Identification and neuroanatomical distribution of immunoreactivity for ...

1 downloads 0 Views 789KB Size Report
for mammalian gonadotropin-releasing hormone (mGnRH) in the brain and neural hypophyseal lobe of the toad Bufo arenarum. Received: 26 January 1998 ...
 Springer-Verlag 1998

Cell Tissue Res (1998) 293:419±425

REGULAR ARTICLE

L.A. Miranda ´ D.A. Paz ´ J.M. Affanni ´ G.M. Somoza

Identification and neuroanatomical distribution of immunoreactivity for mammalian gonadotropin-releasing hormone (mGnRH) in the brain and neural hypophyseal lobe of the toad Bufo arenarum Received: 26 January 1998 / Accepted: 8 April 1998

Abstract The presence and distribution of gonadotropinreleasing hormone (GnRH) in sexually mature specimens of Bufo arenarum was studied by reverse phase/high performance liquid chromatography (RP-HPLC) combined with radioimmunoassay and immunocytochemistry. The analysis of brain extracts with RP-HPLC followed by radioimmunoassay with PBL#45 antiserum showed the presence of only one peak with immunoreactivity for GnRH (ir-GnRH) having the chromatographic and immunological characteristics of mammalian GnRH (mGnRH). This peak was further analyzed with two mGnRH-specific antisera, EL-15 and m1076, yielding serial dilution displacement curves parallel to those obtained with the mGnRH synthetic standard. Immunocytochemical results with the monoclonal antibody LRH13 showed the presence of a terminal nerve-septo-preoptic system with neurons and fibers distributed from the olfactory bulb, septal area, and anterior preoptic area toward the hypothalamus and hypophyseal neural lobe. The main group of ir-GnRH fibers and neurons was identified in the anterior preoptic area. These neurons appear to be the origin of fibers that, after surrounding the preoptic recess, border the dorsal surface of the optic chiasma, extend through the infundibulum, traverse the external layer of the median eminence, and end in the pars nervosa. Key words Gonadotropin-releasing hormone ´ Brain ´ Hypophyseal neural lobe (pars nervosa) ´ Reproduction ´ Bufo arenarum (Anura)

This work was supported by a grant from CONICET (Pict 467 PrØstamo BID 802/OC-AR)

)

L.A. Miranda ( ) ´ D.A. Paz ´ J.M. Affanni ´ G.M. Somoza Instituto de Neurociencia (INEUCI-CONICET). Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón II, Piso 4, (1428) Buenos Aires, Argentina Tel./Fax: +54±1±781±8016; e-mail: [email protected]

Introduction Gonadotropin-releasing hormone (GnRH) is a decapeptide originally identified in ovine and porcine brains because of its ability to stimulate the release of gonadotropins from the pars distalis of the pituitary (Burgus et al. 1972; Matsuo et al. 1971). In vertebrates, GnRH is the main regulator of gonadotropin release from the anterior pituitary lobe. Subsequently, 11 variants have been sequenced in vertebrates and two in protochordates (Powell et al. 1994, 1996; Jimenez-Liæan et al. 1997; Sherwood 1997). In amphibians, three different variants have been identified: mammalian GnRH (mGnRH), chicken GnRH II (cGnRH-II), and a third variant with chromatographic and immunological characteristics similar to those of salmon GnRH (sGnRH) (King and Millar 1986; Sherwood et al. 1986; Cariello et al. 1989; King et al. 1994; Somoza et al. 1996). Other reports have also shown the presence of a post-translational modified variant: hydroxyproline mGnRH (Hyp9mGnRH) (King et al. 1994; Iella et al. 1996). Nevertheless, the functional significance of the different GnRH variants is not known. The neuroanatomical organization of neurons expressing GnRH has recently been reviewed by Muske (1997). Basically, this author has proposed that these neurons are organized into two systems of different embryological origins. One system, the so-called terminal nerve (TN)-septo-preoptic system, originates in the olfactory placode and expresses variants that vary according to species. The most widespread variants are: mGnRH, chicken I GnRH (cGnRH-I), and sGnRH. The other system, named the midbrain system, expresses cGnRH-II and might originate from precursor cells among the ventricular ependyma (Muske 1997). In amphibians, there are few reports on the differential expression of GnRH variants during ontogenetical stages (Crim 1985; King and Millar 1981; D'Aniello et al. 1991, 1995; Di Fiore et al. 1996; Iella et al. 1996). In a previous study, Somoza et al. (1996) have identified only mGnRH in the brain of the metamorphic larvae of Bufo arenarum. These authors have also suggested the presence of an sGnRH-like variant in adult brains.

420

The main objective of this paper has been to identify GnRH variants in sexually mature specimens of Bufo arenarum by reverse phase/high performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA), together with a study by immunocytochemistry (ICC) of their neuroanatomical distribution.

Materials and methods Adult specimens of Bufo arenarum of both sexes were captured in the surroundings of Buenos Aires City during the reproductive season (spring). They were taken to the Instituto de Neurociencia and kept at 25 C for 2 days prior to use. Animals were maintained and killed according to NIH guidelines and following national laws. HPLC and RIAs The toads (7 adult females and 7 adult males) were anesthetized with MS-222 (Sandoz) and killed by decapitation during the morning. Entire brains were immediately removed, frozen on dry ice, and stored at ±70 C. They were extracted, and the extracts were separated on a Lichrospher 100 RP18 (4”250 mm, 5 m, Merck) as previously described (Somoza et al. 1996); 70 fractions were collected. mGnRH, cGnRH-I, cGnRH-II, and sGnRH synthetic standards were chromatographed following tissue-extract chromatography. Fractions were then lyophilized and resuspended in RIA buffer (Somoza et al. 1994) for the radioimmunological detection of GnRH variants. Synthetic mGnRH and sGnRH (Peninsula Laboratories, San Carlos, CA, USA) were iodinated following chloramine-T protocols. RIAs involved the use of the following antibodies. (1) PBL#45 (kindly supplied by Dr. Wylie Vale, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, USA), raised in rabbits against [Lys(8)]-GnRH. This antiserum was used at a final dilution of 1:150 000 with sGnRH as a tracer and standard. It yielded the following cross-reactivities: mGnRH: 100%; cGnRH-I: 133.3%; cGnRH-II: 25.9%; sGnRH: 100%; lamprey GnRH I (lGnRH-I): 24%; dogfish GnRH: 1.58%; other forms were not determined. Fractions immunoreactive for mGnRH (irmGnRH) were pooled and tested by using the following systems. (2) EL-15 (kindly supplied by Dr. W. Ellinwood, Oregon, USA) was raised in rabbits against mGnRH and used at a final dilution of 1:175 000 with mGnRH tracer and standard. This system showed the following cross-reactivities: mGnRH: 100%; cGnRH-I: