Immunohistochemical localization of gonadotropin releasing hormones in the brain and pituitary gland of the Nile perch, Lates niloticus (Teleostei, Centropomidae)

Immunohistochemical localization of gonadotropinreleasing hormones in the brain and pituitary gland of the Nile perch,

Lates niloticus (Teleostei, Centropomidae)

Mostafa A. Mousaa and Shaaban A. Mousab,*

a National Institute of Oceanography and Fisheries, Alexandria, Egyptb Zoology Department, Faculty of Science at Aswan, South Valley University, Aswan, Egypt

Accepted 11 October 2002

Abstract

In the present study we investigated the distribution of gonadotropin-releasing hormones (GnRH) in the brain of Lates niloticus

and their association with different pituitary cell types using immunohistochemical techniques. We found immunoreactive (ir)

chicken GnRH-II (cGnRH-II) and mammalian GnRH (mGnRH) as the main components of the GnRH-ir system within the brain

of the Nile perch. The results indicate that mGnRH and cGnRH are localized in different neurons: mGnRH-ir perikaria were

observed in the preoptic region particularly in the organum vasculosum laminae terminalis (OVLT) and in the nucleus lateralis

tuberis pars posterior (NLTP) of the mediobasal hypothalamus. These cell bodies are located along a continuum of ir-fibers that

could be traced from the olfactory nerve to the pituitary. mGnRH-ir fibers were detected in many parts of the brain (olfactory bulbs,

ventral telencephalon, hypothalamus, and mesencephalon) and in the pituitary. cGnRH-ir cell bodies are restricted to the optic

tract, but few scattered fibers could be detected in different parts of the brain. The pituitary exhibited very few cGnRH-II ir fibers,

contrasting with an extensive mGnRH innervation. Moreover, mGnRH-ir fibers were targeting the three areas of the pituitary

gland: rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI). Double immunolabeling studies showed

GnRH-ir fibers in close proximity with prolactin (PRL)- and adrenocorticotropic hormone (ACTH)-producing cells in the RPD,

growth hormone (GH)-producing cells in the PPD, gonadotropins (GTHs)-producing cells in the PPD in the external border of the

PI, and with somatolactin (SL)- and a-melanocyte stimulating hormone (a-MSH)-producing cells in the PI. Our results showed

direct morphological evidence for a close association of GnRH-ir fibers with the different adenohypophysial cell types. These results

suggest a multiple role of GnRH in the regulation of various pituitary hormones� release.� 2003 Elsevier Science (USA). All rights reserved.

Keywords: GnRH; Immunohistochemistry; Brain; Pituitary gland; Lates niloticus

1. Introduction

Gonadotropin-releasing hormone (GnRH) is a deca-peptide neuroendocrine hormone that is considered to

play important roles in the regulation of teleost repro-

duction, mainly by stimulation of gonadotropin release

from the pituitary gland (Peter et al., 1991). Several

studies have reported that in vertebrates at least two dif-

ferent GnRH forms are expressed within the brain of a

single species, generally one GnRH functions as a neu-rohormone regulating GTH release in the pituitary and

the other form may have a neurotransmitter/neuromod-

ulatory function and is generally localized in areas outside

the hypothalamus or midbrain regions (Muske, 1993). In

the brain of teleosts, chicken cGnRH-II is systematically

found while the second form is either mGnRH (Lescheid

et al., 1995), catfish GnRH (cfGnRH) (Ngamvongchon

et al., 1992), or salmon GnRH (sGnRH) (Ravaglia et al.,1997).

Teleost fishes lack a functional-hypophyseal portal

system but GnRH nerve fibers terminate in the vicinity of

General and Comparative Endocrinology 130 (2003) 245–255

www.elsevier.com/locate/ygcen

GENERAL AND COMPARATIVE

ENDOCRINOLOGY

* Corresponding author. Present address: Shaaban Mousa, Freie

Universitat Berlin, Universitatsklinikum Benjamin Franklin, Klinik

far Anaesthesiologie und operative intensivmedizin, Hindenburgdamm

30, Berlin D-12200, Germany. Fax: +49-30-8445-44-69.

E-mail address: [email protected] (S.A. Mou-

sa).

0016-6480/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0016-6480(02)00611-1

mail to: [email protected]

the pituitary gonadotrophs (Muske, 1993). Recently, agrowing body of data suggests the involvement of GnRH

in regulating the secretion of various pituitary hormones

including GTHs. Some reports have demonstrated a

stimulatory action of GnRH on GH release in vivo and

in vitro (Marchant et al., 1989; Melamed et al., 1995). It

has been shown that GnRH stimulates the release of

PRL from the RPD in tilapia (Oreochromis mossambi-

cus) (Weber et al., 1997). On the other hand, evidencesuggests that SL-producing cells are also regulated by

GnRH in Oncorhynchus mykiss (Kakizawa et al., 1997)

and in Oncorhynchus nerka (Taniyama et al., 2000).

Furthermore, Parhar and Iwata (1994) have observed

GnRH-ir fibers projecting to SL cells in the steelhead

trout, O. mykiss. In addition, direct morphological evi-

dences of a close association of GnRH fibers with GH,

PRL, GTH, and SL-expressing cells were observed in thepejerrey, Odontesthes bonariensis (Vissio et al., 1999).

