2006-Mongiat-RegulatoryPeptides.pdf

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Experimental data supporting the expression of the highly conserved GnRH-II in the brain and pituitary gland of rats

L.A. Mongiat

^a

, M.O. Fernández

^a,c

, V.A.R. Lux-Lantos

^a

, L.G. Guilgur

^b

, G.M. Somoza

^b

, C. Libertun

^a,c,

^⁎

aInstituto de Biología y Medicina Experimental, CONICET, Vuelta de Obligado 2490 (C1428ADN), Ciudad Autónoma de Buenos Aires, Argentina

bInstituto de Investigaciones Biotecnologicas, Instituto Tecnológico Chascomús, CONICET, Universidad de San Martín, Camino de Circunvalación Laguna Km. 6 (B7130IWA), Chascomús, Pcia, Buenos Aires, Argentina

cFacultad de Medicina, Universidad de Buenos Aires, Viamonte 444, Buenos Aires, Argentina Received 24 November 2005; received in revised form 12 April 2006; accepted 28 April 2006

Available online 30 June 2006

Abstract

The second GnRH form, originally identified in chickens (cGnRH-II or GnRH-II), is the most ubiquitous peptide of the GnRH neuropeptide family, being present from jawed fish to human beings. However, the presence of GnRH-II in such an important experimental model as the rat is still an object of discussion. Here we present chromatographic, immunologic and biologic activity evidence supporting the expression of GnRH-II in the rat.

Olfactory bulb, hypothalamus, remnant brain and anterior pituitary from a pool of 50 female adult rats were extracted and subjected to RP-HPLC on a C-18 column. The fractions were collected and evaluated by using two different RIA systems, specific for GnRH-I and GnRH-II respectively.

Under these conditions the GnRH-I standard eluted in fraction 21 (f21) was only detected with the GnRH-I RIA system, whereas the GnRH-II standard was only detected in the fraction 27 (f27) by using a GnRH-II RIA system. In the olfactory bulbs extract, the fractions analyzed by the GnRH-I RIA systems showed a single peak in f21, whereas by using the GnRH-II RIA system a single peak at f27 was observed. In the hypothalamus GnRH-I was detected in f21 meanwhile GnRH-II could not be detected. When the remnant brain and pituitary gland extracts were analyzed, both GnRH forms were detected. To the best of our knowledge, this is the first report concerning GnRH-II detection in a mammalian pituitary.

Serial dilutions of f27 and GnRH-II presented similar displacement of radioiodinated-GnRH-II, demonstrating that both molecules share immunological properties. Moreover, after 60 min stimulation, both f27 and GnRH-II had similar LH and FSH releasing activity in 12 day-old rat pituitary primary cell cultures.

However, we failed to characterize the GnRH-II gene in this model.

These results provide strong evidence for the expression of GnRH-II in the rat brain and pituitary gland.

Keywords:GnRH; GnRH-II; Pituitary; Rat

1. Introduction

Gonadotropin-releasing hormone-I (GnRH-I, mGnRH, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), originally isolated from the mammalian hypothalamus, plays a central role in the development and maintenance of reproductive physiology in vertebrates[1,2]. This peptide is synthesized by

preoptic-hypothalamic neurons and upon release into the portal vessels it reaches the pituitary gland to induce the synthesis and secretion of gonadotropic hormones, which in turn regulate gonadal function.

At present, a total of 24 GnRH variants have been isolated and characterized from vertebrate and protochordate nervous tissue;

all these molecules are decapeptides with a highly conserved structure[3,4].

In addition to this molecular diversity, most vertebrate species express more than one GnRH form. This multiplicity of GnRH variants has been demonstrated in all seven classes of vertebrates[5]. The second molecular form of GnRH originally

⁎ Corresponding author. Instituto de Biología y Medicina Experimental, CONICET, Vuelta de Obligado 2490 (C1428ADN), Ciudad Autónoma de Buenos Aires, Argentina. Tel.: +54 11 4783 2869x225; fax: +54 11 47862564.

