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Endocrinology, doi:10.1210/en.2007-0311
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Endocrinology Vol. 148, No. 12 5822-5830
Copyright © 2007 by The Endocrine Society

Sexual Maturation Modulates Expression of Nuclear Receptor Types in Laser-Captured Single Cells of the Cichlid (Oreochromis niloticus) Pituitary

Takashi Kitahashi, Satoshi Ogawa, Tomoko Soga, Yasuo Sakuma and Ishwar Parhar

School of Medicine and Health Sciences (T.K., S.O., T.S., I.P.), Monash University, 46150 Bandar Sunway, Malaysia; and Department of Physiology (T.K., S.O., T.S., Y.S., I.P.), Nippon Medical School, Sendagi, Tokyo 113-8602, Japan

Address all correspondence and requests for reprints to: Professor Ishwar Parhar, School of Medicine and Health Sciences, Monash University, 46150 Bandar Sunway, Selangor, Malaysia. E-mail: ishwar{at}med.monash.edu.my.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of steroid/thyroid hormones in the regulation of endocrine cells at the level of the pituitary has remained unclear. Therefore, using single-cell quantitative real-time PCR, we examined absolute amounts of transcripts for nuclear receptors [estrogen receptors (ERs) {alpha}, ß, and {gamma}; androgen receptors (ARs) a and b; glucocorticoid receptors (GRs) 1, 2a, and 2b; and thyroid hormone receptors (TRs) {alpha}1, {alpha}2, and ß] in pituitary cells of immature (IM) and mature (M) male tilapia, Oreochromis niloticus. In the two reproductive stages, ACTH cells expressed only ERß, whereas all other pituitary cell types expressed ER{alpha} + ß, and a subpopulation coexpressed ARa, ARb, GR1, GR2b, and TRß but lacked ER{gamma}, GR2a, TR{alpha}1, and TR{alpha}2. IM males had high percentages of LH cells (IM 46.0% vs. M 10.0%), GH cells (IM 23.3% vs. M 7.9%), and prolactin cells (IM 68.8% vs. M 6.0%) with ERß, and TSH cells (IM 19.2% vs. M 0.0%) and MSH cells (IM 25.6% vs. M 0.0%) with ER{alpha} + TRß. A high percentage of FSH cells in IM males expressed ERß (IM 46.9% vs. M 18.8%), and FSH cells in M males showed significantly high GR1 transcripts (IM 76.0 ± 5.0 vs. M 195.0 ± 10.7 copies per cell; P < 0.05), suggesting that FSH cells are regulated differently in the two reproductive stages. Coexpression of ER{alpha} + ß in high percentages of cells of the GH family (GH, IM 43.8% vs. M 14.3%; prolactin, IM 8.3% vs. M 59.7%; somatolactin, IM 22.2% vs. M 42.2%) suggests that the expression of both ERs is important for functionality. Thus, differential coexpression of genes for nuclear receptors in subpopulations of pituitary cell types suggests multiple steroid/thyroid hormone regulatory pathways at the level of the pituitary during the two reproductive stages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL documented that steroid/thyroid hormones regulate the synthesis and release of pituitary hormones [FSH, LH, TSH, GH, prolactin (PRL), somatolactin (SL), ACTH, and MSH] through complex interactions with hypothalamic neurons.

There is evidence that steroid/thyroid hormones can influence the synthesis and release of pituitary hormones in in vitro cultures (1, 2, 3, 4). Furthermore, several studies in teleosts have shown the presence of binding sites for steroid hormones in the pituitary (5), and the presence of glucocorticoid receptor (GR) in FSH and LH cells (6), as well as aromatase in the pituitary (7). These facts suggest direct effects of steroid/thyroid hormones and aromatizable androgen at the level of the pituitary.

The recent discovery of more than one steroid/thyroid hormone receptor (TR) subtype [estrogen receptors (ERs) {alpha}, ß, and {gamma} (8); androgen receptors (ARs) {alpha} and ß (9, 10); GR1, GR2a, and GR2b (11); and TR{alpha}1, TR{alpha}2, and TRß (12)] in vertebrates raises new questions as to whether multiple types or subtypes of transcripts for nuclear receptors are coexpressed in individual pituitary cells. Identification of nuclear receptor expression in pituitary endocrine cells will help to elucidate the mechanisms of action of steroid/thyroid hormones at the pituitary level, which presently remains unclear in vertebrates.

