush, 1971). The o ther compound, 6 - h y d r o x y d o p a m i n e (6-HODA), deple tes noradrenerg ic (NA) and dopaminerg ic (DA) nerve t e rmina l s of the i r t r ansmi t t e r conten t (Ungers tedt , 1971) b y way of a select ive and t r ans i en t degenera t ion of such te rmina l s (Thoenen, Tranzer and Hausler , 1970). Adrenerg ie te rmina ls are more suscept ible to th is compound because the amine is accumula ted b y the i r m e m b r a n e pump and a t t a in s a high in t racel lu lar concent ra t ion (Tranzer, 197l). E lec t ron microscope observa t ions were coupled with NA, DA, and pro lac t in de te rmina t ions in control as well as in pharmacolog ica l ly t r e a t ed animals , in order to provide add i t iona l informat ion concerning the re la t ionship be tween the aminergie inne rva t ion and pro lac t in secret ion.

Mater ia l and Methods Animals: Mugil platanus were obtained in winter froln the Salado River near the 1R.io de

la Plata (sodium concentration of the river ranged from 250 300 mEq/l). Mature animals of both sexes weighingS00 1000 g average were acclimated at 10~ in 100 liter tanks under a 14-hour light, 10-hour dark photoperiod.

Experimental Conditions. For the present study the animals were divided in two experi- mental conditions: (a) animals injected with reserpine and (b) animals injected with 6-hydroxy- dopamine (6-HODA).

a) Reserpine. Animals kept with the same river water injected subcutaneously with reserpine (Serpasol, CIBA, Argentina), with a single dose of 5 mg/kg. Groups of t5 animals each were killed after 2, 5, and 24 hours. Five animals from each group were used for electron microscope studies of their rostral pars distalis (RPD). Pituitaries from another 5 animals from each group were processed for disc electrophoresis and densitometry. Finally another 5 animals were used for DA and NA determinations. One group of 15 animals serving as controls, received a subcutaneous injection of the solvent in which reserpine is usually disolvcd : benzyl alcohol, polyethylenglycol, and distilled water (Olivereau, 1972).

b) 6-HODA. Fifteen animals kept with the same river water were injected intracisternally with 500 t~l of 6-hydroxydopamine hydrobromide solution (Regis Co., Chicago, Ill.) at dosage of 50 mg/kg of free base. The 6-HODA was dissolved in ascorbic acid (1 mg/ml) and adjusted to pH 5.0 with hydrochloric acid. The solution was made up just before use and was kept on ice. The animals received two intracisternal injections, 24 hours apart. Another group of 15 animals was injected with the ascorbic acid vehicle to serve as controls. Pituitaries from 5 animals of each

The Rostral Pars distalis hypophyseos of Mugil platanus 553

group were studied with conventional electron microscopy, while in another 5 RPD, the pituitary prolactin content was determined by disc elcctrophoresis. In the other 5 animals, RPD, DA, and NA concentrations were determined. Rostral pars distalis studied for conven- tional electron microscopy were processed as previously described (Zambrano and Iturriza, 1973). Once the animals were sacrificed, the pituitary gland was removed and placed in a drop of fixative. The RPD containing the prolactin and ACTH cells were carefully dissected from the rest of the gland under the dissecting microscope and processed for final embedding and sectioning.

As each of the remaining pituitaries were removed, each RPD was separated from the rest of the gland. Five RPD from each group were homogenized in Tris-glycine buffer at 0 ~ C and their prolactin content determined by disc electrophoresis and densitometry (Clarke, 1973). Optical density readings of their stained prolactin bands were converted to fzg prolactin using a standard curve prepared with ovine prolactin.

Five RPD from each group were homogenized in cold 0.4 N perchloric acid and the ex- tracts purified before use by column chromatography in Dowex 50 W-x4 resine. Dopamine (DA) was determined fluorometrically according to Laverty and Taylor (1968) and nor- adrenaline (NA) according to tIaggendal (1963).

Data of pituitary prolactin content are presented as means • 1 standard error.

Results

NA and DA. Assays in RPD in Di//erent Experimental Conditions

As shown in Table 1, a f te r 5 hours of a single in jec t ion of reserpine, a consid- erable deple t ion of both N A and D A was de tec ted in t he R P D . This deple t ion was s t a t i s t i ca l ly s ignif icant . Similar ly , in ject ions of 6 -HODA also p roduced a s ignif icant decrease of the two amines in the ros t ra l pars dis ta l is t h a t was even more dramat ic .

