Peroxidase-antiperoxidase Method Research Articles

Immunohistochemical and immunogenetic analysis of ocular adenexal lymphoid proliferations

We examined 20 cases (21 specimens) of ocular adnexal lymphoid proliferations, using the histological, immunohistochemical and molecular genetic methods. The latter two protocols were performed to detect the light chains restriction of immunoglobulin with peroxidase-antiperoxidase (PAP) methods, and the clonality of immunoglobulin heavy chain gene with the hemi-nested polymerase chain reaction (PCR) method, respectively. Although in 8 cases it could not be morphologically determined whether they were neoplastic or not, clonality was revealed in 1 case with immunohistochemistry and in 4 cases with PCR. Two cases showed discordant results between immunohistochemistry and PCR probably due to somatic mutation of the framework region of the immunoglobulin heavy chain gene. Therefore, we, concluded that examination with these methods contribute to a better understanding of the nature of the ocular adnexal lymphoid proliferations. Furthermore, the immunoglobulin gene PCR method is very useful in practical examination, as it can be used with formalin-fixed paraffin-embedded specimens.

Co-localization of serotonin and FMRFamide-like immunoreactivities in the central nervous system of the earthworm, Eisenia Jetida

In the present study co-localization of serotonin and FMRFamide-like immunoreactivities was studied in the central nervous system of the earthworm, Eisenia fetida. Alternate serial sections were stained with antisera raised against serotonin and FMRFamide, using the peroxidase-antiperoxidase method. The immunostained consecutive sections were compared by light-microscopy and on reconstructed images using NIH Image and Neurolucida programs. Although there was a large number of serotonin and FMRFamide-like immunoreactive neurons in the central nervous system of Eisenia, co-localization of immunostaining was relatively rare. In the cerebral ganglion no cells were found that stained for both antisera. In the subesophageal and ventral cord ganglia the neurons reacting for both antisera represented approximately 17-23% and 10-14% of the serotonin- and 7-11% and 9-17% of the FMRF-amide-like immunoreactive cells, respectively. It is suggested, that FMRFamide-related peptides act as neuromodulators and may also influence the activity of serotonergic neurons.

Ontogeny of immunoreactive somatolactin cells in the pituitary of gilthead sea bream (Sparus aurata L., Teleostei).

This is the first report on the identification of somatolactin (SL) cells during the early developmental stages of the teleost fish Sparus aurata. The SL cells were followed from newly hatched until 46 months. SL cells were immunocytochemically identified at light microscopical level with anti-cod SL in the developing pituitary using the peroxidase-antiperoxidase method. SL cells first appeared in newly hatched specimens, in which the pituitary gland lacked the neurohypophysis. They were scarce and located from the middle to the posterior region of the adenohypophysis. As the fish developed, the cells progressively increased in number and surrounded the developing neurohypophysis, which could be distinguised from 12-day-old larvae onwards in the caudal region of the gland. From 51 days onwards, SL cells were found in a discontinuous layer surrounding the neurohypophysis branches that entered the pars intermedia as clustered or isolated cells among non-SL-immunoreactive cells of the pars intermedia, and in the proximal pars distalis. The somatolactin-immunoreactive cells are periodic acid-Schiff-positive only in the adult stages. These data confirm, previous findings concerning the presence of two molecular forms of SL, glycosylated and nonglycosylated, in this species.

Substance P Immunoreactive Elements in the Nervous System of Earthworm (Lumbricus terrestris)

Substance P immunoreactive elements were detected in the nervous system of the earthworm, Lumbricus terrestris using peroxidase-antiperoxidase method. Each part of the central nervous system contains immunoreactive perikarya. The central neuropil shows no stained fibers. Immunopositive nerve cells were found in the stomatogastric ganglia as well as nerve cells and fibers in the wall of the alimentary canal. In the body wall, epidermal sensory cells and fibers surrounding the chaetae display immunoreactivity. The results are compared with those described previously by other authors.

