localization of gnrh molecular forms in the brain, pituitary, and testis of the frog,rana esculenta

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 274:33-40 (1996)

Localization of GnRH Molecular Forms in the Brain, Pituitary, and Testis of the Frog, Rana esculenta

LOREDANA DI MATTEO, MAURO VALLARINO, AND

RICCARDO PIERANTONI Dipartimento di Fisiologia Umana e Funzioni Biologiche Integrate ''I? Bottazzi," Facoltu di Medicina e Chirurgia, Seconda Universita di Napoli, 80138 Naples (L.D., R.P) and Istituto di Anatomia Comparata, Universita di Genova, 16132 Genoa, Italy (M. I?)

ABSTRACT In the amphibian brain four molecular forms of GnRH have been identified so far: mammalian GnRH (m- and hydro~yproline~m-), chicken I1 GnRH (cII), and a salmon (s) GnRH-like peptide. In Rana esculenta, cII- and s-GnRH-like molecules have been partially characterized in the brain extracts using HPLC combined with radioimmunoassay Moreover, since cII-GnRH-like material has been detected in Rana esculenta testis, the present study describes the localization of the above peptides in the brain and testis of the frog. Immunoreactive cII- GnRH and m-GnRH neurons and fibers were identified in the anterior preoptic area (APOA) and in the median septa1 area (MSA). Apopulation of cells located on the dorsal side of the caudal preoptic region was also stained.

Immunopositive fibers were seen to overlap the median eminence before ending within the pars nervosa. Moreover, densely packed fibers made close contact with the vascular complex in the median eminence. Conversely, immunoreactive s-GnRH-like material was absent in APOA and MSA, but weakly scattered elements were detected by the anti-s-GnRH serum in the dorsal side of the caudal preoptic region. Using m-GnRH antiserum, a strong immunopositivity was observed in the median eminence but not within the pars nervosa, indicating that, besides cII-GnRH and s- GnRH-like material, also m-GnRH-like material is present in Rana esculenta brain. In the testis, cells of the interstitial and germinal compartment were detected by anti-cII-GnRH during different periods of the annual cycle. In particular, in October and February interstitial tissue was intensely stained, coinciding with periods of increased androgen production and the onset of the new spermatogenic wave, respectively. o 1996 Wiley-Liss, Inc

In vertebrates, the presence of peptides with gonadotropin releasing activity (GnRH) in hypothalamic and extrahypothalamic areas has been demonstrated. Some peptides (mammalian, m-; chicken I, cI-: chicken 11, cII; salmon, s-; catfish, cf; dogfish, df; lamprey, 11, and lamprey 111, 1111) have been characterized by amino acid sequence, but the list of peptides is still growing (Gautron et al., '91; &ox et al., '94; and references therein). Early studies focused primarily on mammals, with recent attempts to elucidate the localization and the activity of GnRH in nonmammalian species (see Chieffi et al., '91; King and Millar, '91, Pierantoni and Fasano, '91, and references therein). Several observations used heterologous antisera, but only recently data have been obtained working with the appropriate homologous antibod- ies (see Chieffi et al., '91). In this respect bird stud-

0 1996 WILEY-LISS, INC.

ies are in progress to determine the role of cI- and cII-GnRH (Mikami et al., '88; Wilson et al., '89; van Gils et al., '92). In the African catfish brain (Sher- wood et al., '89; Ngamvongchon et al., '92) catfish I and cII-GnRH have been observed, and in goldfish brain two GnRH forms, s-GnRH and cII-GnRH, have been shown to have differential distribution (Yu et al., '88).

The anti-mammalian GnRH has been used to localize GnRH immunoreactivity in brain and extrabrain areas of several amphibian species: Bufo vulgaris (Doerr-Schott and Dubois, '75), Rana esculenta (Goos et al., '76; Fasano et al., '88;

Received November 29. 1993; revision accepted July 17. 1996. Address reprint requests t o Loredana Di Matteo, Dipartimento

di Fisiologia Umana e Funzioni Biologiche Integrate "F. Bottazzi," Facolta di Medicina e Chirurgia, Seconda Universita di Napoli, 80138 Naples, Italy.

34 L. DI MATTE0 ET AL.

Rastogi et al., '901, Xenopus laevis (Doerr-Schott and Dubois, '76; Nozaki and Kobayashi, '79), Rana pipiens (Alpert et al., ,761, Rana catesbeiana (Crim, '851, and Rana ridibunda (Andersen et al., '88). For brain localization, in Rana esculenta anti-cI, anti-cII, and anti-s-GnRH have also been used (D'Aniello et al., '91; di Meglio et al., '91).

High pressure liquid chromatography (HPLC) coupled with radioimmunoassay has shown that in amphibian brain, m-, hydro~yproline~m-, cII-, and a s-GnRH-like peptide are present in vari- ous combinations (Peter, '86; Sherwood et al., '86; Chieffi et al., '91; King and Millar, '91, Conlon et al., '93). In particular, a s-GnRH-like material has been evidenced in Hyla regilla (Sherwood et al., '86) and Xenopus laevis (King et al., '94) brain. In Rana esculenta, cII- and also a s-GnRH-like peptide have been evidenced in brain extracts showing seasonal fluctuations (Cariello et al., '89; Fasano et al., '93). Recently, absence of s-GnRH from Rana esculenta brain has been claimed (Licht et al., '94), opening the possibility that the s-GnRH immunoreactivity previously observed in amphibians (for re- view see King and Millar, '91) is unlikely due to a peptide identical to s-GnRH as suggested by King and coworkers ('94).

Besides brain hypothalamic and extrahypothalamic areas, GnRH immunoreactivity has been found also in sympathetic ganglia and adrenal gland of bullfrog (Eiden et al., ,821, and a cII- GnRH-like material has been detected in the testis of Rana esculenta (Cariello et al., '89).

Therefore, the objective of this study in Rana escuZenta is to localize the forms of GnRH present in the brain, using anti-m-, -cII-, and -s-GnRH. The s-GnRH antiserum has been used with the aim to localize the putative third not yet characterized peptide, similar to s-GnRH, present in the amphibian brain. Furthermore, also in the testis we attempt to localize GnRH-like peptides. The observations are extended to the annual reproductive cycle to determine changes in the distribution pattern.

MATERIALS AND METHODS Intact male frogs, Rana esculenta, were collected

monthly (n = 5 ) during the year. The frogs were stunned, and the brains and the testes were rap- idly dissected and fixed in Bouin's fluid for 24 h at room temperature. The tissues were dehydrated in ethanol, cleared in xylene, and embedded in paraffin. The brains were cut in transverse or sagittal planes at 6 pm and mounted on glass slides coated with chromalum-gelatin. The sections were

processed by indirect immunofluorescence technique using rabbit antisera raised against m-GnRH (Sigma, St. Louis, MO), cII-GnRH, and s-GnRH (Prof. H.J.T. GOOS, University of Utrecht, The Netherlands). Sections were rinsed in 0.05 M phosphate buffer saline (PBS), pH 7.4, and prein- cubated for 20 min in normal goat serum (Dako- patts, Copenhagen, DK) at a dilution 1:50. Thereafter, incubation of adjacent sections was performed at 4°C for 18 h in a moist chamber with GnRH antisera diluted 1:400 and 1:800, respectively, in PBS containing 0.3% Triton X-100 and 1% human serum albumin. The sections were thoroughly rinsed in PBS and incubated for 1 h at room temperature in fluorescein isothiocyanate- conjugated swine anti-rabbit gamma-globulins (Dakopatts, DK) diluted 1 : l O O in PBS. Finally, the sections were rinsed three times in PBS and mounted in a glycerol-PBS (1: 1) solution.

All preparations were examined on a Zeiss epifluorescence microscope. Some sections were counterstained with cresyl violet to localize neuro- anatomically the immunoreactive cells.

Testes were processed by the peroxidase-antiperoxidase technique (PAP). Rehydrated sections were treated for 30 min with (1) HzOz to block endogenous peroxidase, (2) with normal goat serum to reduce nonspecific background and then (3) incubated with the antisera used for brain sections, diluted 1:300 and 1500 at 4" C for 18 h. Afkr washing in PBS, the sections were incubated for 1 h at room temperature with mouse antiserum to rabbit immunoglobulins (Dakopatts, DK) diluted 1 : l O O and subsequently incubated with rabbit peroxidase-antiperoxidase (PAP) complex (Dakopatts, DK). The antigens were visualized using 3,3'- diaminobenzidine tetrahydrochloride (DAB) (Sigma) and 0.3% hydrogen peroxide in PBS solution.