Densitometric Measurement o/ Pituitary Prolactin Content by Disc Electrophoresis

After a single reserp ine inject ion, a slight, nonsigni f icant decrease in p i t u i t a r y pro lac t in conten t was recorded 2 hours pos t in jec t ion (Fig. 1). Af te r 5 hours t he p i t u i t a r y pro lac t in conten t d ropped sha rp ly to a level s t a t i s t i ca l ly lower t han controls. On the o ther hand, 24 hours a f te r inject ion, the pro lac t in content was s t a t i s t i ca l ly h igher t h a n af ter 5 hours. This content was even h igher t h a n in control animals bu t was not s t a t i s t i ca l ly s ignif icant as compared to controls. F ina l ly , the p i t u i t a r y p ro lac t in conten t af ter 6 - t t O D A in jec t ion d roppe d sha rp ly as compared to controls (Fig. 2). Such a d rop was s t a t i s t i ca l ly s ignif icant .

Electron Microscope Observations o / R P D

a) Reserpine. Elec t ron microscope s tudies of the R P D from these fish showed a submicroscope organiza t ion which is s imilar to o ther te leos t fishes (D ha rma mba and Nishioka, 1968; Z a m b r a n o et al., 1974). The n e u r o h y p o p h y s i a l pro jec t ions which pene t r a t e t he R P D appea red as small e longated or c ircular profi les com- p le te ly sepa ra t ed from the ACTH and pro lac t in cells b y a basement membrane and processes of s te l la te cells (Fig. 6). Such pro jec t ions conta in numerous peri- phe ra l ly loca ted nerve f ibers which correspond to t y p e " B " neurosecre to ry fibers of the classif icat ion of Knowles (1965). The t y p e " B " f ibers in the neurohypo- phys ia l pro jec t ions of M. platanus conta in two t y p e s of vesicles: one s imilar to the " c l e a r " synap t i c vesicles p resen t in cholinergic te rminals , and the o ther consist ing

554 D. Zambrano

~ Pituitary prolactin content

c~ n.. n-" o'1 ~n

i/1

P _ r

o ~ u-, 6 r

a.=-

2 n-

6C

hO

20

~\\\\"q

,\\\\~

Contr. 2hr 5hr

RESERPINE

2h hr

,- .-_.-_.- .- ."

Contr. 6-HODA

6-HODA

Fig. 1. Effect of reserpine and 6-HODA on pituitary prolactin content from Mugil cephalus as determined by disc electrophoresis

Table 1. Amine assay in the RPD of Mugil platanus

Group NA DA

~zg/g net weight

Control 0.481 • 0.083 0.453 ~= 0.090 Reserpine 0.100• 0.0152i0.250 6-HODA 0.077~= 0.090 0.052 =~ 0.011

of dense cored vesicles (DGV) or granulated vesicles with a mean diameter of 900-1000/~. I n the rostral pars distalis of M. platanus it was possible to observe type " B " fibers t raversing the basement membrane to become closely inter- mingled with prolactin cells, where they finally make synaptoid contacts (Fig. 2, inset).

Prolactin cells f rom control pituitaries showed numerous dense spherical secretory granules, 2000/~ in diameter (Fig. 2). The few endoplasmic reticulum (ER) cisternae appeared around the nucleus, which has a small nucleolus. Golgi

Fig. 2. I~PD from Mugil after injection of reserpine vehicle. Prolactin cells have numerous secretory granules but the Golgi complexes and endoplasmic reticulum are poorly developed. x 6000. Inset: Synaptoid contact between a type "B" fiber and a prolactin cell. • 13000

Fig. 3. RPD from Mugil 2 hours after a single injection of reserpine. Prolactin cells show slight degranulation. Golgi complexes and endoplasmic reticulum are here more developed than in controls. One prolactin cell (top right) is highly degranulated and contains vacnoled

endoplasmic reticulum. • 7000

556 D. Zambrano

complexes appeared small or were absent. The overall picture leads to the con- clusion that prolactin cells from controls synthesize and secrete their secretory products at a very low rate.

Two hours after reserpine administration, prolactin cells showed several ultrastructural modifications with respect to controls (Fig. 3). The nucleus showed an enlarged nucleolus, usually attached to the nuclear membrane or connected to it by dense granular material. Degranulation was constant, but discrete, and not uniform in all cells. The perinuclear ER cisternae appeared increased in number and usually rather dilated. Sometimes some prolactin cells showed a highly degranulated cytoplasm together with an unusually dilated ER cisternae (Fig. 3, top right).

