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Endocrinology, doi:10.1210/en.2003-0386
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Endocrinology Vol. 144, No. 8 3297-3300
Copyright © 2003 by The Endocrine Society

BRIEF COMMUNICATION

Single-Cell Real-Time Quantitative Polymerase Chain Reaction of Immunofluorescently Identified Neurons of Gonadotropin-Releasing Hormone Subtypes in Cichlid Fish

Ishwar S. Parhar, Satoshi Ogawa, Tomohiro Hamada and Yasuo Sakuma

Department of Physiology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan

Address all correspondence and requests for reprints to: Ishwar S. Parhar, Department of Physiology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan. E-mail: ishwar{at}nms.ac.jp.

Abstract

The regulatory mechanisms controlling gene expression of GnRH subtypes, particularly of the evolutionarily conserved GnRH2, remain speculative. To address this issue, we have successfully coupled the anatomic specificity of immunofluorescently defined "cell picking" with the sensitivity of real-time quantitative RT-PCR (RT-Q-RT-PCR), which enabled us to examine the presence and quantity of GnRH mRNAs in individual neurons. Here, using RT-Q-RT-PCR, we report change in the levels of transcripts of GnRH subtypes in individual neurons harvested from the brain of mature and immature males of tilapia, Oreochromis niloticus. The levels of GnRH1 mRNA per cell and the percentage of neurons expressing GnRH1 transcripts exceeding 0.05 x 102 fg/cell were significantly higher in mature males (44.2%) compared with immature males (4.7%). In contrast, there was no difference in mRNA levels and the percentage of cells expressing GnRH2 and GnRH3 between the two reproductive states. Thus, using a novel approach that enables immunofluorescently labeled single-cell RT-Q-RT-PCR analysis of GnRH neurons, we present evidence that shows preoptic GnRH1 is important for gonadal maturation, whereas GnRH2 and GnRH3 might have supplementary roles in reproductive behaviors or nonreproductive functions. Furthermore, we speculate that the use of this method will allow the identification and quantification of known and unknown genes in single GnRH neurons, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells of GnRH variants in vertebrate species.

GnRH-I (GnRH1) IS EXPRESSED by a restricted group of neurons present in the septo-hypothalamic area, from which they coordinate reproduction and reproductive behavior in vertebrates (1). With the discovery of chicken GnRH II (GnRH2) in the midbrain, it has become increasingly clear that all vertebrate species ranging from fish to humans possess two or, as in advanced teleost, three GnRH subtypes (salmon GnRH, GnRH3) (2, 3, 4). All 16 GnRH variants identified in the animal kingdom participate in some aspect of reproduction, although the precise function and the regulatory mechanisms controlling gene expression of these GnRH subtypes remain speculative.

Because genes do not act in isolation, analyzing expression profiles of many genes in single cells would allow correlation of gene expression to cellular physiology of individual cells. To address this issue, we have initiated a research program that involves the successful combination of harvesting single GnRH cells coupled with real-time quantitative RT-PCR (RT-Q-RT-PCR) (5). The integration of these two technologies will allow the identification and quantification of known and unknown genes in single GnRH neurons, which would greatly facilitate our understanding of the complex interactions that exist within individual cells. As an initial step, we developed a rapid immunofluorescent cell picking procedure that allows harvesting immunoidentified individual GnRH neurons with precision and high preservation of mRNA for analysis. Furthermore, using RT-Q-RT-PCR, we analyzed the functional state of individual neurons of the three GnRH subtypes (GnRH1, GnRH2, GnRH3) in immature and mature males of tilapia, Oreochromis niloticus.

Materials and Methods

Brain slice preparations and GnRH immunofluorescence
Experimental procedures in the present study were performed under the Guidelines of the Animal Care Committee of Nippon Medical School. Tilapia (O. niloticus) were maintained in fresh water at 27 C with a natural photo regime (10 h of light, 14 h of darkness). This cichlid fish exhibits two classes of males: dominant, aggressive, reproductively active; and socially stressed, reproductively inactive subordinates. Dominant mature males (gonadosomatic index = 1.2 ± 0.2; values throughout text are mean ± SEM) and socially stressed immature males (gonadosomatic index = 0.4 ± 0.2) of almost equal size (11.1 ± 1.2 cm; n = 4 each) were anesthetized by immersing in a 0.01% solution of 3-aminobenzonic acid ethyl ester (MS222; Sigma, St. Louis, MO) before they were killed by decapitation between 1400 and 1700 h. The brains were dissected and fixed in 4% buffered paraformaldehyde for 1 h, embedded in 5% gelatin, and then chilled on ice for 30 min. Using a microslicer (Dousaka EM Co., Kyoto, Japan), 500-µm-thick coronal brain slices were made, starting from the rostral-most part of the olfactory bulb (zero point; Fig. 1Go). The brain slices were incubated for 12 h with primary antisera against salmon GnRH (lot 2; 1:3500; courtesy of K. Aida, University of Tokyo, Tokyo, Japan) for brain slices 1000–2000 µm; seabream GnRH (ISP-I; 1:4000) for brain slices 2000–3000 µm, and chicken GnRH-II (ISP-II; 1:3500) for brain slices 4000–5000 µm from the rostral-most part of the olfactory bulb (Fig. 1Go). Brain slices were then incubated at room temperature for 2 h with Cy3-conjugated antirabbit IgG (Jackson ImmunoResearch, West Grove, PA; diluted 1:400), which gave red fluorescence (Fig. 2Go).