The characterization as well as the anatomical distri-

bution of GnRH forms have not been investigated in the

‘‘Nile perch’’ Lates niloticus (Linneaus, 1758). This fish is

the most economically important fish species in Egypt

living in tropical and semitropical waters. It grows fast

but is less salt-tolerant than Lates calcarifer, and attains

190 cm in length with maximum weight of 200 kg. In ourprevious studies, we have identified and anatomically

localized adenohypophysial cell types in this species

(Mousa, 2001). The anatomical localization of different

GnRH forms in the brain and pituitary is important in

understanding their functional role and physiological

relevance. The first aim of this study was to characterize

and investigate the distribution of GnRH forms in the

brain and pituitary. The second aim was to determinewhether there is any association between GnRH-ir fibers

with different pituitary endocrine cell types in Lates nil-

oticus using immunohistochemical techniques.

2. Materials and methods

2.1. Animals

Lates niloticus (20 immature and mature animals of

both sexes) with standard length larger than 20 cm, were

collected alive during the prespawning and spawning

season (July–September) from Lake Nasser.

2.2. Tissue processing

The fishes were anesthetized in a solution (100mg/L)

of tricaine methanosulfonate (MS222, Sandoz) and then

perfused via the ascending aorta with 20ml of normal

saline, followed by 50ml of Bouin�s fluid at 4 �C. Thepituitary gland attached to the brain was immediately

removed and postfixed in Bouin�s fluid for 24 h at 4 �C.The fixed brain and pituitaries were thereafter dehy-

drated through graded ethanol solution, cleared andembedded in paraplast (M.P.: 56–58 �C). Consecutive

median sagittal sections of the brain and the pituitary

gland were made at 4lm thickness.

2.3. Immunohistochemical procedures

2.3.1. Antibodies

Rabbit antisera directed against human ACTH wasobtained from National Institute of Health. The a-MSH

antiserum (Dr. R.M. Dores, University of Denver,

USA). Antisera to chum salmon (Oncorhynchus keta)

hormones; chum salmon GTH IIb subunit (Lot No.

8506), chum salmon GH (Lot No. 8208), chum salmon

PRL (Lot No. 8502), and chum salmon SL (Lot No.

8906) were obtained from Dr. H. Kawauchi (School of

Fisheries Science, Kitasato University, Iwate, Japan).GnRH antisera: mGnRH (83LRF) (G. Tramu, Avenue

des Facult�ees, Talence, France), salmon GnRH (Lot No.

1668) (J.A. King, University of Cap Town, South Af-

rica), cGnRH-II (R€uudiger W. Schulz, University of

Utrecht, Faculty of Biology, The Netherlands), cGnRH-

II (aCII6) (Koichi Okuzawa, National Research Insti-

tute of Aquaculture, Mie, Japan), Lamprey I GnRH

(Lot 21-134), and Lamprey III GnRH (Lot 3952) (StaciaSower, University of New Hampshire, USA).

2.4. Immunohistochemistry

Immunocytochemical staining for the sections of the

pituitary gland and brain was generally performed with

a vectastain ABC (avidin–biotin peroxidase complex)

Kit (Vector Laboratories) as described previously(Mousa and Mousa, 1999). In brief, sections were

deparaffinized in xylene, rehydrated through graded

ethanol, washed in phosphate-buffered saline (PBS; pH

7.4) for two times 10min each. All incubations were

done at 4 �C and PBS was used for washing after each

step. Sections were incubated with the antisera to the

various GnRHs overnight at 4 �C (1:1000 for each of

lGnRH-I, lGnRH-III, sGnRH (1668), cGnRH-II, andmGnRH (83LRF). Thereafter, the sections were incu-

bated with the biotinylated secondary antibody (Vector

Laboratories) for 1 h and with avidin–biotin-conjugated

peroxidase for 45min. Finally, the sections were washed

and stained with 30,30-diaminobenzidine tetrahydro-

chloride (DAB) (Sigma) including 0.01% H2O2 in

0.05M Tris-buffered saline (pH 7.6) for 3–5min. After

the enzyme reaction, the sections were washed in tapwater, dehydrated in alcohol, cleared in xylene, and

mounted in DPX.

2.5. Immunohistochemical double staining

Colocalization studies were performed using double-

label immunostains. Sections stained with anti-GnRH

246 M.A. Mousa, S.A. Mousa / General and Comparative Endocrinology 130 (2003) 245–255

antibody (as previously mentioned) were washed inseveral changes of PBS. Sections were then incubated

with a second primary antibody against a pituitary

hormone (PRL, ACTH, GH, GTHIIb, SL, or a-MSH),

respectively (as described previously by Mousa, 2001)

overnight at 4 �C, washed in PBS, exposed to the bio-

tinylated secondary antibody (Vector Laboratories) for

1 h and with avidin–biotin-conjugated peroxidase for

45min. Finally, the sections were washed and stainedwith 30,30-diaminobenzidine tetrahydrochloride (DAB)

(Sigma) including 0.01% H2O2 in 0.05M Tris-buffered

saline (pH 7.6) for 3–5min. During staining with DAB,

the nickel solution was used to differentiate between the

double immunostaining. The chromogen DAB/Ni used

for the first primary antiserum appeared gray/black,

whereas the one used for the second primary antiserum

appeared brown. After the enzyme reaction, the sectionswere washed in tap water, dehydrated in alcohol, cleared

in xylene, and mounted in DPX.