The Study of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)-like Immunoreactivity in the Brain of a Teleost, Stargazer, Uranoscopus japonicus

Pituitary adenylate cyclase activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal polypeptide family of peptides, consists of a 38-residue (PACAP 38) and a truncated 27-residue (PACAP 27) form that play several roles in mammals. Recently, we isolated a PACAP-like peptide with a sequence highly homologous with that of tetrapod PACAP from the brain of a teleost, the blue spotted-stargazer (Gnathagnus elongatus). In this study, a PACAP-like peptide obtained from the brain of the stargazer, Uranoscopus japonicus, was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis using an anti-PACAP 27 serum. The stargazer PACAP-like peptide and synthetic human PACAPs were reacted with this antiserum. The distribution of PACAP-like immunoreactivity in the stargazer brain was also studied immunohistochemically using the peroxidase-antiperoxidase method. PACAP-like immunoreactive (LI) cells were found in the nucleus preopticus, pars parvicellularis of the hypothalamus. PACAP-LI nerve fibers and terminals were observed in the anterior neurohypophysis and in the rostral pars distalis, and many PACAP-LI nerve fibers were seen in the medulla oblongata. These results suggest that PACAP-like peptide may be involved in pituitary regulation and/or other neural functions in the stargazer brain.

Vasoactive intestinal polypeptide immunoreactive nerves in the gill arch of teleost fish, Carassius auratus L.

The localization of vasoactive intestinal polypeptide (VIP)-immunoreactive (ir) nerve cell bodies and fibers has been studied in the gill arches of goldfish (Carassius auratus, L.) using the peroxidase-antiperoxidase (PAP) immunohistochemical method. It was found that VIP-ir nerve cell bodies are localized in connective tissue on the oral side of the gill arch; these cells were present as single cells, in couples or as small clusters. Moreover, a dense network of VIP-ir fibers was observed beneath the lining epithelium of the raker cushion. The possible involvement of this peptide in mucus secretion in the gill arches of teleost is discussed.

Neuropeptide-containing nerve fibres in the human parotid gland: a semiquantitative analysis using an antibody against protein gene product 9.5.

The occurrence and distribution of neuropeptide-containing fibres in the human parotid gland were examined by the peroxidase-antiperoxidase method with attention to the quality of fixation and the condition of patients. Many fibres immunoreactive for neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) and a moderate number of galanin-positive (GAL) fibres were distributed around the acini. A moderate number of NPY and VIP fibres were distributed around the intercalated ducts. The semiquantitative mean densities (+/- SD) of periacinar NPY, VIP and GAL fibres expressed as a percentage of the total protein gene product (PGP) 9.5 immunoreactive fibres were 75.62 +/- 7.25%, 70.52 +/- 9.33% and 41.76 +/- 5.45%, respectively, whereas those of substance P (SP), calcitonin gene-related peptide (CGRP) and FMRF amide (FMRF) fibres were below 10%. The mean densities of NPY and VIP fibres around the intercalated ducts expressed as the percentage of PGP 9.5 fibres associated with these ducts were 52.37 +/- 6.19% and 59.62 +/- 7.02% respectively. Those of SP, CGRP, GAL, and FMRF fibres were below 10%. The densities of NPY, VIP, SP, CGRP, GAL and FMRF fibres around the striated and excretory ducts were also below 10%. In the vasculature, NPY fibres were the most prominent. Similarly, the mean density of perivascular NPY fibres was 93.76 +/- 2.03%. No somatostatin or leucine or methionine enkephalin immunoreactivity was detected around the acini, duct system or blood vessels. These findings suggest that, in this gland, the periacinar NPY, VIP and GAL fibres may participate in regulating the synthesis of saliva and its secretion and that perivascular peptidergic fibres, especially NPY fibres, may be involved in controlling local blood flow.

Peoscopic diagnosis of flat condyloma and penile intraepithelial neoplasia. Clinical manifestation.