E-mail address:[email protected](C. Libertun).

doi:10.1016/j.regpep.2006.04.012

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reproductive functions. GnRH-I varies across vertebrate classes, it is localized mainly in the preoptic area and is responsible for the central regulation of gonadotropin secretion by the pituitary, whereas GnRH-II is localized in extrahypothalamic brain areas, mainly the midbrain, and has been related to a range of reproductive-related functions[6]. Thus GnRH-II is an ancient GnRH variant and is structurally conserved for over 500 million years of evolution, suggesting that its neuronal functions are of utmost importance. In the musk shrew, mice and marmoset monkeys, GnRH-II acts as a permissive regulator of female reproductive behavior based on energy status, as well as a modifier of short-term food intake[6–9].

The expression of GnRH-II was demonstrated not only in the central nervous system, but also in several different peripheral tissues as well as kidney, pituitary, ovary and testes amongst others[10].

The presence of GnRH-II was suggested by indirect evidence in a diverse set of mammals[5]. Moreover, the confirmation of GnRH-II presence by gene and/or protein sequences was only obtained in a few mammals such asSuncus murinus, (Insectivora, AF107315)[11];Tupaia glis, (Scandentia, U63327)[12],Tricho- surus vulpecula (Diptrodontia, AF193516), Macaca mulatta (Primates, AF097356) [13] and Homo sapiens (Primates, AF036329)[10].

Since the laboratory rat,Rattus norvegicushas been exten- sively proposed as an endocrinological, physiological, behav- ioral and reproductive model, and because the participation of GnRH-II in all these topics has been established in different animal models, we emphasize the relevance in elucidating whether GnRH-II is present in this animal.

Here, by using reverse phase-high performance liquid chromatography (RP-HPLC), radioimmunoassay (RIA) and biological activity tests we provide strong evidence supporting the presence of GnRH-II in the rat brain and pituitary gland.

2. Methods 2.1. Animals

Fifty Sprague–Dawley female rats from the Instituto de Biología y Medicina Experimental (IBYME) colony were used.

The animals were housed in groups in an air-conditioned room, with lights on from 0700 to 1900 h. They were given free access to laboratory chow and tap water. The animals were sacrificed by decapitation according to protocols for animal use, approved by the Institutional Animal Care and Use Committee (IBYME–

CONICET) following the NIH guidelines (Guide for Care and Use of Laboratory Animals). Olfactory bulbs and hypothalamic areas (limited by the optic chiasma, laterally by the hypothalamic fissures, caudally by the mammillary bodies and in depth by the subthalamic sulcus, and including the preoptic-supra-

Frozen rat tissues were powdered in liquid nitrogen and peptide extraction was performed as it was previously described by Montaner et al. [14]. Briefly, powdered tissues were homogenized in acetone:1 N HCl (100:3, v/v) at 4 °C, the mixtures were stirred at 4 °C for 3 h and filtered. The insoluble material was re-extracted in acetone:0.01 N HCl (in 40% of the original volume), stirred for 5 min and filtered. The combined filtrates were treated with petroleum ether (bp 30–60 °C) for five successive times. The extracts were lyophilized and solubilized in 2 ml 0.05 M phosphate buffer.

2.3. Reverse phase-high pressure liquid chromatography (RP-HPLC)

The RP-HPLC protocol was performed as previously described[15]. Briefly, tissue extracts were filtered and injected onto a Supelco (Supelcosil LC-18) analytical column using a Beckman 166 model liquid chromatograph. The samples were applied at the beginning of a 10 min isocratic period of 17%

ACN in 0.25 M triethylammonium formate TEAF (pH 6.5);

ACN was then increased to 24% over a 7 min period and held isocratically for 43 more min. The flow rate was kept at 1 ml/

min and 1 ml fractions were collected. Aliquots of 100μl from each fraction were used for RIA. Before each tissue extract chromatography a blank running was performed and the column was washed successively several times.

Standards of GnRH-I and GnRH-II were injected to evaluate the elution position of each peptide. Both standards were obtained from Peninsula Laboratories Inc. (Belmont, Calif., USA).

2.4. GnRH RIA

The GnRH RIA protocols were performed as already published[14,15]. To do this study two different RIA systems,

Table 1

Primer names and sequences used to amplify genomic sequences for a GnRH-II like gene in the rat

Primers designed for mammalians GnRH-II Primer

name

Sequence direction Position (human

GnRH-II gene) Fo-cII1 5′gct Sct gMt gct RMt gRc Wgc 3′ Forward 2131–2151 Re-cII1 5′gYc Rtg gtc Ytg ScY tcc YMg g 3′ Reverse 2479–2459 mamc-II1 5′cag cac tgg tcc cat ggc 3′ Forward 2174–2191 mamc-II2 5′cat ggc tgg taY cct gga gg 3′ Forward 2186–2205 mamc-II3 5′tgg tcY tgc cct ccY Mgg 3′ Reverse 2475–2459 mamc-II4 5′ctt ggg agg cYa tgR gYa gYc tgg 3′ Reverse 2426–2403 ratS26 5′cga ggg aaa ttg cac gag gcc cga 3′ Reverse 4104–4081 The last column shows the relative positions of these sequences in the human GnRH-II gene sequence (AF036329).

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specific for GnRH-I and for GnRH-II were set up. GnRH-I and GnRH-II standard radioiodination was performed following the cloramine-T method[16]. The RIA systems used here were as follows:

(a) GnRH-I:HU-60 antiserum against GnRH-I was kindly supplied by Dr. Henrik Urbanski[17]. This antiserum was used at 1:100,000 final dilution using synthetic GnRH-I as tracer and standard.

(b) GnRH-II:acII6 antiserum against GnRH-II was a gift from Dr. Kiochi Okuzawa[18], we used this antiserum at 1:100,000 final dilution with synthetic GnRH-II as tracer and standard.

2.5. Adenohypophyseal cells monolayer culture

Anterior pituitary cells were obtained from a 12 day-old female rat as already described[14]. Pituitaries from 12 day-old females were rapidly removed and placed in freshly prepared Krebs-Ringer bicarbonate buffer without Ca²⁺ and Mg²⁺. Pituitaries were cut into small pieces and incubated in 0.2%

trypsin for 30 min. After addition of DNase and lima bean trypsin inhibitor, the fragments were dispersed gently into individual cells and filtered through Nytex mesh. Pituitary cells were plated in 96-well plates (50,000 cells/well) with Dulbec- co's modified Eagle medium, supplemented with 10% horse serum, 2.5% fetal calf serum, 1% MEM nonessential amino acids, fungizone and gentamicin. After 4 days in culture, the cells were washed twice with serum-free DMEM-F12 medium containing 2.2 g/l NaCO3H and 0.1% BSA and then stimulated for 1 h. At the end of the incubation period, the media were removed and stored at−20 °C until LH and FSH were analyzed by RIA after appropriate dilution with 0.01 M phosphate- buffered saline containing 1% egg albumin. Cell cultures were repeated three times, and each experimental group was done in quadruplicate. Results were analyzed by repeated measures ANOVA.

2.6. LH and FSH RIA

LH and FSH were determined in media samples by RIA using kits provided by the NIDDK. Results were expressed in terms of RP3 rat LH and RP2 rat FSH standards. Assay sensitivities were 0.15 ng/ml for LH and 0.6 ng/ml for FSH.

Intra- and interassay coefficients of variation for LH were 7.2 and 11.4% and for FSH 8.0 and 13.2%.

2.7. Search strategy for the GnRH-II gene

Total RNA was prepared from rat midbrain by the GITC phenol-chloroform method [19] using the TRIZOL reagent (GIBCO BRL). Total RNAs were reverse transcribed into

Fig. 2. Characterization of GnRH-I and GnRH-II RIA systems. a) GnRH-I RIA:

The plot shows the mean of three independent displacement curves for each standard peptide, black circles: GnRH-I, gray circles GnRH-II. The D50 values in pg/100μl were 16.0 ± 3.8 for GnRH-I and 288.8 ± 21.3 for GnRH-II. b) GnRH-II RIA: mean values of three independent displacement curves for each standard peptide, black circles: GnRH-I, gray circles GnRH-II. The D50 values in pg/

100μl were 18.7 ± 0.3 for GnRH-II and for GnRH-I there was no displacement observed at the assayed doses.

Fig. 1. Representation of the GnRH-II gene. This figure shows a model of the GnRH-II human gene comprising the 5′UTR, the three GnRH-II exons, their respective introns and the 3′UTR comprising the S26 ribosomal protein. Black arrows correspond to the forward primers, whereas the gray arrows indicate the reverse primers and their positions (not in scale) overlapping the human GnRH-II gene.

