# Comparative distribution of somatostatin, LH-RH, neurophysin, and α-endorphin in the rainbow trout: An immunocytological study

M.P. Dubois, Roland Billard, Bernard Breton, R.E. Peter

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M.P. Dubois, Roland Billard, Bernard Breton, R.E. Peter. Comparative distribution of somatostatin, LH-RH, neurophysin, and α-endorphin in the rainbow trout: An immunocytological study. General and Comparative Endocrinology, 1979, 37 (2), pp.220-232. 10.1016/0016-6480(79)90110-2. hal-01601006

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# Comparative Distribution of Somatostatin, LH-RH, Neurophysin, and α-Endorphin in the Rainbow Trout: An Immunocytological Study

M. P. DUBOIS,* R. BILLARD,† B. BRETON,† AND R. E. PETER‡

*Station de Physiologie de la Reproduction, Institut National de la Recherche Agronomique, 37380 Nouzilly, France, †Laboratoire de Physiologie des Poissons, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France, and ‡Department of Zoology, University of Alberta, Edmonton T6G 2E9, Canada

Accepted November 16, 1978

Using immunofluorescence, evidence of a somatostatin (SRIF)-like antigen has been found in the brain and digestive tract of rainbow trout. In the diencephalon, periventricular SRIF immunoreactive hypendymocytes are located in the region dorsal to the nucleus preopticus (NPO). SRIF immunoreactive perikarya are concentrated anterior to the NPO in the nucleus preopticus periventricularis, scattered in small cells in the nucleus lateralis tuberis (NLT) pars anterior, and in a few cells located in an unnamed nucleus in the dorsomedial hypothalamus. In the pituitary SRIF immunoreactive material is located in the neurohypophysial tissue in the proximal pars distalis. In the gut, SRIF cells have been found in the endocrine pancreas and in the gastric mucosa. Comparatively, material immunoreactive for luteinizing hormone-releasing hormone has the same distribution in the pituitary as SRIF, whereas neurophysin immunoreactivity was found in only the neurophysial tissue of the neurointermediate lobe. A few cells reacting with anti-α-endorphin were seen in the NLT in the pituitary stalk region. All the pars intermedia cells in the neurointermediate lobe react with anti-α-endorphin.

Somatostatin (SRIF), or a related anti-
gen, has been demonstrated by immuno-
cytology in the median eminence of a
variety of vertebrates, including rat, pig,
sheep, fowl, duck, triton, rainbow trout,
and guinea pig (Dubois et al., 1974; Hökfelt
et al., 1974, 1975; Calas et al., 1975; Dubé
et al., 1975). More recently, hypothalamic
centers producing SRIF have been
localized by the same method in the rat
(Dubois and Kolodziejczyk, 1975; Elde and
Parsons, 1975; Alpert et al., 1976; Parsons
et al., 1976), the toads Bufo bufo and
Xenopus laevis (Doerr-Schott and Dubois,
1976a), tadpoles of Alytes obstetricans
(Rémy and Dubois, 1978), the lizard
Lacerta muralis (Doerr-Schott and Dubois,
1976b), and the human fetus (Bugnon et al.,
1976, 1977; Paulin et al., 1976). The im-
munofluorescent localization of SRIF cells

in the diencephalon and their axonal end-
ings in pituitary of the rainbow trout has
been reported briefly previously (Dubois et
al., 1978) and is described in detail here. In
addition, we describe here in detail the im-
munofluorescent localization of neurophy-
sin (NPH), luteinizing hormone-releasing
hormone (LH-RH), and α-endorphin in the
brain and pituitary of rainbow trout.

## MATERIALS AND METHODS

Brains and other tissue samples were ob-
tained from male or female adult rainbow
trout, killed at different times of the year.
Tissues were fixed with Bouin-Hollande,
without acetic acid, containing HgCl₂
(0.5%), and embedded in paraffin. Rehy-
drated sections were investigated by im-
munofluorescence using the indirect
method and a counterstain of the prepa-
rations with Evans blue (0.01%) as described previously (Dubois, 1976). Nomenclature of nuclei in the diencephalon was according to the scheme proposed for *Carassius auratus* by Peter and Gill (1975).

The following antisera were used: anti-SRIF, anti-bovine neurophysin, anti-LHRH, anti-α-endophin. The preparation and specificity of these antibodies have been reported previously (Dubois, 1975; Dubois, 1976; Leonardelli *et al.*, 1973; Follenius and Dubois, 1978).