In order to check the specificity of the immuno- reactions, controls were treated omitting one step of the reactions or by preabsorbing the primary antisera with synthetic homologous peptides ( M) or thyroglobulin, the large protein that was conjugated with cII-GnRH. Additional controls were performed using the primary antisera preabsorbed with heterologous GnRH peptides (Dr. J.A. King and Dr. R.C. deLisle Milton, University of Cape Tbwn, South Africa) (lo-' M). No immunostaining was observed using antisera preabsorbed with homologous GnRH, while the reaction was unaffected when the primary antisera were preabsorbed with heterologous peptides or thyroglobulin. In some experiments the primary antisera were replaced by nonimmunized rabbit

GNRH MOLECULAR FORMS IN RANA ESCULENTA 35

serum or PBS. In this case the immunoreaction was absent.

Anti-cII-GnRH cross-reacts < 1% with s-GnRH, while anti-s-GnRH cross-reacts 5% with cII-GnRH (Zandbergen et al., '92; Fasano et al., '93); no cross- reactivity was evidenced with m-GnRH. Anti-m- GnRH cross-reacts < 1% with s- or cII-GnRH.

RESULTS Brain and pituitary immunocytochemistry A map of cell bodies and fibers immunostained

by anti-m-, -cII-, and -s-GnRH in the brain and pituitary of the frog is given in Figure 1. Differ- ences were not observed when different antiserum dilutions were used. Cells and fibers were found mainly in the forebrain of all specimens examined. The immunofluorescence appeared to be less bright in structures of summer animals, al- though the distribution of the immunoreactivity did not differ from that found in winter animals. Differences in the distribution and intensity of the immunoreaction were evaluated in adjacent sections treated with the different antisera. Using m-GnRH antiserum, the highest number of positive neurons was identified in the anterior preoptic nucleus. These neurons were generally bipolar or pseudounipolar, densely packed medially and in the ventral region (Fig. 2). As second group of positive cells was observed in the medial septal area

(MSA) (Fig. 3). Both the APOA and MSA regions contained numerous immunofluorescent fibers. Rostra1 to these regions, some fibers were seen coursing the basal telencephalon and the lateral regions of the telencephalic ventricles. A great number of fibers were found within the lateral preoptic area in front of the optic chiasma. A strong immunopositivity was observed in the median eminence (ME) (Fig. 4). Immunoreactive fibers were also detected within the penventricular region and the thalamus and through the central gray of the mesencephalon.

Using the cII-GnRH antiserum, the distribution of the immunoreactivity in the APOA and MSA was very similar to that reported with m-GnRH antiserum (Fig. 5 j. In addition, the cII-GnRH antiserum stained a population of scattered cells located in the dorsal side of the caudal preoptic region (Fig. 6). These cells, mainly located medially, were round in shape and showed a thin immunofluorescent cytoplasmic rim. The pattern of the immunoreactive fibers was very similar to that reported above for m-GnRH. At the pituitary level most immunopositive fibers were seen in the ME before ending within the pars nervosa (Fig. 8). The s-GnRH antiserum did not react with cells located in the APOA and MSA regions but weakly stained only scattered elements located in the dorsal side of the caudal preoptic region (Fig. 7),

Tec M

A d A A A A A A & A A

APOA Fig. 1. Schematic parasagittal section through the frog brain

showing the distribution of GnRH-like immunoreactivities. As- GnRH immunoreactive perikarya, As-GnRH immunoreactive fibers; OcII-GnRH immunoreactive perikarya, 0 cII-GnRH immunoreactive fibers; A m-GnRH immunoreactive perikarya, am-GnRH immunoreactive fibers. APOA, anterior preoptic

area; C, cerebellum; dPR, dorsal preoptic region; I, infundibular region; LV, lateral ventricle; ME, median eminence; MSA, medial septal area; OC, optic chiasma; Ppd, pituitary pars distalis; Ppn, pituitary pars nervosa; T, telencephalon; QcM, tectum mesencephali; Th, thalamus.

Fig. 2. m-GnRH immunoreactive perikarya and fibers within the anterior preoptic area (APOA) of Rana esculenta. Sagittal section. x260.