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  		FIG. 1. A schematic illustration showing brain regions used for immunostaining and harvesting GnRH neurons (black dots). A, Caudal-most part of the olfactory bulbs (GnRH3); B, preoptic area (GnRH1); C, midbrain tegmentum (GnRH2). Numbers represent distances in micrometers from zero point (0) at the rostral-most part of the olfactory bulbs.

 


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  		FIG. 2. Photomicrographs of Cy3-labeled immunofluorescent GnRH neurons. A and B, Preoptic neurons (GnRH1); C and D, midbrain neurons (GnRH2); E and F, ganglia at the caudal-most olfactory bulbs (GnRH3); G, harvested cell in the micropipet. Arrows indicate cells before and after harvesting. Scale bar, 100 µm.

 
Harvesting single GnRH neurons
The brain slices were transferred into a culture dish containing lysis buffer (200 mM Tris-HCl, 200 mM NaCl, 1.5 mM MgCl2, 2% sodium dodecyl sulfate at pH 7.5) and observed under a fluorescence microscope (Olympus BX50 WI; Olympus Optical Co. Ltd., Tokyo, Japan) fitted with a micromanipulator (Narishige, Tokyo, Japan). Three different populations of immunolabeled single GnRH neurons were harvested from the brain of immature males (GnRH1, 43; GnRH2, 33; and GnRH3, 48 cells) and mature males (GnRH1, 43; GnRH2, 34; and GnRH3, 48 cells). Micropipettes were fabricated from thin-walled borosilicated glass microcapillaries (1.5-mm outer diameter, Harvard Apparatus Ltd., Edenbridge, Kent, UK), heated, and pulled using a micropipet puller (type PE-2, Narishige). Immunofluorescent GnRH neurons were identified with a x10 water immersion objective lens and WI filter (Olympus BX50). With the help of the micromanipulator, the micropipette was brought in contact with the cell soma. Using a negative pressure, the cell was harvested into the micropipette under visual control (Fig. 2Go) and subsequently expelled into a sterile 1.5-ml reaction tube containing 50 µl of the lysis buffer and stored in -80 C until the total RNA was extracted.

Single-cell RT-Q-RT-PCR for GnRH
The harvested single cell was digested with 1 µg of proteinase K (Gentra Systems, Minneapolis, MN) and 10 U prime ribonuclease inhibitor (Eppendorf, Hamburg, Germany) for an hour at 53 C. The cell lysate was incubated for 1 h at 37 C with 1 U ribonuclease-free deoxyribonuclease I (Promega, Madison, WI) to eliminate genomic DNA. Total RNA was extracted from the cell lysate using ISOGEN (Nippon Gene, Tokyo, Japan) and Mini RNA Isolation Kit (Zymo Research, Orange, CA), and reverse transcribed (RT) using SensiScript reverse transcriptase (QIAGEN, Hilden, Germany).

To confirm the presence and integrity of GnRH mRNA, the single-cell lysate was subjected to RT-PCR. PCRs were performed in a final volume of 20 µl containing GeneAmp 1x PCR Buffer, 160 µM of deoxynucleotide triphosphate, 1.0 U of DNA polymerase (AmpliTaq Gold, Applied Biosystems, Foster City, CA), 250 nM gene-specific primers (G1–6, GP1–2; Table 1Go), and one tenth of a single cell’s RT cDNA. Reaction conditions for PCRs were 94 C for 10 min, 50 cycles at 94 C for 15 sec and 60 C for 15 sec and 72 C for 15 sec, and 72 C for 7 min. Twenty microliters of the reaction mixture were run on a 2% agarose gel and visualized with ethidium bromide (Fig. 3Go). To confirm the sequences, some bands were subcloned into pGEM-T Easy vector (Promega), and both strands of the DNA were sequenced with T7 and SP6 promoter primers (Promega) using an ABI PRISM 310 Genetic Analyzer and Sequence Analysis Software (Applied Biosystems). Several controls were included for the RT-PCR: buffer without harvested cells, no-RT, and non-GnRH cells. For GnRH and non-GnRH cells, glial fibrillary acidic protein (GFAP) primers (GP1–2; Table 1Go) were also included in the PCR protocol. The GenBank accession numbers of the PCR primers (G1–6, GP1–2; Table 1Go) of the three GnRH variants and GFAP in tilapia are as follows: GnRH1, AB101665; GnRH2, AB101666; GnRH3, AB101667; and GFAP, AB109167. RT-Q-RT-PCR was performed in duplicate in 50-µl reaction volumes consisting of 1x TaqMan Universal PCR Master Mix (Applied Biosystems), 300 nM primers (G7, G8, G10, G11, G13, and G14), 200 nM hybridization probe (G9, G12, and G15), and one tenth of a single cell’s RT cDNA or control plasmid DNA using the ABI PRISM 7700 Sequence Detection System (TaqMan PCR, PE Applied Biosystems). The PCR conditions were 95 C for 10 min, followed by 75 cycles at 95 C for 15 sec, 60 C for 1 min.


