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BOR - Papers in Press, published online ahead of print May 26, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028019
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BIOLOGY OF REPRODUCTION 71, 1026–1035 (2004)
DOI: 10.1095/biolreprod.104.028019
© 2004 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Developmental Expression of Three Forms of Gonadotropin-Releasing Hormone and Ontogeny of the Hypothalamic-Pituitary-Gonadal Axis in Gilthead Seabream (Sparus aurata)1

Ten-Tsao Wong3, Yoav Gothilf4, Nilli Zmora3, Katherine E. Kight3, Iris Meiri5, Abigail Elizur6, and Yonathan Zohar2,3

Center of Marine Biotechnology,3 University of Maryland Biotechnology Institute, Baltimore, Maryland 21202 Department of Zoology,4 George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel Israel Oceanographic and Limnological Research,5 National Center for Mariculture, Eilat, Israel Bribie Island Aquaculture Research Center,6 Queensland Department of Primary Industries, Woorim, Queensland, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To address the complexity of the origin of the GnRH system in perciforms, we investigated the ontogenic expression of three GnRHs in gilthead seabream. Using in situ hybridization, chicken (c) GnRH-II mRNA-expressing cells were detected in the hindbrain at 1.5 days postfertilization (DPF) and in the midbrain at 2 DPF and thereafter; the hindbrain signals became undetectable after 10 DPF. Salmon (s) GnRH mRNA-expressing cells were first seen in the olfactory placode at 3 DPF, started caudal migration at 14 DPF, and reached the preoptic areas at 59 DPF. Seabream (sb) GnRH mRNA-expressing cells were first detected in the terminal nerve ganglion cells (TNgc), ventral part of the ventral telencephalon, nucleus preopticus parvocellularis, and thalamus at 39 DPF, and extended to the nucleus preopticus magnocellularis at 43 DPF, ventrolateral hypothalamus at 51 DPF, and nucleus lateralis tuberis and posterior tuberculum at 59 DPF. Coexpression of sbGnRH and sGnRH transcripts was found in the TNgc. Using real-time fluorescence-based quantitative polymerase chain reaction, transcript levels of cGnRH-II and sGnRH were first detected at 1 and 1.5 DPF, respectively, and increased and remained high thereafter. Transcript levels of sbGnRH remained low after first detection at 1 DPF. Furthermore, these GnRH expression profiles were correlated with the expression profiles of reproduction-related genes in which at least four concomitant increases of GnRH, GnRH receptor, gonadotropin, gonadotropin receptor, and Vasa transcripts were found at 5, 8, 14, and 28 DPF. Our data provide an expanded view of the ontogeny of the GnRH system and reproductive axis in perciforms.

developmental biology, early development, follicle-stimulating hormone, gonadotropin-releasing hormone, luteinizing hormone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GnRH is the primary neuropeptide that acts on the pituitary gland to stimulate the synthesis and secretion of gonadotropins, namely FSH and LH. The gonadotropins, in turn, regulate gonadal steroidogenesis, gametogenesis, and gonadal growth, thereby directly influencing reproductive function. In fish, GnRH neurons directly innervate the pituitary [1], and germ cell differentiation in the gonads is correlated with pituitary innervation by these neurons [2]. The development and establishment of this hypothalamic-pituitary-gonadal (HPG) axis determines the reproductive competence of the fish.

Developmental studies of the GnRH system indicated that GnRH-positive cells are first detected in the olfactory placode and subsequently migrate into the terminal nerve (TN) system in mammals, chickens, and amphibians. Using immunocytochemistry (ICC) and in situ hybridization (ISH), it has been demonstrated that GnRH neurons originate from the olfactory placode and migrate in association with the olfactory tract into the forebrain in rodents. Thereafter, they continue to migrate to the preoptic area (POA) and hypothalamus and project their axons to the median eminence [3–5]. A similar migratory route of GnRH neurons was described in the rhesus monkey [6]. In chicken embryos, GnRH cells first appear in the olfactory epithelium, are later found along the olfactory nerve as they enter the forebrain, and continuously migrate to the hypothalamus [7–9]. Olfactory placode ablation resulted in loss of brain GnRH neurons on the surgically ablated side [10]. ICC studies and olfactory placode ablation suggest a similar origin and migratory route for GnRH neurons in amphibians [11, 12]. All the evidence described above indicates that forebrain GnRH originates from one source, the olfactory placode. In addition to the forebrain GnRH system, there is a cluster of chicken (c) GnRH-II expressing cells in the midbrain. Unlike the relatively wide distribution of the forebrain GnRH system, the expression of cGnRH-II is restricted to the midbrain [12–18]. All evidence suggests that these cells have a different embryonic origin than the forebrain GnRH system [12, 19–21].