To demonstrate specificity of staining, the following

controls were included: (1) preabsorption of diluted an-

tibodies with 50lM synthetic GnRH peptides (Peninsula

Laboratories, CA, USA) for 24 h at 4 �C. (2) Omission of

either the primary antisera, the secondary antibodies or

avidin–biotin complex. These control experiments didnot show positive staining for any antibody tested.

3. Results

3.1. Immunocytochemical localization of GnRH forms in

the Nile perch

Specific lGnRH-I, lGnRH-III, and sGnRH immu-

noreactivity was not observed in any cell bodies or nerve

fibers in the brain and pituitary. However, cGnRH-II

and mGnRH are detected in different areas of the brain

of the Nile perch. They are localized in different neu-

rons. The distribution of the cGnRH-II and mGnRH-ir

cell bodies and fibers is illustrated on camera lucida

drawing of longitudinal sections of the brain (Figs. 1Aand B) and micrographs (Figs. 2–4).

3.2. Immunocytochemical localization of mGnRH immu-

noreactive system

The organization of the mGnRH ir system is sum-

marized on a camera lucida drawing of longitudinal

brain sections (Fig. 1A). The main mGnRH-ir areacorresponded to the mediobasal hypothalamus in the

NLTP. In the NLTP of the hypothalamus, two types,

small and large cells, of mGnRH-ir cells are found in

groups. Cells of small size are numerous and have strong

immunoreactivity of mGnRH (Fig. 2D). The rostramost

mGnRH-ir cell bodies were localized at the level of the

preoptic area, often close to the optic tracts as they

emerged from the optic chiasma. Some mGnRH cell

bodies were detected in the OVLT (Fig. 1A). mGnRH-ir

cells in the OVLT were similar to the smallest cells of the

hypothalamus (Fig. 3F). The mGnRH-ir fibers were

particularly abundant in the olfactory bulbs, ventral

telencephalon, hypothalamus, and mesencephalon (Figs.

1A and 2E and F). Bundles of mGnRH-ir fibers tra-versing the NLTP area and ran ventrally reaching the

ventral surface of the hypothalamus entering into the

pituitary stalk (Figs. 3A and B). Heavy mGnRH-ir

fibers entering the neurohypophysis from the ventral

hypothalamus penetrate the different areas of the ade-

nohypophysis (Figs. 3C–E). Preabsorption of mGnRH

antibody with their respective antigen did not produce

any immunoreactivity.

3.3. Immunocytochemical localization of cGnRH-II-im-

munoreactive system

The overall amount of cGnRH-II immunoreactivity

was much lower than that of mGnRH. The only

cGnRH-II-ir cells were detected in the optic tract (Fig.

1B). This area contained a large population of cGnRH-ir cell bodies (Figs. 4A–C). The cGnRH-ir cell bodies

were seen in the optic nerve (ON) (Figs. 4A and B).

These cells were elongated with strong immunoreactiv-

Fig. 1. Schematic illustration of the distribution of GnRH cell bodies

(dots) and fibers (lines) on a sagittal section revealed with: (A)

mGnRH antiserum; (B) cGnRH antiserum. Two antibodies showed

different distribution patterns. C, cerebellum; MO, medulla oblongata;

MT, midbrain tegmentum; NLTP, nucleus lateralis tuberis pars pos-

terior; OB, olfactory bulb; ON, optic nerve; O tec, optic tectum;

OVLT, organum vasculosum laminae terminalis; pit, pituitary gland;

Tel, telencephalon.

M.A. Mousa, S.A. Mousa / General and Comparative Endocrinology 130 (2003) 245–255 247

ity. They aggregated in-groups and showed continuous

layers (Fig. 4D). cGnRH-II-ir fibers were found in many

brain regions (Fig. 1B). The most evident fibers were

seen in the olfactory bulbs (Fig. 4E) which run caudally

and ventrally, and reaching the ventral surface of the

hypothalamus (Fig. 4F). Preabsorption of cGnRH

Fig. 2. Sagittal section through the NLTP, nucleus lateralis tuberis pars posterior and the pituitary gland showing mGnRH immunoreactivity. (A) A

cluster of mGnRH-ir cell bodies in the NLTP and the mGnRH-ir fibers innervated the pituitary gland. (B–E) Magnified portion of (A). (B) Two

types of mGnRH neurons; large neurons (arrowheads) and small ones (arrows), beside mGnRH-ir fibers innervated the RPD. (C) The large

mGnRH-ir neurons exhibiting different shapes and equipped with thick and thin axons. (D) The small mGnRH-ir neurons having strong immu-

noreactivity and equipped with thin axons. (E) Axons from small mGnRH-ir cells form distinct bundle (arrowhead). (F) Sagittal section through

ventral hypothalamus showing intense mGnRH-ir fibers. Bar ¼ 100lm (A); 80lm (B); and 40lm (C)–(F).


antibody with their respective antigen did not produce

any immunoreactivity.