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tion in lysis buffer (100 mM Tris–HCl pH 8.5; 5 mM EDTA pH 8; 200 mM NaCl; 0.2% SDS). Samples were treated with proteinase-K (100 μg/μl, 2 h at 55 °C) and centrifuged.

Supernatants were recovered and 1 vol of phenol–chloroform–

isoamilic alcohol was added, mixed and then 1 vol of TE was added. Aqueous phases were separated after centrifugation and precipitated with 1 vol of 100% EtOH (30 min at −70 °C).

Pellets were obtained by centrifugation at 12,000 rpm, washed with EtOH 70%, allowed to dry and re-suspended in 100μl TE.

Mammalian GnRH-II cDNA sequences were obtained from the GenBank database: H. sapiens (AF036329), M. mulatta (AF097356), S. murinus (AF107315) and T. glis (U63327).

These sequences were aligned by using the Clustal-W algorithm and nested primers for PCR were designed based on the most conserved regions (Table 1,Fig. 1).

Each 25μl PCR reaction contained 2.5 U Taq polymerase (Invitrogen), 2.5 μl buffer, 2.5 mM MgCl2, 0.2 mM deox- ynucleoside triphosphates and 20 pmol of each 5′forward and 3′reverse primer. PCR conditions were: 35 cycles, 94 °C for 30 s, annealing temperatures ranged from 50 to 58 °C (depending on primer pairs) for 30 s and 72 °C for 30 s. The PCR products were visualized on a 1.8% agarose gel, stained with ethidium bromide. Selected bands were isolated (QIA-

GnRH-I and GnRH-II according to their hydrophobic characteristics as already published[14,15]. The elution position for each GnRH synthetic standard after chromatography was in fraction 21 for GnRH-I and in fraction 27 for GnRH-II. The eluted fractions were tested by two different GnRH RIA systems, specific for GnRH-I and GnRH-II respectively. We characterized both RIA systems according to their cross- reactivity against the other GnRH variant. The upper panel of Fig. 2shows the displacement curves for GnRH-I and GnRH-II in the GnRH-I RIA system. The D50values were 16.0 ± 3.8 pg/

100μl for GnRH-I and 288 ± 21.3 pg/100μl for GnRH-II (n= 3, pb0.001). Whereas, the lower panel ofFig. 2shows the cross- reactivity of both GnRH peptides in the GnRH-II RIA system.

Dose-50 value for GnRH-II was 18.7 ± 0.34 pg/100 μl (n= 3), whereas GnRH-I was not able to displace the radiolabeled GnRH-II at any of the assayed doses.

Olfactory bulb extracts were subjected to RP-HPLC on C-18 columns, eluted and assayed by the RIA systems. The GnRH-I RIA showed a single immunoreactive (ir) peak which eluted in the same position the GnRH-I standard did, fraction 21 (Fig. 3a, upper panel). When the same samples were analyzed with the GnRH-II RIA, a major peak in the fraction 27 was observed, corresponding to the elution position of the GnRH-II standard

Fig. 3. a) Olfactory bulb RP-HPLC eluted fractions were subjected to GnRH-I RIA system (upper panel) and GnRH-II RIA system (lower panel). b) Hypothalamus RP-HPLC eluted fractions were subjected to GnRH-I RIA system (upper panel) and GnRH-II RIA system (lower panel). The arrows represent the elution position for the GnRH-I (fraction 21) and GnRH-II standards (fraction 27).

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(Fig. 3a, lower panel). Hypothalamic extracts were also analyzed (Fig. 3b) and ir-GnRH-I was detected in fraction 21 (upper panel). Moreover a minor ir-GnRH peak was observed in fractions 15–16, corresponding to the post-translationally modified [Hyp⁹]GnRH-I as it was previously demonstrated by Montaner et al.[14]. However the GnRH-II RIA analysis was unable to detect any GnRH-II immunoreactive material in these hypothalamic extracts (Fig. 3b, lower panel).

After analyzing the remnant brain extract, two ir-GnRH peaks, corresponding to GnRH-I and GnRH-II elution position, were detected by the respective RIA systems (Fig. 4a).