Following the isolation of three forms of GnRH from the gilthead seabream [22], our laboratory and others have shown that the forebrain GnRH system in adult perciforms consists of two cell populations: cells in the olfactory bulb (OB) and TN express salmon (s) GnRH, whereas a second population located in the POA and hypothalamus expresses seabream (sb) GnRH [18, 23–25]. Studies to date show that teleosts express either two or three different forms of GnRH. The two-GnRH system, consisting of one forebrain GnRH and the ubiquitous midbrain form (cGnRH-II), has been noted in catfish [26], salmon [27], goldfish [28], and zebrafish [29, 30]. Studies in salmon suggested that the forebrain GnRH cells originate from one brain region, the olfactory placode [31, 32]. In contrast, the brain GnRH system represented in the three GnRH model (described in the gilthead seabream and other perciforms) consists of two unique forebrain GnRH peptides in addition to the ubiquitous midbrain cGnRH-II. This raised the question whether these two forebrain GnRH populations (OB-TN vs. POA-hypothalamic GnRHs) originate in the same region, the olfactory placode, or have separate origins. Results from ICC and ISH studies have led to contradicting hypotheses. One suggested that both OB-TN and POA-hypothalamic GnRH neurons originate from the same embryonic source, the olfactory placode [21, 33], whereas the other indicated that the POA-hypothalamic GnRH neurons have a different origin than the OB-TN GnRH neurons [20, 25]. To resolve this conflicting data, ablation studies and additional evidence from different fish models on the ontogeny of the GnRH system are needed.

In the present study, we attempted to address the ontogeny of GnRHs in a three GnRH model species, the gilthead seabream, beginning with the very early developmental stages (before hatching). We applied whole-mount and section ISH to investigate the possible origins of GnRH cells with specific riboprobes corresponding to each GnRH mRNA. Furthermore, we engaged a dual-labeling hybridization strategy in an attempt to determine whether sbGnRH and sGnRH cells share the same origin. In order to accurately quantify the levels of the three GnRH transcripts and understand the ontogeny of the HPG axis in developing seabream, we used real-time fluorescence-based quantitative polymerase chain reaction (quantitative PCR hereafter) to measure the transcript amounts of GnRHs, GnRH receptor (GnRHr), FSH ß-subunit (Fshß), LH ß-subunit (Lhß), FSH receptor (Fshr), LH receptor (Lhr), and Vasa during the first 36 days of development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All animal husbandry and experimentation were conducted in accordance with our Institutional Animal Care and Use Protocols and adhered to the National Research Council's Guide for Care and Use of Laboratory Animals promulgated by the Society for the Study of Reproduction.

Experimental Animal Holding and Sample Collection

Fertilized eggs, collected in the first 2 h of spawning from a single broodstock tank holding about 120 fish, were held in four 200-L conical tanks in our Aquaculture Research Center. Each tank was stocked with around 20 000 eggs (100 eggs/L) and exposed to simulated natural photoperiod conditions and to temperatures of 18° to 20°C. Under these conditions, embryos hatched between 45 and 48 h postfertilization. A published protocol [34] was followed for the animal rearing and feeding. When animals were killed, they were anesthetized in 200 ppm 2-phenoxyethanol and immersed into 4% paraformaldehyde in PBS or frozen in liquid nitrogen. Samples at the development stages of 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 39, 43, 51, and 59 days postfertilization (DPF) were collected. Approximately 40 (10 from each tank) animals were fixed overnight for whole-mount and section ISH, and exactly 20 (five from each tank) animals at each stage were frozen for RNA extraction. For whole-mount ISH, postfixed animals were preserved in methanol at –20°C. For section ISH, whole fixed animals were processed through successive baths of ethanol (50%, 70%, 95%, and 100%) and two xylene baths, and then embedded in paraffin. European sea bass (Dicentrarchus labrax) samples were obtained from Israel Oceanographic and Limnological Research, National Center for Mariculture.

Syntheses of Riboprobes and RNA Standard Preparation for Quantitative PCR

For riboprobes, the gilthead seabream GnRH [35] and European sea bass cGnRH-II [36] cDNA-containing plasmids were linearized and used as templates for anti-sense and sense digoxigenin (DIG) labeled riboprobe synthesis (Roche, Indianapolis, IN) according to the manufacturer's instructions. DIG-riboprobes were purified through a size exclusion column (Chroma Spin-200; BD Biosciences, Palo Alto, CA). For dual-labeling hybridization, fluorescein (FLU)-labeled anti-sGnRH probe was synthesized using a FLU-UTP mixture (Roche).

For RNA standard syntheses, plasmids containing all of the relevant cDNAs including each seabream GnRH [35], Fshß, and Lhß [37], and our recently cloned cDNAs (see Table 1 for GenBank accession numbers) including GnRHr (partial), Fshr (open reading frame), Lhr (open reading frame), and Vasa (partial), were linearized and used as templates for gene-specific RNA standard syntheses. The same protocol described above was followed except a 1 mM UTP instead of the DIG-UTP mixture was used. Total RNA isolated from seabream liver using Tri-reagent (MRC Inc., Cincinnati, OH), a modified acid-phenol extraction method, followed by the DNase treatment served as the standard for 18s RNA. The amount of each RNA standard was determined using a RiboGreen RNA quantification kit (Molecular Probes, Eugene, OR).


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  		TABLE 1. Primers, amplicon sizes, and PCR efficiencies for each transcript in quantitative PCR assays