To address these questions, we examined the expression of genes for multiple nuclear receptor types in individual pituitary cells during sexual maturation in the tilapia, Oreochromis niloticus. We used a novel technique integrating laser capture microdissection (LCM) of single digoxigenin (DIG)-labeled pituitary cells coupled with quantitative real-time PCR that allows precise harvesting of identified cells, high preservation of mRNA for analysis, and high sensitivity for the detection of multiple targets (13). Furthermore, we used immunocytochemistry to confirm the presence of GR and aromatase in pituitary cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Experimental procedures in the present study were performed under the guidelines of the Animal Care Committee of Nippon Medical School, Tokyo. The tilapia used in the present study were maintained in fresh water at 27 ± 1 C with a natural photo regime (14- and 10-h light and dark cycles, respectively).

Tissue preparation and in situ hybridization for pituitary hormones
Immature (IM) (standard length = 6.5 ± 0.3 cm; body weight = 11.6 ± 3.4 g; gonadosomatic index = 0.25 ± 0.04; n = 25) and mature (M) (standard length = 13.7 ± 0.4 cm; body weight = 89.4 ± 6.9 g; gonadosomatic index = 1.23 ± 0.18; n = 20) males were anesthetized by immersing in a 0.01% solution of 3-aminobenzoic acid ethyl ester (Sigma, St. Louis, MO) before they were killed by decapitation. Gonads were fixed in Bouin’s solution, processed through ethanol series, and embedded in Paraplast Plus (Oxford Labware, St. Louis, MO) for histological examination. The brains with pituitaries attached were dissected and fixed in 4% buffered paraformaldehyde for 6 h at room temperature, cryoprotected in 20% sucrose, and embedded in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan). Pituitary sections were cut in sagittal planes (6 µm), mounted onto aminopropyl triethoxy silane-treated glass slides (Matsunami Glass, Tokyo, Japan), and stored at –80 C until use for localization of pituitary cell types.

For DIG in situ hybridization, we used nucleotide sequences of cDNA for tilapia pituitary hormones obtained from the GenBank [FSHß subunit, AF289173; LHß subunit, AY294016; TSHß subunit, AB120769; GH1, M97766; PRL188, M27010; SL, AB120767; proopiomelanocortin (POMC) = ACTH and MSH, AF116240]. Because GH1 and GH2 mRNAs encode an identical polypeptide (14), and PRL188 and PRL177 are colocalized in the same pituitary cells in the tilapia (15), we used GH1 and PRL188 cDNAs to synthesize riboprobes for in situ hybridization. The pGEM-T Easy transcription vector constructs (Promega, Madison, WI) that contained the cDNA were linearized with Spe I or Nco I endonuclease (Nippon Gene, Tokyo, Japan) and used as templates. Sense and antisense riboprobes were synthesized with T7 or SP6 RNA polymerase (Toyobo, Tokyo, Japan) and DIG RNA-labeling mix (Roche Diagnostics, Tokyo, Japan). DIG in situ hybridization was performed on sagittal pituitary sections, as described previously (16, 17).

LCM of pituitary cells
The cell harvesting protocol was the same as that described elsewhere (13, 17, 18). The dehydrated pituitary section was overlaid with a thermoplastic membrane mounted on an optically transparent cap (CapSure Macro LCM Caps; Arcturus, Mountain View, CA). Using a Pix Cell II Laser capture instrument (Arcturus), DIG-identified pituitary cells were microdissected by focal melting of the membrane through laser activation (laser pulse power, 25–65 mW; laser pulse duration, 1.5 msec; laser spot size, 10 µm diameter). Because the laser beam was larger than the diameter of the cells, undesirable tissue was also captured along the periphery of the cells. Heat-pulled borosilicate glass microcapillary pipette (1.5-mm outer diameter, Harvard Apparatus Ltd., Edenbridge, Kent; micropipette puller; Type PE-2, Narishige, Tokyo, Japan) attached to a micromanipulator (Narishige) was used to remove undesirable tissue around the periphery of the single-laser captured cells. By using negative pressure, single cells were then harvested from the LCM cap into the micropipette under visual control, then expelled into a sterile 1.5-ml reaction tube containing 50 µl of the lysis buffer individually, and stored at –80 C until total RNA isolation. For unbiased cell sampling, eight to 10 cells were harvested at random (approximately two cells per alternate section) along the rostral-caudal extent of the whole population of each pituitary cell type in each animal.

RT-PCR for pituitary hormones and nuclear receptors in pituitary cells
The conditions for RT-PCR were similar to those described elsewhere (13, 17, 18). Briefly, the harvested single cell from the pituitary was digested with 1 µg proteinase K (Gentra Systems, Minneapolis, MN) for 1 h at 53 C. The cell lysate was incubated for 1 h at 37 C with 1 U ribonuclease-free DNase I (Promega) to eliminate genomic DNA, and heat denatured at 95 C for 10 min to separate the mRNA from the DIG-labeled riboprobe. Total RNA was extracted from the cell lysate by ISOGEN (Nippon Gene), and reverse transcribed to cDNA in a reaction mixture (20 µl) with 100 pmol random hexamer (Takara, Tokyo, Japan) and 10 U Prime RNase Inhibitor (Eppendorf, Hamburg, Germany) using 40 U SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA).