Peoscopy was performed in order to assess penile lesions in the male sexual partners of 326 women with cervical intraepithelial neoplasia (CIN) or flat condyloma (FC). Each patient was submitted to a careful naked-eye inspection, peoscopy and biopsy of any suspicious lesion which was confirmed histologically and immunohistochemically. A brush cytologic examination of the distal portion of the urethra was also performed. The distribution of penile lesions was as follows: (1) 8 patients with herpes virus infection; (2) 37 patients with condyloma accuminata (CA); (3) 89 patients with FC; (4) 51 patients with FC and CA; (5) 18 patients with penile intraepithelial neoplasia grade 1 (PIN-I); (6) 2 patients with PIN-II; (7) 17 patients with PIN-III; (8) 92 patients with no penile lesions; (9) 7 patients with human papilloma virus infection of papillae coronae glandis, and (10) 5 patients with FC of the distal portion of the urethra. Naked-eye inspection revealed the presence of penile lesions in 39 of 233 patients (16.73%). Peoscopic examination revealed the presence of penile lesions in 233 of 326 patients (71.48%). In 135 of 155 patients the peoscopic findings were in accordance with the histologic diagnosis (87.09%). Immunohistochemical (by indirect peroxidase-antiperoxidase method) detection of virus antigens was positive in 16 of 34 patients (47.03%). It is concluded that peoscopy of the male sexual partners of women with CIN or FC should be performed to better assess the treatment used in the couple.

Expression of RANTES mRNA and protein in airways of patients with mild asthma.

Chemoattractant cytokines (chemokines) such as RANTES, are potent chemoattractants for eosinophils, T lymphocytes, and monocyte/macrophages. We examined RANTES mRNA and protein expression in bronchial biopsies and bronchoalveolar lavage (BAL) cells from patients with mild asthma on beta 2-adrenergic agonist therapy only. Using quantitative polymerase chain reaction (PCR), mean RANTES starting cDNA was 110 +/- 18 fg/pg beta-actin in asthmatics compared with 33.7 +/- 11.0 fg/pg in normals (p < 0.003) but there was no significant difference in RANTES mRNA expression in BAL cells between the two groups. Immunohistochemical staining of cryostat sections of bronchial biopsies with an anti-RANTES antibody using peroxidase antiperoxidase method revealed constitutive staining in airway epithelial cells, airway smooth muscle, and subepithelial cells. However, there was no difference in the number of RANTES-staining cells between normal subjects and asthmatics despite significantly increased numbers of CD4+ T-cells, CD68+ macrophages, and MBP+ eosinophils in mucosal biopsies obtained from asthmatics. Double-staining revealed expression of RANTES in a proportion of CD3+ T lymphocytes. Thus, RANTES is constitutively expressed in the airways and RANTES mRNA is elevated in airways of patients with mild asthma, supporting a role for RANTES in normal and asthmatic airways.

Hepatocellular carcinoma in young patients

The study was based on the biopsy material collected in Eastern coastal region of South Africa with high incidence of primary hepatocellular carcinoma (HCC). Forty-one patients were between 9 and 25 years old of the total number of 474 cases of HCC available for examination. Liver biopsies were fixed in 10% of neutralised formalin, processed to paraffin blocks, cut and stained with hematoxylin and eosin, silver reticulin, Masson trichrome and Prussian blue stains. Representative biopsies of 21 patients younger than 25 years and 56 older than 35 years were in addition examined immunohistochemically with HBsAg antibody, endothelial marker (F VIII-related antigen) and for oncoproteins c-myc and c-erbB-2 using peroxidase-antiperoxidase method. Cirrhotic liver was evident in 41.5% of all patients and in 28% of younger than 25 years. Hemosiderosis of the liver of patients over 35 years was nearly twice as common as in younger than 25 years and showed the opposite relationship to the presence of HBsAg in liver tissue. Oncoprotein expression was also higher in tumor tissue of younger patients. These results indicate the etiological association of HCC with HBV infection, cirrhosis and possibly siderosis of the liver with HCC. Simultaneous expression of oncoproteins and HBsAg indicate the primary importance of viral infection in etiopathogenesis of HCC.

Estrogen Receptor Immunocytochemistry in Endometrial Carcinoma: A Prognostic Marker for Survival

Only a few parameters such as tumor grade and stage are of value in prognosticating disease course in endometrial carcinoma. Biochemical steroid hormone receptor assays could also be useful but are difficult to perform and interpret. Immunocytochemical assay (ICA) might be the method of choice for detecting endometrial receptors. Methods: Frozen tissue from 78 cases of endometrial adenocarcinoma was examined for the presence of estrogen (ER) and progesterone receptors (PgR) with specific monoclonal anti-receptor antibodies and the peroxidase–antiperoxidase method. In over 60 cases, frozen tissue was also assayed for ER and PgR by biochemical means. Results: Fifty-five (71%) of the endometrial carcinomas were ERICA-positive and 55 (71%) PgRICA-positive. Although both ERICA and PgRICA correlated significantly with biochemical ER and PgR only ERICA was predictive of survival. A woman with a negative ERICA was 4 times more likely to die of her disease than if she were ERICA-positive (P= 0.009; mean follow-up, 37.5 months). Three cases ERICA-positive and PgRICA-negative survived while 3 others ERICA-negative and PgRICA-positive died. Conclusion: ERICA, a technique easy to perform and interpret at the community hospital level, appears to provide prognostic information independent of tumor stage and grade. Such information might be of value in planning postoperative therapies for women with endometrial cancer.