In Situ Hybridization

Whole-mount ISH modified from a published procedure [38] was applied in this study. Fixed and methanol-preserved animals were rehydrated through successive baths of methanol (70%, 50%, and 30% in PBS); depigmented with 6% H2O2 in PBS for 30 to 60 min (depending on the stage of the animal); rinsed for 5 min twice with PBTw/BSA (PBS containing 0.1% Tween 20 and 0.2% BSA); and treated with proteinase K (20 µg/ml in PBTw/BSA) for 10–40 min. Samples were postfixed for 30 min in 4% paraformaldehyde/PBS, dipped in cold acetone for 8 min, washed in PBTw/BSA, and prehybridized for 3 h at 65°C in hybridization buffer I (50% formamide, 5x SSC, 5 mM EDTA, 0.1% Tween 20, 0.1% CHAPS, 50 mg/ml heparin, and 1 mg/ml yeast RNA). Hybridization was carried out overnight at 65°C with either DIG-labeled anti-sense or sense (for controls) riboprobe at a concentration of 1 µg/ml in hybridization buffer I. Samples were then washed twice with 50% formamide and 1x SSC at 65°C for 30 min, 1x SSC at 65°C for 15 min, and twice with 0.2x SSC at 65°C for 30 min. Alkaline phosphatase (AP) coupled anti-DIG antibody (Roche) was applied to the samples at 150 mU/ml. After color development using BM Purple AP substrate (Roche), samples were examined under a light microscope (Axioplan 2; Zeiss, Jena, Germany) and computer images were obtained with a digital camera (MDS; Eastman Kodak, Rochester, NY) and processed in Adobe PhotoShop 6.0. No subsequent alterations were made to the images.

Section ISH modified from a published procedure [39] was utilized to investigate the ontogeny of sbGnRH mRNA-expressing cells and the coexpression of sbGnRH and sGnRH mRNAs. Rehydrated sections at 6 µm were incubated with 0.2 M of HCl for 20 min; washed in PBS; treated with proteinase K (10 µg/ml in 50 mM Tris-HCl, pH 7.5, and 50 mM EDTA) for 15 min; and acetylated in 0.1 M triethanolamine-HCl/0.25% (v/v) acetic anhydride. Each section was covered with 500 µl of hybridization buffer II (50% formamide, 5x SSC, 50 µg/ml yeast tRNA, and 50 µg/ml denatured calf thymus DNA) and incubated for 2 h at 58°C. After prehybridization, buffer was replaced with new hybridization buffer II containing 400 ng/ml of denatured probes (DIG-labeled for sbGnRH, FLU-labeled for sGnRH); covered with a cover-slip; and incubated overnight at 58°C. After hybridization, the sections were washed for 30 min in 2x SSC at 25°C and for 1 h each in 2x SSC, 0.4x SSC, and 0.1x SSC at 65°C. AP-coupled anti-DIG antibody and peroxidase (POD) coupled anti-FLU antibody (Roche) were applied at 150 mU/ml of each in buffer I (100 mM Tris-HCl and 150 mM NaCl, pH 7.5). Excess antibodies were removed by two 15-min washes with buffer I. Color development in sections was initiated with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO) and followed by a second color development using BM Purple AP substrate. Sections were examined under a BH-2 light microscope (Olympus, Tokyo, Japan), and computer images were obtained.

Quantification of Transcript Levels of GnRHs, GnRHr, Fshß, Lhß, Fshr, Lhr, and Vasa

Total RNA, isolated from whole animals (pooled 20 animals in each stage up to 36 DPF) using Tri-reagent, was treated with DNase (RNase-free, 2 U in 50 µl final reaction mixture) for 30 min at 37°C and quantified by spectrophotometer (DU 640; Beckman Coulter, Fullerton, CA). RNA standards and total RNA from each sample were reverse-transcribed into cDNA using random hexamers and MMLV reverse transcriptase (Promega, Madison, WI). Triplicate cDNA aliquots (1 ng of total RNA for 18s RNA and 20 ng for the other transcripts) from each sample served as templates in PCR using SYBR Green PCR core reagent (Applied Biosystems, Foster City, CA) containing 200 nM gene-specific primers (Table 1). Amplification reactions were carried out via ABI Prism 7700 Sequence Detection System at 50°C for 4 min, 95°C for 10 min, and 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Copy number in unknown samples was determined by comparing CT (threshold cycles) values [40] to the specific standard (run in every plate) and normalized to the amount of 18s RNA in each sample. To validate the PCR products in quantitative PCR, equally pooled total RNA from samples at 1, 5, 10, and 14 DPF (1 ng for 18s and 20 ng for other transcripts) and pituitary total RNA (1 ng for Lhß only) were used to investigate the paired primers designed for amplifying each transcript. PCR products were separated on a 3% agarose gel.

Statistical Analyses

Data obtained from quantitative PCR for the transcript level of each gene were presented as the mean and standard deviation. For statistical analyses, log transformation was applied to correct problems of unequal variances among data. The results were analyzed over time for each developmental stage (categorized by DPF) using one-way ANOVA. In all cases, significance was accepted at P < 0.01. In order to show the very significant increase in expression level of each gene throughout the developmental stages, a Student t-test was applied to compare the transcript level of each gene at each developmental stage (DPF) with the level from its previous development stage. The developmental stages with very significant increases (P < 0.001) in transcript expression were considered as worthwhile candidates for further scrutiny. Additional examinations were expected to identify the concomitant increase events of the reproduction-related genes during the ontogeny of the HPG axis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In whole-mount ISH and section ISH, anti-sense-probed animals and sections showed very clear hybridization signals in the specific brain areas, whereas sense probes did not show any remarkable labeling. In quantitative PCR assays, the basal line was set between 3 and 15 cycles (default setting) for all transcripts except for 18s RNA (between 3 and 8 cycles). The correlation coefficient for each standard curve in each reaction plate was between 0.96 and 0.99. RNA controls from each sample, in the absence of reverse transcriptase in the cDNA syntheses, showed no significant amplification. The level of detection for each transcript was set at the lowest standard point with a significantly different amplification (significantly lower CT value) than the controls (no template and RNA template controls). The setting for each transcript was 24 copies/reaction for all three GnRHs; 48 copies/reaction for Fshß, Lhß, Fshr, and Lhr; and 480 copies/reaction for GnRHr and Vasa.