3.4. Double immunostaining of mGnRH and different

hormone producing cell types in the pituitary gland

The mGnRH-ir fibers, which are passing through theNLTP area of hypothalamus and the pituitary stalk in-

nervate the three areas of the pituitary gland; RPD, PPD,

and PI (Fig. 3A). The PPD was characterized by the

presence of GH and GTHs positive cells. GTHs-pro-

ducing cells were located in the central area of the PPD

and in the external border of the PI (Figs. 5A–D). The

anti-chum salmon GTHIIb antiserum stained the GTHs-

producing cells in close proximity of mGnRH-ir fibers(Figs. 5E–H). Some mGnRH-ir fibers appear in close

Fig. 3. Sagittal sections through the NLTP, nucleus lateralis tuberis pars posterior and the pituitary gland (A, B), pituitary gland (C–E), and the

OVLT (F) showing mGnRH immunoreactivity. (A) A bundels of mGnRH-ir fibers entering neurohypophysis form ventral hypothalamus (ar-

rowhead) and reaching the three areas of the pituitary gland: RPD, PPD, and PI. (B–E) A magnified portion of (A). (B) Axons of mGnRH-ir neurons

are densely aggregated and passing through the pituitary stalk (arrowhead). (C) A densely labeled fibers are in the PPD. (D) Only few fibers (ar-

rowheads) are labeled in the RPD (E). Extensive innervation of the PI with mGnRH-ir fibers. (F) Bipolar neuron equipped with thin and coarse

axons in the OVLT. Bar ¼ 100lm (A); 80lm (B); and 40lm (C)–(F).


contact with GTH-ir cells. The anti-chum salmon GH

antiserum revealed a group of cells surrounding the neu-

rohypophysis (Fig. 6A). Moreover, bundles of mGnRH-ir fibers are surrounded by a group of GH-ir cells (Figs.

6A and B). Also, mGnRH-ir fibers were seen in intimate

contact with GH-ir cells (Figs. 6C–E). In the RPD, the

anti-chum salmon PRL antiserum stained PRL-positive

cells and most of them located in a compact group. The

double immunostaining revealedmGnRH-ir fiberswithin

PRL-ir cells containing area in the pituitary gland (Fig.

6F). Moreover, GnRH-ir fibers appeared in immediatevicinity with PRL positive cells (Figs. 6G and H). The

anti-human ACTH antiserum stained the ACTH cells.

These cells bordered the neurohypophysis and islets be-

tween PRL cells in the RPD. The mGnRH-ir fibers that

reached the RPD are in close contact with the ACTH

Fig. 4. Sagittal sections through the ON, optic nerve (A–D), OB (E), and NLTP, nucleus lateralis tuberis pars posterior and pituitary (F) showing

cGnRH-II immunoreactivity. (A, B) Many of cGnRH-II-ir cells are distributed in the ON. (C–D) A magnified portions of (A). (C) cGnRH-II-ir cells

were intensely immunostained in the ON. (D) Aggregated groups of cGnRH-II-ir cells formed a continuous layer. (E) cGnRH-II-ir fibers are

distributed in the OB. (F) cGnRH-II-ir fibers are passed through the NLTP and entered the pituitary gland. Bar ¼ 100lm (A, B); 80lm (E); and

40lm (C, D, F).


positive cells (Figs. 7A–C). In the PI, the anti-a-MSH

antiserum stained the a-MSH-producing cells. mGnRH-

ir fibers extending through neurohypophysis were sur-

rounding groups of a-MSH-ir cells and appeared in close

contact with these cells (Figs. 7D–F). Cells immuno-

stained with anti-chum salmon SL antiserum were ex-

clusively located in the PI bordering the neural tissue. The

double immunostaining revealed a close association be-tween mGnRH-ir and SL positive cells (Figs. 7G–I).

4. Discussion

This immunocytochemical study shows that the brain

of Nile perch contains two forms of GnRH like immu-

noreactivity. These two forms are mGnRH and cGnRH.

In this study only immunohistochemical techniques with

antibodies against some GnRH forms were used, thus

we cannot rule out the possible presence of a third formof GnRH, such as seabream (sb) GnRH, salmon (s)

Fig. 5. Sagittal section of the pituitary gland of L. niloticus double immunostained with GTHIIb and mGnRH antisera. (A, B) Double immuno-

staining showing mGnRH-ir fibers reaching the three areas of the adenohypophysis; RPD, PPD, and PI. (C, D) A magnified portion of (A, B)

showing GTH-ir cells in apposition with mGnRH-ir fibers (arrows) in PI (C) and in PPD (D). (E–H) Detail of the close contact between mGnRH-ir

fibers (arrows) and GTH-ir cells. Bar ¼ 100lm (A, B); 80lm (C, D); and 40lm (E–G).


GnRH, dogfish (df) GnRH or novel forms in the Nile

perch. This is in agreement with several other studies

confirming the presence of more than two forms of

GnRH in teleosts (Robinson et al., 2000; Rodriguezet al., 2000; Stefano et al., 2000). mGnRH was more

dominant than cGnRH-II in the whole brain, particu-

larly in the anterior brain and in the pituitary. Since

mGnRH and cGnRH are located in neurons of different

brain areas of the Nile perch, this indicates that

mGnRH and cGnRH are not colocalized. In the Nile

perch, mGnRH and cGnRH-II appear to be synthesized

by distinct neurons which are submitted to differentialregulations and are therefore likely to play different

physiological roles. This is in agreement with other im-

munocytochemical studies that reported a differential

distribution of different forms of GnRH (sGnRH and

Fig. 6. Part of sagittal sections of the pituitary of L. niloticus double immunostained with mGnRH and GH (A–E) or PRL (F–H) antisera. (A) GH-ir

cells in apposition with mGnRH-ir fibers in the PPD. (B) A magnified portion of (A) showing mGnRH-ir fibers surrounding a groups of GH-ir cells.

(C–E) Detail of the intimate contact between mGnRH-ir fibers (arrows) and GH-ir cells. (F) Double immunostaining showing mGnRH-ir fibers

(arrows) within the PRL-ir cells area in the RPD. (G, H) Detail of the close contact between mGnRH-ir fibers (arrows) and PRL-ir cells. Bar ¼ 80lm(A, F); 40lm (B, C, D, E, G, H).


cGnRH-II in Oncorhynchus masu: Amano et al., 1991;

mGnRH and cGnRH-II in Anguilla anguilla: Montero

et al., 1994; sGnRH and lGnRH in Catostomus com-

mersoni : Robinson et al., 2000). In contrast, a colocal-

ization of different forms of GnRH has been indicated inthe brain of Carassius auratus (sGnRH and cGnRH-II:

Kim et al., 1995) and in O. bonariensis (mGnRH and

sGnRH: Stefano et al., 2000). This data may indicate a

different evolution of GnRH systems amongst teleosts,

but some colocalizations could also be due to cross-re-

actions of the antibodies (Muske, 1993). Using specific

radioimmunoassays, a differential regulation of the

brain and pituitary levels of mGnRH and cGnRH-IIwas observed in A. anguilla (Dufour et al., 1993; Mon-

tero et al., 1993). Also, steroid treatments increased the

brain and pituitary mGnRH levels but reduced those of

cGnRH-II (Montero et al., 1993).

In addition, our immunocytochemical results indicate

that cGnRH-II immunoreactivity is much lower than

that of mGnRH. Chicken GnRH-ir cell bodies are de-

tected in the optic tract. Other studies have identified

cGnRH-II-ir cells in the midbrain tegmentum of O.

mossambicus (Parhar, 1997), Oreochromis niloticus

(Parhar et al., 1998), and O. bonariensis (Stefano et al.,

2000). The main function of GnRH is regulating gona-

dotropin release in teleost fish (Peter et al., 1991). Otherpossible roles for GnRH peptides include acting as

neurotransmitters and neuromodulators (Oka and

Matsushima, 1993). cGnRH-II may serve one or both of

these functions in the Nile perch.

Interestingly, we observed a very heavy mGnRH in-

nervation in the digitations of the neurohypophysis at

the level of all areas of the adenohypophysis in the pi-

tuitary gland of the Nile perch. Direct innervation of thepituitary gland by fibers of hypothalamus GnRH neu-

rons is characteristic of the class Osteichthyes (Muske,

1993). Similar immunocytochemical results were ob-

tained in O. bonariensis (Stefano et al., 2000). Other

authors have shown that mGnRH fibers projected into

the PPD and PI, but not into the RPD in other teleosts

(Chiba et al., 1996; Montero et al., 1994; Parhar and

Iwata, 1994). The localization of the mGnRH-ir fibers

Fig. 7. Part of sagittal sections of the pituitary of L. niloticus double immunostained with mGnRH and ACTH (A–C), a-MSH (D–F), or SL (G–I)

antisera. (A) Double immunostaining showing mGnRH-ir fibers (arrows) from ventral hypothalamus reaching the ACTH-ir cells. (B–C) Detail of

mGnRH-ir fibers (arrows) in close contact with ACTH-ir cells. (D) MSH-ir cells are in apposition with mGnRH-ir fibers (arrows). (E, F) Detail of

the close proximity between mGnRH-ir fibers (arrows) and MSH-ir cells. (G) Double immunostaining showing SL-ir cells in apposition with

mGnRH-ir fibers. (H, I) Detail of the intimate contact between mGnRH-ir fibers (arrows) and SL-ir cells. Bar ¼ 80lm (A, D, G); 40lm (B, C, E, F,

H, I).


suggests that mGnRH plays an important role in theneurohormonal regulation of pituitary hormones in the

Nile perch. In the RPD, a close association was observed

between mGnRH-ir fibers and ACTH- and PRL-ex-

pressing cells. Similarly, in O. bonariensis a close asso-

ciation between GnRH-ir fibers and PRL cells has been

described, suggesting GnRH involvement in PRL regu-

lation in this species (Vissio et al., 1999). The presence of

GnRH binding sites in PRL-expressing cells reinforcesthis postulate (Stefano et al., 1999). It has been reported

that GnRH can induce PRL release from the RPD

fragment of the tilapia (O. mossambicus) pituitary gland,

in vitro (Weber et al., 1997). In addition, the injection of

GnRH stimulates the release and synthesis of the ACTH

during the induction of maturation and ovulation in Liza

ramada (Mousa, 1999). The close association between

mGnRH-ir fibers and ACTH and PRL cells in the Nileperch represent morphological evidence of a possible

involvement of GnRH on ACTH and PRL release.