The presence of ir-GnRH was also evaluated using the same approach in rat pituitary extracts. Once again, two ir-GnRH peaks were found after screening with the two homologous radioimmunoassays (Fig. 4b).

In addition, the immunological properties of the ir-GnRH-II fraction (f27) were tested in displacement experiments in comparison to GnRH-II synthetic standard. No differences were found, suggesting that the molecule contained in this fraction possesses a highly similar or identical epitope for this antibody to GnRH-II (Fig. 5).

Using an in vitro pituitary cell static system, after 60 min stimulation, both f27 and GnRH-II synthetic standard showed LH and FSH releasing activity in 12 day-old rat pituitary primary cell cultures. A fraction with no ir-GnRH (f35) showed no effects on gonadotropin release (Fig. 6).

Finally, we decided to look for a partial GnRH-II gene sequence from rat midbrain cDNA. After PCR amplification (primers Fo-cII1 and Re-cII1), we obtained a single band

ranging 280 bp. The expected size of this PCR product based on the mammalian aligned sequences was approximately 220–

245 bp. We purified the amplified band and cloned it to a pGEM Vector-T for further sequencing. Unfortunately, the sequence obtained did not correspond to GnRH-II.

Next, we designed new primer pairs for nested PCRs and used rat genomic DNA as template, to avoid difficulties regarding putative GnRH-II expression patterns.

First, we amplified genomic DNA by using the forward primer mamc-II2 with the outer reverse ratS26. This amplification

Fig. 4. a) Remnant brain (without olfactory bulb and hypothalamus) RP-HPLC eluted fractions were subjected to GnRH-I RIA system (upper panel) and GnRH-II RIA system (lower panel). b) Pituitary RP-HPLC eluted fractions were subjected to GnRH-I RIA system (upper panel) and GnRH-II RIA system (lower panel). The arrows represent the elution position for the GnRH-I (fraction 21) and GnRH-II standards (fraction 27).

Fig. 5. GnRH-II RIA showing parallel displacement for both GnRH-II peptide (ranging from 1 to 1000 pg/100 ml) and serial dilutions of the eluted fraction 27.

The regression values were: for GnRH-II, ord. = 4.06, slope =−3.23,r²= 0.985;

and for f27, ord. = 4.38, slope =−3.04,r²= 0.957.

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product was further employed as a template in a nested PCR (forward primer mamc-II2, reverse primer mamc-II3). The amplified product revealed a main band of approximately 300 bp (being the expected size around 289 bp based on the human GnRH-II gene). This band was purified and used as a template in the following nested PCR (forward primer mamc-II2, reverse mamc-II4) and a single 250 bp PCR product was obtained.

This product was then cloned and submitted for sequencing. The sequence obtained did not match the GnRH-II genes previously reported.

4. Discussion

Although the biological functions of GnRH-II are still practically unknown, the highly conserved structure of GnRH- II, over millions of years of vertebrate evolution, suggests that this neuropeptide plays an essential role. The expression of GnRH-II peptide together with GnRH-I in a single species is well documented in different vertebrate groups, being a decapeptide

chromatographic properties with GnRH-II and it is not detected by a GnRH-I RIA system. Moreover, the ir-GnRH-II material in fraction 27, behaves as the GnRH-II peptide in cross-reactivity assays. These results are in agreement with those reported by Chen et al.[21]when they showed the presence of GnRH-II by RP-HPLC and RIA in rats.

We previously reported that GnRH-II was able to stimulate gonadotropin release in a GnRH-I-like manner in adenohypophyseal cell cultures [14,22]. Our present results show that fraction 27 induces LH and FSH release in in vitro studies, in a pattern that is similar to that of GnRH-II.