Ontogeny of cGnRH-II mRNA-Expressing Cells

Using whole-mount ISH, the cGnRH-II mRNA-expressing cells (purple-blue stain, red arrows in Fig. 1) in developing seabream were first detected at 1.5 DPF; these cells appeared to have a strong expression bilaterally in the hindbrain area (Fig. 1A). Later at 2 DPF, the expression was also seen in the midbrain area in addition to the hindbrain (Fig. 1B). After 2 DPF, there was a continuous expression of cGnRH-II transcripts in the midbrain area of developing seabream (5 DPF, Fig. 1C; 10 DPF, Fig. 1D; 14 DPF, Fig. 1E; 18 DPF, Fig. 1F). However, the expression of hindbrain cGnRH-II decreased and was not detected after 10 DPF (Fig. 1, D, E, and F). In order to confirm this novel expression of cGnRH-II in the hindbrain, we have also investigated the ontogeny of cGnRH-II mRNA-expressing cells in another perciform fish, European sea bass. The detection of cGnRH-II mRNA-expressing cells occurred at 4.5 and 5.5 DPF, and the signals were first seen in the hindbrain (Fig. 1, G and H).



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  		FIG. 1. Ontogenic localization of cGnRH-II mRNA-expressing cells in developing gilthead seabream (A–F) and European sea bass (G, H). Initial expression of cGnRH-II in the hindbrain area at 1.5 DPF (A). A weak expression in the midbrain area in addition to the hindbrain signals at 2 DPF (B). cGnRH-II expression in both midbrain and hindbrain areas at 5 DPF (C). Only midbrain cGnRH-II detected at 10 DPF (D), 14 DPF (E), and 18 DPF (F). The detection of the hindbrain cGnRH-II mRNA-expressing cells in European sea bass at 4.5 DPF (G) and 5.5 DPF (H). Red arrows point to cells (purple-blue stain) expressing cGnRH-II transcripts. Bar = 400 µm

Ontogeny of sGnRH mRNA-Expressing Cells

Using whole-mount ISH, the sGnRH mRNA-expressing cells (purple-blue stain, red arrows in Fig. 2) were first seen in the olfactory areas at 3 DPF (Fig. 2A) and remained in the same areas at 5 and 10 DPF (Fig. 2, B and C). Later, at 14 DPF, two bilaterally caudal migration tracts toward the rostral part of the telencephalon were detected (Fig. 2D). These migration tracts extended continuously and were seen at 18 and 22 DPF (Fig. 2, E and F, respectively). Using section ISH, sGnRH mRNA-expressing cells (brown stain, black arrows in Fig. 3) were detected in the olfactory TN (Fig. 3A) and TN ganglion cells (TNgc; Fig. 3, B and B1), the transition between the OB and the telencephalon, at 39 DPF. At 59 DPF, sGnRH mRNA-expressing cells were not only found in the OB-TN (data not shown) and TNgc (Fig. 3B4), but also in the ventral part of the ventral telencephalon (Vv; Fig. 3C) and POA (Fig. 3D). Among all of the developmental stages we investigated, no other sGnRH mRNA-expressing cells could be detected further caudal in the brain.



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  		FIG. 2. Ontogenic localization of sGnRH mRNA-expressing cells in developing gilthead seabream. The first detection of sGnRH transcripts in the olfactory placode areas at 3 DPF (A). sGnRH signals in the same areas at 5 (B) and 10 (C) DPF. Two bilaterally caudal migration tracts toward the rostral part of the telencephalon at 14 DPF (D). Continuously extending migration tracts at 18 DPF (E) and 22 DPF (F). Red arrows point to cells (purple-blue stain) expressing sGnRH transcripts. Bar = 400 µm



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  		FIG. 3. Ontogenic localization of sGnRH mRNA-expressing cells and sbGnRH mRNA-expressing cells in sagittal brain sections of developing gilthead seabream. Signals of sGnRH mRNA-expressing cells (brown) in the olfactory TN (a migration tract; A) and the TNgc (B, B1) at 39 DPF and later in the Vv (C) and POA (D) at 59 DPF. The expression of sbGnRH transcript (purple-blue) in the NPP (B), Vv, and thalamus (E) at 39 DPF. Coexpression of sGnRH and sbGnRH transcripts in the TNgc (green arrows) at 39 (B1), 43 (B2) 51 (B3), and 59 (B4) DPF. Signals of thalamic sbGnRH at 43 DPF (F). Extension of the POA-hypothalamic sbGnRH to the PM (G) at 43 DPF, to the ventrolateral hypothalamus (H) at 51 DPF, and to the NLT (I) at 59 DPF. Extension of the thalamic sbGnRH to the PT (I) and the detection of Vv sbGnRH (J) at 59 DPF. Black scale bars = 200 µm; red scale bars = 20 µm. Black arrows point to sGnRH cells; red arrows point to sbGnRH cells; green arrows indicate the coexpression of sGnRH and sbGnRH transcripts. Hyp, Hypothalamus; NLT, nucleus lateralis tuberis; NPP, nucleus preopticus parvocellularis; OB, olfactory bulb; OT, optic tectum; PM, nucleus preopticus magnocellularis; POA, preoptic area; PT, posterior tuberculum; Tel, telencephalon; TN, terminal nerve; TNgc, terminal nerve ganglion cell; Vv, ventral part of the ventral telencephalon