In the Nile perch, mGnRH-ir projections reached

GTH-expressing cells in the PPD and the external border

of the PI. They also reached GH-ir cells located close to

the neurohypophysis at the PPD. These observations are

in accordance with those observed in other teleosts spe-

cies (Parhar and Iwata, 1994; Vissio et al., 1999). Inaddition, specific GnRH binding sites were detected in

GTH- and GH-expressing cells inO. bonariensis (Stefano

et al., 1999). The immunolocalization of mGnRH-ir fi-

bers and the parallel regulation of mGnRH and GTH

levels by steroids suggests that mGnRH is the main

peptide involved in the neurohormonal regulation of

pituitary GTH in A. anguilla (Dufour et al., 1993).

Furthermore, in C. auratus the administration of GnRHstimulated the release of GTH and GH from the pitui-

tary gland in vivo and in vitro (Peter et al., 1990). Also,

specific binding of GnRH to somatotrophs was demon-

strated in O. bonariensis (Stefano et al., 1999). The

morphological association between mGnRH fibers and

GTH- andGH-expressing cells suggests a possible role of

mGnRH on GTH and GH secretion in the Nile perch.

Athough the biological function of SL is still largelyunclear, several studies have suggested the involvement

of SL in reproduction (Mousa and Mousa, 2000; Rand-

Weaver et al., 1992), stress (Kakizawa et al., 1995), dark

background adaptation (Zhu and Thomas, 1995), and

other biological events such as energy metabolism,

feeding, and ionoregulation (Kaneko, 1996).

Our double immunostaining revealed a close associa-

tion between mGnRH-ir fibers and SL-ir cells. Little isknown about the regulation of SL secretion. There is

pharmacological and morphological evidence showing

that GnRH could be involved in SL release. Kakizawa

et al. (1997) showed that GnRH stimulated dopamine-

inhibited SL release in vitro. In addition, injection of

GnRH stimulated the release of SL during the induction

of maturation and ovulation in Liza ramada (Mousa,

1999). The immunocytochemical results obtained in O.mykiss (Parhar and Iwata, 1994) and in O. bonariensis

(Vissio et al., 1999) and the present data suggest that

GnRH-ir fibers ending in contact with SL-ir cells can be

the morphological substrate of GnRH action on SL re-

lease and this could be a common pattern in teleost fishes.

The a-MSH has been related to adaptation to a dif-

ferent background color (Zhu and Thomas, 1996) and

stress response (Wendelaar Bonga, 1997). The presentresults identified a close association between mGnRH-ir

fibers and a-MSH-expressing cells in the Nile perch.

Also, an activation of a-MSH cells was obtained during

GnRH induction of oocytes maturation and ovulation

in L. ramada (Mousa, 1999). Taken together, these data

suggest that GnRH may be involved in a-MSH secre-

tion in teleost fishes.

In summary, our results showed that cGnRH-II andmGnRH, which are found in most teleost fish, are the

main components of the GnRH-ir system in the brain of

the Nile perch. The results indicate that mGnRH and

cGnRH-II are localized in different neurons and that

mGnRH-ir fibers are in close association with various

pituitary cells. The present study provides immunocy-

tochemical evidence that mGnRH may have a multiple

function in regulating pituitary hormones secretionother than GTHs in the Nile perch. Our results stimulate

further experimental approaches, such as the effect of

GnRH on hormone secretion of adenohypophysial cells

to confirm the multiple function of GnRH in regulating

the release of different pituitary hormones.

Acknowledgments

The authors are extremely grateful to Drs. H. Ka-

wauchi, R.M. Dores, J.A. King, G. Tramu, R.W.

Schulz, K. Okuzawa, and Stacia Sower for donating the

antisera used in this study and to Prof. Dr. Michael

Sch€aafer for critical review of the manuscript.

References

Amano, M., Oka, Y., Aida, K., Okumoto, N., Kawashima, S.,

Hasegawa, Y., 1991. Immunocytochemical demonstration of

salmon GnRH and chicken GnRH-II in the brain of masu salmon,

Oncorhynchus masou. J. Comp. Neurol. 314, 587–597.

Chiba, A., Sohn, Y.S., Honma, Y., 1996. Distribution of neuropeptide

Y and gonadotropin-releasing hormone immunoreactivities in the

brain and hypophysis of the Ayu, Plectoglossus altivelis (Teleostei).

Arch. Histol. Cytol. 59, 137–148.