To confirm the presence of pituitary hormone transcripts, the single cell’s cDNA was subjected to RT-PCR using gene-specific primers for pituitary hormones (Table 1Go) and one 20th of a single cell’s cDNA solution. The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualized by illumination with UV light. Only those cells that were positive for each pituitary hormone mRNA were used for further analysis (n = 3–9 cells per animal; 26–73 cells per pituitary hormone cell type per age group).


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  		TABLE 1. Sequences of PCR primers and fluorogenic probes used for RT-PCR and real-time PCR

 
To analyze the presence of nuclear receptors in pituitary cells, we cloned and identified partial cDNA sequences of tilapia ERß (GenBank accession no. AB110981), GR1 (GenBank accession no. AB245405), and GR2b (GenBank accession no. AB245406), whereas sequences of other tilapia nuclear receptors were obtained from the GenBank (accession nos.: ER{alpha}, U75604; ER{gamma}, U75605; ARa, AB045211; ARb, B045212; TR{alpha}1, AF302248; TR{alpha}2, AF302249; and TRß, AF302247). One tenth of the single cell’s cDNAs was pooled from 25 samples of each cell type, and then one 12th of the pooled cDNA was subjected to PCR using gene-specific primers (Table 1Go). As positive controls, cloned cDNA for nuclear receptors were subjected to PCR. No-RT samples were also subjected to PCR as negative controls. The PCR products were electrophoresed on a 4% agarose gel, stained with ethidium bromide, and visualized by illumination with UV light.

Real-time PCR of nuclear receptors in pituitary cells
We used cDNA samples of GH, PRL, SL, MSH, and ACTH cells that had been prepared in our previous study (17). Moreover, we harvested FSH, LH, and TSH cells from pituitary sections that were prepared at the same time and stored at –80 C, as we required more cDNA samples. To quantify copies of nuclear receptor transcripts in pituitary cells, cDNAs from single cells were subjected to real-time PCR, which was performed in 10 µl reaction volumes consisting of 1x TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), 300 nM of each forward and reverse nuclear receptor primer, 200 nM TaqMan probe (Table 1Go), and one 12th of a single cell’s cDNA solution or standard cDNA using a ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Amplification was performed at 50 C for 2 min, 95 C for 10 min, 45 cycles of 95 C for 15 sec, and 60 C for 1 min. The absolute amounts of transcripts were determined using several concentrations of standard cDNA (10, 30, 102, 103, 104, and 105 copies) that were reverse transcribed from serially diluted standard RNAs. The copy numbers of transcripts per reaction were multiplied by 12 to obtain the copy numbers of transcripts per cell. For each pituitary cell type, given copies of transcripts per cell were combined to give experimental group means. All values are expressed as mean ± SEM, and statistical comparisons were made between IM and M males using the Student’s t test. P less than 0.01 and less than 0.05 were considered statistically significant.

Immunocytochemistry
Three adult male tilapia were anesthetized by immersing in a 0.01% solution of 3-aminobenzoic acid ethyl ester, and killed by decapitation. The brains with attached pituitaries were dissected out, fixed in Bouin’s solution overnight at 4 C, processed through a graded series of ethanol, and embedded in Paraplast Plus. Serial sections were cut in sagittal planes (15 µm) and processed for nuclear receptor immunocytochemistry as described before (19).

Deparaffinized sections were incubated with primary antisera [anti-tilapia GR1 diluted 1:2000; RACR21 (20), raised against synthetic peptide corresponding to a hydrophilic portion of hormone binding domain of tilapia GR1, which probably recognizes both GR1 and GR2 in tilapia (kindly provided by Dr. Masatomo Tagawa, Kyoto University, Kyoto, Japan), and anti-tilapia aromatase diluted 1:6000 was raised against synthetic peptide corresponding to the C-terminal region of tilapia aromatase (21) (kindly provided by Dr. Masaru Nakamura, University of the Ryukyu, Okinawa, Japan)] for 48 h at 4 C. The specificities of these antisera have been confirmed and reported previously (20, 22, 23). The sections were incubated with biotinylated anti-IgG for 30 min, "ABC" complex for 60 min (Vectastain "ABC" Elite kit; Vector Laboratories, Burlingame, CA), and then treated with 0.05% 3, 3' diaminobenzidine tetrahydrochloride (Sigma) with 0.03% H2O2 for 15 min. Immunoreactivity was seen as brown granular reaction product in certain cells and fibers in the pituitary.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gonadal status of IM and M fish
Gonadal sections stained with cresyl violet showed that IM males possessed only spermatogonia in the testis, whereas M males possessed all stages of germinal cells (spermatogonia, primary spermatogonia, secondary spermatogonia, spermatocyte, spermatid, and spermatozoa), including an abundance of spermatozoa (Fig. 1Go).