Ontogeny of sbGnRH mRNA-Expressing Cells and the Colocalization of sbGnRH and sGnRH mRNA Expression

Because we were not able to detect sbGnRH mRNA-expressing cells by whole-mount ISH, section ISH was utilized to investigate the ontogeny of sbGnRH mRNA-expressing cells on the sagittal sections of whole embryo or brain from 5 to 59 DPF. The first detection of sbGnRH expression (purple-blue stain, red arrows in Fig. 3) occurred at 39 DPF with a strong signal in the nucleus preopticus parvocellularis (NPP; Fig. 3B), two very intense signals in the dorsal and ventral parts of the thalamus and a signal in the Vv (Fig. 3E). Moreover, the hybridization signal of sbGnRH mRNA-expressing cells was also found in the TNgc, where it was colocalized in the same cells expressing sGnRH mRNA by using dual-labeling hybridization (Fig. 3B1, green arrows). This colocalization of sGnRH and sbGnRH transcripts in the TNgc was also found at 43, 51, and 59 DPF (Fig. 3, B2, B3, and B4, respectively). At 43 DPF, the expression of sbGnRH was still seen in the thalamic area (Fig. 3F), whereas the preoptic sbGnRH mRNA-expressing cells extended posterior to the nucleus preopticus magnocellularis (PM; Fig. 3G). At 51 DPF, sbGnRH expression extended to the ventrolateral hypothalamus (Fig. 3H). The thalamic sbGnRH mRNA-expressing cells extended to the posterior tuberculum (PT), whereas the POA-hypothalamic sbGnRH cells reached the nucleus lateralis tuberis (NLT) at 59 DPF (Fig. 3I). The Vv sbGnRH signal was still visible at 59 DPF (Fig. 3J).

Developmental Expression of Three GnRH, GnRHr, Fshß, Lhß, Fshr, Lhr, and Vasa Transcripts

Quantitative PCR assays were established and used for measuring changes in the expression level of each transcript, including the three GnRHs, Type III [41] GnRHr, Fshß, Lhß, Fshr, Lhr, and Vasa. This permitted us to quantify transcript levels of the three GnRHs, as well as to investigate the developmental expression of indicatory genes at the pituitary level (Fshß and Lhß) and gonadal level (Vasa) and of other reproduction-related genes (GnRHr, Fshr, and Lhr). The amplicon size and PCR efficiency for each transcript in quantitative PCR are shown in Table 1. Only one PCR product was seen in quantitative PCR for each transcript (Fig. 4) except Lhß (no PCR product, lane 6) when pooled total RNA (from samples at 1, 5, 10, and 14 DPF) was used. Pituitary total RNA was used to further validate the paired primers designed for amplifying Lhß, and only one PCR product was seen in this reaction (Fig. 4, lane 11). After normalization with the amount of 18s RNA, the results were presented as copy number of transcript per 20 ng of total RNA and/or copy number of transcript per animal. The first detected cGnRH-II transcripts occurred at 1 DPF (Fig. 5A). Expression of cGnRH-II transcripts increased continuously and maintained a relatively high level, peaking at 14 DPF (around 4600 copies/20 ng of total RNA), which was followed by a decreasing trend. The sGnRH transcripts were first detected at 1.5 DPF, increased continuously with a similar trend as seen in cGnRH-II expression from 3 to 12 DPF, remained at a higher level between 12 and 22 DPF, and then followed a decreasing trend after 22 DPF (Fig. 5A). The first detection of sbGnRH transcripts occurred at 1 DPF; however, its relative amount decreased to undetectable levels at 1.5 DPF. Second detection of sbGnRH occurred at 2 DPF, after which the transcript levels of sbGnRH remained low during the first 36 days of development but exhibited significant peaks at 14, 20, and 28 DPF (Fig. 5A). Because the total RNA per animal increased tremendously after 22 DPF (data not shown), the transcript levels of the three GnRHs were also presented as copy number per animal. After this transformation (Fig. 5B), the expression of each GnRH exhibited a trend similar to the ones presented in Figure 5A, except after 24 DPF. When the data were presented as copy number per animal, all three GnRH transcripts exhibited a very significant increase after 24 DPF (Fig. 5B). Indeed presenting the data as copy number per animal eliminates the dilution effect resulting from the dramatic increase of total RNA as the animal grows.



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  		FIG. 4. Quantitative PCR products amplified from each transcript. Using total RNA pooled from developing seabream (at 1, 5, 10, and 14 DPF) with gene-specific primers, PCR products amplified from each transcript, GnRHr (lane 1) to 18s RNA (lane 10) are presented as the order shown in Table 1. Lane 11 is the PCR product from pituitary Lhß transcripts. M, DNA markers (bp)



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  		FIG. 5. Ontogenic expression of three GnRHs during the first 36 days of development. Data are presented as the mean and standard deviation. The amount of each GnRH is presented as copies/20 ng of total RNA (A) or as copies/animal (B). The significant increase (P < 0.001) of each transcript expression at each developmental stage (compared with the value of its previous developmental stage) is indicated by "a" for cGnRH-II, "b" for sGnRH, and "c" for sbGnRH (A), and by "d" for cGnRH-II, "e" for sGnRH, and "f" for sbGnRH (B). The developmental stages with the concomitant expression increases of the reproduction-related genes are indicated by asterisks (*). ND, Not detected