## RESULTS

### *SRIF Immunofluorescence*

A SRIF-like antigen was localized in a number of brain areas. Periventricular hypendymocytes (Fig. 1) located next to the dorsal nucleus preopticus (NPO) were found in some animals. SRIF immunoreactive perikarya were constantly found in three separate hypothalamic nuclei: an unnamed dorsomedial hypothalamic nucleus

(Figs. 2 and 4), nucleus lateralis tuberis (NLT) (Figs. 2 and 5), and nucleus preopticus periventricularis (NPP) (Figs. 2 and 6). There were also isolated SRIF-immunoreactive cells scattered throughout the hypothalamus (Fig. 3). The localization in the dorsomedial hypothalamus (Figs. 2 and 4) consisted of a small group of cells (about 10 per field) at about the level of the ventrolateral part of the ventromedial nucleus. In the NLT (Figs. 2 and 5) reactive cells were found scattered along the walls of the third ventricle, particularly anterior to and in the region of the pituitary stalk. Reactive cells were scarce in the posterior NLT. A dense net of immunoreactive axons was observed at the periphery of the NLT. In the preoptic region two symmetrical dense clusters of SRIF immunoreactive cells (Figs. 2 and 6) were seen overlying the preoptic recess at the periphery of the rostral end of the NPO; the cells were observed in horizontal as well as parasagittal sections of the brain and appeared quite apart from

<figure><img src="image_2.png" /><figcaption>FIG. 1. Sagittal section of the trout brain showing SRIF reactive hypendymal cells in the wall of the IIIrd ventricle, next to the upper (dorsal) part of nucleus preopticus. Note the immunofluorescent cytoplasmic halos, apical poles, and transverse sections of distal ends of the hypendymocytes. × 700.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>FIG. 2. Camera lucida drawings of representative sections from trout brains showing localizations of hypothalamic nuclei containing SRIF cells: (1) Horizontal section crossing the nucleus preopticus (NPO) at the level of the anterior pars parvocellularis. (2) Sagittal section showing the IIIrd ventricle, the pituitary, and the saccus vasculosus. (3) Slightly oblique parasagittal section in which the IIIrd ventricle appears as an anteroposterior cleft parting the NPO pars parvocellularis into two nearly equal halves. (4) Enlargement of the surround field of (3), showing the suprachiasmatic group of SRIF cells in the nucleus preopticus periventricularis, next to the supraoptic recess, in reference to the NPO and the IIIrd ventricle. a, IIIrd ventricle: b, NPO (anterior pars parvocellularis) c, hypothalamic nucleus containing SRIF cells; d, supraoptic recess of the IIIrd ventricle; e, optic chiasma; f, pituitary; g, saccus vasculosus.</figcaption></figure>

the NPO and corresponded to the NPP. In some cases nearly all of the perikarya of the NPP showed a strong immunofluorescent reaction.

SRIF axonal endings were observed in the pars nervosa of the proximal pars distalis (Figs. 12 and 13), but were not observed in the pars nervosa of the rostral pars distalis or the neurointermediate lobe. Immunoreactive endings were not observed in close contact with pituitary cells.

SRIF immunoreactive cells were seen in the digestive tract at two levels: in pancreatic islets and in the digestive mucosa. Brightly reacting cells were found in the endocrine pancreas of trout alevines 1 day after hatching (Fig. 7). In adult trout the gastric mucosa showed small SRIF reactive cells (Fig. 8), similar to those observed in the antrum, pyloric, and duodenal mucosa of mammals and birds (Dubois, 1975; Hökfelt *et al.*, 1975; Parsons *et al.*, 1976).
<figure><img src="image_4.png" /><figcaption>FIG. 3. Isolated SRIF cells scattered in the hypothalamus of the rainbow trout. × 700.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>FIG. 4. Horizontal section of the rainbow trout brain corresponding to Fig. 2 (1). Small nucleus of SRIF localized symmetrically in the dorsomedial hypothalamus. × 700.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>FIG. 5. Sagittal section of the rainbow trout brain corresponding to Fig. 2 (2). Wall of the IIIrd ventricle containing the anterior part of the nucleus lateral tuberis (NLT). A lot of SRIF cells are scattered among small nonreacting cells of the NLT; axonal fragments are seen between the cell bodies. × 700.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>FIG. 6. Parasagittal section of the rainbow trout brain corresponding to Fig. 2 (3,4) showing the suprachiasmatic grouping of SRIF cells in the nucleus preopticus periventricularis. Nearly all the cells in this area react with anti-SRIF contrary to the scattered distribution of SRIF cells in the nucleus lateralis tuberis (Fig. 5). × 560.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>FIG. 7. Cells in the pancreatic islets of a rainbow trout alevine (one day after hatching) reacting with anti-SRIF. × 700.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>FIG. 8. Cells in the gastric mucosa of an adult rainbow trout containing immunoreactive material for SRIF. × 800.</figcaption></figure>
## NPH Immunofluorescence