Dufour, S., Montero, M., Le Belle, N., Bassompierre, M., King, J.A.,

Millar, R.P., Peter, R.E., Fontaine, Y.A., 1993. Differential

distribution and response to experimental sexual maturation of

two forms of brain gonadotropin-releasing hormone in the

European eel, Anguilla anguilla. Fish Physiol. Biochem. 11, 99–106.

Kakizawa, S., Kaneko, T., Hasegawa, S., Hirano, T., 1995. Effects of

feeding, fasting, background adaptation, acute stress, and exhaus-


tive exercise on the plasma somatolactin concentration in rainbow

trout. Gen. Comp. Endocrinol. 98, 137–146.

Kakizawa, S., Kaneko, T., Hirano, T., 1997. Effects of hypothalamic

factors on somatolactin secretion from the organ-cultured pituitary

of rainbow trout. Gen. Comp. Endocrinol. 105, 71–78.

Kaneko, T., 1996. Cell biology of somatolactin. Int. Rev. Cytol. 169,

1–14.

Kim, M.H., Oka, Y., Amano, M., Kobayashi, K., Okuzawa, Y.,

Hasegawa, S., Kawashima, Y., Suzuki, Y., Aida, K., 1995. Immu-

nocytochemical localization of sGnRH and cGnRH-II in the brain

of goldfish, Carassius auratus. J. Comp. Neurol. 356, 72–82.

Lescheid, D.W., Powell, J.F.F., Fisher, W.H., Park, M., Craig, A.,

Bukovskaya, O., Barannikova, I.A., Sherwood, N.M., 1995.

Mammalian gonadotropin-releasing hormone (GnRH) indentified

by primary structure in Russian sturgeon, Acipenser gueldenstaedti.

Regul. Peptides 55, 299–309.

Marchant, T.A., Change, J.P., Nahorniak, C.S., Peter, R.E., 1989.

Evidence that gonadotropin-releasing hormone also functions as a

growth hormone-releasing factor in the goldfish. Endocrinology

124, 2509–2518.

Melamed, P., Eliahu, N., Levavi-Sivan, B., Ofir, M., Farchi-Pisanty,

O., Renteier-Delrube, F., Smal, J., Yaron, Z., Naor, Z., 1995.

Hypothalamic and thyroidal regulation of growth hormone in

tilapia. Gen. Comp. Endocrinol. 64, 396–400.

Montero, M., Le Belle, N., Dufour, S., 1993. R�eetrocontroole positif de

st�eero€iides sexuels sur la synth�eese de mGnRH, une des deux formes

de gonadolib�eerines pr�eesentes chez l�anguille europ�eeenne. Ann.

Endocrinol. 54, 27.

Montero, M., Vidal, B., King, J.A., Tramu, G., Vandesande, F.,

Dufour, S., Kah, O., 1994. Immunocytochemical localization of

mammalian GnRH (gonadotropin-releasing hormone) and chicken

GnRH-II in the brain of the European silver eel (Anguilla anguilla

L.). J. Chem. Neuroanatomy 7, 227–241.

Mousa, M.A., 1999. Hormonal induction of oocyte final maturation

and ovulation in thin-lipped grey mullet, Liza ramada (Risso). Bull.

Nat. Inst. Oceanogr. Fish., A.R.E. 25, 331–355.

Mousa, M.A., 2001. Immunocytochemical identification and distribu-

tion of the cell types in the pituitary gland of the Nile perch, Lates

niloticus (Teleostei, Centropomidae). Bull. Nat. Inst. Oceanogr.

Fish. A.R.E. 27, 62–71.

Mousa, M.A., Mousa, S.A., 1999. Immunocytochemical study on the

localization and distribution of the somatolactin cells in the

pituitary gland and the brain of Oreochromis niloticus (Teleostei,

Cichlidae). Gen. Comp. Endocrinol. 113, 197–211.

Mousa, M.A., Mousa, S.A., 2000. Implication of somatolactin in the

regulation of sexual maturation and spawning ofMugil cephalus. J.

Exp. Zool. 287, 62–73.

Muske, L.E., 1993. Evolution of gonadotropin-releasing hormone

(GnRH) neuronal systems. Brain Behav. Evol. 42, 215–230.

Ngamvongchon, S., Lovejoy, D.A., Fischer, W.H., Craig, A.G.,

Nahorniak, C.S., Peter, R.E., Rivier, J.E., Sherwood, N.M., 1992.

Primary structures of two forms of gonadotropin-releasing hor-

mone, one distinct and one conserved, from catfish brain. Mol. Cell

Neurosci. 3, 17–22.

Oka, Y., Matsushima, T., 1993. Gonadotropin-releasing hormone

(GnRH)-immunoreactive terminal nerve cells have intrinsic rhyth-

micity and project widely in the brain. J. Neurosci. 13, 2161–2176.

Parhar, S.I., 1997. GnRH in tilapia: Three genes, three origins and

their roles. In: Parhar, I.S., Sakuma, Y. (Eds.), GnRH Neurons:

Gene to Behaviour. Brain Shuppan, Japan, pp. 99–123.

Parhar, S.I., Iwata, M., 1994. Gonadotropin-releasing hormone

(GnRH) neurons project to growth hormone and somatolactin

cells in the steelhead trout. Histochemistry 102, 195–203.