Transcripts of Lhß were undetectable through the first 36 days of development. The transcript levels of GnRHr, Fshß (Fig. 6A), Fshr, Lhr, and Vasa (Fig. 6B) were all detectable in our 1 DPF sample, presented as copy number per animal. After their first detection, expression of both GnRHr and Fshß transcripts increased at 1.5 DPF and fluctuated thereafter during the first 36 days of development, resulting in several significant (P < 0.001) expression peaks that occurred concomitantly at 5, 14, and 28 DPF (Fig. 6A). During the first 4 days, the transcript levels of Fshr, Lhr, and Vasa displayed a decreasing trend after their first detection at 1 DPF. Levels of these three transcripts climbed at 5 DPF and fluctuated thereafter, resulting in several significant concomitant expression peaks/increases at 5 DPF for Vasa and Lhr; at 14 DPF for Vasa and Fshr; and at 8, 22, and 28 DPF for all three transcripts (Fig. 6B).



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  		FIG. 6. Ontogenic expression of GnRHr and Fshß transcripts (A), and Fshr, Lhr, and Vasa transcripts (B) during the first 36 days of development. Data are presented as the mean and standard deviation. The amount of each transcript is presented as copies/animal. The very significant increase (P < 0.001) of each transcript expression at each developmental stage (compared with the value of its previous developmental stage) is indicated by "g" for Fshß and "h" for GnRHr (A), and by "i" for Fshr, "j" for Lhr, and "k" for Vasa (B). The developmental stages with the concomitant expression increases of the reproduction-related genes are indicated by asterisks (*). ND, Not detected

After ANOVA (P < 0.01 in all cases) and Student t-test analyses, the data points showing a very significant increase were indicated by letter distinctions as shown in Figures 5, A and B, and 6, A and B. When analyzing the expression patterns of all genes together, our results demonstrate that at least four concomitant increases occurred at the transcript levels of reproduction-related genes in the HPG axis at 5, 8, 14, and 28 DPF (Fig. 5B and 6, A and B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The development and establishment of the GnRH system play a key role in fertility, which has been conclusively demonstrated by two mutations. In the hypogonadal (hpg) mouse, a deletion in the GnRH gene prevents production of the GnRH peptide. As a result, the mice are hypogonadal and infertile. Introduction of an intact GnRH gene into the genome of these mice restores fertility [42, 43]. Another form of genetic hypogonadism, observed in humans, is Kallman syndrome. The infertility associated with this syndrome, caused by a developmental defect in the migration and targeting of GnRH neurons during embryogenesis, can be reversed by administration of synthetic GnRH [44]. This demonstrates that the correct development of the GnRH neurons in the brain is necessary for gonadal development and fertility in the adult.

In this study, we reported the ontogenic development of the three different GnRH mRNA-expressing cells in the gilthead seabream during the first 2 mo of development. The results presented here provide evidence of the novel ontogenic expression of the hindbrain cGnRH-II in vertebrates. Although the hindbrain cGnRH-II expression in lake whitefish has been reported recently [45], no ontogenic study of the hindbrain cGnRH-II has been stated. Most data obtained in fish, amphibians, birds, and mammals [12–18, 20, 25, 33, 46–48] demonstrated that cGnRH-II-expressing neurons were found only in the midbrain. Since our results showed that the hindbrain cGnRH-II mRNA-expressing cells were detected first and no migration tract was found between the hindbrain and midbrain, it suggests that the hindbrain cGnRH-II mRNA-expressing cells originate from the hindbrain area, different from the origin of the midbrain cGnRH-II cells. This novel finding of the hindbrain cGnRH-II in gilthead seabream was further supported by the data obtained from European sea bass. Our results also indicated that hindbrain cGnRH-II was expressed in a transient manner during early development because this early expression of the hindbrain cGnRH-II decreased gradually and became undetectable after 10 DPF. The function of this transient and region-specific expression is unknown. However, as GnRHs are also believed to act as neurotransmitters and neuromodulators [49–51], this transiently expressed cGnRH-II could be very important for early brain development. The temporally and spatially restricted expression of the hindbrain cGnRH-II also indicates that a specific transcriptional control mechanism may be involved in the regulation of its expression. A similar mechanism was suggested in the development of the murine GnRH system. Specifically, in the murine system the suppression of GnRH-I gene transcription in the lateral septum and bed nucleus of the stria terminalis (BNST) in late development may involve the cis- or trans-acting repressor elements present in the promoter of the GnRH-I gene [52].

We first detected sbGnRH mRNA-expressing cells not only in the POA but also, unexpectedly, in the thalamic area at 39 DPF. This nonclassical thalamic sbGnRH expression was distributed in both the dorsal and ventral parts of the thalamus and extended to the PT at 59 DPF. Thalamic GnRH was found in the brain of adult seabream [18] and the three-spined stickleback [53]; however, no ontogenic study of this thalamic GnRH has been reported in fish. The finding of early expression of the thalamic sbGnRH is in agreement with the detection of thalamic GnRH in chicken embryo [54]. It was suggested that this nonclassical GnRH originates from a different brain region than the POA-hypothalamic GnRH in chicken [12, 55] and mouse [52, 56]. In mouse, one study found nonclassical expression of GnRH-I was found in tectum [56], and another found it in the lateral septum, BNST, and tectum [52], during early development. Furthermore, using the Pax-6 null mice that fail to develop an olfactory placode, GnRH-I-immunoreactive cells were still found in the lateral septum, tectum, and BNST but not in the septohypothalamic area [52]. This result demonstrated that these nonclassical GnRH-expressing populations have a different origin from the olfactory placode. Together with the temporal and spatial expression pattern of thalamic sbGnRH, and the lack of any detectable migration tract between the preoptic-hypothalamic area and the thalamus, it is possible that thalamic sbGnRH cells may originate differently from the POA-hypothalamic GnRH cells in gilthead seabream.