Five hours after reserpine injection, prolactin cells showed ultrastructural features of enhanced synthesis and release of their secretory products, although such reaction was not uniform (Fig. 4). While many cells appeared degranulated, others appeared filled with secretory granules. Degranulated cells usually showed a highly hypertrophied ER, which was composed of large dilated cisternae (vacuoles) containing a filamentous material of low electron density. A constant feature was the hypertrophy of the Golgi complexes, which showed numerous related secretory granules. Nucleoli appeared usually enlarged.

Examination of the large dilated cisternae revealed their close association with smaller ones, thus suggesting that the large ones are the result of the confluence of numerous small ones (Fig. 5, small arrows). Of special interest was the finding that reserpine treatment did not produce depletion of the dense core of DGV present in type " B " fibers (Fig. 5, thick arrow).

Although not shown here, after 24 hours of reserpine administration, prolactin cells appeared almost normal. There was still some hypertrophy of the Golgi complexes and the nucleoli were still enlarged, but the cytoplasm were filled with numerous secretory granules.

b) 6-Hydroxydopamine. After two injections of 6-HODA, most type " B " fibers appeared under different degrees of degeneration (Fig. 7). These fibers appeared with a dense cytoplasm, or with vacuoles of different sizes and with swollen mitochondrial

They also showed electron-opaque multilamellate bodies of different sizes. No DGV were found in such neurosecretory fibers. Pituicytes which appeared hyper- trophied and closely associated with the damaged fibers, frequently contained multilamellate bodies (Fig. 7).

Whereas ACTH (Fig. 6) and stellate cells remained unaltered, prolactin cells showed several subcellular features of increased synthesis and release of their secretory products (Fig. 7). This cell type showed fewer secretory granules than was present in controls, although exocytosis was not observed. The Golgi complexes and the rough endoplasmic reticulum appeared hypertrophied. The cisternae of the last organelle usually became dilated, and in some cells they even produced one or two large vacuoles in a single section.

Discussion

The present observations support further evidence that type " B " fibers which innervate pituitary cells of teleost fishes contain catecholamines and proteins. In

Fig. 4. R P D from Mugil 5 hours aff~r a single injection of reserpine. Numerous prolactin cells appear poorly granulated while others show enlarged endoplasmic reticulum with dilated

cisternae. Observe nucleolar hypertrophy. X 3500

Fig. 5. A detail from Fig. 4, a t higher magnification to show three large vacuoles associated to the dilated cisternae of the endoplasmic reticulum. The th in arrow indicates the point of confluence between an E R cisternae with the large vacuole. The thick arrow indicates a type

" B " fiber filled with DGV x 9000

l l Cell. Tiss. Res.

Fig. 6. Contact zone between a neurohypophysial projection (right) and secretory cells from the RPD (left). Numerous type " B " neurosecretory fibers appear a t tached to the basement membrane which is consti tuted by processes originated from stellate cells (SAT). • 9000

Fig. 7. Same as Fig. 6, bu t from an animal injected with 6-HODA. The type " B " fibers in the neurohypophysial projection show striking features of degeneration. Corticotrophic (ACTH)

cells appear unchanged • 7 000

The Rostral Pars distalis hypophyseos of Mugil platanus 559

Fig. 8. Same condition as Fig. 7. Prolactin cells appear highly degranulated and with hyper- trophied rough endoplasmic reticulum and Golgi complexes. Large vacuoles in several pro- lactin cells are evident. A pituicyte (PIT) associated with a capillary in a neurohypophysial

projection has engulfed nerve fibers under different stages of degeneration. • 6000

560 D. Zambrano

whole pituitary extracts from the RPD of M. platanus both NA and DA were detected in constant but relatively low concentrations. Since no catecholamines containing glandular cells have been detected in this region of teleost pituitary gland (Bs et al., 1974), it seems reasonable to speculate that these monoamines are mostly stored in nerve fibers. Our results with 6-HODA treatment indicate that such nerve fibers in the pituitary gland of M. platanus are only type " B " - DGV ones, since only these fibers became selectively destroyed by the drug. Such effect has been shown to occur only in adrenergic systems (Thoenen and Tranzer, 1968), because these systems are the only ones which efficiently accumulate "false" neurotransmitters (Malmfors and Sachs, 1968), a sine qua non condition for subsequent destructive effects (Thoenen e$ al., 1970). Our results confirm previous results that this introduction via the cerebrospinal route is effective in achieving degeneration of central catecholamine-containing systems (Uretsky and Iversen, 1970). Furthermore, Foll6nius (1972) and Zambrano et al. (1972, 1974) have described similar degenerative effects of 6-HODA on " B " fibers of other teleost fishes. Hopkins (1971) found identical effects on the adrenergic innerva- tion of amphibian pars intermedia glandular cells.