Immunocytochemical distribution of locustamyoinhibiting peptide (Lom-MIP) in the nervous system of Locusta migratoria

Locustamyoinhibiting peptide (Lom-MIP) is one of the 4 identified myoinhibiting neuropeptides, isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion complexes of the locust, Locusta migratoria. An antiserum was raised against Lom-MIP for use in immunohistochemistry. Locustamyoinhibiting peptide-like immunoreactivity (Lom-MIP-LI) was visualized in the nervous system and peripheral organs of Locusta migratoria by means of the peroxidase-antiperoxidase method. A total of 12 specific immunoreactive neurons was found in the brain. Processes of these neurons innervate the protocerebral bridge the central body complex and distinct neuropil areas in the proto- and tritocerebrum but not in the deuterocerebrum nor in the optic lobes. The glandular cells of the corpora cardiaca, known to produce adipokinetic hormones, are contacted by Lom-MIP-LI fibers. The corpora allata were innervated by the nervus corporis allati I containing immunoreactive fibers. Lom-MIP-LI cell bodies were also found in the subesophageal ganglion, the metathoracic ganglion and the abdominal ganglia I–IV. In peripheral muscles, Lom-MIP-LI fibers innervate the heart, the oviduct, and the hindgut. In the salivary glands, Lom-MIP-LI was detected in the intracellular ductule of the parietal cells. Possible functions of Lom-MIP are discussed.

Successful treatment of gallbladder carcinoma producing alpha-fetoprotein with segmental adenomyomatosis

The successful treatment of hepatoid adenocarcinoma of the gallbladder with elevated serum alpha-fetoprotein (1243 ng/ml) and segmental adenomyomatosis in a 58-year-old woman is described. The woman had alpha-fetoprotein (AFP)-producing carcinoma of the gallbladder with regional lymph node metastasis and was treated by extended radical resection and postoperative adjuvant chemotherapy. She is alive, showing normal serum AFP concentration and no recurrence, 57 months after surgery. The tumor cells were stained immunohistochemically for AFP by the peroxidase anti-peroxidase method. Serum AFP reactivity to concanavalin A and lentil lectin was similar to the pattern shown in hepatocellular carcinoma. Only a few cases of AFP-producing gallbladder carcinoma have been reported and there have been no reports of long-term survivors. The combination of aggressive radical resection and chemotherapy seems to have been effective for achieving long-term survival without liver metastasis.

Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis.