Fig. 3. Sagittal section through the medial septa1 area (MSA) or Rana esculenta showing the presence of m-GnRH immunoreactive perikarya and fibers (arrows). x260.

m-GnRH immunoreactivity in the median eminence (ME) of Rana esculenta. Sagittal section. x125.

cII-GnRH immunoreactive perikarya and fibers in the anterior preoptic area (APOA) of Rana esculenta. Sagit- tal section (arrows). x260.

Fig. 6. cII-GnRH immunoreactive perikarya (arrows) in the dorsal side of the caudal preoptic region. Transverse section. x260.

Fig. 7. s-GnRH immunoreactive perikarya in the dorsal side of the caudal preoptic region. Ransverse section. x260.

Fig. 8. cII-GnRH immunoreactivity in the infundibular region (I), median eminence (ME), and pars nervosa (PN) of the pituitary. Sagittal section. x125.

Fig. 4.

Fig. 5 .


which also contained cII-GnRH immunoreactive elements. Immunoreactive fibers were found mainly through the basal and caudal infundibu- lum, entering the pituitary stalk, and, as for cII- GnRH (Fig. 81, the fibers ended both in the ME and in the pars nervosa.

!&stis immunocytochemistry Immunopositive GnRH cells were not seen us-

ing anti-m- or anti-s-GnRH sera. Immunopositive cII-GnRH cells were present in the interstitial compartment. The majority of these cells were ir- regular or round in shape (Leydig cells). However, elongated immunopositive cells, difficult to iden- tify, were also found in the interstitial compartment. The intensity of the reaction changed during different periods of the annual cycle, showing strong staining in February-March and October -November (Fig, 9), while the staining intensity decreased in the remaining periods of the year (Fig. 10). Within the seminiferous tubules, primary spermatogonia and spermatids showed strong reaction in some tubules. In particular, immunopositive cysts of round and elongated spermatids were found in April and November (Fig. 11). The Sertoli cells were negative. Reaction was not observed in tissue that had been incubated with the absorbed antiserum, normal serum, or buffered saline (Fig. 12).

DISCUSSION The present investigation shows that GnRH im-

munoreactivity is detectable in the brain and testis of Rand esculenta. In general the distribution seen using anti-cII- and -s-GnRH sera in the brain confirms other data reported for frogs using anti- m-GnRH (Goos et al., '76; Chieffi et al., '91). How- ever, the use of different antisera suggests that the immunoreactivity is localized in different brain areas, and this finding contrasts with the distribution of GnRH previously described (Rastogi et al., '90; D'Aniello et al., '91). The above quoted studies were carried out using anti-m-, anti-cI-, anti-cII-, or anti-s-GnRH, and different distribution of immunoreactivity in the brain of juvenile or adult frogs was not detected (D'Aniello et al., '91). Positive reaction was evidenced in perikarya in the olfactory bulb, MSA, AF'OA, periventricular areas of the thalamus, and interpenduncular nucleus and tegmentum area. In amphibians cI- GnRH-like material has not been isolated from brain extracts using HPLC purification (King et al., '94, and references therein); therefore, the use of anti-cI-GnRH in those species in which this pep-

tide is absent may be misleading, and the over- lapping of immunoreactive areas evidenced with homologous and heterologous antisera may suggest a high degree of cross-reactivity in the con- ditions used for immunocytochemical reactions. Our present results obtained using cII-GnRH antiserum show immunoreactivity in the APOA and MSA. This result is similar to that shown in Rana ridibunda (Conlon et al., '93). Immunopositive s- GnRH elements are only localized in the dorso- caudal preoptic region; the same anatomical areas also contain cII-GnRH cells. The absence of immunoreactivity in the APOA and MSA and the presence of positive elements in the dorso- caudal preoptic region strongly suggest that in this area s-GnRH antiserum does not cross react with cII-GnRH, but it detects a GnRH form with immunological properties of s-GnRH. In this respect, it is necessary to remember that an unidentified molecular form with structural chromatographic and immunological features in common with s- GnRH has been found in Rana esculenta and other amphibian species (Cariello et al., '89; King et al., '94, and references therein). Licht and coworkers ('94) were unable to find GnRH immunoreactivity in three species of ranid frogs, including Runa esculenta. In Xenopus laevis a peptide with similar HPLC, immunological, and biological properties to s-GnRH was identified, but this peptide is unlikely identical to s-GnRH (King et al., '94, and references therein). Therefore, our results (Car- iello et al., '89; Fasano et al,, '93; present data) may be explained by the presence of a GnRH molecular form similar to the s-GnRH peptide (as reported by King et al., '94) not detectable by the antiserum used by Licht et al. ('94). In general, anti-m-GnRH detects immunoreactivity in the same areas stained by anti-cII-GnRH. Anti-m- GnRH also detects fibers confined t o the ME, whereas anti-cII-GnRH stains both the ME (with less intensity) and pars nervosa of the pituitary. The strong immunoreactivity detected in the ME by anti-m-GnRH serum may be ascribed to a mammalian GnRH-like form not previously evidenced (Cariello et al., '89). In this respect, it is important to consider that in amphibian, rat, and human hypothalamus a new GnRH form (hydrox- yprolineg-GnRH) has been recently characterized (Gautron et al., '91; King et al., '94). Moreover, m- GnRH has been isolated from ranid species (Conlon et al., '93; Licht et al., '94) and sequenced in Rana ridibunda (Conlon et al., '93), which is closely related to Rana esculenta.