Figure 1
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  		FIG. 1. Photomicrographs of gonadal sections of IM and M males. A, IM fish. B, M fish. Scale bars, 20 µm. Lc, Leydig cell; psg, primary spermatogonia; sc, spermatocyte; sg, spermatogonia; ssg, secondary spermatogonia; st, spermatid; sz, spermatozoa.

 
Localization of pituitary endocrine cells
DIG in situ hybridization showed localization of pituitary cells in IM and M males in: rostral pars distalis (PRL, TSH, and ACTH cells); proximal pars distalis (FSH, LH, and GH cells); and pars intermedia (SL and MSH cells) (Fig. 2Go). The distribution pattern of pituitary endocrine cells was similar to that in our earlier report (17).


Figure 2
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  		FIG. 2. Photomicrographs of DIG-labeled endocrine cells in midsagittal sections of the pituitary. A, A diagram showing sectioning plane of the pituitary. B, An illustration showing localization of pituitary cell types in a sagittal section at the level of plane I in A. C, FSH cells. D, LH cells. E, TSH cells. Inset, TSH cells at higher magnification. F, GH cells. G, PRL cells. H, SL cells. I, POMC (MSH and ACTH). Inset, ACTH cells at high magnification. J and K, DIG-labeled FSH cells before (J) and after (K) LCM. Arrows indicate the harvested cells. Scale bars, 200 µm in C–I, 40 µm in Inset E, 50 µm in Inset I, and 10 µm in J and K. Note that all photomicrographs except for F were taken from midsagittal sections at the level of plane (I) in A. F was photographed from a more lateral section to the midsagittal plane (II) in A. NeH, Neurohypophysis; PI, pars intermedia.

 
Nuclear receptors in pituitary cells
There was no genomic DNA contamination in the total RNA extracted from harvested single cells of the pituitary (Fig. 3Go). The amplicon sizes of pituitary hormone and nuclear receptor cDNAs were of the expected sizes (Fig. 3Go; see Materials and Methods for GenBank accession numbers). Because GR2a and GR2b are splice variants, the primers used for RT-PCR recognized both transcripts, and the expected amplicon sizes of GR2a and GR2b were 95 and 122 bp, respectively. RT-PCR revealed that pituitary cell types had ER{alpha}, ERß, ARa, ARb, GR1, GR2b, and TRß but lacked ER{gamma}, GR2a, TR{alpha}1, and TR{alpha}2 transcripts (Fig. 3Go). A subpopulation of pituitary cell types coexpressed multiple nuclear receptors (see below for details).


Figure 3
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  		FIG. 3. Composite gel showing existence of mRNAs for pituitary hormones and nuclear receptors in FSH, LH, TSH, GH, PRL, SL, MSH, and ACTH cells taken from IM males. For each cell type, cDNAs from 25 cells were combined and subjected to PCR. Note that PCR primers for GR2b can amplify both splice variants. The POMC transcript encodes a precursor peptide of MSH and ACTH. The sizes of the bands, in base pairs, are given in the right-hand margin. M, Marker, stable 100-bp DNA Ladder (SIGMA GENOSYS, Sapporo, Japan); PC, positive control; RT-, without reverse transcriptase.

 
Quantitative analysis of nuclear receptors in pituitary cells
Real-time PCR showed that 4.7–100% of pituitary cells expressed single or multiple nuclear receptor genes (Table 2Go). A large variation in copies of nuclear receptor transcripts was observed between individual cells (Figs. 4Go, A–C, and 5Go, A–E, left panels). ER{alpha} and/or ERß transcripts were predominant in most pituitary cell types. Absolute copies of ARa and GR2b transcripts in FSH cells, ARa and ARb in LH cells, GR1 in GH cells, and TRß in MSH cells were unquantifiable in M males (Figs. 4Go, A–C, and 5Go, A–E, right panels).