The present paper describes the immunocytochemical and morphometric characteristics of two major cell groups of the suprachiasmatic nucleus (SCN) in the human hypothalamus: the vasopressin (VP) and vasoactive intestinal polypeptide (VIP) neuronal subdivisions. The dimensions (volume and length) and the number of neurons expressing each peptide in the two subdivisions were obtained, as well as the mean diameter of the cell nuclei. All morphometric parameters were studied in relation to sex and age. Brains of 42 human subjects (22 males and 20 females) ranging in age from 10 to 92 years were obtained at autopsy. The hypothalamic area containing the SCN was dissected from each brain, dehydrated, and embedded in paraffin. Serial sections of 6 microns were cut in a coronal plane and stained with thionin for general orientation. To determine the architectonic boundaries of the VP- and VIP-expressing cell populations every 25th section was immunocytochemically stained by means of antibodies against arginine VP or VIP using the peroxidase-antiperoxidase method. The VP- and VIP-expressing cell numbers in the SCN of each subject were estimated by unilaterally counting the number of nuclear profiles with the aid of a Zeiss microscope under x 500 magnification, using a deconvolution procedure and a correction for section thickness. The main portion of the VP positive neurons is located in the dorsomedial part of the SCN and is rostrocaudally longer in females than in males (1.76 +/- 0.12 mm and 1.40 +/- 0.10 mm, respectively). The volume of the VP subdivision is 0.244 +/- 0.017 mm3 and contains 6,890 +/- 520 VP-immunoreactive neurons, with a mean density of about 29,000 neurons/mm3. No significant sexual dimorphism or age-related alterations in the population of VP neurons is found. The VIP positive neurons are mainly located in the ventral and central part of the SCN and extend rostrocaudally in a similar way in females and males (1.07 +/- 0.08 mm and 1.02 +/- 0.11 mm, respectively). The volume of the VIP subdivision is 0.034 +/- 0.004 mm3 and contains 1,700 +/- 140 VIP-immunoreactive neurons, with a mean density of about 63,000 neurons/mm3. An age-dependent sexual dimorphism is observed in the number of VIP-expressing neurons in the SCN: young males have about twice as many VIP neurons as females of the same age, whereas in middle-aged subjects this sexual difference is reversed, and less robust, with females now having about 1.7 times as many VIP neurons as males. In old subjects the difference in VIP cell number between men and women disappears. The present study clearly shows that the population of VP neurons in the human SCN is considerably larger than the population of VIP neurons. Furthermore, the age-related sexual differences in the VIP cell number reinforces the idea that the SCN is not only involved in the timing of circadian rhythms but also in the temporal organization of reproductive functions.

Use of a specific zona pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP integrity and function

To evaluate binding characteristics of a specific zona pellucida (ZP) protein 3 (ZP3) antiserum to human oocytes in order to determine its usefulness as a clinical marker for human ZP integrity and function and its correlation with IVF outcome. Prospectively designed, blinded, internally controlled study. Tertiary care academic center. Patients undergoing IVF therapy who had either total failed fertilization or partial fertilization were studied. Metaphase II oocytes showing absence of pronuclear formation were salt stored 48 hours after insemination and bisected into matching hemizonae using micromanipulation. One hemizona was incubated with AS ZP3-6 (an antiserum generated against a synthetic ZP3 peptide derived from an amino acid sequence that is highly conserved in the structure of ZP3), whereas the matching hemizona was incubated with AS ZP3-7, an antiserum detecting exclusively mouse ZP3 (internal, negative control). Antibody binding was visualized using the peroxidase-antiperoxidase method and diaminobenzidine as color reagent. A total of 104 unfertilized oocytes were evaluated. Analysis of variance showed a significant interaction between gamete factor groups (sperm and oocyte) and antiserum factor. Patients with oocyte factor had significantly lower mean staining scores for the AS ZP3-6-treated hemizonae than patients with sperm factor. These results demonstrate that anomalies of human ZP3 can be identified with AS ZP3-6 and that these ZP abnormalities correlate with fertilization failure during IVF treatment. Thus, this newly developed biomarker may be of clinical significance in the identification of oocyte defects that are associated with fertilization disorders and may help in the decision-making process in the IVF-assisted fertilization setting.

Immunohistochemical studies of neural elements in pancreatic islets of the cat.

The cat pancreas has a complex neural supply comprising adrenergic, cholinergic and peptidergic innervations. To elucidate the localization of neuropeptides and enzymes in neural elements of the cat pancreatic islets, single or double immunohistochemical staining was performed using avidin-biotin-peroxidase complex and/or peroxidase-antiperoxidase methods. In intrapancreatic ganglia, numerous galanin (Gal)- or vasoactive intestinal polypeptide (VIP) immunoreactive (IR) ganglion cells were seen. Many nitric oxide synthase (NOS)- or cholecystokinin (CCK). IR and a few bombesin (Bom)- or substance-P (SR)-IR ganglion cells were also observed. Intrapancreatic ganglion cells were surrounded by numerous VIP- or SP-IR, many Bom- or neuropeptide Y (NPY)-IR, several calcitonin gene-related peptide (CGRP)- or peptide histidine isoleucine IR and a few NOS- or CCK-IR nerve terminals. In pancreatic islets, many VIP-IR nerve terminals were located predominantly around insulin (Ins)-IR cell masses and many tyrosine hydroxylase-IR nerve terminals were demonstrated in the core of Ins-IR cell masses as well as around them. Gal-, NPY-, SP- or CGRP-IR nerve terminals were found much more frequently within Ins-immunonegative cell areas than in Ins-IR cell masses. These observations may provide a morphological basis for the possible transmitting or modulating role of neuropeptides in pancreatic islets of the cat.