Quantitative changes in hypothalamic and/or

38 L. DI MATTE0 ET AL.

Fig. 9. Immunohistochemical localization of cII-GnRH in Fig. 11. Positive immunostaining, obtained using cII- GnRH antiserum, in round and elongated spermatids (arrow- heads) in April testis. x500.

Control testis, in which the primary antiserum was replaced by PBS, showing absence of immuno-

February testis. The interstitial compartment shows numerous immunopositive cells (arrows). x325.

Fig. 10. Immunoreactive cII-GnRH in the interstitium of November testis. Note that few immunopositive cells are present (arrows). x325. reaction. x250.

Fig. 12.


extrahypothalamic m-GnRH content in relation to seasonal reproductive cycle have been described in a few species (King and Millar, '79; Jokura and Urano, '85; Zoeller and Moore, '85; King and Millar, '79; Fasano et al., '88). No seasonal changes have been documented in the topography of ir- GnRH cell bodies and fibers of Rana esculenta (Rastogi et al., 'go), and similar results have been obtained by radioimmunoassay from seasonally grouped frogs (Fasano et al., '88). In the present study we find a weaker immunofluorescence in animals collected in summer, but the regional distribution of the immunoreactivity did not change compared to that found in November animals. The intensity of the immunostaining coincides with data obtained using radioimmunoassay showing changes of GnRH concentration in monthly collected animals (Fasano et al., '93). Moreover, the immunofluorescence was bright within the fibers and terminals and relatively weaker in the perikarya. In this respect, it should be considered that a maturational process in the fibers from a larger molecule present in the cell bodies (Zoeller and Moore, '85) has been supposed also in amphibians (Andersen et al., '88). Whether or not different peptides localized in the same areas are located in the same neuron is difficult to ascertain in absence of double-labeling pro- cedures.

As for the distribution of cII-GnRH-like material in testes, strong staining of the interstitial compartment in February-March and October- November appears concomitantly with the in- crease of androgen production and the onset of the new spermatogenic wave. Groups of interstitial cells, probably Leydig cells, are positive to cII- GnRH antiserum. Immunostaining localization and fluctuations are in accordance with previous data obtained using an anti-m-GnRH (Fasano et al., '88). Its activity, however, was not confirmed in further studies due to the high rate of nonspecific staining (Chieffi et al., '91).

As for positivity of primary spermatogonia, we have no current explanation, but it is no- table that in rat testis such a positivity has also been observed in nuclei (Paul1 et al., '81). More- over, the functional significance of the presence of immunopositive spermatids in two different periods of the reproductive cycle should be in- vestigated.

In conclusion, present results demonstrate a differential localization of GnRH molecular forms in the Rana esculenta brain, suggesting that they play a different role at central level. At testicular level, our results further support previous data

(Chieffi et al., '91; DAntonio et al., '92) on the possible modulation of gonadal activity by a GnRH-like material of local origin.

ACKNOWLEDGMENTS We thank Prof. H.J.T. Goos for the generous gift

of GnRH antisera, Dr. J.A. King and Dr. R.C. deLisle Milton for the generous gift of peptides, and Prof. A. Fasolo for his criticism. This work was supported by grants from CNR and MURST.

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localization of gnrh molecular forms in the brain, pituitary, and testis of the frog,rana esculenta

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