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  		TABLE 2. Percentage of pituitary cells with single or multiple nuclear receptor transcripts

 

Figure 4
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  		FIG. 4. Expression of nuclear receptor genes in FSH, LH, and TSH cells. Left, Graphs showing the expression of nuclear receptor genes in individual cells expressing FSH (A) (IM 49; M 32 cells), LH (B) (IM 50; M 40), and TSH (C) (IM 26; M 33) taken from IM (short bars) and M (triangles) males. The x-axis represents cell identity numbers, and the y-axis represents copies of nuclear receptor transcripts per cell. The short bars and triangles below "zero" are unquantifiable levels of transcripts in positively identified pituitary cells. Right, Histograms showing the average copies of nuclear receptor transcripts per cell that possess a quantifiable amount of nuclear receptor transcripts in cells expressing FSH (A), LH (B), and TSH (C) taken from IM (open bars) and M males (filled bars). *, P < 0.05. by Student’s t test. Cell numbers that possess a quantifiable amount of nuclear receptor transcripts are indicated in the right panels.

 

Figure 5
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  		FIG. 5. Expression of nuclear receptor genes in GH, PRL, SL, MSH, and ACTH cells. Left, Graphs showing the expression of nuclear receptor genes in individual cells expressing GH (A) (IM 73; M 63 cells), PRL (B) (IM 48; M 67), SL (C) (IM 72; M 45), MSH (D) (IM 39; M 40 cells), and ACTH (E) (IM 50; M 43 cells) taken from IM (short bars) and M (triangles) males. The x-axis represents cell identity numbers, and the y-axis represents copies of nuclear receptor transcripts per cell. The short bars and triangles below "zero" are unquantifiable levels of transcripts in positively identified pituitary cells. Right, Histograms showing the average copies of nuclear receptor transcripts per cell that possess a quantifiable amount of nuclear receptor transcript in cells expressing GH (A), PRL (B), SL (C), MSH (D), and ACTH (E) taken from IM (open bars) and M males (filled bars). *, P < 0.05; **, P < 0.01; by Student’s t test. Cell numbers that possess a quantifiable amount of nuclear receptor transcripts are indicated in the right panel.

 
Glycoprotein hormones (FSH/LH/TSH)
FSH cells.
FSH cells had ER{alpha}, ERß, ARa, GR1, and GR2b transcripts (Fig. 3Go). A significant percentage of FSH cells expressed single or multiple nuclear receptors (IM 79.6%, 39 of 49 cells; M 56.3%, 18 of 32 cells) (Table 2Go). The percentage of FSH cells possessing only ERß transcripts was relatively higher in IM than M males (IM 46.9% vs. M 18.8%). Expression of GR1 was seen only in M males (Table 2Go). Of FSH cells in IM and M males, 28.6 and 6.2%, respectively, expressed multiple nuclear receptors. ERß + ARa combination was seen in 14.3% of FSH cells only in IM males. Absolute copies of GR1 transcripts in FSH cells were significantly higher in M than IM males (IM 76.0 ± 5.0 vs. M 195.0 ± 10.7 copies per cell; P < 0.05) (Fig. 4AGo). Absolute copies of ER{alpha}, ERß, ARa, and GR2b were statistically nonsignificant between the two reproductive stages.

LH cells.
LH cells had ER{alpha}, ERß, ARa, ARb, and GR1 transcripts (Fig. 3Go). The total percentage of LH cells expressing single or multiple nuclear receptor was higher in IM males (IM 100%, 50 of 50 cells vs. M 47.5%, 19 of 40 cells) (Table 2Go). A higher percentage of LH cells in IM males expressed ERß (IM 46.0% vs. M 10.0%) and ERß + ARa (IM 20.0% vs. M 0.0%) when compared with those in M males. Absolute copies of ER{alpha}, ERß, ARa, ARb, and GR1 transcripts were statistically nonsignificant between the two reproductive stages (Fig. 4BGo).

TSH cells.
TSH cells had ER{alpha}, ERß, ARa, and TRß transcripts (Fig. 3Go). The total percentage of TSH cells expressing single or multiple nuclear receptors was higher in IM than M males (IM 96.2%, 25 of 26 cells vs. M 39.4%, 13 of 33 cells) (Table 2Go). The percentage of TSH cells expressing TRß alone or in combination with other receptors was higher in IM than M males (IM 88.5% vs. M 3.0%). Absolute copies of transcripts for ER{alpha}, ERß, ARa, and TRß were statistically nonsignificant between the two reproductive stages (Fig. 4CGo).