Unlike mammals, the forebrain of perciform fish contains two different forms of GnRH; thus, sGnRH is distributed in the olfactory nerves, OB, and TN, and sbGnRH is expressed in the POA and hypothalamus [18, 24, 33, 47, 48]. Most studies to date agree that sGnRH cells originate from the olfactory placode in perciforms. However, controversy remains as to the origin of sbGnRH cells, either from a preoptic primordium [20, 25] or from the olfactory placode, the same origin of the sGnRH cells [19, 21]. The results obtained in this study revealed the overlapping distribution of sGnRH mRNA-expressing cells and sbGnRH mRNA-expressing cells in the TNgc and Vv, which favor the hypothesis that preoptic and hypothalamic sbGnRH cells originate from the olfactory placode. Our data showed that sbGnRH transcripts appeared in the TNgc, Vv, and POA at their first detection at 39 DPF. In our experiences, it took longer to see the sbGnRH signals in the TNgc and Vv than those in the POA-hypothalamic areas during color development. Our explanation is that these cells (at the TNgc, Vv, and possibly OB) may express sbGnRH transcripts at relatively lower levels before they arrive in the POA-hypothalamic areas. As such, and due to the sensitivity of the methodology we used in section ISH, we did not find sbGnRH expression either in the OB or as a sequential pattern from the OB to the POA as were found previously in European sea bass [21]. Considered together with the findings in European sea bass, the above data seem to suggest that the developmental origin of the forebrain GnRH neurons in perciform fish is similar to that of other vertebrates; both TN and preoptic GnRH neurons are of olfactory placode origin. Furthermore, using dual-labeling hybridization, we provided evidence of the coexpression of sGnRH and sbGnRH transcripts in the same cell in the TNgc, which further reinforced the possibility that the TN-POA-hypothalamic sbGnRH mRNA-expressing cells share a common origin with sGnRH mRNA-expressing cells. We have also detected the sGnRH mRNA-expressing cells in the Vv and POA at 59 DPF. These cells expressed lower levels of sGnRH transcripts than those found in the olfactory TN at 59 DPF (data not shown).

The TN is thought to play an important role in establishing the migratory route that guides the GnRH neurons toward the forebrain [3]. Interestingly, both sbGnRH and sGnRH mRNA-expressing cells in the early developing seabream extend to the POA through the ventral forebrain, following the course of TN fibers. Thereafter, only the sbGnRH mRNA-expressing cells continue migrating into the ventral and lateral parts of the hypothalamus. It is noteworthy that in the rhesus monkey, two types of GnRH cell populations that originate from the olfactory placode and migrate into the brain have been described. One population starts to migrate earlier than the other. The later migrating cells take their adult position and function to stimulate gonadotropin secretion [57]. Studies on catfish (cf) have also shown that the forebrain cfGnRH-expressing cells can be distinguished into two populations (TN vs. POA-hypothalamic) based on their differences in temporal and spatial appearance and morphology [58]. It is possible that the OB-TN-POA-hypothalamic GnRH system in all vertebrates is comprised of two types of GnRH cells, but the origin of both cell types is the same. However, the case seen in the gilthead seabream and other perciforms is that these two GnRH cell types express two different forms of GnRH (i.e., sGnRH for the OB-TN and sbGnRH for the POA-hypothalamic GnRH system).

Using quantitative PCR, transcripts of cGnRH-II were first detected in the earliest sample collected at 1 DPF (at least 12 h earlier than its first detection by whole-mount ISH), and sGnRH was first seen at 1.5 DPF (36 h earlier than its first detection by whole-mount ISH). The high sensitivity of quantitative PCR can also be seen for the expression patterns of sbGnRH transcripts that were first detected at 1 DPF but, not surprisingly, the expression remained at a lower level compared with the transcript levels of sGnRH and cGnRH-II during the first 36 days of development. These low expression levels explained the reasons that sbGnRH transcripts were undetectable in samples collected before 39 DPF with either whole-mount ISH or section ISH. During the ontogeny of the three GnRHs, sbGnRH represented the lowest expression level (measured by quantitative PCR) and the most delayed GnRH form in the brain (at 39 DPF by section ISH). In perciforms, sbGnRH is considered as the main hypophysiotropic form, the most abundant GnRH in the pituitary, and the form that controls gonadotropin synthesis and release [18, 22, 59, 60]. As such, it would be interesting to know whether the delayed and lower expression level of sbGnRH results in the delayed ontogeny of the gonadotropin expressing system and HPG axis. In order to answer this question, we measured the transcript levels of GnRHr, Fshß, Lhß, Lhr, Fshr, and Vasa during the first 36 days of development. Surprisingly, except for Lhß, transcripts of the other genes were detected as early as 1 DPF. Interestingly, the expression of GnRHr and Fshß transcripts increased significantly at 1.5 DPF, indicating that the embryonic transcription of these two genes started as early as 1.5 DPF. In contrast, a clear decreasing trend was found in the transcript levels of Fshr, Lhr, and Vasa after their first detection (at 1 DPF) until 4 DPF, which suggested that low (or no) activity of embryonic transcription of these three genes occurred during the first 4 days and the decreasing trend resulted from the decay of maternal transcripts. No Lhß transcripts were detected during the entire investigation, which is in agreement with the finding in rainbow trout that FSH-expressing cells develop earlier and FSH but not LH-immunoreactive cells were found in the pituitary of the rainbow trout, when mitosis of germ cells was first detected in the gonad [61].