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  		TABLE 1. GnRH primers and fluogenic probes

 


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  		FIG. 3. Composite gel showing amplicons from three representative preoptic GnRH1 (G1–G6), three midbrain GnRH2 (G7–G12), and three caudal olfactory bulb GnRH3 (G13–G18) neurons taken from immature male. Nested-PCR was necessary to observe GnRH2. With reverse transcriptase (RT+) and without (RT-). BUF, Buffer controls (B1–9). NGC, Non-GnRH cells surrounding GnRH1–3-immunoreactive neurons (C1–C9). M, Marker, a DNA 100-bp-size ladder. The sizes of the bands, in base pairs, is given in the right-hand margin.

 
Absolute values of GnRH transcripts from single GnRH neurons were obtained by establishing a linear amplification curve from serial dilutions (1 fg to 10 pg) of a plasmid DNA ligated with a GnRH cDNA fragment. Concentrations of plasmid DNA were measured at A260. Hybridization primers (G7, G8, G10, G11, G13, and G14) and fluorogenic GnRH probes (G9, G12, and G15) for real-time PCR were optimized using the ABI PRISM Primer Express Software. The number of target copies in each sample was interpolated from its detection threshold (CT) value using the plasmid DNA, normalized against the background fluorescence. Statistical analysis was performed using ANOVA followed by Scheffé’s F test.

Results and Discussion

Using the anterior-most part of the olfactory bulbs as the zero point allowed us to visualize the three populations of GnRH neurons with high precision when coronal sections (500 µm thick) were made of the brain of tilapia (~11.0 cm body length). Each antibody specifically labeled cells in only one of the three brain regions: in the preoptic area (GnRH1), midbrain tegmentum (GnRH2), and at the caudal-most part of the olfactory bulbs (GnRH3) (Fig. 2Go). These results are consistent with our earlier observations using in situ hybridization and immunocytochemistry (6, 7). Furthermore, although only 60–70% of GnRH1- and GnRH3- and 25% of GnRH2-immunoreactive neurons were seen to contain GnRH transcripts by RT-PCR (Fig. 3Go); the more sensitive RT-Q-RT-PCR showed 100% of GnRH1–3 neurons had GnRH transcripts, and the yield of total RNA from a single neuron was sufficient for analysis (Fig. 4Go). The amplicon sizes were approximately 196 (GnRH1), 176 (GnRH2), and 218 bp (GnRH3) (Fig. 3Go), and their sequences were identical with tilapia GnRHs (see Materials and Methods for GenBank accession numbers). There was no genomic DNA contamination in the harvested single cells.



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  		FIG. 4. Graphs showing the distribution of GnRH1, GnRH2, and GnRH3 mRNA levels per cell. Note that all GnRH1–3 neurons have detectable level of mRNA, although a significant number of GnRH2 neurons have low abundance of mRNA. The x-axis represents the value of each individual cell. Black dots, Immature males; red dots, mature males.