GH family (GH/PRL/SL)
GH cells.
GH cells had ER{alpha}, ERß, and GR1 transcripts (Fig. 3Go). The total percentage of GH cells expressing single or multiple nuclear receptors was higher in IM than M males (IM 87.7%, 64 of 73 cells vs. M 34.9%, 22 of 63 cells) (Table 2Go). A large percentage of cells in IM males expressed ERß (IM 23.3% vs. M 7.9%) and ER{alpha} + ß (IM 43.8% vs. M 14.3%). The absolute copies of ER{alpha} and ERß transcripts were statistically nonsignificant between the two reproductive stages (Fig. 5AGo).

PRL cells.
PRL cells had ER{alpha} and ERß transcripts (Fig. 3Go). PRL cells expressed ER{alpha}, ERß, or ER{alpha} + ß (IM 79.2%, 38 of 48 cells; M 83.6%, 56 of 67 cells) (Table 2Go). A higher percentage of PRL cells expressed ERß alone in IM males (IM 68.8% vs. M 6.0%) and ER{alpha} + ß in M males (IM 8.3% vs. M 59.7%). Absolute copies of ER{alpha} transcripts in PRL cells were significantly higher in M males (IM 189.6 ± 45.4 vs. M 1784.1 ± 369.9 copies per cell) (P < 0.01; Fig. 5BGo).

SL cells.
SL cells had ER{alpha} and ERß transcripts (Fig. 3Go). SL cells expressed ER{alpha}, ERß, or ER{alpha} + ß (IM 54.2%, 39 of 72 cells; M 84.4%, 38 of 45 cells) (Table 2Go), but a higher percentage of cells in M males expressed ER{alpha} + ß when compared with IM males (IM 22.2% vs. M 42.2%). Absolute copies of ER{alpha} transcripts (IM 184.8 ± 21.2 vs. M 2911.7 ± 643.9 copies per cell) and ERß transcripts (IM 296.4 ± 76.8 vs. M 997.3 ± 312.0 copies per cell) in SL cells were significantly higher in M males (P < 0.01; Fig. 5CGo).

POMC family (MSH/ACTH)
MSH cells.
MSH cells had ER{alpha}, ERß, and TRß transcripts (Fig. 3Go). The total percentage of MSH cells expressing single or multiple nuclear receptors was higher in IM than M males (IM 94.9%, 37 of 39 cells vs. M 32.5%, 13 of 40 cells) (Table 2Go). MSH cells expressing TRß gene alone or in combination with ER{alpha} or ERß were found only in IM males. Absolute copies of ER{alpha} transcripts were significantly high in IM males (IM 402.4 ± 73.7 vs. M 213.4 ± 32.4 copies per cell; P < 0.05) (Fig. 5DGo).

ACTH cells.
A small percentage of ACTH cells in both reproductive stages had ERß transcripts (IM 10.0%, 5 of 50 cells vs. M 4.7%, 2 of 43 cells) (Table 2Go). Absolute copies of ERß transcript were 345.0 ± 82.0 copies per cell in IM males and 80.2 copies per cell in M males (Fig. 5EGo).

GR and aromatase immunoreactivity in the pituitary
GR immunoreactivity was seen in the proximal pars distalis, where FSH, LH, and GH cells are located, and aromatase immunoreactivity was seen in the fiber terminals of the proximal pars distalis and pars intermedia (Fig. 6Go). Buffer controls did not show any immunoreactivity in the pituitary sections (data not shown).


Figure 6
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  		FIG. 6. GR and aromatase immunoreactivity in a midsagittal section of the pituitary. A, GR immunoreactivity in the proximal pars distalis (FSH/LH/GH cell zone). B, Aromatase immunoreactivity (arrows) in the neurohypophysis of the proximal pars distalis. C, Aromatase immunoreactivity in the neurohypophysis of the pars intermedia (MSH/SL cell zone). Scale bars, 200 µm in A, and 20 µm in B and C. NeH, Neurohypophysis; PI, pars intermedia; PPD, proximal pars distalis; RPD, rostral pars distalis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows coexpression of multiple nuclear receptors in individual pituitary cells, which confirms direct action of steroid/thyroid hormones at the level of the pituitary. In the two reproductive stages, ACTH cells expressed only ERß, whereas all other pituitary cell types expressed ER{alpha} + ß, and coexpressed ARa, ARb, GR1, GR2b, and TRß in a subpopulation (Fig. 7Go). All of the pituitary cell types lacked ER{gamma}, GR2a, TR{alpha}1, and TR{alpha}2. In the GH family, a high percentage of ER{alpha} transcript-containing cells also contained ERß transcripts, indicating the possibility of ER heterodimerization for functionality.