Comparing the significant increases over the time change for each gene expression showed at least four distinguished concomitant increases at 5, 8, 14, and 28 DPF, which may reflect synchronized events in the early ontogeny of the HPG axis. Our results suggest that the ontogeny and organization of the HPG axis may start as early as 5 DPF, become highly activated at 14 DPF, and maintain a more stable development after 28 DPF. Since no Lhß transcripts were detected during the first 36 days of development, it is evident that Fshß, rather than Lhß, plays an important role as the integral messenger in the establishment of the HPG axis. Among the three GnRHs, cGnRH-II seemed to be a major player because of its higher expression level and its appearance at all four concomitant events. Moreover, the other two expression peaks of cGnRH-II transcripts, which occurred at 18 and 22 DPF, were also closely associated with the concomitant expression increases of Fshß, Fshr, and Vasa transcripts. Several lines of evidence from fish including gilthead seabream [60] supported the existence of a cGnRH-II peptide in the pituitary. Hence, cGnRH-II may reach the pituitary and be involved in the ontogeny of FSH-expressing cells.

At the gonadal level, we measured the transcript levels of Vasa, an RNA helicase of the DEAD-box family. In vertebrates, a Vasa-like homologue has been cloned and shown as a marker gene specifically expressed in the germ cell in several animals including fish [62]. There were six expression peaks of Vasa transcripts appearing at 5, 8, 14, 18, 22, and 28 DPF that are closely associated with the expression increases of Fshß and Fshr, rather than Lhr, transcripts (see Fig. 6, A and B). This result reinforces the importance of FSH signaling in the early germ cell development. The paired developing gonads posterior to the swim bladder were first seen at 14 DPF, where few primordial germ cells were found; the gonads grow but remained undifferentiated until 59 DPF (data not shown). This undifferentiated gonad is still found in 4-mo old fish. At 5 mo of age, juvenile seabream gonads contain mixed masculine and feminine primordial germ cells [63, 64]. The concomitant expression changes of the reproduction-related genes during the first 36 days of development may correlate with the gonadal establishment and germ cell proliferation, rather than sex differentiation. Although the Lhß transcripts were undetected throughout the first 36 days of development, the Lhr expression peaked at 5, 8, 22, and 28 DPF. It is possible that Lhß did express but was under the detection limit of our measurement tool, or that FSH can also act via LHR to initiate the signal cascade.

In the present study, our results provided evidence revealing 1) novel ontogeny of cGnRH-II cells in the hindbrain in addition to its classical expression in the midbrain in vertebrates; 2) a possible second origin of sbGnRH cells in the thalamus in addition to the classical TN-POA-hypothalamic sbGnRH expression in fish; and 3) coexpression of sbGnRH and sGnRH in the same cells in the TNgc in a perciform model. Our results support the idea that the POA-hypothalamic sbGnRH cells originate from the olfactory placode, the origin of sGnRH cells. However, taken together with the findings of the ontogenic hindbrain cGnRH-II and the thalamic sbGnRH cells, our data provide an expanded view of the complexity of the origin of the GnRH system in a perciform model. Our results indicate that ontogeny and organization of the reproductive axis may start as early as 5 DPF, become more activated at 14 DPF, and maintain a stable development after 28 DPF. Possibly, cGnRH-II, FSH, and primarily FSHR are the major players involved in this early establishment.


   ACKNOWLEDGMENTS
 
We are grateful to Steven Rogers and Eric Evans at the Aquaculture Research Center, University of Maryland Biotechnology Institute, for maintaining the experimental fish. Thanks are also extended to John Stubblefield for editing this manuscript.


   FOOTNOTES
 
1 Supported by US-Israel Bi-national Agricultural Research and Development (BARD) Foundation grant 3428-03 (Y.G. and Y.Z.); Maryland Sea Grant Fellowship (T.-T.W.); and MDSG award NA46RG0091 (Y.Z.). This is contribution number 04-621 from the Center of Marine Biotechnology, University of Maryland Biotechnology Institute. Back

2 Correspondence: Yonathan Zohar, 701 East Pratt Street, Baltimore, MD 21202. FAX: 410 234 8896; zohar{at}umbi.umd.edu Back

Received: 18 February 2004.

First decision: 13 March 2004.

Accepted: 20 May 2004.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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T.-T. Wong, S. Ijiri, and Y. Zohar
Molecular Biology of Ovarian Aromatase in Sex Reversal: Complementary DNA and 5'-Flanking Region Isolation and Differential Expression of Ovarian Aromatase in the Gilthead Seabream (Sparus aurata)
Biol Reprod, May 1, 2006; 74(5): 857 - 864.
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N. B. Schwartz and J. E. Levine
Ontogeny of gonadotropin-releasing hormone neurons: fishing for clues in medaka.
Endocrinology, March 1, 2006; 147(3): 1074 - 1075.
[Full Text] [PDF]