ARTICLE |
Correspondence to: Gérard Morel, CNRS UMR 5578, Bât. 404, 3ème étage, Université Claude Bernard-Lyon l, 43 Bd 11 Novembre 1918, F69622 Villeurbanne Cedex, France. E-mail: Gerard.Morel@univ-lyonl.fr
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Summary |
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Growth hormone (GH) exerts its immune effects on mature lymphocytes through an autocrine/paracrine mechanism. We investigated the prenatal synthesis of GH mRNA in rat lymphoid organs using the sensitive in situ RT-PCR methodology. We show that GH transcripts are detectable in the thymus and liver of the 18-day fetus. At this stage, all thymocytes are immature and express the GH gene. In fetal liver, GH gene expression was localized in circulating lymphocytes and in hematopoietic cells surrounding GH mRNA-negative hepatocytes. In situ GH gene expression in fetal lymphoid organs was confirmed by in vitro RT-PCR showing that the amplified product from fetal lymphoid tissues was similar to the product obtained from the pituitary. Moreover, GH gene expression was detected in the thymus, spleen, and ileum Peyer's patches of adult rat, with a localization restricted to the lymphocytes and endothelial and smooth muscle cells of blood vessels. The autocrine/paracrine expression of the GH gene by lymphoid and hematopoietic cells during fetal growth might influence the generation of regulatory cells involved in immunity and hematopoiesis. (J Histochem Cytochem 49:347354, 2001)
Key Words: lymphoid organs, hematopoietic cells, rat fetus, growth hormone, in situ RT-PCR
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Introduction |
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PITUITARY GROWTH HORMONE (GH) has a direct role in the proliferation and functions of most immune cells and participates in host defense against microbial infection and tumor formation (
It was assumed for decades that embryonic and fetal growth were independent of maternal or fetal GH. However, although pituitary GH synthesis starts on Day 19 of gestation (
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Materials and Methods |
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Tissue Preparation
Rat tissues were obtained from male Wistar rats (2 months) housed at 22C on a 12-hr light/dark cycle and 18-day-old (E18) fetuses (Iffa Credo; l'Arbresle, France). Food and water were available ad libitum. Whole fetuses were obtained after cesarean section and fetal tissues by microsurgery. For RNA isolation, adult and fetal rat tissues were stored at -80C until used. For in situ RT-PCR, adult male rat tissues and whole fetuses were fixed in 4% paraformaldehyde and paraffin-embedded. Sections 7 µm thick were mounted on in situ RT-PCR glass slides.
Oligonucleotide Primers and Probes
Two 25-mer oligonucleotide primers, HPLC-purified, were synthesized (Eurobio; Les Ulis, France) and used for the RT-PCR. The antisense primer (nt 135160: 5'-CGCAAAGCGGCGACACTTCATGACC-3') is located at the end of the fifth exon and the sense primer (nt 643668: 5'-CCAGTCTGTTTGCCAATGCTGTGCT-3') is in the second exon (
RT-PCR
Total RNA was extracted by the guanidium isothiocyanatephenolchloroform method. One µg of total RNA was primed using polyT and M-MLV reverse transcriptase (Gibco BRL; Cergy Pontoise, France). RT reaction products were amplified by 30 cycles of PCR on a thermal cycler (Perkin) using 1 U/µl Taq DNA polymerase (Eurobio). The hot start method was employed and each cycle consisted of denaturating at 94C for 45 sec, annealing at 69C for 45 sec, and extension at 72C for 1 min, followed by 7 min at 72C. Amplified products were analyzed on a 1.5% agarose gel.