 
Using RT-Q-RT-PCR, a large variation in GnRH1–3 transcript levels was observed within cells, similar to our published study using in situ hybridization (7). Significantly higher levels of GnRH1 mRNA were observed in mature males (range, 0.011–0.19 x 102 fg/cell; mean, 0.051 ± 0.006; n = 43) compared with immature males (range, 0.006–0.053 x 102 fg/cell; mean, 0.023 ± 0.002; P < 0.01; n = 43; Figs. 4Go and 5Go). Furthermore, the percentage of neurons expressing GnRH1 transcripts exceeding 0.05 x 102 fg/cell in mature males was higher (44.2%) compared with immature males (4.7%). From these results, it is apparent that GnRH1 mRNA levels are dependent upon testicular maturation. In addition, developmental studies have shown the expression of preoptic GnRH1 and fiber projections to the pituitary gonadotropes coincide precisely with the onset of gonadal sex differentiation in tilapia (6). GnRH1 neurons might also be socially regulated as suggested in a related fish species (Haplochromis burtoni), with dominant males having larger cells and higher mRNA content than subordinates (8), which remains to be seen in tilapia. In contrast to GnRH1, there was no difference in mRNA levels of GnRH2 (mature males: range, 0.0001–0.18 x 102 fg/cell; mean, 0015 ± 0.006; n = 34; immature males: range, 0.0002–0.2 x 102 fg/cell; mean, 0.0171 ± 0.008; n = 33; Figs. 4Go and 5Go) and GnRH3 (mature males: range, 0.001–0.77 x 102 fg/cell; mean, 0.23 ± 0.03; n = 48; immature males: range, 0.001–0.67 x 102 fg/cell; mean, 0.204 ± 0.028; n = 48; Figs. 4Go and 5Go) between the males. Furthermore, the percentage of neurons expressing GnRH2 transcripts exceeding 0.05 x 102 fg/cell (mature males, 5.9%; immature males, 12.1%) and neurons expressing GnRH3 transcripts exceeding 0.05 x 102 fg/cell (mature males, 70.8%; immature males, 62.5%) were not different between the males. An intriguing outcome of these studies is that no changes in mRNA levels were detected for either GnRH2 or GnRH3. Only rudimentary data support the role of the evolutionarily conserved GnRH2 in reproductive behavior in rodents and primitive mammals (9, 10). From the present results, it is clear that more than 85% of GnRH2 neurons have low abundance of mRNA ("silent cells"); this precludes their role in reproduction, which is supported by their insensitivity to steroid hormones (7), but strengthens support for nonreproductive function such as the control of prolactin release (4). Whether reproduction is regulated at the translational level remains to be seen. Given that GnRH3 neurons are clustered to form ganglia (nucleus olfactoretinalis), using in situ hybridization to observe mRNA change in individual neurons was technically difficult because of the overlap of silver grains from neighboring cells. The present study overcomes this technical problem. At the single-cell level, it is obvious that, even in socially stressed immature males, GnRH3 neurons maintain a significantly high level of the mRNA (Figs. 4Go and 5Go), which gives these animals tactical advantage to participate in reproductive behaviors at any given opportunity (Soga, T., S. Ogawa, Y. Sakuma, and I. Parhar, unpublished observations). The present procedure is a novel approach through which single immunoidentified GnRH neurons can be harvested routinely for gene profiling of inhibitory and stimulatory molecules for quantitative studies, which would greatly facilitate our understanding of the molecular physiology of GnRH variants in vertebrate species.



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  		FIG. 5. Histograms showing GnRH1–3 mRNA per cell. Average values of GnRH mRNA per cell were deduced from the total number of individual cells analyzed for each maturational stage and for each GnRH subtype (mean ± SEM). Statistical analysis showed significant difference in GnRH1 between mature (MM) and immature males (IM).

 

Footnotes

Abbreviations: GFAP, Glial fibrillary acidic protein; RT, reverse-transcribed; RT-Q-RT-PCR, real-time quantitative RT-PCR.

Received March 26, 2003.

Accepted for publication May 27, 2003.

References

  1. Silverman AJ, Livne I, Witkin JW 1994 The gonadotropin-releasing hormone (GnRH) neuronal systems: immunocyto-chemistry and in situ hybridization. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press; 1683–1709
  2. Millar RP 2003 GnRH II and type II GnRH receptors. Trends Endocrinol Metab 14:35–43[CrossRef][Medline]
  3. Terasawa E 2003 Gonadotropin-releasing hormone II: is this neuropeptide important for mammalian reproduction? Endocrinology 144:3–4[Free Full Text]
  4. Parhar IS 2002 Cell migration and evolutionary significance of GnRH subtypes. Prog Brain Res 141:3–17[Medline]
  5. Liss B 2002 Improved quantitative real-time RT-PCR for expression profiling of individual cells. Nucleic Acids Res 30:e89
  6. Parhar IS 1997 GnRH in tilapia: three genes, three origins and their roles. In: Parhar IS, Sakuma Y, eds. GnRH neurons, gene to behavior. Tokyo: Brain Shuppan Publishers; 99–122
  7. Parhar IS, Soga T, Sakuma Y 2000 Thyroid hormone and estrogen regulate brain region-specific messenger ribonucleic acids encoding three gonadotropin-releasing hormone genes in sexually immature male fish, Oreochromis niloticus. Endocrinology 141:1618–1626[Abstract/Free Full Text]
  8. White SA, Nguyen T, Fernald RD 2002 Social regulation of gonadotropin-releasing hormone. J Exp Biol 205:2567–2581[Abstract/Free Full Text]
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  10. Temple JL, Millar RP, Rissman EF 2003 An evolutionarily conserved form of gonadotropin-releasing hormone coordinates energy and reproductive behavior. Endocrinology 144:13–19[Abstract/Free Full Text]



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