Immunohistochemical studies of glycosaminoglycans in nanophthalmic sclera.

It has been observed that there may be some connection between scleral collagen changes, glycosaminoglycan (GAG) changes and nanophthalmic uveal effusion. In a previous paper we reported increased dermatan sulfate (DS) and chondroitin sulfate (CS) contents in nanophthalmos sclera using biochemical analysis. In the present work we used immunohistochemical methods to assess the GAG changes in nanophthalmos sclera. Scleral specimens from three nanophthalmic patients and five controls were processed to immunostaining with peroxidase-antiperoxidase (PAP) methods. Monoclonal antibodies for chondroitin (Ch), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), DS and keratan sulfate (KS) were used. The specimens were observed under a light microscope. Nanophthalmos scleral samples exhibited strong KS staining compared with the control specimens. Both the nanophthalmos and the control samples were stained with anti-Ch, anti-C4S, anti-C6S and anti-DS antibodies to the same intensity. Our results suggest that an increased or abnormal distribution of KS may contribute to abnormalities in collagen fibers.

Parathyroid Hormone-related Peptide: Immunolocalisation in Normal Salivary Glands and in Pleomorphic Adenomas

Parathyroid hormone-related peptide (PTHrP) has been shown to be produced as an early step in the differentiation sequence and is considered to be a marker of some progenitor cells. We investigated the presence and distribution of PTHrP in 7 normal parotid glands and in 18 salivary pleomorphic adenomas (PA). Localisation of PTHrP was studied by immunohistochemistry with a three-step unlabelled peroxidase-antiperoxidase method. In the normal glands PTHrP is found mainly in the basal and dark cells, and to a lesser extent in the light cells of the ducts. In PA the inner layer of tubulo-ductal structures and all cells of the cyst-like structures show strong positivity for PTHrP. Scattered cells in the dense clusters also stain strongly. Virtually all tumour cells in myxoid and chondroid areas are devoid of staining. In clusters of squamous metaplasia, most cells are slightly positive and scattered cells are stained strongly. PTHrP contributes to cellular differentiation and is also related to keratinisation. We suggest that the PTHrP-positive inner layer cells in pleomorphic adenomas represent a step in the squamous differentiation and in the further elaboration of the tubulo-ductal structures.

Glial fibrillary acidic protein (GFAP) immunochemical profile after Junin virus infection of rat cultured astrocytes

Cultured astrocytes derived from newborn rat brain were inoculated with Junin virus (JV) to characterize their response to infection by means of their glial fibrillary acidic protein (GFAP) immunochemical profile. Samples from 1 to 11 days post-inoculation (pi), as well as matched controls, were serially harvested for GFAP labeling by peroxidase-antiperoxidase (PAP) method. It was only at day 3 that significantly greater values of GFAP staining ( P < 0.05) were disclosed by three complementary approaches: image analysis, ELISA and immunoblot densitometry. Since such increase was abolished by Triton X-100 treatment, soluble GFAP fraction appeared responsible for the early though transient enhancement of GFAP immunoreactivity that followed viral inoculation.

Localization of corticotropin-releasing factor immunoreactivity in the brain of the teleost Sparus aurata

The distribution of perikarya and fibers containing corticotropin-releasing factor (CRF) was studied in the brain of the teleost Sparus aurata by immunocytochemistry using the peroxidase-antiperoxidase method. Antisera against rat CRF, arginine vasotocin, and human adrenocorticotropin (ACTH) were used. Most CRF-immunoreactive neurons were located in the nucleus lateralis tuberis, but they were absent from the nucleus preopticus, which only contained arginine vasotocin neurons. Few CRF perikarya were identified in the nucleus preopticus periventricularis and in the mesencephalic tegmentum. A conspicuous bundle of immunoreactive fibers ran along the diencephalic floor and pituitary stalk to end near the cells of the hypophysial pars intermedia. No CRF was seen near the adenohypophysial rostral pars distalis. Our results suggest that, in Sparus aurata, CRF is a releasing factor for melanotropic cells. Its role as a releasing factor for ACTH is discussed.