Previous data with electron microscope autoradiographs have shown the ability of type " B " fibers to selectively accumulate either NA (FollSnius, 1968) or DA (Foll6nius, 1971), thus providing further evidence of the aminergic nature of the type " B " fibers. The present results confirm the participation of pituicytes in the removal of degenerated axons, as has been previously shown in hypo- thalamic neurosecretory systems of other species (for a review, see Dellmann, 1973).

The relatively low concentrations of both NA and DA in the RPD of M. platanus confirm previous suggestions of Zambrano et al. (1971) that type " B " fibers of fish probably contain low concentrations of biologically active amines. For example, incubations of teleost pituitaries with 5-HODA, another "false" neurotransmitter which is usually incorporated as enhanced electron dense core in the DGV, revealed a weaker reaction in type " B " fibers of teleost pituitaries (Zambrano et al., 1971) than in rat hypothalamic neurons (Richards and Tranzer, 1970).

The effects of reserpine on DGV of type " B " fibers of the pituitary gland was first described by Zambrano (1970) in the teleost Gillichthys mirabilis. I t was found that reserpine did not produce a decrease in opacity of the dense cores if aldehydes were used as fixatives. The present results showing that reserpine decreases DA content in Mugil pituitaries with no ultrastructural effects on the density of DGV suggests that a substance, other than the active monoamine, is responsible for the persistence of the dense core of DGV. The suggestion here of a second component in DGV opens the possibility that the so-called releasing factors generally regarded as polypeptides may be present in such terminals associated in the DGV to active monoamines. Zambrano (1970) reached a similar conclusion with histochemical techniques in the pituitary gland of the teleost Gillichthys mirabilis.

The independent ultrastructural and densitometric data indicate that both reserpine and 6-HODA brings about prolactin secretion, probably by removing an inhibitory influence on prolactin secretion. These results confirm previous ones

The Rostral Pars distalis hypophyseos of Mugil platanus 561

with reserpine (Sundararaj and Nayyar, 1969; Sage, 1970) or 6-HODA (Zambrano et al., 1974) that prolactin cells in several teleosts are under inhibitory hypothal- amic control. Our results indicate that type " B " fibers may be the anatomical pathway for this inhibitory control, because type " A " were not affected by 6-HODA.

Since in vivo (see Nagahama et al., 1973; Nagahama et al., 1974) as well as in vitro studies (Sage, 1966, 1968; Ingleton et al., 1973 ; Zambrano et al., 1974) have shown the ability of a low osmotic pressure of the medium to stimulate prolactin secretion, it seems logical to suppose that hypothalamic influence provides a final control which is superimposed on that osmotic stimuli.

Activation of prolactin cells of euryhaline fishes may lead to different sub- cellular changes, which may also vary depending upon the intensity and period of the stimulus. For example, when specimens of Platychthys stellatus were trans- fered from sea water to fresh water (Nagahama et al., 1973) or when the RPD of Tilapia mossambica were incubated for 3 hours in hyposmotic Ringer-Krebs medium (Zambrano et al., 1974) prolactin cells became degranulated and with hypertrophied cisternae of the rough endoplasmic reticulum distended with a granulofilamentous material. Such reaction, which is reminiscent of that found here in M. platanus, is not detected in chronically stimulated prolactin cells. For example, prolactin cells of SW-acclimated Tilapia (Dharmamba and Nishioka, 1968) or M. cephalus (Abraham, 1971) failed to show the subcellular changes of activation reported here.

Finally, although no quantitative estimation was performed on the degree of granulation of prolactin cells under the different conditions reported here, our results show a correlation between the degree of granulation of prolactin cells as evidenced by electron microscopy and pituitary prolaetin content as measured by disc electrophoresis. This correlation suggests that secretory granules do store prolactin hormone, a fact already demonstrated for mammalian secretory granules (Zanini and Giannattasio, 1973).

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