Figure 7
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  		FIG. 7. A diagram summarizing the presence of nuclear receptors in pituitary cell types. PRL, SL, and ACTH cells possess only ERs. All other pituitary cell types possess ERs; in addition, FSH and LH cells also possess ARs and GRs, GH cells GR1, MSH cells TRß, and TSH cells TRß and ARa. Differential coexpression of nuclear receptor genes in subpopulations of pituitary cell types suggests that multiple steroid/thyroid hormone regulatory pathways exist at the level of the pituitary, of which the E2-mediated pathways are the most common in all cell types. F, Cortisol. Arrows from T–E2 represent aromatization of T–E2.

 
Glycoprotein hormone family
In mammals, immunocytochemical studies have shown the presence of ERs and ARs in FSH cells (24, 25), and the regulation of FSH cells by 17ß-estradiol (E2) and testosterone (T) at the pituitary cell level (26). The existence of ER and AR transcripts in FSH cells, shown in the present study, and their response to E2 and T in vitro culture system (27) suggest evolutionally conserved regulatory mechanisms in FSH cells from fish to mammals. The relatively insignificant percentage of FSH cells that possess AR transcripts and the very low copies of AR transcripts in them suggest that the stimulatory effect of T on cultured FSH cells in male tilapia (27) is mediated by aromatization of T to E2. This idea is further supported by the presence of aromatase immunoreactivity in the FSH cell zone (7) (present study) and the existence of half-site of estrogen response element in the 5' flanking region of tilapia FSHß gene (28). The role of AR in FSH cells, if any, is probably limited to IM fish because no AR transcripts were detected in M males.

The presence of GR transcripts and GR immunoreactivity in FSH cells in teleost (6) and mammals (29) indicates direct action of cortisol on FSH cells. As absolute copies of GR transcripts increased in FSH cells during maturation, the inactivation of reproductive functions by cortisol (30) may occur by inhibiting FSH cell activity.

Furthermore, the increased levels of GR and ER{alpha} transcripts in M males suggest that FSH cells have an important role in the M stage, as well as in the IM stage (31), which supports our previous findings of increased GnRH receptors in FSH cells in M males (17).

Our demonstration of ER and AR transcripts in LH cells is consistent with the results of immunocytochemical studies in mammals (24, 25), and suggests that there is a direct effect of E2 and T on LH cells at the pituitary level, as observed in cultured pituitary cells of teleosts (27, 32) and mammals (26).

LH cells in IM and M males had different combinations of nuclear receptors, suggesting steroid hormones use different pathways to regulate IM and M LH cells. IM fish had a large percentage of LH cells that express ERß alone or in combination with ARs when compared with M fish, suggesting the importance of these receptors during IM stages.

In M males, only a small number of LH cells possessed AR transcripts, and the amounts of AR transcripts were below measurable level. In addition, in sexually regressed male tilapia, only T but not nonaromatizable androgen (11-ketotestosterone) had stimulatory effect on LHß gene expression (27). Therefore, as in FSH cells, the effect of T on LHß gene expression could be mediated by the conversion of T to E2, which is supported by the presence of aromatase in the proximal pars distalis (7) (present study), where LH cells reside, and half-sites of estrogen response element in the promoter region of LHß gene.

In mammals, the existence of ER immunoreactivity (33) and ER transcripts (34) has been shown in TSH cells. Furthermore, estrogen (estradiol benzoate) and androgen (T propionate) treatments stimulated TSH release from these cells (35, 36), which suggests that sex steroids can directly regulate TSH cells in vertebrate. The present study reconfirms these findings and provides evidence for direct actions of sex steroids through ER{alpha}, ERß, and ARa in TSH cells in teleosts. The existence of TRß transcripts in TSH cells (37) (present study) and TR immunoreactivity in mammalian TSH cells (37) confirms direct action of thyroid hormones in cultured TSH cells in teleosts (1, 2). The percentage of TSH cells possessing TRß transcripts was relatively higher in IM than M males, whereas no change was seen in TRß transcript levels in the pituitary of M fish (38). Thus, TSH cells and thyroid hormones may play an important role during early maturation. Indeed, it has been shown that thyroid hormones are essential during puberty in sheep (39), and probably in tilapia (17) as well.

GH family
The direct action of E2 on GH cells seen in cultured pituitary of teleost (4) is also supported by the presence of ER transcripts (40) and immunoreactivity (33) in mammalian GH cells. The higher percentage of GH cells possessing ER (ERß and ER{alpha} + ß) transcripts in the IM stage emphasizes the role of E2 in the regulation of GH cells during IM stages. The existence of GH receptors in the gonads of tilapia (41) emphasizes the role of GH in gonadal maturation, and a link between E2/ER and GH cell regulation.