Parhar, I.S., Soga, T., Sakuma, Y., 1998. Quantitative in situ

hybridization of three gonadotropin-releasing hormone encoding

mRNAs in castrated and progesterone treated male tilapia. Gen.

Comp. Endocrinol. 112, 406–414.

Peter, R.E., Habibi, H.R., Chang, J.P., Nahorniak, C.S., Yu, K.L.,

Huang, Y.P., Marchant, T.A., 1990. Actions of gonadotropin-

releasing hormone (GnRH) in the goldfish. In: Epple, A., Scanes,

C.G., Stetson, M.H. (Eds.), Comp Endocrinol. Wiley-Liss, New

York, pp. 393–398.

Peter, R.E., Trudeau, V.L., Sloley, B.D., Peng, C., Nahorniak, C.S.,

1991. Actions of catecholamines, peptides and sex steroids in

regulation of gonadotropin-II in the goldfish. In: Scott, A.P.,

Sumpter, J.P., Kime, D.E., Rolfe, M.S. (Eds.), Reproductive

Physiology of Fish, Fish Symposium 91. University of East Anglia

Printing Unit, Sheffield, pp. 30–34.

Rand-Weaver, M., Swanson, P., Kawauchi, H., Dickhoff, W.W., 1992.

Somatolactin, a novel pituitary protein: purification and plasma

levels during reproductive maturation of coho salmon. J. Endocri-

nol. 133 (3), 393–403.

Ravaglia, M.A., Lo Nostro, F.L., Maggesse, M.C., Guerrero, G.A.,

Somoza, G.M., 1997. Characterization of molecular variants of

GnRH, induction of spermiation and sex reversal using salmon

GnRH-A and domperidone in the protogynous diandric fish,

Symbranchus marmoratus Bloch, (Teleostei, Synbranchidae). Fish

Physiol. Biochem. 16, 425–436.

Robinson, T.C., Tobet, S.A., Chase, C., Waldron, T., Sower, S.A.,

2000. Gonadotropin-releasing hormones in the brain and pituitary

of the teleost, the white sucker. Gen. Comp. Endocrinol. 117, 381–

394.

Rodriguez, L., Carrillo, M., Sorbera, L.A., Soubrier, M.A., Mananos,

E., Holland, M.C., Zohar, Y., Zanuy, S., 2000. Pituitary levels of

three forms of GnRH in the male European sea bass (Dicentrar-

chus labrax, L.) during sex differentiation and first spawning

season. Gen. Comp. Endocrinol. 120, 67–74.

Stefano, A.V., Aldana-Marcos, H.J., Affanni, J.M., Somoza, G.M.,

2000. Gonadotropin-releasing hormone (GnRH) neuronal systems

in the pejerrey, Odontesthes bonariensis (Atheriniformes). Fish


Stefano, A.V., Vissio, P.G., Paz, D.A., Somoza, G.M., Maggese,

M.C., Barrantes, G.E., 1999. Colocalization of GnRH binding sites

with gonadotropin-, somatotropin-, somatolactin-, and prolactin-

expressing pituitary cells of the pejerrey, Odontesthes bonariensis, in

vitro. Gen. Comp. Endocrinol. 116, 133–139.

Taniyama, S., Kitahashi, T., Ando, H., Kaeriyama, M., Zohar, Y.,

Ueda, H., Urano, A., 2000. Effects of gonadotropin-releasing

hormone analog on expression of genes encoding the growth

hormone/prolactin/somatolactin family and a pituitary-specific

transcription factor in the pituitaries of prespawning sockeye

salmon. Gen. Comp. Endocrinol. 118, 418–424.

Vissio, P.G., Stefano, A.V., Somoza, G.M., Maggese, M.C., Paz,

D.A., 1999. Close association of gonadotropin-releasing hormone

fibers and gonadotropin, growth hormone, somatolactin and

prolactin expressing cells in pejerrey, Odontesthes bonariensis. Fish


Weber, G.M., Powell, J.F.F., Park, M., Fisher, W.H., Craig, A.G.,

Rivier, J.E., Nanakon, U., Parhar, I.S., Ngamvongchon, S.,

Grau, E.G., Sherwood, N.M., 1997. Evidence that gonadotropin-

releasing hormone (GnRH) functions as a prolactin-releasing

factor in a teleost fish (Oreochromis mossambicus) and primary

structures for three native GnRH molecules. J. Endocrinol. 155,

121–132.

Wendelaar Bonga, S.E., 1997. The stress response in fish. Physiol. Rev.

77, 591–615.

Zhu, Y., Thomas, P., 1995. Red drum somatolactin: development of a

homologous radioimmunoassay and plasma levels after exposure

to stressors or various backgrounds. Gen. Comp. Endocrinol. 99,

274–288.

Zhu, Y., Thomas, P., 1996. Elevations of somatolactin in plasma and

pituitaries and increased a-MSH cell activity in red drum exposed

to black background and decreased illumination. Gen. Comp.

Endocrinol. 101, 21–31.


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Immunohistochemical localization of gonadotropin releasing hormones in the brain and pituitary gland of the Nile perch, Lates niloticus (Teleostei, Centropomidae)