In Situ Hybridization
Dewaxed sections were treated with 5 µg/ml proteinase K (37C, 15 min) and hybridized overnight at 40C with 30 pmoles of antisense-labeled probe/ml of hybridization buffer as described (
In Situ RT-PCR
Dewaxed sections treated with proteinase K were dehydrated and air-dried. In situ RT reaction buffer (100 µl) contained 50 mM Tris-HCl, 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 1 mM of each deoxynucleotide triphosphate, 1 µM antisense primer, 10 U RNase inhibitor, and 200 U M-MLV. The sections were covered with 40 µl of the reaction buffer, sealed with amplicover discs and amplicover clips, and incubated at 42C for 1 hr. Coverclips were removed and sections were washed in 0.1 M phosphate buffer, dehydrated, and air-dried. In situ PCR reaction buffer (100 µl) contained 10 mM Tris-HCl, 50 mM KCl, 3 mM MgCl2, 0.5 mM of each deoxynucleotide triphosphate, 1 µM of each primer, and 20 U Taq DNA polymerase. The sections were covered with 40 µl of this reaction buffer, sealed, and placed on a PerkinElmer thermal cycler. A total of 25 PCR cycles were performed under the same conditions as for liquid PCR. Sections were then fixed with 4% paraformaldehyde (15 min) and washed as described in the ISH procedure. The sections were then hybridized overnight at 40C with 30 pmoles of sense- and antisense-labeled probes/ml after 3-min denaturation at 94C. Washing steps were done as described for the ISH procedure. Several controls were performed: (a) ISH only, (b) omission of the reverse transcriptase, and (c) omission of the Taq DNA polymerase.
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Results |
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Expression of GH mRNA in Rat Lymphoid Tissues by Liquid RT-PCR Analysis
The high expression of GH gene in pituitary was used as a positive control (Fig 1, Lane 2). The predicted band of 533 bp was found in adult spleen (Fig 1, Lane 3), thymus (Fig 1, Lane 4), ileum Peyer's patches (Fig 1, Lane 5), fetal thymus (Fig 1, Lane 6), and fetal liver (Fig 1, Lane 7). No band was detected in adult liver tissue (Fig 1, Lane 8) or when the Taq polymerase was omitted (Fig 1, Lane 9). We confirmed the identity of the PCR product after Rsa I digestion, giving two predicted bands of 290 and 243 bp (data not shown).
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Localization of GH mRNA by ISH and In Situ RT-PCR
The positive signal indicating the expression of GH gene appears as a dark purple precipitate (Fig 2 Fig 3 Fig 4). The in situ RT-PCR methodology was validated on pituitary sections. The signal obtained (Fig 2A) was stronger compared to the ISH signal (Fig 2B) and was restricted to the somatotrophs, representing about 40% of the pituitary cells (
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At Day 18 of gestation, the majority of GH mRNA-positive cells in the thymus were localized at the periphery of thymic lobes in the fetal lymphocytes, whereas cells in the center exhibited a weak in situ RT-PCR signal (Fig 4A). No signal was detected when the Taq polymerase was omitted in the PCR reaction (Fig 4B). In the fetal liver, strong expression of the GH gene was detected in hematopoietic cells that surrounded large GH mRNA-negative hepatocytes (Fig 4C and Fig 4E). When direct ISH was used for detection of GH gene expression, the positive signal could be detected only within the lymphocytes circulating in the fetal liver sinusoidal spaces (Fig 4D). Finally, we also found that lymphocytes from the intracardiac cavity express the GH gene, in contrast to red blood cells (Fig 4F).
Discussion
In this study, GH gene expression was characterized in fetal and adult rat lymphoid tissues, using RT-PCR and the highly sensitive in situ RT-PCR. The primers used guaranteed the amplification of almost the entire GH transcript. Both sense and antisense primers of GH span two exons, and intronic regions between the primers are approximately 1100 bp. Therefore, amplification of genomic DNA was impossible. The PCR product of 533 bp identified in immune organs is of GH gene origin and is similar to the pituitary product. We identified lymphocytes as the major GH mRNA-positive cells in lymphoid organs of the fetal and adult rats. In the adult thymus, the site of T-lymphocyte differentiation, GH mRNA synthesis was localized mainly in cells of the cortex, although some cells of septa and medulla were GH mRNA-positive. Lymphocytes expressing the GH gene were identified according to their localization in the cortex, where they are in the majority compared to the medulla. In our study, rat GH gene expression could not be detected in reticular or epithelial cells, in contrast to studies on human thymus (
Our study has demonstrated for the first time that the GH gene is expressed by fetal lymphoid tissues. The fetal GH gene was expressed principally in thymic and blood lymphocytes. The demonstration of GH synthesis by fetal lymphocytes is of importance and suggests that the hormone could directly regulate immune and hematopoietic functions during fetal growth. Experiments that strongly suggest no role for GH in fetal development are disputed by the demonstration that GH receptors are expressed in major rat organs at fetal stages E18 (
Received for publication June 29, 2000; accepted October 24, 2000.
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