Our demonstration of GR transcripts in GH cells and GR immunoreactivity in the proximal pars distalis, where GH cells reside, supports direct effects of cortisol on GH gene expression at the pituitary level (3). The stimulatory effect of thyroid hormone on GH gene expression in tilapia pituitary (42), in the absence of TR in GH cells, is probably mediated through ER because thyroid hormones can compete with E2 for the activation of estrogen response element (43).

In vitro studies in teleost show that E2 regulates PRL gene expression (4, 44), which is supported by the presence of ER transcripts (45) and ER immunoreactivity (46) in PRL cells, and estrogen response element in the promoter region of PRL gene in mammals (47). The regulation of PRL cells by E2 also suggests its role in reproduction, besides freshwater adaptation in teleost (48). The large percentage of PRL cells that express only ERß gene during the IM stage, together with the ERß immunoreactivity seen during fetal stages in rodents (46), supports the role of ERß in the regulation of PRL cells in the IM stage. On the contrary, during the M stage, a large percentage of PRL cells possessed ER{alpha} or ER{alpha} + ß transcripts. These results suggest differential usage of ER{alpha} and ERß, which could account for the difference in PRL gene response to E2 in the two reproductive stages in teleost (49). The coexpression of ER{alpha} + ß genes in a high percentage of PRL cells in the M stage suggests a possible need of both ERs for functionality.

The higher percentage of SL cells expressing ER{alpha} + ß genes and the significantly high ER{alpha} and ERß transcript levels in M males accord well with the role of SL cells in sexual maturation (50, 51, 52) and their regulation by sex steroid hormones (4).

POMC family
The primary role of {alpha}-MSH is melanocyte stimulation in amphibians and teleosts. The absence of GR transcripts in tilapia MSH cells suggests that the dynamic change in body color in tilapia under stressful conditions (53) occurs due to direct neural control, and not by plasma cortisol. The significantly high amount of ER{alpha} transcripts in MSH cells in IM males emphasizes the role of MSH in the control of reproduction in tilapia (17), as in mammals (54). An interesting observation is the expression of TRß gene in a large percentage of MSH cells in IM males, which suggests a novel role for the thyroid hormone in the regulation of MSH cells in tilapia.

Interactions between sexual maturation and stress response have been documented in fish (55) and rats (56). However, there is no documentation of GR in ACTH cells of teleost. The absence of GR transcripts in ACTH cells of the tilapia suggests that a hypothalamic pathway may be involved in the regulation of ACTH cells in fish. Alternatively, the amount of GR transcripts may be below a detectable level in teleost, and a regulatory mechanism, similar to that in mammals (29, 57), may exist at the level of ACTH cells. The present results also suggest a possible role in vertebrate species of E2 in the regulation of a subpopulation of ACTH cells through ERß.

In summary, the high sensitivity of our single-cell real-time PCR technique has allowed us to examine the expression of multiple nuclear receptors in single cells in dense layers of heterogenous cell populations, which would have been difficult to examine using traditional methods such as in situ hybridization and immunocytochemistry. Furthermore, this technique has been useful to examine expressions of nuclear receptor genes in TSH and ACTH cells, which otherwise are difficult to study due to their low abundance.

E2 appears to be the main steroid hormone that regulates all pituitary cell types. In addition, there are indications that T might be aromatized to E2 for the control of FSH, LH, and TSH cells in the M stage. Stress hormones could affect growth and reproduction directly at the level of FSH, LH, and GH cells through a GR pathway. A novel finding in the present study is a possible role for TRß in the regulation of TSH and MSH cells during early sexual development.

The coexpression of different combinations of genes for nuclear receptors in subpopulations of pituitary cell types suggests multiple pathways of steroid/thyroid hormone actions at the level of the pituitary.


   Footnotes
 
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant 14580777 (to I.P.) and Grant 4370025 (to Y.S.)], and the Ministry of Health, Labour and Welfare [Grant H16-kagaku-002 (to I.P.)].

The initial part of this project was conducted at the Nippon Medical School, Tokyo, Japan.

The nucleotide sequences reported in this paper have been deposited in the DDBJ/EMBL/GenBank database [accession nos. AB110981 (ERß), AB245405 (GR1), and AB245406 (GR2b)].

Disclosure Statement: The authors have nothing to declare.

First Published Online September 6, 2007

Abbreviations: AR, Androgen receptor; DIG, digoxigenin; E2, 17ß-estradiol; ER, estrogen receptor; GR, glucocorticoid receptor; IM, immature; LCM, laser capture microdissection; M, mature; POMC, proopiomelanocortin; PRL, prolactin; SL, somatolactin; T, testosterone; TR, thyroid hormone receptor.

Received March 6, 2007.

Accepted for publication August 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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