The GnRH-II system is well characterized in teleost fish species where it is represented by groups of neurons located in the posterior diencephalon and in the rostral part of the mesencephalon[16,18,23]. A similar distribution of GnRH-II was reported in amphibians, where this neuropeptide was restricted to the midbrain tegmentum [24]. In birds also, GnRH-II was localized in magnocellular neurons distributed in the rostral end of the mesencephalon, whereas GnRH-II fibers were described in limbic structures and olfactory areas[25,26]. In early evolved mammals, as the musk shrew, GnRH-II immunoreactive cells were found in the periaqueductal area, in the regions of the oculomotor and red nuclei as well as in the basal hypothalamus- median eminence region[11]. In primates, the GnRH-I neurons are in the olfactory region, preoptic area, hypothalamus, and around the lateral ventricle[27], whereas the GnRH-II neurons are in the midbrain[28]and unexpectedly in the hypothalamic region [29]. In humans, GnRH-II was found to be present in neuronal cells that are localized mainly in the periaqueductal area as well as in the oculomotor and red nuclei of the midbrain [21]. Moreover, in a species closely related to the rat, immunocytochemical staining for GnRH-II in mouse brain demonstrated that this peptide was localized in the midbrain as in other vertebrates, as well as in cells surrounding the ventricles and in cells adjacent to the hippocampus[30]. Moreover, in that study, staining of adjacent sections using GnRH-I antibody revealed that the distribution of GnRH-I did not overlap with that of GnRH-II. The results presented here, describe some regional distribution of this GnRH-II-like peptide. However, based on the characteristics of the technique employed, we are unable to differentiate whether the olfactory bulb is a synthesizing or releasing area for this GnRH-II-like peptide.

On the other hand, we did not find any ir-GnRH-II in hypothalamic eluates, contrasting with the in situ hybridization results obtained in rhesus monkey [29]. The reason for this difference could be due to a low number or absence of GnRH-II producing neurons, or that these GnRH-II neurons store the peptide in the synaptic terminals at extrahypothalamic regions.

GnRH-I expression was demonstrated in both human and rat pituitary[31–36]. In this work we could confirm the presence of ir-GnRH-I in this gland and we presented evidence of the

Fig. 6. LH and FSH releasing activity in 12 day-old rat pituitary primary cell cultures. The cells were incubated for 60 min with GnRH-II (10⁻⁹M, 10⁻¹⁰M and 10⁻¹¹M) and with the f27 lyophilized and re-suspended to approximately the same concentrations assayed for the GnRH-II peptide. The f35 is a fraction which has no GnRH immunoreactivity. The plots shows the mean of three independent experiments, **pb0.01 vs. control, *pb0.05 vs. control.

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presence of GnRH-II in the rat pituitary. To the best of our knowledge, this is the first report showing the presence of ir- GnRH-II in a mammalian adenohypophyseal tissue. Recently, Schirman-Hildesheim and collaborators, described that GnRH-I expression is regulated during the estral cycle of the rat, and suggested a physiological role in the preparation of this organ for the midcycle gonadotropin surge via local GnRH– gonadotropin axes[37]. The present results lead us to speculate a more complicated GnRH-I/GnRH-II participation in the local regulation of the pituitary function.

From our point of view, it looks highly unlikely that such an evolutionary and structurally conserved decapeptide such as GnRH-II would have been skipped from rat brain. However, in a different murine model other authors found in a mouse gene data bank that a putative GnRH-II gene, has been silenced[38].

Here, we attempted to clone and characterize a putative GnRH- II gene by different cloning strategies. We designed a group of primers based on the mammalian cDNA sequences available on the gene bank (human, AF036329; rhesus monkey, AF097356;

tree shrew, U63327 and house shrew, AF107315). Our first attempt was performed with midbrain cDNA as template and we were unable to find a GnRH-II-like sequence. The reason of this could be that we did not work during a high expression period for GnRH-II mRNA in this tissue. On the other hand, we were able to detect ir-GnRH-II in peptidic extracts; however in these RP-HPLC/RIA experiments we worked with a pool of female rats at random stages of the estrous cycle. In addition, GnRH-II peptide detection could be associated to storage and accumu- lation, whereas the mRNA may suffer a faster turnover.

Then we performed further attempts to identify a GnRH-II gene working with rat genomic DNA. After a series of nested PCRs, we obtained the respective amplification bands ranging the expected sizes based on the human GnRH-II gene.

However, we failed to obtain a GnRH-II sequence from these PCR products. A possible explanation for this could be that the rat GnRH-II gene sequence is not as well conserved as in other mammals, warranting further studies.

These results provide additional information that suggests the presence of GnRH-II in rat brain. Furthermore, this is the first study providing evidence for the presence of GnRH-II in rat pituitary. We propose that future cDNA cloning strategies or peptide characterization experiments should be undertaken to unequivocally confirm GnRH-II existence in the rat.

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