A strong reaction for anti-bovine NPH was found in the parvocellular (Fig. 9) and magnocellular (Figs. 9 and 10) perikarya of the NPO, in hypothalamic nerve fibers supposedly originating from the NPO, and in axonal endings in the pars nervosa of the neurointermediate lobe (Fig. 11). The distribution of NPH in the neurointermediate lobe did not overlap the distribution of SRIF immunoreactive endings in the proximal pars distalis (Fig. 12).

## LH-RH Immunofluorescence

A moderate but undoubted specific immunofluorescence for LH-RH was seen in the pars nervosa ramifying in the proximal pars distalis (Fig. 14). These axons have the same distribution as SRIF fibers (Fig. 13). We have not been able to demonstrate LH-RH immunoreactivity in the brain of rainbow trout.

### α-Endorphin Immunofluorescence

A few small cells reacting with anti-α-endorphin were seen scattered in the NLT in the pituitary stalk region (Fig. 15). In the pituitary the entire cell population of the neurointermediate lobe reacts with anti-α-endorphin (Fig. 16).

## DISCUSSION

The present results give evidence for immunoreactive SRIF, NPH, and α-endorphin cells in various brain regions, the presence of immunoreactive SRIF, NPH, LH-RH, and α-endorphin in the pituitary, and the presence of SRIF in the gut mucosa and pancreatic islets of rainbow trout. Although the present study is based on tissues taken from both males and females killed at different times of year, we are not able to indicate at present whether there are any annual cycles in the amount of immunoreactivity for any of these substances, or what the most favorable environmental and physiological conditions

might be to demonstrate the presence of these substances.

Immunoreactive SRIF hypendymal cells dorsal to the NPO in the rainbow trout are similar to SRIF-containing hypendymocytes observed in the hypothalamus of porcine and equine fetus (Dubois, 1976, 1978) and in adult mammals (Alpert et al., 1976; Dubois, 1976, 1978). At this point we are unable to identify these cells either as tanycytes or as "C.S.F. contacting neurons" according to Vigh and Vigh-Teichmann (1973). In the trout the evidence for these cells was unsteady and we are presently unable to define the conditions for their demonstration.

The demonstration of SRIF cells in the dorsomedial hypothalamus, NLT and NPP is reliable from one animal to another. No specific physiological function for these various groupings of SRIF cells can be suggested at this point. SRIF cells have been demonstrated in the arcuate nucleus in mammals (Parsons et al., 1976), a homologous location to the NLT. The distribution of SRIF cells in the anterior NLT, next to the sagittal plane of the brain in the walls of the III ventricle, appears more medial than the distribution of the scarce α-endorphin containing cells scattered in the NLT in the pituitary stalk region. Magnocellular perikarya of the NLT pars lateralis and NLT pars anterior were not reactive for α-endorphin in rainbow trout, as in *C. auratus* and *Cyprinus carpio* (Follenius and Dubois, 1978).

In the preoptic region of *C. auratus* the NPP can be distinguished from the NPO in that only the latter stains with paraldehyde fuchsin (Peter and Gill, 1975). A similar division of nuclei occurs in rainbow trout (Peter and Billard, unpublished observations). This distinction of nuclei agrees with the present results of a reaction for NPH in the NPO and not in the NPP. This also agrees with the findings of Goossens et al. (1977) that cells immunoreactive for vasotocin and
<figure><img src="image_10.png" /><figcaption>FIG. 9. Horizontal section of the rainbow trout hypothalamus showing the anterior pars parvocellularis of the nucleus preopticus (NPO) reacting with antineurophysin (the dotted line shows the bounds of the IIIrd ventricle and the NPP reacting with anti-SRIF; a, IIIrd ventricle; b, NPO; c, NPP; d, supraoptic recess). × 300.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>FIG. 10. Part of the nucleus preopticus pars magnocellularis of the rainbow trout showing perikarya reacting with antineurophysin. × 700.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>FIG. 11. Frontal section of the pituitary of the rainbow trout showing axonal endings reacting with antineurophysin localized exclusively in the ramifications of the pars nervosa distributed in the neurointermediate lobe. × 140.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>FIG. 12. Frontal section of the rainbow trout hypothalamus through the pituitary stalk showing the pars nervosa of the pituitary; SRIF immunofluorescent axons follow the ramifications of the pars nervosa in the proximal pars distalis, i, infundibulum; p, pituitary (Adenohypophysis). × 140.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>FIG. 13. Section of the proximal pars distalis showing SRIF immunoreactive axons following ultimate endings of the pars nervosa in the proximal pars distalis. × 700.</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>FIG. 14. Section of the proximal pars distalis, adjacent to Fig. 13, showing the distribution of LH-RF immunoreactive axonal endings. LH-RF and SRIF endings are superposed. × 700.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>FIG. 15. Parasagittal sections of the rainbow trout hypothalamus (next to the midline of the brain) crossing the recess of the IIIrd ventricle separating the nucleus lateralis tuberis from the pituitary, small scarce cells scattered in the nucleus lateralis tuberis and reacting with anti-α-endorphin (dotted line: lateral infundibular recess). × 700.</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>FIG. 16. Section of the neurointermediate lobe of the rainbow trout showing that the whole cell population reacts with anti-α-endorphin. × 140.</figcaption></figure>
isotocin are restricted to the NPO in rainbow trout, goldfish, and the flatfish *Pleuronectes platessa*. Furthermore, the distribution of SRIF cells in the preoptic region in rainbow trout is restricted to the NPP and does not grade into the NPO. In mammals SRIF cells are found in the preoptic and anterior periventricular hypothalamus (Dubois and Kolodziejczyk, 1975; Elde and Parsons, 1975; Hökfelt *et al.*, 1975; Alpert *et al.*, 1976; Parsons *et al.*, 1976; Bugnon *et al.*, 1977), which may in part be homologous to the NPP location in the trout. Contrary to the rainbow trout, in the rat (Dubois and Kolodziejczyk, 1975) and man (Bugnon *et al.*, 1977) certain magnocellular perikarya in the supraoptic and paraventricular nuclei react for both SRIF and NPH.

In the rainbow trout we have observed an overlapping distribution of SRIF and LH-RH immunoreactivity in the neurohypophysial tissue of the proximal pars distalis. The function of SRIF in the teleost pituitary remains open, although it has been suggested to act as an inhibitor of thyrotropin release (Peter and McKeown, 1975). LH-RH has gonadotropin releasing activity in teleost fishes but whether it is the native neurohormone in teleosts remains open (for review: Peter and Fryer, 1979). Although Goos and Murathanoglu (1977) reported LH-RH immunoreactive cell bodies in the area dorsalis pars medialis of the telencephalon in rainbow trout, we were not able to do so. In rainbow trout we found that NPH immunoreactivity was restricted to the neurohypophysial tissue of the neurointermediate lobe of the pituitary and that it did not overlap with the distribution of SRIF and LH-RH in the pituitary. Gill *et al.* (1977) also found immunoreactivity for vasotocin and NPH in the neurohypophysial tissue in the pituitary of the sucker *Catostomus commersoni*. In mammals the distribution of SRIF and NPH in the median eminence have been shown

not to overlap (Dubois and Barry, 1976; Bugnon *et al.*, 1977), but there may be some overlap in the distribution of LH-RH and SRIF in the median eminence (Hökfelt *et al.*, 1974).

## ACKNOWLEDGMENTS

The authors are very indebted to Dr. R. Guillemin, The Salk Institute, San Diego, California, for his kind gifts of somatostatin and endorphin, and to Dr. F. Stutinsky, Institut de Physiologie, Strasbourg, France, for his gift of neurophysin. Thanks are due to Mrs. Chantal Couvrand, Nadine Martinat, and Mr. Terriot for their valuable histological, immunochemical, and photographical assistance. The work was supported in part by NATO Research Grant 1305 and by a NRC-CNRS Scientist Exchange Grant to R.E.P.

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